CHEMBL5732386

Inhibitory Assay: An inhibitory assay was conducted using the previously described p-nitrophenylphosphate assay (Rayapureddi, J. P. et al. Nature 426, 295-298 (2003)). Briefly, ED was incubated in a reaction mixture containing 20 mM MES pH 6, 2 mM MgCl2, 125 μM inhibitor, 3.4 mM para-nitrophenol phosphate (pNPP) and 0.01 μg/μL enzyme. The amount of 4-nitrophenol (pNP) produced was monitored at 405 nM on a BioTek EL808 plate reader. A similar protocol was used to screen for inhibitor activity using hEYA3 and hEYA2(ED).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/L 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.These results were mirrored when an alternate substrate, a 10 amino acid phosphopeptide representing the C-terminus of the known EYA substrate γ-H2AX (Cook, P. J. et al. Nature 458, 591-596, (2009); Krishnan, N. et al. J. Biol. Chem. (2009), the contents of which are incorporated by reference in their entireties). The phospho-peptide KKATQASQEpY (SEQ. ID 5) was obtained from Genscript. Peptide assays were conducted in 20 mM MES pH 6, 2 mM MgCl2, and a range of peptide concentrations from 0 to 300 μM as previously described in the incorporated materials of Rayapureddi, J. P. (2003). IC50 values were then calculated using PRISM software.