Discovery of the Novel mTOR Inhibitor and Its Antitumor Activities In Vitro and In Vivo Free

The phosphoinositide 3-kinase (PI3-K)/Akt/mTOR pathway is a signal transduction cascade that is central to a variety of important physiologic functions, including cell survival, protein synthesis and growth, cell cycle, metabolism, and angiogenesis (1, 2). PI3-K is activated upon growth factors binding to their cognate receptors, resulting in the activation of PI3-K–dependent kinases, which then phosphorylate and activate Akt (3). Phosphorylation of Akt at both serine 473 and threonine 308 is required for its full activation (4). Akt then activates mTOR by direct phosphorylation and inhibition of tuberous sclerosis complex 2, which is a negative regulator of mTOR. In cells, mTOR exists in at least 2 functionally distinct protein complexes, mTORC1 and mTORC2. mTORC1 phosphorylates the p70S6 kinase (p70S6K), which in turn phosphorylates the S6 ribosomal protein and 4E-BP1, leading to protein translation (5). mTORC2 functions in a feedback loop to activate Akt by phosphorylation on serine 473 (6).

The PI3K/Akt/mTOR signal transduction pathway is frequently deregulated in human cancers and thereby has attracted considerable attention as an oncology drug discovery target (7, 8). The most well-characterized inhibitor targeting this pathway is rapamycin and its analogs (also referred as rapalogs), which are currently used with success for treating several types of tumors (9). Previous studies have shown that the rapalogs are allosteric inhibitors that, in complex with FKBP12, target the FKB domain of mTOR (10). They partially inhibit mTOR through allosteric binding to mTORC1, but not mTORC2 (11). However, inhibiting only mTORC1 may not be sufficient for achieving a broad and robust anticancer effect due to the failure to inhibit mTORC2 in some tumor types. Moreover, resistance to treatment with rapamycin/rapalogs has been reported. The resistance has been ascribed, at least in part, to a feedback loop triggered by rapamycin, which leads to activation of Akt through inhibition of p70S6K, thereby, counteracting the antitumor potential of mTOR inhibition (12, 13). Therefore, great interest exists in the development of novel mTOR kinase inhibitors, which may suppress both mTORC1 and 2 or may inhibit both mTOR and PI3-K activities, thereby attenuating Akt activation.

In the present study, we identified a novel mTOR inhibitor from the Natural Product Database (NPD) database. The homologous structure of mTOR was used for virtual database screening, and [(E)-3-(4-(benzo[d][1,3]dioxol-5-yl)-2- oxobut-3-en-1-yl)-3-hydroxyindolin-2-one; designated herein as 3HOI-BA-01], was shown to be most effective, possessing in vitro and in vivo antitumor activity mediated through attenuation of mTOR signaling.

The 3-dimensional structure of mTOR was obtained from the SWISS-MODEL Repository, which is a kind of homology model based on the crystal structure of PI3-K-delta (PDB id 2WXG). Protein-ligand docking was conducted using Glide, which is a high-performance hierarchical docking algorithm (14). The final binding model structure of mTOR–3HOI-BA-01 was generated from Schrodinger induced fit docking (IFD; ref. 15), which merges the predictive power of Prime with the docking and scoring capabilities of Glide for accommodating the possible protein conformational changes upon ligand binding.

3HOI-BA-01 was purchased from ChemBridge or synthesized in house as previously described (16). The structure of the synthesized molecule was confirmed by ¹H NMR and compared with the authentic commercial sample. Rapamycin was purchased from LC Laboratories. Recombinant active kinases, mTOR (1362-end), PI3-K, and Akt, were purchased from Millipore. The inactive p70S6K protein was from SignalChem and Epoxy-activated Sepharose 6B was purchased from GE Healthcare. Phosphorylated p70S6K (T389), p70S6K, phosphorylated mTOR (S2448), mTOR, phosphorylated S6 (S235, 236), S6, and phosphorylated Akt (S473) and Akt were purchased from Cell Signaling Technology. The antibody against β-actin was purchased from Santa Cruz Biotechnology.

