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1.
Molecules ; 27(21)2022 Nov 04.
Article in English | MEDLINE | ID: covidwho-2099669

ABSTRACT

Middle East respiratory syndrome coronavirus (MERS-CoV), belonging to the betacoronavirus genus can cause severe respiratory illnesses, accompanied by pneumonia, multiorgan failure, and ultimately death. CoVs have the ability to transgress species barriers and spread swiftly into new host species, with human-to-human transmission causing epidemic diseases. Despite the severe public health threat of MERS-CoV, there are currently no vaccines or drugs available for its treatment. MERS-CoV papain-like protease (PLpro) is a key enzyme that plays an important role in its replication. In the present study, we evaluated the inhibitory activities of doxorubicin (DOX) against the recombinant MERS-CoV PLpro by employing protease inhibition assays. Hydrolysis of fluorogenic peptide from the Z-RLRGG-AMC-peptide bond in the presence of DOX showed an IC50 value of 1.67 µM at 30 min. Subsequently, we confirmed the interaction between DOX and MERS-CoV PLpro by thermal shift assay (TSA), and DOX increased ΔTm by ~20 °C, clearly indicating a coherent interaction between the MERS-CoV PL protease and DOX. The binding site of DOX on MERS-CoV PLpro was assessed using docking techniques and molecular dynamic (MD) simulations. DOX bound to the thumb region of the catalytic domain of the MERS-CoV PLpro. MD simulation results showed flexible BL2 loops, as well as other potential residues, such as R231, R233, and G276 of MERS-CoV PLpro. Development of drug repurposing is a remarkable opportunity to quickly examine the efficacy of different aspects of treating various diseases. Protease inhibitors have been found to be effective against MERS-CoV to date, and numerous candidates are currently undergoing clinical trials to prove this. Our effort follows a in similar direction.


Subject(s)
Middle East Respiratory Syndrome Coronavirus , Humans , Middle East Respiratory Syndrome Coronavirus/metabolism , Papain/chemistry , Peptide Hydrolases/metabolism , Drug Repositioning , Doxorubicin/pharmacology , Doxorubicin/metabolism
3.
Int J Mol Sci ; 23(18)2022 Sep 19.
Article in English | MEDLINE | ID: covidwho-2071502

ABSTRACT

The chymotrypsin-like cysteine protease (3CLpro, also known as main protease-Mpro) and papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been used as the main targets for screening potential synthetic inhibitors for posterior in vitro evaluation of the most promising compounds. In this sense, the present work reports for the first time the evaluation of the interaction between Mpro/PLpro with a series of 17 porphyrin analogues-corrole (C1), meso-aryl-corrole (C2), and 15 fluorinated-meso-aryl-corrole derivatives (C3-C17) via molecular docking calculations. The impact of fluorine atoms on meso-aryl-corrole structure was also evaluated in terms of binding affinity and physical-chemical properties by two-dimensional quantitative structure-activity relationship (2D-QSAR). The presence of phenyl moieties increased the binding capacity of corrole for both proteases and depending on the position of fluorine atoms might impact positively or negatively the binding capacity. For Mpro the para-fluorine atoms might decrease drastically the binding capacity, while for PLpro there was a certain increase in the binding affinity of fluorinated-corroles with the increase of fluorine atoms into meso-aryl-corrole structure mainly from tri-fluorinated insertions. The 2D-QSAR models indicated two separated regions of higher and lower affinity for Mpro:C1-C17 based on dual electronic parameters (σI and σR), as well as one model was obtained with a correlation between the docking score value of Mpro:C2-C17 and the corresponding 13C nuclear magnetic resonance (NMR) chemical shifts of the sp2 carbon atoms (δC-1 and δC-2) of C2-C17. Overall, the fluorinated-meso-aryl-corrole derivatives showed favorable in silico parameters as potential synthetic compounds for future in vitro assays on the inhibition of SARS-CoV-2 replication.


