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Clinical phenotypes of familial hypobetalipoproteinemia (FHBL) are related to a number of defective apolipoprotein B (APOB) alleles. Fatty liver disease is a typical manifestation, but serious neurological symptoms can appear. In this study, genetic analysis of the APOB gene and ophthalmological diagnostics were performed for family members with FHBL. Five relatives with FHBL, including a proband who developed neurological disorders, were examined. A sequencing analysis of the whole coding region of the APOB gene, including flanking intronic regions, was performed using the next-generation sequencing (NGS) method. Electrophysiological ophthalmological examinations were also done. In the proband and his affected relatives, NGS identified the presence of the pathogenic, rare heterozygous splicing variant c.3696+1G>T. Two known heterozygous missense variants-c.2188G>A, p.(Val730Ile) and c.8353A>C, p.(Asn2785His)-in the APOB gene were also detected. In all patients, many ophthalmologic abnormalities in electrophysiological tests were also found. The identified splicing variant c.3696+1G>T can be associated with observed autosomal, dominant FHBL with coexisting neurological symptoms, and both identified missense variants could be excluded as the main cause of observed clinical signs, according to mutation databases and the literature. Electroretinography examination is a sensitive method for the detection of early neuropathy and should therefore be recommended for the care of patients with FHBL.
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Apolipoproteína B-100 , Hipobetalipoproteinemia Familiar por Apolipoproteína B , Mutación Missense , Enfermedades del Sistema Nervioso , Empalme del ARN , Adulto , Anciano , Sustitución de Aminoácidos , Apolipoproteína B-100/genética , Apolipoproteína B-100/metabolismo , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Hipobetalipoproteinemia Familiar por Apolipoproteína B/diagnóstico por imagen , Hipobetalipoproteinemia Familiar por Apolipoproteína B/genética , Hipobetalipoproteinemia Familiar por Apolipoproteína B/metabolismo , Masculino , Enfermedades del Sistema Nervioso/diagnóstico por imagen , Enfermedades del Sistema Nervioso/genética , Enfermedades del Sistema Nervioso/metabolismoRESUMEN
OBJECTIVE: To comprehensively describe the new syndrome of myoclonus epilepsy and ataxia due to potassium channel mutation (MEAK), including cellular electrophysiological characterization of observed clinical improvement with fever. METHODS: We analyzed clinical, electroclinical, and neuroimaging data for 20 patients with MEAK due to recurrent KCNC1 p.R320H mutation. In vitro electrophysiological studies were conducted using whole cell patch-clamp to explore biophysical properties of wild-type and mutant KV 3.1 channels. RESULTS: Symptoms began at between 3 and 15 years of age (median = 9.5), with progressively severe myoclonus and rare tonic-clonic seizures. Ataxia was present early, but quickly became overshadowed by myoclonus; 10 patients were wheelchair-bound by their late teenage years. Mild cognitive decline occurred in half. Early death was not observed. Electroencephalogram (EEG) showed generalized spike and polyspike wave discharges, with documented photosensitivity in most. Polygraphic EEG-electromyographic studies demonstrated a cortical origin for myoclonus and striking coactivation of agonist and antagonist muscles. Magnetic resonance imaging revealed symmetrical cerebellar atrophy, which appeared progressive, and a prominent corpus callosum. Unexpectedly, transient clinical improvement with fever was noted in 6 patients. To explore this, we performed high-temperature in vitro recordings. At elevated temperatures, there was a robust leftward shift in activation of wild-type KV 3.1, increasing channel availability. INTERPRETATION: MEAK has a relatively homogeneous presentation, resembling Unverricht-Lundborg disease, despite the genetic and biological basis being quite different. A remarkable improvement with fever may be explained by the temperature-dependent leftward shift in activation of wild-type KV 3.1 subunit-containing channels, which would counter the loss of function observed for mutant channels, highlighting KCNC1 as a potential target for precision therapeutics. Ann Neurol 2017;81:677-689.