Non–small cell lung cancer (NSCLC) cell lines, A549, H520, and H1650, and the mouse epidermal cell line, JB6 Cl41, were purchased from American Type Culture Collection. A549 cells were cultured in F-12K containing penicillin (100 U/mL), streptomycin (100 μg/mL), l-glutamine (2 mmol/L), and 10% FBS (Life Technologies). H520 and H1650 cells were cultured in RPMI-1640 containing penicillin (100 U/mL), streptomycin (100 μg/mL), l-glutamine (2.05 mmol/L), and 10% FBS (Life Technologies). JB6 Cl41 cells were cultured in Eagle's Minimal Essential Medium containing penicillin (100 U/mL), streptomycin (100 μg/mL), and 5% FBS. Cells were maintained at 5% CO₂ and 37°C in a humidified incubator. The cells were cytogenetically tested and authenticated before the cells were frozen. Each vial of frozen cells was thawed and maintained for about 2 months (10 passages).

Non–small cell lung cancer cell lines were suspended in Basal medium Eagle medium and added to 0.6% agar, with vehicle or 5, 10, 20 μmol/L 3HOI-BA-01 in base and top layers of 0.3% agar. For JB6 Cl41 cells, the procedure is similar, except that these cells were exposed to EGF (20 ng/mL) during treatment with 3HOI-BA-01 or vehicle. The cultures were maintained at 37°C in a 5% CO₂ incubator for 1 to 2 weeks and then colonies were counted under a microscope using the Image-Pro Plus software (v6.1) program (Media Cybernetics).

A549 cells were seeded on 60 mm plates and treated or not treated with 3HOI-BA-01 for 72 hours. Then cells were fixed in 70% ethanol and stored at −20 °C overnight. Propidium iodine staining of DNA was conducted to determine cell-cycle distribution using a BD FACSCalibur Flow Cytometer (BD Biosciences).

To estimate cytotoxicity, JB6 Cl41 cells were seeded (8 × 10³ cells/well) in 96-well plates and cultured overnight. Cells were then fed with fresh medium and treated with vehicle or 5, 10, 20, 40, or 80 μmol/L 3HOI-BA-01. After culturing for 24 or 48 hours, the cytotoxicity of 3HOI-BA-01 was measured using an MTS assay kit (Promega) according to the manufacturer's instructions.

Western blotting was conducted as previously reported (17). In brief, proteins were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride membranes, which were blocked with nonfat milk and hybridized with specific primary antibodies. The protein bands were visualized using an enhanced chemiluminescence reagent (GE Healthcare) after hybridization with a horseradish peroxidase-conjugated secondary antibody.

Inactive S6K proteins (1 μg) were used as the substrate for an in vitro kinase assay with 250 ng of active mTOR (1362-end). Reactions were carried out in 1 × kinase buffer [25 mmol/L Tris-HCl pH 7.5, 5 mmol/L β-glycerophosphate, 2 mmol/L dithiothreitol (DTT), 0.1 mmol/L Na₃VO₄, 10 mmol/L MgCl₂, and 5 mmol/L MnCl₂) containing 100 μmol/L ATP at 30°C for 30 minutes. Reactions were stopped and proteins were detected by Western blotting.

The PI3-K in vitro kinase assay was conducted as previously described (18). Briefly, 100 ng active PI3-K (Millipore) was incubated with dimethyl sulfoxide (DMSO) or 3HOI-BA-01 for 15 minutes and then reacted with phosphatidylinositol sodium salt (MP Biomedical). Reactions were carried out in kinase buffer containing 50 μmol/L unlabeled ATP with or without 10 μCi of [γ-³²P] ATP at 30°C for 20 minutes. Reactions were terminated and resolved by thin layer chromatography and visualized by autoradiography.

