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1.
PLoS Pathog ; 12(2): e1005428, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26863439

RESUMEN

Hepatitis C virus (HCV) is a positive-strand RNA virus of the Flaviviridae family and a major cause of liver disease worldwide. HCV replicates in the cytoplasm, and the synthesis of viral proteins induces extensive rearrangements of host cell membranes producing structures, collectively termed the membranous web (MW). The MW contains the sites of viral replication and assembly, and we have identified distinct membrane fractions derived from HCV-infected cells that contain replication and assembly complexes enriched for viral RNA and infectious virus, respectively. The complex membrane structure of the MW is thought to protect the viral genome limiting its interactions with cytoplasmic pattern recognition receptors (PRRs) and thereby preventing activation of cellular innate immune responses. Here we show that PRRs, including RIG-I and MDA5, and ribosomes are excluded from viral replication and assembly centers within the MW. Furthermore, we present evidence that components of the nuclear transport machinery regulate access of proteins to MW compartments. We show that the restricted assess of RIG-I to the MW can be overcome by the addition of a nuclear localization signal sequence, and that expression of a NLS-RIG-I construct leads to increased immune activation and the inhibition of viral replication.


Asunto(s)
Hepacivirus/fisiología , Replicación Viral/genética , Transporte Activo de Núcleo Celular , Línea Celular , Membrana Celular/virología , Humanos , Señales de Localización Nuclear/metabolismo , ARN Viral/aislamiento & purificación , Receptores de Reconocimiento de Patrones/inmunología , Proteínas Virales/genética
2.
Gut ; 66(10): 1853-1861, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-27436270

RESUMEN

OBJECTIVE: Silibinin is a flavonolignan that is well established for its robust antiviral activity against HCV infection and has undergone several clinical trials for the management of hepatitis C. Despite its potency, silibinin suffers from poor solubility and bioavailability, restricting its clinical use. To overcome this limitation, we developed highly bioavailable silibinin nanoparticles (SB-NPs) and evaluated their efficiency against HCV infection. DESIGN: SB-NPs were prepared using a nanoemulsification technique and were physicochemically characterised. Infectious HCV culture systems were used to evaluate the influence of SB-NP on the virus life cycle and examine their antioxidant activity against HCV-induced oxidative stress. The safety profiles of SB-NP, in vivo pharmacokinetic studies and antiviral activity against infection of primary human hepatocytes were also assessed. RESULTS: SB-NP consisted of nanoscale spherical particles (<200 nm) encapsulating amorphous silibinin at >97% efficiency and increasing the compound's solubility by >75%. Treatment with SB-NP efficiently restricted HCV cell-to-cell transmission, suggesting that they retained silibinin's robust anti-HCV activity. In addition, SB-NP exerted an antioxidant effect via their free radical scavenging function. Oral administration of SB-NP in rodents produced no apparent in vivo toxicity, and pharmacokinetic studies revealed an enhanced serum level and superior biodistribution to the liver compared with non-modified silibinin. Finally, SB-NP efficiently reduced HCV infection of primary human hepatocytes. CONCLUSIONS: Due to SB-NP's enhanced bioavailability, effective anti-HCV activity and an overall hepatoprotective effect, we suggest that SB-NP may be a cost-effective anti-HCV agent that merits further evaluation for the treatment of hepatitis C.


Asunto(s)
Antioxidantes/farmacología , Hepacivirus/efectos de los fármacos , Silimarina/farmacología , Animales , Antioxidantes/administración & dosificación , Antioxidantes/farmacocinética , Células Cultivadas , Sistemas de Liberación de Medicamentos , Hepacivirus/patogenicidad , Hepatocitos/virología , Humanos , Estadios del Ciclo de Vida/efectos de los fármacos , Masculino , Nanosferas , Ratas , Silibina , Silimarina/administración & dosificación , Silimarina/farmacocinética
3.
PLoS Pathog ; 10(12): e1004556, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25503988

RESUMEN

Influenza is a major cause of morbidity and mortality in immunosuppressed persons, and vaccination often confers insufficient protection. IL-28B, a member of the interferon (IFN)-λ family, has variable expression due to single nucleotide polymorphisms (SNPs). While type-I IFNs are well known to modulate adaptive immunity, the impact of IL-28B on B- and T-cell vaccine responses is unclear. Here we demonstrate that the presence of the IL-28B TG/GG genotype (rs8099917, minor-allele) was associated with increased seroconversion following influenza vaccination (OR 1.99 p = 0.038). Also, influenza A (H1N1)-stimulated T- and B-cells from minor-allele carriers showed increased IL-4 production (4-fold) and HLA-DR expression, respectively. In vitro, recombinant IL-28B increased Th1-cytokines (e.g. IFN-γ), and suppressed Th2-cytokines (e.g. IL-4, IL-5, and IL-13), H1N1-stimulated B-cell proliferation (reduced 70%), and IgG-production (reduced>70%). Since IL-28B inhibited B-cell responses, we designed antagonistic peptides to block the IL-28 receptor α-subunit (IL28RA). In vitro, these peptides significantly suppressed binding of IFN-λs to IL28RA, increased H1N1-stimulated B-cell activation and IgG-production in samples from healthy volunteers (2-fold) and from transplant patients previously unresponsive to vaccination (1.4-fold). Together, these findings identify IL-28B as a key regulator of the Th1/Th2 balance during influenza vaccination. Blockade of IL28RA offers a novel strategy to augment vaccine responses.


