Molecular PharmacologyFrom DNA to Drug Discovery

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MolecularPharmacologyFrom DNA to Drug Discovery

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

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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]615.1′9–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 printmay 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 11.1 Introduction to molecular pharmacology 11.2 Scope of this textbook 21.3 The nature of drug targets 31.4 Future drug targets 71.5 Molecular pharmacology and drug discovery 11

References 12

2 Molecular Cloning of Drug Targets 132.1 Introduction to molecular cloning – from DNA to drug discovery 132.2 ‘Traditional’ pharmacology 142.3 The relevance of recombinant DNA technology to pharmacology/drug

discovery 142.4 The ‘cloning’ of drug targets 152.5 What information can DNA cloning provide? 202.6 Comparing the pharmacologic profile of the ‘cloned’ and the ‘native’

drug target 232.7 Reverse pharmacology illustrated on orphan GPCRs 242.8 Summary 27

References 27

3 G Protein-coupled Receptors 313.1 Introduction to G protein-coupled receptors 313.2 Heterotrimeric G-proteins 363.3 Signal transduction pathways 403.4 Desensitisation and down-regulation of GPCR signalling 443.5 Constitutive GPCR activity 453.6 Promiscuous G-protein coupling 473.7 Agonist-directed signalling 483.8 Allosteric modulators of GPCR function 493.9 Pharmacological chaperones for GPCRs 50

3.10 GPCR dimerisation 513.11 GPCR splice variants 633.12 Summary 67

References 67Useful Web sites 70

4 Ion Channels 714.1 Introduction 714.2 Voltage-gated ion channels 73

v

vi Contents

4.3 Other types of voltage-gated ion channels 894.4 Ligand-gated ion channels 1094.5 Summary 125

References 125

5 Transporter Proteins 1295.1 Introduction 1295.2 Classification 1295.3 Structural analysis of transporters 1325.4 Transporter families of pharmacological interest 1335.5 Transporters and cellular homeostasis 1675.6 Summary 169

References 169

6 Cystic Fibrosis: Alternative Approaches to the Treatment of a Genetic Disease 1756.1 Introduction 1756.2 Cystic fibrosis transmembrane conductance regulator 1796.3 Mutations in CFTR 1836.4 Why is cystic fibrosis so common? 1846.5 Animal models of Cystic fibrosis 1866.6 Pharmacotherapy 1866.7 Gene therapy 1916.8 Conclusion 195

References 196

7 Pharmacogenomics 2017.1 Types of genetic variation in the human genome 2017.2 Thiopurine S-methyltransferase and K+ channel polymorphisms 2027.3 Polymorphisms affecting drug metabolism 2047.4 Methods for detecting genetic polymorphisms 2097.5 Genetic variation in drug transporters 2117.6 Genetic variation in G protein coupled receptors 2157.7 Summary 225

References 225Useful Web sites 226

8 Transcription Factors and Gene Expression 2278.1 Control of gene expression 2278.2 Transcription factors 2298.3 CREB 2338.4 Nuclear receptors 2388.5 Peroxisome proliferator-activated receptors 2408.6 Growth factors 2478.7 Alternative splicing 2478.8 RNA editing 2518.9 The importance of non-coding RNAs in gene expression 257

8.10 Summary 270References 271

9 Cellular Calcium 2779.1 Introduction 2779.2 Measurement of calcium 278

Contents vii

9.3 The exocrine pancreas 2899.4 Calcium signalling in pancreatic acinar cells 2929.5 Nuclear calcium signalling 3039.6 Conclusions 310

References 311

10 Genetic Engineering of Mice 31510.1 Introduction to genetic engineering 31510.2 Genomics and the accumulation of sequence data 31510.3 The mouse as a model organism 31810.4 Techniques for genetic engineering 31910.5 Examples of genetically-engineered mice 33210.6 Summary 334

References 334

11 Signalling Complexes: Protein-protein Interactions and Lipid Rafts 33911.1 Introduction to cell signalling complexes 33911.2 Introduction to GPCR interacting proteins 34011.3 Methods used to identify GPCR interacting proteins 34011.4 Functional roles of GPCR interacting proteins 34511.5 GPCR signalling complexes 34811.6 GPCR and ion channel complexes 35511.7 Ion channel signalling complexes 35611.8 Development of pharmaceuticals that target GPCR interacting proteins 35611.9 Development of pharmaceuticals that target protein-protein interactions 35611.10 Lipid rafts 35711.11 Receptor-mediated endocytosis 36111.12 Summary 364

References 364

12 Recombinant Proteins and Immunotherapeutics 36712.1 Introduction to immunotherapeutics 36712.2 Historical background of immunotherapeutics 36812.3 Basis of immunotherapeutics 36812.4 Types of immunotherapeutics 36912.5 Humanisation of antibody therapy 37212.6 Immunotherapeutics in clinical practice 37612.7 Advantages and disadvantages of immunotherapy 37812.8 The future 37912.9 Summary 380

References 380

Glossary 381

Index 403

Preface

Nottingham Trent University offers a suite of successfulMSc courses in the Biosciences field that are delivered byfull-time, part-time and distance (e-learning) teaching.The authors are members of the Pharmacology teamat Nottingham Trent University and teach extensively onthe MSc Pharmacology and Neuropharmacology courses.The content of this book was inspired by these coursesas there is no comparable postgraduate textbook onmolecular pharmacology and it is a rapidly expandingsubject. The primary aim of this text was to provide aplatform to complement our courses and enhance thestudent experience. Given the breadth and depth of thisvolume it will be of use to students from other institutionsas a teaching aid as well as an invaluable source ofbackground information for post-graduate researchers.The value of this book is enhanced by the researchportfolio of the Bioscience Department and individualauthors who have research careers spanning over 25 years.

This textbook illustrates how genes can influence ourphysiology and hence our pharmacological response todrugs used to treat pathological conditions. Tailoring of

therapeutic drugs is the future of drug design as it enablesphysicians to prescribe personalised medical treatmentsbased on an individual’s genome. The book utilises adrug target-based approach rather than the traditionalorgan/system-based viewpoint and reflects the currentadvances and research trends towards in silico drug designbased on gene and derived protein structure.

The authors would like to thank Prof Mark Darlison(Napier University, Edinburgh, UK) for providing theinitial impetus, inspiration and belief that a book ofsuch magnitude was possible. We would also like toacknowledge the unflagging encouragement and supportof the Wiley-Blackwell team (Nicky, Fiona and Clara)during the preparation of this work. Finally thanks shouldalso be given to the helpful, constructive and positivecomments provided by the reviewers. We hope that youenjoy this book as much as we enjoyed writing it.

