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Introduction: Influenza A virus (IAV) is one of the leading causes of respiratory tract infections in humans, representing a major public health concern. The various types of cell death have a crucial role in IAV pathogenesis because this virus may trigger both apoptosis and necroptosis in airway epithelial cells in parallel. Macrophages play an important role in the clearance of virus particles, priming the adaptive immune response in influenza. However, the contribution of macrophage death to pathogenesis of IAV infection remains unclear. Methods: In this work, we investigated IAV-induced macrophage death, along with potential therapeutic intervention. We conducted in vitro and in vivo experiments to evaluate the mechanism and the contribution of macrophages death to the inflammatory response induced by IAV infection. Results: We found that IAV or its surface glycoprotein hemagglutinin (HA) triggers inflammatory programmed cell death in human and murine macrophages in a Toll-like receptor-4 (TLR4)- and TNF-dependent manner. Anti-TNF treatment in vivo with the clinically approved drug etanercept prevented the engagement of the necroptotic loop and mouse mortality. Etanercept impaired the IAV-induced proinflammatory cytokine storm and lung injury. Conclusion: In summary, we demonstrated a positive feedback loop of events that led to necroptosis and exacerbated inflammation in IAV-infected macrophages. Our results highlight an additional mechanism involved in severe influenza that could be attenuated with clinically available therapies.
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Virus de la Influenza A , Gripe Humana , Humanos , Animales , Ratones , Etanercept , Inhibidores del Factor de Necrosis Tumoral , Apoptosis , MacrófagosRESUMEN
Orally available antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are necessary because of the continuous circulation of new variants that challenge immunized individuals. Because severe COVID-19 is a virus-triggered immune and inflammatory dysfunction, molecules endowed with both antiviral and anti-inflammatory activity are highly desirable. We identified here that kinetin (MB-905) inhibits the in vitro replication of SARS-CoV-2 in human hepatic and pulmonary cell lines. On infected monocytes, MB-905 reduced virus replication, IL-6 and TNFα levels. MB-905 is converted into its triphosphate nucleotide to inhibit viral RNA synthesis and induce error-prone virus replication. Coinhibition of SARS-CoV-2 exonuclease, a proofreading enzyme that corrects erroneously incorporated nucleotides during viral RNA replication, potentiated the inhibitory effect of MB-905. MB-905 shows good oral absorption, its metabolites are stable, achieving long-lasting plasma and lung concentrations, and this drug is not mutagenic nor cardiotoxic in acute and chronic treatments. SARS-CoV-2-infected hACE-mice and hamsters treated with MB-905 show decreased viral replication, lung necrosis, hemorrhage and inflammation. Because kinetin is clinically investigated for a rare genetic disease at regimens beyond the predicted concentrations of antiviral/anti-inflammatory inhibition, our investigation suggests the opportunity for the rapid clinical development of a new antiviral substance for the treatment of COVID-19.
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Antivirales , COVID-19 , Animales , Humanos , Ratones , Antivirales/farmacología , Antivirales/uso terapéutico , SARS-CoV-2 , Cinetina/farmacología , Inflamación/tratamiento farmacológico , Nucleótidos , Replicación ViralRESUMEN
Despite the fast development of vaccines, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still circulating and generating variants of concern (VoC) that escape the humoral immune response. In this context, the search for anti-SARS-CoV-2 compounds is still essential. A class of natural polyphenols known as flavonoids, frequently available in fruits and vegetables, is widely explored in the treatment of different diseases and used as a scaffold for the design of novel drugs. Therefore, herein we evaluate seven flavonoids divided into three subclasses, isoflavone (genistein), flavone (apigenin and luteolin) and flavonol (fisetin, kaempferol, myricetin, and quercetin), for COVID-19 treatment using cell-based assays and in silico calculations validated with experimental enzymatic data. The flavonols were better SARS-CoV-2 inhibitors than isoflavone and flavones. The increasing number of hydroxyl groups in ring B of the flavonols kaempferol, quercetin, and myricetin decreased the 50% effective concentration (EC50) value due to their impact on the orientation of the compounds inside the target. Myricetin and fisetin appear to be preferred candidates; they are both anti-inflammatory (decreasing TNF-α levels) and inhibit SARS-CoV-2 mainly by targeting the processability of the main protease (Mpro) in a non-competitive manner, with a potency comparable to the repurposed drug atazanavir. However, fisetin and myricetin might also be considered hits that are amenable to synthetic modification to improve their anti-SARS-CoV-2 profile by inhibiting not only Mpro, but also the 3'-5' exonuclease (ExoN).
