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1.
Front Immunol ; 13: 834988, 2022.
Article in English | MEDLINE | ID: covidwho-1817941

ABSTRACT

Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibited higher basal levels of activation measured by P-selectin surface expression and had poor functional reserve upon in vitro stimulation. To investigate this question in more detail, we developed an assay to assess the capacity of plasma from COVID-19 patients to activate platelets from healthy donors. Platelet activation was a common feature of plasma from COVID-19 patients and correlated with key measures of clinical outcome including kidney and liver injury, and APACHEIII scores. Further, we identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the FcγRIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions. These data identified these potentially actionable pathways as central for platelet activation and/or vascular complications and clinical outcomes in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect.


Subject(s)
Blood Platelets/immunology , COVID-19/immunology , Complement C5a/metabolism , Receptor, Anaphylatoxin C5a/metabolism , Receptors, IgG/metabolism , SARS-CoV-2/physiology , Thromboembolism/immunology , Adult , Aminopyridines/pharmacology , Cells, Cultured , Female , Hospitalization , Humans , Male , Morpholines/pharmacology , Platelet Activation , Pyrimidines/pharmacology , Severity of Illness Index , Signal Transduction , Syk Kinase/antagonists & inhibitors
2.
Biomolecules ; 11(12)2021 12 18.
Article in English | MEDLINE | ID: covidwho-1581038

ABSTRACT

Hydrogen sulfide (H2S) is a ubiquitous gaseous signaling molecule that has an important role in many physiological and pathological processes in mammalian tissues, with the same importance as two others endogenous gasotransmitters such as NO (nitric oxide) and CO (carbon monoxide). Endogenous H2S is involved in a broad gamut of processes in mammalian tissues including inflammation, vascular tone, hypertension, gastric mucosal integrity, neuromodulation, and defense mechanisms against viral infections as well as SARS-CoV-2 infection. These results suggest that the modulation of H2S levels has a potential therapeutic value. Consequently, synthetic H2S-releasing agents represent not only important research tools, but also potent therapeutic agents. This review has been designed in order to summarize the currently available H2S donors; furthermore, herein we discuss their preparation, the H2S-releasing mechanisms, and their -biological applications.


Subject(s)
Drug Discovery , Gasotransmitters/pharmacology , Hydrogen Sulfide/pharmacology , Animals , Benzenesulfonates/administration & dosage , Benzenesulfonates/metabolism , Benzenesulfonates/pharmacology , Benzenesulfonates/therapeutic use , Chemistry, Pharmaceutical , Gasotransmitters/administration & dosage , Gasotransmitters/metabolism , Gasotransmitters/therapeutic use , Humans , Hydrogen Sulfide/administration & dosage , Hydrogen Sulfide/metabolism , Hydrogen Sulfide/therapeutic use , Morpholines/administration & dosage , Morpholines/metabolism , Morpholines/pharmacology , Morpholines/therapeutic use , Naproxen/administration & dosage , Naproxen/analogs & derivatives , Naproxen/metabolism , Naproxen/pharmacology , Naproxen/therapeutic use , Organothiophosphorus Compounds/administration & dosage , Organothiophosphorus Compounds/metabolism , Organothiophosphorus Compounds/pharmacology , Organothiophosphorus Compounds/therapeutic use
3.
J Virol ; 95(21): e0097521, 2021 10 13.
Article in English | MEDLINE | ID: covidwho-1361966

ABSTRACT

Repurposing FDA-approved inhibitors able to prevent infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could provide a rapid path to establish new therapeutic options to mitigate the effects of coronavirus disease 2019 (COVID-19). Proteolytic cleavages of the spike (S) protein of SARS-CoV-2, mediated by the host cell proteases cathepsin and TMPRSS2, alone or in combination, are key early activation steps required for efficient infection. The PIKfyve kinase inhibitor apilimod interferes with late endosomal viral traffic and through an ill-defined mechanism prevents in vitro infection through late endosomes mediated by cathepsin. Similarly, inhibition of TMPRSS2 protease activity by camostat mesylate or nafamostat mesylate prevents infection mediated by the TMPRSS2-dependent and cathepsin-independent pathway. Here, we combined the use of apilimod with camostat mesylate or nafamostat mesylate and found an unexpected ∼5- to 10-fold increase in their effectiveness to prevent SARS-CoV-2 infection in different cell types. Comparable synergism was observed using both a chimeric vesicular stomatitis virus (VSV) containing S of SARS-CoV-2 (VSV-SARS-CoV-2) and SARS-CoV-2. The substantial ∼5-fold or higher decrease of the half-maximal effective concentrations (EC50s) suggests a plausible treatment strategy based on the combined use of these inhibitors. IMPORTANCE Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing the coronavirus disease 2019 (COVID-2019) global pandemic. There are ongoing efforts to uncover effective antiviral agents that could mitigate the severity of the disease by controlling the ensuing viral replication. Promising candidates include small molecules that inhibit the enzymatic activities of host proteins, thus preventing SARS-CoV-2 entry and infection. They include apilimod, an inhibitor of PIKfyve kinase, and camostat mesylate and nafamostat mesylate, inhibitors of TMPRSS2 protease. Our research is significant for having uncovered an unexpected synergism in the effective inhibitory activity of apilimod used together with camostat mesylate or nafamostat mesylate.


