RESUMO
Neuraminidase (NA) is an ideal target for the development of anti-influenza drugs. In this paper, ZINC06057848 was screened out as a hit compound by docking-based virtual screening and molecular dynamics (MD) simulation. The modification and optimization of hit ZINC06057848 resulted in the discovery of a series of novel 1,3,4-triazole-containing NA inhibitors (5a-5j). Compound 5c exerts the best inhibitory activity (IC50 = 0.11 µM) against NA, which is comparable to the positive control oseltamivir carboxylate (OSC) (IC50 = 0.10 µM). Molecular docking analysis indicates that the good efficacy of inhibitor 5c may be attributed to the furan and triazole rings extending into 430-cavity and the ethylbenzene part occupying the active site. The results of this work may help in the development of new NA inhibitors.
Assuntos
Acetamidas/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Neuraminidase/antagonistas & inibidores , Triazóis/farmacologia , Acetamidas/síntese química , Acetamidas/química , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Neuraminidase/metabolismo , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/químicaRESUMO
Influenza viral proteins Haemagglutinin (HA) and Neuraminidase (NA) are important targets for antiviral design. We analyzed for the first time the anti-HA activity and the NA inhibitory activity of extracts and their fractions from Diospyros anisandra on the influenza AH1N1pdm09 virus. The n-hexane fruit extract exhibited HA inhibitory (HAI) activity, and fraction F3 inhibited the hemagglutination from 12.5 up to 100 µg/ml. Gas chromatography-mass spectrometry analysis (GC-MS) on fraction F3, and the n-hexane fruit extract, identified six compounds that were individually evaluated. Only vitamin E and lupeol showed a slight inhibitory activity on HA at 100 µg/ml. Regarding the NA assays, the presence of fluorescent (coumarin) and antioxidant (α-tocopherol) compounds in the root extract, masked the NA assays when using fluorescence techniques. We concluded that D. anisandra is a promising source of bioactive compounds with diverse properties including anti-HA activity on the influenza AH1N1pdm09 virus.
Assuntos
Diospyros , Influenza Humana , Diospyros/química , Hemaglutininas , Humanos , Neuraminidase/metabolismo , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/farmacologia , Proteínas ViraisRESUMO
Angelica gigas Nakai root contains decursin which exerts beneficial properties such as anti-amnesic and anti-inflammatory activities. Until now, however, the neuroprotective effects of decursin against transient ischemic injury in the forebrain have been insufficiently investigated. Here, we revealed that post-treatment with decursin and the root extract saved pyramidal neurons in the hippocampus following transient ischemia for 5 min in gerbil forebrain. Through high-performance liquid chromatography, we defined that decursin was contained in the extract as 7.3 ± 0.2%. Based on this, we post-treated with 350 mg/kg of extract, which is the corresponding dosage of 25 mg/kg of decursin that exerted neuroprotection in gerbil hippocampus against the ischemia. In addition, behavioral tests were conducted to evaluate ischemia-induced dysfunctions via tests of spatial memory (by the 8-arm radial maze test) and learning memory (by the passive avoidance test), and post-treatment with the extract and decursin attenuated ischemia-induced memory impairments. Furthermore, we carried out histochemistry, immunohistochemistry, and double immunohistofluorescence. Pyramidal neurons located in the subfield cornu ammonis 1 (CA1) among the hippocampal subfields were dead at 5 days after the ischemia; however, treatment with the extract and decursin saved the pyramidal neurons after ischemia. Immunoglobulin G (IgG, an indicator of extravasation), which is not found in the parenchyma in normal brain tissue, was apparently shown in CA1 parenchyma from 2 days after the ischemia, but IgG leakage was dramatically attenuated in the CA1 parenchyma treated with the extract and decursin. Furthermore, astrocyte endfeet, which are a component of the blood-brain barrier (BBB), were severely damaged at 5 days after the ischemia; however, post-treatment with the extract and decursin dramatically attenuated the damage of the endfeet. In brief, therapeutic treatment of the extract of Angelica gigas Nakai root and decursin after 5 min transient forebrain ischemia protected hippocampal neurons from the ischemia, showing that ischemia-induced BBB leakage and damage of astrocyte endfeet was significantly attenuated by the extract and decursin. Based on these findings, we suggest that Angelica gigas Nakai root containing decursin can be employed as a pharmaceutical composition to develop a therapeutic strategy for brain ischemic injury.
