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¹ Institut de Biologie Structurale, Grenoble, France; ² Laboratoire de Chimie Organique Appliquée, Centre National de la Recherche Scientifique, Université Louis Pasteur, Faculté de Pharmacie, Illkirch, France; and ³ Service de Marquage Moléculaire et de Chimie Bio-organique, CEA-Saclay, Gif sur Yvette, France

Requests for reprints: Frank Kozielski, Institut de Biologie Structurale, 41, rue Jules Horowitz, 38027 Grenoble Cedex 01, France. Phone: 33-4-3878-4024; Fax: 33-4-3878-5494. E-mail: Frank.Kozielski{at}ibs.fr

Human Eg5, a member of the kinesin superfamily, plays a key role in mitosis, as it is required for the formation of a bipolar spindle. We describe here the first in vitro microtubule-activated ATPase-based assay for the identification of small-molecule inhibitors of Eg5. We screened preselected libraries obtained from the National Cancer Institute and identified S-trityl-L-cysteine as the most effective Eg5 inhibitor with an IC₅₀ of 1.0 µmol/L for the inhibition of basal ATPase activity and 140 nmol/L for the microtubule-activated ATPase activity. Subsequent cell-based assays revealed that S-trityl-L-cysteine induced mitotic arrest in HeLa cells (IC₅₀, 700 nmol/L) with characteristic monoastral spindles. S-trityl-L-cysteine is 36 times more potent for inducing mitotic arrest than the well-studied inhibitor, monastrol. Gossypol, flexeril, and two phenothiazine analogues were also identified as Eg5 inhibitors, and we found that they all result in monoastral spindles in HeLa cells. It is notable that all the Eg5 inhibitors identified here have been shown previously to inhibit tumor cell line growth in the NCI 60 tumor cell line screen, and we conclude that their antitumor activity may at least in part be explained by their ability to inhibit Eg5 activity.

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⁴ http://dtp.nci.nih.gov/branches/dscb/diversity_explanation.html.

⁵ http://dtp.nci.nih.gov/branches/dscb/mechanistic_explanation.html.

⁶ http://dtp.nci.nih.gov/branches/btb/ivclsp.html.

Received 3/15/04; revised 6/ 8/04; accepted 7/ 6/04.