Osmium Carbonyl Clusters Containing Labile Ligands Hyperstabilize

May 14, 2009 - A study into the possible molecular targets of the osmium carbonyl cluster Os3(CO)10(NCCH3)2 (2) in the ER− breast carcinoma ...
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Osmium Carbonyl Clusters Containing Labile Ligands Hyperstabilize Microtubules Kien Voon Kong, Weng Kee Leong,* and Lina H. K. Lim Department of Chemistry, National UniVersity of Singapore, Kent Ridge, Singapore 117543 ReceiVed February 9, 2009

A study into the possible molecular targets of the osmium carbonyl cluster Os3(CO)10(NCCH3)2 (2) in the ER- breast carcinoma (MDA-MB-231) cell line was carried out. Infrared and 1H NMR analyses of cells treated with 2 showed the formation of carboxylato- and thiolato-bridged clusters from the interaction with intracellular carboxylic acid and sulfhydryl residues. The cytotoxicity of 2 was reduced in the presence of fetal bovine serum, and measurement with Ellman’s reagent as well as fluorescence confocal microscopy with tetramethylrhodamine-5-maleimide staining all demonstrated binding to intracellular sulfhydryl groups leading up to cell disruption. Tubulin-FITC antibody staining of treated cells showed disruption of the microtubules, and a tubulin polmerization assay showed that 2 induced hyperstabilization of the microtubules. Introduction Platinum-based compounds remain among the most successful anticancer drugs, and their success and limitations have prompted the continued search for new metal-based anticancer drugs. One class that has attracted attention is organometallic compounds (1-4). Many of these compounds are known or believed to target DNA, although there are also a number which target proteins or, more specifically, receptors. An example of the latter is SERMs (selective estrogen receptor modulators), which have been investigated particularly by the group of Jaouen (5). One unusual class of metal-based drugs is that of organometallic clusters; these are compounds that contain two or more metals with bonds among them, and some of them have been shown to offer promise as anticancer agents (6-9). We recently reported our evaluation of the anticancer activity of a series of triosmium clusters against five cancer cell lines, including Os3(CO)12 (1), Os3(CO)10(NCMe)2 (2), and Os6(CO)16(NCMe)2 (3) (Figure 1) (10, 11). While 1 showed little cytotoxicity, 2 and 3 exhibited good cytotoxicity against four out of five cancer cell lines tested. Cluster 2, in particular, showed IC50 values of ∼5-10 µM against the four cancer cell lines, as opposed to 18 µM for normal epithelial cells (MCF10A). Of particular interest was that it showed efficacy against both ER+ and ER- breast carcinoma (MCF-7 and MDA-MB231). In those earlier studies, we also showed that the cytotoxicity resulted from the induction of apoptosis and that the activity of the compounds was correlated to the availability of vacant coordination sites on the compunds. For instance, dissociation of the two labile acetonitrile (MeCN) ligands from 2 and 3 would afford two vacant coordination sites on the metal centers. Because the chemistry of 2, in particular, has been extensively explored (12-14), the interesting cytotoxicity exhibited by this class of organometallic compounds prompted us to investigate its biomolecular targets in relation to its known chemistry. We * To whom all correspondence should be addressed. Current address: Division of Chemistry and Biological Chemistry, SPMS-04-01, 21 Nanyang Link, SPMS-CBC-06-07, Singapore 637371. Tel: +65 65927577. Fax: +65 67911961. E-mail: [email protected].

Figure 1. Molecular structure of organometallic clusters 1-3. Short lines denote carbonyl (CO) ligands.

report here our studies carried out with 2, especially with the MDA-MB-231 cell line.