Subject(s)
COVID-19 , Porphyrins , Antiviral Agents/pharmacology , COVID-19/drug therapy , Carbon , Chymotrypsin , Coronavirus 3C Proteases , Fluorine , Humans , Molecular Docking Simulation , Papain , Peptide Hydrolases , Porphyrins/pharmacology , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Quantitative Structure-Activity Relationship , SARS-CoV-2
4.
Biochem J ; 479(20): 2175-2193, 2022 Oct 28.
Article in English | MEDLINE | ID: covidwho-2062282

ABSTRACT

Coronaviruses have been responsible for multiple challenging global pandemics, including coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Papain-like protease (PLpro), one of two cysteine proteases responsible for the maturation and infectivity of SARS-CoV-2, processes and liberates functional proteins from the viral polyproteins and cleaves ubiquitin and ISG15 modifications to inhibit innate immune sensing. Consequently, PLpro is an attractive target for developing COVID-19 therapies. PLpro contains a zinc-finger domain important for substrate binding and structural stability. However, the impact of metal ions on the activity and biophysical properties of SARS-CoV-2 PLpro has not been comprehensively studied. Here, we assessed the impacts of metal ions on the catalytic activity of PLpro. Zinc had the largest inhibitory effect on PLpro, followed by manganese. Calcium, magnesium, and iron had smaller or no effects on PLpro activity. EDTA at a concentration of 0.5 mM was essential for PLpro activity, likely by chelating trace metals that inhibit PLpro. IC50 values for ZnCl2, ZnSO4, and MnCl2 of 0.42 ± 0.02 mM, 0.35 ± 0.01 mM, and 2.6 ± 0.3 mM were obtained in the presence of 0.5 mM EDTA; in the absence of EDTA, the estimated IC50 of ZnCl2 was 14 µM. Tryptophan intrinsic fluorescence analysis confirmed the binding of zinc and manganese to PLpro, and differential scanning calorimetry revealed that zinc but not manganese reduced ΔHcal of PLpro. The results of this study provide a reference for further work targeting PLpro to prevent and treat COVID-19.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Papain/chemistry , Papain/metabolism , COVID-19/drug therapy , Peptide Hydrolases/metabolism , Magnesium , Calcium , Tryptophan , Edetic Acid , Ubiquitin/metabolism , Polyproteins , Ions , Zinc , Iron
5.
J Mol Model ; 28(11): 354, 2022 Oct 12.
Article in English | MEDLINE | ID: covidwho-2059878

ABSTRACT

The papain-like protease (PLpro) from SARS-CoV-2 is an important target for the development of antivirals against COVID-19. The safe drug disulfiram (DSF) presents antiviral activity inhibiting PLpro in vitro, and it is under clinical trial studies, indicating to be a promising anti-COVID-19 drug. In this work, we aimed to understand the mechanism of PLpro inhibition by DSF and verify if DSF metabolites and derivatives could be potential inhibitors too. Molecular docking, DFT, and ADMET techniques were applied. The carbamoylation of the active site cysteine residue by DSF metabolite (DETC-MeSO) is kinetically and thermodynamically favorable (ΔG‡ = 3.15 and ΔG = - 12.10 kcal mol-1, respectively). Our results strongly suggest that the sulfoxide metabolites from DSF are promising covalent inhibitors of PLpro and should be tested in in vitro and in vivo assays to confirm their antiviral action.


Subject(s)
COVID-19 , SARS-CoV-2 , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , COVID-19/drug therapy , Clinical Trials as Topic , Computational Chemistry , Cysteine , Disulfiram/metabolism , Disulfiram/pharmacology , Humans , Molecular Docking Simulation , Papain , Peptide Hydrolases , Protease Inhibitors/chemistry , Sulfoxides
6.
Int J Mol Sci ; 23(19)2022 Sep 24.
Article in English | MEDLINE | ID: covidwho-2043775

