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1.
Nanoscale ; 14(23): 8291-8305, 2022 Jun 16.
Article in English | MEDLINE | ID: covidwho-1873563

ABSTRACT

The envelope (E) protein encoded in the genome of an RNA virus is crucial for the replication, budding and pathophysiology of the virus. In the light of the ongoing pandemic, we explored similarities/differences between SARS-CoV-1 and SARS-CoV-2 E protein ion channels in terms of their selectivity. Further, we also examined the impact of variation of the bath concentration and introduction of potential and concentration gradients across the channel on the binding ratios of sodium and chloride ions for the SARS-CoV-2 E protein. Ion transport is described through the fourth-order Poisson-Nernst-Planck-Bikerman (4PNPBik) model which generalizes the traditional model by including ionic interactions between ions and their surrounding medium and non-ionic interactions between particles due to their finite size. Governing equations are solved numerically using the immersed boundary-lattice Boltzmann method (IB-LBM). The mathematical model has been validated by comparing analytical and experimental ion activity. The SARS-CoV-1 E protein ion channel is found to be more permeable to cationic ions, while the SARS-CoV-2 E protein has similar selectivity for both cationic and anionic species. For SARS-CoV-2, an increase in the bath concentration results in an increase in the binding ratio for sodium ions. Furthermore, the chloride binding ratio increases as the concentration gradient increases. A potential gradient has a minimal effect on the binding ratio. The SARS-CoV-2 E protein was found to support higher ionic currents than the SARS-CoV-1 E protein. Furthermore, the ionic current increased with increasing bath concentrations.


Subject(s)
COVID-19 , SARS-CoV-2 , Chlorides , Humans , Ion Channels , Ion Transport , Sodium
2.
Commun Biol ; 4(1): 1347, 2021 12 01.
Article in English | MEDLINE | ID: covidwho-1852515

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.


Subject(s)
Amantadine/pharmacology , Amiloride/analogs & derivatives , Antiviral Agents/pharmacology , Rimantadine/pharmacology , SARS-CoV-2/drug effects , Viral Proteins/physiology , Amiloride/pharmacology , Ion Channels/physiology
3.
Viruses ; 14(4)2022 03 28.
Article in English | MEDLINE | ID: covidwho-1792421

ABSTRACT

Focusing on the transmembrane domains (TMDs) of viral fusion and channel-forming proteins (VCPs), experimentally available and newly generated peptides in an ideal conformation of the S and E proteins of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and SARS-CoV, gp41 and Vpu, both of human immunodeficiency virus type 1 (HIV-1), haemagglutinin and M2 of influenza A, as well as gB of herpes simplex virus (HSV), are embedded in a fully hydrated lipid bilayer and used in multi-nanosecond molecular dynamics simulations. It is aimed to identify differences in the dynamics of the individual TMDs of the two types of viral membrane proteins. The assumption is made that the dynamics of the individual TMDs are decoupled from their extra-membrane domains, and that the mechanics of the TMDs are distinct from each other due to the different mechanism of function of the two types of proteins. The diffusivity coefficient (DC) of the translational and rotational diffusion is decreased in the oligomeric state of the TMDs compared to those values when calculated from simulations in their monomeric state. When comparing the calculations for two different lengths of the TMD, a longer full peptide and a shorter purely TMD stretch, (i) the difference of the calculated DCs begins to level out when the difference exceeds approximately 15 amino acids per peptide chain, and (ii) the channel protein rotational DC is the most affected diffusion parameter. The rotational dynamics of the individual amino acids within the middle section of the TMDs of the fusion peptides remain high upon oligomerization, but decrease for the channel peptides, with an increasing number of monomers forming the oligomeric state, suggesting an entropic penalty on oligomerization for the latter.


