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Infection by SARS-CoV-2 is associated with uncontrolled inflammatory response during COVID-19 severe disease, in which monocytes are one of the main sources of pro-inflammatory mediators leading to acute respiratory distress syndrome. Extracellular vesicles (EVs) from different cells play important roles during SARS-CoV-2 infection, but investigations describing the involvement of EVs from primary human monocyte-derived macrophages (MDM) on the regulation of this infection are not available. Here, we describe the effects of EVs released by MDM stimulated with the neuropeptides VIP and PACAP on SARS-CoV-2-infected monocytes. MDM-derived EVs were isolated by differential centrifugation of medium collected from cells cultured for 24 h in serum-reduced conditions. Based on morphological properties, we distinguished two subpopulations of MDM-EVs, namely large (LEV) and small EVs (SEV). We found that MDM-derived EVs stimulated with the neuropeptides inhibited SARS-CoV-2 RNA synthesis/replication in monocytes, protected these cells from virus-induced cytopathic effects and reduced the production of pro-inflammatory mediators. In addition, EVs derived from VIP- and PACAP-treated MDM prevented the SARS-CoV-2-induced NF-κB activation. Overall, our findings suggest that MDM-EVs are endowed with immunoregulatory properties that might contribute to the antiviral and anti-inflammatory responses in SARS-CoV-2-infected monocytes and expand our knowledge of EV effects during COVID-19 pathogenesis.
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Zika still poses a threat to global health owing to its association with serious neurological conditions and the absence of a vaccine and treatment. Sofosbuvir, an anti-hepatitis C drug, has shown anti-Zika effects in animal and cell models. Thus, this study aimed to develop and validate novel LC-MS/MS methods for the quantification of sofosbuvir and its major metabolite (GS-331007) in human plasma and cerebrospinal (CSF) and seminal fluid (SF), and apply the methods to a pilot clinical trial. The samples were prepared by liquid-liquid extraction and separated using isocratic mode on Gemini C18 columns. Analytical detection was performed using a triple quadrupole mass spectrometer equipped with an electrospray ionization source. The validated ranges for sofosbuvir were 0.5-2,000 ng/mL (plasma) and 0.5-100 ng/mL (CSF and SF), while for the metabolite they were 2.0-2,000 ng/mL (plasma), 5.0-200 ng/mL (CSF) and 10-1,500 ng/mL (SF). The intra-day and inter-day accuracies (90.8-113.8%) and precisions (1.4-14.8%) were within the acceptance range. The developed methods fulfilled all validation parameters concerning selectivity, matrix effect, carryover, linearity, dilution integrity, precision, accuracy and stability, confirming the suitability of the method for the analysis of clinical samples.
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Infección por el Virus Zika , Virus Zika , Animales , Humanos , Cromatografía Liquida/métodos , Límite de Detección , Plasma , Reproducibilidad de los Resultados , Sofosbuvir , Espectrometría de Masas en Tándem/métodosRESUMEN
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, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) still circulates through variants of concern (VoC) and escape the humoral immune response. SARS-CoV-2 has provoked over 200,000 deaths/months since its emergence and only a few antiviral drugs showed clinical benefit up to this moment. Thus, chemical structures endowed with anti-SARS-CoV-2 activity are important for continuous antiviral development and natural products represent a fruitful source of substances with biological activity. In the present study, agathisflavone (AGT), a biflavonoid from Anacardium occidentale was investigated as a candidate anti-SARS-CoV-2 compound. In silico and enzymatic analysis indicated that AGT may target mainly the viral main protease (Mpro) and not the papain-like protease (PLpro) in a non-competitive way. Cell-based assays in type II pneumocytes cell lineage (Calu-3) showed that SARS-CoV-2 is more susceptible to AGT than to apigenin (APG, monomer of AGT), in a dose-dependent manner, with an EC50 of 4.23 ± 0.21 µM and CC50 of 61.3 ± 0.1 µM and with a capacity to inhibit the level of pro-inflammatory mediator tumor necrosis factor-alpha (TNF-α). These results configure AGT as an interesting chemical scaffold for the development of novel semisynthetic antivirals against SARS-CoV-2.
