Contents

Chapter 1 Ion Channel Drug Discovery: a Historical Perspective 1Brian Cox

1.1 Introduction 11.2 History of Ion Channel Drug Discovery 21.3 Conclusion 13Acknowledgement 13References 13

Chapter 2 High-Throughput Screening 16Alexander Bocker, Sabine Schaertl and Stephen D. Hess

2.1 Introduction 162.2 Goal(S) of HTS Campaigns 182.3 HTS before and after the Cloning of Human Ion

Channels 182.4 A Brief History of Ion Channel HTS Methodologies 20

2.4.1 Atomic Absorption Spectrophotometry 202.4.2 Isothermal Calorimetry 212.4.3 Fluorescence-based Assays 212.4.4 Automated Electrophysiology 22

2.5 Strategies for Selection of Confirmation andSelectivity Assays 222.5.1 Ion Channel HTS – Experience 22

2.6 Library Composition 332.7 Conclusions and Future Perspective 37Acknowledgement 38References 38

RSC Drug Discovery Series No. 39Ion Channel Drug DiscoveryEdited by Brian Cox and Martin Goslingr The Royal Society of Chemistry 2015Published by the Royal Society of Chemistry, www.rsc.org

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Chapter 3 Automated Electrophysiology in Ion Channel DrugDiscovery 42John Dunlop

3.1 Ion Channels as Targets for TherapeuticIntervention 42

3.2 Non-electrophysiological Approaches to IonChannel Screening 43

3.3 Patch Clamp Automation (R)Evolution 443.4 Impact of Automated Ion Channel Screening

Technology 453.5 Drug Discovery Applications 513.6 Concluding Remarks 53References 54

Chapter 4 Structural Understanding of Ion Channels in AtomicDetail 56Phillip J. Stansfeld

4.1 Introduction 564.2 Methods for Resolving Membrane Protein Structures 574.3 Potassium Channels and their Relatives 58

4.3.1 Conserved Pore Architecture 594.3.2 Kir Channels 624.3.3 K2P Channels 644.3.4 Kv Channels 654.3.5 Voltage-gated Sodium (Nav) Channels 66

4.4 Ligand Gated Ion Channels 674.4.1 Glutamate Receptors 674.4.2 Cys-loop Receptors 694.4.3 Ionotropic Purinergic Receptors (P2X) and

their Relatives 714.5 Ion Channels with the Transporter Scaffold 72

4.5.1 Chloride Channels (CLC) 724.5.2 Cystic Fibrosis Transmembrane Regulator

(CFTR) 734.5.3 ATP-Sensitive K1 Channel (KATP) 73

4.6 Conclusions 74References 76

Chapter 5 Voltage-gated Sodium Channels: Structure, Function, andMolecular Pharmacology 83William A. Catterall

5.1 Sodium Channel Function 835.2 Sodium Channel Subunit Structure 84

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5.3 Sodium Channel Genes 865.4 Expression and Localization of Sodium Channel

Subtypes 885.5 Molecular Basis of Sodium Channel Function 885.6 Sodium Channel Pharmacology 895.7 High-resolution Structure of Sodium Channels 90

5.7.1 Structure of NavAb in a Membrane-likeEnvironment 91

5.7.2 The NavAb Voltage Sensor is Activated 915.7.3 Architecture of the Pore of NavAb 925.7.4 The NavAb Activation Gate is Closed 945.7.5 Ion Conductance and Selectivity in NavAb 945.7.6 Fenestrations Provide Hydrophobic Access

to the Pore of NavAb 955.8 Structural Basis for Voltage-dependent Gating 96

5.8.1 The Rosetta Sliding Helix Model of VoltageSensing 96

5.8.2 Slow Inactivation 975.8.3 The Fast Inactivation Gate 99

5.9 Structural Basis for Sodium Channel Pharmacology 995.9.1 Receptor Sites for Pore Blockers 995.9.2 Drug Access in the Resting State 995.9.3 Conformational Change in the Local

