Methods for kinetic evaluation of reversible covalent inhibitors from time-dependent IC50 data†

Abstract

Potent reversible covalent inhibitors are often slow in establishing their covalent modification equilibrium, resulting in time-dependent inhibition. While these inhibitors are commonly assessed using IC₅₀ values, there are no methods available to analyze their time-dependent IC₅₀ data to provide their inhibition (K_i and ) and covalent modification rate (k₅ and k₆) constants, leading to difficulty in accurately ranking drug candidates. Herein, we present an implicit equation that can estimate these constants from incubation time-dependent IC₅₀ values and a numerical modelling method, EPIC-CoRe, that can fit these kinetic parameters from pre-incubation time-dependent IC₅₀ data. The application of these new methods is demonstrated by the evaluation of a known inhibitor, saxagliptin, providing results consistent with those obtained by other known methods. This work introduces two new practical methods of evaluation for time-dependent reversible covalent inhibitors, allowing for rigorous characterization to enable the fine-tuning of their binding and reactivity.