Frontiers in Medicinal Chemistry , Volume (5)

Frontiers in Medicinal Chemistry Bentham Science Publishers Ltd. http://www.bentham.org/fmc

Volume 5, 2009 Contents Preface

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Contributors

vi Central Nervous System

Always Around, Never the Same: Pathways of Amyloid Beta 1 Induced Neurodegeneration Throughout the Pathogenic Cascade of Alzheimer’s Disease Jeroen J.M. Hoozemans, Sidhartha M. Chafekar, Frank Baas, Piet Eikelenboom and Wiep Scheper Novel Neuroprotective Therapies for Alzheimer’s and 15 Parkinsons’s Disease Kirubakaran Shanmugam, Annette E. Maczurek, Megan L. Steele, Obdulio Benavente-García, Julián Castillo and Gerald Münch 58

Improving Memory: A Role for Phosphodiesterases Arjan Blokland, Rudy Schreiber and Jos Prickaerts Biologics Progress in Current Non-Viral Carriers for Gene Delivery

81

Yoko Shoji-Moskowitz, Daisuke Asai, Kota Kodama, Yoshiki Katayama and Hideki Nakashima contd……

Protein Transduction: Cell Penetrating Peptides and Their 98 Therapeutic Applications Kylie M. Wagstaff and David A. Jans Design of Peptide-Based Vaccines for Cancer Geoffrey A. Pietersz, Apostolopoulos

Dodie

S.

Pouniotis

127 and

Vasso

Oncology Inhibitors of Protein: Geranylgeranyl Transferases

167

Farid El Oualid, Gijs A. van der Marel and Mark Overhand Voltage-Gated Sodium Channels: New Targets in Cancer 234 Therapy? Ludovic Gillet, Sébastien Roger, Marie Potier, Lucie Brisson, Christophe Vandier, Pierre Besson and Jean-Yves Le Guennec Antiinfectives Recent Advances in Antiviral Agents: Antiviral Drug 257 Discovery for Hepatitis Viruses Kyuichi Tanikawa Cardiovascular NO-Releasing Hybrids of Cardiovascular Drugs

272

Alma Martelli, Simona Rapposelli, Maria C. Breschi and Vincenzo Calderone Endocrinology Trends in the Development of New Drugs for Treatment of 309 Benign Prostatic Hyperplasia Katarzyna Kulig and Barbara Malawska contd….

Pain, Inflammation, and Immunology Targeting the Prostaglandin D2 Receptors DP1 and CRTH2 350 for Treatment of Inflammation Trond Ulven and Evi Kostenis Enabling Technologies Privileged Structures as Leads in Medicinal Chemistry

381

Luca Costantino and Daniela Barlocco Acetylcholinesterase Reprised: Molecular Modeling with the 423 Whole Toolkit Gerald H. Lushington, Jian-Xin Guo and Margaret M. Hurley

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PREFACE This 5th volume of the book series “Frontiers in Medicinal Chemistry” consists of a compendium of useful reviews on topics of wide interest to researchers in the fields of Medicinal Chemistry, Drug Discovery and Chemical Biology. The contributions are separated into different therapeutic areas, along with two reviews on general enabling technologies. The objective of “Frontiers in Medicinal Chemistry” is to periodically publish collections of comprehensive overviews based on areas of topical interest, written by leading experts in their respective fields. Initial versions of these reviews have been published in the journals Current Medicinal Chemistry, Current Topics in Medicinal Chemistry, and Current Pharmaceutical Design, and the authors have had the opportunity to update their original manuscripts prior to publication in the current volume. The initial section of this volume entails three reviews in the area of Central Nervous System therapeutics. The first by Scheper et al. summarizes current understanding on the role of the
-amyloid peptides in Alzheimer’s disease. The second by Münch and colleagues entails neuroprotection for both Alzheimer’s and Parkinson’s diseases, and the third by Blokland et al. is on the topic of phosphodiesterase inhibition to improve cognition. The Biologics section is next, containing reviews by Shoji-Moskowitz et al. on non-viral approaches for gene therapy, Jans and Wagstaff on peptides that can cross a membrane barrier, and Apostolopoulos and co-workers on peptidic vaccines for cancer. The next section has two reviews in the area of Oncology. The first by Overhand et al. deals with geranylgeranyl transferases, the second by Besson el al. focuses on voltage-gated sodium channels as targets for cancer. There are then four reviews that follow covering separate therapeutic areas. The first is a contribution in the area of Infectious Disease by Tanikawa on hepatitis B and C, Cardiovascular Diseases by Calderon et al. on nitric oxide releasing drugs, Endocrinology covering benign prostatic hypertrophy by Malawska and Kulig, and Inflammation on prostaglandin D2 receptor modulators by Ulven and Kostenis.

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Finally, topics in the last section on Enabling Technologies include the concept of privileged structures in medicinal chemistry by Costantino and Barlocco, and the use of molecular modeling with application to acetylcholinesterase by Lushington et al. We hope that Frontiers in Medicinal Chemistry becomes an invaluable resource for the benefit of researchers in various aspects of therapeutic drug discovery. We wish to thank the authors who have contributed to this volume, and Mahmood Alam, Samina Khan, Qurat-ulAin and Matthew Honan of Bentham Science Publishers for their efforts.

ALLEN B. REITZ Fox Chase Chemical Diversity Center, Inc. Doylestown, PA 18902 USA

M. IQBAL CHOUDHARY International Center for Chemical and Biological Sciences (HEJ Research Institute of Chemistry, Dr. Panjwani Center for Molecular Medicine & Drug Research) University of Karachi Karachi-75270 Pakistan

ATTA-UR-RAHMAN International Center for Chemical and Biological Sciences (HEJ Research Institute of Chemistry, Dr. Panjwani Center for Molecular Medicine & Drug Research) University of Karachi Karachi-75270 Pakistan

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Contributors Jeroen J.M. Hoozemans

Neurogenetics Laboratory, Amsterdam, The Netherlands

Sidhartha M. Chafekar

Neurogenetics Laboratory, Amsterdam, The Netherlands

Frank Baas

Neurogenetics Laboratory, Department of Neurology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Piet Eikelenboom

Department of Neurology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Wiep Scheper

Neurogenetics Laboratory, Amsterdam, The Netherlands

Kirubakaran Shanmugam

Department of Pharmacology, School of Medicine, University of Western Sydney, Campbelltown, Australia

Annette E. Maczurek

Department of Pharmacology, School of Medicine, University of Western Sydney, Campbelltown, Australia

Megan L. Steele

Department of Pharmacology, School of Medicine, University of Western Sydney, Campbelltown, Australia

Obdulio Benavente-García

Furfural Español-Nutrafur, Alcantarilla, Murcia, Spain

Julián Castillo

Furfural Español-Nutrafur, Alcantarilla, Murcia, Spain

Gerald Münch

Department of Pharmacology, School of Medicine, University of Western Sydney, Campbelltown, Australia

Arjan Blokland

Department of Psychology and Neuroscience, EURON, Maastricht University,P.O. Box 616, 6200 MD Maastricht, The Netherlands

Rudy Schreiber

Sepracor, Inc., 84 Waterford Drive, Marlborough MA, 01752, USA

Jos Prickaerts

Department of Psychiatry and Neuropsychology, Brain & Behavior Institute, EURON, Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands

Yoko Shoji-Moskowitz

CREST, JAPAN;FOLIGO Therapeutics, Inc. USA

Daisuke Asai

CREST, JAPAN; Department of Microbiology, St. Marianna University School of Medicine, Japan, Department of Biomedical Engineering, Duke University, USA

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Kota Kodama

CREST, JAPAN; FOLIGO Therapeutics, Inc. USA Creative Research Institution, Hokkaido University, Japan

Yoshiki Katayama

CREST, JAPAN; Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Japan

Hideki Nakashima

CREST, JAPAN; Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Japan

Kylie M. Wagstaff

Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia

David A. Jans

ARC Centre of Excellence for Biotechnology and Development, Australia

Geoffrey A. Pietersz

Bio-organic and Medicinal Chemistry Laboratory, Australia

Dodie S. Pouniotis

Immunology and Vaccine Laboratory, Centre for Immunology, Burnet Institute, Commercial Road, Melbourne, VIC, 3004, Australia

Vasso Apostolopoulos

Immunology and Vaccine Laboratory, Centre for Immunology, Burnet Institute, Commercial Road, Melbourne, VIC, 3004, Australia

Farid El Oualid

Netherlands Cancer Institute, Division of Cell Biology, 1066 CX Amsterdam, The Netherlands

Gijs A. van der Marel

Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands

Mark Overhand

Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands

Ludovic Gillet

U921 Inserm, Nutrition Croissance Cancer; Université de Tours; Faculté de Médecine, 10 Bd Tonnellé, 37032 TOURS France

Sébastien Roger

U921 Inserm, Nutrition Croissance Cancer; Université de Tours; Faculté de Médecine, 10 Bd Tonnellé, 37032 TOURS France

Marie Potier

U921 Inserm, Nutrition Croissance Cancer; Université de Tours; Faculté de Médecine, 10 Bd Tonnellé, 37032 TOURS France

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Lucie Brisson

U921 Inserm, Nutrition Croissance Cancer; Université de Tours; Faculté de Médecine, 10 Bd Tonnellé, 37032 TOURS France

Christophe Vandier

U921 Inserm, Nutrition Croissance Cancer; Université de Tours; Faculté de Médecine, 10 Bd Tonnellé, 37032 TOURS France

Pierre Besson

U921 Inserm, Nutrition Croissance Cancer; Université de Tours; Faculté de Médecine, 10 Bd Tonnellé, 37032 TOURS France

Jean-Yves Le Guennec

U921 Inserm, Nutrition Croissance Cancer; Université de Tours; Faculté de Médecine, 10 Bd Tonnellé, 37032 TOURS France

Kyuichi Tanikawa

Professor Emeritus, Kurume University, President, International Institute for Liver Research, International Institute for Liver Research, Alley II 202, Tsubuku-honmachi 636-1, Kurume 830-0047, Japan

Alma Martelli

Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie,Università degli Studi di Pisa, Via Bonanno, 6, I-56126 Pisa, Italy

Simona Rapposelli

Dipartimento di Scienze Farmaceutiche, Università degli Studi di Pisa,Via Bonanno, 6, I-56126 Pisa, Italy

Maria C. Breschi

Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie,Università degli Studi di Pisa, Via Bonanno, 6, I-56126 Pisa, Italy

Vincenzo Calderone

Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie,Università degli Studi di Pisa, Via Bonanno, 6, I-56126 Pisa, Italy

Katarzyna Kulig

Department of Physicochemical Drug Analysis, Chair of Pharmaceutical Chemistry, Jagiellonian University Medical College, Medyczna 9 str., 30-688 Kraków, Poland

Barbara Malawska

Department of Physicochemical Drug Analysis, Chair of Pharmaceutical Chemistry, Jagiellonian University Medical College, Medyczna 9 str., 30-688 Kraków, Poland

Trond Ulven

Department of Physics and Chemistry, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark

Evi Kostenis

Institute for Pharmaceutical Biology, University of Bonn, Nussallee 6, 53115 Bonn, Germany

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Luca Costantino

University of Modena e Reggio Emilia, Dipartimento di Scienze Farmaceutiche, Via Campi 183, 41100 Modena, Italy

Daniela Barloccob

University of Milano, Dipartimento di Scienze Farmaceutiche “Pietro Pratesi”, Via L. Mangiagalli 25, 20133 Milano, Italy

Gerald H. Lushington

Molecular Graphics and Modeling Laboratory, University of Kansas, Lawrence, KS 66045 USA

Jian-Xin Guo

Molecular Graphics and Modeling Laboratory, University of Kansas, Lawrence, KS 66045 USA

Margaret M. Hurley

Weapons and Materials Research Directorate, U.S. Army Research Laboratory, Aberdeen Proving Ground, MD 21005, USA

Frontiers in Medicinal Chemistry, 2010, 5, 1-14

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Always Around, Never the Same: Pathways of Amyloid Beta Induced Neurodegeneration Throughout the Pathogenic Cascade of Alzheimer’s Disease Jeroen J.M. Hoozemansa, Sidhartha M. Chafekara, Frank Baasa,b, Piet Eikelenboomb and Wiep Schepera,* a

Neurogenetics Laboratory, bDepartment of Neurology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Abstract: There is an increasing amount of evidence showing the importance of intermediate aggregation species of amyloid
(A
) in the pathogenic cascade of Alzheimer’s disease (AD). Different A
assembly forms may mediate diverse toxic effects at different stages of the disease. Mouse models for AD suggest that intraneuronal accumulation of A
oligomers might be involved in AD pathogenesis at a very early stage of the disease. The detrimental effect of oligomeric A
on synaptic efficacy is suggested to be an early event in the pathogenic cascade. Also early neuronal responses as activation of the unfolded protein response are processes likely to be associated with the increased occurrence of oligomeric or low fibrillar A
in AD pathology. In later stages of AD pathology, the fibrillarity of A
increases, concomitantly with a neuroinflammatory response, followed by tau related neurofibrillary changes in end stage pathology. We will review recent findings in in vitro cell models, in vivo mouse models, and post mortem AD brain tissue in view of the effects of different A
peptide species on neurodegeneration during AD pathogenesis. Insight into the role of different A
species during AD pathogenesis is essential for the development of disease modifying drugs and therapeutical strategies.

Keywords: Alzheimer’s disease, amyloid
, aggregation, ER stress, neuroinflammation, neurotoxicity, pathological cascade, unfolded protein response 1. INTRODUCTION Alzheimer’s disease (AD) is characterized by progressive memory loss and other cognitive and behavioral deficits and is the most common cause of dementia. Deposits of aggregated proteins are a prominent neuropathological hallmark of AD: intracellular aggregates of tau in the neurofibrillary tangles and extracellular aggregates of
-amyloid (A
) in the senile plaques. AD thus represents a prime example of a protein folding disease [1, 2]. According to the widely supported amyloid cascade hypothesis, increased levels or a conformational change of A
is the primary cause of AD pathogenesis [3, 4]. A
is formed *Corresponding author: Tel: 0031-20-5664959; Fax: 0031-20-5669312; E-mail: [email protected] Allen B. Reitz / Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.

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by proteolytic cleavage of the amyloid precursor protein (APP) by a combined action of
and -secretases [5]. Important support for the central role of A
in AD comes from the familial variants of the disease. To date, mutations in three genes (APP, Presenilin 1 and Presenilin 2) have been shown to cause AD in an autosomal dominant fashion [6]. All these mutations can directly be linked to altered or increased A
formation, and more specifically to increased A
1-42 levels. Mutations in APP lead to preferential amyloidogenic processing and presenilins play a role in the proteolytic cleavage of APP. In addition, Down’s syndrome patients have a trisomy of chromosome 21, which contains the APP gene and typically develops early onset AD [7]. Stronger evidence for a gene dosage effect comes from a recent report on a family that carries a duplication of the APP locus only, resulting in early onset AD [8]. Alzheimer’s disease has an average duration of 8 years after clinical symptoms occur. During this time but also long before clinical symptoms are apparent, the brain is subject to pathological changes. Study of post mortem human brain tissue has resulted in a pathological staging of Alzheimer’s disease, the Braak score [9, 10]. Although the Braak score does not represent a temporal scale, there is good correlation between the Braak score for neurofibrillary changes and the clinical course of AD as determined by the cognitive status [11, 12]. This suggests that the pathological staging indeed reflects the temporal sequence of events in the pathogenic cascade. The first two stages of the Braak score for neurofibrillary changes, stage I and II, present with mild to severe alteration of the transenthorinal region. Stages III and IV are characterized by the severe involvement of the enthorinal and transenthorinal region, while the isocortex gradually becomes mildly affected. Stages V and VI are marked by the severe occurrence of neurofibrillary changes in the isocortex. While in Braak stage I to III the temporal cortex is almost devoid of neurofibrillary changes, lowfibrillar A
deposits can be detected in these cases, indicating a role for A
very early in the disease. Recent data suggest that oligomeric A
, and especially intracellular localized A
, plays a significant role in AD pathology. However, difficulty in the detection of oligomeric and intracellular A
in AD mouse models and even more so in AD brain tissue, has hampered the study of the role of these A
species in the pathogenic cascade. It is still elusive in what stage of disease these A
oligomers are involved. In view of development of therapeutics directed against oligomeric forms of A
it is crucial to place the A
oligomers in AD pathogenesis and to define associated pathological hallmarks. In this review we will show that AD associated disease mechanisms like the stress response in the endoplasmic reticulum (ER) and neuroinflammation can be placed in different disease stages alongside the occurrence of different A
conformation changes. Compiling information from human brain tissue, mouse models and in vitro studies, we will show that in all these stages A
appears to show a different face with regard to the aggregation state and the cellular processes that are affected. We have combined this in a model explaining the role of the different conformations of A
in disease mechanisms occurring throughout the pathogenic cascade of AD. 2. A
AGGREGATION Electron microscopy and more recently (time-lapse) atomic force microscopy analyses have greatly contributed to visualizing different A
aggregation intermediates that are formed in vitro before formation of the mature 6- to 10-nm diameter fibrils with the characteristic cross-
structure [13-15]. The aggregation conditions in vitro are very artificial, using high concentrations of pure A
, therefore caution is warranted in translating these data directly to the aggregation process in human brain.

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Here we will use the definitions for A
aggregation forms as proposed by Ross and Poirier [16]; oligomeric aggregates are small globular assemblies, protofibrils are soluble fibrilshaped structures, thinner and shorter than a mature fibril and fibrillar aggregates are stable, insoluble and highly structured aggregates. Although oligomers and protofibrils are usually considered as intermediates in fibril formation, there is no definite proof that they are “on” the fibrillization pathway or whether they represent a pathway “off” fibril formation. Fibrillization of A
appears to proceed in two phases: a rate-limiting nucleation step in which a “seed” for further aggregation is formed, followed by an extension phase [17, 18]. Fibril growth in vitro proceeds by the addition of monomers to the ends of the fibrils [12, 19, 20]. The aggregation process is highly dependent on the A
concentration. In vitro, unphysiologically high concentrations are required, but it is likely that in vivo such a high concentration can be achieved locally in intracellular compartments or by binding to proteins or lipids [17]. A
1-42 was shown to nucleate easier than A
1-40 [17], which may be why subtle changes in the A
1-42/1-40 ratio induce AD pathology. Transgenic mice that secrete high levels of A
1-42 in the absence of APP overexpression, show massive amyloid pathology, in contrast to transgenic A
1-40 mice [21]. These experiments underscore the importance of A
1-42 in amyloid deposition, and it was suggested that A
1-42 is required for nucleation in vivo. A very interesting insight in A
aggregation kinetics comes from the Arctic APP mutation. The conversion of the amino acid Glutamic acid to Glycine at position 22 in A
is the only mutation in the A
peptide itself that gives rise to a classical AD phenotype, despite resulting in lower A
1-42 levels [22]. This was shown not to be due to increased fibrillization, but rather to a change in aggregation kinetics that accelerates protofibril formation [23]. The exact nature of the nucleation seed in vivo is not known. It is very likely that in the crowded environment inside neurons or the brain parenchyma, binding of A
to other proteins or lipids contributes to seeding. For example A
bound to the ganglioside GM1 was shown to have a different conformation that appeared to act as a seed for aggregation, thereby greatly enhancing fibril formation [24]. 3. INTRA- VERSUS EXTRA-CELLULAR A
A
is secreted into the extracellular space and ultimately deposited in the extracellular plaques in AD brain. This in combination with the toxicity of extracellular A
in vitro, provides strong evidence for A
exerting its toxic action from the outside of the cell. More recently, the role of intracellular A
has gained a lot of attention and intraneuronal A
is currently viewed as an early event in AD pathogenesis prior to extracellular A
deposition [25]. A
is produced intracellularly in a variety of subcellular compartments, including the endoplasmic reticulum (ER), trans-Golgi network and lysosomes. Therefore, by definition, it spends time inside neurons on its way out. LaFerla and colleagues were the first to describe the presence of intracellular A
in neurons in AD brain [26] and since then several reports showing intraneuronal A
accumulation have been published (reviewed in [27]). In human neurons intracellular oligomeric A
aggregates have been detected [28]. In AD brain, neurons were shown to contain thioflavin Sreactive deposits [26], suggesting accumulation of fibrillar A
although this finding was not confirmed by others [29]. It is not known whether intracellular aggregation of A
is a prerequisite for toxicity. In human post mortem brain A
is most easily detected in the endo-/lysosomal system [30-32], possibly because the acidic environment favours fibrillization. In addition, it has

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been found freely distributed in the perikaryon of neurons, indicating that A
can be translocated from membranous compartments into the cytosol [33]. It is unclear whether A
in the endo-/lysosomal system is derived from re-uptake from the extracellular space or whether it is directly formed in these compartments. In transgenic mice, intraneuronal A
aggregates were found associated with the ER, Golgi and mitochondria [34]. It appears that intracellular A
deposition is dependent on the levels of A
, as intracellular A
pathology is more prominent in models with higher A
production. This may imply that if the concentration is sufficiently high, A
is retained inside the cell, maybe because the high local concentration causes it to aggregate in intracellular compartments. Evidence for a correlation between the level of A
and the site of accumulation comes from studies in a transgenic mouse model that produces very high levels of A
due to the presence of four familial FAD mutations. These mice exhibit intraneuronal rather than extracellular thioflavin-S positive A
aggregates [35]. Interestingly, these mice show more extensive neuronal loss than models that predominantly have extracellular deposits, suggesting that intracellular A
is more toxic. Also a transgenic mouse harboring the Swedish APP mutation, leading to increased A
levels, combined with the Arctic mutation (tg-APPArcSwe model) results in intraneuronal A
aggregation [36]. In this respect it is interesting to mention sporadic Inclusion Body Myositis (sIBM). In sIBM myotubes, A
exclusively forms intracellular inclusion bodies that co-localize with several ER chaperones [37]. The cause of the intracellular localisation of A
deposits in sIBM is not known, but it is likely that the different cellular environment is an important determinant. It is tempting to speculate that the local concentration of intracellular A
is much higher in muscle cells, possibly because their secretory capacity will be lower than that of neurons. In any case, the sIBM example indicates that also in man, conditions can be found where aggregation and deposition of A
is exclusively intracellular with degeneration as result. Intuitively it seems logical that there is equilibrium between extra- and intracellular pools of A
. Intracellular A
decreases with plaque load [29, 32], although more intraneuronal A
correlating with a higher plaque load has also been reported [38]. More direct experimental evidence for a balance between extra- and intracellular A
pools comes from A
vaccination experiments in the triple transgenic AD mouse model. This model shows intracellular A
accumulation before plaque formation [39]. In the same model, immunization results in clearance of extracellular A
before the clearance of intracellular A
and after dissipation of the immunizing antibody the intraneuronal accumulation of A
reappeared before the extracellular A
deposits [40]. Because antibodies obviously can not enter the intracellular space, this points to a direct connection between intra- and extracellular A
pools. 4. MECHANISMS OF A
INDUCED NEURODEGENERATION In vitro, both oligomeric as well as fibrillar preparations of A
induce cell death in virtually any cell in culture, but oligomeric preparations do so more potently. The mechanism underlying effects of A
on cell viability is not exactly known, although a plethora of cellular processes is disturbed by A
. It is likely that several degenerative pathways act simultaneously. Here we will focus on cellular processes that can be attributed to specific A
forms and can be placed in the pathogenic cascade. We will compare recent data from different experimental models in order to show the significance of the different aggregation species in the induction of specific neurodegenerative pathways in the pathological cascade in AD.

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4.1. Synaptic Dysfunction There are several lines of evidence indicating that synaptic dysfunction is one of the first presentations of AD pathology and probably of A
induced toxicity. Synaptic dysfunction in human or transgenic animal brains manifests for example as deficits of neurotransmitters, decreased synapse density and changed electrophysiological parameters (reviewed in [41]). For example the decreasing number of synapses in AD brain correlates better with disease progression than cell loss or plaque load [42]. Especially mouse models for AD have been very instructive in this matter. Many of these models show loss of synapses before plaque formation and the actual loss of neurons [43, 44]. Recent studies using two-photon microscopy elegantly show the manifestation of this in decreased synaptic contacts [45]. These effects are observed before plaque deposition, indicating this may be an effect of nonfibrillar A
. It has been suggested that the earliest cellular changes in AD are caused by intracellular A
. Although there is little doubt that intracellular A
exists, real proof for its role in toxicity is difficult to obtain. A
immunization in a transgenic mouse model that shows intracellular A
accumulation early in the pathogenesis, clears the intracellular A
and cures early cognitive deficits [44]. From these experiments, it is very tempting to conclude a primary role for intracellular A
, however, thusfar experiments really distinguishing between A
acting inside the cell and secreted A
acting from the extracellular space have not been performed. The role of extracellular A
is corroborated by the inhibition LTP by oligomeric A
specifically [46]. This is reversed by passive immunization against oligomeric A
[47]. Oligomeric A
was shown to colocalize with clusters of PSD-95, a marker for post-synaptic terminals, on dendritic arbors of hippocampal cultures [48]. The data further show that A
targets specific synaptic sites, which may provide a basis for the selective loss of synapses in AD. A
may directly affect the presence of receptors in the synaptic cleft, as it has been shown that oligomeric A
induces endocytosis of NMDA receptors [49]. However, other mechanisms as the activation of oxidative/nitrosative stress signaling cascades (reviewed in [50]) or reductions in the levels of proteins involved in synaptic transmission like PSD-95 and the glutamate receptor subunit GluR1 [51] have also been reported. In AD brain, loss of proteins involved in dendritic spine dynamics like the kinase PAK and the spine regulatory protein drebrin is observed [52]. In the same study, oligomeric A
induced loss of PAK activity and drebrin in cultured neurons. In conclusion there are several lines of evidence suggesting that early A
aggregation intermediates are causative to synaptic dysfunction in AD, however this does not exclude a role for intracellular A
. 4.2. Unfolded Protein Response One clear example of aggregation state specific effects is on calcium homeostasis. Oligomeric A
was shown to induce calcium influx [53]. Although the mechanism is unknown, the data suggested that it was not through the formation of pores in the membrane, which had previously been suggested as a mechanism of A
toxicity. It is possible that A
binds to a receptor and thus initiates a signaling cascade. Disturbance of Ca2+ homeostasis could have dramatic effects including defects in synaptic transmission, membrane dynamics and dysfunction of Ca2+ binding proteins. Interestingly, oligomeric A
also induces release of Ca2+ from intracellular stores, i.e. the endoplasmic reticulum [53], which may have severe effects on the function of the ER in protein synthesis and folding. The presence of misfolded proteins in the ER triggers a cellular stress response called the unfolded protein response (UPR) intended to protect the cell against the toxic buildup of misfolded proteins [54, 55]. Recently, our group showed the activation of the UPR in neu-

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rons in AD brain [56]. In the temporal cortex and the hippocampus of AD cases protein levels of the ER chaperone BiP/GRP78 are increased, which is indicative for UPR activation. At the immunohistochemical level intensified staining of BiP/GRP78 was observed in neurons of AD cases. In addition, immunohistochemistry for phosphorylated pancreatic ER kinase (pPERK) and phosphorylated eukaryotic initiation factor 2
(p-eIF2
), a substrate for PERK, indicates activation of the UPR only in AD neurons and not in non-demented control cases Interestingly, we find that Rab6, a protein involved in retrograde trafficking from the Golgi to the ER is upregulated in AD in a manner that correlates strongly with the levels of ER stress [57]. Our data indicate that the Rab6 pathway is a post-ER protein quality mechanism, and therefore it appears that an array of protein quality control in the secretory pathway is activated during AD pathogenesis [27]. Activation of protein quality control in the secretory pathway is in this case not secondary to decreased proteasomal activity, as the latter is observed only in the final stages of AD [58]. Because our data indicate that both activation of the UPR as well as Rab6 upregulation precede tangle formation and occur as early as Braak stage III [57], it is a relatively early response, likely to be mediated by oligomeric or low fibrillar A. Extracellular A peptides can induce the UPR in neuronal cells [59, 60]. In addition, data from our own lab show that oligomeric A, but not fibrillar A induces ER stress [61]. This may imply that if A induces a signaling cascade, and that the signal is transmitted to the ER, possibly via its effects on Ca2+ signaling [62]. An alternative possibility is the uptake of A and concomitant transport to the ER. Our data show preferential uptake of oligomeric A in some cell types, but we found no evidence as yet for actual transport to the ER [63]. Whether the oligomer specific activation of the UPR is caused by disturbed Ca2+ homeostasis remains to be shown. 4.3. Neuroinflammation and Microglial Activation The neuroinflammatory response in AD pathology is closely associated with the deposition of A plaques. Increased levels of cytokines, complement proteins and acute phase proteins can be detected in the vicinity of A plaques [64]. Since there is no apparent influx of leukocytes and immunoglobulins are not detected, it is generally believed that these inflammatory proteins are synthesized locally in the brain [65]. Intracerebral A plaques are associated with complement factors,
1-antichymotrypsin, apolipoprotein E (ApoE), clusterin, serum amyloid P component (SAP) and proteoglycans [66]. The occurrence of these amyloid associated proteins is closely related with the fibrillarity of the A plaques. Some amyloid associated factors, like C1q and ApoE, enhance the aggregation and fibril formation of newly formed A peptides. Since non- to low-grade fibrillar A can have direct neurotoxic effects [67], the binding of amyloid associated factors that enhance fibril formation may be beneficial. A plaques that have accumulated SAP and C1q are associated with clusters of activated microglia [68]. Microglia are considered to be brain-resident macrophages involved in the maintenance of homeostasis within the brain and are rapidly activated in response to neuronal damage and stress [69]. In AD brain, microglia are most likely recruited to remove the A. Cultured microglia can phagocytose A, although their ability to degrade aggregated A is limited [70]. In addition, A associated proteins, such as proteoglycans and complement proteins could also reduce the phagocytosis of A by microglia [71, 72]. While attempting to remove the A, microglia become activated and release reactive oxygen species, pro-inflammatory cytokines, excitotoxins, and proteases, all potentially neurotoxic substances. The A induced microglial activation, as measured by cytokine release is

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reduced by peptides and drugs like minocycline and tetracycline that function as
-sheet breakers [73, 74]. This indicates that the fibrillarity of A
is important for microglial activation. In addition, the neuroinflammatory response and the microglial activation in AD are observed in a disease stage (Braak IV-V) which can be discriminated from other stages in that it involves a reaction to the presence of fibrillar A
. Although the neuroinflammatory response induced by A
could be viewed as a potential contributor to AD neurodegeneration, some inflammatory proteins may have beneficial effects by inducing microglial mediated A
removal [75], or enhancing A
aggregation and fibril formation reducing the levels of neurotoxic non- to low-grade fibrillar Aß. The role of inflammation as a double-edged sword in neurodegenerative disorders attracts much interest in current AD research [76]. This is not surprising because eliminating pathogenic stimuli, such as the removal of fibrillar A
deposits is an essential characteristic of the inflammatory process and interesting from therapeutical point of view. 5. THE MANY FACES OF A
IN THE PATHOLOGICAL CASCADE OF AD For AD therapy it will be important to determine disease stages and to verify at which stage different disease processes are involved. Thusfar, it is not feasible to study pathogenic pathways at the molecular level in living AD patients. The combination of AD human post mortem data, results from AD mouse models, and cellular models can give an indication of which disease mechanisms are involved in different stages of AD pathogenesis. In our own studies we investigated the mid-temporal cortex of non-demented control and AD cases grouped according to the Braak stage for neurofibrillary changes [9]. The activation of the UPR already occurs in Braak stage III-IV and therefore seems a disease mechanism more likely to be related to the occurrence of intra- or extracellular oligomeric A
or low fibrillar A
. Extracellular A
peptides, especially oligomeric A
, induce ER stress in vitro. The increase in UPR activation coincides with the increase of A
deposits, suggesting a direct link between A
and ER stress. In addition, there is a weak colocalization of UPR markers with hyperphosphorylated tau and the increased UPR activation is already present in the temporal cortex when moderate neurofibrillary changes are observed (Braak stage IIIIV). UPR activation increases concomitant with plaques containing hyperphosphorylated tau (neuritic plaques) but precedes the increased incidence of neurofibrillary tangles. In vitro experiments show that induction of the UPR results in activation of glycogen synthase kinase 3
(GSK-3
) [77]. GSK-3
is involved in a remarkably broad spectrum of ADassociated events, especially in the phosphorylation of tau, a prerequisite for the formation of dystrophic neurites and neurofibrillary tangles. The UPR could therefore be directly involved in early A
plaque associated neurofibrillary changes via GSK-3
. The neuroinflammatory response in AD pathology is closely associated with the deposition of A
plaques. The occurrence of amyloid associated proteins is closely related with the fibrillarity of the A
plaques and with the increased occurrence of high fibrillar A
deposits, the neuroinflammatory response is augmented with a central role for activated microglia. This disease stage (Braak IV-V) can be discriminated from previous stages in that it involves a reaction to the presence of fibrillar A
. Although initiated to remove the pathogenic A
aggregates, the neuroinflammatory response could aggravate other disease mechanisms, i.e. increased APP production, A
formation and plaque development, UPR activation, neurofibrillary changes and the formation of neurofibrillary tangles, the latter occurring predominantly in the last stage of the pathological cascade (Braak V-VI).

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Hoozemans et al.

6. THERAPEUTIC OPPORTUNITIES IN THE EARLY STAGES OF AD PATHOLOGY An obvious approach for AD therapy would be to reduce A
production, especially A
1to inhibit fibrillar A
aggregation, and to induce A
clearance. In the next section we will focus on possible therapeutic approaches that target the presence and effects of A
early in the pathological cascade.

42,

Inhibiting the proteolytic cleavage of APP by
- and -secretase inhibitors could block the production of A
, which presumably would slow or halt the progression of the disease. However, much is still elusive on how these secretases are regulated and how they are involved in the pathogenesis of sporadic AD. In addition, the side-effects of -secretase inhibition on for example Notch processing may be detrimental. A variety of experimental studies indicate that a subset of classical non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen, flurbiprofen, indomethacin and sulindac also possess A
1-42-lowering properties in both AD transgenic mice and cell cultures of peripheral, glial and neuronal origin [78-80]. In addition, there are indications that certain NSAIDs inhibit A
1-42 formation by direct modulation of -secretase activity [81]. Recently it is demonstrated that these NSAIDs have an allosteric effect on -secretase by which these drugs selectively reduce A
1-42 but do not affect processing of other -secretase targets [82, 83]. Another approach for therapeutic intervention that could prevent A
aggregation and deposition is immunization with A
peptides or vaccination with antibodies directed against the A
peptide. The first report on immunization of PDAPP transgenic mice with the A
peptide showed reduced amyloid plaque formation [84]. Subsequent reports not only confirmed the potential of immunization strategies to induce clearance of A
, but also indicated the potential of reducing cognitive dysfunction (reviewed by [85]). An immunization approach could work on different conformations of A
and in different pathological stages. First, immunization increases the efflux of A
out of the brain, into the periphery where it is degraded [86]. Peripheral administration of an antibody directed against A
could results in a rapid decrease of oligomeric and low fibrillar A
in the brain in an early stage of the disease. Second, antibodies directed against A
can reduce the cytotoxic effect which A
, especially the oligomeric forms, elicits on neuronal cells [87]. And third, antibodies directed against A
could enhance the clearance of A
by stimulating Fc receptor mediated phagocytosis of A
by microglia in later stages of the disease [88]. Although promising in preclinical studies, the first human trial with A
immunization halted prematurely due to the development of meningoencephalitis in 18 of the 298 patients enrolled in this trial [89]. Also AD transgenic mouse studies have shown, besides a clearance of parenchymal A
deposits, an increase in vascular amyloid and microhemorrhages after passive immunotherapy [90, 91]. This indicates that immunization therapy has to develop towards a strategy where A
is efficiently removed without increasing the toxic A
species at other sites of the brain. In addition, at later stages in AD pathology immunization therapy could be used in combination with agents that suppress the detrimental effects of the neuroinflammatory response [92]. The therapeutic approaches mentioned above are aimed at the synthesis of A
and disrupting the different conformational changes of A
. Another approach is to target the cellular response to the different conformational forms A
. Pharmacological induction of molecular chaperones may protect against aggregate toxicity [93]. The therapeutic potential of such a strategy is best illustrated by treatment of SODG93A mice, a model for amyotrophic lateral sclerosis, with arimoclomol, a co-inducer of heat shock proteins [94]. This treatment

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resulted in decreased neuronal loss and consequently improved motorfunction, as well as increased lifespan. Another way to employ protein quality control to tackle neurodegeneration is via the UPR. Recently, the compound salubrinal was identified as an inhibitor of eIF2
de-phosphorylation and shown to protect against ER stress-induced cell death [95, 96]. Salubrinal is protective in a Parkinson’s disease cell model [97], indicating its potential for the treatment of neurodegenerative diseases. A drawback is that constitutive phosphorylation of eIF2
is unlikely to present a long-term treatment opportunity [98], therefore more selective targeting should be investigated. Valproate, a drug widely prescribed in the treatment of bipolar disorder and epilepsy, has been shown to increase the levels of BiP/GRP78 and other ER chaperones [99, 100]. Although valproate increases ER chaperones it has been reported that ER stress induced cellular dysfunction is reduced by valproate through inhibition of GSK-3
/ [101]. Valproate could work protective in AD reducing ER stress and reducing tau phosphorylation by GSK-3. Currently, clinical trails have been started investigating the effect of valproate on AD patients. A very interesting finding was recently reported by Kondo and colleagues. They report that a transcription factor called OASIS specifically stimulates BiP/GRP78 upregulation (a positive response), but downregulates the ER stress induced cell death pathways [102]. OASIS is specifically induced in astrocytes, but factors with similar properties may exist in neurons as well, and present an ideal therapeutic target by stimulating positive and downregulating negative responses through a single protein. In conclusion, lowering A synthesis or stimulating A removal is promising in the sense that it removes the pathogen, although an undesired side-effect may be the generation of more toxic oligomeric A by dissolving fibrillar A. Alternatively, targeting quality control by either diminishing destructive or stimulating protective responses is attractive as well, because it makes use of the cell’s own mechanisms that have evolved to specifically deal with this problem. However, the presence and role of oligomeric and intracellular A in AD pathology and the disease stage they are involved is still not completely understood. For future therapies it will be necessary to characterize these molecular pathways and place them more precisely in the temporal sequence of the pathological cascade. ACKNOWLEDGEMENTS Work discussed in this review was financially supported by the Internationale Stichting Alzheimer Onderzoek (ISAO grants 02504 and 04503) and a fellowship of the Anton Meelmeijer Center for Translational Research to WS. Part of this work has been supported by a grant from the European Commission FP6 (ADIT, contract n. LSHB-CT-2005511977). REFERENCES [1] [2] [3] [4] [5] [6] [7]

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Webster, S. D.; Yang, A. J.; Margol, L.; Garzon-Rodriguez, W.; Glabe, C. G.; Tenner, A. J. Complement component C1q modulates the phagocytosis of Abeta by microglia. Exp. Neurol., 2000, 161, 127-138. Familian, A.; Boshuizen, R. S.; Eikelenboom, P.; Veerhuis, R. Inhibitory effect of minocycline on amyloid beta fibril formation and human microglial activation. Glia, 2006, 53, 233-240. Takata, K.; Kitamura, Y.; Umeki, M.; Tsuchiya, D.; Kakimura, J.; Taniguchi, T.; Gebicke-Haerter, P. J.; Shimohama, S. Possible involvement of small oligomers of amyloid-beta peptides in 15-deoxy-delta 12, 14 prostaglandin J2-sensitive microglial activation. J. Pharmacol. Sci., 2003, 91, 330-333. Rogers, J.; Strohmeyer, R.; Kovelowski, C. J.; Li, R. Microglia and inflammatory mechanisms in the clearance of amyloid beta peptide. Glia, 2002, 40, 260-269. Wyss-Coray, T. and Mucke, L. Inflammation in neurodegenerative disease--a double-edged sword. Neuron, 2002, 35, 419-32. Song, L.; De Sarno, P.; Jope, R. S. Central role of glycogen synthase kinase-3beta in endoplasmic reticulum stress-induced caspase-3 activation. J Biol Chem, 2002, 277, 44701-44708. Weggen, S.; Eriksen, J. L.; Das, P.; Sagi, S. A.; Wang, R.; Pietrzik, C. U.; Findlay, K. A.; Smith, T. E.; Murphy, M. P.; Bulter, T.; Kang, D. E.; Marquez-Sterling, N.; Golde, T. E.; Koo, E. H. A subset of NSAIDs lower amyloidogenic Abeta42 independently of cyclooxygenase activity. Nature, 2001, 414, 212216. Eriksen, J. L.; Sagi, S. A.; Smith, T. E.; Weggen, S.; Das, P.; McLendon, D. C.; Ozols, V. V.; Jessing, K. W.; Zavitz, K. H.; Koo, E. H.; Golde, T. E. NSAIDs and enantiomers of flurbiprofen target gammasecretase and lower Abeta 42 in vivo. J. Clin. Investig, 2003, 112, 440-449. Morihara, T.; Chu, T.; Ubeda, O.; Beech, W.; Cole, G. M. Selective inhibition of Abeta42 production by NSAID R-enantiomers. J. Neurochem., 2002, 83, 1009-1012. Weggen, S.; Eriksen, J. L.; Sagi, S. A.; Pietrzik, C. U.; Ozols, V.; Fauq, A.; Golde, T. E.; Koo, E. H. Evidence that nonsteroidal anti-inflammatory drugs decrease amyloid beta 42 production by direct modulation of gamma-secretase activity. J. Biol. Chem., 2003, 278, 31831-31837. Beher, D.; Clarke, E. E.; Wrigley, J. D.; Martin, A. C.; Nadin, A.; Churcher, I.; Shearman, M. S. Selected non-steroidal anti-inflammatory drugs and their derivatives target gamma-secretase at a novel site. Evidence for an allosteric mechanism. J. Biol. Chem., 2004, 279, 43419-43426. Lleo, A.; Berezovska, O.; Herl, L.; Raju, S.; Deng, A.; Bacskai, B. J.; Frosch, M. P.; Irizarry, M.; Hyman, B. T. Nonsteroidal anti-inflammatory drugs lower Abeta42 and change presenilin 1 conformation. Nat. Med., 2004, 10, 1065-1066. Schenk, D.; Barbour, R.; Dunn, W.; Gordon, G.; Grajeda, H.; Guido, T.; Hu, K.; Huang, J.; JohnsonWood, K.; Khan, K.; Kholodenko, D.; Lee, M.; Liao, Z.; Lieberburg, I.; Motter, R.; Mutter, L.; Soriano, F.; Shopp, G.; Vasquez, N.; Vandevert, C.; Walker, S.; Wogulis, M.; Yednock, T.; Games, D.; Seubert, P. Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature, 1999, 400, 173-177. Gelinas, D. S.; DaSilva, K.; Fenili, D.; George-Hyslop, P.; McLaurin, J. Immunotherapy for Alzheimer's disease. Proc. Natl. Acad. Sci. USA, 2004, 101 (Suppl 2), 14657-14662. DeMattos, R. B.; Bales, K. R.; Cummins, D. J.; Dodart, J. C.; Paul, S. M.; Holtzman, D. M. Peripheral anti-A beta antibody alters CNS and plasma A beta clearance and decreases brain A beta burden in a mouse model of Alzheimer's disease. Proc. Natl. Acad. Sci. USA, 2001, 98, 8850-8855. McLaurin, J.; Cecal, R.; Kierstead, M. E.; Tian, X.; Phinney, A. L.; Manea, M.; French, J. E.; Lambermon, M. H.; Darabie, A. A.; Brown, M. E.; Janus, C.; Chishti, M. A.; Horne, P.; Westaway, D.; Fraser, P. E.; Mount, H. T.; Przybylski, M.; George-Hyslop, P. Therapeutically effective antibodies against amyloidbeta peptide target amyloid-beta residues 4-10 and inhibit cytotoxicity and fibrillogenesis. Nat. Med., 2002, 8, 1263-1269. Bard, F.; Cannon, C.; Barbour, R.; Burke, R. L.; Games, D.; Grajeda, H.; Guido, T.; Hu, K.; Huang, J.; Johnson-Wood, K.; Khan, K.; Kholodenko, D.; Lee, M.; Lieberburg, I.; Motter, R.; Nguyen, M.; Soriano, F.; Vasquez, N.; Weiss, K.; Welch, B.; Seubert, P.; Schenk, D.; Yednock, T. Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat Med., 2000, 6, 916-9. Orgogozo, J. M.; Gilman, S.; Dartigues, J. F.; Laurent, B.; Puel, M.; Kirby, L. C.; Jouanny, P.; Dubois, B.; Eisner, L.; Flitman, S.; Michel, B. F.; Boada, M.; Frank, A.; Hock, C. 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Novel Neuroprotective Therapies for Alzheimer’s and Parkinsons’s Disease Kirubakaran Shanmugam1, Annette E. Maczurek1, Megan L. Steele1, Obdulio Benavente-García2, Julián Castillo2 and Gerald Münch1,* 1

Department of Pharmacology, School of Medicine, and Molecular Medicine Research Group, University of Western Sydney, Campbelltown, Australia; 2Furfural Español-Nutrafur, Alcantarilla, Murcia, Spain

Abstract: One of the major age-related damaging agents are reactive oxygen species (ROS). The brain is more vulnerable to oxidative stress than other organs as concomitant low activity and capacity of antioxidative protection systems allow for increased exposure of target molecules to ROS. Since neurons are postmitotic cells, they have to live with cellular damage accumulated over many decades. Increased levels of ROS (also termed “oxidative stress”), produced by normal mitochondrial activity, inflammation and excess glutamate levels, are proposed to accelerate neurodegenerative processes characteristic for Alzheimer’s disease and Parkinson’s disease. This review presents evidence for the importance of oxidative stress in the pathogenesis of these diseases and explains the nature of different types of ROS mediating neuronal damage. Furthermore, the potential beneficial effects of neuroprotective treatments, including synthetic and plant deroved antioxidants, energy supplements and anti - glutamatergic drugs are discussed.

Keywords: Oxidative stress, Alzheimer’s disease, Parkinson’s disease, neuroprotection, antioxidants, NMDA receptor antagonists, bioenergetic drugs. 1. NEURODEGENERATIVE DISEASES – A GROWING BURDEN IN THE AGING POPULATION Neurodegenerative diseases are initially characterized by subtle changes in the normal function of neurons, which is followed by overt neuronal dysfunction and cell death in later stage of the disease. Age is the single most important risk factor for the development of many neurodegenerative disorders including Parkinson’s (PD) and Alzheimer’s disease (AD). In 2006, 26.6 million people were living with Alzheimer’s disease worldwide. According to the American Alzheimer’s Association, this number is set to exceed 100 million by 2050, at which time one in 85 people will be living with the disease, due to rapidly ageing populations and longer life expectancy. Patients with neurodegenerative diseases not only suffer emotionally and physically but also represent a significant financial and *Corresponding author: Tel: ++61 2 9852 4718; Fax: ++61 2 9852 7730; E-mail: [email protected] Allen B. Reitz / Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.

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emotional burden for caregivers, society and community. Thus, it is becoming increasingly important to find the cause(s) of neurodegenerative diseases and, based on these findings, develop and introduce novel therapies that prevent such diseases by providing neuroprotection. 2. NEURODEGENERATIVE DISORDERS 2.1. Alzheimer Disease Symptoms Alzheimer’s disease (AD) is a progressive neurodegenerative brain disorder that gradually destroys a person’s memory and ability to learn, make judgments, communicate with the social environment and carry out daily activities. In the course of the disease, shortterm memory is affected first, caused by neuronal dysfunction and cell death in the hippocampus and amygdala. As the disease progresses further, neurons also die in other cortical regions of the brain. At that stage, sufferers often experience dramatic changes in personality and behaviour, such as anxiety, suspiciousness or agitation, as well as delusions or hallucinations. Epidemiology AD prevalence in the different age groups is 1 % (65-69 years), 3% (70-74 years), 6 % (75-79 years), 12% (80-84 years), and 25% (85 & over) [1]. As the society ages, the number of Alzheimer's disease patients will increase by 27% by 2020, 70% by 2030, and nearly 300% by 2050, unless science finds a way to slow the progression of the disease or prevent it. Alzheimer’s disease advances at widely different rates, and therefore the duration of the illness varies between 3 and 20 years. Neuropathology and Biochemistry AD is characterized by two characteristic lesions, amyloid plaques and neurofibrillary tangles, which are present in high numbers in the grey matter of affected brain areas. Neurofibrillary tangles are intracellular deposits formed by hyperphosphorylated and extensively crosslinked tau protein. Tau is a microtubule associated protein that regulates a variety of properties of neuronal microtubules, especially their stability and orientation. In AD, however, tau is hyperphosphorylated and forms fibrillar inclusions. Presumably this lead to neuronal dysfunction by disturbing cytoskeletal functions of neurons resulting in impaired axonal transport processes [2]. Senile plaques are the second characteristic hallmark in Alzheimer disease. These extracellular protein deposits are mainly composed of
-amyloid peptide (A
), which forms
sheeted fibrils and become insoluble. A
is derived from the
-amyloid precursor protein (APP), an integral membrane protein that is processed by
- and -secretases. In a variety of cell culture models, A
was shown to cause toxicity to neurons by various mechanism, many of which involving oxidative stress. For example, 4-hydroxynonenal and malondialdehyde, both markers of lipid peroxidation, were found in the hippocampus of patients with AD. Markers of protein oxidation such as protein nitration are also increased in the hippocampus and neocortex of individuals with AD. The fact that A
binds strongly to metal ions like copper and iron and catalyses the formation of the toxic hydroxyl radical from hydrogen peroxide strongly suggests that may be causally involved in oxidative stress in neurons. Amyloid plaques, which can be present in the brain for decades, are a target for modifica-

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tion with advanced glycation endproducts (AGEs), i.e. sugar or carbonyl compounds attaching to lysine and arginine residues of long-lived proteins. AGEs have been found not only in amyloids, but also in other proteins with slow turnover such as eye lens crystalline or cartilage and skin collagen. In AD, A and AGE are both present in senile plaques and are resistant to degradation by macrophages due to extensive cross-linking. A and AGE are able to chemoattract and activate microglial cells that surround these senile plaques. AGEs and A bind to the same receptor, i.e. the receptor for glycation endproducts (RAGE), and activate signal pathways involved in the induction of oxidative and nitrosative stress and proinflammatory effectors [3]. Inflammation, as evidenced by the activation of microglia and astroglia, is another hallmark of AD. Inflammation, particularly the induction of superoxide production (“oxidative burst”), is an important source of oxidative stress in AD patients. The inflammatory process occurs mainly around the amyloid plaques and is characterized by pro-inflammatory substances released from activated microglia [4]. ROS are the most prominent molecules in the inflammatory process, along with prostaglandins, IL-1, IL-6, M-CSF and TNF-
[5-7]. 2.2. Parkinson’s Disease Epidemiology and Symptoms Parkinson’s disease (PD) is a progressive, degenerative disease of unknown etiology characterised by rhythmic tremor of the limbs, stooped posture, slowness of voluntary movements, and mask-like facial expression. Idiopathic PD is the most common form of parkinsonism, which designates a group of movement disorders that have similar features and symptoms. Parkinson’s disease is called “idiopathic” because its cause is unknown, in contrast to other forms of parkinsonism, where a cause has been established or is suspected. The disease is quite frequent – about 4 people in every thousand have PD. Symptoms of Parkinson’s disease may appear at any age, but the average age of onset is 60. Neuropathology and Biochemistry Parkinson’s results from the degeneration of dopaminergic neurons in the substantia nigra that develop inclusions called Lewy bodies. These deposits are composed of aggregated alpha synuclein and ubiquitin. Ubiquitin is covalently attached to damaged or aged proteins, in order to mark them for degradation in the proteasome. Alpha synuclein is a synaptic protein of unknown function. When dopamine levels are depleted, the neuronal populations in the basal ganglia, to which the dopaminergic neurons project, become unable to coordinate and control the primary motor system, resulting in loss of prompt and smooth movement of limbs and trunk. Parkinson’s disease patients have lost 80% or more of their dopamine-producing cells before symptoms appear, highlighting the great spare capacity normal human beings possess. There is plenty of evidence that oxidative stress is occurring in PD. Partial deficiency of mitochondrial complex I leads to enhanced production of ROS and therefore to an inhibition of complex I [8]. Damaged neurones show reduced mitochondrial cytochrome c-oxidase activity, high concentrations of iron [9] as well as ROS release [10]. An increase in lipid peroxidation products [11], reactive protein carbonyls [12] and DNA oxidation [9] provide further evidence for oxidative stress in PD. Inflammation may also occur in this neurodegenerative disorder, since reactive microglia and activated complement components are found in affected brain regions [13].

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3. MECHANISMS OF NEURODEGENERATION 3.1. Oxidative Stress 3.1.1. Inflammation and Glia-Derived Oxidative Burst One important source of ROS is the oxidative burst of activated macrophages and microglia, which release superoxide via the enzymatic activity of NADPH-oxidase. Under normal circumstances the human body is able to scavenge these radicals but oxidant (particularly superoxide) production is dramatically increased in some inflammatory situations. Such inflammation is mediated through resident brain microglia or immigrated peripheral monocytes and macrophages. Their normal function is to destroy bacteria or virus-infected cells with an oxidative burst of O2.- and its dismutation product H2O2, as well as HOCl and NO [14, 15]. These oxidants are generated by four differente enzymes, i.e. NADPH oxidase, superoxide dismutase, myeloperoxidase and nitric oxide synthase. NADPH oxidase is inactive in resting phagocytes, but exposure to various stimuli (i.e. immune complexes, complement 5a,
-amyloid, AGEs) leads to its activation, i.e. catalysis of the conversion of molecular oxygen to superoxide. 2 O2 + NADPH 2 O2-

+ NADP+ + H+

The oxidation of halide ions to hypohalous acid at the expense of hydrogen peroxide is carried out by the enzyme myeloperoxidase [16]: Cl- + H2O2 OCl-

+ H2O

Hypochlorite can then further react with hypochlorite, ammonia or amines, the precursors of chloramines, a group of microbicidal oxidized halogens [17]. 3.1.2. Superoxide Production by Mitochondria In the human body, 20% of the total oxygen consumption is needed for the brain, which is quite intriguing given that the brain takes only 2% space of the total tissue volume [18]. About 0.15 - 2% of the oxygen consumed in neuronal mitochondria is incompletely reduced to generate superoxide anions by electron leakage from the electron transport chain (Chance, Sies, and Boveris 1979). These superoxide anions are usually converted by the enzyme superoxide dismutase (SOD) to hydrogen peroxide and then further converted to water by catalase or glutathione peroxidase [19]. Neurons in human brain are highly vulnerable to oxidative stress not only because of high oxygen consumption (and therefore high superoxide production), but also due to poor antioxidative mechanisms, particularly catalase activity, in comparison to other organs in the human body. The cell membranes in the brain are especially vulnerable to attack by hydroxl radicals as the double bonds of polyunsaturated fatty acids are easily oxidized. There are many different species of oxygen free radicals including superoxide, hydrogen peroxide and the hydroxyl radical, which are generated by reduction of molecular oxygen in three steps, and their generation and will be described in detail below.

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3.2. Reactive Oxygen Species 3.2.1. Superoxide Superoxide (O2.-) is a precursor for hydroxyl radicals; it is necessary for the formation of hydrogen peroxide and also acts as a reducing agent of transition metals. Most of the superoxide produced in human brain is generated by the electron transport chain of the mitochondria by incomplete reduction of molecular oxygen. eO2

O2._

An increase in O2.- production also occurs when Ca2+ concentrations increase, as is the case during NMDA receptor overstimulation. Lipid metabolism is a second minor pathway for O2.- formation. When Ca2+ enters neurons, phospholipase A2 is activated and arachidonic acid (AA) is formed [20]. Cyclooxygenase then converts AA to prostaglandins, with O2.generated as a by-product [21]. O2- formed within the neurons does not appear to cross plasma membrane [22] and is therefore only found in the intracellular environment following damage to the neuronal membrane. Superoxide is more reactive in organic solutions than in aqueous ones and can therefore damage hydrophobic membranes. Although O2.- on its own may have little significance in terms of toxicity, its conversion to peroxynitrite and hydroxyl radicals greatly increases neurotoxicity. 3.2.2. Hydrogen Peroxide Hydrogen peroxide (H2O2) is not a free radical per se, but belongs to the category of ROS. It is not particularly reactive but it is the main source of hydroxyl radicals in the presence of transition metal ions [23]. It is also involved in the production of hypochlorous acid (HOCl) by neutrophils during oxidative burst [24]. H2O2 is generated in the dismutation reaction during which two molecules of superoxide are converted into one molecule of H2O2 plus molecular oxygen: -

2 O2 + 2 H+

O2 + H2O2

O2, the substrate, reacts with itself to give an oxidized product (i.e. oxygen) and a reduced product (i.e. H2O2). Superoxide dismutase (SOD) is the enzyme responsible for this reaction that exists as a copper-zinc enzyme in the cytoplasm and as manganese isoform in the mitochondria. 3.2.3. Hydroxyl Radical The reactions of the hydroxyl radical, which is the most damaging free radical by far, can have devastating effects in any tissue type. The hydroxyl radical is a third generation species of radical which is derived from hydrogen peroxide. Hydrogen peroxide in turn, is derived from the superoxide radical through the action of SOD, and then reduced to hydroxyl radicals in the presence of transition metals such iron or copper. It reacts with any molecule including macromolecules such as DNA, membrane lipids, proteins, and carbohydrates. In DNA, the hydroxyl radical can induce strand breaks as well as chemical changes in the deoxyribose and in the purine and pyrimidine bases. Damaged proteins, many of them crucial enzymes in neurons, lose their efficiency and cellular function deteriorates. Protein oxidation mediated by hydroxyl radicals has been proposed as an explanation for the functional deficits associated with aging in many tissues and brain aging and neurodegeneration in particular.

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The conversion of hydrogen peroxide to the hydroxyl radical depends on the presence of transition metals such as copper or iron. There is evidence that these metals accumulate in aged brain, and they may therefore contribute to neurodegenerative disorders. Some brain regions and some types of brain cells , for example oligodendrocytes, show above-average concentrations of iron [25] while their antioxidative protection systems are rather weak. Being transition metals, iron can exist in the oxidized (+III) or reduced (+ II) forms and likewise copper as (+II) or (+ I), which makes them important cofactors for many enzymes. Free transition metals may, however, generate ROS via the Fenton reaction. Here, Fe2+ is able to transfer one electron to H2O2, thus producing the hydroxy radical: Fe 2+ + H2O2
Fe3+ + .OH + OH.The Haber-Weiss reaction also can form .OH in an interaction between O2.- and H2O2 in the presence of Fe2+ or Fe3+ [26]. The body has developed chaperones to prevent such metaldependent hydroxyl radical generation. Free copper is generally bound to cytochrome C oxidase, Cu/Zn superoxide dismutase and caeruloplasmin whilst iron is mainly bound to ferritin. This prevents the inadvertent release of the metals and limits their availability to catalyze the Haber-Weiss reaction. 3.2.4. Nitric Oxide Nitric Oxide (NO) is the another radical proposed to be involved in neurodegeneration and is produced by various NO-synthetases. Ca2+ activated neuronal nitric oxide synthases (nNOS, bNOS, cNOS, TypeI) catalyze the oxidation of arginine to citrulline and nitric oxide, utilizing molecular oxygen: Arginine + O2 + NADPH

NO + citrulline + NADP+

In response to increased intracellular Ca2+, nNOS interacts with Ca2+/calmodulin (CaM), forms a complex with the cofactor tetrahydrobiopterin (BH4) and translocates from the plasma membrane to the cytoplasm. The dephosphorylation of nNOS by calcineurin initates the production of NO. A further source of NO is the inducible nitric oxide isoform, which is upregulated in glia in response to pro-inflammatory stimuli present in AD and PD brains. NO then has the capacity to activate several pathways, predominantly cGMP-regulated signalling pathways, via the activation of guanylate cyclase (GC). NO undergoes a further reaction with O2. – radicals, to form peroxynitrite (ONOO-), one of the most potent oxidants in biological systems: NO.

+ O2 -

ONOO-

Peroxynitrite causes nitration of tyrosine, phenylalanine and tryptophan in proteins as well as the oxidation of lipids, DNA and proteins. Lipid peroxidation is a significant problem for neurons, as the destruction of lipidic cell membranes means that ionic gradients can no longer be maintained. Enhanced release of glutamate from presynaptic terminals further exacerbates this damage. The half-life of peroxynitrite is very short, and it decomposes in a liquid environment into further reactive oxygen- and nitrogen species, i.e. hydroxyl radicals and various nitrogen oxides (NxOy). 4. OVERSTIMULATION OF GLUTAMATE RECEPTORS (EXCITOTOXICITY) Overstimulation of glutamate receptors, called excitotoxicity, is a further mechanism of neuronal cell death in neurodegeneration [27]. Glutamate is the major excitatory neurotransmitter in the mammalian CNS and is present in millimolar concentrations in the gray matter. Once released it can act on ionotropic and metatropic receptors, two general recep-

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tors for glutamate. Metabotropic receptors are linked to G-proteins, which activate phospholipase C (PLC) or adenylcyclase (AC). Activated PLC catalyses the cleavage of L-3phosphatidyl-inositol-4,5-bisphosphate (PIP2) into inositol–1,4,5, trisphosphate (IP3) and diacylglycerol (DAG). IP3 is responsible for intracellular release of Ca2+ from the endoplasmic reticulum. Released Ca2+ is involved in the activation of protein kinase C (PKC) in neurons, which then phosphorylates cytosolic proteins [28, 29]. Ionotropic receptors form ion channels, and are subdivided into three further forms according to their respective agonists, i.e. kainic acid (KA), N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). KA and AMPA receptors are thought to subserve primarily rapid excitatory neurotransmission in the CNS and are mainly permeable to Na+ and K + but not to Ca2+. In contrast, NMDA receptors are permeable to Ca2+ and respond more slowly to glutamate. It is therefore believed that they will not function as mediators of rapid synaptic transmission but mediate the neurotoxic effects of glutamate in the adult brain (reviewed in [30]. A characteristic feature of the NMDA receptor is the so-called magnesium block, which must be released in a voltage-dependent manner for activation. It could been shown that glutamate becomes more neurotoxic when intracellular energy levels are reduced [31]. Since ATP levels are decreased in AD, neurons are more sensitive to the neurotoxic action of glutamate, because ATP production and functional Na+, K+-ATPases are necessary to generate a resting potential to maintain the voltage-dependent Mg2+ block of the NMDA receptor channel. Relief of the Mg2+ block enables the excitatory amino acids to act persistently at the NMDA receptor, resulting in its opening and subsequent neuronal damage. Activation of NMDA receptors increases cytoplasmatic calcium levels and subverts of cytoplasmic Ca2+ homeostasis. The increased intracellular Ca2+ concentration leads to enhanced metabolic stress on mitochondria, resulting in excessive oxidative phosphorylation and production of ROS e.g., via the activation of Ca2+dependent nitric oxide synthases[32]. Ca2+ toxicity is also mediated by activation of proteolytic enzymes like calpains which are able to degrade essential proteins, or endonucleases that degrade DNA. O2.- and H2O2 are produced by xanthine oxidase, which arises from proteolytic cleavage of xanthine dehydrogenase by calcium–dependent enzymes to. Morover, Ca2+ activates phospholipase A2 (PLA2) catalyzing the production of arachidonic acid, which in turn, is transformed by cyclooxygenases to prostaglandins, with O2.- formed as a byproduct. Lipton et al. (Lipton et al. 1993) have shown evidence indicating the presence of alternative redox states of NO: the radical form and the ionic form. Radical form of NO easily permeates the cell membrane and reacts with superoxide anion to produce neurotoxicity when it yields peroxynitrite (ONOO
). This suggests that NO is a key substance in NMDA receptor-mediated glutamate neurotoxicity in the CNS although this evidence does not exclude the possible involvement of other mediators in the glutamate-induced neurotoxicity (Akaike et al. 1999). In summary, excitotoxicity also appears to involve oxidative stress which makes an antioxidant therapy a promising alternative to NMDA receptor antagonists. 5. NEUROPROTECTION 5.1 Introduction to the Principles of Neuroprotection As discussed above, the role of oxidative stress in neurodegenerative disorders such as PD and AD is well documented. It should therefore be possible to provide neuroprotection

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by manipulating the redox state of the cells, e.g., by increasing the antioxidant capacity of the cells or by decreasing the pro-oxidative conditions in cells in the human brain. There are several therapeutic approaches for providing such neuroprotection, including supplementation of the patient with antioxidants, energy enhancing or anti-glutamatergic drugs. Antioxidants are used because of their ability to scavenge ROS and thus preventing damage to neurons. Since many plant-derived antioxidants are present in certain foods in different combinations, we will sometimes describe the positive effects of the foods rather than those of the isolated ingredients. In addition, energy supplementation is supposed to neuronal resistance to glutamate and ROS induced damages and initiate repair processes. Anti-glutamatergic drugs will decrease pathophysiological calcium influx and calcium-related injury including mitochondrial dysfunction. In the subsequent sections, a variety of natural and synthetic neuroprotective drugs or foods supplements are described, and evidence for their beneficial effects in cell culture and animal models of AD and PD as well as clinical evidence for their efficacy will be discussed. 5.2. Antioxidants Antioxidants are low molecular weight substances which either block the production of free radicals or neutralize them by absorbing the unpaired electron, thereby turning into a radical that is less reactive. Endogenous antioxidant systems include anti-oxidative enzymes (e.g. glutathione peroxidase and catalase) and thiol based scavengers (e.g. glutathione). However, therapeutic manipulation of anti-oxidative enzymes is at a very early stage, since their expression is genetically determined and therefore difficult to increase by synthetic drugs without changing overall expression profiles. By contrast, exogenous antioxidants such as vitamins C and E have long been supplied with food in order to boost antioxidative defenses in age-related diseases. More and more foods, particularly those containing plantderived antioxidants, are now offered by commercial vendors. Even in the absence of double-blind placebo controlled clinical trials, various antioxidants or antioxidant-rich plant extracts are therefore used as a therapeutic option in PD and AD. 5.1.1. Vitamin C Occurrence Vitamin C (ascorbate) is synthesized from glucose in the liver of most mammals. Guinea pigs, higher primates and humans, however, have lost the ability to synthesize the vitamin and are relying on dietary supplementation [33]. Vitamin C is found in high concentrations in many fruits and vegetables, including citrus fruits, kiwis and tomatoes. The recommended daily allowance of vitamin C is 75 mg. Function and Chemical Reactions Vitamin C is a hydrophilic vitamin and can exist as a partially oxidized ascorbyl free radical or as dehydroascorbic acid (DHA) in the presence of mild oxidants, depending on how many electrons are lost. The latter form is very short-lived (with a half life of about 6 minutes) and it is reduced back to the monoanion form (e.g. by vitamin E), which is the most common form of ascorbate at physiological pH [34]. Hydrolysis of the lactone ring in DHA leads to the irreversible conversion to 2,3-diketo-1-gulonic acid (Fig. 1). Besides its functions in the synthesis of the amino acid hydroxyproline, as major building block of collagen in connective tissue and bones, and in the absorption of iron, ascorbate plays a role as an antioxidant, protecting cells against oxidative stress, particularly in the cytosolic components of the cell, due to its reducing potential of its carbon-carbon double bond. It mediates its antioxidant activity in the cytosol by donating two hydrogens and electrons to ROS like

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peroxyl and hydroxyl radicals, superoxide, singlet oxygen and peroxynitrite [35]. When associated with the plasma membrane, however, it is a powerful reductant of the
tocopherol (vitamin E) radical which is the most active form of tocopherol against peroxyl radicals.

Fig. (1). Oxidation of vitamin c (ascorbic acid) via dehydroascorbic acid as intermediate.

Neuroprotective Effects in Cell Culture and Animal Models Since neurons have a tenfold higher oxidative metabolism than glial cells, vitamin C is an important antioxidant molecule in this cell type, and there is evidence that it also scavenges glutamate-generated ROS [36]. Using human cortical neurons it could be shown that ascorbic acid inhibits Hydrogen peroxide, TNF-
, dopamine or -amyloid peptide induced apoptosis. This occurs through preventing the loss of mitochondrial membrane potential and DNA fragmentation [37]. These findings are concurrent with the results from Lockardt and colleagues, who have shown that vitamin C can attenuate the neurotoxic effect of betaamyloid in rat hippocampal cultures [38]. In PD, an increase in oxidative stress is observed by treatment of patients with levodopa, which is metabolized by monoaminooxidases in a radical-generating reaction. In an appropriate cell culture mode, levodopa proved toxic for the human neuroblastoma cell line NB69. Tocopherol lacks significant preventive effect on levodopa toxicity, but ascorbic acid in millimolar concentrations prevents levodopa toxicity and quinone formation [39]. In a similar study, the role of ascorbic acid on dopamine (DA) oxidation-mediated cytotoxicity was studied using a different cell system, the neuronal cell line PC12. DA cytotoxicity was slightly attenuated by ascorbic acid, whereas the cytotoxicity of 6-hydroxydopamine (6OHDA), a DA oxidation product, was actually potentiated [40]. Neuroprotective Effects in Alzheimer and Parkinson Patients Vitamin C, among other antioxidants, is often recommended for PD and AD patients. Epidemiological studies, however, yielded conflicting results. In the Rotterdam study, 5395

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participants, who did not display any signs or symptoms of AD, were analyzed in respect of the future onset of the disease. It was demonstrated that persons with a higher intake of vitamin C had a reduced risk of developing AD, compared to the ones with lower intake. An intake of 133 mg/day compared to 95 mg/day of vitamin C led to a 34% decrease in the development of AD [41]. In contrast, the Chicago Health and Aging Project (CHAP), in which 3838 healthy persons took part (of whom 815 persons, with the average age of 72 years, completed the study), found no relation between the time of onset of AD and the intake of vitamin C from supplements (Morris et al. 2002). Plasma vitamin C in PD patients are significantly lower than in controls which may evidence that oxidative stress is involved in PD [42]. However, no convincing positive epidemiological studies or clinical trials with vitamin C alone, in PD patients, have been reported so far. 5.1.2. Vitamin E Occurrence Vitamin E (
-tocopherol) is known as an essential nutrient since 1968. It is only produced in plants, particularly in wheat germ, plant oils, nuts and soybeans. The daily requirement is reported as 30 mg/d, but this varies between ages and country. Function and Chemical Reactions Vitamin E is a term for a group of tocopherols and tocotrienols of which
-tocopherol is the most active one. It is lipid-soluble and consists of a chromane ring with an isoprenoid side chain (Fig. 2).

Fig. (2). Chemical structure of vitamin E (tocopherol).

Since it is hydrophobic and concentrated in cell membranes, its main mechanism is the prevention of lipid peroxidation and other radical-driven oxidative events involving fatty acids [43, 44]. Vitamin E is regenerated by vitamin C, as was shown in both liposomal membrane systems and in homogeneous solutions [45]. Neuroprotective Effects in Cell Culture and Animal Models It has been demonstrated that vitamin E has indirect neuroprotective effects through the suppression of microglial activation [46]. LPS-activated microglia showed attenuated expression of inflammatory mediators such as IL-1
, TNF-
or NO when co-cultured with vitamin E. Therefore vitamin E has a therapeutic use in AD, since neurotoxic microglialneuronal interactions are implicated in the pathogenesis in this type of dementia. Further relationship between AD and vitamin E is the prevention of protein oxidation and ROS production induced by -amyloid. It has been shown that this does not occur by inhibiting the A fibril formation but rather by scavenging the A associated free radicals [47].

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In a PD cell culture model, dopamine triggered apoptosis in PC12 cells, only the thiolcontaining compounds, reduced glutathione (GSH), N-acetyl-cysteine (NAC), and dithiothreitol (DTT) were markedly protective, while vitamin E had no effect. The thiol antioxidants and vitamin C but not vitamin E, prevented dopamine autooxidation and production of dopamine-melanin [48]. Neuroprotective Effects in Alzheimer and Parkinson Patients The effect of vitamin E in AD patients was investigated in the Rotterdam study. A daily intake of 15.5 mg of vitamin E compared to an intake less than 10.5 mg/day decreases the risk of developing AD to 43% [41]. This finding was further confirmed in the Chicago Health and Aging Project, where a vitamin E intake of more than 11.4 I.U/d resulted in a 70% reduced risk for AD development compared to an intake of less than 6.21 I.U/d. The positive effect of vitamin E was not only significant, but dose-dependent and seemed to have an increased protective role when taken over a longer period of time [49]. In a further study, the combination of vitamin E and C in AD patients was evaluated. Use of vitamin E and C (ascorbic acid) supplements in combination was associated with reduced AD prevalence (adjusted odds ratio, 0.22; 95% confidence interval, 0.05-0.60) and incidence (adjusted hazard ratio, 0.36; 95% confidence interval, 0.09-0.99). A trend toward lower AD risk was also evident in users of vitamin E and multivitamins containing vitamin C, but no evidence of a protective effect with use of vitamin E or vitamin C supplements alone, with multivitamins alone, or with vitamin B–complex supplements was observed. In a epidemiological study, a total of 371 incident PD cases were ascertained in the Nurses' Health Study, which comprised 76,890 women who were followed for 14 years, and the Health Professionals Follow-Up Study, which comprised 47,331 men who were followed for 12 years. In this study, the risk of PD was significantly reduced among men and women with high intake of dietary vitamin E. Since the beneficial effects were only seen with vitamin E from foods (not from synthetic sources), the authors suggest that other constituents of foods rich in vitamin E may be protective [50]. In contrast, the community-based Rotterdam Study in the Netherlands collected data of 5342 independently living individuals without dementia between 55 and 95 years of age, including 31 participants with PD. In this study, vitamin E was protective, and the association of the relative risk with vitamin E intake was dose dependent [51]. Vitamin E (and deprenyl) was tested by the Parkinson Study Group in the “Deprenyl and Tocopherol Antioxidative Therapy of Parkinsonism” (DATATOP) trial. However, deprenyl delayed the onset of disability associated with early, otherwise untreated Parkinson's disease, however, in contrast to the expectation of the authors, a-tocopherol proved to be ineffective in the DATATOP study [52]. 5.1.3. Lipoic Acid Occurrence Lipoic acid was first identified as a component of several human enzyme systems involved in the conversion of carbohydrates and fats into energy, such as pyruvate dehydrogenase,
-ketoglutarate dehydrogenase. Lipoic acid was not classified as a vitamin because some small amounts can be synthesized by the human body. However, lipoic acid as a therapeutic drug comes from chemically synthesised sources since amounts of 600mg can not be taken up with the normal diet.

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Function and Chemical Reactions Most antioxidants are either water or fat soluble. A few, such as lipoic acid, are both. This dual nature allows lipoic acid to function in both fatty and aqueous environment, an ability that is the reason why lipoic acid is often termed "universal antioxidant." Lipoic acid provides therapeutic benefits in numerous oxidative stress related conditions and diseases including diabetes and heart disease. Lipoic acid and its reduced form, dihydrolipoic acid (DHLA), show the ability to directly quench a variety of reactive oxygen species (singlet oxygen, superoxides, peroxyl and hydroxyl radicals, hypochlorite, and peroxynitrite), inhibit reactive oxygen generators and spare and regenerate other antioxidants. Lipoic acid can also regenerate a variety of antioxidants including glutathione, vitamins C and E, and the mitochondrial antioxidant coenzyme Q10 [53]. Neuroprotective Effects in Cell Culture and Animal Models Lipoic acid (LA) can protect cortical neurons against cell death induced by A
and hydrogen peroxide. It could be shown that this occurs through the Akt signalling pathway [54]. In a further study, the effects of LA and its reduced form, dihydrolipoic acid DHLA, in neuron cultures treated with amyloid beta-peptide (A
25-35), were studied. Pretreatment of dissociated primary hippocampal cultures with LA significantly protected against A
toxicity. In contrast, concomitant treatment of cultures with LA and Fe/H2O2 significantly potentiated the toxicity, most likely because LA has too be reduced to DHLA before exerting a antioxidant effect[55]. Using PC12 cells as a cell model in PD it could been shown, that pretreatment with R-lipoic acid prevents depletion of the thiol compound glutathione (GSH), impairment of mitochondrial complex I and ROS generation [56]. Neuroprotective Effects in Alzheimer and Parkinson Patients Oxidative stress and energy depletion are characteristic biochemical hallmarks of Alzheimer's disease (AD), thus antioxidants with positive effects on glucose metabolism such as lipoic acid should exert positive effects in these patients. Therefore, 600 mg lipoic acid was given daily to patients with AD and related dementias (receiving a standard treatment with acetylcholinesterase inhibitors) in an open study over an observation period of, on average, 337+/-80 days. The treatment led to a stabilization of cognitive functions in the study group, demonstrated by constant scores in two neuropsychological tests (mini-mental state examination: MMSE and AD assessment scale, cognitive subscale: ADAScog) [57]. Despite the encouraging data from cell culture models, no positive study with lipoic acid in PD patients has been reported so far. 5.3. Polyphenols Polyphenols are secondary plant compounds and can be subdivided into different compound classes depending on the substituents attached to the three ring systems. All polyphenols share a common structure with two phenolic rings structures which are capable of scavenging free radicals. Four classes of polyphenols are particulary interesting as antioxidant drugs in human nutrition: Flavanols, Flavonols and Anthocyanidins (Fig. 3). Polyphenols have developed in plants in response to stress, including protection against UV light. Plants often contain several different polyphenols, and it is difficult to judge the most potent antioxidant or neuroprotective compound in a plant derived food or beverage. Therefore, the next chapters can only describe the neuroprotective property of these food or plant preparations rather than been able to attribute these effects to a single polyphenol alone.

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Fig. (3). Chemical structures of polyphenols including three classes of flavonoids (flavonols, flavanols, anthocyanidins) and trihydroxystilben.

5.3.1. Flavanones Occurrence Flavonoids are part of a family of naturally occurring polyphenolic compounds characterized by a common benzo-
-pyrone structure. Flavanones is one of the most important flavonoid family present in vegetables, especially in genus Citrus (family Rutaceae) [58]. Flavanones are the first step on the biosynthesis pathway of all the other flavonoid families. They represent a large number of compounds arising from the various combinations of multiple hydroxyls, methoxyl and O-glycoside group substituents on the basic benzo-
-pyrone (C6-C3-C6) [59]. The concentration of these compounds depends on the age of the plant, and the highest levels are detected in tissues showing pronounced cell divisions [60-64]. These compounds not only play an important physiological and ecological role but are also of commercial interest because of their multitude of applications in the food and pharmaceutical industries [58, 65-68]. Function and Chemical Reaction Epidemiological and animal studies point to a possible protective effect of flavanones against cardiovascular diseases and some types of cancer. Although flavanones have been studied for about 50 years, the cellular mechanisms involved in their biological action are still not completely known. Many of the pharmacological properties of these citrus flavonoids can be linked to the abilities of these compounds to inhibit enzymes involved in cell activation. In vitro, they have demonstrated their capacity to modify the activity of enzymatic systems in mammals (kinases, phospholipases, ATPase, lipooxygenases, cyclooxygenases, phosphodiesterases, etc), a correlation having been observed in some cases between the flavonoid structure and its enzymatic activity [58, 69-73]. The most important structural factors that may condition flavonoid activity are structure oxidation stage, substituents (position, number and nature of groups in both A and B ring of the flavonoid structure), and the presence of glycosylation [58, 74, 75]. Much of the above mentioned effects can be attributed to the abilities of flavanone type flavonoids to interact with the nucleotide binding sites of regulatory enzymes [75]. Research has also shown that citrus flavonoids are

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potent radical scavengers, and thus, are able to help in many aging and degenerative events involving reactive oxygen species [58]. Neuroprotective Effects in Cell Culture and Animal Models Some studies demonstrate the effectiveness of Naringenin (NAR) in preventing the cognitive deficits. Thus, intracerebroventricular (ICV) injection of streptozotocin (STZ) causes cognitive impairment in rats, the STZ-injected rats received NAR (50 mg/kg/day p.o.) starting 1 day pre-surgery for 3 weeks. The learning and memory performance was assessed using passive avoidance paradigm, and for spatial cognition evaluation, radial eight-arm maze (RAM) task was used. The obtained results demonstrate the protection capacity of naringenin and its potential in the treatment of neurodegenerative diseases such as Alzheimer's disease [76]. Amyloid beta protein (Abeta)-induced free radical-mediated neurotoxicity is known as a leading hypothesis for a cause of Alzheimer's disease. Abeta increased free radical production and lipid peroxidation in PC12 nerve cells, resulting in apoptosis and cell death. The protective effect of naringenin, against Abeta-induced neurotoxicity was investigated using PC12 cells. Pretreatment with isolated naringenin and vitamin C prevented the generation of the Abeta-induced reactive oxygen species. Naringenin resulted in the decrease of Abeta toxicity in a manner of concentration dependence [77]. Naringenin inhibited acetylcholinesterase (AChE) inhibitors activity in a dose-dependent manner [78]. Also, the antiamnestic activity of naringenin in vivo was also evaluated using ICR mice with amnesia induced by scopolamine (1 mg/kg body weight). Naringenin, when administered to ICR mice at 4.5 mg/kg body weight, significantly ameliorated scopolamine-induced amnesia as measured in the passive avoidance test. Therefore, these results indicate that micromolecular induced in vitro oxidative cell stress is reduced by naringenin [77, 78]. Although the cause of dopaminergic cell death in Parkinson's disease (PD) remains unknown, oxidative stress has been strongly implicated. Unilateral infusion of 6-OHDA into the medial forebrain bundle produced a significant loss of tyrosine hydroxylase (TH)positive cells in the substantia nigra (SN) as well as a decreased of dopamine (DA) content in the striata in the vehicle-treated animals. Rats pretreated with naringenin showed a clear protection of the number of TH-positive cells in the SN and DA levels in the striata. The ability of naringenin to exhibit neuroprotection in the 6-OHDA model of PD may be related to their antioxidant capabilities and their capability to penetrate into the brain [79]. Peroxynitrite (ONOO-) is a reactive oxidant formed from superoxide (*O2(-)) and nitric oxide (*NO), that can oxidize several cellular components, including essential protein, nonprotein thiols, DNA, low-density lipoproteins (LDL), and membrane phospholipids. ONOOhas contributed to the pathogenesis of Alzheimer's disease. Because of the lack of endogenous enzymes to detoxify ONOO-, developing a specific ONOO- scavenger is remarkably important. Hesperetin can efficiently scavenge authentic ONOO-. Hesperetin exhibited significant inhibition on the nitration of bovine serum albumin (BSA) by ONOO- in a dosedependent manner. Hesperetin also manifested cytoprotection from cell damage induced by ONOO- and ROS [80]. The death of nigral neurons in Parkinson's disease is thought to involve the formation of the endogenous neurotoxin, 5-S-cysteinyl-dopamine. Hesperetin inhibits tyrosinase-induced formation of 5-S-cysteinyl-dopamine. This inhibition was not accompanied by the formation of cysteinyl-hesperetin adducts, indicating that it may inhibit via direct interaction with tyrosinase [81].

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Liquiritigenin, a newly found agonist of selective estrogen receptor-beta, has neuroprotective activity against beta-amyloid peptide (Abeta) in rat hippocampal neurons. Some genes, including B-cell lymphoma/leukemia-2 (Bcl-2), neurotrophin 3 (Ntf-3) and amyloid beta (A4) precursor protein-binding, family B, member 1 (Apbb-1) were regulated by liquiritigenin, consequently, liquiritigenin exhibits neuroprotective effects against Abeta(25-35)-induced neurotoxicity and that it can decrease the secretion of Abeta(1-40) [82]. Flavanones from Sophora flavescens were examined for their inhibitory effects against beta-secretase. Lavandulyl flavanones showed potent beta-secretase (BACE1) inhibitory activities with IC(50)s in the range 2.6–8.4
M, thus, reduced A secretion dosedependently in transfected human embryonic kidney (HEK-293) cells [83]. Neuroprotective Effects in Alzheimer and Parkinson Patiens To our best knowledge, there are no published epidemiological data or clinical trials for flavanones. 5.3.2. Flavones Occurrence Flavones are, probably, the most widely distributed compounds of the flavonoids family on the plant kingdom. Structurally, they are similar to flavanones, and only show, as difference, the characteristic double bond between the carbons 2 and 3 of the generated C-ring [58]. Flavones occur in an almost bewildering array of derivatives that represent a large number of compounds arising from the various combinations of multiple hydroxyls, methoxyl and O-glycoside group substituents on the basic benzo-
-pyrone (C6-C3-C6) [59]. Similarly to flavanones, their formation normally depends on light, so they are mainly concentrated in the outer tissues, being their concentrations directly related with the age of the plant, and the highest levels are also detected in tissues showing pronounced cell growth [60-64]. Flavones are the flavonoids more developed on the pharmaceutical industry, mainly for their cardiovascular and vasoprotective properties [58, 65-68]. Function and Chemical Reaction Animal, epidemiological and pre-clinical studies have shown that flavones possesses a variety of pharmacological activities, including antioxidant, anti-inflammatory, antimicrobial and, also, a possible protective effect some types of cancer. The ability of flavones to inhibit angiogenesis, to induce apoptosis, to prevent carcinogenesis in animal models, to reduce tumor growth in vivo and to sensitize tumor cells to the cytotoxic effects of some anticancer drugs suggests that flavones have cancer chemopreventive and chemotherapeutic potential. Modulation of ROS levels, inhibition of topoisomerases I and II, reduction of NFB and AP-1 activity, stabilization of p53, and inhibition of PI3K, STAT3, IGF1R and HER2 are possible mechanisms involved in the biological activities of the main flavones [84]. Much of the above mentioned effects can be attributed to the abilities of flavanones flavonoids to interact with the nucleotide binding sites of regulatory enzymes [75]. Several flavones are potent radical scavengers, and thus, are able to help in many aging and degenerative events involving reactive oxygen species [58]. A correlation has been observed in some cases between the flavonoid structure and its enzymatic activity [58, 69-73]. The most Important structural factors that may condition flavone activity are structure oxidation stage, substituents (position, number and nature of groups in both A and B ring of the flavonoid structure), and the presence of glycosylation [58, 74, 75]. Several flavonoids attenuate lipopolysaccharide-induced nitric oxide and tu-

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mour necrosis factor production in murine microglia and macrophages and the structureactivity relationships of these flavonoids demonstrated three distinct principles: (i) flavonoid-aglycons are more potent than the corresponding glycosides, (ii) flavonoids with a 4'OH substitution in the B-ring are more potent than those with a 3'-OH-4'-methoxy substitution, (iii) flavonoids of the flavone type (with a C2=C3 double bond) are more potent than those of the flavanone type (with a at C2-C3 single bond) [85]. Neuroprotective Effects in Cell Culture and Animal Models In case of injury or disease, microglia is recruited to the site of the pathology and become activated as evidenced by morphological changes and expression of pro-inflammatory cytokines. Evidence suggests that microglia proliferate by cell division to create gliosis at the site of pathological conditions such as the amyloid plaques in Alzheimer's disease and the substantia nigra of Parkinson's disease patients. The hyperactivation of microglia contributes to neurotoxicity. Related with this consideration, several flavones were as tested by the hypothesis that antioxidant and anti-inflammatory compounds modulate the progression of cell cycle and induce apoptosis of the activated cells [86]. Apigenin, luteolin and polymethoxyflavones are the most studied compounds from this flavone family. Several of these natural flavones act as non-peptidic BACE-1 inhibitors and potently inhibit BACE-1 activity and reduce the level of secreted A
in primary cortical neurons [87]. Apigenininduced cell cycle arrest preferentially in the G2/M phase. Apigenin induces apoptosis significantly in early and late stages. The induction of apoptosis by apigenin was confirmed using TUNEL assay, revealing that 25 microM apigenin significantly increased apoptosis in BV-2 cells [86]. Experiments performed to study the possible effects of exogenously administered apigenin (as 7-O-glucoside) on the cognitive performance in aged and LPS-treated mice (an animal model for AD) using passive avoidance and elevated plus-maze tasks, showed that chronic administration of the apigenin-7-glucoside (5-20 mg/kg i.p.) reverses cognitive deficits in aged and LPS-intoxicated mice which suggests that modulation of cyclooxygenase-2 and inducible nitric synthase by this flavone may be important in the prevention of memory deficits, one of the symptoms related to AD [88]. Recently, apigenin shows its ability to downregulate inflammatory markers and to protect neurons against microglial insult [89]. CD40 signaling is critically involved in microglia-related immune responses in the brain. It is well known that the activation of the signal transducer and activator of transcription (STAT) signaling pathway plays a central role in interferon-gamma (IFN-gamma)-induced microglial CD40 expression, and this expression is significantly induced by IFN-gamma and amyloid-beta (A beta) peptide. Apigenin and luteolin concentration-dependently suppressed IFN-gamma-induced CD40 expression. Apigenin and luteolin also suppressed microglial TNF-alpha and IL-6 production stimulated by IFN-gamma challenge in the presence of CD40 ligation. In addition, apigenin and luteolin markedly inhibited IFN-gamma-induced phosphorylation of STAT1 with little impact on cell survival [90]. Glycogen synthase kinase 3 (GSK-3) dysregulation is implicated in the two AD pathological hallmarks: beta-amyloid plaques and neurofibrillary tangles. GSK-3 inhibitors may abrogate AD pathology by inhibiting amyloidogenic gamma-secretase cleavage of amyloid precursor protein (APP). Luteolin, when applied to the Tg2576 mouse model of AD, decreases soluble Abeta levels, reduces GSK-3 activity, and disrupts PS1-APP association. In addition, we find that Tg2576 mice treated with diosmin, the flavone-glycoside most used in pharmaceuticals, also display significantly reduced Abeta pathology [91]. Luteolin protects dopaminergic neurons from inflammation-induced injury through inhibition of microglial

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activation [92] and also protects rat PC12 and C6 cells against MPP+ induced toxicity via an ERK dependent Keap1-Nrf2-ARE pathway [93]. Short-term supplementation with citrus extracts rich in polymethoxyflavones, mainly tangeretin, protect nigrostriatal dopaminergic neurons to the dopaminergic neurotoxin 6hydroxydopamine lesions in a rat model of Parkinson's disease [94, 95]. Preadministration of tangeretin in mice enhanced expression of glucose-regulated protein (GRP) 78, (a molecular chaperone in the endoplasmic reticulum) in the substantia nigra pars compacta and protected dopaminergic neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a neurotoxin that induces both oxidative and endoplasmic reticulum stress [96]. Nobiletin, probably the most important citrus polymethoxylated flavone, restoring betaamyloid-impaired CREB phosphorylation rescues memory deterioration in Alzheimer's disease model rats [97]. Treatment with 50 mg/kg of nobiletin (i.p.) for the consecutive 7 days before and after brain ischemia significantly inhibited delayed neuronal death in the hippocampal CA1 neurons in a 20-min bilateral common carotid arteries occlusion ischemia. The nobiletin treatment prevented the reduction in CaMKII, MAP2 and GluR1 protein levels in the hippocampal CA1 region, accompanied by restoration of both ERK and CREB phosphorylation and CaMKII auto-phosphorylation [98]. Nobiletin rescues olfactorybulbectomized-induced cholinergic neurodegeneration, accompanied by improvement of impaired memory in olfactory-bulbectomized mice [99]. The administration of nobiletin decreased the A
burden and plaques in the hippocampus of APP-SL 7-5 Tg mice (transgenic mice that overexpress human APP695 harboring the double Swedish and London mutations) [100]. Neuroprotective Effects in Alzheimer and Parkinson Patiens It has been postulated that oxidative stress may play a key role in dementia. Thus, flavonoid intake, mainly flavones, could be associated with a lower incidence of dementia in a cohort of 1367 subjects above 65 years of age (Paquid). A questionnaire was used to evaluate their intake of flavonoids and subjects were followed-up for 5 years between 1991 and 1996 and the estimation of the relative risk of dementia show that the intake of flavonoids is inversely related to the risk of incident dementia [101]. The Rotterdam Study, a population-based, prospective cohort study conducted in the Netherlands included a total of 5395 participants who, at baseline (1990-1993), were aged at least 55 years, free of dementia, and non-institutionalized and had reliable dietary assessment. Participants were re-examined in 1993-1994 and 1997-1999 and were continuously monitored for incident dementia. After a mean follow-up of 6 years, 197 participants developed dementia, of whom 146 had Alzheimer disease; after adjustments, the use of antioxidant supplements with flavonoids was associated with lower risk of Alzheimer disease, however, lesser than vitamins C and E [41]. The Kame project tested whether consumption of fruit and vegetable juices, containing a high concentration of polyphenols, mainly flavone-glycosides, decreases the risk of incident probable Alzheimer's disease in the corresponding cohort, a population-based prospective study of 1836 Japanese Americans in King County, Washington, who were dementia-free at baseline (1992-1994) and were followed through 2001. The hazard ratio for probable Alzheimer's disease was 0.24 (95% confidence interval [CI], 0.09-0.61) comparing subjects who drank juices at least 3 times per week with those who drank less often than once per week with a hazard ratio of 0.84 (95% CI, 0.31-2.29) for those drinking juices 1 to 2 times

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per week (p < 0.01). This inverse association tended to be more pronounced among those with an apo-lipoprotein epsilon-4 allele and those who were not physically active. Conversely, no association was observed for dietary intake of vitamins E, C, or betacarotene or tea consumption. Fruit and vegetable juices may play an important role in delaying the onset of Alzheimer's disease, particularly among those who are at high risk for the disease [102]. In the PAQUID (Personnes Agées Quid) study, the authors prospectively examined flavonoid intake in relation to cognitive function and decline among subjects aged 65 years or older. A total of 1,640 subjects free from dementia at baseline in 1990 and with reliable dietary assessment were re-examined four times over a 10-year period. Cognitive functioning was assessed through three psychometric tests (Mini-Mental State Examination, Benton's Visual Retention Test, "Isaacs" Set Test) at each visit. Information on flavonoid intake was collected at baseline, being the flavones apigenin and luteolin, as glucoside-forms, two of the most abundant. Flavonoid intake was associated with better cognitive performance at baseline (p = 0.019) and with a better evolution of the performance over time (p = 0.046). Subjects included in the two highest quartiles of flavonoid intake had better cognitive evolution than did subjects in the lowest quartile. After 10 years' follow-up, subjects with the lowest flavonoid intake had lost on average 2.1 points on the Mini-Mental State Examination, whereas subjects with the highest quartile had lost 1.2 points [103]. 5.3.3. Flavanols (Catechins, Epicatechins) Occurrence Catechins are flavanols of which epicatechin (EC), (-)-epigallocatechin (EGC) and (-)epicatechin gallate (EGCG) are the main ones present in fresh tea leaves [104]. Daily intakes in humans are up to 100 mg/day. Sources of flavanols are wine, apples, chocolate and black and green tea. One cup (237 ml) of green tea contains typically 30 to 130 mg of EGCG while black tea contains only 0 to 70 mg of EGCG (Fig. 4).

Fig. (4). Chemical structures of catechins (a type of flavanol), main antioxidant ingredients of green tea.

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Function and Chemical Reactions It is documented that catechins scavenge free radicals in vitro systems [105]. Catechins are able to scavenge .NO in vitro [106] as well ONOO- [107], even if the mechanisms of scavenging radicals is still unknown. Singlet oxygen (1O2), O2.-, .OH, and peroxyl radicals . OOH are further radicals that are scavenged by catechins [108-110]. Their superb antioxidant activity is because of a low reduction potential compared to vitamin E, but not to vitamin C. Therefore EGCG and EGC are superior electron donators, which is concurrent with a better antioxidant activity. Neuroprotective Effects in Cell Culture and Animal Models Catechins in green tea are also capable of decreasing lipid peroxidation. Using pulse radiolysis, Bors and Michel have found that catechins rather than flavonols and flavones, are the antioxidative compounds in tea [111]. Catechins increase the activity of glutathione peroxidase and glutathione reductase, while decreasing the levels of lipid hydroperoxidases (LOOH), 4-hydroxysynonenal (4-HNE) and malondialdehyde (MDA) [112].The peroxidase activity of lipoxygenase and cyclooxygenase is one mechanism to increase oxidative stress, which can be inhibited by flavonoids and phenolic antioxidants in vitro [113]. Both enzymes increase oxidative stress because they take part in the synthesis of leukotrienes and prostaglandins, two molecules involved in inflammation. Xanthine oxidase is another enzyme inhibited by catechins. This enzyme reduces O2 to O2.- and H2O2 during the oxidation of xanthine and hypoxanthine to uric acid [104], catechins however inhibit the production of these ROS. But besides the inhibition of enzymes, catechins are also able to induce some enzymes including phase II enzymes for detoxification or antioxidant enzymes such as glutathione peroxidase (GPx)[114], catalase and superoxidase dismutase (SOD) [115]. EGCG has been shown to exert protective effects against A
-induced neurotoxicity and regulate secretory processing of non-amyloidogenic APP. In a cell culture model of AD, EGCG was shown to be able to protect PC12 cells against A
toxicity in a dose-dependent manner. In addition, EGCG enhances (approximately 6-fold) the release of the nonamyloidogenic and neurotrophic form of the amyloid precursor protein (sAPPalpha) into the conditioned media of human SH-SY5Y and PC12 neuroblastoma cells [116]. More recently, green tea catechins have been suggested to have the potential to prevent AD because of their anti-amyloidogenic, anti-oxidative, and anti-inflammatory properties, also activating adaptive cellular stress responses, called "neurohormesis", and suppress disease processes [117]. A further function of catechins in tea is the inhibition of up-regulation of enzymes that propagate inflammation resulting in cellular oxidative stress. For example it has been demonstrated that both green and black tea inhibit LPS-induced iNOS expression in macrophages by inhibiting the transcription factor for iNOS which is NF-
B on its DNA binding activity and through phosphorylation of its inhibitor I
B[118, 119]. A recent study shown that (-)-epigallocatechin-3-gallate exert neurorescue and mitochondrial stabilization actions across multimodal mechanisms [120]. Studies on the bioavailability and brain deposition of these polyphenols are the main elements to clarify their possible capacity as neuroprotective compounds. Long-term administration of green tea catechins [Polyphenon E (PE): 63% of epigallocatechin-3-gallate, 11% of epicatechin, 6% of (-)-epigallocatechin and 6% of (-)-epicatechin-gallate] prevents cognitive impairment in an animal model of AD, rats infused with Abeta1-40 into the cerebral ventricle. PE administration for 26 weeks significantly decreased the Abeta-induced

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increase in the number of reference and working memory errors, with a concomitant reduction of hippocampal lipid peroxide (LPO; 40%) and cortico-hippocampal reactive oxygen species (ROS; 42% and 50%, respectively) [121]. Similarly, repeated daily exposure to grape seed extract catechins was found to significantly increase bioavailability catechin and epicatechin by 253 and 282% relative to animals receiving only a single acute extract dose. Epicatechin and catechin were not detectable in brain tissues of rats receiving a single dose but reached levels of 290.7 +/- 45.9 and 576.7 +/- 227.7 pg/g in brain tissues from rats administered the grape extract for 10 days [122]. The purpose of a further study was to investigate potential neuroprotective effects of tea extracts and possible signal pathway involved in a neuronal cell model of Parkinson's disease. 6-OHDA activated the iron dependent inflammatory redox sensitive nuclear factor-
B in rat pheochromocytoma (PC12) and human neuroblastoma (NB) SH-SY5Y cells, respectively. Immunofluorescence and electromobility shift assays showed increased nuclear translocation and binding activity of NF-
B after exposure to 6-OHDA in NB SH-SY5Y cells, with a concomitant disappearance from the cytoplasm. Introduction of Green tea extract before 6-OHDA inhibited both NF-
nuclear translocation and binding activity induced by this toxin in NB SH-SY5Y cells. The authors conclude that neuroprotection was attributed to the potent antioxidant and iron chelating actions of the polyphenolic constituents of tea extracts, preventing nuclear translocation and activation of NF-
B [123]. In an animal models of Parkinson's disease (PD), the effect of green tea toxicity of 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was investigated in mice. Both tea and the oral administration of EGCG prevented the loss of tyrosine hydroxylase (TH)-positive cells in the substantia nigra (SN) and of TH activity in the striatum. These treatments also preserved striatal levels of dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid (HVA). Also tea plus MPTP and EGCG plus MPTP treatments decreased expressions of neuronal NO synthase (nNOS) at the similar levels of EGCG treatment group [124]. However, new studies show certain controversies, thus short-term supplementation with plant extracts rich in different catechin structures (flavan-3-ols) protect nigrostriatal dopaminergic neurons in rat model of Parkinson's disease. Pretreatment of animals with a cocoa extract rich in procyanidins (polymeric flavan-3-ols; 100 mg/kg/day) significantly attenuated the 6-OHDA-induced dopaminergic loss. However, no significant protection was seen in animals supplemented with a grape seed extract, rich in catechins, (100 mg/kg/day), and a rich catechins cocoa extract (100 mg/kg/day) [95]. Despite the huge and increasing amount of the in vitro studies trying to unravel the mechanisms of action of dietary polyphenols, the research in this field is still incomplete, and questions about bioavailability, biotransformation, synergism with other dietary factors, mechanisms of the antioxidant activity, risks inherent to their possible pro-oxidant activities are still unanswered. Most of all, the capacity of the majority of these compounds to cross the blood-brain barrier and reach brain is still unknown [125]. Neuroprotective Effects in Alzheimer and Parkinson Patients In a study in the northern health region of England, risk factors for presenile AD (onset blow 65 years of age) were analysed in 109 patients. In this study, which was originally aimed to assess the impact of aluminium of dementia, no significant relationship between AD and exposure to tea and antacids was found [126].

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A protective role of tea consumption for PD has been shown only in Asian countries, where green tea is a major beverage. A study in Hong Kong in which 215 PD patients and 313 controls took part found that a regular tea drinking is protective against PD [127]. In a study undertaken in Singapore, the relationship between coffee and tea drinking, cigarette smoking, and other environmental factors and risk of PD among 300 PD and 500 population (ethnic Chinese) was analysed. The authors calculated that 3 cups/day of tea for 10 years would lead to 28% risk reduction of PD [128]. However, a study in France showed the opposite. 140 patients and 240 hospital controls showed an increased risk by 90% to develop this dementia [129]. It is also reported from a study in Spain that there was no significant association between tea consumption and the risk of PD [130]. 5.3.4. Flavonols (Quercetin, Kaempferol) Occurrence Flavonols including quercetin and kaempferol are characterized by a keto group at the 4position in the heterocyclic C ring. They are present in red wine, onions, apples, tea and broccoli and in the leaves of the Gingko biloba tree. In a normal European diet, daily flavonol intakes of 3 – 64 mg have been reported. The Ginkgo tree and has evolved strategies to live for up to 1,000 years [131]. Because of the high antioxidant content of its leaves, the pharmaceutical industry has developed the standardised leaf extract EGb 761 to treat vascular and neurodegenerative diseases. This extract consists of 24% flavone glycosides (also called ginkgosides, mainly quercetin, kaempferol and isorhamnetin glycosides) and 6% lactone terpenes, consisting of ginkgolides A, B, C and J as well as bilobalide. The amount of ginkgolic acids is less than 0.0005%, because it has been described, that 100
M of it causes allergic symptoms and neuronal cell death [132, 133]. Neuroprotective Effects in Cell Culture and Animal Models It has been shown that quercetin or structurally similar flavonoids are potent neuroprotective antioxidants in red wine or related fruit derived foods. It is thought that the presence of 2,3 unsaturation together with an oxo function at position 4 in the ring C, co-planarity of the molecule and a 3’,4’-dihydroxy catechol structure in the B ring are responsible for the antioxidant scavenger activity [134]. The standardised leaf extract EGb 761 is mainly used for the treatment of vascular diseases and for neurodegenerative dementia. The antioxidant effects are mediated by the ginkgosides [135], however terpenes are also thought to contribute via their anti-inflammatory effects [136]. It has been demonstrated in rats that the protein level and activity of antioxidant enzymes is increased by EGb 761 and it is therefore believed that this drug exerts its effect by stabilization of the redox status as by radical scavenging [137], [138]. EGb 761 is also able to scavenge NO [139]. Since increased nNOS and iNOS expression is found in AD, EGb 761 has been proposed as therapeutic treatment for this type of dementia conditions. Oxidative damage caused by superoxide leakage from mitochondrial respiration can be decreased by EGb 761, most likely by increasing the messenger RNA (mRNA) and protein level of subunit I of mitochondrial NADH dehydrogenase. There is also plenty of evidence for neuroprotective activities of Gingko biloba extracts in AD specific disease models. A, the main compound of senile plaques in AD, originates from a larger amyloid precursor protein, the amyloid precursor protein (APP). This protein can then either be cleaved by - and -secretases, as described the neuropathology and biochemistry section of AD, or by
-secretases that release a large and soluble extra cellular domain of APP, the so called
APPs, that represents the non-toxic form of A. Using hippocampal slices cultures, it could be shown that EGb761 increases
APPs release [140].

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EGb761 has also been shown to decrease ROS in a neuroblastoma cell line that stably expresses an AD-associated double mutation, which exhibits both increased secretion and intracellular accumulation of Abeta when stimulated. This effect could also been shown in vivo using C. elegans that constitutively expresses human Abeta. Both, the cell line and C. elegans showed a rise in levels of hydrogen peroxide compared with wild type controls, before stimulating [141]. The effect of the flavonol kaempferol on A
-induced toxicity in PC12 neuroblastoma and T47D human breast cancer cells was investigated and a protective effect of kaempferol (comparable to that observed with estradiol) was observed. Since the effects of the weak estrogen receptor agonists
-estradiol and kaempferol were found to be similar to the effects of the strong estrogen receptor agonist
-estradiol, the authors suggesting a mode of action independent from the nuclear estrogen receptor such as radical scavenging [142]. A beneficial effect of extracts of Ginkgo biloba leaves (EGb) was observed in the Parkinson disease (PD) model induced by MPTP. MPTP was microinjected into substantia nigra of rats to induce a behavior change of rotation. EGb treatment decreased the duration and frequency of the rotation of rats as well as the level of the lipid peroxidation product MDA. In addition, the decrease of DA levels was attenuated which indicates increased viability of dopaminergic neurons [143]. Similar positive effect were observed in the 6-hydroxydopamine (6OHDA) model, where neurotoxicity in the nigrostriatal dopaminergic system of the rat brain was attenuated by the EGb extract [144]. Neuroprotective Effects in Alzheimer and Parkinson Patients A study with 216 patients with mild to moderate degenerative dementia of AD or multiinfarct dementia was carried out. Patients were treated with 240 mg of EGb 761 or placebo for 24 weeks. Using memory functions, independence, coping skills and other parameters as the basis of measures, it was concluded that EGb 761 has clinical efficacy in AD patients [145]. In another study the potential association between the use EGb761 and AD was investigated. A case-control study was nested in a cohort of 1462 community-dwelling elderly woman aged over 75 years. 69 women with AD were compared with 345 women whose cognitive function remained normal. Analysis revealed that fewer women who developed AD had been prescribed EGb761 for at least 2 years but to establish a causal relationship, prospective studies have to be carried out to confirm these findings [146]. 5.3.5. Anthocyanins (Cyanidine and Others) Occurrence Anthocyanins are natural red and blue colours of plants and fruits, including various berries (strawberries, blueberries, raspberries, black currants) and red grapes. The average daily intake in humans is about 200 mg/day. Function and Chemical Reactions Anthocyanins also belong to the class of polyphenols and consist of 3 carbon rings with 2 aromatic (A and B) and one O-heterocyclic ring, which is positively charged (Fig. 3). Neuroprotective Effects in Cell Culture and Animal Models The effects of purple sweet potato anthocyanin (SPA) on lipid peroxidation, 1,1diphenyl-2-picryl-hydrazyl (DPPH) radicals and cognitive deficits were examined. SPA was shown to exhibit DPPH radical scavenging activities and to effectively inhibit lipid peroxidation initiated by Fe2+ and ascorbic acid in rat brain homogenates. Furthermore, SPA

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markedly enhanced cognitive performance, assessed by passive avoidance test in ethanoltreated mice. These results demonstrate that anthocyanin prepared from purple sweet potato exhibits memory enhancing effects, which may be associated with its antioxidant properties [147]. In another study, Fischer 344 rats received dietary supplementation with fruit or vegetable extracts high in anthocyanins (e.g. blueberry (BB) or spinach, respectively). This diet decreased the age-related vulnerability to oxidative stress in these rats as assessed in vivo by examining reductions in neuronal signaling and behavioral deficits and in vitro via H2O2-induced decrements in striatal synaptosomal calcium buffering. Examinations have also revealed that BB supplementations are effective in antagonizing other age-related changes in brain and behavior, as well as decreasing indices of inflammation and oxidative stress in muscles. The authors conclude that anthocyanins show the most efficacy in penetrating the cell membrane and in providing antioxidant protection [148]. A diet rich in blueberries was also shown to prevent behavioural deficits in an AD model, the APP over expressing transgenic mouse. The authors believed that the protective effect is due to the enhancement of redox-sensitive memory-associated neuronal signalling and alterations in neutral sphingomyelin-specific phospholipase C activity [149]. 5.3.6. Isoflavones Occurrence Isoflavones are a subclass of flavonoid characterized by have a 3-phenyl chromen-4-one (3-phenyl-1,4-benzopyrone) structure instead a 2-phenyl chromen-4-one (3-phenyl-1,4benzopyrone) structure tipical of flavanones and flavones. They have a large structure variability and more of than 600 isoflavones have been identified. The primary dietary sources of isoflavones are soybeans (Glycine max) and soyfoods. It is often stated in the literature that many legumes and fruits and vegetables contain isoflavones; however, although these statements are technically correct they are misleading, because the amount in these foods is so small as to be nutritionally irrelevant. In contrast to many phytoallexins (substances that are formed by host tissue in response to physiological stimuli, infectious agents, or their products and that accumulate to levels that inhibit the growth of micro-organisms), isoflavones are always present in significant quantities in soybeans, because one of their primary functions is to stimulate nodulation genes in soil bacteria called Rhizobium. There are 12 different soybean isoflavone isomers. These are the three aglycones genistein (4',5,7-trihydroxyisoflavone), daidzein (4',7-dihydroxyisoflavone), and glycitein (7,4'dihydroxy-6-methoxyisoflavone), their respective
-glycosides genistin, daidzin, and glycitin, and three
-glucosides each esterified with either malonic or acetic acid having the three aglycones the higher concentrations. Red clover (Trifolium pratense) also contains a rich supply of isoflavones and, along with soybeans, is used as a source for the production of isoflavone supplements. Function and Chemical Reactions Many pottial health benefits have been linked to intake of soy products according to epidemiologial investigations [150]. Animal [151] and human [152] studies have also shown that consumption of soy isoflavones has beneficial impacts on the risk factors for cardiovascular disease including lowering liver or blood triglyceride, total and LDL cholesterol levels, increasing HDL cholesterol and the ratio of HDL/LDL cholesterol. Cellular and molecular biology studies have demonstrated that soy components modulate the key transcription factors involved in the regulation of lipid metabolism and their regulated downstream gene expression in animals and in vitro cultured human cells at transcriptional or posttrans-

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lational levels [151]. However the most popular property of isoflavones is their effects like phytoestrogens. The isoflavones are strikingly similar in chemical structure to mammalian estrogens [153]. The phenolic ring is a key structural element of most compounds that bind to estrogen receptors [154]. When the structures of the isoflavone metabolite equol and estradiol are overlaid, they can be virtually superimposed. On the basis of structure alone, it is not surprising that isoflavones bind to estrogen receptors (ER); however, their actions are more those of partial estrogen agonist or antagonist. Isoflavones have been shown be effectives in diseases where hormonal receptors are involved, so can stimulate bone formation in menopauseal women [155]. In cell culture, genistein inhibited proliferation of humanbreast and prostate cancer cells stimulated by epidermal growth factor (EGF) independently of whether the cells expressed estrogen or androgen receptors, respectively [156]. In human studies, isoflavone have been shown reduce risk for breast [157] or prostate cancer [158]. May not be appreciated that the levels of intake of these biologically active phytoestrogens exceed by several orders of magnitude the levels of endogenous estrogens. Typical circulating concentrations of isoflavones can exceed endogenous estradiol concentrations by 100fold in adults [159]. Neuroprotective Effects in Cell Culture and Animal Models Isoflavone genistein show comparable levels of protection that estradiol against A
induced deaths of cultured SH-SY5Y human neuroblastoma cells, which where blocked by an estrogen receptor antagonist, ICI 182,780 [160]. Genistein protects neurons from A induced damage via estrogen receptor-mediated pathway, and at the micromolar level, the neuroprotective effect of genistein is mediated mainly by its antioxidative properties [161], however have been suggest that also other mechanismes such antiinflammatory effects could be involved. Inflammation has been implicated in neurodegenerative disorders such as Alzheimer’s disease (AD). The main inflammatory players in AD are the glial cells which initiate the imflammatory response. One of the earliest neuropathological changes in AD is the accumulation of astrocytes at sites of A deposition. A induces inflammatory mediators, such as cyclooxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS), interleukin 1 (IL-1) and tumor necrosis factor  (TNF-). All these effects were prevented when cells were pretreated with estradiol or genistein, demostrating their antiiflammatory effects in astrocytes culture [162]. Also an increse in PAAR- (peroxisome proliferator activated receptors) expression in astrocytes was detected suggesting that some of the antiinflammatory effects of genistein may be mediated and activated by PAAR-. Isoflavones also may exert these effects via direct modulation of protein and lipid kinase signalling pathways, for example via the inhibition of MAPK signalling cascades which regulate both iNOS and TNF- expression in activated glial cells [163]. In the other hand, related experiments in a primate model of menopause demonstrated that ingestion of soy containing isoflavones was correlated with the suppression of neurodegeneration-relevant phosphorylation of the microtubule-associated protein tau, while intake of Premarin (a hormone re-placement therapy that is commonly prescribed for women) was not correlated. These results indicate that genistein, and probably other related phytoestrogens, have pleiotropic actions, some of which may involve TGFb activity [164]. Neuroprotective Effects in Alzheimer and Parkinson Patients It is known that dementia and neurodegenerative diseases, AD or PD, rates are lower in Asian countries than in western countries. This fact has been associated to asian lifestyle and particularly to asian diet roich in fish and soy products. However, few epidemiological studies have been made to corroborate soy benefits on brain health.

