Table of Contents
Forward by Paul S. Anderson
Preface
Acknowledgements
Chapter 1. Why Enzymes as Drug Targets?
1.1 Enzymes are Essential for Life
1.2 Enzyme Structure and Catalysis
1.3 Permutations of Enzyme Structure During Catalysis
1.4 Other Reasons for Studying Enzymes
1.5 Summary
References
Chapter 2. Enzyme Reaction Mechanisms.
2.1 Initial Binding of Substrate
2.2 Noncovalent Forces in Reversible Ligand Binding to Enzymes
2.2.1 Electrostatic Forces
2.2.2 Hydrogen Bonds
2.2.3 Hydrophobic Forces
2.2.4 van der Waals Forces
2.3 Transformations of the Bound Substrate
2.3.1 Strategies for Transition State Stabilization
2.3.1.1 Approximation
2.3.1.2 Covalent Catalysis
2.3.1.3 Acid/Base Catalysis
2.3.1.4 Conformational Distortion
2.3.2 Enzyme Active Sites are Most Complementary to the Transition State Structure
2.4 Steady State Analysis of Enzyme Kinetics
2.4.1 Factors Affecting the Steady State Kinetic Constants
2.5 Graphical Determination of kcat and KM
2.6 Reactions Involving Multiple Substrates
2.6.1 Bisubstrate Reaction Mechanisms
2.7 Summary
References
Chapter 3. Reversible Modes of Inhibitor Interactions with Enzymes
3.1 Enzyme-Inhibitor Binding Equilibria
3.2 Competitive Inhibition
3.3 Noncompetititive Inhibition
3.3.1 Mutual Exclusivity Studies
3.4 Uncompetitive Inhibition
3.5 Inhibition Modality in Bisubstrate Reactions
3.6 The Value of Knowing Inhibitor Modality
3.6.1 Quantitative Comparisons of Inhibitor Affinity
3.6.2 Relating Ki to Binding Energy
3.6.3 Defining Target Selectivity by Ki Values
3.6.4 Potential Advantages and Disadvantages of Different Inhibition Modalities In Vivo
3.6.5 Knowing Inhibition Modality is Important for Structure-Based Lead Optimization
3.7 Summary
References
Chapter 4. Assay Considerations for Compound Library Screening
4.1 Defining Inhibition, Signal Robustness and Hit Criteria
4.2 Measuring Initial Velocity
4.2.1 End Point and Kinetic Readouts
4.2.2 Effects of Enzyme Concentration
4.2.3 Other Factors Affecting Initial Velocity
4.3 Balanced Assay Conditions
4.3.1 Balanced Conditions for Multisubstrate Reactions
4.4 Order of Reagent Addition
4.5 Use of Natural Substrates and Enzymes
4.6 Coupled Enzyme Assays
4.7 Hit Validation and Progression
4.8 Summary
References
Chapter 5. Lead Optimization and Structrue-Activity Relationships for Reversible Inhibitors
5.1 Concentration-Response Plots and IC50 Determination
5.1.1 The Hill Coefficient
5.1.2 Graphing and Reporting Concentration-Response Data
5.2 Testing for Reversibility
5.3 Determining Reversible Inhibition Modality and Dissociation Constant
5.4 Comparing Relative Affinity
5.4.1 Compound Selectivity
5.5 Associating Cellular Effects with Target Enzyme Inhibition
5.5.1 The Cellular Phenotype Should be Consistent with Genetic Knockout or Knockdown of the Target Enzyme
5.5.2 Cellular Activity Should Require a Certain Affinity for the Target Enzyme
5.5.3 A Build Up of Substrate and/or Diminution of Product for the Target Enzyme Should be Observed in Cells
5.5.4 The Cellular Phenotype Should be Reversed by Cell-Permeable Product or Downstream Metabolites of the Target Enzyme Activity
5.5.5 Mutation of the Target Enzyme Should Lead to Resistance or Hypersensitivity to Inhibitors
