RESUMEN
BACKGROUND: HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) is an incapacitating neuroinflammatory disorder for which no disease-modifying therapy is available, but corticosteroids provide some clinical benefit. Although HAM/TSP pathogenesis is not fully elucidated, older age, female sex and higher proviral load are established risk factors. We investigated systemic cytokines and a novel chronic inflammatory marker, GlycA, as possible biomarkers of immunopathogenesis and therapeutic response in HAM/TSP, and examined their interaction with established risk factors. PATIENTS AND METHODS: We recruited 110 People living with HTLV-1 (PLHTLV-1, 67 asymptomatic individuals and 43 HAM/TSP patients) with a total of 946 person-years of clinical follow-up. Plasma cytokine levels (IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, TNF) and GlycA were quantified by Cytometric Bead Array and 1NMR, respectively. Cytokine signaling and prednisolone response were validated in an independent cohort by nCounter digital transcriptomics. We used multivariable regression, machine learning algorithms and Bayesian network learning for biomarker identification. RESULTS: We found that systemic IL-6 was positively correlated with both age (r = 0.50, p < 0.001) and GlycA (r = 0.45, p = 0.00049) in asymptomatics, revealing an 'inflammaging" signature which was absent in HAM/TSP. GlycA levels were higher in women (p = 0.0069), but cytokine levels did not differ between the sexes. IFN-γ (p = 0.007) and IL-17A (p = 0.0001) levels were increased in untreated HAM/TSP Multivariable logistic regression identified IL-17A and proviral load as independent determinants of clinical status, resulting in modest accuracy of predicting HAM/TSP status (64.1%), while a machine learning-derived decision tree classified HAM/TSP patients with 90.7% accuracy. Pre-treatment GlycA and TNF levels significantly predicted clinical worsening (measured by Osame Motor Disability Scale), independent of proviral load. In addition, a poor prednisolone response was significantly correlated with higher post-treatment IFN-γ levels. Likewise, a transcriptomic IFN signaling score, significantly correlated with previously proposed HAM/TSP biomarkers (CASP5/CXCL10/FCGR1A/STAT1), was efficiently blunted by in vitro prednisolone treatment of PBMC from PLHTLV-1 and incident HAM/TSP. CONCLUSIONS: An age-related increase in systemic IL-6/GlycA levels reveals inflammaging in PLHTLV-1, in the absence of neurological disease. IFN-γ and IL-17A are biomarkers of untreated HAM/TSP, while pre-treatment GlycA and TNF predict therapeutic response to prednisolone pulse therapy, paving the way for a precision medicine approach in HAM/TSP.
Asunto(s)
Infecciones por HTLV-I , Trastornos Motores , Enfermedades Neuroinflamatorias , Femenino , Humanos , Teorema de Bayes , Citocinas , Virus Linfotrópico T Tipo 1 Humano , Interleucina-17 , Interleucina-6 , Leucocitos Mononucleares , Trastornos Motores/virología , Enfermedades Neuroinflamatorias/virología , Infecciones por HTLV-I/complicacionesRESUMEN
Here, we report on the anti-influenza virus activity of the mannose-binding agents Hippeastrum hybrid agglutinin (HHA) and Galanthus nivalis agglutinin (GNA) and the (N-acetylglucosamine) n -specific Urtica dioica agglutinin (UDA). These carbohydrate-binding agents (CBA) strongly inhibited various influenza A(H1N1), A(H3N2), and B viruses in vitro, with 50% effective concentration values ranging from 0.016 to 83 nM, generating selectivity indexes up to 125,000. Somewhat less activity was observed against A/Puerto Rico/8/34 and an A(H1N1)pdm09 strain. In time-of-addition experiments, these CBA lost their inhibitory activity when added 30 min postinfection (p.i.). Interference with virus entry processes was also evident from strong inhibition of virus-induced hemolysis at low pH. However, a direct effect on acid-induced refolding of the viral hemagglutinin (HA) was excluded by the tryptic digestion assay. Instead, HHA treatment of HA-expressing cells led to a significant reduction of plasma membrane mobility. Crosslinking of membrane glycoproteins, through interaction with HA, could also explain the inhibitory effect on the release of newly formed virions when HHA was added at 6 h p.i. These CBA presumably interact with one or more N-glycans on the globular head of HA, since their absence led to reduced activity against mutant influenza B viruses and HHA-resistant A(H1N1) viruses. The latter condition emerged only after 33 cell culture passages in the continuous presence of HHA, and the A(H3N2) virus retained full sensitivity even after 50 passages. Thus, these CBA qualify as potent inhibitors of influenza A and B viruses in vitro with a pleiotropic mechanism of action and a high barrier for viral resistance.
