Evaluation of Enzyme Inhibitors in Drug Discovery: A Guide for Medicinal Chemists and Pharmacologists

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

• ISBN: 978-1-118-48813-3
• Editorial: Wiley–Blackwell
• Encuadernacion: Cartoné
• Páginas: 572
• Fecha Publicación: 16/04/2013
• Nº Volúmenes: 1
• Idioma: Inglés