Milciclib

K-Ras(G12D)LA2 mice

N, 1, 4, 4-tetramethyl-8-(4-(4-methylpiperazin-1-yl)phenylamino)-4, 5-dihydro-1H-pyrazolo[4, 3-h]quinazoline-3-carboxamide

PHA-848125 is a potent, ATP-competitive cyclin A/CDK2 inhibitor with IC50 of 45 nM.

5% dextrose solution

PHA-848125 inhibits, although with lower potency, the activities of cyclin H/CDK7, cyclin D1/CDK4, p35/CDK5, as well as cyclin E/CDK2 and cyclin B/CDK1 with IC50 values of 0.15, 0.16, 0.265, 0.363, 0.398 uM, respectively. [1] Thropomyosin receptor kinase A is also inhibited by PHA-848125 in the same nanomolar range as CDKs. In the most PHA-848125-sensitive cell line, PHA-848125 induces a concentration-dependent G(1) arrest.[2] PHA-848125 also impairs phosphorylation of the retinoblastoma protein at CDK2 and CDK4 specific sites, reduces retinoblastoma protein and cyclin A levels, and increases p21(Cip1), p27(Kip1) and p53 expression. PHA-848125 is added to the cells 48 h after TMZ and cell growth is evaluated after 3 additional days of culture. [2] A drug combination of TMZ, BG and PHA-848125 induces an additive or synergistic effect on cell growth, depending on the cell line. [2] In the absence of BG, the combination is still more active than the single agents in cell lines moderately sensitive to TMZ, but comparable to PHA-848125 alone in the two TMZ-resistant cell lines. When TMZ plus BG are used in combination with PHA-848125 against cultured normal melanocytes, neither synergistic nor additive antiproliferative effects are observed.[2]

2 hours

Melanoma cells are suspended in culture media at a concentration of 2 x 104 cells/mL, dispensed in 50 uL aliquots into flat-bottom 96-well plates and allowed to adhere overnight at 37 C. Graded amounts of PHA-848125 or TMZ are then added to the wells (4 wells per point) in 50 uL of CM and the plates are incubated at 37 C in a 5% CO2 humidified atmosphere for 5 days. The cytotoxic effects of TMZ are also evaluated in combination with the MGMT inhibitor BG. To this end, 10 uM BG is added to the plates 2 hours before TMZ and left in culture for the entire period of cell exposure to the drug. ContS1017rol groups are represented by untreated cells and cells treated with BG or DMSO alone. The growth of the cells treated with BG or DMSO alone does not differ from that of untreated cells. MGMT activity of BG-treated cells is undetectable 2 hours after the addition of the inhibitor PHA-848125 and remained essentially undetectable up to the end of the assay. Normal melanocytes are suspended in MGM at the concentration of 1.6 x 105 cells/mL, plated (50 uL/well) and exposed to TMZ + BG or to PHA-848125 as described for melanoma cells. At the end of the incubation period, cell growth is evaluated by the MTT assay. Briefly, 0.1 mg of MTT (in 20 uL of PBS) is added to each well and cells are incubated at 37 C for 4 hours. Cells are then lysed with a buffer (0.1 mL/well) containing 20% SDS and 50% N, N-dimethylformamide, pH 4.7. After overnight incubation, the absorbance is read at 595 nm using a 3550-UV microplate reader. Cell sensitivity to drug treatment is expressed in terms of IC50 (drug concentration producing 50% inhibition of cell growth, calculated on the regression line in which absorbance values at 595 nm are plotted against the logarithm of drug concentration).

PHA-848125 Chemical Structure

2 years -20CPowder, 2 weeks4Cin DMSO, 2 months-80Cin DMSO