4.2.4. c-Met Kinase Assay

A c-Met kinase assay was performed at the Reaction Biology Corporation (Malvern, PA, USA) using a Kinase HotSpotSM assay platform (www.reactionbiology.com, last accessed at 19 October 2020). Briefly, human c-Met kinase (5–10 mU) and peptide substrate (KKKSPGEYVNIEFG, 20 µM) were prepared in a reaction buffer with a final volume of 25 μL. The compounds were delivered into the reaction, followed ~20 min. later by the addition of a mixture of ATP and [γ-33P-ATP] (specific activity approx. 500 cpm/pmol, concentration as required) to a final concentration of 10 μM. After incubation for 40 min. at 25 °C, the reaction was stopped by the addition of a 3% phosphoric acid solution. Then, the reaction was spotted onto a P30 filtermat, and unbound phosphate was removed by washing 3 times for 5 min in 75 mM phosphoric acid and once in methanol prior to drying and scintillation counting. The background counting derived from the control reactions containing the inactive enzyme was subtracted, and specific kinase activity data were expressed as the percentage of remaining kinase activity in the test compounds compared to the vehicle (dimethyl sulfoxide) reactions. IC50 values and curve fits were obtained using GraphPad Prism 5 software.