One-Pot Measurement of the Kinetic Parameters KI, kinact, and Time-Dependent IC₅₀ for Analysis of Covalent Small Molecule Kinase Inhibitors

To address the need for the analysis of covalent kinase inhibitors in a high-throughput method, we established a protocol for the facile measurement of kinact and KI utilizing a proprietary off-chip mobility shift assay supplied by PerkinElmer. With this electrophoretic technique, these kinetic parameters, which describe covalent inhibitors better than IC50 measurements, are accurately and reproducibly measured. As a proof of concept measurement, an inhibitor of BMX, compound BMX-IN-1, was shown to have a kinact = 0.0298 ± 0.0024 min-1 and a KI = 0.134 ± 0.021 uM. The industry standard for reporting is kinact/KI and was calculated to be 0.222 ± 0.039 uM-1min-1. To direct medicinal chemists optimizing parameters of covalent inhibitors, simulations of the equations used to fit progress curves were performed in MatLab. These revealed the compelling argument to pursue improved KI before kinact and are discussed further. In addition, some limitations of our assay are presented.