Progress in Medicinal Chemistry 39
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Progress in Medicinal Chemistry 39
Editors: F.D. KING,
BSC.,D.PHIL.. C.CHEM., F.R.S.C.
GlnxoSmithKline New Frontiers Science Park (North) Third Avenue Hurlow, Essex CM19 5 A W United Kingdon?
and A.W. OXFORD,
M.A., D.PHIL.
Consultunt in Medicinal Chemistry P.O. Box IS1 Royston SG8 5 Y Q United Kingdom
2002
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1. Pharmaceutical chemistry I. King, F. D. 11. Oxford, A. W. 6 15.1’9 ISBN: 0 444 50959 3
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Preface Five important topics in medicinal chemistry are reviewed in this volume. Chapter 1 provides a comprehensive account of inhibitors of the caspase family of proteolytic enzymes that represent a new class of anti-inflammatory and antiapoptotic agents of potential value in rheumatoid arthritis, brain ischaemia and other central and peripheral indications. The extent to which the peptidic character of early inhibitors has been reduced is also discussed. It is imperative that novel approaches are urgently pursued to overcome the increasing problems of resistance to antibacterial and antiviral agents, especially to HIV. Hence chapter 2 focuses on recent advances in our understanding of the binding of these agents to ribosomal RNA or RNA/ protein complexes as this should provide a major impetus to the design of sinall molecules. Included also is a well documented survey of semi-synthetic and totally synthetic antibiotics and anti HIV agents and their sites of interaction. A plethora of structural types, including many derived from natural products, have been described as inhibitors of the intracellular enzyme acylCoA: cholesterol O-acyltransferase (ACAT) and these are comprehensively reviewed in chapter 3. Although disappointing in hypercholesterolemia, for which they were originally developed, ACAT inhibitors may prove effective as anti-atherosclerotic agents. Strategies to reduce the peptide character of growth hormone secretagogues leading to drugs with improved oral bioavailability and duration of action are described in chapter 4. Encouraging clinical studies with first generation compounds in growth hormone deficient children and adults suggest these agents will fulfil an unmet medical need. The proteolytic enzyme, hepatitis C virus NS3-4A protease, required for viral replication, is one of the most attractive targets for HCV infections. The problems in designing potent inhibitors is lucidly described in chapter 5 and traces the evolution so far of non-peptide inhibitors from peptides.
vi
PREFACE
We are most grateful to all the authors of this volume for committing so much of their time and effort to assessing the extensive literature of their topics and compiling these reviews. We also thank the staff of the publishers for their continuing support and encouragement to the series.
July 2001
F. D. King A.W. Oxford
vii
List of Contributors Fareed Aboul-Ela Department of Structural Biology, RiboTargets Ltd., Granta Park, Abington, Cambridge CB1 6GB, U.K. Mohammad Afshar Department of Drug Design, RiboTargets Ltd., Granta Park, Abington, Cambridge CB1 6GB, U.K. Michael Ankersen Medicinal Chemistry Research I, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Denmark Martin J. Drysdale Department of Chemistry, RiboTargets Ltd., Granta Park, Abington, Cambridge CBI 6GB, U.K. Piotr P. Graczyk Department of Medicinal Chemistry, EISAI London Research Laboratories, University College London, Bernard Katz Building, London WClE 6BT, U.K. Brian R. Krause Pfizer Global Research and Development, Ann Arbor Laboratories, 2800 Plymouth Road, Ann Arbor, MI 48105. U.S.A. Ann D. Kwong Vertex Pharmaceuticals, Inc. 130 Waverly Street, Cambridge, MA 02139. U.S.A. Georg Lentzen Department of Drug Discovery, RiboTargets Ltd., Granta Park, Abington, Cambridge CBl 6GB, U.K.
...
Vlll
LIST OF CONTRIBUTORS
Natalia Matassova Department of Drug Discovery, RiboTargets Ltd., Granta Park, Abington, Cambridge CBI 6GB, U.K. Alastair I. H. Murchie Department of Drug Discovery, RiboTargets Ltd., Granta Park, Abington, Cambridge CBI 6GB, U.K Robert B. Perni Vertex Pharmaceuticals Inc., 130 Waverly Street, Cambridge, MA 02139, U.S.A. Joseph A. Picard Pfizer Global Research and Development, Ann Arbor Laboratories, 2800 Plymouth Road, Ann Arbor, MI 48105, U S A . Drago R. Sliskovic Pfizer Global Research and Development, Ann Arbor Laboratories, 2800 Plymouth Road, Ann Arbor, MI 48105, U.S.A.
Contents List of Contributors Preface
vii V
Caspase Inhibitors as Anti-inflammatory and Antiapoptotic Agents Piotr P. Graczyk
1
RNA as a Drug Target Martin J. Drysdale, Georg Lentzen, Natalia Matassova, Alastair I.H. Murchie, Fareed Aboul-Ela and Mohanimad Afshar
73
ACAT Inhibitors: The Search for a Novel and Effective Treatment of Hypercholesterolemia and Atherosclerosis Drago R. Sliskovic, Joseph A. Picard a n d Brian R. Krause
121
Growth Hormone Secretagogues: Discovery of Small Orally Active Molecules by Peptidomimetic Strategies Michael Ankersen
173
Inhibitors of Hepatitis C Virus NS3.4A Protease: An Overdue Line of Therapy Robert B. Perni and Ann D. Kwong
215
Subject Index Author Index (Vols. 1-39) Subject Index (Vols. 1-39)
257 263 269
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Progress in Medicinal Chemistry - Vol. 39, Edited by F.D. King and A.W. Oxford 0 2002 Elsevier Science B.V. All rights reserved.
1 Caspase Inhibitors as Anti-inflammatory and Ant iapoptotic Agents PIOTR P. GRACZYK Department qf Medicinal Chemistry, EISAI London Research Laboratories, University College London, Bernard Katz Building, London WCIE 6BT. U.K.
INTRODUCTION ROLE OF CASPASES Caspase- I Caspase-2 Caspase-3 Caspases-4 and -5 Caspases-6 and -7 Caspase-8 Caspase-9 Caspases- 10 and - 1 1 Caspases-12, -13. and -14
4 4 6 7 8 8 8 8 9 9
REGULATION OF CASPASES Transcriptional regulation Post translational modification Activation
9 9 II II
INHIBITION OF CASPASES Protein inhibitors P35 I AP CrmA Other serpins: Serp2. P I 9 Non-peptidic small molecules Peptide-derived inhibitors Pharmacophore moiety Non-acidic pharmacophores Peptide backbone (P2-P4) and its simple modifications
14 15 15 15 17 17 18 24 24 30 32
2
CASPASE INHIBITORS
Peptidomimetic modification o / the P2-P4 amino ucids - acyclic strirctiires Peptidomimetic replucement u / the P3 amino acid Peptidomimetic replacemenl of the P2 amino acid Bicyclic peptidomimetic replacements of P2-P3
35 31 39 40
CASPASE INHIBITORS I N VlVO Axotomy/axonal lesions Brain ischaemia Other ischaemia models Brain trauma Excitotoxicity (kainic acid, NMDA, AMPA) Endotoxic shock Amyotrophic lateral sclerosis Multiple sclerosis Parkinson’s disease Huntington’s disease Graft rejection CNS inflammation Peripheral inflammation Hepatitis Shigellosis Meningitis Pneumopathy
43 43 44 45 46 46 41 41 41 48 48 48 48 48 49 49 49 50
CLINICAL STUDIES
50
SUMMARY
51
ACKNOWLEDGEMENT
51
REFERENCES
51
INTRODUCTION Among the enzymes responsible for cleaving a peptide bond (peptidases) exists a group whose proteolytic activity is due to the presence of a cysteine thiol functionality at the active site [I]. Some of them, based on shared sequence homology and a preference for the aspartate residue in the P1 position of their substrates, have been arranged into a family called ‘caspases’ [2]. There are fourteen caspases known at present: caspase- 1 (ICE) [3,4], caspase2 (Nedd2, Ich-1) [5], caspase-3 (CPP32, Yama, apopain) [B], caspase-4 (TX, Ich-2, ICEJI) [7-91, caspase-5 (ICErcl-III, TY) [9], caspase-6 (Mch2) [ 101, caspase-7 (Mch3, ICE-LAP3, CMH-1) [ 11-13], caspase-8 (FLICE, MACH, Mch5) [14-181, caspase-9 (ICE-LAPB, Mchb) [ 19,201, caspase-10 (Mch4) [12, 211, caspase-11 (Ich-3) [22,23], caspase-12 [24], caspase-13 (ERICE) [25], and caspase-14 (MICE) [2628]. The human caspase family members form 3 groups
P.P. GRACZYK
3
based on substrate specificity, structural similarities and function [2,24]. Group I (caspase-1 subfamily) consists of caspases-I, -4, -5, and -13. They function primarily as inflammatory mediators with caspase- 1 being the most extensively studied both in terms of biology [29] and design of its inhibitors [3, 4, 301. The remaining caspases, group I1 (caspases-2, -3, and -7) and group 111 (caspases-6, -8, -9, and -lo), play an important role in apoptosis. Apoptosis is a physiological cell suicide programme which occurs naturally during normal animal development [31, 321. It regulates cell number and morphogenesis. In order to maintain healthy tissues, abnormal cells can also be removed by apoptotic mechanisms. Recently, however, contribution of this type of death to various diseases has become an emerging concept. Within the nervous system, the apoptotic component in slow-onset neurodegenerative diseases such as Alzheimer’s (AD) [33, 341, Huntington’s (HD) [35] and Parkinson’s (PD) [36, 371 disease and amyotrophic lateral sclerosis (ALS) [38] has recently been appreciated [3%43]. An increasing abundance of data suggests that apoptosis may also contribute to acute-onset neurodegeneration which occurs after stroke [40, 441, spinal cord [45-471 and traumatic brain injury [48, 491, and acute bacterial meningitis [50]. Although the rapid neuronal death seen following such insults can be attributed to excitotoxicity [5 I], the apoptotic mechanisms responsible for delayed cell death are supported by characteristic DNA fragmentation in the ischaemic rodent brain [52, 531, studies on the role of apoptosis-regulatory genes in ischaemia [54-571 as well as other histological and biochemical data [58, 591. One may add that certain features of apoptosis may be favoured in the developing versus the adult brain [60]. Apoptotic death may also occur in the course of autoimmune diseases [61] and leukemia [62]. After myocardial ischaemia and reperfusion, cells die by apoptosis, probably due to oxidative stress in the reperfused myocardium [63, 641. Endotoxin-induced liver failure is accompanied by apoptosis of parenchymal cells [65]. The number of apoptotic cells is also increased in renal amyloidosis [66], acute renal failure [67], and HIV-induced nephropathy [68]. Apoptotic mechanisms may contribute to the hair follicle regression [69] and especially hair loss induced by anticancer drugs [70, 711. The exact pathway of cell death, and in particular the relative contribution of apoptosis in the above disorders, still remains a matter of controversy [39, 48, 72-75]. Although alternative, non apoptotic forms of cell death have also been considered [76, 771, the apoptotic machinery has been an attractive target for the design of cell death inhibitors with neuroprotective drugs potentially fulfilling the areas of highest unmet medical needs [7&84]. Since one of the most important hallmarks of apoptosis is thought to be activation of the caspase family of proteases [24, 85481, the search for antiapoptotic caspase inhibitors has become an important area for medicinal chemistry [8%92] and
4
CASPASE INHIBITORS
neuroscience in particular [75, 78, 93-95]. Moreover, new areas of application may emerge in the future as the involvement of caspase-like proteases in apoptotic cell death pathways in plants is also gaining support [96, 971.
ROLE OF CASPASES Studies using knockouts of caspases in mice by homologous recombination have shown that these enzymes play essential roles in development, immune regulation and apoptosis [98]. The role of individual enzymes is discussed below and summarised in Table 1.1 CASPASE-I
The primary role of the prototype enzyme, caspase- 1 (interleukin- 1/?-converting enzyme, ICE) is the control of key steps in inflammatory response and immunity, by activation of the proinflammatory cytokines interleukin- 1p (IL1s) and interleukin-1 8 (IL- 18, formerly interferon-y-inducing factor) [99]. IL- 18 and IL- 1/? are synthesized as inactive precursors which require cleavage into an active molecule. Caspase-1 cleaves the Asp1 16-Alal17 bond in the intracellular inactive precursor 3 1 kDa form of pro-interleukin- 1/? (pro-IL1/?) and releases a 17 kDa active IL-I/? fragment. Mature IL-I/? is then secreted into the extracellular space. One has to note, however, that alternative
Table 1 .I Caspase
SUMMARY DATA FOR CASPASES
Alternutive name
Diseuse
1
ICE
2
Nedd2, Ich-l
3
CPP32, Yarna, apopain
4
TX, Ich-2. ICEreiII ICEJII, TY Mch2 Mch3, ICE-LAP3, CMH-1 FLICE, MACH, Mch5 ICE-LAP6, Mch6 Mch4 Ich-3
Brain trauma, MS, HD, PD, liver injury, AD, ALS, OA, IBD AD, HIV-I infection, Salrnonellosis TBI, heart failure, hepatitis, AD, OA, PD PKD
~
5
6 I 8 9 10
I1 12
13 14
ERICE MICE
~~
HD Hepatitis Hepatitis, HD ALPS MS, ischaemia
P.P. GRACZYK
5
pro-IL-1P processing pathways exist. Extracellular pro-IL- 1 can also be cleaved by cathepsin G, chymotrypsin, elastase, a mast cell chymase, matrix metalloproteinases and granzyme A [ 100, 10I]. Caspase-1 -independent release of IL-IP - induced by Fas ligand has also been described [102]. Some of these alternative pathways can play an important role in vivo since the inflammation induced by turpentine injection in caspase- 1 knockout mice resulted in IL- 1/) expression indistinguishable from that observed in wild type animals [ 1031. As mentioned above, caspase- I is the primary enzyme responsible for cleavage of pro-IL-18 to generate IL-18. This cytokine may contribute to inflammation, gene expression and the synthesis of tumour necrosis factor (TNF), IL-1 and chemokines [104, 1051. However, it has been demonstrated that proteinase-3 is an alternative IL-I 8 -processing enzyme [ 1011. Caspase- 1independent secretion of IL- 18 from macrophages has also been described [ 1061. Despite similarity between IL-18 and IL-1B both in terms of primary amino acid sequence and folding pattern, their functions differ [99, 1011. IL-IP is a key cytokine contributing to inflammation [ 1071 while IL-18 plays an important role in the T-helper cell type 1 (Thl) response, by inducing interferon-y (IFN-y) production in T cells and natural killer (NK) cells. The role of IL-18 modulation in tumours, infections, and autoimmune and inflammatory diseases has recently been reviewed [99, 108, 1091. Although a proinflammatory role of IL-18 in rheumatoid arthritis (RA) [I 101, lung injury or inflammatory bowel disease (IBD) [99] has been suggested, in the opinion of Dayer [ 1 1 I ] blocking IL- 18 production should be approached with caution. Caspase- 1-deficient mice are resistant to lipopolisaccharide (LPS)-induced liver injury [ 1121. Nonetheless, IL-18 release in FasL-induced acute liver injury in Propionibacterium acnes-primed caspase- 1-deficient mice may occur in a caspase- 1-independent manner [ 1 121. Since the secretion of IL- 18 may still be inhibited by caspase inhibitors it has been suggested that some caspases other than caspase-1 may be involved in the processing of IL-18 in FasL-stimulated macrophages [106]. Apart from roles in inflammation and immunoregulation caspase-1 may also be important in the control of apoptosis and neurodegeneration. In 1993, Yuan et al. [ 1 131 recognized a homology between caspase- 1 and Caenorhabditis elegans gene, ced-3, which is necessary for programmed cell death (apoptosis) in the nematode. This finding raised the question as to whether caspase-1 is important for apoptosis in mammals. Initial studies in caspase- 1-deficient mice [ 1 141 and other models [ 1 151 indicated that caspase-1 does not play a major role in apoptotic events. However, subsequent experiments with transgenic mice expressing a dominant-negative mutant of caspase-1 [116, 1171, and with mice deficient in the caspase- 1 gene [ 1 181 have shown that the absence of this enzyme prevents neuronal cell death induced by trophic support withdrawal,
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CASPASE INHIBITORS
and reduces ischaemic brain injury [ 1 191. Furthermore, attenuated brain damage occurs after moderate hypoxia-ischaemia in caspase- 1 knockout mice [ 1201. While the role of caspase- 1 in neuronal injury due to oxygen deprivation in the developing brain has also received significant support [121], it is not clear, however, whether the mechanism of protection is based on reduced apoptosis and/or inflammation. These results indicate that caspase- 1 activity may play an important role in stroke. Indeed, caspase-1-deficient mice exhibit reduced incidence and severity of experimental autoimmune encephalomyelitis (EAE) [122], suggesting that the protease may be involved in central nervous system (CNS) inflammation and in particular in multiple sclerosis (MS), a chronic inflammatory demyelinating disease of the CNS [ 1231. Caspase-1 activation has been demonstrated in the brains of a transgenic mouse model of HD as well as HD human patients [124]. Elimination of caspase-l activity in the mouse model by expression of a dominant-negative caspase-1 mutant delayed the disease progression and mortality [ 1241. Moreover, these transgenic mice exhibit decreased injury following brain trauma [ 1251. Since the expression of the dominant-negative caspase-1 mutant also results in an increased resistance to MPTP-induced neurotoxicity, caspase inhibitors have been suggested for the treatment of Parkinson’s disease (PD) [ 1261. Thus, the pretreatment of ventral mesencephalon cultures with tetrapeptide inhibitors of the caspase-3-like proteases, Z-VAD-fmk or Ac-DEVD-H, specifically inhibited death of dopamine neurones induced by low concentrations of 1 -methyl-4-phenylpyridinium (MPP ) [ 1271, whereas the caspase- 1 inhibitor, Ac-WAD-H, was without effect [ 1271. Caspase-1 appears to promote osteoarthritis (OA). Its presence was confirmed in human articular cartilage, and an increased cellular level has been found in OA tissue [128]. There is also evidence that caspase-1 may be involved in IBD [ 1291. Macrophage apoptosis plays a pivotal role in the course of bacillary dysentery caused by the enterobacteria Shigellu. One prerequisite gene for cytotoxicity is IpaB which activates caspase-1. The released IL-IP initiates a strong inflammatory response and contributes to the pathogenesis of shigellosis, which is one of the major causes of infant mortality in developing countries [ 1301. Contribution of caspase-1 and IL-lB to the pathology of AD is supported by an elevated caspase-1 activity found in post-mortem brains of AD patients [ 1311. Caspase-l activity is also elevated by an average of 81.5% in the spinal cord of humans with ALS [ 1321. +
CASPASE-2
Studies in caspase-2 deficient mice [ 1331 suggest that it acts both as a positive and negative cell death effector. In particular, the role of caspase-2 depends on
P.P.GRACZYK
7
the type of its isoform [134]. Since neurones from caspase-2 null mice are totally resistant to amyloid beta (AD, -42) toxicity, caspase-2 may be important for the pathology of AD [ 1351. It is required for apoptotic death of PC 12 cells and sympathetic neurones following withdrawal of trophic support [ 136, 1371, and oxyhaemoglobin (0xyHb)-induced apoptosis of brain microvessel endothelial cells [ 1381. It is also activated during apoptosis of human CD4 + T-lymphocytes after infection with HIV-I [ 1391, apoptosis of macrophages induced by Salmonella [ 1401, and sepsis-induced apoptosis of thymocytes [141]. Apart from its nuclear distribution, caspase-2 is localised to the Golgi complex, where it is involved in transducing proapoptotic signals [ 1421. CASPASE-3
Although thymocytes of caspase-3 deficient mouse are sensitive to apoptotic stimuli, programmed cell death in the brain is decreased resulting in abnormal development [ 1431. The importance of caspase-3 for neuronal death [6] has been further corroborated by experiments on the traumatic brain injury (TBI) in the rat [ 1441 and a recent finding of caspase-3 upregulation in human brain after traumatic head injury [ 1451. Cleavage of huntingtin by caspase-3 may be a crucial step in aggregate formation and neurotoxicity in HD [146, 1471. It is also the predominant caspase involved in amyloid precursor protein (APP) processing which results in elevated AP peptide formation [148]. This peptide, in turn, may induce PC 12 cell death mediated by caspases [ 1491. Furthermore, amyloidogenic Afi~5-3~as well as prion protein PrPloG126 can dose-dependently activate caspase-3 in rat cortical neurones [ 1501. Rather unexpectedly, however, in brains of patients with AD, the protein level of caspase-3 (and caspases-8 and -9) is decreased [ 1511. Caspase-3 may contribute to apoptotic death following cytokine deprivation in hematopoietic cells [ 1521, apoptosis of thymocytes [153], cardiac myocytes [ 1541, neurones and astrocytes [ 155, 1561, and tumour cell lines in response to various apoptotic stimuli [ 157-1591. Caspase-3 seems to be involved in apoptotic cell death in ischaemic/reperhsed rat heart as the enzyme levels were substantially elevated after regional myocardial ischaemia in ischaemic/reperfused rat heart [63]. Recent evidence suggests that caspase-3 activation occurs also in human heart failure [160]. The enzymes belonging to the caspase-3 subfamily (group II), rather than the caspase- 1 subfamily (group I), play the dominant role in Fas antibody-induced hepatitis [ 16 I]. Activation of caspase-3-like enzyme was also observed during rat liver regeneration following partial hepatectomy [ 1621. Interestingly, however, ischaemic preconditioning of mouse liver offered dramatic protection against prolonged ischaemia through down-regulation of caspase-3 activity [ 1631.
8
CASPASE INHIBITORS
Activation of caspase-3 in dopaminergic neurones in PD precedes and is not a consequence of apoptotic cell death [ 1641. Caspase-3 levels are also increased following optic nerve transection [165]. This enzyme may also play an important role in osteoarthritis since a positive correlation between the levels of chondrocyte apoptosis and levels of caspase-3 was found in the relevant dog model [ 1661. CASPASES-4 AND -5
The Fas-mediated apoptotic pathway seems to be mediated by a caspase-4-like protease [ 1671, although caspase-4, together with caspases-1 and -5, acts primarily as a mediator of inflammation [85]. Greater than seven-fold upregulation of caspase-4 has been observed in cystic kidneys in a mice model of polycystic kidney disease (PKD) [ 1681. CASPASES-6 AND -7
Caspase-6 may be involved in the cleavage of huntingtin thus contributing to aggregate formation and neuronal apoptosis in HD [146]. It has been found that both caspase-3 and caspase-6 are the two major active caspases present in apoptotic cells [ 1571. Caspase-7 seems to be a potential mediator of lovastatin-induced apoptosis in the prostate cancer cell line LNCaP [169]. CASPASE-8
Caspases-8 [ 17, 1701, -9 [ 171, 1721, and possibly - 10 [24] are critical upstream activators of the caspase cascade in some forms of apoptosis. Fas-mediated apoptosis in mice hepatocytes seems to be dependent on activation of caspase-8 [ 1731 and executor caspases-3, and -7 [ 174, 1751. The active form of caspase-8 was found in neurones as early as 6 h after focal stroke induced in rats by permanent middle cerebral artery occlusion (MCAO) [ 1761. Activated caspase8 is also present in the insoluble fraction of affected brain regions from HD patients [177]. An essential role of caspase-8 in HD is supported by the requirement for caspase-8 for the death of primary rat neurones induced by an expanded polyglutamine repeat (479) [ 1771. In addition, insufficient expression of functional caspase-8 was linked to childhood neuroblastomas [ 1781, although this has been questioned [179]. CASPASE-9
Caspase-9, an important activator of caspase-3, plays a critical role in apoptosis induction in axotomized retinal ganglion cells (RGCs) in vivo and is regulated
P.P. GRACZYK
9
following treatment with growth and survival factors [ 1801. It is also released from the intermembrane space (IMS) of mitochondria in an animal model of stroke [ 18 I]. Activation of caspase-9 was also observed in serum-/glucosedeprived cardiac myocytes [ 1541. CASPASES-I0 AND - I 1
A mutation in caspase-10 leading to defective lymphocyte and dendritic cell apoptosis is considered to be the origin of type I1 Autoimmune Lymphoproliferative Syndrome (ALPS) [182]. Caspase- 1 1 is involved in the activation of caspase- 1 in TNF-induced oligodendrocyte cell death [I831 and may play a crucial role in autoimmunemediated demyelination [ 1841. Furthermore, caspase-1 1 may be the initiator caspase responsible for the activation of caspase-3 as well as caspase-1 under certain pathological conditions, e.g., ischaemic insult [ 1851. CASPASES-12, -13, AND -14
Caspase-12 is involved in apoptosis due to stress in the endoplasmic reticulum [ 186, 1871. Overexpression of caspase- 13 induces apoptosis of 293 human embryonic kidney cells and MCF7 breast carcinoma cells [25]. Caspase- 14 is activated during differentiation of keratinocytes and seems to be involved in the formation of human skin [ 188, 1891. REGULATION OF CASPASES Although caspase-independent pathways in the inflammatory response and cell death in some systems have been found [76, 103, 19&195], the important contribution of caspases to neurodegeneration and inflammation has stimulated dynamic research to delineate the mechanisms involved in the regulation of caspase activity [49, 85, 88, 89, 119, 170, 1711. Currently, it is assumed that caspase regulation may occur via transcription, post translational modification, activation, and inhibition. TRANSCRIPTIONAL REGULATION
During the acute stage of EAE, an animal model of MS, both caspase- 1 and IL18 mRNA levels increase [ 1961. Recent studies on the role of caspase-1 in EAE confirmed this upregulation [ 1221. Furthermore, caspase-1 mRNA levels are significantly increased in peripheral blood mononuclear cells (PBMCs) from patients with MS compared with healthy controls ( p < 0.001) [ 1231. However,
10
CASPASE INHIBITORS
an ischaemic insult did not upregulate caspase-1 in the rat [197] and gerbil [ 1981 brain. Stimulation of U937 and HL60 leukemic cells and HT29 colon carcinoma cells with etoposide results in upregulation of caspase-2 and -3 genes and enhances the synthesis of pro-caspases [ 1991. In some of the animal models of ischaemia [ 197, 198, 200,2011 the levels of caspase-2 mRNA are also elevated (2-fold [198] and 3.8-fold [197]). A lateral fluid percussion brain injury in rats (a model of TBI) is similarly accompanied by an increase in caspase-2 mRNA level 12 h after the injury [202]. After permanent MCAO in rat, caspase-3 mRNA level increases 5.8-fold within 24 h [ 1971. An analogous increase both at the mRNA and protein level occurs in rat hippocampal CA1 pyramidal neurones 8-72 h after transient global ischaemia [201, 2031. In contrast, there were no significant changes in the expression of pro-caspase-3 at the protein level over 24 h after MCAO in mice although immunoreactivity due to pro-caspase-3 increased in the penumbra [204]. During transient focal ischaemia in the rat, an elevation in caspase-3 at the mRNA level was observed only in the resistant dorsomedial cortex at 1 day, and has been attributed to reactive changes in resistant brain areas [205]. A recent report suggests that in gerbil brains subjected to two 10min episodes of unilateral common carotid artery occlusion separated by 5 h, caspase-3 mRNA levels increase with a timecourse dependent on the brain area [206]. In another study of ischaemia in Mongolian gerbil caspase-3 and -4 mRNA levels remained constant over 2 days [ 1981. Upregulation of caspase-3 mRNA was also observed after ischaemiareperfusion injury in rat kidney [200], in motor neurones of both neonatal and adult rats after facial motor neurone axotomy [207], and in the rat spinal cord injury model [208]. Nevertheless, the mechanism by which the caspase-3 gene is induced is still not clear [203]. It has been shown that caspase-1 and caspase-3 mRNA upregulation can be inhibited by minocycline [209]. Using a recently reported polymerase chain reaction system, Lin et al. [2 101 have demonstrated IFN-y-induced transcriptional upregulation of caspase-5 in HT-29 colon carcinoma cells in the absence of upregulation at the protein level. In contrast, both caspase-5 mRNA and caspase-5 protein were induced by LPS in the monocytic cell line THP- 1 [2 lo]. Inhibition of endogenous nitric oxide (NO) synthesis in human melanoma cells by aminoguanidine, a specific, inducible nitric oxide synthetase (iNOS) inhibitor, led to upregulation of caspase- 1, caspase-3, and caspase-6 mRNA and eventually to cell death by apoptosis [21 I]. Pro-caspase-8 is constitutively expressed within spinal cord neurones. However, as early as 1.5 h after transient spinal cord ischaemia, an increase in
P.P. GRACZYK
11
caspase-8 (p 18) and caspase-8 mRNA levels could be observed within neurones in intermediate grey matter and in the medial ventral horn, and was followed by caspase-3 activation [212].
POST TRANSLATIONAL MODIFICATION
Caspases can be directly regulated by protein phosphorylation. In particular, Martins et al. suggested that phosphorylation of an active caspase could have an inhibitory effect on the enzyme activity [213]. Indeed, AKT can phosphorylate pro-caspase-9 which leads to inhibition of its proteolytic activity [2 141. Moreover, cytochrome c-induced proteolytic processing of pro-caspase-9 is defective in cytosolic extracts from cells expressing either active Ras or AKT [214]. These results are in contrast with other studies [215] showing that AKT cannot prevent apoptosis induced by microinjection of cytochrome c. ACTIVATION
Caspase proenzymes are normally present in cells [24, 2161 and can be rapidly activated in response to various stresses including DNA damage, UV radiation, heat shock, oxidative stress, or extracellular stimuli which triggers the caspase cascade [24, 85, 170, 2 17, 2 181. Activation pathways for proinflammatory caspases (group I) have not been studied as much as those for caspases involved in apoptosis. A RIP-like kinase, termed CARDIAK, promotes caspase- 1 activation in vitro by interacting with the caspase-1 recruiting domain (CARD) [2 191. Caspase- 1 might also be activated by caspase- I 1 [23]. Studies in vitro suggest that caspase-1 (and -1 1 ) can be activated by caspase-8 [220]. There is some evidence that, upon receipt of a proinflammatory stimulus, an upstream adaptor, RIP2, binds and oligomerizes caspase- 1 zymogen, promoting its autoactivation [221]. However, in the opinion of Zeuner et a/. [220], it is unlikely that self-processing leading to activation represents the pathway occuring in vivo. The association of caspase-1 with RIP2 can be prevented by a recently discovered protein ICEBERG [22 11. Apoptosis involves initiator (group 111) and executioner (group 11) caspases, and is currently interpreted in terms of two basic pathways: the first via death receptors (‘extrinsic pathway’) and the second involving mitochondria (‘intrinsic pathway’) [ 1701 (Figure 1.1). Caspase-8, and possibly caspase-10 are the apical caspases of a cascade triggered by the death receptors such as TNF-Rl (TNFa receptor l), CD95 (Fas/Apo- 1 ) [223], DR3 (APO-3/TRAMP/Wsl-l /LARD), DR4 (TRAIL-Rl) and DR5 (TRAIL-R2) [224]. For instance, upon assembly of the CD95
12
CASPASE INHIBITORS
Figure I . 1. SimpliJied diagram of extrinsic and intrinsic apoptotic pathways.
(Fas/Apo- 1) receptor death-inducing signalling complex (DISC), pro-caspase8 undergoes recruitment by Fas-associated death domain (FADD), an adapter protein, and oligomerization. According to the induced proximity model [225] this leads to an increase in the local concentration of the proenzyme and concomitant self-activation [226, 2271. In an analogous way, after stimulation
P.P.GRACZYK
13
of TNF-RI by TNFa, the receptor recruits an adapter protein TRADD (TNFR1-associated death domain) which then interacts with FADD and pro-caspase8, leading to its activation. Interestingly, activation of caspase-8 induced by various drugs can be mediated by a CD95/Fas-independent mechanism and preceded by activation of (an)other caspase(s) [22&230]. Active caspase-8 activates caspases-3, -7 [231, 2321, and -13 [25] and probably several more [220] via cleavage of the relevant pro-caspases (stepwise for pro-caspase-3 [233]). There is some confusion as to which downstream/executioner caspases are capable of activation of other caspases. Caspase-3 has been shown to subsequently activate caspase-6 [23 1, 2321. However, during apoptosis of cerebellar granule cells (CGCs) induced by withdrawal of trophic support, caspase-6 activated caspase-3 in cellular extracts from non-apoptotic CGCs, whereas caspase-3 failed to activate caspase-6 [234]. Experiments in vitro show, however, that caspase-3 and -6 cleave each other’s precursors quite well [220]. Similarly, caspases-6 and -7 can activate each other [220]. It has also been reported [I671 that death signal transmission from caspase-8 to caspase-3(-like) proteases may involve a caspase-4(-like) protease. Scaffidi et al. have suggested that there are two types of cells in which two different CD95 (Fas/APO-I) signalling pathways exist [235]. In type I cells activation of caspase-8 occurs rapidly via DISC. In type I1 cells activation of caspases-8 and -3 is delayed and occurs downstream of mitochondria. In these cells, DISC formation is strongly reduced [235]. Signalling through death receptors can be inhibited by viral [236] or cellular [237, 2381 proteins known as FLIP, which contain a fragment similar to that present in caspase-8, and therefore are able to compete with pro-caspase-8 for binding with FADD. A similar mechanism has been considered for ARC, an inhibitor of apoptosis expressed in skeletal muscle and heart [239]. Activity of caspase-3 might also be inhibited indirectly by hepatitis B virus-encoded HBx protein [240]. Caspase-9 is the most upstream protease in the mitochondria1 (intrinsic) pathway in which caspase activation is triggered by cytochrome c and dATP [24 1-2431. During apoptosis cytochrome c is released from the mitochondria into the cytosol where it forms a complex involving Apaf-1 (a CED-4 homologue), then dATP [244] and pro-caspase-9 [245]. Once activated in an autocatalytic manner, caspase-9 then activates caspases-3 and -7 [243, 2461. Recent studies, however, indicate that autoproteolysis of pro-caspase-9 is not required, as the expression ofthe zymogen activity is dependent on cytosolic factors [247]. Caspase-3 may subsequently activate caspases-2 and -6 as shown in Figure 1.1 [243]. Caspase-6 may then activate caspases-8 and -10. In addition, the caspase-9 pathway might be modulated by proteins from the Bcl-2/Ced-9 family [248, 2491 and recently discovered Aven [250]. Moreover, activation of
14
CASPASE INHIBITORS
caspase-9 by Apaf-1 can be blocked by an endogenous, alternatively spliced isoform of caspase-9, named caspase-9b, which functions as an endogenous inhibitor of apoptosis [25 11. Importantly, the extrinsic and intrinsic pathways may interact with each other. In particular the mitochondria1 pathway may amplify the receptormediated apoptotic signal [252]. Caspase-8 can cleave the proapoptotic protein BID, to liberate truncated BID (tBID) which translocates to mitochondria and triggers cytochrome c release [253, 2541. Furthermore, caspase-3-catalyzed cleavage of antiapoptotic Bcl-2 produces a carboxyl-terminal Bcl-2 cleavage product able to induce cell death and further activation of downstream caspases [255]. Activated caspases are capable of cleaving many targets, e.g. poly(ADP-ribose) polymerase (PAM) [256, 2571, lamins [258], cell cycle proteins [259] and many other nuclear [260] and cytoplasmic targets [89] including proteins involved in transduction of survival signals [261]. This proteolytic activity eventually contributes to the characteristic morphological changes such as chromatin condensation and DNA fragmentation. It should be noted, however, that the role of caspases -6 and -7 as important downstream effectors has recently been questioned [262]. The actual apoptotic pathway is most probably much more complex [ 157, 2631. Recent results from this laboratory [264,265] and others [32, 73, 76, 266, 2671 indicate that the mode of cell death and activation of specific caspases depend on the cell type and the death stimulus involved. Additional activation of other non-caspase-like proteases [268], including the 24-kDa apoptotic serine protease (AP24), may also be necessary [269, 2701. Furthermore, Nakagawa et al. have identified an additional, caspase- 12-mediated, apoptotic mechanism which is initiated by stress in the endoplasmic reticulum [ I861 and in which caspase-12 may be activated by calpain [271]. However, inhibition of caspase activity in a cell may not be sufficient to keep it alive [272, 2731. Zeuner et al. pointed out that activation of proapoptotic caspases may even occur in the absence of cell death indicating their possible non apoptotic role [222]. Interestingly, FasL, usually connected with death signalling, may also be involved in other pathways, e.g. inflammation [ 1021.
INHIBITION OF CASPASES Caspase inhibition can be achieved using compounds of both natural and synthetic origin. Some of these compounds are proteins which are derived either from viruses (p35, CrmA) or which may also be endogenously expressed in mammals (IAP).
P.P. GRACZYK
15
PROTEIN INHIBITORS
Due to the importance of caspases in many biological processes, several ways to control activity of these enzymes have developed during evolution. A strategy which is based on generating proteins capable of inhibiting the active caspases involves p35, IAP, and CrmA proteins. P35 Organisms fight against viral infections by killing the infected cells. To counteract this defence mechanism viruses evolved antiapoptotic genes such as p35 and IAP [274]. The product of p35 is a 35-kDa protein initially found to suppress virus-induced apoptosis in host cells of Spodopterufbugiperda [275]. Further studies have shown antiapoptotic activity of p35 in many other cell types including cardiomyocytes [276], cancer [277, 2781, neuronal [27%281], host pupal eye cells of the fly Drosophila melunoguster [282], and C. elegans cells normally programmed to die [283]. p35 is a broad range caspase-specific and active site-directed [284] inhibitor capable of interacting with caspases-I, -2, -3, -4 [285], -6, -7, -8, and -10 with Ki values from 0.1 to 9 nM [284]. Interestingly, the p35 protein does not inhibit caspase-9 activity in a cell-free system of mammalian caspase activation [286]. In the opinion of Vier et al. [286] p35 evolved specifically to inhibit effector rather than initiator caspases. Indeed, the presence of an initiator caspase that is resistant to p35 has been suggested [287]. Inhibition of enzymatic activity is accompanied by cleavage [285] of p35 at Asp-87 [284]. It has also been demonstrated that the insect Spodoptera frugiperdu target of the baculovirus p35 is Sf caspase-1 of which the sequence and specific activity is highly related to human caspase-7 and caspase-3 [288,289]. p35 serves as a substrate of Sf caspase-1, which cleaves p35 to similar sized fragments [289]. Recently, Fischer et ul. have determined the crystal structure of p35 and proposed a multistep mechanism for stoichiometric caspase inhibition involving a reactive site loop analogous to that of serpins [290]. IAP Inhibitor of apoptosis proteins (IAPs) have been found in baculoviruses, higher eukaryotes [291, 2921, and mammals [291,293]. The mammalian IAPs include X-IAP (MIHA, hILP), c-IAP1 (MIHB, inhibitor of apoptosis-1), c-IAP2 (MIHC), neuronal apoptosis inhibitory protein (NAIP), the recently discovered survivin [294], apollon [295], livin (IAP-3) [296, 2971, and ML-IAP [298]. Down-regulation of X-IAP following adenoviral antisense expression may induce apoptosis in some types of cancer cells [299].
16
CASPASE INHIBITORS
IAPs seem to have a much narrower spectrum of activity than p35 [300]. Viral IAP, when expressed in mammalian cells, can block caspase-1 and -2-induced apoptosis, but is ineffective against death due to caspase-7 expression [301] and caspase-induced apoptosis in insect cells [288, 3021. IAP can prevent caspase-mediated cleavage of p35 in vivo and inhibit caspase activity upon viral infection or UV irradiation [302]. This was rationalized by assuming that IAPs inhibit the activation of pro-caspases [288, 302, 3031. Thus, the antiapoptotic properties of NF-KB could be due to the upregulation of IAP genes [304, 3051, and subsequent inhibition of pro-caspase-8 activation [305]. However, it is possible that not all IAPs work by the same mechanism [292]. Caspase-inhibitory function of IAP may require homo-oligomerization [306]. Other studies have shown that the members of the human IAP family specifically bind to caspases-3 and -7 [307-3091 with inhibition constants for X-IAP of 0.7 and 0.2 nM, respectively [3 lo], thus suggesting that IAPs may act as direct caspase inhibitors. X-IAP can also inhibit caspase-9 (but not caspases- 1, -6 or -8) [296, 297, 308, 31 1, 3121. In contrast to p35 protein, no cleavage of IAPs occurs during inhibition of caspases, indicating a different mechanism of action [307]. However, during Fas-induced apoptosis, endogenous X-IAP can be cleaved into two fragments comprising baculoviral inhibitory repeat (BIR) 1 and 2 domains and the BIR3 and RING domains (BIR3-Ring) [313]. Recently, it has been found that only the second (BIR2) of the three BIR domains in X-IAP is necessary and sufficient for inhibiting caspases-3 and -7 with apparent inhibition constants (Ki) of 2-5 nM [314]. The fragment of X-IAP encompassing the third BIR domain (BIR3) and RING domain is responsible for specific inhibition of mammalian caspase-9 [313]. This finding agrees with the apoptosis-suppressing activity of a new Spodoptera frugiperda S'IAP in human cells, which occurs by inhibition of a caspase-9 homologue, and requires both BIR and RING domains [315]. The remaining IAPs, i.e. c-IAP1 and c-IAP2 are less potent inhibitors with K,'s against caspases-3 and -7 in the range from 29 to 108 nM [307]. NAIP does not inhibit any of these enzymes and presumably inhibits apoptosis via other targets [307]. Overexpression of survivin occurs in many common types of cancer [294]. Although survivin is capable of inhibiting caspases-3 and -7 (but not caspase8), and promotes survival of 293 cells exposed to diverse apoptotic stimuli [309], its function may also be related to cytokinesis [316]. Recently, its caspase-3 inhibitory activity has been questioned [3 171. Livin can bind caspases-3 and -7 via its BIR domain [297]. It might also interact with both unprocessed and cleaved forms of caspase-9 [297]. Interestingly, IAPs can be inactivated by binding to endogenous proteins like Smac [318]/Diablo [319], which are released into the cytosol when cells undergo apoptosis [32&322].
P.P. GRACZYK
17
Finally, failed caspase inhibition due to the presence of disfunctional NAIP gene has been linked with spinal muscular atrophy (SMA), a genetic disorder characterized by motor neurone loss in the spinal cord [323]. CrmA
CrmA (cytokine response modifier A) is a 38 kDa protein from cowpox virus which belongs to the serpin superfamily of serine proteinase inhibitors. For several years it has been known as a caspase-1 inhibitor [324, 3251. Its potency may be attributed to the sequence LVAD of the reactive site loop, which is close to YVAD recognized by caspase-1, -3 and -4 [326]. CrmA rapidly (1.7 x 1 0 7 ~ - 1s - ~ ) [327] inhibits caspase-1 forming a tight complex with an equilibrium constant for inhibition (K,) of about 0.004-0.010 nM [325, 327, 3281. It is also active against caspase-4 [329] ( K , 1.1 nM) [327], caspase-5 [329] ( K , < 0.1 nM) [327], caspase-8 [23 I] (K, < 0.34; 0.95 nM) [327, 3281, caspase-9 ( K , <2.3nM) [327], and caspase-I0 (K, 17nM) [327]. The in vitro inhibitory activity against caspases-3 and -6 is much lower (K, 1600 and 1300nM, respectively) [327]. However, Ekert et al. pointed out that in vivo activity of CrmA may be limited to inhibition of caspase-1 and/or caspase-8 only [91]. The observed antiapoptotic activity of CrmA in some cell systems [330, 3311 has been interpreted [328] in terms of inhibiting caspase-8, which catalyzes the crucial step in the promotion of cell death. CrmA does not inhibit caspases-2 [5, 3271 and -7 [327, 3281, which may be due to the absence of the P4 aspartate residue (preferred by group I1 caspases) at the CrmA recognition site. Interestingly, after changing the site sequence to DQMD by means of genetic engineering, such an analogue of CrmA could inhibit apoptosis caused by direct activation of caspase-3 [332]. Selectivity of CrmA has also been rationalized based on the crystal structure of its cleaved form [333]. It seems that CrmA is more selective than p35 since CrmA, in contrast to p35, does not suppress developmental cell death in C. elegans [334] and does not inhibit the death of either undifferentiated or neuronally differentiated PC 12 cells induced by growth factor deprivation [28 11. Other serpins: Serp2, PI9 Serp2 is a newly discovered myxoma virus-encoded serpin protein, about 35% identical to CrmA. Similarly to CrmA, Serp2 contains an aspartate residue within the serpin reactive loop [335]. Purified HislO-tagged Serp2 protein is a relatively poor inhibitor of caspase-1, with a Ki of 80nM [335], but perhaps potent enough to suppress an inflammatory response and apoptosis in some systems [336].
18
CASPASE INHIBITORS
An acidic residue (Glu) at the reactive site loop of PI9 (Proteinase Inhibitor 9) might account for its inhibitory activity against caspase-1 (apparent second order rate constant 700 M- Is- I ), caspase-4 and caspase-8 (weaker inhibition) [337]. Caspase-3 is not inhibited, probably due to the presence of Val at P4 of the loop [337]. Several other serpins can interfere with cell death using other mechanisms. They include SPI- 1 (Poxvirus Serine Protease Inhibitor 1), PAI-2 (Plasminogen Activator Inhibitor 2), and PN-I (Protease Nexin I) [338]. NON-PEPTIDIC SMALL MOLECULES
Several small inorganic moieties can inhibit caspases. Nitric oxide (NO) has recently emerged as an important endogenous inhibitor of apoptosis. Produced either extracellularly by NO donors or intracellularly by iNOS, it has been found to prevent apoptosis in hepatocytes initiated by the removal of growth factors, exposure to TNFcl and anti-Fas antibody [33%34 11. Similarly, angiotensin-11-induced apoptosis of human umbilical venous endothelial cells (HUVEC) and caspase-3 activity were completely inhibited by the NO donors sodium nitroprusside and S-nitrosopenicillamine [342]. Further studies involving cell lysates [343] and recombinant enzymes [339, 344-3463 have shown that NO is capable of inhibiting caspases-1 [344, 3461, -2 [346], -3 [339, 3443461, -4, -6, -7, and -8 [341] most probably by direct S-nitrosylation of the caspase cysteine residue, which is reversed by addition of dithiothreitol [339, 3451. Operation of the S-nitrosylation mechanism with regard to caspase-3 inhibition has recently been confirmed in vivo by using exogenous NO donors [347], and for caspase-1 was supported by studies on iNOS knockout mice [348]. However, as pointed out by Briine et al. [343, 3491, it is not clear whether the antiapoptotic properties of NO are entirely due to its caspase-inhibitory action since the enzyme assay conditions do not reflect the situation in a living cell. Other mechanisms of cytoprotection have been considered as well [339, 349, 3501. Recent studies on ischaemia-reperfusion injury in cardiomyocytes indicate that inhibition of NOS results in increased apoptosis [35 11. In the opinion of Weiland et al. endogenous NO synthesis might protect against apoptotic cell death [351]. It should be noted, however, the proapoptotic properties of NO have also been recognized [349, 352-3551, Taking into account the low stability and photosensitivity of NO donors such as sodium nitroprusside and S-nitrosoglutathione, Guo et al. have described N-nitrosoanilines as a new class of caspase-3 inhibitors [356]. The most potent compound (1) exhibits a pseudo first order rate constant of inhibition kobsd of 0.195 min-'. The N-nitrosoaniline pharmacophore has also been introduced into peptide-based inhibitors (see below). In addition, a new class of NO-releasing non-steroidal anti-inflammatory drugs which might
P.P. GRACZYK
19
target caspase- 1 in a cyclooxygenase-independent way, including e.g. NCX401 6 (2), a NO-aspirin derivative, has recently been described [357]. Caspases can also be inhibited by metal ions. Zinc cations (Zn2 )probably act as endogenous inhibitors of apoptosis. It has been found that depletion of intracellular zinc induces protein synthesis-dependent neuronal apoptosis in cortical cultures [358]. Studies in vitro have demonstrated that activity of caspases 3, -6, -7, and -8 is non-selectively abolished by Zn2 in the submicromolar range [359], which is a common characteristic of cysteine proteases. It has been shown that Zn2+ inhibits P A W cleavage at low micromolar concentrations, most probably by inhibiting caspase-3 [360]. Zn2 ions also inhibited the cleavage of the 32-kDa pro-caspase-3 by caspase-9 [36 I]. Nevertheless, excessive exposure to extracellular Zn2+ can induce cell death with characteristics of either apoptosis or necrosis, depending on the intensity of the Zn" exposure [362]. +
+
+
(3)
(4)
Other metal ions can also interact with caspases. In particular, caspase-3 inhibition by cadmium salts may explain their ability to suppress apoptosis of Chinese hamster ovary (CHO KI-BH4) cells induced by hygromycin B and actinomycin D [363]. It might also imply a non-genotoxic mechanism of Cd-related carcinogenesis. Nevertheless, cadmium salts may also induce apoptosis [364]. Phenylarsine oxide (3), PAO, a small organic molecule, can inhibit recombinant human caspases as well as endogenous chicken caspases that are active in S/M extracts [365] due to strong arsenic-thiol interactions [366]. Dithiocarbamates are known to be potent inhibitors of apoptosis in thymocytes [367], leukemic cells [368], and fibroblasts [369] and work by blocking the proteolytic activation of caspases [370, 3711. It has been shown that disulfiram (4), DSF, inhibits caspases-1 and -3 with apparent rate constants of 2.2 x 103MM's-' and 0.45 x 103M-'s-' , respectively. Dithiocarbamates
CASPASE INHIBITORS
20
Enzyme-SH
a
O-
' 0
Enzyme-S-
Michael acceptor Figure 1.2. Michael addition reaction qj' cysteine sulfirr to cc,~-unsuturuted systems
inactivate caspases by mixed disulfide formation between (4) and the enzyme, as has been demonstrated using '%-labelled (4) [37 11. Additional mechanisms of antiapoptotic activity of (4) may include stabilization of mitochondria1 membrane potential and suppression of reactive oxygen species [372]. Many organic compounds which inhibit caspases contain an a,/?-unsaturated system and behave as Michael acceptors with respect to the enzyme active site cysteine -SH group [373]. The enzyme may react with the inhibitor as shown in Figure 1.2 and become irreversibly inactivated. One such compound, 4-hydroxynonenal(5), HNE, a metabolite generated by lipid peroxidation, has been shown to inhibit recombinant human caspase-1 in a dose-dependent manner [374]. However, compound (5) may also induce activation of the caspase cascade [375]. An analogous mechanism is responsible for inhibitory activity of quinones. Studies on bone marrow cell depression caused by benzene have demonstrated that quinone (6), which is a benzene metabolite, can inhibit such cysteine proteases as calpain and caspase-1 [376]. The antiapoptotic activity of hydroquinone (7), HQ, in myeloblasts was also explained by inhibition of caspases-1 and -3 by (6) which is formed by oxidation of (7) [377, 3781. Other quinones may also non-selectively inhibit caspase-3 with ICs0 values down to the low
I
0
OH
COOH
NH CI
P.P.GRACZYK
24 nM
52.5 nM
21
100 nM
130 nM
Figure 1.3. I C f n values [nM] ,for inhibition of recombinanl caspase-3 with selected quinones.
nanomolar range (Figure 1.3) [379]. Studies on peptide derivatives containing a quinone moiety as pharmacophore have also been performed (see below). Xylaric acid (8), L-741,494, isolated from the the fungal genus Xylaria, may serve as another example of a Michael acceptor-based caspase inhibitor [380]. A similar mode of action probably operates for secobatzelline A (9) which has been isolated from marine sponges of the genus Batzella [381]. Secobatzelline A (9) exhibits strong inhibitory activity against caspase-3 (ICsOof about 80 nM). Epoxides (1 0), EI-I 507- I , and (1 l), EI-1507-2, isolated from the culture broth of Streptomyces, show the IC50 values of 0.23 and 0.42 pM against recombinant caspase- 1, respectively [382]. The epoxide functionality also appears in a relatively potent manumycin-related caspase- 1 inhibitor (12), EI1511-3, (ICsO value of 0.09 pM) and its oxidation product (13) (IC50 of 0.07 pM) [383, 3841. Apart from being potential Michael acceptors, compounds (lo), (12), and (13) may also undergo thiophilic epoxide ring opening via the addition of cysteine thiol to the carbonyl group followed by irreversible 1,2-shift (Figure 1.4) [385]. Another epoxide-containing caspase- 1 inhibitor, Pentenocin A (14), IC50 0.575 pM, has been isolated together with its a$carbonyl analogue, Pentenocin B (15), ICs0 0.25 pM, from the broth of Trichoderma hamatum FO-6903 [386]. Peptides containing the epoxide moiety in the pharmacophore will be described in the next section. Many other small molecule caspase inhibitors have appeared primarily in the patent literature: cephem oxides, e.g. (16) [387, 3881, halomethyl amides, e.g. (17), IC50 < 1 pM [389], pyrimidotriazinediones, e.g. (IS), ICS0 74 nM [390], and pyridazines, e.g. (19), kOb$[I] 70 M-ls-' [391, 3921. a-Ketoamides, e.g. (20), have been claimed as inhibitors of caspases involved in cell death [393]. This functionality is also present in pharmacophores used in peptidic structures (see next section). Compound (20) shares a common p-aminobenzenesulfonamide fragment with 5-aminosulfonyl-2-indolones,e.g. (21) [394] and sulfamoylisatins, e.g. (22), which have recently been claimed as potent dual inhibitors of caspases-3 and -7; K, of 15 and 47 nM, respectively for (22) [395, 3961. A co-crystal structure of the complex between (22) and caspase-3 demonstrates that the catalytic cysteine thiol and isatin ketone carbonyl group
CASPASE INHIBITORS
22
&:;
@:: /
/
/
/
M e 0 OH
M e 0 OH
HO
OH
0
Me
.(;I:. 0
0
(14)
(15)
Enzyme-SH
Figure 1.4. One of'several possible modes qf'cuspase inhibition by epoxides (10). (12) and (13)
P.P.GRACZYK
23
CI
d
C(0)NHR
N
PhO
!? 0
form a tetrahedral intermediate, and the S 1 subsite is occupied only by a water molecule [396]. The pyrrolidine ring in (22) is involved in hydrophobic interactions with the S2 pocket of the enzyme, which are responsible for the observed high selectivity [396]. Interestingly, despite the high potency of (22) against caspase-3, its efficacy in a cell-based model of caspase-3-dependent apoptosis was very low (EDSo of about 10 pM). The attenuated potency of the
24
CASPASE INHIBITORS
inhibitor has been attributed by Lee et al. to reversible binding between (22) and cytosolic constituents [396]. The X-ray structures have also been used for design and optimization of other small molecule caspase inhibitors. Albrecht et al. [397] have identified 22 binding sites in caspase- 1, and suggested several pyridine derivatives, e.g. (23) and (24), as possible structures of caspase- 1 inhibitors.
