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Saffarian Delkhosh A, Hadadianpour E, Islam MM, Georgieva ER. Highly versatile small virus-encoded proteins in cellular membranes: A structural perspective on how proteins' inherent conformational plasticity couples with host membranes' properties to control cellular processes. J Struct Biol X 2025; 11:100117. [PMID: 39802090 PMCID: PMC11714672 DOI: 10.1016/j.yjsbx.2024.100117] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2024] [Revised: 12/02/2024] [Accepted: 12/03/2024] [Indexed: 01/16/2025] Open
Abstract
We investigated several small viral proteins that reside and function in cellular membranes. These proteins belong to the viroporin family because they assemble into ion-conducting oligomers. However, despite forming similar oligomeric structures with analogous functions, these proteins have diverse amino acid sequences. In particular, the amino acid compositions of the proposed channel-forming transmembrane (TM) helices are vastly different-some contain residues (e.g., His, Trp, Asp, Ser) that could facilitate cation transport. Still, other viroporins' TM helices encompass exclusively hydrophobic residues; therefore, it is difficult to explain their channels' activity, unless other mechanisms (e.g., involving a negative lipid headgroups and/or membrane destabilization) take place. For this study, we selected the M2, Vpu, E, p13II, p7, and 2B proteins from the influenza A, HIV-1, human T-cell leukemia, hepatitis C, and picorna viruses, respectively. We provide a brief overview of the current knowledge about these proteins' structures as well as remaining questions about more comprehensive understanding of their structures, conformational dynamics, and function. Finally, we outline strategies to utilize a multi-prong structural and computational approach to overcome current deficiencies in the knowledge about these proteins.
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Affiliation(s)
| | | | - Md Majharul Islam
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409, USA
| | - Elka R. Georgieva
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409, USA
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2
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Negi V, Kuhn RJ. A BSL-2 chimeric system designed to screen SARS-CoV-2 E protein ion channel inhibitors. J Virol 2025; 99:e0225224. [PMID: 40304492 PMCID: PMC12090776 DOI: 10.1128/jvi.02252-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2024] [Accepted: 03/12/2025] [Indexed: 05/02/2025] Open
Abstract
A major hindrance to the identification of new drug targets and the large-scale testing of new or existing compound libraries against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is that research on the virus is restricted to biosafety level 3 (BSL-3) laboratories. In such cases, BSL-2 surrogate systems or chimeric and attenuated versions of the virus are developed for safer, faster, and cheaper examination of the stages of the virus life cycle and specific drug targets. In this study, we describe a BSL-2 chimeric viral system utilizing a Sindbis virus background as a tool to study one such target, the SARS-CoV-2 Envelope (E) protein channel activity. This protein is fully conserved between SARS-CoV and SARS-CoV-2 variants of concern (VOCs), except for a threonine to isoleucine mutation in the Omicron variant, making the E ion channel domain an attractive antiviral target for combination therapy. Using a BSL-2-chimeric system, we have been able to show similar inhibition profiles using channel inhibitors as previously reported for E-channel inhibition in authentic SARS-CoV-2. This system has the potential to allow faster initial screening of E-channel inhibitors and can be useful in developing broad-spectrum antivirals against viral channel proteins.IMPORTANCEDespite its importance in viral infections, no antivirals exist against the ion channel activity of the SARS-CoV-2 Envelope (E) protein. The E protein is highly conserved among SARS-CoV-2 variants, making it an attractive target for antiviral therapies. Research on SARS-CoV-2 is restricted to BSL-3 laboratories, creating a bottleneck for screening potential antiviral compounds. This study presents a BSL-2 chimeric system using a Sindbis virus background to study the ion channel activity of the E protein. This novel BSL-2 system bypasses this limitation, offering a safer and faster approach for the initial screening of ion channel inhibitors. By replicating the channel inhibition profiles of authentic SARS-CoV-2 in a more accessible system, this research paves the way for the development of broad-spectrum antivirals against viral channel proteins, potentially expediting the discovery of life-saving treatments for COVID-19 and other viral diseases.
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Affiliation(s)
- Vashi Negi
- Department of Biological Sicences, Purdue University, West Lafayette, Indiana, USA
| | - Richard J. Kuhn
- Department of Biological Sicences, Purdue University, West Lafayette, Indiana, USA
- Purdue Institute of Inflammation, Immunology, and Infectious Disease, Purdue University, West Lafayette, Indiana, USA
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3
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Hong M. Solid-State NMR of Virus Membrane Proteins. Acc Chem Res 2025; 58:847-860. [PMID: 40019485 DOI: 10.1021/acs.accounts.4c00800] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/01/2025]
Abstract
Enveloped viruses encode ion-conducting pores that permeabilize the host cell membranes and mediate the budding of new viruses. These viroporins are some of the essential membrane proteins of viruses, and have high sequence conservation, making them important targets of antiviral drugs. High-resolution structures of viroporins are challenging to determine by X-ray crystallography and cryoelectron microscopy, because these proteins are small, hydrophobic, and prone to induce membrane curvature. Solid-state NMR (ssNMR) spectroscopy is an ideal method for elucidating the structure, dynamics, and mechanism of action of viroporins in phospholipid membranes. This Account describes our investigations of influenza M2 proteins and the SARS-CoV-2 E protein using solid-state NMR.M2 proteins form acid-activated tetrameric proton channels that initiate influenza uncoating in the cell. 15N and 13C exchange NMR revealed that M2 shuttles protons into the virion using a crucial histidine, whose imidazole nitrogens pick up and release protons on the microsecond time scale at acidic pH. This proton exchange is synchronized with and facilitated by imidazole reorientation, which is observed in NMR spectra. Quantitative 15N NMR spectra yielded the populations of neutral and cationic histidines as a function of pH, giving four proton dissociation constants (pKa's). The pKa's of influenza AM2 indicate that the +3 charged channel has the highest time-averaged single-channel conductance; thus the third protonation event defines channel activation. In comparison, influenza BM2 exhibits lower pKa's due to a second, peripheral histidine, which accelerates proton dissociation from the central proton-selective histidine. Amantadine binding to AM2 suppressed proton exchange and imidazole reorientation, indicating that this antiviral drug acts by inhibiting proton shuttling. Solid-state NMR 13C-2H distance measurements revealed that amantadine binds the N-terminal pore of the channel near a crucial Ser31, whose mutation to asparagine causes amantadine resistance in circulating influenza A viruses. A second binding site, on the lipid-facing surface of the protein, only occurs when amantadine is in large excess in lipid bilayers. M2 not only functions as a proton channel but also conducts membrane scission during influenza budding in a cholesterol-dependent manner. Solid-state NMR distance experiments revealed that two cholesterol molecules bind asymmetrically to the surface of the tetrameric channel, thus recruiting the protein to the cholesterol-rich budding region of the cell membrane to cause membrane scission.To accelerate full structure determination of viroporins, we developed a suite of 19F solid-state NMR techniques that measure interatomic distances to 1-2 nm. Using this approach, we determined the atomic structures of influenza BM2, SARS-CoV-2 E, and EmrE, a multidrug-resistance bacterial transporter. pH-induced structural changes of these proteins gave detailed insights into the activation mechanisms of BM2 and E and the proton-coupled substrate transport mechanism of EmrE. The SARS-CoV-2 E protein forms pentameric helical bundles whose structures are distinct between the closed state at neutral pH and the open state at acidic pH. These 19F-enabled distance NMR experiments are also instrumental for identifying the binding mode and binding site of hexamethylene amiloride in E, paving the way for developing new antiviral drugs that target these pathogenic virus ion channels.
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Affiliation(s)
- Mei Hong
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
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Medeiros-Silva J, Pankratova Y, Sučec I, Dregni AJ, Hong M. Polar Networks Mediate Ion Conduction of the SARS-CoV-2 Envelope Protein. J Am Chem Soc 2025; 147:746-757. [PMID: 39726395 DOI: 10.1021/jacs.4c13229] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2024]
Abstract
The SARS-CoV-2 E protein conducts cations across the cell membrane to cause pathogenicity to infected cells. The high-resolution structures of the E transmembrane domain (ETM) in the closed state at neutral pH and in the open state at acidic pH have been determined. However, the ion conduction mechanism remains elusive. Here, we use solid-state NMR spectroscopy to investigate the side chain structure, dynamics, and interactions of five polar residues at the N-terminal entrance of the channel and three polar residues at the C-terminal end. The chemical shifts of the N-terminal Glu8 reveal that the Glu side chain interacts with protons, Ca2+ and two neighboring Thr residues, and adopts distinct motionally averaged conformational ensembles. These polar interactions are sensitive to the presence of negatively charged lipids in the membrane. A T9I mutation, prevalent in the Omicron variants of SARS-CoV-2 E, perturbs these interactions and partially immobilizes the N-terminal segment. Deeper into the channel, two polar residues, Asn15 and Ser16, form interhelical hydrogen bonds in the closed state but become separated by water molecules in the open state. This is manifested by Asn15-Ser16 correlation signals at neutral pH and the loss of these correlations and the appearance of water cross peaks with Ser16 at acidic pH in the presence of Ca2+. Finally, the guanidinium side chain of the C-terminal Arg38 undergoes fast reorientations in the closed state but becomes more restricted in the open state. These results provide evidence for a dynamic and hydrogen-bonded N-terminal polar network that recruits and relays protons and Ca2+ in a lipid-dependent manner. Once inside, the ions permeate past the hydrophobic middle of the transmembrane domain with the help of enhanced hydrophilicity of the C-terminal channel lumen due to the insertion of the Arg38 side chain into the pore.
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Affiliation(s)
- João Medeiros-Silva
- Department of Chemistry, Massachusetts Institute of Technology, 170 Albany Street, Cambridge, Massachusetts 02139, United States
| | - Yanina Pankratova
- Department of Chemistry, Massachusetts Institute of Technology, 170 Albany Street, Cambridge, Massachusetts 02139, United States
| | - Iva Sučec
- Department of Chemistry, Massachusetts Institute of Technology, 170 Albany Street, Cambridge, Massachusetts 02139, United States
| | - Aurelio J Dregni
- Department of Chemistry, Massachusetts Institute of Technology, 170 Albany Street, Cambridge, Massachusetts 02139, United States
| | - Mei Hong
- Department of Chemistry, Massachusetts Institute of Technology, 170 Albany Street, Cambridge, Massachusetts 02139, United States
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Georgiou K, Kolocouris A. Conformational heterogeneity and structural features for function of the prototype viroporin influenza AM2. BIOCHIMICA ET BIOPHYSICA ACTA. BIOMEMBRANES 2025; 1867:184387. [PMID: 39424094 DOI: 10.1016/j.bbamem.2024.184387] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/20/2024] [Revised: 09/18/2024] [Accepted: 10/01/2024] [Indexed: 10/21/2024]
Abstract
The 97-residue influenza A matrix 2 (ΑM2) protein, a prototype for viroporins, transports protons through water molecules and His37. We discuss structural biology and molecular biophysics experiments and some functional assays that have transformed over 40 years our understanding of the structure and function of AM2. The structural studies on ΑM2 have been performed with different conditions (pH, temperature, lipid, constructs) and using various protein constructs, e.g., AM2 transmembrane (AM2TM) domain, AM2 conductance domain (AM2CD), ectodomain-containing or ectodomain-truncated, AM2 full length (AM2FL) and aimed to describe the different conformations and structural details that are necessary for the stability and function of AM2. However, the conclusions from these experiments appeared sometimes ambiguous and caused exciting debates. This was not due to inaccurate measurements, but instead because of the different membrane mimetic environment used, e.g., detergent, micelles or phospholipid bilayer, the method (e.g., X-ray crystallography, solid state NMR, solution NMR, native mass spectrometry), the used protein construct (e.g., AM2TM or AM2CD), or the amino acids residues to follow observables (e.g., NMR chemical shifts). We present these results according to the different used biophysical methods, the research groups and often by keeping a chronological order for presenting the progress in the research. We discuss ideas for additional research on structural details of AM2 and how the present findings can be useful to explore new routes of influenza A inhibition. The AM2 research can provide inspiration to study other viroporins as drug targets.
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Affiliation(s)
- Kyriakos Georgiou
- Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, National and Kapodistrian University of Athens, Panepistimiopolis-Zografou, Athens 157 71, Greece
| | - Antonios Kolocouris
- Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, National and Kapodistrian University of Athens, Panepistimiopolis-Zografou, Athens 157 71, Greece.
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6
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Torres J, Pervushin K, Surya W. Prediction of conformational states in a coronavirus channel using Alphafold-2 and DeepMSA2: Strengths and limitations. Comput Struct Biotechnol J 2024; 23:3730-3740. [PMID: 39525089 PMCID: PMC11543627 DOI: 10.1016/j.csbj.2024.10.021] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2024] [Revised: 10/01/2024] [Accepted: 10/15/2024] [Indexed: 11/16/2024] Open
Abstract
The envelope (E) protein is present in all coronavirus genera. This protein can form pentameric oligomers with ion channel activity which have been proposed as a possible therapeutic target. However, high resolution structures of E channels are limited to those of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), responsible for the recent COVID-19 pandemic. In the present work, we used Alphafold-2 (AF2), in ColabFold without templates, to predict the transmembrane domain (TMD) structure of six E-channels representative of genera alpha-, beta- and gamma-coronaviruses in the Coronaviridae family. High-confidence models were produced in all cases when combining multiple sequence alignments (MSAs) obtained from DeepMSA2. Overall, AF2 predicted at least two possible orientations of the α-helices in E-TMD channels: one where a conserved polar residue (Asn-15 in the SARS sequence) is oriented towards the center of the channel, 'polar-in', and one where this residue is in an interhelical orientation 'polar-inter'. For the SARS models, the comparison with the two experimental models 'closed' (PDB: 7K3G) and 'open' (PDB: 8SUZ) is described, and suggests a ∼60˚ α-helix rotation mechanism involving either the full TMD or only its N-terminal half, to allow the passage of ions. While the results obtained are not identical to the two high resolution models available, they suggest various conformational states with striking similarities to those models. We believe these results can be further optimized by means of MSA subsampling, and guide future high resolution structural studies in these and other viral channels.