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Tratamiento Farmacológico de COVID-19 , Flavonas , Isoflavonas , Flavonas/farmacología , Flavonoides/farmacología , Flavonoles/farmacología , Humanos , Isoflavonas/farmacología , Quempferoles , Simulación del Acoplamiento Molecular , Inhibidores de Proteasas , Quercetina/farmacología , SARS-CoV-2RESUMEN
Infection by SARS-CoV-2 may elicit uncontrolled and damaging inflammatory responses. Thus, it is critical to identify compounds able to inhibit virus replication and thwart the inflammatory reaction. Here, we show that the plasma levels of the immunoregulatory neuropeptide VIP are elevated in patients with severe COVID-19, correlating with reduced inflammatory mediators and with survival on those patients. In vitro, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), highly similar neuropeptides, decreased the SARS-CoV-2 RNA content in human monocytes and viral production in lung epithelial cells, also reducing cell death. Both neuropeptides inhibited the production of proinflammatory mediators in lung epithelial cells and in monocytes. VIP and PACAP prevented in monocytes the SARS-CoV-2-induced activation of NF-kB and SREBP1 and SREBP2, transcriptions factors involved in proinflammatory reactions and lipid metabolism, respectively. They also promoted CREB activation, a transcription factor with antiapoptotic activity and negative regulator of NF-kB. Specific inhibition of NF-kB and SREBP1/2 reproduced the anti-inflammatory, antiviral, and cell death protection effects of VIP and PACAP. Our results support further clinical investigations of these neuropeptides against COVID-19.
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COVID-19 , Péptido Intestinal Vasoactivo , Humanos , Lipopolisacáridos/farmacología , FN-kappa B/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , ARN Viral , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo , SARS-CoV-2 , Factores de Transcripción/metabolismo , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
Despite the development of specific therapies against severe acute respiratory coronavirus 2 (SARS-CoV-2), the continuous investigation of the mechanism of action of clinically approved drugs could provide new information on the druggable steps of virus-host interaction. For example, chloroquine (CQ)/hydroxychloroquine (HCQ) lacks in vitro activity against SARS-CoV-2 in TMPRSS2-expressing cells, such as human pneumocyte cell line Calu-3, and likewise, failed to show clinical benefit in the Solidarity and Recovery clinical trials. Another antimalarial drug, mefloquine, which is not a 4-aminoquinoline like CQ/HCQ, has emerged as a potential anti-SARS-CoV-2 antiviral in vitro and has also been previously repurposed for respiratory diseases. Here, we investigated the anti-SARS-CoV-2 mechanism of action of mefloquine in cells relevant for the physiopathology of COVID-19, such as Calu-3 cells (that recapitulate type II pneumocytes) and monocytes. Molecular pathways modulated by mefloquine were assessed by differential expression analysis, and confirmed by biological assays. A PBPK model was developed to assess mefloquine's optimal doses for achieving therapeutic concentrations. Mefloquine inhibited SARS-CoV-2 replication in Calu-3, with an EC50 of 1.2 µM and EC90 of 5.3 µM. It reduced SARS-CoV-2 RNA levels in monocytes and prevented virus-induced enhancement of IL-6 and TNF-α. Mefloquine reduced SARS-CoV-2 entry and synergized with Remdesivir. Mefloquine's pharmacological parameters are consistent with its plasma exposure in humans and its tissue-to-plasma predicted coefficient points suggesting that mefloquine may accumulate in the lungs. Altogether, our data indicate that mefloquine's chemical structure could represent an orally available host-acting agent to inhibit virus entry.
