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J. Biol. Chem., Vol. 281, Issue 26, 17559-17569, June 30, 2006

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

Dimitrios A. Skoufias, Salvatore DeBonis, Yasmina Saoudi^¶, Luc Lebeau^||, Isabelle Crevel^**, Robert Cross^**, Richard H. Wade, David Hackney, and Frank Kozielski¹

From the 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, ^¶Laboratoire du Cytosquelette, INSERM Unite 366, Commissariat à l'Energie Atomique, 38054 Grenoble Cedex 9, France, ^||Laboratoire de Chimie Organique Appliquée, Institut Gilbert-Laustriat, CNRS, Université Louis Pasteur, Faculté de Pharmacie, 74 Route du Rhin, 67401 Illkirch Cedex, France, ^**Marie Curie Research Institute, Oxted RH8 0TL, United Kingdom, and the Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213

Received for publication, October 31, 2005 , and in revised form, February 15, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 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 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Kinesins form a superfamily of motor proteins with about 14 different subfamilies clearly identified so far. They play important roles in intracellular transport and at different stages of cell division. The driving force behind these processes is ATP hydrolysis.

The roles of different kinesins during cell division make them highly important for understanding fundamental aspects of mitosis and meiosis. In recent years, some of them have appeared as potential targets for anti-cancer drugs (1–3). One of these mitotic kinesins, human Eg5 (HsEg5/KSP), a member of the kinesin-5 family (4), is responsible for the formation and maintenance of the bipolar spindle (5). Eg5 represents an especially attractive target because when inhibited by microinjection with suitable antibodies (5), by RNAi² (6), or by treating cells with specific Eg5 inhibitors (7), it displays a very characteristic mitotic arrest phenotype, i.e. a monoastral spindle with an array of microtubules (MTs) emanating from a pair of nonseparated centrosomes surrounded by chromosomes. Cell-based as well as in vitro assays have led to the discovery of a series of inhibitors that target Eg5 and lead to mitotic arrest and cell death. Among these inhibitors are monastrol, the first Eg5 inhibitor discovered (7), terpendole E, identified from a fungal strain (8), HR22C16, structurally related to monastrol (9), CK0106023, a quinazolinone analogue representing the most potent Eg5 inhibitor identified so far (10), dihydropyrazoles (11), and S-trityl-L-cysteine (STLC) (12). Several of these inhibitors are currently intensively studied as potential anticancer drugs, as tools for studying fundamental processes in mitosis and function of its target (chemical genetics) (13), or simply as a model to understand the mechanisms of inhibition of this important class of proteins (14–17).

By using two small, preselected libraries from the NCI, we have recently identified several new inhibitors of human Eg5 activity, with STLC being the most potent in vitro and in cell-based assays (12). STLC was shown to effectively inhibit the in vitro Eg5 basal and MT-stimulated ATPase activities (IC₅₀ = 1 µM and IC₅₀ = 140 nM, respectively) each at a given protein concentration and to induce mitotic arrest in HeLa cells in the sub-micromolar range (IC₅₀ = 0.7 µM), without visible effects on the MT network, even at high inhibitor concentration. Additionally, we were able to show that STLC and monastrol share a common binding region on Eg5 (18). STLC has been identified by computer-assisted analysis of cytotoxicity data as a mitotic arrest agent (19), and it has been shown to inhibit tumor growth in the NCI 60 tumor cell line screen, making it a potentially very interesting candidate for drug development.

To better understand its inhibition mechanism, we studied the effect of STLC in HeLa and U2OS cells and in vitro by using the recombinantly expressed Eg5 motor domain comprising the first 386 N-terminal residues of the native protein. We describe here the characterization of the interaction of STLC with human Eg5, its mode of inhibition, effects on HeLa cells, and the characteristics of both enantiomers. These results provide a basis for understanding the inhibition mechanism of a molecular motor and for the development of more potent inhibitors.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Fetal bovine serum was bought from Hyclone. Dulbecco's modified Eagle's medium was purchased from Invitrogen. Nocodazole, taxol, and aphidicholine were from Sigma, and STLC was from Novabiochem. 96-Well half-area and 96-well µclear plates were from Greiner BioOne. The anti--tubulin antibody was obtained from Sigma. FITC-conjugated goat anti-rat, FITC-conjugated anti-mouse IgG, and Cy3-conjugated anti-mouse secondary antibodies were bought from Jackson ImmunoResearch, West Grove, PA. The MPM-2 mouse monoclonal antibody was from Dako, Carpinteria, CA. Mant-ADP was purchased from Jena Biosciences, Germany. The chiral chirobiotic T column was from Advanced Separation Technologies Inc., Whippany, NJ.

