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J. Biol. Chem., Vol. 282, Issue 13, 9740-9747, March 30, 2007

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Structure of Human Eg5 in Complex with a New Monastrol-based Inhibitor Bound in the R Configuration^*

Isabel Garcia-Saez, Salvatore DeBonis, Roman Lopez, Fernando Trucco, Bernard Rousseau, Pierre Thuéry^¶, and Frank Kozielski¹

From the Laboratoire des Moteurs Moléculaires, IBS, Institut de Biologie Structurale Jean-Pierre Ebel, CNRS-Commissariat à l'Energie Atomique (CEA)-Université Joseph Fourier, 41 rue Jules Horowitz, F-38027 Grenoble, the Groupe de Chimie Combinatoire et Criblage à haut débit, CEA-Saclay, Service de Marquage Moléculaire et de Chimie Bioorganique, Bat 547, 91191 Gif-sur-Yvette, and ^¶Service de Chimie Moléculaire DSM/DRECAM, CEA/Saclay 91191 Gif-sur-Yvette, France

Drugs that target mitotic spindle proteins have been proven useful for tackling tumor growth. Eg5, a kinesin-5 family member, represents a potential target, since its inhibition leads to prolonged mitotic arrest through the activation of the mitotic checkpoint and apoptotic cell death. Monastrol, a specific dihydropyrimidine inhibitor of Eg5, shows stereo-specificity, since predominantly the (S)-, but not the (R)-, enantiomer has been shown to be the biologically active compound in vitro and in cell-based assays. Here, we solved the crystal structure (2.7Å) of the complex between human Eg5 and a new keto derivative of monastrol (named mon-97), a potent antimitotic inhibitor. Surprisingly, we identified the (R)-enantiomer bound in the active site, and not, as for monastrol, the (S)-enantiomer. The absolute configuration of this more active (R)-enantiomer has been unambiguously determined via chemical correlation and x-ray analysis. Unexpectedly, both the R- and the S-forms inhibit Eg5 ATPase activity with IC₅₀ values of 110 and 520 nM (basal assays) and 150 nM and 650 nM (microtubule-stimulated assays), respectively. However, the difference was large enough for the protein to select the (R)- over the (S)-enantiomer. Taken together, these results show that in this new monastrol family, both (R)- and (S)-enantiomers can be active as Eg5 inhibitors. This considerably broadens the alternatives for rational drug design.

Received for publication, September 15, 2006 , and in revised form, January 10, 2007.

^* This work was supported by grants from Association pour la Recherche sur le Cancer (Contract number 3973), Alliance des Recherches sur le Cancer and Structural Proteomics in Europe (Contract number QLG2-CT-2002-00988). Crystallographic data for the structural analysis of (R)-mon-97 coupled with the (R)-Mosher acid have been deposited at the Cambridge Crystallographic Data Centre, CCDC number 630372. 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 Fig. 1 and Movie 1.

The atomic coordinates and structure factors (code 2IEH) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

¹ To whom correspondence should be addressed: Institut de Biologie Structurale (CEA-CNRS-UJF), Laboratoire de Moteurs Moléculaires, 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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