Asunto(s)
Amaryllidaceae , Herpesvirus Cercopitecino 1 , Subtipo H1N1 del Virus de la Influenza A , Gripe Humana , Aglutininas , Antivirales/farmacología , Glicoproteínas Hemaglutininas del Virus de la Influenza , Humanos , Subtipo H3N2 del Virus de la Influenza A , Virus de la Influenza B , Manosa , Lectinas de Unión a Manosa , Lectinas de Plantas , Replicación ViralRESUMEN
The influenza virus hemagglutinin (HA) mediates membrane fusion after viral entry by endocytosis. The fusion process requires drastic low pH-induced HA refolding and is prevented by arbidol and tert-butylhydroquinone (TBHQ). We here report a class of superior inhibitors with indole-substituted spirothiazolidinone structure. The most active analogue 5f has an EC50 value against influenza A/H3N2 virus of 1â¯nM and selectivity index of almost 2000. Resistance data and in silico modeling indicate that 5f combines optimized fitting in the TBHQ/arbidol HA binding pocket with a capability for endosomal accumulation. Both criteria appear relevant to achieve superior inhibitors of HA-mediated fusion.
Asunto(s)
Antivirales/farmacología , Glicoproteínas Hemaglutininas del Virus de la Influenza/efectos de los fármacos , Indoles/farmacología , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Gripe Humana/tratamiento farmacológico , Compuestos de Espiro/farmacología , Tiazolidinas/farmacología , Animales , Antivirales/síntesis química , Antivirales/química , Perros , Relación Dosis-Respuesta a Droga , Humanos , Concentración de Iones de Hidrógeno , Indoles/química , Células de Riñón Canino Madin Darby/efectos de los fármacos , Células de Riñón Canino Madin Darby/virología , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Estructura Molecular , Replegamiento Proteico/efectos de los fármacos , Compuestos de Espiro/química , Relación Estructura-Actividad , Tiazolidinas/químicaRESUMEN
The possible resistance of influenza virus against existing antiviral drugs calls for new therapeutic concepts. One appealing strategy is to inhibit virus entry, in particular at the stage of internalization. This requires a better understanding of virus-host interactions during the entry process, including the role of receptor tyrosine kinases (RTKs). To search for cellular targets, we evaluated a panel of 276 protein kinase inhibitors in a multicycle antiviral assay in Madin-Darby canine kidney cells. The RTK inhibitor Ki8751 displayed robust anti-influenza A and B virus activity and was selected for mechanistic investigations. Ki8751 efficiently disrupted the endocytic process of influenza virus in different cell lines carrying platelet-derived growth factor receptor ß (PDGFRß), an RTK that is known to act at GM3 ganglioside-positive lipid rafts. The more efficient virus entry in CHO-K1 cells compared to the wild-type ancestor (CHO-wt) cells indicated a positive effect of GM3, which is abundant in CHO-K1 but not in CHO-wt cells. Entering virus localized to GM3-positive lipid rafts and the PDGFRß-containing endosomal compartment. PDGFRß/GM3-dependent virus internalization involved PDGFRß phosphorylation, which was potently inhibited by Ki8751, and desialylation of activated PDGFRß by the viral neuraminidase. Virus uptake coincided with strong activation of the Raf/MEK/Erk cascade, but not of PI3K/Akt or phospholipase C-γ. We conclude that influenza virus efficiently hijacks the GM3-enhanced PDGFRß signalling pathway for cell penetration, providing an opportunity for host cell-targeting antiviral intervention.
Asunto(s)
Gangliósido G(M3)/metabolismo , Gripe Humana/metabolismo , Gripe Humana/virología , Infecciones por Orthomyxoviridae/metabolismo , Orthomyxoviridae/patogenicidad , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal/fisiología , Animales , Células CHO , Línea Celular , Cricetulus , Perros , Células HEK293 , Humanos , Gripe Humana/tratamiento farmacológico , Células de Riñón Canino Madin Darby , Orthomyxoviridae/efectos de los fármacos , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Compuestos de Fenilurea/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/fisiología , Inhibidores de Proteínas Quinasas/farmacología , Quinolinas/farmacología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal/efectos de los fármacos , Internalización del Virus/efectos de los fármacosRESUMEN
Hemagglutinin is the surface protein of the influenza virus that mediates both binding and penetration of the virus into host cells. We here report on the synthesis and structure-activity relationship of some novel N-(1-thia-4-azaspiro[4.5]decan-4-yl)-carboxamide compounds carrying the 5-chloro-2-methoxybenzamide structure, designed as influenza virus fusion inhibitors. The carboxamides (1a-h, 2a-h) have a similar backbone structure as the fusion inhibitors that we reported on previously. Compounds 2b and 2d displayed inhibitory activity against influenza A/H3N2 virus replication (average antiviral EC50 : 2.1 µM for 2b and 3.4 µM for 2d). Data obtained in the hemolysis inhibition assay supported that these compounds act as inhibitors of the influenza virus hemagglutinin-mediated fusion process.