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Ataxia , Disfunción Cognitiva/etiología , Epilepsias Mioclónicas , Calor , Canales de Potasio Shaw/metabolismo , Adolescente , Adulto , Edad de Inicio , Ataxia/complicaciones , Ataxia/diagnóstico por imagen , Ataxia/genética , Ataxia/fisiopatología , Electroencefalografía , Epilepsias Mioclónicas/complicaciones , Epilepsias Mioclónicas/diagnóstico por imagen , Epilepsias Mioclónicas/genética , Epilepsias Mioclónicas/fisiopatología , Femenino , Células HEK293 , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Mutación , Linaje , Canales de Potasio Shaw/genética , Síndrome , Adulto JovenRESUMEN
Advances in gene therapy and genome editing give hope that new treatments will soon be available for inherited eye diseases that together affect a significant proportion of the adult population. New solutions are needed to make genetic diagnosis fast and affordable. This is the first study of such a large group of patients with inherited retinal dystrophies (IRD) and inherited optic neuropathies (ION) in the Polish population. It is based on four years of diagnostic analysis using a broad, targeted NGS approach. The results include the most common pathogenic variants, as well as 91 novel causative variants, including frameshifts in the cumbersome RPGR ORF15 region. The high frequency of the ABCA4 complex haplotype p.(Leu541Pro;Ala1038Val) was confirmed. Additionally, a deletion of exons 22-24 in USH2A, probably specific to the Polish population, was uncovered as the most frequent copy number variation. The diagnostic yield of the broad NGS panel reached 64.3% and is comparable to the results reported for genetic studies of IRD and ION performed for other populations with more extensive WES or WGS methods. A combined approach to identify genetic causes of all known diseases manifesting in the posterior eye segment appears to be the optimal choice given the currently available treatment options and advanced clinical trials.
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PURPOSE: Age-related macular degeneration (AMD) is a complex eye disorder affecting millions worldwide. This article uses deep learning techniques to investigate the relationship between AMD, genetics and optical coherence tomography (OCT) scans. METHODS: The cohort consisted of 332 patients, of which 235 were diagnosed with AMD and 97 were controls with no signs of AMD. The genome-wide association studies summary statistics utilized to establish the polygenic risk score (PRS) in relation to AMD were derived from the GERA European study. A PRS estimation based on OCT volumes for both eyes was performed using a proprietary convolutional neural network (CNN) model supported by machine learning models. The method's performance was assessed using numerical evaluation metrics, and the Grad-CAM technique was used to evaluate the results by visualizing the features learned by the model. RESULTS: The best results were obtained with the CNN and the Extra Tree regressor (MAE = 0.55, MSE = 0.49, RMSE = 0.70, R2 = 0.34). Extending the feature vector with additional information on AMD diagnosis, age and smoking history improved the results slightly, with mainly AMD diagnosis used by the model (MAE = 0.54, MSE = 0.44, RMSE = 0.66, R2 = 0.42). Grad-CAM heatmap evaluation showed that the model decisions rely on retinal morphology factors relevant to AMD diagnosis. CONCLUSION: The developed method allows an efficient PRS estimation from OCT images. A new technique for analysing the association of OCT images with PRS of AMD, using a deep learning approach, may provide an opportunity to discover new associations between genotype-based AMD risk and retinal morphology.
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BACKGROUND: Age-related macular degeneration (AMD) is a multifactorial disease encompassing a complex interaction between aging, environmental risk factors, and genetic susceptibility. The study aimed to determine whether there is a relationship between the polygenic risk score (PRS) in patients with AMD and the characteristics of the retinal vascular network visualized by optical coherence tomography angiography (OCTA). METHODS: 235 patients with AMD and 97 healthy controls were included. We used data from a previous AMD PRS study with the same group. The vascular features from different retina layers were compared between the control group and the patients with AMD. The association between features and PRS was then analyzed using univariate and multivariate approaches. RESULTS: Significant differences between the control group and AMD patients were found in the vessel diameter distribution (variance: p = 0.0193, skewness: p = 0.0457) and fractal dimension distribution (mean: p = 0.0024, variance: p = 0.0123). Both univariate and multivariate analyses showed no direct and significant association between the characteristics of the vascular network and AMD PRS. CONCLUSIONS: The vascular features of the retina do not constitute a biomarker of the risk of AMD. We have not identified a genotype-phenotype relationship, and the expression of AMD-related genes is perhaps not associated with the characteristics of the retinal vascular network.
