ML-264

A potent, selective of KLF5 expression inhibtor, withour effect on kinases associated with the KLF5 pathway; inhibits the proliferation of DLD-1 cells with IC50 of 29 nM, and several other KLF5-expressing cell types as well HCT116, IC50 560 nM; HT29, IC50=130 nM; SW620, IC50=430 nM) ; potently inhibits proliferation of colorectal cancer cells in vitro through modifications of the cell-cycle profile; efficiently inhibits growth of the tumo in an established xenograft mouse model of colon cancer.Colon Cancer Preclinical (In Vitro) :ML264 is highly active (IC50=29 nM is a cell-based assay for proliferation of DLD-1 cells, IC50=81 nM in a cell-based luciferase assay) . ML264 lacks cytotoxicity in the IEC-6 control cell line (IC50>50 μM, <50% inhibition is observed at 100 μM) . Robust activity is also seen in several other KLF5-expressing cell types as well (e.g., HCT116, IC50=560 nM; HT29, IC50=130 nM; SW620, IC50=430 nM) . Western blot analysis shows that ML264 significantly reduces KLF5 expression. The effects of ML264 are tested on the rate of cell proliferation of colon cancer cells lines DLD-1 and HCT116 over 72 hours. ML264 efficiently inhibits the rate of proliferation of both cell lines. A significant decrease in proliferation is evident within 24 hours of treatment and by 72 hours the live cell numbers of ML264-treated and vehicle-treated cells differed by 15- to 30- fold. An MTS assay that allows the quantification of metabolically active cells is performed. ML264 treatment significantly reduces the number of cells undergoing mitosis in DLD-1 cells at 24, 48 and 72 hours. (In Vivo) :Single daily injections of ML264 at 10 mg/kg do not significantly affect tumor growth. However, twice daily injections of ML264 at 10 mg/kg or 25 mg/kg result in significant reductions in tumor growth, and this effect can be detected as early as two days after the first injection. The data also show that there is a concentration-dependent effect of ML264 on the tumor volume. Statistical analysis of tumor growth reveals significant tumor size reduction in mice treated twice daily with ML264 compared to those receiving only vehicle at day 5 and 10.