Assuntos
Angelica/química , Astrócitos/patologia , Benzopiranos/uso terapêutico , Barreira Hematoencefálica/patologia , Butiratos/uso terapêutico , Ataque Isquêmico Transitório/patologia , Extratos Vegetais/uso terapêutico , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Benzopiranos/química , Benzopiranos/farmacologia , Barreira Hematoencefálica/efeitos dos fármacos , Butiratos/química , Butiratos/farmacologia , Gerbillinae , Proteína Glial Fibrilar Ácida/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Imunoglobulina G/metabolismo , Masculino , Neuraminidase/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Extratos Vegetais/farmacologia , Padrões de Referência , Memória Espacial/efeitos dos fármacosRESUMO
Neuraminidase, also known as sialidase, is ubiquitous in animals and microorganisms. It is predominantly distributed in the cell membrane, cytoplasmic vesicles, and lysosomes. Neuraminidase generally recognizes the sialic acid glycosidic bonds at the ends of glycoproteins or glycolipids and enzymatically removes sialic acid. There are four types of neuraminidases, named as Neu1, Neu2, Neu3, and Neu4. Among them, Neu1 is the most abundant in mammals. Recent studies have revealed the involvement of Neu1 in several diseases, including cardiovascular diseases, diabetes, cancers, and neurological disorders. In this review, we center the attention to the role of Neu1 in cardiovascular diseases, including atherosclerosis, ischemic myocardial injury, cerebrovascular disease, congenital heart disease, and pulmonary embolism. We also summarize inhibitors from Chinese herbal medicines (CHMs) in inhibiting virus neuraminidase or human Neu1. Many Chinese herbs and Chinese herb preparations, such as Lonicerae Japonicae Flos, Scutellariae Radix, Yupingfeng San, and Huanglian Jiedu Decoction, have neuraminidase inhibitory activity. We hope to highlight the emerging role of Neu1 in humans and potentially titillate interest for further studies in this area.
Assuntos
Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/enzimologia , Medicamentos de Ervas Chinesas/farmacologia , Neuraminidase/efeitos dos fármacos , Neuraminidase/metabolismo , Medicamentos de Ervas Chinesas/química , Humanos , Estrutura MolecularRESUMO
This study was to assess the possibility of using competitive and slow binding experiments with affinity-based ultrafiltration UPLC-QTof-MS analysis to identify potent bacterial neuraminidase (bNA) inhibitors from the Broussonetia papyrifera roots extract. To isolate unbound compounds from the enzyme-binding complex, the root bark extracts were either incubated in the absence of bNA, in the presence of bNA, or with the time-dependent bNA before the ultrafiltration was performed. Thirteen flavonoids were separated from the target extract, and their inhibitory activities were tested against bNA. The isolated flavonoids exhibited potent inhibition against NA (IC50 = 0.7-54.0 µM). Our kinetic analysis of representative active flavonoids (1, 2, and 6) showed slow and time-dependent reversible inhibition. Additionally, chalcones exhibited noncompetitive inhibition characteristics, whereas flavonols and flavans showed mixed-type behavior. The computational results supported the experimental behaviors of flavonoids 2, 6, 10, and 12, indicating that bounded to the active site, but flavonoids 6 and 10 binds near but not accurately at the active site. Although this is mixed-type inhibition, their binding can be considered competitive.
Assuntos
Broussonetia/química , Flavonoides/química , Raízes de Plantas/química , Chalcona/química , Chalconas/química , Flavonóis/química , Cinética , Neuraminidase/química , Neuraminidase/isolamento & purificação , Neuraminidase/metabolismo , Casca de Planta/química , Extratos Vegetais/química , Polifenóis/química , Prenilação/fisiologiaRESUMO
BACKGROUND: Chagas disease, caused by the parasite Trypanosoma cruzi, represents a worldwide epidemiological, economic, and social problem. In the last decades, the trans-sialidase enzyme of Trypanosoma cruzi has been considered an attractive target for the development of new agents with potential trypanocidal activity. OBJECTIVE: In this work, the aim was to find new potential non-sugar trans-sialidase inhibitors using benzoic acid as a scaffold. METHODS: A structure-based virtual screening of the ZINC15 database was carried out. Additionally, the enzyme and trypanocidal activity of the selected compounds was determined. RESULTS: The results of this work detected 487 compounds derived from benzoic acid as potential transsialidase inhibitors with a more promising binding energy value (< -7.7 kcal/mol) than the known inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA). In particular, two lead compounds, V1 and V2, turned out to be promising trans-sialidase inhibitors. Even though the trypanocidal activity displayed was low, these compounds showed trans-sialidase inhibition values of 87.6% and 29.6%, respectively. CONCLUSION: Structure-based virtual screening using a molecular docking approach is a useful method for the identification of new trans-sialidase inhibitors.