% WCOO 0
/
0
PEPTIDE-DERIVED INHIBITORS
Rational design of an enzyme inhibitor may be performed based on the mode of action of the enzyme and structure of its substrate(s). Since caspases are responsible for cleavage of peptide bonds, peptide-derived substances were the first systematically studied inhibitors of the prototype enzyme, caspase- 1 [3]. Recently, an analogous approach has been applied for the design of inhibitors of other members of the caspase family. Peptide-based inhibitors of caspases consist of two elements: the pharmacophore moiety involved in interactions at the catalytic site and the peptide/peptidomimetic element responsible for recognition at the binding site. PHARMACOPHORE MOIETY
Caspases display almost absolute preference for the aspartate moiety at the PI position of their substrates. Peptides containing P 1 residues such as glutamate or asparagine are not processed well by caspase-1 [3, 3981. The almost absolute preference for the aspartate moiety at P1 has therefore been reflected in almost all of the pharmacophores being based on a properly modified aspartate moiety. For the enzyme to become inactivated, the cysteine sulfur at the active site should engage in some interaction with an electrophilic component of the inhibitor. The simplest example of such a moiety is provided by the frequently used aldehyde functionality (pharmacophore 1, Figure 1.4, which reversibly creates a hemithioacetal structure (25), (Figure 1.6u, X = 0, R = H). Formation of the hemiacetal has been confirmed by X-ray structures of enzyme-inhibitor complexes such as caspase- 1-Ac-YVAD-H [424], caspase- 1-Ac-DEVD-H [425], caspase-3-Ac-DEVD-H [425, 4261, caspase-7-Ac-DEW-H [425], caspase-8-Ac-DEVD-H [425], and caspase-8-Ac-IETD-H [427]. An analogous
P.P. GRACZYK
25
reversible mode of inhibition could be observed for hydrazones (pharmacophore 2, Figure 1.5) and ketones (pharmacophores 3 and 4, Figure 1.5). The chemistry and inhibitory activity of these classes of inhibitors has been thoroughly reviewed by Ator and Dolle [3]. Recently, due to the low bioavailability of peptide-derived inhibitors containing the aspartic aldehyde functionality, prodrug forms have been developed [408, 41 1, 412, 414, 4281. A successful example of this strategy is provided by the Vertex caspase-1 inhibitor, (26), VX-740, which has advanced to Phase I1 clinical trial for rheumatoid arthritis. The original approach, leading to irreversible inhibition, was based on the concept of a quiescent affinity label [429] and employed methyl ketones
)=x
Pharmacophore 1
R
II
Reference [3, 399-4171
0
II [3, 403-4051 H
3
II
Alkyl, phenylalkyl
0
[3, 400, 418-4201 [3, 400, 4211
[3, 4221 0
6
II N,
0
.Y
/H
[411, 4231
Y=alkyl, aryl Figure I 5
Pharniucophores based on aspartule aldehyde&,ketone5 and their derivatives
26
CASPASE INHIBITORS
a)
,COOH
AN&/ H
Enzyme-SH
--.+
,COOH
” .NH
”
S - Enzyme
(28)
(27)
Figure 1.6. Inhibition of cysteine proteases by a) aldehydes, ketones and ,some of’their derivatives 6) cc-substituted methylene keiones.
activated by the presence of an a-substituent which could serve as a leaving group e.g. halogen, acyloxy or aryloxy (pharmacophores 1-5, Figure 1.7). Such derivatives would possibly interact with the enzyme as shown in Figure 1.6b. While the rapid, irreversible mode of action for halogen derivatives (Y = F, CI) has not been questioned, recent data for the other types of activated aspartic ketones suggests a more complex kinetic picture. Brady et al. observed that, in many cases, there is a clear differentiation of a reversible phase of inhibition leading to the hemithioacetal (27) and an irreversible inactivation producing thioether (28) [455, 4561.
(26)
If one assumes the mechanism of inhibition pictured in Figure 1.6b (other mechanisms have also been considered by Brady et al. [456]), then the compounds will behave as inactivators only if the thioether (28) is formed very quickly (ki >> k-,). Ifthe conversion of (27) into (28) occurs slowly (ki < k- ,), the aspartic ketone derivative would exhibit the features of both reversible and irreversible inhibitors. Such compounds are called ‘bimodal’ inhibitors. Finally, if ki M 0 (no thioether (28) is formed) the compound will behave as a reversible inhibitor.
P.P.GRACZYK
Pharniacophorr
Reference
1
F
[406,415,417,430]
2
C1
[431,432]
3
Br
[433,434]
4
-OC(0)alkyl
[435,436]
5
"4
[3, 24, 405, 406, 408, 415,417,423,430, 433,434,437-4391
0 -Z-Alkyl Z = 0, S, N
6 7 8
-S-Aryl -NHS02R
9
-NHCOR
[412,419,436,4404431 [442,443] [412,443] 1441,4431
27
Pharmucnphore 17
Reference
)pubst [405, 4521
\
0
2
18
[411,432]
19
[453,454]
20
[453, 4541
21
13281
22
[4171
FF3
10
[3. 406,4441
d , N
\
0 bh
I1 12
-OP(O)Rz
\
0
13
\o
14
[3,406,415,417, 4341
&:
[3,24,406,417, 438-4481
G S u b s t
4 2
[405, 4521
23
[328,405,4454471
24
14171
[85,449]
25
r4111
Subst
IS
'
x X
16
v
= 0.S, MeS' -ON( R)COR
[408,411,432, 4501 26 W11
~4161
-
Figure 1.7. r-Suhstituents in activated aspartate methylene ketones (2 = C, N, 0,S).
It is difficult to unequivocally predict the mode of inhibition for a particular Y group (Figure 1.66) based on its pK, value. However, Brady et al. [456] identified two binding modes for potential leaving groups Y at the active site of caspase-1 which can be linked with the mode of inhibition. In one of them,
28
CASPASE INHIBITORS
schematically presented in Figure 1.8a, the dihedral angle S-C-C-Y is about 180°, which facilitates the rapid intramolecular nucleophilic substitution and formation of thioether (28). This kind of interaction would be characteristic of inactivators. Reversible inhibitors, on the other hand, would assume the synclinal conformation (Figure 1.8b), in which the departure of Y is difficult. Such a situation exists for alkythiomethyl ketones (pharmacophore 6, Figure I . 7, Z = S). Aliphatic ketones (pharmacophore 3, Figure 1.5), which also behave as reversible inhibitors, represent an exemption to this rule. Although their dihedral angle S-C-C-Y is about 180", and should favour the inactivating mode, the departure of an alkyl carbanion (bad leaving group) cannot occur. Other inhibitors which show a mostly reversible mode of action include arylthiomethyl ketones (pharmacophore 7, Figure I . 7), sulfonylaminomethyl ketones (pharmacophore 8, Figure I . 7 ) [456], aminomethyl ketones (pharmacophore 6, Figure 1.7, Z = N) and their acyl derivatives (pharmacophore 9, Figure 1.7) [441]. Aminomethyl ketones constitute an interesting pharmacophore due to increased water solubility and good inhibitory potency, e.g. Ki 0.37 nM against caspase-1 for (29) [441]. Unfortunately, such secondary amines are plagued by lack of chemical stability. The relevant tertiary amino derivatives such as (30) are potent (caspase-1 Ki 4 nM) and much more chemically stable [441].
As far as the inactivators are concerned most of dichlorobenzoyloxy (DCB) derivatives and (pyrazoly1oxy)methyl (PTP) ketones (pharmacophores 5 and 10, respectively; Figure 1.7) behave in this way [456]. Inactivating pharmacophores 11 [438] and 12 [446] (Figure 1.7) may lead to inhibitors with kobS/[I] of the order of 3 x lo5 M-'s-' . Other structures may exhibit various behaviours depending on the type of the leaving group and specific interactions with the enzyme at its active site [456]. This can be clearly seen in the results of recent studies on the caspase-1 inhibitory activity of acyloxymethyl ketones (pharmacophore 4, Figure 1.7) containing no P2-P4 amino acid residues, Fmoc-D-CH2X, as a function of the S'-P' interactions involving the X residue [457]. No inhibition was observed for X=CH3CO0 and
P.P. GRACZYK
Figure I 8
29
Two hinding modes for aspartare methylene ketones leading to a) inactivafion h) reversible inhibition.
X = Z-L-Ala. While X = Z-L-Pro gave a reversible inhibitor with a Ki of 10 pM, its enantiomer X = Z-o-Pro afforded a relatively potent (130 M-'s-'), selective (no inhibition of chymotrypsin and papain), and irreversibly acting compound [457]. However, most acyloxy- and aryloxymethyl ketones (pharmacophores 4 and 13, Figure 1.7) display bimodal kinetics. The most active among them exhibit rapid inactivation (kobsl[l] of the order of lo6 M- Is- I ) with the diffusion-controlled rate limiting step [458]. Diazomethane derivatives (pharmacophore 14, Figure 1.7) are much less potent (kObs/[I] 16400 M-'s-l) [401]. Despite very potent inhibition of caspases by acyloxymethyl and aryloxymethyl ketones in vitro these pharmacophores are plagued by low stability (ester bond cleavage by esterases) and significant toxicity in vivo as well as low solubility. To address these issues pharmacophores of heterocyclic character 1726 ( F i p r e 1.7) have been developed. For instance, hydroxamate (3 1) exhibits an IC50 value of 3 nM against caspase-1 and 4 nM against caspase-4 [451].
Several types of pharmacophores have been developed which do not belong to the substituted methyl ketone class. Reversibly acting cyano derivatives (pharmacophore 1, Figure 1.9) are not very potent (Ki of less than 1 pM in a caspase- 1 assay) [459]. However, a-ketoamides (pharmacophore 2, Figure 1.9) may afford inhibitors with a Ki of less than 10 nM [92, 4561. An oxadiazole pharmacophore ( 3 , Figure 1.9)has been employed to construct peptide inhibitors of various cysteine proteases, including caspases [460, 46 11. The relevant Ac-YVAD-het derivative was very potent against caspase-8 (Ki < 20 nM),
30
CASPASE INHIBITORS
No
Phamawphore
Reference
No
Pharmacophore
-
n
Reference
x=o.s
,COOH
Figure 1.9. Other aspartate-like pharmacophores: for possible Y groups see Figure I . 7.
whereas Ac-DVED-het potently inhibited both caspases-7 and -8 (Ki< 30 nM) [460, 4611; neither peptide inhibited Granzyme B. Bajusz et al. found that peptidyl L-fl-homoaspartal (32) and its parent Ac-YVAD-H are almost equipotent inhibitors of caspase-1 [464, 4651. By analogy, the relevant homoaspartal based on the DEVD sequence is only 2 times less potent a caspase-3 inhibitor than Ac-DEVD-H [465].
(32)
Recently publishedpeptide irreversibleinhibitorscontaining the N-nitrosoaniline functionality (pharmacophore 5, Figure 1.9; cf small molecule (1)) exhibit second-order rate constants of up to 322 M-'s-' and are claimed to show weak carcinogenicity and almost no mutagenicity [356]. Non-acidic pharmacophores
The remaining carboxylic acid residue in aspartate-based pharmacophores discussed above may contribute to low cell membrane permeability of the inhibitors.
P.P.GRACZYK
31
Therefore, despite the dogma of the absolute preference for the aspartate residue at P1 of peptide-based inhibitors, significant effort has been put into exploring other options. Initial attempts to use diacylhydrazines (pharmacophore 6, Figure 1.9) as the replacement for the P1 aspartate moiety, with the COOH group still present, resulted in 10&1000 times drop in inhibitory activity against caspase- 1 as compared with the parent compounds [446]. Okamoto et al. found that the carboxylic acid moiety could be replaced with a sulfonamide derivative (1 and 2, Figure 1.10) with no significant loss in in vitro potency against caspase- 1 [467]. Furthermore, compound (33) has shown improved cell membrane permeability (ratio of ICsO values for cell-based system/enzyme = 6.1) [467]. Interestingly, in the complex of (33) with caspase1, the sulfonamide group behaves analogously to the carboxylic acid function. The nitrogen atom is deprotonated and forms a salt bridge with the enzyme’s arginine residues [467].
A more radical approach was attempted in our laboratory. We decided to remove the acidic functionality altogether hoping that the gain in membrane permeability would outweigh the expected loss of inhibitory potency. Three types of pharmacophores were studied: quinones (pharmacophores 3 and 4, Figure 1.lo), epoxyquinones (5, Figure 1.10) and epoxyquinols (6, Figure 1.10) [379]. A naphthoquinone-containingpeptide (34) based on the DEVD sequence was able to inhibit caspase-3 with an ICso value of 40 nM (cf parent naphtoquinone ICsO 120 nM). Its benzoquinone analogue (35) was less potent (ICsO value of 0.13 pM) but already some selectivity against cathepsin C (a noncaspase cysteine protease) could be observed (ICso I .69 pM). Selectivity of two isomeric epoxyquinones (36) and (37) was even higher, as both inhibited caspase-3 with an ICso value of 0.25 pM but were inactive against cathepsin C up to a concentration of 5 pM. Compounds (34) and (37) exhibited a slight cell protective effect at concentrations of several micromolar in an assay with rat cerebellar granule neurones induced to become apoptotic by staurosporine. Unfortunately, this was accompanied at higher concentrations by toxicity. The toxic component was eliminated in the epoxyquinol series. Compound (38) inhibited caspase-3 (IC50 0.95 pM) and showed moderate neuroprotection in an experiment in which superior cervical ganglion (SCG) neurones were microinjected with a solution of (38). Interestingly, its epimer (39) was only slightly
CASPASE INHIBITORS
32 Pharmacophore
No
Reference
tHF &
No
Phanacophore
Reference
U
H
f
MenMe
0
PH 6 H
0
Figure 1.10. Non-acidic pharmacophores in caspase inhibitors; for possible Y groups see Figure I . 7.
less active, while isomeric epoxides (40) and (41) were totally inactive against caspase-3. Presumably, the higher activity of (38) and (39) than that of (40) and (41) is due to the epoxyquinol OH group forming hydrogen bonds with Arg-79 and/or Gln-283 at the active site and exposing the correct face of the pharmacophore to attack by the enzyme cysteine sulfur atom. A similar dependency of inhibitory activity on stereochemistry of the epoxide ring was also observed for some epoxyquinones [379]. Peptide backbone (P2-P4) and its simple modifications
The minimum length of a peptide substrate necessary for efficient cleavage by caspases usually spans positions Pl-P4, as was originally described for caspase-1 [401]. However, caspase-2 additionally requires a P5 residue [326]. The requirements for caspase-5 are even greater, as at least nine amino acid residues (Pl-P4 and Pl’-P5/) are needed [92]. Amino acid preferences have been established for caspases by means of positional scanning of synthetic combinatorial libraries (caspase- 1) [29], by using defined peptide sequence variants (caspases-I, -2, -3, -4, -6, and -7) [326], and an analogous method employing combinatorial array of substrates (caspases -1 and -4) [468]. Although there are some differences between the results coming from these approaches, the general features of the preferred
P.P.GRACZYK
33
(35)
(34)
COOH
3 ( NH
(j:o
rCH N J$:::o
0
0
HG
2 - NHJ$o
2-N 0
0
0
34
CASPASE INHlBITORS
sequences are similar. A wide range of amino acids seems to be tolerated at P2 [326]. While there is a similar preference for Glu at P3 for all caspases, the P4 preference varies from Asp (caspases-2, -3, -7) to hydrophobic aromatic (e.g. Trp and Tyr for caspase-1), and hydrophobic aliphatic (e.g. Val, Leu, or Ile for caspases-4, -6, -8, -9, -10) [29, 92, 326, 4681. The preferred sequence for caspase-1, WEHD [29], is different from the previously suggested YVAD [469], and different from those found in its substrates IL-lP (YVHD) and IL-18 (LESD). The relevant aldehyde Ac-WEHD-H appears to be the most potent caspase inhibitor to date (Ki 56 pM), about 14 times more potent than Ac-YVAD-H [29] formerly considered to be the most potent. The inhibitory activities of other commonly used tetrapeptide aldehydes established by Garcia-Calvo et al. [327] are presented in Table 1.2. It is clear that selectivity of these inhibitors is limited, which has serious practical consequences for ‘proof of concept’ experiments. For instance Ac-DEVD-H which contains the preferred recognition motif for caspase-3, can inhibit caspase-8 with Ki in the subnanomolar range. Boc-IETD-H is a broad spectrum inhibitor of group I11 caspases also capable of inhibiting caspase- 1. X-ray structural studies on caspase-inhibitor complexes, including caspase- 1Ac-YVAD-H [424], caspase- 1-Ac-DEVD-H [425] caspase-3-Ac-DEVD-H [425, 4261, caspase-7-Ac-DEVD-H [425], caspase-8-Z-DEVD-H [470], caspase-8-Ac-DEVD-H [425], caspase-8-Ac-IETD-H [427] and caspase-8-ZEVD-dichloromethylketone [47 I] (reviewed recently by Griitter [SS]), provide an explanation for the observed substrate specificities within the caspase family. Many inhibitors with much shorter sequences have also been described, especially in the patent literature. Several examples are presented in Table 1.3 including as a reference Z-VAD-fmk, a compound frequently encountered in the biological literature. This inhibitor exhibits a wide spectrum of inhibitory
Table 1.2. INHIBITORY ACTIVITY OF SELECTED TETRAPEPTIDE ALDEHYDE INHIBITORS AGAINST CASPASES, Ki [nM](FROM [327]) Caspase Group I
Group I1
1
4 5 2 3
I
Group 111
6 8 9 10
Ac- WEHD-H
Ac-YVAD-H
Ac-DEVD-H
0.056 97 43 > 10000 1960 > 10000 3090 21.1 508 330
0.76 362 I63 > 10000 > 10000 > 10000 > 10000 352 970 408
132 205 1710 0.23 1.6 31 0.92 60 12
18
Boc-IETD-H
<6 400 223 9400 195 3280 5.6 1.05 108
27
P.P. GRACZYK
35
Table 1.3. EXAMPLES OF SHORT PEPTIDE-DERIVED INHIBITORS
IC,, (nM] for caspuseCompound
I
2
280" 33 27
0.29"
3
16" 13 10
34 10 19.8 200
4
5
6
7
5.5a
130"
7.1" 37 1500
1320 267
18.4
lga
8
9
Ret
280a 7.6 179
180a
[327] [4 161 [4 171 [4721 [4731 [4091 14741
n.s.-not stated, "-second order inactivation rate [I000 M-'s-']
activity, partly due to suboptimal recognition of its short peptide chain. However, some of the binding energy lost by removal of further amino acids could be compensated by properly chosen N-terminal protection. Examples (42) and (43) show that this approach allows significant selectivity to be achieved even between caspases-3 and -7, which exhibit very similar substrate specificities. A large series of various N-terminal protecting groups has been introduced into tripeptidic aldehydes (pharmacophore 1, Figure 1.5) by researchers from Vertex [428], which allowed them to obtain inhibitors with Ki values against caspase- 1 in the low nanomolar range. Dipeptidic indolecontaining inhibitors, e.g. (48), (IDN1965), achieved Ki values of 17 nM and an inactivation rate of 58800 M-'sK' against caspase-6 [415, 4751. The peptidic character of potent inhibitors may give rise to many problems during interpretation of 'proof of concept' experiments in cell-based assays and in vivo. We found that neither Ac-DEVD-H nor Z-DEVD-H, which are potent inhibitors of caspase-3, protected cerebellar granule neurones from staurosporine-induced apoptosis up to a concentration of 100 pM. In order to learn whether lack of activity is due to insufficient membrane permeability we synthesized the relevant double ester prodrug (49) [379]. This compound was capable of rescuing cerebellar granule neurones from staurosporine induced apoptosis at 100 pM. Interestingly, the relevant trimethyl ester (49, R = Me) was not neuroprotective, probably due to the slow hydrolysis of some COOMe groups as compared with the rate of degradation of the drug in the cell environment.
Peptidomimetic mod$cation of the P2-P4 amino acids - acyclic structures Peptide-based inhibitors usually suffer from low bioavailability and stability under physiological conditions, which is primarily due to the presence of several
CASPASE INHIBITORS
36
(45)
(44)
(46)
(47) COOR
(48)
(49) R = MeC(O)OCH,
peptide bonds. Peptidomimetic modification of P2 and further position(s) may resolve some of these issues [476]. Furthermore, introducing a structural element which mimics the P2 and/or P3 amino acids, and is able to force the secondary structure of the molecule into the preferentially recognized P-sheet conformation [424, 426, 477, 4781, might additionally improve the potency of the inhibitor. Taking advantage of the observation that the P2 NH group is not important for activity against caspase-1 [444], the P2 and P3 amino acids have been
P.P.GRACZYK
37
replaced with various cyclic structures. Compound (47) may serve as one of the simplest examples in which both P2 and P3 amino acids have been replaced with a single phenyl ring. However, the potency of this compound is not high, presumably due to the flat structure of the phenyl ring and suboptimal recognition by the enzyme. Another, much more successful, approach has been developed at Warner-Lambert who patented a series of peptidomimetic compounds in which the P2-P3 segment of peptide inhibitors has been replaced by a properly substituted succinamide moiety [412]. An example from this series, compound (50), exhibits Kiof 0.4 nM against caspase- 1. The peptidic nature of peptide inhibitors can also be reduced by N-alkylation of the P2 NH group [462], which may promote the cis conformation of the amide bond [479, 4801, and therefore affect the conformation of the molecule. Compound (51), based on this principle, showed a Ki of 1.9 nM.
n
Me
Peptidomimetic replacement of the P3 amino acid In order to achieve proper spatial arrangement of the aspartate pharmacophore and the N-terminal fragment another strategy, based on replacing each P2 or P3 amino acid residue individually (Figure 1.11) has been employed by various research groups. Pyridone (scaffold 1, Figure 1.12) [481, 4821 or pyrimidin-6one (scaffold 2, Figure 1.12) [405, 4824841, the scaffolds previously used in the design of human leukocyte elastase inhibitors, were utilized to replace the P3 amino acid during the construction of caspase-1 inhibitors. The relevant pyridone-derived aldehydes were not very active (Ki values of about 1 pM)
38
CASPASE INHIBITORS
CHO OH Ac-YVAD-H Figure 1.11. Creation of cyclic peptidomimetic replacements of P3 and P2 amino acids based on Ac- W A D -H.
[48 11. However, rigidification of the peptide backbone by introduction of R’=Me into the scaffold led to a 30-fold increase in the inhibitory activity [481]. Aryloxymethyl ketones (pharmacophore 13, Figure I. 7), based on the pyridone scaffold with R’=Ph, have shown Ki’s of the order of 1 nM [482].
No
Scaffold
H
O
Reference
R
[408, 450, 466, 481, 4821
No
Scaffold
Reference
4
1408, 41I, 4231
4851 H
0
-
X=N, CH
Figure 1.12. Peptidomimetic replacements of P3 amino acid
P.P.GRACZYK
39
Design of peptidomimetic inhibitors of caspases has been facilitated by recent advances in X-ray crystallography and computational methods [486]. Golec et ul. used an X-ray structure of a complex between caspase-l and pyridone-based irreversible inhibitor to identify a lipophilic pocket near the S2 subsite which could accommodate the appropriate R group (scaffold 1, Figure 1.12) [481]. Introduction of R=PhCH2 led to a 32-fold increase in activity as compared to the parent compound (R = H). Using the the pyrimidin-6-one derivatives (scaffold 2, Figure 1.12; R = aromatic group) Dolle et al. synthesized potent, irreversible inhibitors with inactivation rate constants of the order of up to 3 x 10' M-'s-' [484]. They explained the observed difference in structure-activity relationship (SAR) between these compounds and their peptide parents by a suboptimal interaction of the P3 amide and the enzyme due to conformational constraint imposed by the flat sp2 center in the pyrimidine ring (Figure 1.13) [484]. Non-aromatic rings have also been frequently used to link the P3 side-chain to P2 NH (for examples see Figure 1.12). Subnanomolar activity could be achieved by 1,2-diazepane (52) ( K , 0.9 nM against caspase-1) [414]. The sevenmembered ring was also optimal for lactam-containing inhibitors of caspase- 1, e.g. (53) (R = H, ICs0 1.68 pM against murine caspase-1) [485]. Karanewsky et ul. have also found that, in agreement with the SAR for pyridone-based inhibitors [481], incorporation of alanine in (53), R = Me, instead of glycine (53), R = H, at P2 enhanced binding affinity almost tenfold [485]. 0
Peptidomimetic replacement of the P2 amino acid Representative examples of P2 replacements are collected in Figure 1.14. Linking the P2-NH with the P2 side-chain in Z-VAD-H using five- or six-
40
CASPASE INHIBITORS
Figure I . 13. D(fference between the parent peptide and the pyrimidin-6-one scaffold.
membered ring, as in (54), n = 1, 2 , allowed Karanewsky et al. to achieve an ICso of about 90 nM against murine caspase-1, comparable with that of the parent compound (64 nM) [485]. At the same time, a 13-fold decrease in the IC50value against caspase-3 was observed for the six-membered analogue (54), n = 2 [485].
Bicyclic peptidomimetic replacements of P2-P3
The pyrimidin-6-one scaffold (see above) served as the basis for further drug design. Dolle et al. achieved significant improvement in the inhibitory activity increase in the second-order inactivation against caspase- 1 (about a 3&50% rate constants as compared with the relevant peptides) by using the bicyclic pyridazinodiazepine system, and introducing the relevant sp3 carbon to force the inhibitor into the desired bioactive conformation (Figure 1.15, cf Figure 1.13) [484, 4871. Increasing the acidity of the N-terminal group of the inhibitor
Scaffold 1
Scaffold 2
[408,417,428,4851
[4281
Scaffold 3
14281
Figure 1.14. Peptidomimetic replacements (ll'P2 amino acid.
P.P. GRACZYK
41
led to a further increase in the inactivation rate, e.g. 1.22 x lo6 M-'s-' for ( 5 5 ) [484]. The relevant aldehydes inhibited caspase-1 with an ICs0 in the 1-25 nM range and were about an order of magnitude more active than the parent peptide aldehydes. Compound (56) exhibited stability in ex vivo liver and intestinal slice assays and was selected for in vivo studies in dog [484]. 0
HOOC-S
0
(55) CI 0
CI'
CF,
t,N I
(58)
Y
O
CI
KH
(59)
Many other reversible and irreversible caspase-1 inhibitors based on a pyridazinodiazepine scaffold and others (Figure I,16) have been synthesized. The potent reversible inhibitors (Ki 7.5-90 nM) were very selective and did not inhibit caspase-3 up to a concentration of 1 pM, in contrast to the relevant irreversible inhibitors [423]. Lack of selectivity of irreversibly acting (57) was accompanied by its relatively potent antiapoptotic activity in anti-Fas induced death of U937 cells (ICs0 of 0.8 pM). The reversibly acting (56) was inactive up to a concentration of 20 pM [423]. In contrast to this observation the in vivo activity of an irreversibly acting pyrimidin-6-one (58) was claimed to be much weaker than that of the relevant aldehyde (59), although their in vitro activities are similar [413].
42
CASPASE INHIBITORS
Figure 1.15. Peptidomimetic bicyclic scuflold design by Dolle el ul. (4R41.
Building on their experience with P3 and P2 peptidomimetics (53) and (54) Karanewsky et al. combined the two structural motifs in (60), which exhibited an ICso of 36 nM against caspase-1 [485]. Furthermore, introduction of the P4 aspartate into a similar scaffold gave (61), a very potent and selective
No
Scaffold
Reference
No
Scaffold
0 II
1411, 4231
14501
4
CHz)+3
1450,485, 4881
H
Figure I . 16. Bicyclic peptidomimetic scuffolds
Reference
P.P.GRACZYK
43
caspase-3 inhibitor (ICso of 18 and 1040 nM against caspases-3 and -1, respectively) [485]. H
H
CASPASE INHIBITORS IN VIVO Caspase inhibitors have been evaluated in various models chosen to study their anti-inflammatory and/or antiapoptotic activity. Due to the complex mechanisms involved in these models, the two modes of action may not always be clearly separated e.g. in ischaemia. Furthermore, due to limited specificity of the inhibitors, especially those acting irreversibly, the observed effects of socalled ‘specific’ caspase inhibitors may actually be the result of inhibition of more than a single enzyme. AXOTOMYiAXONAL LESIONS
Caspase inhibition may be achieved using chemical and genetic strategies. The latter approach would correspond to application of protein-type inhibitors. The X-IAP delivered by adenoviral vector at the rat optic nerve stump inhibited secondary cell death after axonal lesions [489]. An adenoviral gene transfer technique was also used to deliver and express NAIP, HIAPl and HAIP2 in motor neurones following axotomy of a peripheral nerve in young rats. The administration of an adv-NAIP, adv-HIAP1 and adv-HIAP2 rescued 3 W O % of motor neurones one week after sciatic nerve axotomy [490]. The effect of the IAP proteins on motor neurone survival decreased with time but was still present 4 weeks post axotomy [490]. Intraocular injection of Z-DEVD-cmk [49 11, Z-DEVD-fmk and Ac-DEVDH [165] in the rat optic nerve transection model results in about a 30% increase in survival of RGCs. Interestingly, Z-VAD-fmk is less effective [491]. The neuroprotective effect of extended (2 week) treatment with
44
CASPASE INHIBITORS
Z-DEVD-cmk decreased over a 4 week period to 24% [492]. This may indicate that a permanent and complete rescue of axotomized RGCs could not be achieved and inhibition of caspases results primarily in a significant delay of secondary death. BRAIN ISCHAEMIA
Both proteins and small molecule inhibitors have shown efficacy in various brain ischaemia models. Virally overexpressed X-IAP protected CA 1 hippocampal neurones following transient forebrain ischaemia [57]. In the rat MCAO model, Z-VAD-DCB reduced the infarct volume by about 50% when it was administered intracerebroventricularly (i.c.v.) starting 30 min before surgery [493]. Injection of Ac-YVAD-cmk (300 ng) i.c.v. 10 min after permanent MCAO in the rat resulted in a reduction of infarct volume both 24 h (36%) and 6 days (25%) after ischaemia. Decreased apoptosis, as measured by nucleosome assay, could be found after 24 h but not 6 days [494]. Ac-YVAD-cmk as well as other irreversibly acting inhibitors, including Z-DEVD-fluoromethylketone (Z-DEVD-fmk) [495] and Z-VAD-fluoromethylketone (Z-VAD-fmk) [496] were active in the corresponding mouse model with Z-VAD-fmk being capable of reducing the infarct volume by 40-50% [495, 4961. This compound was still efficacious if given immediately after reperfusion in a single dose [495]. The more selective AcYVAD-cmk required a higher dose in the pretreatment regimen to show protection while the effectiveness of Z-DEVD-fmk was very low (27% reduction in infarct volume) [495]. One has to emphasise that behavioural deficits after MCAO were improved by all three inhibitors [495]. Hara et al. suggested, therefore, that less selective inhibitors are more likely to show efficacy in ischaemia [495]. This has been supported by very recent studies in the transient cerebral ischaemia model in Mongolian gerbils [497]. The strongest protective effect on CA1 pyramidal cells (66 and 91% at 1 and 10 pg dose, respectively) was exhibited by Z-VAD-fmk infused directly into the right lateral ventricle. The specific caspase- 1 inhibitor, Ac-WEHD-H, showed a smaller efficacy (43 and 57% reduction in infarct volume at 1 and lops), while Ac-DMQD-H, a caspase-3 inhibitor, was active only at the highest dose (34% reduction at 10 pg) [497]. The therapeutic window in the transient MCAO model in terms of the infarct volume and neurological score can be extended to 6 h with Z-VAD-fmk and ZDEVD-fmk given i.c.v. [498]. In another experiment Z-DEVD-fmk blocked delayed cell loss of hippocampal CA1 neurones after transient forebrain ischaemia in the rat [499]. However, it did not prevent impairment of induction of long-term potentiation in post-ischaemic CA 1 cells, suggesting that caspase inhibition alone does not preserve neuronal plasticity [499]. Significant
P.P.GRACZYK
45
neuroprotection (>50%) after delayed treatment (up to 3 h) was also found for Boc-D(0Me)-fmk (BAF) given i.c.v. in a rat model of neonatal hypoxiaischaemia [60]. While the i.c.v. injection of a drug can be used at the concept verification stage during preclinical studies, the ultimate goal is drug efficacy after peripheral administration. Cheng et al. have demonstrated for the first time that systemic, delayed administration of a caspase inhibitor (BAF) can be neuroprotective [60]. Significant neuroprotective properties (up to 57% infarct volume reduction) of intravenously administered Z-VAD-fmk (post treatment 20 mg/kg bolus, followed by continuous infusion) in the transient focal ischaemia model in the rat have recently been claimed by Cytovia researchers [409]. Also, the peripherally administered peptidomimetic (59) (3 x 50 mg/kg) was capable of reducing the lesion size by 60% in the mouse transient MCAO model [413]. Despite the successful use of caspase inhibitors in the above models, White et al. pointed out that therapy for brain ischaemia and reperfusion with a single drug is likely to fail in the clinic. Due to numerous mechanisms involved, successful treatment may require multidrug protocols [500]. It has also been emphasised that if the ischaemically injured cells have suffered mitochondria1 damage sufficient to impair respiration, caspase inhibitors are not expected to offer long-term protection [73]. OTHER ISCHAEMTA MODELS
In the mouse intestinal ischaemia-reperfusion model, subcutaneous administration of Z-VAD-fmk (multiple dosing) diminished the degree of small bowel injury [50I]. Rat liver injury following ischaemidreperfusion can also be prevented by caspase inhibition [502]. In the rat [503] and rabbit [504] myocardial reperfusion injury model administration of various, irreversibly acting caspase inhibitors, e.g. Z-VAD-fmk (3.3 mg/kg) [503], or Ac-WAD-cmk (4.8 mg/kg) [504], before the onset of ischaemia, reduced the myocardial infarct volume by up to 3 1% [504]. This has been attributed to attenuation of cardiomyocyte apoptosis [503]. Furthermore, Ac-YVAD-cmk attenuated ischaemia-induced myocardial disfunction [505]. A recent study of rat myocardial reperfusion injury has shown that Z-VAD-fmk can be cardioprotective even when the drug is administered after the onset of ischaemia [506]. Interestingly, the reversibly-acting inhibitors YVAD-H (3.5 mg/kg) and DEVD-H (3.5 mg/kg), administered intravenously 5 min prior to ischaemia in the rat, did not significantly change the infarct volume, although myocyte DNA fragmentation and caspase activation was inhibited [507]. In another study using this model, a wider range of irreversible caspase inhibitors, including Z-VAD-fmk, Z-IETD-fmk (targeting caspase-8), Z-LEHD-fmk
46
CASPASE INHIBITORS
(caspase-9 inhibitor), and Ac-DEVD-cmk exhibited significant efficacy in limiting the infarct volume [508]. In the opinion of Huang et al. the higher efficacy of irreversibly acting derivatives may be due to their limited selectivity and inhibition of additional non-caspase-related mechanisms [506]. During renal ischaemia-reperfusion in rats, application of Z-D-DCB attenuated the resultant increase in caspase-3 activity [509]. BRAIN TRAUMA
Reduced infarct volume was observed in cold injury-induced brain trauma in mice treated with Z-VAD-fmk [5 lo]. Neuroprotection following brain trauma was also achieved by i.c.v. administration of caspase inhibitors Ac-YVAD-cmk (200 ng; 56% reduction in the lesion volume) and Z-VAD-fmk (480 ng; 53% reduction) [ 1251. A non-significant reduction (20%) in the total lesion volume was detected when Z-VAD-fmk was applied 1 h following trauma [ 1251. EXCITOTOXICITY (KAINIC ACID, NMDA, AMPA)
Neuronal expression of p35 in transgenic mice was found to protect neurones against apoptotic death induced by excitotoxic kainic acid [5 111. When neuronal damage was induced in rat by intraamygdaloid injection of kainic acid (0.1 pg), Z-DEVD-fmk significantly improved neuronal survival within the hippocampal CA3/CA4 regions [5 121. Z-VAD-fmk has shown protection against excitotoxic brain damage due to intrastriatal microinjection of N-methyl-D-aspartate (NMDA) and, to a lesser extent, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate(AMPA) [495]. Another irreversibly acting inhibitor, peptidomimetic pyrimidin-6-one (58), was inactive in an excitotoxic brain damage model in neonatal rats [413]. However, the corresponding aldehyde (59) was able to significantly reduce weight loss of the NMDA-injected hemisphere [413]. In the perinatal rat model of NMDA-induced neurotoxicity, aldehydes DEVD-H and YVAD-H were less potent (40 and 30% protection, respectively) than irreversible Z-VAD-cmk (50% protection) [119]. The best protection was achieved with MK-801 (NMDA receptor antagonist, about 80%) in the pretreatment regimen only, as expected [498]. Interestingly, MK-801 may act in synergy with caspase inhibitors [513, 5141. In the mouse MCAO model, pretreatment with a subthreshold dose of Z-VAD-fmk or Z-DEVD-fmk extended the time window for MK-801 effectiveness by 2 h from 1 h before reperfusion to at least 1 h after reperfusion [5 131. Furthermore, pretreatment with a subthreshold dose of MK-801 extended the time window for Z-DEW-fmk effectiveness from 1 h to at least 3 h after reperfusion [513].
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ENDOTOXIC SHOCK
Systemic administration of Ac-YVAD-H in a murine model of endotoxic shock (induced by LPS) reduced the elevation of IL-lp in the plasma and peritoneal fluid [515]. In the rat model of endotoxemia, pretreatment of rats with the irreversibly acting Ac-YVAD-cmk at a dose of 12.5 pmol/kg significantly reduced mortality from 83 to 33% without, however, modifying the cytokine profiles as compared with the LPS-drug vehicle group [5 161. Furthermore, the survival-promoting effect of (62) (IDN 1529) in the murine model of endotoxic shock could not be linked to inhibition of caspase-1 [517]. In particular, the protective effect of (62) was accompanied by an increase in the serum concentration of caspase- I-derived cytokines such as IL-Ip, IL- la, and IL-18. Despite its very similar structure, another caspase inhibitor (48) (IDN 1965) showed no efficacy [517]. The authors suggested that lethality in endotoxic shock is due to another process different from apoptosis or the release of inflammatory cytokines [5 171.
AMYOTROPHIC LATERAL SCLEROSIS
Z-VAD-fmk given i.c.v. in an ALS transgenic mouse model (mSODlG93A), delayed disease onset and mortality [ 1321. The authors suggest that caspase-1 may mediate early disease processes [ 1321. In the opinion of Friedlander [518], additional caspases are actually involved in the disease and therefore a broad acting inhibitor that blocks all caspases might bring the desired therapeutic action even at the expense of some side-effects.
MULTIPLE SCLEROSIS
Pretreatment of mice with Z-VAD-fmk significantly reduced EAE incidence which suggests that caspase- 1 represents a possible therapeutic target in the acute phase of relapsing-remitting MS [349].
48
CASPASE INHIBITORS PARKINSON’S DISEASE
Adenoviral gene transfer of X-IAP and the glial cell line-derived neurotrophic factor to the striatum rescued dopaminergic neurones in the substantia nigra pars compacta from cell death in a mouse model of PD [519]. HUNTINGTON’S DISEASE
Administration of Z-VAD-fmk (i.c.v) in a mouse model of HD delayed the disease progression and mortality [ 1241. GRAFT REJECTION
Neural grafting constitutes a potential treatment for Parkinson’s disease [520]. Unfortunately, it is plagued by poor survival of grafted dopaminergic neurones (5-1 0%). Ac-WAD-cmk increased the survival of dopaminergic neurones grafted into hemiparkinsonian rats, and improved their functional recovery [521]. The same compound promoted survival of transplants of embryonic striatal cells placed into the excitotoxically lesioned rat striatum (a model of HD) [522]. Apoptosis that occurs in acute rat cardiac allograft rejection could be reduced with ZnClz (1 and 5 mg/kg) in a dose-dependent manner via caspase-3 inhibition [523]. CNS INFLAMMATION
Chronic brain inflammation due to infusion of lipopolisaccharide into the basal forebrain of young rats may serve as a model of degeneration of forebrain cholinergic neurones in Alzheimer’s disease, and is accompanied by upregulation of caspases-3, -8, and -9 [524]. Although administration of Z-VAD-fmk reduced the levels of caspases-3, -8, and -9, the inhibitor did not provide any neuroprotection [524]. PERIPHERAL INFLAMMATION
Z-VAD-DCB inhibited the release of IL-lB by 35 and 55% at 10 and 100 mg/kg, respectively, in a mouse subcutaneous tissue chamber implant model of inflammation [525]. Peptidomimetic (56) was even more potent as IL-lP production was inhibited by >95% at a single 100 mg/kg dose [484]. In a dog model oral bioavailability of (56), established in two dogs, was 12 and 16% [484]. Compound (56) was stable in ex vivo liver and intestinal slice assays, and possessed a plasma clearance rate of about 7 mL/min/kg [484].
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HEPATITIS
Pretreatment of hepatocytes with Ac-DEVD-H, Z-VAD-fmk, or Z-D-DCB inhibited anti-Fas-mediated apoptosis [ 1751. In agreement with this observation, DEVD-H showed a protective effect in the Fas antibody-induced hepatitis model in mice [161]. A shorter peptide inhibitor, Z-VAD-fmk, prevented Fas-mediated hepatic microcirculatory failure and Kupffer cell dyshnction [526]. Interestingly, the very short, non-selective dipeptidic inhibitor Z-VDfmk has been claimed to provide almost 100% protection after 1 h at 0.25 mg/kg dose [409]. By contrast, YVAD-H was ineffective in the hepatitis model [161]. Some of the problems associated with low efficacy of the inhibitors in vivo may be explained by the possibility that attenuation of certain caspase activities is compensated by the activation of other caspases. In particular, caspase-3 and -9-deficient mice were not protected against Fas agonistic antibody (J02) toxicity in an in vivo model of Fas-mediated apoptosis [527]. Deficiency in those caspases, which are essential for the above pathway, unexpectedly resulted in rapid activation of alternative caspases, probably mediated through mitochondria [527], Interestingly, some nitric oxide-releasing compounds can protect against liver damage, probably by non-selective inhibition of caspases-3, -8 and -9 [528]. Caspase-8 itself may be a promising therapeutic target for inhibition of Fasmediated apoptosis. In the relevant mouse model, treatment with IETD-H (10 mg/kg) attenuated the increase in caspase-3 activity and DNA fragmentation by 80-90% and completely prevented haemorrhage and parenchymal cell damage [ 1731. Early release of mitochondria1 cytochrome c and the processing of caspases-3, -8, and -9 was prevented by IETD-H [ 1731. Finally, the issue of low bioavailability of caspase inhibitors could be overcome, for instance, by using nanospheres, as demonstrated in a mouse model of acute hepatitis [529]. SHIGELLOSIS
Hilbi et al. have found that Shigella-induced macrophage cell death could be blocked by caspase inhibitors such as Ac-YVAD-H and Ac-YVAD-cmk [530]. MENR\IGITIS
In a rabbit model of meningitis, injection of Z-VAD-fmk into the cisterna magna immediately after bacterial inoculation significantly reduced spongiform encephalopathy and inhibited neuronal loss and apoptosis of dentate gyms neurones [50]. Importantly, the antiapoptotic effect of the caspase inhibitor could be observed even six days after the combined treatment with Z-VAD-fmk and an antibiotic which was initiated 8 h after the infection [50].
50
CASPASE INHIBITORS PNEUMOPATHY
In a mouse bleomycin-induced pneumopathy model, Z-VAD-fmk decreased the number of apoptotic cells, the pathological grade of lung inflammation and fibrosis, and the hydroxyproline content in lung tissues, suggesting that caspase inhibitors could constitute a new therapeutic approach against lung injury and pulmonary fibrosis [53 13. The antifibrotic potential of Z-VAD-fmk has also been demonstrated in the rat model of lung fibrosis in response to bleomycin, where collagen accumulation was inhibited by 85% [532].
CLINICAL STUDIES Pralnacasan (26), (VX-740/HMR 3480) has advanced to clinical trials (Phase I1 for rheumatoid arthritis; administration p.0.) [533]. Studies in France are being carried out by Aventis and by Vertex in the United States. Clinical trials in osteoarthritis, heart failure and stroke have been planned for 2001. Vertex is also developing another orally active caspase- 1 inhibitor, VX-765. The company is also interested in other caspases, e.g. caspase-9. NO-aspirin (2), (NCX-4016) is being developed by NicOx for cardiovascular pathologies [534] as well as vein graft failure [535]. The compound was in Phase I last year, and Phase I1 trials are expected in 200 1. The compound seems to be well tolerated [536]. Idun is developing antiapoptotic caspase-8 inhibitors. In particular, IDN 6556 entered a Phase I trial involving 75 patients as an agent to treat acute alcoholic hepatitis [537]. Preclinical studies (safety and efficacy in animal models) have also been carried out in the US with IDN 5370 (a broad-spectrum irreversible caspase inhibitor) in a collaboration between Idun and Novartis. Recent press releases indicate that the company is looking into developing caspase inhibitors for the treatment of heart attack and CNS injury [538]. Maxim’s CV-1013, (63), a broad spectrum, irreversible inhibitor, is in preclinical studies for the treatment of hepatitis, stroke and myocardial infarction [539]. The company plans to initiate a Phase I cardiovascular disease study in 2001. Together with CoCensys, Maxim is actively investigating a series of caspase inhibitors for the treatment of neurodegenerative diseases.
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SUMMARY The striking efficacy of Z-VAD-fink in the various animal models presented above may reflect its ability to inhibit multiple enzymes including caspases. In accord with this, more selective, reversible inhibitors usually show low efficacy in multifactorial models such as ischaemia, but may offer some protection against NMDA-induced excitotoxicity and hepatitis. Importantly, caspase inhibitors may exhibit significant activity in vivo even when they are applied post insult. As far as the CNS is concerned, the first systemically active inhibitors have emerged. Functional recovery could be achieved in some ischaemia models, but long-term protection by caspase inhibitors is still being questioned. Recent developments in drug design enabled the first caspase inhibitors to enter the clinic. Although initially directed towards peripheral indications such as rheumatoid arthritis, caspase inhibitors will no doubt eventually be used to target CNS disorders. For this purpose the peptidic character of current inhibitors will have to be further reduced. Small molecule, nonpeptidic caspase inhibitors, which have appeared recently, indicate that this goal can be accomplished. Unfortunately, many fundamental questions still remain to be addressed. In particular, the necessary spectrum of inhibitory activity required to achieve the desired effect needs to be determined. There is also a safety aspect associated with prolonged administration. Therefore, the next therapeutic areas for broader-range caspase inhibitors are likely to involve acute treatment. Recent results with synergistic effects between MK-80 1 and caspase inhibitors in ischaemia suggest that caspase inhibitors may need to be used in conjunction with other drugs. It can be expected that, in the near future, research on caspases and their inhibitors will remain a rapidly developing area of biology and medicinal chemistry. More time, however, may be needed for the first caspase inhibitors to appear on the market. ACKNOWLEDGEMENT
I would like to thank Joanne Taylor, Les Turski, Terence Smith, Hirotoshi Numata and Afzal Khan for helpful comments on the manuscript. REFERENCES I 2
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483
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502 Cursio, R., Gugenheim, J., Ricci, J.E., Crenesse. D., Rostagno, P., Maulon, L., Saint-Paul, M.C., Ferrua, B. and Auberger, A.P. (1999) FASEB J. 13, 253-261. 503 Yaoita, H., Ogawa, K., Maehara, K. and Maruyama, Y. (1998) Circulation 97, 276-281. 504 Holly, T.A., Drincic, A., Byun, Y., Nakamura, S., Harris. K., Klocke, F.J. and Cryns, V.L. (1999) J. Mol. Cell. Cardiol. 31, 170’+1715. 505 Pomerantz, B.J., Reznikov, L.L., Harken, A.H. and Dinarello, C.A. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 2871-2876. 506 Huang, J.-Q., Radinovic, S., Rezaiefar, P. and Black, S.C. (2000) Eur. J . Pharmacol. 402, 139-142. 507 Okamura, T., Miura, T., Takemura, G., Fujiwara, H., Iwamoto, H., Kawamura, S., Kimura, M., Ikeda, Y . ,Iwatate, M. and Matsuzaki, M. (2000) Cardiovasc. Res. 45, 642450. 508 .Mocanu, M.M., Baxter, G.F. and Yellon, D.M. (2000) Br. J. Pharmacol. 130, 197-200. 509 Shi, Y., Melnikov, V.Y., Schrier, R.W. and Edelstein, C.L. (2000) Am. J. Physiol. 279 F509F517. 510 Morita-Fujimura, Y., Fujimura, M., Kawase, M., Murakami, K., Kim, G.W. and Chan, P.H. (1999) J. Cereb. Blood Flow Metab. 19, 634442. 51 1 Viswanath, V., Wu, 2.. Fonck, C., Wei, Q., Boonplueang, R. and Andersen, J.K. (2000) Proc. Natl. Acad. Sci. U.S.A. 97(5), 227G2275. 512 Henshall, D.C., Chen, J. and Simon, R.P. (2000) J. Neurochem. 74, 12151223. 513 Ma, J., Endres, M. and Moskowitz, M.A. (1998) Br. J. Pharmacol. 124, 756-762. 514 Schulz, J.B., Weller, M., Matthews, R.T., Henekd, M.T., Groscurth, P., Martinou, J.-C., Lommatzsch, J., Coelln, R.V., Wullner, U., Loschmann, P.-A,, Bed, M.F., Dichgans, J. and Klockgether, T. (1998) Cell Death Differ. 5 , 847-857. 515 Fletcher, D.S., Agarwal, L., Chapman, K.T., Chin, J., Egger, L.A., Limjuco, G., Luell, S., MacIntyre, D.E., Peterson, E.P., Thornberry, N. and Kostura, M.J.A. (1995) J. Interferon Cytokine Res. 15, 243-248. 516 Mathiak, G., Grass, G., Herzmann, T., Luebke, T., Zetina, C.C., Boehm, S.A., Bohlen, H., Neville, L.F. and Hoelscher, A.H. (2000) Br. J. Pharmacol. 131, 383-386. 517 Grobmyer, S.R., Armstrong, R.C., Nicholson, S.C., Gabay, C., Arend, W.P., Potter, S.H., Melchior, M., Fritz, L.C. and Nathan, C.F. (1999) Mol. Med. 5, 585-594. 518 Friedlander, R.M. in BioWorld Today 04/ 14/2000. 519 Eberhardt, O., Coelln, R.V., Kugler, S., Lindenau, J., Rathke-Hartlieb, S., Gerhardt, E., Haid, S., Isenmann, S., Gravel, C., Srinivasan, A., Bahr, M., Weller, M., Dichgans, J. and Schulz, J.B. (2000) J. Neurosci. 20, 9126-9134. 520 Olanow, C.W., Kordower, J.H. and Freeman, T.B. (1996) Trends Neurosci. 19, 102-109. 521 Schierle, G.S., Hansson, O., Leist, M., Nicotera, P., Widner, H. and Brundin, P. (1999) Nat. Med. 5 , 97-100. 522 Mundt-Petersen, U., Petersen, A., Emgard, M., Dunnett, S.B. and Brundin, P. (2000) Exp. Neurol. 164, 112-120. 523 Kown, M.H., Van der Steenhoven, T., Blankenberg, F.G., Hoyt, G.. Berry, G.J., Tait, J.F., Straws, H.W. and Robbins, R.C. (2000) Circulation 102, 111/228-111/232. 524 Wenk, G. L., McGann, K., Mencarelli, A,, Hauss-Wegrzyniak, B., Del Soldato, P. and Fiorucci. S. (2000) Eur. J. Pharmacol. 402, 77-85. 525 Miller, B. E., Krasney, P. A., Gauvin, D. M., Holbrook, K. B., Koonz, D. J., Abruzzese, R. V., Miller, R. E., Pagani, K. A., Dolle, R. E., Ator, M. A. and Gilman, S. C. (1995) J. Immunol. 154, 1331-1338. 526 Wanner, G.A., Mica, L., Hentze, H., Trentz, 0. and Ertel, W. (1999) Chir. Forum Exp. Klin. Forsch. 6 9 7 4 9 9 . 527 Zheng, T.S., Hunot, S., Kuida, K., Momoi, T., Srinivasan, A,, Nicholson, D.W., Lazebnik, Y. and Flavell, R.A. (2000) Nat. Med. (N. Y.) 6, 1241-1247.
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528 Fiorucci. S., Mencarelli, A,, Palazzetti, B., Del Soldato. P., Morelli, A,, Ignarro, L.J. (2001) Proc. Natl. Acad. Sci. U.S.A. 98(5), 2652-2657. 529 Shibuya. I., Akaike, T. and Watanabe, Y. (2000) Hepatology 32, 13OC-1308. 530 Hilbi, H.,Chen, Y., Thirumalai, K., Zychlinsky, A. (1997) Infect. Iminun. 65(12), 51655 170. 53 I Kuwano. K., Kunitake, R., Maeyama, T., Hagimoto, N., Kawasaki, M., Matsuba, T., Yoshimi, M., Inoshima, I., Yoshida, K. and Hara, N. (2001) Am. J. Physiol. Lung Cell Mol. Physiol. 280, L31GL325. 532 Wang, R., Ibarra-Sunga, 0.. Verlinski, L., Pick, R. and Uhal, B.D. (2000) Am. J. Physiol. 279, L143-LI51. 533 Vertex press release on 4 January, 200 I. 534 NicOx press release on 2 1 October, 2000. 535 NicOx press release on 21 November, 2000. 536 NicOx press release on 2 I February, 200 1. 537 ldun press release on 18 December, 2000. 538 ldun press release on 7 February, 2001. 539 Cai, S.X..Wang, Y., Huang, J.-C., Guastella. J., Wu, Y., Xue, D. and Drewe, J. (2000) Abstr. Pap. - Am. Chem. SOC.220th MEDI-153.
ABBREVIATIONS A/I - amyloid beta AD - Alzheimer's disease ALPS Autoimmune Lymphoproliferative Syndrome ALS - amyotrophic lateral sclerosis AMPA - r-amino-3-hydroxy-5-methyl-4-isoxazolepropionate APP - amyloid precursor protein BAF - Boc-D(0Me)-fmk BIR - baculoviral inhibitory repeat CARD caspase recruiting domain CGC cerebellar granule cell CNS - central nervous system CrmA - cytokine response modifier A DCB - dichlorobenzyloxy DISC - death-inducing signalling complex DNA - deoxyribonucleic acid EAE - experimental autoimmune encephalomyelitis FADD - Fas-associated death domain FasL Fas ligand HD - Huntington's disease HIV - human immunodeficiency virus HUVEC - human umbilical venous endothelial cells IAP - inhibitor of apoptosis protein IBD - inflammatory bowel disease i.c.v. - intracerebroventricular IFN-y - interferon-? IL-18 interleukin-18 IL-I/I - interleukin-I/] IMS - intermembrane space ~
~
-
~
~
72
CASPASE INHIBITORS
iNOS - inducible nitric oxide synthase LPS - lipopolisaccharide MCAO - middle cerebral artery occlusion min minute MPP- - 1-methyl-4-phenylpyridinium MPTP - 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine mRNA - messenger RNA MS -multiple sclerosis NAIP - neuronal apoptosis inhibitory protein NK - natural killer cells NMDA - N-methyl-o-aspartate NO - nitric oxide OA - osteoarthritis OxyHb - oxyhaemoglobin PAI-2 - Plasminogen Activator Inhibitor 2 P A W - poly(ADP-ribose) polymerase PBMC - peripheral blood mononuclear cell PD - Parkinson’s disease PI9 - Proteinase Inhibitor 9 PN-I - Protease Nexin 1 pro-IL-l/3 - pro-interleukin- 1 PKD - polycystic kidney disease PrP - prion protein PTP - (pyrazoly1oxy)methyl RGC - retinal ganglion cell RA - rheumatoid arthritis SAR - structure-activity relationship SMA - spinal muscular atrophy SPI-1 - Poxvirus Serine Protease Inhibitor 1 TBI -traumatic brain injury tBID - truncated BID Thl - T-helper-cell type I TNF - tumour necrosis factor TNF-R1 - TNFa receptor 1 TRADD - TNF-R1-associated death domain. ~
Progress in Medicinal Chemistry - Vol. 39, Edited by F.D. King and A.W. Oxford 0 2002 Elsevier Science B.V. All rights reserved.
2 RNA as a Drug Target MARTIN J. DRYSDALE', GEORG LENTZEN~, NATALIA MAT ASS OVA^, ALASTAIR I. H. MURCHIE~, FAREED ABOUL-ELA3 and MOHAMMAD AFSHAR4 'Departments of Chemistry, 2Drug Discovery, 3Structural Biology and Drug Design, RiboTargets Ltd., Granta Park, Abington, Cambridge, CBI 6GB. U.K.
