CHEMBL3887654

Z′-LYTE Screening Assay: Axitinib was tested using a Z′-LYTE Screening Protocol (Invitrogen, Carlsbad, Calif.). Axitinib was prepared in 100% DMSO, and added to the reaction for a final DMSO concentration of 1%. Inhibitor concentrations varied from 1.0 μM to 0.00003 μM. Typical reactions were 10 μL, contained ABL1 or ABL1 [T315I], ATP (ABL1; 10 μM and ABL1 [T315I]; 5 μM), 2.0 μM Tyr 02 peptide, 10 mM MgCl2, 1.0 mM EGTA, 0.01% BRIJ-35, 1% DMSO in 50 mM HEPES pH 7.5. The mixture was incubated in a 384-well polypropylene plate at room temperature for an hour and terminated by the addition of 5 μL of a 1:64 dilution of the development reagent utilized with Z′-LYTE® Screening Protocol, followed by a 30 second shake.