Evaluation of Enzyme Inhibitors in Drug Discovery – A Guide for Medicinal Chemists and Pharmacologists, Second Edition

<p>FOREWORD TO SECOND EDITION BY CHRISTOPHER T. WALSH xvii<br /> <br /> PREFACE TO SECOND EDITION xix<br /> <br /> FOREWORD TO FIRST EDITION BY PAUL S. ANDERSON xxiii<br /> <br /> PREFACE TO FIRST EDITION xxv<br /> <br /> ACKNOWLEDGMENTS FROM FIRST EDITION xxix<br /> <br /> 1. WHY ENZYMES AS DRUG TARGETS? 1<br /> <br /> Key Learning Points 1<br /> <br /> 1.1 Enzymes Are Essential for Life 2<br /> <br /> 1.2 Enzyme Structure and Catalysis 6<br /> <br /> 1.3 Permutations of Enzyme Structure During Catalysis 12<br /> <br /> 1.4 Extension to Other Target Classes 17<br /> <br /> 1.5 Other Reasons for Studying Enzymes 18<br /> <br /> 1.6 Summary 21<br /> <br /> References 22<br /> <br /> 2. ENZYME REACTION MECHANISMS 25<br /> <br /> Key Learning Points 25<br /> <br /> 2.1 Initial Binding of Substrate 25<br /> <br /> 2.2 Noncovalent Forces in Reversible Ligand Binding to Enzymes 28<br /> <br /> 2.2.1 Electrostatic Forces 28<br /> <br /> 2.2.2 Hydrogen Bonds 28<br /> <br /> 2.2.3 Hydrophobic Forces 29<br /> <br /> 2.2.4 Van der Waals Forces 30<br /> <br /> 2.3 Transformations of the Bound Substrate 30<br /> <br /> 2.3.1 Strategies for Transition State Stabilization 32<br /> <br /> 2.3.2 Enzyme Active Sites Are Most Complementary to the Transition State Structure 36<br /> <br /> 2.4 Steady State Analysis of Enzyme Kinetics 39<br /> <br /> 2.4.1 Factors Affecting the Steady State Kinetic Constants 43<br /> <br /> 2.5 Typical Values of Steady State Kinetic Parameters 46<br /> <br /> 2.6 Graphical Determination of kcat and KM 47<br /> <br /> 2.7 Reactions Involving Multiple Substrates 49<br /> <br /> 2.7.1 Bisubstrate Reaction Mechanisms 49<br /> <br /> 2.8 Summary 54<br /> <br /> References 54<br /> <br /> 3. REVERSIBLE MODES OF INHIBITOR INTERACTIONS WITH ENZYMES 57<br /> <br /> Key Learning Points 57<br /> <br /> 3.1 Enzyme Inhibitor Binding Equilibria 58<br /> <br /> 3.2 Competitive Inhibition 59<br /> <br /> 3.3 Noncompetitive Inhibition 68<br /> <br /> 3.3.1 Mutual Exclusivity Studies 76<br /> <br /> 3.3.2 Noncompetitive Inhibition by Active Site–Directed Inhibitors 80<br /> <br /> 3.4 Uncompetitive Inhibition 82<br /> <br /> 3.5 Inhibition Modality in Bisubstrate Reactions 86<br /> <br /> 3.6 Value of Knowing Inhibitor Modality 88<br /> <br /> 3.6.1 Quantitative Comparisons of Inhibitor Affinity 88<br /> <br /> 3.6.2 Relating Ki to Binding Energy 89<br /> <br /> 3.6.3 Defi ning Target Selectivity by Ki Values 92<br /> <br /> 3.6.4 Potential Advantages and Disadvantages of Different Inhibition Modalities in Vivo 92<br /> <br /> 3.6.5 Knowing Inhibition Modality Is Important for Structure–Based Lead Optimization 95<br /> <br /> 3.7 Enzyme Reactions on Macromolecular Substrates 96<br /> <br /> 3.7.1 Challenges in Inhibiting Protein–Protein Interactions 97<br /> <br /> 3.7.2 Hot Spots in Protein Protein Interactions 99<br /> <br /> 3.7.3 Factors Affecting Protein Protein Interactions 104<br /> <br /> 3.7.4 Separation of Binding and Catalytic Recognition Elements 107<br /> <br /> 3.7.5 Noncompetitive Inhibition by Active Site–Binding Molecules for Exosite Utilizing Enzymes 109<br /> <br /> 3.7.6 Processive and Distributive Mechanisms of Catalysis 110<br /> <br /> 3.7.7 Effect of Substrate Conformation on Enzyme Kinetics 116<br /> <br /> 3.7.8 Inhibitor Binding to Substrates 116<br /> <br /> 3.8 Summary 118<br /> <br /> References 119<br /> <br /> 4. ASSAY CONSIDERATIONS FOR COMPOUND LIBRARY SCREENING 123<br /> <br /> Key Learning Points 123<br /> <br /> 4.1 Measures of Assay Performance 125<br /> <br /> 4.1.1 Calibration Curves 125<br /> <br /> 4.1.2 Total, Background, and Specific Signal 128<br /> <br /> 4.1.3 Defining Inhibition, Signal Robustness, and Hit Criteria 130<br /> <br /> 4.2 Measuring Initial Velocity 133<br /> <br /> 4.2.1 End–Point and Kinetic Readouts 135<br /> <br /> 4.2.2 Effect of Enzyme Concentration 137<br /> <br /> 4.2.3 Other Factors Affecting Initial Velocity 139<br /> <br /> 4.3 Balanced Assay Conditions 142<br /> <br /> 4.3.1 Balancing Conditions for Multisubstrate Reactions 145<br /> <br /> 4.4 Order of Reagent Addition 146<br /> <br /> 4.5 Use of Natural Substrates and Enzymes 148<br /> <br /> 4.6 Coupled Enzyme Assays 154<br /> <br /> 4.7 Hit Validation 156<br /> <br /> 4.7.1 Determination of Hit Reproducibility 156<br /> <br /> 4.7.2 Verification of Chemical Purity and Structure 158<br /> <br /> 4.7.3 Hit Verification in Orthogonal Assays 159<br /> <br /> 4.7.4 Chemical and Pharmacological Tractability 160<br /> <br /> 4.7.5 Promiscuous Inhibitors 162<br /> <br /> 4.7.6 Prioritization of Confirmed Hits 164<br /> <br /> 4.7.7 Hit Expansion 165<br /> <br /> 4.8 Summary 166<br /> <br /> References 166<br /> <br /> 5. LEAD OPTIMIZATION AND STRUCTURE ACTIVITY RELATIONSHIPS FOR REVERSIBLE INHIBITORS 169<br /> <br /> Key Learning Points 169<br /> <br /> 5.1 Concentration Response Plots and IC50 Determination 170<br /> <br /> 5.1.1 The Hill Coefficient 176<br /> <br /> 5.1.2 Graphing and Reporting Concentration Response Data 180<br /> <br /> 5.2 Testing for Reversibility 183<br /> <br /> 5.3 Determining Reversible Inhibition Modality and Dissociation Constant 188<br /> <br /> 5.4 Comparing Relative Affinity 190<br /> <br /> 5.4.1 Compound Selectivity 192<br /> <br /> 5.5 Associating Cellular Effects with Target Enzyme Inhibition 193<br /> <br /> 5.5.1 Cellular Phenotype Should Be Consistent with Genetic Knockout or Knockdown of the Target Enzyme<br /> 194<br /> <br /> 5.5.2 Cellular Activity Should Require a Certain Affinity for the Target Enzyme 194<br /> <br /> 5.5.3 Buildup of Substrate andor Diminution of Product for the Target Enzyme Should Be Observed in Cells<br /> 197<br /> <br /> 5.5.4 Cellular Phenotype Should Be Reversed by Cell–Permeable Product or Downstream Metabolites of the<br /> Target Enzyme Activity 198<br /> <br /> 5.5.5 Mutation of the Target Enzyme Should Lead to Resistance or Hypersensitivity to Inhibitors 199<br /> <br /> 5.6 Summary 200<br /> <br /> References 200<br /> <br /> 6. SLOW BINDING INHIBITORS 203<br /> <br /> Key Learning Points 203<br /> <br /> 6.1 Determining kobs: The Rate Constant for Onset of Inhibition 205<br /> <br /> 6.2 Mechanisms of Slow Binding Inhibition 207<br /> <br /> 6.3 Determination of Mechanism and Assessment of True Affinity 210<br /> <br /> 6.3.1 Potential Clincial Advantages of Slow Off–Rate Inhibitors 217<br /> <br /> 6.4 Determining Inhibition Modality for Slow Binding Inhibitors 217<br /> <br /> 6.5 SAR for Slow Binding Inhibitors 219<br /> <br /> 6.6 Some Examples of Pharmacologically Interesting Slow Binding Inhibitors 220<br /> <br /> 6.6.1 Examples of Scheme B: Inhibitors of Zinc Peptidases and Proteases 220<br /> <br /> 6.6.2 Example of Scheme C: Inhibition of Dihydrofolate Reductase by Methotrexate 226<br /> <br /> 6.6.3 Example of Scheme C: Inhibition of Calcineurin by FKBP–Inhibitor Complexes 229<br /> <br /> 6.6.4 Example of Scheme C When Ki Ki ∗ &lt;&lt; : Aspartyl Protease Inhibitors 231<br /> <br /> 6.6.5 Example of Scheme C When k6 Is Very Small: