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1.
J Med Chem ; 65(1): 876-884, 2022 01 13.
Article in English | MEDLINE | ID: covidwho-1606194

ABSTRACT

Coronavirus disease 2019 (COVID-19) pandemic, a global health threat, was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 papain-like cysteine protease (PLpro) was recognized as a promising drug target because of multiple functions in virus maturation and antiviral immune responses. Inhibitor GRL0617 occupied the interferon-stimulated gene 15 (ISG15) C-terminus-binding pocket and showed an effective antiviral inhibition. Here, we described a novel peptide-drug conjugate (PDC), in which GRL0617 was linked to a sulfonium-tethered peptide derived from PLpro-specific substrate LRGG. The EM-C and EC-M PDCs showed a promising in vitro IC50 of 7.40 ± 0.37 and 8.63 ± 0.55 µM, respectively. EC-M could covalently label PLpro active site C111 and display anti-ISGylation activities in cellular assays. The results represent the first attempt to design PDCs composed of stabilized peptide inhibitors and GRL0617 to inhibit PLpro. These novel PDCs provide promising opportunities for antiviral drug design.


Subject(s)
Aniline Compounds/chemistry , Antiviral Agents/metabolism , Benzamides/chemistry , Coronavirus Papain-Like Proteases/metabolism , Drug Design , Naphthalenes/chemistry , Peptides/chemistry , SARS-CoV-2/enzymology , Aniline Compounds/metabolism , Aniline Compounds/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Benzamides/metabolism , Benzamides/pharmacology , COVID-19/drug therapy , COVID-19/pathology , COVID-19/virology , Cell Line , Cell Survival/drug effects , Coronavirus Papain-Like Proteases/chemistry , Cytokines/chemistry , Drug Evaluation, Preclinical , Humans , Inhibitory Concentration 50 , Naphthalenes/metabolism , Naphthalenes/pharmacology , SARS-CoV-2/isolation & purification , Ubiquitins/chemistry
2.
Molecules ; 26(21)2021 Nov 08.
Article in English | MEDLINE | ID: covidwho-1512511

ABSTRACT

This work describes an untargeted analytical approach for the screening, identification, and characterization of the trans-epithelial transport of green tea (Camellia sinensis) catechin extracts with in vitro inhibitory effect against the SARS-CoV-2 papain-like protease (PLpro) activity. After specific catechin extraction, a chromatographic separation obtained six fractions were carried out. The fractions were assessed in vitro against the PLpro target. Fraction 5 showed the highest inhibitory activity against the SARS-CoV-2 PLpro (IC50 of 0.125 µg mL-1). The untargeted characterization revealed that (-)-epicatechin-3-gallate (ECG) was the most abundant compound in the fraction and the primary molecule absorbed by differentiated Caco-2 cells. Results indicated that fraction 5 was approximately 10 times more active than ECG (IC50 value equal to 11.62 ± 0.47 µg mL-1) to inhibit the PLpro target. Overall, our findings highlight the synergistic effects of the various components of the crude extract compared to isolated ECG.


Subject(s)
Catechin/pharmacology , Coronavirus Papain-Like Proteases/metabolism , Tea/metabolism , Antiviral Agents/chemistry , COVID-19/drug therapy , COVID-19/metabolism , Caco-2 Cells , Camellia sinensis/metabolism , Catechin/analogs & derivatives , Catechin/chemistry , Catechin/metabolism , Coronavirus Papain-Like Proteases/drug effects , Epithelium/drug effects , Epithelium/metabolism , Humans , Mass Spectrometry/methods , Plant Extracts/chemistry , Plant Extracts/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Tea/chemistry , Tea/physiology
3.
Molecules ; 26(21)2021 Oct 30.
Article in English | MEDLINE | ID: covidwho-1488678

ABSTRACT

Papain-like protease is an essential enzyme in the proteolytic processing required for the replication of SARS-CoV-2. Accordingly, such an enzyme is an important target for the development of anti-SARS-CoV-2 agents which may reduce the mortality associated with outbreaks of SARS-CoV-2. A set of 69 semi-synthesized molecules that exhibited the structural features of SARS-CoV-2 papain-like protease inhibitors (PLPI) were docked against the coronavirus papain-like protease (PLpro) enzyme (PDB ID: (4OW0). Docking studies showed that derivatives 34 and 58 were better than the co-crystallized ligand while derivatives 17, 28, 31, 40, 41, 43, 47, 54, and 65 exhibited good binding modes and binding free energies. The pharmacokinetic profiling study was conducted according to the four principles of the Lipinski rules and excluded derivative 31. Furthermore, ADMET and toxicity studies showed that derivatives 28, 34, and 47 have the potential to be drugs and have been demonstrated as safe when assessed via seven toxicity models. Finally, comparing the molecular orbital energies and the molecular electrostatic potential maps of 28, 34, and 47 against the co-crystallized ligand in a DFT study indicated that 28 is the most promising candidate to interact with the target receptor (PLpro).