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Affiliation(s)
- Jaume Torres
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore
| | - Konstantin Pervushin
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore
| | - Wahyu Surya
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore
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Barghash RF, Gemmati D, Awad AM, Elbakry MMM, Tisato V, Awad K, Singh AV. Navigating the COVID-19 Therapeutic Landscape: Unveiling Novel Perspectives on FDA-Approved Medications, Vaccination Targets, and Emerging Novel Strategies. Molecules 2024; 29:5564. [PMID: 39683724 PMCID: PMC11643501 DOI: 10.3390/molecules29235564] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2024] [Revised: 11/21/2024] [Accepted: 11/22/2024] [Indexed: 12/18/2024] Open
Abstract
Amidst the ongoing global challenge of the SARS-CoV-2 pandemic, the quest for effective antiviral medications remains paramount. This comprehensive review delves into the dynamic landscape of FDA-approved medications repurposed for COVID-19, categorized as antiviral and non-antiviral agents. Our focus extends beyond conventional narratives, encompassing vaccination targets, repurposing efficacy, clinical studies, innovative treatment modalities, and future outlooks. Unveiling the genomic intricacies of SARS-CoV-2 variants, including the WHO-designated Omicron variant, we explore diverse antiviral categories such as fusion inhibitors, protease inhibitors, transcription inhibitors, neuraminidase inhibitors, nucleoside reverse transcriptase, and non-antiviral interventions like importin α/β1-mediated nuclear import inhibitors, neutralizing antibodies, and convalescent plasma. Notably, Molnupiravir emerges as a pivotal player, now licensed in the UK. This review offers a fresh perspective on the historical evolution of COVID-19 therapeutics, from repurposing endeavors to the latest developments in oral anti-SARS-CoV-2 treatments, ushering in a new era of hope in the battle against the pandemic.
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Affiliation(s)
- Reham F. Barghash
- Institute of Chemical Industries Research, National Research Centre, Dokki, Cairo 12622, Egypt
- Faculty of Biotechnology, October University for Modern Sciences and Arts (MSA), Cairo 12451, Egypt
| | - Donato Gemmati
- Department of Translational Medicine, University of Ferrara, 44121 Ferrara, Italy
| | - Ahmed M. Awad
- Department of Chemistry, California State University Channel Islands, Camarillo, CA 93012, USA
| | - Mustafa M. M. Elbakry
- Faculty of Biotechnology, October University for Modern Sciences and Arts (MSA), Cairo 12451, Egypt
- Biochemistry Department, Faculty of Science, Ain Shams University, Cairo 11566, Egypt
| | - Veronica Tisato
- Centre Hemostasis & Thrombosis, University of Ferrara, 44121 Ferrara, Italy
| | - Kareem Awad
- Institute of Pharmaceutical and Drug Industries Research, National Research Center, Dokki, Cairo 12622, Egypt;
| | - Ajay Vikram Singh
- Department of Chemical and Product Safety, German Federal Institute for Risk Assessment (BfR), Max-Dohrn-Strasse 8-10, 10589 Berlin, Germany
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8
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Devantier K, Kjær VMS, Griffin S, Kragelund BB, Rosenkilde MM. Advancing the field of viroporins-Structure, function and pharmacology: IUPHAR Review 39. Br J Pharmacol 2024; 181:4450-4490. [PMID: 39224966 DOI: 10.1111/bph.17317] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2024] [Revised: 06/28/2024] [Accepted: 07/07/2024] [Indexed: 09/04/2024] Open
Abstract
Viroporins possess important potential as antiviral targets due to their critical roles during virus life cycles, spanning from virus entry to egress. Although the antiviral amantadine targets the M2 viroporin of influenza A virus, successful progression of other viroporin inhibitors into clinical use remains challenging. These challenges relate in varying proportions to a lack of reliable full-length 3D-structures, difficulties in functionally characterising individual viroporins, and absence of verifiable direct binding between inhibitor and viroporin. This review offers perspectives to help overcome these challenges. We provide a comprehensive overview of the viroporin family, including their structural and functional features, highlighting the moldability of their energy landscapes and actions. To advance the field, we suggest a list of best practices to aspire towards unambiguous viroporin identification and characterisation, along with considerations of potential pitfalls. Finally, we present current and future scenarios of, and prospects for, viroporin targeting drugs.
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Affiliation(s)
- Kira Devantier
- Molecular and Translational Pharmacology, Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
- Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, Copenhagen, Denmark
| | - Viktoria M S Kjær
- Molecular and Translational Pharmacology, Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Stephen Griffin
- Leeds Institute of Medical Research, St James' University Hospital, School of Medicine, Faculty of Medicine and Health, University of Leeds, Leeds, UK
- Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK
| | - Birthe B Kragelund
- Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, Copenhagen, Denmark
| | - Mette M Rosenkilde
- Molecular and Translational Pharmacology, Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
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Volovik MV, Batishchev OV. Viral fingerprints of the ion channel evolution: compromise of complexity and function. J Biomol Struct Dyn 2024:1-20. [PMID: 39365745 DOI: 10.1080/07391102.2024.2411523] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2024] [Accepted: 04/29/2024] [Indexed: 10/06/2024]
Abstract
Evolution from precellular supramolecular assemblies to cellular world originated from the ability to make a barrier between the interior of the cell and the outer environment. This step resulted from the possibility to form a membrane, which preserves the cell like a wall of the castle. However, every castle needs gates for trading, i.e. in the case of cell, for controlled exchange of substances. These 'gates' should have the mechanism of opening and closing, guards, entry rules, and so on. Different structures are known to be able to make membrane permeable to various substances, from ions to macromolecules. They are amphipathic peptides, their assemblies, sophisticated membrane channels with numerous transmembrane domains, etc. Upon evolving, cellular world preserved and selected many variants, which, finally, have provided both prokaryotes and eukaryotes with highly selective and regulated ion channels. However, various simpler variants of ion channels are found in viruses. Despite the origin of viruses is still under debates, they have evolved parallelly with the cellular forms of life. Being initial form of the enveloped organisms, reduction of protocells or their escaped parts, viruses might be fingerprints of the evolutionary steps of cellular structures like ion channels. Therefore, viroporins may provide us a necessary information about selection between high functionality and less complex structure in supporting all the requirements for controlled membrane permeability. In this review we tried to elucidate these compromises and show the possible way of the evolution of ion channels, from peptides to complex multi-subunit structures, basing on viral examples.
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Affiliation(s)
- Marta V Volovik
- Laboratory of Bioelectrochemistry, A.N. Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, Moscow, Russia
| | - Oleg V Batishchev
- Laboratory of Bioelectrochemistry, A.N. Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, Moscow, Russia
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Kinnun JJ, Carrillo JMY, Collier CP, Smith MD, Katsaras J. Amantadine interactions with phase separated lipid membranes. Chem Phys Lipids 2024; 262:105397. [PMID: 38740276 DOI: 10.1016/j.chemphyslip.2024.105397] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2024] [Revised: 05/07/2024] [Accepted: 05/08/2024] [Indexed: 05/16/2024]
Abstract
Amantadine, a small amphilphic organic compound that consists of an adamantane backbone and an amino group, was first recognized as an antiviral in 1963 and received approval for prophylaxis against the type A influenza virus in 1976. Since then, it has also been used to treat Parkinson's disease-related dyskinesia and is being considered as a treatment for corona viruses. Since amantadine usually targets membrane-bound proteins, its interactions with the membrane are also thought to be important. Biological membranes are now widely understood to be laterally heterogeneous and certain proteins are known to preferentially co-localize within specific lipid domains. Does amantadine, therefore, preferentially localize in certain lipid composition domains? To address this question, we studied amantadine's interactions with phase separating membranes composed of cholesterol, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine), and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), as well as single-phase DPhPC (1,2-diphytanoyl-sn-glycero-3-phos-phocholine) membranes. From Langmuir trough and differential scanning calorimetry (DSC) measurements, we determined, respectively, that amantadine preferentially binds to disordered lipids, such as POPC, and lowers the phase transition temperature of POPC/DSPC/cholesterol mixtures, implying that amantadine increases membrane disorder. Further, using droplet interface bilayers (DIBs), we observed that amantadine disrupts DPhPC membranes, consistent with its disordering properties. Finally, we carried out molecular dynamics (MD) simulations on POPC/DSPC/cholesterol membranes with varying amounts of amantadine. Consistent with experiment, MD simulations showed that amantadine prefers to associate with disordered POPC-rich domains, domain boundaries, and lipid glycerol backbones. Since different proteins co-localize with different lipid domains, our results have possible implications as to which classes of proteins may be better targets for amantadine.
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Affiliation(s)
- Jacob J Kinnun
- Department of Chemistry, University of Tennessee, Knoxville, TN 37996, United States.
| | - Jan Michael Y Carrillo
- Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, Oak Ridge, TN 37831, United States
| | - C Patrick Collier
- Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, Oak Ridge, TN 37831, United States
| | - Micholas Dean Smith
- Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996, United States; UT/ORNL Center for Molecular Biophysics, Oak Ridge National Laboratory, Biosciences Division, Oak Ridge, TN 37831, United States
| | - John Katsaras
- Labs and Soft Matter Group, Neutron Scattering Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, United States; Shull-Wollan Center, Oak Ridge National Laboratory, Oak Ridge, TN 37831, United States; Department of Physics and Astronomy, University of Tennessee, Knoxville, TN 37996, United States.
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11
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Sučec I, Pankratova Y, Parasar M, Hong M. Transmembrane conformation of the envelope protein of an alpha coronavirus, NL63. Protein Sci 2024; 33:e4923. [PMID: 38501465 PMCID: PMC10949323 DOI: 10.1002/pro.4923] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2023] [Revised: 01/26/2024] [Accepted: 01/27/2024] [Indexed: 03/20/2024]
Abstract
The envelope (E) proteins of coronaviruses (CoVs) form cation-conducting channels that are associated with the pathogenicity of these viruses. To date, high-resolution structural information about these viroporins is limited to the SARS-CoV E protein. To broaden our structural knowledge of other members of this family of viroporins, we now investigate the conformation of the E protein of the human coronavirus (hCoV), NL63. Using two- and three-dimensional magic-angle-spinning NMR, we have measured 13 C and 15 N chemical shifts of the transmembrane domain of E (ETM), which yielded backbone (ϕ, ψ) torsion angles. We further measured the water accessibility of NL63 ETM at neutral pH versus acidic pH in the presence of Ca2+ ions. These data show that NL63 ETM adopts a regular α-helical conformation that is unaffected by pH and the N-terminal ectodomain. Interestingly, the water accessibility of NL63 ETM increases only modestly at acidic pH in the presence of Ca2+ compared to neutral pH, in contrast to SARS ETM, which becomes much more hydrated at acidic pH. This difference suggests a structural basis for the weaker channel conductance of α-CoV compared to β-CoV E proteins. The weaker E channel activity may in turn contribute to the reduced virulence of hCoV-NL63 compared to SARS-CoV viruses.
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Affiliation(s)
- Iva Sučec
- Department of ChemistryMassachusetts Institute of TechnologyCambridgeMassachusettsUSA
| | - Yanina Pankratova
- Department of ChemistryMassachusetts Institute of TechnologyCambridgeMassachusettsUSA
| | - Mriganka Parasar
- Department of ChemistryMassachusetts Institute of TechnologyCambridgeMassachusettsUSA
| | - Mei Hong
- Department of ChemistryMassachusetts Institute of TechnologyCambridgeMassachusettsUSA
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12
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Qiu Y, Sun Y, Zheng X, Gong L, Yang L, Xiang B. Identification of host proteins interacting with the E protein of porcine epidemic diarrhea virus. Front Microbiol 2024; 15:1380578. [PMID: 38577683 PMCID: PMC10994376 DOI: 10.3389/fmicb.2024.1380578] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2024] [Accepted: 02/29/2024] [Indexed: 04/06/2024] Open
Abstract
Introduction Porcine epidemic diarrhea (PED) is an acute, highly contagious, and high-mortality enterophilic infectious disease caused by the porcine epidemic diarrhea virus (PEDV). PEDV is globally endemic and causes substantial economic losses in the swine industry. The PEDV E protein is the smallest structural protein with high expression levels that interacts with the M protein and participates in virus assembly. However, how the host proteins interact with E proteins in PEDV replication remains unknown. Methods We identified host proteins that interact with the PEDV E protein using a combination of PEDV E protein-labeled antibody co-immunoprecipitation and tandem liquid-chromatography mass-spectroscopy (LC-MS/MS). Results Bioinformatical analysis showed that in eukaryotes, ribosome biogenesis, RNA transport, and amino acid biosynthesis represent the three main pathways that are associated with the E protein. The interaction between the E protein and isocitrate dehydrogenase [NAD] β-subunit (NAD-IDH-β), DNA-directed RNA polymerase II subunit RPB9, and mRNA-associated protein MRNP 41 was validated using co-immunoprecipitation and confocal assays. NAD-IDH-β overexpression significantly inhibited viral replication. Discussion The antiviral effect of NAD-IDH-β suggesting that the E protein may regulate host metabolism by interacting with NAD-IDH-β, thereby reducing the available energy for viral replication. Elucidating the interaction between the PEDV E protein and host proteins may clarify its role in viral replication. These results provide a theoretical basis for the study of PEDV infection mechanism and antiviral targets.
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Affiliation(s)
- Yingwu Qiu
- College of Veterinary Medicine, Yunnan Agricultural University, Kunming, China
- College of Veterinary Medicine, South China Agricultural University, Guangzhou, China
| | - Yingshuo Sun
- College of Veterinary Medicine, South China Agricultural University, Guangzhou, China
| | - Xiaoyu Zheng
- College of Veterinary Medicine, South China Agricultural University, Guangzhou, China
| | - Lang Gong
- College of Veterinary Medicine, South China Agricultural University, Guangzhou, China
| | - Liangyu Yang
- College of Veterinary Medicine, Yunnan Agricultural University, Kunming, China
| | - Bin Xiang
- College of Veterinary Medicine, Yunnan Agricultural University, Kunming, China
- College of Veterinary Medicine, South China Agricultural University, Guangzhou, China
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Li X, Wu Y, Yan Z, Li G, Luo J, Huang S, Guo X. A Comprehensive View on the Protein Functions of Porcine Epidemic Diarrhea Virus. Genes (Basel) 2024; 15:165. [PMID: 38397155 PMCID: PMC10887554 DOI: 10.3390/genes15020165] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2023] [Revised: 01/24/2024] [Accepted: 01/24/2024] [Indexed: 02/25/2024] Open
Abstract
Porcine epidemic diarrhea (PED) virus (PEDV) is one of the main pathogens causing diarrhea in piglets and fattening pigs. The clinical signs of PED are vomiting, acute diarrhea, dehydration, and mortality resulting in significant economic losses and becoming a major challenge in the pig industry. PEDV possesses various crucial structural and functional proteins, which play important roles in viral structure, infection, replication, assembly, and release, as well as in escaping host innate immunity. Over the past few years, there has been progress in the study of PEDV pathogenesis, revealing the crucial role of the interaction between PEDV viral proteins and host cytokines in PEDV infection. At present, the main control measure against PEDV is vaccine immunization of sows, but the protective effect for emerging virus strains is still insufficient, and there is no ideal safe and efficient vaccine. Although scientists have persistently delved their research into the intricate structure and functionalities of the PEDV genome and viral proteins for years, the pathogenic mechanism of PEDV remains incompletely elucidated. Here, we focus on reviewing the research progress of PEDV structural and nonstructural proteins to facilitate the understanding of biological processes such as PEDV infection and pathogenesis.
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Affiliation(s)
- Xin Li
- College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China; (X.L.); (Y.W.); (Z.Y.); (G.L.); (J.L.)