Asunto(s)
Antivirales/farmacología , Compuestos Aza/farmacología , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Compuestos de Espiro/farmacología , Replicación Viral/efectos de los fármacos , Antivirales/síntesis química , Antivirales/química , Compuestos Aza/síntesis química , Compuestos Aza/química , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Estructura Molecular , Compuestos de Espiro/síntesis química , Compuestos de Espiro/química , Relación Estructura-ActividadRESUMEN
T-705 (favipiravir) is a new antiviral agent in advanced clinical development for influenza therapy. It is supposed to act as an alternative substrate for the viral polymerase, causing inhibition of viral RNA synthesis or virus mutagenesis. These mechanisms were also proposed for ribavirin, an established and broad antiviral drug that shares structural similarity with T-705. We here performed a comparative analysis of the effects of T-705 and ribavirin on influenza virus and host cell functions. Influenza virus-infected cell cultures were exposed to T-705 or ribavirin during single or serial virus passaging. The effects on viral RNA synthesis and infectious virus yield were determined and mutations appearing in the viral genome were detected by whole-genome virus sequencing. In addition, the cellular nucleotide pools as well as direct inhibition of the viral polymerase enzyme were quantified. We demonstrate that the anti-influenza virus effect of ribavirin is based on IMP dehydrogenase inhibition, which results in fast and profound GTP depletion and an imbalance in the nucleotide pools. In contrast, T-705 acts as a potent and GTP-competitive inhibitor of the viral polymerase. In infected cells, viral RNA synthesis is completely inhibited by T-705 or ribavirin at ≥50 µM, whereas exposure to lower drug concentrations induces formation of noninfectious particles and accumulation of random point mutations in the viral genome. This mutagenic effect is 2-fold higher for T-705 than for ribavirin. Hence, T-705 and ribavirin both act as purine pseudobases but profoundly differ with regard to the mechanism behind their antiviral and mutagenic effects on influenza virus.
Asunto(s)
Amidas/farmacología , Antivirales/farmacología , Regulación Viral de la Expresión Génica , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Pirazinas/farmacología , Virus Reordenados/efectos de los fármacos , Ribavirina/farmacología , Células A549 , Amidas/química , Animales , Antivirales/química , Embrión de Pollo , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Perros , Humanos , IMP Deshidrogenasa/antagonistas & inhibidores , IMP Deshidrogenasa/genética , IMP Deshidrogenasa/metabolismo , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/crecimiento & desarrollo , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/crecimiento & desarrollo , Subtipo H3N2 del Virus de la Influenza A/metabolismo , Células de Riñón Canino Madin Darby , Mutación/efectos de los fármacos , Pirazinas/química , ARN Viral/antagonistas & inhibidores , ARN Viral/biosíntesis , Virus Reordenados/genética , Virus Reordenados/crecimiento & desarrollo , Virus Reordenados/metabolismo , Ribavirina/química , Análisis de Secuencia de ARN , Relación Estructura-Actividad , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Influenza A and B viruses are highly contagious respiratory pathogens with a considerable medical and socioeconomical burden and known pandemic potential. Current influenza vaccines require annual updating and provide only partial protection in some risk groups. Due to the global spread of viruses with resistance to the M2 proton channel inhibitor amantadine or the neuraminidase inhibitor oseltamivir, novel antiviral agents with an original mode of action are urgently needed. We here focus on emerging options to interfere with the influenza virus entry process, which consists of the following steps: attachment of the viral hemagglutinin to the sialylated host cell receptors, endocytosis, M2-mediated uncoating, low pH-induced membrane fusion, and, finally, import of the viral ribonucleoprotein into the nucleus. We review the current functional and structural insights in the viral and cellular components of this entry process, and the diverse antiviral strategies that are being explored. This encompasses small molecule inhibitors as well as macromolecules such as therapeutic antibodies. There is optimism that at least some of these innovative concepts to block influenza virus entry will proceed from the proof of concept to a more advanced stage. Special attention is therefore given to the challenging issues of influenza virus (sub)type-dependent activity or potential drug resistance.
