In vitro MEK1 Kinase Assay

In vitro MEK1 kinase inhibition by inhibitor analogs were determined using a standard kinase assay reaction and Kinase-Glo luminescent kinase assay kit from Promega (WI, USA). Kinase reactions were carried out with purified recombinant active MEK1-GST (Cat #: M8822, Sigma-Aldrich) and inactive Erk2 (Cat #: PV3314, Thermofisher Scientific) in kinase reaction buffer (ab189135, Abcam) supplemented with 0.25 mM DTT. In brief, inhibitors were pre-incubated with recombinant MEK1 at a final concentration of 4 μg/mL at room temperature for 30 min prior to addition of inactive substrate (Erk2) and ATP at final concentration of 0.025 μg/μL and 10 μM, respectively. Reactions were incubated at room temperature for 2 hours before equal volumes of Kinase-Glo solution were added to each well and incubated for 30 min in the dark. Bioluminescence was measured on an Envision multilabel reader from PerkinElmer. Assays were conducted in triplicate with various inhibitor concentrations each run in duplicate. IC₅₀ data were calculated using GraphPad Prism software (version 7.0a, La Jolla, CA). Data represent three independent experiments with SEM.