S-Tritylcysteine Compounds—Stock solutions of STLC (NSC 83265) and S-trityl-D-cysteine (STDC, NSC 124676) were prepared at 50 mM in Me₂SO and kept at –20 °C. The purity of all compounds used in this study was checked by liquid chromatography coupled to mass spectrometry. To better judge the small differences observed between STLC and STDC by using the in vitro and cell-based assays, we compared the wavelength scans of both solutions (2 µl of each enantiomer stock solution in 98 µl of ethanol) and found that their concentrations were practically identical with a maximal error of ±4.6%. The enantiomeric purity of STLC and STDC was checked by high performance liquid chromatography (HPLC) on a chiral chirobiotic T column (250 x 4.6 mm) using a mixture of acetonitrile/water (85:15 v/v; flow rate, 1 ml/min). The wavelength for the detection of S-trityl compounds was 235 nm. STLC from either NCI or Novabiochem was eluted at t_R 10.9 min, whereas STDC was eluted at t_R 13.5 min. Both STLC and STDC used in this study were judged to be at least 97% enantiomerically pure. In addition STLC obtained from the NCI seemed to be enantiomerically slightly more pure than the compound purchased from Novabiochem (supplemental Fig. 1).

Expression and Purification of Recombinant Proteins—Homo sapiens kinesins containing the catalytic domain were purified as follows: Eg5 (construct HsEg5_2–386 with bound ADP as well as nucleotide-free and XlEg5_1–430), uKHC (a member of the kinesin-1 family), MKLP1 and RabK6 (kinesin-6), KIFC1 (kinesin-14), CENP-E (kinesin-7), KIF2A and KIF2C (kinesin-13), and Kid (kinesin-10) were prepared as described³ (14, 20–22).

Tubulin Purification from Bovine Brain and Polymerization Assay in Vitro—Tubulin was purified from bovine brain using the polymerization-depolymerization method as described (23). The polymerization assays of tubulin (at 40 µM) in the presence and absence of STLC (50 µM) and two controls (40 µM taxol and 10 µM nocodazole) were performed in 96-well half-area plates in a volume of 100 µl at 37 °C (Sunrise photometer, TECAN, Maennesdorf, Switzerland). The buffer used for polymerization of tubulin was 100 mM PIPES, pH 6.75, 1 mM Na-EGTA, 1 mM MgCl₂, and 0.7 mM GTP. All concentrations indicated are final after mixing.

Testing the Specificity of STLC Using Other Human Kinesins—The ATPase activity of kinesins was determined by using the enzyme-coupled assay described by Hackney and Jiang (24). The specificity of STLC was determined by measuring the inhibition of the basal or MT-stimulated ATPase activity in the presence of increasing amounts of STLC using NaCl concentrations that had been optimized individually for each kinesin to maximize their activities.

Testing STLC and STDC for Tight Binding—Measurements of inhibition of the basal Eg5 ATPase activity were performed as described by DeBonis et al. (12). 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 µM) was incubated at room temperature for 25 min with STLC or STDC from 0 to 5 µM. The final Me₂SO concentration in all cases was 2% in a final volume of 30 µl. The mixture containing Eg5 and STLC or STDC was rapidly mixed with 170 µl ATPase buffer previously dispensed into wells of a µclear 96-well plate, and the resulting decrease in absorbance at 340 nm was measured using the 96-well Sunrise photometer. The IC₅₀ values for inhibition of basal Eg5 ATPase activity by increasing amounts of inhibitor were determined in triplicate. In a previous publication (12), we determined initial IC₅₀ values for STLC by fitting the experimental data to Equation 1,

(Eq. 1)

where v is the reaction velocity at different STLC concentrations; v₀ is the control velocity in the absence of inhibitor; A represents the amplitude; [I] indicates the concentration of STLC, and IC₅₀ indicates the median inhibitory concentration. This led to a nonoptimal fit for tight binding STLC. The experimental data were best fitted now to the Morrison Equation 2 (25) for tight binding inhibitors,

(Eq. 2)

where v₀ is the velocity in the absence of inhibitor; v_i is the measured velocity; [E] is equal to the total enzyme concentration; [I] indicates the added inhibitor concentration, and K_i,app indicates the apparent equilibrium inhibition constant, which depend on the type of inhibition.

Stopped-flow Analysis—Stopped-flow analysis of the fast phase of STLC binding to the complex of Eg5 with mant-ADP was performed as described previously for monastrol (14). The experiments were performed at high (300 mM NaCl) and low (25 mM KCl) salt concentrations. Excitation was at 285 nm. The data for the fast phase were fitted to Equation 3 at low STLC concentrations,

(Eq. 3)

where I represents the different STLC concentrations, and k₁ and k_–1 are the binding and release rate constants for Reaction 1,

REACTION 1

and k₁ and k_–1 are the rate constants for binding and release of STLC.

