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

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.