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One of these studies conducted in Hawaii called the Honolulu Heart Study came up with a surprising finding. The study looked at Japanese men residing in Hawaii and aimed to compare diet to risk of dementia. The researchers found that those men who ate tofu most frequently during their mid-40's to mid-60's showed the most signs of mental deterioration in their 70's to early 90's [165]. In this study of over 3000 men, intake of 26 foods, including tofu, was recorded between 1965 and 1967 and again in 1971 to 1974. Cognitive test performance was assessed between 1991 and 1993 and the researchers also looked at brain shrinkage through autopsy data of the men who died during the study. Tofu consumption of just two to four servings per week was associated with poorer test performance and more brain loss. Not only that, but the wives of men who ate tofu also showed more signs of dementia. The results of this study is not agree with that lower dementia rates in Asian countries and when Japanese lifestyle has actually been associated with better cognition in old age. Many have used this as an argument to show that the Hawaii study results must be wrong. But there is a possible biological explanation for the findings. Soybeans contain isoflavones, which are weak estrogens. They fall into the category of estrogen-like compounds known as selective estrogen receptor modulators (SERMS) [166]. This means that they have estrogenic effects in some tissues and anti-estrogenic effects in others. Estrogen may have a positive effect on brain tissue but the researchers of the Hawaii study suggested that isoflavones may have antiestrogenic effects on the brain. Results of three clinical studies suggest soy and isoflavones have beneficial effects on cognition. In the first published study, young adult men and women who consumed a high soy diet for 10 weeks experienced significant improvements in short-term and long-term memory and in mental flexibility [167]. The other two studies found that isoflavone supplements, when taken by postmenopausal women, improve cognitive function [168]. Even with these findings, we really have very little information on how soyfoods consumption might affect cognitive function. 5.3.7. Caffeoyl Compounds Occurrence Caffeoyl compounds are a large numbers of caffeoyl esters mainly sterified with sugars or quinic acid. These caffeoyl derivatives are widely distributed in many plants along the wide world. The main components of this phenolic group are Chlorogenic acid (3caffeoylquinic acid) (CGA) and Rosmarinic acid (RA). CGA widely exist in edible and medicinal plants being some of the most important green coffe and artichoke. RA is found in severals herbs in the Lamiaceae family, such as rosemary, thyme, sage or lemon balm. RA is the main phenolic compound in Rosemery plants [169]. In plants, rosmarinic acid is supposed to act as a preformed constitutively accumulated defence compound. In a retrospective review of the historical role of a number of European herbs we can found that the culinary herbs such as rosemary and sage have been widely used in the improvement of cognition and memory. In the other hand, Green (or raw) coffee is a major source of CGA in nature (5–12 g/100 g) [170]. Function and Chemical Reactions CGA and RA have been shown strong antioxidant activities against DPPH and ABTS radical in vitro models [171, 172]. TEAC value for RA was 1.6 times vs AA [173]. RA is able to protect lipid from peroxidation in the Thiobarbituric (TBARS) assay. In this model,

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malonyldialdehyde formation was delayed when RA was used. The protection factor was 3.24 times vs AA [173]. RA have shown antiinflammatory and antiallergic properties. The oral supplementation of 200 mg of RA to patients with seasonal allergic rhinoconjunctivitis (SAR) during 21 days resulted in asignificantly decreased in responder rates for each symptom. RA also significantly decreased the numbers of neutrophils and eosinophils in nasal lavage fluid [174]. In a parallel animal study RA showed anti-inflammatory activity 5-hours after 12tetradecanoylphorbol 13-acetate (TPA) treatment with marked inhibition of neutrophil infiltration. Up regulation of ICAM-1, VCAM-1 cyclooxygenase-2 (COX-2), KC and MIP-2 by TPA were markedly reduced by pre-treatment with RA. Reactive oxygen radical production detected as thiobarbituric acid reactive substance (TBARS), lipid peroxide (LPO) and 8-hydroxy-2'deoxyguanosine (8OH-dG), by double treatment of TPA was reduced by pretreatment with RA [174]. CGA efficiently suppressed cytotoxicity and loss of GSH caused by peroxides in PC12 cells [175]. CGA showed a excellent elimination of ROS induced by t-BHP, Hydrogen peroxide or ferrous sulfate suggesting that may have protective properties against oxidative stress induced in CNS. CGA is also a potent inhibitor of the microsomal glucose-6phosphate translocase (G6PT), is thought to possess cancer chemopreventive properties. G6PT could regulate in glioma cells the intracellular signalling and invasive phenotype of brain tumor cells, and that can be targeted by the anticancer properties of CGA [176]. Neuroprotective Effects in Cell Culture and Animal Models Very recently the role of caffeic acid (CA) as neuroprotective agent has been studied. Pretreatment of the PC12 cells with 10 and 20 μg/ml of CA, for 1 h prior to A
, significantly reverse the A
-induced neurotoxicity. The suggested CA neuroprotective mechanism is by attenuating of intracellular calcium influx and decreasing tau phosphorylation by the reduction of GSK-3
activation [177]. As CA, also other caffeoyl compounds as RA and CGA have been shown neuroprotective effects in cell culture. RA from sage prevents the neurotoxicity induced by A
42. Significant inhibitory effects were achieved at concentrations of 10-7 M [178]. RA reduced A
42-induced ROS formation and lipid peroxidation in a concentration-dependent manner and also can inhibit the formation of fibrils from A
destabilized preformed A
fibrils in vitro. Therefore, in PC12 cells, RA may inhibit ROS formation directly or indirectly by preventing fibril formation from A
[178]. As was described for CA, the neuroprotective mechanism of RA is based in the inhibition of tau hyperphosphorylation, but probably acting through the inhibition of p38 MAP kinase pathway instead of the inhibition of GSK-3
hyperphosphorylation as was described for CA. CGA also have shown neuroprotective properties in PC12 cells. CGA not only suppressed the generation of ROS, the decrease of activity in Gpx and the decrease of GSH, but also attenuated caspase-3 activation by MeHg, protecting PC12 cells against MeHg-induced apoptosis [179]. The antioxidant and neuroprotective effects of RA in animal model were examined. RA daily administrated to mice at dose of 0,25 mg/kg, after injection of A25
35, prevented induced nitration of proteins, an indicator of peroxynitrite damage, in mice hippocampus. At this dose RA also prevented nitration of proteins and impairment of recognition memory induced by peroxynitrite suggesting that the memory protective effects of RA in the neurotoxicity of A25
35 is due to its scavenging of peroxynitrite. This results suggest that RA can protect against memory impairments observed in AD [180].

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Neuroprotective Effects in Alzheimer and Parkinson Patients Based on a retrospective review of the historical role of a number of European herbs in the improvement of memory and cognition, it was shown that spices such as rosemary, sage or lemon balm might potentially provide a natural remedy for AD [181]. Recently, severals clinical studies has shown that sage or lemon balm are effectives in the management of mild to moderate AD [33-35]. A recent study has shown that lemon balm modulates mood and cognitive performance when administered to young, healthy volunteers [182]. In addition, a parallel, randomized, double-blind placebo-controlled study assessed the efficacy and safety of lemon balm in 42 patients between 65 and 80 years with mild to moderate AD [183]. Subjects were treated for four months. The main efficacy measures were the cognitive subscale of the Alzheimer's Disease Assessment Scale (ADAS-cog) and the Clinical Dementia Rating-Sum of the Boxes (CDR-SB) scores. The CDR-SB provides a consensus-based global clinical measure by summing the ratings from six domains: memory, orientation, judgment, problem solving, community affairs, home and hobbies, and personal care. The results revealed that patients receiving lemon balm extract experienced significant improvements in cognition after 16 weeks of treatment. Improvements were seen on both the ADAS-cog and CDR-SB scores. Moreover, another similar study showed that patients with mild to moderate AD receiving sage extract experienced statistically significant benefits in cognition after 16 weeks of treatment [184]. The clinical relevance of these findings was emphasized by the improvements seen in both the ADAS-cog and CDR-SB measures in the sage extract group on both observed case and intention-to-treat analyses. 5.3.8. Hydroxystilbenes (Resveratrol) Occurrence Resveratrol (3, 4’, 5-trihydroxystilbene) is natural polyphenol, which differs in structure from the flavonoids having only two (aromatic) ring systems (Fig. 3). It is synthesized by plants in response to fungal infection, and helps the plant to survive due to its strong antifungal properties. In human beverages, high amounts of resveratrol are found in red wine – highlighting an interesting fact that fungi indirectly increase the beneficial effect of a food or beverage. Because it is released from the skin of grapes, the fermentation time is a major factor in its effectiveness. White wine (which contains very low levels of anthocyanins) has also a lower content of resveratrol compared to red wine due to its shorter maceration time. Function and Chemical Reactions The effects of resveratrol include inhibition of lipid peroxidation, chelation of copper, free-radical scavenging and anti-inflammatory activity (reviewed [185]). Neuroprotective Effects in Cell Culture and Animal Models Resveratrol also exerts its anti-oxidative action by enhancing the intracellular freeradical scavenger glutathione. Reveratol was able to protect SH-SY5Y neuroblastoma cells from A
and H2O2 induced cell death and was also able to increase the level of reduced glutathione [186]. In a further study, the authors hypothesized that lipoproteins (LP) enhance the A
-mediated toxicity to neurons and that uptake of oxidized LP by neuron leads to an acceleration of intracellular oxidative pathways and deteriorate cell death. Using PC12 cells it could be shown that the combination of Abeta and oxidized LP leads to an enhanced cell

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death. However, this effect could been ameliorated by resveratrol, showing that it acts as a potent neuroprotective antioxidant [187]. Furthermore, the combination of trans-resveratrol and vitamin C and/or E were more effective in protecting cells than was any of these three antioxidants alone [188]. Neuroprotective Effects in Alzheimer and Parkinson Patients Moderated consumption of red wine - despite a high fat intake – is considered to be the cause of a low incidence of cardiovascular disease in France – the so-called “French paradox”. In line with this type of studies, a prospective community study in the Bordeaux area in France has demonstrated that the consumption of red wine significantly decreases the risk of AD. 3777 seniors (65 years and older) were observed in respect of their alcohol consumption and onset of dementia. In moderate drinkers who had 250 - 500 ml of red wine per day, red wine seemed to have a strong favourable effect, since the odds ratio was 0.28 compared to non-drinkers [189]. In a similar study, the Washington Heights Inwood-Columbia Aging Project, the intake of up to three daily servings of wine was associated with a lower risk of AD (hazard ratio=0.55) but intake of liquor, beer, and total alcohol was not associated with a lower risk of AD [190]. 5.4. Energy Supplementation There is evidence that bioenergetic dysfunction plays a role in many neuropathological disorders. Mitochondrial respiratory chain and oxidative phosphorylation system are responsible for the production of ATP and defects in energy production are therefore thought to be a cause for neurodegeneration. Energy depletion and neurodegenerative diseases are linked by various pathways, including free-radical generation, impaired calcium buffering and the mitochondrial permeability transition, all leading to cell death. During the failure of neutralisation of radicals, energy supplementation is another opportunity to ensure enough ATP levels for survival and for the repair of the damage occurring in the cell. Hence the application of bioenergetic drugs, including creatine and pyruvate, are further an option for neuroprotective treatment. 5.4.1. Creatine Occurrence Creatine (CR) can be produced endogenously by various organs such as pancreas, liver and kidney using arginine, glycine and methionine (Fig. 5), but can also be taken up by ingestion of meat products [191] or as a supplement in the form of Creatine monohydrate (CM). Latter is commercially available as a water soluble powder, in capsule or chewable. The daily recommended dose of creatine is about 5-10 g [192].

Fig. (5). Chemical structures of the pro-energetic compounds creatine and pyruvate.

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Function and Chemical Reactions A healthy brain contains approximately 4.5 mM phosphocreatine (PCr) [193], which serves as an energy buffer in muscles and brain through transferring the phosphoryl group to ADP, catalyzed by cytosolic creatine kinase (CK). Neuroprotective Effects in Cell Culture and Animal Models CR can also function together with PCr as an energy buffer between the cytosol and mitochondria. The mitochondrial isoform of CK (MtCK) exists in octameric and dimeric forms. MtCK in its octameric form inhibits the opening of the mitochondrial permeability transition pore (MTP) [194], but dimerizes in the presence of radicals, allowing the MTP to open and impair mitochondrial respiration leading to cell death [195]. It has been shown that creatine attenuate these negative effects [195-197]. Furthermore creatine administration has been found to protect against glutamate and amyloid toxicity in rat hippocampal neurons in vitro, 198]. There is evidence from in vitro and animal experiments that oral creatine supplementation might prevent or slow down neurodegeneration in Huntington’s disease (HD). Neuroprotective Effects in Alzheimer and Parkinson Patients The importance of the substrates of CK, CR, and PCr, for normal brain function in man is emphasized by the fact that patients with genetic CRT-deficiency lack any detectable CR in the brain [199]and have severe neurological phenotypes including hypotonia, developmental speech delay, autism, and brain atrophy [199]. It has been documented that creatine concentrations are decreased in AD [200]. A randomized, double-blind, Phase II futility clinical trial shown that CR should be considered for definitive Phase III trials to determine if they alter the long term progression of Parkinson disease (PD). In 2007, the NIH National Institute of Neurological Disorders and Stroke (NINDS) has announced the launch of a large-scale double-blind, placebo-controlled, phase III clinical trial to learn if the nutritional supplement creatine can slow the progression of Parkinson's disease (PD). Though there is no data of clinical trials for creatine supplementation for AD have been published so far, in two studies, Cr supplementation has been shown to improve mental concentration as well as memory and learning in healthy human subjects. It is possible that this will also be true for early stage AD patients. 5.4.2. Pyruvate Function and Chemical Reactions A common feature of AD and PD is an impairment of glucose metabolism [201]. The resulting decrease in pyruvate (Fig. 5), which is an intermediate in glycolyis, is thought to cause a deficit of energy, which could be ameliorated by supplementation from exogenous sources. Pyruvate acts also as an antioxidant. Pyruvate and other
-ketoacids can react nonenzymatically with H2O2 through a reaction in which carbon dioxide is liberated, and
ketoacid is converted into the corresponding carboxylic acid [202].

R-RCCOOH + H2O2

RCOOH + CO2 + H2O

Pyruvate is shuttled by the H+-monocarbonylate cotransporter between the intracellular and extracellular space and can therefore scavenge H2O2 inside and outside the cell [203, 204]. However, the neuroprotective effects of pyruvate are decreased in high concentration since this leads to intracellular acidification, mainly generated by the H+ co-transport across

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the plasma membrane [205, 206] and to a minor extent, from undissociated pyruvic acid [207]. Neuroprotective Effects in Cell Culture and Animal Models Mechanisms responsible for the neuroprotective effects of pyruvate are controversial. It was suggested that the neuroprotective effect of pyruvate is more likely due to its antioxidant effect rather through its improvement of energy metabolism, because the neuroprotection could be reproduced with other
-ketoacids (which are able to react with H2O2) but not with lactate, another neuronal energy substrate [202]. However, it was also shown that the neuroprotective effect of pyruvate is not related to its ability to undergo a decarboxylation process by counteracting the deep decrease in the neuronal ATP content, indicating the neurons are protected by rescuing the cellular energy charge [208]. It was demonstrated that increased glutamate release such as that in excitotoxity, stimulates glycolysis in astrocytes and that the endproduct pyruvate may contribute to neuronal protection [209]. In addition, glial cells release more of the energy substrate lactate in the presence of glutamate. The release of glutamate from nerve terminals may therefore cause a negative feedback, increasing neurotoxicity [210]. Neuroprotective Effects in Alzheimer and Parkinson Patients Pyruvate administration for therapeutic use may provide benefit for patients, because unlike exogenous catalase, pyruvate and other alpha-ketoacids can cross the blood brain barrier [204, 211]. However, until now clinical trials with pyruvate in AD or PD patients are still missing. 5.5. NMDA Antagonists Function and Chemical Reactions Since excitotoxicity plays a role in AD as described above, several NMDA antagonists have been developed for the treatment of neurodegenerative disorders. Synthetic drugs like memantine, MK-801 or dizocilpine have made into different stages of clinical development, also natural components like kynurenic acid or quinolinic acid. However, the most promising NMDA receptor antagonist so far is memantine (Fig. 6). Memantine acts as a noncompetitive (channel-blocking) antagonist. It inhibits pathopysiological Ca2+ influx without blocking physiological activation of the NMDA receptor, which is important for learning and memory. Although the mode of action is not clearified yet it seems that memantine is combined and released with the ion channel depending on electric potential in the same way as the magnesium ion [212]. The main advantage of memantine is its low-moderate affinity in comparison to phencyclidine (PCP) and MK-801, which are high-affinity NMDA receptors antagonists and are therefore not practical due to adverse side effects such as schizophrenic symptoms [213]. Kynurenic acid is a natural metabolite of tryptophan and can antagonize non-NMDA as well as NMDA receptor activation [214]. Neuroprotective Effects in Cell Culture and Animal Models In a AD animal model, Sprague-Dawley rats received memantine and beta-amyloid 1-40 [Abeta(1-40)]. A
(1-40) injections into hippocampus led to neuronal loss in the CA1 subfield, evidence of widespread apoptosis, and astrocytic and microglial activation and hypertrophy. Memantine treated animals had significant reductions in the amount of neuronal degeneration, pyknotic nuclei and glial fibrillary acidic protein (GFAP) immunostaining as

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compared with vehicle treated animals. These data suggest that memantine, at therapeutically relevant concentrations, can protect against neuronal degeneration induced by A
[215].

Fig. (6). Chemical structure of the NMDA-receptor antagonist memantine, an 1-aminoadamantan derivative.

Memantine was able to abolish toxic effects of glutamate in a rat model with parkinsonian syndromes. Already 5 mg/kg weakend the development of oligokinesea and muscular ricidity induced trough MPTP [216]. Neuroprotective Effects in Alzheimer and Parkinson Patients After successful clinical trials, memantine is now used as a therapeutic drug for AD in the EU and USA. In one of the clinical trials, 166 AD patients were treated with 10 mg/d of memantine. It was shown that there was an improvement in the care dependence subscore on the Behaviorial Rating Scale for Geriatric Patients (BGP) of 3.1 points compared to 1.1 points in the placebo group [206]. These data were consistent with another study, in which Reisberg and coworker analysed 252 patients with moderate-to-severe AD, who were treated with 20 mg of memantine daily for 28 weeks [217]. According to the result of the Clinician’s Interview-Based Impression of Change Plus (CIBIC-Plus) the change from baseline was significantly better with memantine compared to placebo. Two more studies reported statistically significant benefits of memantine. In a US-28-week placebo-controlled clinical trial memantine was determined for clinical efficacy and safety for people with Alzheimer´s disease. 20 mg/day of memantine caused a positive effect on cognition, mood and behaviour, however clinical detectable changes could not be observed [206]. In a further European trial, memantine was investigated in severely demented nursing home patients suffering from AD. Memantine-treated patients showed a functional and global improvement, reduced care dependency, compared to placebo-treated persons [218]. Positive effects of NMDA receptor antagonists such as Memantine have also been reported for PD patients. However, rather than being neuroprotective (which can only be demonstrated in long-term studies), these drugs are rather beneficial in PD patients by alleviating characteristic PD symptoms such as dyskinesia, suggesting an effect on glutamatergic signal transduction. In an open-fashion conducted study, 5 out of 14 PD patients improved their main Parkinson features whilst 6 further patients of this group improved their “off” episodes [219]. In a further case report, memantine was used with three cognitively impaired, dyskinetic Parkinsonian patients, and two seemed to benefit from this medication regarding their dyskinesia [220]. ACKNOWLEDGEMENTS We thank Steven Robinson and Peter Riederer for their helpful discussion. This study was supported by the Alzheimer Forschungs Initiative e.V and the DFG (Mu 1011-13).

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ABBREVIATIONS AA

=

Arachidonic acid

A


=

Beta-amyloid peptide

AC

=

Adenyl cyclase

AD

=

Alzheimer Disease

AGE

=

Advanced Glycation Endproduct

AMPA

=

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

APP

=

Amyloid precursor protein

BB

=

Blueberry

BGP

=

Behaviorial Rating Scale for Geriatric Patients

BH4

=

Tetrahydrobiopterin

cGMP

=

Cyclic Guanyl monophosphate

CHAP

=

Chicago Health and Aging Project

CIBIC-Plus

=

Clinician’s Interview-Based Impression of Change Plus

CK

=

Creatine Kinase

CM

=

Creatine monhohydrate

CR

=

Creatine

DA

=

Dopamine

DAG

=

Diacylglycerol

DATATOP

=

Deprenyl and Tocopherol Antioxidative Therapy of Parkinsonism

DHA

=

Dehydroascorbic acid

DTT

=

Dithiothreitol

DHLA

=

Dihydrolipoic acid

DPPH

=

1,1-diphenyl-2-picryl-hydrazyl

EC

=

Epicatechin (EC)

EGb

=

Ginkgo Biloba

EGC

=

(-)-Epigallocatechin

EGCG

=

(-)-Epicatechin gallate

GC

=

Guanylate cyclase

GSH

=

Glutathione recuced

4-HNE

=

4-hydroxysynonenal

H2O2

=

Hydrogen peroxide

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HOCL

=

Hypochlorous acid

HVA

=

Homovanillic acid

IL-1


=

Interleukin 1 beta

IL-6

=

Interleukin 6

IP3

=

Inositol-1,4,5, trisphosphate

KA

=

Kainic acid

LA

=

Lipoic acid

LOOH

=

Lipid hydroperoxidases

LP

=

Lipoprotein

M-CSF

=

Macrophage-Colony stimulating factor

MDA

=

Malondialdehyde

NADH

=

Nicotine amide dinucleotide

NAC

=

N-acetyl-cysteine

NADH

=

Nicotine amide dinucleotide phosphate

NMDA

=

N-methyl-D-aspartate

MPTP

=

1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine

MtCK

=

Mitochondriale Creatine Kinase

MTP

=

Mitochondrial permeability transition pore

NO

=

Nitric oxide

NxOy

=

Nitrogen oxides

MMSE

=

Mini-mental state examination

=

Superoxide

OCl

=

Hypochlorite

OH

=

Hydroxyl radical

=

6-hydroxy dopamine

=

Peroxinitrite

PCP

=

Phencyclidine

PCr

=

Phosphocreatine

PD

=

Parkinson Disease

PIP2

=

L-3-phosphatidyl-inositol-4,5-bisphosphate

PKA

=

Protein kinase A

PKC

=

Protein kinase C

O2

-

6-OHDA ONOO

-

47

48 Frontiers in Medicinal Chemistry, 2010, Vol. 5

PLC

=

Phospholipase C

RAGE

=

Receptor for AGE

ROS

=

Reactive oxygen species

SN

=

Substantia nigra

SOD

=

Superoxide dismutase

SPA

=

Sweet potato anthocyanin

TH

=

Tyrosine hydrolase

TNF-


=

Tumour necrosis factor- alpha

Shanmugam et al.

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Improving Memory: A Role for Phosphodiesterases Arjan Blokland1,*, Rudy Schreiber2 and Jos Prickaerts3 1

Department of Psychology and Neuroscience, EURON, Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands; 2Sepracor, Inc., 84 Waterford Drive, Marlborough MA, 01752, USA; 3Department of Psychiatry and Neuropsychology, Brain & Behavior Institute, EURON, Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands Abstract: During the last decennia, our understanding of the neurobiological processes underlying learning and memory has continuously improved, leading to the identification of targets for the development of memory-enhancing drugs. Here we review a class of drugs which has more recently been identified: the phosphodiesterase (PDE) inhibitors. An overview is given of the different PDEs that are known and we focus on three PDEs which have been identified as possible relevant targets for memory improvement: PDE2, PDE4 and PDE5. PDEs differ in the substrate, i.e. cyclic adenosine monophosphate (cAMP) and/or cyclic guanosine monophosphate (cGMP), being hydrolyzed. Since these cyclic nucleotides have been suggested to play distinct roles in processes of memory, selective PDE inhibitors preventing the breakdown of cAMP and/or cGMP could improve memory. The present data suggest that PDE4 (cAMP) is involved in acquisition processes, although a possible role in late consolidation processes cannot be excluded. PDE5 (cGMP) is involved in early consolidation processes. Since PDE2 inhibition affects both cAMP and cGMP, PDE2 inhibitors may improve both memory processes. The field of PDEs is highly dynamic and new isoforms of PDEs are still being described. This may lead to the discovery and development of new memory enhancing drugs that selectively inhibit such isoforms. Such drugs may exert their effects only in specific brain areas and hence possess an improved side effect profile.

Keywords: cGMP, cAMP, consolidation, long-term potentiation, cognition enhancer. INTRODUCTION Although memories are always available, it is well known that the ability to recall information from memory is dependent on several factors [1]. For example, the memory for acquired information is better if the context in which the information has been learned is the same as the context in which the memory has to be retrieved (state-dependent learning). Also, the ability to store or retrieve information seems to be dependent on the time of day. This time-of-day effect may be related to circadian rhythms of various biological factors. *Corresponding author: Tel: (31) 43 3881903; Fax: (31) 43 3884125; E-mail: [email protected] Allen B. Reitz / Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.

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Besides these intra-individual differences, there are also great inter-individual differences in the memory performance. Some people can easily and quickly remember ten pages of a complex biochemistry chapter, whereas others need at least ten hours to learn just one page. However, the person who faces difficulties with biochemistry may be much better in remembering the lyrics of a song than a good biochemistry student. Thus, memory as such does not seem to be a stable phenomenon but the performance may fluctuate under influence of various intra- and inter-individual factors under normal physiological conditions. Until now, numerous biological factors have been identified which mediate the memory performance. Neurotransmitters, neuromodulators, intracellular molecules, hormones, plant extracts and nutritional ingredients all improve or impair the memory performance under specific conditions [2-8]. Psychopharmacological, and especially animal experiments, have provided valuable insights into the role of different biological mechanisms that underlie memory functions and the discovery and development of cognitive-enhancing drugs. However, more work is needed to understand the relationship between specific memory functions and the specificity of the biological mechanisms underlying these functions. This information becomes even more relevant considering the impairments in memory functions in aging and pathological conditions. With respect to aging, the relative and total number of aged people will increase considerably in the next decades, which will lead to an increase in the complaints these people are facing. Aside from many other bodily dysfunctions, impairments in memory have a strong negative impact on the daily activities and quality of life of aged people. The loss of cognitive functions is even more serious in pathological conditions like (Alzheimer’s) dementia. But also in depressed and schizophrenic patients prominent memory deficits are present [9, 10] . Cleary, there is a high need for drugs that counteract conditions in which the memory performance is deteriorated. The aim of this paper is twofold: first, to provide a more general overview of memory and the drugs that have been shown to improve performance. A second aim is to review the effects of specific PDE inhibitors on long-term potentiation, a measure of synaptic plasticity and a proposed neurophysiological substrate of memory, and to evaluate their effects on memory functions. MEMORY SYSTEMS As mentioned before, memory is not a static entity. On top of that, there is ample evidence that memory should not be seen as one entity but that it comprises several (sub)systems [11]. In Fig. (1) a schematic representation of the temporal aspects and memory structures is presented. A first dimension in memory systems is the temporal aspect. Neuropsychological studies have provided strong evidence for the existence of a short-term and a long-term memory storage. This short-term storage has also been referred to as the working memory [12]. It has also been proposed that new information first has to be acquired. This stage has been related to the working memory and attention processes. Subsequently, the new memory is stored and it has been suggested that there are two stages of storing new information. This has been referred to as an early consolidation and a late consolidation phase [13, 14]. Both phases are required to store new information in the long-term memory. The long-term memory can be further divided into a procedural and declarative memory system. The latter can be subdivided into a declarative and nondeclarative system. An important note to make is that the memory needs to be retrieved from memory before it can be assured that the new information was stored. This suggests that if a memory cannot

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be recalled this indicates that the memory has not been stored or that the information cannot be retrieved. These two possibilities should always be considered before drawing conclusions in case a subject cannot recall information.

A Information

Early/Late Consolidation Sensory Store (0.5 – 3 s)

Attention

STM/WM (10 – 15 s)

Retrieval

LTM (15 s and longer)

Rehearsal

B Working memory

Visual Sketch Pad

Central Executive

Phonological Loop

Long-Term Memory

Declarative

Episodic

Non-Declarative

Semantic

Fig. (1). Aspects of the memory systems: A) temporal features. Forgetting can occur in the STM/WM due to not attending to the new information. Forgetting can also occur in the LTM. B) Structure of memory. The working memory, according to Baddeley (2003) is composed of two parallel systems which are supervised by the Central Executive. The long-term memory can be divided in different subsystems. The declarative memory system represents facts of a subject and the non-declarative system hold information about skills. The episodic memory is information that is unique for each individual subject whereas the semantic memory is information of general knowledge. STM: Short-term memory; WM: Working memory; LTM: Long-term memory.

The ability of subjects to store information in one of the abovementioned subsystems (temporal aspects and memory systems, see Fig. 1) also seems to depend on the type of memory that has to be stored. Thus, the memory for visual information seems to be different from the memory for spatial, verbal, and emotional information. Taken all these factors into account, memory can be considered as a mosaic pattern having temporal-, memory type-, and modality type-like dimensions.

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NEUROBIOLOGY OF MEMORY There is ample evidence that memory functions are modulated by biological mechanisms and a key question is what might be the best manner to improve memory performance without adverse drug effects. Various classes of drugs improve memory functions in man, and in animal studies the number of drugs with cognition-enhancing properties is even greater. Most of these effects have been observed in different models. One of these models evaluates the effects of drugs in aged subjects suffering from cognitive deficits. Other models involve drug-induced memory impairments, of which amnesia induced by the muscarinic antagonist scopolamine is the best known [15, 16]. But also acute depletion of the precursor for serotonin, tryptophan, and treatment with the glutamatergic antagonist, ketamine, have been used to induce memory impairments in humans [17-19] and animals [20, 21]. There are additional memory deficit models available in animal research. For example, lesions of brain structures that are affected by neurodegenerative diseases can be modeled in animals [2225]. Finally, genetic models have been developed that mimic the pathological stages in Alzheimer’s disease [26, 27]. Each of these models assumes a specific neurobiological deficit underlying the memory impairment in a disease [28-30]. A prominent example is the scopolamine model, which assumes that cognitive deficits in dementia are primarily related to a cholinergic deficit [31]. Role of Neurotransmitters Various neurotransmitters have been implicated in processes of learning and memory. Acetylcholine has received a lot of attention since the early eighties, mainly based on the observation that dementia was associated with a reduction in cholinergic markers related to impairments in cognitive functions in dementia [32]. Triggered by these findings, drugs have been developed which enhance the cholinergic neurotransmission: acetylcholinesterase inhibitors and nicotinic agonists. Acetylcholinesterase inhibitors block the enzyme that breaks down acetylcholine in the synaptic cleft and are marketed to improve cognitive functions in mild to moderate Alzheimer’s disease [33, 34]. It is well known that nicotine improves cognitive functions in man [35, 36]. But both acetylcholinesterase inhibitors and nicotine possess peripheral side effects which hamper their clinical use. Preclinical studies attempted to find drugs that possess a more central mechanism of action to prevent these undesirable side effects. Recently, drugs have been developed that bind to a specific subtype of the nicotine receptors and which appear to have less side effects than nicotine [37]. These drugs have a very promising profile for cognition-enhancement in animal studies [38-40]. Nevertheless, at present no nicotinic treatments are available for humans suggesting that this is not a very successful approach yet. But also other neurotransmitters have been implicated in cognitive functions. In general, for each neurotransmitter studies can be found which support its involvement in memory [41]. However, the effects can, at least to some extent, be differentiated with regards to the specific aspects of cognition which these neurotransmitters affect. Several reviews are available in which these topics are dealt with [2, 8, 42-45]. Noteworthy is that the most widely used drug in the world, caffeine, also has shown to improve memory functions in man [46]. This effect has been linked to the activation of the adenosine receptors. Role of Other Biochemical Mechanisms Glucose improves memory functions consistently in animals and human. Although some evidence supports a peripheral mechanism of action, the exact mechanism still needs to be resolved [7].

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Hormones also modulate memory performance [47], especially ACTH and corticosterone. These stress hormones are particularly involved in emotional memories [48] and related to the age-related decline in memory functions [49]. Finally, there are various plant extracts that have been claimed to improve memory functions, of which the most well known is Ginkgo Biloba [50]. PHOSPHODIESTERASE INHIBITORS PDEs and LTP In addition to the drugs which improve memory performance involving the mechanisms mentioned in the previous section, there is accumulating evidence that inhibition of PDE activity may consist of a particular interesting mechanism for memory enhancement [14, 5153]. This is related to the substrates of PDEs, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Both cyclic nucleotides play an important role in intracellular signaling [54-56], and in processes of neuroplasticity such long term potentiation (LTP). Importantly, in contrast with current cognitive-enhancing drugs, which target single neurotransmitter systems, drugs which intervene with second messenger systems modulate multiple biochemical systems and may therefore offer superior efficacy in the treatment of cognitive disorders. LTP in the hippocampus, a pivotal brain structure for memory consolidation, is considered as a neurophysiological correlate of learning and memory [57]. Briefly, a short-lasting (<1 s) stimulation of the perforant path towards the CA1 stratum radiatum of the hippocampus induces long-lasting (hours to days) effects on synaptic connectivity of neurons in the hippocampus. It is assumed that this long-lasting change in the connectivity between neurons may represent the synaptic plasticity underlying formation of new networks for new memories. Since the discovery of LTP, a vast amount of studies has accumulated in which the molecular mechanisms underlying LTP have been investigated [58-60]. The insights which were gained from these studies provided molecular tools to improve LTP and subsequently memory. Among the various molecules that are involved in LTP, cAMP and cGMP seem to play an important role. A simplified diagram of the processes involved in LTP is presented in Fig. (2), and highlights the presumed role of cAMP and cGMP. Generally, it is postulated that increased levels of cAMP and cGMP would enhance the processes that maintain LTP and thereby augment synaptic plasticity [61-64]. This is the basic principle for the possible mechanism for the use of PDEs in treating memory dysfunctions [see 65]. Based on the electrophysiological properties of LTP, at least two types of LTP have been described: early phase LTP (E-LTP), induced by relative weak electrical stimulation, and late phase LTP (L-LTP), induced by high electrical stimulation. [62, 64, 66, 67]. Below, we will describe the separate contributions of cAMP and cGMP to these types of LTP. However, it is beyond the scope of this review to discuss in detail the pre- and postsynaptic changes resulting in the expression of E- and L-LTP [for discussion, see 14, 68] Postsynaptic, cAMP/protein kinase A (PKA) signaling is not required for the expression of E-LTP [68], but the presynaptic cGMP/protein kinase G (PKG) pathway is involved in E-LTP in the CA1 stratum radiatum [69-71]. The duration of E-LTP is less than 3 h and E-LTP is not dependent on gene expression and protein synthesis. The time-window of E-LTP corresponds with the duration of short-term memory (STM) as defined by some researchers, i.e., as a form of memory which does not require gene expression and protein synthesis [42].

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Post-synaptic

Pre-synaptic Glutamate Ca2+

cGMP sGC

NOS

cAMP cGMP sGC

CaM

Protein synthesis

NO

Fig. (2). Simplified diagram of molecules involved in long-term potentiation (LTP). After the release of glutamate from the presynaptic neuron, it binds to AMPA and NMDA receptors. If the signal is strong enough, calcium (Ca2+) will enter the post-synaptic neuron through the NMDA receptor and activates calmodulin (CaM) which in turn stimulates adenylate cyclase (AC). Subsequently, cAMP is synthesized. Besides activation of CaM, Ca2+ activates constitutive nitric oxide synthase (NOS). After activation of NOS the gaseous molecule nitric oxide (NO) is formed, and can have a dual action. First, NO can diffuse back to the pre-synaptic region and activate soluble guanylate cyclase (sGC). cGC increases cGMP levels which in turn can potentiate the release of glutamate. A second action of NO is the activation of sGC postsynaptically. This can also lead to protein synthesis. These phenomena, including other molecular events, are generally assumed to underlie LTP. PDEs break down cGMP and camp. Selective PDE inhibitors can therefore increase cGMP levels pre-synaptically and/or increase cAMP and cGMP levels post-synaptically.

In the CA1 stratum radiatum, a postsynaptic cAMP/ PKA/cAMP responsive element binding protein (CREB) pathway, a postsynaptic cGMP/PKG pathway, and a postsynaptic cGMP/PKG/CREB pathway are involved in L-LTP [61, 72, 73]. In contrast to E-LTP, LLTP lasts longer than 3 h and may therefore be a candidate for long-term memory (LTM) processes. Taken together, although there is some debate with regard to the exact localization of signaling pathways for both types of LTP in hippocampal neurons, it is assumed that the molecular pathways underlying E-LTP processes are located pre-synaptically. Inspection of Fig. (2) shows that the retrograde NO signal that activates sGC/cGMP presynaptically is likely to underlie E-LTP. Conversely, the postsynaptic cAMP/protein kinase A (PKA) signaling pathway is more likely to be involved in L-LTP. Since E-LTP and L-LTP may represent non-structural and structural changes in neuronal connections, respectively, it is tempting to relate E-LTP to short-term/working memory and L-LTP to long-term memory. Finally, some evidence supports a role for cGMP in L-LTP via a postsynaptic cGMP/PKG/CREB pathway [74]. However, it is beyond the scope of this review to discuss these dual effects of cGMP (see Fig. (2) for pathways). PDEs and Memory The family of cyclic nucleotide PDEs is large, and eleven different isozymes have been identified at present [75, 76]. An overview of these PDEs and some of their characteristics are listed in Table 1. PDE6 and PDE11 appear not relevant targets for memory functions since these enzymes are not present in the brain. Conversely, inhibitors of the other PDEs are expected to increase cAMP and/or cGMP levels in neurons and hence might improve

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memory. Since the hippocampus is the main structure where LTP and memory are closely linked, localization of PDEs in this region is consistent with a potential role in processes of learning and memory. It should be mentioned that LTP is classically related to hippocampal synaptic plasticity, but LTP (and Long-Term Depression, LTD) has also been recorded in other brain regions, including cortical areas [77, 78]. Therefore, relative specificity of the localization of PDE enzymes in hippocampus and/or cortex could be considered as a criterion for selecting a PDE as a drug target. Table 1.

Type

Overview and Properties of Known Enzymes of the Cyclic Nucleotide Phosphodiesterases

Number of

Localization

Description

Substrate

Genes PDE1

3

Selective Inhibitors

brain, heart, smooth

Ca2+ -CaM

muscle

stimulated

cAMP/cGMP

IBMX, calimidazolium, phenethiazines, vinpocetine, SCH51866

PDE2

PDE3

1

2

brain, heart, adrenal

cGMP

cortex

stimulated

brain, heart, smooth

cGMP inhibited

cAMP/cGMP

aptosyn cAMP

muscle, adipose platelets PDE4

4

ubiquitous

EHNA, Bay 60-7550,

cilostamide, milrinone, SK&F 95654

cAMP specific

cAMP

rolipram, rofluminast, Ariflo, HT0712, ibudilast, mesembrine

PDE5

1

brain, lung, platelets,

cGMP specific

cGMP

smooth muscle

zaprinast, sildenafil, vardenafil, tadelafil, SK&F 96231, udenafil, avanafil, SK&F 96231

PDE6

4

rod and cones photo

Photoreceptor

cGMP

(sildenafil)

brain, skeletal muscle,

cAMP high

cAMP

BRL 50481

kidney, T-cells,

affinity

cAMP

?

cGMP

SCH 81566, BAY 73-6691

receptor PDE7

2

liver, testis PDE8

2

brain, liver, testis, ovary

cAMP high affinity

PDE9

1

brain, kidney

cGMP high affinity

PDE10

1

PDE11

1

brain, testis

cAMP-inhibited

cGMP

Papaverine, TP-10, PQ10

skeletal muscle, kidney,

Dual substrate

cAMP/cGMP

(tadelafil)

liver, pituitary Note that the list of tissues in the localization column is not exhaustive.

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As depicted in Tables 1 and 2, PDE1-PDE5 and PDE7-PDE10 enzymes are located in the brain. Inhibition of these enzymes could therefore be considered as a means for improving brain functions. As mentioned above, the hippocampus and the cortex are considered to be the most relevant sites involved in cognitive functions. In Table 2, an overview is given of the localization of different PDEs, and their splice variants, in different brain regions. For a possible target for cognition, high expression in other organs, or high expression in many other brain areas is not preferable. At present a number of studies showed effects of selective PDE inhibitors on memory performance. These include inhibitors of PDE2, PDE4, PDE5 and PDE9. Table 2.

Localization of Different Types of PDEs (Including their Isoforms) in Cortex, Hippocampus and Other Brain Regions. Note that this Table does not Provide Information with Respect to the Level of Expression of the Different PDEs in the Brain

PDE

Cortex

Hippocampus

PDE1

+

+

Rest of Brain thalamus, cerebellum, olfactory bulb, nucleus accumbens, striatum

Species

Reference

rat

[122-124]

mouse human

PDE1B

+

+

olfactory bulb, striatum

mouse

[125]

PDE1B1

-

-

striatum

mouse

[126]

PDE1C

+

+

cerebellum, amygdala

mouse

[127]

PDE2

+

+

forebrain, midbrain, amygdala

rat

[128-131]

PDE3

+

+

throughout brain

rat

[95, 131]

PDE4

+

+

throughout brain

rat

[131, 132]

mouse PDE4A

+

+

olfactory bulb

mouse

[132]

PDE4B

+

+

striatum, hypothalamus, midbrain, cerebellum

mouse

[132]

PDE4D

+

+

hypothalamus, midbrain, cerebellum

mouse

[132]

PDE5

+

+

cerebellum

rat

[129]

mouse PDE7A

+

+

olfactory bulb, cerebellum

PDE7B

-

+

striatum, midbrain

rat

[134]

PDE8B

+

+

olfactory bulb, midbrain

rat

[135]

PDE9A

+

+

rat

[129, 136]

striatum, cerebellum, olfactory bulb, amygdala, midbrain

PDE10

+

+

striatum, cerebellum, substantia nigra

[133]

mouse rat

[137]

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These PDEs differ in their substrate (see Table 1). The specific substrate for PDE4 is cAMP. The substrates for PDE5 and PDE9 inhibitors appear to be cGMP. Finally, both cAMP and cGMP are substrates for PDE2. Studies with cAMP and cGMP analogs support a role for these cyclic nucleotides in memory formation (see below). ROLE OF CAMP AND CGMP IN MEMORY cAMP The effects of direct manipulations of brain cAMP levels have only sporadically been investigated, i.e. to our knowledge there are only few one-trial learning studies with intrahippocampal injections of cAMP analogues. With respect to STM/WM it seems that cAMP/PKA signaling is probably not involved as demonstrated with manipulations downstream of cAMP, i.e. affecting the PKA/CREB pathway, did not impair STM [42], but this appears to be dependent on the memory performance tested [79]. In the passive avoidance task, post-training bilateral intrahippocampal injections of 8bromo-cAMP (1.25 μg/side) enhanced retention performance of rats 24 h after training when given 3 h after training, but not immediately after training [80, 81]. The latter was also found in the object recognition task [82]. Furthermore, rats submitted to the passive avoidance task showed an increase in the amount of cAMP in the hippocampus at 3 h after training, but not immediately after training [80, 81]. Thus, it can be argued that hippocampal cAMP is only involved in late consolidation processes of LTM. cGMP Studies on the injections of cGMP or its analogues into the brain and its effect on memory are also scarce. In rats, bilateral cGMP (10 μg/side) or 8-bromo-cGMP (1.25 μg/side) injection into the hippocampus immediately after training improved passive avoidance retention 24 h later [80, 83]. Likewise, when 8-bromo-cGMP (10 μg/side) was bilaterally injected into the hippocampus immediately after object recognition training, it improved retention performance 24 h later [82]. However, bilateral intrahippocampal injections of 8-bromo-cGMP (1.25 μg/side) had no effect on the retention of passive avoidance performance 24 h after training when given 3 h after training [80]. Furthermore, rats tested in a passive avoidance task showed an increase in the amount of cGMP in the hippocampus only at 0 and 30 min after training [80, 81]. Thus, it appears that cGMP in the hippocampus is involved in early consolidation processes of object and shock information into LTM. Conversely, late consolidation processes appear to be independent of cGMP. EFFECTS OF SPECIFIC PDE INHIBITORS ON MEMORY PDE2 Inhibitors PDE2 hydrolyzes both cAMP and cGMP. At present, only a few studies have been published in which PDE2 inhibitors have been tested in behavioral assays. This is primarily related to lack of specific PDE2 inhibitors. Some studies have investigated the effects of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) on behavior. For example, in one study EHNA was injected intracerebroventricularly and the effects on avoidance learning in rats was measured [84]. The results suggested that the performance of these rats was impaired after EHNA treatment. Although this finding does not support the notion that PDE2 inhibition should be able to improve memory performance, it should be noted that EHNA is also a potent adenosine deaminase inhibitor [e.g. 85]. Consequently, the use of EHNA may not be suitable to test the specific effects of PDE2 inhibition.

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To our knowledge, Bay 60-7550 is the only selective PDE2 inhibitor which has been tested in learning and memory models [86-88]. In vitro experiments showed that this compound increases both cAMP and cGMP levels in cortical neurons and hippocampal fibers (especially in the perforant path), and improved the memory performance of rats in different memory tests [86]. These findings were replicated in further studies and showed that PDE inhibition improved both early- and late-phase consolidation [87, 88]. Effects on STM/WM and LTM Different behavioral tests were used to characterize the effects of Bay 60-7550 on memory performance [86]. At a dose of 3 mg/kg (p.o.) Bay 60-7550 reversed the spatial WM deficit in a T-maze induced by the NMDA antagonist dizocilpine (MK-801) (0.04 mg/kg, i.p.). When given immediately after training in tasks of object or social recognition, Bay 60-7550 improved object recognition in both rats (1 and 3 mg/kg, p.o.) and mice (0.3 and 1 mg/kg, p.o). Likewise, social recognition was improved in rats (1 and 3 mg/kg, p.o.) as well as mice (0.6, 2 and 6 mg/kg, p.o.). These effects of BAY 60-7550 on consolidation processes may be mainly mediated by cGMP and not cAMP, since cGMP, but not cAMP, injections into the hippocampus immediately after training improved LTM in tasks of object recognition and passive avoidance learning. PDE4 Inhibitors Ample evidence supports a role for the cAMP/PKA/CREB pathway in learning and memory processes [89, 90]. PDE4 inhibitors specifically increase cAMP levels and the underlying mechanism for their cognitive-enhancing effects may involve modulation of activity within the cAMP/PKA/CREB pathway [91, 92]. The prototypical PDE4 inhibitor most widely used in cognition studies is rolipram (Table 3). It possesses good brain penetration and a half-life of 1-3 h [93] and in vitro studies showed that cAMP levels increased in hippocampal slices treated with rolipram [94, 95]. One of the perceived liabilities of rolipram is emesis, and second generation PDE4 inhibitors such as cilomilast (Ariflo
) and roflumilast are thought to be less emetic. But these newer PDE4 inhibitors have been developed for peripheral indications, such as respiratory disorders, and so far no data have been published regarding their activity in cognition models. Herein, we review the cognition literature with PDE4 inhibitors and what aspects of memory systems are predominantly modulated (Table 3). Effects on STM/WM Rolipram and HT0712, both given before the training trial, had no effect on STM performance of mice in the object recognition task and contextual fear conditioning [94, 96]. The latter is in agreement with the assumption that cAMP is generally not involved in STM (see previous sections), although it must be realized that it is generally difficult to improve STM/WM in unimpaired animals due to a high level of baseline performance (‘ceiling effect’). Indeed, employing a cholinergic deficiency model in rodents treated with the muscarinic antagonist scopolamine, rolipram attenuated deficits in spatial and non-spatial STM and WM in several behavioral tasks [52, 53, 91]. Rolipram reversed the disruption in reference memory and/or working memory by the glutamate antagonist MK-801 in a radial-arm maze, and reversed the effects of MK-801 in a passive avoidance task [97]. The underlying mechanisms for these effects on STM/WM are not well understood. However, several neurotransmitters involved in learning and STM/WM processes use cAMP as a second

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Table 3.

Blokland et al.

Effects of PDE4 Inhibitors on Learning and Memory Processes

Task

Model

(Memory Process)

(Species)

Treatment

Results

Reference

Object recognition (episodic-like memory,

Scopolamine and time-

Rolipram (0.01-0.1 mg/kg in

MED in scopolamine

[138]

dependent deficit (rat)

time dependent forgetting;

model: 0.1 mg/kg

hippocampus and

0.03-0.3 mg/kg in

MED in time-dependent

rhinal cortex dependent

scopolamine model)

forgetting: 0.03 mg/kg

Rolipram (0.01-0.1 mg/kg)

MED 0.1 mg/kg

[139]

Time dependent

Rolipram (0.03 mg/kg), 0, 1,

Rolipram effective when

[87]

forgetting

3, 6 h after first trial

given 3 h after first trial

Object-Place recogni-

Time dependent

Rolipram (0.03 mg/kg) 3 h

Rolipram effective when

tion (episodic-spatial

forgetting

after first trial

given 3 h after first trial

Morris water maze

Microsphere embolism-

Rolipram 3 mg/kg, ip, 10

Rolipram attenuates

(spatial, hippocampus-

induced cerebral

days, after embolism

acquisition deficit

dependent)

ischaemia

Actute tryptophan depletion (5-HT deficit) (rat)

[88]

memory, hippocampus dependent) [92]

measured at days 7-9

(rat) Normal forgetting

L-454,560 (0.1-1 mg/kg)

MED 0.3 mg/kg

[140]

Radial arm water maze

APP-PS1 Alzheimer

Rolipram 0.1 uM/kg for 3

Improvement when

[101]

(spatial, hippocampus

(mouse)

weeks

tested at 2 months after

Delayed-matching to position in water maze (spatial, hippocampusdependent)

dependent)

3-week treatment

Barnes circular maze

Age-deficit

Rolipram 0.016 mg/kg, ip,

More mice acquire the

(Spatial, hippocampus-

(18 months old mouse)

40 min before training

task and number of

dependent)

[141]

errors is reduced

Radial arm maze

Scopolamine

Rolipram 0.01 – 1 mg/kg, ip,

MED: 0.1 (working

(working & reference

0.5/1.0mg/kg, ip,

45 min before test

memory) and >0.1

memory, hippocampus-

30 min before test

mg/kg (reference

dependent)

(rat)

memory)

Scopolamine 0.5 mg/kg

(±)-Rolipram

MED (working

ip, 30 min before test

0.01 – 1 mg/kg, po

memory):

(rat)

(-)-rolipram

(±)-rolipram 0.02 mg/kg

0.005-1mg/kg, po

(-)-rolipram 0.01 and 0.2

(+)-rolipram

mg/kg (biphasic)

(0.1-50 mg/kg, po)

(+)-rolipram 20/50 mg/kg

[53]

[52]

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Table 3. contd….

Task

Model

(Memory Process)

(Species)

Treatment

Results

Reference

Dizocilpine 0.l mg/kg,

Rolipram 0.01 – 0.1 mg/kg,

MED: 0.05 (working

[99]

ip, 60 min before test

ip, 30 min before test

memory) and 0.1 mg/kg

(rat)

(reference memory)

MEK inhibitor UO126,

Rolipram, 0.05, 0.1, mg/kg,

MED: 0.1 mg/kg

8ug/rat into

ip, 30 min before test

(reference memory)

[142]

hippocampus, given twice: 60 and 30 min before test Passive avoidance

Scopolamine, 3 mg/kg

Given 60 min before

MED:

(inhibitory avoidance

ip, 30 min before

retention test.

(±)-rolipram 0.02 mg/kg

learning, hippocampus

retention test

(±)-rolipram 0.01 – 0.1

(-)-rolipram 0.01 mg/kg

and amygdala

(rat)

mg/kg, po

(+)-rolipram 2 mg/kg;

(-)-rolipram 0.005-0.02

no effect at 10 mg/kg

dependent)

[52]

mg/kg, po (+)-rolipram 0.3-10 mg/kg, po Scopolamine, 1.5

rolipram 10 and 30 mg/kg,

mg/kg, ip, immediately

po, 30 min before training

MED: 30 mg/kg

[143]

MED: 10 mg/kg

[91]

MED:
0.1 mg/kg

[99]

[142]

after training (mouse) Scopolamine, 1 mg/kg,

Rolipram, 1-30 mg/kg, ip,

ip 30 min before

30 min before acquisition

acquisition (mouse) Dizocilpine 0.l mg/kg,

Rolipram 0.1 mg/kg, ip, 30

ip, 60 min before test

min before test

(rat) MEK inhibitor UO126,

Rolipram, 0.1, mg/kg, ip, 30

Reversal retention deficit

8ug/rat into hippocam-

min before test or 30 ug/rat

48-h post training

pus, given twice: 60

into hippocampus, 20 min

and 30 min before test

before test

3-panel runway task

Scopolamine, 0.56

Rolipram, 0.032, 0.1 mg/kg,

MED: 0.1 mg/kg for

(working memory,

mg/kg, ip, 15 min

ip, 30 min before first trial

decrease errors

hippocampus- prefron-

before first trial

tal cortex dependent)

(rat) Cerebral ischemia by

Rolipram, 0.032, 0.1 mg/kg,

MED: 0.1 mg/kg for

four-vessel occlusion

ip, 30 min before first

decrease errors

(rat)

trial (immediately after reperfusion)

[91]

[91]

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Table 3. contd….

Task

Model

(Memory Process)

(Species)

Treatment

Results

Reference

ECS immediately after

Rolipram, 0.1 , 0.32 mg/kg,

MED: 0.32 mg/kg for

[91]

training

ip, just before ECS

decrease errors

(rat) Inhibitory avoidance

1. Protein synthesis

Rolipram 3, 10 mg/kg, ip,

MED 10 mg/kg, given

(hippocampus and

inhibitor anisomycin,

immediately after training or

immediately after

amygdala dependent)

150 mg/kg, sc, 30 min

3 hours after training

training (1 + 2)

[144]

before training 2. Low baseline (mouse) Contextual

Unimpaired

Rolipram 0.03 mg/kg, sc,

Improved retention 24 h

fear-conditioning

(mice)

30 min before training

after training

Impaired TG2576

Rolipram 30 min prior to

Improvement in mutants

Alzheimer mice

training at 0.1 mg/kg ip

and wild type

APP-PS Alzheimer

Rolipram 0.1 uM/kg for 3

Improvement when

mice

weeks

tested 2 months

[94]

(hippocampus and amygdala dependent) [100]

[101]

following 3-week treatment Heterozygous CBP

Rolipram 0.1 mg/kg, ip;

MED: 0.1 mg/kg for

mutants

HT0712 0.001 – 0.5 mg/kg,

both drugs. Improved

(mice)

ip 20 min before training

object recognition at 24

[96]

hours Object retrieval

Increase in complexity

Rolipram (0.003-0.03

(frontal cortex)

(cynomolgus

mg/kg)

MED 0.01 mg/kg

[145]

[103]

macaques) Delayed responding

unimpaired and

rolipram 0.01-100 ug/kg, im

At 0.1 ug/kg, trend for

(prefrontal cortex

age-deficit

1 hour before testing

improvement in young

dependent, working

(rhesus monkeys)

memory)

Aged subjects impaired by 10 ug/kg.

messenger, and an increase in cAMP by rolipram may modulate the activity of these transmitters and their effects on cAMP, subsequently leading to an improvement in STM/WM. For example, cAMP facilitates the release of noradrenaline, dopamine, and serotonin from rat brain slices [98] and potentiates cholinergic activity as it increased tremors induced by the cholinergic agonist oxotremorine in mice [52]. Thus, PDE4 inhibition may lead elevated activity of cholinergic neurons which could explain the effects of rolipram in the scopolamine model. Furthermore, rolipram facilitated the NMDA-induced elevation in cAMP, and it has been suggested that PDE4 is an integral component of the NMDA receptor-mediated transduction pathway involved in memory processes [99]. Such a

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transduction pathway involved in memory processes [99]. Such a mechanism may underlie the reversal of the cognition-impairing effect of dizocilpine by PDE4 inhibitors. Effects on LTM Rolipram or HT0712 treatment of mice 20 min before training in the object recognition task improved retention performance 24 h later [96]. Likewise, the retention performance 24 h after contextual fear learning was improved in mice treated with rolipram 30 min before training [94]. Similar results were obtained with rodents in spatial and non spatial tasks in unimpaired and impaired animals (see Table 3). A preliminary finding of special interest is that rolipram improved memory performance in transgenic mouse models for Alzheimer’s disease [100], even when tested two months after drug treatment [101]. Such persistence of memory is consistent with the hypothesis that activation of the cAMP/PKA/CREB pathway leads to transcription of CREB-dependent genes, synthesis of proteins such as BDNF, and synaptic re-modeling [89, 90] and that such changes provide the ‘wiring’ for memory storage. The finding that PDE4 inhibition can reverse memory performance in different mouse disease models [96, 100, 101] provides strong support that this treatment approach may be effective in Alzheimer's disease. This notion is supported by a recent study showing that A
inhibits the PKA/CREB pathway and this inhibition can be reversed by drugs that enhance cAMP levels [102]. Temporal Features So far, no studies have been reported which specifically address the effects of PDE4 inhibitors on the early stages of information processing such as attention. There has also been a lack of a systematic and profound evaluation of the effects of PDE4 inhibition on acquisition, consolidation and retrieval in tasks of STM/WM or LTM. Rolipram has a short half-life and has typically been given shortly before training or testing (see Table 3), suggesting that PDE4 inhibition improves acquisition and/or consolidation processes involved in LTM. However, some recent studies suggest that rolipram may especially exerts its effects via late phase LTP processes. This was shown in a study in which rolipram showed clear effects when administered 3 h after the acquisition of new information [87, 88]. However, other mechanisms of rolipram cannot be excluded (see above). The activity of rolipram reported across a range of different models and experimental conditions in rodents is remarkable and suggests that PDE4 inhibitors possess robust cognitive-enhancing properties. PDE4 inhibitors can improve STM/WM, likely via modulation of neurotransmitter activity, which might be such that it also improves LTM. On the other hand, consolidation of LTM might also be influenced, presumably via a camp/PKA/CREB pathway. Notwithstanding these intriguing findings in rodents, PDE4 is a clinically invalidated target for the treatment of cognitive disorders and the rationale would be strengthened if more data in higher species such as nonhuman primates became available. In a first study, a trend was found for rolipram to improve delayed responding performance in young – but not old - rhesus monkeys [103]. Clearly further studies in non human primates are needed to address the potential of PDE4 inhibitors as novel cognition enhancers. PDE5 Inhibitors In 1997 it was first described that PDE5 inhibition improves memory processes [104]. Zaprinast was used to inhibit PDE5 and when given immediately after training at a dose of 10 mg/kg (i.p.) it improved the LTM performance of rats in the object recognition task

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[104]. However, we now know that zaprinast also inhibits PDE1, 9, 10 and 11. Since then, more selective PDE5 inhibitors have been developed, except for the pharmacological treatment of erectile dysfunction. Currently, three PDE5 inhibitors are approved for use in several countries; sildenafil (Viagra
), vardenafil (Levitra
) and tadalafil (Cialis
). In vitro experiments demonstrated that sildenafil or vardenafil increased NO-mediated cGMP accumulation in the hippocampus [95, 105, 106]. Both sildenafil and vardenafil have already been tested in LTM models as is discussed below. Effects on LTM Sildenafil treatment immediately after training in the object recognition task improved the retention performance of rats 24 h later with a lowest effective dose of 3 mg/kg (p.o.) [106]. Compared with sildenafil, vardenafil appeared to be even more potent in this respect since it already produced a high discrimination performance at a dose of 0.3 mg/kg (p.o.) [106]. When sildenafil was given 30 min before the first trial it also clearly improved the memory performance [107]. However, the lowest effective dose was increased from 3 mg/kg to 10 mg/kg when changing the administration of sildenafil from after to 30 min before the first trial. In male rats the elimination half-life of sildenafil is about 0.4 h and as a result sildenafil levels quickly decrease as a result of its metabolic clearance by the liver [108]. Thus, 30 min after its administration the 10 mg/kg dose may be close to the level of the 3 mg/kg dose and, consequently, improves consolidation processes of object information. Sildenafil has also been tested in mice. When administered immediately after the training trial of passive avoidance learning, sildenafil improved the retention performance 48 h later with a MED of 3 mg/kg (i.p.) [109]. Zaprinast and vardenafil were also tested in spatial LTM tasks at doses which were effective in object LTM tasks. Zaprinast (10 mg/kg, i.p.) was given immediately after daily learning in the Morris water escape task. Vardenafil (3 mg/kg, i.p.) was tested in a spatial recognition task (Y-maze) and given immediately after training [14]. However, both drugs did not affect consolidation processes of spatial information. A more recent study showed that vardenafil was especially effective when given immediately after the acquisition trial in an object recognition paradigm [87]. This finding suggests that PDE5 inhibition may be especially effective in improving early phase LTP. Effects in Humans The effects of sildenafil (100 mg) on early information processing and memory processes in humans have only sporadically been studied. Thus far, no clear effect on the behavioral measures of attention and verbal recognition memory have been reported [110]. However, there are some methodological considerations since only STM was measured at an already optimal performance allowing a minimal window for treatment effects. Yet, in the same study, attention-related, event-related brain potential (ERP) measures were enhanced by sildenafil. In addition, another study reported that the simple choice reaction time was faster in sildenafil-treated subjects [111]. Thus, early information processing in humans can be influenced by PDE5 inhibition, but these processes as well as memory-related processes clearly need more investigation. Taken together, animal studies indicate that PDE5 inhibitors have the potential to improve early consolidation processes of LTM, although this may be excluded for spatial information. This memory improvement might be mediated by elevations in central cGMP

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levels. Sildenafil treatment in humans suggests that information processing may be improved. CONCLUSIONS Reviewing the existing literature on the effects of PDE inhibitors on memory performance there is a large body of evidence indicating that these drugs can improve the memory performance in animals. This may not be unexpected since the mechanism of action of these drugs can be related to neurochemical processes that are assumed to underlie neuronal plasticity (LTP). There seems good evidence that PDE5 inhibition improves processes of early consolidation [14], which may involve a cGMP/PKG/CREB pathway. cAMP is assumed to be involved in late consolidation [80, 81] and PDE4 inhibition should improve this phase of memory formation. Studies with rolipram suggest that PDE4 inhibition can improve both acquisition and consolidation and it is proposed that the former may predominantly involve cAMP-mediated changes in the activity of acetylcholine, glutamate, and other neurotransmitters. Conversely, consolidation may predominantly involve changes in the cAMP/PKA/ CREB pathway, although it is not clear whether it is early or late consolidation. Along similar lines, since cAMP and cGMP are involved in different pathways underlying different types of LTP, it is likely that each PDE is involved in specific memory processes [65]. Clearly, more studies are needed to demonstrate such a specific effect on memory for PDE4 and PDE5 inhibitors. At present these data have been mostly obtained in animal studies, and only sporadic evidence is available that PDE5 inhibitors appear to affect central processes in man. Human studies, designed to test the memory enhancing effects of PDE inhibitors, are needed to demonstrate a proof of principle for this class of drugs as a possible tool for improving memory in man. It is also important to anticipate on possible on possible side effects of treatment with PDE inhibitors. Since PDEs are widely distributed throughout the body, this is an important point to consider. At present not much information is available since there are no clinical relevant drugs except for rolipram and selective PDE5 inhibitors. With respect to rolipram, some side effects have been reported [eg. headache, nausea, gastric hyper secretion and emesis, see 112]. On the other hand, rolipram has anti-inflammatory activity via suppression of proinflammatory cytokines and other mediators of inflammation [112]. This can be considered as a positive effect. Sildenafil has been reported not to affect cerebral blood flow but does seem to induce headache in healthy control subjects [113]. However, a recent study suggest that the effects of PDE4 or PDE5 inhibition on memory performance are independent on cerebral blood flow and glucose metabolism [88]. Also, there have been various anecdotal reports that sildenafil induces various types of neurological and affective effects [eg. loss of consciousness, anxiety, aggression, see 114]. Interestingly, a recent study indicated that chronic PDE5A inhibition has a positive effect as it prevented and reversed cardiac hypertrophy [115]. Although no clear and consistent negative side effect profile of rolipram and sildenafil are apparent, it is clear that more data are needed to evaluate side effect profile of selective PDE inhibitors. The field of PDEs is still highly novel and new aspects of these PDEs will emerge in the near future. One of these features is the existence of isoforms of PDEs. For example, the PDE4B and PDE4D enzymes seem to have a different distribution in the brain (see Table 2), and may therefore underlie different functions. In a recent study in which PDE4D knock out (-/-), heterozygote (+/-) and wild type mice were tested in models of depression, the PDE4D (-/-) mice showed clear anti-depressive like behavior and these mice were insensitive to rol-

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ipram. [116]. Comparative studies with PDE4B and PDE4A knock out mice will help to address if splice variants of PDE can subserve different behavioral functions. In line with this notion, preliminary data suggest that PDE4D (-/-) mice perform better in several learning tasks [117]. However, recent studies provide no clear interpretations with respect to a specific involvement of these suptypes [118, 119] As stated by Houslay and Adams [120]: “PDE4 genes code for 16 isoforms which play a pivotal role in controlling functionally and spatially distinct pools of cAMP by virtue of their unique intracellular targeting”. Therefore even subtle differences between compounds in their inhibition of PDE4 subtypes may result in a differential pattern of action across cognition models. Evidently, much more research is needed to delineate the specific role of these isoforms and to develop molecules that specifically inhibit these enzymes. In this paper we only reviewed drugs that have been tested in animal models of learning and memory. This is merely based on the availability of specific PDE inhibitors and the rational to test these drugs in these models. Based on the localization of the PDE enzymes in the brain and their distribution in the body, we suggest that PDE2, PDE4 and PDE5 are relevant targets for improving memory functions. Other PDEs, except PDE6 and PDE11, could also be relevant because they are all expressed in areas that are directly or indirectly related to learning and memory. A recent study showed that PDE9 may also be considered as a relevant target [121]. The selective PDE9 inhibitor BAY 73-6691 showed clear memory enhancing effects in different animal models. It should be noted that presently there are no PDEs that are exclusively located in the hippocampus and/or cortex (see Table 2). Nevertheless, animal studies have shown remarkable effects of PDE inhibitors on memory performance thus far. The development of selective PDE inhibitors and specific localization studies of PDEs are needed to evaluate the potential of PDE inhibitors for the development of memory dysfunctions. REFERENCES [1] [2]

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J., 2003, 370, 1-18. van der Staay, F.J.; Rutten, K.; Barfacker, L.; Devry, J.; Erb, C.; Heckroth, H.; Karthaus, D.; Tersteegen, A.; van Kampen, M.; Blokland, A.; Prickaerts, J.; Reymann, K.G.; Schroder, U.H.; Hendrix, M. The novel selective PDE9 inhibitor BAY 73-6691 improves learning and memory in rodents. Neuropharmacology, 2008, 55, 908-918. Yan, C.; Bentley, J.K.; Sonnenburg, W.K.; Beavo, J.A. Differential expression of the 61 kDa and 63 kDa calmodulin-dependent phosphodiesterases in the mouse brain. J. Neurosci., 1994, 14, 973-984. Billingsley, M.L.; Polli, J.W.; Balaban, C.D.; Kincaid, R.L. Developmental expression of calmodulindependent cyclic nucleotide phosphodiesterase in rat brain. Dev. Brain Res., 1990, 53, 253-263. Lal, S.; Sharma, R.K.; McGregor, C.; Macaulay, R.J. Immunohistochemical localization of calmodulindependent cyclic phosphodiesterase in the human brain. Neurochem. Res., 1999, 24, 43-49. Reed, T.M.; Browning, J.E.; Blough, R.I.; Vorhees, C.V.; Repaske, D.R. Genomic structure and chromosome location of the murine PDE1B phosphodiesterase gene. Mamm. Genome, 1998, 9, 571-576. Polli, J.W.; Kincaid, R.L. Expression of a calmodulin-dependent phosphodiesterase isoform (PDE1B1) correlates with brain regions having extensive dopaminergic innervation. J. Neurosci., 1994, 14, 12511261. Yan, C.; Zhao, A.Z.; Bentley, J.K.; Beavo, J.A. The calmodulin-dependent phosphodiesterase gene PDE1C encodes several functionally different splice variants in a tissue-specific manner. J. Biol. Chem., 1996, 271, 25699-25706. Repaske, D.R.; Corbin, J.G.; Conti, M.; Goy, M.F. A cyclic GMP-stimulated cyclic nucleotide phosphodiesterase gene is highly expressed in the limbic system of the rat brain. Neuroscience, 1993, 56, 673-686. Van Staveren, W.C.; Steinbusch, H.W.; Markerink-Van Ittersum, M.; Repaske, D.R.; Goy, M.F.; Kotera, J.; Omori, K.; Beavo, J.A.; De Vente, J. mRNA expression patterns of the cGMP-hydrolyzing phosphodiesterases types 2, 5, and 9 during development of the rat brain. J. Comp. Neurol., 2003, 467, 566-580. Sonnenburg, W.K.; Mullaney, P.J.; Beavo, J.A. Molecular cloning of a cyclic GMP-stimulated cyclic nucleotide phosphodiesterase cDNA. Identification and distribution of isozyme variants. J. Biol. Chem., 1991, 266, 17655-17661. Bolger, G.B.; Rodgers, L.; Riggs, M. Differential CNS expression of alternative mRNA isoforms of the mammalian genes encoding cAMP-specific phosphodiesterases. Gene, 1994, 149, 237-244. Cherry, J.A.; Davis, R.L.: Cyclic AMP phosphodiesterases are localized in regions of the mouse brain associated with reinforcement, movement, and affect. J. Comp. Neurol., 1999, 407, 287-301. Miro, X.; Perez-Torres, S.; Palacios, J.M.; Puigdomenech, P.; Mengod, G. Differential distribution of cAMP-specific phosphodiesterase 7A mRNA in rat brain and peripheral organs. Synapse, 2001, 40, 201214. Sasaki, T.; Kotera, J.; Omori, K. Novel alternative splice variants of rat phosphodiesterase 7B showing unique tissue-specific expression and phosphorylation. Biochem. J., 2002, 361, 211-220. Kobayashi, T.; Gamanuma, M.; Sasaki, T.; Yamashita, Y.; Yuasa, K.; Kotera, J.; Omori, K. Molecular comparison of rat cyclic nucleotide phosphodiesterase 8 family: unique expression of PDE8B in rat brain. Gene, 2003, 319, 21-31. van Staveren, W.C.; Glick, J.; Markerink-van Ittersum, M.; Shimizu, M.; Beavo, J.A.; Steinbusch, H.W.; de Vente, J. Cloning and localization of the cGMP-specific phosphodiesterase type 9 in the rat brain. J. Neurocytol., 2002, 31, 729-741. Seeger, T.F.; Bartlett, B.; Coskran, T.M.; Culp, J.S.; James, L.C.; Krull, D.L.; Lanfear, J.; Ryan, A.M.; Schmidt, C.J.; Strick, C.A.; Varghese, A.H.; Williams, R.D.; Wylie, P.G.; Menniti, F.S. 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Progress in Current Non-Viral Carriers for Gene Delivery Yoko Shoji-Moskowitz1,2,*, Daisuke Asai1,3,4, Kota Kodama1,5, Yoshiki Katayama1,6 and Hideki Nakashima1,3 1

CREST, JAPAN; 2FOLIGO Therapeutics, Inc. USA; 3Department of Microbiology, St. Marianna University School of Medicine, Japan; 4Department of Biomedical Engineering, Duke University, USA; 5Creative Research Institution, Hokkaido University, Japan; 6Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Japan Abstract: Since the viral vector for gene therapy has serious problems, including oncogenesity and other adverse effects, non-viral carriers have attracted a great deal of attention. However, the most critical issue of gene delivery by non-viral carriers is the low-expression efficiencies of the desired gene. In order to apply non-viral carriers for gene therapy in practical clinical usage, the improvement of transfection efficiency is a prerequisite. We will summarize the current progress of non-viral delivery systems for gene therapy. Especially, we will address the applications of cationic lipids (lipoplex) and cationic polymers (polyplex) in vivo. Furthermore, there have been reported a disease-site-specific delivery system which responds to highly activated cellular signals, which is called a drug delivery system based on responses cellular signal (D-RECS). We will also introduce the current progress of D-RECS gene delivery which is activated by HIV protease in only HIV-infected cells.

Keywords: Non-viral carrier, polyplex, lipoplex. 1. INTRODUCTION The progress of post-genomic research has provided further understanding of the genetic background of intractable diseases. Based on these findings, gene-based medicine is now in use. However, the biggest hurdle for gene-based medicine to overcome is the development of non-toxic and efficient delivery systems [1]. Currently, the intramuscularly administration of a naked plasmid DNA allows us to obtain a local transient transgene expression [2]. However, the intravenous injection of the naked DNA has resulted in low levels of gene expression in all major organs [3]. Therefore, the development of efficient delivery systems is urgently needed to treat metastatic diseases. According to data published in the Journal of Gene Medicine, over 1340 clinical trials for gene therapy had been approved worldwide by 2007 [4]. Approximately only 20% of these trials use non-viral vectors. Viral vectors are more efficient in terms of transfection efficiency, yet are plagued by safety concerns [5]. *Corresponding author: Tel: + 1-301-770-0099; Fax: + 1-301-881-7640; E-mail:[email protected] Allen B. Reitz / Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.

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The first gene medicine, type 5 adenoviruses bearing the human wild-type p53 gene (Ad-p53) for the treatment of head and neck cancer, was approved by the State Food and Drug Administration (SFDA) of China. It has been on the market as Gendicine since October 2003. In 2005, SFDA approved Oncolin, adenoviruses with a defective E1B-55 kDa molecule for head and neck cancer, which has been on the market since 2006 [6]. In the past years, however, the safety issues surrounding the viral vectors were brought sharply into focus by the sudden death of an 18-year-old patient in a gene therapy trial [7]. Retrovirus is also used as a vector as well as adenovirus for gene therapy. Despite the successful gene therapy for correcting X-linked severe combined immunodeficiency, 2 out of 10 cases developed leukemia [8, 9]. There is a major concern that the randomness of the integration of retrovirus vectors on chromosomes may lead to activating oncogene, which causes cancer. Therefore, despite their comparatively low efficiency, non-viral vectors have been enthusiastically and vigorously investigated due to their advantageous safety profile. In addition to the safety issue, a recent report on the comparative activity of retroviral and non-viral (liposomal) gene transfer in a mouse model demonstrated that viral vectors have no advantage over the survival period [10]. Several clinical trials of non-viral carriers [11-13] have been evaluated, resulting in encouraging the research of non-viral carriers. The other promising non-viral carrier is a delivery system that responds to cellular signals [14-18]. As shown in Fig. (1), the system, the so-called drug or drug delivery system, which responded to cellular signals (D-RECS), includes a peptide substrate, which can be cleaved by the target enzyme in disease cells. For example, protein kinase C (PKC) alpha is excessively activated in most tumor cells. D-RECS containing cationic peptide substrate, which is phosphorylated by PKC-alpha, coupled with cationic copolymers comprising hydrophilic and neutral polymers in main chain. Under the presence of PKC-alpha, pDNA was released from cationic copolymer with DNA complex in tumor cells [15, 17]. Another example is D-RECS activated by caspase [16]. These reports successfully showed DNA release from D-RECS and DNA complex in the presence of corresponding a peptide substrate that was cleavable only in tumor cells. As a result, the desired DNA expression could gain only in tumor cells, not in normal cells. Recently, D-RECS containing peptide substrate for HIV protease demonstrated a specific transgene expression in HIV-infected cells by responding to HIV protease [19, 20]. This review will address the current progress of non-viral gene delivery systems including D-RECS. 2. NAKED DNA The administration of naked DNA is the most straightforward strategy for gene therapy. The direct administration of plasmid DNA to skeletal muscle cells results in efficient gene expression, which was contrary to expected results [3]. Therefore, the naked DNA has been administered intramuscularly for the purposes of vaccination, where antigen-encoding DNA is administered to produce a protective immune response to the transgene antigenic product [22, 22]. It was thought that this method would not be suitable for prolonged transgene expression. Efficient transfection levels, however, have been obtained on the direct injection of the naked DNA to the liver [23, 24], to solid tumors [25], and to the epidermis and hair follicles [26]. The introduction of bolus injection of a large amount of DNA via an artery improved enough to gain the high level of gene expression [27].

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Normal cells general transcription factors not accessible inhibit transcription

polymer/DNA complex

HIV infection

HIV protease expression

side chain peptide cleavage by HIV protease Infected cells disintegration of polymer/DNA complex permit transcription

Fig. (1). Schematic model of D-RECS responds to HIV protease.

Intriguingly, the direct administration of transgenes using non-viral carriers such as cationic liposomes does not enhance the transfection efficiency [28, 29]. And actually these carriers hinder gene expression [30]. The failure in the enhancement of transfection efficiency by carriers is observed even when DNA tissue clearance is inhibited by the use of cationic liposomes [28]. The deleterious effects of cationic lipids such as N- [1-(2,3dioleoyloxy) propyl]-N,N,N-trimethylammonium methylsulphate (DOTAP) to cells such as erythrocytes [31] and macrophages [32] may be responsible for the lack of transfection efficiency. The molecular mechanism of DNA uptake has remained unknown. Early studies suggested that DNA binding proteins on the cell surface could promote uptake of DNA. They have not been confirmed, however. Recently, it was suggested that DNA is taken up via proteoglycan dependent macropinocytosis [33]. Although DNA transporting proteins in various tissues should be further investigated, this finding would give insights into the better understanding of DNA internalization. The direct injection of DNA without any carriers is the simplest way to administer it, but this method would not be appropriate for metastasized diseases and in anatomically inaccessible sites.

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3. LIPOPLEX AND POLYPLEX The approaches using non-viral carriers involve the combination of nucleic acids with cationic lipids (lipoplexes) and/or cationic polymers (polyplexes). Several major obstacles need to be overcome for the development of non-viral gene delivery systems for clinical use in humans. These obstacles include: transfection efficiency, toxicity, manufacturing process, formulation and bio-physiological stability, and extracellular/intracellular barriers. Recent progress on the development of lipoplex and polyplex is introduced here. 4. LIPOPLEX The first gene delivery cationic liposome with a polar head group (protonated at physiological pH), was introduced in 1987 [34]. Since then, a variety of commercial cationic lipids as in vitro gene delivery reagents, e.g. lipofectin (N- [1-(2,3-di-olyloxy) propyl]-N,N,Ntrimethyl-ammonium chloride, 1,2-dioleoyl- phosphatidylethanolamine-DOPE, 1:1) have been developed. Furthermore, the liposomal gene delivery method has been applied in clinical trials [35, 36]. A large number of cationic lipids have been synthesized and studied as non-viral carriers for gene delivery. The structure-activity relationship studies have shown that increasing the number of amine groups, which is necessity for the transfection competence, per molecule [37], and the distance between the amine groups and the hydrophobic units [38], is advantageous to gene delivery. This arrangement of atoms allows an intimate level of DNA binding in the lipoplex (by increasing contact sites) as well as a dissociation of the bound DNA from the cohesive interaction of the hydrophobic units. Lipoplexes range from 50 nm to just over a micrometer in size. The influence of lipoplex size on the transfection efficacy is contrary to what would be expected. Actually, the larger lipoplexes have been reported as improving the transfection efficiency in vitro. A positive charge of lipoplex is essential for the cell binding prior to the internalization by endocytosis [39]. Some neutral lipids, such as DOPE [40] and cholesterol [41-43], have been incorporated into the cationic lipid bilayer with DOPE to improve in vitro transfection of some cell lines by facilitating endosormal escape [40]. The role of cholesterol, however, is unclear [42, 44]. The use of cationic liposomes to deliver genes increases the level of protein expression obtained on intravenous injection [45, 46]. The complexation of DNA with cationic liposomes also prevents DNA degradation in the plasma [47]. Polyethylene glycol (PEG) lipids into the lipoplexes can contribute to diverting lipoplex from the lung. PEG coating successfully prolongs the circulation time of the lipoplexes and allows the production of the desired protein in the distal tumors [48, 49]. This strategy is reminiscent of that used to divert drug-carrying liposomes from the liver and spleen [50]. In vitro, however, a PEG coating decreases the uptake and gene transfer [51]. Cationic liposomes may also be administered directly to the target site in order to avoid the targeting difficulties that are encountered in the case of intravenous administration. Access to the alveolar epithelium has been achieved via the intratracheal route, resulting in expression of the
-1-antitrypsin [52], the -galactosidase reporter [53] genes and a reduction in the size of pulmonary tumors on the administration of the p53 apoptosisinducing gene [54]. However, intratracheal administration is not routinely applicable in the clinical usage. The application of aerosols [55] made it possible to access the alveolar epithelium. Lipoplexes were effective in preventing degradation of DNA during aerosolisation [56]. Successful gene delivery to the eye will have an enormous impact on the treatment of genetic eye diseases. It has been reported that the efficient transfection to the retinal gan-

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glion cells has been observed on the instillation of lipoplex eye drops with no inflammation [57]. Since cell toxicity of cationic liposomes has been reported, research to reduce the toxicity of these carriers is required. In addition to improving the biological properties of lipidic gene carriers, some studies have focused on improving the stability of lipoplexes employing lyophilisation with the aid of monosaccharide, disaccharide or PEG lipid [28] cryoprotectants. Allovectin is a mixture of plasmid DNA encoding the genes HLA-B7 and 2-microglobulin complexed with a cationic lipid mixture, DMRIE/DOPE (1,2-dimyristyloxypropyl3-dimethyl-hydroxyethyl ammonium bromide/dioleoyl-phosphatidyl-ethanolamine) [58-60]. Allovectin was intratumorally injected to treat Stage III or IV melanoma patients, and showed minor adverse effects. These effects included ecchymosis and pruritis, which were not serious [58]. The cationic liposome showed no severe cytotoxicity. Tumor regression at the injection site was observed in 18% of patients [58]. The mechanism of Allovectin is still not completely defined, however, it is thought that it stimulates the immune system to recognize and destroy cancerous cells. This DNA-based immunotherapy is also encouraging for the clinical usage of cationic liposomes. Currently, phase III clinical trial of Allovectin has been conducted. Most cationic liposomes and DNA complexes form a multilayered structure with DNA sandwiched between cationic lipids. The membrane charge density of the cationic liposomes is one of the parameters to gain transfection efficiency [61]. Further understanding of the supermolecular structure of lipoplex may lead to the design of better non-viral carriers in terms of delivery efficiency. 5. POLYPLEX Polyplex, which has protonated polymer-bearing groups at physiological pH, has been employed as a gene carrier. The electrostatic attraction between the cationic charge on the polymers and the negatively charged DNA results in a particulate complex–the polyplex, which is the transfecting unit. 5.1. Poly-Lysine Based Polymers The first polycation for gene delivery was poly-L-lysine (PLL) conjugated with asialoorosomucoid for hepatocellular gene targeting [62]. Unlike cationic liposomes, many of the early works involving the use of polyplexes utilized ligands to facilitate cellular uptake, e.g. asialoorosomucoid, transferrin [63-65], folate [66], monoclonal antibodies [67-69] and basic fibroblast growth factor [70]. The gene transfer activity of PLL polyplexes without the use of receptor-mediated strategies is poor [71] unless endosomolytic or lysosomotropic agents (e.g. chloroquine) are added [72, 73]. This is an important difference in the biological activity of the amphiphilic cationic lipids and the soluble polymer PLL. Cellular uptake of gene transfer by polyplexes in the absence [73] or presence [74] of targeting ligands is, however, still dependant on the presence of a positively charged polyplex [74]. Various other PLL copolymers have also been shown to transfer genes into mammalian cells such as those incorporating
–tryptophan [72], and graft poly-histidine [76]. The conjugation of histidine to lysine residues of PLL [76] resulted in a transfecting polyplex, which was more efficient than a PLL-chloroquine mixture. This graft copolymer showed an enhancement in activity in the absence of chloroquine because of the additional endosormal buffering capacity

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ity offered by histidine, which is protonated below pH 6. In a similar strategy, gluconylated polyhistidine has also been used to transfer genes to mammalian cells and does not require chloroquine [77]. Hence, the use of histidine residues seems to facilitate an endosormal escape. Although PLL polyplexes prevent the degradation of DNA by the serum nucleases [78] in a similar manner to liposomes [46]; on the intravenous injection, these polyplexes are bound to the plasma proteins and rapidly cleared from the plasma [79] that is similar to cationic liposomes. Polyplex opsonisation by the plasma proteins may be suppressed by coating the polyplexes with a hydrophilic polymer such as hydroxypropyl methacrylic acid. The cellular uptake of the polyplexes may be promoted by the conjugation of targeting ligands such as transferrin [80] or fibroblast growth factor [81] to the surface of the coated polyplexes. To improve the transfection efficiency, reducible linear L-lysine modified copolymer was used instead of high molecular weight PLL. The molecular weight of the copolymer was about 3.2kD. The transfection efficiency of this polyplex was much higher compared to the optimal PLL control [82]. Mannose receptors are abundant on dendritic cells (DCs) and macrophages. To facilitate mannose receptor for DNA internalization, oxidized mannan or reduced mannan conjugated with PLL (mannan-PLL) as a carrier was applied for DNA vaccination [83]. The injection of DNA encoding ovalbumin (OA) complexed with mannan-PLL in tumor-bearing C57BL/6 mice showed sufficient antibody production leading to the inhibition of OVA tumor growth and induced CD4 and CD8 T-cell responses. This method provides efficient internalization via a mannose receptor, which was better than the PLL delivery system alone [83]. 5.2. Polyethylenimine Recently both branched [84, 85] and linear [86] polyethylenimine (PEI) have been introduced as cationic polymers for gene delivery. Unlike PLL, this polymer shows efficient gene transfer without the need for endosomolytic or lysosomotropic agents or any agents facilitating receptor-mediated uptake. PEI is endocytosed by cells and also facilitates endosormal escape [87]. As with all the other non-viral gene delivery systems mentioned above, it has a positive charge for the internalization [84]. Although PEI is quite an efficient gene transfer agent [88], the addition of targeting ligands to this polymer enhances its activity in some cell lines [89-92]. Recently hydrophobised PEI, which has been incorporated within DOPE, egg phosphatidylcholine and dipalmitoyl phosphatidylcholine liposomes, produced an efficient gene transfection, although the activity of this soluble amphiphilic polymer was diminished when it was administered without the liposomal lipids [93]. Unfortunately, PEI, like some of the cationic lipids, has also been reported to be toxic to cells. Ovarian cancer is the most deadly gynecological cancer and most patients are found at a late stage with a lack of effective treatments. PEI containing polyplex conjugated with interleukin-12 (IL-12) plasmid was tested to enhance local and systemic immune responses against ovarian cancer [94]. The intraperitoneal administration of polyplex with IL-12 plasmid increased murine IL-12 and interferon-gamma in ascites fluid, although serum levels remained unchanged. They observed the delay of onset of ascite tumors. Followed by successful animal tests, a phase 1 clinical trial of PEI-gene complex in ovarian cancer showed promising results [13]. Synthetic polyethyleneglycol-PEI-cholesterol conjugated with IL-12 plasmid was administered to women with chemotherapy-resistant recurrent ovarian cancer to enhance immune responses against ovarian cancer. This gene delivery system proved safe

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and demonstrated the reduction of serum CA-125 levels in some patients [13]. Some patients remained at a steady condition. Thus, this system would open a new door to treat chemotherapy-resistant, metastasized, and recurrent cancers. 5.3. Other polymers A transfecting peptide has been prepared from the N-terminal of the human adenovirus fiber protein, which promotes the transport of DNA to the nucleus and improves transfection rates. The cationic liposomes prepared from DOTAP [95], copolymer including PEG and poly (aminoethyl methacrylate) and poly (dimethylamino) ehytlmethacrylate [96], poly (Nethyl-4-vinylpyridinium) polymers [97], poly-histidine [76] polyglycolide (PLG) and chitosan [98] have also been used for gene delivery. Both chitosan [99] and PLG [16] nanoparticles appeared to control the release of DNA and prolong its action both in vitro [99] an in vivo [30]. The use of PLG is a peculiar example of the efficacy of a polymer. It is not protonated and hence not cationic at physiological pH, so that PLG particles actually possess a negative surface charge [30]. Chitosan has also been used via oral administration to achieve an immune response against a peanut allergen [100], which is the first report of an orally active gene delivery system. Cyclodextrin (CD) has been used as the modification of existent therapeutic reagents to protect physical, enzymatic degradation and to improve delivery efficiency [101]. The properties of CD and its toxicology have been well characterized clinically. Thus, CD is an ideal carrier for clinical usage. A cationic CD has a great ability to bind polyanionic polymer such as oligonucleotides and DNA. The conjugation of mannose with dendrimer has been also applied for integration CD showing promising transfection efficiency [102]. Recently, a film containing DNA drew great interest for gene delivery such as a patch vaccination [103]. The multilayered film consisting of poly L-glutamic acid (PLGA) and PLL containing DNA in the presence of the cationic CD demonstrated efficient gene delivery as a carrier. The cationic CD acts as an enhancer of cellular permeability in this system. The cationic CD also preserves DNA activity well and shows no significant DNA release from the film for 6 days. This type of film promotes not only the absorption but also the interfacial delivery of DNA into cells [103]. 5.4. Dendrimers Dendrimers are spherical nanostructures that can encapsulate therapeutic drugs in their interior or attach to the surface. The dendrimer diameter increases linearly per generation, whereas the number of surface groups increases geometrically. Dendrimers consist of core nanoparticles and interior branching, which connect the core to surface groups such as cationic, anionic, neutral, either hydrophobic moiety. Among all, polyamidoamine (PAMAM) dendrimer has been commonly studied. A range of polyamidoamine- [104-108] and phosphorous-containing dendrimers has been studied as gene delivery systems. Terminal amino groups bind to DNA by electrostatic force [106]. Positively charged complexes are advantageous for gene delivery [109]. An increase in the level of terminal amino groups appeared to enhance gene delivery [110]. Dendrimer-gene complexes are presumably internalized by endocytosis and there are also advantages associated with the star shape of the polymer as DNA appears to interact with the surface-primary amines only, leaving the internal tertiary amines available for the neutralization of the acid pH [111] within the endosomal/lysosomal compartment. Following the internalization, the release of polyamidoamine-gene complexes from the endosome has been attributed to the protonation of the internal tertiary nitrogens by endosormal protons, which then results in a swelling of the endosome and the release of the DNA to the cytoplasm [112]. The hydrolytic degradation of polyamidoamine dendrimer

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amide bonds in water or ethanol increased transfection efficacy up to 50-fold, which the authors attribute to the increased flexibility of the polymer on heat degradation [112,113]. This increased flexibility is crucial to the swelling of the endosome [112]. Partially hydrolyzed polyamidoamine dendrimers were found to be more effective as gene transfer agents than branched PEI in the in vivo transfection of the carotid artery of rabbits [113]. Cytotoxicity is a pivotal issue to overcome. Primi et al. investigated the internalization process of PAMAM dendrimers. They provided insights that 500-700nM was effective in cell internalization without significant cytotoxicity [114]. Systemic administration of polypropylemimine dendrimer showed remarkable tumor regression [115]. The polypropylemimine dendrimer was conjugated with transferin which were over-expressed in tumors. Transferrin-bearing polypropylemimine dendrimer resulted in high gene expression, mainly in the tumors. Therapeutic DNA conjugated with transferring-bearing polypropylemimine dendrimer showed significant tumor regression without apparent signs of toxicity. 6. CELLULAR SIGNAL RESPONSE DRUG DELIVERY SYSTEM (D-RECS) The aforementioned targeting strategies often utilize a molecular marker on the cellular surface to promote the efficient transfection of genes to the target cells. However, this socalled active targeting strategy is not always successful because the effective molecular markers are not always available. It is inevitable that genes are delivered to the unwanted sites and it is difficult to control the gene expression itself in the target cells. A novel strategy that can discriminate normal cells and target cells in gene therapy by focusing on differences in intracellular signals such as PKC alpha, I
-B kinase, caspase 3, HIV protease has been reported [14-20, 116-118]. Aberrant activation of certain intracellular signals is often seen in many diseases [119-126]. Thus, if such unusual intracellular signals could be utilized to activate the delivered gene, the cell-selective control of the delivered gene expression could be achieved. Fig. (1) shows a schematic model of D-RECS containing peptide side chains that are cleavable by HIV protease. D-RECS consists of polymer which has cationic peptide chains and responsible DNA. Because of cationic peptide side chains, the polymer conjugate can form a stable polyplex with DNA. Thus, the transcription of DNA is suppressed in the nonactivated condition (normal cells). On the other hand, the cationic peptide is connected to the polymer backbone via a sequence that is specifically cleaved by the target protease, HIV protease. When the conjugate encounters the target protease, the cationic peptide side chain is cleaved by the target protease and the polymer loses the electrostatic interaction with the DNA. As a result, the DNA is released in the target cell and is transcribed (Fig. 1). Thus, DRECS acts as a gene regulator only in cells containing the target protease. Recently, protein kinase C (PKC) was used as a trigger to activate the transgene expression to prove the applicability of D-RECS as a gene carrier [118]. We also apply the strategy of D-RECS in HIV infectious disease [19, 20]. This peptide side chain consists of the consensus substrate sequence for HIV protease, and protein transduction domain sequence of HIV-Tat protein (CPCHIVtat). First of all, we studied whether the peptide substrate is specifically cleaved by HIV protease in cell-free system. DNA was released from CPCHIVtat -DNA complex and was activated under the presence of HIV protease [19]. On the contrary, DNA was not released without HIV protease, which suggested that peptide substrate is cleaved by HIV protease [19]. However, HIV infected cells contain various hydrolytic enzymes. We also confirmed that the substrate peptide used in CPCHIVtat was not digested at all by other potential intracellular proteases, including

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caspase-8 and caspoase-3, using MALDI-TOF-MS and/or reversed-phase HPLC (data not shown). In the cell-free assay system, many factors, such as charged protein and high concentration of salts in the reticulocyte lysate, may influence the stability of the polyplex. Thus, under such conditions, it is likely that a larger C/A charge would be required to suppress the transgene expression. Furthermore, HIV-1 protease-induced transgene expression from CPCHIVtat polyplex in this cell-free system was inhibited by protease inhibitors such as indinavir, saquinavir, and ritnavir [20]. Next, we studied whether this strategy works in HIV-infected cells. Various human T lymphocyte cell lines have been used for HIV infection experiments, including MT-4, CEM, MOLT-4, and Jurkat cells, and all of them are able to work as HIV producer cells. We chose a Jurkat human T lymphocyte cell line because Jurkat cells are not killed by HIV infection, i.e., they can be persistently infected with HIV, like CEM and MOLT-4 cells. Also their transfection efficiency is rather high compared with other human T lymphocyte cell lines. We confirmed that the titer of HIV in the supernatant of the Jurkat cell culture medium was high enough compared with that of MOLT-4 cell, and the concentration of p24 protein was similar in both cell lines (Fig. 2). Fig. (3) showed that CPCHIVtat-DNA was transfected in HIV-infected cells under fluorescent microscopy. As shown in Fig. (4), DNA was released from CPCHIVtat-DNA in HIV infected cells while negative control didn’t reveal transgene expression. Thus, CPCHIVtat-DNA reveals transgene expression responding to HIV-1 protease in HIV infected cells. These results indicated D-RECS can facilitate HIV protease signals in HIV-infected cells to switch on the target gene in only HIV-infected cells. p24 protein (pg/mL)

500 400 300 200 100 0 Jurkat

MOLT-4 Cells

Fig. (2). Cells were infected with HIV at a multiplicity of infection (MOI) of 0.01. HIV-infected cells (3.0 x 105 cells/ml) were placed into 96-well microtiter plates and incubated. After 5 days of culture at 37˚C in a CO2 incubator, the culture supernatant was collected, and the HIV-1 p24 antigen was detected and quantified by the HIV-1 p24 Antigen ELISA (ZeptoMetrix Corp., Buffalo, NY, USA).

7. CONCLUSION The success of gene therapy in China has inspired the medical arena, which had been awaiting it [6]. Unfortunately, non-Chinese researchers are unaware of the progress of gene therapy in China because most of these clinical trials of gene therapy have been reported only in Chinese. Furthermore, it is difficult to access such information and to get relevant comparisons of their clinical data to Western standardized clinical data. Also, the adenovirus receptors are often down-regulated in tumors resulting in insufficient therapeutic efficiency [127]. Another concern of gene therapy using adenovirus vector is that the presence of an adenovirus neutralizing antibody, which would hamper the therapeutic efficacy. It is thought that about 60% of adults have adenovirus neutralizing antibody before treatment. Further-

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more, weakened viruses would alter and gain pathogenesity in the body. Also, patients may develop immune responses to the viruses and quickly eliminate the viral-DNA complex. Such events cause the inefficiency of activating therapeutic DNA. CP(DVP)tat

CPCHIVtat Fluorescein

Marged

(fluorescein and phase contrast)

Fluorescein

Marged

(fluorescein and phase contrast)

(-) HIV (naive)

(+) HIV (HIV-infected)

Fig. (3). Cells were incubated with the polyplexes at a C/A ratio of 1.0 in OptiMEM I medium for 4 h at 37˚C. After washing with PBS, DNA labeled was observed by fluorescence microscopy.

Luciferase activity (RLU/mg protein)

4.0E+08

- HIV + HIV

3.0E+08 2.0E+08 1.0E+08 0.0E+00

charge ratio Polymor

(-) none

+ 1.0

+ 2.0

CPCHIVtat

+ 1.0 CP(DVP)tat

+ 1.0 CPCCVtat

+ 2.0 PLL

Fig. (4). Transgene expression in HIV-infected Jurkat cells following transfection with various polymers. pMetLuc-control condensed with indicated polymers at a charge ratio of +1.0 and/or +2.0 was added and the cells were cultured for two days. None indicates plasmid DNA only. The light unit values represent specific luciferase activity (RLU/mg protein) standardized against total cellular protein content of cell lysates. Data shown are the mean ± SD of duplicate samples and are representative of at least two independent experiments.

As well as viral vectors, non-viral carriers for gene therapy have improved transfection efficiency, which prove that enough transgene expression can be introduced to the target

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site. In this article, we summarize the recent progress of lipoplexes and polyplexes as nonviral carriers for gene delivery. Mostly, non-viral carriers possess a positive charge to facilitate good transfection efficiency. It seems to be advantageous for gene delivery in increasing the number of amine groups and the distance between the amine groups and the hydrophobic units. A positive charge is advantageous to bind to the DNA and to facilitate endosomal escape. Other promising carriers for gene delivery are polyplexes, such as PEI, chitosan, and dendrimers. Major concerns about the toxicity of non-viral carriers have been drastically improved. Recently, PEI with tolerable low toxicity for clinical usage has reported, which is promising. D-RECS is a smart new strategy that can achieve the gene expression only in the desired sites. By using this system, the gene does not express in the normal cell even though the gene is delivered to the normal cell. Cellular signals only in the aberrant cell (e.g. caspase, HIV protease) cleave the carriers as shown in Fig. (1) and release DNA. For clinical usage, the toxicity of the polymer used in this study has to be improved. However, D-RECS can provide the specific targeting that other non-viral carriers do not. As other virus proteaseresponsive D-RECS, coxsackievirus (CV) 2A protease was applied and showed a similar result as HIV infection, which proved D-RECS is applicable for other virus infections [19]. Thus, D-RECS strategy can be useful to establish the universal concept of drug design for virus-infected cell-specific attacks or other cancer treatments. Recently, non-viral gene delivery has become more promising because several clinical trials have been conducted with encouraging results. Moreover, non-viral carriers can be repeatedly injected into the body, which viral vectors often cannot. In the last decade, the development of non-viral carriers for gene therapy has steadily progressed from the bench to clinical trials. If this progress continues at current rates, the coming decade will be bright for gene therapy. ACKNOWLEDGEMENTS This study was partially funded by a grant CREST of Japan Science and Technology Agency, Japan. The authors would like to express their thanks to Mr. Ken Moskowitz for editing this manuscript. REFERENCES [1] [2] [3] [4] [5] [6]

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Boussif, O.; Zanta, M.A.; Behr, J.P. Optimized galenics improve in vitro gene transfer with cationic molecules up to 1000-fold. Gene Ther., 1996, 3, 1074. Chemin, I.; Moradpour, D.; Wieland, S.; Offensperger, W.B.; Walter, E.; Behr, J.P.; Blum, H.E. Liver-directed gene transfer; a linear Polyethylenimine derivative mediates highly efficient DNA delivery to primary hepatocytes in vitro and in vivo. J. Viral. Hepat., 1998, 5, 369. Klemm, A.R.; Young, D.; Lloyd, J.B. Effects of polyethyleneimine on endocytosis and lysosome stability. Biochem. Pharmacol., 1998, 56, 41. Ferrari, S.; Moro, E.; Pettenazzo, A.; Behr, J.P.; Zacchello, F.; Scarpa, M. ExGene 500 is an efficient vector for gene delivery to lung epithelial cells in vitro and in vivo. Gene Ther., 1997, 4, 1100. Kircheis, R.; Kichler, A.; Wallner, G.; Kursa, M.; Ogris, M.; Felzmann, T.; Buchberger, M.; Wagner, E. Coupling of cell-binding ligands to polyethylenimine for target gene delivery. Gene Ther., 1997, 4, 409. Zanta, M.A.; Boussif, O.; Adib, A.; Behr, J.P. In vitro gene delivery to hepatocytes with galactosylated polyethylenimine. Bioconjug. Chem., 1997, 8, 839. Li, B.; Li, S.; Tan, Y.D.; Stolz, D.B.; Watkins, S.C.; Block, L.H.; Huang, L. Lyophilization of cationic lipid-protamine-DNA (LPD) complexes. J. Pharm. Sci., 2000, 89, 355. Li, S.; Tan, Y.; Viroonchatapan, E.; Pitt, B.R.; Huang, L. Targeted gene delivery to pulmonary endothelium by anti-PECAM antibody. Am. J. Physiol. Lung Cell Mol. Physiol., 2000, 278, L504. Yamazaki, Y.; Nango, M.; Matsura, M.; Hasegawa, Y.; Hasegawa, M.; Oku, N. Polycation liposomes, a novel nonviral gene transfer system, constructed from cetylated polyethylenimine. Gene Ther., 2000, 7, 1148. Fewell, J.G.; Matar, M..; Rice, J.S.; Brunhoeber, E., Slobodkin, G.; Pence, C.; Worker, M.; Lewis, D.H.; Anwer, K. Treatment of disseminated ovarian cancer using nonviral interleukin-12 gene therapy delivered intraperitoneally. J. Gene Med., 2009, 11,78. Zhang, F.; Andreassen, P.; Fender, P.; Geissler, E.; Hernandez, J.F.; Chroboczek, J. A transfecting peptide derived from adenovirus fiber protein. Gene Ther., 1999, 6, 171. Dufresne, M.H.; Elasabahy, M.; Leroux, J.C. Characeterization of polyion complex micelles designed to address the challenges of oligonucleotides delivery. Pharm. Res., 2008, 25, 2083 . Kabanov, A.V.; Astafieva, I.V.; Maksimova, I.V.; Lukanidin, E.M.; Georgiev, G.P.; Kabanov, V.A. Efficient transformation of mammalian cells using DNA interpolyelectrolyte complexes with carbon chain polycations. Bioconjug. Chem., 1993, 4, 448. Murata, J.; Nagae, H.; Ohya, Y.; Ouchi, T. Design of macromolecular biological response modifier by immobilizing of D-glucose of muramyl dipeptide on carboxymethyl-dextran having mannose branches. J. Biomater. Sci. Polym. Ed., 1997, 8, 931. Erbacher, P.; Zou, S.; Bettinger, T.; Steffan, A.M.; Remy, J.S. Chitosan-based vector/DNA complexes for gene delivery: biophysical characteristics and transfection ability. Pharm. Res., 1998, 15, 1332. Roy, K.; Mao, H.Q.; Huang, S.K.; Leong, K.W. Oral gene delivery with chitosan—DNA nanoparticles generates immunologic protection in a murine model of peanut allergy. Nat. Med., 1999, 5, 387.

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Protein Transduction: Cell Penetrating Peptides and Their Therapeutic Applications Kylie M. Wagstaff 1 and David A. Jans2,* 1

Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia; 2ARC Centre of Excellence for Biotechnology and Development, Australia Abstract: Cell penetrating proteins or peptides (CPPs) have the ability to cross the plasma membranes of mammalian cells in an apparently energy- and receptorindependent fashion. Although there is much debate over the mechanism by which this “protein transduction” occurs, the ability of CPPs to translocate rapidly into cells is being exploited to deliver a broad range of therapeutics including proteins, DNA, antibodies, oligonucleotides, imaging agents and liposomes in a variety of situations and biological systems. The current review looks at the delivery of many such molecules by various CPPs, and their potential therapeutic application in a wide range of areas. CPP ability to deliver different cargoes in a relatively efficient and non-invasive manner has implications as far reaching as drug delivery, gene transfer, DNA vaccination and beyond. Although many questions remain to be answered and limitations on the use of CPPs exist, it is clear that this emerging technology has much to offer in a clinical setting.

Keywords: Cell penetrating protein or peptide, CPP, protein transduction. 1. INTRODUCTION A large proportion of current therapeutics require the intracellular delivery of biologically active compounds to mediate effects in the cytoplasm, nucleus and other organelles. The greatest barrier to this process is the plasma membrane itself. Although numerous strategies to overcome this in vitro have been devised, including microinjection and electroporation, they have extremely limited in vivo applications. Furthermore, large molecules usually enter cells through receptor-mediated endocytosis, which, due to subsequent fusion with the cellular lysosomal compartment, results in their degradation. Thus, methods to increase the bioavailability of compounds in vivo, especially those that can facilitate cell entry without the requirement for endocytosis, are particularly sought after. In recent years, numerous proteins and peptides have been found to enter intact cells in an apparently energy- and receptor-independent fashion [1, 2]. This process of translocation across plasma membranes, also termed protein transduction or “cellular internalisation”, has already been utilised to deliver numerous cargoes to a large number of cell types and tissues, both in vitro and in vivo.

*Corresponding author: Tel: +613 99029341; Fax: # + 613 99029500; E-mail: [email protected] Allen B. Reitz / Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.

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Molecules that can undergo protein transduction are known as Cell Penetrating Proteins or Peptides (CPPs), an exponentially expanding class of molecules [3]. The first CPP was discovered independently by two laboratories, which found that the trans-activating transcriptional activator (Tat) from Human Immunodeficiency Virus 1 (HIV-1) could be efficiently taken up from the surrounding media by numerous cell types in culture [4, 5]. Since then, translocational ability has been established for the Homeodomain of Antennapedia (Antp, a transcription factor from Drosophila) [6, 7], Herpes Simplex Virus 1 protein 22 (VP22) [8], Flock house Virus coat protein (FHVC) [9], the core histones [10, 11] and a multitude of others. Numerous peptides, either derived from these, or generated synthetically such as Transportan [12], have also shown strong protein transduction ability. More specifically, distinct short domains, typically less than 20-30 amino acids, which are highly rich in basic residues, mediate protein transduction [1]. Such domains are known as Protein Transduction Domains (PTDs), but their mechanism of action is still poorly understood. The present review seeks to outline the nature of some of the better-understood CPPs and their potential mechanisms of cellular entry. The main focus is the application of CPPs to mediate the cellular delivery of a wide range of therapeutic molecules and the various advantages of this system, such as the ability to deliver efficiently to a broad range of cell types and tissues. It is clear that CPPs represent an exciting new prospect for drug delivery, in a range of clinical contexts. 2. SPECIFIC CELL-PENETRATING PROTEINS It has been proposed that PTDs have evolved specifically to facilitate the translocation into cells of certain proteins and peptides with intracellular bioactivity [13], where they may play a role in host response to intracellular infections, or in infection itself in diseases such as the PrP related disorder Creutzfeldt-Jacob [14], where the cellular uptake mechanism of the infectious protein particle is unknown. In favour of the former idea, various naturally occurring antimicrobial peptides (eg, magainin or buforin) are known to penetrate plasma membranes rapidly [15], resulting in subsequent killing of the infecting organism either through cell lysis or inhibition of functions through binding to DNA and/or RNA targets. This implies that membrane translocation of specialised proteins may be an important mechanism to protect against infectious agents. In terms of pathogens, VP22 (see Section 2.3), shown to mediate intercellular trafficking between neighbouring cells, both in vitro and in vivo, plays an important role in HSV-1 infection [8, 16]. As more CPPs are discovered it has become clear that there are at least two distinct classes of PTD, as highlighted in Table (1). One class consists of Arginine (Arg) rich peptides such as Antp and Tat, whilst the other is composed of amphipathic helical peptides, such as Transportan and model amphipathic peptide, in which Lysine (Lys) is the predominant positively charged residue. The next sections describe some of the more commonly studied and most interesting CPPs, most of which will then be discussed in detail in the subsequent sections with respect to their mechanisms of action and potential therapeutic applications. 2.1. Trans-activating Transcriptional Activator (Tat) HIV-1 Tat is an 86 amino acid transcription activating protein. It has numerous domains, including a highly basic region (residues 49-57, containing 6 Arg and 2 Lys residues), which is important for nuclear localisation and RNA binding [17]. The original Tat peptide used to mediate the cellular entry of proteins (Tatp) was Tat (residues 37-72) [18], including the basic domain as well as an amphipathic
-helical region (residues 38-49) [19]. The
-helix

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Examples of CPP/PTD Sequences

CPP

Sequence*

Length (Residues)

Reference

11

[22]

16

[6]

8

[50]

A) Arginine Rich Peptides Tatp

YGRKKRRQRRR57

Antp (Penetratin)

RQIKIWFQNRRMKWKK

Arg8

RRRRRRRR

pVEC

58#

18

[42]

50

17

[9]

MAP

KLALKLALKALKAALKLA

18

[44]

Transportan

GWTLNSAGYLLGKINLKALAALAKKIL

27

[12]

Transportan-10

AGYLLGKINLKALAALAKKIL

21

[30]

KALA

WEAKLAKALAKALAKHLAKALAKALKACEA

30

[65]

ppTG1

GLFKALLKLLKSLWKLLLKA

20

[61]

27

[98]

HIV Rev

LLIILRRRIRKQAHAHSK

635

TRQARRNRRRRWRERQR

B) Amphipathic Helical Peptides (Lysine Rich)

C) Non-basic Chimeric Peptide MPG

GALFLGFLGAAGSTMGAWSQPKSKRKV

*

Single letter amino acid code Residue 58 of the homeodomiain; corresponds to residue 356 of the full length Antennapedia protein. Abbreviations: CPP, Cell Penetrating Peptide; PTD, Protein Transduction Domain; Tat, Trans-acting transcriptional activator; Antp, pAntennapedia; pVEC, VE-cadherin-derived cell-penetrating peptide; HIV, Human Immunodeficiency Virus; MAP; Model Amphipathic peptide.