Motility Assays—STLC was resuspended at 50 µM in 100% Me₂SO and subsequently diluted in Me₂SO prior to addition to the motility buffer, to keep a constant 2% Me₂SO concentration. Before loading in the chamber, Eg5 was diluted 3-fold to 5 µM using buffer A (80 mM PIPES, pH 6.9, 100 mM NaCl, 2 mM Mg(OAc)₂, 1 mM Na-EGTA, 5 mM 1,4-dithiothreitol), supplemented with 1 mM ATP and 0.1 mg/ml casein. The chamber was incubated 5 min at room temperature and then washed with buffer X containing 1 mM ATP. Finally, in vitro assembled pig brain MTs at 0.075 µM were injected in buffer X containing 1 mM ATP and 20 µM paclitaxel. Both the STLC inhibition and the Me₂SO inhibition were confirmed to be fully reversible on washout.

Cell Culture—HeLa and U2OS cells were grown as monolayer cultures in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and maintained in a humidified incubator at 37 °C in 5% CO₂. Stock solutions of 1 mg/ml nocodazole and 50 mM aphidicholine were prepared in Me₂SO and kept at –20 °C until use.

Immunofluorescence Microscopy—Cells were left to adhere for at least 36 h on poly-D-lysine-coated glass in 24-well plates before the addition of the drugs. Following incubation with drugs for 8 h, cells were fixed with 1% paraformaldehyde/PBS at 37 °C for 3 min, followed by an incubation in 100% methanol at –20 °C for 5 min and then washing with PBS for 5 min. After two additional 5-min washes, fixed cells were incubated with YL1/2 anti--tubulin and anti--tubulin for 1 h and then with FITC-conjugated goat anti-rat and Cy3-conjugated anti-mouse secondary antibodies for 30 min and counterstained with propidium iodide. Images were collected with a, MRC-600 laser scanning confocal apparatus (Bio-Rad) coupled to a Nikon Optiphot microscope.

Flow Cytometric Analysis—Cells treated as above were collected and fixed in 90% methanol at –20 °C for at least 10 min, followed by three washes with PBS. Two-dimensional flow cytometric analysis was carried out using the MPM-2 mouse monoclonal antibody, a specific mitotic marker (26), and propidium iodide, a marker of DNA content. Following incubation with MPM-2, cells were labeled with FITC-conjugated anti-mouse IgG secondary antibodies and propidium iodide as described previously (27). Data were collected using a FACScan flow cytometer (BD Biosciences) using Cellquest software, and only gated cells were taken into account.

Live Cell Imaging and Video Microscopy—For time-lapse microscopy, HeLa cells were plated in glass-bottom dishes covered with a CO₂-permeable membrane and then placed inside the video microscopy platform equipped with an incubator enabling the regulation of the temperature and the CO₂ level. Time lapse Z series images (Z = 3) were collected with an inverted motorized microscope (Axiovert 200M, Zeiss) controlled by MetaMorph software (Universal Imaging, Downingtown, PA). Tubulin-GFP-marked cells were observed with a plane neofluar objective (63 x 1.4 NA), and fluorescent images were acquired with a CoolSnap HQ charge-coupled device camera (Roper Scientific, Trenton, NJ) every 5 min for 8 h for the control without inhibitor and up to a maximum of 38 h in presence of 5 or 10 µM STLC under low illumination to avoid cell damage. The acquisition time was 100 ms. For each Z series images, the best focus was chosen before the reconstitution of the movie.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Comparison between STLC and STDC—STLC is a non-natural derivative of the -amino acid cysteine (Fig. 1), which incorporates a single chiral center and consequently exists as two enantiomers (D and L). The enantiomeric purity of STLC and STDC was determined by HPLC on the chiral chirobiotic T column (supplemental Fig. 1). STLC from either the NCI (Fig. 1A) or Novabiochem (Fig. 1B) was eluted at t_R 10.9 min, whereas STDC was eluted at t_R 13.5 min (Fig. 1C). The difference in retention time between the two enantiomers was more evident when the two were mixed and then separated (Fig. 1D). The STLC and STDC used in this study were both judged to be at least 97% enantiomerically pure.

To determine whether one of the enantiomers is more active than the other, we measured the inhibition of basal- and MT-activated ATPase activities using Eg5 concentrations of 0.7 and 0.073 µM, respectively, and quantified the number of monoastral spindles when incubating HeLa cells with STLC (Fig. 2). Both enantiomers inhibit the basal Eg5 ATPase activity with IC₅₀ values of 403 nM for STLC and 611 nM for STDC (Fig. 2A), and the MT-activated ATPase activity with an IC₅₀ of 286 ± 11 nM for STLC and 240 ± 28 nM for STDC (Fig. 2B). Phenotype-based assays with HeLa cells also showed that the two enantiomers are nearly equally potent (Fig. 2C). Within the first 8 h of incubation, both enantiomers gave a concentration-dependent accumulation of mitotic cells with MTs emanating from a single spindle aster (Fig. 2C, upper panels) with IC₅₀ values of 700 and 750 nM for STLC and STDC, respectively (Fig. 2D). Immunofluorescence microscopy using a -tubulin antibody as a centrosomal marker revealed the two centrosomes (four -tubulin spots) had failed to separate in the center of the monoasters (Fig. 2C, lower panels). This monoaster phenotype has been described previously to result from inhibition of Eg5 activity either after injection of Eg5 antibodies (5) or after inhibition of Eg5 activity by specific inhibitors like monastrol (7), terpendole E (8), HR22C16 (9), or CK0106023 (10). Both enantiomers were also equally potent in inhibiting cell cycle progression at G₂/M. Two-dimensional FACScan analysis (using propidium iodide for DNA content and the mitotic marker MPM2 antibody) revealed that addition of STLC and STDC in asynchronously cycling HeLa cells leads to the accumulation of cells, in a concentration-dependent manner, with a 4 N DNA content (Fig. 2, E and F). After 24 h of incubation with the drugs at concentrations equal to and above 5 µM, more than 80% of the cells are arrested in G₂/M phase (Fig. 2G). Interestingly, after a 24-h incubation period and even at high concentrations the drugs do not appear to be cytotoxic. Furthermore, 80% of total cells were mitotic because they were positive for the mitotic marker MPM2 (Fig. 2H). These data suggest that STLC and STDC are potent mitotic inhibitors. Quantitatively similar mitotic arrest data were obtained with the U2OS tumor cell line (data not shown).

