Methyl rosmarinate is an allosteric inhibitor of SARS-CoV-2 3 CL protease as a potential candidate against SARS-cov-2 infection

Hongtao Li; Meng Sun; Fuzhi Lei; Jinfeng Liu; Xixiang Chen; Yaqi Li; Ying Wang; Jiani Lu; Danmei Yu; Yueqiu Gao; Jianrong Xu; Hongzhuan Chen; Man Li; Zhigang Yi; Xiao He; Lili Chen

doi:10.1016/j.antiviral.2024.105841

Methyl rosmarinate is an allosteric inhibitor of SARS-CoV-2 3 CL protease as a potential candidate against SARS-cov-2 infection

Antiviral Res. 2024 Apr:224:105841. doi: 10.1016/j.antiviral.2024.105841. Epub 2024 Feb 24.

Authors

Hongtao Li¹, Meng Sun², Fuzhi Lei³, Jinfeng Liu⁴, Xixiang Chen¹, Yaqi Li⁵, Ying Wang⁶, Jiani Lu¹, Danmei Yu¹, Yueqiu Gao⁷, Jianrong Xu¹, Hongzhuan Chen¹, Man Li⁸, Zhigang Yi⁹, Xiao He¹⁰, Lili Chen¹¹

Affiliations

¹ Shanghai Frontiers Science Center of TCM Chemical Biology, Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China.
² Department of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, Nanjing, 210009, China.
³ Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, and Shanghai Institute of Infectious Disease and Biosecurity, Fudan University, Shanghai, China.
⁴ Department of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, Nanjing, 210009, China; Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, Shanghai Frontiers Science Center of Molecule Intelligent Syntheses, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai, 200062, China.
⁵ The National and Local Joint Engineering Laboratory of Animal Peptide Drug Development, College of Life Sciences, Hunan Normal University, Changsha 410081, China; Institute of Interdisciplinary Studies, Hunan Normal University, Changsha 410081, China; Peptide and small Molecule Drug R&D Plateform, Furong Laboratory, Hunan Normal University, Changsha 410081, Hunan, China; DP Technology, Beijing, China.
⁶ The National and Local Joint Engineering Laboratory of Animal Peptide Drug Development, College of Life Sciences, Hunan Normal University, Changsha 410081, China; Institute of Interdisciplinary Studies, Hunan Normal University, Changsha 410081, China; Peptide and small Molecule Drug R&D Plateform, Furong Laboratory, Hunan Normal University, Changsha 410081, Hunan, China.
⁷ Department of Hepatopathy, Shuguang Hospital, Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China; Laboratory of Cellular Immunity, Shuguang Hospital, Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China; Institute of Infectious Diseases of Integrated Traditional Chinese and Western Medicine, China.
⁸ Laboratory of Cellular Immunity, Shuguang Hospital, Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China. Electronic address: liman121000@shutcm.edu.cn.
⁹ Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, and Shanghai Institute of Infectious Disease and Biosecurity, Fudan University, Shanghai, China; Shanghai Public Health Clinical Center, Fudan University, Shanghai, China. Electronic address: zgyi@fudan.edu.cn.
¹⁰ Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, Shanghai Frontiers Science Center of Molecule Intelligent Syntheses, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai, 200062, China; New York University-East China Normal University Center for Computational Chemistry, New York University Shanghai, Shanghai, 200062, China. Electronic address: xiaohe@phy.ecnu.edu.cn.
¹¹ Shanghai Frontiers Science Center of TCM Chemical Biology, Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China; Longhua Hospital Shanghai University of Traditional Chinese Medicine, 725 South Wanping Road, Shanghai, 200032, China. Electronic address: llchen@shutcm.edu.cn.

PMID: 38408645
DOI: 10.1016/j.antiviral.2024.105841

Abstract

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been ongoing for more than three years and urgently needs to be addressed. Traditional Chinese medicine (TCM) prescriptions have played an important role in the clinical treatment of patients with COVID-19 in China. However, it is difficult to uncover the potential molecular mechanisms of the active ingredients in these TCM prescriptions. In this paper, we developed a new approach by integrating the experimental assay, virtual screening, and the experimental verification, exploring the rapid discovery of active ingredients from TCM prescriptions. To achieve this goal, 4 TCM prescriptions in clinical use for different indications were selected to find the antiviral active ingredients in TCMs. The 3-chymotrypsin-like protease (3CL^pro), an important target for fighting COVID-19, was utilized to determine the inhibitory activity of the TCM prescriptions and single herb. It was found that 10 single herbs had better inhibitory activity than other herbs by using a fluorescence resonance energy transfer (FRET) - based enzymatic assay of SARS-CoV-2 3CL^pro. The ingredients contained in 10 herbs were thus virtually screened and the predicted active ingredients were experimentally validated. Thus, such a research strategy firstly removed many single herbs with no inhibitory activity against SARS-CoV-2 3CL^pro at the very beginning by FRET-based assay, making our subsequent virtual screening more effective. Finally, 4 active components were found to have stronger inhibitory effects on SARS-CoV-2 3CL^pro, and their inhibitory mechanism was subsequently investigated. Among of them, methyl rosmarinate as an allosteric inhibitor of SARS-CoV-2 3CL^pro was confirmed and its ability to inhibit viral replication was demonstrated by the SARS-CoV-2 replicon system. To validate the binding mode via docking, the mutation experiment, circular dichroism (CD), enzymatic inhibition and surface plasmon resonance (SPR) assay were performed, demonstrating that methyl rosmarinate bound to the allosteric site of SARS-CoV-2 3CL^pro. In conclusion, this paper provides the new ideas for the rapid discovery of active ingredients in TCM prescriptions based on a specific target, and methyl rosmarinate has the potential to be developed as an antiviral therapeutic candidate against SARS-CoV-2 infection.

Keywords: Active ingredients; Methyl rosmarinate; SARS-CoV-2 3CL(pro); Traditional Chinese medicine (TCM); Virtual screening.

MeSH terms

Antiviral Agents / pharmacology
COVID-19*
Humans
Molecular Docking Simulation
Peptide Hydrolases
Protease Inhibitors / pharmacology
Rosmarinic Acid
SARS-CoV-2

Substances

Rosmarinic Acid
Peptide Hydrolases
Antiviral Agents
Protease Inhibitors