Subject(s)
COVID-19 , Ion Channels , Molecular Dynamics Simulation , Viral Fusion Proteins , Amino Acids , Humans , Ion Channels/ultrastructure , Peptides/chemistry , SARS-CoV-2 , Viral Fusion Proteins/ultrastructure
4.
Cells ; 11(6)2022 03 08.
Article in English | MEDLINE | ID: covidwho-1760407

ABSTRACT

A distinct set of channels and transporters regulates the ion fluxes across the lysosomal membrane. Malfunctioning of these transport proteins and the resulting ionic imbalance is involved in various human diseases, such as lysosomal storage disorders, cancer, as well as metabolic and neurodegenerative diseases. As a consequence, these proteins have stimulated strong interest for their suitability as possible drug targets. A detailed functional characterization of many lysosomal channels and transporters is lacking, mainly due to technical difficulties in applying the standard patch-clamp technique to these small intracellular compartments. In this review, we focus on current methods used to unravel the functional properties of lysosomal ion channels and transporters, stressing their advantages and disadvantages and evaluating their fields of applicability.


Subject(s)
Ion Channels , Lysosomal Storage Diseases , Humans , Intracellular Membranes/metabolism , Ion Channels/metabolism , Ions/metabolism , Lysosomal Storage Diseases/metabolism , Lysosomes/metabolism , Patch-Clamp Techniques
5.
Math Biosci Eng ; 19(4): 3269-3284, 2022 01 21.
Article in English | MEDLINE | ID: covidwho-1667425

ABSTRACT

Research on the relationship between drugs and targets is the key to precision medicine. Ion channel is a kind of important drug targets. Aiming at the urgent needs of corona virus disease 2019 (COVID-19) treatment and drug development, this paper designed a mixed graph network model to predict the affinity between ion channel targets of COVID-19 and drugs. According to the simplified molecular input line entry specification (SMILES) code of drugs, firstly, the atomic features were extracted to construct the point sets, and edge sets were constructed according to atomic bonds. Then the undirected graph with atomic features was generated by RDKit tool and the graph attention layer was used to extract the drug feature information. Five ion channel target proteins were screened from the whole SARS-CoV-2 genome sequences of NCBI database, and the protein features were extracted by convolution neural network (CNN). Using attention mechanism and graph convolutional network (GCN), the extracted drug features and target features information were connected. After two full connection layers operation, the drug-target affinity was output, and model was obtained. Kiba dataset was used to train the model and determine the model parameters. Compared with DeepDTA, WideDTA, graph attention network (GAT), GCN and graph isomorphism network (GIN) models, it was proved that the mean square error (MSE) of the proposed model was decreased by 0.055, 0.04, 0.001, 0.046, 0.013 and the consistency index (CI) was increased by 0.028, 0.016, 0.003, 0.03 and 0.01, respectively. It can predict the drug-target affinity more accurately. According to the prediction results of drug-target affinity of SARS-CoV-2 ion channel targets, seven kinds of small molecule drugs acting on five ion channel targets were obtained, namely SCH-47112, Dehydroaltenusin, alternariol 5-o-sulfate, LPA1 antagonist 1, alternariol, butin, and AT-9283.These drugs provide a reference for drug repositioning and precise treatment of COVID-19.


Subject(s)
COVID-19 , Drug Repositioning , COVID-19/drug therapy , Humans , Ion Channels , Neural Networks, Computer , SARS-CoV-2
6.
Cells ; 11(1)2021 12 24.
Article in English | MEDLINE | ID: covidwho-1580995

ABSTRACT

The lamellar body (LB) of the alveolar type II (ATII) cell is a lysosome-related organelle (LRO) that contains surfactant, a complex mix of mainly lipids and specific surfactant proteins. The major function of surfactant in the lung is the reduction of surface tension and stabilization of alveoli during respiration. Its lack or deficiency may cause various forms of respiratory distress syndrome (RDS). Surfactant is also part of the innate immune system in the lung, defending the organism against air-borne pathogens. The limiting (organelle) membrane that encloses the LB contains various transporters that are in part responsible for translocating lipids and other organic material into the LB. On the other hand, this membrane contains ion transporters and channels that maintain a specific internal ion composition including the acidic pH of about 5. Furthermore, P2X4 receptors, ligand gated ion channels of the danger signal ATP, are expressed in the limiting LB membrane. They play a role in boosting surfactant secretion and fluid clearance. In this review, we discuss the functions of these transporting pathways of the LB, including possible roles in disease and as therapeutic targets, including viral infections such as SARS-CoV-2.