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Células Epiteliales Alveolares/efectos de los fármacos , Antivirales/farmacología , Cloroquina/farmacología , Mefloquina/farmacología , SARS-CoV-2/efectos de los fármacos , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Células Epiteliales Alveolares/virología , Línea Celular , Reposicionamiento de Medicamentos/métodos , Humanos , Serina Endopeptidasas/genética , Internalización del Virus/efectos de los fármacos , Tratamiento Farmacológico de COVID-19RESUMEN
Influenza A virus (IAV) is the main etiological agent of acute respiratory tract infections. During IAV infection, interferon triggers the overexpression of restriction factors (RFs), the intracellular antiviral branch of the innate immune system. Conversely, severe influenza is associated with an unbalanced pro-inflammatory cytokine release. It is unclear whether other cytokines and chemokines released during IAV infection modulate RFs to control virus replication. Among the molecules enhanced in the infected respiratory tract, ligands of the CCR5 receptor play a key role, as they stimulate the migration of inflammatory cells to the alveoli. We investigated here whether ligands of the CCR5 receptor could enhance RFs to levels able to inhibit IAV replication. For this purpose, the human alveolar basal epithelial cell line (A549) was treated with endogenous (CCL3, CCL4 and CCL5) or exogenous (HIV-1 gp120) ligands prior to IAV infection. The three CC-chemokines tested reduced infectious titers between 30% to 45% upon 24 hours of infection. Eploying RT-PCR, a panel of RF mRNA levels from cells treated with CCR5 agonists was evaluated, which showed that the SAMHD1 expression was up-regulated four times over control upon exposure to CCL3, CCL4 and CCL5. We also found that IAV inhibition by CCL5 was dependent on PKC and that SAMHD1 protein levels were also increased after treatment with CCL5. In functional assays, we observed that the knockdown of SAMHD1 resulted in enhanced IAV replication in A549 cells and abolished both CCL5-mediated inhibition of IAV replication and CCL5-mediated cell death inhibition. Our data show that stimuli unrelated to interferon may trigger the upregulation of SAMHD1 and that this RF may directly interfere with IAV replication in alveolar epithelial cells.
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Virus de la Influenza A , Gripe Humana , Quimiocina CCL5 , Humanos , Proteína 1 que Contiene Dominios SAM y HD , Replicación ViralRESUMEN
BACKGROUND: Current approaches of drug repurposing against COVID-19 have not proven overwhelmingly successful and the SARS-CoV-2 pandemic continues to cause major global mortality. SARS-CoV-2 nsp12, its RNA polymerase, shares homology in the nucleotide uptake channel with the HCV orthologue enzyme NS5B. Besides, HCV enzyme NS5A has pleiotropic activities, such as RNA binding, that are shared with various SARS-CoV-2 proteins. Thus, anti-HCV NS5B and NS5A inhibitors, like sofosbuvir and daclatasvir, respectively, could be endowed with anti-SARS-CoV-2 activity. METHODS: SARS-CoV-2-infected Vero cells, HuH-7 cells, Calu-3 cells, neural stem cells and monocytes were used to investigate the effects of daclatasvir and sofosbuvir. In silico and cell-free based assays were performed with SARS-CoV-2 RNA and nsp12 to better comprehend the mechanism of inhibition of the investigated compounds. A physiologically based pharmacokinetic model was generated to estimate daclatasvir's dose and schedule to maximize the probability of success for COVID-19. RESULTS: Daclatasvir inhibited SARS-CoV-2 replication in Vero, HuH-7 and Calu-3 cells, with potencies of 0.8, 0.6 and 1.1 µM, respectively. Although less potent than daclatasvir, sofosbuvir alone and combined with daclatasvir inhibited replication in Calu-3 cells. Sofosbuvir and daclatasvir prevented virus-induced neuronal apoptosis and release of cytokine storm-related inflammatory mediators, respectively. Sofosbuvir inhibited RNA synthesis by chain termination and daclatasvir targeted the folding of secondary RNA structures in the SARS-CoV-2 genome. Concentrations required for partial daclatasvir in vitro activity are achieved in plasma at Cmax after administration of the approved dose to humans. CONCLUSIONS: Daclatasvir, alone or in combination with sofosbuvir, at higher doses than used against HCV, may be further fostered as an anti-COVID-19 therapy.