Experimental Procedures Os3(CO)12 (1) was purchased from Oxkem, and the osmium carbonyl clusters Os3(CO)10(NCCH3)2 (2) (15) and Os6(CO)16(NCCH3)2 (3) (16) were prepared from 1 according to reported procedures. All other chemicals were purchased from commercial sources and used as supplied. Cell Culture. Experimental cultures of the cell lines MDA-MB231, MCF-7, and MCF-10A were obtained from the American Type Culture Collection (ATCC) and cultured in tissue culture dishes (Nunc Inc., Naperville, IL). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Grand Island, NY) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamate (Gibco Laboratories), and 1% penicillin-streptomycin (Gibco Laboratories) at 37 °C in a 5% CO2 atmosphere unless otherwise stated. Cell cultures were maintained in an antibiotic-free condition during cell growing and experiments. Phosphate buffer saline (PBS) was obtained from first BASE. Infrared Spectroscopy of Treated Cells and Cell Fractions. All infrared spectra were recorded in the solid state with a Shimadzu Prestige-21 FTIR spectrometer at a spectral resolution of 4 cm-1 on CaF2 windows. MDA-MB-231 cells (2 × 107) were incubated (24 h) with 2 and serum-free DMEM, washed with PBS (2 mL), and then scraped in the presence of 200 µL of lysis buffer (Triton X-100, 5 M NaCl, 0.5 M EDTA, and 10% NP40) (Sigma-Aldrich) supplemented with 1× protease inhibitor (Pierce Biotechnology) and phosphatase inhibitors [50 µM okadaic acid (Sigma) and 200 mM sodium vanadate (Sigma)]. The mixture was vortexed (1 min every half hour) for 2 h and centrifuged (13000g) at 4 °C. The supernatant and pellet were saved as protein and nonprotein fractions, respectively.

10.1021/tx900056a CCC: $40.75  2009 American Chemical Society Published on Web 05/14/2009

Osmium Carbonyl Clusters Hyperstabilizes Microtubules For the compartment spectra, the cells (2 × 107) were lysed with a Chemicon compartment separation kit and separated into four fractions, namely, cytoplasmic, nuclear, membrane, and organelles. Briefly, the treated cells were homogenized in sucrose gradient buffer at moderate speed and centrifuged (20000g) at 4 °C for 20 min. The supernatant was saved as the cytoplasmic fraction, while the pellet was washed with cold sucrose gradient buffer and centrifuged again as above. The supernatant so obtained was saved as the nuclear fraction, and the procedure was repeated to yield the membrane fraction as the supernatant and a pellet that contained organelles, granules, and cytoskeleton, which was labeled as the organelles fraction. All of these compartments were then freezedried. NMR Spectroscopy of Treated Cells. Approximately 2 × 1010 cells (MDA-MB-231) treated with 40 µM 2 for 24 h was accumulated. They were rinsed with PBS (3 × 20 mL, 5 min each), resuspended in chloroform (10 mL), and vortexed (5 min) before homogenizing. The chloroform layer was then collected and vacuum-dried, and CDCl3 (3 mL) was added to the dried residues and transferred to an NMR tube. The 1H NMR spectrum was recorded on a Bruker ACF300 NMR spectrometer; chemical shifts reported are referenced against the residual proton signals of the solvents. Time-of-Flight Secondary Ion Mass Spectrometric (ToFSIMS) Analysis of Treated Cells and Tubulins. MDA-MB-231 cells (∼3 × 104) cultured on silicon wafers in six-well cell culture plates were incubated (24 h) with 2 (40 µM in DMEM), washed with PBS (3 × 1 mL), and dried under an argon flow before ToFSIMS analysis. ToF-SIMS analyses were carried out on an IONTOF SIMS 4 instrument, using bunched 69Ga+ ion pulses with an impact energy of 25 keV. Immunoprecipitation was also applied to selectively precipitate tubulin protein from total cell proteins for ToF-SIMS analysis. Briefly, MDA-MB-231 cells (20 × 106) were incubated (24 h) with 2 and washed with cold PBS (20 mL), and then, 2 mL of RIPA buffer supplemented with protease inhibitor (Peirce Biotechnology) was added. Cells were harvested after incubation (30 min) on ice, and then, the harvested solution, including cells debris, was centrifuged (12000 rpm, 400g) at 4 °C for 15 min, and the supernatant was collected as the protein fraction. This protein fraction (500 µL) was mixed gently with R-tubulin antibody (10 µL, Sigma) and incubated (3 h) at 4 °C. Meanwhile, Protein A/G Plus-Agarose IP reagent (50 µL, Santa Cruz) suspended in RIPA solution containing R-tubulin antibody (300 µL) was incubated (3 h) and centrifuged (12000 rpm) for 1 min, and the supernatant was collected. To this was added the protein fraction containing the R-tubulin antibody, and it was then incubated (3 h) and centrifuged (12000 rpm) at 4 °C for 10 min, and the supernatant was collected as “other proteins”. The pellet was washed with RIPA buffer (1 mL), and the centrifugation and washing were repeated twice. The pellet was then collected as “tubulin protein”. Silicon wafers were immersed (1 min) in the “other proteins” or “tubulin protein” solutions and then dried under an argon flow before ToF-SIMS analysis. Effect of FBS and Cysteine on Drug Efficacy. Typically, a solution of 2 in dimethyl sulfoxide (DMSO) was prepared and then diluted 1000 times to the desired concentration for treatment. Cells were seeded in growth medium at the same initial density, allowed to adhere and grow for 24 h to 80% confluence, washed once with serum-free DMEM medium, and then serum-starved for 6 h before treatment with a solution of 2 at the indicated concentrations together with DMEM and 10% FBS. These served as controls. For treatment under FBS-free condition, cells were seeded, grown, and treated using growth medium without FBS. A cysteine-free treatment condition was achieved using cysteine-free DMEM (Invitrogen). After treatment, to each well was added 20% of Cell Titer 96 Aqueous One Cell Proliferation Assay (Promega) and then incubated in a 37 °C incubator with 5% CO2 for 2 h. The absorbance intensities at 490 nm were then measured, and the cell proliferation relative to the control sample was calculated. Each sample was analyzed in triplicate.