ABSTRACT

In late 2019, a new coronavirus (CoV) caused the outbreak of a deadly respiratory disease, resulting in the COVID-19 pandemic. In view of the ongoing pandemic, there is an immediate need to find drugs to treat patients. SARS-CoV-2 papain-like cysteine protease (PLpro) not only plays an important role in the pathogenesis of the virus but is also a target protein for the development of inhibitor drugs. Therefore, to develop targeted inhibitors, it is necessary to analyse and verify PLpro sites and explore whether there are other cryptic binding pockets with better activity. In this study, first, we detected the site of the whole PLpro protein by sitemap of Schrödinger (version 2018), the cavity of LigBuilder V3, and DeepSite, and roughly judged the possible activated binding site area. Then, we used the mixed solvent dynamics simulation (MixMD) of probe molecules to induce conformational changes in the protein to find the possible cryptic active sites. Finally, the TRAPP method was used to predict the druggability of cryptic pockets and analyse the changes in the physicochemical properties of residues around these sites. This work will help promote the research of SARS-CoV-2 PLpro inhibitors.


Subject(s)
COVID-19 , Papain , Amino Acid Sequence , COVID-19/drug therapy , Coronavirus Papain-Like Proteases , Humans , Pandemics , Papain/metabolism , SARS-CoV-2 , Solvents
7.
J Med Chem ; 65(19): 12562-12593, 2022 10 13.
Article in English | MEDLINE | ID: covidwho-2036741

ABSTRACT

Viral pathologies encompass activation of pro-oxidative pathways and inflammatory burst. Alleviating overproduction of reactive oxygen species and cytokine storm in COVID-19 is essential to counteract the immunogenic damage in endothelium and alveolar membranes. Antioxidants alleviate oxidative stress, cytokine storm, hyperinflammation, and diminish the risk of organ failure. Direct antiviral roles imply: impact on viral spike protein, interference with the ACE2 receptor, inhibition of dipeptidyl peptidase 4, transmembrane protease serine 2 or furin, and impact on of helicase, papain-like protease, 3-chyomotrypsin like protease, and RNA-dependent RNA polymerase. Prooxidative environment favors conformational changes in the receptor binding domain, promoting the affinity of the spike protein for the host receptor. Viral pathologies imply a vicious cycle, oxidative stress promoting inflammatory responses, and vice versa. The same was noticed with respect to the relationship antioxidant impairment-viral replication. Timing, dosage, pro-oxidative activities, mutual influences, and interference with other antioxidants should be carefully regarded. Deficiency is linked to illness severity.


Subject(s)
COVID-19 , SARS-CoV-2 , Angiotensin-Converting Enzyme 2 , Anti-Inflammatory Agents , Antioxidants/pharmacology , Antioxidants/therapeutic use , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Cytokine Release Syndrome , Dipeptidyl Peptidase 4 , Furin , Humans , Papain , RNA-Dependent RNA Polymerase , Reactive Oxygen Species , Serine , Spike Glycoprotein, Coronavirus/metabolism
8.
Molecules ; 27(18)2022 Sep 14.
Article in English | MEDLINE | ID: covidwho-2033067

ABSTRACT

The SARS-CoV-2 targets were evaluated for a set of FDA-approved drugs using a combination of drug repositioning and rigorous computational modeling methodologies such as molecular docking and molecular dynamics (MD) simulations followed by binding free energy calculations. Six FDA-approved drugs including, Ouabain, Digitoxin, Digoxin, Proscillaridin, Salinomycin and Niclosamide with promising anti-SARS-CoV-2 activity were screened in silico against four SARS-CoV-2 proteins-papain-like protease (PLpro), RNA-dependent RNA polymerase (RdRp), SARS-CoV-2 main protease (Mpro), and adaptor-associated kinase 1 (AAK1)-in an attempt to define their promising targets. The applied computational techniques suggest that all the tested drugs exhibited excellent binding patterns with higher scores and stable complexes compared to the native protein cocrystallized inhibitors. Ouabain was suggested to act as a dual inhibitor for both PLpro and Mpro enzymes, while Digitoxin bonded perfectly to RdRp. In addition, Salinomycin targeted PLpro. Particularly, Niclosamide was found to target AAK1 with greater affinity compared to the reference drug. Our study provides comprehensive molecular-level insights for identifying or designing novel anti-COVID-19 drugs.


