Development of an RHEB-Targeting Peptide To Inhibit mTORC1 Kinase Activity

Abstract In cancer, the mechanistic/mammalian target of rapamycin complex-1 (mTORC1) is hyperactivated to promote survival under adverse conditions. The kinase activity of mTORC1 is activated by small-GTPase RHEB-GTP. Therefore, a new modality to inhibit mTORC1 activity has emerged, through intercepting RHEB. However, due to the relatively large contact area involved in the interaction between RHEB and mTORC1, facilitating this inhibition through small molecules has been challenging. Here, we report the development of a peptide that can inhibit the RHEB–mTORC1 interaction. The peptide, P1_WT, was designed based on the α-helix (aa 101–115) of the N-heat domain of mTOR to interact with switch II of RHEB. P1_WT bound to RHEB (KD = 0.14 μM) and inhibited RHEB-mTORN‑heat interaction (IC50 = 0.33 μM) in vitro. Consequently, P1_WT inhibited mTORC1 activity at a sub-micromolar level (IC50 ∼ 0.3 μM). P1_WT was predicted to be cell-permeable due to the rich content of arginine (23%), enhancing the intracellular translocation. These results show that P1_WT is a potential compound to further develop inhibitors for mTORC1 by intercepting RHEB from mTORC1.