Abstract
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" IC(50) 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 k(inact) and K(I) values. Herein we report EPIC-Fit, a new method to determine k(inact) and K(I) values from global fitting of Endpoint Pre-incubation IC(50) data that can be implemented using Microsoft Excel. Experimental characterization of a known tissue transglutaminase inhibitor, AA9, using EPIC-Fit provided k(inact) and K(I) 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.