Subject(s)
COVID-19/metabolism , Ion Channels/metabolism , Lung/metabolism , Membrane Transport Proteins/metabolism , Pulmonary Surfactants/metabolism , COVID-19/virology , Humans , Lung/virology , Organelles/metabolism , Organelles/virology , Pulmonary Alveoli/metabolism , Pulmonary Alveoli/virology , SARS-CoV-2/physiology
7.
Eur J Pharmacol ; 915: 174670, 2022 Jan 15.
Article in English | MEDLINE | ID: covidwho-1549763

ABSTRACT

Hydroxychloroquine (HCQ) is a derivative of the antimalaria drug chloroquine primarily prescribed for autoimmune diseases. Recent attempts to repurpose HCQ in the treatment of corona virus disease 2019 has raised concerns because of its propensity to prolong the QT-segment on the electrocardiogram, an effect associated with increased pro-arrhythmic risk. Since chirality can affect drug pharmacological properties, we have evaluated the functional effects of the R(-) and S(+) enantiomers of HCQ on six ion channels contributing to the cardiac action potential and on electrophysiological parameters of isolated Purkinje fibers. We found that R(-)HCQ and S(+)HCQ block human Kir2.1 and hERG potassium channels in the 1 µM-100 µM range with a 2-4 fold enantiomeric separation. NaV1.5 sodium currents and CaV1.2 calcium currents, as well as KV4.3 and KV7.1 potassium currents remained unaffected at up to 90 µM. In rabbit Purkinje fibers, R(-)HCQ prominently depolarized the membrane resting potential, inducing autogenic activity at 10 µM and 30 µM, while S(+)HCQ primarily increased the action potential duration, inducing occasional early afterdepolarization at these concentrations. These data suggest that both enantiomers of HCQ can alter cardiac tissue electrophysiology at concentrations above their plasmatic levels at therapeutic doses, and that chirality does not substantially influence their arrhythmogenic potential in vitro.


Subject(s)
Antimalarials/chemistry , Antimalarials/pharmacology , Heart/drug effects , Hydroxychloroquine/chemistry , Hydroxychloroquine/pharmacology , Ion Channels/drug effects , Action Potentials/drug effects , Animals , Arrhythmias, Cardiac/chemically induced , Electrocardiography , Electrophysiologic Techniques, Cardiac , Ether-A-Go-Go Potassium Channels , Humans , Membrane Potentials/drug effects , Patch-Clamp Techniques , Purkinje Fibers/drug effects , Rabbits , Stereoisomerism
8.
Commun Biol ; 4(1): 1347, 2021 12 01.
Article in English | MEDLINE | ID: covidwho-1545655

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.


Subject(s)
Amantadine/pharmacology , Amiloride/analogs & derivatives , Antiviral Agents/pharmacology , Rimantadine/pharmacology , SARS-CoV-2/drug effects , Viral Proteins/physiology , Amiloride/pharmacology , Ion Channels/physiology
9.
Front Immunol ; 12: 767319, 2021.
Article in English | MEDLINE | ID: covidwho-1538373

ABSTRACT

The importance of innate immune cells to sense and respond to their physical environment is becoming increasingly recognized. Innate immune cells (e.g. macrophages and neutrophils) are able to receive mechanical signals through several mechanisms. In this review, we discuss the role of mechanosensitive ion channels, such as Piezo1 and transient receptor potential vanilloid 4 (TRPV4), and cell adhesion molecules, such as integrins, selectins, and cadherins in biology and human disease. Furthermore, we explain that these mechanical stimuli activate intracellular signaling pathways, such as MAPK (p38, JNK), YAP/TAZ, EDN1, NF-kB, and HIF-1α, to induce protein conformation changes and modulate gene expression to drive cellular function. Understanding the mechanisms by which immune cells interpret mechanosensitive information presents potential targets to treat human disease. Important areas of future study in this area include autoimmune, allergic, infectious, and malignant conditions.