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A subset of ophthalmic imaging examination results from 334 patients were subjected to reanalysis to identify a specific group of patients with pigment epithelial detachment (PED) in at least one eye. Overall, we found a subgroup of 47 patients manifesting PED and studied their genotypes in comparison to those of patients with age-related macular degeneration without PED and healthy controls. We established a polygenic risk score that allowed the explanation of 16.3% of the variation within the disease. The highest predictive value was achieved for a model consisting of six non-coding variants: rs760306 (BEST1), rs148662546 (BEST1), rs11569560 (C3), rs74600252 (GUCA1B), rs2240688 (PROM1), and rs185507582 (TCF4). The risk of PED occurrence was found to be the highest in the first tercile, showing a 7.89-fold higher risk compared to the third tercile for AMD without PED (95% CI: 2.87; 21.71, p < 0.001) and a 7.22-fold higher risk compared to the healthy controls (95% CI: 2.60; 20.06, p < 0.001). In addition, we focused on rare variants in targeted genes. The rare variants' burden was compared among the groups, but no statistical significance was observed in the number of rare variants, predicted functional effects, or pathogenicity classification.
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Degeneración Macular , Desprendimiento de Retina , Humanos , Epitelio Pigmentado de la Retina , Degeneración Macular/genética , Desprendimiento de Retina/genética , Factores de Riesgo , BestrofinasRESUMEN
The aim of the present study was to analyze the relationship of age-related macular degeneration (AMD) progression with clinical characteristics, demographic, and environmental risk factors that would affect disease development. In addition, the influence of three genetic AMD polymorphisms (CFH Y402H, ARMS2 A69S, and PRPH2 c.582-67T>A) on AMD progression was investigated. In total, 94 participants with previously diagnosed early or intermediate AMD in at least one eye were recalled for an updated re-evaluation after 3 years. The initial visual outcomes, medical history, retinal imaging data, and choroidal imaging data were collected to characterize the AMD disease status. Among the AMD patients, 48 demonstrated AMD progression, and 46 showed no disease worsening at 3 years. Disease progression was significantly associated with worse initial visual acuity (OR = 6.74, 95% CI = 1.24-36.79, p = 0.03) and the presence of the wet AMD subtype in fellow eyes (OR = 3.79, 95%CI = 0.94-15.2, p = 0.05). In addition, a higher risk of AMD progression appeared in the patients with active thyroxine supplementation (OR = 4.77, CI = 1.25-18.25, p = 0.002). The CC variant of CFH Y402H was associated with AMD advancement compared to the TC+TT phenotype (OR = 2.76, 95% CI: 0.98-7.79, p = 0.05). Identifying risk factors of AMD progression may lead to earlier intervention and better outcomes, preventing the expansion of the late stage of the disease.
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Age-related macular degeneration (AMD) is a common retina degenerative disease with a complex genetic and environmental background. This study aimed to determine the polygenic risk score (PRS) stratification between the AMD case and control patients. The PRS model was established on the targeted sequencing data of a cohort of 471 patients diagnosed with AMD and 167 healthy controls without symptoms of retinal degeneration. The highest predictive value to the target dataset was achieved for a 22-variant model with a p-value lower than threshold PT = 0.0123. The median PRS for cases was higher by 1.1 than for control samples (95% CI: (−1.19; −0.85)). The patients in the highest quantile had a significantly higher relative risk of developing AMD than those in the lowest reference quantile (OR = 35.13, 95% CI: (7.9; 156.1), p < 0.001). The diagnostic ability was investigated using ROC analysis with AUC = 0.76 (95% CI: (0.72; 0.80)). The polygenic susceptibility to AMD may be the starting point to expand AMD diagnostics based on rare highly penetrant variants and investigate associations with disease progression and treatment response in Polish patients in future studies.
