Fragment-based Drug Discovery Lessons and Outlook Methods & Principles in Medicinal Chemistry Series

From its origins as a niche technique more than 15 years ago, fragment-based approaches have become a major tool for drug and ligand discovery, often yielding results where other methods have failed. Written by the pioneers in the field, this book provides a comprehensive overview of current methods and applications of fragment-based discovery, as well as an outlook on where the field is headed.

The first part discusses basic considerations of when to use fragment-based methods, how to select targets, and how to build libraries in the chemical fragment space. The second part describes established, novel and emerging methods for fragment screening, including empirical as well as computational approaches. Special cases of fragment-based screening, e. g. for complex target systems and for covalent inhibitors are also discussed. The third part presents several case studies from recent and on-going drug discovery projects for a variety of target classes, from kinases and phosphatases to targeting protein-protein interaction and epigenetic targets.

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
György G. Ferenczy and György 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 Jörg 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 Rüdisser, Eric Vangrevelinghe, and Jürgen 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