Molecular From DNA to Discovery Companion website This book is accompanied by a companion website: www.wiley.com/go/dickenson/dnamolecular The website includes: Figures and Tables from the book for downloading Molecular Pharmacology From DNA to

John Dickenson, Fiona Freeman, Chris Lloyd Mills, Shiva Sivasubramaniam and Christian Thode Nottingham Trent University

A John Wiley & Sons, Ltd., Publication This edition first published 2013 © 2013 by John Wiley & Sons, Ltd

Wiley-Blackwell is an imprint of John Wiley & Sons, formed by the merger of Wiley’s global Scientific, Technical and Medical business with Blackwell Publishing.

Registered office: John Wiley & Sons, Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK

Editorial offices: 9600 Garsington Road, Oxford, OX4 2DQ, UK The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK 111 River Street, Hoboken, NJ 07030-5774, USA

For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com/wiley-blackwell.

The right of the author to be identified as the author of this work has been asserted in accordance with the UK Copyright, Designs and Patents Act 1988.

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought.

Library of Congress Cataloging-in-Publication Data

Molecular pharmacology : from DNA to drug discovery / John Dickenson ... [et al.]. p. ; cm. Includes index. ISBN 978-0-470-68444-3 (cloth) – ISBN 978-0-470-68443-6 (pbk.) I. Dickenson, John. [DNLM: 1. Molecular Targeted Therapy. 2. Pharmacogenetics–methods. 3. Drug Delivery Systems. 4. Drug Discovery. QV 38.5] 615.19–dc23 2012034772

A catalogue record for this book is available from the British Library.

Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books.

Typeset in 9.25/11.5pt Minion by Laserwords Private Limited, Chennai, India.

First Impression 2013 Contents

Preface ix Abbreviations x 1 Introduction to Drug Targets and Molecular Pharmacology 1 1.1 Introduction to molecular pharmacology 1 1.2 Scope of this textbook 2 1.3 The nature of drug targets 3 1.4 Future drug targets 7 1.5 Molecular pharmacology and drug discovery 11 References 12 2 Molecular Cloning of Drug Targets 13 2.1 Introduction to molecular cloning – from DNA to drug discovery 13 2.2 ‘Traditional’ pharmacology 14 2.3 The relevance of recombinant DNA technology to pharmacology/drug discovery 14 2.4 The ‘cloning’ of drug targets 15 2.5 What information can DNA cloning provide? 20 2.6 Comparing the pharmacologic profile of the ‘cloned’ and the ‘native’ drug target 23 2.7 Reverse pharmacology illustrated on orphan GPCRs 24 2.8 Summary 27 References 27 3 G Protein-coupled Receptors 31 3.1 Introduction to G protein-coupled receptors 31 3.2 Heterotrimeric G-proteins 36 3.3 Signal transduction pathways 40 3.4 Desensitisation and down-regulation of GPCR signalling 44 3.5 Constitutive GPCR activity 45 3.6 Promiscuous G-protein coupling 47 3.7 -directed signalling 48 3.8 Allosteric modulators of GPCR function 49 3.9 Pharmacological chaperones for GPCRs 50 3.10 GPCR dimerisation 51 3.11 GPCR splice variants 63 3.12 Summary 67 References 67 Useful Web sites 70 4 Ion Channels 71 4.1 Introduction 71 4.2 Voltage-gated ion channels 73

v vi Contents

4.3 Other types of voltage-gated ion channels 89 4.4 -gated ion channels 109 4.5 Summary 125 References 125 5 Transporter Proteins 129 5.1 Introduction 129 5.2 Classification 129 5.3 Structural analysis of transporters 132 5.4 Transporter families of pharmacological interest 133 5.5 Transporters and cellular homeostasis 167 5.6 Summary 169 References 169 6 Cystic Fibrosis: Alternative Approaches to the Treatment of a Genetic Disease 175 6.1 Introduction 175 6.2 Cystic fibrosis transmembrane conductance regulator 179 6.3 Mutations in CFTR 183 6.4 Why is cystic fibrosis so common? 184 6.5 Animal models of Cystic fibrosis 186 6.6 Pharmacotherapy 186 6.7 Gene therapy 191 6.8 Conclusion 195 References 196 7 201 7.1 Types of genetic variation in the human genome 201 7.2 Thiopurine S-methyltransferase and K+ channel polymorphisms 202 7.3 Polymorphisms affecting 204 7.4 Methods for detecting genetic polymorphisms 209 7.5 Genetic variation in drug transporters 211 7.6 Genetic variation in G protein coupled receptors 215 7.7 Summary 225 References 225 Useful Web sites 226 8 Transcription Factors and Gene Expression 227 8.1 Control of gene expression 227 8.2 Transcription factors 229 8.3 CREB 233 8.4 Nuclear receptors 238 8.5 Peroxisome proliferator-activated receptors 240 8.6 Growth factors 247 8.7 Alternative splicing 247 8.8 RNA editing 251 8.9 The importance of non-coding RNAs in gene expression 257 8.10 Summary 270 References 271 9 Cellular Calcium 277 9.1 Introduction 277 9.2 Measurement of calcium 278 Contents vii

9.3 The exocrine pancreas 289 9.4 Calcium signalling in pancreatic acinar cells 292 9.5 Nuclear calcium signalling 303 9.6 Conclusions 310 References 311 10 Genetic Engineering of Mice 315 10.1 Introduction to genetic engineering 315 10.2 Genomics and the accumulation of sequence data 315 10.3 The mouse as a model organism 318 10.4 Techniques for genetic engineering 319 10.5 Examples of genetically-engineered mice 332 10.6 Summary 334 References 334 11 Signalling Complexes: Protein-protein Interactions and Lipid Rafts 339 11.1 Introduction to cell signalling complexes 339 11.2 Introduction to GPCR interacting proteins 340 11.3 Methods used to identify GPCR interacting proteins 340 11.4 Functional roles of GPCR interacting proteins 345 11.5 GPCR signalling complexes 348 11.6 GPCR and ion channel complexes 355 11.7 Ion channel signalling complexes 356 11.8 Development of pharmaceuticals that target GPCR interacting proteins 356 11.9 Development of pharmaceuticals that target protein-protein interactions 356 11.10 Lipid rafts 357 11.11 -mediated endocytosis 361 11.12 Summary 364 References 364 12 Recombinant Proteins and Immunotherapeutics 367 12.1 Introduction to immunotherapeutics 367 12.2 Historical background of immunotherapeutics 368 12.3 Basis of immunotherapeutics 368 12.4 Types of immunotherapeutics 369 12.5 Humanisation of antibody therapy 372 12.6 Immunotherapeutics in clinical practice 376 12.7 Advantages and disadvantages of immunotherapy 378 12.8 The future 379 12.9 Summary 380 References 380 Glossary 381 Index 403

