Structural Basis for Coronaviral Main Proteases Inhibition by the 3CLpro Inhibitor GC376

The main protease (M^pro) of coronaviruses participates in viral replication, serving as a hot target for drug design. GC376 is able to effectively inhibit the activity of M^pro, which is due to nucleophilic addition of GC376 by binding covalently with Cys145 in M^pro active site. Here, we used fluorescence resonance energy transfer (FRET) assay to analyze the IC₅₀ values of GC376 against M^pros from six different coronaviruses (SARS-CoV-2, HCoV-229E, HCoV-HUK1, MERS-CoV, SARS-CoV, HCoV-NL63) and five M^pro mutants (G15S, M49I, K90R, P132H, S46F) from SARS-CoV-2 variants. The results showed that GC376 displays effective inhibition to various coronaviral M^pros and SARS-CoV-2 M^pro mutants. In addition, the crystal structures of SARS-CoV-2 M^pro (wide type)-GC376, SARS-CoV M^pro-GC376, MERS-CoV M^pro-GC376, and SARS-CoV-2 M^pro mutants (G15S, M49I, S46F, K90R, and P132H)-GC376 complexes were solved. We found that GC376 is able to fit into the active site of M^pros from different coronaviruses and different SARS-CoV-2 variants properly. Detailed structural analysis revealed key molecular determinants necessary for inhibition and illustrated the binding patterns of GC376 to these different M^pros. In conclusion, we not only proved the inhibitory activity of GC376 against different M^pros including SARS-CoV-2 M^pro mutants, but also revealed the molecular mechanism of inhibition by GC376, which will provide scientific guidance for the development of broad-spectrum drugs against SARS-CoV-2 as well as other coronaviruses.