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

Abstract

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.