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1.
Front Immunol ; 13: 947272, 2022.
Article in English | MEDLINE | ID: covidwho-2005872

ABSTRACT

The newly emerged severe acute respiratory syndrome (SARS) coronavirus-2 (SARS-CoV-2) can result in dysregulated interferon (IFN) responses that contribute to disease severity. The papain-like protease of SARS-CoV-2 (SCoV2-PLpro) has been previously reported to attenuate IFN responses, but the underlying mechanism is not fully understood. In this study, we found that SCoV2-PLpro potently suppressed IFN production and signaling induced by Sendai virus as well as RIG-I-like receptor (RLR) signaling pathway components, including RIG-I, MAVS, TBK1, TRAF3, TRAF6, and IRF3. SCoV2-PLpro exhibited different specificity and efficiency than SARS-CoV PLpro, with the former exerting a greater inhibitory effect on the RIG-I- and TRAF3-mediated IFN response but a weaker effect on the MAVS-mediated IFN response. Furthermore, we showed that SCoV2-PLpro significantly reduced K63-ubiquitination of RIG-I, MAVS, TBK1, TRAF3, TRAF6, and IRF3 and K48-ubiquitination of IκBα, which are known critical for the innate immune signal transduction. The deubiquitinating (DUB) activity of SCoV2-PLpro required a catalytic residue cysteine 111 (C111) but not the UBL domain. Notably, by utilizing the DUB-defective C111 mutant, we demonstrated that SCoV2-PLpro targeted RLR signaling pathway regulators via deubiquitination-dependent and -independent mechanisms, with the inhibitory activities of RIG-I and TBK1 correlating with DUB function, whereas the antagonism effects on MAVS, TRAF3, TRAF6, and IRF3 independent on DUB activity. Overall, our results reveal that SCoV2-PLpro evolves differential IFN antagonism activity from SCoV1-PLpro and it targets multiple key RLR signaling pathway components via various mechanisms, providing insights into SARS-CoV-2 pathogenesis and clues for developing antiviral therapies for COVID-19.


Subject(s)
Coronavirus Papain-Like Proteases , DEAD Box Protein 58 , Receptors, Immunologic , SARS-CoV-2 , Signal Transduction , COVID-19 , Coronavirus Papain-Like Proteases/metabolism , DEAD Box Protein 58/metabolism , Humans , Receptors, Immunologic/metabolism , SARS-CoV-2/enzymology , Ubiquitination
2.
Mar Drugs ; 20(3)2022 Feb 28.
Article in English | MEDLINE | ID: covidwho-1715534

ABSTRACT

Several natural products recovered from a marine-derived Aspergillus niger were tested for their inhibitory activity against SARS CoV-2 in vitro. Aurasperone A (3) was found to inhibit SARS CoV-2 efficiently (IC50 = 12.25 µM) with comparable activity with the positive control remdesivir (IC50 = 10.11 µM). Aurasperone A exerted minimal cytotoxicity on Vero E6 cells (CC50 = 32.36 mM, SI = 2641.5) and it was found to be much safer than remdesivir (CC50 = 415.22 µM, SI = 41.07). To putatively highlight its molecular target, aurasperone A was subjected to molecular docking against several key-viral protein targets followed by a series of molecular dynamics-based in silico experiments that suggested Mpro to be its primary viral protein target. More potent anti-SARS CoV-2 Mpro inhibitors can be developed according to our findings presented in the present investigation.


Subject(s)
Antiviral Agents/pharmacology , Chromones/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Animals , Antiviral Agents/isolation & purification , Aspergillus niger/chemistry , Chlorocebus aethiops , Chromones/isolation & purification , Coronavirus 3C Proteases/metabolism , Coronavirus Papain-Like Proteases/metabolism , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Molecular Docking Simulation , Protease Inhibitors/isolation & purification , RNA Helicases/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
3.
Sci Rep ; 12(1): 2145, 2022 02 09.
Article in English | MEDLINE | ID: covidwho-1692555

