Compounds were added to plates in duplicate and the kinase assay was incubated. Plates were cleaned, peptide calculator blocked and washed before anti Phospho p53 antibody was put into the plates and incubated. To reduce non specific binding dishes were washed ahead of incubation with HRP conjugated goat anti rabbit IgG secondary antibody.
Extra antibody that was for this phosphorylated GST p53 protein was detected with TMB substrate reagent. Plates were developed and the reaction was stopped before absorbance was determined. Compounds that inhibited ATM kinase activity in ELISA assays, were recognized regarding inhibition of ATM/ATR kinases employing in vitro kinase assays. Western blotting utilising the anti Phospho p53 antibody was employed as a of ATM/ATR inhibition. Expanded analysis of CP466722 against a commercially available panel of kinases was done by Upstate. HeLa or Even A T cells were incubated for 24h and plated in triplicate. Cells were pre treated: DMSO, CP466722 or KU55933 ahead of IR. Cells were incubated for 4h following IR before press was removed, cells washed, trypsinsed, counted and re coated in the absence HC-030031 of drug and incubated for 10 days. Ahead of colony counting, cells were rinsed, stained, cleaned and dried.
Identified numbers were counted together surviving nest, data were calculated as percentage surviving cities relative to control plates SE. Huge amounts of purified protein will be required to work High Throughput Screens to identify small molecule inhibitors of ATM. For that reason, a led Gene expression display based approach was followed where a collection of 1500 compounds was selected based on known kinase inhibitor templates and determined kinase pharmacophores from the Pfizer exclusive chemical file. These compounds were screened having an in Hedgehog inhibitor vitro ELISA assay, with possible inhibitors being identified by a reduced capacity of purified ATM kinase to phosphorylate GST p53 substrate. Ingredients identified by this assay were put through an in vitro kinase assay to screen out false positives.
Being an ATM chemical in tissue culture models this assessment method revealed the compound CP466722 as an applicant for characterization. Inhibitory actions against abl and src kinases were known in this in vitro screen, though the ATM related kinase, ATR, wasn’t inhibited by CP466722 in vitro.
As an preliminary evaluation of cellular effects of exposure to CP466722, no negative effects on cell viability were observed in primary and hTERT immortalized human diploid fibroblasts or in a number of human tumor cell lines, despite continuous exposure for 72 hours.