#

forming domain was originally presumed to be critical for Tat protein transduction, because shorter peptides, such as Tat (37-58) or (47-58), possessed weaker translocating ability [18]. Subsequently, however, structure-activity relationship studies of peptides derived from Tatp demonstrated the involvement of the complete basic domain (Tat (48-60)) [20]. Since any deletion within residues 49-57 led to a reduced uptake [21], the generally accepted Tat PTD peptide is now regarded as residues 47-57 [22]. This is supported by mutational studies, whereby deletion or substitution of the three C-terminal Arg residues [23], or Ala substitution of any of the cationic residues between residues 48-57, results in dramatic decreases in cellular uptake [24]. 2.2. Homeodomain of Antennapedia (Antp)/Penetratin Derived from the third helix of the Antennapedia protein homeodomain from Drosophila, Antp [6, 7] is one of the most widely studied and best-understood CPPs. Homeodomains are regions of a specific sequence of 60 amino acids, found in the homeoprotein class of transcription factors that are responsible for binding to DNA [25]. The homeodomain is structured into 3
-helices, and although the entire 60-amino acid domain is capable of

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translocation, this is mediated entirely by amino acids in the third helix [6]. Specifically, the Antp PTD, also commonly known as Penetratin, consists of residues 43-58 of the homeodomain [13, 26] (see Table 1). 2.3. Herpes Simplex Virus Type 1 (HSV-1) protein 22 (VP22) VP22, one of the major structural components of HSV-1, has the ability to transport between cells [8]. In cells expressing VP22, the protein is found predominantly in the cytoplasm, where it colocalises with microtubules [27]. From here, it is exported from the cell and then taken up by surrounding cells, where it can accumulate in the nucleus [8, 16]. Within the HSV-1 virus life cycle, VP22 has many different functions and, by utilising a series of Green Fluorescent Protein (GFP)-tagged deletion constructs, these functions have been mapped to specific regions of the polypeptide as follows: intercellular transport, amino acids 81-195; binding and reorganisation/stabilisation of the cytoskeleton, amino acids 159267; inhibition of cytoskeleton collapse, amino acids 81-121; and nuclear targeting and facilitation of intercellular transport, amino acids 267-301 [27, 28]. 2.4. Transportan The 27 amino acid long Transportan is a chimeric CPP containing 12 functional amino acids from the N-terminus of galanin (a neuropeptide), bound to the membrane interacting wasp venom peptide, Mastoporan, via a Lys residue [12]. Transportan predominantly resides in membranous structures inside cells [29], but it can be taken into the nucleus where it appears to concentrate in the nucleoli [12]. Deletion analogues of Transportan, such as Transportan-10 which lacks the first 6 amino acid residues, have also been generated and retain much of the translocational ability [30] (see Table 1). 2.5. Model Amphipathic Peptide (MAP) The 18 amino acid model amphipathic peptide (MAP) is
-helical in structure and displays extensive uptake into several cell types [31]. This peptide has been shown to translocate across plasma membranes in both energy-dependent and energy-independent fashion (see Section 3). In kinetic studies comparing the cellular uptake and cargo delivery of several CPPs, MAP showed both the fastest uptake and the highest cargo delivery efficiency, followed by Transportan, Tat (48-60) and then Antp (43-58) [32]. By examining the influence of various CPPs on cellular 2-((3)H)deoxyglucose-6-phosphate leakage, they also demonstrated that the membrane-disturbing potential of the peptides correlated with their hydrophobic moment. 2.6. Histones One of the most recent additions to the CPP family are the core histone proteins [33]. The primary unit of DNA compaction in eukaryotic cells is the nucleosome core particle, where 146 bp of DNA are wrapped twice around a protein core, consisting of two of each of the four core histones; H2A, H2B, H3 and H4 [34]. The DNA is electrostatically bound to this histone octamer [35] and the intervening DNA between each nucleosome condensed further through the action of a fifth histone, H1 [36]. Due to their DNA binding and packaging ability histones have been examined as potential gene therapy vectors [37, 38], under the assumption that they enter cells via classical endocytotic mechanisms (see Section 3.1 below). Recently, however, it was shown that histone proteins are capable of direct translocation across plasma membranes [33]. H2A and H4 posses the strongest translocational ability, with H3 not far behind, while H2B demonstrated the lowest amount of cell-penetrating

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potential, both in whole cells and in uni- and multi-lamellar liposomes [10, 33]. Interestingly, core histones were also shown to penetrate the plasma membrane of petunia protoplasts, the first demonstration of the activity of CPPs in plant cells [11]. Importantly, histones of the linker variety (histone H1 and its homologues) have also been investigated for their CPP activity [39, 40], with one recent report demonstrating that nuclear delivery of proteins mediated by protein transduction through Histone-H1o could be significantly enhanced by the inclusion of a dynein-association sequence, resulting in enhanced binding to the microtubule network [41], suggesting that traffic through the cytoplasm is also an important factor in nuclear delivery of CPP cargo. 2.7. Other Cell Penetrating Proteins/Peptides Apart from those mentioned above, many other CPPs have been discovered or designed. These include; pVEC, an 18 amino acid peptide derived from the murine cell adhesion molecule vascular endothelial cadherin (amino acids 613-635), shown to be efficiently taken up by cells and accumulated predominantly in the nucleus [42]; and the PreS2-domain of the hepatitis B virus surface antigens, which translocate into cells through energy-dependent mechanisms, and subsequently localise diffusely throughout the cytoplasm [43]. Interestingly, the 26 amino acid peptide VT5, exhibits extensive cellular internalisation even though it forms a water soluble, amphipathic -sheet conformation [44], whilst D-substituted analogues with no propensity to form -sheets, show only ~5% translocation ability compared to VT5 itself. This is in stark contrast to the “classical”
-helical amphipathic CPPs (see Table 1B), but the authors suggest that VT5 internalisation may not follow the same precise pathway, as its uptake is temperature-, energy- and pH-dependent (see Section 3). Importantly, the N-terminal portion of the mouse prion protein (PrP), which has a strong tendency towards aggregation and forms -sheets in the presence of phospholipids, has also been shown to act as a CPP [14]. 3. MECHANISMS OF MEMBRANE TRANSLOCATION 3.1. Conventional Cellular Entry Mechanisms There are many ways in which a molecule can gain entry into a cell, some of which result in degradation of the ingested substance. The cellular uptake pathways, summarised in Table 2, include receptor-mediated endocytosis, pinocytosis, macropinocytosis and potocytosis (for a review see [45]). Since the pathways involve very different uptake mechanisms, the effects of inhibitors vary greatly (see Table 2), with their actions used extensively to define the pathways. Receptor-mediated endocytosis (see Table 2A) involves the recognition of a ligand by a cell surface receptor. Cellular uptake is then mediated through invagination of the plasma membrane to form a vacuole. This invagination is energy-dependent and relies upon clathrin, which lines the membrane ‘pits’ that are central to the process and mediates the invagination process and also involves microtubules and actin filaments. Pinocytosis (see Table 2B) is the mechanism through which a cell samples its extracellular environment by the budding of small vesicles from the cell membrane into the cytoplasm. This mechanism allows the cell to gain a representative sample of the molecules and ions in the extracellular fluid, or to replenish nutrients that may be in short supply. Only small molecules are engulfed by pinocytosis, which occurs continuously in almost all cell types.

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Macropinocytosis (see Table 2C), on the other hand, allows cells to engulf much larger molecules. In this regard, it is similar to receptor-mediated endocytosis, but is not reliant on a specific receptor. Instead, the formation of the endocytotic vesicles occurs in response to stimulation of a cell-signalling cascade involving RhoGTPases, resulting in the closure of lamellipodia at the sites of membrane ruffling to form large, irregular vesicles. The membrane ruffling in this process is primarily actin-driven and does not require clathrin. Macropinosomes have been known to undergo acidification and enter the endosomal pathway, although this is not always the case. Potocytosis (see Table 2D), also known as caveolae-mediated uptake, is a mechanism by which both small and large molecules can be sequestered and transported by caveolae, flaskshaped specialised regions of the plasma membrane which are characterised by the filamentous coat of caveolins that line their inner surface. They differ from endosomes in that they are not associated with clathrin and can deliver the internalised molecule to four different parts of the cell, bypassing the lysosome. There is some evidence to suggest that potocytosis is temperature independent [46]. 3.2. Receptor-independent Uptake The uptake of CPPs (see Table 2E) does not appear to require specific cell-surface receptors [13]; rather the overall charge of the peptide appears to be important. Indeed, structural studies of the Tat PTD have demonstrated the general importance of Arg residues in mediating translocation [47] (see Section 2.1). Added to this, the retro and inverso forms of Tatp, synthesised with either a reversed linear orientation or containing D-amino acids respectively, show translocational ability comparable to wild type peptide [9, 24], implying that the very specific conformational/steric constraints typical of ligand-receptor interactions do not apply to Tat. Similarly, D-enantiomers of Antp and pVEC show no decrease in internalisation, with the D-enantiomer of (Arg)8 actually showing enhanced uptake [24, 42, 48]. 3.3. Energy-dependent or Energy-independent Uptake Originally, the uptake of Tat, Antp and others was believed to occur in non-saturable, dose-dependent fashion. Protein translocation was initially viewed as non-endocytotic in light of the fact that no decrease in uptake efficiency is seen at 4ºC, when compared to 37ºC, implying an energy-independent translocation mechanism (see Table 2E) [20, 49]. Low temperature was thought to inhibit all forms of cell entry, but potocytosis has also been proposed to be a temperature insensitive uptake mechanism, although there is little direct evidence to support this [46]. In favour of a non-endocytotic mechanism of uptake are reports that showed no effect on CPP uptake in the presence of inhibitors of endocytosis [33, 50], including the microtubule disrupting agents nocodazole, taxol and colchicine; the filament disrupting agent cytochalasin D; brefeldin A, an inhibitor of trans-Golgi transport; the phosphatidylinositol-3 kinase inhibitor Wortmanin; the caveolae formation inhibitor nystatin; and chloroquine, an inhibitor of the acidification of endocytotic vesicles [33, 50-52] (see Table 2). Translocation also occurred in ATP-depleted cells and in cells incubated with 0.5 M sucrose, conditions that block endocytosis/pinocytosis [33]. Recently however, the energy-independence of CPP uptake has come into question. Some studies have applied a trypsin digestion step after application of the peptide, prior to visualisation of CPP localisation, the idea being that trypsin will remove any extracellular proteins that may interfere with the peptide, as well as removing any residual surface-bound

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Summary of Conventional Cell Entry Pathways Compared to Protein Transduction Name

A Receptor-mediated endocytosis

eg. Ligands such as Transferrin, Epidermal Growth Factor (EGF).

B Pinocytosis

eg. Molecules such as Low Density Lipoproteins (LDLs), Iron.

C Macropinocytosis

eg. Fibroblast Growth Factor (FGF)

Description

Characteristics

Ingestion of ligands after interaction with specific cell surface receptors into intracellular endosomes. These undergo acidification and eventually fuse with lysosomes to degrade the internalised material

Dependent upon:

Small molecules, usually liquids are ingested from the extracellular fluid through vesicles budding intracellularly from the plasma membrane.

Dependent upon:

Also important for membrane recycling

Caveolin

Engulfment of large macromolecule by invagination of the membrane through actin-driven ruffling

Inhibited by

Cell Surface Receptor Clathrin

Brefeldin A (Clathrin inhibitor).

Kinases

Wortmanin (PI-3K* inhibitor)

Acidic Compartment

Chloroquine (prevents

Energy

acidification of endosomes)

Temperature

Sodium azide, sodium fluoride

Cytoskeleton

(metabolic inhibitors) 4ºC

Independent of: Caveolin

Energy

Nocodazole, taxol, colchicine and cytochalasin D (target the cytoskeleton)

Temperature

Sodium azide, sodium fluoride (metabolic inhibitors)

Tubulin

4ºC Colchicine (targets tubulin)

Independent of: Cell-Surface Receptors

Others

Clathrin

Bacitracin and monensin (antibiotics that inhibit pinocytosis, thought to be related to Ca+/Mg+ binding ability)

Acidic Compartment Kinases

Yeast mannans and phosphorylated sugars (inhibit pinocytosis possibly through competition)

Dependent on: Actin

Cytochalasin D (targets actin)

RhoGTPases

RhoGTPase inhibitors, such as C. difficile toxin B.

Energy Temperature

Sodium azide, sodium fluoride (metabolic inhibitors) 4ºC

Independent of: Cell Surface Receptors Clathrin Caveolin

Others ethylisopropylamiloride (EIPA) and dimethylamiloride (DMA) (inhibit the formation of macropinosomes)

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Table 2. contd….

Name D Potocytosis

eg. Retinol Binding Protein, Simian Virus 40 (SV40)

Description Transport of small and large molecules into cells, bypassing the lysosome. Delivery to four internal cellular destinations

Characteristics

Inhibited by

Dependent upon: Caveolae

V-type proton ATPases Lipid Rafts Energy

Independent of: Temperature (?)

Sterol-binding agents such as nystatin, filipin and digitonin (disassemble caveolae) Activators of protein kinase C such as PMA* (inhibit caveolae internalization) Bafilomycin A1, (inhibits vacuolar ATPases)

Sodium azide, sodium fluoride (metabolic inhibitors)

Cell Surface Receptors Clathrin Actin E Protein Transduction

eg. Transportan, Histones

Direct translocation through cell membrane. Precise mechanism unknown

Dependent upon%

Unknown

Basic residues Amphipathic
-helices Heparin Sulfate

Independent of: Energy Temperature# Cell Surface Receptors Clathrin Caveolin

%

See Section 3 See however [48, 51] *Abbreviations: PI-3K, Phosphotidinositol-3-Kinase; PMA phorbol-12-myristate-13-acetate

#

peptide not displaced by washing [52]. Using this protocol, a strong reduction in Tatp uptake was observed, as measured by Fluorescence Assisted Cell Sorting (FACS) [53]. This indicates that previous FACS measurement of the internalisation of CPPs may have overestimated the extent of translocation by including surface-bound peptides in the analysis [52]. Further to this, low temperature incubation followed by trypsin treatment showed little to no cellular internalisation of CPPs. This implies that the previously reported temperatureinsensitivity of CPP uptake may in fact be an artefact, due to extracellular binding of peptide, rather than membrane translocation [53]. Studies using the trypsin approach demonstrated that the kinetics of Tat uptake fits with those of cell entry of molecules that are nor-

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mally taken up by endocytosis. In accordance with this, one study found that the delivery of Tat-oligonucleotide complexes was mediated by endocytosis, as it was inhibited at 4ºC, and in the presence of metabolic inhibitors such as sodium azide, sodium fluoride and antimycin A, as well as increased in the presence of chloroquine [54] (see Table (2)). Fluorescence microscopic examination observed temperature dependent localisation of either Tatp or Antp bound to oligonucleotides in perinuclear vesicles having the hallmarks of endosomes, indicative of endocytosis [55]. It is worth noting at this point that this study utilised trypsin to remove adherent cells prior to FACS, in a similar fashion to the above study. In general, the contribution of extracellular binding of peptide, classical endocytosis and true protein transduction mechanisms to the observed cellular uptake of CPPs is not entirely clear. Whether the afore-mentioned effects hold true for all of the diverse CPP family members remains to be seen. 3.4. Is Translocation Merely an Artefact of Fixation? Recently, it has been reported that both VP22 and Histone H1 can adhere to the cell membrane of living cells, with membrane disruption upon fixation allowing them to relocate to the cell cytoplasm and nucleus [14]. Such a change in localisation upon fixation was also seen for Tatp and Tat-mini-antigen fusion protein constructs [53, 56]. Even paraformaldehyde, which is a relatively mild means to fix cells, has been shown to result in an artefactually high level of translocation [53]. This suggests that the protein transduction observed in many cases may simply be an artefact of cell fixation, not reflecting the live cell situation. This does not explain the induction of biological effects after uptake of biologically active molecules fused to CPPs, however. An elegant example is experiments using a transducing, modified, apoptosis-promoting caspase-3 protein, Tat-Casp3, that has HIV proteolytic cleavage sites substituted for its endogenous ones [57]. Tat-Casp3 was shown to transduce nearly 100% of cells, but remains inactive in uninfected cells. In HIV-infected cells, however, Tat-Casp3 is processed into an active form by the HIV protease, resulting in efficient apoptosis of such cells, without affecting the uninfected cell population [57]. This is a clear demonstration of the intracellular functionality of a CPP-delivered protein, in the absence of cell fixation procedures. Furthermore, numerous reports have demonstrated the uptake of CPPs and/or the corresponding biological response in living cells by fluorescence microscopy and in vivo experiments [10, 33, 58, 59]. Nonetheless, it is clear that fixation should be avoided if at all possible when dealing with CPPs, and that demonstration of an expected biological outcome, such as the expression of a protein or downregulation of a gene, can be considered as the gold standard for confirming intracellular delivery of the attached cargo [60, 61]. 3.5. Important Features Required for Membrane Translocation When comparing the physico-chemical characteristics of the various CPPs, a consistent finding is the high content of basic amino acids and a resultant positive charge [61]. As discussed briefly above, cationic residues have been shown to be critical for protein transduction [62, 63], but positive charge alone is not enough. Using polyArg peptides of various lengths, Futaki et al. [9] demonstrated that there is an optimal number of residues (~8) for efficient translocation, with greater chain lengths not aiding, but rather hindering uptake, as shown for the (Arg)16 peptide, which shows no cellular uptake. It was therefore concluded that the guanidinium group of Arg was therefore important for translocation [24, 47], and further, that the distance between this group and the peptide backbone is critical [9, 24].

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This of course, does not account for the strong translocating ability of Lys-rich peptides with little or no Arg content, such as MAP or Transportan [12, 44] (see Table 1B). Secondary structure is also important for translocation, with many CPPs adopting, or predicted to adopt, an
-helical structure, including KALA and the Tatp [64, 65]. This does not, however, appear to be a critical requirement. Although the parental Antennapedia homeodomain adopts and maintains an
-helical conformation, Antp has been shown to adopt a -sheet conformation in the presence of a charged lipid monolayer [66]. Similarly, an amphipathic -sheet peptide displayed potent PTD qualities, in that it remained in soluble form and was efficiently taken up into cells [44]. Consistent with reports that
-helix conformations can be maintained and even promoted by the presence of SDS micelles [13, 67], Transportan has been observed to adopt a random coil conformation when in aqueous solutions, but becomes mainly
-helical in the presence of SDS [68]. Similarly hCT(9-32), a CPP derived from human calcitonin is unstructured in aqueous solutions, but adopts two short helical stretches when bound to dodecylphosphocholine micelles [69]. Pep-1, a synthetic CPP with an
-helical trytophan-rich N terminus, has been shown to interact with both micelles and protein cargo via its tryptophan-rich domain [70]; a C-terminal cysteamine group was found to offer additional anchorage to the membrane surface, leading to more efficient cellular translocation, a property also seen with hCT(9-32) mutants [69, 70]. Yet further studies maintain that Tat fusion proteins must first be unfolded in order to promote efficient uptake by cells [71, 72]; unfolding may favour access to the cell membrane to promote internalisation. 3.6. Proposed Models of Membrane Translocation Three models have been put forward to explain the mechanism of cellular uptake utilised by CPPs. The inverted micelle model (Fig. (1A)) was proposed from NMR studies on the interaction of Antp with phospholipid membranes [49]. The first step of translocation involves the positively charged CPP interacting with, or recruiting, the negatively charged heads of the membrane phospholipids into the membrane vicinity. A reorganisation of the lipid bilayer around the CPP then occurs, aided by interactions of the hydrophobic residues of the CPP with the membrane. This induces the formation of an inverted micelle, in which the hydrophilic centre pocket entraps the CPP. Intracellular release is then affected by an analogous set of reactions in reverse. Although this model may explain translocation of some CPPs, it is not sufficient for peptide/proteins that do not contain the hydrophobic amino acids necessary to mediate the lipid interactions of the translocation process (eg. polyArg) [13]. Moreover, the large size of some of the transported cargoes (eg. a 45 nm diameter ferromagnetic particle [73]) argues strongly against the formation of such particles. Also, intermediate membrane structures such as those shown in Fig. (1A) have never been observed microscopically [52]. There is some evidence, however, to suggest that the entry of Antp into liposomes involves a trans-membrane electrical potential, which may act in a similar fashion to the initial steps of this model [74], where it was shown that the positively charged peptide initially accumulated on the surface of the membrane. This asymmetrical distribution of the peptide between the outer and inner surfaces of the charged lipid bilayer led to a transmembrane electrical field, which presumably altered the lateral and curvature stresses acting within the membrane and resulted in an electroporation-like permeabilisation of the membrane. Further binding of the CPP caused the electrical field to reach a critical threshold value, ultimately inducing CPP uptake, presumably via the formation of inversely curved membrane structures, similar to those proposed in the inverted micelle model [75].

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A

Wagstaff and Jans

B

CPP

C

(a) (b)

(a) CPP

CPP

(c) (b)

(c) CPP

CPP

(d)

(c) CPP

CPP

CPP

(d) CPP

(d)

CPP

(e)

Fig. (1). Proposed models of membrane translocation mechanisms: A is based on NMR studies, B and C are based on the membrane translocation of antimicrobial peptides [49, 74, 75]. A. Inverted micelle model: The positive charges present in the CPP interact with and recruit negatively charged phospholipids in the cell membrane (a). The hydrophobic part of the CPP then interacts with the membrane (b), generating an inverted micelle (c). The hydrophilic cavity can then accommodate the protein and allow release into the cytoplasm (d). B. ‘Carpet’ model: (a) The CPP first interacts through its positive charges with the negative charges of the phospholipids, causing a change in secondary structure as the basic residues turn themselves towards the membrane. (b) The CPP then rotates, allowing hydrophobic residues to interact with the hydrophobic core of the membrane. (c) A small disruption in the lipid membrane then permits the CPP to be internalised, presumably followed by membrane closure (d). C. Pore-formation model: The CPP first interacts with the membrane as per B (a/b). Amphipathic
helices in the CPPs then form into bundles (c), thus creating a pore within the membrane (d), where the hydrophobic residues interact with the lipid membrane and the hydrophilic residues line the pore, allowing other peptides to pass through. (e) The pore formation is speculated to be transient.

The carpet model (Fig. (1B)) is based on the known translocation mechanism of some antimicrobial peptides [76], but has been proposed to apply to CPP translocation in the case of certain CPPs, based mainly on their toxicity at high concentrations [13, 76]. The CPP is proposed to interact initially with the negatively charged phospholipids; this causes a change in the CPP secondary structure as the basic residues turn towards the membrane surface. The CPP then rotates so that its hydrophobic residues can interact with the hydrophobic membrane core. Finally, a small disruption of the lipid membrane surface occurs, allowing the peptide to pass through and into the cytoplasm, leaving the membrane to presumably close behind it. Although this mechanism has been suggested for CPP entry, its reliance on hydrophobic residues seems to preclude its application to most CPPs, as they are predominantly basic (see Table 1). It has clear value as a mechanism by which antimicrobial peptides exert their toxicity, however [13, 76].

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As for the carpet model, the pore formation model (Fig. (1C)), is based on one of mechanisms by which antimicrobial peptides appear to weaken bacterial membranes [77]. Amphipathic
-helices within the CPPs are proposed to form bundles on the membrane surface. Following this, the outwardly facing hydrophobic residues interact with the lipid membrane, allowing the inwardly facing hydrophilic residues to form a pore through which other peptides can pass. Some amphipathic basic peptides such as mastoparan and magainin have been shown to form helical structures in the cell membrane and to assemble as complexes to form pores [50, 78-80]. This pore formation leads to a leakage of the intracellular contents and resultant high cytotoxicity. HIV Rev (34-50), which contains an Arg-rich basic domain similar to Tat, has been suggested to form helical structures in the membrane, much like mastoparan [9]. However, no significant membrane perturbation was seen when cells were treated with this, or a number of other Arg-rich peptides [50] and no significant cytotoxicity was associated with internalisation of these peptides. This indicates that if this mechanism is a means by which CPPs enter cells, then the pores must be transient and the membrane must repair itself afterwards. Although all three mechanisms potentially explain membrane translocation, none are satisfying in terms of explaining the translocation by all CPPs, and none of them are able to satisfactorily describe how these peptides can translocate cargoes 100-fold their own size [13]. Clearly, much further research is required to establish the mechanism of CPP translocation through membranes, and discount the very real possibility that CPPs use multiple different mechanisms (see above and below) and that they may behave differently when complexed with cargo molecules. To this end, evidence in favour of other mechanisms of uptake is beginning to mount. 3.7. Enhanced Endosomal Escape Overwhelming support for endocytotic uptake of CPPs has been generated recently, at least for Tatp and Antp. It was demonstrated that Tatp or Antp bound to oligonucleotides enter cells in a mechanism indistinguishable from endocytosis [55]. Similarly, plasmid DNA translocation into cells mediated by oligomers of Tatp, was found to occur by endocytosis [54]. Several reports have shown that Tatp demonstrated enhanced binding to the cell membrane, subsequently resulting in endocytosis and accumulation in endosomes [56, 61, 63]. Tat-fusion proteins also seem to enter the cell via an endosomal pathway, but circumvent degradation in the lysosome and are ultimately sequestered in the nuclear periphery [81]. In support of this, the synthetic CPP, ppTG1, has demonstrated extremely efficient membrane translocation in HeLa cells, which appeared to be due to endosomal uptake combined with enhanced liposomal escape as demonstrated by in vitro liposomal leakage assays [82]. These studies demonstrated that ppTG1 forms a random coil structure at neutral pH, but in acidic conditions, such as those found in late endosomes, it converts to an amphipathic
helix and inserts into the liposomal membrane to cause membrane disruption and leakage of vesicle contents. Therefore, it seems possible that rather than follow the mechanisms illustrated in Fig. (1), the cationic residues of Tatp initially confer interaction with negative charged moieties at the membrane surface, such as phospholipids or extracellular proteins. Following this, Tatp is internalised through conventional endocytosis, subsequent to which it manages to circumvent lysosomal degradation and escapes into the cytoplasm [52]. Whether these endosomolytic properties, rather than protein transduction, hold true for other CPPs is yet to be shown.

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3.8. Multiple Mechanisms of Membrane Translocation In contrast to the above, much evidence supports the idea that many CPPs do not enter the cell via endocytosis [33, 50], consistent with more than one mechanism being involved in the translocation of CPPs. It may be that the apparently conflicting endocytosis versus non-endocytosis reports are explicable in terms of the fact that both endocytotic and nonendocytotic mechanisms work in parallel, both contributing to translocation to differing extents, depending on the CPP [13, 60]. Alternatively, considering the vast physicochemical diversity among the different classes of CPPs, it is possible that distinct cellular uptake mechanisms operate for different CPPs [13]. For example, Tat-mediated intracellular delivery may proceed via energy-dependent macropinocytosis, with subsequent enhanced endosomal escape [83], whilst other CPPs seem to penetrate cells via charge interactions and hydrogen bonding, without an energy requirement [1, 84]. In all cases, it seems clear that direct contact between the CPP and the cell surface are essential for successful cellular uptake [1]. The role of cell surface proteoglycans such as heparin sulfate, in membrane translocation, and whether they may act like cell surface receptors for the endocytotic/ macropinocytotic uptake remains unclear [50, 85, 86]. 4. THERAPEUTIC APPLICATION OF CPPS Since the cell membrane represents a major barrier to the delivery of numerous pharmaceuticals, CPPs are very promising potential delivery vectors. They have already been utilised to deliver a wide range of cargoes into mammalian cells, both in vitro and in vivo [1]. CPPs often have many desirable features for cellular delivery, such as efficacy in vivo, nuclear targeting, applicability in a wide variety of cell types and no apparent cargo size restrictions or immunogenic, antigenic or inflammatory properties [13]. They also appear to be able to deliver cargo such as peptide nucleic acids into bacterial cells [87] and can efficiently translocate mycoplasma membranes [10]. These properties make CPPs ideal for use in a wide range of therapeutic applications, some of which are covered in the remaining sections. 4.1. Cargo Considerations 4.1.1. Cargo Size CPPs have been used to deliver numerous classes of cargo, including DNA, proteins, drugs and nanoparticles, most of which are many times larger than the CPP itself [1]. However, it has not yet been conclusively determined whether the coupling of a CPP to a cargo, particularly a sizeable one, interferes with the translocation mechanism [88]. Some studies suggest that the coupling of a CPP to a high-molecular weight cargo forces it into an endocytotic route of entry, regardless of whether this is the case for the CPP alone [54, 61, 85, 89]. In such a circumstance, the CPP would therefore appear to mediate mainly the binding of the CPP/cargo to the cell membrane [88]. 4.1.2. Coupling of CPP to Cargo An important consideration is the nature of the CPP-cargo attachment, some common examples of which are illustrated in Fig. (2). The link between the two is usually a covalent bond, with, in these cases, the orientation of the CPP and cargo major considerations [88]. A common approach is the use of suitable amino acid side chains such as the thiol group of Cys or the amino group of Lys [12, 90]. Of these, disulfide bonds are the most common and have the added benefit of being rapidly reduced in the intracellular environment, enabling

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release of the cargo [12]. Other stable, covalent linkages that have been employed include amide, thioether, thiolmalmeide, thiazolidine, oxmine and hydrazine linkages, as well as bifunctional cross-linkers such as SMCC [88, 90-92]. If the cargo is a peptide/protein, then the two can be produced as a single chimeric protein in bacteria [92, 93].

Covalent Linkages Genetic Linkage

A

Non-Covalent Linkages

Side-chain Linkages

B

F H

CPP

CPP

Cargo

C

N

CPP Cargo

O

C

G CPP

S

S

Cargo

CPP

Cargo

D H S

CPP

N H

Cargo

E CPP

S

SMCC

Cargo

Fig. (2). Common methods of CPP cargo attachment; Covalent bonding techniques include: A. Generation of fusion proteins in bacteria B. Peptide bond formation (similar to A, but for synthetic peptide CPPs and cargo) C. Disulfide bridge formation D. Thiazoline ring formation E. Bifunctional linker molecules, such as SMCC (Succinimidyl 4-(N-maleimidomethyl)cyclohexane1-carboxylate). Non-covalent bonding techniques include: A. Electrostatic interactions between cargo (usually nucleic acid) and CPP B. “Piggy-back” attachment: Non-covalent attachment of a large cargo molecule to a smaller cargo (represented by the dark circle), that is covalently attached to the CPP.

Non-covalent attachments can also be employed, most commonly for nucleic acid delivery. In this case, an electrostatic interaction between the positively charged CPP and the negatively charged nucleotide-phosphate backbone effects complexation [1, 90]. Alternatively cargo and CPP can be cross-linked individually to biotin and streptavidin, and subsequent attachment conferred by the streptavidin-biotin interaction (Fig. (2G)) [94]. When utilising non-covalent bonding to deliver cargo by CPP, an important consideration is the stoichiometry of chemical complexation and physical formulation and how this may affect translocation into the cell [88]. 4.2. Application of CPPs in Gene Therapy To treat genetic disorders through introducing therapeutically active genes that overcome debilitating loss-of-function mutations has long been a goal for medical science. A major limitation, however, is the delivery of DNA across biological membranes, including both the plasma membrane and subsequently the nuclear membrane, whilst minimising toxicity to the cell. Although viral gene delivery vectors have been commonly used due to their efficient gene transfer capabilities, many safety considerations have hampered their use as

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therapeutics, including pathogenicity, oncogenicity and stimulation of immunological responses in the host [95]. Non-viral vectors, on the other hand, are relatively safe to use, but their inefficiency is an ongoing problem. CPP-based delivery systems have emerged as strong possibilities to facilitate non-viral gene therapy; they have also been applied to the enhancement of viral gene therapy, with limited success [96]. 4.2.1. Synthetic CPPs for DNA delivery To overcome the inefficient gene transfer mediated by non-viral based vectors, synthetic CPP delivery systems have been developed. Synthetic CPPs have been designed which can both condense DNA and transport it across the lipid bilayer into the cell, either directly, in the case of amphipathic CPPs, or via endosomes, in which case the CPP mediates plasmid release by destabilising the endosomal lipid bilayer at low pH [97]. Some of these, such as the synthetic membrane destabilising KALA peptide [65] and (LARL)6 [98], have proved relatively successful in the transfection of different cell lines in vitro. However, the increased cytotoxicity of these peptides and their inability to mediate DNA transfer into primary cell lines has limited their use in vivo. New basic amphipathic CPPs have been designed, with gene delivery to animals being achieved using a single-component peptide vector, although this system most likely utilises endosomal uptake and endosomolytic activity for enhanced entry to the cell [82]. These peptides (designated ppTG1 and ppTG20) were found to be superior to KALA in cell culture experiments, leading to transfection efficiencies comparable to polyethylenimine (PEI) and Lipofectin, especially at low DNA doses. Structure-function studies suggest that the high gene transfer efficiency of these peptides correlates with their ability to form
-helical conformations at low pH, which is largely influenced by the nature of the hydrophobic amino acids within the peptides [64, 65, 82]. This conformation enables the CPP to interact with the endosomal membrane, causing release of the plasmid into the cytoplasm [60, 82]. This is in agreement with earlier reports that demonstrate that cationic
-helical peptides bound to DNA are internalised via the endocytotic pathway in vitro [98] (see also Section 3.7). As electrostatic interactions between the DNA and the CPP may interfere with the protein transduction process, synthetic peptides, designed as branching complexes were also investigated [99]. The 8-tat peptide, consisting of 8 arborising Tat moieties, was capable of transfecting both dividing and non-dividing endothelial cells, but unfortunately these levels, especially in non-dividing cells, were well below those obtained using the commercial transfection reagent LipofectAMINE. 4.2.2. Delivery of Oligonucleotides by CPP Much of the work in terms of nucleic acid delivery by CPPs has thus far focused on the delivery of oligonucleotides (ONs), and in particular antisense ONs for gene silencing [60]. Antisense therapies are based on ONs that can hybridise, in sequence-specific fashion, with complimentary mRNA strands in the cytoplasm, causing translational arrest or the recruitment of RNaseH [100]. The end result is the downregulation/silencing of gene expression. As the cellular uptake of ONs themselves is relatively poor, coupling to CPPs can significantly increase their efficiency [2, 97]; for a review on the preparation of ON-peptide conjugates, see [101]. Peptides are most commonly coupled to ONs via electrostatic binding interactions [65, 97], rather than hydrophobic or covalent bonding, although di-sulfide linkages have also been used [12, 58], and may in fact have advantages as the linkage can be readily reduced in the intracellular environment [12, 32]. ONs have been modified in may ways, with changes to the sequence, chemical group modification and the use of nucleotide analogues resulting in varying degrees of antisense

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activity. Above all, it has been shown that modification of the sugar-phosphate backbone of the ON plays an important part, not only in the gene silencing, but also in efficient membrane translocation [62]. Improvements to ON modifications are constantly being developed [100], the use of ONs containing nucleotide analogues overcoming many of the degradation issues plaguing naturally occurring ONs. For example, morpholinos, which are modified ONs, have standard nucleic acid bases, but are bound to morpholine rings instead of deoxyribose rings and linked through phosphorodiamidate groups instead of phosphates [102]. This makes them completely resistant to nuclease degradation and prevents them from activating innate immune responses. Several CPPs, bound to ONs have been shown to be efficiently delivered into cultured mammalian cells [12, 97, 103, 104] with genes such as c-myc, bcl-2 and glyceraldehyde 3phosphate dehydrogenase (GAPDH) [105-107] having been targeted in this fashion. In an elegant study, VP22 (residues 159-301) was found to assemble with ONs into regular spherical particles, termed Vectosomes, that entered cells efficiently in 2-4 hours and were stable in the cell cytoplasm for several days [108]. Remarkably, when these cells were exposed to a bright light, the Vectosomes were disrupted and released the protein and ON into the nucleus and cytoplasm within seconds. This process was captured by time-lapse microscopy and was later demonstrated to be just as effective in vivo in the delivery of anti-c-Raf1 ONs to subcutaneous tumours implanted into nude mice [104]. One drawback to this process may be that VP22 can elicit an immune response in immunocompetent hosts, which may either neutralise the effect of repeated injections or prime an antitumoural effect favourable to tumour regression [104]. A sophisticated approach to examine the delivery of ONs for anti-sense therapy involves the blockade of an aberrant alternative splice site with an ON, thereby inducing the correct translation of a reporter gene, namely either GFP or firefly luciferase [55, 62, 109]. This technique has been utilised either in cultured mammalian cells, or in transgenic mice to examine ON delivery to organs in vivo [62]. In both cases, peptide-ON conjugates targeted to the aberrant splice site, but not mismatched controls, causing an increase in reporter gene expression, which is not observed with ON alone. The use of PNAs (Peptide Nucleic Acids) is common in CPP-mediated antisense delivery, possibly because the peptide backbone of the PNA allows the CPP-PNA construct to be synthesised as a single polypeptide [60, 101]. There is evidence to suggest, however, that synthesis of the peptide-PNA in this fashion may interfere either with the CPPs’ ability to translocate across membranes, or with the PNA-mRNA interaction [60]. Therefore, more often than not, PNAs are coupled to CPPs via a disulfide bridge [29, 110]. Both Transportan and Antp (43-58) were used in this way to deliver an antisense PNA complementary to the human galanin receptor type 1 mRNA in vitro and in vivo [12], resulting in significant suppression of the galanin receptor expression in cultured Bowes melanoma cells, as well as in the spinal cord of rats, leading to a modification of the pain response. This approach has also been extended such that the CPP is coupled to the antisense PNA via DOTA (a derivative of 1,4,7,10-tetraazacyclododecane-N,N’,N’,N’’-tetraacetic acid), which incorporates macrocyclic radiometal chelates into the conjugate, enabling it to be traced using radioimaging approaches [105]. CPPs conjugated via electrostatic interactions to short interfering RNA (siRNA) have also been used to promote robust down-regulation of target mRNAs [107]. 4.2.3. Suicide Gene Therapy Approaches Suicide gene therapy is a widely exploited approach for the treatment of cancers and other hyperproliferative disorders [1]. It involves the introduction into target cells of a gene

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encoding an enzyme that converts a harmless prodrug into a potent cytotoxic agent [111]. Whilst various enzyme/prodrug combinations have been developed, the most widely used is the HSV-1 thymidine kinase (TK)/ganciclovir (GCV) combination [111, 112]. Despite its popularity, clinical successes have been hampered by the inefficiency of TK gene transfer and its limited effect on bystander cells [1]. Amplification of the cytotoxicity by co-transfer of the TK gene fused to a CPP has proved very successful. In cell culture, VP22-promoted spreading of TK resulted in killing of virtually all cells in cultures that only had a transfection efficiency of 27.5% [111]. This demonstrates that VP22 enhances both the intercellular trafficking and killing effect of TK, especially in the lower range of GCV concentrations. Similar results were seen using the Tat PTD [112], indicating that fusion to CPPs may constitute a viable approach in the optimisation of TK suicide gene therapy, towards its efficacious use in a clinical setting. 4.2.4. Plasmid Delivery by CPP The report that Tat PTD, when expressed as a fusion protein on the surface of recombinant
-phage particles, can induce significant expression of marker genes, with no harmful effect on the cells [51], introduced the possibility of using PTDs for the in vivo delivery of therapeutic genes [1]. One recent study added polyLys residues to the Tat PTD (Tat-pK) to enhance gene delivery [113]. The resultant polypeptide possessed DNA binding ability and was able to mediate delivery of a reporter gene to cultured cells. Significantly, since delivery was enhanced in the presence of chloroquine or ammonium chloride, it appears clear that uptake of the plasmid DNA is via the endocytotic pathway (see Table 2A), rather than protein transduction. Previously, however, it was demonstrated that various viral derived peptides, including Tat (48-60) and FHV coat (35-49), were able to transfect Cos-7 cells as efficiently as polyLys or poly-Arg [9, 114]. Further, N-terminal stearylation of these peptides, including octoArg peptides, resulted in a 100-fold increase in transfection efficiency, that was not further enhanced by chloroquine [114]. This implies that CPPs modified to include this fatty acid group, can mediate cell entry of DNA via a pathway that is not endocytosis driven, although it should be stressed that it is not clear whether this modified CPP is taken up by the same mechanism as unmodified CPPs. Another study demonstrated that Tat (47-57) alone has DNA-binding ability sufficient to induce efficient transfection of plasmid DNA into cultured mammalian cells, which however appeared to be mediated by the endocytotic pathway [89]. Moreover, intravenous injection of the positively charged DNA-Tat complexes into C57BL/6 mice resulted in very low levels of gene expression in the liver. This was attributed to inactivation of the DNA-Tat complexes by serum proteins in vivo [89]. Finally, one group have generated a novel peptide-based delivery vector (MPG), derived from a chimera of the hydrophobic fusion peptide of HIV-1 gp41 and the hydrophilic nuclear localisation sequence of SV40 large tumour antigen (T-ag), that is non-cytotoxic, insensitive to serum, and importantly, able to deliver plasmids efficiently into several cell lines, in less than 1 hour [115]. MPG interacts strongly with nucleic acids, most likely forming a peptide cage around them, which can both stabilise and protect them from degradation in cell culture medium. Although this peptide has been studied quite extensively [97, 107, 115, 116], it has yet to be tested in whole animal experiments, which of course is essential to confirming its potential in therapeutic applications. As most of these studies were performed with either the Tatp or full length Tat protein and seemed to involve endocytotic-mediated uptake, further work in this area is required to

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demonstrate conclusively that DNA can be efficiently delivered by protein transduction both in vitro and, more importantly, in vivo. It may be the case, that although CPPs can mediate the cellular uptake of smaller cargoes by protein transduction, coupling to plasmid DNA may result in complexes that can only enter the cell via endocytosis, or some other mechanism. VP22, however, has been utilised to increase the efficiency of myocardial gene therapy [1]. Based on the ability of VP22 to mediate intercellular trafficking of proteins to which it has been genetically linked [117, 118], DNA encoding a VP22-LacZ fusion protein, was injected into rat myocardium in vivo [16]. Histochemical staining showed expression of
Gal in the cytoplasm of cardiomyocytes surrounding the site of injection in both LacZ and VP22-LacZ injected hearts. Interestingly, in VP22-LacZ samples only, cardiomyocytes surrounding this region showed
-Gal staining in their nuclei, which was presumed to be due to secondary intercellular trafficking of VP22-LacZ into neighbouring cells and accumulation in the nucleus. Although this approach is not strictly VP22-mediated gene delivery, it highlights other potential uses of CPPs to enhance the gene therapy process. Targeting of fusion proteins specifically to the nucleus may be a limitation of this technique, but one can imagine it would be useful in enhancing the delivery of nuclear acting proteins, such as transcription factors [16]. Using this approach DNA encoding VP22 fused to the tumour suppressor gene p27 demonstrated significant spreading in cell culture, whilst intratumoural injection of the naked DNA resulted in significantly enhanced antitumour activity, compared to p27 alone-encoding plasmids [104]. This demonstrates that VP22 has important application in cancer gene therapy, as part of the encoded reporter gene product, in delivering the therapeutic gene product to bystander cells in the target tissue. Due to their CPP activity and also their ability to bind to and compact DNA, protecting it from degradation by nucleases, histones have been investigated by many as potential vectors for non-viral delivery of DNA [119]. In particular, histone H2A and H3 have been utilised for gene delivery to intact mammalian cells in culture [37, 120-122]. More recently, engineered histone H2B proteins, optimised for binding to the cellular nuclear import machinery, were demonstrated to deliver DNA to intact cells more efficiently than traditional liposomal techniques, either as monomers, or in a dimeric complex with histone H2A [123]; a process coined histone-mediated transduction (HMT). In similar fashion it has also been demonstrated that histone proteins engineered for efficient nuclear targeting can deliver DNA in reconstituted chromatin form, resulting in gene delivery to intact cells that is ~6fold more efficient than liposome-mediated transfection [124]. These reports highlight the importance of enhancing the nuclear delivery of DNA delivered by CPPs [119]. For an indepth review of the use of histone proteins in gene delivery see [119]. 4.2.5. Further Considerations for Gene Delivery 4.2.5.1. DNA compaction/Protection A major disadvantage with the use of non-viral gene delivery vectors is that the DNA is susceptible to degradation by nucleases, thereby greatly decreasing the likelihood that the therapeutic DNA will reach target cell nuclei in intact form [125]. DNA delivery by CPP does not appear to be immune from this issue, especially when the PTD is coupled to the nucleic acid through disulfide bonding [58], rather than complexed to it through either electrostatic or hydrophobic interactions. However, it has been demonstrated that oligomers of the Tat (47-57) peptide can compact DNA into nanometric particles and protect it against

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nuclease degradation [54]. Compacting the DNA should have the added benefit of facilitating easier passage into the cell, but this may not always be the case. It has also been argued that electrostatic binding of the peptide to the DNA [65, 89] may lead to particles that are inappropriate for membrane penetration, i.e. particles that are simply too big [54]. 4.2.5.2. Transport to the Nucleus In order for therapeutic DNA to be expressed, it must first gain entry to the nucleus. Molecules > 45kDa in size such as DNA are excluded from the nucleus unless they possess a specific targeting signal, known as a nuclear localisation signal (NLS) [126, 127]. These NLSs mediate specific interactions with the nuclear import machinery, and in particular with members of the importin (Imp) protein superfamily [128-130]. NLSs are usually comprised of one or two short stretches of basic amino acids [131], also typically important in the process of protein transduction (see above; Table(1)) [1]. It is thus not surprising that many CPPs, including the Tat PTD [17] and Antp, also retain NLS function that may aid in the delivery of DNA to the nucleus. To this end, it has been shown that Tat (47-57), complexed to DNA, promoted 3-fold more efficient transfection in cell-cycle arrested cells, when compared to PEI transfection in proliferating cells [54]. This data, along with confocal images of the Tat-DNA complexes in cells, suggests that the Tat NLS is involved in the enhancement of gene transfer in this system. The use of NLSs to enhance non-viral gene therapy has been quite extensively investigated [125]. Linking the T-ag NLS via a PNA to plasmid DNA encoding GFP or LacZ led to an 8-fold increase in the uptake of the plasmid, when delivered by PEI [132]. Due to the high content of basic residues in the T-ag NLS (PKKKRKV132), it was suggested that the Tag NLS may itself function as a CPP [60], but the T-ag NLS-PNA has been shown not to be sufficient to mediate cellular uptake of the vector on its own [132]. Despite this, conjugation of efficient, optimised NLSs to CPPs is an exciting possibility to enhance the gene transfer mediated by these proteins. Indeed, the MPG peptide, which contains the T-ag NLS as part of its construction, enters the cell in endocytosis-independent fashion and effectively mediates interaction with Imps to effect nuclear targeting [107]. In contrast, a mutated non-functional version of this peptide, had significantly reduced nuclear import potential, but could still be utilised effectively in the delivery of siRNA to the cytoplasm of cultured mammalian cells. Fig. (3) depicts an optimised scheme for the efficient delivery of plasmid DNA into cell nuclei, incorporating both CPP and NLS activity, with the end result being increased transcription of the therapeutic gene due to elevated nuclear amounts of the DNA. A system similar to that depicted has been developed recently, with histone H2B serving as the DNA binding and compacting agent, as well as mediating the cellular entry, by including an optimised version of the T-ag NLS in the engineered histone, very efficient gene delivery was achieved [123, 124]. 4.2.5.3. Cell-type Specific CPPs A potentially major drawback in the use of CPPs in vivo is their lack of specificity, in that they are able to transduce almost every cell type tested in vitro and are distributed to multiple organs in vivo [59, 60]. Ideally pharmaceuticals should be delivered to specific target cells only, but thus far, there is little evidence for the cell specificity of CPP-mediated translocation. In the past, cell type specificity has been achieved by coupling cell-type specific ligands to the non-viral gene delivery vector [133, 134], but this of course results in entry of the vector into cells via endocytosis rather than translocation.

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Plasmid DNA

1 Plasmid

DNA

CPP

2

Plasmid DNA

CPP

Extracellular

3

Cytoplasm Plasmid

Plasmid

DNA

DNA

CPP

4

CPP

Importin

Resistant to Degradation

5 Ran-GTP

CPP Plasmid DNA

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6

Fig. (3). Scheme for CPP-mediated nuclear gene delivery. (1) The CPP binds to a therapeutic plasmid and condenses it. The CPP then binds to the plasma membrane (2) and mediates translocation through it into the cytoplasm (3). Once inside the cytosol, the plasmid is subjected to possible degradation by cytosolic nucleases, from which it is protected by its condensation. (4) Importin binding is mediated by the NLS moiety contained within the CPP or covalently bonded to it, followed by translocation into the nucleus. (5) Within the nucleus, Ran-GTP binding to the importin effects release of the plasmid DNA and the CPP, leading to (6) enhanced gene expression due to the increased nuclear concentration of the therapeutic gene. Inside the nucleus the plasmid DNA may need to be decondensed before it is transcribed.

Recently, one PTD was shown to specifically transduce synovial fibroblasts and was able to cause specific cell death by delivering apoptotic agents exclusively to these cells [135], demonstrating that cell specificity can occur for some CPPs, although the mechanism behind this remains a mystery. As always, when dealing with non-viral gene therapy, tissue specific promoters, such as the transthyretin promoter for expression in hepatocytes [136], and in situ administration of the compound through direct intratumoural or intramuscular injections [104] to the target site can be explored [137].

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4.3. Delivery of Proteins Many varied therapeutic techniques require the delivery of proteins into cells. Traditionally this has been a difficult task to accomplish, especially in vivo [1]; the use of CPPs greatly facilitates this process. Perhaps the best example of CPP-mediated transduction of a biologically active protein is that of a Tatp-
-gal fusion protein [59]. Intraperitoneal (i.p.) injection of this protein induced
-gal activity in all samples assayed, including blood cells, liver, kidney, lung, heart muscle, spleen and even the brain. Protein delivery across the blood brain barrier is usually restricted to small (< 6 amino acids) highly lipophilic peptides, whereas the
-gal fusion protein is over 480 kDa in size, indicating that Tatp was not only able to pass through the blood brain barrier, but was capable of taking large molecules across with it. The blood-brain barrier was confirmed to be intact in these animals, in stark contrast to control mice [59], which were injected with protamine, a protein usually found in sperm that is well known to break down the blood-brain barrier [138]. In another example, the Tat PTD was fused to apoptin, a protein that reportedly induces apoptosis specifically in cancer cells, but not in normal cells [139]. Tat-apoptin protein was efficient at transducing both normal and tumour cells, but remained in the cytoplasm in normal cell lines and did not mediate apoptotic killing of these cell types. In cancer cell lines however, it migrated into the nucleus, resulting in apoptosis after 24 hours. This demonstrates how the fusion of tissue specific functional proteins to CPPs may result in celltype specificity of the induced effect, if not of the delivery vector itself (see also Section 4.2.5.3) and that, clearly, Tat CPP can transport protein into cells in active form. Tat CPP significantly enhances intracellular protein delivery to a wide variety of cell types including all blood cells, bone marrow stem cells, fibroblasts, osteoclasts, osteosarcoma cells and keratinocytes [1, 72, 93, 140]. Other examples of protein delivery using Tatp include delivery of horseradish peroxidase, RNaseA and domain III of Pseudomonas exotoxin A into a wide variety of cell types in vitro [18] and enhancement of intestinal absorption of insulin in an in vitro model system [141]. Tatp has also been used to deliver Bcl-xL, a well-characterised death-suppressing molecule of the Bcl-2 family, in vivo [142]. Bcl-xL is expressed in embryonic and adult neurons of the CNS and may play a critical role in preventing neuronal apoptosis that occurs during brain development or results from diverse pathologic stimuli, including cerebral ischemia [143]. i.p. injection of Tat-Bcl-xl fusion proteins into murine models of stroke transduced brain cells within 1-2 hours and decreased cerebral infarction in a dose-dependent manner. In addition, it attenuated ischemia-induced caspase-3 activation in ischemic neurons [142], demonstrating its ability as a neuroprotectant. Protein delivery by CPP has not been restricted to Tatp. VP22 has been used to deliver the human papilloma virus (HPV) E2 protein to target cells [144]. E2 overexpression in cervical cancer cells can induce growth arrest and/or apoptosis and therefore has applications in cervical cancer therapy [1]. When VP22-E2 expressing Cos-7 cells were seeded into cultures of HPV-transformed cervical carcinoma cells, VP22-E2 was delivered to the nonproducing cells and induced apoptosis, implying that local delivery of VP22-E2 cells may be useful in the treatment of cervical cancer and other HPV-associated diseases [144]. The feasibility of this in vivo in a clinical setting remains to be tested. Tat-mediated protein delivery has also shown potential as a prophylactic vaccine and therapeutic. Proteins cannot usually enter the cytosol to access the MHC class I processing pathway, making it difficult to induce class-I restricted cytotoxic T-lymphocyte (CTL) re-

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sponses [1, 145]. Tat conjugated antigenic compounds, such as Tat-ovalbumin conjugates, however, have been reported to be taken up by antigen-presenting cells, processed and displayed on the cell surface, resulting in effective killing of the target cells by antigen specific CTLs [145, 146]. CPP-mediated delivery of proteins has potential uses in the treatment of many other diseases/disorders such as in the treatment of tumours or inflammatory conditions, many of which are currently under study [1, 143, 147]. 4.4. Delivery of Other Cargo by CPP Apart from DNA and protein delivery, CPPs are being investigated as potential delivery vectors for a whole host of other therapeutics, only some of which can be mentioned here. For instance, Tatp has been utilised for the delivery of anti-tumour antibodies (Ab), where it significantly enhanced Ab-cell surface interaction and cellular uptake [148]. Similar or slightly enhanced results were seen with other CPPs in an analogous study [91]. Tatp (37-72) tethered to anti-tetanus toxin Ab was efficiently taken up by cells, where it showed a moderate ability to neutralise the tetanus toxin [149]. This has therapeutic applications, as tetanus toxin is known for its very slow degradation rate, especially once inside cells of the nervous system [1, 149]. Unfortunately, a biodistribution study on Tat (44-57)-Ab conjugates showed that, despite efficient cellular uptake, these had greatly a reduced tumour targeting performance, compared to unmodified antibody [150]. This reinforces the need for careful consideration of CPP-cargo conjugation techniques. Tat has also been used to deliver inhibitory peptides to cells, such as peptide inhibitors of the c-Jun N-terminal Kinase (JNK), which have been shown to potently protect against cerebral ischaemia [151]. Delivery of a short, 11aa peptide inhibitor of JNK by Tatp resulted in significant protection of the cells from cell death by necrosis [152]. In a similar vein the Tat PTD has also been used to deliver the C3 exoenzyme, a bacterial toxin that is a highly specific inhibitor of the Rho family of G proteins, to cardiac myocytes, resulting in abnormal cellular projections, and indicating a role for Rho in the maintenance of cellular morphology [153]. These studies indicate the potential use for CPPs to deliver inhibitory compounds to cells with numerous therapeutic applications. Liposomes have been used to decrease the toxicity of drugs and to enhance their half-life and solubility. However, they have display very slow cell penetration, which hampers their therapeutic use [143]. Conjugation of Tatp or Antp onto the surface of liposomes appears to enhance their cellular delivery dramatically, such that they display fast and efficient translocation into the cell cytoplasm [154-156]. Fluorescent microscopic observation of markers trapped inside the liposomes and incorporated into the liposomal membrane demonstrated that the liposomes remain intact in the cytoplasm for the first few hours and migrate slowly towards the nucleus [154]. Eventually they release their contents into the cytoplasm [154], but it is clear that this intracellular release step needs to be significantly improved in order for CPP-liposome complexes to be a viable delivery approach to achieve levels of pharmaceuticals sufficient for a clinical setting [156]. Tatp-liposomes have also been used as gene delivery vectors, resulting in a relatively high transfection efficiency in vitro, with low cytotoxicity, and robust ectopic gene expression [154]. CPPs have also been used to deliver peptide-based imaging agents such as Oxotechnetium V and Oxorhenium V into cellular compartments, achieving intracellular concentrations sufficiently high to carry out imaging and radiotherapy [157]. Tatp can also significantly improve the intracellular uptake of paramagnetic nanoparticles and labels, that can

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easily be detected by magnetic resonance imaging (MRI) [73, 158]. This technique was so sensitive as to enable single cells to be detected in tissue samples [73]. Clearly, CPPs have a vast array of possible applications in clinical settings. Although optimisation of these therapeutic techniques is required, the results already obtained in in vitro and in vivo settings promises much for the future in this regard. CONCLUSION CPPs are already being utilised with great success. They have shown extraordinary potential in a range of therapeutic applications, both in vitro and in vivo. This is in large part due to the ease with which they can be coupled to numerous classes of cargoes, as well as their reasonable efficiency in cargo delivery and relatively low cytotoxicity. The field of gene therapy in particular, stands to profit most from CPP technology, in that the applications for its use in this setting are the most varied. The application of CPPs in drug and DNA delivery, already underway, does not require precise definition of the mechanism by which CPPs mediate cell entry, but it is becoming increasingly clear that there is more than one mechanism for CPP uptake at work. Endocytosis, endosomolytic activity, macropinocytosis and true protein transduction all seem to contribute to the uptake of different members of the CPP family. Whatever the mechanism, CPPs are clearly likely to be invaluable tools in the not too distant future for the therapeutic delivery of macromolecules. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]

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Design of Peptide-Based Vaccines for Cancer Geoffrey A. Pietersz1, Dodie S. Pouniotis2† and Vasso Apostolopoulos2,* 1

Bio-organic and Medicinal Chemistry Laboratory, 2Immunology and Vaccine Laboratory, Centre for Immunology, Burnet Institute, Commercial Road, Melbourne, VIC, 3004, Australia

Abstract: The immune system responds efficiently to bacteria, viruses and other agents however, the immune response to cancers is not as effective. In most cases other than specific genetic rearrangements leading to non-self proteins such as in leukemia and idiotypes in lymphoma, tumor associated proteins are self proteins and are not recognized by the immune system to prevent malignancy. In most cancers, patients develop antibodies and/or CTL-precursors to tumor associated antigens but are not effective in generating a therapeutic immune response. Adjuvants have been used with either whole tumors, subunits or peptides with the aim of increasing their immunity. Whole tumor antigens have certain advantages associated with it, such as ready availability as recombinant proteins, potential epitopes that can be presented by a number of MHC class I/II alleles and antibody development. The methods of identification of CD8 and CD4 epitopes either by use of epitope prediction algorithms or use of transgenic mice has made the use of defined synthetic peptides more attractive. The possibility to synthesize long peptides and introduce multiple epitopes (CD4 or CD8) from single or multiple antigens makes peptide a viable alternative to whole proteins. As an alternative to totally synthetic peptide constructs or polymers, polytopes have been generated by genetic engineering methods. In addition, to deliver immunogens to and to activate DC, receptor-mediated delivery of peptides using antibodies, cytokines and carbohydrates have been used. This review will encompass the various strategies, preclinical and clinical applications in designing peptide-based vaccines for cancer.

Keywords: Peptide, vaccine, mimotope, MAP, cancer, multiepitope, tumor associated antigens. INTRODUCTION The first vaccine was developed in 1796 when Dr Edward Jenner performed the first vaccination with infectious fluid from the hands of milkmaids infected with cowpox into the arm of a healthy boy. The boy showed symptoms of cowpox infection, then recovered and was later infected with smallpox but did not show any clinical signs of smallpox. This principle of using a less harmful infectious organism to cross protect against a harmful infectious agent spurred the field of vaccination and resulted in highly effective inactivated *Corresponding author: Tel: +61 3 9282-2100; Fax: +61 3 9282-2111; E-mail: [email protected] †Current address: School of Medical Sciences, RMIT University, Melbourne, VIC, Australia Allen B. Reitz / Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.

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(flu, cholera, plague, and hepatitis A), live/attenuated (flu, yellow fever, measles, rubella) or toxoid (tetanus and diphtheria) vaccines against infectious diseases. Vaccines have contributed to the eradication of smallpox - the last case in Ethiopia in 1976 and in 1980, the WHO announced that vaccines had been successful at eradicating smallpox from the world, one of the most contagious and deadly diseases known to man. Incidence of other diseases such as rubella, polio, measles, mumps, chickenpox, and typhoid has also decreased significantly over the past 100 years. The use of immunotherapy to combat cancer originated from the initial attempts of Dr William Coley in the late 19th century who used bacterial extracts (Coley’s toxin) to treat patients with advanced cancer [1, 2] Coley’s toxin was remarkably effective with a strong febrile response crucial for regression. A retrospective study compared the 10 year survival rates of patients treated with Coley’s toxin with those treated with modern treatments and concluded that despite the vast amounts of money spent on developing new therapies, patients receiving modern treatments did not fare any better than those receiving Coley’s toxin more than 100 years ago. Interestingly BCG is the only bacterial vaccine in use and the only effective treatment for superficial bladder cancer when given intravesically. Despite all the efforts on research and clinical trials there is still no effective cancer vaccine. The discovery that tumors express antigens that can be targeted by cytotoxic T lymphocytes (CTL) is the fundamental concept behind developing current immunotherapy [3]. The generation of a CD8 T cell response is necessary for the eradication of tumors. However, to expand and sustain the CTL response, CD4+ T cell help is crucial. There is a large number of tumor associated antigens (TAA) identified and used as targets for immunotherapy. These belong to several classes a) those not expressed by normal cells such as viral antigens [human papillomavirus-16 (E6, E7). HBV (HbsAg, HBVPresS2, HbcAg), EBV (gp340/320, EBNA-3A)], idiotypes of B cell lymphomas b) products of rearranged genes (Bcr-Abl, EGFRvIII) or mutated genes (p53, ras p21), c) cancer-testis antigens (MAGE, NY-ESO) or d) antigens with low expression on normal cells and elevated expression on tumor cells (MUC1, carcinoembryonic antigen, HER-2/neu, p53). Various approaches such as whole tumor cells, recombinant proteins, peptides and delivery systems such as antigen presenting cell (APC) targeting, microparticulate sytems, adjuvants, pulsed dendritic cells (DC), DNA, RNA and recombinant viruses have all been used for vaccination. Immunization with whole proteins have some advantages, as they potentially have multiple CTL epitopes and T helper cell epitopes not restricted to one HLA type. However, since most TAAs are self antigens T cells that recognize the high affinity immunodominant epitopes may have been deleted during lymphocyte development. Rationally designed peptide vaccines may be able to overcome some of the limitations of other modalities of vaccination. Multiepitope vaccines incorporating, multiple CTL epitopes encompassing HLA-A2, A3, A24 and promiscous T helper epitopes from one or more antigens together with an appropriate adjuvant or danger signal are prerequisites for better cancer vaccines. Using information from MHC/peptide crystal structures low affinity subdominant CTL epitopes maybe also be optimized. In this review we will discuss novel TAAs and the design of synthetic peptide vaccines with particular reference to the most recent literature. Approaches that have been used to deliver multiepitope vaccines and the use of B/T cell mimotopes to overcome peripheral tolerance to TAA will also be discussed.

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IDENTIFICATION OF MHC CLASS I AND CLASS II EPITOPES The specificity of MHC class I molecules has been determined using pool sequencing of eluted peptides, binding studies and/or phage display library analysis [4]. These results indicated the presence of conserved/consensus anchor residues amongst the high affinity binding peptides. In the process however low-to-medium affinity peptides or medium-high affinity non-canonical peptides were missed in this analysis. As a result the identification of such non-canonical peptides were not predictable from the protein primary sequence. Given that it is impractical to test all possible overlapping peptides of proteins, numerous programs have been developed to predict peptides with the potential to bind to MHC molecules based on the canonical anchor motifs known for high affinity peptide binding [4-5]. Systematic screening from TAA for potential MHC class I epitopes, using the programs (SYFPEITHI [5]; MHCPEP [6]; HLA-BIND [7]) is a relatively simple process. However, most of these peptides identified by these programs do not induce T cell responses. Furthermore, noncanonical binding peptides are not predicted / identified using these programs. New methods / programs are emerging which are able to predict an array of peptides which can bind nonconventionally to MHC class I molecules. EpiMatrix and Conservatrix search for unique or multi-HLA restricted (promiscuous) T cell epitopes and identifies epitopes that are conserved across variant strains of the same pathogen [8]. Quantitative structure-activity relationship (QSAR) methods have been used to identify peptides which bind to more than one allele [9, 10]. A new method was developed for predicting MHC binding of peptides based on peptide property models constructed using biophysical parameters of the constituent amino acids and a training set of known binders [11]. This method has been constructed based on the tumor associated antigens, MART-1, S-100, MBP, CD63, MUC1, p53, cyclin B1, HER-2/neu and CEA and a number of low-medium affinity peptides were identified which were usually missed in other standard prediction programs. In addition to using standard programs to predict binding of peptides to MHC class I, programs have been developed which determine which peptide sequences are cleaved in the proteosome, are transported through TAP and bind to MHC class I [12]. Likewise, Immune Epitope Database (IEDB) (web: http://tools.immuneepitope.org/main/index.html) provides a collection of tools for the prediction and analysis of immune epitopes (T and B cell epitope prediction). The T cell epitope prediction tools includes the prediction of peptides binding to MHC class I and MHC class II molecules based on their IC50 values. In addition, a tool is available which predicts epitopes based upon proteasomal processing, TAP transport and MHC binding to produce peptides intrinsic potential of being a T cell epitope. IEDB also includes NetChop, a predictor of proteasomal processing and NetCTL [13] which predicts CTL epitopes in a protein sequence (web server: http://tools.immuneepitope.org/main/index.html). Thus, the peptides identified would most likely induce CTL responses compared to peptides that have been identified to only bind to MHC class I. Furthermore, the method PepScope has been established and identifies many potential T cell epitopes based on new anchor motifs which would normally be missed with current prediction programs [14] In addition to MHC class I prediction programs, there are numerous methods which identify MHC class II binding peptides. RANKPEP [15], Gibbs motif sampler [16] and TEPITOPE [17] are commonly used. More recently, a program was developed based on the average relative binding matrix method that predicts IC50 values of different length peptides and to bind to MHC class II molecules (web server: http://epitope.liai.org:8080/matrix) [18]. Furthermore, MULTIPRED was designed to predict peptides binding to multiple MHC class I and II alleles. This method also allows the prediction of peptides that promiscuously bind to multiple HLA alleles within one supertype using hidden Markov models and artificial neural network methods (http://antigen.i2r.a-star.edu.sg/multipred/) [19]. SMM-align [20], was developed to

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identify a preference for hydrophobic or neutral amino acids at the anchors which is more superior to TEPITOPE which favors basic amino acids at most anchor positions. Much work is required in (i) identifying non-canonical peptides from proteins and (ii) accurately identifying peptides from prediction programs which bind to MHC class I/II and induce immune responses. STRUCTURE BASED MODIFICATIONS OF CLASS I AND CLASS II EPITOPES Peptide epitope modifications can enhance peptide immunogenicity by improving binding and stability. Tolerance can be overcome by selecting low affinity peptides and modifying the ‘anchor’ residues to increase binding affinity. Alternatively, the TCR contact residues can be modified to improve T cell activation, Fig. (1).

Fig. (1). Side view of peptides bound to MHC class I, H-2Kb. Peptide side chains which point ‘down’, bind into pockets (A-F), peptide side chains which point ‘up’, interact with the T cell receptor (TCR). Modifications can be made to the ‘up-pointing’ amino acids or to the ‘down-pointing’ amino acids.

‘Anchor’ Residue Modification Tumor antigens are self antigens, and therefore tolerance is one of the limiting factors. Self antigens are poorly immunogenic for CTL. High vaccination efficiency has been demonstrated for low-affinity epitopes, derived from the murine telomerase reverse transcriptase. Anchor modifications (Fig. (1)) improved MHC binding affinity and exhibited potent anti-tumor immunity with no autoimmune responses in mice [21]. Enhanced CTL activation has been achieved with ‘anchor’ residue modification of the melanoma antigen MART-127-35 (AAGIGILTV) to LAGIGILTV [22]. Furthermore, mutations of the MART126-35 peptide epitope in the central position (positions 31-33) induced high affinity ligands to HLA-A2 and amplified the responses of a T cell MART-126-35 specific clone [23]. The MUC1-8 peptide (SAPDTRPA) binds to murine MHC class I, H2-Kb with low affinity, of which the crystal structure is known [24]. Mutations to MUC1-8 peptide to result in MUC185F8L (SAPDFRPL) bound to H2-Kb with high affinity, induce high avidity CTL in C57BL/6 mice and overcame tolerance in MUC1 transgenic mice [25]. Recently, Ran1, tumor specific antigen CTL epitope was mutated to Ran1 1Y, where, position 1 was replaced with Y, elicited stronger CTL and bound to HLA-A2 with high affinity compared to wild type peptide [26]. Making anchor motif modifications to include high affinity anchors, does not necessarily indicate that the peptide will bind with high affinity to the MHC as one must consider proximal/distal amino acids [27]. Changes in the side chain of P8 of peptides TLTSCNTSL and TLTSCNTSV were shown to influence the orientation of Arg97 from the

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HLA-A2 binding groove. Due to steric hindrance, Arg97 orientated towards the N-terminus of the peptide TLTSCNTSL, thus facilitating binding when a hydrogen bond donor/acceptor is placed at position P3. In contrast, Arg97 orientated towards the C-terminus when the small chain from valine was present at position P8, as for TLTSCNTSV. Anchor modifications may therefore be complicated in this manner and require rational alterations to peptide sequences, using molecular modeling techniques, in order to successfully achieve the desired effect. In an interesting study, the human Her-2/neu CTL peptide, [hHer-2/neu (435443)] was injected into HDD transgenic mice [28]. The murine Her-2/neu (435-443) peptide [mHer-2/neu 9(435)] differs from the human peptide by one amino acid at position 4. The [hHer-2/neu (435-443)] peptide induced strong CTL against the murine peptide, and induced strong protective and therapeutic immunity against an adoptively transferred HLAA2/Her-2/neu tumor [28]. Thus, enhanced immunity to the xenoantigen was induced compared to the self antigen in mice, and demonstrates that a one amino acid variation alters immune responses and overcomes tolerance in this mouse model. A number of T cell epitopes derived from infectious pathogens or pathogens which cause cancer are highly variable leading to peptides no longer capable of binding to MHC molecules or TCR thereby facilitating immune evasion. Naturally occurring peptide variants have been described in a number of diseases including viral diseases, malaria and cancer. One such example is of an EBV strain carrying an HLA A2-restricted epitope variant of LMP-1, which has been shown to be prevalent in nasopharyngeal carcinomas (NPCs). The variant has two natural mutations at anchor residues of an L to an F and an M to an I at positions 2 and 5 respectively[29]. The mutant peptide bound to HLA-A2 with lower affinity, CTL lysis was abrogated and IFN-gamma cytokine responses were not induced. These results show that EBV isolates from NPCs is dominated by an HLA A2-restricted natural variant of LMP-1, which would allow the virus to resist immune recognition and may in part contribute to the prevalence of NPC in these populations [29]. TCR contact Residue Modifications (Altered Peptide Ligands) Peptides with amino acid substitutions which interact with the TCR are known as altered peptide ligands (APLs), Fig. (1). TAA are usually weakly immunogenic and their peptides bind to MHC class I with low affinity. APLs can be designed to increase their immunogenicity and to induce stronger T cell responses than the native peptide epitope, to act as a super-agonist. APLs may exhibit similar or optimal MHC anchor motifs and have equivalent MHC binding as the cognate peptide, however, APLs influence the type of potency of T cell responses by modulating intracellular signaling and phosphorylation patterns involved in T cell activation [30]. An example of APL is the HLA-A2 peptide (CAP1, YLSGANLNL) from carcinoembryonic antigen (CEA) overexpressed on colorectal, gastric and pancreatic cancers [31-32]. Substitution studies in the TCR contact residues, CAP1-6D (YLSGADLNL), significantly enhanced CAP1 specific CTL responses with unaltered MHC binding [33]. In initial clinical trials immunization with CAP1-6D, induced enhanced CTL responses in patients compared to CAP1 peptide [34]. However, in a recent clinical trial, CAP1-6D, generated low-affinity CD8+ T cells which did not recognize CEAexpressing colorectal carcinoma cells, even though these T cells were reactive against the CAP1-6D peptide and to a lesser affinity to the CAP1 native peptide [35]. This study demonstrates, that further work is required to analyse tumor cross-recognition prior to any clinical usage of APL as anti-cancer vaccines. In addition, a single amino acid substitution of MART127-35 (AAGIGILTV) (substitution of Leu in position 1) enhances MART127-35 T cell activity, by inducing IFN-g and IL-2 in vitro and these T cells are insensitive to inhibitory effects of MART127-35 antagonist peptides [22]. Mutated gp100209-217 peptides with preferred

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HLA-A2 anchor residues bound with higher affinity to HLA-A2 as compared to the native gp100 peptide [36]. CTL lines generated from patients immunized with gp100 peptide showed enhanced IFN-gamma secretion in the presence of the mutated peptides[36]. CD4 T cells to lymphoblastic leukemia antigen TEL/AML proliferate specifically to TEL/AML as well as Th1 cytokine secretion are enhanced in the presence of APLs [37]. Thus, superagonist APLs can be of use for anti-leukemic immunotherapy. Single mutations to a peptide are known to change the entire conformation of the peptide and thus, altering the conformation of peptide-MHC contact residues or the TCR contact residues. Modifications at anchor residues have been shown to dramatically influence the conformation of the MHC peptidegroove and have profound effects on TCR interactions [38]. It is important to be cautious in designing modifications to peptides for cancer immunotherapy to improve them of higher affinity, since this can effect T cell reactivity. To overcome these problems, solid-phase epitope recovery method has been used to determine reactive peptides with immunogenic properties of interest [39]. APLs have also been used (antagonists) for autoimmune diseases [4046] and for infectious diseases [47]. More recently, molecular simulation was used to optimise the melanoma immunodominant epitope NY-ESO-1(157-165) by substituting TCR contact residues. A W to F substitution resulted in an enhanced ability to induce crossreactive CTL responses with the wild type peptide and lysis of NY-ESO-1-expressing tumor cells [48]. Thus, APLs can be used in conjunction with molecular modeling/simulation and binding studies to enhance T cell responses. MIMOTOPE VACCINE DESIGN B Cell Mimotopes Potential B cell epitopes may be identified using computer-based analysis of hydrophilicity, hydrophobicity, antigenic index, and surface probability based on the protein sequence [49-50]. Since there is only limited correlation between predicted and actual epitopes, epitope scanning methods such as PepScan with anti-sera need to be utilized [51-52]. Most vaccines are designed with the aim to generate cellular responses to destroy tumors. However, it is becoming increasingly evident that the presence of systemic antibodies that recognize tumor antigens such as MUC1 may offer a survival advantage in patients with cancer [53]. Passively administered naked mouse, human, chimeric or humanized antibodies that fix complement or have ADCC activity have shown some promise and the most impressive results have been generated with the antibodies, Trastuzumab (Herceptin®) and Rituximab (Rituxan®) [54-57]. An immunoconjugate of CD33 and calicheamycin has also given good responses in patients with acute myelogenic leukemia and is approved by the FDA[58]. As a means of generating antibodies to tumor associated proteins by active immunization, patients may be injected with tumor proteins with various adjuvants. However, in some cases responses are weak possibly due to tolerance to the protein [59]. An alternative approach that may generate greater antibody responses is to use antibody mimotopes. Mimotopes are peptides that bind to the paratope of antibodies. Mimotopes may be identified with the use of combinatorial synthetic peptide libraries or random peptide libraries displayed on bacteriophage [60-61]. In the latter approach peptide libraries are displayed fused to the bacteriophage coat proteins pIII or pVIII. Phage libraries with peptides of lengths 6-43 amino acids (aa) have been generated [62-63]. Using the process of successive panning on a particular target, specific bacteriophages that bind the target can be isolated. Since the phenotype of the phage is linked to the genotype, the specific peptide sequence of the specific phage can be identified by DNA sequencing. Several groups have identified mimotopes that

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bind to the humanized anti-HER-2/neu antibody, Trastuzumab. The mimotope H98 (LLGPYELWELSH) was identified by panning a 12-mer peptide library with Trastuzumab as the target [64]. A GST-H98 fusion protein was able to block the binding of Trastuzumab to HER-2/neu and mice immunized with GST-H98 generated antibodies which bound to H98 as well as to HER-2/neu. In another study, 5 mimotopes of Trastuzumab were isolated from a constrained 10-mer library of which C-QMWAPQWGPD-C was the best candidate for immunogenicity studies [65]. Immunization of BALB/c mice with the constrained peptide resulted in antibody that bound to HER-2/neu expressing SK-BR-3 cells [65]. Three different anti-HER-2/neu antibodies (Ab2, Ab4 and Ab5) were used to pan against a 12-mer constrained (XCX8 CX) and a 15-mer linear random phage display library. Phage selected for binding to Ab2 bound Ab2 and not to Ab4 and Ab5. Some phage isolated from panning on Ab4 specifically reacted with Ab2 rather than Ab4 and some with Ab5 suggesting that the antibodies may have overlapping domains. All three peptides selected from the screening of the constrained library on Ab2 (VCQPWDHNSICN, SCQPWDAPARCE and HCLPRDRMGQCH) were capable of inhibiting the binding of Ab2 antibody to HER-2/neu expressing T47D cells [66]. Anti-CD20 antibody rituximab was panned with a phage library displaying 7-mer cyclic peptides and peptides with consensus sequence A(S)NPS was isolated [67]. The consensus sequence overlapped the CD20 170ANPS173 sequence and these peptides inhibited the binding of rituximab to CD20 +ve cells. Mice immunized with KLHlinked peptides generated sera that blocked rituximab binding to CD20 +ve cells. Interestingly, not all mimotopes were capable of generating specific antibodies and is influenced by the surrounding amino acids of the mimotope motif [68]. Mimotopes are extremely useful when tumor specific antibodies are available but the antigen is unknown or difficult to isolate and purify. An antibody against, MG7 antigen (Ag), a gastric cancer TAA, is used as a test for gastric cancer and to monitor treatment efficacy [69]. A linear 9-mer and cyclic 9-mer library were screened against the monoclonal antibody to MG7-Ag. Panning lead to groups of peptides sharing the consensus sequences PLX0-2S, SAVR and XRMX. MHC class I and class II epitope prediction programs indicated that all the peptides could potentially bind several HLA molecules. A MG7-Ag mimotope (KPHVHTKGGGS) was incorporated with the universal T helper epitope, PADRE (AKFVAAWTLKAAZ) into a pcDNA3.1 plasmid for transfection into Salmonella typhimurium [70]. Oral administration of the Salmonella resulted in MG7-Ag specific antibody and partially protected mice from MG7-Ag expressing EAC tumor cells. Similar results were observed with an alternative oral vaccine utilizing a fusion gene of the mimotope to HbcAg gene [71]. Furthermore, a prime-boost vaccination strategy utilizing priming with the oral DNA vaccination followed by boosting with an adenovirus construct incorporating the MG7-Ag mimotope induced more efficient T cell responses and protection of mice from a tumor challenge [72]. A recent study identified peptide mimics of the melanoma cell-adhesion molecule (Mel-CAM) which when coupled to tetanus toxoid and injected into BALB/c mice induced antibodies which cross-reacted with Mel-CAM [73-74]. In addition, mimotopes of high-molecular weight melanoma-associated antigen (HMW-MAA) identified by screening a 9-mer phage library on anti-HMW-MAA antibody 225.28S share a consensus sequence which displays partial homology to the HMW-MAA. When the mimotope was fused to albumin binding protein and used to immunize mice, antibodies were generated that lyzed 518A2 melanoma cells in ADCC assays. Similar B-cell mimotopes have been identified for prostate specific membrane antigen, CAMPATH-1 (CD52), MUC1, CEA and Mgb1-Ag [75-77]. B cell mimotope identification is important for peptide based vaccines.

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T Cell Mimotopes Several strategies can be used to identify T cell epitopes from tumor associated antigens (TAA), (i) isolation of MHC molecules from tumor cells and eluted peptides fractionated by HPLC and identifying T cell stimulatory peptides by sequencing, (ii) Use of CTL clones and expression libraries from tumor cell cDNA and (iii) synthesizing overlapping peptides from known protein sequences and identifying peptides that stimulate antigen specific T cells. Similarly to the identification of B cell mimotopes using combinatorial libraries or phage display libraries, T cell mimotopes can be identified using antigen-specific T cell clones. To identify T cell stimulatory peptides using the combinatorial library approach, pools of 9 aa peptides are made that have each of the 9 positions randomized with the 19 aa (excluding Cys) and each position with a single aa and the other 8 positions randomized with the 19 aa to result in 171 pools. The target cells are pulsed with the pools and subjected to lysis by the CTL clone. Pools with the specific aa that cause lysis by the CTL defines that position with the particular aa. This method has been used to identify T cell mimotopes of the CD8+ T cell clone specific for cutaneous T cell lymphoma (CTCL) [78]. Two HLA-B8-restricted mimotopes PVKTKDIKL and PVKTKDIKL were identified and T cell lines were generated by stimulating peripheral blood mononuclear cells (PBMC) which were able to lyze autologous EBV pulsed cells as well as MyLa tumor cells. A mixture of these two mimotopes were used with tuberculin to immunize two HLA-B8 CTCL patients [79]. Mimotope specific T cell frequencies reached 0.21 and 0.52% in two patients and mimotope reactive T cells isolated from patients lyzed MyLa target cells and mimotope pulsed EBV-transformed B cells. Peptide display libraries cannot be used to identify T cell mimotopes since T cells recognize peptides in complex with MHC class I. To enable this, single chain peptide MHC molecules have been expressed on phage via the pIII protein, on yeast and insect cells [80]. The most promising method is the baculovirus based system where a random peptide library of 9-10-peptides is linked to the N-terminus of 2-microglobulin via a flexible linker expressed with membrane bound MHC class I H-2Dd heavy chain [81]. The libraries can be screened with fluorescently labeled soluble
TCR using flow cytometry to identify T cell mimotopes. This method was used to identify a mimotope for a T cell, reactive to an unknown peptide in context with H-2Dd. The unknown peptide (AGATRWCRL) was identified from the homology of the mimotope (TGPTRWCRL) with a murine homolog of the Drosophila protein, spinster. A similar method has been used for baculovirus display of MHC class II I-Ab-peptide complex [82]. Interestingly the identified mimotope (FEAQRARRAARVD) for one of the TCRs had high homology to the peptide (FEAQKAKANKAV) used for immunization. T cell mimotope identification using libraries is a powerful method to use when designing peptide based vaccines. Carbohydrate Mimotopes Carbohydrate antigens as immunogens are not ideal being T cell independent antigens and induce IgM antibodies. Use of carrier proteins convert carbohydrate antigens to T cell dependent antigens [83-84]. The concept of using peptide mimotopes of carbohydrate is attractive because of the possibility of generating both cellular and humoral responses. Carbohydrates such as sialyl-Lewis X, Lewis X and Lewis Y are upregulated on a variety of cancers and vaccination against these with a carbohydrate mimotope is advantageous [8586]. A peptide mimic (GGIYWRYDIYWRYDIYWRYD) of sialyl-Lewis X was identified and used as a Multiple Antigenic Peptide (MAP, see below, Fig. (2)) to immunize BALB/c mice [87-88]. A combination of IL-12 with MAP induced CTL in mice which protected

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them against Lewis X expressing Meth A tumor cell challenge. In addition, the peptide DAHWESWL was identified as a mimic of the gal
(1, 3)gal epitope by screening a peptide library on IB4 lectin [89-90]. This peptide and sugar was also mimicked by the MUC1 VNTR peptide APDTRPAPGS Fig. (3). Furthermore the CTL that recognized MUC1 also recognized the gal
(1.3)gal mimic DAHWESWL, Fig. (1) [91-94]. Neuroblastoma and melanoma cells express the weakly immunogenic GD2 ganglioside that induces IgM antibody responses in patients. Currently, GD2 linked to KLH is used for active immunotherapy of melanoma [95]. Screening a 15-mer phage display library with the 14G2a anti-GD2 antibody resulted in several peptides of which peptide 47 (EDPSHSLGLDVALFM) had the greatest inhibition of 14G2a binding to GD2+ neuroblastoma cells [96]. A more active analog 47-LDA (EDPSHSLGLDAALFM) was designed based on molecular modeling of peptide with 14G2a antibody. A DNAvaccine expressing the 47-LDA epitope inhibited the growth of GD2-expressing NSX neuroblastoma in mice [97]. O (CH2)4

HOOC

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Fig. (2). Structure of 2-branch, 4-branch and 8-branch MAP for use in cancer vaccine studies.

Recently, it was demonstrated that the original 14G2a antibody crossreacted with the CD166 adhesion molecule and the CD8 T cells generated with the 47-LDA mimotope was recognizing a cross-reactive epitope within the CD166 molecule [98]. Carbohydrate, T cell and B cell mimotopes are great prerequisites in the design of peptide based vaccines for cancer immunotherapy. MULTIEPITOPE VACCINES An ideal vaccine for cancer encompass a means of overcoming tumor antigen escape variants, epitopes capable of priming MHC class I and class II responses and restricted to several HLA alleles. Use of several TAAs with a suitable adjuvant could satisfy this criteria.

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However, development of such a vaccine may be hindered by the difficulty in being approved by regulatory agencies and possibly commercial licencing issues of antigens. In addition, it is likely that there will be a selective advantage for immunodominant epitopes to be presented in preference to the subdominant epitopes. The immunodominant epitopes would most likely be those of higher affinity and since tumor antigens are in most cases autoantigens and tolerance to self antigen may prevent the generation of strong immune responses. An elegant method to overcome these problems is to design a vaccine based on minimal CTL and T helper epitopes from one or more antigens. The so called ‘polytope’ vaccines can be made by genetic engineering methods or using chemical synthesis. In it’s simplest form polytopes are made by synthesis of linear tandem peptides with or without spacer amino acids separating the minimal epitopes. The branched multiepitope peptide complexes have been most used in vaccines developed for infectious diseases but equally applicable to vaccines for cancer.

Fig. (3). Molecular model of MUC1 peptide (yellow; APDTRPAPG) and MUC1 mimic (red; DAWHESWL) in complex with MHC class I (green). In the model, a charged residue (R-P5 in the MUC1 peptide and E-P5 in the mimic peptide) point down into the groove of the MHC. All though, the amino acid sequence of the peptides (P4, P6/P7) vary at the TCR interface, the volume occupied by each of the amino acids is similar.

Linear multiepitopes linked in tandem Within the sequence of human papillomavirus-16 antigen there is a continuous short peptide sequence that contains a CTL epitope (H-2Db), T helper epitopes (I-Ab and I-Eb) and a pan specific B cell epitope (E744-62, QAEPDRAHYNIVTFCCKCD) [99]. Transcutaneous immunization of mice with the E744-62 peptide with CpG and cholera toxin protected mice from an E7 expressing tumor challenge [100]. Thymidylate synthetase (TS) is an intracellular enzyme overexpressed in cancer cells. Three HLA-A2 peptides were identified by MHC class I epitope prediction algorithms and binding to T2 cells. CTL lines were derived from HLA-A2 PBMC[101]. A 28-mer peptide was synthesized in tandem containing all three epitopes and mice were immunized which delayed the growth of TS transfected EL-4/HHD tumor [101]. In addition, the 20 aa tandem repeat sequence of MUC1 contains a B cell epitope (APDTR) and a number of CTL epitopes [102-104]. An oxidized mannan conjugate of a MUC1 GST fusion protein consisting of 5 tandem repeat sequences was highly immunogenic in mice and capable of eradicating MUC1 expressing tumors in C57BL/6, DBA/2 and BALB/c mice[105-144]. These conjugates [115-117]have been used in clinical trials by injection [118]or by ex vivo pulsed DC[119]. Amphipathic peptides such as fuseogenic peptides from HIV TAT protein, penetratin from Drosophila Antennapedia or cationic peptides

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such as poly-arginine synthesized in tandem with CTL epitopes generate good cellular responses in mice[113, 120-123]. Lu and co-workers utilized synthetic peptides with the HIV1 Tat-derived Trojan peptide in tandem with 3 different CTL epitopes to generate strong CTL responses to all three epitopes in mice when given with CpG [124]. Another amphipathic peptide pep-1 (KETWWETWWTEWSQPKKKRKV) non-covalently linked with peptides or proteins was used to facilitate transfer of a multi-epitope peptide consisting three HLA-A2 restricted epitopes Her-2/neu369, Her-2/neu435 and Her-2/neu789 peptides separated by double arginine spacers across the cell membrane [125]. When administered in HHD HLA-A2 mice with a promiscuous T-helper epitope from tetanus toxoid and MDP primed CTL responses in mice 2-6 fold higher compared to the multiepitope peptide alone. The polycationic poly amino acid Poly-L-arginine simply mixed with TRP-2181-188 H-2Kbrestricted tyrosinase-related protein 2 induced sustained T cell responses in mice [126]. Linear multiepitopes linked in tandem induce immune responses in mice and is a method being tested in human clinical trials. Multiepitopes Linked in Branches Via a Poly-L-Lysine Core Branched peptides known as Multiple Antigenic Peptides (MAP) was initially used for antibody production based on a lysine core capable of incorporating 4 or 8 B cell epitopes in one synthetic complex, Fig. (2). Since these are of high molecular weight they did not need any adjuvants for antibody production. The use of the MAP approach in cancer has not been as prevalent as it’s use in vaccines for infectious diseases. The early MAPs had only identical peptides in either 4 or 8 branches [127]. However, using orthogonal protection schemes, MAPs with 2 or 3 different peptides can be incorporated into the complex [128]. Lipoproteins are components of bacterial cell walls and these have been shown to bind to TLR-2 [129]. The lipids are also involved in translocation of antigen into the cell. Lipid containing peptides can provide the necessary danger signals to prime effective immune responses. A lipid core peptide based on polylysine with 4 minimal CTL epitopes from either OVA257-264 (SIINFEKL) or LCMV33-41 and three C12 lipoamino acids activated DC (increased expression of CD86 and CD40) [130]. The lipid core peptide with SIINFEKL protected mice from an OVA positive tumor challenge but in combination with OVA to provide CD4 help. A totally synthetic vaccine incorporating TLR-2 targeting lipid S-[2, 3-bis(palmitoyloxy) propyl]cysteine (Pam2Cys) with a T helper epitope, a CTL epitope and a B cell epitope [131], activated NF-kB-dependent gene activation via TLR-2 and matured DC. One particular construct incorporating the OVA CD8 epitope and a T helper epitope from the fusion protein of the morbillivirus canine distemper virus protected 50% of immunized mice from B16-OVA tumors [131]. Human malignant gliomas express a unique mutation in the gene that encodes the epidermal growth factor receptor. As a result of this mutation a unique epitope is expressed by gliomas and not on normal tissues which is a potential target for immunotherapy [132]. A MAP incorporating the unique epitope (LEEKKGNYVVTDHC) was used to immunize rats that generated a humoral response. Use of MAP with GM-CSF resulted in cellular responses (CTL, IFN
) and reduced the growth of F98EGFRvIII tumors. In the early studies MAPs were equipped with a B cell epitope because they were primarily designed for generating antibodies. However, for modern vaccines the ability to prepare MAPs with different multiple epitopes is desirable. By utilizing of orthogonally protected lysines, MAPs with B cell epitopes as well as a T helper epitopes have been synthesized [133]. Another approach to generate multiepitope constructs is by copolymerization of acryloyl peptides with acrylamide or another acryloyl aa [134-135]. This methodology enables the use of multiple acryloyl peptides to result in large molecular weight peptide poly-

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mers. As an alternative to recombinant protein complex multiepitope proteins could be synthesized using orthogonal ligation strategies [136-138]. A multitude of chemical ligation procedures are available to selectively ligate prefunctionalized unprotected CTL, T helper or B cell epitopes, Fig. (4A). Several studies have also investigated the yield of ligation reaction, stability of the various linkages, the arrangement of epitopes and their immunogenicity [139]. In these studies epitopes ligated via disulphide linkages gave the lowest yields and were weakly immunogenic. Chemoselective ligation chemistry was used to design a branched oxime-linked peptide containing two copies of the MUC1 peptide and a universal T cell epitope, Fig. (4A) [140]. The 20 aa MUC1 sequence was incorporated in two orientations one with a free amino terminal and the other with a free carboxy terminal. A monomeric peptide and a linear dimeric peptide did not induce antibody responses but only the branched peptide with the oppositly oriented MUC1 peptide generated antibodies which reacted with the non-glycosylated MUC1. In addition to peptides, carbohydrate antigens are also targets of immune responses. Tn, TF and sTn are carbohydrate antigens expressed on breast, prostate, lung and pancreatic cancers [141]. Vaccines that target these have been developed by conjugating the glycopeptides to carriers such as BSA, KLH or OSA. Novel scaffolds, regiospecifically addressable functionalized templates (RAFTs) can be used for design of these vaccines. RAFTs are composed of a backbone cyclized decapeptide with two proline-glycine
-turns and stabilizes the conformation in solution, Fig. (4C). A conjugate containing 4 Tn analogs and a CD4 T helper epitope from type I poliovirus (KLFAVWKITYKDT) was recognized by anti-Tn monoclonal antibodies indicating that the conjugate mimics the natural display of Tn antigens present on repeat S/T repeats of mucins [142]. Furthermore, the conjugates also stimulated poliovirus specific T cell hybridomas when presented by DC. By using suitable orthogonal protecting groups and/or chemoselective ligation procedures, the positions occupied by the B cell epitopes maybe replaced with other epitopes. RECENTLY IDENTIFIED TAA CTL EPITOPES Mesothelin is overexpressed in mesothelioma, pancreatic and ovarian cancers [143]. Two naturally expressed mesothelin specific HLA-A2 CTL have been demonstrated to lyze pancreatic and ovarian tumor cells in vitro [144]. In addition, agonist peptides of these HLA-A2 epitopes are able to bind with higher affinity to HLA-A2 molecules and the CTL lyze mesothelin expressing tumor cells more efficiently as well as induce increased numbers of specific CTLs in vitro from healthy individuals and cancer patients [144]. Adipophilin is a protein involved in lipid homeostasis of adipocytes and macrophages and is selectively overexpressed in some renal cell carcinoma (RCC) and more importantly is found at very low levels on normal tissues [145]. Only recently, CTLs were generated after pulsing with a previously identified HLA-A2+ restricted CTL epitope, SVASTITGV in vitro using DCs derived from HLA-A2+ healthy donors and T2 cells [146]which recognized endogenously expressed adipophilin protein in RCC, malignant melanoma, breast cancer, and multiple myeloma [147]. Moreover, CTL clones generated from chronic lymphocytic leukaemia (CLL) and plasma cell leukaemia patients with DCs pulsed with peptide SVASTITGV were able to lyze autologous leukaemia cells [147]. Papillomavirus binding factor (PBF) was recently identified using a cDNA expression cloning procedure as an osteosarcoma antigen recognized by autologous CTLs and a HLAB55 restricted 12-mer peptide, CTACRWKKACQR [148]. Prevalence of this epitope is particularly low and much effort is required to identify HLA-A2 and HLA-24 restricted epitopes to be more clinically applicable.

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H2C

O

H2C

NH2

O

N P H

H N

O P

NH2

HN

H

O

Hydrazone

H N

NH2

HN

HN

P X

XH

X= S Thiazolidine X= O Oxazolidine

O

O

O

NH2

HN

N H Br

NH2

N P

O

HN

Oxime

O

O

H

139

NH

S O

O

O

SH

NH2

HN

NH2

HN SH

S

Thioether

NH2

S

H N

O

B O A-Muc

H N

Disulphide

O H N

N H O

N

O

N

O

PADRE

O Muc

HN O B

B

BK

BK X

C G P

K

K T

X= Ala OH OH

P

K G

H HO B-epitope =

H O H H

H AcHN O O

O

O KLFAVWKITYKDT-NH2

T-epitope =

O

N

Fig. (4). A. Various chemoselective ligation strategies. B. Multiepitope peptide incorporating MUC1 and T helper epitopes. C. Example of a RAFT scaffold.

Ring finger protein 43 (RNF43) is expressed by colon cancer cells and HLA-A2 (ALWPWLLMA and ALWPWLLMAT) and HLA-A24 (NSQPVWLCL) restricted epitopes have been identified after generation of CTL clones [149]. These CTL clones were able to lyze targets pulsed with the specific peptides as well as tumor cells naturally expressing

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RNF43 and it was clearly shown that the HLA-A24+ epitope induced enhanced tumor lysis compared to HLA-A2+ epitopes [149]. A more recent summary of recently identified T cell epitope peptides is shown in Table 1. Table 1.

Summary of Newly Identified T Cell Epitopes

Antigen

Epitope

HLA Type

Refs

EGFR

853-861 ITDFGLAKL

HLA-A*201

[214]

HER-2/neu

828-836 QIAKGMSYL

HLA-A*201

[215]

Ribosomal protein L19

133-141 KNKRILMEH

HLA-A*31012

[216]

CEA

CEA.24, LLTFWNPPTTAKLTI CEA.488 RTTVKTITVSAELPK

HLA-DR

[217]

MUC4

P01204

HLA-A*0201

[218]

Papillomavirus binding factor

412-420 ALPSFQIPV

HLA-A*0201, HLA-A*0206

[219]

Cyclooxygenase-2

P479 479–487, ALYGDIDAV

HLA-A*0201 & HLA-A*03

[220]

CML66

70-78 WYQDSVYYI 76-84 YYIDTLGRI

HLA-A24 HLA-A*2402

[221222]

MAGE-A4

284–293 YVKVLEHVVR; MAGE-A4 284–294 (YVKVLEHVVRV;

HLA-DPB1*0501 HLADRB1*1403-

[223]

NY-ESO-1

60-APRGPHGGAASG2

HLA-B7

[224]

HIF prolyl hydroxylase-3 (HIFPH3)

295-303 RYAMTVWYF

HLA-A24

[225]

PAX5

311-319 TLPGYPPHV

HLA-A2

[226]

Matrilysin (MMP-7)

96-107 SLFPNSPKWTSK

HLA-A3

[227]

Heparanase

525-533 (PAFSYSFFV, Hpa525), 277-285 (KMLKSFLKA, Hpa277), and 405-413 (WLSLLFKKL, Hpa405)

HLA-A2

[228]

Glypican-3

GPC3(144-152) FVGEFFTDV GPC3(298-306) EYILSLEEL

HLA-A*0201, HLA-A*2402

[229]

Cytochrome P450 1B1

CYP240 LVDVMPWLQY

HLA-A1, HLA-B35

[230]

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CLINICAL TRIALS WITH PEPTIDE BASED VACCINES Peptide vaccines incorporate defined tumor specific T cell epitopes. There are a number of newly defined CD8+ and CD4+ T cell epitopes derived from tumor antigens overexpressed on tumor cells being used for preclinical studies and are currently being used for the treatment of malignant melanoma as well as other cancers in Phase I, II and III clinical trials [150]. These vaccines attempt to induce activation and expansion of antigen-specific CD8+ and CD4+ T cells in the context of MHC class I and II molecules, respectively which can subsequently destroy tumor cells. Although peptide immunization often leads to the induction of strong T-cell responses, it has not been effective against established tumors in patients. A major challenge in developing peptide vaccines against cancer is breaking tolerance to tumor associated antigens which are self-proteins. In addition, sufficient CD4+ T helper responses are required for effective and lasting responses. To date most clinical trials have used MHC class I restricted peptides in combination with an adjuvant and no significant correlation between antigen specific CD8+ T cell expansion and the generation of protective immune responses has been shown (Table 2-4). Adjuvants The identification of tumor antigens has spurred the development of more efficient adjuvants and novel delivery systems for cancer immunotherapy (summarized in Tables 2-4). Immunologic adjuvants are used to generate effective tumor immunity by overcoming tolerance to tumor antigens which in most circumstances are self-antigens. GM-CSF acts as a growth factor to activate and stimulate DCs [151]. Intradermal immunization with GM-CSF results in activation and infiltration of DCs and enhances peptide-MHC complex formation [152]. GM-CSF has been shown in previous human clinical trials to enhance CD8+ T cell responses against tumor-specific epitopes such as tyrosinase and gp100 compared to incomplete Freund’s adjuvant (IFA) [153, 154]. It has also been suggested that GM-CSF can enhance antigen processing and presentation of full length cancer proteins [155]. DC based immunization represents a promising approach for the immunotherapy of cancer. It was recently reported in a review by Banchereau and colleagues that after summarizing 6 melanoma based clinical trials using DCs loaded with melanoma antigen, resulted in total 9.5% tumor regression compared to 4.6% for other protocols including peptide vaccination, viral vectors and tumor cells [156]. However there are a number of considerations when using DCs as adjuvants in human clinical trials including the presence of diverse human DC subsets able to induce different immune responses in vivo [157-158], DC maturation [159], migration [160], antigen loading [160-162] and host related factors. The optimal conditions required to prepare DCs to induce the most effective and appropriate immune response remains to be defined. Bacterial DNA containing CpG motifs can trigger DCs [163]. CpG has also been shown to induce functional maturation of DCs loaded with tumor antigens and is effective in tumor vaccinations and has shown protection against lethal tumor challenges in mice [164]. Flt3 ligand (FL) is an important haemopoietic cytokine and injection of FL into humans has shown the most significant increases in DC precursors [165]. Numerous clinical trials have employed FL to increase DC numbers and some significant clinical responses have been observed [34]. There are currently many preclinical and clinical studies evaluating the potential of FL as an adjuvant for cancer immunotherapy. Interleukin 2 (IL-2) was first recognized as a promising immunotherapeutic agent after high dose administration of IL-2 in cancer patients resulted in tumor regression [166-168]. The action of IL-2 is different to previously discussed adjuvants as it does not have a direct effect on cancer cells but instead alters the host immune response and supports the effector functions induced by T cell responses [169]. Toll-like receptor (TLR) ligands have been shown to induce DC

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maturation as well as enhance antigen presentation for activation of CTLs and natural killer (NK) cells. Numerous ligands for TLRs have been identified and are currently being used for clinical trials for melanoma. Imiquimod is a compound which binds to TLR-7 and TLR8 and activates macrophages and DCs and stimulates secretions of pro-inflammatory cytokines [170]. A list of adjuvants and peptides used in human clinical trials in the last 2 years are summarized in (Table 1, 2). Clinical Trials with Novel TAA Peptides Numerous TAA have been identified and the immunogenicity of their MHC class I and class II epitopes identified and studied in preclinical and clinical trials. These have been extensively reviewed in the past literature [171]. In this section we will review preclinical and clinical studies of recently identified TAA (Table 2-4). NY-ESO-1 NY-ESO-1 is expressed by a broad range of human tumors including melanoma, breast, lung, prostate and bladder but not normal tissues except the testis and is considered the most immunogenic of the cancer testis antigens [172-173]. There has been several MHC class I but only a few class II restricted tumor epitopes identified in the NY-ESO-1 gene and immunization of these epitopes in humans can elicit antibody and CTL responses in vivo [174178]. MHC class I and II restricted T cell epitopes have been identified by in vitro studies using overlapping long peptides spanning the NY-ESO-1 sequence. A number of new MHC class I restricted CTL epitopes have been identified recently and provide opportunities for new tumor associated antigens to be tested in preclinical and clinical studies. Two new epitopes (p92–104, LAMPFATPMEAEL) and (p94–102, MPFATPMEA) were identified after binding efficiently to HLA-B35 molecules and was presented and recognized by its corresponding polyclonal tumor infiltrating lymphocyte melanoma clone generated from melanoma invaded lymph nodes of stage III patients [179]. Peptide 92-104 was also presented by HLA-B51 whereas peptide 94-102 was presented by HLA-B51 as well as HLACw3 [179]. Other MHC restricted CTL epitopes to NY-ESO-1 have been generated in vitro. A HLA-A24 restricted epitope, p158-166 was identified using a computer based epitope prediction program as well as using peptide binding assays with subsequent induction of specific CTLs from HLA-A24 healthy donors and the CTL in vitro killing of NY-ESO-1 expressing carcinomas. This finding was particulary important since HLA-A24 expressing individuals dominate in certain countries including Japan and provides additional tumor antigens to be used for peptide based vaccination [180]. NY-ESO-1 specific p158-166 was also shown to induce CTLs against colon carcinoma cell line WiDr, gastric carcinoma cell lines MKN7/28 and esophageal carcinoma cell line TE8 [180]. As well as identifying novel NY-ESO-1 specific tumor epitopes, immunodominant CTL epitopes are also been engineered to improve efficacy and reduce induction of heterogenous immune responses based on the x-ray crystallography structure of HLA-A2 complexed to NY-ESO p157-165. Two peptide analogues of NY-ES0 p157-165 were synthesized with the C-terminal cysteine residue was substituted to an alanine or a serine [181]. Substitution with alanine resulted in stability of the HLA-A2, NY-ESO p157-165 complex as well as maintaining specific CTL recognition from peptide vaccinated melanoma patients [181]. In neuroblastoma patients, NY-ESO-1-specific immune responses were observed for CD4+ and CD8+ T cells after in vitro stimulation with the HLA-A2 restricted peptide NY-ESO-1 p157-165 and were also able to recognize NY-ESO-1 expressing neuroblastoma cells [182].

Peptide Based Vaccine Design

Table 2.

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Summary of Immunological Adjuvants used in Peptide Based Clinical Trials from 2004-05

Adjuvants

Peptide Based Vaccine

Immunological Efficacy

Clinical Efficacy

Adverse Reactions

Refs

GM-CSF

HLA-A1, A2, A3 restricted gp100 and tyrosinase peptides, tetanus helper peptide and montanide ISA-51

DTH, 67% antibody responses, 33% vaccine specific cell mediated responses

2 patients showed objective clinical responses 2 patients had stable disease

Vitiligo (15% patients)

[152]

Pool of eight peptides derived from the complimentarity determining regions of human antip53 antibodies

All subjects had DTH to 2 or more peptides.Antipeptide ab 4/6. No T cell response to p53.

No adverse reactions reported

[231]

DCs pulsed with HLA-A2 or HLA-A2 restricted carcinoembryonic antigen peptides

70% CEA specific T cells

20% patients had stable disease for at least 12 weeks

No adverse reaction reported

[60]

DCs were pulsed with CEA-derived, HLAA24-restricted 9-mer peptide (CEA652)

Most patients showed DTH and positive in vitro CTL response to CEA652 peptide

Long term stable disease or marked decreases in the serum CEA level were observed in some patients

No adverse reactions reported

[232]

DCs loaded with a cocktail of HLA-A2 restricted wild-type and modified p53 peptides

50% induced specific T cell responses against modified and unmodified p53 peptides

33% disease stabilization

No adverse reactions reported

[233]

DCs loaded with HLA-A2 restricted hTERT 540 peptide (ILAKFLHWL) + KLH

57% showed hTERT specific CTL responses

Partial tumor regression in 1 patient

No adverse reactions reported

[234]

DCs loaded with peptides from the melanoma antigens MAGE-3.A2, tyrosinase, gp100, and MART-1 + KLH

DTH

No clinical responses

No adverse reactions reported

[235]

DC

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Table 2 contd….

Adjuvants

Peptide Based Vaccine

Immunological Efficacy

Clinical Efficacy

Adverse Reactions

Refs

DC

DCs loaded with HLA-A1, A2, A3 restricted gp100 and tyrosinase peptides

11% and 13% peptide specific CTL in peripheral blood (PBL) and sentinel immunized node (SIN), respectively

1 patient showed objective clinical response

No adverse reaction reported

[152]

CpG 7909

CpG 7909 mixed with melanoma antigen A (Melan-A; identical to MART-1) analog peptide and IFA

100%patients exhibited rapid and strong antigenspecific T cell responses

No toxicity or adverse reactions reported

[236]

Flt3 ligand

Influenza (Flu), Melan-A (Mel), tyrosinase (Tyr), and NY-ESO-1 peptides + imiquimod+ Flt3 ligand

Increases in immature CD11c+ and CD123+ PMBC DCs

Some patients developed clinically significant toxicities related to Flt3

[237]

E75 HLA-A2 epitope from HER-2/neu + Flt3 ligand as a systemic vaccine adjuvant

Increased DC in peripheral blood DTH responses No significant peptide specific T-cell responses were detected by ELIspot

2 patients developed grade III skin reaction and grade II autoimmune hypothyroidism

[238]

IL-2

HLA-A1, A2, A3 restricted gp100 and tyrosinase peptides, tetanus helper peptide and IL-2 adminstered daily beginning day 7 (group1) or day 28(group2)

36% of peripheral blood and 38% of sentinenel immunized node in group 1, and in 53% of PBL and 83% of SINs in group 2.

Disease-free survival estimates at 2 years were 39% for group 1 and 50% for group 2.

No toxicity or adverse reactions reported

[239]

Interferonalpha

Melan-A/MART-1:2635(27L) and gp100:209-217 (210M)

5/7 enhancement of CD8+ anti-peptide responses. Increased frequency of effectors and effector memory cells.

3/7 stable disease.

Pleiotropic clinical and hematological effects

[240]

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Table 2 contd….

Adjuvants

Peptide Based Vaccine

Immunological Efficacy

TLR ligands

Imiquimod, a Toll-like receptor-7 ligand with influenza (Flu), Melan-A (Mel), tyrosinase (Tyr), and NY-ESO-1 peptides + Flt3 ligand

Increased circulating peptide-specific CD8+ T-cells compared to Flt3 ligand

Table 3.

Clinical Efficacy

Adverse Reactions

Refs

67% adverse reactions related to their cancer

[237]

Summary of Peptide Based Vaccines used in Human Clinical Trials from 2004-05

Cancer Candidate

Type of Cancer

HLA Type

Personalized peptide vaccine

Pancreatic cancer

HLA-A24 HLA-A2

Melan A

Melanoma

8 HLA-A2

Four gp100and tyrosinasederived peptides

Adjuvant

Clinical Responses

Refs

7/11 DTH increased cellular and humoral responses

[241]

CpG 7909, incomplete Freund’s adjuvant

Antigen specific CD8 T cells (>3%) Effector memory T cells – IFN gamma, granzyme B, perforin

[236]

HLA-A1 HLA-A2 HLA-A3

Tetanus helper peptide plus IL-2

IFNgamma responses in PBMC and sentinel lymph node (~40%) Disease free at 2 years ~39-50%

[239]

Individualized peptide vaccination

Prostate cancer

HLA-A24

Low dose estramustine

Decrease PSA DTH reaction at injection site

[242]

WT1 peptide-based immunotherapy

breast or lung cancer, myelodysplastic syndrome, or acute myeloid leukemia

HLA-A24

Montanide ISA51 adjuvant

18/26 received 3 or more vaccinations 12/20 showed reduction in tumor size, tumor markers, blast cells correlation between CTL and clinical responses

[243]

CTL precursororiented peptide vaccine

Advanced colorectal cancer

10 HLAA24

5/10 patients showed increases antigen specific T cells 7/10 patients showed Aantipeptide IgG 3/10 DTH response 1/10 partial clinical response

[244]

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Table 3. contd….

Cancer Candidate

Type of Cancer

HLA Type

Adjuvant

Clinical Responses

Refs

Pool of eight peptides derived from the complimentarity determining regions (CDRs) of human anti-p53 antibodies

Advanced malignancy

6/14 completed trial

GM-CSF

DTH Lots of antibodies and some cellular responses No T cell responses to p53

[231]

bcr-abl-derived fusion peptide vaccine

Chronic myelogenous leukaemia

Quillaja saponaria (QS-21)

14/14 DTH, CD4 proliferation 11/14 IFN gamma ELISPOT

[245]

ESO-1:165V (HLA-A2) NY-ESO-1:161-180 (HLA-DP4)

Metastatic melanoma

Antigen specific T cells to ESO-1:165V more than NY-ESO1:161-180

[189]

Influenza peptide NY-ESO-1 Melan A Tyrosinase

27 metastatic and high-risk melanoma

Flt3 ligand 8 patients had imiquimod (TLR7 ligand) applied topically

Increase CD11c+ and CD123+ Peptide specific CD8+ T cells in imiquimod vaccination patients

[237]

MUC1 peptide (100mer) (5xrepeat regions)

16 advanced pancreatic cancer

SB-AS2

Increase CD8+ T cells Increases total MUC1 antibody2 patients alive after 5 years

[246]

T-helper epitope derived from the melanoma differentiation antigen

Resected, high-risk metastatic melanoma

HLA-DR4

Th1 and Th2 T cell proliferation to epitope Secrete granzyme B, cytolysis

[247]

Carcinoembryonic antigen (CEA) peptides

10 late-stage colorectal carcinoma

HLA-A2 HLA-A24

Increase CEA specific T cells (70%) 2/10 stable disease for 12 weeks

[248]

CTL precursororiented peptide vaccine

10 advanced colorectal carcinoma

HLA-A24

5/10 patients showed increases antigen specific T cells 7/10 patients showed Aantipeptide IgG 3/10 DTH response 1/10 partial clinical response

[244]

HLA-A2 HLA-DP4

Autologous DC + TNFalpha

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Table 3. contd….

Cancer Candidate

Type of Cancer

HLA Type

Adjuvant

Clinical Responses

Refs

CEA652 9mer peptide

CEA-expressing metastatic gastrointestinal or lung adenocarcinomas

HLA-A24

Autologous DC (GM-CSF, IL-4)

DTH responses in most patients In vitro CTL response to CEA652 peptide after therapy

[232]

Cocktail of three wild-type and three modified p53 peptides

6 progressive advanced breast cancer

HLA-A2

Autologous DC

2/6 stable disease 3/6 specific T-cell responses against modified and unmodified p53 peptides

[233]

hTERT I540 peptide

Advanced breast and prostate carcinoma

HLA-A2

Ex-vivo DC and KLH

4/7 induced antigen specific responses 1/8 partial tumor regression

[234]

MAGE-3.A2, tyrosinase, gp100, and MART-1

14 AJCC stage IV melanoma

Non matured DC + KLH

5/14 stable disease 50% patients had responses to MART-1 peptide and a third to the other melanoma peptides

[235]

Table 4.

Summary of Peptide Based Vaccines used in Human Clinical Trials

Cancer Candidate

Type of Cancer

HLA Type

Adjuvant

Clinical Responses

Refs

MAGE, MART1/MelanA, gp100 and tyrosinase helper T-cell epitope peptides

Stage IIIB to IV melanoma

At least on HLA-DR1, DR4, -DR11, DR13 or – DR15

Montanide ISA-51

Proliferation responses to peptides in 81% patients.Objective clinical responses in 2/17 (1-3.9+ yrs), stable disease in 2/17 (1.8-4.6+ yrs).

[249]

CEA, HER2, p53, MAGE and PADRE (IDM-2101)

Non-small cell lung cancer

HLA-A2

Montanide ISA-51

One year survival was 60% and median survival time 17.3 months. 1 complete and 1 partial response. Survival longer in patients with an anti-peptide response

[250]

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Table 4. contd….

Cancer Candidate

Type of Cancer

HLA Type

Adjuvant

Clinical Responses

Refs

Gp100, MART1, tyrosinase, MAGE-3.A2, AMGE-A10 & NA17

Metastatic melanoma

HLA-A2

DC ± IL-2

Intranodal injection. 2/9 PR, 3 SD in groups receiving IL2 and 2 SD in groups witout IL2. No difference in survival between groups that receive IL2 or not.

[251]

NY-ESO-1b

Ovarian cancer - after primary surgery and chemotherapy

HLA-A2

Montanide ISA51

3/4 NY-ESO-1+ve patients had T-cell immunity by tetramer & ELISpot, 6/9 media progression-free survival 13 months, 3/9 complete remission at 25, 38 and 52 months. T cell immunity in patients with or without NY-ESO-1 positive tumors.

