Discovery of Novel Leucyl-tRNA Synthetase (LRS)-Targeted Mammalian Target of Rapamycin Complex 1 (mTORC1) Inhibitors

Abstract

Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that incorporates both intracellular and extracellular signals and plays a crucial role in cell metabolism, growth, proliferation and autophagy. mTOR exists in two structurally and functionally different multi-protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). In particular, overactive mTORC1 reported to be associated with various human diseases such as diabetes, neurodegeneration and cancers. Rapamycin and its analogs which are well known allosteric inhibitor of mTORC1, binds to the FK506-binding protein 12 (FKBP12) and interacts with the FKBP12-rapamycin binding (FRB) domain in mTORC1. Although rapamycin and its analogues have been developed for the use of anti-cancer agents, these agents have limitations that only partially inhibit mTORC1 activity and rapamycin alone is not sufficient to control the mTORC1 activity in many pathological conditions. Thus, small molecules targeting other possible regulators can offer alternative strategy to overcome rapamycin resistance in anti-cancer agents. Leucyl-tRNA synthetase (LRS) has been reported to be a possible mediator of intracellular amino acids signaling to mTORC1. LRS is a member of the class І aminoacyl-tRNA synthetase (ARSs) family that catalyzes the ATP-dependent ligation of leucine to cognate tRNA in protein biosynthesis. Recent studies indicate that LRS may act as a leucine sensor for the mTORC1 pathway. Given that mTORC1 is associated with cell proliferation and tumorigenesis, the LRS-mediated mTORC1 pathway may offer an alternative strategy in anticancer therapy. We have developed one of the leucinol analogs, (S)-4-isobutyloxazolidine-2-one as a LRS-targeted mTORC1 inhibitors. This compound inhibited downstream phosphorylation of mTORC1 by blocking leucine-sensing ability of LRS, without affecting the catalytic activity of LRS. In addition, it exhibited cytotoxicity against rapamycin-resistant colon cancer cells, suggesting that LRS has the potential to serve as a novel therapeutic target. Next, we developed leucyladenylate sulfamate derivatives as LRS-targeted mTORC1 inhibitors. We demonstrated that (S)-2-hydroxy-4-methylpentanoyl adenylate sulfamate selectively inhibited LRS-mediated mTORC1 activation and exerted specific cytotoxicity against colon cancer cells with a hyperactive mTORC1. Furthermore, we replaced the adenylate group with a N-(3,4-dimethoxybenzyl)benzenesulfonamide or a N-(2-phenoxyethyl)benzenesulfonamide groups that can maintain specific binding, but has more favorable physicochemical properties such as reduced polarity and asymmetric centers. Among these simplified analogues, we discovered that the compound and its constrained analogue effectively inhibited S6K phosphorylation in a dose-dependent manner and exhibited cancer cell specific cytotoxicity against six different types of cancer cells. In our continuing efforts to expand our in-house library of simplified leucyladenylates analogues, we decided to design new scaffolds based on the gefitinib structure. We devised new series of simplified structures by introducing N-(3-chloro-4-fluorophenyl) quinazolin-4-amine instead of adenine group and various linker structures to replace 5-O-sulfamoylribose. We demonstrated that compounds with sulfonamide, ethyl alcohol or ethyl amino linker in new series of simplified adenylate structure showed potent inhibition. Furthermore, these compounds showed general cytotoxicity against various types of cancer cell lines, suggesting that they have a potential as effective anticancer agent.