MK-1775 was prepared in a vehicle of 0.5% methylcellulose solution and was dosed p.o.24 h after dosing DNA-damaging agents.F or 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 ? 100 if ?T > 0 or ? 100 if ?T < 0.?T was the change in mean tumor volume to the initial tumor volume for the treatment group, and ?C was the change in mean tumor MEK Inhibitor selleck volume to the initial tumor volume for the vehicle control group.Ti was the initial tumor volume of the treatment group.For all biomarker assays, tumors were isolated 8 h after MK-1775 administration.The CDC2 protein was solubilized by homogenizing cells in a buffer containing 1% NP40 and 0.1% Triton X-100 and was detected by Western blotting with an anti?p-CDC2Y15 specific antibody.For pHH3 immunohistochemistry, tumors were fixed in 10% formalin, paraffin embedded, and sectioned.Sec tions were incubated with rabbit polyclonal anti-pHH3 Ser10 antibody followed by incubation with biotinylated goat anti-rabbit IgG antibody and then with streptavidin/horseradish peroxidase.
Signal was detected by development with peroxidase substrate.Immunostained area was quantified using Image Pro Plus software.N ecrotic regions of the tumor were excluded from the analysis.The percentage of area positively immunostained in each tumor was calculated as the percentage of the total field area.
For p-CDC2Y15 measurements in skin, tissue was fixed and sectioned as described above for tumor tissue.Skin tissue sections were probed with the same antibody used for Western blots.Detection of captured antibodies was done as with pHH3 immunohistochemistry.Results Olaparib selleck MK-1775 Inhibits Phosphorylation of CDC2 at Tyr15 and Abrogates the G2 DNA Damage Checkpoint in a Dose-Dependent Manner A high-throughput screening was done with a small chemical compound library to find potent inhibitors of Wee1 kinase in enzymatic assay.Modification of the initial hit compounds by leveraging the information on structureactivity relationships led to the identification of a potent and selective small-molecule inhibitor of Wee1 kinase, MK-1775 , with an IC50 value of 5.2 nmol/L in in vitro kinase assays.A n increasing linear relationship was observed between the IC50 value of MK-1775 and ATP concentration in an enzyme assay, suggesting that MK-1775 inhibited Wee1 kinase in an ATP-competitive manner.MK-1775 is highly selective against other serine/threonine or tyrosine kinases.