RESUMEN
Viral proteases play a crucial role in viral infection and are regarded as promising targets for antiviral drug development. Consequently, biosensing methods that target viral proteases have contributed to the study of virus-related diseases. This work presents a ratiometric electrochemical sensor that enables highly sensitive detection of viral proteases through the integration of target proteolysis-activated in vitro transcription and the DNA-functionalized electrochemical interface. In particular, each viral protease-mediated proteolysis triggers the transcription of multiple RNA outputs, leading to amplified ratiometric signals on the electrochemical interface. Using the NS3/4A protease of the hepatitis C virus as a model, this method achieves robust and specific NS3/4A protease sensing with sub-femtomolar sensitivity. The feasibility of this sensor was demonstrated by monitoring NS3/4A protease activities in virus-infected cell samples with varying viral loads and post-infection times. This study provides a new approach to analyzing viral proteases and holds the potential for developing direct-acting antivirals and novel therapies for viral infections.
Asunto(s)
Técnicas Electroquímicas , Proteolisis , Proteasas Virales/metabolismo , Hepatitis C/enzimología , Técnicas Electroquímicas/métodos , Humanos , Línea CelularRESUMEN
Hepatitis C virus (HCV) life cycle is highly dependent on cellular proteins for viral propagation. In order to identify the cellular factors involved in HCV propagation, we previously performed a protein microarray assay using the HCV nonstructural 5A (NS5A) protein as a probe. Of â¼9,000 human cellular proteins immobilized in a microarray, adenosylhomocysteinase like 1 (AHCYL1) was among 90 proteins identified as NS5A interactors. Of these candidates, AHCYL1 was selected for further study. In the present study, we verified the physical interaction between NS5A and AHCYL1 by both in vitro pulldown and coimmunoprecipitation assays. Furthermore, HCV NS5A interacted with endogenous AHCYL1 in Jc1-infected cells. Both NS5A and AHCYL1 were colocalized in the cytoplasmic region in HCV-replicating cells. siRNAmediated knockdown of AHCYL1 abrogated HCV propagation. Exogenous expression of the siRNA-resistant AHCYL1 mutant, but not of the wild-type AHCYL1, restored HCV protein expression levels, indicating that AHCYL1 was required specifically for HCV propagation. Importantly, AHCYL1 was involved in the HCV internal ribosome entry site-mediated translation step of the HCV life cycle. Finally, we demonstrated that the proteasomal degradation pathway of AHCYL1 was modulated by persistent HCV infection. Collectively, these data suggest that HCV may modulate the AHCYL1 protein to promote viral propagation.
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Hepacivirus/metabolismo , Hepatitis C/enzimología , Proteínas no Estructurales Virales/metabolismo , Hepacivirus/genética , Hepacivirus/crecimiento & desarrollo , Hepatitis C/genética , Hepatitis C/virología , Interacciones Huésped-Patógeno , Humanos , Unión Proteica , Proteínas no Estructurales Virales/genéticaRESUMEN
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused over 1.4 million deaths worldwide. Repurposing existing drugs offers the fastest opportunity to identify new indications for existing drugs as a stable solution against coronavirus disease 2019 (COVID-19). The SARS-CoV-2 main protease (Mpro) is a critical target for designing potent antiviral agents against COVID-19. In this study, we identify potential inhibitors against COVID-19, using an amalgam of virtual screening, molecular dynamics (MD) simulations, and binding-free energy approaches from the Korea Chemical Bank drug repurposing (KCB-DR) database. The database screening of KCB-DR resulted in 149 binders. The dynamics of protein-drug complex formation for the seven top scoring drugs were investigated through MD simulations. Six drugs showed stable binding with active site of SARS-CoV-2 Mpro indicated by steady RMSD of protein backbone atoms and potential energy profiles. Furthermore, binding free energy calculations suggested the community-acquired bacterial pneumonia drug ceftaroline fosamil and the hepatitis C virus (HCV) protease inhibitor telaprevir are potent inhibitors against Mpro. Molecular dynamics and interaction analysis revealed that ceftaroline fosamil and telaprevir form hydrogen bonds with important active site residues such as Thr24, Thr25, His41, Thr45, Gly143, Ser144, Cys145, and Glu166 that is supported by crystallographic information of known inhibitors. Telaprevir has potential side effects, but its derivatives have good pharmacokinetic properties and are suggested to bind Mpro. We suggest the telaprevir derivatives and ceftaroline fosamil bind tightly with SARS-CoV-2 Mpro and should be validated through preclinical testing.
