RESUMEN
Influenza viruses (IV) exploit a variety of signaling pathways. Previous studies showed that the rapidly accelerated fibrosarcoma/mitogen-activated protein kinase/extracellular signal-regulated kinase (Raf/MEK/ERK) pathway is functionally linked to nuclear export of viral ribonucleoprotein (vRNP) complexes, suggesting that vRNP export is a signaling-induced event. However, the underlying mechanism remained completely enigmatic. Here we have dissected the unknown molecular steps of signaling-driven vRNP export. We identified kinases RSK1/2 as downstream targets of virus-activated ERK signaling. While RSK2 displays an antiviral role, we demonstrate a virus-supportive function of RSK1, migrating to the nucleus to phosphorylate nucleoprotein (NP), the major constituent of vRNPs. This drives association with viral matrix protein 1 (M1) at the chromatin, important for vRNP export. Inhibition or knockdown of MEK, ERK or RSK1 caused impaired vRNP export and reduced progeny virus titers. This work not only expedites the development of anti-influenza strategies, but in addition demonstrates converse actions of different RSK isoforms.
Asunto(s)
Virus de la Influenza A/metabolismo , Gripe Humana/virología , Ribonucleoproteínas/metabolismo , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Núcleo Celular/virología , Humanos , Virus de la Influenza A/genética , Gripe Humana/genética , Gripe Humana/metabolismo , Sistema de Señalización de MAP Quinasas , Señales de Exportación Nuclear , Ribonucleoproteínas/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismoRESUMEN
Cardiac glycosides (CGs) are natural compounds traditionally used for the treatment of heart disorders, and recently new therapeutic possibilities were proposed. Their antitumor reports and clinical trials have notably enhanced, including those targeted for lung cancer, the most lethal type that lacks of new treatment agents, instigating the research of these molecules. The CGs studied here, named C10 {3ß-[(N-(2-hydroxyethyl)aminoacetyl]amino-3-deoxydigitoxigenin} and C18 (3ß-(aminoacetyl)amino-3-deoxydigitoxigenin), are semisynthetic derivatives prepared from digitoxigenin scaffold. Both compounds demonstrated high cytotoxicity for different cancer cell lines, especially H460 lung cancer cells, and their cytotoxic effects were deeply investigated using different methodological approaches. C10 induced cell death at lower concentrations and during shorter periods of treatment than C18, and increased the number of small and irregular nuclei, which are characteristics of apoptosis. This type of cell death was confirmed by caspase-3/7 assay. Both compounds reduced H460 cells proliferative potential by long-term action, and C10 showed the strongest potential. Moreover, these compounds induced a significant decrease of the area and viability of H460 spheroids providing preclinical favorable profiles to develop new chemotherapeutic agents.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis , Proliferación Celular , Digitoxigenina/análogos & derivados , Digitoxigenina/química , Digitoxigenina/farmacología , Neoplasias Pulmonares/patología , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Células Tumorales CultivadasRESUMEN
Human herpesviruses are among the most prevalent pathogens worldwide and have become an important public health issue. Recurrent infections and the emergence of resistant viral strains reinforce the need of searching new drugs to treat herpes virus infections. Cardiac glycosides are used clinically to treat cardiovascular disturbances, such as congestive heart failure and atrial arrhythmias. In recent years, they have sparked new interest in their potential anti-herpes action. It has been previously reported by our research group that two new semisynthetic cardenolides, namely C10 (3ß-[(N-(2-hydroxyethyl)aminoacetyl]amino-3-deoxydigitoxigenin) and C11 (3ß-(hydroxyacetyl)amino-3-deoxydigitoxigenin), exhibited potential anti-HSV-1 and anti-HSV-2 with selectivity index values > 1,000, comparable with those of acyclovir. This work reports the mechanism investigation of anti-herpes action of these derivatives. The results demonstrated that C10 and C11 interfere with the intermediate and final steps of HSV replication, but not with the early stages, since they completely abolished the expression of the UL42 (ß) and gD (γ) proteins and partially reduced that of ICP27 (α). Additionally, they were not virucidal and had no prophylactic effects. Both compounds inhibited HSV replication at nanomolar concentrations, but cardenolide C10 was more active than C11 and can be considered as an anti-herpes drug candidate including against acyclovir-resistant HSV-1 strains.
