| Assay Method Information | |
| | Enzymatic Assay |
| Description: | The inhibitory activity of compounds against DNA-PK was determined by TR-FRET measuring a fluorescent labelled peptide substrate converting to a phosphorylated product. All assays were performed in black Greiner 384 well low volume plates (Greiner), in a total reaction volume of 6 μL and 0.5% (v/v) final DMSO concentration. Full length human DNAPK protein, Fluorescein-P53 (Ser15) Peptide Substrate (Fluorescein-EPPLSQEAFADLWKK) and LanthaScreen™ Tb-anti-phospho-p53 [pSer15] Antibody Kit were purchased from Thermo Fisher Scientific. Initially DNA-PK protein was incubated with compound for 30 minutes at room temperature in reaction buffer (50 mM HEPES pH 7.5, 0.01% Brij-35, 10 mM MgCl2, 1 mM EGTA, 1 mM DTT, 10 μg/ml Calf Thymus DNA). The reaction was then initiated by the addition of ATP and Fluorescein-P53 (Ser15) Peptide Substrate. The kinase reaction (10 μM ATP, 1.6 μM peptide substrate) was quenched after 60 minutes by the addition of 6 μl of stop buffer containing 20 mM EDTA, 4 nM Tb anti-phospho-p53 [Serl5] Antibody. The reaction was incubated for a further hour and the plates were read on Spark 20M (Tecan). Data was analyzed and the concentration of compound producing 50% inhibition of the respective kinase (IC50) was calculated using four-parameter logistic fit with XLfit. |
| Affinity data for this assay | |
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