Subject(s)
Immunity, Innate/immunology , Macrophages/immunology , Mechanotransduction, Cellular/immunology , Neutrophils/immunology , Signal Transduction/immunology , Animals , Cytokines/immunology , Cytokines/metabolism , Humans , Ion Channels/immunology , Ion Channels/metabolism , Macrophages/metabolism , Neutrophils/metabolism , TRPV Cation Channels/immunology , TRPV Cation Channels/metabolism
10.
J Cell Physiol ; 237(2): 1521-1531, 2022 02.
Article in English | MEDLINE | ID: covidwho-1490820

ABSTRACT

Mechanical forces can modulate the immune response, mostly described as promoting the activation of immune cells, but the role and mechanism of pathological levels of mechanical stress in lymphocyte activation have not been focused on before. By an ex vivo experimental approach, we observed that mechanical stressing of murine spleen lymphocytes with 50 mmHg for 3 h induced the nuclear localization of NFAT1, increased C-Jun, and increased the expression of early activation marker CD69 in resting CD8+ cells. Interestingly, 50 mmHg mechanical stressing induced the nuclear localization of NFAT1; but conversely decreased C-Jun and inhibited the expression of CD69 in lymphocytes under lipopolysaccharide or phorbol 12-myristate 13-acetate/ionomycin stimulation. Additionally, we observed similar changes trends when comparing RNA-seq data of hypertensive and normotensive COVID-19 patients. Our results indicate a biphasic effect of mechanical stress on lymphocyte activation, which provides insight into the variety of immune responses in pathologies involving elevated mechanical stress.


Subject(s)
Lymphocyte Activation/immunology , Stress, Mechanical , Animals , Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/metabolism , Biomarkers/metabolism , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , COVID-19/complications , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Comorbidity , Gene Expression Regulation/drug effects , Humans , Hypertension/complications , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Ion Channels/metabolism , Lectins, C-Type/metabolism , Lipopolysaccharides/pharmacology , Lymphocyte Activation/drug effects , Lymphocyte Activation/genetics , Male , Mice, Inbred C57BL , NFATC Transcription Factors/metabolism , Protein Transport/drug effects , Proto-Oncogene Proteins c-jun/metabolism , Signal Transduction/drug effects , Tetradecanoylphorbol Acetate/pharmacology
11.
Viruses ; 13(11)2021 10 27.
Article in English | MEDLINE | ID: covidwho-1488757

ABSTRACT

The current COVID-19 pandemic has highlighted the need for the research community to develop a better understanding of viruses, in particular their modes of infection and replicative lifecycles, to aid in the development of novel vaccines and much needed anti-viral therapeutics. Several viruses express proteins capable of forming pores in host cellular membranes, termed "Viroporins". They are a family of small hydrophobic proteins, with at least one amphipathic domain, which characteristically form oligomeric structures with central hydrophilic domains. Consequently, they can facilitate the transport of ions through the hydrophilic core. Viroporins localise to host membranes such as the endoplasmic reticulum and regulate ion homeostasis creating a favourable environment for viral infection. Viroporins also contribute to viral immune evasion via several mechanisms. Given that viroporins are often essential for virion assembly and egress, and as their structural features tend to be evolutionarily conserved, they are attractive targets for anti-viral therapeutics. This review discusses the current knowledge of several viroporins, namely Influenza A virus (IAV) M2, Human Immunodeficiency Virus (HIV)-1 Viral protein U (Vpu), Hepatitis C Virus (HCV) p7, Human Papillomavirus (HPV)-16 E5, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) Open Reading Frame (ORF)3a and Polyomavirus agnoprotein. We highlight the intricate but broad immunomodulatory effects of these viroporins and discuss the current antiviral therapies that target them; continually highlighting the need for future investigations to focus on novel therapeutics in the treatment of existing and future emergent viruses.


