PDF file, 67K, Supplementary Figure 4. Effects of scheduling and exposure times on the ability of two different CHK1 inhibitors to potentiate gemcitabine cytotoxicity in SW620 cells in vitro. A, Effect of different contact times on the capacity of SAR-020106 to enhance gemcitabine cytotoxicity in SW620 cells. Gemcitabine was continuously present (96h) at an IC50 concentration and SAR- 020106 was added at the start of treatment over a range of concentrations for the intervals shown (i.e. 0-T1, 0-T2, 0-T3, etc). Growth delay and potentiation were measured using an SRB assay as described in Materials and Methods. Potentiation Index (PI) was expressed as a percentage of the maximum PI. Values are mean�SE, for n=3-5 independent experiments. B, Treatment of synchronized SW620 cells with gemcitabine and SAR-020106. Cells were synchronized with nocodazole (100ng/ml x 16h). Eight hours following release (at G1/S boundary) cells were treated with a fixed concentration of gemcitabine (GI50) for 96h combined with different concentrations of SAR-020106 for 0-96, 0-48 or 0-24h. Values are mean�SE for 3 independent determinations. C, Effects of contact time on the potentiation of gemcitabine cytotoxicity in synchronized compared to asynchronous SW620 cells. See B and Materials and Methods for further details. Values are mean�SE, n=3-5. D, Effects of different contact times and treatment schedules on the ability of CCT244747 to enhance gemcitabine cytotoxicity in SW620 cells. Values are mean�SE, for 3-7 independent experiments.
ARTICLE ABSTRACT
Purpose: Many tumors exhibit defective cell-cycle checkpoint control and increased replicative stress. CHK1 is critically involved in the DNA damage response and maintenance of replication fork stability. We have therefore discovered a novel potent, highly selective, orally active ATP-competitive CHK1 inhibitor, CCT244747, and present its preclinical pharmacology and therapeutic activity.Experimental Design: Cellular CHK1 activity was assessed using an ELISA assay, and cytotoxicity a SRB assay. Biomarker modulation was measured using immunoblotting, and cell-cycle effects by flow cytometry analysis. Single-agent oral CCT244747 antitumor activity was evaluated in a MYCN-driven transgenic mouse model of neuroblastoma by MRI and in genotoxic combinations in human tumor xenografts by growth delay.Results: CCT244747 inhibited cellular CHK1 activity (IC50 29–170 nmol/L), significantly enhanced the cytotoxicity of several anticancer drugs, and abrogated drug-induced S and G2 arrest in multiple tumor cell lines. Biomarkers of CHK1 (pS296 CHK1) activity and cell-cycle inactivity (pY15 CDK1) were induced by genotoxics and inhibited by CCT244747 both in vitro and in vivo, producing enhanced DNA damage and apoptosis. Active tumor concentrations of CCT244747 were obtained following oral administration. The antitumor activity of both gemcitabine and irinotecan were significantly enhanced by CCT244747 in several human tumor xenografts, giving concomitant biomarker modulation indicative of CHK1 inhibition. CCT244747 also showed marked antitumor activity as a single agent in a MYCN-driven neuroblastoma.Conclusion: CCT244747 represents the first structural disclosure of a highly selective, orally active CHK1 inhibitor and warrants further evaluation alone or combined with genotoxic anticancer therapies. Clin Cancer Res; 18(20); 5650–61. ©2012 AACR.
