Progress in Medicinal Chemistry, Volume 37

Progress in Medicinal Chemistry 37

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Progress in Medicinal Chemistry 37

Editors:

F.D. KING, B.SC.,

D . P H I L . , C.CHEM., F . R . S . C .

SmithKline B r t d a n i Phurniuceuticuls Neu. Frontiers Science Purk (North) Tliird Aivnue Hcir.loi~~, Ewe.\- CM19 5 A W United Kingdom

Consultunt in Merlicinul Clieniistrj3 P. 0.Box 151 Rqvston SG8 5 Y Q U n i t d Kingdoin

2000

ELSEVIER AMSTERDAM . L A U S A N N E * N E W YORK*OXFORD~SHANNON~SINGAPORE~TOKYO

ELSEVIER SCIENCE B.V. Sara Burgerhartstraat 25 P.O. Box 21 I. 1000 AE Amsterdam,The Netherlands

02000 Elsevier Science B.V. All rights reserved This work is protected under copyright by Elsevier Science,and the following terms and conditions apply to its use: Photocopying Single photocopies of single chapters may be made for personal use as allowed by national copyright laws. Permission of the Publisher and payment of a fee is required for all other photocopying, including multiple or systematic copying, copying for advertising or promotional purposes, resale, and all forms of document delivery. Special rates are available for educational institutions that wish to make photocopies for non-profit educational classroom use. Permissions may he sought directly from Elsevier Science Rights & Permissions Department, PO Box 800.Oxford OX5 IDX, UK; phone: (+44) 1865 843830, fax: (+a) 1865 853333, e-mail: permissions(a,elsevier.co.uk. You may also contact Rights & Permissions directly through Elsevier’s home page (http:/ /www.elsevier.nl), selecting first ‘Customer Support’, then ‘General Information’. then ‘Permissions Query Form’. In the USA, users may clear permissions and make payments through the Copyright Clearance Center. Inc., 222 Rosewood Drive, Danvers. MA 01923, USA; phone: (978) 7508400. fax: (978) 7504744, and in the UK through the Copyright Licensing Agency Rapid Clearance Service (CLARCS), 90 Tottenham Court Road, London WIP OLP, UK; phone: (+44) 171 631 5555; fax: (+44) 171 631 5500. Other countries may have a local reprographic rights agency for payments. Derivative Works Tables of contents may be reproduced for internal circulation. but permission of Elsevier Science is required for external resale or distribution of such material. Permission of the Publisher is required for all other derivative works, including compilations and translations. Electronic Storage or Usage Permission ofthe Publisher is required to store or use electronicallyany material contained in this work, including any chapter or part of a chapter. Except as outlined above. no part of this work may be reproduced, stored in a retrieval system or transmitted in any form or by any means. electronic, mechanical, photocopying, recording or otherwise, without prior written permission of the Publisher. Address permissions requests to: Elsevier Science Rights & Permissions Department, at the mail. fax and e-mail addresses noted above. Notice No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular. independent verification of diagnoses and drug dosages should be made. First edition 2000 Library of Congress Cataloging in Publication Data Please refer to card number 62-2712 for this series.

ISBN: 0 444 50294 7 ISBN Series: 0 7204 7400 0

@ The paper used in this publication meets the requirements of ANSIlNISO 239.48-1 992 (Permanence of Paper). Printed in The Netherlands.

Preface This volume reviews recent advances in five important areas of medicinal chemistry which will be of interest both to chemists and to scientists of other disciplines engaged in medicines research and development. We have included accounts of successful drug discovery programmes, disease targets of unmet medical need, and recent progress in new technologies which are considered by many to hold the key to future developments in medicinal chemistry. The style and organisation of chapters follow a similar pattern to previous volumes but references, where appropriate, now include website addresses of the World Wide Web. We are most grateful to our contributors for their appraisal of the extensive literature of their topics and to the staff of our publishers for their continuing help and encouragement. July 1999

Dr. F. D. King Dr. A.W. Oxford

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vii

Contents Preface

V

The Impact of Genomics on Drug Discovery Lee J. Beeley, Ph.D., D. Malcolm Duckworth, Ph.D. and Christopher Southan, Ph.D. Smith Kline Beecham Phurmaceuticuls, New Frontiers Science Purk, Third Avenue, Harlow, Essex CM19 5AW, U K.

1

CCK-B Antagonists in the Control of Anxiety and Gastric Acid Secretion Mark S. Chambers, Ph.D. and Stephen R. Fletcher, Ph.D. The Neuroscience Reseurch Centre, Merck, Sharp und Dohme Research Lahorutories, Terlings Purk, Harlow, Essex, CM20 2QR, U K.

45

Application of High-throughput Screening Techniques to Drug Discovery Brian Cox, Jane C. Denyer, Alastair Binnie, Mary C . Donnelly, Brian Evans, Darren V. S. Green, Jane A. Lewis, Tom H. Mander, Andy T. Merritt, Martin J. Valler and Stephen P. Watson. Divisions of Discovery Teclznology, Medicinal Sciences und Biounalysis und Metubolism. Gluxo Wellcome Medicines Research Centre, Gunnels Wood Road, Stevenuge, Herts, S G l 2 N Y , UK.

83

Development of Neurosteroid-Based Novel Psychotropic Drugs Doodipala S. Reddy and Shrinivas K. Kulkarni Departrnent of Phurmucology, University Institute of’Phurmaceutical Sciences, Punjub University, Chundigurh 160 014, Indiu

135

Benzolblpyranols and Related Novel Antiepileptic Agents Neil Upton and Mervyn Thompson SmithKlinP Beccham Pharmuceuticuls, New Frontiers Science Purk, Harlow, Essex, CM19 5AW, U.K.

177

~

Subject Index

20 1

Author Index (Vols. 1-37)

205

Subject Index (Vols. 1-37)

21 1

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Progress in Medicinal Chemistry Vol. 37, Edited by F.D. King and A.W. Oxford 02000 Elsevier Science B.V. All rights reserved. ~

1 The Impact of Genomics on Drug Discovery LEE J. BEELEY* Ph.D., D. MALCOLM DUCKWORTH Ph.D. and CHRISTOPHER SOUTHAN Ph.D. SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow: Essex CM19 5 A K U.K.

INTRODUCTION

2

TARGETS Target Identification Dutuhase niiningjbr new targets Experimental approuche.s Target Validation Tiirget prqfiling - niRNA Espression Target profiling - Proteins Coniparurive Genomics - use qfmodel organisms Target Structure Sequences with low or no homology Structur~ilgenotnics Honiology or comparative models Structure hasedgenome comparisons

4

17 18 19

COMBINATORIAL CHEMISTRY

19

SCREENING

22

CASE HISTORIES G Protein-coupled Receptors Ligiind identijcution h,y dutuhase searching Ligunds identified by fishing Kinases Proteases Orphan proteases Ctrthepsrn K

23 23 24 24 26 28 28 29

(*now Hzer Central Research, Sandwich, Kent CT13 9NJ, U.K.) 1

4

4 7 7 8 11 13 16 17

2

THE IMPACT OF GENOMICS ON DRUG DISCOVERY

FUTURE PERSPECTIVES

32

GLOSSARY

33

WEB GLOSSARY

35

ACKNOWLEDGEMENTS

31

REFERENCES

37

INTRODUCTION The successful incorporation of genomics into the drug discovery process is critical to the long term survival of pharmaceutical companies. It has been heralded by many people as a technology that will revolutionise the industry. Ultimately this may happen, but in the interim, it is purely a rich source of additional information that, if used correctly, will fuel innovation. Since the complete genome of Haemophilus injluenzae was published in 1995 [l], the sequencing of a wide range of organisms, from bacteria to man, has continued apace. A preliminary version of 90% of the human genome may be available as early as Spring 2000 with the final, high-quality sequence completed by 2003 [2]. Since only 1OYo of the human genome is complete at the time of writing no one can predict how many human genes there will be, but the estimates are from 80,000 to 100,000. It is important to note that even when the sequencing is completed, a substantial fraction of the genes, possibly up to 50%, will have neither predictable biochemical nor physiological functions. From a drug discovery perspective, only a proportion will be amenable to pharmacological exploitation. Based on model organism data in eukaryotes, those that are exploitable may represent approximately 20% of the genome, or about 20,000 genes. Using existing technology, large pharmaceutical companies can only progress 50-100 new targets a year. It is clear therefore, that to identify which of these are genes relevant to disease, we have either to employ more reliable methods of target selection and be more innovative, or industrialisation of the drug discovery process has to take place. The discovery of a drug has hitherto always depended on a combination of creative thinking, good science, serendipity and, often, an individual prepared to champion its progression. Unfortunately, drug discovery has always had a high attrition rate. A key goal therefore, is to reduce this attrition rate by transforming the discovery of a drug into a high-throughput, rational

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process. Genomics is a key enabling technology in this transformation, but other technologies must also be in place to ensure success. To some, a drug discovery approach based on genomics uses gene targets discovered solely by the bioinformatic analysis (mining) of databases, but it goes beyond this. It encompasses targets discovered by characterizing mutant genes responsible for interesting phenotypes in animal models and other model organisms, and disease genes determined by association or linkage studies in man. Several terms are now becoming the hallmark of genomics-based research, in particular, bioinformatics, combinatorial chemistry, chemical diversity, high-throughput screening (HTS), reporter assays, DNA microarrays, proteomics and target validation. Some of these platform technologies are now in place on a scale required to improve the efficiency of the drug discovery process. Others are in the process of being developed. Schematics such as that in Figure 1.1 summarise some of the key components of the genomics-based drug discovery process. There are many genomics to drug discovery paradigms but they broadly fall into three categories (Figure 1.2). The paradigms can be differentiated by the means used to validate the target and the stage at which hit optimisation takes place. In paradigm 1, a database derived target is progressed through a range of validation steps, which enhance the confidence that a drug should work, thereby justifying the use of medicinal chemistry resource

High-Throughput Screen

I

+

n

CANDIDATES

Chemical Databanks

I

V

Figure 1.1. Cloning of targets genes followed by protein expression and conjiguration into HighThroughput Screens (HTS) i.s carried out in parallel with the generation of chemical databanks using combinutorial chemistry. In addition ongoing target validation methods are used to increase the confidence in the disease relevance of the target gene. Hits from the HTS are optimised and trunsformed into cundidate drugs.

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THE IMPACT OF GENOMICS ON DRUG DISCOVERY

Genomic Drug Discovery Paradigms 1 2 3 (Reverse Genetics) (Forward Genetics) (Industrialisation) Large Number of Database Genes

Bioinformaticsl Database Genes

Model Organism/ Pharmacogenatics Disease Genes

Target Validation

Bioinformatics

High-Throughput CloninglExpression

Chemical Databanks

Chemical Databanks

Massively Diverse Chemical Databanks

Hit Compounds HTS

+

I

HTS Hit Compounds

Ultra HTS Tool Compounds that can validate target gene

Figure 1.2. Three basic purudignis underlying the genonlics to drug discovery process. Paradigms I and 2 result in HTS hits that require chemical modification befi)re u tool compound is discovered, whereas the hitherto hypothetical industrialisation puradigm (3) would yield tools direct from the HTS.

to optimise the hit. In the second paradigm, gene targets are chosen on the basis that they are known to be involved in the disease process, in either animal models or man. In paradigm 3, the vast scale of the process and the massive diversity of the chemical libraries, ensures that without further modification, the lead obtained from HTS can be used to validate the target. TARGETS TARGET IDENTIFICATION

Database mining for new targets

Database mining is significantly less resource intensive than any of the wet cloning methods and can quickly lead to the discovery of many potential targets in protein families that have a proven track record for yielding drugs. These new targets will have little or no validation of their disease relevance. The key information is stored in nucleotide and protein databases. To date, of the expected 80,000 - 100,000 human gene products, around 8,000 cDNAs have been annotated in GenBank [3]. The protein sequences derived from these cDNAs can be accessed from two main databases: SWISSPROT/TrEMBL [4] and PIR [5]. The remaining genes are contained in nu-

L.J. BEELEY ETAL.

5

cleotide databases of expressed sequence tags (ESTs) or in high-throughput genomic (HTG) sequences. The majority of human cDNAs now have at least partial representation in EST databases. ESTs from a range of organisms are being generated in many laboratories as a means of identifying the coding regions of DNA. They are derived from cDNA libraries and yield the partial sequences of mRNA in a particular tissue or cell line. Overlapping ESTs form contigs (assemblies) that potentially code for the expressed proteins. A significant proportion of the ESTs is in proprietary nucleotide databases. These include the commercial human collections from Incyte ( > 2.5 million), Human Genome Sciences ( >2 million) and the public domain collection, dbEST (1.2 million), which can be accessed via the National Center for Biotechnology Information (NCBI) (http:/ /www.ncbi.nlm.nih.gov/). The HTG sequences, derived from genomic DNA, are the product of the worldwide Human Genome Project [6] (http://www.ncbi.nlm.nih.gov/ HUGO/). These are large sequences of chromosomal DNA (up to 200,000 nucleotide bases long) comprising coding and non-coding regions. When these sections of genomic DNA are completely finished they are transferred to the primate division of GenBank. Annotated protein sequences in genomic DNA are transferred to TrEMBL and eventually to SWISS-PROT. The EST and genome sequencing strategies complement one another and, although EST sequencing is reaching a plateau, the Human Genome Project will provide very valuable additional information. A vast amount of data will also be available from the genomic sequences of many other species e.g. mouse, fruit fly, nematode and various bacteria (Table I . 1). There are a further 75 genomes of eukaryotic or bacterial origin currently being sequenced (See KEGG: Kyoto Encyclopaedia of Gene and Genomes, http:/ /www.genome.ad.jp/kegg/java/org-proj.htm1; TIGR: The Institute of Genomic Research, http: / /www.tigr.org/tigrhome/tdb/mdb/mdb.html; NCBI Entrez Genomes, http: / /www.ncbi.nlm.nih.gov/Entrez/Genome/ org.html). Irrespective of the source of a new gene, a putative coding region can be used to interrogate a protein database using statistically based search algorithms, such as the BLAST [28] or the FASTA programs [29]. Statistically significant similarity of the protein sequence to a protein of known function, or the presence of particular amino acid motif, may allow it to be classified with respect to potential biochemical function. The successful use of ESTand genomic sequence databases is exemplified by the recent discovery of two genes, one for a G protein-coupled receptor (GPCR), GABAbR2, and the other, an ion channel, 5HT3b. Following the discovery of the GABAbRl receptor in 1997 by expression

6

THE IMPACT OF GENOMICS ON DRUG DISCOVERY Table 1. I . STATUS OF GENOMES O F SEQUENCED

Category

Proteobacteria

Organisms or Species

Escherichia coli K- 12 Haeniopliilus injirenzae Helicohacter pylori 26695 Helicohacterpylori J99 Rickettsia pro wazekii Bacillus suhtilis Gm+ve bacteria Mycoplasma genitalium My coplasma pneumoniae Mycohactericini tuherculosis H37Rv Chlamydia Chlamydia truchomatis Spirochete Borrelia hurgdorferi Treponema pallidum Oxygen-reducing bacteria Aqiiifex aeolicus Cyanobacteria Synecliocystis sp. PCC6803 Archaea Methanococcus jannaschii Metlzanohacterium thermoautotrophicuni delta H Archaeoglohusfulgidus Pyrococcus horikosliii OT3 Parasite Plasmodium falciparum Chr 2 Yeast Succharomyees cerevisiae Scliizosaccharomy ces pomhe Higher plants Arahidopsis thaliana Rice Oryza sntiva Nematode Caenorhahditis elegans Fruit fly Drosophila melanogaster Fish Fugu ruhripes Mouse Mus musculus Human Homo sapiens

Completed Size 0RF.s (Mb)

Reference

1997 1995 1997 1999 1998 I997 I995 1996 1998

4.64 1.83 1.67 1.64 1.1 I 4.21 0.58 0.82 4.41

4,289 1,717 1,566 1,495 834 4,100 467 677 3,918

1998 1997 1998 1998 I996

1.04 0.91 1.14 1.55 3.57

894 1,256 1,031 1,522 3,166

[I51 [16] [I71 [I81 [19,20]

1996

1.66

1,770

[21]

1997

1.75

1,869

[22]

1997 1998 1998

2.18 1.74 1.oo

2,407 1,979

[23] [24, 251

1997

12.07 6,286

[26]

14

1998 1999

70 450 97 165

2004 2003

400 3,000 3,000

-30,000 19000 [27]

cloning [30], several groups working in parallel have found a second subtype [31-331. One of the groups [32] identified ESTs that covered the coding region of the gene from the N-terminus to the C-terminus of protein, which would have greatly facilitated the cloning of the gene. Whereas EST databases have been the main source of novel genes so far,

L.J. BEELEY ETAL.

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the recent increase in HTG sequence has led to a valuable new source of genes that have low levels of expression. By mining high-throughput genomic sequences, a long sought subtype of the 5HT3 ion channel has been identified [34]. This ion channel is one component of the multimeric target for several drugs that are used for treatment of chemotherapy-induced emesis. These drugs were found using cellular and tissue assays that contain the endogenous proteins in the form of heteromers. Hitherto, only one of the components had been cloned, but the cloning of this second component has already led to a greater understanding of the different pharmacology observed in a variety of tissues. Experimental approaches Conventional molecular biology has yielded many new targets through homology and expression cloning as well as the more resource intensive positional cloning. The strategies behind these different cloning methods are as follows: a) Homology cloning. A strategy for isolating a gene whose gene product is of a known family and for which probes can be designed on the basis of a close homologue, e.g. Beta-3 adrenoceptor [35]. b) Expression cloning. A strategy for isolating a gene whose gene product is completely unknown, which utilises a known molecule or protein that binds with good affinity to the target gene product, e.g. GABAb receptor [301. c) Positional cloning. A strategy for isolating a gene whose gene product is completely unknown, which starts from a knowledge of its chromosomal location, e.g. Leptin [36]. These cloning strategies are listed in order of complexity and this is reflected in the resources required to achieve the goal. Positional cloning is the most resource intensive, but often provides groundbreaking results that can spawn other areas of interesting research. Unfortunately, positional cloning is a ‘molecular blind date’ with respect to outcome. All too frequently, positional cloning reveals a disease relevant gene that is of unknown biochemical function, or a gene whose protein product is in a class that has hitherto proved difficult to exploit pharmacologically. TARGET VALIDATION

What do we mean by target validation? Validation usually means that the gene has been shown to be relevant to the target disease, thereby giving confidence that modulation of the activity of the gene product will ameliorate

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THE IMPACT OF GENOMICS ON DRUG DISCOVERY

the disease state. A validated target could be described as one in which there is reasonable confidence that it is relevant to disease and therefore justifies significant medicinal chemistry effort. Unfortunately, the activities that give the best validation are also those that are most resource intensive. A key part of the target validation process is the application of functional genomic technologies, that is, a range of high-throughput methodologies aimed at elucidating the biochemical function and physiological roles of gene products. These methodologies are in three main areas: mRNA profiling, protein profiling and comparative genomics. All three approaches are powerfully complementary. The development of these technologies to the genome-wide scale is so recent that there are few published examples of drug targets identified exclusively from these approaches. However, data from a range of public and proprietary experimental systems have already unequivocally established their value for drug discovery. Cancer, for example, has become a testing ground for functional genomic approaches. Target projiling - mRNA Expression

As described in the foregoing sections, bioinformatic analyses will be able to infer the biochemical roles for many gene products but a substantial fraction will have little or no data attached to them. The answers to the key biological and drug discovery orientated questions for these genes can only be supplied by the results of high-throughput, mRNA profiling experiments. These questions include: (a) which of the possible splice variants of a given gene product are being expressed? (b) in which cells and tissues are they expressed? (c) for which groups of genes can similar patterns of co- or anti-regulation be observed? and (d) how do these expression pattern change with other variables? (e.g. mouse versus man, or health versus disease). Although one of the earliest approaches, the EST collections are a valuable form of mRNA profiling. Despite many technical caveats, these collections broadly represent a surrogate mRNA profile summed across the tissues used to make the cDNA collections. In NCBI dbEST, these span a range where many genes are represented only by single ESTs whereas the most abundant are represented by up to several thousand (e.g. alpha globin with 3,418 ESTs). One important application of the ESTapproach has been organised by the Cancer Genome Anatomy Project (CGAP) (http: / / www.ncbi.nlm.nih.gov/CGAP/). The goal of this work is to discover known and new human genes that may be causatively or diagnostically associated with cancer. Towards this end, the consortium has now sequenced over 400,000 ESTs from normal and cancerous tissues. These can be interrogated by electronic subtraction to highlight genes that may be specifically up or

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down regulated in these cancer tissues [37]. The large-scale EST aspect is part of a comprehensive transcript approach that includes microarray methods to compare the transcription profile of normal versus tumour tissue. DNA microarrays consist of microscopic immobilised dots of cDNA clones or synthetic oligonucleotides representing different genes. When these arrays are probed with fluorescently labelled DNA, reverse transcribed from an RNA sample, the individual dot will light up if the gene transcript matches (i.e. will hybridise with) one of the immobilised clones. This technology has recently been reviewed in depth [38]. Current operational limits fall within the ranges of 10,000-50,000 genes. immobilised as cDNAs at 1,000/cm2,0.5-5 pg of mRNA for sample preparation and a 2 to 3 order of magnitude detection range. One particularly elegant new method of sample preparation is laser capture microdissection (LCM) carried out under direct microscopic visualisation. This facilitates mRNA preparations from selected human cell populations from complex tissue sections [39]. Different research groups have clearly demonstrated the utility of microarrays for looking at gene transcript regulation in systems of direct relevance to human disease and drug discovery. One group utilised mRNA from cultured macrophages, chondrocyte cell lines, primary chondrocytes, and synoviocytes [40]. The work examined the expression profiles for selected cytokines, chemokines, DNA binding proteins, and matrix-degrading metalloproteinases selected for their probable pathogenic relevance. It also included a set of 1,000 unknown genes from blood cells. Comparisons between rheumatoid arthritis and inflammatory bowel disease tissues verified the involvement of some of the pre-selected set and revealed novel participation of the cytokine interleukin 3, the chemokine Gro-a and the metalloproteinase matrix metallo-elastase in both diseases. From the peripheral blood library, tissue inhibitor of metalloproteinase 1, ferritin light chain, and manganese superoxide dismutase were identified as genes expressed differentially in rheumatoid arthritis compared with inflammatory bowel disease. Using the same technology a different group has investigated the effect of serum addition on fibroblast proliferation [41]. A total of 8,631 genes were arrayed for this experiment, of which approximately half were ESTs from as yet uncharacterised gene products. Analysis of the results by a clustering and display method revealed unexpectedly diverse patterns of co-ordinated regulation of groups of genes. The inclusion within these groups of numerous genes with known roles in wound healing is a reassuring validation of this methodology. An example of interpretable co-regulation was provided by furin, a prohormone-processing protease required for one of the processing steps in the generation of active endothelin. This was induced in parallel

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THE IMPACT OF GENOMICS ON DRUG DISCOVERY

with induction of the gene encoding the precursor of endothelin-1 . Conversely, expression of CALLA/CD 10, a membrane metalloprotease that degrades endothelin-1, was reduced, along with other peptide mediators of acute inflammation, providing an example of anti-regulation. Of particular relevance to drug discovery was the finding of over 200 previously unknown genes whose expression was regulated in specific temporal pattergs during the response of fibroblasts to serum, and by implication, could include potential drug targets for wound healing. The application of mRNA profiling approaches to drug discovery presents several problems. However sophisticated the measurement of mRNA, changes may have no causative relationship to the phenomena being studied, be they physiological changes, pathological processes or drug effects. A large proportion of the genome will contain genes whose primary function is developmental but these may still be transcriptionally active and may have a secondary but important function in some adult tissues. Another subset of the genome will contain pseudogenes that give rise to aberrant, partial and/or untranslatable transcripts. Perhaps the biggest question mark over mRNA profiling is that whole areas of biochemical activity (signalling cascades, phosphorylation events, protein cleavage, proteidprotein interactions, protein ligand/substrate/inhibitor interactions) take place directly at the protein level and will be undetectable at the mRNA level. A further difficulty is that signal quantitation on microarrays is still more relative than absolute, making comparisons between experiments difficult. The problem of data overload has already emerged. Even when the absolute quantitation of all genes, all transcript variants, all common alleles, from all tissues, and across all time points, is technically achieved, it is still not clear which data analysis schemes will best reveal a robust set of drug intervention points. In this connection, it has been suggested that public microarray data sets will need a fundamentally new kind of scientific publishing. Result sets too large for conventional manuscript formats will have to be presented as web-based data [42]. Regardless of the caveats, microarray based mRNA profiling is continuously developing and is likely to extend to all genes by the time the human genome draft sequence is available. There is no doubt that genome-wide expression analysis will become an essential part of the both drug target discovery process and the mechanistic evaluation of candidate compounds [43]. Although some pharmaceutical companies are establishing microarray projects from scratch in-house, there is already a big choice of external suppliers for all of the components necessary to apply this technology to drug discovery programmes [44]. These range from the front end supply of mRNA from human pathological samples from tissue banks, through to a

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choice of representative gene clone sets, and extends to image and data analysis systems that can be interfaced with an in-house LIMS (laboratory information management system). Target profiling Proteins ~

The term proteomics is used to describe technologies for the high-throughput profiling of the individual protein components of cells or tissues analogous to that described for mRNA. The central technique of two dimensional (2D) gel electrophoresis is well established [45]. Polypeptides separated by charge in the first dimension and size in the second dimension are visualised on a gel, usually by direct staining or fluorescent dyes. Individual gels are currently capable of resolving over 2,000 protein spots. Some immediate differences are apparent by comparison with microarray methodology. The disadvantages are (a) protein spots have to be identified by a post-separation analysis. In contrast, microarrays will usually be spotted with pre-identified gene products; and (b) reproducability and pattern recognition become key problems for comparing 2D gels between different experimental systems. The advantages are (a) the 2D gels can be used not only for cells and tissues, but also for body fluids (even including ear exudates and tears), subcellular fractions and fully differentiated cells where no RNA is present (e.g. platelets); (b) protein spot characterisation can identify post-translational modifications; and (c) the spot pattern provides crude molecular weight and protein iso-electric point data for the individual polypeptides. The identification of low abundance spots on 2D gels has been greatly facilitated by recent advances in analytical mass spectrometry [45]. It is now possible to identify proteins present in femtomole quantities on gels and these procedures are being automated by both commercial and academic laboratories. The likelihood of connecting mass spectrometry data to human cDNA, already much increased with the expansion of EST collections, should become a certainty with completion of the genome [46]. However, 2D gels still have a long way to go to approach the detection range of microarrays, and the current detection limit, achievable with proteins incorporating radioactive methionine, is still orders of magnitude lower than the identification limit. Other major problems with 2D gels are the poor resolution of large, basic, hydrophobic, or small proteins ( < 3KD). To overcome some of these inherent difficulties, it is likely that 2D gels will increasingly be substituted and/or complimented by multi-dimensional chromatographic approaches to protein profiling [47]. These are scaleable over many orders of magnitude, and when miniaturised into microfluidic

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THE IMPACT OF GENOMICS ON DRUG DISCOVERY

chips directly connected on-line for MS analysis, have higher direct detection sensitivity for proteins. Many advantages accrue from fluidic handling of native protein samples as against samples that end up denatured and immobilised in the polyacrylamide matrix of 2D gels. In addition to the full range of analytical options, such as Edman degradation, mass spectrometric analysis, and amino acid analysis, eluted proteins can be captured on affinity matrices. A straightforward scale-up of multidimensional chromatography could even provide enough of an enzyme drug target for preliminary substrate testing. Extending this idea, the intriguing possibility arises that it could be feasible to move immediately to screening, thereby bypassing the in vitro expression bottleneck. The maturity of proteome technology is evidenced by the proliferation of on-line 2D gel maps from many biological sources. For example, the most recent release of the SWISS-2DPAGE database of proteins identified on 2-D gels (release 9.0) contains 544 entries in 22 reference gels. These include human maps for liver, plasma, HepG2 cells, red blood cells, lymphoma, HepG2 secreted proteins, cerebrospinal fluid, a macrophage-like cell line, erythroleukemia cells, platelets, kidney, promyelocytic leukemia cells, colorectal epithelia cells, and colorectal adenocarcinoma cells (http: / /www.expasy.ch/ch2d/). This web site also links to other centres with human tissue or cell line 2D maps, which provide useful baseline studies for a range of human pathologies. From a drug discovery perspective, as already described for microarrays, the study of cancer provides a good opportunity for identifying disease specific proteins by comparing normal and diseased tissue from the same patient. For example, proteomic studies have been reported on neuroblastomas, human breast proteins from normal and tumour sources, lung tumours, colon tumours and bladder tumours. With regard to the latter, keratinocyte cells have been a very useful surrogate for the study of fresh bladder squamous cell carcinomas (SCC) cells as they closely resemble keratinocytes both in morphology and protein expression profiles (http: / /biobase.dk/cgi-bin/celis) [48]. By collating information on differentiation markers, tissue distribution and secreted or peripheral proteins into a master database, it has been possible to identify protein markers that define the degree of differentiation of these lesions. In addition, the calcium binding and chemoattractant protein, psoriasin, which is expressed specifically by some differentiated tumour cells, can be detected as a disease specific marker in the urine of SCC patients. Further studies are in progress to find markers for pre-malignant lesions by applying proteomic technology, in combination with immunohistochemistry,to the analysis of tumours and

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urothelial tissue biopsies obtained from patients that have undergone removal of the bladder due to invasive disease. Proteomics has also found a niche in examining mechanisms of drug action [49]. The authors, in this case from a commercial organisation, Large Scale Biology Corporation (http:/ /www.lsbc.com), have developed a database describing effects of many drug compounds in rodent liver. This database can detect, classify, and characterise a broad range of liver toxicity mechanisms. The observed drug effects range from broad, for example sex steroids that alter levels of 45'!4 of all liver proteins, to specific, such as hepatic hydroxymethyl glutaryl coenzyme A reductase inhibitors, which affect only six spots. Additional studies from the same group using the proteomic approach are already revealing general patterns. These include: (a) most compounds tested cause observable changes in liver protein abundance measurements; (b) the protein changes are often consistent across compound structural classes; and (c) where compound-specific protein changes have been identified they can often be related to known molecular mechanisms. This ability of proteomics to be both a mechanistic sensor and classifier of drug effects at the protein level is obviously very applicable to toxicology. However, the methodology clearly can be applied to evaluate compounds directed towards new drug targets, even at the early stages where tool compounds may become available directly from HTS. Directly compared data from the mRNA profiling and protein profiling approaches in the same experimental model will give complimentary information on the biochemical relevance of the changes observed. To date only one publication has addressed this by comparing 2D spot and EST frequencies in liver [50].As might have been expected from a theoretical standpoint, the results highlight a significant difference in the mRNA and protein abundance results. Secreted proteins were more abundant (as mRNAs) than cellular proteins, suggesting that mRNAs for secreted proteins may have shorter half-lives than mRNAs for cellular enzymes. This implies the latter are more frequently regulated at the translational level, and hence, mRNA profiling in liver may quantitatively highlight secreted proteins over cellular molecules. This represents an advantage of the mRNA approach relative to protein detection in the search for novel cytokines and other secreted proteins, but a disadvantage in the characterisation of cellular metabolic and control processes. Comparative Genomics - use of model organisms

Cross species comparison of genes is one of the most powerful means of

14

THE IMPACT OF GENOMICS ON DRUG DISCOVERY

validating a target. Experiments can be done in yeast (Saccharomyces cerevisiae), nematode (Caenorhabditis elegans), fruit fly (Drosophila melanogaster) and mouse (Mus musculus) that are not possible in humans, which may determine the biochemical and sometimes the biological role of a specific gene. Although the physiological role of a particular gene may vary between species, many metabolic and signalling pathways are conserved. Many prokaryotic organisms have been completely sequenced, but only two eukaryotic organisms, yeast and nematode, are complete. A third, the fruit fly, will be finished this year. Complete genome sequences, in conjunction with phenotypic data from large-scale mutagenesis experiments, are a powerful combination for functional genomics. Yeast has many genes that are homologues of human disease genes. However, with the exception of enzymes, the organism is either not representative of, or is devoid of, some families of proteins that have proved very successful drug targets, such as GPCRs and nuclear hormone receptors. The nematode and the fruit fly, on the other hand, have a similar repertoire of these targets to man. On this basis, although yeast has proved to be a test-bed for comparative eukaryotic genomics, the nematode and fruit fly, albeit more resource intensive, hold greater promise. The power of functional genomics increases even further with the mouse. Comparison of mouse with human gene sequences offers the potential for detailed analysis, for example, of regulatory elements. There are several good examples of the use of data from model organisms as an effective means of probing the function of novel human genes. Work with nematode in order to identify key components involved in apoptosis (programmed cell death) resulted in the identification of several genes that may have important implications in Alzheimer’s disease and cancer. The finding of mammalian homologues for three of the nematode cell death genes, ced-3, ced-4 and ced-9 is a case in point [51, 521. Over 120 human cDNAs showing significant homology to genes that cause fruit fly mutant phenotypes have been identified (so-called ‘DRESDrosophila related expressed sequences, http: / /www.tigem.it/LOCAL/ drosophila/dros.html). These have been mapped in mouse and man, and expression scanned by in situ hybridisation [53, 541. Fruit fly genes, such as those involved in circadian rhythm [55], have proved useful in identifying mammalian homologues that are potentially new molecular targets for the treatment of sleep disorders in man. In the reverse situation, many human positionally cloned disease genes have homologues in nematode and yeast [56]. This may allow the use of the multicellular nematode to investigate gene function. It is important when interrogating databases for species homologues, to

L.J.BEELEY ETAL.

15

distinguish between orthologues and paralogues. An orthologue is the equivalent gene in another organism, whereas a paralogue is a close family member in the same species. In order to carry out meaningful comparative functional genomics, it is necessary to study the phenotype caused by the disruption of the equivalent gene. This is not always easy to determine without a complete description of all the genes in both organisms under comparison. There are many examples where a species orthologue has been described only later to be superseded by another gene that is a better candidate for the role. In addition to sequence data, supporting evidence for true orthology can be obtained in mammalian species from the relative chromosomal location and tissue distribution. The process of genome cross referencing has been given a further boost by the establishment of a public database (http: / /www.ncbi.nlm.nih.gov/ XREFdb/) which systematically identifies novel ESTs that are related to genes in model organisms. The ESTs are located onto human and mouse genomic maps, which allows the cross referencing of model organism genes with mapped human and mouse phenotypes [57]. This should aid the identification of human disease genes via the ‘positioned candidate’ approach, a half-way house between positional cloning and EST analysis. Working with model organisms provides valuable information on biochemical function and pathways. For clues about physiological function, the mouse represents the best mammalian model organism for human genome interpretation, with essentially the same genome size and genetic constitution, and similar physiology. There are two ways that the mouse has provided important information on the function of several important human genes. The first is through positional cloning of mutated genes responsible for mouse models of human disease, for example, the obese mouse. In the second, phenotypic analysis following a targeted mutation can strongly support the association of a particular gene with a disease process. A recent example of such a mutation is the protein tyrosine phosphatase 1b (PTP1b), thought to be involved in insulin signalling [%I. The recent work appears to support this hypothesis [59]. Attempts are being made to use the potent chemical mutagen, ethyl nitroso urea (ENU), for saturation mutagenesis in the mouse, combined with phenotypic or genotypic screens. An example of one such mutagenesis programme is underway at the MRC Mammalian Genetics Unit at Harwell, in collaboration with SmithKline Beecham Pharmaceuticals, Imperial College, London and Queen Mary and Westfield College, London (http:// www.mgu.har.mrc.ac.uk/mutabase/).Phenotypic analysis will result in mouse models of human disease, but identifying the primary mutation will essentially involve positional candidate cloning. In order for saturation mu-

16

THE IMPACT OF GENOMICS ON DRUG DISCOVERY

tagenesis in the mouse to be useful for disease-related gene identification, on a large scale, a technology breakthrough in genotypic analysis and positional cloning is needed. Approaches amenable to high-throughput are based on the various gene trapping strategies that have been developed to disrupt genes in embryonic stem (ES) cells [60-661. ES cells can be grown for many weeks, but still retain the capacity to differentiate, for example into nerve cells and blood vessels. When injected into mouse blastocysts they are able to participate in the formation of all tissues. Recently, sequence based screens have been developed to identify genes disrupted as a result of provirus integration in ES cells [67, 681. The process, called ‘tagged sequence mutagenesis’, entails sequencing a short stretch of the DNA from each disrupted gene and identifying the sequence by database searches, which can be automated. Large numbers of ES cells are analysed and frozen down to produce a library of mutated cells, which may then be used to generate mutant mice. Many genes, however, are temporally as well as spatially regulated. Analysing gene function using ES cell technology can therefore be confounded by an early embryonic lethal phenotype, precluding gene function analysis in the adult. From a pharmaceutical perspective, this is disappointing, as a gene that might potentially be useful as a drug target often has a different but vital function during embryonic development. For a more relevant assessment of the function of a gene, inducible gene modifications are required, but this technology is still in its nascent stage. TARGET STRUCTURE

The availability of a 3D structure of a protein, or protein domain, is of immense benefit to the medicinal chemist. Better still for drug design is a 3D structure with a small molecule bound. However, medicinal chemists involved in drug design programmes targeting multi-transmembrane spanning proteins, for example, GPCRs, ion channels or transporter proteins, have to accept that the chances of obtaining an experimentally derived 3D structure on the target are still remote. Can medicinal chemists expect to have more 3D structures at their disposal because of genomics? The answer is definitely yes. There are more protein primary sequences available and there have been key developments in structure determination methodology. For membrane proteins, however, progress is still very slow. A key issue is how to deal with the large number of sequences that have no known sequence homologues and therefore cannot be classified.

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Sequences with low or no homology

As more gene sequences become available it will be clear that large new families of proteins will emerge that cannot be classified on the basis of sequence homology or structural predictions. Dealing with these unknowns is important. For any unknown gene thought to be of particular interest due to chromosomal or tissue localisation, or association with a particular disease, a clue to function may be gained from secondary or tertiary structure predictions of the deduced protein sequence. Many proteins with no significant sequence similarity share both a common structural motif, or fold, and common function. Detection of such similarities in advance of structure determination can be addressed by fold recognition or ‘threading’ [69, 701. Here the sequences for proteins of unknown structure are compared with a database of known structures. The compatibility of the sequence with each structure is assessed with the expectation that the best fit will resemble the correct structure. Cytokines with a four helix bundle structure, for example, frequently have very low homology [7 I], but they interact with a family of receptors that signal through homologous proteins [72]. Structural genomics

One of the long-term goals of structural genomics would be to solve the structures of all the 80,000-1 00,000 expressed human proteins. This has prompted initiatives for high-throughput structure determination, which are known as structural genomics projects [73, 741. The initiatives will capitalise on the recent advances in protein crystallography, NMR spectroscopy and data processing [75,76]. As of April 1999, the number of all the entries in the protein data bank (PDB) (http: / /www.rcsb.org/pdb/index.html) with released atomic coordinates was 9,63 1. This number reduces approximately 3-fold and 10-fold when redundancy and homology, respectively, are taken into account. How many more structures must be solved to have sufficient data to predict with some degree of accuracy all of the protein structures within the genome? Firstly, we can consider the number of folds that are currently represented by known proteins. A fold is defined as a recurring spatial arrangement and connectivity of secondary structures forming the protein core. In 1992, the estimate for the number of different folds was around 1,000 [77]. As defined by the Structural Classification of Proteins (SCOP) [78, 791, 506 fold variations are known (SCOP v. 1.38). The PDB therefore can be considered to represent approximately half of all the expected protein folds. Secondly, the number of structures needed for experimental structure

18

THE IMPACT OF GENOMICS ON DRUG DISCOVERY

determination can be reduced using sequence-based analyses to assemble proteinslprotein domains into classes, for example PFAM [80], such that a 3D structure of one member of each class should be enough to build homology models for the others. Thirdly, the current rate of determination of new folds is reducing even though new structures are emerging. This infers that a greater number of structures will be needed to fill the remaining fold space. From these considerations, it is estimated that 10,000 newly solved structures will be needed to predict all the protein structures within the genome PlI. Homology or comparative models

Where a related structure is known, homology or comparative models of the protein may be generated. Although homology modelling is largely the domain of the computational scientist, advances have been made that have brought automation to the process of model assembly. SWISS-MODEL (http:/ /www.expasy.ch/swissmod/SWISS-MODEL.html) and the SwissPdb viewer have been designed as internet-based tools for comparative modelling [82, 831. Typical of the large scale increases in all aspects of genomics, SWISS-MODEL has been put to the test in a large scale homology modelling project, 3Dcrunch, analysing the 200,000 public protein sequences in the SWISS-PROT and TREMBL databases. This project was a collaboration between Silicon Graphics Inc. and international bioinformatics research organisations. The resulting 3D structure co-ordinates for 64,000 protein sequence entries are stored in individual files and have been made accessible via the World Wide Web (http: I 1www.expasy.chlswissmodl SWISS-MODEL.htm1). The hope is that the predictions will eventually be compared with experimentally determined structures. With regard to membrane bound proteins, the opportunities for homology modelling are limited. For seven transmembrane spanning proteins such as GPCRs, the only co-ordinates available are for bacteriorhodopsin [84], which is not G protein-coupled and has no sequence homology with any of the GPCR superfamily of proteins. However, using the information on bacteriorhodopsin in conjunction with sequence alignments, experimental and preliminary structural data on a true GPCR, rhodopsin, it is possible to build homology models of other GPCRs [85, 861. Given the recent successes yith the determination of the first two structures of ion channels (at 3 to 4 A resolution), that of the KcsA potassium channel from the bacteria Streptomyces lividam [87] and a gated mechanosensitive ion channel from Mycobacterium tuberculosis [88], it seems likely that homology models of other ion channel subunits may soon be forthcoming.

L.J. BEELEY ETAL.

19

Structure bused genome conzpcirisons

The increase in the number of protein sequences emerging from the wide range of prokaryotic and eukaryotic genome sequencing projects has led to structural analyses of observed protein folds in species representing the three superkingdoms of life: archaea, bacteria and eukaryotes [89, 901. These studies have revealed that the distribution of common folds is very similar in archaea and bacteria, but distinct in eukaryotes. In all organisms, the Ploop NTP hydrolases are the most abundant fold. Many folds of multicellular organisms are associated with cellular communication, for example, protein kinases (catalytic core). Some of the most common folds in vertebrates, such as globins or zinc-fingers, are rare or absent in bacteria. The bacterial top ten fold ranking shows a preponderance of folds associated with metabolic enzymes, in particular glycolytic enzymes, and one that is unique to bacteria, that for beta-Iactamases and D-Ala carboxypeptidases. These enzymes perform functions associated with the structure of the bacterial cell wall. Viruses share with other organisms folds associated with essential viral functions (polymerases, acid proteases and ribonucleases) but have few of the folds associated with metabolic enzymes, for example, TIM barrels, Rossman folds and NTP hydrolases [91]. Given that the number of complete genomes for eukaryotes is low, these studies have revealed that the most common folds seen for each of the superkingdoms are already present in SCOP. The expectation is that further genomes will not change the ranking significantly. A further feature of these analyses is that knowing the species of origin of an unknown sequence might give clues as to its fold. COMBINATORIAL CHEMISTRY This is now established as one of the key platform technologies of modern drug discovery. Few in the pharmaceutical industry, at the beginning of this decade, would have believed that this technology would become such an integral part of the medicinal chemist’s approach to synthesis. It is essential not only as a means of rapidly increasing compound collections for lead generation by HTS but also for the rapid optimisation of those leads into potential development compounds. The combinatorial mixture approach to synthesis and screening started with peptide chemistry in the 1980s. The concept owes much to Houghten who realised that screening a combinatorial library was in essence mimicking what occurs in the normal environment of all biological interactions.