Subject(s)
COVID-19 , Proscillaridin , Antiviral Agents/chemistry , Cysteine Endopeptidases/chemistry , Digitoxin , Digoxin , Drug Repositioning , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Niclosamide , Ouabain , Papain/metabolism , RNA-Dependent RNA Polymerase , SARS-CoV-2
9.
Comput Biol Med ; 149: 106049, 2022 10.
Article in English | MEDLINE | ID: covidwho-2007624

ABSTRACT

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections generate approximately one million virions per day, and the majority of available antivirals are ineffective against it due to the virus's inherent genetic mutability. This necessitates the investigation of concurrent inhibition of multiple SARS-CoV-2 targets. We show that fortunellin (acacetin 7-O-neohesperidoside), a phytochemical, is a promising candidate for preventing and treating coronavirus disease (COVID-19) by targeting multiple key viral target proteins. Fortunellin supports protective immunity while inhibiting pro-inflammatory cytokines and apoptosis pathways and protecting against tissue damage. Fortunellin is a phytochemical found in Gojihwadi kwath, an Indian traditional Ayurvedic formulation with an antiviral activity that is effective in COVID-19 patients. The mechanistic action of its antiviral activity, however, is unknown. The current study comprehensively evaluates the potential therapeutic mechanisms of fortunellin in preventing and treating COVID-19. We have used molecular docking, molecular dynamics simulations, free-energy calculations, host target mining of fortunellin, gene ontology enrichment, pathway analyses, and protein-protein interaction analysis. We discovered that fortunellin reliably binds to key targets that are necessary for viral replication, growth, invasion, and infectivity including Nucleocapsid (N-CTD) (-54.62 kcal/mol), Replicase-monomer at NSP-8 binding site (-34.48 kcal/mol), Replicase-dimer interface (-31.29 kcal/mol), Helicase (-30.02 kcal/mol), Papain-like-protease (-28.12 kcal/mol), 2'-O-methyltransferase (-23.17 kcal/mol), Main-protease (-21.63 kcal/mol), Replicase-monomer at dimer interface (-22.04 kcal/mol), RNA-dependent-RNA-polymerase (-19.98 kcal/mol), Nucleocapsid-NTD (-16.92 kcal/mol), and Endoribonuclease (-16.81 kcal/mol). Furthermore, we identify and evaluate the potential human targets of fortunellin and its effect on the SARS-CoV-2 infected tissues, including normal-human-bronchial-epithelium (NHBE) and lung cells and organoids such as pancreatic, colon, liver, and cornea using a network pharmacology approach. Thus, our findings indicate that fortunellin has a dual role; multi-target antiviral activities against SARS-CoV-2 and immunomodulatory capabilities against the host.


Subject(s)
COVID-19 , SARS-CoV-2 , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , COVID-19/drug therapy , Cytokines , Endoribonucleases , Flavonoids , Glycosides , Humans , Methyltransferases , Molecular Docking Simulation , Molecular Dynamics Simulation , Papain , Phytochemicals/pharmacology , RNA
10.
Chembiochem ; 23(19): e202200327, 2022 10 06.
Article in English | MEDLINE | ID: covidwho-1999838

ABSTRACT

Emerging variants of SARS-CoV-2 and potential novel epidemic coronaviruses underline the importance of investigating various viral proteins as potential drug targets. The papain-like protease of coronaviruses has been less explored than other viral proteins; however, its substantive role in viral replication and impact on the host immune response make it a suitable target to study. This review article focuses on the structure and function of the papain-like protease (PLpro ) of SARS-CoV-2, including variants of concern, and compares it to those of other coronaviruses, such as SARS-CoV-1 and MERS-CoV. The protease's recognition motif is mirrored in ubiquitin and ISG15, which are involved in the antiviral immune response. Inhibitors, including GRL0617 derivatives, and their prospects as potential future antiviral agents are also discussed.


