A selective Chk1 inhibitor
CHIR-124, a selective inhibitor, inhibits Chk1 with IC50 value of 0.3nM 2,000-fold more potently than Chk2 with IC50 value of 0.7μM. CHIR-124 also potently targets other kinases such as PDGFR with IC50 value of 6.6nM and FLT3 with IC50 value of 5.8nM [1].
CHIR-124 abrogates the S and G2-M checkpoints induced by topoisomerase I poisons and selectively sensitizes tumors lacking p53 function to undergo mitotic death. In addition, CHIR-124 enhances the antitumor effect of irinotecan in tumor xenografts by inhibiting the G2-M checkpoint and inducing apoptosis.
In vitro, the effect of a matrix of camptothecin and CHIR-124 combinations in a number of human cancer cell lines, including breast carcinoma (MDA-MB-231and MDAMB-435) and colon carcinoma (SW-620 and Colo205), all of which are mutant for p53. When cells were simultaneously exposed to a matrix of different concentration combinations of CHIR-124 and SN-38 for 48 h, significant synergy or >10% deviation from additivity was observed in the concentration ranges of ≥4.2×108 mol/L for SN-38 and≥ 6.0×108 mol/L for CHIR-124. Compared to IR alone, the number of mitotic cells increased dramatically in p53-/- HCT116 cells after concomitant Chir-124 exposure, while no such effect was observed in p53-sufficient WT HCT116 cells. Chir-124 was able to radiosensitize HCT116 cells that lack checkpoint kinase-2 (CHK2) or that were deficient for the spindle checkpoint protein Mad2. Additionally, Chir-124 could radiosensitize tetraploid cell lines, which were resistant to DNA damaging agents. Radiosensitization mediated by Chir-124 is greatly influenced by the p53 and cell cycle checkpoint system [1, 2].
In vivo, severe combined immunodeficient mice harboring MDA-MD-435 tumor xenografts were randomized into the treatment of 10 mg/kg CHIR-124, 20 mg/kg CHIR-124, 10 mg/kg CHIR-124 with 5 mg/kg CPT-11, or 20 mg/kg CHIR-124 with 5 mg/kg CPT-11. CPT-11 was given i.p. four times daily ×5 on days 1 to 5, while CHIR-124 was given orally four times daily ×6 on days 2 to 7 in captisol. Tumors harvested from mice sacrificed on day 4 of treatment were examined for apoptosis by terminal deoxynucleotidyl transferase-mediated nick-end labeling staining and for mitotic index by immunofluorescence labeling with phospho-histone H3 antibody in a similar study [1].
参考文献:
[1].?Tse AN, Rendahl KG, Sheikh T, et al. CHIR-124, a novel potent inhibitor of Chk1, potentiates the cytotoxicity of topoisomerase I poisons in vitro and in vivo. Clinical Cancer Research, 2007, 13(2): 591-602.
[2].?Tao YG, Leteur C, Yang CY, et al. Radiosensitization by Chir-124, a selective CHK1 inhibitor Effects of p53 and cell cycle checkpoints. Cell Cycle, 2007, 8(8): 1196-1205.
Kinase experiment: | 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 kinase reaction buffer containing a final concentration of 30 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 2 mM DTT, 4 mM EDTA, 25 mM β-glycerophosphate, 5 mM MnCl2, 0.01% bovine serum albumin, 1.35 nM CHK1 kinase domain, 0.5 μM peptide substrate, and 1 μM unlabeled ATP, plus 5 nM 33P γ-labeled ATP (specific activity =2,000 Ci/mmol). Reactions and detection of the phosphate transfer are carried out by a radioactive method. |
Animal experiment: | Severe combined immunodeficient mice harboring MDA-MD-435 tumor xenografts are randomized into the following treatment groups of 10: vehicle (captisol) alone, 5 mg/kg CPT-11, 10 mg/kg CHIR-124, 20 mg/kg CHIR-124, 5 mg/kg CPT-11 plus 10 mg/kg CHIR-124, or 5 mg/kg CPT-11 plus 20 mg/kg CHIR-124. Treatment is initiated on the day after randomization (day 1). CPT-11 is given i.p. daily (four times daily) ×5 on days 1 to 5, whereas CHIR-124 is given orally four times daily ×6 on days 2 to 7 in captisol. Percent tumor growth inhibition is defined as % T/C, where T = the treatment group mean, and C = the control group mean. In a similar study, tumors harvested from mice sacrificed on day 4 of treatment are examined for apoptosis by terminal deoxynucleotidyl transferase-mediated nick-end labeling staining and for mitotic index by immunofluorescence labeling with phospho-histone H3 antibody. |
参考文献: [1]. Tse AN, et al. CHIR-124, a novel potent inhibitor of Chk1, potentiates the cytotoxicity of topoisomerase I poisons in vitro and in vivo. Clin Cancer Res, 2007, 13(2 Pt 1), 591-602. | |
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