20

THE IMPACT OF GENOMICS ON DRUG DISCOVERY

Here one is observing specific responses of some compounds from a natural mixture despite the presence of many others [92]. Bunin and Ellman’s communication in 1992 on the solid phase synthesis of 1,4-benzodiazepines, demonstrated that a technique previously used for peptide synthesis was applicable also to the synthesis of small molecules [93]. Coincidentally, in 1992, Human Genome Sciences was also established. This was the key event that started the commercialisation of EST databases and which opened up a new approach to identifying biological targets for drug discovery. Both events can be considered to have had a profound effect on the way drug discovery is undertaken. There have been major advances in this technology in many areas, such as, increasing the range of synthetic methodology, robotics and automation, combinatorial library design and deconvolution, for example see [94-961 (http:/ / w w w . ~ z . c o ~ ) . The increase in number and range of biological targets has necessitated that more compounds be made to screen against them. In many cases now, it is no longer possible to have each chemist producing on average fifty compounds of high purity each year at an estimated cost of around f4,500 per compound, whilst combinatorial chemistry applied by one chemist over one month, can produce 3,300 compounds for around f 8 per compound P71. The demand for more compounds has resulted in the introduction of automation and robotics in many chemistry laboratories. This has been an area of successful collaboration between the pharmaceutical industry and technology companies [94]. Process optimisation is one of several factors addressed by the MyriadTMsynthesizer, which was created by The Technology Partnership - now Mettler-Toledo Myriad Ltd (http: / /www.ttpgroup. co.uk/ttp/myriad.htm) in collaboration with a consortium of pharmaceutical companies including SmithKline Beecham, Pfizer, BASF, Novartis, Merck, Takeda and Chiroscience. The system is based on a series of robotic processing modules that allow the synthesis process to be split into efficient, high-throughput units. Myriad automates solid and solution phase chemistry both for compound libraries or parallel synthesis of single compounds. The chemist can also develop chemistry on a Myriad Personal SynthesizerTM, which is fully compatible with the total Myriad system. Argonaut Technologies (http: / /www,argotech.com) have also formed a consortium with major pharmaceutical companies (Merck & Co., Abbott Laboratories, Ariad Pharmaceuticals, Genetics Institute, Rhone-Poulenc Rorer, DuPont, Merck KgAA, Astra and Sumitomo) to develop an automated library synthesizer, TridentTM,capable of synthesizing in parallel 192 compounds, either by solid or solution phase. ~

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Deconvolution of combinatorial libraries, in order to identify the active components, has led to much ingenuity in tagging methodology [98-1011. The trend now is to use non-invasive coding strategies, such as molecular tags, since it is synthetically less complex. Libraries having two to four varying substituent groups, encoding from a few hundred to several thousand components, can be designed to be self-coded such that each component has a unique molecular weight. Such a property allows library analysis to be undertaken by mass spectrometry [ 1021. This elegant solution removes any restrictions on the manner in which the library is synthesized or screened. However, such an approach is not always possible. A different strategy uses radiofrequency tags to record encoding and other relevant information along the synthetic pathway [99, 1031. The tags are used to identify individual MicroKanTMor MicroTubeTM reactors, and were developed by Irori (http: / /www.irori.com) for use in solid phase ‘split and pool’ synthesis [104]. It is important that compound collections are maximised to increase the success rate of HTS. One feature of collections that became apparent with the advent of HTS was the clustering of compounds into categories that reflected a company’s drug discovery history, and the consequent absence of compounds that would produce hits against a wider range of targets. Clearly, new biological targets are likely to augment this problem. The solution has been to involve computational chemists in an examination of compound collections and chemical databases, in order to identify how well these cover a variety of physicochemical features, such as lipophilicity, shape and flexibility [ 105, 1061. The coverage across these sets of properties has become known as diversity. This diversity inforination can be used to design combinatorial libraries with the features of ‘drug-like’ molecules, tailored to lead generation or lead optimisation [I 07-1091. The ‘drug-like’ concept was a key feature of the first non-peptide benzodiazepine combinatorial library of Ellman [93]. Whilst much effort has been spent on increasing the scope of combinatorial chemistry, the focus of many groups has been to synthesize libraries that will yield biologically active agents acting at chemically exploitable protein targets, for example, GPCRs, proteases and kinases. A comprehensive survey has been undertaken of biologically active chemical libraries appearing in the literature between 1992 and 1997 [ 1 101. Many of these examples (approximately 70%) used alpha-amino acid synthons in their construction. Carbohydrates are a further set of biologically active molecules displaying molecular diversity and are amenable to design by combinatorial chemistry [ 1 1 11. Not only do glycoconjugates play a fundamental role in biological recognition, but monosaccharides have also been used as molecular scaffolds

22

THE IMPACT OF GENOMICS ON DRUG DISCOVERY

for beta-turn mimetics during the design of non-peptide antagonists of somatastatin [ l 121. SCREENING The drug discovery screening process was starting to change in the 1980s because of the demands brought on by the growth and impact of molecular biology. This saw the emergence of single protein entities as biological targets, a shift that greatly increased the ratio of in vitro to in vivo screening, and was viewed as a more rational approach to the treatment and understanding of disease processes. Genomics has now dramatically increased the number of single molecular targets and has added impetus to the enormous progress made in screening methodology during the last decade. The result of this is that HTS is now at the heart of drug discovery. Paradoxically, with the rationalisation of target choice, the generation of the leads to initiate chemical programmes has became more of a chance process. Two factors will be important to successful outcomes: (a) the quality of leads for optimisation and (b) the time taken to generate the hits. Hit rates in screening should be increased by expanding compound collections with a wider diversity of components, and results should be produced at a realistic speed through new technology and assay development. Robotics and automation are absolutely essential for sample preparation and the screening process itself. Significant progress has been made in assay detection methods to enable greater throughput, for example, scintillation proximity assays, fluorescence based techniques [ I 13-1 151. In 1998 HTS laboratories worked with an average of 18 disease targets per year. This is expected to rise to 23 per year in 2000 [116]. The rise in screen numbers can be met by the move towards miniaturisation, which will increase the number of assay wells on the same footprint from a current standard of 96 to 384, 1,536 and probably beyond. Currently, the average number of microplate well readdweek stands at 55,000, and this will increase by an order of magnitude to 347,000 by 2003 [116]. This rise in throughput will inevitably come at increased cost, largely due to the amount of all the reagents involved. Three further factors that impact on drug discovery deserve comment: (a) assay work-up times can run to several months in order to ensure that screens are robust and response variability is kept to a minimum; (b) despite the increased number of screens carried out, it is unlikely that many companies will have the range of screens in place to test for selectivity over a wide range of protein targets. Thus, there is a need for companies that can provide

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an extensive cross screening service, for example, CEREP (http:/ /www. cerep.fr/Utilisateur/index.asp);and (c) given the enormous progress made in HTS, attention must be paid to trends of successes and failures emerging from the results. Inevitably, this proprietary information is not often disclosed since it is possible to infer from it information about company compound collections. Pfizer, however, were prepared to publish some general observations of their experience with over 150 high-throughput screens. Their experiences indicate that the chances of finding a good lead were high for enzyme and receptor targets (GPCRs, steroid superfamily, ligand-gated ion channels) whereas hits that interrupt cytokine or growth factor interactions. that is protein-protein interactions, were generally unsuccessful [ 1 171. CASE HISTORIES Having considered the key technologies that are linked with genomics, the following case histories illustrate how some of these have been applied to protein targets that are recognised as pharmaceutically exploitable. G PROTEIN-COUPLED RECEPTORS

The GPCRs are one of the largest superfamilies ofgenes with over 800 members from a range of species. This protein family has a proven record of delivering good drug targets with many hundreds of drugs launched over the last three decades. These include beta-blockers (adrenergic receptors), antiulcer drugs (histamine receptors), anti-psychotics (serotonergic, dopaminergic receptors) and anti-migraine compounds (serotonergic receptors). For many of the new GPCRs, however, their sequence similarity to another family member does not guarantee the identity of the ligand that activates it. These are known as ‘orphan’ receptors. The challenge is to pair each orphan with an activating ligand, since without a ligand, there is as yet limited opportunity to screen for a small molecule agonist or antagonist. SmithKline Beecham have identified over 100 human orphan receptors that are distributed throughout the GPCR evolutionary tree, the majority of which have been identified by extensive bioinformatic analysis of EST databases [118]. Some of these receptors are selectively expressed in a range of therapeutically relevant tissues and have the potential to be a source of targets for drug discovery.

24

THE IMPACT OF GENOMICS ON DRUG DISCOVERY

Ligand identiJication by dutubuse searching Several approaches have been used to try and find ligands for orphan receptors. One method is to search DNA sequence databases for homologues of known ligands, particularly neuropeptides, which act through GPCRs. Rat cortistatin, which has similarity to somatostatin, was initially discovered by the isolation of a region-specific brain mRNA [ 1 191. The peptide binds to all five known somatostatin receptors and is of particular interest because it acts as a regulator of neuronal activity and sleep. A gene that encoded a human cortistatin-like peptide with significant homology to rat preprocortistatin cDNA, was discovered by identifying ESTs from a foetal brain cDNA library [120]. Although the entire human amino acid sequence has about 55% identity to rat preprocortistatin, the predicted mature peptides are well conserved. The most important difference is the presumed dibasic amino acid cleavage sites which leads to a 14-mer in rat, CST-14 (PCKNFFWKTFSSCK) and a 17-mer in humans, hCS-17 (DRMPCRNFFWKFTSSCK). The natural occurrence of the peptides needs to be confirmed in native tissues; however, biological results suggest that hCS-17 and rat CS-14 have similar sleep modulating activity [120]. Ligunds iclentified by,fishing

A common approach for identifying ligands of orphan receptors is to use reverse pharmacology. This involves a process known as ligand fishing [ 1 18, 1211. It is important to check for receptor expression, for example, by use of antibodies, and to know which intracellular second messenger system forms the basis of the functional coupling. The choice of functional assay is important when robust screening is needed. It is now possible to engineer GPCR signal transduction through a common pathway involving phospholipase C and Ca2+mobilisation, by the co-expression of the receptor with promiscuous G-proteins G a l s /16, or with chimeric G,-proteins such as GqiS,and thereby take advantage of fluorescence based screening techniques that rely on calcium ions. The screening process for activating ligands follows the following sequence. Banks of potential ligands (typically small molecules, peptides) are assayed first. This is followed by assays using biological extracts obtained from tissues, biological fluids and cell supernatants. A further option is to transfect human GPCRs into yeast from which the endogenous GPCRs have been genetically deleted, and link the receptors to the yeast signal transduction system. Activation of the signal transduction system can be configured to measurable changes in yeast growth or a colorimetric response. By

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engineering thc yeast to express both the receptor and libraries of secreted random peptides, any autocrine activation of the receptor can be detected. This assay technique can be configured into a high-throughput screen for natural peptide ligands or surrogates [ 122, 1231. This technique was successfully used to identify surrogate agonists for the human formyl peptide receptor like-1 (FPRL-I) receptor. Stimulation of the receptor with the surrogate agonists resulted in neutrophil activation and suggests that the receptor functions as a component of the inflammatory response [124]. Further examples of successful pairings of receptors to ligands are shown in Table 1.2. The list includes instances of pairing known ligands with orphan receptors and of pairing novel ligands with orphan receptors. In all cases, the discovery opens up avenues for exploring the biology of the receptor, in vitro and in vivo. The important factor for the drug industry is that a receptor-ligand pairing allows the configuration of a high-throughput screen for identification of agonists and antagonists. These compounds can then Table 1.2. EXAMPLES OF PAIRED G PROTEIN-COUPLED RECEPTORS AND LIGANDS Ligand

Therapeutic Relevance

ORLI (opioid receptor like) CGRP (calcitonin gene-related peptide) receptor

Nociceptin (orphanin FQ) (FGGFTGARKSARKLANQ) CGRP

C3a receptor

Anaphylatoxin C3a

hGR3/GPR 10

EDGI. EDG3

Prolactin releasing peptides (SRAHQHSMEIRTPDINPAW YAGRGIRPVGRFamide and TPDINPAWYAGRGIRPVGR Famide) Sphingosine-I -phosphate

Pain therapy, anxiety, learning Cardiovascular system, migraine, non-insulin dependent diabetes Inflammatory disorders H yperprolactinaemia

EDG2, EDG4

Lysophosphatidic acid

OX 1 . 0 x 2 APJ

Orexin-A, orexin-B Apelins, e.g. apelin- 13 (QRPRLSHKGPMPF) MMK-I (LESIFRSLLFRVM) A5 (SLLWLTCRPWEAM)

FPRL- I

Wound healing, tissue regeneration Wound healing, tissue regeneration Eating disorders HIV infection, immune response Inflammatory responses

Reference

26

THE IMPACT OF GENOMICS ON DRUG DISCOVERY

support biological studies that may determine possible disease relevance of the receptors. KINASES

There are estimated to be 2,000 kinases encoded in the human genome. Since they are fundamental in processes such as cell signalling and cell cycle control, genetic aberrations can eventually lead to disease, typically cancer. Of particular interest are the protein kinases that phosphorylate serine, threonine or tyrosine residues. They are known to be inhibited by small molecules and the proteins crystallise sufficiently well for 3D structures to be determined. For these reasons, kinases are considered attractive and important drug targets. Cyclin-dependent kinases (CDKs) play a central role in the timing of cell division. They are negatively regulated by phosphorylation and by the action of cyclin kinase inhibitors (CKIs). These CKIs are versatile negative regulators of CDK function and have potential roles in development, checkpoint control and tumour suppression. In addition to the natural inhibitors, small molecule, selective inhibitors have been developed. The work of Shultz and co-workers is an example of kinase inhibitor design that utilises combinatorial chemistry to design potent inhibitors of CDKs, protein crystallography to explore the structural basis of binding and selectivity, and comparative genomics in order to understand the cellular effects of the inhibitors [I 371. The starting point was the purine CKI, olomoucine (l), which exhibited good selectivity but only moderate inhibition [ICso 7pM] of a subset of the CDK family of protein kinases. After examination of the binding mode in the kinase ATP binding site, a combinatorial chemistry approach was used to modify the substituents at positions 2 , 6 and 9 of the purine base. Out of the library of compounds came the potent inhibitor purvalanol B (2). Purvalanol B has an ICso against the complex of CDK2-cyclin A of 6nM, which corresponds to a 3 orders of magnitude potency increase over olomoucine, and a 30-fold increase over flavopiridol(4), one of the most potent and selective CDK2 inhibitors known. Against 22 purified human kinases tested, purvalanol B showed a high degree of kinase selectivity and only a subset of CDKs were significantly inhibited (cdc2-cyclin B, CDK2-cyclin A, CDK2cyclin E, CDKSp35). No inhibition was observed against other kinase subfamilies, for example, extracellular regulated kinases, c-jun, PKC isoforms, cyclic AMP kinases, casein kinase, Raf kinase, GSK3fi or insulin-receptor tyrosine kinase. The structural basis of the selectivity and affinity of these inhibitors was determined by obtaining a crystal structure of human CDK2-

21

L.J. BEELEY ETAL.

0

(3)

(4)

Kinrise structures

purvalanol B at 2.05A resolution. Purvalanol B was estimated to have 86% complimentarity of fit in the ATP binding site by comparing surface areas. The purine inhibitors were tested in cell growth assays. However, in order to gain some understanding of the effects of the inhibitors on cellular pathways, it was necessary to turn to yeast. This was possible because of the existence of a more membrane permeable analogue, purvalanol A (3), which had an IC50against the yeast CDK, Cdc28p, of 80nM compared with a potency of greater than l pM for purvalanol B. Using microarrays of yeast mRNAs, different inhibitor classes could be compared with each other and also against yeast strains with non-functioning CDKs brought about by genetic mutation. The effects on the mRNA levels of over 6,200 yeast genes were investigated using high-density oligonucleotide expression systems. Inhibitors were assayed in parallel for their effects on yeast gene expression profiles and the outcomes compared. Of nine transcripts down regulated by the inhibitors, five were associated with cell cycle progression. One set of genes affected was those involved in phosphate metabolism, which showed an increase in expression. Increases in transcript levels were also seen for many genes involved in cellular metabolism. Although yeast cdc28p is the intended target of both purvalanol A and flavopiridol, more than half of the mRNA changes that result from ex-

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THE IMPACT OF GENOMICS ON DRUG DISCOVERY

posure to the two compounds are distinct, for example, they have differential effects on ribosomal proteins. The reasons for this are not exactly clear. The effects of genetic mutations of yeast cdc28p were also compared with the chemical inhibition. Only two of the cell cycle genes that changed in response to the small molecules were affected by genetic mutations. Metabolic genes were affected, as were genes involved in stress signalling. There were a number of genes that showed significant changes only in the genetic mutants, including a protein with transmembrane domains (YOLl55C), a metabolic gene (ACH1) and a variety with unknown function. Clearly, these yeast experiments demonstrate the differing transcript profiles from compounds with ostensibly similar in vitro activity. In the future, and in a mammalian context, different profiles will be useful for evaluating the selectivity of drug candidates and, in addition, may point to further proteins that may make alternative drug candidates. PROTEASES

This category of enzymes have been successful targets for drug discovery, for example, in the development of anti-bacterial agents (bacterial serine proteases are inhibited by p-lactam antibiotics) and more recently, in anti-viral therapy (HIV protease inhibitors). As for non-infective disorders in humans, relatively few successful drugs have emerged. The zinc-metallo enzyme, angiotensin-converting enzyme (ACE), is a key example where inhibitors have proved to be therapeutically useful antihypertensive drugs. Industry efforts to market inhibitors of other metalloproteases, and serine proteases such as elastase and thrombin, may well prove fruitful in the not too distant future [ 1381. Orphan proteases Based on figures available for yeast and nematode, it is estimated that the human genome will contain somewhere between 800-1,000 proteases. Currently, the public databases contain just over 200 human protease sequences and the genome projects will therefore generate several hundred novel vertebrate sequences. The majority of these will be orphans, i.e. no experimental data will be available on their substrates, biochemical roles, or natural inhibitors in vivo. The final count of protease inhibitors in the genome is likely to be 10-20% of the protease numbers. Whilst it can be straightforward to assign a new protease primary sequence to one of the general mechanistic classes of proteases (serine, aspartic, cysteine, metallo, exoprotease, endoprotease) on the basis of homology,

L.J. BEELEY ETAL.

29

bioinformatic analysis alone will not allow the identification of the in vivo substrate(s), inhibitors, biochemical function or pathological significance. If characterization data are available for at least one member of an orthologous family, it is reasonable to infer that biochemical function is likely to be conserved for truly orthologous sequences in human, mouse and rat. Extrapolation of function from paralogous relationships, even between close homologues in the same species, is more hazardous because the evolutionary persistence of paralogues implies functional divergence. There are specific approaches to characterize substrates for orphan proteases. It is necessary to make a clear distinction between surrogate substrates that can be turned over by the enzyme in vitro, and physiological or ‘real’ substrates which are cleaved in vivo. Surrogate substrate determinations centre on the testing of peptide libraries, produced either by combinatorial synthetic methods [ 1391.or as a phage display [ 1401. Once the appropriate peptide P-P’ residue specificities are found, it may be possible to narrow down the candidate in vivo substrates, but these preferences cannot be searched with any precision. Surrogate peptide substrates can be used to develop a fluorogenic HTS assay. These assays can produce tool inhibitor compounds that may be specific enough to probe the function of the protease in cellular systems or animal models. Physiological substrate determination for an orphan protease presents a much more difficult problem. Many proteases show broad and overlapping specificities, particularly between close paralogues, and ‘one protease: one inhibitor: one substrate’ is the exception rather than the rule. In addition, specificity may not be an inherent property of the enzyme but can be derived from the co-localisation of active protease and substrate in particular tissues or subcelluar compartments. When considering proteases as drug targets, there is an additional problem created when the pathology arises from ‘inappropriate’ protease activity on substrates not cleaved under normal conditions. There are instances of the reverse problem, that is, orphan substrates for which the protease is unknown. Myelin-associated glycoprotein is an example where its proteolysis has been implicated in demyelinating diseases. By a range of experiments, including peptidolysis assays, the authors have narrowed down the candidate protease to an extracellular, cystatin-C inhibitable, cathepsin-L like enzyme [141]. Cathepsin K This is one example of protease discovery and inhibitor design that encompasses several aspects of the genomics-driven drug discovery process. In humans the skeleton is maintained and renewed by a bone remodelling process

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THE IMPACT OF GENOMICS ON DRUG DISCOVERY

in which a balance is sustained between osteoblasts (bone building cells) and osteoclasts (bone absorbing cells). If osteoclast activity outstrips osteoblast activity, the skeleton becomes porous and weak and osteoporosis may develop. Osteoclasts are specialised rare cells embedded in the bone matrix. It was reasoned that any novel genes identified in osteoclasts would have a high probability of being involved in the bone resorption process. ESTs were generated from the cDNA osteoclast library in a collaboration between SmithKline Beecham and Human Genome Sciences. A novel protease was identified and classified as a member of the papain family of cysteine proteases, based on similarity to published sequences, and named cathepsin K (EC 3.4.22.38). It was shown to be highly specific to the osteoclast library. Approximately 4% of the 9,000 ESTs encoded the protease, and there were few examples of the protease appearing in ESTs from other cDNA libraries. The osteoclast specificity was confirmed by localisation studies. Cathepsins B, L and S could not be detected in osteoclasts [142]. The full-lengthclone of cathepsin K was expressed and generated a protease of 329 amino acids, 56% identical to the preproenzyme form of human cathepsin S . Further evidence to validate the protease as a drug target was provided by gene localisation and antisense studies. The gene localises to chromosome lq21 [143, 1441. Genetic studies have shown that this is the candidate gene for pycnodysostosis, an autosomal recessive osteochondrodysplasia characterized by osteosclerosis and short stature [ 1451. Antisense studies have also shown that osteoclastic pit formation is inhibited in a concentration-dependent fashion [146]. Further work has shown that the likely key in vivo substrate is cortical bone collagen [147]. Cathepsin K is synthesized and secreted from the cell as an inactive proenzyme that is converted to its mature form by proteolytic cleavage of a 99 amino acid propeptide from the amino terminus. This process is autocatalytic and occurs at low pH [148], consistent with the boneopitenvironment. Crystal structures have been determined at between 2-3A for both procathepsin K [149] and cathepsin K complexed with mechanism based inhibitors [ 150, 1511. Prior to the structure determination of cathepsin K, papain was,chosenas a surrogate for the structure based design of cathepsin K inhibitors because it had reasonable sequence similarity and the structure was known. The binding mode of leupeptin (Ac-Leu-Leu-Arg-H)(9,a peptide aldehyde inhibitor of papain, was known to be on the S side of the active site of the enzyme. A related aldehyde (6), however, was found to bind in the opposite s’ direction. By overlaying the two crystal structures, potent inhibitors of peptidic nature were designed (7), which span both sides of the active site [ 152,

31

L.J. BEELEY ETAL.

HzNYNH

PhCH,O

PhCH,O

1q N . . N I N , E , P H N

H

H

0

0

(10)

Catkepsin K structures

OCH,Ph

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THE IMPACT OF GENOMICS ON DRUG DISCOVERY

1531. Potent, non-peptide analogues were then synthesized (8) [154], (9) [155] and (10 ) [156], based on a strategy involving iterative cycles of structurebased design, inhibitor synthesis and evaluation, and crystallographic structure determination. These inhibitors have potent antiresorptive activity both in vitro and in vivo, and are therefore promising leads for therapeutic agents to the treatment of osteoporosis. FUTURE PERSPECTIVES Articles appear regularly in the scientific and popular press describing how genomics will lead to an explosion in our knowledge of human genetics, and how this will impact on our understanding of diseases. Consequently, most pharmaceutical companies are now resourcing either ‘in house’ or external activities with the aim of exploiting the linkage of genetic variation with disease. Although a greater understanding of genetic variation in a defined population will lead to better targeted therapies, research activities in this area are expensive and drug companies will have to make prudent decisions on which directions should be followed. These key decisions will have to be based on an understanding of how genetics will impact on the drug discovery process. Will genetics be a means to quicker, larger scale validation of targets, or merely become an aspect of the developability process? Investments such as that by Roche in deCODE Genetics [1’57], who unveiled a five-year, $200 million research pact in 1998 that aims to decipher the genetic causes of a dozen common diseases, would suggest the former, but the reality may be nearer the latter. Gene variation caused by single nucleotide polymorphisms (SNPs) is a topic of much discussion and databases of genome-wide SNP scans will soon be available [158]. SNPs in the coding region of a gene may give rise to an amino acid change or they can be silent. Not all the variants will cause functional changes; however, those that do, may lead to a significant variation in response to drug treatment depending upon population frequency. In the not too distant future, this may require the co-development of a diagnostic kit to ensure appropriate patient selection, or require a switch to another drug with a different mechanistic approach. The production of SNP data means that somewhere between 2003 and 2005, we will not only have access to the entire genome but also to all the common polymorphisms that define the genetic variation between populations and individuals. We should then have the information we need to move from a position of having a drug discovery process with a proven track record of delivering quality medicines to one that promises even more.

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GLOSSARY Algorithm - A detailed sequence of actions embodied in a computer program. Biochemical pathway - A network of interacting molecules responsible for a specific biochemical function. Bioinformatics - The application of computer and statistical techniques to the management of genome information. This includes the analysis of DNA sequences, protein sequences, structure prediction and genome comparisons. BLAST- Basic Local Alignment Search Tool. A heuristic sequence comparison algorithm used to search sequence databases for optimal local alignments to a query. CGAP - Cancer Genome Anatomy Project. A programme established to decipher the molecular anatomy of a cancer cell. Comparative genomics The study of comparing complete genome sequences, or use of model organisms to understand gene function. DNA microarrays -Technology for parallel processing and analysis of thousands of DNA segments. Used to monitor expression patterns of mRNAs and detecting mutation patterns in genomic DNAs. Domain - A portion of a protein that can independently fold into a stable tertiary structure. Often associated with a function. Proteins may comprise a single domain e.g. globins, epidermal growth factor (EGF), or consist of many e.g. plasminogen, EGF receptor. Each domain is described by its fold, EMBL Sequence Database - Database of nucleotide sequences maintained by the European Bioinformatics Institute (EBI), a European outpost of the European Molecular Biology Laboratory (EMBL). EST- Expressed Sequence Tag. A short sequence read of a cDNA (complementary DNA) clone. FASTA - A heuristic sequence comparison algorithm used to search sequence databases for optimal local alignments to a query. Fold - A recurring spatial arrangement and connectivity of secondary structures forming a protein core, e.g. four helix bundle (two alpha-alpha units), Rossman fold (beta-alpha-beta-alpha-beta). Functional genomics - The study of obtaining an overall picture of genome functions. Includes the expression profiles at the mRNA level, the protein level and comparative genomics. GenBank - A nucleotide database maintained by the NCBI. Genetics -The study of the patterns of inheritance. ~

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THE IMPACT OF GENOMICS ON DRUG DISCOVERY

Genome -All the genetic material in the chromosomes of a particular organism. The size is generally given as its total number of nucleotide base pairs. Genomics -The study of genome information. Homologous Sequences - Sequences that are related by divergence from a common ancestor. It is not a synonym for similarity. HTG sequence - High-throughput genomic sequence. Sequences derived from genome sequencing projects. Part of a chromosome, generally up to 200,000 nucleotide base pairs, comprising coding and non-coding regions. Motifs - Simple arrangements of two or three consecutive secondary structure elements e.g. two alpha helices joined by a loop region, observed in DNA binding proteins and calcium binding proteins (EF hand). Also known as supersecondary structures. mRNA expression profile -The identities and absolute or relative expression levels of mRNAs that characterise a particular cell type or physiological, developmental or pathological state. Multdimensional chromatography - This is the combining of separation modes with orthogonal selectivity performed consecutively. A common example would be size fractionation in the first dimension, an ion exchange charge separation in the second dimension, and fractionation by hydrophobicity for the third dimension. NCBI - National Center for Biotechnology Information. nr database - Non-redundant database of protein or DNA sequences available from the NCBI. Oligonucleotide chips - High-density arrays of oligonucleotide probes that are synthesized by manufacturing techniques similar to those for silicon chips. Used to detect, for example, variations of DNA sequence patterns by hybridisation studies. See DNA microarrays. Orthologues - Homologous sequences in different species that arose from a common ancestor gene during speciation. They may or may not be responsible for a similar function. See Paralogues. Paralogues - Homologous sequences in the same species that share a common evolutionary ancestor, which diverged by gene duplication. See Orthologues. PDB database - Protein Data Bank (http:/ /www.rscb.org/pdb/index.html). The repository of solved protein structures. Protein family - Groups of sequences that show similarity throughout their length. Protein superfamily - Groups of several families that are related by (divergent) evolution and usually still share some functional elements.

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Proteomics - Technologies for the high-throughput profiling of the individual protein components of cells or tissues. Single Nucleotide Polymorphisms (SNP) - Nucleotide variability at a single nucleotide position in the genome, within a population. In the gene-coding region these may be silent (no amino acid change) or non-silent (amino acid change). Supersecondary Structures - see Motifs, Folds. SWISS-PROT - A curated protein sequence database that provides a high level of annotations, a minimal level of redundancy and a high level of integration with other databases. It is maintained collaboratively by the Department of Medical Biochemistry at the University of Geneva and the European Bioinformatics Institute (EBI). TrEMBL - A supplement of SWISS-PROT that contains all the translations for EMBL nucleotide sequence entries not yet integrated into SWISSPROT. See SWISS-PROT. Wormpep - A periodically updated compilation of the protein sequences for predicted Cuenorhubditis elegans genes from the nematode genome project. Maintained by The Sanger Centre, U.K.

WEB GLOSSARY

‘DRES’ - Drosophila related expressed sequences: a resource provided by the Telethon Institute of Genetics and Medicine (TIGEM), Italy. By EST database searching, human cDNAs homologous to Drosophila mutant genes have been identified and mapped (http: / /www.tigem.it/LOCAL/ drosophila/dros. html). Biobase: the Danish Centre for Human Genome Research‘s 2-D PAGE Databases at the University of Aarhus, which are being developed for functional genome analysis in health and disease-and contain data on proteins identified on various reference maps (http: / /biobase.dk/cgi-bin/celis). CGAP - Cancer Genome Anatomy Project: a programme established to achieve a comprehensive molecular characterisation of normal, precancerous and malignant cells (http: / /www.ncbi.nlm.nih.gov/CGAP/). Combinatorial chemistry: data resource (http: / / w w w . ~ z . c o ~ ) . Human Genome Sequencing Index: the NCBI site that provides a service to members of the international consortium to support coordination and tracking of the Human Genome Project (HGP) (http: / /www.ncbi.nlm. nih.gov/HUGO/). KEGG: Kyoto Encyclopaedia of Gene and Genomes: is an effort to computerize current knowledge of molecular and cellular biology in terms of the in-

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THE IMPACT OF GENOMICS ON DRUG DISCOVERY

formation pathways that consist of interacting molecules or genes and to provide links from the gene catalogues produced by genome sequencing projects. The KEGG project is being undertaken in the Institute for Chemical Research, Kyoto University as part of the Japanese Human Genome Program (http: / /www.genome.ad.jp/kegg/kegg.html). Large Scale Biology Corporation (LSB): a biotechnology company focused on the discovery of protein-level changes underlying pharmaceutical mechanisms and disease processes (http: / /www.lsbc.com). MRC Mammalian Genetics Unit, Harwell - mouse mutation database: ENU mutagenesis programme. A large scale mouse genetics programme to identify new phenotypes of relevance to human disease (http: / / www.mgu.har.mrc.ac.uk/mutabase/). National Center for Biotechnology Information (NCBI): the site that supports and distributes a variety of databases for the medical and scientific communities, for example GenBank, and also supports search and retrieval systems that provides users with integrated access to sequence, mapping, taxonomy, and structural data (http: / /www.ncbi.nlm.nih.gov/). NCBI Entrez Genomes: the NCBI page for genomes (http: / /www.ncbi.nlm. nih.gov/Entrez/Genome/org. html). Protein Data Bank (PDB): the single international repository for the processing and distribution of 3-D structure data of biological macromolecules determined experimentally by X-ray crystallography and NMR (http: / / www.rcsb.org/pdb/index.html). SWSS3DPAGE: contains data on proteins identified on various 2-D PAGE reference maps (http: / /www.expasy.ch/ch2d/). SWISS-MODEL: an automated comparative protein modelling server (http:/ /www.expasy.ch/swissmod/SWISS-MODEL. html). TIGR: The Institute of Genomic Research: a not-for-profit research institute with interests in structural, functional, and comparative analysis of genomes and gene products in viruses, eubacteria, pathogenic bacteria, archaea, and eukaryotes (both plant and animal), including humans (http: / /www.tigr.org/index.html). Wormpep: a periodically updated compilation of the protein sequences for predicted Caenorhabditis elegans genes from the nematode genome project, maintained by The Sanger Centre, U.K. (http: / /www.sanger.ac.uk/ Projects/C.elegans/wormpep). XREF a database for cross-referencing the genetics of model organisms with mammalian phenotypes (http: / /www.ncbi.nlm.nih.gov/XREFdb/).

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ACKNOWLEDGEMENTS We would like to thank the many colleagues within SmithKline Beecham for their helpful discussions and comments.

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2 CCK-B Antagonists in the Control of Anxiety and Gastric Acid Secretion MARK S. CHAMBERS, Ph.D. and STEPHEN R. FLETCHER, Ph.D. The Neuroscience Research Centre, Merck, Sharp and Dohme Research Laboratories, Terlings Park, Harlow, Essex, CM20 2QR, U K.

INTRODUCTION

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ANXIETY

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ANTI-SECRETORY ACTION

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CLINICAL RESULTS TO DATE

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CLASSES OF CCK-B ANTAGONISTS Benzodiazepines Benzazepines Peptoids Hybrids Ureidomethylcarbamoylphenylketones Dibenzobicyclo[2.2.2]octaneand bicycloheteroaromatic derivatives

49 49 63 64 66 71 73

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REFERENCES

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INTRODUCTION Since the first isolation of Cholecystokinin (CCK) by Mutt and Jorpes in 1968 and its subsequent characterization as a 33 amino acid peptide [ 11, the hormone has been recognized as a significant modulator of both the central nervous system (CNS) and gut function. It is now known that CCK constitutes a number of biologically active peptides formed from a 1 15 amino acid precursor protein. Active forms include CCK-58, CCK-39, CCK-33, 45

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sulphated CCK-8 and CCK-7, unsulphated CCK-8 and CCK-7, CCK-5 and CCK-4 [2]. The total amount of CCK in the mammalian CNS is higher than that of any other regulatory peptide [3]. Two receptor subtypes, CCKA and CCK-B, which mediate the many biological functions of CCK have been identified to date. The human CCK-A [4] and CCK-B [5-71 receptors have both been cloned and shown to belong to the G-protein-coupled receptor (GPCR) superfamily. Signal transduction is mediated via activation of phospholipase C and the formation of inositol 1,4,5-triphosphate (IP3) and 1,2-diacylglycerol [8]. At the outset, the CCK-A (alimentary) subtype was considered to be located solely in the periphery and CCK-B (brain) subtype within the CNS but autoradiography studies subsequently demonstrated the presence of CCK-A receptors in discrete brain regions [9] and cloning showed that a single gene encodes for both the CCK-B (brain) receptor and the peripheral, gastrin receptor in man [7]. What remains indisputable is that CCK-A receptors are more abundant in the periphery whilst CCK-B predominates in the brain. CCK-A receptors in the gut control pancreatic enzyme secretion, gall bladder contraction, gastric emptying and intestinal motility [lo], whilst CCK-A receptors located within the CNS have been postulated to play a significant role in neuropsychiatric disorders [8, 1I]. CCK-B receptors are more widely distributed in the CNS and have been implicated in modulation of anxiety [ 12-14], panic disorder [ 15-1 71, depression [ 181, nociception [ 19-22] and satiety [ 10,231.Therapeutic targets related to CCK-B include anxiety, analgesia and schizophrenia via modulation of the centrally located receptors. More recently treatment of gastric secretory disorders and GI tumors via modulation of peripheral CCK-B receptors has been aggressively pursued [lo, 24,251. Through the innovative efforts of medicinal chemists over the past fifteen years, a range of non-peptide receptor antagonists have been identified which should enable the physiological roles of both CCK-A and CCK-B receptors to be clarified. Such approaches have been widely reviewed [26-311. In this chapter advances in the use of CCK-B receptor antagonists in the control of anxiety and gastric acid secretion will be described. ANXIETY There have been many studies reported in which administration of CCK peptide agonists has been demonstrated to produce anxiogenic-like effects in animals leading to the conclusion that CCK antagonists might find utility as anxiolytics [32]. Such studies have, however, been hampered by the lack of effective animal models. Not all investigators have observed anxiogenic

M.S. CHAMBERS A N D S.R. FLETCHER

41

effects and recent more stringent studies with CCK-B receptor antagonists have failed to provide evidence of anti-anxiety effects in animals [33, 341. It would appear that the only effective way of evaluating the therapeutic potential of CCK-B receptor antagonists as anxiolytics is through clinical studies in man [35]. ANTI-SECRETORY ACTION The peptide hormone gastrin stimulates acid secretion and mucosal growth in the stomach. Animal studies have shown that inactivation of gastrin by antibodies serves to reduce acid secretion induced by a range of pharmacological stimuli [36,37] and it is well established that CCK-B receptor antagonists such as L-365,260 (1) can inhibit gastric acid secretion in animals [38-411. CCK-B receptor antagonists may, therefore, have therapeutic use in peptic ulcer disease as well as several other diseases such as diabetes mellitus, Zollinger-Ellison syndrome, gastrinomas and G-cell hyperplasia, where elevated gastrin levels are observed [3 11. CLINICAL RESULTS TO DATE Several clinical investigators have shown that CCK produces anxiety-like symptoms in man. In 1990 it was reported that CCK-4 given intravenously produced panic-like symptoms in patients with panic disorder [42]. Since CCK-4 was known to be a selective CCK-B receptor agonist this suggested there was a role for CCK-B receptor antagonists in anxiety. It was later demonstrated that this effect can be blocked with the CCK-B receptor antagonist L-365,260 (Figure 2.2) [ 161. Lines et al. demonstrated the ability of L-365,260 to block induced panic in man acutely. Pre-treatment of healthy volunteers with L-365,260 was also shown to reverse the autonomic and anxiogenic effects of pentagastrin [43]. CI-988 (2) has been evaluated in healthy volunteers and symptoms of CCK-4 induced panic attack were relieved at 100 mg p.0. [44]. On the other hand, more recent findings by Kramer et al. of a lack of clinical efficacy in patients with panic disorder following repeated dosing of L-365,260 [45]and failure of CI-988 to antagonise CCK-4 induced panic in patients with panic disorder [46] are less encouraging. It is thought that unequivocal evidence for the use of CCK-B receptor antagonists as anxiolytics using L-365,260 and CI-988 has not been forthcoming because of associated formulation and pharmacokinetic issues [47]. Clinical data have been reported on the effect of two CCK-B receptor an-

48

CCK-B ANTAGONISTS

tagonists on gastric acid secretion. Revel et al. showed that spiroglumide (3) infused i.v. in the range of 1-7.5 mg/kg/h dose dependently and competitively antagonised gastrin-stimulated gastric acid secretion [48]. In Phase I1 studies, spiroglumide infused at 7.5 mg/kg/h decreased both basal and meal-stimulated gastric acid secretion [49]. However, although bioavailable, the poor CCK-B affinity and selectivity of spiroglumide (CCK-B IC50, 1,400 nM; CCK-A ICs0, 13,500 nM) [50] precluded development as a treatment for peptic ulcer. Researchers at Merck, using L-365,260 (CCK-B ICS0 8.5 nM, CCK-A IC50 740 nM), found that the inhibition of gastric acid secretion was both modest and of short duration and concluded that L-365,260 was unlikely to be clinically useful for this purpose [51]. Despite the disappointing clinical results with CCK-B receptor antagonists obtained to date, effects have been observed on induced anxiety and on gastric acid secretion in man (Figure 2. I ) . Second-generation compounds with enhanced potency and improved pharmacokinetics are clearly required.

(1) L-365,260

P

(3)Spiroglumide

Figure 2.1. CCK-B receptor antagonists which have been evaluated in man.

M.S. CHAMBERS A N D S.R. FLETCHER

49

CLASSES OF CCK-B ANTAGONISTS BENZODIAZEPINES

Faced with the formidable challenge of identifying low molecular weight, non-peptide structures displaying selectivity for the CCK subtypes, Merck scientists used natural product screening techniques to identify asperlicin (4) as the first CCK receptor antagonist. The innovative discovery of 1,4benzodiazepine-based CCK-A receptor antagonists from asperlicin initially led to the development of MK-329 (5, devazepide) which entered clinical trials for the treatment of satiety [52]. Further studies then led to the identification of 1,4-benzodiazepines that bind selectively to CCK-B/gastrin receptors [53]. Particularly striking was the influence of the absolute configuration at C-3 of the benzodiazepine on subtype selectivity, with the R-stereochemistry proving optimal for CCK-B affinity [54]. The first-generation benzodiazepine L-365,260 ( I ) has been evaluated in the clinic as described above. The compound had to be specially formulated in order to achieve adequate levels of oral bioavailability, mainly as a consequence of the low aqueous solubility of the crystalline form (<0.002 mg/mL, pH 7.4). Extensive studies have since been carried out by the Merck group and others to identify effective second-generation benzodiazepine-based CCK-B receptor antagonists with improved water solubility.

(4) Asperlicin

( 5 ) MK-329

CCK-B ANTAGONISTS

50

Table 2.1. RECEPTOR BINDING AFFINITIES AND SOLUBILITIES OF ACIDIC 3-(PHENYLUREID0)-1,4-BENZODIAZEPINES

IC,, (nM)"

'

Compd

R

R

(1) (6) (7)

Me Me Me

Me C02H

(8)

iBu

J> J)

CCK-B

CCK-A

Solubility (mg1mL. (PW i

8.5

740 1600 580

< 0.002 (7.4)

6.7 1 .o

0.14

1400

4.7 3.7

(7.4) (7.4)

1.4

(7.0) (8.0)

H

111

H

0.27

(9)

Me

980

0.41 1.7

(7.4) (8.0)

H alCSorepresents the concentration producing half-maximal inhibition of specific binding of ['2SI]-BoltonHunter CCK-8 to CCK-receptors in guinea pig cortical membranes (CCK-B) or the rat pancreas (CCK-A).

An initial approach was to append acidic solubilising groups to the phenyl ring of L-365,260 [55]. High affinity at the CCK-B subtype was retained within this series (Table 2.1). Development of an ex vivo binding assay using [1251]Bolton Hunter CCK-8s enabled brain penetration to be estimated for the lead tetrazole (8), in mouse, following i.v. administration [56]. This showed the compound to be poorly brain penetrant with a braixplasma ratio of 0.02 (cJ: L-365,260, ratio 0.48). Subsequently a significant discovery was made within Merck with the finding that the phenyl group of L-365,260 may be effectively replaced by a cyclohexyl group giving L-708,474 (10) [57]. This

51

M.S. CHAMBERS AND S.R. FLETCHER Table 2.2. RECEPTOR BINDING OF 5-CYCLOALKYL- 3-(PHENYLUREIDO)I ,4-BENZODIAZEPINES

Cot1rpd.

R

3 stereo

CCK-B

CCK-A

(1)

Ph

R

8.5

740

Cyclohexyl

R

0.28

1800

Cyclohexyl Cyclopentyl Cyclobutyl

S R,S R,S

L-365,260 (10)

L-708,474 (11)

(12) (13)

170 16 30

0.46 27 400

"ICio represents the concentration producing half-maximal inhibition of specific binding of ['251]-Bolton Hunter CCK-8 to CCK-receptors in guinea pig cortical membranes (CCK-B) or the rat pancreas (CCK-A).

change led to enhanced CCK-B receptor affinity and selectivity (Table 2.2). Whilst the physical properties were not improved over L-365,260 a new avenue of research was opened up since further modifications led to high affinity CCK-B receptor antagonists with vastly improved aqueous solubility. Initial studies based on the introduction of acidic groups onto the aromatic ring of L-708,474 floundered, in the same manner as the approach based on L-365,260, because of poor brain penetration. However, by developing a strategy of flanking an acidic acylsulphonamide group with lipophilic groups it was possible to achieve significant brain penetration [58]. Thus L-736,309 (21) proved to be equipotent with L-708,474, was bioavailable in rat (F, 14%) and had significantly improved aqueous solubility (Table 2.3). The reduced brain penetration relative to L-365,260, however, ultimately precluded further development. A more detailed investigation of the influence of pKa on solubility and brain penetration was carried out utilising an amino tetrazole moiety in which the pKa could be modulated over a range of 4.5-6 (Table2 . 4 ) [59]. Using the ex vivo binding method described earlier to estimate receptor occupancy, this study showed conclusively that even weakly acidic derivatives poorly penetrate the brain. These findings led to a focused investigation of basic derivatives in an effort to enhance aqueous solubility whilst maintaining brain penetrability.

52

CCK-B ANTAGONISTS

Table 2.3. RECEPTOR BINDING OF ACIDIC 5-CYCLOHEXYL- 3-(PHENYLUREID0)1,4-BENZODIAZEPINES

Compd.

R

3 stereo

Me COzH CONHSOzMe CONHS02iPr CONHS02iPr CONHS02Ph CONHS02Ph CONHSO,-o-tolyl S02NHCOMe

CCK-B

0.28

1800

1.4

2.8 1.7 12 1300 11 > 6000 1600

0.58 0.3 0.78 0.59 0.73 0.27

RS

CCK-A

0.36

19

Solubility (mg/mLI i10 ng/mL

1.1 1.3

0.74 1.2 -

0.37 0.41 0.26

aIC50represents the concentration producing half-maximal inhibition of specific binding of ['2SI]-BoltonHunter CCK-8 to CCK-receptors in guinea pig cortical membranes (CCK-B) or the rat pancreas (CCK-A).

A range of amidines were prepared in which the cyclohexane ring of L-708,474 was replaced with a variety of N-linked cyclic amines (Table 2.5) [60]. The azabicyclic derivative L-740,093 (40) has high CCK-B receptor affinity (CCK-B ICs0 g.p. 0.1 nM) whilst also displaying excellent receptor subtype selectivity (CCK-A/CCK-B, 16,000) [39]. Comparable high affinity was observed in cells expressing the human CCK-B receptor (hCCK-B ICso 0.49 nM), and the compound has been shown to be an antagonist. Unlike L-365,260, L-740,093 behaves as an insurmountable antagonist of the human CCK-B receptor attributed, partly, to slow dissociation from the receptor [61]. In addition to having increased potency compared to L-365,260, amidine L-740,093 has superior physical properties and enhanced aqueous solubility as the hydrochloride salt (solubility, 0.15 mg/mL; Log P,octanoll pH 7.4 aqueous buffer, 4.7; pKa, 7.1) [60]. Thus L-740,093 represents a considerable improvement as a clinical candidate over L-365,260 and continued progress will be monitored with interest.

