Contributors XV
Preface XXI
A Personal Foreword XXIII
Part I The Concept of Fragment-based Drug Discovery 1
1 The Role of Fragment-based Discovery in Lead Finding 3
Roderick E. Hubbard
1.1 Introduction 3
1.2 What is FBLD? 4
1.3 FBLD: Current Practice 5
1.3.1 Using Fragments: Conventional Targets 5
1.3.2 Using Fragments: Unconventional Targets 13
1.4 What do Fragments Bring to Lead Discovery? 14
1.5 How did We Get Here? 16
1.5.1 Evolution of the Early Ideas and History 16
1.5.2 What has Changed Since the First Book was Published in 2006? 16
1.6 Evolution of the Methods and Their Application Since 2005 19
1.6.1 Developments in Fragment Libraries 21
1.6.2 Fragment Hit Rate and Druggability 22
1.6.3 Developments in Fragment Screening 23
1.6.4 Ways of Evolving Fragments 23
1.6.5 Integrating Fragments Alongside Other Lead-Finding Strategies 23
1.6.6 Fragments Can be Selective 24
1.6.7 Fragment Binding Modes 25
1.6.8 Fragments, Chemical Space, and Novelty 27
1.7 Current Application and Impact 27
1.8 Future Opportunities 28
References 29
2 Selecting the Right Targets for Fragment-Based Drug Discovery 37
Thomas G. Davies, Harren Jhoti, Puja Pathuri, and Glyn Williams
2.1 Introduction 37
2.2 Properties of Targets and Binding Sites 39
2.3 Assessing Druggability 41
2.4 Properties of Ligands and Drugs 42
2.5 Case Studies 43
2.5.1 Case Study 1: Inhibitors of Apoptosis Proteins (IAPs) 44
2.5.2 Case Study 2: HCV-NS3 46
2.5.3 Case Study 3: PKM2 47
2.5.4 Case Study 4: Soluble Adenylate Cyclase 49
2.6 Conclusions 50
References 51
3 Enumeration of Chemical Fragment Space 57
Jean-Louis Reymond, Ricardo Visini, and Mahendra Awale
3.1 Introduction 57
3.2 The Enumeration of Chemical Space 58
3.2.1 Counting and Sampling Approaches 58
3.2.2 Enumeration of the Chemical Universe Database GDB 58
3.2.3 GDB Contents 59
3.3 Using and Understanding GDB 61
3.3.1 Drug Discovery 61
3.3.2 The MQN System 62
3.3.3 Other Fingerprints 63
3.4 Fragments from GDB 65
3.4.1 Fragment Replacement 65
3.4.2 Shape Diversity of GDB Fragments 66
3.4.3 Aromatic Fragments from GDB 68
3.5 Conclusions and Outlook 68
Acknowledgment 69
References 69
4 Ligand Efficiency Metrics and their Use in Fragment Optimizations 75
Gyorgy G. Ferenczy and Gyorgy M. Keserû
4.1 Introduction 75
4.2 Ligand Efficiency 75
4.3 Binding Thermodynamics and Efficiency Indices 78
4.4 Enthalpic Efficiency Indices 81
4.5 Lipophilic Efficiency Indices 83
4.6 Application of Efficiency Indices in Fragment-Based Drug Discovery Programs 88
4.7 Conclusions 94
References 95
Part II Methods and Approaches for Fragment-based Drug Discovery 99
5 Strategies for Fragment Library Design 101
Justin Bower, Angelo Pugliese, and Martin Drysdale
5.1 Introduction 101
5.2 Aims 102
5.3 Progress 102
5.3.1 BDDP Fragment Library Design: Maximizing Diversity 103
5.3.2 Assessing Three-Dimensionality 103
5.3.3 3DFrag Consortium 104
5.3.4 Commercial Fragment Space Analysis 105
5.3.5 BDDP Fragment Library Design 108
5.3.6 Fragment Complexity 111
5.3.6.1 Diversity-Oriented Synthesis-Derived Fragment-Like Molecules 113
5.4 Future Plans 114
5.5 Summary 116
5.6 Key Achievements 116
References 116
6 The Synthesis of Biophysical Methods In Support of Robust Fragment-Based Lead Discovery 119
Ben J. Davis and Anthony M. Giannetti
6.1 Introduction 119
6.2 Fragment-Based Lead Discovery on a Difficult Kinase 121
6.3 Application of Orthogonal Biophysical Methods to Identify and Overcome an Unusual Ligand: Protein Interaction 127