View larger version (10K): [in this window] [in a new window]   FIG. 1. Chemical structure of tritylcysteine. The molecule contains a chiral center and exists as D-(left) and L-enantiomers (right).

STLC as a Specific Inhibitor of Mitotic Progression—We were also interested to know if STLC was able to block cells in a different phase of the cell cycle. U2OS cells were exposed to STLC, following presynchronization with aphidicholine at early G₁/S before centrosome duplication (Fig. 3C). U2OS cells, 8 h following aphidicholine release in the presence of STLC, passed through the S phase and arrived at G₂/M synchronously where they remained blocked for the next 24 h. Identical behavior was obtained with the MT assembly inhibitor nocodazole (data not shown). These results suggest that STLC does not inhibit cell cycle progression at S or G₂ phases and that indeed is a specific inhibitor of mitotic progression.

View larger version (32K): [in this window] [in a new window]   FIG. 2. Comparison of STLC and STDC. A, inhibition of basal Eg5 ATPase activity by STLC (open squares) and STDC (open circles). B, inhibition of MT-activated Eg5 ATPase activity by STLC (open circles) and STDC (open squares). C, double-immunofluorescence microscopy of cells stained with anti--tubulin for MTs and anti--tubulin for centrosomes showing induction of monoastral spindles following treatment with STLC and STDC. D, induction of monoastral spindles in the presence of STLC and STDC. HeLa cells were treated at the indicated concentrations of STLC (solid bars) and STDC (open bars) for 8 h and then were fixed and stained for immunofluorescence microscopy. The percentage of monoastral spindles from the total mitotic cells were then determined. Cell cycle distribution of HeLa cells treated with STLC (E) and STDC (F) by two-dimensional FACScan analysis. 24 h of incubation of cells leads to concentration-dependent accumulation of cells with 4 N DNA content. G, quantitative representation of the histograms in E and F of the % of cells with 4 N DNA. H, quantitative representation of the histograms in E and F of cells positive for the mitotic marker MPM2 indicating a concentration-dependent accumulation of mitotic cells (STLC, solid bars; STDC, open bars).

View larger version (17K): [in this window] [in a new window]   FIG. 3. STLC blocks cell cycle specifically in mitosis. HeLa cells (A) and U2OS (B) cells were analyzed by FACScan analysis following incubation with STLC (5 µM) at the indicated times. C, U2OS cells presynchronized with aphidicholine for 18 h at the G1/S boundary of the cell cycle. Following aphidicholine washout, cells were treated with 5µM STLC. Samples for FACScan analysis at the indicated times were collected. Cells reached G2/M phase 8 h following release from the G1/S block and remained blocked at G2/M for the next 24 h in the presence of STLC.

View larger version (29K): [in this window] [in a new window]   FIG. 4. STLC is a reversible mitotic inhibitor. Mitotic HeLa cells were collected by shake off (time 0 h) following 18 h of incubation with either 5 µM STLC or 40 ng/ml nocodazole (NOC) or with both drugs. Flow cytometric analysis of released cells was carried out for the indicated times following release into medium from STLC (A) and NOC (B). C, cells following coincubation with STLC and NOC were released from NOC only. Cells released from STLC exited from mitosis in the same time course as cells released from NOC. The continued presence of STLC in NOC and STLC coincubated cells results in efficient M block. D, quantitative analysis of the histograms in A–C, of the % of cells with 4 N DNA cells following release from the various treatments. (Release from STLC, solid bars; from NOC, open bars) and cells in the continued presence of STLC after NOC washout of coincubated cells (gray bars).