Subject(s)
Immunomodulation , Ion Channels/metabolism , Viroporin Proteins/metabolism , Virus Diseases/drug therapy , Viruses/metabolism , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Autophagy , Host-Pathogen Interactions , Human Immunodeficiency Virus Proteins/chemistry , Human Immunodeficiency Virus Proteins/metabolism , Immune Evasion , Inflammasomes/immunology , Oncogene Proteins, Viral/chemistry , Oncogene Proteins, Viral/metabolism , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/metabolism , Viral Proteins/chemistry , Viral Proteins/metabolism , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/metabolism , Viral Structural Proteins/chemistry , Viral Structural Proteins/metabolism , Viroporin Proteins/chemistry , Virus Diseases/immunology , Virus Diseases/virology , Viruses/drug effects , Viruses/immunology , Viruses/pathogenicity
12.
Viruses ; 13(10)2021 10 15.
Article in English | MEDLINE | ID: covidwho-1470997

ABSTRACT

We report the in vitro efficacy of ion-channel inhibitors amantadine, memantine and rimantadine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In VeroE6 cells, rimantadine was most potent followed by memantine and amantadine (50% effective concentrations: 36, 80 and 116 µM, respectively). Rimantadine also showed the highest selectivity index, followed by amantadine and memantine (17.3, 12.2 and 7.6, respectively). Similar results were observed in human hepatoma Huh7.5 and lung carcinoma A549-hACE2 cells. Inhibitors interacted in a similar antagonistic manner with remdesivir and had a similar barrier to viral escape. Rimantadine acted mainly at the viral post-entry level and partially at the viral entry level. Based on these results, rimantadine showed the most promise for treatment of SARS-CoV-2.


Subject(s)
Amantadine/pharmacology , Antiviral Agents/pharmacology , COVID-19/drug therapy , Memantine/pharmacology , Rimantadine/pharmacology , SARS-CoV-2/drug effects , A549 Cells , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Animals , Cell Line, Tumor , Chlorocebus aethiops , Denmark , Drug Repositioning , Humans , Ion Channels/antagonists & inhibitors , Vero Cells
13.
ACS Chem Biol ; 15(9): 2331-2337, 2020 09 18.
Article in English | MEDLINE | ID: covidwho-1387140

ABSTRACT

We report on using the synthetic aminoadamantane-CH2-aryl derivatives 1-6 as sensitive probes for blocking M2 S31N and influenza A virus (IAV) M2 wild-type (WT) channels as well as virus replication in cell culture. The binding kinetics measured using electrophysiology (EP) for M2 S31N channel are very dependent on the length between the adamantane moiety and the first ring of the aryl headgroup realized in 2 and 3 and the girth and length of the adamantane adduct realized in 4 and 5. Study of 1-6 shows that, according to molecular dynamics (MD) simulations and molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) calculations, all bind in the M2 S31N channel with the adamantyl group positioned between V27 and G34 and the aryl group projecting out of the channel with the phenyl (or isoxazole in 6) embedded in the V27 cluster. In this outward binding configuration, an elongation of the ligand by only one methylene in rimantadine 2 or using diamantane or triamantane instead of adamantane in 4 and 5, respectively, causes incomplete entry and facilitates exit, abolishing effective block compared to the amantadine derivatives 1 and 6. In the active M2 S31N blockers 1 and 6, the phenyl and isoxazolyl head groups achieve a deeper binding position and high kon/low koff and high kon/high koff rate constants, compared to inactive 2-5, which have much lower kon and higher koff. Compounds 1-5 block the M2 WT channel by binding in the longer area from V27-H37, in the inward orientation, with high kon and low koff rate constants. Infection of cell cultures by influenza virus containing M2 WT or M2 S31N is inhibited by 1-5 or 1-4 and 6, respectively. While 1 and 6 block infection through the M2 block mechanism in the S31N variant, 2-4 may block M2 S31N virus replication in cell culture through the lysosomotropic effect, just as chloroquine is thought to inhibit SARS-CoV-2 infection.