Assuntos
Ácido Benzoico/química , Ácido Benzoico/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Neuraminidase/antagonistas & inibidores , Trypanosoma cruzi/enzimologia , Ácido Benzoico/metabolismo , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/metabolismo , Simulação de Acoplamento Molecular , Neuraminidase/química , Neuraminidase/metabolismo , Conformação Proteica , Termodinâmica , Trypanosoma cruzi/efeitos dos fármacos , Interface Usuário-ComputadorRESUMO
This study was designed to explore the bioactive ingredients in the extracts of Fallopia denticulata (C.C. Huang) Holub, a medicinal plant grown in China, which exhibits the best neuraminidase (NA) inhibition activity. Three fractions of ethyl acetate, ethanol, and water were tested on NA inhibition assay, and the best one was conducted by ultra-performance liquid chromatography-time-of-flight mass spectrometry in the negative and positive modes to analyze the metabolic components. The results revealed the identification of the following 21 compounds: 3 organic acids, 11 flavonoids, 1 coumarin, and 6 others, such as ß-daucosterol, gallic acid, and syringic acid, of which 12 compounds were discovered for the first time in F. denticulata. In addition, we used the molecular docking technique to support the anti-NA activity of each compound in the best extract. The results confirmed that the two better bioactive compounds were (-)-epicatechin gallate and (+)-catechin. Therefore, F. denticulata could be used as a potential material for new anti-influenza drugs.
Assuntos
Medicamentos de Ervas Chinesas , Inibidores Enzimáticos , Fallopia/química , Neuraminidase/antagonistas & inibidores , Plantas Medicinais/química , Catequina , China , Cromatografia Líquida de Alta Pressão , Medicamentos de Ervas Chinesas/análise , Medicamentos de Ervas Chinesas/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Ácido Gálico , Espectrometria de Massas , Simulação de Acoplamento Molecular , Neuraminidase/metabolismoRESUMO
Petasites japonicus have been used since a long time in folk medicine to treat diseases including plague, pestilential fever, allergy, and inflammation in East Asia and European countries. Bioactive compounds that may prevent and treat infectious diseases are identified based on their ability to inhibit bacterial neuraminidase (NA). We aimed to isolate and identify bioactive compounds from leaves and stems of P. japonicas (PJA) and elucidate their mechanisms of NA inhibition. Key bioactive compounds of PJA responsible for NA inhibition were isolated using column chromatography, their chemical structures revealed using 1 H NMR, 13 C NMR, DEPT, and HMBC, and identified to be bakkenolide B (1), bakkenolide D (2), 1,5-di-O-caffeoylquinic acid (3), and 5-O-caffeoylquinic acid (4). Of these, 3 exhibited the most potent NA inhibitory activity (IC50 = 2.3 ± 0.4 µM). Enzyme kinetic studies revealed that 3 and 4 were competitive inhibitors, whereas 2 exhibited non-competitive inhibition. Furthermore, a molecular docking simulation revealed the binding affinity of these compounds to NA and their mechanism of inhibition. Negative-binding energies indicated high proximity of these compounds to the active site and allosteric sites of NA. Therefore, PJA has the potential to be further developed as an antibacterial agent for use against diseases associated with NA.
Assuntos
Clostridium perfringens/enzimologia , Inibidores de Glicosídeo Hidrolases/farmacologia , Neuraminidase/antagonistas & inibidores , Petasites/química , Extratos Vegetais/farmacologia , Ácido Quínico/análogos & derivados , Sesquiterpenos/farmacologia , Inibidores de Glicosídeo Hidrolases/química , Inibidores de Glicosídeo Hidrolases/isolamento & purificação , Cinética , Estrutura Molecular , Neuraminidase/metabolismo , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Ácido Quínico/química , Ácido Quínico/isolamento & purificação , Ácido Quínico/farmacologia , Sesquiterpenos/química , Sesquiterpenos/isolamento & purificaçãoAssuntos
Ácidos Carbocíclicos/uso terapêutico , Antivirais/uso terapêutico , Farmacorresistência Viral , Guanidinas/uso terapêutico , Vírus da Influenza B/genética , Influenza Humana/diagnóstico , Adulto , Dibenzotiepinas/uso terapêutico , Humanos , Indonésia , Vírus da Influenza B/isolamento & purificação , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Japão , Masculino , Testes de Sensibilidade Microbiana , Morfolinas/uso terapêutico , Mutação , Neuraminidase/metabolismo , Piranos/uso terapêutico , Piridonas/uso terapêutico , Ácidos Siálicos/uso terapêutico , Triazinas/uso terapêutico , Zanamivir/uso terapêuticoRESUMO
