Skoufias, D., DeBonis, S., Saoudi, Y., Lebeau, L., Crevel, I., Cross, R., Wade, R., Hackney, D. and Kozielski, F. (2006) S-trityl-L-cysteine is a reversible, tight binding inhibitor of the human kinesin Eg5 that specifically blocks mitotic progression. Journal of Biological Chemistry, 281(26), pp. 17559-17569. (doi: 10.1074/jbc.M511735200)
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Abstract
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 G(2) 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,K-app < 150 nM at 300mM NaCl and 600 nM at 25 mM KCl). S-Trityl-L-cysteine binds more tightly than monastrol because it has both an similar to 8-fold faster association rate and similar to 4-fold slower release rate (6.1 mu M-1 s(-1) and 3.6 s(-1) for S-trityl-L-cysteine versus 0.78 mu M-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.
Item Type: | Articles |
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Status: | Published |
Refereed: | Yes |
Glasgow Author(s) Enlighten ID: | Kozielski, Professor Frank |
Authors: | Skoufias, D., DeBonis, S., Saoudi, Y., Lebeau, L., Crevel, I., Cross, R., Wade, R., Hackney, D., and Kozielski, F. |
College/School: | College of Medical Veterinary and Life Sciences > School of Cancer Sciences |
Journal Name: | Journal of Biological Chemistry |
Journal Abbr.: | J Biol Chem. |
Publisher: | American Society for Biochemistry and Molecular Biology, Inc. |
ISSN: | 0021-9258 |
ISSN (Online): | 1083-351X |
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