CHEMBL3705648

Inhibition Assay: Compounds were screened for their ability to inhibit ATR kinase using a radioactive-phosphate incorporation assay. Assays were carried out in a mixture of 50 mM Tris/HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT. Final substrate concentrations were 10 uM [gamma-33P]ATP (3 mCi 33P ATP/mmol ATP, Perkin Elmer) and 800 uM target peptide (ASELPASQPQPFSAKKK).Assays were carried out at 25 C. in the presence of 5 nM full-length ATR. An assay stock buffer solution was prepared containing all of the reagents listed above, with the exception of ATP and the test compound of interest. 13.5 uL of the stock solution was placed in a 96 well plate followed by addition of 2 uL of DMSO stock containing serial dilutions of the test compound (typically starting from a final concentration of 15 uM with 3-fold serial dilutions) in duplicate (final DMSO concentration 7%). The plate was pre-incubated for 10 minutes at 25 C.