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Biflavonoides , Tratamiento Farmacológico de COVID-19 , Humanos , SARS-CoV-2 , Proteasas 3C de Coronavirus , Biflavonoides/farmacología , Péptido Hidrolasas , Antivirales/química , Inhibidores de Proteasas/químicaRESUMEN
The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has put an enormous pressure on human societies, at both health and economic levels. Early diagnosis of SARS-CoV-2, the causative agent of 2019 coronavirus disease (COVID-19), has proved an efficient method to rapidly isolate positive individuals and reduce transmission rates, thus alleviating its negative impact on society's well-being and economic growth. In this work, through a coordinated and centralized effort to monitor SARS-CoV-2 circulation in companies from the State of Rio de Janeiro, Brazil, we have detected and linked an early rise of infection rates in January 2022 to the introduction of the Omicron variant of concern (VoC) (BA.1). Interestingly, when the Omicron genomic isolates were compared to correlates from public datasets, it was revealed that introduction events were multiple, with possible migration routes mapping to: Mali; Oman and United States; and Italy, Latin America, and United States. In addition, we have built a haplotype network with our genomic dataset and found no strong evidence of transmission chains, between and within companies. Considering Omicron's particularly high transmissibility, and that most of our samples (>87%) arose from 3 out of 10 companies, these findings suggest that workers from such environments were exposed to SARS-CoV-2 outside their company boundaries. Thus, using a mixed strategy in which quick molecular diagnosis finds support in comprehensive genomic analysis, we have shown that a successfully implemented occupational health program should contribute to document emerging VoC and to limit the spread of SARS-CoV-2 at the workplace.
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Finding antivirals for SARS-CoV-2 is still a major challenge, and many computational and experimental approaches have been employed to find a solution to this problem. While the global vaccination campaigns are the primary driver of controlling the current pandemic, orally bioavailable small-molecule drugs and biologics are critical to overcome this global issue. Improved therapeutics and prophylactics are required to treat people with circulating and emerging new variants, addressing severe infection, and people with underlying or immunocompromised conditions. The SARS-CoV-2 envelope spike is a challenging target for viral entry inhibitors. Pindolol presented a good docking score in a previous virtual screening using computational docking calculations after screening a Food and Drug Administration (FDA)-approved drug library of 2400 molecules as potential candidates to block the SARS-CoV-2 spike protein interaction with the angiotensin-converting enzyme 2 (ACE-2). Here, we expanded the computational evaluation to identify five beta-blockers against SARS-CoV-2 using several techniques, such as microscale thermophoresis, NanoDSF, and in vitro assays in different cell lines. These data identified carvedilol with a K d of 364 ± 22 nM for the SARS-CoV-2 spike and in vitro activity (EC50 of 7.57 µM, CC50 of 18.07 µM) against SARS-CoV-2 in Calu-3 cells. We have shown how we can apply multiple computational and experimental approaches to find molecules that can be further optimized to improve anti-SARS-CoV-2 activity.