Asunto(s)
Inmunidad Adaptativa/efectos de los fármacos , Linfocitos B/patología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/farmacología , Gripe Humana/patología , Interleucinas/fisiología , Linfocitos T/patología , Inmunidad Adaptativa/inmunología , Inmunidad Adaptativa/fisiología , Adulto , Anciano , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Proliferación Celular , Femenino , Antígenos HLA-DR/metabolismo , Humanos , Huésped Inmunocomprometido , Inmunoglobulina G/metabolismo , Técnicas In Vitro , Vacunas contra la Influenza/inmunología , Gripe Humana/metabolismo , Gripe Humana/prevención & control , Interferones , Interleucina-4/metabolismo , Interleucinas/genética , Interleucinas/farmacología , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple/genética , Proteínas Recombinantes/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Células TH1/patología , Células Th2/patología , Receptores de Trasplantes
4.
Cytokine ; 78: 27-36, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26615570

RESUMEN

Recently, differences in the levels of various chemokines and cytokines were reported in patients with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) as compared with controls. Moreover, the analyte profile differed between chronic ME/CFS patients of long duration versus patients with disease of less than 3years. In the current study, we measured the plasma levels of 34 cytokines, chemokines and growth factors in 100 chronic ME/CFS patients of long duration and in 79 gender and age-matched controls. We observed highly significant reductions in the concentration of circulating interleukin (IL)-16, IL-7, and Vascular Endothelial Growth Factor A (VEGF-A) in ME/CFS patients. All three biomarkers were significantly correlated in a multivariate cluster analysis. In addition, we identified significant reductions in the concentrations of fractalkine (CX3CL1) and monokine-induced-by-IFN-γ (MIG; CXCL9) along with increases in the concentrations of eotaxin 2 (CCL24) in ME/CFS patients. Our data recapitulates previous data from another USA ME/CFS cohort in which circulating levels of IL-7 were reduced. Also, a reduced level of VEGF-A was reported previously in sera of patients with Gulf War Illness as well as in cerebral spinal fluid samples from a different cohort of USA ME/CFS patients. To our knowledge, we are the first to test for levels of IL-16 in ME/CFS patients. In combination with previous data, our work suggests that the clustered reduction of IL-7, IL-16 and VEGF-A may have physiological relevance to ME/CFS disease. This profile is ME/CFS-specific since measurement of the same analytes present in chronic infectious and autoimmune liver diseases, where persistent fatigue is also a major symptom, failed to demonstrate the same changes. Further studies of other ME/CFS and overlapping disease cohorts are warranted in future.


Asunto(s)
Síndrome de Fatiga Crónica/sangre , Interleucina-16/sangre , Interleucina-7/sangre , Factor A de Crecimiento Endotelial Vascular/sangre , Adulto , Biomarcadores/sangre , Estudios de Casos y Controles , Quimiocinas/sangre , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/sangre , Masculino , Persona de Mediana Edad , Análisis Multivariante
5.
J Hepatol ; 62(3): 541-8, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25450204

RESUMEN

BACKGROUND & AIMS: A vaccine against hepatitis C virus (HCV) is unavailable and cost-effective antivirals that prevent HCV infection and re-infection, such as in the transplant setting, do not exist. In a search for novel and economical prophylactic agents, we examined the antiviral activity of saikosaponins (SSa, SSb2, SSc, and SSd) from Bupleurum kaoi root (BK) as entry inhibitors against HCV infection. METHODS: Infectious HCV culture systems were used to examine the effect of saikosaponins on the complete virus life cycle (entry, RNA replication/translation, and particle production). Antiviral activity against various HCV genotypes, clinical isolates, and infection of primary human hepatocytes were also evaluated. RESULTS: BK and the saikosaponins potently inhibited HCV infection at non-cytotoxic concentrations. These natural agents targeted early steps of the viral life cycle, while leaving replication/translation, egress, and spread relatively unaffected. In particular, we identified SSb2 as an efficient inhibitor of early HCV entry, including neutralization of virus particles, preventing viral attachment, and inhibiting viral entry/fusion. Binding analysis, using soluble viral glycoproteins, demonstrated that SSb2 acted on HCV E2. Moreover, SSb2 inhibited infection by several genotypic strains and prevented binding of serum-derived HCV onto hepatoma cells. Finally, treatment with the compound blocked HCV infection of primary human hepatocytes. CONCLUSIONS: Due to its potency, SSb2 may be of value for development as an antagonist of HCV entry and could be explored as prophylactic treatment during the course of liver transplantation.


