Binding Mode Assay

To determine the mechanism of action of the hit compound with respect to ATP and methionine, IC₅₀ values were determined using SPA assays as described above. Final compound concentrations in each IC₅₀ reaction ranged from 200 to 0.4 μM. Competition with ATP was determined at 25, 50, 100, 250, 500, 1000 μM ATP while the methionine concentration was 75 μM. Competition with the amino acid was determined at 25, 50, 100, 200, 300 μM methionine while the ATP concentration was 2.5 mM. Positive controls contained only DMSO (2 μl) without compound.