Anesthetic Receptor Site during SlowInactivation 100

5.10 Looking Ahead 100References 101

Chapter 6 AMPA Receptor Positive Allosteric Modulators – a CaseHistory 105Simon E Ward

6.1 Introduction 1056.1.1 Ionotropic Glutamate Receptors 1056.1.2 AMPA Receptors (AMPARs) 1076.1.3 AMPAR Positive Allosteric Modulators 109

6.2 Clinical Landscape 1096.3 Discovery Landscape and Choice of Screening

Methodology 1116.3.1 Benzamides 1116.3.2 Benzothiadiazines 1136.3.3 Phenethyl Sulfonamides 113

6.4 Selection of Screening Platforms and Cascades 1146.5 Integration of X-ray Crystallography 1186.6 Ion Channel Lead Optimisation Case History 119

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6.7 Challenges in Lead Optimisation and Selection ofClinical Discovery Candidate 128

6.8 Future Perspectives 130References 131

Chapter 7 The Discovery of Novel Inhaled ENaC Blockers for theTreatment of Cystic Fibrosis Lung Disease 135Catherine Howsham and Henry Danahay

7.1 Introduction 1357.2 ENaC: Structure, Function and Regulation 1367.3 ENaC: Evidence for a Role in Respiratory Function

and Disease 1397.4 Inhaled ENaC Blockers for the Treatment of CF Lung

Disease 1417.5 Inhaled CAP Inhibitors for the Treatment of CF Lung

Disease 1477.6 Mucosal Hydration and the Future for ENaC-based

Therapies 149References 149

Chapter 8 The Therapeutic Potential of Small-molecule Modulatorsof the Cystic Fibrosis Transmembrane ConductanceRegulator (CFTR) Cl� Channel 156Jia Liu, Gerta Cami-Kobeci, Yiting Wang, Pissared Khuituan,Zhiwei Cai, Hongyu Li, Stephen M. Husbands andDavid N. Sheppard

8.1 Introduction 1568.2 The Pathophysiology of CFTR 157

8.2.1 The Physiology of CFTR 1578.2.2 Cystic Fibrosis 1598.2.3 Secretory Diarrhoea 1618.2.4 Autosomal Dominant Polycystic Kidney Disease 161

8.3 CFTR Structure and Function 1628.4 Restoration of CFTR Function 165

8.4.1 CFTR Correctors 1658.4.2 CFTR Potentiators 1678.4.3 CFTR Corrector-potentiators 1708.4.4 Towards the Therapeutic Application of

CFTR Correctors and Potentiators 1718.5 Inhibition of CFTR Function 172

8.5.1 CFTR Inhibitors and Secretory Diarrhoea 1738.5.2 CFTR Inhibitors and ADPKD 1768.5.3 Towards the Therapeutic Application of

CFTR Inhibitors 177

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8.6 Conculsion 178Acknowledgements 179References 179

Chapter 9 TRPV1 Antagonism: From Research to Clinic 186Mark S. Nash, J. Martin Verkuyl and Gurdip Bhalay

9.1 Introduction 1869.2 Preclinical Perspectives on TRPV1 187

9.2.1 Expression in Disease Models 1879.2.2 Knockout Phenotype 1889.2.3 TRPV1 Antagonism 191

9.3 Clinical Perspectives on TRPV1 1939.3.1 TRPV1 Expression and Human Disease 1949.3.2 Genetic Associations 196

9.4 The Search for TRPV1 Antagonists 1979.4.1 TRPV1 Agonists as Analgesics 1989.4.2 TRPV1 Antagonists as Analgesics 1989.4.3 First Generation TRPV1 Antagonists 2009.4.4 Conclusions on Clinical Experience with

TRPV1 Antagonists 2089.5 Second Generation TRPV1 Antagonists and

Regulation of Body Temperature 2109.5.1 Abbott Pharmaceuticals 2119.5.2 Astellas Pharma 2129.5.3 Gruenenthal 2129.5.4 PharmEste 2129.5.5 Recent Developments 213