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Tratamiento Farmacológico de COVID-19 , COVID-19/enzimología , Cefalosporinas/química , Proteasas 3C de Coronavirus , Reposicionamiento de Medicamentos , Hepacivirus/enzimología , Hepatitis C/tratamiento farmacológico , Simulación de Dinámica Molecular , Oligopéptidos/química , SARS-CoV-2 , Cefalosporinas/uso terapéutico , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/química , Hepatitis C/enzimología , Humanos , Oligopéptidos/uso terapéutico , SARS-CoV-2/química , SARS-CoV-2/enzimología , CeftarolinaRESUMEN
The nonstructural (NS) protein NS3/4A protease is a critical factor for hepatitis C virus (HCV) maturation that requires activation by NS4A. Synthetic peptide mutants of NS4A were found to inhibit NS3 function. The bridging from peptide inhibitors to heterocyclic peptidomimetics of NS4A has not been considered in the literature and, therefore, we decided to explore this strategy for developing a new class of NS3 inhibitors. In this report, a structure-based design approach was used to convert the bound form of NS4A into 1H-imidazole-2,5-dicarboxamide derivatives as first generation peptidomimetics. This scaffold mimics the buried amino acid sequence Ile-25` to Arg-28` at the core of NS4A21`-33` needed to activate the NS3 protease. Some of the synthesized compounds (Coded MOC) were able to compete with and displace NS4A21`-33` for binding to NS3. For instance, N5-(4-guanidinobutyl)-N2-(n-hexyl)-1H-imidazole-2,5-dicarboxamide (MOC-24) inhibited the binding of NS4A21`-33` with a competition half maximal inhibitory concentration (IC50) of 1.9 ± 0.12 µM in a fluorescence anisotropy assay and stabilized the denaturation of NS3 by increasing the aggregation temperature (40% compared to NS4A21`-33`). MOC-24 also inhibited NS3 protease activity in a fluorometric assay. Molecular dynamics simulations were conducted to rationalize the differences in structure-activity relationship (SAR) between the active MOC-24 and the inactive MOC-26. Our data show that MOC compounds are possibly the first examples of NS4A peptidomimetics that have demonstrated promising activities against NS3 proteins.
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Hepatitis C/enzimología , Péptidos y Proteínas de Señalización Intracelular/química , Peptidomiméticos/química , Inhibidores de Serina Proteinasa/química , Proteínas no Estructurales Virales/química , Peptidomiméticos/síntesis química , Inhibidores de Serina Proteinasa/síntesis química , Proteínas no Estructurales Virales/antagonistas & inhibidoresRESUMEN
Flaviviridae infections represent a major global health burden. By deciphering mechanistic aspects of hepatitis C virus (HCV)-host interactions, one could discover common strategy for inhibiting the replication of related flaviviruses. By elucidating the HCV interactome, we identified the 17-beta-hydroxysteroid dehydrogenase type 12 (HSD17B12) as a human hub of the very-long-chain fatty acid (VLCFA) synthesis pathway and core interactor. Here we show that HSD17B12 knockdown (KD) impairs HCV replication and reduces virion production. Mechanistically, depletion of HSD17B12 induces alterations in VLCFA-containing lipid species and a drastic reduction of lipid droplets (LDs) that play a critical role in virus assembly. Oleic acid supplementation rescues viral RNA replication and production of infectious particles in HSD17B12 depleted cells, supporting a specific role of VLCFA in HCV life cycle. Furthermore, the small-molecule HSD17B12 inhibitor, INH-12, significantly reduces replication and infectious particle production of HCV as well as dengue virus and Zika virus revealing a conserved requirement across Flaviviridae virus family. Overall, the data provide a strong rationale for the advanced evaluation of HSD17B12 inhibition as a promising broad-spectrum antiviral strategy for the treatment of Flaviviridae infections.
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17-Hidroxiesteroide Deshidrogenasas/metabolismo , Hepacivirus/fisiología , Hepatitis C/enzimología , Ácido Oléico/farmacología , Replicación Viral/efectos de los fármacos , 17-Hidroxiesteroide Deshidrogenasas/genética , Animales , Chlorocebus aethiops , Células HeLa , Células Hep G2 , Hepatitis C/genética , Humanos , Células Vero , Replicación Viral/genéticaRESUMEN
Two lignans including a new one, five flavonoids and five coumarins were isolated from the whole plant of Viola philippica (synonymised as Viola yedoensis Makino). The new compound was structurally determined as (7R,8S,8'S) -3,3'-dimethoxy- 4,4',9-trihydroxy- 7,9'-epoxy-8,8'-lignan 9-O-rutinoside by analysis of its NMR, MS and CD spectroscopic data. The known compounds were characterised by comparing their NMR and MS data with those reported. Among the known compounds, 5-hydroxy-4'-methoxyflavone-7-O- rutinoside, 6,7-di-O-ß-D- glucopyranosylesculetin, and 7R,8S-dihydrodehydrodiconiferyl alcohol 4-O-ß-D- glucopyranoside were isolated and identified from this genus for the first time. Of these compounds, 5-hydroxy-4'-methoxyflavone-7-O-rutinoside and (7R,8S,8'S) -3,3'-dimethoxy- 4,4',9-trihydroxy- 7,9'-epoxy-8,8'-lignan 9-O-rutinoside were potently active against α-glucosidase, while the two dimeric coumarins, 5, 5'-bi (6, 7-dihydroxycoumarin) and 6,6',7,7'-tetrahydroxy-5,8'-bicoumarin potently inhibited HCV protease.
