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

METHODS: Human pancreatic cancer cells PSN-1 and MiaPaCa-2 were treated with Berzosertib (80 nmol/L) and gemcitabine (100 nM), XRT (6 Gy), and the expression levels of target proteins were detected by Western Blot.
RESULTS: Berzosertib reduced phosphorylated Ser345-Chk1. Berzosertib did not inhibit ATM, Chk2 or DNA-PK phosphorylation in response to radiation, which further supports the selectivity of Berzosertib for ATR. [1]
METHODS: Osteosarcoma cells MNNG/HOS and 143B were treated with Berzosertib (0-100 µM) for 48 h. Cell viability was measured by MTT assay.
RESULTS: Berzosterib caused a dose-dependent decrease in MNNG/HOS and 143B cell viability. [2]

METHODS: To assay antitumor activity in vivo, Nude mice bearing PSN-1 xenografts were treated with Berzosertib (60 mg/kg, once daily by gavage) and XRT (6 Gy, once) for six days.
RESULTS: Berzosertib alone had no effect on tumor growth, but XRT plus Berzosertib administered for six or four days more than doubled the TV600 of XRT alone. [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].