Asunto(s)
Antivirales/farmacología , Orthomyxoviridae/efectos de los fármacos , Orthomyxoviridae/fisiología , Internalización del Virus/efectos de los fármacos , Transporte Activo de Núcleo Celular/efectos de los fármacos , Endocitosis/efectos de los fármacos , Fusión de Membrana/efectos de los fármacosRESUMEN
In order to obtain self assembling, multivalent ligand for influenza virus hemagglutinin α-N-acetylneuraminyl-(2-6)-D-galactopyranose has been synthesized and bonded to a water soluble fullerene derivative using 1,3-dipolar cycloaddition click reaction. The aggregating amphiphilic compound did not inhibit the influenza virus hemagglutinin, but it proved to be an inhibitor of its neuraminidase with a 50% inhibitory concentration of 81 µM.
Asunto(s)
Hidrocarburos Aromáticos con Puentes/farmacología , Disacáridos/farmacología , Fulerenos/farmacología , Hemaglutininas/metabolismo , Neuraminidasa/antagonistas & inhibidores , Orthomyxoviridae/metabolismo , Hidrocarburos Aromáticos con Puentes/síntesis química , Hidrocarburos Aromáticos con Puentes/química , Disacáridos/síntesis química , Disacáridos/química , Relación Dosis-Respuesta a Droga , Fulerenos/química , Ligandos , Estructura Molecular , Neuraminidasa/metabolismo , Orthomyxoviridae/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
In order to obtain new, cluster-forming antibiotic compounds, teicoplanin pseudoaglycone derivatives containing two lipophilic n-octyl chains have been synthesized. The compounds proved to be poor antibacterials, but, surprisingly, they exhibited potent anti-influenza virus activity against influenza A strains. This antiviral action was related to inhibition of the binding interaction between the virus and the host cell. Related analogs bearing methyl substituents in lieu of the octyl chains, displayed no anti-influenza virus activity. Hence, an interaction between the active, dually n-octylated compounds and the lipid bilayer of the host cell can be postulated, to explain the observed inhibition of influenza virus attachment.
Asunto(s)
Antivirales/farmacología , Orthomyxoviridae/efectos de los fármacos , Teicoplanina/farmacología , Animales , Antivirales/síntesis química , Antivirales/química , Sitios de Unión/efectos de los fármacos , Perros , Relación Dosis-Respuesta a Droga , Membrana Dobles de Lípidos/metabolismo , Células de Riñón Canino Madin Darby/efectos de los fármacos , Células de Riñón Canino Madin Darby/metabolismo , Células de Riñón Canino Madin Darby/virología , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Relación Estructura-Actividad , Teicoplanina/síntesis química , Teicoplanina/químicaRESUMEN
Human T-Lymphotropic Virus type-1 (HTLV-1) is a unique retrovirus associated with both leukemogenesis and a specific neuroinflammatory condition known as HTLV-1-Associated Myelopathy (HAM). Currently, most proposed HAM biomarkers require invasive CSF sampling, which is not suitable for large cohorts or repeated prospective screening. To identify non-invasive biomarkers for incident HAM in a large Brazilian cohort of PLwHTLV-1 (n=615 with 6,673 person-years of clinical follow-up), we selected all plasma samples available at the time of entry in the cohort (between 1997-2019), in which up to 43 cytokines/chemokines and immune mediators were measured. Thus, we selected 110 People Living with HTLV-1 (PLwHTLV-1), of which 68 were neurologically asymptomatic (AS) at baseline and 42 HAM patients. Nine incident HAM cases were identified among 68 AS during follow-up. Using multivariate logistic regression, we found that lower IL-10, IL-4 and female sex were independent predictors of clinical progression to definite HAM (AUROC 0.91), and outperformed previously suggested biomarkers age, sex and proviral load (AUROC 0.77). Moreover, baseline IL-10 significantly predicted proviral load dynamics at follow-up in all PLwHTLV-1. In an exploratory analysis, we identified additional plasma biomarkers which were able to discriminate iHAM from either AS (IL6Rα, IL-27) or HAM (IL-29/IFN-λ1, Osteopontin, and TNFR2). In conclusion, female sex and low anti-inflammatory IL-10 and IL-4 are independent risk factors for incident HAM in PLwHTLV-1,while proviral load is not, in agreement with IL-10 being upstream of proviral load dynamics. Additional candidate biomarkers IL-29/IL-6R/TNFR2 represent plausible therapeutic targets for future clinical trials in HAM patients.
