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J. Biol. Chem., Vol. 281, Issue 26, 17559-17569, June 30, 2006

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S-Trityl-L-cysteine Is a Reversible, Tight Binding Inhibitor of the Human Kinesin Eg5 That Specifically Blocks Mitotic Progression^*

Dimitrios A. Skoufias, Salvatore DeBonis, Yasmina Saoudi^¶, Luc Lebeau^||, Isabelle Crevel^**, Robert Cross^**, Richard H. Wade, David Hackney, and Frank Kozielski¹

From the Laboratoire des Protéines du Cytosquelette and Laboratoire de Moteurs Moléculaires, Institut de Biologie Structurale (Commissariat à l'Energie Atomique-CNRS-UJF), 41 Rue Jules Horowitz, 38027 Grenoble Cedex 01, France, ^¶Laboratoire du Cytosquelette, INSERM Unite 366, Commissariat à l'Energie Atomique, 38054 Grenoble Cedex 9, France, ^||Laboratoire de Chimie Organique Appliquée, Institut Gilbert-Laustriat, CNRS, Université Louis Pasteur, Faculté de Pharmacie, 74 Route du Rhin, 67401 Illkirch Cedex, France, ^**Marie Curie Research Institute, Oxted RH8 0TL, United Kingdom, and the Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213

Human Eg5, responsible for the formation of the bipolar mitotic spindle, has been identified recently as one of the targets of S-trityl-L-cysteine, a potent tumor growth inhibitor in the NCI 60 tumor cell line screen. Here we show that in cell-based assays S-trityl-L-cysteine does not prevent cell cycle progression at the S or G₂ phases but inhibits both separation of the duplicated centrosomes and bipolar spindle formation, thereby blocking cells specifically in the M phase of the cell cycle with monoastral spindles. Following removal of S-trityl-L-cysteine, mitotically arrested cells exit mitosis normally. In vitro, S-trityl-L-cysteine targets the catalytic domain of Eg5 and inhibits Eg5 basal and microtubule-activated ATPase activity as well as mant-ADP release. S-Trityl-L-cysteine is a tight binding inhibitor (estimation of K_i,app <150 nM at 300 mM NaCl and 600 nM at 25 mM KCl). S-Trityl-L-cysteine binds more tightly than monastrol because it has both an 8-fold faster association rate and 4-fold slower release rate (6.1 µM^–1 s^–1 and 3.6 s^–1 for S-trityl-L-cysteine versus 0.78 µM^–1 s^–1 and 15 s^–1 for monastrol). S-Trityl-L-cysteine inhibits Eg5-driven microtubule sliding velocity in a reversible fashion with an IC₅₀ of 500 nM. The S and D-enantiomers of S-tritylcysteine are nearly equally potent, indicating that there is no significant stereospecificity. Among nine different human kinesins tested, S-trityl-L-cysteine is specific for Eg5. The results presented here together with the proven effect on human tumor cell line growth make S-trityl-L-cysteine a very attractive starting point for the development of more potent mitotic inhibitors.

Received for publication, October 31, 2005 , and in revised form, February 15, 2006.

^* This work was supported by Contract 5197 from Association pour la Recherche sur le Cancer, Alliance Des Recherches sur le Cancer, and Contracts 03 013690 02 and 03 013690 01 from the Région Rhône-Alpes. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2 and Movies S1 and S2.

¹ To whom correspondence should be addressed: Institut de Biologie Structurale, 41 Rue Jules Horowitz, 38027 Grenoble, France. Tel.: 33-4-3878-4024; Fax: 33-4-3878-5494; E-mail: Frank.kozielski{at}ibs.fr.

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