Asunto(s)
Antivirales/farmacología , Cardenólidos/farmacología , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 2/efectos de los fármacos , Aciclovir/farmacología , Animales , Antivirales/síntesis química , Cardenólidos/síntesis química , Chlorocebus aethiops , Evaluación Preclínica de Medicamentos , Farmacorresistencia Viral , Infecciones por Herpesviridae/tratamiento farmacológico , Humanos , Células VeroRESUMEN
Spondias mobin leaves have been traditionally used for treating cold sores. The study investigated the mechanism of antiherpes action of S. mombin extract, fractions, and geraniin. Different concentrations of samples were used to evaluate the in vitro antiherpes activity (anti-HSV-1) in virucidal, post-infection, attachment, and penetration assays. The mechanism of action of geraniin was investigated considering the glycoproteins gB and gD of HSV-1 surface as potential molecular targets. Molecular docking simulations were carried out for both in order to determine the possible binding mode position of geraniin at the activity sites. The binding mode position was posteriorly optimized considering the flexibility of the glycoproteins. The chemical analysis of samples was performed by LC-MS and revealed the presence of 22 substances, which are hydrolysable tannins, O-glycosylated flavonoids, phenolic acids, and a carbohydrate. The extract, tannin-rich fraction and geraniin showed important in vitro virucidal activity through blocking viral attachment but showed no relevant inhibition of viral penetration. The in silico approaches demonstrated a high number of potential strong intermolecular interactions as hydrogen bonds between geraniin and the activity site of the glycoproteins, particularly the glycoprotein gB. In silico experiments indicated that geraniin is at least partially responsible for the anti-herpes activity through interaction with the viral surface glycoprotein gB, which is responsible for viral adsorption. These results highlight the therapeutic potential of S. mombin anti-herpes treatment and provides support for its traditional purposes. However, further studies are required to validate the antiviral activities in vivo, as well as efficacy in humans.
Asunto(s)
Anacardiaceae , Antivirales , Herpes Simple , Herpesvirus Humano 1 , Herpesvirus Humano 2 , Humanos , Simulación del Acoplamiento Molecular , Extractos Vegetales , Hojas de la Planta , Células Vero , Replicación ViralRESUMEN
Influenza virus infections represent a major public health issue by causing annual epidemics and occasional pandemics that affect thousands of people worldwide. Vaccination is the main prophylaxis to prevent these epidemics/pandemics, although the effectiveness of licensed vaccines is rather limited due to the constant mutations of influenza virus antigenic characteristics. The available anti-influenza drugs are still restricted and there is an increasing viral resistance to these compounds, thus highlighting the need for research and development of new antiviral drugs. In this work, two semisynthetic derivatives of digitoxigenin, namely C10 (3ß-((N-(2-hydroxyethyl)aminoacetyl)amino-3-deoxydigitoxigenin) and C11 (3ß-(hydroxyacetyl)amino-3-deoxydigitoxigenin), showed anti-influenza A virus activity by affecting the expression of viral proteins at the early and late stages of replication cycle, and altering the transcription and synthesis of new viral proteins, thereby inhibiting the formation of new virions. Such antiviral action occurred due to the interference in the assembly of viral polymerase, resulting in an impaired polymerase activity and, therefore, reducing viral replication. Confirming the in vitro results, a clinically relevant ex vivo model of influenza virus infection of human tumor-free lung tissues corroborated the potential of these compounds, especially C10, to completely abrogate influenza A virus replication at the highest concentration tested (2.0 µM). Taken together, these promising results demonstrated that C10 and C11 can be considered as potential new anti-influenza drug candidates.
