Kien Tran, Hugo Lavoie, Amal Wahhab, Damien Garrido, Chang Hwa Jo, Marc-André Poupart, T. Arya, Alexandre Beautrait, Ryan C. Killoran, Cédric Dicaire-Leduc, Éric Bonneil, Michael J. Osborne, Doris A. Schuetz, Faraz Shaikh, Pierre Thibault, Matthew J. Smith, Anne Marinier, Marc Therrien
Despite therapeutic advances against RAS mutations in cancer, acquired resistance frequently arises. Several secondary mutations at the binding sites effectively confer resistance to both Switch-II inhibitors and cyclophilin-A molecular glues. This underscores the need for RAS inhibitors that engage alternative binding pockets or operate through novel mechanisms. Here, we report the design of 10-mer macrocyclic peptides that mimic the FG-loop of the NS1 monobody, which targets the allosteric α4-α5-β6 surface of H/KRAS to disrupt RAS clustering and downstream signaling. These noncovalent inhibitors bind to H/KRAS with equivalent potencies, regardless of nucleotide state or the presence of oncogenic mutations (G12D, G12V, G13R, Q61K), and their binding site was confirmed by NMR and X-ray crystallography. Furthermore, covalent analogs targeting Cys118 were shown to label RAS in vitro and in complete cell lysates. Finally, we demonstrated that the key pharmacophores are connectable, providing a foundation for the development of smaller allosteric H/KRAS inhibitors.