The neuraminidase (NA) inhibitor (NAI) oseltamivir (OST) is the most widely used influenza antiviral drug. Several NA amino acid substitutions are reported to reduce viral susceptibility to OST in in vitro assays. However, whether there is a correlation between the level of reduction in susceptibility in vitro and the efficacy of OST against these viruses in vivo is not well understood. In this study, a ferret model was utilised to evaluate OST efficacy against circulating influenza A and B viruses with a range of in vitro generated 50% inhibitory concentrations (IC50) values for OST. OST efficacy against an A(H1N1)pdm09 and an A(H1N1)pdm09 virus with the H275Y substitution in neuraminidase was also tested in the macaque model. The results from this study showed that OST had a significant impact on virological parameters compared to placebo treatment of ferrets infected with wild-type influenza A viruses with normal IC50 values (~1 nM). However, this efficacy was lower against wild-type influenza B and other viruses with higher IC50 values. Differing pathogenicity of the viruses made evaluation of clinical parameters difficult, although some effect of OST in reducing clinical signs was observed with influenza A(H1N1) and A(H1N1)pdm09 (H275Y) viruses. Viral titres in macaques were too low to draw conclusive results. Analysis of the ferret data revealed a correlation between IC50 and OST efficacy in reducing viral shedding but highlighted that the current WHO guidelines/criteria for defining normal, reduced or highly reduced inhibition in influenza B viruses based on in vitro data are not well aligned with the low in vivo OST efficacy observed for both wild-type influenza B viruses and those with reduced OST susceptibility.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A Subtipo H3N2 , Vírus da Influenza B , Infecções por Orthomyxoviridae , Oseltamivir , Animais , Feminino , Masculino , Substituição de Aminoácidos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Furões , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/metabolismo , Vírus da Influenza B/genética , Vírus da Influenza B/metabolismo , Macaca fascicularis , Macrolídeos , Mutação de Sentido Incorreto , Neuraminidase/genética , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/patologia , Oseltamivir/farmacologiaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav. (Umbelliferae family) is an herbaceous, perennial plant native to northern and eastern Asia. The root of A. dahurica has traditionally been used under the name "Bai Zhi" as a medicinal plant for colds, dizziness, ulcers, and rheumatism. Moreover, it is also an important ingredient of various prescriptions, such as Gumiganghwal-Tang, for the common cold and influenza. AIM OF THE STUDY: Even though various biological activities of the root of A. dahurica have been reported along with its chemical components, the detailed mechanism of how it exerts anti-influenza activity at the compound level has not been studied. Therefore, we investigated the anti-influenza properties of furanocoumarins purified by bioactivity-guided isolation. MATERIALS AND METHODS: Bioactivity-guided isolation from a 70% EtOH extract of the root of A. dahurica was performed to produce four active furanocoumarins. The inhibition of cytopathic effects (CPEs) was evaluated to ascertain the antiviral activity of these compounds against influenza A (H1N1 and H9N2) viruses. The most potent compound was subjected to detailed mechanistic studies such as the inhibition of viral protein synthesis, CPE inhibition in different phases of the viral replication cycle, neuraminidase (NA) inhibition, antiapoptotic activity using flow cytometry, and immunofluorescence. RESULTS: The bioactivity-guided isolation produced four active furanocoumarins, isoimperatorin (1), oxypeucedanin (2), oxypeucedanin hydrate (3) and imperatorin (4) from the n-BuOH fraction. Among them, compound 2 (followed by compounds 1, 4 and 3) showed a significant CPE inhibition effect, which was stronger than that of the positive control ribavirin, against both H1N1 and H9N2 with an EC50 (µM) of 5.98 ± 0.71 and 4.52 ± 0.39, respectively. Compound 2 inhibited the synthesis of NA and nucleoprotein (NP) in a dose-dependent manner. In the time course assays, the cytopathic effects of influenza A-infected MDCK cells were reduced by 80-90% when treated with compound 2 for 1 and 2 h after infection and declined drastically 3 h after infection. The level of viral NA and NP production was markedly reduced to less than 20% for both proteins in compound 2 (20 µM)-treated cells compared to untreated cells at 2 h after infection. In the molecular docking analysis, compound 2 showed a stronger binding affinity for the C-terminus of polymerase acidic protein (PAC; -36.28 kcal/mol) than the other two polymerase subunits. Compound 2 also exerted an antiapoptotic effect on virus infected cells and significantly inhibited the mRNA expression of caspase-3 and Bax. CONCLUSION: Our results suggest that compound 2 might exert anti-influenza A activity via the inhibition of the early phase of the viral replication cycle, not direct neutralization of surface proteins, such as hemagglutinin and NA, and abnormal apoptosis induced by virus infection. Taken together, these findings suggest that furanocoumarins predominant in A. dahurica play a pivotal role in its antiviral activity. These findings can also explain the reasons for the ethnopharmacological uses of this plant as an important ingredient in many antiviral prescriptions in traditional Chinese medicine (TCM).
