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

Abstract

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.

Keywords

Main protease / Ephedra / Ephedra non-alkaloid fraction / Covalent inhibitors / Synergistic effect

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Funding

National Key Research and Development Program of China(2022YFC3502000)

Basic Public Welfare Research Program of Zhejiang Province(LGF22H280012)

Zhejiang Provincial TCM Science and Technology Plan Project(2023ZR064)

Zhejiang Provincial TCM Science and Technology Plan Project(GZY-ZJ-KJ-24004)

Zhejiang Provincial TCM Science and Technology Plan Project(2024ZL007)

Zhejiang Provincial TCM Science and Technology Plan Project(2022ZB017)

Medical Science and Technology Project of Zhejiang Province(2022495401)

Medical Science and Technology Project of Zhejiang Province(2021KY040)

Medical Science and Technology Project of Zhejiang Province(2022KY069)

Zhejiang Provincial Key Projects in Chinese Medicine(2020ZZ003)

Zhejiang Provincial Key Projects in Chinese Medicine(2021ZZ001)

Shanghai Science and Technology Innovation Action Plans(21S21900600)

Zhejiang Province "Ten Thousand People Plan" Science and Technology Innovation Leading Talents Project(2020R52029)