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.
(© 2023. The Author(s).)