PHA-767491 HCl

2 years -80 in solvent

Intravenous or oral administration twice a day

HeLa, MCF7, HCT-116, U2OS, A2780, K562, SF-539, SF-268, Ovcar8, SW480, COLO205, HCT-15, Jurkat, PC3, and NHDF

ca.50 mg/kg

10 nM, 10 nM, 10 nM, 10 nM, 10 nM, 10 nM

Administration of PHA-767491 twice a day for 5 days significantly inhibits the growth of HL60 xenograft in a dose-dependent manner with TGI of 50% and 92% at dose of 20 mg/kg and 30 mg/kg, respectively, the effect of which is also marked in A2780, Mx-1, and HCT-116 xenograft models as well as the DMBA-induced mammary carcinomas, and correlates with Cdc7 inhibition and subsequently decreased phosphorylation of Mcm2 at the Cdc7-dependent site Ser40 [1]

In vitro kinase assays, The inhibition of Cdc7 and Cdk9 by PHA-767491 (IC50) is determined using the strong anion exchanger (Dowex 1-X8 resin, formate form)-based assay. For each enzyme, the absolute Km values for ATP and the specific substrate are initially determined, and each assay is then run at optimized ATP/33P-gamma-ATP mix (2Km) and substrate (5Km) concentrations. Cdc7 kinase assay is performed in a buffer containing 50 mM Hepes pH 7.9, 15 mM MgCl2, 2 mM beta- glycerylphosphate, 0.2 mg/mL BSA, 1 mM DTT, 3 uM Na3VO4, 2Km ATP/33P-gamma-ATP mix, 5Km Mcm2 (aa 10-294), 37 nM of recombinant Cdc7/Dbf4 and increasing concentration of PHA-767491 in a final volume of 30 uL, and incubated for 1 hour at 25 C. Cdk9 kinase assay is performed using 50 nM of recombinant Cdk9/cyclin T in 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 3 uM Na3VO4, 2Km ATP/33P-gamma-ATP mix, 5Km RNA polymerase CDT peptide and increasing concentration of PHA-767491 in a final volume of 30 uL, and incubated for 1 hour at 25 C. After incubation, an amount of 150 uL of resin/formate (pH 3.0) is added to stop the reaction and capture unreacted 33P-gamma-ATP, separating it from the phosphorylated substrate in solution. After 1 hour of rest, a volume of 50 uL supernatant is transferred to Optiplate 96-well plates. After the additon of 150 uL of Microscint 40, the radioactivity is counted in the TopCount.

DMSO 24 mg/mL, Water <1 mg/mL, Ethanol <1 mg/mL

2-(pyridin-4-yl)-6, 7-dihydro-1H-pyrrolo[3, 2-c]pyridin-4(5H)-one