Exploiting Bridged Conformations for Precise Molecular Recognition in the Design of KRAS-Selective, Orally Available Macrocyclic Peptides

Abstract Macrocyclic peptides are attracting attention as a promising therapeutic modality for addressing intracellular targets that are traditionally difficult to address with conventional small molecules or antibodies. In this study, we present the chemical optimization process behind the development of AUBE00 (AP7400, compound 30), a KRAS-selective inhibitor based on a scaffold similar to LUNA18, an orally bioavailable macrocyclic peptide RAS inhibitor. Achieving KRAS isoform selectivity is exceptionally challenging due to the nearly identical backbone structures of KRAS, HRAS, and NRAS (Cα RMSD < 1.1 Å). The success of this study relied on two critical factors: (1) achieving conformational control of the flexible N-alkyl group through a bridging structure linked to the adjacent side chain, which enabled the exploitation of subtle interaction energy differences among the isoforms; and (2) overcoming the trade-off between improved KRAS selectivity and reduced permeability associated with P-glycoprotein (P-gp) substrate liability by employing a modified Caco-2 membrane permeability assay to recover oral bioavailability. Our results demonstrate that a conformational rigidification through a bridged scaffold, implemented while preserving membrane permeability, is a highly effective strategy for achieving isoform selectivity. This study establishes a generalizable design principle for orally bioavailable macrocyclic peptides targeting intracellular proteins that require mutation or isoform selectivity.