Journal Article

In vitro selection of macrocyclic peptide inhibitors containing cyclic γ2,4-amino acids targeting the SARS-CoV-2 main protease

Terasaka, Naohiro, Suga, Hiroaki, Miura, Takashi, Schofield, Christopher J, Lukacik, Petra, Walsh, Martin A, Mikolajek, Halina, Owen, C. David, Strain-Damerell, Claire, Malla, Tika R, Salah, Eidarus, Katoh, Takayuki, Kawamura, Akane, Brewitz, Lennart, McDonough, Michael A, Tumber, Anthony

2023

Overview

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 γ2,4-amino acids resulted in the discovery of very potent inhibitors of the SARS-CoV-2 main protease (Mpro). Two kinds of cyclic γ2,4-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 Mpro 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 Mpro: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 Mpro enabled production of a variant with a 5-fold increase in potency.In vitro screening of a ribosomally synthesized macrocyclic peptide library containing cyclic γ2,4-amino acids (cγAA) afforded the discovery of potent inhibitors of the SARS-CoV-2 main protease (Mpro). A co-crystal structure revealed the contribution of this cγAA to Mpro binding and the proteolytic stability of these macrocycles.

• Publisher: Nature Publishing Group
• Subject: Alanine / Amino acids / Binding / Carboxylic acids / COVID-19 / Crystal structure / Inhibitors / Libraries / Natural products / Peptide inhibitors / Peptides / Protease / Proteinase / Proteolysis / Severe acute respiratory syndrome coronavirus 2 / Stability / Substrate inhibition