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Figure 2. Molecular structure of Os3(µ-H)(CO)10(µ-O2CR), Os3(µH)(CO)10(µ-Cl), and Os3(µ-H)(CO)10(µ-SR). Short lines denote carbonyl (CO) ligands.

Annexin-V and PI Staining for Flow Cytometry. The percentage of cells actively undergoing apoptosis was determined using annexin V-PE-based immunofluorescence, as described previously (17). Briefly, cells were plated in six-well culture plates at concentrations determined to yield 80% confluence within 24 h and treated under the conditions described above. After 24 h of treatment, the cells were harvested and then double-labeled with annexin V-FITC and PI, as described by the manufacturer (Becton Dickinson). The cells were analyzed using a FACScan instrument equipped with a FACStation running Cell Quest software (Becton Dickinson, San Jose, CA). All experiments were performed in duplicate and yielded similar results. Determination of Cell Sulfhydryl with DTNB (Ellman’s Reagent). The amount of sulfhydryl in cells was determined according to the literature method (18). MDA-MB-231 cells were seeded in triplicate in six-well plates and allowed to adhere, serum starved for 6 h, and then supplemented with the indicated concentrations of 2 in serum-free DMEM for 24 h. The cells were washed with serum-free DMEM (3 × 2 mL) and PBS (2 mL), harvested, and dispersed in PBS (800 mL), and 5,5′-dithiobis-(2nitrobenzoic acid) (DTNB, 200 mL of a 10 mM solution) was then added. After 40 min at room temperature, the solution and its absorbance at 412 nm were determined. All readings were relative to the control. Fluorescence Labeling. Cells were grown as described above and treated with the indicated concentrations of 2 or 3 for 24 h, fixed with formaldehyde (10%, 30 min at -20 °C), and then permeabilized with Triton X-100 (0.1% in PBS, 5 min) (USB Corp). They were then incubated in PBS solution containing tetramethylrhodamine-5-maleimide (TRM, 10 µM, 25 min at 22 °C) (Invitrogen). Nuclei were stained concomitantly with 4,6-diamidino2-phenylindole (DAPI, 1 mg mL-1 in methanol, 5 min at 37 °C). The samples were then washed with PBS (3 × 3 mL), mounted with Fluorosave (Calbiochem) mounting medium, and analyzed using a Leica SP5 fluorescence confocal microscope. Immunofluorescence. Cells were grown on coverslips (60% confluence), incubated (24 h) with solutions (40 µM) of 2 or 3, washed with PBS (3 × 2 mL), fixed with 5% formaldehyde in PBS (20 min, room temperature), aspirated, rinsed with PBS (3 × 2 mL), and permeabilized with 0.1% Triton X-100 solution (100 mL). The cells were blocked with blocking buffer (2 mL, 5% goat serum, 1 h at room temperature), and then, the cell monolayers were incubated (overnight at 4 °C) with monoclonal antibody tubulin conjugated with FITC (100 µL, 2 mg mL-1) (Sigma-Aldrich). After removal of the antibody solution, the cells were mounted with Fluorosave mounting medium. The coverslip slides were sealed with commercial nail polish. Immunofluorescence was detected using a Leica SP5 fluorescence confocal microscope. Cell Cycle Analysis by Flow Cytometry. Cells were plated in six-well flat bottom plates with 80% confluence after 24 h. They were then incubated with 1 or 2 for 24 h, trypsinized, washed with cold PBS (2 × 2 mL), resuspended in 70% ethanol, and then incubated (30 min at room temperature). After this, they were spun down at 2000 rpm (400 g) for 5 min, washed once, and resuspended again with PBS containing 1% FBS (100 µL). This was then incubated (37 °C for 15 min) after the addition of RNase (20 µL), after which PI (50 µL) was added, and the sample was analyzed by flow cytometry within 1 h, with a Dako CyAn ADP highperformance research flow cytometer. Tubulin Polymerization Assay. A Cytoskeleton tubulin polymerization assay kit (catalog no. CDS03 and BK006) was used for