M.S. CHAMBERS A N D S.R. FLETCHER

53

TAble 2.4. RECEPTOR BlNDING OF 3-((TETRAZOL-5-YLAMINO)PHENYLUREIDO)1,4-BENZODIAZEPIN ES

R

n

R'

1 0 0 0 0 0 1 0 0 0 0

H H H Me Me -CH*-CHZH H H Me Me

CCK-B

CCK-A

pKa

Log D

7.5 5.7 0.58 1.1 20

3000 3000 3000 3000 3000 4100 153 200 400 800 1600

5.9 5.1

1.47 0.89 1.14 1.37 1.58 1.38

~

Ph Ph Ph Ph Ph PI1 Cyclohexyl Cyclohexyl Cyclohexyl Cyclohexyl Cyclohexyl

H H Me H Me

0.11

H H Me H Me

0.28 0.2 0.05 0.07 0.30

5.4 5.7 4.8

I .85

"lCso represcnts the concentration producing half-maximal inhibition of specific binding of ['Z51]-BoltonHunter CCK-8 to CCK-receptors in guinea pig cortical membranes (CCK-B) or the rat pancreas (CCK-A).

Whilst a number of issues associated with L-365,260 were addressed with L-740,093. only a modest impact was made on the short half-life observed in vivo (L-365,260, Tllzrat 0.6 h) [62]. Further modifications based on L-708,474 were therefore carried out. The 4-piperidino derivative (42) displayed relatively poor affinity for the CCK-B receptor (Tuble 2.6). The 2-piperidinyl derivatives, on the other hand, led to potent selective CCK-B antagonists (Tublc 2.6), and through judicious choice of substituents, compounds with increased half-lives compared to L-365,260 were identified, for example (47) (TI,2rat, 2.3 h) and (48)(TIi2rat, 3.4 h). Interestingly, the increase in half-life in rat was found to be due to increased volume of distribution rather than reduced clearance [62]. Further studies are required to obtain a suitable successor to L-365,260 and L-740,093. Scientists at Yamanouchi have replaced the N-1 methyl substituent in the C-5 phenyl- 1,6benzodiazepine series with a 1-aroylmethyl group, and identified the o-tolyl analogue YM022 (50) as a very high affinity CCK-B antago-

54

CCK-B ANTAGONISTS

Pi (50)YM022

nist that has significantly enhanced selectivity for gastrinlCCK-B receptors in rat brain over the rat pancreatic CCK-A receptor compared to L-365,260 (1) [63] (Tabfe2.7). YM022 (50) was found to be 500-fold more potent than (1) at inhibiting pentagastrin-stimulated gastric acid secretion in anaesthetized rats when given i.v. [63] and bad antisecretory and antiulcer activities comparable with the H2 antagonist famotidine [38]. Replacing the o-tolyl group with tert-butyl gave compound (5 1) having similar potency in the rat gastric acid secretion model when dosed i.v. (Table 2.8) but which was more potent than YM022 when administered i.d. [(51), 80% inhibition; YM022, 16% inhibition at 0.3 pmol/kg i.d.1 [64]. This enhancement in in vivo potency is noteworthy since the tert-butyl analogue (51) has 5-fold lower affinity in v i m at the gastrin/CCK-B receptor than YM022. Replacing the C-5 phenyl group present in (51) with 2-pyridyl(52) resulted in only a small decrease in potency in the rat gastric acid secretion model following i.v. administration. Significantly, however, (52) showed enhanced oral bioavailability in rat (C,,, 571 ng/mL at 10 mg/kg p.0.) compared to (51) (C,,, 81 ng/mL). The pyridyl analogue was subsequently evaluated in the Heidenhain pouch dog model [65], and demonstrated 97% inhibition of pentagastrin-induced gastric acid secretion when dosed orally at 3.0 pmol/kg [66]. Further exploration in this series involved replacement of the methyl substituent on the phenylurea ring with weakly basic groups such as methylamino YF476

(51)

X = CH;R = Me

(52)X = N; R = Me (53)X = N;R = NHMe YF476 (54) X = N;R = NMez

M.S. CHAMBERSAND S.R. FLETCHER

55

Table 2.5. RECEPTOR BINDING AFFINITIES O F 5-AMINO-1,4-BENZODIAZEPINES

R

-0 43

-0 -0 -0

-a a a

3 sterco

CCK-B 137

CCK-A 480

5.7

17

1.25

10

1.35

> 3000

144

0.53

0.1

27

7.9

20

1604

6.5

"ICso represents the concentration producing half-maximal inhibition of specific binding of [''SI]-Bolton Hunter CCK-8 to CCK-receptors in guinea pig cortical membranes (CCK-B) or the rat pancreas (CCK-A).

(53) and dimethylamino (54) [67]. Anilines (53) and (54) retained the in vitro binding profile of the previous analogues and had comparable potency in the pentagastrin-induced gastric acid secretion model in rats following i.v. dosing (Table 2.8). Moreover these derivatives had much improved in vivo activity in dogs when dosed orally. Compounds (53) and (54) were equipotent in the dog and an EDso value of 0.01 mg/kg (21 nmol/kg) was reported for

CCK-B ANTAGONISTS

56

Table 2.6. RECEPTOR BINDING AFFINITIES O F 5-CYCLOALKYLAMINO1,4-BENZODIAZEPINES

Compd.

R

R’

Stereo

CCK-B

CCK-A

Me

3R,S

2300

> 3000

nPr

3 R, S,2’SR

41

> 3000

nPr

3 R. S,2’RS

36

> 3000

nPr

3R.S.2’R.Y

54

2090

nPr

3R.S.2’RS

nPr

3R.S.2’RS

15

1510

Me

3 R. S,2’RS

26

3540

Me

3 R ,S.2’RS

12

1390

1.5

3090

alC,o represents the concentration producing half-maximal inhibition of specific binding of [1251]-BoltonHunter CCK-8 to CCK-receptors in guinea pig cortical membranes (CCK-B) or the rat pancreas (CCK-A).

M.S. CHAMBERS A N D S.R. FLETCHER

57

Table 2.7. COMPARISON O F T H E IN VITRO A N D IN VIVO PROFILE OF YM022 A N D L-365,260

Ki ( n M ) clnrlpcl.

CCK-B"

CCK-A/'

A/B

EDso (nniol/kg)"

(50) YM022 ( I ) L-365,260

0.068 19

63 5400

926 284

7.8 4230

"Ki value determined for the inhibition of["51]-CCK-8 binding to gastriniCCK-B receptors in mouse brain. 'Ki value determined for the inhibition of ['H]-L-364.718 binding to CCK-A receptors in rat pancreatic tissue. 'ED5" value obtained using the pentagastrin-induced gastric acid secretion model in anaesthetized rats following i.v. dosing.

Table 2.8. IN VITRO A N D IN VIVO PROFILES OF T H E YAMANOUCHI I,4-BENZODIAZEPINES

(50) YM022 (51) (52) (53) YF476 (54)

0.1 I 0.52 0.44 0.10 0.20

I46 Ill 470 502 I13

1327 213 I068 5020 565

7.8 5.7 16 8.0 II

"1C5,,value for displacement of ["'I]-CCK-8 from gastrin/CCK-B receptors from rat brain. 'IC5,, value for displacement of [jH]-L-364,7 18 from CCK-A receptors from rat pancreas. 'Inhibition of pentagastrin-induced gastric acid secretion in anaesthetized rats following i.v. dosing.

Table 2.9. INHIBITION O F PENTAGASTRIN-INDUCED GASTRIC ACID SECRETION IN HEIDENHAIN POUCH DOGS

Compd.

(50) Y M022

(52) (53)YF476 (54)

Inlrihition in Heidenlzuin poucli ciog.s,f~1111111~i~igp. (1. do.riiig

72'K at 3.0 pnolikg EDjo = 1.9 pmol/kg 97'5, at 3.0 ,uniol/kg 65'5, at 0.03 /mol/kg ED50 = 0.021 pmolikg 77% at 0.03 pmol/kg

Inlrihition in Heidiwliuinpoucli dogs ,followingi. v. dosing EDso = 0.026 pmol/kg Not determined ED5,) = 0.018 pnol/kg ED5"= 0.014 pmol/kg

58

CCK-B ANTAGONISTS

(53), approximately 90-fold more potent than YM022 (Table 2.9). Both compounds displayed good duration of action, maintaining complete inhibition of acid secretion for more than 6 h after oral administration at 100 nmol/ kg. The EDso values of the anilines (53) and (54), following i.v. dosing in the dog, differed only slightly from those obtained orally, indicating excellent oral bioavailability in dog. Following consideration of their physico-chemical properties compound (53) was chosen as a development candidate, and is reported to be undergoing clinical investigation for the treatment of gastro-oesophagal reflux disease. A series of potent CCK-B antagonists based on a 1,5-benzodiazepin-2,4dione skeleton possessing a C-3 ureido [68] or carbamate substituent have been identified by workers at Glaxo [69, 701. Sterically large groups at N-1 are important for achieving high CCK-B receptor affinity and good selectivity over the CCK-A receptor, and 1-adamantylmethyl was found to be optimal [71]. The N-5 phenyl group present in (55) may be replaced with bulky alkyl groups such as cyclohexyl(56) and 3-methylbutyl(58) and high CCKB affinity and subtype selectivity is retained [72] (Table 2.20). Generally, for those compounds unsubstituted on the benzofused ring, the ureas have higher CCK-B affinity and improved subtype selectivity compared to the carbaTable 2.10. CCK-B AND CCK-A AFFINITIES OF THE GLAXO

1,5-BENZODIAZEPINEDIONES

pKi

Compd.a

R

X

CCK-Bh

CCK-A'

A/B

(55) (56)

Ph Cyclohexyl Cyclohexyl 3-Methylbutyl

NH NH

8.64 8.62 7.37 8.86

6.15 6.25 5.96 6.54

309 234 26 209

(57) (58)

0

NH

"Compounds are racemic at C-3. bpKi value measured by displacement of [3H]-CCK-8Sbound on guinea pig brain CCK-B receptors. 'pKi value measured by displacement of [3H]-CCK-8S bound on rat pancreas CCK-A receptors.

M.S. CHAMBERS A N D S.R. FLETCHER

59

mates. As was found earlier in the 1,4-benzodiazepine series, resolution of the stereogenic centre at C-3 is crucial for optimum CCK-B subtype selectivity [73, 741. The best compound from this series, the N-1 (I-adamanty1)methyl analogue GV150013X (59), was found to antagonize CCK-4-induced contractions in guinea-pig ileum longitudinal myenteric plexus (GPILMP) with a pKB value of 8.9 [75].In guinea-pig gall bladder tissue GVI 50013X antagonized CCK-8s-induced contractions with a pKB of 5.8. These data correspond well with the radioligand binding data obtained in guinea pig cortex for CCK-B (pKi 9.15) and rat pancreas for CCK-A (pKi 5.83) [75]. GV150013X was tested in several animal models of anxiety including the mouse lightldark box and the marmoset 'human' threat test and was shown to have potent and long lasting anxiolytic activity [76]. An EDSoof 0.05 pglkg p.0. was observed in the mouse model, and the duration of action (6 h at 0.3 pglkg p.0.) was found to be longer than other compounds tested at equieffective doses (e.g. PD 134308, <2 h at 0.3 pg/kg p.0.; diazepam, < 4 h at 1 mglkg p.0.). In view of the in vitro and in vivo profile, GVI 50013X was selected for exploratory development in the treatment of panic attacks and anxiety. In order to improve the physico-chemical properties of the 1,5-benzodiazepines a morpholinoethyl substituent was introduced at the N-5 position to give GV I9 1 869X (60).This derivative was reported to have improved solubility compared to GV150013X and be a potent and selective CCK-B antagonist (CCK-B pKi 9.4, human temporal cortex; CCK-A pKi 5.7, rat pancreas) [77]. GV 191869X demonstrated dose-dependent anxiolytic activity in both the mouse lightldark box and in the marmoset 'human threat' test models. In the mouse an EDso value of 0.002 pglkg was calculated. In both models GVI 9 1869X maintained a significant effect in the dose range 0.01 pg/kg to 10 pgikg [77]. In view of the impressive in vitvo and in vivo profile and the wide dose range of anxiolytic activity demonstrated in animal models GV191869X would appear to be an attractive candidate to test the hypothesis that CCK-B antagonists are anxiolytic in man. Researchers at Glaxo working on the gastrin antagonist programme have further utilized the 1,5-benzodiazepin-2,4-dioneframework to identify

60

CCK-B ANTAGONISTS

GR199114X (61), a potent, selective and orally bioavailable CCK-B/gastrin receptor antagonist [78]. The N-5 phenyl-l,5-benzodiazepine(62) was identified early in the programme as being more efficacious than L-365,260 at blocking pentagastrin-stimulated gastric acid secretion in isolated rat gastric mucosa. Fluorination of the N-5 and urea phenyl rings produced compound (63) which has a similar in vitro profile to (62) (Tuble2. l l ) ,but much improved oral bioavailability (F, 68% rat). However, the difluorinated derivative (63) has decreased potency and selectivity at the cloned human CCKB [79] and human CCK-A [80] receptors. In parallel with findings in the 1,4-benzodiazepine series [57], saturation of the N-5 phenyl ring to give the cyclohexane analogue resulted in an increase in CCK-B affinity. Resolution Table 2.11, FUNCTIONAL ACTIVITY AND BINDING AFFINITY O F THE GLAXO

1,5-BENZODIAZEPINEDIONES

Coinpd.

R

X

RGM"

GPP

CCK-B'

CCK-A"

(1) L-365,260 (+ /-)-(62) (+/-)-(63)

Ph

H F

7.6 8.5 8.6

1.7 7.3 6.8

1.9

7.3

8.8

7. I

"pKBvalue for blocking pentagastrin-stimulated acid secretion from isolated rat gastric mucosa (RGM).bpKBvalue for blocking pentagastrin-stimulated acid secretion from guinea pig isolated ileum longitudinal muscle-myenteric plexus (GPI). 'pKi value determined at the human CCK-B receptor, isolated from a human temporal cortex cDNA library and stably transfected into a HeLa cell line. dpKi value determined at the human CCK-A receptor, isolated from a human gallbladder cDNA library and transiently transfected into a COSM6 cell line.

61

M.S. CHAMBERS AND S.R. FLETCHER

of the racemic material provided the active enantiomer (6 1, GVI 99 1 14X) which was found to have S-stereochemistry at C-3. This compound has good affinity and selectivity for the CCK-B receptor (T'ble2. I I ) , is 23% orally bioavailable in rat and has good activity in vivo, inhibiting gastric acid secretion in the pentagastrin-stimulated gastric fistula rat model when administered either i.v. (60% inhibition at 0.3 mg/kg) or i.g. (intra gastric; 67% inhibition at 0.3 mgikg). The Shionogi group have developed a series of symmetrical IJ-benzodiazepin-2,Cdione CCK-B antagonists. Such compounds are of course achiral and therefore, unlike the unsymmetrical 1,5- and 1,4-benzodiazepines, require no optical resolution or stereoselective synthesis. Symmetrical 1 5 benzodiazepines, reported by Glaxo [72], bearing an unsubstituted or metuTable 2.12. CCK-B A N D CCK-A RECEPTOR AFFlNlTIES OF THE GLAXO SYMMETRICAL 1,s-BENZODIAZEPINES

- __

R

pKi

R'

CCK-B'

CCK-A~

A/B

H

8.6

7.2

26

H

7.8

6.6

11

N Me?

8.6

7. I

32

yMe Me ____

~

~~

.'lCi,, value for displacement of ['HI-CCK-gS bound on guinea pig brain CCK-B receptors. hlCio value for displacement of ['H]-CCK-8S bound on rat pancreas CCK-A receptors.

62

CCK-B ANTAGONISTS

(68) R =

'y\N HN.~

(69) R = SCH2C02Et (70) R = SCH2C02H

dimethylamino substituted phenylurea ring and bulky alkyl substituents at N-1 and N-5 for example (64)-(67), had either relatively low affinity for both CCK-A and CCK-B receptors or relatively poor CCK-B subtype selectivity (Table 2.12). Shionogi found the introduction of a carbonylmethyl substituent at N-1 and N-5 (such as cyclopropylcarbonylmethyl) and a hydrophilic group on the phenylurea ring (such as carboxyl) provided potent and selective CCK-B receptor antagonists (Table 2.13) [8 13. The tetrazolyl derivaTable 2.13. CCK-B A N D CCK-A RECEPTOR AFFINITIES A N D INHIBITORY EFFECT ON GASTIC ACID SECRETION FOR THE SHIONOGI SYMMETRICAL 1,5-BENZODIAZEPINES Gastric acid inhibition''

Compd. (68) (69) (70) (1)

L-365.260 (501 YM022

CCK-B 1

6 2 16

0.89

i.d. (nig/kg)

CCK-A

A/B

1300 460 500 14100

1300 71 250 909

0.22 0.06 0.20 0.104

15

84

0.107

"ICs0represents the concentration producing half maximal inhibition of binding of ['HI-CCK8s to CCK-receptors in mouse cortical membranes (CCK-B) or the mouse pancreas (CCK-A). hInhibition of pentagastrin-induced gastric acid secretion in anaesthetized rats following intraduodenal (id.) dosing.

63

M.S. CHAMBERS AND S.R. FLETCHER

tive (68) has high CCK-B receptor subtype affinity and selectivity, but in vivo the optimum compound from the series was the ester (69) which demonstrated potent inhibition of pentagastrin-induced gastric acid secretion in anaesthetized rats with an EDs0 value of 0.06 mg/kg i.d. In fact the ester (69) was more effective in this assay than either L-365,260 or YM022, the latter of which has > 20-fold higher CCK-B affinity in vitro. BENZAZEPINES

Using L-365,260 as a starting point, Pfizer selected a 5-phenyl-3-ureidobenzazepin-2-one skeleton to provide a mimic of the important structural features for CCK-B receptor recognition. The Pfizer group produced two compounds for advanced evaluation in anxiolytic studies, CP-212,454 (7 1) [82] and CP-310,713 (72) [83]. However, CP-212,454 has poor oral bioavailabil,CONHtBu

(71) R = Ph, X = CI; CP-212,454

(72) R = Cyclohexyl, X = C 0 2 H CP-310,713

ity in the rat and dog, possibly as a result of its low aqueous solubility, and attempts to provide a suitable formulation for clinical studies proved unsuccessful [84]. The focus of the programme thus turned to identifying a more water-soluble derivative, and the potassium salt of the carboxylic acid derivTable 2.14. CCK- RECEPTOR AFFINITY AND AQUEOUS SOLUBILITY OF THE PFIZER BENZAZEPINES Aq.solubility (mg/mL)

Compd.

CCK-B"

CCK-A~

(71) CP-212,454 (72) CP-3 10,713

0.48

I76

367

0.10

1400

14000

Selectivity

0.0002 4.8'

"Binding affinity for the CCK-B receptor in guinea pig cortex using ['251]-Bolton-HunterCCK-8 as the radioligand. bBinding affinity for the CCK-A receptor in guinea pig pancreas using ['**1]-Bolton-HunterCCK-8 as the radioligand."Solubilityof the potassium salt in unbuffered water.

64

CCK-B ANTAGONISTS

ative CP-3 10,713 has much improved aqueous solubility compared to CP212,454 (Table 2.14). Despite displaying very potent activity in blocking CCK-4 induced cardiovascular changes in guinea-pigs, in a CCK-4 induced panic attack model in monkey high doses of CP-310,713 were required for efficacy. This discrepancy between the concentrations of drug required for efficacy in the animal models was attributed to low CNS penetration of CP310,713. In view of the efficacy and bioavailability problems Pfizer have reportedly terminated the project [84]. PEPTOIDS

Parke-Davis used the peptide tetragastrin (CCK-4) as a starting point for the rational design of non-peptide ligands for the CCK-B receptor. The resultant structures were designated ‘dipeptoids’ [85,861. Having established that the Trp and Phe residues were necessary to impart micromolar affinity for the CCK-B receptor [87], a strategy was adopted to optimise the N-terminus and then incorporate the resultant substituent into the molecule during the C-terminus optimisation procedure. This approach depended on the structure-activity relationship (SAR) being additive, which had been proposed because these structures have sufficient flexibility to allow the C-terminus side-chain to find its optimal energy minima at the CCK-B receptor independent of the N-terminus. Examination of the N-terminus SAR revealed that cycloalkyl or bulky substituents were preferred [87] and that the 2-adamantyl group was optimum. Following incorporation of the N-terminal(2-adamanty1oxy)carbonyl group into the general structure, an SAR study of the C-terminus was carried out. The preferred compound to emerge from this study was PD134308 (CI988) [88]. CI-988 (2) demonstrated potent anxiolytic effects in several animal models of anxiety, including the rat elevated plus maze, marmoset ‘human threat’ test and the rat social interaction models. In the mouse black/white box assay CI-988 was active following either subcutaneous (s.c.) or oral dosing with a MED of 100 ng/kg S.C. [89]. In view of this anxiolytic profile, CI988 was evaluated in the clinic as a potential anxiolytic agent as discussed earlier. The lack of anxiolytic effects in humans could possibly be due to the relatively poor brain penetration of CI-988 [47] or the low systemic exposure in man [44]. The poor oral bioavailability (1-3‘%,) in rat [90], monkey [91, 921 and humans also highlighted the need for a second-generation dipeptoid with an improved pharmacokinetic profile. The low bioavailability was attributed to poor absorption and efficient hepatic extraction and compounds were sought with a lower molecular weight than CI-988 (MW 614) since it was envisaged this would improve the absorption and brain penetration. An-

M.S. CHAMBERS A N D S.R. FLETCHER

65

other issue to arise with CI-988 was that comparison of binding affinities obtained using antagonist and agonist radioligands suggested the compound was a partial agonist at the CCK-A receptor. Functional efficacy was then demonstrated in stimulation of amylase secretion in rat pancreatic acinar cells [93]. Subsequently, other workers reported (3-988 to behave as a CCK-B/gastrin agonist in the isolated vascularly perfused rat stomach [94]. Workers at Parke-Davis have undertaken studies to address these issues. Following a structural analysis of (21-988 it was observed that the central portion containing the indole moiety was essential for high affinity [95], although the C-terminus could be manipulated and CCK-B receptor affinity maintained. Following a QSAR analysis of phenyl substitution the parafluorophenyl dipeptoid (73) was identified as a higher affinity analogue than CI-988 with three orders of magnitude selectivity for the CCK-B subtype [96] (Tuble2.15).The S-configuration at the substituted phenethylamide centre was necessary for the optimal in vifro profile. A more extensive programme of C-terminus modification ultimately produced the (2-hydroxycyclohexyl)amino analogue CI-1015 (74) lacking the carboxylic acid group previously thought to be crucial for optimum potency. CI-I0 15 in v i v o produced a potent inhibition of pentagastrin-induced gastric acid secretion in Ghosh and Schild rats, comparable to CI-988 (Table 2.15). Following oral administration CI-1015 showed an anxiolytic-like profile in the elevated rat X-maze (MED 1.0 pg/kg) and in the mouse light/dark box test (MED 10 pg/kg). CI-1015 demonstrated a significant improvement in oral bioavailability compared to CI-988, and an optimal value of 28% in rat was achieved when (21-1015 was dosed as a solution in 50% (w/v) (hydroxypropy1)-P-cyclodextrin. Using an ex-vivo binding measurement the bloodbrain permeability of the more lipophilic CI-1015 was found to be significantly enhanced relative to CI-988 and compared favourably with L-

(74) CI-101s

66

CCK-B ANTAGONISTS

Table 2.15. IN VITRO AND IN VZVO ACTIVITY OF THE PARKE-DAVIS DIPEPTOIDS ICJO(~W"

Anti-secretory activity

Compd.

CCK-B

CCK-A

A/B

EDtidmdkgl

(2) CI-988 (74) CI-1015 (73)

1.7 3.0 0.08

4300 2900 75

2529 967 936

0.2 0.3

"IC50 represents the concentration producing half-maximal inhibition of specific binding of ['251]-BoltonHunter CCK-8 to CCK-receptors in the mouse cerebral cortex (CCK-B) or the rat pancreas (CCK-A). bED50value for inhibition of pentagastrin-induced gastric acid secretion in Ghosh and Schild rats following i.v. dosing.

365,260 (Table 2.26). Compound CI-1015 was chosen for clinical evaluation on the basis of the improved pharmacokinetic profile and enhanced brain penetration, and ought to be a better tool than CI-988 with which to evaluate the anxiolytic potential of CCK-B receptor antagonists. Table 2.16. BRAIN LEVELS OF CI-988, CI-1015 AND L-365,260 DETERMINED BY EX VIVO BINDING FOLLOWING Z.K DOSING brain level (pmol/forehrain)ajier injection Compd."

5 min

10 min

20 min

(1) L-365,260 (2) CI-9Sb (74) (21-1015

115

61 BLDC 154

33 8 166

10 177

aL-365,260and CI-1015 were dosed i.v. at 1 rng/kg. bCI-988 was dosed i.v. at 10 mg/kg. 'Below the level of detection (approx. Ipmol/forebrain)

HYBRIDS

Parke-Davis have developed a quinazolinone series of CCK-B antagonists based on two important pharmacophores from existing series of CCK-B ligands. Combination of the phenylurea moiety of the 1,4-benzodiazepines [54]with a quinazolinone core developed by Eli Lilly [97] produced a novel class of selective and orally active CCK-B antagonists (Figure 2.2). Early work in this series suggested that the length of the linking group connecting the urea portion and the quinazolinone skeleton was a critical determinant of CCK-B receptor affinity, with a single spacer group present in (76) being

M.S. CHAMBERS A N D S.R. FLETCHER

61

optimum (Table 2.17) [98]. Replacing the methylene linker with an amino group (78) produced a dramatic increase in CCK-B affinity and subtype selectivity [99]. Following a programme of optimization a t the N-3 position it was found that 3-isopropoxyphenyl was the preferred substituent for binding and selectivity.This substituent was also optimal in the original quinazoh o n e series developed by Lilly [97]. An SAR study of the urea terminus revealed that electron-withdrawing groups at the meta-position of the phenylurea ring further enhanced CCK-B affinity (Table 2.18) [99]. Representative compounds from this hybrid series were evaluated for functional activity in virro, using the guinea-pig stomach strip assay [loo]. All compounds demonstrated full CCK-B/gastrin antagonist activity, reversibly antagonizing pentagastrin-evoked increases in intracellular calcium levels. The terr-butyl ester (79) and the dimethylamino-substituted quinazolinone (82) were evaluated in vivo in the rat elevated X-maze test [loll. Both compounds displayed dose-dependent anxiolytic activity with an MED of 1

Figure 2.2. Design of the Parke-Duvis quinazolinones.

68

CCK-B ANTAGONISTS Table 2.17. EFFECT O F THE LINKING GROUP ON THE CCK-B AND CCK-A RECEPTOR AFFINITY IN THE PARKE-DAVIS QUINAZOLINONE SERIES

Compcl.

X

CCK-B

CCK-A

A/B

(75) (76) (77) (78)

-

> 1000

> 1000

-

I40 585 14

980 1630 3430

CH2 CHzCHz NH

7 2.8 24 5

"1CSorepresents the concentration producing half-maximal inhibition of specific binding of ['251]-Bolton Hunter CCK-8 to CCK-receptors in the mouse cerebral cortex (CCK-B) or the rat pancreas (CCK-A). Table 2.18. CCK-B AND CCK-A RECEPTOR AFFINITIES O F THE PARKE-DAVIS META-SUBSTITUTED QUINAZOLINONES

d

Cotnpd

R

R'

CCK-B

CCK-A

A/B

(79) (80)

iPrO iPrO iPrO NMe2

COztBu NO2 CN CN

2960 2560 5350 7100

1557 2560 4458 507

-

-

1.9 I .o I .2 14 9.3b

(81) (82)

(83)

aIC50represents the concentration producing half-maximal inhibition of specific binding of ['251]-Bolton Hunter CCK-8 to CCK-receptors in the mouse cerebral cortex (CCK-B) or the rat pancreas (CCK-A). blCso for inhibition of ['2SI]-labelledCCK-8s binding with mouse brain membranes. See Reference 97.

M.S. CHAMBERS A N D S.R. FLETCHER

69

H

mg/kg following oral administration. However, in the same assay, the Lilly quinazolinone (83), which has 5-fold lower affinity than the ester (79) and similar affinity to the nitrile (82), was found to be 10-fold more potent ( M E D 0.1 mg/kg) than both hybrid compounds. The ester had negligible oral bioavailability in the rat (F, < 5%) whereas the nitrile was estimated to be 22% bioavailable following oral administration in dimethylacetamide/ PEG400i 5%) dextrose water ( 1 0:40:50). Scientists at Daiichi Pharmaceutical Co. identified a series of phenoxyacetic acid derivatives as highly potent gastrin/CCK-B receptor antagonists [ 1021. Using molecular mechanics calculations to estimate the stable conformations of the potent CCK-B antagonists YM022 (50) [lo31 and RP 72540 (84) [I 041, several common structural features were identified. In particular it was considered that the C-5 phenyl ring of the benzodiazepine YM022 may overlay the methoxyl moiety on the N-phenyl ring of the ureidoacetamide RP 72540. Aware that in the 1,4-benzodiazepine series the C-5 phenyl ring of L-365,260 could be replaced with more bulky groups such as homopiperidinyl and azabicyclo[3.2.2]nonanylto provide a significant increase in CCK-B affinity and selectivity [60], Daiichi researchers speculated that the methoxyl substituent of RP 72540 could be replaced with larger groups to produce higher affinity analogues. This approach led to the phenoxyacetamides DZ-35 14 (85) and DA-3797 (86), both of which have high affinity for the human gastriniCCK-B receptor and > 200-fold selectivity over the human CCK-A subtype (Tuble 2.19). Introduction of oxygenated substituents onto the phenyl-

(84)RF' 72540

70

CCK-B ANTAGONISTS Table 2.19. CCK-B AND CCK-A RECEPTOR AFFINITIES OF THE DAIICHI PHENOXYACETIC ACID DERIVATIVES

GO lnW Isomer"

Compd. (84) RP72540 (50) YM022 (85) DZ-3514 (86) DA-3797 (87) DA-3934 (88)

R

gastrinh

CCK-A"

A/B

1.6

> 1000

> 625

0.33

20

61

3-

Me

0.8

I78

223

2-

Me

0.9

210

233

2-

CH2C02H

0.4

877

2193

2-

CH2C02Me

1.1

660

600

aPosition of attatchment of the oxyacetamide substituent. 'ICSo value for displacement of ['251]gastrin binding to human gastrin receptor. 'ICSOvalue for displacement of ['251]CCK-8 binding to human CCK-A receptor.

ureido ring in the phenoxyacetic acid series afforded the carboxylic acid derivative DA-3934 (87) and the corresponding methyl ester (88), both of which have high affinity for the human gastrin/CCK-B receptor and high selectivity Table 2.20. ANTI-SECRETORY ACTIVITY O F THE YAMANOUCHI PHENOXYACETIC ACID DERIVATIVES

Compd.

i.d. route (mg/kg)

i. v. route ( @ k g )

(50) YM022 (87) DA-3934 (88)

1.5 5.2 1.9

1.3 12.5 NT'

"EDSovalue for inhibition of pentagastrin-induced gastric acid secretion in anaesthetized rats. 'Not tested.

M.S. CHAMBERS A N D S.R.FLETCHER

71

over the human CCK-A receptor [105]. DA-3934 and the ester (88) were found to inhibit pentagastrin-induced gastric acid secretion in anaesthetized rats in a dose-dependent manner when administered either i.d. or i.v. (Table 2.20). EDso values of 5.2 mg/kg i.d. and 12.5 ,ug/kg i.v. were reported for DA-3934. The ester (88) had 3-fold higher in vivo activity compared to DA3934 despite having lower affinity for the gastrin/CCK-B receptor, and had similar anti-secretory activity compared toYM022 when dosed i.d. It was proposed that the ester functions as a pro-drug. U REIDOMETHYLCARBAMOYLPHENY LKETONES

A series of ureidomethylcarbamoylphenylketones have been developed by Shionogi [ 1061 as selective CCK-B receptor antagonists for potential use as inhibitors of gastric acid secretion. This series of compounds, exemplified by the urea (89), was derived by cleavage of the C-3/N-4 bond of the 1,4benzodiazepine L-365,260 (Figure 2.3). An SAR study revealed that metasubstitution on the phenylurea ring offered increased potency compared to paw-substitution. In terms of CCK-B subtype selectivity the optimal phenylurea and carboxamido N-substituents were carboxylic acid and tert-butoxycarbonylmethyl respectively. The highlight of the series was S-0509 (90), which is reportedly 120-fold selective for CCK-B receptors over CCK-A.

(1) L-365.260

a

d3

(89)

Figure 2.3. Design of the Shionogi ureidomethylcarbarnoylphenylketonesby C-3/N-4bond cleavage of L-365,26O.

12

CCK-B ANTAGONISTS

(90) R= CO2H $0509

(91)R=

qN-\ N’ H

S-0509 was shown to be a potent inhibitor of gastric acid secretion induced by pentagastrin in anaesthetized rats with an ED50 value of 0.014 mg/kg id. The tetrazole (91) had the most impressive in vitro profile but in vivo displayed poor potency in the gastric acid secretion model (Table 2.21). When administered i.v. S-0509 inhibited pentagastrin-stimulated acid secretion with an ED50 of 0.001 mg/kg, some 200-fold more potent than L-365,260 (ED50 3.0 mg/kg). In addition S-0509 inhibited basal gastric acid secretion (ED503.0mg/kg) when given orally 30 min prior to pylorus ligation, whereas L-365,260 is reported to have no significant effect on basal acid secretion in the rat [107]. It was suggested that since L-365,260 and S-0509 have comparable affinity in vitro, the increase in in vivo efficacy of S-0509 may arise from this compound having a chain structure rather than the cyclised structure of L-365,260. The selectivity of S-0509 for the CCK-B receptor over other receptors was not disclosed. The presence of a carboxylic Table 2.21. CCK-B AND CCK-A RECEPTOR AFFINITIES A N D INHIBITORY EFFECT ON GASTIC ACID SECRETION FOR THE SHIONOGI UREIDOMETHYLCARBAMOYLPHENYLKETONES Ic.50( n W “

Gastrir acid inhibition

Compd.

CCK-B

CCK-A

A/B

(90) S-0509 (91) (1) L-365.260

24 8 16

2813 1650 14100

120 206 909

EDSOi. d. (mg/kg) 0.014 >0.3 0.104

‘ICso represents the concentrationproducing half maximal inhibition of [3H]-CCK-8Sto CCKreceptors in mouse cortical membranes (CCK-B) or the mouse pancreas (CCK-A). bInhibition of pentagastrin-induced gastric acid secretion in anaesthetizedrats followingi d . administration

M.S. CHAMBERS A N D S.R. FLETCHER

73

acid moiety on the phenylurea ring would be expected to suppress penetration of the blood-brain barrier, and indeed this was confirmed using the rat tail flick test for enhancement of morphine analgesia [lo81 in which S-0509 had a negligible effect. Thus S-0509 has good selectivity for the peripheral effects of gastrin antagonism compared to the behavioral effects mediated by CCK-B receptors in the CNS. No pharmacokinetic data was reported for S-0509. D I BENZOBICY CL0[2.2.2]OCTAN E A N D BICYCLOHETEROA ROM ATIC DERIVATIVES

Scientists at the James Black Foundation have designed a series of selective CCK-B receptor antagonists based on a consideration of the topography of CCK-4. Rigid structures were sought as replacements for the peptide backbone of tetragastrin which would retain the desired stereoelectronic features obtained from molecular mechanics calculations and fluorescence studies [ 1091. Incorporation of a dibenzobicyclo[2.2.2]octane(BCO) skeleton within the CCK-4 framework produced a series of compounds with submicromolar affinity at the CCK-B receptor and > 30-fold selectivity over the CCK-A receptor [ 1091.This class of compounds is exemplified by the proline derivative (92) with a pKi of 7.4 at the CCK-B receptor and 500-fold selectivity over the CCK-A receptor (Table 2.22). Manipulation of the acidic terminus and replacement of the proline residue with phenylalanine produced the 3,5-dicarboxyanilide (93) with enhanced CCK-B affinity and subtype selectivity [l lo]. A structure-activity study based on the anilide (93) revealed that a carboxylic acid group is crucial for high CCK-B affinity and that conversely alTable 2.22. CCK-B A N D CCK-A RECEPTOR AFFINITIES FOR T H E DIBENZOBICYCL0[2.2.2]OCTANE (BCO) A N D BICYCLIC HETEROAROMATIC DERIVATIVES pKi" Con1pd

CCK-B/gastuin

CCK-A

(92) (93) (94) (95) (96) (97)

7.4 8.8 8.4 8.6 9.0 8.3

4.6 5.7 5.7 5.8 5.4 5.8

"pKi value determined for the inhibition of ['251]-Bolton Hunter CCK-8S to CCK-receptors in mouse cortical homogenates (CCK-B) o r the guinea pig pancreas (CCK-A).

CCK-B ANTAGONISTS

74

&

CONHX

X=

*

(93)

x=

(94)

x=

(95)

x=

w

9, a

tering the chain length of the phenylalanine residue is detrimental to binding. The adamantylmethyl group could, however, be replaced with other hydrophobic substituents such as cycloheptylmethyl(94) or 2-naphthylmethyl (95) with no loss of affinity or selectivity. Interestingly, representative compounds from this series showed species-variation when examined in rat and dog models [28]. For example, the anilide (93) was reasonably potent in antagonizing pentagastrin-stimulated gastric acid secretion when administered i.v. to Ghosh and Schild anaesthetized rats, but was 700-fold less active when given i.v. to chronic gastric fistula dogs (Table2.23). This discrepancy prompted further modification of the series to identify a replacement for the BCO framework. This search resulted in a series of ortho-disubstituted bicyclic heteroaromatic derivatives [ 1 1 11 which maintained the activity and selectivity demonstrated by (93), but gave more consistent results in vivo. For example, the indole (96) was found to be as potent as the BCO analogue (93) in the pentagastrin-stimulated gastric acid secretion model in anaesthetized rats, but more importantly, had similar potency in the rat and dog gastric acid secretion models (Table2.23). Compounds (96) and (97) were demonstrated to have increased potency in the acid secretion models compared to L-365,260. A definitive reason for the discrepancy observed in vivo with the BCO-derivatives and not the heteroaromatic bicyclic compounds was not provided, although pharmacokinetic factors or interspecies variation in

75

M.S. CHAMBERS A N D S.R. FLETCHER Table 2.23. INHIBITORY EFFECT ON PENTAGASTRIN-STIMULATED GASTRIC ACID SECRETION BY THE DIBENZOBlCYCLO[2.2.2]OCTANE A N D BICYCLIC HETEROAROMATIC DERIVATIVES Gastric fistula dog

Ghosh and Schild rai Compd.

dose"

% inhibitionh

dose"

%inhibitionc

(93) (96) (97) ( 1 ) L-365.260

0.025 0.025 0.025 1 .o

79 97 61 46

17.5 0.1 0.1 4.0

49 79 83 70

"Compound dose in pmol/kg. Materials dosed by i. bolus. bPeak percentage inhibition relative to an infused. submaximal acid secretory dose of 0.1 pg/kg/min of pentagastrin. 'Peak percentage inhibition relative to an infused, submaximal acid secretory dose of pentagastrin. This dose varies for each animal. 11.

CCK-B/gastrin receptors was proposed as a possible explanation. Pharmacokinetic data for these compounds is not disclosed, but it is known that there is interspecies variation in CCK-B/gastrin receptors which, whilst having no effect on agonist affinities, do influence the affinity of non-peptide antagonists such as L-363,260. In particular Beinborn et af. have demonstrated that it is the valine residue at position 319 in the human receptor which is the critical binding determinant of non-peptide antagonist activity [I 121. The rat receptor also has valine at this crucial position. In the canine receptor (which has lower affinity for L-365,260 compared to human and rat receptors) it is the leucine residue at position 355 which is the critical binding determinant. Indeed, replacing valine 319 with a leucine residue decreases the affinity for L-365,260 some 20-fold relative to the wild type human receptor, whilst increasing the affinity for the benzodiazepine-based CCK-A antagonist L-364,718 [112].

(96)X = CH (97)X= N

76

CCK-B ANTAGONISTS

CONCLUSION AND PROSPECTS Extensive studies had been carried out, and still continue, in an effort to evaluate the potential of CCK-B antagonists in the control of anxiety and gastric acid secretion. Clinical data for compounds evaluated to date suggest a lack of efficacy in anxiety and that a reduced effect on gastric acid secretion is achieved compared with H2-antagonists. However, the weak CNS and peripheral effects observed may be attributable to deficiencies in the compounds evaluated. In addition, development of positron-emission tomography (PET) [113] and other imaging techniques has now advanced and complete receptor occupancy may be ensured in future studies. Evaluation of the potential of CCK-B antagonists in other therapeutic areas has continued. Recent reports suggest a role for CCK-B antagonists in the treatment of schizophrenia [I 14-1 161 and in modulation of opioid analgesia [I 171 whilst studies with CCK-B agonists imply a role in memory disorders [118]. The discovery and development of second-generation compounds will add impetus to these studies. REFERENCES 1 Mutt,V. and Jorpes. JE. (1968) Eur. J. Biochem. 6, 156-162. 2 Rehfeld, J.F. and Nielsen, F.C. (1995) in Cholecystokinin and Anxiety: from Neuron to Behaviour (Bradwejn, J. and Vasar, E., eds.) pp.33-56, Springer Verlag-R.G. Landes Co., Austin. 3 Rehfeld, J.F. (1985) J. Neurochem. 44. 1-10. 4 de Weerth, A.. Pisegna, J.R., Huppi, K. and Wdnk, S.A. (1993) Biochem. Biophys. Res. Commun. 194.81 1-818. 5 Song, I., Brown, D.R., Wiltshire, R.N., Gantz, I., Trent, J.M. and Yamada, T. (1993) Proc. Natl. Acad. Sci. U.S.A. 90,9085-9089. 6 Ito, M.. Matsui, T., Taniguchi, T., Tsukamoto, T.. Murayama, T., Arima, N., Nakata, H.. Chiba,T. and Chihara. K. (1993) J. Biol. Chem. 268, 18300-18305. 7 Lee, Y.M., Beinborn, M., McBride, E.W., Lu, M., Kolakowski, L.F.J. and Kopin, AS. (1993) J. Biol. Chem. 268,8164-8169. 8 Shlik, J.,Vasar, E. and Bradwejn, J. (1997) CNS Drugs 134-52. 9 Moran, T.H., Robinson, P.H., Goldrich, M.S. and McHugh, P.R. (1986) Brain Res. 362, 175-179. 10 DAmato, M., Makovec, F. and Rovati, L.C. (1994) Drug News Perspect. 7.87-95. I I Schiantdrelh, P. (1993) Pharmacol. Rev. 28, 1-9. 12 Lydiard, B.R. (1994) Clin. Chem. 40,3 15-3 18. 13 Hamon, M. (1994) Trends Pharmacol. Sci. 15,36-39. 14 Harro, J., Vasar, E. and Brddwejn, J. (1993) Trends Pharmacol. Sci. 14,244249. 15 Bradwejn, J., Koszycki, D., Payeur, R., Bourin, M. and Borthwick, H. (1992) Am. J. Psychiatry 149,962-964.

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Progress in Medicinal Chemistry - Vol. 37, Edited by ED. King and A.W. Oxford 02000 Elsevier Science B.V. All rights reserved.

3 Application of High-throughput Screening Techniques to Drug Discovery BRIAN cox“,JANEc.DENYER, ALASTAIR BINNIE~, MARY C. DONNELLY“, BRIAN EVANS, DARREN V. S. GREEN, JANE A. LEWIS, TOM H. MANDERd, ANDY T. MERRIT‘I“, MARTIN J. VALLER and STEPHEN P. WATSON. Divisions of Discovery Technology,“Medicinal Sciences and “Bioanalysisand Metabolism. G b x o Wellcome Medicines Research Centre, Gunnels Wood Road, Stevenage, Herts, SGI 2 N x UK. ’Current address: Discovery Technologies, Lead Discovery, Bristol Myers-Squibb Company, Pharmaceutical Research Institute, 5 Research Parkway, Walling$ord, CTO6492-7660, USA. “Current address: EvoTech BioSystems AG, Schnachenhurgalle 114, 22525, Hamburg, Germany.