In vitro Akt1 and 2 kinase assays were conducted in accordance with the manufacturer's instructions. In brief, an active Akt1 or Akt2 protein (30 ng) and substrate (30 μmol/L) were incubated with or without 3HOI-BA-01 for 30 minutes at 30°C. Afterward, 20 μL aliquots were transferred onto p81 paper and washed 3 times with 0.75% phosphoric acid for 5 minutes per wash and once with acetone for 5 minutes. Radioactive incorporation was determined using a scintillation counter (LS6500; Beckman Coulter).

Recombinant human mTOR (1362-end; 200 ng) or cell lysates (1 mg) were incubated with 3HOI-BA-01-Sepharose 6B beads (or Sepharose 6B beads alone as a control; 100 μL, 50% slurry) in the reaction buffer [50 mmol/L Tris (pH 7.5), 5 mmol/L EDTA, 150 mmol/L NaCl, 1 mmol/L DTT, 0.01% Nonidet P-40, 2 μg/mL bovine serum albumin, 0.02 mmol/L phenylmethylsulfonyl fluoride, and 1 μg/mL protease inhibitor mixture]. After incubation with gentle rocking overnight at 4°C, the beads were washed 5 times and proteins bound to the beads were analyzed by Western blotting.

Athymic nude mice [Cr:NIH (S), NIH Swiss nude, female, 6-week-old] were obtained from Harlan Laboratories and maintained under “specific pathogen-free” conditions based on the guidelines established by the University of Minnesota Institutional Animal Care and Use Committee. Mice were divided into different groups (n = 8 of each group). A549 lung cancer cells (3 × 10⁶/0.2 mL 1× PBS) were injected subcutaneously into the right flank of each mouse. 3HOI-BA-01 was prepared once a week and protected from light and kept at 4°C. 3HOI-BA-01 or vehicle control was administered by intraperitoneal injection 3 times a week. Tumor volumes were measured 3 times a week and the body weights were measured once a week. Mice were monitored until tumors reached 1 cm³ total volume, at which time, mice were euthanized and tumors extracted and were analyzed by Western blotting.

All quantitative data are expressed as means ± SD. and significant differences were determined by the Student t test or one-way ANOVA. A probability value of P < 0.05 was used as the criterion for statistical significance.

With the purpose of identifying novel inhibitors of mTOR kinase activity, we conducted an intensive molecular docking analysis using Glide v5.7 (14) to screen 60,000 compounds from the NPD against the structure of the homology model of mTOR, which was based on the crystal structure of PI3-K-delta (PDB id 2WXG; ref. 19). The top-ranked 100 compounds were redocked into mTOR with the IFD (15) protocol implemented in Schrodinger Suite 2011. Finally, 13 commercially available compounds were selected from the top-ranked list of 100 and were tested experimentally. 3HOI-BA-01 (Fig. 1A) dramatically inhibited neoplastic cell transformation (data not shown) and was selected for further study. According to the docking result (Fig. 1B), 3HOI-BA-01 formed 4 hydrogen bonds with the mTOR protein. One hydrogen bond involved the backbone atoms of the hinge residue Val2240 and the other 3 hydrogen bonds were formed with the side chain atoms of Lys2187, Tyr2225, and Asp2357, respectively. In addition, 1 phenyl ring formed strong π–π stacking interactions with the hydroxyphenyl ring of Tyr2225. These results indicate that 3HOI-BA-01 may inhibit mTOR by directly binding with that protein.

To determine whether 3HOI-BA-01 inhibits mTOR kinase activity, we first conducted an in vitro kinase assay with recombinant mTOR in the presence of various concentrations of 3HOI-BA-01. Wortmannin, a well-known inhibitor of both PI3-K and mTOR, was used as a positive control in this assay. The phosphorylation of p70S6K (Thr389), an mTOR substrate, was strongly suppressed by 3HOI-BA-01 in a concentration-dependent manner. A very low concentration of 3HOI-BA-01 (1 μmol/L; Fig. 2A) caused a 21% inhibition of mTOR kinase activity and 3HOI-BA-01 at 5 μmol/L caused a 67% inhibition, and the highest concentration (20 μmol/L) of 3HOI-BA-01 resulted in a 95% inhibition. These results clearly indicate that 3HOI-BA-01 is an inhibitor of mTOR kinase activity.