- Zhaoqing Branch Center of Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology, Zhaoqing 526238, China
| | - Yiwan Wu
- College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China; (X.L.); (Y.W.); (Z.Y.); (G.L.); (J.L.)
- Zhaoqing Branch Center of Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology, Zhaoqing 526238, China
| | - Zhibin Yan
- College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China; (X.L.); (Y.W.); (Z.Y.); (G.L.); (J.L.)
- Zhaoqing Branch Center of Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology, Zhaoqing 526238, China
| | - Gen Li
- College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China; (X.L.); (Y.W.); (Z.Y.); (G.L.); (J.L.)
| | - Jun Luo
- College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China; (X.L.); (Y.W.); (Z.Y.); (G.L.); (J.L.)
| | - Shile Huang
- Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130-3932, USA
- Department of Hematology and Oncology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130-3932, USA
- Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130-3932, USA
| | - Xiaofeng Guo
- College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China; (X.L.); (Y.W.); (Z.Y.); (G.L.); (J.L.)
- Zhaoqing Branch Center of Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology, Zhaoqing 526238, China
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14
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Chen YM, Lu CT, Wang CW, Fischer WB. Repurposing dye ligands as antivirals via a docking approach on viral membrane and globular proteins - SARS-CoV-2 and HPV-16. BIOCHIMICA ET BIOPHYSICA ACTA. BIOMEMBRANES 2024; 1866:184220. [PMID: 37657640 DOI: 10.1016/j.bbamem.2023.184220] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/24/2023] [Revised: 08/21/2023] [Accepted: 08/24/2023] [Indexed: 09/03/2023]
Abstract
A series of dye ligands are docked to three different proteins, E and 3a of severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) and E6 of human papilloma virus type 16 (HPV-16) using three different software. A four-level selection algorithm is used based on nonparametric statistics of numerical key values such as the "rank" derived from (i) averaged estimated binding energies (EBEs) and (ii) absolute EBE value of each of the software, (iii) frequency of ranking and (iv) rank of the area-under-curve values (AUCs) from decoy docking. A series of repurposing drugs and known antivirals used in experimental studies are docked for comparison. One dye ligand is ranked best for all proteins using the selection algorithm levels i - iii. Another three dye ligands are ranked top for the proteins individually when using all four levels.
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Affiliation(s)
- Yi-Ming Chen
- Institute of Biophotonics, School of Biomedical Science and Engineering, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Ching-Tai Lu
- Institute of Biophotonics, School of Biomedical Science and Engineering, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Chia-Wen Wang
- Institute of Biophotonics, School of Biomedical Science and Engineering, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Wolfgang B Fischer
- Institute of Biophotonics, School of Biomedical Science and Engineering, National Yang Ming Chiao Tung University, Taipei, Taiwan.
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15
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Miura K, Suzuki Y, Ishida K, Arakawa M, Wu H, Fujioka Y, Emi A, Maeda K, Hamajima R, Nakano T, Tenno T, Hiroaki H, Morita E. Distinct motifs in the E protein are required for SARS-CoV-2 virus particle formation and lysosomal deacidification in host cells. J Virol 2023; 97:e0042623. [PMID: 37830820 PMCID: PMC10617393 DOI: 10.1128/jvi.00426-23] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2023] [Accepted: 07/18/2023] [Indexed: 10/14/2023] Open
Abstract
IMPORTANCE Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19), has caused a global public health crisis. The E protein, a structural protein found in this virus particle, is also known to be a viroporin. As such, it forms oligomeric ion channels or pores in the host cell membrane. However, the relationship between these two functions is poorly understood. In this study, we showed that the roles of E protein in virus particle and viroporin formation are distinct. This study contributes to the development of drugs that inhibit SARS-CoV-2 virus particle formation. Additionally, we designed a highly sensitive and high-throughput virus-like particle detection system using the HiBiT tag, which is a useful tool for studying the release of SARS-CoV-2.
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Affiliation(s)
- Koya Miura
- Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University, Aomori, Japan
| | - Youichi Suzuki
- Department of Microbiology and Infection Control, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Osaka, Japan
| | - Kotaro Ishida
- Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University, Aomori, Japan
| | - Masashi Arakawa
- Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University, Aomori, Japan
| | - Hong Wu
- Department of Microbiology and Infection Control, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Osaka, Japan
| | - Yoshihiko Fujioka
- Department of Microbiology and Infection Control, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Osaka, Japan
| | - Akino Emi
- Department of Microbiology and Infection Control, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Osaka, Japan
| | - Koki Maeda
- Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University, Aomori, Japan
| | - Ryusei Hamajima
- Laboratory of Structural and Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya University, Aichi, Japan
| | - Takashi Nakano
- Department of Microbiology and Infection Control, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Osaka, Japan
| | - Takeshi Tenno
- Laboratory of Structural and Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya University, Aichi, Japan
- BeCellBar LLC, Nagoya, Aichi, Japan
| | - Hidekazu Hiroaki
- Laboratory of Structural and Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya University, Aichi, Japan
- BeCellBar LLC, Nagoya, Aichi, Japan
| | - Eiji Morita
- Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University, Aomori, Japan
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16
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Medeiros-Silva J, Dregni AJ, Somberg NH, Duan P, Hong M. Atomic structure of the open SARS-CoV-2 E viroporin. SCIENCE ADVANCES 2023; 9:eadi9007. [PMID: 37831764 PMCID: PMC10575589 DOI: 10.1126/sciadv.adi9007] [Citation(s) in RCA: 16] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/24/2023] [Accepted: 09/08/2023] [Indexed: 10/15/2023]
Abstract
The envelope (E) protein of the SARS-CoV-2 virus forms cation-conducting channels in the endoplasmic reticulum Golgi intermediate compartment (ERGIC) of infected cells. The calcium channel activity of E is associated with the inflammatory responses of COVID-19. Using solid-state NMR (ssNMR) spectroscopy, we have determined the open-state structure of E's transmembrane domain (ETM) in lipid bilayers. Compared to the closed state, open ETM has an expansive water-filled amino-terminal chamber capped by key glutamate and threonine residues, a loose phenylalanine aromatic belt in the middle, and a constricted polar carboxyl-terminal pore filled with an arginine and a threonine residue. This structure gives insights into how protons and calcium ions are selected by ETM and how they permeate across the hydrophobic gate of this viroporin.
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Affiliation(s)
| | - Aurelio J. Dregni
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | | | - Pu Duan
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
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17
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Weis N, Bollerup S, Sund JD, Glamann JB, Vinten C, Jensen LR, Sejling C, Kledal TN, Rosenkilde MM. Amantadine for COVID-19 treatment (ACT) study: a randomized, double-blinded, placebo-controlled clinical trial. Clin Microbiol Infect 2023; 29:1313-1319. [PMID: 37353078 PMCID: PMC10284620 DOI: 10.1016/j.cmi.2023.06.023] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2023] [Revised: 06/18/2023] [Accepted: 06/19/2023] [Indexed: 06/25/2023]
Abstract
OBJECTIVES The COVID-19 pandemic has revealed a severe need for effective antiviral treatment. The objectives of this study were to assess if pre-emptive treatment with amantadine for COVID-19 in non-hospitalized persons ≥40 years or adults with comorbidities was able to prevent disease progression and hospitalization. Primary outcomes were clinical status on day 14. METHODS Between 9 June 2021 and 27 January 2022, this randomized, double-blinded, placebo-controlled, single-centre clinical trial included 242 subjects with a follow-up period of 90 days. Subjects were randomly assigned 1:1 to either amantadine 100 mg or placebo twice daily for 5 days. The inclusion criteria were confirmed SARS-CoV-2 infection and at least one of (a) age ≥40 years, age ≥18 years and (b) at least one comorbidity, or (c) body mass index ≥30. The study protocol was published at www. CLINICALTRIALS gov (unique protocol #02032021) and at www.clinicaltrialregister.eu (EudraCT-number 2021-001177-22). RESULTS With 121 participants in each arm, we found no difference in the primary endpoint with 82 participants in the amantadine arm, and 92 participants in the placebo arm with no limitations to activities, respectively, and 25 and 37 with limitations to activities in the amantadine arm and the placebo arm, respectively. No participants in either group were admitted to hospital or died. The OR of having state severity increased by 1 in the amantadine group versus placebo was 1.8 (CI 1.0-3.3, [p 0.051]). On day 7, one participant was hospitalized in each group; throughout the study, this increased to five and three participants for amantadine versus placebo treatment (p 0.72). Similarly, on day 7, there was no difference in the status of oropharyngeal swabs. Most participants (108 in each group) were SARS-CoV-2 RNA positive (p 0.84). CONCLUSION We found no effect of amantadine on disease progression of SARS-CoV-2 infection.
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Affiliation(s)
- Nina Weis
- Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Copenhagen, Denmark; Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Signe Bollerup
- Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Copenhagen, Denmark
| | - Jon Dissing Sund
- Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Copenhagen, Denmark
| | - Jakob Borg Glamann
- Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Copenhagen, Denmark
| | - Caroline Vinten
- Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Copenhagen, Denmark
| | - Louise Riger Jensen
- Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Copenhagen, Denmark
| | - Christoffer Sejling
- Department of Public Health, Section of Biostatistics, University of Copenhagen, Copenhagen, Denmark
| | | | - Mette Marie Rosenkilde
- Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
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18
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Sučec I, Mammeri NE, Dregni AJ, Hong M. Rapid Determination of the Topology of Oligomeric α-Helical Membrane Proteins by Water- and Lipid-Edited Methyl NMR. J Phys Chem B 2023; 127:7518-7530. [PMID: 37606918 PMCID: PMC10893779 DOI: 10.1021/acs.jpcb.3c05295] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/23/2023]
Abstract
Single-span oligomeric α-helical transmembrane proteins are common in virus ion channels, which are targets of antiviral drugs. Knowledge about the high-resolution structures of these oligomeric α-helical bundles is so far scarce. Structure determination of these membrane proteins by solid-state NMR traditionally requires resolving and assigning protein chemical shifts and measuring many interhelical distances, which are time-consuming. To accelerate experimental structure determination, here we introduce a simple solid-state NMR approach that uses magnetization transfer from water and lipid protons to the protein. By detecting the water- and lipid-transferred intensities of the high-sensitivity methyl 13C signals of Leu, Val, and Ile residues, which are highly enriched in these membrane proteins, we can derive models of the topology of these homo-oligomeric helical bundles. The topology is specified by the positions of amino acid residues in heptad repeats and the orientations of residues relative to the channel pore, lipids, and the helical interface. We demonstrate this water- and lipid-edited methyl NMR approach on the envelope (E) protein of SARS-CoV-2, the causative agent of the COVID-19 pandemic. We show that water-edited and lipid-edited 2D 13C-13C correlation spectra can be measured with sufficient sensitivity. Even without resolving multiple residues of the same type in the NMR spectra, we can obtain the helical bundle topology. We apply these experiments to the structurally unknown E proteins of the MERS coronavirus and the human coronavirus NL63. The resulting structural topologies show interesting differences in the positions of the aromatic residues in these three E proteins, suggesting that these viroporins may have different mechanisms of activation and ion conduction.
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Affiliation(s)
- Iva Sučec
- Department of Chemistry, Massachusetts Institute of Technology, 170 Albany Street, Cambridge, MA 02139
| | - Nadia El Mammeri
- Department of Chemistry, Massachusetts Institute of Technology, 170 Albany Street, Cambridge, MA 02139
| | - Aurelio J. Dregni
- Department of Chemistry, Massachusetts Institute of Technology, 170 Albany Street, Cambridge, MA 02139
| | - Mei Hong
- Department of Chemistry, Massachusetts Institute of Technology, 170 Albany Street, Cambridge, MA 02139
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19
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Surya W, Tavares-Neto E, Sanchis A, Queralt-Martín M, Alcaraz A, Torres J, Aguilella VM. The Complex Proteolipidic Behavior of the SARS-CoV-2 Envelope Protein Channel: Weak Selectivity and Heterogeneous Oligomerization. Int J Mol Sci 2023; 24:12454. [PMID: 37569828 PMCID: PMC10420310 DOI: 10.3390/ijms241512454] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2023] [Revised: 07/27/2023] [Accepted: 08/02/2023] [Indexed: 08/13/2023] Open
Abstract
The envelope (E) protein is a small polypeptide that can form ion channels in coronaviruses. In SARS coronavirus 2 (SARS-CoV-2), the agent that caused the recent COVID-19 pandemic, and its predecessor SARS-CoV-1, E protein is found in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), where virion budding takes place. Several reports claim that E protein promotes the formation of "cation-selective channels". However, whether this term represents specificity to certain ions (e.g., potassium or calcium) or the partial or total exclusion of anions is debatable. Herein, we discuss this claim based on the available data for SARS-CoV-1 and -2 E and on new experiments performed using the untagged full-length E protein from SARS-CoV-2 in planar lipid membranes of different types, including those that closely mimic the ERGIC membrane composition. We provide evidence that the selectivity of the E-induced channels is very mild and depends strongly on lipid environment. Thus, despite past and recent claims, we found no indication that the E protein forms cation-selective channels that prevent anion transport, and even less that E protein forms bona fide specific calcium channels. In fact, the E channel maintains its multi-ionic non-specific neutral character even in concentrated solutions of Ca2+ ions. Also, in contrast to previous studies, we found no evidence that SARS-CoV-2 E channel activation requires a particular voltage, high calcium concentrations or low pH, in agreement with available data from SARS-CoV-1 E. In addition, sedimentation velocity experiments suggest that the E channel population is mostly pentameric, but very dynamic and probably heterogeneous, consistent with the broad distribution of conductance values typically found in electrophysiological experiments. The latter has been explained by the presence of proteolipidic channel structures.
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Affiliation(s)
- Wahyu Surya
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore;
| | - Ernesto Tavares-Neto
- Laboratory of Molecular Biophysics, Department of Physics, Universitat Jaume I, 12080 Castellon, Spain; (E.T.-N.); (M.Q.-M.); (A.A.)
| | - Andrea Sanchis
- Laboratory of Molecular Biophysics, Department of Physics, Universitat Jaume I, 12080 Castellon, Spain; (E.T.-N.); (M.Q.-M.); (A.A.)
| | - María Queralt-Martín
- Laboratory of Molecular Biophysics, Department of Physics, Universitat Jaume I, 12080 Castellon, Spain; (E.T.-N.); (M.Q.-M.); (A.A.)
| | - Antonio Alcaraz
- Laboratory of Molecular Biophysics, Department of Physics, Universitat Jaume I, 12080 Castellon, Spain; (E.T.-N.); (M.Q.-M.); (A.A.)
| | - Jaume Torres
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore;
| | - Vicente M. Aguilella
- Laboratory of Molecular Biophysics, Department of Physics, Universitat Jaume I, 12080 Castellon, Spain; (E.T.-N.); (M.Q.-M.); (A.A.)
