Mirror-image mRNA display uncovers isoform-selective D-peptide macrocycles targeting a cryptic KRAS pocket

SUMMARY Activating KRAS mutations drive millions of cancers diagnosed worldwide, 1 yet for decades this oncoprotein was deemed “undruggable”, reflecting the challenge of discovering molecules capable of perturbing its complex biological functions, and of translating these discoveries into effective cancer therapeutics. 2 Recent advances propelled by innovative screening have identified diverse modalities that bind at or near the switch-II pocket (SII-P) of RAS proteins, including molecular glues, 3 macrocyclic peptides, 4 fragment-derived small molecules, 5 and approved therapies that covalently target KRAS G12C . 6,7 Unfortunately, resistance to approved therapies has emerged, 8,9 highlighting the need for molecules that engage new or underexploited binding sites on RAS oncoproteins with mechanisms complementary to established SII-P inhibitors. 10,11 Here we show that mirror-image mRNA display 12 enabled the discovery of all-D macrocyclic peptide ligands targeting a cryptic RAS back pocket (CRB-P). 13 These ligands engage KRAS(OFF) and KRAS(ON) with equal affinity, exploit a single-residue difference among isoforms to bind KRAS selectively, and successfully inhibit oncogenic signaling in KRAS-mutant cells through a mechanism distinct from SII-P binders. Mirror-image screening directly afforded nanomolar peptide ligands stable toward cellular proteolysis and delivered probes targeting distinct epitopes not accessible by homochiral peptide-display methods. Together, these findings establish the CRB-P as a specifically druggable and mechanistically differentiated site on KRAS with potential for combination with emerging RAS-targeting therapies and substantiate mirror-image mRNA display as a strategy for discovering stable all-D macrocyclic peptides targeting previously inaccessible epitopes on challenging targets.