Allosteric mutations impact the catalytic activity and oligomeric state of the main protease of coronavirus.

The COVID-19 pandemic has underscored the urgent need for effective antiviral strategies. A promising target is the 3CL protease of severe acute respiratory syndrome coronavirus 2, a crucial enzyme for viral replication. This study explores the structural and functional roles of specific amino acids essential for enzyme activation and dimerization, revealing potential allosteric sites as novel drug targets. Although current drug development efforts primarily focus on active site inhibitors that disrupt enzymatic function, the emergence of drug resistance necessitates alternative targets for inhibiting proteolytic activity. Our findings highlighted the comparative analysis between active (F140 and C145) and allosteric (N28 and R298) site residues, which play critical roles in enzymatic function. Mutations in these residues disrupt essential interactions hindering enzymatic activity and oligomerization as confirmed by in silico and in vitro analyses. Notably, these allosteric sites can be potential targets for modulating activity; where a single amino acid mutation disrupts enzyme activity and oligomeric form. Collectively, identifying inhibitors against these allosteric sites paves the way for precise and enduring antiviral drugs against SARS-CoV-2, as these sites offer enhanced specificity and reduced risk of drug resistance compared to the active site inhibitors.