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20
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Breitinger U, Sedky CA, Sticht H, Breitinger HG. Patch-clamp studies and cell viability assays suggest a distinct site for viroporin inhibitors on the E protein of SARS-CoV-2. Virol J 2023; 20:142. [PMID: 37422646 PMCID: PMC10329798 DOI: 10.1186/s12985-023-02095-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2022] [Accepted: 06/08/2023] [Indexed: 07/10/2023] Open
Abstract
BACKGROUND SARS-CoV-2 has caused a worldwide pandemic since December 2019 and the search for pharmaceutical targets against COVID-19 remains an important challenge. Here, we studied the envelope protein E of SARS-CoV and SARS-CoV-2, a highly conserved 75-76 amino acid viroporin that is crucial for virus assembly and release. E protein channels were recombinantly expressed in HEK293 cells, a membrane-directing signal peptide ensured transfer to the plasma membrane. METHODS Viroporin channel activity of both E proteins was investigated using patch-clamp electrophysiology in combination with a cell viability assay. We verified inhibition by classical viroporin inhibitors amantadine, rimantadine and 5-(N,N-hexamethylene)-amiloride, and tested four ivermectin derivatives. RESULTS Classical inhibitors showed potent activity in patch-clamp recordings and viability assays. In contrast, ivermectin and milbemycin inhibited the E channel in patch-clamp recordings but displayed only moderate activity on the E protein in the cell viability assay, which is also sensitive to general cytotoxic activity of the tested compounds. Nemadectin and ivermectin aglycon were inactive. All ivermectin derivatives were cytotoxic at concentrations > 5 µM, i.e. below the level required for E protein inhibition. CONCLUSIONS This study demonstrates direct inhibition of the SARS-CoV-2 E protein by classical viroporin inhibitors. Ivermectin and milbemycin inhibit the E protein channel but their cytotoxicity argues against clinical application.
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Affiliation(s)
- Ulrike Breitinger
- Department of Biochemistry, German University in Cairo, Main Entrance of Al Tagamoa Al Khames, New Cairo, 11835, Egypt.
| | - Christine Adel Sedky
- Department of Biochemistry, German University in Cairo, Main Entrance of Al Tagamoa Al Khames, New Cairo, 11835, Egypt
| | - Heinrich Sticht
- Division of Bioinformatics, Institute for Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany
| | - Hans-Georg Breitinger
- Department of Biochemistry, German University in Cairo, Main Entrance of Al Tagamoa Al Khames, New Cairo, 11835, Egypt
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21
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Oliveira-Mendes BBR, Alameh M, Ollivier B, Montnach J, Bidère N, Souazé F, Escriou N, Charpentier F, Baró I, De Waard M, Loussouarn G. SARS-CoV-2 E and 3a Proteins Are Inducers of Pannexin Currents. Cells 2023; 12:1474. [PMID: 37296595 PMCID: PMC10252541 DOI: 10.3390/cells12111474] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2023] [Revised: 05/10/2023] [Accepted: 05/18/2023] [Indexed: 06/12/2023] Open
Abstract
Controversial reports have suggested that SARS-CoV E and 3a proteins are plasma membrane viroporins. Here, we aimed at better characterizing the cellular responses induced by these proteins. First, we show that expression of SARS-CoV-2 E or 3a protein in CHO cells gives rise to cells with newly acquired round shapes that detach from the Petri dish. This suggests that cell death is induced upon expression of E or 3a protein. We confirmed this by using flow cytometry. In adhering cells expressing E or 3a protein, the whole-cell currents were not different from those of the control, suggesting that E and 3a proteins are not plasma membrane viroporins. In contrast, recording the currents on detached cells uncovered outwardly rectifying currents much larger than those observed in the control. We illustrate for the first time that carbenoxolone and probenecid block these outwardly rectifying currents; thus, these currents are most probably conducted by pannexin channels that are activated by cell morphology changes and also potentially by cell death. The truncation of C-terminal PDZ binding motifs reduces the proportion of dying cells but does not prevent these outwardly rectifying currents. This suggests distinct pathways for the induction of these cellular events by the two proteins. We conclude that SARS-CoV-2 E and 3a proteins are not viroporins expressed at the plasma membrane.
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Affiliation(s)
| | - Malak Alameh
- L’institut du Thorax, Nantes Université, CNRS, INSERM, F-44000 Nantes, France; (B.B.R.O.-M.); (M.A.)
- Labex Ion Channels, Science and Therapeutics, F-06560 Valbonne, France
| | - Béatrice Ollivier
- L’institut du Thorax, Nantes Université, CNRS, INSERM, F-44000 Nantes, France; (B.B.R.O.-M.); (M.A.)
| | - Jérôme Montnach
- L’institut du Thorax, Nantes Université, CNRS, INSERM, F-44000 Nantes, France; (B.B.R.O.-M.); (M.A.)
| | - Nicolas Bidère
- Team SOAP, CRCI2NA, INSERM, CNRS, Nantes Université, Université d’Angers, F-44000 Nantes, France
- Equipe Labellisée Ligue Contre le Cancer, F-75006 Paris, France
| | | | - Nicolas Escriou
- Institut Pasteur, Université Paris Cité, Département de Santé Globale, F-75015 Paris, France
| | - Flavien Charpentier
- L’institut du Thorax, Nantes Université, CNRS, INSERM, F-44000 Nantes, France; (B.B.R.O.-M.); (M.A.)
| | - Isabelle Baró
- L’institut du Thorax, Nantes Université, CNRS, INSERM, F-44000 Nantes, France; (B.B.R.O.-M.); (M.A.)
| | - Michel De Waard
- L’institut du Thorax, Nantes Université, CNRS, INSERM, F-44000 Nantes, France; (B.B.R.O.-M.); (M.A.)
- Labex Ion Channels, Science and Therapeutics, F-06560 Valbonne, France
| | - Gildas Loussouarn
- L’institut du Thorax, Nantes Université, CNRS, INSERM, F-44000 Nantes, France; (B.B.R.O.-M.); (M.A.)
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22
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Poggio E, Vallese F, Hartel AJW, Morgenstern TJ, Kanner SA, Rauh O, Giamogante F, Barazzuol L, Shepard KL, Colecraft HM, Clarke OB, Brini M, Calì T. Perturbation of the host cell Ca 2+ homeostasis and ER-mitochondria contact sites by the SARS-CoV-2 structural proteins E and M. Cell Death Dis 2023; 14:297. [PMID: 37120609 PMCID: PMC10148623 DOI: 10.1038/s41419-023-05817-w] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2023] [Revised: 04/11/2023] [Accepted: 04/18/2023] [Indexed: 05/01/2023]
Abstract
Coronavirus disease (COVID-19) is a contagious respiratory disease caused by the SARS-CoV-2 virus. The clinical phenotypes are variable, ranging from spontaneous recovery to serious illness and death. On March 2020, a global COVID-19 pandemic was declared by the World Health Organization (WHO). As of February 2023, almost 670 million cases and 6,8 million deaths have been confirmed worldwide. Coronaviruses, including SARS-CoV-2, contain a single-stranded RNA genome enclosed in a viral capsid consisting of four structural proteins: the nucleocapsid (N) protein, in the ribonucleoprotein core, the spike (S) protein, the envelope (E) protein, and the membrane (M) protein, embedded in the surface envelope. In particular, the E protein is a poorly characterized viroporin with high identity amongst all the β-coronaviruses (SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-OC43) and a low mutation rate. Here, we focused our attention on the study of SARS-CoV-2 E and M proteins, and we found a general perturbation of the host cell calcium (Ca2+) homeostasis and a selective rearrangement of the interorganelle contact sites. In vitro and in vivo biochemical analyses revealed that the binding of specific nanobodies to soluble regions of SARS-CoV-2 E protein reversed the observed phenotypes, suggesting that the E protein might be an important therapeutic candidate not only for vaccine development, but also for the clinical management of COVID designing drug regimens that, so far, are very limited.
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Affiliation(s)
- Elena Poggio
- Department of Biology, University of Padova, Padova, Italy
| | - Francesca Vallese
- Department of Anesthesiology, Columbia University Irving Medical Center, New York, NY, USA
- Department of Physiology and Cellular Biophysics, Columbia University, New York, NY, USA
| | - Andreas J W Hartel
- Department of Electrical Engineering, Columbia University, New York, NY, USA
| | - Travis J Morgenstern
- Department of Molecular Pharmacology and Therapeutics, Columbia University Irving Medical Center, New York, NY, USA
| | - Scott A Kanner
- Doctoral Program in Neurobiology and Behavior, Columbia University Vagelos College of Physicians and Surgeons, New York, NY, USA
| | - Oliver Rauh
- Membrane Biophysics, Department of Biology, Technical University of Darmstadt, Darmstadt, Germany
| | - Flavia Giamogante
- Department of Biomedical Sciences, University of Padova, Padova, Italy
| | - Lucia Barazzuol
- Department of Biomedical Sciences, University of Padova, Padova, Italy
| | - Kenneth L Shepard
- Department of Electrical Engineering, Columbia University, New York, NY, USA
| | - Henry M Colecraft
- Department of Physiology and Cellular Biophysics, Columbia University, New York, NY, USA
- Department of Molecular Pharmacology and Therapeutics, Columbia University Irving Medical Center, New York, NY, USA
- Doctoral Program in Neurobiology and Behavior, Columbia University Vagelos College of Physicians and Surgeons, New York, NY, USA
| | - Oliver Biggs Clarke
- Department of Anesthesiology, Columbia University Irving Medical Center, New York, NY, USA
- Department of Physiology and Cellular Biophysics, Columbia University, New York, NY, USA
| | - Marisa Brini
- Department of Biology, University of Padova, Padova, Italy
- Study Center for Neurodegeneration (CESNE), University of Padova, Padova, Italy
| | - Tito Calì
- Department of Biomedical Sciences, University of Padova, Padova, Italy.
- Study Center for Neurodegeneration (CESNE), University of Padova, Padova, Italy.
- Padova Neuroscience Center (PNC), University of Padova, Padova, Italy.
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23
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Fam MS, Sedky CA, Turky NO, Breitinger HG, Breitinger U. Channel activity of SARS-CoV-2 viroporin ORF3a inhibited by adamantanes and phenolic plant metabolites. Sci Rep 2023; 13:5328. [PMID: 37005439 PMCID: PMC10067842 DOI: 10.1038/s41598-023-31764-9] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2022] [Accepted: 03/16/2023] [Indexed: 04/04/2023] Open
Abstract
SARS-CoV-2 has been responsible for the major worldwide pandemic of COVID-19. Despite the enormous success of vaccination campaigns, virus infections are still prevalent and effective antiviral therapies are urgently needed. Viroporins are essential for virus replication and release, and are thus promising therapeutic targets. Here, we studied the expression and function of recombinant ORF3a viroporin of SARS-CoV-2 using a combination of cell viability assays and patch-clamp electrophysiology. ORF3a was expressed in HEK293 cells and transport to the plasma membrane verified by a dot blot assay. Incorporation of a membrane-directing signal peptide increased plasma membrane expression. Cell viability tests were carried out to measure cell damage associated with ORF3a activity, and voltage-clamp recordings verified its channel activity. The classical viroporin inhibitors amantadine and rimantadine inhibited ORF3a channels. A series of ten flavonoids and polyphenolics were studied. Kaempferol, quercetin, epigallocatechin gallate, nobiletin, resveratrol and curcumin were ORF3a inhibitors, with IC50 values ranging between 1 and 6 µM, while 6-gingerol, apigenin, naringenin and genistein were inactive. For flavonoids, inhibitory activity could be related to the pattern of OH groups on the chromone ring system. Thus, the ORF3a viroporin of SARS-CoV-2 may indeed be a promising target for antiviral drugs.
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Affiliation(s)
- Marina Sherif Fam
- Department of Biochemistry, German University in Cairo, Main Entrance of Al Tagamoa Al Khames, New Cairo, New Cairo, 11835, Egypt
| | - Christine Adel Sedky
- Department of Biochemistry, German University in Cairo, Main Entrance of Al Tagamoa Al Khames, New Cairo, New Cairo, 11835, Egypt
| | - Nancy Osama Turky
- Department of Biochemistry, German University in Cairo, Main Entrance of Al Tagamoa Al Khames, New Cairo, New Cairo, 11835, Egypt
| | - Hans-Georg Breitinger
- Department of Biochemistry, German University in Cairo, Main Entrance of Al Tagamoa Al Khames, New Cairo, New Cairo, 11835, Egypt
| | - Ulrike Breitinger
- Department of Biochemistry, German University in Cairo, Main Entrance of Al Tagamoa Al Khames, New Cairo, New Cairo, 11835, Egypt.
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24
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Oh CK, Nakamura T, Beutler N, Zhang X, Piña-Crespo J, Talantova M, Ghatak S, Trudler D, Carnevale LN, McKercher SR, Bakowski MA, Diedrich JK, Roberts AJ, Woods AK, Chi V, Gupta AK, Rosenfeld MA, Kearns FL, Casalino L, Shaabani N, Liu H, Wilson IA, Amaro RE, Burton DR, Yates JR, Becker C, Rogers TF, Chatterjee AK, Lipton SA. Targeted protein S-nitrosylation of ACE2 inhibits SARS-CoV-2 infection. Nat Chem Biol 2023; 19:275-283. [PMID: 36175661 PMCID: PMC10127945 DOI: 10.1038/s41589-022-01149-6] [Citation(s) in RCA: 15] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2022] [Accepted: 08/24/2022] [Indexed: 12/12/2022]
Abstract
Prevention of infection and propagation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a high priority in the Coronavirus Disease 2019 (COVID-19) pandemic. Here we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin-converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 spike protein, thereby inhibiting viral entry, infectivity and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and, thus, the spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model and, thus, provide a novel avenue to pursue therapy.
