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

Area of Science:

• Biochemistry
• Molecular Biology
• Cancer Research

Background:

• Mechanistic/mammalian target of rapamycin complex-1 (mTORC1) is hyperactivated in cancer, promoting cell survival.
• mTORC1 activity is regulated by the small-GTPase RHEB-GTP, making it a target for inhibition.
• Inhibiting the RHEB-mTORC1 interaction is challenging for small molecules due to the large interface.

Purpose of the Study:

• To develop a novel peptide-based inhibitor targeting the RHEB-mTORC1 interaction.
• To assess the efficacy of the designed peptide in inhibiting mTORC1 activity.

Main Methods:

• Peptide design based on the mTOR N-heat domain's α-helix.
• In vitro binding assays to determine RHEB-peptide affinity (K_D).
• Inhibition assays to measure the peptide's effect on RHEB-mTOR^N-heat interaction (IC₅₀) and mTORC1 activity.

Main Results:

• The peptide P1_WT demonstrated binding to RHEB with a K_D of 0.14 μM.
• P1_WT inhibited the RHEB-mTOR^N-heat interaction with an IC₅₀ of 0.33 μM.
• P1_WT effectively inhibited mTORC1 activity at a sub-micromolar level (IC₅₀ ≈ 0.3 μM) and is predicted to be cell-permeable.

Conclusions:

• P1_WT is a potent inhibitor of the RHEB-mTORC1 interaction.
• The peptide shows promise as a lead compound for developing new mTORC1-targeting cancer therapeutics.
• Targeting RHEB offers a viable strategy for inhibiting hyperactivated mTORC1 signaling in cancer.