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Cumarinas/farmacología , Flavonoides/farmacología , Inhibidores de Glicósido Hidrolasas/farmacología , Hepatitis C/enzimología , Lignanos/farmacología , Inhibidores de Proteasas/farmacología , Viola/química , Dicroismo Circular , Cumarinas/química , Flavonoides/química , Inhibidores de Glicósido Hidrolasas/química , Lignanos/química , Espectroscopía de Resonancia Magnética , Estructura Molecular , Inhibidores de Proteasas/químicaRESUMEN
Sphingosine-1-phospate is a potent bioactive lipid metabolite that regulates cancer progression. Because sphingosine kinase 1 and sphingosine kinase 2 (SPHK 1/2) are both essential for sphingosine-1-phospate production, they could be a therapeutic target in various cancers. Peretinoin, an acyclic retinoid, inhibits post-therapeutic recurrence of hepatocellular carcinoma via unclear mechanisms. In this study, we assessed effects of peretinoin on SPHK expression and liver cancer development in vitro and in vivo. We examined effects of peretinoin on expression, enzymatic and promoter activity of SPHK1 in a human hepatoma cell line, Huh-7. We also investigated effects of SPHK1 on hepatocarcinogenesis induced by diethylnitrosamine using SPHK1 knockout mice. Peretinoin treatment of Huh-7 cells reduced mRNA levels, protein expression and enzymatic activity of SPHK1. Peretinoin reduced SPHK1 promoter activity; this effect of peretinoin was blocked by overexpression of Sp1, a transcription factor. Deletion of all Sp1 binding sites within the SPHK1 promoter region abolished SPHK1 promoter activity, suggesting that peretinoin reduced mRNA levels of SPHK1 via Sp1. Additionally, diethylnitrosamine-induced hepatoma was fewer and less frequent in SPHK1 knockout compared to wild-type mice. Our data showed crucial roles of SPHK1 in hepatocarcinogenesis and suggests that peretinoin prevents hepatocarcinogenesis by suppressing mRNA levels of SPHK1.
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Antineoplásicos/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Retinoides/farmacología , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Dietilnitrosamina/toxicidad , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hepatitis C/tratamiento farmacológico , Hepatitis C/enzimología , Hepatitis C/genética , Humanos , Hígado/metabolismo , Cirrosis Hepática/enzimología , Cirrosis Hepática/genética , Cirrosis Hepática/virología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/genética , Ratones Noqueados , Ratones Transgénicos , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Esfingolípidos/genética , Esfingolípidos/metabolismoRESUMEN
BACKGROUND: The ratio of serum γ-glutamyl transferase (GGT) to alanine aminotransferase (ALT) (GGT/ALT) is a marker for evaluating effects to antivirotic treatment and a helpful predictive factor for the prognosis of Child-Pugh A hepatocellular carcinoma (HCC) patients after surgery. The relationship between the incidence of postoperative acute kidney injury (AKI) and preoperative GGT/ALT is studied in hepatectomized hepatitis B- or C- associated HCC patients. METHODS: A total of 253 hepatitis B or C virus-related HCC patients undergoing hepatectomy between September 2012 and August 2016 at our hospital were included in the retrospective study. Serum ALT and GGT value were recorded, and the GGT/ALT was computed. AKI was defined that based on the "Kidney Disease Improving Global Outcomes (KDIGO) criteria". RESULTS: AKI was observed in 22 (8.7%) patients. Mean GGT/ALT of patients with AKI was significantly higher than in those without it (6.0 vs 2.1, P<0.001). Multivariate analysis revealed an increase in GGT/ALT as an independent risk factor for AKI in hepatitis B- or C- associated HCC patients, particularly in patients with Barcelona Clinic Liver Cancer (BCLC) stage 0 or A staged HCC (odds ratio (OR) 1.400, P<0.001). Multivariate analysis showed that ALT (OR 0.966, P=0.044) was somewhat inversely associated with the incidence of AKI in hepatitis B- or C- associated HCC patients. The best cutoff point of GGT/ALT was 2.92. Multivariate analysis showed that preoperative GGT/ALT ≥2.92 predicted poor prognosis of postoperative AKI in patients with HCC after hepatectomy (odds ratio 17.697, P<0.001). After propensity score matching, preoperative GGT/ALT ≥2.92 remained an independent risk factor for AKI in HCC patients (OR 13.947, P=0.003). CONCLUSIONS: The GGT/ALT of patients with AKI was significantly higher than those without it. Evaluation of GGT/ALT before surgery can be a helpful predictive tool for postoperative AKI in hepatitis B- or C- associated HCC patients undergoing hepatectomy, particularly in patients with BCLC stage 0 or A staged HCC. Hepatitis B- or C- associated HCC patients with low ALT especially within the normal range may have a high risk of AKI. However, the reason remains to be elucidated.
