The COVID-19 pandemic is a public health and economic disaster. The viral main protease (M^pro) is essential for SARS-CoV-2 replication and thus is a compelling target for small-molecule antivirals. Computational docking and structure-guided drug-design strategies rely on atomic scale understanding of the active site traditionally derived from X-ray crystallographic data collected at cryogenic temperatures. This technique is limited by cryo-artifacts and an inability to locate the functional hydrogen atoms in enzyme active sites. Neutrons are an ideal probe to observe the protonation states of ionizable amino acids at near-physiological temperature, such as the non-canonical Cys-His catalytic dyad of M^pro. Our studies using neutron diffraction produced crucial insights for inhibitor design and clinical drug repurposing efforts.

X-ray crystal structures of M^pro collected at near-physiological temperatures brought rapid insights into the reactivity of the catalytic cysteine, malleability of the active site, and binding modes with clinical protease inhibitors. Structural plasticity of the ligand binding site observed in room-temperature M^pro suggests the model is relevant for supercomputer docking studies. The catalytic cysteine can be trapped in the rare peroxysulfenic acid oxidation state at physiological pH signifying high reactivity. Three clinical HCV antivirals are low-micromolar inhibitors of SARS-CoV-2 M^pro by binding to the active site cavity where active site malleability enables adaptation to the size of inhibitor moieties.

We determined the SARS-CoV-2 M^pro neutron structure to provide direct observation of protonation states of a catalytic site in a cysteine protease for the first time. At rest, the catalytic Cys-His dyad of M^pro exists in the reactive zwitterionic state instead of the anticipated neutral dyad state. This research is providing real-time data for atomistic design of M^pro inhibitors to combat the current COVID-19 pandemic and prepare for future pathogenic coronavirus threats.

Kevin Weiss

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