Foreword xiii
Preface xv
Acknowledgments xix
Epigraph xxi
1 The History of Molecular Medicine 1
1.1 Disease and Treatments in Antiquity 2
1.2 Natural Products as Medicines 2
1.3 Isolation of Active Ingredients and Chemical Synthesis 4
1.4 The Receptor Theory of Drug Action 9
1.5 Early Chemotherapy: Attempts to Mimic Nature 15
1.6 Linus Pauling Establishes Sickle Cell Anemia as a Molecular Disease 19
1.7 ACE Inhibitors: An Early Example of Targeted Therapeutics 21
1.8 Genetic Alterations in Disease 26
1.9 Structure-Based Drug Design 28
1.10 Recombinant DNA and Protein Engineering 31
1.11 Modern Drug Discovery 35
1.12 Summary 38
References 39
Further Reading 43
2 Molecular Targeted Therapeutic Modalities 45
2.1 Candidate Drug Profile 48
2.2 Biologic Therapeutics 49
2.2.1 Therapeutic Antibody Discovery 52
2.2.1.1 Target Selection and Antibody Generation 52
2.2.1.2 Hit-Finding and Hit-to-Lead 53
2.2.1.3 Lead Optimization 54
2.3 Small-Molecule Drugs 55
2.3.1 Target Druggability 55
2.3.2 Reversible Inhibitors/Antagonists 56
2.3.2.1 Orthosteric and Allosteric Binding Sites 61
2.3.2.2 Inhibitor Modality is Not an Indication of Inhibitor-Binding Site 63
2.3.3 Reversible Agonists and Activators 66
2.3.4 Reversible Inhibitors of Proteinâ"Protein Interactions 71
2.3.5 Small-Molecule Modulators of Protein Homeostasis 77
2.3.5.1 Small-Molecule Correctors and Potentiators of Misfolded Proteins 79
2.3.5.2 Inducers of Proteinâ"Protein Interactions 82
2.3.6 Irreversible Covalent Inactivators 89
2.3.6.1 In Vitro Analysis of Irreversible Covalent Inactivators 91
2.3.6.2 KRAS G12C Inactivators: A Case Study 95
2.4 Antibodyâ"Drug Conjugates (ADCs) 98
2.5 Summary 100
References 100
3 Target Selection 107
3.1 Disease Relevance 108
3.1.1 Genetic Alterations in Disease 110
3.1.1.1 Differences in Orthologue Sequences 110
3.1.1.2 Gene Amplification and Protein Overexpression 113
3.1.1.3 Target Mutations in Disease 114
3.1.1.4 Chromosomal Translocations in Disease 116
3.1.1.5 Synthetic Lethality 117
3.1.1.6 Disease Association Versus Disease Causality 121
3.2 Selective Essentiality and Therapeutic Index 122
3.2.1 RNA-Silencing Methods for Target Validation 123
3.2.1.1 RNAi 124
3.2.1.2 CRISPR-Cas 9 128
3.2.2 Genetic Versus Disease Vulnerability Target Validation Funnels 130
3.3 Actionability 132
3.3.1 Existing Structures and/or Ligands 132
3.3.2 Target Classes 133
3.3.3 Sequence Alignment and Homology Modeling 133
3.3.4 Other Computational Methods 135
3.3.5 Target Vitality and Competition with Endogenous Ligands 135
3.4 Unmet Medical Need 138
3.5 DHX9: An Illustrative Example of Target Validation 140
3.6 Summary 145
References 145
4 Hit-Finding and Hit-Validation 151
4.1 Properties of Small-Molecule Therapeutics 152
4.2 Hit-Validation Criteria 153
4.3 Screening Strategies 155
4.4 Protein Constructs for Screening 156
4.5 Compound Sources for Screening 158
4.5.1 Diversity Libraries 158
4.5.2 Fragment-Based Libraries 159
4.5.3 DNA-Encoded Libraries (DEL) 160
4.5.4 Target Class-Specific Libraries 162
4.5.5 Mechanism-Guided Design 163
4.5.6 Structure-Based Design and Computational Docking Methods 168
4.6 Assay Formats 169
4.6.1 Binding Assays 169
4.6.1.1 Competitive Displacement Assays 171
4.6.2 Assays of Target Function 174
4.6.3 Solution Conditions for Assays 177
4.7 Measures of Assay Performance 178
4.7.1 Calibration Curves 179
4.7.2 Time-Dependent Background 180
4.7.3 The Z â² Statistic 181
4.7.4 Definition of Hits 182
4.7.5 Post-Screening Prioritization of Reproducible Hits 183
4.8 Orthogonal Assays and Mode of Modulation 186
4.9 Confirmation of Chemical Purity and Identity 188
4.10 Hit Expansion 188
4.11 Hit-Validation Funnel 189
4.12 Summary 191
References 191
5 From Hits to Leads 197
5.1 The Project Matrix Team 198
