A naturally occurring G11S mutation in the 3C-like protease from the SARS-CoV-2 virus dramatically weakens the dimer interface.

The 3C-like protease (3CLpro ) is crucial to the replication of SARS-CoV-2, the causative agent of COVID-19, and is the target of several successful drugs including Paxlovid and Xocova. Nevertheless, the emergence of viral resistance underlines the need for alternative drug strategies. 3CLpro only functions as a homodimer, making the protein-protein interface an attractive drug target. Dimerization is partly mediated by a conserved glycine at position 11. However, some naturally occurring SARS-CoV-2 sequences contain a serine at this position, potentially disrupting the dimer. We have used concentration-dependent activity assays and mass spectrometry to show that indeed the G11S mutation reduces the stability of the dimer by 600-fold. This helps to set a quantitative benchmark for the minimum potency required of any future protein-protein interaction inhibitors targeting 3CLpro and raises interesting questions regarding how coronaviruses bearing such weakly dimerizing 3CLpro enzymes are capable of replication.