CHEMBL5736865

Radiometric Assay: Enzyme assay using full length recombinant active form of wild-type BTK and BTK-C481S was measured as described previously (Anastassiadis T, et al., Nat. Biotechnol. 29(11):1039-45 (2011)). Compounds were tested in 10-point dose IC50 mode with 3-fold serial dilution starting at 1 or 10 μM concentration. BTK kinase activity was assayed in a buffer solution containing 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO. Compounds in 100% DMSO were mixed with kinase (8 nM wild-type BTK or 5 nM BTK-C481S mutant) with substrate into the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range) and incubated for 20 min at room temp. The reaction was initiated by adding 10 μM ATP containing 33P-ATP into the mixture and incubated for 2 hours at room temperature. Kinase activity was detected by P81 filter-binding 33P radioisotope based radiometric method. All compounds were tested in duplicate. The raw data was fit to a 4-parameter logistic model to derive the IC50 value for kinase activity inhibition.