Danshensu inhibits SARS-CoV-2 by targeting its main protease as a specific covalent inhibitor and discovery of bifunctional compounds eliciting antiviral and anti-inflammatory activity

Ruyu Wang; Xuwen Chen; Hongtao Li; Xixiang Chen; Donghui Sun; Danmei Yu; Jiani Lu; Yuanyuan Xie; Qian Zhang; Jianrong Xu; Weidong Zhang; Hongzhuan Chen; Shunying Liu; Lili Chen

doi:10.1016/j.ijbiomac.2023.128623

Danshensu inhibits SARS-CoV-2 by targeting its main protease as a specific covalent inhibitor and discovery of bifunctional compounds eliciting antiviral and anti-inflammatory activity

Int J Biol Macromol. 2024 Feb;257(Pt 2):128623. doi: 10.1016/j.ijbiomac.2023.128623. Epub 2023 Dec 7.

Authors

Ruyu Wang¹, Xuwen Chen², Hongtao Li¹, Xixiang Chen¹, Donghui Sun², Danmei Yu¹, Jiani Lu¹, Yuanyuan Xie³, Qian Zhang⁴, Jianrong Xu³, Weidong Zhang⁵, Hongzhuan Chen¹, Shunying Liu⁶, Lili Chen⁷

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 Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200062, China.
³ Shanghai Frontiers Science Center of TCM Chemical Biology, Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; Academy of Integrative Medicine, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201203, China.
⁴ Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, China.
⁵ Shanghai Frontiers Science Center of TCM Chemical Biology, Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; Shanghai Institute of Infectious Diseases and Biosafety, Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
⁶ Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200062, China. Electronic address: syliu@sist.ecnu.edu.cn.
⁷ Shanghai Frontiers Science Center of TCM Chemical Biology, Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China. Electronic address: llchen@shutcm.edu.cn.

PMID: 38070810
DOI: 10.1016/j.ijbiomac.2023.128623

Abstract

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a serious threat to human. Since there are still no effective treatment options against the new emerging variants of SARS-CoV-2, it is necessary to devote a continuous endeavor for more targeted drugs and the preparation for the next pandemic. Salvia miltiorrhiza and its active ingredients possess wide antiviral activities, including against SARS-CoV-2. Danshensu, as one of the most important active ingredients in Salvia miltiorrhiza, has been reported to inhibit the entry of SARS-CoV-2 into ACE2 (angiotensin-converting enzyme 2)-overexpressed HEK-293T cells and Vero-E6 cells. However, there is a paucity of information regarding its detailed target and mechanism against SARS-CoV-2. Here, we present Danshensu as a covalent inhibitor of 3-chymotrypsin-like protease (3CL^pro) against SARS-CoV-2 by the time-dependent inhibition assay (TDI) and mass spectrometry analysis. Further molecular docking, site-directed mutagenesis, circular dichroism (CD) and fluorescence spectra revealed that Danshensu covalently binds to C145 of SARS-CoV-2 3CL^pro, meanwhile forming the hydrogen bonds with S144, H163 and E166 in the S1 site. Structure-based optimization of Danshensu led to the discovery of the promising compounds with good inhibitory activity and microsomal stability in vitro. Due to Danshensu inhibiting lung inflammation in the mouse model, we found that Danshensu derivatives also showed better anti-inflammatory activity than Danshensu in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Thus, our study provides not only the clue of the efficacy of Salvia miltiorrhiza against SARS-CoV-2, but also a detailed mechanistic insight into the covalent mode of action of Danshensu for design of covalent inhibitors against SARS-CoV-2 3CL^pro, highlighting its potential as a bifunctional molecule with antivirus and anti-inflammation.

Keywords: Anti-inflammation; Bifunctional molecule; Covalent inhibitor; Danshensu; SARS-CoV-2 3CL(pro).

MeSH terms

Animals
Antiviral Agents / chemistry
COVID-19*
Humans
Lactates*
Mice
Molecular Docking Simulation
Peptide Hydrolases / pharmacology
Protease Inhibitors / pharmacology
SARS-CoV-2*
Viral Nonstructural Proteins / chemistry

Substances

3,4-dihydroxyphenyllactic acid
Viral Nonstructural Proteins
Antiviral Agents
Peptide Hydrolases
Protease Inhibitors
Lactates