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1.
Int J Mol Sci ; 24(10)2023 May 11.
Article in English | MEDLINE | ID: covidwho-20244460

ABSTRACT

The papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a critical role in the proteolytic processing of viral polyproteins and the dysregulation of the host immune response, providing a promising therapeutic target. Here, we report the structure-guide design of novel peptidomimetic inhibitors covalently targeting SARS-CoV-2 PLpro. The resulting inhibitors demonstrate submicromolar potency in the enzymatic assay (IC50 = 0.23 µM) and significant inhibition of SARS-CoV-2 PLpro in the HEK293T cells using a cell-based protease assay (EC50 = 3.61 µM). Moreover, an X-ray crystal structure of SARS-CoV-2 PLpro in complex with compound 2 confirms the covalent binding of the inhibitor to the catalytic residue cysteine 111 (C111) and emphasizes the importance of interactions with tyrosine 268 (Y268). Together, our findings reveal a new scaffold of SARS-CoV-2 PLpro inhibitors and provide an attractive starting point for further optimization.


Subject(s)
COVID-19 , Peptidomimetics , Humans , Peptidomimetics/pharmacology , HEK293 Cells , SARS-CoV-2 , Peptide Hydrolases , Protease Inhibitors/pharmacology , Antiviral Agents/pharmacology , Antiviral Agents/chemistry
2.
Angewandte Chemie ; 134(52):1-9, 2022.
Article in English | Academic Search Complete | ID: covidwho-2172438

ABSTRACT

The coronavirus papain‐like protease (PLpro) plays an important role in the proteolytic processing of viral polyproteins and the dysregulation of the host immune response, providing a promising therapeutic target. However, the development of inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) PLpro is challenging owing to the restricted S1/S2 sites in the substrate binding pocket. Here we report the discovery of two activators of SARS‐CoV‐2 PLpro and the identification of the unique residue, cysteine 270 (C270), as an allosteric and covalent regulatory site for the activators. This site is also specifically modified by glutathione, resulting in protease activation. Furthermore, a compound was found to allosterically inhibit the protease activity by covalent binding to C270. Together, these results elucidate an unrevealed molecular mechanism for allosteric modulation of SARS‐CoV‐2 PLpro and provid a novel site for allosteric inhibitors design. [ FROM AUTHOR]

3.
Angew Chem Int Ed Engl ; : e202212378, 2022 Oct 29.
Article in English | MEDLINE | ID: covidwho-2094147

ABSTRACT

The coronavirus papain-like protease (PLpro ) plays an important role in the proteolytic processing of viral polyproteins and the dysregulation of the host immune response, providing a promising therapeutic target. However, the development of inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro is challenging owing to the restricted S1/S2 sites in the substrate binding pocket. Here we report the discovery of two activators of SARS-CoV-2 PLpro and the identification of the unique residue, cysteine 270 (C270), as an allosteric and covalent regulatory site for the activators. This site is also specifically modified by glutathione, resulting in protease activation. Furthermore, a compound was found to allosterically inhibit the protease activity by covalent binding to C270. Together, these results elucidate an unrevealed molecular mechanism for allosteric modulation of SARS-CoV-2 PLpro and provid a novel site for allosteric inhibitors design.

4.
Eur J Med Chem ; 231: 114130, 2022 Mar 05.
Article in English | MEDLINE | ID: covidwho-1654357

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3CL protease (3CLpro) has been regarded as an extremely promising antiviral target for the treatment of coronavirus disease 2019 (COVID-19). Here, we carried out a virtual screening based on commercial compounds database to find novel covalent non-peptidomimetic inhibitors of this protease. It allowed us to identify 3 hit compounds with potential covalent binding modes, which were evaluated through an enzymatic activity assay of the SARS-CoV-2 3CLpro. Moreover, an X-ray crystal structure of the SARS-CoV-2 3CLpro in complex with compound 8, the most potent hit with an IC50 value of 8.50 µM, confirmed the covalent binding of the predicted warhead to the catalytic residue C145, as well as portrayed interactions of the compound with S1' and S2 subsites at the ligand binding pocket. Overall, the present work not merely provided an experiment-validated covalent hit targeting the SARS-CoV-2 3CLpro, but also displayed a prime example to seeking new covalent small molecules by a feasible and effective computational approach.


