CCT245737 is an orally active, selective Chk1 inhibitor with an IC50 of 1.3 nM, and is >1,000-fold selective over CHK2 and CDK1.

Chk1:1.4 nM (Cell-free)

METHODS: K562 cells were treated with CCT245737 (0.01, 0.05, 0.1, 0.5, 1, 2, 5, 24 hours), and the total expression of CHK1 protein was detected by Western Blot; K562 cells treated with VP16 (5 μM), CCT245737 (5 μM) or the combination of VP16 and CCT245737 (0.01, 0.05, 0.1, 0.5, 1, 2, 5, 24 hours) were analyzed by Western blot.
RESULTS Increasing CCT245737 concentrations did not significantly reduce total CHK1 protein levels, and CHK1 was only inhibited at a high concentration of CCT245737 (5 μM); CCT245737 could effectively inhibit CHK1 activation by inhibiting VP16-induced CHK1 autophosphorylation at Ser296 and promoting the accumulation of DNA damage in VP16-treated K562 cells. [3]

METHODS: CCT245737 (10 mg/kg, intravenous/oral) was administered to mice and the pharmacokinetics in vivo were tested.
RESULTS The peak plasma concentration of CCT245737 after intravenous injection was 4 μmol/, the half-life was 2.86 h, the AUC was 9.96 μmol.h/L, the plasma clearance was 2.1 L/h/kg, and the distribution volume was large (0.19 L); the equivalent oral dose was almost the same as the AUC, indicating complete oral bioavailability (F = 105%). [1]

Commercial in vitro 33P radiometric kinase assays is carried out against 124 human kinases using 10 μM CCT245737 at ATP concentrations corresponding to the kinase Km, ATP [2].

Cytotoxicity is determined as the drug concentration that gives 50% inhibition of tumour cell proliferation (GI50) using a 96 h Sulforhodamine B (SRB) assay. Inhibition of intracellular CHK1 activity is measured using a cell-based ELISA for the abrogation of an etoposide-induced G2 checkpoint (mitosis induction assay, MIA). The IC50 for G2 checkpoint abrogation (MIA) is determined in the presence of nocodazole using UCN01 as a positive control. The activity index (AI) is used as a measure of the compounds ability to induce mitosis relative to its toxicity (i.e., ratio of MIA IC50: 96 h SRB GI50). Routine potentiation studies are carried out using a fixed concentration (GI50) of either gemcitabine or SN38 in combination with a range of CCT245737 concentrations to determine the combination GI50 of CCT245737. The ability of CCT245737 to enhance gemcitabine or SN38 cell killing is expressed as a potentiation index (PI) equal to the ratio of the GI50 for CCT245737 alone versus the combination GI50 for CCT245737. PI values > 1 indicate the potentiation of the genotoxic activity. In addition, a series of experiments is carried out using fixed, non- or minimally toxic concentrations of CCT245737 (≤GI20) with a range of different concentrations of gemcitabine or SN38 to determine the extent to which CCT245737 enhances drug cytotoxicity compared with the genotoxic agent alone, i.e. conventional PI (ratio GI50 genotoxic alone: GI50 genotoxic combined with non-toxic CCT245737 concentration, Con PI)[2].

Human HT29 colorectal carcinoma cells are injected s.c into the flanks of female NCr athymic mice 6-8 weeks of age. Dosing commenced 5 days after transplantation when tumours reach a mean diameter of 5.5 mm. Gemcitabine (100 mg/kg i.v.) is dosed in saline on days 0, 7 and 14 and compounds 4 (CCT245737) and 41 (150 mg/kg p.o.) in 10% DMSO 20% PEG 400, 5% Tween 80, 65% water, 24 and 48 h after each dose of gemcitabine. Tumours are measured and body weights recorded three times weekly. Animals are culled on an individual basis when tumours reach a predetermined humane endpoint (mean diameter <15 mm)[1].