Subject(s)
Coronavirus Papain-Like Proteases/metabolism , SARS-CoV-2/drug effects , Virus Replication/drug effects , Antiviral Agents/pharmacology , COVID-19/drug therapy , COVID-19/metabolism , Computer Simulation , Coronavirus Papain-Like Proteases/drug effects , Drug Evaluation, Preclinical/methods , Humans , Ligands , Molecular Docking Simulation , Molecular Dynamics Simulation , Papain/metabolism , Peptide Hydrolases/metabolism , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , Protease Inhibitors/pharmacology , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity
4.
Nat Immunol ; 22(11): 1416-1427, 2021 11.
Article in English | MEDLINE | ID: covidwho-1475314

ABSTRACT

Ubiquitin-like protein ISG15 (interferon-stimulated gene 15) (ISG15) is a ubiquitin-like modifier induced during infections and involved in host defense mechanisms. Not surprisingly, many viruses encode deISGylating activities to antagonize its effect. Here we show that infection by Zika, SARS-CoV-2 and influenza viruses induce ISG15-modifying enzymes. While influenza and Zika viruses induce ISGylation, SARS-CoV-2 triggers deISGylation instead to generate free ISG15. The ratio of free versus conjugated ISG15 driven by the papain-like protease (PLpro) enzyme of SARS-CoV-2 correlates with macrophage polarization toward a pro-inflammatory phenotype and attenuated antigen presentation. In vitro characterization of purified wild-type and mutant PLpro revealed its strong deISGylating over deubiquitylating activity. Quantitative proteomic analyses of PLpro substrates and secretome from SARS-CoV-2-infected macrophages revealed several glycolytic enzymes previously implicated in the expression of inflammatory genes and pro-inflammatory cytokines, respectively. Collectively, our results indicate that altered free versus conjugated ISG15 dysregulates macrophage responses and probably contributes to the cytokine storms triggered by SARS-CoV-2.


Subject(s)
COVID-19/immunology , Cytokines/metabolism , Inflammation/immunology , Macrophages/immunology , SARS-CoV-2/physiology , Ubiquitins/metabolism , Cell Differentiation , Coronavirus Papain-Like Proteases/metabolism , Cytokines/genetics , Gene Knockdown Techniques , HeLa Cells , Humans , Immune Evasion , Immunity, Innate , Influenza A virus/physiology , Influenza, Human/immunology , Pluripotent Stem Cells/cytology , Ubiquitination , Ubiquitins/genetics , Zika Virus/physiology , Zika Virus Infection/immunology
5.
Phys Chem Chem Phys ; 23(40): 22957-22971, 2021 Oct 20.
Article in English | MEDLINE | ID: covidwho-1462045

ABSTRACT

The identification of chemical compounds able to bind specific sites of the human/viral proteins involved in the SARS-CoV-2 infection cycle is a prerequisite to design effective antiviral drugs. Here we conduct a molecular dynamics study with the aim to assess the interactions of ivermectin, an antiparasitic drug with broad-spectrum antiviral activity, with the human Angiotensin-Converting Enzyme 2 (ACE2), the viral 3CLpro and PLpro proteases, and the viral SARS Unique Domain (SUD). The drug/target interactions have been characterized in silico by describing the nature of the non-covalent interactions found and by measuring the extent of their time duration along the MD simulation. Results reveal that the ACE2 protein and the ACE2/RBD aggregates form the most persistent interactions with ivermectin, while the binding with the remaining viral proteins is more limited and unspecific.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/metabolism , Coronavirus 3C Proteases/metabolism , Coronavirus Papain-Like Proteases/metabolism , Ivermectin/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Antiviral Agents/chemistry , Binding Sites , Coronavirus 3C Proteases/chemistry , Coronavirus Papain-Like Proteases/chemistry , G-Quadruplexes , Humans , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Ivermectin/chemistry , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Binding , Protein Domains , RNA/genetics , RNA/metabolism , SARS-CoV-2
6.
Int J Mol Sci ; 22(19)2021 Oct 02.
Article in English | MEDLINE | ID: covidwho-1444233

