BMS-265246 is a potent and selective CDK1/2 inhibitor. Its chemical name is (4-[R-(2,3-Dihydro-benzo[1,4]dioxin-6-yl)-1H-indol-1-yl]-1H-pyrazolo[3,4-d] pyrimidine-6-amine), and it functions by targeting CDK1 and CDK2 to potentially disrupt cell cycle progression.

CDK4-CyclinD:230 nM, CDK2-CyclinE:9 nM, CDK1-CyclinB:6 nM

CDK1/Cyclin B1 Kinase Assay: Kinase reactions consists of 100 ng of baculovirus expressed GST-CDK1/cyclin B1 complex, 1 μg histone H1, 0.2 μCi 33P γ-ATP, 25 μM ATP in 50 μL of kinase buffer (50 mM Tris, pH 8.0, 10 mM MgCl2, 1 mM EGTA, 0.5 mM DTT). Reactions are incubated for 45 min at 30 °C and stopped by the addition of cold trichloroacetic acid (TCA) to a final concentration of 15%. TCA precipitates are collected onto GF/C unifilter plates using a Filtermate universal harvester, and the filters are quantitated using a TopCount 96 well liquid scintillation counter. Dose response curves are generated to determine the concentration required to inhibit 50% of kinase activity (IC50). BMS265246 is dissolved at 10 mM in DMSO and evaluated at six concentrations, each in triplicate. The final concentration of DMSO in the assay equals 2%. IC50 values are derived by nonlinear regression analysis and have a coefficient of variance (SD/mean, n = 6) = 16%.

HCT-116 cells are plated onto 96-well dishes. For each well, the cell density is calculated by counting the number of objects (cells) per field of view, and averaging across all fields for a given well. For a treatment compound, cell density is converted to a percentage relative to the plate-averaged cell density from DMSO treatment (i.e., 100% corresponds to the average cell density for DMSO treatment). Logistic regression curve fits are done using TIBCO Spotfire, and the concentration at which the curve crosses 50% is reported as the EC50 of BMS-265246.(Only for Reference)