Discovery of a nasal spray steroid, tixocortol, as an inhibitor of SARS-CoV-2 main protease and viral replication.

Coronaviruses rely on the viral-encoded chymotrypsin-like main protease (M^pro or 3CL^pro) for replication and assembly. Our previous research on M^pro of SARS-CoV-2 identified cysteine 300 (Cys300) as a potential allosteric site of M^pro inhibition. Here, we identified tixocortol (TX) as a covalent modifier of Cys300 which inhibits M^pro activity in vitro as well as in a cell-based M^pro expression assay. Most importantly TX inhibited SARS-CoV-2 replication in ACE2 expressing HeLa cells. Biochemical analysis and kinetic assays were consistent with TX acting as a non-competitive inhibitor. By contrast, TX was a weaker inhibitor and modifier of C300S M^pro, confirming a role for Cys300 in inhibition of WT M^pro but also providing evidence for an additional Cys target. TX pivalate (TP), a prodrug for TX that was previously marketed as a nasal spray, also inhibited SARS-CoV-2 replication in HeLa-ACE2 cells at low micromolar IC₅₀s. These studies suggest that TX and/or TP could possibly be repurposed for the prevention and/or treatment of SARS-CoV-2 infection.