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We synthesized new tropolone derivatives substituted with cyclic amines: piperidine, piperazine or pyrrolidine. The most active anti-helicase compound (IC50=3.4 microM), 3,5,7-tri[(4'-methylpiperazin-1'-yl)methyl]tropolone (2), inhibited RNA replication by 50% at 46.9 microM (EC50) and exhibited the lowest cytotoxicity (CC50)>1 mM resulting in a selectivity index (SI=CC50/EC50)>21. The most efficient replication inhibitor, 3,5,7-tri[(4'-methylpiperidin-1'-yl)methyl]tropolone (6), inhibited RNA replication with an EC50 of 32.0 microM and a SI value of 17.4, whereas 3,5,7-tri[(3'-methylpiperidin-1'-yl)methyl]tropolone (7) exhibited a slightly lower activity with an EC50 of 35.6 microM and a SI of 9.8.
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Antivirales/química , Antivirales/farmacología , ADN Helicasas/metabolismo , Hepacivirus/efectos de los fármacos , Tropolona/análogos & derivados , Tropolona/farmacología , Replicación Viral/efectos de los fármacos , Antivirales/síntesis química , Línea Celular Tumoral , ADN Helicasas/antagonistas & inhibidores , ADN Helicasas/química , Sinergismo Farmacológico , Hepacivirus/enzimología , Hepatitis C/tratamiento farmacológico , Humanos , Interferón gamma/farmacología , Modelos Moleculares , Mutación , ARN Helicasas/antagonistas & inhibidores , ARN Helicasas/química , ARN Helicasas/metabolismo , ARN Viral/antagonistas & inhibidores , ARN Viral/metabolismo , Ribavirina/farmacología , Tropolona/síntesis química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismoRESUMEN
The non-structural protein 3 (NS3) of Hepatitis C virus (HCV) is a bifunctional enzyme with RNA-dependent NTPase/RNA helicase and serine protease activities, and thus represents a promising target for anti-HCV therapy. These functions are performed by two distinct moieties; the N-terminal protease domain and the C-terminal helicase domain that further folds into three structural subdomains. To obtain lower molecular mass proteins suitable for nuclear magnetic resonance studies of helicase-inhibitor complexes, helicase domains 1, 2, and 1+2 devoid of a hydrophobic beta-loop were overexpressed and purified. Circular dichroism studies were carried out to confirm the secondary structure content and to determine thermodynamic parameters describing the stability of the proteins. Both thermal and GuHCl-induced unfolding experiments confirmed the multidomain organization of the helicase. The unfolding transition observed for domain 1+2 was in agreement with the model of two well-resolved successive steps corresponding to the independent unfolding of domains 1 and 2, respectively. In the case of the full-length helicase, the presence of domain 3 remarkably changed the transition profile, leading to fast and irreversible transformation of partially unfolded protein.
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Hepacivirus/genética , Proteínas no Estructurales Virales/química , Dicroismo Circular , Clonación Molecular , Calor , Espectroscopía de Resonancia Magnética , Conformación Molecular , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , ARN Helicasas/química , TermodinámicaRESUMEN
A new class of compounds--acridone derivatives--was tested using the direct fluorometric helicase activity assay to determine the inhibitory properties of the derivatives towards the NS3 helicase of Hepatitis C virus (HCV). The compounds were also tested as putative transcription inhibitors of in vitro transcription based on the DNA-dependent T7 RNA polymerase. Most of the acridone derivatives tested were transcription inhibitors; however, only four of them inhibited the NS3 helicase at low concentrations (IC(50) from 3 microM to 20 microM) and were therefore selected for further studies on the mechanism of inhibition. The acridone derivatives probably act via intercalation into double-stranded nucleic acids but they may also interact directly with viral enzymes. Selected carboxamides were tested in the subgenomic HCV replicon system. Two of the compounds: N-(pyridin-4-yl)-amide and N-(pyridin-2-yl)-amide of acridone-4-carboxylic acid are efficient RNA replication inhibitors with selectivity indexes of 19.4 and 40.5, respectively, proving that the acridone derivatives may be regarded as potential antiviral agents.
