Disulfiram inhibits coronaviral main protease by conjugating to its substrate entry site

Coronaviruses (CoV) are highly pathogenic single-strand RNA viruses. CoV infections cause fatal respiratory symptoms and lung injuries in humans and significant economic losses in livestock. Since the SARS-2 outbreak in 2019, the highly conserved main protease (M^pro), also termed 3-chymotrypsin-like protease (3CL^pro), has been considered an attractive drug target for treating CoV infections. M^pro mediates the proteolytic cleavage of eleven sites in viral polypeptides necessary for virus replication. Here, we report that disulfiram, an FDA-approved drug for alcoholic treatment, exhibits a broad-spectrum inhibitory effect on CoV M^pros. Analytical ultracentrifugation and circular dichroism analyses indicated that disulfiram treatment blocks the dimeric formation of SARS and PEDV M^pros and decreases the thermostability of SARS, SARS-2, and PEDV M^pros, whereas it facilitates the dimerization and stability of MERS M^pro. Furthermore, mass spectrometry and structural alignment revealed that disulfiram targets the Cys44 residue of M^pros, which is located at the substrate entrance and close to the catalytic His41. In addition, molecular docking analysis suggests that disulfiram conjugation interferes with substrate entry to the catalytic center. In agreement, mutation of Cys44 modulates the disulfiram sensitivity of CoV M^pros. Our study suggests a broad-spectrum inhibitory function of disulfiram against CoV M^pros.