Asunto(s)
Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Hepacivirus/fisiología , Ácido Oleanólico/análogos & derivados , Saponinas/farmacología , Internalización del Virus/efectos de los fármacos , Animales , Antivirales/aislamiento & purificación , Antivirales/toxicidad , Bupleurum , Línea Celular , Hepatitis C/prevención & control , Hepatocitos/efectos de los fármacos , Hepatocitos/virología , Humanos , Trasplante de Hígado , Masculino , Ácido Oleanólico/aislamiento & purificación , Ácido Oleanólico/farmacología , Ácido Oleanólico/toxicidad , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacología , Ratas , Ratas Sprague-Dawley , Saponinas/aislamiento & purificación , Saponinas/toxicidad , Virión/efectos de los fármacos , Replicación Viral/efectos de los fármacos
6.
PLoS Pathog ; 9(10): e1003744, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24204278

RESUMEN

Hepatitis C virus (HCV) infection induces formation of a membranous web structure in the host cell cytoplasm where the viral genome replicates and virions assemble. The membranous web is thought to concentrate viral components and hide viral RNA from pattern recognition receptors. We have uncovered a role for nuclear pore complex proteins (Nups) and nuclear transport factors (NTFs) in the membranous web. We show that HCV infection leads to increased levels of cytoplasmic Nups that accumulate at sites enriched for HCV proteins. Moreover, we detected interactions between specific HCV proteins and both Nups and NTFs. We hypothesize that cytoplasmically positioned Nups facilitate formation of the membranous web and contribute to the compartmentalization of viral replication. Accordingly, we show that transport cargo proteins normally targeted to the nucleus are capable of entering regions of the membranous web, and that depletion of specific Nups or Kaps inhibits HCV replication and assembly.


Asunto(s)
Hepacivirus/fisiología , Hepatitis C/metabolismo , Membranas Intracelulares/metabolismo , Poro Nuclear/metabolismo , Replicación Viral/fisiología , Transporte Activo de Núcleo Celular/genética , Línea Celular , Hepatitis C/genética , Hepatitis C/patología , Humanos , Membranas Intracelulares/virología , Poro Nuclear/genética , Poro Nuclear/patología , Poro Nuclear/virología
7.
Proc Natl Acad Sci U S A ; 107(40): 17339-44, 2010 Oct 05.
Artículo en Inglés | MEDLINE | ID: mdl-20823220

RESUMEN

Antiviral drugs targeting viral proteins often result in prompt selection for resistance. Moreover, the number of viral targets is limited. Novel antiviral targets are therefore needed. The unique characteristics of fusion between virion envelopes and cell membranes may provide such targets. Like all fusing bilayers, viral envelopes locally adopt hourglass-shaped stalks during the initial stages of fusion, a process that requires local negative membrane curvature. Unlike cellular vesicles, however, viral envelopes do not redistribute lipids between leaflets, can only use the energy released by virion proteins, and fuse to the extracellular leaflets of cell membranes. Enrichment in phospholipids with hydrophilic heads larger than their hydrophobic tails in the convex outer leaflet of vesicles favors positive curvature, therefore increasing the activation energy barrier for fusion. Such phospholipids can increase the activation barrier beyond the energy provided by virion proteins, thereby inhibiting viral fusion. However, phospholipids are not pharmacologically useful. We show here that a family of synthetic rigid amphiphiles of shape similar to such phospholipids, RAFIs (rigid amphipathic fusion inhibitors), inhibit the infectivity of several otherwise unrelated enveloped viruses, including hepatitis C and HSV-1 and -2 (lowest apparent IC(50) 48 nM), with no cytotoxic or cytostatic effects (selectivity index > 3,000) by inhibiting the increased negative curvature required for the initial stages of fusion.


