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In Silico and In Cell Hybrid Selection of Nonrapalog Ligands to Allosterically Inhibit the Kinase Ac

In Silico and In Cell Hybrid Selection of Nonrapalog Ligands to Allosterically Inhibit the Kinase Activity of mTORC1

Cancer-specific metabolic alterations hyperactivate the kinase activity of the mammalian/mechanistic target of rapamycin (mTOR) for overcoming stressful environments. Rapalogs, which allosterically inhibit mTOR complex 1 (mTORC1), have been approved as anticancer agents. However, the immunosuppressive side effect of these compounds results in the promotion of tumor metastasis, thereby limiting their therapeutic efficacy. They first report a nonrapalog inhibitor, WRX606, identified by a hybrid strategy of in silico and in cell selections. Their studies showed that WRX606 formed a ternary complex with FK506-binding protein-12 (FKBP12) and FKBP–rapamycin-binding (FRB) domain of mTOR, resulting in the allosteric inhibition of mTORC1. WRX606 inhibited the phosphorylation of not only the ribosomal protein S6 kinase 1 (S6K1) but also eIF4E-binding protein-1 (4E-BP1). Hence, WRX606 efficiently suppressed tumor growth in mice without promotion of metastasis. These results suggest that WRX606 is a potent lead compound for developing anticancer drugs discovered by in silico and in cell methods.


Metabolic reprogramming or cancer-specific metabolic alterations are mediated by oncogenic signals in cancer cells. The abnormal signals in cancer cells accelerate the activities of mTOR complex 1 (mTORC1) and/or mTOR complex 2 (mTORC2) to overcome the stressful cancer microenvironment. mTORC1 controls protein synthesis and cell proliferation in response to the environmental levels of nutrients, energy, and growth factors via the phosphorylation of the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) and the ribosomal protein S6 kinase 1 (S6K1).



In silico selection of ligands to form a ternary complex with FKBP12 and FRB. (A) In silico selection of FKBP12/FRB binding ligands from three different libraries prepared from ZINC15 database. (B) Scatter plot of the top 103 hits of each library, showing that the search efficacy for higher affinity ligands comes from larger molecular weights (H-bond: hydrogen-bond energy; Da: Dalton). (C–E) Chemical structures of the primary top-scoring ligands from the indicated libraries. (F) A schematic drawing of a principle of NanoBiT in cell assay. FKBP-SmBiT and FRB-LgBiT plasmids were cotransfected into HEK293 cells. Luminescence was observed when a FKBP–ligand–FRB ternary complex was formed because it reconstructs the luciferase consisting of SmBiT and LgBiT proteins. (G) Primary selection of FKBP12-FRB-binding ligands by NanoBiT cell assay using HEK293 cell line. The result shows the mean of three independent experiments (n = 5 replicates each) ± SD; the signal is normalized to the background; ordinary one-way ANOVA was used: ****P < 0.0001; *P < 0.05; ns, P > 0.05. (H) Close-up view of the docked position of ZINC8593606 (WRX606; cyan) at the FKBP12-binding site (light blue ribbon). Hydrogen bonds are represented as red dotted lines (hydrogen bond between Y26 and D37 for receptor stability). (I) Lateral view of the FKBP12–WRX606–FRB ternary complex. (J) Close-up view of WRX606 at the FRB-binding site (buff ribbon).


On the other hand, mTORC2 regulates cell survival, cell cycle, and cytoskeleton organization by phosphorylating the protein kinases AKT, PKC, and SGK.Rapamycin was isolated from Streptomyces hygroscopicus as an antifungal macrolide.Later, it was found to suppress the immune response in mammals by forming a ternary complex with FKBP12, a ubiquitous prolyl isomerase, and a variety of immune cells.Therefore, rapamycin was first approved as an immunosuppressant for transplantation to prevent allograft rejection.Moreover, rapamycin showed to inhibit tumor growth in mammals through the allosteric inhibition of mTORC1.The FKBP12–rapamycin-binding (FRB) domain recruits S6K1 to the catalytic site for phosphorylation. Thus, FKBP12–rapamycin complex inhibits the recruitment by occupying the FRB domain, leading to the allosteric inhibition of mTORC1. In contrast, the FKBP12–rapamycin complex does not access the FRB domain of mTORC2, because of the steric hindrance due to the proximity of the components.These structure-dependent properties of rapamycin limit its therapeutic effect because of the incomplete inhibition of 4E-BP1 phosphorylation as a substrate-dependent inhibitor.