John Dickenson, Fiona Freeman, Chris Lloyd Mills, ShivaSivasubramaniam and Christian Thode.

ix

Abbreviations

[Ca2+]i intracellular free ionised calciumconcentration

[Ca2+]n nuclear free ionised calciumconcentration

[Ca2+]o extracellular free ionised calciumconcentration

2-APB 2-aminoethoxydiphenyl borate4EFmut DREAM 4th EF hand mutant DREAM5F-BAPTA 1,2-bis(2-amino-5,6-diflurophenoxy)

ethane-N,N,N′,N′-tretracacetic acid5-HT 5-hydroxytyrptamine / serotoninAAV adeno-associated virusABC ATP-binding cassette (transporter)AC adenylyl cyclaseACC mitochondrial ADP/ATP carrier

(transporter)ACh acetylcholineACS anion-cation subfamilyAD Alzheimer’s diseaseADAR adenosine deaminase acting on RNA

(1, 2 or 3)ADCC antibody-dependent cellular

cytotoxicityADEPT antibody-directed enzyme pro-drug

therapyADHD attention deficit hyperactivity disorderAF1/2 transcriptional activating function

(1 or 2)Ala alanine (A)AM acetoxylmethylAMPA α-amino-3-hydroxy-5-methylis

oxazole 4-propionic acidApo- apolipoproteins (A, B or C)APP amyloid precursor proteinAQP aquaporins

ARC channels arachidonic acid regulated Ca2+

channelsArg arginine (R)ASIC acid sensing ion channelsASL airways surface liquidAsn asparagine (N)Asp aspartic acid (D)ATF1 activation transcription factor 1ATP adenosine triphosphateAV adenovirusAβ amyloid β peptideBAC bacterial artificial chromosomeBBB blood brain barrierBCRP breast cancer resistant proteinBDNF brain-derived neurotrophic factorBKCa big conductance Ca2+-activated K+

channelsBLAST Basic Local Alignment Search Toolbp base pairsBRET bioluminescence resonance energy

transferBrm/brg1 mammalian helicase like proteinsBTF basal transcription factorsBZ benzodiazepineCa-CaM Ca2+-calmodulinCaCC calcium activated chloride channelcADPr cyclic adenosine diphosphoriboseCaM calmodulinCaMK calcium-dependent calmodulin kinasecAMP cyclic adenosine 3′,5′ monophsophateCaRE calcium responsive elementcatSper cation channels in spermCaV voltage-gated Ca2+ channelsCBAVD congenital bilateral absence of the vas

deferens

x

Abbreviations xi

CBP CREB binding proteinCCCP carbonyl cyanide

m-chlorophenylhydrazoneCCK cholecystokininCDAR cytosine deaminase acting on RNAcDNA complementary DNACDR complementarily-determining regionCF cystic fibrosisCFP cyan fluorescent proteinCFS colony stimulating factorsCFTR cystic fibrosis transmembrane

conductance regulatorcGMP cyclic guanosine 3′,5′ monophosphateCHF congestive heart failureCHO Chinese hamster ovary cell lineCICR calcium induced calcium releaseCIF calcium influx factorClC chloride channelCMV cytomegalovirusCNG cyclic nucleotide-gated channelCNS central nervous systemCNT concentrative nucleoside transporterCOS CV-1 cell line from Simian kidney cells

immortalised with SV40 viralgenome

COX cyclooxygenases (1, 2 or 3)CPA monovalent cation/proton antiporter

super familyCpG cytosine-phosphate-guanine regions in

DNACPP cell penetrating peptide (transporter)CRE cAMP responsive elementCREB cAMP responsive element binding

proteinCREM CRE modulatorCRF corticotropin-releasing factorCRM chromatin remodelling complexCRTC cAMP-regulated transcriptional

co-activator familyCSF cerebral spinal fluidCTD C terminal domainCTL cytotoxic T lymphocyteCYP cytochrome P450

Cys cysteine (C)DAG diacylglycerolDAX1 dosage-sensitive sex reversal gene/TFDBD DNA-binding domainDC dicarboxylateDHA drug:H+ antiporter family

(transporter)

Dlg1 drosophila disc large tumoursuppressor

DNA deoxyribonucleic acidDOPA dihydroxyphenylalanineDPE downstream promoter elementDRE downstream regulatory elementDREAM DRE antagonist modulatordsRNA double-stranded RNAEBV Epstein Barr virusEGF epidermal growth factorEGFR epidermal growth factor receptorEGTA ethylene glycol tetraacetic acidELISA enzyme linked immunosorbent assayENaC epithelial sodium channelEPO erythropoietinER endoplasmic reticulumERK extracellular-signal-regulated kinaseseRNA enhancer RNAERTF oestrogen receptor transcription factorES cells embryonic stem cellsESE exon splicing enhancerESS exon splicing silencerEST expressed sequence tagFab antibody binding domainFACS fluorescent-activated cell sortingFc constant fragment of the monoclonal

antibodiesFEV1 forced expiratory volume in 1 secondFGF-9 fibroblast growth factorFIH factor inhibiting HIFFISH fluorescence in situ hybridisationFOXL2 fork-head box proteinFRET fluorescence resonance energy transferFXS fragile-X syndromeG3P glucose-3-phosphateGABA gamma-aminobutyric acidGAT GABA transportersGC guanylyl cyclaseGFP green fluorescent proteinGIRK G-protein-gated inwardly rectify K+

channelGln glutamine (Q)GlpT sn-glycerol-3-phosphate/phosphate

antiporterGltPh Pyrococcus horikoshii glutamate

transportersGlu glutamic acid (E)GLUT glucose transportersGly glycine (G)GLYT glycine transporters

xii Abbreviations

GMP guanosine monophosphateGPCR G protein coupled receptorGPN glycyl-L-phenylalanine-2-

napthylamideGRK G-protein coupled receptor kinaseGST Glutathione S-transferaseH+ hydrogen ion; protonHAD histone deacetylasesHAMA human anti-murine antibodiesHAT histone acetyltransferasesHCF host cell factorHCN hyperpolarisation-activated cyclic

nucleotide-gated channelsHDL high density lipoproteinHIF hypoxia inducible factorHis histidine (H)HMG high mobility groupHMIT H+/myo-inositol transporterhnRNP nuclear ribonucleoproteinsHOX homeoboxHPLC high-performance liquid

chromatographyHRE hypoxia response elementsHsp70 heat shock protein of the 70 kilodalton

familyHSV herpes simplex virusHSV-tk herpes simplex virus thymidine kinaseHTS high-throughput screeningHtt HuntingtinIBMX 3-isobutyl-1-methylxanthineIcrac calcium release activated Ca2+ channelICSI intra-cytoplasmic sperm injectionIfs interferonsIg immunoglobulinsIGF-1 insulin-like growth factor-IiGluR ionotropic glutamate receptorIHD ischaemic heart diseaseIL-10 interleukin-10Ile isoleucine (I)INN international non-proprietary namesINR initiator elementINSL3 insulin-like factor 3IP3 inositol 1,4,5-triphosphateIP3R IP3 receptoriPLA2β β isoform of Ca2+ independent

phospholipase A2

IRT immunoreactive trypsinogenIsc short circuit currentISE introns splicing enhancerISS introns splicing silencerK2P two-pore potassium channels

K3K4 HMT histone methyl transferaseKATP ATP-sensitive K+ channelskb kilobaseKCa Ca2+-activated K+ channelsKCC K+-Cl− co-transporterKChIP K+ channel interacting proteinKCO K+ channel openersKd Ca2+ dissociation constantKG G-protein gated K+ channelsKID kinase-inducible domainKir inwardly rectifying K+ channelsKV voltage-gated K+ channelLacY lactose:H+ symporterLBD ligand binding domainsLDL low density lipoproteinLeu leucine (L)LeuTAa Aquifex aeolicus leucine transporterLGIC ligand-gated ion channellncRNA long non-coding RNALPS lipopolysaccharidelys lysine (K)Mab monoclonal antibodiesMAC membrane attack complexMAPK mitogen-activated protein kinaseMATE multidrug and toxic compound

extrusion superfamily (transporter)Mb megabaseMCT mono carboxylate transportersMCU mitochondrial Ca2+ uniporterMDR multidrug resistance (transporter)MDR1 multidrug resistant transporter 1Met methionine (M)MFP periplasmic membrane fusion protein

family (transporter)MFS major facilitator superfamily

(transporter)MHC histocompatibility complexmiRNA microRNAmPTP mitochondrial permeability transition