4
INTRODUCTION
74
HUMAN IMMUNODEFICIENCY VIRUS (HIV-I) TatjTAR Structure based design Amrdino 2.5-diphphenylfurunsand D-ghCUl anulogites High throughput screening Others Rev/RRE Amrnoglyco~ i d eunalogttes Dragdamidme analoguer
75 75 71 79 81 83 83 84 85
THE BACTERIAL RIBOSOME Ribosomal function Iniliation' Elongation Terminution Ribosome binding antibiotics Antibiotics binding to the 30s Subunit Antibiotics binding to the 50s Subirnit Established sites qJ interaction New sites of interaction
88 89 90 90 92 93 93 100 101 108
SUMMARY
1 I4
REFERENCES
1 I4
13
RNA AS A DRUG TARGET
74
INTRODUCTION The central role of RNA in the transcription/translation process makes it an attractive target for small molecule drug design. In the anti-bacterial field it has been long established that several classes of drugs obtained from natural sources have been shown to work by binding to RNA or RNA/protein complexes. These include the thiopeptides exemplified by thiostrepton (l), the aminoglycosides such as paromomycin (2) and macrolides including erythromycin A (3) and clarithromycin (4). However, it is only relatively recently that RNA has been considered as a drug target for small molecules [l]. The elucidation of RNA structures, revealing a diversity of threedimensional folds [2] with specific interactions to effector ligands provides many potential drug targets. The recent publications describing the X-ray structures of both the 30s [3] and 50s [4] subunits of the bacterial ribosome, including a range of ternary complexes with existing antibiotics, provides a major impetus to the area.
Me
(1) Thiostrepton .OH
( 2 ) Paromomycin
M.J. DRYSDALE ET A L .
R
(3) R = H Erythromycin A (4) R
=
Me Clarithromycin
The majority of work which has been carried out in terms of both small molecule research and structural studies on RNA are in the areas of viral disease, particularly the human immunodeficiency virus (HIV- 1) and in the antibacterial arena. Given the issues of increasing resistance to the current HIV- 1 and antibacterial therapies, the need for novel mechanistic approaches to these diseases is pressing. RNA targets may well provide some of the answers to these problems and will be the focus of this review.
HUMAN IMMUNODEFICIENCY VIRUS (HIV- 1) The current classes of agents utilized in the treatment of HIV-1 infections are dogged by a number of problems, including undesirable side-effects [ 5 ] and resistance mutations emerging and spreading rapidly [ 6 ] . The hunt is on for alternative targets. HIV-1 gene expression is dependent upon the interaction of two virally encoded proteins, Tat and Rev, with their respective RNA substrates. Binding of Tat to the trans-activation response region (TAR) RNA activates transcription of viral mRNA by promoting efficient elongation of HIV transcripts [ 7 ] .The RevResponse-Element (RRE) RNA serves as the Rev binding site and stimulates the export of unspliced viral RNA from the nucleus [ S ] . Thus the interactions between these RNA structures and their regulatory proteins are essential for efficient viral replication and offer novel mechanisms for inhibition. Tat/TAR
HIV-1 Tat is a small basic protein ranging in length between 86 and 101 amino acids in different HIV-1 isolates. Several functional regions have been
16
RNA AS A DRUG TARGET
identified within this protein including a basic region (amino acids 48-59) containing six arginines and two lysines, and is the signature of this class of RNA-binding proteins [9]. Binding of basic peptide mimics of this region to TAR induces a conformational change in the RNA molecule. First proposed by footprinting [lo] and circular dichroism (CD) studies [ l l , 121, this conformational change was confirmed by determination of high resolution NMR structures of the free (Figure 2. l a ) and peptide-bound form (Figure 2.16) [ 131. In fact it was observed by NMR that the conformational change in TAR can be induced by argininamide [ 14, 151 and that the argininamide-induced conformation of TAR is very similar to that induced by peptides containing the full basic domain of Tat [ 131.
Figure 2.la.
TAR RNA in its unbound conformation.
M.J. DRYSDALE ET AL.
77
Figure 2.lb. TAR RNA in its peptide (Tat) bound conformation as measured by NMR studies
~31.
Structure based design Using the knowledge of the structural requirements of these basic peptides to bind to and induce the conformational change in TAR, workers at Novartis in a collaboration with the Medical Research Council’s Laboratory of Molecular Biology in Cambridge, designed a 3.2 million hybrid D-peptide/peptoid library on solid phase in order to identify low molecular weight inhibitors [ 161. Of the 20 amino acids and peptoid side-chains incorporated, the majority were heavily biased towards basic moieties. This bias was introduced because electrostatic
78
RNA AS A DRUG TARGET
interactions had been shown to be a major source of binding affinity as well as cation-x stacking interactions being important for specificity. Five positions were randomized in the synthetic scheme, and these were always capped by the four C-terminal residue sequence composed of D-LyS-D-LyS-D-Arg-D-PrONH~. In this study, binding was measured by gel electrophoresis mobility shift assay. The 20 sub-libraries were tested for their ability to disrupt the complex formed between recombinant Tat protein and [ Y - ~ ~ P I T Aduplex. R After five deconvolution cycles, a single compound with high affinity for TAR RNA, CGP64222 (5) was identified. CGP64222 was reported to require a concentration of 12nM to inhibit formation of the Tat/TAR complex by 50%. NMR studies showed that this compound induced the same conformational change in TAR RNA as that reported for ADP-1 [13], a Tat derived peptide. CGP64222 was shown to have activity in a novel cellular system called the fusion-induced gene stimulation (FIGS) assay with an ICs0 = 3-5 pM. In addition, this compound has inhibitory effects on HIV-lLAVreplication in acutely infected human lymphocytes and totally suppresses supernatent reverse transcriptase at 30 pM.
(5)CGP64222
The same Novartis group also designed a series of novel Tat/TAR inhibitors using structural information to guide their design principle [17]. The best compound exemplified was the acridine analogue CGP40336 (6). This molecule serves to exemplify the design features of this series which comprise a polyaromatic/heterocyclic moiety with the potential for stacking interactions, a feature providing positive charges for interaction with the phosphate backbone on RNA, and a spacer to connect the two. Generally, varying the spacer length from C 2 4 5 was unimportant in activity terms, but utilising an amide
M.J. DRYSDALE E T A L .
79
containing linker abrogated activity 10 to 100-fold. It also appeared that the length of the carbon chains linking the tertiary amine to the primaries did not affect activity significantly. These compounds were tested in the same gel shift and FIGS assays as described above and CGP40336 was shown to have CDSo=22nM in the gel shift assay and an ICso= 1 pM in the FIGS assay. Verification that the inhibitory activity of these compounds was due to interaction with RNA was shown by Rnase A footprinting experiments. It was seen that both protection from and enhancement of Rnase A cleavage in TAR in the presence of CGP40336 compared to TAR on its own, was similar to that observed with the Tat peptide complexed to TAR. In addition 2D NMR studies showed the same pattern of RNA signals for the CGP40336/TAR complex as that seen with Tat derived peptides.
(6) CGP40336
Amidino 2,5-diphenylfurans and D-glucal analogues Amidino 2,5-diphenylfurans have been shown to be active against Pneumocystis carinii pathogen (PCP), one of the most common causes of mortality in AIDS patients. A small series of these analogues (7-10) have been investigated for their activity against TAR RNA [ 181. In the previously described gel-shift assay, ICso values were measured at I , 5, 7 and 12 pM for compounds (7), (8), (9) and (10) respectively. Examination of the ability of these ligands to alter the thermal denaturing profile of TAR as a direct measure of binding gave the same rank order of affinities as the gel-shift assay. However in the FIGS assay, only compound (7) showed any selectivity of activity over toxicity, with an ICso = 1 pM and a selectivity index (SI) of 10. Interestingly in terms of anti-PCP activities, the compounds rank in the order (10)>(8)>>(9)>(7). Correlations between anti-PCP effects and binding to DNA or DNA-binding proteins have been seen [19], suggesting that requirements for RNA binding compared to DNA binding are quite different in this series.
RNA AS A DRUG TARGET
80
0
HNYNH
f-fN"
A series of compounds derived from tri-0-acetybglucal were described in the patent literature by workers from Allelix Biopharmaceuticals [20]. Shown in Table 2.1, the OMe compound with undefined stereochemistry at R' (1 1) and the a-OEt anomer (12) have the best activity with ICso values of 0.051 and 0.046 pM respectively. Extending this side-chain as either a polyether (13) or introducing an additional basic centre (14) results in a loss in activity. Introduction of additional hydroxyl fbnctionality at R2 and R3 to give (15) was detrimental to activity. Additionally, removal of the anomeric substituent completely abrogated activity by at least 1000-fold in the R3 substituted Table 2.1. ACTIVITY OF D-GLUCAL DERIVED GUANIDINE ANALOGUES IN A Tat/TAR ELECTROPHORETIC GEL-MOBILITY SHIFT ASSAY
Compound
R'
(11) (12) (13) (14) (15) (16) (17) (18)
H OMe H a-OEt u - O ( C H ~ ) ~ - O ( C H ~ ) ~ O EH~ u-O(CH~)~NHC(NH)NH~H a-OMe OH H H H H H H
R2
'R
Gel-shqt IC,, (pM)
H H H H OH OH OMe O(CH2)3NHC(NH)NHZ
0.05 1 0.046 0.5 0.17 1.35 55 185 110
M.J. DRYSDALE ET AL.
81
analogues ( 1 6 18). The loss of activity of the tris-guanidino compounds (14) and ( 1 8) compared to the bis-guanidines ( 1 1) and (12), shows that simply populating such structures with more basic centres does not necessarily lead to an increase in binding to RNA. High throughput screening A group at Parke-Davis have described a complete therapeutic programme targeting TAR RNA, where they utilize high throughput screening to identify inhibitors of the Tat/TAR complex [21]. As well as the previously described gel shift assay, they described two other assay types suitable for an HTS campaign towards this target. Firstly, a scintillation proximity assay (SPA) was outlined using '251-Tat12 and biotinylated TAR RNA. Secondly they described a high throughput filtration assay using 32P-labelledTAR RNA and unlabelled Tat peptide. In this assay, the protein adheres to the membrane material and so protein-bound RNA is also captured whilst the free RNA passes through the membrane. Thus in the presence of a competitor ligand the quantitation of counts on the membrane compared to controls give a value for ligand binding. The SPA and filtration assays were utilized in the parallel screening of a 150,000 compound collection, where the compounds were screened as mixtures with each component at 20 yM. A hit rate of 1-2% was claimed, and upon deconvolution, one-third of these had IC5,, values < 50 pM. In a later publication [22] the same authors identified aminoglycosides (exemplified by neomycin (19)), quinoxaline2,3-diones (e.g. (20)) and 2,4-diaminoquinazolines (e.g. (21)) as three classes of hit structures from their HTS campaign. values as measured in the gelshift assay were quoted at 0.92yM, 1.3pM and lOyM for (19), (20) and (21) respectively.
In order to determine whether these compounds bound directly to the RNA component in these assays, electrospray ionisation mass spectrometry
82
RNA AS A DRUG TARGET
(ESI-MS) was used. Molecular weights corresponding to TAR alone and the 1: 1 complexes of TAR and ligands (19), (20) and (21) were observed by ESIMS. Under similar conditions, no complexes with a Tat4o peptide were found for any of these molecules. Further studies using chemical and enzymatic footprinting studies elucidated the region of TAR targeted by each inhibitor. Compound (2 1) was further characterized for cellular activity. HeLa cells constitutively expressing Tat, were transfected by a plasmid (pHIVlacZ) containing a promoter domain from the HIV-1 3'-LTR and a lacZ reporter gene. The HIV-1 LTR promoter containing TAR RNA can be bound and activated by Tat. The lac2 gene is controlled by the LTR promoter and the products of expression, p-galactosidases, can be quantified. In this assay compound (21) has a dose response effect with an IC5,, measured at 19 pM. No apparent cellular toxicity was observed at concentrations below 100pM. In a related cell line, the HIV-1 promoter is replaced by the cytomeglovirus (CMV) promoter to measure TAR independent gene expression. Compound (21) showed no effects on the extent of gene expression driven by the CMV promoter in contrast to that observed with LTR-activated lacZ expression. HIV-1 replication studies were carried out in both OM-10.1 and Ul cell lines. In OM-10.1 cells, (21) has an EC=,o=4pM and a toxic concentration (TC)50 value of 54pM. In the U1 cell line, an ECso of 16pM was measured.
The group at ISIS pharmaceuticals have utilized a related high throughput SPA assay to that described above to identify novel piperazinyl polyazacyclophane scaffolds [23] and polyazadipyridinocyclophanes [24] inhibitors of the Tat/TAR interaction. This group used a solution phase combinatorial approach. In this strategy, the secondary amines in (22) and (24) (R' -R4 = H), are treated with a mixture of alkylating agents to give a mixture of 625 compounds in each case. For the piperazinyl polyazacyclophane analogues, the library depicted by (23) gave an IC50= 0.08 pM, and in the case of the polyazadipyridinocyclophanes (25) and (26), IC50 values of 0.08 pM and 0.09 pM respectively were measured. In each case, related libraries with less polar or
M.J. DRYSDALE ET AL.
83
fewer basic functionalities were less effective at disrupting the Tat/TAR interaction. NH (24) R1-R*=H
0
NC
II
NH
Others
Hoechst 33258 (27), a bisbenzimidazole derivative, was shown by a variety of techniques to bind to TAR [25]. Using a combination of UV absorption studies, thermal denaturation, circular dichroism (CD), electrical linear dichroism (ELD) and RNase A footprinting, this ligand was shown to be a tight binder of TAR with specific contacts in the bulge region of the RNA.
( 2 7 ) Hoechst 33258 Rev/RRE
RRE RNA is a 234-nucleotide RNA sequence located within the env gene of HIV and is regulated by binding to Rev protein [26, 271. Biochemical studies have identified a high affinity Rev binding site in the stem-loop region of stem IIB of RRE [28, 291. Model constructs based on this region of RNA, and the Arg-rich binding domain of the Rev protein (Rev34-50and related peptides) have been used in a number of studies to look at small molecule interactions in this system.
RNA AS A DRUG TARGET
84
Aminoglycoside analogues Using methods such as surface plasmon resonance (SPR) [30], Neomycin B (28), has been shown to be the most effective inhibitor of Rev/RRE binding with an ICso in the region 0.1-1 pM. Closely related analogues such as paromomycin (2) have been shown to be significantly less active by this method. SPR is a useful technique that allows direct observation of ligand-RNA interactions and can be used to test for specificity. 5TRQARRNRRRRWRERQR
ooc 0 OH
H2N
(28) Neornycin B
(29) RhdRev
In a more recent study [31,, a tetramethylrhodamine labellei Rev34-50 peptide, RhdRev (29) was prepared which upon binding to RRE results in an increase in fluorescence anisotropy of RhdRev. Competition by an antagonist results in decreased fluorescence anisotropy and dissociation constants measured. Ten aminoglycosides were studied and their Kd values are shown in Table 2.2. Consistent with previous data, neomycin B is the most potent compound with a Kd = 4.4pM. All the other aminoglycosides had Kd values
Table 2.2. DISSOCIATION CONSTANTS OF AMINOGLYCOSIDES FOR RRE IIB RNA Aminoglycoside
Kd (PM)
Gentamycin Hygromycin B Kanamycin A Kanamycin B Neomycin B (28) Nearnine (30) Parornomycin (2) Ribostamycin Streptomycin Tobramycin
34.6 248 46.0 14.4 4.38 20.8 34.5 87.5 200 15.5
M.J. DRYSDALE ET AL.
85
Table 2.3. DISSOCIATION CONSTANTS OF AROMATIC RING SUBSTITUTED NEAMINE ANALOGUES FOR REZE IIB RNA
Inh ibiiors
Kd (@)!
20.8 141 40.6 3.6 0.240 0.753 0.277
>I0 pM. Of note is the value for neamine (30), Kd = 20.8 pM. Modification of this simplified scaffold by incorporation of groups capable of intercalating with the groove or stacking with bases (3 1-36) results in inhibitors with significantly improved activity (Table 2.3). The loss of activity of (31) and (32) compared to (30) suggests that the benzoyl and 2-naphthoyl groups may not stack well, and that substitution of the primary amine is detrimental to binding. However, introduction of the anthracene-9-carbonyl group (33) results in a compound with six-fold higher activity than the parent neamine (& = 3.6 pM vs. 20.8pM). Introduction of a pyrene unit had even more significant effects. Depending on linker length, Kd values of 0.240, 0.753 and 0.277 pM respectively were measured for (34-36) respectively. In a related communication [32], neomycin B was linked to 9-aminoacridine to give (37). In a gel-shift mobility assay IC50 values for (28) and (37) were measured at 5.9 and 0.65 pM. This communication also described a fluorescence anisotropy assay related to that described above, where a C-terminal fluorescein-labeled Rev34-50 analogue was displaced from RRE by inhibitors. In this assay paradigm, competition experiments gave IC50 values of 0.8 pM and 0.015 pM for (28) and (37) respectively. Additionally enzymatic footprinting studies in this system confirmed direct binding of (37) to RRE RNA.
Diarylamidine analogues A series of diphenylfuran bisamidines related to structures (7-10) were reported to inhibit Rev/= binding [33]. ICS0values were measured in a gelshift assay and compound (38) (Table 2.4), ICsO<1 pM, was the most potent inhibitor in this series. Interestingly, shortening the chain length linking the N,N-dimethy lamine to the amidine was detrimental to activity, comparing (38) to (39), and removal of the N,N-dimethylamine completely, abrogates activity by up to >lOO-fold as shown with (40).
RNA AS A DRUG TARGET
86
(30) R = H
(34)R =
(33)R=
(
'
3
6
)
R / -/ 4
/
Table 2.4 DIPHENYLFURAN BISAMIDINES IN GEL-SHIFT Rev/RRE ASSAY
Compound
R
Gel-shift IC,, (pM) <1
t-
NH(CH,),NMe,
5
100
M.J. DRYSDALE E T A .
87
In a later study by the same group a broader SAR was described utilizing (38) as the starting point for modification [34]. The data for these analogues are outlined in Table 2.5 with neomycin B (28) and (38) used as the reference compounds. It should be noted that the IC50 values in Table 2.5 are higher than those reported previously for (28) [35] and ( 3 8 ) (Table 2 . 4 ) and is due to an increased assay stringency in this study. Replacement of the amidines by a non-basic amide (41) resulted in a serious loss of Table 2.5 Rev/RRE INHIBITORS MEASURED BY GEL-SHIFT ASSAY
0
CH
0
CH
NH
CH
-Q
(43)
S
CH
-Q
(44 )
0
N
43-
(45)
0
N
c-
1-3
0
CH
c-
<1
'NA - Not applicable
NH(CHJ2NMe,
3-10
+ NH(CH,),NMe,
c-
NH(CH,J>NMe2
NH(CHJ,NMt,
NH(CHJ,NMe,
NH(CH,),NMe,
>30
3-1 0
1-3
88
RNA AS A DRUG TARGET
activity, with very little inhibition seen at 30pM. A range of modifications of the central furan core was reported. The pyrrole (42) was significantly less active than the furan (38), with an ICSo>30pM. However, the thiophene (43) had similar activity to (38) and the oxazole (44) was an improvement with an ICso between 1 and 3pM. Replacing one of the phenyl rings of the oxazole (44) with a furan ring gave (45) which was equipotent. However, in the case of (46), introduction of the fused biaryl benzimidazole as a phenyl replacement gave the most potent compound in this series with an 1 pM. This increase in activity may originate from the hydrogen bonding ability of the ring nitrogens, the shape of the system and/or the increased stacking interaction from the benzimidazole ring. A more detailed study of (46) using enzymatic footprinting, UV-visible and fluorescence spectroscopy, SPR and a series of 2-D NMR experiments indicated that this compound binds to RRE in a highly structured and cooperative complex at a 2:l (46) to RRE ratio [36]. A very closely related series of compounds (47) has also been recently described in the patent literature [37]. Structures such as (47) have a 1,3relationship between the central ring and the amidine side-chain compared to the 1,4-substitution pattern seen in examples such as (38) and (4244). Of the analogues described in the patent, compound (48) was the most potent with an ICSo=0.75pM as measured in an inhibition filter binding assay of the Rev/RRE interaction.
THE BACTERIAL RIBOSOME: A BILLION YEAR OLD ANTIBIOTIC TARGET The development of protein biosynthesis must necessarily be regarded as one of the earliest and most significant biological events. In all living organisms
M.J. DRYSDALE ET AL.
89
decoding of the genetic information is performed by special cellular components - ribosomes. The ribosome is a large macromolecule with a complex and asymmetrical tertiary structure. Ribosomes from different kingdoms vary in the size and number of components. All ribosomes have two subunits made of both ribosomal proteins (r-proteins) and ribosomal RNA (rRNA), which accounts for two-thirds of their mass. Ribosomes are ubiquitous in the cell, making up about 80% of the cellular RNA. The bacterial ribosome from Escherichiu coli (70s) is the most extensively characterized. It has a molecular mass of 2.8 MDa and consists of about 4,500 RNA nucleotides and 55 proteins. The small (30s) subunit has one rRNA molecule (16s) and 21 r-proteins. The large (50s) subunit is made of two rRNAs (23s and 5s) and 34 different proteins. The three-dimensional structure of the ribosome was exhaustively studied by neutron scattering, electron microscopy and by footprinting and cross-linking approaches [38-42]. The structures of 70s ribosome [43,44], 30s [3a, 3c, 451 and 50s [4a, 461 ribo:omal ybunits tave been resolved recently by X-ray crystallography at 5.5 A, 3.4 A and 2.4 A resolution, respectively. To build a protein, the ribosome needs the following: (i) a message which codes for a protein; (ii) the protein building blocks, amino acids; (iii) energy. All these three components (information, material and energy) meet each other on the ribosome. The ribosome itself contains an enzymatic activity - it catalyses the formation of a peptide bond and, therefore, performs a polymerization of aminoacyl residues into the polypeptide chain of a protein. It is evident that the detailed information about the three-dimensional structure of the translational components and their relative movements during the protein synthesis can be revealed only by a combination of structural and functional analysis. The crystal structures of the ribosome with bound antibiotics [3b, 4b, 471, initiation factor IF1 [48], mRNA and tRNAs [43, 441 have revealed a remarkable example of interactions between these molecules. Cryoelectron microscopy reconstructions of the functional complexes modelling different steps of protein synthesis give low resolution structural details of the interactions between ribosome, translation factors, mRNA and tRNAs during protein synthesis never seen before [39, 40, 4!&52]. The protein synthetic machinery is a target for a large number of antibiotics, naturally occurring low molecular weight metabolic compounds. The ribosome appears to be the major target for them. Therefore the knowledge of the molecular basis of protein biosynthesis is important in understanding the precise mechanism of the inhibitory action of different drugs [53]. RIBOSOMAL FUNCTION
Ribosomes were discovered in 1940, a minimal model for a translational cycle was established in the early 60s, and there are many reviews and papers on the
90
RNA AS A DRUG TARGET
subject ofribosomal structure and function [41,6547,73,79,81] although much remains to be learned about the detailed mechanism of translation. Protein synthesis on the ribosome can be divided into the three major steps - initiation, elongation and termination. Polymerizing one amino acid after another into the peptide, the ribosome interacts with a large number of ligands such as mRNA, tRNAs (deacylated, aminoacyl- and peptidyl-), different protein factors etc. There are three major functional sites on the ribosome-A (acceptor-), P (peptidyl-) and E (exit). During translation the ribosome undergoes significant conformational changes and, by doing so, synchronizes the order of actions of all participants.
Initiation The first step of translation is the formation of the initiation complex ready to enter the elongation phase of translation. Initiation of protein synthesis in bacteria involves several steps for bringing together the 70s ribosome, the initiator Wet-tRNAme' and the translation initiation region of the mRNA. The conserved polypurine sequence, which is located 3-1 0 nucleotides in the 5'direction from the first codon ('Shine-Dalgarno' (SD) sequence, [54]) interacts with the complementary (anti-SD) sequence at the 3'-end of 16s rRNA (nucleotides 1535-1539 in archaea). It helps to expose the first codon (AUG) into the ribosomal P-site where it binds fMet-tRNAme'. Three initiation factors - IFI, IF2 and IF3 are required for the formation of the correct initiation complex. All three factors participate in the formation of 30s initiation complex between 30S, initiator tRNA and mRNA. IF2 binds to the 30s subunit and promotes the initiation codon-dependent binding of Met-tRNAme' [55]. IF2 stimulates the association of 30s and 50s subunits as well [56]. During the transition from the 30s to the 70s initiation complex, initiation factors IF1 and IF3 are ejected from the ribosome, IF2 hydrolyzes GTP and dissociates from the ribosome. The acceptor end of initiator Wet-tRNAtMc' is finally fitted into the P-site and becomes a substrate for the ribosomal peptidyltransferase [57-591. As a result the first peptide bond can be formed between W e t and the aminoacyl residue of the aa-tRNA bound to the second coding triplet in the A-site [55c].
Elongation The sequence of events during protein chain elongation has been uncovered on the basis of biochemical, footprinting and cross-linking data obtained by many groups over the last 30 years [41, 42, 6 M 2 , 791. During the elongation phase, the ribosome polymerizes amino acids from aminoacyl-tRNAs according to a mRNA template. The template is moved by one codon after each round of elongation. The simplified scheme of the elongation cycle is shown in Figure 2.2. It consists of three major steps. Elongation of a peptide chain
M.J. DRYSDALE ET AL.
91
begins when a ribosome contains mRNA, peptidyl-tRNA (or initiator tRNA) in the P site and a free codon in the A site (see before). During the first step, an aminoacyl-tRNA (aa-tRNA) is delivered by the elongation factor Tu (EF-Tu) to the ribosome as a ternary complex EF-Tu/GTP/aa-tRNA. If aa-tRNA does correspond to the mRNA codon exposed to the A-site, decoding takes place and
Figure 2.2. Elongation cycle on the ribosome. The siniplifed scheme of the translational elongation cycle. 70s ribo.some is divided into 30s and 50s subunits. Three tRNA binding sites - the A (aminoac.vl-). P (pepti&-) and E (exit) sites are represented as rectangles. tRNAs are shown as vertical bars (deucyluted tRNA is a bar with OH-group on a top) and amino acid by a small ball. mRNA i s represented as a red line bound to the 30s subunit. Initial!v the P site is,filled with peptidyl tRNA. Ternmy complex aa-tRNA* elongation factor Tu (EF-Tu)* GTP binds to the ribosomal A site. Following mdon recognition. GTP is hydrolyied, EF-Tu(GDP) is released, and aa-tRNA enters the A site. Trcmspeptidation takes place immediate!v. Elongation factor C (EF-G) * GTP hinds to 70S, h,vdro!v,ses GTP and promotes the translocation. As a result ofthis movement E - and P-sites are.firl1.v occupied by tRNAs, and an empty A-site is able to accept aa-tRNA.
92
RNA AS A DRUG TARGET
aa-tRNA binds to the vacant A site in a codon-dependent manner. After the codon recognition GTP hydrolysis takes place, EF-Tu undergoes drastic conformational changes and leaves the ribosome as EF-Tu/GDP. After accommodation the 3’-ends of two tRNAs (P-site bound peptidyl-tRNA and A-site bound aa-tRNA) are arranged in the close vicinity, and the peptide bond forms immediately. Transpeptidation is an internal enzymatic function of the ribosome, it happens even in the absence of translational factors. Extensive biochemical, genetic and crystallographic data suggest that peptide bond formation takes place in the so-called peptidyltransferase centre of the 50s ribosomal subunit which is formed mainly by a part of highly conserved domain V of 23s rRNA [4b, 43, 44, 61, 63, 641. During the peptide bond formation the nucleophilic primary amine of the aa-tRNA in the A-site attacks the electron deficient aminoacyl ester bond of the peptidyl tRNA in the P-site. It should been mentioned that instead of aa-tRNA just a 3’-end fragments of aa-tRNA, (even the aminoacyl ester of adenosine) or the antibiotic puromycin can participate as an acceptor (nucleophil-) substrate in the ribosome-catalyzed reaction of transpeptidation [4b, 651. As a result, the growing peptide chain is transferred to the A-site yielding a deacylated tRNA in the P site and a peptidyl-tRNA in the A site. The elongation cycle is completed by translocation, which is catalyzed by elongation factor G (EF-G) with GTP. Each time a new peptide bond has been formed, the mRNA together with peptidyl-tRNA and deacylated tRNA bound to it is moved along the ribosome by one codon. During translocation, the peptidyl-tRNA is displaced from the A site to the P site, while the P site-bound deacylated tRNA is moved to the E (exit-) site and spontaneousely dissociates from the ribosome [66, 671. Termination The ribosome starts translation on the initiator codon AUG, reads mRNA one triplet after another and finally reaches UAA, UAG or UGU codon. These are terminator codons - there are no aa-tRNAs that correspond to them. The protein chain elongation stops there. Three protein factors (RF1, RF2 and RF3) are involved in the termination in prokaryotes. When the terminator codon appears in the ribosomal A-site, RF1 and RF2 recognize it and activate the peptidyl hydrolase centre of the ribosome. As a result the ester bond linking the nascent peptide to the 3’-end of the tRNA in the P-site bound peptidyl-tRNA is hydrolysed [68, 691. After peptidyl-tRNA hydrolysis the RF3 binds to the ribosome and in a GTP-dependent way and catalyses the dissociation of RF1 and RF2 from the ribosomal A-site [70]. All bound components - mRNA and tRNA, leave the ribosome, 70s dissociates into the subunits which can start a new cycle.
M.J. DRYSDALE E T A .
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RIBOSOME BINDING ANTIBIOTICS
A large number of antibiotics inhibit protein synthesis and the majority of these drugs act on the ribosome. In all cases studied so far, ribosomal RNA plays the key role in binding and recognition of these antibiotics. Despite the enormous size of the ribosome (Figures 2.3, 2.4 and 2.5), only a relatively small number of different antibiotic binding sites have been identified. These sites coincide with the key functional regions of the ribosome (Tables 2.6 and 2.7). NOWthat X-ray structures of ribosome-antibiotic complexes have become available, there is a realistic prospect for the rational design of improved and novel antibiotics. In particular, utilizing the X-ray co-ordinates, a global and detailed picture of known antibiotics binding to both the 30s (Figures 2.6 and 2.7), and the 50s (Figures 2.8 and 2.9) subunits can be obtained. Antibiotics binding to the 30s subunit
Many aminoglycoside antibiotics like paromomycin (2), neomycin (19), kanamycin and hygromycin B (49) act by binding to the 30s decoding site and cause misreading of mRNA resulting in the incorporation of the wrong amino acid. Best studied is the interaction of paromomycin (2) with the decoding centre of 16s RNA (Figure 2.3). Structural analysis by NMR [7 11 and recently X-ray crystallography [3b] of paromomycin (2) bound to 16s rRNA have led to a detailed understanding of the paromomycin-rRNA interaction. Kinetic studies revealed how paromomycin (2) causes misincorporation of amino acids by altering the conformation of the decoding site [72]. Hygromycin B (49), a universal translational inhibitor, binds close to the paromomycin binding site. The X-ray structure of the 30s-hygromycin B complex [47] reveals a binding site on helix 44 partially overlapping but distinct from the paromomycin site.
HO.
(49)
94
RNA AS A DRUG TARGET
Figure 2.3. Secondary structure representation of 16s rRNA, highlighting antibiotic binding sites. Four aniinoglycosides (paromomycin [3b]), blue, hygromycin B [47] light green, and streptomycin [47], black and spectinomycin [3b], red) are included, along with tetracycline [47] (dark green primary site, and pink - secondary site) and pactamycin [47] (yellow). Residues which are within five angstroms of the ligand in question [3b. 471, as defined by the X-ray coordinates, are highlighted for each antibiotic binding site. This Figure and Fig. 2.4 and 2.5 are mod$cations o f a diagram from the website of Robin R. Gutell, University of Texas. U.S.A..http://www.rna.icmb.utexas.edu/, Gutell R. R.. Cunnone J. J., Shang Z., Du Y. and Serra M. J. (2000) J. Mol. Biol. 304, 335-354.
M.J. DRYSDALE ET A L .
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Secondary Structure: large subunit ribosomal RNA - 3 half
Figuse 2.4. Secondary structure representation oj'the 23s rRNA. 3' half: highlighting chemical or enqmatic footprints associated with three classes of antibiotics. The lincosumides [ I 2 71, on relestiretin (red), nzacrolides [I281 jrom en~thromycin(blue) [I231 and oxazolidinones [I 13c] illustrated by linrzolid (vellow).
96
RNA AS A DRUG TARGET
-
Secondary Structure: large subunit ribosomal RNA 5’ half
Figure 2.5. Secondary structure representation of’ the 23s rRNA, 5’ half: highlighting resistance mutation sites associated with two classes of antibiotics. The thiazole [61b, 1261 site is based on data for thiostrepton (green) and oxazolidinones [113c] illustrated by linezolid (vellow).
M.J. DRYSDALE ET AL.
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Table 2.6 SELECTIVITY AND BINDING SITE OF 30s RIBOSOMAL SUBUNIT BINDING ANTIBIOTICS
Antibiotic Paromomycin (2)
Class
Aminoglycoside/ Aminocyclitol Hygromycin B (49) Aminoglycoside/ Aminocyclitol Streptomycin ( S O ) Aminoglycoside/ Aminocyclitol Spectinomycin (5 I ) Aminoglycoside/ Aminocyclitol Tetracycline (52) Tetracycline Pactamycin (53) Pactamycin group Kasugamycin (54) Aminoglycoside/ Aminocyclitol Peptide Edeine (55)
Selectivit))' Site of interaction
Reference'
Pro
Helix 44 (decoding site)
[3b]
Pro/eu
Helix 44
[471
Pro
A site
[3b1
Pro
Helix 34
[3b1
Pro Pro/eu Pro
Helix 34, Helix 31 (A site) Helices 23b, 24a P site
[47] [471 [821
Pro/eu
P site
[791
'Pro = prokaryote specific; eu = eukaryote specific; Pro/eu = active against both pro- and eukaryotes 'The references refer to identifications of the site of action of the ribosome
98
RNA AS A DRUG TARGET
Table 2.7 SELECTIVITY AND BINDING SITE OF 50s RIBOSOMAL SUBUNIT BINDlNG ANTIBIOTICS Antibiotic
Class
Selectrvity’ Site of interaction
Chloramphenicol (57) Erythromycin (3) ABT-773 (62) Lincomycin (64) Dalfopristin (65) Quinupristin (66) Pleuromutilin (69) Puromycin (56) Blasticidin S (86) Cycloheximide (87) Narciclasine (88) Anisomycin (89) T2 toxin (90) Thiostrepton ( 1 ) Linezolid (68) Viomycin (67) Evemimicin
Chloramphenicol group Macrolide Ketolide Lincosamide Streptogramin A Strcptogramin B Pleuromutilins Nucleoside derivative Nucleoside derivative Glutarimide Alkaloid Pyrrolidine derivative Sesquiterpene Thiazole peptide Oxazolidinone Peptide Oligosaccharide
Pro Pro Pro Pro Pro
Pro Pro Proleu Proleu Eu Proleu Proleu Eu Pro Pro Pro Pro
Reference2
PTC3 1851 PTC3 ~231 PTC’ [104, 1051 PTC’ ~ 4 1 PTC’ [881 PTC? ~851 PTC3 [ 1241 PTC3 [4b1 PTC3 1851 PTC’ [ 1251 PTC3 ~ 5 1 PTC3 [851 PTC3 1851 GAR4 [ 1261 E site (LI binding site) [113] Helix 38 [ I 101 Helices 89, 91 [1121
’Pro = prokaryote specific; Eu = eukaryote specific; pro/eu = active against both pro- and eukaryotes 2The references refer to identifications of the site of action of the ribosome 3PTC peptidyl transferase center 4GAR GTPase activating region ~
~
R = Ii Edeiiir A
(55)
Streptomycin (50) is an aminoglycoside that binds to a site in close proximity to the decoding centre. Like paromomycin (2), streptomycin (50) causes misreading but also interferes with the binding of aminoacyl-tRNA to the A site. X-ray crystallography [3b] shows how helices 27, 18 and 44 together with ribosomal protein S 12 build the complex streptomycin binding site. Streptomycin (50) probably acts by interfering with a conformational switch
M.J. DRYSDALE E T A L
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Figure 2.6. Ghhal view of the 30s ribosomal sub-unit structure (dark blue) with 6 antibiotics bound: spectinomycin (red), tetracycline (light blue), streptomycin (white), paromomycin and hygromycin B (green). pactamycin (orange).
that occurs during translation in helix 27 [73], thus fixing the 30s subunit in an error-prone conformation. Spectinomycin (5 1) is an aminocyclitol antibiotic that binds to the top of helix 34 of 16s RNA. It does not promote misreading but inhibits tRNA translocation by interfering with EF-G function [74]. Tetracyclines (52) were the first broad-spectrum antibiotics and have been extensively used in medicine [75]. Tetracycline (52) blocks binding of aminoacyl-tRNA to the A site [76 and references therein]. The binding of tetracycline (52) to a primary and multiple secondary sites has been described [77, 781. A primary and secondary binding site have now been identified by X-ray crystallography [47] and correspond to nucleotide positions identified by chemical footprinting [79]. The primary site is close to the binding site for A site tRNA whereas the secondary site is in the helix 27 switch region. Pactamycin ( 5 3 ) is a universal translational inhibitor that acts on the initiation step. It has been shown to be in contact with universally conserved positions (3693 and C795 [47]. Pactamycin (53) inhibits initiation and interferes with P-site binding during initiation [SO] and might interfere with the function
100
RNA AS A DRUG TARGET
Figure 2.7. Ribbon representation of the RNA structure of the decoding region of the 30s with the 6 antibiotics, from Figure 2.6 represented in van der Waals spheres.
of IF-3, which binds to the pactamycin binding site [81]. The aminoglycoside kasugamycin (54) and the oligopeptide edeine ( 5 5 ) have been mapped to sites close to but distinct from the pactamycin site [79, 82, 831. Antibiotics binding to the 50s subunit Most antibiotics acting on the 50s subunit target the peptidyl transferase centre (PTC). The PTC is made up entirely of RNA and is formed by a complex folding of the central loop of domain V of 2 3 s rRNA (Figure 2.4). Drugs binding to the PTC show a huge diversity of chemical structures as well as modes of action. Binding sites for many antibiotics have been mapped by
M.J. DRYSDALE ET AL.
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Figure 2.8 Global view of the 50s ribosomal subunit structure (green). The RNA bases that have been shown to inreract with antibiotics celesticetin (red) and erythromycin (2) (blue) are also represented wich vuii der W a d sspheres. Overlappinginteraction regionsf o r the 2 antibioticsare sho wn in white. Note the clustering of the contacts around the ‘peptidyltransferase center’ of the ribosome.
chemical footprinting and cross-linking [64, 84-9 11 and reveal characteristic contacts to the central loop of domain V in 23s rRNA (Figure 2.4).
Established
sites
of interaction
Puromycin (56), a universal inhibitor, mimicks the 3’-end of aminoacyl-tRNA and binds to the 50s A site. The peptidyl transferase catalyzes the formation of peptidyl-puromycin which is released from the ribosome. Puromycin (56) has been extensively used in the biochemical studies of peptidyl transferase reaction and in the affinity labelling of the components of the PTC.
I02
RNA AS A DRUG TARGET
Figure 2.9. Ribbon diagram of the RNA structure of the 50s structure focused on the peptidvl transferase centre.
Chloramphenicol (57), like many other PTC inhibitors, contains residues mimicking a peptide bond, but its mechanism of action is distinct from puromycin (56). It probably acts by disturbing the proper orientation of the CCAend of A-site tRNA in the PTC [76].
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A large group of antibiotics interacting with the PTC are the macrolides which all contain a lactone ring of 12-16 atoms. The larger members of this group like spiramycin and tylosin (both with a 16-membered lactone ring) inhibit the peptidyl transferase activity. The smaller macrolides including erythromycin (3), do not directly inhibit peptidyl transferase but promote dissociation of peptidyl-tRNA [92]. Compounds of this type such as erythromycin (3) and clarithromycin (4) are well established orally active antibiotics which are safe and effective treatments of respiratory tract infections [93]. However, erythromycin (3) is rapidly degraded under acidic conditions in the stomach and results in poor oral bioavailability and gastrointestinal (GI) side-effects [94]. Though clarithromycin (4) has better acid-stability and therefore improved oral bioavalability, pharmacokinetics and GI tolerence, all 14-, 15- and 16-membered macrolides have several drawbacks. They are inactive against macrolide, lincosamide and streptogramin B (MLSB)resistant streptococci and Streptococcus pneumoniue [93, 951 with resistance of S. pneumoniue to erythromycin increasing from 5% to 40% in recent years [96].
a
(58) Azithromycin
'
(59) A-66321
/
N
(60)X = NH, Hh4R3004 (RU004) (61) Telithromycin
I04
RNA AS A DRUG TARGET
Additionally, macrolides with the exception of azithromycin (58), are weakly active against Haemophilius injuenzae [93, 971.
(63)2-Fluoro-6-0-propargylKetolide (62).
A number of modifications of macrolides have been made, but only the series exemplified by the carbamate A-6632 1 (59), showed moderate activity against MLSB- resistant strains [98]. A major step forward in this area arrived with the identification of the ketolide structure, whereby the cladinose moiety was removed and the 3-keto group introduced. This led to the identification of HMR3004 (60) [99] as the prototype ketolide which showed potent activity against a wide range of macrolide resistant strains in vitro as well as having in vivo efficacy when administered by the oral route in a number of infection models [99]. NMR studies using 2D transferred nuclear Overhauser effect spectroscopy (TRNOESY) of (60) in solution and bound to E. coli ribosomes were carried out [loo]. This study showed that HMR3004 (60) does interact with ribosomes, specifically to the 50s subunit. Further studies in this series led to the identification of HMR 3647, telithromycin (61) [ 1011, which is now in late stage clinical trials. Telithromycin was very active against erythromycin-resistant (MLSB constitutive type) S. pneumoniae, very potent against both resistant and sensitive Gram positive pathogens (MJC’s between 0.024.15 pg/mL) and comparible to azithromycin against H. inpuenzae.In acute lethal infection models, telithromycin was effective against a range of erythromycin sensitive and resistant strains [ 1011. TRNOESY experiments have confirmed binding of telithromycin to ribosomes [ 1021. Structurally related analogues have been described by workers at Abbott, where elaboration of the 6-hydroxyl substituent led to the identification of ABT-773 (62) [ 1031. In mouse protection tests, ABT-773 demonstrated improved efficacy against macrolide-susceptible strains as compared to the reference macrolide azithromycin and telithromycin. ABT-773 was also an improvement on the same compounds in the rat lung infection model, against
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Table 2.8 IN VIVO EFFICACY OF ABT-773 (62), TELITHROMYCIN (61) AND AZITHROMYCIN (58) IN RAT LUNG INFECTION MODELS (EDSo, mg/kg/day)
S.pneumoniae
S.pneumoniae 5649'
6303" Conipound
MIC (pg/mL)
EDro (95% CL)
ABT-773 Telithromycin Azithromycin
0.004 0.004 0.12
<0.63 2.3 6.0
MIC (/ig/mL) 0.25 0.5 16
S.pneumoniae 639@
EDso (95% CL)
MIC (pg/mL)
EDSO (95% CL)
7.0 25.8 78.7
0.015 0.125 2128
1.6 26.7 >I00
"S.pneumoniue 6303 is an erythromycin-susceptible strain, S.pneurnoniae 5649 is an effluxresistant strain, and S. pneumoniae 6396 is a MLSn-resistant strain. CL, confidence limits.
a macrolide-susceptible strain, S. pneumoniae 6303, an efflux-resistant strain, S. pneumoniue 5649, and an MLSB-resistant strain S. pneumoniae 5649, Table 2.8. Preliminary radioisotope studies showed that ABT-773 competed with erythromycin (3) or itself for ribosomal binding sites on the surface of the ribosome [ 1041. Chemical footprinting studies with DMS indicated that ABT773 protected the same nucleotides A2058 and A2059 in domain V of 23s RNA, similarly to erythromycin [105]. Additionally this compound was able to bind to methylated ribosomes, though at lower affinities than with wild-type ribosomes, and accumulated in S. pneumoniae strains with the efflux-resistant phenotype [ 1051, thus circumventing two significant modes of resistance seen with conventional macrolides. A related series of 2-fluoro-6-0-propargyl- 1 1,12-carbamate ketolides (63) have also been reported [106], where R=aryl or acyl-aryl. No biological activity of these analogues was reported in this communication. Recently, the mycarose sugar moiety present in tylosin and spiramycin has been found to be essential for inhibition of peptide bond formation [91]. The linconiycins, exemplified by lincomycin (64), bind to a site overlapping with the chloramphenicol and macrolide binding site and biochemically share many properties with the macrolides [76].
RNA AS A DRUG TARGET
106
A very interesting group of PTC inhibitors are the streptogramins which are produced as a mixture of two unrelated compounds, type A and type B. They act synergistically, binding to distinct sites in the PTC region. A mixture of the modified streptogramins A and B, Dalfopristin (65)/Quinupristin (66) has recently been approved as Synercid'" .
le
6'
The GAR region is located in domain I1 of 23s rRNA and includes the bases 105&1108. This region is the site at which the ribosomal protein LI 1 binds. In
the secondary structure, this region contains a number of RNA motifs, including a helical junction and two stem loops [107, 1081. This relatively small region of the RNA can fold independently and adopt a similar structure to that found in the w$ole ribosome. A co-crystal of the L11 RNA complex has been solved to 2.5 A resolution [107, 1081. In the presence of L11 protein the RNA folds to adopt a complex fold, in which the two helical domains associate, hinging about the helical junction. The two stem loops of the GAR RNA include the bases A 1067 and A 1095 that have been identified by chemical footprinting techniques as positions on the RNA involved in the binding of the thiazole antibiotics thiostrepton ( 1) and micrococcin. Thiostrepton exhibits exquisite selectivity in in vitro translation assays (Figure 2.10); it is inactive in eukaryotic translation but has an ICso of 50nM for prokaryotic translation. Thiostrepton ( I ) binding affects ribosome function by inhibiting tRNA translocation and also turnover of ribosome dependent GTP hydrolysis by elongation factor G(EFG) [66]. In contrast micrococcin has been reported to increase the rate of ribosome dependant GTP hydrolysis by elongation factor G(EF-G) [ 1091. Thiostrepton and micrococcin are relatively large cyclic peptide-derived molecules (thiostrepton for example has a molecular weight of 1600). They
M.J. DRYSDALE ET AL.
107
Ecoli translation 1nM
00
I
-85
I
I
-7 5
-6 5
.---
-
-1
-55
I
-4 5
[log Thiostrepton ]
RRL(eukatyot1c) translation
-8
-6 -5 [Log Thiostrepton ]
-7
Figure 2.10. hhibitory uctivity profile of thiostrepton ( I ) in an in vitro coupled transcription/ trunslation luci/krase assay. The assyv MJUS either performed with an (A) prohiyotic (E. coli MRE60Oj or (B) eukatyotic (rabbit rrticulocvte ly.sute) translation system. Translation is qirantiJied by measuring the luminescence activity ojthe translation product Firefly luciferuse. Data obtained were from three independent meusurements. The IC,, value for bacterial translation was obtained fromfitting the data to a sigmoidul dose-response equation. In the eukaryotic translation system, the highest titrution point corresponds to 50 p M thiostrepton.
,
are highly insoluble, which makes them inappropriate for general use as antibiotics. Nevertheless, the activities of these compounds validate the GTPase centre as a selective ribosomal target for the development of translational inhibitors. Another translocation inhibitor is the basic peptide Viomycin (67) which binds to helix 38 of 23s RNA [110] to a site involved in subunit association [ 1 1 I].
RNA AS A DRUG TARGET
108
H , N ~
New sites of interaction
New sites of drug interaction on the 50s subunit have recently been identified. Evernimicin is an oligosaccharide antibiotic that interferes with the function of initiation factor IF-2 [112]. It binds to a novel site on the 50s subunit involving helices 89 and 91 and ribosomal protein L16.
F (68)
The oxazolidinones represent a completely new class of ribosome binding antibiotics. They are the first chemically synthesized class of ribosome-binding antibiotics identified to date which have reached the clinic. The oxazolidinone linezolid ( Z y v ~ x " ~(68) ) [113a, b] has recently been approved as a drug for treatment of gram-positive infections. The exact mechanism of action of the oxazolidinones is unclear and their likely mode of action emphasises the subtle forces that contribute to a functional bacterial ribosome. Like the majority of ribosome binding antibiotics studied so far, the main ribosomal target for the oxazolidinones appears to be ribosomal RNA. Binding sites of oxazolidinones have been identified on 70s by both U.V. induced cross-linking and DMS footprinting to both 16s and 23s rRNA [ I 13cJ. These direct binding experiments have distinguished highly conserved residues in domain V of 23s rRNA adjacent to the binding site of protein L l and close to the 3'-end of E (exit) site bound deacylated tRNA. Interestingly, rRNA mutations that confer resistance to the drug linezolid are also localised in domain V, close to the peptidyl transferase centre [114]. The oxazolidinones are the subject of extensive literature [ 1 13d and references therein] and will not be reviewed further.
M.J. DRYSDALE E T A L .
109
(69) Pleuromutilin
(70)Tiamulin
Pleuromutilins (exemplified by pleuromutilin(69)), were initially shown to bind specifically to the 50s ribosomal subunit [ 1 151 and selectively inhibit bacterial synthesis by interaction at a unique site on the prokaryotic ribosome. Liberation of the free OH at C-14 leads to inactive compounds, but further modification of the C- 14 was investigated [ 1 161. Replacing the hydroxy group of the glycolic ester at C-14 by an alternative heteroatom-linked group gave improved anti-microbial activity and led to the discovery oftiamulin (70) which is used as a vetinary antibiotic. More recently a range of 14-O-carbamoyl(71,72) derivatives have been described in the patent literature [ 1 17, 1181, where it was suggested that reduction of the C-12 ally1 group to ethyl had no effect on activity. The later patent [ 1 181, a selection from the first [ 1 171, describes a series of azabicyclic carbamoyloxy mutilin derivatives, exemplified by the 1-aza-bicyclo[2,2,2]oct-4-yl analogues (73, 74). No data was supplied with this filing. A later patent [ 1 191 describes analogues where R4 and some combination of Ra and/or Rbare aza-mono- or bicyclic moieties (Figure 2.11). Those reported with data are tiamulin-like (75-77) and are revealed in Table 2.9. They all show an improved profile, compared to pleuromutilin and tiamulin, against a small panel of Gram positive and negative strains, with the exception of (76) against Haemophilus influenzae. Compound (77) is a significant improvement over the standard compounds in this report. Most recently the novel isoxazoline analogues [ 1201 have been described (Figure 2.12). The most favoured R' substituents, 1aza-bicyclo[2,2,2]oct-4-yl (78, 79), 1-methylpiperidin-4-yl (80) and ex0-8methyl-8-aza-bicyclo[3,2,l]oct-3-yl(8 I ) are described as having MICs in the range 0.0pg/mL against Staph. aureus Oxford and Strep. pneumoniae (R6). Pleuromutilin analogues are active against organisms commonly encountered in community infections, particularly those of the respiratory tract and are reported to have no clinically relevant cross resistance with other classes of antibiotics. However, they have poor oral bioavailability due to hepatic metabolism, primarily mediated by CYP450 hydroxylation of the cyclopentanone ring. A recent meeting report [ 12 I ] described a binding assay which used the fluorescent pleuromutilin analogue (82) binding to 70s ribosomes obtained from E. coli. Compound (82) was used to investigate modifications of the
RNA AS A DRUG TARGET
I10
cyclopentanone ring of the methoxyphenyl compound (83). All modifications carried out resulted in a significant loss of activity against ribosome binding and the 3 strains of bacteria reported on. At the same meeting [122] utilising two cross-linking probes, the azide, pal-1 (84) and the enone, pal-2 (85), the site of interaction of pleuromutilins on the 50s subunit was described. Pal-1 interacts with domain V of E. coli 23s RNA in the vicinity of the peptidyl transferase centre (PTC). Specifically the interaction was with bases G206 I , A2062, A2407, U2408 and G2502 which are located at the PTC. This described some overlap with the chloamphenicol binding site, but three of these are specific to pleuromutilins. In addition to 23s RNA interactions, pal-1 crosslinked and radio-labelled the ribosomal proteins L2, L13, L27 and L28, whilst pal-2 was seen to interact with L4 as well as L27 and L28. All of these proteins have been implicated in the activity of the PTC.
R 1=vinyl, El (71) R1=vinyl
(72)
R'= Et
R'
= vinyl,
0
Et
(73) R1= vinyl
(74) R'= Et
(78) R2= vinyl (79) R2= Et
In addition to those described above there are a number of other antibiotics which have been shown to interact with the 50s subunit. These are described in Table 2.7 and their structures are outlined in Figure 2.13.
M.J. DRYSDALE E T A L .
Rz = vinyl, Et; R' Figure 2. I I
= H,
111
OH
Generic structure of plruromiitilin analogues from ref.' [I 191.
Figure 2.12. Generic structure of isoxazoline carboxylate derivatives of mutilin from re/.' [120].
RNA AS A DRUG TARGET
112
Table 2.9 ANTIBACTERIAL PROPERTIES OF PLEUROMUTILIN DERIVATIVES
Compound
R'
Pleuromutilin (69) Tiamulin (70)
NA' Et,
dN-L
cy
(75)
'
E.c.'
H.i.'
2
8
>64
2
0.5
0.25
0.25
%4
2
0.125
< 0.06
< 0.06
16
< 0.06
< 0.06
64
< 0.06
< 0.06
s.p.
S.U.'
0.25
32
M.c.'
< 0.06
< 0.06
"TO
(77)
0.5
0.5
< 0.06
'Activities are given as minimum inhibitory concentrations (MIC) in pg/mL, and were determined using a standard broth dilution method in microtitre; S.a. = Staphylococcus uureus Oxford; S.p. =Streptococcus pneumoniue 1629; E.c. = Escherichiu coli DCO; H i . = Huemophilus influenzue QI;M.c. = Morarellu 'NA Not applicable. ~
M.J. DRYSDALE ET A L .
113
Figure 2.13. Additional structures of ribosome binding antibiotics whose site of action and specificicv are descrihed in Table 7 above.
1 I4
RNA AS A DRUG TARGET
SUMMARY In the antiviral and antibacterial area, increasing drug resistance means that there is an ever growing need for novel approaches towards structures and mechanisms which avoid the current problems. The huge increase in high resolution structural data is set to make a dramatic impact on targeting RNA as a drug target. The examples of the RNA binding antibiotics, particularly the totally synthetic oxazolidinones, should help persuade the sceptics that clinically useful, selective drugs can be obtained from targeting RNA directly.
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M.J. DRYSDALE E T A L .
