Leucine (Leu), an essential amino acid, is known to stimulate protein synthesis in the skeletal muscle via mTOR complex 1 (mTORC1) activation. However, the intrinsic contribution of other amino acids to Leu-mediated activation of mTORC1 signaling remains unexplored. This study aimed to identify amino acids that can promote mTORC1 activity in combination with Leu and to assess the effectiveness of these combinations in vitro and in vivo. We found that tyrosine (Tyr) enhanced Leu-induced phosphorylation of S6 kinase (S6K), an indicator of mTORC1 activity, although it exerted no such effect individually. This booster effect was observed in C2C12 cells, isolated murine muscle, and the skeletal muscles of mice orally administered the amino acids. To explore the molecular mechanisms underlying this Tyr-mediated booster effect, the expression of the intracellular Leu sensors, Sestrin1 and 2, was suppressed, and the cells were treated with Leu and Tyr. This suppression enabled Tyr alone to induce S6K phosphorylation and enhanced the booster effect, suggesting that Tyr possibly contributes to mTORC1 activation when Sestrin-GAP activity toward Rags 2 (GATOR2) is dissociated through Sestrin knockdown or the binding of Sestrins to Leu. Collectively, these results indicate that Tyr is a key regulator of Leu-mediated protein synthesis.

Background: Antibody–drug conjugates (ADCs) have emerged as an important class of targeted anticancer therapeutics; however, their efficacy can be limited by intrinsic or acquired resistance to their cytotoxic payloads. Pharmacological potentiation of payload activity may therefore provide a complementary strategy for improving ADC efficacy. Nobiletin, a polymethoxyflavone derived from citrus peel, has been reported to enhance the cytotoxic effects of microtubule-targeting agents, but its poor aqueous solubility and low bioavailability restrict its in vivo application. Objective: In this study, we investigated whether nobiletin and its bioavailability-enhanced formulation, Nob-MeHes ASD, an amorphous solid dispersion of nobiletin and methyl hesperidin, could potentiate the activity of the microtubule inhibitor DM1 and a DM1-containing ADC. Methods: The effects of nobiletin and Nob-MeHes ASD on DM1- and αEpCAM-DM1-induced cytotoxicity were evaluated using cell viability assays, cell cycle analysis, and mouse xenograft models. Findings: Nobiletin enhanced DM1-induced cytotoxicity in DLD1 colorectal cancer and BxPC-3 pancreatic cancer cells and increased the proportions of cells in the G2/M and Sub-G1 populations. Nob-MeHes ASD retained this potentiating activity in vitro and, when combined with DM1, significantly suppressed tumor growth in a BxPC-3 xenograft model. Nobiletin also enhanced the cytotoxic activity of αEpCAM-DM1, an EpCAM-targeting DM1-conjugated ADC, in EpCAM-positive cancer cells, although potentiation was also observed in HUVEC. In contrast, neither αEpCAM-DM1 alone nor its combination with nobiletin showed appreciable cytotoxicity in EpCAM-knockout DLD1 cells. Furthermore, Nob-MeHes ASD potentiated αEpCAM-DM1 activity in vitro and enhanced tumor growth suppression in a DLD1 xenograft model. Although changes in serum biochemical parameters were observed, further studies are required to establish the safety of the combination. Conclusions: These findings provide proof of concept that a bioavailability-enhanced phytochemical may serve as a pharmacological potentiator of DM1-containing ADC therapy, supporting further investigation of this combination strategy.