Cys44 of SARS-CoV-2 3CL^pro affects its catalytic activity

SARS-CoV-2 encodes a 3C-like protease (3CL^pro) that is essential for viral replication. This cysteine protease cleaves viral polyproteins to release functional nonstructural proteins, making it a prime target for antiviral drug development. We investigated the inhibitory effects of halicin, a known c-Jun N-terminal kinase inhibitor, on 3CL^pro. Mass spectrometry and crystallographic analysis revealed that halicin covalently binds to several cysteine residues in 3CL^pro. As expected, Cys145, the catalytic residue, was found to be the most targeted residue by halicin. Secondly, Cys44 was found to be modified, suggesting a potential inhibitory role of this residue. A mutant protease (Cys44Ala) was generated to further understand the function of Cys44. In silico and enzymatic assays showed that the mutation significantly reduced the stability and activity of 3CL^pro, indicating the importance of Cys44 in maintaining the active conformation of the protease. Differential scanning fluorimetry assays confirmed this evidence, showing a reduced thermal stability of the mutant compared to the wild-type protease. Our results highlight the potential of halicin as a multi-target inhibitor of 3CL^pro and underline the importance of Cys44 in the function of the protease. These findings contribute to the development of effective antiviral therapies against COVID-19 by targeting critical residues in 3CL^pro.