Published OnlineFirst March 27, 2013; DOI: 10.1158/1535-7163.MCT-12-1241 Molecular Cancer Chemical Therapeutics Therapeutics Discovery of the Novel mTOR Inhibitor and Its Antitumor Activities In Vitro and In Vivo Hua Xie1,3, Mee-Hyun Lee1, Feng Zhu1, Kanamata Reddy1, Zunnan Huang1, Dong Joon Kim1, Yan Li1, Cong Peng1, Do Young Lim1, Soouk Kang1, Sung Keun Jung1, Xiang Li1, Haitao Li1, Weiya Ma1, Ronald A. Lubet2, Jian Ding3, Ann M. Bode1, and Zigang Dong1 Abstract The phosphoinositide 3-kinase (PI3-K)/Akt and mTOR signaling pathway plays a critical role in cell survival and proliferation and is often aberrantly activated in many types of cancer. The mTOR kinase protein, one of the key molecules in this pathway, has been shown to be an important target for cancer therapy. In the present study, a ligand docking method was used to screen for novel scaffold mTOR inhibitors. Sixty thousand compounds in the Natural Product Database were screened against the mTOR homologous structure, and 13 commercially available compounds listed in the top-ranked 100 compounds were selected for further examin- ation. Compound [(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 then selected for further study of its antitumor activity. An in vitro study has shown that 3HOI-BA-01 inhibited mTOR kinase activity in a dose-dependent manner by directly binding with mTOR. In a panel of non–small cell lung cancer cells, the compound also attenuated mTOR downstream signaling, including the phosphorylation of p70S6K, S6, and Akt, resulting in G1 cell-cycle arrest and growth inhibition. Results of an in vivo study have shown that intraperitoneal injection of 3HOI-BA-01 in A549 lung tumor–bearing mice effectively suppressed cancer growth without affecting the body weight of the mice. The expression of downstream signaling molecules in the mTOR pathway in tumor tissues was also reduced after 3HOI-BA-01 treatment. Taken together, we identified 3HOI-BA-01 as a novel and effective mTOR inhibitor. Mol Cancer Ther; 12(6); 950–8. Ó2013 AACR. Introduction mTORC1 phosphorylates the p70S6 kinase (p70S6K), The phosphoinositide 3-kinase (PI3-K)/Akt/mTOR which in turn phosphorylates the S6 ribosomal protein pathway is a signal transduction cascade that is central and 4E-BP1, leading to protein translation (5). mTORC2 to a variety of important physiologic functions, including functions in a feedback loop to activate Akt by phosphor- cell survival, protein synthesis and growth, cell cycle, ylation on serine 473 (6). metabolism, and angiogenesis (1, 2). PI3-K is activated The PI3K/Akt/mTOR signal transduction pathway is upon growth factors binding to their cognate receptors, frequently deregulated in human cancers and thereby has resulting in the activation of PI3-K–dependent kinases, attracted considerable attention as an oncology drug which then phosphorylate and activate Akt (3). Phosphor- discovery target (7, 8). The most well-characterized inhib- ylation of Akt at both serine 473 and threonine 308 is itor targeting this pathway is rapamycin and its analogs required for its full activation (4). Akt then activates (also referred as rapalogs), which are currently used with mTOR by direct phosphorylation and inhibition of tuber- success for treating several types of tumors (9). Previous ous sclerosis complex 2, which is a negative regulator studies have shown that the rapalogs are allosteric inhi- of mTOR. In cells, mTOR exists in at least 2 functionally bitors that, in complex with FKBP12, target the FKB distinct protein complexes, mTORC1 and mTORC2. 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 Authors' Affiliations: 1The Hormel Institute, University of Minnesota, Austin, Minnesota; 2National Institutes of Health, National Cancer Institute, effect due to the failure to inhibit mTORC2 in some tumor Maryland; and 3State Key Laboratory of Drug Research, Shanghai Institute types. Moreover, resistance to treatment with rapamycin/ of Materia Medica, Chinese Academy of Sciences, Shanghai, China rapalogs has been reported. The resistance has been H. Xie, MH. Lee, and F. Zhu contributed equally to this work. ascribed, at least in part, to a feedback loop triggered by Corresponding Author: Zigang Dong, The Hormel Institute, University of rapamycin, which leads to activation of Akt through Minnesota, 801 16th Avenue NE, Austin, MN 55912. Phone: 507-437-9600; inhibition of p70S6K, thereby, counteracting the antitu- Fax: 507-437-9606; E-mail: [email protected] mor potential of mTOR inhibition (12, 13). Therefore, great doi: 10.1158/1535-7163.MCT-12-1241 interest exists in the development of novel mTOR kinase Ó2013 American Association for Cancer Research. inhibitors, which may suppress both mTORC1 and 2 or 950 Mol Cancer Ther; 12(6) June 2013 Downloaded from mct.aacrjournals.org on September 25, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst March 27, 2013; DOI: 10.1158/1535-7163.MCT-12-1241 Discovery of mTOR Inhibitor 3HOI-BA-01 Antitumor Activities may inhibit both mTOR and PI3-K activities, thereby authenticated before the cells were frozen. Each vial of attenuating Akt activation. frozen cells was thawed and maintained for about 2 In the present study, we identified a novel mTOR inhib- months (10 passages). itor from the Natural Product Database (NPD) database. The homologous structure of mTOR was used for virtual Anchorage-independent cell transformation assay database screening, and [(E)-3-(4-(benzo[d][1,3]dioxol- Non–small cell lung cancer cell lines were suspended in 5-yl)-2- oxobut-3-en-1-yl)-3-hydroxyindolin-2-one; desig- Basal medium Eagle medium and added to 0.6% agar, nated herein as 3HOI-BA-01], was shown to be most with vehicle or 5, 10, 20 mmol/L 3HOI-BA-01 in base and effective, possessing in vitro and in vivo antitumor activity top layers of 0.3% agar. For JB6 Cl41 cells, the procedure is mediated through attenuation of mTOR signaling. similar, except that these cells were exposed to EGF (20 ng/mL) during treatment with 3HOI-BA-01 or vehicle. Materials and Methods The cultures were maintained at 37 C in a 5% CO2 incu- Computational modeling bator for 1 to 2 weeks and then colonies were counted The 3-dimensional structure of mTOR was obtained under a microscope using the Image-Pro Plus software from the SWISS-MODEL Repository, which is a kind of (v6.1) program (Media Cybernetics). homology model based on the crystal structure of PI3-K- delta (PDB id 2WXG). Protein-ligand docking was con- Cell-cycle analysis ducted using Glide, which is a high-performance hierar- A549 cells were seeded on 60 mm plates and treated or chical docking algorithm (14). The final binding model not treated with 3HOI-BA-01 for 72 hours. Then cells were structure of mTOR–3HOI-BA-01 was generated from fixed in 70% ethanol and stored at À20 C overnight. Schrodinger induced fit docking (IFD; ref. 15), which Propidium iodine staining of DNA was conducted to merges the predictive power of Prime with the docking determine cell-cycle distribution using a BD FACSCalibur and scoring capabilities of Glide for accommodating the Flow Cytometer (BD Biosciences). possible protein conformational changes upon ligand binding. MTS assay To estimate cytotoxicity, JB6 Cl41 cells were seeded (8 Â Reagents 103 cells/well) in 96-well plates and cultured overnight. 3HOI-BA-01 was purchased from ChemBridge or Cells were then fed with fresh medium and treated with synthesized in house as previously described (16). The vehicle or 5, 10, 20, 40, or 80 mmol/L 3HOI-BA-01. After structure of the synthesized molecule was confirmed by culturing for 24 or 48 hours, the cytotoxicity of 3HOI-BA- 1HNMRandcomparedwiththeauthenticcommercial 01 was measured using an MTS assay kit (Promega) sample. Rapamycin was purchased from LC Laborato- according to the manufacturer’s instructions. ries. Recombinant active kinases, mTOR (1362-end), PI3-K, and Akt, were purchased from Millipore. The Western blot analysis inactive p70S6K protein was from SignalChem and Western blotting was conducted as previously reported Epoxy-activated Sepharose 6B was purchased from GE (17). In brief, proteins were resolved by SDS-PAGE and Healthcare. Phosphorylated p70S6K (T389), p70S6K, transferred onto polyvinylidene difluoride membranes, phosphorylated mTOR (S2448), mTOR, phosphorylated which were blocked with nonfat milk and hybridized S6 (S235, 236), S6, and phosphorylated Akt (S473) and with specific primary antibodies. The protein bands were Akt were purchased from Cell Signaling Technology. visualized using an enhanced chemiluminescence reagent The antibody against b-actin was purchased from Santa (GE Healthcare) after hybridization with a horseradish Cruz Biotechnology. peroxidase-conjugated secondary antibody. Cell culture mTOR in vitro kinase assay Non–small cell lung cancer (NSCLC) cell lines, A549, Inactive S6K proteins (1 mg) were used as the substrate H520, and H1650, and the mouse epidermal cell line, JB6 for an in vitro kinase assay with 250 ng of active mTOR Cl41, were purchased from American Type Culture (1362-end). Reactions were carried out in 1 Â kinase buffer Collection. A549 cells were cultured in F-12K containing [25 mmol/L Tris-HCl pH 7.5, 5 mmol/L b-glyceropho- penicillin (100 U/mL), streptomycin (100 mg/mL), L- sphate, 2 mmol/L dithiothreitol (DTT), 0.1 mmol/L Na3 glutamine (2 mmol/L), and 10% FBS (Life Technolo- VO4, 10 mmol/L MgCl2, and 5 mmol/L MnCl2) contain- gies). H520 and H1650 cells were cultured in RPMI-1640 ing 100 mmol/L ATP at 30C for 30 minutes. Reactions containing penicillin (100 U/mL), streptomycin (100 were stopped and proteins were detected by Western mg/mL), L-glutamine (2.05 mmol/L), and 10% FBS (Life blotting.