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COVID-19 , Preparaciones Farmacéuticas , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , Carbamatos , Chlorocebus aethiops , Humanos , Imidazoles , Pirrolidinas , ARN Viral , SARS-CoV-2 , Sofosbuvir/farmacología , Valina/análogos & derivados , Células VeroRESUMEN
Infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been associated with leukopenia and uncontrolled inflammatory response in critically ill patients. A better comprehension of SARS-CoV-2-induced monocyte death is essential for the identification of therapies capable to control the hyper-inflammation and reduce viral replication in patients with 2019 coronavirus disease (COVID-19). Here, we show that SARS-CoV-2 engages inflammasome and triggers pyroptosis in human monocytes, experimentally infected, and from patients under intensive care. Pyroptosis associated with caspase-1 activation, IL-1ß production, gasdermin D cleavage, and enhanced pro-inflammatory cytokine levels in human primary monocytes. At least in part, our results originally describe mechanisms by which monocytes, a central cellular component recruited from peripheral blood to respiratory tract, succumb to control severe COVID-19.
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Atazanavir (ATV) has already been considered as a potential repurposing drug to 2019 coronavirus disease (COVID-19); however, there are controversial reports on its mechanism of action and effectiveness as anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Through the pre-clinical chain of experiments: enzymatic, molecular docking, cell-based and in vivo assays, it is demonstrated here that both SARS-CoV-2 B.1 lineage and variant of concern gamma are susceptible to this antiretroviral. Enzymatic assays and molecular docking calculations showed that SARS-CoV-2 main protease (Mpro) was inhibited by ATV, with Morrison's inhibitory constant (Ki) 1.5-fold higher than GC376 (a positive control) dependent of the catalytic water (H2Ocat) content. ATV was a competitive inhibitor, increasing the Mpro's Michaelis-Menten (Km) more than sixfold. Cell-based assays indicated that different lineages of SARS-CoV-2 is susceptible to ATV. Using oral administration of ATV in mice to reach plasmatic exposure similar to humans, transgenic mice expression in human angiotensin converting enzyme 2 (K18-hACE2) were partially protected against lethal challenge with SARS-CoV-2 gamma. Moreover, less cell death and inflammation were observed in the lung from infected and treated mice. Our studies may contribute to a better comprehension of the Mpro/ATV interaction, which could pave the way to the development of specific inhibitors of this viral protease.
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Viruses are obligate intracellular parasites that make use of the host metabolic machineries to meet their biosynthetic needs. Thus, identifying the host pathways essential for the virus replication may lead to potential targets for therapeutic intervention. The mechanisms and pathways explored by SARS-CoV-2 to support its replication within host cells are not fully known. Lipid droplets (LD) are organelles with major functions in lipid metabolism, energy homeostasis and intracellular transport, and have multiple roles in infections and inflammation. Here we described that monocytes from COVID-19 patients have an increased LD accumulation compared to SARS-CoV-2 negative donors. In vitro, SARS-CoV-2 infection were seen to modulate pathways of lipid synthesis and uptake as monitored by testing for CD36, SREBP-1, PPARγ, and DGAT-1 expression in monocytes and triggered LD formation in different human cell lines. LDs were found in close apposition with SARS-CoV-2 proteins and double-stranded (ds)-RNA in infected Vero cells. Electron microscopy (EM) analysis of SARS-CoV-2 infected Vero cells show viral particles colocalizing with LDs, suggestive that LDs might serve as an assembly platform. Pharmacological modulation of LD formation by inhibition of DGAT-1 with A922500 significantly inhibited SARS-CoV-2 replication as well as reduced production of mediators pro-inflammatory response. Taken together, we demonstrate the essential role of lipid metabolic reprograming and LD formation in SARS-CoV-2 replication and pathogenesis, opening new opportunities for therapeutic strategies to COVID-19.
