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• W2057523896 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. 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. 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 (1Wood K.W. Cornwell W.D. Jackson J.R. Curr. Opin. Pharmacol. 2001; 1: 370-377Crossref PubMed Scopus (256) Google Scholar, 2Miyamoto D.T. Perlman Z.E. Mitchison T.J. Shirasu-Hiza M. Prog. Cell Cycle Res. 2003; 5: 349-360PubMed Google Scholar, 3Bergnes G. Brejc K. Belmont L. Curr. Top Med. Chem. 2005; 5: 127-145Crossref PubMed Scopus (118) Google Scholar). One of these mitotic kinesins, human Eg5 (HsEg5/KSP), a member of the kinesin-5 family (4Lawrence C.J. Dawe R.K. Christie K.R. Cleveland N. Dawson S.C. Endow S.A. Goldstein L.S. Goodson H.V. Hirokawa N. Howard J. Malmberg R.L. McIntosh J.R. Miki H. Mitchison T.J. Okada Y. Reddy A.S. Saxton W.M. Schliwa M. Scholey J.M. Vale R.D. Walczak C.E. Wordeman L. J. Cell Biol. 2004; 167: 19-22Crossref PubMed Scopus (561) Google Scholar), is responsible for the formation and maintenance of the bipolar spindle (5Blangy A. Lane H.A. d'Herin P. Harper M. Kress M. Nigg E.A. Cell. 1995; 83: 1159-1169Abstract Full Text PDF PubMed Scopus (775) Google Scholar). Eg5 represents an especially attractive target because when inhibited by microinjection with suitable antibodies (5Blangy A. Lane H.A. d'Herin P. Harper M. Kress M. Nigg E.A. Cell. 1995; 83: 1159-1169Abstract Full Text PDF PubMed Scopus (775) Google Scholar), by RNAi 2The 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. 2The 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. (6Weil D. Garcon L. Harper M. Dumenil D. Dautry F. Kress M. BioTechniques. 2002; 33: 1244-1248Crossref PubMed Scopus (97) Google Scholar), or by treating cells with specific Eg5 inhibitors (7Mayer T.U. Kapoor T.M. Haggarty S.J. King R.W. Schreiber S.L. Mitchison T.J. Science. 1999; 286: 971-974Crossref PubMed Scopus (1598) Google Scholar), 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 (7Mayer T.U. Kapoor T.M. Haggarty S.J. King R.W. Schreiber S.L. Mitchison T.J. Science. 1999; 286: 971-974Crossref PubMed Scopus (1598) Google Scholar), terpendole E, identified from a fungal strain (8Nakazawa J. Yajima J. Usui T. Ueki M. Takatsuki A. Imoto M. Toyoshima Y.Y. Osada H. Chem. Biol. 2003; 10: 131-137Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), HR22C16, structurally related to monastrol (9Hotha S. Yarrow J.C. Yang J.G. Garrett S. Renduchintala K.V. Mayer T.U. Kapoor T.M. Angew. Chem. Int. Ed. Engl. 2003; 42: 2379-2382Crossref PubMed Scopus (135) Google Scholar), CK0106023, a quinazolinone analogue representing the most potent Eg5 inhibitor identified so far (10Sakowicz R. Finer J.T. Beraud C. Crompton A. Lewis E. Fritsch A. Lee Y. Mak J. Moody R. Turincio R. Chabala J.C. Gonzales P. Roth S. Weitman S. Wood K.W. Cancer Res. 2004; 64: 3276-3280Crossref PubMed Scopus (243) Google Scholar), dihydropyrazoles (11Cox C.D. Breslin M.J. Mariano B.J. Coleman P.J. Buser C.A. Walsh E.S. Hamilton K. Huber H.E. Kohl N.E. Torrent M. Yan Y. Kuo L.C. Hartman G.D. Bioorg. Med. Chem. Lett. 2005; 15: 2041-2045Crossref PubMed Scopus (138) Google Scholar), and S-trityl-l-cysteine (STLC) (12DeBonis S. Skoufias D. Robin G. Lebeau L. Lopez R. Margolis R. Wade R.H. Kozielski F. Mol. Cancer Ther. 2004; 3: 1079-1090PubMed Google Scholar). 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) (13Canman J.C. Cameron L.A. Maddox P.S. Straight A. Tirnauer J.S. Mitchison T.J. Fang G. Kapoor T.M. Salmon E.D. Nature. 2003; 424: 1074-1078Crossref PubMed Scopus (200) Google Scholar), or simply as a model to understand the mechanisms of inhibition of this important class of proteins (14DeBonis S. Simorre J.P. Crevel I. Lebeau L. Skoufias D.A. Blangy A. Ebel C. Gans P. Cross R. Hackney D.D. Wade R.H. Kozielski F. Biochemistry. 