In Vitro | In Vitro Activity: NU7441 (also called KU-57788) is a highly potent, selective and ATP-competitive DNA-PK inhibitor with IC50 of 14 nM. It also inhibits PI3K with IC50 value of 5 μM in cell-free assays. NU7441 showed no inhibition effect on the DNA-PK-related enzymes ATM and ATR at concentration of 100 μM. For mTOR and PI3K, NU7441 exhibited inhibition activities with IC50 values of 1.7 and 5 μM, respectively, which were about 100-fold higher than the IC50 value of DNA-PK. NU7441 sensitized HeLa cells to ionizing radiation in vitro, with dose modification factors of 2.5 at 10% survival being observed at 0.5 microM. Kinase Assay:NU7441 (also called KU-57788) is a highly potent, selective and ATP-competitive DNA-PK inhibitor with IC50 of 14 nM. It also inhibits PI3K with IC50 value of 5 μM in cell-free assays. Cell Assay: SW620, LoVo, V3-YAC and V3 cells; The effect of NU7441 on cellular survival following exposure to etoposide, doxorubicin, and ionizing radiation is measured in SW620, LoVo, V3, and V3-YAC cells by clonogenic assays. Briefly, growing cells in six-well plates or 6-cm dishes are exposed to etoposide or doxorubicin with or without NU7441 (0.5 or 1.0 μM) for 16 hours. For radiosensitization studies, NU7441 is added to the cells 1 hour before irradiation. V3 and V3-YAC cells are exposed to γ-irradiation (3.1 Gy/min 137Cesium). SW620 and LoVo are exposed to X-irradiation (2.9 Gy/min at 230 kV, 10 mA) due to the equipment available. After irradiation, the cells are incubated with or without NU7441 for a further 16 hours. Cells are then harvested by trypsinization, counted, and seeded into 10-cm diameter Petri dishes at densities varying from 100 to 105 per dish in drug-free medium for colony formation. Colonies are stained with crystal violet after 10 to 14 days and counted with an automated colony counter. The survival reduction factor (SRF) is calculated as the surviving fraction of cells in the absence of NU7441 divided by the surviving fraction of cells in the presence of NU7441 for any given dose or concentration of cytotoxic agent. The dose modification ratio (DMR90) is calculated as the concentration/dose of cytotoxic agent required to kill 90% of the cells in the absence of NU7441 divided by the concentration/dose of cytotoxic agent required to kill 90% of the cells in the presence of NU7441. NU7441 increases the persistence of γH2AX foci after ionizing radiation–induced or etoposide-induced DNA damage. NU7441 (0.5 μM or 1 μM) appreciably increases G2-M accumulation induced by ionizing radiation, etoposide, and doxorubicin in both SW620 and LoVo cells. NU7441 causes persistence of doxorubicin- and ionising radiation-induced DNA double-strand break and also slightly decreases homologous recombination activity DNA-PK-proficient M059-Fus-1 and DNA-PK-deficient M059 J human tumour cells. NU7441 inhibits UV-induced RPA p34 hyperphosphorylation in a dose-dependent manner both in cells lacking and cells expressing polymerase η. NU7441 increases levels of fludarabine-induced γH2AX foci and correspondingly decreased fludarabine-induced cell death in chronic lymphocytic leukemia cells. NU7441 also inhibits mitoxantrone-induced DNA-PKcs autophosphorylation and repair in chronic lymphocytic leukemia cells. |