62 63 64 65 66 67 68
69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93
94
117
Matassova, N.B., Savelsbergh, A,, Rodnina, M.V. and Wintermeyer, W. (2000) Mol. Cell 6, 501-505; (f) Savelsbergh, A., Matassova, N.B., Rodnina, M.V. and Wintermeyer, W. (2000) J. Mol. Biol. 300, 951-961. Noller, H.F. (2000) FASEB J. 7, 87-89. Green, R., Samaha, R.R. and Noller, H.F. (1997) J. Mol. Biol. 266, 4GSO. Green, R., Switzer, C. and N o h , H.F. (1998) Science (Washington, D.C.) 280, 286289. Crick, F.H. (1968) J. Mol. Biol. 383, 67-79 Rodnina, M.V., Savelsbergh, A,, Matassova, N.B., Katunin, V.I., Semenkov, Y.P. and Wintermeyer, W. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9586-9590. Rodnina, M.V., Stark, H., Savelsbergh, A., Wieden, H-J., Mohr, D., Matassova, N., Peske, F. and Wintermeyer, W. (2000) Biol. Chem. 381, 377-387. (a) Buckingham, R.H., Grentzmann, G. and Kisselev, L. (1997) Mol. Microbiol. 24,449-456; (b) Janosi, L., Mottagui-Tabar, S., Isaksson, L.A., Sekine, Y., Ohtsubo, E., Zhang, S., Goon, S., Nelken. S., Shuda, M. and Kaji, A. (1998) EMBO J. 17, 1141-1151. Ganoza, M.C. (1966) Cold Spring Harb. Symp. Quant. Biol. 31, 273-278. Freistroffer, D.V., Pavlov, M.Y., MacDougall. J., Buckingham, R.H., Ehrenberg, M. (1997) EMBO J. 16, 4126-4133. Fourmy, D., Recht, M.I., Blanchard, S.C. and Puglisi, J.D. (1996) Science (Washington, D.C.) 274, 1367-1371. Pape, T., Wintermeyer, W. and Rodnina, M.V. (2000) Nat. Struct. Biol. 7, 104-107. Lodmell, J.S. and Dahlberg, A.E. (1997) Science (Washington, D.C.) 277, 1262-1267. Bilgin, N., Richter, A.A., Ehrenberg, M., Dahlberg, A.E. and Kurland, C.G. (1990)EMBO J. 9, 735-739. Chopra, I., Hawkey, P.M. and Hinton, M. (1992) J. Antimicrob. Chemother. 29, 245-277. Gale, E.F., Cundliffe, E., Reynolds, P.E., Richmond, M.H. and Waring, M.J. (1981) The Molecular Basis of Antibiotic Action. Wiley, London. Epe, B., Woolley, P. and Homig, H. (1987) FEBS Lett. 213, 443447. Kolesnikov. I.V., Protasova, N.Y. and Gudkov, A.T. (1996) Biochimie 78, 8 6 W 7 3 . Moazed. D. and Noller, H.F. (1987) Nature (London) 327, 38%394. Cohen, L.B.,Goldberg, I.H. and Hemer, A.E. (1969) Biochemistry 8, 1327-1335. Moazed, D., Samaha. R.R., Gualerzi, C. and Noller, H.F. (1995) J. Mol. Biol. 248, 207-210. Woodcock, J., Moazed, D., Cannon, M., Davies, J. and Noller, H.F. (1991) EMBO J. 10, 309%3 103. Vila-Sanjurjo, A,, Squires, C.L. and Dahlberg, A.E. (1999) J. Mol. Biol. 293, 1-8. Douthwaite. S. (1992) Nucleic Acids Res. 20, 47174120. Rodriguez-Fonseca, C., Amils, R. and Garrett, R.A. (1995) J. Mol. Biol. 247, 224-235. Leviev, I.G., Rodriguez-Fonseca, C., Phan, H., Garrett, R.A., Heilek, G., Noller, H.F. and Mankin, A.S. (1994) EMBO J. 13. 1682-1686. Xiong, L., Shah, S., Mauvais, P. and Mankin, AS. (1999) Mol. Microbiol. 31, 633439. Porse, B.T. and Garrett, R.A. (1999) J. Mol. Biol. 286, 375387. Kirillov, S.V., Porse, B.T. and Garrett, R.A. (1999) RNA 5, 1003-1013. Kuechler, E., Steiner, G. and Barta, A. (1988) Methods Enzymol. 164, 361-372. Poulsen, S.M., Kofoed, C. and Vester, B. (2000) J. Mol. Biol. 304, 471481. Menninger, J.R. and Otto, D.P. (1982) Antimicrob. Agents Chemother. 21, 81 1-818. (a) (1993) Macrolides: Chemistry, Pharmacology and Clinical Uses (Bryskier, A.J., Butzler, J-P., Neu, H.C. and Tulkens, P.M. eds.), Arnette Blackwell, Paris; (b) (1995) New Macrolides, Azalides and Streptogramins in Clinical Practice (Neu, H.C., Young, L.S., Zinner, S.H. and Acar, J.F. eds.), Marcel Dekker, New York. (a) Chu, D.T.W. (1995) Exp. Opin. Invest. Drugs 4,65-; (b) Lartey, P.A. and Perun, T. (1993) in Studies in Natural Products Chemistry (Rahman, A,, ed.), pp. 155185, Elsevier Science, Amsterdam.
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95 (a) Acar, J.F. and Goldstein, F.W. (1995) in Ref. 93(b) pp. 4 1 4 9 ; (b) Shah, P.M. and Bryskier, A.J. (1993) in Ref. 93(b) pp. 143166. 96 (a) Neu, H.C. (1992) Science (Washington, D.C.) 257, 1064-1073; (b) Appelbaum, P.C. (1992) Clin. Infect. Dis. 15, 77-83; (c) Friedland, I.R. and McCracken, G.H. (1994) New Engl. J. Med. 331, 377-382; (d) Goldstein, F.W. and Acar, J.F. (1996) J. Antimicrob. Chemother. 38, 71-84. 97 Hardy, D.J.. Hensey, D.M., Beyer, J.M., Vojtko. C., McDonald, E.J. and Fernandes, P.B. (1988) Antimicrob. Agents Chemother. 32, 1710-1719. 98 Fernandes, P.B.. Baker, W.R., Freiberg, L.A., Hardy, D.J. and McDonald, E.J. (1989) Antimicrob. Agents Chemother. 33, 78-81. 99 Agouridas, C., Denis, A,, Auger, J-M., Benedetti, Y., Bonnefoy, A,, Bretin, F., Chantot, J-F., Dussarat, A., Fromentin, C., Gouin D’Ambrieres, S., Lachaud, S . , Laurin, P., Le Martret, O., Loyau, V. and Tessot, N. (1998) J. Med. Chem. 41, 408&4100. 100 Bertho, G., Gharbi-Benarous, J., Delaforge, M., Lang, C., Parent, A. and Girault, J-P. (1998) J. Med. Chem. 41, 3373-3386. 101 Denis, A,, Agouridas, C.. Auger, J-M., Benedetti, Y., Bonnefoy, A,, Bretin, F., Chantot, J-F., Dussarat, A., Fromentin, C., Gouin D’Ambrieres, S., Lachaud, S . , Laurin, P., Le Martret, 0.. Loyau, V., Tessot, N., Pejac, J-M. and Perron, S . (1999) Bioorg. Med. Chem. Lett. 9, 3 0 7 s 3080. I 02 Evrard-Todeschi, N., Gharbi-Benarous, J., Gaillet, C., Verdier, L. Bertho, G., Lang, C., Parent, A. and Girault, J-P. (2000) Bioorg. Med. Chem. 8, 1579-1597. 103 Or, Y.S., Clark, R.F., Wang, S., Chu, D.T.W., Nilius, A.M., Flamm, R.K., Mitten. M., Ewing, P., Alder, J. and Ma, Z. (2000) J. Med. Chem. 43, 104S1049. 104 Cao, Z., Hammond, R., Pratt, S., Saiki, A,, Lerner, C. and Zhong, P. (1999) 39th Intersci. Conf. Antimicrob. Agents Chemother., Abstr. 2 135. 105 Copobianco, J.O., Cao, Z., Shortridge, V.D., Ma, Z.. Flamm, R.K. and Zhong, P. (2000) Antimicrob. Agents Chemother. 44, 1562-1 567. I 06 Phan, L.T., Clark, R.F., Rupp, M., Or, Y.S., Chu, D.T.W. and Ma, 2. (2000) Org. Lett. 2, 295 1-2954. 107 Wimberly, B.T., Guymon, R., McCutcheon, J.P., White, S.W. and Ramakrishnan, V.A. (1999) Cell 97, 491-502. 108 Conn, G.L., Draper, D.E., Lattman, E.E. and Gittis, A.G. (1999) Science (Washington, D.C.) 284, 1171-1174. I 09 Cundliffe E, Thompson J. (1981) Eur. J. Biochem. 118, 47-52. 110 Moazed, D. and Noller, H.F. (1987) Biochimie 69, 87%884. 1 1 1 Menyman, C., Moazed, D., Daubresse, G. and Noller, H.F. (1999) J. Mol. Biol. 285, 107-1 13. I12 Belova, L., Tenson, T., Xiong, L., McNicholas, P.M.and Mankin A.S. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 3726-3731. 113 (a) Barrett, J. F. (2000) Curr. Opin. Investig. Drugs I , 181-187; (b) French, G. (2001) Int. J. Clin. Pract. 55, 59-63; (c) Matassova, N.B., Rodnina, M.V., Endermann, R., Kroll, H.P., Pleiss, U., Wild, H. and Wintermeyer W. (1999) RNA 5, 939-946; (d) Tucker, J.A., Allwine, D.A., Grega, K.C., Barbachyn, M.R., Klock, J.L., Adamski, J.L., Bnckner, S.J., Hutchison, D.K., Ford, C.W., Zurenko, G.E., Conradi, R.A., Burton, P.S. and Jensen, R.M. (1998) J. Med. Chem. 41, 3727-3735. 114 Xiong, L., Kloss, P., Douthwaite, S., Andersen, N.M., Swaney, S., Shinabarger, D.L. and Mankin, A S . (2000) J. Bacteriol. 182, 5325-5331. 1 I5 Nurse, K., Sterner, T., Ferguson, D., Takle, A,, Davies, S., Ham, P., Dean, D., Hunt, E., Woodnutt, G., King, F., Meek, T. and Hegg, L. (1999) Nucleic Acids Symposium Series 41, 11-12.
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Progress in Medicinal Chemistry - Vol. 39, Edited by F.D. King and A.W. Oxford 0 2002-Elsevier Siience B.V. All rights reserved.
3 ACAT Inhibitors: The Search for a Novel and Effective Treatment of Hypercholesterolemia and Atherosclerosis DRAG0 R. SLISKOVIC, JOSEPH A. PICARD AND BRIAN R. KRAUSE*
Pfizer Global Research and Development, Ann Arbor Laboratories, 2800 Plymouth Road, Ann Arbor, Michigan, 48105, U.S.A. *Current address: Esperion Therapeutics, Inc. Ann Arbor, Michigan, 48 108, U.S.A.
INTRODUCTION
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ACAT PHYSIOLOGY Intestine Liver Macrophages
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ACAT PHARMACOLOGY Intestine Liver Arterial wall
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REGULATION OF ACAT
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ACAT ISOFORMS AND TRANSGENIC MOUSE MODELS
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EVOLUTION OF ACAT INHIBITORS
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CLASSES OF ACAT INHIBITORS Amides Ureas Imidazole/imidazolines Natural products
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CLINICAL EXPERIENCE
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REFERENCES
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INTRODUCTION It has been recognized for most of this century that feeding cholesterol-rich diets to rabbits results in atherosclerosis, and that atherosclerotic plaques contain an abundance of cholesterol [I]. The connection between elevated blood cholesterol and atherosclerosis led to the supposition that reducing blood cholesterol will lead to reduced morbidity and inortality from atherosclerotic disease and, in particular, coronary heart disease (CHD). Clinical trials with a variety of sterols, food substances and vitamins, as well as different types of fats, were conducted to find the best ways for lowering blood cholesterol. In fact, the oldest of the remedies is still widely used, namely, high-dose nicotinic acid. Later, in 1953, it was noted that plasma cholesterol was low in farmers exposed to certain insecticides and this led eventually to the development of clofibrate [2]. Although many different clofibrate-type compounds soon appeared with some desirable features, and a unifying molecular mechanism now exists for these drugs [3], none of them are particularly efficacious with regard to cholesterol reduction, especially low density lipoprotein-cholesterol (LDL-cholesterol). They have been most useful for triglyceride reduction and to some extent, elevation of high density lipoprotein-cholesterol (HDL-cholesterol). Thus, other drugs were sought, resulting in the emergence of compounds that inhibit specific steps in the cholesterol biosynthetic pathway. This emphasis led to the discovery of the HMG-CoA reductase inhibitors, now the dominant class of drugs widely used for the treatment of hypercholesterolemia. Several reviews have appeared comparing the pharmacology of these agents [4-8]. The present review describes the biology and pharmacology of another class of potential cholesterol-lowering agents, namely, inhibitors of the intracellular enzyme, acyl-CoA:cholesterol 0-acyltransferase (ACAT). ACAT catalyzes the conversion of unesterified (free) cholesterol to cholesteryl esters in virtually every tissue, especially liver, intestine and the endocrine organs, and thereby plays a major role in intracellular cholesterol homeostasis. The focus of the present review will be on lipoprotein-producing tissues, with an additional emphasis on specific cells within developing atherosclerotic lesions. The biology of ACAT is briefly discussed for these tissues, followed by a comprehensive evaluation of ACAT inhibitors that have emerged. The information provided attempts to build upon previous reviews on this subject [9-121.
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ACAT PHYSIOLOGY INTESTINE
It was recognized as early as 1909 that some dietary cholesterol is absorbed and that esterification occurs during the absorptive process. Goodman reviewed this early history [13]. There has been considerable debate over the years as to which enzyme is responsible for the formation of cholesteryl esters destined for lymphatic transport within intestinally-derived lipoproteins. Initially ( 1 96G-1980) it was thought that a pancreatic enzyme (pancreatic cholesteryl esterase, pCEH, EC 3.1.1.13) was responsible. Data suggested that pCEH was not only able to hydrolyze cholesteryl esters within the intestinal lumen, but also catalyze the reverse reaction, i.e., the synthesis of cholesteryl esters, after itself gaining access to the mucosal cell by transfer across the brush border membrane. However, Haugen and Norum identified ACAT in intestinal microsomes using rats and suggested that ‘ACAT may play a role in true absorption of cholesterol from the gut’ [14]. Subsequent studies demonstrated the presence of intestinal ACAT activity in other species and its regulation by dietary cholesterol, fats and other sterols [15]. The site of ACAT activity in the intestine (villus cells of the jejunum) coincided with the site where cholesterol absorption takes place [ 161. Moreover, a correlation between intestinal ACAT activity and the amount of cholesteryl esters in mesenteric lymph was described [17]. Norum and his colleagues later estimated from the specific activity of the enzyme in human mucosal biopsies that there was more than enough activity present to account for all of the cholesteryl esters found in chylomicrons [18]. Careful studies by Field reevaluated the role of pCEH. He found that pCEH, as detected within the cytoplasm of enterocytes, was actually a contaminant of the cytosolic fraction and represented pancreatic enzyme that was trapped in the mucus within the lumen, as opposed to a bone fide intracellular enzyme [19]. There is also evidence that pCEH in the liver may be a more important determinant of the plasma cholesterol concentration in some species [20]. These data, together with the subsequent observation that mice lacking the pCEH gene absorb cholesterol normally [2 13, shifted attention towards ACAT as the cholesterolesterifying enzyme within intestinal cells. More recently it has been demonstrated that the increased cholesterol absorption observed in diabetic rats (streptozotocin (STZ)-treated) is associated with increased intestinal ACAT activity [22, 231 and that the exaggerated postprandial lipemia in STZ-diabetic rats is ACAT-mediated [24]. Thus, the physiological evidence that ACAT is involved in the cholesterol absorptive process is quite convincing, especially in experimental animals.
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Mukherjee et al. first described liver cholesterol esterifying activity requiring ATP and CoA, in 1958 [25]. Almost all ofthe early work on liver ACAT was done using rats and it has been known for 50 years that rat liver can accumulate massive amounts of cholesteryl esters, mainly cholesteryl oleate, when the animals are challenged with dietary cholesterol. Initial attempts to characterize liver ACAT, utilizing radiolabelled fatty acid with a CoA generating system, demonstrated induction by dietary cholesterol, but no circadian rhythm [26]. Erickson and colleagues studied the regulation of rat liver ACAT using radiolabelled oleoylCoA, a water-soluble substrate with minimal endogenous dilution [27]. They were able to confirm the stimulation by dietary cholesterol, but also showed that ACAT activity exhibited a circadian rhythm similar to that for HMG-CoA reductase. Moreover, liver ACAT was enhanced by 25-hydroxycholesterol and inhibited by progesterone. These same perturbations also decrease (25-hydroxycholesterol) and increase (progesterone) the output of cholesterol into bile in intact rats [28], thus demonstrating in vivo that liver ACAT is a determinant of biliary cholesterol secretion in rats. Studies using isolated hepatocytes and radiolabelled oleic acid as substrate also demonstrated the stimulatory effect of 25-hydroxycholesterol (and mevalonolactone) and clearly showed for the first time that changes in liver ACAT are reflected in the amount of cholesteryl esters secreted within very low density lipoproteins (VLDL) [29]. Although early attempts using radiolabelled cholesterol or fatty acid failed to detect significant ACAT activity in human liver samples [30, 311, subsequent attempts were successful using labelled oleoyl CoA and endogenous cholesterol as reactants. Thus, Erickson and Cooper found specific activities in the range of those reported for human skin fibroblasts and human intestine, with highest values from fatty livers [32]. Interestingly, the ACAT activities from human liver were similar to rabbit liver, but much lower than those in rats. The ACAT values of Erickson and Cooper for human liver were later confirmed using liver biopsies from patients undergoing cholecystectomy [33]. It was also demonstrated that liver ACAT was reduced in patients with gallstones [34], consistent with the observation in rats that inhibitors of ACAT (e.g., progesterone) increase biliary cholesterol output. However, other determinants of gallstone formation (bile acid and phospholipid content of bile) were not determined in this study. The use of the human hepatoblastoma-derived cell line, HepG2, has been appropriately used to further explore the function of ACAT in human liver. ACAT activity, measured in microsomes from HepG2 cells, was similar to the activities found in human liver [35] and increased when the availability of endogenous cholesterol substrate was increased by the addition of mevalonolactone. In 1990 Cianflone et al. demonstrated that cholesteryl ester synthesis
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and the secretion of apolipoprotein B (apoB) could be stimulated in HepG2 cells by the addition of oleic acid [36]. Others [37] showed that changes in cellular neutral lipids with 25-hydroxycholesterol were reflected in corresponding changes in the composition of secreted low density lipoproteins, as reported earlier for rat hepatocytes [38]. In HepG2 cells there was a significant positive correlation between the cellular cholesteryl ester content and the rate of apoB secretion [37]. The cumulative data support the notion that ACAT activity can determine the amount of cholesteryl esters secreted in the form of VLDL, analogous to its function in determining the ester content of intestinal chylomicrons. Moreover, the suggestion has been made that the availability of cholesteryl esters determines in large part how much apoB is degraded within hepatocytes [39].
MACROPHAGES
Lofland et al. noted from studies using perfused pigeon aortas that it was the sterol ester fraction that showed the greatest increase as the artery progressed from ‘undiseased to the diseased state’ [40]. Later work showed that the cholesteryl oleate fraction in pigeon aortic lipids increased from 45% to 70% of total sterol esters [41] and that cholesterol was esterified by transfer of fatty acyl-CoA (preferentially oleic acid) to cholesterol in a reaction that required ATP [42]. The reaction was identical to the one described by Goodman et al. for liver ACAT [ 131. Similar results were later found by Hashimoto et al. [43] using rabbit aortas, who went on to show that there was ‘hyperactivity of the cholesterol-esterifying system’ in aortas from atherosclerotic rabbits [44]. This was later ascribed to both an increased amount of enzyme [45] and to an increase in the microsomal free cholesterol pool [46]. Continued research soon focused on which cell types within the arterial microsomal preparations were responsible for the esterifying activity. It is now recognized that a major cell type within the arterial wall that contains ACAT, and that is very important with respect to atherogenesis, is the macrophage. Macrophages are found at all stages of lesion formation and have the ability to recognize and take up large quantities of cholesteryl esters in the form of lipoproteins. Candidate lipoproteins include beta-migrating VLDL, aggregated LDL, oxidized LDL and nitrated LDL. The receptors involved, termed scavenger receptors, are primarily CD-36 and SR-A (scavenger receptor Class A) as shown in Figure 3.1. After uptake, these lipoproteins are delivered to lysosomes where the cholesteryl esters are hydrolyzed. The liberated unesterified free cholesterol then enters the cytoplasm and is re-esterified by ACAT. The constant turnover of cholesterol via the ester is termed the ‘cholesteryl ester cycle’, and was described in detail by Brown and Goldstein in 1983 [47]. Inhibition of ACAT disrupts this cycle
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preventing the re-esterification of free cholesterol. The free cholesterol is then removed from the cell by efflux acceptors, such as HDL, that return the free cholesterol to the liver.
ACAT PHARMACOLOGY INTESTINE
The site of ACAT within the proximal intestine, and its induction by dietary cholesterol, implicated ACAT as the enzyme responsible for the formation of cholesteryl esters destined for chylomicrons. As suggestive as it is, the correlation between lymph cholesteryl ester content and ACAT activity did not prove that the esters were ACAT-derived. One approach that was rapidly used by many to test this assumption was to use specific inhibitors of ACAT to see if they block cholesterol absorption. Heider was the first to accomplish this task using cholesterol-fed rabbits as the experimental model [48] and this was soon followed by many others using a variety of inhibitors. Inhibition of cholesterol absorption was demonstrated in other species (rats, hamsters) by a variety of methods (e.g., lymph-fistula model, dual-isotope method, fecal isotope method, etc). However, it became apparent that the ability of ACAT inhibitors to block cholesterol absorption did not seem to apply to higher species, specifically nonhuman primates and humans. Moreover, in a human model of intestinal cells (CaCo-2 cells) the activity of ACAT did not regulate the uptake of cholesterol [49], nor did ACAT inhibitors alter the total amount of cholesterol secreted on the basolateral side of the monolayers; only the percentage of cholesterol in the esterified form was altered [50]. These results, coupled with the failure of ACAT inhibitors to lower plasma cholesterol in clinical trials [5 1, 521, strongly suggest that ACAT activity in the intestine of humans, unlike other species, is not a determinant of the extent of cholesterol absorption. It is possible that nonhuman primates and humans do not have the necessary machinery to pump out excess free cholesterol into the intestinal lumen in the presence of an ACAT inhibitor, but rather it is secreted into chylomicrons. In this regard, the ATP cassette transporter 1 (ABCl or ABCA1) has been postulated to play a role in the efflux of cholesterol out of enterocytes into the lumen, and this seems to be absent or significantly diminished in non-human primates [53]. LIVER
ACAT inhibitors have also been used to assess the role of the liver enzyme in governing plasma lipid levels. To this end, the inhibitors CI-976 (1) [54] and
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ACAT INHIBITORS: TREATMENT OF HYPERCHOLESTEROLEMIA
DuP 128 (2) [55] were administered to animals fed cholesterol-free diets and both compounds lowered plasma cholesterol concentrations. Others used ACAT inhibitors in perfused livers [56] or HepG2 cells [36, 57, 581 demonstrating decreases in the secretion of cholesteryl esters and apoB, qualitatively similar to the results found in early studies using progesterone. In addition, it was observed that the inhibition of ACAT by DuP 128 (2) increased bile acid synthesis in hamster hepatocytes [59]. The ACAT inhibitor, avasimibe (CI1011, (3)) also increases bile acid synthesis, owing to an increased expression of cholesterol 7-alpha hydroxylase [60].
(3) CI-1011, Avasirnibe
Overall, the use of ACAT inhibitors in these various model systems have strengthened the contention that hepatic ACAT can play a major role in determining the composition of secreted lipoproteins and the extent of conversion of cholesterol into bile acids for excretion. However, data using HepG2 cells suggests that some (CL 277082 (4), CI-1011 (3)) but not all (SaH 58-035 (9, 447C88 (6)) ACAT inhibitors decrease apoB secretion [57, 61431. The differences among these structurally diverse inhibitors with respect to apoB secretion were apparently not due to potencies against ACAT and remain to be explained [64]. Some ACAT inhibitors (CP-113818 (7), PD 138142-15 (8)) but not others (CI-976 (1)) also decreased apoB secretion from perfused monkey livers [56]. Of note is the down-regulation of LDL uptake noted in HepG2 cells with 447C88 (6) [62] and SaH 58-035 (5) [65], although changes in LDL receptor mRNA in HepG2 cells were not observed for either CI-1011 (3) or DuP 128 (2) [57]. In vivo LDL apoB secretion is decreased, at least in miniature swine, with DuP 128 (2) [ 5 5 ] , and even more so with CI-1011 (3) [66]. Since LDL does not increase or accumulate in animals administered ACAT inhibitors, most pharmaceutical scientists assume that downregulation of LDL receptors in
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(8) Cl-999, PD 138142-15
the liver is not occurring. In fact, CT-101 1 (3) has been shown to drastically reduce plasma LDL-C in animal models by actually increasing the clearance of LDL (casein-fed rabbits [67]), or the production of LDL (pigs [66]). Compound-specific differences in apoB secretion or LDL clearance in vitro and/or in vivo may relate to access of these lipophilic compounds to compartments within hepatocytes responsible for the degradation of apoB and/or the accumulation of free cholesterol. Since this would be difficult to assess, the attractiveness of in vivo pharmacological profiles in a variety of species have driven the development of these compounds into the clinical arena. ARTERIAL WALL
In 1974 Morin et al. evaluated the potential of several diverse compounds for inhibition of cholesterol esterification using microsomes from swine arteries [68]. This group appears to be the first 'to explore the possible value of future in vivo administration of these compounds as inhibitors of cholesterol ester
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formation during the induction of experimental atherosclerosis' - hence the beginning of the search for arterial wall ACAT inhibitors. It was soon realized that the cholesteryl ester-rich foam cells which accumulate in lesions, are derived from circulating monocytes, and that macrophages play a major role in many stages of atherosclerosis [69-72]. Brown and Goldstein also clearly showed that when ACAT was inhibited by progesterone in macrophages, the total amount of cholesteryl esters decreased due to continued ester hydrolysis [47]. Hence, in vitro 'foam cell' formation could be inhibited. Thus, although most effort in the area focused on identifying ACAT inhibitors to lower plasma cholesterol, it became apparent that any inhibitor with sufficient bioavailability
A
(1 1) HL-004, TS-962
(13) PD 132301-2
I
N(CH3)z .HCI
(14) FR145237
CI
flcH
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and potency had the potential to directly inhibit foam cell formation in vivo to prevent or slow atherogenesis. In 1985 Schmitz and coworkers showed that incubation of mouse peritoneal macrophages with acetylated LDL in the presence of progesterone, SaH 58-035 ( 5 ) , or octimibate reduced the incorporation of radiolabelled oleic acid into cholesteryl esters, and importantly, induced an increase in the binding of HDL and cholesterol efflux [73]. Other fatty acid amides (e.g., (21-976 (1)) and ureas (CL 277,082 (4)) were found to inhibit ACAT in macrophages, but detailed studies using this cell type did not appear until 1995 when Kogushi et al. compared the inhibitory effects of E5324 (9) and (21-976 (1) [74]. After extensive characterization of several macrophage cell lines loaded with different forms of lipoproteins, they showed that in the human macrophage cell line, THP-I , these compounds were equipotent at inhibiting ACAT. Another inhibitor, NTE-122 (lo), not only potently inhibited ACAT in THP-1 cells, but also resulted in enhanced HDL-induced cholesterol efflux [75]. These results were in agreement for those found with the inhibitor HL-004 (1 1). This compound is very potent in mouse peritoneal macrophages (ICso = 89 nM) and free cholesterol only accumulates to a major extent if HDL is not present [76]. Interestingly, NTE-122 (10) was most potent using human cell lines and was more potent than E5324 (9) and CI-976 (1). Others reported that E5324 (9) was a relatively weak inhibitor in 5774 macrophages [77]. Recently, another bioavailable inhibitor, F 1251 1 (12) was found to be more potent than CI-976 (l), PD 132301-2 (13), CI-loll (3) and DuP 128 (2), but equal to CP 113,818 (7), for inhibiting ACAT in THP-1 cells [78]. Although it is difficult to compare results between laboratories, it seems that the rank order of potency in humanderived macrophages is F125 1 1 (1 2) = CP- 1 138 18 (7) = HL-004 (1 1) > NTE122 (10)>C1-976 (l)>CI-IOII (3)>E5324 (9). Human monocyte-derived macrophages are different from other macrophage cell lines in that they are capable of releasing (effluxing) cholesterol into the medium in the absence of acceptors like HDL [79]. Rodriguez et al. used this system to demonstrate that SaH 58-035 (5) and CI-976 ( I ) could reduce the amount of cellular cholesterol, both by increasing free cholesterol efflux and by decreasing uptake of acetylated LDL [go]. These same results have now been found with CI-I01 1 (3) [81]. The finding that ACAT inhibitors do not cause accumulation of free cholesterol in human macrophages, and are not toxic in vitro as previously shown for rodent macrophages [82], clearly has implications for future clinical trials. Many laboratories have reported the effects of ACAT inhibitors on atherosclerotic lesions in experimental animals, but most early work failed to control for changes in plasma cholesterol, and thus could not demonstrate a direct effect of the drugs on ACAT in cells of the arterial wall, such as the report using HL-004 (1 1) [83]. The demonstration that CL 277082 (4) could decrease the cholesteryl
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ester content of macrophage-rich granulomas in rabbits represented one approach, but effects independent of changes in plasma cholesterol were equivocal [84]. This approach also assumes that accumulation of cholesteryl esters in granulomas was a reasonable model for arterial lesions. Another laboratory utilized a protocol that employed a novel feeding paradigm in rabbits with bioavailable inhibitors, CI-976 (1) [85] andCI-1011 (3) [86]. This approach proved to be convincing, and others used variations for FR145237 (14) [87] and E5324 (9) [88]. Differences in lesion endpoints could be shown between control and drugtreated groups with the same plasma cholesterol level, highly suggestive that ACAT inhibitors that reach the arterial wall in sufficient amounts can alter atherosclerosis progression. In other studies with Watanabe rabbits, plasma cholesterol was not altered by the ACAT inhibitors E5324 (9) [89] and FR 145237 (14) [90], yet the cholesterol content and the percentage area of surface involvement of the aortic arch and thoracic aorta were reduced by greater than 50%. The above experimental data suggests that systemically available ACAT inhibitors with sufficient potency can directly inhibit atherogenesis in experimental animals. Although most evidence is derived from rabbit models of atherosclerosis, similar supportive data has been obtained in hamsters [9 1, 921 and transgenic mice [93] for the inhibitor, CI-1011 (3). The consequences of inhibiting ACAT in cells of the arterial wall are only beginning to emerge and may involve more than the expected decrease in cholesteryl ester content. For instance, Bocan et al. provided evidence that with ACAT inhibition, macrophage area decreases due to a decrease in macrophage number, and consequently, the activity of macrophage-derived matrix metalloproteinases (MMPs) decreases [86]. MMPs have been implicated in plaque rupture due to their ability to degrade collagen. For this reason, ACAT inhibitors may be viewed as potential ‘plaque stabilizing’ agents. Others have found that ACAT inhibitors can decrease endothelium-induced relaxations to acetylcholine in thoracic aortic rings from cholesterol-fed rabbits [78]. Such results imply possible functional consequences of ACAT inhibition that may relate to changes in haemodynamics. These and other effects of ACAT inhibition, both structural and functional, need to be explored further. REGULATION OF ACAT The increase in ACAT activity observed in intestine, liver and arterial wall in cholesterol-fed animals has been assumed to be due primarily to increased substrate (cholesterol) supply, and to some extent, the availability of acyl-CoA binding protein (ACBP) which donates acyl-CoA to ACAT [94]. The availability of the human cDNA clone [95] not only led to the cloning of the homologues for mouse [96], hamster [97], rabbit [98], yeast [99, 1001 and rat
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[ l o l l , it also led to the production of a specific antibody (DM-10) by immunizing rabbits with the recombinant fusion protein (GST segment plus the first 131 amino acids). Using this antibody, the amount of ACAT protein was not altered by added sterol to the medium of a variety of human cell lines (fibroblasts, HepG2) as well as CHO cells [102]. Others reported that ACAT mRNA levels do not change with cholesterol loading in rabbit primary hepatocytes [ 1031. These results confirmed the importance of cholesterol, specifically cholesterol in the endoplasmic reticulum, as the major regulator of ACAT activity. However, others using the same human clone showed that gene expression was upregulated in human monocytes during differentiation into macrophages, and that the changes in mRNA were associated with increases in functional ACAT protein [104, 1051. Increases in ACAT mRNA were also reported in liver and aorta in cholesterol-fed rabbits using a partial cDNA derived from the human clone sequence [98]. The combined results using available reagents suggests that ACAT activity is elevated in macrophages of the atherosclerotic lesion due to both enhanced substrate availability and most likely, an increased transcription rate, or decrease in ACAT message turnover. Such regulation would allow the recognized accumulation of cholesteryl esters to form fatty streaks and unstable shoulder regions of atherosclerotic plaque. ACAT ISOFORMS AND TRANSGENIC MOUSE MODELS Soon after the description of the first ACAT gene, designated ACAT-1, the cDNA, which was isolated from a macrophage library, was used to generate homozygous knockout mice. Disruption of ACAT- 1 surprisingly did not alter liver or plasma cholesterol concentrations. Moreover, cholesterol absorption was unchanged. But the peritoneal macrophages from these knockout animals failed to accumulate large amounts of cholesteryl esters in the presence of acetylated LDL [106]. This led to the logical hypothesis that another ACAT might exist in liver and intestine, and soon thereafter a second ACAT gene was cloned independently, designated ACAT-2, for mice [ 1071, monkeys [ 1081 and human [109]. The distribution of ACAT-1 and ACAT-2 is different in human, however, as opposed to mouse and monkey. In the latter two species, ACAT-2 message is prominent in liver and intestine, whereas ACAT-I is ubiquitous. In humans, the major isoform in the intestine is ACAT-2, like the other mammals, but ACAT-1 is the major ACAT isoform in human liver [ 110, 11 11. This suggests that an inhibitor that is selective for ACAT-2 may lower plasma lipids in animals but not in humans. It would also lack direct activity against the macrophages in the arterial wall. Conversely, a selective ACAT- 1 inhibitor would not only conceivably lower plasma lipids but also have direct effects on macrophage ACAT in the arterial wall.
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Thus far, however, ACAT inhibitors with significant preference for one isoform over the other have not been described for the human ACAT. The availability of the ACAT-1 knockout model has been recently used to test the hypothesis as to whether absence of ACAT-1 would alter atherosclerosis in mice. To this end, the ACAT-1 knockout animals have been crossed with mouse strains that develop atherosclerotic lesions. In one study in which the cross was made onto an LDL receptor knockout strain, there was extensive accumulation of free cholesterol in tissues, and an unexplained decrease in plasma cholesterol [ 11&112]. For these reasons changes in lesion endpoints could not be ascribed solely to the absence of ACAT-1. Therefore, the approach was taken to obtain macrophages from the ACAT-1 knockout animals and introduce them into the LDL receptor deficient mice. Surprisingly, this manipulation resulted in more atherosclerosis and the accumulation of free cholesterol, however, the plaques contained reduced numbers of macrophages [113]. It is probable that the accumulation of free cholesterol, as a result of the lack of ACAT, led to macrophage death, and this in turn to a proinflammatory state, as suggested by the authors [113]. No analysis of HDL or HDL composition was provided, and possibly this would have provided clues as to why there was an apparent lack of sufficient cholesterol efflux under these conditions. The study is consistent with the aforementioned work with ACAT inhibitors in the sense that fewer macrophages were present when cholesterolfed rabbits were treated, however, in rabbits accumulation of free cholesterol was not observed. Moreover, in another study in which ACAT-1 was knocked out in all cells in LDL receptor deficient mice, not just macrophages, there was a reduction in atherosclerosis [114]. This would be more analogous to treatment with an ACAT inhibitor, as other cells adjacent to the macrophages cannot compensate with an enhancement of ACAT activity or expression. Taken together, it appears that the extreme situation described by Fazio et al. in which cholesterol efflux mechanisms are overwhelmed, may not be analogous to the treatment by inhibitors and in humans, inhibition of ACAT-1 would also alter lipoprotein composition, which could conceivably alter favourably cholesterol efflux capacity. Nonetheless, the results cannot be ignored, and at the very least clinical evaluation of inhibitors should proceed cautiously, potentially with monitoring of skin cholesterol as a surrogate. EVOLUTION OF ACAT INHIBITORS Medicinal chemists have been searching for inhibitors of ACAT for almost twenty years. Initial efforts in the search for potent ACAT inhibitors concentrated on the discovery of a purely ‘intestinal agent’, i.e., a nonabsorbable drug that does not
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CH3 0 (CWSCH3 H
(15) melinamide, ArtesB
(16) SaH 57-118
reach the systemic circulation and therefore has a lower potential for inducing toxicity. In addition, since this agent would act by inhibiting a specific enzyme, the doses would theoretically be much lower than the more traditional cholesterol absorption inhibitors (resins, cholesterol sequestrants, plant sterols) that act by altering lumenal events. The prototype compounds for this intestinal approach are the fatty acid amides e.g., melinamide (15), SaH 57-1 18 (16), SaH 58-035 ( 5 ) . These compounds inhibit cholesterol absorption and lower plasma cholesterol in rodents at high doses [ 1 151. As expected, the absorption of these extremely lipophilic compounds is highly variable and dependent on the vehicle used [ 1 161. In human clinical studies, several grams of SaH 57-1 18 (15) were required to inhibit cholesterol absorption and it was noted that the effect was greatest in patients who are hyperabsorbers of dietary cholesterol [117]. This variability coupled with dosing regimens involving oil vehicles and questionable efficacy led to disappointing experiences in the clinic with the result that it is now widely acknowledged that intestinal ACAT inhibition will not result in a significant lowering of plasma lipids. In addition, agents that regulate cholesterol biosynthesis (specifically HMG-CoA reductase inhibitors) have become a highly effective and preferred method of lipid regulation. As a result, the focus of ACAT inhibitor research has shifted away from nonabsorbed intestinal ACAT inhibitors to bioavailable inhibitors that affect ACAT at peripheral sites where it is further implicated in hypercholesterolemia and ultimately atherosclerosis. It had been noted that enhancing the absorption of some ACAT inhibitors, or using parenteral absorption, have led to increased efficacy, suggesting the possibility that the amount of unchanged drug in the liver may be important. Efforts were then undertaken to identify compounds with greater bioavailability, by decreasing the lipophilicity and increasing the hydrophilicity of these compounds. As previously described, inhibition of ACAT in the liver results in a decreased secretion of atherogenic apoB containing particles and an increased
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elimination of hepatic cholesterol. All of the non-pharmacological evidence suggested that liver ACAT was a worthwhile drug target, especially in dyslipidemias thought to be caused by an overproduction of apo B (e.g., familial combined hyperlipidemia). It has been mentioned that ACAT is responsible for the esterification and storage of cholesterol in macrophages and it has been hypothesized that inhibition of ACAT in the artery wall may prevent the formation of the fatty streak and the development of the clinically significant fibrous lesions. Several of the early ACAT inhibitors have been evaluated in animal models of atherosclerosis and evaluated for their ability to alter lesion extent and the cholesterol ester content of the lesion. However, demonstrating the effect of directly inhibiting ACAT at the artery wall has been complicated by the fact that the antiatherosclerotic activity observed could be indirectly associated with plasma cholesterol lowering and not the direct inhibition of arterial ACAT. A greater vascular benefit may be achieved by directly inhibiting arterial ACAT with a systemically bioavailable inhibitor. The first bioavailable ACAT inhibitor which showed direct anti-atherosclerotic effects was CI-976 (1). This compound was the result of an extensive SAR investigation on a series of fatty acid anilides derived from oleic acid. After amide hydrolysis was identified as a major problem with the series, a number of sterically hindered amides was prepared. From these series of analogues, (21-976 (1) was chosen for further development. It produced both significant reductions in non-HDL-cholesterol and elevations in the HDL-cholesterol in cholesterol-fed rats [ 1181. In a separate study, it was shown to be a competitive inhibitor of ACAT with respect to oleoyl-CoA [ 1 191. Additionally, it was found to produce marked reductions in atherosclerotic lesions in cholesterol-fed rabbits. This rabbit model is unique in that it uses a relatively low amount of dietary cholesterol and that both regression of a pre-established lesion (iliac-femoral) and progression of a lesion (thoracic aorta) can be evaluated in the same animal. This anti-atherosclerotic effect was observed at a dose level where plasma cholesterol levels were unchanged. This result clearly suggests that the compound may have a direct effect at the arterial wall [85]. It had previously been shown that CI-976 (1) was well absorbed with 29% bioavailability in rats when given as a 50 mg/kg suspension, however, lower bioavailability was observed in the monkey (5%) [ 1201. When dosed at 25 mg/kg to hypercholesterolemic rabbits, blood levels of 0.3 pg/mL (0.77 pM) were achieved. This value compared favorably to the median inhibiting concentration (0.61 pM,0.24 pg/mL) for ACAT inhibition in isolated mouse peritoneal macrophages [85]. However, the corollary to achieving oral bioavailability with a compound is that the chances of producing target organ toxicities increase. The first report of potential target organ toxicity with an orally bioavailable ACAT inhibitor originated with studies on PD 132301-2 (13). Administration of this compound
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to beagle dogs for two weeks at doses ranging from MOOmg/kg/day resulted in significant decreases (6&80%) in adrenal total and esterified cholesterol. At the 12 and 25 mg/kg doses, there was near complete suppression of the ACTH response and electrolyte disturbances characteristic of adrenocortical insuficiency. Unexpectedly, adrenocortical degeneration, including necrosis in the zona fasciculata and zona reticularis, was observed at all doses [121]. Similar findings were seen in the guinea pig [122] and cynomolgus monkey [ 1231. Further mechanistic studies in guinea pig adrenocortical cells in culture, suggested that the observed toxicity was not as a result of ACAT inhibition, but of ATP depletion resulting from direct inhibition of mitochondria1 respiration [ 1241. Similar effects were seen with other bioavailable ACAT inhibitors. PD 138142-15 (8) was the first water soluble ACAT inhibitor described in the literature. In beagle dogs, plasma concentrations of this compound increased with increasing dose: however,
I
(21)
(22) FRI 90809 R = S02CH3 (23) FR186485 R = CH3
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plasma concentrations were significantly lower during week 12 than those on day 1, possibly due to autoinduction. This compound caused a decrease in adrenal cholesterol esters and a non-reversible zonal atrophy and degeneration of the adrenal gland [ 1251. In addition, RP 73 163 (1 7) dosed to guinea pigs at doses of 100, 300 and 1000mg/kg for 3 weeks produced dose-related histopathologic changes in the adrenal cortex consisting of diffise coarse vacuolation of zona fasciculata cells similar to that observed in dogs with PD 132301-2 (13). These authors also concluded that the adrenal toxicity was not related to ACAT inhibition, since the pharmacologically inactive enantiomer of RP 73 163 (17) also caused similar toxic effects. Further mechanistic studies suggested that both RP 73 163 (1 7) and its enantiomer were metabolized to a common product, which was an inhibitor of adrenal cholesterol 17a-reductase activity. The authors speculated that this inhibition of cholesterol 17~-reductase activity may be the source of the coarse vacuolation produced by Rp 73163 (17), although ACAT inhibition could not be ruled out [126, 1271. It was also disclosed that FR 145237 (14), a potent ACAT inhibitor possessing direct antiatherosclerotic activity at the artery wall [90] also produced adrenal toxicity in normal rabbits after a single i.v. dose [128]. Surprisingly, FR 145237 (14) did not induce adrenal toxicity in LDL-receptor deficient WHHL rabbits, despite producing adrenal drug concentrations equivalent to those found in the normal rabbits. These authors speculated that, because of the 3-D structural similarity between FR 145237 (14) and PD 132301-2 (13), the toxicity induced by FR 145237 (14) may not be due to ACAT inhibition but to inhibition of mitochondria1 respiration. Another study in rabbits demonstrated severe cellular damage with XP767 (18) at doses between 10 and lOOmg/kg. However, a structurally related analogue, XR920 ( 1 9), produced no adrenal changes, despite having similar in vitro potency to XP767 (18) [ 1291. Long-term incubation of potent ACAT inhibitors (SaH 58-035 ( 5 ) and CP-113,818 (7)) with cholesterol-enriched mouse peritoneal macrophages led the authors to conclude that ACAT inhibition in these cells increased cell toxicity due to the build-up of intracellular free cholesterol concentrations. This was supported by the observations that cell toxicity paralleled the increase in intracellular free cholesterol concentrations and that removal of free cholesterol by the addition of extracellular cholesterol ‘acceptors’ or by blocking intracellular sterol transport relieved the ACAT inhibitor-induced toxicity. Thus, it was proposed that ACAT inhibition in vivo could induce cell death by destabilization of the plasma membrane upon cholesterol enrichment, unless sufficient cholesterol acceptors were present [82]. Despite these findings of adrenal toxicity with systemically available ACAT inhibitors, results in ACAT deficient transgenic mice support the position that the observed toxicity is not related to ACAT inhibition, since these animals
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developed normally and had no evidence of adrenal dysfunction although their adrenal cholesteryl esters were markedly reduced [ 1061. It has been possible to identify potent and bioavailable ACAT inhibitors that are not toxic to the adrenal gland. The adrenal glands of guinea pigs given compound (20) at doses of 10 to lOOmg/kg had minimal to mild increases in coarse vacuolation and reduced fine vacuolation of zona fasciculata cortical cells. There was no evidence of adrenal cortical necrosis at any dose. Since the beagle dog has been reported to be the most sensitive species for evaluating the adrenal effects of ACAT inhibitors [ 1301, compound (20) was evaluated in this model and compared with the tetrazole amide (21), a potent bioavailable ACAT inhibitor previously shown to be non-toxic to the adrenal gland in guinea pigs [13 I]. Adrenal toxicity was observed in dogs given (20) at 100 mg/kg for three weeks. These changes were not observed in dogs administered (2 1) at the same dose for the same duration of treatment. Plasma drug blood levels for compounds (20) and (21) were determined by HPLC. Both compounds achieved blood levels far in excess of the necessary to inhibit macrophage ACAT. These levels were highly variable and entirely consistent with the very lipophilic nature of the compounds. For compounds (20) and (21), blood levels of 200-690 ng/mL and 24&3 10 ng/mL were achieved, respectively. Thus, even though both compounds achieved blood levels adequate to inhibit ACAT in the periphery, only one compound (20) was shown to be adrenotoxic [132]. Additionally, FR190809 (22) and FR186485 (23) caused no adrenal necrosis in rabbits at a single dose of 5 mg/kg i.v. [ 1331. Thus, it has been demonstrated in the most sensitive species (dog) that it is possible to identify potent ACAT inhibitors, such as (2 1 ), that are bioavailable and not adrenotoxic. The search for effective ACAT inhibitors has evolved from the non-absorbable cholesterol absorption inhibitors that lower plasma cholesterol to compounds that inhibit the enzyme in peripheral tissues and thus may act as agents that affect the disease process directly. CLASSES OF ACAT INHIBITORS AMIDES
The search for inhibitors of this enzyme using traditional medicinal chemistry techniques, such as protein crystallization and X-ray crystallography, has been hampered by an inability to produce pure enzyme. This is not surprising since both of the isoforms, ACATl and ACAT2, are membrane bound enzymes with multiple transmembrane domains. A recent publication has compared the membrane topology of ACATl and ACAT2 [ 1341. Each enzyme has five.
I40
ACAT INHIBITORS: TREATMENT OF HYPERCHOLESTEROLEMIA
transmembrane domains with the N-terminus of each enzyme residing in the cytosol and the C-terminus is in the lumen of the endoplasmic reticulum (ER). The highest sequence similarity between the three ACAT gene family members (DGAT being the third) is in the cytoplasmic region containing the Asp-TrpTrp-Asn (DWWN) sequence. It has been speculated that this is the acyl-CoA binding site. An alternative model of membrane topology for ACATl has shown that seven transmembrane domains are present in ACAT 1 [ 1351. At the present time, this is the most complete picture we have of the structure of the ACAT enzyme. Such a sparcity of knowledge prevents the construction of a pharmacophore model for ACAT inhibition and the consequent de novo design of potent and specific inhibitors. Likewise, an understanding of the structural requirements for ACAT inhibition, fiom the wide variety of structural types of inhibitors available, remains unclear. Thus, ACAT inhibitors have historically been designed as substrate mimics. Since the function of ACAT is to transfer a fatty acid molecule from acyl-CoA to cholesterol, it is not surprising that fatty acid amides were initially identified as ACAT inhibitors. In fact, the only marketed ACAT inhibitor, melinamide (15) (available only in Japan as Artes'" ), is a simple linoleic acid amide. Early amide inhibitors such as CI-976 (1) [85], CP-113818 (7) [136] and SaH-58,035 (5) [137] all have long chain alkyl groups presumably interacting with the enzyme in a similar fashion to the natural substrate fatty acyl-CoA. A series of compounds has been reported that are structurally very similar to CP-113,818 (7). The difference being that the thioether side chain of CP113,818 (7) has been incorporated into a dihydrothiopyran ring system as exemplified by XP767 (18) and XR920 (19) [ 1291. These compounds were potent against ACAT derived from rat liver microsomes, with ICsO's of 32nM and 42 nM respectively. Interestingly, these two compounds displayed different pharmacological profiles. They displayed slight differences when ACAT inhibition was measured in rabbit adrenal cells (IC50 = 13 nM for XP767 (1 8) and ICso = 84 nM for XR920 (19)). The difference was even more pronounced in a model that measures the inhibition of the formation of human foam cells. In that screen, XP767 (1 8) remained very potent (ICso = 12 nM) while XR920 (19) was considerably less potent (ICsO= 708 nM). Additionally, as discussed above, XP767 (1 8) demonstrated adrenal toxicity while XR920 (19) did not. One of the more thoroughly studied amide ACAT inhibitors, HL-004 (1 1) (also recently identified as TS-962 [92]) is an acetanilide that features a longchain thio-alkyl group. This compound is potent against ACAT from a variety of cellular sources, producing an ICs0 of 1.7nM in rabbit intestinal microsomes, 2.2nM in rabbit liver microsomes and 7.9nM in microsomes from rabbit aorta [138]. A recent literature report shows that HL-004 (1 1) inhibits ACAT in HepG2 cells causing a decrease in cholesterol ester content [139]. In
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mouse peritoneal macrophages, HL-004 ( I 1) also caused a decrease in cholesterol esters and an increase in free cholesterol. When HDL was included in the culture, an increased efflux of free cholesterol was observed [76]. In addition to these in vitro potencies, hypolipidemic and anti-atherosclerotic effects have been demonstrated in a variety of animal models. In stroke-prone spontaneously hypertensive rats fed a high fat diet, HL-004 ( 1 I ) showed a dose dependent hypocholesterolemic effect as well as a decrease in arterial fat deposits [140]. Similar hypolipidemic effects were observed in normal rats fed a high fat diet. In this study, the lipid regulation was shown to be due to inhibition of cholesterol absorption in the intestine and ACAT inhibition in the liver [141]. In cholesterol-fed rabbits, HL-004 ( 1 1) was administered for 12 weeks at a dose of 25 mg/kg and it normalized total plasma cholesterol levels to the levels observed in normal chow fed rabbits. This reduction of total plasma cholesterol levels resulted in a decrease of atherosclerotic lesions observed in these animals [83]. A similar effect was reported in cholesterol-fed hamsters where HL-004 ( I 1) at a dose of 15 mg/kg normalized cholesterol levels and also decreased aortic lesion coverage. Interestingly, that protocol also included a lower dose cohort (approximately 1.5 mg/kg) that did not normalize plasma cholesterol levels yet still gave a decrease in aortic lesion coverage, indicative of a direct affect of this ACAT inhibitor at the artery wall [92]. Several research groups are investigating similar amides containing a long chain thioether group. For example, F 12511 (12) is a very potent ACAT inhibitor that is structurally very similar to HL-004 (1 1). The IC50 for F 1251 1 (12) is 59nM in rat liver microsomes and 41 nM in rabbit intestinal microsomes. It was also very potent in human cell lines, with an IC50 of 3nM in HepG2 cells, 7 nM in CaCo-2 cells, and 71 nM in THP-I cells [142]. Warner Lambert followed up its initial amide ACAT inhibitor (CI-976 (1)) with several very potent series of amides, including malonamide-esters like compound (20) (IC50= lOnM in rat liver microsomes) [I431 and heterocyclic amides such as compound (21) (IC5,= lOnM in rat liver microsomes) [131]. A recent report disclosed a series of indoline based amide ACAT inhibitors as exemplified by compound (24) [144]. Interestingly, in these compounds, the long chain alkyl group is not directly attached to the amide nitrogen as is usually the case with other fatty acid anilide ACAT inhibitors. These indoline compounds are potent ACAT inhibitors, compound (24) has an lC50 in rabbit intestinal microsomes of 90 nM. Additionally, compound (24) was shown to be bioavailable (C,,, = 716 ng/mL after 10 mg/kg dose in dogs) and to possess antioxidant properties that would presumably protect LDL from oxidation. Another series of amide ACAT inhibitors has been disclosed wherein the same branched alkyl group of CI-976 (1) was used but oxygen containing
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CH3
HCI
O Y N H (CH3)7CH3 t-Bu (24)
heterocycles were used in place of the trimethoxy aniline of CI-976 (1). TEI6522 (25) [I451 and TEI-6620 (26) [I461 are representative examples. Both TEI-6522 (25) and TEI-6620 (26) were potent against rabbit intestine and liver ACAT (Table 3.1). TEI-6620 (26) however, was a more potent inhibitor of foam cell formation as measured in rat peritoneal macrophages. TEI-6522 (25) and TEI-6620 (26) were both very efficacious in a cholesterol fed rat model where a 0.3 mg/kg dose caused 66% (TEI-6522) and 71% (TEI6620) reductions in total cholesterol after 3 days of dosing. Compound (27), a polyunsaturated acid anilide, was described as a more potent ACAT inhibitor than HL-004 (1 1) and CI-976 (1) when screened against ACAT from human sources [ 1471. Thus, in CaCo-2 cells, compound (27) had an ICso of 43 nM (cf. HL-004 (1 I), ICso = 1 13 nM and CI-976 (l), ICso = 838 nM). In HepG2 cells, compound (27) had an ICs0 = 36 nM while HL-004 (1 1) and
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(32) EAB-309
HCI
I
HCI
(33) YM17E
Table 3 . I . IN-VITRO COMPARISON OF TEI-6522 AND TEI-6620
Conipound TEI-6522 (25) TEI-6620 (26)
nM IC,, b Rabbit intestinal niicrosornes 13
20
Rabbit liver microsornes
Rai foam cell ,formation
16 9
160 30
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ACAT INHIBITORS: TREATMENT OF HYPERCHOLESTEROLEMIA
CI-976 (1) had IC50 values of 89 and 926 nM, respectively. Similar results were seen in microsomes from U937 cells where compound (27) had an IC50of 35 nM and the ICso values for HL-004 (1 1) and (21-976 (1) were 130 nM and 6 19 nM, respectively. Interestingly, in canine intestinal and liver microsomes, compound (27) had only micromolar potency (ICSo’s= 7.2 and 3.5 pM, respectively) while HL-004 (1 1) was still potent (IC50’s = 281 and 141 nM). This demonstrates that species differences in ACAT can often be dramatic and compounds can be made with differing selectivities across the various forms of ACAT. Even though compound (27) is potent in vitro, it is a lipophilic anilide and one would expect it to be a poorly bioavailable compound that may not reach the desired tissues. UREAS
Ureas represent a logical isosteric replacement for amides and indeed, a wide variety of ureas have been examined as ACAT inhibitors. DuP 128 (2) is a tri-substituted urea that has been extensively studied. DuP 128 (2) is a potent ACAT inhibitor = 10 nM against rat liver microsomes) that was designed to inhibit ACAT in the intestine and act as a cholesterol absorption inhibitor [ 1481. In 1994, initial phase I clinical results were reported for this drug [52]. In this clinical study, patients received 900, 1,800, or 3,60Omg/day for 7 weeks. At week 7, cholesterol absorption was measured using a dual isotope method. When compared to the baseline measurement of cholesterol absorption, the pooled DuP 128 (2) patients showed a modest 14% decrease in cholesterol absorption. At week 6, serum lipid concentrations were determined and DuP 128 (2) lowered total cholesterol by 3.9% (pooled across the 3 doses). Consequently, focus turned to identifying bioavailable ACAT inhibitors that could inhibit the enzyme in the liver and in the macrophages at the arterial wall [ 1491. Although it is a urea, DuP 128 (2) served as a starting point for a whole series of imidazole compounds that are discussed in a separate section below. FR145237 (14) represents another series of potent urea ACAT inhibitors (IC50 = 7.5 nM in rabbit intestinal microsomes) that showed anti-atherosclerotic effects in cholesterol fed rabbits. This compound would appear to be bioavailable as it gave a decrease in atherosclerotic lesion coverage in Watanabe heritable hyperlipidemic (WHHL) rabbits without significant effects on plasma lipids [90]. Although this compound had promising in vivo activity, it also displayed adrenal toxicity in rabbits and development was discontinued [ 1281. FR182980 (28) [ 1501 is also a potent ACAT inhibitor in vitro (ICso = 30 nM in rabbit intestinal microsomes), however, it too demonstrated adrenal toxicity in rabbits and dogs [ 15 11. A thorough investigation of a variety of urea ACAT inhibitors from this series revealed an apparent correlation between potent inhibition of foam cell formation in murine peritoneal macrophages and the observed adrenal toxicity [133]. As a result of this work, a series of second
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generation urea ACAT inhibitors were identified as exemplified by FR190809 (22) and FR186485 (23). These compounds were potent against ACAT from rabbit intestines (ICsO’s= 45 nM and 62 nM, respectively) but they were less potent inhibitors of foam cell formation in mouse peritoneal macrophages (240nM). These compounds and several others in this series with a similar activity profile did not exhibit significant adrenal toxicity [ 1521. Thus, the authors were able to use the mouse peritoneal macrophage model to screen for ACAT inhibitors with potential adrenal toxicity. A combination ACAT inhibitor/anti-oxidant, T-259 1 (29) has been designed and shown to be a potent inhibitor of foam cell formation in mouse peritoneal macrophages and 5774 (mouse macrophage) cells [ 1531. Interestingly, compound (29) was potent against ACAT in the 5774 cells (ICs0 = 67 nM) but was only modestly potent against ACAT from rabbit intestine (ICsO= 260 nM), liver (ICsO= 4,600 nM) and aorta (ICsO= 4,100 nM). T-259 1 (29) was also shown to be effective in protecting LDL from oxidation. FCE27677 (30) represents another very potent urea ACAT inhibitor [ 1541. This compound is potent against ACAT from a variety of sources; rabbit intestine (ICso = 9.3 &), rabbit aorta (ICsO= 7.0nM), rabbit liver (ICso = 32.8 nM), rat liver (ICsO= 38.9 nM), mouse liver (ICsO= 65.7 nM), monkey liver (ICso = 71.7 nM), HepG2 (human hepatoma, IC50= 30.0 nM), and 5774-A1 (mouse macrophages, IC50 = 30.2 nM). In addition, to this potent in vitro activity, compound (30) is an effective lipid lowering agent in several animal models where it suppressed the secretion of ApoB-100 containing lipoproteins from the liver [155]. TMP-153 (3I ) is another potent urea ACAT inhibitor that has been reported [ 1561. TMP- 153 (3 1) is very potent against liver and intestinal ACAT from a variety of animal species. For example, it has an IC50 of 9 n M in rat liver microsomes and 6.4nM in rat intestinal microsomes. This compound also displays potent lowering of plasma cholesterol in vivo with an ED50 of 0.25mg/kg when dosed for one week to cholesterol-fed rats [157]. E5324 (9) is a urea ACAT inhibitor that has progressed into clinical trials. This compound has ICsO’sof 53 nM, 160nM and 190 nM in microsomes prepared from rabbit intestine, liver and aorta respectively [74]. The latest reports on E5324 (9) indicated that it had anti-atherosclerotic effects both in a cholesterol fed rabbit model [88] and in WHHL rabbits [89]. In the latter model, the anti-atherosclerotic effect was observed without significant lowering of plasma lipid levels indicating direct inhibition of ACAT at the artery wall. EAB-309 (32) is also a potent urea ACAT inhibitor [I581 with an ICso of 5nM against ACAT from rat liver microsomes. This compound was not detected in the plasma or liver of rats receiving an oral dose of 10mg/kg. In fact, using [I4C] labelled EAB-309, autoradiography showed that EAB-309 (32) was completely localized at the intestine. Thus, EAB-309 (32) is not absorbed and
I46
ACAT INHIBITORS: TREATMENT OF HYPERCHOLESTEROLEMIA
was shown to lower plasma cholesterol primarily by inhibition of intestinal ACAT when dosed in vivo [159]. Some researchers have pursued high molecular weight, bis-ureas as ACAT inhibitors. YM17E (33) is a potent ACAT inhibitor (ICso in rabbit liver microsomes = 45 nM), which has shown some side-effects (diarrhoea) in dogs [ 1601. Despite the high molecular weight, it does have a favourable pharmacokinetic profile which affords it good bioavailability in rats [161]. As a result, this compound has been advanced into clinical trials. In an initial clinical study, YM17E (33) was shown to lower serum cholesterol levels by 25%, but only at the highest dose of 300 mg b.i.d. and diarrhoea was observed in all patients [ 1621. A very similar bis-urea has also been reported (NTE-122, (10)) as a very potent ACAT inhibitor with ICsOvalues of 7.6 nM, 4.4 nM and 9.6 nM in microsomes from rabbit small intestine, liver and aorta, respectively [163]. To support the hypothesis that NTE-122 (10) will have clinical effects at the three major targets of ACAT intervention (intestine, liver, and aortic macrophages), a series of papers document its effects in the appropriate human cell lines. In CaCo-2 cells, NTE-122 (10) was shown to have an ICSo of 4.7nM and it markedly decreased the secretion of cholesterol esters from these cells [ 1641. In HepG2 cells, NTE- 122 (1 0) has an ICso of 6.0 nM and it markedly decreased the concentrations of cellular and secreted cholesterol esters [165]. In THP-1 cells, NTE-122 (10) has an ICso of 0.88nM and again, it markedly decreased the concentrations of cellular cholesterol esters and it appeared to increase the HDL promoted efflux of free cholesterol [75]. A unique approach to designing a substrate based urea ACAT inhibitor is exemplified by F1394 (34). Like many other urea ACAT inhibitors, this compound has a long chain alkyl group, which presumably mimics the fatty acid natural substrate. The other side of the urea in this compound is a pantothenic acid moiety that is designed to mimic the terminal end of acyl-CoA. Thus, this compound represents a concerted effort to mimic the coenzyme A-fatty acid
0 OH
n
rsK(CHz).CH=CH(CHz),CH3
a d e n o s i n e - 3 1 - ~NHN ~ phosphoric 0 oleoyl-CoA acid-5'-pyroph osphorlc acld
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substrate of ACAT. The result is a very potent inhibitor against ACAT from a variety of sources; rat liver microsomes = 6 nM), rabbit intestinal microsomes ( ICso = 1 I nM) 5774 mouse macrophage cells (IC50 = 32 nM) [ 1661 and HepG2 cells (ICs0 = 42 nM) [ 1671. Additionally, this compound has been reported to display antiatherosclerotic effects in cholesterol fed rabbits [ 1681. This compound is reported to still be in clinical development [ 1691. Like the prototypical fatty-acid anilides, most of the urea ACAT inhibitors are very lipophilic with clog P values well above 6 and little aqueous solubility. This results in compounds that are poorly absorbed. To address this issue, Warner Lambert had prepared PD132301-2 (13). This compound had good aqueous solubility (0.7 mg/mL) when compared to the lipophilic traditional ACAT inhibitors [170] and this resulted in moderate bioavailability in rats (34%) and dogs (28%) [171]. Interestingly, sulfonyl ureas (a well known series of oral hypoglycemic agents) are completely absorbed after oral administration [ 1721. Based upon this structural template, several series of sulfonylated and acylated ureas have been prepared incorporating structural features from known ACAT pharmacophores. Thus, a series of acylated ureas (and related carbamates) as exemplified by compounds (35-38) were reported in 1994 [173]. Table 3.2 shows the ACAT inhibitory activity measured in rabbit intestinal microsomes and the change in plasma total cholesterol (TC) measured in an acute, cholesterol-fed rat model. The series of acylated carbamates, as exemplified by compound (37), typically gave more potent inhibitors in vitro. In vivo, the TC concentrations for all three series of carbamates (compounds (36), (37) and (38)) were decreased -20% as compared to cholesterol-fed control animals. Interestingly, the aminocarbonyl urea series (exemplified by compound (35))gave little or no lipid lowering in vivo.