2003; 42: 338-349Crossref PubMed Scopus (169) Google Scholar, 15Maliga Z. Kapoor T.M. Mitchison T.J. Chem. Biol. 2002; 9: 989-996Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 16Luo L. Carson J.D. Dhanak D. Jackson J.R. Huang P.S. Lee Y. Sakowicz R. Copeland R.A. Biochemistry. 2004; 43: 15258-15266Crossref PubMed Scopus (51) Google Scholar, 17Cochran J.C. Gatial J.E. III Kapoor Gilbert T. M.S.P. J. Biol. Chem. 2004; 280: 12658-12667Abstract Full Text Full Text PDF Scopus (131) Google Scholar).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 (12DeBonis S. Skoufias D. Robin G. Lebeau L. Lopez R. Margolis R. Wade R.H. Kozielski F. Mol. Cancer Ther. 2004; 3: 1079-1090PubMed Google Scholar). 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 (18Brier S. Lemaire D. DeBonis S. Forest E. Kozielski F. Biochemistry. 2004; 43: 13072-13082Crossref PubMed Scopus (107) Google Scholar). STLC has been identified by computer-assisted analysis of cytotoxicity data as a mitotic arrest agent (19Paull K.D. Lin C.M. Malspeis L. Hamel E. Cancer Res. 1992; 52: 3892-3900PubMed Google Scholar), 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 PROCEDURESMaterials—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-γ-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 × 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 tR 10.9 min, whereas STDC was eluted at tR 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 HsEg52–386 with bound ADP as well as nucleotide-free and XlEg51–430), 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 described 3S. 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. (14DeBonis S. Simorre J.P. Crevel I. Lebeau L. Skoufias D.A. Blangy A. Ebel C. Gans P. Cross R. Hackney D.D. Wade R.H. Kozielski F. Biochemistry. 2003; 42: 338-349Crossref PubMed Scopus (169) Google Scholar, 20Crevel I. Lockhart A. Cross R.A. J. Mol. Biol. 1997; 273: 160-170Crossref PubMed Scopus (79) Google Scholar, 21Kozielski F. Svergun D.I. Zaccai G. Wade R.H. Koch M.H.J. J. Biol. Chem. 2001; 276: 1267-1275Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 22Garcia-Saez I. Yen T. Wade R.H. Kozielski F. J. Mol. Biol. 2004; 340: 1107-1116Crossref PubMed Scopus (51) Google Scholar).Tubulin Purification from Bovine Brain and Polymerization Assay in Vitro—Tubulin was purified from bovine brain using the polymerization-depolymerization method as described (23Robley C. Williams J.R. Lee J.C. Methods Enzymol. 1982; 85: 376-385Crossref PubMed Scopus (405) Google Scholar). 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 (24Hackney D.D. Jiang W. Methods Mol. Biol. 2001; 164: 65-71PubMed Google Scholar). 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. (12DeBonis S. Skoufias D. Robin G. Lebeau L. Lopez R. Margolis R. Wade R.H. Kozielski F. Mol. Cancer Ther. 2004; 3: 1079-1090PubMed Google Scholar). 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 (12DeBonis S. Skoufias D. Robin G. Lebeau L. Lopez R. Margolis R. Wade R.H. Kozielski F. Mol. Cancer Ther. 2004; 3: 1079-1090PubMed Google Scholar), we determined initial IC50 values for STLC by fitting the experimental data to Equation 1, v/v0=100−(A×([I]/([I]+IC50)(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 (25Morrison J.F. Biochim. Biophys. Acta. 1969; 185: 269-286Crossref PubMed Scopus (712) Google Scholar) for tight binding inhibitors, vi/v0=1−(([E]+[I]+Ki,app)−([E]+[I]+Ki,app)2−4[E][I])/2[E](Eq. 2) where v0 is the velocity in the absence of inhibitor; vi is the measured velocity; [E] is equal to the total enzyme concentration; [I] indicates the added inhibitor concentration, and Ki,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 (14DeBonis S. Simorre J.P. Crevel I. Lebeau L. Skoufias D.A. Blangy A. Ebel C. Gans P. Cross R. Hackney D.D. Wade R.H. Kozielski F. Biochemistry. 