Asunto(s)
Antivirales , Proteínas Virales/metabolismo , Internalización del Virus/efectos de los fármacos , Virus/efectos de los fármacos , Animales , Antivirales/química , Antivirales/metabolismo , Antivirales/farmacología , Línea Celular , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 1/patogenicidad , Herpesvirus Humano 1/ultraestructura , Humanos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Lípidos de la Membrana/metabolismo , Estructura Molecular , Virión/metabolismo , Virión/patogenicidad , Virión/ultraestructura , Virus/patogenicidad , Virus/ultraestructura
8.
Anal Chem ; 84(18): 7603-6, 2012 Sep 18.
Artículo en Inglés | MEDLINE | ID: mdl-22931544

RESUMEN

We describe a single-cycle DNA aptamer selection strategy that is able to obtain high affinity aptamers (K(d) of sub-nM) directly from a protein blotted on membrane. The key to the success of this strategy is the unique use of DNase I digestion to remove unwanted ssDNA from the membrane, leaving only the strongest bound aptamers. A crude Hepatitis B virus core protein (HBcAg) was separated using polyacrylamide gel electrophoresis (PAGE) and electro-blotted onto a polyvinylidene fluoride (PVDF) membrane. The membrane strip containing HBcAg and a second membrane strip containing human serum proteins were coincubated with a ssDNA library consisting of ∼10 copies each of 10(15) random sequences. Unbound and weakly bound sequences were efficiently removed from the membrane containing HBcAg using DNase I digestion and gradient wash with urea buffers. The remaining ssDNA bound to the target consisted of approximately 500 molecules, from which two aptamers with high affinity (K(d) ∼100 and 200 pM) were identified. This technique can be potentially used for selection of aptamers directly from multiple proteins that are separated by gel electrophoresis from a biological mixture.


Asunto(s)
Aptámeros de Nucleótidos/química , Desoxirribonucleasas/metabolismo , Antígenos del Núcleo de la Hepatitis B/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Aptámeros de Nucleótidos/metabolismo , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , ADN de Cadena Simple/metabolismo , Electroforesis en Gel de Poliacrilamida , Antígenos del Núcleo de la Hepatitis B/aislamiento & purificación , Polivinilos/química
9.
Nat Commun ; 13(1): 6992, 2022 11 16.
Artículo en Inglés | MEDLINE | ID: mdl-36385011

RESUMEN

Interferons induced early after SARS-CoV-2 infection are crucial for shaping immunity and preventing severe COVID-19. We previously demonstrated that injection of pegylated interferon-lambda accelerated viral clearance in COVID-19 patients (NCT04354259). To determine if the viral decline is mediated by enhanced immunity, we assess in vivo responses to interferon-lambda by single cell RNA sequencing and measure SARS-CoV-2-specific T cell and antibody responses between placebo and interferon-lambda-treated patients. Here we show that interferon-lambda treatment induces interferon stimulated genes in peripheral immune cells expressing IFNLR1, including plasmacytoid dendritic cells and B cells. Interferon-lambda does not affect SARS-CoV-2-specific antibody levels or the magnitude of virus-specific T cells. However, we identify delayed T cell responses in older adults, suggesting that interferon-lambda can overcome delays in adaptive immunity to accelerate viral clearance in high-risk patients. Altogether, interferon-lambda offers an early COVID-19 treatment option for outpatients to boost innate antiviral defenses without dampening peripheral adaptive immunity.


Asunto(s)
Tratamiento Farmacológico de COVID-19 , Interferones , Humanos , Anciano , SARS-CoV-2 , Anticuerpos Antivirales , Antivirales/farmacología , Antivirales/uso terapéutico , Linfocitos T
10.
BMC Pharmacol Toxicol ; 22(1): 61, 2021 10 21.
Artículo en Inglés | MEDLINE | ID: mdl-34674775

RESUMEN

BACKGROUND: The emergence and rapid spread of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) in thelate 2019 has caused a devastating global pandemic of the severe pneumonia-like disease coronavirus disease 2019 (COVID-19). Although vaccines have been and are being developed, they are not accessible to everyone and not everyone can receive these vaccines. Also, it typically takes more than 10 years until a new therapeutic agent is approved for usage. Therefore, repurposing of known drugs can lend itself well as a key approach for significantly expediting the development of new therapies for COVID-19. METHODS: We have incorporated machine learning-based computational tools and in silico models into the drug discovery process to predict Adsorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) profiles of 90 potential drugs for COVID-19 treatment identified from two independent studies mainly with the purpose of mitigating late-phase failures because of inferior pharmacokinetics and toxicity. RESULTS: Here, we summarize the cardiotoxicity and general toxicity profiles of 90 potential drugs for COVID-19 treatment and outline the risks of repurposing and propose a stratification of patients accordingly. We shortlist a total of five compounds based on their non-toxic properties. CONCLUSION: In summary, this manuscript aims to provide a potentially useful source of essential knowledge on toxicity assessment of 90 compounds for healthcare practitioners and researchers to find off-label alternatives for the treatment for COVID-19. The majority of the molecules discussed in this manuscript have already moved into clinical trials and thus their known pharmacological and human safety profiles are expected to facilitate a fast track preclinical and clinical assessment for treating COVID-19.