Assuntos
Angelica , Antivirais/farmacologia , Células Epiteliais/efeitos dos fármacos , Furocumarinas/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H9N2/efeitos dos fármacos , Infecções por Orthomyxoviridae/tratamento farmacológico , Extratos Vegetais/farmacologia , Angelica/química , Animais , Antivirais/isolamento & purificação , Apoptose/efeitos dos fármacos , Efeito Citopatogênico Viral/efeitos dos fármacos , Cães , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células Epiteliais/virologia , Furocumarinas/isolamento & purificação , Interações entre Hospedeiro e Microrganismos , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H9N2/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H9N2/metabolismo , Células Madin Darby de Rim Canino , Simulação de Acoplamento Molecular , Neuraminidase/antagonistas & inibidores , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Extratos Vegetais/isolamento & purificação , Raízes de Plantas , Replicação Viral/efeitos dos fármacosRESUMO
Turkey adenovirus 3 (TAdV-3) is the causative agent of an immune-mediated disease in turkeys, haemorrhagic enteritis, through targeting B lymphocytes. In the present study, we investigated the role of sialic acid in TAdV-3 entry and characterized the structural components of TAdV-3 receptor(s) on RP19, B lymphoblastoid cells. Removal of the cell-surface sialic acids by neuraminidases or blocking of sialic acids by wheat germ agglutinin lectin reduced virus infection. Pre-incubation of cells with Maackia amurensis lectin or Sambucus nigra agglutinin resulted in virus reduction, suggesting that TAdV-3 uses both α2,3-linked and α2,6-linked sialic acids as attachment receptor. Virus infectivity data from RP19 cells treated with sodium periodate, proteases (trypsin or bromelain) or metabolic inhibitors (dl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, tunicamycin, or benzyl N-acetyl-α-d-galactosaminide) indicated that N-linked, but not O-linked, carbohydrates are part of the sialylated receptor and they are likely based on a membrane glycoprotein, rather than a glycolipid. Furthermore, our data, in conjunction with previous findings, implies that the secondary receptor for TAdV-3 is a protein molecule since the inhibition of glycolipid biosynthesis did not affect the virus infection, which was rather reduced by protease treatment. We can conclude that terminal sialic acids attached to N-linked membrane glycoproteins on B cells are used for virus attachment and are essential for successful virus infection.
Assuntos
Glicoproteínas/metabolismo , Interações Hospedeiro-Patógeno , Receptores Virais/metabolismo , Siadenovirus/fisiologia , Ácidos Siálicos/metabolismo , Infecções por Adenoviridae/metabolismo , Infecções por Adenoviridae/virologia , Animais , Linhagem Celular , Ativação Enzimática , Citometria de Fluxo , Neuraminidase/metabolismo , Ligação Viral , Replicação ViralRESUMO
Pinus densiflora was screened in an ongoing project to discover anti-influenza candidates from natural products. An extensive phytochemical investigation provided 26 compounds, including two new megastigmane glycosides (1 and 2), 21 diterpenoids (3-23), and three flavonoids (24-26). The chemical structures were elucidated by a series of chemical reactions, including modified Mosher's analysis and various spectroscopic measurements such as LC/MS and 1D- and 2D-NMR. The anti-influenza A activities of all isolates were screened by cytopathic effect (CPE) inhibition assays and neuraminidase (NA) inhibition assays. Ten candidates were selected, and detailed mechanistic studies were performed by various assays, such as Western blot, immunofluorescence, real-time PCR and flow cytometry. Compound 5 exerted its antiviral activity not by direct neutralizing virion surface proteins, such as HA, but by inhibiting the expression of viral mRNA. In contrast, compound 24 showed NA inhibitory activity in a noncompetitive manner with little effect on viral mRNA expression. Interestingly, both compounds 5 and 24 were shown to inhibit nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in a dose-dependent manner. Taken together, these results provide not only the chemical profiling of P. densiflora but also anti-influenza A candidates.