Asunto(s)
Biomarcadores , Virus Linfotrópico T Tipo 1 Humano , Interleucina-10 , Carga Viral , Humanos , Femenino , Masculino , Brasil/epidemiología , Virus Linfotrópico T Tipo 1 Humano/inmunología , Interleucina-10/sangre , Biomarcadores/sangre , Persona de Mediana Edad , Adulto , Infecciones por HTLV-I/inmunología , Infecciones por HTLV-I/sangre , Infecciones por HTLV-I/diagnóstico , Provirus , Estudios de Cohortes , Paraparesia Espástica Tropical/sangre , Paraparesia Espástica Tropical/inmunología , Paraparesia Espástica Tropical/virología , IncidenciaRESUMEN
We report on a new anti-influenza virus agent, SA-19, a lipophilic glycopeptide derivative consisting of aglycoristocetin coupled to a phenylbenzyl-substituted cyclobutenedione. In Madin-Darby canine kidney cells infected with influenza A/H1N1, A/H3N2, or B virus, SA-19 displayed a 50% antivirally effective concentration of 0.60 µM and a selectivity index (ratio of cytotoxic versus antiviral concentration) of 112. SA-19 was 11-fold more potent than unsubstituted aglycoristocetin and was active in human and nonhuman cell lines. Virus yield at 72 h p.i. was reduced by 3.6 logs at 0.8 µM SA-19. In contrast to amantadine and oseltamivir, SA-19 did not select for resistance upon prolonged virus exposure. SA-19 was shown to inhibit an early postbinding step in virus replication. The compound had no effect on hemagglutinin (HA)-mediated membrane fusion in an HA-polykaryon assay and did not inhibit the low-pH-induced refolding of the HA in a tryptic digestion assay. However, a marked inhibitory effect on the transduction exerted by retroviral pseudoparticles carrying an HA or vesicular stomatitis virus glycoprotein (VSV-G) fusion protein was noted, suggesting that SA-19 targets a cellular factor with a role in influenza virus and VSV entry. Using confocal microscopy with antinucleoprotein staining, SA-19 was proven to completely prevent the influenza virus nuclear entry. This virus arrest was characterized by the formation of cytoplasmic aggregates. SA-19 appeared to disturb the endocytic uptake and trap the influenza virus in vesicles distinct from early, late, or recycling endosomes. The aglycoristocetin derivative SA-19 represents a new class of potent and broad-acting influenza virus inhibitors with potential clinical relevance.
Asunto(s)
Antivirales/farmacología , Citoplasma/virología , Glicopéptidos/farmacología , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza B/efectos de los fármacos , Animales , Antivirales/química , Línea Celular , Citoplasma/efectos de los fármacos , Perros , Glicopéptidos/química , Humanos , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/fisiología , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/fisiología , Virus de la Influenza A/fisiología , Virus de la Influenza B/fisiología , Gripe Humana/tratamiento farmacológico , Gripe Humana/virología , Estructura Molecular , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Here, we report on the anti-SARS-CoV-2 activity of PRO-2000, a sulfonated polyanionic compound. In Vero cells infected with the Wuhan, alpha, beta, delta or omicron variant, PRO-2000 displayed EC50 values of 1.1 µM, 2.4 µM, 1.3 µM, 2.1 µM and 0.11 µM, respectively, and an average selectivity index (i.e. ratio of cytotoxic versus antiviral concentration) of 172. Its anti-SARS-CoV-2 activity was confirmed by virus yield assays in Vero cells, Caco2 cells and A549 cells overexpressing ACE2 and TMPRSS2 (A549-AT). Using pseudoviruses bearing the SARS-CoV-2 spike (S), PRO-2000 was shown to block the S-mediated pseudovirus entry in Vero cells and A549-AT cells, with EC50 values of 0.091 µM and 1.6 µM, respectively. This entry process is initiated by interaction of the S glycoprotein with angiotensin-converting enzyme 2 (ACE2) and heparan sulfate proteoglycans. Surface Plasmon Resonance (SPR) studies showed that PRO-2000 binds to the receptor-binding domain (RBD) of S with a KD of 1.6 nM. Similar KD values (range: 1.2 nM-2.1 nM) were obtained with the RBDs of the alpha, beta, delta and omicron variants. In an SPR neutralization assay, PRO-2000 had no effect on the interaction between the RBD and ACE2. Instead, PRO-2000 was proven to inhibit binding of the RBD to a heparin-coated sensor chip, yielding an IC50 of 1.1 nM. To conclude, PRO-2000 has the potential to inhibit a broad range of SARS-CoV-2 variants by blocking the heparin-binding site on the S protein.
