VE-821 (ATR Inhibitor IV) is a selective ATP competitive inhibitor of ATR( Ki/IC50: 13/26 nM in cell-free assays).

ATR:13 nM (Ki, cell free)

VE-821 在对ATR的选择性上表现出色，对其相关的PIKKs，包括ATM、DNA依赖性蛋白激酶（DNA-PK）、哺乳动物雷帕霉素靶蛋白和磷酸肌醇3激酶-γ的交叉反应性极小（其Kis分别为16 μM、2.2 μM、>1 μM和3.9 μM）。VE-821在经羟基脲处理的HT29癌细胞中阻断了H2AX的磷酸化，但对用新霉素治疗的M059J或HT144细胞线无影响[1]。VE-821显著提高了PSN-1、MiaPaCa-2和原发性PancM胰腺癌细胞对放射和吉西他滨的敏感性，无论是在正常氧还是缺氧条件下。VE-821通过ATR抑制显著阻止了癌细胞中由辐射引起的G2/M阶段阻滞[2]。在OVCAR-8细胞中，VE-821（1和4 μM）增强了由拓扑替康和顺铂诱导的Ser139位点上H2AX的磷酸化。VE-821未能阻断由吉西他滨、拓扑替康或顺铂触发的ATR介导的Ser345 Chk1或Ser296自磷酸化[3]。

The ability of compounds (e.g., VE-821) to inhibit ATR, ATM or DNAPK kinase activity is tested using a radiometric-phosphate incorporation assay. A stock solution is prepared consisting of the appropriate buffer, kinase, and target peptide. To this is added the compound of interest, at varying concentrations in DMSO to a final DMSO concentration of 7%. Assays are initiated by addition of an appropriate [g-33P]ATP solution and incubated at 25°C. Assays are stopped, after the desired time course, by addition of phosphoric acid and ATP to a final concentration of 100 mM and 0.66 μM, respectively. Peptides are captured on a phosphocellulose membrane, prepared, and washed six times with 200 μL of 100 mM phosphoric acid, prior to the addition of 100 μL of scintillation cocktail and scintillation counting on a 1450 Microbeta Liquid Scintillation Counter. Dose-response data are analyzed using GraphPad Prism software [4].

Clonogenic survival assays were performed as described before. Briefly, logarithmically growing cells were plated in triplicate in 6-well tissue culture dishes under oxic (21% O2) or hypoxic conditions (0.5% O2) using an InVivo2 300 chamber. Cells were incubated for 6 h before irradiation under oxia or hypoxia using tightly sealed chambers. The target O2 level was achieved within 6 h of gassing and maintained during irradiation, as confirmed by an OxyLite oxygen probe. Cells irradiated under hypoxia were exposed to normoxia at 1 h post-irradiation. As standard, VE-821 (1 μM) was added 1 h prior to irradiation (6 Gy) and was washed away 72 h after irradiation. For the chemotherapy experiments, cells were initially exposed to increasing concentrations of gemcitabine (5, 10 and 20 nM) for 24 h before addition of the VE-821 (1 μM) for another 72 h. The effect of triple combination of irradiation with VE-821 and gemcitabine was examined as well. Cells were incubated for 10–21 d until colonies were stained with 0.5% crystal violet and counted in a CellCount automated colony counter. Clonogenic survival was calculated and data were fitted in GraphPad Prism 4.0 [2].