In vitro selection of macrocyclic peptide inhibitors containing cyclic γ^2,4-amino acids targeting the SARS-CoV-2 main...

γ-Amino acids can play important roles in the biological activities of natural products; however, the ribosomal incorporation of γ-amino acids into peptides is challenging. Here we report how a selection campaign employing a non-canonical peptide library containing cyclic γ^2,4-amino acids resulted in the discovery of very potent inhibitors of the SARS-CoV-2 main protease (M^pro). Two kinds of cyclic γ^2,4-amino acids, cis-3-aminocyclobutane carboxylic acid (γ¹) and (1R,3S)-3-aminocyclopentane carboxylic acid (γ²), were ribosomally introduced into a library of thioether-macrocyclic peptides. One resultant potent M^pro inhibitor (half-maximal inhibitory concentration = 50 nM), GM4, comprising 13 residues with γ¹ at the fourth position, manifests a 5.2 nM dissociation constant. An M^pro:GM4 complex crystal structure reveals the intact inhibitor spans the substrate binding cleft. The γ¹ interacts with the S1' catalytic subsite and contributes to a 12-fold increase in proteolytic stability compared to its alanine-substituted variant. Knowledge of interactions between GM4 and M^pro enabled production of a variant with a 5-fold increase in potency.