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Acridonas/farmacología , Antivirales/farmacología , Ácidos Carboxílicos/farmacología , Inhibidores Enzimáticos/farmacología , Hepacivirus/efectos de los fármacos , Virus ARN/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Acridonas/síntesis química , Antivirales/síntesis química , Ácidos Carboxílicos/síntesis química , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Fluorometría , Hepacivirus/enzimología , Concentración 50 Inhibidora , ARN Helicasas/antagonistas & inhibidores , ARN Helicasas/metabolismo , Relación Estructura-ActividadRESUMEN
The non-structural protein 3 (NS3) of hepatitis C virus (HCV) is a highly promising target for anti-HCV therapy because of its multiple enzymatic activities, such as RNA-stimulated nucleoside triphosphatase, RNA helicase and serine protease. The helicase domain of NS3 as well as domain 2 of the helicase were expressed in a baculovirus system to obtain in high yield active proteins for prospective studies of complexes of the helicase with its inhibitors. A novel direct fluorometric test of helicase activity with a quenched DNA substrate, 3' labeled with a Cy3 dye and 5' labeled with a Black Hole Quencher, was developed and optimal reaction conditions established. This test based on fluorescence resonance energy transfer is simple and fast. It allows for direct measurements of enzyme activity, circumventing laborious and complicated radioactive techniques that are poorly reproducible. The results obtained encourage us to propose this new fluorescent assay as a method enabling high throughput screening of anti-helicase compounds.
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Fluorometría/métodos , Hepacivirus/enzimología , Proteínas no Estructurales Virales/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Baculoviridae/genética , Dominio Catalítico , Línea Celular , Magnesio/química , Magnesio/metabolismo , Manganeso/química , Manganeso/metabolismo , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spodoptera/citología , Especificidad por Sustrato , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genéticaRESUMEN
A new goup of acridone derivatives, obtained by reaction of acridone-4-carboxylic acid derivatives with aromatic amines, was tested to determine the inhibitory properties toward the NS3 helicase of hepatitis C virus (HCV). Six compounds inhibited the NS3 helicase at low concentrations (IC(50) from 1.5 to 20 microM). The acridone derivatives probably act via intercalation into double-stranded nucleic acids with a strong specificity for double-stranded RNA, although an interaction with the enzyme cannot be excluded. Testing in the subgenomic HCV replicon system revealed that compounds 10 and 13 are efficient RNA replication inhibitors, with EC(50) of 3.5 and 1 microM and therapeutic indexes of >28 and 20, respectively. Compound 16, with EC(50) < 1 microM and TI > 1000, is extremely specific and practically noncytotoxic at the concentrations tested, proving that the acridone derivatives may be regarded as potential antiviral agents. Although the mechanism of action of 16 in the replicon system remains unclear, it is the key lead compound for further development of anti-HCV drugs.
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Acridonas/síntesis química , Antivirales/síntesis química , Hepacivirus/efectos de los fármacos , ARN Helicasas/antagonistas & inhibidores , Proteínas no Estructurales Virales/antagonistas & inhibidores , Acridonas/farmacología , Acridonas/toxicidad , Antivirales/farmacología , Antivirales/toxicidad , Línea Celular Tumoral , Farmacorresistencia Viral , Hepacivirus/genética , Hepacivirus/fisiología , Humanos , Modelos Moleculares , Mutación , ARN Viral/genética , Replicón , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
The development of techniques based on fluorescence has made it possible to create new types of assays that represent an advantageous alternative to old tests relying on radioactivity. Such a novel approach has been applied to develop a high-throughput assay to measure the helicase activity of the hepatitis C virus (HCV) NS3 protein and the inhibitory potential of several classes of compounds. The NS3 helicase is one of the most promising targets of anti-HCV-oriented screening of compounds due to the urgent need for more effective and tolerable drugs. The 96- or 384-well microplate assay that we developed is based on the use of a quenched double-stranded DNA substrate labeled with a fluorophore (Cy3 or FAM) and with a Black Hole Quencher 1 or 2. It allows for direct (real-time) measurements of substrate unwinding and inhibition of unwinding by anti-helicase compounds. After a few modifications of buffers and assay conditions this method can be applied to various variants of HCV helicase and other proteins with helicase activities.