Selective COX2 Inhibitors 234<br /> <br /> 6.7 Summary 242<br /> <br /> References 243<br /> <br /> 7. TIGHT BINDING INHIBITION 245<br /> <br /> Key Learning Points 245<br /> <br /> 7.1 Effects of Tight Binding Inhibition on Concentration Response Data 246<br /> <br /> 7.2 The IC50 Value Depends on Ki app and [E]T&nbsp; 248<br /> <br /> 7.3 Morrison s Quadratic Equation for Fitting Concentration Response Data for Tight Binding Inhibitors 253<br /> <br /> 7.3.1 Optimizing Conditions for Ki app Determination Using Morrison s Equation 255<br /> <br /> 7.3.2 Limits on Ki app Determinations 256<br /> <br /> 7.3.3 Use of a Cubic Equation When Both Substrate and Inhibitor Are Tight Binding 257<br /> <br /> 7.4 Determining Modality for Tight Binding Enzyme Inhibitors 258<br /> <br /> 7.5 Tight Binding Inhibitors Often Display Slow Binding Behavior 261<br /> <br /> 7.6 Practical Approaches to Overcoming the Tight Binding Limit in Determining Ki 263<br /> <br /> 7.7 Enzyme–Reaction Intermediate Analogues as Examples of Tight Binding Inhibitors 266<br /> <br /> 7.7.1 Bisubstrate Analogues 271<br /> <br /> 7.7.2 Testing for Transition State Mimicry 272<br /> <br /> 7.8 Potential Clinical Advantages of Tight Binding Inhibitors 277<br /> <br /> 7.9 Determination of [E]T Using Tight Binding Inhibitors 279<br /> <br /> 7.10 Summary 282<br /> <br /> References 282<br /> <br /> 8. DRUG TARGET RESIDENCE TIME 287<br /> <br /> Key Learning Points 287<br /> <br /> 8.1 Open and Closed Systems in Biology 288<br /> <br /> 8.2 The Static View of Drug Target Interactions 292<br /> <br /> 8.3 Conformational Adaptation in Drug Target Interactions 294<br /> <br /> 8.3.1 Conformational Selection Model 294<br /> <br /> 8.3.2 Induced–Fit Model 296<br /> <br /> 8.3.3 Kinetic Distinction Between Conformational Selection and Induced–Fit Mechanisms 297<br /> <br /> 8.4 Impact of Residence Time on Natural Receptor Ligand Function 300<br /> <br /> 8.4.1 Immune Response 300<br /> <br /> 8.4.2 Control of Protease Activity by Natural Inhibitors 302<br /> <br /> 8.5 Impact of Drug Target Residence Time on Drug Action 304<br /> <br /> 8.5.1 Mathematical Defi nition of Residence Time for Different Mechanisms of Drug Target Interaction 304<br /> <br /> 8.5.2 Impact of Residence Time on Cellular Activity 305<br /> <br /> 8.5.3 Impact on Effi cacy and Duration in Vivo 309<br /> <br /> 8.5.4 Temporal Target Selectivity and Drug Safety 316<br /> <br /> 8.6 Experimental Measures of Drug Target Residence Time 318<br /> <br /> 8.6.1 Kinetic Analysis of Approach to Equilibrium 318<br /> <br /> 8.6.2 Jump–Dilution Experiments 319<br /> <br /> 8.6.3 Separation Methods 321<br /> <br /> 8.6.4 Spectroscopic Differentiation 322<br /> <br /> 8.6.5 Immobilized Binding Partner Methods 324<br /> <br /> 8.7 Drug Target Residence Time Structure Activity Relationships 325<br /> <br /> 8.7.1 Structural Changes Associated with Conformational Adaptation 326<br /> <br /> 8.7.2 Thermodynamics of Drug Target Complex Dissociation 328<br /> <br /> 8.7.3 A Retrograded Induced–Fit Model of Drug Target Complex Dissociation 332<br /> <br /> 8.8 Recent Applications of the Residence Time Concept 334<br /> <br /> 8.9 Limitations of Drug Target Residence Time 338<br /> <br /> 8.10 Summary 340<br /> <br /> References 341<br /> <br /> 9. IRREVERSIBLE ENZYME INACTIVATORS 345<br /> <br /> Key Learning Points 345<br /> <br /> 9.1 Kinetic Evaluation of Irreversible Enzyme Inactivators 346<br /> <br /> 9.2 Affinity Labels 350<br /> <br /> 9.2.1 Quiescent Affinity Labels 351<br /> <br /> 9.2.2 Potential Liabilities