9.6 Other TRP’s Involved in Pain 2149.6.1 The TRPV Family 214

9.7 Other TRP Channels 2179.7.1 TRPM3 2179.7.2 TRPM8 2189.7.3 TRPA1 218

9.8 Conclusion 2209.9 Important Questions Remaining in TRPV1 Research 221References 221

Chapter 10 Open Access to the KCNQ Channel: Retigabine andSecond Generation M-current Openers 238Johannes Krupp, Anthony M. Rush, Britt-Marie Swahn andMartin Main

10.1 Introduction 23810.2 KCNQ (Kv7) Potassium Channel Family 239

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10.3 KCNQ Channels Underlie M-current 24010.4 Retigabine: Discovery, Molecular Target and

Mechanism of Action 24110.5 Retigabine in Preclinical Models 24210.6 Retigabine in Clinical Trials 24310.7 Molecular Pharmacology of Retigabine: Options

for Improvement 24510.8 Chemistry and Preclinical Drug Discovery of

KCNQ Openers 24510.9 Mapping the Site of Molecular Interaction

of Retigabine and other KCNQ ChannelOpeners 248

10.10 Future KCNQ Channel Openers: Lead GenerationApproaches 250

References 252

Chapter 11 The Therapeutic Potential of hERG1 K1 Channels forTreating Cancer and Cardiac Arrhythmias 258John Mitcheson and Annarosa Arcangeli

11.1 Introduction 25811.1.1 hERG Channel Family Members and

Alternative Isoforms 25911.1.2 hERG1 Channel Gating 26411.1.3 hERG1 Channel Structure 265

11.2 Physiological and Pathophysiological Rolesof hERG1 26711.2.1 Repolarisation of the Cardiac Action

Potential 26711.2.2 Neuronal and Smooth Muscle Cell

Excitability 26811.3 hERG1 as an Antitarget 268

11.3.1 Drug Induced VentricularArrhythmias 269

11.3.2 Medicinal Chemistry Strategies forAvoiding hERG Channel Block 272

11.4 hERG1 as a Target 27411.4.1 hERG1 Channels as Potential Drug Targets

in Oncology 27411.4.2 Therapeutic Potential of hERG1 Activators

as Antiarrhythmic Compounds 28311.5 Conclusions 284References 284

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Chapter 12 Does Nature do Ion Channel Drug Discovery Betterthan Us? 297Richard J. Lewis, Irina Vetter, Fernanda C. Cardoso,Marco Inserra and Glenn King

12.1 Voltage-gated Calcium Channels 29912.2 Inhibition of Cav2.2 by Small Molecules and

Natural Products 30212.2.1 Natural Cav2.2 Inhibitors from Cone Snail

Venoms 30212.2.2 Small Molecule Cav2.2 Inhibitors 303

12.3 Voltage-gated Sodium Channels 30412.4 Inhibition of Nav by Small Molecules and Natural

Products 30612.4.1 Natural Nav1.7 Inhibitors from Venoms 30612.4.2 Small Molecule Nav1.7 Inhibitors 307

12.5 Voltage-gated K1 Channels 30712.6 Peptide Modulators of Kv1.3 30812.7 Small Molecule Modulators of Kv1.3 30912.8 Outlook 309References 310

Chapter 13 Antibodies as Ion Channel Modulators 320Wilson Edwards and Alan D. Wickenden

13.1 Introduction 32013.2 Modifying Ion Channel Function with Antibodies 323

13.2.1 Direct Modulation of Channel Function byAntibodies 324

13.2.2 Other Mechanisms of Antibody-mediatedChannel Modulation 326

13.3 Current Status 32713.4 Challenges 329

13.4.1 Discovery of Functional Antibodies 32913.4.2 Biodistribution 332

13.5 Fusion Proteins 33413.6 Conclusion 335References 336

Chapter 14 Ion Channel Drug Discovery: Future Perspectives 341Martin Gosling

14.1 Introduction 34114.2 Channels, Channels, Channels: How Many

and What Do They Do? 342

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14.3 Ion Channel Modulators: More, Better. . .Different? 34714.4 Concluding Remarks 352Acknowledgements 352References 352

Subject Index 355

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