Preface

Nottingham Trent University offers a suite of successful therapeutic is the future of as it enables MSc courses in the Biosciences field that are delivered by physicians to prescribe personalised medical treatments full-time, part-time and distance (e-learning) teaching. based on an individual’s genome. The book utilises a The authors are members of the Pharmacology team drug target-based approach rather than the traditional at Nottingham Trent University and teach extensively on organ/system-based viewpoint and reflects the current the MSc Pharmacology and courses. advances and research trends towards in silico drug design The content of this book was inspired by these courses based on gene and derived protein structure. as there is no comparable postgraduate textbook on The authors would like to thank Prof Mark Darlison molecular pharmacology and it is a rapidly expanding (Napier University, Edinburgh, UK) for providing the subject. The primary aim of this text was to provide a initial impetus, inspiration and belief that a book of platform to complement our courses and enhance the such magnitude was possible. We would also like to student experience. Given the breadth and depth of this acknowledge the unflagging encouragement and support volume it will be of use to students from other institutions of the Wiley-Blackwell team (Nicky, Fiona and Clara) as a teaching aid as well as an invaluable source of during the preparation of this work. Finally thanks should background information for post-graduate researchers. also be given to the helpful, constructive and positive The value of this book is enhanced by the research comments provided by the reviewers. We hope that you portfolio of the Bioscience Department and individual enjoy this book as much as we enjoyed writing it. authors who have research careers spanning over 25 years. This textbook illustrates how genes can influence our John Dickenson, Fiona Freeman, Chris Lloyd Mills, Shiva and hence our pharmacological response to Sivasubramaniam and Christian Thode. drugs used to treat pathological conditions. Tailoring of

ix Abbreviations

2+ 2+ [Ca ]i intracellular free ionised calcium ARC channels arachidonic acid regulated Ca concentration channels 2+ [Ca ]n nuclear free ionised calcium Arg arginine (R) concentration ASIC acid sensing ion channels 2+ [Ca ]o extracellular free ionised calcium ASL airways surface liquid concentration Asn asparagine (N) 2-APB 2-aminoethoxydiphenyl borate Asp aspartic acid (D) 4EFmut DREAM 4th EF hand mutant DREAM ATF1 activation transcription factor 1 5F-BAPTA 1,2-bis(2-amino-5,6-diflurophenoxy) ATP adenosine triphosphate ethane-N,N,N ,N -tretracacetic acid AV adenovirus 5-HT 5-hydroxytyrptamine / serotonin Aβ amyloid β peptide AAV adeno-associated virus BAC bacterial artificial chromosome ABC ATP-binding cassette (transporter) BBB blood brain barrier AC adenylyl cyclase BCRP breast cancer resistant protein ACC mitochondrial ADP/ATP carrier BDNF brain-derived neurotrophic factor 2+ + (transporter) BKCa big conductance Ca -activated K ACh acetylcholine channels ACS anion-cation subfamily BLAST Basic Local Alignment Search Tool AD Alzheimer’s disease bp base pairs ADAR adenosine deaminase acting on RNA BRET bioluminescence resonance energy (1, 2 or 3) transfer ADCC antibody-dependent cellular Brm/brg1 mammalian helicase like proteins cytotoxicity BTF basal transcription factors ADEPT antibody-directed enzyme pro-drug BZ benzodiazepine therapy Ca-CaM Ca2+-calmodulin ADHD attention deficit hyperactivity disorder CaCC calcium activated chloride channel AF1/2 transcriptional activating function cADPr cyclic adenosine diphosphoribose (1 or 2) CaM calmodulin Ala alanine (A) CaMK calcium-dependent calmodulin kinase AM acetoxylmethyl cAMP cyclic adenosine 3 ,5 monophsophate AMPA α-amino-3-hydroxy-5-methylis CaRE calcium responsive element oxazole 4-propionic acid catSper cation channels in sperm 2+ Apo- apolipoproteins (A, B or C) CaV voltage-gated Ca channels APP amyloid precursor protein CBAVD congenital bilateral absence of the vas AQP aquaporins deferens

x Abbreviations xi

CBP CREB binding protein Dlg1 drosophila disc large tumour CCCP carbonyl cyanide suppressor m-chlorophenylhydrazone DNA deoxyribonucleic acid CCK cholecystokinin DOPA dihydroxyphenylalanine CDAR cytosine deaminase acting on RNA DPE downstream promoter element cDNA complementary DNA DRE downstream regulatory element CDR complementarily-determining region DREAM DRE antagonist modulator CF cystic fibrosis dsRNA double-stranded RNA CFP cyan fluorescent protein EBV Epstein Barr virus CFS colony stimulating factors EGF epidermal growth factor CFTR cystic fibrosis transmembrane EGFR epidermal growth factor receptor conductance regulator EGTA ethylene glycol tetraacetic acid cGMP cyclic guanosine 3 ,5 monophosphate ELISA enzyme linked immunosorbent assay CHF congestive heart failure ENaC epithelial sodium channel CHO Chinese hamster ovary cell line EPO erythropoietin CICR calcium induced calcium release ER endoplasmic reticulum CIF calcium influx factor ERK extracellular-signal-regulated kinases ClC chloride channel eRNA enhancer RNA CMV cytomegalovirus ERTF oestrogen receptor transcription factor CNG cyclic nucleotide-gated channel ES cells embryonic stem cells CNS central nervous system ESE exon splicing enhancer CNT concentrative nucleoside transporter ESS exon splicing silencer COS CV-1 cell line from Simian kidney cells EST expressed sequence tag immortalised with SV40 viral Fab antibody binding domain genome FACS fluorescent-activated cell sorting COX cyclooxygenases (1, 2 or 3) Fc constant fragment of the monoclonal CPA monovalent cation/proton antiporter antibodies

super family FEV1 forced expiratory volume in 1 second CpG cytosine-phosphate-guanine regions in FGF-9 fibroblast growth factor DNA FIH factor inhibiting HIF CPP cell penetrating peptide (transporter) FISH fluorescence in situ hybridisation CRE cAMP responsive element FOXL2 fork-head box protein CREB cAMP responsive element binding FRET fluorescence resonance energy transfer protein FXS fragile-X syndrome CREM CRE modulator G3P glucose-3-phosphate CRF corticotropin-releasing factor GABA gamma-aminobutyric acid CRM chromatin remodelling complex GAT GABA transporters CRTC cAMP-regulated transcriptional GC guanylyl cyclase co-activator family GFP green fluorescent protein CSF cerebral spinal fluid GIRK G-protein-gated inwardly rectify K+ CTD C terminal domain channel CTL cytotoxic T lymphocyte Gln glutamine (Q) CYP cytochrome P450 GlpT sn-glycerol-3-phosphate/phosphate Cys cysteine (C) antiporter DAG diacylglycerol GltPh Pyrococcus horikoshii glutamate DAX1 dosage-sensitive sex reversal gene/TF transporters DBD DNA-binding domain Glu glutamic acid (E) DC dicarboxylate GLUT glucose transporters DHA drug:H+ antiporter family Gly glycine (G) (transporter) GLYT glycine transporters xii Abbreviations

GMP guanosine monophosphate K3K4 HMT histone methyl transferase + GPCR G protein coupled receptor KATP ATP-sensitive K channels GPN glycyl-L-phenylalanine-2- kb kilobase napthylamide 2+ + KCa Ca -activated K channels − GRK G-protein coupled receptor kinase KCC K+-Cl co-transporter GST Glutathione S-transferase KChIP K+ channel interacting protein H+ hydrogen ion; proton KCO K+ channel openers HAD histone deacetylases Kd Ca2+ HAMA human anti-murine antibodies + HAT histone acetyltransferases KG G-protein gated K channels HCF host cell factor KID kinase-inducible domain + HCN hyperpolarisation-activated cyclic Kir inwardly rectifying K channels + nucleotide-gated channels KV voltage-gated K channel HDL high density lipoprotein LacY lactose:H+ symporter HIF hypoxia inducible factor LBD ligand binding domains His histidine (H) LDL low density lipoprotein HMG high mobility group Leu leucine (L) HMIT H+/myo-inositol transporter LeuTAa Aquifex aeolicus leucine transporter hnRNP nuclear ribonucleoproteins LGIC ligand-gated ion channel HOX homeobox lncRNA long non-coding RNA HPLC high-performance liquid LPS lipopolysaccharide chromatography lys lysine (K) HRE hypoxia response elements Mab monoclonal antibodies Hsp70 heat shock protein of the 70 kilodalton MAC membrane attack complex family MAPK mitogen-activated protein kinase HSV herpes simplex virus MATE multidrug and toxic compound HSV-tk herpes simplex virus thymidine kinase extrusion superfamily (transporter) HTS high-throughput screening Mb megabase Htt Huntingtin MCT mono carboxylate transporters IBMX 3-isobutyl-1-methylxanthine MCU mitochondrial Ca2+ uniporter 2+ Icrac calcium release activated Ca channel MDR multidrug resistance (transporter) ICSI intra-cytoplasmic sperm injection MDR1 multidrug resistant transporter 1 Ifs interferons Met methionine (M) Ig immunoglobulins MFP periplasmic membrane fusion protein IGF-1 insulin-like growth factor-I family (transporter) iGluR ionotropic glutamate receptor MFS major facilitator superfamily IHD ischaemic heart disease (transporter) IL-10 interleukin-10 MHC histocompatibility complex Ile isoleucine (I) miRNA microRNA INN international non-proprietary names mPTP mitochondrial permeability transition INR initiator element pore INSL3 insulin-like factor 3 mRNA messenger RNA