of Affinity Labels as Drugs 356<br /> <br /> 9.3 Mechanism–Based Inactivators 358<br /> <br /> 9.3.1 Distinguishing Features of Mechanism–Based Inactivation 360<br /> <br /> 9.3.2 Determination of the Partition Ratio 366<br /> <br /> 9.3.3 Potential Clinical Advantages of Mechanism–Based Inactivators 367<br /> <br /> 9.3.4 Examples of Mechanism–Based Inactivators as Drugs 368<br /> <br /> 9.4 Use of Affi nity Labels as Mechanistic Tools 375<br /> <br /> 9.5 Summary 380<br /> <br /> References 380<br /> <br /> 10. QUANTITATIVE BIOCHEMISTRY IN THE PHARMACOLOGICAL EVALUATION OF DRUGS 383<br /> <br /> Key Learning Points 383<br /> <br /> 10.1 In Vitro ADMET Properties 384<br /> <br /> 10.1.1 Exponential Decay Processes and the Definition of Half–Life 385<br /> <br /> 10.1.2 Caco–2 Cell Permeability as a Surrogate for Intestinal Absorption 387<br /> <br /> 10.1.3 Whole Blood or Plasma Stability 390<br /> <br /> 10.1.4 Plasma Protein Binding 392<br /> <br /> 10.1.5 Metabolism of Xenobiotics in the Liver 397<br /> <br /> 10.1.6 Hepatocyte, S9, and Microsome Stability 400<br /> <br /> 10.1.7 CYP450 Mediated Metabolism 403<br /> <br /> 10.1.8 Cytochrome P450 Inhibition 408<br /> <br /> 10.1.9 hERG Inhibition 416<br /> <br /> 10.2 In Vivo Pharmacokinetic Studies 426<br /> <br /> 10.2.1 General Considerations and Curve Fitting Parameters 426<br /> <br /> 10.2.2 Kinetic Models of Drug PK 432<br /> <br /> 10.2.3 Absorption and Bioavailability 444<br /> <br /> 10.2.4 Factors Affecting PK Parameters 445<br /> <br /> 10.2.5 Allometric Scaling of Drug Pharmacokinetics 451<br /> <br /> 10.3 Metabolite Identification 453<br /> <br /> 10.4 Measures of Target Occupancy 454<br /> <br /> 10.4.1 Radiometric Imaging 455<br /> <br /> 10.4.2 Ex Vivo Determination of Target Occupancy 457<br /> <br /> 10.4.3 Pharmacodynamic Measures of Target Engagement 459<br /> <br /> 10.5 Summary 465<br /> <br /> References 466<br /> <br /> APPENDIX 1 KINETICS OF BIOCHEMICAL REACTIONS 471<br /> <br /> A1.1 The Law of Mass Action and Reaction Order 471<br /> <br /> A1.2 First–Order Reaction Kinetics 475<br /> <br /> A1.3 Second–Order Reaction Kinetics 478<br /> <br /> A1.4 Pseudo First–Order Reaction Conditions 479<br /> <br /> A1.5 Approach to Equilibrium: An Example of the Kinetics of Reversible Reactions 480<br /> <br /> APPENDIX 2 DERIVATION OF THE ENZYME LIGAND BINDING ISOTHERM EQUATION 483<br /> <br /> APPENDIX 3 SERIAL DILUTION SCHEMES 487<br /> <br /> APPENDIX 4 RELATIONSHIP BETWEEN [I ]IC50 AND PERCENTAGE INHIBITION OF ENZYME ACTIVITY WHEN<br /> h = 1 491<br /> <br /> APPENDIX 5 PROPAGATION OF UNCERTAINTIES IN EXPERIMENTAL MEASUREMENTS 493<br /> <br /> A5.1 Uncertainty Propagation for Addition or Subtraction of Two Experimental Parameters 493<br /> <br /> A5.2 Uncertainty Propagation for Multiplication or Division of Two Experimental Parameters 494<br /> <br /> A5.3 Uncertainty Propagation for Multiplication or Division of an Experimental Parameter by A Constant 494<br /> <br /> A5.4 Uncertainty Propagation for an Experimental Parameter Raised by an Exponent 494<br /> <br /> A5.5 Uncertainty Propagation for a General Function of Experimental Parameters 494<br /> <br /> Reference 495<br /> <br /> APPENDIX 6 USEFUL PHYSICAL CONSTANTS AT DIFFERENT TEMPERATURES 497<br /> <br /> APPENDIX 7 COMMON RADIOACTIVE ISOTOPES USED IN STUDIES OF ENZYMES 499<br /> <br /> APPENDIX 8 COMMON PREFIXES FOR UNITS IN BIOCHEMISTRY 501<br /> <br /> APPENDIX 9 SOME AROMATIC RING SYSTEMS COMMONLY FOUND IN DRUGS 503<br /> <br /> APPENDIX 10 RESIDUAL PLOTS 505<br /> <br /> INDEX 509</p>