Ceralasertib (AZD6738) is an orally active, and selective ATR kinase inhibitor with IC50 of 1 nM.

In four Kras mutant cell lines: H23, H460, A549, and H358,AZD6738 inhibits ATR kinase activity and impairs cell viability. AZD6738 strongly synergizes with cisplatin in an ATM-deficient NSCLC cell line in vitro[1]. In p53 or ATM defective cells, AZD6738 treatment resulted in replication fork stalls and accumulation of unrepaired DNA damage, as evidenced by γH2AX and 53BP1 foci formation, which was carried through into mitosis, resulting in cell death by mitotic catastrophe[2].

The combination of 50 mg/kg AZD6738 and cisplatin resulted in a 75.5% mean tumor growth inhibition (TGI) of H460 xenografts at day 14 (P ≤ 0.0001 compared to vehicle). Growth of tumors treated with the combination was also significantly different from that of tumors treated with cisplatin or AZD6738 alone [1]. In LoVo derived mouse xenograft, a combination of AZD6738 (50?mg/kg)?+?IR (2?Gy) can be adjusted to avoid toxicity while still maintaining efficacy[3].

General procedure for the EC50 test: DDR1 is induced by 2 Gg/ml doxycycline for 48 hrs prior to DDR1 activation by rat tail collagen I. The DDR1 over-expressed U2OS is pre-treated by media containing each concentration of the compound for 1 hr and treated by changing the media to the EC50 test media containing 10 Gg/ml collagen and each concentration of the compound for 2 hrs. Each cells is washed with cold PBS three times and lysed with the lysis buffer (50 mMTris, pH 7.5, 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 5 mM EDTA, 100 mMNaF, 2 mM Na3VO4, 1 mM PMSF, 10 Gg/ml aprotinin, and 10 Gg/ml leupeptin). The activation of DDR1 is quantified by density using program ImageJ to determine EC50 following Western blot using anti-activated human DDR1b (Y513).

Cells are treated in white walled, clear bottom 96-well plates with the indicated doses of AZD6738, cisplatin, gemcitabine, or combination for 48 h. ATP levels are assessed as surrogate measure of viability was assessed using the CellTiter-Glo Luminescent Cell Viability Assay and Safire 2 plate reader. Raw data are corrected for background luminescence prior to further analysis. For AZD6738 treatment, log dose response curves are generated in GraphPad Prism 6 by nonlinear regression (log(inhibitor) vs. response with variable slope) of log-transformed (x = log(x)) data normalized to the mean of untreated controls. GI values, defined as the dose X at which Y = 50%, were extrapolated from dose response curves.