6.4 Direct Comparison of Orthogonal Screening Methods Against a Well-Characterized Protein System 131
6.5 Conclusions 135
References 136
7 Differential Scanning Fluorimetry as Part of a Biophysical Screening Cascade 139
Duncan E. Scott, Christina Spry, and Chris Abell
7.1 Introduction 139
7.2 Theory 140
7.2.1 Equilbria are Temperature Dependent 140
7.2.2 Thermodynamics of Protein Unfolding 142
7.2.3 Exact Mathematical Solutions to Ligand-Induced Thermal Shifts 143
7.2.4 Ligand Binding and Protein Unfolding Thermodynamics Contribute to the Magnitude of Thermal Shifts 145
7.2.5 Ligand Concentration and the Magnitude of Thermal Shifts 147
7.2.6 Models of Protein Unfolding Equilibria and Ligand Binding 148
7.2.7 Negative Thermal Shifts and General Confusions 150
7.2.8 Lessons Learnt from Theoretical Analysis of DSF 151
7.3 Practical Considerations for Applying DSF in Fragment-Based Approaches 152
7.4 Application of DSF to Fragment-Based Drug Discovery 154
7.4.1 DSF as a Primary Enrichment Technique 154
7.4.2 DSF Compared with Other Hit Identification Techniques 159
7.4.3 Pursuing Destabilizing Fragment Hits 166
7.4.4 Lessons Learnt from Literature Examples of DSF in Fragment-Based Drug Discovery 168
7.5 Concluding Remarks 169
Acknowledgments 169
References 170
8 Emerging Technologies for Fragment Screening 173
Sten Ohlson and Minh-Dao Duong-Thi
8.1 Introduction 173
8.2 Emerging Technologies 175
8.2.1 Weak Affinity Chromatography 175
8.2.1.1 Introduction 175
8.2.1.2 Theory 177
8.2.1.3 Fragment Screening 179
8.2.2 Mass Spectrometry 185
8.2.2.1 Introduction 185
8.2.2.2 Theory 186
8.2.2.3 Applications 186
8.2.3 Microscale Thermophoresis 187
8.2.3.1 Introduction 187
8.2.3.2 Theory 189
8.2.3.3 Applications 189
8.3 Conclusions 189
Acknowledgments 191
References 191
9 Computational Methods to Support Fragment-based Drug Discovery 197
Laurie E. Grove, Sandor Vajda, and Dima Kozakov
9.1 Computational Aspects of FBDD 197
9.2 Detection of Ligand Binding Sites and Binding Hot Spots 198
9.2.1 Geometry-based Methods 199
9.2.2 Energy-based Methods 201
9.2.3 Evolutionary and Structure-based Methods 202
9.2.4 Combination Methods 202
9.3 Assessment of Druggability 203
9.4 Generation of Fragment Libraries 205
9.4.1 Known Drugs 206
9.4.2 Natural Compounds 207
9.4.3 Novel Scaffolds 208
9.5 Docking Fragments and Scoring 209
9.5.1 Challenges of Fragment Docking 209
9.5.2 Examples of Fragment Docking 210
9.6 Expansion of Fragments 212
9.7 Outlook 214
References 214
10 Making FBDD Work in Academia 223
Stacie L. Bulfer, Frantz Jean-Francois, and Michelle R. Arkin
10.1 Introduction 223
10.2 How Academic and Industry Drug Discovery Efforts Differ 225
10.3 The Making of a Good Academic FBDD Project 226
10.4 FBDD Techniques Currently Used in Academia 228
10.4.1 Nuclear Magnetic Resonance 229
10.4.2 X-Ray Crystallography 230
10.4.3 Surface Plasmon Resonance/Biolayer Interferometry 231
10.4.4 Differential Scanning Fluorimetry 232
10.4.5 Isothermal Titration Calorimetry 232
10.4.6 Virtual Screening 232
10.4.7 Mass Spectrometry 233
10.4.7.1 Native MS 233
10.4.7.2 Site-Directed Disulfide Trapping (Tethering) 234
10.4.8 High-Concentration Bioassays 234
10.5 Project Structures for Doing FBDD in Academia 235
10.5.1 Targeting p97: A Chemical Biology Consortium Project 235
10.5.2 Targeting Caspase-6: An Academic–Industry Partnership 236
10.6 Conclusions and Perspectives 239
References 240