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Lesión Renal Aguda/diagnóstico , Alanina Transaminasa/sangre , Carcinoma Hepatocelular/enzimología , Hepatitis B/enzimología , Hepatitis C/enzimología , Neoplasias Hepáticas/enzimología , gamma-Glutamiltransferasa/sangre , Lesión Renal Aguda/enzimología , Lesión Renal Aguda/etiología , Lesión Renal Aguda/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Carcinoma Hepatocelular/complicaciones , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/cirugía , Femenino , Hepatectomía/efectos adversos , Hepatitis B/complicaciones , Hepatitis B/patología , Hepatitis B/cirugía , Hepatitis C/complicaciones , Hepatitis C/patología , Hepatitis C/cirugía , Humanos , Riñón/enzimología , Riñón/patología , Riñón/cirugía , Neoplasias Hepáticas/complicaciones , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/cirugía , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Periodo Posoperatorio , Pronóstico , Estudios Retrospectivos , Factores de RiesgoRESUMEN
HCV infection can decrease NAD+/NADH ratio, which could convert lipid metabolism to favor HCV replication. In hepatocytes, quinolinate phosphoribosyl transferase (QPRT) catabolizes quinolinic acid (QA) to nicotinic acid mononucleotide (NAMN) for de novo NAD synthesis. However, whether and how HCV modulates QPRT hence the lipogenesis is unknown. In this work, we found QPRT was reduced significantly in livers of patients or humanized C/OTg mice with persistent HCV infection. Mechanistic studies indicated that HCV NS3/4A promoted proteasomal degradation of QPRT through Smurf2, an E3 ubiquitin-protein ligase, in Huh7.5.1 cells. Furthermore, QPRT enzymatic activity involved in suppression of HCV replication in cells. Activation of QPRT with clofibrate (CLO) or addition of QPRT catabolite NAD both inhibited HCV replication in cells, probably through NAD+-dependent Sirt1 inhibition of cellular lipogenesis. More importantly, administration of CLO, a hypolipidemic drug used in clinics, could significantly reduce the viral load in HCV infected C/OTg mice. Take together, these results suggested that HCV infection triggered proteasomal degradation of QPRT and consequently reduced de novo NAD synthesis and lipogenesis, in favor of HCV replication. Hepatic QPRT thus likely served as a cellular factor that dampened productive HCV replication.
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Hepacivirus/fisiología , Hepatitis C/enzimología , Hepatitis C/virología , Pentosiltransferasa/metabolismo , Animales , Línea Celular Tumoral , Humanos , Lipogénesis , Ratones , NAD/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas no Estructurales Virales/metabolismo , Replicación ViralRESUMEN
PURPOSE: Monitoring of hepatitis C virus (HCV) treatment response is performed by serial HCV RNA measurements; however, this may not be useful for predicting treatment success or failure with oral direct-acting antiviral agent (DAA) therapies. Liver enzyme levels, which are elevated in chronic HCV and tend to decline on therapy, may serve as a more logistically and economically feasible alternative for monitoring treatment response. SOURCE: The Ottawa Hospital Viral Hepatitis Clinic patients (n=219), receiving interferon-free oral DAA treatments, were assessed for liver enzymes and HCV RNA levels at baseline, week 4 and ≥12 weeks post-treatment. Suppression cut points used for this analysis were ALT ≤ 40U L-1 and AST ≤ 30U L-1. The primary outcome was week 12 sustained virologic response (SVR). By our analysis, all indicators had strong PPV (>90%) but limited NPV (.
