Evaluation of Enzyme Inhibitors in Drug Discovery: A Guide for Medicinal Chemists and Pharmacologists / Edition 1

Evaluation of Enzyme Inhibitors in Drug Discovery: A Guide for Medicinal Chemists and Pharmacologists / Edition 1

by Robert A. Copeland

Vital information for discovering and optimizing new drugs

"Understanding the data and the experimental details that support it has always been at the heart of good science and the assumption challenging process that leads from good science to drug discovery. This book helps medicinal chemists and pharmacologists to do exactly that in the realm of enzyme

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Vital information for discovering and optimizing new drugs

"Understanding the data and the experimental details that support it has always been at the heart of good science and the assumption challenging process that leads from good science to drug discovery. This book helps medicinal chemists and pharmacologists to do exactly that in the realm of enzyme inhibitors."
—Paul S. Anderson, PhD

This publication provides readers with a thorough understanding of enzyme-inhibitor evaluation to assist them in their efforts to discover and optimize novel drug therapies. Key topics such as competitive, noncompetitive, and uncompetitive inhibition, slow binding, tight binding, and the use of Hill coefficients to study reaction stoichiometry are all presented. Examples of key concepts are presented with an emphasis on clinical relevance and practical applications.

Targeted to medicinal chemists and pharmacologists, Evaluation of Enzyme Inhibitors in Drug Discovery focuses on the questions that they need to address:

  • What opportunities for inhibitor interactions with enzyme targets arise from consideration of the catalytic reaction mechanism?
  • How are inhibitors evaluated for potency, selectivity, and mode of action?
  • What are the advantages and disadvantages of specific inhibition modalities with respect to efficacy in vivo?
  • What information do medicinal chemists and pharmacologists need from their biochemistry and enzymology colleagues to effectively pursue lead optimization?

Beginning with a discussion of the advantages of enzymes as targets for drug discovery, the publication then explores the reaction mechanisms of enzyme catalysis and the types of interactions that can occur between enzymes and inhibitory molecules that lend themselves to therapeutic use. Next are discussions of mechanistic issues that must be considered when designing enzyme assays for compound library screening and for lead optimization efforts. Finally, the publication delves into special forms of inhibition that are commonly encountered in drug discovery efforts, but can be easily overlooked or misinterpreted.

This publication is designed to provide students with a solid foundation in enzymology and its role in drug discovery. Medicinal chemists and pharmacologists can refer to individual chapters as specific issues arise during the course of their ongoing drug discovery efforts.

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Product Details

Publication date:
Methods of Biochemical Analysis Series, #46
Edition description:
Older Edition
Product dimensions:
6.48(w) x 9.35(h) x 0.80(d)

Related Subjects

Table of Contents




1. Why Enzymes as Drug Targets?

1.1 Enzymes Are Essentials 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.


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 Bond 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 Graphical Determination of kcat and KM

2.6 Reactions Involving Multiple Substates.

2.6.1 Bisubstrate Reaction Mechanisms.

2.7 Summary.


3. Reversible Modes of Inhibitor Interactions with Enzymes.

3.1 Enzyme-Inhibitor Binding Equilibria.

3.2 Competitive Inhibition.

3.3 Noncompetitive Inhibition.

3.3.1 Mutual Exclusively Studies.

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 Organization.

3.7 Summary.


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.

4.3 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 and Progression.

4.8 Summary.


5. Lead Optimization and Structure-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 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.


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 Methotresate.

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.


7. Tight Binding Inhibitors.

7.1 Effects of Tight Binding Inhibition Concentration-Response Data.

7.2 The IC50 Value Depends on Kiapp and [E]T.

7.3 Morrison’s Quadratic Equation for Fiting 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 Determine Ki.

7.7 Enzyme-Reaction Intermediate Analogues as Example 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.


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.

8.3.2 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.


Appendix 1. Kinetic 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.


Appendix 2. Derivation of the Enzyme-Ligand Binding Isotherm Equation.


Appendix 3. Serial Dilution Schemes.


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