Asunto(s)
Antivirales , COVID-19 , Chlorocebus aethiops , Animales , Humanos , Antivirales/farmacología , Enzima Convertidora de Angiotensina 2 , Células CACO-2 , Células Vero , SARS-CoV-2 , Unión Proteica , Glicoproteína de la Espiga del CoronavirusRESUMEN
A microwave-assisted three-component one-pot cyclocondensation method was applied for the synthesis of novel N-(1-thia-4-azaspiro[4.5]decan-4-yl)carboxamide compounds carrying an adamantyl moiety. The structures of the compounds were confirmed by spectral and elemental analysis. All compounds were evaluated for antiviral activity against influenza A (H1N1 and H3N2) and influenza B virus in MDCK cell cultures. The compounds displayed a confined structure-activity relationship. The N-(2,8-dimethyl-3-oxo-1-thia-4-azaspiro[4.5]dec-4-yl)adamantane-1-carboxamide 3b was the most potent inhibitor [antiviral EC(50): 1.4 µM against influenza A/H3N2 virus]. Its strong inhibitory effect in a virus hemolysis assay supports that 3b acts as an influenza virus fusion inhibitor by preventing the conformational change of the influenza virus hemagglutinin at low pH.
Asunto(s)
Adamantano/análogos & derivados , Antivirales/síntesis química , Compuestos Aza/síntesis química , Orthomyxoviridae/efectos de los fármacos , Compuestos de Espiro/síntesis química , Adamantano/síntesis química , Adamantano/química , Adamantano/farmacología , Amidas/síntesis química , Amidas/química , Amidas/farmacología , Animales , Antivirales/química , Antivirales/farmacología , Compuestos Aza/química , Compuestos Aza/farmacología , Pollos , Perros , Humanos , Subtipo H1N1 del Virus de la Influenza A , Subtipo H3N2 del Virus de la Influenza A , Virus de la Influenza B , Células de Riñón Canino Madin Darby , Microondas , Orthomyxoviridae/fisiología , Compuestos de Espiro/química , Compuestos de Espiro/farmacología , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
In previous investigations, we identified a class of 1,3,4-thiadiazole derivatives with antiviral activity. N-{3-(Methylsulfanyl)-1-[5-(phenylamino)-1,3,4-thiadiazole-2-yl]propyl}benzamide emerged as a relevant lead compound for designing novel influenza A virus inhibitors. In the present study, we elaborated on this initial lead by performing chemical synthesis and antiviral evaluation of a series of structural analogues. During this research, thirteen novel 1,3,4-thiadiazole derivatives were synthesized by the cyclization of the corresponding thiosemicarbazides as synthetic precursors. The structures and the purities of the synthesized compounds were confirmed through chromatographic and spectral data. Four L-methionine-based 1,3,4-thiadiazole derivatives displayed activity against influenza A virus, the two best compounds being 24 carrying a 5-(4-chlorophenylamino)-1,3,4-thiadiazole moiety and 30 possessing a 5-(benzoylamino)-1,3,4-thiadiazole structure [antiviral EC50 against influenza A/H3N2 virus: 4.8 and 7.4 µM, respectively]. The 1,3,4-thiadiazole derivatives were inactive against influenza B virus and a wide panel of unrelated DNA and RNA viruses. Compound 24 represents a new class of selective influenza A virus inhibitors acting during the virus entry process, as evidenced by our findings in a time-of-addition assay. Molecular descriptors and in silico prediction of ADMET properties of the active compounds were calculated. According to in silico ADMET and drug similarity studies, active compounds have been estimated to be good candidates for oral administration with no apparent toxicity considerations.
Asunto(s)
Antivirales/síntesis química , Metionina/química , Tiadiazoles/química , Antivirales/química , Antivirales/farmacología , Diseño de Fármacos , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/fisiología , Virus de la Influenza B/efectos de los fármacos , Virus de la Influenza B/fisiología , Relación Estructura-Actividad , Tiadiazoles/síntesis química , Tiadiazoles/farmacología , Internalización del Virus/efectos de los fármacosRESUMEN
A new class of N-(1-thia-4-azaspiro[4.5]decan-4-yl)carboxamide inhibitors of influenza virus hemagglutinin (HA)-mediated membrane fusion that has a narrow and defined structure-activity relationship was identified. In Madin-Darby canine kidney (MDCK) cells infected with different strains of human influenza virus A/H3N2, the lead compound, 4c, displayed a 50% effective concentration of 3 to 23 muM and an antiviral selectivity index of 10. No activity was observed for A/H1N1, A/H5N1, A/H7N2, and B viruses. The activity of 4c was reduced considerably when added 30 min or later postinfection, indicating that 4c inhibits an early step in virus replication. 4c and its congeners inhibited influenza A/H3N2 virus-induced erythrocyte hemolysis at low pH. 4c-resistant virus mutants, selected in MDCK cells, contained either a single D112N change in the HA2 subunit of the viral HA or a combination of three substitutions, i.e., R220S (in HA1) and E57K (in HA2) and an A-T substitution at position 43 or 96 of HA2. The mutants showed efficiency for receptor binding and replication similar to that of wild-type virus yet displayed an increased pH of erythrocyte hemolysis. In polykaryon assays with cells expressing single-mutant HA proteins, the E57K, A96T, and D112N mutations resulted in 4c resistance, and the HA proteins containing R220S, A96T, and D112N mutations displayed an increased fusion pH. Molecular modeling identified a binding cavity for 4c involving arginine-54 and glutamic acid-57 in the HA2 subunit. Our studies with the new fusion inhibitor 4c confirm the importance of this HA region in the development of influenza virus fusion inhibitors.