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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
Herpes simplex virus type-1 (HSV-1) infection causes several disorders, and acyclovir is used as a reference compound. However, resistant strains are commonly observed. Herein, we investigate the effects of N-heterocyclic compounds (pyrazolopyridine derivatives), named ARA-04, ARA-05, and AM-57, on HSV-1 in vitro replication. We show that the 50% effective concentration (EC50) values of the compounds ARA-04, ARA-05, and AM-57 were 1.00 ± 0.10, 1.00 ± 0.05, and 0.70 ± 0.10 µM, respectively. These compounds presented high 50% cytotoxic concentration (CC50) values, which resulted in a selective index (SI) of 1000, 1000, and 857.1 for ARA-04, ARA-05, and AM-57, respectively. To gain insight into which step of the HSV-1 replication cycle these molecules would impair, we performed adsorption and penetration inhibition assays and time-of-addition experiments. Our results indicated that ARA-04 and ARA-05 affected viral adsorption, while AM-57 interfered with the virus replication during its α- and γ-phases and decreased ICP27 content during initial and late events of HSV-1 replication. In addition, we also observed that AM-57 caused a strong decrease in viral gD content, which was reinforced by in silico calculations that suggested AM-57 interacts preferentially with the viral complex between a general transcription factor and virion protein (TFIIBc-VP16). In contrast, ARA-04 and ARA-05 interact preferentially in the proteins responsible for the viral adsorption process (nectin-1 and glycoprotein). Thus, our results suggest that the 1H-pyrazolo[3,4-b]pyridine derivatives inhibit the HSV-1 replicative cycle with a novel mechanism of action, and its scaffold can be used as a template for the synthesis of promising new molecules with antiviral effects, including to reinforce the presented data herein for a limited number of molecules.
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Herpes Simple , Infecciones por Herpesviridae , Herpesvirus Humano 1 , Aciclovir/farmacología , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , Chlorocebus aethiops , Herpes Simple/tratamiento farmacológico , Infecciones por Herpesviridae/tratamiento farmacológico , Herpesvirus Humano 1/fisiología , Pirazoles , Piridinas/farmacología , Piridinas/uso terapéutico , Células Vero , Replicación ViralRESUMEN
Coronavirus disease 2019 (COVID-19) has affected over 400 million people worldwide, leading to 6 million deaths. Among the complex symptomatology of COVID-19, hypercoagulation and thrombosis have been described to directly contribute to lethality, pointing out platelets as an important SARS-CoV-2 target. In this work, we explored the platelet proteome of COVID-19 patients through a label-free shotgun proteomics approach to identify platelet responses to infection, as well as validation experiments in a larger patient cohort. Exclusively detected proteins (EPs) and differentially expressed proteins (DEPs) were identified in the proteomic dataset and thus classified into biological processes to map pathways correlated with pathogenesis. Significant changes in the expression of proteins related to platelet activation, cell death, and antiviral response through interferon type-I were found in all patients. Since the outcome of COVID-19 varies highly among individuals, we also performed a cross-comparison of proteins found in survivors and nonsurvivors. Proteins belonging to the translation pathway were strongly highlighted in the nonsurvivor group. Moreover, the SARS-CoV-2 genome was fully sequenced in platelets from five patients, indicating viral internalization and preprocessing, with CD147 as a potential entry route. In summary, platelets play a significant role in COVID-19 pathogenesis via platelet activation, antiviral response, and disease severity.
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The SARS-CoV-2 pandemic has had a social and economic impact worldwide, and vaccination is an efficient strategy for diminishing those damages. New adjuvant formulations are required for the high vaccine demands, especially adjuvant formulations that induce a Th1 phenotype. Herein we assess a vaccination strategy using a combination of Alum and polyinosinic:polycytidylic acid [Poly(I:C)] adjuvants plus the SARS-CoV-2 spike protein in a prefusion trimeric conformation by an intradermal (ID) route. We found high levels of IgG anti-spike antibodies in the serum by enzyme linked immunosorbent assay (ELISA) and high neutralizing titers against SARS-CoV-2 in vitro by neutralization assay, after two or three immunizations. By evaluating the production of IgG subtypes, as expected, we found that formulations containing Poly(I:C) induced IgG2a whereas Alum did not. The combination of these two adjuvants induced high levels of both IgG1 and IgG2a. In addition, cellular immune responses of CD4+ and CD8+ T cells producing interferon-gamma were equivalent, demonstrating that the Alum + Poly(I:C) combination supported a Th1 profile. Based on the high neutralizing titers, we evaluated B cells in the germinal centers, which are specific for receptor-binding domain (RBD) and spike, and observed that more positive B cells were induced upon the Alum + Poly(I:C) combination. Moreover, these B cells produced antibodies against both RBD and non-RBD sites. We also studied the impact of this vaccination preparation [spike protein with Alum + Poly(I:C)] in the lungs of mice challenged with inactivated SARS-CoV-2 virus. We found a production of IgG, but not IgA, and a reduction in neutrophil recruitment in the bronchoalveolar lavage fluid (BALF) of mice, suggesting that our immunization scheme reduced lung inflammation. Altogether, our data suggest that Alum and Poly(I:C) together is a possible adjuvant combination for vaccines against SARS-CoV-2 by the intradermal route.