The docking results indicated that 3HOI-BA-01 formed a good interaction with the mTOR active site, which may be the basis of the direct binding of 3HOI-BA-01 to mTOR, resulting in the inhibition of mTOR kinase activity. To confirm this idea, we conducted an in vitro binding assay using 3HOI-BA-01–conjugated Sepharose 6B beads or control Sepharose 6B beads. No obvious band was observed when the mTOR protein was incubated with Sepharose 6B beads, whereas a clear band was seen when mTOR was incubated with 3HOI-BA-01–conjugated Sepharose 6B beads (Fig. 2B), indicating that 3HOI-BA-01 directly binds to recombinant mTOR in vitro. We then conducted an ex vivo pull-down assay in A549 human lung cancer cells, and similar results were also obtained with an A549 cell lysate (Fig. 2C). These results indicated that 3HOI-BA-01 binds directly to mTOR protein and thus inhibits mTOR kinase activity.

We then examined the effect of 3HOI-BA-01 on EGF-induced neoplastic transformation of JB6 Cl41 cells. 3HOI-BA-01 significantly inhibited EGF-promoted neoplastic transformation in a dose-dependent manner (Fig. 3A). 3HOI-BA-01 at 1, 5, 10, or 20 μmol/L caused a decrease of 18, 19, 62, or 98% of control, respectively (Fig. 3B). The inhibition of colony formation by 3HOI-BA-01 was not due to cytotoxicity because the effective concentration range for suppressing cell transformation did not affect JB6 Cl41 cell viability (Fig. 3C). Because anchorage-independent growth ability is an ex vivo indicator and a key characteristic of the transformed cell phenotype (20), these results suggest that 3HOI-BA-01 can reduce the malignant potential of JB6 Cl41 cells induced by EGF.

To test whether 3HOI-BA-01 exerts its activity by inhibiting mTOR activity in JB6 Cl41 cell, we treated cells with 3HOI-BA-01 and examined several key signaling molecules, including those in the RAS/RAF/MEK and PI3-K/Akt/mTOR pathways, which are frequently deregulated in human malignancies. Western blot results showed that 3HOI-BA-01 dose dependently suppressed the phosphorylation of p70S6K and S6 ribosomal protein (S6), which are downstream signaling molecules of mTOR (Fig. 3D). However, 3HOI-BA-01 had no effect on the phosphorylation of other molecules, including EGFR, RAF, MEK, and ERKs (data not shown). This result suggested that the mTOR signaling pathway accounts for the inhibition of cell transformation by 3HOI-BA-01.

Previous studies suggested that the mTOR signaling pathway is highly activated in human lung cancer (21–23). Therefore, we examined the effect of 3HOI-BA-01 in a panel of NSCLC cells. First, we investigated the effects of 3HOI-BA-01 on anchorage-independent cell growth and the results showed that 3HOI-BA-01 significantly inhibited A549 cell growth in soft agar in a concentration-dependent manner (Fig. 4A). Colony formation was inhibited by more than 50% after treatment with 3HOI-BA-01 at a concentration of 10 μmol/L, and almost no colonies were formed at 20 μmol/L (Fig. 4A). Moreover, we also examined the effect of 3HOI-BA-01 on the growth of several other lung cancer cell lines, including H520 and H1650. Results showed that 3HOI-BA-01 dose dependently inhibited the growth of each cell line in soft agar (Fig. 4B and C).

We then investigated the effect of 3HOI-BA-01 on downstream targets of mTOR in A549 cancer cells. Western blot results showed that mTORC1-mediated phosphorylation of p70S6K (T389) and S6 (S235, 236) was substantially decreased dose dependently with 3HOI-BA-01 treatment (Fig. 4D). Meanwhile, mTORC2-mediated phosphorylation of Akt (S473) was also inhibited in a dose-dependent manner (Fig. 4D). Moreover, results also showed that the phosphorylation of mTOR (S2448) was attenuated at the higher concentration (20 μmol/L) of 3HOI-BA-01 (Fig. 4D).