ABSTRACT

The most common host entry point of human adapted coronaviruses (CoV) including SARS-CoV-2 is through the initial colonization in the nostril and mouth region which is responsible for spread of the infection. Most recent studies suggest that the commercially available oral and nasal rinse products are effective in inhibiting the viral replication. However, the anti-viral mechanism of the active ingredients present in the oral rinses have not been studied. In the present study, we have assessed in vitro enzymatic inhibitory activity of active ingredients in the oral mouth rinse products: aloin A and B, chlorhexidine, eucalyptol, hexetidine, menthol, triclosan, methyl salicylate, sodium fluoride and povidone, against two important proteases of SARS-CoV-2 PLpro and 3CLpro. Our results indicate only aloin A and B effectively inhibited proteolytic activity of PLpro with an IC50 of 13.16 and 16.08 µM. Interestingly, neither of the aloin isoforms inhibited 3CLpro enzymatic activity. Computational structural modelling of aloin A and B interaction with PLpro revealed that, both aloin isoforms form hydrogen bond with Tyr268 of PLpro, which is critical for their proteolytic activity. Furthermore, 100 ns molecular dynamics (MD) simulation studies predicted that both aloin isoforms have strong interaction with Glu167, which is required for PLpro deubiquitination activity. Our results from the in vitro deubiquitinase inhibition assay show that aloin A and B isomers exhibit deubiquitination inhibitory activity with an IC50 value of 15.68 and 17.51 µM, respectively. In conclusion, the isoforms of aloin inhibit both proteolytic and the deubiquitinating activity of SARS-CoV-2 PLpro, suggesting potential in inhibiting the replication of SARS-CoV-2 virus.


Subject(s)
Coronavirus Papain-Like Proteases/metabolism , Emodin/analogs & derivatives , SARS-CoV-2/enzymology , Animals , Binding Sites , COVID-19/pathology , COVID-19/virology , Cell Survival/drug effects , Chlorocebus aethiops , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/metabolism , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Emodin/chemistry , Emodin/metabolism , Emodin/pharmacology , Humans , Molecular Dynamics Simulation , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Protein Isoforms/pharmacology , SARS-CoV-2/isolation & purification , Vero Cells
4.
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/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 , COVID-19 Drug Treatment
5.
Angew Chem Int Ed Engl ; 61(9): e202113617, 2022 02 21.
Article in English | MEDLINE | ID: covidwho-1565164

ABSTRACT

The main protease (Mpro ) and papain-like protease (PLpro ) play critical roles in SARS-CoV-2 replication and are promising targets for antiviral inhibitors. The simultaneous visualization of Mpro and PLpro is extremely valuable for SARS-CoV-2 detection and rapid inhibitor screening. However, such a crucial investigation has remained challenging because of the lack of suitable probes. We have now developed a dual-color probe (3MBP5) for the simultaneous detection of Mpro and PLpro by fluorescence (or Förster) resonance energy transfer (FRET). This probe produces fluorescence from both the Cy3 and Cy5 fluorophores that are cleaved by Mpro and PLpro . 3MBP5-activatable specificity was demonstrated with recombinant proteins, inhibitors, plasmid-transfected HEK 293T cells, and SARS-CoV-2-infected TMPRSS2-Vero cells. Results from the dual-color probe first verified the simultaneous detection and intracellular distribution of SARS-CoV-2 Mpro and PLpro . This is a powerful tool for the simultaneous detection of different proteases with value for the rapid screening of inhibitors.


Subject(s)
Color , Coronavirus 3C Proteases/metabolism , Coronavirus Papain-Like Proteases/metabolism , Fluorescent Dyes/chemistry , Protease Inhibitors/pharmacology , SARS-CoV-2/enzymology , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Fluorescence Resonance Energy Transfer , HEK293 Cells , Humans
6.
J Am Chem Soc ; 143(48): 20095-20108, 2021 12 08.
Article in English | MEDLINE | ID: covidwho-1531986