poremRNA messenger RNAMSD membrane spanning domainMTF modulatory transcription factorsMyc myc oncogeneNAADP nicotinic acid adenine dinucleotide

phosphatenAChR nicotinic acetylcholine receptorsNAD+ nicotinamide adenine dinucleotideNADP+ nicotinamide adenine dinucleotide

phosphate

Abbreviations xiii

NALCN sodium leak channel non-selectiveprotein channel

NAT natural antisense transcriptNaV voltage-gated Na+ channelsNBD nucleotide binding domainncRNA non-coding RNAneoR neomycin resistanceNES nuclear endoplasmic spaceNFAT nuclear factor of activated T cellsNFκB nuclear factor kappa of activated B

cellsNHA Na+/H+ antiportersNhaA Escherichia coli Na+/H+ antiporterNHE Na+/H+ exchangerNKCC sodium potassium 2 chloride

cotransporterNM nuclear membraneNMDA N-methyl-D-aspartateNMR nuclear magnetic reasonanceNO nitric oxideNPA Asn-Pro-Ala motifNPC nuclear pore complexNR nucleoplasmic reticulumNR-HSP nuclear receptor-heat shock protein

complexNRSE neuron restrictive silencer elementNSS neurotransmitter sodium symporter

(transporter)nt nucleotideNTD N- terminal domainNVGDS non viral gene delivery systemsOA- organic anionOAT organic anion transportersOCT organic cation transportersOct/OAP octomer/octomer associated proteinsOMF outer membrane factor family

(transporter)ORCC outwardly rectifying chloride channelORF open-reading frameOSN olfactory sensory neuronsOxlT oxalate:formate antiporterPax paired box gene/TFpCa -log10 of the Ca2+ concentrationPCR polymerase chain reactionPD potential differencePDE phosphodiesterasePDZ PSD95-Dlg1-zo-1 (protein motif)PEPT dipeptide transportersPG prostaglandinsPGC-1α peroxisome proliferator-activated

receptor α, co-activator 1α

PGE2 prostaglandin E2

P-gp permeability glycoprotein(transporter)

Phe phenylalanine (F)Pi inorganic phosphatePI3 phosphatidylinositol 3-kinasesPIP2 phosphatidylinositol 4,5-bisphosphatePKA protein kinase APKC protein kinase CPLC phospholipase CPLCβ β isoform of phospholipase CpLGICs pentameric ligand-gated ion channelsPM plasma membranePMCA plasma membrane Ca2+ ATPasePP1 protein phosphatase 1PPAR peroxisome proliferator-activated

receptors (α, β, δ, or γ)PPRE PPAR response elementpRB retinoblastoma proteinPro proline (P)PSD95 post synaptic density protein-95Q1/Q2 glutamine-rich domains (1 or 2)RaM rapid mode uptakeRAMP receptor-activity modifying proteinRas rat sarcoma (causing factor)RBC red blood cellREST repressor element-1 transcription

factorRFLP restriction fragment length

polymorphismrhDNase recombinant human DNaseRICs radio-immunoconjugatesRIP receptor-interacting proteinRISC RNA-induced silencing complexRLF relaxin-like factorRNA pol RNA polymerasesRNA ribonucleic acidRNAi RNA interferenceRND resistance-nodulation-cell division

(transporter)ROS reactive oxygen speciesrRNA ribosomal RNARSPO1 R-spondin-1RT-PCR reverse-transcription polymerase

chain reactionRXR retinoic acid receptorRyR ryanodine receptorsSAM intraluminal sterile α motifSBP substrate binding proteinSer serine (S)

xiv Abbreviations

SERCA sarco/endoplasmic reticulum Ca2+

ATPaseShh sonic hedgehog homolog gene/TFsiRNA short interfering RNASKCa small conductance Ca2+-activated K+

channelsSLC solute carrier superfamily

(transporter)SMN survival of motor neurons proteinSMR small multidrug resistance superfamily

(transporter)snoRNA small nucleolar RNASNP single nucleotide polymorphismsnRNA spliceosomal small nuclear RNASOC store operated Ca2+ channelSox9 SRY-related HMG box-9 gene/factorSR sarcoplasmic reticulumSRC-1 steroid receptor co-activator-1.SREBP sterol regulatory element-binding

proteinsSRY sex-determining region YSSS solute sodium symporter (transporter)STAT signal transducer and activator of

transcription (1, 2 or 3)STIM stromal interaction moleculeSUG-1 suppressor of gal4D lesions −1SUMO small ubiquitin like modifierSUR sulfonylureas receptorSW1/SNF switching mating type/sucrose

non-fermenting proteinsTAD transactivation domainTAP transporters associated with antigen

processingTCA tricarboxlyic acidTCR T cell receptorTDF testis-determining factorTEAD TEA domain proteinsTEF transcription enhancer factorTESCO testis-specific enhancer of Sox9TGF transforming growth factorTGN trans-Golgi networkTH tyrosine hydroxylaseThr threonine (T)

TIF-1 transcription intermediary factorTIRF total internal reflection fluorescence

imagingTMAO trimethylamine N-oxideTMD transmembrane domainTMS transmembrane segmentsTNFs tumour necrosis factorsTPC two pore calcium channelsTPEN N,N,N′,N′-tetrakis(2-

pyridylmethyl)ethylenediamineTrk tyrosine kinase receptor (A, B or C)tRNA transfer RNATRP transient receptor potential channelsTrp tryptophan (W)TTX tetrodotoxinTyr tyrosine (Y)TZD thiazolidinedioneUbi ubiquitinationUTR untranslated regionVal valine (V)VDAC voltage dependent anion channelVEGF vasculoendothelial growth factoVFT venus flytrapvGLUT vesicular glutamate transporterVHL von Hippel-Lindau proteinVIP vasoactive intestinal peptideVLDL very low density lipoproteinVm membrane potentialVOCC voltage-operated calcium channelsWNT4 wingless-type mouse mammary

tumour virus integration siteYAC yeast artificial chromosomeYFP yellow fluorescent proteinYORK yeast outward rectifying K+ channelZAC zinc-activated channelZo-1 zonula occludens-1 protein

POST-FIXesChimeric antibodies – xiMabs

Human antibodies – muMbsHumanised antibodies – zumabMonoclonal antibodies – oMabs

1 Introduction to Drug Targetsand Molecular Pharmacology

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

1.1 Introduction to molecularpharmacology

During the past 30 years there have been significantadvances and developments in the discipline of molecularpharmacology – an area of pharmacology that is con-cerned with the study of drugs and their targets at themolecular or chemical level. Major landmarks during thistime include the cloning of the first G-protein coupledreceptor (GPCR) namely the β2-adrenergic receptor in1986 (Dixon et al., 1986). This was quickly followed by thecloning of additional adrenergic receptor family genes andultimately other GPCRs. The molecular biology explo-sion during the 1980s also resulted in the cloning of genesencoding ion channel subunits (e.g. the nicotinic acetyl-choline receptor and voltage-gated Na+ channel) andnuclear receptors. The cloning of numerous drug targetscontinued at a pace during the 1990s but it was not untilthe completion of the human genome project in 2001 thatthe numbers of genes for each major drug target familycould be determined and fully appreciated. As would beexpected, the cloning of the human genome also resultedin the identification of many potentially new drug targets.The completion of genome projects for widely used model

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.

organisms such as mouse (2002) and rat (2004) has alsobeen of great benefit to the drug discovery process.