IP3 inositol 1,4,5-triphosphate MSD membrane spanning domain IP3R IP3 receptor MTF modulatory transcription factors β β 2+ iPLA2 isoform of Ca independent Myc myc oncogene phospholipase A2 NAADP nicotinic acid adenine dinucleotide IRT immunoreactive trypsinogen phosphate

Isc short circuit current nAChR nicotinic acetylcholine receptors ISE introns splicing enhancer NAD+ nicotinamide adenine dinucleotide ISS introns splicing silencer NADP+ nicotinamide adenine dinucleotide

K2P two-pore potassium channels phosphate Abbreviations xiii

NALCN sodium leak channel non-selective PGE2 prostaglandin E2 protein channel P-gp permeability glycoprotein NAT natural antisense transcript (transporter) + NaV voltage-gated Na channels Phe phenylalanine (F) NBD nucleotide binding domain Pi inorganic phosphate ncRNA non-coding RNA PI3 phosphatidylinositol 3-kinases neoR neomycin resistance PIP phosphatidylinositol 4,5-bisphosphate NES nuclear endoplasmic space 2 PKA protein kinase A NFAT nuclear factor of activated T cells NFκB nuclear factor kappa of activated B PKC protein kinase C cells PLC phospholipase C β β NHA Na+/H+ antiporters PLC isoform of phospholipase C NhaA Escherichia coli Na+/H+ antiporter pLGICs pentameric ligand-gated ion channels NHE Na+/H+ exchanger PM plasma membrane NKCC sodium potassium 2 chloride PMCA plasma membrane Ca2+ ATPase cotransporter PP1 protein phosphatase 1 NM nuclear membrane PPAR peroxisome proliferator-activated NMDA N-methyl-D-aspartate receptors (α, β, δ,orγ) NMR nuclear magnetic reasonance PPRE PPAR response element NO nitric oxide pRB retinoblastoma protein NPA Asn-Pro-Ala motif Pro proline (P)

NPC nuclear pore complex PSD95 post synaptic density protein-95 NR nucleoplasmic reticulum Q1/Q2 glutamine-rich domains (1 or 2) NR-HSP nuclear receptor-heat shock protein RaM rapid mode uptake complex RAMP receptor-activity modifying protein NRSE neuron restrictive silencer element Ras rat sarcoma (causing factor) NSS sodium symporter RBC red blood cell (transporter) REST repressor element-1 transcription nt nucleotide factor NTD N- terminal domain RFLP restriction fragment length NVGDS non viral gene delivery systems polymorphism OA- organic anion rhDNase recombinant human DNase OAT organic anion transporters RICs radio-immunoconjugates OCT organic cation transporters RIP receptor-interacting protein Oct/OAP octomer/octomer associated proteins RISC RNA-induced silencing complex OMF outer membrane factor family RLF relaxin-like factor (transporter) RNA pol RNA polymerases ORCC outwardly rectifying chloride channel RNA ribonucleic acid ORF open-reading frame RNAi RNA interference OSN olfactory sensory neurons RND resistance-nodulation-cell division OxlT oxalate:formate antiporter (transporter) Pax paired box gene/TF ROS reactive oxygen species 2+ pCa -log10 of the Ca concentration rRNA ribosomal RNA PCR polymerase chain reaction RSPO1 R-spondin-1 PD potential difference RT-PCR reverse-transcription polymerase PDE phosphodiesterase chain reaction PDZ PSD95-Dlg1-zo-1 (protein motif) RXR retinoic acid receptor PEPT dipeptide transporters RyR ryanodine receptors PG prostaglandins SAM intraluminal sterile α motif PGC-1α peroxisome proliferator-activated SBP substrate binding protein receptor α, co-activator 1α Ser serine (S) xiv Abbreviations

SERCA sarco/endoplasmic reticulum Ca2+ TIF-1 transcription intermediary factor ATPase TIRF total internal reflection fluorescence Shh sonic hedgehog homolog gene/TF imaging siRNA short interfering RNA TMAO trimethylamine N-oxide 2+ + SKCa small conductance Ca -activated K TMD transmembrane domain channels TMS transmembrane segments SLC solute carrier superfamily TNFs tumour necrosis factors (transporter) TPC two pore calcium channels SMN survival of motor neurons protein TPEN N,N,N ,N -tetrakis(2- SMR small multidrug resistance superfamily pyridylmethyl)ethylenediamine (transporter) Trk tyrosine kinase receptor (A, B or C) snoRNA small nucleolar RNA tRNA transfer RNA SNP single nucleotide polymorphism TRP transient receptor potential channels snRNA spliceosomal small nuclear RNA Trp tryptophan (W) SOC store operated Ca2+ channel TTX tetrodotoxin Sox9 SRY-related HMG box-9 gene/factor Tyr tyrosine (Y) SR sarcoplasmic reticulum TZD thiazolidinedione SRC-1 steroid receptor co-activator-1. Ubi ubiquitination SREBP sterol regulatory element-binding UTR untranslated region proteins Val valine (V) SRY sex-determining region Y VDAC voltage dependent anion channel SSS solute sodium symporter (transporter) VEGF vasculoendothelial growth facto STAT signal transducer and activator of VFT venus flytrap transcription (1, 2 or 3) vGLUT vesicular glutamate transporter STIM stromal interaction molecule VHL von Hippel-Lindau protein SUG-1 suppressor of gal4D lesions −1 VIP vasoactive intestinal peptide SUMO small ubiquitin like modifier VLDL very low density lipoprotein

SUR sulfonylureas receptor Vm membrane potential SW1/SNF switching mating type/sucrose VOCC voltage-operated calcium channels non-fermenting proteins WNT4 wingless-type mouse mammary TAD transactivation domain tumour virus integration site TAP transporters associated with antigen YAC yeast artificial chromosome processing YFP yellow fluorescent protein TCA tricarboxlyic acid YORK yeast outward rectifying K+ channel TCR T cell receptor ZAC zinc-activated channel TDF testis-determining factor Zo-1 zonula occludens-1 protein TEAD TEA domain proteins TEF transcription enhancer factor TESCO testis-specific enhancer of Sox9 POST-FIXes TGF transforming growth factor Chimeric antibodies – xiMabs TGN trans-Golgi network Human antibodies – muMbs TH tyrosine hydroxylase Humanised antibodies – zumab Thr threonine (T) Monoclonal antibodies – oMabs Introduction to Drug Targets 1 and Molecular Pharmacology