[252]

WT1 peptide-based immunotherapy

Gynecological cancer

HLA-A24

Montanide ISA51 adjuvant

12 weekly injections 3/12 stable disease, 9/12 progressive disease

[253]

Her-2/neu

Metastatic breast cancer

Not restricted

Influenza virosomes

IM day 1, 28 & 56 8/10 increase peptide-specific antibody Increased IL-2, IFNgamma, TNFalpha

[254]

Her-2/neu (GP2)

Disease free, lymph node – ve breast cancer patients

HLA-A2

GM-CSF

Dose escalation. In vivo GP2-specific T cells increased from pre-vaccination: 0.8%-1.6%. Epitope spreading

[255]

HER2

Various metastatic tumors

B ell epitope and measles virus T cell epitope

Muramyl dipeptide/ SEPPIC ISA 720

Dose escalation.MTD 3 mg. 4 patients had stable disease, 2 partial responses & 11 progressive disease.

[256]

P53

Ovarian cancer

Not specified

Montanide ISA51

P53-specific T cells in all patients. T cells were CD4+ and produced Th1 and Th2 cytokines.Stable disease evaluated by CA-125 and CT scans in 2/20.

[257]

Vascular endothelial growth factor 2

Metastatic pancreatic cancer

HLA-A*2402

Montanide ISA 51

Dose escalation. CTL in 11/18 patients. Median overall survival time = 8.7 months.

[258]

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Table 4. contd….

Cancer Candidate

Type of Cancer

HLA Type

Adjuvant

Clinical Responses

Refs

HPV-16 E6/E7

Vulvar intraepithelial neoplasia

No restriction

Montanide ISA 51

At 12 months 15/19 had clinical responses with complete responses in 9/19.Strong CD4 and CD8 T cell responses in patients with complete response

[259]

Bcr-Abl fusion peptide

Chronic myeloid leukemia

No restriction

Montanide ISA 51

3/10 1-log reduction of Bcr-Able transcript levels

[260]

PSA, PSCA, PSMA, Survivin, Prostein, TRP, Flu matrix peptide

Prostate cancer-rising PSA but no detectable desease

HLA-A*0201

Montanide ISA51 + imiquimod, GMCSF, mucin-1mRNA/protamine, local hyperthermia or no adjuvant

Analysis based on PSA levels: 4/19 increased PSA, 2/11 stable PSA during vaccination, 3/19 decreased PSA (all received TLR-7 agonist), 11/19 increased PSA levels.

[261]

Mucin 1

Pancreatic & biliary cancer after resection of primary tumor.

No restriction

Dendritic cells

Transient increase of FoxP3+CD4+ cells after each immunization. 4/12 patients alive at 4-year follow up.

[262]

Survivin

Urothelial cancer

HLA-A24

Montanide ISA 51

Increased CTL precursor frequency in 5/9 patients. Small tumor reduction in 1 patient.

[263]

Survivin

Breast cancer

HLA-A*2402

± Montanide ISA 51

Increased peptide-specific CTL frequency in peptide + IFA groups. No clinical responses

[264]

CD4+ T cells play an important role in the induction and maintenance of adequate CD8+ T cell mediated anti-tumor responses. Therefore, identification of MHC class II restricted tumor antigenic epitopes is of major importance for the development of effective immunotherapies and has been subject to a great deal of study over the past few years. It was noted that polyclonal CD4+ T cell responses reacted to known and unknown NY-ESO-1 epitopes in 11 out the 13 melanoma patients tested while no healthy donors showed any responses [183]. The most immunogenic of the CD4+ epitopes tested was NY-ESO-1 peptide 80-109 containing restricted by HLA-DP4, HLA-DR, HLA-DR7 as well as a peptide showing diverse HLA class II binding [183-184]. Peptide 87–111 bound to HLA-DRB1 (DRB1-0101, DRB1-0401, DRB1-0701, DRB1-1101, DRB1-1501, DRB5-0101) [185]. In addition, p87–

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111 and p87-101 was able to stimulate both HLA-DR and HLA-DP4 restricted CD4+ T cells from patient PBMCs. These patient specific NY-ESO-1 p87-111 and p87-101 CD4+ T cells were subsequently able to recognize autologous melanoma cell lines and autologous DCs loaded with NY-ESO-1 protein or transfected with NY-ESO-1 cDNA and stimulate both Th1 and Th2 CD4+ T cell responses [185]. Two novel CD4+ T cell epitopes were also identified after patients were immunized with monocyte derived dendritic cells pulsed with full-length NY-ESO-1 protein formulated with ISCOMATRIX [186]. HLA-DR2 restricted peptide (NY-ESO-1186–99) was dominant in one patient sample and suggested that the previously identified immunodominant HLA-DP4 restricted NY-ESO-1 p157-170 epitope was not the immunodominant epitope presented and others should be considered for inclusion in vaccine preparations [186]. Recently, CD4+ T cell clones were generated from a patient with NY-ESO-1 expressing synovial sarcoma by stimulation. These generated CD4+ clones were generated by peptide NY-ESO-1 p49-66 and p55-72 stimulation and was shown to be HLA-DQ B1 03011 restricted. Furthermore, the CD4+ clones stimulated with p49-66 showed cross-reactivity with p55-72 but not visa versa [187]. As well as reacting to the specific peptides, the NY-ESO-1 peptide reactive CD4+ T cell clones reacted against the naturally presented NY-ESO-1. A clinical trial using peptides p157–167 (SLLMWITQCFL), p157–165 (SLLMWITQC), and p155–163 (QLSLLMWIT) in 33% DMSO and GM-CSF were injected in HLA-A2+ patients with progressing NY-ESO-1 expressing metastatic tumors of different types [175177]. Both seronegative and seropositive patients induced antigen specific CD8+ IFN-
T cell responses and developed DTH responses [175-177, 188]. There have been suggestions that synthetic peptide immunization derived from NY-ESO-1 may not be the most effective method of inducing anti-tumor responses in melanoma patients. Patients with metastatic melanoma injected with HLA-A2 restricted peptide, p165V, a HLA-DP4 restricted peptide p161-180 or both peptides given in parallel did not induce significant antigen specific CD8+ T cells with any of the vaccination regimes [189]. Subsequent clinical trials using NY-ESO1 have used recombinant proteins and not peptides for melanoma patients. NY-ESO-1 protein formulated with the ISCOMATRIX adjuvant induced antibody responses, strong delayed-type hypersensitivity reactions and circulating NY-ESO-1 epitope specific CD8+ and CD4+ T cells [190]. A peptide based vaccine consisting of five defined melanoma associated peptides, including three overlapping NY-ESO-1 peptides, SLLMWITQCFL p157-167, SLLMWITQC p157-165, and QLSLLMWIT p155-163; as well as a tyrosinase internal peptide and Melan-A/MART-1 analogue peptide ELAGIGILTV in the presence of Flt3 ligand induced potent CD8+ and CD4+ T cells at the site of injection and the CD8+ T cells expanded and could also recognize natural tumor antigen, produce IFN-
and kill in a cytotoxicity assay in patients with melanoma or resected stage II, III, or IV melanoma [191]. It is clear that NY-ESO-1 antigen is immunogenic in human clinical trials whether administered as protein or peptide. Mammaglobin-A Mammaglobin-A is a novel breast cancer associated antigen and is overexpressed in 80% of primary and metastatic breast tumors [192-193]. Levels of Mammaglobin-A are maintained among well, moderately and poorly differentiated breast tumors [194] and mammaglobin gene expression on leukapheresis products of high-risk breast cancer patients is an indicator of poor prognosis [195]. Only recently it was shown that mammaglobin-A can exist in two forms in breast tumor tissue and the high molecular weight form correlated negatively with tumor grade and proliferation rate [196]. Mammaglobin-A is largely restricted to mammary epithelium, thus, a clear understanding of T cell mediated immunity to

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mammaglobin is important in designing specific peptide based vaccines against breast cancer [197]. Breast cancer patients have a significantly higher frequency of mammaglobin-Areactive T cells as compared to normal individuals. Eight HLA-A3 restricted epitopes identified using MHC class I binding prediction program detected high (Mam-A3.3, Mam-A3.5, Mam-A3.6, Mam-A3.7) and low (Mam-A3.1, Mam-A3.2, Mam-A3.4, Mam-A3.8) affinity peptides [198]. CTL were observed against peptides low affinity peptides but not high affinity peptides. Mam-A3.1 was recognized as the immunodominant epitope [198]. In addition, HLA-A2 restricted epitopes have also been identified [199]. CTL recognized peptides MamA2.1, Mam-A2.2, Mam-A2.3, Mam-A2.4 and Mam-A2.7, however Mam-A2.2 was also recognized by T cells from healthy individuals [199]. When CTL lines were generated from HLA-A2 healthy individuals, they recognized MamA2.1, Mam-A2.2, Mam-A2.3 and MamA2.4 but not Mam-A2.7 and were able to kill UACC-812 breast cancer cell lines in vitro. DC transduced with a Tat-mammaglobin fusion protein stimulated antigen-specific CD4+ and CD8+ T cells in mice and further suggested the potential development of Mammaglobin-A as a vaccine candidate for breast cancer [200]. EphA2 EphA2 is a transmembrane receptor tyrosine kinase that is up-regulated on many aggressive tumor cells [201-202]. EphA2 is overexpressed and functionally altered on a number of cancers and promotes metastatic disease. In normal cells, EphA2 localizes to sites of cell-tocell contact and functions as a negative regulator of cell growth [203]. EphA2 is an attractive candidiate for cancer immunotherapy as it is overexpressed on a number of tumors (breast, prostate, colon, lung) at high levels relative to surrounding epithelium and the highest levels are found on the most aggressive tumor cells [201, 202]. Interestingly, when EphA2 binds to its receptor, ephrinA1, it negatively regulates tumor cell growth and migration, however, EphA2 does not bind to its receptor on cancer cells and this does not affect its enzymatic activity [204]. Inhibitory antibodies have been designed to bind to EphA2 which results in its autophosphorylation and degradation and inhibits cancer cell growth [205]. In renal cell carcinoma (RCC) patients, CD4+ and CD8+ specific EphA2 T cell responses are detectable where the presence of CD8+ T cells is inversely correlated to the presence of disease in patients and increases after patients receive treatment [206]. CD4+ T cell responses have also been correlated with disease progression where the more advanced forms of RCC skew towards a Th2 response. Although the presence of CD8+ T cells in RCC was associated with disease-free stage, HLA-A2 restricted EphA2883–891 specific CD8+ responses in RCC patients did not, however, it was noted that in 3 HLA-A2+ RCC patients with stage I disease, antigen-specific CD8+ T cells increased after curative surgery [206]. In addition, EphA2663–677 specific Th1 specific CD4+ T cell responses were increased in Stage I RCC patients while Th2/Tr1 specific CD4+ T cell responses were consistently presence in Stage IV RCC patients [206]. Two HLA-A2-restricted epitopes (EphA258 and EphA2550) have been described. These epitopes have a high affinity for HLA-A2, trigger CTL in HHD mice and in vitro in healthy humans and patients with prostate cancer [207]. Furthermore, in mouse models, immunization with H-2Kb-binding mEphA2682–689, and I-Abbinding mEphA230–44 loaded DC in C57BL/6 mice induced specific CTL lysis in vitro. Mice immunized with peptides, EphA2671–679, EphA2682–679, or EphA230–44 peptides showed protection against a EphA2 negative B16 tumor challenge and induced significant suppression of lung metastases after challenge with B16-BL6 tumor cells [208]. Mechanism of EphA2 protection was due to inhibition of vascular endothelial growth factor (VEGF) induced angiogenesis.

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HCA587 The HCA587 gene belongs to MAGE-C subfamily with a large terminal exon encoding a protein of 373 aa and is highly expressed on human hepatocellular carcinoma (HCC) tissues [209]. HCA587 is not expressed on normal tissues except germ cells in testis and Purkinji cells in cerebellum. HCA587 protein is expressed on 37.1% from 71 HCC samples [210]. High levels of mRNA HCA587 have been detected in RCC patients [211]. It was also suggested that higher HCA587 protein levels correlated with poorly differentiated HCC tumors and HCA587 specific antibodies were only found in patients with poorly differentiated tumors [210]. Healthy individuals immunized with autologous DC loaded with HCA587 protein induce specific T cell responses [212]. HCA587-specific CD8+ and CD4+ T cell proliferation was detected as well as intracellular IFN-
expression by PBMCs [212]. Six HCA587-derived peptides that bind to HLA-A2 have been described [213]. DC pulsed with high affinity binding HLA-A2 restricted HCA587 peptide p317-325, stimulated CD8+ T cells in the presence of IL-2 and IL-6 and generated HLA-A2 restricted CD8+ T cells against HCA587 p317-325 in healthy donors [213]. CONCLUSION A number of tumor associated antigens have been characterized, with some of the more recent ones described here. It is clear that they all induce cellular and humoral responses in patients when delivered in an appropriate manner and are good targets for tumor immunotherapy studies. These antigens are either unique to a particular tumor type or are expressed at an elevated level on tumor cells compared to normal cells. There are also universal tumor antigens such as telomerase reverse transcriptase and survivin that are ideal targets particularly if combined with specific TAA. Many of these antigens or minimal epitopes together with a variety of adjuvants have been tested in preclinical studies and clinical studies. Nevertheless, the outcomes from the vaccine clinical trials is not impressive. Recently, much effort has been directed towards the identification of new TAA, using protein profiling, DNA arrays and prediction of epitopes in silico. Designing peptide based vaccines is a challenge, however, a number of delivery methods show promise in both preclinical and clinical settings. Synthetic chemical methods are available to design peptide vaccines incorporating various features to optimize immunogenicity such as minimal epitopes from multiple antigens (T helper, CTL and B cell epitopes, TLR activating compounds). Even the optimal vaccine may need to be administered with various cytokines that modulate T cell responses and overcome inhibitory signals in the microenvironment of tumors. ABBREVIATIONS aa

=

Amino acid

Ag

=

Antigen

APC

=

Antigen presenting cell

APL

=

Altered peptide ligand

CEA

=

Carcinoembrionic antigen

CTL

=

Cytotoxic T lymphocytes

DC

=

Dendritic cells

MAP

=

Multiple Antigenic Peptide

PBMC

=

Peripheral blood mononuclear cells

Peptide Based Vaccine Design

TAA

=

Tumor associated antigen

TLR

=

Toll-like receptor

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ACKNOWLEDGEMENTS VA was supported by an NH&MRC R. Douglas Wright Fellowship (223316), NHMRC project grant 223310 and Beauties and the Beast. GAP was supported by NHMRC project grant 266818 and Cancer Council Victoria. In addition, all authors were supported by Burnet Institute at Austin. The authors would like to thank Dr Eliada Lazoura and Dr Gareth Chelvanayagam for preparation of part of - Fig. (1 and 3) respectively. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8]

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The impact of imiquimod, a Toll-like receptor-7 ligand (TLR7L), on the immunogenicity of melanoma peptide vaccination with adjuvant Flt3 ligand. Cancer Immun., 2004, 4, 9. McNeel, D. G.; Knutson, K. L.; Schiffman, K.; Davis, D. R.; Caron, D.; Disis, M. L., Pilot study of an HLA-A2 peptide vaccine using flt3 ligand as a systemic vaccine adjuvant. J. Clin. Immunol., 2003, 23, 6272. Slingluff, C. L., Jr.; Petroni, G. R.; Yamshchikov, G. V.; Hibbitts, S.; Grosh, W. W.; Chianese-Bullock, K. A.; Bissonette, E. A.; Barnd, D. L.; Deacon, D. H.; Patterson, J. W.; Parekh, J.; Neese, P. Y.; Woodson, E. M.; Wiernasz, C. J.; Merrill, P. Immunologic and clinical outcomes of vaccination with a multiepitope melanoma peptide vaccine plus low-dose interleukin-2 administered either concurrently or on a delayed schedule. J. Clin. Oncol., 2004, 22, 4474-4485. Di Pucchio, T.; Pilla, L.; Capone, I.; Ferrantini, M.; Montefiore, E.; Urbani, F.; Patuzzo, R.; Pennacchioli, E.; Santinami, M.; Cova, A.; Sovena, G.; Arienti, F.; Lombardo, C.; Lombardi, A.; Caporaso, P.; D'Atri, S.; Marchetti, P.; Bonmassar, E.; Parmiani, G.; Belardelli, F.; Rivoltini, L. Immunization of stage IV melanoma patients with Melan-A/MART-1 and gp100 peptides plus IFN-alpha results in the activation of specific CD8(+) T cells and monocyte/dendritic cell precursors. Cancer Res., 2006, 66, 4943-4951.

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USA., 2004, 101, 13885-13890. Sato, Y.; Maeda, Y.; Shomura, H.; Sasatomi, T.; Takahashi, M.; Une, Y.; Kondo, M.; Shinohara, T.; Hida, N.; Katagiri, K.; Sato, K.; Sato, M.; Yamada, A.; Yamana, H.; Harada, M.; Itoh, K.; Todo, S. A phase I trial of cytotoxic T-lymphocyte precursor-oriented peptide vaccines for colorectal carcinoma patients. Br. J. Cancer, 2004, 90, 1334-1342. Cathcart, K.; Pinilla-Ibarz, J.; Korontsvit, T.; Schwartz, J.; Zakhaleva, V.; Papadopoulos, E. B.; Scheinberg, D. A. A multivalent bcr-abl fusion peptide vaccination trial in patients with chronic myeloid leukemia. Blood, 2004, 103, 1037-1042. Ramanathan, R. K.; Lee, K. M.; McKolanis, J.; Hitbold, E.; Schraut, W.; Moser, A. J.; Warnick, E.; Whiteside, T.; Osborne, J.; Kim, H.; Day, R.; Troetschel, M.; Finn, O. J. Phase I study of a MUC1 vaccine composed of different doses of MUC1 peptide with SB-AS2 adjuvant in resected and locally advanced pancreatic cancer. Cancer Immunol. Immunother., 2005, 54, 254-264. Wong, R.; Lau, R.; Chang, J.; Kuus-Reichel, T.; Brichard, V.; Bruck, C.; Weber, J. Immune responses to a class II helper peptide epitope in patients with stage III/IV resected melanoma. Clin. Cancer Res., 2004, 10, 5004-5013. Liu, K. J.; Wang, C. C.; Chen, L. T.; Cheng, A. L.; Lin, D. T.; Wu, Y. C.; Yu, W. L.; Hung, Y. M.; Yang, H. Y.; Juang, S. H.; Whang-Peng, J. Generation of carcinoembryonic antigen (CEA)-specific T-cell responses in HLA-A*0201 and HLA-A*2402 late-stage colorectal cancer patients after vaccination with dendritic cells loaded with CEA peptides. Clin. Cancer Res., 2004, 10, 2645-2651. Slingluff, C. L., Jr.; Petroni, G. R.; Olson, W.; Czarkowski, A.; Grosh, W. W.; Smolkin, M.; ChianeseBullock, K. A.; Neese, P. Y.; Deacon, D. H.; Nail, C.; Merrill, P.; Fink, R.; Patterson, J. W.; Rehm, P. K. Helper T-cell responses and clinical activity of a melanoma vaccine with multiple peptides from MAGE and melanocytic differentiation antigens. J. Clin. Oncol., 2008, 26, 4973-4980. Barve, M.; Bender, J.; Senzer, N.; Cunningham, C.; Greco, F. A.; McCune, D.; Steis, R.; Khong, H.; Richards, D.; Stephenson, J.; Ganesa, P.; Nemunaitis, J.; Ishioka, G.; Pappen, B.; Nemunaitis, M.; Morse, M.; Mills, B.; Maples, P. B.; Sherman, J.; Nemunaitis, J.J. Induction of immune responses and clinical efficacy in a phase II trial of IDM-2101, a 10-epitope cytotoxic T-lymphocyte vaccine, in metastatic nonsmall-cell lung cancer. J. Clin. Oncol., 2008, 26, 4418-4425. Hersey, P.; Halliday, G. M.; Farrelly, M. L.; DeSilva, C.; Lett, M.; Menzies, S. W. Phase I/II study of treatment with matured dendritic cells with or without low dose IL-2 in patients with disseminated melanoma. Cancer Immunol. Immunother., 2008, 57, 1039-1051. Diefenbach, C. S.; Gnjatic, S.; Sabbatini, P.; Aghajanian, C.; Hensley, M. L.; Spriggs, D. R.; Iasonos, A.; Lee, H.; Dupont, B.; Pezzulli, S.; Jungbluth, A. A.; Old, L. J.; Dupont, J. Safety and immunogenicity study of NY-ESO-1b peptide and montanide ISA-51 vaccination of patients with epithelial ovarian cancer in high-risk first remission. Clin. Cancer Res., 2008, 14, 2740-2748. Ohno, S.; Kyo, S.; Myojo, S.; Dohi, S.; Ishizaki, J.; Miyamoto, K.; Morita, S.; Sakamoto, J.; Enomoto, T.; Kimura, T.; Oka, Y.; Tsuboi, A.; Sugiyama, H.; Inoue, M., Wilms' tumor 1 (WT1) peptide immunotherapy for gynecological malignancy. Anticancer Res., 2009, 29, 4779-4784. Wiedermann, U.; Wiltschke, C.; Jasinska, J.; Kundi, M.; Zurbriggen, R.; Garner-Spitzer, E.; Bartsch, R.; Steger, G.; Pehamberger, H.; Scheiner, O.; Zielinski, C.C. A virosomal formulated Her-2/neu multipeptide vaccine induces Her-2/neu-specific immune responses in patients with metastatic breast cancer: a phase I study. Breast Cancer Res. Treat., 2010, 119, 673-83. Carmichael, M. G.; Benavides, L. C.; Holmes, J. P.; Gates, J. D.; Mittendorf, E. A.; Ponniah, S.; Peoples, G. E. Results of the first phase 1 clinical trial of the HER-2/neu peptide (GP2) vaccine in disease-free breast cancer patients: united States Military Cancer Institute Clinical Trials Group Study I-04. Cancer , 2009. Kaumaya, P. T.; Foy, K. C.; Garrett, J.; Rawale, S. V.; Vicari, D.; Thurmond, J. M.; Lamb, T.; Mani, A.; Kane, Y.; Balint, C. R.; Chalupa, D.; Otterson, G. A.; Shapiro, C. L.; Fowler, J. M.; Grever, M. R.; BekaiiSaab, T. S.; Carson, W. E. 3rd, Phase I active immunotherapy with combination of two chimeric, human epidermal growth factor receptor 2, B-cell epitopes fused to a promiscuous T-cell epitope in patients with metastatic and/or recurrent solid tumors. J. Clin. Oncol., 2009, 27, 5270-5277.

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Inhibitors of Protein: Geranylgeranyl Transferases Farid El Oualid1, Gijs A. van der Marel2 and Mark Overhand2,* 1

Netherlands Cancer Institute, Division of Cell Biology, 1066 CX Amsterdam, The Netherlands; 2Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands Abstract: The enzyme protein:geranylgeranyl transferase-1 (PGGT-1 or GGTase-I) catalyzes the geranylgeranylation of cysteine residues near the C-termini of a variety of proteins, including most monomeric GTP binding precursor proteins belonging to the Rho, Rac and Rap subfamilies. These proteins are involved in signaling pathways controlling important processes such as cell differentiation and growth. In the framework of the development of therapeutics against disorders associated with aberrant cell proliferation, the interference with these signal transduction cascades has been a major focus of investigation. For instance, PGGT-1 inhibitors have shown promise in the treatment of cancer, smooth muscle hyperplasia as well as parasitic infections, such as malaria. In this chapter, we discuss the structural and mechanistic aspects of the protein:geranylgeranyl transferases and their importance with respect to the terpene metabolism. In view of the latter, several terpene based proteomic probes have been developed and applied. An extensive summary of reported inhibitors of PGGT-1, classified as natural products, peptide substrate (Ca1 a2L box), terpene substrate (geranylgeranyl pyrophosphate) and others, is presented. The few known inhibitors of the other geranylgeranylating enzyme, protein:geranylgeranyl transferase-2 (PGGT-2) also known as Rab geranylgeranyl transferase are also included.

Keywords: Protein: geranylgeranyl transferase-1 and -2, peptidomimetics, Ca1a2L box, small GTP binding proteins, signal transduction, aberrant cell proliferation, anti-cancer agents, functional proteomics. 1. INTRODUCTION Numerous cytosolic polypeptides are post-translationally modified before they reach their mature state as functional proteins [1]. Protein prenylation is a post-translational event that facilitates the anchoring of certain proteins to cellular membranes (Scheme 1). Examples of post-translationally prenylated proteins are (small) GTP binding proteins that function as molecular switches in signaling pathways, involving cell growth and differentiation. The wide interest in the interference of prenylation is primarily based on the finding that several prenylated small GTP binding proteins are involved in the malignant transformation of cells. *Corresponding author: Tel: 0031715274483; Fax: 0031715274407; E-mail: [email protected] Allen B. Reitz / Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.

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Acetyl CoA

HMG CoA synthase

El Oualid et al.

growth factor receptor tyrosine kinase

cholesterol lowering agents (statins)

HMG CoA HMG CoA reductase

P

Y Y

NF1/GAP

P Ras GDP

Ras GTP SOS1

O O O O O P P O- O-

O

OH

-O

mevalonate

GPP

membrane anchoring agents against osteoporosis (bisphosphonates)

FPP synthase

S

O O O O O P P squalene synthase OO FPP

cholesterol lowering agents

Rho

N H

OMe

additional lipid modifications possible (S-palmitoylation & N-myristoylation)

sterols (e.g. cholesterol)

O

GGPP

O O O O P P O- O-

mature protein

O

Golgi

squalene

GGPP synthase

subject of this chapter

Gab/SHC GRB2/SHP2 complex

OH

activation

- cytoskeletal organization (TIAM1, Rac) - survival (PI3K, PDK1, Akt) - cell proliferation (BRAF, ARAF, RAF1, MEK, ERK) - vesicle trafficking (RALGDS, RGL, RGL2, RaI, PLD) - Calcium signalling (PLCε, PKC)

isoprenylcysteine carboxyl methyltransferase

endoplasmic reticulum

anti-tumor agents anti-tumor agents

proteolyic cleavage a1a2L tripeptide

PGGT-1

drug de deve elopment for agents against: cancer, smooth muscle hyperplasia, multiple sclerosis parasitic infections, osteoporosis, hepatitis C virus infection

S

Rho N H

a1a2L

O

Scheme 1. Part of the terpene metabolism involved in geranylgeranylation of precursor proteins (e.g. Rho) - potential and existing drug targets are indicated [6d]. In contrast to H-Ras, N-Ras and K-Ras4a, K-Ras4B is not palmitoylated. Instead it uses a polybasic lysine-rich sequence near its C-terminal cysteine for additional interactions with the plasma membrane. Although isoprenylation is a major determinant for proper functioning of most GTPases, subsequent post-translational modifications are also important. These post-translational modifications comprise a number of steps. First, there is the proteolytic cleavage of the a1a2X tripeptide by the protease Rce. Next, the formed C-terminal carboxylic group is methylated by isoprenylcysteine carboxyl methyltransferase. In the case of H-Ras, N-Ras and K-RasA, upstream cysteine residues are then palmitoylated through the action of a palmitoyl transferase, adding additional hydrophobicity and thus promote further association to the cell membrane. K-RasB on the other hand, contains a stretch of upstream located positively charged lysine residues which allow K-RasB to interact with the negatively charged heads of the lipid bilayer. It is believed that this electrostatic interaction substitutes for the palmitoyl interaction present in H-Ras, NRas and K-RasA.

The first step in the post-translational modification of GTP binding proteins involves the covalent attachment of a farnesyl (C15) - or a geranylgeranyl (C20) chain to the thiol functionality of the cysteine residue in the Ca1a2X motif, a C-terminal tetrapeptide consensus sequence. Precursor proteins with X= methionine, serine, alanine or glutamine, are normally farnesylated by protein:farnesyl transferase (PFT), while geranylgeranylation by protein:geranylgeranyl transferase-1 (PGGT-1) occurs preferentially for X= leucine [2]. The most important and abundant small GTP binding proteins involved in human tumorigenesis are members of the Ras family [3]. The observation that certain small peptide sequences inhibit PFT [4a], thereby preventing the prenylation of oncogenic Ras precursor

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proteins and thus their projected signaling malfunction [4b], initiated a search for effective PFT inhibitors [4]. However, it was found that upon blocking PFT, N-Ras and the most abundant human oncogenic Ras precursor protein K-RasB can be geranylgeranylated through the action of PGGT-1 [5]. This identifies both PFT and PGGT-1 as important targets for the development of anti-tumor agents aimed at disabling prenylation [6]. In addition, PGGT-1 inhibitors are potentially valuable agents for the treatment of smooth muscle hyperplasia [7] in relation to restenosis/atherosclerosis [8], multiple sclerosis [9], parasitic infections [10], osteoporosis [11], and hepatitis C virus infection [12]. 2. MECHANISM OF ACTION AND STRUCTURE OF PGGT-1 The majority of the prenylated proteins in eukaryotic cells are geranylgeranylated. Polypeptide substrates of mammalian PGGT-1 (ec 2.5.1.59) include the -subunits of nine of the 12 heterotrimeric GTP binding proteins and the majority of small GTP binding precursor proteins belonging to the Rho, Rac and Rap subfamilies [13]. In the prenylation reaction, PGGT-1 uses geranylgeranylpyrophosphate (GGPP) as donor substrate and a Zn2+ ion as co-factor. The structure of mammalian PGGT-1 has been established recently with X-ray analysis [14a]. PGGT-1 is a heterodimeric protein consisting of a 48 kDa
-subunit and a 43 kDa
subunit [15], with both subunits consisting of helical domains (Fig. 1, top box). The helices of the
-subunit are arranged in
-helical hairpin pairs, forming a crescent that wraps around the -subunit having an interface area that exceeds 3300 Å2. The -subunit forms a compact, globular,
-
barrel domain with a central funnel-shaped cavity. The substrate binding region involves part of the interface between the
- and  subunits and extends into the central funnel-shaped cavity of the -subunit, which is lined with hydrophobic residues. A zinc ion is positioned at the top of this active site funnel.

CVIL peptide funnel shaped active site Zn2+ catalytic triangle

β-subunit GGPP

β-subunit α-subunit

Fig. (1). X-ray structure of mammalian PGGT-1 (pdb 1N4P, 1N4Q, 1N4R and 1N4S). The left part shows a top view of the
-subunit with the central funnel shaped active site and the Zn2+ binding site highlighted.

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Based on the X-ray analysis of a series of PGGT-1 substrate/inhibitor complexes, in combination with the previously obtained molecular insight in the mechanism of action [16] of the related enzyme PFT (ec 2.5.1.58,
-subunit identical, -subunit 25% homology), a working model of PGGT-1 has been postulated [14a]. The catalytic cycle commences with the binding of GGPP (A in Scheme 2), with its C20 chain inserted in the hydrophobic funnelshaped cavity and its diphosphate moiety placed at the
- subunit interface. The terminal isoprene moiety binds to the enzyme in an angle of ~ 90o with respect to the rest of the chain, thus adopting a distinct conformation (A in Scheme 2). Once loaded with GGPP, the enzyme can bind to the (poly)peptide substrate that adopts an extended conformation (B in Scheme 2). The carboxylic acid functionality of the Ca1a2L sequence, as well as the C=O of the a2 residue, are involved in hydrogen bonding interactions with the enzyme. The side chain of the a1 residue projects into the solvent, while the a2 side chain makes extensive hydrophobic contacts with the enzyme and the terminal isoprene unit. The large contact area (140 Å2) between the two substrates is the likely origin for the observed ordered substrate binding and the non-productive binding of Ca1a2L containing peptides in the absence of GGPP [17]. The side chain functionality of the cysteine residue of the consensus sequence coordinates the zinc ion as its thiolate [17d]. Subsequently, the terpene chain is presumed to undergo a conformational change (rotation about the C6-C7-C8-C9 dihedral angle), reorienting the diphosphate moiety and the first two isoprene units attached to it. The position of the -phosphate of the diphosphate moiety changes only slightly, while its -phosphate is repositioned in such a manner that C1 is in close proximity to the cysteine thiolate (C’ in Scheme 2). The thiolate can now form a thioether linkage by a nucleophilic attack on C1, (B
C’
C in Scheme 2), releasing pyrophosphate [2c, d]. Upon binding of the next GGPP substrate, the formed prenylated product is shifted to a secondary binding site. In this binding mode, the leucine residue remains in its binding pocket, but the Ca1a2L sequence adopts a -turn conformation creating a hydrogen bonding interaction between the cysteine C=O and the NH of the leucine (D in Scheme 2). Binding of the next protein substrate or interaction with the next enzyme in the post-translational cascade facilitates the release of the geranylgeranylated product. There is no evidence that the enzyme undergoes any conformational changes during the proposed catalytic cycle. This proposed mechanism of action of PGGT-1 forms the basis to understand the manner by which the enzyme selects its two substrates. The terpene binding site of PGGT-1 is always occupied during the catalytic cycle, either by GGPP or the geranylgeranyl moiety of the prenylated product. It has been shown that farnesyl pyrophosphate (FPP) is not able to displace GGPP from its binding site, and only GGPP can displace the prenylated product (CD, Scheme 2) [14a]. Selection of the polypeptide substrate is more complex and involves the recognition of the side chain of the terminal X residue of the Ca1a2X box by surface complementarity of the so-called “specificity pocket” (Fig. 2) [14]. The hydrophobic “specificity pocket” of the enzyme discriminates against polar side chains, while the shape and volume of the pocket further restrict the variety of terminal residues able to productively occupy this pocket. The leucine side chain ideally fits in this pocket. However, other terminal amino acids having hydrophobic side chains can also be accommodated. Weak substrates for PGGT-1 are Ca1a2X sequences with X= phenylalanine or methionine. It can be envisioned that polypeptide substrates terminating in the sequence Ca1a2 M that are normally farnesylated, will be geranylgeranylated upon selective inhibition of PFT. However, using kinetic measurements it was recently shown that increased hydrophobicity of the X-residue of a Ca1a2X substrate results in increased product dissociation from PGGT-1 and thereby in a higher turn-over rate. This study indicates that the peptide substrate specificity is determined by its reactivity rather than its binding affinity [14b].

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peptide

GGPP

PGGT-1

H N

R O Zn2+

H O H

Zn2+

O N H S

6 P

O

O

O

O

O

N H

O

8.2

OO

O

H N

171

Oα P O

7

8

O β O P OO-

O P OO-

Peptide-GG

A

B

3

S

R

O N

O

H

NH O

Zn2+

H N

O

O

O-

O

H O H

P

O O

O

P OO-

N H Sδ−

H N

O

O

O

N H

O

6 7 8

O β P O-

O O

O

H δ+ C O- H 1 α O − P δ

Zn2+

OO

H N

R N H

OH N

R O

O N H S

H N O

O

O

O-

N H

Zn2+

D

B'

GGPP

C

Scheme 2. Schematic representation of proposed mechanism of action of PGGT-1. His201α

interacts with Asp269β, Cys271β, His321β

H

interacts with Thr49β, Phe53β, Leu320β, 3rd & d 4th isoprene GGPP, X-amino acid

O

Ile & Val preferred over Leu, Phe, Tyr, Thr, Met

H Zn2+

δ− S N H

any amino acid possible

exposed to solvent

H N O

a2

O

a1

N H

H N O

Arg173β

H

O O

X

O

H

Gln167α

"Specificity Pocket" interacts with Thr49β, His121β, Ala123β, Phe174β

Leu preferred over Phe, Met, Ile, Val

Fig. (2). Binding interactions of polypeptide substrate in the active site of PGGT-1.

3. NATURAL INHIBITORS OF PGGT-1 (TABLE 1) To date a few natural inhibitors of PGGT-1 have been identified. The simplest in terms of structure are three thiosulfinates isolated from garlic (entries 1-3, Table 1). These were found inhibit bovine PGGT-1 in the micromolar range (entries 1-3, Table 1) [18]. Although their mode of action has not been elucidated, it would not be surprising when it involved coordination of a sulfur functionality to the Zn2+ ion in the active site of prenylating enzymes [19].

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Table 1.

El Oualid et al.

Natural Inhibitors of PGGT-1. (H-Ras is a Substrate of PFT and K-Ras is a Substrate for Both PFT and PGGT-1) IC50 in Vitro

Compound

IC50 in

PGGT-1 S S

bovine:

bovine:

K-Ras: 13.3 μM

43 μM

1800 μM

H-Ras: 27 μM

S

bovine:

bovine: 540

K-Ras: 36.3 μM

O

57 μM

μM

H-Ras: 25 μM

S

bovine:

bovine: 400

K-Ras: 22.5 μM

O

53 μM

μM

H-Ras: 30 μM

human:

proliferation

1.1 - 80 μM

of 6 breast cancer

S

2

S

S N N S

human:

OH

17 μM

O

OH

Refs

[18]

[18]

[18]

Inhibition

O

4

Cultured Cells

O

1

3

PFT

Gliotoxin

(depends on

cell lines:

conc. DTT

38 nM - 985 nM;

in assay)

Mean: 289 ±

[20a]

328 nM O O

N

5

O

human:

N

90.3 ± 8.0 μM

S

human: nd

[21]

nd

nd

[24]

nd

nd

[24]

nd

nd

[24]

748 ± 263 μM

O

O HO

OH

6

19

C. albicans:

18

1.9 μM

17 Corticatic acid A

O

7

19

OH

HO

18

OH 17

C. albicans: 3.3 μM

Corticatic acid D

OH

O

8

C. albicans:

19

HO

18 17 Corticatic acid E

7.3 μM

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Table 1. contd…. IC50 in Vitro

Compound

PGGT-1

IC50 in PFT

Cultured Cells

Refs

Br NH2

HN O NH OH H

HN

9

Br NH

O

C. albicans:

O

Br

N H

HN

NH

3.9 μM

nd

nd

[25]

HN

OH

Br Massadine

NH2

S. cervisiae: O HO

10

OH

O

14

O O

O

OH

15

10 μM

S. cervisiae:

C. albicans:

MIC= 0.43 μM

5 μM

C. albicans:

C. neoformans:

MIC= 0.43 μM

3 μM

OH O O

OH

nd

A. fumigatus: Citrafungin B

O

C. neoformans:

[27a]

MIC= 6.8 μM

2.5 μM

A. fumigatus:

human:

MIC= 13.7 μM

0.46 μM S. cervisiae: O

OH O 14

HO O

11

O

O

OH

15

OH O O

OH

Citrafungin A

O

HO

O

S. cervisiae:

C. albicans:

MIC= 13.7 μM

5 μM

C. albicans:

C. neoformans:

MIC= 6.9 μM

nd

3 μM

C. neoformans:

A. fumigatus:

MIC= 55 μM

2.6 μM

A. fumigatus:

human: 0.90 μM

MIC= 55 μM

[27a]

O O

12

15 μM

S. cervisiae:

O

S. cervisiae:

HO O

O

0.5 μM

nd

128 μg/mL C. albicans:

[28a]

128 μg/mL 4-'Hydroxy-3'-methoxy-(+)-S-mitorubrin

HO

O

O

S. cervisiae: No inhibition at

13 OH O

O

> 10 μM

nd

128 μg/mL C. albicans: > 128 μg/mL

Penicillone

[28a]

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Table 1. contd….

IC50 In Vitro

Compound

PGGT-1

IC50 in PFT

Cultured Cells

Refs

O OH O

14

O

S. cervisiae:

HO

150 μM (+ DTT)

O

O

nd

[28a]

(-)-R-mitorubrin

O

O

O

O

O

S. cervisiae:

S. cervisiae


DTT: 2 μM,


DTT: nd

>16 μg/mL

+ DTT: 25 μM

+ DTT: 388

C. albicans:

human: 36 μM (
DTT)

μM

> 32 μg/mL

15 O Purpactin C

S. cervisiae: [28a]

O

S. cervisiae:
DTT

+ DTT

4 μg/mL

S. cervisiae:

1 μM

15 μM

C. albicans:

C. albicans:

nd

41 μM

O O OH

O

16

O

O

C. neoformans: 7.6 μM 25 μM

O (-)-Lunatoic acid

S. cervisiae:

8 μg/mL


DTT: nd

C. neoformans:

A. fumigatus:

26 μM 49 μM

+ DTT:

4 μg/mL

human:

nd

146 μM

170 μM

A. fumigatus:

[28a]

64 μg/mL O

%inhib.@

n

100 μM

OH

17

O R n=1, R= OMe: 3-(4'-geranyloxy-3'methoxyphenyl)-2-trans propenoic acid

n=1, R= OMe: 55 ± 14 μM

13.4 ± 6.4

n=1, R= H: 39 ± 9.5 μM

12.7 ± 23.0

n=2, R= H: 28 μM

16.2

n=2, R= OMe: 66 μM

11.0

nd

[28c]

The fungal (Gliocladium fimbriatum) metabolite gliotoxin (entry 4), another sulfur containing compound with an epidithiodiketopiperazine core, has not only been identified as an inhibitor of mammalian PGGT-1, but also showed low toxicity and potent anti-tumor activity against lymphosarcoma cells and various breast cancer cell lines (Table 1) [20a]. The observation that gliotoxin shows a higher inhibitory activity in vivo than in vitro has been attributed to the (necessary) use of DTT during the biological assay. DTT causes gliotoxin to be reduced and the reduced form of gliotoxin is known to be inactive [20b]. The high cellular antiproliferative activity and limited toxicity [21a], resulted in the synthesis of several structurally related compounds [21b] of which one (entry 5) was found to exhibit inhibitory activity against (recombinant human) PGGT-1. It is believed that gliotoxin acts as a redox active toxin and forms mixed disulfides with accessible thiol residues on proteins [21c]. As the gliotoxin derivative in entry 5 contains no disulfide its mode of action must be different. Aimed at identifying inhibitors of fungal PGGT-1, several research groups have reported on the use of screening compounds originating from natural sources. For example, one of

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the most prevalent human fungal pathogens is Candida albicans. The crystal structure of C. albicans PGGT-1 (CaPGGT-1) complexed with GGPP shows that there are enough structural differences with mammalian PGGT-1 to make selective CaPGGT-1 inhibitors possible [22]. Corticatic acids A, D and E [23], named after the sponge Petrosia corticata from which they were isolated, exhibited inhibition of fungal PGGT-1 activity (entries 6-8, Table 1) [24]. Interestingly, these polyacetylenic acids possess a carboxylic acid and hydroxyl functionality located at the opposing termini of a long, mainly hydrophobic chain. It is therefore highly feasible that these compounds inhibit PGGT-1 by acting as mimics of GGPP. Massadine, a compound isolated from the marine organism Stylissa aff. massa (entry 9, Table 1) [25] was also found to be an inhibitor of fungal PGGT-1. This unusual polycyclic compound contains two cationic cyclic guanidines and two dibrominated pyrrole units and can be viewed as an oxygenated congener of dimeric oroidin derivates [26]. Although no data about its mode of action is available, it is not excluded that the 1,2 dibromovinyl moiety interacts with the zinc ion of the enzyme. The polyketides citrafungins A and B (entries 10 and 11), which are metabolites of the alkylcitrate family, were found to inhibit PGGT-1 from various pathogenic fungal species with equal efficiency [27a]. However, both compounds also proved to be inhibitors of human PGGT-1. In recent years, three different approaches have been published to synthesize citrafungin A [27c-e]. Furthermore, it was shown that the free carboxylic acid functionalities are essential for the inhibitory potency. The carboxylic acid arrangement of these citrafungins is somewhat reminiscent of zaragozic acid precursors [27b] and suggests that other enzymes, including squalene synthase, may be targeted. Note that the overall structure of the citrafungins resembles that of GGPP and that its inhibition mode may be based on binding in the GGPP pocket. A set of natural inhibitors of fungal PGGT-1 were identified during fungal fermentation screening [28a]. 4-‘Hydroxy-3’-methoxy-(+)-S-mitorubrin (Table 1, entry 12) and penicillone (Table 1, entry 13) were isolated from Penicillum citrinum. (-)-R-Mitorubrin (Table 1, entry 14) and purpactin C (Table 1, entry 15) were isolated from Talaromyces flavus and (-)lunatoic acid (entry 16) was isolated from Curvularia sp. Compounds such as depicted in entries 12, 14 and 16 belong to a structurally diverse family of natural products called the azaphilones [28b]. These natural products contain a highly oxygenated bicyclic core and are believed to serve as an electrophile, trapping the target enzyme. The electrophilic character of these compounds is also made clear by the DTT mediated inactivation of their inhibitory activity (Table 1, + DTT values). The prenyloxyphenylpropanoids depicted in entry 17 (Table 1) [28c] are derivatives of 3-(4'-geranyloxy-3'methoxyphenyl)-2-trans propenoic acid, a secondary metabolite isolated from the bark of the small Australian plant Acronychia baueri Schott. Interest in compounds from this plant stems from cancer chemoprevention by dietary feedingin rats and other inhibitory effects on cancer development [28d]. 4. INHIBITORS BASED ON THE CA1A2L BOX (TABLE 2) A major class of PGGT-1 inhibitors is based on the C-terminal end of its (poly)peptide substrate. As (tetra)peptides are metabolically unstable and have poor bioavailability, they have little therapeutic potential. With the objective to overcome these drawbacks, attention was focused on the design, synthesis and evaluation of peptidomimetics. Replacement of the natural peptide bonds by amine linkages is a valuable approach to prevent degradation of

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Ca1a2L analogs by proteases. However, such modification may negatively influence the binding affinity of these analogues for the enzyme. For instance, studies have shown that the amide bond between residues a2 and L in the Ca1a2L analogs cannot be omitted as its carbonyl functionality is involved in hydrogen bonding [14a]. The finding that the binding affinity with the enzyme is not greatly affected by incorporation of a variety of hydrophobic amino acids in the a1 and a2 positions of the Ca1a2L box indicates that the central part of PGGT-1 inhibitors can contain a hydrophobic dipeptide isostere [29]. Functionalized benzoic amino acids pipecolic acids, sugar amino acids and urea derivatives have been used to replace the central two amino acid residues. As the Ca1a2L terminating substrates adopt an extended or a turn conformation on interaction with the enzyme (see Scheme 2), mimics of the a1a2 dipeptide, predisposed to adopt either conformation, have been investigated. Both the side chain and the COOH group of leucine in the Ca1a2L sequence are important with respect to binding to the enzyme and are preserved in most inhibitors. To facilitate transport through the cell membrane, several inhibitors contain a C-terminal methyl ester as prodrug moiety. It is believed that cytosolic esterases transform the ester into the corresponding carboxylic acid. Additionally, the aminoethane thiol functionality of a Ca1a2L analog can potentially be replaced by another zinc coordinating functionality, such as an imidazole or pyridine group. The data summarized in Table 2 provides the structures of the compounds having the general characteristics of Ca1a2L based inhibitors, and where available their IC50 values in an enzyme-assay, as well as in cultured cells towards PGGT-1 and PFT. It is important to note that the IC50 values are dependent on several variables in the assay, such as the specific activity of the purified enzymes and substrate concentrations. This indicates that only the IC50 values of compounds within a specific series can be compared. Throughout the text, the PFT/PGGT-1 ratio is indicative of the selectivity of a specific compound, however in view of the relative nature of the IC50 values as discussed above, one must keep in mind that these selectivity ratios are not absolute values. Table 2.

Ca1a2L Based Inhibitors. (H-Ras is a Substrate for PFT, Rap1A is a Substrate for PGGT-1, K-Ras is a Substrate for Both Enzymes) IC50 In Vitro

Compound

PGGT-1

IC50 in PFT

Cultured Cells

Refs

HS

H2N

H N

R= H: 135 O H N

1 R=H: GGTI-279 R=CH3: GGTI-279

OR

O

R= H: 418

± 36 nM

± 184 nM

R= CH3: 6733

R= CH3: 897

± 2311 nM

± 183 nM

nd

[29a]

HS

H2N

R= H: nd

H N

O H N

2 R=H: GGTI-287 R=CH3: GGTI-286

O

R= H: 7.3 ± 2.6 nM OR

R= CH3: 240 ± 65 nM

21 ± 11 nM

R= CH3: H-Ras:

R= CH3: 183

10000 nM;

± 104 nM

K-Ras: 2 μM; Rap1A: 2000 nM

[29a] [5a]

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177

Table 2. contd…. IC50 In Vitro

Compound

PGGT-1

IC50 in PFT

Cultured Cells

Refs

HS H N

H2N

O H N

3 GGTI-2115

OH

32 ± 6 nM

56 ± 23 nM

nd

[29a]

41 ± 10 nM

47 ± 13 nM

nd

[29a]

O

HS H N

H2N

O H N

4

OH GGTI-2117

O

HS

5

H N

H2N

H N

R=H: GGTI-297 R=CH3: GGTI-298

R= H: 56 ± 20 nM

O OR

R= CH3: nd

R= H: 203 ± 71 nM R= CH3: nd

O

HN

>20000 mM

[29a]

Rap1A: 3000 nM

R= H: H-Ras:

H N

>10000 nM;

O

N

H N

6 R=H: GGTI-2132 R=CH3: GGTI-2146

R- H: nd R= CH3: H-Ras:

OR

R= H: 90 nM

R= H:1300 nM

Rap1A: >10000 nM

R= CH3: nd

R= CH3: nd

R= CH3: H-Ras:

O

[29b]

>30000 nM; Rap1A: 10000 nM

HN H N

R= H: nd

O

N

H N

7 R=H: GGTI-2154 R=CH3: GGTI-2160

OR

R= H: 21 nM

R= H: 5600 nM

R= CH3: H-Ras:

R= CH3: nd

R= CH3: nd

>30000 nM;

O

[29c]

Rap1A: 300 nM

HN H N N

H N

8

GGTI-2144

O

CO2H

150 nM

900 nM

H-Ras: >100000 nM Rap1A: >100000 nM

[29b]

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Table 2. contd…. IC50 In Vitro

Compound

PGGT-1

O

N

IC50 in PFT

OR

R= H: 32 ± 11nM

R= H: 303 ± 57 nM

Rap1A: >10000 nM

R= CH3: nd

R= CH3: nd

R= CH3: H-Ras: >10000

O

R=H: GGTI-2157 R=CH3: GGTI-2158

Refs

R= H: H-Ras: >10000 nM;

O H N

9

Cultured Cells

[29b]

nM; Rap1A: >10000 nM

GGTI-2163 O H N

10

OH

N NH

6100 nM

46000 nM

400 nM

50000 nM

H-Ras: >100000 nM Rap1A: >100000 nM

[29b]

O

N H

GGTI-2139 O H N

11

N H N

OH

H-Ras: >100000 nM Rap1A: >100000 nM

[29b]

O

H N

R= H: H-Ras: >30000 μM; H N

N

R=H: GGTI-2133 R=CH3: GGTI2147

R= H:38 ± 9 nM

O

12

H N

R= CH3: nd

OR

R= H: 5400 ± 750

Rap1A: 10000 μM

nM

R= CH3: H-Ras:

R= CH3: nd

>30000 nM;

O

[29b]

Rap1A: 500 nM

R= H: H-Ras: >100000 nM; 13

O

N H

HN

H N

N

R= H: 10000 nM

Rap1A: >100000 nM

R= CH3: nd

R= CH3: nd

R= CH3: H-Ras:

[29b]

>10000 nM

O

R=H: GGTI-2151 R=CH3: GGTI2152

N

OR

R= H: 44 ± 10 nM

Rap1A: 10000 nM

H N

O

14

4000 nM

H N OH

GGTI-2145 O

100000 nM

nd

[29b]

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Table 2. contd…. IC50 In Vitro

Compound

PGGT-1

IC50 in PFT

Cultured Cells

Refs

R= H: H-Ras: >30000 nM;

N H N

O

15

H N R=H: GGTI-2159 R=CH3: GGTI2160

OR

R= H: 210 nM

R= H: 8000 nM

R= CH3: nd

R= CH3: nd

O

Rap1A: 20000 nM R= CH3: H-Ras:

[29b]

>30000 nM; Rap1A: 500 nM

R= H: H-Ras: >30000 nM

N H N O

16

H N R=H: GGTI-2164 R=CH3: GGTI2165

OR

R= H: 1600 nM

R= H: 58000 nM

R= CH3: nd

R= CH3: nd

O

Rap1A: >30000 nM R= CH3: H-Ras:

[29b]

>30000 nM; Rap1A: 20000 nM

L or D HS

OH

L,L trans:

O H N

17

H N

H2N

O O

OH

L,L cis :
1000 μM

O

L or D

HS

O H N

H2N

trans: 206 ± 34 μM

O H N

O

O

nd

nd

[32]

nd

[33]

nd

[35a]

nd

[35a]

D,L-cis : 69 ± 20 μM

cis or trans

18

68 ± 16 μM

cis: 57 ± 18 μM OH

trans: 321 ± 18 μM cis: 14 ± 6 μM

O cis or trans

O

O

N

19

N

HN

N

OR

N H

O

O L or D

O

O

N

20

HN

N

N

OR N H

O

O L or D

D-Phe:

D-Phe:

R= H: 6300 nM

R= H: >10 μM

R= CH3: >10 μM

R= CH3: >10 μM

L-Phe:

L-Phe:

R= H: 170 nM

R= H: >10 μM

R= CH3: 4500 nM

R= CH3: >10 μM

D-Leu:

D-Leu:

R= H: 2700 nM

R= H: >10 μM

R= CH3: >10 μM

R= CH3: >10 μM

L-Leu:

L-Leu:

R= H: 580 nM

R= H: >10 μM

R= CH3: >10 μM

R= CH3: >10 μM

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Table 2. contd…. IC50 In Vitro

Compound

O

21

O

N HN

PGGT-1

N

N

OH

N H

O

IC50 in PFT

Cultured Cells

Refs

6425 nM

>10 μM

nd

[35a]

3350 nM

>10 μM

nd

[35a]

R= H: 760 ± 96 nM

R= H: 8150 nM

R= H and R= CH3:

R= CH2CH3:

R= CH2CH3:

H-Ras: >10000 nM ;

18000 nM

8850 nM

Rap1A: >10000 nM

O

S O

22

O

N HN

N

N

OH

N H

O

O

O N

N

O

NH

N OR

23 O

R1

24a 24b

O N

HN

O

N

H N

N O

R1 =R 2= H:

R1 =R 2= H:

R1 =R 2= H:

450 ± 95 nM

300 ± 220 nM

H-Ras and Rap1A: >10 μM

R1 =H, R 2 = CH 3 :

R1 =H, R 2 = CH 3 :

R1 =H, R 2 = CH 3 :

4450 nM

2350 nM

H-Ras and Rap1A: 10 μM

R1 = CH 3, R 2 = H:

R1 =R 2= H:

R1 = CH 3, R 2= H:

OR2

230 ± 140 nM

800 ± 310 nM

H-Ras and Rap1A: >10 μM

S

R1 =R 2= CH 3 :

R1 =R 2= CH 3 :

R1 = R 2 = CH 3 :

8±1.2 μM

>10 μM

H-Ras and Rap1A: >10 μM

[35b]

[35b]

R1 =R 2= H: H-Ras and Rap1A: >10 μM R1

25a

R1 =H, R 2 =CH 3 : 2.4 ± 2

N

HN

O

μM R 1 = CH 3, R 2 = H:

H N

N

N

25b

R1 =R 2= H: 62 ± 14 μM

O

OR2

O

9.5 ± 2.0 μM R1 =R 2= CH 3 : 1.8 ± 1.2 μM

R1= Me, R2= H: GGTI-2418

R1 =R 2= H: 4.4 ± 2 μM

R1 =H, R 2 = CH 3 :

R1 =H, R 2 = CH 3 : >10

H-Ras: > 10 μM,

μM

Rap1A: 850 nM

R1 =R 2=H: 53 μM±11

R1 = CH 3, R 2= H:

μM

H-Ras and Rap1A: >10 μM

R1 =R 2= CH 3 : >10 μM

[35b]

R1 = R 2 = CH 3 : H-Ras: >10 μM Rap1A: 400 ± 100 nM

R= H: H-Ras,

O

26

N

HN N

O H N

N O

OR

R= H: 25 ± 13 nM

R= H: >10 μM

Rap1A: >10 μM

R= CH 3 : 8150 nM

R= CH 3 : >10 μM

R= CH 3 : H-Ras: >10 μM, Rap1A: 4000 nM

[35b]

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Table 2. contd…. IC50 In Vitro

Compound

IC50 in

PGGT-1

PFT

Cultured Cells

R= H: 0.52 μM ± 0.13 μM

R= H: 22 μM ± 11 μM

Rap1A: >10 μM

R= CH 3 : >10 μM

R= CH 3 : >10 μM

R= CH 3 : H-Ras,

R= H: H-Ras,

O

27

N

HN

O H N

N

N

Refs

OR

[35b]

Rap1A: >10 μM

O

R= H: H-Ras,

O N

HN

28

O H N

N

N

OR

R= H: 5.5 μM ± 0.15 μM

R= H: 29.5 μM

R= CH3: >10 μM

R= CH3: >10 μM

O

O

29

H N

N

OR

R= H: 440 ± 180 nM

R= H: >10 μM

Rap1A: >10 μM

R= CH3: 65 μM

R= CH3: >10 μM

R= CH3: H-Ras: >10 μM

R= H: H-Ras,

O N

HN

O H N

N

N

[35b]

Rap1A: >15 μM

O

30

[35b]

Rap1A:

R= H: H-Ras, O

N

R= CH3: H-Ras, >10 μM

N

HN

Rap1A: >10 μM

R= H: 14 ± 6.4 μM

R= H: 4.8 ± 1.1 μM

Rap1A: >10 μM

R= CH3: 24 ± 13 μM

R= CH3: 24 μM

R= CH3: H-Ras: 50 μM,

[35b]

Rap1A: 0.6 μM

OR2

O

R1= F, R2= H: H-Ras,

R1

O

31

N

HN N

O H N

N O

OR2

R1= F, R2= H:

R1=F, R2=H:

Rap1A: >10 μM.

7.1 ± 4.3 μM

130±58 μM

R1= F, R2= CH3:

R1= F, R2= CH3:

R1= F, R2= CH3:

H-Ras: >50 μM,

8 μM

>10 μM

Rap1A: 0.7 μM

R1= R2= H:

R1= R2= H:

R1=R2=H: H-Ras, Rap1A: >10 μM.

0.68 ± 0.12 μM

480 μM

R1= H, R2= CH3:

R1= H, R2= CH3:

12 μM

>10 μM

R1= H, R2= CH3: H-Ras, Rap1A: >10 μM

[35b]

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Table 2. contd…. IC50 In Vitro

Compound

PGGT-1

IC50 in PFT

Cultured Cells

Refs

O

32

N

HN

O

6.1 μM

H N

N

N

>10 μM

OH

H-Ras and Rap1A: >10 μM

[35b]

O

C. albicans C. albicans

O

HS

H N

33 N

H2N

R= OH: < 5 nM, R

O

R= OCH3: 5 nM R= Alanine: 10 nM

O

C. albicans

R= OH, R= OCH3:

R= OH: 2000 nM

MIC and MFC

R= OCH3: nd

>128 μg/mL

R= Alanine:

R= Alanine

3000 nM

MIC= 1 μg/mL,

[36a] [36b]

MFC= 2 μg/mL

Human

34

H N

H3N

Cys-Val-Ile-Leu-OH

1.4 ± 0.5 μM

>100 μM

nd

[36c]

All : >100 μM

nd

[36c]

O

Cys-Val-Ile-Leu-OH HN n

O

n=3: 0.7 ± 0.05 μM

R

n= 5: 0.6 ± 0.06 μM

R

NH3

35

R= CH2Ph

NH

O

O

O O

NH3

n= 11: 1 ± 0.5 μM R= H n=11: 0.6 ± 0.03 μM

H3N R

The first family of Ca1a2L analogs use amino- or phenolbenzoic acid as a hydrophobic dipeptide isostere of the central a1a2 region (Table 2, entries 1-16) [29a-c]. In the parent compound, the terminal cysteine moiety is connected to the para-amino benzoic acid unit via an amine linkage (entry 1, R= H). The parent compound and its corresponding methyl ester derivative were tested against both PGGT-1 and PFT to investigate their selectivity. The parent compound showed a slight preference for inhibition of PGGT-1. Surprisingly, the methyl ester derivative was also active in the enzyme assay, having a lower activity and reverse selectivity. The inhibitory potency and selectivity of these para-amino benzoic acid derived compounds has been modulated by variation of the substitution pattern of the amino benzoic acid moiety. Introduction of a phenyl - or a naphtyl group at the ortho position of the para-amino benzoic acid moiety (entries 2 and 5, R= H) improved the inhibitory potency

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to a larger extent than the selectivity. By virtue of the enhanced cell permeability, their methyl ester derivatives interfered with both farnesylation and geranylgeranylation in cultured cells. Besides introducing phenyl substituents, the alkyl side-chain of the neighboring amino acid was varied, without improvement of the activity (entries 3-4). A further attempt to improve the biological profile of these compounds was made by the replacement of the amino ethane thiol moiety by other zinc binders such as the imidazole or pyridine group. Replacement of the amino ethane thiol functionality by an imidazole group (entry 6) of the ortho phenyl substituted compounds resulted in reduction of activity. Introduction of one methyl substituent on the aromatic ring had a positive effect on both the potency and selectivity (entry 7), but a second methyl substituent led to diminished inhibitory activity (entry 8). The nature of the linker between the zinc binding element and the other part of the molecule influenced the activity, as exemplified by the para-benzoic acid derivatives depicted in entries 6, 9 and 10. Also the position of the zinc binding moiety proved to be of prime importance as ortho and meta derivatives of the amino benzoic acid region were substantially less active than their para substituted counterparts (entries 10 and 11). This finding may be explained by the violation of the principle of isosteric dipeptide replacement [30]. A set of similar variations was also executed with the naphthyl analogs (entries 12-16). From these compounds the imidazole-napthyl derivative (entry 12, R= H) was found to be highly selective for PGGT-1 (PFT/PGGT-1= 142). Its methyl ester prodrug (entry 12, R= CH3) showed good potency in vivo [29c, i]. Sugar amino acids [31], as conformationally restricted dipeptide isosteres, were also used to replace the a1a2 part of the tetrapeptide consensus sequence. A series of sugar amino acid containing Ca1a2L based analogs was constructed by variation of the stereochemistry of the dideoxy sugar core (entries 17 and 18). To investigate the effect of the stereochemistry of the side chains of the adjacent amino acid residues leucine and cysteine, a series of eight derivatives was synthesized from which the analog shown in entry 17 proved to be the most active PGGT-1 inhibitor [32]. In order to improve the biological activity of the sugar amino acid containing compounds, by masking the secondary hydroxyl functionality with a hydrophobic benzyl group. From a series of compounds, the cis configured sugar amino acid containing derivative (entry 18) was found to be the most potent, also inhibiting PFT [33]. Based on simple energy-minimizing molecular modeling studies and promising results obtained with a Ca1a2L derived compound [34] in the growth inhibition of breast carcinoma and tumor regression in H-Ras transgenic mice, a set of 2,4-quinazolindione analogs was constructed (Table 2, entries 19-22) in which the nature and configuration of the C-terminal amino acid was varied [35a]. The most potent (IC50= 170 nM) and selective (PFT/PGGT-1> 59) PGGT-1 inhibitor in this series is depicted in entry 19 (R= H) and contains a natural Lleucine residue. In line with these studies, a wide range (Table 2, entries 23-32) of Ca1a2L analogs was developed in which the piperazin-2-one was employed as a1a2 mimic [35b]. After varying the Zn2+ binding imidazole moiety, length, the piperazin-2-one scaffold and the C-terminal residue (entries 23-32), entry 25b (GGTI-2418) was found to be the most active (IC50= 9.5 ± 2.0 nM) and selective (PFT/PGGT-1> 5500) among these set of compounds. This high potency and selectivity of entry 25b was retained in vivo when its methyl ester prodrug was tested. Modeling studies of entry 25b in the recently reported crystal structure of PGGT-1 [14a] revealed that the spacer does not make any significant binding interactions and thus must play a conformational role. Furthermore, as the Zn2+ ion binding pocket in PGGT-1 is small [14a], it cannot accommodate large groups (such as benzyl)

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on the imidazole functionality. The methyl group, however, was found to fit nicely in the pocket and is well tolerated. Entries 28 - 32 are analogs of entry 25b. Although in vitro and in vivo potency was not enhanced significantly, replacing the phenylalanine of entry 25b for a para-fluoro-phenylalanine group (entry 31) resulted in a three-fold improvement in selectivity. As part of a program aimed at developing anti-fungal drugs, the 4-phenyl piperidine moiety was used as a1a2 analog in the Ca1a2L motif (entry 32) [36a]. Note that the 4-phenyl piperidine is not a true dipeptide isostere as the overall length of the backbone lacks one atom [30]. Although the carboxylic acid and methyl ester derivative of entry 32 were very potent (IC50= <5 nM), neither displayed any in vivo potency against a clicical isolate of C. albicans. Apparently, cell permeability was still very low and in order to enhance this, an alanine conjugate was synthesized. It was reasoned that the in vivo potency could be enhanced by converting the pipecolic acid derivatives in such a way that they can utilize the active transport systems present in fungal cells, thus increasing bioavailability [36b]. As anticipated, this derivative showed an improved in vivo antifungal activity. Furthermore, it retained its in vivo selectivity since only the membrane localization of PGGT-1 substrate Rho1p was inhibited whereas that of the PFT substrate Ras1p was unaffected. Besides the conventional approach of targeting interior binding cavities, potent and selective PGGT-1 inhibitors (with regard to PFT) were reported that target both the interior and the exterior of PGGT-1 (entries 34 and 35) [36c]. Here a bivalent hybrid compound (Fig. 3) consists of: (1) a surface binding mode which was modelled to target the acidic surface of PGGT-1; (2) the CVIL tetrapeptide as an interior targeting device. These hybrids were not geranylgeranylated by PGGT-1 which suggests that the external art prohibits the tetrapeptide from adopting a correct conformation for catalytic turnover (Scheme 2). linker

exterior binding motif

+ + + + + + PGGT-1

CVIL peptide

Fig. (3). Bivalent inhibitors that target the exterior and interior surface of PGGT- [36c].

5. PGGT-1 INHIBITORS BASED ON GGPP (TABLE 3) The design of a second major class of PGGT-1 inhibitors is based on the structure of its terpene substrate GGPP (Table 3). As the GGPP moiety is amphiphilic in nature, with its polar head group and lipophilic tail, most of the reported GGPP mimics also have an amphiphilic character. Because the pyrophosphate moiety is metabolically unstable, several isosteres of this functionality have been developed. Although beneficial in terms of inducing interactions with the enzyme, inhibitors bearing pyrophosphate isosteres with multiple negative charges are generally poorly taken up by cells. To circumvent this drawback a prodrug approach can be followed. The structures of the GGPP based inhibitors, in accord with these general characteristics, are shown in Table 3. It is important to note that only the potency of

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compounds within a series of Table 3 can be compared, as the conditions under which the IC50 values were obtained may differ from laboratory to laboratory. Where available, the IC50 values in cultured cells and the IC50 values in PFT are also given. Table 3.

GGPP Based Inhibitors

IC50 In Vitro

Compound

PGGT-1 E-isomer: 66 ± 8

3

nM. Inhibition of

1-E

1

1-Z

3

P

O

2

Rap1A processing:

OH

H N O

OH

20500 ± 3100 nM. Z-isomer: 255 ± 35

O

IC50 in PFT

E-isomer: 3500 ± 700 nM

Cultured Cells

Refs

E-isomer: Proliferation of PC-3 prostate

[37] [38]

cancer cells: [39c]

Z-isomer: 18500 ± 2600

nM

> 10000 nM

Inhibition of Rap1A

No effect

PC-3 prostate

processing:

on farne-

cancer cells:

40200 ± 6200 nM

sylation

35100 ± 4600

nM Proliferation of

3 O

H N

2

P

O O

OH

O

[37]

nM

3 OH

H N

3

P

O O

OH

4-Z

3

OH

H N

1

P

O 2

O

>10000 nM

E-isomer:

E-isomer:

146 ± 20 nM

>10000 nM

nd

[38]

O

3 4-E

151 ± 30 nM

OH

O

[38] nd

Z-isomer:

Z-isomer:

1000 ± 130 nM

>10000 nM

[39c]

3 O H N

5

P

O

OH

39 ± 5 nM

OH

560 ± 70 nM

735 ± 80 nM

nd

[39b]

nd

[39b]

nd

[38]

OH O

O

3

OH O

H N

6

P

O O

1000 ± 115 nM

OH

3 O 7

O P

N H

OH

OH

275 ± 40 nM

5000 ± 1000 nM

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Table 3. contd….

IC50 In Vitro

Compound

PGGT-1

IC50 in PFT

Cultured Cells

> 10000 nM

nd

Refs

3 O

O

8

P

N H

OH

3 O

9-Z

O

1

9-E 3

P

N H

2

111 ± 15 nM

[38]

OH

OH

OH

E-isomer:

E-isomer:

566 ± 75 nM

>10000 nM

Z-isomer:

Z-isomer:

764 ± 73 nM

>10000 nM

1.5 ± 0.25 μM

0.579 ± 0.070 μM

[38] nd [39c]

3 O

O

10 N H

P

OH

nd

[39b]

OH

3

Inhibition of O

O

11 O

N H

O S

Rap1A

NH2

processing:

No effect on farnesylation

28.2 ± 4.7 μM

Proliferation of PC-3 prostate cancer cells:

[37]

22.2 ± 4.5 μM

3 O

12

O

P

P OH

OH

OH

6 ± 0.8 μM

0.442 ± 0.065 μM

nd

[39a]

6 ± 0.075 μM

> 10 μM

nd

[39a]

E-isomer:

E-isomer:

500 ± 80 nM

>10000 nM

Z-isomer:

Z-isomer:

5000 ± 650 nM

1250 ± 160 nM

3 O OH O

13

P

P

OH OH

3 O OH O

14-E

P

14-Z

P

OH OH

[39a] nd [39c]

3

15 O

O

O

P

P

OH

OH

[39a] OH 0.02 ± 0.003 μM

0.638 ± 0.048 μM

nd [41]

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Table 3. contd….

IC50 In Vitro

Compound

PGGT-1

IC50 in PFT

Cultured Cells

Refs

n= 0 strong inhibition of DNA synthesis; no 16 17

2

+K-O

O

O-K+

P

O-K+

18

O

n

19

O-K+

P O

20

n= 0: >100 μM

1.08±0.23 μM

n= 1: 1.15±0.13 μM

0.58±0.45 μM

viability HSM cells

n= 2: 0.98±0.01 μM

5.27±1.28 μM

n=1: moderate

n= 3: 20.1±5.7 μM

14.7±3.0 μM

inhibition of

n= 4: 18.2±5.9 μM

8.2±1.6 μM

inhibition of cell [40] [8a]

DNA synthesis; no inhibition of cell viability HSM cells n=2-4: nd

2 n

21 22 23

+K-O

P

O

O

n= 2: 15.9±2.7 μM,

18.6±1.6 μM

n= 3: 45.8±8.2 μM,

38.5±8.0 μM

n= 4: 22.8±4.8 μM,

82.9±7.9 μM

nd

O P

O-K+

[40]

O

Strong inhibition 2

O

24

P

of DNA synthesis

O O

O-K+

P

O-K+

6.5±0.8 μM

0.93±0.23 μM

O-K+

and very strong inhibition of cell

[40]

viability in human smooth muscle cells

2

Very strong

O-K+ P

25

inhibition of DNA O-K+

39.7±0.9 μM

O P

O

13.5±1.1 μM

viability in human

O-K+

26 P

O

[8a]

smooth muscle cells

O-K+

2

[41]

synthesis and cell

O-K+

O-K+

P

P

O

O

O-K+

No inhibition of 3.60±0.35 μM

1.81±0.38 μM

O-K+

DNA synthesis and cell viability human

[8a]

smooth muscle cells

O-K+ O O

Strong inhibition of

O

DNA synthesis

2

O P O P O O O O

27

O O P O O O O O O

O

inactive

inactive

(IC50= 1 – 10 μM); no inhibition cell viability of human smooth muscle cells

[8a]

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Table 3. contd….

IC50 In Vitro

Compound

IC50 in

Refs

PGGT-1

PFT

Cultured Cells

3.87±1.24 μM

3.99±0.84 μM

nd

[8b]

33.3±3.2 μM

56.2±5.0 μM

nd

[8a]

11.5 ± 0.6 μM

96 ± 16 μM

nd

[8a]

40.2 ± 1.5 μM

449 ± 0.8 μM

nd

[42]

136 ± 9 μM

190 ± 23 μM

nd

[43]

20.8 ± 0.2 μM

>1000 μM

nd

[43]

401 ± 8 μM

>1000 μM

nd

[43]

229 ± 15 μM

854 ± 80 μM

nd

[43]

2

O-K+

28 2 O

O-K+

P

P

O

O

P O-K+ O-K+

O-K+

2

O-K+

29

P 2 O

O

P

O

O-K+

P

O-K+

O

O OH

30

OH

2 O O O

31

O

O O

32

O O O

33

O

2

O H O

34

O

O

35

O O

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Table 3. contd….

IC50 In Vitro

Compound

PGGT-1

IC50 in PFT

Cultured Cells

Refs

O-Na+ F

NO2

12.5 ± 2.0 μM

36 F

O

6.3 ± 1.11 μM

nd

[43]

F

2

O-Na+ F

NO2

O

F

37

2.9 μM

6.2 μM

nd

[44]

7.5 ± 0.8 μM

>100 μM

nd

[44]

nd

[44]

F +K-O

O

O-K+

P

2

38

O-K+ O-K+ P

O n

O

OH F

NO2

39 O

O

F

H

OH

O O-Na+

40 O

F F R

O O

2

42 O 2

43 O 2

45 ± 12 μM

F

F

41

8.5 ± 1.1 μM

P

O

P O-

O

O

P

P

O

O-

O-

O

O

O-

R= vinyl: 715 nM

R= allyl:

R= allyl:

846 nM

453 nM

Ki= 3.0 μM

nd

nd

Ki= 3.0 μM

nd

nd

[45a] nd

[45b] [45d]

O

O-

P

R= vinyl: nd Km= 1750 nM

O

P O-

O-

[45a] [45b]

[45a] [45b]

O-

[45a] O-

Ki= 3.7 μM

nd

nd [45b]

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Table 3. contd….

IC50 In Vitro

Compound

O

44

P O

O-

2

PFT

Cultured Cells

Ki= 6.1 μM

nd

nd

O

P O

IC50 in

PGGT-1

Refs

[45a] O-

[45b]

O-

Inhibition growth of HRas-F transfected cells: 45

OH

nd

nd

18.0 ± 4.1 μM;

[45a]

Inhibition growth of H-

[45b]

Ras-GG transfected cells:

2

13.9 ± 2.1 μM; Inhibition growth of H-Ras-F transfected nd

46

OH 2

nd

cells: >25μM

[45a]

Inhibition growth of

[45b]

H-Ras-GG transfected cells: 4.6±1.9 μM

One of the first examples of GGPP-based inhibitors is the phosphono(acetamido)oxy terpene shown as entry 1-E in Table 3 [37]. This compound has two negative charges in its polar head group, and was found to potently and selectively inhibit PGGT-1 (PFT/PGGT-1 50), to partially prevent the geranylgeranylation of Rap1A and to inhibit the proliferation of prostate cancer cells. Its ethylphosphonate monoester derivative was less efficient (entry 2). When the E-double bond of the phosphono(acetamido)oxy terpene in entry 1-E was substituted for a Z-double bond (entry 1-Z), this lead to a ~4-fold decrease in inhibitory potency and a slight drop in selectivity (PFT/PGGT-1 > 40) [39c]. Docking studies revealed that the compound in entry 1-E binds to PGGT-1 in a similar fashion as GGPP. Transforming the double from an E to Z configuration resulted in an interaction between the terminal phosphonic acid group and lysine residue 311. Interestingly, this lysine residue interacts with the
-phosphate of GGPP (Scheme 2) during the repositioning of C1 to the cysteine thiolate (Scheme 2) [14a]. With the aim to improve the selectivity and/or potency of the parent compound (entry 1E), two
-alkylated derivatives were prepared (entries 3 and 4-E) [38] and in entry 4-Z, a Z double bond was introduced [39c]. Both modifications, however, lead to a loss of inhibitory potency. The one carbon homolog of entry 1-E (entry 5) showed comparable potency for PGGT-1, but was less selective with regard to PFT. The
-carbonyl substituted carboxylic acid derivative (entry 6) proved to be less active [39]. In addition, replacing the acetamidooxy linkage with an amide linkage (entries 7-10) did not improve the inhibitory activity either. In the case of entry 9-E, inhibitory potency and selectivity was not greatly affected by the introduction of a Z-double bond (entry 9-Z) [39c].

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An alternative pyrophosphate mimic is the [[(aminosulfonyl)amino]carbonyl]oxy group depicted in the compound shown in entry 11. This compound was found to be a potent inhibitor of PGGT-1, with an activity in cultured cells comparable to that of the parent compound in entry 1-E. It is likely that due to the diminished negative charge, this pyrophosphate mimic is more cell permeable. Next, a series of phosphinatephosphonate derivatives was investigated (entries 12-15). These compounds possess an additional negative charge as compared to the compounds shown in entries 1-10 (Table 3). While the compound depicted in entry 12 proved to be more effective against PFT, its double -methylated derivative (entry 13) had an increased selectivity for PGGT-1. The diethyl analog (entry 14-E) was even more active and selective (PFT/PGGT-1 > 20). The corresponding Z-double bond analog (entry 14-Z) [39c] showed a 10-fold decrease in PGGT-1 inhibitory activity and a dramatic loss of selectivity (PFT/PGGT-1 ~
). Docking studies revealed that entry 14-Z fits very well in the (shorter) FPP pocket of PFT and that the diethyl-phosphinatephosphonate interacts with PFT in a similar fashion as PFT. Introducing an ether linkage (entry 15) in parent compound entry 12, resulted in a sharp rise of PGGT-1 inhibitory potency and selectivity (PFT/PGGT-1
30). Branched phosphonate terpene derivatives with increasing chain length are depicted in entries 16-20. [40] [41a]. To probe the importance of negative charge and flexibility, cyclic derivatives (entries 21-23) were constructed. Although one would expect that increasing the length would have a beneficial effect on selectivity [14a], the concurrent increasing conformational flexibility may result in non-specific interactions with PGGT-1 and PFT. In general, these cyclic derivatives were found to be less active than their non-cyclic counterparts (entries 18-20), indicating that the diminished negative charge plays an important role in these compounds. The most potent PGGT-1 inhibitor (IC50= 0.98 ± 0.01 μM) of the branched and cyclic derivatives in this series is depicted in entry 18 (PFT/PGGT-1 ~ 5). Other structurally related phosphor containing derivatives, including phosphonatephosphates, bisphosphonates, triphosphonates and di-prenylated analogs, were found to serve as inhibitors of both PGGT-1 and PFT (entries 24-29) [41]. The prodrug depicted in entry 27 was found to be readily taken up by smooth muscle cells and inhibit their proliferation without effecting cell viability. This prodrug proved to be more promising as an anti-restenosis lead compound than the compounds of entry 24 and 25, as the latter two showed toxic sideeffects. The two carboxylic acid funtionalities in the chaetomellic acid derivative shown in entry 30 mimic the negatively charged pyrophosphate moiety [42]. This compound inhibited PGGT-1 with an IC50 of 11.5 μM and showed a moderate selectivity for PGGT-1 (PFT/PGGT-1 ~8). Hydrophobic cyclic anhydride derivatives, also based on chaetomellic acid, are depicted in entries 31-35 [43]. The most potent and selective compound in this series is shown in entry 33 (PFT/PGGT-1 >48). Interestingly, these cyclic anhydrides are more active than their corresponding dicarboxylic acid or ester derivatives, suggesting a different role for the anhydride part than serving as a mimic of the negatively charged pyrophosphate moiety. Furthermore, it is not clear whether the anydride is stable to hydrolysis in vivo. It would be interesting to investigate the biological activity of a derivative of entry 32 in which the two exo-cyclic double bounds are reduced, as the biological activity of such a compound can be directly correlated with the compound shown in entry 33.

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Prenylated nitro- and carboxylic acid functionalized fluorophenol ethers (entries 36-39) were also developed as potential inhibitors of prenylating enzymes [44]. Both the ring –OH and fluor atoms were found to be important for inhibitory activity. Incorporation of an epoxide on the C2-C3 double bond in the compound shown in entry 37 resulted in a much more selective PGGT-1 inhibitor (entry 38). Interestingly, this epoxygeranyl compound was designed to act as a potential suicide substrate by alkylating the C-terminal cysteine residue of a peptide substrate. Unfortunately, no evidence for this was reported. Rather than designing a GGPP based inhibitor bearing a pyrophosphate mimic, a alternative interesting approach entails the modification of the terpene chain. Therefore, a series of C3 and C7 modified terpene pyrophosphates were developed to act as suicide substrates of PGGT-1 (entries 40-44) [45a,b,d]. These GGPP substrate analogs were designed to delocalize the positive charge generated at C1 in the transition state after removal of the pyrophosphate moiety (Scheme 2), thus creating a stabilized electrophilic centre which can potentially react with a nearby nucleophile in the active site of the enzyme. In the case of entry 41, a striking observation was that the 7-vinylGGPP analog acts as a modest PGGT-1 inhibitor (IC50= 1 μM) while the 7-allylGGPP analog is a highly potent alterantive GGPP substrate (Km= 27 nM, GGPP Km= 3 nM). Furthermore, the structure-activity relationship data of isoprenoids targeted at PGGT-1 did not correlate well with that of PFT. Because the pyrophosphate moiety is metabolically labile, their alcohol precursors (entries 45 and 46), which are presumably phosphorylated in vivo by kinases [45c], were evaluated in cultured cells. The prenyl alcohols were shown to act as in situ dual inhibitors of PGGT-1 and PFT. 6. MISCELLANEOUS INHIBITORS (TABLE 4) The strategies to develop PGGT-1 inhibitors discussed before are based on the mimicking of either of its two substrates. An interesting, but largely unexplored, strategy is to develop bisubstrate inhibitors of PGGT-1. With this aim in mind, a series of derivatives of the Ca1a2L based inhibitor shown in entry 17 of Table 2 was equipped with fatty acyl chains of different lengths [46]. The best inhibitors of these series, having a one or three carbon wamino acid spacer and the trans configuration of the sugar amino acid core are depicted in entries 1 and 2 of Table 4. Although based on a bisubstrate rationale and the observation that the inhibitory activity improved notably compared to the non-lipidated compound, it was not proven that these type of lipopeptide derivatives indeed function as bisubstrate inhibitors. Table 4. Other type of PGGT-1 Inhibitors (H-Ras and HDJ2 are PFT Substrates, Rap1A is a PGGT-1 Substrate and K-Ras is a Substrate for Both Enzymes) IC50 In Vitro PGGT-1 PFT

Compound

IC50 in Cultured Cells

Refs

nd

[46]

O SH

HN

1

O

OH H N

N H O

O H N OH

O O

12.7 ± 1.3 μM

nd

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Table 4. contd….

IC50 In Vitro PGGT-1 PFT

Compound

IC50 in Cultured Cells

Refs

H N O

SH

O

OH

N H

O H N

H N

2

12.3 ± 1.0 μM

nd

nd

[46]

8.1 ± 1.2 μM

nd

nd

[47a]

3.8 ± 0.9 μM

nd

nd

[47a]

OH

O

O

O

NH2 O

3 O

H N

N H

OH

N H

O

O NH2

NH2

4 O

N H

O H N

OH

N H

O

O NH2

PFT binding: IC50= 3 nM Cl N

5

N

N

98 nM

N

2 nM

O N L-778,123

Rap1A: MIC=1000 nM, EC50= 6760 nM; Ki-Ras: EC50= 6300 nM, HDJ2: EC50= 92 nM,

[48] [50b]

H-Ras: IC90= 0.1 μM

Cl

18 nM

O

6

N

7 nM

Rap1A: MIC=300 1000 nM

N

N N

O

Cl

N N O

[48a]

H-Ras: IC90= 0.3 μM,

N

7

PFT binding: IC50= 110 nM

N

N O N

809 nM

9 nM

nd

[48c]

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Table 4. contd….

IC50 In Vitro PGGT-1 PFT

Compound

Cl

Cl O

8

N

0.7 nM

N

7 nM

N N

O

IC50 in Cultured Cells

Refs

PFT binding: IC50= 41 nM

[48a]

Rap1A: MIC= 300 nM

N

Cl

Cl

9

20 nM

O N

4 nM

N

[48a]

Rap1A: nd

N N

PFT binding: IC50= 93 nM

O N

Cl

Cl

10

15 nM

O N

19 nM

N

PFT binding: IC50= 350 nM

[48a]

Rap1A: nd

N N

O N

O

Cl

11

62 nM

O N

32 nM

N

PFT binding: IC50= 311 nM

[48a]

Rap1A: nd

N N

O N

Cl O

12

N

N

N N

O N

199 nM

1 nM

PFT binding: IC50= 48 nM Rap1A: nd

[48a]

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Table 4. contd….

IC50 In Vitro PGGT-1 PFT

Compound

Cl O

13

N

290 nM

4 nM

IC50 in Cultured Cells

Refs

nd

[48a]

N

N N

O N

OH

O

Cl O

14

N

1.3 nM

2.5 nM

[48a]

Rap1A: MIC= 3000 nM

N

N N

PFT binding: IC50= 8 nM

O N

Cl S N

15

52 nM

N

11 nM

[48a]

Rap1A: MIC=1000 nM

N N

PFT binding: IC50= 17 nM

O N

Cl O N

16

12 nM

N

18 nM

PFT binding: IC50= 255 nM

[48a]

Rap1A: nd

N O N

Cl O N

17

N

65 nM

1230 nM

nd

[48a]

136 nM

670 nM

nd

[48a]

N O N

18

O N

N

N O N

Cl

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Table 4. contd….

IC50 In Vitro PGGT-1 PFT

Compound

O N

19

N

155 nM

CF3

5770 nM

IC50 in Cultured Cells

Refs

nd

[48a]

N O N

Inhibited PFT in a

Cl N

N

1390 nM

N

20

12 nM

O

Inhibited PFT in a

Cl N

N

210 nM

N

8.7 nM

O

Cl

Cl N

Cl

cell-based assay: 193 nM

[49]

PGGT-1: nd

N

N

Inhibited PFT in a 8450 nM

N

22

[49]

PGGT-1: nd

N

21

cell-based assay: 1600 nM

17 nM

cell-based assay: 576 nM

[49]

O

PGGT-1: nd

N

Cl

Inhibited PFT in a N

Cl

N

3030 nM

N

23

35 nM

O N

[49]

PGGT-1: nd

Cl Cl N

N

Inhibited PFT in a 1340 nM

N

24

cell-based assay: 437 nM

15 nM

cell-based assay: 600 nM

[49]

O

F

PGGT-1: nd

N Cl N

F3C

N

2680 nM

N

25

Inhibited PFT in a 410 nM

cell-based assay: 662 nM

[49]

O

PGGT-1: nd

N

Cl N

F3C

N

N

26

3500 nM O N

F3C

1870 nM

nd

[49]

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Table 4. contd….

IC50 In Vitro PGGT-1 PFT

Compound

Cl N

27

N

Refs

Inhibited PFT in a 140 nM

N

IC50 in Cultured Cells

5.7 nM

cell-based assay: 29 nM

[49]

O

F3C

PGGT-1: nd

N

Cl O

N

28

N

1860 nM

N

392 nM

nd

[49]

O

F3C

N

Cl N

29

N

N

Inhibited PFT in a 2300 nM

N

19 nM

cell-based assay: 250 nM

[49]

O

Cl

PGGT-1: nd

N

Cl N

30

N

N

560 nM

N

F3C

52 nM

nd

[49]

O

O

N

Cl N

31

N

N

Inhibited PFT in a 509 nM

N

14 nM

cell-based assay: 159 nM

[49]

O

F3C

PGGT-1: nd

N

Cl

Inhibited PFT in a

O N

32

N

70 nM

N

1.6 nM

cell-based assay: 12 nM

[49]

O

F3C

PGGT-1: nd

N

Inhibited PFT in a Cl

cell-based assay: 1.2 nM

O

33

F3C

151 nM

N N N

N O

0.17 nM

PFT: HDJ2, EC50= 28 nM PGGT-1: Rap1A, EC50= 3400 nM

[49]

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Table 4. contd….

IC50 In Vitro PGGT-1 PFT

Compound

IC50 in Cultured Cells

Refs

n=1: Inhibited PFT in a N

cell-based assay: 0.29 nM O

34 N

N

N

n=1: 301 nM

n=1:0.1 nM

n=2: 16 nM

n=2:0.25 nM

n=2: Inhibited PFT in a

[49] [50]

cell-based assay: 0.19 nM

N O

n

Inhibition of Rap1A processing: 10000 nM

PGGT-1: nd Br O

Inhibited PFT in a 161 nM

35 N

N

1.2 nM

N

cell-based assay: 2.0 nM

[49]

PGGT-1: nd

N O

CF3 O

Inhibited PFT in a

36

15.6 nM N

N

2.1 nM

N

cell-based assay: 1.1 nM

[49]

PGGT-1: nd

N O

O

37

N

N

509 nM

8 nM

nd

[49]

2480 nM

10.3 nM

nd

[49]

6.1 nM

cell-based assay: 0.81 nM

N N O O

O

38 O

F3C

SH O

N S

Inhibited PFT in a

O N

O

4.8 nM

39 N N N

PGGT-1: nd

[49]

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Table 4. contd….

IC50 In Vitro PGGT-1 PFT

Compound

IC50 in Cultured Cells

Refs

Inhibited PFT in a F3C

cell-based assay: 0.48 nM

O N

O

60 nM

40

3.1 nM

N N

PFT: HDJ2, EC50= 6.9 nM

N

PGGT-1: Rap1A, EC50= 800 nM

O

PFT binding IC50= 3.3 nM

[49]

N N

41

3780 nM

O

N

2.2 nM

[50b]

PGGT-1: Rap1A, MIC= 30000 nM

N N

F N N O

42

2750 nM

<1 nM

PFT binding IC50= 1 nM

[50b]

450 nM

<1 nM

PFT binding IC50= 0.3 nM

[50b]

O

N N

N Cl N N

O

43 O

N N

N Br N N O

44

100 nM

0.45 nM

502 nM

0.26 nM

O

N N

PFT binding IC50= 1.1 nM PGGT-1 (Rap1A) MIC= 10000 nM

[50b]

N

N N

O

45 N

O

N N

PFT binding IC50= 0.33 nM

[50b]

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Table 4. contd….

IC50 In Vitro PGGT-1 PFT

Compound

IC50 in Cultured Cells

Refs

N O

O

124 nM

N

0.66 nM

46 N

N

PFT binding IC50= 1.2 nM PGGT-1: Rap1A, MIC= 1000 nM

[50b]

N

N O

O

107 nM

N

0.1 nM

47 N

PFT binding IC50= 0.59 nM

[50b]

PGGT-1: Rap1A, MIC= 1000 nM

N N

N O

O

97 nM

N

0.31 nM

48 N

PFT binding IC50= 0.44 nM

[50b]

PGGT-1: Rap1A, MIC= 1000 nM

N N

N O

O

49

212 nM

N

N

0.5 nM

N

PFT binding IC50= 1.4 nM PGGT-1: Rap1A, MIC= 3000 nM

[50b]

N

N O

O

50

55 nM

N

N

38 nM

PFT binding IC50= 10 nM PGGT-1: Rap1A, MIC= 1000 nM

N N

[50b]

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Table 4. contd….

IC50 In Vitro PGGT-1 PFT

Compound

IC50 in Cultured Cells

Refs

N O

O

51

N

N

2.4 nM

364 nM

534 nM

645 nM

N

PFT binding IC50= 170 nM PGGT-1: Rap1A, MIC= 300 nM

[50b]

N

N O

O

52

N

N

nd

[50b]

N N

N O

O

53

238 nM

0.66 nM

N

N

PFT binding IC50= 2.2 nM PGGT-1: Rap1A, MIC= 10000 nM

[50b]

N N

N O

54

O

2.1 nM

N

N

19 nM

PFT binding IC50= 28 nM

[50b]

PGGT-1: Rap1A, MIC= 100 nM N N

PFT binding IC50= 5.7 nM

N

N

N

N O

1.7 nM

55 O

N

1.6 nM

PGGT-1: Rap1A, MIC= 30 nM, EC50= 140 nM, Ki-Ras= 1065 nM HDJ2: EC50= 12 nM

[50b]

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Table 4. contd….

IC50 In Vitro PGGT-1 PFT

Compound

IC50 in Cultured Cells

Refs

OH N

PFT binding IC50= 40 nM

N OH

N

O

N

1050 nM

56

6.8 nM

O

PGGT-1: Rap1A EC50= 690 nM, Ki-Ras= 1515 nM

[50b]

HDJ2 EC50= 10 nM

N

O N

N O

N

O

N

46 nM

57

0.5 nM

PFT binding IC50= 0.86 nM

[50b]

PGGT-1: Rap1A, MIC= <300 nM

O

N

O N N

N

O

O

N

420 nM

58

24 nM

PFT binding IC50= 20 nM

[50b]

PGGT-1: Rap1A, MIC=3000 nM

O

N

N

N

N

N

O O

59

N H

43 nM

20 nM

5.5 nM

1.1 nM

O

PFT binding IC50= 30 nM PGGT-1: Rap1A, MIC= 100 nM

[50b]

N

H N

2 N

N N

O

HDJ2 EC50= 4.3 nM

60 O

N

in vitro t1/2= 2 h 92 % bioavailability

[51a]

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Table 4. contd….

IC50 In Vitro PGGT-1 PFT

Compound

IC50 in Cultured Cells

Refs

H N

2 N

N N

O

61

35 ± 23 nM

1.2 nM

HDJ2: EC50= 5.5 nM

[51a]

32 nM

1.5 nM

HDJ2: EC50= 6.9 nM

[51a]

39 nM

1.2 nM

nd

[51a]

7.1 nM

2.9 nM

nd

[51b]

25 nM

5.3 nM

nd

[51c]

O

N

2

N

N

N N

O

62 O

N

2

N

N

N N

O

63 O

N

N N

N

O

64 O

N

N N

R

N

O

65 O

N

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Table 4. contd….

IC50 In Vitro PGGT-1 PFT

Compound

IC50 in Cultured Cells

Refs

N N

S

N

O

66

5.8 nM

1.4 nM

nd

[51c]

560 ± 60 nM

61 nM

nd

[51c]

1200 nM

41 nM

nd

[51c]

1.3 nM

20 nM

nd

[51c]

100 nM

280 nM

nd

[51c]

8.9 nM

1.9 nM

nd

[51c]

O

N

N N

N

O

67 O

N HN N

N

O

68 O

N

HN N

N

O

69 O

N HN N

N

70 O

N

O N N N

71 O

N

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Table 4. contd….

Compound N

IC50 In Vitro PGGT-1 PFT

IC50 in Cultured Cells

Refs

[51c]

N

N H

O

72

12 nM

13 nM

nd

10 ± 1 nM

0.6 nM

nd

17 nM

2.0 nM

nd

[51c]

8 nM

0.19 nM

nd

[51c]

10000 nM

1800 nM

nd

[51c]

13 ± 5 nM

1.2 nM

nd

[51c]

O

N

N

N

N

O

73

[51c]

O

N N

N OH

N

O

74 O

N N

N NH2

N

O

75 O

N N

N O

N

O

76 O

N N

N OH

N

O

77 O

N

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Table 4. contd….

IC50 In Vitro PGGT-1 PFT

Compound

N

IC50 in Cultured Cells

Refs

nd

[51c]

N NH2

N

O

78

5.1 ± 2.0 nM

0.38 nM

O

N

N

N

NH2 N S

HDJ2: EC50= 2.1 ± 0.9 nM

S

O

79

3.6 nM

0.06 nM

O

N

NH2 N

HDJ2: EC50= 11 nM

R

O

S

80

4.5 nM

1.5 nM

O

N S

O

R

81

13 nM

0.36 nM

O

N O

R

82

R

54 nM O

N

[51c]

HDJ2: EC50= 120 nM

NH2 N

Rap1A: EC50= 140 nM K-Ras: EC50= 230 nM

N

N

[51c]

HDJ2: EC50= 6 nM

NH2 N

Rap1A: EC50= 720 nM K-Ras: EC50= 1900 nM

N

N

[51c]

K-Ras: EC50= 340 ± 50 nM

N

N

Rap1A: EC50= 470 ± 20 nM

7.6 nM

Rap1A: EC50= 3200 nM K-Ras: EC50= 8000 nM

[51c]

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Table 4. contd….

IC50 In Vitro PGGT-1 PFT

Compound N

IC50 in Cultured Cells

Refs

nd

[51c]

N NH2

N

O H

83

92 nM

11 nM

O

N

n= 0, HDJ2: EC50= 1.5 nM N

N NH2

N

O

n=0: 30 nM n

84

n=2: 2.9 nM n=3: 1.4 nM

O

N

n=0: 0.41 nM n=2: 0.46 nM n=3: 0.12 nM

Rap1A: 87 nM; K-Ras: 220 nM n= 2, HDJ2: EC50= 0.9 nM

[51c]

Rap1A: 220 nM; K-Ras: 100 nM n= 3, HDJ2: EC50= 4 nM Rap1A: 250 nM; K-Ras: 320 nM

N

N NH2

N

O

HDJ2: EC50= 1.5 nM

85

2.1 nM

0.3 nM

Rap1A: EC50= 87 nM

[51c]

K-Ras: EC50= 220 nM

O

N N

N NH2

N

O

HDJ2: EC50= 3.1 nM CF3

86

2.5 nM

0.19 nM

Rap1A: EC50= 47 nM

[51c]

K-Ras: EC50= 207 nM

O

N

N

N NH2

N

O

87

HDJ2: EC50= 11 nM 0.81 nM

O

N

0.18 nM

Rap1A: EC50= >1000 nM K-Ras: EC50= 1290 nM

[51c]

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Table 4. contd….

IC50 In Vitro PGGT-1 PFT

Compound

IC50 in Cultured Cells

Refs

Br H N

88

N

256 nM

>10000 nM

nd

[52]

460 nM

>10000 nM

nd

[52]

90 nM

nd

nd

[52]

860 nM

nd

nd

[52]

1070 nM

nd

nd

[52]

1100 nM

nd

nd

[52]

N O

F H N

89

N N O

F H N

90

N HO

N O

O

H N

91

N HO

N O

O

F H N N

92 HO

N S O

O

O O F

H N N

93 HO O

N

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Table 4. contd….

IC50 In Vitro PGGT-1 PFT

Compound

H N

Refs

[52]

S

N

94

IC50 in Cultured Cells

HO

1070 nM

nd

nd

human: 40000 nM

human:

C. albicans:

>1000000 nM

No antifungal activity

nd

nd

[53a]

C. albicans: 31 nM

nd

nd

[53a]

C. albicans: 73 nM

nd

nd

[53a]

C. albicans: 35 nM

nd

nd

[53a]

nd

No anti-fungal activity

[53a]

N

O SH O O

N

O

95

SH

C. albicans: 17 nM

OH

S

O

n

O O

SH

O

N

n= 2: 240 nM

S

O

O O

97

SH O

N

OH

S

O

98

C. albicans: n= 1: 1300 nM

96

[53a]

MIC= >120 μg/mL

O O

N H

SH

O

O

99

H N

N

N H O

N H Cl Cl

human: > 10000 nM

O H N

100 N H O

O

N N H

C. albicans: 10 nM

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Table 4. contd….

IC50 In Vitro PGGT-1 PFT

Compound

IC50 in Cultured Cells

Refs

[53c]

[53d]

Cl Cl

N

IC50= 8 nM

IC50> 2 μM

Inhibits localization of Ras-CVLL and Rho proteins

IC50= 5.6 μM

nd

nd

IC50= 5.6 μM IC50= 43 μM (2 in vitro assays performed)

Little to no activity (details in Supporting Information ref 53d)

IC50= 4.2 μM

nd

nd

[53d]

IC50= 5.6 μM IC50= 35 μM (2 in vitro assays performed)

Little to no activity (details in Supporting Information ref 53d)

IC50= 3 μM

[53d]

IC50= 4.0 μM

nd

nd

[53d]

O

N H N

101 O N

NH2

O S

O NH2

O

102

S

N

N H

N

N O

O

103

O

O

N

NH2

S

N

N H

N

104

O N Cl N

N O

N

O

105

N N H

N

N

[53d]

N H

N

Cl

N

IC50= 3 μM

O

N

O

106

N

O H N Cl O

N N H

N

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Table 4. contd….

IC50 In Vitro PGGT-1 PFT

Compound

N

O

107

H N

Cl

N

N

IC50= 4.4 μM

nd

IC50= 3.8 μM

Little to no activity

IC50 in Cultured Cells

Refs

nd

[53d]

N H O

Cl

O O N H

HN

108

IC50= 35 μM (2 in vitro assays performed)

O Cl

(details in Supporting Information ref 53d)

IC50= 2 μM

[53d]

Cl

Inhibition RhoA geranylgeranylation R= OH: 0.5 μM

Cl

O

109

S N

R

O

Inhibition KRas4B geranylgeranylation R= OH: 0.9 μM

O

Cl O

S

110

Cl

O

Inhibition RhoA geranylgeranylation R= H: 0.3 μM Inhibition KRas4B geranylgeranylation R= H: 2 μM

N HO O

R= OH: Inhibits geranylgeranylation of Rheb-CSVL (HEK 293 cells) R= H: > 50 μM

S H N

N

O

[53f]

R= NH2: 11 μM

> 50 μM

Inhibits geranylgeranylation of Rheb-CSVL (HEK 293 cells)

[53e] [53f]

Inhibition K562 leukemia cells: O

R

[53e]

R= OH: 20 μM, R= OEt: >25 μM,

Cl

111

Inhibition K562 leukemia cells:

O

nd

nd

R= OMe: 10.5 μM, R= NH2: 2.2 μM No inhibition of H-Ras farnesylation and Rab5 geranylgeranylation

[53f]

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Table 4. contd….

Compound

IC50 In Vitro PGGT-1 PFT

IC50 in Cultured Cells

R= NH2: 1 μM

Inhibition K562 leukemia cells: R= OMe: 10.5 μM,

Cl

112

O

O

R O

N H

S N

O

R3

R2

R1

O

R1= Me, R 2= Cl, R 3= H: PGGT-1: 8.7 μM, PGGT-2: 4.7 μM R1= Cl, R 2= H, R3= Cl: PGGT-1: 8.9 μM, PGGT-2: 3.1 μM

S N

O

HO O

[53f]

R= NH2: 2.2 μM

O

113

Nd

O

Refs

all: > 100 μM

[53f]

> 100 μM

[53f]

R1= R 2= H, R3= Cl: PGGT-1:

S O

2.4 μM, PGGT-2: 7.0 μM R3

R2

R1

O

114

PGGT-1: 5.1 μM

S

O

N

O

PGGT-2: 7.0 μM

HO S

Several research groups have reported the synthesis and screening of compound libraries to find novel inhibitors of PGGT-1. For instance, an optimization procedure was used to generate a series of amphiphilic amides and select for PGGT-1inhibition [47a]. Two novel lipopeptides were discovered to inhibit the enzyme in the low micromolar range (entries 3 and 4, Table 4). These lipopeptides can serve as starting point for the development of more potent derivatives using rational design. Although the mode of action of these lipopeptides has not been determined, it is worth mentioning that these compounds have a structural resemblance with known bisubstrate inhibitors of PFT [47b-d]. A large library of compounds was generated around the N-arylpiperazinone motif (entries 5-87) [48-51]. The parent N-arylpiperazinone compound (entry 5) was found to be a potent inhibitor of the prenylating enzymes with a 50-fold preference for PFT over PGGT-1. Although based on the Ca1a2L motif, the mode of inhibition of entry 5 and analogues compounds, was found to be rather complex and involve Ca1a2L, GGPP or mixed type competitive behavior. Furthermore, the inhibitory potency of GGPP competitive compounds such as entry 5 and 8, was influenced by anions such as ATP and dithiophosphates [48b]. Interestingly, these GGPP competitive compounds became Ca1a2L competitive in the absence of anions. A hypothesis for this so called anion effect [48c] may be based on a model suggested by Scholten and co-workers when they found this anion dependance in which anions

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compete with the pyrophosphate group of GGPP for binding to PGGT-1. When the anion binds the pyrophosphate pocket, this offers opportunities for inhibitors such as entry 5 to bind in the empty geranylgeranyl pocket. In relation to this remarkable switching behavior of the inhibition mechanism, it would be interesting to evaluate the influence of bisubstrate inhibitors based on entry 5, on the inhibitory potency. Aryloxy derivatives of entry 5 were synthesized in order to investigate whether the potency of this dual PFT/PGGT-1 inhibitor could be enhanced. Introduction of various functional groups adjacent to the nitrile group of the terminal aromatic moiety, resulted in the aryloxy derivative depicted in entry 6. This compound showed a 5-fold improvement in inhibitory potency against PGGT-1. Swapping the nitrile and aryloxy functionality of entry 6, resulting in the compound shown in entry 7, had a detrimental effect on PGGT-1 inhibitory potency. A series of chlorinated derivatives of the compound shown in entry 6 were prepared (entries 8-10). The ortho chlorinated derivative depicted in entry 8 was shown to be the most active for inhibition of PGGT-1, with improved selectivity (PFT/PGGT-1= 10). A series of related aryloxy analogs, and one phenylthio analog, are shown in entries 11-16. The best dual inhibitor of this series is the compound depicted in entry 14. Despite a high in vitro activity, the compounds tested for in vitro potency against pure PGGT-1 substrate Rap1A (entries 5, 6, 8 and 14), indicate that cell permeability of these compounds was not significantly enhanced in comparison to the parent compound shown in entry 5. When the nitrile functionality in entry 6 was removed (entries 17-19), this resulted in an improved selectivity for PGGT-1. In line with this, several related N-(ortho-chloro) arylpiperazinone biaryl derivatives, in which the para-cyanophenylgroup was replaced by various biaryl groups (entries 20-33), were constructe [49]. Selectivity for PGGT-1 was not improved by these modifications, although the para-trifluormethylphenyl group (entries 27, 32 and 33) was found to be a good substitute for the para nitrile group. The paratrifluormethylphenyl derivative depicted in entry 33 showed in vivo inhibition of geranylgeranylation (inhibition of Rap1A geranylgeranylation), correlating well with its in vitro data. A further derivatisation of the parent N-arylpiperazinone compound in entry 5 was accomplished via cyclization (entries 34-40). The rationale behind this modification is based on the observation that entry 5 was found to adopt a folded conformation when bound to PFT and that macrocyclization had a significant effect on potency and selectivity [50a]. The 17-membered ring macrocycle displayed in entry 34 (n= 2), proved to be a potent inhibitor of both PGGT-1 and PFT and furthermore exhibited excellent in vivo potency against PFT. Replacing the nitrile group by different groups led to the discovery of the most potent PGGT-1 inhibitor and most balanced dual inhibitor of this series (entry 39). In order to improve the PGGT-1 inhibitory potency of these macrocyclic compounds, a series of 16-membered ring macrocycles with various benzyl derivatives was synthesised (entries 41-59) [50b]. Biological evaluation of these derivatives led to the finding of the selective PGGT-1 inhibitor shown in entry 51 (PGGT-1/PFT= 150). Reduction of the triple bond of the compound shown in entry 51 gave the compound depicted in entry 55, which proved to be a very potent dual inhibitor both in vitro as in vivo. Kinetic analysis of these macrocycles showed that the mode of action of these macrocycles ranged from not GGPP competitive (and thus presumable Ca1a2L competitive) to mixed type. Besides the piperazinone linker, the 3-aminopyrrolidinone group has also been used in the development of macrocyclic PFT/PGGT-1 inhibitors (entries 60-63) [51a]. These cyclic

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and rigid compounds were constructed with the aim to improve the plasma half-life and potency of their lineair counterparts by lowering the amount of possible conformers sensitive toward oxidative metabolism. Drug metabolism studies revealed that the benzylic carbon of the (cyanobenzyl)imidazole functionality was sensitive toward oxidative metabolism and simple steric blockade (i.e. methylation) of the benzylic carbon did not solve this problem. The dual inhibitor in entry 60 proved to have a good plasma half-life (2 h) in dogs and high bioavailability. X-ray analysis of a ternary complex of entry 60 and PFT•FPP revelead that residues involved in binding of this molecule are quite similar to those found in PGGT-1. The cyanobenzyl substituent is stacked against the isoprenoid chain and the imidazole ring is ligated to the catalytic Zn2+ ion. An interesting difference is that in PFT the naphtyl residue binds in a more aromatic pocket (Trp102, Trp106, Trp303 and Tyr361) and in PGGT-1 this pocket is less aromatic (Thr102, Phe106, Trp303 and Leu361). The caprolactams shown in entry 64-87 are a structurally related class of dual inhibitors. Several of these compounds (entry 79-87) were shown to be potent inhibitors in vivo [51b,c]. X-ray analysis of entry 79 and PFT•FPP suggest that the cyano group interacts with Arg202(PFT) which is preserved in PGGT-1 (Arg173). As the previous mentioned aminopyrrolidinones, the central phenyl ring interacts with the same hydrophobic pocket formed by Trp102, Trp106, Trp303 and Tyr361. The aliphatic part of the azepinone interacts in PFT with three amino acids: Ala151, Trp102 and Ser99. In PGGT-1 these residues are all preserved (Ala123, Trp102 and Ser99, respectively). The ethyl group interacts with Leu96 and Trp106 which in PGGT-1 are Leu43 and Phe53 respectively. Reduction of the lactam carbonyl (entry 69
entry 70) to an amine had, especially for PGGT-1, a detrimental effect on inhibition potency. From the crystal structure it became evident that this amide bond was exposed to solvent and it was suggested that introduction of an amine at that position could have unfavorable electrostatic interactions with Arg202. As this residue is preserved in PGGT 1 this supports the decrease in potency as observed for PFT. From the in vivo experiments, no clear relationship between in vivo and in vitro inhibitory potency could be observed. In addition, the compounds in entry 79 and entry 86 exhibited severe toxicity in mice, poor oral bioavailability and a rapid clearance [6a]. Thus, although these compounds showed promising in vitro potency, their bad pharmacokinetic properties proved to be a major drawback. As part of program to develop potent inhibitors of fungal PGGT-1, diverse libraries of methane amino imidazoles were synthesised (Table 4, entry 88-94) [52]. Not surprisingly, the imidazole constitutes an important part of these molecules (Zn2+ ligation), as replacing it with other heterocycles (thiazole, pyridine) resulted in loss of activity. Next, it was reasoned that a carboxylic group in the proximity of the imidazole ring may interact with residues of the nearby pyrophosphate pocket. Using solid phase chemistry, a range of -amino acid compounds were synthesised and the most potent PGGT-1 inhibitor was identified as the fluoro derivative in entry 90. This compound showed a 5-fold improvement in inhibitory potency compared to the parent compound in entry 89. The (lead) aromatic thiazoline derivative in entry 95 originates from random screening of compound libraries [53a]. It is a potent (IC50= 17 nM) inhibitor of C. albicans PGGT-1, with a high selectivity against human PGGT-1 and PFT (IC50= 40 μM and >1000 μM, respectively). Unfortunately, in vivo activity was poor and in order to improve this, the metabolically labile dithiotreitol group, which is believed to interact with the catalytic Zn2+ ion, was replaced by analogs (entries 96-100).

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In the same bio-assay the depsipeptide derivative in entry 98 proved to be even more active. An interesting observation was that despite its potent in vitro activity, the compound in entry 98 still did not show any anti-fungal activity against C. albicans. The authors attribute this discrepancy between in vitro and in vivo potency to the same phenomenon of crossprenylation seen in mammalian cells [5]. In line with this hypothesis, Kurtz and co-workers had shown before that C. albicans in which PGGT-1 activity was absent or inhibited, could still be viable [53b]. These results underscore the significance of dual inhibition in certain therapeutic strategies. The pyrazole-based inhibitor in entry 101 (GGTI-DU40) was developed by structural optimization of a chemical library screening hit [53c]. According to kinetic data, this very potent and selective PGGT-1 inhibitor targets the Ca1a2X binding pocket. It also proved to be effective in vivo as it caused mislocalization of Ras-CVLL at an concentration of 2 μM and inhibited the geranylgeranylation of Rho and Rap1. Its high affinity and selectivity for PGGT-1 can be attributed to several interaction sites. An important interaction was found to involve the sulfur atom; substituting this for an amine resulted in a
250 fold loss in activity. Interestingly, whereas the monomeric G-proteins Ras, Rho and Rap1 are affected by this inhibitor, membrane localization of the heterotrimeric G-protein G
 was only inhibited partially. This selective effect might be attributed to different protein half-lives and indicates that selective inhibition of specific geranylgeranylated proteins is possible. A powerful approach toward novel bioactive compounds entails using quantitative structure-activity relationship modelling in combination with virtual screening. Recently this method was used to identify new PGGT-1 inhibitors [53d]. Using 48 known PGGT-1 inhibitors with 2 different scaffolds, a quantitative structure-activity relationship model was developed and subsequently used to virtually screen 9.5 million compounds. An initial set of 47 compounds with moderate to high predicted activity was identified (Fig. 3). Of this set, 7 members (entries 102 – 108) with novel scaffolds were chosen for enzymatic characterization. As predicted, the 7 compounds inhibited PGGT-1 (IC50 values between 4 – 6 μM). When four compounds were tested against PFT, no to little activity could be measured. By screening a collection of heterocycles for their ability to inhibit the geranylgeranylation of K-Ras4B or RhoA by PGGT-1, Kwon and co-workers identified a dihydropyrrole (1, Scheme 3) and tetrahydropyridine (2, Scheme 3) as powerful PGGT-1 inhibitory lead compounds [53e]. The phosphine mediated annulation of allenoates and N-tosylimines [53e] can be used to synthesise analogs of these compounds. By translating this chemistry into a new solid-phase based synthesis (Scheme 3), a library of 4288 compounds could be constructed. From this library two new and more potent PGGT-1 inhibitors were identified, (entries 109 and 110), both exhibiting in vitro and in vivo activity. Kinetic data revealed that these inhibitors were competetive with respect to the protein substrate [53f]. In order to enhance in vivo potency, the carboxylic acid of entry 109 was modified (entries 111  112). Introducing an L-phenylalanine residue resulted in a 3  9 fold improvement of the inhibition of K562 leukemia cell proliferation. Interestingly, besides selective PGGT-1 inhibitors, several compounds were found to be dual PGGT-1/2 inhibitors (entries 113  114). These compounds share a characteristic six-atom tail linked to the pyrrolidine core by a thioether. By selecting for this feature, 23 compounds were identified which showed preferential inhibition of PGGT-2. This selection of compounds (Table 5) is described in the section about PGGT-2.