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COVID-19/complicaciones , Mediadores de Inflamación/metabolismo , Inflamación/etiología , Gotas Lipídicas/patología , SARS-CoV-2/aislamiento & purificación , Animales , COVID-19/inmunología , COVID-19/patología , COVID-19/virología , Estudios de Casos y Controles , Chlorocebus aethiops , Humanos , Inflamación/metabolismo , Inflamación/patología , Células Vero , Replicación ViralRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is already responsible for far more deaths than previous pathogenic coronaviruses (CoVs) from 2002 and 2012. The identification of clinically approved drugs to be repurposed to combat 2019 CoV disease (COVID-19) would allow the rapid implementation of potentially life-saving procedures. The major protease (Mpro) of SARS-CoV-2 is considered a promising target, based on previous results from related CoVs with lopinavir (LPV), an HIV protease inhibitor. However, limited evidence exists for other clinically approved antiretroviral protease inhibitors. Extensive use of atazanavir (ATV) as antiretroviral and previous evidence suggesting its bioavailability within the respiratory tract prompted us to study this molecule against SARS-CoV-2. Our results show that ATV docks in the active site of SARS-CoV-2 Mpro with greater strength than LPV, blocking Mpro activity. We confirmed that ATV inhibits SARS-CoV-2 replication, alone or in combination with ritonavir (RTV) in Vero cells and a human pulmonary epithelial cell line. ATV/RTV also impaired virus-induced enhancement of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) levels. Together, our data strongly suggest that ATV and ATV/RTV should be considered among the candidate repurposed drugs undergoing clinical trials in the fight against COVID-19.
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Antivirales/farmacología , Sulfato de Atazanavir/farmacología , Betacoronavirus/efectos de los fármacos , Citocinas/metabolismo , Ritonavir/farmacología , Animales , Sulfato de Atazanavir/química , Betacoronavirus/patogenicidad , Betacoronavirus/fisiología , COVID-19 , Muerte Celular/efectos de los fármacos , Chlorocebus aethiops , Proteasas 3C de Coronavirus , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/metabolismo , Infecciones por Coronavirus/patología , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Quimioterapia Combinada , Humanos , Inflamación/metabolismo , Inflamación/virología , Lopinavir/farmacología , Simulación del Acoplamiento Molecular , Monocitos/virología , Pandemias , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/metabolismo , Neumonía Viral/patología , Inhibidores de Proteasas/farmacología , SARS-CoV-2 , Células Vero , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacos , Tratamiento Farmacológico de COVID-19RESUMEN
BACKGROUND: Malaria-triggered lung injury can occur in both severe and non-severe cases. Platelets may interact with parasitized erythrocytes, leukocytes and endothelium. These interactions can lead to microvessel obstructions and induce release of inflammatory mediators. Induction of the haem oxygenase enzyme is important in the host's response to free haem and to several other molecules generated by infectious or non-infectious diseases. In addition, an important role for the haem oxygenase-1 isotype has been demonstrated in experimental cerebral malaria and in clinical cases. Therefore, the present work aims to determine the influence of haem oxygenase in thrombocytopaenia and acute pulmonary injury during infection with Plasmodium berghei strain NK65. METHODS: C57BL/6 mice were infected with P. berghei and analysed 7-10 days post-infection. For each experiment, Cobalt Protoporphyrin IX/CoPPIX or saline were administered. Bronchoalveolar lavage fluid was used for total and differential leukocyte count and for protein measurement. Lungs were used for histological analyses or for analysis of cytokines and western blotting. The lung permeability was analysed by Evans blue dye concentration. Platelet-leukocyte aggregate formation was assayed using the flow cytometer. RESULTS: Plasmodium berghei NK65 infection generated an intense lung injury, with increased levels of inflammatory mediators, oedema, and cell migration into the lung. Plasmodium berghei infection was also accompanied by marked thrombocytopaenia and formation of platelet-leukocyte aggregates in peripheral blood. Treatment with the HO-1 inducer cobalt protoporphyrin IX (CoPPIX) modified the inflammatory response but did not affect the evolution of parasitaemia. Animals treated with CoPPIX showed an improvement in lung injury, with decreased inflammatory infiltrate in the lung parenchyma, oedema and reduced thrombocytopaenia. CONCLUSION: Data here presented suggest that treatment with CoPPIX inducer leads to less severe pulmonary lung injury and thrombocytopaenia during malaria infection, thus increasing animal survival.
