Discovery and characterization of naturally occurring covalent inhibitors of SARS-CoV-2 M^pro from the antiviral herb Ephedra

The Chinese herb Ephedra (also known as Mahuang) has been extensively utilized for the prevention and treatment of coronavirus-induced diseases, including coronavirus disease 2019 (COVID-19). However, the specific anti-SARS-CoV-2 compounds and mechanisms have not been fully elucidated. The main protease (M^pro) of SARS-CoV-2 is a highly conserved enzyme responsible for proteolytic processing during the viral life cycle, making it a critical target for the development of antiviral therapies. This study aimed to identify naturally occurring covalent inhibitors of SARS-CoV-2 M^pro from Ephedra and to investigate their covalent binding sites. The results demonstrated that the non-alkaloid fraction of Ephedra (ENA) exhibited a potent inhibitory effect against the SARS-CoV-2 M^pro effect, whereas the alkaloid fraction did not. Subsequently, the chemical constituents in ENA were identified, and the major constituents’ anti-SARS-CoV-2 M^pro effects were evaluated. Among the tested constituents, herbacetin (HE) and gallic acid (GA) were found to inhibit SARS-CoV-2 M^pro in a time- and dose-dependent manner. Their combination displayed a significant synergistic effect on this key enzyme. Additionally, various techniques, including inhibition kinetic assays, chemoproteomic methods, and molecular dynamics simulations, were employed to further elucidate the synergistic anti-M^pro mechanisms of the combination of HE and GA. Overall, this study deciphers the naturally occurring covalent inhibitors of SARS-CoV-2 M^pro from Ephedra and characterizes their synergistic anti-M^pro synergistic effect, providing robust evidence to support the anti-coronavirus efficacy of Ephedra.