Subject(s)
Antiviral Agents/pharmacology , Benzamidines/pharmacology , Esters/pharmacology , Guanidines/pharmacology , Hydrazones/pharmacology , Morpholines/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Pyrimidines/pharmacology , SARS-CoV-2/drug effects , Serine Endopeptidases/metabolism , Animals , COVID-19/drug therapy , Cell Line, Tumor , Chlorocebus aethiops , Drug Synergism , Humans , Phosphoinositide-3 Kinase Inhibitors/pharmacology , Protease Inhibitors/pharmacology , SARS-CoV-2/physiology , Vero Cells , Virus Internalization
4.
Antiviral Res ; 194: 105158, 2021 10.
Article in English | MEDLINE | ID: covidwho-1340541

ABSTRACT

It is more than 20 years since the neuraminidase inhibitors, oseltamivir and zanamivir were approved for the treatment and prevention of influenza. Guidelines for global surveillance and methods for evaluating resistance were established initially by the Neuraminidase Inhibitor Susceptibility Network (NISN), which merged 10 years ago with the International Society for influenza and other Respiratory Virus Diseases (isirv) to become the isirv-Antiviral Group (isirv-AVG). With the ongoing development of new influenza polymerase inhibitors and recent approval of baloxavir marboxil, the isirv-AVG held a closed meeting in August 2019 to discuss the impact of resistance to these inhibitors. Following this meeting and review of the current literature, this article is intended to summarize current knowledge regarding the clinical impact of resistance to polymerase inhibitors and approaches for surveillance and methods for laboratory evaluation of resistance, both in vitro and in animal models. We highlight limitations and gaps in current knowledge and suggest some strategies for addressing these gaps, including the need for additional clinical studies of influenza antiviral drug combinations. Lessons learned from influenza resistance monitoring may also be helpful for establishing future drug susceptibility surveillance and testing for SARS-CoV-2.


Subject(s)
Antiviral Agents/therapeutic use , Influenza, Human/drug therapy , Animals , Antiviral Agents/adverse effects , Antiviral Agents/pharmacology , Dibenzothiepins/pharmacology , Drug Resistance, Viral , Enzyme Inhibitors/pharmacology , Humans , Influenza, Human/virology , Knowledge , Morpholines/pharmacology , Neuraminidase/therapeutic use , Oseltamivir/pharmacology , Pyridones/pharmacology , SARS-CoV-2/drug effects , Triazines/pharmacology , Virus Replication/drug effects , Zanamivir/pharmacology
5.
Nat Commun ; 12(1): 1876, 2021 03 25.
Article in English | MEDLINE | ID: covidwho-1152854

ABSTRACT

Viruses hijack host cell metabolism to acquire the building blocks required for replication. Understanding how SARS-CoV-2 alters host cell metabolism may lead to potential treatments for COVID-19. Here we profile metabolic changes conferred by SARS-CoV-2 infection in kidney epithelial cells and lung air-liquid interface (ALI) cultures, and show that SARS-CoV-2 infection increases glucose carbon entry into the TCA cycle via increased pyruvate carboxylase expression. SARS-CoV-2 also reduces oxidative glutamine metabolism while maintaining reductive carboxylation. Consistent with these changes, SARS-CoV-2 infection increases the activity of mTORC1 in cell lines and lung ALI cultures. Lastly, we show evidence of mTORC1 activation in COVID-19 patient lung tissue, and that mTORC1 inhibitors reduce viral replication in kidney epithelial cells and lung ALI cultures. Our results suggest that targeting mTORC1 may be a feasible treatment strategy for COVID-19 patients, although further studies are required to determine the mechanism of inhibition and potential efficacy in patients.


