Applied Biophysics for Drug Discovery

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 xiii1 Introduction 1Donald HuddlerReferences 32 Thermodynamics in Drug Discovery 7Ronan O’Brien, Natalia Markova, and Geoffrey A. Holdgate2.1 Introduction 72.2 Methods for Measuring Thermodynamics of Biomolecular Interactions 82.2.1 Direct Method: Isothermal Titration Calorimetry 82.2.2 Indirect Methods: van’t Hoff Analysis 82.2.2.1 Enthalpy Measurement Using van’t Hoff Analysis 82.3 Thermodynamic‐Driven Lead Optimization 92.3.1 The Thermodynamic Rules of Thumb 92.3.2 Enthalpy–Entropy Compensation 102.3.3 Enthalpy–Entropy Transduction 132.3.4 The Role of Water 142.4 Enthalpy as a Probe for Binding 152.4.1 Thermodynamics in Fragment‐Based Drug Design (FBDD) 152.4.2 Experimental Considerations and Limitations When Working with Fragments 162.4.3 Enthalpic Screening 172.5 Enthalpy as a Tool for Studying Complex Interactions 172.5.1 Identifying and Handling Complexity 172.6 Current and Future Prospects for Thermodynamics in Decision‐Making Processes 24References 253 Tailoring Hit Identification and Qualification Methods for Targeting Protein–Protein Interactions 29Björn Walse, Andrew P. Turnbull, and Susan M. Boyd3.1 Introduction 293.2 Structural Characteristics of PPI Interfaces 293.3 Screening Library Properties 313.3.1 Standard/Targeted Libraries/DOS 313.3.2 Fragment Libraries 333.3.3 Macrocyclic and Constrained Peptides 333.3.4 DNA‐Encoded Libraries 343.4 Hit‐Finding Strategies 343.4.1 Small‐Molecule Approaches 363.4.2 Peptide‐Based Approaches 383.4.3 In Silico Approaches 393.5 Druggability Assessment 393.5.1 Small Molecule: Ligand‐Based Approaches 413.5.2 Small Molecule: Protein Structure‐Based Approaches 413.6 Allosteric Inhibition of PPIs 423.7 Stabilization of PPIs 433.8 Case Studies 433.8.1 Primary Peptide Epitopes 433.8.1.1 Bromodomains 443.8.2 Secondary Structure Epitopes 463.8.2.1 Bcl‐2 463.8.2.2 p53/MDM2 473.8.3 Tertiary Structure Epitopes 473.8.3.1 CD80–CD28 483.8.3.2 IL‐17A 483.9 Summary 49References 504 Hydrogen–Deuterium Exchange Mass Spectrometry in Drug Discovery - Theory, Practice and Future 61Thorleif Lavold, Roman Zubarev, and Juan Astorga‐Wells4.1 General Principles 614.2 Parameters Affecting Deuterium Incorporation 634.2.1 Primary Sequence 634.2.2 Intramolecular Hydrogen Bonding 634.2.3 Solvent Accessibility 634.2.4 pH Value 634.3 Utilization of HDX MS 644.3.1 Binding Site and Structural Changes Characterization upon Ligand Binding 644.3.1.1 Protein Stability - Biosimilar Characterization 644.4 Practical Aspects of HDX MS 654.4.1 Labeling 664.4.1.1 Deuterium Oxide and Protein Concentration 664.4.1.2 Ligand/Protein Ratio 664.4.1.3 Incubation–Labeling Time 664.4.1.4 Careful Preparation of the Control Sample 664.4.2 Sample Analysis 664.4.3 Data Analysis 674.5 Advantages of HDX MS 674.6 Perspectives and Future Application of HDX MS 68References 695 Microscale Thermophoresis in Drug Discovery 73Tanja Bartoschik, Melanie Maschberger, Alessandra Feoli, Timon André, Philipp Baaske, Stefan Duhr, and Dennis Breitsprecher5.1 Microscale Thermophoresis 735.1.1 Theoretical Background 745.1.2 Added Values for Small‐Molecule Interaction Studies 765.1.2.1 Size‐Change Independent Binding Signals 765.1.2.2 Difficult Targets and Assay Conditions 785.1.2.3 Detection of Aggregation and Other Secondary Effects 805.1.2.4 Quantification of Thermodynamic Parameters by MST 805.2 MST‐Based Lead Discovery 825.2.1 Single‐Point Screening 825.2.2 Secondary Affinity‐Based Fragment Screening by MST 855.2.3 Hit Identification and Affinity