Assuntos
Antivirais/química , Inibidores Enzimáticos/química , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Pinus/química , Extratos Vegetais/química , Animais , Antivirais/farmacologia , Sítios de Ligação , Cães , Inibidores Enzimáticos/farmacologia , Flavonoides/análise , Células Madin Darby de Rim Canino , Camundongos , Neuraminidase/antagonistas & inibidores , Neuraminidase/química , Neuraminidase/metabolismo , Extratos Vegetais/farmacologia , Ligação Proteica , Células RAW 264.7 , Terpenos/análise , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Influenza virus is one of the most important human pathogens, causing substantial seasonal and pandemic morbidity and mortality. Houttuynia cordata is a traditionally used medicinal plant for the treatment of pneumonia. Flavonoids are one of the major bioactive constituents of Houttuynia cordata. PURPOSE: This study was designed to investigate the therapeutic effect and mechanism of flavonoid glycosides from H. cordata on influenza A virus (IAV)-induced acute lung injury (ALI) in mice. METHODS: Flavonoids from H. cordata (HCF) were extracted from H. cordata and identified by high-performance liquid chromatography. Mice were infected intranasally with influenza virus H1N1 (A/FM/1/47). HCF (50, 100, or 200 mg/kg) or Ribavirin (100 mg/kg, the positive control) were administered intragastrically. Survival rates, life spans, weight losses, lung indexes, histological changes, inflammatory infiltration, and inflammatory markers in the lungs were measured. Lung virus titers and neuraminidase (NA) activities were detected. The expression of Toll-like receptors (TLRs) and levels of NF-κB p65 phosphorylation (NF-κB p65(p)) in the lungs were analysed. The effects of HCF on viral replication and TLR signalling were further evaluated in cells. RESULTS: HCF contained 78.5% flavonoid glycosides. The contents of rutin, hyperin, isoquercitrin, and quercitrin in HCF were 8.8%, 26.7%, 9.9% and 31.7%. HCF (50, 100 and 200 mg/kg) increased the survival rate and life span of mice infected with the lethal H1N1 virus. In H1N1-induced ALI, mice treated with HCF (50, 100 and 200 mg/kg) showed lesser weight loss and lower lung index than the model group. The lungs of HCF-treated ALI mice presented more intact lung microstructural morphology, milder inflammatory infiltration, and lower levels of monocyte chemotactic protein 1 (MCP-1), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α) and malondialdehyde (MDA) than in the model group. Further investigation revealed that HCF exerted antiviral and TLR-inhibitory effects in vivo and in vitro. HCF (50, 100 and 200 mg/kg) reduced lung H1N1 virus titers and inhibited viral NA activity in mice. HCF (100 and 200 mg/kg) elevated the levels of interferon-ß in lungs. HCF also decreased the expression of TLR3/4/7 and level of NF-κB p65(p) in lung tissues. In vitro experiments showed that HCF (50, 100 and 200 µg/ml) significantly inhibited viral proliferation and suppressed NA activity. In RAW 264.7 cells, TLR3, TLR4, and TLR7 agonist-stimulated cytokine secretion, NF-κB p65 phosphorylation, and nuclear translocation were constrained by HCF treatment. Furthermore, among the four major flavonoid glycosides in HCF, hyperin and quercitrin inhibited both viral replication and TLR signalling in cells. CONCLUSION: HCF significantly alleviated H1N1-induced ALI in mice, which were associated with its dual antiviral and anti-inflammatory effects via inhibiting influenzal NA activity and TLR signalling. among the four major flavonoid glycosides in HCF, hyperin and quercitrin played key roles in the therapeutic effect of HCF.
Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/virologia , Antivirais/farmacologia , Flavonoides/farmacologia , Houttuynia/química , Vírus da Influenza A Subtipo H1N1/patogenicidade , Lesão Pulmonar Aguda/metabolismo , Animais , Antivirais/química , Cães , Flavonoides/química , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/fisiologia , Células Madin Darby de Rim Canino , Camundongos Endogâmicos BALB C , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/metabolismo , Receptores Toll-Like/metabolismo , Fator de Transcrição RelA/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Influenza A virus (IAV) is still a major health threat. The clinical manifestations of this infection are related to immune dysregulation, which causes morbidity and mortality. The usage of traditional medication with immunomodulatory properties against influenza infection has been increased recently. Our previous study showed antiviral activity of quercetin-3-O-α-L-rhamnopyranoside (Q3R) isolated from Rapanea melanophloeos (RM) (L.) Mez (family Myrsinaceae) against H1N1 (A/PR/8/34) infection. This study aimed to confirm the wider range of immunomodulatory effect of Q3R on selective pro- and anti-inflammatory cytokines against IAV in vitro, to evaluate the effect of Q3R on apoptosis pathway in combination with H1N1, also to assess the physical interaction of Q3R with virus glycoproteins and RhoA protein using computational docking. METHODS: MDCK cells were exposed to Q3R and 100CCID50/100 µl of H1N1 in combined treatments (co-, pre- and post-penetration treatments). The treatments were tested for the cytokines evaluation at RNA and protein levels by qPCR and ELISA, respectively. In another set of treatment, apoptosis was examined by detecting RhoA GTPase protein and caspase-3 activity. Molecular docking was used as a tool for evaluation of the potential anti-influenza activity of Q3R. RESULTS: The expressions of cytokines in both genome and protein levels were significantly affected by Q3R treatment. It was shown that Q3R was much more effective against influenza when it was applied in co-penetration treatment. Q3R in combination with H1N1 increased caspase-3 activity while decreasing RhoA activation. The molecular docking results showed strong binding ability of Q3R with M2 transmembrane, Neuraminidase of 2009 pandemic H1N1, N1 and H1 of PR/8/1934 and Human RhoA proteins, with docking energy of - 10.81, - 10.47, - 9.52, - 9.24 and - 8.78 Kcal/mol, respectively. CONCLUSIONS: Quercetin-3-O-α-L-rhamnopyranoside from RM was significantly effective against influenza infection by immunomodulatory properties, affecting the apoptosis pathway and binding ability to viral receptors M2 transmembrane and Neuraminidase of 2009 pandemic H1N1 and human RhoA cellular protein. Further research will focus on detecting the detailed specific mechanism of Q3R in virus-host interactions.
