CHEMBL5736627

HTRF Kinease-TK Assay: The potency of a compound inhibiting wild type and exemplary mutant ROS1 kinases was determined using CisBio's HTRF Kinease-TK assay technology. The assays contained 5 nM wild type ROS1 (SignalChem—Cat. No. R14-11G), 5 nM G2032R ROS1 (SignalChem—Cat. No. R14-12BG), 5 nM L2026M ROS1 (Array Biopharma, p1965), or 5 nM D2033N ROS1 (Array Biopharma, p1994). Each kinase is incubated with 250 nM TK-substrate biotin (CisBio, Cat. No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM MOPS [pH 7.4], 5 mM MgCl2, 0.005% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were prepared in a four-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 120-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.3 nM Sa-XL665 and 1× TK-Ab-Cryptate in HTRF detection buffer (CisBio, Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC is determined using no test compound and 0 POC is determined in the absence of enzyme.