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Bioensayo/métodos , Fluorometría/métodos , Proteínas no Estructurales Virales/metabolismo , Bioensayo/instrumentación , ADN/química , ADN/genética , ADN/metabolismo , Fluorometría/instrumentación , Genoma Viral , Conformación de Ácido Nucleico , Proteínas no Estructurales Virales/antagonistas & inhibidoresRESUMEN
Hepatitis C virus (HCV) infections represent one of the major and still unresolved health problems because current therapy is effective in only 50-80% of cases, depending on viral genotype. A large group of amidinoanthracyclines, with decreased acute toxicity and cardiotoxicity compared to the parent antibiotics, was tested in a high-throughput fluorometric HCV helicase assay. Here, we report the selection of more than 50 potent inhibitors of helicase activity that inhibit the enzyme with IC(50) values in the range of 0.03- 10 mum; four of these compounds are the most potent inhibitors of helicase activity described in the literature. The activity of these inhibitors is highly dependent on their chemical structure, mainly on the substituent at the amidino carbon atom and on the orientation of the hydroxyl group at the 4 inch position of the daunosamine moiety. The most effective compounds act not solely via intercalation into the double-stranded DNA substrate, but also compete with the enzyme for access to the substrate, impeding formation of the active helicase complex. Selected amidinoanthracyclines were tested in the subgenomic HCV replicon system. These experiments confirmed the antiviral activity of two selected inhibitors (EC(50) values below 0.2 mum with selectivity indices of 19 and 33) and proved that they may be considered as potential anti-HCV drugs.
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Amidas/química , Antraciclinas/química , Antraciclinas/farmacología , Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Amidas/metabolismo , Antraciclinas/metabolismo , Línea Celular Tumoral , Humanos , Concentración 50 InhibidoraRESUMEN
Hepatitis C virus (HCV) chronic infections represent one of the major and still unresolved health problems because of low efficiency and high cost of current therapy. Therefore, our studies centered on a viral protein, the NS3 helicase, whose activity is indispensable for replication of the viral RNA, and on its peptide inhibitor that corresponds to a highly conserved arginine-rich sequence of domain 2 of the helicase. The NS3 peptide (p14) was expressed in bacteria. Its 50% inhibitory activity in a fluorometric helicase assay corresponded to 725 nM, while the ATPase activity of NS3 was not affected. Nuclear magnetic resonance (NMR) studies of peptide-protein interactions using the relaxation filtering technique revealed that p14 binds directly to the full-length helicase and its separately expressed domain 1 but not to domain 2. Changes in the NMR chemical shift of backbone amide nuclei ((1)H and (15)N) of domain 1 or p14, measured during complex formation, were used to identify the principal amino acids of both domain 1 and the peptide engaged in their interaction. In the proposed interplay model, p14 contacts the clefts between domains 1 and 2, as well as between domains 1 and 3, preventing substrate binding. This interaction is strongly supported by cross-linking experiments, as well as by kinetic studies performed using a fluorometric assay. The antiviral activity of p14 was tested in a subgenomic HCV replicon assay that showed that the peptide at micromolar concentrations can reduce HCV RNA replication.
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Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Péptidos/farmacología , ARN Helicasas/antagonistas & inhibidores , Replicón/efectos de los fármacos , Proteínas no Estructurales Virales/química , Secuencia de Aminoácidos , Antivirales/química , Línea Celular Tumoral , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Hepacivirus/enzimología , Hepacivirus/genética , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Péptidos/química , ARN Helicasas/química , ARN Viral/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Hepatitis C virus (HCV) is considered one of the most dangerous pathogens since about 3% of the world population is HCV-infected and the virus is a major cause of hepatitis, cirrhosis, and liver carcinoma. A need for a more efficient therapy prompted us to investigate new class of compounds, such as tropolone derivatives that possess antiviral, antibacterial, and antifungal activities. To synthesize bromo- and morpholinomethyl-analogues of tropolone, the previously reported methods were modified. The influence of new derivatives on the activity of the helicase and NTP-ase of HCV was investigated. The most potent inhibitory effect in the fluorometric helicase assay was exerted by 3,7-dibromo-5-morpholinomethyltropolone, for which the IC50 value was at low micromolar range. All the morpholino-derivatives had inhibitory activities higher than those of the non-modified analogues. Low toxicity in a yeast-based toxicity assay indicates that these compounds could be further modified to develop potent inhibitors of the HCV helicase and of viral replication.