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Results

Figure 3. Effect of sulfhydryl biomolecules on the cytotoxicity of 2 to MDA-MB-231 cells. Cells were incubated (24 h) with different concentrations of 2 together with DMEM and FBS (red square), DMEM but no FBS (blue square), and FBS and cysteine-free DMEM (yellow square).

the tubulin polymerization study. Briefly, 10 µL of general tubulin buffer (80 mM PIPES, pH 6.9, 2 mM MgCl2, and 0.5 mM EGTA) containing 2, 3, or taxol was pipetted into the prewarmed 96 well plate. Tubulin (defrosted to room temperature from -80 °C and then placed on ice before use) was diluted with tubulin polymerization buffer [750 µL of general tubulin buffer, 250 µL of tubulin glycerol buffer (15% glycerol in general tubulin buffer), and 10 µL of 1 mM GTP] to a final concentration of 3 mg mL-1. Diluted tubulin (100 µL) was added into the wells containing 2 or taxol. Diluted tubulin (100 µL) mixed with general tubulin buffer (10 µL) served as control. The absorbance at 340 nm was read immediately with a Biotek Synergy 4 microplate reader.

Both MCF-7 and MDA-MB-231 cells were treated with 40 µM solutions of 2 and lysed, and the pellets were analyzed by infrared spectroscopy. The infrared spectra showed strong absorptions in the carbonyl stretching region (2200-1950 cm-1), which were different from that of 2, indicating the presence of an osmium carbonyl species different from 2 (Figure S1 of the Supporting Information). MDA-MB-231 cells that have been treated with 2 were also fractionated into six major fractions (nuclear, cytoplasmic, organelles, membrane, protein, and nonprotein) via sucrose density gradient centrifugation. Infrared spectroscopic analysis showed that 2 was translocated into all six fractions (Figure S2 of the Supporting Information). The complexity of the IR spectra suggests that mixtures of osmium carbonyl species were present, but their identity cannot be obtained from an analysis of the IR spectra. The intracellular carbonyl cluster adducts were also characterized by 1H NMR spectroscopy (Figure S3 of the Supporting Information). The major problem is the low sensitivity of NMR spectroscopy, which necessitated the use of a large number of cells (∼2 × 1010). While the organic region of the 1H NMR spectrum was rather complicated and unresolved, three resonances (at -10.43, -14.29, and -16.11 ppm) were clearly visible in the metal hydride region. These could be assigned to the cluster species Os3(µ-H)(CO)10(µ-O2CR), Os3(µ-H)(CO)10(µCl), and Os3(µ-H)(CO)10(µ-SR), respectively (Figure 2) (19-21) and are indicative of the reaction of 2 with cellular carboxylic acid groups, chloride ions, and sulfhydryl groups, respectively, as it is known to react with compounds containing such

Figure 4. Detection of early and late apoptotic MDA-MB-231 cells after staining with annexin V-FITC and PI. Cells were incubated for 24 h with (a) DMEM only (control); (b) FBS, DMEM, and 2; (c) FBS, cysteine-free DMEM, and 2; and (d) DMEM and 2. The concentration of 2 used was 10 µM.

