Fig. 1

AZD7648 is a potent radiosensitizer in vitro. a Optimization of screening hit 1 into the potent and selective DNA-PK inhibitor, AZD7648. b Western blot analysis of A549 cells treated with 8 Gy IR ± 1 h pre-treatment with increasing concentrations of AZD7648. c High-content imaging analysis of indicated DNA damage markers in A459 cells. Cells were treated with 2 Gy IR ± AZD7648 pre-treatment (1 h) and immunofluorescently stained at indicated time points. Graphs represent mean foci, number of cells, micronuclei per cell or relative nuclear fragmentation from two independent experiments (n = 2, ±SEM). Black dotted lines indicate AZD7648 cellular IC₅₀ concentration (91 nM). Representative images are shown for 2 Gy IR ± 1.25 μM AZD7648 at 72 h (scale bars, 25 μM). D represents DMSO vehicle-treated controls. d Cell cycle analysis was performed based on DAPI staining intensity of nuclei detected by imaging analysis. Graphs represent cell cycle distribution (percentage population) from a representative experiment (n = 2). N/A (non-assigned) represents cell populations where signal intensities exceeded the threshold to accurately determine the cell cycle phase. Dotted lines indicate AZD7648 cellular IC₅₀ concentration (91 nM). e Colony formation assays performed with A549 or NCI-H1299 cells treated with an ionizing radiation dose response ± AZD7648. Graphs represent mean surviving fraction normalized to the single-agent activity of AZD7648. Data were fitted to the linear quadratic model (mean ± SD (n = 2); unpaired t-test where P ≤ 0.05 is significant). Mean dose enhancement factor values (DEF₃₇) are shown. DAPI 4′,6-diamidino-2-phenylindole, DMSO dimethyl sulfoxide, SD standard deviation, SEM standard error of the mean