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APPLICATION OF SOLID PHASE SYNTHESIS TO BEAD BASED SCREENING Beads and bead based synthesis Split-mix library synthesis Tagging and encoding Bead based screening A successful example of bead based screening using differential release

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Bead hosed .screening in a lrwn fiirniat Beud bused screening in solution ussciys

SAMPLE SELECTION Ethnomedical sample selection Chemoinformatics (%enioii?/i,rnrrrric:cin the lrrrtlgenerrrtionprocess Molc.ctilnr ciescriprors Selc.crionmethods l+iiiiiuiion

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APPLICATION OF HTS TECHNIQUES TO DRUG DISCOVERY

BIOASSAY DESIGN Biochemical assays Scintillution proximity u.s.suys Fluorescence-bused uppromhes ELISA and ion eschunge separution u.s.suy.s Cellular functional assays HIGH-THROUGHPUT SCREENS FOR EVALUATING ABSORPTION, DISTRIBUTION. METABOLISM AND EXCRETION (ADME) PROPERTIES Approaches to screening In vitro studies: absorption In vitro studies: metabolism Computational techniques

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HTS AUTOMATION Fully automated screening robotics Rohot arm on truck Enclosure Liquid-hundling instruments Detection instruments Incuhutors Plute input/output buffers ( ‘hotels’) Otlier instrunzents Gerierul utilit~.’function.s Sojilwre

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DATA HANDLING FOR HTS Logistics of HTS data handling Bioassay data analysis: overview Typical stages in the analysis of HTS data Generution ofsanipk records Creation ofthe esperini~ntrilpro,ocol Crecition of’esperiments Iniplementation cflesperinients Rmv dutu ltunciling Quu1it.y control, error review Generution of’output Other outputs

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REFERENCES

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INTRODUCTION The process of uncovering candidate lead compounds through either targeted or diversity-based high-throughput screening (HTS) has undergone

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rapid evolution in recent years with the advent of combinatorial chemistry, new assay technologies, miniaturized formats and fully automated hardware platforms [l-61. It is now possible for tens of thousands of samples to be tested per day in an intensive search for compounds active at disease targets. The traditional approach to prosecuting targets in HTS up to the mid 1990s involved testing crude natural product extracts predominantly on the scale of a few thousand samples per week. The arrival of combinatorial chemistries, such as bead based techniques and the ability to hold and access compounds in large automated stores, has led to a dramatic increase in the diversity and quantity of samples available for screening. Many assay formats have been miniaturized, enabling more efficient, higher throughput and cost-effective screening campaigns to be run routinely. In addition, there have been marked improvements in the capacity, versatility and reliability of screen robots to carry out the primary screening process. This chapter will overview the key technologies, tactics and processes which enable the discovery of compounds active at disease targets through HTS in Glaxo Wellcome (GW). The impact of high-throughput techniques in the downstream progression of hits from an HTS campaign through to the optimization of lead series will also be discussed (e.g. array synthesis and high-throughput biometabolism assays). Recent developments in the industry will be highlighted, and future directions discussed. APPLICATION OF SOLID PHASE SYNTHESIS TO BEAD BASED SCREENING The introduction of automation and the increasing levels of miniaturization in the high-throughput screening arena at the start of the 1990s provided the impetus for the development of combinatorial chemistry in drug discovery [7,7a]. At Glaxo (now Glaxo Wellcome), as in many other pharmaceutical companies, an investment was made into combinatorial chemistry techniques, equipment and the generation of large libraries to meet this need. The belief was that large diversity libraries would give excellent coverage of pharmacological ‘space’ and hence provide active compounds against most, if not all, new targets being developed for high-throughput screening. Moreover, these compounds would be provided by combinatorial chemistry, and as such would contain many attributes desired for lead compounds [8]. This might include a desirable molecular weight, good hydrophobicity profile, and pharmacophorically biased functionality (so called privileged structures based on past experience, for example benzodiazepines) which could all be delivered by careful selection of the monomers and chemistry. Addi-

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tionally, the fact that the compounds were generated as part of a combinatorial library would mean good tractability as lead compounds. Many companies developed in-house automation approaches to prepare these large libraries. Commercial equipment such as Advanced Chemtech (ACT) equipment also became commonplace in the industry, supporting the preparation of larger solid phase libraries through split-mix approaches. Initially reliant on amidic chemistry, these approaches have now expanded to a wide range of chemistries and functional motifs. Bead based screening (BS) has the capacity to radically transform high throughput compound screening, both in terms of efficiency and throughput. Indeed screening on, or directly from, beads, allied to a suitable tagging/encoding system, is the only effective means of screening which can respond to the remarkable chemical efficiency of split-mix library synthesis. The aim of this section is to broadly overview the principles of bead based library screening, to outline the advantages of bead based library screening and to illustrate the power of BS with a worked example from our own laboratories. This necessarily also involves a brief introduction to the principles of synthesis on resin beads, split-mix library synthesis and tagging/encoding of beads. For the purposes of this section bead based screening will cover those approaches in which the bead based format is key to the screening protocol, this includes both screening of compounds still attached to their bead supports and the screening of compounds cleaved prior to screening. However, it does not include the use of solid phase/bead based methods as simply another means of preparing compounds for conventional screening. Whilst the section is not fully comprehensive and it is acknowledged that there are variants and related methodologies which have not been covered, it is hoped that it will serve as a good overview of the principles and strengths of bead based screening and stimulate readers interest in a rapidly burgeoning area of technology. The limited space available only allows the discussion of broad principles and strategies and some familiarity with the basic concepts of libraries and solid phase synthesis has been assumed. For more technical detail the reader is referred to several excellent recent reviews of the primary literature. BEADS A N D BEAD BASED SYNTHESIS

Initially used to make peptides, but more latterly small molecules, synthesis on resin bead supports is now commonplace [9-121. This form of solid supported synthesis can be used simply as an alternative to the conventional synthesis of compounds in the solution phase, over which it can have many practical and chemical advantages. However, it can also be used in more ver-

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satile ways, such as in the synthesis of large collections of compounds or libraries, where it has many other distinct advantages over conventional solution methods, for example, in the split-mix protocol (see below). Typically beads suitable for synthesis of libraries for bead based screening are derived from cross-linked polystyrene resin and are between 90 and 250pm in diameter (just visible to the naked eye). The number of beads in a given mass or volume of resin is determined by the average bead size, for example for 150pm diameter beads there are ca. 500 beads per milligram of bulk resin. The beaddresin display a linker to which building blocks can be sequentially attached, to effectively grow molecules. When a synthesis is complete cleavage at the linker releases the molecule(s) from the bead@). Beads can be handled in bulk either dry (i.e. free from solvent) or as a slurry in a suitable solvent which facilitates dispensing of beads and transfer between reaction vessels and containers. The quantity of beads dispensed can be determined and controlled either by weighing of dry beads or by transferring aliquots of beads in a suspension of neutral buoyancy in a solvent of suitable density (Figure 3. I ) . Beads suitable for synthesis of libraries for bead based screening typically have a compound loading of between 0.1 and 0.4mmol/ g, given the bead dimensions outlined above, this corresponds to about 1 nanomole of compound per bead for the higher loading.

-

B

A

@-Linker

*Linker-A

+

*Linker-AB

C --C

*Linker-ABC

Cleave

ABC

Figure 3. I . Sequence of solidphase synihesis.

SPLIT-MIX LIBRARY SYNTHESIS

The split-mix approach to library synthesis is now in widespread use throughout the chemical and pharmaceutical communities [9-121. The beauty and attraction of the split-mix approach is that it is synthetically very efficient, allowing the synthesis of very large numbers of compounds for comparatively few chemical steps. All the possible combinations from sets of building blocks can be prepared in a modest number of chemical reactions. Whilst the split-mix protocol is synthetically efficient affording libraries as compound/bead mixtures any one individual bead only ever displays a single compound/combination of building blocks. It is this combination of synthetic efficiency and single compound-single bead nature which bead based screening exploits to such good effect. For the purposes of this chapter split-mix refers to synthesis on resin beads. However, in prin-

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ciple the split-mix approach can be used for any discrete, separable solid support, for example crowns or pins [ 131. Whilst superficially simple, split-mix synthesis exploits chemical and statistical principles in a subtle manner and is probably best illustrated by way of example. Figure 3.2 depicts the split-mix synthesis of a library by sequential coupling of three sets of n chemical building blocks, A, B and C to resin. A bulk resin sample is evenly distributed (i.e. ‘split’) into a number (n) of reaction vessels (RVs). A different building block Ai is coupled to the sample of resin in each of the different RVs. The resin from all of the RVs is then combined (‘mixed’) to give one mass of beads. This mass of beads can be statistically described as e A I -n but any individual bead will carry a single compound derived from the specific building block Ai to which the RV from which it was derived was exposed and can be described as C-Ai. The bead mass is then evenly split again into the RVs, such that each RV now contains the statistical mixture *Al-,, a different building block B is then coupled to the resin in each different RV. In any one RV the beads will all have been exposed to the same building block, e.g. Bj, thus the beads in that RV can be statistically described as e A I-nBjbut any individual bead will bear only one specific A building block and can thus be described as o-AiBj. The resins from each RV are then recombined to give one mass of beads with the statistical description eAI.,,BI.,but with any individual bead displaying a single A and a single B building block and thus described as o-AiBj. The process is then repeated, the statistical mixture is evenly re-distributed into the RVs and the third set of building blocks C added. As before in any one RV. the beads will display a single C building block, e.g. Ck and be statistically represented as eAI-nBl-nCk,but any individual bead will also display a single A and B, i.e. G A ~ B ~ CThe ~ . library can then be either; (a) retained in this form, i.e. as n pools of beads where each pool has a specific known C and mixture of all A and B combinations, i.e. eAI.nBI-nCkr or (b) the contents of the RVs can be pooled again to afford the entire library as one bead mass containing all of the possible combinations of A, B and C, statistically reprewith again any one bead carrying a single building sented by eAl..nB1-nC1.n block combination, C-AiBjCk. The library contains all possible combinations of the building blocks A, B, and C. i.e. n3 compounds. However, each of the n RVs has been used only three times so the total number of chemical transformations is 3n. Thus IOAs, lOBs and lOCs would afford 1,000 compounds for only 30 reactions; this represents the incredible synthetic efficiency of the split-mix protocol, the relative efficiency increasing as n increases. Any number of split-mix cycles can be used (within certain limitations -

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89 0

Starling Bulk Resin

0

Spllt Starling Resin into n RVS

a

a

0

0

0 0

*A,

*4 04

*a,

*A"

0

Add Building blocks A One Disllnct A 10 Each RV

0%

0

0%

0-4

Combins all resin from all RVs

Combine all resin lrom all RVs

Keep Llbrary as n Pools. each wllh a known C but s l l i s l l c d mlxtures of A and 8. or recomblno lo afford a 61n~Ie pool with il slallrtlcal mlxturc 01 all posrlble buildingblock comblnatlons

-

T

*X

SLIIIItIcaI Dercripllon of the Beads In a PwVRV

*A,.~w,.~

O X

S l l e of any Speelflc Slngls &ad h a P w l

04fF.

Figure 3.2. Schematic description of split-mix synthesis of a three component library,

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APPLICATION OF HTS TECHNIQUESTO DRUG DISCOVERY

see below) to make in principle many millions of compounds. Similarly n, the number of RVs into which the beads are distributed, can be varied for each cycle depending on the desired/available numbers of building blocks. The split-mix protocol relies on the number of beads used being very much greater than the number of compounds to be made and the number of building blocks to be used. Thus at each ‘split’each RV receives a genuine statistical and even mixture of the possible compound combinations at that stage, and in the final library all possible building block combinations will be evenly represented. However, if the number of beads and the number of building block combinations becomes comparable, problems arise. In such a scenario each RV no longer receives an even and representative distribution of building block combinations to that point, indeed some combinations may be missing from some RVs, resulting in missing combinations in the final library. Thus a simple lOxlOxl0 library could not be made on just 1,000 beads, the statistics of mixing and splitting would result in a large number of combinations being absent and a correspondingly large number being over represented. However, if 100,000 beads are used, there are in principle 100 beads for each compound (100 so-called library equivalents) and the statistics of split-mix ensure that all combinations will be adequately represented. Whilst this point may seem facile it is important and does become very relevant when making very large combinatorial libraries. The statistics of split-mix synthesis have been rigorously examined and analysed [14]. TAGGING AND ENCODING

The key to bead based screening approaches is the ability to determine the identity of a compound expressed from a single resin bead. Whilst split-mix synthesis generates libraries in a highly efficient manner, by definition, it generates the corresponding libraries as mixtures of compounds. This presents challenges for screening in the conventional manner, most particularly in identifying the component of a mixture which is responsible for an active screening result. However, bead based screening approaches can overcome these difficulties by exploiting the fact that, whilst split-mix affords mixtures of compounds, each bead expresses a single individual compound. Thus screening of single beads, or the compounds derived from single beads, corresponds to screening of single compounds, side-stepping the problems of split-mix synthesis affording mixtures of compounds/beads. However, whilst BS effectively allows the screening of single compounds from a single bead, there remains the problem of the identity of the compounds on or derived from the single beads. If screening from a single bead gives an assay positive the corresponding compound needs to be identified.

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Whilst the gross statistical composition of the pool from which the bead originated is known, the identity of the compound on any single bead is not specifically known. Whilst in principle the identity of a compound derived from a single bead could be determined by classical analytical techniques this is not possible in practice, principally for reasons of sensitivity, but also for a number of other technical reasons. To overcome these problems tagging/encoding strategies [9-121 are typically employed to allow determination of the identity of the compounds derived from single beads. The principles of coding/tagging are straightforward. During the splitmix library synthesis, the addition of each building block is accompanied by the addition of a corresponding tagging/encoding fragment, either before the chemical step or prior to the subsequent mixing step. In the above library synthesis example as each building block Ai is added a tagging fragment TagAi, uniquely corresponding to this building block, is added before the resin is subsequently mixed. This process is repeated at the subsequent steps with TagBi for building block Bi and TagCi for building block Ci. Any individual bead will now possess a compound made up from a single combination of building blocks and an associated tag sequence with a specific tag corresponding to each specific building block (Figure 3.3). Tags have properties which facilitate their analysis. Thus the identity of the compound on a single bead can be determined simply by analysing the tagging sequence. Numerous different systems of tagging have been developed. Most systems grow a tagging sequence alongside the library compounds, but all tagging systems incorporate features which facilitate the analysis of the very small amounts of tagging molecules released from a single bead. Systems developed include tags based on DNA which are read by amplification of a DNA sequence by PCR [15]; amide based isotopic ratio codes [ 161 sensitized to, and read by, mass spectroscopy; biarylcarbene derived codes which are read by gas chromatography [ 171 and amide codes which are read by hydrolysis of the amides to afford the corresponding amines which are derivatised by a Dansyl group and analysed by fluorescence HPLC [ 151.Whilst the coding methods may vary they are all designed to have the same effect, namely to allow the determination of the identity of a compound expressed form a single bead. (To be strictly correct the code seTagA,.,TagBl.,TagC,.,

+Al-nB1-nC1-n

Statistical Description of a Tagged Library Figure 3.3

TagAiTagBjTagCk

+AiBjCk

A Tagged Single Bead

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APPLICATION OF HTS TECHNIQUES TO DRUG DISCOVERY

quence records the chemical history of the individual bead, i.e. which building blocks to which it has been exposed). BEAD BASED SCREENING

Bead based screening, split-mix solid supported synthesis, and encoding combine to offer a formidable all round package. The split-mix protocol allows the efficient synthesis of large numbers of compounds, bead based screening allows their screening as, effectively, single compounds, and encoding allows the determination of the identity of the single compounds which give rise to assay activity. There are a number of differing formats and approaches to bead based screening. It would be beyond the scope of this chapter to review all the different approaches in detail, it is intended therefore, to focus mainly on the principles involved. Whilst bead based screening exploits the single bead-single compound nature of split-mix library synthesis, it is important to stress that single bead screening usually only represents the final phase of screening a library and takes place on a modest number of single beads after an initial phase of screening in pools. The amount of compound attached to and released from a single bead is relatively small (ca. Inm see above) and has corresponding consequences on assay format and sensitivity. For example a total of lnm of material cleaved from one bead into a volume of 1 OOpL gives a nominal concentration of 1Opmol/L. Bead based screening can be performed either with compounds still attached to the bead, or compounds can be cleaved from the bead into solution prior to assay. There are strengths and weaknesses associated with each of these two approaches. In general it is preferred to screen libraries of small drug like molecules off bead [9], in solution, where the whole of the molecule is exposed to the assay target and the potential behaviour of a molecule in vivo is more accurately reflected. For large peptidic libraries the on bead format has often been preferred [9]. However, both on and off bead screening exploit the same basic principles. Most forms of bead based screening require the distribution or ‘picking’of single resin beads. This can be done by hand, which is both demanding and tedious, but there are also a number of different automated systems to perform this function. However, discussion of this technology, along with other aspects of the hardware associated with bead based screening, is beyond the scope of this chapter. Before proceeding to describe BS formats it is also important to highlight some of the relevant statistical aspects [I41 of BS and to introduce the con-

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cept of Library Equivalents. The split-mix approach produces a library as a statistical mixture (see above) but provided that the number of beads is high relative to the number of compounds, all possible members of a library (i.e. all possible building block combinations) will be present in an even distribution. Statistical considerations also play a role in the screening of a split-mix library. If a pool of beads contains n theoretical building block combinations (i.e. n compounds) and a sample of only n beads is taken from this pool for screening in a particular assay, statistics dictate that it would be highly unlikely that the n beads correspond to one example of each of the n possible compounds. Detailed statistical analysis reveals that in a sample of 3n beads there is a 95% probability of there being at least one bead bearing a copy of each possible compound, and in a sample of 4n beads a 99% probability of a copy of each compound (with the corresponding statistical probability of there being no examples of some potential compounds). The 3n and 4n beads are referred to as respectively 3 and 4 library equivalents. Suffice it to say that in screening from pools of beads, library equivalence can be extremely important. Additionally, the total number of equivalents in a library determines its possible extent of usage. For example a 20,000 member library prepared on 3.2g of a resin at 500 beads per mg would only allow screening at 4 library equivalents in 20 assays ( I .6 million beads, 80 beads per compound). Beud bused screening in u lawn forniut

The lawn format [I91 is conceptually the most straightforward example of bead based screening (Figure 3.4). In this format a pool of encoded beads at the appropriate number of library equivalents is thinly dispersed across a lawn assay plate (for example a bacteriocidal assay). The library compounds can then be cleaved from the beads (for example by photolysis of a photolabile linker) and allowed to diffuse into the assay medium, or screening can Pick Active Bead Read Code Determine Compound Identity

Assay Plate

Figure 3.4. Srheriwtir illustrution of heud bused screening in luiin format.

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APPLICATION OF HTS TECHNIQUES TO DRUG DISCOVERY

take place directly on the bead without cleavage. Those beads that display, or have released active molecules, are easily identified through the assay readout (e.g. a bacterial killing zone). Any beads associated with assay activity can then be removed from the assay plate and the codes read to determine the identity of the active molecules. These molecules can then be resynthesised in bulk, and their activity (hopefully) confirmed and more thoroughly and accurately investigated. If screening is performed at the 4 library equivalents level, on average there will be 4 beads bearing a copy of any single compound. Thus in principle one might expect to identify the same compound as active from 4 different beads. Such a finding is reassuring and indicative of genuine activity in a compound. However, the statistical nature of the split-mix protocol manifests itself at this point. There are on average four copies of each compound in 4 library equivalents of beads. However, the 4 library equivalents only ensures that there is a 99.6% chance of 97% of the compounds being represented once. Hence there will be some compounds which will only occur once or twice and an accompanying number which occur more than four times. Thus there should be a spread in the number of copies of an active compound which are identified. It is unlikely, but possible, that only a single copy of an active compound will be identified. There is a greater chance that multiple copies will be identified and a significant possibility of the number of copies of a compound identified exceeding the number of library equivalents screened. The lawn assay format clearly illustrates the strengths of the bead-based approach to screening. Large numbers of compounds can be screened, but, in the absence of very high hit rates, any activity is associated with single compounds from single beads. Bead based screening in solution assays Bead based screening in solution based assays [20] is more complex than the lawn format but exploits the same basic principles. In the first phase of screening (Figure 3.5)the appropriate number of library equivalents (e.g. 4) is removed from a bulk sample of beads. These beads can either be used as a single pool, or subdivided into smaller pools of beads, factors such as the nominal number of compounds in the pool, the assay volume etc. will determine if or how subdivision is effected. The compounds are then cleaved (e.g. by treatment of an acid labile linker with trifluoroacetic acid) from the bead pool(s) into solution. The resulting solutions can then be assayed as appropriate. If one of these solution pools shows activity in an assay it reflects one or more of the compounds present in the original bead pool being active.

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To identify the active compound(s) it is necessary to return to the initial bulk bead sample for a second phase of screening at the single bead level (Figure 3.5). The appropriate number of library equivalents (e.g. 3 or 4) of fresh beads is taken from the bulk sample and distributed as single beads. These compounds are cleaved individually into solution from these single beads and the product from each individually assayed. In this manner the activity of the original bead pool is assigned to individual compounds. The codes of the beads of the active compounds can then be read and the identity of the active compounds determined. Again statistical factors manifest themselves in the single bead screening. There is a remote chance on returning to the bulk bead sample that no bead bearing a copy of the compound responsible for the activity in the screening of pools is selected. If 3 library equivalents are selected there is a 55% probability that 95% of the compounds will be represented once. When 4 library equivalents are used, there is a 99.6% probability that 97% of the compounds will be represented once. Again, as in the lawn format, it is most likely that multiple copies of active compounds will be identified. Library dimensions determine how efficient or attractive this form of library screening is. For example, if 10 building blocks are used in each stage of the synthesis of the three component library in Figure 3.2 this generates a library of 1,000 compounds. If the beads are not recombined at the last step this corresponds to 10 pools of 100 compounds. Screening at 4 library equivalents 400 beads would then have to be removed from each final bulk sample for the first phase of screening in any given assay. These could either be cleaved directly and screened as a mixture of nominally 100 compounds, Phase 2 Activity in pool(s) from phase 1 Return to Onginal Libraty Pool Taka 4n Library Equivalentsof Beads Distributeas Single Beads Cleave Single Beads Screen Single Comound Solutions

Phew 1 Take 4n Library Equivalentsof Beads Cleave into Solution and screen mixture or, Splll Inti sub pools cleave and test mixtures

.... .... .... .... .... ....

Active Band Read Code

Figure 3.5.Bead based screening in solution assc~ys.

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APPLICATION OF HTS TECHNIQUES TO DRUG DISCOVERY

or subdivided into smaller pools, e.g. 16 pools of ca. 25 beads and cleaved and screened accordingly. However, if assay activity is observed it could arise from any one of the 100 compounds in the original pool, thus to identify the compounds responsible for assay activity 300 (3 library equivalents) or 400 (4library equivalents) beads have to be distributed and assayed singly. Thus the smaller the library pool size the smaller the number of single beads which must be distributed to identify the active component, but the less efficient is the library synthesis and first phase of screening. Conversely, the larger the pool size the more efficient is the library synthesis and the first phase of screening, but the more single beads have to be distributed to identify active compounds in the second phase. The optimum balance between these factors is dependent on a number of different parameters, e.g. capacity for distributing single beads and assay hit rate. Bead based screening in solution can be further improved if the bead linker structure is modified to enable compounds to be cleaved in two distinct incremental steps (Figure 3.6). This allows the compound to be cleaved from the same set of beads at each of the two phases of screening, and brings a number of other advantages (see below). The standard split-mix protocol of Figure 3.2 is easily adapted to allow the synthesis of libraries in the form of Figure 3.6, in which the compounds are attached to the bead through two different linkers. These linkers are orthogonal, that is they are cleaved under mutually exclusive conditions, such that the linker A can be cleaved to release its copy of the compound whilst leaving the other copy still attached via linker B. Linker B can then be subsequently cleaved to release a second copy of the compound. An example of a pair of orthogonal linkers would be a photolabile linker and a TFA labile linker cleaved by, respectively, UV light and trifluoroacetic acid (Figure 3.7). Libraries built in such a twin orthogonal linker format offer distinct advantages for bead based screening. In the first phase of screening a pool of beads is cleaved at linker A to release the first compound copies into solution but leaving the second copies still attached via linker B. The resulting pool is then assayed. However, if there is any assay activity in the pool there is no longer any need to return to the original library for fresh beads. The pool of initially cleaved beads can simply be distributed as single beads and, by cleavage of linker B, release Linker A---Corrpwod

clflavags 1

Linker 0--cO-nd

Unkesr A

T'dOf

+Compound

Cleavage2

__I

Unhr 0 - - C o m d

Figure 3.6. Incrernenttrlly rleavuhlefi~rtnat.

Tw+w"'

+Compound ulker 8

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91

the second copy of the compound into solution for screening as single compounds. When activity from a single compound is identified the identity of the compound can be determined by code reading as described previously. The beauty of the twin linker format is that in the second phase it is only necessary to singly distribute the beads from the pool that gave activity in the first phase. In this format a bead-based library can be screened in sub pools of any desired size, and the second phase requires single bead distribution from only the sub pools which lead to activity. Thus, in the above example if activity is found in a pool of 100 compounds as 400 beads, it is necessary to distribute 400 single beads even if the screening is performed in sub pools of 25 beads. However, in the twin linker format if a sub pool of 25 beads leads to activity, only those 25 beads need be distributed as singles in the second phase. Moreover, in the above example of the 1,000 member library derived from Figure 3.2 it is now advantageous to recombine the resin after the addition of building blocks C to give a single bead mass of 1,000 compounds. Provided the requisite overall number of library equivalents is used, the library can then be assayed in whatever pool size is most convenient, because the second phase only ever requires return to the initial cleaved pool. The ability to recombine the library at the last step offers numerous advantages. Storing and cataloguing the library becomes trivial, but more importantly the screening pools will now be a genuine statistical sample of the library. They will not be composed of molecules which all contain a common final building block which can lead to problems with additive effects from weakly active individual similar compounds leading to misleading screening results. The considerations of library equivalence are still important in this form of bead based screening. It is essential that the appropriate number of library

Phase 2 Distribute Single bead5 from active well in phase 1 Cleave at Linker B Screen Solutions

Entire Library

E

Phase 1 Take 4n Libraly Equivalents of Beads Split into small pools Cleave at Linker A Screen Solution Mixture

Figurr 3.7. B e d hasedscreening with incremental compound release.

Acllve Bead Read Code

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APPLICATION OF HTS TECHNIQUES TO DRUG DISCOVERY

equivalents of beads be screened in the first phase to ensure that all of the possible compounds are in fact screened. However, it is not a consideration in the second phase of screening as the same beads are used. Again a statistical distribution of multiple copies of actives compounds will be identified. A number of different approaches to incremental compound release for bead based screening have been examined. Tiered release from a single linker has been developed [21] and a system of two linkers that cleave at different pH’s [22] has been used. In our own laboratories we use a twin orthogonal linker system in which the first linker is cleaved under acidic conditions and the second linker photolytically. We have used this system to successfully identify a number of interesting biologically active molecules by bead based screening, The example below serves to illustrate the use of this system but more importantly as a prototypical example of the use of bead based screening. A SUCCESSFUL EXAMPLE OF BEAD BASED SREENING USING DIFFERENTIAL RELEASE

160 Micron Tentage1 beads with a loading of OSmMol/g of resin are differentiated by coupling Boc glycine and Fmoc glycine (Fmoc = 9-Fluorenylmethoxycabonyl) to the amino groups in a 9:l ratio. The Fmoc group is removed and the first code is added. The Boc group is removed with TFA and a photocleavable linker and an acid cleavable linker are added in a 1:1 ratio (Figure 3.8). A 1,296 member library was constructed by removing the Fmoc from each linker followed by addition of 36 Fmoc protected amino acids, in 36 separate reaction vessels, to the encoded beads. The beads were combined and the Fmoc groups were removed followed by coupling of an Fmoc protected benzodiazepine core. Portions of the beads were split into 36 reaction vessels, the Fmoc group was removed and 36 carboxylic acids were coupled. A code was added to identify each of the acids. All the beads were mixed and stored as one pool of randomly mixed beads (Figure 3.9). About 4,000 beads (3 library equivalents) from the library were distributed as pools of approximately 25 beads. Compounds were released from the acid cleavable linker and screened against the CCKa receptor at a concentration of 2.5pM per individual component. Single beads were picked from 9 wells that showed greater than 80% inhibition of binding of CCK-8 to the CCKa receptor. The remaining 50% of the compound was released with light and the discrete compounds were screened at 2.5pM in the assay. Three compounds were identified from more than one bead. These were resynthesized, purified and characterized as the discrete samples GW529578,

99

B. COX ETAL.

-

*NH,

Boc-GlydGly-Fmoc

\

AIIoc-

0

7-

RZ

Alloc-

4

0

N-R’

OMe

0 Figure 3.8. Construction of diybnliufed resin.

GW52958 1X and GW52957 1X. Concentration response curves were measured for each sample and the IC50 determined (Figure 3.10) Identification of compounds from fully eficoded differential release libraries avoids costly and inefficient chemical deconvolutions. Mixing all the beads after completion of the synthesis and randomly redistributing the beads reduces the possibility of additive effects in the first round of screening. COMBINATORIAL APPROACHES TO LEAD OPTIMIZATION The development of automated synthesizers has generally been targeted at equipment for the production of larger libraries for lead identification. However, the power of the automation did not go unnoticed by chemists working

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APPLICATION OF HTS TECHNIQUES TO DRUG DISCOVERY

Figure 3.9. Library bared around a benrodiurepine core

GW 529578X IC, 622nM

GW52950l X IC,, 639nM

H*N

GW52957lX IC, 575nM

Figure 3.10. lC.7,,Determination on purified compounds.

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101

on the optimization of leads for medicinal chemistry projects. Initially the preparation of libraries to aid optimization was only carried out by the specialist users of the equipment. The complexity of the equipment and the need for specialist operation therefore limited its use. Although various pharmaceutical companies explored the potential to introduce synthesizers into general laboratory conditions there was an initial reluctance to use these by many project chemists. The need to invest in long periods of familiarization with software and equipment that might only be used once every few months was the main cause of this apparent lack of enthusiasm. In essence, the wrong processes were being targeted, as optimization chemistry typically demands preparation of smaller libraries (1 0’s to 100’s) and in greater quantities and quality than for lead discovery. Thus the automated systems found use for projects in rapid evaluation of new lead series or back up compounds through larger libraries, a phase often short lived and widely spaced, but were not suitable for the optimization of key series and compounds. The development of equipment to support the latter stage of the optimization process has now gone some way to meet these needs. Smaller compound numbers and an option for the chemist to evaluate in real time the status of the chemistry, and therefore intervene if necessary, typically characterize this type of equipment. Much of the equipment has relied on the use of technology to increase throughput whilst maintaining processes as familiar as possible to typical practices. Thus simple multiple equivalents of round bottom flasks for reflux or cooling, such as the Stem block (Figure 3.Z1), allow the same processes to be repeated in a parallel format, though requiring the same level of chemist handling as before. Similar blocks for solid phase would include the HiTops system (Figure 3.12) on the microtitre format, or more recently the J-KEM system (Figure 3.13).These still typically require attention from chemists for all processes and handling. More sophisticated systems such as the those from Bohdan (Figure 3.14) or the Quest series (e.g. the 210, Figure 3.15) from Argonaut have automated certain processes, typically those of washing cycles, though they still retain simplified control systems and the ability to manually override and interject into synthesis when required. More complex automated synthesizers have been developed, such as the Argonaut trident (Figure 3.16)or Technology partnerships Myriad personal synthesizers (Figure 3.17).Designed to give greater control over a wide range of reaction conditions and allow much more complicated chemistry, they also claim strength in the reproducibility of reaction and reaction conditions both for repeat operations and for transfer to other compatible equipment. However, these pieces of equipment are typically complicated systems that may require expert users. Moreover, for the costs involved the return on the

I02

APPLICATION OF HTS TECHNIQUES TO DRUG DISCOVERY

Figure 3.12. HiTops system.

Figure 3.11. The Stem block.

Figure 3.13. The J-KEM system (Photograph courtesy of J-KEM Scient$c Inc.).

Figure 3.15. Argonaut Quest series (210).

Figure 3.14. The Bohdan system (Photograph courtesy of Bodhan Automation Inc.).

Figure 3.16. Argonaut trident (Photograph courtesy of Argonaut Technologies Inc.).

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Figure 3.17. Myriad personal synthesiser (Photograph courtesy of Meitler-ToledoMyriad Lid).

Figure 3.18. Radley's multiple reaction station for stirrer-hot plate (Photograph courtesy of R. B. Radley t Co. Lid).

Figure 3.19. Multiple tip pipette system.

Figure 3.20. Multiple shot pipette system.

Figure 3.21. Genevac system.

Figure 3.22. Micromass LC-MS system.

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APPLICATION OF HTS TECHNIQUES TO DRUG DISCOVERY

Figure 3.24. Chiron system.

number of compounds synthesized on some of these machines can be disappointing. In complete contrast to these examples, very simple approaches can also have great impact on medicinal chemistry. Simple carousel reaction blocks economically convert single stirrers to multiple reaction stations (Figure 3.18).The use of multiple tip (Figure 3.19), and multiple shot pipette systems (Figure 3.20), long in use in biological applications, have allowed rapid synthesis in microtitre and other spatially addressed formats. Developments for solvent removal (for example Genevac systems, Figure 3.21)and analysis (for example micromass LC-MS systems, Figure 3.22) have ensured that bottlenecks are not created at related parts of the overall synthesis process.

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Data acquisition and handling now remains as a key target for developments, especially in the arena of visualization tools. Perhaps the epitome of technology development to support ‘comfortable’ working practices for lead optimization has been the IRORI (Figure 3.23) and more recently Chiron (Figure 3.24) rf (radiofrequency) tagging technologies. Designed to allow full use of split-mix techniques to produce discrete compounds, these systems are specifically built to allow the use of standard glassware and processes, but deliver the numbers of compounds only accessible by solid phase split-mix. Already in widespread use within the pharmaceutical industry, these packaging and monitoring processes for solid phase chemistry have perhaps done more to revolutionize the use of combinatorial techniques in optimization chemistry than any other. Anyone interested in more up to date and live references to this field are recommended to access one or more of the available internet sites focusing on combinatorial technologies [23]. Any reference to specific equipment in this section does not indicate a preference for that equipment, but is rather just an illustration of the style and type of equipment. SAMPLE SELECTION ETHNOMEDICAL SAMPLE SELECTION

The origin of samples tested in high-throughput screening has varied considerably over the last 15 years. At the outset, samples screened were mainly natural products - and predominantly filamentous bacteria - in origin. Over recent years, with the advent of highly sophisticated automated methodologies for the rapid generation of large numbers of synthetic molecules, a wealth of synthetic chemical diversity is available for evaluation. Inevitably, this means that the relative significance of natural products as input material may diminish. However, natural product materials can still offer access to otherwise untapped chemical diversity. In fact, analysis of the topselling pharmaceuticals between 1985 and 1995 shows that around half the top 20 medicines have active ingredients with a chemical structure based on a compound found originally in nature [24]. Various issues have to be considered when taking a decision on whether to utilize natural materials in a screening programme. The feasibility of using any natural materials in drug discovery is dependent upon ensuring that effective procurement strategies are in place. The key objectives of the United Nations Convention on Biodiversity, 1992, are to ensure the conservation of biological diversity, the sustainable use of its components and to imple-

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ment fair and equitable sharing of benefits arising from its use. The Convention emphasizes the concepts of the sovereignty of states over genetic resources and their obligation to facilitate access. The contracting parties are expected to establish measures for benefit sharing in the event of commercial utilization. This involves collaboration between the collector, the source country and the industrial partner. It is now standard practice to draw up a legal agreement to cover these issues and many companies have issued policy statements describing their and their collaborators obligations in this area (for example Glaxo Wellcome Policy Statement, 1997). There are several strategies that may then be adopted for tapping into a natural materials collection in order that the most appropriate samples are tested in a biological target, given that it may sometimes not be desirable to test very large numbers of samples. If numbers are not significantly restricted then random acquisition and preparation of available species is a possible strategy, whereby the likelihood of success in identifying a compound which possesses interesting biological activity is increased by testing large numbers of samples across a wide range of target screens. Alternatively, one may look at a diverse collection of natural product materials, with the hypothesis that taxonomic diversity of samples will be reflected in the chemical diversity of extracts subsequently prepared. A more focussed approach depends on having prior knowledge about selected natural product samples, which may suggest that they contain particular chemical classes of interest or that they possess desirable biological properties. This strategy can be considered under two headings - chemical targeting and biological targeting. The chemical approach utilizes natural materials as sources of specific compounds of interest to a particular disease area, or as potential sources of chemical classes that could be of relevance to a given target. Alternatively, the biology driven strategy may be thought of more as a disease-driven process. Natural product samples, particularly plant or macrofungi, can be selected for biological evaluation using some type of information associated with them that suggests their relevance for evaluation in a given therapeutic target. Perhaps the most striking observations available are ethnobotanical reports of traditional medicinal uses of plants. A significant number of commercially available orthodox medicines were discovered by following leads provided from indigenous knowledge [25]. For example, Digitalis purpurea has long been used in traditional medicine to treat dropsy, a swelling of the body caused by inadequate pumping of the heart [26].This led to the identification of digitoxin and related cardiac glycosides and ultimately to the discovery of digoxin in the related Digitalis lanata. There is a wealth of information relating to the use of Papaver somniferum (opium poppy) as a medicine

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and psychoactive drug prior to the isolation of the well known opiate alkaloids such as morphine and codeine [27]. Ephedrine from Ephedra species is widely used as a bronchodilator. It’s source, Ephedra species, has been used for thousands of years in Chinese traditional medicine to treat colds and coughs. Cinchona species, the source of quinine, and Artemisia annua, from which the antimalarial compound artemisinin was identified, have again both long been reported to be used in traditional medicine for treatment of fevers and malaria [28]. The anticancer agent, taxol, was identified from the Pacific Yew, Tuxus brevifolia [29]. Yew trees have long been known to be highly toxic, a possible clue to the cytotoxic potential of its constituent. Numerous other examples may be cited to show the relevance of ethnobotanical observations in providing clues to selecting plants that may yield biologically active compounds e.g. [25], [30], and [36]. A variety of approaches can be taken in order to gather the ethnobotanical information needed to select plants of particular relevance for a given disease target. Some research groups rely on developing a network of ethnobotanists who work closely with indigenous colleagues and traditional doctors in various countries. The outcome of this approach is a low number of plant samples identified for evaluation in the laboratory and detailed information on their use. Many groups, however, prefer to utilize published information sources. A key example of a database of great potential in this area is the NAPRALERT (Natural Products Alert) database [313. This system is maintained at the University of Illinois at Chicago. It contains over 120,000 references relating to reports of biological activity in the scientific literature, ethnobotanical reports and phytochemical data. It is possible to search for plant species that are reported to be utilized for selected diseases of interest. A set of extracts ‘targeted’ at a given assay or group of assays related to a given disease area may then be assembled. In some cases a generic sample preparation and extraction protocol will be undertaken. However, if in depth analysis is required on a small number of samples it may be desirable to prepare extracts of the plant using a similar methodology to that employed in the traditional medicinal use. If biological activity is detected, the extracts will be subjected to isolation and purification techniques in order to isolate and chemically characterize the active component. CHEMOINFORMATICS

Chemoinformutics in the lead generation process Chemoinformatics is a relatively new term [32], which combines several disciplines traditionally found in the pharmaceutical industry; computational

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chemistry, information science and statistics [33]. What is the relevance of these information-based methods to what could be considered the nemesis of a rational approach to lead generation? To answer this question, it is necessary to look at the lead generation process from a sample centric viewpoint. Figure 3.25 is an illustration of what the screening process attempts to do, which is to filter a large number of candidate samples down to the most interesting. The first thing to realise from this view of the process is that there are many more accessible molecules than can ever be tested in a HTS assay. Estimates of the number of ‘drug-like’molecules average around lo4’ [34] (for comparison, physicists compute the number of seconds since Big Bang to be lo”), whilst there are lo7 molecules registered by CAS and the number of unique compounds (excluding library samples) offered to GW for purchase in the last 4 years exceeds l million. So, one use for chemoinformatics is to aid in the selection of compounds to pass from the ‘accessible’ chemical space into the ‘available’chemical space, to optimize the diversity of the corporate compound collection. This can involve combinatorial library design, the selection of compounds for purchase or involvement in compound exchanges with other companies. Moving down the filter, it is often necessary for a subset of the corporate collection to be selected for screening in a particular assay. This may be due to the limited assay capacity, the cost of the assay, or a mismatch between the size of the sample collection and the throughput of the assay systems. Many companies use the concept of a ‘representative’ set of compounds, which is perhaps 10-20% of the collection, and is derived with the intention of covering all of the chemical classes present. It is also common to collate samples of a particular type, and screen them against families of proteins. For example, one might collect all the benzodiazepines together and screen them against 7-transmembrane receptors. At Glaxo Wellcome we term this biased screening. Finally, there may be very specific knowledge about the target such as a crystal structure, and there are many ways of using 3D information to identify lists of the most likely compounds to bind to a target protein. We term this focussed screening. We can view these activities as positive selection, that is we really want to screen those particular compounds. With several years experience of screening large compound collections, many groups have seen the need to develop a deselection strategy, that is to find compounds that we do not want to screen because they may be reactive (detergents, alkylating agents, dyes and so on) or synthetically intractable [35-371. Although most chemists are quite capable of rejecting undesirable compounds by eye, with large collections of compounds this is impossible, and thus these techniques must be auto-

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Accessible Compounds

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)Compound purchase, synthesis or exchange C

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Corporate Samples (e accessible samples) belection for screen Screen B

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Figure 3.25. A sutnple-centric v i m of the screening process. Chenioinjormutics systems ure essent i d j i i r the processes outlined in hold lines.

mated to run on whole databases. Once developed and deployed these are usually referred to collectively asfilters. If a pooling strategy is employed, then the above techniques can be applied to direct the chemical make-up of the pools. To avoid additive effects, each pool may be designed to contain diverse structures, whilst other strategies may be used to aid deconvolution, or to keep pairs of compounds which may react together, apart [38]. All of the above roles involve sample selection of some kind. However, there are other valuable roles to consider. Compound selectivity is often a key criterion for sample progression, and the ability to look over all the assays that a certain sample has been tested in, is an essential part of a chemoinformatics function. In addition, a chemist may wish to look at the results of a chemical series to see if a compound is the only active member of a series or if there is a spread of activities. And of course most companies now collect metrics on the performance of sample types. These may be thought of as decision support tools, in that the science is minimal but the data analysis is very necessary. Finally, there is the possibility that informatics might change research processes. The concept of itemtive or seguentiul screening is challenging the received wisdom of ‘blitz’ screening [39,40].The idea is to screen either a fo-

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cussed or representative set of samples, and then use the information generated to feed a selection method which suggests a new set of samples to be screened. Several iterations may be used until the required number of hits are found, or no more hits are generated. Molecular descriptors

To do any of the sample selection processes it is necessary to describe the molecules such that they can be compared and searched for. The most common descriptors used [41] are 2D or 3D fingerprints, topological indices and physical properties. Fingerprints are commonly employed in chemical database systems, where they are used to speed up, say, a substructure search. A fingerprint describes features in a molecule by setting particular bits ‘on’ to reflect atom or bond types and connectivities found in the molecule. Typically between 100-2,000 bits are used to describe molecules. 2D fingerprints therefore tend to encode the chemical structure of the molecule, whereas 3D fingerprints generally try to encode the pharmacophore information contained in the molecule. Figure 3.26 illustrates the conformationally flexible 3-point pharmacophores used by the ChemX system. These fingerprints are generally very long, often between lo5 and lo6 bits in length. Topological descriptors are derived from the ‘molecular graph’ of the compound. These descriptors encode information about the level of branching, ring content and such like. As these descriptors are very quick to calculate, they are often used as a ‘quick and dirty’approximation to more sophisticated concepts, such as molecular shape and flexibility [42]. Physical properties are perhaps the most familiar descriptors to the medicinal chemist. Molecular weight, log P, molar refractivity, rotatable bonds and the like are easily calculable and widely used. Indeed, it has become clear [43-461 that the most effective library design methodology must take into account these whole molecule descriptors otherwise diversity based libraries often have little resemblance to the molecular properties of known drugs. For compound filters, bespoke sets of substructural fragments, corresponding to functional groups deemed unsuitable by chemists [35, 361, are often used in concert with physical properties. Selection methods

As with descriptors, there is a battery of selection methodologies available. A crude classification can be employed, defining techniques as those that describe only the set of molecules in hand, and those that describe a whole chemical space in which the molecules of interest are placed. Clustering

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Pharmacophore point A

Figure 3.26. The ChernX 3 0 , 3-point pharinacophore keys. Each pharmacophorir point A, Band C can he any one of'srven diflererent pharnTacophore types (acid. basic N , quaternary N, donor, acceptor. aromatic, hydrophobic). The distances are typically partitioned into 31 bins. In this standard configuration the,fingerprintis 848K bits long.

techniques [47] are very popular, particularly for the construction of representative sets, and provide the best example of the first category. Clustering attempts to divide a set of molecules into a series of subsets, in which all the members are similar to each other, but different to other clusters. There are two types of clustering, hierarchical and non-hierarchical. Briefly, hierarchical clustering keeps and attempts to optimize the relationships between clusters, whilst non-hierarchical does not. As a consequence, hierarchical methods are much more computationally intensive, and are often not applicable to extremely large sets of compounds. Any clustering method depends on the ability to calculate the similarity of one molecule to another. For fingerprints, the most commonly similarity index is the Tanimoto coefficient, which ranges from 1 (identical) to zero (no similarity), and is simply the number of bits set by molecule A and molecule B, divided by the total number of bits set by molecule A or molecule B: TanimotoAB

AandB AorB

=-

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For non-binary descriptors, the similarity index is most often the Euclidean distance. Similarity indices have different behaviours that can affect selections and have been the subject of much study [48,49]. Clustering is a very good way of describing a set of molecules. What it is not good at is describing what you do not have, which is necessary for effective compound acquisition. For example, how long should a company continue to purchase new samples? In this situation, it is desirable to describe chemical space, and to place the corporate samples in it, the objective of compound acquisition being to fill the space. Here, a common practice is to use a smaller number of whole molecule descriptors to construct a low-dimensional chemical space [50]. Each axis (descriptor) of the space is binned into a number of divisions, to define partirions. The group at Rhone-Poulenc Rorer have used 6 descriptors (to reflect hydrophobicity, polarity, hydrogen bond acceptor and donor capability, flexibility and shape); each divided into 2-4 partitions, giving a chemical space of 576 bins [51].With these bins defined, it is easy to identify the types of molecules missing from the collection and then to search for molecules to make or purchase to enhance the set. A problem with the partitioning approach is that the space quickly becomes vast, for example just 6 descriptors in 10 partitions requires 1 million molecules to fill it. Hence, many groups have used statistical tools such as factor analysis to reducejhe data from many descriptors down to a manageable number [52]. An alternative approach to this problem is provided by the use of 3D pharmacophore fingerprints as described above, where each of the 848K unique pharmacophores can be thought of as an individual partition [45], [51]. A final point in favour of partitioning is that it enables a definition of diversity, complete diversity being when all the partitions are filled. The key to the choice of both descriptor and selection method is the goal of the experiment. For example, should the requirement be to select a representative set of the chemical classes in the corporate collection, clustering using 2D fingerprints would be a sensible choice. Should the objective be to define a set of diverse combinatorial libraries that should be applicable across multiple screens, then a partitioning approach may be more appropriate. Validation

Although many combinations of descriptors and selection methodologies are available, not all may be relevant. The most important property of a descriptor set is that it exhibits ‘neighbourhood behaviour’ [53], that is, it defines molecules with the same biological activity as similar. It is then the job

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of the selection method to group these molecules together and to separate them from the rest of the data set. Several studies have been reported [54-571, following the same general validation strategy: a data set of compounds and known activities are subjected to several types of descriptors and selection methods. The effectiveness of the combinations is measured by how closely the separation of the molecule reflects the biological data and by how much the methodology improves over random selection. These studies have illustrated the effectiveness of a combination of Ward’s clustering (a hierarchical method) with 2D fingerprints. Current research is now focussed on providing methods that improve on this ‘gold standard’. Chemoinformatics is an essential part of the lead generation process. The effective combination of statistics, computational chemistry and information science has already provided methods that can be used to effectively select sets of compounds for screening, synthesis and acquisition. This is a rapidly changing field that is set to be more influential, and impact on the work of all chemists working in the lead generation area. BIOASSAY DESIGN The requirements of today’s lead discovery operations has led to the development of a specialized discipline, bioassay design, focussing on the development of assays which are sensitive, robust, efficient, time- and cost-effective enough to support high-throughput screening campaigns. The evolution of these techniques is going hand in hand not only with emerging technologies in biological sciences but also with developments in HTS technology. Typically, HTS bioassays are designed to work in 96 or 384 well microtitre plates that can be handled in fully automated screening robots. Future developments are looking to run these assays in even higher format, in 1536 plates. The following sections give an overview of some of the more commonly used biochemical and whole cell bioassays in HTS campaigns in Glaxo Wellcome, and point out the key aspects in design features which make them readily amenable to screening. BIOCHEMICAL ASSAYS

Biochemical assays are built with defined molecular entities and output information about the interaction of a chemical entity with the defined target. They may involve the binding of a ligand to a receptor, the catalysis of a reaction by an enzyme, the interaction of two proteins, a protein with DNA

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or a carbohydrate with a protein. The biological components of the assay are derived from either natively expressing or recombinant cells and supplied to the assay either within crude cell fractions or as highly purified preparations. Preferred assays for HTS are homogeneous, involving one or more addition steps of reagents into a 96 or 384 well plate containing pre-dispensed test compounds, one or more short ( < 2h) incubation times, then a detection step in the appropriate plate reader. Scintillation proximity assays A large proportion of HTS campaigns use the scintillation proximity assay (SPA) format [58]. SPA has been designed for radioisotopic detection of bound radioisotopes to scintillant coated beads to circumvent the need for separation of free from bound isotope and addition of liquid scintillant before scintillation counting. The principle of the scintillation proximity assay (SPA) is shown in Figure 3.27. For light emission to occur, the isotope must be in close proximity to the bead for the emitted radiation to activate the scintillant. In receptor-binding studies, the receptor (either purified or as a membrane preparation) is bound to the SPA bead. Upon addition of the radiolabelled ligand, only that which binds to the receptor is brought sufficiently near the bead for light emission to occur. The consequence of this is that ligand-receptor interactions can be quantified without the need for separation of bound from free ligand, and the whole assay can be performed in a homogeneous format. A similar principle is employed with the Flashplate (NEN-DuPont) [59]. Here, the interior of each well of a microtitre plate is coated with a thin layer of polystyrene-based scintillant. The wells may then be coated with the target receptor and radiolabelled ligand binding detected through activation of the scintillant. The scintillant proximity approach has been used successfully in screening against enzymes, receptors, protein-protein and protein-DNA interactions. With an environmental and safety drive to limit radio-isotopic usage, other formats are preferable to detecting binding or enzyme catalysis. Changes in colour or fluorescence of a substrate are often used to measure hydrolysis by enzymes. Fluorescence-based approaches For fluorescence-based binding assays in homogeneous format, three assay techniques are available to quantify bound from unbound species. Fluorescence resonance energy transfer (FRET) assays can be designed using donor and acceptor molecules where the absorption maximum of the acceptor is

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matched to the emission peak of the donor [60]. Here, for example, the donor and the acceptor may be tagged to regions either side of an enzymatic cleavage site on a substrate molecule, or onto the molecular pair in a protein-protein or receptor-ligand interaction. Alternatively, FRET can be designed to use internally quenched fluorescence, such that hydrolysis or separation of the fluorophore with the quencher increases the fluorescence signal. A problem with FRET assays lies in the discrimination of the specific signal from the background fluorescence from plates or test. Homogeneous time-resolved fluorescence (HTRF) addresses this by introducing a timegating on the specific fluorescent signal of interest [6 1,621. Here, the donor molecule is europium cryptate (EuK), which exhibits a fluorescent signal that is longer-lived than the fluorescent noise from plates/compounds. The acceptor molecule is cross-linked allophycocyanine (XL665) which by itself exhibits a short-lived signal at 665nm. When XL665 is in close proximity to europium cryptate, the energy transfer results in an amplified and long-lived signal at 665nm. The specific signal from the interaction of the two molecules can thus be resolved from background noise by measurement of the signal after the background signal has decayed (Figure 3.28). Fluorescence polarization is another technique applicable to homogeneous format HTS assays [63]. The technique utilized fluorescently tagged molecules and indirectly measures the size of molecules through their ability to polarize light as they rotate in space. Small fluorescent molecules rotate rapidly during the fluorescent lifetime, scattering the emitted light randomly, resulting in a low degree of polarization. Large fluorescent molecules rotate more slowly, resulting in a higher degree of polarized emitted fluorescence.