Table 3.2. COMPARISON OF ACYLATED UREAS AND CARBAMATES
Compound
X
Y
(35) (36) (37) (38)
NH NH 0 0
NH 0
0 S
Rabbit intestinal A CAT ICw (W)
> 5.0 0.98 0.1 I 3.2
96 A plasma cholesterol (30mgfkg dose) 0 -25 -24 -20
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ACAT INHIBITORS: TREATMENT OF HYPERCHOLESTEROLEMIA
A series of sulfonated ureas and carbamates was also prepared [ 1741 and the in vitro ACAT inhibition and in vivo TC lowering of several analogues is shown in Table 3.3 Compound (42) from the oxysulfonyl carbamate series was the most active compound in vivo, despite relatively weak ACAT inhibition in vitro. Compound (42) had little aqueous solubility, but the sodium salt ((8), CI-999) had more desirable physical properties [175]. The partition coefficient (log P) of CI-999 (8), as measured by the shake flask method, was 2.98 at pH 7.4. The aqueous solubility of compound (8) at pH values between 6.87 and 9.85 was a constant 2 1,000 pg/ml. This combination of low lipophilicity and aqueous solubility was unique in the ACAT inhibitor field and it resulted in a bioavailable compound. For example, a single 50mg/kg dose of CI-999 (8) in chow-fed rats gave a plasma concentration of 17 pg/ml, 3 hours postdose [175]. C1-999 (8) was, however, chemically unstable in acidic solutions. The degradation involved cleavage of the carbamate group and so a series of more stable acyl sulfamates was designed resulting in the identification of ((3), CI1011, avasimibe). Like the oxysulfonyl carbamates, these compounds were initially thought to be weak ACAT inhibitors in the microsomal assay, but effective hypocholesterolemic agents in vivo. For example, CI-1011 (3) had an ICso of 12pM in rat liver microsomes. However, CI-1011 (3) is highly membrane bound and since ACAT is an integral membrane protein, the ACAT inhibition of CI-1011 depends upon its concentration within the membrane. Therefore, the inhibitory activity for this compound is highly dependent on the concentration of microsomes used in the assay. Thus, the ICs0 of CI-1011 (3) was found to be 0.7 pM when 0.2 mg/mL of microsomes
Table 3.3. SULFONYLATED UREAS AND CARBAMATES
Compound
X
Y
Rabbit intestinn1 ACA T Icso (PW
(39) (40) (41) (42) (43)
NH NH 0 0 S
NH 0 NH 0 0
II 8.7 25.8 9.4 8.7
% ! A plasma cholesterol (30 mg f kg dose)
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are used instead of the standard 1 mg/mL. Restoring the membrane concentration by adding reconstituted lipids back to the media gave an IC50 value close to 12.0pM. In addition to this observation, it has been shown that this compound also binds avidly to bovine serum albumin (BSA) which is present in the assay at a concentration of 50 pM. Thus, as with the membrane binding, the ICsO values are overestimated because of the BSA binding. At 20pM BSA, the ICS0 in IC-21 macrophages was 0.76pM, this value decreased to 60nM when no BSA was in the assay. Collectively, these data indicate that CI-loll (3) is indeed a potent ACAT inhibitor and that the previous microsoma1 data is more a measure of the affinity of CI-1011 (3) for BSA than of ACAT inhibition. In vivo, CI-101 1 (3) gave a 73% decrease in TC in the cholesterol-fed rat model. The anti-atherosclerotic activity of CI-1011 (3) was evaluated in an injured, cholesterol-fed rabbit model. In this model, CI-1011 (3) decreased mean plasma total cholesterol levels relative to dietary intervention alone by 70%. Plasma C1-1011 (3) Cmax levels of 66-302 ng/mL were achieved. Liver total cholesterol and cholesteryl ester (CE) content were reduced by 83% and 92%, respectively, and the CE content of the thoracic aorta and the iliac-femoral artery were reduced by 39% and 36%, respectively. CI-1011 (3) reduced the percent lesion coverage of the thoracic aorta from 34% in control animals to 2&22% in the drug-treated animals. This data shows that CI-1011 (3) affects the CE enrichment, size, monocyte-macrophage content and complexity of the developing atherosclerotic lesions. Another interesting feature of this ACAT inhibitor is its ability to demonstrate hypolipidemic effects in chow fed rats [ 1761 and cynomologous monkeys [ 1771. Avasimibe (3) has been progressed to the clinic and an initial report has indicated that it lowered both VLDL and triglyceride levels [ 1781.
IMIDAZOLE/IMIDAZOLINES
The 4,5-diphenylimidazole ring system was initially developed in the series of ureas that resulted in DuP 128 (2) described above. The disappointing clinical results with DuP 128 (2) led to a re-evaluation of the imidazole series with emphasis on improving both bioavailability and ACAT inhibition at the liver and macrophages. A great deal of SAR has been done around the DuP 128 series [148, 1791. One of the early analogues to emerge from this series of diphenyl imidazoles was compound (44). This compound gave more potent ACAT inhibition in 5774 macrophage cells (I& = 3 nM) than observed in rat liver microsomes ( ICsO= 80 nM) [ 1801. Unfortunately, the lack of significant bioavailability was still an issue with these compounds.
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ACAT INHIBITORS: TREATMENT OF HYPERCHOLESTEROLEMIA
h
Interestingly, an oxidative rearrangement of the 4,5-diphenylimidazoles gave a series of imidazolines [ 1811. These compounds were modestly potent ACAT inhibitors in vitro as exemplified by compound (45),which is reported to have an ICs0 values of 1.4-2.0 pM against rat liver microsomes and 1 1.&I 9.7 pM in 5774 cells. Despite the moderate ACAT inhibitory activity, this compound was very efficacious in vivo. A 10mg/kg oral dose in cholesterol-fed hamsters gave a 63% decrease in plasma cholesterol concentrations and in cholesterol-fed rabbits, a 5 mg/kg oral dose resulted in a 75% reduction in plasma cholesterol levels [ 182, 1831. The activity in this series of compounds is stereospecific such that the stereoisomer shown (compound (45)) has the potencies described above, while its enantiomer has an IC50 of 55 pM against rat liver microsomes and gave only a 12% lowering of plasma cholesterol in the cholesterol-fed rabbit model. Examination of crystal structures of chiral enantiomers in this series demonstrated that the R enantiomers (such as compound (45)) overlay nicely with the 3-D structure of cholesterol while the S enantiomers do not. The phenyl ring that is directly attached to the imidazoline core overlays with the A ring of cholesterol while the benzamide ring occupies similar space to the alkyl side chain of cholesterol. The authors propose that these compounds are acting as a cholesterol mimics in contrast to most of the other ACAT inhibitors in de-
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velopment which were designed as fatty-acyl-CoA mimics [ 1841. The authors also suggest that the dramatic in vivo potency, despite modest ACAT inhibition in vitro, may be explained by this ‘cholesterol mimic’ interacting at other enzymes or receptors in the complex system of lipid regulation. Another imidazole compound that has been in development as an ACAT inhibitor is RP-73 163 (1 7). This drug is a potent ACAT inhibitor = 86 nM against rat liver enzyme) [ 1261, that was also bioavailable in a variety of animal species [ 1851. Development of this compound was subsequently discontinued when adrenal toxicity was observed in dogs. A related series of 4,s-diphenylimidazoles were examined, exemplified by compound (46) [ 1861. These compounds were designed as back-ups to RP-73 163 (17) that would improve the in vitro activity and presumably address the issue of adrenal toxicity in this series. NATURAL PRODUCTS
One of the growth areas in the search for ACAT inhibitors is the identification of novel structural types of microbial origin, which are shown to inhibit ACAT to varying degrees. These compounds may provide additional insight into the pharmacophore necessary for ACAT inhibition, and allow the preparation of potentially more potent analogues. (47), R’=R2=R3=Bz (48). R’=R2=R3=s-Bu (49), R’=i-Pr; R2=R3=s-Bu (50),R’=R2=R3=i-Pr (51), R‘=R2=i-Pr; R3=s-Bu (52),R’=R2=i-Pr; R3=i-Bu (53). R’=i-Pr; R2=i-Bu; R3=s-Bu (54), R’=i-Bu, R2=R3=s-Bu
(56).R=CH?OH (57). R=CHO
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ACAT INHIBITORS: TREATMENT OF HYPERCHOLESTEROLEMIA
(59) R’ = H. R2 = CH2CH=C(CH&
(60)R’ = CH3, R2 = CH(OH)CHzCH(CH3)2 = H,R2 = CH2CH=C(CH3)2 (diastereomer of 59) (61) R’
CH3
Fungal strains Fusarium spp. FO-740 and FO-1305 were shown to produce a number of cyclodepsipeptide antibiotics, which were subsequently shown to inhibit ACAT. The active compounds were beauvericin (47) from the former strain and seven enniatins (A (48), A1 (49), B (50), B1 (51), D (52), E (53) and F (54)). All of these compounds inhibited ACAT using rat liver microsomes with ICso values of 3 (47), 22 (48), 49 (49), 113 (50), 73 (51), 87 (52), 57 (53), 40 (54) pM,respectively. In this study, for comparison, the ICso for CL-277082 was 6.6pM. These compounds were also shown to inhibit cholesteryl ester formation in J 774 macrophages, the IC50for (47) was 0.17 pM, the IC50values of the enniatins ranged from 0.28-1.1 pM. Whether the ACAT inhibition displayed by these compounds is independent of the ionophoric activity is still unclear [ 187, 1881. Purpactins A, B (55, 56) and C (57), isolated from the fermentation broth of Penicillium pupurogenom (FO-608), were shown to be modestly potent inhibitors (ICs0 ’s range from 121-126pM) of ACAT using rat liver microsomes. In J 774 macrophages, (55) was 100-fold more potent in inhibiting cholesteryl ester formation, although cell cytotoxicity was observed [189, 1901. AS-186a, b, c, d and g (58, 55, 59-61) were isolated from the cultured broth of Penicillium asperosporum KY1635 and shown to inhibit ACAT from rabbit liver microsomes with IC50’s of 22.9, 8.2, 11.5, 12.4 and 13.9 pM, respectively. AS-1 86b was shown to be identical in structure to purpactin A [191]. Glisoprenins A (62) and B (63) were isolated from the fermentation broth of Gliocladium sp. FO-1513 and shown to be inhibitors of ACAT (ICso =46 and 61 pM, respectively) using rat liver microsomes. In a cellular assay using J 774 macrophages, the compounds were much more potent, with ICso values of 1.2 and 0.57 pM, respectively. Preliminary in vivo testing with (63) showed that, when administered orally at a dose of 50 mg/kg to hamsters, cholesterol absorption was inhibited by 25% [ 192, 1931.
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(67) R'=CH*OH; R~=CHO (68) R'=COOH; R2=CH0
(69)R'=CH~OH;R~=COOH
Acaterin (64) was the first ACAT inhibitor isolated from a bacterial source. It was isolated from a culture broth of Pseudomonas sp. A92. In the presence of oxidized LDL, (64) inhibited cholesteryl ester synthesis in J 774 macrophages with an ICSO of 45pM, and in rat liver microsomes kinetic studies showed the inhibition (ICs0 = 120 pM) to be non-competitive with respect to oleyl-CoA and reversible [ 1941. The absolute configuration of acaterin has been determined and the synthesis of all the four possible stereoisomers has been accomplished [ 1951. A closely related compound, AS-I83 (65), was isolated from the culture broth of a fungus, Scedosporium sp. SPC-I 5549. This compound inhibited ACAT, using rat liver microsomes with an ICSOof 0.94pM. It also displayed cellular activity, inhibiting cholesteryl ester formation in Hep G2, CaCo-2 and THP-1 cells with ICsO values of 18.1, 25.5, and 34.5pM,respectively [196]. Lateritin (66) was isolated from Gibberella lateritium I F 0 7188 and shown to inhibit ACAT from rat liver microsomes with an ICSOof 5.7pM in a time-dependant and irreversible fashion [ 1971. Helminthosporol (67) and related compounds (68,
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ACAT INHIBITORS: TREATMENT OF HYPERCHOLESTEROLEMIA
69) were isolated from a culture of Cochliobolus sativus I F 0 7259, and showed to be weak inhibitors of rat liver microsomal ACAT (ICSo=0.5, 1.5, 1 .O mM, respectively) [ 1981. The terpendoles A-D (70-73) were isolated from the culture broth of a fungal strain FO-2546, which was subsequently classified as a new genus, Albophoma yamanashiensis. Each compound contains indole and diterpene moieties and they are structurally related to the known fungal metabolites, emindole SB and paspaline. Terpendole C (72) was the most potent ACAT inhibitor with an ICSO= 2.1 pM in a rat liver microsome assay. Terpendoles D, A and B had ICs0’s of 3.2, 15.1 and 26.8pM, respectively. These compounds were also evaluated for their activity in cellular assays. Using 5774 macrophages, terpendole D (73) had an IC50= 48 nM and exhibited the widest margin between cell toxicity and ACAT inhibition [199, 2001. Using a different culture, terpendoles E-L (74-8 I ) were isolated and characterized. These analogue were much weaker ACAT inhibitors with ICSO’s between 39-388 pM [201].
(70) Terpendole A R =
30
k
(72) Terpendole C R =
(74) Terpendole E R = CH3
r
,
(75) Terpendole F R = CH20H (76) Terpendole G R = CHO
Epi-cochlioquinone A (82) was isolated from the fermentation broth of Stachybotrys bisbyi and shown to inhibit ACAT with an I G O of 1.7 pM. It also inhibited cholesterol absorption in rats by 50% at a dose of 75 mg/kg [202].
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(71) Terpendole B R = H; R' = H; R2 = H
(73) Terpendole D R = H; R' = OH; R2 =
(78) Terpendole I R = OH; R' = OH; R2 = H (79) Terpendole J R = OH; R' = OH; R2 =
(77) Terpendole H
(80) Terpendole K
(81) Terpendole L
Gypsetin (83) was isolated from the fermentation broth of Nannizzia gypsea vat-.Incurvatu and shown to competively inhibit ACAT with an ICs0 of 18 pM.
It also inhibited cholesterol ester formation in cultured macrophages with an ICg) of 0.65 pM [203, 2041.
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ACAT INHIBITORS: TREATMENT OF HYPERCHOLESTEROLEMIA
HO (82) Epi-cochlioquinone A
(85) lsochrornophilone IV R=
0
P ,
HO
R = Ac
(87) lsochrornophilone VI
Azaphilones are yellow and orange plant pigments produced by fungi, several new members of this class, named isochromophilones 111-VI (84-87) were isolated from the culture broth of Penicillium multicolor and shown to weakly inhibit ACAT with ICso’s between 5&100 pM [205]. One of the most exciting series of compounds shown to potently inhibit ACAT are the pyripyropenes. These have attracted much attention in the field due to their extremely potent ACAT inhibition and the challenges of structural elucidation, chemical synthesis and the development of structure-activity relationships. Initially, four compounds (pyripyropenes A-D ( 8 8 H 9 1)) were isolated from Aspergillus fumigatus FO- 1289. These compounds were shown to potently inhibit ACAT, derived from rat liver microsomes with ICs~’sof 58, 1 17, 53 and 268 nM, respectively [206]. Pyripyropene A was also shown to be
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R’
(88)Pyripyropene A: R’= R2= R3= OAc, R4 = OH (89) Pynpyropene B: R’= OC(0)CH2CH3, R2= R3= OAc, R4= OH (90) Pyripyropene C: R’= OAc, R2= OC(O)CH2CH3, R3= OAc, R4= OH (91) Pyripyropene D: R1= R2= OAc, R3= OC(O)CHzCH3, R4= OH (92) Pyripyropene E: R’= R2= H, R3= OAc, R4= H (93) Pyripyropene F: R’= R2= H, R3= OC(O)CH&H3, R4= H (94) Pyripyropene G: R’= R2= H. R3= OAc, R4= OH (95) Pyripyropene H: R’= R2= H, R3= OC(0)CH2CH3, R4= OH (96) Pyripyropene I: R’= R2= R3= OC(0)CH2CH3, R4= OH (97) Pyripyropene J: R’= OAc, R2= R3= OC(0)CH2CH3, R4= OH (98) Pyripyropene K: R’= OC(0)CH2CH3, R2= OAc. R3= OC(0)CH2CH3, R4= OH (99) Pyripyropene L: R‘= R2= OC(O)CH&H3, R3= OAc, R4= OH (100) Pyripyropene M: R’= OAc, R2= OC(0)CH2CH3, R3= OAc. R4= H (101) Pyripyropene N: R1= OC(0)CHpCH3, R2= H, R3= OC(O)CH2CH3, R4= OH (102) Pyripyropene 0: R’= OAc, R2= H, R3= OAc, R4= H (103) Pyripyropene P: R’= OC(0)CH2CH3,R2= H, R3= OAc, R4= H (104) Pyripyropene Q: R’= OC(O)CH2CH3, R2= H, R3= OAc, R4= OH (105) Pyripyropene R: R’= OAc, R2= H, R3= OC(0)CH2CH3, R4= H
effective at reducing cholesterol absorption in hamsters by 3 2 4 6 % at doses of 25-75 mg/kg [207]. The structural elucidation of pyripyropenes A-D has been reported [208] and the relative and absolute steroechemistry of pyripyropene A has been determined [209] and this led to the first total synthesis of (+)pyripyropene A (88) in 1995 [2 lo]. Interestingly, a mutant strain of Aspergillus fumigatus, FO- 1289-2501, produced 14 new pyripyropenes, E-R (92-105), which were also shown to be potent ACAT inhibitors [211, 2121. The simplest member of this family, pyripyropene E (92), has been synthesized as its natural ( + )-enantiomer using a biomimetic approach and farnesyl acetate as a starting material [213]. Pyripyropene E was proved to be identical to GERI-BPOO1, reported by Bok et ul [214, 2151. A similar biomimetic approach to that previously mentioned gave racemic GERI-BPOO1 [216]. Another novel structural type, GERI-BP002-A ( 106) was isolated from Aspergillus fumigatus F93 and shown to be a weak inhibitor of ACAT (50% inhibition at 50pM) [217]. Modification of the pyripyropenes has yielded some of the most potent ACAT
,
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ACAT INHIBITORS: TREATMENT OF HYPERCHOLESTEROLEMIA
(106) GERI-BPOOZ-A
inhibitors synthesized so far. Pyripyropene A has 4 hydroxyl groups (acetylated at positions 1-, 7-, and 11-, free at -13) available for chemical modification. Removal of all the acetyl groups led to a complete loss of ACAT inhibitory activity. Modification, by incorporating a longer acyl chain, at positions 1-,1 1and 13- led, in general, to weaker inhibitors. However, the 7-0-acyl derivatives showed potent ACAT inhibition. The 7-0-valeryl derivative has an ICS0= 13 nM. Introduction of a methanesulfonyl group at the 1 1 -position gave a compound with an ICs0 = 19 nM that showed a dose-dependent inhibition of cholesterol absorption in hamsters with an ED50 = 10 mg/kg, tenfold more potent than pyripyropene A [218, 2191. In addition, studies have shown that cyclization to form 1,ll-cyclic acetal derivatives can yield highly potent inhibitors. The benzylidene analogue (107) has an ICS0= 6 nM and was as effective as pyripyropene A at inhibiting cholesterol absorption in the hamster [220]. Further studies have shown that the pyridine-pyrone moiety is essential for ACAT inhibitory activity [221]. Microbes have not been the sole source of novel ACAT inhibitors. Many new structural types are being isolated from plant materials prevalent in the field of herbal medicine. Ginseng has been used as a herbal medicine in Korea, Japan and China for centuries. It has been shown that ginseng lowered both total and LDL-cholesterol in both monkey and human, while raising HDL-cholesterol [222, 2231. A number of different compounds have been extracted from ginseng samples and shown to inhibit ACAT. The polyacetylene analogues (108-1 11) were
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R,-) HO
(108) Panaxynol R = (109) Panaxydol R =
nCHde u ( C H 2 ) 6 C H 3
w
(1 10) Panaxydiol R =
(1 11) Panaxytriol R =
H6
(112) R = (113) R =
(115)R=
\
OH
A
c
O OH
V
(116) R =
0 OAc (114)R=
(117) R =
J OH
(118)R= H O -
isolated from Panax ginseng and shown to inhibit ACAT with IC~O’Sof 94, 80, 45 and 79 pM, respectively [224]. A number of closely related polyacetylenes ( 1 12-1 18) were isolated from the root of Gymnaster koraiensis, an endemic plant in Korea. As well as displaying anti-proliferative activity against a number of cancer cells and inhibiting nitric oxide production, these compounds also inhibited ACAT with ICs0’s in the 35-55 pg/mL range [225]. Methanol extracts of Korean ginseng roots produced ginseng saponins which weakly inhibited ACAT. However, acid hydrolysis produced the deglycosylated sapogenins ( 1 19-126) that inhibited ACAT with IC~O’Sof 10, 6, 30, 24, 55, 12, 25, and 28 pM, respectively [226]. Similarly, a total of 17 triterpenoid saponins were isolated from the leaves of Ilex kudincha, a herbal tea used in the treatment of obesity in the People’s Republic of China. Most of the compounds
160
ACAT INHIBITORS: TREATMENT OF HYPERCHOLESTEROLEMIA
(1 19) (20R)-ProtopanaxadioI R = H, R1=
~a,
(120) (20~)-~rotopanaxatrio1 R =OH, R'=
H%,*,
4.
(121) (20S)-ProtopanaxadioI R = H, R'=
HO
\
(122) (20S)-Protopanaxatriol R = OH, R'=
(123) (2OR)-PanaxadiolR = H. R'= (124) (20R)-Panaxatriol R = OH, R'= (125) (20s)-Panaxadiol R
CH3%,,
cH%,.
H, R'= CH3
(126) (20s)-PanaxatriolR = OH, R'=
\
CH3\
inhibited ACAT weakly with the most potent compound (127) having an IC50 of 44 pM [227, 2281. The leaves of Magnolia obavata have been used as a stomachic herb in Korea, China and Japan for centuries. A methanolic extract of the leaves was found to contain a number of lignans that inhibited ACAT. One of them, obovatol (128), inhibited ACAT derived from rat liver microsomes with an ICsO of 42 pM [229]. In a separate study, 15 lignans were isolated from the fruits of Schizandra chinensis, the leaves of Machilus thunbergii, and the flower buds of Magnolia denudata. They were identified as gomisins, schizandrin, wuweizisu, schizantherin, licarins and machilin and were found to inhibit ACAT with ICso values of 25-200 pM. Gomisin N (129) is the most potent inhibitor with an IC50 of 25 pM [230].
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-
(127) llekudinol B
(128) Obovatol
CH3O
OH OH (129) Gomisin N (130) Kudingoside A R =
(131) Kudingoside B R =
&"
Ku-Ding-Cha (Ligustrum pedunculare REHD) is drunk as a tea in the southern and western regions of China. Two monoterpene glycosides, kudingosides A (130) and B (13 1) were isolated and shown to inhibit ACAT with ICso values of 2.7 and 2.8 mM, respectively [23 I]. Another herbal tea, OKGK, has also been shown to inhibit intestinal ACAT activity but not hepatic ACAT activity [232]. Naringenin, a citrus bioflavonoid, was shown to lower significantly plasma total cholesterol in rats fed a high cholesterol diet and 0.1% naringenin supplementation. It was also shown that naringenin inhibited the activities of both ACAT and HMG-CoA reductase [233]. Glucobrassicin a secondary plant metabolite found in cruciferous vegetables such as broccoli, cabbage and cauliflower undergoes autolysis during
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ACAT INHIBITORS: TREATMENT OF HYPERCHOLESTEROLEMIA
mastication to indole-3-carbinol, which is then oligomerized in the acidic environment of the stomach to yield a variety of oligomers that have been shown to weakly inhibit ACAT [234]. It has been reported that the pentacyclic triterpene ester (132), found in rabbit and human tissue, irreversibly inhibits microsomal ACAT (ICs0 = 20 pM). This compound is structurally similar to known plant terpenes and since it is not synthesized endogenously in the rabbit, the presence of (132) in various tissues may be due to the exogenous dietary absorption of plant triterpenes [235].
CLINICAL EXPERIENCE To date, the clinical experience with ACAT inhibitors has been disappointing [236]. Early clinical trials used the non-absorbable intestinal ACAT inhibitors. The only marketed ACAT inhibitor, melinamide (Artes” ) (15) reduced total cholesterol by 20% in hypercholesterolemic patients after 12 months administration at a dose of 2.25 g/day [237]. Both SaH 57-1 18 (16) and SaH 58-035 (5) were studied in Phase I clinical trials and found to be safe at doses as high as 5 g/day and they also reduced cholesterol absorption by 70% [ 1 171. In contrast to this finding, when the tri-substituted urea, CL 277082 (4) was dosed to eight patients at a dose of 750mg/day, there was no effect on cholesterol absorption or any plasma lipoprotein despite attaining significant blood levels [5 I]. The closely related, DuP 128 (2) was dosed to 30 patients for seven weeks and gave a poorly defined dose response for cholesterol absorption inhibition (%17.5% decrease) at doses between 90&3,600 mg/day. In addition, there were insignificant reductions in total (3.9%) and LDL-cholesterol (4.9%) [52]. The disubstituted urea, BW447A (6) was well tolerated by human volunteers at all doses (25-800 mg/day), however, the plasma levels of drug were barely quantifiable. Dosing to eight patients for 10 days at a dose of 200 mg twice daily gave no significant changes in any lipid parameter [238]. In single and repeated oral dose studies at 150mg b.i.d., YMl7E (33) produced no significant changes in serum cholesterol levels but all patients receiving the drug developed diarrhoea. Extensive SAR studies on the disubstituted urea class of inhibitors identified E5324 (9) as a systemically bioavailable ACAT inhibitor. This compound was shown to be orally bioavailable in rats (43%) and dogs (29%) and when dosed to healthy volunteers for eight days at a dose of 200 mg/day t.i.d., blood levels of 2 I00 ng/mL were achieved at day seven. Modest reductions (14%) in LDLcholesterol were observed along with similar reductions in HDL-cholesterol [239]. Interestingly, avasimibe (3) lowered both VLDL and triglycerides by approximately 1 6 3 2 % at doses ranging from 5&500mg/day in an eight-week study in 130 patients with combined hyperlipidemia [240].
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As can be seen from these studies the true utility of an ACAT inhibitor may not be as a lipid modulating agent, but as a true anti-atherosclerotic agent acting at the artery wall at the site of the disease. The challenge will be to demonstrate clinical efficacy at the artery wall without the benefit of a plasma marker of atherosclerotic disease
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Progress in Medicinal Chemistry Vol. 39, Edited by F.D. King and A.W. Oxford 0 2002 Elsevier Science B.V. All rights reserved ~
4 Growth Hormone Secretagogues: Discovery of Small Orally Active Molecules by Peptidomimetic Strategies MICHAEL ANKERSEN Medicinal Chemistry Research I. Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Denmark
INTRODUCTION Reverse drug discovery Release of growth liornione from the pituitary Discovery of peptidic growth hormone secretagogues
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CHEMISTRY OF GROWTH HORMONE SECRETAGOGUES Benzolactam-based GHSs Thiourea-. oxindole- and quinazolinone-based GI-ISs Spiropiperidine-based GHSs (MK677 analogues) Peptidic GHSs leading to smaller analogues Peptidomimetic GHSs derived from ipamorelin N- Termini ~J-NUI und o-Phe nioieiies C- Termini
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DISCOVERY STRATEGIES OF PEPTIDOMIMETIC GROWTH HORMONE SECRETAGOGUES
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MECHANISM OF ACTION OF GROWTH HORMONE SECRETAGOGUES
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CLINICAL STUDIES WITH GROWTH HORMONE SECRETAGOGUES Acute GH releasc GH deficiency (GI ID)
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Elderly Obesity Catabolism Sleep enhancement Side- and adverse-effects
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INTRODUCTION Since peptides are essential to many pharmacological processes they are often of great interest in the drug-discovery process [l, 21. However, major drawbacks for peptides as drugs are their poor pharmacokinetic properties, such as poor oral bioavailability and short biological half-lives. It is therefore one of the most challenging goals of modern medicinal chemistry to find rational principles for transforming the information provided by peptide ligands into lowmolecular-weight non-peptide molecules that bind to a target receptor. Such compounds are called peptidomimetics [ 3 4 ] . Low molecular weight peptidomimetics should elicit similar pharmacodynamic effects as the native peptide, but are expected to possess desirable pharmacokinetic properties superior to natural peptides, including good oral activity and long duration of action. Such compounds are therefore considered more useful targets for the drugdiscovery process. In principle, two types of peptidomimetics can be depicted. In the first one, the proposed pharmacophoric groups of the native peptide are kept in the correct spatial arrangement, but the peptide backbone is replaced with a non-peptidic scaffold. Such a scaffold may either be reduced amides (i.e. methyleneamines), N-methylated amides, D-aminoacids or any other organic scaffold not related to a regular peptide bond. The second type does not necessarily hold any of the proposed pharmacophoric elements and may be structurally very distinct from the native peptide. The binding mode of such peptidomimetics is often quite different from the binding mode of the native peptide, but the pharmacological consequence is the same. Today, an extensive pool of peptidomimetic antagonists of G-protein coupled peptide receptors are known (more than 100) [7], while the number of nonpeptidic agonists is limited to agonists for angiotensin I1 [8], cholecystokinin [9], bradykinin [lo], opioid [l I], arginine vasopressin [ 121 and somatostatin receptors [13, 141. Such a limited number of agonists compared to antagonists does not necessarily indicate that it is more difficult to discover or design agonists than antagonists, but is rather a consequence of the shifl in screening method in going from a binding to a hnctional assay; a change which took place at the beginning of the nineties. Amongst the agonists only the opioids were discovered before 1990.
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In addition to the above-mentioned agonists of G-protein coupled peptide receptors peptidomimetic growth hormone secretagogues (GHSs) were also discovered during the nineties [ 15-19]. REVERSE DRUG DISCOVERY
A typical drug discovery process starts with the identification of a natural ligand (e.g. a peptide, protein or neurotransmitter) followed by identification of the corresponding receptor. When such a system has been identified, pharmacological studies are initiated in various species, including man, and depending on the outcome of such studies, the screening and design of synthetic ligands is initiated. With respect to GHSs, this sequence has been reversed. Firstly, a number of synthetic ligands with the ability to release growth hormone (GH) were identified in the late seventies, then clinical studies with the synthetic compounds were carried out in the late eighties, a corresponding receptor was published in the mid nineties and the endogenous ligand was finally identified in 1999. Bowers has previously referred to this process as ‘reverse pharmacology’, but it may be more accurately described as ‘reverse drug discovery’ as it refers to a more comprehensive process than just pharmacology [ 161. RELEASE O F GROWTH HORMONE FROM THE PITUITARY
Human growth hormone (hGH), a 191 amino acid peptide, is a pleiotropic hormone interacting with most tissues in the human body [20]. The most prominent effects of hGH are the growth promoting effect, the protein anabolic effect and the lipolytic effect. Since 1985, when supplies of recombinant hGH became freely available, it has been possible to investigate the metabolic effect of hGH rather than just the growth-promoting effects, which by now are well characterized [ 2 11. At present, data suggest beneficial effects of hGH treatment in a number of indications, such as osteoporosis [22], some conditions characterized by nitrogen-wasting (e.g. AIDS, chronic dialysis or glucocorticoid treated patients) [23], complicated fracture, cardiomyopathy and obesity [24, 251. Growth hormone (GH) is synthesized and released from somatotrophs (i.e. GH releasing cells) in the anterior pituitary [26]. Both GH synthesis and GH release from somatotrophs are under tight control by two hypothalamic hormones; growth hormone releasing hormone (GHRH) [27] which stimulates the synthesis and release of GH, and somatostatin [28] which inhibits GH release. In addition to the effect of GHRH, a new class of compounds, called growth hormone secretagogues (GHS), is able to induce GH release from the pituitary [29].
I76
GROWTH HORMONE SECRETAGOGUES
An endogenous ligand of the GHS pathway is ghrelin [30], a 28 amino acid 0-n-octanoylated peptide, which was identified more than 20 years later than the first synthetic compounds. Ghrelin has been shown to be released from the stomach and is able to release GH in vitro as well as in vivo. Additionally, ghrelin has shown high affinity to a G-protein coupled receptor, termed GHS 1A receptor, which was shown in 1995 to correspond to the receptor for the synthetic GHSs. Recently, it has also been shown that adenosine has high affinity for the GHS 1A receptor. However, adenosine does not release GH, either in vitro or in vivo, and the pharmacological consequence of this interesting observation has still to be elucidated [3 11. It is widely believed that ghrelin and the synthetic GHSs elicit their effect at both the hypothalamic and pituitary level and work synergistically with GHRH [32]. Release of growth hormone causes an increase in insulin-like growth factor-1 (IGF-I) plasma levels via the liver, but GH and the subsequent release of IGF-I is presumably autoregulated via a negative feedback mechanism at the hypothalamic level (see Figure 4.1). All these factors in concert are believed to be responsible for the pulsatile release of GH. The proposed negative feedback may, in addition, explain the observed attenuation of GH release observed after chronic administration of GHSs in several species. OI L - C H ,
GSSFLSPEHQKAQQRKESKKPPAKLQPR Ghrelin
As the present hGH therapy cannot be administered by the oral route, it is desirable to develop orally bioavailable drugs with the capability of inducing an effect similar to that of hGH. Such drugs might mediate their effect through the GHS pathway. An added advantage of such drugs might be a hGH release profile that mimics the natural pulsatile pattern better than recombinant hGH. In previous literature, the terms GHRP (growth hormone releasing peptides) as well as GHS were used to describe compounds acting via the growth hormone secreting pathway. Most often the term GHRP referred to peptidic, while GHS referred to nonpeptidic growth hormone secretagogues. In this review, all compounds acting via the growth hormone secretagogue pathway will be termed ‘GHS’ and the term peptidic GHSs and peptidomimetic GHSs will be used to distinguish those compounds with peptidic nature from those with nonpeptidic structures. A number of other modulators of growth hormone such as GHRH, opiates, galanin etc. may in principle be considered to be GHSs, but these will not be named as such in this review since they act through a different mechanism.
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Figure 4.1. Regulation of GH release via the pituitaty-hypothalumus axis by GHS.
DISCOVERY OF PEPTIDIC GROWTH HORMONE SECRETAGOGUES
During their work with met-enkephalin analogues, Bowers, Momany and colleagues discovered a series of compounds that released GH in primary cultures of rat pituitary cells [33]. This group of compounds, represented in Figure 4.2 by GHRP-I (l), GHRP-2 (2) and GHRP-6 (3), released GH in a distinct manner from that of GHRH as demonstrated by the use of specific antagonists for GHRH and GHSs [34-36). While GHRH increases CAMPin somatotrophs [37], GHSs activate the PLCpathway directly leading to an increase in intracellular calcium [38, 391. GHSs and GHRH in combination have an additive or even synergistic effect on the secretion of GH from the somatotrophs [32, 181, supporting the existence of two separate receptor-signalling systems. It has been shown that GHSs act directly on somatotrophs to cause GH release and to potentiate the effects of GHRH [40].
CHEMISTRY OF GROWTH HORMONE SECRETAGOGUES The very low oral bioavailability of the original peptidic GHSs (Figure 4.2) led to an extensive search for new compounds with improved pharmacokinetic profiles. A number of structurally different GHSs have already been discovered.
178
GROWTH HORMONE SECRETAGOGUES
Ala-His-D-2Nal-Ala-Trp-D-Phe-Lys-NH2 (1) GHRP-1
D-Ala-D-2Nal-Ala-Trp-D-Phe-Lys-NH, (2) GHRP-2 His-D-Trp-Ala-Trp-D-Phe-Lys-NH, (3) GHRP-6 Figure 4.2. Some of the mostprominent members of the original class ofpeptidic GHSs discovered by Bowers, Momany and colleagues [33].
These compounds have in principle been found through directed screening of large compound libraries containing ‘preferred pharmacophoric elements’ or through a peptidomimetic strategy based on the original peptidic GHSs, e.g. GHW-1 (1). It is worth noting that all work with synthetic GHSs had been carried out before the identification of the endogenous ligand, ghrelin, and therefore no structural input from ghrelin has been included in the design of these compounds. The various classes of compounds with GH releasing properties will be described in the following sections (for excellent reviews see Smith et al. [ 181 and Nargund et al. [15]). BENZOLACTAM-BASED GHSS
The first peptidomimetic GHS was described by a group from Merck directed by Roy Smith [39]. The work of Momany and Bowers [4144] on structureactivity relationships (SAR) suggested that the basic amine at the N-terminus of the peptidic GHSs (i.e. position 1 at e.g. GHRP-1 (1)) was essential for GH releasing activity. Furthermore, a number of aromatic amino acids were favoured, and the use of D-tryptophane (~-Trp)at position 2 converted the original met-enkephalin into a GHS. This information led to the general hypothesis of a pharmacophore that ‘two aromatic groups and one amino group in the right spatial arrangement’ were essential for GH releasing ability. An extensive screening programme was initiated at Merck based on this hypothesis. In 1993 it was reported that directed screening using a rat pituitary cell assay [39] (see later for details on assay and EC50 values) resulted in the identification of a low potency lead compound L-158,077 (4) (ECm = 2000 nM), a racemic biarylic carboxylic acid [45]. Replacement of the carboxylic acid group by a tetrazole moiety increased potency and subsequent
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synthesis of its R-enantiomer afforded the first potent peptidomimetic GHS, L692,429 ( 5 ) . Although the in vitro potency (ECso = 60 nM) of L-692,429 (5) was significantly lower than that of GHRP-6 (3) (ECso = 2 nM), this nonpeptidyl structure attracted much attention and encouraged a number of research groups to develop orally active GHSs. A systematic investigation of L-692,429 (5) by several groups [46-551 led to a number of analogues with improved potency. These included the hydroxypropyl analogue, L-692,585 (6) [46], a number of ester isosteres (7) [54] and the naphtholactam NNC 26-0610 (8) [55], which all showed a 2 to 20-fold increase in potency compared to L-692,429 ( 5 ) .
(4) L-158,077
(5) L-692,429
(6) L-692,585
None of these compounds showed oral bioavailability better than 5%. However, DeVita et al. [53] have recently described two benzolactam-derivatives, (9) and L-739,943 (lo), which showed good oral bioavailability. Thus, the carboxamide (9) was orally active in dogs at a dose of 5 mg/kg, and L-739, 943 showed oral activity at 0.5 mg/kg. The oral bioavailability of L-739,943 was found to be 24% in dogs.
7%
H3C NH,
(7)
(8) NNC 26-0610
GROWTH HORMONE SECRETAGOGUES
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(9)
(10) L-739,943
THIOUREA-, OXINDOLE- AND QUINAZOLINONE-BASED GHSS
A completely different type of compound, the thiourea based GHS, was disclosed in 1997. Although these compounds were only weakly active (e.g. (1 1) EC50 = 1800 nM)) [56], the series confirmed the hypothesis that a scaffold holding two aromatic groups and an amino function would prove sufficient to obtain GH releasing activity and represents the smallest and simplest active GHS known. However, so far no really potent thiourea derivative has been reported, but recently a structurally related class of compounds, the oxindole derivatives, was disclosed. The closest analogue of this class to the thiourea (1 1) is the oxaindole (1 2). Although this compound has been reported to be inactive, the slightly modified analogue (1 3) has shown very good potency, in vitro as well as in vivo and in addition has good oral bioavailability in rats [57]. Another very potent type of compound is the quinazolinone (14), which has an ECso of 0.63 nM for the secretion of GH from cultured rat pituitary cells [58]. SPIROPIPERIDINE-BASED GHSS (MK677 ANALOGUES)
Nonpeptide camphorsulfonamide GHSs, discovered by directed screening, were disclosed in 1996 from the Merck group [59]. Structure-activity relationship studies around a moderately active camphorbased GHS, (1 5) (ECso = 300 nM), led to the discovery of (16) (ECs0 = 90 nM). Interestingly, this benefited from the use of a 2(R)-hydroxypropyl group as previously seen in L-692,585 (6). Meanwhile, derivatizing a spiroindanylpiperidine led to the spiroindolyl derivative, MK677 (1 7), which was the first GHS with high
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in vivo potency and good oral bioavailability (> 60%) in beagle dogs. MK677 (17) has now undergone clinical studies in a number of indications (see later). Based on the structure of MK677 (1 7) a number of closely related analogues have beenreported [59-711, includingL-163,540(18) [60],CP-424,391(19) [61], (20) [62], (21) [60] and most recently LY444711 (22) [72]. Also a number of related tripeptides such as the Europeptide compound EP 51 389 (23) [73] and (24) [62] with an aminoisobutyryl group (Aib) in the Nterminus, as in MK677 (17), have been described to have in vivo potency similar to MK677 (17) and GHRP-6 (3).
GROWTH HORMONE SECRETAGOGUES
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(17) MK677
(18) L-163.540
(20) L-163,255
(21) L-163,833
(19) CP-424,391
(22) LY444711
:zi5r H
g
H,
0 H '
(23)EP51389
PEPTIDIC GHSs LEADING TO SMALLER ANALOGUES
In parallel with the rational screening and optimization effort, which was mainly carried out at Merck and has been previously reviewed [15, 181, a number of other groups including those at Europeptide, Genentech and Novo Nordisk took a different approach to identify orally active GHSs.
M. ANKERSEN
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To gain an understanding of the pharmacophoric and metabolic stability requirements, a large number of peptides have been prepared and the main outcome of this effort was the identification of hexarelin (25) [74] by Europeptides and ipamorelin (26) [75] by Novo Nordisk. Hexarelin is a very close analogue of GHRP-6 (3), in which ~ - T r pat position 2 of GHRP-6 is replaced by ~-(2-Me)-Trp.This synthetic amino acid has previously been used in a luteinizing hormone-releasing hormone (LHRH) analogue [76] found to increase metabolic stability and the metabolic stability was also increased in hexarelin [74]. Ipamorelin (26) was derived from GHRP-1 (1) using a traditional peptide truncation/amino acid substitution strategy, combined with distance geometry calculations based on various peptidic GHSs and L-692,429 (5) [75,77]. A simple superposition ofGHRP-1 (I), L-692,429 and ipamorelin is shown in Figure 4.3. As illustrated the central dipeptide alanine-tryptophane of GHRP- 1 (1) seemed superfluous and was deleted, and ipamorelin was derived by subsequent replacement of the alanyl (Ala) moiety in the N-terminus with Aib [75, 771.
(25)Hexarelin (H-HisD.(Z-Me~TrpAla-TrpDPhsL~NH,)
(26)Ipamorelin(KAib-His-DNal-DPhsLys-NY)
Although the size of ipainorelin compared to GHRP- 1 (1) was reduced by about 25%, the in vitro and in vivo potency was retained. Hexarelin and ipamorelin have been shown to release GH in a number of species, including human [78, 791. In addition, a number of peptide-like compounds were reported in which one (or several) of the amino acids was replaced by unnatural amino acids (such as 3-aminomethylbenzoic acid or isonipecotic acid). Such compounds included
GROWTH HORMONE SECRETAGOGUES
184
Superfluity \
1
1 Y'
-
CHRP- I L-692,429 ipamorelin
'
Aromatic Area
Aromatic Area
Figure 4.3. Superposition of GHRP-I ( I ) , L-692,429 (5) and ipumorelin (26). The primary amino group in the N-terminal of (I), (5) and (26). the aromatic moiety of O-Nu/of ( I ) and (26) and the benzoluctam moiety of (5).and the aromatic moiety of o-Phe of(1) and (26) and the biaryl moiety of (5) have been overlapped.
NNC 26-0235 (27) [77] and G-7039 (28) [80]. These two compounds showed very high in vitro potency (EC5o = 0.5 nM for (27) and ECso = 0.18 nM for (28)) and both compounds were very active with respect to GH release in rats, but neither of them showed satisfactory oral bioavailability [77, 8 11. Hypothesizing that the lack of oral bioavailability was mainly attributable to the size and the number of hydrogen-bonding sites [82] of these molecules, the two groups independently identified analogues of the peptidic GHSs using a step-by-step peptidomimetic approach.
(27) NNC 26-0235
(28) G-7039
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The Novo Nordisk group searched for the smallest structure based on NNC 26-0235 (27) with the minimum pharmacophoric requirements and identified NNC 26-0323 (29) [77]. Although this compound was only moderately active in vitro it represented a new GHS, discovered via a peptidomimetic approach, with good oral bioavailability in rats ( z 20%) [77]. The Genentech group used (3-7039 (28) as a template and replaced the three amino acids in the C-terminus (~-Nal-Phe-Lys-NH2) with ~-Trp-ol and obtained a small truncated analogue (3-7502 (30). G-7502 (30) was very active in rats with an EDs0 0.8 mg/kg after i.v. administration, but the oral bioavailability has not been reported [80].
(29) NNC 26-0323
(30)G-7502
PEPTIDOMIMETIC GHSs DERIVED FROM IPAMORELIN
The small analogue NNC 26-0323 (29) showed only moderate potency in vitro, but due to its relatively high oral bioavailability, it confirmed the strategy for the identification of oral bioavailable compounds and created the structural basis for a new class of small molecules [77, 831. Thus, the strategy for identifying orally active GHSs at Novo Nordisk was to use ipamorelin (26) as a template and reduce the size of the molecule and the number of hydrogen bond acceptors and donors via a step-by-step peptidomimetic process [83]. This strategy is in accordance with what is now known as the 'rule of five' [84] which states that a compound is unlikely to be orally bioavailable if two or more of the following four rules apply: (i) the molecular weight exceeds 500; (ii) the number of hydrogen donors exceeds 5 (expressed as the sum of OH and NH bonds); (iii) the number of hydrogen acceptors exceeds 10 (expressed as the sum of N's and 0's) and (iv) the calculated log P (clogP) exceeds 5 . On considering these rules we had a number of concerns about ipamorelin (26) because the molecular weight was 7 1 1, 1 1 hydrogen atoms were potential
186
GROWTH HORMONE SECRETAGOGUES
H-bond donors, 14 atoms were potential H-bond acceptors and the clogP was about 0.5 [85]. The lysine residue in the C-terminus, the imidazole moiety of the histidine and the secondary amides in the backbone all contributed to the high number of hydrogen bonding sites. The N-terminal amino group also contributed but this group was thought to be crucial for activity. The size and number of hydrogen bonding sites of ipamorelin (26) was reduced by removing the lysine in the C-terminus and methylating the remaining backbone amides. Interestingly, the resulting compound (3 1) showed high in vitro potency with an EC50 of 3.8 nM.
The next step was to replace the dipeptidyl Aib-His moiety in the Nterminus with a more lipophilic group. Compound (32) was obtained by replacing this moiety with a 3-aminomethylbenzoyl group, as used in NNC 26-0235 (27) and NNC 26-0323 (29). This compound showed only a 10-fold decrease of in vitro activity (EC5o = 40 nM) despite the fact that four heteroatoms and thereby potential H-bond acceptors or donors, were removed. Meanwhile, previous results in the SAR of ipamorelin (26) showed that the two methyl groups at the N-terminus for this series of compounds were important for in vitro activity [75]. Preparation of the desired aminoisopropylbenzoyl N-terminus was attempted without success, but instead the aminomethylbenzoyl moiety of (32) was replaced with a group derived from an aminopentenoic acid where two methyl groups were introduced. This peptidomimetic GHS, "703 (33), showed in vitro potency at 5 nM (EC50) and ED50 in pigs of 155 nmol/kg (= 0.08 mg/kg) after i.v. administration, high specificity with respect to ACTH and cortisol release and oral bioavailability in dogs was measured to be more than 30% [86]. Interestingly, "703 [83, 861, complied well with the 'rule of five' (MW=529; HB donors = 3; HB acceptors = 7; clogP = 4.2).
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(33) "703
It is important to stress that although the 'rule of five' formed the basis of the peptidomimetic strategy, some inconsistencies in the series of "703 (33) analogues were observed. The majority of analogues of "703 (33) complied with the rule and did indeed show good oral bioavailability, but a few compounds, for reasons unknown, did not show any oral bioavailability [83]. In the course of discovering "703 a large number of close analogues with modifications in various positions were prepared by classical as well as combinatorial methods [83, 87, 881. In these studies, the backbone amides were fixed and the N-terminus, the D-naphthylalanyl (D-Nal), the D-phenylalanyl(D-Phe) moiety and the C-terminus were replaced with various groups (Scheme 4. I).
Hydrocarbons
N-terminal
D-Nal moiety
D-Phe
Cterminal
moiety
Scheme 4.1. Schematic illustration and the terminology used to describe each building block of "703 (33) unulogues.