ABSTRACT

Considering the current dramatic and fatal situation due to the high spreading of SARS-CoV-2 infection, there is an urgent unmet medical need to identify novel and effective approaches for prevention and treatment of Coronavirus disease (COVID 19) by re-evaluating and repurposing of known drugs. For this, tomatidine and patchouli alcohol have been selected as potential drugs for combating the virus. The hit compounds were subsequently docked into the active site and molecular docking analyses revealed that both drugs can bind the active site of SARS-CoV-2 3CLpro, PLpro, NSP15, COX-2 and PLA2 targets with a number of important binding interactions. To further validate the interactions of promising compound tomatidine, Molecular dynamics study of 100 ns was carried out towards 3CLpro, NSP15 and COX-2. This indicated that the protein-ligand complex was stable throughout the simulation period, and minimal backbone fluctuations have ensued in the system. Post dynamic MM-GBSA analysis of molecular dynamics data showed promising mean binding free energy 47.4633 ± 9.28, 51.8064 ± 8.91 and 54.8918 ± 7.55 kcal/mol, respectively. Likewise, in silico ADMET studies of the selected ligands showed excellent pharmacokinetic properties with good absorption, bioavailability and devoid of toxicity. Therefore, patchouli alcohol and especially, tomatidine may provide prospect treatment options against SARS-CoV-2 infection by potentially inhibiting virus duplication though more research is guaranteed and secured.


Subject(s)
Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Endoribonucleases/antagonists & inhibitors , SARS-CoV-2/enzymology , Sesquiterpenes/pharmacology , Tomatine/analogs & derivatives , Viral Nonstructural Proteins/antagonists & inhibitors , Antiviral Agents/pharmacology , COVID-19/drug therapy , COVID-19/virology , Coronavirus 3C Proteases/metabolism , Coronavirus Papain-Like Proteases/metabolism , Endoribonucleases/metabolism , Enzyme Inhibitors/pharmacology , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , SARS-CoV-2/drug effects , Tomatine/pharmacology , Viral Nonstructural Proteins/metabolism
7.
Sci Rep ; 11(1): 18851, 2021 09 22.
Article in English | MEDLINE | ID: covidwho-1434149

ABSTRACT

In this pandemic SARS-CoV-2 crisis, any attempt to contain and eliminate the virus will also stop its spread and consequently decrease the risk of severe illness and death. While ozone treatment has been suggested as an effective disinfection process, no precise mechanism of action has been previously reported. This study aimed to further investigate the effect of ozone treatment on SARS-CoV-2. Therefore, virus collected from nasopharyngeal and oropharyngeal swab and sputum samples from symptomatic patients was exposed to ozone for different exposure times. The virus morphology and structure were monitored and analyzed through Atomic Force Microscopy (AFM), Transmission Electron Microscopy (TEM), Atomic Absorption Spectroscopy (AAS), and ATR-FTIR. The obtained results showed that ozone treatment not only unsettles the virus morphology but also alters the virus proteins' structure and conformation through amino acid disturbance and Zn ion release from the virus non-structural proteins. These results could provide a clearer pathway for virus elimination and therapeutics preparation.


Subject(s)
COVID-19/drug therapy , Ozone/pharmacology , SARS-CoV-2/chemistry , SARS-CoV-2/drug effects , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/metabolism , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Humans , Microscopy, Electron, Transmission , Protein Structure, Secondary/drug effects , Protein Structure, Tertiary/drug effects , SARS-CoV-2/ultrastructure , Time Factors , Viral Envelope/chemistry , Viral Envelope/drug effects , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/metabolism , Zinc/chemistry , Zinc/metabolism
8.
Cell Rep ; 36(13): 109754, 2021 09 28.
Article in English | MEDLINE | ID: covidwho-1401298

ABSTRACT

The SARS-CoV-2 papain-like protease (PLpro) is a target for antiviral drug development. It is essential for processing viral polyproteins for replication and functions in host immune evasion by cleaving ubiquitin (Ub) and ubiquitin-like protein (Ubl) conjugates. While highly conserved, SARS-CoV-2 and SARS-CoV PLpro have contrasting Ub/Ubl substrate preferences. Using a combination of structural analyses and functional assays, we identify a molecular sensor within the S1 Ub-binding site of PLpro that serves as a key determinant of substrate specificity. Variations within the S1 sensor specifically alter cleavage of Ub substrates but not of the Ubl interferon-stimulated gene 15 protein (ISG15). Significantly, a variant of concern associated with immune evasion carries a mutation in the S1 sensor that enhances PLpro activity on Ub substrates. Collectively, our data identify the S1 sensor region as a potential hotspot of variability that could alter host antiviral immune responses to newly emerging SARS-CoV-2 lineages.