Figure 3.27. Principle ofthe SPA assaj. Rarlioligunclmust be brought in close proxiniity to the bead. illrough binding to the su~$i:fircehound receptor,,fbrlight emissicin to occur.

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This technique can be used to follow, for example, the enzymatic conversion of large molecules to small ones. ELISA and ion exchange separation assays

With the drive towards using homogeneous formats for biochemical targets, the number of more complex assay formats (ELISA, ion exchange separation) is diminishing. ELISAs (enzyme-linked immunosorbent assay) are highly sensitive, applicable across target classes and are widely used in biochemical research laboratories. The drawback for ELISA's for HTS are that multiple steps are involved in the assay, and these include wash steps that require a complex plate handling protocol and very large volumes of wash buffer. In most cases, ELISA's can be effectively redeveloped into homogeneous (SPA or HTRF) assay formats. Some non-homogeneous radioisotopic ion exchange separation assays have been run as screens in Glaxo Wellcome using fully automated robotic systems. Here, ion exchange was used for enzyme assays where a charge difference existed between the substrate and the product and no other assay technique was applicable. These assays followed the enzymatic conversion of a positively charged, radiolabelled substrate to a neutral radiolabelled product. After incubation of enzyme, substrate and test compound, the neutral product was separated from the charged substrate by addition of a strongly cationic resin slurry to bind and sediment the charged substrate. Supernatant, containing the radiolabelled product was then transferred to another plate, mixed with scintillation cocktail and counted.

Figure 3.28. Principle of the HTRFassay. Antibodies lubelled with europium cryptate (EuK) and cross-linked allophycocyanine (APC) bind to antigenic sites on a molecule. In the absence of binding, excitation at 337nm procluces only u short-lived 665nm signal from the APC. Only when both antibodies are bound is a specific, long-lived 665mn sigtiul getieruted from the APC niolecule through non-rudiutive and energy tramfer from the excited EuK.

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CELLULAR FUNCTIONAL ASSAYS

As well as biochemical targets, a range of cellular functional assays are used in HTS campaigns. These are designed to report on the degree of agonism or activation of a compound at a receptor or ion channel or the degree to which compounds modulate intracellular transcription events. Alternatively, viability assays can be designed to indicate compound toxicity. In cellular reporter assays, the molecular interaction of interest is detected through the transcription of a readily quantifiable enzyme or protein either endogenously expressed or genetically engineered into the cell [64]. Reporter genes commonly used are secreted placental alkaline phosphatase (SPAP), chloramphenicol acetyltransferase (CAT), firefly luciferase, renilla luciferase, /?-galactosidase, 8-glucuronidase and green fluorescent protein (GFP). In Glaxo Wellcome, the SPAP reporter system, in which the substrate p-nitrophenol phosphate is converted to the yellow product p-nitrophenol, has recently been shown in-house to be compatible with a miniaturized, 1536 plate format [65].The measurement of rapid kinetics changes in fluorescence in mammalian cells for HTS has been enabled for HTS by the introduction of FLIPR, a fluorescence plate reader equipped with integral 96 or 384 pipetting heads [66]. Using this instrument, all wells in a 96 or 384 well plate can be imaged simultaneously. Functional assays measuring the intracellular changes in ionic concentration can be performed using cell lines loaded with fluorescent ion indicators (e.g. Flu03 for Ca2+)or voltage-dependent dyes (e.g. DiBAC(4)3). These assays are applicable for quantification of cell surface receptor activation (through intracellular Ca signalling) or ion channel activity (using ion dyes/ voltage dyes) [67,68]. Finally, antimicrobial toxicity screens are routinely run as HTS campaigns. Optical density or colour changes using a redox indicator are the most frequently used assay methodologies. Both these techniques are readily amenable to HTS. HIGH-THROUGHPUT SCREENS FOR EVALUATING ABSORPTION, DISTRIBUTION, METABOLISM AND EXCRETION (ADME) PROPERTIES It is now recognised that selection of screening hits based on activity only leads to development problems downstream. A high-throughput screening campaign biased towards potency means that we are selecting optimized ligands but poor drug candidates. Hence, the need to bring physicochemical, pharmacokinetic and toxicity optimization into the high-throughput envi-

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ronment has become a major challenge within the pharmaceutical industry [69]. Drug metabolism will have to adapt and integrate with the new processes driving drug design. APPROACHES TO SCREENING

The main focus of developing high-throughput ADME screening is on in vitro models of absorption and metabolism. There are advantages to using in vitro systems rather than in vivo in terms of cost, ease of automation and, most importantly, isolation of the mechanism and consequent determination of relationships between molecular structure and the extent of absorptionimetabolism. IN VITRO STUDIES: ABSORPTION

Most models of absorption involve the use of cultured, immortalised, intestinal epithelial monolayers; most commonly a human intestinal cell-line, Caco2 [70,71]. The constraints in the current methodology surrounding preparation and use of these cells limit the screen throughput. Recent developments in mass spectrometry, with respect to instrumentation costs and analysis software, have shown great potential in maximizing throughput. Samples can be pooled into cassettes, either before or after experiments. The mixtures of components are then separated and analysed rapidly on single quadrapole mass spectrometers that measure selected parent ions. IN VITRO STUDIES: METABOLISM

The need for a consistent, validated, high-throughput metabolism screen is clear, as rapid metabolism is one of the prime reasons why compounds fail to achieve useful systemic levels. Microsomal preparations from either animal or human liver have frequently been used as a test vector to examine metabolic stability within a series of molecules of interest. However, drug metabolism is a multifactorial process. None of the in vitro preparations used contain all of the potential metabolising enzymes in vivo. The various enzymes decay relatively rapidly in vitro and at different rates, so the timeframe in which to examine and differentiate between compounds is usually short. Microsomal preparations can also be highly inconsistent in their preparation or, in the case of human material, their source. Consequently, even though the incubation methods used easily lend themselves to the application of robotics, a standard preparation for the regular screening of large numbers of molecules is not easily

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achieved. However, the use of recombinant techniques is allowing us to gain an understanding of the metabolism of xenobiotics by human enzymes without the constraints of tissue supply. Baculovirus, E. Coli and yeast expression systems are potential rich sources of the pure P450 isoforms that can be used in metabolic stability screens. Another important factor which limits the throughput in these studies is the need for specific analytical methodology for each compound examined and the need to isolate and identify unknown metabolites to understand which sites on a molecule are the most vulnerable. Generic readouts for ADME screens have so far proved elusive. However, LC-MS and LC-MSMS have enabled us to reduce the time for both method development and analytical run time. Coupled with the use of robotic, pattern recognition software and 96-well plate format sample handling, the analytical throughput of the mass spectrometer is increasing dramatically. COMPUTATIONAL TECHNIQUES

If we understand the physicochemical principles that determine ADME properties, we can predict the behaviour of molecules using computational methods. There is currently little confidence in predictions of metabolic route or rate, or absorption, based on chemical structure. Alongside modern developments in chemistry and screening, automated ADME screens will result in an explosion of information and Information Technology will underpin our greater ability to predict kinetic outcome and design drugs. Large sets of marketed molecules have already been studied using modern computational modelling tools to derive ‘sets of rules’ that predict pharmacokinetic outcomes [8,72]. The construction of predictive model systems based on the very limited structural information available on specific metabolising enzymes [73-761 shows some promise. Correlation of the sites of metabolism by cyp2D6 with areas of high frontier electron density is one approach that has been tried [77,78]. Alternatively, many workers are modelling the active site of the cyp450 enzymes with a view to predicting metabolic susceptibility and/ or interactions. In summary, the techniques employed in the field of drug metabolism and pharmacokinetics are undergoing a radical change to meet the needs imposed by the new technologies of chemical synthesis and HTS. The eventual introduction of HT ADME screens should greatly aid the understanding of factors underpinning the kinetic behaviour of drugs. Hopefully, prediction and virtual drug design will be the outcome of mechanism based screens for ADME parameters.

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HTS AUTOMATION Most large pharmaceutical companies and many medium-sized companies who employ HTS as part of their discovery efforts use automation in performing screens. Many repetitive tasks in performing assays in microtitre plates are now performed by bench-top instruments. Glaxo Wellcome, along with a number of other major pharmaceutical companies have recently invested tens of millions of pounds in building large highly-integrated automated systems for HTS [79,80] and for compound storage and retrieval. The development of automated HTS has been made possible because of the adoption by the screening community of an approximately standard disposable assay vessel: the microtitre plate. The typical appearance of microtitre plates is shown in Figure 3.29. Until recently, high-throughput screening has been conducted exclusively using the 96-well microtitre plate as the assay format. 96-Well plates are widely available from many manufacturers and distributors, and are designed to cater for a wide variety of assay formats. Recently, higher density 384-well and 1536-well plates have become available. 384-Well assays are now a standard format for screening in Glaxo Wellcome and assays are being miniaturized with a view to running them in 1536 format. To support the use of the microtitre plate in screening, a wide range of instruments and devices is commercially available to perform liquid handling, incubation, washing, centrifugation, sealing, and detection. Liquid-hand-

Figure 3.29. 96,384 and 1536-well microtitre plutes.

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ling instruments in regular use range from entirely manually-operated pipetting devices, single or multi-channel, through to fully automated robotic sample processors, which can be used in stand-alone mode on a bench-top, or can be integrated into robotic systems. All the conventional devices are based on either some form of syringe-pump technology to provide volumetric precision, or on multi-channel peristaltic pumps that offer higher speed at the expense of some precision. Many reading instruments are available specifically for use with microtitre plates. The conventional instruments are broadly based upon light emission/detection techniques: prompt fluorescence, optical density, luminescence, scintillation events and fluorescence polarization, are typical. Excitation is generally achieved with a xenon flash source or laser source; detection is performed by one or more photomultiplier tube(s), which may be cooled to reduce electronic noise, or by a solidstate CCD chip camera, which might also be cooled to reduce noise. FULLY AUTOMATED SCREENING ROBOTICS

One of the large-scale automated systems in operation at Glaxo Wellcome is shown in Figure 3.30. Systems of this general type are to be found in most high-throughput screening groups in large pharmaceutical companies today. The key components of this type of system are described in the following sections: Robot urin on truck

The robot arm is an industrial-type multi-axis arm. Three manufacturer’s products are in widespread use: the CRS arm, the Sagian-Beckman Orca, and the Zymark Zymate. All three products are very capable. In practice, most customers will not directly choose the arm themselves; instead they will choose a preferred system integrator, which will tend to work with one of the standard arms. The track on which the robot runs is usually a servomotor driven belt, chain or transmission cable, mounted either directly on the worktable surface or in a slot between two halves of the worktable. Track lengths from Im to 6m have been employed, with 3-4m being about average. Enclosure

The enclosure is normally an aluminium-framed clear acrylic cabinet with sliding or hinged doors, mounted on or around the worktable and enclosing the entire system. Within the European Union, all robotic systems of this type must be contained in an enclosure that only allows access via power-

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Figure 3.30. Large fully-integrated screening robot system Clockwisefrom top left: 1. Robot visiringplate hotel. Note plute washer and bar-code scanner in upper Iejt ofpicture. 2. Robot visiting one of two scintillation counters. Disposuble tip carousel on right ofpicture. 3. Robot visiting plate hotel. 37C incubutor on lefi, delidding station top lefi ofpicture. 4. Robot visiting colorimetrr. Fluorinieter in background.

interlocked doors, such that the robot at least is powered whenever a door is opened. Liquid-handling instruments

Most systems will have a number of liquid-handling instruments of the type described above: typically there will be at least a robotic sample processor, one or more common reagent dispensers and possibly a 96-channel replicator. Associated with each of these, usually mounted below the worktable, will be reagent storage refrigerators and liquid waste receptacles.

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Detection instrunients

All the most common detection instruments can be integrated; it is now very rare for a new instrument to be launched which is not described by its supplier as ‘fully robot-friendly’, meaning that it is mechanically compatible with robot grippers and provided with a communications port, through which it can respond to remotely-sent commands and also send out status and diagnostic data and, of course, assay data. Many systems currently have a number of different detection instruments on-board; the need for this is now diminishing to some extent with the advent of multi-label readers. Incubutors

A typical system will include some capacity for 37°C incubation, usually using a conventional incubator that has been modified to allow robot access. Room temperature incubation of plates is usually achieved simply by leaving p1a;es on an open hotel shelf for thc required time. Plate input/output buffkrs (‘hotels’)

In the simplest applications plates are loaded into the system by individually placing them on large arrays of static shelves, usually called ‘hotels’. This is very reliable and simple to operate, and is still the most widespread plate input system. However, hotels take up a lot of valuable system space, which restricts the total plate capacity. It is becoming increasingly common to see high-capacity plate input buffers (e.g. an automated stacker or large carousel) in which the plate shelves are not static but instead move under automated control by the system controller. Completed plates are either returned to their starting position on the hotel, if there might be any further use for them, or are discarded into a bin mounted below the worktable. Other instruments

In addition to the instruments described above, individual systems will usually have instruments specific to their intended application. For example, systems designed for cellular assays will certainly have a plate washer and systems designed for radioactive applications might have a plate sealer for improved safety.

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Generul utility,functions A system will usually offer a number of ancillary functions. For example, almost all systems have a plate-shaker for one or more plates, which may be a linear shaker or, more usefully, an orbital shaker. A stand-alone barcode scanner will usually be integrated, unless the laboratory barcode code type and position are fully compatible with any barcode readers integral to the reading instruments on the system. Most systems allow for the use of plate lids, which are removed for liquid handling operations and detection either by an array of suction cups or by the robot arm itself, which deposits the lid on a hotel shelf or local lid station. The lids are replaced during incubations to minimise evaporation.

Software The software which controls all of the system’s activities consists of a number of distinct areas: the programming interface, the scheduling engine, the robot control system, the device drivers, the native instrument control software, and the local data storage system. The programming interface allows the user to specify the sequence of tasks that must be performed on the plates, and all of the parameters that exactly define how those tasks are carried out. Programming interfaces may be highly graphical, menu-based, or script-based, depending on the age of the system. The scheduling engine defines the sequence and timing by which the system will execute its tasks, issue commands to and receive status information from all of the peripheral devices. The robot control system drives the robot and track itself, under commands from the scheduler. Each instrument is controlled via a specifically written device driver, which allows some (or preferably all) of the instrument’s stand-alone functions to be available to the automated system. In some cases the driver will directly control the instrument’s functions, particularly for simple devices such as common reagent dispensers, incubators or shakers. Where an instrument has its own native control software, as is usually the case for complex instruments like robotic sample processors or detection instruments, then it is normal for all of the programming of that instrument’s operations to be performed using the native control software. In these cases the instrument driver’s tasks will broadly be limited to starting a named protocol at the correct time (i.e. when the plate@)are in position) and then receiving a ‘protocol finished’ signal back from the instrument. Assay data from the detection instruments is usually stored in a local directory, with the data from each plate associated with a date and time stamp and the plate barcode identity. After or during a

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run, these files can be inspected and then uploaded into a corporate data management system for further data reduction. DATA HANDLING FOR HTS LOGISTICS OF HTS DATA HANDLING

A typical high-throughput screen involves screening 200-500K wells (containing discrete or pooled samples at a given concentration) against the biological target of interest. From the first test through a primary screen, active wells are identified and the associated sample flagged for re-testing. Discrete samples positive at retest go forward for concentration-response profiling whereas pooled or natural product samples are deconvoluted (or for natural products, fractionated) to identify the active constituent prior to concentration-response profiling. A major issue that arises in any HTS operation is the logistical aspect of dealing with a large sample inventory. Most pharmaceutical companies maintain a database containing information about the availability of discrete compounds and natural product samples. In an HTS environment, sample numbers in the order of 60,000 are required per day so many companies with a specialization in HTS rely on a dedicated store of dissolved samples, often in solution in DMSO, to enable fast accession and dispensing. The primary sample format in such stores is either the micronic tube (in an 8 x 12 rack format) or a 96 or 384 deep-well microtitre plate or block. The advantage of an array format is that it permits parallel sample handling with 96 channel pipetting devices, such as the Matrix Platemate. When samples are grouped together for the purposes of replica dispensing, there is a clear advantage in incorporating an identifier to the source plate or block. HTS inventory systems achieve much of their efficiency by treating plates or blocks of samples as individual entities for tracking purposes. Each plate typically has an identifier, which in turn, references a plate map showing the identities of the individual samples together with their location. Another key feature of HTS data handling systems is the close association between functions responsible for sample supply (tracking and inventory) and functions responsible for processing assay data generated by the screen. After samples have been tested and data analysed, those meeting the criteria for further progression must be accessed again from the store for further study. For this to be an efficient process, access to the sample ordering/delivery system must be seamless. Commercial and bespoke systems place a pre-

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mium on the close association between both functionalities, often to the extent of implementing both under a common user interface. Examples of commercial systems following this pattern are MDL Screen@(MDL Information Systems Inc.) and ActivityBase@(ID Business Solutions Ltd). BIOASSAY DATA ANALYSIS: OVERVIEW

The analysis of bioassay data encompasses a wide range of techniques that vary in their scope and objectives. Although data analysis focussed on HTS can share functionality with other areas of biology, there are key features, tailored to the demands of HTS, which set the field apart. The first phase of an HTS, for any given sample, is generally in the form of a single test at a single concentration. The progression criterion for acceptance into the next stage in screening is usually defined as activity (e.g. inhibition, in a screen looking for inhibitors) above a predefined level. Deriving the height of the hurdle that a sample must clear in order to be progressed is key to the validity of an HTS. An integral aspect of the screen development will be the association between the potency of known standard compounds in a recognised disease model at multiple concentrations and their effect at a single concentration. The aim is to achieve a protocol with a combination of test concentration and cut-off value that has the best probability of yielding the required level of novel hits of a desired activity. The activity of unknown samples is generally normalized; i.e. expressed as a proportion of other experimental conditions or control samples that serve to define the maximum and minimum response possible in the bioassay. Control samples are generally present on each plate and hence provide a means to minimize plate to plate variation in results. This also allows the progression criterion (cut-offvalue) to be defined in percentage terms and be equally valid for all the plates in a screen. Samples identified for progression are then passed to the sample handling function. Often the next stage is a confirmatory retest, where one or more aliquots of the original active sample are re-tested in a new plate at the same concentration. For data purposes, both replicates should confirm the original result to be acceptable for progression. Hence each replicate is compared individually without averaging. Where selectivity in a further assay (greater or lesser activity) is part of the progression path, a further retest assay may be carried out, at a relatively high scale, on the secondary target. The samples that reliably pass this test may progress to a more conventional biology analysis at this stage. Concentration-response analysis on a range of sample concentrations bears many similarities with conventional biology. Non-linear curve fitting

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programmes such as Grafit@(Erithacasus Software Ltd), Fig.P and other functions integrated into full data analysis suites such as ActivityBase are commonly employed. Unlike research programmes studying defined series of molecules, where the properties of interaction can be more precisely defined, it is common practice in screening to use a generic curve fitting algorithm such as the 4 parameter logistic (4pl) because of the diversity of structural types and behaviours which an HTS programme reveals. In many cases, the mode of interaction is undefined at this stage and is best approximated overall by the 4pl fit or constrained versions of the same. TYPICAL STAGES IN THE ANALYSIS OF HTS DATA

The progression of data in an analysis system can vary widely. Many systems in use today are custom applications, written in-house to fulfil the needs of the HTS operation. This also means that details are hard to obtain and compare. Many of the features discussed below are exemplified in the Glaxo Wellcome custom data analysis system, Blitz. Generation ojsample records As described, this is a key feature of the inventory system. The records, which are created along with plates containing samples, would typically associate the sample and all of its descriptive elements (identifier, structure, molecular weight, etc.) with the plate, the position within the plate and, in some cases, the destination screen for the samples. Creation ofthe experimental protocol Most HTS systems require a protocol definition that contains information about aspects of the work to be carried out. This partly consists of parameters to facilitate the analysis (e.g. dilution ratio) but also captures more general information which can permit future searching of data (i.e. for indexing and records purposes). In some circumstances, in laboratories dealing with a relatively defined set of studies, this takes the place of conventional written laboratory records (forming a so-called LIMS or Laboratory Information Management System) but generally written records are also maintained for intellectual property reasons. Typical information stored at this stage would be the target name, responsible individual or group and associated study or therapeutic area. Some systems allow documents and other supporting files to be associated at this stage.

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Creation of experiments

Within the limits of the experimental protocol it is conventional to define the format of experiments which constitute the HTS. Information collected at this stage would typically refer to aspects such as the plate format of the samples to be tested (96 well, 384 well or other format) and the generic layout of the samples and controls within each plate in the experiment. A graphical interface editor at this stage makes this straightforward. Common practice for a 96 well plate is to define 80 wells (columns 1 to 10) and the remaining 16 wells (columns 1 1 and 12) for control materials; to consist of at least a low (no response) and high (maximum response) control. The meaning of each of these well types is predefined within the data analysis system. The calculation used to derive the desired result can be selected here, or in some systems is inherent in the subsequent analysis. For a single concentration (discrete) assay the cut-off value to be exceeded to indicate a positive would be entered here. Other elements logged at this stage would be the volume and concentration of samples and the solvent used. Implementation of experiments

Data analysis systems differ in how this stage is handled. At least one system requires barcode scanning to register test samples for the upcoming experiment. Barcoding is commonly used where sample generation and hit identification are carried out by separate functions within an organisation so as to ensure accurate tracking of samples. More generically, a specific number of samples are selected at this point for subsequent association with data. Raw data handling In any HTS operation there is likely to be a wide variety of instrumentation used to measure the signal associated with the assay. For many reasons (manufacturer preference, signal type, i.e. CPM or DPM for radioactive counters, O D units for colourimeters, fluorescence units for fluorimeters etc.) the format of the data presented as the output for a plate varies. To cope with this, most data analysis systems will incorporate some type of template editor which allows the user to construct an automatic process to convert a ‘raw’data file from a given reader into a uniform format which the remainder of the system can use. For example, blank lines and superfluous data labels and text could be deleted. For any given experiment, it is then sufficient to reference the template which matches the reader, and the conversion is enabled automatically. At this stage, the system has been supplied

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with sufficient information to carry out the remainder of the analysis automatically. This can be performed in an ‘on-line’ way whereby a user initiates the analysis by locating data files appropriate for a given experiment. Alternatively this can take place in an ‘off-line’ way, where the system itself monitors for the generation of new data files in a predefined directory and initiates an analysis as the data arrives. Quulity control, error review

When the calculations have taken place it is necessary to be able to view the data and check the validity of all values. This type of quality control is implemented in a variety of ways, but a graphical representation is convenient. A minimal requirement is the ability to invalidate a result that is abnormal in some way. Other aspects of quality control, showing intra-plate and plate to plate variability can be implemented. Generation of output Depending on the system in use, output may be defined as a formatted list of active samples, a sample order supplied to an inventory system to source a subsequent test phase or a graphical presentation of IC50 curves. Systems differ in the focus placed on these aspects; some are very closely coupled with sample handling systems, some work independently using the sample identity as the common currency. Other outputs At any stage in an HTS process it is useful to be able to obtain an overall summary of data and results. Essentially all data handling systems have at their core a relational database of some type, usually Oracle@ based, which contains all of the information. This gives a huge scope for extracting information as required, by means of SQL based tools. A variety of third party software solutions exist to allow for summaries, updates, and specific types of ad hoc query to be predefined. The scope of these types of query is essentially unlimited, but careful thought must be given to the speed of response. This can be dramatically affected by the internal model structure of the database and this must be taken into account when designing queries.

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CONCLUDING REMARKS The past decade has seen a revolution in the lead discovery process, through the introduction of combinatorial chemistry techniques, improved bioassay technologies, screening automation and data handling systems. For the millennium, the process is poised for development towards a more efficient, higher capacity process, smarter in its ability to use past screen information to refine and expand chemical sampling. For bioassay design, the future will encompass more specific mechanistic assay formats, generating a higher quality of screen information. Assay technology will be compatible with more miniaturized formats (1 536 or beyond), allowing for an increased number of compounds to be screened in a more cost-effectiveway. As the quality of the primary assay data improves, so will the capabilities of the informatics groups. This will result in the routine re-use of information for more efficient refinement of hits, in terms of potency, selectivity and pharamacokinetics, and synthesis of new lead series based on information gleaned from primary assays. Drug metabolism will adapt to integrate these new processes so that ADME parameters can be a major determinant of good leads becoming good medicines. REFERENCES I 2 3 4 5 6 7 7a 8 9 10 11 12

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Progress in Medicinal Chemistry - Vol. 37, Edited by F.D. King and A.W. Oxford Q 2000 Elsevier Science B.V. All rights reserved.

4 Development of Neurosteroid-Based Novel Psychotropic Drugs DOODIPALA S. REDDY and SHRINIVAS K. KULKARNI* Depurtment of 'Pharmacology, University Institute of Pharmuceuticul Sciences, Punjub University, Chandigurh - 160 014, India

INTRODUCTION Identification and detection of neurosteroids Biosynthetic pathways Mitochondria1 DBI receptors

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MECHANISMS OF ACTION GABA-A receptor modulation NMDA receptor modulation Sigma receptor modulation Glycine receptor modulation Voltage-gated calcium channel modulation Nicotinic receptor modulation Steroid receptor modulation

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THERAPEUTIC POTENTIAL Stress Epilepsy Anxiety Learning and memory Aggression Depression Alcohol-related behaviours Sleep Food intake Drug tolerance and dependence ACTH mechanisms Pain and migraine Ageing and neurodegeneration

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*Correspondence to be addressed to Prof. Kulkarni 135

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CONCLUSIONS

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INTRODUCTION The term ‘neurosteroid’ denotes a steroid that is synthesized in the central nervous system (CNS), either de novo from cholesterol or from steroid hormone precursors, and which accumulates in the nervous system to levels that are at least in part independent of steroidogenic gland secretion rates. The steroid may be synthesized by classical endocrine tissues but have an action on neural tissues and participate in neuronal signalling. The term ‘neuroactive steroid’ on the other hand may refer to either an endogenous or a synthetic steroid that rapidly alters CNS excitability [ 1,2]. Selye first described the sedative-anaesthetic activity of progesterone (1) and deoxycorticosterone (DOC, 2) six decades ago and suggested that their ring A-reduced metabolites were extremely potent agents [3, 41. In subsequent years, several steroid anaesthetics were synthesized and introduced into medicine including for example, alphaxolone (3), which has been used extensively in the clinic [6]. It was hypothesized that such steroids induce anaesthesia by augmenting inhibitory neurotransmission in the CNS. However, it has become apparent that the mechanisms responsible for the rapid effects of steroids on neuronal excitability are unrelated to their classical intracellular receptors that regulate gene expression [5].During the last decade several steroids have been characterized as neuro(active) steroids including progesterone, its metabolite allopregnanolone (AP, 4) (3a-hydroxy-5apregnan-20-one) and the deoxycorticosterone metabolite, allotetrahydrodeoxy-corticosterone (THDOC, 5 ) (3a,21-dihydroxy-5a-pregnan-20-one), pregnenolone, pregnenolone sulphate (PS, 6) (5-pregnen-31-01-20-one sulphate), dehydroepiandrosterone (DHEA), and dehydroepiandrosterone sulphate (DHEAS, 7) (d5-androsten-3j-ol- 17-one sulphate) [7-91 (see Figure 4.1). Glucocorticoids, androgens and oestrogens are probably not neurosteroids because they disappear from the brain after gonadectomy and adrenalectomy [101. An understanding of the role played by neurosteroids in behavioural and neurochemical activities is just beginning to unfold and they have been implicated in diverse physiological and neurological disorders. Consequently, there is increasing interest in the pharmacology of this group of compounds

D.S. REDDY AND S.K. KULKARNI

&

0

/

(1) Progesterone

HO""

H3

0&OH / (2) Deoxycorticosterone

H

(3) Alphaxolone

(4) Allopregnanolone

HO"'

H (5) Allotetrahydro-DOC

(7) DHEA-sulphate

(6) Pregnenolone sulphate

(8) Pregnanolone

Figure 4.1. Cliemiculs1ructure.sof neuro(uc*five)steroids. (Continued on ne.ufpuge)

137

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N EUROSTEROID-BASEDPSYCHOTROPIC DRUGS

(9) 7a-Hydroxyprogesterone

(11) Tetrahydro-DOC

(13) Alphadolone

(1 0) 5a-Dihydroprogesterone

(12) 7a-Hydroxy-DOC

(14) RU-5135

Figure 4.1. (continued)

as they have been proposed as endogenous ligands at avariety of neurotransmitter receptors in the brain. This article briefly reviews the current state of neurosteroid research, in particular their biosynthetic pathways and regulation of synthesis, putative receptor mechanisms and their implications for the development of new drugs. IDENTIFICATION AND DETECTION OF NEUROSTEROIDS

Lack of suitable and reproducible methodology for the separation and detection of neurosteroids in plasma and brain has greatly hampered investigation of their neurochemistry. Nevertheless, the new gas chromatography/ negative ion chemical ionisation mass spectrometry methodology now permits accurate assessment of pregnenolone, progesterone and 5a- and Sfl-reduced metabolites, simultaneously, in a single sample [ 111. After partial

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purification by high performance liquid chromatography (HPLC), neurosteroids are derivatized with heptafluorobutyric acid anhydride or FLOROX reagent. Ionisation in the mass spectrometer in its negative ion chemical ionisation mode allows identification by each steroid’s unique fragmentation spectrum in addition to quantification of the compound at concentrations as low as femtomolar [ 121. Pregnenolone is obtained from whole rat brain by extraction with ethyl acetate followed by silica gel column chromatography. The fraction obtained is derivatized with I-dimethylarninonaphthalene-5-sulphonylhydrazine (dansylhydrazine) or 4-(N,N-dimethylaminosulphonyl)-7-hydrazino-2,1,3benzoxadiazole, and the derivative is separated by successive preparative HPLC with fluorescence detection [ 131. BIOSYNTHETIC PATHWAYS

There is much evidence to support the view that the brain is a steroidogenic organ and synthesizes a variety of neurosteroids. For example, abundant quantities of pregnenolone, DHEA, their sulphate esters and fatty acid esters have been found in rat brain at concentrations independent of their plasma levels [ I , 141. Moreover, accumulation of several 3a-hydroxy-neurosteroids in the brain was demonstrated in rats, rabbits, monkeys and human beings [2, lo]. These steroids appeared to be independent of gonadal and adrenal synthesis, since they persist after adrenalectomy and gonadectomy or after pharmacological suppression of adrenal and gonadal secretions. Myelin of the white matter in the CNS is made up of a particular type of glial cells, differentiated into oligodendrocytes and astrocytes. Glial cells play an important role in the repair of the CNS and astrocytes in the brain control in part, the extracellular environment and neuron function by their implication in ion transport, neurotransmitter metabolism and synthesis of neurotrophic factors. Therefore, glial cells may be involved in the biosynthesis and metabolism of neurosteroids. Several neurosteroids have been shown to be formed de nova in mammalian brain via classical steroid metabolic pathways [8,15] (Figure 4.2). Cytochrome P450 catalysed side-chain cleavage (P45OS,J of cholesterol to pregnenolone has been demonstrated in mitochondria of glial cultures of oligodendrocyte-rich embryonic rat brain [161. Incubation of primary cultures of rat forebrain glial cultures with a precursor to cholesterol led to the formation of cholesterol itself, pregnenolone, progesterone and 20-hydroxy-pregnenolone [ 16,171. Biochemical, immunohistochemical and molecular biological studies have also revealed the presence of P450,,, enzyme activity in mammalian brain [ 181. It is found mainly in the cerebral cortex,

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CHOLESTEROL

1

Cytochrome P450,,

PREGNENOLONE

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Cytochrome P450,,,

7-a-HYDROXYPREGNENOLONE

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Sulphatase

dehydrogenme/isomerase

i

P4501,

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SuIphatme

* SULPHATE

SULPHATE

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5a-Reductme

21 -Hydroxylme

DIHYDROPROGESTERONE

!

3a-Hydroxysteroid oxidoreductme

*

I

CORTICOSTERONE

I

3a-Hydroxyteroid oxidoreductme

ALLOTETRAHYDRO-DOC

Figure 4.2. Biochemical pathways of neurosteroid synthesis in ihe brain (Modifiedfrom Ref: 2 with the permission of Prous Publishers).

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amygdala, hippocampus and mid brain and is expressed in both the central and peripheral nervous systems of the developing rat and mouse foetus, as well as throughout the adult brain [ 191, suggesting that neurosteroids are biosynthesized during embryogenesis and development of the nervous system. It has been shown that the brain can convert pregnenolone into progesterone, a d4-ox0 steroid. This conversion has been reported to occur in homogenates of discrete brain areas, as well as in cultured glial cells and neurons [20.21]. This key enzymatic step is catalysed by the enzyme 3P-hydroxysteroid dehydrogenase/d5-A4 isomerase (3P-HSD). Expression of a 3P-HSD mRNA has been reported in neurons of the olfactory bulb, striatum, cortex, thalamus, hypothalamus, septum, hippocampus and cerebellum [22]. The presence of 3P-HSD in rat brain provides additional evidence for the local biosynthetic pathway for neurosteroids. Enzymes present in the limbic regions of the brain and in glial cell culture have been shown to catalyse the oxidation of pregnenolone to progesterone [23]. In addition, mixed cultures of neurons and glia reduce progesterone to AP (4) via the intermediate Sa-pregnane-3,20-dione [24], which is formed by Sa-reductase activity present in both neurons and glial cells [25]. Further reduction of 5a-pregnan-3,20-dione to AP is reported to occur in astrocytes of rat brain by 3a-steroid hydroxysteroid oxido-reductase (3a-HSOR) [26]. AP can be measured as late as three weeks after gonadectomy and adrenalectomy of adult male rats [l l ] indicating the presence of 3a-HSOR in the brain. The metabolic pathway of steroids involving the Sa-reductase has been further characterized in the brain [27]. The greatest amount of 3a-HSOR activity is found in the pituitary and hypothalamus, with lesser amounts in the cerebellum, thalamus, midbrain medulla and pineal gland. The highest amount of Scc-reductaseactivity is found in the pituitary, followed by the hypothalamus, medulla, thalamus, midbrain, Cerebellum and pineal gland [28]. Both 5a-reductase and 3a-HSOR activities are influenced by the estrous cycle with peak activities at proestrus, estrus and metestrus. Changes in enzymatic activity appear to inversely correlate with plasma estradiol levels, and can be mimicked by overiectomy/oestrogen replacement, suggesting that both enzymes and substrates for neurosteroid synthesis may be modulated in vivo by the hormones present [29]. It has been demonstrated that rat brain contains mRNA for 1 lg-hydroxylase (P45o1la), encoding the 1 Ig-hydroxylase that converts 11-deoxycortisol to cortisol [30]. The concentrations of P4501I p mRNA are greatest in the cortex, with lesser amounts in amygdala, hippocampus and midbrain. Unlike P450,,, mRNA, there are gender differences in P4501I B mRNA concentra-

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tions with expression in the female greater than in the male hippocampus [28]. P45011p mRNA may be expressed in neurons, rather than in glia. The lack of P450-11 aldosterone synthase mRNA expression suggests that the rat brain does not synthesize aldosterone. The adult rat brain does not have 17a-hydroxylase activity and P45OI7pmRNA [30]. Thus, rat brain cannot convert pregnenolone or progesterone into 17-hydroxylated steroids. However, DHEA concentrations in brain persist long after removal of gonads and adrenals [311 suggesting there are alternative pathways for its biogenesis in the brain. Furthermore, it has been demonstrated in vitro that, as in steroidogenic glands, the brain converts DHEA to androstanedione [32]. Brain slices or homogenates, as well as cultured new-born rat glial cells, foetal neurons and astroglial cells incubated with radioactive DHEA or pregnenolone yielded large quantities of very polar metabolites identified as 38-androstanediols. Neurosteroids DHEA and pregnenolone are converted by rat brain microsomes into the corresponding 7a-hydroxylated derivatives through a cytochrome P450 7a-hydroxylase enzyme in a NADPH-dependent fashion [33, 341. Neurosteroidogenesis in other neural tissues has recently received much attention. For example, biochemical and immunocytochemical studies revealed the presence of cytochrome P450,,, enzyme in the retinal ganglion cell layer, and abundant quantities of pregnenolone, progesterone, AP, DHEA, THDOC and other 17-hydroxysteroidsindicating the presence of a steroidogenic pathway within the retina [35, 361. The retina is currently under development as an in vitro model system to study neurosteroidogenesis in the CNS. Myelination in the peripheral nervous system is carried out by Schwann cells in place of oligodendrocytes and therefore Schwann cells share with oligodendrocytes the capacity to produce neurosteroids. Biochemical and immunocytochemical studies have demonstrated the neurosteroidogenesis process in the sciatic nerve of adult male rats and mice which is independent of adrenal and gonadal sources [37]. However, the physiological significance of these findings remains unclear. MITOCHONDRIAL DBI RECEPTORS

Although the biosynthesis of neurosteroids in the brain has been widely investigated, little is currently known concerning the regulation of neurosteroid synthesis and metabolism. The rate-limiting step in neurosteroid biosynthesis is the conversion of cholesterol to pregnenolone. This reaction is catalysed by the enzyme P450,,,, which is located on the inner mitochondria1

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membrane. Several laboratories have established that it is the rate at which cholesterol is transported to the inner mitochondrial membrane, and not P450,,, activity itself, that limits the rate of pregnenolone synthesis [38]. A receptor that is located on the outer mitochondrial membrane participates in the regulation of intramitochondrial cholesterol transport [39]. Diazepam-binding inhibitor (DBI), an endogenous 9-kDa and 86-amino acid peptide, binds to this receptor and stimulates steroidogenesis by facilitating cholesterol transport to the inner mitochondrial membrane [40 - 421. Mitochondrial DBI receptor is a heterooligomeric complex that has high-affinity recognition sites for the isoquinoline carboxamide, PK 1 1 195 (1 5), the imidazopyridine, alpidem, the benzodiazepine, 4'-chlordiazepam [39, 421, and the 2-arylindoleacetamide FGIN- 1-27 (16) [43]. The mitochondrial DBI receptor consists of protein subunits of 18, 30, and 32 kDa [39, 441. It is the 18-kDa subunit that binds to PK11195 and this has been purified and cloned [45] and has since been shown to complex with the mitochondrial voltage-dependent anion carrier (VDAC) and the adenine nucleotide carrier protein [44]. Four to six molecules of the mitochondrial DBI receptor may form a pore within the mitochondria in association with VDAC, possibly in a 5:l stoichiometry [46]. VDAC is located in mitochondrial membrane contact sites and therefore may bridge the outer and inner membranes of the mitochondria.