1.1 Introduction to molecular pharmacology 1 1.4 Future drug targets 7 1.2 Scope of this textbook 2 1.5 Molecular pharmacology and drug discovery 11 1.3 The nature of drug targets 3 References 12

organisms such as mouse (2002) and rat (2004) has also 1.1 Introduction to molecular been of great benefit to the drug discovery process. pharmacology The capacity to clone and express genes opened up access to a wealth of information that was simply not avail- During the past 30 years there have been significant able from traditional pharmacology-based approaches advances and developments in the discipline of molecular using isolated animal tissue preparations. In the case pharmacology – an area of pharmacology that is con- of GPCRs detailed expression pattern analysis could be cerned with the study of drugs and their targets at the performed using a range of molecular biology tech- molecular or chemical level. Major landmarks during this niques such as in situ hybridisation, RT-PCR (reverse time include the cloning of the first G-protein coupled transcriptase-polymerase chain reaction) and Northern β receptor (GPCR) namely the 2-adrenergic receptor in blotting. Furthermore having a cloned GPCR gene in a 1986 (Dixon et al., 1986). This was quickly followed by the simple DNA plasmid made it possible for the first time cloning of additional adrenergic receptor family genes and to transfect and express GPCRs in cultured cell lines. ultimately other GPCRs. The molecular biology explo- This permitted detailed pharmacological and functional sion during the 1980s also resulted in the cloning of genes analysis (e.g. second messenger pathways) of specific encoding ion channel subunits (e.g. the nicotinic acetyl- receptor subtypes in cells not expressing related sub- + choline receptor and voltage-gated Na channel) and types, which was often a problem when using tissue nuclear receptors. The cloning of numerous drug targets preparations. Techniques such as site-directed muta- continued at a pace during the 1990s but it was not until genesis enable pharmacologists to investigate complex the completion of the human genome project in 2001 that structure-function relationships aimed at understanding, the numbers of genes for each major drug target family for example, which amino acid residues are crucial for could be determined and fully appreciated. As would be ligand binding to the receptor. As cloning and expres- expected, the cloning of the human genome also resulted sion techniques developed further it became possible in the identification of many potentially new drug targets. to manipulate gene expression in vivo.Itisnowcom- The completion of genome projects for widely used model mon practice to explore the consequences of deleting a

Molecular Pharmacology: From DNA to Drug Discovery, First Edition. John Dickenson, Fiona Freeman, Chris Lloyd Mills, Shiva Sivasubramaniam and Christian Thode. © 2013 John Wiley & Sons, Ltd. Published 2013 by John Wiley & Sons, Ltd.

1 2 Chapter 1

Cell-based studies Animal-based studies • Transfected cells • Gene knockout • Reporter gene assays • Gene knockin • Protein-protein interactions • Disease models • GFP-based assays

Drug Targets

Expression patterns Structure-function studies • In situ hybridization • Site-directed mutagenesis • RT-PCR • X-ray crystallography • Northern blotting

Figure 1.1 Molecular pharmacology-based methods used to interrogate drug targets. specific gene either from an entire genome (knockout) studies, which are already used for structural studies of or from a specific tissue/organ (conditional knockout). other drug targets such as kinases, would be the ability It is also possible to insert mutated forms of genes into to obtain GPCR dynamics and ligand activation data an organism’s genome using knockin technology. These which is not possible using X-ray based methods. Some transgenic approaches allow molecular pharmacologists of the molecular pharmacology based approaches used to study developmental and physiological aspects of gene to interrogate drug targets are outlined in Figure 1.1. function in vivo and in the case of gene knockin techniques Despite this increased knowledge of drug targets to develop disease models. obtained during the molecular biology revolution, there The molecular biology revolution also enabled the has been a clear slowdown in the number of new drugs development of novel approaches for studying the reaching the market (Betz, 2005). However, since it takes complex signal transduction characteristics of pharma- approximately 15 years to bring a new drug to market cologically important proteins such as receptors and it may be too early to assess the impact of the human ion channels. These include reporter gene assays, green genome project on drug discovery. In 2009 the global fluorescent protein (GFP) based techniques for visual- pharmaceutical market was worth an estimated $815 ising proteins in living cells and yeast two hybrid-based billion. However during the next few years a major assays for exploring protein-protein interactions. You problem facing the pharmaceutical industry is the loss will find detailed explanations of these and other current of drug patents on key blockbusters. The hope for the molecular-based techniques throughout this textbook. future is that the advances in molecular pharmacology witnessed during the last decade or so will start to deliver Another major breakthrough in the 2000s was the new blockbuster therapeutics for the twenty-first century. development of methods that allowed high resolution structural images of membrane-associated proteins to be obtained from X-ray crystallography. During this time 1.2 Scope of this textbook the first X-ray structures of GPCRs and ion channels were reported enabling scientists to understand how As briefly detailed above there have been numerous such proteins function at the molecular level. Indeed exciting developments in the field of molecular phar- crystallography is an important tool in the drug discovery macology. The scope of this textbook is to explore aspects process since crystal structures can be used for in silico of molecular pharmacology in greater depth than cov- drug design. More recently researchers have used NMR ered in traditional pharmacology textbooks (summarised spectroscopy to obtain a high-resolution structural in Figure 1.2). Recent advances and developments in β information of the 2-adrenergic receptor (Bokoch et the four major human drug target families (GPCRs, al., 2010). A distinct advantage of NMR-based structural ion channels, nuclear receptors and transporters) are Introduction to Drug Targets and Molecular Pharmacology 3

Transgenic mouse Pharmacogenomics technology Chapter 7 Chapter 10

Cloning of Gene therapy drug targets Chapter 6 DNA Chapter 2

Transcription factor Transcription based drugs Chapter 8

RNA

Anti-sense oligonucleotides Interference RNA Translation Chapter 8 Conventional Drug targets GPCRs (chapter 3) Recombinant proteins Ion channels (chapter 4) Antibody-based Protein Transporters (chapter 5) therapies Nuclear receptors (chapter 8) Chapter 12

Figure 1.2 Drug targets within the central dogma of molecular biology. To date the majority of conventional therapeutics target a relatively small group of protein families that include G-protein coupled receptors, ion channels, and transporters. Novel therapeutic strategies include blocking translation of mRNA into protein using anti-sense oligonucleotide and/or RNA interference technology. Gene transcription can also be targeted via the activation/inhibition of nuclear receptor function. The chapters covering these topics are indicated. covered in separate chapters (Chapters 3–5 and 8). The The information from the project, which is freely avail- molecular targets of anti-infective drugs (anti-bacterial able on the internet, will enable scientists to understand and anti-viral) whilst of great importance are not covered how genetic variations contribute to risk of disease and in this book. Other chapters deal with the cloning of drug drug response. Finally, we take an in depth look at the targets (Chapter 2) and transgenic animal technology role of calcium in the cell, looking at techniques used to (Chapter 10). The concept of gene therapy is explored measure this important second messenger (Chapter 9). in a case study-based chapter which looks at current and possible future treatment strategies for cystic fibrosis, the commonest lethal genetic disease of Caucasians (Chapter 1.3 The nature of drug targets 6). Another major development in molecular pharma- cology has been the discipline of pharmacogenomics: the How many potential drug targets are there in the human study of how an individual’s genetic makeup influences genome? This is an important question often asked by their response to therapeutic drugs (Chapter 7). These the pharmaceutical industry since they are faced with naturally occurring variations in the human genome the task of developing novel therapeutics for the future. are caused predominantly by single nucleotide polymor- When the draft sequence of the human genome was com- phisms (DNA variation involving a change in a single pleted in 2001 it was estimated to contain approximately nucleotide) and there is a major research consortium 31,000 protein-coding genes. However since its comple- aimed at documenting all the common variants of the tion the number of human protein-coding genes has human genome (The International HapMap project). been continually revised with current estimates ranging 4 Chapter 1

G-protein coupled receptors (26.8%)

Ion channels (13.4%)

Nuclear receptors (13%)

Transporters (2.7%)

Others (44.1)