5.2 Target Potency and Selectivity 200
5.2.1 Comparative Measures of Target Potency 203
5.2.2 Selectivity Profiling 205
5.2.3 Iterative Structural Biology 207
5.2.4 Solubility and Lipophilicity 210
5.3 Cellular Assays 212
5.3.1 Caco-2 Permeability and Active Efflux 213
5.3.2 Pharmacodynamic Assays 214
5.3.3 Phenotypic Assays 218
5.3.4 Intervening Biological Responses 221
5.3.5 Characteristics of Useful Cellular Assays 222
5.3.6 Positive and Negative Cell Types 224
5.4 ADME Properties 225
5.4.1 Plasma Stability 225
5.4.2 Plasma Protein Binding 227
5.4.3 Microsomal Stability: An Initial Indication of Hepatic Metabolism 229
5.4.4 Assessment of hERG Channel Inhibition 230
5.5 Chemical Tractability 232
5.5.1 Chemical Stability 233
5.5.2 Synthetic Route(s) 233
5.5.3 Patent Assessment 234
5.6 Lead Series 234
5.6.1 The Elements of Translational Chemical Biology 235
5.7 Case Studies 240
5.7.1 DOT1L Inhibitors for Mixed Lineage Leukemia 240
5.7.2 METTL3 Inhibitors for Acute Myeloid Leukemia (AML) 242
5.7.3 EZH2 Inhibitors for Treatment of Non-Hodgkinâs Lymphoma 244
5.8 Summary 247
References 247
6 Lead Optimization 251
6.1 Typical Criteria for Development Candidate Declaration 252
6.2 Continued Optimization of Target Potency, Selectivity, and Cellular Potency 252
6.2.1 Measuring Target Potency Under Tight-Binding Conditions 252
6.2.2 The Impact of Protein Conformational Dynamics on SAR 256
6.2.3 Extended Surveys of Target Selectivity 260
6.2.3.1 Secondary Pharmacology Screening 261
6.2.3.2 Assessing Affinity for Target Orthologues 262
6.2.4 Optimization of Potency and Durability of Cellular Pharmacology 266
6.2.5 Expanded Survey of Additional Cell Lines 269
6.3 In Vitro ADME Properties 271
6.3.1 CYP450-Mediated Metabolism 272
6.3.2 CYP450 Inhibition and Drugâ"Drug Interactions 274
6.3.2.1 Time-Dependent CYP450 Inhibition (TDI) 276
6.3.3 CYP450 Induction 277
6.3.4 Major Metabolite Identification 278
6.4 In Vivo ADME Properties 280
6.4.1 Oral Bioavailability 280
6.4.2 Pharmacokinetic Profile of Orally Bioavailable Drugs 280
6.4.3 Doseâ"Exposure Proportionality 282
6.4.4 Relating Plasma Proteinâ"Bindingâ"Adjusted Exposure to Cellular EC 50 or EC 90 282
6.4.5 Relating PK and PD Time Courses 283
6.4.6 Compartmental Modeling of Pharmacokinetics 285
6.5 In Vivo Drug Efficacy 289
6.5.1 Mouse Models of Efficacy in Cancer Drug Discovery 291
6.5.1.1 Subcutaneous (Ectopic) Xenografts 291
6.5.1.2 Relating Tumor Growth Inhibition to Systemic and Intratumoral Drug Exposure and PD 299
6.5.1.3 Case Studies 299
6.6 Allometric Scaling and Predicted Human Dose 309
6.7 Preliminary Safety Assessment in Two Species 312
6.8 Summary 313
References 314
Appendix 1 Kinetic and Equilibrium Assessment of Receptorâ"Ligand Interactions 319
A1.1 The Equilibrium Dissociation Constant, K d 320
A1.2 The Kinetics of Approach to Equilibrium 321
A1.3 Kinetics of Irreversible or Pseudo-Irreversible Processes 323
A1.4 Binding Measurements at Equilibrium 324
A1.4.1 Derivation of the Hillâ"Langmuir Isotherm 324
A1.4.2 Competition Among Ligands for a Common Target Receptor 326
A1.5 Extension to Other Pharmacological Situations 327
Appendix 2 Investigational New Drug (IND) Applications 331
A2.1 Types of IND Applications 332
A2.2 Key Content of an IND Application 333
A2.3 Review by FDA 334
A2.4 IND-Enabling Studies 335
A2.4.1 CMC Studies 335
A2.4.1.1 Drug Substance 335
A2.4.1.2 Drug Product 336
A2.4.1.3 Manufacturing Process 336
A2.4.1.4 Specifications and Quality Control 336
A2.4.1.5 Stability Testing 336
A2.4.1.6 Environmental Impact Considerations 337
A2.4.2 Pharmacology, Pharmacokinetics, and Toxicity Studies 337
A2.4.2.1 Pharmacology Studies 337
A2.4.2.2 PK Studies 337
A2.4.2.3 General Toxicity Studies 338
A2.4.2.4 Specialized Toxicity Studies 339
References 340
Index 341