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Antivirales/administración & dosificación , Antivirales/uso terapéutico , Hepacivirus/efectos de los fármacos , Hepacivirus/patogenicidad , Hepatitis C/tratamiento farmacológico , Hepatitis C/enzimología , Administración Oral , Adulto , Femenino , Hepatitis C/patología , Humanos , Hígado/enzimología , Hígado/virología , Masculino , Persona de Mediana Edad , ARN Viral/genética , Resultado del TratamientoRESUMEN
Hepatitis C virus (HCV) subverts the cellular DEAD-box RNA helicase DDX6 to promote virus infection. Using polysome gradient analysis and the subgenomic HCV Renilla reporter replicon genome, we determined that DDX6 does not affect HCV translation. Rather expression of the subgenomic HCV Renilla luciferase reporter at late times, as well as labeling of newly synthesized viral RNA with 4-thiouridine showed that DDX6 modulates replication. Because DDX6 is an effector protein of the microRNA pathway, we also investigated its role in miR-122-directed HCV gene expression. Similar to sequestering miR-122, depletion of DDX6 modulated HCV RNA stability. Interestingly, miR-122-HCV RNA interaction assays with mutant HCV genomes sites and compensatory exogenous miR-122 showed that DDX6 affects the function of miR-122 at one particular binding site. We propose that DDX6 facilitates the miR-122 interaction with HCV 5' UTR, which is necessary for stabilizing the viral genome and the switch between translation and replication.
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ARN Helicasas DEAD-box/metabolismo , Hepacivirus/metabolismo , Hepatitis C/enzimología , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , ARN Viral/metabolismo , Regiones no Traducidas 5' , Línea Celular Tumoral , ARN Helicasas DEAD-box/genética , Hepacivirus/genética , Hepatitis C/genética , Hepatitis C/metabolismo , Hepatitis C/virología , Humanos , MicroARNs/genética , Proteínas Proto-Oncogénicas/genética , ARN Viral/genéticaRESUMEN
Grazoprevir is a potent pan-genotype and macrocyclic inhibitor of hepatitis C virus (HCV) NS3/4A protease and was developed for treating chronic HCV infection. In HCV genotype (GT) 1a, grazoprevir maintains potent activity against a majority of NS3 resistance-associated amino acid substitutions, including the highly prevalent and naturally occurring Q80K polymorphism that impacts simeprevir, another NS3/4A protease inhibitor. The basis for an unexpected difference in the clinical impact of some NS3 substitutions was investigated. Phenotypic analysis of resistance-associated substitutions identified in NS3 from GT1a-infected patients who failed therapy with grazoprevir (in combination with elbasvir, an inhibitor of HCV NS5A protein) showed that positions 56, 156, and 168 in NS3 were most impactful because they diminished protein-inhibitor interactions. Although an amino acid substitution from aspartic acid to alanine at position 168 (D168A) reduced the potency of grazoprevir, its combination with R155K unexpectedly nullified this effect. Molecular dynamics and free-energy surface studies indicated that Asp-168 is important in anchoring Arg-155 for ligand binding but is not critical for Lys-155 because of the inherent flexibility of its side chain. Moreover, modeling studies supported a strong direct cation-heterocycle interaction between the Lys-155 side chain of the double substitution, R155K/D168A, and the lone pair on the quinoxaline in grazoprevir. This unique interaction provides a structural basis for grazoprevir's higher potency than simeprevir, an inhibitor to which the double substitution confers a significant reduction in potency. Our findings are consistent with the detection of R155K/D168A in NS3 from virologic failures treated with simeprevir but not grazoprevir.
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Hepacivirus/enzimología , Simulación de Dinámica Molecular , Mutación Missense , Quinoxalinas/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Amidas , Sustitución de Aminoácidos , Carbamatos , Línea Celular Tumoral , Ciclopropanos , Hepacivirus/genética , Hepatitis C/tratamiento farmacológico , Hepatitis C/enzimología , Hepatitis C/genética , Humanos , Quinoxalinas/uso terapéutico , Simeprevir/química , Simeprevir/uso terapéutico , Sulfonamidas , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
From the earliest times, medicine has focused on finding the most suitable and effective treatment for every patient. At present, a dynamic development of diagnostic methods and techniques for designing new drugs allows to create therapies for many diseases at the molecular level. Among the many drugs appearing on the medical market every year, special attention should be paid to those whose action is based on the inhibition of proteolytic enzyme activity. Protease inhibitors are a diverse group of biologically active molecules for which antiviral, antimicrobial, antifungal, antiparasitic or anticancer effects have been documented. Successes in the treatment of HIV infection, hepatitis C and influenza diseases certainly encourage researchers to look for new inhibitors that could be used in new therapies. This paper provides an overview of selected information on enzyme inhibitors, especially protease inhibitors, which are already registered medicines and substances that are promising candidates for medical use.