11 Site-Directed Fragment Discovery for Allostery 247
T. Justin Rettenmaier, Sean A. Hudson, and James A. Wells
11.1 Introduction 247
11.2 Caspases 249
11.2.1 Tethered Allosteric Inhibitors of Executioner Caspases-3 and -7 249
11.2.2 Tethering Inflammatory Caspase-1 250
11.2.3 Tethered Allosteric Inhibitors of Caspase-5 251
11.2.4 General Allosteric Regulation at the Caspase Dimer Interface 252
11.2.5 Using Disulfide Fragments as “Chemi-Locks” to Generate Conformation-Specific Antibodies 253
11.3 Tethering K-Ras(G12C) 254
11.4 The Master Transcriptional Coactivator CREB Binding Protein 256
11.4.1 Tethering to Find Stabilizers of the KIX Domain of CBP 256
11.4.2 Dissecting the Allosteric Coupling between Binding Sites on KIX 257
11.4.3 Rapid Identification of pKID-Competitive Fragments for KIX 258
11.5 Tethering Against the PIF Pocket of Phosphoinositide-Dependent Kinase 1 (PDK1) 259
11.6 Tethering Against GPCRs: Complement 5A Receptor 261
11.7 Conclusions and Future Directions 263
References 264
12 Fragment Screening in Complex Systems 267
Miles Congreve and John A. Christopher
12.1 Introduction 267
12.2 Fragment Screening and Detection of Fragment Hits 268
12.2.1 Fragment Screening Using NMR Techniques 270
12.2.2 Fragment Screening Using Surface Plasmon Resonance 271
12.2.3 Fragment Screening Using Capillary Electrophoresis 272
12.2.4 Fragment Screening Using Radioligand and Fluorescence-Based Binding Assays 273
12.2.5 Ion Channel Fragment Screening 275
12.3 Validating Fragment Hits 276
12.4 Fragment to Hit 279
12.4.1 Fragment Evolution 280
12.4.2 Fragment Linking 281
12.5 Fragment to Lead Approaches 281
12.5.1 Fragment Evolution 282
12.5.2 Fragment Linking 284
12.6 Perspective and Conclusions 285
Acknowledgments 287
References 287
13 Protein-Templated Fragment Ligation Methods: Emerging Technologies in Fragment-Based Drug Discovery 293
Mike Jaegle, Eric Nawrotzky, Ee Lin Wong, Christoph Arkona, and Jorg Rademann
13.1 Introduction: Challenges and Visions in Fragment-Based Drug Discovery 293
13.2 Target-Guided Fragment Ligation: Concepts and Definitions 294
13.3 Reversible Fragment Ligation 295
13.3.1 Dynamic Reversible Fragment Ligation Strategies 295
13.3.2 Chemical Reactions Used in Dynamic Fragment Ligations 296
13.3.3 Detection Strategies in Dynamic Fragment Ligations 299
13.3.4 Applications of Dynamic Fragment Ligations in FBDD 301
13.4 Irreversible Fragment Ligation 311
13.4.1 Irreversible Fragment Ligation Strategies: Pros and Cons 311
13.4.2 Detection in Irreversible Fragment Ligation 311
13.4.3 Applications of Irreversible Fragment Ligations in FBDD 313
13.5 Fragment Ligations Involving Covalent Reactions with Proteins 316
13.6 Conclusions and Future Outlook: How Far did We Get and What will be Possible? 319
References 320
Part III Successes from Fragment-based Drug Discovery 327
14 BACE Inhibitors 329
Daniel F. Wyss, Jared N. Cumming, Corey O. Strickland, and Andrew W. Stamford
14.1 Introduction 329
14.2 FBDD Efforts on BACE1 333
14.2.1 Fragment Hit Identification, Validation, and Expansion 333
14.2.2 Fragment Optimization 333
14.2.3 From a Key Pharmacophore to Clinical Candidates 340
14.3 Conclusions 346
References 346
15 Epigenetics and Fragment-Based Drug Discovery 355
Aman Iqbal and Peter J. Brown
15.1 Introduction 355
15.2 Epigenetic Families and Drug Targets 357
15.3 Epigenetics Drug Discovery Approaches and Challenges 358
15.4 FBDD Case Studies 359