The capacity to clone and express genes opened upaccess to a wealth of information that was simply not avail-able from traditional pharmacology-based approachesusing isolated animal tissue preparations. In the caseof GPCRs detailed expression pattern analysis could beperformed using a range of molecular biology tech-niques such as in situ hybridisation, RT-PCR (reversetranscriptase-polymerase chain reaction) and Northernblotting. Furthermore having a cloned GPCR gene in asimple DNA plasmid made it possible for the first timeto transfect and express GPCRs in cultured cell lines.This permitted detailed pharmacological and functionalanalysis (e.g. second messenger pathways) of specificreceptor subtypes in cells not expressing related sub-types, which was often a problem when using tissuepreparations. Techniques such as site-directed muta-genesis enable pharmacologists to investigate complexstructure-function relationships aimed at understanding,for example, which amino acid residues are crucial forligand binding to the receptor. As cloning and expres-sion techniques developed further it became possibleto manipulate gene expression in vivo. It is now com-mon practice to explore the consequences of deleting a

1

2 Chapter 1

Animal-based studies• Gene knockout• Gene knockin• Disease models

Drug Targets

Cell-based studies• Transfected cells• Reporter gene assays• Protein-protein interactions• GFP-based assays

Expression patterns• In situ hybridization• RT-PCR• Northern blotting

Structure-function studies• Site-directed mutagenesis• X-ray crystallography

Figure 1.1 Molecular pharmacology-based methods used to interrogate drug targets.

specific gene either from an entire genome (knockout)or from a specific tissue/organ (conditional knockout).It is also possible to insert mutated forms of genes intoan organism’s genome using knockin technology. Thesetransgenic approaches allow molecular pharmacologiststo study developmental and physiological aspects of genefunction in vivo and in the case of gene knockin techniquesto develop disease models.

The molecular biology revolution also enabled thedevelopment of novel approaches for studying thecomplex signal transduction characteristics of pharma-cologically important proteins such as receptors andion channels. These include reporter gene assays, greenfluorescent protein (GFP) based techniques for visual-ising proteins in living cells and yeast two hybrid-basedassays for exploring protein-protein interactions. Youwill find detailed explanations of these and other currentmolecular-based techniques throughout this textbook.Another major breakthrough in the 2000s was thedevelopment of methods that allowed high resolutionstructural images of membrane-associated proteins to beobtained from X-ray crystallography. During this timethe first X-ray structures of GPCRs and ion channelswere reported enabling scientists to understand howsuch proteins function at the molecular level. Indeedcrystallography is an important tool in the drug discoveryprocess since crystal structures can be used for in silicodrug design. More recently researchers have used NMRspectroscopy to obtain a high-resolution structuralinformation of the β2-adrenergic receptor (Bokoch etal., 2010). A distinct advantage of NMR-based structural

studies, which are already used for structural studies ofother drug targets such as kinases, would be the abilityto obtain GPCR dynamics and ligand activation datawhich is not possible using X-ray based methods. Someof the molecular pharmacology based approaches usedto interrogate drug targets are outlined in Figure 1.1.

Despite this increased knowledge of drug targetsobtained during the molecular biology revolution, therehas been a clear slowdown in the number of new drugsreaching the market (Betz, 2005). However, since it takesapproximately 15 years to bring a new drug to marketit may be too early to assess the impact of the humangenome project on drug discovery. In 2009 the globalpharmaceutical market was worth an estimated $815billion. However during the next few years a majorproblem facing the pharmaceutical industry is the lossof drug patents on key blockbusters. The hope for thefuture is that the advances in molecular pharmacologywitnessed during the last decade or so will start to delivernew blockbuster therapeutics for the twenty-first century.

1.2 Scope of this textbook

As briefly detailed above there have been numerousexciting developments in the field of molecular phar-macology. The scope of this textbook is to explore aspectsof molecular pharmacology in greater depth than cov-ered in traditional pharmacology textbooks (summarisedin Figure 1.2). Recent advances and developments inthe four major human drug target families (GPCRs,ion channels, nuclear receptors and transporters) are

Introduction to Drug Targets and Molecular Pharmacology 3

Transcription

Translation

PharmacogenomicsChapter 7

Gene therapyChapter 6 DNA

RNA

Protein

Cloning ofdrug targets

Chapter 2

GPCRs (chapter 3)Ion channels (chapter 4)Transporters (chapter 5)Nuclear receptors (chapter 8)

Conventional Drug targets

Transcription factorbased drugs

Chapter 8

Recombinant proteinsAntibody-basedtherapiesChapter 12

Anti-sense oligonucleotidesInterference RNAChapter 8

Transgenic mousetechnologyChapter 10

Figure 1.2 Drug targets within the central dogma of molecular biology. To date the majority of conventional therapeutics target arelatively small group of protein families that include G-protein coupled receptors, ion channels, and transporters. Novel therapeuticstrategies 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 topicsare indicated.

covered in separate chapters (Chapters 3–5 and 8). Themolecular targets of anti-infective drugs (anti-bacterialand anti-viral) whilst of great importance are not coveredin this book. Other chapters deal with the cloning of drugtargets (Chapter 2) and transgenic animal technology(Chapter 10). The concept of gene therapy is exploredin a case study-based chapter which looks at current andpossible future treatment strategies for cystic fibrosis, thecommonest lethal genetic disease of Caucasians (Chapter6). Another major development in molecular pharma-cology has been the discipline of pharmacogenomics: thestudy of how an individual’s genetic makeup influencestheir response to therapeutic drugs (Chapter 7). Thesenaturally occurring variations in the human genomeare caused predominantly by single nucleotide polymor-phisms (DNA variation involving a change in a singlenucleotide) and there is a major research consortiumaimed at documenting all the common variants of thehuman genome (The International HapMap project).

The information from the project, which is freely avail-able on the internet, will enable scientists to understandhow genetic variations contribute to risk of disease anddrug response. Finally, we take an in depth look at therole of calcium in the cell, looking at techniques used tomeasure this important second messenger (Chapter 9).

1.3 The nature of drug targets

How many potential drug targets are there in the humangenome? This is an important question often asked bythe pharmaceutical industry since they are faced withthe task of developing novel therapeutics for the future.When the draft sequence of the human genome was com-pleted in 2001 it was estimated to contain approximately31,000 protein-coding genes. However since its comple-tion the number of human protein-coding genes hasbeen 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 ofDecember 2005. Drugs that target ligand-gated ion channels and voltage-gated ion channels have been grouped together as ionchannels. Drug targets grouped together as others encompass 120 different specific targets many of which are enzymes. Data takenfrom Overington et al. (2006).

between 20,000 and 25,000. Of these it is predicted thatabout 3000 are feasible protein drug targets. In 2005 itwas calculated that about 100 drug targets account forall prescription drugs. On this basis there is obviouslyconsiderable scope for the development and discovery ofnovel drug targets to treat disease. At present the classicaldrug targets include GPCRs (Chapter 3), ion channels(Chapter 4), nuclear receptors (Chapter 8), transporters(Chapter 5) and enzymes. These important classical drugtargets, whilst briefly covered in this Introduction, areextensively covered in later chapters. The distribution ofdrug targets expressed as a percentage of total productsapproved by the Food and Drug Administration (FDA;agency in the USA responsible for approving drugs fortherapeutic use) is illustrated in Figure 1.3.