Osmium Carbonyl Clusters Hyperstabilizes Microtubules

Figure 5. Assay for intracellular sulfhydryl groups using Ellman’s reagent for MDA-MD-231 (left) and MCF-7 (right) treated with different concentrations of 2 in the presence and absence of FBS.

functional group to afford these cluster species (19-22). However, the chloro-bridged species may be an artifact resulting from the decomposition of some triosmium cluster species during workup and/or data acquisition, as we have found that standing a solution of 2 in CHCl3 overnight, or incubating it with PBS, affords this species. This is corroborated by the results from a ToF-SIMS analysis of cells treated with 2, which showed mass fragments that could be assigned to [Os3H(CO)n(S)]- and [Os3H(CO)n(O2C)]- (n e 6) (Figure S10 of the Supporting Information). The effect of sulfhydryl biomolecules, viz. cysteine (Cys) and FBS, on the cytotoxicity of 2 was assessed against the MDAMB-231, MCF-7, and MCF-10A cell lines; the results were similar for all three cell lines, and that for MDA-MB-231 is shown in Figure 3. The results show that 2 reduced viability on these cell lines in a concentration-dependent manner (10). However, its cytotoxicity was significantly depressed by FBS (by ∼70-100%) and slightly by L-cysteine (by ∼40-80%). Flow cytometry using FITC-conjugated annexin V and propidium iodide (PI) staining of these treated cells showed that the early apoptosis cell population (FITC stained) was reduced from ∼46% in a serum-free environment to ∼24% in the

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presence of FBS (Figure 4). The effect of L-cysteine was less, as its absence afforded an early apoptosis cell population of ∼34%. The interaction of 2 with intracellular sulfhydryl groups was measured with Ellman’s reagent, which showed that the intracellular sulfhydryl groups were reduced in a concentrationdependent manner (Figure 5). The percentage of intracellular sulfhydryl groups was markedly higher in the presence of FBS. The interaction of 2 with sulfhydryl groups was also visualized via fluorescence confocal microscopy with tetramethylrhodamine5-maleimide (TRM). MDA-MB-231 cells stained with TRM following 24 h of treatment with 2 showed attenuated fluorescence as compared to control (Figure 6). Destruction of cell structure was also observed at the higher concentration (40 µM). When TRM staining was carried out at 4 °C, with omission of the permeabilization step, 8 h of incubation with 2 was sufficient to lead to attenuation of the fluorescence; no destruction of cell structure was observed. MDA-MB-231 cells treated with 2 or 3 stained with tubulinFITC antibody showed disruption in the morphology of the microtubules (Figure 7). The treated cells also showed a condensed DNA and spindle structure at higher concentration. Fractional DNA content analyses of MDA-MB-231 cells treated with 1 or 2 revealed an accumulation of cells in the G2 phase for the latter but not the former (Figure S8 of the Supporting Information). Similarly, a large population of apoptotic cells was observed with 2; an early apoptotic cell population reached 46%, as compared to about 5 and 3% for 1 and control, respectively (Figure S9 of the Supporting Information). The effect of 2 and 3 on tubulin polymerization was compared with taxol (10 µmol). An increase in absorbance at 340 nm indicates an increase in tubulin polymerization (23), and the results showed that 2 induced an increase in tubulin polymerization at both 10 and 100 µM concentrations (Figure 8). The tubulin polymerization ability of 3, on the other hand, was very low.

Discussion As indicated in the Introduction, the reactivity of compound 2 has been well-studied, and the spectroscopic, primarily IR

Figure 6. Fluorescence confocal microscopic images of MDA-MB-231 cells stained with TRM (red) and DAPI (blue): after 24 h of incubation with (a) DMSO (control), (b) 20 µM 2, and (c) 40 µM 2 and stained at 4 °C with no membrane permeabilization, after 8 h of incubation with (d) DMSO (control) and (e) 40 µM 2. Arrow indicates disrupted cells.

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Figure 7. Fluorescence confocal microscopic images of MDA-MB-231 cells stained with tubulin-FITC antibody after 24 h of incubation with (a) DMSO (control), (b) 20 µM solution of 2, (c) 20 µM solution of 3, (d) 40 µM solution of 2, and (e) 40 µM solution of 3.

Figure 8. Tubulin polymerization assays with 2 and 3. Plot of absorbance at 340 nm vs treatment time.

and 1H NMR, characteristics of many of its reaction products are also well-established. The major spectroscopic advantages associated with transition metal carbonyl compounds are the strong CO stretching vibrations and, particularly for cluster compounds, the very upfield chemical shifts (negative of -5 ppm) for 1H resonances of metal hydrides. The latter characteristic is a great advantage in that this chemical shift region is clear of interference from organic resonances, making it possible to gain useful information despite the practical difficulties associated with the low sensitivity of NMR spectroscopy. Thus, infrared and 1H NMR analyses of the cells treated with 2 clearly showed that it has reacted with some cellular species, albeit they were not localized, and, together with the ToF-SIMS results, suggested that 2 formed carboxylato- and thiolatobridged clusters by interacting with intracellular carboxylic acid and sulfhydryl residues. Sulfhydryl groups are a very probable target since many toxic metals and metal-based drugs are known to act through binding to them. For instance, mercury and arsenic bind to sulfhydrylrich receptors on the cellular surface (24, 25), while the efficacy of platinum anticancer drugs has been attributed to the high affinity of platinum for sulfhydryl groups (26). Similarly, 2 is known to show a high affinity for sulfhydryl groups (22). That binding to sulfhydryl groups is responsible for the efficacy of 2 is supported by the reduced cytotoxicity in the presence of