Subject(s)
COVID-19/pathology , Citric Acid Cycle/physiology , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Mechanistic Target of Rapamycin Complex 1/metabolism , Protein Kinase Inhibitors/pharmacology , Animals , Benzamides/pharmacology , Cell Line , Chlorocebus aethiops , Glucose/metabolism , Glutamine/metabolism , HEK293 Cells , Humans , Lung/metabolism , Lung/virology , Morpholines/pharmacology , Naphthyridines/pharmacology , Pyrimidines/pharmacology , Pyruvate Carboxylase/biosynthesis , SARS-CoV-2/metabolism , Vero Cells , Virus Replication/drug effects
6.
PLoS Pathog ; 17(2): e1009371, 2021 02.
Article in English | MEDLINE | ID: covidwho-1138592

ABSTRACT

Morbilliviruses, such as measles virus (MeV) and canine distemper virus (CDV), are highly infectious members of the paramyxovirus family. MeV is responsible for major morbidity and mortality in non-vaccinated populations. ERDRP-0519, a pan-morbillivirus small molecule inhibitor for the treatment of measles, targets the morbillivirus RNA-dependent RNA-polymerase (RdRP) complex and displayed unparalleled oral efficacy against lethal infection of ferrets with CDV, an established surrogate model for human measles. Resistance profiling identified the L subunit of the RdRP, which harbors all enzymatic activity of the polymerase complex, as the molecular target of inhibition. Here, we examined binding characteristics, physical docking site, and the molecular mechanism of action of ERDRP-0519 through label-free biolayer interferometry, photoaffinity cross-linking, and in vitro RdRP assays using purified MeV RdRP complexes and synthetic templates. Results demonstrate that unlike all other mononegavirus small molecule inhibitors identified to date, ERDRP-0519 inhibits all phosphodiester bond formation in both de novo initiation of RNA synthesis at the promoter and RNA elongation by a committed polymerase complex. Photocrosslinking and resistance profiling-informed ligand docking revealed that this unprecedented mechanism of action of ERDRP-0519 is due to simultaneous engagement of the L protein polyribonucleotidyl transferase (PRNTase)-like domain and the flexible intrusion loop by the compound, pharmacologically locking the polymerase in pre-initiation conformation. This study informs selection of ERDRP-0519 as clinical candidate for measles therapy and identifies a previously unrecognized druggable site in mononegavirus L polymerase proteins that can silence all synthesis of viral RNA.


Subject(s)
Antiviral Agents/pharmacology , Enzyme Inhibitors/pharmacology , Measles virus/drug effects , Measles/drug therapy , Morpholines/pharmacology , Piperidines/pharmacology , Pyrazoles/pharmacology , RNA, Viral/biosynthesis , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Animals , Chlorocebus aethiops , Measles/metabolism , Measles/virology , Measles virus/enzymology , Mutation , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , Vero Cells
7.
PLoS Biol ; 19(2): e3001091, 2021 02.
Article in English | MEDLINE | ID: covidwho-1102372

ABSTRACT

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.


Subject(s)
COVID-19 Vaccines , COVID-19/diagnosis , COVID-19/virology , Reverse Genetics , SARS-CoV-2/genetics , A549 Cells , Angiotensin-Converting Enzyme 2/metabolism , Animals , Chlorocebus aethiops , Codon , Humans , Hydrazones/pharmacology , Mice , Morpholines/pharmacology , Open Reading Frames , Plasmids/genetics , Pyrimidines/pharmacology , Serine Endopeptidases/metabolism , Vero Cells , Viral Proteins/metabolism
8.
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
10.
Cells ; 10(1)2020 12 27.
Article in English | MEDLINE | ID: covidwho-1011425

ABSTRACT

The PIKfyve inhibitor apilimod is currently undergoing clinical trials for treatment of COVID-19. However, although apilimod might prevent viral invasion by inhibiting host cell proteases, the same proteases are critical for antigen presentation leading to T cell activation and there is good evidence from both in vitro studies and the clinic that apilimod blocks antiviral immune responses. We therefore warn that the immunosuppression observed in many COVID-19 patients might be aggravated by apilimod.


