CHEMBL5733865

Inhibition Assay: The compounds were then tested using full-length human recombinant, purified EYA3 and pNPP as a substrate. Compounds were dissolved in DMSO and diluted as needed. IC50 values were determined by adding varying amounts of inhibitor (0-400 μM) to reaction mixtures containing 20 mM MES pH 6, 2 mM MgCl2, 2% DMSO, 3.4 mM pNPP, and 0.01 μg/μL enzyme. Reactions were incubated at 30° C. for 30 minutes and quenched with 100 mM EDTA pH 10. IC50 values were then calculated directly from regression curves using PRISM software. All reported values are the mean of two independent experiments.