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Lesión Pulmonar Aguda/tratamiento farmacológico , Hemo-Oxigenasa 1/farmacología , Malaria/complicaciones , Proteínas de la Membrana/farmacología , Sustancias Protectoras/farmacología , Trombocitopenia/tratamiento farmacológico , Lesión Pulmonar Aguda/etiología , Animales , Líquido del Lavado Bronquioalveolar/química , Femenino , Recuento de Leucocitos , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Plasmodium berghei/fisiología , Trombocitopenia/etiologíaRESUMEN
BACKGROUND: Neuraminidase inhibitors (NAIs) are the only class of antivirals in clinical use against influenza virus approved worldwide. However, approximately 1-3% of circulating strains present resistance mutations to oseltamivir (OST), the most used NAI. Therefore, it is important to catalogue new molecules to inhibit influenza virus, especially OST-resistant strains. Natural products from tropical plants used for human consumption represent a worthy class of substances. Their use could be stimulated in resource-limited setting where the access to expensive antiviral therapies is restricted. METHODS: We evaluated the anti-influenza virus activity of agathisflavone derived from Anacardium occidentale L. RESULTS: The neuraminidase (NA) activity of wild-type and OST-resistant influenza virus was inhibited by agathisflavone, with IC50 values ranging from 20 to 2.0 µM, respectively. Agathisflavone inhibited influenza virus replication with EC50 of 1.3 µM. Sequential passages of the virus in the presence of agathisflavone revealed the emergence of mutation R249S, A250S and R253Q in the NA gene. These changes are outside the OST binding region, meaning that agathisflavone targets this viral enzyme at a region different than conventional NAIs. CONCLUSION: Altogether our data suggest that agathisflavone has a promising chemical structure for the development of anti-influenza drugs.
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Anacardium/química , Biflavonoides/farmacología , Inhibidores Enzimáticos/farmacología , Neuraminidasa/antagonistas & inhibidores , Orthomyxoviridae/efectos de los fármacos , Fitoquímicos/farmacología , Animales , Biflavonoides/química , Biflavonoides/aislamiento & purificación , Supervivencia Celular/efectos de los fármacos , Perros , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Células de Riñón Canino Madin Darby/efectos de los fármacos , Células de Riñón Canino Madin Darby/virología , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Neuraminidasa/metabolismo , Orthomyxoviridae/enzimología , Fitoquímicos/química , Fitoquímicos/aislamiento & purificación , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
Malaria is an infectious disease of major worldwide clinical importance that causes a variety of severe, or complicated, syndromes including cerebral malaria, which is often fatal. Leukocyte integrins are essential for host defense but also mediate physiologic responses of the innate and adaptive immune systems. We previously showed that targeted deletion of the αD subunit (αD-/-) of the αDß2 integrin, which is expressed on key leukocyte subsets in mice and humans, leads to absent expression of the integrin heterodimer on murine macrophages and reduces mortality in mice infected with Plasmodium berghei ANKA (P. berghei ANKA). To further identify mechanisms involved in the protective effect of αD deletion in this model of severe malaria we examined wild type C57BL/6 (WT) and αD-/- mice after P. berghei ANKA infection and found that vessel plugging and leukocyte infiltration were significantly decreased in the brains of αD-/- animals. Intravital microscopy demonstrated decreased rolling and adhesion of leukocytes in cerebral vessels of αD-/- mice. Flow cytometry analysis showed decreased T-lymphocyte accumulation in the brains of infected αD-/- animals. Evans blue dye exclusion assays demonstrated significantly less dye extravasation in the brains of αD-/- mice, indicating preserved blood-brain barrier integrity. WT mice that were salvaged from P. berghei ANKA infection by treatment with chloroquine had impaired aversive memory, which was not observed in αD-/- mice. We conclude that deletion of integrin αDß2 alters the natural course of experimental severe malaria, demonstrating previously unrecognized activities of a key leukocyte integrin in immune-inflammatory responses that mediate cerebral involvement.