Assuntos
Antivirais , Glicosídeos , Vírus da Influenza A Subtipo H1N1 , Myrsine/química , Compostos Fitoquímicos , Quercetina/análogos & derivados , Animais , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Citocinas/metabolismo , Cães , Glicosídeos/química , Glicosídeos/metabolismo , Glicosídeos/farmacologia , Células Madin Darby de Rim Canino , Simulação de Acoplamento Molecular , Neuraminidase/química , Neuraminidase/metabolismo , Compostos Fitoquímicos/química , Compostos Fitoquímicos/metabolismo , Compostos Fitoquímicos/farmacologia , Quercetina/química , Quercetina/metabolismo , Quercetina/farmacologia , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismoRESUMO
The stems of Dryopteris crassirhizoma, one of the main components of Lianhua-Qingwen Formula (LQF) was traditionally used for heat-clearing and detoxifying. Dryocrassin ABBA is a key antiviral component in the herbal medicine while the compound is hard to get in large amounts with the features of homologous compounds, polyphenol groups, and low contents. Therefore, the present work aims to seek influenza H7N9 virus inhibitors from natural source by synthesis of dryocrassin ABBA and its analogues. As a result, total synthesis of the compound was achieved in nine steps with an over-all yield of 4.6%. Neuraminidases (NAs) inhibitory activities of the synthesized product and its analogues were evaluated afterward. Comparing with the positive control, OSV (9.6⯵M), it was very exciting that dryocrassin ABBA and its analogues (b5 and e2) showed better NAs inhibitory activity against Anhui H7N9 with IC50 values of 3.6⯵M, 2.5⯵M and 1.6⯵M. For the highly resistant Shanghai N9, these compounds can also show medium inhibitory activities. Docking results indicated the direct interaction of synthesized 3 hits with the key K294 by hydrogen bonds, but no direct interaction of OSV with the key K294 was observed in Shanghai N9. This study suggested that dryocrassin ABBA and its analogues especially AB, which consisted of polyphenol groups may have beneficial effects on treating avian influenza H7N9 virus.
Assuntos
Antivirais/farmacologia , Compostos de Benzilideno/farmacologia , Cicloexanonas/farmacologia , Farmacorresistência Viral/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Subtipo H7N9 do Vírus da Influenza A/efeitos dos fármacos , Neuraminidase/antagonistas & inibidores , Antivirais/síntese química , Antivirais/química , Compostos de Benzilideno/síntese química , Compostos de Benzilideno/química , Cicloexanonas/síntese química , Cicloexanonas/química , Relação Dose-Resposta a Droga , Dryopteris/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Subtipo H7N9 do Vírus da Influenza A/enzimologia , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Neuraminidase/metabolismo , Relação Estrutura-AtividadeRESUMO
Toddalia asiatica (Linn.) Lam. is a medical plant traditionally used to treat coughs, fevers, and various diseases. Alkaloids are the main active ingredients in Toddalia asiatica (Linn.) Lam., but traditional methods for screening and separation are complex and labor-intensive. In this work, an efficient strategy was developed to rapidly screen, identify, and separate neuraminidase inhibitors from Toddalia asiatica (Linn.) Lam. Ultrafiltration, high performance liquid chromatography, and time-of-flight mass spectrometry were employed for rapid screening and identification of neuraminidase inhibitors. A two-phase solvent system comprising n-hexane/ethyl acetate/methanol/water (5:5:3:7, v/v) was then selected for separation by high-speed counter-current chromatography. A sample loading of 200 mg and a stepwise flow rate were achieved by increasing the flow rate from 2 to 4 mL/min after 4 h. Three main fluoroquinoline alkaloids (haplopine, skimmianine, and 5-methoxydictamnine) along with two coumarins were obtained via one-step separation and their structures were determined by mass spectrometry and nuclear magnetic resonance. In vitro assays revealed skimmianine with half-maximal inhibitory concentration of 16.2 ± 0.7 µmol/L was selected as the potential highest neuraminidase inhibitor. The results suggest that ultrafiltration high performance liquid chromatography-mass spectrometry combined with high-speed counter-current chromatography is efficient for the screening and isolation of neuraminidase inhibitors from complex natural products.
