| Description: |
PF 477736 is a potent, selective ATP-competitive inhibitor of Chk1, with a Ki of 0.49 nM, 100-fold selectivity versus Chk2 (Ki, 47 nM). |
| Target: |
Chk1:0.49 nM (Ki)
Chk2:47 nM (Ki)
CDK1:9.9 μM (Ki) |
| In Vivo: |
PF 477736 (PF-00477736, 4-60 mg/kg, i.p.) once a day (s.i.d.) or twice a day (b.i.d.) treatment alone exhibits no antitumor activity in human colon Colo205 xenograft model. However, PF 477736 dependently enhances the antitumor activity of a MTD of gemcitabine. PF 477736 (15 and 30 mg/kg) induces histone H3 phosphorylation and DNA damage and increases apoptosis in Colo205 tumor samples from in vivo xenograft model[1]. |
| In Vitro: |
PF 477736 (PF-00477736, 0.01-1 μM) abrogates the camptothecin-induced DNA damage checkpoint in a dose-dependent manner in CA46 cells. PF 477736 (180, 360, 540 nM) enhances gemcitabine-induced cytotoxicity in a time- and dose-dependent manner in HT29 cells as determined by cell survival assay. PF 477736 (360 nM) in combination with gemcitabine significantly decreases the phosphorylation of CDK1, consistent with checkpoint abrogation and cell entry into mitosis[1]. PF 477736 (PF-00477736) poorly inhibits CDK1 activity, with Ki of 9.9 μM[1][2]. |
| Cell Assay: |
HT29 or human umbilical vein endothelial cells are treated with gemcitabine (15 nM) or camptothecin (25 nM) for 16 h. PF 477736 (PF-00477736) is subsequently added at varying concentrations. At 4 to 48 h after the addition of PF 477736, the drug-containing medium is removed and cells are incubated in drug-free medium. When the vehicle-treated control cells are 90% confluent (8 days from plating), cells are harvested and counted by Coulter counter[1]. |
| Animal Administration: |
Chemotherapy agents or PF 477736 (PF-00477736) are administered by i.p. injection when tumors are 100 to 150 mm3 in volume over the designated treatment schedules. Gemcitabine is administered over a range of doses including the maximum tolerated dose (MTD) in mice according to a once every 3 days for four treatments (q3d × 4) schedule. PF 477736 is administered over a range of doses (4-60 mg/kg) according to the q3d × 4 schedule beginning 24 h after gemcitabine. MTD of PF 477736 is determined to be 40 mg/kg considering the severity of the behavioral response on i.p. administration and body weight loss of 5% to 10%. For cytotoxic agents, MTD is the occurrence of mean body weight loss of 5% to 10%[1]. |
| References: |
[1]. Blasina A, et al. Breaching the DNA damage checkpoint via PF-00477736, a novel small-molecule inhibitor of checkpoint kinase 1. Mol Cancer Ther. 2008 Aug;7(8):2394-404
[2]. Ashwell S, et al. Keeping checkpoint kinases in line: new selective inhibitors in clinical trials. Expert Opin Investig Drugs. 2008 Sep;17(9):1331-40. |
