Dual inhibition of coronavirus M^proand PL^proenzymes by phenothiazines and their antiviral activity

ABSTRACT Coronavirus (CoV) replication requires efficient cleavage of viral polyproteins into an array of non-structural proteins involved in viral replication, organelle formation, viral RNA synthesis, and host shutoff. Human CoVs (HCoVs) encode two viral cysteine proteases, main protease (M pro ) and papain-like protease (PL pro ), that mediate polyprotein cleavage. Using a structure-guided approach, a phenothiazine urea derivative that inhibits both SARS-CoV-2 M pro and PL pro protease activity in vitro was identified. In silico docking studies also predicted binding of the phenothiazine to the active sites of M pro and PL pro from distantly related alphacoronavirus, HCoV-229E (229E) and the betacoronavirus, HCoV-OC43 (OC43). The lead phenothiazine urea derivative displayed broad antiviral activity against all three HCoVs tested in cell culture infection models. It was further demonstrated that the compound inhibited 229E and OC43 at an early stage of viral replication, with diminished formation of viral replication organelles and the RNAs that are made within them, as expected following viral protease inhibition. These observations suggest that the phenothiazine urea derivative inhibits viral replication and may broadly inhibit proteases of diverse coronaviruses. Graphical Abstract Highlights Coronavirus cysteine proteases M pro and PL pro are targets for novel antiviral agents Phenothiazine ureas inhibit SARS-CoV-2 M pro and PL pro protease activity Some phenothiazine ureas inhibit replication of diverse coronaviruses with minimal cytotoxicity Phenothiazine ureas inhibit early stages of coronavirus replication consistent with failure of viral polyprotein cleavage