A phenothiazine urea derivative broadly inhibits coronavirus replication via viral protease inhibition

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 was identified. In silico docking studies also predicted the binding of the phenothiazine urea to the active sites of structurally similar M^pro and PL^pro proteases from distantly related alphacoronavirus, HCoV-229 E (229 E), and the betacoronavirus, HCoV-OC43 (OC43). The lead phenothiazine urea derivative displayed broad antiviral activity against all three HCoVs tested in cellulo. It was further demonstrated that the compound inhibited 229 E 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 readily inhibits viral replication and may broadly inhibit proteases of diverse coronaviruses.