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Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/uso terapéutico , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Antivirales/farmacología , Antivirales/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/enzimología , Hepatitis C/tratamiento farmacológico , Hepatitis C/enzimología , Humanos , Gripe Humana/tratamiento farmacológico , Gripe Humana/enzimologíaRESUMEN
Nucleic acid amplification test (NAT), which was introduced by the Japanese Red Cross Society in October 1999, began to be performed for screening of blood transfusion formulations in Japan in August 2014. In this study, the precision of immunological screenings of hepatitis B (HBsAg, HBcAb, and HBsAb), hepatitis C (HCVAb), and human immunodeficiency (HIVAb) virus antigens in donated blood were evaluated. In addition, the sensitivity of the alanine aminotransferase (ALT) test for detection of the hepatitis B and C viruses was re-evaluated. Immunological screenings showed high precision of detecting the viral antigens. In contrast, the ALT test showed much lower precision of detecting the presence of the hepatitis B and C viruses. Results of the NAT and immunological screenings revealed that ALT levels in donors were more strongly correlated with their levels of gammaglutamyltranspeptidase (γGTP) and body mass index (BMI), than with the results of NAT and immunological screening. Our study indicates that elevated level(s) of ALT, were more likely to be associated with lifestyles factors such as high intake of alcohol or obesity than with infection. Therefore, ALT may be excluded as surrogate markers of HBV, HCV, and HIV in donated blood.
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Alanina Transaminasa/sangre , Hepatitis B/sangre , Hepatitis C/sangre , Virus de Hepatitis/aislamiento & purificación , Adulto , Biomarcadores/sangre , Femenino , Anticuerpos Antihepatitis/sangre , Antígenos de la Hepatitis/sangre , Hepatitis B/enzimología , Hepatitis B/inmunología , Hepatitis B/virología , Hepatitis C/enzimología , Hepatitis C/inmunología , Hepatitis C/virología , Humanos , Masculino , Persona de Mediana Edad , Técnicas de Amplificación de Ácido Nucleico , Curva ROC , Análisis de RegresiónRESUMEN
Domain II of the nonstructural protein 5 (NS5A) of the hepatitis C virus (HCV) is involved in intermolecular interactions with the viral RNA genome, the RNA-dependent RNA polymerase NS5B, and the host factor cyclophilin A (CypA). However, domain II of NS5A (NS5ADII) is largely disordered, which makes it difficult to characterize the protein-protein or protein-nucleic acid interfaces. Here we utilized a mass spectrometry-based protein footprinting approach in attempts to characterize regions forming contacts between NS5ADII and its binding partners. In particular, we compared surface topologies of lysine and arginine residues in the context of free and bound NS5ADII. These experiments have led to the identification of an RNA binding motif (305RSRKFPR311) in an arginine-rich region of NS5ADII. Furthermore, we show that K308 is indispensable for both RNA and NS5B binding, whereas W316, further downstream, is essential for protein-protein interactions with CypA and NS5B. Most importantly, NS5ADII binding to NS5B involves a region associated with RNA binding within NS5B. This interaction down-regulated RNA synthesis by NS5B, suggesting that NS5ADII modulates the activity of NS5B and potentially regulates HCV replication.