Subject(s)
Adamantane/pharmacology , Influenza A virus/drug effects , Influenza, Human/prevention & control , Ion Channels/antagonists & inhibitors , Molecular Probes/chemistry , Viral Matrix Proteins/antagonists & inhibitors , Adamantane/analogs & derivatives , Adamantane/chemistry , Adamantane/metabolism , Betacoronavirus/drug effects , Binding Sites , COVID-19 , Cells, Cultured , Chloroquine/pharmacology , Coronavirus Infections/drug therapy , Coronavirus Infections/prevention & control , Genetic Variation , Humans , Influenza A virus/chemistry , Influenza A virus/genetics , Influenza, Human/drug therapy , Kinetics , Molecular Probes/metabolism , Pandemics/prevention & control , Pneumonia, Viral/drug therapy , Pneumonia, Viral/prevention & control , Protein Binding , SARS-CoV-2 , Structure-Activity Relationship , Virus Replication/drug effects
14.
J Chem Inf Model ; 61(8): 4011-4022, 2021 08 23.
Article in English | MEDLINE | ID: covidwho-1327180

ABSTRACT

Target-based design and repositioning are mainstream strategies of drug discovery. Numerous drug design and repositioning projects have been launched to fight the ongoing COVID-19 pandemic. The resulting drug candidates have often failed due to the misprediction of their target-bound structures. The determination of water positions of such structures is particularly challenging due to the large number of possible drugs and the diversity of their hydration patterns. To answer this challenge and help correct predictions, we introduce a new protocol HydroDock, which can build hydrated drug-target complexes from scratch. HydroDock requires only the dry target and drug structures and produces their complexes with appropriately positioned water molecules. As a test application of the protocol, we built the structures of amantadine derivatives in complex with the influenza M2 transmembrane ion channel. The repositioning of amantadine derivatives from this influenza target to the SARS-CoV-2 envelope protein was also investigated. Excellent agreement was observed between experiments and the structures determined by HydroDock. The atomic resolution complex structures showed that water plays a similar role in the binding of amphipathic amantadine derivatives to transmembrane ion channels of both influenza A and SARS-CoV-2. While the hydrophobic regions of the channels capture the bulky hydrocarbon group of the ligand, the surrounding waters direct its orientation parallel with the axes of the channels via bridging interactions with the ionic ligand head. As HydroDock supplied otherwise undetermined structural details, it can be recommended to improve the reliability of future design and repositioning of antiviral drug candidates and many other ligands with an influence of water structure on their mechanism of action.


Subject(s)
COVID-19 , Drug Design , Antiviral Agents/pharmacology , Drug Repositioning , Humans , Ion Channels , Ligands , Pandemics , Reproducibility of Results , SARS-CoV-2 , Viral Matrix Proteins/metabolism
15.
PLoS Pathog ; 17(5): e1009519, 2021 05.
Article in English | MEDLINE | ID: covidwho-1232468

