High performance enzyme kinetics of turnover, activation and inhibition for translational drug discovery

ABSTRACT

Introduction: Enzymes are the macromolecular catalysts of many living processes and represent a sizable proportion of all druggable biological targets. Enzymology has been practiced just over a century during which much progress has been made in both the identification of new enzymes and the development of novel methodologies for enzyme kinetics.

Areas covered: This review aims to address several key practical aspects in enzyme kinetics in reference to translational drug discovery research. The authors first define what constitutes a high performance enzyme kinetic assay. The authors then review the best practices for turnover, activation and inhibition kinetics to derive critical parameters guiding drug discovery. Notably, the authors recommend global progress curve analysis of dose/time dependence employing an integrated Michaelis-Menten equation and global curve fitting of dose/dose dependence.

Expert opinion: The authors believe that in vivo enzyme and substrate abundance and their dynamics, binding modality, drug binding kinetics and enzyme’s position in metabolic networks should be assessed to gauge the translational impact on drug efficacy and safety. Integrating these factors in a systems biology and systems pharmacology model should facilitate translational drug discovery.

KEYWORDS:

• High performance enzyme assay
• Michaelis-Menten enzyme kinetics
• quasi-steady-state approximation
• enzyme activation
• enzyme inhibition
• active site titration
• binding kinetics
• binding modality
• target abundance
• drug discovery
• pharmacodynamics
• global progress curve analysis
• global curve fitting
• closed forms of Michaelis-Menten equation

Article highlights

• High performance enzyme kinetics assay for drug discovery should employ pathophysiogically relevant enzymes, substrates and cofactors, be robust, have large dynamic potency range, and reveal both binding kinetics and modality.

• Steady and non-steady state enzyme turnover kinetics should be best handled by the progress curve analysis that complements the traditional initial velocity and replot analysis. This is made possible by the now available integrated, closed form of the Michaelis-Menten equation.

• Practical guidance for experimental design and data analysis is provided to reliably determine enzyme turnover parameters, enzyme activation and inhibition kinetics and active site concentrations using global curve fitting approach. Commonly used equations for data analysis are tabulated and explained.

• Impacts of in vivo enzyme and substrate abundance, binding kinetics and modality, and enzyme’s network position are opined for greater awareness in translational drug discovery.

This box summarizes key points contained in the article.

Acknowledgments

The authors thank C. Barbieri, G. Schroeder and P. Tawa for their critical reading of the manuscript. We thank M. Webb for the management support.

Declaration of interest

The authors are employees of Merck Sharp & Dohme Corp, a subsidiary of Merck Co & Inc, Kenilworth, NJ USA. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Additional information

Funding

The authors are supported by Merck Sharp & Dohme Corp, a subsidiary of Merck Co & Inc, Kenilworth, NJ USA.