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Ciclofilina A/metabolismo , Hepacivirus/metabolismo , Hepatitis C/enzimología , Hepatitis C/virología , ARN Viral/metabolismo , Proteínas no Estructurales Virales/metabolismo , Secuencias de Aminoácidos , Ciclofilina A/genética , Regulación Viral de la Expresión Génica , Hepacivirus/química , Hepacivirus/genética , Hepatitis C/genética , Interacciones Huésped-Patógeno , Humanos , Unión Proteica , Dominios Proteicos , ARN Viral/genética , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genéticaRESUMEN
BACKGROUND: Mannan-binding lectin (MBL) - associated serine protease 2 (MASP-2) co-activates the lectin pathway of complement in response to several viral infections. The quality of this response partly depends on MASP2 gene polymorphisms, which modulate MASP-2 function and serum levels. In this study we investigated a possible role of MASP2 polymorphisms, MASP-2 serum levels and MBL-mediated complement activation in the susceptibility to HIV/AIDS and HBV/HCV coinfection. METHODS: A total of 178 HIV patients, 89 (50%) coinfected with HBV/HCV, 51.7% female, average age 40 (12-73) years, and 385 controls were evaluated. MASP-2 levels and MBL-driven complement activation were evaluated by enzyme-linked immunosorbent assay and 11 MASP2 polymorphisms from the promoter to the last exon were haplotyped using multiplex sequence-specific PCR. RESULTS: Genotype distribution was in Hardy-Weinberg equilibrium and differed between HIV+ patients and controls (P=0.030), irrespective of HBV or HCV coinfection. The p.126L variant, which was associated with MASP-2 levels <200ng/mL (OR=5.0 [95%CI=1.3-19.2] P=0.019), increased the susceptibility to HIV infection (OR=5.67 [95%CI=1.75-18.33], P=0.004) and to HIV+HBV+ status (OR=6.44 [95%CI=1.69-24.53, P=0.006). A similar association occurred with the ancient haplotype harboring this variant, AGCDV (OR=2.35 [95%CI=1.31-4.23], P=0.004). On the other hand, p.126L in addition to other variants associated with low MASP-2 levels-p.120G, p.377A and p.439H, presented a protective effect against AIDS (OR=0.25 [95%CI=0.08-0.80], P=0.020), independently of age, sex, hepatic function and viral load. MASP-2 serum levels were lower in HIV+ and HIV+HBV+ patients than in controls (P=0.0004). Among patients, MASP-2 levels were higher in patients with opportunistic diseases (P=0.001) and AIDS (P=0.004). MASP-2 levels correlated positively with MBL/MASP2-mediated C4 deposition (r=0.29, P=0.0002) and negatively with CD4+ cell counts (r=-0.21, P=0.018), being related to decreased CD4+ cell counts (OR=5.8 [95%CI=1.23-27.5, P=0.026). CONCLUSIONS: Genetically determined MASP-2 levels seem to have a two-edge effect in HIV and probably HCV/HBV coinfection, whereas low levels increase the susceptibility to infection, but on the other side protects against AIDS.
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Infecciones por VIH/genética , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/genética , Síndrome de Inmunodeficiencia Adquirida/enzimología , Síndrome de Inmunodeficiencia Adquirida/genética , Síndrome de Inmunodeficiencia Adquirida/inmunología , Adolescente , Adulto , Anciano , Niño , Coinfección/enzimología , Coinfección/genética , Coinfección/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Predisposición Genética a la Enfermedad/genética , Genotipo , Infecciones por VIH/enzimología , Infecciones por VIH/inmunología , Hepatitis B/enzimología , Hepatitis B/genética , Hepatitis B/inmunología , Hepatitis C/enzimología , Hepatitis C/genética , Hepatitis C/inmunología , Humanos , Masculino , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/inmunología , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa Multiplex , Polimorfismo de Nucleótido Simple , Adulto JovenAsunto(s)
Antivirales/farmacología , Hepatitis C/tratamiento farmacológico , Janus Quinasa 2/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Antivirales/química , Productos Biológicos/química , Productos Biológicos/farmacología , Línea Celular , Diseño de Fármacos , Descubrimiento de Drogas , Hepacivirus/enzimología , Hepacivirus/genética , Hepatitis C/enzimología , Hepatitis C/virología , Hong Kong , Humanos , Simulación del Acoplamiento Molecular , Inhibidores de Proteínas Quinasas/química , Relación Estructura-ActividadRESUMEN
AIM: To elucidate the mechanism(s) by which S-adenosyl-L-methionine (SAM) decreases hepatitis C virus (HCV) expression. METHODS: We examined the effects of SAM on viral expression using an HCV subgenomic replicon cell culture system. Huh7 HCV-replicon cells were treated with 1 mmol/L SAM for different times (24-72 h), then total RNA and proteins were isolated. cDNA was synthesized and real time-PCR was achieved to quantify HCV-RNA, superoxide dismutase 1 and 2 (SOD-1, SOD-2) catalase, thioredoxin 1, methionine adenosyltransferase 1A and 2A (MAT1A, MAT2A) expression, and GAPDH and RPS18 as endogenous genes. Expression of cellular and viral protein was evaluated by western-blot analysis using antibodies vs HCV-NS5A, SOD-1, SOD-2, catalase, thioredoxin-1, MAT1A, MAT2A, GAPDH and actin. Total glutathione levels were measured at different times by Ellman's recycling method (0-24 h). Reactive oxidative species (ROS) levels were quantified by the dichlorofluorescein assay (0-48 h); Pyrrolidin dithiocarbamate (PDTC) was tested as an antioxidant control and H2O2 as a positive oxidant agent. RESULTS: SAM exposition decreased HCV-RNA levels 50%-70% compared to non-treated controls (24-72 h). SAM induced a synergic antiviral effect with standard IFN treatment but it was independent of IFN signaling. In addition, 1 mmol/L SAM exposition did not modify viral RNA stability, but it needs cellular translation machinery in order to decrease HCV expression. Total glutathione levels increased upon SAM treatment in HCV-replicon cells. Transcriptional antioxidant enzyme expression (SOD-1, SOD-2 and thioredoxin-1) was increased at different times but interestingly, there was no significant change in ROS levels upon SAM treatment, contrary to what was detected with PDTC treatment, where an average 40% reduction was observed in exposed cells. There was a turnover from MAT1A/MAT2A, since MAT1A expression was increased (2.5 fold-times at 48 h) and MAT2A was diminished (from 24 h) upon SAM treatment at both the transcriptional and translational level. CONCLUSION: A likely mechanism(s) by which SAM diminish HCV expression could involve modulating antioxidant enzymes, restoring biosynthesis of glutathione and switching MAT1/MAT2 turnover in HCV expressing cells.