Subject(s)
Antiviral Agents/adverse effects , COVID-19/drug therapy , Hydrazones/adverse effects , Morpholines/adverse effects , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors/adverse effects , Pyrimidines/adverse effects , SARS-CoV-2/drug effects , Antiviral Agents/pharmacology , COVID-19/immunology , Humans , Hydrazones/pharmacology , Morpholines/pharmacology , Peptide Hydrolases/metabolism , Phosphoinositide-3 Kinase Inhibitors/pharmacology , Protease Inhibitors/pharmacology , Pyrimidines/pharmacology , Serine Endopeptidases/metabolism
11.
Proc Natl Acad Sci U S A ; 117(34): 20803-20813, 2020 08 25.
Article in English | MEDLINE | ID: covidwho-695945

ABSTRACT

Virus entry is a multistep process. It initiates when the virus attaches to the host cell and ends when the viral contents reach the cytosol. Genetically unrelated viruses can subvert analogous subcellular mechanisms and use similar trafficking pathways for successful entry. Antiviral strategies targeting early steps of infection are therefore appealing, particularly when the probability for successful interference through a common step is highest. We describe here potent inhibitory effects on content release and infection by chimeric vesicular stomatitis virus (VSV) containing the envelope proteins of Zaire ebolavirus (VSV-ZEBOV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (VSV-SARS-CoV-2) elicited by Apilimod and Vacuolin-1, small-molecule inhibitors of the main endosomal phosphatidylinositol-3-phosphate/phosphatidylinositol 5-kinase, PIKfyve. We also describe potent inhibition of SARS-CoV-2 strain 2019-nCoV/USA-WA1/2020 by Apilimod. These results define tools for studying the intracellular trafficking of pathogens elicited by inhibition of PIKfyve kinase and suggest the potential for targeting this kinase in developing small-molecule antivirals against SARS-CoV-2.


Subject(s)
Betacoronavirus/drug effects , Ebolavirus/drug effects , Heterocyclic Compounds, 4 or More Rings/pharmacology , Morpholines/pharmacology , Phosphatidylinositol 3-Kinases , Triazines/pharmacology , Virus Internalization/drug effects , Animals , Betacoronavirus/physiology , COVID-19 , Cells, Cultured , Coronavirus Infections , Ebolavirus/physiology , Gene Editing , Humans , Hydrazones , Pandemics , Phosphoinositide-3 Kinase Inhibitors/pharmacology , Pneumonia, Viral , Pyrimidines , SARS-CoV-2 , Viral Envelope Proteins/genetics
12.
Nature ; 586(7827): 113-119, 2020 10.
Article in English | MEDLINE | ID: covidwho-672174

ABSTRACT

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of the severe pneumonia-like disease coronavirus disease 2019 (COVID-19)1. The development of a vaccine is likely to take at least 12-18 months, and the typical timeline for approval of a new antiviral therapeutic agent can exceed 10 years. Thus, repurposing of known drugs could substantially accelerate the deployment of new therapies for COVID-19. Here we profiled a library of drugs encompassing approximately 12,000 clinical-stage or Food and Drug Administration (FDA)-approved small molecules to identify candidate therapeutic drugs for COVID-19. We report the identification of 100 molecules that inhibit viral replication of SARS-CoV-2, including 21 drugs that exhibit dose-response relationships. Of these, thirteen were found to harbour effective concentrations commensurate with probable achievable therapeutic doses in patients, including the PIKfyve kinase inhibitor apilimod2-4 and the cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825 and ONO 5334. Notably, MDL-28170, ONO 5334 and apilimod were found to antagonize viral replication in human pneumocyte-like cells derived from induced pluripotent stem cells, and apilimod also demonstrated antiviral efficacy in a primary human lung explant model. Since most of the molecules identified in this study have already advanced into the clinic, their known pharmacological and human safety profiles will enable accelerated preclinical and clinical evaluation of these drugs for the treatment of COVID-19.


Subject(s)
Antiviral Agents/analysis , Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Drug Evaluation, Preclinical , Drug Repositioning , Pneumonia, Viral/drug therapy , Pneumonia, Viral/virology , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Alveolar Epithelial Cells/cytology , Alveolar Epithelial Cells/drug effects , Betacoronavirus/growth & development , COVID-19 , Cell Line , Cysteine Proteinase Inhibitors/analysis , Cysteine Proteinase Inhibitors/pharmacology , Dose-Response Relationship, Drug , Drug Synergism , Gene Expression Regulation/drug effects , Humans , Hydrazones , Induced Pluripotent Stem Cells/cytology , Models, Biological , Morpholines/analysis , Morpholines/pharmacology , Pandemics , Pyrimidines , Reproducibility of Results , SARS-CoV-2 , Small Molecule Libraries/analysis , Small Molecule Libraries/pharmacology , Triazines/analysis , Triazines/pharmacology , Virus Internalization/drug effects , Virus Replication/drug effects
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