Figure 1.3 The molecular targets of prescribed drugs. The data is expressed as a percentage of all FDA approved drugs as of December 2005. Drugs that target ligand-gated ion channels and voltage-gated ion channels have been grouped together as ion channels. Drug targets grouped together as others encompass 120 different specific targets many of which are enzymes. Data taken from Overington et al. (2006). between 20,000 and 25,000. Of these it is predicted that including GPCR dimerisation. Many of these exciting about 3000 are feasible protein drug targets. In 2005 it advances have revealed new pharmaceutical approaches was calculated that about 100 drug targets account for for targeting GPCRs such as inverse , allosteric all prescription drugs. On this basis there is obviously modulators, biased agonists and bivalent ligands that considerable scope for the development and discovery of target GPCR heterodimers. Since the completion of the novel drug targets to treat disease. At present the classical human genome project it has emerged that the total drug targets include GPCRs (Chapter 3), ion channels number of human GPCRs may be as high as 865, which (Chapter 4), nuclear receptors (Chapter 8), transporters would account for approximately 3.4% of total pre- (Chapter 5) and enzymes. These important classical drug dicted protein-coding genes (assuming a total of 25,000). targets, whilst briefly covered in this Introduction, are For many cloned GPCRs the endogenous ligand(s) are extensively covered in later chapters. The distribution of unknown (so called ‘orphan’ GPCRs) and the identi- drug targets expressed as a percentage of total products fication of these orphan receptor ligands is the focus approved by the Food and Drug Administration (FDA; of drug discovery programmes within the pharmaceuti- agency in the USA responsible for approving drugs for cal industry. The process of GPCR de-orphanisation is therapeutic use) is illustrated in Figure 1.3. addressed in Chapter 2. In Chapter 11 the concept that GPCRs interact with a host of accessory proteins that G-protein coupled receptors (GPCRs) are important in modulating many aspects in the life GPCRs represent the largest single family of pharmaceu- of a GPCR including the formation of signalling com- tical drug target accounting for approximately 30% of the plexes will be explored. Indeed, targeting such GPCR current market. Their primary function is to detect extra- signalling complexes with drugs that disrupt protein- cellular signals and through heterotrimeric G-protein protein interactions is another exciting avenue for future activation trigger intracellular signal transduction cas- drug development not only in the field of GPCRs but also cades that promote cellular responses (Figure 1.4). Whilst in other areas of signal transduction. their share of the overall drug market is likely to fall in the future they still represent ‘hot’ targets for drug dis- Ion channels covery programmes. GPCRs are conventionally targeted Ion channels represent important drug targets since they using small molecules (typically less than 500 Da) that are involved in regulating a wide range of fundamental are classified as agonists (receptor activating) or antag- physiological processes. Indeed, at present they are the onists (inhibit receptor function by blocking the effect second largest class of drug target after GPCRs. They of an agonist). Some key examples of drugs that target operate the rapid transport of ions across membranes GPCRs are listed in Table 1.1. Chapter 3 will explain in (down their electrochemical gradients) and in doing so detail many of the recent developments in GPCR struc- trigger plasma and organelle membrane hyperpolari- ture, function, pharmacology and signal transduction sation or depolarisation. They are also potential drug Introduction to Drug Targets and Molecular Pharmacology 5

Agonist Agonist Agonist GPCR Antagonist Biased agonist

γ γ γ α β α β α β

GTP GDP GDP Arrestin G-protein signalling No (a) (b) Arrestin G-protein signalling signalling (c) Agonist

Agonist Antagonist γ α β

GTP

G-protein Allosteric signalling modulators (d) (e) Figure 1.4 G-protein coupled receptors as drug targets. (a) GPCRs can be targeted using selective synthetic agonists which trigger receptor activation thus enabling G-protein coupling and subsequent cell signalling responses. (b) antagonists can be used to block the binding of an thus preventing receptor activation. (c) GPCRs can also trigger G-protein independent cell signalling pathways which are dependent on arrestin binding to the activated receptor. Biased agonists are being developed that specifically promote the activation of arrestin-dependent signalling pathways. (d) bivalent ligands targeting specific GPCR heterodimers. (e) GPCRs can also be targeted using allosteric modulators which bind to sites on the receptor that are distinct from the agonist (orthosteric) binding site.

Table 1.1 G-protein coupled receptors as drug targets.

GPCR Drug (brand name) Agonist/Antagonist Condition/use

Histamine H2 receptor Famotidine (Pepcidine) Antagonist Stomach ulcers α 1-adrenergic receptor Doxazosin (Cardura) Antagonist Hypertension GnRH1 receptor Leuprorelin (Lupron) Agonist Prostate cancer 5-HT1D receptor Sumatriptan (Imigran) Agonist Migraine μ-opioid receptor Fentanyl (Sublimaze) Agonist Analgesic

Abbreviations: GnRH, gonadotropin-releasing hormone. targets for the treatment of rare monogenic hereditary channels may rival GPCRs as drug targets in the future disorders caused by mutations in genes that encode (Jiang et al., 2008). Other major developments include ion channel subunits. Such conditions termed ‘ion the first 3D resolution of ion channel structure by X-ray channelopathies’ include mutations in sodium, chloride crystallography, which was reported for the voltage-gated and calcium channels that cause alterations in skeletal potassium channel in 2003 (MacKinnon et al., 2003). muscle excitability. The understanding of ion channel Despite these important advances in the understanding diversity and complexity increased significantly following of ion channel diversity and structure very few new ion the completion of the human genome project which channel drugs have reached the market during the last identified over 400 genes encoding ion channel subunits. decade. Some key examples of ion channels as drug Given this number of genes it has been suggested that ion targets are shown in Table 1.2. 6 Chapter 1

• • ATP-gated Table 1.2 Ion channels as drug targets. 5-HT3 receptor • Nicotinic ACh receptor channels (P2x) + Ion channel Drug (brand name) Condition/use K+ K Voltage-gated Ca2+ Amlodipine Hypertension channel (Norvasc) and angina Voltage-gated Na+ Phenytoin Epilepsy channel (Dilantin) ATP-sensitive K+ Glibenclamide Type II diabetes channel (Glimepride)

GABAA receptor Benzodiazepines Anxiety (Diazepam) Cl− Na+/Ca2+ Na+/Ca2+ Na+/Ca2+ 5-HT receptor Ondansetron Nausea and 3 • Glycine receptor • Ionotropic (Zofran) vomiting • GABAA receptor Glutamate receptors (a)

+ Ion channels are broadly classified into two main K 2+ + groups (Figure 1.5). Firstly there are ligand-gated ion Ca channels Na channels channels or ionotropic receptors which open when activated by an agonist binding to a specific ion channel subunit. Examples of this class include the nicotinic acetylcholine receptor, GABAA receptor, glycine receptor, 5-HT3 receptor, ionotropic glutamate receptors, and ATP-gated channels. The second group which includes K+ channels voltage-gated or voltage-operated ion channels are Ca2+ Na+ opened by other mechanisms including changes in plasma (b) membrane potential. Examples include voltage-gated 2+ + + Figure 1.5 Ion channel classification. (a) Ligand-gated ion Ca ,Na,andK channels. The molecular structure channels comprise a family of multi-subunit transmembrane and classification of ion channels together with their use proteins that are activated by a diverse set of ligands (indicated as drug targets will be explored in detail in Chapter 4. by the orange circle) that include amino acids (glycine, glutamate and GABA), 5-hydroxytryptamine (5-HT), Nuclear receptors acetylcholine (ACh), and ATP. (b) voltage-gated channel Nuclear receptors are a large family of transcription channels, which are also multi-subunit proteins, open in factors that play a pivotal role in endocrine function. response to local changes in membrane potential. In contrast to other families of transcription factor the activity of nuclear receptors (as their name suggests) receptors is small in comparison to GPCRs they are is specifically regulated by the binding of ligands the target of approximately 13% of all prescribed drugs. (Figure 1.6). Such ligands, which are small and lipophilic, For example, the chronic inflammation associated with include steroid hormones (glucocorticoids, mineralo- asthma can be suppressed by inhaled glucocorticoids and corticoids, androgens, oestrogens and progestogens), oestrogen-sensitive breast cancer responds to treatment thyroid hormones (T3 and T4), fat soluble vitamins D with the oestrogen tamoxifen. The and A (retinoic acid) and various fatty acid derivatives. structure, classification, signal transduction mechanisms Since the completion of the human genome sequencing and therapeutic uses of nuclear receptor targeting drugs project 48 members of the human nuclear receptor will be explored in detail in Chapter 8. family have been identified. However, for many nuclear receptors the identity of the ligand is unknown. These Neurotransmitter transporters ‘orphan’ nuclear receptors are of significant interest The concentration of some within to the pharmaceutical industry since they may lead to the synaptic cleft is tightly regulated by specific the discovery of novel endocrine systems with potential plasma membrane-bound transporter proteins. These therapeutic use. Whilst the total number of nuclear transporters, which belong to the solute carrier Introduction to Drug Targets and Molecular Pharmacology 7

Cell membrane

hormone

HSP

NR dimer

HSP/NR NR/hormone Nuclear pore complex complex Modulation of gene transcription

nucleus

cytoplasm GRE

Figure 1.6 Type I nuclear receptor-mediated signal transduction. In the absence of hormone (e.g. glucocorticoid) the nuclear receptor (NR) is located in the cytoplasm bound to a heat shock protein (HSP). Hormone binding triggers dissociation of the HSP from the NR/HSP complex, dimerisation of the NR and translocation to the nucleus. Once in the nucleus the NR dimer binds to a specific DNA sequence known as glucocorticoid response element (GRE) and modulates gene transcription.

