Riviciclib hydrochloride (P276-00) is a novel inhibitor of CDK1, CDK4, and CDK9, with IC50 values of 79 nM, 63 nM, and 20 nM, respectively, currently in Phase 2/3.

CDK2-CyclinE:2.500 μM, CDK6-CyclinD1:0.396 μM, CDK4-CyclinD1:63 nM, CDK1-CyclinB:79 nM, CDK9-CyclinT1:20 nM, CDK9-CyclinH:2.900 μM, CDK2-CyclinA:0.224 μM

Riviciclib shows 40-fold selectivity toward Cdk4-D1, compared with Cdk2-E[1]. 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[1]. Riviciclib can down-regulate cyclin D1 and Cdk4 in an ATP- competitive manner and decrease Cdk4-specific pRb Ser780 phosphorylation. Riviciclib also induces apoptosis by actving cellular caspase-3 activity and DNA ladder formation[1].

Riviciclib, administered i.p. at 50 mg/kg daily for 20 treatments can significantly induce growth inhibition of murine colon cancer (CA-51). However, in murine lung carcinoma model (Lewis lung), an increased dose of 60 mg/kg (30 mg/kg twice daily) administered every alternate day i.p. for 7 treatments shows significant inhibition in the growth[2]. And it also inhibit the growth of human colon carcinoma HCT-116 xenograft and human non-small cell lung carcinoma H-460 xenograft[2]. Efficacy Studies show its maximum tolerated dose is 78 mg/kg/d[2].

Cdk4-D1/Cdk2-E enzyme 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.

The cells 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. (Only for Reference)