Adavosertib (MK-1775) is a selective inhibitor of the cell cycle regulatory protein Wee1 (IC50=5.2 nM). Adavosertib has antitumor activity that blocks the cell cycle and inhibits tumor cell proliferation.

MCF7 cells:1.1 μM, NCI-H1299 cells:0.2837 nM, MM1.S cells:0.31 μM, HEK-293T cells:0.29 μM, MDA-MB-231 cells:0.26 μM, Daoy cells:150 nM, PC3 cells:8 μM, LNCaP cells:0.5 μM, A-427 cells:78 nM, NCI-H23 cells:122 nM, MV4-11 cells:95 nM, BT-549 cells:0.49 μM, Wee1:5.2 nM (cell free)

Kinase reaction was conducted with 10 μmol/L ATP, 1.0 μCi of [γ-33P]ATP, and 2.5 μg of poly(Lys, Tyr) as a substrate at 30°C for 30 min. Radioactivity incorporated into the substrate was trapped on MultiScreen-PH plates and was counted on a liquid scintillation counter [1].

Tumor cells were cultured in 96-well plates and incubated with DNA-damaging agents for 24 h, then with MK-1775 and nocodazole for additional 8 h. For p-CDC2Y15 assay, cells were lysed and subjected in a colorimetric ELISA to determine the amounts of p-CDC2Y15 (1:100) and total CDC2 (1:200). For phospho-histone H3 (pHH3), cells were fixed with methanol, stained with anti-pHH3 specific antibody and bound antibody was stained with Alexa Fluor 488 goat anti-rabbit antibody. Images were acquired with an INCell Analyzer 1000 [1].

Subcutaneous xenograft tumors were formed by injection of the human cancer cell lines in the hind flank of immunodeficient nude rats (F344/NJcl-rnu). To facilitate tumor formation, cells were injected in medium containing Matrigel, a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma. Gemcitabine, carboplatin, and cisplatin were dissolved or diluted in saline and were dosed i.v. MK-1775 was prepared in a vehicle of 0.5% methylcellulose solution and was dosed p.o. 24 h after dosing DNA-damaging agents. For efficacy studies, tumor volumes were measured with a caliper every 3 d and body weights were determined each weekday. Statistical analysis was done using repeated-measure ANOVA followed by Dunnett's test for relative tumor volume. T/C (%) was calculated as (ΔT/ΔC) × 100 if ΔT > 0 or (ΔT/TI) × 100 if ΔT < 0. ΔT was the change in mean tumor volume to the initial tumor volume for the treatment group, and ΔC was the change in mean tumor volume to the initial tumor volume for the vehicle control group. Ti was the initial tumor volume of the treatment group [1].