Asunto(s)
Antivirales/toxicidad , Tratamiento Farmacológico de COVID-19 , Descubrimiento de Drogas , Reposicionamiento de Medicamentos , Animales , Antivirales/efectos adversos , Captopril/uso terapéutico , Cardiotoxinas/toxicidad , Catecoles/uso terapéutico , Biología Computacional , Sistema Enzimático del Citocromo P-450/metabolismo , Descubrimiento de Drogas/métodos , Humanos , Indometacina/uso terapéutico , Linezolid/uso terapéutico , Hígado/efectos de los fármacos , Ratones , Modelos Biológicos , Nitrilos/uso terapéutico , Ratas , Reproducción/efectos de los fármacos , Programas Informáticos , Ácido Valproico/uso terapéutico
11.
Am J Physiol Gastrointest Liver Physiol ; 299(4): G844-54, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20651006

RESUMEN

Although multiple determinants for hepatitis C virus (HCV) infection are known, it remains partly unclear what determines the human specificity of HCV infection. Presumably, the presence of appropriate entry receptors is essential, and this may explain why HCV is unable to infect nonhuman hepatocytes. However, using mice with chimeric human livers, we show in this study that the presence of human hepatocytes, and therefore human entry receptors, is not sufficient for HCV infection. In successfully transplanted SCID/Alb-uPA mice, infection with HCV is reliable only when ∼70-80% of the liver consists of human hepatocytes. We show that chimeric mice, which are hard to infect with HCV, have significant groups of human hepatocytes that are readily infected with hepatitis B virus. Thus it is unlikely that the lack of infection with HCV can simply be attributed to low hepatocyte numbers. We investigated whether the humanization of lipoprotein profiles is positively associated with infection success. We show that the lipoprotein profiles of chimeric mice become more human-like at high levels of engraftment of human hepatocytes. This and expression of markers of human lipoprotein biosynthesis, human apolipoprotein B (ApoB) and cholesterol ester transfer protein (CETP), show a strong positive correlation with successful infection. Association of HCV in the blood of chimeric mice to ApoB-containing lipoproteins is comparable to association of HCV in patient serum and provides further support for a critical role for ApoB-containing lipoproteins in the infectious cycle of HCV. Our data suggest that the weakest link in the HCV infection chain does not appear to be the presence of human hepatocytes per se. We believe that HCV infection also depends on the presence of sufficient levels of human lipoproteins.


Asunto(s)
Hepacivirus/fisiología , Hepatitis C/metabolismo , Hepatocitos/trasplante , Lipoproteínas/sangre , Animales , Trasplante de Células , Quimera , Hepatitis B/metabolismo , Virus de la Hepatitis B/fisiología , Humanos , Lipoproteínas/metabolismo , Ratones , Ratones SCID , Replicación Viral
12.
Hepatology ; 49(3): 745-52, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19072827

RESUMEN

UNLABELLED: Anti-hepatitis C virus (HCV) drug development has been challenged by a lack of experience with inhibitors inclusive of in vitro, animal model, and clinical study. This manuscript outlines activity and correlation across such a spectrum of models and into clinical trials with a novel selective nonstructural protein 5B (NS5B) polymerase inhibitor, HCV796. Enzyme assays yielded median inhibitory concentration (IC(50)) values of 0.01 to 0.14 microM for genotype 1, with half maximal effective concentration (EC(50)s) of 5 nM and 9 nM against genotype 1a and 1b replicons. In the chimeric mouse model, a 2.02 +/- 0.55 log reduction in HCV titer was seen with monotherapy, whereas a suboptimal dose of 30 mg/kg three times per day in combination with interferon demonstrated a 2.44 log reduction (P = 0.001 versus interferon alone) Clinical outcomes in combination with pegylated interferon and ribavirin have revealed additive efficacy in treatment naïve patients. Abnormal liver function test results were observed in 8% of HCV-796 patients treated for over 8 weeks, resulting in suspension of further trial activity. CONCLUSION: The RNA-dependent RNA polymerase inhibitor HCV796 demonstrated potent anti-HCV activity consistently through enzyme inhibition assays, subgenomic replicon, and chimeric mouse studies. Strong correlations of outcomes in the mouse model were seen with subsequent clinical trials, including a plateau in dose-related antiviral activity and additive impact from combination therapy with interferon. These outcomes demonstrate the utility of the range of in vitro and in vivo models now available for anti-HCV drug development and support the potential utility of polymerase inhibitors in future combination therapies for HCV treatment.