Selection of WRX606 analogs based on the common scaffold. (A) Chemical structures of the common scaffold (the substitution group highlighted in red color) and the 13 analogs clustered into groups based on the substitution position (showed only the functional group clusters in dashed squares). (B) In cell selection of the secondary screen ligands for FKBP12/FRB binding using NanoBiT in HEK293 cell line. The result shows the mean of three independent experiments (n = 5 replicates each) ± SD; Ordinary one-way ANOVA was used: ****P < 0.0001; **P < 0.01; ns, P > 0.05. (C) Dose-dependent binding of the selected ligands and rapamycin at the FKBP12/FRB interface by NanoBiT cell assay. The signal is normalized to the background. The result shows the mean of two independent experiments (n = 5 replicates each) ± SD. (D) Close-up view of WRX601 (purple) at the FKBP12-binding site (light blue ribbon). Hydrogen bonds are shown as red dotted lines (hydrogen bond between Y26 and D37 for receptor stability). (E) Lateral view of FKBP12–WRX601–FRB ternary complex. (F) Close-up view of WRX601 at the FRB-binding site (buff ribbon).



In contrast, the catalytic inhibitors for mTOR, such as Torin1, directly bind the ATP-binding sites to inhibit kinase activity. However, preclinical trials showed that such an inhibition effect on the mTORC1 or mTORC2 was soon tolerated.Like rapamycin, the catalytic inhibitors are not exempt from typical drawbacks during long-term dosing, which may result in mTORC2 activation. In addition, inhibitors with dual inhibition mode for PI3K and mTOR, such as PI-103, showed cytotoxicity for normal cells, suggesting a limited therapeutic range.Thus, a new inhibitor is needed to address the drawbacks of the current mTOR inhibitors.


WRX606 binds the FKBP12/FRB interface more strongly than WRX601. (A–C) 2D representations showing the binding mode of rapamycin (A), WRX606 (B), or WRX601 (C) with FKBP12 (light blue) and FRB (buff) at the FKBP12/FRB interface (PDB ID: 1FAP). Ligand portions interacted with FRB are shown in red, and hydrogen bonds are shown as red dashes. (D) Close-up view of the ligand-binding site at the FKBP12 side showing residues (D37, I56, and Y82; magenta) involved in hydrogen bonding to ligands. *K73 (blue) as a negative control residue is also depicted. (E) Lateral view of the ternary complex with an indication of the ligand-binding site. (F) Close-up view of the ligand-binding site at the FRB side showing the residues (S2035, Y2038, F2039, T2098, W2101, Y2104, and Y2105; magenta) involved in ligand interaction. *E2052 (blue) as a negative control is also depicted. (G) Point mutagenesis at FKBP12 or FRB-binding sites of FKBP12-SmBiT or FRB-LgBiT plasmids to confirm the binding modes of WRX606 and WRX601 along with rapamycin using NanoBiT assay. Four point mutants of FKBP12 (D37A, I56A, *K73A, or Y82A) and seven point mutants of FRB (S2035A, Y2038A, F2039A, *E2052A, T2098A, W2101A, Y2104A, or Y2105A) were prepared. *K73A and *E2052A served as negative control mutant residues located away from the ligand-binding site. The results are shown as a percent of the wild-type groups treated with the indicated compound. The results represent the mean of three independent experiments (n = 3 replicates each) ± SD; two-way ANOVA was used: ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, P > 0.05. (H) Superposition of WRX601-FRB on WRX606-FRB showing the difference in binding modes. Y2038 and Y2104 (cyan) are only involved in binding to WRX606.


WRX606 inhibits S6K1 and 4E-BP1 phosphorylation. (A) Schematic representation of AlphaLISA assay principle. Antibodies specific for a target protein and a phosphorylated target protein are immobilized on acceptor and donor beads, respectively. Fluorescence is observed when the beads come close to each other upon binding to the target protein. (B) AlphaLISA measurements of all the selected ligands for the inhibition of mTORC1 kinase activity. HeLa cells were treated with a single dose (1 μM) of the ligands or rapamycin for 1 h under starvation conditions. Two-way ANOVA was used: ****P < 0.0001; ***P < 0.001; *P < 0.05; ns, P > 0.05. (C) Inhibition curves of T389p-S6K1 of prestarved MCF-7 cells treated with WRX601, WRX606, or rapamycin for 1 h. (D) Inhibition curves of T37/46p-4E-BP1 of the same cells as in (C). Results represent the mean of two independent experiments as a percent of the controls (n = 3 replicates each) ± SD. (E) Summary of IC50 values of (C and D).


