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 most, and perhaps all, eukaryotic cells. Several Kinesin-5 proteins, notably the human version, HsEg5, are targets of a constantly expanding group of small-molecule inhibitors, which hold promise both as tools to probe mechanochemical transduction and as anti-cancer agents. Although most such compounds are selective for HsEg5 and closely related Kinesin-5 proteins, some, such as NSC 622124, exhibit activity against at least one kinesin from outside the Kinesin-5 family. Here we show NSC 622124, despite identification in a screen that yielded inhibitors now known to target the HsEg5 monastrol-binding site, does not compete with 14C-monastrol for binding to HsEg5, and is able to inhibit the basal and microtubule-stimulated ATPase activity of the monastrol-insensitive Kinesin-5, KLP61F. NSC 622124 competes with microtubules, but not ATP, for interaction with HsEg5, and disrupts the microtubule binding of HsEg5, KLP61F and Kinesin-1. 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 microtubule-binding site of kinesin proteins. Further, unlike compounds previously reported to target the kinesin microtubule-binding site, NSC 622124 does not produce any enhancement of basal ATPase activity, and thus acts solely as a negative regulator through interaction with a site traditionally viewed as a binding region for positive regulators (i.e., microtubules). Our work emphasizes the concept that microtubule-dependent motor proteins may be controlled at multiple sites by both positive and negative effectors.

Small Molecule Screen for Candidate Antimalarials Targeting Plasmodium Kinesin-5

“…2B). These basal catalytic rates were comparable with the 0.18 s Ϫ1 rate of the native human Eg5 motor in our hands (24,34,38,44). Moreover, equivalent rates were obtained with either the NADHcoupled assay, which monitors the decay of the 340-nm absorbance of NADH upon ADP production, or with the malachite green assay, which monitors dye interaction with phosphate ion and is monitored instead at 620 nm.…”

“…These control reactions established a noise baseline and never recorded a value in excess of 0.009 s Ϫ1 (average ϭ 0.004 Ϯ 0.002 s Ϫ1 (n ϭ 6)); control data are independent of, yet consistent with, prior laboratory publications (24,38). Data were analyzed using IGOR Pro software (Wavemetrics Inc.).…”

“…and another that measures P i concentration. The first is the NADH-coupled ATPase assay (22), which was successfully utilized in previous screens for human Eg5 inhibitors (35,38,48,49) and has long been used in our laboratory (24,32,34,38,44). This assay monitors ADP concentration through a coupled reaction that results in the oxidation of NADH to NAD ϩ , detectable by decreasing absorbance at 340 nm.…”

Allosteric Drug Discrimination Is Coupled to Mechanochemical Changes in the Kinesin-5 Motor Core

“…In Vitro ATPase Assays-Basal ATPase activities were measured with a coupled enzymatic assay (31) with 5 M motor protein and 1 mM MgATP in a SpectraMax 2E spectrophotometer for high throughput kinetic measurements (32). Reactions were supplemented with monastrol (0 -600 M; Sigma) or STC (0 -200 M; Sigma) as appropriate, and control reactions contained equivalent DMSO.…”

Loop 5-directed Compounds Inhibit Chimeric Kinesin-5 Motors

“…In particular, human Eg5 kinesin (HsEg5) 2 forms a homotetramer that is capable of cross-linking adjacent microtubule bundles during spindle formation and is essential for mitotic progression. High-throughput screens for small chemical inhibitors of mitosis have often yielded compounds that target this kinesin in particular.…”