Asunto(s)
Antivirales/farmacología , Azepinas/farmacología , Hemaglutininas Virales/metabolismo , Virus de la Influenza A/efectos de los fármacos , Internalización del Virus/efectos de los fármacos , Sustitución de Aminoácidos/genética , Animales , Antivirales/química , Azepinas/química , Línea Celular , Perros , Hemaglutininas Virales/química , Hemaglutininas Virales/genética , Humanos , Virus de la Influenza B/efectos de los fármacos , Concentración 50 Inhibidora , Modelos Moleculares , Estructura Molecular , Mutación Missense , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
Favipiravir (T-705) is a broad-spectrum antiviral drug that inhibits RNA viruses after intracellular conversion into its active form, T-705 ribofuranosyl 5'-triphosphate. We previously showed that T-705 is able to significantly inhibit the replication of chikungunya virus (CHIKV), an arbovirus transmitted by Aedes mosquitoes, in mammalian cells and in mouse models. In contrast, the effect of T-705 on CHIKV infection and replication in the mosquito vector is unknown. Since the antiviral activity of T-705 has been shown to be cell line-dependent, we studied here its antiviral efficacy in Aedes-derived mosquito cells and in Aedes aegypti mosquitoes. Interestingly, T-705 was devoid of anti-CHIKV activity in mosquito cells, despite being effective against CHIKV in Vero cells. By investigating the metabolic activation profile, we showed that, unlike Vero cells, mosquito cells were not able to convert T-705 into its active form. To explore whether alternative metabolization pathways might exist in vivo, Aedes aegypti mosquitoes were infected with CHIKV and administered T-705 via an artificial blood meal. Virus titrations of whole mosquitoes showed that T-705 was not able to reduce CHIKV infection in mosquitoes. Combined, these in vitro and in vivo data indicate that T-705 lacks antiviral activity in mosquitoes due to inadequate metabolic activation in this animal species.
RESUMEN
To suppress serious influenza infections in persons showing insufficient protection from the vaccines, antiviral drugs are of vital importance. There is a need for novel agents with broad activity against influenza A (IAV) and B (IBV) viruses and with targets that differ from those of the current antivirals. We here report a new small molecule influenza virus inhibitor referred to as CPD A (chemical name: N-(pyridin-3-yl)thiophene-2-carboxamide). In an influenza virus minigenome assay, this non-nucleoside compound inhibited RNA synthesis of IAV and IBV with EC50 values of 2.3 µM and 2.6 µM, respectively. Robust in vitro activity was noted against a broad panel of IAV (H1N1 and H3N2) and IBV strains, with a median EC50 value of 0.20 µM, which is 185-fold below the 50% cytotoxic concentration. The action point in the viral replication cycle was located between 1 and 5 h p.i., showing a similar profile as ribavirin. Like this nucleoside analogue, CPD A was shown to cause strong depletion of the cellular GTP pool and, accordingly, its antiviral activity was antagonized when this pool was restored with exogenous guanosine. This aligns with the observed inhibition in a cell-based IMP dehydrogenase (IMPDH) assay, which seems to require metabolic activation of CPD A since no direct inhibition was seen in an enzymatic IMPDH assay. The combination of CPD A with ribavirin, another IMPDH inhibitor, proved strongly synergistic. To conclude, we established CPD A as a new inhibitor of influenza A and B virus replication and RNA synthesis, and support the potential of IMPDH inhibitors for influenza therapy with acceptable safety profile.
