Evaluation of Enzyme Inhibitors in Drug Discovery

Name: Evaluation of Enzyme Inhibitors in Drug Discovery | A Guide for Medicinal Chemists and Pharmacologists
Brand: Wiley
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A Guide for Medicinal Chemists and Pharmacologists

Robert A. Copeland(Autor*in)

Wiley (Verlag)

2. Auflage

Erschienen am 31. Januar 2013

572 Seiten

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978-1-118-54028-2 (ISBN)

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Inhalt

Cover
Title page
Copyright page
Dedication
Contents
Foreword to Second Edition
Preface to Second Edition
Foreword to First Edition
Preface to First Edition
Acknowledgments (From First Edition)
Epigraph
CHAPTER 1: Why Enzymes as Drug Targets?
Key Learning Points
1.1 Enzymes Are Essential for Life
1.2 Enzyme Structure and Catalysis
1.3 Permutations of Enzyme Structure During Catalysis
1.4 Extension to Other Target Classes
1.5 Other Reasons for Studying Enzymes
1.6 Summary
References
CHAPTER 2: Enzyme Reaction Mechanisms
Key Learning Points
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
2.3.2 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 Typical Values of Steady State Kinetic Parameters
2.6 Graphical Determination of kcat and KM
2.7 Reactions Involving Multiple Substrates
2.7.1 Bisubstrate Reaction Mechanisms
2.8 Summary
References
CHAPTER 3: Reversible Modes of Inhibitor Interactions with Enzymes
Key Learning Points
3.1 Enzyme-Inhibitor Binding Equilibria
3.2 Competitive Inhibition
3.3 Noncompetitive Inhibition
3.3.1 Mutual Exclusivity Studies
3.3.2 Noncompetitive Inhibition by Active Site-Directed Inhibitors
3.4 Uncompetitive Inhibition
3.5 Inhibition Modality in Bisubstrate Reactions
3.6 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 Enzyme Reactions on Macromolecular Substrates
3.7.1 Challenges in Inhibiting Protein-Protein Interactions
3.7.2 Hot Spots in Protein-Protein Interactions
3.7.3 Factors Affecting Protein-Protein Interactions
3.7.4 Separation of Binding and Catalytic Recognition Elements
3.7.5 Noncompetitive Inhibition by Active Site-Binding Molecules for Exosite Utilizing Enzymes
3.7.6 Processive and Distributive Mechanisms of Catalysis
3.7.7 Effect of Substrate Conformation on Enzyme Kinetics
3.7.8 Inhibitor Binding to Substrates
3.8 Summary
References
CHAPTER 4: Assay Considerations for Compound Library Screening
Key Learning Points
4.1 Measures of Assay Performance
4.1.1 Calibration Curves
4.1.2 Total, Background, and Specific Signal
4.1.3 Defining Inhibition, Signal Robustness, and Hit Criteria
4.2 Measuring Initial Velocity
4.2.1 End-Point and Kinetic Readouts
4.2.2 Effect of Enzyme Concentration
4.2.3 Other Factors Affecting Initial Velocity
4.3 Balanced Assay Conditions
4.3.1 Balancing 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
4.7.1 Determination of Hit Reproducibility
4.7.2 Verification of Chemical Purity and Structure
4.7.3 Hit Verification in Orthogonal Assays
4.7.4 Chemical and Pharmacological Tractability
4.7.5 Promiscuous Inhibitors
4.7.6 Prioritization of Confirmed Hits
4.7.7 Hit Expansion
4.8 Summary
References
CHAPTER 5: Lead Optimization and Structure-Activity Relationships for Reversible Inhibitors
Key Learning Points
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 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 Buildup of Substrate and/or Diminution of Product for the Target Enzyme Should Be Observed in Cells
5.5.4 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
Key Learning Points
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 Clincial 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 Calcineurin 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 Inhibition
Key Learning Points
7.1 Effects of Tight Binding Inhibition 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 Determinations
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 Enzyme-Reaction Intermediate Analogues as Examples of Tight Binding Inhibitors
7.7.1 Bisubstrate Analogues
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: Drug-Target Residence Time
Key Learning Points
