Berzosertib (VE-822) is an ATR inhibitor (Ki<0.2 nM) that also inhibits ATM (Ki=34 nM). Berzosertib has antitumor activity and has been used in trials investigating the treatment of ovarian tumors, plasmacytoid tumors of the ovary, solid tumors in adults, advanced solid tumors, and advanced solid tumors.

ATR:19 nM

方法: 人胰腺癌细胞 PSN-1 和 MiaPaCa-2 用 Berzosertib (80 nmol/L) 和 gemcitabine (100 nM)、XRT (6 Gy) 处理，使用 Western Blot 检测靶点蛋白表达水平。
结果: Berzosertib 减少了磷酸化 Ser345-Chk1。Berzosertib 在辐射反应中没有抑制 ATM、Chk2 或 DNA-PK 磷酸化，这进一步支持了 Berzosertib 对 ATR 的选择性。[1]
方法: 骨肉瘤细胞 MNNG/HOS 和 143B 用 Berzosertib (0-100 µM) 处理 48 h，使用 MTT assay 检测细胞活力。
结果: Berzosterib 导致 MNNG/HOS 和 143B 细胞存活率呈剂量依赖性下降。[2]

方法: 为检测体内抗肿瘤活性，用 Berzosertib (60 mg/kg，灌胃给药，每天一次) 和 XRT (6 Gy，一次) 治疗携带 PSN-1 异种移植物的 Nude 小鼠，给药六天。
结果: 单独使用 Berzosertib 对肿瘤生长没有影响，但 XRT 加 Berzosertib 给药 6 天或 4 天，使单独使用 XRT 的 TV600 增加了一倍多。[1]

A375 cells are pre-treated with 1 μM JNK-IN-8 for the indicated amounts of time. Remove the medium and wash 3 times with PBS. Resuspend the cell pellet with 1 mL Lysis Buffer (1% NP-40, 1% CHAPS, 25 mM Tris, 150 mM NaCl, Phosphatase Inhibitor Cocktail, and Protease Inhibitor Cocktail). Rotate end-to-end for 30 min at 4°C. Lysates are cleared by centrifugation at 14000 rpm for 15 min in the Eppendorf. The cleared lysates gel filtered into Kinase Buffer (0.1% NP-40, 20 mM HEPES, 150 mM NaCl, Phosphatase Inhibitor Cocktail, Protease Inhibitor Cocktail) using Bio-Rad 10DG colums. The total protein concentration of the gel-filtered lysate should be around 5-15 mg/mL. Cell lysate is labeled with the probe from ActivX at 5 μM for 1 hour. Samples are reduced with DTT, and cysteines are blocked with iodoacetamide and gel filtered to remove excess reagents and exchange the buffer. Add 1 volume of 2X Binding Buffer (2% Triton-100, 1% NP-40, 2 mM EDTA, 2X PBS) and 50 μL streptavidin bead slurry and rotate end-to-end for 2 hours, centrifuge at 7000 rpm for 2 min. Wash 3 times with 1X Binding Buffer and 3 times with PBS. Add 30 μL 1X sample buffer to beads, heat samples at 95°C for 10 min. Run samples on an SDS-PAGE gel at 110V. After transferred, the membrane is immunoblotted with JNK antibody[1].

VE-822 is dissolved in DMSO and stored, and then diluted with appropriate media before use[1]. Gemcitabine (10 nM) is added 24 h pre-XRT and is replaced with fresh medium before addition of VE-822. PSN-1 cells are treated with VE-822 (80 nM) for 1 h before, through to 18 h after, XRT (6 Gy). Apoptosis is analyzed 48 h after XRT by flow cytometry using an Annexin V-FITC kit with PI[1].