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Antioxidantes/metabolismo , Antivirales/farmacología , Glutatión/biosíntesis , Hepacivirus/efectos de los fármacos , Hepatitis C/tratamiento farmacológico , Hepatocitos/efectos de los fármacos , Metionina Adenosiltransferasa/metabolismo , S-Adenosilmetionina/farmacología , Replicación Viral/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hepacivirus/genética , Hepacivirus/crecimiento & desarrollo , Hepatitis C/enzimología , Hepatitis C/genética , Hepatocitos/enzimología , Interacciones Huésped-Patógeno , Humanos , Metionina Adenosiltransferasa/genética , Estrés Oxidativo/efectos de los fármacos , ARN Viral/biosíntesis , Factores de Tiempo , TransfecciónRESUMEN
Hepatitis C virus (HCV) activates PI3K-Akt signaling to enhance entry and replication. Here, we found that this pathway also increased HCV translation. Knocking down the three Akt isoforms significantly decreased, whereas ectopic expression increased HCV translation. HCV translation upregulation by Akt required their kinase activities because Akt kinase-dead mutants downregulated HCV translation; and was dependent on PI3K activity since it was sensitive to PI3K inhibitor wortmannin. The viral 3'UTR was not involved in translation upregulation by Akt. HCV NS5A increased Akt phosphorylation/activity and HCV translation in the absence of the viral 3'UTR. Sterol regulatory element-binding proteins (SREBPs) were the downstream effectors of the PI3K-Akt pathway in regulating HCV translation because Akt1 and Akt2 activated both SREBP-1 and SREBP-2, whereas Akt3 upregulated SREBP-1. Knocking down SREBPs significantly decreased, while ectopic expression of SREBPs increased HCV translation. Taken together, we showed that the PI3K-Akt signaling pathway positively regulates HCV translation through SREBPs.
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Hepacivirus/genética , Hepatitis C/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Viral/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Hepacivirus/metabolismo , Hepatitis C/enzimología , Hepatitis C/genética , Hepatitis C/virología , Interacciones Huésped-Patógeno , Humanos , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , ARN Viral/metabolismo , Transducción de Señal , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genética , Regulación hacia ArribaRESUMEN
GBF1 is a host factor required for hepatitis C virus (HCV) replication. GBF1 functions as a guanine nucleotide exchange factor for G-proteins of the Arf family, which regulate membrane dynamics in the early secretory pathway and the metabolism of cytoplasmic lipid droplets. Here we established that the Arf-guanine nucleotide exchange factor activity of GBF1 is critical for its function in HCV replication, indicating that it promotes viral replication by activating one or more Arf family members. Arf involvement was confirmed with the use of two dominant negative Arf1 mutants. However, siRNA-mediated depletion of Arf1, Arf3 (class I Arfs), Arf4 or Arf5 (class II Arfs), which potentially interact with GBF1, did not significantly inhibit HCV infection. In contrast, the simultaneous depletion of both Arf4 and Arf5, but not of any other Arf pair, imposed a significant inhibition of HCV infection. Interestingly, the simultaneous depletion of both Arf4 and Arf5 had no impact on the activity of the secretory pathway and induced a compaction of the Golgi and an accumulation of lipid droplets. A similar phenotype of lipid droplet accumulation was also observed when GBF1 was inhibited by brefeldin A. In contrast, the simultaneous depletion of both Arf1 and Arf4 resulted in secretion inhibition and Golgi scattering, two actions reminiscent of GBF1 inhibition. We conclude that GBF1 could regulate different metabolic pathways through the activation of different pairs of Arf proteins.