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Affiliation(s)
- Chang-Ki Oh
- Departments of Molecular Medicine and Neuroscience, Neurodegeneration New Medicines Center, La Jolla, CA, USA
| | - Tomohiro Nakamura
- Departments of Molecular Medicine and Neuroscience, Neurodegeneration New Medicines Center, La Jolla, CA, USA
| | - Nathan Beutler
- Department of Immunology and Microbiology, Scripps Research Institute, La Jolla, CA, USA
| | - Xu Zhang
- Departments of Molecular Medicine and Neuroscience, Neurodegeneration New Medicines Center, La Jolla, CA, USA
| | - Juan Piña-Crespo
- Departments of Molecular Medicine and Neuroscience, Neurodegeneration New Medicines Center, La Jolla, CA, USA
| | - Maria Talantova
- Departments of Molecular Medicine and Neuroscience, Neurodegeneration New Medicines Center, La Jolla, CA, USA
| | - Swagata Ghatak
- Departments of Molecular Medicine and Neuroscience, Neurodegeneration New Medicines Center, La Jolla, CA, USA
| | - Dorit Trudler
- Departments of Molecular Medicine and Neuroscience, Neurodegeneration New Medicines Center, La Jolla, CA, USA
| | - Lauren N Carnevale
- Departments of Molecular Medicine and Neuroscience, Neurodegeneration New Medicines Center, La Jolla, CA, USA
| | - Scott R McKercher
- Departments of Molecular Medicine and Neuroscience, Neurodegeneration New Medicines Center, La Jolla, CA, USA
| | - Malina A Bakowski
- Calibr, a division of the Scripps Research Institute, La Jolla, CA, USA
| | - Jolene K Diedrich
- Departments of Molecular Medicine and Neuroscience, Neurodegeneration New Medicines Center, La Jolla, CA, USA
| | - Amanda J Roberts
- Animal Models Core, Scripps Research Institute, La Jolla, CA, USA
| | - Ashley K Woods
- Calibr, a division of the Scripps Research Institute, La Jolla, CA, USA
| | - Victor Chi
- Calibr, a division of the Scripps Research Institute, La Jolla, CA, USA
| | - Anil K Gupta
- Calibr, a division of the Scripps Research Institute, La Jolla, CA, USA
| | - Mia A Rosenfeld
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA
| | - Fiona L Kearns
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA
| | - Lorenzo Casalino
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA
| | - Namir Shaabani
- Department of Immunology and Microbiology, Scripps Research Institute, La Jolla, CA, USA
| | - Hejun Liu
- Department of Integrative Structural and Computational Biology, Scripps Research Institute, La Jolla, CA, USA
| | - Ian A Wilson
- Department of Integrative Structural and Computational Biology, Scripps Research Institute, La Jolla, CA, USA
| | - Rommie E Amaro
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA
| | - Dennis R Burton
- Department of Immunology and Microbiology, Scripps Research Institute, La Jolla, CA, USA
| | - John R Yates
- Departments of Molecular Medicine and Neuroscience, Neurodegeneration New Medicines Center, La Jolla, CA, USA
| | | | - Thomas F Rogers
- Department of Immunology and Microbiology, Scripps Research Institute, La Jolla, CA, USA
- Division of Infectious Diseases, Department of Medicine, University of California, San Diego, La Jolla, CA, USA
| | | | - Stuart A Lipton
- Departments of Molecular Medicine and Neuroscience, Neurodegeneration New Medicines Center, La Jolla, CA, USA.
- Department of Neurosciences, School of Medicine, University of California, San Diego, La Jolla, CA, USA.
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25
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COVID-19 signalome: Potential therapeutic interventions. Cell Signal 2023; 103:110559. [PMID: 36521656 PMCID: PMC9744501 DOI: 10.1016/j.cellsig.2022.110559] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2022] [Revised: 11/21/2022] [Accepted: 12/08/2022] [Indexed: 12/14/2022]
Abstract
The COVID-19 pandemic has triggered intensive research and development of drugs and vaccines against SARS-CoV-2 during the last two years. The major success was especially observed with development of vaccines based on viral vectors, nucleic acids and whole viral particles, which have received emergent authorization leading to global mass vaccinations. Although the vaccine programs have made a big impact on COVID-19 spread and severity, emerging novel variants have raised serious concerns about vaccine efficacy. Due to the urgent demand, drug development had originally to rely on repurposing of antiviral drugs developed against other infectious diseases. For both drug and vaccine development the focus has been mainly on SARS-CoV-2 surface proteins and host cell receptors involved in viral attachment and entry. In this review, we expand the spectrum of SARS-CoV-2 targets by investigating the COVID-19 signalome. In addition to the SARS-CoV-2 Spike protein, the envelope, membrane, and nucleoprotein targets have been subjected to research. Moreover, viral proteases have presented the possibility to develop different strategies for the inhibition of SARS-CoV-2 replication and spread. Several signaling pathways involving the renin-angiotensin system, angiotensin-converting enzymes, immune pathways, hypoxia, and calcium signaling have provided attractive alternative targets for more efficient drug development.
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26
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Dregni AJ, McKay MJ, Surya W, Queralt-Martin M, Medeiros-Silva J, Wang HK, Aguilella V, Torres J, Hong M. The Cytoplasmic Domain of the SARS-CoV-2 Envelope Protein Assembles into a β-Sheet Bundle in Lipid Bilayers. J Mol Biol 2023; 435:167966. [PMID: 36682677 PMCID: PMC9851921 DOI: 10.1016/j.jmb.2023.167966] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2022] [Revised: 12/23/2022] [Accepted: 01/11/2023] [Indexed: 01/21/2023]
Abstract
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) envelope (E) protein forms a pentameric ion channel in the lipid membrane of the endoplasmic reticulum Golgi intermediate compartment (ERGIC) of the infected cell. The cytoplasmic domain of E interacts with host proteins to cause virus pathogenicity and may also mediate virus assembly and budding. To understand the structural basis of these functions, here we investigate the conformation and dynamics of an E protein construct (residues 8-65) that encompasses the transmembrane domain and the majority of the cytoplasmic domain using solid-state NMR. 13C and 15N chemical shifts indicate that the cytoplasmic domain adopts a β-sheet-rich conformation that contains three β-strands separated by turns. The five subunits associate into an umbrella-shaped bundle that is attached to the transmembrane helices by a disordered loop. Water-edited NMR spectra indicate that the third β-strand at the C terminus of the protein is well hydrated, indicating that it is at the surface of the β-bundle. The structure of the cytoplasmic domain cannot be uniquely determined from the inter-residue correlations obtained here due to ambiguities in distinguishing intermolecular and intramolecular contacts for a compact pentameric assembly of this small domain. Instead, we present four structural topologies that are consistent with the measured inter-residue contacts. These data indicate that the cytoplasmic domain of the SARS-CoV-2 E protein has a strong propensity to adopt β-sheet conformations when the protein is present at high concentrations in lipid bilayers. The equilibrium between the β-strand conformation and the previously reported α-helical conformation may underlie the multiple functions of E in the host cell and in the virion.
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Affiliation(s)
- Aurelio J Dregni
- Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, United States
| | - Matthew J McKay
- Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, United States
| | - Wahyu Surya
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore
| | - Maria Queralt-Martin
- Laboratory of Molecular Biophysics. Department of Physics. Universitat Jaume I. 12080 Castellón, Spain
| | - João Medeiros-Silva
- Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, United States
| | - Harrison K Wang
- Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, United States
| | - Vicente Aguilella
- Laboratory of Molecular Biophysics. Department of Physics. Universitat Jaume I. 12080 Castellón, Spain
| | - Jaume Torres
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore
| | - Mei Hong
- Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, United States.
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27
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In Silico Screening of Drugs That Target Different Forms of E Protein for Potential Treatment of COVID-19. Pharmaceuticals (Basel) 2023. [DOI: 10.3390/ph16020296] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/17/2023] Open
Abstract
Recently the E protein of SARS-CoV-2 has become a very important target in the potential treatment of COVID-19 since it is known to regulate different stages of the viral cycle. There is biochemical evidence that E protein exists in two forms, as monomer and homopentamer. An in silico screening analysis was carried out employing 5852 ligands (from Zinc databases), and performing an ADMET analysis, remaining a set of 2155 compounds. Furthermore, docking analysis was performed on specific sites and different forms of the E protein. From this study we could identify that the following ligands showed the highest binding affinity: nilotinib, dutasteride, irinotecan, saquinavir and alectinib. We carried out some molecular dynamics simulations and free energy MM–PBSA calculations of the protein–ligand complexes (with the mentioned ligands). Of worthy interest is that saquinavir, nilotinib and alectinib are also considered as a promising multitarget ligand because it seems to inhibit three targets, which play an important role in the viral cycle. On the other side, saquinavir was shown to be able to bind to E protein both in its monomeric as well as pentameric forms. Finally, further experimental assays are needed to probe our hypothesis derived from in silico studies.
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28
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Priyathilaka TT, Laaker CJ, Herbath M, Fabry Z, Sandor M. Modeling infectious diseases of the central nervous system with human brain organoids. Transl Res 2022; 250:18-35. [PMID: 35811019 PMCID: PMC11185418 DOI: 10.1016/j.trsl.2022.06.013] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/25/2022] [Revised: 06/15/2022] [Accepted: 06/21/2022] [Indexed: 10/17/2022]
Abstract
Bacteria, fungi, viruses, and protozoa are known to infect and induce diseases in the human central nervous system (CNS). Modeling the mechanisms of interaction between pathogens and the CNS microenvironment is essential to understand their pathophysiology and develop new treatments. Recent advancements in stem cell technologies have allowed for the creation of human brain organoids, which more closely resembles the human CNS microenvironment when compared to classical 2-dimensional (2D) cultures. Now researchers can utilize these systems to investigate and reinvestigate questions related to CNS infection in a human-derived brain organoid system. Here in this review, we highlight several infectious diseases which have been tested in human brain organoids and compare similarities in response to these pathogens across different investigations. We also provide a brief overview of some recent advancements which can further enrich this model to develop new and better therapies to treat brain infections.
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Affiliation(s)
- Thanthrige Thiunuwan Priyathilaka
- Department of Pathology and Laboratory Medicine, School of Medicine and Public Health, University of Wisconsin Madison, Madison, Wisconsin
| | - Collin James Laaker
- Neuroscience Training Program, University of Wisconsin Madison, Madison, Wisconsin
| | - Melinda Herbath
- Department of Pathology and Laboratory Medicine, School of Medicine and Public Health, University of Wisconsin Madison, Madison, Wisconsin
| | - Zsuzsanna Fabry
- Department of Pathology and Laboratory Medicine, School of Medicine and Public Health, University of Wisconsin Madison, Madison, Wisconsin
| | - Matyas Sandor
- Department of Pathology and Laboratory Medicine, School of Medicine and Public Health, University of Wisconsin Madison, Madison, Wisconsin.
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29
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Zhang Y, Chen Y, Zhou J, Wang X, Ma L, Li J, Yang L, Yuan H, Pang D, Ouyang H. Porcine Epidemic Diarrhea Virus: An Updated Overview of Virus Epidemiology, Virulence Variation Patterns and Virus-Host Interactions. Viruses 2022; 14:2434. [PMID: 36366532 PMCID: PMC9695474 DOI: 10.3390/v14112434] [Citation(s) in RCA: 61] [Impact Index Per Article: 20.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2022] [Revised: 10/31/2022] [Accepted: 11/01/2022] [Indexed: 11/06/2022] Open
Abstract
The porcine epidemic diarrhea virus (PEDV) is a member of the coronavirus family, causing deadly watery diarrhea in newborn piglets. The global pandemic of PEDV, with significant morbidity and mortality, poses a huge threat to the swine industry. The currently developed vaccines and drugs are only effective against the classic GI strains that were prevalent before 2010, while there is no effective control against the GII variant strains that are currently a global pandemic. In this review, we summarize the latest progress in the biology of PEDV, including its transmission and origin, structure and function, evolution, and virus-host interaction, in an attempt to find the potential virulence factors influencing PEDV pathogenesis. We conclude with the mechanism by which PEDV components antagonize the immune responses of the virus, and the role of host factors in virus infection. Essentially, this review serves as a valuable reference for the development of attenuated virus vaccines and the potential of host factors as antiviral targets for the prevention and control of PEDV infection.
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Affiliation(s)
- Yuanzhu Zhang
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Sciences, Jilin University, Changchun 130062, China
| | - Yiwu Chen
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Sciences, Jilin University, Changchun 130062, China
| | - Jian Zhou
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Sciences, Jilin University, Changchun 130062, China
| | - Xi Wang
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Sciences, Jilin University, Changchun 130062, China
| | - Lerong Ma
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Sciences, Jilin University, Changchun 130062, China
| | - Jianing Li
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Sciences, Jilin University, Changchun 130062, China
| | - Lin Yang
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Sciences, Jilin University, Changchun 130062, China
| | - Hongming Yuan
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Sciences, Jilin University, Changchun 130062, China
- Chongqing Research Institute, Jilin University, Chongqing 401120, China
| | - Daxin Pang
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Sciences, Jilin University, Changchun 130062, China
- Chongqing Research Institute, Jilin University, Chongqing 401120, China
- Chongqing Jitang Biotechnology Research Institute Co., Ltd., Chongqing 401120, China
| | - Hongsheng Ouyang
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Sciences, Jilin University, Changchun 130062, China
- Chongqing Research Institute, Jilin University, Chongqing 401120, China
- Chongqing Jitang Biotechnology Research Institute Co., Ltd., Chongqing 401120, China
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30
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Surya W, Torres J. Oligomerization-Dependent Beta-Structure Formation in SARS-CoV-2 Envelope Protein. Int J Mol Sci 2022; 23:13285. [PMID: 36362071 PMCID: PMC9658050 DOI: 10.3390/ijms232113285] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2022] [Revised: 10/28/2022] [Accepted: 10/28/2022] [Indexed: 08/13/2023] Open
Abstract
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic. In SARS-CoV-2, the channel-forming envelope (E) protein is almost identical to the E protein in SARS-CoV, and both share an identical α-helical channel-forming domain. Structures for the latter are available in both detergent and lipid membranes. However, models of the extramembrane domains have only been obtained from solution NMR in detergents, and show no β-strands, in contrast to secondary-structure predictions. Herein, we have studied the conformation of purified SARS-CoV-2 E protein in lipid bilayers that mimic the composition of ER-Golgi intermediate compartment (ERGIC) membranes. The full-length E protein at high protein-to-lipid ratios produced a clear shoulder at 1635 cm-1, consistent with the β-structure, but this was absent when the E protein was diluted, which instead showed a band at around 1688 cm-1, usually assigned to β-turns. The results were similar with a mixture of POPC:POPG (2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine/3-glycerol) and also when using an E-truncated form (residues 8-65). However, the latter only showed β-structure formation at the highest concentration tested, while having a weaker oligomerization tendency in detergents than in full-length E protein. Therefore, we conclude that E monomer-monomer interaction triggers formation of the β-structure from an undefined structure (possibly β-turns) in at least about 15 residues located at the C-terminal extramembrane domain. Due to its proximity to the channel, this β-structure domain could modulate channel activity or modify membrane structure at the time of virion formation inside the cell.