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Ar

HO TFA

S

N

O

Bu3P or PPh3, 60oC

O

Ar

Ar S

Ar

S

O

O

N-tosyltolualdimine O

O N

F

R 5: dihydropyrroles

O

N

HO

O O

R TFA

R-SH

O 1

O O

O

R

R

O

Ar

S

N

3

IC50 RhoA= 200 μM IC50 KRas4B= 250 μM

Ar

HO

R

S

6:pyrrolidines

O

R

resin bound allenoates O

R

O

TFA O

S

O

N

HO

O

Ar

N

O

HO

R

Bu3P, rt O

N-tosyltolualdimine 2

S

Ar

O

N

7:tetrahydropyridines

Ar

O R-SH

O

TFA

R

O

Ar

S

O 4

IC50 RhoA= 120 μM IC50 KRas4B= 80 μM

Ar

S

F

N

HO R

S

O Ar

8:piperidines

Scheme 3. Solid phase synthesis of small-molecules PGGT-1/2 inhibitors by phosphine catalysed ring-formation between allenoates and N-tosylimines. Table 5.

Inhibitors of PGGT-2. (H-Ras is a Substrate for PFT, Rap1A and RhoA are Substrates for PGGT-1 and Rab6 is a Substrate for PGGT-2)

Entry

Compound

IC50 In Vitro

Inhibition Potency in vivo

Refs

Br

PFT: >100 μM

O

1

O S

PGGT-1: >50 μM

OH N

[53f]

PGGT-2: 4.5 μM

O O

S

Br

m=2, n=4
PGGT-2: 3.6 μM O

2

OH

O S

N

O

S m

n

m=2, n=5
no effect on H-Ras

m=1, n=5
PGGT-2: 4.8 μM

and Rap1

m=2, n=5
PGGT-2: 2.1 μM

prenylation.

m=5, n=5
PGGT-2: 2.2 μM

Causes

All: PFT: >100 μM, PGGT-1: >50 μM

mislocalization of Rab5b

[53f]

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Table 5. contd….

Entry

Compound

IC50 In Vitro

Inhibition Potency in vivo

Refs

Inhibits proliferation of J774 macrophage cells with IC50= 30 μM. No inhibition of

O OH

P

H-Ras/Rap1A (J774

OH

3

PGGT-2: 600 μM

OH N

HO

cells). Complete

[59a]

inhib. Rab6

O

geranylgeranylation. IC50 Rab1a inhibition= 31.9 ± 2.1 μM O P

N

4

OH

Rab1a inhibition

OH

IC50= 1.3 ± 0.22 μM

OH N

HO

[59b]

O

m= 6
PFT: 98 ± 5 μM, PGGT-1: 97 ± 31 μM, O

5

PGGT-2: 2.8 ± 0.4 μM

O H N

N

O

m

H N

N H

and 8.8 ± 0.7 μM m= 9
PFT: nd, PGGT-1:

O

[59c]

>100 μM, PGGT-2:

N N OH

4.7 ± 1.0 μM and

HN

2.8 ± 0.1 μM R= Tyr, PFT: nd,

N

PGGT-1: >100 μM, PGGT-2: HN

O

6

O

H N

N

O

R= His, PFT: 35 ± 5.8 μM, O

R

N

9

H N

N H

6.3 ± 0.7 μM and 4.3 ± 0.4 μM [59c]

PGGT-1: 60 ± 5.3 μM, PGGT-2: OH

11 ± 1.2 μM and 10 ± 0.9 μM

N

O

7

HN

N H

O R

R= Tyr

O

H N

N H

9

H N

PGGT-2: 7.2 ± 0.3 μM [59c] R= His

O

PGGT-2: 5.2 ± 0.8 μM NH2 OH

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Table 5. contd….

Entry

Compound

IC50 In Vitro

Inhibition Potency in vivo

Refs

R1= HO, R2 =Tyr O

PGGT-2: 22.7 ± 1.8 μM

O H N

8

H N

R1

O

R1= HONH, R2 =Tyr

N R2

O

O N

R1= HONH, R2 =Trp N H

O

PGGT-2: 5.2 ± 0.7 μM

O H N

9

[59c]

PGGT-2: 9.0 ± 1.0 μM

HO O

N H

H N

O

PGGT-2: 4.1 ± 0.3 μM

O

OH

[59c]

OH

Inhibition of RhoA and Rab6 processing: IC50= 1000 μM and 500 μM, respectively. 10

OH (S)-perillyl alcohol

No inhibition of PFT in NIH3T3 cells.

[59f] [59g]

At a conc. of 1000 μM, PGGT-1 was inhibited by 62% and PGGT-2 by 72%.

7. PROTEIN:GERANYLGERANYL TRANSFERASE-2 (PGGT-2) The second geranylgeranylating enzyme, protein:geranylgeranyl transferase-2 (PGGT-2, GGTase-II or RabGGTase, ec 2.5.1.60) is responsible for the post-translational modification of cysteine residues of Rab precursor proteins having CCXX, XCXC or XXCC (X= any amino acid) C-terminal consensus sequences [54]. After further processing, Rab precursor proteins are involved in the communication between organelles through vesicular transport [55]. Mammalian PGGT-2 is composed of a 60 kDa
-subunit, which is structurally similar to PFT and PGGT-1, and a 38 kDa-subunit (Fig. 5A). The crystal structure of (rat) PGGT2 is shown in Fig. (5) [56b] and, as in PGGT-1, the active site extends into a central funnelshaped cavity of the -subunit with a Zn2+ ion positioned at the top. The
-subunit contains two additional distinct modules whose function is unknown: an immunoglobuline-like (Iglike) domain and a leucine-rich repeat (LRR) domain. What is known is that they have no interactions within the catalytic ternary complex and are not found in PGGT-2 of lower eukaryotes [56]. It is however plausible that these domains are involved in interactions with proteins or intracellular membranes. The structure of the PGGT-2•REP•Rab complex has

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not been elucidated yet. Using the solved structures of the REP-1•Rab7 and REP-1
PGGT
2 complexes, a model was constructed (Fig. 5B) [57f]. Compared to PFT and PGGT-1, PGGT-2 has a rather complex mode of action. It requires the Rab escort protein (REP) for its catalytic activity (Scheme 4) and Rab protein specificity [57]. Two pathways towards the active REP
Rab
PGGT-2 complex have been reported (Scheme 4). In the first pathway (Fig. 4, pathway 1) [57g], unmodified Rab precursor protein is recruited by REP to form a REP
Rab complex, which is subsequently tightened by two binding regions (i.e. the CBR in REP and CIM in Rab, Scheme 4). It is believed that this interaction is responsible for directing the C-terminal portion of a Rab protein to the PGGT-2 active site. This is in line with the observation that PGGT-2 does not bind its peptide substrate by interacting with amino acid residues and as a consequence can process >60 Rab proteins with unrelated C-termini (Scheme 2) [57]. The REP
Rab then binds PGGT-2 resulting in the active complex. In the alternative pathway (Scheme 4, pathway 2) [57h], GGPP binding to PGGT-2 promotes the binding of REP and subsequently the Rab protein. After formation of the GGPP•PGGT-2•REP•Rab complex, the most N-terminal cysteine residue of the Rab precursor concensus sequence is geranylgeranylated in the active site of PGGT-2 [16], mediated by the catalytic Zn2+ ion via a similar mechanism as that of PGGT-1 [58]. Next, the geranylgeranyl thioether moiety may be sequestered in a 7.5 Å diameter hydrophobic tunnel of the enzyme, followed by translocation of the mono-prenylated product from the site of catalysis. This alternative binding mode of the mono-prenylated product is thought to be associated with the binding of the next GGPP substrate. The thiolate of the second unmodified cysteine residue can now interact with the Zn2+ ion to undergo isoprenylation. The catalytic cycle can now start over again by release of the product.

Scheme 4. Schematic representation of the proposed mechanism of action of PGGT-2.

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N Y

R2 R4

R3

O

N N

R1

N N

R4

R3

C, N

O, CH2

H N

R1

N

N N

O R2

R3

O

R1 O

R1

R2

Fig. (4). General structures of novel scaffolds in the initial set of 47 virtial PGGT-1 inhibitors identified by quantitative structure-activity relationship-virtual screening.

PGGT-2 is developing into a powerful drug target for several reasons. Besides Rab dysfunction being linked to thrombic disorders, genetic disorders, osteoporosis and cancer [55, 59, 60], overexpression of PGGT-2 itself has been found in several cancer types. In addition to its complex mode of action, the development of PGGT-2 inhibitors requires several characteristics to be taken into account. First, PGGT-2 has a relatively large active site when compared to PFT and PGGT-1. Because of the conserved nature of the active-site in the three prenyl transferases, libraries that were originally targeted against PFT and PGGT-1 represent a powerful source of potential PGGT-2 inhibitors. This cross-reactivity is based on the conserved nature of the active-site in the three prenyl transferases. As outlined in the PGGT-1 section (entries 109 – 144, Table 4), a set of pyrrolidines (entries 1 and 2, Table 5) were identified that showed selective inhibition of PGGT-2. A competition experiment with the 3rd compound of entry 3 (Table 5) revealed that it inhibited PGGT-2 by competing with the protein substrate (in this case Rab7) and not GGPP. Although it is currently unclear how these compounds work exactly, the characteristic lipid tail is important for PGGT-2 selectivity. The phosphonate depicted in entry 3, 2-[3-pyridinyl]-1-hydroxyethylidene-1,1phosphonocarboxylic acid (3-PEHPC, Table 5) [59a] is a potent PGGT-2 inhibitor that disrupts the membrane localization of Rab proteins in osteoclasts. It is derived from risedronate (Actonel®), which is used in the clinic for the treatment of osteoporosis. A recent study showed that 3-PEHPC prevents the development of myeloma bone disease in vivo [59b]. Note that risedronate inhibits Rab geranylgeranylation indirectly by targeting FPP synthase (Scheme 1). By replacing the pyridine group of entry 1 for an imidazole[1,2-a]pyridine moiety (entry 4), the inhibition of Rab1a geranylgeranylation was improved 25-fold [59c]. Overall, the phosphonocarboxylates in entries 3 and 4 are potent lead compounds for the development of higher affinity analogs that can be applied for the treatment of thrombotic disorders or osteoporosis. The inhibitory potency of these phosphonocarboxylates was found to be based on preventing the second geranylgeranylation event of PGGT-2 (Scheme 4) [59c], with monogeranylgeranylation resulting in mislocalization and loss-of-function (e.g. in the case of Rab5). Although double geranylgeranylation is required for the majority of Rab proteins, biological processes regulated by monogeranylgeranylated Rab proteins (Rab8, Rab13, Rab18, Rab23) are not affected by these compounds. In addition, the phosphonocarboxylates were found to be non-competitive with regard to Rab-substrate and GGPP. This suggests that these compounds bind in a pocket that serves to accommodate the mono-geranylgeranylated cysteine, an event which is required for binding the second geranylgeranyl lipid.

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A

Ig-like domain α-subunit

β-subunit

α-subunit

funnel shaped Active site

LRR domain

β-subunit

LRR domain Ig-like domain

B

Rab7 REP1 REP1 Rab7

α-subunit

CBR-CIM interaction

funnel shaped active site

α-subunit

β-subunit

C-terminus is positioned for entrance into active site

Ig-like domain Ig-like domain

LRR domain

β-subunit

Fig. (5A). X-ray structure of rat PGGT-2 with the characteristic Ig-like and LRR domain bound to the
-subunit (pdb 1DCE). The right part shows a top view of the -subunit with the central funnel shaped active site and the Zn2+ and pyrophosphate binding site highlighted. Graphics were generated with PYMOL. (5B). Left: Side view of a PGGT-2•REP•Rab model constructed by superimposing the structures of REP-1•Rab7 (pdb 1VGO) and REP-1•PGGT-2 (pdb 1LTX). Right: top view. Graphics were generated with PYMOL.

By using the natural tripeptide PFT inhibitor Pepticinnamin E (Scheme 5, isolated from Streptomyces OH-4652) as a scaffold, a large set of analogs was synthesised by Fmoc solid

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phase peptide synthesis (Scheme 5) and screened for PGGT-2 inhibitory activity [59d]. In entries 4
9 a selection of potent PGGT-2 inhibitors is shown. The upper member of entry 8 was used to determine the first crystal structure of a PGGT-2
inhibitor complex (a truncated PGGT-2 protein was used). A graphical representation of the interactions in this complex is depicted in Fig. (6). The compounds were also found to inhibit the prenylation of Rab7 in COS-7 cells overexpressing this protein. Using this initial tripeptide library as a starting point, additional analogs were synthesised and tested [59e]. The compounds of this second library were classified into tripeptides with (1) a free C-terminal carboxylic/ hydroxamic acid and lipophilic N-terminus; (2) a free C-terminal carboxylic acid and heterocyclic N-terminus; (3) a C-terminal amine or N-heterocycle and lipophilic N-terminus. Fig. (7) depicts structural features that were found in the most potent PGGT-2 inhibitors. 1. removal Fmoc

O NHFmoc

O

2. coupling building block

R2

Cl OH Cl

3. acidic cleavage in the case of 2-ClTrityl linker

O

or H N O

O

O N

N

O O

O

3. oxidative/nucleophilic cleavage in the case of aryl hydrazine linker

2-Cl-Trityl linker

NH HN O

OH

O O

H N

Pepticinnamin E (Streptomyces OH-4652)

O

R3

O

H N

NHFmoc

N H

H N

R6

N

R1 R2

R2

O

R4

R5

O

Aryl hydrazine linker

Scheme 5. Solid phase synthesis of peptide based PGGT-2 inhibitors. protrudes in GGPP binding pocket OH O Tyr241


HO

O H N

H N

N O

O O

N HO

NH2 N H

protrudes in protein binding pocket

HO

HN

N H

Arg144


Tyr97


Fig. (6). Structural binding of first member of entry 8 (Table 5) with a truncated PGGT-2.

The monoterpene (S)-perillyl alcohol (entry 10) [59f,g] has anticancer activity via the dual inhibition of PGGT-1 and PGGT-2, but does not interfere with PFT activity. 8. PROTEOMICS AND THE PRENYLOME Although numerous precursor proteins which are targeted by PGGT-1, PGGT-2 or PFT have been identified, the interplay of their mature counterparts with respect to cell function

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and dysfunction is generally not well understood on the functional protein level. Recently, an azide modified FPP analog, designed to function as a substrate for PFT [61], was used for the production of tagged farnesylated proteins which subsequently could be analyzed on a functional level by a specific chemical reaction of the azide moiety. Via this functional proteomics approach [62], 18 prenylated proteins were identified, including several with novel farnesylation motifs [61a]. In addition, the effects of inhibitors of PFT on the farnesylation of these proteins could be followed. Extension of this approach, by the application of modified GGPP substrates [63], will not only lead to the identification [64] of currently unknown geranylgeranylated proteins, but will also show the effects of selective and dual inhibitors on the functional prenylated protein level and will allow correlation of geranylgeranylation and farnesylation events, in relation to disease. In Fig. (8), GGPP analogs are depicted that are used to tag and identify geranylgeranylated proteins by means of a chemical reactive residue [63f-k]. HO

N H

N

HO HO N

HN

HN

N R

R

R

R 4-11

N

N

N H

R2

O H N

H2N

N H

O H N

N H

R1

R O

O

R3

O

Fig. (7). Structural features found in the most potent tripeptidic PGGT-2 inhibitors of ref. [59de], entries 4 – 9 Table 5. O2N OH

OH

N3

O

O

N H

N N

Azido-GGPP

H H

OH N H

O

P

P

O

O

OH

OH

OH

O

OH O

P O

OH O

O

NH

HN

OH O

NBD-GGPP

O S

P

P O

O

P

OH

O

O "Biotin-GGPP"

"Alkyne-GGPP"

Fig. (8). Chemical tools to tag and identify geranylgeranylated proteins for proteomics.

9. CONCLUSIONS Three prenylating enzymes have been identified, purified and characterised by X-ray analysis: PFT, PGGT-1 and PGGT-2. Until now, PGGT-1 and in particular PFT have

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become important targets for rational treatment in a number of diseases in which aberrant cell behavior can be linked to prenylated proteins. Although major progress has been made, inhibition of prenylation remains an active area of research. There is the complex interplay of the prenylating enzymes, underscored by the fact that PFT and PGGT-1 share several common structural and functional features. This has important consequences such as the PGGT-1 mediated geranylgeranylation of PFT substrates upon inhibition of PFT [5]. As such dual inhibition of both enzymes seems important for a good clinical effect. As total inhibition of prenylation may give rise to toxic side-effects [65], the challenge is to develop dual prenyl transferase inhibitors which do not exhibit severe toxicity. Another interesting approach, which might prove to be the most effective anti-cancer strategy, is to administer prenyl transferase inhibitors in combination with other cytotoxic agents [29c] [66]. Understanding how interfering in the prenylating process [6d] might affect certain physiological processes is further complicated by evidence form in vitro and in vivo studies, that certain prenyl transferase inhibitors do not solely inhibit the Ras signalling pathway. An interesting emerging suspect in this context is the proteasome [6ef]. The PGGT-1 inhibitor GGTI-298 (entry 5, Table 2), for example, was found to also inhibit proteasome chymotrypsin activity in vitro [6e]. It is too early to state what this all means exactly, but it does highlight the importance of detailed in vivo studies. Then there is the involvement of PGGT-2. Despite its more complex mechanism of action, compared to PFT and PGGT-1, there is evidence that suggests that undesired side-effects of some PFT inhibitors are caused by the simultaneous inhibition of PGGT-2 [67]. The emergence of functional proteomics holds promise to obtain detailed insight about the levels of farnesylated and geranylgeranylated proteins in the absence or presence of selected inhibitors. In view of the complexity of isoprenoid transferase drug targeting, the development and use of modified prenyl pyrophosphate substrates (Table 6) is a promising (proteomics) approach for analysing the prenylome [63h]. This will not only be valuable for the search for novel biomarkers in clinical diagnostics, but also for the discovery of interesting pharmaceutical targets and their therapeutics in relation to various disorders or diseases in which prenylated proteins play a key role. ABBREVIATIONS CBR

=

C-terminus binding region

CIM

=

CBR interacting motif

DTT

=

Dithiotreitol

EC50

=

Fmoc

=

9-fluorenylmethoxycarbonyl

FPP

=

Farnesyl pyrophosphate

GGPP

=

Geranylgeranyl pyrophosphate

GTP

=

Guanosine triphosphate

HSM cells

=

Human smooth muscle cells

IC50

=

Concentration of an inhibitor required for 50% inhibition

Concentration of an agonist required for 50% of the maximal possible response for that agonist

Inhibitors of Protein: Geranylgeranyl Transferases

Frontiers in Medicinal Chemistry, 2010, Vol. 5

Ig

=

Immunoglobuline

LRR

=

Leucine-rich repeat

MIC

=

Minimum inhibitory concentration

MFC

=

Minimal fungicidal concentration

PFT

=

Protein: farnesyl transferase

PGGT-1

=

Protein: geranylgeranyl transferase-1

PGGT-2

=

Protein: geranylgeranyl transferase-2

REP

=

Rab escort protein

225

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nocarboxylate inhibitor of Rab geranylgeranyl transferase that specifically prevents Rab prenylation in osteoclasts and macrophages. J. Biol. Chem., 2001, 276, 48213-22. (b) Lawson, M.A.; Coulton, L.; Ebetino, F.H.; Vanderkerken, K.; Croucher, P.I. Geranylgeranyl transferase type II inhibition prevents myeloma bone disease. Biochem. Biophys. Res. Comm., 2008, 377, 453-57. (c) Baron, R.A.; Tavaré, R.; Figueiredo, A.C.; Baewska, K.M.; Kashemirov, B.A.; McKenna, C.E.; Ebetino, F.H.; Taylor, A.; Rogers, M.J.; Coxon, F.P.; Seabra, M.C. Phosphonocarboxylates Inhibit the Second Geranylgeranyl Addition by Rab Geranylgeranyl Transferase. J. Biol. Chem., 2009, 284, 6861-8. (d) Guo, Z.; Wu, Y.-W.; Tan, K.-T.; Bon, R.S.; Guiu-Rozas, E.; Delon, C.; Nguyen, U.T.; Wetzel, S.; Arndt, S.; Goody, R.S.; Blankenfeldt, W.; Alexandrov, K.; Waldmann, H. Development of selective RabGGTase inhibitors and crystal structure of a RabGGTase-inhibitor complex. Angew. Chem. Int. Ed., 2008, 47, 3747-50. (e) Tan, K.-T.; Guiu-Rozas, E.; Bon, R.S.; Guo, Z.; Delon, C.; Wetzel, S.; Arndt, S.; Alexandrov, K.; Waldmann, H.; Goody, R.S.; Wu, Y.-W.; Blankenfeldt, W. Design, Synthesis, and Characterization of Peptide-Based Rab Geranylgeranyl Transferase Inhibitors. J. Med. Chem., 2009, 52, 8025-37. (f) Coxon, F. P.; Ebetino, F.H.; Mules, E.H.; Seabra, M. C.; McKenna, C.E.; Rogers, M. J. Phosphonocarboxylate inhibitors of Rab geranylgeranyl transferase disrupt the prenylation and membrane localization of Rab proteins in osteoclasts in vitro and in vivo. Bone. 2005, 37, 34958. (g) Ren, Z.; Elson, C.E.; Gould, M.N. Inhibition of type I and type II geranylgeranyl-protein transferases by the monoterpene perillyl alcohol in NIH3T3 cells. Biochem. Pharm., 1997, 54, 113-20. (a) Olkkonen, V.M.; Ikonen, E. Mechanisms of disease - Genetic defects of intracellular-membrane transport. New Engl. J. Med., 2000, 343, 1095-1104. (b) Aridor, M.; Hannan, L.B. Traffic jam: A compendium of human diseases that affect intracellular transport processes. Traffic, 2001, 1, 836-51. (c) Pereira-Leal, J.B.; Hume, A.N.; Seabra, M.C. Prenylation of Rab GTPases: molecular mechanisms and involvement in genetic disease. FEBS Lett., 2001, 498, 197-200. (a) Kho, Y.; Kim, S.C.; Jiang, C.; Barma, D.; Kwon, S.W.; Cheng, J.; Jaubergs, J.; Weinbaum, C.; Tamanoi, F.; Falck, J.; Zhao, Y. A tagging-via-substrate technology for detection and proteomics of farnesylated proteins. Proc. Natl. Acad. Sci. U.S.A., 2004, 101, 12479-84. (b) El Oualid, F. Design, Synthesis and biological evaluation of Peptidomimetic prenyl transferase inhibitors. PhD Thesis Leiden University: Leiden, 2005; chapter 6, pp. 117
26. (a) Kannicht, C. Posttranslational Modification of Proteins: Tools for Functional Proteomics, Methods in Molecular Biology, 2002, Vol. 194. (b) M. Baumann, S. Meri Techniques for studying protein heterogeneity and post-translational modifications. Exp. Rev. Prot. 2004, 1, 207-17. (c) van Swieten, P.F.; Leeuwenburgh, M.A.; Kessler, B.M.; Overkleeft, H.S. Bioorthogonal organic chemistry in living cells: novel strategies for labeling biomolecules. Org. Biomol. Chem., 2005, 3, 20-7. (a) Bukhtiyarov, Y.E.; Omer, C.A.; Allen, C.M. Photoreactive analogs of prenyl diphosphates as inhibitors and probes of human protein farnesyl transferase and geranylgeranyltransferase type-I. J. Biol. Chem., 1995, 270, 19035-40. (b) Turek, T.C.; Gaon, I.; Gamache, D.; Distefano, M.D. Synthesis and evaluation of benzophenone-based photoaffinity labeling analogs of prenyl pyrophosphates containing stable amide linkages. Bioorg. Med. Chem. Lett., 1997, 7, 2125-30. (c) Quellhorst Jr., G.J.; Allen, C.M.; WesslingResnick, M. Modification of Rab5 with a photoactivatable analog of geranylgeranyl diphosphate. J. Biol. Chem., 2001, 276, 40727-33. (d) Liu, X.-H.; Prestwich, G.D. Didehydrogeranylgeranyl (Delta Delta GG): A fluorescent probe for protein prenylation. J. Am. Chem. Soc., 2002, 124, 20-1. (e) Dursina, B.-E.; Reents, R.; Niculae, A.; Veligodsky, A.; Breitling, R.; Pyatkov K.; Waldmann, H.; Goody, R.S.; Alexandrov, K. A genetically encodable microtag for chemo-enzymatic derivatization and purification of recombinant proteins. Prot. Exp. Pur., 2005, 39, 71-81. (f) Chan, L.N.; Hart, C.; Guo, L.; Nyberg, T.; Davies, B.S.J.; Fong, L.G.; Young, S.G.; Agnew, B.J.; Tamanoi, F. A novel approach to tag and identify geranylgeranylated proteins. Electrophoresis, 2009, 30, 3598-3606. (g) Charron, G.; Wilson, J.; Hang, H.C. Chemical tools for understanding protein lipidation in eukaryotes. Curr. Opion. Chem. Biol., 2009, 13, 382-91. (h) Nguyen, U.T.T.; Guo, Z.; Delon, C.; Wu, Y.W.; Deraeve, C.; Fraenzel, B.; Bon, R.S.; Blankenfeldt, W.; Goody, R.S.; Waldmann, H.; Wolters, D.; Alexandrov, K. Analysis of the eukaryotic prenylome by isoprenoid affinity tagging. Nat. Chem. Biol., 2009, 5, 227-35. (i) Hosokawa, A.; Wollack, J.W.; Zhang, Z.; Chen, L.; Barany, G. Evaluation of an alkyne-containing analogue of farnesyl diphosphate as a dual substrate for protein-prenyltransferases. Int. J. Pept. Res. Ther., 2007, 13, 345-54. (j) Wu, Y-W.; Tan, K-T.; Waldmann, H.; Goody, R.S.; Alexandrov, K. Interaction analysis of prenylated Rab GTPase with Rab escort protein and GDP dissociation inhibitor explains the need for both regulators. Proc. Natl. Acad. Sci. U.S.A., 2007, 104, 12294-99. (k) Wu, Y-W.; Alexandrov, K.; Brunsveld, L. Synthesis of a fluorescent analogue of geranylgeranyl pyrophosphate and its use in a high-throughput fluorometric assay for Rab geranylgeranyltransferase. Nat. Prot., 2007, 2, 2704-11. (l) Dursina, B.; Reents, R.; Delon, C.; Wu, Y.; Kulharia, M.; Thutewohl, M.; Veligodsky, A.; Kalinin, A.; Evstifeev, V.; Ciobanu, D.; Szedlacsek, S.E.; Waldmann, H.; Goody, R.S.; Alexandrov, K. Identification and specificity profiling of protein prenyltransferase inhibitors using new fluorescent phosphoisoprenoids. J. Am. Chem. Soc., 2006, 128, 2822-35.

Inhibitors of Protein: Geranylgeranyl Transferases [64]

[65] [66]

[67]

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(a) Buser, C.A.; Dinsmore, C.J.; Fernandes, C.; Greenberg, I.; Hamilton, K.; Mosser, S.D.; Walsh, E.S.; Williams, T.M.; Koblan, K.S. High-performance liquid chromatography/mass spectrometry characterization of Ki4B-Ras in PSN-1 cells treated with the prenyltransferase inhibitor L-778,123. Anal. Biochem., 2001, 290, 126-37. (b) Durek, T.; Alexandrov, K.; Goody, R.S.; Hildebrand, A.; Heinemann, I.; Waldmann, H. Synthesis of fluorescently labeled mono- and diprenylated Rab7 GTPase. J. Am. Chem. Soc., 2004, 126, 16368-78. (c) Goossens, L.; Deweer, S.; Pommery, J.; Hénichart, J.-P.; Goossens, J.-F. Spectroscopic study of fluorescent peptides for prenyl transferase assays. J. Pharm. Biomed. Anal., 2005, 37, 417-22. (d) Tong, H.; Holstein, S.A.; Hohl, R.J. Simultaneous determination of farnesyl and geranylgeranyl pyrophosphate levels in cultured cells. Anal. Biochem., 2005, 336, 51-9. (a) McGuire, T.F.; Sebti, S.M. Geranylgeraniol potentiates lovastatin inhibition of oncogenic H-Ras processing and signaling while preventing cytotoxicity. Oncogene, 1997, 14, 305-12. (b) Banerjee, S.; McGeady, P. Inhibitors of Protein Prenylation: An Overview. Curr. Enz. Inhib., 2005, 1, 183-206. Martin, N.E.; Brunner, T.B.; Kiel, K.D.; DeLaney, T.F.; Regine, W.F.; Mohiuddin, M.; Rosato, E.F.; Haller, D.G.; Stevenson, J.P.; Smith, D.; Pramanik, B.; Tepper, J.; Tanaka, W.K.; Morrison, B.; Deutsch, P.; Gupta, A.K.; Muschel, R.J.; Gillies McKenna, W.; Bernhard, E.J.; Hahn, S. M. A phase I trial of the dual farnesyltransferase and geranylgeranyltransferase inhibitor L-778,123 and radiotherapy for locally advanced pancreatic cancer. Clin. Cancer Res., 2004, 10, 5447-54. Lackner, M.R.; Kindt, R.M.; Carroll, P.M.; Brown, K.; Cancilla, M.R.; Chen, C.; de Silva, H.; Franke, Y.; Guan, B.; Heuer, T.; Hung, T.; Keegan, K.; Lee, J.M.; Manne, V.; O’Brien, C.; Parry, D.; Perez-Villar, J.J.; Reddy, R.K.; Xiao, H.; Zhan, H.; Cockett, M.; Plowman, G.; Fitzgerald, K.; Costa, M.; RossMacdonald, P. Chemical genetics identifies Rab geranylgeranyl transferase as an apoptotic target of farnesyl transferase inhibitors. Cancer Cell, 2005, 7, 325-36.

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Voltage-Gated Sodium Channels: New Targets in Cancer Therapy? Ludovic Gillet, Sébastien Roger, Marie Potier, Lucie Brisson, Christophe Vandier, Pierre Besson* and Jean-Yves Le Guennec+ U921 Inserm, Nutrition Croissance Cancer, Université de Tours, Faculté de Médecine, 10 Bd Tonnellé, 37032 TOURS France Abstract: Early detection and treatment of cancers have increased survival and improved clinical outcome. The development of metastases is often associated with a poor prognostic of survival. Finding early markers of metastasis and developing new therapies against their development is a great challenge. Since a few years, there is more evidence that ion channels are involved in the oncogenic process. Among these, voltage-gated sodium channels expressed in non-nervous or non-muscular organs are often associated with the metastatic behaviour of different cancers. The aim of this review is to describe the current knowledge on the functional expression of voltage-gated sodium channels and their biological roles in different cancers such as prostate, breast, lung (small cells and non-small cells) and leukaemia. As a conclusion, we develop conceptual approaches to understand how such channels can be involved in the metastatic process and conclude that blockers targeted toward these channels are promising new therapeutic solutions against metastatic cancers.

Keywords: Cancers, voltage-gated sodium channels, TTX, metastasis, invasiveness, motility, galvanotaxis. INTRODUCTION Cancers are among the leading causes of death in the world and as such represent a major problem of public health. This is the reason why governments, pharmaceutical companies, industrials and academics are all so intensely involved in a better understanding of the etiologies of the diseases. Such efforts aim at the improvement in the early detection of tumours in order to propose more efficient treatments. These could be effective in replacement of or in association with other therapies used presently. One of the new promising fields of investigation is the pharmacology of ion channels which are known to be involved in different aspects of the carcinogenic process. Among ion channels, voltage-gated sodium channels (VGNaC) make the topic of this review. These channels consist of a highly processed
subunit, which is approximately 260 kDa, associated with at least two auxiliary  subunits as displayed in Fig. (1) [1]. Sodium channels in the adult central nervous system contain 2 plus either 1 or 3 subunits, while sodium channels in adult skeletal muscle have only *Corresponding author: Tel: (+33) 2 47 36 60 24; Fax: (+33) 2 47 36 62 26; E-mail: [email protected] +present address: U637 Inserm, Physiopathologie Cardiovasculaire; Université Montpellier-2; CHU Arnaud de Villeneuve; 371 avenue du Doyen Gaston Giraud, 34295 MONTPELLIER France

Allen B. Reitz / Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.

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the 1 subunit. The pore-forming
subunit is sufficient for functional expression, but the channel and current density, kinetics and voltage-dependence of gating are modified by the  subunits [2]. A new 2-related subunit called 4 was recently discovered [3]. A mutation of this protein has been shown to induce a sustained activity of the channel responsible for cardiac arrhythmias (long QT syndrome) [4]. Ten genes encoding VGNaC
subunits have been identified and nine of these have been functionally studied in expression systems and constitute a single family named NaV1 according to their phylogeny and are designated NaV1.1 to NaV1.9 [5,6]. The remaining isoform, NaX, shows a structure diverging from the NaV1 family and seems to be gated by sodium concentration and not by voltage. Based on their tetrodotoxin (TTX) sensitivity, VGNaC are classified as TTX-sensitive or TTX-S (NaV1.1 to NaV1.4, NaV1.6 and NaV1.7) and TTX-resistant or TTX-R (NaV1.5, NaV1.8 and NaV1.9). VGNaC are classically described as critical elements of action potential initiation and propagation in excitable cells because they are responsible for the initial depolarisation of the membrane. However, these channels are also known to be expressed in non excitable cells such as T-lymphocytes [7].

Fig. (1). Subunit structure of the voltage-gated sodium channels. The secondary structures of the
and  subunits of the voltage-gated sodium channels are illustrated as transmembrane folding diagram. Cylinders represent probable alpha helical segments. The different important parts of the channel are indicated (voltage-sensing, pore, modulation, inactivation and interaction with drugs). P indicates sites of demonstrated protein phosphorylation by protein kinase A (circles) and protein kinase C (diamond). Sites of binding of
- and -scorpion toxins are shown. Reprinted from [1] with permission from Elsevier.

To investigate the expression of proteins like ion channels, techniques such as RT-PCR and immunoblot allow to determine the presence of mRNA and their translation into proteins. However, one state-of-the-art technique exists, namely the patch-clamp technique [8], which allows to quantify the activity of these proteins. Displacement of ions from one com-

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partment to another leads to current generation due to the electric charges borne by ions. The patch-clamp technique uses a glass pipette as an electrode to measure the electric current flowing through ion channels. Since the flux of ions depends mainly on passive diffusion, changes in current amplitude directly reflect the changes in channel conformation and thus activity. The two major configurations to measure currents flowing through all the channels of an isolated cell, and thus called whole cell recording, are the "ruptured patch" (the membrane patch at the tip of the pipette is broken and the intracellular content is replaced by the medium present in the pipette) and the "perforated patch" (antibiotics such as amphotericin B present in the pipette medium form pores, into the membrane trapped at the tip of the pipette, to get an electrical access to the interior of the cell while reducing the dilution of intracellular soluble molecules). To study some aspects of cancers, cultured cell lines are generally used. Different aspects of their biology can refer to different aspect of the disease. For example, cell proliferation in vitro can be an index of the kinetics of tumour proliferation in vivo. The facilities with which the cells migrate through filters, covered or not with Matrigel
(mimicking the extracellular matrix) are considered as indexes of invasion and migration, respectively. These parameters are both involved in the metastatic properties of the cells. Even if metastatic properties do not depend on cell invasiveness only, extracellular matrix invasion is a prerequisite to get metastases. The evaluation of the capacity of cells to produce metastases can be assessed by injecting the cells into nude mice. Understanding this aspect of the disease is very important because it is the main cause of death by cancer. Metastasis can be summarised as a process whereby cells escaping from a primary tumour enter circulation (blood or lymph), migrate and become lodged at tissue-specific or non-specific sites where they proliferate to form secondary tumours. To escape the primary tumour and to set in a distant site, tumour cells need to produce, or induce host cells to produce, proteases (e.g. matrix metalloproteases, MMP and/or cystein cathepsins) to degrade the extracellular matrix. PROSTATE CANCER PCa is the most frequently diagnosed male cancer and the second most common cause of death of men from cancer in Western societies [9]. The first study of a sodium current measured in patch-clamp conditions was made in a rat prostate cancer cell line [10]. In this paper, the authors describe the activity of sodium channels only in a highly metastatic cell line, Mat-Ly-Lu, but not in the weakly metastatic one, AT-2. This current is fully blocked by 1
M TTX suggesting that it is a TTX-S. Proliferation was not affected by TTX. Since the currents were recorded in the metastatic cell line, the authors investigated its possible involvement in the metastatic process using an in vitro Matrigel
invasion chamber assay. Incubation of the Mat-Ly-Lu cells with 600 nM TTX for 48 h reduced their invasive capacity by 33%. In contrast, TTX had no significant effect on the invasiveness of the AT-2 cell line. Later on, the same group performed similar experiments in human prostate cancer cell lines, PC-3 and LNCaP, and found similar results [11]. It must be underlined that in both studies [10, 11], the authors reported that not all cells investigated in patch-clamp possess functional sodium channels. This led these authors to further investigate the relationship between protein expression and invasiveness [12]. This study was performed in both rat and human cell lines in which the expression of VGNaC was assessed by flow cytometry using a fluorescein-labelled polyclonal antibody. Weakly metastatic cell lines such as AT-2 and LNCaP displayed almost no expression of VGNaC. On the opposite, highly invasive cell lines such as PC3 and Mat-Ly-Lu displayed bimodal frequency distribution profiles with a subpopulation of cells expressing high levels of sodium channel protein. Comparison of the degree of sodium channels expression with the capacity of the different cell lines to invade

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Matrigel
shows a significant positive correlation. In addition, two transfected cell lines, obtained by transfecting genomic DNA from rat prostatic tumour cells into a recipient benign cell line (non invasive), were found to express significantly elevated levels of sodium channel protein compared to the original cell line. This enhanced expression was correlated with increased invasiveness in vitro. The only uncorrelated observation in this study is that PNT2 cell line, which is reported to be benign, contains numerous sodium channels. This expression could be subsequent to the transfection with the SV40 genome. It also could mean that cells need more channels than the amount expressed in PNT2 to become invasive [12]. It has also been speculated that the presence of the channels gives the potential for invasion but this is prevented by local factors or the need for other essential criteria such as accumulation of other mutations [12]. Whatever the reason, it must be noticed that, to our knowledge, there was no attempt to determine the functional activity of the channels in PNT2 cells. Bennet et al. (2004) compared a weakly metastatic human cell line, LNCaP, and two increasingly tumorogenic daughter cell lines, C4 and C4-2. They showed that the increasing invasiveness of the C4 and C4-2 cell lines was related with an increasing level of VGNaC proteins measured by immunobloting [13]. TTX at 1
M completely reversed the difference in invasiveness between C4, C4-2 and LNCaP cell lines. Moreover the transient expression of the skeletal isoform (NaV1.4) of the channel in the 3 cell lines increased their invasive capacity in a TTX-sensitive manner. From their work, it can be concluded that sodium channels expressed are sufficient for prostate cancer cell invasiveness. TTX Sensitivity of VGNaC In a very elegant study, the electrophysiological and pharmacological properties of MatLy-Lu cells current have been characterised [14]. The only powerful blocker found was TTX with an IC50 of about 18 nM (see Table 1). Such sensitivity makes this channel a TTXS. Isoform(s) of the Channel(s) The question of the isoforms expressed was addressed both in Mat-Ly-Lu (rat) and PC3 (human) cell lines. These cells express mRNA coding for the skeletal isoform SkM1 now named NaV1.4 [15]. In situ hybridation experiments indicated that the levels of mRNA expression were very variable from cell to cell in Mat-Ly-Lu cells. In contrast, this signal was more uniform in AT-2 cells although at much lower levels. In the same vein, LNCaP cells also expressed NaV1.4 transcript. In that study, they did not find any mRNA expression for NaV1.1 (also called RB I), NaV1.2 (RB II), NaV1.3 (RB III), Nax (NaG/SCL-11) and NaV1.6 (Na6) in any of the cell lines tested. It was concluded that all cell lines expressed VGNaC mRNA although the channel was functional only in highly metastatic cell lines [15]. However, the authors noticed that the pharmacological properties of the functional protein were particular since 10
M
-conotoxin was needed to completely block the current while nanomolar concentrations are usually enough to block the skeletal sodium current (see also [14]). This surprising result led the authors to re-investigate this point using other molecular biology methods (degenerate primer screening and semi-quantitative PCR) on the same cell lines [16]. They found that the main mRNA isoform expressed in Mat-Ly-Lu and PC3 cells was NaV1.7 and this was thus likely to be the main source of the functional VGNaC detected. Other isoforms are observed such as NaV1.1, NaV1.2, NaV1.4 (see Table 1 and Fig. 2A). Recently, Nakajima et al. (2009) showed that silencing genes coding NaV1.6 and NaV1.7 inhibited endocytosis and invasiveness in PC-3 cells [17].

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Gillet et al.

List of the Non-Central Nervous System Cancers in which VGNaC have been Studied

Cancer Type

Prostate

Cell Lines

PC-3

mRNA

TTX

Functional

VGNaC

VGNaC in

Expression

Sensitivity

Expression

Role

Biopsies

NaV1.7

8.6 nM

fNaV1.7

Invasion

Yes

NaV1.2

Motility (G)

(IH)

NaV1.3

Motility

References

[11, 16, 34]

(WH) LnCaP

NaV1.2

No current

Mat-Ly-Lu

NaV1.7

(rat)

NaV1.1

Motility (G)

NaV1.4

Motility

18 nM

fNaV1.7

[11, 16] Invasion

? [10, 14, 16]

(WH) AT-2 (rat)

No current

NaV1.1 NaV1.4

---

---

[10, 16]

No current

---

---

[31]

fNaV1.5

Invasion

NaV1.9 Normal

PNT-2

?

MDA-MB-231

NaV1.7

(immortalised) Breast


2
M

NaV1.5

Yes (RT-PCR)

[44, 50, 55]

---

[44]

NaV1.6 MCF-7

NaV1.5

No current

NaV1.6

---

NaV1.7

Normal (long term

MDA-MB-468

?

HMEC

NaV1.6

primoculture)

Cervix

No current ?

Tiny current

[44] Unknown

Personal

NaV1.7

(non inva-

data

NaV1.2

sive cells)

(not shown)

JP, 354, 085

NaV1.2

(primary

NaV1.7

cultures)

NaV1.4

5-15 nM

Current

?

Yes

[73]

(RT-PCR)

NaV1.6 Melanoma

C8161, C8146

?

6 nM

Current

?

?

[72]

Malignant Pleural

Primary MPM

NaV1.7

16 nM

Current

Motility

Yes

[74]

cells

NaV1.6

[64, 65]

Mesenthelioma

NaV1.2 Small-Cell

H146

?

215 nM

Current

?

?

Lung Cancer

H128

?

?

Current

?

?

[64]

H69

?

?

Current

Endocytosis

?

[64,66]

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Table 1. contd…. Cancer Type

Non-Small-Cell

Cell Lines

H460

Lung Cancer

mRNA

TTX

Functional

VGNaC

VGNaC in

Expression

Sensitivity

Expression

Role

Biopsies

NaV1.7


10 nM

NaV1.7

Invasion

NaV1.6

NaV1.6

Yes

References

[67]

(RT-PCR)

NaV1.5 NaV1.3 Calu-1

NaV1.7


5 nM +

NaV1.6


1
M

NaV1.5

NaV1.5

NaV1.7

NaV1.1

NaV1.6

NaV1.2

NaV1.1

NaV1.3

NaV1.2

NaV1.9

NaV1.3

Invasion

[67]

Invasion

[67]

NaV1.8 H23

NaV1.7


10 nM

NaV1.6

NaV1.7 NaV1.6

NaV1.5 Normal

A549

(immortalised)

NaV1.6

No current

---

---

[67]

No current

---

---

[67]

No current

---

---

[67]

Current

?

?

[68]

No current

---

---

[69]

NaV1.7

Normal (immortalised)

NL20

NaV1.7 NaV1.6 NaV1.2 NaV1.3

BEAS-2B

NaV1.5 NaV1.7 NaV1.1 NaV1.6

Leukaemia

K562

?

?

K562 K562/ADM

?

<100 nM

Current

?

?

[69]

CCRF-CEM

?

<150 nM

Current

?

?

[70]

CEM/VLB100

?

<150 nM

Current

?

?

[70]

Jurkat

NaV1.5


900 nM

NaV1.5

Invasion

NaV1.7

[71]

NaV1.9 Cell lines indicates the human cancer cell lines used (except when "rat" is indicated in brackets); mRNA expression indicates the mRNA coding for the channels sorted in order of decreasing intensity as determined by semi-quantitative RT-PCR; TTX sensitivity indicates the constant of inhibition of the current by TTX; functional expression indicates the isoform of the channel which likely bears the current measured in patch-clamp. If a current was recorded but the isoform not determined then it is indicated "Current"; VGNaC role indicates the role of the channel in vitro as determined after inhibition by TTX. G indicates motility assessed by galvanotaxis; WH indicates motility assessed by wound-healing assay; VGNaC in biopsies indicates the presence or not of the mRNA (RT-PCR) or protein (immuno-histochemistry, IH) in biopsies obtained from patients. "?": not determined or unknown.

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weakly metastatic

highly metastatic

A

rat

NaV

NaV

1.7

1.1

1.4

1.9

1.2

1.7

1.1

1.4

1.9

1.2

human

NaV NaV

1.7

1.2

1.3

1.7

1.2

1.3

B

Fig. (2). VGNaC in prostatic cancer cells. A, expression levels of the various VGNaC
subunits indicated below the bars. In each case, the vertical axis denotes the approximate level of expression with respect to mRNA levels in the highly metastatic counterpart. B, Schematic diagram of the possible mechanisms modulating membrane vesicle recycling involving Na+ influx resulting from VGNaC (VGSC) activity. The scheme illustrates possible down-stream and microdomain effects of rising intracellular Na+. Intracellular sodium can affect intracellular calcium and pH. Both modulations of homeostasis could in turn modify the activity of kinases involved in exocytoses. Abbreviations: ER, endoplasmic reticulum; IP3/Ryn, inositol triphosphate and/or ryanodine receptor. The "kinase" may be protein kinase A, C or CaM kinase II. A from [16] and B from [28]. Reprinted with permission of Wiley-Liss Inc, a subsidiary of John Wiley & Sons, Inc.

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Interestingly, NaV1.4 has been transiently expressed in LNCaP, C4 and C4-2 cells lines [13]. In each cell line, transient expression of the protein induced a large increase in invasiveness which was completely reversed by 1
M TTX. This result confirms the role played by sodium channel in the invasion process. Above all, this result indicates that, even if NaV1.7 seems to be the channel primarily involved in invasive properties of rat and human prostate cancer cell lines, other isoforms of the channel can render cells invasive. Biological Consequences of Sodium Channels Activity To address this important question, the group of Mustafa Djamgoz (Imperial College, London, UK) tested three different cellular processes important to PCa metastasis: galvanotaxis, motility and invasion. I - Galvanotaxis is the ability of cells to respond to an electric field by moving directionally. Such a property is known to be involved in a number of basic biological process such as embryonic development [18] but also in other physiological conditions such as wound healing [19]. To evaluate this property, Mat-Ly-Lu and AT-2 cells were exposed to exogenous direct-current electric fields of physiological strength [20, 21]. In these conditions, it has been shown that highly metastatic Mat-Ly-Lu cells responded to the application of an electric field by migrating towards the cathode. AT-2 cells gave no such response. The migration of Mat-Ly-Lu cells was blocked by 1
M TTX while 10
M veratridine, a sodium channel opener, enhanced it. This study thus showed that galvanotaxis of metastatic cells is under the control of the VGNaC activity. The precise mechanisms involved in this particular case are not understood but some hypotheses have been put forward based on other cases (see [22]). II - Motility is an integral component of the metastatic cascade and involves sub-cellular mechanisms like changes in cell volume, cell-matrix interaction, cytoskeletal elements and ion channels activity. In 1999, Fraser et al. [23] found that VGNaC activity played a significant role in determining the morphological development of Mat-Ly-Lu cells. Later on, the same group completed this observation by showing that lateral motility of these cells, assessed using a "wound-heal" assay, was significantly reduced with 1
M TTX [24]. Quite surprisingly, 20
M veratridine did not affect motility of these cells while aconitine (100
M) and ATXII (25 pM), two other "sodium-openers", increased it. At this concentration, veratridine did not affect cell proliferation while at 50
M, proliferation was reduced [25]. The lack of veratridine effect could be due to its interaction with other ion channels [24]. III – Before cells migrate into the local circulation and then interact with other particular tissues to form a secondary tumour, the extracellular matrix has to be digested. This is done by proteases such as matrix metalloproteases (MMPs) (see [26] for review) and/or cystein cathepsins [27]. In vitro, this property, called invasiveness, is evaluated through the potency of cells to migrate through filters coated with a film of Matrigel
. An indirect way to evaluate the capacity of cells to release proteases by exocytosis is to measure the secretory membrane activity. This was done by Mycielska et al. who measured the uptake of horseradish peroxidase (HRP) [28]. Since the recycling of membrane constituents requires close matching between the levels of endocytosis and exocytosis, HRP uptake is a marker of secretory membrane activity. Mat-Ly-Lu cells have a higher activity than AT-2 cells [28]. This difference is abolished in presence of 1
M TTX while veratridine did not increase the activity in Mat-Ly-Lu cells. To explain this surprising result, it has been proposed that the VGNaC would be working at maximal efficiency [28]. From these results, different hypotheses were

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proposed to link the VGNaC activity to the increased membrane trafficking as illustrated on Fig. (2B). One hypothesis was that Na+ influx could disrupt intracellular pH which would lead to a release of Ca2+ from intracellular stores. This Ca2+ could then activate protein kinase C and/or CaM kinase II and lead to phosphorylation of the actin cytoskeleton thereby allowing secretion and endocytosis to occur. An alternative hypothesis is that a direct action of Na+ on adenylate cyclase could alter levels of cAMP which activates protein kinase A. The changes in actin cytoskeleton phosphorylation would similarly allow secretion and endocytosis. Since veratridine affects VGNaC dependent parameters such as galvanotaxis without affecting others like motility and membrane secretory activity, it appears that the relationship between the channels activity and the final effects is rather complex as is the dependence of membrane secretory activity when analysed by fractal methods [29]. This might mean that motility and secretory activities are already maximal and thus cannot be enhanced by an increase in VGNaC activity. One of the major functions of normal prostate gland is the synthesis, accumulation and secretion of large amounts of citrate [30]. During the progression of malignancy, the intermediate metabolism of citrate is altered. This can have important implications on cellular bioenergetics, cell growth and apoptosis, angiogenesis [30]. The mechanism of citrate release by human normal prostatic cells, PNT2-C2, has been studied [31]. They found that these cells functionally express an electrogenic citrate transporter which mediates the cotransfert of 1 citrate (trivalent anion) alongside 4 K+ out of the cells. A similar study was also performed in a highly metastatic cell line, PC-3M [32]. The K+/citrate cotransporter found in PNT2-C2 cells was also found in PC-3M cells. However, PC-3M cells also expressed a novel Na+-dependent citrate transporter. Interestingly, the expression of this transporter was controlled by the VGNaC activity: treatment of the cells with 1
M TTX induced a reduction of the activity of the Na+-dependent transporter while the K+-dependent transporter activity increased. The contribution of the citrate transporters to metastatic disease has to be understood to determine their interplay with VGNaC activity. The diagram shown on Fig. (2B), underlines a very important unresolved question: how does sodium enter the cells through the channel? Usually, VGNaC are opened following a depolarisation leading to the triggering of an action potential. In the case of cancer cells, it has been proposed that metastatic prostate cancer cells membrane are "potentially electrically excitable" since they express both sodium and potassium voltage-gated channels [33]. However, such action potentials have never been observed, spontaneously or triggered, weakening such a hypothesis. An explanation might arise in view of some electrophysiological properties of the channel (see the Breast cancer section). Regulation of Prostate Cancer VGNaC Cancer cells are very sensitive to the chemical conditions of the in vivo environments encountered during tumorigenesis and metastatic spread (see in [33]). Among them Nerve Growth Factor (NGF) and Epidermal Growth Factor (EGF) have been associated with prostate cancer progression and have also been found to up-regulate VGNaC expression (see [33]). Such factors can be found in the serum used to grow cells in culture. Using Mat-LyLu cells, it was shown that, depending on the serum concentration used to grow cells, the electrophysiological properties of the current varied, suggesting both transcriptional and post-transcriptional regulations of the channel. Interestingly, the sensitivity of the current to TTX also varied [33]. This has been interpreted by the authors as a possible regulation of the expression of the different mRNA isoforms. This finding is of importance since weakly

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metastatic cells often express VGNaC transcripts (see Table 1). It is thus possible that changes in the surrounding environment, such as an increase in growth factors, can amplify the functional expression of VGNaC rendering the cells more invasive. Expression of VGNaC in Patients Up-regulations of VGNaC in PCa have been shown in two studies. In the first one, an antibody directed against an intracellular epitope of VGNaC was used on tissue microarray slides of 80 clinical PCa specimens and 4 normal prostate specimens [34]. Out of 80 PCa specimens, 44 showed higher levels of VGNaC compared to normal, with 14 (of 44) showing increased focal staining in scattered individual cells or small group of cells. It thus appears that the observations obtained in prostate cancer cell lines are pertinent with those made in PCa tumour specimens: in prostate cancer, an increased level of expression of the VGNaC is observed. Recently, another study went further by showing that the level of expression of the mRNA coding for NaV1.7 is related with the grade of PCa: the higher the grade, the higher the expression of NaV1.7 mRNA [35]. Thus, the expression of NaV1.7 has been proposed to be a potential novel marker for human prostate cancer [35]. These results led some research groups to develop new molecules targeted against VGNaC in order to block their activity (see [36] for review). This underlines the interest these channels might have as new therapeutic targets. The problems with these studies are that: (i) the VGNaC isoform tested (NaV1.2) has not been shown to be involved in cell invasion [37] and (ii) the cellular function evaluated (proliferation) is not affected by the functional VGNaC expression [36, 37]. BREAST CANCER Among women, breast cancer is the most common cancer and the first cause of death. Death occurs primarily after the development of metastasis. Until the beginning of the XXIst century, the studies on ion channels and breast cancer focused mainly on potassium channels and their involvement in proliferation [38-42]. Also, the role of the Ca2+ -activated chloride channel, coded by the CLCA2 gene, was also studied with regard to tumour metastasis [43]. It was found that it was a tumour repressor. All these studies showed that ion channels can be involved in breast cancer genesis. In the beginning of the 2000’s, two different research groups, our group in Tours and the one of Mustafa Djamgoz in London, showed at the same time that VGNaCs were expressed in a highly metastatic breast cancer cell line, MDA-MB-231 [44, 45]. As shown on Fig. (3A), the electrophysiological features of the current are not particular except for the voltage activating threshold (voltage at which the channel starts opening) which is more positive than in the case of the cardiac current. This is proposed to be related to the foetal vs adult isoform (see below), but could also be due to a particular arrangement with auxiliary
subunits. Indeed, it is known that co-expression of
1 with the NaV1.5 pore-forming subunit in HEK293 cells induces a positive shift of the activation and inactivation voltage relations curves, associated with a slowing of the reactivation kinetic [46]. Interestingly, this subunit has shown to be functionally expressed in MDA-MB-231 cells [47]. The current recorded in MDA-MB-231 cells was not observed in two other cell lines, MCF-7 and MDA-MB-468. MCF-7 cells are weakly invasive in vitro and non-metastatic in nude mice. MDA-MB-468 cells have an invasive capacity similar to that of MDA-MB-231 cells [44] but are less metastatic [48]. Like in PCa, it seems that VGNaC participates to invasion in vivo leading to higher metastatic potentials.

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B

A

C

Fig. (3). VGNaC in breast cancer cells. A, sodium current-voltage relationship of MDA-MB-231 cells. The inset on the left shows a family of currents recorded from a cell depolarised, from a holding potential of –100 mV, from –50 to +20 mV by 5 mV increments. B, relative effects of 30
M TTX on Matrigel
invasion of three different breast cancer cell lines as compared to control. C, influence of the patch-clamp configuration on sodium current-voltage relationship. The inset shows an example of currents recorded from cells depolarised at –10 mV from a holding potential of –100 mV in perforated patch (PP) and ruptured patch (RP). A and B from [44] reprinted with permission from Elsevier; C from [55] with kind permission of Springer Science and Business Media.

Pharmacological and Electrophysiogical Profile of VGNaC The pharmacology of the sodium channel expressed in MDA-MB-231 cells is rather particular [44, 49]. While its TTX sensitivity is consistent with a TTX-R isoform of the channel (see Table 1), the channel is surprisingly also sensitive to some calcium channel blockers with IC50 of 53.2 ± 3.6
M for diltiazem and 37.6 ± 2.5
M for verapamil, which are concentrations classically used to block calcium channels [49]. The VGNaC mainly expressed in these cells is the foetal isoform of NaV1.5, coming from alternative splicing [50]. This could explain the particular pharmacology of the channel towards calcium channels blockers, while the TTX sensitivity is the same than the adult isoform [49,51]. This finding

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could also bring another explanation why verapamil was beneficially used along with conventional chemotherapy [52]. In that study, verapamil was used as a chemoresistance inhibitor. We propose that it could also have worked as a VGNaC blocker. Electrophysiological properties for this neonatal isoform have recently been compared to those of the adult NaV1.5, and several changes such as a more positive activation threshold and slower activation / inactivation kinetics were associated to this neonatal splicing. This is mainly linked to the substitution of one aa; the aspartate in position 211 into a lysine residue [51]. Isoform(s) of the Channel(s) Semi-quantitative RT-PCR showed that NaV1.5, NaV1.6 and NaV1.7 mRNA are expressed in MDA-MB-231 cells [50, 53]. The low TTX sensitivity of the channel (IC50
2
M [44]) strongly suggests that the main isoform functionally expressed is NaV1.5. However, a minor expression of a functional TTX-S isoform has been also suggested [50]. Biological Consequences of Sodium Channels Activity ? To evaluate the role of this channel, the specific blocker of the VGNaC, TTX was initially used. Since the NaV1.5 is a TTX-R channel, 30
M of TTX was used to block all the channels [44]. This elevated concentration of TTX has never been shown to affect other proteins activity, or more generally, other biological activity than VGNaC. Neither cell proliferation nor migration was affected by the blockade of VGNaC. On the other hand, in vitro invasion was reduced by about 30% in MDA-MB-231 cells and it was not changed in MCF7 and MDA-MB-468 cells, which do not express such functional channels (Fig. 3B). Similar results were obtained in a more recent study using 10
M TTX [50] and by specifically down regulating NaV1.5 expression using RNA interference [54, 55]. Conversely, invasion was increased by the "sodium-opener" veratridine [55] reinforcing the potential role of VGNaC in invasion. It thus seems that the activity of the channels participates in the degradation of the Matrigel. Other properties involved in the metastatic process such as galvanotaxis, exocytosis and lateral motility have been shown to be under the control of the VGNaC activity [50]. Regarding cell migration through filters with calibrated pores, results are discrepant. In one study, no effect of 30
M TTX on transwell migration through 8
M diameter pores was reported [44]. Moreover, we performed wound-healing assays with MDA-MB-231 cells and found no effect of TTX (unpublished data). In contrast, the group of Djamgoz found that transwell migration through 12
M diameter pores was inhibited partially by 10
M TTX [50]. Since a similar reduction was observed in an in vitro invasion assay, the results obtained by Fraser et al. [50] suggest that the main effects of VGNaC activity are exerted through modifications of cell motility while in the case of Roger et al. [44], the only effect is through the modulation of the capacity of cells to degrade Matrigel. This apparent discrepancy has currently no obvious explanation. In agreement with our cell invasion observations, we recently demonstrated that NaV1.5 activity controls cathepsin B and S through a regulation of perimembrane pH [55]. Such a mechanism, which also applies to the human lung cancer cell line H460 [55] and the prostatic cancer cell line PC3 (unpublished data), can explain the reduced invasion observed. We also showed that while NaV1.5 is not involved in the regulation of breast cancer cell proliferation per se, it regulates the growth of cancer cell colonies [55]. Thus we can postulate that VGNaC could also be involved in primary tumour growth.

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Regulation of Breast Cancer VGNaC The sodium channel expressed in MDA-MB-231 cells can be regulated but the precise mechanisms are still unknown. The configuration of the patch-clamp used to study the channel was reported to affect some electrophysiological characteristics of the sodium current (Fig. 3C) [57]. The ascending limb of the I-V curve (from -60 to -10 mV) was shifted leftward in ruptured patch compared to perforated patch conditions and this was associated with slower kinetics of recovery of the channel from inactivation. Interfering effects on signalling pathways with the patch-clamp configuration have been observed in cardiac cells and have been attributed to intracellular content dilution [58]. The observations made in the breast cancer cell line were thus interpreted as a consequence of the dilution of a factor involved in a signalling pathway such as the PKC pathway, induced by the intracellular perfusion in ruptured patch-clamp. Other dedicated experiments have to be performed to evaluate how the expression of the channel is regulated by environmental factors such as growth factors. At the transcriptional level, it has been recently shown that the PKA signalling pathway could increase the expression of the neonatal underscript NaV1.5 [47]. Expression of VGNaC in Patients The relationship between the expression of the channel mRNA with lymph node invasion has been studied [50]. It was found that patients with lymph node invasion, determined using anatomo-pathological techniques, expressed mRNA for NaV1.5 while those who were negative for lymph node invasion did not express mRNA for this VGNaC. This correlation was not observed for NaV1.6 and NaV1.7. The immunohistochemical staining of human breast tissues for the NaV1.5 indicates no staining in normal tissues while a strong heterogeneous staining was observed in cancerous tissues [50]. These findings suggest that the expression of neonatal NaV1.5 can be used as a prognostic factor of metastatic development. The development of a neonatal specific antibody directed against the neonatal isoform of NaV1.5 has been undertaken [56]. These results strongly suggest that the observation made in a highly metastatic cancer cell line, MDA-MB-231 are not restricted to a cell line but can be transposed to the evolution of the disease. The particular isoform of the channel expressed, associated with its unusual pharmacology suggests that a particular treatment targeted toward this channel, without side effects on cardiac function, can be developed. The unanswered question is still: how are VGNaC involved in the invasive phenotype of these cells ? We proposed that, since it has never been possible to record an action potential, the membrane potential of MDA-MB-231 cells would have to be in a particular range of the sodium channel activity. Indeed, at that potential (-29 ± 2 mV, n = 65 cells), the sodium channel is partially activated and not fully inactivated [44]. The resulting ionic current is called a window current. What is important is that, even without any action potential, the partial opening of the channel leads to a continuous entry of sodium into the cell. This entry might be responsible for the increased intracellular sodium concentration observed in biopsies [59] which in turn can modulate other homeostases such as calcium or proton. After those changes, the consequences on cell physiology can be the same as the one presented on Fig. (2B), which concerns prostate cancer cells. LUNG CANCERS Lung cancer is the most common cancer over the world, with more than one million cases of death a year. It is divided in two major histopathological groups: non-small cell lung cancer and small cell lung cancer which occur with frequencies of 80% and 20% re-

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spectively [60]. Small cell lung cancer affects mainly non smokers and is characterised by rapidly proliferating tumours and poor prognosis. This cancer is often associated with a neuromuscular autoimmune disease characterised by the insufficient release of acetylcholine from the motor nerve terminal [61] and known as the Lambert-Eaton myasthenic syndrome. Non-small cell lung cancer is the very widespread kind of lung cancer which is associated with smoking. The occurrence of metastases, initially appearing in lymph nodes and then in other organs such as lung itself, brain, bone, bone marrow, liver, adrenal glands, is always a marker of poor prognosis. Small Cells Small-cell lung cancer cells have been shown to be neuroendocrine-like cells capable of producing action potentials [62]. This discovery has been reinforced by others who suggested that small-cell lung cancer cells derived from primitive endodermal cells which differentiate into neuroendocrine cells [63]. Later, the ionic channels expressed in different cell lines (H128, H69 and H146) were characterised and VGNaC were found to be expressed in all of them [64]. These VGNaC and a calcium current also present were able to elicit action potentials [65]. Both channels were blocked by autoimmune antibodies responsible for the Lambert-Eaton syndrome. The sodium channels expressed in H146 cells were weakly sensitive to TTX (Ki = 215 nM) (see Fig. 4A) [65]. From their analysis of electrophysiological and pharmacological characteristics, they determined that only one population of TTX-resistant sodium channels was expressed. Since these cells are neuroendocrine-like and since they are able to produce action potentials, it is tempting to assume that they are involved in excitation-secretion coupling function. However, as noticed by Blandino et al. [65], the resting membrane potential of the H146 cells was approximately -44 mV, a level sufficient to inactivate most of the Na+ channels (see Fig. 4B). Either the resting membrane potential of this kind of cells is actually more hyperpolarised in tumours than it was measured in H146 or these sodium channels are not involved in the elicitation of action potentials [65]. In that latter case, we propose an alternative hypothesis described in the conclusion section. In a recent study, it has been observed that in H69 and other small-cell lung cancer cell lines (H209 and H510), 100 nM TTX reduced Horse Radish Peroxidase uptake suggesting a role for VGNaC in the exocytic activity [66]. However, the presence of functional VGNaC has not been undertaken in H209 and H510 cells and other properties involved in the metastatic phenotype have not been evaluated. Non Small Cells 4 cancer cell lines (H23, H460, A549, Calu-1) and 2 non-cancerous epithelial cell lines (NL-20, BEAS-2B) were tested [67]. While the non-cancerous cells did not have functional channels as measured in patch-clamp conditions, three cancer cell lines (H23, H460 and Calu-1) expressed functional channels with different electrophysiological and pharmacological properties (see Table 1) [67]. TTX Sensitivity of VGNaC The most striking difference between the cell lines concerns the isoforms expressed functionally, as determined by their TTX sensitivity. This sensitivity was in the nanomolar range in H23 and H460 cells. Thus the protein isoforms belonged to TTX-S in these cell lines. For Calu-1 cells, there were clearly two kinds of channels expressed: TTX-R and

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TTX-S. In contrast with the other cancers described in this paper, the heterogeneous sensitivity to TTX indicates heterogeneity in isoforms expression between cell lines.

A

B

Fig. (4). VGNaC in small cell lung cancer cells. Voltage dependent sodium currents recorded from H146 cells. A, whole-cell currents were evoked from depolarizing test potentials of 40 ms duration varying from –30 to +10 mV with 10 mV increments. The holding potential was –80 mV. Left, current observed in control conditions and the right panel illustrates current traces obtained in presence of 100 nM TTX. B, Pseudo-steady-state activation (m
) and inactivation (h
) parameters of INa. From [65] with kind permission of Springer Science and Business Media .

Isoforms of the Channels Semi-quantitative RT-PCR experiments indicated that all the studied cell lines (cancerous and non-cancerous) expressed mRNA for the NaV1.6 and NaV1.7 isoforms which are both TTX-S. In the case of H23 and H460 cells, NaV1.7 transcripts were more abundant and were possibly responsible for the expressed functional channels. However, a possible expression of NaV1.6 cannot be rejected. These cells express mRNA for the NaV1.5 isoform but the TTX sensitivity of the current suggests that it was not functionally expressed. Calu-1 cells expressed transcripts for all the isoforms except for NaV1.4. The most abundant mRNA was, in descending order, those of NaV1.7, NaV1.6, NaV1.5, NaV1.1 and NaV1.3. The particular pharmacology of the current, which is clearly both TTX-R and TTX-S, indicates that

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the NaV1.5 is functionally expressed. With regard to TTX-S, the situation is more complicated since mRNA for NaV1.6, NaV1.7 and NaV1.1 are comparably expressed and a possible participation of NaV1.3 cannot be rejected. Knowing that non-cancerous cells, NL-20 and BEAS-2B, express mRNA for NaV1.6 and NaV1.7, plus NaV1.5 for the latter, like the cancerous cell lines, but did not have functional proteins, it is tempting to speculate that these cells have the potential to functionally express these VGNaC but they miss something to do it. Biological Control of Sodium Channels Activity The effect of
M TTX on proliferation, migration and Matrigel
invasion was tested on the 6 different cell lines [67]. We followed the same protocols that we previously used on breast cancer cell lines [44]. In all cell lines, TTX had no effect on proliferation and migration. An effect was only observed when TTX was applied and Matrigel
invasion evaluated. In that case, a reduction of invasion between 40 to 50% was observed in H23, H460 and Calu-1 cells which functionally express the VGNaC. Calu-1 cells express functional TTX-S and TTX-R VGNaC. Application of different concentrations of TTX blocking TTXS or TTX-S + TTX-R VGNaC led to a gradual reduction of in vitro invasion. This indicates that both isoforms of channels are involved in the invasion properties of the cells. Expression of VGNaC in Patients In an unpublished study, we performed semi-quantitative RT-PCRs on 10 cancerous and associated non-cancerous lung biopsies collected from the same patients. The mRNA coding for the 9 isoforms of NaV were tested. In contrast to what could be expected from the cell line experiments, we did not observe an up-regulation of NaV mRNA expression. Rather, there is a tendency for a down-regulation. These results indicate that mRNA expression was modified during the disease. Nothing is known about their translation in proteins and VGNaC activity. This is of importance since we found that only VGNaC activity is important in the invasion process. Cross-talks have been described between the functioning of VGNaC and mRNA production. For example, the application of 1
M TTX to rat prostatic cancerous cells Mat-Ly-Lu increased the production of mRNA coding for the VGNaC [32]. Thus, we can hypothesise that a reduction in mRNA can result in a decreased channel activity but also that it is a consequence of an increased channel activity. Additional experiments at the functional level, using patch-clamp, are needed to elucidate this question. LEUKAEMIA There are few studies about the role of VGNaC in leukaemia. The first one was a brief description showing that the leukaemia cancer cell line K562 expressed a VGNaC [68]. During the same period of time, another research group, using the same cell line, did not find this current except in particular conditions in which cells became resistant to anticancerous drugs (MDR phenotype) [69]. This current was blocked by nM TTX (Ki<100 nM). While the functional expression of the sodium current was clearly associated with the MDR phenotype, it was not involved in the resistance to chemotherapy since 1
M TTX had no effect on vincristine (an anticancerous drug) uptake. Following this work, leukaemia cell lines from T-cell origin were studied: CCRF-CEM and a MDR variant CEM/VLB100 [70]. Contrarily to the work of Yamashita et al. [69], they found that a sodium current was observed in both cell lines and as such was not associated with the MDR phenotype. Here again, the current was sensitive to TTX (Ki < 150 nM).

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In conclusions of these three studies, there is no clear link between VGNaC activity and the MDR phenotype. Moreover, the role of VGNaC, if there is one, is not described in these studies. Interestingly, a recent study showed that 8-9% of Jurkat cells, a cell line derived from leukemic T-lymphocytes, expressed a sodium current [71]. This study confirmed the presence of a sodium current on T-lymphocytes and T-lymphocytes-derived cell lines [7]. This current is blocked by TTX with a Ki around 900 nM and as such can be classified as TTX-resistant. RT-PCR analysis indicates the presence of mRNA for at least 4 isoforms of VGNaC, two of them being TTX-R, NaV1.5 and NaV1.9. NaV1.8 was not detected and for the authors, the protein which is most likely expressed is NaV1.5. The mRNA coding for NaV1.6 and NaV1.7 were also observed but to a lesser extent. Interestingly, Matrigel
invasion assays on Jurkat cells were performed [71]. In control conditions, about 8% of the cells were invasive, close to the percentage of cells expressing an electrophysiologically detectable sodium current. Following treatment with 10
M TTX, invasiveness was decreased by approximately 93% (see Fig. 5). This suggests that the observation of a sodium current in leukaemia cells might not be due to a neo-synthesis of VGNaC but to an up-regulation of already expressed channels (see conclusions for development). GENERAL CONCLUSIONS As shown on Table 1, functional expression of VGNaC (mainly NaV1.7, NaV1.6 and NaV1.5) is often associated with invasive properties of some cancer cell lines, and while their function is still unknown, sodium currents have also been characterized in melanoma [72] and in primary cultures of human cancers such as cervix cancer and pleural mesenthelioma [73, 74]. VGNaC are also expressed in biopsies from metastatic patients. VGNaC are classically described as being responsible of the excitability and propagation of the action potential. This is why the presence of VGNaC is often interpreted as an indication of possible excitability whatever the tissue in which it is found. However, in cancer cells, the membrane potential is less negative than it is in excitable cells. This strongly suggests that the channels must be partially inactivated. The membrane potential of cancer cells is often, if not always, located in a window of voltage where there is a continuous entry of sodium owing to a partial activation and incomplete inactivation of VGNaC (see Fig. 4). This led us to hypothesise that such an entry of sodium could be responsible for an increase in intracellular calcium which can then enhance the release of proteases [44], in accordance with results obtained on prostatic cancer cells [28]. However, we recently showed that the cystein cathepsin expression (at the RNA and protein levels) and secretion are not altered by NaV activity [55]. Another hypothesis is a regulation of the intracellular pH (pHi) and perimembrane pH (pHp) by NaV activity. Intracellular sodium regulates pHi mainly through the activity of the sodium-proton exchangers (NHE) and the sodium-bicarbonate exchangers. Indeed, it is known that many proteins involved in the metastatic process are tightly regulated by the surrounding pH [28]. In the case of PCa, VGNaC activity regulates a specific sodium-dependent citrate transport [32]. However, this is one aspect of the physiology which is particular to the prostate functioning. The regulation of invasion by VGNaC activity strongly suggests that there is a regulation of proteases or growth factors release and/or activity which is common to all the cancers described. Recently, the role of the acidic proteases cysteine cathepsins, mainly B and S, has been highlighted [55]. Beside its effect on motility and invasion, the sodium channels can have other roles which are important in the process of metastatic tumour development. Indeed it is often reported that among highly invasive cell lines (as determined in vitro), those which express the VGNaC are more metastatic when injected in nude mice than are those which lack the

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VGNaC. This feature might be brought by the adherence properties of auxiliary -subunits such as the 2 [75] and 1 [47]. Indeed, these subunits can help migrating cells to settle in specific metastatic site (e.g. lung, bones) and then invade this tissue to develop the secondary tumour.

B

A

C

Fig. (5). VGNaC in leukemic cells (Jurkat cells). A, From a holding potential of –100 mV, a 30 msec test pulse to –10 mV every 10 sec elicits a rapid inward current which is not very sensitive to 200 nM (i) and 2
M (ii) TTX. B, Semi-quantitative PCR gel images for (i) NaV1.5, (ii) NaV1.6, (iii) NaV1.7 and (iv) NaV1.9 products amplified from Jurkat cells. PCR cycle numbers for given bands are indicated above the gels.
denotes the mis-transcribed/exon-skipped form of NaV1.6. C, Matrigel invasiveness by Jurkat cells under control conditions (dark bar) and following treatment with 10
M TTX (light bar). The asterisks (***) indicate that the difference was statistically significant (p<0.001). From [70] with the permission of the authors.

A question arises considering that VGNaC can be expressed in different cancer cells and in very few "normal" cells. Does this expression represent dedifferentiation towards a more embryonic phenotype? It has been proposed that embryonic genes, which are silent in the

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cells of the mature organ, are re-expressed in cancer cells [76]. In line with this hypothesis, the splice variant of VGNaC expressed in prostate and breast cancers are neonatal (see [77]). This hypothesis is also reinforced by the observation that cultured normal cells (smooth muscle cells, retinal pigment epithelial cells) express VGNaC while freshly isolated cells do not [78-82]. From the literature regarding the role of VGNaC in cancer cells, it seems that its main role is to participate in the invasive capacity of the cells. We can thus assume that VGNaC are expressed in cells during particular events of embryogenesis. For example, during the embryonic development, cells need to migrate and invade to find the nest where the organ can develop. The invasion before nesting can be due to the production of proteases which are, at least partially, under the control of a VGNaC. After the nesting of the cells, the no longer necessary genes encoding the VGNaC can be switched off. Mutations during carcinogenesis might lead to the switch on of the silent genes. It is known that some regions of the chromosomes are more sensitive to mutations like deletions. For example, it has been reported that in renal, breast and lung carcinomas, there are frequent deletions in chromosome 3p21.3 [83, 84]. The 3p21 region contains numerous genes including NaV1.5 and NaV1.9 (see [5]). It is thus possible that the probability to have mutations leading to the oncogenic process is higher in chromosome regions corresponding to genes active during the fœtal period. If this hypothesis is true, it could explain the fœtal-splice form expressed in some cancer cells and the overall specificity of the isoform expressed in some cancers (for example NaV1.5 in breast cancer and NaV1.7 in prostate cancers). In conclusion, VGNaC are found in several metastatic cancers and their functional expression may represent a more generic involvement in physiological and pathophysiological invasive processes. These ionic channels probably interfere with calcium homeostasis which in turn modifies signalling pathways involved in invasiveness, a prerequisite to metastasis formation. As such, these channels, as fœtal isoforms, represent a promising new field of investigations to develop new specific blockers to fight some metastatic cancers. ACKNOWLEDGEMENTS The results on the expression of VGNaC in cancerous lung tissues were obtained thanks to collaboration with Yves Gruel and Jerôme Rollin (U618 Inserm, University of Tours). We are indebted to Philippe Bougnoux for sharing with us his knowledge in oncology and to Yves Gruel, Jérôme Rollin, Jacques Goré, Karine Mahéo, Marie-Lise Jourdan, Sophie Vibet and Aurélia Barascu for helpful discussions on cellular physiology of cancerous cells. The work on VGNaC is supported by "La Ligue contre le Cancer Région Centre" and the Canceropole Grand-Ouest. ABBREVIATIONS VGNaC

=

Voltage-gated sodium channel

TTX

=

Tetrodotoxin

TTX-S

=

Tetrodotoxin-sensitive channel

TTX-R

=

Tetrodotoxin-resistant channel

PCa

=

Prostate cancer

MMP

=

Matrix metalloproteases

I-V

=

Current-voltage

Voltage-Gated Sodium Channels

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Recent Advances in Antiviral Agents: Antiviral Drug Discovery for Hepatitis Viruses Kyuichi Tanikawa* Kurume University, International Institute for Liver Research, Alley II 202, Tsubuku-honmachi 636-1, Kurume 830-0047, Japan Abstract: Hepatitis B virus (HBV)- or hepatitis C virus (HCV)- associated liver diseases are now one of the important health problems in the world because of the high numbers of patients and the serious consequences. Recently, however, relatively effective treatments with antiviral agents have become available. Interferon (IFN) and several nucleotide analogs (lamivudine, adefovir, entecavir and tenofovir) are now approved for treatment of HBV-associated liver diseases and they have been shown to be fairly effective. The goal of treatments for HBVassociated liver disease is to achieve a clinical cure in as short a period as possible without producing resistance mutation of the virus. In the case of HCV-associated liver diseases, Pegylated IFN (Peg IFN) + ribavirin combination therapy is the standard and most effective treatment with a sustained response of 60-70%. The goal of the treatments for these liver diseases is to induce the complete eradication of the infected virus and at present new anti HCV drugs targeting the molecular segments of the virus are under development. It is expected that the complete eradication of infected virus will be possible in most cases in the near future.

Keywords: HBV, HCV, HDV, liver disease, anti-hepatitis virus agent. INTRODUCTION At present, five different types of hepatitis viruses have been recognized: Hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV). HAV and HEV are both transmitted orally, and induce acute forms of hepatitis which are self-limited and therefore do not require antiviral agents for their treatment. There are presently no antiviral agents available for the treatment of HDV infection, which requires co-infection with HBV. Therefore, this article will focus mainly on HBV and HCV infections, especially chronic infections. Worldwide, it is estimated that 350 million people are infected with HBV [1] and 170 million people with HCV [2]. Chronic HBV and HCV infections are a major cause of endstage liver disease and hepatocellular carcinoma (HCC). A considerable number of patients with these chronic viral infections die of end-stage liver disease and HCC. Therefore, *Corresponding author: Tel: +81-942-31-1231; Fax: +81-942-31-1232; E-mail: [email protected]

Allen B. Reitz / Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.

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chronic HBV or HCV infection is one of the most important viral infections in the world today. 1. GENERAL ASPECTS OF PATHOGENESIS AND INDICATIONS FOR TREATMENT WITH ANTIVIRAL AGENTS IN HBV OR HCV-ASSOCIATED LIVER DISEASES HBV-induced liver diseases are considered to be the result of a host immune response of HBV-specific cytotoxic T cells to hepatocytes infected with HBV, because HBV itself has no cytopathic effects. Therefore, the magnitude of the hepatocyte injury is dependent on both the viral load and the host immune response. In HBV infection in adults, a strong host immune response to HBV occurs that is able to eliminate infected hepatocytes almost completely, and thus, acute HBV hepatitis in adults is self limited and generally needs no antiviral agents for its treatment. However, antiviral agents could be administered in the early stage of HBV fulminant hepatitis. Antiviral agents could also be indicated in rare cases in which the acute infection is protracted. On the other hand, chronic HBV infection, which generally requires treatment with antiviral agents during its clinical course, occurs as the result of vertical transmission of HBV at birth or infection during the first 2-3 years after birth. Such vertical transmission can be prevented successfully by HBV vaccine or by a combination of HBV vaccine and HBIG (hepatitis B immunoglobulin) at birth. Persistent infection with high viral load is established because of an immature host immunoresponse to HBV in newborn infants when infected with HBV. The clinical courses of such HBV carriers with no liver injuries are characterized by episodes of a host immune response to the HBV with acute inflammatory changes. Most such episodes end in a clinically cured state with decreased viral load. However, some continue to have persistent inflammatory changes and are diagnosed with chronic hepatitis around the age of 20 or over. Such chronic hepatitis continues for years and finally ends naturally with a clinical cure in most cases. However, 10 to 20% of chronic hepatitis progresses without a clinical cure to liver cirrhosis. Such progressive changes tend to be seen in patients 30 to 35 years of age or older. Thus, there are two treatment strategies for chronic HBV hepatitis: one is to accelerate a clinical cure by IFN, the other is to inhibit viral replication by the use of antiviral agents such as nucleoside analogs (lamivudine, adefovir, entecavir and tenofovir) to prevent progression to a more advanced stage of liver disease. Such antiviral agents are also indicated in cases of active liver cirrhosis as a means of preventing fatal liver failure. In the treatment of chronic HBV liver diseases, not only effective antiviral agents but also effective agents to enhance the host immunological response to HBV are of great importance. However, although HBV vaccine in combination with antiviral agents has been tried for this purpose, the results have not been satisfactory. It is impossible to eradicate HBV completely from HBV carriers because the virus genome is integrated into the DNA of the hepatocyte. In fact, even in clinically cured cases where positive HBc antibody is the only marker of disease, a small amount of HBV is retained in the hepatocytes [3]. This is shown by the fact that HBV hepatitis develops in liver transplant patients who receive donor livers positive only for HBc antibody [4]. One of the important clinical challenges associated with HBVassociated liver disease is the fact that a considerable number of patients develop HCC and lose their lives. Finding ways to prevent HCC occurrence is therefore an important clinical

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problem. HCC is primarily associated with liver cirrhosis, thus, inhibiting the progression to cirrhosis by use of antiviral agents could be an important therapy. It was recently reported that lamivudine, a commonly used antiviral agent, was able to prevent HCC occurrence to some degree [5]. HCV infection occurs mainly via the intravenous route, namely through medical treatments such as blood transfusions and also via drug abuse, and it is estimated that there are 170 million HCV carriers worldwide, mostly in developed countries. The most important characteristic of HCV infection is that once HCV infection occurs most infected persons become persistent carriers. In acute HCV hepatitis, complete clearance of the virus is observed in 20-40% of infected persons as a result of vigorous host immunoreactions to HCV. However, 60-80% of infected persons become persistent carriers. Thus, IFN treatment is indicated when acute hepatitis C tends to be protracted in the clinical course for preventing a transition to chronic hepatitis [6]. Most of these HCV carriers do not experience any episodes of acute hepatitis and the infection is unapparent. Once chronic hepatitis is established, most patients progress gradually to liver cirrhosis and finally to HCC without a natural cure. Once liver cirrhosis occurs, most cases develop HCC (annual occurrence: 7%) [7]. It is unclear why most infected persons become persistent carriers once HCV infection occurs, and why so many patients with chronic hepatic injuries go on to develop HCC. One suggested mechanism is as follows: in HCV infection, not only the hepatocytes but also lymphoid cells such as dendritic cells are probably infected with HCV [8]. Dendritic cells infected with HCV become dysfunctional [9], and this eventually results in insufficient HCV-specific cytotoxic T cell activity [10]. Dysfunction of HCV-specific cytotoxic T cells may thus be responsible for the persistence of HCV infection. It has also been shown that HCV NS3/4A serine protease blocks the phosphorylation and effector action of IFN regulatory factor-3, which is an important cellular antiviral signaling molecule [11, 12]. Such HCV induced changes allow HCV infection to be persistent. In addition, it has been found that oxidative stress is induced in hepatocytes infected with HCV [13, 14]. Oxidative stress is known to be one of the leading causative factors in carcinogenesis [15]. Thus, oxidative stress in hepatocytes infected with HCV for a long period of time is considered one of the important causative factors in HCV-associated HCC. These seem to explain the reasons for persistent infection and high frequency of HCC in HCV infection [16] (Fig. 1). Approximately 30% of HCV carriers are asymptomatic, namely, they are positive for HCV RNA, but show normal liver function. At present, it is recommended that such patients receive treatments to clear the infected virus because a considerable number of such asymptomatic patients eventually develop chronic liver injuries [17, 18]. 2. TARGETS OF ANTIVIRAL AGENTS Recently, much progress has been made in clarifying the processes of viral replication in host cells, especially with regard to the enzyme systems necessary for viral replication. This has made possible the development of new antiviral agents especially targeting these enzyme activities. Hepatitis virus-associated liver diseases have become a global health problem and the development of new anti HBV and anti HCV agents has been progressing worldwide.

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Mechanism of HCV-related liver injuries HCV Infection

Infection

Persistent infection

Hepatocyte Oxidative stress

(IFN ）

（HCV specific T cell ）

Chronic hepatitis （Cell injuries）

Liver cirrhosis （DNA damages）

HCC

Lymphoid cells Functional disorders Dendritic cells NK cells T cells

（NK cell Immunological tumor surveillance ）

Fig. (1). Mechanism of HCV-related liver injuries.

At present, however, there are no anti hepatitis virus agents that target the processes of virus adhesion or entry to host cells following uncapping. So far, ARA-AMP, adenine arabinoside 5’-monophosphate, has been the first adenosine analog to be tested clinically that targets the transcription and replication of viral DNA in chronic HBV hepatitis. Side effects, however, have limited the clinical usefulness of this agent. At present, entecavir or tenofovir has been moved from lamivudine or adefovir to the first-line oral antiviral medication, based on the results of double-blind randomized trials for HBV-associated liver diseases as specific inhibitors of HBV replication [19], and IFN + ribavirin combination treatment is considered the treatment of choice for chronic HCV hepatitis. IFN is presently used to treat both HBV-and HCV-associated liver diseases. IFN itself is produced primarily from the cell infected with virus and lymphocytes as an antiviral cytokine in various viral infections. Administration of IFN is therefore expected to be effective against all kinds of viral infections. However, IFN is being used clinically at present mostly for chronic HBV and HCV infections, and is an important therapeutic modality in these diseases. Clinically available IFNs at present are IFN
, IFN, consensus IFN and Pegylated IFN. The functional mechanisms of IFN involve suppression of viral replication through phosphorylation of the protein synthesis initiation factor e1F2 after a serine / threonine protein kinase activity is activated. This inhibits the initiation of translation of viral protein. In addi-

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tion, IFN enhances immune activities which promote clearance of infected virus. In fact, the virus cannot be eradicated without stimulating immune activity. For this reason, HBV vaccine [20] is occasionally used in combination with antiviral agents and thymosin alpha 1 (Aadaxin, Sci Clone Pharmaceuticals) [21] to treat HCV infection. However, the efficacy of this therapy has not been confirmed. 3. ANTIVIRAL AGENTS FOR HBV AND HCV-ASSOCIATED LIVER DISEASES i) IFN IFN was first discovered in 1954 by Nagano and Kojima and was also independently found by Issacs and Lindenmann who named this cytokine IFN. IFN is produced in the body when viral infection occurs to control the infection. Thus, IFN has natural antiviral effects including inhibition of viral replication. In addition, IFN also has numerous other functions, for example, inhibition of cell proliferation, antitumor effects, immune modulating effects, induction of cell differentiation, and so on. IFN was not available clinically for some years after its discovery because of difficulty in mass producing IFN as a therapeutic agent. However, progress in cell culture techniques and genetic engineering has made it possible to mass produce IFN, and relatively inexpensive recombinant IFNs are now clinically available. IFN molecules first bind to receptors on the surface of the cell, inducing gene expression and the production of gene products. Specifically, IFN binding to receptors activates tyrosin phosphorylation enzymes. Genes expressed by IFN produce 2-5A synthetase, a main source of the antiviral effects of IFN. Besides its antiviral effects, IFN also has experimentally and clinically proven antitumor effects [22, 23]. At present, most of the HCC we see are associated with HBV or HCV infection, and their mechanisms of carcinogenesis have been partially clarified. Therefore, IFN treatment is important, not only for the treatment of hepatitis, but also for the prevention of HCC. IFN shows a direct antitumor effect by its inhibition of cell proliferation, and by enhancement of adhesion factors on the cell surface. In addition, IFN produces indirect antitumor effects by the activation of antitumor effector cells such as NK cells. In the case of HBV-associated liver injuries, as it is impossible to eradicate all of the infected viruses, the goal of IFN treatment is to enhance host immunity while decreasing viral load. Namely, the goal is to achieve a clinically cured state. In the case of HCV-associated liver diseases, IFN is generally administered in order to completely eradicate HCV from patients. Eradication efficacy is improved by combination therapy of ribavirin with IFN [24]. Today there are two main types of IFN available for clinical use, IFN
and IFN
and both have similar antiviral effects. However, at present, IFN
is more widely used because of a lack of recombinant IFN. Another new recombinant IFN has been developed that consists of 166 amino acid residues including the sequences most often observed among the 14 subtypes of IFN
. Thus, this IFN is called “Consensus IFN” (Amgen), and its antiviral effects are shown to be higher than those of regular recombinant IFN
[25]. In addition, a long-acting “Peg IFN” is now available. This is an IFN
in which peptides are combined with polyethylene glycol polymers. Administration of Peg IFN once a week is sufficient to maintain an effective level of IFN in the blood. There are two types of Peg IFN: Peg IFN
2a (Roche) and Peg IFN
2b (Schering Plough). The side effects of Peg IFN are

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similar to those of regular IFN. A minimum of one year of IFN treatment is required for HCV chronic hepatitis patients of genotype I with a high viral load. In such cases Peg IFN is especially useful. Recently, Albinterferon [26], a novel fusion protein of human albumin and IFN
-2b, has now been in clinical trial and its administration once two to four weeks seems to be sufficient for maintenance of IFN level. IL-29, IFN-related cytokine, also inhibits HCV replication by inducing a cellular antiviral response similar to that of IFN [27]. Lacteron [28], controlled-release formulation of unpegylated recombinant IFN in microspheres, has also been evaluated and both are now in clinical trials. ii) Lamivudine Lamivudine (Glaxo), -2’, 3’-dideoxy-3’-thiacytidine, is deoxycytidin analog in its pure (-)enantiomeric form which differs from naturally occurring (+) enantiomers. Thus, lamivudine is not taken up into human DNA and may be given to patients for long periods without serious side effects. Lamivudine is shown to have antiviral effects only against HBV and HIV. This is because lamivudine specifically inhibits virus-encoded RNA dependent DNA polymerases (reverse transcriptase) that are essential for viral replication. The antiviral effects of this agent are much stronger than those of gancichvir or ara-AMP. The primary mode of action involves inhibition of viral DNA synthesis through incorporation of lamivudine into newly synthesized HBV DNA, resulting in chain termination. Competitive inhibition of HBVencoded DNA polymerase has also been shown. Lamivudine is absorbed rapidly after oral administration and is phosphorylated to its 5’triphosphate derivative, which is shown to have potent antiviral effects [29]. This agent is given once a day orally at a dose of 100mg because the triphosphate has a 17-19 hour serum half-life. About 70% of the lamivudine is excreted to urine without any changes. This agent is the first specific antiviral therapy to become available for the treatment of chronic hepatitis B. It has few side effects even after long administration, however, HBV polymerase coded gene mutation (YMDD mutation) occurs frequently when lamivudine is administered for longer than one year [30]. In addition, post-treatment flare is often seen when the drug is discontinued, so patients must be followed carefully at the time of discontinuation. iii) Adefovir dipivoxil (ADV) Adefovir, (Gilead Science), a potent inhibitor of HBV replication, is a synthetic nucleoside analog of adenosine 5’-monophosphate. ADV, the oral prodrug of adefovir, is rapidly converted to adefovir by esterase in the intestine or serum, and is then transported to cells and phosphorylated to the active diphosphate form which acts as an alternative substrate for an inhibitor of HBV polymerase. In addition, incorporation of this agent into the viral DNA results in chain termination. Efficacy of daily oral administration of this drug at a dose of 10mg is similar to that of lamivudine, and adefovir generates a significant response in lamivudine-resistant chronic hepatitis B [31]. Very few ADV resistance mutations have been reported [32]. Adefovir, at a daily dose of 10mg, is well tolerated, however, this drug is cleared primarily by excretion of unchanged drug into the urine, and special care is required in the case of renal dysfunction.

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iv) Entecavir Entecavir, (Bristol-Meyers Squibb), is a nucleoside analog of deoxyguanosine, which is a very potent inhibitor of HBV DNA polymerase. The antiviral efficacy of this drug has been reported to be superior lamivudine [33, 34]. Only a few resistant mutations have been reported [35]. The reduction in HBV DNA and serum ALT at a dose of 0.5mg daily is significantly better than with lamivudine. This agent is at present most frequently given to HBC associated liver injuries as the first line drug. v) Tenofovir Disoproxil Fumarate Tenofovir DF, the oral prodrug of tenofovir, is a nucleotide analog that inhibits viral polymerases by direct bindings, and after incorporation into DNA, by termination of the DNA chain due to the absence of a requisite 3’ hydroxl on the tenofovir molecule. Based on the results of two double-blind randomized trials which showed a superiority of tenofovir at a daily dose of 300mg compared to adefovir at a daily dose of 100mg [36], this agent is now approved and licenced in the US and other countires. Tenofovir resistance was not detected in any of patients after up to 96 weeks treatment. The recommended dose of tenofovir is 300mg daily. However, dose adjustments should be made in patients with impaired renal function. vi) Telbivudine Telbivudine (
-L-2’-deoxythynindine) is an orally bioavailable L-nucleoside with potent and specific anti-HBV activity. A significant higher proportion of HbeAg-positive patients receiving telbivudine than of those receiving lamivudine had a therapeutic or histologic response [37]. However, telbivudine is associated with a high rate of resistance and telbivudine monotherapy has a limited role in the treatment of hepatitis B. vii) Ribavirin Ribavirin, developed in 1972, is a nucleoside analog of guanosine with wide antiviral activities. At present, IFN and ribavirin combination therapy is a standard treatment for chronic hepatitis C. However, the antiviral effects of single administration of this agent against chronic hepatitis C are not known. The antiviral mechanisms of this agent are also not clear. Inhibition of viral RNA polymerase has been suggested. Recently antiviral effects through immunomodulatory changes have been also been proposed because of an observed change in Th1/Th2 balance [38]. The most serious problem in the use of ribavirin is hemolytic anemia, which occurs in almost all cases administered. About 90% of orally administered ribavirin enters the bloodstream via the intestine and then spreads throughout the body. Ribavirin incorporated into erythrocytes accumulates in these cells and is not excreted to the outside because of the absence of dephosphorylating enzymes in the erythrocyte. Thus, agents with no such side effects are needed as replacements for ribavirin.

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viii) Levovirin, Viramidine These agents are derivatives of ribavirin and have no side effects such as hemolytic anemia [39]. Levovirin (LCN Pharmaceuticals), an L-enantiomer of ribavirin, is excreted in nonphosphorylated form to the urine and is therefore does not accumulate in erythrocytes. This agent has no antiviral activities and has no effect on viral polymerase until it is phosphorylated. However, like ribavirin it induces Th1/Th2 modulation, which stimulates HCV-specific T cell proliferation. Viramidine (LCN Pharmaceuticals) has a carboxamidin structure and differs from ribavirin, which is a carboxamide, as regards intra cellular transport. However, this agent is converted to ribavirin rapidly and thus has antiviral and immunomodulatory effects similar to ribavirin. A question of interest at present is whether combination therapies of IFN with these ribavirin derivatives will have antiviral effects similar to those of IFN-ribavirin combination therapy, but with fewer side effects. ix) Other Nucleoside Analogs for HBV; Emtricitabine, Clevudine A clinical trial of emtricitabine (Gilead Sience), 25-200mg for 48 weeks, showed antiviral effects. However, like lamivudine, resistance mutations were observed in the 100mg and 200mg groups [40]. And in the case of clevudine (Triangle Pharmaceutical) a 2.5-3.0 log 10 copies/ml decrease of viral load was noted in a clinical trial after 4 weeks [41]. x) Non-Nucleoside Analogs for HBV Bay 41-4109 (Bayer Health Care), which belongs to the heteroaryl dihydroprimidine family, has shown a potent antiviral activity to HBV in in vitro study. This agent exerts its antiviral effects by inhibiting nucleocapsid maturation through binding to core protein in the process of HBV replication [42]. xi) New Anti HCV Agents Based on Molecular Structures The HCV genome consists of a 5’ untranslated region, 4 structural proteins, 6 nonstructural proteins and a 3’ untranslated region, and these HCV gene sequences or HCV proteins are considered ideal drug targets [43] (Fig. 2). Drug development has been delayed because of a lack of effective cell culture systems and small animal models for HCV. However, a HCV subgenomic replicon system using hepatoma cells [44] has now been developed that makes it possible to screen candidate drugs that target the gene products. In addition, elucidation of the crystalline structure of HCV proteins such as serine protease (NS3 protease) and RNA-dependent RNA polymerase (NS5B) has also led to the development of new anti HCV drugs. a) Anti HCV Drugs which Target the 5’ Untranslated Region The 5’ untranslated region has been a good target for the development of new drugs because of the important roles it plays in HCV protein translation or HCV replication. ISIS-14803 (ISIS Pharmaceuticals Inc.) is an antisense DNA that consists of a 20-base phosphorothioate oligonucleotide [45]. Heptazyme (RPI, Boulder) is an HCV specific ribozyme, designed to cleave the HCV-IRES. Both are now being evaluated in Phase I or II trials.

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HCV genome and target sites of antiviral agents

IRES

NS3/4A serine protease

NS2/3 protease

Core

E1

E2 P7 NS2

NS3

NS4A/NS4B

NS5A/NS5B

3’UTR

5’UTR NS3 helicase

RNA dependent RNA polymerase

Fig. (2). HCV genome and target sites of antiviral agents.

b) Anti HCV Drugs which Target NS3 NS3 encodes a serine protease and its crystalline structure has been defined. Extensive studies to develop new anti HCV drugs targeting NS3 are being carried out at present. New agents which target the NS3 helicase are also being studied. BILN 2061 (Boehringer Ingelheim) is a very specific serine protease inhibitor which showed few side effects in clinical trials and is expected to become a useful anti HCV drug in future [46]. Telaprevir (VX-950, Vertex Pharmaceutical) is another NS3 protease inhibitor that is now being evaluated in clinical trials [47]. Two phase II trial, PROVE1 and PROVE2 have been carried out with triple combination with Telaprevir, Peg IFN-2a and ribavirin. In PROVE1, SVR was acheved in 61% of studied HCV patients for 24 weeks and 67% for 48 weeks. In PROVE2, SVR was achieved in 68 and 62% by 12 or 24 weeks with the triple combination therapy compared with 48% of SVR in the standard of case [48]. Boceprevir (SHC 503034), a novel and potent oral HCV protease inhibitor, was evaluated in combination with Peg IFN
-2b for genotype 1 nonresponders and this preliminary studies suggest a potent new therapeutic option for nonresponder patients [49]. c) Anti HCV Drugs which Target NS5B R1626, prodrug of NS5B polymerase inhibitor R1479, evaluated with a 4-week administration in combination of Peg IFNa-2a + ribavirin in HCV genotype 1 patients showed that 74% of patients treated with triple combination therapy showed undetectable HCV RNA. However, high frequency of Grade 4 neutropenia was observed [50]. There are other several polymerase inhibitors in preclinical or early clinical studies. d) Other Agents for HCV Infection Nitazoxanide, widely used as an antiparasite agent for cryplosporidiosis and helminthic infection has been recently shown to be effective on HBV or HCV mutants and combination

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of this agent with IFN + ribavirin showed that SVR rate was 79%, which was better than that of the standard care of the patients of 50% [51]. Furthur clinical trials are on going. Cyclosporine A has been used for posttransplantation and various autoimmune diseases. It has been reported that cyclosporin A inhibits intracellular HCV replication and cyclosporin D [52] and N1M811 [53], non-immunosuppressive cyclosporin analogs, also induced a HCV suppression. DEB10-025, a non-immunosuppressive cyclosporin analog in combination with Peg IFN
2a showed early HCV-RNA decline compared with that of Peg IFN monotherapy in a double-blind, placebo-control trial [54]. xii) Prenylation Inhibitor for HDV Infection HDV infection is mostly seen along the Mediterranean coast. The HDV RNA is enveloped with HBsAg, the envelope of HBV and HDV infection requires co-infection with HBV. Anti HBV drugs such as lamivudine have been administrated for HDV infection without satisfactory clinical effects. However, anti HDV effects have recently been demonstrated in HBV model mice by inhibiting the prenylation of L-HDAg necessary for the formation of HDV particles. A clinical trial of this prenylation inhibitor has shown no serious side effects and it is expected to become the first effective anti HDV drug [55]. 4. PRESENT STATUS OF ANTIVIRAL TREATMENTS FOR VIRAL HEPATITIS At present, IFN preparations are the most commonly administered antiviral treatments for HBV or HCV associated liver diseases. These diseases are now one of the most important health problems in the world because of the high numbers of patients and the serious or even fatal consequences. In the case of HBV-associated liver diseases, most cases end their clinical course naturally without active treatments, and only 10 to 20% of these patients progress to liver cirrhosis or HCC, which are eventually fatal if untreated. Therefore active treatments such as the use of antiviral agents must be given to those patients. However, at present it is difficult to recognize at the beginning stage of chronic HBV hepatitis which patients will be naturally cured and which will progress to liver cirrhosis and HCC. Thus, it is hard to determine when antiviral drug treatment should be started. However, antiviral drugs would be strongly indicated for chronic hepatitis of stage F2 or F3, which is associated with progressive hepatic changes, and also for liver cirrhosis, which shows progressive hepatic changes and may be expected to lead to hepatic failure. In addition, antiviral drugs are indicated for episodes of acute flare in the clinical course. Recently, reactivation of hepatitis has been paid much attention because of serious outcome in HBV carriers treated with immunosuppressive drugs [56]. The anti HBV drugs that are clinically available at present, besides IFN, are nucleotide analogs such as lamivudine, adefovir, entecavir, telbudine or tenofovir which can now be administered without serious side effects. However, patients treated with lamivudine for more than one year often develop an YMDD mutation which reduces the effectiveness of the drug. Presently adefovir is given to those patients with or without lamivudine with good results. At present, entecavir or tenofovir is first-line oral anti viral medication because of its high antiviral effects and because it produces few resistant mutations. In any case, a complete eradication of HBV from the body is impossible because of the nature of HBV replication processes. The goal of treatments for HBV-associated liver diseases is to induce a state of clinical cure by long-term administration of antiviral drugs to decrease viral load on one

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hand, and by using IFN or HBV vaccine to enhance immunoresponse on the other. Most chronic liver diseases based on mother to infant transmission can now be prevented by control of transmission at birth. Therefore, if such controls are implemented thoroughly and treatments with recently developed anti HBV drugs are widely applied, it will eventually be possible to conquer HBV-associated liver disease, not only in developed countries, but also in developing countries. In the case of HCV-associated liver diseases, antiviral treatments have been carried out only for HCV-associated chronic liver diseases. However, such treatments are now being considered also for non-symptomatic HCV carriers with normal serum ALT because these carriers often develop liver injuries in future. Thus, at present antiviral treatments to eradicate infected HCV are indicated for all HCV carriers. Today, Peg-IFN + ribavirin combination therapy is the standard and most effective treatment for HCV-associated liver diseases and over 50% of chronic hepatitis C patients with genotype I and high viral load are sustained responders to this combination therapy. Therefore, the complete response rate of HCV carriers for the eradication of the virus is totally around 60-70%. This is a very exciting result. However several problems are also associated with these treatments. First, IFN based treatments should be continued at least for one year. Additionally, both IFN and ribavirin have considerable side effects. More specifically, hemolytic anemia is observed in all patients treated with ribavirin. Thus, it is hoped that ribavirin derivatives that do not induce hemolytic anemia will become clinically available in the near future. Moreover, new anti HCV drugs which target specific segments of the HCV genome are also expected to be clinically useful in combination with IFN + ribavirin therapy and several drugs are now expected to be clinically used in the very near future. As mentioned earlier, once HCV infection occurs, natural clearance of the viruses is rare, and once chronic hepatitis is established few patients are naturally cured, with most patients progressing to liver cirrhosis and finally to HCC. Japanese studies show that 7% of liver cirrhosis patients develop HCC annually. In other words, all HCV-associated liver cirrhosis patients can be expected to develop HCC within about 10 years. As a result, it is suggested that all HCV carriers should receive antiviral treatment for the eradication of HCV. To this end it is hoped that safer, more effective, and less expensive antiviral agents will become available in the very near future. ABBREVIATIONS ADV

=

Adefovir

ALT

=

Alanine aminotransferase

AMP

=

Adenosine monophosphate

DNA

=

Deoxyribonucleic acid

HAV

=

Hepatitis A virus

HBc

=

Hepatitis B core

HBV

=

Hepatitis B virus

HBIG

=

Hepatitis B immunoglobulin

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HCC

=

Hepatocellular carcinoma

HCV

=

Hepatitis C virus

HDV

=

Hepatitis D virus

HEV

=

Hepatitis E virus

HIV

=

Human immunodeficiency virus

IFN

=

Interferon

IRES

=

Internal ribosome entry site

NS

=

Non-structural protein

Peg IFN

=

Pegylated IFN

RNA

=

Ribonucleic acid

Th1, Th2

=

Helper T cell 1, Helper T cell 2

YMDD

=

Aminoacid sequence: tyrosin-methionin-aspartate-aspartate

K. Tanikawa

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NO-Releasing Hybrids of Cardiovascular Drugs Alma Martelli1, Simona Rapposelli2, Maria C. Breschi1 and Vincenzo Calderone1,* 1

Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Università degli Studi di Pisa, Via Bonanno, 6, I-56126 Pisa, Italy; 2 Dipartimento di Scienze Farmaceutiche, Università degli Studi di Pisa, Via Bonanno, 6, I-56126 Pisa, Italy Abstract: Nitric oxide (NO) is an endogenous compound, playing a fundamental role in the modulation of the function of cardiovascular system, where it induces vasorelaxing and antiplatelet responses mainly through the stimulation of guanylate cyclase and the increase of cGMP. Many drugs of common and old clinical use (for example, glycerol trinitrate and all the vasodilator nitrites and nitrates) act via the release of exogenous NO, thus mimcking the effects of the endogenous factor. In the last years, a revision of the “one-compound-one-target” paradigm led pharmacologists and medicinal chemists to develop new classes of molecules, joining more pharmacodynamic properties. Among them, this innovative pharmacological/pharmaceutical strategy produced hybrid drugs, with a dual mechanism of action: a) the slow release of nitric oxide and b) an other fundamental pharmacodynamic profile. These drugs have been obtained with the insertion of appropriate NO-donor chemical groups (i.e. nitrate esters, nitrosothiols, etc.), linked to a known drug, through a variable spacer moiety. These new pharmacodynamic hybrids present the advantage of joining to a main mechanism of action (for example, cyclooxigenase inhibition, beta-antagonism or ACE-inhibition) also a slow release of NO, useful either to reduce the adverse side effects (for example, the gastrotoxicity of NSAIDs) or to improve the effectiveness of the drug (for example, conferring direct vasorelaxing and antiplatelet effects to an ACE-inhibitor). This review wishes to present the chemical features of NO-releasing hybrids of cardiovascular drugs and to explain the pharmacological improvements conferred by the addiction of the NO-donor properties.

Keywords: Nitric oxide, NO-donor, hybrid drugs, cardiovascular drugs. INTRODUCTION The pharmacological treatment of many pathological states often requires the action of different and complementary pharmacodynamic mechanisms. In the clinical practice, this is *Corresponding author: Tel: +39-(0)50-2219589; Fax: +39-(0)50-2219609; E-mail: [email protected] Allen B. Reitz / Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.

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frequently achieved through the administration of “cocktails”, composed by more drugs possessing different mechanisms of action. In the last years, the “one-drug-one-target” paradigm has been shelved and numerous multi-target drugs have been projected and synthesised. Such compounds share two (or more) desired pharmacodynamic properties, ensured by the presence of overlapping or conjugated pharmacophores. Compared with a pharmacological “cocktail”, the use of only one multi-target drug presents some advantages, such as an easier prediction of pharmacokinetic/pharmacodynamic relationships and an improved compliance by the patient, due to the administration of a single medicine [1]. The release of nitric oxide (NO) is the mechanism of action accounting for the pharmacological features and clinical applications of drugs, such as the “old” vasodilators nitrites and nitrates, because of the fundamental roles played by this small molecule in the cardiovascular system. More recently, the NO-releasing ability has been individuated as an interesting property to be added to molecules already possessing another pharmacodynamic pattern. This strategy led to the development of important multi-target drugs, interesting pharmacodynamic hybrids joining to a “native” mechanism of action a NO-donor property, addressed to reduce possible side-effects (for example, the gastrotoxicity of aspirin) or improve the therapeutic impact (e.g., the increase of antiplatelet activity of aspirin, again). This review focuses on the fundamental biological properties of NO at the cardiovascular level. Moreover, the chemical features of the most commonly used NO-donor moieties and the mechanisms ensuring the release of NO are shown. Finally, the main classes of multi-target drugs, showing the conjugation of a NO-donor group to a “native” molecule acting at the cardiovascular level, are described by the chemical and pharmacological points of view. ENDOTHELIAL NITRIC OXIDE Vascular endothelium plays a fundamental role in the regulation of several aspects of blood circulation. It, following both mechanical and chemical stimuli, biosynthesises and releases many endogenous compounds, which control and modulate the tone of vascular smooth muscle cells, their proliferation and several steps involved in the process of emostasis. In many pathological states, endothelial dysfunction is known to account for several aspects involved in the pathogenesis of cardiovascular disorders [2]. Among the heterogeneous compounds produced by endothelial cells, the most relevant one is probably a small molecule, originally described as endothelium-derived relaxing factor (EDRF) [3] identified as nitric oxide [4-6]. In the endothelial cell, NO is biosynthesised by the endothelial Ca2+dependent constitutive enzyme NO-synthase (e-NOS) from L-arginine [7]. Besides the wellknown endogenous and exogenous compounds (such as acetylcholine, bradykinin, calcium ionophore A23187, etc.) accounting for a chemical stimulation of the release of endothelial NO, blood flow and shear stress are the mechanical physiological triggers for the production and release of endothelial NO [8, 9] and an endothelium-mediated vasorelaxing effect induced by blood flow has been demonstrated in animal and human vessels [10-14]. Conversely, the inhibition of eNOS causes vasocontractile effects [15-18] and determines a significant reduction of perfusion flow in isolated perfused tissues [19]. This endothelium-mediated vasorelaxing effect is principally due to the NO-induced activation of

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cytosolic guanylate cyclase in the vascular smooth muscle cell, with a consequent raise of intracellular concentration of cGMP [20, 21]. Besides, other vasorelaxing mechanisms, such as a direct activation of muscular potassium channels by NO [22], have been described. To remark the fundamental role played by the vasorelaxing effect of endothelial NO in the regulation of blood pressure within the physiological range, it is noteworthy that often most cardiovascular diseases are associated with an impairment of the vasodilator function of endothelium [23-25]. Indeed, the NO-dependent vasorelaxing effects of acetylcholine are decreased in patient affected by essential hypertension, as well as in aged humans [26-29]. Furthermore, specific physiological functions of particular vascular districts, such as the corpus cavernosum, are ensured by NO, whose release induced by NANC (non-adrenergic non-cholinergic) neural influence, with the consequent vasorelaxing effects, is a key-step involved in penis erection [30]. Besides its fundamental vasorelaxing effect, NO controls an another important aspect of cardiovascular system: the platelet function. In particular, NO, produced by the endothelial cells but also by the platelet themselves, reduces the platelet adhesion and aggregation [31-34]. NO is also involved in the regulation of the modelling of the vascular structure, through both direct and indirect mechanisms. Indeed, the platelet adhesion on the site of an endothelial lesion determines the proliferation of vascular smooth muscle cells, due to the release of platelet-derived factors [35]. Therefore, the anti-platelet function of NO represents per se an indirect anti-proliferative function. Furthermore, NO inhibits the biosynthesis of MCP-1 (monocytes chemoattractive protein) and, consequently the adhesion of monocytes to the vascular wall, where they could release proliferative factors and cytokines [36]. More recently, a possible role of NO in the process of “ischemic preconditioning” has been extensively debated. Although, to date, the different experimental approaches on different animal species do not allow a consistent unitary theory on the exact role played by endogenous NO and on the specific contributes of the different isoforms of NO synthase [37, 38], there are clear and unequivocal evidences that the administration of exogenous NO-donors determines a significant reduction of the myocardial damage in ischemia-injured hearts from different animal species [39-43]. This experimental evidence lets us foresee a further intriguing positive aspect for a rationale use of NO-releasing molecules as potential anti-ischemic drugs. NO-DONOR DRUGS The prototypical class of NO-releasing derivatives is represented by organic nitrates and nitrites, such as glyceryl trinitrate, isosorbide dinitrate or 5-mononitrate and amyl nitrite, able to release NO after a metabolic bio-transformation, or other molecules able to release spontaneously NO with a temperature dependent mechanism, such as sodium nitroprusside (Fig. 1). All these compounds can be viewed as pro-drugs, which, through the release of exogenous NO, activate the same metabolic pathway of endogenous NO [44] and thus can exhibit all its biological properties. Nevertheless, their use is substantially confined into the only sphere of pathological situations requiring a rapid significant vasorelaxing effect, because of their short half-life (i.e. a rapid and massive release of NO). In the last two decades, two different biochemical activations for organic nitrates were hypothesised and then described: the enzymatic and the non-enzymatic ones. The enzymatic pathway, has its primary location on plasma membrane of muscular or endothelial cells [45].