STLC Does Not Influence MT Dynamics Either in Vitro or in Cell-based Assays—In order to exclude the possibility that STLC may have additional MT assembling or stabilizing/destabilizing properties, we coincubated HeLa cells with STLC and nocodazole and washed out either both drugs or nocodazole only. We reasoned that if STLC had MT-stabilizing properties then MTs in the presence of STLC should have been resistant to nocodazole. Furthermore, if STLC had MT depolymerization properties, MTs should not polymerize following nocodazole washout in the presence of STLC. The results shown in Fig. 5 indicated that MT stability in mitotic and interphase cells was not sensitive to the presence of STLC. Following a 4-h incubation with nocodazole in the absence or in the presence of STLC, mitotic cells had only very few short MT asters (Fig. 5A). Following nocodazole washout, mitotic cells formed normal bipolar spindles within the first 30 min, and by 60 min cells were exiting mitosis. Under nocodazole washout conditions in the presence of STLC, monoastral spindles were assembled readily, with a dense MT network nucleating from the center of the cells where the duplicated centrosomes were located. In interphase treated cells, STLC did not affect the interphase MT network and did not affect the presence of few nocodazole-resistant MTs (Fig. 5B). 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. These results were confirmed by performing in vitro assays, observing the polymerization of tubulin alone and in the presence of either nocodazole, taxotere, or STLC over a time range of several hours. Even at high STLC concentration (50 µM) no significant influence on MT dynamics was observed in vitro (supplemental Fig. 2).

View larger version (43K): [in this window] [in a new window]   FIG. 5. STLC does not inhibit MT polymerization and stability in cells. HeLa cells were treated with either STLC (5 µM) or nocodazole (0.1 µg/ml) or with both drugs. Following a 4-h exposure, drugs were washed out using either medium or STLC and fixed and stained for immunofluorescence microscopy with an anti--tubulin antibody at the indicated times. Images of mitotic (A) and interphase cells (B) of the various treatments are shown.

STLC and STDC Are Tight Binding Inhibitors of Eg5—Having investigated the effect of STLC in cell-based assays, we wanted to clarify in more detail the mechanism by which STLC inhibits Eg5. To test the possibility that STLC and STDC are tight binding inhibitors of human Eg5, we preincubated Eg5 at different protein concentrations with increasing amounts of both inhibitors and determined the IC₅₀ values. Preincubation is necessary because tight binding inhibitors are often characterized by a slow onset of inhibition (28). The dose-response plot of the fractional ATPase velocities of Eg5 in the absence of MTs as a function of STLC and STDC concentrations at different Eg5 concentrations is shown in Fig. 7, A and B. For STLC, the IC₅₀ values increase with increasing Eg5 concentrations, spanning IC₅₀ values from 0.2 to 1.83 µM. The calculated slope from the plot of IC₅₀ values as a function of Eg5 concentration is 0.36, in acceptable agreement with the theoretical value of 0.5 (Fig. 7C). The Y intercept gives an estimate of the K_i,app of 140 nM. The IC₅₀ values for STDC are also concentration-dependent with a slope of 0.40, with an estimate of 23 nM for the K_i,app. We conclude that both enantiomers are potent tight binding inhibitors of human Eg5.

View larger version (118K): [in this window] [in a new window]   FIG. 6. HeLa cells stably expressing GFP--tubulin were observed by live cell imaging in the absence (A) or in the presence of 10µM STLC (B). Duplicated centrosomes in untreated cells from the moment they were first visible were able to fully separate, and cells completed mitosis successfully within 50 min. In contrast, in STLC-treated cells duplicated centrosomes were unable to fully separate and cells remained blocked in mitosis (see supplemental movies S1 and S2).

The amplitude of the fast phase is 10% of the total decrease, and the rate of the fast phase increases with increasing STLC as indicated in Fig. 8. The fast phase likely corresponds to two-step binding of STLC with an STLC-induced conformational change in the initial ternary complex of Eg5, mant-ADP, and STLC that produces a small change in the efficiency of the FRET. The bimolecular rate for net STLC binding is approximately the same in high and low salt at 6 µM^–1 s^–1. There is indication of saturation in the rate of the fast phase at high STLC, although the maximum rate cannot be reliably estimated. When assayed at 0.1 µM Eg5 to maintain approximate pseudo-first order conditions with an STLC:Eg5 ratio 6, the rate of STLC dissociation from the ternary complex is 3.7 s^–1 as defined by the intercept in the absence of STLC at 25 mM KCl (Fig. 8B, inset). The rates of 6 µM^–1 s^–1 and 3.7 s^–1 for STLC binding from the fast phase of the fluorescent transient predict a K_d of 0.62 µM for STLC binding to Eg5·mant-ADP, which is in good agreement with the IC₅₀ of 0.64 µM obtained from the slow phase in Fig. 7A at 25 mM KCl.

Tight Binding Versus Slow Tight Binding Inhibitor—Many tight binding inhibitors are characterized by a slow onset of inhibition. They bind slowly (or time-dependent) to their target enzyme with the four different modes of interactions between inhibitor and enzyme to be distinguished. The fast bimolecular association rate of 6.1 µM^–1 s^–1 for STLC indicates that there is no time-dependent component that might characterize STLC as a slow tight binding inhibitor. We therefore measured the progress curve of Eg5 activity (phosphate release) in the absence and presence of STLC initiating the reaction by adding 0.9 µM Eg5 to a mixture of 1 mM ATP and 175 nM inhibitor. Over a period of 10 min, uninhibited as well as inhibited Eg5 displays a linear progress curve (Fig. 8C), confirming that STLC is a tight binding and not a slow tight binding inhibitor of Eg5.