188
GROWTH HORMONE SECRETAGOGUES
To study the structural requirements for each building block of which "703 (33) was composed (see Scheme 4.I ) more than twenty different non-polar 'dipeptidomimetic' amino acids were used to form N-termini. Four D-amino acids were used as D-Nal analogues, four aromatic o-amino acids as D-Phe analogues and four different C-termini were employed in various combinations. N- Termini
The successful replacement of the Aib-His moiety of (3 1) by (2E)-5-methyl-5aminohex-2-enoyl (as in "703 (33)), suggested that the distance between the amide carbonyl and the amino group combined with conformational constraints induced by the double bond and the geminal methyl groups, respectively, were crucial for retaining high in vitro potency [87]. Based on this observation a series of compounds with a variety of N-termini were prepared to clarify the role of the double bond and the effect of steric hindrance around the amino moiety as well as the distance between the carbonyl group and the amino group [87, 891. Selected "703 (33) analogues from this study are shown in Table 4.1. They include different N-termini, either with more flexible ether linkages (as in (33a) and (33b)), with steric hindrance around the amino group (as in (33c), (33d) and (33e)) or with shorter or more constraint backbones (as in (330 and (33g)). The conclusion from this study was that the original Nterminus containing a vinyl residue may be replaced with several other constrained N-termini, while a shorter and more fixed distance between the amino group and the carbonyl group was prohibited [87]. D-Nal and D-Phe moieties A 6 x 4 x 4 combinatorial library of "703 (33) analogues was prepared with modification at the N-terminus, the D-Nal position and the D-Phe position to further explore the influence of the various aromatic substructures of "703 (33) [83, 881. In this study six N-termini, four o-Nal analogues and four D-Phe analogues were combined in a 96-member library (Figure 4.4, see ref. 88 for details). A sky-line view of the relative in vitro potencies of the 96-member library is illustrated in Figure 4.5. It shows that a ~-(4-phenyl)phenylalanyl moiety (B4 in Figure 4.4) may replace the D-Nal moiety in most combinations and that a Dthienylalanyl (C1 in Figure 4.4) moiety may replace the D-Phe moiety. Several of the compounds in the library showed improved in vitro potency [88], and the study showed that certain fragments were only favourable in certain defined combinations. While certain building blocks showed very poor receptor interaction in most combinations, the same particular building block may in a defined structural arrangement show very good potency. Thus, the study clearly
M. ANKERSEN
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Table 4.1. SELECTED "703 (33) ANALOGUES WITH VARIOUS N-TERMINI AND THEIR RESPECTIVE POTENCIES IN A RAT PITUITARY CELL ASSAY
85
'Mean of 1-5 separate experiments.
demonstrated that a much larger number of combinations might be needed to rank particular building blocks against each other [88]. C- Termini Variation of the C-terminal methylamino group in "703 (33) was also explored with several other replacements [83, 90, 911, such as hydrazino [90], ethylamino, cyclopropylmethylamino, (2-tetrahydrofuranyl)methylamino and (ZR)-2-hydroxypropylamino groups in various combinations [83]. None of these groups produced significant improvement of potencies in vitro. However, a number of these analogues were evaluated in pigs or dogs with respect to GH releasing ability and NNC 26-0722 (34) in particular showed significant improvement of GH release in pigs after i.v. administration [83]. A number of hydrazides, such as NNC 26-1167 (35) also showed increased efficacy in dogs after oral administration at 2.C2.8 mg/kg compared to "703 (33) [90].
GROWTH HORMONE SECRETAGOGUES
190
/c"'
OOH H , N ~ C o o H
0-COOH
H2N /
/
H2N
.p'
PI
OH
HN I CH,
HF:CH,
0
f HYCHI
0
HN CH, I
0 OH
,POMe
HN
CH,
0
Figure 4.4. The three sets of building blocks AI-A6, BI-B4 and CI-C4 that constituted the 96 mem- her combinatorial libranj The building block A I - A 6 represent N-termini, BI-B4 represent D-Nu1unalogue.9 and CI -C4 represent D-Phe unulogues.
The in vivo potency (EDso ) in pigs of NNC 26-0722 (34) [83] was found (33) [86]. As noted, NNC to be 31 nmolkg versus 150 nmol/kg for "703 26-0722 (33) contains the (29-hydroxypropyl group, which previously has been shown to improve the in vitro potency in both the benzolactam derived compounds, (6) [46], and in the camphor-based compounds, (12) [59]. In these two compounds the (25')-hydroxypropyl group serves as an extension of the N-terminal amino group, whereas in NNC 26-0722 (34) it serves as an extension of the C-terminal amide. Since the attachment of this fragment series increases the in vivo but not the in vitro potency, as is to the "703 the case in the benzolactam and the camphor series, it is most likely that the
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Figure 4.5. 3-Dimensional graphic illustration I$ the potencies of the 96 growth hormone secretugogues prepired as a total comhinatoricrl lihrury. A I-A6 represent six N-termini, BI-B4 represent ,four u-Nu1 replacements and CI-C2 represent u-Phe replacements. The y-axis does not show the e.wct potency. hut insteuci expresses how many times the compound is more potent than I0 nM (i.e. higher y-mi.s more potenc.~).In this wu-v the most potent compound is visually Javoured.
(2S)-hydroxypropyl group in NNC 26-0722 improves the pharmacokinetic properties, rather than participates in receptor recognition. While the intention of using (29-hydroxypropyl as a C-terminus was to increase potency by adding an additional pharmacophoric group (which was not successful), the introduction of hydrazido and thienylalanyl groups in the C-terminus, as in NNC 26-1 167 ( 3 3 , was intended to address more specifically the pharmacokinetic profile of the compound. It has previously been suggested
(34)NNC 26-0722
(35) NNC 26-1 167
GROWTH HORMONE SECRETAGOGUES
192
that the relatively long plasma elimination half-life of MK677 (16) is related to the observed receptor-desensitization and magnitude of GH-induced IGF-I release [62]. For instance, chronic dosing of MK677 (16) in humans results in down regulation of the GH release but still exhibits prolonged elevation of the IGF-I level [92]. This has been associated with the relatively long plasma elimination half-life of MK677 (16) ( t l p 6 h). In this context it is noteworthy that short acting compounds such as GHRP-2 (2) have demonstrated efficacy with regard to growth rate in children despite the absence of a sustained IGF-I elevation [93]. A long half-life has also been observed with "703 in dogs (tip 4 h) and therefore a search was initiated for compounds in this series with similarly good in vitro and in vivo potencies, good oral bioavailability but a significantly shorter half-life. Since the half-life is related to the volume of distribution, and the volume of distribution is believed to be related to the fat/ water partition [94], it was hypothesized that a slight increase in hydrophilicity could lead to the desired change in pharmacokinetic parameters [90]. Modification of the C-terminus, which included the incorporation of a polar group, led to the identification of a series of analogues of "703 with hydrazides in the C-terminus, where in particular NNC 26- 1 167 (35) had a significantly lower volume of distribution and shorter elimination half-life than "703. It was speculated that this may also explain the relatively higher in vivo potency observed for NNC 26-1 167 (35) [90].
-
-
DISCOVERY STRATEGIES OF PEPTIDOMIMETIC GROWTH HORMONE SECRETAGOGUES Summarized above are the different strategies that have been used to discover various growth hormone secretagogues. The Merck group used a 'rational screening' approach based on the general pharmacophoric hypothesis that two aromatic groups and an amino group were essential for activity [15]. This approach was combined with a procedure suggested by Evans that a useful way of designing receptor agonists and antagonists was to apply and derivatize frequently occurring structural units. These recurring core units are the socalled 'privileged structures' [95]. Presumably a number of receptors contain similar binding sites whose size and hydrophobic properties are well suited to accommodate these privileged structures. If recurring structural units bind near receptor 'active sites' then derivatization with amino acids and small peptides might afford agonists or antagonists for peptide receptors [ 15 and references therein]. Merck succeeded twice in identifying peptidomimetic GHSs useful as clinical candidates using the 'privileged structure' approach. Firstly, they discovered the benzolactam, L-692,429 (5) which contained two privileged structures; the benzolactam moiety from CCK antagonists [96] and
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the biaryl tetrazole moiety from angiotensin antagonists [97]. Secondly, they identified MK677 ( 16) in which the spiroindanylpiperidine fragment has previously been described to bind to the oxytocin [98, 991, NK- 1, NK-2 [ 100, 101, 1021 and a-receptors [103, 1041. A 'rational design' approach was employed both at Genentech and Novo Nordisk using a step-by-step peptidomimetic procedure to identify peptidomimetic GHSs from the peptidic GHSs as templates. The Genentech group developed a cyclic analogue of GHRP-2 (2), G-7203 (36), which served as template for extensive medicinal chemistry culminating in the identification of G-7502 (29) [80], while the Novo Nordisk group used ipamorelin (26) [75, 771 as template and from there developed the oral bioavailable "703 (33) [86].
(36)G-7203
Although the Merck group used a different approach than Genentech and Novo Nordisk to identify GHSs there are a number of structural features common to most of the compounds [15, 1051. Such features include, beside the two aromatic groups and one N-terminal amino group which so far have formed the general pharmacophoric hypothesis, a stereocenter, which is seen in the linear compounds as a D-amino acid and in the benzolactams in the Rconfiguration on the lactam-ring [39]. Although a high degree of structural dissimilarity is present, particularly between MK677 (17) and the peptidic GHSs, but also between MK677 (17) and "703 (33), this stereocenter is conserved in all GHSs. A receptor with very high affinity for MK677 (16) has been cloned [ 1061. This receptor has subsequently also been shown to have high affinity for the endogenous ligand, ghrelin. Since most of the work with synthetic GHSs has been carried out without knowing the structure of ghrelin, a direct SAR comparison between the endogenous ligand and the various classes of GHSs, and an
194
GROWTH HORMONE SECRETAGOGUES
investigation of the binding mode of the natural ligand and the various GHSs, have not yet been carried out. However, Howard et al. [lo51 have used sitedirected mutagenesis and molecular modelling in an attempt to classify GHFW6 (3), L-692,585 (6) and MK677 (17) with respect to binding at the GHS 1A receptor. This study suggests that the three compounds have some common binding epitopes (e.g. E124), but that the binding sites might be partially overlapping, and in conclusion that 'there are distinct regions of the receptor that are selective for particular classes of agonists' [ 1051. Assuming this hypothesis is correct, it is tempting to speculate whether "703 (33) binds to the same binding epitopes as MK677 (17) (or 3 or 6). Obviously "703 as well as MK677 possess the proposed pharmacophoric groups, but the flexibility of "703 differs from MK677. MK677 is a very compact compound and its flexibility is primarily centered around the O-benzylserine moiety, while "703 has more flexibility around its backbone. However, if the aromatic moieties of "703 and MK677 (i.e. D-Nal to 0benzylserine and D-Phe to spiroindolyl) are superimposed and assuming the Nterminal amino group of the two compounds interact with, for example, El24 of the GHS 1A receptor, a possible 3D-pharmacophore model may be as depicted in Figure 4.6. This model may be further extended to hold an additional binding site for the lysine moiety of, for example, GHRP-6 (3). Meanwhile further evidence suggests that "703 and MK677 do not necessarily recognize the GHS 1A receptor in the same manner (see later) [86]. PHARMACOLOGY OF GROWTH HORMONE SECRETAGOGUES The principal screening assay for GHSs has been a functional method based on primary cultures of rat pituitary cells [107]. In this assay, the GH releasing cells, somatotrophs of the pituitary, are isolated and cultured. The test compound is added to cells at various concentrations and the secreted GH is measured, giving a value of potency (indicated in Table 4.2 as ECS0) and efficacy (Emax; not included in Table 4.2). This assay has been shown to demonstrate GH release in a reliable and dose-dependent manner [86]. As shown in Table 4.2 most of the GHSs described, with a few exceptions such as L-692,429 (5) and NNC 26-0323 (30), show EC50 values in the low nanomolar range. All compounds in Table 4.2 show 100% efficacy compared to GHRP-6 and are consequently full agonists. A number of GHSs have been tested in various animal models, including rats [41], dogs [I081 and pigs [109]. Since different animal models have been used, a direct in vivo comparison between the compounds is not possible. Most of the peptidic GHSs are very potent in rats, while most of
M. ANKERSEN Figure 4.6. Docking of "703 (33) and MK677 (I 7) into a proposed 3-dimensional pharmacophore model, based on the assumption that MV- 703 (33) and MK677 (17) elicit their efect through the same binding epitopes and that El24 of the human GHS 1A receptor interacts with the N-terminal amino group.
195
I96
GROWTH HORMONE SECRETAGOGUES
Table 4.2. IN VITRO POTENCY AND ORAL BIOAVAILABILITY OF A NUMBER OF SELEC-TED GROWTH HORMONE SECRETAGOGUES. VALUES ARE FROM VARIOUS RESEARCH GROUPS AND MAY NOT BE DIRECTLY COMPARABLE Compound
GHRP-I GHRP-2 GHRP-6 Hexarelin (Europeptides) lpamorelin (Novo Nordisk) NNC 26-0235 (Novo Nordisk) (3-7039 (Genentech) (3-7502 (Genentech) NNC 26-0323 (Novo Nordisk) "703 (Novo Nordisk) NNC 26-0722 (Novo Nordisk) L-692,429 (Merck) L-692,585 (Merck) NNC 26-0610 (Novo Nordisk) (+)-I (Sumitomo) MK677 (Merck) L-163,540 (Merck) CP-424,39 1 (Pfizer) LY4447 I I (Eli Lilly) EP 51319 (Europeptides)
EC50
(nM)
.fj,,>W9,
Reference
1.1
I .8 2.2 2.3 1.3 0.5 0.2 10.6 265 2.7 8.9 60 3.0 8.0 7.1 1.3 1.6 3.0 1.1 NA
Z=
5 (dog)
sz 10
(dog)
20 (rat) 35 (dog) 25 (dog) zz 4 (dog) 30 (rat) > 60 (dog) 29 (dog) 12 (rat) 44 (dog) 65 (rat) > 35 (dog)
ECSo= concentration needed to induce half-maximal stimulation of GH release in viiro; fp, = oral bioavailability, measured as [AUC,, x dosei,]/[AUCi, x dose,,]. Since the data are collected from various sources, no SEM's are given.
the peptidomimetic GHSs have very weak activity in this species, and in that respect the lysine group may be of particular importance for potency in the rat. However, most of the peptidomimetic GHSs as well as the peptidic GHSs are potent in larger animals such as pigs and dogs [75, 861. Ipamorelin (26), hexarelin (25), GHRP-2 (2) and GHW-6 (3) are equipotent in pigs after i.v. bolus administration at doses below 0.001 mg/kg, while MK677 (17) and "703 (33) are 10 to 30-fold less potent [86]. However, the oral bioavailability of MK677 and "703 in dogs is above 30%, while it is less than 5% for the peptidic GHSs. In a recent publication by Hansen et al. [86] the in vitro potency (EC50) based on GH release from pituitary cells was compared with in vivo potency (ED50) in pigs and binding affinity (Ki) to the GHS 1A receptor of a number of GHSs. The data from this study are summarized in Table 4.3. It is relevant to comment on the discrepancies between the three sets of data. In the rat pituitary cell based in vitvo assay, GHRP-2 (2), GHRP-6 ( 3 ) ,
M. ANKERSEN
197
Table 4.3. IN VITRO POTENCY FROM A RAT PITUITARY CELL BASED ASSAY (EC,,), IN VlVO POTENCY IN PIGS (EDSO)AND BINDING AFFlNITlES AT THE GHS IA RECEPTOR EXPRESSED IN COS-7 CELLS (K,) USING [35S]MK677AS RADIOLIGAND Cbnzpund
ECSO
EDw
K,
GHRP-2 GHRP-6 Hexarelin lpamorelin "703 MK677
1.8 f0.5' 2.2 0.3" 1.8 k 0.4" 1.3 i0.4" 2.7 i 1.4" 0.4 f 0.2$. a
0.6 k 0.2' 3.9 k 1.4b 2.0 f0.2" 2.3 0.03' 155 f 2 3 a 46 6"
0.6 f0.2" 0.9 f0.4a I .8 & 0.3" 63.4 f4.5' 50.0 & 2. l a 0.3 0.07a
*
*
*
*
EC5o = concentration needed to induce half-maximal stimulation of GH release in vitro; EDso = concentration needed to induce half-maximal stimulation of GH release in vivo in pigs; K, converted from ICs,, (concentration needed to induce half-maximal inhibition of MK677) using ChengPrusoff equation (K, = ICso/[ 1 + ligand/l(d], & = 0.2 nM). [35S]MK677 is used as radioligand.[ 1101 'The EC50 values for MK677 in this table is not in accordance with Table 4.2. The data in Table 4.2 is gathered from different sources, while EC,,l-values in Table 3 are from the same experiment. a Data from Hansen et al. [86]. Data from Raun et ul. [75].
'
hexarelin (25), ipamorelin (26), "703 (33) and MK677 (17), demonstrate similar potencies [86]. In the binding studies [ 1 lo], MK677, GHRP-2, GHRP-6 and hexarelin all show binding affinities in the sub-nanomolar range, while ipamorelin (26) and "703 have about 200-fold weaker binding affinity than MK677. This is not in accordance with in vivo potencies in pigs, where, in particular, ipamorelin is much more potent than MK677 [75, 861. These observations will also be discussed later. SPECIFICITY OF GHSs
Many studies have addressed the effect of GHSs in various animals and most of these have focused on their specificity toward GH-release. An early study with L692,429 (5) demonstrated high potency in dogs after i.v. administration [l 111. As expected, the IGF-I levels were increased (IGF-I is secondary to GH), but also ACTH and cortisol levels were increased (cortisol may be released as a consequence of ACTH release) [112], while there were no effects on prolactin, insulin and thyroxine. Elevation of ACTH and cortisol levels have also been seen with some of the other GHSs [ 113, 114, 1151. However, it is interesting to note that "703, in contrast to hexarelin and MK677, did not dose-dependently induce cortisol increases in pigs (see Figure 4.7), suggesting that this phenomenon is not a general effect of all GHSs [86]. This observation will also be discussed later.
198
GROWTH HORMONE SECRETAGOGUES
Figure 4.7. The release of cortisol of "703. hexarelin and MK677 afler i.v. adniinistration to pigs. The doses are approximately equivalent to E D ~ O10 , x ED50 and 100 x ED50 ofthe respective conipound with respect to GH release [91].
A study by Hickey et al. [ 1 151 with once-daily oral dosing of MK677 over 2 weeks in dogs, showed a sustained increase in IGF-I levels, while both GH and cortisol were normalized at the end of the 2-week study. This attenuation of GH and cortisol after repeated administration may be explained by negative feedback inhibition by IGF-I (see Figure 4.1). CENTRAL ACTIONS OF GHSs
The mechanism of action underlying the GH releasing activity of GHSs has been suggested to involve antagonism of somatostatinergic pathways at both the pituitary and hypothalamic levels as well as the stimulation of GHRH secreting neurons [ 1 161. Data show that GHSs activate distinct subpopulations of hypothalamic arcuate neurons in rats and mice, as reflected by increased c-fos activity in cell nuclei [117, 118, 1191. Recent studies by Dickson et al. [ 120, 1211 suggest that the arcuate neurones activated by systemic or intracerebroventricular injections of GHRP-6 (3) are mainly of the neurosecretory type and contain GHRH or NPY. As mentioned above, the activity of most GHSs is not fully specific. A slight stimulatory effect on prolactin, ACTH/cortisol levels as well as influences on the control of sleep and food intake have been demonstrated [ 111, 122-1241. These actions could take place via activation of specific receptors at levels other than the hypothalamo-pituitary system. In agreement with this hypothesis, binding sites of GHSs have been detected in the forebrain of rat, pig and human [125-1271. Indeed, expression ofthe GHS-R 1A receptor is seen in the anterior
M. ANKERSEN
I99
hypothalamus, suprachiasmatic nucleus, supraoptic nucleus, ventromedial hypothalamus, arcuate nucleus, dentate gyrus, tuberomamillary nucleus, para compacte of substantia nigra, the ventral tegmental area, dorsal raphe nuclei, and median raphe nuclei [128]. These areas of the brain are implicated in such different hnctions as learning, memory, motor control, reinforcement behaviour, nociception and feeding. The expression of the GHS 1A receptor in brain regions not generally associated with GH release is intriguing and suggests a broader physiological significance for the role of the natural ligand of the GHS 1A receptor [ 181. In addition to the GHS 1A receptor that has been identified and cloned, an alternative receptor has been suggested by Ong, McNicoll and colleagues [ 1291. This receptor, on the basis of binding studies in various tissues, has been shown to have high binding affinity for hexarelin, but not MK677 (1 7). The receptor has not yet been isolated or cloned. MECHANISM OF ACTION OF GROWTH HORMONE SECRETAGOGUES In order to understand the mechanism of action of GHSs, it is important to look at the distribution of the GHS 1A receptor and projection of some of the neurons involved in GH and ACTH/cortisol release in the hypothalamus and pituitary. Figure 4.8 shows a simple illustration of some of the neurons involved in GH and ACTH/cortisol release. As previously stated, GH is released from the pituitary under tight control of the two hypothalamic hormones, GHRH and somatostatin. GHRH neurons are located in the arcuate nucleus. In an in situ hybridization experiment in rats treated with MK677 (17) and GHRP-6 (3), approximately 25% of neurons showed an increase in c-fos activating expressed of GHRH mRNA and about 50% expressed NPY mRNA [ 12 I]. It is therefore generally believed that the GHS 1A receptor in the hypothalamus is located at the GHRH containing neurons and NPY containing neurons. The activated NPY containing neurons may be split into at least two sub-populations, one that is projecting to somatostatin neurons in the periventricular nucleus (PeVN) and another which is projecting to the CRF neurons in the paraventricular nucleus (PVN) [121]. Thus, the involvement of the GHSs in GH release may be caused by an activation of the GHS 1A receptor directly on the somatotrophs and an activation of the GHRH neurons in the arcuate nucleus. Activation of the NPY neurons projecting to PeVN will activate somatostatin (SS) neurons, which then will cause inhibition of the GH release. Additionally, the negative feedback from GH at the hypothalamic level [130], as well as IGF-I released via stimulation of the GH receptor in the liver [131, 1321, will contribute to
200
GROWTH HORMONE SECRETAGOGUES
Figure 4.8. Simple schematic illustration ofpart ojthe neural network that is involved in GH and cortisol release via the hypothalamic-pituitavy axis. ti has been shown that the GHSs acts at both the hypothalamic and the pituitary level as illustrated. GHSR = GHS I A receptor.
inhibition through the somatostatin neurons. This concerted action of GHRH, NPY, SS and IGF-I will, together with a number of other minor factors, influence the pulsatile pattern of GH release. The sub-population of NPY containing neurons, which is projecting to PVN, may also be activated via the GHS 1A receptor and further via CRF in the PVN and ACTH in the pituitary. Cortisol may be released from the adrenal gland [ 1 1 11. This general mechanism of the GHSs may explain both the GH release and the cortisol release that have been observed for some GHSs, such as MK677 (17) and hexarelin (25). But it is interesting to note that compounds, such as "703 (33) and ipamorelin (26) do not release any cortisol. Whether this is caused by a difference in pharmacokinetic factors (such as different blood-brain barrier penetration or different volume of distribution etc), different receptor activation mechanism or by receptor heterogeneity is still unclear (note the discrepancies between binding and functional response in Table 4.3). However, in the light of the recent publication by Ong et al. [129], claiming a second GHS receptor involved in the mechanism, some of these discrepancies may be explained by such a subtype.
M. ANKERSEN
20 1
It is most likely that for a while there will be only speculative explanations for the mechanism of action of the various GHSs, but the clinical potential of any GHS may not be realized until its effectiveness in eliciting GH release is fully characterized and until the mechanism by which it elicits GH release is well understood. The identification of ghrelin as an endogenous ligand to the GHS 1A receptor will undoubtedly be of utmost importance in this research.
CLINICAL STUDIES WITH GROWTH HORMONE SECRETAGOGUES Clinical research has predominantly been carried out with GHRP-I , GHRP-2, GHRP-6, hexarelin, ipamorelin, L-692,429, and MK677. Of these, only MK677 has shown good oral bioavailability. A number of clinical studies performed with various GHSs are summarized in Table 4.4 ACUTE GH RELEASE
The acute GH release in man stimulated by GHSs has been studied extensively following several routes of administration (i.e. parenteral, intranasal, oral). When 18 healthy men were given i.v. boluses of GHRP-6 a maximum effect was observed at 1 pg/kg (68.7f15.5 ng hGH/ml) [32]. In the same study it was observed that submaximal doses of GHRP-6 and 1 pg/kg of GHRH increased GH levels more than 1 pg/kg of GHRH alone indicating that GHRP-6 and GHRH stimulate GH secretion synergistically, and that GHRP-6 acts through a different mechanism than GHRH (Figure 4.9). Additionally, it was shown in this study that prolactin (PRL) and cortisol levels rose while there were no significant changes in luteinizing hormone (LH) and thyroid-stimulating hormone (TSH) levels. This increase in serum PRL and cortisol has also been observed after treatment of healthy and obese subjects with MK677, but only PRL remained raised after chronic treatment [ 1331. Studies in healthy elderly humans with an oral once-daily 25 mg dose of MK677 increased mean 24h GH concentration by almost 97*23% and serum IGF-I levels were increased from 141k21 ngiml into the normal range of young adults (219f21 ng/ml). These increases were sustained for at least 28 days [ 1341. GH DEFICIENCY (GHD)
Long-term studies have been performed with GHRP-2, GHRP-6, hexarelin and MK677 in GH-deficient children and GH-deficient adults following intravenous, subcutanous, intranasal and oral administration.
Table 4.4. SELECTED INDICATIONS WHICH HAVE BEEN EXPLORED WITH GHSs. REFERENCES ARE GIVEN IN SQUARE BRACKETS Compound Acute GH response GHD Children GHRP-1
Bowers [138], [139] Laron [I411 Robinson [I401 Mericq [I421
GHW-2
Bowers [139]
Pihoker [93] Tuilpakov [143] Mericq [ 1441
GW-6
Ilson [I481 Penalva [I491 Maccario [I501
Pombo [151]
Hexarelin
Ghigo [78]
Laron [154], [155] Loche [I561 Klinger [ 1351
GHD Adults
Elderly
Leal-Cerro [152]
Obese
Catabolic States
Sleep Improvement
Frieboes [I231
Cordido [153]
Ipamorelin Gobburu [ 1571 "703
Zdravkovic [ 1581
L-692,429 Gertz [159] MK677
Chapman [ 1341
Aloi [160]
Yu [ 1621
Kirk [I711
Gertz [161]
Chapman [ 1371 Chapman [ 1341 Svensson [ 1331 Murphy [ 1741 Plotkin [ 1701
Copinshi [ 1751
M.ANKERSEN
203
200
180
160
0
100
I
80
c3
.1 CHRP
010
e-e
1 CHRP
5319
2590
lCHRH
.l CHRP + 1
CHRH
10065
120
Y
v
5)o
0-0
e-•
n
\
GH-WCkl/Lw
put=
0-0
I40 -I
w/LpBw 0-0
60 40
20
0
-60 -40 -20 0
20
40
60
80
100 120 140 160 180 200 220 240
1
iv bolus
TIME (rnin)
Figure 4.9. Comparative GH responses in individual subjects. GH responses to various doses of GHRP, GHRP ph4s GHRH. und 1.0 mglkg GHRH in two normal men (copied with permission,from Bowers et al. [SZ]).
In a long-term clinical study of children with stunted growth it was shown that the growth rate increased from 5.3 to 7.4 c d y e a r with intranasal hexarelin (60 pg/kg). Although the mean hGH peak dropped from 23.5 to 11.3 ng/ml after 7 days, probably due to desensitization and remained at that level for 6 months of treatment, it did not affect the observed increase in growth rate [ 1351. GHRP-2 given subcutanously (0.3-3 .O pg/kg/day) to prepubertal GH-deficient children over 6 months increased growth rate significantly but without increasing the corresponding serum IGF-I level [136], in contrast to oral administration of MK677 over 4 days in GH-deficient adults, where 24 h hGH and serum IGF-I was increased in all subjects [137]. Whether this difference is due to a different selectivity or different pharmacokinetic profile of these two compounds is still unclear. ELDERLY
The release of GH changes dramatically throughout life from being high at birth, slightly decreased during childhood, increased during puberty and then slowly declines in adulthood (Figure 4.10) [163-1681. The advantage of
204
GROWTH HORMONE SECRETAGOGUES
Figure 4.10. Influence of age on GH releuse given as relative mean 2 4 h GH concentrations throughout lije.
increasing the level of GH in the elderly has recently been demonstrated in healthy elderly men, where a once-daily GH treatment for 3 months was able to increase lean body mass, muscle mass and thigh strength [169]. GHSs offer great potential in reversing the decline of GH during ageing and it has been shown in a double blind study in 104 elderly men that oral administration of MK677 over 9 weeks was able to significantly increase serum GH, IGF-I and prolactin, whereas cortisol remained unchanged. An increase in muscle strength and bone turnover was noted [ 1701. OBESITY
In obese individuals spontaneous growth hormone secretion is attenuated and this can be reversed by weight loss and fasting. When L-692,429 was administered at 0.2 or 0.75 mg/kg i.v. on three separate occasions after overnight fasting to 12 healthy obese, young men, the growth hormone release was significantly increased compared to both placebo and the corresponding administration of 1 pg/kg GHRH. Whereas in non-obese subjects, the GH release of L-692,429 was blunted by feeding, the GH release in obese subjects was similar in the fasted and fed condition, indicating that obesity represents a metabolic condition which is sensitive to GHSs [171].
M. ANKERSEN
205
When obese subjects with B M b 3 0 kg/m2 were treated with MK677 for eight weeks, the fat-free mass increased by 3 kg in the treated group vs. 0.2 kg in the placebo control group [133]. CATABOLISM
Catabolic states are usually precipitated by dietary energy restriction, excessive energy utilization through exercise, surgery, chronic glucocorticoid treatment and ageing. The anabolic property of GH suggests that it may be useful in the treatment of catabolic disorders, and GH treatment has been shown to partially reverse a number of catabolic conditions, including glucocorticoid induced catabolism [ 1721. In order to determine whether GHSs can reverse the steroid suppression of GH, nine young healthy men were treated with prednisolone for four days and then with 0.2 or 0.75 mg/kg L-692,429. The study demonstrated that the GHS was able to reverse the inhibition of GH secretion by a high dose ofglucocorticoid, and points to the potential use of GHSs in mild to medium catabolic states induced by chronic steroid treatment [ 16I , 1731. This may have a particularly important role in chronic inflammatory diseases in children, such as asthma, juvenile arthritis or inflammatory bowel disease. In these conditions, which are frequently controlled by chronic steroid treatment, growth retardation is a prominent feature. To investigate whether MK677 can reverse diet induced protein catabolism a study was carried out with eight healthy men, who were calorie restricted for 2 weeks and treated with MK677 for the last 7 days. This study suggests that MK677 can reverse diet-induced nitrogen wasting [ 1741. SLEEP ENHANCEMENT
Most of the clinical studies have in one way or another been related to results obtained with recombinant hGH, and it is likely that the effects observed with GHSs are due to release of GH. It is therefore very interesting that it has been reported that GHSs may have a special influence on the sleep pattern of normal young subjects. An increase in stage D2 sleep was observed after repetitive subcutaneous administration of GHRP-6 [ 1231, and an increase in the duration of D4 sleep and rapid eye movement sleep was observed after prolonged oral administration of MK677 [ 1751. SIDE- AND ADVERSE-EFFECTS
As has been seen in animal models, administration of MK677 causes a significant release of cortisol and prolactin. However, the cortisol release was only transient and after four days of once-daily dosing orally the release of cortisol was normalized.
206
GROWTH HORMONE SECRETAGOGUES
Also fasting glucose and insulin concentrations have been shown to increase after chronic treatment of MK677, but it is still unclear whether this will be of clinical significance, since this is also known to occur in treatments with hGH and changes in body composition may eventually counteract the insulin resistance [ 1761. CONCLUSION AND FUTURE PERSPECTIVES Recent advances in the discovery and development of growth hormone secretagogues have provided us with a better understanding of the chemistry, mechanism and pharmacology behind the secretion of growth hormone. The cloning of a unique G-protein-coupled receptor with high affinity for MK677 (17) and its presence in the pituitary, hypothalamus and other parts of the CNS indicates the existence of an endogenous ligand. The recent identification [ 1291, yet not isolation, of an alternative receptor with high affinity for hexarelin, but low affinity for MK677 (1 7), suggests the existence of GHS receptor subtypes. This may partly explain the discrepancy in potencies in various in vitro and in vivo models that have been observed for different classes of compounds, and suggests that several pharmacologically different categories of GHSs may exist. The presence of such receptors in brain and periphery, taken together with the effect of GHSs on sleep and food intake, suggests that the role of these receptors and their corresponding endogenous ligand may be much more profound than GH release [ 181. Most of the work described here has been carried out without knowing the identity of an endogenous ligand but now that Ghrelin has been identified as such a ligand this will further broaden our understanding of growth hormone secretion and other neuroendocrine pathways. The rapid response to exogenous GHSs in the majority of idiopathic GHdeficient children suggests that this condition may largely be due to a deficiency in endogenous GHS production. The very same mechanism may underlie some symptoms characteristic of the elderly population who also seem to respond to exogenous GHS treatment by marked GH secretion. The discovery and development of a selective orally active GHS for clinical use in these conditions, and possibly in other indications such as obesity, osteoporosis and catabolic states, therefore seems to fulfill an unmet medical need. Today, a large number of non-peptidic antagonists for G-protein coupled peptide receptors have been discovered, but only very few non-peptidic agonists are known [7]. The growth hormone secretagogues can now be added to this group of non-peptidic agonists for G-protein coupled peptide receptors.
M. ANKERSEN
207
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19 20 21 22 23 24 25 26
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121 122 123 I24 125 126 I27 128 I29 130 131 132 133 134
135 136 137 138 139 140 141 142 143 144 145
GROWTH HORMONE SECRETAGOGUES Dickson, S.L., Doutrelantviltart, 0. and Leng, G. (1995) J. Endocrinol. 146, 51%528. Dickson, S.L. (1994) J. PhysioL-London 475P, PI37-Pl37. Dickson, S.L., Leng, G., Dyball, R.E.J. and Smith, R.G. (1995) Neuroendocrinology 61,3&43. Dickson, S.L., Doutrelantviltart, O., Dyball, R.E.J. and Leng, G. (1996) J. Endocrinol. 151, 323-33 1. Dickson, S.L. and Luckman, S.M. (1997) Endocrinology 138, 771-777. Locke, W., Kirgis, H.D., Bowers, C.Y. and Abdoh, A.A. (1995) Life Sci. 5 5 , 1347-1352. Frieboes, R.M., Murck, H., Maier, P., Schier, T., Holsboer, F., Steiger, A. (1995) Neuroendocrinology 61, 58&589. Arvat, E., Divito, L., Maccagno, B., Broglio, F., Boghen, M.F., Deghenghi, R., Camanni, F. and Ghigo E. (1997) Peptides 18, 885-892. Codd, E.E., Yellin, T. and Walker, R.F. (1988) Neuropharmacology 27, 1019-1025. Veeraragavan, K., Sethumadhavan, K. and Bowers C.Y. (1992) Life Sci. 50, 114%1155. Muccioli, G., Ghe, C., Ghigo, M.C., Papotti, M., Arvat, E., Boghen, M.F., Nilsson, M.H.L.. Deghenghi, R., Ong, H. and Ghigo, E. (1998) J. Endocrinol. 157, 99-106. Guan, X.M., Yu, H., Palyha, O.C., McKee, K.K., Feighner, S.D., Sirinathsinghji, D.J., Smith, R.G., Van der Ploeg, L.H. and Howard, A.D. (1997) Mol. Brain Res. 48, 23-29. Ong, H., Mcnicoll, N., Escher, E., Collu, R., Deghenghi, R., Locatelli, V., Ghigo, E., Muccioli, G., Boghen, M. and Nilsson, M. (1998) Endocrinology 139, 432435. Minami, S., Kamegai, J., Sugihara, H., Suzuki, N. and Wakabayashi, I. ( 1998) Endocr. J. 45, S 1-26, Yamashita, S. and Melmed, S. (1986) Endocrinology 118, 176182. Harel, 2.and Tannenbaum, G.S. (1992) Endocrinology 131, 758-764. Svensson, J., Lonn, L., Jansson, J.O., Murphy, G., Wyss, D., Krupa, D., Cerchio, K., Polvino, W., Gertz, B., Boseaus, I., Sjostrom, L. and Bengtsson, B.A. (1998) J. Clin. Endocrinol. Metab. 83, 362-369. Chapman, I.M., Bach, M.A., Van Cauter, E., Fanner, M., Krupa, D., Taylor, A.M., Schilling, L.M., Cole, K.Y., Skiles, E.H., Pezzoli, S.S., Hartman, M.L., Veldhuis, J.D., Gonnley, G.J. and Thorner, M.O. (1996) J. Clin. Endocrinol. Metab. 81, 4249-4257. Klinger, B., Silbergeld, A,, Deghenghi, R., Frenkel, J. and Laron, Z. (1996) Eur. J. Endocrinol. 134, 716719. Mericq, V., Cassorla, F., Salazar, T., Avila, A., Iniguez, G., Bowers, C.Y. and Meniam, G.R. (1998) J. Clin. Endocrinol. Metab. 83, 23552360. Chapman, I.M., Pescovitz, O.H., Murphy, G., Treep, T., Cerchio, K.A., Krupa, D., Gertz, B., Polvino, W.J., Skiles, E.H., Pezzoli, S.S. and Thorner, M.O. (1997) J. Clin. Endocrinol. Metab. 82, 34553463. Bowers, C.Y., Alster, D.K. and Frentz, J.M. (1992) J. Clin. Endocrinol. Metab. 74, 292-298. Bowers, C.Y. (1993) J. Pediatr. Endocrinol. 6, 21-31. Robinson, B.M., Friberg, R.D., Bowers, C.Y. and Barkan, A.L. (1992) J. Clin. Endocrinol. Metab. 75, 1121-1 124. Laron, Z., Bowers, C.Y., Hirsch, D., Almonte, A.S., Pelz, M., Keret, R. and Gil-Ad, 1. (1993) Acta Endocrinol. 129, 424426. Mericq, V., Cassorla, F., Garcia, H., Avila, A,, Bowers, C.Y. and Memam, G.R. (1995) J. Clin. Endocrinol. Metab. 80, 1681-1 684. Tuilpakov, A.N., Bulatov, A.A., Peterkova, V.A., Elizarova, G.P., Volevodz, N.N. and Bowers, C.Y. (1995) Metab. Clin. Exp. 44, 119%1204. Mericq, V., Cassorla, F., Salazar, T., Avila, A,, Iniguez, G., Bowers, C.Y. and Merriam, G.R. (1996) J. Invest. Med. 44, 103A. Van den Berg, G., Veldhuis, J.D., Frolich, M. and Roelfsema, F. (1996) J. Clin. Endocrinol. Metab. 8 I , 246g2467.
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146 Van den Berghe, G., De Zegher, F., Veldhuis, J.D., Wouters, P., Awouters, M., Verbruggen, W., Schetz, M., Venvaest, C., Lauwers, P., Bouillon, R. and Bowers, C.Y. (1997) J. Clin. Endocrinol. Metab. 82, 59C599. 147 Van den Berghe, G., De Zegher. F., Baxter, R.C., Veldhuis, J.D., Wouters, P., Schetz, M., Venvaest, C., Van der Vorst, E., Lauwers, P., Bouillon, R. and Bowers, C.Y. (1998) J. Clin. Endocrinol. Metab. 83, 30’+3 I . 148 Ikon, B.E., Jorkasky, D.K., Cumow, R.T. and Stote, R.M. (1989) J. Clin. Endocrinol. Metab. 69, 212-214. 149 Penalva, A,, Pombo, M., Carballo, A,, Barreiro, J., Casanueva, F.F. and Dieguez, C. (1993) Clin. Endocrinol. 38, 87-91. 150 Maccario, M., Arvat, E., Procopio, M., Gianotti, L., Grottoli, S., Imbimbo, B.P., Lenaerts, V., Deghenghi, R., Camanni, F. and Ghigo, E. (1995) Metab. Clin. Exp. 44, 134-138. 151 Pombo, M., Barreiro. I., Penalva, A,, Mallo, F., Casanueva, F.F. and Dieguez, C. (1995) Acta Pediatrica 84. 904-908. 152 Lealxerro, A,, Garcia, E., Astorga, R.. Casanueva, F.F. and Dieguez, C. (1995) Eur. J. Endocrinol. 132, 712-715. 153 Cordido, F., Penalva, A,, Dieguez, C. and Casanueva, F.F. (1993) J. Clin. Endocrinol. Metab. 76, 81%823. 154 Frenkel, J., Silbergeld, A,, Deghenghi, R. and Laron, Z. (1995) J. Pediatr. Endocrinol. Metab. 8, 4 3 4 5 . 155 Laron, Z.. Wang, X.L., Klinger, B., Silbergeld, A. and Wilcken, D.E.L. (1997) J. Endocrinol. 136. 377-381. 156 Loche, S.. Cambiaso, P., Carta, D., Setzu, S., Imbimbo, B.P., Borrelli, P., Pintor, C. and Cappa, M. (1995) J. Clin. Endocrinol. Metab. 80, 674-678. 157 Gobburu, J.V., Agerso, H., Jusko, W.J. and Ynddal, L. (1999) Pharm. Res. 16, 1412-1416. 158 Zdravkovic. M., Sogaard, B., Ynddal, L., Christiansen, T., Agerso, H., Thomsen, M.S., Falch, J.E. and Ilondo, M.M. (2000) Growth Horm. IGF Res. 10, 193-198. 159 Gertz, B.J.. Barrett, J.S., Eisenhandler, R., Krupa, D.A., Wittreich, J.M., Seibold, J.R. and Schneider, S.H. (1993) J. Clin. Endocrinol. Metab. 77, 1393-1397. 160 Aloi, J.A., Gertz, B.J., Hartman, M.L., Huhn. W.C.. Pezzoli, S.S., Wittreich, J.M., Krupa, D.A. and Thorner, M.O. (1994) J. Clin. Endocrinol. Metab. 79, 943949. 161 Gertz, B.J., Sciberras, D.G., Yogendran, L., Christie, K., Bador, K., Krupa, D., Wittreich, J.M. and James, 1. (1994) J. Clin. Endocrinol. Metab. 79, 745-749. 162 Yu, H., Cassorla, F., Tiulpakov, A,, Shi, Y.-F., Setian, N., Bercu, B., Arango, A,, Kletter, G., Pescovitz, 0.. DiMartino, J., Krupa, D., Cambria, M., Kanojia, P. and Bach, M.A. (1998) Proceedings of 80th Annual Meeting Endocrine Society, New Orleans, U.S.A., OR24-6. 163 Ho, K.Y.. Evans, W.S., Blizzard, R.M., Veldhuis, J.D., Merriam, G.R., Samojlik, E., Furlanetto, R., Rogol, A.D., Kaiser, D.L. and Thomer, M.O. (1987) J. Clin. Endocrinol. Metab. 64, 51-58. 164 Iranmancsh, A,, Lizarralde, G. and Veldhuis, J.D. (1991) J. Clin. Endocrinol. Metab. 73, I 08 1-1088. 165 Martha Jr, P.M., Rogol, A.D., Veldhuis. J.D., Kemgan, J.R., Goodman, D.W. and Blizzard, R.M. (1989) J. Clin. Endocrinol. Metab. 69, 563-570. 166 Rose, S.R., Municchi, G., Barnes, K.M., Kamp, G.A., Uriarte, M.M., Ross, J.L., Cassorla, F. and Cutler Jr., G.B. (1991) J. Clin. Endocrinol. Metab. 73, 428435. 167 Vermeulen, A. (1987) J. Clin. Endocrinol. Metab. 64, 884-888. 168 Zadik, Z., Chalew, S.A., McCarter Jr, R.J., Meistas, M. and Kowarski, A.A. (1985) J. Clin. Endocrinol. Metab. 60, 5 13-5 16. 169 Welle, S.. Thomton, C., Statt, M. and McHenry, B. (1996) J. Clin. Endocrinol. Metab. 81, 323F3243.
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170 Murphy, G., Bach, M., Plotkin, D., Bolognese, J., Cerchio, K., Gormley, G. and Gertz, B. (1996) J. Bone Miner. Res. 11 (Suppl.), M668. 171 Kirk, S.E., Gertz, B.J., Schneider, S.H., Hartman, M.L., Pezzoli, S.S., Wittreich, J.M., Krupa, D.A., Seibold, J.R. and Thorner, M.O. (1997) J. Clin. Endocrinol. Metab. 82, 115&1159. 172 Horber, F.F. and Haymond, M.W. (1990) J. Clin. Invest. 86, 265-272. 173 Davies, U.M., Rooney, M., Preece, M.A., Ansell, B.M. and Woo, P. (1994) J. Rheumatol. 21, 153-158. 174 Murphy, M.G., Plunkett, L.M., Gertz, B.J., He, W., Wittreich, J., Polvino, W.M. and Clemmons, D.R. (1998) J. Clin. Endocrinol. Metab. 83, 320-325. 175 Copinschi, G . , Van Onderbergen, A., L’Hermite-Baleriaux, M., Mendel, C.M., Caufriez, A,, Leproult, R., Bolognese, J.A., De Smet, M., Thorner, M.O. and Van Cauter, E. (1996) J. Clin. Endocrinol. Metab. 81, 277&2782. 176 Johannsson, G., Marin, P., Lonn, L., Ottosson, M., Stenlof, K., Bjorntorp, P., Sjostrom, L. and Bengtsson, B.A. (1997) J. Clin. Endocrinol. Metab. 82, 727-734.
Progress in Medicinal Chemistry - Vol. 39, Edited by F.D. King and A.W. Oxford 0 2002 Elsevier Science B.V. All rights reserved.
5 Inhibitors of Hepatitis C Virus NS3.4A Protease: An Overdue Line of Therapy ROBERT B. PERNI and ANN D. KWONG Vertex Pharmaceuticals Inc., 130 Waverly Street, Cambridge, MA 02139, U.S.A.
INTRODUCTION - THE NEED FOR PROTEASE INHIBITORS The disease Current therapies Why target the NS3.4A protease?
216 216 217 218
THE PROBLEM The hepatitis C viral pedigree NS3*4A protease structure and function The nature of the NS3-NS4A interaction
219 219 220 220
THE TOOLS Enzymatic assays Surrogate cells assays Replication assays Animal models
223 223 224 225 225
HEPATITIS C NS304A PROTEASE INHIBITORS HCV subsites and nomenclature General drug design considerations
226 226 226
PEPTIDIC INHIBITORS Non-substrate based inhibitors Substrate-based inhibitors Warheads Aldehydes Boronutes r-Ketoacids. umides and diketones The PI specificity pocket P2 variants and solvent shielding P3, P4 substituents P3 indolinyl derivatives Cvclic derivatives
228 228 229 232 232 234 235 237 239 240 24 1 24 I 215
HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY
216
P’ region based inhibitors
243
NON PEPTIDIC INHIBITORS Screening leads
243 243
SYNTHESIS OF NS3*4A PROTEASE INHIBITORS General comments Boronates Aldehyde synthesis a-Keto acids and derivatives Novel PI carboxylic acids P3 Indolinyl derivatives
245 245 245 245 246 248 249
THE FUTURE: SUMMARY AND OUTLOOK
249
REFERENCES
250
INTRODUCTION: THE NEED AND THE CASE FOR PROTEASE INHIBITORS THE DISEASE
In the world today an essentially unnoticed epidemic rages [ M I . Hepatitis C virus has infected approximately 3% of the world’s population. The majority of these patients are in third world countries though a significant number of infections occur in industrialized nations. Much of the initial spread of the disease was via transfusion of contaminated blood and until 1989 the infection was referred to as non-A, non-B hepatitis as the hepatitis C virus had not yet been identified [ 5 ] . Screening of blood supplies was initiated and the rate of new infections has dropped significantly from 180,000 new cases annually in the United States in the 1980s to approximately 28,000 [6, 71. Unfortunately, while the blood supply has been largely cleared of the hepatitis C virus, in western countries other modes of transmission, mostly parenteral, continue to spread the disease [4, -1. These include intravenous drug use, tatooing, skin-piercing and perinatal transmission. In addition there is a significant fraction of infected individuals where the mode of transmission is unknown [8, 91. Hepatitis C disease progression is exceedingly slow. Typical duration from the time of infection to symptomatic disease runs in the order of 20-30 years. Even with this long duration it is estimated that >50% of infected individuals are asymptomatic and do not seek medical intervention even though the risk of incurring significant liver damage is high. The long-term use of
R.B. PERNI AND A.D. KWONG
217
interferodribavirin as front-line therapy and the associated side-effects (persistent flu-like symptoms, depression) also discourages patient compliance. The prognosis for infected individuals varies greatly with age, duration of infection, viral genotype and alcohol use among other factors [lo]. Hepatitis C virus is an infectious agent related to thejaviridae family of viruses. A small percentage of infected patients spontaneously clear the virus (<15%) while the remaining population progresses to a chronic infected state [ l 11. The physical manifestations of this chronic state vary widely from being completely asymptomatic to fulminant liver disease, though deaths from fulminant HCV disease are rare. Fatalities occur from HCV associated chronic liver disease, e.g., cirrhosis, hepatocellular carcinoma since the HCV virus does not appear to be directly cytopathic to the liver [12, 131. There are an estimated 8,00&10,000 deaths annually from HCV derived disease, about 0.2% of the estimated 4 million infected individuals, and this figure is expected to triple over the next twenty years [14]. As a public health problem, the healthcare costs are high. HCV induced cirrhosis and carcinoma are responsible for most of the liver transplants currently performed [4, 151. Though a study has recently shown that the current standard of care, the interferon/ribavirin combination therapy, is cost effective relative to interferon alone, the costs are still high [16, 171. The major reservoir of the virus is the liver but several recent reports have shown evidence for the presence of replicating virus in the bone marrow [ 181 and peripheral mononuclear blood cells, however the observed levels are very low [ 191. Unchecked, hepatitis C viral particles are turned over at a rapid rate. Up to 10I2 particles are produced per day and the half-life of these virions is approximately 2.7 h. Clearance of the virus varies significantly because the rate of infected cell death varies widely among individuals. Cell death tl/2 range from less than 2 days to 70 days [2&22]. Regardless of extra-hepatic pools of virus, the evidence indicates that the overwhelming predominance is in the liver. CURRENT THERAPIES
Current therapies revolve around immunological manipulation by interferon-a (IFN-a) often in tandem with a nucleoside antiviral, ribavirin [23-261. While this type of therapy has improved somewhat with the introduction of polyethyleneglycolated (PEG)- interferon [27-291, the treatment is still arduous for the patient and often not successful in producing a long-term sustained response. The response rate has also been shown to vary with virus genotype and race. An additional issue for interferon therapy is the high cost of treatment [3]. Indeed while PEG-interferon has recently been given regulatory approval, the standard interferon-ribivarin combination therapy produced a sustained
218
HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY
response in less than 50% of patients. A definition of response has been slow to be adopted [30]. A recent preliminary report describes the possible benefits of using a combination of interferon-cr plus zinc [3 11. Research is ongoing in many groups on a number of non-immunological approaches to the eradication of the hepatitis virus which have been reviewed [32, 331. For example, amantadine, an established antiviral agent, has been studied but has been shown to be relatively ineffective with significant sideeffects [34]. Ribozyme therapy is another option being investigated. Ribozymes (catalytic RNA molecules) that target HCV RNA may be introduced via gene introduction and expression [35] or delivered as an ordinary drug. A manufactured ribozyme, heptazyme, is currently in clinical trials [36]. Heptazyme is designed to selectively cleave viral RNA and consequently inhibit replication. The inhibition of the internal ribosomal entry site (IRES) has been receiving significant attention. The IRES is required for translation and consequently may present a useful target. So far, IRES inhibitors that have been reported appear to function as intercalators [37]. This research is in the early preclinical stages. Simultaneous, multiple lines of attack may be more likely to be successful since the hepatitis C virus exists in at least six known genotypes (I-VI) and an increasing number of subtypes. This situation is the result of a high mutation rate and the constant production of quasi-species. Even within a single individual the virus mutates rapidly and evolves into a large number of viral variants. A potential anti-viral agent must have the ability to interfere with replication regardless of genotype or subtype. The clinical situation and outlook for hepatitis C patients has been recently summarized [38, 391.
WHY TARGET THE NS3*4A PROTEASE?
In a patient population where the majority of patients are chronically infected and asymptomatic and the prognoses are unknown, an effective drug must possess significantly fewer side-effects than currently available treatments. Since therapy is more likely to be more effective before the onset of symptoms of hepatitis and liver damage the patient treatment burden must be very low. Unlike IFN-u, protease inhibitors have the potential to fulfill this need. The hepatitis C non-structural protein3 (NS3) is a proteolytic enzyme required for processing of the viral polyprotein and consequently viral replication. Fortunately, despite the huge number of viral variants associated with HCV infection, the active site of the NS3 protease remains highly conserved thus making its inhibition an attractive mode of intervention. Recent successes in the treatment of HIV with protease inhibitors support the concept that the inhibition of the NS3 is a key target in the battle against HCV.
R.B. PEW1 AND A.D. KWONG
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THE PROBLEM THE HEPATITIS C VIRAL PEDIGREE
The hepatitis C virus is aflaviridae type RNA virus and was not identified until 1989 [40]. The hepatitis C viral genome is enveloped and contains a single strand RNA molecule composed of circa 9600 base pairs. The RNA encodes a polypeptide comprised of approximately 30 10 aminoacids. Significant efforts in the study of the molecular virology of the hepatitis C virus have resulted in some understanding of the viral replicative process. This area has been thoroughly reviewed [41-43]. The HCV polyprotein is processed by viral and host peptidases into 10 discreet peptides (Figure 5.1) which serve a variety of functions [44]. There are three structural proteins, C , El and E2. The P7 protein is of unknown function and is comprised of a highly variable sequence. There are six nonstructural proteins. NS2 is a zinc-dependent metalloproteinase that functions in conjunction with a portion of the NS3 protein. NS3 incorporates two cataytic functions (separate from its association with NS2): a serine protease at the N-terminal end which requires NS4A as a cofactor [45] and an ATP-ase dependent helicase function at the carboxy terminus. NS4A is a tightly associated but non-covalent cofactor of the serine protease [4&49]. The function of NS4B is currently unknown. NSSA may be involved in immunomodulation and NSSB functions as the viral RNA dependent RNA polymerase (RdRP). The NS3a4A protease is responsible for cleaving four sites on the viral polyprotein [SO]. The sequence location of the protease activity was determined in 1995 [Sl]. The NS3-NS4A cleavage is autocatalytic, occurring in cis. The remaining three hydrolyses, NS4A-NS4B, NS4B-NSSA and NSSA-NSSB all occur in trans (Figure 5.2).
- - --
Structural proteins
HzN-
NS3-cofactor
Protease
-
C-EI-EZ-P7-NSZ-NS3-NS4A-NS4B-NS5A-NSSB-COOH
n
H2N-[Protease-181-AA]-[Helicase]-COOH
Figure 5.1. HCV polyprotein structure.
Polymerase
220
HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY
IIII cis
H2N-C-El
-E2-P7-NS2-NS3 NS4A NS4B NSBA NSBB-
trans trans trans
Figure 5.2. HCV NS3 mediated polyprotein cleavage sites.
NS3-4A PROTEASE STRUCTURE AND FUNCTION
NS3 is a serine protease which is structurally classified as a chymotrypsin like protease. The distinguishing features as determined by X-ray analysis are two structural domains containing a twisted /?-sheet and a characteristic chymotrypsin-like fold (Figure 5.3) [52-541. Various forms of the enzyme have been studied including full length as well as truncated forms which exclude the carboxy-terminus helicase region of NS3. Truncated enzyme usually consists of amino acids 1-181. Recently an active single chain N S 3 4 A construct was described [55,56]. NMR structural analyses have also been carried out [57]. The NS3 protein is thought to be localized at least partially in the cellular nuclei although it also appears to be difisely distributed in the cytoplasm [58, 591. THE NATURE OF THE NS3-NS4A INTERACTION
While the NS3 serine protease possesses proteolytic activity by itself, the HCV protease is not an efficient enzyme in terms of catalyzing polyprotein cleavage [60]. It has been shown that a central hydrophobic region of the NS4A protein is required for this enhancement [61]. For example, in the presence of NS4A the cleavage of NS5A/5B is increased by over 20-fold (Table 5. I). The NS4A protein is most likely membrane associated and anchors the NS3*4A complex [62]. Evidence suggests that the NS4A protein serves to make the NS3 active site more rigid [63, 641. Indicative of the growth of this topic an increasing number of detailed reports and reviews on the structure and function of the NS3*4A complex are appearing [65-67]. NS4A peptides have been prepared synthetically and have been engineered into a soluble form while maintaining activity and presumably the secondary structure [62]. In addition, mutated versions of an NS4A peptide have been investigated and activity has been restored by biotinylation [68]. The function and sequence of NS4A is well conserved among HCV genotypes Structurally this implies that the NS3-4A binding motif is also conserved ~91.
