developed compounds targeting this pathway, which has been approved for the treatment of advanced kidney cancer. Two other rapamycin derivatives RAD-001[6] and AP-23573 are currently in phase III clinical trials. With an increasing understanding of the feedback loops exiting in the PI3K-Akt-mTOR pathway, it has been recognized that inhibition on one output costs at the expense of activation of the others. For instance, rapamycin monotherapy activates Akt through inhibition of an mTOR-dependent retrograde signal [7]. For this reason, there is a growing consensus that the inhibition of combined components in the PI3K-Akt-mTOR pathway likely reflects a mechanistic rationale for the therapeutic options. As a matter of fact, many of the most effective PI3K-Akt- mTOR-targeted cancer therapies owe their activity to inhibition of dual or multiple components in this pathway, such as NVP-BEZ235 (a dual pan-PI3K/mTOR inhibitor) and SF-1126 (inhibitor of all isoforms of PI3K and other PIKK family members), which are now entering into Phase I/II clinical trials [4]. Given the severity of most cancers and their high unmet clinical need, together with the fact that the PI3K-Akt-mTOR axis represents an important emerging class of drug targets, we designed and synthesized a series of hybrids of a-methylene-c- lactones and 2-phenyl indoles with the aim of discovery of new inhibitor(s) targeting PI3K-Akt-mTOR pathway [8]. Of them, S9 stood out by functioning as a dual PI3K and mTOR inhibitor and also a microtubule destabilizer. The converged outcome of both cytostatic and cytotoxic identities of S9, together with its appreciable in vivo antitumor profiles, help prove to be a promising novel chemical template for the development of axis inhibitors. Results S9 inhibits PI3k-Akt-mTOR signaling S9 restrains PI3k-Akt-mTOR signaling stimulated by EGF in cancer cells. We have previously reported that S9 potently down-regulated phosphorylation of Akt, mTOR, p70S6K and 4E- BP1 upon stimulation with EGF in Rh30 cells [8]. To gain insight of the effect of S9 on the PI3k-Akt-mTOR signaling, Rh30 cells were exposed to various concentrations of S9 followed by EGF stimulation. Although located in the upstream of this pathway, phosphorylation of PDK-1 at Ser 241 was insensitive to serum deprivation or EGF stimulation (Fig 1A), which is consistent with the observation reported elsewhere [9]. Phosphorylation of PDK-1 was slightly depressed in the presence of 40 mM S9. In contrast, S9 significantly inhibited phosphorylation of Akt at both Ser 473 and Thr 308 (Fig 1A). Compared with Ser473, Thr308 phosphorylation was more vulnerable to S9 treatment, which might be due to phosphorylation of Ser 473 prior to the phosphorylation at Thr 308 and is important for the recognition and activation of Akt by PDK-1 [10]. Similarly, S9 abrogated phosphorylation of mTOR at Ser 2448 and its downstream proteins p70S6K at Thr 389 and 4E-BP1 at Thr 37/64 and Thr 70 in a concentration-dependent manner. The inhibitory activity of S9 on the PI3K-Akt-mTOR signaling was also confirmed using ovary carcinoma SKOV-3 cells (Fig 1B). Inactivation of the PI3K- Akt-mTOR pathway was also reflected by decrease in the kinase activities of PI3K, Akt and mTOR in the RH30 cells after S9 treatment (Fig S1). S9 blocks Akt cellular translocation and cell membrane ruffling. When cell is stimulated with growth factor, such as insulin, insulin-like growth factor (IGF) and EGF, Akt aggregates around cell membrane responding to recruitment by PIP3, which was a catalytic product of activated PI3K. Activation of PI3K also induces cell membrane ruffling [11]. Once the activity of PI3K is harshly prohibited, aggregation of Akt and membrane ruffling S9 Targets PI3K/mTOR/Tubulin disappears even under growth factor stimulation. As shown in Figure 