Allosteric Regulation and Inhibition of Coronavirus 3CLpro Revealed by HDX‐MS

Abstract Coronavirus (CoV) infections have caused contagious and fatal respiratory diseases in humans worldwide. CoV 3‐chymotrypsin‐like proteases (3CLpro or Mpro) play an important role in viral maturation, and maintenance of their dimeric conformation is crucial for viral activity. Therefore, allosterically regulated dimerization of 3CLpro can be employed as a drug development target. Here, we investigated the allosteric regulatory mechanism of 3CLpro dimerization by using hydrogen/deuterium exchange coupled with mass spectrometry (HDX‐MS) technology. We found that the FLAG tag directly coupled to the N‐finger of 3CLpro significantly increased HDX kinetics at the dimer interface, and 3CLpro transformed from a dimer to a monomer. The 3CLpro mutants of SARS‐CoV‐2, which are monomeric, also exhibited increased deuterium exchange. Binding of the allosteric inhibitor Gastrodenol to most betacoronavirus 3CLpros led to increased allosteric deuterium exchange, resulting in the monomeric conformation of the CoV 3CLpro upon binding. Molecular dynamics (MD) simulation analysis further indicated the molecular mechanism of action of Gastrodenol on CoV 3CLpro: binding of Gastrodenol to SARS‐CoV‐2 3CLpro destroyed the hydrogen bond in the dimer interface. These results suggest that Gastrodenol may be a potential broad‐spectrum anti‐betacoronavirus drug.