In vitro activity: ML264 is a novel and selective inhibitor of Krüppel-like factor 5 (KLF5) that potently inhibits the proliferation of colorectal cancer cells in vitro through modifications of the cell-cycle profile. 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). It lacks cytotoxicity in the IEC-6 control cell line (IC50 >50 μM,<50% inhibition was observed at 100 μM). Robust activity was 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). ML264 does not inhibit kinases associated with the KLF5 pathway, as determined using a panel of 47 selected kinases. Western blot analysis shows that ML264 significantly reduces KLF5 expression. These results demonstrate KLF5 target specificity. An NCI60 panel study using ML264 revealed that it induces death of most colon cancer cell lines, with cytotoxicity toward several other tumor cell lines as well. ML264 is chemically stable, unreactive with glutathione, has suitable aqueous solubility, is highly stable to mouse, rat, and human hepatic microsomes, has favorable properties associated with drug-likeness, and does not inhibit CYP enzymes. Due to these properties in concert with its high cellular potency and selectivity, ML264 is a good candidate for in vivo anticancer studies, with great potential for use in long time-course in situ studies aimed to elucidate the role of KLF5 as a regulator of cellular proliferation and tumor formation in the intestinal epithelium. ML264 and its analogues may hold a promise as a novel therapeutic agent to curb the development and progression of colorectal cancer.

Kinase Assay: 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.

Cell Assay: For cell proliferation experiments, DLD-1 and HCT116 cells are treated with 10 μM ML264 or with vehicle (DMSO). Live cells are collected at 24, 48 and 72 hours post treatment and their numbers are determined by counting using a Coulter counter. Each experiment is done in triplicate. In MTS assay, DLD-1 and HCT116 cells are treated with 10 μM ML264 or with vehicle (DMSO). After 24, 48, and 72 hours of incubations, 20 μL of MTS solution is added to each well and an analysis is performed. The measurement of the control (cells with medium and DMSO) is defined as 100% and the results from other measurements are calculated accordingly. Each experiment is done in sextuplicate. A cell cycle progression assay is performed. Each experiment is done in triplicate. The apoptosis rate is determined using the Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit with analysis by flow cytometry. Each experiment is done in triplicate.