S9 Targets PI3K/mTOR/Tubulin Figure 4. S9 disturbs the microtubular structure by binding to tubulin directly and inhibiting tubulin polymerization. A) S9 disturbs the cell skeleton structure. SK-OV-3 cells were exposed to 5 mM S9 for 24 h and stained with anti-tubulin antibody (green) and DAPI (blue). Scale bar: 30 mm. Data shown are representative of at least three independent experiments. B) Inhibition on purified tubulin polymerization by S9. Purified pork tubulin was incubated with S9, vincristine (VCR) or colchicine (COL) or vehicle in reaction buffer. The optical density (OD) at 340 nm was measured with a spectrophotometer every 1 min till the OD value became stable. C). Disruption secondary structure of tubulin by S9. Purified tubulin and 25 mM S9 were co-incubated in reaction buffer for 1 h. The circular dichroism (CD) spectra were recorded. The characterized absorbance wave lengths were 209/222 nm for a–helix and 196 nm for b–sheet. D) S9 (left panel) and colchicines (right panel) bind to tubulin directly. Purified tubulin was immobilized on sensor chip. Association and dissociation rate constants and real-time binding capacity were recorded by Surface Plasmon Resonance (SPR). The Kd values were computed by Biacore evaluation Version 3.0 software. E) Competitive binding of S9 to colchicine-binding sites on tubulin but not vinblastine-binding sites. Tubulin was co-incubated with indicated concentrations of S9 or vincristine for 1 h, then colchicine was added (left panel). Right panel, tubulin was co-incubated with S9 or vinblastine (VLB) for 1 h followed by addition of BODIPY-FL-vinblastine. The fluorescence was measured by spectrofluorometer. All assays were repeated twice and representative data were shown. doi:10.1371/journal.pone.0004881.g004 fluorescence BODIPY FL-vinblastine while the fluorescence reduced in the presence of vinblastine (Fig. 4E, right panel). These findings provide the evidence that S9 directly binds to colchicine-binding site but not vinblastine-binding site of tubulin. The superimposed binding models of S9 and colchicine in tubulin were next performed (Fig. 5). Despite their strikingly difference in structures, the conformation of S9 occupies similar Cartesian space in the colchicine site. Moreover, S9 hydrophobi- PLoS ONE | www.plosone.org 6 March 2009 | Volume 4 | Issue 3 | e4881