ARRY-429520, a Novel KSP Inhibitor with Potent Anti-Proliferative Activity in Leukemic Cell Lines.

Kinesins are eukaryotic microtubule-associated motor proteins. There are over 40 known kinesins, approximately 15 of which are closely associated with mitosis. Kinesin Spindle Protein (KSP), also known as Eg5, is a mitotic kinesin that is a required enzyme in mitosis (prophase / prometaphase). This protein plays a key role in the formation of the bipolar spindle, particularly related to its role in centrosome maturation/separation. Because KSP is expressed predominately in proliferating cells and is absent from postmitotic neurons, its inhibition should not produce the peripheral neuropathy associated with traditional microtubule disruption agents (taxanes and vinca alkaloids). We report here the in vitro characterization of a potent KSP inhibitor, ARRY-429520, a member of a series of KSP inhibitors discovered and optimized by structure-based design. ARRY-429520 inhibits human KSP, with an IC50 of 6 nM, by a mechanism which was demonstrated to be uncompetitive with respect to ATP and noncompetitive with respect to tubulin. It was shown to arrest cells in mitosis as measured by FACs analysis as well as the accumulation of phospho- histone H3, with an EC50 of 1.5 nM. Furthermore, this compound was demonstrated to be antiproliferative, with EC50s between 0.3 nM and 6.5 nM against a panel of human tumor cell lines, including various leukemia lines (K-562, KU-812, HL-60, KG-1, MOLT3, MOLT4). In addition, ARRY-429520’s potency in MDR-overexpressing cell lines was minimally impacted as compared to paclitaxel. Cellular imaging studies demonstrate that the normal mitotic spindle configuration was disrupted, with the formation of monopolar spindles, a hallmark feature of KSP inhibition, at single digit nanomolar concentrations of ARRY-429520. Markers of mitotic arrest and apoptosis were demonstrated in tumor xenografts from animals treated with ARRY-429520.