5.6 Summary
References
Chapter 6. Slow Binding Inhibitors
6.1 Determining kobs: The Rate Constant for Onset of Inhibition
6.2 Mechanisms of Slow Binding Inhibition
6.3 Determination of Mechanism and Assessment of True Affinity
6.3.1 Potential Clinical Advantages of Slow Off-Rate Inhibitors
6.4 Determining Inhibition Modality for Slow Binding Inhibitors
6.5 SAR for Slow Binding Inhibitors
6.6 Some Examples of Pharmacologically Interesting Slow Binding Inhibitors
6.6.1 Examples of Scheme B: Inhibitors of Zinc Peptidases and Proteases
6.6.2 Example of Scheme C: Inhibition of Dihydrofolate Reductase by Methotrexate
6.6.3 Example of Scheme C: Inhibition of Calcinurine by FKBP-Inhibitor Complexes
6.6.4 Example of Scheme C When Ki* << Ki: Aspartyl Protease Inhibitors
6.6.5 Example of Scheme C When k6 is Very Small: Selective COX2 Inhibitors
6.7 Summary
References
Chapter 7. Tight Binding Inhibitors
7.1 Effects of Tight Binding Inhibitors on Concentration-Response Data
7.2 The IC50 Value Depends on Kiapp and [E]T
7.3 Morrison's Quadratic Equation for Fitting Concentration-Response Data for Tight Binding Inhibitors
7.3.1 Optimizing Conditions for Kiapp Determination Using Morrison's Equation
7.3.2 Limits on Kiapp Determination
7.3.3 Use of a Cubic Equation when Both Substrate and Inhibitor are Tight Binding
7.4 Determining Modality for Tight Binding Enzyme Inhibitors
7.5 Tight Binding Inhibitors Often Display Slow Binding Behavior
7.6 Practical Approaches to Overcoming the Tight Binding Limit in Determining Ki
7.7 Reaction Intermediate Analogs as Examples of Tight Binding Inhibitors
7.7.1 Bisubstrate Analogs
7.7.2 Testing for Transition State Mimicry
7.8 Potential Clinical Advantages of Tight Binding Inhibitors
7.9 Determination of [E]T Using Tight Binding Inhibitors
7.10 Summary
References
Chapter 8. Irreversible Enzyme Inactivators
8.1 Kinetic Evaluation of Irreversible Enzyme Inactivators
8.2 Affinity Labels
8.2.1 Quiescent Affinity Labels
8.2.2 Potential Liabilities of Affinity Labels as Drugs
8.3 Mechanism-Based Inactivators
8.3.1 Distinguishing Features of Mechanism-Based Inactivation
8.3.1.1 Inhibition Must be Time Dependent
8.3.1.2 Inactivation Kinetics Must be Saturable
8.3.1.3 Substrate Must Protect Against Inactivation
8.3.1.4 Inactivation Must be Irreversible
8.3.1.5 The Stoichiometry of Inactivation Must be < 1:1 with Enzyme
8.3.1.6 Inactivation Must Require Catalysis
8.3.1.7 Inactivation Must Occur Prior to Release of the Active Species from the Enzyme
8.3.2 Determination of the Partition Ratio
8.3.3 Potential Clinical Advantages of Mechanism-Based Inactivators
8.3.4 Examples of Mechanism-Based Inactivators as Drugs
8.4 Use of Affinity Labels as Mechanistic Tools
8.5 Summary
References
Appendix 1. Kinetics of Biochemical Reactions
A1.1 The Law of Mass Action and Reaction Order
A1.2 First Order Reactions
A1.3 Second Order Reactions
A1.4 Pseudo-first Order Reaction Conditions
A1.5 Approach to Equilibrium: An Example of the Kinetics of Reversible Reactions
References
Appendix 2. Derivation of the Enzyme-Ligand Binding Isotherm Equation
References
Appendix 3. Serial Dilution Schemes