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.