CVT-313 (NG-26) is a potent, selective, reversible, and ATP-competitive inhibitor.

CDK2:0.5 μM

CVT-313已显示在抑制其他激酶方面的作用，但其IC50值相较于CDK2 (IC50=0.5 μM)显著较高，具体包括CDK1 (IC50=4.2 μM)、CDK4 D1 (IC50=215 μM)以及MAPK/PKA/PKC (IC50>1.25 mM)。研究表明，CVT-313在5-20 μM浓度下对细胞增殖有显著影响[1]。CVT-313是从嘌呤类似物库中筛选出的一种强效CDK2抑制剂，体外IC50为0.5 μM。其抑制作用与ATP竞争性相关(Ki=95 nM)，且表现出对于非相关ATP依赖性丝氨酸/苏氨酸激酶无影响的选择性。当添加至CDK1或CDK4时，需CVT-313浓度分别增至8.5倍和430倍才能实现酶活性的半最大抑制。通过使用正常及肿瘤人类/鼠类细胞系，对CVT-313对细胞增殖的影响进行了测量，其生长抑制的IC50范围为1.25至20 μM[2]。

For kinase assays, purified CDC5L(295-795)-His6 is mixed with [γ-32P]ATP, COS-7 cell extract, and incubated in 100 μL 20 mM HEPES, pH 7.5, 50 mM NaCl, 2 mM MnCl2, 10 mM MgCl2, 0.5% NP-40, 0.5 mM PMSF, 5 mM benzamidine hydrochloride, 5 mM NaF, 1 mM NaVO3 and the specific inhibitor at 30°C for 10 minutes. Cell extract as a source of kinase activity is prepared from subconfluent, serum-stimulated COS-7 cells lysed in 20 mM HEPES-NaOH, pH 7.5, 50 mM NaCl, 1% Triton X-100, 10% glycerol, protease and phosphotase inhibitors. Phosphorylated proteins are separated by electrophoresis in 15% polyacrylamide-SDS gels. Specific inhibitors included 20 μM staurosporine, 10 μM genistein, 1 μM CVT-313, 10 μM Rp-MB-cAMPS and 50 μM PD98059[1].

CVT313 is prepared in DMSO and stored, and then diluted with appropriate medium before use[2]. MRC-5 cells are grown in Dulbecco's modified Eagle's medium containing 5% fetal calf serum. CVT313 (0, 5, 10, 15 μM) is added to exponentially growing cells in tissue culture. Cell population is measured. Proliferation assays are carried out using the nonradioactive CellTiter 96 kit after 48-h exposure. For FACS analysis of DNA content, cells are trypsinized, fixed in 70% ice-cold ethanol, and treated with 0.1 mg/mL RNase A and 40 μg/mL propidium iodide for 1 h at 37°C[2].