Determination of Small‐Molecule Binders to p38 Alpha Kinase 87References 876 SPR Screening: Applying the New Generation of SPR Hardware 93Kartik Narayan and Steven S. Carroll6.1 Platforms for Screening 936.2 SensiQ Pioneer as a “OneStep” Solution for Hit Identification 956.3 Deprioritization of False Positives Arising from Compound Aggregation 996.4 Concluding Remarks 103References 1047 Weak Affinity Chromatography (WAC) 107Sten Ohlson and Minh‐Dao Duong‐Thi7.1 Introduction 1077.2 Theory of WAC 1097.3 Virtual WAC 1107.4 Equipment and Procedure 1117.5 Validation of WAC 1137.6 Applications 1147.6.1 Inhibitors for Cholera Toxin 1157.6.2 Drug/Hormone: Protein Binding 1157.6.3 Analysis of Stereoisomers 1197.6.4 Carbohydrate Analysis with Antibodies and Lectins 1207.6.5 Fragment Screening 1217.6.6 Membrane Proteins 1227.7 Conclusions and Future Perspectives 124Acknowledgments 125References 1258 1D NMR Methods for Hit Identification 131Mary J. Harner, Guille Metzler, Caroline A. Fanslau, Luciano Mueller, and William J. Metzler8.1 Introduction 1318.2 NMR Methods for Quality Control 1318.2.1 Compound DMSO Stock Concentration Determination 1328.2.2 Compound Solubility Measurements in Aqueous Buffer 1348.2.3 Compound Structural Integrity 1368.2.4 Protein Reagent Characterization 1368.3 NMR Binding Assays 1368.3.1 Saturation Transfer Difference Assay 1388.3.2 T2 Relaxation Assay 1408.3.3 WaterLOGSY Assay 1418.3.4 19F Displacement Assay 1428.4 Multiplexing 1438.5 Specificity 1448.6 Automation 1468.7 Practical Considerations for NMR Binding Assays 1468.7.1 Compound Libraries 1468.7.2 Tube Selection and Filling 1478.7.3 Buffers 1488.7.4 Targets 1498.7.5 Experiment Selection 1508.8 Conclusions 151References 1519 Protein‐Based NMR Methods Applied to Drug Discovery 153Alessio Bortoluzzi and Alessio Ciulli9.1 Introduction 1539.2 Chemical Shift Perturbation 1549.2.1 Using Chemical Shift Perturbation to Study a Binding Event Between a Protein and a Ligand 1549.2.2 Tackling the High Molecular Weight Limit by Reducing Transverse Relaxation and by Selective Labeling Patterns 1569.2.3 CSP as Tool for Screening Campaigns 1579.2.4 Structure–Activity Relationship by NMR 1609.3 Methods for Obtaining Structural Information on Protein–Ligand Complex 1609.3.1 SOS‐NMR 1619.3.2 NOE‐Matching 1629.3.3 Paramagnetic NMR Spectroscopy 1629.4 Recent and Innovative Examples of Protein‐Observed NMR Techniques Applied Drug Discovery 1639.4.1 An NMR‐Based Conformational Assay to Aid the Drug Discovery Process 1639.4.2 In‐Cell NMR Techniques Applied to Drug Discovery 1659.4.3 Time‐Resolved NMR Spectroscopy as a Tool for Studying Inhibitors of Posttranslational Modification Enzymes 1669.4.4 Protein‐Observed 19F NMR Spectroscopy 1689.5 Conclusions and Future Perspectives 170References 17010 Applications of Ligand and Protein‐Observed NMR in Ligand Discovery 175Isabelle Krimm10.1 Introduction 17510.2 Ligand‐Observed NMR Experiments Based on the Overhauser Effect 17610.2.1 Transferred NOE, ILOE, and INPHARMA Experiments 17610.2.1.1 Principle of the Transferred 2D 1H‐1H NOESY Experiment 17610.2.1.2 Fragment‐Based Screening Using 2D Tr‐NOESY Experiment 17810.2.1.3 Elucidation of the Active Conformation of the Ligand Using 2D 1H‐1H NOESY Experiment 17810.2.1.4 Design of Protein Inhibitors Using Interligand NOEs 17810.2.1.5 Identification of the Ligand Binding Site and Binding Mode Using INPHARMA 17810.2.1.6 Design of Protein Inhibitors Using INPHARMA with Protein–Peptide Complexes 17910.2.1.7 Experimental Conditions of the 2D 1H‐1H NOESY Experiment 17910.2.2 Saturation