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

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 (Mpro) 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 Mpro 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 Mpro 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 Mpro. The urgent need for novel antivirals extends beyond managing current COVID‐19 infections to preparing for future zoonotic outbreaks. The SARS‐CoV‐2 main protease (Mpro) is essential for viral replication and a validated antiviral target. Through optimization of a peptide inhibitor identified via mRNA display, a cyclic peptide with enhanced cell permeability and antiviral activity was developed.