New chemical tools for investigating human mitotic kinesin Eg5

We have designed and synthesized a series of monastrol derivatives, an allosteric inhibitor of Eg5, a motor protein responsible for the formation and maintenance of the bipolar spindle in mitotic cells. Sterically demanding structural modifications have been introduced on the skeleton of the parent drug either via a multicomponent Biginelli reaction or a stepwise modification of monastrol. The ability of these compounds to inhibit Eg5 activity has been investigated using two in vitro steady-state ATPase assays (basal and microtubule-stimulated) as well as a cell-based assay. One compound in the series appeared more potent than monastrol by a fivefold factor. Three other compounds that were unable to inhibit Eg5 ATPase activity in vitro proved potent Eg5 inhibitors in the cell-based assay. The results obtained led to the identification of structure–activity relationships further used to design an affinity matrix that can be used for fast and efficient purification of Eg5 from crude lysate of eukaryotic cells.

Monastrol is a specific allosteric inhibitor of Eg5, a kinesin responsible for the formation and maintenance of the bipolar spindle. In contrast to anticancer drugs that perturb mitosis and all target tubulin, it does not display any neurotoxicity and thus is thought to have antitumor potential. A series of analogs have been synthesized and tested for inhibition of Eg5 in vitro and for arresting mitosis of cultured cells. One compound was more potent than parent monastrol by a fivefold factor. Structure–activity relationships led to the preparation of an affinity matrix for unprecedentedly fast and efficient purification of full-length Eg5 from eukaryotic cells. The question of monastrol-targets other than Eg5 is discussed.Figure optionsDownload as PowerPoint slide