Evaluation of enzyme inhibitors in drug discovery : A guide for medicinal chemis ts & pharmacologists (Methods of biochemical analysus Volume 46)

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.

Foreword.

Preface.

Acknowledgments.

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.

References.

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 k_cat andK<,sub>,M

2.6 Reactions Involving Multiple Substates.

2.6.1 Bisubstrate Reaction Mechanisms.

2.7 Summary.

References.

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 K<,sub>,i to Binding Energy.

3.6.3 Defining Target Selectivity by K<,sub>,i 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.

References.

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.

References.

5. Lead Optimization and Structure Activity Relationships for Reversible Inhibitors.

5.1 Concentration Response Plots and IC<,sub>,50 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...