Specificity of S-Trityl-L-cysteine—Is the mitotic arrest phenotype observed with these inhibitors uniquely due to the inhibition of human Eg5 activity? Is human Eg5 the only target? The specificity of STLC was tested using other members of the H. sapiens kinesin superfamily as follows: conventional kinesin (construct HK379) as the prototype for a plus-end directed molecular motor involved in intracellular transport (29–30); the kinetochore-associated motor CENP-E (31), essential for some aspects of kinetochore MT attachments; MKLP1 and RabK6, essential for cytokinesis (32–33); KIFC1, a C-terminal kinesin-related protein that opposes the activity of Eg5 (34); Kid, required for chromosome congression (35); and KIF2A and KIF2C, two kinesins with an internal motor domain (36), were similarly tested. The results are summarized in Table 1. Among the nine different human kinesins tested, only Eg5 is significantly inhibited by STLC.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The mitotic process is highly dynamic, and any drug that alters or inhibits this process is of potential interest as an anticancer agent (1). The secondary effects produced by certain classes of antitumor drugs such as taxanes (taxol and taxotere) and vinca alkaloids (vinblastine, vincristine, and vinorelbine), which have tubulin and MTs as their primary targets, indicate the need to search for new mitotic targets and better drugs. 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). We have previously identified STLC as an inhibitor of human Eg5 motor protein (12) and report here a more detailed characterization of the mechanism of its inhibitory action.

View larger version (18K): [in this window] [in a new window]   FIG. 7. A, dose-response plot of fractional velocities as a function of increasing STLC concentrations at seven different Eg5 concentrations using the inhibition of the basal Eg5 ATPase activity. The Eg5 concentrations used are as follows: = 0.175 µM, = 0.35 µM, = 0.7 µM, = 1.4 µM, = 2.8 µM, • = 3.5 µM, and = 4.2 µM. Each data point is the average of three measurements, taken in parallel and using the same buffer. Error bars represent ± S.D. B, dose-response plot of fractional velocities as a function of increasing STDC concentrations at four different Eg5 concentrations using the inhibition of the basal Eg5 ATPase activity. The Eg5 concentrations used are: = 0.35 µM, = 0.7 µM, = 1.4 µM, = 2.8 µM. Each data point is the average of three measurements, taken in parallel and using the same buffer. Error bars represent ± S.D. C, plot of IC50 values for STLC (•) and STDC () obtained from the curves represented in A and B, as a function of Eg5 concentration.

Evidence from Drosophila embryos and cells following antibody microinjection and RNAi depletion of the Drosophila Eg5-like protein Klp61F showed that centrosomes initially separate and then collapse together in a kinesin-14-driven process, specifically during prometaphase (37–39). Similar antagonistic behavior between Eg5 and HSET motors were observed in mammalian cells (34). Our live cell imaging results with STLC-treated cells did not show any evidence of a bipolar spindle formation and did not show any evidence of full centrosome separation, although an initial but very short distance centrosome separation was detected very early in prophase. One explanation for this discrepancy is that it is possible that there are still some low but undetectable levels of Klp61F following RNAi that may permit the formation of a bipolar spindle, but these low levels are unable to sustain it. One advantage of using specific chemical inhibitors is that depending on the concentration used one can be sure that all the target protein is inhibited. Alternatively, subtle differences in the properties in the motors and/or the pathways of spindle assembly between Drosophila and mammals may exist.

View larger version (14K): [in this window] [in a new window]   FIG. 8. Mant-ADP release from Eg5 at different salt concentrations. A, slow phase of ADP release. Mant-ADP was chased from Eg5 by rapidly mixing with 200 µM ATP and different STLC concentrations (0 to 16 µM). The Eg5 concentration used was 0.1 µM, and data were fitted with a mutual depletion model. The rate of ADP release is salt-dependent; the presence of 25 mM KCl (filled triangles) gives 90% fractional inhibition at saturating STLC concentration with a Kd of 0.64 µM. In the presence of 300 mM NaCl (filled diamonds), the maximal inhibition is 98% with a Kd of 0.13 µM. B, kinetics of fast phase of fluorescence decrease on addition of STLC to Eg5·mant-ADP. A complex of Eg5·mant-ADP was formed by adding a 10-fold excess of mant-ATP to Eg5 and incubating for >10 min at room temperature to allow equilibration of nucleotide at the Eg5-binding site. This complex was then mixed in the stopped-flow apparatus with STLC and ATP (0.2 mM final concentration), and the rate for the initial fast phase was determined. The buffer was A25 with 25 mM KCl (open triangles) or 300 mM NaCl (open circles), and the Eg5 concentration was 0.4 µM. The inset shows results extending the lower STLC concentrations obtained with an Eg5 concentration of 0.125 µM Eg5 in the presence of 25 mM KCl. C, progress curve of Eg5 activity in the absence (filled circles) and presence (filled squares) of STLC followed over a 10-min time period. The STLC concentration was 175 nM. Linear plots are obtained in both cases indicating that STLC is a tight but not slow tight binding inhibitor of Eg5 activity.

