Kinetics of Enzyme Action - Basic Principles for Drug Hunters

Course Code: KNTX

Overview

This course covers the essentials of enzyme kinetic analysis emphasizing concepts of importance in drug discovery. It provides an introduction to enzymology, assay methods, and free-energy diagrams; and also covers simple reversible inhibition and time-dependent inhibition; transition state rate theory and TS analog inhibitors; kinetics and inhibition of two substrate enzymatic reactions; screening for inhibitors of enzymatic reactions; and, probes of mechanism.

When and Where

Key Topics

• Enzymology, Assay Methods, and Free-Energy Diagrams
• Concepts of Simple Reversible Inhibition
• Time-Dependent Inhibition
• Transition State Rate Theory and TS Analog Inhibitors
• Kinetics of Two Substrate Enzymatic Reactions
• Inhibition of Two-Substrate Enzymatic Reactions
• Screening for Inhibitors of Enzymatic Reactions
• Probes of Mechanism

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Course Instructors

Ross Stein is an Associate Professor in the Department of Neurology at Harvard Medical School and Director of the Laboratory for Drug Discovery in Neurodegeneration of the Harvard Center for Neurodegeneration and Repair. Dr. Stein has been a practicing enzymologist for nearly 30 years and has published over 90 research papers on no less than a dozen different enzyme systems. Before joining Harvard in 2001, he spent 20 years building and running Enzymology and Biochemistry departments in the pharmaceutical and biotech industry.

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Who Should Attend?

• Chemists, biochemists, and molecular biologists who use enzyme kinetics in their day-to-day drug discovery efforts.
• Medicinal chemists who need to understand the basic principles of enzyme kinetics to interpret SAR data.
• Research managers who need a refresher on this area of critical importance to drug discovery.

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How You'll Benefit

• Design and develop assays for one- and two-substrate enzyme reactions.
• Determine the kinetic mechanism of two-substrate enzymes.
• Determine Ki values for enzyme inhibitors.
• Determine the kinetic mechanism of inhibition for one- and two-substrate enzyme reactions.
• Determine precise estimates of Ki values for tight-binding inhibitors.
• Determine the kinetics of inhibition for slow-binding inhibitors.
• Design transition state analog inhibitors.
• Design screens to identify inhibitors of one- and two-substrate enzymes.

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Agenda

Introduction (Enzymology, Assay Methods, and Free-Energy Diagrams)

• The Goal of Enzymology - Elucidation of Mechanism
• Assays Methods - General Comments
• Time Courses of Enzyme-Catalyzed Reactions
• Substrate-Concentration Dependence of Initial Velocity
• Kinetic Consequences of Complex Formation During Enzyme Catalysis
• Free-Energy Diagrams
• Free-Energy Diagrams - Prolyl Isomerization

Concepts of Simple Reversible Inhibition

• Enzymes and Inhibitors - Simple Mass Action
• Basics of Reversible Inhibition
• Inhibition of One Substrate Enzymes - Classical Mechanisms
• Inhibition of One Substrate Enzymes - General Mechanism
• Determination of Inhibition Mechanism - General Method
• How to Handle Steady-State Rate Data with Multiple Independent Variables
• Method of Replots
• Mixed Inhibition of the ATPase Activity of cdk5/p25 by APS
  Screen for Modulators of UCH-L1
• LDN-091946 Is an Uncompetitive Inhibitor of UCH-L1
• Tight-Binding Inibition
• Inhibition of UCH-L1 by Ubiquitin Aldehyde
• Substrate and Product Inhibition

Time-Dependent Inhibition

• Primer of Chemical Kinetics
• Time-Dependent Enzyme Inhibition
• Progress Curve Analysis for Time-Dependent Inhibition
• Independent Determination of koff
• Inhibition of Human Transglutaminase by LDN-27219
• Slow-Binding Inhibition of g-Glutamyl Transpeptidase by g-boroGlu
• Inhibition of UCH-L1 by Ubiquitin Aldehyde

Transition State Rate Theory and TS Analog Inhibitors

• Transition State Rate Theory
• Enzyme Catalysis and Transition State Theory
• Transition State Analog Inhibition
• Inhibition of Thermolysin by Peptide Phosphonamidates
• Inhibition of Elastase by Peptide-Based Trifluormethyl Ketones
• Inhibition of Purine Nucleoside Phosphorylase

Kinetics of Two Substrate Enzymatic Reactions

• Rapid Equilibrium, Sequential Mechanisms
• Kinetic Analysis of Two-Substrate Reactions - General
  Redux – How to Handle Steady-State Data with Multiple Independent Variables
• Kinetic Analyis of Two-Substrate Reactions - Method of Replots
• Replots for Random Addition of Substrates
• Replots for Ordered Addition of Substrates
• Ping-Pong Mechanism
• Kinetic Mechanism for cdk5/p25 Kinase
• Acyl-Transfer Enzymology
• Burst Kinetics in Acyl Hydrolases

Inhibition of Two-Substrate Enzymatic Reactions

• Inhibition of Two Substrate Reactions – General Principles
• Inhibition of Random, Two Substrate Reaction
• Inhibition of Ordered, Two Substrate Reaction

Screening for Inhibitors of Enzymatic Reactions

• General Concepts
• Choice of Concentration of Substrate(s)
• Balancing the Steady-State for a Single-Substrate Reaction
• Balancing the Steady-State for an Ordered Mechanism
• Balancing the Steady-State for a Random Mechanism

Additional Topics: Probes of Mechanism

• pH-Dependencies of Enzyme Catalysis
• Temperature-Dependencies of Enzyme Catalysis
• Kinetic Isotope Effects as Mechanic Probes

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Course Fees

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