(15) PK 11195

F* 'c

(17) FGIN-1-29

(16) FGIN-1-27

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High affinity mitochondrial DBI receptor ligands such as alpidem and 4’chlordiazepam at nanomolar concentrations, stimulate pregnenolone synthesis in glia and neurons [41,42]. It seems possible that this is the mechanism by which these ligands produce behavioural effects in animals [47]. PK11195 does not show marked behavioural effects in rats and thus is characterized as an antagonist of mitochondrial DBI receptor modulated steroidogenesis. Recently, a new class of ligands, 2-arylindole-3-acetamides such as FGIN-1-27 (16) and FGIN-1-29 (17), that bind with high affinity and specificity to the mitochondria1 DBI rcccptors have been shown to elicit antineophobic and anxiolytic effects via stimulating pregnenolone formation in the brain [47,48]. MECHANISMS OF ACTION Neurosteroids exert their effects rapidly and thus could not involve steroid receptor-mediated activation of gene transcription [5]. Data generated during the past decade have demonstrated that the rapid inhibition of CNS excitability by neurosteroids results from their selective interaction with the major inhibitory and excitatory neurotransmitter receptors (Table 4. I ) . Table 4.I POTENTIAL NEUROTRANSMITTER RECEPTOR SITES FOR NEUROACTIVE STEROID MODULATION O F BRAIN FUNCTION

Progesterone Epipregnanolone Allopregnanolone Ganaxolone Minaxolone Pregnenolone PS DHEAS Deoxycorticosterone THDOC Alphaxolone Alphadolone RU 5135 THCC

GABA-A

NMDA

Glycine

PAM^

NCA

NCA NCA

NCA PAM PAM PAM NCA NCA NCA

PAM^

PAM PAM PAM NCA PAM

VGCC

Sigma

NCA NCA

PAM PAM

NCA NCA

Nicotinic

NCA NCA NCA NCA

PAM NCA N CA

aVGCC; Voltage-gated Ca2+channel bInactiveper se but metabolites are positive modulators of the receptor ‘PAM’ Positive allosteric modulator ‘NCA’ Non-competitive antagonist

NCA

PAM PAM

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Figure 4.3. Structure of GABA-A receptor coniples. Prc~p~poserl molecular sitesfor GA BA, convul(CON VS), horhiturutes (BARBIT), henzodiuzepines (BZD) und neur0steroid.s (NS) are slio~t~n, lioweever their esmt locutions on GABA-A rcwqtor ure not known (Reprodured,fronl Ref.' 2 with the perniission of Prous Puhlisliers). .writs

GABA-A RECEPTOR MODULATION

The GABA-A receptor, a member of the ligand-gated ion channel superfamily, is a heterooligomeric protein consisting of multiple homologous membrane-spanning subunits (a, /I. y. 6, E and n) that form an integral chloride channel [49]. The four a-helical regions that span the plasma membrane form the channel for chloride ions (Figure 4.3). In mammals, the 13 known subtypes of GABA-A receptor subunits have been categorized within four y1-3 and 6). These subunits are thought to asstructural classes semble in different pentameric complexes, with most functional receptors containing a//)/;) or Cc//j/G subunit combinations, which exhibit the properties of native GABA-A receptors. Variations in subunit composition can have profound effects on the sensitivity of GABA-A receptors to modulatory agents such as barbiturates and benzodiazepines. Electrophysiological and ligand binding experiments showed that neurosteroids also interact with the GABA-A receptors. Alphaxolone (3) was shown to augment inhibitory events mediated by GABA in spinal cord and olfactory neurons, and potentiated the effects of GABA when the GABA

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was applied to slices of rat cuneate nucleus, and enhanced the binding of a GABA-A receptor agonist, [3H]-muscimol [50]. Low concentrations of alphaxolone (30 nM) markedly potentiated the amplitude of membrane currents elicited by GABA, whereas higher concentrations (1 pM) resulted in a direct bicuculline-insensitive chloride current, in a manner similar to that produced with barbiturates [5 13. Betaxolone, the inactive 3B-hydroxy epimer of alphaxolone, failed to augment GABA-activated chloride conductance, suggesting that neurosteroids, unlike barbiturates and anticonvulsant drugs, act stereoselectively and in a rather specific manner in augmenting GABAactivated chloride ion conductance. Submicromolar concentrations of AP(4) and THDOC (5) inhibited specific binding of [35S]-TBPS(t-butylbicyclophosphorothionate) to crude synaptosomal membranes, enhanced the binding of [3H]-benzodiazepines to GABA-A receptors, and directly stimulated 36Cl-uptake in synaptoneurosomes in a picrotoxin sensitive manner [52]. However, their parent steroids, progesterone and deoxycortisone (2), failed to modulate GABA-induced chloride currents except in high concentrations. AP and THDOC augmented GABA-A receptor mediated 36Cl-flux in synaptoneurosomes at nanomolar concentrations [53], and dose-dependently and reversibly enhanced GABA-induced chloride currents in bovine chromaffin cells [54]. There is considerable evidence to suggest that neurosteroids act as allosteric agonists of the GABA-A receptor and indirectly potentiate GABA-activated chloride conductance in low concentrations and directly stimulate chloride conductance in higher concentrations [55]. The GABA-A receptor active steroids increase the average duration of the channel in the open state and increase the frequency of single channel openings. In the latter respect, their molecular mechanism resembles that of the benzodiazepines, where the promotion of the long open-state is similar to that produced by the barbiturates [55]. Structure-activity requirements for neurosteroids interacting with the neurosteroid binding site on the GABA-A receptor have revealed a marked stereoselectivity for both binding and receptor activity [56, 571. The requirements are the 3a-hydroxy configuration at C-3 on the steroid A ring and the presence of a keto group at either (2-20 of the pregnane steroid side-chain or C-17 of the androstane ring system. The P-epimers were 40% less active in augmenting GABA-receptor-mediated 36Cl- uptake than either AP or THDOC [58]. Introduction of the 2B-morpholinyl moiety may confer water solubility for pregnane steroids without loss of GABA-A receptor activity [55]. Molecular modelling provided a rationale for the observation that the configuration of the hydroxyl group at C-3 is a greater determinant of anaesthetic potency than the stereochemistry of the A/B ring fusion at C-5. The

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electrophysiological results identified steric restrictions on the space occupied by 5a- and 5fi-reduced steroid modulators of GABA-A receptors. These regions were close to the steroid C-5, C-10, and possibly C-4 positions [59]. Furthermore, molecular modelling of neurosteroids indicated the presence of multiple steroid recognition sites associated with the GABA-A receptor complex. Further evidence suggests that neurosteroids bind to a specific site on the GABA-A receptor complex, distinct from the benzodiazepine, barbiturate or convulsant recognition sites (Figure 4.3). AP can enhance GABA-A receptor-mediated chloride currents even in the presence of maximal effective concentrations of pentobarbital [54, 581. However, together they exhibit synergistic but nonadditive effects on the inhibition of [35S]-TBPSbinding [60]. A steroid recognition site has been shown to be functionally coupled to the GABA-A receptor [61]. There is an excellent correlation between the pharmacological and electrophysiological interactions of these positive steroid modulators with GABA-A receptor complexes [62]. Neurosteroids markedly enhance the benzodiazepine agonist [“HI-flunitrazepam binding, but do not affect benzodiazepine antagonist [3H]-flumazenil or benzodiazepine inverse agonist [3H]-FG 7 142 binding to the GABA-A receptor complex [63, 641. However, differences in these effects across brain regions have also been reported [65]. Neuroactive steroids which are positive allosteric modulators of the GABA-A receptor complex have been termed ‘epalons’, and the steroid binding site has been also named as an ‘epalon recognition site’ [66, 671. The presence of multiple binding sites for epalons has been suggested based on electrophysiological and binding assays [62,68]. The epalon AP-induced enhancement of [3H]-muscimol binding may result from an increased density of high affinity [3H]-muscimol binding sites and an increased affinity of [3H]-muscimol for low affinity binding sites. This binding occurs via both one- and two-component mechanisms in a stereoselective and structure specific manner. Although the exact location and nature of the neurosteroid binding site is currently unknown, neurosteroids AP and THDOC have been proposed as endogenous ligands of the GABA-A receptor complex. In recent years, the identification, cloning and sequencing of the neurosteroid binding site on the GABA-A receptor has emerged as the most challenging and high priority task in many neuropharmacology laboratories. Harrison and colleagues have constructed two sets of chimeras between alphaxolone-sensitive GABA-A receptor m2- or P,-subunits and the alphaxolone-insensitive glycine receptor a1-subunit to determine the structural domains important for the modulatory actions of neuroactive steroids [69]. The site of action for neurosteroids appears to be different from that for vo-

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latile anaesthetics and ethanol, but is identified on the N-terminal side of the middle of transmembrane-2 of the GABA-A receptor. Neurosteroids may differentially modulate recombinantly expressed GABA-A receptors. AP enhanced the GABA-activated chloride currents greater than that of flunitrazepam in human embryonic kidney cells which are transfected with various combinations of GABA-A receptor subunit cDNAs [70]. A broad biphasic concentration-response relationship which was observed with a single form of the GABA-A receptor (a1+PI +?I), suggests the existence of multiple affinity states for neurosteroid binding to a given GABA-A isoreceptor [71]. Expression of y2-subunits with either a l +PI or a2+/31-subunitsyields GABA-A receptors that are markedly more sensitive to steroids than those containing the a3-subunit [72], suggesting that neurosteroidal modulation of GABA-A receptors is markedly influenced by the a- and y2-subunit types expressed. Expression of the cc3-subunit with /3+y2, resulted in receptors that are more steroid sensitive than those containing the a l - and cc2-subunits[73]. Coexpression of the yz-subunit with aand 8-subunits results in GABA-A receptors that are more neurosteroid sensitive. However, further studies suggest that the 8-subunit does not influence the pharmacology of the neurosteroid site of the GABA-A receptor complex [74, 751. In addition, 6- and &-subunitsthat can assemble with c1- and 8-subunits may inhibit neurosteroid modulation or confer an insensitivity at GABA-A receptors [76,77]. Neurosteroids increase the probability of GABA-A receptor chloride channels being open when steady-state activation is induced by low agonist concentrations. Although neurosteroids potentiate inhibitory synaptic transmission by increasing the duration of the inhibitory post-synaptic conductance, the mechanisms of neurosteroid modulation of GABAergic synaptic transmission cannot be predicted easily from studies of neurosteroid effects on steady-state, low agonist-activated currents, because synaptic currents are produced by transient jumps in high GABA concentrations that induce a desensitising response [78]. Furthermore, neurosteroid modulation of GABA-gated currents under nonequilibrium recording conditions has been characterized using fast applications of agonist to nucleated patches of rat cerebellar slices [79]. THDOC potentiates the inhibitory postsynaptic transmission via the prolongation of the slow deactivation. The alteration of kinetics of entry and exit from desensitised states is the basis of the allosteric modification of GABA-A receptors by neurosteroids [go]. Neuroactive steroids with partial agonist activity have also been reported [81]. Previous studies with Scl-THDOC (5) and its stereoisomer 58-THDOC (1 1) revealed important differences in potency, efficacy and regional selectivity at the GABA-A receptor complex, accounted for by a difference in the

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spatial orientation of the steroid A-ring [65] (Figure 4.4). In contrast to AP and 5a-THDOC, 58-THDOC has limited efficacy as an allosteric modulator of [35S]-TBPSbinding, and antagonizes the action of AP and Sa-THDOC at GABA-A receptors in vitro and in vivo [82]. Thus, 5fi-THDOC represents a partial agonist that may serve as tool for the elucidation of the physiological role of endogenous neurosteroids. The sulphated neurosteroids such as PS and DHEAS, act as non-competitive antagonists of the GABA-A receptor and inhibit GABA-induced chloride channel opening [83-861, while pregnenolone acts as an inverse agonist at the GABA-A receptor [87]. Low micromolar concentrations of PS and DHEAS antagonise GABA receptor-mediated 36Cl- uptake in synaptoneurosomes and chloride conductance in cultured neurons [88]. Both steroids have been reported to antagonize GABA-A receptor-mediated currents by reducing channel opening frequency [89], analogous to BZD receptor inverse agonists. PS, at micromolar concentrations, interacts with the GABA-A receptor complex as a picrotoxin-like antagonist [88]. PS binds to a convulsant picrotoxin-TBPS recognition site and inhibits pentobarbitalenhanced benzodiazepine binding and GABA agonist-stimulated chloride influx. Although the proposed PS binding site has been shown to be on the a I subunit of the GABA-A receptor [90], further details of this steroid-binding site are currently unknown. In contrast to PS, micromolar concentrations of DHEAS reduce [‘HI-flunitrazepam binding [91]. Pregnenolone, the desulphated form of PS, is inactive as a modulator of the GABA-A receptor [88], whereas DHEA inhibits GABA-induced currents, albeit 3-4 times less potently than DHEAS [9 I]. PS exhibits mixed GABA-agonist/antagonist properties, while DHEAS is devoid of these effects. Although PS and DHEAS inhibit each others binding, their specific sites of binding appear distinct. While [’HI-PS binding to H

0

HO HO 5wTHDOC

5p-THDOC

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NEUROSTEROID-BASED PSYCHOTROPIC DRUGS

high ai€inity sites is inhibited primarily by picrotoxin, and minimally by barbiturates, the binding of [3H]-DHEAS is robustly inhibited by barbiturates, but not by picrotoxin [MI. These data suggest that the sites of action for PS may be the same or proximal to those for convulsants, whereas DHEAS may act at sites close or identical to those where barbiturates act at the GABA-A receptor. DHEAS, but not DHEA, competitively inhibits the binding of [35S]-TBPSto rat brain membranes and it displaces [35S]-TBPS binding from its binding sites with a profile similar to other convulsants and non-competitive antagonists of GABA-A receptors such as picrotoxin, TBPS and pentylenetetrazole [92]. These data indicate that DHEAS, but not DHEA, interacts competitively with the picrotoxin/TBPS site and this interaction may be responsible for the non-competitive inhibition of GABA responses. In contrast to in v i m studies, only a limited number of in vivo studies have shown the effects of chronic treatment with neurosteroids on the GABA-A receptor. A recent study indicated that chronic treatment (10 pM, 48 h) ) epipregnanolone (EP) (3c(,5[1) eliminated the potenwith AP (4) ( 3 4 5 ~and tiation by EP of [3H]-flunitrazepam binding in chick whole-brain neurons [93]. Further investigation revealed that chronic AP treatment (2 pM, 5 days) produced down regulation of the GABA-A receptors, heterologous uncoupling and decreased heterologous efficacy at the GABA-A receptor complex in mammalian cortical neurons [94, 951. Electrophysiological experiments showed decreased GABA-induced currents with AP and pentobarbital after chronic AP treatment in cortical cells [96], suggesting that the GABA response and its potentiation by barbiturates and neurosteroids are attenuated after chronic neurosteroid treatment. NMDA RECEPTOR MODULATION

N-Methyl-D-aspartate (NMDA) receptor is an ionotropic L-glutamate receptor. It contains multiple binding sites for various ligands at which transmitters, cotransmitters, and pharmacological agents can act either to regulate channel gating or to modulate the channel-gating effects of other ligands. These include an agonist-binding site which also binds competitive antagonists, and binding sites for glycine, Zn2+,Mg2+ ions and phencyclidine. The responses of the NMDA receptor are positively modulated by glycine and glycine may be required for receptor function [97]. Sulphated neurosteroids PS and DHEAS have been shown to be potent allosteric agonists at the NMDA receptor complex [98]. PS can potentiate NMDA-mediated responses, as assessed by electrophysiological recording [99] or measurements of NMDA-induced increases in intracellular Ca2+

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[ 1001 in cultured neurons. PS selectively augments NMDA receptormediated, glut amate-induced, depolarisation in chick spinal cord neurons, while inhibiting the GABA, glycine, and non-NMDA response. PS potentiation of the NMDA response is unaffected by the presence of maximal glycine indicating that the steroid modulatory site is distinct from the glycine modulatory site [98]. Furthermore, it was found that PS markedly augments NMDA receptor-mediated elevations in the intracellular calcium ion concentration in cultured rat hippocampal neurons [7]. PS and its derivatives potentiated, while 3a-hydroxy-5p-pregnan-20-one sulphate inhibited NMDA-induced elevations in intracellular Ca2+ [ 1011. PS has also been shown to potentiate the NMDA-mediated increase in intracellular Ca2+in cultured chick cortical neurons [102].Despite the potentiation of NMDA-induced inward currents and intracellular Ca2+, PS does not potentiate the neurotoxic effects of NMDA in cultured rat cortical or hippocampal neurons. PS modulates the NMDA receptor by increasing the fractional open time of NMDA-activated channels, by increasing the frequency of opening and the duration of channel opening. Systemic progesterone administration can inhibit NMDA responses in rat cerebellar neurons [103]. The non-competitive interaction of these steroids with the NMDA receptor and a high degree of structural specificity suggest the presence of one or more steroid recognition sites on the NMDA receptor complex. SIGMA RECEPTOR MODULATION

Sigma (0) receptors, a distinct family of receptors, are present in high density in the brain [104]. Binding and bioassay studies provided evidence for the existence of at least two subtypes, denoted el and c2 [105].The most commonly used c ligands, including haloperidol and DTG [ 1,3-di-(2-tolyl)guanidine], are non-specific ligands of c receptors, whereas drugs such as (+)-pentazocine; PD- 144,418 [ 1-propyl-5-(3-p-tolyl-isoxazol-5-yl)-1,2,3,6tetrahydropyridine]; (+)-SKF- 10,047 (N-allylnormetazocine); BMY- 14802 [a-( 4-fluorophenyl)-4-( 5-fluoro-2-pyrimidiny1)-1-piperazine butanol]; and NE-100 [N-dipropyl-2-(4-methoxy-3-(2-phenylethoxy)phenyl)-ethylamine monohydrochloride], are selective for a lreceptors [106]. The elsite was recently purified and the cDNA was cloned, which showed there was a shared homology with fungal sterol C& isomerase [107]. al-Receptor agonists potentiate several NMDA-mediated responses [ 1061, suggesting an interaction between NMDA and c receptors. Radioligand and neuropsychopharmacological studies revealed that several neurosteroids, including progesterone, pregnenolone, DHEAS and PS interact with a receptors [108, 109, 1 lo].A crossed pharmacology between

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the effects of o1 ligands and neurosteroids was recently decribed, DHEAS and PS behaving as agonists and progesterone as an antagonist [ 1 1 1, 1 121. DHEAS is reported to potentiate, whereas PS inhibits the NMDA-induced [3H]-norepinephrinerelease from preloaded hippocampal slices, both the effects are sensitive to t~ receptor antagonists haloperidol and BD-1063 { 1-[2(3,4-dichlorophenyl)ethyl]-4-methylpiperazine}, as well as to progesterone [l 1 11. DHEA potentiates the NMDA-evoked electrical activity of hippocampal neurons, an effect that could be blocked by the o Iantagonists haloperidol and NE-100, as well as by progesterone [I 121. DHEAS produces antiamnesic effects in a similar manner to selective c1 receptor agonists [113]. This interaction with the t~ systems could constitute a possible mechanism for the neurosteroidal non-genomic effects in cognitive and other behavioural processes. The possibility that neurosteroids may act as endogenous ligands for the t~~ receptors remains open to further investigation. GLYCINE RECEPTOR MODULATION

Glycine is the potent inhibitory neurotransmitter of spinal cord interneurons. Low micromolar concentrations of PS blocks the strychnine-sensitive glycine receptors [114,115] and rapidly and reversibly inhibits the glycine-induced current in a dose-dependent manner (ECSo3.7 pM) in cultured chick spinal cord neurons. It appears to act as a competitive antagonist of the glycine receptor since it induces a parallel, rightward shift of the glycine-dose response curve and the response is neither voltage- nor agonist-dependent. Progesterone also inversely modulates glycine-induced currents [2]. However, neither AP nor alphaxolone have any effect on glycine-activated chloride currents. The neurosteroidal blockade of glycine-mediated chloride channel receptors may also, in part, contribute to the excitatory properties of the steroids and to neuronal homeostasis. VOLTAGE-GATED CALCIUM CHANNEL MODULATION

Voltage-gated calcium channels (VGCC), which are gated by the electrical potential across the plasma membrane, regulate the influx of Ca2+ ions. They have been subclassified as L, N, P and T channels. Low micromolar concentrations of THDOC, DHEA and pregnenolone rapidly and reversibly block VGCC in adult mammalian hippocampal neurons [ 116, 1 171. Tail current analysis shows that THDOC appears to be a reversible selective ligand that depresses the w-conotoxin sensitive portion of the calcium currents. Both pregnenolone and PS, but not progesterone, depress the calcium currents in isolated guinea pig hippocampal CAI neurons [I 181. Pregnenolone,

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THDOC and PS, but not pregnenolone acetate, inhibited a fraction of the calcium channel current in a voltage-dependent manner when perfused extracellularly. THDOC primarily inhibits the w-conotoxin-sensitive or Ntype calcium channel current, while pregnenolone inhibits both the w-conotoxin and the nifedipine-sensitive calcium channel currents. Furthermore, the calcium channel current inhibition may involve a pertussis toxin-sensitive G-protein-coupled mechanism associated with the activation of protein kinase C [ 1 171. In vivo administration of estradiol and PS, but not DHEAS, inhibits the Mg2+/Ca2+-ATPaseactivity in cortex and cerebellum [ 1201. Thus, modulation of calcium channels by neurosteroids may participate in the regulation of synaptic processes such as modulation of neuronal activity and neurotransmitter release. NICOTINIC RECEPTOR MODULATION

3a-Hydroxy-neurosteroids may also affect brain nicotinic acetylcholine receptor function since nicotine-induced seizures are antagonized by pretreatment with allopregnanolone, and the enhancement by this neurosteroid of paired-pulse inhibition in the hippocampal slice is partially reversed by nicotine [ 1201. Progesterone is an allosteric inhibitor of both native and recombinantly expressed nicotinic receptors [ 121, 1221. Further studies revealed that progesterone is a weak inhibitor, while the two A-ring reduced metabolites of progesterone are potent allosteric inhibitors of brain nicotinic receptors [ 1231. However, the neurophysiological significance of such interactions remains unclear. STEROID RECEPTOR MODULATION

In addition to their interactions at neurotransmitter receptors, neurosteroids may also act at classical intracellular steroid receptors and affect gene transcription [ 1241. However, transcriptional activation of the steroid receptors requires intracellular oxidation of the neurosteroids into progestin or glucocortioid receptor-active Sa-pregnane steroids. THERAPEUTIC POTENTIAL STRESS

The cerebral cortical levels of selected neurosteroids are altered by acute stress paradigms such as forced swimming, handling manoeuvres that pre-

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cede sacrifice, carbon dioxide inhalation and foot shock [125-1271. These different stresses are known to differentially affect the type of neuroactive steroid. Foot shock also increases in a time-dependent manner the brain cortical pregnenolone, progesterone and THDOC levels [ 1281. Stress increases AP and THDOC concentrations in the brain [125] to levels at which they can activate GABA-A receptors. Neurosteroids may directly inhibit CRF and subsequent ACTH release via GABA-A receptors, as THDOC administration attenuates corticosterone elevation in response to mild stress, and AP alters the expression and release of CRF in the rat hypothalamus [129]. The stress-induced increase in brain PS levels may suppress the inhibitory input of GABA and contribute to the heightened arousal which is characteristic of early stages of stress [88]. Thus, endogenous neurosteroids may participate in the regulation of the HPA axis and facilitate the recovery of homeostasis following stressful stimuli. Thus, there may be an interplay between PS, a GABA antagonist, and allopregnanolone, a GABA agonist in stress homeostasis. Neurosteroids may, therefore, be exploited for their potential therapeutic application in stress and related disorders. Although acute stress increases neurosteroid levels, the mechanisms by which neurosteroids influence physiological stress remain unclear. Recently, we reported the effects of progesterone, a pregnane neurosteroid precursor and 4’-chlordiazepam, a high affinity mitochondrial DBI receptor agonist, on the immobilisation stressinduced behavioural responses in mice, and compared them with triazolam, a short acting benzodiazepine [ 1301. Chronic progesterone treatment (1 mg/kg x 9 days) produced a significant antistress effect which was blocked by GABA-A antagonists picrotoxin and bicuculline but not by flumazenil, a specific benzodiazepine antagonist. 4’-Chlordiazepam produced marked antistress effects in a flumazenil-insensitive manner, but was blocked by pre-treatment with PK11195, a selective partial agonist of the mitochondrial DBI receptor, and with bicuculline [130]. Although sedation may be an impediment to the therapeutic use of neurosteroids, there are differences between various neurosteroids in the extent to which motor impairment is produced at antistress doses, indicating that the design of specific neurosteroids may have great potential for use in stress-related conditions. EPILEPSY

It has been known for many years that progesterone [4] and certain structurally related 3,20-pregnenediones and 3,20-pregnanediones are able to protect against pentylenetetrazole (PTZ)-induced seizures in animals as are the synthetic A-ring reduced pregnanes 2~-morpholino-5~,3a-pregnanolone

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and alphaxolone [88]. The endogenous metabolites of progesterone, 3a,5apregnanolone and 3a,5p-pregnanolone, exhibit potent anticonvulsant activity against seizures induced by the GABA-A receptor antagonists PTZ, bicuculline, and picrotoxin [131-1331. The anticonvulsant activity of neurosteroids highly correlate with their ability to potentiate the GABA-A receptorevoked chloride currents [ 1341.These neurosteroids exhibit good therapeutic indices in mice, protecting against PTZ-induced seizures with ED50 values of approximately 3.0 mg/kg i.p. and produce locomotor impairment in the rota-rod ataxia test (TDSO)at approximately 20 mg/kg [135]. Lower doses of neurosteroids protect against electrically induced [ 1361 and drug-induced [120, 1371seizures as well as the seizures seen during withdrawal from ethanol [ 1381. Alphaxolone and AP potentiated the antiseizure efficacy of flurazepam in incremental electroconvulsive shock-induced seizures which is consistent with the in vitro allosteric interactions [139, 1401. Neurosteroids at non-sedative doses protect mice against NMDA-induced seizures and mortality [141]. AP and its 3P-methyl analogue, ganaxolone (1 8) as well as diazepam and phenobarbital dose-dependently protected against PTZ-induced clonic convulsions, but dizocilpine, a NMDA receptor antagonist was ineffective. In contrast to diazepam and phenobarbital, neuroactive steroids and dizocilpine were efficacious against the cocaine- and NMDA-induced seizures [ 1401, indicating a unique and broad spectrum anticonvulsant activity. Several clinical studies on the association between seizures and menses in female epileptics led to the speculation that cyclical variations in seizure susceptibility (catamenial epilepsy) may be correlated with changes in ovarian steroid levels [ 1321.The seizure susceptibility of women with catamenial epilepsy is better correlated with levels of the GABA-A receptor active progesterone metabolite, 5a-pregnan-3~,20a-diolthan oestrogen [ 1421. Neurosteroids protect animals against hypoxic convulsions by interacting with GABA-A receptors [ 1431. Therefore, neurosteroids may be considered for further evaluation in conditions such as neuroplasticity and neurodegenerative disease, as well as in the treatment of cerebral ischemia. However, several neurosteroids such as AP and its analogues, are unsuitable as therapeutic agents because they are readily oxidised at the 3a-position [ 1441, resulting in compounds that are inactive at neuronal but potentially active at hormonal steroid receptors [55, 1241. Recently, a 3P-methyl-substituted analogue of allopregnanolone, ganaxolone ( 18), has been synthesized and characterized as a potent anticonvulsant and steroid modulator of GABA-A receptors [ 145,1461. Several lines of evidence indicate that ganaxolone is a high-affinity, stereoselective, positive allosteric modulator of the GABA-A receptor complex that exhibits potent anticonvulsant activity

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(18) Ganaxolone

(19) Minaxolone

across a range of animal procedures. 3b-Substitution, which in part prevents metabolism of the 3a-hydroxy moiety, has been suggested to enhance the bioavailability of pregnane steroids without altering their primary pharmacological properties [66]. Ganaxolone is currently in phase I11 clinical trials as a neurosteroid-based antiepileptic drug. Another derivative of AP, minaxolone (19), (2b,3a,5a,1 1~)-2-ethoxy-3-hydroxy-1 1-N,N-dimethyl-aminopregnane-20-one, is currently under preclinical development [ 1471. Chronic treatment with minaxolone in mice resulted in the development of tolerance to its sedative effects. In contrast to this, acute or chronic administration of sulphated neurosteroids PS and DHEAS may produce excitatory actions on neurons [148] and elicit convulsant [149] and proconvulsant activity [150,1513, indicating a role for sulphated neurosteroids in epileptogenesis. Both PS and DHEAS act as agonists at central sigma receptors and exert a facilitatory action on NMDA-mediated glutametergic neurotransmission [ 1 10, 1 1 I]. ANXIETY

Neurosteroids, like other positive allosteric modulators of the GABA-A receptor complex, also produce anxiolytic, sedative-hypnotic and anaesthetic actions [ 152,1531. AP reduced anxiety-related behaviours in animals in several different tests at a variety of ages. Neurosteroids, AP, THDOC, as well as the synthetic steroid anaesthetic alphaxolone, were potent anxiolytics in

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several animal models of anxiety such as the elevated plus-maze test [154-1561, the conflict test [135, 152, 1561, the light/dark transition test [ 135,1521,the open field test [ 1571, the burying behaviour test [ 1581, an ultrasonic vocalisation test after brief maternal separation [ 1591and the mirrored chamber test [160,161]. DHEA is anxiolytic in the plus-maze test [162], whereas pregnenolone and its sulphate ester PS have been shown to be anxiogenic in adult mice in this test [ 1631. Progesterone and 4’-chlordiazepam produce dose-dependent anxiolytic responses in the mirrored chamber and elevated plus-maze tests [ 161,1621. Further attempts were made to determine the potential mechanisms mediating the anxiolytic/anxiogenic effects of neurosteroids. The AP-, progesterone- and 4’-chlordiazepam-elicited anxiolytic behaviour was blocked by picrotoxin, but not by flumazenil. In contrast to this, the anxiolytic effect of PS was not blocked by picrotoxin. The 4’-chlordiazepam-induced anxiolytic effect was prevented by pre-treatment with PK11195, indicating the role of GABA-A and mitochondria1 DBI receptors in the anxiolytic effects of neurosteroids and 4’-chlordiazepam, respectively [ 1601. PS injected systemically can cross the blood-brain barrier without being hydrolysed to the more lipophilic pregnenolone, and can thus be taken up by the brain [ 1641. PS may increase AP levels in various brain regions. This effect could be blocked by inhibition of Sa-reductase using SKF-10511 or by inhibition of 3a-HSOR with indomethacin [ 1651. Interestingly, the specific serotonin reuptake inhibitor, fluoxetine, also dose-dependently increases AP levels in the brain [ 1661, suggesting that a fluoxetine stimulation of brain AP biosynthesis might be operative in the anxiolytic and antidysphoric actions of this drug. LEARNING A N D MEMORY

Neurosteroids have been reported to modulate learning and memory processes in young, aged rodents and in pharmacological models of amnesia. PS infused into the basal magnocellular nucleus enhanced memory performance, whereas AP disrupted memory [ 1671. Pregnenolone, PS, DHEA, and DHEAS increased memory when injected systemically [168,169], centrally [170,171] or into the amygdala [172]. PS has also been reported to enhance learning in the Morris water maze [ 1731 and improves acquisition and retention in a food search task [174]. In contrast to this, post-training administration of AP reduced retention of memory in a conditioned odour task in rat pups [ 1751. PS and DHEAS produced attenuating effects on the muscarinic receptor antagonist, scopolamine [ 1761 and non-competitive NMDA receptor antagonist, dizocilpine [ 1 13,1771, competitive NMDA

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receptor antagonist, 3-(+)-2-(carboxypiperazin-4-yl)-propyl-l-phosphonic acid (CPP) [178], and ethanol [179] induced amnesia in various cognitive paradigms in rodents. Although the precise mechanism by which neurosteroids affect learning and memory is unknown, it is interesting to hypothesize that memory loss associated with many neurological diseases and normal developmental processes in humans, including ageing, may be a result of altered neurosteroidogenesis. Normal ageing is associated with a decline in cognitive and other brain functions. Apart from the degeneration of cholinergic neurons, there is evidence to suggest that the cognitive dysfunction associated with ageing is characterized by decreased levels of DHEA and DHEAS [90,180]. Neurosteroids are also implicated in Alzheimer’s disease [90,18I]. Administration of DHEA and DHEAS improved performance in the retention of memory in aged animals [182], indicating a possible pathological role for neurosteroids in dementia related cognitive disorders. Pregnenolone, dehydroepiandrosterone, and their sulphate esters, dose-dependently attenuated the memory deficits induced by 825-35 peptide, a protein implicated in Alzheimer’s disease and the effects of the neurosteroids were blocked by the cr receptor antagonist haloperidol and by progesterone [ 1831. In a modified passiveavoidance paradigm, neurosteroids PS or DHEAS facilitate acquisition and consolidation rather than retrieval of memory processes, independent of state-dependent effects on learning [184]. Inhibitors of steroid sulphatase, an enzyme which converts sulphated steroids into free steroids, may alter the metabolism of neurosteroids and may modulate brain function. The steroid sulphatase inhibitor estrone-3-0-sulphamate and p-0-(sulphamoy1)-N-tetradecanoyltyramine [185,1861 potentiated the antiamnesic effect of DHEAS, suggesting that increasing the levels of endogenous sulphated neurosteroids via the inhibition of steroid sulphatase activity may enhance learning and memory function. Further developments in this field would augment the therapeutic potential of sulphatase inhibitors in the treatment of cognitive disorders. The exact mechanism and even specificity of the memory enhancing effect of neurosteroids is currently not known. It is thought that steroid hormones bind to specific receptors in the cytoplasm, which translocate into the nucleus and finally regulate gene transcription or affect translational efficiency and protein stability. These effects are postulated to be involved in long-lasting learning and memory storage. Alternately, it has been hypothesized that a cell membrane receptor mechanism@)may also be involved in the effects of neurosteroids in learning and memory processes. Neurosteroids have been shown to affect the activity of neurotransmitter systems which are involved in the learning and memory processes. PS and DHEAS are

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negative allosteric modulators of the GABA-A receptor [2, 881 and positive modulators of NMDA receptor-mediated responses [ 101,1021.Recently, increasing evidence indicates that the neurosteroids PS and DHEAS act as agonists at central cr receptors and exert a facilitatory action on NMDAmediated glutamatergic neurotransmission [ 1061. DHEAS attenuates dizocilpine-induced learning impairment via 01 receptors [ 1 131, which would further support our argument for a receptor-mediated signalling mechanism in the nootropic and antiamnesic effect of neurosteroids. On the basis of these studies it is speculated that a direct interaction between neurosteroids and crI receptors could constitute a possible mechanism for the neurosteroidal non-genomic effects in learning and memory processes. The effects of neurosteroids on learning and memory processes may involve, at least in part, an interaction with central cr receptors, since they could be significantly blocked by concurrent administration of haloperidol, a prototypical cr receptor antagonist, or NE- 100, a selective cr1 receptor antagonist [ 1 13,1841. In view of the importance of nitric oxide (NO) as a critical mediator in learning and memory processes and long-term potentiation, we studied the possible involvement of a neuronal NO mechanism in the nootropic and antiamnesic effects of neurosteroids PS and DHEAS on ageing- and dizocilpine-induced cognitive dysfunction in mice [ 1871. The allosteric agonist actions of neurosteroids PS and DHEAS at NMDA receptors play an important role in the cognitive process. PS and DHEAS, at doses of 1-20 mg/ kg, significantly attenuated the amnesia induced in mice by systemic administration of dizocilpine in a step-down type of passive-avoidance paradigm, and did not themselves affect mnemonic capacity. Preadministration of L-NAME, a NO synthase inhibitor, significantly blocked the PS- and DHEAS-induced attenuation of dizocilpine-induced amnesia [ 1871. Furthermore, this effect of L-NAMEwas completely reversed by L-arginine, a competitive substrate for NO synthase, confirming that it was a specific effect mediated through inhibition of central NO synthase. In addition, the learning impairment observed in aged (1 6 months old) mice could be significantly ameliorated by PS and DHEAS. These ameliorative effects of neurosteroids may involve at least partly a NO mechanism, since they could be significantly prevented by preadministration of L-NAME, and the effect of LNAME was reversed by L-arginine [ 1871. Taken together, these results strongly indicate a NO-dependent mechanism in the nootropic and antiamnesic activity of neurosteroids, particularly in the long-term memory processes. Despite their possible involvement with NO, neurosteroids such as progesterone or its metabolite allopregnanolone, however, do not significantly affect blood pressure. Acute administration of progesterone (1 mg/kg i.p.)

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[224a] or allopregnanolone (0.5 mg/kg i.p.) [224b] had no effect on arterial blood pressure. However, neurosteroids may produce transient marginal changes in arterial pressure by indirect mechanisms by acting at central nuclei involved in cardiovascular regulation. For example, allosteric potentiation of GABA-A receptor function by allopregnanolone in nuclei involved in the baroreflex pathways may modulate autonomic regulation of blood pressure in conditions such as during pregnancy. Similarly, both threshold pressure and saturation pressure for inhibition of rostra1 ventrolateral medulla neurones were decreased after administration of allopregnanolone [224cl. AGGRESSION

Aggression includes a spectrum of different types of intrusive and offensive behaviour which is under strong hormonal control. THDOC and its precursor DOC dose-dependently inhibit aggressive behaviour in animals [ 188,1891. DHEAS was also shown to inhibit aggressiveness of castrated males against lactating females [ 1901. DHEA and methyl-DHEA inhibit the aggressive behaviour of castrated male mice against lactating female intruders [191]. Both molecules produce a marked and significant decrease of PS concentrations in the brain of treated castrated mice, indicating that, by decreasing PS levels in the brain, DHEA might increase GABAergic tone and control aggressiveness. Adult female mice also display an aggressive behaviour towards lactating intruders [ 1921, which is independent of ovarian hormones. However, females which had been androgenized at birth, then treated when adult with DHEA were much less aggressive towards lactating intruders. Hence, neurosteroids have been suggested to have a role in the regulation of aggressive behaviour. DEPRESSION

Analysis of the cerebrospinal fluid levels of neuroactive steroids in healthy volunteers suffering from depression revealed that pregnenolone is decreased in subjects with affective illness, particularly during episodes of active depression [ 1931. DHEA administration to patients with Alzheimer’s disease [ 1941, benign senescent and memory deficits [195] and emotional disturbance [ 1961 resulted in improvements in mood, energy, confidence, interest and activity levels. DHEA improves the well-being in middle-aged and elderly healthy individuals [ 1971, while DHEAS improves depression ratings, as well as aspects of memory performance in middle-aged and elderly patients with major depression and low basal plasma DHEA/DHEAS levels

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[198]. Moderate doses of DHEAS or PS show an antidepressant-like effect in the Porsalt’s immobility test, which is sensitive to NE-100, a putative cr1 receptor antagonist, or progesterone, a neurosteroid cr receptor antagonist [ 1991.This crossed pharmacology between antidepressant-like effects of neurosteroids and crl receptor agonists [200] suggest a role for central cr receptors in the antidepressant-like effects of neurosteroids. ALCOHOL-RELATED BEHAVIOURS

There is accumulating evidence that the effects of ethanol and neurosteroids are interactive [201]. Prenatal neonates exposed to alcohol exhibited a marked shift to the right in the anxiolytic dose-response curve when administered i.c.v. injections of AP [165]. This decreased sensitivity may indicate that prenatal alcohol exposure causes a decrease in density of the GABA-A receptors involved in the stress response, resulting in a reduction in the sensitivity to the neurosteroid. Similar shifts in sensitivity were seen with PS in prenatal alcohol-exposed neonatal rats [165], partly because of a possible conversion of PS to AP via 5wreductase. Adult rats and mice chronically exposed to ethanol have increased sensitivity to the anticonvulsant effects of AP [ 138,2021, and the anxiolytic effects of ethanol were blocked by the excitatory neurosteroid DHEAS [ 1631. Furthermore, neurosteroids have been implicated in the gender differences in human alcoholism [203]. The neurosteroid AP protects against the increased seizure susceptibility associated with ethanol-withdrawal [ 137, 2041. In contrast to benzodiazepines, ethanol withdrawing rats are sensitised to the anticonvulsant effects of AP and THDOC [202]. Chronic administration of DHEAS, but not DHEA, blocks the development of ethanol dependence [205]. Thus, neuroactive steroids or their analogues may have promise in the therapeutic intervention for alcoholism and alcohol withdrawal. SLEEP

PS, when injected intracerebroventricularly or intraperitoneally, shortened the sleeping time produced by pentobarbital [206] in rats. Systemic administration of low doses of pregnenolone produced several rapid effects on human sleep-EEG [87], including improvement in the quality of sleep as shown by an increase in slow-wave sleep, a higher sleep efficiency and a trend to decreased intermittent wakefulness. In contrast to midazolam, DHEA increases EEG delta activity during non-rapid eye movement sleep in the rat [207]. DHEAS suppresses hippocampal recurrent inhibition and synchronises neuronal activity to theta rhythms [208]. Based on these data, the ac-

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tions of pregnenolone have been attributed to its partial inverse agonist modulation of GABA-A receptors. Moreover, DHEA has also been shown to increase rapid eye movement sleep [209]. In a pilot study, the soporific effect of the neurosteroid pregnanolone (8), a metabolite of progesterone and stereoisomer of AP (4), was studied in human volunteers [210]. Although there was a correlation between the soporific effect and plasma concentrations of pregnanolone, the study had limited value because of the lack of a placebo control. Intraperitonial administration of progesterone produces dose-dependent hypnotic effects as it shortens sleep latency, decreases time spent in wakefulness and rapid eye movement sleep (REMS) and selectively promotes preREMS [211-2141 . Administration of micronised progesterone (300 mg, orally) to male subjects has been shown to reduce non-REMS latency, to promote stage 2 sleep, to slightly suppress slow-wave sleep and to decrease EEG activity in the lower frequencies and enhance activity in the frequencies > 15 Hz during non-REMS [214]. CCD-3693 (20), a new orally active synthetic neuroactive steroid, appears to be more intrinsically efficacious in promoting non-REM sleep than the benzodiazepines without the accompanying rebound wakefulness after the subsiding non-REM sleep-promoting effect [2 15a], suggesting a distinct sedative-hypnotic profile with a promising potential for insomnia. Although the neurosteroid AP affects sleep in a benzodiazepine-like fashion there is no evidence yet to suggest that endogenous AP regulates sleep under physiological conditions [215bl. FOOD INTAKE

Consistent with their activity at the GABA-A receptor complex, neurosteroids produce hyperphagic effects in satiated male rats [216]. In a similar manner to triazolam, AP, progesterone and 4’-chlordiazepam produce a dose-dependent hyperphagic effects in 24 h food-deprived animals [217]. In contrast to this, moderate doses of PS and DHEAS produce a hypophagic effect. The AP-, progesterone- and 4’-chlordiazepam-induced hyperphagic

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effect was blocked by picrotoxin, but not by flumazenil. The hypophagic effect of DHEAS was reversed by dizocilpine, an NMDA receptor antagonist, but was resistant to muscimol, a selective GABA-A receptor agonist, suggesting that the inhibitory effect of DHEAS on food intake may be dependent on an interaction with a NMDA receptor-mediated mechanism [217]. In contrast to this, the PS-induced hypophagic response was resistant to dizocilpine, but sensitive to muscimol indicating that the hypophagic effect of PS could, at least partly, be mediated via an antagonist interaction with the GABA-A receptor. Preadministration of PS or DHEAS blocks the hyperphagic actions of AP [217], consistent with their in vitro interactions at the GABA-A receptor complex and suggests a possible endogenous role for neurosteroids in the regulation of food intake. It is therefore hoped that future studies may lead to the development of neurosteroid-based anorectic or hyperphagic agents for therapeutic use. However, little is known as yet whether the hyperphagic effects of neurosteroids are affected by natural variations such as the stage of estrous and gender of the animal. Our study reveals that the neurosteroid AP is more potent in diestrous females, whereas the benzodiazepine triazolam exhibits significantly high hyperphagic potency in estrous females [2 181. The extent of anorexia following DHEAS appears similar in male and female rats. DRUG TOLERANCE AND DEPENDENCE

Chronic coadministration of neurosteroids, AP, PS or DHEAS, followed by morphine prevents the development of tolerance to the antinociceptive effect of morphine and suppresses the naloxone-precipitated withdrawal jumps in morphine dependent animals [219, 2201. These effects are not seen with DHEA-acetate. However, acute treatment with these neurosteroids was not associated with any decrease in withdrawal jumping behaviour in morphine-dependent mice. These data support a role for neurosteroids in the development of tolerance to and dependence on morphine and suggest the potential utility of specific neuroactive steroids in its treatment. Coadministration of neurosteroids, AP or PS but not DHEAS may abolish the development of tolerance and attenuated withdrawal-induced anxiety and hyperlocomotion due to triazolam [221]. Concomitant progesterone, or 4’-chlordiazepam also prevented the development of tolerance and significantly augmented the recovery from withdrawal-induced anxiety and hyperlocomotion to diazepam [2211. Thus coadininistration of specific neurosteroids prevents the development of tolerance to benzodiazepines and augments the recovery from withdrawal of chronic benzodiazepine treatment.

