Ro 3306 is a CDK1 inhibitor that inhibits CDK1, CDK1/cyclin B1, and CDK2/cyclin E (Ki=20/35/340 nM) selectively and ATP-competitively. Ro-3306 exhibits antitumor activity, inhibits cell cycle arrest, and induces apoptosis.

SGK:497 nM(Ki), ERK:1980 nM(Ki), CDK1:20 nM(Ki), PKCδ:318 nM(Ki)

方法：卵巢癌细胞 OVCA-429 和 OVCAR-3 用 Ro 3306 (1-2.5 nM) 处理 9 天，使用 crystal violet assay 检测细胞活力。
结果：OVCA-429 和 OVCAR-3 细胞在高剂量浓度的 Ro 3306 下长达 9 天的生长速率较低。使用 2.5 µM Ro 3306 处理，OVCA-429 和 OVCAR-3 细胞的生长率在第 9 天分别降低了 75.3% 和 87.7%。[1]
方法：人肿瘤细胞系 HCT116、SW480 和 HeLa 用 Ro 3306 (9 µM) 处理 20 h，使用 Flow cytometry 检测细胞周期。
结果：用 Ro 3306 对增殖的人类肿瘤细胞处理 20 h 导致 G2/M 期细胞周期的完全阻断。[2]

方法：为检测体内抗肿瘤活性，将 Ro 3306 (4 mg/kg) 和 Cisplatin (4 mg/kg) 腹腔注射给携带卵巢癌肿瘤 OVCAR-3 的 BALB/c nude 小鼠，每四天一次，持续四周。
结果：与 Ro 3306 或 Cisplatin 治疗相比，联合治疗有效抑制了肿瘤生长。[1]

CDK assay: The activity of CDK1 cyclin B1, CDK1 cyclin A, CDK2 cyclin E, and CDK4 cyclin D is measured by a homogeneous time-resolved fluorescence assay in a 96-well format. The assay buffer contained 25 mM Hepes, 6.25 mM MgCl2, 0.003% Tween 20, 0.3 mg/mL BSA, 1.5 mM DTT, and ATP as follows: 162 μM (CDK1), 90 mM (CDK2), or 135 μM (CDK4). CDK1 and CDK2 buffer contained 10 mM MgCl2. Test compounds are diluted in assay buffer to 3-fold their final concentration in 20 μL, and the reaction is started by the addition of a 40 μL assay buffer containing the pRB substrate (0.185 μM). The plates are incubated at 37°C for 30 min with constant agitation, and the reaction is terminated by the addition of 15 μL of 1.6 μM anti-phospho pRB antibody (Ser-780) in 25 mM Hepes, 24 mM EDTA, and 0.2 mg/mL BSA. After an additional 30 min of incubation with shaking, 15μL of 3 nM Lance-Eu-W1024-labeledanti-rabbitIgG and 60 nM Alophycocyanin-conjugated anti-His-6 antibody in 25 mM Hepes, and 0.5 mg/mL BSA is added and incubated for 1 h. The plates are read in the Victor-V multi- label reader at excitation 340 nm and emission 615 nm and 665 nm. The IC50 values are calculated from the readings at 665 nm and normalized for Europium readings at 615 nm. Ki values are calculated according to the equation: Ki= IC50/(1 + S/Km ), where S is the ATP concentration in the assay and Km is the Michaelis-Menten constant for ATP. The inhibitory activity against the panel of kinases is determined by the IMAP assay technology.

Log phases cells (25,000) are seeed in 96-well plates and incubated in a 37℃ incubator with CO2, After 24 h, different concentrations of RO-3306 are administered to determine the drug concentrations required to achieve a 50% growth inhibition (IC50). MTT (20 μL, 5 mg/mL stock solution in saline) is added to each well and the cells are incubated for 4 h. Supernatants are removed and formazan crystals from viable cells are solubilized with 200 μL anhydrous DMSO. The absorbance is detected with a 550 model microplate reader at the 565 nm wavelength.(Only for Reference)