Targeting the H/KRAS α4-β6-α5 Allosteric Lobe with Macrocyclic Peptides

Area of Science:

• Oncology
• Molecular Biology
• Drug Discovery

Background:

• Acquired resistance to RAS inhibitors is a significant challenge in cancer therapy.
• Existing inhibitors targeting Switch-II or using molecular glues are often circumvented by secondary RAS mutations.

Purpose of the Study:

• To design novel H/KRAS inhibitors that overcome resistance mechanisms.
• To explore alternative binding pockets and mechanisms for RAS inhibition.

Main Methods:

• Design and synthesis of 10-mer macrocyclic peptides mimicking the NS1 monobody FG-loop.
• Biochemical assays to assess binding affinity and inhibition of H/KRAS.
• NMR and X-ray crystallography to confirm the binding site.
• Development of covalent analogs targeting Cys118.

Main Results:

• Macrocyclic peptides effectively bind to H/KRAS, irrespective of nucleotide state or common oncogenic mutations (G12D, G12V, G13R, Q61K).
• The binding site was validated using NMR and X-ray crystallography.
• Covalent analogs demonstrated RAS labeling in vitro and in cell lysates.
• Key pharmacophores were identified as connectable for developing smaller inhibitors.

Conclusions:

• Macrocyclic peptides targeting the allosteric α4-α5-β6 surface represent a promising strategy against resistant RAS mutations.
• The identified pharmacophores provide a basis for developing next-generation, smaller allosteric H/KRAS inhibitors.