Discovery of Antiviral Cyclic Peptides Targeting the Main Protease of SARS-CoV-2 via mRNA Display

Antivirals that specifically target SARS-CoV-2 are needed to control the COVID-19 pandemic. The main protease (M^pro) is essential for SARS-CoV-2 replication and is an attractive target for antiviral development. Here we report the use of the Random nonstandard Peptide Integrated Discovery (RaPID) mRNA display on a chemically cross-linked SARS-CoV-2 M^pro dimer, which yielded several high-affinity thioether-linked cyclic peptide inhibitors of the protease. Structural analysis of M^pro complexed with a selenoether analogue of the highest-affinity peptide revealed key binding interactions, including glutamine and leucine residues in sites S₁ and S₂, respectively, and a binding epitope straddling both protein chains in the physiological dimer. Several of these M^pro peptide inhibitors possessed antiviral activity against SARS-CoV-2 in vitro with EC₅₀ values in the low micromolar range. These cyclic peptides serve as a foundation for the development of much needed antivirals that specifically target SARS-CoV-2.