FBS, as well as the corresponding flow cytometry results. This interaction is also more specifically measured with Ellman’s reagent, which demonstrates that 2 indeed binds to intracellular sulfhydryl groups, and this interaction is reduced in the presence of FBS. Fluorescence confocal microscopy with tetramethylrhodamine-5-maleimide (TRM), which couples covalently to free sulfhydryl groups, is generally considered to be highly specific; any interaction of 2 with sulfhydryl groups will block coupling of the maleimide (27). Thus, the attenuated fluorescence observed for MDA-MB-231 cells stained with TRM following treatment, and destruction of cell structure observed at the higher concentration, further corroborate binding to sulfhydryl groups. TRM staining at 4 °C with omission of the permeabilization step allows for differentiation between binding to intracellular sulfhydryl groups and binding to sulfhydryl groups on the surface of the cell membrane (27). Our observations suggest that although 2 binds to surface sulfhydryl groups, cell disruption is the result of attack on intracellular rather than surface sulfhydryl groups. We have also been able to rule out glutathione as a target, as we have found that 2 did not react with it under physiological (37.0 °C and 24 h) conditions to afford any thiolato-bridged cluster species. That 2 interacts with sulfhydryl groups suggests disruption of the tubulin function or structure as a possible target. Tubulin is the major protein component of microtubules, and it is known

Osmium Carbonyl Clusters Hyperstabilizes Microtubules

to be the target of numerous antimitotic drugs (28-30), as disruption of the microtubule dynamics can ultimately trigger the mitochondrial apoptotic pathway (31). The 3D structure of tubulin shows the presence of about 20 sulfhydryl groups, which are heterogeneously distributed (32-34) and which are targeted by several sulfhydryl binding agents (35-37). However, while compounds such as colchicine bind to a sulfhydryl site on tubulin to prevent the normal polymerization of microtubules (38, 39), taxanes such as paclitaxel (taxol) and docetaxel (taxotere) act via the hyperstabilization of microtubules (40); the interaction of the taxanes with tubulin is via a combination of hydrogen bonds and hydrophobic interactions (41-44). The interaction of 2 with tubulins is supported by the tubulinFITC antibody staining of treated cells, which showed disruption of the microtubule structures. The condensed DNA and spindle structure observed at a higher concentration is also consistent with the inhibition of microtubule formation and cell cycle arrest. The latter, investigated via fractional DNA content analyses, revealed the induction of G2/M arrest by 2 but not 1, which is consistent with our earlier observations (10, 11) and the wellknown greater reactivity of the former. Consistent with these results, 2 showed similar tubulin polmerization ability as taxol. As mentioned above, identification of the type of interaction between 2 and tubulins was also attempted through ToF-SIMS analysis of the tubulins obtained by immunoprecipitation from cells treated with 2. The results suggested that 2 interacted with both COOH and SH groups (Figure S10 of the Supporting Information), but this was not confined to only the tubulins; similar products also appeared in other cellular components.

Conclusion In this study, we have shown spectroscopically and via biochemical studies that the osmium cluster 2 interacts with intracellular carboxylate and sulfhydryl residues. One consequence of its binding to sulfhydryl residues is that 2 interacts with tubulins, leading to hyperstabilization of the microtubules. It is therefore possible that this is linked to the cytotoxicity of 2. To establish this point, however, it would be interesting to explore if these results are also seen at doses below the IC50. Acknowledgment. Financial support from the University Academic Research Fund (Grant R-143-000-267-112) and a Research Scholarship under the MedChem Programme to K.V.K. are gratefully acknowledged. Supporting Information Available: IR, 1H NMR, and ToFSIMS spectral data, cytotoxicity data, fluorescence images, and other biochemical analyses. This material is available free of charge via the Internet at http://pubs.acs.org.

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