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COVID-19 , Vacunas Virales , Adyuvantes Inmunológicos , Compuestos de Alumbre , Animales , Linfocitos T CD8-positivos , Vacunas contra la COVID-19 , Humanos , Inmunoglobulina G , Ratones , Poli I-C , SARS-CoV-2 , Glicoproteína de la Espiga del CoronavirusRESUMEN
The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to extra caution in workplaces to avoid the coronavirus disease 2019 (COVID-19). In the occupational environment, SARS-CoV-2 testing is a powerful approach in providing valuable information to detect, monitor, and mitigate the spread of the virus and preserve productivity. Here a centralized Occupational Health Center provided molecular diagnosis and genomic sequences for companies and industries in Rio de Janeiro, Brazil. From May to August 2021, around 20% of the SARS-CoV-2 positive nasopharyngeal swabs from routinely tested workers were sequenced and reproduced the replacement of Gamma with Delta variant observed in regular surveillance programs. Moreover, as a proof-of-concept on the sensibility of the occupational health genomic surveillance program described here, it was also found: i) the primo-identification of B.1.139 and A.2.5 viral genomes in Brazil and ii) an improved dating of Delta VoC evolution, by identifying earlier cases associated with AY-related genomes. We interpret that SARS-CoV-2 molecular testing of workers, independent of symptom presentation, provides an earlier opportunity to identify variants. Thus, considering the continuous monitoring of SARS-CoV-2 in workplaces, positive samples from occupation health programs should be regarded as essential to improve the knowledge on virus genetic diversity and VoC emergence.
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BACKGROUND: Critically ill 2019 coronavirus disease (COVID-19) patients under invasive mechanical ventilation (IMV) are 10 to 40 times more likely to die than the general population. Although progression from mild to severe COVID-19 has been associated with hypoxia, uncontrolled inflammation, and coagulopathy, the mechanisms involved in the progression to severity are poorly understood. METHODS: The virome of tracheal aspirates (TA) from 25 COVID-19 patients under IMV was assessed through unbiased RNA sequencing (RNA-seq), and correlation analyses were conducted using available clinical data. Unbiased sequences from nasopharyngeal swabs (NS) from mild cases and TA from non-COVID patients were included in our study for further comparisons. RESULTS: We found higher levels and differential expression of human endogenous retrovirus K (HERV-K) genes in TA from critically ill and deceased patients when comparing nasopharyngeal swabs from mild cases to TA from non-COVID patients. In critically ill patients, higher HERV-K levels were associated with early mortality (within 14 days of diagnosis) in the intensive care unit. Increased HERV-K expression in deceased patients was associated with IL-17-related inflammation, monocyte activation, and an increased consumption of clotting/fibrinolysis factors. Moreover, increased HERV-K expression was detected in human primary monocytes from healthy donors after experimental SARS-CoV-2 infection in vitro. CONCLUSION: Our data implicate the levels of HERV-K transcripts in the physiopathology of COVID-19 in the respiratory tract of patients under invasive mechanical ventilation. Video abstract.