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Affiliation(s)
| | - Jaume Torres
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore
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31
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Kolocouris A, Arkin I, Glykos NM. A proof-of-concept study of the secondary structure of influenza A, B M2 and MERS- and SARS-CoV E transmembrane peptides using folding molecular dynamics simulations in a membrane mimetic solvent. Phys Chem Chem Phys 2022; 24:25391-25402. [PMID: 36239696 DOI: 10.1039/d2cp02881f] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
Here, we have carried out a proof-of-concept molecular dynamics (MD) simulation with adaptive tempering in a membrane mimetic environment to study the folding of single-pass membrane peptides. We tested the influenza A M2 viroporin, influenza B M2 viroporin, and protein E from coronaviruses MERS-Cov-2 and SARS-CoV-2 peptides with known experimental secondary structures in membrane bilayers. The two influenza-derived peptides are significantly different in the peptide sequence and secondary structure and more polar than the two coronavirus-derived peptides. Through a total of more than 50 μs of simulation time that could be accomplished in trifluoroethanol (TFE), as a membrane model, we characterized comparatively the folding behavior, helical stability, and helical propensity of these transmembrane peptides that match perfectly their experimental secondary structures, and we identified common motifs that reflect their quaternary organization and known (or not) biochemical function. We showed that BM2 is organized into two structurally distinct parts: a significantly more stable N-terminal half, and a fast-converting C-terminal half that continuously folds and unfolds between α-helical structures and non-canonical structures, which are mostly turns. In AM2, both the N-terminal half and C-terminal half are very flexible. In contrast, the two coronavirus-derived transmembrane peptides are much more stable and fast helix-formers when compared with the influenza ones. In particular, the SARS-derived peptide E appears to be the fastest and most stable helix-former of all the four viral peptides studied, with a helical structure that persists almost without disruption for the whole of its 10 μs simulation. By comparing the results with experimental observations, we benchmarked TFE in studying the conformation of membrane and hydrophobic peptides. This work provided accurate results suggesting a methodology to run long MD simulations and predict structural properties of biologically important membrane peptides.
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Affiliation(s)
- Antonios Kolocouris
- Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, National and Kapodistrian University of Athens, Panepistimiopolis, 15771, Greece.
| | - Isaiah Arkin
- Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Edmond J. Safra Campus Givat-Ram, Jerusalem, 91904, Israel
| | - Nicholas M Glykos
- Department of Molecular Biology and Genetics, Democritus University of Thrace, University Campus, Alexandroupolis, 68100, Greece.
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Amantadine and Rimantadine Inhibit Hepatitis A Virus Replication through the Induction of Autophagy. J Virol 2022; 96:e0064622. [PMID: 36040176 PMCID: PMC9517723 DOI: 10.1128/jvi.00646-22] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Hepatitis A virus (HAV) infection is a major cause of acute viral hepatitis worldwide. Furthermore, HAV causes acute liver failure or acute-on-chronic liver failure. However, no potent anti-HAV drugs are currently available in the clinical situations. There have been some reports that amantadine, a broad-spectrum antiviral, suppresses HAV replication in vitro. Therefore, we examined the effects of amantadine and rimantadine, derivates of adamantane, on HAV replication, and investigated the mechanisms of these drugs. In the present study, we evaluated the effects of amantadine and rimantadine on HAV HM175 genotype IB subgenomic replicon replication and HAV HA11-1299 genotype IIIA replication in cell culture infection systems. Amantadine and rimantadine significantly inhibited HAV replication at the post-entry stage in Huh7 cells. HAV infection inhibited autophagy by suppressing the autophagy marker light chain 3 and reducing number of lysosomes. Proteomic analysis on HAV-infected Huh7 cells treated by amantadine and rimantadine revealed the changes of the expression levels in 42 of 373 immune response-related proteins. Amantadine and rimantadine inhibited HAV replication, partially through the enhancement of autophagy. Taken together, our results suggest a novel mechanism by which HAV replicates along with the inhibition of autophagy and that amantadine and rimantadine inhibit HAV replication by enhancing autophagy. IMPORTANCE Amantadine, a nonspecific antiviral medication, also effectively inhibits HAV replication. Autophagy is an important cellular mechanism in several virus-host cell interactions. The results of this study provide evidence indicating that autophagy is involved in HAV replication and plays a role in the HAV life cycle. In addition, amantadine and its derivative rimantadine suppress HAV replication partly by enhancing autophagy at the post-entry phase of HAV infection in human hepatocytes. Amantadine may be useful for the control of acute HAV infection by inhibiting cellular autophagy pathways during HAV infection processes.
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In Silico Evaluation of Hexamethylene Amiloride Derivatives as Potential Luminal Inhibitors of SARS-CoV-2 E Protein. Int J Mol Sci 2022; 23:ijms231810647. [PMID: 36142556 PMCID: PMC9503309 DOI: 10.3390/ijms231810647] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2022] [Revised: 09/01/2022] [Accepted: 09/01/2022] [Indexed: 11/24/2022] Open
Abstract
The coronavirus E proteins are small membrane proteins found in the virus envelope of alpha and beta coronaviruses that have a high degree of overlap in their biochemical and functional properties despite minor sequence variations. The SARS-CoV-2 E is a 75-amino acid transmembrane protein capable of acting as an ion channel when assembled in a pentameric fashion. Various studies have found that hexamethylene amiloride (HMA) can inhibit the ion channel activity of the E protein in bilayers and also inhibit viral replication in cultured cells. Here, we use the available structural data in conjunction with homology modelling to build a comprehensive model of the E protein to assess potential binding sites and molecular interactions of HMA derivatives. Furthermore, we employed an iterative cycle of molecular modelling, extensive docking simulations, molecular dynamics and leveraging steered molecular dynamics to better understand the pore characteristics and quantify the affinity of the bound ligands. Results from this work highlight the potential of acylguanidines as blockers of the E protein and guide the development of subsequent small molecule inhibitors.
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34
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Sinha M, Zabini D, Guntur D, Nagaraj C, Enyedi P, Olschewski H, Kuebler WM, Olschewski A. Chloride channels in the lung: Challenges and perspectives for viral infections, pulmonary arterial hypertension, and cystic fibrosis. Pharmacol Ther 2022; 237:108249. [PMID: 35878810 DOI: 10.1016/j.pharmthera.2022.108249] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2022] [Revised: 07/06/2022] [Accepted: 07/11/2022] [Indexed: 10/16/2022]
Abstract
Fine control over chloride homeostasis in the lung is required to maintain membrane excitability, transepithelial transport as well as intra- and extracellular ion and water homeostasis. Over the last decades, a growing number of chloride channels and transporters have been identified in the cells of the pulmonary vasculature and the respiratory tract. The importance of these proteins is underpinned by the fact that impairment of their physiological function is associated with functional dysregulation, structural remodeling, or hereditary diseases of the lung. This paper reviews the field of chloride channels and transporters in the lung and discusses chloride channels in disease processes such as viral infections including SARS-CoV- 2, pulmonary arterial hypertension, cystic fibrosis and asthma. Although chloride channels have become a hot research topic in recent years, remarkably few of them have been targeted by pharmacological agents. As such, we complement the putative pathophysiological role of chloride channels here with a summary of their therapeutic potential.
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Affiliation(s)
- Madhushri Sinha
- Experimental Anaesthesiology, Department of Anaesthesiology and Intensive Care Medicine, Medical University of Graz, Auenbruggerplatz 5, 8036 Graz, Austria.
| | - Diana Zabini
- Department of Physiology, Neue Stiftingtalstrasse 6/V, 8010 Graz, Austria.
| | - Divya Guntur
- Experimental Anaesthesiology, Department of Anaesthesiology and Intensive Care Medicine, Medical University of Graz, Auenbruggerplatz 5, 8036 Graz, Austria.
| | - Chandran Nagaraj
- Ludwig Boltzmann Institute for Lung Vascular Research, Neue Stiftingtalstraße 6, 8010 Graz, Austria.
| | - Peter Enyedi
- Department of Physiology, Semmelweis University, Tűzoltó utca 37-47, 1094 Budapest, Hungary.
| | - Horst Olschewski
- Department of Internal Medicine, Division of Pulmonology, Medical University of Graz, Auenbruggerplatz 15, 8036 Graz, Austria.
| | - Wolfgang M Kuebler
- Institute of Physiology, Charité - Universitätsmedizin Berlin, Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany.
| | - Andrea Olschewski
- Experimental Anaesthesiology, Department of Anaesthesiology and Intensive Care Medicine, Medical University of Graz, Auenbruggerplatz 5, 8036 Graz, Austria; Ludwig Boltzmann Institute for Lung Vascular Research, Neue Stiftingtalstraße 6, 8010 Graz, Austria.
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35
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The Genetic Stability, Replication Kinetics and Cytopathogenicity of Recombinant Avian Coronaviruses with a T16A or an A26F Mutation within the E Protein Is Cell-Type Dependent. Viruses 2022; 14:v14081784. [PMID: 36016406 PMCID: PMC9415719 DOI: 10.3390/v14081784] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2022] [Revised: 08/09/2022] [Accepted: 08/11/2022] [Indexed: 11/25/2022] Open
Abstract
The envelope (E) protein of the avian coronavirus infectious bronchitis virus (IBV) is a small-membrane protein present in two forms during infection: a monomer and a pentameric ion channel. Each form has an independent role during replication; the monomer disrupts the secretory pathway, and the pentamer facilitates virion production. The presence of a T16A or A26F mutation within E exclusively generates the pentameric or monomeric form, respectively. We generated two recombinant IBVs (rIBVs) based on the apathogenic molecular clone Beau-R, containing either a T16A or A26F mutation, denoted as BeauR-T16A and BeauR-A26F. The replication and genetic stability of the rIBVs were assessed in several different cell types, including primary and continuous cells, ex vivo tracheal organ cultures (TOCs) and in ovo. Different replication profiles were observed between cell cultures of different origins. BeauR-A26F replicated to a lower level than Beau-R in Vero cells and in ovo but not in DF1, primary chicken kidney (CK) cells or TOCs. Genetic stability and cytopathic effects were found to differ depending on the cell system. The effect of the T16A and A26F mutations appear to be cell-type dependent, which, therefore, highlights the importance of cell type in the investigation of the IBV E protein.
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36
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Schoeman D, Cloete R, Fielding BC. The Flexible, Extended Coil of the PDZ-Binding Motif of the Three Deadly Human Coronavirus E Proteins Plays a Role in Pathogenicity. Viruses 2022; 14:v14081707. [PMID: 36016329 PMCID: PMC9416557 DOI: 10.3390/v14081707] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2022] [Revised: 07/22/2022] [Accepted: 07/29/2022] [Indexed: 02/04/2023] Open
Abstract
The less virulent human (h) coronaviruses (CoVs) 229E, NL63, OC43, and HKU1 cause mild, self-limiting respiratory tract infections, while the more virulent SARS-CoV-1, MERS-CoV, and SARS-CoV-2 have caused severe outbreaks. The CoV envelope (E) protein, an important contributor to the pathogenesis of severe hCoV infections, may provide insight into this disparate severity of the disease. We, therefore, generated full-length E protein models for SARS-CoV-1 and -2, MERS-CoV, HCoV-229E, and HCoV-NL63 and docked C-terminal peptides of each model to the PDZ domain of the human PALS1 protein. The PDZ-binding motif (PBM) of the SARS-CoV-1 and -2 and MERS-CoV models adopted a more flexible, extended coil, while the HCoV-229E and HCoV-NL63 models adopted a less flexible alpha helix. All the E peptides docked to PALS1 occupied the same binding site and the more virulent hCoV E peptides generally interacted more stably with PALS1 than the less virulent ones. We hypothesize that the increased flexibility of the PBM in the more virulent hCoVs facilitates more stable binding to various host proteins, thereby contributing to more severe disease. This is the first paper to model full-length 3D structures for both the more virulent and less virulent hCoV E proteins, providing novel insights for possible drug and/or vaccine development.
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Affiliation(s)
- Dewald Schoeman
- Molecular Biology and Virology Research Laboratory, Department of Medical Biosciences, University of the Western Cape, Private Bag X17, Bellville, Cape Town 7535, South Africa;
| | - Ruben Cloete
- South African Medical Research Council Bioinformatics Unit, South African National Bioinformatics Institute, University of the Western Cape, Private Bag X17, Bellville, Cape Town 7535, South Africa;
| | - Burtram C. Fielding
- Molecular Biology and Virology Research Laboratory, Department of Medical Biosciences, University of the Western Cape, Private Bag X17, Bellville, Cape Town 7535, South Africa;
- Correspondence:
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37
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Rani I, Kalsi A, Kaur G, Sharma P, Gupta S, Gautam RK, Chopra H, Bibi S, Ahmad SU, Singh I, Dhawan M, Emran TB. Modern drug discovery applications for the identification of novel candidates for COVID-19 infections. Ann Med Surg (Lond) 2022; 80:104125. [PMID: 35845863 PMCID: PMC9273307 DOI: 10.1016/j.amsu.2022.104125] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2022] [Revised: 07/05/2022] [Accepted: 07/06/2022] [Indexed: 11/23/2022] Open
Abstract
In early December 2019, a large pneumonia epidemic occurred in Wuhan, China. The World Health Organization is concerned about the outbreak of another coronavirus with the powerful, rapid, and contagious transmission. Anyone with minor symptoms like fever and cough or travel history to contaminated places might be suspected of having COVID-19. COVID-19 therapy focuses on treating the disease's symptoms. So far, no such therapeutic molecule has been shown effective in treating this condition. So the treatment is mostly supportive and plasma. Globally, numerous studies and researchers have recently started fighting this virus. Vaccines and chemical compounds are also being investigated against infection. COVID-19 was successfully diagnosed using RNA detection and very sensitive RT-PCR (reverse transcription-polymerase chain reaction). The evolution of particular vaccinations is required to reduce illness severity and spread. Numerous computational analyses and molecular docking have predicted various target compounds that might stop this condition. This paper examines the main characteristics of coronavirus and the computational analyses necessary to avoid infection.
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Affiliation(s)
- Isha Rani
- MM College of Pharmacy, Maharishi Markandeshwar (Deemed to be University), Mullana, Haryana, India
| | - Avjit Kalsi
- MM School of Pharmacy, MM University, Sadopur, Ambala, Haryana, India
| | - Gagandeep Kaur
- Chitkara School of Pharmacy, Chitkara University-Baddi, Himachal Pradesh, India
| | - Pankaj Sharma
- Apotex Research Pvt. Ltd, Bangalore, Karnataka, India
| | - Sumeet Gupta
- MM College of Pharmacy, Maharishi Markandeshwar (Deemed to be University), Mullana, Haryana, India
| | - Rupesh K. Gautam
- MM School of Pharmacy, MM University, Sadopur, Ambala, Haryana, India
| | - Hitesh Chopra
- Department of Pharmaceutics, Chitkara College of Pharmacy, Chitkara University, Punjab, India
| | - Shabana Bibi
- Yunnan Herbal Laboratory, College of Ecology and Environmental Sciences, Yunnan University, Kunming, 650091, Yunnan, China
- The International Joint Research Center for Sustainable Utilization of Cordyceps Bioresources in China and Southeast Asia, Yunnan University, Kunming, 650091, Yunnan, China
| | - Syed Umair Ahmad
- Department of Bioinformatics, Hazara University, Mansehra, Pakistan
| | - Inderbir Singh
- Department of Pharmaceutics, Chitkara College of Pharmacy, Chitkara University, Punjab, India
| | - Manish Dhawan
- Department of Microbiology, Punjab Agricultural University, Ludhiana, 141004, Punjab, India
- Trafford College, Altrincham, Manchester, WA14 5PQ, UK
| | - Talha Bin Emran
- Department of Pharmacy, BGC Trust University Bangladesh, Chittagong, 4381, Bangladesh
- Department of Pharmacy, Faculty of Allied Health Sciences, Daffodil International University, Dhaka, 1207, Bangladesh
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38
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Xia X, Cheng A, Wang M, Ou X, Sun D, Mao S, Huang J, Yang Q, Wu Y, Chen S, Zhang S, Zhu D, Jia R, Liu M, Zhao XX, Gao Q, Tian B. Functions of Viroporins in the Viral Life Cycle and Their Regulation of Host Cell Responses. Front Immunol 2022; 13:890549. [PMID: 35720341 PMCID: PMC9202500 DOI: 10.3389/fimmu.2022.890549] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2022] [Accepted: 05/10/2022] [Indexed: 11/13/2022] Open
Abstract
Viroporins are virally encoded transmembrane proteins that are essential for viral pathogenicity and can participate in various stages of the viral life cycle, thereby promoting viral proliferation. Viroporins have multifaceted effects on host cell biological functions, including altering cell membrane permeability, triggering inflammasome formation, inducing apoptosis and autophagy, and evading immune responses, thereby ensuring that the virus completes its life cycle. Viroporins are also virulence factors, and their complete or partial deletion often reduces virion release and reduces viral pathogenicity, highlighting the important role of these proteins in the viral life cycle. Thus, viroporins represent a common drug-protein target for inhibiting drugs and the development of antiviral therapies. This article reviews current studies on the functions of viroporins in the viral life cycle and their regulation of host cell responses, with the aim of improving the understanding of this growing family of viral proteins.