8.1 Open and Closed Systems in Biology
8.2 The Static View of Drug-Target Interactions
8.3 Conformational Adaptation in Drug-Target Interactions
8.3.1 Conformational Selection Model
8.3.2 Induced-Fit Model
8.3.3 Kinetic Distinction Between Conformational Selection and Induced-Fit Mechanisms
8.4 Impact of Residence Time on Natural Receptor-Ligand Function
8.4.1 Immune Response
8.4.2 Control of Protease Activity by Natural Inhibitors
8.5 Impact of Drug-Target Residence Time on Drug Action
8.5.1 Mathematical Definition of Residence Time for Different Mechanisms of Drug-Target Interaction
8.5.2 Impact of Residence Time on Cellular Activity
8.5.3 Impact on Efficacy and Duration in Vivo
8.5.4 Temporal Target Selectivity and Drug Safety
8.6 Experimental Measures of Drug-Target Residence Time
8.6.1 Kinetic Analysis of Approach to Equilibrium
8.6.2 Jump-Dilution Experiments
8.6.3 Separation Methods
8.6.4 Spectroscopic Differentiation
8.6.5 Immobilized Binding Partner Methods
8.7 Drug-Target Residence Time Structure-Activity Relationships
8.7.1 Structural Changes Associated with Conformational Adaptation
8.7.2 Thermodynamics of Drug-Target Complex Dissociation
8.7.3 A Retrograded Induced-Fit Model of Drug-Target Complex Dissociation
8.8 Recent Applications of The Residence Time Concept
8.9 Limitations of Drug-Target Residence Time
8.10 Summary
References
CHAPTER 9: Irreversible Enzyme Inactivators
Key Learning Points
9.1 Kinetic Evaluation of Irreversible Enzyme Inactivators
9.2 Affinity Labels
9.2.1 Quiescent Affinity Labels
9.2.2 Potential Liabilities of Affinity Labels as Drugs
9.3 Mechanism-Based Inactivators
9.3.1 Distinguishing Features of Mechanism-Based Inactivation
9.3.2 Determination of the Partition Ratio
9.3.3 Potential Clinical Advantages of Mechanism-Based Inactivators
9.3.4 Examples of Mechanism-Based Inactivators as Drugs
9.4 Use of Affinity Labels as Mechanistic Tools
9.5 Summary
References
CHAPTER 10: Quantitative Biochemistry in the Pharmacological Evaluation of Drugs
Key Learning Points
10.1 In Vitro Admet Properties
10.1.1 Exponential Decay Processes and the Definition of Half-Life
10.1.2 Caco-2 Cell Permeability as a Surrogate for Intestinal Absorption
10.1.3 Whole Blood or Plasma Stability
10.1.4 Plasma Protein Binding
10.1.5 Metabolism of Xenobiotics in the Liver
10.1.6 Hepatocyte, S9, and Microsome Stability
10.1.7 CYP450 Mediated Metabolism
10.1.8 Cytochrome P450 Inhibition
10.1.9 hERG Inhibition
10.2 In Vivo Pharmacokinetic Studies
10.2.1 General Considerations and Curve Fitting Parameters
10.2.2 Kinetic Models of Drug PK
10.2.3 Absorption and Bioavailability
10.2.4 Factors Affecting PK Parameters
10.2.5 Allometric Scaling of Drug Pharmacokinetics
10.3 Metabolite Identification
10.4 Measures of Target Occupancy
10.4.1 Radiometric Imaging
10.4.2 Ex Vivo Determination of Target Occupancy
10.4.3 Pharmacodynamic Measures of Target Engagement
10.5 Summary
References
APPENDIX 1: Kinetics of Biochemical Reactions
A1.1 The Law of Mass Action and Reaction Order
A1.2 First-Order Reaction Kinetics
A1.3 Second-Order Reaction Kinetics
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
APPENDIX 4: Relationship between [I]/iC50 and Percentage Inhibition of Enzyme Activity When h = 1
APPENDIX 5: Propagation of Uncertainties in Experimental Measurements
A5.1 Uncertainty Propagation for Addition or Subtraction of Two Experimental Parameters
A5.2 Uncertainty Propagation for Multiplication or Division of Two Experimental Parameters
A5.3 Uncertainty Propagation for Multiplication or Division of an Experimental Parameter by A Constant
A5.4 Uncertainty Propagation for an Experimental Parameter Raised by an Exponent
A5.5 Uncertainty Propagation for a General Function of Experimental Parameters
Reference
APPENDIX 6: Useful Physical Constants at Different Temperatures
APPENDIX 7: Common Radioactive Isotopes Used in Studies of Enzymes
APPENDIX 8: Common Prefixes for Units in Biochemistry
APPENDIX 9: Some Aromatic Ring Systems Commonly Found in Drugs
APPENDIX 10: Residual Plots
References
Index

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