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

SARS-CoV-2 encodes a 3C-like protease (3CL) 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. Mass spectrometry and crystallographic analysis revealed that halicin covalently binds to several cysteine residues in 3CL. 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, 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 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.