Subject(s)
COVID-19 , Papain , Aniline Compounds , Antiviral Agents/chemistry , Benzamides , COVID-19/drug therapy , Coronavirus Papain-Like Proteases , Humans , Naphthalenes , Papain/chemistry , Papain/metabolism , Peptide Hydrolases/metabolism , Protease Inhibitors/pharmacology , SARS-CoV-2 , Ubiquitin/metabolism , Viral Proteins/chemistry
11.
Commun Biol ; 5(1): 805, 2022 08 11.
Article in English | MEDLINE | ID: covidwho-1991680

ABSTRACT

SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, facilitating cleavage of the viral polypeptide chain, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to support coronaviruses in evading the host's innate immune responses. We identified three phenolic compounds bound to PLpro, preventing essential molecular interactions to ISG15 by screening a natural compound library. The compounds identified by X-ray screening and complexed to PLpro demonstrate clear inhibition of PLpro in a deISGylation activity assay. Two compounds exhibit distinct antiviral activity in Vero cell line assays and one inhibited a cytopathic effect in non-cytotoxic concentration ranges. In the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections.


Subject(s)
Antiviral Agents , COVID-19 , Allosteric Site , Antiviral Agents/pharmacology , COVID-19/drug therapy , Coronavirus Papain-Like Proteases , Humans , Papain/metabolism , Peptide Hydrolases/metabolism , SARS-CoV-2
12.
J Biol Chem ; 298(8): 102250, 2022 08.
Article in English | MEDLINE | ID: covidwho-1991134

ABSTRACT

Rubella, a viral disease characterized by a red skin rash, is well controlled because of an effective vaccine, but outbreaks are still occurring in the absence of available antiviral treatments. The Rubella virus (RUBV) papain-like protease (RubPro) is crucial for RUBV replication, cleaving the nonstructural polyprotein p200 into two multifunctional proteins, p150 and p90. This protease could represent a potential drug target, but structural and mechanistic details important for the inhibition of this enzyme are unclear. Here, we report a novel crystal structure of RubPro at a resolution of 1.64 Å. The RubPro adopts a unique papain-like protease fold, with a similar catalytic core to that of proteases from Severe acute respiratory syndrome coronavirus 2 and foot-and-mouth disease virus while having a distinctive N-terminal fingers domain. RubPro has well-conserved sequence motifs that are also found in its newly discovered Rubivirus relatives. In addition, we show that the RubPro construct has protease activity in trans against a construct of RUBV protease-helicase and fluorogenic peptides. A protease-helicase construct, exogenously expressed in Escherichia coli, was also cleaved at the p150-p90 cleavage junction, demonstrating protease activity of the protease-helicase protein. We also demonstrate that RubPro possesses deubiquitylation activity, suggesting a potential role of RubPro in modulating the host's innate immune responses. We anticipate that these structural and functional insights of RubPro will advance our current understanding of its function and help facilitate more structure-based research into the RUBV replication machinery, in hopes of developing antiviral therapeutics against RUBV.


Subject(s)
Peptide Hydrolases , Rubella virus , Amino Acid Motifs , Papain/chemistry , Peptide Hydrolases/chemistry , Peptide Hydrolases/metabolism , Protein Folding , Protein Structure, Tertiary , Rubella virus/chemistry , Rubella virus/enzymology
13.
Virology ; 574: 18-24, 2022 09.
Article in English | MEDLINE | ID: covidwho-1984216

ABSTRACT

The global scourge of COVID-19 is a serious threat to public health, but effective therapies remain very limited for this disease. Therefore, the discovery of novel antiviral agents is urgently needed to fight against COVID-19. In the lifecycle of SARS-CoV-2, the causing pathogen of COVID-19, papain-like protease (PLpro) is responsible for the cleavage of polyprotein into functional units as well as immune evasion of vaccines. Hence, PLpro has been regarded as an attractive target to develop antiviral agents. Herein, we first developed a robust and simple sandwich-like fluorescence polarization (FP) screening assay for the discovery of PLpro inhibitors, and identified anacardic acid as a novel competitive inhibitor against PLpro in vitro with an IC50 value of 24.26 ± 0.4 µM. This reliable FP screening assay could provide a prospective avenue for rapid discovery of antiviral agents targeting PLpro in a large-scale screening.