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Antígenos CD11/metabolismo , Cadenas alfa de Integrinas/metabolismo , Malaria/fisiopatología , Animales , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Edema Encefálico/metabolismo , Edema Encefálico/fisiopatología , Antígenos CD11/fisiología , Cloroquina/metabolismo , Modelos Animales de Enfermedad , Inflamación/metabolismo , Cadenas alfa de Integrinas/fisiología , Integrinas/inmunología , Integrinas/metabolismo , Recuento de Leucocitos , Leucocitos/metabolismo , Leucocitos/fisiología , Macrófagos/metabolismo , Malaria/genética , Malaria Cerebral/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Plasmodium berghei/metabolismoRESUMEN
Yellow fever virus (YFV) is a member of the Flaviviridae family. In Brazil, yellow fever (YF) cases have increased dramatically in sylvatic areas neighboring urban zones in the last few years. Because of the high lethality rates associated with infection and absence of any antiviral treatments, it is essential to identify therapeutic options to respond to YFV outbreaks. Repurposing of clinically approved drugs represents the fastest alternative to discover antivirals for public health emergencies. Other Flaviviruses, such as Zika (ZIKV) and dengue (DENV) viruses, are susceptible to sofosbuvir, a clinically approved drug against hepatitis C virus (HCV). Our data showed that sofosbuvir docks onto YFV RNA polymerase using conserved amino acid residues for nucleotide binding. This drug inhibited the replication of both vaccine and wild-type strains of YFV on human hepatoma cells, with EC50 values around 5 µM. Sofosbuvir protected YFV-infected neonatal Swiss mice and adult type I interferon receptor knockout mice (A129-/-) from mortality and weight loss. Because of its safety profile in humans and significant antiviral effects in vitro and in mice, Sofosbuvir may represent a novel therapeutic option for the treatment of YF. Key-words: Yellow fever virus; Yellow fever, antiviral; sofosbuvir.
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Antivirales/farmacología , Farmacorresistencia Viral , ARN Viral/efectos de los fármacos , Sofosbuvir/farmacología , Fiebre Amarilla/tratamiento farmacológico , Virus de la Fiebre Amarilla/efectos de los fármacos , Animales , Chlorocebus aethiops , Modelos Animales de Enfermedad , Células Hep G2 , Humanos , Ratones , Ratones Noqueados , ARN Viral/sangre , ARN Viral/genética , Células Vero , Fiebre Amarilla/sangre , Fiebre Amarilla/patología , Fiebre Amarilla/virología , Virus de la Fiebre Amarilla/genéticaRESUMEN
Chikungunya virus (CHIKV) causes a febrile disease associated with chronic arthralgia, which may progress to neurological impairment. Chikungunya fever (CF) is an ongoing public health problem in tropical and subtropical regions of the world, where control of the CHIKV vector, Aedes mosquitos, has failed. As there is no vaccine or specific treatment for CHIKV, patients receive only palliative care to alleviate pain and arthralgia. Thus, drug repurposing is necessary to identify antivirals against CHIKV. CHIKV RNA polymerase is similar to the orthologue enzyme of other positive-sense RNA viruses, such as members of the Flaviviridae family. Among the Flaviviridae, not only is hepatitis C virus RNA polymerase susceptible to sofosbuvir, a clinically approved nucleotide analogue, but so is dengue, Zika, and yellow fever virus replication. Here, we found that sofosbuvir was three times more selective in inhibiting CHIKV production in human hepatoma cells than ribavirin, a pan-antiviral drug. Although CHIKV replication in human induced pluripotent stem cell-derived astrocytes was less susceptible to sofosbuvir than were hepatoma cells, sofosbuvir nevertheless impaired virus production and cell death in a multiplicity of infection-dependent manner. Sofosbuvir also exhibited antiviral activity in vivo by preventing CHIKV-induced paw edema in adult mice at a dose of 20 mg/kg of body weight/day and prevented mortality in a neonate mouse model at 40- and 80-mg/kg/day doses. Our data demonstrate that a prototypic alphavirus, CHIKV, is also susceptible to sofosbuvir. As sofosbuvir is a clinically approved drug, our findings could pave the way to it becoming a therapeutic option against CF.