G-protein coupled receptors (GPCRs)GPCRs represent the largest single family of pharmaceu-tical drug target accounting for approximately 30% of thecurrent market. Their primary function is to detect extra-cellular signals and through heterotrimeric G-proteinactivation trigger intracellular signal transduction cas-cades that promote cellular responses (Figure 1.4). Whilsttheir share of the overall drug market is likely to fall inthe future they still represent ‘hot’ targets for drug dis-covery programmes. GPCRs are conventionally targetedusing small molecules (typically less than 500 Da) thatare classified as agonists (receptor activating) or antag-onists (inhibit receptor function by blocking the effectof an agonist). Some key examples of drugs that targetGPCRs are listed in Table 1.1. Chapter 3 will explain indetail many of the recent developments in GPCR struc-ture, function, pharmacology and signal transduction

including GPCR dimerisation. Many of these excitingadvances have revealed new pharmaceutical approachesfor targeting GPCRs such as inverse agonists, allostericmodulators, biased agonists and bivalent ligands thattarget GPCR heterodimers. Since the completion of thehuman genome project it has emerged that the totalnumber of human GPCRs may be as high as 865, whichwould account for approximately 3.4% of total pre-dicted protein-coding genes (assuming a total of 25,000).For many cloned GPCRs the endogenous ligand(s) areunknown (so called ‘orphan’ GPCRs) and the identi-fication of these orphan receptor ligands is the focusof drug discovery programmes within the pharmaceuti-cal industry. The process of GPCR de-orphanisation isaddressed in Chapter 2. In Chapter 11 the concept thatGPCRs interact with a host of accessory proteins thatare important in modulating many aspects in the lifeof a GPCR including the formation of signalling com-plexes will be explored. Indeed, targeting such GPCRsignalling complexes with drugs that disrupt protein-protein interactions is another exciting avenue for futuredrug development not only in the field of GPCRs but alsoin other areas of signal transduction.

Ion channelsIon channels represent important drug targets since theyare involved in regulating a wide range of fundamentalphysiological processes. Indeed, at present they are thesecond largest class of drug target after GPCRs. Theyoperate the rapid transport of ions across membranes(down their electrochemical gradients) and in doing sotrigger plasma and organelle membrane hyperpolari-sation or depolarisation. They are also potential drug

Introduction to Drug Targets and Molecular Pharmacology 5

GTP

(a)

Agonist

G-proteinsignalling

GDP

(b)

AntagonistAgonist

Agonist Antagonist

Allostericmodulators

GPCR

GTP

(e)

Agonist

(d)

ArrestinGDP

(c)

Biased agonistAgonist

Arrestinsignalling

NoG-proteinsignalling

G-proteinsignalling

γβαγβα

γβα

γβα

Figure 1.4 G-protein coupled receptors as drug targets. (a) GPCRs can be targeted using selective synthetic agonists which triggerreceptor activation thus enabling G-protein coupling and subsequent cell signalling responses. (b) antagonists can be used to blockthe binding of an endogenous agonist thus preventing receptor activation. (c) GPCRs can also trigger G-protein independent cellsignalling pathways which are dependent on arrestin binding to the activated receptor. Biased agonists are being developed thatspecifically promote the activation of arrestin-dependent signalling pathways. (d) bivalent ligands targeting specific GPCRheterodimers. (e) GPCRs can also be targeted using allosteric modulators which bind to sites on the receptor that are distinct fromthe 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 ulcersα1-adrenergic receptor Doxazosin (Cardura) Antagonist HypertensionGnRH1 receptor Leuprorelin (Lupron) Agonist Prostate cancer5-HT1D receptor Sumatriptan (Imigran) Agonist Migraineμ-opioid receptor Fentanyl (Sublimaze) Agonist Analgesic

Abbreviations: GnRH, gonadotropin-releasing hormone.

targets for the treatment of rare monogenic hereditarydisorders caused by mutations in genes that encodeion channel subunits. Such conditions termed ‘ionchannelopathies’ include mutations in sodium, chlorideand calcium channels that cause alterations in skeletalmuscle excitability. The understanding of ion channeldiversity and complexity increased significantly followingthe completion of the human genome project whichidentified over 400 genes encoding ion channel subunits.Given this number of genes it has been suggested that ion

channels may rival GPCRs as drug targets in the future(Jiang et al., 2008). Other major developments includethe first 3D resolution of ion channel structure by X-raycrystallography, which was reported for the voltage-gatedpotassium channel in 2003 (MacKinnon et al., 2003).Despite these important advances in the understandingof ion channel diversity and structure very few new ionchannel drugs have reached the market during the lastdecade. Some key examples of ion channels as drugtargets are shown in Table 1.2.

6 Chapter 1

Table 1.2 Ion channels as drug targets.

Ion channel Drug (brand name) Condition/use

Voltage-gated Ca2+

channelAmlodipine

(Norvasc)Hypertension

and anginaVoltage-gated Na+

channelPhenytoin

(Dilantin)Epilepsy

ATP-sensitive K+

channelGlibenclamide

(Glimepride)Type II diabetes

GABAA receptor Benzodiazepines(Diazepam)

Anxiety

5-HT3 receptor Ondansetron(Zofran)

Nausea andvomiting

Ion channels are broadly classified into two maingroups (Figure 1.5). Firstly there are ligand-gated ionchannels or ionotropic receptors which open whenactivated by an agonist binding to a specific ion channelsubunit. Examples of this class include the nicotinicacetylcholine receptor, GABAA receptor, glycine receptor,5-HT3 receptor, ionotropic glutamate receptors, andATP-gated channels. The second group which includesvoltage-gated or voltage-operated ion channels areopened by other mechanisms including changes in plasmamembrane potential. Examples include voltage-gatedCa2+, Na+, and K+ channels. The molecular structureand classification of ion channels together with their useas drug targets will be explored in detail in Chapter 4.

Nuclear receptorsNuclear receptors are a large family of transcriptionfactors that play a pivotal role in endocrine function.In contrast to other families of transcription factor theactivity of nuclear receptors (as their name suggests)is specifically regulated by the binding of ligands(Figure 1.6). Such ligands, which are small and lipophilic,include steroid hormones (glucocorticoids, mineralo-corticoids, androgens, oestrogens and progestogens),thyroid hormones (T3 and T4), fat soluble vitamins Dand A (retinoic acid) and various fatty acid derivatives.Since the completion of the human genome sequencingproject 48 members of the human nuclear receptorfamily have been identified. However, for many nuclearreceptors the identity of the ligand is unknown. These‘orphan’ nuclear receptors are of significant interestto the pharmaceutical industry since they may lead tothe discovery of novel endocrine systems with potentialtherapeutic use. Whilst the total number of nuclear

(b)

Na+ channels

Na+

Ca2+ channelsK+

K+ channelsCa2+

• 5-HT3 receptor• Nicotinic ACh receptor

(a)

• Glycine receptor• GABAA receptor

K+K+

• Ionotropic Glutamate receptors

Cl− Na+/Ca2+ Na+/Ca2+ Na+/Ca2+

• ATP-gatedchannels (P2x)

Figure 1.5 Ion channel classification. (a) Ligand-gated ionchannels comprise a family of multi-subunit transmembraneproteins that are activated by a diverse set of ligands (indicatedby the orange circle) that include amino acids (glycine,glutamate and GABA), 5-hydroxytryptamine (5-HT),acetylcholine (ACh), and ATP. (b) voltage-gated channelchannels, which are also multi-subunit proteins, open inresponse to local changes in membrane potential.

receptors is small in comparison to GPCRs they arethe target of approximately 13% of all prescribed drugs.For example, the chronic inflammation associated withasthma can be suppressed by inhaled glucocorticoids andoestrogen-sensitive breast cancer responds to treatmentwith the oestrogen receptor antagonist tamoxifen. Thestructure, classification, signal transduction mechanismsand therapeutic uses of nuclear receptor targeting drugswill be explored in detail in Chapter 8.