Subject(s)
COVID-19 , SARS-CoV-2 , Antiviral Agents/pharmacology , Coronavirus Papain-Like Proteases , Fluorescence Polarization , Humans , Papain , Peptide Hydrolases , Prospective Studies
14.
Neurotox Res ; 40(5): 1553-1569, 2022 Oct.
Article in English | MEDLINE | ID: covidwho-1966190

ABSTRACT

Since the appearance of SARS-CoV-2 and the COVID-19 pandemic, the search for new approaches to treat this disease took place in the scientific community. The in silico approach has gained importance at this moment, once the methodologies used in this kind of study allow for the identification of specific protein-ligand interactions, which may serve as a filter step for molecules that can act as specific inhibitors. In addition, it is a low-cost and high-speed technology. Molecular docking has been widely used to find potential viral protein inhibitors for structural and non-structural proteins of the SARS-CoV-2, aiming to block the infection and the virus multiplication. The papain-like protease (PLpro) participates in the proteolytic processing of SARS-CoV-2 and composes one of the main targets studied for pharmacological intervention by in silico methodologies. Based on that, we performed a systematic review about PLpro inhibitors from the perspective of in silico research, including possible therapeutic molecules in relation to this viral protein. The neurological problems triggered by COVID-19 were also briefly discussed, especially relative to the similarities of neuroinflammation present in Alzheimer's disease. In this context, we focused on two molecules, curcumin and glycyrrhizinic acid, given their PLpro inhibitory actions and neuroprotective properties and potential therapeutic effects on COVID-19.


Subject(s)
COVID-19 , Curcumin , COVID-19/drug therapy , Glycyrrhizic Acid , Humans , Ligands , Molecular Docking Simulation , Pandemics , Papain/chemistry , Papain/metabolism , Peptide Hydrolases/metabolism , SARS-CoV-2 , Viral Proteins/chemistry , Viral Proteins/metabolism
15.
Acta Biochim Biophys Sin (Shanghai) ; 54(8): 1133-1139, 2022 Aug 25.
Article in English | MEDLINE | ID: covidwho-1964135

ABSTRACT

The coronavirus papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for viral polypeptide cleavage and the deISGylation of interferon-stimulated gene 15 (ISG15), which enable it to participate in virus replication and host innate immune pathways. Therefore, PLpro is considered an attractive antiviral drug target. Here, we show that parthenolide, a germacrane sesquiterpene lactone, has SARS-CoV-2 PLpro inhibitory activity. Parthenolide covalently binds to Cys-191 or Cys-194 of the PLpro protein, but not the Cys-111 at the PLpro catalytic site. Mutation of Cys-191 or Cys-194 reduces the activity of PLpro. Molecular docking studies show that parthenolide may also form hydrogen bonds with Lys-192, Thr-193, and Gln-231. Furthermore, parthenolide inhibits the deISGylation but not the deubiquitinating activity of PLpro in vitro. These results reveal that parthenolide inhibits PLpro activity by allosteric regulation.


Subject(s)
COVID-19 , Coronavirus Papain-Like Proteases , Antiviral Agents/pharmacology , COVID-19/drug therapy , Humans , Interferons , Lactones , Molecular Docking Simulation , Papain/chemistry , Papain/metabolism , Peptide Hydrolases/metabolism , SARS-CoV-2 , Sesquiterpenes , Sesquiterpenes, Germacrane , Ubiquitin/metabolism
16.
Int J Mol Sci ; 23(14)2022 Jul 15.
Article in English | MEDLINE | ID: covidwho-1938839

ABSTRACT

In the SARS-CoV-2 lifecycle, papain-like protease PLpro cuts off the non-structural proteins nsp1, nsp2, and nsp3 from a large polyprotein. This is the earliest viral enzymatic activity, which is crucial for all downstream steps. Here, we designed two genetically encoded fluorescent sensors for the real-time detection of PLpro activity in live cells. The first sensor was based on the Förster resonance energy transfer (FRET) between the red fluorescent protein mScarlet as a donor and the biliverdin-binding near-infrared fluorescent protein miRFP670 as an acceptor. A linker with the PLpro recognition site LKGG in between made this FRET pair sensitive to PLpro cleavage. Upon the co-expression of mScarlet-LKGG-miRFP670 and PLpro in HeLa cells, we observed a gradual increase in the donor fluorescence intensity of about 1.5-fold. In the second sensor, both PLpro and its target-green mNeonGreen and red mScarletI fluorescent proteins separated by an LKGG-containing linker-were attached to the endoplasmic reticulum (ER) membrane. Upon cleavage by PLpro, mScarletI diffused from the ER throughout the cell. About a two-fold increase in the nucleus/cytoplasm ratio was observed as a result of the PLpro action. We believe that the new PLpro sensors can potentially be used to detect the earliest stages of SARS-CoV-2 propagation in live cells as well as for the screening of PLpro inhibitors.


