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

ATR:1 nM, HT29 cells:2.6 μM (GI50), DYRK (pS421):10.8 μM, A549 cells:2 μM, PI3Kδ:6.8 μM, DYRK:10.8 μM

METHODS: 276 different tumor cell lines were treated with Ceralasertib for 3 days and cell viability was measured by MTS assay.
RESULTS: For most cell lines, the median GI50 for 50% growth inhibition (1.47 µmol/L) was higher than the IC90 for ATR cells, with only 13% of the cell lines having a GI50 below the median, and 30% below 1 µmol/L. Hematological cell lines (median GI50= 0.82 µmol/L) generally showed increased sensitivity compared to solid tumor cells (median GI50= 1.68 µmol/L). [1]
METHODS: Colorectal cancer cells HT29 were treated with trifluridine (70 µM) and Ceralasertib (0.5 µM) for 48 h, and the expression levels of target proteins were measured by Western Blot.
RESULTS: The trifluridine+Ceralasertib group inhibited Chk1 phosphorylation in HT29 cells at 48 h compared to the trifluridine group. Therefore, it was confirmed that Ceralasertib inhibited Chk1 phosphorylation. In HT29 and HCT116 cells, DNA damage was more severe in the trifluridine+Ceralasertib group than in the trifluridine group, which could be confirmed by the increased level of γH2A expression. [2]

METHODS: To assay anti-tumor activity in vivo, Ceralasertib (10-50 mg/kg, 10% DMSO+40% Propylene Glycol+50% deionized water) was orally administered to LoVo, Granta-519, NCI-H23, or 549 xenograft-carrying athymic nude mice bearing LoVo, Granta-519, NCI-H23 or 549 xenografts once daily for 14-28 days.
RESULTS: LoVo and Granta-519 showed dose-dependent efficacy, with significant TGI at 50 mg/kg, moderate activity at 25 mg/kg, and no activity at 10 mg/kg. Significant antitumor activity was also observed in NCI-H23 but not in A549 model. [1]

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.

H2O: < 1 mg/mL (insoluble or slightly soluble)

Ethanol: 39 mg/mL (94.54 mM), Sonication is recommended.

DMSO: 250 mg/mL (606.05 mM), Sonication is recommended.

10% DMSO+40% PEG300+5% Tween 80+45% Saline: 7.6 mg/mL (18.42 mM), Solution.