Discovery and characterization of the covalent SARS-CoV-2 3CL pro inhibitors from Ginkgo biloba extract via integrating chemoproteomic and biochemical approaches.

Background: The 3C-like proteases (3CL ^pro s) are cysteine-rich homodimeric proteins and can be covalently modified by numerous natural and synthetic compounds, which in turn, block the proteolytic activity or the formation of enzymatically active dimeric forms. Although herbal medicines have been widely used to treat COVID-19, identification of the key herbal constituents that can covalently modify the 3CL ^pro s in β-coronaviruses (CoVs) remains a big challenge.
Aims: To construct a comprehensive approach for efficient discovering the covalent SARS-CoV-2 3CL ^pro inhibitors from herbal medicines. To decipher the key anti-SARS-CoV-2 3CL ^pro constituents in Ginkgo biloba extract 50 (GBE50) and to study their anti-SARS-CoV-2 3CL ^pro mechanisms.
Methods: SARS-CoV-2 3CL ^pro inhibition assay including time-dependent inhibition assays and inactivation kinetic analyses were conducted using a fluorescence-based biochemical assay. The constituents in GBE50 were analyzed by UHPLC-Q-Exactive Orbitrap HRMS. The peptides modified by herbal constituents were characterized by using nanoLC-MS/MS.
Results: Following testing the anti-SARS-CoV-2 3CL ^pro effects of 104 herbal medicines, it was found that Ginkgo biloba extract 50 (GBE50) potently inhibited SARS-CoV-2 3CL ^pro in dose- and time-dependent manners. A total of 38 constituents were identified from GBE50 by UHPLC-Q-Exactive Orbitrap HRMS, while 26 peptides modified by 18 constituents were identified by chemoproteomic profiling. The anti-SARS-CoV-2 3CL ^pro effects of 18 identified covalent inhibitors were then validated by performing time-dependent inhibition assays. The results clearly demonstrated that most tested constituents showed time-dependent inhibition on SARS-CoV-2 3CL ^pro , while gallocatechin and sciadopitysin displayed the most potent anti-SARS-CoV-2 3CL ^pro effects.
Conclusion: Collectively, GBE50 and some constituents in this herbal product could strongly inhibit SARS-CoV-2 3CL ^pro in dose- and time-dependent manner. Gallocatechin and sciadopitysin were identified as potent SARS-CoV-2 3CL ^pro inhibitors, which offers promising lead compounds for the development of novel anti-SARS-CoV-2 drugs.
Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests.
(Copyright © 2023. Published by Elsevier GmbH.)