Subject(s)
COVID-19 , SARS-CoV-2 , Coronavirus Papain-Like Proteases , HeLa Cells , Humans , Papain/metabolism , SARS-CoV-2/genetics
17.
Int J Mol Sci ; 23(10)2022 May 23.
Article in English | MEDLINE | ID: covidwho-1934115

ABSTRACT

Inhibition of the papain-like protease (PLpro) of SARS-CoV-2 has been demonstrated to be a successful target to prevent the spreading of the coronavirus in the infected body. In this regard, covalent inhibitors, such as the recently proposed VIR251 ligand, can irreversibly inactivate PLpro by forming a covalent bond with a specific residue of the catalytic site (Cys111), through a Michael addition reaction. An inhibition mechanism can therefore be proposed, including four steps: (i) ligand entry into the protease pocket; (ii) Cys111 deprotonation of the thiol group by a Brønsted-Lowry base; (iii) Cys111-S- addition to the ligand; and (iv) proton transfer from the protonated base to the covalently bound ligand. Evaluating the energetics and PLpro conformational changes at each of these steps could aid the design of more efficient and selective covalent inhibitors. For this aim, we have studied by means of MD simulations and QM/MM calculations the whole mechanism. Regarding the first step, we show that the inhibitor entry in the PLpro pocket is thermodynamically favorable only when considering the neutral Cys111, that is, prior to the Cys111 deprotonation. For the second step, MD simulations revealed that His272 would deprotonate Cys111 after overcoming an energy barrier of ca. 32 kcal/mol (at the QM/MM level), but implying a decrease of the inhibitor stability inside the protease pocket. This information points to a reversible Cys111 deprotonation, whose equilibrium is largely shifted toward the neutral Cys111 form. Although thermodynamically disfavored, if Cys111 is deprotonated in close proximity to the vinylic carbon of the ligand, then covalent binding takes place in an irreversible way (third step) to form the enolate intermediate. Finally, due to Cys111-S- negative charge redistribution over the bound ligand, proton transfer from the initially protonated His272 is favored, finally leading to an irreversibly modified Cys111 and a restored His272. These results elucidate the selectivity of Cys111 to enable formation of a covalent bond, even if a weak proton acceptor is available, as His272.


Subject(s)
COVID-19 , Protons , COVID-19/drug therapy , Coronavirus Papain-Like Proteases , Humans , Ligands , Papain/metabolism , Peptide Hydrolases/metabolism , SARS-CoV-2
18.
Viruses ; 14(7)2022 Jun 27.
Article in English | MEDLINE | ID: covidwho-1911658

ABSTRACT

The ubiquitin proteasome system (UPS), particularly its deubiquitinating enzymes (DUBs), play a key role in the replication cycle of coronaviruses. The SARS-CoV-2 papain-like protease (Plpro) is known to process the viral polyproteins to form the replicase transcriptase complex and to counteract the host viral response. Recently, it was shown that this viral protease can also act as a deubiquitinating enzyme. In this study, we demonstrate that certain DUB-Inhibitors (DIs) interfere with SARS-CoV-2 replication. The DIs PR-619 and HBX41108 restrict SARS-CoV-2 in both Vero B4 and human Calu-3 lung cells where cells were infected with a Multiplicity of Infection (MOI) of 0.02. An in vitro protease assay using recombinant Plpro and Amido-4-methylcoumarin (AMC)-conjugated substrate revealed that PR-619 and HBX41108 are able to block the protease at concentrations where the interventions restricted virus replication. In contrast, DIs that do not inhibit Plpro had no influence on virus replication, which indicated that the protease might be at least one major target. Future vertical studies that would gain more insights into the mechanisms of how DUBs effect the replication of SARS-CoV-2 will further validate them as a potential therapeutic target.