Subject(s)
Coronavirus Papain-Like Proteases/metabolism , Coronavirus Papain-Like Proteases/ultrastructure , SARS-CoV-2/genetics , Amino Acid Sequence/genetics , Binding Sites/genetics , COVID-19/genetics , COVID-19/metabolism , Coronavirus Papain-Like Proteases/genetics , HEK293 Cells , Humans , Papain/chemistry , Papain/metabolism , Peptide Hydrolases/chemistry , Peptide Hydrolases/metabolism , Protein Binding/genetics , SARS-CoV-2/metabolism , Substrate Specificity/genetics , Ubiquitin/metabolism , Ubiquitins/metabolism , Viral Proteins/metabolism
9.
Int J Mol Sci ; 22(6)2021 Mar 15.
Article in English | MEDLINE | ID: covidwho-1389394

ABSTRACT

SARS-CoV-2 currently lacks effective first-line drug treatment. We present promising data from in silico docking studies of new Methisazone compounds (modified with calcium, Ca; iron, Fe; magnesium, Mg; manganese, Mn; or zinc, Zn) designed to bind more strongly to key proteins involved in replication of SARS-CoV-2. In this in silico molecular docking study, we investigated the inhibiting role of Methisazone and the modified drugs against SARS-CoV-2 proteins: ribonucleic acid (RNA)-dependent RNA polymerase (RdRp), spike protein, papain-like protease (PlPr), and main protease (MPro). We found that the highest binding interactions were found with the spike protein (6VYB), with the highest overall binding being observed with Mn-bound Methisazone at -8.3 kcal/mol, followed by Zn and Ca at -8.0 kcal/mol, and Fe and Mg at -7.9 kcal/mol. We also found that the metal-modified Methisazone had higher affinity for PlPr and MPro. In addition, we identified multiple binding pockets that could be singly or multiply occupied on all proteins tested. The best binding energy was with Mn-Methisazone versus spike protein, and the largest cumulative increases in binding energies were found with PlPr. We suggest that further studies are warranted to identify whether these compounds may be effective for treatment and/or prophylaxis.


Subject(s)
Antiviral Agents/chemistry , Metals/chemistry , Methisazone/chemistry , Molecular Docking Simulation , SARS-CoV-2/chemistry , Antiviral Agents/metabolism , COVID-19/drug therapy , Calcium/chemistry , Calcium/metabolism , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/metabolism , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Drug Design , Humans , Iron/chemistry , Iron/metabolism , Magnesium/chemistry , Magnesium/metabolism , Manganese/chemistry , Manganese/metabolism , Metals/metabolism , Methisazone/metabolism , Models, Molecular , Molecular Dynamics Simulation , Protein Binding , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Zinc/chemistry , Zinc/metabolism
10.
Chempluschem ; 86(7): 972-981, 2021 07.
Article in English | MEDLINE | ID: covidwho-1384145

ABSTRACT

We report the synthesis and characterization of a fullerene-steroid hybrid that contains H2 @C60 and a dehydroepiandrosterone moiety synthesized by a cyclopropanation reaction with 76 % yield. Theoretical calculations at the DFT-D3(BJ)/PBE 6-311G(d,p) level predict the most stable conformation and that the saturation of a double bond is the main factor causing the upfield shielding of the signal appearing at -3.13 ppm, which corresponds to the H2 located inside the fullerene cage. Relevant stereoelectronic parameters were also investigated and reinforce the idea that electronic interactions must be considered to develop studies on chemical-biological interactions. A molecular docking simulation predicted that the binding energy values for the protease-hybrid complexes were -9.9 kcal/mol and -13.5 kcal/mol for PLpro and 3CLpro respectively, indicating the potential use of the synthesized steroid-H2 @C60 as anti-SARS-Cov-2 agent.