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Affiliation(s)
- Xiaoyan Xia
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Anchun Cheng
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Mingshu Wang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Xumin Ou
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Di Sun
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Sai Mao
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Juan Huang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Qiao Yang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Ying Wu
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Shun Chen
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Shaqiu Zhang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Dekang Zhu
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Renyong Jia
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Mafeng Liu
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Xin-Xin Zhao
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Qun Gao
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu City, China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
| | - Bin Tian
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China.,Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu City, China
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39
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Kuzmin A, Orekhov P, Astashkin R, Gordeliy V, Gushchin I. Structure and dynamics of the SARS-CoV-2 envelope protein monomer. Proteins 2022; 90:1102-1114. [PMID: 35119706 DOI: 10.1002/prot.26317] [Citation(s) in RCA: 22] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2021] [Revised: 01/09/2022] [Accepted: 01/31/2022] [Indexed: 12/11/2022]
Abstract
Coronaviruses, especially severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), present an ongoing threat to human wellbeing. Consequently, elucidation of molecular determinants of their function and interaction with the host is an important task. Whereas some of the coronaviral proteins are extensively characterized, others remain understudied. Here, we use molecular dynamics simulations to analyze the structure and dynamics of the SARS-CoV-2 envelope (E) protein (a viroporin) in the monomeric form. The protein consists of the hydrophobic α-helical transmembrane domain (TMD) and amphiphilic α-helices H2 and H3, connected by flexible linkers. We show that TMD has a preferable orientation in the membrane, while H2 and H3 reside at the membrane surface. Orientation of H2 is strongly influenced by palmitoylation of cysteines Cys40, Cys43, and Cys44. Glycosylation of Asn66 affects the orientation of H3. We also observe that the monomeric E protein both generates and senses the membrane curvature, preferably localizing with the C-terminus at the convex regions of the membrane; the protein in the pentameric form displays these properties as well. Localization to curved regions may be favorable for assembly of the E protein oligomers, whereas induction of curvature may facilitate the budding of the viral particles. The presented results may be helpful for a better understanding of the function of the coronaviral E protein and viroporins in general, and for overcoming the ongoing SARS-CoV-2 pandemic.
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Affiliation(s)
- Alexander Kuzmin
- Research Center for Molecular Mechanisms of Aging and Age-related Diseases, Moscow Institute of Physics and Technology, Dolgoprudny, Russia
| | - Philipp Orekhov
- Research Center for Molecular Mechanisms of Aging and Age-related Diseases, Moscow Institute of Physics and Technology, Dolgoprudny, Russia.,Faculty of Biology, M.V. Lomonosov Moscow State University, Moscow, Russia.,Faculty of Biology, Shenzhen MSU-BIT University, Shenzhen, China
| | - Roman Astashkin
- Research Center for Molecular Mechanisms of Aging and Age-related Diseases, Moscow Institute of Physics and Technology, Dolgoprudny, Russia.,Institut de Biologie Structurale (IBS), Université Grenoble Alpes, CEA, CNRS, Grenoble, France
| | - Valentin Gordeliy
- Research Center for Molecular Mechanisms of Aging and Age-related Diseases, Moscow Institute of Physics and Technology, Dolgoprudny, Russia.,Institut de Biologie Structurale (IBS), Université Grenoble Alpes, CEA, CNRS, Grenoble, France.,Institute of Biological Information Processing (IBI-7: Structural Biochemistry), Forschungszentrum Jülich GmbH, Jülich, Germany.,JuStruct: Jülich Center for Structural Biology, Forschungszentrum Jülich GmbH, Jülich, Germany
| | - Ivan Gushchin
- Research Center for Molecular Mechanisms of Aging and Age-related Diseases, Moscow Institute of Physics and Technology, Dolgoprudny, Russia
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40
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Medeiros-Silva J, Somberg NH, Wang HK, McKay MJ, Mandala VS, Dregni AJ, Hong M. pH- and Calcium-Dependent Aromatic Network in the SARS-CoV-2 Envelope Protein. J Am Chem Soc 2022; 144:6839-6850. [PMID: 35380805 DOI: 10.1021/jacs.2c00973] [Citation(s) in RCA: 30] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
The envelope (E) protein of the SARS-CoV-2 virus is a membrane-bound viroporin that conducts cations across the endoplasmic reticulum Golgi intermediate compartment (ERGIC) membrane of the host cell to cause virus pathogenicity. The structure of the closed state of the E transmembrane (TM) domain, ETM, was recently determined using solid-state NMR spectroscopy. However, how the channel pore opens to mediate cation transport is unclear. Here, we use 13C and 19F solid-state NMR spectroscopy to investigate the conformation and dynamics of ETM at acidic pH and in the presence of calcium ions, which mimic the ERGIC and lysosomal environment experienced by the E protein in the cell. Acidic pH and calcium ions increased the conformational disorder of the N- and C-terminal residues and also increased the water accessibility of the protein, indicating that the pore lumen has become more spacious. ETM contains three regularly spaced phenylalanine (Phe) residues in the center of the peptide. 19F NMR spectra of para-fluorinated Phe20 and Phe26 indicate that both residues exhibit two sidechain conformations, which coexist within each channel. These two Phe conformations differ in their water accessibility, lipid contact, and dynamics. Channel opening by acidic pH and Ca2+ increases the population of the dynamic lipid-facing conformation. These results suggest an intricate aromatic network that regulates the opening of the ETM channel pore. This aromatic network may be a target for E inhibitors against SARS-CoV-2 and related coronaviruses.
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Affiliation(s)
- João Medeiros-Silva
- Department of Chemistry, Massachusetts Institute of Technology, 170 Albany Street, Cambridge, Massachusetts 02139, United States
| | - Noah H Somberg
- Department of Chemistry, Massachusetts Institute of Technology, 170 Albany Street, Cambridge, Massachusetts 02139, United States
| | - Harrison K Wang
- Department of Chemistry, Massachusetts Institute of Technology, 170 Albany Street, Cambridge, Massachusetts 02139, United States
| | - Matthew J McKay
- Department of Chemistry, Massachusetts Institute of Technology, 170 Albany Street, Cambridge, Massachusetts 02139, United States
| | - Venkata S Mandala
- Department of Chemistry, Massachusetts Institute of Technology, 170 Albany Street, Cambridge, Massachusetts 02139, United States
| | - Aurelio J Dregni
- Department of Chemistry, Massachusetts Institute of Technology, 170 Albany Street, Cambridge, Massachusetts 02139, United States
| | - Mei Hong
- Department of Chemistry, Massachusetts Institute of Technology, 170 Albany Street, Cambridge, Massachusetts 02139, United States
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41
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Oh CK, Nakamura T, Beutler N, Zhang X, Piña-Crespo J, Talantova M, Ghatak S, Trudler D, Carnevale LN, McKercher SR, Bakowski MA, Diedrich JK, Roberts AJ, Woods AK, Chi V, Gupta AK, Rosenfeld MA, Kearns FL, Casalino L, Shaabani N, Liu H, Wilson IA, Amaro RE, Burton DR, Yates JR, Becker C, Rogers TF, Chatterjee AK, Lipton SA. Targeted protein S-nitrosylation of ACE2 as potential treatment to prevent spread of SARS-CoV-2 infection. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2022:2022.04.05.487060. [PMID: 35411336 PMCID: PMC8996617 DOI: 10.1101/2022.04.05.487060] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
Prevention of infection and propagation of SARS-CoV-2 is of high priority in the COVID-19 pandemic. Here, we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 Spike protein, thereby inhibiting viral entry, infectivity, and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and thus spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E-protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model, and thus provide a novel avenue for therapy.
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42
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Nahhas AF, Nahhas AF, Alshaikh AA, Webster TJ. Inhibiting Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Variants: Targeting the Spike and Envelope Proteins Using Nanomaterial Like Peptides. J Biomed Nanotechnol 2022; 18:1121-1130. [PMID: 35854452 DOI: 10.1166/jbn.2022.3307] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused significant death, economic crisis, and the world to almost completely shut down. This present study focused on targeting the novel SARS-CoV-2 envelope protein, which has not been frequently mutating, and the S protein with a much larger peptide capable of inhibiting virus-mammalian cell attraction. In doing so, molecular dynamics software was used here to model six peptides including: NapFFTLUFLTUTE, NapFFSLAFLTATE, NapFFSLUFLSUTE, NapFFTLAFLTATE, NapFFSLUFLSUSE, and NapFFMLUFLMUME. Results showed that two of these completely hydrophobic peptides (NapFFTLUFLTUTE and NapFFMLUFLMUME) had a strong ability to bind to the virus, preventing its binding to a mammalian cell membrane, entering the cell, and replicating by covering many cell attachment sites on SARS-CoV-2. Further cell modeling results demonstrated the low toxicity and suitable pharmacokinetic properties of both peptides making them ideal for additional in vitro and in vivo investigation. In this manner, these two peptides should be further explored for a wide range of present and future COVID-19 therapeutic and prophylactic applications.
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Affiliation(s)
- Alaa F Nahhas
- Biochemistry Department, College of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia
| | - Alrayan F Nahhas
- Biochemistry Department, College of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia
| | - Abdulrahman A Alshaikh
- Internal Medicine Department, College of Medicine, King Abdulaziz University, Jeddah 21589, Saudi Arabia
| | - Thomas J Webster
- Department of Chemical Engineering, College of Engineering, Northeastern University, Boston, MA 02115, United States
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43
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Breitinger U, Farag NS, Sticht H, Breitinger HG. Viroporins: Structure, function, and their role in the life cycle of SARS-CoV-2. Int J Biochem Cell Biol 2022; 145:106185. [PMID: 35219876 PMCID: PMC8868010 DOI: 10.1016/j.biocel.2022.106185] [Citation(s) in RCA: 20] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2021] [Revised: 02/15/2022] [Accepted: 02/21/2022] [Indexed: 12/12/2022]
Abstract
Viroporins are indispensable for viral replication. As intracellular ion channels they disturb pH gradients of organelles and allow Ca2+ flux across ER membranes. Viroporins interact with numerous intracellular proteins and pathways and can trigger inflammatory responses. Thus, they are relevant targets in the search for antiviral drugs. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) underlies the world-wide pandemic of COVID-19, where an effective therapy is still lacking despite impressive progress in the development of vaccines and vaccination campaigns. Among the 29 proteins of SARS-CoV-2, the E- and ORF3a proteins have been identified as viroporins that contribute to the massive release of inflammatory cytokines observed in COVID-19. Here, we describe structure and function of viroporins and their role in inflammasome activation and cellular processes during the virus replication cycle. Techniques to study viroporin function are presented, with a focus on cellular and electrophysiological assays. Contributions of SARS-CoV-2 viroporins to the viral life cycle are discussed with respect to their structure, channel function, binding partners, and their role in viral infection and virus replication. Viroporin sequences of new variants of concern (α–ο) of SARS-CoV-2 are briefly reviewed as they harbour changes in E and 3a proteins that may affect their function.
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Affiliation(s)
- Ulrike Breitinger
- Department of Biochemistry, German University in Cairo, New Cairo, Egypt
| | - Noha S Farag
- Department of Microbiology and Immunology, German University in Cairo, New Cairo, Egypt
| | - Heinrich Sticht
- Division of Bioinformatics, Institute for Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Germany
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44
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Ashour NA, Abo Elmaaty A, Sarhan AA, Elkaeed EB, Moussa AM, Erfan IA, Al-Karmalawy AA. A Systematic Review of the Global Intervention for SARS-CoV-2 Combating: From Drugs Repurposing to Molnupiravir Approval. Drug Des Devel Ther 2022; 16:685-715. [PMID: 35321497 PMCID: PMC8935998 DOI: 10.2147/dddt.s354841] [Citation(s) in RCA: 28] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2021] [Accepted: 02/26/2022] [Indexed: 02/05/2023] Open
Abstract
The rising outbreak of SARS-CoV-2 continues to unfold all over the world. The development of novel effective antiviral drugs to fight against SARS-CoV-2 is a time cost. As a result, some specific FDA-approved drugs have already been repurposed and authorized for COVID-19 treatment. The repurposed drugs used were either antiviral or non-antiviral drugs. Accordingly, the present review thoroughly focuses on the repurposing efficacy of these drugs including clinical trials experienced, the combination therapies used, the novel methods followed for treatment, and their future perspective. Therefore, drug repurposing was regarded as an effective avenue for COVID-19 treatment. Recently, molnupiravir is a prodrug antiviral medication that was approved in the United Kingdom in November 2021 for the treatment of COVID-19. On the other hand, PF-07321332 is an oral antiviral drug developed by Pfizer. For the treatment of COVID-19, the PF-07321332/ritonavir combination medication is used in Phase III studies and was marketed as Paxlovid. Herein, we represented the almost history of combating COVID-19 from repurposing to the recently available oral anti-SARS-CoV-2 candidates, as a new hope to end the current pandemic.
