ATR/ATM kinases can phosphorylate a broad and overlapping catalogue of several thousand substrates in DNA damage signaling. The kinase activity of ATR, activated by single-stranded DNA (ssDNA) coated with replication protein A (RPA), is increased at damaged replication forks and resected DNA double-strand breaks (DSBs). AZD6738 is a highly selective and potent inhibitor of ATR kinase activity with IC50 value of 1nM in isolated enzyme assay and showed inhibitory effect on ATR kinase-dependent cellular Chk1 phosphorylation with IC50 value of 74nM. AZD6738 impaired viability of the Kras mutant cell line, H23, H460, A549 and H358, with the lowest GI50 and greatest maximal inhibition in H460 and H23 cells (1.05μM, 88.0% and 2.38μM, 86.2%, respectively). Meanwhile, induced activation of ATM, as evident by increased pATM-S1981, stabilized p53 and expression of p21 and p27 can be observed in p53-wildtype H460 and A549 cells treated with 1μM AZD6738. A marked increase in pH2A.X-ser139 and cleaved PARP, indicative of accumulation of DNA damage and induction of apoptosis with greater sensitivity in cell viability assay, can be observed in H23 and H460 cells treated with AZD6738. Combination of AZD6738 (1μM) and cisplatin caused accumulation of cells in early S-phase and at the G1/S border, as well as dramatic cell death of H23 and H460 cells, suggesting its potent synergy with cisplatin in ATM-deficient cells independent of the ATM-p53 signaling pathway. Consistent with the in vitro study, daily administration of AZD6738 at dose of 25mg/kg for 14 consecutive days enhanced the therapeutic efficacy of cisplatin (3mg/kg) and caused rapid regression of ATM-deficient H23 tumors[1].
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