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 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 Ki,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.