1C, addition of IGF triggered Akt to aggregate on membrane in EGFP-Akt-stably-transfected CHO cells deprived of serum, demonstrated as highly fluorescent foci near the membrane (blue arrow). Membrane ruffling (white arrow) was also observed in these cells after IGF treatment. However, exposure of cells to S9 before IGF stimulation abrogated Akt translocation and membrane ruffling (Fig. 1C). The granule intensity in each cell was measured and statistically analyzed by IN Cell Analyzer workstation version 3.2. As shown in Figure 1D, S9 reduced the intensity of Akt granule in a concentration-dependent manner. S9 also inhibited PI3K activity and block membrane ruffling in Rh30 cells (Fig S2). S9 inhibits the catalytic activities of PI3K and mTOR. As S9 inhibits the PI3K-Akt-mTOR pathway signaling cascade, PI3K kinase assays were first carried out to determine whether S9 inhibits the catalytic activity of PI3K. PI3K were activated with EGF and immunoprecipitated from Rh30 cells, and kinase assays were performed in the presence of S9. As shown in Figure 2A, the ability of PI3K to generate PIP3 decreased after S9 treatment in a concentration-dependent manner and the activity decreased to 53.2% in the presence of 1 mM S9. Since S9 is capable of inhibiting the activity of PI3K, we detected whether S9 inhibits the activity of mTOR, which bears a highly homologous COOH- terminal catalytic domain with PI3K. As shown in Figure 2B, phosphorylation of 4E-BP1 by mTOR was greatly down-regulated by S9 treatment. 1 mM S9 decreased mTOR kinase activity by 58.7%. Thus, S9 is a new dual inhibitor of PI3K and mTOR. Rapamycin and its derivatives, which specifically inhibit mTOR, induced rapid Akt (Ser473) phosphorylation both in cancer cells and in clinical patient tumors [7], presumably due to inhibition of a mTOR-dependent retrograde signal [12]. This effects evoked impugnation for rapamycin and its derivatives’ application in clinical cancer therapy. As shown in Figure 2C, rapamycin treatment induced up-regulation of Akt phosphoryla- tion at Ser 473 in Rh30 cells, with no influence on phosphory- lation at Thr 308, which was consistent with previous report [13]. Although S9 is able to inhibit the activity of mTOR, S9 treatment alone does not induce hyper-phosphorylation of Akt at Ser 473 (Fig. 2C). Moreover, co-current treatment with S9 and rapamycin completely attenuated the hyper-phosphorylation of Akt at Ser 473 induced by rapamycin (Fig. 2C), indicating S9 could circumvent activation of Akt triggered by mTOR inhibition. S9 blocks ATM- and ATR-regulated DNA repair pathways. ATM, ATR and DNA-PK, which are involved in DNA damage response, also belong to the phosphoinositide 3- kinase-related kinases (PIKK) family and share highly homologous catalytic domain with PI3K and mTOR. We next determined the effect of S9 on the activities of ATM, ATR and DNA-PK by measuring the level of phosphorylated H2AX (cH2AX). H2AX could be quickly phosphorylated by ATM, ATR and DNA-PK upon DNA strand breaks induced by CPT [14]. As shown in Fig 2D, Rh30 cells displayed a robust increase in cH2AX level after exposure to CPT for 1 h. Co-treatment with S9 significantly abrogated the up-regulation levels of cH2AX at concentrations above 50 mM, indicating that S9 also inhibited the kinase activity of ATM, ATR or/and DNA-PK in Rh30 cells. To determine the selectivity of S9 on kinases, we tested the effect of S9 on 9 tyrosine kinases and 21 serine/threonine kinases. We found that S9 possessed weak or no inhibition on the tested kinase panel (Table S1), indicating S9 is selective against PI3K and mTOR, and against ATM, ATR and DNA-PK as well. S9 potentially binds to PI3K and mTOR. Based on the facts that most PI3K inhibitors apparently bind to the ATP- PLoS ONE | www.plosone.org 2 March 2009 | Volume 4 | Issue 3 | e4881