K858, a Novel Inhibitor of Mitotic Kinesin Eg5 and Antitumor Agent, Induces Cell Death in Cancer Cells

Abstract

The aim of this study was to investigate the mechanism of inhibition of Eg5 (kinesin spindle protein), a mitotic kinesin that plays an essential role in establishing mitotic spindle bipolarity, by the novel small molecule inhibitor K858. K858 was selected in a phenotype-based forward chemical genetics screen as an antimitotic agent, and subsequently characterized as an inhibitor of Eg5. K858 blocked centrosome separation, activated the spindle checkpoint, and induced mitotic arrest in cells accompanied by the formation of monopolar spindles. Long-term continuous treatment of cancer cells with K858 resulted in antiproliferative effects through the induction of mitotic cell death, and polyploidization followed by senescence. In contrast, treatment of nontransformed cells with K858 resulted in mitotic slippage without cell death, and cell cycle arrest in G 1 phase in a tetraploid state. In contrast to paclitaxel, K858 did not induce the formation of micronuclei in either cancer or nontransformed cells, suggesting that K858 has minimal effects on abnormalities in the number and structure of chromosomes. K858 exhibited potent antitumor activity in xenograft models of cancer, and induced the accumulation of mitotic cells with monopolar spindles in tumor tissues. Importantly, K858, unlike antimicrotubule agents, had no effect on microtubule polymerization in cell-free and cellbased assays, and was not neurotoxic in a motor coordination test in mice. Taken together, the Eg5 inhibitor K858 represents an important compound for further investigation as a novel anticancer therapeutic. [Cancer Res 2009;69(9):3901-9]

A Novel Eg5 Inhibitor (LY2523355) Causes Mitotic Arrest and Apoptosis in Cancer Cells and Shows Potent Antitumor Activity in Xenograft Tumor Models

“…Sources of the cancer cell lines used for the in vivo xenograft tumor model studies at Kyowa Hakko Kirin as summarized in Table 1 1998. These cancer cell lines at Kyowa Hakko Kirin were not authenticated and were maintained as previously described (32).…”

“…RNAi, compound handling and treatment, immunoblotting, flow cytometry, and immunofluorescence microscopy were performed as previously described (32,33). Assays for ATPase activity of kinesins and in vitro microtubule polymerization and depolymerization were also done as previously described (32).…”

“…Assays for ATPase activity of kinesins and in vitro microtubule polymerization and depolymerization were also done as previously described (32).…”

“…Antitumor efficacy in subcutaneous xenograft tumor-bearing mice with 10 mice per treatment group from either established cancer cell lines or fragments of human tumor explants was evaluated as tumor volume by serial caliper measurements and was calculated as described previously (32,34). The p388 syngeneic tumor model was developed for high-content imaging analysis using female BDF1 mice, weighing 20 to 23 g, which were purchased from Charles River and acclimated in-house for one week before their use in experiments.…”

“…We describe here the discovery and characterization of LY2523355, a selective allosteric inhibitor of human Eg5, derived through an extensive structure-activity relationship program from a precursor compound identified by a phenotype-based screening for mitotic arrest of HCT116 cells (32). We demonstrate that inhibition of Eg5 in cancer cells by LY2523355 causes rapid apoptosis following SAC-dependent mitotic arrest.…”