In vitro selection of macrocyclic peptide inhibitors containing cyclic γ2,4-amino acids targeting the SARS-CoV-2 main protease
Takashi Miura, Tika R. Malla, David Owen, Anthony Tumber, Lennart Brewitz, M.A. McDonough, E. Salah, Naohiro Terasaka, Takayuki Katoh, Petra Lukacik, Claire Strain‐Damerell, Halina Mikolajek, Martin Walsh, Akane Kawamura, Christopher J. Schofield, Hiroaki Suga
Abstract
Abstract 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 (1 R ,3 S )-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.