A Discovery Of A Small Molecule Mtorc1 Allosteric Inhibitor Targeting Cancer Cells

The allosteric inhibition of the mechanistic target of rapamycin complex 1 (mTORC1), the central growth regulator that is highly activated in cancer cells, is unique for rapamycin and rapalogs. Upon binding to 12-kDa FK506-binding protein (FKBP12), the FKBP12-rapamycin specifically complexed with mTORC1 and inhibits the mediated downstream signals incorporated in metabolic processes. Since rapamycin has a large molecular weight and a poor solubility, it does not completely inhibit mTORC1 due to the pharmacokinetic limitations. It just shows a cytostatic effect in the treatment of a few benign cancers which results in tumors re-growth. In order to overcome these limitations, we have looked for a novel potential small molecule mTORC1 allosteric inhibitor by in silico and in vitro methods. Firstly, we carried out the in silico selection using Virtual Ligand Screening (VLS) for 105 compounds to select ligands with the high calculated scores toward FRB/FKBP12 interface as a receptor (PDB ID: 1fap). After further score calculations, we selected the top 5 compounds for the next selection step. We also prepared FKBP-rapamycin binding domain (FRB) of the mTORC1 and FKBP12, by using pET15b expression vector and BL21(DE3) E. coli. For the molecular-level protein: protein interaction (PPI) measurement, we used AlphaLISATM system (PerkinElmer, USA) using the prepared proteins. In addition, we checked the kinase activity using AlphaLISA® p-P70 S6K (T389) assay kit (PerkinElmer, USA), as well as Western blotting against p-S6 (S235/236) and p-4E-BP1 (T37/46). Finally, we performed the cell cytotoxicity assay of the selected compound against cancer and noncancer cell lines. The docking simulations showed that the residues involved in the interaction between FRB/FKBP12 binding site and rapamycin are partially overlapped with those of the screened compounds which stabilized the formation of FRB:Ligand:FKBP12 ternary complex. We also measured the inhibitory effect of the selected compounds against the ternary complex formation by rapamycin and two of them, ZNC1 and ZNC5, showed interactivity by IC50 of 30 nM and 41 nM, respectively. On the other hand, only ZNC5 promoted the formation of the ternary complex by a KD value of 1.7 nM. In addition, ZNC5 inhibited the kinase activity not only in the starvation condition by IC50 of 1.2 nM but also in the feeding-induced mTORC1 activation condition with IC50 of 3.5 nM. Moreover, it has been observed that the ZNC5 inhibited kinase activity of mTORC1 in the cancer cells while no effect on the normal cells. In this study, we discovered the ZNC5, a new allosteric inhibitor to the kinase of mTORC1, through the in silico and in vitro hybrid selection. The smaller size of ZNC5 comparing to rapamycin is probably advantageous for better pharmacokinetics in vivo. Most importantly, ZNC5 specifically inhibited the growth of cancer cells over normal cells, while no such specificity observed by rapamycin. Citation Format: Raef Shams, Yoshihiro Ito, Hideyuki Miyatake. A discovery of a small molecule mTORC1 allosteric inhibitor targeting cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 662.