Subject(s)
Androsterone/chemistry , Antiviral Agents/chemistry , Fullerenes/chemistry , Molecular Docking Simulation , SARS-CoV-2/metabolism , Antiviral Agents/metabolism , Binding Sites , COVID-19/pathology , COVID-19/virology , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/metabolism , Density Functional Theory , Humans , Protein Binding , SARS-CoV-2/isolation & purification , Static Electricity , Thermodynamics
11.
Molecules ; 26(17)2021 Aug 24.
Article in English | MEDLINE | ID: covidwho-1374471

ABSTRACT

The emergence of COVID-19 continues to pose severe threats to global public health. The pandemic has infected over 171 million people and claimed more than 3.5 million lives to date. We investigated the binding potential of antiviral cyanobacterial proteins including cyanovirin-N, scytovirin and phycocyanin with fundamental proteins involved in attachment and replication of SARS-CoV-2. Cyanovirin-N displayed the highest binding energy scores (-16.8 ± 0.02 kcal/mol, -12.3 ± 0.03 kcal/mol and -13.4 ± 0.02 kcal/mol, respectively) with the spike protein, the main protease (Mpro) and the papainlike protease (PLpro) of SARS-CoV-2. Cyanovirin-N was observed to interact with the crucial residues involved in the attachment of the human ACE2 receptor. Analysis of the binding affinities calculated employing the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) approach revealed that all forms of energy, except the polar solvation energy, favourably contributed to the interactions of cyanovirin-N with the viral proteins. With particular emphasis on cyanovirin-N, the current work presents evidence for the potential inhibition of SARS-CoV-2 by cyanobacterial proteins, and offers the opportunity for in vitro and in vivo experiments to deploy the cyanobacterial proteins as valuable therapeutics against COVID-19.


Subject(s)
Antiviral Agents/pharmacology , Bacterial Proteins/pharmacology , COVID-19/drug therapy , Coronavirus Protease Inhibitors/pharmacology , Antiviral Agents/therapeutic use , Bacterial Proteins/therapeutic use , Bacterial Proteins/ultrastructure , COVID-19/virology , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/metabolism , Coronavirus 3C Proteases/ultrastructure , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Coronavirus Papain-Like Proteases/metabolism , Coronavirus Papain-Like Proteases/ultrastructure , Coronavirus Protease Inhibitors/therapeutic use , Coronavirus Protease Inhibitors/ultrastructure , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Binding , Protein Interaction Mapping , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/metabolism , Spike Glycoprotein, Coronavirus/ultrastructure , X-Ray Diffraction
12.
Nutrients ; 13(8)2021 Aug 16.
Article in English | MEDLINE | ID: covidwho-1360797

ABSTRACT

Hesperidin (HD) is a common flavanone glycoside isolated from citrus fruits and possesses great potential for cardiovascular protection. Hesperetin (HT) is an aglycone metabolite of HD with high bioavailability. Through the docking simulation, HD and HT have shown their potential to bind to two cellular proteins: transmembrane serine protease 2 (TMPRSS2) and angiotensin-converting enzyme 2 (ACE2), which are required for the cellular entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results further found that HT and HD suppressed the infection of VeroE6 cells using lentiviral-based pseudo-particles with wild types and variants of SARS-CoV-2 with spike (S) proteins, by blocking the interaction between the S protein and cellular receptor ACE2 and reducing ACE2 and TMPRSS2 expression. In summary, hesperidin is a potential TMPRSS2 inhibitor for the reduction of the SARS-CoV-2 infection.


Subject(s)
COVID-19/drug therapy , Hesperidin/chemistry , Hesperidin/pharmacology , SARS-CoV-2/drug effects , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/metabolism , COVID-19/virology , Cell Line, Tumor , Chlorocebus aethiops , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/metabolism , Humans , Molecular Docking Simulation , SARS-CoV-2/metabolism , Serine Endopeptidases/chemistry , Serine Endopeptidases/drug effects , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
13.
Mol Inform ; 40(8): e2100028, 2021 08.
Article in English | MEDLINE | ID: covidwho-1345038

