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*
ROBERT A. COPELAND, PhD, is Executive Vice President andChief Scientific Officer at Epizyme, Inc., a biopharmaceuticalcompany in Cambridge, Massachusetts. He is on the Editorial Boardof The Journal of Biological Chemistry and a memberof the Faculty of 1000. Dr. Copeland has contributed more than 175publications to the scientific literature and holds eightU.S.-issued patents. He has authored several books in proteinscience and enzymology, including Enzymes: A PracticalIntroduction to Structure, Mechanism, and Data Analysis, SecondEdition (Wiley).
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