NSC 622124 inhibits human Eg5 and other kinesins via interaction with the conserved microtubule-binding site.
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 for probing mechanochemical transduction and as anticancer 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 [(14)C]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 alpha3 and alpha5 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.
Learman
Sarah S
SS
Department of Biological Sciences, 2119 Derring Hall, Virginia Tech, Blacksburg, Virginia 24061-0406, USA.
Kim
Catherine D
CD
Stevens
Nathaniel S
NS
Kim
Sunyoung
S
Wojcik
Edward J
EJ
Walker
Richard A
RA
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000818:Animals; D001665:Binding Sites; D017124:Conserved Sequence; D004330:Drosophila / Q000187:drug effects; D029721:Drosophila Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D016547:Kinesins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D008870:Microtubules / Q000187:drug effects* / Q000378:metabolism*; D008982:Molybdenum / Q000494:pharmacology*; D009068:Movement / Q000187:drug effects; D010087:Oxides / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017433:Protein Structure, Secondary; D011743:Pyrimidines / Q000494:pharmacology; D013871:Thiones / Q000494:pharmacology
D029721:Drosophila Proteins; C550252:K6Mo18O62P2; C470243:KIF11 protein, human; C084034:Klp61F protein, Drosophila; D008869:Microtubule-Associated Proteins; D010087:Oxides; D011743:Pyrimidines; D013871:Thiones; C400223:monastrol; D008982:Molybdenum; D000251:Adenosine Triphosphatases; D016547:Kinesins