Asunto(s)
Antivirales/farmacología , Cardenólidos/farmacología , Virus de la Influenza A/efectos de los fármacos , Gripe Humana/tratamiento farmacológico , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Antivirales/química , Cardenólidos/química , Humanos , Conformación Molecular , ARN Polimerasa Dependiente del ARN/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND: Phytochemical profiles of two Brazilian native fruits - pitanga (red and purple) and araçá (yellow and red) - as well as strawberry cultivars Albion, Aromas and Camarosa, blackberry cultivar Tupy and blueberry cultivar Bluegen cultivated in Brazil were characterized for total phenolic content and total anthocyanin content by liquid chromatography coupled to a photodiode array and a quadrupole time-of-flight mass spectrometer. Radical scavenging, antiherpes and cytotoxic activities of these berry extracts were also evaluated. RESULTS: Blueberry presented the highest total anthocyanin content (1202 mg cyanidin-O-glucoside equivalents kg-1 fresh fruit), while strawberry cultivar Aromas presented the highest total phenolic content (13 550 mg gallic acid equivalents kg-1 fresh fruit). Liquid chromatographic-mass spectrometric analysis resulted in the identification of 21 anthocyanins. To the best of our knowledge this is the first report of cyanidin-O-glucoside in yellow and red Araçá fruit and the first time eight anthocyanins have been reported in pitanga fruits. DPPH and ABTS assays showed that blueberry cultivar Bluegen, blackberry cultivar Tupy and pitanga (red and purple) showed the most promising antiradical activities, respectively. No relevant cytotoxicity against three cancer cell lines or antiherpes activity was detected under the experimental conditions tested. CONCLUSION: Total anthocyanin content of all fruits had a strong positive correlation with their free radical scavenging activity, suggesting anthocyanins contribute to the antioxidant potential of these fruits. © 2018 Society of Chemical Industry.
Asunto(s)
Antocianinas/química , Frutas/química , Antioxidantes/química , Arándanos Azules (Planta)/química , Arándanos Azules (Planta)/crecimiento & desarrollo , Brasil , Cromatografía Líquida de Alta Presión , Fragaria/química , Fragaria/crecimiento & desarrollo , Frutas/crecimiento & desarrollo , Fenoles/química , Rubus/química , Rubus/crecimiento & desarrollo , Espectrometría de Masas en TándemRESUMEN
Millions of people use contaminated water sources for direct consumption. Chlorine is the most widely disinfection product but can produce toxic by-products. In this context, natural and synthetic compounds can be an alternative to water disinfection. Therefore, the aim of this study was to assess the inactivation of human adenovirus by N-chlorotaurine (NCT), bromamine-T (BAT) and Grape seed extract (GSE) in water. Distilled water artificially contaminated with recombinant human adenovirus type 5 (rAdV-GFP) was treated with different concentrations of each compound for up to 120 min, and viral infectivity was assessed by fluorescence microscopy. The decrease in activity of the compounds in the presence of organic matter was evaluated in water supplemented with peptone. As results, NCT and GSE inactivated approximately 2.5 log10 of adenovirus after 120 min. With BAT, more than 4.0 log10 decrease was observed within 10 min. The oxidative activity of 1% BAT decreased by 50% in 0.5% peptone within a few minutes, while the reduction was only 30% for 1% NCT in 5% peptone after 60 min. Organic matter had no effect on the activity of GSE. Moreover, the minimal concentration of BAT and GSE to kill viruses was lower than that known to kill human cells. It was concluded that the three compounds have potential to be used for water disinfection for drinking or reuse purposes.