NO-Releasing Hybrids of Cardiovascular Drugs O

Frontiers in Medicinal Chemistry, 2010, Vol. 5 O 2N

NO2

O

H

H O

O

NO2 O

O

O

H H

NO2

Glyceryl trinitrate H

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O

NO2

Isosorbide dinitrate

H

H

O

O NO

O

H H

O

Amyl nitrite

NO2

Isosorbide 5-mononitrate

2-

NO NC

CN .

Fe2+

2Na+

CN

NC CN

Sodium nitroprusside

Fig. (1). Chemical structures of the “classical” NO-donors glyceryl trinitrate, isosorbide dinitrate, isosorbide 5-mononitrate, amyl nitrite and sodium nitroprusside.

Although the enzymes responsible for the in vivo denitration and reduction of organic nitrates have not still identified, different potential enzymes have been purposed for this role, such as glutathione-S-transferase [46], cytochrome P-450 like enzymes [47], and according to recent studies, also mitochondrial aldehyde dehydrogenase (ALDH2) seems able to mediate denitration of classical nitrates [48]. Glutathione-S-transferase is responsible for the conversion of glyceryl trinitrate (GTN) in its active metabolites 1,2-GDN and 1,3-GDN [49]. Such enzyme probably uses the reduced thiolic group of glutathione, which is bound to the catalytic active site, to release nitroso acid and the glutathione oxidated form. The nitroso acid then is reduced to NO, by protonation, or by the formation of of S-nitrosothiol [50]. As concerns the non-enzymatic pathway, in the late ’60, Needleman and coworkers [51, 52] introduced the concept of organic nitrates as “prodrugs” which need intermediates to act their mechanism of action. According to this hypothesis, molecules containing SH groups, such as cysteine or glutathione, appeared indispensable for the conversion of organic nitrates into NO or S-nitrosothiols [53]. Today it is suggested that organic nitrates, interacting with sulfhydryl groups, produce NO or S-nitrosothiols; these intermediates, activating guanylate cyclase, produce cGMP which determines vasodilatation. A recent study focused the attention on the mechanism of cytochrome P450 reductase (CYP450R)-mediated nitric oxide and nitrosothiol generation from organic nitrates. According to this study cytochrome P450 reductase catalyses the bioactivation of organic nitrate through reduction to form the intermediate organic nitrite, which is converted to NO and nitrosothiols in a thiol-dependent reaction. This series of experiments was performed both on rat liver microsomes (containing the CYP450R-CYP450 complex) and on purified recombinant CYP450R. The presence of NADPH, compared with NADH, results in a much

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more efficient reducing substrate, as electron donor, to support the CYP450R-mediated GTN/isosorbide dinitrate (ISDN) reduction to produce nitrite, thanks to its better substrate affinity for CYP450R. The CYP450R flavin site inhibitor, diphenyleneiodonium, inhibits the NO2 – generation, whereas the CYP450 inhibitor clotrimazole does not inhibit this first step but greatly inhibits NO2 – -dependent NO generation. Therefore, CYP450R catalyzes organic nitrate reduction, producing nitrite, whereas CYP450 seems to mediate further nitrite reduction to NO. However nitrite-dependent NO generation contributed <10% of the CYP450R-CYP450 mediated NO generation from organic nitrates suggesting that nitrite is not the primary precursor of NO in the process of microsomal CYP450R-CYP450-mediated organic nitrate biotransformation but is the precursor of both NO and nitrosothiols. In fact it is well known that sulphydryl compounds are needed in GTN activation and that the repeated administration of GTN causes sulphydryl depletion and consequent tolerance to further vasodilatation. This study shows that the presence of L-cysteine triggered significant NO generation from recombinant CYP450R-mediated reduction of GTN/ISDN, whereas no detectable NO was generated without the addition of thiols. In contrast, with microsomal CYP450R, external thiols were not required for NO generation, as the sulphydryl groups in the microsomal proteins may serve to reduce organic nitrite to NO. So the proposed mechanism of CYP450R-CYP450-mediated biotransformation of organic nitrate is the following: 1st Step R-O-NO2 + NADPH + H+ CYP450R

R-O-NO + NADP+ + H2O

2nd Step R-O-NO + H2O

R-O-H + HNO2

3rd Step HNO2 + H+

CYP450

NO +H2O

4th Step 2R-O-NO + 2R’-SH

2NO + 2ROH + R’-S-S-R’

5th Step R-O-NO + R’-SH

R-OH +R’-S-NO

According to this mechanism, an organic nitrite (R-O-NO) is the initial product in the process of CYP450R-mediated organic nitrate biotransformation and is the precursor of NO and nitrosothiol. In the absence of thiols, organic nitrite undergoes hydrolysis to form nitrite. However in the presence of thiols, either NO or nitrosothiols can be formed [54]. Besides the organic nitrates and nitrites, which require a metabolic biotransformation for activity [55], there are direct NO-donors able to release NO spontaneously from a nitroso or nitrosyl moiety. Although metabolic processes, due to NADPH-generating systems located in the cell membrane, seem to be involved in an increased release of NO from sodium nitroprusside (SNP), these “facilitating” processes are not required as a necessary condition for the NOrelease [56]. Therefore SNP is generally classified as an agent belonging to the class of spontaneous NO-donors [57-59]. In its molecule (Fig. 1), a square bipyramidal structure, a nitrosyl group is linked to a ferrous ion connected also with five cyanide anions [57]. NO is released from this complex at physiological pH. However, the clinical employment of

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this drug, as a vasodilator, is limited by the toxicity due to the formation of thiocyanate and the need of parenteral administration [58]. Moreover, SNP exhibits the induction of an enhanced hydrogen peroxide-mediated cytotoxicity, probably due to the release of cyanide and intracellular residual iron complexes [60]. Diazeniumdiolates, also called NONOates (Fig. 2), possess the ability to release directly NO as a radical. Their efficacy was demonstrated in reversing cerebral vasospasm, reducing pulmonary vascular pressure and their coupling with metallic stents is studied in order to reduce the risk of restenosis following angioplasty surgical interventions [61]. R1

ON

N+

R2 R3 is ionically bound

Pathway B: H+

R3 R1

OR3

N

Pathway A: metabolism and/or hydrolysis

R3 is covalently bound R3

R1 H + 2 NO

N R2

H+ Pathway C: H+

ON+

N R2

O-

N

H 2N

H N

H 2N

N

(CH2)4 N -O

N+ N

-O

N+

O- Na+ DEA-NONOate

H 2N

spermine NONOate

N + O- Na

NH2 H N N N -O

N+ N O- Na+

MAHMA NONOate

-O

N+ N O- Na

+

DETA NONOate

Fig. (2). The two mechanisms accounting for the release of NO from a generic diazeniumdiolate (NONOate): if the O-R3 bond is ionic, a H+-catalysed reaction leads to the release of NO (pathway A); if the O-R3 bond is covalent, a first biotransformation leads to an intermediate derivative (pathway B), which in turn is converted into NO by a H+-catalysed reaction (pathway C). The chemical structures of some NONOates, commonly used as NO-donors, are also shown.

Among the direct NO-donors there are two classes of heterocyclic NO-releasing drugs, furoxanes and sydnonimines (Fig. 3), which require a facilitator agent to release NO. These cofactors are represented by thiol-derivatives for furoxanes and by oxidants for sydnonimines. The most known sydnonimine, the 3-morpholinosydnonimine (SIN-1), deriving from molsidomine, represents the combination of morpholine and a sydnonimine; it has been hypothesised that this zwitterionic compound, at physiological pH, decomposes into NO. and

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superoxide anion [62]. Nevertheless, other theories suggest that the composition of SIN-1 could lead to nitrogen oxide products, other than NO; these products (such as peroxynitrite) could be responsible for toxicity [60]. O N-

N

O

CH3

N+ N

O

O

Molsidomine O NH-

N N+ N

O

3-Morpholino sydnonimine (SIN-1)

O R1

O R2

N+

N O

O-

R1

-O

R2

N+

N O

Furoxan derivatives

Fig. (3). NO-releasing heterocyclic moieties: molsidomine, SIN-1 and generic structures of furoxans.

As concerns furoxanes (1,2,5-oxadiazole 2-oxide derivatives), they are often employed as NO-donor moieties linked to “native” drugs in order to add cGMP-mediated vasodilating and antiplatelet properties [63, 64]. In the last years, our group described the synthesis and the pharmacological evaluation of some benzoic and benzylic nitrooxy derivatives, which are NO-donors in themselves but can also be seen as useful linkers for multi-target drugs able to release NO (Fig. 4). The NO-mediated vasorelaxing effects of these compounds were tested on endotheliumdenuded isolated rat aortic rings pre-contracted with KCl and the pharmacological results indicate a clear correlation between slight structural modification, such as the insertion of (a) methyl group(s) into the nitrooxymethyl chain or into the aromatic ring, or a change in the position of the nitrooxy group itself, and the rate of the NO release [65]. Finally, the S-nitrosothiols must be mentioned, a class of NO-donating compounds which represent a source of circulating endogenous NO releasing spontaneously NO and NO+ (nitrosonium) [66]. S-nitrosothiols may also be able to improve transmembrane transfer of NO, realised by the cell-surface protein disulfide isomerase (PDI) from a S-nitrosothiol source [67]. Moreover, S-nitrosothiols seem to be involved as intermediates, in the organic nitrate mechanism of action. In fact, organic nitrates first interact with sulfhydryl groups to produce NO (or S-nitrosothiols) which in turn activate guanylate cyclase to produce cGMP (which causes vasodilation). This biochemical cascade, albeit not indispensable for the mechanism of nitrates, seems to be chemically very favourable and rapid [45]. Besides, members of this class, as S-nitroso-N-acetylpenicillamine (SNAP), show some advantages with respect to organic nitrates or SNP, in fact they exhibit a lower capacity to induce oxidative stress or tolerance in vascular cells [68].

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R2 O2NO

X R

R3

R1

R R2

O2NO X R1

R3

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X

R

R1

R2

R3

C

H

H

H

COOH

C

Me

H

H

COOH

C

H

Me

Me

COOH

N

H

-

H

COOH

C

H

H

H

OH

C

H

H

H

CH2OH

C

Me

H

H

CH2OH

C

H

H

H

COOH

C

Me

H

H

COOH

C

H

H

H

CH2OH

C

Me

H

H

CH2OH

279

Fig. (4). NO-donor linkers with a carboxy, phenolic or hydroxymethyl function.

Indeed, the induction of tolerance seems to be one of the most relevant problems, related with a long-term administration of many NO-releasing drugs. Although the comprehension of the exact mechanisms accounting for the development of such a tolerance is still controversial, it is accepted that this represents a multifactorial phenomenon involving several (and not completely clarified) processes. Besides an early hypo-responsiveness (known as pseudotolerance), mainly due to counter-regulatory mechanisms, such as an increased production of endogenous vasoactive compounds, a long-term administration of organic nitrates, and in particular glyceryl trinitrate, has been related with many intrinsic alterations, leading to a more significant loss of responsiveness (tolerance) [69-78]. More in general, the tolerance to a NO-releasing agent is a complex phenomenon, probably linked to the involvement of several aspects, depending both on the chemical nature of the drug and on the triggering of “proximal” and “distal” biological mechanisms. The “proximal” events are those related to the alteration of one or more steps possibly involved in the bio-transformation of the drug, leading to the release of NO, but also those related to the metabolic fate of the released NO. Instead, the “distal” mechanisms are those more closely related to the possible reduction of the NO-induced rise of cGMP. As concerns the “proximal” ones, the induction of tolerance by organic nitrates has been studied, taking glyceryl trinitrate as the most commonly used NO-donor reference compound. A significant role of a decreased activity of enzymes, such as aldehyde deydrogenase 2 (ALDH2) and cytochrome P-450 (both probably involved in the bio-activation of glyceryl trinitrate), has been suggested as a cause of tolerance. Indeed, ALDH activity was reduced in in vitro models of vascular tolerance and ALDH-inhibiting drugs could produce a reduced response to glyceryl trinitrate [48]. However, other in vivo studies led to controversial results [79], indicating that a decreased enzymatic activity of ALDH2 can play a role in the development of tolerance, but this role is not dominant. Instead, an important role played by a decreased activity of cytochrome P-450 has been proposed, because the pharmacological

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induction of this enzymatic system could reverse the development of tolerance caused by glyceryl trinitrate [80], while a vitamin E-deficiency, related to a decrease of cytochrome P450 expression (but also to an increased oxidative stress) caused an acceleration in the development of tolerance to glyceryl trinitrate [81]. Nitrate tolerance seems to derive, at least in part, also from an intracellular sulphydryl depletion; this event reduces the metabolic conversion of organic nitrates to NO and thereby vasodilation [45]. But some experimental evidences show that non-sulphydryl compounds, like enalapril [82] and hydralazine [83] can reverse or prevent the occurrence of in vivo nitroglycerine-induced tolerance, indicating that the sulfhydryl depletion-theory itself is not sufficient to explain the mechanisms of nitrate tolerance. Of course, the development of tolerance mechanisms, related to a reduced bioactivation of organic nitrates, can satisfactorily explain the cross-tolerance between two drugs which share a common metabolic activation pathway. On the contrary, this can not account for the cross-tolerance observed also between an organic nitrate and a spontaneous NO-donor, (or a drug able to release NO through different activation pathway). For example, a reduced responsiveness to SIN-1 has been observed in vascular preparations obtained from rabbits in which tolerance was induced by glyceryl trinitrate [84]. Moreover, in the “tolerant” vessels, the authors found high levels of superoxide levels and increased amounts of nitrotyrosine, a stable metabolite of peroxynitrite. This experimental evidence led to hypothesise a significant role of an accelerated inactivation of NO, as a possible cause of such a cross-tolerance [84]. Indeed, increased levels of superoxide and of peroxynitrite (readily produced by the reaction of surperoxide with NO) have been observed in rat vessels made tolerant by glyceryl trinitrate. Consistently, tolerance was reduced by pre-treatment with several antioxidant, such as ascorbic acid and uric acid [85]. Finally, also the “distal” mechanisms, such as a decreased enzymatic activity of guanylate cyclase [86] and/or an increased hydrolytic degradation of cGMP [87], can lead to a hypo-responsiveness to NOdonors. Of course, such mechanisms can perfectly participate to the multifactorial phenomenon of tolerance to organic nitrate and can also explain the tolerance observed for spontaneous NO-releasing agents. Indeed, even if the tolerance induced by non-nitrate NOdonors is generally considered to be of lower magnitude with respect to that induced by organic nitrates, relatively high concentrations (0.1-10 μM) of SNP determined a significant desensitisation of soluble guanilate cyclase and the development of cross-tolerance with authentic NO [88]. However, the development of this desensitisation was not observed after treatment with lower concentrations (1 nM) of SNP [88]. It is interesting to note that some particular effects of NO-donor drugs, such as the anti-platelet one, seems to be resistant to the development of tolerance [89]. The problem of tolerance for NO-donors, whose whole comprehension needs further experimental data, has been authoritatively reviewed by some recent and excellent papers [59,90]. NO-DONOR HYBRIDS In the last years, the knowledge of the biochemical and pharmacological properties of nitric oxide led to the effort in design of new hybrid molecules, which couple a “native” well-known drug with a NO-donor moiety (Fig. 5). This innovative approach furnished a variegated sample of new chemical entities which conserve the therapeutic efficacy of the “parent” drug, enriched through the nitric oxide activity, moreover this “synergism” often resulted in a reverse of their side effects. Among the NO-donor hybrids an increasing interest was aroused by the NO-releasing cardiovascular drugs, such as aspirin, statins,
and -blockers, Ca2+-antagonists, K ATP-

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openers, ACE-inhibitors and sartans; the goal of these hybrid molecules is the improvement of their cardiovascular actions and, sometimes, even the elision of their adverse effects thanks to NO properties. Native + drug

Native drug

Linker

Linker

+

NO-donor moiety

NO-donor moiety

Fig. (5). Theoretical template for the synthesis of a pharmacodynamic hybrid. A NO-releasing function is generally bound to a “native” drug, directly or often through a linker moiety.

NO-ASPIRINS One of the most characterised classes of NO-donors hybrids is represented by NONSAIDs (NO-donating non steroidal anti-inflammatory drugs) which, synthesised by esterifiation of the parent drug (a NSAIDs drug) with a NO-donor chain, maintain the original anti-inflammatory properties and show, through the nitric oxide action, a marked reduction of gastrolesivity. In fact, NO determines a protective effect on gastric mucosa attributable to several pharmacodynamic mechanism such as the assurance of mucosal blood flow, the stimulation of the mucus production, the inhibition of leukocyte adhesion and of enzymatic caspase [91-94]. As concerns the pharmacokinetic, the NO-NSAIDs may be assimilated to organic nitrates because they also require metabolic activation before releasing NO. Although the enzymes involved are not yet identified, there are a lot of evidences about a role of esterases in the cleavage of NO from the parent drug [95-97]. Nevertheless, even if the involvement of esterases is compatible with the chemical structure of NO-NSAIDs (the NO-releasing moiety is linked to the “native” molecule by an ester bond) also cytochrome P450 seems to act in the NO-NSAIDs breakdown [98]. Moreover, several different experiments on the rate of the enzymatic catabolism, both in vitro and in vivo, indicate that NO-NSAIDs have a NO-release slower than the “classical” NO-donors, such as SNP or SNAP [99]. This gradual release may be the key to explain why, in experimental animals, NO-NSAIDs do not alter systemic arterial blood pressure even when administered intravenously, in large dose, whereas an equimolar dose of the “classical” NO-donors causes a deep hypotension [95]. A recent study of Knaus’ group shows the development and the biological evaluation of a new serie of NO-NSAIDs. Hybrid NO-NSAIDs possessing a 1-(pyrrolidin-1-yl) diazen-1ium-1,2-diolate or 1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate moiety linked by a onecarbon methylene spacer to the carboxylic acid group of “native” aspirin (Fig. 6), ibuprofen and indomethacin were synthesised and characterised as NSAID and NO-donors. Although these new molecules did not show any inhibitory activity against COX1 and COX2, they result in a decreasing of carrageenan-induced rat paw edema similar to that exhibited by the “native” drugs and in improving of classical NSAIDs ulcerogenity. As concerns the NO-release an increased effect was observed in tests carried out in presence of guinea pig serum with respect to those in phosphate buffer solution.

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ONO2 O

O

O O

O O

O

O ONO2

O CH3

O

NCX 4040

O

ONO2

O

CH3

CH3

NCX 4060

NCX 4016

O O

O

HCl OH

N O

O O

ONO2

ONO2

O O

O O O

CH3 O

CH3

CH3

Aspirin NCX 4018

NCX 4050

O

CH3

O N O

O

N+

N

N O

O-

N N+

CH3

O-

O O

O

O CH3

O

CH3

Diazenumdiolate-Aspirin derivatives

Fig. (6). Structures of several NO-releasing hybrids, obtained from the “native” drug aspirin. The hydrolytic cleavage of the linkers (carrying the NO-donor moiety) leads to the release of the “native” drug, without any structural alteration.

All these considerations seem to suggest that such compounds are prodrugs which require a metabolic activation reaction (esterase-mediated ester cleavage) to be active [100]. The NO-releasing aspirin derivatives represent a new class of nitric esters in which a nitrate group is coupled to the carboxylic moiety of acetyl salicylic acid (ASA) through a variety of spacers (aliphatic, aromatic or heterocyclic ones) (Fig. 6) which result in conferring different pharmacokinetic or pharmacodynamic properties to the new hybrid [91]. The nitroaspirin NCX 4016 [3-(nitrooxymethyl)-phenyl 2-(acetyloxy)-benzoate] is the lead compound of this new category of anti-inflammatory and antithrombotic drugs [101] because this chimerical compound is able to maintain the anti-inflammatory action without the gastrointestinal toxicity and to show new cardiovascular properties due to the NO-donor moiety.

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Aspirin is the most known NSAID but also its use for the prevention and the treatment of such cardiovascular diseases, such as thrombosis, is well established [102,103]. The antithrombotic effect of aspirin is due to the irreversible inhibition of COX-1 which determines a reduction in the production of the pro-aggregatory TxA2 by platelets and of the antiaggregatory PGI2 by vascular endothelial cells, but while the endothelial cells are able to synthesise new COX-1 enzyme, platelets do not show this possibility, therefore the final balance is shifted towards the inhibition of platelet aggregation [104]. However, aspirin blocks only one pathway of platelet activation thereby leaving the others unaffected and this may limit its effectiveness like antithrombotic agent [105,106]. NO is well known as antiaggregatory agent, in fact it suppresses platelet activation by activating guanylate cyclase (GC), thereby increasing the conversion of GTP to cGMP, enhancing calcium ATPasedependent refilling of intracellular calcium stores and inhibiting the activation of phosphoinositide 3-kinase (PI3K). As a consequence, intracellular calcium (Ca2+) flux is inhibited, leading to suppression of P-selectin expression and of the active conformation of glycoprotein IIb/IIIa (GPIIb/IIIa) required for binding fibrinogen [107]. Hence, nitroaspirin could represent a good solution for ASA lacks, because its mechanism of action results from the sum of COX-mediated effects, such as inhibition of biosynthesis of inflammatory prostaglandins and of platelet aggregation, and NO-mediated effects (cGMP-dependent and independent) such as vasodilation, inhibition of platelet aggregation, leukocyte adherence to the vascular endothelium, inflammatory cytokine synthesis (through nitrosylation of caspase) and apoptosis (through nitrosylation of caspase and possibly regulation of mitochondrial function) [108]. This synergistic activity of the two pharmacophoric moieties, aspirin and NO, potentiates the pharmacological profile of NCX 4016, in comparison with the “parent” drug, and gives to NCX 4016 the status of completely new chemical entity. The effectiveness of NCX 4016 on platelets and on other cellular and metabolic functions relevant to thrombosis has been characterised in in vitro studies [109]. According to these studies, NCX 4016 shows a wider range of action than ASA and inhibits platelet aggregation induced by both aspirin-sensitive and aspirin-insensitive mechanism [110]. In fact, NCX 4016 is able to inhibit platelet aggregation induced not only by arachidonic acid and collagen, inhibited also by aspirin, but also the aggregation induced by U46619, a thromboxane analogue, and by thrombin, which are ASA-insensitive [93,110-114]. Since also classic NO donors are able to inhibit the platelet aggregation induced by U46619 and thrombin, it seems clear that the NO-moiety of NCX 4016 greatly contributes to the antiplatelet property [93,110,112-115]. Moreover NCX 4016 is able, like SNP but differently from aspirin, to decrease platelet adhesion to collagen under flow conditions and to prevent shear stress-induced platelet activation [112,116,117]. These data are interesting, because adhesion is the first step in the pathway of platelet activation which leads to thrombus formation and only few of the antiplatelet drugs today available are able to prevent it, and a high shear stress like that which occurs in stenosed or atheromatous arteries is retained responsible for the in vivo platelet activation. Remaining in the vascular district, a possible clinical application of NCX 4016 seems to be the prevention of restenosis due to neointimal hyperplasia, which represent the main long-term complication of percutaneous transluminal coronary angioplasty (PTCA), the most frequent treatment to resolve the coronary atherosclerotic stenosis [118]. Restenosis is often associated with an impairment of NO-dependent pathways: the modulation of the NOenzymatic pathway could influence the development of restenosis, i.e., L-NAME, (L-N
nitroarginine methyl ester, an inhibitor of NO synthase) stimulates neointimal hyperplasia.

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Moreover, NO controls also other pathophysiological responses occurring during atherosclerotic restenosis: inflammatory adhesion, vascular reactivity, endothelial permeability and regulation of smooth muscle cell proliferation linked with the vasculature remodelling. Thereby, as expected, in a model of restenosis in hypercholesterolaemic mice, NCX 4016 has been found to reduce restenosis more efficiently than aspirin and at lower doses [119,120]. Nitroaspirin could represent also a new approach for the treatment of saphenous vein graft failure which could occur after an autologous saphenous vein coronary artery bypass grafting (CABG) and which are represented by thrombosis, increasing of media thickening and neointima formation, and proliferation and migration of vascular smooth muscle cells (VSMC). Aspirin affects thrombosis but not the other responses which are instead inhibited by NO, so a NO-releasing aspirin could be a strategy to contrast all these aspects [121]. Finally, an intriguing application for NCX 4016 seems to be represented by a cardioprotective role in myocardial ischemia-reperfusion process. Aspirin is presently used for reducing mortality from acute myocardial infarct but its effects are most likely due to prevention of re-infarction rather than a direct cardioprotective effect, i.e. on arrhythmias [122]. NCX 4016 reduces infarct size in several animal models but recently its activity has been well characterised in rats. NCX 4016 showed remarkable cardioprotection in rats submitted to myocardial ischemia/reperfusion, as was evident in the reduction of ventricular premature beats and in the incidence of ventricular tachycardia and fibrillation; these arrhythmias were reduced dose-dependently, resulting in survival of all rats treated with higher dose of NCX 4016. Also the infarct size was restricted proportionally to the dose of NCX 4016 and this action was associated with biochemical data such as diminution of both plasma creatine phosphokinase and cardiac myeloperoxidase activities [123]. An exhaustive overview on the interesting pharmacological properties and on the relatively safe toxicological profile of NCX 4016, emerging from both pre-clinical and the first clinical studies has been recently published [124]. In this article no mention is done about the possible induction of tolerance. However, a bidirectional cross-tolerance between NCX 4016 and glyceryl trinitrate has been described in an in vitro study [125]. NO-STATINS Inhibitors of 3-hydroxy-3methylglutaryl CoA reductase, also called statins, are the most employed drugs in the treatment of hypercholesterolemia. The efficacy of statins in reducing low-density lipoprotein-cholesterol levels has been confirmed by several clinical trials [126]. However, in the last years, the theory of the “pleiotropic effects” of statins is raising. According to this hypothesis, there are other mechanisms of action, beyond lipid-lowering activity, which participate to the beneficial effects of statins in atherosclerotic disease [127,128]. In fact, statins are able to elicit an increase in endothelial NO production which leads to an anti-inflammatory action at the endothelium level and to an inhibition of VSMC proliferation [129]; but often, especially in those diseases like atherosclerosis and diabetes, there is an impairment of endothelium function which reduce the production of endogenous NO, so NO-releasing statins could offer an alternative source of NO for the therapies of these pathologies. A recent study on the NO derivatives of pravastatin (NCX 6550) and fluvastatin (NCX 6553) (Fig. 7) shows that the NO-donor moiety significantly potentiates the nonlipid-lowering mechanism of action of native statins. For example, as concerns the restoration of endothelial function, NO-releasing statins determine a reduction in both smooth muscle cells proliferation and inflammatory events, beyond those induced by statins alone in atherosclerosis [130]. A chronic treatment with the nitropravastatin derivative, NCX 6550,

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is able to improve the endothelial dysfunction also in spontaneously hypertensive rats (SHR), a model with marked vascular dysfunction and normal levels of cholesterol [131]. Moreover the NO moiety brings additional properties to the native statins, as demonstrated by NCX 6550 which showed the capacity to inhibit platelet aggregation in vitro, and to reduce mortality in the thromboembolism mouse model [132]. Finally, the last derivative, the NO-releasing atorvastatin (NCX 6560) (Fig. 7), has been recently compared with atorvastatin in cell-based assays as well as in normal and hypercholesterolaemic mice. These studies show a retained HMG-CoA reductase inhibitory activity but, at the same time, in a model of platelet pulmonary thromboembolism in mice, NCX 6560, significantly reduced mortality, while an equimolar dose of atorvastatin was ineffective. Moreover in LDLR (Low Density Lipoprotein Receptor) knock-out mice fed with hyperlipidemic diet, NCX 6560, but not atorvastatin, significantly reduced ex vivo platelet adhesion to collagen under high shear rate [133]. O

O O

COOH H

OH

O

COO(CH2)4ONO2

H

OH

HO

OH

OH

HO Pravastatin

NO-Pravastatin (NCX 6550)

OH

OH

N

OH N

COOH

COO(CH2)4ONO2

F

F

Fluvastatin

NO-Fluvastatin (NCX 6553)

O2NO(CH2)4O

HOOC OH

OH

F

OH N

i-Pr

PhNH

C O

OH

F

OH N

i-Pr

Ph O Atorvastatin

PhNH

Ph O

NO-Atorvastatin (NCX 6560)

Fig. (7). Chemical structures of pravastatin, fluvastatin and atorvastatin, with their corresponding NOreleasing hybrid derivatives.

More in particular, it has been observed that, in hyperlipidemic mice, NCX 6560 was more effective than atorvastatin in lowering serum cholesterol; furthermore its ability to

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induce vasodilation, cGMP formation and to reduce epinephrine-induced platelet pulmonary thromboembolism in mice was confirmed. Finally this new molecule seems to exert also an anti-inflammatory activity reducing iNOS expression and TNF
release in LPS (lipopolysaccharide)-trated macrophages [134]. NO-SILDENAFIL Sildenafil is a selective inhibitor of phosphodiesterase type 5 (PDE5) and it is the pioneer drug in the oral therapy of erectile dysfunction. Its mechanism of action consists in the inhibition of a cGMP phosphodiesterase V subtype, particularly present in penile smooth muscle; in this way, it prevents cGMP hydrolysis, thus allowing a prolonged signalling actions of NO [135]. In patients with stable angina, co-administration of sildenafil with organic nitrates produces significant reductions in blood pressure, greater than those induced by nitrates alone. On the basis of these findings, sildenafil should not be associated to a therapy with nitrates because it could lead to an exacerbation of negative and potentially life-threatening cardiovascular consequences [136]. O O

N

O

Me N N

S N

N H

N Me

n-Pr O

OEt Sildenafil

O

O

Me N

N

N

S N

N H

N Me

.HNO3 n-Pr

OEt (NO-SILDENAFIL) (NCX 911)

Fig. (8). Structures of sildenafil and its nitrate salt derivative, a well-representative example of effective NO-releasing hybrid, based on a simple chemical manipulation.

Although the association of sildenafil with NO-donors seems to be deleterious, in last years a nitroderivative of sildenafil, NCX-911 (Fig. 8), has been synthesised. By a chemical point of view, it could be probably considered as a simplest and effective example of pharmacodynamic hybrid: the NO-donor property has been conferred to the “native” sildenafil through the conversion in its corresponding nitrate salt. A lot of studies were carried out on this new molecule: thus, NCX-911 was found to be as potent as sildenafil in inducing relaxation of rabbit corpus cavernosum, but in the presence of L-NAME (a NO synthase inhibitor) the potency of NCX-911 was not altered, while sildenafil potency decreased fivefold. This result suggests that NO-releasing PDE5 inhibitors could potentially be more useful than PDE5 inhibitors in the treatment of erectile dysfunction, in conditions of reduced availability of endogenous NO [137]. For example, in diabetes the endogenous NO is significantly decreased and a comparative study between NCX-911 and sildenafil on the anococcygeus muscle of diabetic rats showed that the potency of NCX-911 in reducing the phenylephrine-induced tone was not altered, while that of sildenafil was significantly

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reduced [138]. Another cardiovascular disease, hypercholesterolaemia, promotes erectile dysfunction through increased superoxide formation and negation of NO bioactivity in cavernosal tissue. Although the origin of this mechanism was still not explained, the effect of sildenafil citrate and NCX-911 was studied on cavernosal tissue of hypercholesterolaemic rabbits. The results of this study seem to suggest that NO donating sildenafil may be therapeutically more beneficial than conventional sildenafil in treating erectile dysfunction with an oxidative stress-related aetiology [139]. NO-RELEASING ANTI-ADRENERGIC DRUGS In the cardiovascular district, the effects of the adrenergic system are mainly mediated by the subtypes 1 and 2 of its
and  receptors. In particular
1 receptors, when activated, are able to exert vasoconstriction at peripheral level through the transductional system in which IP3 and DAG are involved as second messengers. The use of
1-antagonist as vasodilators is exercised since several years but, in the last decade, new NO-donor
1-antagonists were synthesised by replacing the furan ring of prazosin (a well known
1-blocker) with a furoxan derivative (Fig. 9), to obtain a series of well balanced hybrids in which the vasodilation mediated by
1-antagonism was integrated with the NO-mediated one [140]. Of course, this strategy led to a structural change of the “native” drug, rather than to a cleavage of the “native” prazosin, following the release of NO. Nevertheless, among the adrenergic receptors, the principal target for antihypertensive therapy is undoubtedly represented by the  ones. As concerns the cardiovascular system, the 1 receptors are principally distributed at cardiac level and their activation determine a positive inotropic, chronotropic, batmotropic and dromotropic effect; the 2 receptors, instead, are responsible of the vasodilation in the skeletal muscle district but their involvement as antihypertensive target is limited. Besides, the 1 receptors are widely expressed also at the level of renal macula densa, where their block prevents the release of renin and as a consequence, contrasts the formation of angiotensin II through the renin-angiotensin system (RAS). Thereby, selective 1-blockers exert their antihypertensive effects through several pharmacodynamic patterns, and today they are commonly used not only to counteract hypertension, thanks to the reduction of cardiac activity and the block in renin release, but also in angina pectoris, thanks to the reduction of cardiac consumption of O2, and in cardiac arrhythmias. Recently, in order to potentiate some 1-blockers activities, such as the lowering effects on the blood pressure and the decreasing in oxygen consumption, several hybrid molecules were synthesised, in which different NO-moieties (furoxanes, 3-nitrooxypivaloyl acid, Nacetyl- D-penicillamine) were linked to chemical structures, closely related with propranolol (Fig. 9) [141,142]. Some of these new hybrid molecules were well-balanced and exert both the
-blocking and the NO-releasing actions in the same range of concentration, but in general, compared with propranolol, the hybrid formation lowers the affinity for -receptors, in particular for the 2-type, to give an increase in 1/ 2 selectivity [142]. The association of a classic nitrovasodilators with -blockers has been often employed in clinical practice, in order to reduce the initial increase of pheripheral resistence, due to the block of -adrenoceptors. However the chronic use of classical nitrovasodilators rapidly leads to the phenomenon of tolerance.

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O

O

C

C

N N

H3CO

R

N O

N

N

H3CO

N

N N

N

O

O

N

H3CO

H3CO NH2

NH2 Prazosin

NO-releasing-prazosin analogue

CH3

R X

O

N H

CH3

O

N H

OH

Propranolol

O

NO-releasing-propranolol analogue

CH3

CH3 O

N+ O

N

OH

N H

*

O

CH3

H

N OH H

CH3

OH

O O O2NO

CH3

Metoprolol

*

ONO2 H PF9404C

CH3 F O

N H

F

CH3

OH O O2NO

Nipradilol

N OH

H

O OH

Nebivolol

Fig. (9). Chemical structure of prazosin, propranolol and metoprolol (respectively,
1 blocker and -blockers) and their NO-releasing analogues. When the NO molecules are metabolised, lead to new compounds. Nipradilol possesses a nitrooxy group, conferring NO-releasing properties. Nebivolol does not possess any NO-releasing moiety, nevertheless it is able to induce an increased release of endothelial NO and thus, nebivolol shows pharmacological effects which can be assimilated to those produced by a hybrid NO--blocker.

An interesting new hybrid -blocker is represented by a S-S enantiomer of a metoprololrelated derivative, PF9404C, studied and developed in order to correct the initial high peripheral resistance occourring in hypertensive patients treated with -blockers. On the other hand, due the -blocker activity, it could prevent the increase of heart rate and catecholamine levels which represent the reaction to a fall of peripheral resistance and blood pressure.

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In a previous study PF9404C was compared with rapid NO-donors producing an immediate but transient vasorelaxation and what was new is that, on the contrary, it seems able to evoke a slowly developed but sustained relaxation [143]. Moreover, although classic nitrovasodilators were often associated with
-blockers as an efficient therapeutic approach in coronary heart disease, the phenomenon of tolerance, rising in chronic treatment, could not be ignored. A recent study demonstrates that if compared with nitroglycerin, PF9404C shows much lesser tolerance. This lower induction of tolerance by PF9404C could be explained through its slower release of NO [144]. Nipradilol (3,4-dihydro-8-(2-hydroxy-3-isopropyl-amino)-propoxy-3-nitroxy-2H-1benzopyran) (Fig. 9) is a nonselective adrenoceptor blocking agent with a weak
adrenoreceptor blocking activity and direct vasodilating property due to a nitro group contained within its molecular structure [145]. Nipradilol showed beneficial effects on cardiac remodelling, ischemic heart diseases and blood pressure control [146, 147], and its involvement on both intracellular calcium mobilization and the content of cGMP was proposed to partially explain its pharmacological action [148]. In a recent study it was hypothesised that mechanical stress-induced activation of intracellular signal transduction cascades is controlled by nipradilol in human aortic smooth muscle cells (HASMC) [149]. In fact, in endothelial cells, mechanical stresses are able to stimulate one or more phosphorylation cascades leading to an activation of mitogen-activated protein kinases (MAPKs) [150]. Also hypertension and angioplasty are rapid inducers of MAPKs activation in vivo [151]. These findings confirm that nipradilol has an action in modulating intracellular signal transduction pathway, i.e. extracellular signal-regulated kinase (ERK) cascade under high atmospheric pressure on HASMC [149], but classical
-blockers are reported to lack an inhibitory effect on SMC proliferation [152]. Therefore, the anti-proliferative effect of nipradilol on HASMC was thought to be caused by the release of NO itself [149]. Different studies suggest that nipradilol is a potent NOS stimulator in endothelial cells with NO releasing action, so this drug could represent a substitute for injured endothelial cells with a possible role in the treatment of endothelial dysfunctions occurring in atherosclerosis, hyperlipidemia and hypertension [153, 154]. Finally, as concerns the pharmacokinetic profile, a direct measurement of nipradilolderived NO in the vascular wall of canine femoral arteries has demonstrated that nipradilol is metabolised to NO in large part through a metabolic process involving thiols with a baseto-peak reaction time of about 1,5 minute [155]. Finally, nipradilol results involved also in a cardioprotective mechanism of action called preconditioning. Preconditioning represents a protective system in which a brief period of ischemia, preceding a most important ischemia, determines a markedly reduced infarct size. This physiological endogenous mechanism can be mimicked through the administration of some exogenous drugs, such as ATP-sensitive potassium-channel openers, adenosine, etc. [156-158]. Nitric oxide is retained to be involved in the mechanism of preconditioning [159], and recent studies have shown that NO is a requisite for cofactor in the preconditioning response generated by the administration of ATP-sensitive potassium-channel openers [160]. After a coronary surgical intervention a lot of patients receive
-blockers, but
adrenoceptors blockade alone prevents preconditioning, so the proposal to administer nipradilol may simultaneously offer a
-blocking action and a NO-mediated preconditioning. As concerns mechanical function, nipradilol may exert its effect by increasing coronary flow after the reperfusion [161].

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This property shown in the cardiac district has been demonstrated also at the brain level. In fact in a recent study the neuroprotective effect of nipradilol on hypoxic-ischemic brain injury of neonatal rats has been demonstrated. In particular it has been observed that the extent of the infarct area in the brain was significantly reduced by injection of a 1
M solution of nipradilol. The denitro-nipradilol (3,4-dihydro-3-hydroxy-8-(2-hydroxy-3isopropylamino)propoxy-2H-1-benzopyran) that does not release NO, did not show the neuroprotective effect on neonatal neurons [162]. Although nebivolol is not a molecule able to release exogeous NO (Fig. 9), it is able to induce an increased release of endothelial NO (as better explained below). Even if it can not be considered as a true “bifunctional” drug, nevertheless it shows pharmacological effects which can be assimilated to those produced by a hybrid NO-
-blocker. Nebivolol is a racemic mixture of equal amounts of the two enantiomers D an L, which belongs to the class of the high cardioselective, non-hybrid, -adrenergic receptor antagonists. Its peculiarity is that nebivolol possesses vasodilator properties, which are not due to the block of adrenergic receptors on smooth muscle cells. This vasodilator response is attributed to L-nebivolol and experimental evidence indicates that it is due to endothelium-dependent mechanism involving NO and the L-arginine/NO pathway [163-165]. Recent in vitro findings show that nebivolol relaxes vascular smooth muscle by mechanism involving the NO-cyclic GMP system [166]; these observations are predictive of in vivo effects in patients and support the findings that nebivolol reverses endothelial dysfunction in patients with essential hypertension [167]. Finally, nebivolol is also able to inhibit vascular smooth muscle cell proliferation by mechanisms involving NO but not cyclic GMP [168]. NO-RELEASING DIHYDROPYRIDINES The 1,4-dihydropyridine (DHP) Ca2+-antagonists, such as nifedipine, nitrendipine, amlodipine, are widely used in the treatment of hypertension and ischemic heart diseases. Their principal mechanism of action consists in the inhibition of Ca2+-influx through voltage-dependent L-type calcium channels (VDCCs) in vascular smooth muscle [169,170]. Besides the effects on the vascular smooth muscle, there are studies which reported that amlodipine may release nitric oxide from canine coronary microvessels through modulating the actions or formation of kinins; however, the mechanism by which calcium antagonists release NO is still unclear because there is no known receptor for Ca2+-antagonists in endothelial cell (ECs) [171] and most ECs lack VDCCs, so endothelial depolarization fails to increase the intracellular Ca2+ concentration through VDCCs [172]. Recently, it has been found that vascular smooth muscle and endothelial cells are tightly coupled via myo-endothelial communication and that electrical signals can be conducted each other [173,174]. This finding suggests that the modulation of electrical signals in smooth muscle by DHPs could affects membrane potential in ECs [175]. In order to potentiate their vasorelaxing properties, DHP Ca2+-antagonists were structurally modified by the addition of furoxan moieties which act as NO-donors. So a new series of 4-phenyl-1,4-dihydropyridines, bearing furoxan moieties at the ortho or meta position of the phenyl ring was synthesised (Fig. 10) and pharmacologically characterised, allowing to distinguish the well balanced hybrid molecules from that having a Ca2+-antagonist or NOdonor dominant profile [176]. Finally, in the last years, also the properties of NO-donor Ca2+-agonists were investigated: in fact, when an appropriate group, such as the nitro group, is inserted in one of the two ester functions of the 1,4-dihydropyridine structure, the resulting two enantiomers display opposite pharmacological profile as in Bay K 8644, in which the (-)-S-antipode is a

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potent agonist at L-type Ca2+-channels while the (+)-R-antipode is a weak antagonist [177]. Ca2+-agonists are positive inotropic agents so they are potentially useful for the treatment of the congestive heart failure (CHF), but their capacity to increase Ca2+ levels in vascular smooth muscle represent a limit because it leads to vasoconstriction. The combination of these 1,4-DHPs able to activate L-type Ca2+channels with a NO-donor moiety (2nitrooxyethyl esters, furoxanes or diazeniumdiolates) (Fig. 10) could represent a strategy to overcome this adverse effect and therefore to realize a new class of hybrid compounds which show a positive inotropism devoid of vasoconstriction [178-181].

NO2 H3COOC

COOCH3

H 3C

N H

CH3

Nifedipine

R

R

OCH2

N N N

H3COOC

O

O

OCH2

COOCH3

H 3C

N H

H3COOC

COOCH3

H 3C

CH3

NO-releasing-nifedipine analogue meta series

N

N H

CH3

NO-releasing-nifedipine analogue ortho series

O

CF3 NO2

O N H Bay K8644

R O

O

O NO2

O

N N

O2NO

O

CF3 NO2

O N H

N H NO-releasing Bay K8644-analogues

Fig. (10). Chemical structures of the dihydropyridines nifedipine and Bay K8644 (respectively, calcium antagonist and calcium-agonist), and of their NO-releasing analogues. When the NOdihydropyridines are metabolised, lead to new compounds.

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NICORANDIL Nicorandil, N-(2-hydroxyethyl)-nicotinamide nitrate ester (Fig. 11), is an hybrid antianginal drug that possesses the characteristics of both an ATP-sensitive potassium channel (KATP) opener and a NO-donor. Nicorandil exerts its vasodilating effect through a dual mechanism of action: as a nitrovasodilator it activates the guanylate cyclase increasing the cGMP formation and these events lead to a relaxation of vascular smooth muscle. As a KATP opener nicorandil determines the hyperpolarisation of the surface membrane which causes the closure of voltage-dependent ion channels and the reduction of free intracellular calcium ions and even this pathway leads to a vasodilatory effect. Due to its KATP opener profile, nicorandil dilates peripheral and coronary resistence arterioles while the nitrate moiety allows it to dilate systemic veins and epicardial coronary arteries. So nicorandil increases coronary blood flow, reduces preload and afterload [182,183] and exerts an anti-anginal effect comparable with the nitroglycerin one [184,185]. However nitrate therapy might induce the development of tolerance, increase vascular sensitivity to vasoconstrictors [186] and deteriorate endothelial function [187]. Indeed nicorandil, due to its dual mechanism of action, is able to determine vasorelaxation even in nitrate-tolerant blood vessel through the opening of KATP channel [184]. H N

N

ONO2 O Nicorandil

Fig. (11). Chemical structure of nicorandil. This KATP-activator shows a nitrooxy group, conferring the NO-donor property, as an additive pharmacodynamic feature.

Moreover the clinical trial “Impact of Nicorandil in Angina” (IONA study) showed that nicorandil improves the prognosis of patients with stable angina pectoris by reducing the frequency of acute coronary syndrome (ACS) [188]. Recent studies seem to suggest that this reduction is possible because nicorandil may be able to inhibit intracoronary thrombus formation through modification of the type-1 plasminogen activator inhibitor (PAI-1) [189]. In last years, several studies demonstrated that the activation of mitochondrial KATP (mito KATP) channel by diazoxide is able to inhibit apoptosis induced by oxidative stress in cardiac myocytes; the same studies found that also nicorandil exerts an anti-apoptotic action through the activation of mitoKATP [190,191]. More recent experimental findings, obtained through the coapplication of the mitoKATP channel antagonist 5-hydroxydecanoate (5-HD) and of the inhibitor of soluble guanylate cyclase ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), on cultured myocytes treated with hydrogen peroxide and nicorandil, seems to indicate that also the nitrate-like effect contributes to the inhibition by nicorandil of apoptosis induced by oxidative stress in cardiac myocytes [192]. Finally, nicorandil is known as a cardioprotective agent acting in the ischemic preconditioning through the opening of mitoKATP channels, however the exact mechanism of action is still unclear, probably involving several components [193,194]. For example, a key role seems to emerge for NO which, according to recent data, selectively activates mitoKATP channels through the protein kinase C (PKC) translocation from the cytosolic to the mitochondrial fraction [195]. The pivotal role of PKC, in cardioprotection upon ischemiareperfusion has been reported [196-198]. Therefore, probably nicorandil exerts its anti-

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ischemic effect through synergistic mitoKATP channel opening generated by a direct effect, together with NO-donor activation of PKC; this dual mechanism of action could explain the greater effectiveness of nicorandil in comparison with other pure KATP channel openers [195]. NO-ACE INHIBITORS ACE inhibitors, such as captopril, enalapril etc. represent a first-choice class of drugs in the pharmacotherapy of hypertension and congestive heart failure: they act on renin angiotensin system (RAS) preventing the conversion of angiotensin I to angiotensin II (AII) by the Angiotensin Converting Enzyme (ACE). In last years, in order to potentiate the ACE inhibitors properties, a S-nitrosylated derivative of captopril, S-nitrosocaptopril (Fig. 12), was synthetised and characterised [199]. Its NO-donor and ACE inhibiting actions emerge after the homolytic cleavage of the S-NO bond, under physiological conditions [200]. The pharmacological properties of SNOcap have been described in systemic vessels, in vitro [201,202] and in vivo [203,204]. Due to the dual mechanism of action, S-nitrosocaptopril is effective in hypertension, platelet aggregation, congestive heart failure and pulmonary hypertension, potentiating the effect of the native captopril [205]. This improved mechanism of action is due not only to the sum of the two components. Recently, Ackermann et al. showed that NO and NO-releasing substances can competitively inhibit ACE [206] and this finding was confirmed by other studies on porcine iliac arteries [205] and on rats pulmonary artery [205], comparing SNOcap with the parent drug, captopril. Finally, the toxicity of SNOcap was examined in rodents: the absence of adverse effects in subchronic toxicity studies makes SNOcap a good candidate for further clinical trials, but it could cause a severe hypotension when overdosed [208]. CH3

CH3 O

S

O

SH

NO N

N

COOH

Captopril

COOH

S-nitroso-Captopril

COOEt CH3 N

COOEt CH3

N H

N

O

N H

O O O

Enalapril

O(CH2)3ONO2

OH NO-Enalapril (NCX-899)

Fig. (12). Chemical structures of the ACE-inhibitor drugs captopril and enalapril and of their NOreleasing hybrids. In such hybrids, the removal of the NO-donor moieties leads to the original “native” drugs, without any structural alteration.

More recently, a new compound, NCX 899, a NO-releasing derivative of enalapril (Fig. 12) was synthetised and characterised, evaluating its action on cardiomyopathic hamsters with heart failure. NCX 899 is a slow NO-donor and the cleavage of the native molecule from the NO-moiety is due to the action of esterases. If compared with enalapril alone, NCX

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899, appears more effective in enhancing vascular effects, increasing left ventricular contractility and preventing unfavourable remodeling, consistently with a vascular delivery of exogenous NO which determines an improvement in endothelial function and a reduction of vascular resistance [209]. Moreover, in a recent study, the pharmacokinetics and pharmacodynamics of NCX 899 was evaluated in male beagles: the results showed that NCX 899 presented pharmacokinetic characteristics similar to the native drug enalapril and it maintained the ACE-inhibiting profile but it was also effective in protecting against the raise of arterial blood pressure and the concomitant bradycardia induced by an i.v. administration of the NO-synthase inhibitor, LNAME [210]. NO-SARTANS In the thriving panorama of the cardiovascular NO-releasing drugs, a new class of potential antihypertensive agents, NO-Sartans, (pharmacodynamic hybrids which show the dual profile of a typical AT1-antagonist and of a slow NO-donor) has been recently reported [211]. Like ACE-inhibitors, sartans, such as Losartan, act on the RAS, not interfering with the ACE, in fact their mechanism of action consists in the antagonism of the angiotensin II receptor AT1. Sartans block the angiotensin II action in a potentially more complete way than ACE-inhibitors, as it is known that besides ACE, some other enzymes exist which determine the angiotensin II production [212]. Moreover, sartans do not prevent ACE from hydrolysing bradykinin, a kinine able to stimulate the endothelial nitric oxide release but also to act on the cough center and/or nucleus [213,214]. This represents an advantage in comparison with ACE-inhibitors, because sartans reduce the incidence of bradykinin-induced coughing; but for the same reason they are lacking of NO-mediated effects. Thereby, the development of a new class of hybrids in which appropriate NO-donor moieties of aromatic or aliphatic nature, are added to the “native” losartan molecule (Fig. 13), conferring NO-mediated but bradykininindependent additional properties, seems to be a winning idea. In a recent study, the synthesis and a preliminary pharmacological characterisation of these dual molecules were presented. These two pioneers nitroderivatives of losartan (bringing an aromatic or an aliphatic linker), were submitted to a pharmacological evaluation which showed that both the NOsartans exhibited AT1-antagonist and NO-releasing properties. In particular, these two compounds induced full vasorelaxing effects which were strongly inhibited by ODQ, as expected in the case of an NO release effect and were able to induce a parallel rightward shift of the concentration-contractile response curves for angiotensin II, with antagonist potencies comparable to that exhibited by losartan. Moreover, a further in vivo investigation, on the aromatic derivative showed that its antihypertensive action was not negatively influenced by the structural modification, since its antihypertensive effects were comparable to those exhibited by losartan and captopril, in spontaneously hypertensive rats (SHR). As concerns the pharmacokinetic profile, it has been suggested that the NO-release from the aromatic derivative is the first step and precedes the hydrolysis of the ester link between losartan and the aromatic NO-donor moiety [211]. After a preliminary work, in which we reported two prototypical NO-sartans, possessing the characteristics of AT1-antagonist and “slow NO-donor”, our group focuses on the modulation of the rate of NO release from NO-sartans in order to strengthen the anti-hypertensive activity of the native drug and to ensure additional effects, such as the

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antiplatelet and anti-ischemic ones. In order to obtain a collection of NO-sartans, showing different rates of NO release, new NO-donor moieties have been linked to losartan (Fig. 13) or its active metabolite (EXP 3174) (Fig. 14). In this study all the synthesised compounds exhibited again both AT1-antagonist and NO-mediated vasorelaxing properties, with a wide range of NO-releasing rates. Further Cl N O2NO N

N

N

NH N

Losartan

Cl

O2NO

N O

Cl

O2NO N

N

O

N

NH

N

O

N

N

N

O

N

NH N

Cl

O2NO

O2NO

N N

Cl

O N

N

O

N

N

NH

O

N

N

N

O

N

NH N

O2NO Cl N O

Cl

O2NO N

N

O

N

N

NH

O

N

N

N

O

N

NH N

Cl

O2NO

Cl

N O O

N N

N N

NH N

O2NO N

N N

NH N

Fig. (13). Chemical structure of the AT1-antagonist losartan and of its NO-releasing hybrids. The hydrolytic cleavage of the linkers (carrying the NO-donor moiety) leads to the release of the “native” drug.

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pharmacological investigations confirmed that hybrids possess, on SHR, anti-hypertensive and cardiac anti-hypertrophic effects similar to those of the reference AT1-blocking or ACE-inhibiting drugs. On an experimental model of cardiac ischemia-reperfusion, the ischemic injury was not reduced by losartan, while an NO-releasing derivative of losartan induced a marked decrease of ischemic damage [215]. Moreover NO-sartans showed also a significant anti-platelet property both on rat [215] and on human platelet rich plasma (unpublished data). Cl N HO N

N O

NH

N

N

EXP 3174

Cl O2NO Cl

O2NO

N

O2NO

Cl N

O

N

O

N

O N

N O

N

O

NH

N

N O

N

N

NH

N N

NH N

N

Fig. (14). Series of new dual molecules in which EXP 3174 is linked to different molecular portions bearing a nitric ester moiety.

N N

N O

N

C

OH

Telmisartan

N N

N N

O

C

O

N

ONO2

Fig. (15). Molecular structure of the NO-releasing derivative of telmisartan.

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Later a chinese research group carried out the synthesis and the pharmacological characterisation of a NO-releasing derivative of another AT1-antagonist such as telmisartan. The new hybrid drug, named WB1106, is composed of telmisartan as native molecule and a pyridinic linker bringing the nitrooxy group conjugated by an esteric bond between the carboxylic group of telmisartan and the linker hydroxylic group (Fig. 15). The same vasorelaxing protocol carried out for our original NO-sartans indicates the WB1106 presents an efficacy value similar to its losartan-based analogue and a potency index almost in the micromolar range. Even for this compound the in vivo anti-hypertensive activity has been evaluated on SHR by the tail-cuff method using the native sartan and an ACE-inhibitor as reference drug and even WB1106 like our pioneer NO-sartans caused a lowering of systolic blood pressure values of SHR, leading these values near to the typical systolic blood pressure of a normotensive rat. Moreover, WB1106, in contrast to equimolar telmisartan, significantly attenuates body weight gains and improves glucose tolerance in high-fat and carbohydrate-fed rats [216]. NO-ANTIDIABETIC DRUGS Diabetes mellitus is a multifactorial disease associated with a number of microvascular (retinopathy, neuropathy, and nephropathy) and macrovascular (ischemic heart disease, cerebrovascular disease and peripheral vascular diseases) complications [217-219]. During the development of diabetes several biochemical (oxidative stress, modified low-density lipoprotein cholesterol, impaired NO production and chronic inflammation) and mechanical (low shear stress and hypertension) factors converge against the endothelium, resulting in endothelial dysfunction and vascular inflammation. So all these harmful stimuli associated with, and derived from, the state of insulin resistance and type 2 diabetes, markedly enhance endothelial dysfunction, which determines a NO reduced biosynthesis by endothelium providing the pathophysiological basis for a great cardiovascular risk [220]. On the basis of these remarks, our group worked on the design, the synthesis and the pharmacological evaluation of a new class of hybrid molecules, coupling the hypoglycaemic activity of an insulin secretagogue, with the several beneficial properties of NO such as the vasorelaxing, anti-platelet/anti-thrombotic and cardioprotective activities. In order to obtain an hybrid molecule in which the NO-donor moiety is conjugated to the native drug by an in vivo easily hydrolisable bond such as the esteric one, 4-transhydroxyglibenclamide (Gli-OH), active metabolite of glibenclamide (an insulin secretagogue widely used for therapy of type 2 diabetes) was selected because of the presence of an alcoholic hydroxy group, useful for the esteric bond (Fig. 16). The two nitrooxy-derivatives of the active metabolite of glibenclamide (4-transhydroxy-glibenclamide) underwent pharmacological studies aimed to demonstrate additional vasorelaxing NO-mediated effects conferred through the conjunction of NO-donor moiety. At the same time, these new hybrid molecules were evaluated by other experimental protocols on human pancreatic islets aimed to verify the preservation of the insulinotropic response. These preliminary studies showed that the nitrooxy-derivatives of glibenclamide, like glibenclamide and Gli-OH possess, on human islets, insulinotropic effects comparable to that exhibited by glibenclamide. Furthermore, these hybrid drugs showed NO releasing properties (not shown by glibenclamide and Gli-OH), which can be considered as a

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complementary pharmacodynamic feature, of potentially great usefulness for cardiovascular complications associated with the diabetic status such as endothelial dysfunction [221, 222]. Cl

H N OCH3 O

H N

S O

H N

O O

Glibenclamide

Cl

H N H N

OCH3 O

H N

S O

O O OH

4-trans-hydroxy-glibenclamide

Cl Cl H N

H N H N

OCH3 O

S O

H N

OCH3 O

H N

O

O

H N

S

O O O

O

O

O O

ONO2

ONO2

Fig. (16). Molecular structures of reference drugs such as glibenclamide and 4-trans-hydroxyglibenclamide and of the new hybrid compounds.

CONCLUSION In these last years, a progressively increasing interest of medicinal chemistry towards the project of pharmacodynamic hybrids led to a relatively wide availability of “bi-functional” drugs, in which a “native” molecule (already showing a given pharmacological pattern) has been linked with a NO-donor moiety. There are two main aims, representing the rational basis of such a strategy: the reduction of side effects and/or the improvement of the overall pharmacological effectiveness. As concerns NO-releasing hybrids of cardiovascular drugs, this latter aspect could be achieved by conferring complementary effects to a “native” drug (for example, the adding of NO-mediated vasorelaxing activity to a
1-blocker, which, per se, can not be considered a vasodilator) or by strengthening the main pharmacodynamic profile of the “native” drug (for example, the dual vasorelaxing activities of NO-calcium antagonists and nicorandil, which could lead to a synergistic, or at least additive, effectiveness). In the other hand, it should be remarked that the adding of NO-mediated biological properties can not be viewed only as a beneficial contribution, but it could confer an increase of the side effects typical of the “native” drug, such as an exacerbation of the hypotensive response. Furthermore, excessive levels of NO are known to cause direct

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toxic effects, through its interactions with enzymes (such as cytochrome c oxidase or ribonucleotide reductase), [223, 224] and to react with reactive oxygen species, generating “aggressive” agents (such as peroxinitrite, for example) responsible for a wide indirect cytotoxicity of NO [225]. Therefore, even if the qualitatively and quantitatively significant presence of the two mechanisms of action represents, itself, the necessary and implicit condition for a bifunctional pharmacodynamic hybrid, this original strategy imposes a fundamental problem: the correct balance of the two pharmacodynamic properties. In other words, the release of NO must be accurately balanced with respect to the other mechanism of action, in order to strengthen the vasorelaxing activity of the “native” drug and/or to add positive cardiovascular effects (such as the antiplatelet and anti-ischemic ones), without exasperating side effects, such as an excessive hypotensive response and, mainly, without causing an “overdose” of NO. In our opinion, this above aspect, i.e. the ideal balancing, represents the most challenging issue for the medicinal chemists and pharmacologists, working on the design, synthesis, characterisation and development of NO-releasing pharmacodynamic hybrids. REFERENCES [1] [2] [3] [4] [5] [6]

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Trends in the Development of New Drugs for Treatment of Benign Prostatic Hyperplasia Katarzyna Kulig and Barbara Malawska* Department of Physicochemical Drug Analysis, Jagiellonian University Medical College, Medyczna 9 str., 30-688 Kraków, Poland Abstract: Benign prostatic hyperplasia (BPH) is a common condition in aging men that is characterized by nonmalignant enlargement of the prostate gland, and is frequently accompanied by urinary obstruction, and lower urinary tract symptoms (LUST). Currently pharmacotherapy of BPH is based on two classes of drugs:
1-adrenoceptor (
1-AR) antagonists and 5
-reductase inhibitors. It has been shown that
1-AR antagonists reduce symptom scores and increase peak urinary flow rates in BPH. Of particular importance for BPH therapy are uroselective
1-AR antagonists for which the hypotensive related side-effect caused by
1-AR blockade is reduced. 5
-Reductase inhibitors reduce prostate volume and symptom scores, while increasing peak urinary flow rates. This review describes new
1-AR antagonists and 5
-reductase inhibitors in the treatment of BPH and is updating paper published in Current Medicinal Chemistry (Katarzyna Kulig & Barbara Malawska, 2006, 13, 3395-3416). The new
1-AR antagonists represent various structures such as quinazolines, phenylethylamines, piperidines, and arylpiperazines. 5
-Reductase inhibitors are classified into two groups: steroidal and non-steroidal. The newer non-steroidal inhibitors include derivatives of benzo[c]quinolizinones, benzo[f]quinolonones, piperidones and carboxylic acids. Besides the development of new compounds belonging to the above mentioned groups, new agents for BPH treatment are sought among combined 5
-reductase/
1-AR inhibitors, endothelins, androgen receptors antagonists, growth factors, estrogens and phosphodiesterase isoenzymes as well as several phytomedicines, used for prevention and treatment of prostate disorders. These new agents can be used for the design of future targets and development of new drugs in the treatment of BPH. The discovery of a number of active leads may also ultimately help in developing new safe and effective drugs.

Keywords: Benign prostatic hyperplasia,
1-adrenoceptor antagonists, 5
-reductase inhibitors. INTRODUCTION Benign prostatic hypertrophy (BPH), alternatively called benign prostatic hyperplasia, is a significant medical problem in the male population. BPH is a progressive enlargement of *Corresponding author: Tel: ++ 48 12 620 54 64; Fax: ++ 48 12 657 02 62; E-mail: [email protected] Allen B. Reitz / Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.

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the prostate gland, characterized by symptoms of bladder outlet obstruction in men that affects the patient’s quality of life. The prevalence of histologically identifiable BPH for 60 year old males is greater than 50%, and nearly 100% in men aged 80 years or older, although symptoms are not always present. Despite several hypotheses, the etiology of BPH remains unknown [1,2]. Up until a decade or so ago, the choice of treatment for BPH was largely one of watchful waiting, transurethral resection of the prostate (TURP) or open prostatectomy. Although most agree that surgery remains the most effective treatment for complicated or severe symptomatic BPH, its invasive nature and potential side effects have led to the search for nonsurgical alternatives. A number of medical strategies have been attempted to treat BPH, but only two of them have achieved widespread acceptance, namely: inhibition of sympathetic tone in the prostate and bladder neck and suppression of androgen stimulation of prostatic growth. The symptomatic improvement produced by these drugs makes it possible to delay or eliminate surgery [3,4]. The first evidence of pharmacological treatment of BPH dates back to biblical times. In those days the balm of Glieard (or Mecca balsam), a yellowish aromatic myrthlike resin from Arabian trees was used [5]. Nowadays, various phytomedicines are used for prevention and treatment of BPH. Their mechanisms of action, long-term efficacy and safety profiles are subjects of many investigations and comparisons involving traditional forms of treatment. More detailed information on phytomedicines used in the therapy of BPH is presented in other reviews [6,7]. Currently, the pharmacotherapy of BPH is based on the two classes of compounds: 1adrenoceptors (1-AR) antagonists and 5-reductase inhibitors. 1-AR antagonists are used to treat the dynamic component of BPH, while 5-reductase (5-R) inhibitors are administered to reduce the prostatic mass. Introduction into therapy of BPH the above mentioned compounds significantly reduced the prevalence of surgical treatment. The symptomatic improvement produced by these compounds makes possible to delay or eliminate surgery. Besides these two groups of compounds, new agents for BPH are sought among endothelins-receptor antagonists, androgen receptors antagonists, growth factors, estrogens and phosphodiesterase inhibitors, vitamin D analogs and antiproliferative strategies [2]. New 1AR antagonists as well as 5 -reductase inhibitors for the treatment of BPH have been reviewed during recent years [8-11]. The aim of this review is to illustrate trends in the development of new drugs for treatment of BPH, taking into consideration the compounds currently used and those at various stages of pharmacological investigation. 
ADRENERGIC RECEPTOR ANTAGONISTS Receptors for adrenaline and noradrenaline designated as adrenoceptors (AR), have been studied for almost a century, and have provided important targets for many drugs. The adrenergic receptor belongs to the superfamily of G-protein coupled receptors which transduce signals across the cell membrane, and are classified into three principal families: 1, 2 and . To date, -AR has been characterized as 1A-, 1B- and 1D and possesses high affinity for prazosin (Fig. (1)). The 1-AR antagonists act on the dynamic component of bladder outlet obstruction, presumably by affecting the contraction of prostate and urethral smooth muscle produced by sympathetic nerve stimulation [12-14]. It has been shown that the 1A-AR subtype is the predominant receptor involved in human prostate physiology, and consequently 1A-AR antagonists are an effective group for the treatment of benign prostatic hyperplasia. Additionally, recent data suggest that the acti-

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vation of the
1A-AR subtype may be responsible for ischemia-induced cardiac arrhythmia. Recently, the role of the
1B-AR subtype in the regulation of blood pressure has been researched, whereas potential therapeutic use of the
1D-AR subtype has not been firmly established. Some evidence suggests that
1D-AR may play a role in the control of blood pressure because of their involvement in the contraction of vessels. Due to the
1D-AR being predominant in the destrusor muscle, their relevant role in the control of the symptoms associated with BPH is also postulated [15]. A number of
1 selective antagonists representing different structural classes of compounds was disclosed during the last decade. These include: quinazolines, phenylethylamines, piperidines, arylpiperazines, and others [16-18]. In this review, recent development of
1 selective antagonists in the treatment of BPH is presented. O O N

N

MeO

N

N

MeO

O

N

O

N

N MeO

N MeO

Terazosin

Prazosin

NH2

NH2

O O N

MeO

H N

N

N

O

N MeO NH2

O

Alfuzosin

MeO

N

N

O

N MeO NH2

Doxazosin

Fig. (1).

Quinazolines Derivatives of quinazoline such as prazosin, doxazosin, terazosin and alfuzosin are among the oldest
1-AR antagonists used in clinical practice Fig. (1). The structure of these compounds differs through a fragment attached to the quinazoline 2-side chain. Doxazosin, terazosin and alfuzosin are chiral compounds. Although it was found that their enantiomers differ in the affinity for
1-AR versus
2-AR receptors (enantiomer (R) has shown greater selectivity), these compounds are marketed as racemic mixtures [8]. Prazosin, the prototype
1-AR antagonist does not distinguish between these
1-AR subtypes [pKi 9.7 (
1a), 9.6 (
1b) and 9.5 (
1d)]. Due to its relatively short half-life this compound has to be administered twice a day which is not ideal for BPH, where long-term sustained effects are required. Being non-selective, the
1-AR antagonist prazosin may also induce (by antagonizing vascular
1B-AR) orthostatic hypertension which often manifests itself as dizziness and/or headache. However, the influence of prazosin on
1B-AR may prove beneficial in patients suffering from both hypertension and BPH. Like prazosin, doxazosin [pKi 8.5 (
1a), 9.0 (
1b) and 8.4 (
1d)] and terazosin [pKi 8.2 (
1a), 8.7 (
1b) and 8.6 (
1d)] are non-selective
1-AR antagonists, and due to their affinity for
1B-AR and
1D-AR the subtypes these drugs can also cause orthostatic hypertension. In contrast to prazosin, doxazosin and terazosin are longer-acting, with elimination half-lives of 12 – 20 h “Fig. (1)”.

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Alfuzosin is another quinazoline derivative used for BPH treatment. It has been shown in a study performed on human-cloned
1-AR that alfuzosin is a non-selective
1-AR [pKi 8.0 (
1a), 8.0 (
1b) and 8.5 (
1d)], but its functional assay and experimental animal model have indicated that it may be a uroselective compound. Similarly to prazosin, alfuzosin has a relativly short half-life [18]. The observation that modifying both piperazine and furan rings of prazosin may afford antagonists that are able to differentiate among
1-AR subtypes was the basis for further investigations within this group “Fig. (2)”. The replacement of the furan ring of prazosin by various substituted phenyl groups has yielded several active compounds [19]. Derivative (1) with a substitution (at the 3 position of the phenyl group by a CHO moiety) displayed good
1A/
1D versus
1B affinity profile [pKb 7.79 (
1A), 6.92 (
1B) and 8.07 (
1D)] and could be a promising drug for BPH treatment [20]. It was also found that the replacement of the piperazine ring of prazosin with
, -alkanediamine yields compounds which are able to differentiate among
1-AR subtypes. This result was the basis for further modification of the structure of these analogues in an attempt to improve their selectivity [20]. Based on these investigations several hybrid structures which combine both prazosin and benextramine were synthesized. Among the obtained compounds, the most promising were derivatives (2) [pKb 8.17 (
1A), 7.22 (
1B) and 8.47 (
1D)] and (3) [pKb 8.48 (
1A), 7.41 (
1B) and 9.30 (
1D)] which may serve as prototypes for the design of competitive
1A/
1D-AR antagonists, a selectivity profile postulated to be of use in BPH treatment due to actions on both prostate and bladder [20]. NH2 MeO

MeO

N N

N

N CHO (1)

O

NH2 MeO

MeO

N N

N

N O

H N

N H

(2)

2

NH2 MeO

MeO

N N

N H N

N

N H O (3)

Fig. (2).

2

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With the aim of widening the biological profile of prazosin-related compounds, further modifications leading to molecules with multiple biological activities were undertaken. In order to obtain compounds endowed with both 1-AR antagonist and antioxidant properties the fuoryl moiety of prazosin was replaced with lipoyl fragments of lipoic acid, its lower homologues or naphthoquinone. The choice of lipoic acid and its homologues was dictated by the observation that lipoic acid is known as a universal antioxidant. Among the synthesized compounds, derivative (4) [pKi 9.54 (1a), 9.51 (1b) and 9.41 (1d)] in which the piperazinyl-quinazoline nucleus of prazosin was linked to 1,2-dithiolanemethylcarbonyl, displayed an affinity towards all 1-AR subtypes comparable to its parent compound, and no remarkable subtype selectivity was observed. The highest potency towards 1d-AR subtype and a 20-fold increase in selectivity for 1d-AR relative to 1a-AR was displayed by compound (5) [pKi 9.09 (1a), 9.27 (1b) and 9.31 (1d)], in which the 1,2-dithiolane ring was directly connected to the carbonyl group. A comparable biological activity and selectivity profile was displayed by naphthoquinone derivative (6) [pKi 8.90 (1a), 9.17 (1b) and 9.03 (1d)]. Additionally, compound (6) showed the highest antiproliferative and antioxidant effects [21] “Fig. (3)”. NH2

NH2 MeO

MeO

N

N

MeO

N

MeO

N N

(4) n = 1 (5) n = 0

N

O N

S n O

N

S

(6) O

Fig. (3).

Phenethylamines Phenethylamines, typified by tamsulosin and silodosin can be described as the most closely related compounds to endogenous agonist noradrenalin (from a structural point of view) Fig. (4). Tamsulosin shows selectivity for 1a-AR and 1d-AR over 1b-AR [pKi 9.70 (1a), 8.90 (1b) and 9.80 (1d)], which means it is effective in BPH treatment without reducing blood pressure and for orthostatic hypotension. Tamsulosin is a chiral compound and it was found that stereochemistry at the methyl-bearing carbon atom has a significant impact on its pharmacological profile. (S) (+) Tamsulosin displays a greater level of subtype selectivity, albeit with at least 10-fold reduction in potency compared with its (R)(-) counterpart. Additionally, its relatively long half-life time (5-10 hours) enables it to be administered just once a day, which is beneficial in BPH [22]. The indoline derivative silodosin is a drug which is structurally related to tamsulosin. The trifluoroethyl group in silodosin has been inserted presumably to block O-dealkylation, a primary metabolic pathway of tamsulosin. Like tamsulosin, its (R) enantiomer is a more potent 1a-AR antagonist. Silodosin binds to the cloned human 1a
AR with pKi equal to 10.44; its affinity to 1b-AR (pKi = 7.68) and 1d-AR (pKi = 8.70) is 583- and 56-fold lower (respectively). This compound is in a pre-registered phase in Japan for the treatment of dysuria associated with BPH Additionally, silodosin have no effect on heart rate or on the electrocardiogram “Fig. (4)”. The cardiovascular profile of silodosin suggests therefore that it is a safe and safe and well-tolerated drug [22-25].

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H N O H2N

O

H N

S

O

O O

N

CF3

O MeO

HO

O

NH2

Silodosin

Tamsulosin

Fig. (4).

In the search for new
1A-AR antagonists among phenethylamines several compounds have been synthesized in which the linker between the aromatic ring and the basic amine is truncated (JTH-601) or extruded (WB-4101) “Fig. (5)”. The first one, JHT-601, maintains an excellent level of binding selectivity [(pKi 9.4 (
1a), 8.9 (
1b) and 8.9 (
1d)] and has shown higher selectivity for canine prostate both in vitro and in vivo. Being a selective
1aAR antagonist this compound is expected to be a drug for the treatment of urinary outlet obstruction caused by benign prostatic hypertrophy with a minimum effect on the cardiovascular system [8, 26]. The second compound, WB-4101 [pKi 9.8 (
1a), 8.6 (
1b) and 9.6 (
1d)] has become a lead molecule, and has been the subject of intensive investigations aimed at identifying its pharmacological features, and improving its affinity and selectivity. As a result, a variety of analogues have been studied and characterized for their affinity for cloned human
1-AR subtypes. Replacing the 2,6-dimethoxyphenyl substituent of WB-4101 with orto-methoxy-1-naphthyl leads to compound (7), whose enantiomers [(S)-(7) (pKi 8.80 (
1a) 7.80, (
1b) and 8.18 (
1d)) and (R)-(7) (pKi 8.34 (
1a), 7.07 (
1b) and 7.65 (
1d))] have lower affinity for
1a-AR than the parent compound, but with a more specific binding profile. Similarly, replacing the benzodioxane moiety with naphthodioxane or tetrahydronaphthodioxane leads to compounds (8) and (9), whose (S) enantiomers [(S)-(8) (pKi 7.47 (
1a), 6.05 (
1b) and 6.38 (
1d); (S)-(9) ((pKi 7.60 (
1a), 6.24 (
1b) and 6.47 (
1d))] display good
1a-AR selectivity with respect to the other two subtypes of AR and 5-HT1A receptors [27] “Fig. (6)”. Other detailed investigations of WB-4101 analogues have shown that the two oxygen atoms of benzodioxane play different roles in the binding with the receptor. The oxygen atom at position 1 seems to contribute to receptor binding, and replacing it with a carbonyl group does not modify the binding profile of the obtained compound, while substituting a sulfur atom or a methylene group results in significant decrease in potency. On the other hand, the oxygen atom at position 4 plays a structural role without significant importance in receptor affinity or binding profile [28, 29]. OMe O

O N OH OH

JHT-601

O

MeO H N O WB 4101

OMe

Fig. (5).

Taking into consideration the above facts, several modifications have been proposed. The insertion of a phenyl ring or a para-tolyl moiety at the 3-position trans relationship with the 2-side chain results in phendioxan or mephendioxan derivatives “Fig. (7)”. These compounds display high affinity for native
1A-AR relative to both
1B-AR and
1D-AR subtypes. The expansion of these studies has led to several compounds in which the oxygen

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atom at position 4 of benzodioxan was replaced by a phenylmethine group, and the resulting compound was the subject of further modifications, such as the exchange of the oxygen atom at position 1 with a sulfur atom, a carbonyl group or a methylene unit. Such investigations have led to compound (10) [pKi 10.30 (
1a), 10.40 (
1b) and 10.05 (
1d)] which is nonselective in binding assays but which has proven a potent and selective
1D-AR antagonist in a functional study (relative to
1B-AR), and may be a useful lead for the design of more selective ligands for
1D-AR “Fig. (6)” [28]. OMe O

*

O

O

O

N H (7)

*

O N H MeO

O

MeO

(8) OMe OMe

O *

O

O

N H

O

MeO

N H MeO

O

(10)

(9) O N H

O

MeO

O

N H

O

O

O (12)

(11)

Fig. (6).

Further work in the search for new
-AR antagonists among structures related to WB4101 has led to several 2,2-diphenyl-1,3-dioxolanyl derivatives. The most interesting compounds in this group, (11) [pKi 7.61 (
1a), 7.34 (
1b) and 8.03 (
1d)] and (12) [pKi 7.43 (
1a), 7.20 (
1b) and 7.94 (
1d)], exhibit high affinity and selectivity profile, and can at least be used in functional studies of the
1d-AR “Fig. (6)” [29]. Considering that the stereochemically defined insertion of a phenyl ring in position 3 of WB-4101 (phendioxan) or in position 4 of its metylene bioisostere was resulted in significant modulation of
1-AR subtype selectivity, several cis and trans 5- or 6- substituted 1,4dioxane derivatives were synthesized “Fig. (7)”. The results obtained showed that the 6 substituted 1,4-dioxane nucleus is a suitable scaffold for selective
1D-AR antagonist. The most interesting results were obtained with compound (13) whose highest pKb value of 8.32 for
1D-AR subtype was not significantly different from WB-4101 [pKb 9.51 (
1A-AR), 8.16 (
1B-AR), 8.80 (
1D-AR)] but in contrast to parent compounds was
1D-AR selective [pKb 6.65 (
1A-AR), 6.86 (
1B-AR)] “Fig. (8)” [28, 30]. Piperidines Indoramin [pKi 8.4 (
1a), 7.4 (
1b) and 6.8 (
1d)] belongs to the first generation of piperidine-based
1-AR antagonists used in therapy of BPH. Interaction of indoramin with other receptors, in particular with serotonin and histamine causes sedation, its major side effect. The major challenge in this group was the synthesis new
1-AR antagonists in which

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poly-pharmacology displayed by indoramin would be reduced [8] “Fig. (9)”. The replacement of the terminal indol group of indoramine by a phenyl ketone yielded the secondgeneration compound SNAP-1069 [pKi 7.8 (
1a), 6.7 (
1b) and 6.1 (
1d)]. SNAP-1069 displayed a 10-fold increase in selectivity for
1a-AR over
1b-AR and
1d-AR but its disadvantage is a broad spectrum of activities [8].

O

O

MeO H N

O

O

MeO H N

O

O

OMe

OMe Mephendioxan

Phendioxan

Fig. (7). O

O

H N

O OMe

(13)

Fig. (8). H N N

N

O

O

O N H

N H Indoramin

SNAP-1069

Fig. (9).

Further work involving the piperidine class has focused on the calcium channel blocker (S)(+) niguldipine [pKi 9.8 (
1a), 7.26 (
1b) and 7.00 (
1d)]. It was found that niguldipine is a potent antagonist for
1-AR with a 100-fold selectivity increase for
1a-AR versus the other
1-AR subtypes. Several modifications yielded compound (14) [pKi 9.63 (
1a), 7.87 (
1b) and 7.18 (
1d)] with preserved potency and selectivity for
1a-AR but attenuated calcium channel activity. One significant drawback of compound (14), however, is its poor oral bioavailability in rats (5%), which may be due to the oxidative metabolic conversion of the dihydropyridine moiety into a pyridine [8, 31] “Fig. (10)”. In order to reduce such oxidative metabolism, and to improve the pharmacokinetic profile, several compounds in which the dihydropyridine ring was replaced by dihydropyrimidinone or dihydropyridine have been synthesized [31-35]. It has been observed that the introduction of a dihydropyrimidinone ring into the molecule offers two logical sites for attaching the piperidine-containing side chain. The dihydropyrimidinone derivative (15) [pKi 9.30 (
1a), 7.55 (
1b) and 7 (
1d)], containing a 4-methoxycarbonyl-4-phenylpiperidine moiety in the side chain, displays better binding affinity than its dihydropyridine analogue [31]. Further systematic modifications of (15) have led to the discovery of SNAP-6201 [pKi 9.70 (
1a), 6.59 (
1b) and 6.46 (
1d)]. This compound showed high affinity and selectivity

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for the
1a-AR subtype. In the anesthetized dog model, SNAP-6201 showed 40-fold higher selectivity for reducing the inhibited intraurethal pressure versus diastolic blood pressure [35] “Fig. (11)”. In vitro human liver microsomes and in vivo rat and dog investigations of SNAP-6201 have enabled its metabolic pathway to be defined. It was found that the predominant (> 50%) metabolic pathway led to formation of an inactive
1a-AR antagonist which showed negligible cross-reactivity at several other G-protein coupled receptor, and Ltype calcium channel activity compound (16), and 4-methoxycarbonyl-4-phenylpiperidine (17). Metabolite (17) was found to be an μ-opioid agonist, and it is also a close analogue of the well-known μ-opioid meperidine. Having a long plasma half-life (>12 h) in rats and dogs, compound 17 may lead to opioid agonist liabilities upon chronic administration of SNAP-6201 [35] “Fig. (12)”. NO2 NO2

O

O

O

O

N

N H

N

O

O O N H

N H (14)

(S)(+) Niguldipine

Fig. (10). F

NO2

F

O

O

O

O

N N H

H2N

N

N H

O N N H

O O

(15)

N

N H O

O

O

O

SNAP-6201

Fig. (11). F F

F F O

O

O

O +

O H2N

H2N

N N H

N H

N N H

O O SNAP-6201

N H

N O

HN

OH (16)

O

O

O

O

O N

Meperidine

Fig. (12).

(17)

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The focus of investigation within this research was on minimizing the metabolic formation of compound (17). This was achieved by modification of the linker and replacement of the piperidine portion with other piperidines which do not exhibit μ-opioid activity. Replacement of the 4-methoxycarbonyl group of piperidine with a cyano or methyl group yields compounds (18) [pKi 9.15 (
1a), 6.21 (
1b) and 5.89 (
1d)] and (19) [pKi 9.30 (
1a), 6.36 (
1b) and 6.19 (
1d)] (essentially inactive at the μ-opioid receptor), which are selective (>880-fold)
1a-AR antagonists over
1b-AR and
1d-AR subtypes, and show good selectivity over several other recombinant human G-protein-coupled receptors. They also show good functional potency in isolated human prostate tissues. In addition, compound (18) exhibited good uroselectivity in in vivo experiments in dogs, similar to SNAP-6201 [35] “Fig. (13)”. F

F F F O

O H2N

N

N H

N N H

O O

N

O (18)

O

N

N H O

N

N H O (19)

Fig. (13).

Success of the above modifications suggests that the exact structure of the central heterocycle is not critical, and that another mode of attachment of the piperidine-containing side chain via amide bond formation of dihydropyrimidinedione C-5 carboxylate might also provide a potent and selective compound. This strategy was based on the lead structure (20), which has pKi values of 8.57, 6.27 and 5.82 versus
1a-AR,
1b-AR and
1d-AR, respectively. Many of these compounds have been synthesized and evaluated in vivo. It has also been found that they are more potent than terazosin in both the rat model of prostate, and the dog model of intra-urethral pressure, without significant effect on blood pressure. The most promising was compound (21) [pKi 9.62 (
1a), 7.39 (
1b) and 6.59 (
1d)], which was selective for
1a-AR over
1b-AR and
1d-AR. This compound was found to have adequate bioavailability (>20 %) and half-life (>6h) in both rats and dogs, and has the potential to relieve the symptoms of BPH without eliciting effects on the cardiovascular system [32] “Fig. (14)”. Enhanced binding affinity within the group of dihydropyrimidinones was also obtained by restriction of the flexibility of the linker between the piperidine and dihydropyrimidinone heterocycles. This strategy resulted in preparation of several cyclopentan derivatives having sub-nanomolar potency and selectivity for
1a-AR. The highest level of activity was displayed by compound (22) [pKi 9.62 (
1a), 6.88 (
1b) and 7.79 (
1d)] [36]. A second possibility for optimizing pharmacokinetic parameters displayed by dihydropyridine derivatives involves substituting a dihydropyrimidine template. The obtained compounds exhibit good binding affinity (< 1 nM) and selectivity (> 300- fold) for
1a-AR over
1b-AR,
1d-AR and
2-AR. Most of them also displayed negligible affinity for the rat Ltype calcium channel. Of most interest in this series was compound (23), whose (+) enantiomer displayed [pKi 9.40 (
1a), 7.00 (
1b) and 6.74 (
1d)] a good binding profile. It also exhibited 100 fold selectivity for
1a-AR over
1b-AR,
1d-AR, and
2-AR, and 1000 fold se-

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lectivity over the rat L-type calcium channel. In functional assays compound (23) displayed high binding affinity for the isolated human prostate (pKi 9.48) and much lower binding affinity for the isolated human aorta (pKi 6.96) [37]. F NO2

F

O O

O N H

N N

N H

N N H

O

O

O

N H

O

O

O

F

(21)

(20)

F

F F

O

F

O

O

NH

N

O N H

N

N

O

N

N

O N H

O

N

(23)

(22)

Fig. (14).

It was also found that the oxazolidinone derivatives constitute a new subgroup of
1AAR antagonists. The most active member of this group is SNAP-7915 [pKi 9.77 (
1A), 6.92 (
1B) and 6.91 (
1D)]. Investigation of the four possible diastereomers indicated that only SNAP-7915 ((+) trans) binds to
1A-AR with the highest affinity and selectivity in radioligand binding. This compound was also found to be a potent antagonist in a number of in vitro and in vivo functional assays, and exhibited long plasma half-life (6h in rats and >12 h in dogs), as well as good oral bioavailabitity (25% in rats and 74% in dogs). These results led to singling out SNAP-7915 for further study [38, 39] “Fig. (15)”. F F

O N

N H

N

O O SNAP-7915

Fig. (15).

F

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Another potential modification within the 4-aryldihydropyridine-containing subgroup of the
1a-AR antagonist was based on assessment of the structural components housed within compound (24) [pKi 8.44 (
1a), 6.15 (
1b) and 4.96 (
1d)]. It was found that metabolic liabilities associated with this structure could be reduced by replacing 4aryldihydropyridine with dihydrocinnamic acid, without altering the remaining core structure “Fig. (16)”. As a result, a series of phenylacetamides has been identified as potent and selective
1a-AR antagonists. In contrast to the known 4-aryl dihydropyridine-containing
1a-AR antagonists, this group of compounds has lower molecular weight and exhibits an improved in vitro pharmacokinetic profile. Among the obtained compounds the most interesting one was the (+) enantiomer of (25) [pKi 8.70 (
1a), 5.85 (
1b) and 5.68 (
1d)], which exhibit high selectivity for
1a-AR, and acceptable pharmacokinetics [40] “Fig. (17)”. NO2

O

N H

N

O

O

O

N

X

N H

Ar

N H

(24)

Ar

O

O

Fig. (16). O

O

F N

N H

N

N

F O N

N

(25)

(26)

Fig. (17).

The results obtained within the research summarized have become a starting point for further investigations aiming at improving their pharmacokinetic properties. It was decided that this goal could be achieved by incorporation of pyrolidin-2,5-dione into the molecule. The synthesized cyclic imides, exemplified by compound (26) [pKi 8.77 (
1a), 5.72 (
1b) and 5.82 (
1d)], show 1100 and 880 fold selectivity versus
1b–AR and
1d-AR subtypes respectively. It has also been shown that their pharmacokinetic profile is modestly improved in comparison to the precursor phenylacetamides “Fig. (17)” [41]. In order to find
1a–AR/
1d–AR selective antagonist as new drugs for the treatment of BPH/LUTS a series of (phenylpiperidinyl)cyclohexylsulfonamides was designed. The compounds obtained (27-31) showed equal affinity for both
1a-AR and
1d-AR adrenoceptor subtypes, with very good selectivity over the
1b-AR subtype (Table 1) “Fig. (18)”. It was found that the displayed by then selectivity profile provides a great improvement over the commercial drug tamsulosine [42].

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Table 1.

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Binding Profiles of Compounds (27-31) (Ki [nM]) [42]

Compound

Configuration

Ki
1a [nM]

Ki
1b [nM]

Ki
1d [nM]

cis

2.2

144

0.77

trans

28

148

40

cis

3.0

470

7.3

trans

1.4

1385

87

cis

0.91

141

2.0

trans

11

133

34

cis

1.8

123

2.2

trans

21

368

23

cis

3.3

299

1.6

trans

43

1487

53

27

28

29

30

31

O O

O O N

O

R

O

S

N

S

OMe

NH

NH

OMe (29)

(27) 3,4-diMeO (28) 3,4-diF

F3C

O O

O

O O N

S

O OMe

N

NH

S

OMe

NH OMe

OMe

F (31)

(30)

Fig. (18).

Arylpiperazines Arylpiperazine represents the pharmacophore moiety with 5-HT1A/
1 selectivity modulated by the type and position of the substituent in the arylpiperazine moiety, by the nature of the second heterocyclic system and by the length of the spacer connecting these two elements. 5-Methylurapidyl [pKi 9.20 (
1a), 7.40 (
1b) and 8.0 (
1d)] is classified into the first generation of
1-AR antagonists among arylpiperazines “Fig. (19)”. A significant drawback connected with this compound is its affinity for the 5-HT1A receptor [8, 43]. In the search for new
1-AR antagonists several 3-(4-arylpiperazin-1-yl-alkyl)-uracils have been synthesized. The obtained compounds exhibited affinity for
1A-AR subtypes,

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with considerable selectivity over 1B–AR and 1D-AR in both radioligand binding and functional studies. The most active compound (32) [pKi 8.9 (1A), 7.1 (1B), 7.2 (1D)] has a similar in vitro profile in human and dog microsomes. Additionally, its metabolite (33) [pKi 8.3 (1A), 6.6 (1B), 6.9 (1D)] also showed nanomolar activity for 1
AR and 30 fold selectivity for 1B-AR and 1D-AR subtypes. Pharmacokinetic studies of (32) indicated that this compound has good bioavailability (over 75%). The half-life of compound (33) is four times longer than for (32) (10 – 11 h versus 2.5 – 2.8 h). Compounds (32) and (33) demonstrated uroselectivity in in vivo preclinical studies (intraurethal pressure induced by hypogastric nerve stimulation and phenylephrine-induced diastolic blood pressure). The results of this study have led to clinical trials of compound (32) for treatment of symptomatic BPH. However, development of this compound was discontinued due to the lack of clinically significant symptomatic improvements [44] “Fig. (19)”. F

F

CF3

O

N H N

N

O

O N

H N

O

N N

N

O

N

N OH O

O

O

H OH N H

O

N

N

5-Methylurapidyl

CF3

(33)

(32)

Fig. (19).

Another strategy in search for new 1-AR antagonists was the synthesis of B8805-033, whose structure combines two known 1-ARs; 5-methylurapidyl and flesinoxan [pKi 6.68 (1A), 5.54 (1B) and 5.94 (1D)]. In contrast to its parent compound, B8805-033 [pKi 7.71 (1A), 5.16 (1B) and 5.49 (1D)] is an 1-AR antagonist with up to 1000-fold functional selectivity for 1A-AR over 1B-AR and 1D-AR. The obtained results indicated that this compound may not only merit further functional investigations of 1-ARs, but also serve as a useful drug for the treatment of BPH [45] “Fig (20)”. O F OH H N O

N

O

O N

OH

O

H

N

N

N H

O N

O

Flesinoxan B8805-033

Fig. (20).

In the course of studies in the field of new 1-AR ligands a series of pyrrolo[3,2d]pyrimidine-2,4-diones was obtained. The most interesting compounds in this group, (34) [pKi 9.34 (1)] and (35) [pKi 7.93 (1)] and (36) [pKi 7.76 (1)], are characterized by 1-AR affinity and an outstanding selectivity over the serotoninergic 5-HT1A and dopaminergic D1 and D2 receptors. These compounds were selected for an additional study aiming at determination of their functional effects and affinity for the 1-AR subtype [46] “Fig. (21)”. Based on these investigations newer 1-AR ligands containing 1H-pyrrolo[2,3d]pyrimidine-2,4(3H, 7H)-dion have been synthesized. Among the obtained compounds derivatives (37) [pKb 7.25 (1A), 7.50 (1B) and 7.78 (1D)], (38) [pKb 7.48 (1A), 8.03 (1B) and 7.69 (1D)] and (39) [(pKb 6.41 (1A), 6.64 (1B) and 7.66 (1D)] were selective for 1-

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AR over 5-HT1A and dopaminergic D1 and D2 receptors. In particular, compound (39), endowed with affinity in the nanomolar range for
1-ARs, showed the best profile in terms of selectivity toward other tested receptors, and, in functional assays, a preference for
1DAR [47] “Fig. (22)”. H N

H N

O Cl

Cl

N

N H

N

O

N

N H

Cl

O

N

Cl

O

N

(34)

N

(35)

H N

O N

N H

N

Cl

O

N (36)

Fig. (21).

O

N N

N N H

N H

O Cl

N

O O

N H

(37)

O

N H

N H

N H

O

Cl

(38)

N N

N

Cl

N N

O

Cl

(39)

Fig. (22).

Naftopidil is an
1-AR antagonist which acts more selectively on the lower urinary tract than on blood vessels, and it has been marketed in Japan since 1999 for the treatment of symptomatic BPH. This compound shows high affinity for the
1A-AR and
1D-AR subtypes, with predominant affinity for the later. A comparison of naftopidil, (11), and (12) prompted to design of compound (40) which could be considered as a rigid analogue of naftopidil. “Fig. (23)”. The compound (40) shows the highest affinity toward
1a–AR and
1d–AR adrenoceptor subtypes (pKi
1a–AR = 9.58, pKi
1d–AR = 9.09) and selectivity over 5-HT1A receptors (
1a–AR/5-HT1A = 100,
1d–AR/ 5-HT1A = 26). In functional experiments it behaves as a potent competitive
1a–AR and
1d–AR adrenoceptor antagonist (pKb
1a–AR = 8.24; pKb
1d–AR = 8.14), whereas at 5-HT1A receptor is a potent partial agonist (pD2 = 8.30) [48].

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MeO MeO

O N N

HO

O

N N

O

Naftopidil

(40) R

O H N

O

O

(11) R=OMe (12) R=H

Fig. (23).

A number of studies from different group [49-51] have demonstrated that compounds with an open-chain linker between arylpiperazinyl and isoindole-1,3-dione-2-yl fragment binds to
1-AR with high affinity but have very limited subtype selectivity. It was found that bridging siter 1 and 4 of n-butyl linker in compounds such as NAN-190 (Ki [nM] 0.26 (
1a), 0.96 (
1b), 0.30 (
1d)) that is, the incorporation of a cyclohexyl ring into such compounds, will retain binding affinity and improve selectivity among the three
1-AR subtypes. The most interesting binding profile for a compounds belonging to a series of 1arylpiperazinyl-4-cyclohexylamine derived isoinole-1,3-diones was displayed by compound (41) (Ki [nM] 2.7 (
1a), 1082 (
1b), 0.30 (
1d)) [52] “Fig. (24)”. O

O

O

O Cl

N

N

N

N

N

N Cl

O NAN-190

O (41)

Fig. (24).

New compounds for the treatment of BPH/LUTS which characterize improved metabolic stability was found in the series of (arylpiperazinyl)-cyclohexylsulfonamides. Among these compounds several selective ligands for the
1a-AR and
1d-AR (cis-42, cis43, and cis-44) [Ki [nM] (cis-42) 1.2 (
1a), 86 (
1b), 0.35 (
1d); Ki [nM] (cis-43) 1.6 (
1a), 109 (
1b), 1.0 (
1d); Ki [nM] (cis-44) 1.0 (
1a), 78 (
1b), 0.75 (
1d)] was found. Additionally it is worth to note that compound cis-42 was also stable against HML metabolism [53, 54]. “Fig. (25)”. As a continuation of above presented results several (phenylpiperazinyl)cyclohexylureas were prepared [55]. The binding affinities and selectivities of these compounds were evaluated in cloned human
1a-AR,
1b-AR and
1d-AR adrenergic receptor subtypes. It was found several compounds shoved equally potent affinity for both
1a-AR and
1d-AR adrenoceptor sybtypes. Two of these compounds (45 and 46) [Ki [nM] (trans45) 5.1 (
1a), 71 (
1b), 1.5 (
1d); Ki [nM] (trans-46) 3.6 (
1a), 60 (
1b), 1.6 (
1d)] also had 14- to 46-fold selectivity versus the
1a-AR. “Fig. (26)” Although from the perspective of
1a/1d-AR antagonism, urea analogues are not as selective as earlier developed sulfonamide

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analogues their unique configuration-activity-relationship opens the door to design new
1AR antagonist pharmacophore [55]. F3C O

O O N

N

O

S

O

OMe

O

NH

N

N

S

OMe

NH

OMe

OMe

(42)

(43) Cl O

O O N

N

S NH MeO

(44)

Fig. (25).

O

O

O

O

NH N

N

NH N

NH

N

NH

Cl

F

(45)

(46)

F

Fig. (26).

MISCELLANEOUS STRUCTURES A structurally novel series of
1-AR antagonists possesses a benz[e]isoindole unit attached to a pyrimidinedione heterocycle via an alkyl chain. 6-Methoxy substitution on the benz[e]isoindole portion, R, R stereochemistry, and a two-carbon linker were found to be optimal for
1-AR. Compound (47) [pKi 9.16 (
1a), 7.45 (
1b) and 8.42 (
1d)] showed the highest selectivity in the radioligand binding assays (50-fold) and in in vitro functional studies (40-fold), as well as uroselectivity (3200-fold). Being both an
1a-AR and an
1d-AR, compound (47) has the potential to not only improve the objective symptoms of BPH such as urinary flow rate but to also to alleviate the subjective symptoms through antagonism of the
1D-AR receptor at the level of the bladder smooth muscle [56] “Fig. (27)”. H O

H

O O

N

N

N O (47)

N H

N

O

N

H (48) Cl

Fig. (27).

O

S

N

H

N

O

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Further investigations within this group have shown that the 6-methoxybenz[e]isoindole unit can also be attached to a variety of bicyclic heterocycles via a two-carbon alkyl chain. A derivative of 6,7-dimethoxyquinazoline-4-on, i.e. compound (48) [pKi 9.57 (
1A), 7.81 (
1B) and 8.11 (
1D)], showed the highest selectivity in radioligand binding assays (57-fold), in in vitro functional studies (80-fold) and in in vivo prostate selectivity (almost 1000-fold) [57]. Nitric oxide NO
is an important biological messenger that elicits a wide range of physiological effects on the cardiovascular system, central and peripheral nervous system, and the immune system. Based on this, hybrid compounds which join the structure of the REC15/2739 uroselective
1-AR antagonist with an NO
donor have been designed. Thus, compound (49) [pKi 9.14 (
1a), 8.19 (
1b) and 8.41 (
1d)] is able to relax tissue contracted by NA because of its
1-AR antagonist activity and its abilities to release NO under experimental conditions. The results of these findings might be of interest for further in vivo studies on their potentialities in the treatment of BPH [58] “Fig. (28)”.

N H N

O O

N

O

N

O N+ ON

(49)

Fig. (28).

INHIBITORS OF 5
-REDUCTASE Enzyme Mechanism Benign prostatic hyperplasia is the most important pathology related to abnormal formation of 5
-dihydrotestosterone (DHT). Other androgen-dependent disorders and diseases where DHT is implicated include prostate cancer, acne and androgenic alopecia in men and hirsutism in women. DHT is produced by the reduction of testosterone (T) under catalysis of the steroid 5
-reductase (5
-R; EC 1.3.99.5) which is a membrane-bound, NADPHdependent enzyme [59]. Steroid 5
-reductase is a system of two isoenzymes (5
R-1 and 5
R-2) which catalyzes the selective, irreversible reduction of 4-ene-3-oxosteroids to the corresponding 5
-3-oxosteroids “Fig. (29)”. These two isozymes of 5
-reductase have been cloned, characterized and localized in different organs and tissues. The type 2 isozyme have been found mainly in the prostate, genital skin, seminal vesicles and in the dermal papilla, while the type 1 isozyme have been found in non-genital skin and hair follicles. Therefore, inhibitors of 5
-reductase can be used as drugs lowering the DHT level, and selective inhibitors of type 2 which reduce high DHT levels in the prostate. This might be a promising strategy for the treatment of prostate diseases [60]. More recently with development of genome-wide expression profile analyses a third type of 5
-reductase enzyme (type 3) has been identified in a hormone-refractory prostate cancer cells (HRPC). This enzyme also converts T to DHL in HRPC cells in a similar way

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to type 1 enzyme. Type 3 isoenzyme has been recognized as a ubiquitos enzyme in mammals [61, 62]. R

R

5
R NADPH

O

O H 5
-H 3-oxosteroid

4-ene-3-oxosteroid

O

HO

O

OH

OH

OH

testosterone

H

H (50)

DHT

the intermediate enolate

Fig. (29).

The kinetic mechanism of testosterone reduction presented in “Fig. (30)” involves the formation of a complex between the enzyme and NADPH, followed by the formation of a complex with the substrate testosterone. There are three types of inhibitors interacting with different enzyme complexes. Type A inhibitors interact with the free enzyme and are competitive with the cofactor (NADPH) and the substrate (T). Type B inhibitors interact with the enzyme-NADPH complex and are competitive with the substrate. Type C inhibitors interact preferentially with the enzyme-NADPH + complex and are generally uncompetitive versus the substrate. NADPH

T

E . NADPH

Enzyme

E . NADPH T

H+

Inhibitor (I) E. I (type A)

NADP+

DHT

E . NADPH I (type B, competitive vs. T)

E . NADPH+.

DHT

E . NADP+

Enzyme

Inhibitor (I) E . NADP+.

I

(type C, uncompetitive vs. T)

Fig. (30).

The design of new 5
-R inhibitors of type B is based on the concept of transition state (TS) of the enzymatic process. The enzyme binding should be greater for compounds that mimic the transition state of the enzymatic process, and thus result in higher inhibition. Accordingly, two possible transition states have been postulated: the “substrate-like” TS in which the hybridization of C-3, C-4 and C-5 are similar to those of the intermediate, and the “product-like” TS in which the hybridization of C-3, C-4 and C-5 are similar to those of the enol form of DHT [60] “Fig. (31)”.

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dTestosterone

Kulig and Malawska

E

HO

E+

O

DHT

H H+ NADPH

+ + E

+

O

E

O H

O

H

H2N

O

H

H

H2N N

+ N

R

R

"Substrate like" TS

"Product like" TS

Fig. (31).

Steroidal 5-Reductase Inhibitors 5-Reductase inhibitors have been studied for over 25 years as potential drugs which might be used in the treatment of diseases where DHT is implicated [63]. These compounds can be classified into two groups: steroidal and non-steroidal inhibitors. A number of steroidal inhibitors of 5-reductase have been identified and their SAR studies have been presented earlier [8-10]. It has been shown, that the following elements are essential in the steroidal pharmacophore: the key A-ring lactam which acts as a transition state mimic of the intermediate enolate (50), and the lipophilic C17-substituent, which significantly enhances potency via binding at a pocket of a receptor [8]. The structure of steroidal inhibitors was based on the testosterone skeleton, which has been modified:
by the introduction of a nitrogen atom in the A ring (4-azasteroids), in the B ring (6azasteroids), and at position 10 (10-azasteroids);
by the introduction of a double bond into the above described structures. Inhibitors with a 4-azasteroid structure are competitve versus testosterone and similar to the DHT enol; they also mimic the “product-like” TS. Inhibitors with a 6-azasteroid structure are competitive inhibitors similar to 10-azasteroid, which possess an enone structure in the A ring and act as “substrate-like” TS analogs “Fig. (32)”. In the 4-azasteroid series the first potent inhibitor of human 5-R with in vivo efficacy was finasteride. Finasteride and other close structural analogs are slow-offset, essentially irreversible inhibitors “Fig. (33)”. The reduction of the
1 A-ring of finasteride to an enolate and subsequent alkylation by NADP+ has led to the formation of a very stable and potent enzyme-NADP-dihydrofinasteride adduct. This complex might act as a potent bi-substrate inhibitor, and therefore finasteride possesses good in vitro and in vivo activity. Finasteride is

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the first potent discovered inhibitor of 5-reductase of type 2 with only weak in vitro activity versus the 5-reductase type 1 isozyme, which has been marketed for the treatment of BPH. It is a time-dependent inhibitor of the type 2 and type 1. The kinetic studies indicated that, the interactions between finasteride and 5
R isozymes are processes conducted by the two-step mechanism. The first step describing by the inhibition constant Ki is rapid and the second step describing by the inhibition constant k3 is slow. The kinetic parameters for the inhibition of 5-R by finasteride are as follows: value pKi = 6.44 and 7.16 for type 1 and type 2 5-R respectively; the rate constant k3/Ki of 4 x 103 M-1s-1and 3 x 105 M-1s-1 (pH 7.0, 37oC) for type 1 and type 2 5-R, respectively [64]. Many clinical studies have shown that finasteride (at a 5 mg/day dosage) caused a 65-80 % lowering of plasma DHT levels; however it has only shown moderate clinical efficacy in BPH patients [8]. R2

R2

E+

O

N R1

E

H

N+

O

R1

H "Product like" TS analogue

4-azasteroid

R2

R2

E+ O

E

N

N+

O

R1

R1

"Substrate like" TS analogue

6-azasteroid

R2

R2

N

E+

N+ E

O 10-azasteroid

O "Substrate like" TS analogue

Fig. (32).

Later studies indicated the presence of a small amount of 5-R-1 in the prostate gland, and therefore the use of dual inhibitors was developed as a new hypothesis for better reduction in DHT levels. This resulted in discovery and market introduction of a new, very potent dual inhibitor, dutasteride, which was approved by FDA in 2002 for the treatment of BPH “Fig. (33)” [65]. Discovery and clinical development of dutasteride have been reviewed very recently by Stephen Frye [66]. Dutasteride, like finasteride, is a derivative of the 4azasteroidal skeleton with the most lipophilic 2,5-difluorophenyl substituent in the amide fragment in the C-17 position. The kinetic parameters for the inhibition of 5-R by dutasteride are as follows: value pKi = 8.22 and 8.15 for type 1 and type 2 5-R respectively;

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the rate constant k3/Ki of 1.8 x 105 M-1s-1and 6.8 x 105M-1s-1 (pH 7.0, 37oC) for type 1 and type 2 5
-R respectively. These data show that dutasteride is 60-fold more potent than finasteride in its initial Ki versus type 1 5
-R and about 5-fold more rapid in inhibition the enzyme. This drug is able to reduce the serum DHT concentration by about 90% and to reduce the total gland size by 25% [67]. Clinical studies confirmed the hypothesis that the nearly total reduction in DHT levels through inhibition of 5
-R isozymes, can prevent the progression of BPH, and thus dutasteride is safe and effective in reducing prostate volume and associated symptoms, improving flow rates and reducing the risk of acute urinary retention. Clinical 4-year trials in men with BPH showed that dutasteride is well tolerated during long-term use for the treatment of symptomatic BPH [68]. O

O

H N

H N

CF3

F3C O

O

N

N H

H Finasteride

Dutasteride

Fig. (33).

The both 5
-R inhibitors, finasteride and dutasteride have been shown to reduce the risk of acute urinary retention and BPH-related surgery, to reduce significantly prostate volume, therefore these drugs are more effective amongst men with enlarged prostates (>/ 30 ml) [69, 70]. The role of 5
-R inhibitors in the chemoprevention of prostate cancer has been also studied. Finasteride prevented or delayed the onset of cancer, however also increased risk of high-grade prostate cancer. The role of dutasteride as a chemopreventive agent is under investigation [71]. In the 10-azasteroid series the most potent in vitro inhibition of human 5
-reductases was observed for 17-(N-tert-butyl)carbamoyl derivative (51), which is a dual inhibitor toward type I (IC50 = 127 nM) and type II (IC50 = 37 nM) isoenzymes [72]. Preliminary studies performed on 10-azasteroids have excluded any time dependence in inhibition activity of these compounds [59, 72]. Structural modification of compound (51) has led to a new series of different 17-[N-(phenyl)methyl/phenyl-amido]-substituted 10-azasteroids, as three pairs of 5
-H/5-H epimers. These compounds were less active than 17-carbamoyl-substituted 10-azasteroid (51); however, compounds (52) (IC50 – 279 and 2000 nM towards isoenzyme I and II respectively) and (53) (IC50 = 913 and 247 nM towards isoenzymes I and II respectively) possess dual inhibitory activity. It is interesting to note that in the same series, 5-H isomers were more potent than the corresponding 5
-H compounds. Two active compounds (52) and (53) represent the potent aza-steroidal inhibitors of the 5
-reductase enzyme, with 5-H stereochemistry “Fig. (34)”. These results indicate that the presence of a substituent at C-17 can modulate selectivity toward 5
-reductase of type I and II in the steroidal skeleton [73]. As a consequence of the important observation that progesterone and deoxycortisone inhibit the synthesis of dihydrotestosterone by competing with 4-en-3-one function of the testostene for 5
-reductase enzyme, several of 4- and 6-halo-progesterone analogues were obtained (54, 55). These compounds were found to be potent antiandrogenic when tested

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against gonadectomized hamster seminal vehicles and were also found to be inhibitors of 5
-reductase [74]. “Fig. (35)”. O

R

H N

N

N

N

O

(52) R = H (53) R = -CO-Ph

O

(51) D9(11)/ D9(8), 9:1

H

Fig. (34). O

O

F

O

O X (54) R=OCOCH3; X=Cl (55) R=OCOCH3; X=Br

O

O

OR

X (56)

Fig. (35).

A range of 4-bromo-17-substituted-4-pregnane-3,20-diones were also synthesized and evaluated as 5
-reductase inhibitors on gonadectomized hamster seminal vesicle and flank organ. Small diameter of the pigmented flank organ and graet reduction in the weight of seminal vesicle has been found with compound having p-fluorobenzyloxy group (56) indicating that presence of more electronegative substituent at C-17 and halogen at C-4 enhances the androgenic activity as well as 5
-reductase inhibitory activity [75] “Fig. (35)”. Further promising 5
-reductase inhibitors were also found among pregnan derivatives (57-63). The compounds obtained exhibited much higher 5
-reductase inhibitory activity in hamster prostate [IC50 [nM] 0.065 (57); 0.06 (58); 0.92 (59); 0.06 (60); 3.0 (61); 3.5 (62); 3.2 (63)], than finasteride (IC50 8.5 nM). Although these steroidal derivatives have different functional groups, they all have one fragment in common, which is
, -unsaturated C-20carbonyl group. It is postulated that their inhibition of enzyme 5
-reductase is bades on the Michael type addition reaction of the enzyme 5
-reductase to the steroidal enone or dienone. The results from this study indicated that the first step in the inhibition of the enzyme 5
-reductase consists in the formation of an enzyme-steroid activated complex. In a subsequent step, the nucleophilic portion of the enzyme (amino group) attacks the conjugated double bond of the steroid in the Michael type addition reaction to form an irreversible adducts. This concept explains very well the high 5
-reductase inhibitory activity of the steroidal derivatives (57-63) which are Michael acceptors [76, 77] “Fig. (36)”. Potent 5
-reductase inhibitors were obtained by dienone derivatives (64), which found to have high 5
-reductase inhibitory activity showing IC50 value of 0.5nM [78] “Fig. (37)”.

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O

O

O

O O

O

O O

O

O

(57)

(59)

(58)

O

O

O

O O

F

O O

O

X

(60)

(61) X=F (62) X=Br (63) X=Cl

Fig. (36).

