Fitting of kinact and KI Values from Endpoint Pre-incubation IC50 Data

Experiments comprising a "pre-incubation" phase, where enzyme is incubated with inhibitor prior to the addition of assay substrate, are commonly used to evaluate covalent inhibitors, often via discontinuous or "endpoint" IC50 assays. However, due to the lack of mathematical tools to describe its biphasic time-dependent nature, this experiment has thus far been unable to provide kinact and KI values. Herein we report EPIC-Fit, a new method to determine kinact and KI values from global fitting of Endpoint Pre-incubation IC50 data that can be implemented using Microsoft Excel. Experimental characterization of a known tissue transglutaminase inhibitor, AA9, using EPIC-Fit provided kinact and KI values with strong correlations to the values determined by other, previously established methods of evaluation. This unprecedented method serves to finally include time-dependent pre-incubation endpoint assays in the medicinal chemist's toolbox for rigorous characterization of irreversible inhibitors.