A novel assay based on pre-equilibrium titration curves for the determination of enzyme inhibitor binding kinetics

Bernard Noppen,Anouk Vanbelle,Marc Vanhove,Alan W Stitt

doi:10.1007/s00249-021-01554-0

Abstract

Selection of pharmacological agents based on potency measurements performed at equilibrium fail to incorporate the kinetic aspects of the drug–target interaction. Here we describe a method for screening or characterization of enzyme inhibitors that allows the concomitant determination of the equilibrium inhibition constant in unison with rates of complex formation and dissociation. The assay is distinct from conventional enzymatic assays and is based on the analysis of inhibition curves recorded prior to full equilibration of the system. The methodology is illustrated using bicyclic peptide inhibitors of the serine protease plasma kallikrein.

Highlights

The intrinsic potency of a given drug is generally expressed on the basis of the affinity for its molecular target, and metrics such as IC50’s, KD’s or Ki’s are broadly used to select and prioritize lead compounds
Such parameters reflect an affinity measured at equilibrium and fail to describe the kinetic aspects of the drug–target interaction and the time-dependent changes in target engagement
The kinetics of drug–target interaction can be represented by the rate of complex formation (kon) and dissociation (koff)

Summary

Introduction

The intrinsic potency of a given drug is generally expressed on the basis of the affinity for its molecular target, and metrics such as IC50’s, KD’s or Ki’s are broadly used to select and prioritize lead compounds. We report a method to study the interaction of enzyme inhibitors with their cognate target based on the analysis of pre-equilibrium inhibition curves.

Results

Conclusion