R.B. PERNI AND A.D. KWONG
Figure 5.3. Structure
22 1
the HCV NS3 protease comple.xed with NS4A (light stick structure) and the ED WCCSMSY substrate (dark stick structure).
of
Since the NS4A peptide affects the prime-side and non-prime side portions of the enzyme differently, the enzymatic interactions with inhibitors differ according to their binding location. A model is shown in Figure 5.4 [60]. The enzyme kinetics have been studied in detail [60]. The NS4A cofactor improves the intrinsic catalytic efficiency to the point of producing viable viral replication but relative to similar viral proteases, the competency is still low. The cleavage rates of a number of natural sequences were determined in the absence or presence of NS4A. The results are shown in Table 5.1. The formation of the three component (NS3 NS4A cofactor inhibitor) complex, is strongly supported by the determination of active site occupancy by measurement of fluorescence energy transfer [70]. An X-ray and CD study of the ternary complexes demonstrated that even for inhibitors that bind with varying potencies, the protein undergoes a
+
+
222
HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY
Table 5.1. KINETIC PARAMETERS FOR THE HYDROLYSIS OF SYNTHETIC PEPTIDE SUBSTRATES BY t-NS3 IN THE PRESENCEIABSENCE OF 4A PEPTIDE Substrate
kcu,
*
0.6 0.007 0.18f0.01 0 . 2 6 i 0.005 0.05 f 0.0002 ND ND 0.2 f 0.005 0.012 f0.002
5A/5B H-EDVV(Abu)C*SMSY-OH 4A/4B H-DEMEEC*SQHLPYI-OH 4B/5A H-ECTTPC*SGSWLRD-OH 5A-pNA
H-EDVV(Abu)C*(p-Nitroanilide)
KWI
kmtlKrn
32i2 270 f 38 16Oi11 805 f 73 ND ND 1010i 157 1080f 167
20,000 700 1,600 60 110 4 200 10
The asterisks denote the site of hydrolysis; Abu denotes L-aminobutyric acid.
I
4A
Ki, I
Ea4A.I
-
J
I
€-4A
-
I€*4A*J
Kj(with 4A)
Ki(with 4A)
€*4A Figure 5.4. Kinetic model for the HCV NS3 and its interaction with NS4A. non-prime-side inhibitors (I) and prime-side inhibitors (4.
R.B. PERNl AND A.D. KWONG
223
rearrangement of tertiary structure to accommodate the inhibitor molecules in an identical fashion. In other words the CD spectra for all the compounds studied were superimposable [7 1, 721. This conformational change implies that an induced fit mechanism is operative in inhibitor binding. The binding of the NS4A to the NS3 also occurs via an induced fit. This results in a partial reorganization of the NS3 to a conformation suitable for efficient binding to substrate. Despite detailed experiments and an ever-increasing understanding of the subtleties of the enzyme fimction, published values for kinetic parameters vary significantly. There are several reasons for this. Several forms of the enzyme have been employed (i.e., NS3, NS31431, native and protease domain). The enzyme itself is very sensitive to a number of experimental variables including pH, salt concentration, and even the specific detergent used. The active site of the HCV NS3 protease is very shallow by comparison to other proteases [52]. For example, the NS3 lacks the high flaps surrounding the active site which exist in thrombin and that serve to immobilize the substrate as well as exclude solvent from the active site.
THE TOOLS ENZYMATIC ASSAYS
The p-nitroaniline (pNA) derived assay has been the mainstay of protease compound screening and has been shown to be useful for the NS3*4A protease as well [60]. The substrate probe is usually an NS5A derived protein incorporating the non-prime side of the NS5 with a p-nitroaniline terminus at the cleavage site. The pNA assay can utilize a 181-amino acid version of the NS3 that lacks the helicase domain or, alternatively, the full-length protein, although the full-length NS3 poses handling problems. Excess exogenous NS4A is added to the reaction containing NS3 alone. This is a high throughput assay that is usually run in 96 well plates. Another spectrophotometric method uses an 0-4-phenylazaphenylester (PAP) coupled peptide, AcDTEDVVP(Nva)-OPAP. This system allows for continuous monitoring of the cleavage reaction. Similar to the pNA assay, this system can be formatted in 96-well plates [73]. Product formation from the polyprotein processing can be monitored directly using HPLC methodology. An assay has been described that uses a synthetic 20-residue peptide to mimic the NS5A/5B cleavage site [74]. A closely related alternative uses the NSSA-NS5B substrate itself with a similar detection method [75]. This methodology is also suited for time course experiments.
224
HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY
NS3e4A enzyme inhibition can also be measured using a scintillation proximity assay [76]. The cleavage protein is biotinylated and tritiated to give ~~o~~~-DEMEECASHLPYK[~~O The ~ ~assay O~~ monitors ~ - ~ Hthe ] -cleaNH~. vage of substrate and shows a decreasing signal as the hydrolysis proceeds and the amount of tritium in proximity to the biotin (i.e., in the same molecule) decreases. This is an automatable high-throughput assay.
SURROGATE CELL ASSAYS
The development of secondary in vivo bioassays to test whether the inhibitor and its derivatives can function in a cellular environment is a critical step in the drug development process. Ideally, one would test whether a potential NS3/4A protease inhibitor inhibits HCV replication in cultured cells infected with HCV. Unfortunately, the testing of candidate antiviral molecules has been limited by the lack of a robust in vitro system for growing HCV in cultured cells. Surrogate cell assays with readouts such as luciferase or alkaline phosphatase activity have been developed which are amenable for screening compounds in 96-well format on a large scale and provide meaningful SAR information. Several cell-based reporter systems have been developed for testing the activity of NS3 protease inhibitors. Hirowatari and co-workers [77] have developed a system in mammalian cells in which NS3 protease cleavage is required for detection of chloroamphenicol acetyltransferase (CAT) activity. Conversely, Song and co-workers developed a system in yeast in which inhibition of NS3 protease cleavage increases B-galactosidase activity [78]. Cho et al. describe an assay that incorporates a NS3.4A-SEAP (secreted alkaline phosphatase) chimeric gene which has been shown to be relatively simple and fast [79]. A drawback for this assay is that it is carried out in yeast cells and not in mammalian cells. Such differences in cellular penetration and transport may provide misleading results. Researchers at Vertex have described a novel method for determining activity of inhibitory drug candidates against a protease [go]. This method uses a multi-domain fusion protein comprised of a protease cleavage site which is used to monitor protease activity in a cell via a reporter (such as luciferase) gene expression system. In the presence of the hepatitis C NS304A serine protease, the expression of a reporter gene is significantly reduced. Because of the low fidelity of RNA-dependent RNA polymerases, the development of resistance to antiviral drugs could become a major hurdle in small-molecule antiviral therapy for HCV as has been the case for anti-HIV therapy. In the absence of a robust HCV viral replication system, several groups have developed recombinant HCV viruses with other RNA viruses such that the
R.B. PERNl AND A.D. KWONG
225
replication of the chimeric or surrogate virus is dependent on the activity of the HCV NS3 protease domain. Several different RNA virus backbones have been used resulting in the development of chimeric sindbis virus [79, 811, chimeric polio virus [82], and chimeric bovine viral diarrhoea virus [83]. Another surrogate system that is being developed for in vitro cell culture and as an animal model is the virus which is most closely related to HCV, namely GB Virus-B (GBV-B), which can infect tamarins [84]. REPLICATION ASSAYS
Over the last ten years there have been numerous descriptions of HCV replication systems in cell culture. These have been summarized and reviewed [MI. Both peripheral blood neutrophils and hepatocytes are thought to be replication sites of the virus, with the majority of the virus coming from the liver [19]. The source of HCV for these experiments has been sera or plasma collected from infected people. The type of cells which have been reported in use ranges from animal cell lines to human hepatic and haemopoietic cell lines and primary liver cells to chimpanzee primary liver cells. Some of the cell lines reported to support HCV replication include MT-2 cells [86, 871, HPB-Ma cells [88-901, Daudi cells [88, 911, HepG2 cells [92], and Huh7 cells [93]. In most cases, the replication rate is low and sporadic. HCV signal has been shown to persist in some cases for greater than a year. In addition to immortalized cell lines, researchers have investigated the use of in vitro infection with HCV of primary adult human hepatocytes [94, 951, and primary fetal human hepatocytes [96, 971. The recent publication of a HCV replicon construct [98, 991 has initiated a flurry of work to characterize the system [loo, 1011, to format the replicon into a useable assay [102], and subsequently to evolve the replicon into a true replication assay. To date no truly reproducible infectious virus replication assay in human cells has been described which is suitable for drug discovery. ANIMAL MODELS
There is currently no validated system to mimic human infection in animals though a number of possible models are in development. There is a chimpanzee infection model [ 103-1 051 but this model is expensive and impractical for use with the large number of animals required to obtain statistically meaningful results. The Trimera mouse is a SCID mouse derived model wherein a section of human liver tissue is implanted in the mouse kidney capsule after the animal has been irradiated [106]. The model suffers from the drawback that the
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HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY
infection is relatively transient and that if a drug has a delayed effect this model may not give a positive result. The extensive homology the GB virus B shares with HCV has prompted its development as a chimeric HCV model in tamarins [107]. Infectivity has been demonstrated in the tamarin and the model awaits testing with therapeutic agents. HEPATITIS C NS3*4A PROTEASE INHIBITORS HCV SUBSITES AND NOMENCLATURE
The nomenclature for protease sub-sites has been standardized [ 1081. Residues on the N-terminal side of the catalytic site of the protease are counted sequentially from the catalytic site, S1, S2, etc. Similarly, residues on the Cterminus side of the catalytic site are referred to sequentially beginning at the catalytic site as Sl’, S2’ etc. Residues on substrates or inhibitors are given the complementary designations P1, P2, P1’ P2’ etc, analogously as described above for the enzyme. A summary of subsites and properties for HCV protease is shown in Figure 5.5. GENERAL DRUG DESIGN CONSIDERATIONS
Though there are a number of therapeutic molecular targets being investigated as previously discussed, the vast majority of the effort appears to be focused on the HCV protease. Inhibition of proteases has become a fashionable research area. This may be due at least in part to the successful application of this strategy against the human immunodeficiency virus coupled with the omnipresence of proteolytic enzymes in a vast number of biological processes [109].
Salt Bridge
Solvent
Lipophillc
.
Solvent
Llpophilic
Small Llpophillc
Figure 5.5. HCV NS3 non-prime side sub-site characteristics
R.B. PERNl AND A.D. KWONG
221
Table 5.2. ACTIVE-SITE ANALYSIS OF VARIOUS PROTEASES Enzyme
Inhibitor
MW
K,
Buried Surface
NS3-4A Elastase ICE Thrombin S. Grieseus Protease HIV-1 Protease
Hexapeptide Aldehyde Tetrapeptide CMK Tetrapeptide Aldehyde NAPAP Tetrapeptide Aldehyde Amprenavir (VX-478)
725 521 493 510 412 406
1 pM 20 n M 9 nM 40nM 15 nM 0.6 nM
849 867 868 968 769 1092
1.2 I .7 1.8 1.9 1.9 2.2
One measure of the effectiveness of protease binding can be derived from a calculation of the buried surface area of an inhibitor. That is the surface area of a bound inhibitor covered by the protein and not accessible to solvent. The calculation can be normalized to area per unit molecular weight. One can easily see from Table 5.2 that it is extremely difficult for the NS3 to cover much of a bound inhibitor unlike other common proteases such as thrombin or HIV protease [ 1 101. The shallow binding groove of the NS3 protease poses particular challenges to the design of peptidyl and peptidomimetic inhibitors. The natural recognition length of ten amino acid residues is a starting point but not a practical end. In addition, the hydrophobic nature of most of the protein groove does not provide tight binding handles for an inhibitor to latch onto. High-energy covalent and/or ionic interactions occur only at the catalytic site or at the terminii of the recognition groove [65, 11 11. A drug designer is left with essentially two obvious possibilities: a covalent inhibitor that reacts reversibly or irreversibly with the catalytic serine or a noncovalent inhibitor that incorporates a strong electrostatic interaction with the active site. In both cases the inhibitor would be truncated to a minimum size that includes hydrophobic groups that are complementary to the hydrophobic sub-sites near the catalytic triad of the NS3. The shallow binding trough creates a situation where a small molecule does not have sufficient contact with the enzyme for strong binding. As mentioned, the hydrophobic nature of the groove with the exceptions of the termini gives few opportunities for energetically strong interactions. The net effect is that the resultant inhibitor binding interaction is weak. While acidic groups at the termini provide potent inhibitors from electrostatic attraction, the presence of multiple, spatially distant, carboxylates, makes these compounds unlikely to penetrate cells and are, therefore, unlikely to be effective drugs. Detailed structural studies primarily by NMR have shown subtle shielding effects wherein the highly solvent exposed active site is shielded from solvent by the catalytic histidine. This arrangement allows for better binding equilibria - i.e., slower off rates. Unfortunately, exploiting this finding has been difficult.
228
HEPATITIS C VIRUS AN OVERDUE LlNE OF THERAPY
An additional complicating factor is the tendency of the protein to undergo surface conformational changes upon binding of inhibitors [62, 112, 1131. This induced fitting phenomenon is particularly true for NS3 in the absence of the NS4A cofactor. These conformational changes are diminished, though not eliminated, by complexation with NS4A. This is further evidence that the NS4A cofactor partially pre-organizes the NS3 conformation to an arrangement more favourable to binding substrates. Even in the presence of NSIA, sub-sites undergo conformational changes to better accommodate differing substituents thus accounting for the relative promiscuity of this enzyme. From the drug design standpoint this ability to adopt conformations specific to an inhibitor implies that inhibitors can be designed which closely resemble the natural substrate.
PEPTIDIC INHIBITORS NON-SUBSTRATE BASED INHIBITORS
A number of peptides not based on the natural substrates of the NS3*4A protease have been studied as NS3a4A inhibitors. Eglin C, is a natural peptide isolated from Hirudo medicinalis. It has been found to inhibit a number of serine proteases [ 1141 and was studied as a basis for HCV NS3 inhibitor optimization. Eglin C is comprised of 70 amino acid residues and exhibits several advantageous characteristics. Eglin C is resistant to both acidic and thermal denaturation. Inhibition of the NS3 protease is thought to occur via the interaction of the active site binding loop with the protease catalytic site (residues 3%49). A total of 20 variants of Eglin C were studied. The most potent variant was found to be the ELEMS modification at P5, P4, P3, P2’ and P3’ respectively (Figure 5.6). The optimized peptide exhibited an = 0.06 pM whereas the parent Eglin C was inactive at 180 pM. Another approach to the design of peptidic inhibitor that does not invoke the natural substrate is based on the sequence ofan HCV minibody [ 1 151. A minibody
P6
3.
+
P1
Residue 39 Elain C -E-G-S-P-V-T-L-D-L-R-Y-
Figure 5.6. Eglin C optimization.
P4‘
3.
49
ECYI >180 uM
R.B. PERNI AND A.D. KWONG
229
Table 5.3. ALANINE SCAN OF MINIBODY INHIBITION AT 1 yM
(26) (27) (28) (29)
(30) (31) (32)
Minihody inhibitor
Mbic H2 loop sequence
96 Inhibition of NS3 activity
Mbic Mbic.2 Mbic.3 Mbic.34 Mbic.4 Mbic.45 Mbic.6
GIEELD GAEELD GIAELD GIAALD GIEALD GIEAAD GIEELA
45 40 30 I 20 22 37
is a minimized version of an antibody hypervariable domain sequence. The minibody is composed of two loops, H1 and H2, that can be modified by mutagenesis and the resulting polypeptides screened for HCV NS3 inhibitory activity. An alanine scan was carried out on the H2 loop and the results are summarized in Table 5.3. Monoclonal antibodies (Mabs) have been studied as potential inhibitors of NS3 [ 1161. A Ki of 39 nM was observed for a Mab designated 8D4. Interestingly, although binding appears to be competitive with respect to substrate, this binding was decreased by the addition of NS4A. In addition, the binding of the Mab to the NS3 only inhibits the in cis processing of the NS3-4A cleavage and does not affect downstream in trans cleavages. SUBSTRATE BASED INHIBITORS
Much of the current drug-design effort revolves around evolved peptidic inhibitors derived from the natural occurring substrates. Such an approach has proved fruitful against other targets and there exists a rapidly growing body of patent literature in this area [ 1 17, 1 IS]. Ideally, with increased understanding of the structure-activity relationships, these compounds will evolve into smaller, less peptidic (and more drug-like) analogues [ 1191. For the NS3*4A protease this evolution has been extremely difficult to achieve. Experiments were initially performed, beginning with the natural ten-amino acid sequence, spanning P6 to P4’, to determine the extent to which each substrate residue contributed to the overall affinity of inhibitor for the protein [60]. Using a non-cleavable active-site analogue, incorporating a tetrahydroisoquinoline P2, Landro has quantified the dramatic reduction in potency observed by the truncation of either end of the decamer as shown in Table 5.4. Simple removal of the P4’ tyrosine results in an immediate 80-fold reduction in K,. Continued prime-side truncation of the P3’ serine and P2’ norleucine had
230
HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY
Table 5.4. EFFECTS OF PRIME-SIDE AND NON-PRIME SIDE TRUNCATIONS ON K, OF DECAPEPTIDE INHIBITORS
Compound
K, f&)’
Pepride Sequence
H-Glu-Asp-Val-Val-Leu-Cys-Tic-Nle-Ser-Tyr-OH H-Glu-Asp-Val-Val-Leu-Cys-Tic-Nle-Ser-OH
H-Glu-Asp-Val-Val-Leu-Cys-Tic-Nle-OH
0.34 27 17
H-Glu-Asp-Val-Val-Leu-Cys-Tic-OH H-Asp-Val-Val-Leu-Cys-Tic-Nle-Ser-Tyr-OH
14 4.4
H-Val-Val-Leu-Cys-Tic-Nle-Ser-Tyr-OH H-Val-Leu-Cys-Tic-Nle-Ser-Tyr-OH
79 500 2000
H-Leu-Cys-Tic-Nle-Ser-Tyr-OH
Experiments performed with NS3 in the presence of added kk4A.
less effect. The same trend was observed on the non-prime side. Removal of the glutamic and aspartic acids at P6 and P5, respectively, reduced the Ki more than 200-fold. It has been determined that the products of the natural substrate cleavage reaction are themselves inhibitors of the NS3 protease [ 1201. The data obtained from this study are consistent with the observations by Landro. Potencies for these inhibitors range from ICs0= 1-150 pM for NS4A derived inhibitors (Table 5.5) and 2.1-5.5 pM for NS5A based compounds (Table 5.6). In both NS4A and NS5A based derivatives removal of acidic residues at P6 and P5 results in significant decreases in binding affinities (Table 5.6).
Table 5.5. NS4A PEPTIDES, P5-P6 TRUNCATIONS compound
P6
P5
P4
P3
P2
PI
ICS, (PM)
(9) (10) (1 1)
AcAsp
Glu AcGlu
(12) (13) (14)
SUC
Met Met AcMet Met Met Met
Glu Glu Glu Glu Glu Glu
Glu Glu Glu Glu Glu Glu
CysOH CysOH CysOH CysOH CysOH CysOH
21 150 1.3 77 69
Glu SUC Glut
1
23 1
R.B. PERNI AND A.D. KWONG Table 5.6. NSSA PEPTIDES, P5-P6 REPLACEMENTS Conipound
P6
P5
P4
P3
P2
P1
ICSO (PM)
(15)
AcGlu SUC Glut AcGlu AcAsp
Asp Asp Asp Asp Glu
Val Val Val Val Val
Val Val Val Val Val
Abu Abu Abu Cys Cys
CysOH CysOH CysOH CysOH CysOH
2.8 4.6 5.5 5.3 2.1
(16) (17) (18) (19)
These structures have subsequently been the starting point for optimization studies [ l l l , 1211. The results shown in Table 5.6 also agree well with those from truncation studies and both approaches lead drug designers to the same question. How to design a reversible inhibitor that binds tightly in the catalytic site but does need to extend out to P6 and P4’? From these studies the answer is not clear. Acidic terminal groups coupled with hydrophobic residues capable of efficiently filling surface subsites clearly provides significant binding. The concept of peptidomimetic inhibitor design was validated as a useful methodology in the HIV field. Using the natural substrate as a starting point for inhibitor design greatly increases the likelihood of identifying compounds that bind to the enzyme active site with high specificity for its target. For hepatitis C the decapeptide recognition sequences would need to be truncated to more drug-like dimensions, on the order of P1’ to P4 as a practical maximum for a deliverable drug (Figure 5.7). The SAR delineated in TubZes 5.3-5.6 is indicative of the intrinsic difficulty of inhibiting this enzyme. The right hand terminus of peptidic inhibitors may contribute to the poor potencies observed, since the carboxylic acid represents a non-covalent
Figure 5.7. Generalized inhibitor design.
232
HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY Table 5.7. OPTIMIZED PRODUCTS
Compound
P6
P5
P4
P3
P2
PI
(20) (21) (22) (23) (24) (25)
AcAsp
Glu AcGlu
AcGlu
Asp AcGlu
Dif Dif AcDif Dif Dif AcDif
Glu Glu Glu Ile Ile Ile
Cha Cha Cha Cha Cha Cha
CysOH CysOH CysOH CysOH CysOH CysOH
G o (IN)
0.05 1.4 30 0.06 2.4 100
warhead. The electrostatic characteristic of the terminal carboxylate does not compensate for the covalency achieved for warheads such as aldehydes or other active carbonyl compounds, for example. WARHEADS
Studies of functional groups that can react covalently with the catalytic serine of the NS3 have focussed almost entirely on reversible systems. A survey of warheads clearly demonstrates the inferior binding ability of the non-covalent carboxylate (33) and is summarized in Table 5.8 [122]. Simple aldehydes provide significant improvement relative to the carboxylic acid. Also evident from Table 5.8 the ketoamide warhead is the best among the limited group. Dicarbonyl compounds, particularly ketoamides are ubiquitous protease inhibitor warheads. Series of a-keto derivatives, ketoamides, esters and acids and diketones have been reported and found to be potent warheads (vide infra) [123, 1241. Other warheads commonly studied as protease inhibitors have been found to be surprisingly ineffective. For example the trifluoroketone (35) is more than 20 times less active than the corresponding aldehyde. Aldehydes Aldehydes are the simplest reversible warheads. Though they are highly reactive toward attack by the proteolytic serine they are problematical because of the inherent instability of aliphatic aldehydes. A series of peptidic aldehydes have been studied and the SAR remains consistent with previous truncation studies of petides with carboxylate termini (Table 5.9). An aldehyde derived from an E D W scaffold (50) shows less than a 14-fold loss of potency relative to the bis-carboxylate containing analogue (5 1) [ 1 171.
R.B. PERNI AND A.D. KWONG
233
Table 5.8. ACTIVATED CARBONYL WARHEADS Compound
Peptide aldehyde sequence'
ICsn (PM)
17
(33)
1.1
(34)
22
(35)
12
0.64
(37)2
'Lower case letters denote
D
amino acids. 2cc-Carbon is racemic
This is a significantly smaller decrease in potency than the 150-fold decrease observed for non-covalent inhibitors (1) and (6) when the P6 and P5 acids are removed (Table 5.4).
HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY
234
Table 5.9. PROTEASE INHIBITORY ACTIVITY OF PEPTIDE ALDEHYDES
Compound
Peptide aldehyde sequence
IC50
Ac-Val-Val-Abu-Cys-H Ac-Val-Val- Abu-Abu-H Ac-Asp-Val-Val-Nva-H Ac- Asp-Val-Val- Abu-Cys-H Ac-Asp-Val-Val- Abu-Abu-H Ac-Asp-Val-Val- Abu-Nva-H Ac-Glu-Asp-Val-Val-Abu-Cys-H Ac-Glu-Asp-Val-Val-Abu-Abu-H Ac-Glu-Asp-Val-Val- Abu-Nva-H Ac-Glu-Asp-Val-Val- Abu-Abu-H Ac-Glu- Asp-Val-Val- Abu-Nva-H Ac-Glu-Asp-Val-Val-Abu-DAbuH
>i00 >i00 >100 >100 >100
fM.
100 2&30 30-40 1&12 5.5
12.4 >loo
Boronates Boronic acid analogues of peptidomimetic inhibitors have also been investigated. Boronates have been previously utilized as warheads for the inhibition of serine proteases [ 1251 and consequently anti-HCV petide boronic acids have been described [ 126-1281. Though boronates are reversible inhibitors the formation of an 0-B bond between protein and inhibitor results in very tight binding. Boronic acid (52) displays in vitro potency of 38 nM versus the NS3e4A protease [129].
R.B. PERNI AND A.D. KWONG
235
a-Ketoacids, amides and diketones The a-ketoacid warhead has been identified as particularly efficient in binding to the NS3*4A complex [ 1301. Detailed mechanistic studies have demonstrated that peptidomimetic inhibitors with ketoacid warheads are slow binding inhibitors which occupy the NS3 active site in the same mode as ketoacids interact with thrombin or trypsin [130, 1311.
COOH I
F-F
(53)
(54)
f
The X-ray crystal structures of tripeptides (53) and (54) show that the tetrahedral intermediate of the keto moiety does not bind in the oxy anion hole, as would be expected based on other serine proteases such as thrombin. Instead, both carboxylate oxygens are hydrogen bound to the oxyanion hole residues Ser-139 and Gly-137 (Figure 5.8). The ketone derived hydroxyl forms a hydrogen bond to His-57 [ 1311. This particularly tight binding motif has allowed the truncation to a tripeptide scaffold to retain significant binding potency though a terminal charge group is present. Possibly more significant than a-keto-acids, from the perspective of a potential drug, are cc-ketoamides. This warhead incorporates all positive attributes of the keto-acids in a neutral moiety that will more likely be able to penetrate
236
HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY
138
/
Gly 137
.vvn
Figure 5.8. u-Ketoacid binding motif:
cells. A number of derivatives have been prepared [123, 1321. The P1’ region has been shown to accommodate a variety of spatially configured and sized groups. Excellent potency is achieved for non-substituted amide (55) in Table 5.10. Similarly, potency is observed for an inhibitor with an amide substituent extending to the prime side of the enzyme. The stereochemistry of
Table 5.1 0. a-KETOAMIDES: PRIME-SIDE SAR I
(55)
(56)
(57)
I
/
4
1100
231
R.B. PERNI AND A.D. KWONG
the prime side group plays a crucial role as demonstrated by the dramatic 250fold decrease in binding affinity for (57) relative to (56) simply by inverting the methyl group (Table 5.10) [ 1321. Similar results are obtained for a-diketones though, in general, poorer potency is observed [ 1231. Compound (58) exhibits ICso = 4.8 pM, about 1000-fold less potent than the corresponding ketoamide (55). Poorer binding and increased synthetic complexity relative to ketoamides and ketoacids make the diketone series of inhibitors less attractive.
THE P1 SPECIFICITY POCKET
When the catalytic effects of substitution of the PI site on the 5A/5B substrate were examined it became apparent that this site serves as a specificity pocket for substrates as it does for other serine proteases [60]. These data are summarized in Table 5.11. It is clear that significant bulk and/or basicity is detrimental to the efficiency of proteolytic catalysis as measure by the K,,,/K, ratio.
Table 5.1 1. CATALYTIC EFFECTS OF P1 SUBSTlTUTIONS ON 5A/5B SUBSTRATE HYDROLYSIS
CYS Aha SMeCysb Thr Ala Val Leu TYr ASP hPheC 2-WaphthAld
0.61 f 0 . 2 0.06 f 0.003 0.049 f 0.003 0.024 f 0.002 0.022 f 0.005 0.005 f 0.0004
32f2 11Of18 130*20 145 f 25 815i205 550 f 67
20000 580 385 165 25 9 5
aAminoburyric acid, bS-Methylcysteine, ‘homophenylalanine, d2-naphthyl alanine
-
>700 >700 >700 >700
23 8
HEPATITIS C VIRUS A N OVERDUE LINE OF THERAPY
Table 5.12. PI SUBSTITUTION
AcDDlVPO JH
Ri
Compound
R'
Ic50
CH2SH CH2SCH3 CH2CH2SCH3 CH2OH CH2NHz CHJ CH2CH-j CH2CH2CH3 CH2CH(CH3)2 CH2CH2CH2CH3
28 160 500 >loo0
(@)!
> 1000 750 250 150
780 190 630 800 500
The HCV NS3 P1 specificity pocket differs from that of most serine proteases. Unlike thrombin for example, basic groups are deleterious to binding to NS304A allowing for good selectivity versus the clotting enzymes. The small shallow pocket is defined by the Leu-135, Phe-154, and Ala-157 side-chains [52, 531. Phe-154 is primarily responsible for the observed specificity by interacting with the cysteine thiol found in trans cleavage sites [133]. Table 5.12 presents a brief survey of small replacement groups for the P1 cysteine found in the natural substrate. Interestingly non-polar groups function at least as well as polar substituents. Ethyl (65), and N-propyl (66) derivatives demonstrate the best potencies in this, albeit, limited series. Surprisingly, despite the poor activity of (69), a compound possessing a gemdimethyl substitution, it was found that a cyclopropyl P1 moiety is advantageous [ 1191. This result has been exploited [ 118, 134-1361 with a non-covalent carboxylate warhead. The cyclopropyl PI (73) provides a 3-fold improvement in binding relative to a norvaline P1 group (72). An optimized hexapeptide inhibitor (74) with larger hydrophobic groups at P2 and P4, displays nanomolar potency.
R.B. PERNI AND A.D. KWONG
239
COOH
(72) Ki=150pM COOH
(73) Ki = 54 p FOOH
M
8
s
(74) Ki = 0.013 pM
The cyclopropyl P1 group has also been combined with a boronate warhead [128]. The boromate was only slighly more potent than the corresponding carboxylate. P2 VARIANTS AND SOLVENT SHIELDING
Recent NMR calculations on an a-ketoacid containing peptidomimetic inhibitor (53) show that a leucine side-chain at P2 has a particular stabilizing effect on the catalytic His-57 imidazole to Asp-81 carboxylate hydrogen bond [ 1 121. This stabilization appears to be effected by the shielding of that site from solvent exposure. This effect can be exploited by the use of large hydrophobic substituents at the P2 position and may be part of the reason for the good potency observed for the naphthylmethylether proline derivative (74). Cyclohexylalanine at P2 has been shown to be superior to smaller side-chains such as leucine [ 1211. As peptidomimetic inhibitors have evolved in recent years the cyclohexylalanine group (cha) has given way to 0-substituted 4-hydroxylproline as the P2 residue of choice. The 0-benzyl group is among the most studied substituent ( e g (50)) but compounds incorporating larger groups are displaying excellent inhibitory potencies. Compound (75) with a tricyclic proline
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HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY
substituent possesses an ICs0 of less than 500 nM against the NS3-4A [118]. Truncation to tripetide derivatives was effective in this series maintaining significant potencies [ 1341.
P3, P4 SUBSITUENTS
Both P3 and P4 subsites benefit from the presence of hydrophobic substitution in regard to enzyme inhibition. The P4 SAR clearly shows the effects of hydrophobic substitution (Table 5.13) [ 12 11. The Dif (diphenylalanine) containing inhibitor (81) is the most potent but from a practical perspective, is very expensive. Cheaper alternatives are available for substitution at P4 but the trade-off is a reduction in potency. Although a Glu residue at P3 is close to optimal, non-charged side-chains are obviously preferred since charged groups hinder cellular penetration. Small hydrophobic amino acids such as valine or iso-leucine have been shown to be comparable to the Glu at P3. D-amino acids are unacceptable at both P3 and P4 [121].
Table 5.13. P4 OPTIMIZATION
Compound
Ac-Asp-Glu-X-Glu-Cha-Cys
~CSO(FW
X Val Nleu Cha Ileu Leu Dif
0.330 0.224 0.140 0.122 0.118 0.055
R.B. PERNI AND A.D. KWONG
24 1
P3 Indolinyl derivatives A particularly interesting class of inhibitors contains a novel P3 group, a 2-substituted indolyl moiety. These compounds incorporating an a-ketoacid warhead display surprising potency despite the presence, in some cases, of isomeric mixtures [72, 1371. The SAR is summarized in Table 5.14.
Cyclic derivatives
A series of novel macrocyclic peptidomimetics have been studied [ 1381. Compound (89) incorporates a 15-membered ring structure tying PI to P3. Cellular activity is claimed for this series of inhibitors in Huh-7 cells. These cells express Table 5.14. P3 INDOLINYL DERIVATIVES
Y-F
F
Compound
R
ICsu (PM)
Isomer Ratio
50
single
92
1.5:l:l:l
16
1:l:l
45
1:l:I:l
5
single
0.8
>10:1
H
gc?z H
H
I
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HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY
a portion of the HCV polyprotein from NS3 through NSSA ending with the first six amino acids of NSSB and utilizes SEAP as the reporter construct [93].
Table 5.15. PI’-REGIONBASED INHIBITORS Compound
Peptide Sequence
Ac-Asp-Glu-Dif-Ile-Cha-Cys-Pro-Nle-Ser-Leu-NH2 Ac-Asp-Glu-Dif-Ile-Cha-Cys-Pro-Nle-Gln-Leu-NH2 Ac-Asp-Glu-Dif-Ile-Cha-Cys-Pro-Nle-Hyp-Leu-NH2 Ac-Asp-Glu-Dif-Ile-Cha-Cys-Pro-Nle-Asp-Leu-NH2 Ac-Asp-Glu-Dif-Ile-Cha-Cys-Pro-Cha-Ser-Leu-NH2 Ac-Asp-Glu-Dif-Ile-C ha-Cys-Pro-Cha-(D)-Trp-Leu-NH2 Ac-Asp-Glu-Dif-lle-C ha-Cys-Pro-Cha-Gln-Leu-NH2 Ac-Asp-GIu-Dif-Ile-Cha-Cys-Pro-Cha-Hyp-Leu-NH2 Ac-Asp-Glu-Dif-lle-C ha-Cys-Pro-ChaAsp-Leu-NH2 Ac-Asp-Glu-Dif-Ile-Cha-Cys-Pro-H~f-Gln-Leu-NH~ Ac-Asp-Glu-Di f-Ile-Cha-C y s-Pro-Hof-Hyp-Leu-NH2 Ac-Asp-Glu-Di f-Ile-Cha-Cy s-Pro-Hof-Asp-Leu-NH2 Ac-Asp-Glu-Dif-lle-Cha-Cys-Pro-Phg-Asp-Leu-NH2
Ic50
(nM)
64 32 26 1.8
23 820 14 11
1.3 18 15 1.8 7
Table 5.16. OPTIMIZED PI’-BASEDINHIBITORS Compound
Peptide Sequence Ac-Glu-A~p-Val-Val- Abu-Cys-Pro-Nle-Ser-NH~
I G n (nM)
8500 876 3100 64 Ac-Asp-Glu-Dif-Ile-Cha-Cys-Pro-Cha-Ser-Leu-NH2 23 Ac-Asp-Glu-Dif-Ile-Cha-Cys-Pro-Cha-Asp-Leu-NH2 1.3 Ac-Asp-D-Glu-Leu-Ile-Cha-Cys-Pro-Cha-Asp-Leu-NH2 <0.2 Ac-Asp-D-Glu-Leu-Ile-Cha-Cys-Pro-Cha-Asp-Leu-Pro-Tyr-Ly~-NH~ <0.2
Ac-Asp-Glu-Dif-Ile-Cha-Cys-Pro-Nle-Ser-Tyr-NH2 Ac-Asp-Glu-Dif-Ile-C ha-Cys-NH2 Ac-Asp-Glu-Dif-Ile-Cha-Cys-Pro-Nle-Ser-Leu-NH2
243
R.B. PERNI AND A.D. KWONG PI’ REGION BASED INHIBITORS
While the S’ region of the NS3 polypeptide is essential for catalytic function it is less important relative to the S-region in regard to substrate binding. This is clear from the truncation studies previously discussed. Specifically, that study showed that removal of P2’ and P3’ from the substrate had little effect on Ki. A recent study has determined optimal peptidic substitution to provide optimal prime side binding in subsites little used by the natural substrate [139]. This study, while not actually evolving drug-like compounds, nevertheless demonstrates the possibility that peptidomimetic compounds that bind on the prime-side represent potential therapeutics. NON-PEPTIDIC INHIBITORS SCREENING LEADS
Significant efforts have also been expended in identifying non-peptidic small molecule inhibitors of the HCV NS304A protease. These compounds have almost always been identified from natural product library screening. Several structural classes have been described but there is a distinct lack of structural diversity even among the classes. Binding has usually been shown to be nonspecific. In some cases multiple inhibitor molecules appear to associate with the enzyme simultaneously. Several diarylamide derivatives, for example (1 11) and (1 12), that incorporate a naphthyl ether moiety have been identified [ 1401. The compounds display reasonable selectivity for HCV NS304A over a number of other serine proteases.
(111)
(112)
CI
Thiazolidine derivatives such as (1 13) and (1 14) are related to the diarylamide series of compounds exemplified by (1 1 1) and (1 12) and have also been shown to inhibit NS304A protease. The haloaryl groups common to both series of compounds are intriguing but the significance of this observation is thus far unknown.
244
HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY
(113) JCm = 2.3 pglmL
(114) IC5,= 3.2 pM
Modest potency has been observed for a series of 15 such inhibitors. Although potency varies for various double bond substitutions from low single digit pM to >50 pg/mL no clear structural relationships are observed [140, 1411. A series of arylalkylidene rhodanine based inhibitors of NS3*4A, for example (1 15), have been reported [ 1421. These rhodanine derivatives resemble a combination of the diary1 amides and thiazolidines described previously. Interestingly these compounds appear to be at least as effective as chymotrypsin and plasmin inhibitors.
H3C0
K 0
,OCHJ
T0
Identified from natural product screening is the fungal metabolite (1 16) [ 1431. Not many HCV protease inhibitors have been reported from the screening of natural product libraries but (1 16) exhibits potency against the NS3 enzyme comparable to the diarylamides and the thiazolidines. Acetylation of the hemiketal reduced potency approximately twofold. OH
(116) ICs0= 3.8 pg/mL
R.B. PERNI AND A.D. KWONG
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SYNTHESIS OF NS304A PROTEASE INHIBITORS GENERAL COMMENTS
The vast majority of the synthetic work has been carried out on peptidomimetic inhibitors. Some of the non-peptidic inhibtors are natural products (e.g., hemiketal 116) [ 1431 and some are commercially available (e.g., diary1 amides) [140]. Clever syntheses of warhead hnctionalities, which allow coupling to polymer supports, have allowed for the partially automated synthesis of the peptidomimetics analogues. As may be expected, the syntheses of peptidomoimetic inhibitors are generally long with low overall yields. This situation may result in lengthy process development programmes and commercialization/cost issues similar to those that originally beset HIV programmes.
BORONATES
A solid phase procedure was used to prepare the boronate series of peptidomimetic inhibitors. The route shown in Figure 5.9 provides the PI-warhead synthon in moderate yields [126]. The resin-based chemistry allows for the preparation of directed libraries of boronate inhibitors. The solid supported amino acid is elaborated using a standard deprotection-coupling sequence. A final acid-mediated hydrolysis frees the completed molecule. This route was also utilized for a solution phase synthesis of boronic acid inhibitors with only minor modifications.
ALDEHYDE SYNTHESIS
A number of groups have prepared aldehydic inhibitors and almost all routes are based on the Weinreb amide-reduction strategy. A typical solution phase methodology is shown in Figure 5.10 wherein a protected amino acid is converted to a Weinreb amide followed by lithium aluminium hydride reduction [129]. The P1 amino aldehyde is protected as a dimethylacetal and standard coupling protocols are carried out. A final deprotection step affords the polypeptide aldehyde. Aldehydic inhibitors are also readily prepared via efficient solid-supported chemistry. A SynPhase-MD-1 polymeric support was used (Figure 5.11). An fmoc protected aminoaldehyde is converted to an aminal via a tether to a polymeric support [ 1441. Peptide elaboration followed by simple TFA deprotection gives the aldehydic inhibitor.
HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY
246
Figure 5.9. Synthesis of boronic acid inhibitors.
R
HCI"H(OCHs)CH3 HOBT, NEM. CHzClz EtN=C=N(CH&N(CH&
-
R
PCH3 1 ) LiAIH, / THF
Fmocx!%N'CH30
c
2) HCI / MeOH
Figure 5.10. Solution-phase aldehyde synthesis.
u-KETO ACIDS AND DERIVATIVES
There are numerous methodologies available for preparing a-keto-acids, esters and amides. As might be expected, all are not equally amenable to the preparation of peptidomimetic derivatives. Two recent syntheses appear useful.
R.B. PERNI AND A.D. KWONG
241
Figure 5.11. Solid-phase aldehyde synthesis
A method starting with aldehydes derived from aminoacids has been found useful and reasonably efficient [123]. The route allows the coupling of a previously assembled tetramer to an a-hydroxy synthon. The aldehyde is condensed with acetone cyanohydrin and the resulting adduct is subsequently hydrolyzed. Following P 1 amide formation the hydroxyamide is coupled with the tetrapeptide. A final Dess-Martin oxidation provides the target compound. The DessMartin periodate has been found to be one of the few oxidants which effectively oxidizes the hydroxyl group without destroying the rest of the molecule.
L
TEA, CHzCIz
BOP / DlEA / DMF
2) HCI/ HzO/ dioxane 3) (BOC)~O / NazCO3/H20
1) HCI /dioxane
Boc-Asp(tBu)-Glu(tBu)-Val-Val-Pro’ 2) tetrapeptide BOP / D I E N DMF
1) Dess-Martin Oxidation
H-Asp-Glu-Val-Val-Pro’ 2) TFA / CHZCI2
Figure 5.12. a-Ketoamide synthesis
HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY
248
DHQPAL
T
O
C
H
3
AOCH, 1) Hz Pd IC
KzIOSOZ(OH)~I CbrNH
MeOH
Cbz-NCI Na H,O-iPrOH
1 Des-Martin Oxidation
2) TFA / CHzCIz
.Ir
2) Tetrapeptide BOP / DIEA / DMF
t
-
i
0
~
Figure 5.13. Alternative a-kefoamide synthesis
An alternative method begins with an olefin and a Sharpless aminohydroxylation protocol for the initial formation of the hydroxy ester intermediate [123]. For the target compound shown in Figure 5.13, a high enantiomeric excess of 95% was obtained after recrystallization of the Sharpless product. Ester hydrolysis and coupling affords the hydroxyamide. The convergent synthesis again culminates in the usual Dess-Martin oxidation/deprotection step. Novel PI carboxylic acids The novel ally1 cyclopropyl P 1 derivatives are prepared according to the route shown in Figure 5.14 [ 1 18, 1341. The condensation of 1,4-dibromo-2-butene with a protected glycine aldehyde gives the cyclopropane carboxylic acid. While the procedure is relatively economical a mixture of isomers is invariably obtained. After separation of diastereomers, the racemic product is resolved to its 1R-2S isomer via an enzymatic reaction with Alcalase.
HCI'H2NCH2C02Et NazSO, I TBME I Et3N
-
srN-co2Et B
r
A
B
r
LiOtBu I toluene I RT
then H30* then NaOH
Figure 5.14. Cycloprop,yl PI .synthesis.
*
R . B . PERNI AND A.D. KWONG
249
P3 INDOLINYL DERIVATIVES
Inhibitors incorporating the novel P3 capping substituent, the 2-indolinyl group, are prepared in a straightforward manner starting with commercially available indoline-2-carboxylic acid [72]. Protection followed by a-alkylation affords the capping synthon which is subsequently coupled to the peptide in standard fashion (Figure 5.15).
THE FUTURE: SUMMARY AND OUTLOOK One cannot separate the medicinal chemistry research into HCV NS3*4A protease inhibitors from the virological research devoted to the development of assays and models. The lack of cellular and animal model systems has hampered progress in the field. This is beginning to change. The construction of an HCV replicon [98], for example, should lead to assay systems that include most of the HCV genome in cell culture. Unfortunately, the development of animal models is progressing more slowly. While the chimpanzee model is generally accepted as the most reliable, it is still not validated. The cost associated with such primate models as well as ethical considerations preclude its use for screening large numbers of molecules. While clear progress in inhibitor design is being made, much work remains to be done. Inhibitory potencies against the NS3*4A protease are improving rapidly particularly with peptide-based inhibitors but the most potent compounds are still not drug-like: they are large, charged, and unlikely to penetrate cells. Removing the charge will provide compounds that are likely to improve cell penetration but will be overall less potent, very hydrophobic and
Figure 5.15. fndofinvl P3 .synthesis
250
HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY
consequently will be difficult to formulate into a suitable dosage form. While cellular model systems are becoming available no clear demonstration of protease inhibition in cellular or animal systems has been published. Non-peptic inhibitors are even further from the finishing line. Library screening has yielded a number of classes of inhibitory compounds but the potencies are less than those of peptidomimetic compounds. In addition, for the majority of non-peptidic inhibitors, simple inhibition kinetics are not observed. In some cases more than one inhibitor molecule is involved in binding to a single protease moiety. Moreover, the SAR has not been easily exploitable as evidenced by the lack of improved activity in analogues. Given this backdrop, structure-based drug design is clearly the best bet. On the brighter side, the recent advances in cellular replication systems, X-ray, NMR and SAR analyses are allowing researchers to begin to crack HCV’s secrets. The understanding of the subtleties of the enzyme function and structure will, in the not too distant future, yield useful, marketable protease inhibitors which will revolutionize the treatment of hepatitis C in the same manner that HIV protease inhibitors revolutionized therapy for AIDS patients. REFERENCES 1 Alberti, A,, Chemello, L. and Benvegnu, L. (1999) J. Hepatol. 31 (Suppl I), 17-24. 2 Hagedorn, C.H. and Rice, C.M. (2000) Curr. Top. Microbiol. Immunol. Vol. 242. Springer, Berlin, New York. 3 Lavanchy, D. (1999) J. Viral Hep. 6, 35-47. 4 Steedman, A., Sarbah, M.D. and Younossi, Z.M. (2000) J. Clin. Gastroenterol. 30, 125-143. 5 Feinstone, S.M., Kapikian, A.Z., Purcell, R.H., Alter, M.J. and Holland, P.V. (1975) New Engl. J. Med. 292, 767-770. 6 Schreiber, G.B., Busch, M.P., Kleinman, S.H. and Korelitz, J.T. (1996) New Engl. J. Med. 334, 1685-1690. 7 Alter. M.J., Kruszon-Moran, D. and Nainan, O.V. (1999) N. Engl. J. Med. 341, 556562. 8 Lam, N.P. (1999) Am. J. Health-Syst. Pharm. 56, 961-973. 9 MacDonald, M., Crofts, N. and Kaldor, J. (1996) Epidemiologic Rev. 18, 137-148. 10 Niederau, C., Lange, S . , Heintges, T., Erhardt, A., Buschkamp, M., Hurter, D., Nawrocki, M., Kruska, L., Hensel, F., Petry, W. and Haussinger, D. (1998) Hepatology 28, 1687-1695. 11 Seef, L.B. (1997) Hepatology 26(Suppl. l ) , 21S28S. 12 Huang, L. and Koziel, M. (2000) Curr. Opin. Immun. 16, 55%564. 13 Kew, M.C. (1994) FEMS Microbiol. Rev. 14,211-220. 14 National Institutes of Health Consensus Development Statement. Management of Hepatitis C. (1997) Hepatology 26(Supplement I), 2’SIOS. 15 KoK R.S. (2000) Am. J. Gastroenter. 95, 1392-1393. 16 Malone, D.C. (2000) Formulatory 35, 681. 17 Wong, J.B., Poynard. T., Ling, M.H., Albrecht, J.K. and Pauker, S.G. (2000) Am. J. Gastroenter. 95, 1524-1530. 18 Radkowski, M.. Kubicka, J., Kisiel, E., Cianciara, J., Nowicki, M., Rakela, J. and Laskus, T. (2000) Blood 95, 39863989.