Transfer Difference Experiment 18010.2.2.1 Principle of the STD Experiment 18010.2.2.2 Detection of Interactions and Library Screening by STD 18010.2.2.3 Epitope Mapping by STD 18110.2.2.4 Affinity Measurement by STD 18110.2.2.5 Quantitative STD Using CORCEMA 18310.2.2.6 Experimental Conditions 18310.2.3 WaterLOGSY Experiment 18410.2.3.1 Principle of the WaterLOGSY Experiment 18410.2.3.2 Screening and Affinity Measurement by WaterLOGSY 18410.2.3.3 Epitope Mapping and Water Accessibility in Protein–Ligand Complexes by WaterLOGSY 18410.2.3.4 Experimental Conditions 18510.3 Protein‐Observed NMR Experiments: Chemical Shift Perturbations 18510.3.1 Principle 18510.3.2 Affinity Measurement Using CSPs 18610.3.3 Localization of Binding Sites Using CSPs 18610.3.3.1 Chemical Shift Mapping 18610.3.3.2 J‐Surface Modeling 18710.3.4 Comparison of CSPs from Analogous Ligands 18710.3.5 Back‐Calculation of Ligand‐Induced CSPs for Ligand Docking 18710.3.5.1 CSP‐Based Post‐Docking Filter 18910.3.5.2 CSP‐Guided Docking 18910.4 Conclusion 189Acknowledgments 191References 19111 Using Biophysical Methods to Optimize Compound Residence Time 197Geoffrey A. Holdgate, Philip Rawlins, Michal Bista, and Christopher J. Stubbs11.1 Introduction 19711.2 Biophysical Methods for Measuring Ligand Binding Kinetics 19711.3 Measuring Structure–Kinetic Relationships: Some Example Case Studies 20011.4 Effects of Conformational Dynamics on Binding Kinetics 20111.5 Kinetic Selectivity 20411.6 Mechanism of Binding and Kinetics 20711.7 Optimizing Residence Time 20711.8 Role of BK in Improving Efficacy 20911.9 Effect of Pharmacokinetics and Pharmacodynamics 21011.10 Summary 212References 21312 Applying Biophysical and Biochemical Methods to the Discovery of Allosteric Modulators of the AAA ATPase p97 217Stacie L. Bulfer and Michelle R. Arkin12.1 p97 and Proteostasis Regulation 21712.2 Structure and Dynamics of p97 21812.3 Drug Discovery Efforts against p97 22212.4 Uncompetitive Inhibitors of p97 Discovered by High‐Throughput Screening 22312.4.1 Biochemical MOA Studies 22312.4.2 Surface Plasmon Resonance 22512.4.3 Nuclear Magnetic Resonance 22612.4.4 Cryo‐EM Defines the Binding Site for an Uncompetitive Inhibitor of p97 22812.4.5 Effect of Inhibitors on p97 PPI and MSP1 Disease Mutations 23112.5 Fragment‐ Based Ligand Screening 23112.5.1 Targeting the ND1 Domains 23212.5.2 Targeting the N‐Domain 23312.6 Conclusions 234References 23413 Driving Drug Discovery with Biophysical Information: Application to Staphylococcus aureus Dihydrofolate Reductase (DHFR) 241Parag 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. Withka13.1 Introduction 24113.2 Results and Discussion 24513.2.1 Protein Dynamics of SA WT and S1 Mutant DHFR in Apo and Bound States 24513.2.2 Protein Backbone 15N, 13C, and 1H NMR Resonance Assignments 24613.2.3 Protein Residues Show Severe Line Broadening due to Conformational Exchange 24613.2.4 R2 Relaxation Dispersion NMR Experiments 24813.2.5 Kinetic Profiling of DHFR Inhibitors 25113.2.6 Characterization of SA WT and S1 Mutant DHFR–TMP Interactions in Solution 25313.2.7 Prospective Biophysics Library Design 25413.3 Conclusion 258References 25914 Assembly of Fragment Screening Libraries: Property and Diversity Analysis 263Bradley C. Doak, Craig J. Morton, Jamie S. Simpson, and Martin J. Scanlon14.1 Introduction 26314.2 Physicochemical Properties of Fragments 26514.3 Molecular Diversity and Its Assessment 26814.4 Experimental Evaluation of Fragments 27414.5 Assembling Libraries for Screening 27514.6 Concluding Remarks 279References 280Index 285