15.4.1 BRD4 (Bromodomain) 360
15.4.2 EP300 (Bromodomain) 363
15.4.3 ATAD2 (Bromodomain) 364
15.4.4 BAZ2B (Bromodomain) 364
15.4.5 SIRT2 (Histone Deacetylase) 365
15.4.6 Next-Generation Epigenetic Targets: The “Royal Family” and Histone Demethylases 366
15.5 Conclusions 367
Abbreviations 368
References 368
16 Discovery of Inhibitors of Protein–Protein Interactions Using Fragment-Based Methods 371
Feng Wang and Stephen W. Fesik
16.1 Introduction 371
16.2 Fragment-Based Strategies for Targeting PPIs 372
16.2.1 Fragment Library Construction 372
16.2.2 NMR-Based Fragment Screening Methods 373
16.2.3 Structure Determination of Complexes 374
16.2.4 Structure-Guided Hit-to-Lead Optimization 375
16.3 Recent Examples from Our Laboratory 376
16.3.1 Discovery of RPA Inhibitors 377
16.3.2 Discovery of Potent Mcl-1 Inhibitors 378
16.3.3 Discovery of Small Molecules that Bind to K-Ras 379
16.4 Summary and Conclusions 382
Acknowledgments 383
References 384
17 Fragment-Based Discovery of Inhibitors of Lactate Dehydrogenase A 391
Alexander L. Breeze, Richard A. Ward, and Jon Winter
17.1 Aerobic Glycolysis, Lactate Metabolism, and Cancer 391
17.2 Lactate Dehydrogenase as a Cancer Target 392
17.3 “Ligandability” Characteristics of the Cofactor and Substrate Binding Sites in LDHA 394
17.4 Previously Reported LDH Inhibitors 395
17.5 Fragment-Based Approach to LDHA Inhibition at AstraZeneca 398
17.5.1 High-Throughput Screening Against LDHA 398
17.5.2 Rationale and Strategy for Exploration of Fragment-Based Approaches 399
17.5.3 Development of Our Biophysical and Structural Biology Platform 400
17.5.4 Elaboration of Adenine Pocket Fragments 404
17.5.5 Screening for Fragments Binding in the Substrate and Nicotinamide Pockets 405
17.5.6 Reaching out Across the Void 407
17.5.7 Fragment Linking and Optimization 408
17.6 Fragment-Based LDHA Inhibitors from Other Groups 410
17.6.1 Nottingham 410
17.6.2 Ariad 413
17.7 Conclusions and Future Perspectives 417
References 419
18 FBDD Applications to Kinase Drug Hunting 425
Gordon Saxty
18.1 Introduction 425
18.2 Virtual Screening and X-ray for PI3K 426
18.3 High-Concentration Screening and X-ray for Rock1/2 427
18.4 Surface Plasmon Resonance for MAP4K4 428
18.5 Weak Affinity Chromatography for GAK 429
18.6 X-ray for CDK 4/6 430
18.7 High-Concentration Screening, Thermal Shift, and X-ray for CHK2 432
18.8 Virtual Screening and Computational Modeling for AMPK 433
18.9 High-Concentration Screening, NMR, and X-ray FBDD for PDK1 434
18.10 Tethering Mass Spectometry and X-ray for PDK1 435
18.11 NMR and X-ray Case Study for Abl (Allosteric) 436
18.12 Review of Current Kinase IND’s and Conclusions 437
References 442
19 An Integrated Approach for Fragment-Based Lead Discovery: Virtual, NMR, and High-Throughput Screening Combined with Structure-Guided Design. Application to the Aspartyl Protease Renin 447
Simon Rudisser, Eric Vangrevelinghe, and Jurgen Maibaum
19.1 Introduction 447
19.2 Renin as a Drug Target 449
19.3 The Catalytic Mechanism of Renin 451
19.4 Virtual Screening 452
19.5 Fragment-Based Lead Finding Applied to Renin and Other Aspartyl Proteases 455
19.6 Renin Fragment Library Design 464
19.7 Fragment Screening by NMR T1ρ Ligand Observation 469
19.8 X-Ray Crystallography 473
19.9 Renin Fragment Hit-to-Lead Evolution 475
19.10 Integration of Fragment Hits and HTS Hits 476
19.11 Conclusions 479
References 480
Index 487
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