Inhibition of mTOR can result in many effects on cells, including cell-cycle arrest and apoptosis. Therefore, we examined whether 3HOI-BA-01 affects cell-cycle progression and apoptosis. Results revealed that 3HOI-BA-01 treatment led to G₁ cell-cycle arrest (Fig. 4E). However, no change was observed in sub-G₁ population after treatment with 3HOI-BA-01 (data not shown), indicating that it has no effect on apoptosis. These results indicated that, as an mTOR inhibitor, 3HOI-BA-01 could induce cell-cycle arrest without affecting apoptosis, which is quite similar to the effects of other reported mTOR inhibitors (24).

The inhibition of Akt (S473) and mTOR (S2448) could result not only from mTORC2 inhibition, but also from inhibition of upstream PI3-K or Akt. Thus, we further examined the effects of 3HOI-BA-01 on PI3-K or Akt in vitro kinase activity. Class IA PI3-Ks, consisting of PI3-K-α, PI3-K-β, and PI3-K-δ, are reportedly implicated in human cancer (25–27). Our results showed that 3HOI-BA-01 suppressed PI3-K-α, PI3-K-β, and PI3-K-δ in vitro activity in a concentration-dependent manner (Fig. 5A). However, the concentration (10 or 20 μmol/L) that inhibits PI3-K is higher than that required for mTOR inhibition. In addition, we examined the activity of 3HOI-BA-01 in Akt1 and Akt2 in vitro kinase assays. Only 30% or less inhibition was observed even at 20 μmol/L 3HOI-BA-01 (Fig. 5B), indicating that Akt1 and Akt2 may not be major targets of 3HOI-BA-01. These results have shown that 3HOI-BA-01 targets both mTOR and PI3-K, with PI3-K being much less sensitive.

To explore the antitumor activity of 3HOI-BA-01 in vivo, A549 cancer cells were innoculated into the right flank of individual athymic nude mice. Mice were then administered vehicle or 3HOI-BA-01 (10 or 40 mg/kg body weight) by intraperitoneal injection, 3 times a week for 29 days. The results showed that treatment of mice with 10 or 40 mg/kg body weight of 3HOI-BA-01 significantly suppressed A549 tumor growth by 58% and 76%, respectively, relative to the vehicle-treated group (Fig. 6A, P < 0.01). Moreover, mice seemed to tolerate treatment with these doses of 3HOI-BA-01 without the loss of body weight compared with vehicle-treated group (Fig. 6B). The effect of 3HOI-BA-01 on mTOR protein targets was evaluated by Western blot analysis after 29 days of treatment. The expression of phosphorylated S6, as well as the phosphorylation of Akt (S473), were markedly decreased after treatment with 3HOI-BA-01 (Fig. 6C). These data indicated that A549 lung tumor development was suppressed by 3HOI-BA-01 through inhibition of the PI3-K/mTOR signaling pathway.

The aim of this study was to identify novel scaffold inhibitors of mTOR using molecular docking methods. We combined 2 docking methods, Glide and IFD, for molecular docking in this study. Glide adopts a hierarchical docking algorithm and efficiently improves docking speed (14). However, the protein structure is regarded as a rigid body and protein flexibility is ignored. With IFD, the side chains in the binding pocket are allowed to move so as to simulate the induced fit effect of the protein structure upon ligand binding (15). The disadvantage of IFD is the high computational cost and low speed. To trade off accuracy and speed, Glide was used for the initial screening and IFD was used for refinement of top-ranked compounds. On the basis of these 2 methods, 60,000 compounds in the NPD were screened against the mTOR homologous structure and several compounds were identified as potential mTOR inhibitors. 3HOI-BA-01, a compound belonging to the 3-hydroxy-2-oxindole group of compounds, was confirmed to inhibit mTOR kinase activity and exert antitumor activity.