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Adrenocorticotrophin (ACTH) is known to produce a variety of neurobehavioral effects such as disruption of cognitive processes, regulation of growth and development of the CNS, plasticity and recovery of function after central or peripheral nerve damage, and modulation of social and behavioural activity [222, 2231. One possible mechanism for these effects includes an ACTH-facilitated synthesis and release of adrenocorticosteroids and neurosteroids in adrenal cortex and brain, respectively, and subsequent modulation of neurotransmitter receptor and second messenger systems [2]. Biochemical and behavioural pharmacological studies strongly indicate the involvement of neurosteroid [224b] and NO mechanisms [225] in the corticotrophin-induced behavioural activity, particularly without affecting the adrenal or cardiovascular responsiveness, which may have implications in neurosteroidogenesis. PAIN A N D MIGRAINE

There is evidence that 5a-reduced progesterone and androgen metabolites may mediate changes in pain sensitivity. Neurosteroid AP is the most effective of the analgesic neuroactive steroids [226]. AP increases tail-flick latencies in male mice [227], attenuates mechanical visceral pain [228] and elevates vocalisation threshold in ovarectomized females [229]. Furthermore, the increase in endogenous AP over the estrous cycle and pregnancy can be correlated with the increase in tail-flick latencies [230]. Moderate doses of progesterone and 3a-androstanediol produce analgesic effects in part via membrane actions within the medial basal hypothalamus and preoptic area, respectively [2311. The concept of employing neurosteroids as anti-migraine agents is supported by literature reports. For example, AP was investigated for anti-migraine activity in an animal model of cephalic pain in guinea pigs and compared with valproic acid (2-propylpentanoic acid) [232]. AP like valproate, which enhances GABA synthesis and blocks degradation, was found to block neurogenic inflammation within the meninges via a GABA-A receptor-mediated mechanism. Progesterone has also been reported to be effective in the acute and prophylactic treatment of migraine [233,234]. Its effectiveness has been shown to be mediated by its reduced metabolite AP. Data from a prospective study of pregnant women with a diagnosis of migraine indicated that 79% of 484 patients experienced improvement in headache recurrence during pregnancy [235], consistent with the notion that it is the increase in endogenous AP during pregnancy which is responsible for this

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apparent therapeutic effect. The synthetic agent, ganaxolone, is currently being evaluated in a phase I1 clinical trial for the acute treatment of migraine headache. AGEING AND NEURODEGENERATION

Ageing is accompanied by a progressive and universal decline of the physiological functions involved in maintaining the body’s composition and homeostasis during action, emotion and eventually, response to aggression. DHEA has been implicated in the ageing processes [236]. DHEAS shows the highest concentrations of all steroids ( > 1 pgiml) in the body [236] but after 25-30 years of age, it starts to decline [180]. This suggests that a decrease in neurosteroid levels may contribute to the process of ageing and that administering this compound may have beneficial effects in old animals. A positive correlation has thus been found between the performances of 2year-old rats in a two-trial recognition task and the concentration of PS in the hippocampus. Animals which performed best had the highest PS levels [29]. Administration of DHEA or DHEAS significantly improved memory processes in aged rodents [ 1821 and remarkably increased the mood in men and women of advancing age [ 1971. PS is synthesized in the nervous system and is a major neurosteroid in rat brain. Significantly lower levels of PS were found in the hippocampus of aged rats [237]. Evaluation of spatial memory performances of aged rats in the Morris water maze and Y-maze indicated that those animals with lower memory deficit had the highest PS levels, whereas no relationship was found with the PS content in other brain areas. Moreover, the memory deficit of cognitively impaired aged rats was transiently corrected after either intraperitoneal or bilateral intrahippocampal injection of PS [237]. PS is both a GABA-A receptor antagonist and a positive allosteric modulator at the NMDA receptor, and may reinforce neurotransmitter systems that decline with age. Indeed, intracerebroventricular injection of PS was shown to stimulate acetylcholine release in the adult rat hippocampus. It is therefore proposed that the hippocampal content of PS plays a physiological role in preserving and enhancing cognitive abilities in old animals, possibly via an interaction with central cholinergic systems. Thus, neurosteroids should be further explored in the context of prevention and/or treatment of age-related memory disorders. Steroids profoundly influence neuronal and glial activity and plasticity during adulthood. Neurosteroids may provide neurotrophic support and play a role in neuronal regeneration. DHEA or DHEAS enhances the survival and differentiation of neurons prepared from embryonic mouse brain

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[238]. Progesterone increases the survival of motorneurons following axotomy [239] and reduces cerebral oedema, secondary neuronal degeneration and behavioural impairment that accompany contusion of the rat frontal cortex [240]. Pregnenolone reduced histopathological changes of the neurons, spared the tissue from secondary injury and increased the recovery from spinal cord injury [241]. Progesterone increases the expression of myelin proteins in cultured rat oligodendrocytes [242], while pregnenolone, PS and DHEAS regulate the morphology of astroglia in the hippocampal cultures from adult rats [243]. Progesterone, synthesized by Schwann cells, promotes the formation of new myelin sheaths after lesion of the mouse sciatic nerve by activating the expression of specific myelin genes through intracellular receptors [ 10, 371. As astrocytes can synthesize progesterone from pregnenolone, this neurosteroid may be an autocrine regulator of astroglial proliferation after injury. Apart from effects on myelin in regenerating nerves, progesterone may play a role in the continuous renewal of the constituents of myelin sheaths [244]. In cultures of glial cells prepared from neonatal rat brain, progesterone increases the number of oligodendrocytes expressing the myelin basic protein and the 2’,3’-cyclic nucleotide-3’-phosphodiesterase. After cryolesion of the male mouse sciatic nerve, blocking the local synthesis or action of progesterone impairs remyelination of the regenerating axons, whereas administration of progesterone to the lesion site promotes the formation of new myelin sheaths [lo]. Progesterone also protects against brain damage following transient middle cerebral artery occlusion and improves physiological and neurological function [245]. In addition, allopregnanolone, PS and DHEAS exhibit cytoprotective properties at different cellular sites in the late phase of cell injury [246]. Thus, neurosteroids may play an important role during development and regeneration of the nervous system, and specific neurosteroids may be therapeutically useful in activating or accelerating the reparative response of both neurons and glial cells to injury. TOXICITY There are a number of potential side-effects associated with neurosteroids that could be use limiting in their therapeutic applications. For example, systemic administration of 3q5a-reduced derivatives of progesterone acting as positive allosteric modulators of the action of GABA at GABA-A receptors may produce profound sedation, motor impairment or ataxia [130,160]. In addition, protracted use of neuroactive steroids may trigger complex DNA transcription modifications in neuronal[124] and glial cells [37], and induce

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tolerance liability, which would limit their use in neuropsychiatric and cognitive disorders [93 - 951. Acute or chronic administration of sulphated neurosteroids may produce convulsant or proconvulsant effects [ 150,151] which would also severely restrict their clinical application. However, such potential neurotoxic manifestations have so far not been seen in the clinic but further studies are clearly warranted to establish the safety of neurosteroids. It should be possible to overcome most of these problems by developing drugs that affect selectively some rate-limiting steps of brain neurosteroid biosynthesis, which unlike that of peripheral endocrine tissues, is not under pituitary control. Only when the mechanisms of currently available neurosteroids are clearly understood will the approaches to exploit neurosteroids for therapeutic purposes be designed effectively and targeted successfully. CONCLUSIONS In the light of the above reports, it appears that the family of neurosteroids, including AP, THDOC, progesterone, PS, DHEAS and the mitochondria1 DBI receptor ligands 4’-chlordiazepam, PIC11195 and FGIN-1-27 are currently being widely used as tools for designing new therapeutic strategies and for neurosteroid structure-based drug development. Although neurosteroids have been found to either positively or negatively modulate various excitatory or inhibitory neurotransmitter receptors such as GABA-A, glycine, NMDA, calcium channels, and sigma receptors, the nature of the binding sites remains obscure at this time. Perhaps novel neurosteroid analogues acting preferentially at a particular receptor will promote research in this direction. Thus, the future holds great promise for research on neurosteroids and their receptor mechanisms as targets for the development of new agents with anxiolytic, anticonvulsant, anorectic, antiamnesic and anti-addiction properties. REFERENCES 1 Baulieu. E-E. (1981)in Steroid Hormone Regulation of the Brain (Fuxe, K., Gustafson, J.A. and Wetterberg, L., eds), Wenner-Green Center International Symposium Series, Vol.

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Progress in Medicinal Chemistry Vol. 37, Edited by F.D. King and A.W. Oxford Q 2000 Elsevier Science B.V. All rights reserved ~

5 Benzo[b]pyranols and Related Novel Antiepileptic Agents NEIL UPTON, Ph.D. and MERVYN THOMPSON, Ph.D. SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Hurlo w,Essex, CMI 9 5 A W8 U.K .

INTRODUCTION The epilepsies and unmet medical need Pathophysiological basis of seizures and mechanism of action of established anticonvulsant drugs Strategies for identifying new anticonvulsant drugs

178 178

EMERGENCE OF T H E BENZOPYRANS A N D SERENDIPITY Early in vivo SAR studies Identification of the SB-204269 binding site SAR of benzopyrans

182

PHARMACOLOGY OF SB-204269 Mouse and rat electroshock seizure models (MESTand MES) Other seizure models Mouse and rut PTZ infusion tests Geneticall,yaudiogenic seizure-prone DBA2 mice Aniygdulu-kindled seizures in rut In vitro high potussiuni rut hippocumpul brain slice model of epileptiform discharges Profile of SB-204268 in animal seizure models Assessment of potential CNS and cardiovascular side-effects Additional pharmacological actions Therapeutic potential of SB-204269

187 187 191 191 191 192 192 193 193 194 195

FUTURE CHEMICAL STRATEGIES High-throughput screening (HTS) and SAR of tetrahydroisoquinolines

196 196

CONCLUSION

198

ACKNOWLEDGEMENTS

198

REFERENCES

198 177

i79 180

182 183

184

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INTRODUCTION THE EPILEPSIES A N D UNMET MEDICAL NEED

The collective term, epilepsy, is used to describe a diverse group of neurological disorders that are characterised by abnormal hypersynchronous discharges of a large population of cerebral neurones resulting in seizures. At this time three major categories of the epilepsies are recognized: A Idiopathic (no underlying cause other than a hereditary predisposition) B Cryptogenic (presumed to be symptomatic, but the aetiology is not known) C Symptomatic (considered to be the consequence of known or suspected disorders of the CNS) [I]. In terms of seizure types, the following classification [2] has been adopted: a) Partial (focal, local) seizures 0 simple or complex Partial seizures account for 40-50%" of all seizures. They have local onset (e.g. in structures such as the temporal lobes) and may involve motor, sensory, autonomic or psychic symptoms with no loss of consciousness (simple partial seizures) or cognitive, affective or psychomotor symptoms with loss of consciousness (complex partial seizures). b) Generalized seizures 0 tonic-clonic (grand mal) 0 absence (petit mal) 0 myoclonic 0 status epilepticus Tonic-clonic seizures constitute 30-40% of the total and, like the other types of generalised seizures, involve abnormal electrical activity which spreads simultaneously throughout both hemispheres of the brain usually with loss of consciousness. A significant proportion of partial seizures progress to have secondary generalisation. Estimates of prevalence indicate that epilepsy is the most common serious neurological condition with around 500,000 sufferers in the UK alone who have epileptic seizures [3]. Of these, as many as 20-30% do not have their seizures fully controlled with currently available anticonvulsant drugs [4] and prognosis for some epileptic syndromes of childhood/adolescence (e.g. Lennox-Gastaut and West syndromes) and partial seizures in adults is especially poor [5]. Unfortunately, even when effective, dose-limiting adverse effects (most commonly neurological impairment) and idiosyncratic reactions are all too common with existing anticonvulsants which can seriously impair their therapeutic utility [4,6]. Considering this evidence as a whole, there is

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clearly a major unmet medical need, particularly in the most refractory types of epilepsy, for agents with enhanced efficacy and safety. It was against this background that we embarked upon our studies with novel benzopyrans in an attempt to identify new improved anticonvulsant agents. PATHOPHYSIOLOGICAL BASIS OF SEIZURES A N D MECHANISM OF ACTION OF ESTABLISHED ANTICONVULSANT DRUGS

Although our understanding of the biochemical processes underlying the different types of seizure disorders is still far from complete, recent advances in neurobiology have yielded significant insights into the cellular and molecular dysfunctions leading to seizure propagation and termination. Formerly, it was widely believed that one single cellular mechanism was involved in the generation of seizures but this idea had to be modified when it became recognised that the mechanisms underlying generalised absence seizures were clearly different from those which led to partial seizures (for an excellent review see [7]). Progress in this area has also been accelerated by detailed study of the mechanisms of action of established anticonvulsant drugs [8]. In view of the complex aetiology of the epilepsy disorders, it is probably not surprising that multiple and overlapping pharmacological effects have been described for anticonvulsant drugs. However, of the many agents that are reportedly at various stages of clinical evaluation, the vast majority can be classified into one or more of three mechanistic categories [8]: a) enhancement of inhibitory (principally GABA-mediated) processes b) reduction of excitatory (particularly glutamate-mediated) transmission c) modulation of membrane cation conductance (Na+ or Ca2+). In addition, modulation of potassium channels is considered a particularly attractive target for future exploitation [8-lo]. Table 5.1 summarises the most compelling theories of how established and examples of the newer generation of anticonvulsant drugs are thought to exert their effects, although it is recognized that this area is still the subject of ongoing debate.

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Table 5.1. PROPOSED MECHANISMS O F ACTION OF ESTABLISHED AND NEWER ANTICONVULSANT DRUGS Drug

Inhibitory Transmission

Estublishedagents Carbamazepine Phenytoin Valproate Ethosuximide Phenobarbital Benzodiazepines Newer agents Lamotrigine Oxcarbazepine Zonisamide Felbamate Topiramate Gabapentin Vigabatrin Tiagabine Levetiracetam

Excitatory Transmission

Ion Channels

Other

TGABA TGABA TGABA

TGABA TGABA TGABA(?)

lglutamate

1glutamate lglutamate

-1 Na' JNa+ INa' /Na+ -1 Na' lNa+(?)

Novel CNS binding site

TGABA TGABA

SB-204269

Novel CNS binding site Novel CNS binding site

7 or 1signifies that function is increased or decreased, respectively. From references [5, 8.9.1 1-1 31.

STRATEGIES FOR IDENTIFYING NEW ANTICONVULSANT DRUGS

Presently, the most popular approach to anticonvulsant drug discovery encompasses current knowledge regarding both the basic pathophysiological events responsible for epilepsy and also the mode of action of established anticonvulsant drugs. The main focus remains on symptomatic treatments, although it is hoped that the emphasis may shift in the future towards disease modifying drugs as the underlying causes of seizure generation become better understood. Despite the growing trend towards rational drug design, tiagabine is one of the few examples to date where this strategy has been successful and identification through empirical animal models is still an important source for new drugs [8]. In addition, the discovery of SB-204269

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(discussed later in this review) also illustrates the value of empirical models in selecting mechanistically novel anticonvulsant agents. Swinyard and Loscher have been at the forefront of validating experimental seizure models and in defining appropriate selection criteria for identifying new anticonvulsant drugs. For many years, the traditional maximal electroshock seizure (MES) and subcutaneous PTZ (pentylenetetrazole, Metrazole@) tests [ 141 have been the mainstay of anticonvulsant testing with the more sensitive, so-called, threshold models [5,8] becoming increasingly popular in recent years. Table 5.2 describes some of the more comTable 5.2. PROPOSED TEST HIERARCHY FOR INITIAL EVALUATION OF ANTICON V U LSANTS Models of.. .gritnar), seizures . .peneralised ,

Threshold for tonic seizures induced by corneal electroshock in mice or rats (also known as maximal electroshock seizure threshold [MEST] test) Thresholds for myoclonic, clonic and tonic seizures induced by intravenous infusion of PTZ in mice or rats Maximal electroshock seizure (MES) test with tonic hindlimb seizures induced by supramaximal corneal stimulation in mice or rats Subcutaneous (s.c.) PTZ test with clonic convulsions in mice or rats Mod~'I.sofpartial seizures with or without secondary generalisation a ) Amygdala-kindled seizures in rats; typically recording seizure severity (focal and secondary generalised) and duration b) In vitro high K + rat hippocampal brain slice model measuring spontaneous electrographic discharges Genetic models

Animals either exhibit spontaneously occurring recurrent seizures (e.g. genetically prone absence epilepsy rats) or are genetically susceptible to undergoing seizures in response to specific sensory stimulation (e.g. audiogenic seizures in DBA2 mice) Models for detecting neurologicul impairment and other aclverse effects a) Accelerating rotdrod in mice or rats b) Irwin profile screen in mice or rats

General coninienis a) The MES model is also considered to have some predictive value for partial seizures b) Comparison of drug effects in threshold and supramaximal models enables determination of whether anticonvulsant activity results primarily from elevation of seizure threshold or from reduction of seizure spread c) Drug administration during kindling development can be used for detection of potential antiepileptogenic effects

Based on [5.8,14].

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monly used models for the initial characterisation of potential anticonvulsants, including SB-204269. EMERGENCE OF THE BENZOPYRANS AND SERENDIPITY EARLY IN VIVO SAR STUDIES COR2 HN

(-) Levcromakalim 3S,4R

(1) R' = EWG, lipophilic group

(k)Cromakalim

R2 = various

Levcromakalim was the first ATP-sensitive potassium channel opener whose antihypertensive activity is a consequence of hyperpolarisation of vascular smooth muscle cells [ 15-1 71. The question was - would an opener of neuronal ATP-sensitive potassium channels prevent excessive CNS excitability and therefore have anticonvulsant activity? This initial premise was supported by literature reports that cromakalim or levcromakalim, when administered intracerebroventricularly,inhibit both chemically-induced seizures in rodents [18,19] and spontaneous seizures in genetically epileptic rats [20]. In contrast to these effects, it was found that cromakalim when administered systemically (orally or intraperitoneally) did not inhibit seizure activity in a mouse maximal electroshock threshold (MEST) model, even at a dose of 10 mg/kg which is much greater than that required for antihypertensive activity [17]. Although the seizure models are different, a possible explanation for the lack of effect was poor CNS penetration. However, when more lipophilic, brain penetrant analogues (1) of levcromakalim retaining the 3S,4R stereochemistry were prepared, they were found to maintain the antihypertensive effects but possess little or no anticonvulsant activity [21]. Typical examples were the fluorobenzamides such as (1) where R' = 6-CN, R2 = 3-FPh. Surprisingly, it was found that the corresponding trans 3R,4S enantiomer showed good anticonvulsant activity in the mouse MEST model, while being devoid of antihypertensive properties. A subsequent communication confirmed that a 4s configuration of the fluorobenzamide is crucial in conferring anticonvulsant activity without

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antihypertensive activity [22]. Although the 3-hydroxyl group appears essential for anticonvulsant activity, since the corresponding racemic chroman is inactive, its stereochemistry is not important. These early data strongly indicated that the mechanism of the anticonvulsant action of compounds such as (1) was unlikely to involve the modulation of ATP-sensitive potassium channels. It was clearly important to gain a fuller understanding of structure-activity relationships (SAR) within the series. Accordingly, this was carried out and analogues such as the 4-fluorobenzamide ('3) (SB-204269) were prepared with markedly enhanced anticonvulsant potency in vivo [23, 241.

-F

(3) SB- 204269

(4) 88-204268

IDENTIFICATION OF THE SB-204269 BINDING SITE

In order to facilitate an understanding of the mechanism of action of these anticonvulsant benzopyrans, [3H]-SB-204269was prepared by [3H] hydrogenolysis of the corresponding 2,5-dibromobenzamide of (3) and was found to define a specific binding site in the brains of several species including mouse [12], rat, cat, dog, marmoset and man [13]. In rat forebrain membranes, ['HI-SB-204269 binding was of moderate affinity (KD 32 nM) and fairly high density (B,,, 250 fmoles/mg protein). The binding was heterogeneously distributed, with the high levels observed in the cerebral cortex, hippocampus and dentate gyrus being of particular interest because of the association of these brain regions with the initiation or maintenance of seizure activity. Significantly, specific binding could not be detected in several rat peripheral tissues (e.g. liver, heart or kidney) [ 131. Extensive pharmacological characterisation has illustrated the unique nature of the ['HI-SB-204269 binding site as a mechanistic target. Binding was highly enantioselective, since in competition studies the enantiomers (+)-(3) (3R,4S) and (-)-(4) (SB-204268 3S,4R) had pKi values of 7.3 and <4.5,respectively. None of a wide range of pharmacological standards in-

184

BENZO[b]PY RANOLS AND RELATED NOVEL ANTIEPILEPTIC AGENTS

cluding both established (diazepam, phenytoin, carbamazepine, valproate and phenobarbitone) and newer (gabapentin, lamotrigine, vigabatrin and levetiracetam) anticonvulsant agents have shown any affinity (at up to 100 pM) to the ['HI-SB-204269 binding site. In addition, although SB-204269 and analogues are structurally, but not stereochemically, related to the benzopyran class of ATP-sensitive potassium channel openers, none of the openers from this or other chemical classes (levcromakalim, aprikalim, or pinacidil) showed any affinity for the binding site [ 131. Several lines of evidence now support the relevance of this novel ['HI-SB204269 site to the anticonvulsant properties of SB-204269. Thus, the affinity of the binding site for SB-204269 (48 nM) is similar to the brain concentration at which threshold anticonvulsant activity of the compound is observed in the rat MEST test [ 13, 231. The enantioselectivity of the binding site also parallels the lack of in vivo anticonvulsant activity of SB-204268 [23] and other 3S,4R enantiomers [22, 241. In addition, the relative affinities of the whole series of benzopyrans prepared correlated well with their anticonvulsant activity in rodent models [ 131. The mechanistic novelty and selectivity of the anticonvulsant benzopyrans is further highlighted by a series of additional in vitro radioligand binding and functional studies [23] showing that SB-204269, unlike other anticonvulsant agents, does not appear to interact with Na+ channels, or GABAergic or glutamatergic pathways (see Table 5.1). SAR OF BENZOPYRANS A N D SELECTION OF SB-204269

Following the discovery of the ['H]-SB-204269 binding site, the screening strategy was revised. Prior to in vivo studies, compounds were initially screened for their ability to displace ['H]-SB-204269 in the rat forebrain and only potent compounds were examined in vivo, initially in the mouse MEST model at a dose of 10 mg/kg p.0. (see Tables 5.3 and 5.4, compounds 2 to 24). Selected compounds were examined further in the equivalent model in the rat and those of particular interest taken forward to the more stringent rat suprathreshold MES model [23]. An SAR analysis of compounds related to (3) was carried out, initially on racemic compounds (Table 5.4). Other electron-withdrawing 6-substituents known to be beneficial in potassium channel openers such as nitro (17), phenylsulfonyl(18) and pentafluoroethyl (15) [ 151 gave compounds with no improvement over the cyano (14) compound (Table 5.3)[24]. Thus, not only is there stereochemical differentiation from potassium channel openers SAR, but also there are clear differences in the optimal nature of the 6-substituent, 6-acetyl being particularly beneficial. Homologation to 6-propionyl

185

N. UPTON AND M. THOMPSON

__

Table 5.3. BIOLOGICAL DATA FOR 4S-AMIDO-2H-BENZO[B]PY RAN-3-OLS COR2

HN

R'

R'

/'H]-SB-204269 Binding" pKi

RodentMESXh % increase in seizure tlireshoid at 10 mg/kgp.o. Mouse at l h post dose

Rut at 4h post dose

110* loo*

~

(2) 3R ( 3 ) 3R ( 5 )3R ( 6 )3 R (7) 3R (8) 3R (9) 3R (10)3R (11)3R (12) 3R (13)3R

COMe COMe COMe COMe COMe COMe COMe COMe COMe COMe COMe

Ph 4-FPh 2-ClPh 3-CIPh 2-C1,4-FPh 3-CI. 4-FPh 2,3-diCIPh 3-pyridyl 2-pyrazinyl 3-thienyl 2-C1(3-thienj(I)

7.3 1.3 7.7 8.0 1.5 7.9 8.0 6.5 4.8 6.8 7.8

I40* 90* 160* 120* I so* 30* -1511s 50* 95 *

Nd 600* 480* 945' 1080' 850* 1070* Nd Nd Nd 520*

(14) 3R (15)3R (16)3S

CN CzFS COMe

4-FPh 4-FPh 2-CIPh

6.0 6.3 7.5

40* -8ns 80*

Nd Nd Nd

Procedure as detailed in [ 131; 'Procedure as detailed in [23, 271;

* p<0.05, ns = non-significant; Nd = not determined.

(19) resulted in little loss in affinity whereas replacement by 6-benzoyl (20) was clearly detrimental. The data suggested that the carbonyl of the 6-acetyl group is likely to be involved in a key interaction with the binding site. As the 6-acetyl moiety appeared to be crucial in conferring high affinity, its position in the benzopyran ring was investigated. Affinity was reduced by around 100-fold when it was moved to any of the alternative aromatic ring positions (e.g. Tublr 5.4 compounds 21-23). It was also demonstrated (e.g. compounds 25, 26) that the orientation of this 6-acetyl group is crucial for optimal activity, further substantiating the hypothesis that it is intimately involved in a key specific interaction with the binding site [25]. Incorporation of other halogens at all the positions of the benzamide

186

BENZO[b]PYRANOLS AND RELATED NOVEL ANTIEPILEPTIC AGENTS

Table 5.4. BlOLOGICAL DATA FOR (k) TRANS 4-AMIDO-2H-BENZO[B]PYRAN-3-OLS CORz HN 4OH

Cpd

R'

6-NO2 6-SO2Ph 6-COEt 6-COPh 5-COMe 7-COMe 8-COMe 6-COMe a

R2

Rodent MEST' % increase in seizure thresholdat 10 mg/kgp.o.

[-'H]-SB-204269 Bindingap Ki

4-F-Ph 4-F-Ph 4-F-Ph 4-F-Ph 4-F-Ph 4-F-Ph 4-F-Ph Cyclohexyl

6.2 5.8 6.8 6. I 5.1 5.5

5.0 5.9

Mouse at l h post dose

Rat at 4h post dose

4s * -7ns 3s* Nd Nd Nd Sns Nd

2ns Nd Nd Nd Nd Nd Nd Nd

Procedure as detailed in [13]; Procedure as detailed in [23, 271;

* p
F

Me

\

Me

Me

(25)pKi e 5.0

(26)pKi 7.6

group in (2) resulted in the retention of activity and 2-C1(5) and 3-C1(6) had enhanced affinity. The effects of combinations of halogen substituents were also explored and some, for example, 2-C1,4-F (7), resulted in both high affinity and particularly good in vivo activity [26].In general, inversion of stereochemistry of the hydroxyl at C-3 led to a similar or marginal reduction in affinity (e.g. cis (16) 1261).

187

N. UPTON A N D M. THOMPSON

Replacement of the benzamide group by a heteroaryl amide provided some interesting results [26]. For nitrogen containing heterocycles, 3-pyridyl (10) had thirty-fold lower affinity than 3-chloro (6) and 2-pyrazinyl (1 1) had very low affinity. The unsubstituted 3-thienyl(l2) showed modest affinity but the introduction of a 2-chloro substituent (13) resulted in a ten-fold increase in affinity up to the same level as seen with 2-chloro benzamide (5). Clearly, there appears to be an appropriately positioned hydrophobic pocket in the binding site. However, replacement of the benzamide moiety by cyclohexylamide (24) caused a reduction in potency, suggesting a role for the phenyl ring other than that of a purely lipophilic binding interaction. PHARMACOLOGY OF SB-204269 MOUSE A N D RAT ELECTROSHOCK SEIZURE MODELS (MEST & MES)

On the basis of the above in vitro and in vivo SAR determinations, SB-204269 was the first example selected for extensive evaluation in both experimental models of epilepsy and also tests of potential neurological deficits. The un-

d y

CI

-N

Pi

Diazepam

Carbamazepine

9

H,N

Lamotrigine

CO,H

Gabapentin

Ph

HNKNH 0

Phenytoin AniiconvuDtrni Coinpounds Evnlu~itedin Pii~~rtnucological Models Descrihed in Tahle 5.5

188

BENZO[h]PYRANOLS AND RELATED NOVEL ANTIEPILEPTIC AGENTS

Table 5.5. ANTICONVULSANT PROPERTIES OF SB-204269 AND STANDARDS IN THE MOUSE AND RAT MEST MODELS Treutment

Pretest Time

% Increase in Seizure Threshold

(mins) Mouse

-

Diazepam

1.25 2.5 5

30"/60h

10

I8* 38* 88* I It*

20 40

Carbamazepine

2.5 5

15a/60b

10

15 20

Lamotrigine

0.5

Gabapentin

5

11

44* 255* 648 * 1200* 8

35* 346* 1144* 143* 1035* 11

120

84* 158* 413*

20 40 0.1 0.3 1 3 10

30 100

18*

43*

10

SB-204269

110* 265* 334*

29* 61* 1 l6*

16*

2.5 3 5 1.5 10 20

14

38*

I

60' I 360b

1

Rut

60"/240b 40* 46* 89* 102* 150* 243*

38* 531 1 l3* 288* 553* 961*

a: mouse b: rat; Adapted from [12,23]; n = 12-16 per treatment group except for lamotrigine in the rat where n = 28. Statistical analysis was conducted by the method of Litchfield and Wilcoxon [40]. * p < 0.05 compared to vehicle-treated control group. Data were generated at the time of peak effect for the respective agents.

N. UPTON AND M. THOMPSON

189

ique pharmacological profile observed is described below. Comparative data are shown for several established anticonvulsant drugs. In the mouse MEST test [27], SB-204269 (0.3-100 mg/kg P.o.), carbamazepine (2.5-1 5 mg/kg P.o.), diazepam (1.25-10 mg/kg P.o.), lamotrigine (1-20 mg/kg p.0.) (Table 5.5) and phenytoin (2:5-30 mg/kg p.0.) (data not shown) all produced significant (p <0.05) and dose-related increases in the threshold for tonic hindlimb extensor seizures, thereby illustrating the anticonvulsant properties of these agents. SB-204269 (0.1-30 mg/kg p.0.) also produced dose-related anticonvulsant properties in the rat MEST model where the compound was found to be comparable to the best of the standards (carbamazepine and lamotrigine) tested regarding the level of efficacy observed (Table 5.5). Moreover, in terms of the minimum significantly effective dose (MED), SB-204269 ( 9 0.1 mg/kg p.0.) was clearly.more potent than lamotrigine (1 mg/kg P.o.), carbamazepine (5 mg/kg P.o.), diazepam (5 mg/kg p.0.) and gabapentin (10 mg/kg p.0.) [23]. The compound had a rapid onset of effect (d 15 min) with a.long duration of action (12-24 h) which corresponds to the extensive plasma half-life (4-6 h) apparent in this species (Yeulet et al., unpublished observations). The peak effect (-600% increase in seizure threshold) was observed at 4 h (Table 5.6). The impressive anticonvulsant properties of SB-204269 observed in the rat MEST model remained unchanged after repeated administration ( 10 Table 5.6. TIME-COURSE OF THE ANTICONVULSANT PROPERTIES OF SB-204269 (10 MG/KG P.O.) IN THE RAT MEST MODEL Pretevt Time (hours)

0.25 0.5 I 2 4 6 8 12 24

‘%Increasein Seizure Threshold Experirnmt I

Experiment 2

48* 90* 176* 424* 570* 489* Nd Nd Nd

Nd Nd Nd Nd 623* Nd 392* 292* 0

n = 1416 per time-point; Nd = not determined. The time-course study was performed in two separate experiments. Statistical analysis was conducted by the method of Litchfield and Wilcoxon [40]. * p < 0.05 compared to vehicle-treated control group.

190

BENZO[b]PYRANOLS AND RELATED NOVEL ANTIEPILEPTIC AGENTS

mg/kg P.o., twice daily for 7d). Thus, there was a 840% versus a 790% increase in seizure threshold following single or chronic treatment, respectively (Figure 5. l), and brain concentrations of the compound measured after acute (1.01 k 0.16 pmol/kg) or chronic (0.97 k 0.14 pmol/kg) administration were also similar. Together these findings indicate a lack of either pharmacodynamic or pharmacokinetic tolerance development upon chronic dosing of SB-204269. In addition, there was no evidence of rebound withdrawal 12-48 h following cessation of chronic treatment in terms of either a lowering of seizure threshold (Figure 5.1) or weight loss. Consistent with previous observations (see Table 5.6), SB-204269 still produced anticonvulsant activity 12h after the last dose in the withdrawal study. Tolerance Phase *

Days 1-7 treatment (bid,; p.0.) Day8treatment (pa)

Vehicle

+

Withdrawal Phase

1

Vehicle

+

SB 2WL. 10 mg/kg

+

Vehicle SB204269 SB204269 10mg/kg 10mg/kg

* 80

.r

Days 1-7treatment Vehicle (b.i.d.; p.0.) Timeafterlastdose (h)

-

SB 204269 10 m&g

12

24

36

48

71

n = 14- I 5 per dose group. Statistical comparisons were made using one-way ANOVA followed by Newman Keuls test. NS: Non significant; * : p < 0.05 compared to vehicle-treated control group. (ns): Non significant compared to the acute SB 204269 group

Figure 5.1. Tolerance and withdrawal studies with SB-204269 in the rat MEST model.

As is reflected by the EDSovalues (shown in parentheses) determined in the more stringent rat MES model, SB-204269 (6.3 mg/kg P.o.,4 h pretest) once again showed a good level of potency compared to diazepam (56.4 mg/kg P.o., 1 h pretest), carbamazepine (12.0 mg/kg P.o., 1 h pretest) and lamotrigine (6.1 mg/kg p . ~ .6, h pretest). SB-204269 also produced a high level of efficacy as indicated by its ability to completely inhibit tonic hindlimb extensor seizures at a dose of 30 mg/kg p.0. [23].

N. UPTON AND M. THOMPSON

191

OTHER SEIZURE MODELS

Mouse and rat PTZ infusion tests

In mice, SB-204269 was found to produce potent inhibition (MED of 3 mg/ kg P.o., l h pretest) of tonic hindlimb extensor seizures induced by intravenous infusion of the chemical convulsant agent PTZ (pentylenetetrazole; for methods see [27]). This finding is consistent with the ability of the compound to prevent tonic hindlimb extensor seizures resulting from electrical stimulation (see above). In contrast, even at the very high dose of 100 mg/ kg P.o., SB-204269 had no effect on myoclonic seizures produced by PTZ infusion (Table 5.7). Similarly, in rats SB-204269 was found to inhibit PTZ-induced tonic forelimb extensor seizures (MED of 10 mg/kg p.0.) but to have no effect (at doses up to 30 mg/kg p.0.) on myoclonic seizures (Table 5.7) [23]. Unlike SB204269, lamotrigine has been reported to actually exacerbate PTZ-induced myoclonus at high dose levels [28]. Geneticully audiogenic seizure-prone DBAZ mice SB-204269 (0.3-3 mglkg P.o.,1 h pretest) produced potent dose-related anticonvulsant activity in DBA2 mice subjected to a continuous sound level of 96-106 decibels (for method see [29]) with complete protection against Table 5.7. ANTICONVULSANT PROPERTIES OF SB-204269 AND DIAZEPAM IN THE MOUSE AND RAT PTZ INFUSION MODELS Treorrnent

Dose (mg/kgp.o.)

Pretest Time (niins)

Diazepam SB-204269

2.5"/7.5b 0.3 I 3 10 30 100

60"/60b 60"/240b

%I

Increase in Seizure Threshold

Myoclonus

Tonus/Death

Mouse

Rat

80**

14** Nd 1

3 -2 -3 -3 10 4

0

0 2 Nd

Mouse

Rat

108**

51**

41 50 151**

Nd 7 38 260** 260** Nd

130**

84** 157**

a: mouse; b: rat; n = 11-12 per dose group, methods as from [23,27]; Nd = not determined. Statistical analysis was performed using 2-tailed Mann Whitney U test following significant Kruskal-Wallis I-way ANOVA; ** p <0.01 compared to vehicle-treated control group.

192

BENZO[h]PYRANOLS AND RELATED NOVEL ANTIEPILEPTIC AGENTS

hindlimb tonic extension seizures at doses 6 1 mg/kg p.0. In contrast, audiogenic-evoked clonic seizures were not inhibited at doses up to 3 mg/kg p.0. The overall profile of activity of SB-204269 in DBA2 mice and in the rodent PTZ infusion and electroshock (MEST & MES) tests (i.e. abolition of tonic extensor seizures with no effect on myoclonus) strongly suggests that the compound primarily produces anticonvulsant activity by preventing seizure spread [30]. Furthermore, the inability of SB-204269 to change the myoclonic seizure threshold in mice or rats indicates that the compound may be unlikely to either inhibit or exacerbate absence seizures in the clinic [5,14]. Amygdala-kindled seizures in rat Repeated application of low level (subconvulsive) electrical stimulations of discrete brain regions such as amygdala, results in a progressive intensification of behavioural seizures, a phenomenon known as kindling [5]. Following administration of SB-204269 (30 mg/kg P.o., 2 h prior to each stimulus), a median of 15 (range 9 - 621) daily electrical stimulations were required to produce the first full kindled seizure in rats. This represented a slight, but non significant (p >0.05), increase above the number of daily stimulations needed to reach the same seizure stage in vehicle-treated control animals (median 13; range 7-19). However, at the conclusion of the study on day 21, 100% (10 out of 10) of control animals, but only 53% (9 out of 17) of SB-204269-treated rats, had exhibited 3 consecutive full kindled seizures (a statistically significant difference, p < 0.05; 2-tailed Fishers exact test). This study indicates that SB-204269 inhibits the evolution of amygdalakindled seizures in the rat and thereby indicates potential efficacy against the progressive process leading to epileptogenesis. In vitro high potassium rut hippocampal bruin slice model of epileptiform discharges In an in vitro rat hippocampal slice model evoking spontaneous electrographic discharges analogous to those observed during clinical focal seizures, SB-204269 produced a marked concentration-dependent decrease in the number of epileptiform afterdischarges recorded in the CA1 region [23]. The initial trigger stimulus was unaffected, thereby reflecting the compound’s ability to retard seizure spread. SB-204269 produced a 50% inhibition of afterdischarges at a concentration of approximately 0.2 pM and was therefore at least ten-fold more potent than carbamazepine (IC503.1 pM) or lamotrigine (ICsO3.4 pM) in this mod-

N. UPTON A N D M. THOMPSON

193

el. Importantly, in control preparations, SB-204269 had no effect on normal synaptic activity at concentrations up to 50 pM [23].

PROFILE OF SB-204268 IN ANIMAL SEIZURE MODELS

In marked contrast to SB-204269, the corresponding 3S,4R enantiomer, SB204268 (4), did not produce significant anticonvulsant activity in the rat MEST (10 mg/kg P.o.), PTZ infusion (10 mg/kg p.0.) or high potassium hippocampal slice (up to 3 pM)models at the doses tested [23].

ASSESSMENT OF POTENTIAL CNS AND CARDIOVASCULAR SIDE-EFFECTS

Because dose-related CNS side effects limit the use of all currently available anticonvulsant drugs [4,6], the potential for SB-204269 to cause neurological deficits such as sedation and motor incoordination was fully investigated. The rat accelerating rotarod test is particularly suited for detecting such properties [311 and in this model SB-204269 did not impair performance even at doses as high as 200 mg/kg p.0. In marked contrast, carbamazepine and lamotrigine produced largely dose-related impairments of rotarod performance both with a MED of 80 mg/kg p.0. [23]. SB-204269 was also found to be devoid of potential CNS side-effects in a wide range of additional behavioural models. For example, SB-204269 was inactive at doses up to 300 mg/kg p.0. in the mouse Irwin profile screen [32] which uses close observation and simple behavioural manipulations to score approximately 40 indicators of behavioural, neurological and autonomic drug effects. Moreover, at very high doses the compound did not impair rat spontaneous locomotor activity (100 mg/kg p . ~ . ) short , term (100 mg/kg p.0.) or spatial memory (50 mg/kg P.o.), nor did it potentiate the CNS depressant actions of ethanol (200 mg/kg p.0.) or pentobarbitone (200 mg/kg P.o.),or have any effect on cortical EEG in conscious rats (50 mg/kg p.0.) (unpublished observations). This lack of behavioural or neurological impairment for SB-204269 is reflected in the compound’s superior therapeutic index (MED in the rat rotarod test/EDSo in MES test) of >31 as compared to equivalent ratios of only 7 and 13 for carbamazepine and lamotrigine, respectively *[23]. Despite the fact that SB-204269 is structurally derived from a series of hypotensive agents related to levcromakalim which exert their actions by opening ATP-regulated potassium channels (see Emergence of the Benzopyrans and Serendipity), there is no evidence to suggest that this compound acts in

194

BENZO[b]PYRANOLS AND RELATED NOVEL ANTIEPILEPTICAGENTS

a similar manner as indicated by its inability to lower arterial blood pressure (diastolic and systolic) or heart rate in conscious rats at doses of 30 or 100 mg/kg p.0. [23].

ADDITIONAL PHARMACOLOGICAL ACTIONS

In view of its pronounced activity in models of seizures, it was hypothesized that SB-204269 may also have potential utility in other CNS disorders in which excessive neuronal excitability could play a role. Of particular interest, therefore, are the observed effects of SB-204269 in animal anxiety paradigms. In the social interaction test [33], SB-204269 (3-30 mg/kg p.0.) was found to increase the level of active social contact between pairs of rats placed in a brightly lit unfamiliar observation arena, without changing concomitant locomotor activity scores (Table 5.8). This profile of activity for SB-204269 is consistent with that of the benzodiazepine anxiolytic agent, chlordiazepoxide (CDP; 5 mg/kg p.0.).

Chlordiazepoxide (CDP)

Similarly, SB-204269 (3 mg/kg p.0.) also produced an anxiolytic-like action in an elevated X-maze test [34] where the compound enhanced the proportion of time spent by rats on the more aversive open arms of the maze (Table 5.9). In contrast to the X-maze test, SB-204269 (10 and 30 mg/kg p.0.) had no effect in the Geller-Seifter shock conflict model of anxiety [35].

195

N. UPTON AND M. THOMPSON

Table 5.8. ANXIOLYTIC PROPERTIES OF SB-204269AND CDP IN THE RAT SOCIAL INTERACTION TEST UNDER HIGH LIGHT/FAMILIAR CONDITIONS Mean Values Treatment

Vehicle Vehicle CDP

SB-204269

Dose ( m d k g p .0.)

Pretest Tiniv (minutes)

Social Interaction (seconds)

Locomotor Activity Score

5 3 10 30

60 240 60 240 240 240

61.6 65.1 158.0"'

652 589 663 636 673 619

107.0'

109.0' 115.0"

n = 12pairs per treatment group; for CDP statistical analysis was performed using Students t test; for SB-204269statistical analysis was performed using Dunnett's t test following significant 1way ANOVA; * p <0.05;"; p tO.01 compared to corresponding vehicle-treated control group. There were no significant effects on locomotor activity. Table 5.9. ANXIOLYTIC PROPERTIES OF SB-204269AND CDP IN THE RAT ELEVATED X-MAZE TEST TreatmenP ( m d k g p .0.)

Median Values X Open/Total

Vehicle (Iml/kg) CDP (20) SB 204269 (3)

End of Open Arm

En tries

Time

22.5 41** 35*

4 17*

14.5

Entries

Time (s)

1

6 44* 34.5

6.5* 4

Total Line Crossings

60 78 62

n=l1-12 per treatment group; ': 240 minute pretest. Statistical analysis was performed using Mann Whitney U test following significant Kruskal-Wallis 1-way ANOVA; * p (0.05; ** p tO.O1 compared to vehicle-treated control group.

THERAPEUTIC POTENTIAL OF SB-204269

We have found SB-204269 to be an orally-effective anticonvulsant with excellent potency and efficacy, relative to standards, in a wide range of in vivo models employing a variety of convulsant stimuli (electrical, chemical or audiogenic) and in vitro rodent seizure preparations (see earlier). The efficacy profile and mode of action of SB-204269 observed in these seizure models is strongly indicative of therapeutic potential for symptomatic control of

196

BENZO[h]PYRANOLS AND RELATED NOVEL ANTIEPILEPTIC AGENTS

partial epilepsy (the most refractory type in adults) and generalized tonicclonic seizures [5,8,14]. Together these seizure types constitute 70-90% of all seizures. In addition, SB-204269 appeared to slow the development of amygdala-kindled seizures in rats indicating that the compound may be able to inhibit epileptogenesis. Importantly, the anticonvulsant properties of the compound showed a long duration of action and no loss of efficacy following repeated administration (see Figure 5.1). Current therapies for epilepsy disorders possess many disadvantages particularly regarding the induction of neurological deficits, but in wide-ranging behavioural studies SB-204269 was remarkably devoid of neurotoxic actions. This beneficial characteristic is manifest in the impressive therapeutic ratio for the compound which is clearly superior to the equivalent ratios determined for existing anticonvulsants. SB-204269 is dissimilar to other reported anticonvulsant drugs in terms of its profile of effects in the extensive range of mechanistic assays evaluated to date and evidence now strongly suggests that the anticonvulsant action of SB-204269 is related to a specific interaction at its own stereoselective binding site in the CNS. These findings highlight the unique nature of the mechanism of action of SB-204269, a property that may correlate with the lack of neurological impairment seen in vivo and could well enhance the prospects of this agent for treating patients with currently refractory epilepsy. As well as the compound’s marked anticonvulsant properties, SB-204269 has other potential therapeutic actions that could prove of additional benefit in the treatment of epilepsy patients. For example, SB-204269 exhibits efficacy in the rat social interaction and elevated X-maze models of anxiety. There is now convincing evidence that anxiety often co-exists with various forms of epilepsy and it has been proposed that in such instances, agents effective for both conditions should be considered [36]. In view of this overall pharmacological profile, the decision was made to evaluate SB-204269 as a potential therapy for epilepsy patients with refractory partial seizures (with or without secondary generalisation). The compound has progressed to Phase I1 of clinical development [37]. FUTURE CHEMICAL STRATEGIES HIGH-THROUGHPUT SCREENING (HTS) A N D SAR OF TETRAHYDROISOQUINOLINES

We wished to identify alternative structural classes in order to fully exploit this novel binding site and capitalise on the superior anticonvulsant profile

197

N. UPTON AND M. THOMPSON

afforded by its modulation. High-throughput screening of the SB compound bank using the [3H]-SB-204269binding assay in rat forebrain revealed the two isomeric tetrahydro-isoquinolinyl (THIQ) benzamides, 5-substituted (27) (pKi 7.8) and 8-substituted (31) (pKi 6.1) (Table 5.10). The former already has a 3-fold higher affinity than SB-204269 but, unfortunately, only gave a moderate increase in seizure threshold when examined in vivo in the mouse MEST test at a dose of 10 mg/kg p.0. Lead optimisation provided compounds such as (27)-(34) with an improved in vivo anticonvulsant profile [38, 391. Attachment of the benzamide moiety at either the 5- or 7-position (28, 29) was much preferred over attachment at the 6- or 8-position (30, 31) of the THIQ nucleus. Interestingly, the presence of a 2-methoxy substituent, essential for high affinity in the Sseries, was not crucial for the 7-series and molecular modelling studies were used to rationalise all these findings [38, 391.

