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

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 (γ(1)) and (1R,3S)-3-aminocyclopentane carboxylic acid (γ(2)), 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 γ(1) 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 γ(1) 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. [Image: see text]