Title Details

Donald Huddler, Widener University Delaware Law School, Wilmington, USA.

Edward R. Zartler is Chief Scientific Officer at Quantum Tessera Consulting, LLC, USA.

List of Contributors xiii

1 Introduction 1
Donald Huddler

References 3

2 Thermodynamics in Drug Discovery 7
Ronan O’Brien, Natalia Markova, and Geoffrey A. Holdgate

2.1 Introduction 7

2.2 Methods for Measuring Thermodynamics of Biomolecular Interactions 8

2.2.1 Direct Method: Isothermal Titration Calorimetry 8

2.2.2 Indirect Methods: van’t Hoff Analysis 8

2.2.2.1 Enthalpy Measurement Using van’t Hoff Analysis 8

2.3 Thermodynamic‐Driven Lead Optimization 9

2.3.1 The Thermodynamic Rules of Thumb 9

2.3.2 Enthalpy–Entropy Compensation 10

2.3.3 Enthalpy–Entropy Transduction 13

2.3.4 The Role of Water 14

2.4 Enthalpy as a Probe for Binding 15

2.4.1 Thermodynamics in Fragment‐Based Drug Design (FBDD) 15

2.4.2 Experimental Considerations and Limitations When Working with Fragments 16

2.4.3 Enthalpic Screening 17

2.5 Enthalpy as a Tool for Studying Complex Interactions 17

2.5.1 Identifying and Handling Complexity 17

2.6 Current and Future Prospects for Thermodynamics in Decision‐Making Processes 24

References 25

3 Tailoring Hit Identification and Qualification Methods for Targeting Protein–Protein Interactions 29
Björn Walse, Andrew P. Turnbull, and Susan M. Boyd

3.1 Introduction 29

3.2 Structural Characteristics of PPI Interfaces 29

3.3 Screening Library Properties 31

3.3.1 Standard/Targeted Libraries/DOS 31

3.3.2 Fragment Libraries 33

3.3.3 Macrocyclic and Constrained Peptides 33

3.3.4 DNA‐Encoded Libraries 34

3.4 Hit‐Finding Strategies 34

3.4.1 Small‐Molecule Approaches 36

3.4.2 Peptide‐Based Approaches 38

3.4.3 In Silico Approaches 39

3.5 Druggability Assessment 39

3.5.1 Small Molecule: Ligand‐Based Approaches 41

3.5.2 Small Molecule: Protein Structure‐Based Approaches 41

3.6 Allosteric Inhibition of PPIs 42

3.7 Stabilization of PPIs 43

3.8 Case Studies 43

3.8.1 Primary Peptide Epitopes 43

3.8.1.1 Bromodomains 44

3.8.2 Secondary Structure Epitopes 46

3.8.2.1 Bcl‐2 46

3.8.2.2 p53/MDM2 47

3.8.3 Tertiary Structure Epitopes 47

3.8.3.1 CD80–CD28 48

3.8.3.2 IL‐17A 48

3.9 Summary 49

References 50

4 Hydrogen–Deuterium Exchange Mass Spectrometry in Drug Discovery - Theory, Practice and Future 61
Thorleif Lavold, Roman Zubarev, and Juan Astorga‐Wells

4.1 General Principles 61

4.2 Parameters Affecting Deuterium Incorporation 63

4.2.1 Primary Sequence 63

4.2.2 Intramolecular Hydrogen Bonding 63

4.2.3 Solvent Accessibility 63

4.2.4 pH Value 63

4.3 Utilization of HDX MS 64

4.3.1 Binding Site and Structural Changes Characterization upon Ligand Binding 64

4.3.1.1 Protein Stability - Biosimilar Characterization 64

4.4 Practical Aspects of HDX MS 65

4.4.1 Labeling 66

4.4.1.1 Deuterium Oxide and Protein Concentration 66

4.4.1.2 Ligand/Protein Ratio 66

4.4.1.3 Incubation–Labeling Time 66

4.4.1.4 Careful Preparation of the Control Sample 66

4.4.2 Sample Analysis 66

4.4.3 Data Analysis 67

4.5 Advantages of HDX MS 67

4.6 Perspectives and Future Application of HDX MS 68

References 69

5 Microscale Thermophoresis in Drug Discovery 73
Tanja Bartoschik, Melanie Maschberger, Alessandra Feoli, Timon André, Philipp Baaske, Stefan Duhr, and Dennis Breitsprecher

