A practical guide for the assay-dependent characterisation of irreversible inhibitors.

Irreversible targeted covalent inhibitors, in the past regarded as inappropriately reactive and toxic, have seen a recent resurgence in clinical interest. This paradigm shift is attributed to the exploitation of the two-step mechanism, in which a high affinity and selectivity (, low ) scaffold binds the target and only then does a pendant low intrinsic reactivity warhead react with the target (moderate ). This highlights the importance of evaluating inhibitors by deriving both their and values.

Fitting of and Values from Endpoint Pre-incubation IC 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" IC 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 and values. Herein we report EPIC-Fit, a new method to determine and values from global fitting of Endpoint Pre-incubation IC data that can be implemented using Microsoft Excel.