NSC 622124 inhibits human Eg5 and other kinesins via interaction with the conserved microtubule‐binding site

Abstract

Kinesin‐5 proteins are essential for formation of a bipolar mitotic spindle in eukaryotic cells. Notably, the human Kinesin‐5 motor, HsEg5, is a target for an expanding group of small‐molecule inhibitors that hold promise both as tools to probe mechanochemical transduction and as anti‐cancer agents. Although most such compounds are selective for HsEg5, some exhibit activity against at least one kinesin from outside the Kinesin‐5 subfamily. Here we show NSC 622124, despite identification in an inhibitor screen targeting the HsEg5 monastrol‐binding site, does not compete with 14C‐monastrol for binding to HsEg5, and inhibits the basal and microtubule (MT)‐stimulated ATPase activity of the monastrol‐insensitive KLP61F. NSC 622124 competes with MTs, but not ATP, for interaction with HsEg5, and disrupts MT binding of other kinesins as well. Proteolytic degradation of an HsEg5▸NSC622124 complex revealed that segments of the α3 and α5 helices map to the inhibitor‐binding site. Overall, our results demonstrate that NSC 622124 targets the conserved MT‐binding site of kinesins. Further, NSC 622124 does not produce any enhancement of basal ATPase activity, and only acts as a negative regulator through interaction with a site traditionally viewed as a binding region for positive regulators. Our work emphasizes that MT‐dependent motor proteins may be controlled at multiple sites by both positive and negative effectors.