View larger version (19K): [in this window] [in a new window]   FIG. 9. Inhibition of X. laevis Eg5 motility by STLC. The inhibition of X. laevis Eg5 motility by increasing amounts of STLC is shown as black circles, and the gliding motility after washout of the inhibitor is represented by closed triangles. The IC50 value for this experiment has been determined to be 0.5 µM.

Interestingly, our conclusion that STLC is a mitotic inhibitor is supported by previous studies, which evaluated the differential cytotoxicity data of different small molecule inhibitors using the COMPARE program (19). STLC was evaluated to be a mitotic inhibitor even though its target and potential mode of action were completely unknown. Pharmacological data for STLC available from the NCI (40–42) reveal that STLC is a strong cytostatic drug with a mean inhibition growth concentration GI₅₀ of 1.31 µM for the 60 different tumor cell lines. STLC has been also tested in the past by the NCI for potential antitumor activity using in vivo mice tumor models (43). More than 20 different models have been tested. In addition, data on human lung LX-1 xenografts as well as for toxicity testing on nontumored animals are available. In all cases STLC was found to inhibit tumor growth at doses that were not lethal to the mice. The NCI data taken together with our data show that STLC is a strong mitotic inhibitor because of its ability to specifically inhibit Eg5 motor activity thus making STLC a very interesting candidate for an antitumor agent.

STLC Is a Tight Binding Inhibitor of Eg5—Our analysis of the biochemical data on the mechanism of inhibition of Eg5 motor activity by STLC provides firm evidence that STLC is a tight binding inhibitor of Eg5. In a recent paper (12), we determined the IC₅₀ of inhibition of the basal Eg5 ATPase activity to be 1.0 µM (determined at a Eg5 concentration of 2.6 µM). The IC₅₀ value for the inhibition of MT-stimulated ATPase activity, however, was 0.14 µM (Eg5 concentration = 0.073 µM), a factor of about 7 lower. One possible explanation for this effect is that STLC also interacts with MTs thus modifying the MT-stimulated ATPase activity of Eg5. We tested this possibility and dismissed it. In both the earlier and the later work, the IC₅₀ values obtained were close to the concentrations of Eg5 used during the tests, indicating tight binding of the inhibitor to Eg5. Further work showed that the IC₅₀ values vary as a function of the Eg5 concentration at a fixed substrate concentration, confirming that in fact STLC is a tight binding inhibitor of Eg5. This provides a plausible explanation for the observed differences between the IC₅₀ values of basal versus MT-stimulated ATPase activities. The inhibition of the MT-stimulated Eg5 ATPase activity is simply measured at a much lower protein concentration than that for the basal Eg5 ATPase activity, resulting in a much lower IC₅₀ value (1 µM versus 140 nM). STLC also effectively inhibits ADP release from Eg5 alone. The results in Fig. 8 indicate that STLC forms a ternary complex with Eg5·ADP and that the tighter binding of STLC to the ADP complex with Eg5, compared to monastrol, is due both to faster association and slower release (6.1 µM^–1 s^–1 and 3.6 s^–1 for STLC versus 0.78 µM^–1 s^–1 and 15 s^–1 for monastrol) (14). STLC thus appears to work by a similar mechanism as monastrol but to bind much tighter.

Both STLC and STDC appear to be tight binding inhibitors. The two enantiomers are almost equally potent inhibitors of basal and MT-stimulated Eg5 ATPase activity. Cell-based assays showed that both enantiomers were nearly equally potent in inhibiting bipolar spindle assembly and that both blocked cells reversibly in the M phase of the cell cycle. In contrast to monastrol, whose two enantiomers display different activity profiles, it appears that there is no strong selectivity for the two enantiomers in inhibiting Eg5 motor activity. Because both enantiomers are nearly equally potent, STLC analogues with no chiral center should be potent as well, making the synthesis of such inhibitors easier, because no enantioselective synthesis or enantiomer separation is required. This is currently under investigation.

STLC Reversibly Inhibits MT Gliding—STLC also inhibits X. laevis Eg5 gliding activity with an IC₅₀ value of 0.5 µM, which is very close to the IC₅₀ for inducing mitotic arrest in the HeLa cell-based assay (0.7 µM). Washout experiments after motility assays and FACScan analysis have revealed that STLC is a reversible inhibitor of Eg5 activity, comparable to monastrol (7, 12), quinazolinone (10), and terpendole E (8).

