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

CDK1-CyclinB:79 nM, CDK9-CyclinT1:20 nM, CDK4-CyclinD1:63 nM

Riviciclib对Cdk4-D1显示出比对Cdk2-E[1]高40倍的选择性。它对多种人类癌症细胞系展现出强大的抗增殖效果，包括HCT-116、U2OS、H-460、HL-60、HT-29、SiHa、MCF-7、Colo-205、SW-480、PC-3、Caco2、T-24，其IC50范围从300到800 nmol/L，且相较于正常成纤维细胞，对癌细胞显示出高度的选择性[1]。Riviciclib能以与ATP竞争的方式下调cyclin D1和Cdk4，并减少Cdk4特异性的pRb Ser780磷酸化。Riviciclib还通过激活细胞caspase-3活性和DNA阶梯形成诱导凋亡[1]。

Riviciclib通过腹膜注射给药方式，每日50 mg/kg，连续治疗20次，可显著抑制小鼠结肠癌(CA-51)的生长。然而，在小鼠肺癌模型(Lewis lung)中，通过每隔一天腹膜注射60 mg/kg（每日两次，每次30 mg/kg），共治疗7次，也显示出显著的生长抑制效果[2]。此外，Riviciclib还能抑制人类结肠癌HCT-116异种移植物和人类非小细胞肺癌H-460异种移植物的生长[2]。有效性研究表明，其最大耐受剂量为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)