Asunto(s)
Adenovirus Humanos/efectos de los fármacos , Desinfectantes/farmacología , Desinfección/métodos , Agua Dulce/virología , Extracto de Semillas de Uva/farmacología , Sulfonamidas/farmacología , Taurina/análogos & derivados , Inactivación de Virus/efectos de los fármacos , Infecciones por Adenoviridae/virología , Adenovirus Humanos/crecimiento & desarrollo , Adenovirus Humanos/fisiología , Humanos , Taurina/farmacologíaRESUMEN
In recent years, new therapeutic possibilities were proposed for cardiac glycosides traditionally used to treat heart diseases, such as anticancer and antiviral activities. In this sense, this work aimed to synthesize the readily accessible 3ß-azido-3-deoxydigitoxigenin (5) from digitoxigenin (1). Two new series of compounds were obtained from derivative (5): (i) O-glycosyl trizols through click chemistry with propargyl glycosides; and (ii) compounds substituted in the alpha carbonyl position with different residues linked via an amino-group. All obtained derivatives have their chemical structures confirmed, and their anti-herpes (against HSV-types 1 and 2 replication) and cytotoxic (against PC3, A549, HCT-8 and LNCaP cell lines) activities evaluated. Compounds 10 and 11 exhibited the most promising results against HSV-1 (KOS and 29-R strains) and HSV-2 (333 strain) replication with SI valuesâ¯>â¯1000. Both compounds were also the most cytotoxic for the human cancer cell lines tested with IC50 values similar to those of paclitaxel. They also presented reduced toxicity toward non-cancerous cell lines (MRC-5 and HGF cells). Promising compounds were tested in regard to their ability to inhibit Na+/K+-ATPase. The inhibition rate correlates suitably with the bioactivity demonstrated by those both compounds against the different human cancer cells tested as well as against HSV replication. Moreover, the results showed that specific chemical features of compound 10 and 11 influenced the bioactivities tested. In summary, it was possible to obtain novel digitoxigenin-derivatives with remarkable cytotoxic and anti-herpes activities as well as low toxicity and high selectivity. In this way, they could be considered potential molecules for the development of new drugs.
Asunto(s)
Antineoplásicos/química , Antivirales/química , Digitoxigenina/farmacología , Infecciones por Herpesviridae/tratamiento farmacológico , Muerte Celular/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Química Clic , Digitoxigenina/análogos & derivados , Digitoxigenina/síntesis química , Ensayos de Selección de Medicamentos Antitumorales , Glicósidos/química , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 2/efectos de los fármacos , HumanosRESUMEN
Oral bioavailability of C-glycosyl flavonoid enriched fraction of Cecropia glaziovii (EFF-Cg) is limited due to its chemical complexity. The purpose of this study is the prospective evaluation of chitosan buccal films impregnated with EFF-Cg-loaded nanospheres as a drug delivery system for labial herpes treatment or for buccal administration. EFF-Cg-loaded PLGA nanospheres were prepared by double emulsion solvent evaporation technique. Nanoparticles were embedded into buccoadhesive chitosan films in different concentrations in order to obtain nanocomposite films. Films were characterized in term of morphology, mechanical properties and water absorption test. Furthermore a cytotoxicity assay was analyzed to evaluate the biocompatibility of systems. The results obtained from these analyses revealed that nanocomposite films present transparent appearance in all composition and Scanning Electron Microscopy (SEM) images show a continuous and compact section structure. Compared to the control film, mechanical responses of nanocomposites presented lower tensile strength values and no significant effect on the elongation at break. Dynamic Mechanical Analysis (DMA) tests indicated that increasing of NP concentration caused decreased stiffness and an increased of glass transition temperature values. Direct cytotoxicity test shows that nanoparticles and chitosan films not induce cytotoxic effect. Given the promising results, the study concludes that the developed buccal film impregnated with EFF-Cg-loaded nanospheres could be a promising approach for effective delivery of EFF-Cg.
RESUMEN
In this work is reported a novel and promising approach for the preparation of C-glycosylflavonoid enriched fraction of Cecropia glaziovii (EFF-Cg) loaded PLGA nanoparticles (NP) with antiherpes properties. The purpose of this study was to evaluate and to compare the effect of two nonionic surfactants (poloxamer 188 (PLU) and polyvinyl alcohol (PVA)), and also an emulsion stabilized by solid particles of cellulose nanocrystal (CNC) in place of surfactants. The characterization of these nanoparticles was in terms of size, polydispersity index, zeta potential, morphology, thermogravimetric analysis (TGA), loading capacity and percent yield. Since TGA analysis revealed thermo stability especially for NP-PLU, this formulation was selected for the evaluation of drug release profile, cytotoxicity and antiherpes activity. The drug delivery profile demonstrated a sustained release through the polymer structure and a significant reduction of the polymer molecular weight at 21-day period. The cytotoxicity of these nanoparticles was determined on Vero cells, and the selected formulation did not exhibit cytotoxicity even at the highest tested concentration. The results demonstrated a potential antiherpetic effect of the EFF-Cg loaded NP at 48h of testing. In summary, EFF-Cg loaded NP exhibited a promising system for the effective drug delivery in the treatment of herpes infections.