ABSTRACT

The COVID-19 pandemic caused by the SARS-CoV-2 has mobilized scientific attention in search of a treatment. The cysteine-proteases, main protease (Mpro) and papain-like protease (PLpro) are important targets for antiviral drugs. In this work, we simulate the interactions between the Mpro and PLpro with Ebselen, its metabolites and derivatives with the aim of finding molecules that can potentially inhibit these enzymes. The docking data demonstrate that there are two main interactions between the thiol (-SH) group of Cys (from the protease active sites) and the electrophilic centers of the organoselenium molecules, i. e. the interaction with the carbonyl group (O=C… SH) and the interaction with the Se moiety (Se… SH). Both interactions may lead to an adduct formation and enzyme inhibition. Density Functional Theory (DFT) calculations with Ebselen indicate that the energetics of the thiol nucleophilic attack is more favorable on Se than on the carbonyl group, which is in accordance with experimental data (Jin et al. Nature, 2020, 582, 289-293). Therefore, organoselenium molecules should be further explored as inhibitors of the SARS-CoV-2 proteases. Furthermore, we suggest that some metabolites of Ebselen (e. g. Ebselen diselenide and methylebselenoxide) and derivatives ethaselen and ebsulfur should be tested in vitro as inhibitors of virus replication and its proteases.


Subject(s)
Azoles/pharmacology , COVID-19/drug therapy , Coronavirus Papain-Like Proteases/metabolism , Organoselenium Compounds/pharmacology , Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , Viral Matrix Proteins/metabolism , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Azoles/chemistry , Azoles/metabolism , COVID-19/metabolism , Catalytic Domain/drug effects , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Drug Discovery , Humans , Isoindoles , Molecular Docking Simulation , Organoselenium Compounds/chemistry , Organoselenium Compounds/metabolism , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , Viral Matrix Proteins/antagonists & inhibitors
14.
J Pharm Pharm Sci ; 24: 390-399, 2021.
Article in English | MEDLINE | ID: covidwho-1329250

ABSTRACT

PURPOSE: SARS-CoV-2 infection is associated with substantial mortality and high morbidity. This study tested the effect of angiotensin II type I receptor blocker, losartan, on SARS-CoV-2 replication and inhibition of the papain-like protease of the virus. METHODS: The dose-dependent inhibitory effect of losartan, in concentrations from 1µM to 100µM as determined by quantitative cell analysis combining fluorescence microscopy, image processing, and cellular measurements (Cellomics analysis) on SARS-CoV-2 replication was investigated in Vero E6 cells. The impact of losartan on deubiquitination and deISGylation of SARS-CoV-2 papain-like protease (PLpro) were also evaluated.  Results: Losartan reduced PLpro cleavage of tetraUbiquitin to diUbiquitin.  It was less effective in inhibiting PLpro's cleavage of ISG15-AMC than Ubiquitin-AMC.  To determine if losartan inhibited SARS-CoV-2 replication, losartan treatment of SARS-CoV-2 infected Vero E6 was examined. Losartan treatment one hour prior to SARS-CoV-2 infection reduced levels of SARS-CoV-2 nuclear protein, an indicator of virus replication, by 80% and treatment one-hour post-infection decreased viral replication by 70%. CONCLUSION: Losartan was not an effective inhibitor of deubiquitinase or deISGylase activity of the PLpro but affected the SARS-CoV-2 replication of Vero E6 cells in vitro.  As losartan has a favorable safety profile and is currently available it has features necessary for efficacious drug repurposing and treatment of COVID-19.


Subject(s)
Angiotensin II Type 1 Receptor Blockers/pharmacology , Antiviral Agents/pharmacology , Losartan/pharmacology , SARS-CoV-2/drug effects , Animals , COVID-19/drug therapy , Chlorocebus aethiops , Computational Biology , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Coronavirus Papain-Like Proteases/metabolism , Deubiquitinating Enzymes/antagonists & inhibitors , Deubiquitinating Enzymes/metabolism , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Ubiquitin/metabolism , Vero Cells , Virus Replication/drug effects
15.
Nucleic Acids Res ; 49(13): 7695-7712, 2021 07 21.
Article in English | MEDLINE | ID: covidwho-1298980

ABSTRACT

The multidomain non-structural protein 3 (Nsp3) is the largest protein encoded by coronavirus (CoV) genomes and several regions of this protein are essential for viral replication. Of note, SARS-CoV Nsp3 contains a SARS-Unique Domain (SUD), which can bind Guanine-rich non-canonical nucleic acid structures called G-quadruplexes (G4) and is essential for SARS-CoV replication. We show herein that the SARS-CoV-2 Nsp3 protein also contains a SUD domain that interacts with G4s. Indeed, interactions between SUD proteins and both DNA and RNA G4s were evidenced by G4 pull-down, Surface Plasmon Resonance and Homogenous Time Resolved Fluorescence. These interactions can be disrupted by mutations that prevent oligonucleotides from folding into G4 structures and, interestingly, by molecules known as specific ligands of these G4s. Structural models for these interactions are proposed and reveal significant differences with the crystallographic and modeled 3D structures of the SARS-CoV SUD-NM/G4 interaction. Altogether, our results pave the way for further studies on the role of SUD/G4 interactions during SARS-CoV-2 replication and the use of inhibitors of these interactions as potential antiviral compounds.


