title: S-trityl-L-cysteine is a reversible, tight binding inhibitor of the human kinesin Eg5 that specifically blocks mitotic progression.
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,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 approximately 8-fold faster association rate and approximately 4-fold slower release rate (6.1 microM(-1) s(-1) and 3.6 s(-1) for S-trityl-L-cysteine versus 0.78 microM(-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 IC(50) 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.
authors.affiliation: 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.
authors.forename: Dimitrios A
authors.initials: DA
authors.lastname: Skoufias
authors.forename: Salvatore
authors.initials: S
authors.lastname: DeBonis
authors.forename: Yasmina
authors.initials: Y
authors.lastname: Saoudi
authors.forename: Luc
authors.initials: L
authors.lastname: Lebeau
authors.forename: Isabelle
authors.initials: I
authors.lastname: Crevel
authors.forename: Robert
authors.initials: R
authors.lastname: Cross
authors.forename: Richard H
authors.initials: RH
authors.lastname: Wade
authors.forename: David
authors.initials: D
authors.lastname: Hackney
authors.forename: Frank
authors.initials: F
authors.lastname: Kozielski
mesh_terms: D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001859:Bone Neoplasms; D002417:Cattle; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D003545:Cysteine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D015195:Drug Design; D006367:HeLa Cells; D006801:Humans; D016547:Kinesins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008938:Mitosis / Q000187:drug effects* / Q000502:physiology; D012516:Osteosarcoma; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D008941:Spindle Apparatus / Q000187:drug effects / Q000502:physiology; D013237:Stereoisomerism; D062367:ortho-Aminobenzoates / Q000378:metabolism / Q000494:pharmacology
chemical_list: C470243:KIF11 protein, human; D011994:Recombinant Proteins; D062367:ortho-Aminobenzoates; C003724:3-tritylthio-L-alanine; D000244:Adenosine Diphosphate; C081523:3'-O-(N-methylanthraniloyl)adenosine 5'-diphosphate; D016547:Kinesins; D003545:Cysteine