We first examined the inhibitory activity of 3HOI-BA-01 using an in vitro mTOR kinase assay. 3HOI-BA-01 could potently suppress mTOR activity in a concentration-dependent manner. We further conducted in vitro binding and ex vivo pull-down assays and confirmed the direct binding between 3HOI-BA-01 and the mTOR protein. In addition, 3HOI-BA-01 inhibited the phosphorylation of mTOR downstream molecules in cancer cells, including the mTORC1 substrates p70S6K (T389) and S6 (S235,236) and the mTORC2 substrate Akt (S473), resulting in the inhibition of the growth of several types of human lung cancer cells. Moreover, results of an in vivo study using a xenograft mouse model further confirmed that 3HOI-BA-01 suppressed mTOR's protein targets in tumor tissues resulting in inhibition of tumor growth in vivo. Therefore, 3HOI-BA-01 inhibits mTOR kinase activity both in vitro and in vivo by directing binding with mTOR, which suppressed tumor growth.

Intriguingly, 3HOI-BA-01 showed inhibitory activity on the phosphorylation of Akt on S473 both in tumor cells and in the xenograft mouse model tumor tissue, indicating an advantage of this compound over rapalogs in Akt inhibition. Rapalogs have been reported to increase the phosphorylation of Akt on S473 in cancer cells and patients treated with these drugs eventually become refractory partly due to Akt activation (28, 29). Previous studies showed that mTORC1, but not mTORC2, is sensitive to rapamycin. The activation of Akt by rapalogs is mainly due to the disruption of a negative feedback loop, which is dependent on IGF-IR/insulin receptor substrate 1 (IRS-1), and involves the S6K-mediated suppression of upstream signaling (28, 29). mTORC1 inhibitors abrogate this feedback suppression, thus resulting in Akt activation. In the present study, we have shown that 3HOI-BA-01 inhibits the phosphorylation of mTORC1 downstream targets, including p70S6K and S6, and the mTORC2 downstream target, Akt. Moreover, this compound also attenuated PI3-K activity at a higher concentration. Therefore, the inhibition of the phosphorylation of Akt (S473) by 3HOI-BA-01 may be the result of both mTORC2 inhibition and of PI3-K inhibition.

Taken together, using a molecular docking method, we identified 3HOI-BA-01 as a novel mTOR/PI3K dual inhibitor, possessing antitumor activity in vitro and in vivo. Allosteric inhibitors of mTOR have been reported to increase the phosphorylation of Akt. Thus, 3HOI-BA-01, as an mTOR inhibitor, possesses a potential advantage and deserves further development for cancer prevention and therapeutic use. In addition, our observations also underscore the importance of testing 3HOI-BA-01 in combination with known and conventional chemotherapeutic drugs.

No potential conflicts of interest were disclosed.

Conception and design: H. Xie, M. Lee, F. Zhu, Z. Huang, Z. Dong

Development of methodology: H. Xie, M. Lee, F. Zhu, Z. Huang, W. Ma, Z. Dong

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H. Xie, M. Lee, D. Kim, D.Y. Lim, S. Kang, A.M. Bode

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): H. Xie, M. Lee, F. Zhu, Z. Huang, Y. Li, C. Peng, D.Y. Lim, H. Li, A.M. Bode, Z. Dong

Writing, review, and/or revision of the manuscript: H. Xie, M. Lee, Z. Huang, J. Ding, A.M. Bode

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S.K. Jung, X. Li, Z. Dong

Study supervision: F. Zhu, Z. Dong

Synthesized chemicals: K. Reddy

The authors thank Tonya Poorman for secretarial assistance in submitting this manuscript.

This work was supported by The Hormel Foundation and NIH grants CA120388, R37 CA081064, ES016548, and NCI Contract No. HHSN-261200533001 (N01-CN53301).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.