Asunto(s)
Antivirales/uso terapéutico , Benzofuranos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Hepacivirus/efectos de los fármacos , Hepatitis C/tratamiento farmacológico , Hígado/efectos de los fármacos , Hígado/virología , Sulfonamidas/uso terapéutico , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Antivirales/farmacología , Benzofuranos/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Quimioterapia Combinada , Inhibidores Enzimáticos/farmacología , Hepacivirus/fisiología , Hepatocitos/trasplante , Humanos , Interferón alfa-2 , Interferón-alfa/uso terapéutico , Hígado/patología , Ratones , Ratones SCID , Polietilenglicoles , Proteínas Recombinantes , Replicón/efectos de los fármacos , Ribavirina/uso terapéutico , Sulfonamidas/farmacología
13.
Bioorg Med Chem Lett ; 20(22): 6790-3, 2010 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-20863701

RESUMEN

Various 2,3'-anhydro analogs of 5-substituted 1-(2-deoxy-ß-d-lyxofuranosyl)uracils (10-15) and a related 1-(3-O-mesyl-2-deoxy-ß-d-lyxofuranosyl) pyrimidine nucleoside analog (18) have been synthesized for evaluation as a new class of potential anti-HBV agents. The compounds 10, 12, and 15 demonstrated most potent anti-HBV activities against duck HBV (DHBV) and human HBV with EC(50) values in the range of 2.5-10 and 5-10 µg/mL, respectively, at non-toxic concentrations (CC(50) = > 200 µg/mL). The nucleoside 18 also demonstrated significant anti-HBV activity against DHBV with an EC(50) value of 2.5 µg/mL, however, it was less active against HBV in 2.2.15 cells (EC(50) = > 10 µg/mL).


Asunto(s)
Antivirales/farmacología , Virus de la Hepatitis B/efectos de los fármacos , Nucleósidos de Pirimidina/farmacología , Nucleósidos de Pirimidina/química
14.
Bioorg Med Chem ; 18(21): 7542-7, 2010 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-20869253

RESUMEN

Chronic infections with hepatitis B virus (HBV) and hepatitis C virus (HCV) lead to serious liver diseases worldwide. Co-infection with HBV and HCV is very common and is associated with increased risk of liver pathogenesis, liver cancer, and liver failure. Several 5-substituted 3'-fluoro (or chloro) (1-4, 6, 7, 17-19) and 2',3'-difluoro 2',3'-dideoxynucleosides (15 and 16) were synthesized and evaluated for in vitro antiviral activities against duck hepatitis B virus (DHBV), human hepatitis B virus, and hepatitis C virus. Of these compounds 4, 7, 17, and 19 demonstrated moderate anti-HBV activity, and 2, 4, 7, 8, and 19 were weak inhibitors of HCV. Although 5-iodo derivative (7) was most inhibitory against HCV, it exhibited a reduction in cellular RNA levels in Huh-7 cells. The 5-hydroxymethyl-3'-fluoro-2',3'-dideoxyuridine (4) and 1-(3-chloro-2,3-dideoxy-ß-d-erythro-pentofuranosyl)-5-fluorouracil (19) provided the most inhibition of both viruses without cytotoxicity.


Asunto(s)
Antivirales/síntesis química , Didesoxinucleósidos/química , Hepacivirus/efectos de los fármacos , Virus de la Hepatitis B/efectos de los fármacos , Animales , Antivirales/química , Antivirales/farmacología , Línea Celular Tumoral , ADN Viral/metabolismo , Didesoxinucleósidos/síntesis química , Didesoxinucleósidos/farmacología , Virus de la Hepatitis B del Pato/efectos de los fármacos , Humanos , ARN Viral/metabolismo
15.
bioRxiv ; 2020 Jul 20.
Artículo en Inglés | MEDLINE | ID: mdl-32743577

RESUMEN

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes COVID-19, utilizes angiotensin-converting enzyme 2 (ACE2) for entry into target cells. ACE2 has been proposed as an interferon-stimulated gene (ISG). Thus, interferon-induced variability in ACE2 expression levels could be important for susceptibility to COVID-19 or its outcomes. Here, we report the discovery of a novel, primate-specific isoform of ACE2, which we designate as deltaACE2 (dACE2). We demonstrate that dACE2, but not ACE2, is an ISG. In vitro, dACE2, which lacks 356 N-terminal amino acids, was non-functional in binding the SARS-CoV-2 spike protein and as a carboxypeptidase. Our results reconcile current knowledge on ACE2 expression and suggest that the ISG-type induction of dACE2 in IFN-high conditions created by treatments, inflammatory tumor microenvironment, or viral co-infections is unlikely to affect the cellular entry of SARS-CoV-2 and promote infection.

16.
J Virol ; 82(16): 8013-21, 2008 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-18524822

RESUMEN

Duck hepatitis B virus (DHBV) is a model virus for human hepatitis B virus (HBV), which infects approximately 360 million individuals worldwide. Nucleoside analogs can decrease virus production by inhibiting the viral polymerase; however, complete clearance by these drugs is not common because of the persistence of the HBV episome. HBV DNA is present in the nucleus as a covalently closed circular (cccDNA) form, where it drives viral transcription and progeny virus production. cccDNA is not the direct target of antiviral nucleoside analogs and is the source of HBV reemergence when antiviral therapy is stopped. To target cccDNA, six different zinc finger proteins (ZFP) were designed to bind DNA sequences in the DHBV enhancer region. After the binding kinetics were assessed by using electrophoretic mobility shift assays and surface plasmon resonance, two candidates with dissociation constants of 12.3 and 40.2 nM were focused on for further study. The ZFPs were cloned into a eukaryotic expression vector and cotransfected into longhorn male hepatoma cells with the plasmid pDHBV1.3, which replicates the DHBV life cycle. In the presence of each ZFP, viral RNA was significantly reduced, and protein levels were dramatically decreased. As a result, intracellular viral particle production was also significantly decreased. In summary, designed ZFPs are able to bind to the DHBV enhancer and interfere with viral transcription, resulting in decreased production of viral products and progeny virus genomes.