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COVID-19 , Retrovirus Endógenos , Enfermedad Crítica , Retrovirus Endógenos/genética , Humanos , Inflamación , Sistema Respiratorio , SARS-CoV-2RESUMEN
Accumulating evidence into the pathogenesis of COVID-19 highlights a hypercoagulability state with high risk of life-threatening thromboembolic complications. However, the mechanisms of hypercoagulability and their link to hyperinflammation remain poorly understood. Here, we investigate functions and mechanisms of platelet activation and platelet-monocyte interactions in inflammatory amplification during SARS-CoV-2 infection. We used a combination of immunophenotyping, single-cell analysis, functional assays, and pharmacological approaches to gain insights on mechanisms. Critically ill patients with COVID-19 exhibited increased platelet-monocyte aggregates formation. We identified a subset of inflammatory monocytes presenting high CD16 and low HLA-DR expression as the subset mainly interacting with platelets during severe COVID-19. Single-cell RNA-sequencing analysis indicated enhanced fibrinogen receptor Mac-1 in monocytes from patients with severe COVID-19. Monocytes from patients with severe COVID-19 displayed increased platelet binding and hyperresponsiveness to P-selectin and fibrinogen with respect to tumor necrosis factor-α and interleukin-1ß secretion. Platelets were able to orchestrate monocyte responses driving tissue factor (TF) expression, inflammatory activation, and inflammatory cytokines secretion in SARS-CoV-2 infection. Platelet-monocyte interactions ex vivo and in SARS-CoV-2 infection model in vitro reciprocally activated monocytes and platelets, inducing the heightened secretion of a wide panel of inflammatory mediators. We identified platelet adhesion as a primary signaling mechanism inducing mediator secretion and TF expression, whereas TF signaling played major roles in amplifying inflammation by inducing proinflammatory cytokines, especially tumor necrosis factor-α and interleukin-1ß. Our data identify platelet-induced TF expression and activity at the crossroad of coagulation and inflammation in severe COVID-19.
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COVID-19 , Trombofilia , Trombosis , Plaquetas/metabolismo , Citocinas/metabolismo , Humanos , Inflamación/patología , Interleucina-1beta/metabolismo , Monocitos/metabolismo , SARS-CoV-2 , Tromboinflamación , Tromboplastina/metabolismo , Trombosis/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
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
Coronavirus disease 2019 (COVID-19) is currently a worldwide emergency caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). In observational clinical studies, statins have been identified as beneficial to hospitalized patients with COVID-19. However, experimental evidence of underlying statins protection against SARS-CoV-2 remains elusive. Here we reported for the first-time experimental evidence of the protective effects of simvastatin treatment both in vitro and in vivo. We found that treatment with simvastatin significantly reduced the viral replication and lung damage in vivo, delaying SARS-CoV-2-associated physiopathology and mortality in the K18-hACE2-transgenic mice model. Moreover, simvastatin also downregulated the inflammation triggered by SARS-CoV-2 infection in pulmonary tissue and in human neutrophils, peripheral blood monocytes, and lung epithelial Calu-3 cells in vitro, showing its potential to modulate the inflammatory response both at the site of infection and systemically. Additionally, we also observed that simvastatin affected the course of SARS-CoV-2 infection through displacing ACE2 on cell membrane lipid rafts. In conclusion, our results show that simvastatin exhibits early protective effects on SARS-CoV-2 infection by inhibiting virus cell entry and inflammatory cytokine production, through mechanisms at least in part dependent on lipid rafts disruption.
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Tratamiento Farmacológico de COVID-19 , Regulación hacia Abajo/efectos de los fármacos , Inflamación/tratamiento farmacológico , Microdominios de Membrana/efectos de los fármacos , SARS-CoV-2/patogenicidad , Simvastatina/farmacología , Animales , COVID-19/virología , Modelos Animales de Enfermedad , Humanos , Inflamación/virología , Pulmón/virología , Ratones , Ratones Transgénicos , Replicación Viral/efectos de los fármacosRESUMEN
SARS-CoV-2 has an exonuclease-based proofreader, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RNA-dependent RNA polymerase and the exonuclease could overcome this deficiency. Here we report the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of Pibrentasvir, RNAs terminated with the active forms of the prodrugs Sofosbuvir, Remdesivir, Favipiravir, Molnupiravir and AT-527 were largely protected from excision by the exonuclease, while in the absence of Pibrentasvir, there was rapid excision. Due to its unique structure, Tenofovir-terminated RNA was highly resistant to exonuclease excision even in the absence of Pibrentasvir. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment.