(SLC) transporter family, facilitate the movement of ligand-gated ion channels and voltage-gated ion channels neurotransmitter either back into the pre-synaptic neu- (Figure 1.3). It is likely that the market share of these ron or in some cases into surrounding glial cells. There are classical drug targets will shrink as new drug targets and two major subclasses of plasma-membrane bound neuro- approaches are developed in the future. transmitter transporter: the SLC1 family which transports glutamate and the larger SLC6 family which transports Protein kinases dopamine, 5-HT, noradrenaline, GABA and glycine It is predicted that protein kinases (and lipid kinases), (Figure 1.7). Both SLC1 and SLC6 families facilitate neu- one of the largest gene families in eukaryotes, will become rotransmitter movement across the plasma membrane major drug targets of the twenty-first century. Protein by secondary active transport using extracellular Na+ ion phosphorylation is reversible and is one of the most concentration as the driving force. As might be expected common ways of post-translationally modifying protein drugs that target neurotransmitter transporters have a function. It regulates numerous cellular functions includ- wide range of therapeutic applications such as treatment ing cell proliferation, cell death, cell survival, cell cycle for depression, anxiety and epilepsy. Indeed, neuro- progression, and cell differentiation. The enzymes that transmitter transporters are the target for approximately catalyse protein phosphorylation are known as protein one-third of all psychoactive drugs (see Table 1.3). The kinases, whereas the enzymes that carry out the reverse molecular structure and classification of neurotransmit- dephosphorylation reaction are referred to as phos- ter transporters and their value as important current and phatases (Figure 1.8a). The human genome encodes for future drug targets will be discussed in detail in Chapter 5. 518 protein kinases and approximately 20 lipid kinases. The predominant sites of protein phosphorylation are the hydroxyl groups (−OH) in the side chains of the 1.4 Future drug targets amino acids serine, threonine and tyrosine (Figure 1.8b). When a phosphate group is attached to a protein it intro- At present more than 50% of drugs target only four duces a strong negative charge which can alter protein major gene families, namely GPCRs, nuclear receptors, conformation and thus function. 8 Chapter 1

Pre-synaptic Pre-synaptic SLC1 nerve terminal SLC6 nerve terminal

GPCR GPCR

Ligand-gated Ion channel Ligand-gated Ion channel

Post-synaptic Post-synaptic nerve terminal nerve terminal

(a) (b) Figure 1.7 Neurotransmitter transporter classification. (a) Glutamate released into the synaptic cleft activates both ion channels (ionotropic glutamate receptors) and GPCRs (metabotropic glutamate receptors) located on the post-synaptic membrane. Released glutamate is subsequently removed from the extracellular space by SLC1 transporters located on pre-synaptic membranes. (b) Dopamine, 5-hydroxytryptamine (5-HT), γ-aminobutyric acid (GABA), noradrenaline and glycine released into the synaptic cleft activate specific ligand-gated ion channels and/or GPCRs located on the post-synaptic membrane. These released neurotransmitters are subsequently transported back into the pre-synaptic nerve terminal via SLC6 transporters. For clarity specific vesicular transporters responsible for transporting neurotransmitters from the cytoplasm into synaptic vesicles have been omitted. Figure adapted from Gether et al. (2006). Trends in Pharmacological Sciences 27: 375–383.

Enzymes Table 1.3 Transporters as drug targets. Enzymes are the drug target for approximately 50% of Transporter Drug Condition/ all prescribed drugs. Some key examples are listed in (brand name) use Table 1.4. However, because of their diverse nature they will not be the focus of a specific chapter in this book. 5-HT transporter (SERT) Sertraline Antidepressant (Zoloft) It is also important to remember that many prescribed Dopamine transporter Cocaine Drug of abuse drugs target bacterial and viral enzymes for the treatment (DAT) of infectious disease and HIV. Also many enzymes, whilst Noradrenaline Bupropiona Antidepressant not direct drug targets, play important roles in drug transporter (NET) (Welbrutin) metabolism for example cytochrome P450 enzymes. GAT-1 (GABA) Tiagabine Epilepsy Protein kinases are classified according to the amino acid they phosphorylate and are grouped into two main aAffinityforDATaswell types: serine/threonine kinases and tyrosine kinases. In Introduction to Drug Targets and Molecular Pharmacology 9

ATP ADP P

Protein Kinase

Protein Protein + ADP Protein phosphatase

(a)

OH OH

− O C O C O

H2 H2 CH COH CH COP O

− NH2 NH2 O Serine Phospho-serine

OH OH − O OCCH3 O C CH3

CH CH OH CH CH OOP

O− NH2 NH2 Threonine Phospho-threonine

OH OH

− OC O C O

H2 H2 CH C OH CH C O P O

NH2 NH2 O− Tyrosine Phospho-tyrosine

(b) Figure 1.8 Reversible protein phosphorylation. (a) Protein kinases transfer a phosphate group (P) from ATP to the target protein altering its biological activity. The removal of phosphate from a phosphorylated protein is catalysed by protein phosphatases. (b) Phosphate groups are transferred to the amino acids serine, threonine and tyrosine. both cases ATP supplies the phosphate group with the regulating many aspects of cell physiology it is not sur- third phosphoryl group (γ; gamma phosphate) being prising that dysfunction in the control of protein kinase transferred to the hydroxyl group of the acceptor amino signalling is associated with major diseases such as can- acid. Examples of serine/threonine kinases include pro- cer, diabetes and rheumatoid arthritis. These alterations tein kinase A (PKA; activated by the second messenger in protein kinase and in some cases lipid kinase function cyclic AMP) and protein kinase C (PKC; activated by the arise from over-activity either due to genetic mutations second messenger diacylglycerol). Examples of tyrosine or over-expression of the protein. It is estimated that kinases include tyrosine kinase linked receptors for insulin up to 30% of all protein targets currently under inves- and epidermal growth factor and non-receptor tyrosine tigation by the pharmaceutical industry are protein or kinases such as Src and JAK (Janus-associated kinase). lipid kinases. Indeed, there are approximately 150 protein Given the prominent role of protein phosphorylation in kinase inhibitors in various stages of clinical development, 10 Chapter 1

Table 1.4 Enzymes as drug targets. Table 1.6 Small-molecule protein kinase inhibitors approved for clinical use. Enzyme Drug Condition/use (brand name) Drug (brand name) Targets Use ® HMG-CoA Statins Used to lower Imatinib (Gleevec ) c-Abl-kinase, Chronic myeloid reductase blood cholesterol c-Kit leukaemia ® levels Gefitinib (Iressa ) EGFR Various cancers ® Phosphodiesterase Sildenafil Erectile dysfunction Sunitinib (Sutent )PDGFR, Renal cell type V (Viagra) and hypertension VEGFR carcinoma ® Cyclo-oxygenease Aspirin Analgesic and anti- Dasatinib (Sprycel ) c-Abl-kinase, Various cancers inflammatory Src ® Angiotensin- Captopril Hypertension Everolimus (Afinitor )mTORa Various cancers converting (Capoten) a enzyme Serine/threonine kinase. Abbreviations: EGFR, epider- Dihydrofolate Methotrexate Cancer mal growth factor receptor; mTOR, mammalian target of reductase rapamycin; PDGFR, platelet-derived growth factor receptor; VEGFR, vascular endothelial growth factor receptor.

