Figure 5. The binding mode of S9 within tubulin. The protein is represented by cartoon. The residues interacting with the compounds are shown in sticks. S9 and colchicine are also represented by sticks and colored in cyan and yellow, respectively. All of the structural diagrams were prepared using PyMOL (http://pymol.sourceforge.net). doi:10.1371/journal.pone.0004881.g005 cally interacts with Val 181, Cys 241, Val 318, Lys 352 and Ile 378, which interact similarly with colchicine. Of particular note, the MOMO group, lying in the side chain of the phenylindole, formed a hydrogen bond with Lys 254, which might offer a conformation for S9 to place effectively in tubulin. In contrast, S7, in which MOMO group is replaced by H [8], has no effect on tubulin polymerization (data not shown). S9 disrupts tumor cell growth in vitro and in vivo S9 induces rapid apoptosis in human cancer cells. Because S9 displayed potent inhibition on PI3K-Akt- mTOR pathway as well as tubulin polymerization, the ability of S9 to induce apoptosis was examined in various tumor cells. Cells were subjected to annexin-V-PI doubling staining after exposure to 10 mM S9 for 8 h. As shown in Figure 6C, tumor cells underwent rapid apoptosis after exposure to S9 for 8 h. PI3K inhibitors usually induce G1 phase arrest rather than apoptosis. Rapid apoptosis induced by S9 might represent its combinatorial effect on PI3K-Akt-mTOR pathway and microtubule cytoskeleton. S9 inhibits proliferation of a panel of tumor cells in vitro. The anti-proliferative activity of S9 was measured with SRB assay against a panel of human tumor cells, including oral epidermoid cancer, lung cancer, liver cancer, colon cancer, rhabdomyosarcoma, ovarian cancer, osteosarcoma, breast cancer, cervix cancer, stomach cancer, prostate cancer and leukemia cells. S9 displayed potent activity against the proliferation of a broad spectrum tumor cells with a mean IC50 of 9 mM (Fig 6A). Wortmannin, a well-known PI3K inhibitor, was taken as a reference drug with a mean IC50 of 16 mM. Although both S9 and wortmannin target PI3K pathway, they displayed different anti-proliferation profiles. For example, S9 were more potent in K562 and U2OS cells than wortmannin. This advantage S9 Targets PI3K/mTOR/Tubulin was also observed in the mini-set of colon cancer cell lines (Fig. 6A). Detailed IC50s were shown in Table S2. The anti-proliferative activity in five pair of MDR cells, Gleevec-resistant K562/G02 and rapamycin resistant Rh30/Rapa cells was also evaluated. Remarkably, S9 demonstrated similar inhibition in drug resistant cancer cells and respective parental cells, with the resistant factors around 1(Fig 6B). Of particular note, S9 remains active against MDR cells unlike other tubulin- interacting drugs such as colchicine and vincristine (Fig 6B). S9 inhibits the growth of human cancer xenografts on nude mice. The anti-tumor activity of S9 in vivo was evaluated using human rhabdomyosarcoma Rh30 and human hepatoma SMMC 7721 xenografts models. After 14-day treatment of S9, the relative tumor volume (RTV) of control group for Rh30 xenograft was 9.24, while the RTV of group received S9 administration (50 mg/kg) reduced to 5.26. Similar inhibitory effect was also observed in SMMC7721 xenograft, with a T/C value of 0.52 (Fig. 6D). The body weights of nude mice reduced slightly after administration of 50 mg/ml S9 for two weeks (data not shown), indicating S9 held some extent of toxicity. However, S9-induced toxicity was tolerable because less than 10% reduction in body weight was observed on day 14 compared to day 0. Discussion S9, among a series of hybrids of a-methylene-c-lactones and 2- phenyl indoles [8], stood out for its potent activity against PI3K- Akt-mTOR signaling [8]. In this study, we identified its cellular targets and characterized its mechanism of action. S9 was found to abrogate EGF-activated PI3K-Akt-mTOR signaling cascade and Akt translocation via its dual inhibition of both PI3K and mTOR. S9 was further noted to inhibit the polymerization of tubulin and thus cause M phase arrest. Such cooperation between blockade of PI3-Akt-mTOR signaling and tubulin cytoskeleton contributes to its anti-proliferative activity observed in vitro against a panel of tumor cells including MDR tumor cells and in vivo antitumor activity in mice bearing human tumor xenografts. We found that S9 potently inhibited the kinase activity of PI3K and mTOR, as well as DNA repair-related PIKK family members and no effects on 30 tested kinases. Therefore, S9 was identified as a selective dual inhibitor against PI3K and its downstream kinase mTOR. PI3K and mTOR belong to the PIKK family, bearing a significant homologous COOH-terminal catalytic domain. Mo- lecular docking displayed that a-methylene-c-lactones moiety represents a key element for binding to the ATP pocket of both PI3Ka and mTOR, via hydrophobic interaction and hydrogen bonds. Interestingly, such a a-methylene-c-lactone moiety also harbors in wortmannin, which has been verified to be indispens- able for its PI3K inhibition and anticancer activity [23]. This notion helps substantiate the importance of a-methylene-c-lactone moiety not only on inhibition of PI3K but also on mTOR as well. Inhibitors of mTOR are currently being tested clinically and have been somewhat disappointing as monotherapy against cancer. Encouragingly, S9 significantly inhibited EGF-triggered PI3K-Akt-mTOR signaling transduction and notably, circum- vented rapamycin-induced hyperphosporylation of Akt. These findings provide pharmacological evidence that retrograde-depen- dent activation of Akt can be overcome by dual inhibition of PI3K and mTOR, which in turn underscore that inhibition of PI3K and mTOR in combination should be more effective than inhibition of mTOR alone. As an inhibitor of PI3K-Akt-mTOR pathway, it is out of our expectation that S9 arrested tumor cells in M phase, which was attributed to its disturbance of microtubule cytoskeleton. The PLoS ONE | www.plosone.org 7 March 2009 | Volume 4 | Issue 3 | e4881