5.1 Microscale Thermophoresis 73

5.1.1 Theoretical Background 74

5.1.2 Added Values for Small‐Molecule Interaction Studies 76

5.1.2.1 Size‐Change Independent Binding Signals 76

5.1.2.2 Difficult Targets and Assay Conditions 78

5.1.2.3 Detection of Aggregation and Other Secondary Effects 80

5.1.2.4 Quantification of Thermodynamic Parameters by MST 80

5.2 MST‐Based Lead Discovery 82

5.2.1 Single‐Point Screening 82

5.2.2 Secondary Affinity‐Based Fragment Screening by MST 85

5.2.3 Hit Identification and Affinity Determination of Small‐Molecule Binders to p38 Alpha Kinase 87

References 87

6 SPR Screening: Applying the New Generation of SPR Hardware 93
Kartik Narayan and Steven S. Carroll

6.1 Platforms for Screening 93

6.2 SensiQ Pioneer as a “OneStep” Solution for Hit Identification 95

6.3 Deprioritization of False Positives Arising from Compound Aggregation 99

6.4 Concluding Remarks 103

References 104

7 Weak Affinity Chromatography (WAC) 107
Sten Ohlson and Minh‐Dao Duong‐Thi

7.1 Introduction 107

7.2 Theory of WAC 109

7.3 Virtual WAC 110

7.4 Equipment and Procedure 111

7.5 Validation of WAC 113

7.6 Applications 114

7.6.1 Inhibitors for Cholera Toxin 115

7.6.2 Drug/Hormone: Protein Binding 115

7.6.3 Analysis of Stereoisomers 119

7.6.4 Carbohydrate Analysis with Antibodies and Lectins 120

7.6.5 Fragment Screening 121

7.6.6 Membrane Proteins 122

7.7 Conclusions and Future Perspectives 124

Acknowledgments 125

References 125

8 1D NMR Methods for Hit Identification 131
Mary J. Harner, Guille Metzler, Caroline A. Fanslau, Luciano Mueller, and William J. Metzler