Asunto(s)
ADN Circular/genética , Virus de la Hepatitis B del Pato/metabolismo , Ingeniería de Proteínas/métodos , ARN Viral/metabolismo , Transcripción Genética , Dedos de Zinc/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Núcleo Celular/virología , Pollos , Elementos de Facilitación Genéticos , Cinética , Datos de Secuencia Molecular , Resonancia por Plasmón de Superficie
17.
Int Immunol ; 20(1): 89-104, 2008 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-18006878

RESUMEN

Hepatitis C virus (HCV) is a devastating human pathogen, yet there is no vaccine available for this virus. From studies with acute or chronic HCV-infected humans and chimpanzees, T-cell responses against HCV-derived conserved non-structural antigens have been correlated with viral clearance. In this study, recombinant adenoviral vectors containing HCV-derived NS4, NS5a or NS5b genes were employed to endogenously express the HCV antigens in human dendritic cells (DCs). The DCs expressing these HCV antigens exhibited normal phenotype and function. Intriguingly, we found that the DCs expressing HCV NS4, NS5a or NS5b antigens were able to significantly stimulate autologous T cells obtained from uninfected healthy individuals. These T cells produced various cytokines and proliferated in an HCV antigen-dependent manner. Evidence of both CD4(+) and CD8(+) T-cell responses generated in vitro against HCV NS4, NS5a or NS5b were obtained. HCV NS4 was much less stimulatory for CD4(+) and CD8(+) T cells than NS5. Further, in secondary assays, the CD4(+) T cells primed in vitro exhibited HCV antigen-specific proliferative responses against recombinant protein antigens. In summary, we provide conclusive evidence of in vitro stimulation of CD4(+) and CD8(+) T cells from HCV-naive individuals against HCV antigens NS4, NS5a and NS5b. The studies with naive T cells represent early events in the induction of cellular immune responses, which most likely govern the outcome of HCV infection. These studies have significant implications in designing vaccines for HCV infection in both prophylactic and therapeutic settings.


Asunto(s)
Donantes de Sangre , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Hepacivirus/inmunología , Activación de Linfocitos/inmunología , Proteínas no Estructurales Virales/inmunología , Adulto , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Hepatitis C/prevención & control , Humanos , Persona de Mediana Edad , Vacunas contra Hepatitis Viral , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo
18.
J Immunol Methods ; 445: 15-22, 2017 06.
Artículo en Inglés | MEDLINE | ID: mdl-28274837

RESUMEN

Type III interferons (IFN-lambdas) are important antiviral cytokines that also modulate immune responses acting through a unique IFN-λR1/IL-10R2 heterodimeric receptor. Conflicting data has been reported for which cells express the IFN-λR1 subunit and directly respond to IFN-λs. In this study we developed a novel method to measure IFN-λ3 binding to IFN-λR1/IL-10R2 on the surface of cells and relate this to a functional readout of interferon stimulated gene (ISG) activity in various cell lines. We show that Huh7.5 hepatoma cells bind IFN-λ3 at the highest levels with the lowest Kd(app), translating to the highest induction of various ISGs. Raji and Jurkat cell lines, representing B and T cells, respectively, moderately bind IFN-λ3 and have lower ISG responses. U937 cells, representing monocytes, did not bind IFN-λ3 well and therefore, did not have any ISG induction. Importantly, knockdown of IFNLR1 in Huh7.5 cells decreased our binding signal proportionally and reduced ISG induction by up to 93%. IFN-λ3 responsiveness increased over time with maximal ISG responses seen at 24h for all but one gene. These data confirm our new IFN-λ3 binding assay can be used to quantify IFN-λ receptor surface expression on a variety of cell types and reflects IFN-λ3 responsiveness.


