In vitro activity: Ro-3306 also inhibited CDK1/cyclin A complexes with Ki of 110 nM. In full agreement with this model, treatment of proliferating human cancer cells (HCT116, SW480, and HeLa) with RO-3306 for 20 h led to a complete block of the cell cycle in the G2/M phase. Treatment of growing AML cells with RO-3306 induced G2/M-phase cell cycle arrest and apoptosis in a dose- and time-dependent manner. RO-3306 acts cooperatively with Nutlin-3 to induce mitochondrial apoptosis in a cell cycle-independent fashion. RO-3306 downregulated expression of the antiapoptotic proteins Bcl-2 and survivin and blocked p53-mediated induction of p21 and MDM2. The GV-arrest effect of RO-3306 was reversible: when RO-3306-arrested COCs were subsequently cultured for 24h in the absence of RO-3306, 76.19 ± 2.68% of these oocytes reached the MII stage after 44 h of in vitro maturation, a rate similar to that of non-treated control oocytes (79.08 ± 3.23%). Furthermore, RO-3306-treated oocytes transferred to drug-free media did not differ significantly from controls (P>0.05) with respect to cleavage and blastocyst formation upon parthenogenetic activation.

Kinase Assay: The activity of CDK1/cyclin B1, CDK1/cyclin A, CDK2/cyclin E, and CDK4/cyclin D was 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 μM (CDK2), or 135 μM (CDK4). CDK1 and CDK2 buffer contained 10 mM MgCl2. Test compounds were diluted in assay buffer to 3-fold their final concentration in 20 μl, and the reaction was started by the addition of a 40-μl assay buffer containing the pRB substrate (0.185 μM). The plates were incubated at 37°C for 30 min with constant agitation, and the reaction was terminated by the addition of 15 μl of 1.6 μM anti-phospho pRB antibody (Ser-780; Cell Signaling Technology, Beverly, MA) in 25 mM Hepes/24 mM EDTA/0.2 mg/ml BSA. After an additional 30 min of incubation with shaking, 15 μl of 3 nM Lance-Eu-W1024-labeled anti-rabbit IgG and 60 nM Alophycocyanin-conjugated anti-His-6 antibody (PerkinElmer Life Sciences) in 25 mM Hepes/0.5 mg/ml BSA was added and incubated for 1 h. The plates were read in the Victor-V multilabel reader (PerkinElmer Life Sciences) at excitation 340 nm and emission 615 nm and 665 nm. The IC50 values were calculated from the readings at 665 nm and normalized for Europium readings at 615 nm. Ki values were 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. Cell assay [1]: All cell lines used in this study were purchased from American Type Culture Collection (ATCC, Manassas, VA) and grown in the recommended media supplemented with 10% heat-inactivated FBS (Invitrogen) in a humidified environment with 5% CO2. Drugs were dissolved in DMSO and kept at 20°C as 10 mM stock solutions. For cell cycle analysis, 106 cells were grown in 75 cm2 tissue-culture flasks.

Cell Assay: In viability assays, RO-3306 induced a significant shift to the left of the AZ12253801 dose-response curve, with 2.4-fold reduction in GI50, comparable to RAD51 depletion or inhibition. At sub-50nM AZ12253801 concentrations there was more striking sensitization, with 13-fold reduction in GI80. Paralleling effects of RAD51 knockdown, RO-3306 did not affect AZ12253801 sensitivity of PC3 or LNCaP cells, but did sensitize 22Rv1 cells.

Proc Natl Acad Sci U S A. 2006 Jul 11;103(28):10660-5; Cancer Sci. 2009 Jun;100(6):1128-36; Anim Reprod Sci. 2014 Jan 30;144(3-4):102-8.