This study aimed to discover a new allosteric inhibitor for mTORC1 using a hybrid strategy involving in silico and in cell selections.Crystallographic studies have revealed the binding mode of rapamycin to FKBP12 and FRB at the atomic level .Based on the structures, virtual libraries from the ZINC15 database were screened by intercoordinate mechanics (ICM)-based docking and further assayed by split luciferase-based in cell assay.As a result, they identified 13 analogs with a common scaffold composed of benzodioxol and quinazoline-2,4-dione groups linked by a butanamide linker. One of the 13 compounds, abbreviated as WRX606 (“WRX” + “number of the last three digits of ZINC8593606”, and hereafter also termed for other ZINC compounds), promoted ternary complexation with FKBP12 and FRB. They further elucidated the molecular mechanism of WRX606 binding using steered molecular dynamics (SMD)and in cell-based point mutagenesis.



WRX606 is more effective than WRX601 in mTORC1 activation conditions. (A) Time-dependent inhibition of T389p-S6K1 in HeLa cells by a single dose (1 μM) of WRX606, WRX601, or rapamycin under activation conditions. (B) Time-dependent inhibition of T37/46p-4E-BP1 in HeLa cells by a single dose (1 μM) of WRX606, WRX601, or rapamycin under activation conditions. The results are indicated as percent of the controls (nontreated cells). The result is representative of two independent experiments (mean ± SD; n = 3). Two-way ANOVA was used: ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, P > 0.05. (C and D) Dose-dependent inhibitory effect of WRX606 (C) or WRX601 (D) on T389p-S6K1 in prestarved HeLa cells treated for 6 h with or without insulin stimulation (1 h); results are representative of two independent experiments (n = 1). (E and F) Dose-dependent inhibitory effect of WRX606 (E) or WRX601 (F) on T37/46p-4E-BP1 under the same condition as in (C or D).


Antitumor effect of WRX606. (A–C) Cytotoxic effect of WRX606 (A) and WRX601 (B) on two different cancer cell lines (HeLa and MCF-7). (C) Summary of IC50 values of WRX606 and WRX601. The results are shown as percent of control (nontreated cells). Experimental data are representative of two independent experiments each (mean ± SD; n = 3). (D–G) Antitumor effect of WRX606 in tumor bearing mice. (D) Design of the animal experiment. (E) Line graphs showing the inhibitory effects of WRX606 (25 mg/kg/day; orally), rapamycin (RAP; 25 mg/kg/day; IP), and PBS control on tumor growth (mean ± SEM; n = 5). (F) Numbers of metastatic tumor colonies of 4T1 cells in the lungs of mice in the experimental groups. The lungs were fixed with Bouin’s solution, by which normal tissues stained yellow and the tumor colonies stained whitish (see photos in Figure S10B). (G) Tumor increment possibilities 10 days after the final treatment, shown as a fold increase in tumor volume (D20/D10). Correlation analysis or unpaired t test was used: ****P < 0.0001; ***P < 0.001; *P < 0.05; ns, P > 0.05.


The results suggested that the quinazoline-2,4-dione and butanamide groups of WRX606 made hydrogen bonds with FKBP12, while benzodioxol and 3-chlorophenylamino groups interacted with FRB via hydrophobic interactions. These interactions enabled the formation of the FKBP12–WRX606–FRB ternary complex, which allosterically inhibited mTORC1, resulting in tumor growth suppression.

  1. In Silico and In Cell Hybrid Selection of Nonrapalog Ligands to Allosterically Inhibit the Kinase Activity of mTORC1 Raef Shams, Akihiro Matsukawa, Yukari Ochi, Yoshihiro Ito, and Hideyuki Miyatake Journal of Medicinal Chemistry Article ASAP DOI: 10.1021/acs.jmedchem.1c00536