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

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 IC₅₀ value of 1.44 mM. We observed that N8-A24 reduced M^pro dimerization with an IC₅₀ 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.