Asunto(s)
Antivirales/farmacología , Inhibidores Enzimáticos/farmacología , IMP Deshidrogenasa/antagonistas & inhibidores , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza B/efectos de los fármacos , Ribavirina/farmacología , Línea Celular , Sinergismo Farmacológico , Humanos , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/clasificación , Gripe Humana/tratamiento farmacológicoRESUMEN
In this study, a series of 10 quinoline analogues was evaluated for their in vitro antiviral activity against a panel of alpha- and beta-coronaviruses, including the severe acute respiratory syndrome coronaviruses 1 and 2 (SARS-CoV-1 and SARS-CoV-2), as well as the human coronaviruses (HCoV) 229E and OC43. Chloroquine and hydroxychloroquine were the most potent with antiviral EC50 values in the range of 0.12-12 µM. Chloroquine displayed the most favorable selectivity index (i.e. ratio cytotoxic versus antiviral concentration), being 165 for HCoV-OC43 in HEL cells. Potent anti-coronavirus activity was also observed with amodiaquine, ferroquine and mefloquine, although this was associated with substantial cytotoxicity for mefloquine. Primaquine, quinidine, quinine and tafenoquine only blocked coronavirus replication at higher concentrations, while piperaquine completely lacked antiviral and cytotoxic effects. A time-of-addition experiment in HCoV-229E-infected HEL cells revealed that chloroquine interferes with viral entry at a post-attachment stage. Using confocal microscopy, no viral RNA synthesis could be detected upon treatment of SARS-CoV-2-infected cells with chloroquine. The inhibition of SARS-CoV-2 replication by chloroquine and hydroxychloroquine coincided with an inhibitory effect on the autophagy pathway as visualized by a dose-dependent increase in LC3-positive puncta. The latter effect was less pronounced or even absent with the other quinolines. In summary, we showed that several quinoline analogues, including chloroquine, hydroxychloroquine, amodiaquine, ferroquine and mefloquine, exhibit broad anti-coronavirus activity in vitro.
Asunto(s)
Antimaláricos/farmacología , Antivirales/farmacología , Infecciones por Coronavirus/tratamiento farmacológico , Coronavirus/efectos de los fármacos , Quinolinas/farmacología , Animales , Chlorocebus aethiops , Cloroquina/farmacología , Coronavirus Humano 229E/efectos de los fármacos , Coronavirus Humano OC43/efectos de los fármacos , Humanos , Hidroxicloroquina/farmacología , SARS-CoV-2/efectos de los fármacos , Células Vero , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Tratamiento Farmacológico de COVID-19RESUMEN
The influenza virus hemagglutinin (HA) is an attractive target for antiviral therapy due to its essential role in mediating virus entry into the host cell. We here report the identification of a class of N-benzyl-4,4,-disubstituted piperidines as influenza A virus fusion inhibitors with specific activity against the H1N1 subtype. Using the highly efficient one-step Ugi four-component reaction, diverse library of piperidine-based analogues was synthesized and evaluated to explore the structure-activity relationships (SAR). Mechanistic studies, including resistance selection with the most active compound (2) demonstrated that it acts as an inhibitor of the low pH-induced HA-mediated membrane fusion process. Computational studies identified an as yet unrecognized fusion inhibitor binding site, which is located at the bottom of the HA2 stem in close proximity to the fusion peptide. A direct π-stacking interaction between the N-benzylpiperidine moiety of 2 and F9HA2 of the fusion peptide, reinforced with an additional π-stacking interaction with Y119HA2, and a salt bridge of the protonated piperidine nitrogen with E120HA2, were identified as important interactions to mediate ligand binding. This site rationalized the observed SAR and provided a structural explanation for the H1N1-specific activity of our inhibitors. Furthermore, the HA1-S326V mutation resulting in resistance to 2 is close to the proposed new binding pocket. Our findings point to the N-benzyl-4,4,-disubstituted piperidines as an interesting class of influenza virus inhibitors, representing the first example of fusion peptide binders with great potential for anti-influenza drug development.
Asunto(s)
Antivirales/farmacología , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Piperidinas/farmacología , Animales , Antivirales/síntesis química , Antivirales/química , Perros , Relación Dosis-Respuesta a Droga , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Humanos , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Células de Riñón Canino Madin Darby/efectos de los fármacos , Células de Riñón Canino Madin Darby/virología , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Piperidinas/síntesis química , Piperidinas/química , Relación Estructura-ActividadRESUMEN
The antiviral drug T-705 (favipiravir) and its non-fluorinated analogue T-1105 inhibit the polymerases of RNA viruses after being converted to their ribonucleoside triphosphate (RTP) metabolite. We here compared the activation efficiency of T-705 and T-1105 in four cell lines that are commonly used for their antiviral evaluation. In MDCK cells, the levels of T-705-RTP were markedly lower than those of T-1105-RTP, while the opposite was seen in A549, Vero and HEK293T cells. In the latter three cell lines, T-1105 activation was hindered by inefficient conversion of the ribonucleoside monophosphate to the ribonucleoside diphosphate en route to forming the active triphosphate. Accordingly, T-1105 had better anti-RNA virus activity in MDCK cells, while T-705 was more potent in the other three cell lines. Additionally, we identified a fourth metabolite, the NAD analogue of T-705/T-1105, and showed that it can be formed by nicotinamide mononucleotide adenylyltransferase.