2003; 42: 338-349Crossref PubMed Scopus (169) Google Scholar). 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, kobs=k−1+k1[I](Eq. 3) where I represents the different STLC concentrations, and k1 and k–1 are the binding and release rate constants for Reaction 1, E·mant-ADP+I⇆E·mant-ADP·I(Eq. 4) 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-α-tubulin and anti-γ-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 (26Davis F.M. Tsao T.Y. Fowler S.K. Rao P.N. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2926-2930Crossref PubMed Scopus (501) Google Scholar), 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 (27Andreassen P.R. Skoufias D.A. Margolis R.L. Methods Mol. Biol. 2004; 281: 213-225PubMed Google Scholar). 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 × 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.RESULTSComparison between STLC and STDC—STLC is a non-natural derivative of the α-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 tR 10.9 min, whereas STDC was eluted at tR 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 γ-tubulin antibody as a centrosomal marker revealed the two centrosomes (four γ-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 (5Blangy A. Lane H.A. d'Herin P. Harper M. Kress M. Nigg E.A. Cell. 1995; 83: 1159-1169Abstract Full Text PDF PubMed Scopus (775) Google Scholar) or after inhibition of Eg5 activity by specific inhibitors like monastrol (7Mayer T.U. Kapoor T.M. Haggarty S.J. King R.W. Schreiber S.L. Mitchison T.J. Science. 1999; 286: 971-974Crossref PubMed Scopus (1598) Google Scholar), terpendole E (8Nakazawa J. Yajima J. Usui T. Ueki M. Takatsuki A. Imoto M. Toyoshima Y.Y. Osada H. Chem. Biol. 2003; 10: 131-137Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), HR22C16 (9Hotha S. Yarrow J.C. Yang J.G. Garrett S. Renduchintala K.V. Mayer T.U. Kapoor T.M. Angew. Chem. Int. Ed. Engl. 2003; 42: 2379-2382Crossref PubMed Scopus (135) Google Scholar), or CK0106023 (10Sakowicz R. Finer J.T. Beraud C. Crompton A. Lewis E. Fritsch A. Lee Y. Mak J. Moody R. Turincio R. Chabala J.C. Gonzales P. Roth S. Weitman S. Wood K.W. Cancer Res. 2004; 64: 3276-3280Crossref PubMed Scopus (243) Google Scholar). 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).FIG. 2Comparison 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-β-tubulin for MTs and anti-γ-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 Large Image Figure ViewerDownload Hi-res image Download (PPT)Duration and Fate of Mitotic Arrest—Longer incubation times of HeLa cells (Fig. 3A) with STLC showed that the G2/M block persisted for as long as 72 h. FACScan analysis of treated cells revealed that when starting before 48 h there is accumulation of the sub-G1 population of cells, indicative of apoptotic cells, which is more prominent at 72 h. Although they block in mitosis after prolonged incubation with STLC, U2OS cells, in contrast to HeLa cells, experience adaptation and mitotic slippage and proceed to the next cell cycle. After 72 h, there is a peak of 8N cells suggesting that the cells were able to proceed to a second round of DNA replication (Fig. 3B). A sub-G1 population of U2OS cells was also apparent.FIG. 3STLC 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 Large Image Figure ViewerDownload Hi-res image Download (PPT)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.Reversibility of STLC—We also examined whether the inhibition of Eg5 activity by STLC in mitotically arrested cel" @default.
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• W2057523896 title "S-Trityl-L-cysteine Is a Reversible, Tight Binding Inhibitor of the Human Kinesin Eg5 That Specifically Blocks Mitotic Progression" @default.
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