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| | Inhibition Assay |
| Description: | 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 M [ -33P]ATP (3mCi 33P ATP/mmol ATP, Perkin Elmer) and 800 M 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 L of the stock solution was placed in a 96 well plate followed by addition of 2 L of DMSO stock containing serial dilutions of the test compound (typically starting from a final concentration of 15 M with 3-fold serial dilutions) in duplicate (final DMSO concentration 7%). The plate was pre-incubated for 10 minutes at 25 ° C. and the reaction initiated by addition of 15 L [ -33P]ATP. |
| Affinity data for this assay | |
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