Remote Residue Substitution and Macrocyclization Preorganize a β‐Hairpin Peptide and Enhance β‐Catenin Binding

Abstract

Peptide‐based inhibitors are promising tools to target challenging protein interfaces, yet improving their affinity and drug‐like properties is still a major hurdle. This holds particularly true for β‐sheet motifs with only limited strategies available to robustly stabilize their conformation. Previously, we reported linear β‐hairpin peptide l12 with micromolar affinity for β‐catenin, a central effector of aberrant Wnt signaling in cancer. Here, we show that a single N4V substitution markedly enhances binding, despite not directly contacting the protein surface as evidenced by obtained X‐ray structures and molecular dynamics simulations. Notably, the substitution combines additively with head‐to‐tail cyclization yielding a peptide with strongly increased affinity. This mechanistic study presents structural and computational analyses revealing that the affinity improvements arise from solution‐state preorganization and β‐hairpin stabilization mediated by an altered intramolecular hydrogen bond network. Our findings highlight the critical role of conformational preorganization in peptide‐protein recognition, demonstrating that optimizing solution‐state properties can unlock substantial affinity gains that are not obvious from crystal structures alone.