8.1 Introduction 131

8.2 NMR Methods for Quality Control 131

8.2.1 Compound DMSO Stock Concentration Determination 132

8.2.2 Compound Solubility Measurements in Aqueous Buffer 134

8.2.3 Compound Structural Integrity 136

8.2.4 Protein Reagent Characterization 136

8.3 NMR Binding Assays 136

8.3.1 Saturation Transfer Difference Assay 138

8.3.2 T2 Relaxation Assay 140

8.3.3 WaterLOGSY Assay 141

8.3.4 19F Displacement Assay 142

8.4 Multiplexing 143

8.5 Specificity 144

8.6 Automation 146

8.7 Practical Considerations for NMR Binding Assays 146

8.7.1 Compound Libraries 146

8.7.2 Tube Selection and Filling 147

8.7.3 Buffers 148

8.7.4 Targets 149

8.7.5 Experiment Selection 150

8.8 Conclusions 151

References 151

9 Protein‐Based NMR Methods Applied to Drug Discovery 153
Alessio Bortoluzzi and Alessio Ciulli

9.1 Introduction 153

9.2 Chemical Shift Perturbation 154

9.2.1 Using Chemical Shift Perturbation to Study a Binding Event Between a Protein and a Ligand 154

9.2.2 Tackling the High Molecular Weight Limit by Reducing Transverse Relaxation and by Selective Labeling Patterns 156

9.2.3 CSP as Tool for Screening Campaigns 157

9.2.4 Structure–Activity Relationship by NMR 160

9.3 Methods for Obtaining Structural Information on Protein–Ligand Complex 160

9.3.1 SOS‐NMR 161

9.3.2 NOE‐Matching 162

9.3.3 Paramagnetic NMR Spectroscopy 162

9.4 Recent and Innovative Examples of Protein‐Observed NMR Techniques Applied Drug Discovery 163

9.4.1 An NMR‐Based Conformational Assay to Aid the Drug Discovery Process 163

9.4.2 In‐Cell NMR Techniques Applied to Drug Discovery 165

9.4.3 Time‐Resolved NMR Spectroscopy as a Tool for Studying Inhibitors of Posttranslational Modification Enzymes 166

9.4.4 Protein‐Observed 19F NMR Spectroscopy 168

9.5 Conclusions and Future Perspectives 170

References 170

10 Applications of Ligand and Protein‐Observed NMR in Ligand Discovery 175
Isabelle Krimm

10.1 Introduction 175

10.2 Ligand‐Observed NMR Experiments Based on the Overhauser Effect 176

10.2.1 Transferred NOE, ILOE, and INPHARMA Experiments 176

10.2.1.1 Principle of the Transferred 2D ¹H‐¹H NOESY Experiment 176

10.2.1.2 Fragment‐Based Screening Using 2D Tr‐NOESY Experiment 178

10.2.1.3 Elucidation of the Active Conformation of the Ligand Using 2D 1H‐1H NOESY Experiment 178

10.2.1.4 Design of Protein Inhibitors Using Interligand NOEs 178

10.2.1.5 Identification of the Ligand Binding Site and Binding Mode Using INPHARMA 178

10.2.1.6 Design of Protein Inhibitors Using INPHARMA with Protein–Peptide Complexes 179

10.2.1.7 Experimental Conditions of the 2D 1H‐1H NOESY Experiment 179

10.2.2 Saturation Transfer Difference Experiment 180

10.2.2.1 Principle of the STD Experiment 180

10.2.2.2 Detection of Interactions and Library Screening by STD 180

10.2.2.3 Epitope Mapping by STD 181

10.2.2.4 Affinity Measurement by STD 181

10.2.2.5 Quantitative STD Using CORCEMA 183

10.2.2.6 Experimental Conditions 183

10.2.3 WaterLOGSY Experiment 184

10.2.3.1 Principle of the WaterLOGSY Experiment 184

10.2.3.2 Screening and Affinity Measurement by WaterLOGSY 184

10.2.3.3 Epitope Mapping and Water Accessibility in Protein–Ligand Complexes by WaterLOGSY 184

10.2.3.4 Experimental Conditions 185

10.3 Protein‐Observed NMR Experiments: Chemical Shift Perturbations 185

10.3.1 Principle 185

10.3.2 Affinity Measurement Using CSPs 186

10.3.3 Localization of Binding Sites Using CSPs 186

10.3.3.1 Chemical Shift Mapping 186

10.3.3.2 J‐Surface Modeling 187

10.3.4 Comparison of CSPs from Analogous Ligands 187

10.3.5 Back‐Calculation of Ligand‐Induced CSPs for Ligand Docking 187

10.3.5.1 CSP‐Based Post‐Docking Filter 189

10.3.5.2 CSP‐Guided Docking 189

10.4 Conclusion 189

Acknowledgments 191

References 191

11 Using Biophysical Methods to Optimize Compound Residence Time 197
Geoffrey A. Holdgate, Philip Rawlins, Michal Bista, and Christopher J. Stubbs

11.1 Introduction 197

11.2 Biophysical Methods for Measuring Ligand Binding Kinetics 197

11.3 Measuring Structure–Kinetic Relationships: Some Example Case Studies 200

11.4 Effects of Conformational Dynamics on Binding Kinetics 201

11.5 Kinetic Selectivity 204

11.6 Mechanism of Binding and Kinetics 207

11.7 Optimizing Residence Time 207

11.8 Role of BK in Improving Efficacy 209

11.9 Effect of Pharmacokinetics and Pharmacodynamics 210

11.10 Summary 212

References 213

12 Applying Biophysical and Biochemical Methods to the Discovery of Allosteric Modulators of the AAA ATPase p97 217
Stacie L. Bulfer and Michelle R. Arkin

12.1 p97 and Proteostasis Regulation 217

12.2 Structure and Dynamics of p97 218

12.3 Drug Discovery Efforts against p97 222

12.4 Uncompetitive Inhibitors of p97 Discovered by High‐Throughput Screening 223

12.4.1 Biochemical MOA Studies 223

12.4.2 Surface Plasmon Resonance 225

12.4.3 Nuclear Magnetic Resonance 226

12.4.4 Cryo‐EM Defines the Binding Site for an Uncompetitive Inhibitor of p97 228

12.4.5 Effect of Inhibitors on p⁹⁷ PPI and MSP1 Disease Mutations 231

12.5 Fragment‐ Based Ligand Screening 231

12.5.1 Targeting the ND1 Domains 232

12.5.2 Targeting the N‐Domain 233

12.6 Conclusions 234

References 234

13 Driving Drug Discovery with Biophysical Information: Application to Staphylococcus aureus Dihydrofolate Reductase (DHFR) 241
Parag Sahasrabudhe, Veerabahu Shanmugasundaram, Mark Flanagan, Kris A. Borzilleri, Holly Heaslet, Anil Rane, Alex McColl, Tim Subashi, George Karam, Ron Sarver, Melissa Harris, Boris A.Chrunyk, Chakrapani Subramanyam, Thomas V. Magee, Kelly Fahnoe, Brian Lacey, Henry Putz, J. Richard Miller, Jaehyun Cho, Arthur Palmer III, and Jane M. Withka

13.1 Introduction 241

13.2 Results and Discussion 245

13.2.1 Protein Dynamics of SA WT and S1 Mutant DHFR in Apo and Bound States 245

13.2.2 Protein Backbone ¹⁵N, ¹³C, and ¹H NMR Resonance Assignments 246

13.2.3 Protein Residues Show Severe Line Broadening due to Conformational Exchange 246

13.2.4 R2 Relaxation Dispersion NMR Experiments 248

13.2.5 Kinetic Profiling of DHFR Inhibitors 251

13.2.6 Characterization of SA WT and S1 Mutant DHFR–TMP Interactions in Solution 253

13.2.7 Prospective Biophysics Library Design 254

13.3 Conclusion 258

References 259

14 Assembly of Fragment Screening Libraries: Property and Diversity Analysis 263
Bradley C. Doak, Craig J. Morton, Jamie S. Simpson, and Martin J. Scanlon

14.1 Introduction 263

14.2 Physicochemical Properties of Fragments 265

14.3 Molecular Diversity and Its Assessment 268

14.4 Experimental Evaluation of Fragments 274

14.5 Assembling Libraries for Screening 275

14.6 Concluding Remarks 279

References 280

Index 285