Two purchase PF-562271 distinct siRNAs directed against CHK1 were tested in order to minimize the likelihood of off target effects. With both siRNAs, GM6914 cells were more sen sitive to CHK1 knockdown than the corrected cell line. For CHK1 1 siRNA, the uncorrected cells exhibited only 42% viability after siRNA treatment, while the corrected cells showed 76% viability. For CHK1 2 siRNA, the uncorrected cells exhibited only 40% viabil ity after siRNA treatment, whereas the corrected cells showed 65% viability. Of note, we previ ously reported that FA deficient cell lines are hypersensi tive to ATM inhibition. The magnitude of the FA specific killing by CHK1 silencing reported here is compa rable to that observed for ATM silencing. A Western blot was performed to confirm silencing of CHK1 and better characterize the molecular nature of the FA CHK1 interaction.

Silencing with the CHK1 1 siRNA resulted in decreased CHK1 pro tein levels and increased phospho Inhibitors,Modulators,Libraries H2Ax levels, particu larly in the FANCA deficient cells. This result suggests that the CHK1 and FA genes function in compen satory manner to maintain genome integrity. Consistent with this model, the GM6914 FANCA cor rected cell line demonstrated enhanced FANCD2 monou biquitination following Inhibitors,Modulators,Libraries knockdown of CHK1, suggesting that the FA pathway is activated fol lowing loss of CHK1 function. FA deficient cells are hypersensitive to pharmacologic CHK1 inhibition To further safeguard against siRNA off target effects, we wished to confirm our observation using a pharmacologic inhibitor of CHK1.

Recent studies have indicated that some of the small molecular inhibitors initially thought to be CHK1 specific possessed activities against related kinases. As more small molecule Inhibitors,Modulators,Libraries kinase inhibitors are subjected to detailed scrutiny, it is becoming increasingly clear that absolute specificity remains elusive. Neverthe less, specificity of each Inhibitors,Modulators,Libraries inhibitor class has improved with each generation of refinement. We searched the literature for a CHK1 inhibitor with high specificity and identified G?6976. In a study where the specificities of 65 com monly used small molecule kinase inhibitors were tested for inhibition of a panel of 80 purified protein kinases, G?6976 was shown to exhibit relative specificity against CHK1. At sub micro molar concentration, the specificity of G?6976 against CHK1 was over 40 fold that of CHK2, Inhibitors,Modulators,Libraries 100 fold that of MAPKAP K2, and 30 fold that of MKK1 and MKK2.

We, therefore, examined FANCA, FANCG, and FANCD2 mutant and paired isogenic corrected cell lines and com pared the sensitivity of the lines to G?6976. In each case, the FA pathway deficient cell line was more sen sitive to G?6976. The LC50 for the FA deficient cell lines dig this ranged 250 500 nM whereas the LC50 for the isogenic FA proficient cell lines ranged 1 2 uM.