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Antivirales/uso terapéutico , Fiebre Chikungunya/tratamiento farmacológico , Virus Chikungunya/efectos de los fármacos , Virus Chikungunya/patogenicidad , Sofosbuvir/uso terapéutico , Replicación Viral/efectos de los fármacos , Animales , Animales Recién Nacidos , Artralgia/tratamiento farmacológico , Artralgia/virología , Fiebre Chikungunya/virología , Humanos , Masculino , RatonesRESUMEN
Zika virus (ZIKV) causes significant public health concerns because of its association with congenital malformations, neurological disorders in adults, and, more recently, death. Considering the necessity to mitigate ZIKV-associated diseases, antiviral interventions are an urgent necessity. Sofosbuvir, a drug in clinical use against hepatitis C virus (HCV), is among the FDA-approved substances endowed with anti-ZIKV activity. In this work, we further investigated the in vivo activity of sofosbuvir against ZIKV. Neonatal Swiss mice were infected with ZIKV (2 × 107 PFU) and treated with sofosbuvir at 20 mg/kg/day, a concentration compatible with pre-clinical development of this drug. We found that sofosbuvir reduced acute levels of ZIKV from 60 to 90% in different anatomical compartments, such as the blood plasma, spleen, kidney, and brain. Early treatment with sofosbuvir doubled the percentage and time of survival of ZIKV-infected animals. Sofosbuvir also prevented the acute neuromotor impairment triggered by ZIKV. In the long-term behavioural analysis of ZIKV-associated sequelae, sofosbuvir prevented loss of hippocampal- and amygdala-dependent memory. Our results indicate that sofosbuvir inhibits ZIKV replication in vivo, which is consistent with the prospective necessity of antiviral drugs to treat ZIKV-infected individuals.
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Antivirales/farmacología , Sofosbuvir/farmacología , Infección por el Virus Zika/tratamiento farmacológico , Virus Zika/fisiología , Animales , Animales Recién Nacidos , Antivirales/administración & dosificación , Chlorocebus aethiops , Memoria , Ratones , ARN Viral , Reflejo de Enderezamiento , Sofosbuvir/administración & dosificación , Células Vero , Replicación Viral/efectos de los fármacos , Infección por el Virus Zika/mortalidadRESUMEN
Zika virus (ZIKV) is a member of the Flaviviridae family, along with other agents of clinical significance such as dengue (DENV) and hepatitis C (HCV) viruses. Since ZIKV causes neurological disorders during fetal development and in adulthood, antiviral drugs are necessary. Sofosbuvir is clinically approved for use against HCV and targets the protein that is most conserved among the members of the Flaviviridae family, the viral RNA polymerase. Indeed, we found that sofosbuvir inhibits ZIKV RNA polymerase, targeting conserved amino acid residues. Sofosbuvir inhibited ZIKV replication in different cellular systems, such as hepatoma (Huh-7) cells, neuroblastoma (SH-Sy5y) cells, neural stem cells (NSC) and brain organoids. In addition to the direct inhibition of the viral RNA polymerase, we observed that sofosbuvir also induced an increase in A-to-G mutations in the viral genome. Together, our data highlight a potential secondary use of sofosbuvir, an anti-HCV drug, against ZIKV.