Lately, a novel class of inhibitors for human and hamster steroid 5
-reductase was proposed for steroida carbamates (65). In comparison to “classical esters” these compounds have a longer half-life and are hydrolyzed slowly. The determined for compound (65) IC50 value 10 nM is similar to that of finasteride [79] “Fig. (37)”. O O H N

O F O

O

O (64)

(65)

O

O H N

O O O X

O

X

O O

(66) (67) (68) (69)

X=Cl X=Br X=I X=H

(70) X=H (71) X=F (72) X=Br

Fig. (37).

Working on similar lines some novel 4,16-pregnadiene-6,20-dione derivatives were synthesized and evaluated as 5
-reductase inhibitors. It has been demonstrated that compounds

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containing chlorine (66), bromine (67), iodine (68) atoms, and without any substituent (69) in the ester moiety at C-3 produce a significant decrease of the prostate weight in castrated animals treated with testosterone. Therefore, it was proposed that the ester moiety at C-3 is functioning as a pharmacophore, enriched by the presence of halogens in these steroidal derivatives leading to the increase in the inhibition of 5
-reductase enzyme. Compounds (67) was found to be the most potent with IC50 value of 1.8 nM while compounds (66) and (68) showed values 14 and 10 nM, respectively. Compound (69) was not that active when compared to halogenated compounds [80] “Fig. (37)”. Recently, several C-6 substituted and unsubstituted pregnane derivatives as potential 5
reductase inhibitors were synthesized. It has been found that steroids that lack of a chlorine atom in C-6 (70-72) exhibited a high capacity for inhibition of the activity of 5
-reductase (IC50 in the range of 25-63 nM) than finasteride [81] “Fig. (37)”. Non-Steroidal Inhibitors Several non-steroidal inhibitors have been discovered during last years. Non-steroidal inhibitors belong to a different class of compounds, and can be classified according to their structure. The structure of non-steroidal inhibitors is also based on the structure of natural substrates, which can be modified:
by removal one or two rings from the (aza)steroid structure;
by replacing a ring with an aromatic one. These modifications enable obtaining different structures of non-steroidal inhibitors such as: a. benzo[c]quinolizinones b. benzo[f]quinolonones, c. piperidones, d. carboxylic acids. In this review, new original compounds are presented. Benzo[c]quinolizinones (73) have been obtained from 10-azasteroids by removing the D ring, and substitution of the C ring with an aromatic one. Non-steroidal inhibitor derivatives of benzo[c]quinolizinones have been studied by Antonio Guarna and co-workers, and many potent and selective 5
-R-1 inhibitors have been reported in this group of compounds [8286]. Upon observation that the use of dual 5
R-1 and 5
R-2 inhibitors is a more effective therapeutic model for completely reducing the circulating DHT, the search for dual inhibitors in the series of benzo[c]quinolizinones has developed. Recently, some potent dual inhibitors of 5
-reductases 1 and 2, based on the benzo[c]quinolizinone structure, with IC50 values ranging between 93 and 166 nM for both isozymes has been reported [86]. The most potent was compound (74) (IC50 values = 93 nM, 5
R-1 and IC50 = 119 nM 5
R-2) having the F atom in the para position of the phenol moiety. A structure-activity relationship (SAR) study indicated that in this series of derivatives of benzo[c]quinolozin-3-ones the presence of the F atom in the ester moiety at position 8 was the most important for inhibition activity “Fig. (38)”. Benzo[f]quinolonones have been obtained from 4-azasteriods by removal of the D ring and substitution of the C ring with an aromatic one. Non-steroidal inhibitor derivatives of benzo[f]quinolonone have been summarized in an earlier review [84]. This group of com-

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pounds can be divided into two series: hexahydro derivatives (75), which possess an unsaturation at position 4a-10a, and octahydro derivatives (76) “Fig. (39)”. F O R O N

N

O

O CH3

CH3

(73)

(74)

Fig. (38). X

Cl

X H

R2

O

N

O

N

R1

R1

(75)

(76)

O H

N H CH3 (77)

Fig. (39).

A SAR study indicated that octahydro derivatives (76) are more potent inhibitors than the corresponding 4a-10a unsaturated compound (75). The most active compound (for inhibition of 5
-reductase of type 1) in this group is derivative (77) (LY 191704) with Cl (chlorine) atom at position 8 and a methyl group at position 4 (IC50 = 8 nM, 5
R-1 in cultured Hs68 human foreskin fibroblast cells). Compound LY 191704 (77) has progressed to human clinical trials as the most potent inhibitor in the series of octahydro-derivatives. Some compounds analogs to (77) have been synthesized, and the role of the B-ring and lactam functionality of the tricyclic analogues has been investigated by A.D. Abell [84, 88]. The obtained results indicate that tricyclic thiolactams (79) (IC50 type 1 = 377 nM, type 2 = 40 μM) and (81) (IC50 type 1 = 183 nM, type 2 = 40 μM) are, like their lactam analogs, selective for type 1 steroid 5
-reductase. However, these thiolactams are less active than the corresponding lactams [(78), IC50 type 1 = 120 nM; (80), IC50 type 1 = 17 nM]. It is also worth noting, that a chloro substituent in the C-ring is favored for type 1 activity in both series (79/82 and 81/83), as is the absence of a double bond in the B-ring (78/80 and 79/81) “Fig. (40)”. Piperidones (Bicyclic Lactams and Thiolactams) Compounds derived from 4-azasteroids are bicyclic lactams “Fig. (41)”. Removal of two rings (the B and D steroidal rings) from 4-azasteroids resulted in piperidones with some potency and selectivity towards 5
R-1. The highest potency was associated with the presence of a chlorine atom on the aromatic ring of (84) (IC50 = 1690 nM towards 5
R-1). The corresponding thiolactam (85) (IC50 = 3360 nM, 5
R-1) was less active than lactam (84). The substituent on the aryl ring in the structure of bicyclic lactams (84) influenced inhibitory potency and selectivity towards 5
R-1 and 5
R-2 isoenzymes. Introduction of a styryl (or aza) substituent enhanced type 2 activity, and also type 1 activity: compounds (86), IC50

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= 320 nM type 1; IC50 = 579 nM type 2) and (87) IC50 = 107 nM type 1, IC50 = 617 nM type 2) proved to be good dual isozyme inhibitors [88]; however these new compounds (78-87) were less active than the steroidal dual inhibitor dutasteride. Cl

Cl

H

S X

N CH3

N H CH3

N

S

CH3

(78) X = O (79) X = S

(82) X = O (83) X = S

(80) X = O (81) X = S

Fig. (40). Cl

O

N

S

N

CH3

CH3

(84)

(85)

N

O

Cl

N

N

Ph

Ph

O

CH3

N CH3

(86) trans

(87)

Fig. (41).

Inhibition against the type 1 isoenzyme was observed for compound (88) (63% inhibition of type 1 activity). It was also found the presence of a long carbon chain enhanced the inhibitory activity of compounds (89) and (90) (43% and 33% inhibition of type 1 activity, respectively). This results provides further evidence for the presence of a lipophilic pocket in the enzyme active site that tolerates bulky hydrophobic groups at the terminus of the inhibitor which is not interacting with residues responsible for the enzyme catalysed reduction [89] “Fig. (42)”. Carboxylic Acids Derivatives of carboxylic acids have been designed as analogs of the steroidal carboxylic acids, possessing inhibitory activity for 5
-reductase. Episteride is the (steroidal) androstenecarboxylic acid, which has demonstrated significant reduction on DHT in animal models of BPH. Episteride is potent versus 5
-R2 (pKi = 9.74), and is a week versus 5
-R1 [8]. It acts as an uncompetitive inhibitor and their steroidal structure mimics the enolate intermediate (50), where the anionic carboxylate ion serves as a mimic for the enolate oxygen in binding to the active site. Clinically it reduces DHT to a lesser extent than finasteride (25%

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at 0.4 mg and 54% at 160 mg) [90]. Therefore many efforts directed toward clinical trials are with finasteride and than with dutasteride. O S

O O

O

N 15 H

n

O

N H

O O

O

O

N H

N (88)

O

N

(89) n=1 (90) n=2

Fig. (42).

Several non-steroidal aryl acids have been obtained by removal of two or more rings compared to parent steroidal compounds. Tricyclic aryl acid (91) has been identified by A.I. Abell [88]; (91) type 1 Ki, app = 26 nM, type 2 gave 20 % inhibition at 10 μM “Fig. (43)”. O NHBu R

O

(91) R = Br OH

Episteride

HOOC

(92) R =

Ph

Fig. (43).

Similarly to lactam (87), introduction of a styryl substituent into the tricyclic aryl acid resulted in compound (92) (IC50 = 152 nM, type1; IC50 = 340 nM type 2), which showed good dual isozyme inhibitory properties with significantly enhanced type 2 activity compared to the acid (91). Other carboxylic steroidal inhibitors of 5-
-reductase with the estradiol skeleton have been discovered earlier by Holt and co-workers [91]. Compound (93) which was a potent type 2 5
-reductase inhibitor with IC50 = 2 nM has provided a template for the design of new non-steroidal inhibitors “Fig. (44)”. D. Lesuisse [92, 93] described a new family of non-steroidal 5-
-reductase inhibitors, obtained by replacing the steroid (skeleton) backbone to estrone with a biphenyl moiety. The schematic structure of these compounds is presented below (94) “Fig. (44)”. Biphenyl derivatives of 4-carboxylic acid, and the 4-carboxamidomethyl group displayed inhibition of 5-
-reductase in the nanomolar range. A SAR study revealed that introduction of substituents such as fluorine and a nitro group in the ortho position in the phenyl ring with an amido group increased the inhibition effect (96, 97). In contrast, monosubstitution at the ortho position of the second aromatic ring had a detrimental effect on inhibition. Compounds (96) and (97) improved inhibition by 10 fold over unsubstituted derivatives (95) (IC50 = 830 nM). These compounds (96) (IC50 = 71 nM) and (97) (IC50 = 9.8 nM displayed better inhibition than the reference finasteride (IC50 = 40 nM).

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O N

HOOC (93)

O R6 R1

N

O N

R5 HOOC

O

R1 X

R3

R2

R2

R4 HOOC

(94)

(95) R1 = R2 = H (96) R1 = R2 = iso-Pr (97) R1 = F, R2 = NO2

R1 - R6: different substituents such as H, Cl, F, Br atom NO2, NH2, CN, alkyl; X = -, CH2, CH(Me), OCH2

O O

HOOC (98)

HOOC (99)

Fig. (44).

R. N. Hartmann discovered several new classes of nonsteroidal inhibitors of 5
-R such as acids, 1-H-quinolin-2-ones, and pyridones [94–101]. In the series of 4’-substituted biphenyl-4-carboxylic acids, the most active compounds towards human type 2 isozyme were the cyclohexyl compound (98) and the dicyclohexylmethyl compound (99), with IC50 values of 0.64 and 0.22 μM, respectively. These compounds showed only marginal activity towards the human type 1 isozyme [98]. N-substituted piperidine-4-(benzylidene-4carboxylic acids) (100) represent derivatives of carboxylic acids as mimics of the steroidal substrate. In contrast to steroidal inhibitors which bear a lipophilic substituent in the 17 position, these acids have the R substituent located outside the plane of the molecule [97] “Fig. (45)”. Among the compounds tested in rats, N-diphenylacetyl (101) (IC50 = 3.44 and 0.37 μM for type 1 and 2, respectively), and N-diphenylcarbamoyl-piperidine-4-(benzylidene-4carboxylic acids) (102) (IC50 = 0.54 and 0.69 μM for type 1 and 2, respectively) displayed the best inhibition towards both isozymes. Compound (103) showed strong inhibition toward type 2 human and rat enzyme (IC50 = 60 and 80 nM) but only moderate activity versus type 1 enzyme (IC50 = approximately 10 μM for rat and human enzyme). A SAR study showed that very bulky and flexible substituents in a position which corresponds to the 17 position of the steroidal substrate are optimal for high activity. These results are in agreement with a hypothesis postulating the existence of a hydrophobic pocket in the active

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site, proposed earlier by others authors [8, 102, 103]. Compounds tested in vivo showed significant inhibition (33-47%) of the prostate weight increase in castrated testosteronetreated rats. However, this in vivo activity (for example 42 % for compound (103)) was not very strong in comparison to finasteride (86 %), on the other hand compound (103) showed oral activity in vivo in rats by reducing the testosterone-induced stimulation of the prostatic weight. Structural modifications of active inhibitors (compounds 101-103) led to the discovery of the most potent inhibitors of isozyme of type 2. In the dicyclohexylacetyl series the introduction of fluorine substituent at the benzene ring (104), exchange of the carboxy group for a carboxymethyl moiety (105), and combination of both structural modifications (106), resulted in highly active inhibitors of the human type 2 izosyme, comparable to the activity of the steroidal reference finasteride (IC50 (nM) values: (104) 11, (105) 6, (106) 7; finasteride 5 respectively) “Fig. (45)”.

R

R

R

N HOOC O

(102)

(101)

(100) N-substituted piperidine-4-(benzylidene- 4-carboxylic acids

X No (103) (104) (109) (110) (111) N ROOC

R

-H

-H

-CH3

X

-H

-F

-H

O

-CH3 -F

-H

(112) -CH3

-OCH3 -OCH3

R

HOOC

N

(105) (106)

R=H R=F

O

Fig. (45).

Molecular modeling studies on non-steroidal inhibitors of 5
reductase generate a hypothetical enzymatic active site [104]. The 3D pharmacophore model of type 2 inhibitors described by Chen et al. [105] using compounds (107) and (108) consists of two hydrogen bond acceptors (HBA1 and HBA2) and three hydrophobic groups (HP1-3) “Fig. (46)”. The results obtained by Hartmann [96] demonstrated that the benzoic acid moiety is a very appropriate mimic of the steroidal A ring. Additionally, the hydrophobic pocket, which also accommodates the substituents in the 17-position of the steroidal inhibitors, is limited in size. The increase in activity observed for the exchange of the carboxy group for a

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carboxymethyl moiety indicated that the protein shows some conformational flexibility in this part of the active site. As continuation of these studies the methyl esters (109, 110, 112) of series of N-substituted 4-benzylidenepiperidine-4’-carboxylic acid (103, 104, 111) were investigated as potential pro-drugs of 5
reductase inhibitors [94]. Carboxylic acids (103, 104 and 111) exhibited good type 2 potency and weak inhibitory potency towards type 1 5
R isoenzyme. Surprisingly, corresponding methyl esters (110 - 112) showed weak-tomediocre inhibitory potencies toward type 2 isoenzyme in the BPH assay and exhibited good-to-excellent type 1 potency “Fig. (45)”. HP2 HBA1

HP3

O O HO

HO O

O

O

(108)

HP1 (107)

HBA2

Fig. (46).

Methyl esters of N-(dicyclohexyl)acetyl-piperidine-4-(benzylidene-4-carboxylic acids) were stable in a buffered salt solution (pH 7.4), Caco-2 cells, and human plasma, whereas all esters were hydrolized into the corresponding acids in benign prostatic hyperplasia tissue homogenate, displaying their 5
R type 2 inhibitory potencies. These properties of esters (109, 110, 112) opened a new therapeutic strategy for dual inhibition of 5
R type 1 and 2 proposed by R.W. Hartmann et al. [101] “Fig. (47)”, Table 2. Ester (hybrid inhibitor)

PERIPHERY (liver, skin) as Drug

TARGET ORGAN (prostate)

as Precursor

good cell pereation

Ester

no hydrolysis

Ester Drug for 5
R1

Fig. (47).

good cell permeation

Ester

hydrolysis

Acid Drug for 5
R2

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Table 2.

Kulig and Malawska

Inhibition of Human 5
R Type 1 and Type 2 methyl ester of N-(dicyclohexyl)acetylpiperidine-4-(benzylidene-4-carboxylic acids) [101] IC50 (nM)

Comp. BPHa Isoenzyme 2 Microsomes

DU145b Isoenzyme 1 Whole Cells

DU145c Isoenzyme 1 Cell Free

103

60

>10,000

>10,000

104

11

>10,000

>10,100

109

>10,000

2000

6860

110

7500

2551

1494

111

130

>10,000

>10,000

112

>10,000

434

206

finasteride

5

41

202

Derivatives of benzoic acid have been reported by Igarashi et al. [106] as potent human 5
R inhibitors (113). Those derivatives were modified by the introduction of alkyl- or arylaminocarbonyl groups (which are also present in the 17 -position of most steroidal inhibitors) into the flexible benzophenone skeleton. In these new 4-(4-alkyl- and phenylaminocarbonyl)benzoyl)benzoic acid derivatives, the most active inhibitor for the human type 2 isozyme was 4-(4-(phenylaminocarboyl)benzoyl)benzoic acid, compound (114) (IC50 = 0.82 μM) [99] “Fig. (48)”. Compounds with an aliphatic (tert-butyl, di-iso-propyl, and dicyclohexyl) aminocarbonyl substituent displayed weak-to-moderate inhibitory activity (13 – 55%), while compound (114) with a phenylaminocarbonyl substituent showed higher inhibitory activity (86% inhibition). This data indicated that an aromatic ring in this position is important for potency. O O H N

HO HOOC O Cl

O (113)

(114)

Fig. (48).

Novels substituted benzoyl benzoic acids and phenylacetic acids were synthesized based on the template structure (99) and were evaluated for the inhibition of rat and human steroid 5
-reductase isozymes 1 and 2. The phenylacetic acid derivatives were more potent than the analogues benzoic acids. Bromianation in the 4-position of the phenoxy moiety led to the strongest inhibitor of the series against human 5
-reductase 2 [(115); IC50 = 5 nM], which was equipotent to finasteride while compounds (116) (IC50 = 23 nM) and (117) (IC50 = 27 nM) were also found to be potent inhibitors against 5
-reductase type 2 [107] “Fig. (49)”.

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341

O R

O HO

O (115) Y=Me, R=H (116) Y=H, R=H (117) Y=H, R=Br

Fig. (49).

Other Structures Compound (118) derivatives with a lactam ring have been reported as mediocre inhibitors of the human type 2 isozyme (IC50 = 13μM) [94]. 6-Arylsubstituted 1-H-quinolin-2ones [95] have been designed as conformationally restricted analogues of lactam (118). The most active inhibitor for the human type 2 izosyme was compound (119) (pKi 6.09), without displaying considerable inhibition of the human type 1 isoform. The N-methyl derivative of compound (22) has been identified as a type 1 selective inhibitor (120) IC50 = 510 nM with loss of type 2 activity. Non-steroidal compounds mimicking the steroidal A and C ring are represented by 5aryl substituted 1-methyl-2-pyridones. In this series the best compound was N(dicyclohaxyl)-4-(1,2-dihydro-1-methyl-2-oxopyrid-5-yl)-benzamide (121), exhibiting an IC50 value for the human-type enzyme of 10 μM. The obtained results confirmed earlier data that the introduction of hydrophobic and bulky substituents, such as dicyclohexyl, would increase inhibitory activity. They also support the hypothesis of the existence of a hydrophobic pocket in the active site of 5
R, as proposed by others “Fig. (50)”. O

O

N

N

O

O

N

N R (119) R = H

(118)

(120) R = CH3

O

O

N CH3

(121)

Fig. (50).

Novel type 1 5
-reductase inhibitors were designed and synthesized by using 3,3diphenylpentane skeleton as a substitute for the usual steroid skeleton. The most potent

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among compounds obtained was 4-(3-(4-N-methylacetamido)phenyl)pentan-3-yl)phenyldibenzylcarbamate (122) which was found to be a competitive 5
-reductase type 1 inhibitor with the IC50 value of 0.84 μM [108] “Fig. (51)”.

O

O N

N (122)

Fig. (51).

COMBINATION THERAPY As hormonally based treatment is aimed at reducing the volume of glandular tissue and adrenoreceptor blockade is aimed at decreasing tone in the stromal and capsular tissue, the concept of combing these agents to obtain increased clinical benefits has obvious appeal. Based on these findings, compound Z-350 (which possesses dual action i.e. that of an
1AR antagonist and of a steroid 5
-reductase inhibitor) has been obtained. Z-350 is a nonselective
1-AR antagonist [pKi 7.82 (
1A), 7.29 (
1B)] with no affinity for 2- and 3-AR, muscarinic, H1 histamine, ET1, ET2 and androgen receptors. It has shown weak dopamine D2, serotonin 5-HT1,
2A-
2B- and 1-AR receptor affinity. Z-350 is a non-competitive inhibitor of rat prostatic steroid 5
-reductase with pIC50 values of 8.42. The obtained results indicate that this compound can be a candidate for treatment of BPH [109] “Fig. (52)”. O

O

N

O

O N O

N

OH

O

N N

O

OH

Z-350 O

Antrasentan (ABT-627)

O

Fig. (52).

ENDOTHELIN-RECEPTOR ANTAGONISTS The discovery of endothelin as the pre-eminent vasoconstrictor has opened new therapeutic strategies for many diseases, including BPH and prostate cancer. Further investigations have shown that endothelin receptor A (ETA) and enothelin receptor B (ETB) are members of the seven-transmembrane G-protein-coupled superfamily. ETA and ETB receptors are both found in the normal prostate. ETA receptor binding sites have been found predominantly in the stromal component of the prostate, whereas ETB binding sites are

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predominant in epithelial luminal cells. Taking the above into consideration, endothelin receptor antagonists became a target for many groups [110]. Based on these findings, Abbott Laboratories developed compound ABT-627 (atrasentan). Atrasentan is a very potent and selective ETA receptor antagonist (pKi = 10.47; 1800fold selectivity increase) blocking the biologic effects of entothelin-1 in a host of in vivo and in vitro systems. In addition, atrasentan inhibited ET-1 mediated biochemical response in the BPH-1 cell line obtained from a 60-year-old BPH patient. In phase II and III clinical trials of atrasentan, disease progression was delayed in some men. The results indicate that this new compound may help convert advanced prostatic cancer to a more chronic disease [111, 112] “Fig. (52)”. Growth Factor Understanding of the precise vascular endothelin growth factor (VEGF) expression and regulation in the human prostate may have implications for potential treatment of BPH and prostate cancer [113]. SUMMARY The presented review summarizes ongoing medicinal chemistry investigations in search for new drugs for treatment of BPH. BPH is a common condition in older men, resulting in chronic lower urinary tract symptoms (LUTS). The resulting symptoms of urinary obstruction are a result of two important components: dynamic and mechanical. The dynamic component of BPH is related to prostatic smooth muscle contraction and depends on
adrenceptors. The second, mechanical component is related to increased prostatic mass, which depends on DHT. Hence,
1-AR antagonists used to treat the dynamic component of BPH, and 5
-reductase inhibitors used to reduce the prostatic mass, have been described. Some of the newer compounds represent structural modifications of pre-existing drugs or active leads. New
1-AR antagonists belong to several different chemical classes such as quinazolines, phenylethylamines, piperidines, arylpiperazines and miscellaneous structures. Inhibitors of 5
-reductase are classified into two groups: steroidal and non-steroidal. During recent years several chemical classes of non-steroidal inhibitors have been discovered, such as derivatives of benzo[c]quinolizinones, benzo[f]quinolonones, piperidones and carboxylic acids. Besides these two most important groups of compounds, new agents for BPH have been sought among endothelin receptor antagonists, androgen receptor antagonists, growth factors, estrogens and phosphodiesterase isoenzymes. These new agents can be used for the design of future targets and development of new drugs in the treatment of BPH. The discovery of a number of active leads may also ultimately help in synthesizing new safe and effective drugs. ABBREVIATIONS BPH

=

Benign prostatic hyperplasia

TURP

=

Transurethral resection of the prostate

LUTS

=

Lower urinary tract symptoms

AR

=

Adrenoceptor

SAR

=

Structure-activity-relationships

HLM

=

Human liver microsomal

HRPC

=

Refrectory prostate cancer cells

344 Frontiers in Medicinal Chemistry, 2010, Vol. 5

DHT

=

5
-dihydrotestosterone

ETA

=

Endothelin receptor A

ETB

=

Endothelin receptor B

VEGF

=

Vascular endothelin growth factor

5-HT

=

5-hydroxytryptamine (serotonin)

T

=

Testosterone

5
-R

=

5
-reductase

Kulig and Malawska

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-reductase: phenoxybenzoic acid derivatives. Chem. Pharm. Bull., 1999, 47, 1073-1080. Salem, O. I. A.; Frotscher, M.; Scherer, C.; Neugebauer, A.; Biemel, K.; Streiber, M.; Maas, R.; Hartmann, R. W. Novel 5
-reductase inhibitors: synthesis, structure-activity studies, and pharmacokinetic profile of phenoxybenzoylphenyl acetic acids. J. Med. Chem., 2006, 49, 748-759. Hosoda, S.; Hashimoto, Y. 3,3-Diphenylpentane skeleton as a steroid skeleton substitute: novel inhibitors of human 5
-reductase. Bioorg. Med. Chem. Lett., 2007, 17, 5414-5418. Fakuda, Y.; Fakuta Y.; Higashino, R.; Ogishima, M.; Yoshida, K.; Tamaki, H.; Takei, M. Z-350, a new chimera compound possesing
1-adrenoceptor antagonistic and steroid 5
-reductase inhibitory actions. Naunym-Schmiedeberg’s Arch Pharmacol., 1999, 359, 433-438. Anderson K-E.; Chapple C.R.; Hfner K. Future drugs for the treatment of benign prostatic hyperplasia. World J. Urol., 2002, 19, 436-442. Ray, A.; Hegde, L.G.; Chugh, A.; Gupta, J. B. Endothelin-receptor antagonists: current and future perspectives. Drug Discov. Today, 2000, 5, 455-464. Nelson, J.B. Endothelin receptor antagonists. World J. Urol., 2005, 23, 19-27. Ravindranath, N.; Wion, D.; Brachet, P.; Djakiew D. Epidermal growth factor modulates the expression of vascular endothelial growth factor inthe human prostate. J. Androl., 2001, 22, 423-443.

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Targeting the Prostaglandin D2 Receptors DP1 and CRTH2 for Treatment of Inflammation Trond Ulven1,* and Evi Kostenis2 1

Department of Physics and Chemistry, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark; 2Institute for Pharmaceutical Biology, University of Bonn, Nussallee 6, 53115 Bonn, Germany Abstract: The involvement of prostaglandin D2 (PGD2) in inflammatory diseases like allergy and asthma is well established, and blocking the effect of this mediator represents and interesting therapeutic approach for the treatment of such diseases. PGD2 is now known to act through two seven-transmembrane (7TM) receptors, DP1 (previously DP) and CRTH2 (DP2), which are also activated by several endogenous metabolites from the arachidonic acid cascade, making the regulatory system highly complex. There has recently been a considerable effort aimed at developing antagonists of the PGD2 receptors for treatment of inflammatory conditions like asthma and rhinitis, and especially CRTH2 has received much attention since its identification as the second high affinity PGD2 receptor in 2001. A number of potent and selective antagonists are now available for both receptors. This review will briefly discuss the biological background and validation of DP1 and CRTH2 as targets for antiinflammatory drugs, and then highlight developments in medicinal chemistry which have appeared in journals and patent applications in the last few years, and which have brought us closer to therapeutic applications of PGD2 receptor antagonists in various indications.

Keywords: Prostaglandin D2, DP1, DP2, CRTH2, inflammation, asthma, allergy, drug discovery. INTRODUCTION Release of arachidonic acid (1, Fig. (1)) from the cell membrane by phospholipase A2 is the initial step in a cascade which, controlled by specific enzymes, leads to biosynthesis of a large number of eicosanoids. These act in complex interplay with various receptors to regulate numerous physiological responses, including inflammation. The arachidonic acid cascade divides into two pathways, one controlled by the lipoxygenases and leading to production of leukotrienes, hydroxyeicosatetraenoic acids and lipoxins, and the other controlled by the cyclooxygenases COX-1 and COX-2, responsible for converting arachidonic acid into the central intermediate prostaglandin H2 (PGH2, 2), which is further converted by various specific enzymes into prostaglandin D2 (PGD2, 3), prostaglandin E2 (PGE2, 4), prostaglandin F2
(PGF2
, 5), prostacyclin (PGI2, 6) and thromboxane A2 (TXA2, 7), each acting through one or several G protein-coupled seven transmembrane (7TM) receptors. The individual prostanoids undergo transformation to various metabolites with biological activity, but *Corresponding author: Tel: +45 6550 2568; E-mail: [email protected] Allen B. Reitz / Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.

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much work remains to firmly associate individual prostanoids with specific physiological functions. Products from both the lipoxygenase and the cyclooxygenase pathways are implicated in inflammatory reactions in a highly complex fashion, and the individual messenger molecules may have both and pro- and antiinflammatory effects, although the overall outcome of the cascades is largely proinflammatory. For example, therapeutic inhibition of the cyclooxygenase pathway by non-steroidal antiinflammatory drugs (NSAIDs) results in antiinflammatory effects in most cases, but is also known to exacerbate inflammation in pathological conditions such as inflammatory bowel disease [1] or asthma [2]. Since individual prostaglandins exert diverse and potent biological effects on almost all organs, interfering with the biosynthesis of a whole cascade of messenger molecules might be regarded as a rather crude therapeutic approach. Indeed, both non-selective and COX-2 selective NSAIDs have been associated with serious adverse effects [3], most likely due to simultaneous inhibition of wound-healing and antiinflammatory prostaglandins distinct from the targeted proinflammatory prostaglandins. Thus, specifically targeting the prostanoid most relevant for a particular disease may represent a more successful strategy for developing efficacious drugs with fewer side-effects, especially for therapeutic intervention with chronic diseases. Many studies have established a link between PGD2 and allergic asthma. Already 25 years ago Roberts and co-workers discovered that the production of PGD2 increases in patients with mastocytosis [4], and that activation of mast cells with anti-IgE stimulates its production [5]. The observations that PGD2 is released into the airways of asthmatic subjects after antigen challenge [6], has a bronchoconstrictive effect [7], that intratracheal administration of PGD2 causes accumulation of eosinophils in the tracheal lumen of dogs [8], and that it is released from skin during allergic reactions [9], have all contributed to the establishment of PGD2 as an important mediator in allergy and asthma [10]. These early observations are supported by more recent studies, showing that mice overexpressing lipocalin-type prostaglandin D synthase (PGDS) which were immunized with ovalbumin (OVA) had a higher production of PGD2 than sensitized wild-type mice after challenge and were more susceptible to development of inflammatory symptoms such as eosinophil infiltration and cytokine production [11]. Furthermore, polymorphisms in the hematopoietic PGDS correlating with the occurrence of asthma were identified in a Japanese population [12]. A correlation between polymorphisms in lipocalin-type PGDS and the occurrence of atherosclerosis in Japanese hypertensive patients is also reported [13]. It is not surprising that such convincing connection between PGD2 and asthma or allergy has triggered interest in both PGDS and the PGD2 receptors as targets for development of novel and superior therapeutics to combat these diseases. The medicinal chemistry effort aimed at targeting the inhibition of PGD2 effects was initiated in the 1980s when specific molecular targets for PGD2 remained elusive, and has now culminated in the production of a number of small molecule antagonists. This review will provide an overview of the PGD2-biology that is relevant to the roles of PGD2 exerted through interaction with DP1 and CRTH2 in inflammatory diseases, discuss the currently known endogenous and synthetic agonists as tools to explore receptor function, and consider the various chemotypes of PGD2-modulating compounds that have so far become available and which are expected to bring us closer to therapeutic applications of PGD2 receptor antagonists in managing inflammatory diseases. This review is an updated version of a review published in 2006 [14]. The progress within development of new antagonists that has appeared in the literature and the most important results connecting the PGD2 receptors to inflammatory disease has been included, but no attempts have been made to review compounds and results that only have appeared in

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patent applications since 2006. Other reviews on PGD2 antagonists and their therapeutic potential have appeared which may also be of interest to the readers [15-20]. Lipid bilayer Phospholipase A2 O OH Lipoxygenases

Cyclooxygenases O

Leukotrienes & lipoxins

H

OH

O

Arachidonic acid (1)

O H HO PGH2 (2)

O HO

H

O O

OH

H

O HO

OH

H

O

OH O

OH

H

H

O

HO

H

H

HO

HO

HO

HO

PGE2 (4)

PGD2 (3)

O

H

H

HO

OH

O

O

HO

PGF2 (5)

H

PGI2 (6)

TXA2 (7)

O

HO OH H

O

HO HO

H

OH

HO

H

H

OH

OH

OH

O

O

O

HO

H

O H

OH

HO TXB2 O

O

H

O

O O

H


12-PGD2 (9)

DK-PGD2 (8)

HO

OH H

OH

9, 11-PGF2 (12)

PGJ2 (11) O

O

H O

HO

HO

O

O H

HO

H

H

OH

OH

HO 11-Dehydro-TXB2 (15)

O

O

HO 15d-PGD2 (10)


12-PGJ2 (13) O H

OH

O 15d-PGJ2 (14)

Fig. (1). The arachidonic acid cascade, including metabolites with activity on DP1 and/or CRTH2.

THE D PROSTANOID RECEPTOR 1 (DP1) The first receptor for PGD2 to be cloned and characterized was the 7TM receptor DP1, which is Gs-coupled and thus stimulates cAMP production (Fig. (2)) [21, 22]. The receptor has been identified as the mediator of PGD2-induced platelet aggregation [23], vasodilation [24], bronchodilation [25], reduction of intraocular pressure [26], and sleep [27]. The role of the receptor in inflammation seems to be more complex. Matsuoka and co-workers observed decreased asthmatic response in DP1-deficient mice compared to wild type mice upon challenge in an OVA-induced asthma model, suggesting that DP1-antagonists may have antiasthmatic potential [28]. This hypothesis is supported by two reports of selective DP1 an-

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tagonists capable of alleviating asthmatic responses (vide infra) [29, 30]. However, other studies have found that PGD2 inhibits airway dendritic cell migration and suppresses asthma through DP1 [31, 32] and that selective DP1 agonists ameliorate inflammatory effects in various murine models [33-35], suggesting a more complex picture in which PGD2 may also exert antiinflammatory effects via the same receptor. Single-nucleotide polymorphisms (SNPs) in the DP1-encoding gene resulting in low transcriptional efficiency correlate negatively with the occurrence of asthma in both white and black populations, suggesting that the receptor contributes to the asthmatic response [36]. The role of DP1 in allergy and asthma has been reviewed previously [37].

CRTH2

 

PLC

Gi

DP



AC



Gs

cAMP

IP3 PKA

Gene regulation

Ca2+

Fig. (2). Prostaglandin D2 receptor signaling pathways. The PGD2 receptors CRTH2 and DP1 are coupled to different G proteins. CRTH2 is coupled to Gi
proteins, and agonist activation results in an inhibition of adenylyl cyclase (AC) followed by a decrease in intracellular cyclic adenosine monophosphate (cAMP). In addition, G subunits which are liberated upon Gi
activation stimulate the phospholipase C (PLC) cascade leading to generation of inositolphosphates (IP) and release of Ca2+ from intracellular stores. The DP1 receptor is coupled to Gs
proteins, which stimulate AC and induce a rise in intracellular cAMP, leading to gene regulation. The overall effect on PGD2 on the intracellular cAMP level will therefore be regulated by the balance between the expression of the two receptors on the cell surface [38, 39].

CHEMOATTRACTANT RECEPTOR-HOMOLOGOUS MOLECULE EXPRESSED ON TH2 CELLS (CRTH2 / DP2) Although DP1 mediates a number of the physiological responses to PGD2, there were other responses, in particular related to migration of inflammatory cells, which could not be explained by activation of DP1, and several studies with selective agonists and antagonists indicated the existence of a second prostaglandin D2 receptor [26, 40, 41]. In 1999 the gene GPR44 was reported to encode for a G protein-coupled receptor with high similarity to chemoattractant receptors [42]. Concomitantly Nagata and coworkers identified a biomarker for Th2 cells which they termed ‘chemoattractant receptor-

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homologous molecule expressed on Th2 cells’ or ‘CRTH2’ [43], and which turned out to be identical to the G protein-coupled receptor encoded by the GPR44 gene. Besides being selectively expressed on Th2 cells over Th1 cells, CRTH2 was also found highly expressed on eosinophils and basophils, and cells transfected with CRTH2 were found to respond to messengers secreted by mast cells, corroborating the notion that the receptor could play a role in immune responses [44]. The mast cell derived messenger molecule capable of activating CRTH2 turned out to be PGD2, thus a second receptor for this important allergic mediator was identified [45]. Monneret and co-workers independently discovered a new prostaglandin D2 receptor on eosinophils which, contrary to DP1, inhibits cAMP formation. They found that the new receptor mediates the chemoattractant effect of PGD2 on eosinophils, and that it induces actin polymerization, CD11b expression and L-selectin shedding [38]. In analogy to the EP receptor nomenclature they coined the name ‘DP2’ for the new receptor, which turned out to be identical with CRTH2. Thus, the receptor is currently known under two names, one referring to its endogenous ligand, and the other referring to the fact that its protein sequence is distinct from the other prostanoid receptors and instead exhibits higher homology with chemoattractant receptors like the N-formyl peptide receptors. Without any preference otherwise, the receptor is referred to by its first name, CRTH2, herein. CRTH2 is coupled to Gi and thus inhibits cAMP production, but activation of this receptor also leads to increased intracellular calcium levels (Fig. (2)) [38, 45]. The opposite effect of DP1 and CRTH2 on the cAMP level is particularly interesting when both receptors are present on the same cell, which is the case for eosinophils and other immune cells [38, 39]. The overall cellular response to PGD2 will then to some degree be regulated by the expression of the individual receptors on the cell surface. The conjecture that the receptors might have opposite physiological function lays close [38-39], but we still have limited understanding of how these opposing signals elicited by the same ligand in the same cell are integrated and affects the overall response during a complex inflammatory process. While selective CRTH2 agonists mediate chemotaxis and degranulation, DP1 appears to delay apoptosis of eosinophils, suggesting that both receptors could play a role in mediating the proinflammatory effect of PGD2 on eosinophils [46]. Studies with DP1 knock-out mice have indicated that DP1 indirectly promotes eosinophilia via Th2 cells [28]. On the other hand, Spik and co-workers found that CRTH2 activation increased eosinophil recruitment to the inflammatory site and the pathology of atopic dermatitis and allergic asthma in mouse models, whereas DP1 activation rather ameliorated asthmatic pathology [34]. The only report suggesting a role of CRTH2 in allergic inflammation which is not clearly proinflammatory describes OVA-immunized CRTH2 knock-out mice showing enhanced eosinophil recruitment compared to wild-type mice, apparently as a result of increased IL-5 production in the knock-out animals [47]. The fact that CRTH2 is expressed on Th2 cells, eosinophils, and basophils and mediates chemotaxis of these cells in response to the inflammatory mediator PGD2 convincingly indicates that the receptor could be of interest in treatment of various inflammatory conditions. Th2 cells are known as prime mediators of allergic asthma, driving IgE response and eosinophilia [48], and eosinophil-deficient mice sensitized with OVA are protected from characteristic asthmatic symptoms [49, 50]. Basophils are also involved in allergy and asthma [51], and evidence suggests that CRTH2 is primarily responsible for the proinflammatory effect of PGD2 on these leukocytes [52]. Furthermore, single nucleotide polymorphisms in the CRTH2 gene of Chinese children and an African American population have shown high

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correlation with the incidence of asthma [53, 54], and SNPs in the CRTH2 gene of German children were found to influence the development of allergic sensitization and asthma [55]. Allergic asthma is not the only potential indication for PGD2 receptor modulators. Expression studies identified both DP1 and CRTH2 in the nasal mucosa of subjects suffering from polyposis, indicating that both receptors can play a role in rhinitis and upper airway inflammation [56]. CRTH2 levels on circulating Th2 cells are increased in patients with atopic dermatitis [57]. The expression of CRTH2 and CCR3 on Th2 cells during septic shock was found to be very low, suggesting that CRTH2 might have a role also in this serious condition [58]. Eosinophil infiltration in the corneal epithelium, recruited by PGD2 through CRTH2, is important to the pathogenesis of allergic conjunctivitis, thus, CRTH2 antagonists may have therapeutic potential in this disease [59]. Both DP1 and CRTH2 are present on osteoblasts, and it is indicated that CRTH2 activation could lead to anabolic response in bones [60]. Although numerous lines of evidence reinforce the relevance and contribution of PGD2 and its specific receptors to the regulation of inflammatory responses, further studies are necessary to support the high clinical expectations and unveil whether this novel class of therapeutics will offer any benefit to existing drugs. PGD2 RECEPTOR AGONISTS Endogenous DP1 and CRTH2 agonists Prostaglandin D2 (3, Fig. (1)) was the first agonist to be identified for both DP1 and CRTH2, activating both receptors with EC50 values in the low nanomolar range [45, 61]. The cellular fate of PGD2 giving rise to various active metabolites distinguished by their preferences for specific PGD2 receptors makes the total picture more complex. The enzymatically-derived PGD2 metabolite 13,14-dihydro-15-keto-prostaglandin D2 (DK-PGD2, 8) is as potent as PGD2 on CRTH2 but devoid of activity on DP1 [45, 61]. DK-PGD2 was found to induce migration in eosinophils, basophils and Th2 cells with only slightly lower potency than PGD2, thus demonstrating that this response is mediated through CRTH2 [45]. The metabolites
12-PGD2 (9), formed by isomerization of PGD2 by albumin, and 15deoxy-
12,14-PGD2 (10) are also potent and selective CRTH2 agonists [62]. Another PGD2 metabolite, PGJ2 (11), is equipotent with PGD2 on DP1 but an order of magnitude less potent on CRTH2 [45, 61]. The metabolite 9,11-PGF2 (12) acts as a low-potency agonist on CRTH2 [63].
12-PGJ2 (13), the most abundant PGD2 metabolite in plasma, is a selective CRTH2 agonist only slightly less potent than PGD2 [45, 61], and with the same ability to induce eosinophil chemotaxis through CRTH2 [64]. The PGD2 metabolite 15-deoxy-
12,14-PGJ2 (15d-PGJ2, 14) is as potent as PGD2 on CRTH2 and 100-fold selective over DP1 [61, 62]. 15d-PGJ2 is also involved in eosinophil chemotaxis, but at the very low concentrations generated in vivo it appears to act through PPAR by priming for eotaxin-induced chemotaxis rather than by direct CRTH2 agonism [65]. The prostaglandins PGE2 (4) and PGF2 (5) have also proven to be moderate to lowpotency agonists of DP1 [66], and PGF2 is also a low-potency CRTH2 agonist [38, 63]. The stable TXA2 metabolite 11-dehydro-TXB2 (15), originally thought to be biologically inactive, was also found to be a full CRTH2 agonist, although with a potency 500-fold lower than PGD2. Elevated levels of 11-dehydro-TXB2 have been observed in asthma and COPD, and it cannot be excluded that thromboxanes contribute to lung inflammation through activation of CRTH2 by 11-dehydro-TXB2 [67].

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Synthetic DP1 agonists The synthetic prostanoid BW 245C (16, Fig. (3)), initially described as a potent and selective inhibitor of platelet aggregation [68], was later found to be a highly potent (Ki 0.3 nM, EC50 0.3 nM) and selective DP1 agonist, with >100-fold selectivity over all other prostanoid receptors including CRTH2 [45, 61, 66]. This compound is still the most commonly used selective DP1 agonist. The selective agonists DK-PGD2 and BW 245C have both been instrumental in the early phase of mapping the roles of CRTH2 and DP1 in inflammation. There are also a number of other synthetic prostanoids exhibiting selective DP1 agonism. The racemic compound L-644,698 (17) can be mentioned as an outstanding example, being a subnanomolar full DP1 agonist (Ki 0.9 nM, EC50 0.5 nM) with at least 300-fold selectivity over other prostanoid receptors, and devoid of affinity to CRTH2 at 20 μM concentration [61, 66]. TS-022 (18) is a DP1 agonist with weak affinity and no activity on CRTH2, which was developed as an anti-pruritic drug for patients with atopic dermatitis [69]. O

O Cl O

OH

HN

OH S

O S

N

OH HO

N

O O

HO

HO BW 245C (16)

HO TS-022 (18)

L-644,698 (17)

Fig. (3). Synthetic prostanoid DP1 agonists.

Synthetic CRTH2 agonists Whereas (15S)-15-methyl-PGD2 (19, Fig. (4)) is a moderately potent and moderately selective CRTH2 agonist, (15R)-15-methyl-PGD2 (20) is an order of magnitude more potent than PGD2 on CRTH2 and exhibits no activity on DP1 at 10 μM concentration [70]. This observation prompted the synthesis of (15R)-PGD2 (21) to investigate if inversion of stereochemistry at C-15 is sufficient to convert PGD2 into a selective CRTH2 agonist. This turned out to be the case, since the ability of 21 to induce CRTH2-mediated actin polymerization on eosinophils was comparable to PGD2, and the compound is a potent promoter of eosinophil migration (EC50 2 nM), whereas its ability to stimulate cAMP production in platelets, an effect mediated by DP1, was almost absent [71, 72]. O

O OH

OH

O

OH

OH

O 19

OH

O OH

OH

O HO 20

Fig. (4). Synthetic prostanoid CRTH2 agonists.

21

HO

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Prostaglandin D2, 15d-PGJ2 and 20 were all found to induce eosinophil infiltration into lungs of rats, whereas the selective DP1 agonist BW 245C was completely inactive, corroborating the conclusion that PGD2 and 15d-PGD2 induce airway eosinophilia through CRTH2, and further validating CRTH2 as an interesting target for treatment of asthma [73]. Several non-prostanoid CRTH2 agonists are also known. Screening of common NSAIDs on CRTH2 led to identification of indomethacin (22, Fig. (5)) as a potent agonist, inducing Ca2+ mobilization in CRTH2 transfected cells (EC50 50 nM), and inducing chemotaxis in eosinophils, basophils and Th2 cells, an effect which could be blocked by CRTH2 specific antibodies. Interestingly, the compound is able to displace [3H]PGD2 only at much higher concentrations in a competition binding assay. The other NSAIDs acemetacin, sulindac, diclofenac and aspirin exhibited no functional activity and very low or no affinity on CRTH2 [74]. Indomethacin also induces shape change, L-selectin shedding, CD11bupregulation and respiratory burst in eosinophils as well as basophil shape change through specific CRTH2 agonistic activity [75]. The agonistic activity of indomethacin on CRTH2 prompted further investigation of NSAIDs and related structures. The metabolite 5desmethyl-indomethacine (23) was found to possess agonistic activity similar to indomethacin [76]. Sulindac sulfide (24) also exhibited low affinity (Ki 3.4 μM) and agonistic activity on CRTH2, and an order of magnitude higher affinity to DP1 (Ki 360 nM) [61, 76]. Sitedirected mutagenesis in the putative binding site of CRTH2 identified residues His-106 in TM-III, Lys-209 in TM-V, generally presumed to interact with the carboxylic moiety, and Glu-268 in TM-VI as well as Arg-178 in the second extracellular loop as important for binding of PGD2. Of these, only the latter two were found to be important for binding of indomethacin. Mutation of Tyr-261 to Phe was detrimental to the binding of indomethacin, but did not affect binding of PGD2 or DK-PGD2 [77]. O O

O

O OH

F OH

MeO

OH

HO

F

N

O N OH

N

N

S N

Cl O

Cl

O

Cl

O MeS

Indomethacin (22)

5-DMI (23)

Sulindac sulfide (24)

L-888,607 (25)

K376 (26)

Fig. (5). Non-prostanoid CRTH2 agonists.

The prostanoid agonists above are not ideal for exploration of CRTH2 because of chemical and metabolic instability, and indomethacin is unsuitable because of other pharmacological effects including COX-1 and COX-2 inhibition. Gervais and co-workers reported a stable full CRTH2 agonist, L-888,607 (25, Ki 0.8 nM), which is chemically related to indomethacin but exhibited excellent selectivity over the other prostanoid receptors, a broad selection of chemokine receptors, anaphylatoxin receptors and the cyclooxygenases. The compound was also found to possess satisfactory pharmacokinetic properties [78]. The tetrahydroquinoline agonist K376 (26) represents an unrelated structural class. This is a moderately potent (Ki 91 nM) partial agonist identified from a series which appears to consist mostly of antagonists (vide infra) [79].

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PGD2 RECEPTOR ANTAGONISTS DP1 antagonists The prostanoid-inspired compound BW A868C (27, Fig. (6)) was found to behave as a selective, competitive DP1 antagonist on human platelets [80]. More recent studies in recombinant expression systems revealed partial agonism of BW A868C, indicating a somewhat diverging functional behavior on natural and transfected cells, possibly due to differences in the concentration of the receptor on the cell surfaces [22, 66]. The compound has no activity on CRTH2 [70]. A recent study found that both BW A868C (27) and the TP/CRTH2 antagonist ramatroban (45, Fig. (10)) inhibited guinea pig and human eosinophil migration, suggesting a role of both DP1 and CRTH2 in eosinophil trafficking [81]. Another early DP1 antagonist is AH6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid), although its utility is limited due to low potency and interaction with other receptors [82]. O O

O OH

O OH

OH

OH

O R

NH N O

O

N N H

BW A868C (27)

S O

HO

NH O

NH

S

O

O

O

MeO S-1452 (28)

S 29

30 R=F S-5751 (31) R=OH

Fig. (6). Synthetic prostanoid DP1 antagonists.

Screening of the compound libraries at Shionogi for DP1 binding resulted in identification of S-1452 (28, IC50 0.6 μM), previously known to be a TP antagonist. The affinities for DP1 were similar for both enantiomers, whereas the (+)-enantiomer of 28 was 25-fold less potent on TP, and was thus chosen for optimization. This effort resulted in 29, binding with IC50 of 24 nM to DP1 receptors on human platelet membranes and inhibiting cAMP production in platelets with IC50 of 52 nM. The compound was active in rhinitis, conjunctivitis (ED50 1.6 and 6.6 mg/kg, respectively) and asthma guinea pig models after oral administration (10 mg/kg) [83, 84]. Exploration of an alternative 6,6-dimethylbicyclo[3.1.1]heptane scaffold, also previously known from TP antagonists, resulted in 30 and S-5751 (31), both exhibiting DP1 antagonism with IC50 values in the (sub)nanomolar range. Both 30 and 31 were highly active in guinea pig rhinitis, conjunctivitis and asthma models after oral administration (3-10 mg/kg) [29, 85], and 31, which is >1000-fold selective over CRTH2, also exhibited activity in allergic sheep (30 mg/kg p.o.) [86]. Compound S-5751 (31) has been in clinical trials for oral treatment of bronchial asthma, but was discontinued as it failed to meed the primary endpoint. In the phase IIa Proof of Concept studies, which included 400 asthmatic patients, no statistically significant difference between high dose 31, low dose 31 and placebo was found on lung function, measured as forced expiratory volume in one second (FEV1) (http://www.shionogi.co.jp/ir_en/news/detail/e_061027.pdf). Even though indomethacin (22, Fig. (5)) has no activity on DP1, Torisu and co-workers succeeded in converting this compound to a low potency DP1 antagonist by replacing the 4chloro by a 4-butoxy substituent (32, Fig. (7)). This compound served as the starting point in

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the discovery of a new series of DP1 antagonists, and by moving the 3-acetic acid moiety of 32 to the 4-position on the indole (33), a derivative with 200-fold selectivity over the EP receptors in the binding assay was realized [87, 88]. Further optimization afforded antagonists with (sub)nanomolar potency. Several analogs also came out with high affinity to EP2 in a counter-screen on the EP receptors. The most potent and selective compound 34 was found to inhibit PGD2-induced conjunctival vascular permeability in a guinea pig model with an oral dose of 0.3 mg/kg [30]. Compound 35, which was 3-fold less potent than 34 towards human DP1 in the cell-based assays and equipotent in binding on mouse DP1, was found equipotent with 34 in the PGD2-induced conjunctivitis model, and significantly reduced OVA-induced vascular permeability after oral administration of 10 mg/kg, whereas 34 did not reach significance at this dose. Compound 35 also exhibited favorable pharmacokinetic properties, with plasma half-life exceeding 7 hours [89]. These results support DP1 antagonists as potentially interesting antiinflammatory agents, however, CRTH2 is not among the disclosed counter-screen receptors, thus it is not possible to exclude that the compounds might also act on this receptor. O O

O

OH OH

OH

MeO

R1 N

N O

32

O

N O

O O

O 33

R2

O N 34 R1=F, R2=H 35 R1=H, R2=Me

Fig. (7). Indomethacin-derived DP1 antagonists.

A patent application from Ono Pharmaceuticals discloses indomethacin-derived indoles similar to the compounds in Fig. (7). The application is exemplified with combinatorial libraries containing compounds with the 3-acetic acid and the 2-methyl of indomethacin conserved, methoxy, methyl, chloro, fluoro or no substituent in the 5-position, and with aromatic carboxamides or sulfonamides in the 1-postion appended with substituents generally similar to those of 34 and 35. The patent application is written in Japanese, but it appears form a translated abstract that the compounds are dual DP1/CRTH2 antagonists also acting on CRTH2 with Ki
10 μM [90]. Merck Frosst scientists identified a series of 2-substituted N-benzylbenzimidazole DP1 antagonists in an HTS campaign. Optimization starting with compound 36 (Ki 73 nM, Fig. (8)) resulted in identification of the ethenylene linker (37, Ki 24 nM) and the ethylene linker (38, Ki 38 nM) as optimal. The latter compound exhibited higher affinity to DP1 in the presence of human serum albumin and was devoid of affinity to TP [91]. The resemblance of this compound series to prostanoid structures, especially the synthetic prostanoid L-644,698 (17, Fig. (3)), sparks the thought that hydrolysis of the methyl ester might produce even more potent compounds. However, the free carboxylic acid analog of 36 exhibited low affinity to DP1 (Ki 3.5 μM), indicating that this is not the case. Merck Frosst has filed a number of patent applications on ring-fused indole PGD2 antagonists related to the compounds in Fig. (9). Ring-fused indole PGD2 receptor antagonists of the type 39 and 40 and substituted analogs, closely related to the CRTH2 agonist L-888,607 (24, Fig. (5)), are claimed to for treatment of allergic rhinitis, nasal congestion

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and asthma. In vitro assays for all prostanoid receptors except CRTH2 are described, leading to the presumption that the compounds are DP1 antagonists. Procedures for in vivo evaluations are also described, but levels of activity are not disclosed [92]. The CRTH2 affinities of the racemic 39 and 40 are given elsewhere to Ki 6 and 80 nM, respectively, but the nature of their functional activity is not revealed [78]. A patent application on related mesylpyrrolopyridine DP1 antagonists like 41 has also appeared, but activity levels are not disclosed [93]. O

O OMe

OMe

S

N

O OMe

N

N

N N

N

36

37

Cl

38

Cl

Cl

Fig. (8). Benzimidazole DP1 antagonists.

N

N

O

N OH

S

OH

OH

N

S

S Cl

Cl 39

O

S Cl

O

40

41

O

F O

O

N

OH S

O

N

N OH

S

O

O

Cl

O

42

Cl 43

S

OH

O

O MK-0524 (44)

Cl

Fig. (9). Ring-fused indole PGD2 modulators.

Compounds 42 and 43 are potent DP1 antagonists binding with Ki 1.7 and 2.1 nM, respectively, and they are two orders of magnitude more potent than their enantiomers [94]. The compounds are also related to the CRTH2 agonist L-888,607 (25), and it is notable that the (S)-configuration at the stereocenter at the acetic acid moiety of L-888,607 is opposite to the (R)-configurations of 42 and 43. Whereas L-888,607 displays 3000-fold selectivity for CRTH2 over DP1 in a binding assay (0.8 vs. 2331 nM), the enantiomer L-888,291 does not show significant selectivity (48 vs. 40 nM), and Gervais et al. notes that the (S)-configuration in general prefers CRTH2 whereas (R)-configuration at this stereocenter produces compounds with preference for DP1 [78].

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A patent application related to prevention of niacin-induced DP1-mediated flushing indicates that a number of related structures, including 41-44, bind to DP1 with Ki values in the range between 0.4 and 16.3 nM, and that the affinities of the same compounds to CRTH2 are in the Ki range 180 to above 20,000 nM [95], thus, these compounds appear to exhibit appreciable selectivity for DP1 over CRTH2. In general, it appears that this structural class may encompass compounds with affinity for both DP1 and CRTH2, as well as both agonists and antagonists. The 5-fluoro-8-mesyl analog MK-0524 (44), also known as laropiprant, is a potent DP1 antagonist (IC50 0.57 nM) with 5-fold selectivity over TP and >100-fold selectivity over the remaining prostanoid receptors, and with favorable bioavailability and pharmacokinetic properties [96, 97]. The compound effectively suppresses the DP1-mediated niacin-induced flushing, which is a disturbing side-effect in treatment of dyslipidemia with niacin [98, 99]. Merck intended to market combination therapy containing niacin and laropiprant in the US for treatment of elevated cholesterol, but the application was rejected by the US Food and Drug Administration, apparently on basis of a risk/benefit analysis. The same combination therapy was however approved only two months later by the European Medicines Agency, and is now marketed in Europe as Tredaptive. Laropiprant (44) has also been in phase II Proof of Concept studies for treatment of both asthma and allergic rhinitis. The asthma study included 100 patients that were treated with placebo or laropiprant (300 mg/day) for the first 3 weeks followed by montelukast (10 mg/day) alone or in addition to laropiprant (300 mg/day) for the last 2 weeks in a doubleblinded cross-over study, and the primary endpoint was FEV1. No significant difference between laropiprant and placebo or between montelukast alone and montelukast with laropiprant was found, whereas the difference between montelukast and placebo was significant [100]. The allergic rhinitis study included 767 patients that were treated with placebo, laropiprant (25 or 100 mg/day for 2 weeks) or the H1 antihistamine cetirizine (10 mg/day for 2 weeks), and the primary end point was the Daytime Nasal Symptoms Score (DNSS). No significant difference between placebo and laropiprant was found, whereas the difference between placebo and cetirizine was significant [100]. Together with the outcome of the clinical trials with S-5751, these results imply a serious setback to the strategy of DP1 antagonists for treatment of asthma and allergy. CRTH2 Antagonists Much effort has been directed towards identifying CRTH2 antagonists the last few years, mainly focused at identifying new therapeutic agents for asthma and atopic conditions. Indomethacin (22, Fig. (5)) and ramatroban (45, Fig. (10)) have frequently served as leads for identification of new selective compounds. Other compound classes derived from screening campaigns and rational approaches have also been reported. Ramatroban (45) is a compound with established efficacy against allergic rhinitis and has been marketed in Japan for this indication [101, 102]. The compound was originally developed as a TP antagonist for treatment of thrombosis and coronary artery disease [103], and was later shown to inhibit both TXA2- and PGD2-induced bronchoconstriction [104, 105]. Recent studies revealed that ramatroban is also a potent CRTH2 antagonist [106], and although some evidence supports a role of TP in asthmatic and allergic reactions [107], it seems likely that the efficacy of ramatroban is mediated at least in part through an antagonistic effect at CRTH2.

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Being an orally available compound with favorable overall pharmacokinetic properties [108], ramatroban has been important in both in vitro and in vivo studies to explore the contribution of CRTH2. The compound was found to attenuate PGD2-induced eosinophil migration in vitro [106]. The CRTH2-selective agonist DK-PGD2 (8, Fig. (1)) was found to dose-dependently induce circulation of eosinophils, lymphocytes and neutrophils in rats, effects which were sensitive to inhibition by ramatroban [109]. Intratracheal administration of PGD2, DK-PGD2, or indomethacin attracts the eosinophils into the airways, an effect which was blocked by ramatroban in all cases, whereas BW 245C (15) had no influence on the eosinophil count in the BAL fluid. Pretreatment with IL-5 was necessary to mobilize the eosinophils into circulation [110]. Ramatroban was found active in a fluorescein isothiocyanate (FITC) induced contact hypersensitivity mouse model. The effect appeared to be due to CRTH2 antagonism, as the TP antagonist ridogrel and the DP1 antagonists BW A868C (27) were without effect, thus indicating that CRTH2 antagonists could be of potential value for the treatment of atopic dermatitis [111]. The effect of ramatroban on FITC sensitized mice was confirmed in another study, where CRTH2-deficient mice confirmed the CRTH2-mediated mechanism [112]. Ramatroban is not the ideal tool compound for studies of CRTH2 because it is an equally potent antagonist on TP (Ki (CRTH2) 4.3 nM, Ki (TP) 4.5 nM). N-Methylation of the sulfonamide (46, Ki (CRTH2) 1.9 nM, Ki (TP) 3000 nM) and shortening of the carboxylic acid chain by one methylene group (47, Ki (CRTH2) 0.5 nM, Ki (TP) 540 nM) both resulted in several hundred-fold loss of activity on TP, whereas activity on CRTH2 was preserved or increased. Combining both modifications produced a highly potent CRTH2 antagonist with remarkable selectivity over TP (TM-30089, 48, Ki (CRTH2) 0.6 nM, Ki (TP) >10000 nM). The compounds were also found to be devoid of activity on DP1. Compounds 46-48 were tested as racemic mixtures, implying that both enantiomers of these compounds lack activity on TP or DP1 [113]. O

O

OH

OH

N

N

O

O

OH

N

N

S

N

O

S

O O

N

R

R

Ramatroban (45) R=H 46 R=Me

N

O

O

F

OH

N

F 47 R=H TM-30089 (48) R=Me

S

O O

N

Me

49

S

O

Me

F

50

F

Fig. (10). Ramatroban and selective CRTH2 antagonist analogs.

Upon more detailed pharmacological study of these compounds, it was discovered that analogs 47 and 48 with the shorter carboxylic acid chain behaved as insurmountable antagonists on CRTH2 on both transfected cells and human eosinophils, whereas the longchain analogs ramatroban (45) and 46 behaved as classic competitive antagonists [114]. It is still uncertain how the insurmountable behavior of 47 and 48 will affect the ability of the compounds to modulate inflammation in vivo. TM30089 (48) acted as a potent inhibitor of

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PDG2-induced release of eosinophils from guinea pig bone marrow [115]. A study with sensitized mice found that both the CRTH2-selective antagonist TM 30089 (48) and the dual CRHT2/TP antagonist ramatroban (45) attenuated asthmatic pathology to the same degree [116]. Merck has filed a patent application where the where the indole ring of TM30089 has been inverted vertically (49), along with two other close analogs [117]. No activity data was disclosed by Merck, but scientists at Amira Pharmaceutical has synthesized the compound, along with the isoindole and several aza-analogs of 48 and 49, and they have tested all new analogs head to head with these two compounds, a favored indomethacin analog from Oxagen (80, Fig. (15)) and a favored pyrimidylacetic acid from Actimis (97, Fig. (20)) on CRTH2 in binding with and without serum albumin, in an eosinophil shape change assay, on the prostanoid receptors DP1, TP and IP, and on CYP enzymes, to give a valuable direct comparison between some leading compounds in the field [118]. Compound 49 shoved an IC50 of 6 nM compared to 1 nM for 48 in this study. Of the new analogs, only compound 50 exhibited high activity in the range of the inspirational compounds, whereas most analogs showed much lower affinity [118]. Robarge and co-workers have investigated a compound series with horizontally inverted tetrahydrocarbazole scaffold of ramatroban, i.e. with the arylsulfonamide substituent moved from the 3- to the 6-position on the tetrahydrocarbazole scaffold. The parent compound 51 (Fig. (11)) was found to be equipotent with ramatroban in a binding assay. Replacement of the sulfonamide by carboxamide, methylamine or urea resulted in loss of affinity. Removal of the 4-fluoro substituent or replacement by chloro, methyl, hydroxy, phenoxy, thienyl, 2fluoro, 3-fluoro, 2,4-difluoro or 3,4-difluoro also resulted in compounds with lower affinity. Screening of a number of different acidic side chains resulted in identification of the acetic acid side chain as the optimal (52, Ki 30 nM), giving an order of magnitude more potent compounds. All other modifications lead to lower or no affinity to CRTH2, including acetic acid isosteres like ethylenesulfonic acid, ethylenephosphoric acid, elongation to butyric or pentanoic acid, the constrained analogs acrylic acid or 2-, 3- or 4-phenylcarboxylic acid, or methyl substituents on the acetic or propionic acid chain. The tetrahydrocarbazole was then investigated further with the optimized acidic side chain. Analogs with 3-methyl (53, Ki 13 nM) and 3-phenyl (54, Ki 50 nM) substitution or expansion to a 7-membered ring (55, Ki 20 nM) resulted in compounds with preserved affinity and paralleled antagonistic activity. Counter-screen of the more potent compounds demonstrated >400-fold selectivity over TP [119]. O

O

OH

OH

N

N R

( )n O

O N

S

N

O

S

O

H

H 51 F

52 R=H, n=1 53 R=Me, n=1 54 R=Ph, n=1 55 R=H, n=2

Fig. (11). Selective CRTH2 antagonists derived from ramatroban.

F

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In two Japanese patent applications Shionogi claims the chemical space around ramatroban and discloses a wide range of example compounds, especially around the 1-acetic acid analogs, including compounds in Fig. (10) and Fig. (11). Other representative examples with disclosed binding affinities are shown in Fig. (12) and include substitution of the 4-fluorophenyl by 2-thienyl (56, IC50 4.9 nM), ring-opened analogs to form indoles (57, IC50 15 nM), constrained analogs (58, IC50 4.5 nM), “inverted” tetrahydrocarbazoles like the ketone 59 (IC50 7.8 nM), ring-expanded analogs like the 4-hydroxysulfonamide 60 (IC50 3.6 nM), ring-opened “inverted” compounds (61, IC50 19 nM), and carbazoles (62, IC50 4.5 nM). Most of the more active compounds share the substructure 3-(2-arylsulfonylaminoethyl)pyrrol-1-ylacetic acid [120, 121]. O

O

O

O

N

OH

N

OH

OH

N

F O N

N

O

S

S

N

S

57

N

O

58

N

O

O

OH

OH

N

N

OH

R O S

O

F

F

O

OH

N

S

59

F O

N

O

O

O O

H S

56

OH

F O

H

N

O O

N

S

O

O O

N

S

O

N

S

O

H R 60

61 OH

62 F

F

63 R=H 64 R=T F

Fig. (12). CRTH2 antagonists related to ramatroban.

The related compound 63 is a high affinity CRTH2 antagonist (pKi 9.2, pIC50 8.4). As an antagonist tracer was required to investigate certain observations (vide infra), this compound was selected as a suitable candidate. The tritiated compound 64 exhibited a pKD of 9.0 [122]. AstraZeneca early on disclosed indomethacin analogs as CRTH2 antagonists which were claimed for use in treatment of asthma and COPD. Antagonistic activities in the range pA2 5.8-6.8 are disclosed for a few compounds, for example 65 and 66 (Fig. (13)), both with pA2 values of 6.8 [123, 124]. The following patent applications claim similar compounds with the indole scaffold flipped to the same orientation as the ramatroban-derived compounds above. An aromatic moiety is attached directly at the 3-position, for example 67 (pIC50 8.15) and 68 [125], or via a sulfur atom, like for 69 (pIC50 8.35) [126]. The compounds are claimed for treatment of asthma and rhinitis. The following patent applications on similar compounds include examples where the aromatic 3-substituent is attached via a sulfone (70, pIC50 8.1), sulfoxide [127] or oxygen (71, pIC50 7.95) [128] linker, and with amide, sulfonamide and heteroaromatic substituents on the 4-, and 5-position of the indole scaffold, including one example with sub-nanomolar receptor affinity (72, pIC50 9.4) [129]. The

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discovery of this compound series was recently described, and it appears that compound 68 has a preferred profile, with IC50 2.6 nM, a bioavailability of 76% in rats and 100% in dogs, and a half life of 1.7 h in rats and 5.3 h in dogs [130]. The compound effectively blocks eosinophil and basophil migration from guinea pig bone marrow [131]. A patent application from the same company describes analogous indazole, pyrrolopyridines and benzimidazolones with no activity information except that all compounds bind to CRTH2 with IC50 <10 μM [132]. O

O

OH

OH

N Cl

OH

N

F

N

66

O

OH

N

N

N

65

O

N

67

OPh

N

68 S

N

N

PhO O O

O

O

OH

N

OH

N Cl

S

N

F3C O

S

OMe

O

O Cl

O 69

OH

N

Cl

70

OH

S

N

S

71

72

O

O

Fig. (13). CRTH2 antagonists related to indomethacin.

Scientists at Oxagen have disclosed of a series of 5-fluoro-2-methyl-1-arylsulfonamideindoleacetic acids with CRTH2 antagonist properties identified using indomethacin as starting point (Fig. (14)). The two compounds most potent in the functional assay on CRTH2 transfected cells, 73 (Ki 29 nM, IC50 (Ca-flux) 26 nM) and 74 (Ki 68 nM, IC50 (Ca-flux) 19 nM), were tested further in leukocyte assays. Compound 73 exhibited poor activity when tested in an eosinophil shape change assay in the presence of 10% fetal calf serum, which was ascribed to poor solubility and high serum protein binding. Compound 74, on the other hand, inhibited PGD2 induced eosinophil shape change and Th2 cell chemotaxis with IC50 values of 74 and 67 nM, respectively. The compound further exhibited high selectivity over DP1 and a panel including more than 85 other receptors, enzymes and ion channels, was stable to human and rat microsomes, did not inhibit or induce selected CYPs, was clean in Ames test, and was found to be in possession of favorable pharmacokinetic properties [133]. N

O

O

O S 75 R=

OH

F

OH

F

N

N

S

O O 73 R=Cl 74 R=SO2Me

R

O

N H

O

N

R

78 77 R=

N

N

76 R=

S O

OH

N H

Fig. (14). Indomethacin-derived CRTH2 and/or DP1 antagonists.

F

S O

F

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A patent application discloses additional 1-sulfonamide-indole-3-acetic acid CRTH2 antagonists, most of which were confirmed to be selective over DP1 and TP [134]. Another patent application discloses compounds of the same general formula ranging from pure CRTH2 antagonists (75, Ki (CRTH2) 15 nM, Ki (DP1) >10000 nM), via dual antagonists (76, Ki (CRTH2) 65 nM, Ki (DP1) 150 nM), to predominant DP1 antagonists (77, Ki (CRTH2) 3050 nM, Ki (DP1) 74 nM), all claimed to be of use in treatment of asthma, rhinitis and atopic dermatitis [135]. A patent application from Novartis claiming pyrrolopyridine analogs similar to the compounds in Fig. (14) also emerged around the same time [136]. The preferred compound 78 has Ki (CRTH2) 30 nM, an oral availability of 54% and a half-life of 1.8 hours in rats [137]. Yet another patent application from Oxagen describes indole-3-acetic acids with 1arylalkyl substituents. Several of the compounds bind to CRTH2 with high affinity, have appreciable selectivity over DP1, are functional antagonists and inhibit PGD2-induced eosinophil shape change and migration, but do not affect eotaxin, 5-oxo-ETE, IL-5, C5a or LTB4 induced eosinophil migration. One example is 79 (Ki (CRTH2) 5.3 nM, IC50 (Ca-flux) 48 nM, Ki (DP1) 3785 nM, Fig. (15)) [138]. Oxagen has also filed a patent application disclosing similar compounds with flipped indole nucleus like 80 (Ki (CRTH2) 9 nM, IC50 (Ca-flux) 30 nM) and 81 (Ki (CRTH2) 6 nM, IC50 (Ca-flux) 79 nM) [139]. The four Oxagen applications were reviewed by Norman in 2005, who correctly points out that the numerous recent patent applications on indoleacetic acid CRTH2 antagonists are making the chemical space around this scaffold highly congested [140]. An additional patent application on a distinct variation in the same compound class recently appeared from Actelion, disclosing Knoevenagel-type indole-1-acetic acid derivatives together with binding affinities and antagonistic activities from a FLIPR assay for several examples, the most potent being 82 (IC50 (binding) 7 nM, IC50 (FLIPR) 48 nM) [141]. A Merck group has explored a related spiro-variation, and identified 83 (Ki 37 nM) and 84 (Ki 10 nM) as having pharmacokinetic properties suitable for further pharmacological evaluation [142]. O

O

OH

N

F

N

OH

N

N

F

80 R=H 81 R=F F

OH

N

F

79

O

O

OH

N

O O

OMe Cl

R

N

N 82 R

N O

83 R=H 84 R=Cl

O F

Fig. (15). Indole and indolinone CRTH2 antagonists.

Mathiesen and co-workers identified the compounds 85 and 86 (Fig. (16)), which selectively antagonize PGD2-induced
-arrestin signaling of CRTH2 without interfering with G protein signaling. The effect is specific for CRTH2, and the compounds do not compete with PGD2 but rather increase the number of available PGD2-binding sites. This is notable since 85 and 86 were identified as analogs of indomethacin and ramatroban which both displace PGD2 from CRTH2. Although the chemical prerequisites for the inhibition of one intracellular signaling pathway without affecting another have not been revealed, this finding might open the way to new signaling pathway specific inhibitors [143]. Mimura and co-workers have described the molecular pharmacology of the tetrahydroquinoline CRTH2 antagonists K117 (87, Ki 5.5 nM) and K604 (88, Ki 11 nM) (Fig. (17)), as well as the CRTH2 agonist K376 (26, Ki 91 nM) in Fig. (5). The compounds exhibit no activity on a panel of relevant receptors and enzymes, and they inhibit PGD2-

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induced eosinophil chemotaxis but not chemotaxis induced by the CCR3 agonist eotaxin [79]. These compounds differ from the other disclosed CRTH2 antagonists in that a carboxylic acid or other acidic group is not required for activity. This compound series was discovered independently by several companies and patent applications containing numerous examples emerged from Millennium [144, 145], Warner-Lambert [146-148] and Tularik [149]. A recent paper described the carboxylic acid substituted 89 as the most potent representative (IC50 3 nM in binding assay with [3H]PGD2 and IC50 0.77 nM in an eosinophil shape change assay), and 90 as a less potent representative (IC50 25 nM in binding and IC50 50 nM in eosinophil shape) with favorable oral bioavailability (38%), clearance (0.73 L/h/kg) and half-life (5.1 h) in rats [150]. F O OH MeO N

S

HN

O

O O HO 85

OEt

86

N H

Fig. (16). CRTH2 antagonists specific for the
-arrestin signaling pathway. O

O

N

O

N

O OH

N

N

O

N O K117 (87)

N

N

O K604 (88)

N

O

O

89

90

OCF3

Fig. (17). Tetrahydroquinoline CRTH2 antagonists.

7TM Pharma reported a new rational approach to discover ligands for target receptors using CRTH2 as a model system. The exercise led to the identification of a large number of diverse compounds with affinities down to 20 nM. Many of the hits make up suitable starting points for optimization, and compound 91 (IC50 1.9 μM, Fig. (18)) is a disclosed example of one such starting point [151]. Cl

O

S

O

N H

OH

MeO 91

Fig. (18). 2-Arylthiazolidine-4-carboxylic acid CRTH2 antagonist.

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Several compound series have appeared which contain a carboxylic acid attached to a benzene or similar aromatic ring system by a short linker, like methylene, ethylene or methyleneoxy. Tularik has filed a patent application on one such compound series. The example compounds in general consist of two benzene rings connected by a sulfonamide. A third aromatic ring is attached via a one-atom linker ortho to the N-substitution and is further substituted by a carboxylic acid attached by a short linker. Compounds 92 and 93, shown in Fig. (19), are representative examples, both with IC50 <1 μM in a binding assay [152]. The preferred compound from the series, AMG 009 (94), is a dual antagonist with IC50 (CRTH2) 3 nM and IC50 (DP1) 12 nM, acceptable bioavailability (28, 60 and 39% in rat, dog and cyno monkey, respectively) and clearance (1.2, 0.77 and 0.27 L/h/kg in rat, dog and cyno, respectively), and dose-dependently reduce airway constriction in PGD2-challenged guinea pigs, with a significant effect reached at 10 mg/kg subcutaneous dose. Following these observations, the compound has been progressed to clinical trials [153]. O O

O

N H

OH OH

O O

O

NH

O

O

O

O

OH

N H

O

S

O

NH O

S

NH

S

OMe Cl

92

AMG-009 (94)

93

Cl

Fig. (19). Arylsulfonamide CRTH2 antagonists.

Bayer has filed patent applications on a series of CRTH2 antagonists exemplified in Fig. (20). The example compounds consist of substituted 2-benzyl-6-aminopyrimidyl-5-acetic acids, and the activity intervals disclosed in the patent application indicate that 95-97 inhibit PGD2-induced Ca2+-mobilization with IC50 <0.5 nM [154]. Compound 98 is an example from a related imidazopyrimidineacetic acid series, and inhibits PGD2-induced Ca2+mobilization with IC50 <10 nM [155]. The compound AP768 is a member of this series, and was progressed to phase I clinical trials by the spin-out company Actimis (http://www. actimis.com/actimisBI6172008.pdf). Cl H N

O

OH

F3C

R H N

N

O N

OH

N

Cl

Cl H N

Cl

O

N

O

N

N

O

OH

N

O N

N

O 95

N

96 R=Cl 97 R=NMe2

98

Fig. (20). Pyrimidine-5-acetic acid CRTH2 antagonists.

Several papers have appeared from Actimis where a compound stated to be a potent and selective CRTH2 antagonist referred to as “Compound A”, presumably identical to AP768, have been studied with promising results in several mouse models of inflammation, including a model of airway hyperreactivity [156]. The compound is stated to be protected by the patent mentioned above [155], placing it in the same class as the compounds of Fig. (20). It is regrettable that the otherwise carefully performed and very interesting study has been

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published without giving the exact structure of the central compound, since it makes it essentially impossible for independent researchers to verify the results. AstraZeneca has filed a patent application on phenoxyacetic acids with small 4substituents and aromatic 2-substituents like 99 (pIC50 8.0) and 100 (pIC50 8.2) in Fig. (21) [157]. In another patent application the aromatic 2-substituent is attached via an oxygen, sulfur, nitrogen or sp3-hybridized carbon atom, as for 101 (pIC50 8.0) and 102 (pIC50 9.0) [158]. Novartis has filed a patent application on phenoxyacetic acids with saturated carbocyclic 2-substituents. The activities of five compounds are disclosed, the most potent being 103 with Ki 60 nM in an SPA assay and IC50 139 nM in a cAMP assay [159]. The compound inhibited CRTH2-mediated eosinophil shape change with an IC50 of 0.25 μM, and had an oral bioavailability of 58% and a half-life of 78 minutes in rats [160]. O O OH

OH

OH

O

OH

O

O

O OH

O Cl

N

Cl

O

O

N

99

O

S

100

S

F3C

O

O Cl

F3C

N

O

O

O

Cl

H N 101

O

102 O

S

103 O

O

S O

Fig. (21). Phenoxyacetic acid CRTH2 antagonists.

7TM Pharma has filed a patent application on phenoxyacetic acids with small 4substituents and aromatic 2-substituents attached via a linker composed of a carbonyl group and 5-membered heterocyclic ring like pyrazole (104-106, Fig. (22)), a 5-membered heterocyclic ring only (107) or in combination with methylene groups (108), which all display IC50 values <0.5 μM in both binding and antagonist assays and inhibit PGD2-induced shape change in human eosinophils [161]. Compound 104 exhibited IC50 values of 1.5 nM in the binding assay, 24 nM in an antagonist assay, high selectivity over a broad panel of receptors and enzymes, an oral bioavailability of 84% and a half-life of 2.7 hours in rats, and significant reduction of peribronchial eosinophilia and goblet cell hyperplasia in mice after 5 mg/kg oral administration [162]. O

O

O

O OH

OH

OH

OH

O

O

O

O O

R

O

Br

O

Br

Br

N N

N 104 R=Br 105 R=Et

N

N

O

S

N

N 106

107

OMe

108

O

Cl

Fig. (22). Phenoxyacetic acid CRTH2 antagonists.

In another patent application 7TM Pharma claims thiazoleacetic acids with a 4-aryl substituent and an aryl-substituted 2-benzyl group like the compounds in Fig. (23) [163].

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The benzyl compound 109 inhibited [3H]PGD2 binding with IC50 3.7 nM,
-arrestin recruitment with IC50 66 nM and cAMP production with IC50 12 nM [164]. The benzyhydryl compound 110 exhibited an IC50 of 3.6 nM in the binding assay and 31 nM an antagonist assay, an oral bioavailability of 81% and a half-life of 5.8 h in rats [165]. The pyrimidine analog 111 has IC50 1.4 nM in the binding assay and 12 nM in the antagonist assay [166, 167]. F O

OH

O

S

O

OH

N

N

Cl

OH

N

S

N

MeO 109

110

OPh

F

111

F

F

Fig. (23). Thiazoleacetic acid and 2-benzamidylbenzoic acid CRTH2 antagonists.

7TM Pharma has also filed a patent application on a series of substituted 2-(arylamide) benzoic acids. Compounds 112 and 113 (Fig. (24)), both binding to CRTH2 with IC50 <0.5 μM, are representative examples [168]. O

OH

O

Cl

H N

N H

F3CO

O

OH

S H N

S O

112

O

H N

O

O 113

O

Fig. (24). 2-Benzamidylbenzoid acid CRTH2 antagonists. CONCLUSION AND OUTLOOK There is compelling evidence that PGD2 plays an important role in modulation of inflammation through activation of the receptors DP1 and CRTH2, and development of compounds acting on one or both of these receptors thus represent a novel and highly interesting approach for treatment of various inflammatory conditions, in particular asthma and allergic diseases. Although the involvement of DP1 in inflammation is established, the evidence is ambiguous regarding whether DP1 activation results in a pro- or antiinflammatory response, and the role of the receptor appears to depend on various factors. Several selective DP1 antagonists have proven efficacious in animal models of various inflammatory conditions, and a couple of compounds have also reached the clinic. The recent failures of the potent and selective DP1 antagonists S-5157 and laropiprant to reach primary endpoints in clinical trials for treatment of asthma and allergic rhinitis does however curb the expectations of new antiinflammatory therapeutics by pure DP1 antagonism. Of the two PGD2 receptors, the role of CRTH2 in inflammation appears much clearer, as a large number of studies conclude that the receptor mediates proinflammatory responses, and consequently point at CRTH2 antagonists as an interesting new class of potential antiinflammatory drugs. Indomethacin and ramatroban were the first non-prostanoid compounds to be identified as CRTH2 ligands, acting as agonist and antagonist, respectively, and a

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large part of the currently known CRTH2 antagonists are structurally related to these two compounds, although several non-indole compound series are also known. The established efficacy and safety profile of the dual TP/CRTH2 antagonist ramatroban against rhinitis indicate that CRTH2 antagonists could represent a safe and efficacious alternative for treatment of allergic conditions. Selective CRTH2 antagonists have exhibited efficacy in several animal models of inflammation [116, 156, 162]. The activity on development of CRTH2 antagonists for treatment of asthma and allergy has been continued at least at the same level as when the first version of this review appeared in 2006. Several companies are currently evaluating CRTH2 antagonists in clinical trials, and some have reached phase II, but the results from these trial have still not appeared in the litterature. All results continue to support CRTH2 as an interesting antiinflammatory target, and the results from clinical trials are awaited with excitement. ABBREVIATIONS 15d-PGJ2

=

15-Deoxy-
12,14-prostaglandin J2

7TM

=

Seven trans-membrane

cAMP

=

Cyclic adenosine monophosphate

CCR3

=

CC-Chemokine receptor 3

COX-1

=

Cyclooxygenase-1

COX-2

=

Cyclooxygenase-2

CRTH2

=

Chemoattractant receptor-homologous molecule expressed on Th2 cells (DP2)

COPD

=

Chronic obstructive pulmonary disease

CYP

=

Cytochrome P450

DK-PGD2 =

13,14-Dihydro-15-keto-prostaglandin D2

DNSS

=

Daytime Nasal Symptoms Score

DP1

=

D prostanoid receptor 1 (previously DP)

DP2

=

D prostanoid receptor 2 (CRTH2)

EP

=

E prostanoid receptors

FEV1

=

Forced expiratory volume in 1 second

FITC

=

Fluorescein isothiocyanate

GPCR

=

G protein-coupled receptor

HTS

=

High-throughput screening

IgE

=

Immunoglobulin E

IL-5

=

Interleukin-5 (also known as CXCL-5)

NSAID

=

Non-steroidal antiinflammatory drug

372 Frontiers in Medicinal Chemistry, 2010, Vol. 5

OVA

=

Ovalbumin

PGD2

=

Prostaglandin D2

PGDS

=

Prostaglandin D synthase

PGF2


=

Prostaglandin F2


PGJ2

=

Prostaglandin J2

PPAR

=

Peroxisome proliferator-activated receptor 

SNP

=

Single-nucleotide polymorphism

Th1

=

T helper type-1 cell

Th2

=

T helper type-2 cell

TP

=

T prostanoid / thromboxane A2 receptor

TX

=

Thromboxane

TXA2

=

Thromboxane A2

Ulven and Kostenis

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Privileged Structures as Leads in Medicinal Chemistry Luca Costantinoa,* and Daniela Barloccob a

University of Modena e Reggio Emilia, Dipartimento di Scienze Farmaceutiche, Via Campi 183, 41100 Modena, Italy; b University of Milano, Dipartimento di Scienze Farmaceutiche “Pietro Pratesi”, Via L. Mangiagalli 25, 20133 Milano, Italy

Abstract: Among the strategies that can lead to the discovery of new drugs, the identification and use of privileged structures, molecular fragments that are able to interact with more than one target, gained particular attention, in an attempt to find new drugs in a shorter time with respect to other strategies. These structures, that have been identified mainly by empirical observations, can target only a given protein family, or can be able to interact with more, unrelated targets. This review deals with structures not covered in recent papers on this topic, and emphasizes the importance of understanding the structure-target relationships, that confer the privileged status.

Keywords: Privileged structures, drug discovery, carbohydrates, benzodiazepines, biphenyls, pirrolynones. INTRODUCTION Despite the recent developments in small scale and high throughput synthesis on solid supports and in solution for the generation of focused libraries, the total output of new pharmaceutical entities has not increased significantly and there is still great interest in new strategies for drug discovery. Besides the screening of sets of compounds by selected biological assays, the modification and improvement of existing active molecules, the selective optimisation of side activities (the SOSA approach [1]), is very attractive, since it deals with compounds that are “druglike” in humans. In addition, the use of “privileged structures” emerged as a possible way to accelerate drug discovery, especially for targets with unknown 3D structure; see for example the G-protein coupled receptors, with only one 3D structure, namely bovine rhodopsin, known to date. The term “privileged structure” was introduced by Evans in 1988 in relation to the heterocycle 1,4-benzodiazepine-2-one, defined as “a single molecular framework able to provide ligands for diverse receptors” [2]. After this discovery, it was noted that some libraries of organic molecules have significantly higher hit rates than typical high-throughput screening results. For example, from an array of 136 spirohydantoins and 132 spiropyrrolo-pyrrols as novel ligands for the neurokinin receptor NK-1 receptor conjugated with the “needle” for *Correspondence Author Tel: 0039-059-2055125; Fax 0039-059-2055131; E-mail: [email protected] Allen B. Reitz / Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.

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NK-1 receptor, the 3,5-bistrifluoromethylphenyl moiety, 97 (hit rate 71%) and 91 (hit rate 69%) high affinity ligands were identified, respectively [3]; thus these spirocompounds, present also in derivatives able to target other G-Protein coupled receptors (GPCRs), have been classified as “privileged structures”. Also spiropiperidines belong to this class of privileged structures; they display activity against a number of GPCRs and have been shown to impart drug-like properties [4, 5]. Many privileged structures have been identified simply by empirical observations; on the other hand, attempts were made using computational methods to classify the known drugs in order to highlight the privileged nature of their molecular skeletons: the results indicate that a large proportion of drug molecules (24%) are based on a small number of molecular frameworks that could be classified as privileged [6]. Several examples of privileged structures include phenyl-substituted monocycles such as biphenyls, diphenylmethane derivatives, 1,4-dihydropyridines, fused (6-7) ring systems such as benzodiazepines, fused (6-6) ring systems such as chromones, quinoxalines/quinazolines, 2-benzoxazolones and fused (5-6) ring systems such as indoles, benzimidazoles and benzofurans [5-11], but there is also incertainty in the identifications of these structures; for example the benzodiazepines are an example of a GPCR privileged substructure either by itself or because they can be considered as an example of a constrained diphenylmethane moiety; both moieties are highly represented in CMC database [6] (compound 1, Fig. 1). While many classes of privileged compounds such as 1,4-dihydropyridines, biphenyls and benzodiazepines here reported and others are highly represented into marketed drugs [5,8], and produce leads, reviewed in [5], with enhanced drug-like properties, other structures such as pyrrolinone compounds were classified as privileged owing to their rigid framework able to direct functional groups in a well defined space, giving new hits for the future development of leads. O N NH

H N

N O 5-terms heterocycle 1 MK-239 CCK-A antagonists

2 aromatic or heteroaromatic 5-terms

Fig. (1). Examples of privileged structures.

In contrast to the “privileged structure” the terminus “needle” was described in the literature as a fragment of an active molecule showing very specific interactions with one particular biological target [12]. Thus one criterion that can be applied to classify a structure or a substructure as privileged is its size (molecular scaffold) relative to the molecule as a whole. The understanding of the molecular determinants that underlie the privileged structuretarget relationships should be the key to apply the full potential of the privileged structure concept in the design of novel targeted compounds libraries. However, the reason for which these compounds are “privileged” is not completely clear. This status arises not only from

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the basis of their versatile chemistry approach, because it was pointed out that, while this is true for compounds possessing a 2-aminothiazole core, it is not true for five-membered heterocycles with a conserved 1,2-diphenyl substitution pattern [10] (general formula 2, Fig. 1). Only recently, for a set of class A GPCRs, a good correlation was found between conservation patterns of residues in the receptor binding pocket (suggesting the existence of a “privileged subpocket”) and the privileged structure fragments of the ligands, e.g. 2phenylindoles, spiropiperidine indane and 2-tetrazole biphenyls. The selectivity versus different GPCRs could be conferred by the presence of several moieties surrounding the privileged core of the compounds, that are able to interact with more variable residues located near the privileged subpocket [13]. Other Authors suggested that, although structures of the GPCRs and their ligand complexes are not yet available, there is evidence of a preferred turn conformation of GPCR-ligands that mediate bioactivity. In fact, a large number of synthetic non-peptidic and peptidomimetic GPCR ligands have been developed, and in many cases they are mimetics of peptide turns [14]. Thus, even in absence of a clear understanding of the reason for this status, the use of the “privileged structures” could provide an empirical alternative both to blind screening attempts and structure-based drug design for the development of new, selective compounds. Natural products can possess the “privileged structure” characteristics, and some recent successful examples will be reviewed. In general, natural products are considered to contain scaffolds with the potentiality to be privileged structures because in many cases they are synthesized by biological systems to specifically interact with protein targets. Natural products differ from synthetic substances under several aspects: they tend to contain fewer nitrogen, halogen or sulfur but more oxygen atoms; they are likely to contain a larger number of rings and more chiral centers [15-17]; thus natural product scaffolds can be used to explore a significant portion of drug-relevant pharmacophoric space. After a period of decline, the role of natural products in drug discovery has undergone a reinassance in the past 5 years, for several reasons; among them, one factor has been the failure of competing technologies such as combinatorial chemistry, to deliver new drug leads in significant numbers, and the understanding that the structures of drugs we use today more closely resemble those of natural products. In fact a multivariate comparison (41 different properties have been considered) of the chemical space occupied by thousand of molecules from three different classes, molecules from combinatorial synthesis, natural products and drug molecules revealed a strikingly good correlation of clinically approved drug molecules with natural compounds, but not with combinatorial output [18]. Thus, since it appears that structural scaffolds of evolutionary selected natural products represent the biologically relevant and prevalidated fractions of chemical structure space explored by nature so far, it is obvious that research tried to start with natural products in order to find new leads. A recent systemical structural classification of natural products has been done (SCONP) on the basis of all of the 193,939 structures from the CRC Dictionary of Natural Products. This classification arranges the scaffolds present in natural compounds in a tree-like fashion and provides a powerful tool for the design of natural product-derived compound collections; these scaffolds were reduced to 25,000, that were clustered in a “parent-child” relationships [19, 20]. The scaffolds are divised into three classes, namely nitrogen heterocycles, carbocycles and oxygen heterocycles, of which two to four ring systems are most

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common. This allowed easy selection of related structures to base library creation for focused screening. It also facilitates the selection of chemically simpler scaffolds that could be expected to maintain the ability to support the desired biological activity. At the same time, the proteins, biological targets of small molecules, were classified into clusters (Protein Structure Similarity Clustering, PSSC) on the basis of the structural similarity around the ligand binding site, since only the combination of fold and sequence determines the binding properties of a protein; the scaffold of a given ligand for one member of the cluster thus can be expected to be a prevalidated starting point for the synthesis of ligands for the other members of the protein cluster [21, 22]. These concepts have been summarized in the overall strategy called Biology-Oriented Synthesis (BIOS). They are fundamentally similar to the privileged structure concept, but BIOS has the added dimension of using protein clustered by three-dimensional shape around the ligand-binding sites as the basis for subsequent screening. Examples of this strategy have been reported, in the field of 11
-hydroxysteroid dehydrogenase type 1, Cdc25A phosphatase and acetylcholinesterase (AChE), three enzymes that display similar ligand binding site and thus, following PSSC, can be grouped into the same cluster. Yohimbine alkaloids (compounds 3-5, Fig. 2) were identified by screening as Cdc25A phosphatase inhibitors; their basic pentacyclic scaffold can be assigned to the indole branch of the SCONP tree, leading to the synthesis of the library 6 (Fig. 2). The screening for phosphatases inhibition led to the discovery of potent and selective inhibitors among a panel of phosphatases, with high hit rates at comparably small library size [23]. H

N N H

H

N

H

N H

H

H

OH

HO

O

H O

O

O Ajmalicine

Yohimbine

4

3

R H

N N H

O O

H

N

R

O N

H O

O

O

O

O

R 6

Reserpine 5

Fig. (2). Design of an term indoloquinolizidine library of phosphatase inhibitors.

In order to find a previously undescribed class of selective and potent inhibitors of 11
hydroxysteroid dehydrogenase (
HSD) type 1, a combined application of SCONP and PSSC was applied. The natural product acid glycyrrhetinic ((compound 7, Fig. 3), inhibitor of 11
HSD1 and 11
HSD2, was analyzed by NP tree; brachiation in the direction of re-

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duced complexity led to a subset two- and three-ring systems. Dysidiolide 8, Fig. (3), is a known natural product inhibitor of Cdc25A, a member of the cluster of proteins together with Cdc25A phosphatase; its structure embodies the octahydronaphtalene scaffold, that has been identified as possible library scaffold by the brachiation approach (NP tree). A library based on this scaffold (162 compounds) was synthesized; among the compounds obtained, 30 compounds showed IC50 values between 0.31-9.1 μM, while 4 were in the nM range; compound 9 (Fig. 3) was the best compound obtained [19].

H HOOC

O

OH

O O

OH

O Dysidiolide 8

H HO

H

Glycirrhetinic acid

Sulfiricin

7

10 decalin derivatives

O IC50 11
HSD1 = 0.31 μM IC50 AChE = 5.3 μM OH 9

O IC50 AChE = 3.7 μM

OH 11

Fig. (3). Design of decalin-based libraries and activities of the most potent compounds.

Analogously, the decalin core motiv of the natural products Dysidiolide 8, Fig. (3), together with Sulfiricin 10, Fig. (3), both inhibitors of Cdc25A phosphatase, served as structural guiding fort the synthesis of a decalin scaffold-based compound library in order to find an AChE inhibitor, an enzyme belonging to the same PSSC as Cdc25A. Of the 162

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compounds thus obtained, the hit rate in AchE inhibition screen amounts to about 2% (IC50 values less than 20 μM). Fig. (3) report the structures of the most active compounds thus obtained, 9 and 11 [24]. At the same time, a new recent approach for lead generation was developed, called Fragment-Based Drug Design (FBDD). It is based on screening in order to identify a key fragment, or set of fragments, that binds to the desired target. Since the fragments are small by design, their binding affinities will be low and, thus, optimization is necessary [25]. At present, several characteristics for fragment library creation have been highlighted, such as the range of physicochemical properties of the fragments, the aqueous solubility, the chemical diversity among these, the chemical tractability for their further development, drug likeness of the fragments and, interestingly, the sampling of privileged medicinal chemistry scaffolds (libraries of such fragments, inspired by the privileged structure concept, are commercially available under the name of “optimers”, http://www.arraybiopharma.com). Given the recognized importance of molecular fragments as source of activity information both in compound and library design [26] computational approaches were undertaken in order to highlight the distribution characteristics of fragments of molecules. Thus, for a number of target classes, attempts have been made to identify privileged substructures that are associated with class-directed compound activity [27, 28] and analyses of large data sets have been used to identify combinations of molecular fragments that are highly recurrent in synthetic compounds [29-31]. More recently, a systematic computational selectivity profile analysis of the BindingDB database, that contains about 31,000 compound entries with about 57,000 activity measurements, was performed. More than 200 molecular scaffolds have been identified that are selective for established target families [32]; these scaffolds could have high potential to yield target-selective compounds. No attempts were made, however, in order to quantify the privileged structures previously identified that are present in this dataset. Several reviews appeared on privileged structures [5-10]. This review deals with structures not covered (or covered only partially) in other reviews, able to interact with a given protein family or present in compounds interacting with more families, expanding the concept introduced earlier by Waldmann [33, 34]. Some of these previously discovered structures [9] were used recently for the design of libraries that target GPCRs. For example, the first potent and selective small-molecule melanocortin-4 receptor (MC4R) agonist resulted from the optimization of the lead that contained a spiroindoline privileged structure [35]. Other potent and selective piperazine-based MC4R agonists were discovered by a screening of another GPCR privileged structure, an arylpiperazine scaffold [36] which was optimized [37-39] and further developed [40, 41]. PRIVILEGED STRUCTURES ABLE TO INTERACT WITH A GIVEN PROTEIN FAMILY The use of “privileged structures” for the discovery of compounds able to interact with a given protein family is very attractive. The analysis of the human genome highlighted about 400 proteins able to become potential targets for the development of new drugs. A large percentage of them can be clustered into families, such as G-protein coupled receptors, serine, threonine and tyrosine kinases, serine cysteine, aspartic and metallo proteases, ion channels and nuclear hormone receptors; the use of “privileged structures” could allow the discovery in a shorter time of compounds able to interact with members of the same family.

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a) Nakijiquinones Nakijiquinones are the only known naturally occurring inhibitors of the Her-2/Neu receptor tyrosine kinase; a library of 56 analogues of this lead structure was synthesized and screened for its inhibitory activity against other receptor tyrosine kinases involved in cell signalling and proliferation, such as the vascular endothelial growth factor receptors (VEGFR1-3), the Tie-2 receptor, the insulin-like growth factor 1 receptor (IGF1R) and the epidermal growth factor receptor (ErbB-1). Whereas none of the natural products exhibited significant inhibitory activity against the new set of receptor kinases, six members of the library of analogues were identified as selective kinase inhibitors in the low micromolar range [42]. b) 2-arylindoles Another example of compounds interacting with members of the same family of proteins is represented by 2-arylindoles, ligands for GPCRs. While the indole ring is one of the most ubiquitous heterocyclic structure found in nature, literature data indicated that 2-arylindole scaffold is scarcerly reported among pharmaceutical compounds. Only recently, it was discovered that 2-arylindole derivatives afforded good ligands for GPCRs. After the discovery, through Merck in-house screening for binding affinity on the rat Gonadotropin Releasing Hormone (GnRH) receptor, a nonpeptidyl GnRH receptor antagonist with a 2-arylindole core was identified [43] and optimized [44]; other derivatives of this class were found to exert high affinity antagonism at the h5HT2a receptor [45]. Then, scientists at Merck synthesized a 128,000-member library; the compound pools were then screened across a panel of 16 GPCRs, including many receptor subtypes. Activity was observed against every receptor, and selectivity was highly dependent on the pattern of 2-arylindole substitution [46]. Nonpeptide agonists of each of the five somatostatin receptors were identified at Merck starting from a cyclic hexapeptide somatostatin agonist (L-363,377, compound 12, Fig. 4); its 3-D model was used as a probe in a search of the Merck sample collection database, and L-264,930 (compound 13, Fig. (4), Ki = 100 nM at sst2 receptor subtype), which possesses the spiropiperidine privileged structure, was discovered as a new lead. L-264,930 is tripartite in structure with an aromatic moiety, a Trp moiety and a diamine moiety, making it quite amenable to a combinatorial chemistry approach. From this effort, among compounds of other chemical classes selective for diffferent receptor subtypes, L-803,087 and L817,818 (compounds 14 and 15, Fig. 4) were discovered, that are selective for sst4 and sst5 receptors respectively [47]. c) Pyridone Derivatives The pyridone or the pyrimidinone substructure (Fig. 5) is often found in Ser protease and Cys protease inhibitors as P3-P2 mimetic, and it was described for the first time in 1994 [48]. This scaffold was proposed as an achiral peptidomimetic constraint for the Ala-Pro dipeptide portion of the peptidic trifluoromethylketone inhibitor of porcine pancreatic elastase, able to reproduce the critical hydrogen bond interactions between the
-sheet of the enzyme and the peptidic inhibitor [49, 50]. PRIVILEGED STRUCTURES ABLE TO INTERACT WITH A GIVEN PROTEIN FOLD The use of privileged structures can also be logically extended to proteins belonging to diverse families, and with different functions but possessing the same folds. Proteins are modularly built from a limited set of approximately 1000 structural domains; protein fami-

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lies can have similar folds even if they catalyse different chemical reactions, owing to different arrangements of the active site residues. As an example, leukotriene A4 hydrolase/aminopeptidase (LTA4H), which has the same fold as M1 metallopeptidases and it is evolutionary related to it, is inhibited by the peptidase inhibitor bestatin though this enzyme catalyzes a very different reaction [33, 34]. Thus, if a ligand for a given domain is known, this could be considered as starting material for the design and synthesis of ligands for proteins that possess that fold, irrespective from the function of that protein [51]. OH NH O

O N H

H N

H2N

O O

N O

NH

H N

N H

NH

H N

NH O

N O

O OH

NH2

13 L 264,930

12 L-363,377

HN NH2

NH2

NH HN

HN

O

O O

O

O

O NH2

F N H

N H F

15

14

L-817,818

L-803-087

Fig. (4). 2-Arylindoles active at somatostatin receptors. X R2

O N

N H

R1

O

X = CH, N

Fig. (5). Pyridones “privileged structures” present in Ser and Cys protease inhibitors.

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2,6,9-Substituted Purines The purine scaffold is very common among pharmacologically and physiologically active compounds. In particular the 2,6,9-substituted purines were initially discovered as Kinase inhibitors, and, despite their molecular mechanism of action (they are competitive inhibitors with respect to ATP) they showed to be rather selective, essentially inhibiting cyclin-dependent kinases CDK1, CDK2, CDK5, CDK7, CDK9 and the MAP kinases Erk1 and Erk2, but not CDK4 and CDK6 [52]. SAR studies showed that the CDK inhibitory properties were limited to 2,6,9-trisubstituted purine subfamily [52, 53], such as olomucine and roscovitine (compounds 16 and 17, Fig. 6).

NH

Cl

NH N

N

N HN

N

N

N

N HN

OH

NH

N

16

N

OH

N

17

Olomucine

Roscovitine

HN

N

N

OH 18 Purvalanol A

O N

N RHN

N

N R

19 19a NU 2058 R = H SO2NH2 19b NU 6102 R =

Fig. (6). 2,6,9-Substituted purines as Kinase inhibitors.

The crystal structures with CDK2 showed that although olomucine (16) occupied the ATP binding pocket of CDK2, its purine ring and that of ATP were not oriented in the same manner; it is rotated of almost 160° relative to that of the adenosine ring of ATP; isopentenyladenine was positioned in yet a third orientation. Roscovitine, purvalanol (18) and other purines are oriented like olomucine, while the apparently related O6-cyclohexylmethylguanines NU 2058 (19a) and Nu 6102 (19b, Fig. 6) were found to bind to CDK2 in a different way [52]. Thus, even if these compounds are very similar, slight modifications around the core of the compounds led to very different binding modes.

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A 2,6,9-substituted purine library developed for the discovery of CDK2 inhibitors was successfully tested also on sulfotransferases. Sulfotransferases and kinases catalyses the transfer of a sulforyl group from the universal sulfate donor 3’-phosphoadenosine-5’phosphosulfate (PAPS) to a hydroxy (or amino) group of the acceptor molecule, while kinases catalyses a similar anion group transfer reaction using ATP as a phosphoryl donor, thus recognizing adenosine-based substrates; moreover, the hydrophobic adenine binding pockets of Estrogen sulfotransferase (EST) and heparin N-sulfotransferase are similar to those of several kinases. Several members of this library of CDK inhibitors were found to be active and selective on carbohydrate sulfotransferase [54] and on EST [55]. 2,6,9Trisubstituted purines were found also to inhibit
-aryl sulfotransferase IV [56], confirming the interest in testing compounds on structurally similar proteins. PRIVILEGED STRUCTURES ABLE TO INTERACT WITH MORE THAN A GIVEN PROTEIN FAMILY a) Carbohydrates Monosaccharides are attractive scaffolds because they are readily available, chiral, conformationally rigid, highly functionalised; they are stable in gastric acid and to glycosidases and display the hydroxy groups in a well-defined three dimensional arrangment as vectors for the introduction of different side chains; owing to their characteristics, several papers dealt with these compounds as “privileged structures”. In fact it has been shown that properly functionalised
D-glucose scaffold can bind diverse GPCRs [57-64] and modulation of receptor and receptor subtype affinities can be achieved using diastereomeric and enantiomeric monosaccharide scaffolds as a means to structural and biological diversity. Specific conformations of peptides have been known as the key determinants of recognition in a number of signalling processes in biological systems. In 1987 it was suggested that nonpeptidal mimetics of beta turns can be generated via the attachment of appropriate side chains to monosaccharide scaffolds typified by glucose [61]. Following this approach, a fully substituted
D-glucose was designed to bind a receptor of the cyclic tetradecapeptide hormone somatostatin-14 (SRIF) (Fig. 7) because it was noted that there is no evidence for a direct interaction of the amide backbone of peptide hormones with their receptors [65]. SRIF, beta adrenergic and Substance P (SP) receptors utilize G-protein mediated signal transduction. It was noted that the bioactive conformation of SRIF and of its cyclic hexapeptide analog L-363,301 (20, Fig. 7) contain a
-turn, which resulted to be important for its activity.
-D-glucose scaffold mimicks the peptide backbone of L-363,301 and three of the four side chains of the beta-turn (Phe7, Trp8 and Lys9) were positioned on the sugar scaffold. Moreover, subtle modifications on a sugar scaffold can generate various levels of specificity and potency against different receptors and receptor subtypes. The glycoside 21a binds somatostatin receptor subtypes with a Ki of 15 μM, and displays the highest affinity at subtype 4 (sst4) [60]; 21a was also found to bind the NK1 receptor of Substance P (SP) as an antagonist, with a Ki of 150 nM [61]; it has been hypothesized that its ability to bind the SRIF, SP and
2-adrenergic receptors reflects not only a similarity among the three corresponding GPCRs, but also the high degree of pseudosimmetry in the sugar scaffold [65]. Compound 21b is
2 adrenergic antagonist with an IC50 of 3 μM [61]. Moreover, the SAR of 21b resembles those of the cyclic hexapeptide L-363,301 (20). In fact, replacing the Phe7 of 20 and 21b (leading to 23) by His enhances potency [63]. Conversely, 23 binds sst4, but not the NK1 receptor [63]. Nevertheless, compound 24a has a

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Ki of 27 nM at the NK1 receptor [62], but does not bind sst4, and the corresponding free amine has a comparable affinity (Ki 66 nM) at the NK1 receptor. Thus analogues of 21a were selective for either SRIF or NK1 receptors. On the basis of the results obtained, a new glucoside (comp. 24b, Fig. 7) was created which has a high affinity at human SRIF receptor (Ki = 53 nM) at sst-4 [63]. These results underline the importance of this scaffold, which is useful because adequate specificity and potency can be achieved efficiently via modulation of its substituents. Later, catechol was identified as the simplest scaffold that can replace the 
D-glucose ring, while maintaining the side chain topology of a  turn [66]. Compound 22a binds the sst4 receptor with a Ki value of 2.02 μM, very similar to 21a (Ki = 1.65 μM); the derivative 22b, while showing the same activity at sst4, displayed a 9-fold affinity enhancement at the sst2 receptor.

O

O O

N O

O N H

NH

H N

N H

NH

H N

O R

O OH

O

NH2

O

O

NH2

O

SRIF agonist L-363,301

20

NH

21 21a R = OBn 21b R = H HN N

O

R O

O

O

NH

O

NH

O

O

NH2

NH2

22a R = H 22b R = CH2-C6H5

23

O

R2

O R1

O

O O

NH

O

N H

R3

24a: R1 = H; R2 = CH2-C6H5; R3 = -COCH3 24b: R1 =

R2 =

HN

N

Fig. (7). SRIF agonist and monosaccharide-based mimetics.

R3 = H N

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Other D-glucose derivatives were found to exhibit similar efficacy as hapalosin, a cyclic depsipeptide isolated from the cyanobacterium Hapalosiphon welwitschii, in antagonizing multidrug resistence-associated protein (MRP) [64]. Monosaccharides are an useful scaffold also for the development of HIV-1 protease (aspartic protease) inhibitors (Fig. 8). In order to develop a peptidomimetic inhibitor of the HIV-protease, the inhibitor 25, containing the hydroxyethylene isoster common to many Asp protease inhibitors [67], was used as a lead. Molecular modeling experiments showed that the
-D-mannopyranoside ring has good overlapping with the peptide-like backbone and the hydroxyethylene isostere of the inhibitor bound to HIV protease and could be a replacement for part of the peptide that binds to enzyme. In Fig. (8) is reported the inhibitor 25 in the bound conformation and the
-D-mannopiranoside-based HIV-protease inhibitor 26, provided with an IC50 value in the micromolar range [68]. S2

NHAc OH NH

S1'

S1

O N HN

25 HIV protease inhibitor in its enzyme bound conformation.

O S2'

NHAc

S3 O S2 HN

P2 O P1'

O

P3 26

OH O P1

O

S1

O

S1'

hydroxyethylene isostere mimic

Fig. (8).
-D-Mannopyranoside based HIV protease inhibitor.

Beta-D-mannopiranoside based HIV-protease inhibitor

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The cyclic hexapeptide including the bioactive Leu-Asp-Thr motif is a potent inhibitor of the Mad-CAM-1/
47 ligand integrin interaction (compound 27, Fig. 9). The bioactive conformation of these cyclic peptides consists of two -turns with the D-proline in the i+1 position of a II’-turn and aspartic acid in the i+1 position of a I and/or II-turn. The sugar scaffold present in compound 28 is able to reproduce the activity of 27 by presenting the pharmacophoric LTD side chain mimetics in a similar spatial orientation [69].

COOH

O H N

COOH O

HN

O

O O

O

O

O

NH O

O

N

O

O HO

28

27

Fig. (9). Carbohydrate-based Mad-CAM-1/
47 ligand integrin inhibitor.

Pyranoses were used also as templates to design thrombin inhibitors [70] (Fig. 10). Thrombin is a key Serine protease enzyme of the coagulation cascade and possesses high specificity for Arg-containing peptide sequences. Thus 6-guanidino hexoses (compounds 29, 30) have been proposed as conformationally restricted Arg mimetics. To judge the selectivity of these thrombin inhibitors, the compounds were tested against the related but less discriminating Ser protease trypsin. Inhibitors 29 and 30 have Ki of 0.9 and 1.1 μM, respectively; 29 is selective (>227) for thrombin vs trypsin, underlining the ability of guanidinohexoses as Arg mimetics highly functionalizable. HN HN

HN NH2

NH2

HN O

HO

O

HO HO NH SO2 OBn

29

HO NH SO2 OBn

30

Fig. (10). Carbohydrate-based thrombin inhibitors.

New scaffolds [71-75] such as sugar aminoacids (general formula 31, Fig. 11), carbohydrate molecules bearing both amino and carboxy functional groups on the regular sugar framework, were developed. In these compounds aminoacids, sugars and nucleosides are amalgamated to produce nature-like structural entities with multifunctional groups anchored

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on a single ensemble. These scaffolds exist in nature in the form of neuraminic and muramic acids as cell wall components and also in some natural products. This sugar aminoacid is a conformationally restricted dipeptide isostere (in Fig. (11) is reported the structure of a sugar aminoacid 31 and the corresponding dipeptide 32) that can be used to replace two adjacent aminoacids, inducing a -turn in linear peptides [75]. OH HO

OH

H2N

OH O O 31 O

H2N

R OH

N H O 32

Fig. (11). Sugar aminoacids and the corresponding dipeptide unit.