ABSTRACT

Chemical modifications of native proteins can affect their stability, activity, interactions, localization, and more. However, there are few nongenetic methods for the installation of chemical modifications at a specific protein site in cells. Here we report a covalent ligand directed release (CoLDR) site-specific labeling strategy, which enables the installation of a variety of functional tags on a target protein while releasing the directing ligand. Using this approach, we were able to label various proteins such as BTK, K-RasG12C, and SARS-CoV-2 PLpro with different tags. For BTK we have shown selective labeling in cells of both alkyne and fluorophores tags. Protein labeling by traditional affinity methods often inhibits protein activity since the directing ligand permanently occupies the target binding pocket. We have shown that using CoLDR chemistry, modification of BTK by these probes in cells preserves its activity. We demonstrated several applications for this approach including determining the half-life of BTK in its native environment with minimal perturbation, as well as quantification of BTK degradation by a noncovalent proteolysis targeting chimera (PROTAC) by in-gel fluorescence. Using an environment-sensitive "turn-on" fluorescent probe, we were able to monitor ligand binding to the active site of BTK. Finally, we have demonstrated efficient CoLDR-based BTK PROTACs (DC50 < 100 nM), which installed a CRBN binder onto BTK. This approach joins very few available labeling strategies that maintain the target protein activity and thus makes an important addition to the toolbox of chemical biology.


Subject(s)
Agammaglobulinaemia Tyrosine Kinase/chemistry , Fluorescent Dyes/chemistry , Ligands , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Adenine/analogs & derivatives , Adenine/chemistry , Adenine/metabolism , Agammaglobulinaemia Tyrosine Kinase/metabolism , Catalytic Domain , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/metabolism , Half-Life , Humans , Piperidines/chemistry , Piperidines/metabolism , Proteolysis , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Pyrimidines/chemistry , Pyrimidines/metabolism , SARS-CoV-2/enzymology
7.
Infect Genet Evol ; 96: 105155, 2021 12.
Article in English | MEDLINE | ID: covidwho-1525880

ABSTRACT

The present study aimed to predict the binding potential of carbon nanotube and nano fullerene towards multiple targets of SARS-CoV-2. Based on the virulent functions, the spike glycoprotein, RNA-dependent RNA polymerase, main protease, papain-like protease, and RNA binding domain of the nucleocapsid proteins of SARS-CoV-2 were prioritized as the molecular targets and their three-dimensional (3D) structures were retrieved from the Protein Data Bank. The 3D structures of carbon nanotubes and nano-fullerene were computationally modeled, and the binding potential of these nanoparticles to the selected molecular targets was predicted by molecular docking and molecular dynamic (MD) simulations. The drug-likeness and pharmacokinetic features of the lead molecules were computationally predicted. The current study suggested that carbon fullerene and nanotube demonstrated significant binding towards the prioritized multi-targets of SARS-CoV-2. Interestingly, carbon nanotube showed better interaction with these targets when compared to carbon fullerene. MD simulation studies clearly showed that the interaction of nanoparticles and selected targets possessed stability and conformational changes. This study revealed that carbon nanotubes and fullerene are probably used as effectual binders to multiple targets of SARS-CoV-2, and the study offers insights into the experimental validation and highlights the relevance of utilizing carbon nanomaterials as a therapeutic remedy against COVID-19.


Subject(s)
Fullerenes/metabolism , Nanotubes, Carbon , SARS-CoV-2/metabolism , Viral Proteins/chemistry , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/metabolism , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/metabolism , Fullerenes/chemistry , Fullerenes/pharmacokinetics , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Nanotubes, Carbon/chemistry , Phosphoproteins/chemistry , Phosphoproteins/metabolism , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Viral Proteins/metabolism
8.
Int J Mol Sci ; 22(22)2021 Nov 17.
Article in English | MEDLINE | ID: covidwho-1524024

ABSTRACT

The worldwide outbreak of COVID-19 was caused by a pathogenic virus called Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Therapies against SARS-CoV-2 target the virus or human cells or the immune system. However, therapies based on specific antibodies, such as vaccines and monoclonal antibodies, may become inefficient enough when the virus changes its antigenicity due to mutations. Polyphenols are the major class of bioactive compounds in nature, exerting diverse health effects based on their direct antioxidant activity and their effects in the modulation of intracellular signaling. There are currently numerous clinical trials investigating the effects of polyphenols in prophylaxis and the treatment of COVID-19, from symptomatic, via moderate and severe COVID-19 treatment, to anti-fibrotic treatment in discharged COVID-19 patients. Antiviral activities of polyphenols and their impact on immune system modulation could serve as a solid basis for developing polyphenol-based natural approaches for preventing and treating COVID-19.


