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Evaluation of Enzyme Inhibitors in Drug Discovery: A Guide for Medicinal Chemists and Pharmacologists, 2nd Edition

ISBN: 978-1-118-48813-3
572 pages
March 2013
Evaluation of Enzyme Inhibitors in Drug Discovery: A Guide for Medicinal Chemists and Pharmacologists, 2nd Edition (111848813X) cover image

Offers essential guidance for discovering and optimizing novel drug therapies

Using detailed examples, Evaluation of Enzyme Inhibitors in Drug Discovery equips researchers with the tools needed to apply the science of enzymology and biochemistry to the discovery, optimization, and preclinical development of drugs that work by inhibiting specific enzyme targets. Readers will applaud this book for its clear and practical presentations, including its expert advice on best practices to follow and pitfalls to avoid.

This Second Edition brings the book thoroughly up to date with the latest research findings and practices. Updates explore additional forms of enzyme inhibition and special treatments for enzymes that act on macromolecular substrates. Readers will also find new discussions detailing the development and application of the concept of drug-target residence time.

Evaluation of Enzyme Inhibitors in Drug Discovery begins by explaining why enzymes are such important drug targets and then examines enzyme reaction mechanisms. The book covers:

  • Reversible modes of inhibitor interactions with enzymes
  • Assay considerations for compound library screening
  • Lead optimization and structure-activity relationships for reversible inhibitors
  • Slow binding and tight binding inhibitors
  • Drug-target residence time
  • Irreversible enzyme inactivators

The book ends with a new chapter exploring the application of quantitative biochemical principles to the pharmacologic evaluation of drug candidates during lead optimization and preclinical development.

The Second Edition of Evaluation of Enzyme Inhibitors in Drug Discovery continues to offer a treatment of enzymology applied to drug discovery that is quantitative and mathematically rigorous. At the same time, the clear and simple presentations demystify the complex science of enzymology, making the book accessible to many fields— from pharmacology to medicinal chemistry to biophysics to clinical medicine.

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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

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ROBERT A. COPELAND, PhD, is Executive Vice President and Chief Scientific Officer at Epizyme, Inc., a biopharmaceutical company in Cambridge, Massachusetts. He is on the Editorial Board of The Journal of Biological Chemistry and a member of the Faculty of 1000. Dr. Copeland has contributed more than 175 publications to the scientific literature and holds eight U.S.-issued patents. He has authored several books in protein science and enzymology, including Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis, Second Edition (Wiley).

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