Neurotransmitter transportersThe concentration of some neurotransmitters withinthe synaptic cleft is tightly regulated by specificplasma membrane-bound transporter proteins. Thesetransporters, which belong to the solute carrier

Introduction to Drug Targets and Molecular Pharmacology 7

Cell membrane

NR dimer

Nuclear pore

Modulation ofgene transcription

hormone

HSP/NRcomplex

HSP

NR/hormonecomplex

cytoplasm

nucleus

GRE

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

(SLC) transporter family, facilitate the movement ofneurotransmitter either back into the pre-synaptic neu-ron or in some cases into surrounding glial cells. There aretwo major subclasses of plasma-membrane bound neuro-transmitter transporter: the SLC1 family which transportsglutamate and the larger SLC6 family which transportsdopamine, 5-HT, noradrenaline, GABA and glycine(Figure 1.7). Both SLC1 and SLC6 families facilitate neu-rotransmitter movement across the plasma membraneby secondary active transport using extracellular Na+ ionconcentration as the driving force. As might be expecteddrugs that target neurotransmitter transporters have awide range of therapeutic applications such as treatmentfor depression, anxiety and epilepsy. Indeed, neuro-transmitter transporters are the target for approximatelyone-third of all psychoactive drugs (see Table 1.3). Themolecular structure and classification of neurotransmit-ter transporters and their value as important current andfuture drug targets will be discussed in detail in Chapter 5.

1.4 Future drug targets

At present more than 50% of drugs target only fourmajor gene families, namely GPCRs, nuclear receptors,

ligand-gated ion channels and voltage-gated ion channels(Figure 1.3). It is likely that the market share of theseclassical drug targets will shrink as new drug targets andapproaches are developed in the future.

Protein kinasesIt is predicted that protein kinases (and lipid kinases),one of the largest gene families in eukaryotes, will becomemajor drug targets of the twenty-first century. Proteinphosphorylation is reversible and is one of the mostcommon ways of post-translationally modifying proteinfunction. It regulates numerous cellular functions includ-ing cell proliferation, cell death, cell survival, cell cycleprogression, and cell differentiation. The enzymes thatcatalyse protein phosphorylation are known as proteinkinases, whereas the enzymes that carry out the reversedephosphorylation reaction are referred to as phos-phatases (Figure 1.8a). The human genome encodes for518 protein kinases and approximately 20 lipid kinases.The predominant sites of protein phosphorylation arethe hydroxyl groups (−OH) in the side chains of theamino acids serine, threonine and tyrosine (Figure 1.8b).When a phosphate group is attached to a protein it intro-duces a strong negative charge which can alter proteinconformation and thus function.

8 Chapter 1

SLC6SLC1

GPCR GPCR

(a) (b)

Pre-synapticnerve terminal

Pre-synapticnerve terminal

Ligand-gatedIon channel

Post-synapticnerve terminal

Ligand-gatedIon channel

Post-synapticnerve terminal

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. Releasedglutamate 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 synapticcleft activate specific ligand-gated ion channels and/or GPCRs located on the post-synaptic membrane. These releasedneurotransmitters are subsequently transported back into the pre-synaptic nerve terminal via SLC6 transporters. For clarity specificvesicular 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.

Table 1.3 Transporters as drug targets.

Transporter Drug(brand name)

Condition/use

5-HT transporter (SERT) Sertraline(Zoloft)

Antidepressant

Dopamine transporter(DAT)

Cocaine Drug of abuse

Noradrenalinetransporter (NET)

Bupropiona

(Welbrutin)Antidepressant

GAT-1 (GABA) Tiagabine Epilepsy

aAffinity for DAT as well

EnzymesEnzymes are the drug target for approximately 50% ofall prescribed drugs. Some key examples are listed inTable 1.4. However, because of their diverse nature theywill not be the focus of a specific chapter in this book.It is also important to remember that many prescribeddrugs target bacterial and viral enzymes for the treatmentof infectious disease and HIV. Also many enzymes, whilstnot direct drug targets, play important roles in drugmetabolism for example cytochrome P450 enzymes.

Protein kinases are classified according to the aminoacid they phosphorylate and are grouped into two maintypes: serine/threonine kinases and tyrosine kinases. In

Introduction to Drug Targets and Molecular Pharmacology 9

OH

OH

CH

Serine Phospho-serine

Phospho-threonine

Phospho-tyrosine

Threonine

Tyrosine

C OH

NH2

NH2

H2

O

CH CH OH

CH3O C

OH

OHC

NH2

H2CH

O C

OH

C

NH2

H2CH

O C

C

OH

CH C O O

O−

O−

O O

O−

O−

P

P

O O

O−

O−

P

NH2

H2

O C

OH

CH CH

NH2

CH3O C

Protein

Protein Kinase

Protein

PATP ADP

+ ADP

(a)

(b)

Proteinphosphatase

Figure 1.8 Reversible protein phosphorylation. (a) Protein kinases transfer a phosphate group (P) from ATP to the target proteinaltering 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 thethird phosphoryl group (γ; gamma phosphate) beingtransferred to the hydroxyl group of the acceptor aminoacid. Examples of serine/threonine kinases include pro-tein kinase A (PKA; activated by the second messengercyclic AMP) and protein kinase C (PKC; activated by thesecond messenger diacylglycerol). Examples of tyrosinekinases include tyrosine kinase linked receptors for insulinand epidermal growth factor and non-receptor tyrosinekinases such as Src and JAK (Janus-associated kinase).Given the prominent role of protein phosphorylation in

regulating many aspects of cell physiology it is not sur-prising that dysfunction in the control of protein kinasesignalling is associated with major diseases such as can-cer, diabetes and rheumatoid arthritis. These alterationsin protein kinase and in some cases lipid kinase functionarise from over-activity either due to genetic mutationsor over-expression of the protein. It is estimated thatup to 30% of all protein targets currently under inves-tigation by the pharmaceutical industry are protein orlipid kinases. Indeed, there are approximately 150 proteinkinase inhibitors in various stages of clinical development,

10 Chapter 1

Table 1.4 Enzymes as drug targets.

Enzyme Drug(brand name)

Condition/use

HMG-CoAreductase

Statins Used to lowerblood cholesterollevels

Phosphodiesterasetype V

Sildenafil(Viagra)

Erectile dysfunctionand hypertension

Cyclo-oxygenease Aspirin Analgesic and anti-inflammatory

Angiotensin-convertingenzyme

Captopril(Capoten)

Hypertension

Dihydrofolatereductase

Methotrexate Cancer

Table 1.5 Selected small-molecule protein kinase inhibitorsin clinical development.

Drug Protein kinase target Use

AZD 1152 Aurora B Kinase Various cancersNP-12 Glycogen synthase kinase

3 (GSK3)Alzheimer’s

diseaseBay 613606 Spleen tyrosine kinase

(Syk)Asthma

INCB-28050 Janus-associated kinase1/2 (JAK1/2)

Rheumatoidarthritis

BMS-582949 p38 mitogen-activatedprotein kinase (p38MAPK)

Rheumatoidarthritis

some of which are highlighted in Table 1.5. Whilst proteinkinases are important new human drug targets they arealso present in bacteria and viruses and thus representpotential targets for infectious disease treatment.