Subject(s)
COVID-19/virology , Coronavirus Papain-Like Proteases/metabolism , G-Quadruplexes , Protein Interaction Domains and Motifs , SARS-CoV-2 , Amino Acid Sequence , Coronavirus Papain-Like Proteases/chemistry , Humans , Ligands , Models, Molecular , Protein Binding , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrum Analysis , Structure-Activity Relationship , Virus Replication
16.
Biochem J ; 478(13): 2517-2531, 2021 07 16.
Article in English | MEDLINE | ID: covidwho-1290988

ABSTRACT

The COVID-19 pandemic has emerged as the biggest life-threatening disease of this century. Whilst vaccination should provide a long-term solution, this is pitted against the constant threat of mutations in the virus rendering the current vaccines less effective. Consequently, small molecule antiviral agents would be extremely useful to complement the vaccination program. The causative agent of COVID-19 is a novel coronavirus, SARS-CoV-2, which encodes at least nine enzymatic activities that all have drug targeting potential. The papain-like protease (PLpro) contained in the nsp3 protein generates viral non-structural proteins from a polyprotein precursor, and cleaves ubiquitin and ISG protein conjugates. Here we describe the expression and purification of PLpro. We developed a protease assay that was used to screen a custom compound library from which we identified dihydrotanshinone I and Ro 08-2750 as compounds that inhibit PLpro in protease and isopeptidase assays and also inhibit viral replication in cell culture-based assays.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Drug Evaluation, Preclinical , SARS-CoV-2/enzymology , Small Molecule Libraries/pharmacology , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Aniline Compounds/pharmacology , Animals , Benzamides/pharmacology , Chlorocebus aethiops , Coronavirus Papain-Like Proteases/genetics , Coronavirus Papain-Like Proteases/isolation & purification , Coronavirus Papain-Like Proteases/metabolism , Drug Synergism , Enzyme Assays , Flavins/pharmacology , Fluorescence Resonance Energy Transfer , Furans/pharmacology , High-Throughput Screening Assays , Inhibitory Concentration 50 , Naphthalenes/pharmacology , Phenanthrenes/pharmacology , Quinones/pharmacology , Reproducibility of Results , SARS-CoV-2/drug effects , SARS-CoV-2/growth & development , Small Molecule Libraries/chemistry , Vero Cells , Virus Replication/drug effects
17.
Molecules ; 26(13)2021 Jun 22.
Article in English | MEDLINE | ID: covidwho-1288958

ABSTRACT

Spanish flu, polio epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases caused by RNA viruses. The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands affordable and reliable assays for testing antivirals. To test inhibitors of viral proteases, we have developed an inexpensive high-throughput assay based on fluorescent energy transfer (FRET). We assayed an array of inhibitors for papain-like protease from SARS-CoV-2 and validated it on protease from the tick-borne encephalitis virus to emphasize its versatility. The reaction progress is monitored as loss of FRET signal of the substrate. This robust and reproducible assay can be used for testing the inhibitors in 96- or 384-well plates.


Subject(s)
Antiviral Agents/pharmacology , Fluorescence Resonance Energy Transfer/methods , High-Throughput Screening Assays/methods , Protease Inhibitors/pharmacology , RNA Viruses/enzymology , COVID-19/drug therapy , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/genetics , Coronavirus Papain-Like Proteases/metabolism , Drug Evaluation, Preclinical , Encephalitis Viruses, Tick-Borne/enzymology , Fluorescent Dyes/chemistry , Humans , RNA Helicases/antagonists & inhibitors , RNA Helicases/chemistry , RNA Helicases/genetics , RNA Helicases/metabolism , SARS-CoV-2/enzymology , Serine Endopeptidases/chemistry , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
18.
J Phys Chem Lett ; 12(23): 5608-5615, 2021 Jun 17.
Article in English | MEDLINE | ID: covidwho-1263456