--

Table 5.10. BIOLOGLCAL DATA FOR COMPOUNDS (27) - (34)

MeO,

(27)- (33)

-

(34) Rodent M E S F

Cpd

lsorner

R

[-'H]-SB-204269 Binding" p Ki

Mouse at I k post-dose

Rat at 4h ~~ost-do.~e

-

7.3 7.8 7.7 7.6 6.4 6.1 8.2 7.6 8.9

loo*** 25* 140*** 130*** 4ns Nd 120*** 130*** Nd

570*** Nd 330*** 1 lo*** Nd Nd 1120*** 1370*** 1200*** PPOI

4-NH2. 5-C1 4-But 4-Bu' 4-But 4-NH2,S-CI 4-OPr'. 5-CI 4-Pr1,5-CF3 3-CF3,4-OMe

a Procedure as detailed in [13]; % Increase in seizure threshold at 10 mg/kg P.o., procedures as detailed in [23. 271; Statistical analysis conducted according to [40] * p<0.05, ** p
198

BENZO[h]PYRANOLS AND RELATED NOVEL ANTIEPILEPTIC AGENTS

Compound (34) was particularly impressive being one of the most potent (pKi 8.9) and efficacious compounds identified (with an EDs0 value of 0.67 mg/kg P.o., 4 h pretest, determined in the more stringent rat MES model) which holds promise for the future of this chemical series. CONCLUSION

SB-204269 is the prototype of a chemically and mechanistically novel class of anticonvulsant agents. Its ability to selectively inhibit experimental seizures affords the promise of a safe and effective treatment for patients with refractory epilepsy and, potentially, in other disorders (e.g. mania) in which anticonvulsant drugs have also proven beneficial. In addition, SB-204269 and analogues have defined a new stereoselective CNS binding site which appears to play an important role in the regulation of neuronal excitability. It will be fascinating in the future to identify the precise molecular nature of this unique mechanistic target. ACKNOWLEDGEMENTS The authors wish to thank Kellie Darker for help with the manuscript preparation, Jenny Roberts and Hugh Herdon for binding and autoradiographic studies and Tania Stean for seizure and anxiolytic model data. REFERENCES 1 Commission on Classification and Terminology of the International League Against

Epilepsy (1989) Epilepsia 30,389-399. 2 Commission on Classification and Terminology of the International League Against Epilepsy (1981) Epilepsia 22,489-501. 3 Leach, J.P. and Brodie, M.J. (1995) Seizure 4, 5-17. 4 Brodie, M.J. (1990) Lancet 336, 350-354. 5 Loscher, W. and Schmidt, D. (1988) Epilepsy Res. 2, 145-181. 6 Kalviainen, R., Keranen, T. and Riekkinen, P.J. (1993) Drugs 46, 1009-1024. 7 Heinemann, U., Draguhn, A. and Meiekord, H. (1996) in The Treatment of Epilepsy (Shorvon, S., Dreifuss, F., Fish, D. and Thomas, D., eds.), pp. 3-19, Blackwell Science Ltd. 8 Upton, N. ( I 994) Trends Pharmacol. Sci. 15,456-463. 9 Rogawski, M.A. and Porter, R.J. (1990) Pharmacol. Rev. 42,223-286. 10 Loscher, W. (1998) Eur. J. Pharmacol. 342, 1-13. 1 1 Noyer, M., Gillard, M., Matagne, A., Henichart, J.-P.and Wulfert, E. (1995) Eur. J. Pharmacol. 286. 137-146.

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12 Herdon, H., Jerman, J., Stean, T., Chan, W., Middlemiss, D. and Upton, N. (1996) Eur. J. Pharmacol. 314. R7-R8. 13 Herdon, H.J.. Jerman, J.C.. Stean, T.O., Middlemiss, D.N., Chan, W.N., Vong, A.K., Evans, J.M.,Thompson, M. and Upton, N. (1997) Brit. J. Pharmacol. 121, 1687-1691. 14 Swinyard. E.A., Woodhead, J.H., White, H.S. and Franklin, M.R. (1989) in Antiepileptic Drugs, (Levy, R., Mattson, R., Meldrum, B., Penry. J.K. and Dreifuss, F.E., eds.), 3rd Edn., pp. 85-102, Raven Press Ltd, New York. 15 Evans, J.M., Hamilton,T.C., Longman, S.D. and Stemp, G. (1996) in Potassium Channels and their Modulators: From Synthesis to Clinical Experience, 1st Edn., Taylor and Francis Ltd., London. 16 Ashwood, V.A., Cassidy, F., Coldwell, M.C., Evans, J.M., Hamilton, T.C., Howlett, D.R., Smith, D.M. and Stemp, G. (1990) J. Med. Chem. 33,2667-2672. 17 Ashwood,V.A., Buckingham, R.E., Cassidy, F., Evans, J.M., Faruk, E.A., Hamilton,T.C., Nash. D.J., Stemp, G. and Willcocks, K. (1986) J. Med. Chem. 29,21942201, 18 Gandolfo, G., Gottesman, C.. Bidard. J.-N. and Lazdunski, M. (1989) Eur. J. Pharmacol. 159,329-330. 19 Del Pozo. E.. Barrios, M. and Baeyens, J. M. (1990) Pharmacol. and Toxicol. 67, 182-184. 20 Gandolfo, G., Romettino, S.. Gottesman, C., van Luitjtelaar, G., Coenen, A,, Bidard, J.-N. and Lazdunski, M. (1989) Eur. J. Pharmacol. 167, 181-183. 21 Blackburn, T.P.. Buckingham, R.E., Chan, W.N., Evans, J.M., Hadley, M.S., Thompson, M: Upton, N., Stemp, G. andVong, A.K. (1995) Bioorg. Med. Chem. Lett. 5, 1163-1166. 22 Brown. T.H.. Campbell, C.A., Chan, W.N., Evans, J.M., Martin, R.T., Stean, T.O., Stemp, G., Stevens, N.C.. Thompson, M., Upton, N. and Vong, A.K. (1995) Bioorg. Med. Chem. Lett. 5. 2563-2566. 23 Upton, N., Blackburn,T.P., Campbell,C.A., Cooper, D., Evans, M.L., Herdon, H.J., King, P.D., Ray, A.M., Stean, T.O., Chan, W.N., Evans, J.M. and Thompson, M. (1997) Br. J. Pharmacol. 121. 1679-1686. 24 Chan. W.N.. Evans, J.M., Hadley, M.S., Herdon, H.J., Jerman, J.C., Morgan, H.K.A., Stean, T.O., Thompson, M., Upton, N. and Vong, A.K. (1996) J. Med. Chem. 39, 4537-4539. 25 Chan, W.N., Evans, J.M., Hadley, M.S., Herdon. H.J., Morgan, H.K.A., Thompson, M. and Upton, N. (1997) Bioorg. Med. Chem. Lett. 7, 1573-1576. 26 Chan, W.N., Evans, J.M.. Hadley. M.S., Herdon, H.J., Jerman, J.C., Parsons, A.A., Read, S.J.. Stean, T.O., Thompson, M., Upton, N. and Ward, R.W. (1998) Bioorg. Med. Chem. Lett. 8,2903-2906. 27 Upton. N., Stean, T., Middlemiss, D., Blackburn, T. and Kennett, G. (1998) Eur. J. Pharmacol. 359.33-40. 28 Miller, A.A.. Sawyer, D.A.. Roth, B., Peck, A.W., Leach, M.J., Wheatley, P.L., Parsons, D.N. and Morgan. R.J.1. (1986) in New Anticonvulsant Drugs (Meldrum, B.S. and Porter. R., eds.), pp. 165-177, John Libbey, London. 29 Fuller. J.L. and Sjursen, F.H. (1967) Heredity 58, 135-140. 30 Piredda, S.G.. Woodhead, J.H. and Swinyard, E.A. (1985) J. Pharmacol. Exp. Ther. 232, 741-745. 31 Jones. B.J. and Roberts, D.J. (1968) J. Pharm. Pharmacol. 20,302-304. 32 Irwin, S. ( I 968) Psychopharmacologia 13,222-257. 33 Kennett. G.A., Wood, M.D., Glen. A,, Grewal, S. Forbes, I.T., Cadre, A. and Blackburn, T.P. (1994)Br. J. Pharmacol. 111,797-802. 34 Blackburn, T.P.. Baxter, G.S., Kennett, G.A., King, ED.. Piper, D.C., Sanger, G.J., Thomas, D.R., Upton, N. and Wood, M.D. (1993) Psychopharmacology 110,257-264.

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35 Kennett, G.A., Bailey, F., Piper, D.C., and Blackburn. T.P. (1995) Psychopharmacology 118, 178-182. 36 Nickell, P.V. and Uhde, T.W. (1991) in Epilepsy and Behaviour (Devinsky, 0. and Theodore, W.H., eds.), pp. 67-84, Wiley-Liss Inc. 37 SCRIP (1998) 2329, 19. 38 Chan, W.N., Hadley, M.S., Harling, J.D., Herdon, H.J., Jerman, J.C., Orlek, B.S., Stean, T.O., Thompson, M. and Upton, N. (1999) Bioorg. Med. Chem. Lett. 9,285-290. 39 Harling, J.D. Trends in Medicinal Chemistry, SMR Meeting, London, Dec., 1998. 40 Litchfield, J.T. and Wilcoxon, F.A. (1949) J. Pharmacol. Exp. Ther. 96,99-113.

Subject Index Allopregnanolone (AP), 136, 137, 144, 146, 147. 150, 152, 15&-157, 160-163 Allotetrahydrodeoxycorticosterone (THDOC). 136, 137. 144, 146, 147, 152-156. 160 Alphadolone, 144 Alphaxolone, 136, 137, 146. 152, 155, 156 Alpidem, 143 Alzheimer’s disease, neurosteroids and. 158, 165 Anticonvulsant drugs, evaluation of, 180-1 82 identification of, I8 1 mechanistic categories, 180 structures, 187 Anti-secretory activity, CCK-B antagonists and. 41 Anxiety. CCK-B antagonists and, 46 Apoptosis. genes regulating, 14 Aprikalim. 184 Asperlicin. 49 Bead based library synthesis, 86 Bead based screening (BS), 86,92-98 differential release. 98.99 in lawn format. 93 in solution assays, 94 incrementally cleavable format, 98 twin orthogonal linker format, 96.97 Benzazepines, as CCK-B antagonists, 63,64 Benzo[b]pyranols. SAR studies, 184-187 Benzodiazepine libraries, 20.21, 98, 100 Benzodiazepines, I80 1,4-Benzodiazepines, as CCK-B antagonists, 49-57 1,s-Benzodiazepines. as CCK-B antagonists, 58-63 Betaxolone. 146 Bioinformatics, 4. 18. 33 Calcium channels, modulation by neurosteroids, I52 Carbamazepine, 180, 188-1 90 Cathepsin K, cysteine protease, 29-32 CCD-3693, 162 CCK-4.46.47.64. 73

CCK-4, actions of, 47 CCK-A receptors, 46 CCK-B antagonists, anti-anxiety activity, 46 anti-secretory activity, 47, 57.62, 66, 70, 72, 74, 75 benzazepines, 49-64 clinical studies, 48 dibenzobicyclo[2.2.2]octanes,73-75 hybrid derivatives, 66-71 peptoids, 64-66 ureidomethylcarbamoylphenylketones. 71-73 CCK-B receptors, 46 Chem X 3D, 3-point pharmacore keys, 110 Chemoinformatics, 107 lead generation using, 107-1 10 molecular descriptors, 1 10 selection methods, 1 10-1 12 validation, 112 4’-Chlordiazepam, mitochondria1 DBI agonist, 143, 154, 157. 162, 163 Chlordiazepoxide, 194 Cholecystokinin (CCK), 45 CI-1015,65,66 CI-988,47. 64-66 Cloning methods, 7 Combinatorial chemistry, 5 , 19 Combinatorial synthesis equipment, 86, 101-105 CP-212,454, 63.64 CP-310,713,63,64 Cromakalim, 182 Cyclin dependent kinase (CDK) inhibitors, 26 Cytokines, 9, 17 DA-3797,69,70 DA-3934,69,70 Deconvolution of combinatorial libraries, 21 Dehydroepiandrosterone (DHEA), 136, 157, 158, 162, 165 Dehydroepiandrosterone sulphate (DHEAS) 136, 137, 144, 149, 151-153, 156-163. 165, 166 Deoxycorticosterone (DOC) 136, 144, 160

202

SUBJECT INDEX

Devazepide (MK-329), 49 Diazepam binding inhibitor (DBI) receptor, 143 Diazepam, 187, 188, 190, 191 Dibenzobicyclo[2.2.2]octanes,as CCK-B antagonists, 73-75 Diversity, 3, 21 DNA microarrays, 9,33 Drug discovery paradigms, 3 Drug targets, 4, 13,23, 26,28 DZ-3514,69,70 ELISA and ion exchange separation assays, 1 I6 Epalons. 147 Epilepsy, aetiology, 179 categories of, 178 drugs for, 179, 180 experimental models for, 180-182 neurosteroids and, 154-1 56 Epipregnanolone, 144 ESTdatabases, 5,8, 15,23,30 Ethosuximide, 180 Expression sequence tags (ESTs), 4 - 1 1, 15, 24,30,33 Felbamate, 180 FGIN-1-27,143,144 FGIN-1-29, 143, 144 Flavopiridol, CDK inhibitor, 26 Fluorescence resonance energy transfer (FRET) assays, 1 14-1 16 Functional genomic technologies, 8 Furin. prohormone processing protease, 9 GABA-A receptors, modulation by neurosteroids, 145-1 50 Gabapentin, 180,187-189 Ganaxolone, 144, 155, 156 Gastrin, 47,48 Genomes, archae, 2,6, 19, 36 bacteria, 5,6, 19,28,36 fish, 6 fruit fly (Drosophila melanogaster),6, 14 haemophilus influenzae, 2,6 human, 2,5,28, 35 mouse (mus musculus), 6, 14, 36 nematode (C. elegans), 6, 14,28,36 sequencing, 2, 5 , 16, 34

yeast (Saccaron?ycescerevisiae), 6, 14,24, 28 Genomic targets, identification of, 4 mRNA profiling, 8-1 I , 14,34 protein profiling, 1 1-13 structure, 16 validation, 7 Genomics, comparative, 13-16, 18,26,33 functional, 14, 33 structural, 17-18 G-protein coupled receptors, 7, 14, 18, 23-26 CCK-B receptors, 46 GR199114X, 60,61 GV150013X, 59 GV191869,59 GW529571X, 99 GW529578,98 GW529581X, 99 High-throughput genomic sequences (HTG), 5,7, 34 High throughput screening (HTS), 3,22,25 anticonvulsants, 196 Hippocampal slice model, 192 Homogeneous time-resolved fluorescence assays, 11 5 5-HT3breceptor, 5,7 HTS automation, 120-1 29 robotics and, 121-125 HTS bioassay design, I 13 fluorescence assays, 114 scintillation proximity assays, 114 HTS biochemical assays, I13 ELISA and ion exchange separation, 116 fluorescence energy transfer (FRET), 114-1 I6 fluorescence polarization, 115 homogeneous time-resolved fluorescence (HTRF), I 15 scintillation proximity, 114 HTS cellular functional assays, 117 HTS data handling, 125 bioassay data analysis, 126-129 logistics, 125 HTS evaluation of ADME properties, 117-120 computational, I19 in vitro for absorption and metabolism, 1 18 HTS sample selection, 105 clustering, 111-1 12

SUBJECT INDEX ethnomedical, 105 filters, 109 partitioning. 112 HTS screening. 22-23 biased, 108 focused, 108 iterative, 109 Human genome project, 5 Human genome. 2 Ion channels, 16. 18, 23 Kinases, 19. 26-28 L-364,718,75 L-365,260,47-54, 57, 60, 62, 63. 66. 69, 71, 72,75 L-708,474,49-51, 53 L-736,309. 51 L-740,093, 52.53.55 Lamotrigine 180, 187-1 90 Lead generation, chemoinformatics in, 107-1 10 Lead optimisation. combinatorial approaches, 99 Levetiracetam. I80 Levcromakalim, 182 Levcromakalim, brain penetrating analogues, 182 Library equivalents. 90, 93-98 Library synthesis, bead-based, 86 linkers, 94,98 split-mix. 87-90 Ligand identification. by database searching, 24 fishing, 24 Linkers in combinatorial synthesis, acid labile, 94 photochemically labile. 98 Metalloproteinases, 9 Microtitre plates. 120 Migraine, neurosteroids and, 164 Minaxolone, 144. 156 MK-329.49 Models of seizure activity. amygdala-kindled, I92 audiogenic, 191 electroshock, 187-1 90 PTZ infusion test, 191

20 3

Molecular descriptors for sample selection, 110 2/~-Morpbolino-Sa,3cc-pregnanolone,I 54 mRNA expression (profiling), 8-1 I , 14, 34 mRNA expression, microarrays, 3.9-12, 27, 33 Natural Products Alert database (NAPRALERT), 107 Natural products libraries, 105-107 Neurosteroids, anorectic activity, I62 anticonvulsant activity, 154 antidepressant activity, 160 anti-migraine activity, 164 anxiolytic activity, 156 assay and identification. 138 biosynthesis, 139-142 drug dependence and, 163 effects on neuronal injury, 166 hypnotic activity, 161 receptors modulated by, 144 sedative activity, 161 structure-activity relationships, 146 structures, 137, 138 therapeutic potential, 153-166 toxicity, 166 Neuroactive steroids, structures, 137, 138, 156, 162 Nucleotide databases, 4,5,33,35 Olomoucine, CDK inhibitor, 26 Orphan proteases. 28 Orphan receptors, 23-26 Orthologues, 15. 34 Oxcarbazepine, 180 Paralogues, I5,34 PD134308 (CI-988), 47,59,64,66 Peptoids, as CCK-B antagonists, 64-66 Phenobarbital, 180 Phenoxyacetic acid derivatives, as CCK-B antagonists, 69-71 Phenytoin, 180, 187, 189 Pinacidil, 184 PKll195, 143, 144 Platform technologies, 3, 19 Potassium channel openers, 182 Sc(-Pregnan-3a,20c-diol, 155 Pregnanolone 137, 162

204

SUBJECT INDEX

Pregnenolone sulphate (PS), 136, 137, 144, 149, 151-154, 156, 157, 159-163, 165, 166 Pregnenolone, 136, 137, 149. 152, 154, 158, 166 Progesterone, 136, 144. 152-154, 157, 158, 162, 166 Proteases, 19.28-32 Protein databases, 4, 12, 17.34, 35 Protein folds, 17-19, 33 Proteomics. 11-1 3, 35 Purvalonol B, CDK inhibitor. 26-27 Quinazolinones, as CCK-B antagonists, 66-69 RP72540,69,70 RU5135, 138, 144 S-0509,7 1-73 38-204268, 183, 193 ['H]-SB-204269,183 SB-204269 anxiolytic properties, 194, 195 anticonvulsant properties, 187-193 cardiovascular and CNS effects, 193 pharmacology, 183-195 stereoselective binding site. 183, 184 structure, 183 therapeutic potential, 195 Single nucleotide polymorphisms (SNPs), 32, 35 Somatostatin receptor hgdnds, 22, 24

Spiroglumide, 48 Steroid binding site on GABA-A receptors, 147 Steroid sulphatase inhibitors, 158 Tagged sequence mutagenesis, 16 Tagging and encoding in combinatorial synthesis, 90-92 Tanimoto coefficient, similarity index, 1I 1 Target validation, 7 Tetrahydroisoquinolines,as anticonvulsants, 196-198 Tiagabine, 180 Topiramate, 180 United Nations Convention on Biodiversity, 105

Ureidomethylcarbamoylphenylketones,as CCK-B antagonists, 71-73 Valproate, 180, 184 Vigabatrin, 180 Voltage gated calcium channels, 152 YF476,54,57 Y M022, 53, 54, 57, 58,62, 63,69, 70 Zollinger-Ellison syndrome, CCK-B antagonists and, 47 Zonisamide, 180

Cumulative Index of Authors for Volumes 1-37 The volunir nirnihrr, (yetrr of publication) and puge number are given in thut order. Adams, S.S.. 5 ( 1967)59 Agrawal, K.C.. 15 (1978)321 Albrecht,W.J., 18 (1981)135 Allain, H., 34 (1997)1 Allen, N.A., 32 (1995)157 Allender, C.J., 36 (1999)235 Andrews, P.R.. 23 (1986)91 Ankier, S.I., 23 (1986)121 Bailey. E., 11 (1975)193 Ballesta. J.P.G., 23 (1986)219 Banting, L., 26 ( I 989)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)I 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)1 Beisler, J.A., 19 (1975)247 Bell, J.A., 29 (1992)239 Belliard. S., 34 ( I 997)I Benfey, B.G.. I 2 ( 1975)293 Bentuk-Ferrer, D.. 34 (1997)I Bernstein, P.R., 31 (1994)59 Binnie, A,, 37 (2000)83 Black, M.E., 11 (1975)67 Blandina, P., 22 (1985)267 Bond. P.A., I 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 (1975)247 Brooks. B.A., I I (1975)193

Brown, J.R.. 15 ( I 978)125 Brunelleschi, S., 22 (1985)267 Bruni. A,. 19 (1982)1 1 1 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(1 965)171; 7 ( 1970)

229;11 (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)1 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.. 31 (1994)297;33 (1996)99 Cox, B., 37 (2000)83 Crossland. J., 5 (1967)251 Crowshaw, K.,IS (1978)357 Cushman, D.W., 17 (1980)41 Cuthbert, A.W., 14 (1977)1 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)176 Davies, R.V., 32 (1995)115 De,A., 18(1981)117 De Clercq, E., 23 (1 986)I87 De Gregorio, M.. 21 (1984)1 1 1 De Luca, H.F., 35 (1998)1 Demeter, D.A., 36 (1999)169 Denyer, J.C., 37 (2000)83 Derouesni, C.. 34 (1997)I

206

CUMULATIVE AUTHOR INDEX

Dimitrakoudi, M., 1 1 (1975) 193 Donnelly, M.C., 37 (2000) 83 Draffan, G.H., 12 (1975) 1 Drewe, J.A., 33 (1996) 233 Dubinsky, B., 36 (1999) 169 Duckworth, D.M., 37 (2000) 1 Duffield, J.R., 28 (1991) 175 Durant, G.J., 7 (1970) 124 Edwards, D.I., 18 (1981) 87 Edwards, P.D., 31 (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 Feigenbaum, J.J., 24 (1987) 159 Feuer, G., 10 (1974) 85 Finberg, J.P.M., 21 (1984) 137 Fletcher, S.R., 37 (2000) 45 Floyd, C.D., 36 (1999) 91 Frank, H., 27 (1990) I Francois, I., 31 (1994) 297 Freeman, S., 34 (1997) 11 1 Fride, E., 35 (1998) 199 Gale, J.B., 30 (1993) 1 Garrdtt, C.J., 17 (1980) 105 Gill, E.W., 4 (1965) 39 Ginsburg, M., l(1961) 132 Goldberg, D.M., 13 (1976) 1 Gould, J., 24 (1987) 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) I Griffiths, K., 26 (1989) 299 Groenewegen, W.A., 29 (1992) 217 Groundwater, P.W.. 33 (1996) 233 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

Halliday, D., 15 (1978) 1 Hammond, S.M., 14 (1977) 105; 16 (1979) 223 Ham0r.T.A.. 20 (1983) 157 Hanson, P.J., 28 (1991) 201 Hanus, L., 35 (1998) 199 Hargreaves, R.B., 31 (1994) 369 Harris, J.B., 21 (1984) 63 Hartley, 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 Heinisch, G., 27 (1990) 1; 29 (1992) 141 Heller, H., l(1961) 132 Heptinstall, S., 29 (1992) 21 7 Herling, A.W., 31 (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 Hooper, M., 20 (1983) 1 Hopwood, D., 13 (1976) 27 I Hosford, D., 27 (1990) 325 Hubbard, R.E., 17 (1980) 105 Hughes, R.E., 14 (1977) 285 Hugo,W.B., 31 (1994) 349 Hulin, B., 31 (1994) 1 Humber, L.G., 24 (1987) 299 Hunt, E., 33 (1996) 99 Imam, S.H., 21 (1984) 169 Ireland, S.J., 29 (1992) 239 Jacques, L.B., 5 (1967) 139 James, K.C., 10 (1974) 203 Jiszbertnyi, J.C., 12 (1975) 395; 14(1977) 181 Jenner, ED., 11 (1975) 193 Jewers, K., 9 (1973) 1 Jindal, D.P., 28 (1991) 233 Jones, D.W., 10 (1974) 159 Judd,A., 11 (1975) 193 Judkins, B.D., 36 (1999) 29 Kadow, J.F.. 32 (1995) 289 Kapoor,V.K., 16(1979)35; 17(1980) 151;22 (1985) 243 Kawato,Y., 34 (1997) 69 Kelly, M.J., 25 (1988) 249

CUMULATIVE AUTHOR INDEX 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 Kirst. H. A.. 30 (1993) 57; 31 (1994) 265 Kitteringham, G.R., 6 (1969) 1 Knight, D.W., 29 (1992) 217 K0bayashi.Y.. 9 (1973) 133 Koch, H.P., 22 (1985) 165 Kopelent-Frank, H.. 29 (1992) 141 Kramer, M.J., 18 (1981) 1 Krogsgaard-Larsen, P.. 22 (1985) 67 Kulkami, S.K., 37 (2000) 135 Kumar, M., 28 (1991) 233 Lambert. P.A., I5 (1978) 87 Launchbury, A.P., 7 (1970) 1 Law. H.D., 4 (1965) 86 Ldwen, A,. 33 (1996) 53 Lawson, A.M. 12 (1975) 1 Leblanc, C.. 36 (1999) 91 Lee. C.R., 11 (1975) 193 Lenton, E.A.. 11 (1975) 193 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 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) 1 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 Matthews, R.S., I0 (1974) 159 Matsumoto. J.. 27 (1990) 123

207

Maudsley, D.V., 9 (1973) 133 May, P.M., 20 (1983) 225 McCague, R., 34 ( I 997) 203 McLelland, M.A., 27 (1990) 51 McNeil, S., 11 (1975) 193 Mechoulam, R., 24(1987) 159; 35 (1998) 199 Meggens, A.A.H.P., 33 (1996) 185 Megges, R., 30 (1993) 135 Merritt, A.T., 37 (2000) 83 Michel, A.D.. 23 (1986) I Miura. K., 5 (1967) 320 Moncada, S., 21 (1984) 237 Monkovic, I., 27 (1990) 297 Montgomery, J.A., 7 ( I 970) 69 Moody, G.J., 14 (1977) 51 Morris, A., 8 (1971) 39; 12 (1975) 333 Munawar, M.A., 33 (1996) 233 Murphy, F., 2(1962) I ; 16(1979) 1 Musallan, H.A., 28 (1991) 1 Musser, J.H., 22 (1985) 293 NatoK 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 (1987) 1 Ondetti, M.A., 17 (1980) 41 Ottenheijm, H.C.J., 23 (1986) 219 Oxford, A.W., 29 ( I 992) 239 Paget, G.E.,4(1965) 18 Palatini, P., 19 (1982) 1I1 Palazzo,G.,21 (1984) 111 Palfreyman, M.N.. 33 (1996) 1 Palmer, D.C., 25 (1988) 85 Pdrkes, M.W:, l(1961) 72 Parnham, M.J., 17 (1980) 185 Parratt, J.R., 6 (1969) 11 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 Petrow,V., 8(1971) 171 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

208

CUMULATIVE AUTHOR INDEX

Price, B.J., 20 (1983) 337 Prior, B., 24 (1987) I Procopiou, P.A., 33 (1996) 331 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., 11 (1975) 67 Richardson, P.T., 24 ( I 987) I Roberts, L.M., 24 (1987) 1 Roe, A.M., 7 (1970) 124 Rose, H.M., 9 (1973) 1 Rosen, T., 27 (1990) 235 Rosenberg, S.H., 32 (1995) 37 Ross, K.C., 34 (1997) 1 1 1 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., I 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 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) 1 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., I (1961) 187 Smith, H.J., 26 (1989) 253; 30 (1993) 327 Smith, R.C., 12 (1975) 105 Smith,W.G., l(1961) I ; lO(1974) 11

Solomons, K.R.H., 33 (1996) 233 Sorenson, J.R.J., 15 (1978)211; 26(1989) 437 Souness, J.E., 33 (1996) 1 Southan, C., 37 (2000) 1 Spencer, P.S.J.. 4 (1965) 1; 14(1977) 249 Spinks, A., 3 (1963) 261 Stihle, L., 25 (1988) 291 Steiner. K.E., 24 (1987) 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) I Sullivan, M.E., 29 (1992) 65 Suschitzky, J.L., 21 (1984) 1 Swain, C.J., 35 (1998) 57 Swallow, D.L., 8 (1971) 1I9 Sykes, R.B., 12 (1975) 333 Talky, J.J., 36 (1999) 201 Taylor, E.C., 25 (1988) 85 Taylor, E.P.. I (1961) 220 YdylOr, s G., 3 1 ( 1994)409 Tegner, C., 3 (1963) 332 Terasawa, H., 34 (1 997) 69 Thomas, G.J., 32 (1995) 239 Thomas, I.L., 10 (1974) 245 Thomas. J.D.R.. 14 (1977) 51 Thompson, E.A., 11 (1975) 193 Thompson, M., 37 (2000) 177 Tilley, J.W., 18 (1981) 1 Traber, R., 25 (1988) 1 Tucker, H., 22 (1985) 121 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 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) 1 Vyas, D.M., 32 (1995) 289 Waigh, R.D., 18 (1981) 45 Wajsbort, J., 21 (1984) 137 Walker, R.T., 23 (1986) 187

CUMULATIVE AUTHOR INDEX Walls, L.P., 3 ( I 963) 52 Walz, D.T.. 19 (1982) I Waring, W.S., 3 ( 1963) 26 1 Watson, N.S.. 33 (1996) 331 Watson, S.P., 37 (2000) 83 Wedler. F. C., 30 ( 1 993) 89 Weidmann, K.. 31 (1994) 233 Weiland. J.. 30 (1993) I35 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., l(1961) 34 Wikstrom, H., 29 (1992) 185 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.. 1 1 (1975) 119 Wold, S., 25 (1989) 291 Wood, E.J.. 26 (1989) 323 Wright, I.G., 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 Zee-Cheng, R.K.Y., 20 (1983) 83 Zon, G., 19 (1 982) 205 Zylicz, 2..23 (1986) 219

209

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Cumulative Index of Subjects for Volumes 1-37 The volume nuniher, (year qfpuhlication) arid!wge nuinher are given in that order.

Adamantane. amino derivatives, 18 (1981) 1 Adenosine triphosphate, 16 (1979) 223 Adenylate cyclase. 12 ( 1 975) 293 Adipose tissue, 17 ( I 980) 105 Adrenergic blockers, u-. 23 (1986) 1 p-. 22 (1985) 121 u2-Adrenoceptors. antagonists, 23 (1986) 1 Adrenochrome derivatives, 9 (1973) 275 Adriamycin, 15 (1978) 125; 21 (1984) 169 AIDS, drugs for, 31 (1994) 121 Aldehyde thiosernicarbazonesas antitumour agents. I5 (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) I ; 22 (1985) 293 Amidines and guanidines, 30 (1993) 203 Aminoadamantane derivatives, 18 (1981) 1 Aminopterins as antitumour agents, 25 (1988) 85 8-Aminoquinolinesas antimalarial drugs, 28 (1991) 1 Analgesic drugs, 2 (1962) 43; 4 (1965) 171; 7 ( 1970) 229; 14 ( I 977) 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 Antiarrhythmic drugs, 29 (1992) 65 Anti-arthriticagents. 15(1978)211; 19(1982) 1;36(1999)201 Antibacterial agents, 6 (1969) 135; 12 (1975) 333; 19 (1982) 269; 27 (1990) 235; 30 (1993) 203; 3 I (1994) 349; 34 (1997) 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 ( 1994) 297; 33 ( 1996) 99

macrolide, 30 (1993) 57; 32 (1995) 157 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) 177 Antidepressant drugs, 15 (1978) 261; 23 (1986) 121 Antiemetic drugs, 27 (1990) 297; 29 (1992) 239 Antiemetic action of 5-HT3 antagonists, 27 (1990) 297; 29 (1992)239 Antiepileptic drugs, 37 (2000) 177 Antifilarial benzimidazoles, 25 (1988) 233 Antifolates as anticancer agents, 25 (1988) 85; 26 (1989) 1 Antifungal agents, 1 (1961) 220 Antihyperlipidaemic agents, 1 I (1975) I19 Anti-inflammatory action of cyclooxygenase2 (COX-2) inhibitors, 36 (1999) 201 of thalidomide, 22 (1985) 165 of 5-lipoxygenase inhibitors, 29 (1992) 1 Anti-inflammatory agents, 5 (1967) 59; 36 (1999) 201 Antimalarial 8-aminoquinolines, 28 (1991) 1 Antimicrobial agents for sterilization, 34 ( I 997) 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) 1;36(1999)201 Antisecretory agents, 37 (2000) 45 Antithrombotic agents, 36 (1999) 29 Antitumouragents,9(1973) 1; 19(1982) 247; 20(1983)83;23(1986)219;24(1987) 1; 24 (1987) 129; 25 (1988) 35; 25 (1988) 85; 26(1989)253;26(1989)299;30(1993)1; 32(1995) 1:32(1995)289;34(1997)69

212

CUMULATIVE SUBJECT INDEX

Antitussive drugs, 3 (1963) 89 Anti-ulcer drugs, ofplant origin, 28 (1991) 20 1 ranitidine, 20 (1983) 67 synthetic, 30 (1993) 203 Antiviral agents, 8 (1971) 119; 23 (1986) 187; 36(1999) 1 Anxiolytic agents, pyrido[l,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, 21 (1984) 1 : 3 1 ( I 994) 369; 31 (1994)409; 33 (1996) 1 ATPase inhibitors, gastric, H+/K+- 31 (1994) 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 I -Benzazepines, medicinal chemistry of, 27 (1990) 123 Benzimidazole carbamates, antifilarial. 25 (1988) 233 Benzisothiazole derivatives, 18 (1981) 117 Benzodiazepines, 20 (1983) 157: 36 (1999) 169 Benzo[b]pyranol derivatives, 37 (2000) 177 Biocides, aldehydes, 34 (1997) 149 mechanisms of resistance, 35 (1998) 133 British Pharmacopoeia Commission, 6 (1969) 1 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 (1989) 253 azides and, 31 (1994) 121 camptothecin derivatives, 34 (1997) 69

endocrine treatment of prostate, 26 (1989) 299 retinoids in chemotherapy, 30 (1993) 1 Cannabinoiddrugs, 24 (1987) 159; 35 (1998) 199 Carbapenem antibiotics, 33 (1996) 99 Carcinogenicity of polycyclic hydrocarbons, 10 (1974) 159 Cardiotonic steroids, 30 (1993) 135 Cardiovascular system, effect of azides, 31 (1994) 121 effect of endothelin, 31 (1994) 369 4-quinolones as antihypertensives, 32 (1995) 115 renin inhibitors as antihypertensive agents, 32 (1995) 37 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 tripositiveelements as, 28 (1991) 41 Chemotherapy of herpes virus, 23 (1985) 67 Chemotopography of digitalis recognition matrix, 30 (1993) 135 Chiral synthesis. 34 (1997) Cholesterol-lowering agents, 33 (1996) 331 Cholinergic receptors, 16 (1976) 257 Chromatography, 12 (1975) 1; 12 (1975) 105 Chromone carboxylic acids, 9 (1973) 65 Clinical enzymology, 13 (1976) 1 Collagenases, synthetic inhibitors, 29 (1992) 27 1 Column chromatography, 12 (1975) 105 Combinatorial chemistry, 36 (1999) 91 Computers in biomedical education, 26 (1989) 323 Medlars information retrieval, 10 (1974) I Copper complexes, 15 (1978) 211; 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) 201

CUMULATIVE SUBJECT lNDEX Cyclophosphamide analogues, 19 (1982) 205 Cyclosporins as immunosuppressants. 25 (1 988) I ; 33 ( 1996) 53 Data analysis in biomedical research, 25 (1988)291 Diaminopyrimidines, 19 (1982) 269 Digitalis recognition matrix, 30 (1993) 135 Diuretic drugs, I (1961) 132 DNA-binding drugs, 16 (1979) 151 Dopamine D2 receptor agonists, 29 (1992) I85 Doxorubicin, 15 (1978) 125; 21 (1984) 169 Drug-receptor interactions, 4 (1965) 39 Drugs, transmitters and peptides. 23 (1986) 91 Elastase, inhibition, 31 (1994) 59 Electron spin resonance, 12 (1975) 191 Electrophysiological (Class Ill) agents for arrhythmia. 29 (1992) 65 Enantiomers, synthesis of, 34 (1997) 203 Endorphins, I7 ( 1980) 1 Endothelin inhibition. 31 (1994) 369 Enkephalin-degrading enzymes, 30 (1993) 327 Enkephalins, 17 ( 1980) 1 Enzymes, inhibitors of, 16 (1979) 223; 26 (1989) 253; 29 (1992) 271; 30 (1993) 327; 31 (1994)59; 31 (1994)297;32(1995)37; 32(1995)239; 36(1999) I; 36(1999)201 Enzymology, clinical use of. I0 (1976) I in pharmacology and toxicology, 10 (1974)

II Erythromycin and its derivatives, 30 ( 1 993) 57; 31 (1994) 265 Feverfew, medicinal chemistry of the herb, 29 (1992)217 Fibrinogen antagonists, iis antithrombotic agents, 36 (1999) 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) I Formaldehyde. biocidal action, 34 (1997) 149

213

Free energy, biological action and linear, 10 ( I 974) 205 GABA, heterocyclic analogues, 22 (1985) 67 GABAA receptor ligands, 36 ( I 999) 169 Gastric H+/K+-ATPase inhibitors, 31 (1994) 233 Gas-liquid chromatography and mass spectrometry, 12 (1975) 1 Genornics, impact on drug discovery, 37 (2000) 1 Glutaraldehyde, biological uses, 13 ( 1976) 27 1 as sterilizing agent, 34 (1997) 149 Gold, immunopharmacology of, 19 (1982) I Guanidines. 7 (1970) 124; 30 (1993) 203 Halogenoalkylamines, 2 (1962) 132 Heparin and heparinoids, 5 (1967) 139 Herpes virus, chemotherapy, 23 (1985) 67 Heterocyclic analogues of GABA, 22 ( 1 985) 67 Heterocyclic carboxaldehyde thiosemicarbazones, 16 (1979) 35; 32 (1995) 1 Heterosteroids, 16 (1979) 35; 28 (1991) 233 High-throughput screening techniques, 37 (2000) 83 Histamine, H2-antagonists, 20 (1983) 337 receptors, 24 (1987) 30 release, 22 ( 1985) 26 secretion, calcium and, 19 (1982) 59 Histidine decarboxylases, 5 (1967) 199 HIV proteinase inhibitors, 32 (1995) 239 Hydrocarbons, carcinogenicity of, 10 (1974) 159 Hypersensitivity reactions, 4 (1965) 1 Hypoglycaemicdrugs, l(1961) 187; 18 (1981) 191;24(1987)209; 30(1993)203;31 (1994) 1 Hypotensive agents, 1 (1961) 34; 30 (1993) 203; 31 (1994) 409; 32 (1995) 37; 32 (1995) 1 I5 Immunopharmacology ofgold, 19 (1982) 1 Immunosuppressant cyclosporins. 25 ( 1988) I India, medicinal research in, 22 (1985) 243 Influenza virus sialidase, inhibitors of, 36 ( 1 999) 1

CUMULATIVE SUBJECT INDEX

214

Information retrieval, 10 (1974) 1 lnotropic steroids, design of, 30 (1993) 135 Insulin, obesity and, 17 (1980) 105 Ion-selective membrane electrodes, 14 (1977) 51

Ion transfer, 14 (1977) 1 Irinotecan, anticancer agent, 34 (1997) 68 Isotopes, in drug metabolism, 9 (1973) 133 stable, I S (1978) 1 Kappa opioid non-peptide ligands, 29 (1992) 109; 35 ( I 998) 83 Lactam antibiotics, 12 (1975) 395; 14 (1977) 181

/3-Lactamase inhibitors, 31 (1994) 297 Leprosy, chemotherapy, 20 (1983) I Leukocyte elastase inhibition, 31 (1994) 59 Ligand-receptor binding, 23 (1986) 41 Linear free energy, 10 (1974) 205 5-Lipoxygenase inhibitors and their antiinflammatory activities, 29 (1992) 1 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; 31 (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 (1985) 267 peptide regulation of, 27 (1990) 143 Medicinal chemistry, literature of, 6 (1969) 266 Medlars computer information retrieval, 10 (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 (1988) 85; 26 (1989) 1 Microcomputers in biomedical education, 26 (1989) 323 Molecularly imprinted polymers, preparation and use of, 36 (1999) 235 Molybdenum hydroxylases, 24 (1987) 85 Monoamine oxidase inhibitors, 21 (1984) 137 Multivariate data analysis and experimental design, 25 (1988) 291 Neuraminidase inhibitors, 36 (1999) 1 Neurokinin receptor antagonists, 35 ( I 998) 57 Neuromuscular blockade. 2 (1 962) 88; 3 (1963) 1; 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 Nickel(I1) chloride and sulphate, biological effects, 27 (1990) 189 Nitriles, synthesis of, 10 (1974) 245 Nitrofurans, 5 (1967) 320 Kitroimidazoles, cytotoxicity of, 18 (1981) 87 NMR spectroscopy, 12 (1975) 159 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, 17 (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 Paclitaxel, anticancer agent, 32 (1995) 289

CUMULATIVE SUBJECT INDEX Parasitic infections, 13 (1976)159;30 (1993)

203 Parasympathomimetics, 1 1 (1975)1 Parenteral nutrition, 28 (1991)175 Parkinsonism, pharmacotherapy of, 9 (1973)

191;21 (1984)137 Patentingofdrugs. 2(1962) I ; 16 (1979)1 Peptides, antibiotics, 5 (1967)I enzymic, 31 ( 1 994)59 hypoglycaemic, 31 ( 1994) 1 mast cell regulators, 27 (1990)143 opioid. 17 (1980)1 Pharmacology of Alzheimer’s disease, 34

(1997)1 Pharmacology of Vitamin E, 25 ( I 988)249 Phosphates and phosphonates as prodrugs, 34 (1997)1 I 1 Phospholipids, 19 (1982)11 1 Photodecomposition of drugs, 27 (1990)51 Platelet-aggregating factor, antagonists, 27 ( 1990)325

Platelet aggregation. inhibitors of, 36 (1999)

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 ( 1 984)63 Prodrugs based on phosphates and phosphonates, 34(1997)1 1 1 Prostacyclins, 21 (1984)237 Prostaglandins. 8 (1971)317;15 (1978)357 Proteinases, inhibitors of, 31 (1994)59;32

(1995)37;32 (1995)239

Pseudomoncts aeruginosa, resistance of, 12

(1975)333;32 (1 995)157

Psychotomimetics. 1 1 ( I 975)91 Psychotropic drugs. 5 (1967)251: 37 (2000)

135

Purines, 7 (1970)69 Pyridazines, pharmacological actions of, 27

(1990)1;29(1992)141

Pyrimidines, 6 ( 1969)67:7 ( 1 970)285;8

(1971)61:19(1982)269 Quantum chemistry. 1 1 (1975)67 Quinolines, &amino-, as antimalarial agents, 28(1991)1

215

4-Quinolones as antibacterial agents, 27

(1990)235 as potential cardiovascular agents, 32

(1995)115

Radioligand-receptor binding, 23 (1986)417 Ranitidine and H2-antagonists, 20 (1983)337 Rauwolfia alkaloids, 3 (1963)146 Recent drugs, 7 (1970)1 Receptors, adrenergic, 22 (1985)121;23 (1986)1 cholecystokinin 37 (2000)45 fibrinogen, 36 (1999)29 histamine, 24 (1987)29 neurokinin, 35 (1998)57 opioid, 35 (1998)83 Renin inhibitors, 32 (1995)37 Ricin, 24 ( I 987) 1 Screening tests, 1 (1961)1 Serine protease inhibitors, 31 (1994)59 Snake venoms, neuroactive, 21 (1984)63 Sodium cromoglycate analogues, 21 (1984)1 Sparsomycin, 23 (1986)219 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, /I-adrenomimetic activity, 18 (1981)45 Tetrazoles, 17 (1980)151 Thalidomide as anti-inflammatory agent, 22 (1985)165 Thiosemicarbazones, biological action, 15 (1978)321;32 (1995)1 Thromboxanes, 15 ( 1978)357 Tilorone and related compounds, 18 (1 981) 135 Toxic actions, mechanisms of, 4(1 965) 18 Tranquillizers, 1 (1961)72 1,2,3-Triazines,medicinal chemistry of, 13 (1976)205

216

CUMULATIVE SUBJECT INDEX

Tripositive elements, chelation of, 28 (1991) 41 Trypanosomiasis, 3 (1963) 52 Venoms, neuroactive snake, 21 (1984) 63

Virus diseases ofplants, 20 (1983) 119 Viruses, chemotherapy of, 8 (1971) 119; 23 ( I 986) 187; 32 (1995) 239; 36 (1999) I Vitamin D3 and its medical uses, 35 (1998) 1 Vitamin E, pharmacology of, 25 (1988) 249