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).

Foreword to Second Edition xvii
Christopher T. Walsh

Preface to Second Edition xix

Foreword to First Edition xxiii
Paul S. Anderson

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 Defining 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 Affinity 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 Kiapp 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 Kiapp Determination Using Morrison's Equation 255

7.3.2 Limits on Kiapp 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 Definition 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 Efficacy 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