In Vitro Selection of Macrocyclic α/β(3)-Peptides against Human EGFR

[Image: see text] Here, we report ribosomal construction of thioether-macrocyclic α/β(3)-peptide libraries in which β-homoglycine, β-homoalanine, β-homophenylglycine, and β-homoglutamine are introduced by genetic code reprogramming. The libraries were applied to the RaPID (Random nonstandard Peptides Integrated Discovery) selection against human EGFR to obtain PPI (protein–protein interaction) inhibitors. The resulting peptides contained up to five β(3)-amino acid (β(3)AA) residues and exhibited outstanding binding affinity, PPI inhibitory activity, and proteolytic stability, which were attributed to the β(3)AAs included in the peptides. This showcase work has demonstrated that the use of such β(3)AAs enhances the drug-like properties of peptides, providing a unique platform for the discovery of de novo macrocycles against a protein of interest.