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

Ya-Ni Zhang; Guang-Hao Zhu; Wei Liu; Yuan Xiong; Qing Hu; Xiao-Yu Zhuang; Gui-Hua Jia; Wei-Dong Zhang; Guang-Bo Ge

doi:10.1016/j.phymed.2023.154796

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

Phytomedicine. 2023 Jun:114:154796. doi: 10.1016/j.phymed.2023.154796. Epub 2023 Mar 29.

Authors

Ya-Ni Zhang¹, Guang-Hao Zhu¹, Wei Liu², Yuan Xiong¹, Qing Hu³, Xiao-Yu Zhuang¹, Gui-Hua Jia¹, Wei-Dong Zhang¹, Guang-Bo Ge⁴

Affiliations

¹ Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
² Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China.
³ Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; Clinical Pharmacy Center, Cancer Center, Department of Pharmacy, Zhejiang Provincial People's Hospital, Affiliated People's Hospital, Hangzhou Medical College, Hangzhou, Zhejiang 310014, China.
⁴ Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China. Electronic address: geguangbo@shutcm.edu.cn.

Abstract

Background: The 3C-like proteases (3CL^pros) 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^pros 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.

Keywords: Anti-CoV agents; Chemoproteomic profiling; Covalent inhibitors; Ginkgo biloba extract 50 (GBE50); SARS-CoV-2 3CLpro.

MeSH terms

Antiviral Agents / pharmacology
COVID-19*
Humans
Peptides
Plant Extracts
SARS-CoV-2*
Tandem Mass Spectrometry

Substances

Antiviral Agents
Ginkgo biloba extract
Peptides
Plant Extracts
3C-like proteinase, SARS-CoV-2