Table 1.5 Selected small-molecule protein kinase inhibitors A useful approach for assessing the therapeutic poten- in clinical development. tial of novel drug targets is the number of approved Drug Protein kinase target Use patents for each target (Zheng et al., 2006). The level of patents gives an indication of the degree of interest AZD 1152 Aurora B Kinase Various cancers in that particular target and hence likelihood of success- NP-12 Glycogen synthase kinase Alzheimer’s ful drugs being developed. Future targets with a high 3 (GSK3) disease number of US-based patents include matrix metallopro- Bay 613606 Spleen tyrosine kinase Asthma teinases (MMPs) as a target for cancer treatment. MMPs (Syk) INCB-28050 Janus-associated kinase Rheumatoid are proteases which break down the extracellular matrix 1/2 (JAK1/2) arthritis thus facilitating cancer cell invasion and metastasis. Other BMS-582949 p38 mitogen-activated Rheumatoid targets include phosphodiesterase 4 (PDE4), caspases and protein kinase (p38 arthritis integrin receptors. Only time will tell whether any of MAPK) these novel targets result in the development of effective therapeutics. For further reading on the identification and characteristics of future drug targets see the review by Zheng et al. (2006). some of which are highlighted in Table 1.5. Whilst protein kinases are important new human drug targets they are also present in bacteria and viruses and thus represent Therapeutic oligonucleotides potential targets for infectious disease treatment. In addition to the development of small-molecule-based Since the launch of imatinib in 2001 several other drugs there are several other approaches to treat human small-molecule protein kinase inhibitors have success- disease including the exciting prospect of therapeutic fully made it to the market place as novel anti-cancer oligonucleotides (anti-sense and RNA interference based) treatments (Table 1.6). The majority of these drugs are as tools for gene silencing and the continued quest for tyrosine kinase inhibitors and in some cases function as gene therapy-based techniques. These molecular biology- multi-kinase inhibitors (e.g. sunitinib) targeting PDGFR based strategies for combating human disease will be (proliferation) and VEGFR (angiogenesis) dependent sig- addressed later in Chapter 8. nalling responses. Monoclonal antibodies are also used Another new class of drugs are short single-stranded to block the increased tyrosine kinase linked receptor oligonucleotides (DNA or RNA based) that have been activity that is associated with many forms of cancer and selectively engineered to target specific intracellular pro- thesewillbediscussedinChapter12. teins (Dausse et al., 2009). These oligonucleotides which Introduction to Drug Targets and Molecular Pharmacology 11 fold into defined three-dimensional structures are known approach may also be combined with computer-based as aptamers or ‘chemical antibodies’. They are gener- ‘virtual screening’ approaches. For example structure- ated and repeatedly selected through a method known based virtual screening involves the use of 3D protein as SELEX (Systematic Evolution of Ligands by Exponen- structures, many of which are now widely available via tial Enrichment). Essentially the process begins with the public databases, to assess whether a ligand can inter- synthesis of a large oligonucleotide library, containing act or dock with the protein of interest. This can be randomly generated sequences of fixed length, which is linked with ligand-based virtual screening which involves screened for binding to the target protein usually by in silico screening of chemical libraries for compounds affinity chromatography. Those that bind are repeatedly that display similar structural features associated with the selected using stringent elution conditions that ulti- binding of the ligand to the target. As indicated above mately result in the identification of the tightest binding structure-based virtual screenings rely on the availabil- sequences. These high affinity sequences can be chemi- ity of accurate 3D structures of the drug target. The cally modified to increase their affinity and effectiveness as discipline of structural biology uses a range of biophys- potential therapeutic oligonucleotides. The first aptamer- ical techniques including X-ray crystallography, NMR based drug approved by the US Food and Drug Admin- spectroscopy and electron cryo-microscopy to determine istration (FDA) targets the VEGFR and is used to treat protein structure. The latter is an emerging technique age-related macular degeneration. Several other aptamer that can be used to determine the 3D structure of macro- oligonucleotides are also undergoing clinical trials. molecular complexes that are too large to be studied using X-ray crystallography and/or NMR spectroscopy. So far in this section we have briefly covered some of the up- 1.5 Molecular pharmacology and drug and-coming techniques that can used to interrogate drug discovery target structure and screen drug targets for lead com- pounds. There is also a drive towards the development The process of drug discovery is a long and costly process novel cell-based and animal-based models that are more with new drugs taking up to 12 years to reach the clinic. representative of human physiology and hence more Many novel molecular pharmacology-based techniques suitable for drug screening. For example, 3D ‘organ- play important roles in the process of drug discovery otypic’ cell microarrays are currently being developed and development. A problem faced by many pharma- that will allow drug screening in a system that is close ceutical companies is the huge task of screening their to the in vivo environment of cells. In summary, we vast chemical libraries (in some cases this can exceed one million compounds) against an increasing number of possible drug targets. The development, in the early • In vivo target validation • High-throughput screening 1990s, of high-throughput screening (HTS) technology - DNA knockout • Fragment-based screening using 96-well microtiter plates enabled the drug screening - RNA knockout • Virtual screening (in silico) - Protein knockout • Novel-cell based assays process to be miniaturised and automated. Using such methodology it became possible to screen up to 10,000 Drug compounds per day. However during the last decade Target Lead discovery & ADMET 384-well microtiter plates and more recently 1536-well discovery optimization screening microtiter plate-based assays have been developed that allow for screening of up to 200,000 compounds a day • Gene cloning • Combinatorial chemistry (ultra-high-throughput screening). Since the screening • Structural biology of large chemical libraries is expensive several alterna- - X-ray crystallography tive strategies to increase the chances of success have - NMR spectroscopy been introduced in recent years. One such approach has Phase III Phase II Phase I been the introduction of fragment-based screening (FBS) Drug clinical clinical clinical Approval or fragment-based lead discovery (FBLD). This involves trials trials trials screening the with small libraries of chemical fragments (molecular weights around 200 Da) Figure 1.9 Schematic representation of the drug discovery with the aim of identifying scaffolds or ‘chemical back- process. ADMET (Adsorption, Distribution, Metabolism, bones’ that can be developed into lead compounds. This and studies). 12 Chapter 1 are witnessing exciting times in the process of drug dis- Dixon RA, Kobilka BK, Strader DJ, Benovic JL, Dohlman HG, covery with the continued development of in silico and Frielle T, Bolanowski MA, Bennett CD, Rands E, Diehl RE, nanotechnology-based methods and the introduction of Munford RA, Slater EE, Sigal IS, Caron MG, Lefkowitz RJ novel cell-based screening models. Has there ever been a and Strader CD (1986). Cloning of the gene and cDNA better time to be a molecular pharmacologist? A schematic for mammalian beta-adrenergic receptor and homology with rhodopsin. Nature 321: 75–79. representation of the drug discovery process is shown Gether U, Andersen PH, Larsson OM and Schousboe A (2006). in Figure 1.9. Neurotransmitter transporters: molecular function of impor- tant drug targets. Trends in Pharmacological Sciences 27: References 375–383. Jiang Y, Lee A, Chen J, Ruta V, Cadene M, Chait BT and Betz UAK (2005). How many genomics targets can a portfolio MacKinnon R (2003). X-ray structure of a voltage-dependent + support? Drug Discovery Today 10: 1057–1063. K channel. Nature 423: 33–41. Bokoch MP, Zou Y, Rasmussen SGF, Liu CW, Nygaard R, Kaczorowski GJ, McManus OB, Priest BT and Garcia ML (2008). Rosenbaum DM, Fung JJ, Choi H-J, Thian FS, Kobilka TS, Ion channels as drug targets: the next GPCRs. Journal General Puglisi JD, Weis WI, Pardo L, Prosser RS, Mueller L and Physiology 131: 399–405. Kobilka BK (2010). Ligand-specific regulation of the extra- Overington JP, Al-Lazikani B and Hopkins AL (2006). How cellular surface of a G protein coupled receptor. Nature 463: many drug targets are there? Nature Reviews 5: 993–996. 108–112. Zheng CJ, Han LY, Yap CW, Ji ZL, Cao ZW and Chen YZ Dausse E, Da Rocha Gomes S and Toulme J-J (2009). Aptamers: (2006). Therapeutic targets: progress of their exploration and a new class of oligonucleotides in the drug discovery pipeline. investigation of their characteristics. Pharmacological Reviews Current Opinion in Pharmacology 9: 602–607. 58: 259–279. Molecular Cloning of Drug 2 Targets

