Exploiting Hydrophobic Amino Acid Scanning to Develop Cyclic Peptide Inhibitors of the SARS‐CoV‐2 Main Protease with Antiviral Activity

Abstract The development of novel antivirals is crucial not only for managing current COVID‐19 infections but for addressing potential future zoonotic outbreaks. SARS‐CoV‐2 main protease (M pro ) is vital for viral replication and viability and therefore serves as an attractive target for antiviral intervention. Herein, we report the optimization of a cyclic peptide inhibitor that emerged from an mRNA display selection against the SARS‐CoV‐2 M pro to enhance its cell permeability and in vitro antiviral activity. By identifying mutation‐tolerant amino acid residues within the peptide sequence, we describe the development of a second‐generation M pro inhibitor bearing five cyclohexylalanine residues. This cyclic peptide analogue exhibited significantly improved cell permeability and antiviral activity compared to the parent peptide. This approach highlights the importance of optimizing cyclic peptide hits for activity against intracellular targets such as the SARS‐CoV‐2 M pro .