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97 98 99 I00 101 I02 I03
253
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I07 108 109 110
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I12 1 I3 1 I4 1 I5
I I6 I17 118
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123 124
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140 141 142 143 144
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Subject Index A-6632 I , macrolide antibiotic, 104 ABT-773, S0S ribosomal binding site, 98 ketolide antibiotic, 104 rat lung infections and, 98, 104, 105 ACAT inhibitors, adrenal toxicity, 137, 138, 144, IS1 classes, 139-162 clinical studies, 127, 14&l46, 149, 162 effect on secretion of apoB, 128 evolution of, 13&139 Iiypolipidemic effects in animal models. 141, 149 pharmacophores, 140 plaque stabilizing agents, 130-1 32 ACAT, acyl-CoA:cholesterol 0-acyl transferase, 123 isoforms, 133 pharmacology, 127-132 physiology, 123-1 27 regulation. 132 transgenic mouse models, 133, 134 ACAT-I, 133, 139, 140 ACAT-2, 133, 139 Acaterin, ACAT inhibitor, 153 ACTH, elevation by GHSs, 197, 199 Alanine scan, 229 Alcaiase, 248 Aldehydes, warheads for HCV protease inhibitors, 232, 245 Alzheimer’s disease, effect of caspase-1 in, 6 Amantadine, 2 18 Amidino 2,5-diphenylfurans, anti- PCP activity, 7 9 4 1 Amyioid precursor protein processing, 7 Amyotrophic lateral sclerosis, 3, 47 Anisomycin, 50s ribosomal binding site, 98 pyrrolidine antibiotic, 113 Apaf-I, CED-4 homologue, 13, 14 Apollon, apoptosis inhibitory protein, 15 Apoptosis. autoimmune diseases and, 3 definition of, 3 neurodegenerative diseases and, 3 pathways, 12, 14
role of caspases, 5-9 ARC, apoptosis inhibitor, 13 Aren, 13 AS-183, ACAT inhibitor, 153 AS-I 86, ACAT inhibitor, 152 Aspartate methylene ketones, caspase inhibitors, 27, 30 Atherosclerosis, I22 experimental models, 132, 136, 141, 144 macrophages and, 125,130 Autoimmune lymphoproliferative syndrome, caspase-I0 and, 9 Avasimibe, CI-101 I , ACAT inhibitor, 128, 131, 132, 148, 149, 162 Azithromycin, macrolide antibiotic, 104 rat lung infections and, 105 Bacterial ribosomes, function, 89 structure, 89 Beauvericin, ACAT inhibitor, 152 Bimodal inhibitors of caspases, 26 BIR, 16 Blasticidin S, 50s ribosomal binding site, 98 nucleoside antibiotic, 113 Boronates, warheads for HCV protease inhibitors, 234, 245 Brain ischaemia, model for, 44 Buried surface area of protease inhibitors, 227 BW-447A, 447C88, ACAT inhibitor, 128, 162 Cadmium salts, inhibitors of apoptosis, 19 CARDIAK, RIP-like kinase, 1 I Caspase inhibitors, aspartate methylene ketones, 27, 30 cadmium salts, 19 clinical studies, 50 dithiocarbamates, I9 epoxides, 21, 22 in vivo models for, 43-50 Michael acceptors, 20 nitric oxide donors, I8 non-peptidic, 18 peptide derived, 3 4 4 3 257
258
SUBJECT INDEX
pharmacophores, 24-32 phenyl arsine oxide, 19 proteins, 15-1 8 zinc salts, 19 Caspases, classification, 2, 3 regulation of, role in disease, 4 9 role in neurodegenerative diseases, 3 Cathepsin C, 31 Celesticetin, 23s rRNA binding site, 95 CGP40336, Tat/TAR inhibitor, 78 CGP64222, Tat/TAR inhibitor, 78 Chimpanzee model of HCV infection, 225 Chloramphenicol, 50s ribosomal binding site, 98, 102 Cholesteryl ester cycle, 125 CI-976, ACAT inhibitor, 127, 128, 131, 132, 136, 140, 144 CI-999, PD 138142-15, ACAT inhibitor, 128, 132, 148 CL 277,082, ACAT inhibitor, 128 131, 162 Clarithromycin, 74, 103 Clofibrate, hypocholesterolemic agent, 122 Cortisol, release by GHS, 197-200 CP-113,818, ACAT inhibitor, 128, 131, 138, 140 CP-424,391, spiropiperidine-based GHS, 181, 196 CPP32, caspase-3, 4, 7 C m A , caspase-1 inhibitor, 15, 17 CV 1013, irreversible caspase inhibitor, 50 Cycloheximide, 50s ribosomal binding site, 98 glutarimide antibiotic, 1 I3 Cytochrome c, caspase activator, 11, 13 Dalfopristin, 50s ribosomal binding site, 98, 106 macrolide antibiotic, 106 Dess Martin oxidation, 247 Diablo, 16 2,4-Diaminoquinolines, Tat/TAR inhibitors, 8 1 Diarylamides, NS3.4 protease inhibitors, 243 a-Dicarbonyl derivatives, warheads for HCV protease inhibitors, 235-237, 246 Diphenylfuran bisamidines, Rev/RRE binding inhibitors, 8 S 8 8 DISC, death inducing signalling complex, 12, 13
Dithiocarbamates, caspase inhibitors, I9 DuP 128, ACAT inhibitor, 128, 131, 144, 149, 162 E 5324, ACAT inhibitor, 131, 132, 145, 162 Em-309, ACAT inhibitor, 145 Edeine, 30s ribosomal binding site, 97, 100 Eglin C, peptidic HCV protease inhibitor, 228 El-1507-1, caspase inhibitor, 21 EI-1507-2, caspase inhibitor. 2 1 Enniatin antibiotics, ACAT inhibitors, 152 EP 5 1389, peptidic GHS, 182, 196 Epi-cochlioquinone A, ACAT inhibitor, 154 Epoxides, caspase inhibitors, 2 1 Epoxyquinones, caspase inhibitors, 32 ERICE, caspase-13, 4, 9 Erythromycin A, 50s ribosomal binding site, 98 macrolide antibiotic, 74, 103 Erythromycin, 23s rRNA binding site, 95 Evemimicin, 50s ribosomal binding site, 98, 108 Experimental autoimmune encephalomyletis, 6 animal model of multiple sclerosis, 9 caspase-3 levels in, 10 focal stroke induced, 8 Extrinsic apoptotic pathways, 12 F 12511, ACAT inhibitor, 131, 141 F 1394, ACAT inhibitor, 146 FADD, fas-associated death domain, 12, 13 FCE 27677, ACAT inhibitor, 145 FIGS, fusion-induced gene stimulation assay, 78 Flaviridue viruses, 217, 219 FLICE, caspase-8, 4, 8 FLIP, 13 Foam cells, inhibition of formation, 126, 129 FR 145237, 132, 136, 138, 144 FR 182980, ACAT inhibitor, 144 FR 186485, ACAT inhibitor, 139, 145 FR 186485, ACAT inhibitor, 145 FR 190809, ACAT inhibitor, 139, 145
G protein coupled peptide receptors, agonists 174 (3-7039, peptidomimetic GHS, 184, 196 G-7203, peptidomimetic GHS, 193
SUBJECT INDEX (3-7502, peptidomimetic GHS, 193 G-7502, peptidomimetic GHS, 185, 196 GB Virus-B, 225, 226 GEM-BP001, ACAT inhibitor, 157 GERI-BP002-A, ACAT inhibitor, 157 Ghrelin, endogenous peptidic GH secretagogue, 176 Ghrelin, receptor, 193 GHRP-I, peptidic growth hormone secretagogue, 177, 182, 196, 201, 202 GHRP-2, peptidic growth hormone secretagogue, 177, 182, 192, 196, 197, 201-203 GHRP-6, peptidic growth hormone secretagogue, 177, 179, 194, 196199, 201,202 GHS IA receptor, I76 GHS 1 A receptor, adenosine and, I76 binding sites, 194, 199 Glisoprenins, ACAT inhibitors, 152 Gomisin N, ACAT inhibitor, 162 Growth hormone releasing hormone, actions, 175-177 binding sites, I99 Growth hormone secretagogues, assays, I94 benzolactam based, I7&180 binding sites, 198, 199 central actions, I98 clinical studies, 175, 192, 201-206 evolution of, 177, 192 indications for, 202 mechanism, I99 peptidic, 182-1 85 peptidomimetic, I8S192 pharmacology, 194-20 I pharmacophore, 178, 192, 193 receptor subtypes, 199, 200 screening assays, 194 specificity, 197. 198 spiropiperidine based, 180 Gypsetin, ACAT inhibitor, 155 HCV minibody, 228, 229 Helminthosporol. ACAT inhibitor, 153 Hepatitis C virus disease, 2 16, 2 17 animal models, 225 current treatment, 217, 218 prevalence, 2 16, 2 I7
259
prognosis, 2 17 Hepatitis C virus NS3.4A protease inhibitors, 226 drug design, 226, 227, 231 macrocyclic peptidomimetics, 24 1 non-peptidic, 243, 244 PI specificity pocket, 237-239 peptidic non-substrate based, 228 substrate based, 229-232 synthesis of, 245-249 warheads, 232 Hepatitis C virus NS3.4A protease, active site, 218, 223, 227, 228 enzymatic assays, 223 NS4A cofactor, 220-223 polyprotein cleavage sites, 219 replication assays, 225 structure and function, 216, 219, 220 subsites and nomenclature, 226 surrogate cell assays, 224 Hepatitis C virus polyprotein, 219 Hepatitis C virus, animal models, 225 genome, 2 19 genotypes, 2 18 minibody, 229 polyprotein, 21 9 subsites and nomenclature, 226 Hepatitis, caspase inhibitors in, 50 mouse model, 49 Heptazyme, 2 18 Hexarelin, peptidic GHS, 183, 196, 197, 200-202 HIV-I Tat, 75 HL-004, TS-962, ACAT inhibitor, 131, 140, 141, 144 HMG-CoA reductase inhibitors, 122 HMR 3004, macrolide antibiotic, 104 HMR 3647, macrolide antibiotic, 104 Hoechst 33258, Tat/TAR inhibitor, 83 Human growth hormone, actions, 175 clinical studies, 175 regulation of release, 175 therapeutic applications, 175 Human immunodeficiency virus, 4, 75-84 Huntingdon’s disease, 3 caspase activation and, 6 caspase-3 in, 7 caspase-6 and, 8
260
SUBJECT INDEX
caspase-8 and, 8 mouse model, 48 4-Hydroxynonenal, caspase inhibitor, 20 Hygromycin B, 30s ribosomal binding site, 93, 97, 99 Hygromycin B. aminoglycoside antibiotic, 97 ICE, caspase-I, 4 6 ICEBERG, 1 1 IDN 1529, caspase inhibitor, 47 IDN 1965, caspase inhibitor, 35, 47 IDN 5370, irreversible caspase inhibitor, 50 IDN 6556, caspase-8 inhibitor, 50 Inducible nitric oxide synthesis, regulation by caspase inhibitors, 10 Insulin-like growth factor-I, 176 GH secretagogue-induced release, I97 GH-induced release, 192 Interferon-ribaverin, treatment for hepatitis C, 217 Interleukin-18, release by caspase-1, 4. 5 role in disease, 46, Interleukin-I p, 4 release by caspase- I , 5 role in Alzheimer's disease, 6 role in shigellosis, 6 Intrisic apoptotic pathways, 12 Ipamorelin, peptidic GHS, 183, 196, 20C202 IRES, internal ribosomal entry site, 218 Ischaemia, raised caspase-3 levels in, 10 Isochromophilones, ACAT inhibitors, 156 Kanamycin, aminoglycoside antibiotic, 93 Kasugamycin, 305 ribosomal binding site, 97. 100 a-Ketoamides, caspase inhibitors, 21 Kudingosides, ACAT inhibitors, 161 L-158,077, benzolactam based GHS, 178 L- 163,540, spiropiperidine-based GHS, 181, 196 L-692,429, benzolactam based GHS, 179, 182, 194, 196, 197, 202, 204, 205 L-692,585, benzolactam based GHS, 179, 194, 196 L-739,743, benzolactam based GHS, I79 L-739,943, benzolactam based GHS. I79 Lateritin, ACAT inhibitor, 153
Lincoinycin S, lincosamide antibiotic, 98, 105 Lincomycin. 50s ribosomal binding site, 98 Lincosamides, 23s rRNA binding site, 95 Linezolid, 23s rRNA binding site, 95 50s ribosomal binding site, 98 oxazolidinone antibiotic, 108 ribosomal binding sites, 108 Lipinski rule of five, 185 Lipolisaccharide, 48 Liver cirrhosis, hepatitis C induced, 216, 217 Livin, apoptosis inhibitory protein, 15, 16 LY444711, spiropiperidine-based GHS, 181, I96 Melinamide, ACAT inhibitor, 135, 140, 162 Meningitis, rabbit model, 49 MICE, caspase-14,4, 9 Michael acceptors, caspase inhibitors, 20 Micrococcin, thiazole antibiotic, 105 Middle cerebral artery occlusion, caspase inhibitors in, 44 caspase-3 mRNA levels and, 10 caspase-8 and, 8 focal stroke and, 8 MK 677, spiropiperidine-based GHS, 180, 181, 192, 194, 196-199, 201-205 MK 801, synergistic effects with caspase inhibitors, 46, 5 I Multiple sclerosis, animal model, 9, 47 caspase - I 1 and. 4 Narciclasine, 50s ribosomal binding site, alkaloid antibiotic, 113 Naringenin, ACAT inhibitor, 161 NCX-4016, caspase inhibitor, SO caspase-l inhibitor, 19, 50 Neomycin B, Rev/RRE binding inhibitor, 84 Neomycin, aminoglycoside antibiotic, 93 Tat/TAR inhibitor, 8 1 Nitric oxide donors, caspase inhibitors, 18 endogenous inhibitor of apoptosis, I8 NMDA, 46 "703, activity, 196-198, 200, 202 analogues, 187-192 binding to GHS 1 A receptor, 194 peptidomimetic GHS, 186 pharmacophore model, 194, 195 NNC 26-0235, peptidomimetic GHS, 184, 196
SUBJECT INDEX NNC-26-0323, peptidomimetic GHS, 185, 194, 196 NNC 26-0610. benzolactam based GHS, 179, 196 NNC 26-0722, peptidomimetic GHS, 189, 191, 196 NNC 26-1 167. peptidomimetic GHS, 189, 191, 192 NPY neurons, effects on growth hormone release, 199 NS3.4A complex. 220, 235 NS4A. cofactor for NS3.4 protease, 22C223, 248 NTE-122, ACAT inhibitor, 131, 146 Obovatol, ACAT inhibitor, 160 Osteodrthritis, caspasc inhibitors in, 50 caspase-l and. 6 caspase-3 and. 8 Oxazolidinones. 23s rRNA binding site, 95 70s ribosomal binding site, 108 antibiotics, 96. 108 Oxyanion hole, of HCV proteases, 235 P' region based inhibitors of HCV NS3, 242, 243 PI specifity pocket, 237-239 P2 substituents, 239 P3 substituents. 240, 241, 249 P4 substituents, 240 Pactamycin, 30s ribosomal binding site, 97, 99 Panaxadiols. ACAT inhibitors, 160 Parkinson's disease, 3 caspase inhibitors and, 6 caspase-3 activation and. 8 Paromomycin, I 6S rRNA binding site, 93 30s ribosomal binding site, 97, 99 aminoglycoside antibiotic, 93 PCP, Pneimwcj:s/i.s curinii pathogen, 79 ID ' 132301-2, 131, 136, 147 PD 138142-15, ACAT inhibitor, 128, 137 PEG-interferon. 2 I7 Pcntenocin A. caspase-l inhibitor, 2 I Peptidic GHSs. SAR for. 178 Peptidyl transfer centre, 50s subunit target for antibiotics, 100 Phenyl arsine oxide, caspasc inhibitor, I9 Pleuroniutilin, 50s ribosomal binding site, 98, 109. 110
26 1
analogues, 10%-112 antibiotic, I09 pNA assay for protease inhibitors, 223 Polyacetylenes. ACAT inhibitors, 158, 159 Polyazadipyridinocyclophanes,Tat/TAR inhibitors, 8 1 Pralnacasan, VX-740, caspase inhibitor, 25, 50 Prion protein, activation of caspase-3, 7 Privileged structures, 192 Prolactin, release by GHS, 201 Protein synthesis, ribosomal, 9&93 Protopanaxadiols, ACAT inhibitors, I60 Puromycin, 50s ribosomal binding site, 98, 101 nucleoside antibiotic, 98 Purpactin antibiotics, ACAT inhibitors, I52 Pyripyropropenes, ACAT inhibitors, 1 5 6 159 Quiescent affinity label, 25 Quinones, caspase inhibitors, 2 I , 3 1 Quinoxaline-2, 3-diones, Tat/TAR inhibitors, XI Quinupristin, 50s ribosomal binding site, 996, 106 niacrolide antibiotic, 106 Rev/RRE binding inhibitors, 83-88 function, 89 structure, 89 RhdRev. 84 Rheumatoid arthritis, caspase inhibitors in, 5 I Ribavirin, 2 17 16s rRNA, antibiotic binding sites, 94 23s rRNA, antibiotic binding sites, 95, 96 30s Ribosomal sub-unit, interaction with antibiotics, 100 30s Ribosomal sub-unit, structure, 99 50s Ribosomal sub-unit, interaction with antibiotics, 102 50s Ribosomal sub-unit, structure, 101 Ribozymes, 218 RP 73 163, ACAT inhibitor, 138, 15 I
S.pnerrrnoniue pathogens, 104, 105 SaH 57-118, ACAT inhibitor, 135, 162 SaH 58-035, ACAT inhibitor, 128, 131, 135, 138, 140, 162 Scintillation proximity assay, 224, 225 Secobatzelline A, caspase inhibitor, 2 I Sharpless amino hydroxylation, 248
262
SUBJECT INDEX
Smac, 16 Solid-phase peptide synthesis, 245 Somatostatin, effect on growth hormone release, 198, 199 Spectinomycin, 16s binding, 99 30s ribosomal binding site, 97, 99 aminoglycoside antibiotic, 93 Spiramycin, 105 macrolide antibiotic, 103 Streptogramins, macrolide antibiotics, 106 Streptomycin. 30s ribosomal binding site, 97,99 aminoglycoside antibiotic, 93 Survivin, apoptosis inhibitory protein, 15. 16 T2 toxin, 50s ribosomal binding site, 98 sesquiterpene antibiotic, 1 13 T-2591, ACAT inhibitor/anti-oxidant, 145 Tamarin model of HCV infection, 225, 226 TAR, 75 Tat, 75 Tat/TAR inhibitors, 2,4-diaminoquinolines, 8 1 polyazadipyridinocyclophanes,8 1 quinoxaline-2, 3-diones, 8 I TEI-6522, ACAT inhibitor, 142, 143 TEI-6620, ACAT inhibitor, 142, 143 Telithromycin, binding to ribosomes, 103 macrolide antibiotic, 103, 104 rat lung infections and, 105
Terpendoles, ACAT inhibitors, 154 Tetracycline, 30s ribosomal binding site, 97,99 Thiostrepton, 23s rRNA binding site, 96 50s ribosomal binding site, 98, 106 thiopeptide antibiotic, 74, 96 Tiamulin, pleuromutilin antibiotic, 109 TMP-153, ACAT inhibitor, 145 Trimera mouse, model for HCV infection, 225 Tylosin, macrolide antibiotic, 103, 105 Viomycin, 23s ribosomal binding site, 107 50s ribosomal binding site, 98 peptidic antibiotic, 108 VX-740, caspase-l inhibitor, 25 Weinreb amide, 245 XP 767, ACAT inhibitor, 138, 140 XR 920, ACAT inhibitor, 138, 140 Xylaric acid, caspase inhibitor, 21 YM 17E, ACAT inhibitor, 146, 162 Zinc salts, caspase inhibitors, 19 Z-VAD-fmk, inhibitor of cytotoxic brain damage, 46 tetrapeptide caspase inhibitor, 6, 34, 43, 46, 47, 50, 51
Cumulative Index of Authors for Volumes 1-39 The volunie niiniher, (veur qf’publicution) und puge number are given in /ha/ order.
Aboul-Ela, F.. 39 (2002) 73 Adams, J.L., 38 (2001) I Adams, S.S.. 5 ( 1967) 59 Afshar, M.. 39 (2002) 73 Agrawal, K.C.. 15 (1978) 321 Albrecht, W.J.. 18 (1981) 135 Allain, H., 34 (1997) I Allen, N.A., 32 (1995) 157 Allender. C.J., 36 (1999) 235 Andrews, P.R., 23 (1986) 91 Ankersen, M., 39 (2002) 173 Ankier, S.I.. 23 (1986) 121 Arrang, J.-M., 38 (2001) 279 Badger, A.M., 38 (2001) 1 Bailey, E., I 1 (1975) 193 Ballesta, J.P.G.. 23 (1986) 219 Banting, L.. 26 (1989) 253; 33 (1996) 147 Barker, G., 9 (1973) 65 Barnes, J.M., 4 (1965) 18 Barnett, M.I., 28 (1991) 175 Batt. D.G., 29 ( 1992) 1 Beaumont, D., 18 (1981) 45 Beckett, A.H., 2 (1962) 43; 4 (1965) 171 Beckman, M.J.. 35 (1998) 1 Beddell, C.R., I7 (1980) 1 Beedham, C., 24 (1987) 85 Beeley, L.J., 37 (2000) I Beisler, J.A., I9 ( 1975) 247 Bell, J.A., 29 (1992) 239 Belliard, S., 34 ( 1997) I Benfey, B.G., 12 (1975) 293 Bentue-Ferrer, D., 34 (1997) I Bemstein, P.R., 3 1 (1994) 59 Binnie, A,, 37 (2000) 83 Black, M.E., I I ( 1975) 67 Blandina. P., 22 (1985) 267 Bond. P.A., 1 1 (1975) 193 Bonta, I.L.. 17 (1980) 185
Booth, A.G., 26 (1989) 323 Boreham, P.F.I., 13 (1976) 159 Bowman, W.C., 2 (1962) 88 Bradner, W.T., 24 (1987) 129 Bragt. P.C., 17 (1980) 185 Brain, K.R., 36 (1999) 235 Branch, S.K., 26 (1989) 355 Braquet, P., 27 (1990) 325 Brezina, M., 12 (1 975) 247 Brooks, B.A., I I (1975) 193 Brown, J.R.. 15 (1978) 125 Brunelleschi. S., 22 (1985) 267 Bruni, A., 19 (1982) I 1 I Buckingham, J.C., 15 (1978) 165 Bulman, R.A., 20 (1983) 225 Carman-Krzan, M.. 23 (1986) 41 Cassells, A.C., 20 (1983) 119 Casy, A.F., 2 (1962) 43; 4 (1965) 171; 7 (1970) 229; I I (1975) 1; 26 (1989) 355 Casy, G., 34 (1997) 203 Caton, M.P.L., 8 (1971) 217; 15 (1978) 357 Chambers, M.S.,37 (2000) 45 Chang, J., 22 (1985) 293 Chappel, C.I., 3 (1963) 89 Chatterjee, S., 28 (1991) I Chawla, A.S., 17 (1980) 151; 22 (1985) 243 Cheng, C.C., 6 (1969) 67; 7 (1970) 285; 8 (1971) 61; 13 (1976) 303; 19 (1982) 269; 20 (1983) 83; 25 (1988) 35 Clark, R.D., 23 (1986) 1 Cobb, R., 5 (1967) 59 Cochrane, D.E., 27 (1990) 143 Coulton, S., 3 1 ( 1 994) 297; 33 (1996) 99 Cox, B., 37 (2000) 83 Crossland, J., 5 (1967) 251 Crowshaw, K.,15 (1978) 357 Cushman, D.W., 17 (1980) 41 Cuthbert, A.W., 14 (1977) I 263
264
CUMULATIVE AUTHOR INDEX
Dabrowiak, J.C., 24 (1987) 129 Daly, M.J., 20 (1983) 337 D’Arcy, P.F., 1 (1961) 220 Daves, G.D., 13 (1976) 303; 22 (1985) I Davies, G.E., 2 (1962) I76 Davies, R.V., 32 (1995) 115 De Clercq, E., 23 (1986) 187 Dc Gregorio, M., 21 (1984) 11 I De Luca, H.F., 35 (1998) I De, A,, 18 (1981) 117 Demeter, D.A., 36 (1999) 169 Denyer, J.C., 37 (2000) 83 Derouesnb, C., 34 (1997) 1 Dimitrakoudi, M., 11 (1975) 193 Donnelly, M.C., 37 (2000) 83 Draffan, G.H., 12 (1975) I Drcwe, J.A., 33 (1996) 233 Drysdale, M.J., 39 (2002) 73 Dubinsky, B., 36 (1 999) 169 Duckworth, D.M., 37 (2000) I Duffield, J.R., 28 (1991) 175 Durant, G.J., 7 (1970) 124 Edwards, D.I., 18 (1981) 87 Edwards, P.D., 3 I (1994) 59 Eldred, C.D., 36 (1999) 29 Ellis, G.P., 6 (1969) 266; 9 (1973) 65; 10 (1974) 245 Evans, B., 37 (2000) 83 Evans, J.M., 31 (1994) 409 Falch, E., 22 (1985) 67 Fantozzi, R., 22 (1985) 267 Feigcnbaum, J.J., 24 (1987) 159 Feucr, G., 10 (1974) 85 Finberg, J.P.M., 21 (1984) 137 Fletcher, S.R., 37 (2000) 45 Floyd, C.D., 36 (1999) 91 FranCois, I., 3 I (1994) 297 Frank, H., 27 (1990) 1 Freeman, S., 34 (1997) 11 1 Fride, E., 35 (1998) 199
Gale, J.B., 30 (1993) 1 Gancllin, C.R., 38 (2001) 279 Gdrbarg, M., 38 (2001) 279 Garratt, C.J., 17 (1980) 105 Gill, E.W., 4 (1965) 39
Ginsburg, M., 1 (1961) 132 Goldberg, D.M., 13 (1976) I Gould, J., 24 (1987) 1 Graczyk, P.P., 39 (2002) 1 Graham, J.D.P., 2 (1962) 132 Green, A.L., 7 (1970) 124 Green, D.V.S., 37 (2000) 83 Greenhill, J.V., 27 (1990) 51; 30 (1993) 206 Griffin, R.J., 31 (1994) 121 Griffiths, D., 24 (1987) 1 Griffiths, K., 26 (1 989) 299 Groencwcgen. W.A., 29 (1992) 217 Groundwater, P.W., 33 (1996) 233 Guile, S.D., 38 (2001) I15 Gunda, E.T., 12 (1975) 395; 14 (1977) 181 Gylys, J.A., 27 (1990) 297 Hacksell, U., 22 (1985) 1 Hall, A.D., 28 (1991) 41 Hall, S.B., 28 (1991) 175 Halldin, C., 38 (2001) 189 Halliday, D., 15 (1978) 1 Hammond, S.M., 14 (1977) 105; 16 (1979) 223 Hamor, T.A., 20 (1983) 157 Hanson, P.J., 28 (1991) 201 Hanus, L.. 35 (1998) 199 Hargreaves, R.B., 31 (1994) 369 Hams, J.B., 21 (1984) 63 Hartlcy, A.J., 10 (1974) 1 Hartog, J., 15 (1978) 261 Heacock, R.A., 9 (1973) 275; 11 (1975) 91 Heard, C.M., 36 (1999) 235 Hcinisch, G., 27 (1990) I ; 29 (1992) 141 Heller, H., I (1961) 132 Hcptinstall, S., 29 (1992) 217 Herling, A.W.. 3 I (1994) 233 Hider, R.C., 28 (1991) 41 Hill, S.J., 24 (1987) 30 Hillen. F.C., 15 (1978) 261 Hino, K., 27 (1990) 123 Hjeds, H., 22 (1985) 67 Holdgatc, G.A., 38 (2001) 309 Hooper, M., 20 (1983) 1 Hopwood, D., 13 (1976) 271 Hosford, D., 27 (1990) 325 Hubbard, R.E., 17 (1980) 105 Hughes, R.E., 14 (1977) 285 Hugo, W.B., 31 (1994) 349
CUMULATIVE AUTHOR INDEX I-lulin, B., 31 (1994) I Humber, L.G., 24 ( 1987) 299 Hunt, E., 33 (1996) 99 Ijzerman, A.P., 38 (2001) 61 Imam, S.H., 21 (1984) 169 Ince. F., 38 (2001) I15 Ingall, A.H., 38 (2001) I15 Ireland, S.J., 29 (1992) 239 Jacques, L.B., 5 (1967) 139 James, K.C., 10 (1974) 203 Jiszberenyi. J.C.. 12 (1975) 395; 14 (1977) 181 Jcnner. F.D., 1 I (1975) 193 Jewers, K., 9 ( 1973) 1 Jindal, D.P.. 28 (1991) 233 Jones, D.W., 10 (1974) 159 Judd. A,, I 1 ( 1975) 193 Judkins, B.D., 36 (1999) 29 Kadow, J.F., 32 (1995) 289 Kapoor, V.K., 16 (1979) 35; 17 (1980) 151; 22 ( 1 985) 243 Kawato, Y., 34 ( 1997) 69 Kelly, M.J.. 25 ( 1988) 249 Kendall, H.E., 24 (1987) 249 Kennis, L.E.J., 33 (1996) 185 Khan, M.A., 9 (1973) 117 Kiefel, M.J., 36 (1999) 1 Kilpatrick, G.J., 29 (1992) 239 Kindon, N.D., 38, (2001) I15 Kirst, H.A., 30 ( 1993) 57; 3 I (1994) 265 Kitteringham, G.R., 6 (1969) I Knight, D.W., 29 (1992) 217 Kobayashi, Y., 9 (1973) 133 Koch, H.P., 22 (1985) 165 Kopelent-Frank, H., 29 (1992) 141 Kramcr, M.J., I8 (1981) 1 Krause. B.R., 39 (2002) 121 Krogsgaard-Larsen, P., 22 ( 1 985) 67 Kulkami, S.K., 37 (2000) 135 Kumar, M., 28 (1991) 233 Kumar, S., 38 (2001) I Kwong, A.D.. 39 (2002) 215 Lambert, P.A., 15 (1978) 87
265
Launchbury, A.P., 7 (1970) 1 Law, H.D., 4 (1965) 86 Lawen, A., 33 (1996) 53 Lawson, A.M.. 12 (1975) 1 Leblanc, C., 36 (1999) 91 Lee, C.R., I 1 (1975) 193 Lee, J.C., 38 (2001) 1 Lenton, E.A., 1 I (1975) 193 Lentzen, G., 39 (2002) 73 Levin, R.H., 18 (1981) 135 Lewis, A.J., 19 (1982) 1; 22 (1985) 293 Lewis, D.A., 28 (1991) 201 Lewis. J.A., 37 (2000) 83 Lien, E.L., 24 (1987) 209 Ligneau, X., 38 (2001) 279 Lin, T.-S., 32 (1995) I LIU,M.-C., 32 (1995) 1 Lloyd, E.J., 23 (1986) 91 Lockhart, I.M., 15 (1978) 1 Lord, J.M., 24 (1987) 1 Lowe, LA., 17 (1980) I Lucas, R.A., 3 (1963) 146 Lue, P., 30 (1993) 206 Luscombe, D.K., 24 (1987) 249
Mackay, D., 5 (1967) 199 Main, B.G., 22 (1985) 121 Malhotra, R.K., 17 (1980) 151 Manchanda, A.H., 9 (1973) 1 Mander, T.H., 37 (2000) 83 Mannaioni, P.F., 22 (1985) 267 Martin, I.L., 20 (1983) 157 Martin, J.A., 32 (1995) 239 Masini, F., 22 (1985) 267 Matassova, N., 39 (2002) 73 Matsumoto, J., 27 (1990) 123 Matthews, R.S., 10 (1974) 159 Maudsley, D.V., 9 (1973) 133 May, P.M., 20 (1983) 225 McCaguc, R., 34 (1997) 203 McLelland, M.A., 27 (1990) 5 1 McNeil. S., 11 (1975) 193 Mcchoulam, R., 24 (1987) 159; 35 (1998) 199 Meggens, A.A.H.P., 33 (1996) 185 Megges, R., 30 (1993) 135 Meghani, P., 38, (2001) 115 Merritt, A.T., 37 (2000) 83
266
CUMULATIVE AUTHOR INDEX
Michel, A.D., 23 (1986) 1 Miura, K.. 5 (1967) 320 Moncada, S., 21 (1984) 237 Monkovic, I., 27 (1990) 297 Montgomery, J.A., 7 (1970) 69 Moody, G.J., 14 (1977) 51 Morris, A,, 8 (1971) 39; 12 (1975) 333 Mortimore, M.P., 38 (2001) I15 Munawar, M.A., 33 (1996) 233 Murchie, A.I.H., 39 (2002) 73 Murphy, F., 2 (1962) 1; 16 (1979) 1 Musallan, H.A., 28 (1991) 1 Musser, J.H., 22 (1985) 293 Natof, I.L., 8 (1971) 1 Neidle, S., 16 (1979) 151 Nicholls, P.J., 26 (1989) 253 Nodiff, E.A., 28 (1991) 1 Nordlind, K., 27 (1990) 189 Nortey, S.O., 36 (1999) 169 O’Hare, M., 24 ( I 987) 1 Ondetti, M.A., 17 (1980) 41 Ottenheijm, H.C.J., 23 (1986) 219 Oxford, A.W., 29 (1992) 239 Paget, G.E., 4 (1965) 18 Palatini, P., 19 (1982) I 1 1 Palazzo. G., 21 (1984) I 1 I Palfreyman, M.N., 33 (1996) I Palmer, D.C., 25 (1988) 85 Parkes, M.W., 1 (1961) 72 Parnham, M.J., 17 (1980) 185 Parratt, J.R., 6 (1969) 1 I Patel, A,, 30 (1993) 327 Paul, D., 16 (1979) 35; 17 (1980) 151 Pearce, F.L., 19 (1982) 59 Peart, W.S., 7 (1970) 215 Perni, R.B., 39 (2002) 215 Petrow, V., 8 (1971) 171 Picard, J.A., 39 (2002) 121 Pike, V.W., 38 (2001) 189 Pinder, R.M., 8 (1971) 231; 9 (1973) 191 Ponnudurai, T.B., 17 (1980) 105 Powell, W.S., 9 (1973) 275 Power, E.G.M., 34 (1997) 149 Price, B.J., 20 (1983) 337 Prior, B., 24 (1987) I
Procopiou, P.A., 33 (1996) 33 I Purohit, M.G., 20 (1983) 1 Ram, S., 25 (1988) 233 Reckendorf, H.K., 5 (1967) 320 Reddy, D.S., 37 (2000) 135 Redshaw, S., 32 (1995) 239 Rees, D.C., 29 (1992) 109 Reitz, A.B., 36 (1999) 169 Repke, K.R.H., 30 (1993) 135 Richards, W.G., 1 1 (1975) 67 Richardson, P.T., 24 (1987) I Roberts, L.M., 24 (1987) 1 Roe, A.M.. 7 (1970) 124 Rose, H.M., 9 (1973) I Rosen, T., 27 (1990) 235 Rosenberg. S.H., 32 (1995) 37 Ross, K.C., 34 (1997) 111 Roth, B., 7 (1970) 285; 8 (1971) 61; 19 (1982) 269 Russell, A.D., 6 (1969) 135; 8 (1971) 39; 13 (1976) 271; 31 (1994) 349; 35 (1998) 133 Ruthven, C.R.J., 6 (1969) 200 Sadler, P.J., 12 (1975) 159 Sampson, G.A., 1 I (1975) 193 Sandler, M., 6 (1969) 200 Sarges, R., 18 (1981) 191 Sartorelli, A.C., 15 (1978) 321; 32 (1995) 1 Schiller, P.W., 28 (1991) 301 Schmidhammer, H., 35 (1998) 83 Schon, R., 30 (1993) 135 Schunack, W., 38 (2001) 279 Schwartz, J.-C., 38 (2001) 279 Schwartz, M.A., 29 (1992) 271 Scott, M.K., 36 (1999) 169 Sewell, R.D.E., 14 (1977) 249; 30 (1993) 327 Shank, R.P., 36 (1999) 169 Shaw, M.A., 26 (1989) 253 Sheard, P., 21 (1984) I Shepherd, D.M., 5 (1967) 199 Silver, P.J., 22 (1985) 293 Silvestrini, B., 21 (1984) 111 Singh, H., 16 (1979) 35; 17 (1980) 151; 22 (1985) 243; 28 (1991) 233 Skotnicki, J.S., 25 (1988) 85 Slater, J.D.H., 1 (1961) 187 Sliskovic, D.R., 39 (2002) 121
CUMULATIVE AUTHOR INDEX Smith, H.J., 26 (1989) 253; 30 (1993) 327 Smith, R.C., 12 (1975) 105 Smith, W.G., I (1961) I ; lO(1974) I I Solomons, K.R.H.. 33 (1996) 233 Sorenson, J.R.J., 15 (1978) 21 I ; 26 (1989) 437 Souness, J.E., 33 (1996) I Southan, C., 37 (2000) 1 Spencer, P.S.J., 4 (1965) 1; 14 (1977) 249 Spinks, A., 3 (1963) 261 StBhle, L., 25 (1988) 291 Stark, H., 38 (2001) 279 Steiner, K.E., 24 ( 1 987) 209 Stenlake, J.B., 3 (1963) 1; 16 (1979) 257 Stevens, M.F.G.. 13 (1976) 205 Stewart. G.A., 3 (1963) 187 Studer, R.O., 5 ( 1963) 1 Sullivan, M.E., 29 (1992) 65 Suschitzky, J.L., 21 (1984) 1 Swain, C.J., 35 (1998) 57 Swallow, D.L., 8 (1971) 119 Sykes, R.B., 12 (1975) 333 Talley, J.J., 36 (1999) 201 Taylor, E.C., 25 (1988) 85 Taylor, E.P., 1 (1961) 220 Taylor, S.G., 3 I ( 1 994) 409 Tegner, C., 3 (1963) 332 Terasawa, H., 34 (1997) 69 Thomas, G.J., 32 (1995) 239 Thomas, I.L., 10 (1974) 245 Thomas, J.D.R., 14 (1977) 5 1 Thompson, E.A.. I 1 (1975) 193 Thompson, M., 37 (2000) 177 Tilley, J.W., 18 (1981) 1 Timmerman, H., 38 (2001) 61 Traber, R., 25 ( I 988) I Tucker, H., 22 (1985) I21 Tyers, M.B., 29 (1992) 239 Upton, N.. 37 (2000) 177 Valler, M.J., 37 (2000) 83 Van den Broek, L.A.G.M., 23 (1986) 219 Van Dijk, J., 15 (1978) 261 Van Muijlwijk-Koezen, J.E., 38 (2001) 61 Van Wart, H.E., 29 (1992) 271 Vincent, J.E.. 17 (1980) 185
Volke. J., 12 (1975) 247 Von Itzstein, M., 36 (1999) 1 Von Seeman, C., 3 (1963) 89 Von Wartburg, A., 25 (1988) I Vyas, D.M., 32 (1995) 289 Waigh, R.D., 18 (1981) 45 Wajsbort, J., 21 (1984) 137 Walker, R.T., 23 (1986) 187 Walls, L.P., 3 (1963) 52 Walz, D.T., 19 (1982) 1 Ward, W.H.J., 38 (2001) 309 Waring. W.S., 3 (1963) 261 Watson, N.S., 33 (1996) 33 1 Watson, S.P.. 37 (2000) 83 Wedler, F.C., 30 (1993) 89 Weidmann, K., 3 I (1994) 233 Weiland, J., 30 (1993) 135 West, G.B., 4 (1965) 1 Whiting, R.L., 23 (1986) 1 Whittaker, M., 36 (1999) 91 Whittle, B.J.R., 21 (1984) 237 Wiedling, S., 3 (1963) 332 Wien, R., 1 (1961) 34 Wikstrom, H., 29 (1992) 185 Wikstrom, H.V., 38 (2001) 189 Wilkinson, S., 17 (1980) 1 Williams, D.R., 28 (1991) 175 Williams, J.C., 31 (1994) 59 Williams, K.W., 12 (1975) 105 Williams-Smith, D.L., 12 (1975) 191 Wilson, C., 31 (1994) 369 Wilson, H.K., 14 (1977) 285 Witte, E.C., I I (1975) 119 Wold, S., 25 (1989) 291 Wood, E.J., 26 (1989) 323 Wright, I.C., 13 (1976) 159 Wyard, S.J., 12 (1975) 191 Yadav, M.R., 28 (1991) 233 Yates, D.B., 32 (1995) 115 Youdim, M.B.H., 21 (1984) 137 Young, P.A., 3 (1963) 187 Young, R.N., 38 (2001) 249 Zee-Cheng, R.K.Y., 20 (1983) 83 Zon, G., 19 (1982) 205 Zylicz, Z., 23 (1986) 219
267
This Page Intentionally Left Blank
Cumulative Index of Subjects for Volumes 1-39 Tlw volume numhes, (veur qfpuhlicution) and puge number use given in that order.
ACAT inhibitors, 39 (2002) 121 Adamantane, amino derivatives, 18 (1981) 1 Adenosine A? receptor Iigands, 38 (2001) 61 Adenosine triphosphate, i 6 ( 1 979) 223 Adenylate cyclase, 12 (1975) 293 Adipose tissue. 17 (1980) 105 Adrenergic blockers, ci, 23 (1986) 1 p-, 22 (1985) I21 c+Adrenoceptors. antagonists, 23 ( 1986) 1 Adrenochrome derivatives, 9 (1973) 275 Adrianiycin. 15 (1978) 125; 21 (1984) 169 AIDS, drugs for, 31 (1994) 121 Aldehyde thioseinicarbazones as antitumour agents, 15 (1978) 321; 32 (1995) I Aldehydes as biocides, 34 (1997) 149 Aldose reductase inhibitors, 24 (1987) 299 Alzheimer's disease, chemotherapy of, 34 (1997) I ; 36 (1999) 201 Allergy, chemotherapy of, 21 (1984) 1; 22 ( I 985) 293 Amidines and guanidines, 30 (1993) 203 Aminoadaniantane derivatives, I8 (1981) I Aminopterins as antitumour agents, 25 (1988) 85
8-Aminoquinolines as antimalarial drugs, 28 (1991) 1 Analgesic drugs, 2 (1962) 43; 4 (1965) 171; 7 (1970) 229; 14 (1977) 249 Anaphylactic reactions, 2 (1962) 176 Angiotensin. 17 (1980) 41; 32 (1995) 37 Anthraquinones, antineoplastic, 20 (1983) 83 Antiallergic drugs, 21 (1984) I ; 22 (1985) 293; 27 (1990) 34 Antiapoptotic agents, 39 (2002) I Antiarrhythmic drugs, 29 (1992) 65 Anti-arthritic agents, 15 (1978) 211; 19 (1982) I ; 36 (1999) 201 Anti-atherosclerotic agents, 39 (2002) 121
Antibacterial agents, 6 ( 1969) 135; 12 ( I 975) 333; 19 (1982) 269; 27 (1990) 235; 30 (1993) 203; 31 (1994) 349; 34 (1997); 39 (2002) 73 resistance to, 32 (1995) 157; 35 (1998) 133 Antibiotics, antitumour, 19 (1982) 247; 23 (1986) 219 carbapenem, 33 (1996) 99 p-lactam, 12 (1975) 395; 14 (1977) 181; 31 ( I 994) 297; 33 (1996) 99 macrolide, 30 (1993) 57; 32 (1995) 157; 39 (2002) 73 mechanisms of resistance, 35 (1998) 133 polyene, 14 (1977) 105; 32 (1995) 157 resistance to, 31 (1994) 297; 32 (1995) 157; 35 (1998) 133 Anticancer agents - see Antibiotics, Antitumour agents Anticonvulsant drugs, 3 (1963) 261; 37 (2000) I77 Antidepressant drugs, 15 (1978) 261; 23 (1986) 121 Antiemetic drugs, 27 (1990) 297; 29 (1992) 239 Antiemetic action of 5-HT3 antagonists, 27 ( 1 990) 297; 29 (1992) 239 Antiepileptic drugs, 37 (2000) 177 Antifilarial benzimidazoles, 25 (1988) 233 Antifolates as anticancer agents, 25 (1988) 85; 26 (1989) I Antifungal agents, 1 (1961) 220 Antihyperlipidaeniic agents, 1 1 (1975) I19 Anti-inflammatory action of cyclooxygenase-2 (COX-2) inhibitors, 36 (1999) 201 of thalidomide, 22 ( 1 985) 165 of 5-lipoxygenase inhibitors, 29 (1992) 1 of p38 MAP kinase inhibitors, 38 (2001) 1
269
CUMULATIVE SUBJECT INDEX
270
Anti-inflammatory agents, 5 (1967) 59; 36 (1999) 201; 38 (2001) I ; 39 (2002) 1 Antimalarial 8-aminoquinolines, 28 (1991) I Antimicrobial agents for sterilization, 34 (1997) 149 Antineoplastic agents, a new approach, 25 (1988) 35 anthraquinones as, 20 (1983) 83 Antipsychotic drugs, 33 (1996) 185 Anti-rheumatic drugs, 17 (1980) 185; 19 (1982) I; 36 (1999) 201 Antisecretory agents, 37 (2000) 45 Antithrombotic agents, 36 (1 999) 29 Antitumour agents, 9 (1973) I ; 19 (1982) 247; 20 (1983) 83; 23 (1986) 219; 24 (1987) I ; 24 (1987) 129; 25 (1988) 35; 25 (1988) 85; 26 (1989) 253; 26 (1989) 299; 30 (1993) I ; 32 (1995) I ; 32 (1995) 289; 34 (1997) 69 Antitussive drugs, 3 (1963) 89 Anti-ulcer drugs, ofplant origin, 28 (1991) 201 ranitidine, 20 (1983) 67 synthetic, 30 (1993) 203 Antiviral agents, 8 (1971) 119; 23 (1986) 187; 36 (1999) I ; 39 (2002) 215 Anxiolytic agents, pyrido[ I ,2-a]benzimidazoles as, 36 (1999) 169 Anxiolytic agents, CCK-B antagonists as, 37 (2000) 45 Aromatase inhibition and breast cancer, 26 (1989) 253; 33 (1996) 147 Aspartic proteinase inhibitors, 32 (1995) 37; 32 ( 1995) 239 Asthma, drugs for, 2 I ( 1984) I ; 3 1 ( 1994) 369; 31 (1994)409;33(1996) 1;38(2001)249 ATPase inhibitors, gastric, H / K +, 3 1 ( I 994) 233 Azides, 31 (1994) 121
+
Bacteria, mechanisms of resistance to antibiotics and biocides, 35 (1998) 133 Bacterial and mammalian collagenases: their inhibition, 29 (1992) 271 1-Benzazepines, medicinal chemistry of, 27 (1990) 123 Benzimidazole carbamates, antifilarial, 25 (1 988) 233 Benzisothiazole derivatives, 18 (1981) 117 Benzodiazepines, 20 (1983) 157; 36 (1999) 169
Benzo[b]pyranol derivatives, 37 (2000) 177 Biocides, aldehydes, 34 (1 997) 149 mechanisms of resistance, 35 (1998) 133 British Pharmacopoeia Commission, 6 (1969) I Bronchodilator and antiallergic therapy, 22 (1985) 293 Calcium and histamine secretion from mast cells, 19 (1982) 59 Calcium channel blocking drugs, 24 (1987) 249 Camptothecin and its analogues, 34 (1997) 69 Cancer, aromatase inhibition and breast, 26 ( I 989) 253 azides and, 3 1 (1994) 12 1 camptothecin derivatives, 34 (1997) 69 endocrine treatment of prostate, 26 (1989) 299 retinoids in chemotherapy, 30 (1993) 1 Cannabinoid drugs, 24 (1987) 159; 35 (1998) 199 Carbapenem antibiotics, 33 (1996) 99 Carcinogenicity of polycyclic hydrocarbons, I0 (1974) 159 Cardiotonic steroids, 30 ( 1993) 135 Cardiovascular system, effect of azides, 3 1 (1994) 121 effect of endothelin, 3 1 (1 994) 369 4-quinolones as antihypertensives, 32 (1995) I I5 renin inhibitors as antihypertensive agents, 32 (1995) 37 Caspase inhibitors, 39 (2002) 1 Catecholamines, 6 (1969) 200 CCK-B antagonists, 37 (2000) 45 Cell membrane transfer, 14 (1977) 1 Central nervous system, drugs, transmitters and peptides, 23 (1986) 91 Centrally acting dopamine D2 receptor agonists, 29 (1992) 185 Chartreusin, 19 (1982) 247 Chelating agents, 20 (1983) 225 tripositive elements as, 28 (1991) 41 Chemotherapy of herpes virus, 23 (1985) 67 Chemotopography of digitalis recognition matrix, 30 ( I 993) 135 Chiral synthesis, 34 (1997) Cholesterol-lowering agents, 33 (1996) 33 I ; 39 (2002) 121
CUMULATIVE SUBJECT INDEX Cholinergic receptors, 16 (1976) 257 Chromatography, 12 (1975) I; 12 (1975) 105 Chromone carboxylic acids, 9 (1973) 65 Clinical enzymology, 13 (1976) I Collagenases, synthetic inhibitors, 29 (1992) 27 I Column Chromatography, 12 (1975) 105 Combinatorial chemistry, 36 (1999) 91 Computers in biomedical education. 26 ( 1 989) 323 Medlars information retrieval, 10 (1974) I Copper complexes. 15 (1978) 21 I ; 26 (1989) 43 7 Coronary circulation, 6 (1969) 1 1 Coumarins, metabolism and biological actions, 10(1974)85 Cyclic AMP, 12 (1975) 293 Cyclooxygenase-2 (COX-2) inhibitors, 36 ( 1999) 20 I Cyclophosphamide analogues, 19 ( 1982) 205 Cyclosporhs as immunosuppressants, 25 (1988) I ; 33 (1996) 53
27 I
in phamiacology and toxicology, 10 ( 1974) II Erythromycin and its derivatives, 30 (1993) 57; 3 I ( 1994) 265 Feverfew, medicinal chemistry of the herb, 29 (1992) 217 Fibrinogen antagonists, as antithrombotic agents, 36 ( 1 999) 29 Flavonoids, physiological and nutritional aspects, 14 (1977) 285 Fluoroquinolone antibacterial agents, 27 (1990) 235 mechanism of resistance to, 32 (1995) 157 Folic acid and analogues, 25 (1988) 85; 26 (1989) 1 Formaldehyde, biocidal action, 34 (1997) 149 Free energy, biological action and linear, I0 ( 1974) 205
Data analysis in biomedical research, 25 (1988) 29 1 Dianiiiiopyrimidines, 19 ( 1 982) 269 Digitalis recognition matrix, 30 (1993) 135 Diuretic drugs, 1 (1961) 132 DNA-binding drugs. 16 ( 1979) 15 I Dopainine D2 receptor agonists, 29 (1992) 185 Doxorubicin, 15 (1978) 125; 21 (1984) 169 Drug-receptor interactions, 4 (1965) 39 Drugs, transmitters and peptides. 23 (1986) 91
GABA, heterocyclic analogues, 22 ( 1 985) 67 GABAA receptor ligands, 36 (1999) 169 Gastric H /K + -ATPase inhibitors, 3 I ( 1994) 233 Gas-liquid chromatography and mass spectrometry. 12 (1975) 1 Genomics, impact on drug discovery, 37 (2000) 1 Glutaraldehyde, biological uses, 13 (1976) 271 as sterilizing agent, 34 (1997) 149 Gold, immunopharmacology of, 19 (1982) 1 Growth hormone secretagogues, 39 (2002) 173 Guanidines, 7 (1970) 124; 30 (1993) 203
Elastase, inhibition, 31 (1994) 59 Electron spin resonance, I2 ( 1975) 19 I Electrophysiological (Class 111) agents for arrhythmia. 29 (1992) 65 Enantiomers. synthesis of, 34 (1997) 203 Endorphins, 17 ( 1980) I Endothelin inhibition, 31 (1994) 369 Enkephalin-degrading enzymes, 30 ( 1 993) 327 Enkephalins, 17 (1980) 1 Enzymes, inhibitors of, 16 (1979) 223; 26 (1989) 253; 29 (1992) 271; 30 (1993) 327; 3 1 ( I 994) 5Y; 3 I ( 1994) 297; 32 (1995) 37; 32 ( 1995) 239; 36 ( 1999) I ; 36 (1999) 201; 38 (2001) I ; 39 (2002) 215 Enzymology, clinical use of, 10 (1976) I
Halogenoalkylarnines, 2 (1962) 132 Heparin and heparinoids, 5 (1967) 139 Hepatitis C virus NS3.4 protease, inhibitors of, 39 (2002) 215 Herpes virus, chemotherapy, 23 ( 1985) 67 Heterocyclic analogues of GABA, 22 (1985) 67 Heterocyclic carboxaldehyde thiosemicarbazones. 16 (1979) 35; 32 (1995) I Heterosteroids, 16 (1979) 35; 28 (1991) 233 High-throughput screening techniques, 37 (2000) 83 Histamine, H3 ligands, 38 (2001) 279 H2-antagonists, 20 (1983) 337 receptors, 24 (1987) 30; 38 (2001) 279
+
272
CUMULATIVE SUBJECT INDEX
release, 22 (1985)26 secretion, calcium and, 19 (1982)59 5-HT1,4 receptors, radioligands for in vivo studies, 38 (2001)189 Histidine decarboxylases, 5 (1 967) 199 HIV proteinase inhibitors, 32 (1995)239 Hydrocarbons, carcinogenicity of, 10 (1974)
159
Hypersensitivity reactions, 4 (1965)1 Hypocholesterolemic agents, 39 (2002)12I Hypoglycaemic drugs, 1 ( I961 ) 187;I8 ( I981)
191;24 (1987)209;30 (1993)203;31 (1994)1 Hypotensive agents, I (1961)34;30 (1993) 203;31 (1994)409;32 (1995)37;32 (1995)115
lnimunopharmacology of gold, 19 (1982)1 Immunosuppressant cyclosporins, 25 ( 1988) 1 India, medicinal research in, 22 (1 985) 243 Influenza virus sialidase, inhibitors of, 36 (1999)1 Information retrieval, 10 (1974)1 lnotropic steroids, design of, 30 (1993)135 Insulin, obesity and, 17 (1980)105 Ion-selective membraneelectrodes, 14( I 977)51 Ion transfer, 14 (1977)1 Irinotecan, anticancer agent, 34 ( I 997)68 Isothermal titration calorimetry, in drug design,
38 (2001)309
Isotopes, in drug metabolism, 9 (1973)133 stable, 15 (1978) I Kappa opioid non-peptide ligands, 29 (1992)
109;35 (1998)83 Lactam antibiotics, 12 (1975)395;14 (1977)
181
p-Lactamase inhibitors, 31 (1994)297 Leprosy, chemotherapy, 20 ( 1983) 1 Leukocyte elastase inhibition, 31 (1 994) 59 Leukotriene D4 antagonists, 38 (2001)249 Ligand-receptor binding, 23 (1986)4I Linear free energy, 10 (1974)205 5-Lipoxygenase inhibitors and their anti-inflammatory activities, 29 (1992)I Literature of medicinal chemistry, 6 (1969)266 Lithium, medicinal use of, 11 (1975)193
Local anaesthetics, 3 (1963)332 Lonidamine and related compounds, 21 (1984) 111 Macrolide antibiotics, 30 (1993)57;3 1 (1994)
265
Malaria, drugs for, 8 (1971)231;19 (1982) 269;28 (1991)1 Manganese, biological significance, 30 ( 1993)
89
Manufacture of enantiomers of drugs, 34
(1997)203
Mass spectrometry and glc, 12 (1975)1 Mast cells, calcium and histamine secretion, 19
(1982)59
cholinergic histamine release, 22 (1 985)
267
peptide regulation of, 27 (1990)143 Medicinal chemistry, literature of, 6 (1969)266 Medlars computer information retrieval, I0 (1974)1 Membrane receptors, 23 (1986)41 Membranes, 14 (1977)I ; 15 (1978)87;16 ( 1979)223
Mercury (11) chloride, biological effects, 27
(1990)189
Methotrexate analogues as anticancer drugs, 25 ( 1 988)85;26 (1 989) 1 Microcomputers in biomedical education, 26
(1989)323 Molecularly imprinted polymers, preparation and use of, 36 (1999)235 Molybdenum hydroxylases, 24 (1 987)85 Monoamine oxidase inhibitors, 21 (1984)137 Montelukast and related leukotriene D4 antagonists, 38 (2001)249 Multivariate data analysis and experimental design, 25 (1988)291 Neuraminidase inhibitors, 36 (1999)1 Neurokinin receptor antagonists, 35 (1998)57 Neuromuscular blockade, 2 (1962)88;3 (1963) I ; 16 (1979)257 Neurokinin receptor antagonists, 35 (1998)57 Neurosteroids, as psychotropic drugs, 37
(2000)135
Next decade [the 197O’s],drugs for, 7 (1970)
215
CUMULATIVE SUBJECT INDEX Nickel(1I) chloride and sulphate, biological effects, 27 (1990) 189 Nitriles, synthesis of, 10 (1974) 245 Nitrofurans, 5 (1967) 320 Nitroimidazolcs, cytotoxicity of, 18 (1981) 87 NMR spectroscopy, I2 ( I 975) I59 high-field, 26 (1989) 355 Non-steroidal anti-inflammatory drugs, 5 (1967) 59; 36 (1999) 201 Non-tricyclic antidepressants, 15 (1978) 39 C-Nucleosides. 13 (1976) 303; 22 (1985) 1 Nutrition, total parenteral, 28 (1991) 175 Obesity and insulin, 17 (1980) 105 Ondansetron and related 5-HT3 antagonists, 29 ( 1992) 239 Opioid peptides, I7 ( 1980) 1 receptor antagonists, 35 (1998) 83 receptor-specific analogues, 28 (1991) 301 Opioid receptor antagonists, 35 (1998) 83 Organophosphorus pesticides, pharmacology of.8(1971) 1 Oxopyranoazines and oxopyranoazoles, 9 (1973) 117
P2 Purinoreceptor ligands, 38 (2001) 115 p38 MAP Kinase inhibitors, 38 (2001) 1 Paclitaxel, anticancer agent, 32 (1995) 289 Parasitic infections, 13 (1976) 159; 30 (1993) 203 Parasympathomimetics, I 1 (1975) 1 Parenteral nutrition, 28 (1991) 175 Parkinsonism, pharniacotherapy of, 9 ( 1973) 191; 21 (1984) 137 Patenting of drugs, 2 (1962) 1; 16 ( 1979) I Peptides, antibiotics, 5 (1967) 1 enzymic, 3 I ( 1994) 59 hypoglycaemic, 3 1 ( 1994) I mast cell regulators, 27 ( I 990) 143 opioid, 17 (1980) 1 Pharmacology of Alzheimer’s disease, 34 (1997) I Pharmacology of Vitamin E, 25 ( I 988) 249 Phosphates and phosphonates as prodrugs, 34 (1997) 1 I I Phospholipids. 19 ( 1 982) 1 I 1 Photodecomposition of drugs. 27 (1990) 51 Platelet-aggregating factor, antagonists, 27 ( I 990) 325
273
Platelet aggregration, inhibitors of, 36 (1 999) 29 Platinum antitumour agents, 24 (1987) 129 Polarography, 12 (1975) 247 Polycyclic hydrocarbons, 10 (1974) 159 Polyene antibiotics, 14 (1977) 105 Polypeptide antibiotics, 5 (1967) 1 Polypeptides, 4 (1965) 86 from snake venom, 21 (1984) 63 Positron emission tomography (PET), 38 (2001) 189 Prodrugs based on phosphates and phosphonates, 34 (1997) 1 I 1 Prostacyclins, 21 (1984) 237 Prostaglandins, 8 (1971) 317; 15 (1978) 357 Proteinases. inhibitors of, 3 1 (1994) 59; 32 (1995) 37; 32 (1995) 239 Pseudomonas aeruginosa, resistance of, 12 (1975) 333; 32 (1995) 157 Psychotomimetics, 1 I (1975) 9 I Psychotropic drugs, 5 ( I 967) 25 1 ; 37 (2000) I35 Purines. 7 ( 1 970) 69 Pyridazines, pharmacological actions of, 27 (1990) I ; 29 (1992) 141 Pyrimidines, 6 (1969) 67; 7 (1970) 285; 8 (1971)61; 19 (1982)269 Quantuin chemistry, 1 1 ( 1 975) 67 Quinolines, 8-amino-, as antimalarial agents, 28 (1991) 1 4-Quinolones as antibacterial agents, 27 ( 1990) 235 as potential cardiovascular agents, 32 (1995) I I5 Radioligand-receptor binding, 23 (1986) 417 Ranitidine and H2-antagonists, 20 (1983) 337 Rauwolfia alkaloids, 3 (1963) 146 Recent drugs, 7 ( 1 970) 1 Receptors, adenosine, 38 (2001 ) 61 adrenergic, 22 ( 1 985) 12 1 ; 23 ( I 986) I cholecystokinin, 37 (2000) 45 fibrinogen, 36 (1999) 29 histamine, 24 (1987) 29; 38 (2001) 279 neurokinin, 35 (1998) 57 opioid, 35 ( 1 998) 83 purino, 38 (2001) 115
274
CUMULATIVE SUBJECT INDEX
Renin inhibitors, 32 (1995) 37 Ricin, 24 (1987) 1 RNA as a drug target, 39 (2002) 73 Single photon emission tomography (SPET), 38 (2001) 189 Screening tests, 1 (1961) 1 Serine protease inhibitors, 3 I (1 994) 59 Serotonin 5-HTtA radioligands, 38 (2001) 189 Snake venoms, neuroactive, 21 (1984) 63 Sodium cromoglycate analogues, 2 1 (1984) I Sparsomycin, 23 ( 1986) 2 I9 Spectroscopy in biology, 12 (1975) 159: 12 (1975) 191; 26 (1989) 355 Statistics in biological screening, 3 (1963) 187; 25 (1988) 291 Sterilization with aldehydes, 34 (1997) 149 Steroids, hetero-, 16 (1979) 35; 28 (1991) 233 design of inotropic, 30 (1993) 135 Synthesis of enantiomers of drugs, 34 (1997) 203
Tetrahydroisoquinolines, P-adrenomimetic activity, 18 (1981) 45 Tetrahydronaphthalenes, b-adrenomimetic activity, 18 ( I 98 I ) 45
Tetrazoles, 17 (1980) 151 Thalidomide as anti-inflammatory agent, 22 (1985) 165 Thiosemicarbazones, biological action, I5 (1978) 321; 32 (1995) 1 Thromboxanes, 15 (1978) 357 Tilorone and related compounds, 18 (198 1 ) 135 Toxic actions, mechanisms of, 4 (1965) 18 Tranquillizers, 1 (1961) 72 I ,2,3-Triazines, medicinal chemistry of, 13 (1976) 205 Tripositive elements, chelation of, 28 (1 99 I ) 41 Trypanosomiasis, 3 (1 963) 52
Venoms, neuroactive snake, 21 (1984) 63 Virus diseases of plants, 20 (1983) 119 Viruses, chemotherapy of, 8 (1971) 119; 23 (1986) 187; 32 (1995) 239; 36 (1999) I ; 39 (2002) 215 Vitamin D3 and its medical uses, 35 (1998) 1 Vitamin E, pharmacology of, 25 (1988) 249
