S-Trityl-L-cysteine Is a Reversible, Tight Binding Inhibitor of the Human Kinesin Eg5 That Specifically Blocks Mitotic Progression

Isabelle Crevel,Frank Kozielski,Richard H Wade,Dimitrios A Skoufias,David Hackney,Yasmina Saoudi,Salvatore Debonis,Robert Cross,Luc Lebeau

doi:10.1074/jbc.m511735200

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.

Highlights

The roles of different kinesins during cell division make them highly important for understanding fundamental aspects of mitosis and meio
Interphase cells reformed a dense MT network within the first 30 min following nocodazole washout even in the presence of STLC, comparable with that of the control untreated cells. These data suggest that STLC at concentrations that induce monoastral spindles does not affect centrosome MT nucleating activity and neither affects MT assembly nor MT stability
Human Eg5 belongs to a group of mitotic motor proteins with a high interest as potential anticancer drug targets because it is involved in establishing and maintaining the bipolar spindle by contributing the forces necessary for the separation of the duplicated centrosomes [5]

Summary

EXPERIMENTAL PROCEDURES

Materials—Fetal bovine serum was bought from Hyclone. Dulbecco’s modified Eagle’s medium was purchased from Invitrogen. The polymerization assays of tubulin (at 40 ␮M) in the presence and absence of STLC (50 ␮M) and two controls (40 ␮M taxol and 10 ␮M nocodazole) were performed in 96-well half-area plates in a volume of 100 ␮l at 37 °C (Sunrise photometer, TECAN, Maennesdorf, Switzerland). To optimize the signal for basal Eg5 activity at low protein concentration, measurements were performed in the presence of 300 mM NaCl. Eg5 at seven different concentrations (0.175, 0.35, 0.7, 1.4, 2.8, 3.5, and 4.2 ␮M) was incubated at room temperature for 25 min with STLC or STDC from 0 to 5 ␮M. In vitro assembled pig brain MTs at 0.075 ␮M were injected in buffer X containing 1 mM ATP and 20 ␮M paclitaxel Both the STLC inhibition and the Me2SO inhibition were confirmed to be fully reversible on washout. For each Z series images, the best focus was chosen before the reconstitution of the movie

RESULTS

DISCUSSION

Kinesin family