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Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , Exonucleasas/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , SARS-CoV-2/efectos de los fármacos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Secuencia de Aminoácidos , Anilidas/farmacología , Animales , Secuencia de Bases , Bencimidazoles/farmacología , COVID-19/virología , Línea Celular Tumoral , Chlorocebus aethiops , Sinergismo Farmacológico , Exonucleasas/genética , Exonucleasas/metabolismo , Humanos , Prolina/farmacología , Pirrolidinas/farmacología , ARN Viral/genética , ARN Viral/metabolismo , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/fisiología , Valina/farmacología , Células Vero , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacos , Replicación Viral/genéticaRESUMEN
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-19Asunto(s)
COVID-19 , Defensa Civil , Control de Enfermedades Transmisibles , Investigación Biomédica Traslacional , COVID-19/epidemiología , COVID-19/prevención & control , Defensa Civil/organización & administración , Defensa Civil/tendencias , Control de Enfermedades Transmisibles/métodos , Control de Enfermedades Transmisibles/organización & administración , Control de Enfermedades Transmisibles/tendencias , Barreras de Comunicación , Desarrollo de Medicamentos , Predicción , Salud Global/normas , Salud Global/tendencias , Humanos , Cooperación Internacional , Salud Pública , SARS-CoV-2 , Investigación Biomédica Traslacional/métodos , Investigación Biomédica Traslacional/normas , Investigación Biomédica Traslacional/tendenciasRESUMEN
Introduction: Cell entry of SARS-CoV-2 causes genome-wide disruption of the transcriptional profiles of genes and biological pathways involved in the pathogenesis of COVID-19. Expression allelic imbalance is characterized by a deviation from the Mendelian expected 1:1 expression ratio and is an important source of allele-specific heterogeneity. Expression allelic imbalance can be measured by allele-specific expression analysis (ASE) across heterozygous informative expressed single nucleotide variants (eSNVs). ASE reflects many regulatory biological phenomena that can be assessed by combining genome and transcriptome information. ASE contributes to the interindividual variability associated with the disease. We aim to estimate the transcriptome-wide impact of SARS-CoV-2 infection by analyzing eSNVs. Methods: We compared ASE profiles in the human lung cell lines Calu-3, A459, and H522 before and after infection with SARS-CoV-2 using RNA-Seq experiments. Results: We identified 34 differential ASE (DASE) sites in 13 genes (HLA-A, HLA-B, HLA-C, BRD2, EHD2, GFM2, GSPT1, HAVCR1, MAT2A, NQO2, SUPT6H, TNFRSF11A, UMPS), all of which are enriched in protein binding functions and play a role in COVID-19. Most DASE sites were assigned to the MHC class I locus and were predominantly upregulated upon infection. DASE sites in the MHC class I locus also occur in iPSC-derived airway epithelium basal cells infected with SARS-CoV-2. Using an RNA-Seq haplotype reconstruction approach, we found DASE sites and adjacent eSNVs in phase (i.e., predicted on the same DNA strand), demonstrating differential haplotype expression upon infection. We found a bias towards the expression of the HLA alleles with a higher binding affinity to SARS-CoV-2 epitopes. Discussion: Independent of gene expression compensation, SARS-CoV-2 infection of human lung cell lines induces transcriptional allelic switching at the MHC loci. This suggests a response mechanism to SARS-CoV-2 infection that swaps HLA alleles with poor epitope binding affinity, an expectation supported by publicly available proteome data.