ABSTRACT

SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose transmembrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle, including virion assembly, budding, release, and pathogenesis. Here, we describe the structure and dynamics of full-length SARS-CoV-2 E protein in hexadecylphosphocholine micelles by NMR spectroscopy. We also characterized its interactions with four putative ion channel inhibitors. The chemical shift index and dipolar wave plots establish that E protein consists of a long transmembrane helix (residues 8-43) and a short cytoplasmic helix (residues 53-60) connected by a complex linker that exhibits some internal mobility. The conformations of the N-terminal transmembrane domain and the C-terminal cytoplasmic domain are unaffected by truncation from the intact protein. The chemical shift perturbations of E protein spectra induced by the addition of the inhibitors demonstrate that the N-terminal region (residues 6-18) is the principal binding site. The binding affinity of the inhibitors to E protein in micelles correlates with their antiviral potency in Vero E6 cells: HMA ≈ EIPA > DMA >> Amiloride, suggesting that bulky hydrophobic groups in the 5' position of the amiloride pyrazine ring play essential roles in binding to E protein and in antiviral activity. An N15A mutation increased the production of virus-like particles, induced significant chemical shift changes from residues in the inhibitor binding site, and abolished HMA binding, suggesting that Asn15 plays a key role in maintaining the protein conformation near the binding site. These studies provide the foundation for complete structure determination of E protein and for structure-based drug discovery targeting this protein.


Subject(s)
Amiloride/pharmacology , COVID-19/drug therapy , Coronavirus Envelope Proteins/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , Amiloride/pharmacokinetics , Animals , Antiviral Agents/pharmacology , Binding Sites/drug effects , COVID-19/virology , Chlorocebus aethiops , Coronavirus Envelope Proteins/chemistry , Humans , Ion Channels/metabolism , Nuclear Magnetic Resonance, Biomolecular , Protein Binding/drug effects , Protein Conformation/drug effects , Protein Domains , Vero Cells , Virus Assembly/drug effects
17.
Acta Crystallogr D Struct Biol ; 77(Pt 4): 391-402, 2021 Apr 01.
Article in English | MEDLINE | ID: covidwho-1172082

ABSTRACT

Viral infection compromises specific organelles of the cell and readdresses its functional resources to satisfy the needs of the invading body. Around 70% of the coronavirus positive-sense single-stranded RNA encodes proteins involved in replication, and these viruses essentially take over the biosynthetic and transport mechanisms to ensure the efficient replication of their genome and trafficking of their virions. Some coronaviruses encode genes for ion-channel proteins - the envelope protein E (orf4a), orf3a and orf8 - which they successfully employ to take control of the endoplasmic reticulum-Golgi complex intermediate compartment or ERGIC. The E protein, which is one of the four structural proteins of SARS-CoV-2 and other coronaviruses, assembles its transmembrane protomers into homopentameric channels with mild cationic selectivity. Orf3a forms homodimers and homotetramers. Both carry a PDZ-binding domain, lending them the versatility to interact with more than 400 target proteins in infected host cells. Orf8 is a very short 29-amino-acid single-passage transmembrane peptide that forms cation-selective channels when assembled in lipid bilayers. This review addresses the contribution of biophysical and structural biology approaches that unravel different facets of coronavirus ion channels, their effects on the cellular machinery of infected cells and some structure-functional correlations with ion channels of higher organisms.


Subject(s)
Computational Chemistry , Ion Channels/chemistry , SARS-CoV-2/chemistry , Viral Proteins/chemistry , COVID-19/virology , Protein Conformation
18.
Clin Transl Sci ; 14(3): 1133-1146, 2021 05.
Article in English | MEDLINE | ID: covidwho-1096723