Since the launch of imatinib in 2001 several othersmall-molecule protein kinase inhibitors have success-fully made it to the market place as novel anti-cancertreatments (Table 1.6). The majority of these drugs aretyrosine kinase inhibitors and in some cases function asmulti-kinase inhibitors (e.g. sunitinib) targeting PDGFR(proliferation) and VEGFR (angiogenesis) dependent sig-nalling responses. Monoclonal antibodies are also usedto block the increased tyrosine kinase linked receptoractivity that is associated with many forms of cancer andthese will be discussed in Chapter 12.

Table 1.6 Small-molecule protein kinase inhibitorsapproved for clinical use.

Drug (brand name) Targets Use

Imatinib (Gleevec®) c-Abl-kinase,c-Kit

Chronic myeloidleukaemia

Gefitinib (Iressa®) EGFR Various cancersSunitinib (Sutent®) PDGFR,

VEGFRRenal cell

carcinomaDasatinib (Sprycel®) c-Abl-kinase,

SrcVarious cancers

Everolimus (Afinitor®) mTORa Various cancers

aSerine/threonine kinase. Abbreviations: EGFR, epider-mal growth factor receptor; mTOR, mammalian target ofrapamycin; PDGFR, platelet-derived growth factor receptor;VEGFR, vascular endothelial growth factor receptor.

A useful approach for assessing the therapeutic poten-tial of novel drug targets is the number of approvedpatents for each target (Zheng et al., 2006). The levelof patents gives an indication of the degree of interestin that particular target and hence likelihood of success-ful drugs being developed. Future targets with a highnumber of US-based patents include matrix metallopro-teinases (MMPs) as a target for cancer treatment. MMPsare proteases which break down the extracellular matrixthus facilitating cancer cell invasion and metastasis. Othertargets include phosphodiesterase 4 (PDE4), caspases andintegrin receptors. Only time will tell whether any ofthese novel targets result in the development of effectivetherapeutics. For further reading on the identificationand characteristics of future drug targets see the reviewby Zheng et al. (2006).

Therapeutic oligonucleotidesIn addition to the development of small-molecule-baseddrugs there are several other approaches to treat humandisease including the exciting prospect of therapeuticoligonucleotides (anti-sense and RNA interference based)as tools for gene silencing and the continued quest forgene therapy-based techniques. These molecular biology-based strategies for combating human disease will beaddressed later in Chapter 8.

Another new class of drugs are short single-strandedoligonucleotides (DNA or RNA based) that have beenselectively engineered to target specific intracellular pro-teins (Dausse et al., 2009). These oligonucleotides which

Introduction to Drug Targets and Molecular Pharmacology 11

fold into defined three-dimensional structures are knownas aptamers or ‘chemical antibodies’. They are gener-ated and repeatedly selected through a method knownas SELEX (Systematic Evolution of Ligands by Exponen-tial Enrichment). Essentially the process begins with thesynthesis of a large oligonucleotide library, containingrandomly generated sequences of fixed length, which isscreened for binding to the target protein usually byaffinity chromatography. Those that bind are repeatedlyselected using stringent elution conditions that ulti-mately result in the identification of the tightest bindingsequences. These high affinity sequences can be chemi-cally modified to increase their affinity and effectiveness aspotential therapeutic oligonucleotides. The first aptamer-based drug approved by the US Food and Drug Admin-istration (FDA) targets the VEGFR and is used to treatage-related macular degeneration. Several other aptameroligonucleotides are also undergoing clinical trials.

1.5 Molecular pharmacology and drugdiscovery

The process of drug discovery is a long and costly processwith new drugs taking up to 12 years to reach the clinic.Many novel molecular pharmacology-based techniquesplay important roles in the process of drug discoveryand development. A problem faced by many pharma-ceutical companies is the huge task of screening theirvast chemical libraries (in some cases this can exceedone million compounds) against an increasing numberof possible drug targets. The development, in the early1990s, of high-throughput screening (HTS) technologyusing 96-well microtiter plates enabled the drug screeningprocess to be miniaturised and automated. Using suchmethodology it became possible to screen up to 10,000compounds per day. However during the last decade384-well microtiter plates and more recently 1536-wellmicrotiter plate-based assays have been developed thatallow for screening of up to 200,000 compounds a day(ultra-high-throughput screening). Since the screeningof large chemical libraries is expensive several alterna-tive strategies to increase the chances of success havebeen introduced in recent years. One such approach hasbeen the introduction of fragment-based screening (FBS)or fragment-based lead discovery (FBLD). This involvesscreening the biological target with small libraries ofchemical fragments (molecular weights around 200 Da)with the aim of identifying scaffolds or ‘chemical back-bones’ that can be developed into lead compounds. This

approach may also be combined with computer-based‘virtual screening’ approaches. For example structure-based virtual screening involves the use of 3D proteinstructures, many of which are now widely available viapublic databases, to assess whether a ligand can inter-act or dock with the protein of interest. This can belinked with ligand-based virtual screening which involvesin silico screening of chemical libraries for compoundsthat display similar structural features associated with thebinding of the ligand to the target. As indicated abovestructure-based virtual screenings rely on the availabil-ity of accurate 3D structures of the drug target. Thediscipline of structural biology uses a range of biophys-ical techniques including X-ray crystallography, NMRspectroscopy and electron cryo-microscopy to determineprotein structure. The latter is an emerging techniquethat can be used to determine the 3D structure of macro-molecular complexes that are too large to be studied usingX-ray crystallography and/or NMR spectroscopy. So farin this section we have briefly covered some of the up-and-coming techniques that can used to interrogate drugtarget structure and screen drug targets for lead com-pounds. There is also a drive towards the developmentnovel cell-based and animal-based models that are morerepresentative of human physiology and hence moresuitable for drug screening. For example, 3D ‘organ-otypic’ cell microarrays are currently being developedthat will allow drug screening in a system that is closeto the in vivo environment of cells. In summary, we

• In vivo target validation - DNA knockout - RNA knockout - Protein knockout

• High-throughput screening• Fragment-based screening• Virtual screening (in silico)• Novel-cell based assays

• Gene cloning• Structural biology - X-ray crystallography - NMR spectroscopy

• Combinatorial chemistry

Targetdiscovery

Drugdiscovery &screening

Leadoptimization

ADMET

Phase Iclinicaltrials

Phase IIclinicaltrials

Phase IIIclinicaltrials

DrugApproval

Figure 1.9 Schematic representation of the drug discoveryprocess. ADMET (Adsorption, Distribution, Metabolism,Excretion and Toxicity studies).

12 Chapter 1

are witnessing exciting times in the process of drug dis-covery with the continued development of in silico andnanotechnology-based methods and the introduction ofnovel cell-based screening models. Has there ever been abetter time to be a molecular pharmacologist? A schematicrepresentation of the drug discovery process is shownin Figure 1.9.

References

Betz UAK (2005). How many genomics targets can a portfoliosupport? Drug Discovery Today 10: 1057–1063.

Bokoch MP, Zou Y, Rasmussen SGF, Liu CW, Nygaard R,Rosenbaum DM, Fung JJ, Choi H-J, Thian FS, Kobilka TS,Puglisi JD, Weis WI, Pardo L, Prosser RS, Mueller L andKobilka BK (2010). Ligand-specific regulation of the extra-cellular surface of a G protein coupled receptor. Nature 463:108–112.

Dausse E, Da Rocha Gomes S and Toulme J-J (2009). Aptamers:a new class of oligonucleotides in the drug discovery pipeline.Current Opinion in Pharmacology 9: 602–607.