Subject(s)
COVID-19 Drug Treatment , SARS-CoV-2 , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Humans , Peptide Hydrolases , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology
5.
Nat Commun ; 12(1): 3623, 2021 06 15.
Article in English | MEDLINE | ID: covidwho-1270656

ABSTRACT

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) urgently needs an effective cure. 3CL protease (3CLpro) is a highly conserved cysteine proteinase that is indispensable for coronavirus replication, providing an attractive target for developing broad-spectrum antiviral drugs. Here we describe the discovery of myricetin, a flavonoid found in many food sources, as a non-peptidomimetic and covalent inhibitor of the SARS-CoV-2 3CLpro. Crystal structures of the protease bound with myricetin and its derivatives unexpectedly revealed that the pyrogallol group worked as an electrophile to covalently modify the catalytic cysteine. Kinetic and selectivity characterization together with theoretical calculations comprehensively illustrated the covalent binding mechanism of myricetin with the protease and demonstrated that the pyrogallol can serve as an electrophile warhead. Structure-based optimization of myricetin led to the discovery of derivatives with good antiviral activity and the potential of oral administration. These results provide detailed mechanistic insights into the covalent mode of action by pyrogallol-containing natural products and a template for design of non-peptidomimetic covalent inhibitors against 3CLpros, highlighting the potential of pyrogallol as an alternative warhead in design of targeted covalent ligands.


Subject(s)
Coronavirus 3C Proteases/drug effects , Pyrogallol/chemistry , Pyrogallol/isolation & purification , Pyrogallol/pharmacology , SARS-CoV-2/drug effects , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Coronavirus Papain-Like Proteases , Drug Design , Flavonoids , HEK293 Cells , Humans , Kinetics , Ligands , Molecular Dynamics Simulation , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Viral Nonstructural Proteins/chemistry , COVID-19 Drug Treatment
6.
Acta Pharmacol Sin ; 41(9): 1167-1177, 2020 Sep.
Article in English | MEDLINE | ID: covidwho-691161

ABSTRACT

Human infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) and there is no cure currently. The 3CL protease (3CLpro) is a highly conserved protease which is indispensable for CoVs replication, and is a promising target for development of broad-spectrum antiviral drugs. In this study we investigated the anti-SARS-CoV-2 potential of Shuanghuanglian preparation, a Chinese traditional patent medicine with a long history for treating respiratory tract infection in China. We showed that either the oral liquid of Shuanghuanglian, the lyophilized powder of Shuanghuanglian for injection or their bioactive components dose-dependently inhibited SARS-CoV-2 3CLpro as well as the replication of SARS-CoV-2 in Vero E6 cells. Baicalin and baicalein, two ingredients of Shuanghuanglian, were characterized as the first noncovalent, nonpeptidomimetic inhibitors of SARS-CoV-2 3CLpro and exhibited potent antiviral activities in a cell-based system. Remarkably, the binding mode of baicalein with SARS-CoV-2 3CLpro determined by X-ray protein crystallography was distinctly different from those of known 3CLpro inhibitors. Baicalein was productively ensconced in the core of the substrate-binding pocket by interacting with two catalytic residues, the crucial S1/S2 subsites and the oxyanion loop, acting as a "shield" in front of the catalytic dyad to effectively prevent substrate access to the catalytic dyad within the active site. Overall, this study provides an example for exploring the in vitro potency of Chinese traditional patent medicines and effectively identifying bioactive ingredients toward a specific target, and gains evidence supporting the in vivo studies of Shuanghuanglian oral liquid as well as two natural products for COVID-19 treatment.


Subject(s)
Betacoronavirus/drug effects , Coronavirus Infections , Drugs, Chinese Herbal , Flavanones , Flavonoids , Pandemics , Pneumonia, Viral , Virus Replication/drug effects , Administration, Oral , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Betacoronavirus/physiology , COVID-19 , Chlorocebus aethiops , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/pharmacology , Enzyme Assays , Flavanones/chemistry , Flavanones/pharmacokinetics , Flavonoids/chemistry , Flavonoids/pharmacokinetics , Humans , Pneumonia, Viral/drug therapy , Pneumonia, Viral/virology , SARS-CoV-2 , Vero Cells , Virus Replication/physiology
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