Identification of Bioactive Small Molecule Inhibitors of Separase.

Separase, a cysteine protease of the CD-clan, triggers chromosome segregation during mitosis by cleaving the cohesin ring entrapping the two sister chromatids. Deregulated separase activity is associated with aneuploidy, a hallmark of most human cancers. In fact, separase is highly overexpressed in many solid cancers making it an attractive chemotherapeutic target. To identify small molecules capable of inhibiting separase in its complex cellular environment, we established a highly sensitive assay to quantify separase activity in cells and screened a 51,009-member library for separase inhibitors. In vitro assays confirmed that the identified compounds efficiently inhibited separase, while not affecting caspase-1, another CD-clan protease structurally related to separase. Importantly, HeLa-cells with compromised separase activity displayed severe chromosome segregation defects upon compound treatment confirming that the identified inhibitors are bioactive in tumor tissue culture cells. Structure-activity relationship studies succeeded in the optimization of the most promising inhibitor. Overall, this study demonstrates the feasibility of identifying separase-specific inhibitors, which serve as promising lead compounds for the development of clinically relevant separase inhibiting drugs.