These molecules have been used in recent years as novel monomeric entities as dipeptide isosteres in peptidomimetics, giving enkephalin [71] (Fig. 12a), somatostatin [75] analogs and integrin (Fig. 12b) antagonists. They are also present in enzyme inhibitors, such as Ras farnesyl transferase (Fig. 12c) and ribonucleotide reductase (Fig. 12d). Integrins are a superfamily of heterodimeric transmembrane glycoproteins that function in cellular adhesion, migration and signal transduction. To date, 17
-subunits and 8 subunits have been identified, which associate selectively to form at least 23 integrins [76]. Two members of this family, the
v3 and
IIb3 (vitronectin) receptor, have been studied extensively; the
v3 plays a key role in angiogenesis, tumor metastasis and acute renal failure, while the
IIb3 mediates platelet aggregation. Both receptors recognize the consensus sequence Arg-Gly-Asp (RGD) of their natural ligands [76, 77]. Several scaffolds “privileged structures” have been successfully investigated for the discovery of nonpeptide
v3 antagonists: they include carbohydrates, biphenyls and benzodiazepines. The antagonists at the
v3 receptor share two common structural features that are determinants for receptor recognition: a carboxylate and a guanidinium-like moiety, mimicking the Arg and Asp side chains of the adhesive RGD sequence and the selectivity arises mainly from the conformation of this motif [78, 79], besides to the sequences flanking the RGD triad. This modifies the distance between Arg and Asp side chains; the turn types (type II’ and Type I -turns) incorporating the RGD sequence modify this distance and hence the specificity for integrins [79]. Cyclic RGD-furanoid sugar aminoacid peptides (35) have been shown to be useful
v3 and
IIb3 inhibitors [80]; pyranoid sugar aminoacids (36,37) were used to replace the two aminoacids D-Phe-Val of the cyclic peptide specific
v3 integrin antagonist, cyclo RGDf(N-Me)V. It is known that the backbone conformation of these two residues resemble

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a II’ turn to force the RGD sequence into a
v3 selective conformation [81];
v3 and
IIb3 inhibitors were thus obtained [82]. a) Leu-Enkephalin analogs HO

O H N

H2N

O H N

N H

O

OH

N H

O

Leu-Enkephalin

O

33 HO HO

OH O

H N

H2N

H N

O

O

Sugar-derived Leu-Enkephalin analogues

OMe

N H

O

O

34

b) Integrins inhibitors

O

OBn

Arg-Gly-Asp BnO

OBn OBn

BnO

OBn

NH

O

O

O HO

HN

OH 35

O

O HN

Asp-Gly-Arg

Asp-Gly-Arg 37

36

c) Protein farnesyl transferase inhibitor HS O A H2N

HS

H N A

O

HO

OH S

O

H N

H2N

O

O CAAX sequence 38

H N O

Ras FTase inhibitor 39

OH S

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Fig. (12). contd….

d) Ribonucleotide reductase inhibitors

COOH O O O

COOH

NH

H N

AcHN

O

OH

O

H N

N H

OH

Peptidic inhibitor of RR

O

O

40

COOH O

O H

COOH

O NH

AcHN

O

H N

N H

O

Sugar-based RR inhibitor

O OH

O 41

Fig. (12). Sugar aminoacids as dipeptide isosteres.

The enzyme Ras farnesyltransferase (FTase) modifies Ras family of proteins, that regulate the transduction of biological information from the plasma membrane to the nucleus; its inhibition may provide a new target in cancer chemotherapy. In order to be active, FTase attaches a farnesyl group to the Cys residue of the Ras carboxy terminal sequence CAAX (C, Cys, A, any aliphatic aminoacid, X, Ser or Met); this reaction is followed by the removal of the AAX tripeptide. Potent FTase inhibitors can be obtained by replacing the AA moiety by a rigid spacer, e.g. a sugar aminoacid, in the CAAX motif [73] (compound 39, Fig. 12c). Ribonucleotide reductase (RR) is an enzyme that plays a critical role in regulating DNA replication, and it is a target for the design of antiviral and cancer chemiotherapeutical agents. Type I RRs, such as mammalian RR, can be inhibited by peptides corresponding to the C-terminus of the R2 subunit such as 40. Compound 41 (Fig. 12d), the tetrahydropyranbased inhibitor, is also active [74].

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Given the biological relevance of both the
D-glucose and the benzodiazepine scaffolds, an hybrid scaffold was designed (compounds 42-44, Fig. 13). The comparison of the minimized structures of these new scaffold originated by the fusion of D-glucose and benzodiazepine scaffolds showed that they have the potential for increased sampling of receptor space relative to the classical benzodiazepines [83, 84]. However, no biological data of compounds containing these scaffolds are available yet. O X1

NH

O

OMe

X1

O 42

NH

X2

O

X2

O

N H

O

OMe

O 43

X1 = NO2; X2 = NH X1 = H; X2 = NH X1 = NO2; X2 = O

O

X1 = N; X2 = CH X1 = CH; X2 = N

O X

NH

OMe O

N H O 44

O X = NO2 X=H

Fig. (13). Hybrid scaffolds between benzodiazepines and sugars.

b) Biphenyls This substructure is found in 4.3% of all known drugs, targeting both GPCR and non GPCRs. In examining the binding of 10080 compounds to 11 different proteins by NMR, Fesik et al. demonstrated that compounds containing a biphenyl motif bound to 50% of the examined proteins, second only to a carboxylic acid group [85]. The Comprehensive Medicinal Chemistry Database (1996) listed the following distinct therapeutic classes for molecules containing this framework: antiamebic, antifungal, antiinfective, term antihypercholesterolemic, antihyperlipoproteinemic, antirheumatic, analgesic, antiinflammatory, antithrombotic, uricosuric, and antiarrhytmic [8]. Biphenyl-containing compounds are enzyme inhibitors (Ser protease: Factor Xa; DNA-dependent protein kinase, oxidosqualene cyclase) and are able to interact with GPCRs (ET antagonists, Histamine H3

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antagonists) and non GPCRs (Integrin
v3 inhibitors, Potassium channels modulators) [8, 86, 87]. Recently it was also shown that this substructure is present in Follicle stimulating hormone receptor agonists [88] and in Gram (+) antibacterial compounds [89]. Some compounds containing this moiety were discovered on the basis of SOSA approach, and not on the basis of the biphenyl moiety as privileged structure. A successful SOSA approach identified the antibacterial sulfonamide sulfathiazole as a ligand of the endothelin ETA receptor and its optimisation to the selective and potent compound BMS207940 [90, 91] (compound 45, Fig. 14). N

O

O HN O2 S

O

N

N H

45 BMS 207940

Fig. (14). Endothelin ETA ligand BMS 207940.

There is crystallographic evidence of the preference for biphenyls-containing inhibitors of matrix metallo proteinases (compound 46, Fig. 15). Cl O H N

O O

S O

NHOH 46

Fig. (15). MMP inhibitor.

Matrix metalloproteinases (MMPs) are a family of structurally related zinc-containing enzymes that mediate the breakdown of connective tissue and are therefore targets for therapeutic inhibitors in many inflammatory, malignant and degenerative diseases. The needle is the hydroxamic acid moiety, able to chelate the active-site Zn++. The inhibitors of these enzymes very often possess a P1’ biphenyl substituent able to interact with the S1’ pocket, which is a dominant factor for inhibitor binding [92]. The preference for biphenyls was rationalized by the degree of flexibility about the aromatic linkage or because it is simple in size and shape and may conform to a wide variety of protein pockets.

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In the search of inhibitors for Neurokinin (a family of peptide neurotransmitters and neuromodulators implicated in a variety of biological disorders), the biphenyl structure was selected (Fig. 16) because it can place the two aromatic rings of compounds of general formula 47, postulated pharmacophore elements for the NK1 receptor in the correct orientation for binding, and also for the ease of rapid optimisation of the biological activity through aryl-aryl coupling reaction [93]. Several compounds of general formula 48 exhibit potent affinities for both NK1 and NK2 receptors. CF3 R3

R1

O

O

N

NR4R5

N F3C

NR1R2

O

O R2 48

47

Fig. (16). NK inhibitors based on the biphenyl scaffold.

More interestingly, the biphenyl moiety was proposed, on the basis of its substitution pattern, as a -strand mimetic, as
-helix mimetic or as a -turn structure. A -strand mimetic is very useful in order to obtain compounds able to interact with proteins. In fact it is known that specific conformations of peptides are the key determinants of recognition in a number of signalling processes in biological system. The active sites of all five protease classes (metallo, serine, cysteine, aspartic and threonine proteases) and other proteins such as major histocompatibility complex (MHC) proteins and transferases [94] were found to recognize peptidic and non-peptidic ligands in an extended beta strand conformation [95]. Biphenyl has been incorporated into inhibitor design as extended dipeptide mimics (Fig. 17) of Ras farnesyl transferase (compound 49, Fig. 17). It has been recognized that CAAX tetrapeptides inhibit FTase in vitro; thus, in order to improve cellular uptake and stability toward proteases, a series of 4-amino-3’-carboxybiphenyl derivatives as mimics of the ValIle-Met tripeptide but with restricted conformational flexibility was designed [96]. Other enzyme inhibitors that contain this moiety targeted Factor Xa (compound 50, Fig. 17); it binds this Ser proteases in an extended conformation [97]; this scaffold is present also in
41 integrin antagonists [98]. HS H2N

H N

H2N

O O

COOH HN O

49

50 O H2N NH

Fig. (17). Ras FTase and Factor Xa inhibitors.

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Based on theoretical arguments, 2,6,3’,5’-substituted biphenyl analogues (51, Fig. 18) were proposed as protein
-helix mimetics, able to superimpose the side chains of the residues i, i+1, i+2, i+3 and i+4 [99]. The importance of this scaffold lies on the fact that several cases are reported in which
-helix recognition is evidenced by crystal structure analysis and which highlight the potential relevance of
-helix mimetic scaffolds for therapeutic applications [99]. However, to date there is no crystallographic evidence of an interaction of a biphenyl with an
-helix recognition site; moreover, the 2,6,3’,5’-substitution pattern is very uncommon. Most of the CMC biphenyl drugs are mono- or disubstituted biphenyls and the ortho and para positions are the most frequent substitution sites as observed in angiotensin AT1 GPCR antagonists [99]. Only terpenyl derivatives, that contain a biphenyl unit, were reported to be
-helix mimics, able to reproduce the projection of functionality on the surface on an
-helix. This structure is present in inhibitors (52a) of protein-protein interactions, and in particular between calmodulin and small muscle myosin light chain kinase [100]. Moreover, on the basis of the interaction between the tumor suppressor p53 in an helical conformation and the hydrophobic cleft of its target, the protein hDM2, Hamilton’s group discovered other terpenyl derivatives based on
-helix mimicry (52b and 52c, Fig. 18) as potent Bcl-xl-BAK and p53-hDM2 interaction inhibitors antagonists [101-103]. Moreover, a series of alternative
-helix mimetic scaffolds with more drug-like character than terpenyl, based on polyamides and biphenyldicarboxamide 53 (Fig. 18) were discovered as Bcl-xL-Bak interaction inhibitors [103, 104]. R1 HOOC

R2

O

i+3 i+4

i

R3

R NH OR

R4

OR

R5

i+1 51 R6

O

52

NRR 53 O

52a: R1 = R3 = H

R2 =

R4 =

R5 =

O

R6 =

O O

O OH

52b: R1 =

R2 = R5 =

R3 = H

R4 =

R6 =

O O 52c: R1 =

OH

OH

O OH

R2 =

R3 = R4 = R5 = CH3

R6 =

OH

Fig. (18). Biphenyl-containing scaffolds as a mimic of
-helix.

While a recent molecular dynamics simulation predicted a relatively poor beta turn stabilisation/imitation potential for biphenyl structures with a substitution pattern as in 54 (Fig. 19) [105], other biphenyls have been successfully utilized as beta-turn mimetics [106] in the field of somatostatin analogs. The peptide hormone Somatostatin contains a -turn in its bioactive conformation. A potent cyclic hexapeptide analogue (MK-678) contains the

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bioactive conformation. A potent cyclic hexapeptide analogue (MK-678) contains the sequence Tyr-DTrp-Lys-Val, of which the central DTrp-Lys dipeptide forms the i+1 and i+2 residues of a II’-turn necessary for high affinity receptor binding. A new scaffold based on a biphenyl is capable of presenting a tetrapeptide sequence in a -turn conformation, and the compound DJS 811 (compound 55, Fig. 19) [106] thus obtained has an affinity towards SSTR2B and SSTR5 high as 1 nM. The discovery of a cyclic peptide
V3 antagonist (c-RGDfV, 56) with known 3-D structure bodes well for the use beta-turn mimetic templates in the development of vitronectin receptor antagonists. On this basis, new biphenyl series (general formula 57) of small molecule RGD mimetics with potent vitronectin (
v3) receptor antagonism were discovered (general formula 58). Docking experiments showed a similar binding mode of these compounds with the cyclic pentapeptide c-RGDf(N-Me)V [107, 108]. In the search for orally bioavailable
IIb3 antagonists, Boehringer identified fradafiban (BIBU-52, 59, Fig. 19) which has an IC50 = 80 nM. This compound was designed using the assumption that RGDX ligands formed either a - or -turn when bound to the receptor and that a -lactam could be used as a scaffold which, however, represented only a part of the turn structure [109]. O Tyr-D-Trp COOH

NH2

N H

Val-Lys

55

54

DJS811

COOH

COOH H N

cRGDfV

H N

HN

O R2

56

HN

NH 57

NH2

58

NH H2N

O HN 59 BIBU-52

COOH O

Fig. (19). Compounds incorporating a biphenyl scaffold as a -turn mimetic.

SO2

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As the biphenyl moiety is known to be a privileged motif for binding to GPCRs, recently a library of these derivatives was designed specifically to be cross-screened against a range of 7-TM receptor targets. Selective compounds as 5-ht5A receptor antagonist were obtained [110]. Another approach, termed Substrate Activity Screening (SAS) has been considered in order to find in a short time protein tyrosine phosphatase PtpB inhibitors encoded by Mycobacterium tubercolosys. This approach is based on the conversion of the optimized substrates in inhibitors by direct replacement of the phosphate with known phosphate isosteres. On the basis of the structures thus discovered, it was recognized the importance for this enzyme of the biphenyl moiety, which is present in the most potent inhibitor thus discovered, which is also the most active inhibitor of this enzyme reported to date [111]. The same moiety was also recognized to be very important for the obtainement of compounds able to inhibit protein tyrosine phosphatase 1B [112]. Moreover, fragment-based drug discovery (FBDD) was successful in the design of high affinity inhibitors of metalloproteinases, a family of Zn++-dependent endopeptidases that are implicated in various diseases, including arthritis and tumor metastasis. Optimization of the initial biphenyl-based fragment led to ABT-518, wchich showed excellent oral antitumor efficacy in animal trials and was approved for phase 1 clinical trials [113]. Another successful result, based on FBDD, was obtained in the field of inhibitors against the antiapoptotic BCL-2 family of proteins (starting from the biphenyl fragment, the optimized product, ABT-737, showed potent antitumor effect in animal models [114] c) 1,4-Benzodiazepines The class of benzodiazepines (BZD), present also in a natural compound, Asperlicin, was called for the first time “privileged structure” owing to its ability to bind to CCK, gastrin and central BZD receptors. These compunds are able to bind a great number of target belonging not only G-protein coupled receptors, but also to enzymes. In addition, they can modulate ion channels [8,9]. Interestingly, the addition of a single methyl group to a BZD substituent converted a CCK-A antagonist to an agonist; similar modification of a biphenyl substituent produced partial agonism in the all antagonists series. The same phenomenon happens with benzodiazepines as ligands for K+ ion channel: the delayed rectifier K+ current modulator L-364,373 activates Iks, the slowly activating delayed rectifier K+ current, an important modulator of cardiac action potential repolarization [115], while another benzodiazepine compound, (L-735,821) is a potent and selective blocker of cardiac Iks [116]. Apparently, the appended functionalities of these privileged structures bind in receptor regions which are quite sensitive to the expression of either agonist or antagonist activity [9]. 1,4-BZD has been introduced internally to the peptide sequence Ac-DEVD-H (Ac-AspGlu-Val-Asp-CHO, compound 60, Fig. 20), a semispecific inhibitor of caspase-3, as a conformational constraints. Caspase-3 is a member of the caspase family of cysteine proteases which plays an important role in many human disorders; the aldehyde functional group reversibly forms a covalent bond with the thiol of the active-site Cys. The introduction of the 1,4-BZD as a P3-P2 dipeptide replacement led to the discovery of a potent and specific inhibitor (61) of caspase-3 [117]. The BZD nucleus was proposed as -turn or as an
-helix mimetic. According to its property to act as a -turn mimetic, the 1,4-BZD nucleus was then selected in order to mimic the C7 turn and the extended Gly conformation of the potent cyclic peptide SKF

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107260 during the search for nonpeptide antagonists of
IIb3 (Fig. 21). Of the two conformers of SKF 107260, the major, which contains a turn at the Arg residue, an extended conformation of the Gly, and a C7 turn at the Asp was chosen for exploration. The 1,4-BZD nucleus was then selected in order to mimic the C7 turn and the extended Gly conformation, reported in a two-dimensional representation in 62, resulting in several potent antagonists like SB-208651 (63) and G-6249 (64) [76, 118]. Benzodiazepinones thus are able to appropriately position the acidic and basic groups of the RGD framework [76]. COOH O H N

O H N

N H

O

H

O HO COOH

N H

H N

O

COOH

O

N

O

N

N H

O

COOH

HN

O

O

O

P1

P3

61

60 Ac-DEVD-H

Fig. (20). Benzodiazepine as a conformational constraints of the peptide AcDEVD-H.

O

HN

HN H N

H2N

H N

NH

O

O

COOH

N H

O 62

63 N

H2N

N H

O NH

SB-208651

O

N

COOH O

N 64 N O

G-6249 COOH

H2N NH

Fig. (21). Portion of the peptide SKF 107260 containing the RGD sequence and examples of active centrally-constrained RGD mimetics whose design followed the conformation of RGD sequence.

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Two selective compounds (65, 66) based on the guanidine mimetic groups are reported in Fig. (22). Probably
v3 recognizes a shorter Arg to Asp distance than
IIb3. Thus the benzodiazepine nucleus is very versatile as a Gly-Asp mimetic, as potent nonpeptide antagonists for the BZD series can be designed with selectivity for either
IIb3 or
v3 simply by altering the length and nature of the Arg mimetic [119]. Docking studies on
v3 with benzodiazepines and cyclo RGDf(N-Me)V were recently reported that highlight the binding mode of these compounds [120]. O

O N

N

N

O N H

HN

N

N

O

NH N H

COOH

COOH

66

65

SB 223245
IIb3 Ki = 30000 nM
v3 Ki = 2 nM

SB 214857
IIb3 Ki = 2.5 nM
v3 Ki = 10340 nM

Fig. (22). Influence of the guanidine mimetic on the integrin receptor selectivity.

The BZD scaffold is present also in RAS-farnesyl transferases (R-FT) inhibitors (Fig. 23). Inhibitors of FTases have been developed by mimicking the carboxy terminal CAAX motif (where C is a Cys residue, A is any aliphatic aminoacid and X is usually Met, Ser or Ala) which is the signal for farnesylation of RAS proteins. The inhibitor 67 possesses a benzodiazepine template which, though appearing unrelated to the original peptides, contains the necessary groups positioned on a novel nonpeptide scaffold to serve as topographical mimetic [121]. HS N

N

O

N

H2N O HOOC

H N O

SMe

67 BZA-2B

Fig. (23). Ras FTase inhibitor with the BZD that replaces the AA in the CAAX motif.

In the design of beta turn peptidomimetics, it is advantageous to have a rigid molecule that could mimic any beta turn structure and position the four side chains (for the i, i+1, i+2, i+3 residues) in their proper orientation as determined by X-ray crystallography of beta turn sequences in known proteins. Benzodiazepines show a remarkable fit to each one of the beta turn examples found in proteins. Confirming this hypothesis, a four aminoacid sequence, which is known to adopt a beta turn conformation, can be substituted by the benzodiazepine nucleus placed in a peptidic sequence, still retaining the beta turn conformation and the po-

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sitioning of the side chain functionality [122]. When BZD beta turn mimic was incorporated into a biologically active peptide (Gramicidin S) that contains beta turns, the compound thus obtained exerts an equivalent biological activity. Thus physical (NMR studies) and biological models of BZD peptidomimetics support the role of this molecular unit as an effective beta turn replacement [123]. The 1,4-benzodiazepin-2-one framework, which is a good mimetic of a number of
-turn types, was selected as nonpeptide leads on the basis of the template HOE 140, a Bradykinin (BK) B2 receptor antagonist, characterized by C terminal
-turns comprising residues 6-9 (68, Fig. 24). Elevated levels of the endogenous hormonal nonapeptide Bradykinin have been implicated in numerous pathophysiological processes, many of which are believed to be mediated by the B2 class of BK receptors. Two of the compounds of general formula 69 have been found to exhibit moderate Ki values at the BK B2 receptors [124]. Ser6 D-Tic7

HO H-(D)-Arg-Arg-Pro-Hyp-Gly-Thi-

N

N H O

NH H2N

O

H N

N H

COOH O

H N

Oic8 H

Arg9 68 HOE 140 C-terminus
-turn

Projects towards D-Tic7 in (R) enantiomer

Basic -Link function

N

N O

Hydrophobic side chain

69

Mimics Arg9

Mimic Oic8

Fig. (24). Benzodiazepine scaffold as a mimic of the bradykinin B2 receptor antagonist, HOE 140.

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On the basis of the importance of beta turn conformation for biological recognition of peptides and proteins, the concept of Garland and Dean [125] was applied in order to find scaffolds with a maximum overlay with a -turn conformation in order to design -turn non peptide scaffolds [94]; spiropiperidine nucleus was thus selected in order to target somatostatin receptors (general formula 70, Fig. 25), of which ligands have been studied as turn peptides [47, 59]; the derivatives such as 71 showed activity to SST2 receptors [126]. A similar strategy led to a library of tetrahydro-1,4-benzodiazepin-2-one [127] (general formula 72, Fig. 25), but no biological results are available at the moment.

O O

R3

R2

R1

N

N

O

O O

O

NH

N

N

N

N O

R4

NH2

71

70

H N

R1

Beta-turn scaffold mimic

O N R3 R2

O

72

Fig. (25). Elaboration of beta turns non-peptide scaffolds.

However, on the basis of the -sheet bioactive conformation of SRIF-14, a benzodiazepine derivative 73 (Fig. 26), with a different substitution pattern from that reported above [127] was discovered as non-peptide mimic of somatostatin [128]. The versatility of this scaffold is evidenced by its ability to act also as an
-helix mimetic and to act in this case as protein-protein interaction inhibitor. This activity is particularly important because it was recognized that many proteins exert their biological roles as components of complexes, and their functions are often determined by these reciprocal interactions. Thus, the availability of compounds able to interfere with such specific interactions is important to influence their function. HDM2 (compound 74a, Fig. 27) binds to an
helix transactivation domain of p53, inhibiting its tumor suppressive function. Antagonists were discovered by screening, and the crystal structure of the optimized derivative bound to HDM2 revealed that the benzodiazepine compound is able to act as
-helix mimetic [129134]. The optimized derivative, 74b, is orally bioavailable [131].

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O N NH

N O

73 NH2

Fig. (26). Benzodiazepine mimic of the -sheet bioactive conformation of somatostatin. R1

O

N Cl N

I O

R

Cl 74 74a R = COOH

R1 = H

74b R = CH3

R1 =

OH O

Fig. (27). Benzodiazepine
-helix mimetic.

d) Benzopyran Derivatives A library of more than 10.000-membered natural product-like was based on the 2,2dimethyl benzopyran template [135-137] (General formula 75, Fig. 28). O Y X 75 Original 10,000 member screening library

Fig. (28). Benzopyran derivatives.

This structural motif is found in numerous natural products with diverse biological activities, suggesting its capability to interact with a variety of cellular targets. This template is present in more than 4000 compounds including natural products and designed structures.

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Moreover, this template was considered owing to its ability to accommodate a great degree of diversity via solid phase split and pool synthesis, it contains a certain degree of rigidity which allow to explore the space in a well-defined fashion, it is sufficiently lipophilic to ensure membrane penetration and finally the molecular weight of the final compounds is less than 500. According to the hypothesis, this scaffold proved able to produce, after optimisation, very active compounds which interact with several, unrelated targets. Following the screening of the library, the most active were optimised; several highly active compounds were thus obtained against methicillin-resistent Staphylococcus Aureus [138]. The original libraries furnished also leads as nuclear receptors Farnesoid X receptor (FXR, a transcriptional sensor for bile acids, the primary product of cholesterol metabolism) activators; these compounds were systematically optimised employing parallel solutionphase synthesis and solid phase synthesis to provide four classes of compounds that potently activate FXR. Some of these compounds are the most potent FXR agonists reported to date in cell-based assays [139]. A number of benzopyran-based inhibitors of NADH:ubiquinone oxidoreductase were also discovered. NADH:ubiquinone oxidoreductase (complex 1) is the first of three enzymes located in the cell’s inner mitochondrial membrane which form the electron transport chain that carries electrons from NADH to molecular oxygen during oxidative phosphorilation. The evaluation of these inhibitors in a number of cancer types and cell lines resulted in the identification of several potent cytostatic and non-cytotoxic compounds [140]. The 2-phenylchromone (flavone) derivatives contain the benzopyran nucleus and display also a remarkable array of biochemical and pharmacological actions, some of which suggest that certain members of this group of compounds may significantly affect the function of multiple mammalian cellular systems [141,142]. They inhibit a huge amount of enzymes such as Histidine decarboxylase and DOPA decarboxylase, Hyaluronidase, lactic dehydrogenase and pyruvate kinase, Catechol-O-methyl transferase, aldose reductase, Protein kinase C, Protein tyrosine kinase, phospholipase A2, Adenosine triphosphatases, Phospholipase C, Ornithine decarboxylase, amylase, sialidase, adenylate cyclase, cyclic nucleotide phosphodiesterase, RNA and DNA polymerases, topoisomerase, reverse transcriptase, glutathione S-transferase, epoxide hydrolase, glyoxalase, lipooxygenase and cyclooxygenase, aromatase and xanthine oxidase. Very few libraries of these compounds were prepared [143-146]. Some naturally occurring flavonoids, as well as the flavone nucleus itself, were found to be ligands for the central BZD receptors. The compounds obtained showed a wide spectrum of pharmacological profiles, ranging from full agonists, to partial agonists and antagonists [145]. The flavonoids are able to interact with proteins able to recognize the adenine ring; they interact with the adenosine receptors [147] and are able to interact with the ATP-binding site of the kinases, and inhibit estrogen sulfotransferase too [143]. In particular Quercetin (3,5,7,3’,4’-pentahydroxyflavone), the most studied member of this class of compounds, is able to act as a competitive inhibitor with respect to ATP for Adenosine triphosphatases [141], cyclic nucleotide phosphodiesterase [141], P-glycoprotein [148], protein kinase C [149] and protein tyrosine kinase [150,151]. However the importance of quercetin as a lead is under discussion because it was shown to interfere with in vitro assay methods adopted for kinases. Quercetin is able to form molecular aggregates, as determined by dynamic light scattering and electron microscopy, with particle size of 30400 nm diameter; these particles represent the inhibiting entity in numerous enzyme assays

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[152, 153]. Nevertheless, quercetin has been a good starting point for the development of selective inhibitors of protein tyrosine kinase p56lck over protein serine/threonine kinases [151]. Despite the fact that there are more than 500 protein kinases in the human genome and that there is a striking conservation of the core ATP-binding phosphotransfer domain (ATP grasp fold) among the members of the protein kinase superfamily, several selective ATPcompetitive inhibitors of cyclin-dependent kinases were discovered in this class of compounds. The cyclin-dependent kinases are Serine/threonine protein kinases, which are the driving force behind the cell cycle and cell proliferation. Two classes of inhibitors of the CDKs, the flavonoids and the purines (olomucine, roscovitine) have been discovered. The flavone derivative L868276 (deschloroflavopyridol, a derivative of the natural product rohitukine (76, Fig. 29) isolated from Dysoxylum binectariferum [154]) was discovered as a specific inhibitor of CDK2 and its chlorinated form, flavopyridol (77, Fig. 29), is currently in phase II as a drug against breast tumors [155]; derivatives of this compound were shown to be CDK1 selective inhibitors [156]. OH

O

OH

O Cl

HO

O

HO

O

OH

N 76

OH

N

77

Fig. (29). Structure of rohitukine and flavopyridol.

The crystal structure of a complex between CDK2 and L868276 [157], flavopyridol [156] and of the 2,6,9-substituted purine derivative roscovitine [158] revealed the structural basis for the inhibition. The benzopyan ring occupies approximately the same region as the purine ring of ATP. Structural requirements of other ATP-competitive CDK inhibitors are reported in [159]. These structures show that the chromenone moiety of the inhibitor acts as a mimetic of the purine moiety of ATP, the cofactor of the enzyme. In particular, the 4-keto and 5hydroxy groups of the compound establish the same bidentate hydrogen bonds with the backbone of CDK2 residues Leu83 and Glu81 as the nitrogen atoms at positions 1 and 6 of the purine moiety of ATP. Moreover, the piperidinyl moiety interacts with conserved residues of the catalytic cleft that are normally used to bind the ribose ring and the phosphate chain of ATP. The recent crystal structure of the flavonol Fisetin complexed with CDK 6 showed that this compound, like other flavonoids, was found to bind in the ATP-binding pocket; however it is rotated of about 180° with respect to deschloroflavopyridol in its complex with CDK2. These different binding orientations agree with previous reports of various binding modes of related CDK inhibitors [160]. e) Cyclic Peptides Head-to-tail cyclic peptides have been reported to bind to multiple, unrelated classes of receptors with high affinity; they may therefore be considered to be privileged structures [161-163].

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Ellmann and collaborators synthesized a library of -turn (78) mimetics of the general structure 79 (Fig. 30) incorporating the side chain display observed in the critical -turn present in somatostatin; this research led to the discovery of derivatives (see for example 80) with high potency and selectivity towards hSST5 receptors [164, 165]. O Ri+1

Ri+2

N H

Ri+1

O

HN O

O N H

HN

O

S

Ri+3

R1

Ri+2

N

n

Ri+3

79 78

NH NH2

O N H S

O N

80

hSST5: 87 nM

Fig. (30). Somatostatin -turn mimetic.

An interesting case of cyclic peptides is represented by the cyclic dipeptide piperazine2,5-dione (Fig. 31) and its derivatives. R R

N

O

O

N

R

R

Fig. (31). Piperazine 2,5-dione scaffold.

The importance in medicinal chemistry of these compounds has been recently reviewed [166]. They possess a broad range of activities, including inhibition at integrin
v3 receptors [166], occupying part of its proximal hydrophobic pocket and forming a hydrogen bonding network (-sheet) with Gly216 [167]. A library based on this scaffold was created and active tryptase (Ser protease with trypsin-like properties) inhibitors were discovered [168]. The crystal structure of a DKP-containing compound bound to trypsin (a Ser protease enzyme) has been studied demonstrating the contribution of the DKP to the formation of an appropriate -sheet with 216Gly [168].

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Other applications on
-triptase are reported in [169, 170]. f) Pyrrolinones On the basis of the concept that the fixation of a ligand in a beta strand conformation should produce high affinity inhibitors even for proteases of unknown structure, provided that substrate specificity and protease type is known [94], the 3,5-linked homochiral pyrrolinone scaffold 82 (Fig. 32) has been successfully proposed as an alternative to amide-derived peptidomimetic inhibitors (81, Fig. 32) of proteolytic enzymes. Pyrrolinone motif is generated by NH displacement from peptide backbone; intrastrand hydrogen bonding of the NH proton with the carbonyl of the neighboring pyrrolinone ring stabilize the
-strand conformation and reduce solvation [171] (Fig. 32). According to the hypothesis, N-unsubstituted 3,5-linked polypyrrolin-4-one scaffold furnished biologically potent mimetics of
-strand. O

R H N

N H

O N H

R

O

R

81

H O

O

N R

R

R HN

O

H

N

82

Fig. (32). Elaboration of the pyrrolinones as peptide mimetics.

As an example, potent pyrrolinone-based inhibitors of proteolytic enzymes (HIV-1 protease [171-173]) were discovered (84, Fig. 33), on the basis of the importance of
-strand bound conformations for inhibitors of the HIV protease. Then, starting from the structure of L-682,679 (83), a potent inhibior of HIV protease, was obtained by replacing its P1’-P3’ tripeptide sequence by a bis(pyrrolinone) bearing the three requisite side chains [173]. The intramolecular H-bonds may account, at least in part, for the improved membrane transport of this compound, compared with their peptidal counterpart, due to a decreased energy penalty for desolvation [173].

OH R

O O

OH

O

H N

H N O

83

NH2

N H O

R

O

HN

H N

O O

NH2 O

84

Fig. (33). HIV protease Inhibitor as a model for the pyrrolinone-based Inhibitors.

HN

O

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An analogous approach led to potent enzymes (renin [174] and matrix metallo proteases [175]) inhibitors. This scaffold was also useful for the construction of pyrrolinone-peptide hybrids as ligand for the class II major histocompatibility complex (MHC) protein HLA-DR1 [176]; on the basis of the X-rays structure of the HA 306-318 peptide bound to the MHC protein HLA-DR1, the bispyrrolinone has been introduced as a viable replacement for the tetrapeptidic sequence VKQN (residues 309-312) of HA 306-318, mimicking the peptidal
-strand. This scaffold showed also to be useful as a functional mimetic of peptide
-turn. Numerous studies revealed that the biologically active pharmacophore of somatostatin (SRIF) comprises a type I
-turn projecting the essential Phe7, Trp8 and Lys9 side chains in the right manner to interact with the GPCR [177]. On the basis of molecular modeling studies on the potent SRIF mimetic L-363,301 (20, Fig. 34), a substituted bispyrrolinone was discovered (85), with high affinity at hsst4 and hsst5, (Fig. 34) demonstrating that the pyrrolinone scaffold can be employed to generate also functional mimetics of peptide
-turns. However, owing to difficult in synthesis, a naphthyl group was introduced instead of the indole ring of L-363,301. H2N

HN

O

HN

O

L 363,301 O

HN

O

HN

20 85

Fig. (34). Discovery of sst receptors agonists on the basis of the SRIF mimetic l-363,301.

Interestingly, it was noted that the 3,5-linked polypyrrolin-4-one scaffold may provide access also to other conformations than
-strand, including those analogoes to helices, when N-methylated. This greatly expands the scope of this scaffold for the development of low molecular weight ligands for biologically important macromolecules [178]; no biological data are available yet for these compounds. CONCLUSIONS The term “privileged structure” has been used in several ways, designing compounds highly represented in the overall bioactive compound population, or scaffold able to present functional groups in a favourable arrangement. While many classes of privileged compounds such as 1,4-dihydropyridines, biphenyls and benzodiazepines here reported and others produce leads with enhanced drug-like properties, other structures such as pyrrolinone compounds were classified as privileged owing to their rigid framework able to direct functional groups in a well defined space. Privileged structures can be structural elements of compounds exerting an effect on more than one target protein, or they can also be used for target gene family of proteins that have in common molecular recognition elements. Several researches dealing with privileged structures, here reported, highlighted the reason for their

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privileged status, even if these studies are limited to few classes of compounds, but the results obtained can help in a more rationale use of these structures in drug discovery. Thus, even if some scaffolds here reported lead to compounds that do not possess drug-like features, the discovery of a hit can however open the way to the development of new leads. Moreover, the combination of the concept of privileged structure with the combinatorial chemistry approach can allow a faster identification of a new lead. In alternative to the use of privileged structures, several studies appeared dealing with the preparation of libraries based on the concept of diversity-oriented synthesis [179-181]. Following this approach, libraries of compounds with rich skeletal and stereochemical diversity were created in order to gain a diverse display of chemical information in three dimensional space. However, no results are available yet regarding the success of this approach. Therefore, until the utility of this approach will be proven, the use of “privileged structures” in the discovery of new drugs can be succesfully employed in order to find new leads. Moreover, it has been shown recently that computational approaches have been able to identify scaffolds and/or privileged fragments among active compounds, and attempts were made in order to select suitable scaffolds for natural products in order to increase the number of active compounds present into designed libraries; taken together, and considering the emerging role of the structure-based drug design in the modern drug discovery process, it appears that the integration of these concepts will be the basis of the drug discovery for the next years. REFERENCES [1] [2]

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41 integrin receptor interactions through library synthesis and screening. Bioorg. Med. Chem. Lett., 1998, 8, 2297-2302. Horton, D.A.; Bourne, G.T.; Smythe, M.L. Exploring privileged structures: the combinatorial synthesis of cyclic peptides. J. Comp. Aided Mol. Drug Des., 2002, 16, 415-431. Li, J.; Murray, C.W.; Waszkowycz, B.; Young, S.C. Targeted molecular diversity in drug discovery: integration of structure-based design and combinatorial chemistry. Drug Discov. Today, 1998, 3, 105118. del Fresno, M.; Fernandez-Forner, D.; Miralpeix, M.; Segarra, V.; Ryder, H.; Royo, M.; Albericio, F. Combinatorial approaches towards the discovery of new tryptase inhibitors. Bioorg. Med. Chem. Lett., 2005, 15, 1659-1664. Schaschke, N.; Dominik, A.; Mastchiner, G.; Sommerhoff, C.P. Bivalent inhibition of -Tryptase: distance scan of neighboring subunits by dibasic inhibitors. Bioorg. Med. Chem. Lett., 2002, 12, 985988. Royo, M.; Van Den Nest, W.; del Fresno, M.; Frieden, A.; Yahalom, D.; Rosenblatt, M.; Chorev, M.; Albericio, F. olid-phase syntheses of constrained RGD scaffolds and their binding to the
v3 integrin receptor. Tetrahedron Lett., 2001, 42, 7387-7391. Smith, A.B.; Hirschmann, R.; Pasternak, A.; Akaishi, R.; Guzman, M.C.; Jones, D.R.; Keenan, T.P.; Sprengler, P.A.; Darke, P.L.; Emini, E.A.; Holloway, M.K.; Schleif, W.A. Design and synthesis of peptidomimetic inhibitors of HIV-1 protease and renin: evidence for improved transport. J. Med. Chem., 1994, 37, 215-218. Smith, A.B.; Hirschmann, R.; Pasternack, A.; Yao, W.; Sprengeler, P.A.; Holloway, M.K.; Kuo, L.C.; Chen, Z.; Darke, P.L.; Schleif, W.A. An orally bioavailable pyrrolinone inhibitor of HIV-1 protease: computational analysis and X-ray crystal structure of the enzyme complex. J. Med. Chem., 1997, 40, 2440-2444. Smith, A.B.; Hirschmann, R.; Pasternak, A.; Guzman, M.C.; Yokoyama, A.; Sprengeler, P.A.; Darke, P.L.; Emini, E.A.; Schleif, W.A. Pyrrolinone-based HIV protease inhibitors: design, synthesis, and antiviral activity: Evidence for improved transport. J. Am. Chem. Soc., 1995, 117, 11113-11123. Smith, A.B.; Akaishi, R.; Jones, D.R.; Keenan, T.P.; Guzman, M.C.; Holcomb, R.C.; Sprengeler, P.A.; Wood, J.L.; Hirschmann, R.; Holloway, M.K. Design and synthesis of nonpeptide peptidomimetic inhibitors of renin. Biopolymers, 1995, 37, 29-53. Smith, A.B.; Nittoli, T.; Sprengeler, P.A.; Duan, J.J.-W.; Liu, R.-Q.; Hirschmann, R.F. Design, synthesis, and evaluation of a pyrrolinone-based matrix metalloprotease inhibitor. Org. Lett., 2000, 2, 3809-3812. Smith, A.B.; Benowitz, A.B.; Sprengeler, P.A.; Barbosa, J.; Guzman, M.C.; Hirschmann, R.; Schweiger, E.J.; Bolin, D.R.; Nagy, Z.; Campbell, R.M.; Cox, D.C.; Olson, G.L. Design and synthesis of a competent pyrrolinone-peptide hybrid ligand for the Class II Major Histocompatibility Complex Protein HLA-DR1. J. Am. Chem. Soc., 1999, 121, 9286-9298. Smith, A.; Charnley, A.K.; Mesaros, E.F.; Kikuchi, O.; Wang, W.; Benowitz, A.; Chu, C.L.; Feng, J.J.; Chen, K.H.; Lin, A.; Cheng, F.C.; Taylor, L.; Hirschmann, R. Design, synthesis, and binding affinities of pyrrolinone-based somatostatin mimetics. Org. Lett., 2005, 7, 399-402.

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Acetylcholinesterase Reprised: Molecular Modeling with the Whole Toolkit Gerald H. Lushington1,*, Jian-Xin Guo1 and Margaret M. Hurley2 1

Molecular Graphics and Modeling Laboratory, University of Kansas, Lawrence, KS 66045 USA; 2Weapons and Materials Research Directorate, U.S. Army Research Laboratory, Aberdeen Proving Ground, MD 21005, USA Abstract: Molecular modeling efforts aimed at probing the structure, function and inhibition of the acetylcholinesterase enzyme have abounded in the last 15 years, largely due to the system's importance to medical conditions such as myasthenia gravis, Alzheimer's disease and Parkinson's disease, and well as its famous toxicological susceptibility to nerve agents. The complexity inherent in such a system with multiple complementary binding sites, critical dynamic effects and intricate mechanisms for enzymatic function and covalent inhibition, has led to an impressively diverse selection of simulation techniques being applied to the system, including quantum chemical mechanistic studies, molecular docking prediction of noncovalent complexes and their associated binding free energies, molecular dynamics conformational analysis and transport kinetics prediction, and quantitative structure activity relationship modeling to tie salient details together into a coherent predictive tool. Effective drug and prophylaxis design strategies for a complex target like this requires some understanding and appreciation for all of the above methods, thus it makes an excellent case study for multi-faceted pharmacological modeling. This paper reviews a sample of the more important studies on acetylcholinesterase and helps to elucidate their interdependencies. Potential future directions are introduced based on the special methodological needs of the acetylcholinesterase system and on emerging trends in molecular modeling.

Keywords: Acetylcholinesterase, Alzheimer's disease, organophosphorus nerve agents, molecular docking, molecular dynamics, quantum chemistry, QSAR, COMBINE I. INTRODUCTION One of the most catalytically efficient enzymes in nature is acetylcholinesterase (AChE) [1], a serine hydrolase found in the neuromuscular junctions of all known animals. AChE is responsible for deacylating excess quantities (post neural discharge) of the neurotransmitter acetylcholine (ACh) in the synaptic cleft. As such, normal AChE function is critical to a wide range of biological processes, most notably including vital central nervous system, cardiac and respiratory functions. AChE has garnered substantial interest in the medical community over the past several decades for a variety of reasons. AChE has been targeted for reversible inhibition in pursuit *Corresponding author: E-mail: [email protected] Allen B. Reitz / Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.

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of treatments for myasthenia gravis (MG) [2] and Alzheimer's disease (AD) [3]. In the former case, the primary symptom of MG is muscle weakness caused by autoimmune attack on the nicotinic receptors found in voluntary muscles, leading to diminished capacity to receive ACh-mediated instructions. Selective inhibition of a sizeable percentage of available AChE receptor sites leads to increased ACh-persistence in the cleft, thus enhancing the opportunity for successful ACh transmission. For the latter, a precise etiological characterization of AD causality remains elusive, however one key symptom is degradation in synaptic efficiency that appears to be somewhat alleviated by the similar mechanism of AChE-inhibiting / AChbolstering therapy applied in MG treatment [4]. Early hypotheses holding that the synaptic inefficiency might actually be a cause of the disease rather than just an effect [5] have proven inadequate in explaining many observations, however, increasing evidence suggests that although AChE-inhibition therapy tends to provide temporary cognitive improvements in AD patients, the tangible benefits toward reversing or even slowing the progression of the disease remain under debate [6, 7]. Diminished AChE activity is also a dysfunction that has a variety of health impacts. Onset of Parkinson's disease (PD), for example, correlates with reduced AChE function in muscarinic receptors in the temporal cortex [8]. A much more obvious issue, however, is the direct link between organophosphorus (OP) and carbamate toxicity and AChE inhibition, providing a biochemical basis for many pesticide formulations as well as most of the nerve agent class of chemical weapons. Not surprisingly, recent studies have suggested an important possible link between OP exposure and elevated risk of PD [9]. Whether the medical point of interest has to do with targeting AChE for inhibition, in the case of MG or AD treatment, or trying to prevent this from occurring (i.e., PD and nerve therapy and prophylaxis), there is a strong motivation for wanting to understand aspects of the AChE activity and deactivation in atomic-level detail. Given the complexity of the system that will be elaborated upon in greater detail in the next section, there is little wonder that molecular modeling methods and other chemical informatics techniques have been applied extensively to the study of this enzyme; in trying to derive as much insight into a variety of different aspects of the system as possible, numerous different computational methods have been applied. This review aims to examine a reasonable selection of these different methods and their intended rhetorical value. Dynamic aspects of the enzyme system itself, and of ligand-enzyme conjugates have been studied extensively via molecular dynamics (MD) simulations, of which a representative sample are discussed in Section III. Optimization of non-covalent inhibition strategies, pursued extensively in the search for non-toxic treatments for MG and AD, has made substantial contributions via molecular docking simulations, some of which are discussed in Section IV. Many aspects of covalent inhibition cannot be modeled via classical simulations and thus require quantum chemical (QC) treatment, which has also been applied toward understanding the underlying enzymatic and inhibitive reaction mechanisms, as well as other electronic structural effects governing enzyme activity. This is reviewed in Section V. Finally, given the multifaceted nature of the AChE inhibition problem and the fact that the potency of a given ligand may depend on any combination of (or all of) the aforementioned considerations, it is proving necessary and valuable to develop statistical methods for assessing the relative importance of given properties and phenomena. Although this review is primarily focused on receptor-based analysis, various quantitative structure activity relationship (QSAR) methods do play significant roles in understanding aspects of the protein structure, function and modulation and some QSAR examples are thus described in Section VI. Some speculations regarding the impact of emerging computational methodologies are discussed in Section VII.

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In the four years since the original version of this article was published in Current Topics in Medicinal Chemistry [10], AChE has remained a very active target for receptor-based modeling. A fairly comprehensive survey of relevant publications spanning the years 20062009 suggests that the foci of these research activities is oriented primarily toward pharmacological / enzymological analysis. As with other targets for which crystal structures exist, a major fraction of the recent AChE publications have applied standard ligand docking techniques [11-35] of relevance to Section IV, while a modest number entail more rigorous and time consuming MD [36-49] that are required for explicit representation of the dynamic effects discussed in Section III. An even smaller number of studies address covalent bond formation (as per Section V) via QC [50-52] or mixed quantum-classical methods [53-58]. A small number of studies exploited multiple techniques, including the use of docking simulations to derive starting conformations for MD analyses [59, 60] or as the basis for QC [61] or mixed quantum-classical [62] studies of the ligand-receptor complex. Some of the above studies assimilate the modeling and simulation data toward more comprehensive understanding of the scaffold-specific pharmacophore via QSAR methods [14, 16, 21, 27, 50] similar to those originally introduced in Section VI. While purely ligand-based QSAR is somewhat peripheral to the scope of this review (our interest is primarily in AChE pharmacology and toxicology as a function of receptor structure and function), the research it embodies is complementary to our analyses and thus it is worthwhile to track the vibrant collection of new research in this area [63-82]. Finally, among the methods proposed speculatively in Section VII as emerging options for AChE modeling, one in particular has gained traction as a practical option: finite element (FE) modeling, which has served as the basis for three subsequent studies [83-85]. Other methods discussed prognosticatively in the 2006 paper have yet to be applied in a meaningful sense but, as will be addressed later, some surprising new modeling strategies have also emerged. II. ACETYLCHOLINESTERASE STRUCTURE, FUNCTION AND INHIBITION The amenability of AChE to detailed studies of structure-function relationships has been greatly augmented by the publication of numerous well-resolved crystal structures of the enzyme in the apo form and in various states of inhibition. The two most widely available crystal structures are those of the Torpedo californica (tcAChE) [86] and Mus Musculus (mAChE) [87], although in recent years the human form (huAChE) has also been published [88] and is depicted in Fig. (1). AChE tends to be very well conserved across species, with huAChE and mAChE sequences exhibiting 88% identity and 97% homology, while even the great evolutationary divergence between humans and Torpedo californica affords 57% identity and 86% homology over the aligned portion of their sequences. Therefore, while we will focus primarily on huAChE in this review as the system of primary pharmacological interest (residue numbering will be according to huAChE except as noted), the vast majority of assertions derived from any one of the above three structural models should hold reasonably well for the other two. AChE monomer units such as the one depicted in Fig. (1A), are classifed according to the Cluster-Architecture-Topology-Homologous Superfamily scheme as

-sandwiches [89]. The two
-rich masses (one of which is on the left side of Fig. (1A), while the other is near the bottom-center) provide sites for the cysteine-cysteine disulphide bridging that leads to dimerization and tetramerization of AChE, thereby optimizing the enzyme's hydrophilicity and catalytic efficacy [90]. Within a coil-rich mass between the two
-rich associative lobes lies the main enzymatic functionality of AChE, whose main features are shown in greater detail in Fig. (1B). The view in Fig. (1B) is oriented obliquely above a gorge whose mouth is demarcated by a peripheral anionic site (PAS), coloured dark blue, comprised

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mostly of hydrophobic residues (Tyr72, Tyr124, Trp286, Tyr341) but also an anionic Asp74. The hydrophobic contact surface plus a single anionic point appears to be an ideal match for attracting the cholinic end of ACh which combines a hydrophobic moiety with a quaternary (i.e., electrophilic) amine.

Fig. (1). Crystal structure of human AChE showing A) secondary structure, and B) active site features. Secondary structural features in A) are colored in standard form (yellow = -sheets, magenta =
helices, cyan = coils). In B), the catalytic triad residues are colored red, the oxyanion hole is green, acyl pocket is cyan, the peripheral active site is blue, Glu202 is magenta, Trp86 is yellow and the mobile loop is purple. The approximate extent of the gorge is depicted in B) as a brown dotted line.

At the base of the gorge lies a classic serine-protease catalytic triad comprised of three residues: Ser203, Glu334 and His447 (shown in red) that collectively serves to deacylate ACh via the mechanism illustrated in Fig. (2). The charge buffering role played by His447 is enhanced by prior extraction of the H proton on the imidazole ring by Glu334. Reaction irreversibility is believed to be facilitated by the nearby Glu202 which may serve the function of attracting the imidazole's H (corresponding to the protonated N in Fig. (2), step 4) during the deacylation (Fig. (2), step 5). The role of Glu202 will be discussed in more detail later. Proximal to Ser 203, one finds two glycines (121, 122) and an alanine (204) whose backbone amido protons co-orient in such a fashion as to provide a strong H-bond donating site amenable to spatially fixing a small nucleophile, such as an ester carbonylic oxygen. In the specific case of ACh, these three residues, collectively referred to as the oxyanion hole, assist in deacylation by selectively orienting esters so as to render the ACh central carbon favorable to nucleophilic attack by Ser 203 (Fig. (2); step 1), and also favorable for subsequent removal of choline (Fig. (2); step 2). Across the base of the gorge from the catalytic triad one notes the presence of a hydrophobic Trp 86 residue (rendered yellow in Fig. (1B)). The presence of the aromatic Trp side chain appears to be well suited to accommodating the hydrophobic but positively charged (CH3)3NCH2 – tail of ACh during the reactive process. Also of relevance, is the acyl pocket shown as cyan residues (Phe 295, Phe 297, Phe 338) in Fig. (1B), whose primary role appears to be in housing the acyl methyl group in the activated ACH-AChE complex. Given the fact that this region comprises one of the few

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major differences in the of AChE receptor structure versus that of the otherwise closely related butyrylcholinesterase (BChE) enzyme (Phe295
Leu 295; Phe297
Ile297), it has been postulated as a primary source of AChE and BChE enzymatic selectivity [91]. Specifically, the smaller hydrophobic groups in the BChE acyl pocket would be expected to yield a larger chamber capable of processing the larger substrate molecules (e.g., butyrylcholine and benzoylcholine) that cannot be admitted into the AChE receptor. Glu 202

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Fig. (2). Reaction diagram illustrating the putative six step deacylation of ACh by AChE. R corresponds to ACh’s (CH3)3NCH2 moiety, Ser, His and Glu indicate serine (203), histidine (447) and glutamine (202,334) AChE receptor residues.

AChE's impressive enzymatic efficiency suggests that the various structural features described above likely produce an optimal mechanism for attracting, trapping and decomposing the ACh substrate, as well as expelling the choline and acetic acid products. It is broadly assumed that much of the efficiency is conferred by an ideal spatial relationship between the different features, whereby 1) the PAS and Trp86 respectively attract and guide

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the (CH3)3NCH2 moiety into place, 2) the oxyanion hole and acyl pocket collectively anchor the ester into a position suitable for nucleophilic substitution, 3) proton shuttling within the triad induces highly effective nucleophilic attack onto the ester center, 4) Glu202 effects a conformational rearrangement of His447 to prevent premature back-sliding of the shuttled proton (although the exact role of Glu202 remains a matter of debate, as will be discussed later), and 5) the spacious nature of the gorge permits the presence of ample water molecules, of which one is required to effect deacylation. It has also been speculated that a number of electrostatic and dynamic effects may enhance catalytic efficiency. Chief among them is the presence of a highly mobile Cys69-Cys96 loop that is believed to engage in gorge gating selectivity mechanisms [87]. The fact that Trp 86 seems to be both a structural keystone of this mobile loop [92] as well as a key lipophilic component of the active site suggests a possible role as a gating trigger that signals the loop to occlude the gorge exit thus preventing the departure of unreacted ACh. Another so-called back-door hypothesis exists, claiming that a concerted post-complexation rearrangement (centered in the Trp86, Val132 and Gly448 region) facilitates departure of the bulky choline leaving group from gorge by an alternate route [15, 93], thus reducing the likelihood that the choline will linger in the region of Trp86 or the PAS and hinder admission of subsequent ACh substrate. Originally postulated as a result of molecular modeling studies, the concept has not been incontravertibly established. Subsequent mutagenesis studies argue against the possible mechanism [94], but later crystallographic evidence [95] and kinetic data [96] provide support for it. Researchers have also postulated the existence of an efficient mechanism to guide the substrate down the extensive gorge to the active site [97]. Detailed studies of the electrostatics of several forms of AChE and BChE [98-100] have demonstrated the conservation of key residues lining the gorge. These residues are postulated to produce a distinct electrostatic profile which facilitates guidance of the substrate down the gorge and subsequent ejection of the leaving group. The very features on which AChE relies for its highly efficient ACh enzymatic processing can be very effectively turned against the system by choice of inhibitors capable of exploiting the various binding sites and subsites in the AChE receptor. Given the physicochemical diversity of these sites and subsites, it is not surprising that an extensive variety of different chemicals exhibit some tendency toward AChE inhibition. Inhibition data within a single online repository exists for compounds from each of the following families: organophosphorus (OP) compounds, carbamates, alkaloids, haloketones, coumarins, ketomalonates, acridines, piperidines, xanthins, benzyl/phenyl amine/amide derivatives, tryptamines, bipyridines, alkylpyridnium oligomers, bis- (and other) quaternary amines, polyethylene ester oligomers, quinolines, quinazolines, triazolines, phenothiazines, curariniums, diazepines, steroids, opioids, fluorides, numerous peptides, and even some quaternary platinum species [101]. Many of the above compounds, especially those with substantial hydrophobic or electrophilic groups, noncovalently inhibit the PAS and/or the Trp86 choline-binding site. However, the strongest AChE inhibitors appear to be species that covalently bind to the esteratic Ser203, especially OP compounds or carbamates. The so-called 'nerve agents', of deadly inhibitive efficacy are OP species with the general formula shown in Fig. (3). The most powerful OP nerve agents effectively mimic the ACh ester structure that is so conducive to binding within the AChE esteratic subsite. Inhibitive OP species possess a phosphonyl group with an electrostatic and steric profile similar to that of an ester (thus being wellaccommodated by the oxyanion hole), and an electrophilic phosphorus apparently even more amenable to Ser203 nucleophilic attack than the native ACh substrate. Effective in-

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hibitors typically possess a small aliphatic group (R) that fits the contours provided by the acyl pocket and a larger hydrophobic group (R') suitable for interacting with the Trp86 choline-binding site. From Fig. (4), however, we see that the final disposition of OP ligands differs from the catalytic ACh decomposition process in that water is not a sufficiently strong nucleophile as to effect a process analogous to the deacylation in steps 4-6 of Fig. (2). Despite its relatively high electronegativity, the X group in OP nerve agents does not appear to detract from the ligand’s efficacy for initial complexation and, as is seen in steps 2,3 of Fig. (4), subverts the normal AChE catalysis by serving as the preferred leaving group instead of the OR group (as would be analogous to ACh). This produces a phosphorylated active site that is inherently resistant to the normal hydrolysis reaction (steps 4-6 in Fig. (2)) that would otherwise permit restoration of the AChE enzyme function. Although the phosphorylation is still technically reversible at this stage, the AChE receptor has been rendered largely inaccessible to ACh, thus providing the principle source of OP toxicity. Some OP agents are capable of undergoing a further dealkylation reaction, illustrated in steps 4,5 of Fig. (4). This reaction, generally referred to as ‘aging’, creates a resonance stabilized inhibition state that can be considered irreversible for most practical purposes [102]. While therapeutic species (primarily oximes) have been developed that can dephosphorylate OP poisoned AChE enzymes from the point described by step 3 in Fig. (4), most aged nerve agents aged states remain resistant to chemical remediation. O

O R

R

P O R'

X S

P O X

R'

R

Fig. (3). General structure of S- and R-stereoisomers for OP nerve agents. R and R’ are usually alkyl groups. X is generally an electronegative group (often a fluoride). Note that the sp3-hybridized ester O may be substituted with S in some formulations (i.e., thioesters).

III. DYNAMIC PROPERTIES AND EFFECTS The most prolific body of work corresponding to molecular dynamics simulations of AChE corresponds to that produced by the McCammon group at the University of California at San Diego. In addition to numerous contributed articles on AChE dynamics, of which some will be discussed shortly, the McCammon group has produced a review article [103] that establishes an important premise for the importance of MD simulations to the understanding of AChE function. Namely, they point out that the structure of the gorge is so deep and narrow that the rigid models corresponding to most available crystal structures do not afford sufficient channel width to admit the known substrate and active site inhibitors. The facts that a) AChE actually does allow both substrate and inhibitors into the furthest depths of the cavity and b) the enzyme is viewed as one of the most catalytically efficient systems in the known proteome [1] clearly suggests that dynamic effects play a major role in facilitating substrate access to the catalytic site. Unraveling the precise nature of this dynamic facilitation has been a major source of research interest over the past fifteen years. To the best of our knowledge, the first published simulations focusing on AChE structural dynamics include the following three papers dating from 1994: 1) an important but relatively uncontroversial comparison of a static AChE crystal structure and its dynamic

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virtual analog [104], 2) the aforementioned (see previous section) ground breaking study in which prediction of the presence of a back door exit channel adjacent to the known gorge was first made [93], and 3) the first detailed assessment of the role of the mobile Cys69Cys96 loop [105]. The first of these papers provided enlightening insight into the degree of enzyme-wide structural rearrangement that is possible upon transition from an apo state to inhibition, while the second and third raised the possibility of specific dynamic features, such as transient channels for the evacuation of product molecules and trafficking of solvent, that might not have been readily intuited from the crystal structure alone. Ser Ser

Ser

R

O

R O

O O

O H P

P P

O

O:

R'

R' X

O H

X

: His

O

R' H

His

His R X 3.

2.

1.

Ser Ser O O P P

O

R'

O

O

R' O

H

His

H

His

R

R 5.

4.

Fig. (4). Reaction diagram illustrating the putative phosphorylation and aging reactions for organophosphorus reactions with the AChE active site. Ser and His refer to the serine and histidine residues in the AChE catalytic triad (numbered Ser 203 and His 447, according to huAChE sequence).

While the acceptance of a back door hypothesis within the experimental community has been a gradual process [94], further MD studies within the McCammon group [106-110], and by other researchers [105], were fairly quick to confirm the feature and have since carefully characterized it. Later studies also postulated the presence of a side door (around Thr75, Leu76, and Thr83) [109], which has been subsequently validated by the McCammon group [110]. In the apo form of the enzyme, it appears that the back door is relatively occluded, only spending about 1% of its existence open broadly enough to admit or emit a water molecule (~1.4 Å radius), while the side door remains shut effectively 100% of the time [108]. When ligated or inhibited, however, the doors are predicted to be much more accessible. With fasciculin bound across the PAS, for example, the back door was found to remain open (a radius of at least 1.4 Å) upwards of 18% of the time, and the side door 13% of the time. It has been hypothesized that these alternate doors not only serve to expedite solvent and product exchange, but possibly also act as an adaptive biological defense mechanism that may permit some degree of ACh processing to occur even when the gorge entry is fully occluded [109]. This may offer a reasonable explanation for continued deacy-

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lation activity in the face of extensive fasciculin-AChE inhibition [111, 112]. Some evidence exists for the possible existence of as many as two other transient portals to the active site cavity [108], suggesting an inherently very porous gorge. It must be additionally noted these dynamics studies address not only the question of ingress and egress of ACh and inhibitors into the gorge, but also pertain to the critical question of solvation within the gorge and diffusion of waters to the active site [98-100]. Detailed analysis of MD simulations suggest that the process of opening and closing of the back and side doors is not merely a localized phenomenon, but entails significant concerted motion across much of the AChE monomeric unit [108]. Interestingly, while Trp86 is generally considered to be a critical residue for the back door, essentially forming part of the mouth of the open portal [93], it is also strongly correlated to side door motion, being one of the residue side chains that moves the most upon side door opening / closing [108]. There does not, however, appear to be a simple correlation between to the relative states (i.e., open vs. closed) of the side and back doors. During a MD simulation with PAS-bound fasciculin, they coexisted in an open (OO) state 8% of the time, were both closed (CC) approximately 77% of time and in the remaining 15% of the observed time steps one was found to be open and the other closed (OC or CO) [108]. Despite this apparent independence, it is still possible that both aperture states depend at least in part on the nature of ligand interactions with Trp86, with different contact conformations favoring each of the four different (OO, OC, CO or CC) states. Since Trp86 has also been identified as possibly performing a gorge gateswitching role through action on the mobile Cys69-Cys96 loop [87], evidence appears to point to this hydrophobic / basic residue as serving (and perhaps helping to coordinate) multiple important enzyme regulatory functions. If so, it may prove to be a very attractive inhibition or mutagenesis target for enzyme modulation. Dynamic modeling has played an important role in the original identification of the mobile loop [104], and characterizing its motion and function [92, 105, 113]. Named because of its apparent status as the most conformationally mobile portion of the AChE molecule, the flexibility of the Cys69-Cys96 loop was first noted from MD analysis, and was thus postulated to serve a role analogous to that observed in lipases [114, 115] as a transitory cap on the active site [104]. Kovach et al. used this loop's mobility as rationale for the existence of the various alternate product channels (e.g., back door, etc.), suggesting that loop motion uncovered transient apertures between the three main permanent channels in the enzyme: the primary gorge, a side channel capable of accommodating cationic products such as choline, and a cavity admitting nucleophiles such as the deacylation leaving group [105]. Subsequent MD studies have supported this assessment, suggesting that the binding of a lipophilic cation (e.g., choline or the phosphorylation leaving group) to Trp86, induces partial unwinding of the loop up into the gorge, opening an aperture suitable for cation evacuation [92]. The importance of the mobile loop has been effectively validated by enzyme kinetic studies as a key participant in allosteric modulation of AChE catalytic activity [116]. Motion of additional residues had also been postulated as being of key importance to binding [117]. Carlacci [118] and Olsen et al. [119] have studied the motion of the acyl pocket loop (residues TcAChE 287-290) through a variety of techniques, including MD and Monte Carlo (MC) simulation. This work analyzed alternative conformations of the acyl pocket loop residues and their influence on substrate binding. Residue Arg289 in particular was singled out as playing a critical role in stabilizing these alternative conformations. Additionally, the role of the solvent was recognized as being integral to this phenomenon. The MD work in particular provided a critical analysis of results using different solvation models. Interestingly, movement of the acyl pocket loop was also detected upon phosphorylation

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by Zeev-Ben-Mordehai et al. [120] in a non-traditional approach that used a morphing procedure to visualize and detect conformational changes in acetylcholinesterase crystal structures. Another major contribution to our net understanding of AChE function and inhibition that MD studies have made is in the area of dynamic AChE-ligand interaction effects. In addition to the aforementioned study of PAS-binding fasciculin-AChE complexes [110] and more recent work on this system by the same group [45, 121], there has been research on the dynamics of the non-covalent active-site binding species such as huperzine and related analogs [46,106], precomplexation studies on ACh and various of its analogs [122], and examinations of PAS-binding inhibitors (e.g., Axillaridine A [37], the Alzheimers drug propidium [123], tacrine analogs [124, 125], and of the novel tacrine-huperzine hydrid huprine-X [126, 127]). As discussed in previous paragraphs in this section, research on fasciculin binding was originally geared toward elucidation of connections between ligand binding and the formation of the back and side door conduits [122] although, being an exceptionally large and influential ligand, it has since generated interest as a subject for gauging the induced-fit ligand-binding effects on the overall protein structure [45]. Another more modest inducedfit phenomenon was identified in the study of ACh analogs, whereby analysis of the different receptor conformers suggested that the presence of the ligand in the active site of AChE not only stabilizes the H-bonding interactions within the catalytic triad but also spatially tightens both subsites, thus collectively enhancing the binding environment [122]. Among the enzyme-inhibitor MD studies, many were intended primarily to supplement static molecular docking evaluation of noncovalent bonding interactions between ligand and enzyme [122], which is a topic to be dealt with primarily in the next section. However, the tacrine analog and huprine-X studies also yielded important insight from their MD simulations that merits some elaboration here. In the tacrine studies, simulations on increasingly hydrophobic analogs suggest that such hydrophobicity produces important changes in the binding mode away from the gorge binding location of tacrine itself, exhibiting simultaneous lipophilic stacking with Trp84 and Phe330 (according to tcAChE numbering; huAChE = Trp86, Tyr337) and yielding an inhibitive mechanism arising more from PAS-binding interactions, thus raising the prospect of tailoring inhibitors toward simultaneous complexation with both subsites [124, 125]. Conversely, when modeling huprine-X it was found that the amalgamation of chemical features from the noncovalent esteratic active site inhibitor huperzine and the gorge binding tacrine led to an extremely strongly binding noncovalent inhibitor spanning both the nucleophilic active site and the lipophilic gorge [126, 127]. It was also noted that huprine-X was found to bind significantly more strongly to tcAChE than to huAChE, thus highlighting the importance of the Phe to Tyr mutation (tcAChE: Phe 330 / huAChE: Tyr337) distinguishing the two structures [126, 127]. Both the tacrine analog and the huprine-X studies demonstrated the analytical power of MD methods for effective pharmacophore mapping within a complex, multifunctional receptor such as that of AChE. Advanced molecular dynamics techniques have also been brought to bear upon the issue of large scale collective motions vs local fluctuations in transport within the cholinesterase system. Xu et al. [128] used steered molecular dynamics (SMD) to analyze Huperzine A binding and unbinding. This delineated multiple transient structural effects that nonetheless appear to play an important role. These include the formation (and breakage) of a hydrogen bond network surrounding Asp72 (tcAChE numbering, which appear vital to movement of Huperzine A in and out of the active site), rotation of the peptide bond between Gly117 and Gly118, and the importance of buried water. This was later compared to the steered molecu-

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lar dynamics of E2020 unbinding from AChE [129], where the peripheral anionic site provides the major resistance to removal of the E2020 from the gorge, and where aromatic residues lining the gorge are postulated to form a conveyor belt. Advanced molecular dynamics techniques have also been used to study gorge penetration for smaller ligands. Tetramethylammonium (TMA) has been a subject of multiple studies due to its resemblance to acetylcholine. Umbrella potential sampling was used to study dynamics of TMA within the mAChE bottleneck region [130] and found evidence of largescale gorge opening, as well as important small fluctuations in aromatic sidechains. This was later corroborated by the metadynamics simulations of Branduardi et al. [131], who additionally highlighted the importance of cation-
interactions. MD simulations have also been used to probe precomplexation interaction between AChE and known covalent inhibitors such as soman [132, 133]. These studies were quite effective in rationalizing the specific receptor-ligand interactions that stabilize and facilitate nerve agent reactions in the active site. In comparing Figs. (2) and (4), one notes that the standard ACh processing involves the usual series of proton transfer reactions and yields neutral products (Fig. (2)), however the OP covalent inhibition reaction involves the successive release of two ions (Fig. (4)) and, in this respect, might be expected to be less energetically favorable than an exchange of neutral species. To address this dilemma, a MD simulation of a fully solvated tetracoordinate phosphonate adduct (based on Soman inhibition of AChE) provided a trajectory whose ligand-enzyme interactions suggested a scenario whereby His447 might remain positively charged (rather than neutralizing the Glu334 anion) and thus effect a charge stabilization on the anionic leaving group (Fig. (4), step 3) [132, 133]. In a commensurate fashion, Trp86, Phe338 and Glu201 were estimated to collectively attract and stabilize the lipophilic and electrostatic complement within a prospective carbenium leaving group and thus facilitate the aging portion of the covalent inhibition (Fig. (4), step 5) [132, 133]. Although these assertions have not yet been definitively validated through spectroscopic or quantum chemical means, they remain generally accepted tenets of AChE inhibition theory. In recent years the logical next step in using MD simulations to address the issues associated with covalently inhibited AChE has also recently been taken, with studies probing the interactions of covalent enzyme reactivators with the covalently inhibited complex [47, 48]. These are important preliminary studies for the discovery of new techniques for toxicological countermeasures in that they provide insight into the specific molecular aspects of known reactivators (e.g., HI-6 [48] and pralidoxime and deazapralidoxime [47]) that facilitate therapeutic entry into the gorge so as approach the inhibited catalytic site in a manner conducive to covalent regeneration. However without the capacity to probe the relative therapeutic propensity for engaging in the actual covalent process of reactivation, it is possible to envision further methodological enhancements. While crystallographic and NMR representations of ligand-protein complexes are generally regarded as the gold standard basis for receptor-based design and analysis, the reputation of the MD simulation as a key tool in deriving important insight from the static experimental models is continually growing. A pair of recent publications arising from the eminent Silman/Sussman crystallographic group at the Weizmann Institute explored the capacity of MD simulations to expose empirically plausible AChE receptor conformers that might not have been apparent from crystallographic resolution alone [42, 43]. For a number of conformations that had been assumed to arise exclusively from ligand-influenced induced fit (i.e., they had been observed in ligated AChE, but rotamer plot analyses implied inaccessi-

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bility from the apo structure), MD simulations revealed reasonable apo state sampling of these states under normal physiological conditions [42]. The implication of this, especially for relatively flexible receptors such as AChE, is that a static apo state representation of a binding site may be inadequate to assess which specific ligands might fit into the cavity and/or give misleading suggestions as to the nature of the specific ligand-receptor interactions that might be observed upon binding. Their suggestion was that simulations be considered as an adjunct to standard crystallographic receptor-based design, so that a fuller manifold of available receptor conformations can be explored [43]. IV. NON-COVALENT INHIBITION One of the most active areas of AChE biochemical research has been in the pursuit and design of strong binding but reversible inhibitors [134], with primary applications being improved AD and MG treatment, as well as development of new prophylaxis schemes for mitigating nerve agent toxicity. The spatial structure of the AChE peripheral and catalytic binding sites, well understood from extensive X-ray crystallographic analysis (see Section II) and MD simulations (Section III), imposes substantial specificity in ligand conformation and the orientation upon binding, thus making AChE a tempting target for receptor-based design and optimization of potential inhibitors. Substantial effort has thus been expended in search of novel AChE inhibitors that are both potent and sufficiently selective as to minimize collateral impact on other serine hydrolase enzymes. A strong desire exists for robust high throughput virtual screening techniques capable of sifting through the broad range of chemical space potentially suitable for AChE inhibition. This has placed a pronounced focus on development of docking algorithms capable of exploiting the wealth of available structural data for the purpose of rapidly and reliably identifying strong non-covalent inhibitors. Early docking work often placed a primary emphasis on obtaining correct conformational and orientational predictions for the bound ligands. Work done by Yamamoto et al. [135, 136], for example, was mainly focused on predicting binding modes for a series of benzylamino AChE inhibitors. They successfully identified that the Trp84, located near the bottom of the binding pocket in Torpedo californica AChE, is the quaternary ammonium binding site for acetylcholine, and Trp279, in the peripheral hydrophobic site, is the binding site for other aromatic ring of the ligands (Trp86 and Trp286 respectively in huAChE numbering). Structural predictions alone, however, do not provide sufficient insight for informed structure-based design. Given a capacity for correct conformational predictions, the next logical focus lies in accurate assessment of ligand binding affinities. In silico, this is generally attempted by devising a scoring function capable of inferring a free energy of interaction from the structure of the ligand-receptor complex. Due to the complexity of the interaction between ligands and AChE, many classical docking scores appear to exhibit little or no significant correlation with experiment. Test calculations on a homogeneous set of noncovalent AChE inhibitors via a selection of commonly used generic docking score functions (including Chem-Score [137], Drug-Score [138], FlexX-Score [139], G-score [140], and PMF-score [141]) demonstrated a consistent failure in predictivity, with no single method achieving a correlation better than R2 = 0.13, and a specially trained linear combination of the five only producing R2 = 0.26 [142]. More recent studies using next-generation scoring models such as are implemented in GOLD [143] have yielded marginally better results [27] which might be adequate for general SAR analysis. Docking practitioners would generally be advised to recognize the challenges associated with the complex and dynamic nature of

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AChE, however, and perform careful validation analyses before accepting quantitative guidance from generic score functions. The main shortcoming of generic docking score functions is typically an inability to reproduce entropic effects in systems where receptor flexibility plays an important role in driving the interaction [144]. Traditionally, the most popular scheme for incorporating aspects of receptor flexibility into the model has been to accumulate multiple enzyme conformers corresponding to different snapshots in a MD simulation and to dock ligands into this manifold of static conformers. To the best of our knowledge, the first studies on AChE inhibitors employing this paradigm were those of Pang and Kozikowski, who performed docking simulations for huperzine A and E2020 (aricept) into a series of 69 AChE conformers chosen from MD studies so as to approximate the receptor flexibility space available to an inhibiting ligand [135, 136]. In the case of E2020, ligand flexibility was also incorporated in an ad hoc fashion by docking 1320 distinct ligand conformers into the receptor manifold. They found that E2020 spans the entire binding cavity of AChE, with the ammonium group interacting mainly with Trp84, Phe330 and Asp72 (tcAChE numbering; corresponding huAChE residues are Trp86, Tyr337, Asp74), the phenyl group interacting mainly with Trp84 and Phe330, and the indanone moiety interacting mainly with Tyr70 and Trp279 (tcAChE numbering; huAChE = Tyr72, Trp286). Huperzine A was found to have a similar binding mode to E2020. These structural predictions compare well with definitive results obtained from subsequent X-ray crystallographic studies [86, 145], however no conclusions were reported in terms of resulting inhibition scores of these two systems relative to known ligand affinities across any control sets. A more thorough study bridging the capacities of MD and docking simulations, already introduced in Section III, was that of Kua and coworkers who combined multiple docking simulations with extensive MD calculations in the study of AChE interacting with its natural substrate ACh, assorted ACh analogs and choline [122]. In this case, they made no attempt to focus on unique characteristic receptor conformers, but rather docked the ligands via AutoDock [146] into a collection of 1998 receptor models corresponding to the enzyme conformer observed at 1 ps intervals of 1 ns MD simulations of a) the apo-AChE structure, and b) the AChE - ACh complex. From the resulting set of docking simulations, they retained the top scoring pose from each run (for each ligand) and discarded those conformers that placed neither the ligand's head nor tail in the its correct binding pocket. Averaged docking free energies and inhibition constants were obtained for each ligand from the resulting manifold. These average inhibition constants correlate well with experimental k(cat)/K(M) values, as well as with experimental AChE binding affinities for a related series of trimethylammonio trifluoroacetophenone (TMTFA) inhibitors. As discussed earlier, a bound ACh appears to have a stabilizing effect on the both the catalytic triad as well as on coupling between the active and peripheral sites, leading to a free energy gain of 0.7 kcal/mol for ACh relative to that obtained for the static structure [122]. Binding of the substrate tail group to the anionic subsite appears to depend on both the size and the presence of a positive charge within the tail group. The removal of the positive charge leads to a loss of 1 kcal/mol in binding affinity. Substituting a hydrogen for the tail's methyl group results in both an incremental loss in docking energy as well as a decrease in the percentage of structures found to bind in an orientation suitable for catalysis. While the combination of MD and docking analysis yields both reasonably accurate binding affinity predictions and substantial physicochemical insight into the factors affecting binding, a major drawback of the method is heavy computational cost that renders the strategy less suitable for virtual screening, and cumbersome for the more speculative (i.e.,

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trial and error) schemes of receptor-based design. However, given the wealth of reliable experimental inhibition data across a fairly broad selection of AChE inhibitors, another powerful tool is available. Namely, the assumption that similar compounds should have similar binding conformations paves the way for the ligand-field methods such as Comparative Molecular Field Analysis (CoMFA) to be used for estimating activity. The earliest application of ligand-field technique to the AChE inhibition problem, to the best of our knowledge, appears to be the work of Cho et al. who employed templates obtained from crystal structures of AChE inhibited by edrophonium (EDR) and 9-amino1,2,3,4-tetrahydroacridine (THA) to align a set of 60 structurally diverse known inhibitors [147]. These aligned structures were then used as the basis for generating a region-focused CoMFA model. Strong correlation was obtained relative to AChE assay IC50 data, with a leave-one-out (LOO) cross validated correlation Q2=0.73. A more recent study by Bernard and coworkers used a hybrid method to estimate the binding affinity of 82 N-benzylpiperidine derivatives by flexibly docking them into the mouse AChE active site and establishing a 3D QSAR model based on this ensemble via CoMFA analysis [148]. This methodology established an important contrast to conventional CoMFA studies wherein compounds are typically fitted to the conformation established by a single reference; the docked structures conformationally respond to the known (albeit static) receptor structure, thus providing a more reasonable rendition of the spatial and electrostatic effects that define the pharmacophore. The resulting CoMFA model, which attained a cross validated LOO correlation of Q2=0.75 relative to an in vitro mouse AChE assay, was subsequently applied to analysis of another series of 29 N-benzylpiperidine derivatives whose inhibitory activity data corresponded to experimental conditions differing from those present for the training set (human AChE; different incubation times and pH). Reasonable agreement between predicted and experimental activity data was achieved (R=0.90), in spite of these important differences, demonstrating an inherent extensibility in the model relative to environmental effects, as well as similarity in AChE function / response across species. In a study of 42 aminopyridazine compounds, Sippl et al. also used a docking strategy for aligning ligand structures as a precursor to the model generation [149]. The aligned conformations were then taken as the basis for a three dimensional (3D) QSAR analysis applying the GRID/GOLPE method [150,151]. A model of high quality was obtained using the GRID water probe, as confirmed by the cross-validation method (Q2(LOO) = 0.937, Q2(leave-50%-out) = 0.910). The validated model, together with the information obtained from the predicted AChE-inhibitor complexes, was applied as a paradigm for designing novel inhibitors. Seven of the prospective inhibitors were subsequently synthesized and tested, and were found to be highly active. While CoMFA methods have a proven track record for generating models capable of reliable inhibition predictions, they are not an ideal basis for conceptual structure-based design strategies in that they produce a fairly abstract pharmacophore map that provides only vague notions of how to refine ligands in order to improve affinity. Of more intuitive value is a model that highlights specific atomic interactions between the receptor and ligand, and permits a ranking of those effects according to importance. In our group, a receptor-specific scoring function was developed for the purpose of predicting binding affinities for human AChE (huAChE) inhibitors and unraveling the underlying interactions [142]. This method is conceptually similar to CoMFA models such as those of Bernard et al. [148] with ligand conformations derived from explicit docking into the receptor target. It is more properly characterized as a Comparative Binding Energy (COMBINE) type approach [152], how-

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ever, in that it replaces CoMFA's standard grid-based molecular field corresponding to a probe atom with an estimate of binding affinity in terms of 3D QSAR, using enthalpic interaction terms between the ligand and the AChE residues as primary descriptors. This resulting model thus entails a statistically trained weighted sum of electrostatic and van der Waals (VDW) interactions between ligands and the receptor residues. Within a 53 ligand training set composed of piperidinium, pyridinium, benzisoxazole and E2020 derivatives, Guo et al. produced a model that yielded a strong correlation (R2 = 0.89) between computed and experimental inhibition constants [142]. Leave-one-out cross-validation also indicated high internal predictive power (Q2 = 0.72), and analysis of a separate 16-compound test set of similar chemical constituency as the training set also produced very good correlation with experiment (R2 = 0.69). The residue-specific scoring function is highly amenable to deconvolution, thus permitting the identification and characterization of important ligand-receptor interactions. The resulting list of residues making the most important electrostatic and VDW contributions within the main active site, gorge area, acyl binding pocket, and peripheral site is largely consistent with available X-ray crystallographic and site-directed mutagenesis data and also provides new insight, supporting the concept of a gating role for Tyr337 in huAChE analogous to that of Phe330 in tcAChE as predicted in prior MD calculations [153]. Interestingly, within this COMBINE model, Tyr337 registers not as one of the dominant hydrophobic interactions (as would clearly be expected for Phe330 in tcAChE) but rather as an electrostatic contributor. This subtle difference among residues serving an otherwise analogous role between tcAChE and huAChE may prove to be a lead toward to devising inhibitors capable of differentiating between AChE from mammalian and other sources. Another recent study employing COMBINE methodology was carried out by MartinSantamaria and coworkers, exploring ligand binding modes within the tcAChE catalytic active site to identify the key residues that modulate the inhibitive potential of a collection of tacrine-, huprine-, and dihydroquinazoline-based acetylcholinesterase inhibitors [153]. The resulting set of interaction enthalpic descriptors, optimized via partial least-squares (PLS) fitting across a unique training set containing the full set of compounds, produced a model exhibiting both strong correlation and predictivity (R2 = 0.91 and Q2 = 0.76, using 4 principal components) across the entire set of ligands. Greater internal predictivity (Q2 = 0.81 and standard deviation of errors in prediction (SDEP) = 0.25 for 3 principal components) was achieved when restricting the training (and corresponding testing) set to just the collection of huprines. The authors uncovered a pronounced correlation between the affinity trends among the inhibitors and their corresponding receptor desolvation term. This may prove to be a simple and powerful descriptor for intuiting new and improved AChE inhibitors. Such COMBINE methods, with their valued attributes of computational efficiency, accuracy, and mechanistic insight, may prove to be vital tools in the pursuit of high throughput virtual screening of irreversible non-covalent AChE inhibitors. Results from past virtual screening studies on AChE have not been overwhelmingly successful. A recent study by Mizutani and Itai [154] screened a virtual library of about 160 000 commercially available compounds against the tcAChE X-ray crystallographic structure. A total of 1551 potential inhibitors were identified. Among the 114 virtual hits that could be purchased and assayed, 35 molecules spanning a reasonable selection of chemical space showed inhibitory activities with IC(50) values less than 100 μM. Thirteen compounds had IC(50) values between 0.5 and 10 μM, most of which displayed substantial chemical uniqueness relative to known inhibitors. While the fact that this exhaustive study uncovered potential new scaffolds for inhibitor discovery is in itself an important contribution, none of the identified and validated

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inhibitors remotely approach the known subnanomolar efficacy of various non-covalent inhibitors identified through purely in vitro screens [155]. Furthermore, the relatively high false positive ratio (69%) suggests relatively poor scoring, and may also imply a sizeable false negative rate that is leaving potentially viable chemical space untapped. It would be interesting to apply a more accurate scoring technique such as a COMBINE-based assessment to a collection of this size V. COVALENT BONDING EFFECTS The scientific literature abounds with examples of computational studies of enzyme catalysts, amply demonstrating that some level of quantum theory must be used to properly model reactive chemistry occurring at the enzyme active site. This is especially vital for AChE, where multiple reaction mechanisms are of immediate interest. Although we have discussed in detail the function of cholinesterase inhibitors that involve non-covalent interaction, a number of systems (ranging from therapeutically useful to toxicologically perilous) exist which function through binding at the active site Serine 203. Among these covalent binders, OP compounds appear to be unique in being posited to undergo an additional side chain dealkylation (a reaction commonly known as ‘aging’) which greatly exacerbates their toxicity and can yield effectively irreversible product states. Fortunately, a number of compounds of potential therapeutic value have been proposed to facilitate the OP-AChE dephosphorylation reaction (prior to any possible aging step), thus restoring enzyme functionality. A full appreciation and the basis for improving upon current antidotal therapeutics requires a thorough understanding of the mechanisms of the underlying reactive processes and their incumbent physicochemical dependencies. The use of quantum models to elucidate such mechanistic detail evokes problematic issues of model truncation, the effect of missing residues, solvation, and lack of the enzymatic environment in toto. To avoid the computational demands of quantum mechanical models involving the thousands of atoms in present in such a biological system, mixed quantum mechanical /classical mechanical (QM/MM) models have proven to afford a reasonably efficient and realistic alternative. QM/MM algorithms enjoyed a period of rapid development during the 1990s, and many of the major quantum software packages have implemented some form of QM/MM in an attempt to facilitate mainstream study of biomolecular systems. AChE was recognized early in the development process as a candidate enzyme likely to benefit from use of an enlarged model. Vasilyev published a very effective QM/MM study [156] (utilizing semiempirical PM3 theory and the OPLS forcefield), demonstrating the strong influence of hydrogen bonding within the catalytic triad on the configuration of the bare enzyme, as well as on the acylation and phosphorylation reactions. This work demonstrated the stabilization by the enzymatic environment of the tetrahedral intermediate (TI) formed during acylation. Particular attention was paid to the role of the oxyanion hole residues, although these residues were not included in the quantum layer of the model system. Several truncated but fully quantum mechanical models for the acylation of the AChE active site were used in conjunction with Brownian dynamics studies by Wlodek et al. [157]. Four models of the active site were utilized to further investigate the role of Glu202 and the oxyanion hole. These models consisted of: 1) WI (model I Wild Type Enzyme): the substrate, the sidechains of the active site triad, and Glu202 sidechain 2) MI (model I mutant): WI model with no Glu202

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3) WII: (model II Wild type) WI plus ammonia groups representing backbone amide of Gly121, Gly122, Ala204 4) MII: (model II mutant) WII with Glu202 sidechain replaced by Gln As is often the case with extremely truncated models, it was necessary to impose constraints by fixing a selection of backbone atoms, including the amide nitrogens of each oxyanion hole residue and the C
for all other residues. Energy profiles were generated for formation of the tetrahedral intermediate (TI) at the HF/3-21G level for all 4 model systems. Although there were definite questions raised by the truncation of the active site and the level of the quantum treatment, these results confirmed the critical role of the oxyanion hole in stabilization of the TI. It was additionally suggested that Glu202 stabilizes the transition state of the acylation via electrostatic interactions with the His447 imidazole ring. Fuxreiter and Warshel studied AChE acylation through calculation of activation free energies using the empirical valence bond (EVB) and an all-atom free energy perturbation (FEP) approach [158], in conjunction with the PDLD/S-LRA method. This work treated the acylation as a two-step process consisting of an initial proton transfer from the active site Ser203 to His447 and a subsequent nucleophilic attack by acetylcholine on the serine. The importance of the ionization state of both nearby glutamates Glu202 and Glu334 was demonstrated, and the significance of oxyanion hole residues was again confirmed. The effect of the enzymatic environment was determined to be beneficial to both steps of the mechanism. The overall decrease on the activation barrier was determined to be ~10-15 kcal/mol relative to an aqueous reference reaction, ostensibly in line with the catalytic effect observed experimentally. Vagedes et al. [159] followed this work with an EVB simulation of the deacylation reaction. This group studied two different mechanisms for the reaction: a two-step process (proton transfer from water to the active site His447 followed by nucleophilic attack of the remaining hydroxide ion on the acetylated Ser203 to produce the tetrahedral intermediate of the deacylation step) and a concerted (simultaneous proton transfer and nucleophilic attack) mechanism were studied. Preliminary to the EVB analysis, the investigators performed Monte Carlo simulations to study the protonation states of all 145 titratable groups in the AChE, and reached the conclusion that Glu202 is protonated in both the free and acylated enzyme. The EVB work determined an energy barrier lowering of 11-12 kcal/mol in AChE relative to an aqueous reference reaction, and again demonstrated a sizable effect from Glu202 and the oxyanion hole residues. Both the concerted and stepwise mechanisms were accelerated in a similar fashion by the enzyme. The investigators recommended the concerted mechanism through comparison to experimental kinetic data. More explicit quantum detail on the acylation reaction was provided by Zhang et al. [160], who used a pseudobond ab initio QM/MM approach with HF/3-21G for the quantum layer and the AMBER95 forcefield for the remainder of the enzyme (Additional single point calculations were carried out at MP2/6-31+G* and B3LYP/6-31+G* for points of interest along the reaction profile). Two different models were used for the quantum layer, a smaller model consisting of the ACh and the active site serine and histidine sidechains, as well as a larger model that also included the active site Glu334. A concerted mechanism was proposed (simultaneous acylation of active site Ser203 and proton transfer from Ser203 to His447), and a transition state obtained which resulted in a 10.5 kcal/mol potential energy barrier for the large quantum model at MP2/6-31+G* (12.6 kcal/mol for the smaller system). This result was determined to be consistent with experimental data. As there were minimal energy differences between the small and large quantum models, it was proposed

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that the Glu334, while providing a vital electrostatic interaction as well as forming a short hydrogen bond with the His447, did not interact via a charge-relay mechanism. The role of the oxyanion hole was again confirmed, suggesting continued existence of two hydrogen bonds, between the ACh and the Gly121 and Gly122 backbones, throughout the course of the reaction. Formation of a weak hydrogen bond between ACh and Ala204 occurred as the reaction proceeded. The role of the notorious Glu202, while important, was less clear and left to future work. The use of QM/MM was expanded past the acylation reaction to predict binding free energies for O,O’-1,4-xylene bispilocarpic acid diester C6 as well as the inhibitors bambuterol and TMTFA. This study included both AChE and BChE, and undertook to understand the differences in the active site regions of the two enzymes [161]. This work, done at the AM1/Dreiding FF level, used a large quantum layer consisting of all residues within 5 Å of the active site Ser oxygen (13 residues for AChE, including the active site, oxyanion hole, Glu199 (tcAChE numbering), as well as additional residues). The thrust of the analysis was aimed at differentiating between BChE and AChE. The role of conformational flexibility of the Phe330 residue (tcAChE numbering) was discussed, and the direct role of solvent in the active site proposed. Additional quantum work has been performed on reactions within the active site related to enzyme phosphorylation as well, although in some cases the role of the quantum simulation was primarily to provide input (refined adduct structure and charges) to further classical simulations. Bencsura et al. [132, 162] utilized this technique to optimize the side chains of the active site serine (and neighboring two residues), as well as assorted adducts mimicking soman, aged soman, and ACh. This work utilized the MNDO method to generate refined structures and charges, which were then employed in molecular mechanics simulations. Evidence was provided again for stabilization provided by the oxyanion hole and catalytic His, and proposed the role of Glu202 as a driving force in the aging reaction, by “pushing” the alkoxy group. This work also provided an analysis of the role of stereochemistry (PR vs PS soman) in active site inhibition, and was expanded further later. A similar approach [163] has been applied to VX, albeit using a very truncated active site model and a numerical basis, and energy partitioning techniques have been applied to a truncated sarin-AChE system at both B3LYP and MP2 levels, identifying the importance of Glu199 (tcAChE numbering) in accordance with previous results [58]. Further quantum mechanical and QM/MM exploration of the phosphorylation reaction was performed by Hurley et al. [164] The mixed quantum/classical work explored the dependence of the catalytic triad configuration on model details such as force field choice, model size, and quantum theory level. Multiple quantum models were used in the fully QM work, which studied the binding of the simulant methyl methyl phosphonofluoridate to the active site. Small models were used which consisted of the substrate and the active site sidechains. The expanded quantum models treated the entirety of the oxyanion hole residues in an ab initio fashion, some of the first work to do so. Analysis of the transition state for the small model performed at the B3LYP/6-31G* level demonstrated several points. The transition state barrier was calculated to be ~15 kcal/mol, which was approximately 15 kcal/mol lower than the concomitant aqueous reaction, fully in line with previously cited work on the related deacylation reaction [164]. The transition state itself corresponded to a concerted mechanism (expanded past that of previous work), demonstrating agent binding simultaneous with dual proton transfer across the active site (Ser -> His and His-> Glu), an argument in favor of including the full active site in the quantum model. It must be noted that the product state in this work was a pentacoordinate phosphorus adduct (later work demon-

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strated the role of solvent in removal of the leaving group on OP binding [165]). Additionally, results from the expanded quantum model demonstrated not just hydrogen bond formation, but appreciable charge transfer upon binding between the OP compound and the oxyanion hole residues, a telling argument for inclusion of the oxyanion hole in the quantum model. Short hydrogen bonds within the active site were found in both the reactant and product states, and calculated distances were in good agreement with experimental results. The role of Glu202 has been a recurring theme throughout this work, yet it remains a question that has not been definitively answered by simulation or experiment. This mobile glutamate has been nominated for everything from a somewhat passive role in electrostatic stabilization to active participation (“pushing”) in reactions including both binding and aging. Additionally, X-ray crystallographic work on tcAChE [166] demonstrated mobility in the active site His440 (huAChE = His447), revealing that it could move from a position binding to the active site Glu327 to the Glu199 (huAChE = Glu330, Glu202), or alternatively reorient the other side of the imidazole ring from binding to the active site Ser200 (huAChE = Ser203) to the OP sidearm. This was furthered by subsequent proton NMR data [167]. Later quantum work [168,169] made an initial attempt to address some of these issues. The model used was expanded from earlier work to include the full catalytic triad residues (rather than sidechain models), the full oxyanion hole residues, Glu202, and an aged DEFP (OP compound). This work, performed at the B3LYP/6-31G** level, studied relative energetics on variation of the binding pattern of the active site histidine, and demonstrated the feasibility of the mobile His447 schema. An additional insight was provided upon analysis of the calculated proton NMR signal and hydrogen bond distances within the active site of the above models. Experimental NMR data and previous molecular dynamics work [132,162] had suggested a shortening of the hydrogen bonding within the active site upon binding. It was demonstrated in the work of Hurley et al. that this in fact will only occur in quantum models containing the Glu202 residue [168, 169]. It must be noted that this is the case even in configurations where His447-Glu334 is the primary hydrogen bond (rather than His447-Glu202). It is apparent from this, that the effect of Glu202 on His447 has its subtle influences as well as its overt ones. This result merits further exploration. While the relative degree of QM/MM enzymological research activity has declined in the years following our initial review of the AChE modeling field, some productive applications have been reported recently including studies aimed at validating QM/MM methods relative to observed reaction kinetics [53] and investigating the covalent susceptibility of AChE h447i mutants [54, 55]. In addition to these, a fascinating prospect was raised with the report in the abstracts of the 232nd ACS National Meeting, San Francisco, CA, United States, Sept. 10-14, 2006 (Y. Cheng, X. Cheng, and J.A. McCammon) of a method that merged a QM/MM representation of the AChE gorge into a FE continuum model of the whole enzyme to produce a relative computationally efficient model of AChE theoretically capable of representing gorge diffusion processes in addition to receptor-specific covalent adduction or catalysis events. The abstract suggests good agreement with experimental kinetics data on wild type and h447i mutant receptors, but the research has thus far not appeared in peer-reviewed literature. Another intriguing development with respect to covalent AChE modeling was the study by Mochizuki et al. [51] who computed a single point quantum chemical calculation on an entire tcAChE monomeric unit complexed with E2020 (Aricept) via a correlated ab initio method known as fractional molecular orbital MP2 (FMO-MP2). The calculation, involving a total of 8409 atoms and 46831 basis functions, exploited the exceptional parallel computing scalability of the FMO-MP2 method, and was performed in less than an hour by parsing

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out the computations over 1024 processors [51]. Very few researchers have dedicated access to that magnitude of computing power, but it is nonetheless quite striking that such a simulation is now possible. While Aricept is not a particularly interesting choice as a test case (it does not interact covalently with the receptor), it is easy to envision possible calculations on covalently binding inhibitors or reactivators that might circumvent difficulty issues in QM/MM models (e.g., artificial choice of reactive region, boundary effects, etc.) and enable more rigorous assessment of the potentially role of non-local effects such as polarization and proton mobility on the resulting chemical kinetics. VI. UNIFYING THE MODELS Despite the recent achievement of a fully quantum mechanical model of an AChE-ligand complex [51], such calculations remain too computationally intensive to pursue on a regular basis, thus practical studies on AChE are still best accomplished via the assimilation of information from multiple different techniques. The application of computational models to reproduce experimentally determined conformational details of the ligand-receptor complex has traditionally proven quite straightforward, both for noncovalent inhibitors (as in Section IV) and covalent species (Section V). Translating the results of computational studies into accurate prediction of in vitro and in vivo efficacy has proven to be significantly more challenging for classes of covalent inhibitors than for non-covalent, however. In addition to the successful structure-based methods discussed in Section IV, quantitative structure-activity relationship (QSAR) analysis has proven reasonably effective in assessing and predicting non-covalent AChE inhibition. A number of early QSAR studies explored the function of various ligand substituent groups via simple charge- and shape-based descriptors, achieving reasonable reproduction of experimental in vitro AChE inhibition results [170,171]. Tong et al. developed a CoMFA model for a series of 1-benzyl-4[2-(Nbenzoylamino)ethyl]piperidine derivatives and N-benzylpiperidine benzisoxazoles, finding a strong correlation between the inhibitory activity of those N-benzylpiperidines and both steric and electronic aspects of the ligand set [172]. Hasegawa and coworkers applied a genetic algorithm-based region selection scheme to refine the CoMFA model, obtaining a model with improved predictivity [173]. Sulea et al., pursued the topic in a somewhat different manner [174], deriving a structure-activity relationship as a function of an ensemble of different ligand conformers, each optimized at the AM1 level [175] via the adjusted multiconformational minimal steric / topology difference (MTD-ADJ) method. The resulting topological pharmacophore maps corresponded well with the spatial constraints apparent in crystallographic receptor structures. Prediction of toxicity trends among covalent AChE inhibitors such as OP compounds has proven much more difficult, largely because of other complex, target-nonspecific interactions that tend to occur in vivo. Eldred and Jurs evaluated an array of different descriptors that might be appropriate for encoding the topological, electronic and geometrical features for a set of 54 OPs with acute oral acute oral mammalian toxicity [176]. Given serious limitations in the body of experimental information regarding the important structural features driving interactions between OP compounds and mammalian AChE, they used a genetic algorithm scheme to find subsets of descriptors that would support a high quality computational neural network (CNN) model capable of linking the structural features to activity trends. A non-linear CNN model was determined that yielded strong correlation relative to experiment. A principle drawback of such a nonlinear model, however, is that is does not provide clear insight into the mechanism underlying the OP toxicity, especially since some

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of the descriptors used in their study have at best an abstract relationship with the actual mechanism and are not readily connected with precise physicochemical processes. Other QSAR studies on covalent / toxic inhibition have focused more on concrete physicochemical parameters with tangible relationships to the OP - AChE inhibitive process. Yazal et al. used a pharmacophore model to describe the inhibition arising from a small set of 8 specific OP compounds, ultimately determining that OP inhibitive potential seems to hinge on the availability of one ligand hydrogen bond acceptor (corresponding to the phosphonyl group to be anchored within the oxyanion hole) and 2-3 ligand hydrophobic sites [177]. More recently, Zhao et al. used CoMFA to study the acute housefly toxicity for a set of 35 OP compounds and determined that the length of ligand's alkyl group, and the electronegativity of the various substituent groups on the P center have significant effects on the AChE activity, but the overall hydrophobicity of the OP molecule appears to have minimal influence [178]. This is in keeping with wide-spread consensus that steric and electronic properties of the OP species play a dominant role in the covalent inhibition of AChE. Bernard and coworkers also explored the interaction between OP compounds and AChE by using CoMFA for a set of 35 compounds with conformations obtained from docking calculations [179]. Somewhat surprisingly given the non-covalent (Michaelis complex) basis for the ligand conformational ensemble, a reasonable correlation (Q2 = 0.70) was found relative to in vitro AChE assay data arising from a variety of different experimental sources. While such QSAR studies based on correlations with in vitro data do have demonstrable value in deriving insight into conditions responsible for effective AChE inhibition, true OP toxicity assessment is only really accessible from in vivo studies. Frequently, experimental in vitro data has been found to exhibit poor and even minimal correlation with in vivo toxicity studies, primarily because of contributing effect of metabolic biotransformation present in the latter [180]. To complicate matters, it has been found that OP metabolites may produce a greater toxic response than their parent compounds. Furthermore, even within the AChE receptor cavity itself, the binding energy of the Michaelis OP-AChE complex is only one of several tangible factors affecting the OP inhibition, since phosphorylation and aging reactions are critical aspects of the inhibition process and may be expected to manifest some differential properties across even a relatively similar family of inhibitors. In the recent work, we have been applied information from a number of different computational methodologies and sources in pursuit of a simple model that both improves the basic mechanistic understanding of OP toxicity and yields reasonable toxicity predictions [181]. Within our training set of 30 known covalent AChE inhibitors (all OPs), 21 were known to act primarily through well characterized metabolites, whereas the other nine were believed to act directly on AChE in their parent form. For the former subset, cytochrome P450 metabolic chemical reactions were quantified approximately as enthalpy-driven processes by including the quantum chemical (AM1) electronic energies of the parent compound and metabolite in the QSAR. Additional absorption, distribution, and excretion effects were accounted for via simple physicochemical parameters. The Michaelis complex binding affinity was incorporated via CoMFA calculations. Finally, OP phosphorylation and aging reactions were approximated enthalpically, again via inclusion of reactant and product energy terms in the QSAR expression. This simple yielded a strong correlation relative to experimental acute toxicity data (R2=0.72), and provided valuable information about the relative weights of these empirical terms (and their corresponding mechanistic processes) in the net toxic process. Further refinement of the model is envisioned through consideration of factors such as ligand solubility, receptor desolvation, processes stabilizing the phosphorylation and aging leaving groups and so forth.

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In the four years since the original version of this review was published, the general protocols by which most researchers pursue model integration and development has adhered closely to conventional canonical QSAR techniques, with most studies selecting molecular descriptors from a relatively narrow slate of options, and applying some variant of PLS method to train linear combinations of these descriptors into a form that predicts macroscopic experimental observables. Modest new developments have been made in the area of developing new descriptors for model generation, including the application of shape signatures [27], and simplex-informational descriptors [69]. However, more progress has been achieved in refining the protocols by which models are trained and refined. Our suggestion in 2006 of the importance in applying feature selection methods to such studies has (not surprisingly given the popularity of feature selection in other applications) begun to take hold, as is evinced by a number of studies performed in recent years [75, 79]. The value of feature selection is greater simplicity: by eliminating descriptors that, alone or in linear combination, tend to duplicate trends already apparent in other descriptors or combinations thereof, one arrives at a more compact and informationally efficient body of molecular attributes from which to train models. Since model training algorithms rarely yield exact solutions for the best fit to experimental data when faced with a large feature basis, careful reduction of the descriptor set often improves the odds of arriving at a more predictive and reliable final model. The other way to potentially improve the quality of the final model can be to employ training algorithms of greater sophistication than PLS, examples of which include support vector machine [27] and neural networks [79-81,176] techniques. VII. FUTURE DIRECTIONS At the present time (ca. 2010) an ideal theoretical model of an enzymatic system might be envisioned as an atomic level representation that accounts for all relevant external environment effects, evolves dynamically over biologically meaningful time periods as a function of plausible biological temperature variations, permits substrate or inhibitors (or both, in a competitive fashion) to enter the model under biologically reasonable transport conditions, supports dynamic chemical reactivity in a realistic time frame and permits products to leave the system at similarly reasonable rates. For AChE. such a model would probably entail a fully solvated active form (i.e., tetrameric) AChE, thus amounting to a coupled quantum-classical molecular dynamics simulation involving on the order of 105 atoms (of which perhaps several hundred would require treatment at quantum mechanical levels) running over a period of at least μ-seconds (the time to process an ACh substrate molecule) and perhaps extending up to the seconds (aging half life for soman) time frame. Needless to say, such a simulation is computationally far in excess of current capabilities and is unlikely to become viable soon, even under the most optimistic interpretations of Moore's Law. In the foreseeable future, therefore, human ingenuity will continue to be our most valuable resource as we uncover increasingly sophisticated methods for approximating such complex and lengthy mesoscopic phenomena. Such a vision of computational simulation of acetylcholinesterase-related biological phenomena on the grand scale is given in a 2004 publication of Tai [182]. For the time being, more practical methods such as variants of QSAR will undoubtedly remain important computational tools for enzyme inhibition research. In the previous section, some near-term plans were discussed for the development and refinement of predictively robust AChE toxicity QSAR models. Attaining reliable and physically intuitive descriptors, in this case, is proposed via detailed analysis of the system through higher level modeling techniques such as quantum chemistry and MD. The basic assumption behind this approach is that many important phenomena governing the enzymatic process (e.g., chemi-

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cal reaction mechanisms, intra-receptor diffusion, etc.) occur in a fundamentally similar fashion across a broad range of potential inhibitors, and that the differences in behavior from one instance to another can be distilled down to simple differences in the structure and properties of the ligands themselves as elucidated from detailed atomic simulations that are localized in space (i.e., only covering a single AChE unit for MD, or even just a fragment of a unit for quantum) and, if dynamic, occurring over a relative short (ns) time scale. A similar strategy of integrating results from detailed atomic level simulations may also prove useful in further refining our COMBINE representation of non-covalent binding. One of the most important limitations in the current model is the approximate treatment of receptor-related dynamic effects, incorporated empirically within statistically trained weights factors applied to the relative enthalpic contribution of different receptor residues to the net ligand binding affinity. In reality, it is very difficult to extract all relevant relaxation-related entropic contributions from a system without some explicit account of the real torsional motions from whence they originate. One possible scheme for recovering a substantial portion of these dynamic effects in a computationally viable fashion is by isolating characteristic receptor conformers from the trajectory of a MD simulation and expressing each ligandreceptor complex not as a static single structure, but rather as a linear combination of multiple different complex conformers. As discussed in Section IV, prior work by Kua et al. [122] sought to implement such a multi-conformational receptor model, although the conformer selection used (fixed-period snapshots every 1 ps over the course of a 1 ns simulation) has the deficiencies of extensive redundancy (typically sampling numerous very similar conformers) and of not ensuring the inclusion of rare-event conformers that are often critical to biochemical processes [183]. Induced-fit modeling techniques may afford a reasonable mechanism for addressing the question of specific receptor conformations relevant to the dynamic binding process, although their suitability for describing AChE has been a source of interested debate [43, 45]. Another alternative that may endow potentially greater discrimination of key receptor dynamics would be to consider using an intelligent featureselection based technique [184] to extract from MD trajectories a small targeted set of structurally unique receptor conformers that are chosen to optimize coverage of conformational space. Flexibly docking ligands into this more modest-sized but also more diverse ensemble of receptor conformers should enhance our ability to approximate a true flexible docking simulation and entail only a small fraction of the computational cost of a more arbitrary sampling. While QSAR methods, including CoMFA, COMBINE, newer analogs such as the informational field and simplex models [69], and hitherto unpublished future variants, will undoubtedly continue to provide valuable predictions for a wide range of inhibitive issues, they remain limited in their ability to predict phenonema or behavior too far beyond the breadth of their empirical programming. This is to say that a model parametrized under one set of conditions (e.g., type of chemical inhibitive mechanism or physical binding mode) may yield predictions of questionable accuracy when extrapolated to another disparate set of conditions (e.g., different mechanism or binding mode, mutated receptor, significant differences in ligand chemical functionality or binding mode, etc.). In this respect, such methods can be viewed as somewhat inflexible. The desire to probe AChE modulation under diverse conditions of inhibition, mutation, environment, etc., has perpetuated a strong interest in more methodologically flexible models capable of reliably predicting response to variable or unusual stimuli. For this reason, MD and quantum chemical simulations (and combinations thereof) will almost certainly remain important tools of choice in that they are highly adaptive, and thus generate modes that effectively respond to situations that may not have been anticipated and empirically encoded into the original paradigm.

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The drawback with both quantum mechanical methods and MD simulations is computational cost. Treating an entire enzyme quantum mechanically at an accuracy level sufficient for qualitative reproduction of even fairly basic phenomena is very challenging, and while computational capacity is gradually rising to the task it is our assumption that complex geometry searches (e.g., transition states, internal reaction coordinations, shallow local minima, etc.) and quantum MD studies of reasonable length (more than a few ns) will remain impractical for the foreseeable future and that scientific progress will continue to depend as much on new algorithmic tricks and judicious computational shortcuts as on brute-force large scale computations. Conversely, for classical MD simulations it is already very feasible to model entire proteins via fully atomic models, however the time scales in which to resolve many phenomena of interest (frequently μs for ligand diffusion, loop restructuring or receptor relaxation) remain far beyond our current capacity (tens of ns). One wonders, therefore, what sort of refinements and modifications will be made to these techniques in order to enhance their adequacy for simulating biologically meaningful systems, properties and phenomena? The time scale issue is probably the single most challenging barrier to a biologically realistic treatment of deep / narrow gorge enzymes such as AChE. In such cases, steric constraints make it increasingly probable that particle diffusion will be the primary rate determining step in such embedded receptor structures. The corresponding diffusion kinetics are frequently governed by rare event phenomena whose incidences are most readily ascertained through computationally demanding MD simulations. Conventional MD simulations of systems as large as proteins currently tend to be limited to the tens of nanoseconds time scale, at which level they frequently produce inadequate sampling of all potentially relevant rare events. Accelerated MD methods may yield a reasonable solution to this dilemma, although the precise method of acceleration must be carefully validated against known physical behaviors before any degree of trust may be placed in the predictions for hitherto uncharacterized processes. Temperature accelerated molecular dynamics for ligand diffusion into an inherently flexible system (as is the case for many enzyme receptors including the AChE active site) may, in addition to speeding up physically reasonable processes, induce unphysical motion and thus compromise simulation accuracy. Other methods require advance specification of a subset of the accessible potential energy surface to be explored, ultimately prejudicing the system to intuitively straightforward conformers and thus falling into the methodological inflexibility trap that we seek to avoid. Other accelerated methods, however, may well prove suitable. One such method is that of Hamelberg et al. [185] wherein barriers are softened via bias potentials. Initial signs that this scheme reproduces known equilibrium structures and identifies plausible rare event conformers make it an encouraging prospective mode of analysis for intra-cavity ligand diffusion and prediction of complementary receptor motion [185]. As discussed earlier, continuum methods based on a FE continuum representation of the protein structure may help to significantly reduce the structural degrees of freedom that must be accounted for in a dynamics simulation and thus permit access to more ambitious time scales. The pioneering work of Song et al., which demonstrated a reasonable representation of larger length scales through a novel application of continuum (i.e., non-atomistic) mechanics [186], has slowly been gaining traction. The incumbent FE solution of the steady-state Smoluchowski equation for modeling has been applied to model ligand diffusion through the gorge of both monomeric and tetrameric AChE and thereby estimating diffusion-controlled ligand binding rate constants [83-85,186,187]. This promising approach has yielded reaction rate calculations in reasonable agreement with experimental data at far less computational cost than analogous Brownian Dynamics (BD) simulations.

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Incorporation of reactive processes entails another set of challenges. QM/MM formalisms provide a promising paradigm for taking simultaneous account of whole protein structural properties and localized reactive or polarizational phenomena. People have envisioned applying such techniques toward the treatment of concurrent dynamic and reactive phenomena so as to permit receptor dynamics to exert their due influence on reactive processes. Careful design and validation is likely still required before effective application is made to the AChE system, however, in that broadly available IMOMM-based methods have exhibited significant deficiencies in their ability to reproduce essential interactions in AChE-OP complexes [164]. It is likely that such problems can be largely mitigated through development of carefully tuned polarization schemes for softening the quantum-classical boundary, and through careful analysis of forces across the boundary to ensure that the quantum and classical regimes are in an appropriate balance. Even given a microscropically robust QM/MM formalism, however, there are major practical challenges to be addressed before dynamic reactive simulations are viable across a reasonable spread of biologically relevant time scales. The task of recomputing a wavefunction at every time step in a lengthy trajectory is currently impractical, even for relatively modest sized quantum shells. Integration of classical MD with a Carr Parrinello representation of the quantum region provides somewhat enhanced efficiency [188] by virtue of intelligently extrapolating an estimated wavefunction from step to step, however the simulation length has not, to our knowledge, yet been extended beyond the picosecond time domain and thus is of only limited applicability. A possible solution to this dilemma may be achieved at some point by selectively switching on the localized quantum mechanical representation only at times when it is likely to have a tangible impact on the accuracy of the structural evolution. The main kinetic limitation for most chemical reactions tends to be a low probability of achieving conformations suitable to activated complex formation. Once the trajectory zeroes in on an activated complex, most chemical reactions occur relatively quickly if accorded sufficient energy along the reactive vibrational mode. At this point we are not yet aware of adaptive methods that have been published and made available for simulations of enzyme systems, however it is possible that they will ultimately provide a reliable basis for dynamic reactive simulations on biologically useful time scales. The aforementioned QM/MM-FE hybrid model discussed in a 2006 ACS National Meeting abstract (Cheng et al.) may bring the task closer to practical viability. The inherent complexity of the AChE enzyme system and its importance to a number of critical health related issues have colluded to yield a vibrant subdiscipline of computational enzymology that is driving the development of innovative, increasingly reliable and efficient simulation techniques that are transforming the computational chemistry field. The fact that a Google search (ca. January, 2010) on the terms "acetylcholinesterase" and "simulation" or "computational" or "modeling" yields approximately 114,000 hits is a testament to the current importance of the topic. While the breadth of computational research in the AChE modeling arena is too great to fully capture in a single review article, we hope to have provided a reasonable overview of some of the more important contributions and their potential implications for future therapeutics discovery. ACKNOWLEDGEMENTS This work was supported in part by a sub-award from NIH grant #5 P20 RR016475-03, and by a Department of Defense Joint Services Science and Technology Base subcontract #DAAD16-02-P-0188. The authors would like to thank J. B. Wright and Alex Balboa for their advice and assistance with manuscript preparation.

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