ABSTRACT

Papain-like protease (PLpro) from SARS-CoV-2 plays essential roles in the replication cycle of the virus. In particular, it preferentially interacts with and cleaves human interferon-stimulated gene 15 (hISG15) to suppress the innate immune response of the host. We used small-angle X-ray and neutron scattering combined with computational techniques to study the mechanism of interaction of SARS-CoV-2 PLpro with hISG15. We showed that hISG15 undergoes a transition from an extended to a compact state after binding to PLpro, a conformation that has not been previously observed in complexes of SARS-CoV-2 PLpro with ISG15 from other species. Furthermore, computational analysis showed significant conformational flexibility in the ISG15 N-terminal domain, suggesting that it is weakly bound to PLpro and supports a binding mechanism that is dominated by the C-terminal ISG15 domain. This study fundamentally improves our understanding of the SARS-CoV-2 deISGylation complex that will help guide development of COVID-19 therapeutics targeting this complex.


Subject(s)
Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/metabolism , Cytokines/chemistry , Cytokines/metabolism , Interferons/metabolism , SARS-CoV-2/metabolism , Ubiquitins/chemistry , Ubiquitins/metabolism , Coronavirus Papain-Like Proteases/genetics , Cytokines/genetics , Humans , Neutron Diffraction , Protein Conformation , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , Scattering, Small Angle , Ubiquitins/genetics , X-Ray Diffraction
19.
Biophys Chem ; 276: 106610, 2021 09.
Article in English | MEDLINE | ID: covidwho-1252522

ABSTRACT

In the new millennium, the outbreak of new coronavirus has happened three times: SARS-CoV, MERS-CoV, and SARS-CoV-2. Unfortunately, we still have no pharmaceutical weapons against the diseases caused by these viruses. The pandemic of SARS-CoV-2 reminds us the urgency to search new drugs with totally different mechanism that may target the weaknesses specific to coronaviruses. Herein, we disclose a computational evaluation of targeted oxidation strategy (TOS) for potential inhibition of SARS-CoV-2 by disulfiram, a 70-year-old anti-alcoholism drug, and predict a multiple-target mechanism. A preliminary list of promising TOS drug candidates targeting the two thiol proteases of SARS-CoV-2 are proposed upon virtual screening of 32,143 disulfides.


Subject(s)
Alcohol Deterrents/chemistry , Antiviral Agents/chemistry , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Disulfiram/chemistry , Protease Inhibitors/chemistry , SARS-CoV-2/chemistry , Alcohol Deterrents/pharmacology , Antiviral Agents/pharmacology , COVID-19/drug therapy , Catalytic Domain , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/metabolism , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/genetics , Coronavirus Papain-Like Proteases/metabolism , Disulfiram/pharmacology , Drug Repositioning , Gene Expression , Humans , Kinetics , Molecular Docking Simulation , Oxidation-Reduction , Protease Inhibitors/pharmacology , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Quantum Theory , SARS-CoV-2/enzymology , Substrate Specificity , Thermodynamics
20.
PLoS One ; 16(5): e0251910, 2021.
Article in English | MEDLINE | ID: covidwho-1234593

ABSTRACT

The COVID-19 disease has infected and killed countless people all over the world since its emergence at the end of 2019. No specific therapy for COVID-19 is not currently available, and urgent treatment solutions are needed. Recent studies have found several potential molecular targets, and one of the most critical proteins of the SARS-CoV-2 virus work machine is the Papain-like protease (Plpro). Potential inhibitors are available, and their X-ray crystallographic structures in complex with this enzyme have been determined recently. However, their activities against this enzyme are insufficient and need to be characterized and improved to be of clinical values. Therefore, in this work, by utilizing the Supervised Molecular Dynamics (SuMD) simulation method, we achieved multiple unbinding events of Plpro inhibitors, GRL0617, and its derivates, and captured and understood the details of the unbinding pathway. We found that residues of the BL2 loop, such as Tyr268 and Gln269, play major roles in the unbinding pathways, but the most important contributing factor is the natural movements and behavior of the BL2 loop, which can control the entire process. We believe that the details found in this study can be used to refine and optimize potential inhibitors like GRL0617 and design more efficacious inhibitors as a treatment for the SARS-CoV-2 virus.


Subject(s)
COVID-19/drug therapy , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Protease Inhibitors/pharmacology , SARS-CoV-2/enzymology , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Binding Sites/drug effects , COVID-19/enzymology , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/metabolism , Crystallography, X-Ray , Humans , Molecular Dynamics Simulation , Papain/metabolism , Protease Inhibitors/chemistry , Protein Binding/drug effects , SARS-CoV-2/drug effects
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