| In Vitro | In vitro activity: P276-00 shows 40-fold selectivity toward Cdk4-D1, compared with Cdk2-E. It shows potent antiproliferative effects against various human cancer cell lines, including HCT-116, U2OS, H-460, HL-60, HT-29, SiHa, MCF-7, Colo-205, SW-480, PC-3, Caco2, T-24 with an IC50 ranging from 300 to 800 nmol/L, and is found to be highly selective for cancer cells as compared with normal fibroblast cells. P276-00 can down-regulate cyclin D1 and Cdk4 in an ATP- competitive manner and decrease Cdk4-specific pRb Ser780 phosphorylation. P276-00 also induces apoptosis by actving cellular caspase-3 activity and DNA ladder formation.
Kinase Assay: The Cdk4-D1/Cdk2-E enzyme assay is run in 96-well format using Millipore Multiscreen filtration plates. All assay steps are done in a single filter plate. The filtration wells are prewetted with 100 μL of kinase buffer [50 mmol/L HEPES (pH, 7.5), 10 mmol/L MgCl2, 1 mmol/L EGTA], and then the solution is removed by vacuum. With filter plate on vacuum manifold, 50 μL GST-Rb bound to GSH-Sepharose beads in kinase buffer (0.5 μg GST-Rb/50 μL) is added to each well, and vacuum is applied to the filter plate. About 25 μL of a reaction mix containing ATP (cold + hot) and 4× phosphatase inhibitor mix (40 μmol/L unlabeled ATP, 10 μCi/mL γ32P-ATP, 40 mmol/L h-glycerophosphate, 4 mmol/L DTT, 0.4 mmol/L NaF, 0.4 mmol/L sodium orthovanadate) diluted in kinase buffer is added to each well. The test compound (4×final concentration in kinase buffer) or kinase buffer alone (control) is then added in an additional 25 μL volume. To each well, 50 μL (100 ng) of human Cdk4-D1/Cdk2-E enzyme in kinase buffer is added to initiate the reaction, which is allowed to continue for 30 min at 30°C. When the reaction is completed, vacuum is applied again, and the plate is washed with the TNEN buffer [20 mmol/L Tris (pH, 8.0), 100 mmol/L NaCl, 1 mmol/L EDTA, 0.5% nonidet-P40] thrice; the filter plate is air-dried and is placed in a Multiscreen adapter plate. Packard Microscint-O cocktail (30 μL) is added, and the plate is covered with a Top-Seal A film. Quantitation of 32P-GST-Rb in 96-well filter plates is carried out by Top Count scintillation counter. All compounds are tested initially at 1 μmol/L concentration. Compounds showing more than or equal to 50% inhibition are further profiled for IC50 determination.
Cell Assay: The cells (HCT-116, U2OS, H-460, HL-60, HT-29, SiHa, MCF-7, Colo-205, SW-480, PC-3, Caco2, T-24) are seeded at a density of 3,000-5,000 cells per well, depending on cell type in 180 μL of culture medium in 96-well plate and incubated overnight to allow the cells to adhere. Varying concentrations of compounds are added to the wells and incubated for 48 h at 37°C. 3H-thymidine (0.25 μCi) is added to each well, and incorporation of the radiolabel is allowed to proceed for 5 to 7 h. Following this incubation, cells are harvested onto GF/B unifilter plates using a Packard Filtermate Universal harvester, and the plates are counted in a Packard Top Count 96-well liquid scintillation counter. |
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