Subject(s)
COVID-19 , SARS-CoV-2 , Coronavirus Papain-Like Proteases , Deubiquitinating Enzymes , Humans , Papain , Peptide Hydrolases , Protease Inhibitors/pharmacology , Virus Replication
19.
Med Res Rev ; 42(6): 2126-2167, 2022 11.
Article in English | MEDLINE | ID: covidwho-1905913

ABSTRACT

The rising pandemic caused by a coronavirus, resulted in a scientific quest to discover some effective treatments against its etiologic agent, the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). This research represented a significant scientific landmark and resulted in many medical advances. However, efforts to understand the viral mechanism of action and how the human body machinery is subverted during the infection are still ongoing. Herein, we contributed to this field with this compilation of the roles of both viral and human enzymes in the context of SARS-CoV-2 infection. In this sense, this overview reports that proteases are vital for the infection to take place: from SARS-CoV-2 perspective, the main protease (Mpro ) and papain-like protease (PLpro ) are highlighted; from the human body, angiotensin-converting enzyme-2, transmembrane serine protease-2, and cathepsins (CatB/L) are pointed out. In addition, the influence of the virus on other enzymes is reported as the JAK/STAT pathway and the levels of lipase, enzymes from the cholesterol metabolism pathway, amylase, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and glyceraldehyde 3-phosphate dehydrogenase are also be disturbed in SARS-CoV-2 infection. Finally, this paper discusses the importance of detailed enzymatic studies for future treatments against SARS-CoV-2, and how some issues related to the syndrome treatment can create opportunities in the biotechnological market of enzymes and the development of new drugs.


Subject(s)
COVID-19 , Alanine Transaminase/pharmacology , Amylases/pharmacology , Angiotensins/pharmacology , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Aspartate Aminotransferases/pharmacology , COVID-19/drug therapy , Cathepsins/pharmacology , Cholesterol , Human Body , Humans , Janus Kinases/pharmacology , Lactate Dehydrogenases , Lipase/pharmacology , Papain/pharmacology , SARS-CoV-2 , STAT Transcription Factors/pharmacology , Serine Proteases/pharmacology , Signal Transduction
20.
Comput Biol Chem ; 99: 107721, 2022 Aug.
Article in English | MEDLINE | ID: covidwho-1906916

ABSTRACT

Papain like protease (PLpro) is a cysteine protease from the coronaviridae family of viruses. Coronaviruses possess a positive sense, single-strand RNA, leading to the translation of two viral polypeptides containing viral structural, non-structural and accessory proteins. PLpro is responsible for the cleavage of nsp1-3 from the viral polypeptide. PLpro also possesses deubiquitinating and deISGlyating activity, which sequesters the virus from the host's immune system. This indispensable attribute of PLpro makes it a protein of interest as a drug target. The present study aims to analyze the structural influences of ligand binding on PLpro. First, PLpro was screened against the ZINC-in-trials library, from which four lead compounds were identified based on estimated binding affinity and interaction patterns. Next, based on molecular docking results, ZINC000000596945, ZINC000064033452 and VIR251 (control molecule) were subjected to molecular dynamics simulation. The study evaluated global and essential dynamics analyses utilising principal component analyses, dynamic cross-correlation matrix, free energy landscape and time-dependant essential dynamics to predict the structural changes observed in PLpro upon ligand binding in a simulated environment. The MM/PBSA-based binding free energy calculations of the two selected molecules, ZINC000000596945 (-41.23 ± 3.70 kcal/mol) and ZINC000064033452 (-25.10 ± 2.65 kcal/mol), displayed significant values which delineate them as potential inhibitors of PLpro from SARS-CoV-2.


Subject(s)
COVID-19 , Papain , Coronavirus Papain-Like Proteases , Humans , Ligands , Molecular Docking Simulation , Molecular Dynamics Simulation , Papain/chemistry , Papain/genetics , Papain/metabolism , Peptide Hydrolases/genetics , Peptide Hydrolases/metabolism , SARS-CoV-2
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