ABSTRACT

We applied a set of in silico and in vitro assays, compliant with the Comprehensive In Vitro Proarrhythmia Assay (CiPA) paradigm, to assess the risk of chloroquine (CLQ) or hydroxychloroquine (OH-CLQ)-mediated QT prolongation and Torsades de Pointes (TdP), alone and combined with erythromycin (ERT) and azithromycin (AZI), drugs repurposed during the first wave of coronavirus disease 2019 (COVID-19). Each drug or drug combination was tested in patch clamp assays on seven cardiac ion channels, in in silico models of human ventricular electrophysiology (Virtual Assay) using control (healthy) or high-risk cell populations, and in human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. In each assay, concentration-response curves encompassing and exceeding therapeutic free plasma levels were generated. Both CLQ and OH-CLQ showed blocking activity against some potassium, sodium, and calcium currents. CLQ and OH-CLQ inhibited IKr (half-maximal inhibitory concentration [IC50 ]: 1 µM and 3-7 µM, respectively) and IK1 currents (IC50 : 5 and 44 µM, respectively). When combining OH-CLQ with AZI, no synergistic effects were observed. The two macrolides had no or very weak effects on the ion currents (IC50  > 300-1000 µM). Using Virtual Assay, both antimalarials affected several TdP indicators, CLQ being more potent than OH-CLQ. Effects were more pronounced in the high-risk cell population. In hiPSC-derived cardiomyocytes, all drugs showed early after-depolarizations, except AZI. Combining CLQ or OH-CLQ with a macrolide did not aggravate their effects. In conclusion, our integrated nonclinical CiPA dataset confirmed that, at therapeutic plasma concentrations relevant for malaria or off-label use in COVID-19, CLQ and OH-CLQ use is associated with a proarrhythmia risk, which is higher in populations carrying predisposing factors but not worsened with macrolide combination.


Subject(s)
Antimalarials/adverse effects , Arrhythmias, Cardiac/chemically induced , COVID-19/drug therapy , Chloroquine/adverse effects , Hydroxychloroquine/adverse effects , Off-Label Use , SARS-CoV-2 , Animals , CHO Cells , Cricetulus , Dose-Response Relationship, Drug , Electrocardiography/drug effects , Humans , Ion Channels/drug effects
19.
Cell Calcium ; 94: 102360, 2021 03.
Article in English | MEDLINE | ID: covidwho-1064903

ABSTRACT

Ion channels are necessary for correct lysosomal function including degradation of cargoes originating from endocytosis. Almost all enveloped viruses, including coronaviruses (CoVs), enter host cells via endocytosis, and do not escape endosomal compartments into the cytoplasm (via fusion with the endolysosomal membrane) unless the virus-encoded envelope proteins are cleaved by lysosomal proteases. With the ongoing outbreak of severe acute respiratory syndrome (SARS)-CoV-2, endolysosomal two-pore channels represent an exciting and emerging target for antiviral therapies. This review focuses on the latest knowledge of the effects of lysosomal ion channels on the cellular entry and uncoating of enveloped viruses, which may aid in development of novel therapies against emerging infectious diseases such as SARS-CoV-2.


Subject(s)
Antiviral Agents/therapeutic use , COVID-19/virology , Ion Channels/physiology , Lysosomes/virology , SARS-CoV-2/physiology , Viral Envelope/physiology , Virus Internalization , Virus Uncoating , Aminopyridines/pharmacology , Aminopyridines/therapeutic use , Antiviral Agents/pharmacology , Drug Design , Endocytosis , Endosomes/metabolism , Endosomes/virology , Heterocyclic Compounds, 3-Ring/pharmacology , Heterocyclic Compounds, 3-Ring/therapeutic use , Humans , Hydrazones/pharmacology , Hydrazones/therapeutic use , Ion Channels/classification , Lysosomes/enzymology , Lysosomes/metabolism , Models, Biological , Morpholines/pharmacology , Morpholines/therapeutic use , Pyrimidines/pharmacology , Pyrimidines/therapeutic use , Vacuolar Proton-Translocating ATPases/physiology , Virus Internalization/drug effects , Virus Uncoating/drug effects
20.
Stem Cell Res ; 52: 102219, 2021 04.
Article in English | MEDLINE | ID: covidwho-1062599

ABSTRACT

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). By late October 2020, more than 43 million cases of infections, including over 1.15 million deaths, have been confirmed worldwide. This review focuses on our current understanding of SARS-CoV-2 from the perspective of the three-dimensional (3D) structures of SARS-CoV-2 viral proteins and their implications on therapeutics development against COVID-19.


Subject(s)
COVID-19/drug therapy , COVID-19/epidemiology , Antibodies, Viral/chemistry , Genome, Viral , Humans , Ion Channels/chemistry , Protein Conformation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry
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