CHIR-124 is a quinolone-based small molecule that is structurally unrelated to other known inhibitors of Chk1. CHIR-124 potently and selectively inhibits Chk1 in vitro. CHIR-124 interacts synergistically with topoisomerase poisons (e.g., camptothecin or SN-38) in causing growth inhibition in several p53-mutant solid tumor cell lines. CHIR-124 abrogates the SN-38-induced S and G(2)-M checkpoints and potentiates apoptosis in MDA-MD-435 breast cancer cells. CHIR-124 treatment can restore the level of cdc25A protein, which is normally targeted by Chk1 for degradation following DNA damage, indicating that Chk1 signaling is suppressed in the presence of CHIR-124. In an orthotopic breast cancer xenograft model, CHIR-124 potentiates the growth inhibitory effects of irinotecan by abrogating the G(2)-M checkpoint and increasing tumor apoptosis. The combination of gemcitabine and CHIR-124 in the multicellular tumor spheroid model enhanced the sensitivity to the gemcitabine antiproliferative effect in correlation with an increase in DNA damage and apoptosis. CHK1 inhibition by Chir-124 sensitizes HCT116 cancer cells to radiation.