Asunto(s)
Citometría de Flujo , Interleucinas/análisis , Receptores de Interferón/genética , Sitios de Unión , Línea Celular Tumoral , Humanos , Interferones , Interleucinas/inmunología , Receptores de Interferón/inmunología
19.
J Med Chem ; 49(6): 2049-54, 2006 Mar 23.
Artículo en Inglés | MEDLINE | ID: mdl-16539393

RESUMEN

Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. The main clinical limitation of a current antiviral drug for HBV, lamivudine, is the emergence of drug-resistant viral strains upon prolonged therapy. A group of 5-, 6-, or 5,6-substituted acyclic pyrimidine nucleosides with a 1-[(2-hydroxyethoxy)methyl] moiety were synthesized and evaluated for antiviral activities. The target compounds were prepared by the reaction of silylated uracils possessing a variety of substituents at the C-5 or C-6 positions or both with 1,3-dioxolane in the presence of potassium iodide and chlorotrimethylsilane by a convenient and single-step synthesis. Among the compounds tested, 5-chloro and 5-bromo analogues possessing an acyclic glycosyl moiety were the most effective and selective antiviral agents in the in vitro assays against wild-type duck HBV (EC50=0.4-2.2 and 3.7-18.5 microM, respectively) and human HBV-containing 2.2.15 cells (EC50=4.5-45.4 and 18.5-37.7 microM, respectively). These compounds were also found to retain sensitivity against lamivudine-resistant HBV containing a single mutation (M204I) and double mutations (L180M/M204V). The compounds investigated did not show cytotoxicity to host HepG2 and Vero cells, up to the highest concentration tested. The results presented here confirm and accentuate the potential of acyclic pyrimidine nucleosides as anti-HBV agents and extend our previous observations. We herein report the capability of acyclic pyrimidine nucleosides to inhibit the replication of both wild-type and drug-resistant mutant HBV.


Asunto(s)
Antivirales/síntesis química , Virus de la Hepatitis B/efectos de los fármacos , Nucleósidos de Pirimidina/síntesis química , Animales , Antivirales/química , Antivirales/farmacología , Línea Celular , Chlorocebus aethiops , Farmacorresistencia Viral , Patos/virología , Virus de la Hepatitis B/genética , Humanos , Lamivudine/farmacología , Mutación , Nucleósidos de Pirimidina/química , Nucleósidos de Pirimidina/farmacología , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacos
20.
J Med Chem ; 49(12): 3693-700, 2006 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-16759112

RESUMEN

Hepatitis B virus (HBV) is the most common cause of chronic liver disease worldwide. Development of drug resistance against clinical anti-HBV drug lamivudine due to long-term use and rebound of viral DNA after cessation of treatment has been a major setback of the current therapy. We have synthesized a series of pyrimidine nucleosides possessing a variety of substituents at the C-5 position, and a 1-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl] flexible acyclic glycosyl moiety at the N-1 position, that have the ability to mimic the natural 2'-deoxyribosyl moiety. Some of these potential antiviral compounds included variations at both C-5 and C-6 positions of the uracil base. Other variations of the uracil derivatives were the 6-aza congeners. 4-Amino and 4-methoxy pyrimidine derivatives were also made. Compounds in which the base moiety was substituted by 5-chloro- (25), 5-(2-bromovinyl)- (32), or 5-bromo-6-methyl- (37) groups possess significant activity against duck-HBV, wild-type human HBV (2.2.15 cells), and lamivudine-resistant HBV containing single and double mutations. No cytotoxicity was seen in host HepG2 and Vero cells, up to the highest concentration tested. The anti-HBV activity exhibited by compounds 25, 32, and 37 was superior for human HBV and comparable for DHBV to that of the corresponding purine nucleoside, ganciclovir. Further, they were only 10-15-fold less inhibitory against human HBV in 2.2.15 cells than the reference drug, lamivudine. Other compounds in the series were moderately inhibitory against DHBV and wild-type human HBV. The size of the halogen and the electronegativity of the substituents at the 5- and 6-positions are important for antiviral activity toward HBV. These compounds were also evaluated for their antiviral activity for West Nile virus, respiratory syncytial virus, SARS-coronavirus, and hepatitis C virus. They were generally inactive in these antiviral assay systems (at concentrations up to 100 microg/mL). 1-[(2-Hydroxy-1-(hydroxymethyl) ethoxy)methyl]-5-fluorocytosine (34) showed some inhibitory activity against hepatitis C virus. Taken together, these data support our previous observations that the 5-substituted pyrimidine nucleosides containing acyclic glycosyl moieties have potential to serve as a new generation of potent, selective, and nontoxic anti-HBV agents for wild-type and lamivudine-resistant mutant HBV.


Asunto(s)
Antivirales/síntesis química , Desoxirribosa/química , Virus de la Hepatitis B/efectos de los fármacos , Nucleósidos de Pirimidina/síntesis química , Animales , Antivirales/química , Antivirales/farmacología , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Farmacorresistencia Viral , Patos/virología , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/aislamiento & purificación , Humanos , Lamivudine/farmacología , Imitación Molecular , Mutación , Nucleósidos de Pirimidina/química , Nucleósidos de Pirimidina/farmacología , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacos
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