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Affiliation(s)
- Nada A Ashour
- Department of Clinical Pharmacology, Faculty of Pharmacy, Horus University-Egypt, New Damietta, 34518, Egypt
| | - Ayman Abo Elmaaty
- Department of Medicinal Chemistry, Faculty of Pharmacy, Port Said University, Port Said, 42526, Egypt
| | - Amany A Sarhan
- Department of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy, Horus University-Egypt, New Damietta, 34518, Egypt
| | - Eslam B Elkaeed
- Department of Pharmaceutical Sciences, College of Pharmacy, AlMaarefa University, Ad Diriyah, 13713, Riyadh, Saudi Arabia
| | - Ahmed M Moussa
- Department of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy, Horus University-Egypt, New Damietta, 34518, Egypt
| | - Ibrahim Ali Erfan
- Department of Pharmacology and Biochemistry, Faculty of Pharmacy, Horus University-Egypt, New Damietta, 34518, Egypt
| | - Ahmed A Al-Karmalawy
- Department of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy, Horus University-Egypt, New Damietta, 34518, Egypt
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45
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Yavarian J, Zebardast A, Latifi T. The role of severe acute respiratory syndrome coronavirus 2 viroporins in inflammation. ADVANCES IN HUMAN BIOLOGY 2022. [DOI: 10.4103/aihb.aihb_108_21] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/04/2022] Open
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46
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Hu Y, Xie X, Yang L, Wang A. A Comprehensive View on the Host Factors and Viral Proteins Associated With Porcine Epidemic Diarrhea Virus Infection. Front Microbiol 2021; 12:762358. [PMID: 34950116 PMCID: PMC8688245 DOI: 10.3389/fmicb.2021.762358] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2021] [Accepted: 10/26/2021] [Indexed: 11/17/2022] Open
Abstract
Porcine epidemic diarrhea virus (PEDV), a coronavirus pathogen of the pig intestinal tract, can cause fatal watery diarrhea in piglets, thereby causing huge economic losses to swine industries around the world. The pathogenesis of PEDV has intensively been studied; however, the viral proteins of PEDV and the host factors in target cells, as well as their interactions, which are the foundation of the molecular mechanisms of viral infection, remain to be summarized and updated. PEDV has multiple important structural and functional proteins, which play various roles in the process of virus infection. Among them, the S and N proteins play vital roles in biological processes related to PEDV survival via interacting with the host cell proteins. Meanwhile, a number of host factors including receptors are required for the infection of PEDV via interacting with the viral proteins, thereby affecting the reproduction of PEDV and contributing to its life cycle. In this review, we provide an updated understanding of viral proteins and host factors, as well as their interactions in terms of PEDV infection. Additionally, the effects of cellular factors, events, and signaling pathways on PEDV infection are also discussed. Thus, these comprehensive and profound insights should facilitate for the further investigations, control, and prevention of PEDV infection.
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Affiliation(s)
- Yi Hu
- Laboratory of Animal Disease Prevention and Control and Animal Model, Hunan Provincial Key Laboratory of Protein Engineering in Animal Vaccines, College of Veterinary Medicine, Hunan Agricultural University, Changsha, China
| | - Xiaohong Xie
- Hunan Engineering Research Center of Livestock and Poultry Health Care, Colleges of Veterinary Medicine, Hunan Agricultural University, Changsha, China
| | - Lingchen Yang
- Laboratory of Animal Disease Prevention and Control and Animal Model, Hunan Provincial Key Laboratory of Protein Engineering in Animal Vaccines, College of Veterinary Medicine, Hunan Agricultural University, Changsha, China
| | - Aibing Wang
- Laboratory of Animal Disease Prevention and Control and Animal Model, Hunan Provincial Key Laboratory of Protein Engineering in Animal Vaccines, College of Veterinary Medicine, Hunan Agricultural University, Changsha, China.,PCB Biotechnology, LLC, Rockville, MD, United States
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47
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Ahmadi K, Zahedifard F, Mafakher L, Einakian MA, Ghaedi T, Kavousipour S, Faezi S, Karmostaji A, Sharifi-Sarasiabi K, Gouklani H, Hassaniazad M. Active site-based analysis of structural proteins for drug targets in different human Coronaviruses. Chem Biol Drug Des 2021; 99:585-602. [PMID: 34914204 DOI: 10.1111/cbdd.14004] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2021] [Revised: 11/03/2021] [Accepted: 12/09/2021] [Indexed: 11/30/2022]
Abstract
Seven types of Coronaviruses (CoVs) have been identified that can cause infection in humans, including HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, SARS-CoV, HCoV-MERS, and SARS-CoV-2. In this study, we investigated the genetic structure, the homology of the structural protein sequences, as well as the investigation of the active site of structural proteins. The active site of structural proteins was determined based on the previous studies, and the homology of their amino acid sequences and structure was compared. Multiple sequence alignment of Spike protein of HCoVs showed that the receptor-binding domain of SARS-CoV-2, SARS-CoV, and MERS-CoV was located at a similar site to the S1 subunit. The binding motif of PDZ (postsynaptic density- 95/discs large/zona occludens-1) of the envelope protein, was conserved in SARS-CoV and SARS-CoV-2 according to multiple sequence alignment but showed different changes in the other HCoVs. Overall, Spike protein showed the most variation in its active sites, but the other structural proteins were highly conserved. In this study, for the first time, the active site of all structural proteins of HCoVs as a drug target was investigated. The binding site of these proteins can be suitable targets for drugs or vaccines among HCoVs.
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Affiliation(s)
- Khadijeh Ahmadi
- Infectious and Tropical Diseases Research Center, Hormozgan Health Institute, Hormozgan University of Medical Sciences, Bandar Abbas, Iran
| | - Farnaz Zahedifard
- Drug discovery and Evaluation unit, Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic
| | - Ladan Mafakher
- Thalassemia & Hemoglobinopathy Research center, Health research institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Mohammad Ali Einakian
- Food Health Research Center, Hormozgan University of Medical Sciences, Bandar Abbas, Iran
| | - Tayebeh Ghaedi
- Infectious and Tropical Diseases Research Center, Hormozgan Health Institute, Hormozgan University of Medical Sciences, Bandar Abbas, Iran
| | - Soudabeh Kavousipour
- Molecular Medicine Research Center, Hormozgan Health Institute, Hormozgan University of Medical Sciences, Bandar Abbas, Iran
| | - Sobhan Faezi
- Department of Microbiology, School of Medicine, Guilan University of Medical Sciences, Rasht, Iran
| | - Afsaneh Karmostaji
- Infectious and Tropical Diseases Research Center, Hormozgan Health Institute, Hormozgan University of Medical Sciences, Bandar Abbas, Iran
| | - Khojasteh Sharifi-Sarasiabi
- Infectious and Tropical Diseases Research Center, Hormozgan Health Institute, Hormozgan University of Medical Sciences, Bandar Abbas, Iran
| | - Hamed Gouklani
- Infectious and Tropical Diseases Research Center, Hormozgan Health Institute, Hormozgan University of Medical Sciences, Bandar Abbas, Iran
| | - Mehdi Hassaniazad
- Infectious and Tropical Diseases Research Center, Hormozgan Health Institute, Hormozgan University of Medical Sciences, Bandar Abbas, Iran
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48
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Toft-Bertelsen TL, Jeppesen MG, Tzortzini E, Xue K, Giller K, Becker S, Mujezinovic A, Bentzen BH, B Andreas L, Kolocouris A, Kledal TN, Rosenkilde MM. Amantadine has potential for the treatment of COVID-19 because it inhibits known and novel ion channels encoded by SARS-CoV-2. Commun Biol 2021; 4:1347. [PMID: 34853399 PMCID: PMC8636635 DOI: 10.1038/s42003-021-02866-9] [Citation(s) in RCA: 37] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2020] [Accepted: 11/02/2021] [Indexed: 12/16/2022] Open
Abstract
The dire need for COVID-19 treatments has inspired strategies of repurposing approved drugs. Amantadine has been suggested as a candidate, and cellular as well as clinical studies have indicated beneficial effects of this drug. We demonstrate that amantadine and hexamethylene-amiloride (HMA), but not rimantadine, block the ion channel activity of Protein E from SARS-CoV-2, a conserved viroporin among coronaviruses. These findings agree with their binding to Protein E as evaluated by solution NMR and molecular dynamics simulations. Moreover, we identify two novel viroporins of SARS-CoV-2; ORF7b and ORF10, by showing ion channel activity in a X. laevis oocyte expression system. Notably, amantadine also blocks the ion channel activity of ORF10, thereby providing two ion channel targets in SARS-CoV-2 for amantadine treatment in COVID-19 patients. A screen of known viroporin inhibitors on Protein E, ORF7b, ORF10 and Protein 3a from SARS-CoV-2 revealed inhibition of Protein E and ORF7b by emodin and xanthene, the latter also blocking Protein 3a. This illustrates a general potential of well-known ion channel blockers against SARS-CoV-2 and specifically a dual molecular basis for the promising effects of amantadine in COVID-19 treatment. We therefore propose amantadine as a novel, cheap, readily available and effective way to treat COVID-19.
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Affiliation(s)
- Trine Lisberg Toft-Bertelsen
- Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
- Department of Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Mads Gravers Jeppesen
- Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
- Synklino ApS, Charlottenlund, Denmark
| | - Eva Tzortzini
- Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, National and Kapodistrian University of Athens, Panepistimioupolis-Zografou, Athens, Greece
| | - Kai Xue
- Department of NMR-based structural biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
| | - Karin Giller
- Department of NMR-based structural biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
| | - Stefan Becker
- Department of NMR-based structural biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
| | - Amer Mujezinovic
- Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Bo Hjorth Bentzen
- Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Loren B Andreas
- Department of NMR-based structural biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
| | - Antonios Kolocouris
- Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, National and Kapodistrian University of Athens, Panepistimioupolis-Zografou, Athens, Greece
| | | | - Mette Marie Rosenkilde
- Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
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49
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Kolokouris D, Kalenderoglou IE, Kolocouris A. Inside and Out of the Pore: Comparing Interactions and Molecular Dynamics of Influenza A M2 Viroporin Complexes in Standard Lipid Bilayers. J Chem Inf Model 2021; 61:5550-5568. [PMID: 34714655 DOI: 10.1021/acs.jcim.1c00264] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Ion channels located at viral envelopes (viroporins) have a critical function for the replication of infectious viruses and are important drug targets. Over the last decade, the number and duration of molecular dynamics (MD) simulations of the influenza A M2 ion channel owing to the increased computational efficiency. Here, we aimed to define the system setup and simulation conditions for the correct description of the protein-pore and the protein-lipid interactions for influenza A M2 in comparison with experimental data. We performed numerous MD simulations of the influenza A M2 protein in complex with adamantane blockers in standard lipid bilayers using OPLS2005 and CHARMM36 (C36) force fields. We explored the effect of varying the M2 construct (M2(22-46) and M2(22-62)), the lipid buffer size and type (stiffer DMPC or softer POPC with or without 20% cholesterol), the simulation time, the H37 protonation site (Nδ or Νε), the conformational state of the W41 channel gate, and M2's cholesterol binding sites (BSs). We report that the 200 ns MD with M2(22-62) (having Nε Η37) in the 20 Å lipid buffer with the C36 force field accurately describe: (a) the M2 pore structure and interactions inside the pore, that is, adamantane channel blocker location, water clathrate structure, and water or chloride anion blockage/passage from the M2 pore in the presence of a channel blocker and (b) interactions between M2 and the membrane environment as reflected by the calculation of the M2 bundle tilt, folding of amphipathic helices, and cholesterol BSs. Strikingly, we also observed that the C36 1 μs MD simulations using M2(22-62) embedded in a 20 Å POPC:cholesterol (5:1) scrambled membrane produced frequent interactions with cholesterol, which when combined with computational kinetic analysis, revealed the experimentally observed BSs of cholesterol and suggested three similarly long-interacting positions in the top leaflet that have previously not been observed experimentally. These findings promise to be useful for other viroporin systems.
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Affiliation(s)
- Dimitrios Kolokouris
- Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis-Zografou, Athens 15771, Greece
| | - Iris E Kalenderoglou
- Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis-Zografou, Athens 15771, Greece
| | - Antonios Kolocouris
- Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis-Zografou, Athens 15771, Greece
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50
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Kamel WA, Kamel MI, Alhasawi A, Elmasry S, AlHamdan F, Al-Hashel JY. Effect of Pre-exposure Use of Amantadine on COVID-19 Infection: A Hospital-Based Cohort Study in Patients With Parkinson's Disease or Multiple Sclerosis. Front Neurol 2021; 12:704186. [PMID: 34690911 PMCID: PMC8529185 DOI: 10.3389/fneur.2021.704186] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2021] [Accepted: 09/07/2021] [Indexed: 11/28/2022] Open
Abstract
Background: Amantadine has been proposed to inhibit E-channel conductance in reconstituted lipid bilayers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We aimed to study whether patients on amantadine have altered risks of contracting COVID-19 infection. Methods: We conducted a hospital-based, observational, retrospective cohort study using data for patients on amantadine supported by data given by the patients through an online questionnaire. We included registered amantadine users in our hospital for 6 months or more on March 1, 2020, and non-amantadine users to act as the control group. We used forced entry, multiple logistic regression models to estimate adjusted ORs for amantadine adjusting for the confounders. Findings: Between September 1, 2019, and March 1, 2020, 212 patients with Parkinson's disease (PD) or multiple sclerosis (MS) received greater than one equal to two prescriptions of amantadine. We selected a random sample of diagnoses which matched 424 patients of non-amantadine users (1:2) as a control group (424 patients). Between March 1, 2020, and March 1, 2021, 256 patients responded to our online questionnaire, 87 patients were on amantadine (group I), and 169 patients were not (control group, group II). COVID-19 disease infection proved to be 5.7 and 11.8% in group I and II patients, respectively. Increased odds of COVID-19 in multivariable-adjusted models were associated with old age and history of contact with COVID cases. Amantadine was associated with a significantly reduced risk of COVID-19 disease infection (adjusted OR 0.256, 95% CI 0.074–0.888). Interpretation: Amantadine is associated with a reduced risk of COVID-19 infection after adjusting for a broad range of variables. History of contact with COVID cases and old age are risk factors for COVID-19 infection. Therefore, we recommended randomized clinical trials investigating amantadine use for the prevention of COVID-19.
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Affiliation(s)
- Walaa A Kamel
- Neurology Department, Faculty of Medicine, Beni-Suef University, Beni-Suef, Egypt.,Neurology Department, Ibn-Sina Hospital, Kuwait City, Kuwait
| | - Mohmed I Kamel
- Occupational and Environmental Medicine, Alexandria University, Alexandria, Egypt
| | - Almunther Alhasawi
- Internal Medicine and Infectious Diseases Consultant, Infectious Disease Hospital, Kuwait City, Kuwait
| | - Sameh Elmasry
- Internal Medicine and Infectious Diseases Consultant, Infectious Disease Hospital, Kuwait City, Kuwait
| | - Fajer AlHamdan
- Internal Medicine Department, Al-Sabah Hospital, Kuwait City, Kuwait
| | - Jasem Y Al-Hashel
- Neurology Department, Ibn-Sina Hospital, Kuwait City, Kuwait.,Department of Medicine, Faculty of Medicine, Kuwait University, Kuwait City, Kuwait
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