Design and Evaluation of Peptide Inhibitors Targeting the Dimerization of SARS‐CoV‐2 Main Protease

Abstract The severe acute respiratory syndrome virus 2 (SARS‐CoV‐2) seriously impacted public health. The evolutionarily conserved viral chymotrypsin‐like main protease (M pro ) is an important target for anti‐SARS‐CoV‐2 drug development. Previous studies have shown that the eight N‐terminal amino acids (N8) of SARS‐CoV M pro are essential for its dimerization, and are used to design inhibitors against SARS‐CoV M pro dimerization. Here, we established a simple readout assay using SDS‐PAGE and Coomassie blue staining to measure inhibitory activity of N8 peptide derived from SARS‐CoV‐2 M pro . To optimize its inhibitory effect, we then modified the side‐chain length, charge, and hydrophilicity of the N8 peptide, and introduced a mutated M pro recognition sequence. As a result, we obtained a series of potent peptide inhibitors against SARS‐CoV‐2 M pro , with N8‐A24 being the most efficient with an IC 50 value of 1.44 mM. We observed that N8‐A24 reduced M pro dimerization with an IC 50 value of 0.86 mM. Molecular docking revealed that N8‐A24 formed hydrogen bond interactions with critical dimeric interface residues, thus inhibiting its dimerization and activity. In conclusion, our study not only discovers a series of peptide inhibitors targeting the SARS‐CoV‐2 M pro dimerization, but also provides a promising strategy for the rational design of new inhibitors against COVID‐19.