2.1 Introduction to molecular cloning – 2.6 Comparing the pharmacologic profile of the from DNA to drug discovery 13 ‘cloned’ and the ‘native’ drug target 23 2.2 ‘Traditional’ pharmacology 14 2.7 Reverse pharmacology illustrated on orphan GPCRs 24 2.3 The relevance of recombinant DNA technology to pharmacology/drug discovery 14 2.8 Summary 27 2.4 The ‘cloning’ of drug targets 15 References 27 2.5 What information can DNA cloning provide? 20

that time: Supported by Sanger’s rapid DNA sequencing 2.1 Introduction to molecular cloning – method (Sanger et al., 1977), it offered a range of novel from DNA to drug discovery approaches for pharmacological studies, sufficient biolog- ical material – and data of previously unknown quantity Over four decades ago, the discovery and characterisation and quality. DNA as a starting material was the new focal of molecular tools, in the form of DNA ligases (Zim- point that led to the molecular cloning of targets for merman et al., 1967), restriction endonucleases (Linn endogenous ligands as well as therapeutic drugs, and thus and Arber, 1968; Smith and Wilcox, 1970; Danna and facilitated drug discovery (see 2.3 below). The technical Nathans, 1971) and reverse transcriptases (Baltimore, 1970; Temin and Mizutani, 1970) provided the platform possibilities seemed infinite. for the emerging recombinant DNA technology (Cohen Consequently, molecular cloning sparked a global race et al., 1972, 1973; Jackson et al., 1972), an array of applica- amongst scientists for the identification of DNAs of major tions used to cut, join, amplify, modify and express DNA drug targets, the molecular nature of which was often fragments. Molecular cloning, in this context, refers to unknown back then! This work revealed a surprising the process that introduces an isolated piece of DNA into diversity of drug targets and complexity of intracellular a vector (recombination) and generates multiple copies signalling cascades. It was frequently accompanied by (clones) of it. (It should not be confused with cloning of the realisation that a single ‘known’ drug target indeed animals or cells!) existed in a multitude of subtypes, which traditional Recombinant DNA technology quickly transformed investigations failed to distinguish. And it highlighted the the field of pharmacology, as it overcame several tech- necessity to develop subtype-specific drugs. However, the nical limitations faced with traditional pharmacology at initial DNA cloning process could now be complemented

Molecular Pharmacology: From DNA to Drug Discovery, First Edition. John Dickenson, Fiona Freeman, Chris Lloyd Mills, Shiva Sivasubramaniam and Christian Thode. © 2013 John Wiley & Sons, Ltd. Published 2013 by John Wiley & Sons, Ltd.

13 14 Chapter 2 by expression studies in host cells, where previously- Thus, an observed response could be due to the activation identified or potential novel drug targets could be func- of more than one protein, such as a receptor (a so-called tionally and pharmacologically characterised in vitro.It mixed response), and this might go undetected. It might also marked the beginning of reverse pharmacology. be possible to detect the presence of multiple targets if The chapter Molecular Cloning of Drug Targets starts the experimenter had access to one or more selective with a brief overview of traditional pharmacology, antagonists. However, a given antagonist might inhibit followed by technical insights into the main methods, a response that was due to the activity of multiple pro- which nowadays are used routinely in molecular teins, but blocking all of them. Famous examples, where cloning. It concludes with a particular example, where ‘traditional’ pharmacology has erroneously postulated molecular cloning enabled the identification of a natural the existence of too few subtypes exist among G-protein endogenous ligand for a receptor. (The interested reader coupled receptors (GPCRs; i.e. muscarinic acetylcholine is referred to Sambrook and Russel’s (2001) Molecular receptors and dopamine receptors) and ligand-gated ion rd Cloning: A Laboratory Manual,(3 ed) CSH Press, for channels (LGICs; i.e. GABAA receptors; see section 2.5 detailed protocols). for details). Conversely, a single GPCR could give rise to different responses, depending on the cell type and the intracellular signalling pathways that it coupled to via dif- 2.2 ‘Traditional’ pharmacology ferent guanine nucleotide binding proteins (G-proteins).

‘Traditional’ pharmacology refers here to the character- isation of ‘native’ drug targets, that is, proteins in their 2.3 The relevance of recombinant DNA natural molecular environment, such as in cells, tissues technology to pharmacology/drug and organs. Typically employed techniques include: discovery • electrophysiological recordings from cells; these permit the study of ion fluxes across the plasma membrane (see The acquisition and successive use of sequence informa- Chapter 4), for example in response to the exposure to tion plays a central role in recombinant DNA technology. ligands. Although the first – perhaps explorative – experiments • radioligand binding to tissue extracts and isolated mem- are often limited to the isolation, cloning and identi- branes; a radiolabelled molecule, whether the natural fication of a relatively short DNA fragment, they can set ligand or another compound, can be used to determine the foundation for a number of more elaborate studies, the binding affinity of a drug for a particular target in such as genetic engineering (see Chapter 10), with access its native state. to physiological information of a different quality. The • receptor autoradiography; the incubation of thin tissue initially-obtained cloned nucleotide sequence, though, sections with a radiolabelled ligand is used to obtain sig- can already provide the deduced primary amino-acid nals (autoradiographs) on X-ray films; their pattern and sequence of a drug target, a map of recognition sites intensity reveals qualitative (location) and quantitative for restriction endonucleases, or some limited structural information (amount) about a drug target in situ. information. For instance (see 2.5), many drug targets • ‘classical’ preparations; for example, isolated tissue (e.g. LGICs or GPCRs) are membrane-bound, and the strips (such as muscle fibres) can be maintained in tis- results of molecular cloning can reveal the number of their sue baths for the purpose of measuring a drug-induced membrane-spanning domains or indicate their topology response (e.g. contraction). (i.e. whether the amino- and carboxy-termini are intra- • enzyme assays; the activity of a particular enzyme is cellular or extracellular). This information is important recorded in response to different drug concentrations. because we do not currently have the ability to purify Despite the negative connotation that the word ‘tra- them in sufficient amounts for crystallographic studies. ditional’ may hold for some, this approach still offers By searching appropriate databases (e.g. http://blast an important means of investigation, frequently supple- .ncbi.nlm.nih.gov/Blast.cgi, http://www.ebi.ac.uk/embl) menting the data from molecular cloning. However, prior with a cloned sequence, it is also possible to find to the advent of molecular cloning studies, ‘traditional’ homologous sequences, either in the same or a different pharmacology on its own had several disadvantages. For species. These sequence comparisons may indicate the example, cells, tissues, and organs may contain more than full length of the open-reading frame of a partially-cloned one target for any given compound that lacks selectivity. fragment or even help to identify protein families.