Dixon RA, Kobilka BK, Strader DJ, Benovic JL, Dohlman HG,Frielle T, Bolanowski MA, Bennett CD, Rands E, Diehl RE,Munford RA, Slater EE, Sigal IS, Caron MG, Lefkowitz RJand Strader CD (1986). Cloning of the gene and cDNAfor mammalian beta-adrenergic receptor and homology withrhodopsin. Nature 321: 75–79.

Gether U, Andersen PH, Larsson OM and Schousboe A (2006).Neurotransmitter transporters: molecular function of impor-tant drug targets. Trends in Pharmacological Sciences 27:375–383.

Jiang Y, Lee A, Chen J, Ruta V, Cadene M, Chait BT andMacKinnon R (2003). X-ray structure of a voltage-dependentK+ channel. Nature 423: 33–41.

Kaczorowski GJ, McManus OB, Priest BT and Garcia ML (2008).Ion channels as drug targets: the next GPCRs. Journal GeneralPhysiology 131: 399–405.

Overington JP, Al-Lazikani B and Hopkins AL (2006). Howmany drug targets are there? Nature Reviews 5: 993–996.

Zheng CJ, Han LY, Yap CW, Ji ZL, Cao ZW and Chen YZ(2006). Therapeutic targets: progress of their exploration andinvestigation of their characteristics. Pharmacological Reviews58: 259–279.

2 Molecular Cloning of DrugTargets

2.1 Introduction to molecular cloning –from DNA to drug discovery 13

2.2 ‘Traditional’ pharmacology 14

2.3 The relevance of recombinant DNAtechnology 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 illustratedon orphan GPCRs 24

2.8 Summary 27

References 27

2.1 Introduction to molecular cloning –from DNA to drug discovery

Over four decades ago, the discovery and characterisationof molecular tools, in the form of DNA ligases (Zim-merman et al., 1967), restriction endonucleases (Linnand Arber, 1968; Smith and Wilcox, 1970; Danna andNathans, 1971) and reverse transcriptases (Baltimore,1970; Temin and Mizutani, 1970) provided the platformfor the emerging recombinant DNA technology (Cohenet al., 1972, 1973; Jackson et al., 1972), an array of applica-tions used to cut, join, amplify, modify and express DNAfragments. Molecular cloning, in this context, refers tothe process that introduces an isolated piece of DNA intoa vector (recombination) and generates multiple copies(clones) of it. (It should not be confused with cloning ofanimals or cells!)

Recombinant DNA technology quickly transformedthe field of pharmacology, as it overcame several tech-nical limitations faced with traditional pharmacology at

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.

that time: Supported by Sanger’s rapid DNA sequencingmethod (Sanger et al., 1977), it offered a range of novelapproaches for pharmacological studies, sufficient biolog-ical material – and data of previously unknown quantityand quality. DNA as a starting material was the new focalpoint that led to the molecular cloning of targets forendogenous ligands as well as therapeutic drugs, and thusfacilitated drug discovery (see 2.3 below). The technicalpossibilities seemed infinite.

Consequently, molecular cloning sparked a global raceamongst scientists for the identification of DNAs of majordrug targets, the molecular nature of which was oftenunknown back then! This work revealed a surprisingdiversity of drug targets and complexity of intracellularsignalling cascades. It was frequently accompanied bythe realisation that a single ‘known’ drug target indeedexisted in a multitude of subtypes, which traditionalinvestigations failed to distinguish. And it highlighted thenecessity to develop subtype-specific drugs. However, theinitial DNA cloning process could now be complemented

13

14 Chapter 2

by expression studies in host cells, where previously-identified or potential novel drug targets could be func-tionally and pharmacologically characterised in vitro. Italso marked the beginning of reverse pharmacology.

The chapter Molecular Cloning of Drug Targets startswith a brief overview of traditional pharmacology,followed by technical insights into the main methods,which nowadays are used routinely in molecularcloning. It concludes with a particular example, wheremolecular cloning enabled the identification of a naturalendogenous ligand for a receptor. (The interested readeris referred to Sambrook and Russel’s (2001) MolecularCloning: A Laboratory Manual, (3rd ed) CSH Press, fordetailed protocols).

2.2 ‘Traditional’ pharmacology

‘Traditional’ pharmacology refers here to the character-isation of ‘native’ drug targets, that is, proteins in theirnatural molecular environment, such as in cells, tissuesand organs. Typically employed techniques include:• electrophysiological recordings from cells; these permit

the study of ion fluxes across the plasma membrane (seeChapter 4), for example in response to the exposure toligands.

• radioligand binding to tissue extracts and isolated mem-branes; a radiolabelled molecule, whether the naturalligand or another compound, can be used to determinethe binding affinity of a drug for a particular target inits native state.

• receptor autoradiography; the incubation of thin tissuesections with a radiolabelled ligand is used to obtain sig-nals (autoradiographs) on X-ray films; their pattern andintensity reveals qualitative (location) and quantitativeinformation (amount) about a drug target in situ.

• ‘classical’ preparations; for example, isolated tissuestrips (such as muscle fibres) can be maintained in tis-sue baths for the purpose of measuring a drug-inducedresponse (e.g. contraction).

• enzyme assays; the activity of a particular enzyme isrecorded in response to different drug concentrations.Despite the negative connotation that the word ‘tra-

ditional’ may hold for some, this approach still offersan important means of investigation, frequently supple-menting the data from molecular cloning. However, priorto the advent of molecular cloning studies, ‘traditional’pharmacology on its own had several disadvantages. Forexample, cells, tissues, and organs may contain more thanone target for any given compound that lacks selectivity.

Thus, an observed response could be due to the activationof more than one protein, such as a receptor (a so-calledmixed response), and this might go undetected. It mightbe possible to detect the presence of multiple targets ifthe experimenter had access to one or more selectiveantagonists. However, a given antagonist might inhibita response that was due to the activity of multiple pro-teins, but blocking all of them. Famous examples, where‘traditional’ pharmacology has erroneously postulatedthe existence of too few subtypes exist among G-proteincoupled receptors (GPCRs; i.e. muscarinic acetylcholinereceptors and dopamine receptors) and ligand-gated ionchannels (LGICs; i.e. GABAA receptors; see section 2.5for details). Conversely, a single GPCR could give rise todifferent responses, depending on the cell type and theintracellular signalling pathways that it coupled to via dif-ferent guanine nucleotide binding proteins (G-proteins).

2.3 The relevance of recombinant DNAtechnology to pharmacology/drugdiscovery

The acquisition and successive use of sequence informa-tion plays a central role in recombinant DNA technology.Although the first – perhaps explorative – experimentsare often limited to the isolation, cloning and identi-fication of a relatively short DNA fragment, they can setthe foundation for a number of more elaborate studies,such as genetic engineering (see Chapter 10), with accessto physiological information of a different quality. Theinitially-obtained cloned nucleotide sequence, though,can already provide the deduced primary amino-acidsequence of a drug target, a map of recognition sitesfor restriction endonucleases, or some limited structuralinformation. For instance (see 2.5), many drug targets(e.g. LGICs or GPCRs) are membrane-bound, and theresults of molecular cloning can reveal the number of theirmembrane-spanning domains or indicate their topology(i.e. whether the amino- and carboxy-termini are intra-cellular or extracellular). This information is importantbecause we do not currently have the ability to purifythem in sufficient amounts for crystallographic studies.

By searching appropriate databases (e.g. http://blast.ncbi.nlm.nih.gov/Blast.cgi, http://www.ebi.ac.uk/embl)with a cloned sequence, it is also possible to findhomologous sequences, either in the same or a differentspecies. These sequence comparisons may indicate thefull length of the open-reading frame of a partially-clonedfragment or even help to identify protein families.