Johansen-Leete, JasonUllrich, SvenFry, SarahFrkic, RebeccaBedding, Max J.Aggarwal, AnupriyaAshhurst, AnnelieseEkanayake, KasuniMahawaththa, MithunMini Sasi, VishnuLuedtke, StephanieFord, DanielO'Donoghue, AnthonyPassioura, TobyLarance, MarkOtting, GottfriedTurville, StuartJackson, ColinNitsche, ChristophPayne, Richard2022-08-022022-08-022041-6520http://hdl.handle.net/1885/270118Antivirals that specifically target SARS-CoV-2 are needed to control the COVID-19 pandemic. The main protease (Mpro) 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 Mpro dimer, which yielded several high-affinity thioether-linked cyclic peptide inhibitors of the protease. Structural analysis of Mpro 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 Mpro 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.We acknowledge the ARC Centre of Excellence for Innovations in Peptide and Protein Science (CE200100012 to GO, CJJ and RJP) for funding and the John A. Lamberton Research Scholarship (to JJ-L) and Research Training Program from the Australian government (to JJ-L, SEF and MJB) for PhD scholarships. CN acknowledges ARC funding (DE190100015, DP200100348).application/pdfen-AU© 2022 The Author(s). Published by the Royal Society of Chemistryhttps://creativecommons.org/licenses/by-nc/3.0/202210.1039/D1SC06750HCreative Commons Attribution-NonCommercial 3.0 Unported Licence