Specificity of STLC—Zhu et al. (44) performed a functional analysis of all human kinesins using RNA interference. Among the 41 different kinesins investigated, at least 12 were shown to be involved in mitosis and cytokinesis. Three of them, Eg5, KIF2A, and KIFC1, were shown to be important for bipolar spindle formation. Only Eg5 and KIF2A, when depleted, displayed a similar phenotype (monoastral spindles for Eg5 and monopolar or asymmetric bipolar spindles for KIF2A) and subsequently mitotic arrest, confirming previous results for KIF2A (45). The other 10 mitotic kinesins displayed other phenotypes related to their involvement in chromosome segregation, anaphase spindle dynamics, and cytokinesis. Therefore, we investigated whether monoastral spindles could be due to KIF2A inhibition by measuring its ATPase activity in the presence of STLC. However, in vitro STLC did not inhibit KIF2A activity. In addition, we always observed monopolar but never asymmetric bipolar spindles. STLC did not inhibit any of the other kinesins tested. We therefore conclude that STLC induces mitotic arrest by specifically inhibiting human Eg5 and that it does not inhibit other human mitotic kinesins tested to date. Whether STLC inhibits kinesins involved in intracellular transport remains to be shown.

Antagonistic Effects of Eg5 Inhibitors with Other Agents—Marcus et al. (46) recently reported an antagonistic effect when treating cells with a combination of taxol and HR22C16 (9), leading to a significant decrease of cells in mitotic arrest compared with either drug alone. They argued that a combination of drugs targeting different proteins may not be favorable. An antagonistic effect has also been observed when combining irofulven, a molecule that inhibits DNA synthesis, with STLC or other antimitotic agents (47). It will be interesting to test whether a combination of STLC with taxol or taxotere leads to similar results.

Organosulfur Compounds and Mitotic Arrest—A recent publication investigated the effects of a series of garlic-derived as well as synthetic organosulfur compounds, including STLC, on colon cancer cells (48). They show that STLC specifically induces G₂/M cell cycle arrest and subsequently apoptosis through spindle impairment but without disrupting MT structure or dynamics, excluding the possibility that its binds directly to tubulin, in agreement with our study. Apoptotic cell death induced by STLC occurs after 48 h and involves a detectable but weak caspase-3 activation and poly(ADP-ribose) polymerase cleavage, a moderate loss of mitochondrial membrane potential but not JNK1 activation, or release of cytochrome c. Xiao et al. (48) therefore argue that STLC might induce apoptosis through a caspase-independent pathway.

In conclusion, the accumulated cell-based and in vitro data suggest that STLC is a small cell-permeable inhibitor, which tightly and specifically binds to Eg5, a mitotic motor of the kinesin superfamily, but does not interfere with MT dynamics. STLC binds to the Eg5·ADP complex forming an ADP·Eg5·STLC ternary complex that leads to the inhibition of ADP release from Eg5. Consequently, it cannot undergo further ATP-driven conformational changes resulting in the inhibition of Eg5-induced gliding apart of antiparallel spindle MTs. Finally, the duplicated centrosomes cannot fully separate, and cells arrest in mitosis with the characteristic monastral spindle phenotype. Prolonged mitotic arrest eventually leads to apoptotic cell death, probably also through the involvement of a caspase-independent pathway.

	FOOTNOTES

 
^* This work was supported by Contract 5197 from Association pour la Recherche sur le Cancer, Alliance Des Recherches sur le Cancer, and Contracts 03 013690 02 and 03 013690 01 from the Région Rhône-Alpes. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2 and Movies S1 and S2.

¹ To whom correspondence should be addressed: Institut de Biologie Structurale, 41 Rue Jules Horowitz, 38027 Grenoble, France. Tel.: 33-4-3878-4024; Fax: 33-4-3878-5494; E-mail: Frank.kozielski{at}ibs.fr.

² The abbreviations used are: RNAi, RNA interference; Me₂SO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate; mant-ADP, 2'-3'-O-(N-methylanthraniloyl)adenosine-5'-diphosphate; MTs, microtubules; NOC, nocodazole; PBS, phosphate-buffered saline; NCI, National Cancer Institute; PIPES, 1,4-piperazinebis(ethanesulfonic acid); STLC, S-trityl-L-cysteine; STDC, S-trityl-D-cysteine; HPLC, high pressure liquid chromatography; FACS, fluorescence-activated cell sorter.

³ S. Tcherniuk, I. Garcia-Saez, C. Calmettes, D. Blot, S. Brier, D. Skoufias, E. Neumann, J. F. Conway, S. DeBonis, G. Gyapay, R. H. Wade, S. Cusack, and F. Kozielski, submitted for publication.

	ACKNOWLEDGMENTS

 
We thank the NCI, National Institutes of Health (Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis), for providing us with some of the STLC and STDC used in this study. The anti--tubulin antibody was a generous gift from Dr. J V. Kilmartin, Medical Research Council, Cambridge, UK. We thank Dr. Tim J. Yen (Fox Chase Cancer Center, Philadelphia) for the stable HeLa -tubulin-GFP cell line.

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