CHIR-124

CHIR-124 is a quinolone-based small molecule that is structurally unrelated to other known inhibitors of Chk1. CHIR-124 interacts synergistically with topoisomerase poisons (e.g., Camptothecin or SN-38) in causing growth inhibition in a variety of cancer cell lines, including breast carcinoma (MDA-MB-231 and MDA-MB-435) and colon carcinoma (SW-620 and Colo205), all of which contains the mutant p53 gene. CHIR-124 abrogates the SN-38-induced S and G2-M checkpoints and potentiates apoptosis in MDA-MD-435 breast cancer cells. The abrogation of the G2-Mcheckpoint and induction of apoptosis by CHIR-124 are enhanced by the loss of p53. ^[1] CHIR-124 also potently targets other kinases such as PDGFR and Flt3 with IC50 of 6.6 nM and 5.8 nM, respectively. ^[2]

CHIR-124 potentiates the growth inhibitory effects by abrogating the G₂-M checkpoint and increasing tumor apoptosis in an orthotopic breast cancer xenograft model.

• Chk1 Assay

  For the Chk1 assay, the kinase domain is expressed in Sf9 insect cells, and a biotinylated cdc25c peptide containing the consensus Chk1/Chk2 phosphorylation site (*)(biotin-[AHX]SGSGS*GLYRSPSMP-ENLNRPR[CONH2]) is used as the substrate. A dilution series of CHIR-124 is mixed with a kinasereaction buffer containing a final concentration of 30 mM Tris-HCl(pH 7.5), 10 mM MgCl₂, 2 mM DTT, 4 mM EDTA, 25 mMβ-glycerophosphate, 5 mM MnCl₂, 0.01% bovine serum albumin, 1.35 nM CHK1 kinase domain, 0.5 μM peptide substrate, and 1 AM unlabeled ATP, plus 5 nM ³³Pγ-labeled ATP (specific activity = 2,000 Ci/mmol). Reactions and detection of the phosphate transfer are carried out by a radioactive method. Reactions are incubated at room temperature for 1 to 4 hours and the phosphorylated peptide captured on streptavidin-coated microtiter plates containing stop reaction buffer (25 mM EDTA [ethylenediaminetetraacetic acid], 50 mMHEPES, pH 7.5). Phosphorylated peptide is measured with the DELFIA TRF system using a Europium-labeled anti-phosphotyrosine antibody PT66. The concentration of CHIR-124 for IC50 is calculated using nonlinear regression with XL-Fit data analysis software.

• Cell lines

  MDA-MB-231, MDA-MB-435, SW-620, and COLO 205 cells

• Concentrations

  0-2350 nM, dependent on cell types

• Incubation Time

  48 hours

• Method

  MDA-MB-231, MDA-MB-435, SW-620, and COLO 205 cells in log-phase are plated into 96-well microplates. CHIR-124 is serially diluted in the presence of six different concentrations of Camptothecin or 0 nM camptothecin. Camptothecin is also serially diluted in the absence of CHIR-124. CHIR-124 is added to cells in 96-well dishes and incubated at 37 °C for 48 hours. Each treatment condition is done in triplicate. Cell proliferation is monitored by the 3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), inner salt assay. MTS inner salt is added to the microplates, which are incubated for another 3 hours, and absorbance at 490 nm is read on a plate reader. The concentrations of each drug in the combinations required to produce 50% inhibition are plotted to generate the isoboles. Isobologram analysis of drug interaction is based the equation of Loewe additivity (1= D _A /IC50, _A + D_B/IC50, _B), where IC50, _A and IC50, _B are the concentrations of drugs to result in 50% inhibition for each drug alone, and D_A and D_B are concentrations of each drug in the combination that yield 50% overall inhibition. A diagonal line indicating Loewe additivity is included in each graph. Data points that fall below the line indicate synergy, whereas those that fall above the line will indicate antagonism

• Animal Models

  MDA-MB-435 cells are implanted in the mammary fat pad of 8- to 10-week-old female immunodeficient mice.

• Dosages

  10 mg/kg or 20 mg/kg

• Administration

  CHIR-124 is given orally four times daily × 6 on days 2 to 7 in captisol.