Subject(s)
Antiviral Agents/therapeutic use , COVID-19/prevention & control , Polyphenols/therapeutic use , Antiviral Agents/chemistry , Antiviral Agents/metabolism , COVID-19/virology , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/metabolism , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Coronavirus Papain-Like Proteases/metabolism , Humans , Plants, Medicinal/chemistry , Plants, Medicinal/metabolism , Polyphenols/chemistry , Polyphenols/metabolism , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/metabolism
9.
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/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 , COVID-19 Drug Treatment
10.
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/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 , COVID-19 Drug Treatment
11.
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
12.
J Med Chem ; 65(4): 2940-2955, 2022 02 24.
Article in English | MEDLINE | ID: covidwho-1475245

ABSTRACT

Antiviral agents that complement vaccination are urgently needed to end the COVID-19 pandemic. The SARS-CoV-2 papain-like protease (PLpro), one of only two essential cysteine proteases that regulate viral replication, also dysregulates host immune sensing by binding and deubiquitination of host protein substrates. PLpro is a promising therapeutic target, albeit challenging owing to featureless P1 and P2 sites recognizing glycine. To overcome this challenge, we leveraged the cooperativity of multiple shallow binding sites on the PLpro surface, yielding novel 2-phenylthiophenes with nanomolar inhibitory potency. New cocrystal structures confirmed that ligand binding induces new interactions with PLpro: by closing of the BL2 loop of PLpro forming a novel "BL2 groove" and by mimicking the binding interaction of ubiquitin with Glu167 of PLpro. Together, this binding cooperativity translates to the most potent PLpro inhibitors reported to date, with slow off-rates, improved binding affinities, and low micromolar antiviral potency in SARS-CoV-2-infected human cells.


Subject(s)
Antiviral Agents/pharmacology , COVID-19 Drug Treatment , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Binding Sites/drug effects , COVID-19/metabolism , Coronavirus Papain-Like Proteases/isolation & purification , Coronavirus Papain-Like Proteases/metabolism , Crystallography, X-Ray , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/chemistry , Humans , Microbial Sensitivity Tests , Microsomes, Liver/chemistry , Microsomes, Liver/metabolism , Models, Molecular , Pandemics , Surface Plasmon Resonance , Tumor Cells, Cultured
13.
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
14.
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/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 , COVID-19 Drug Treatment
15.
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 Treatment , 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
16.
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
17.
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
18.
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 , 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 , COVID-19 Drug Treatment
19.
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 Treatment , 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
20.
Mol Biotechnol ; 64(1): 1-8, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1366408

ABSTRACT

Because of the essential roles of SARS-CoV-2 papain-like protease (PLpro) in the viral polyprotein processing and suppression of host immune responses, it is a crucial target for drug discovery against COVID-19. To develop robust biochemical methodologies for inhibitor screening against PLpro, extensive characterization of recombinant protein is important. Here we report cloning, expression, and purification of the recombinant SARS-CoV-2 PLpro, and explore various parameters affecting its stability and the catalytic activity. We also report the optimum conditions which should be used for high-throughput inhibitor screening using a fluorogenic tetrapeptide substrate.


Subject(s)
Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/metabolism , High-Throughput Screening Assays/methods , Antiviral Agents/pharmacology , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Coronavirus Papain-Like Proteases/isolation & purification , Coumarins/chemistry , Coumarins/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Dimethyl Sulfoxide/chemistry , Dynamic Light Scattering , Edetic Acid/chemistry , Enzyme Stability , Fluorometry/methods , Hydrogen-Ion Concentration , Osmolar Concentration , Peptides/chemistry , Peptides/metabolism , Temperature
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