Biochemical Characterization of a Class of Inhibitors of the Human Mitotic Kinesin KSP with a Novel Mechanism of Action

The motor protein kinesin spindle protein (KSP, also known as Eg5) plays a critical role in the establishment of the mitotic spindle. KSP is essential for the proper separation of spindle poles during mitosis and inhibition results in mitotic arrest and the formation of characteristic monoaster spindles. Prolonged mitotic arrest leads to apoptosis in tumor cells and tumor growth inhibition in xenograft mouse tumor models. Kinesin inhibitors are expected to be general antiproliferative agents effective in most cancers currently treated with anti-microtubule agents. Several distinct classes of KSP inhibitors have been described previously in the public and patent literature; those that have been characterized as to mechanism of inhibition share a common induced-fit allosteric binding site. In a high-throughput screen for inhibitors of KSP, a novel class of thiazole-containing inhibitors was identified. Unlike the previously described allosteric KSP inhibitors, the thiazoles described here show ATP competitive kinetic behavior and are presumed to bind within the nucleotide binding pocket. Although they bind to a pocket that is highly conserved across kinesins, these molecules exhibit significant selectivity for KSP over other kinesins and other ATP utilizing enzymes. Several of these compounds are active in cells and produce a phenotype similar to that observed with previously published allosteric inhibitors of KSP.