Assuntos
Inibidores Enzimáticos/análise , Inibidores Enzimáticos/isolamento & purificação , Neuraminidase/antagonistas & inibidores , Extratos Vegetais/química , Raízes de Plantas/química , Rutaceae/química , Alcaloides/antagonistas & inibidores , Alcaloides/metabolismo , Cromatografia Líquida de Alta Pressão , Distribuição Contracorrente , Inibidores Enzimáticos/farmacologia , Estrutura Molecular , Neuraminidase/metabolismo , Extratos Vegetais/farmacologiaRESUMO
Influenza neuraminidase is responsible for the escape of new viral particles from the infected cell surface. Several neuraminidase inhibitors are used clinically to treat patients or stockpiled for emergencies. However, the increasing development of viral resistance against approved inhibitors has underscored the need for the development of new antivirals effective against resistant influenza strains. A facile, sensitive, and inexpensive screening method would help achieve this goal. Recently, we described a multiwell plate-based DNA-linked inhibitor antibody assay (DIANA). This highly sensitive method can quantify femtomolar concentrations of enzymes. DIANA also has been applied to high-throughput enzyme inhibitor screening, allowing the evaluation of inhibition constants from a single inhibitor concentration. Here, we report the design, synthesis, and structural characterization of a tamiphosphor derivative linked to a reporter DNA oligonucleotide for the development of a DIANA-type assay to screen potential influenza neuraminidase inhibitors. The neuraminidase is first captured by an immobilized antibody, and the test compound competes for binding to the enzyme with the oligo-linked detection probe, which is then quantified by qPCR. We validated this novel assay by comparing it with the standard fluorometric assay and demonstrated its usefulness for sensitive neuraminidase detection as well as high-throughput screening of potential new neuraminidase inhibitors.
Assuntos
DNA/química , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Oseltamivir/análogos & derivados , Ácidos Fosforosos/química , Antivirais/química , Antivirais/farmacologia , Inibidores Enzimáticos/química , Humanos , Vírus da Influenza A/enzimologia , Vírus da Influenza A/fisiologia , Influenza Humana/tratamento farmacológico , Influenza Humana/enzimologia , Influenza Humana/virologia , Neuraminidase/antagonistas & inibidores , Neuraminidase/metabolismo , Oseltamivir/química , Reprodutibilidade dos Testes , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismoRESUMO
Neuraminidase (NA) is a glycoside hydrolase that has been proposed as a potential therapeutic target for influenza. Thus, the identification of compounds that modulate NA activity could be of great therapeutic importance. The aim of this study is to develop a drug discovery tool for the identification of novel modulators of NA from both compound libraries and natural plant extracts. NA was immobilized onto the surface of magnetic beads and the inherent catalytic activity of NA-functionalized magnetic beads was characterized. Based on the enzymatic activity (hydrolysis ratio), the inhibitory activities of 12 compounds from plant secondary metabolites were screened, and the desired anti-NA activities of flavonoids were certified. Ligand fishing with the immobilized enzyme was optimized using an artificial test mixture consisting of oseltamivir, lycorine and matrine prior to carrying out the proof-of-concept experiment with the crude extract of Flos Lonicerae. The combination of ligand fishing and HPLC-MS/MS identified luteolin-7-O-ß-D-glucoside, luteolin, 3,5-di-O-caffeoylquinic acid and 3,4-di-O-caffeoylquinic acid as neuraminidase inhibitory ligands in Flos Lonicerae. This is the first report on the use of neuraminidase functionalized magnetic beads for the identification of active ligands from a botanical matrix, and it sets the basis for the de novo identification of NA modulators from complex biological mixtures.
Assuntos
Produtos Biológicos/farmacologia , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/análise , Magnetismo , Microesferas , Neuraminidase/metabolismo , Bibliotecas de Moléculas Pequenas , Produtos Biológicos/química , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Processamento de Imagem Assistida por Computador , Cinética , Ligantes , Lonicera , Extratos Vegetais/análise , Reprodutibilidade dos Testes , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Artemisia scoparia Waldst and Kit is a famous traditional Chinese medicine widely distributed in Xinjiang, China. Flavonoids extracted from it exhibits inhibitory activities against several influenza virus strains. Despite this fact, the antiviral properties of CST, one of such flavonoids, against the influenza virus has not been reported. Thus, the aim of this study is to investigate the anti-influenza virus efficacy and antiviral mechanism of CST. METHODS: The inhibitory activity of CST against influenza viruses was assessed by using viral titers and performing Western blot, qRT-PCR, and immunofluorescence assays in Madin-Darby canine kidney (MDCK) cells and a human monocytic cell line (THP-1). The mechanism of CST against influenza virus was analyzed by hemagglutination inhibition (HI) assay, neuraminidase (NA) inhibition assay, and Western blot. RESULTS: CST reduced viral titers and influenza A virus (IAV) RNA and protein synthesis in a dose-dependent manner. Mechanistically, CST had no inhibitory effect on the attachment and release processes of the viral life cycle, as indicated by the HI and NA assays. Conversely, the CST-mediated inhibition of IAV is possibly linked to the inactivation of the NF-κB/p65 signal pathway. CST also suppressed the activation of JNK MAPK and P38 MAPK in vitro. In line with NF-κB/p65 inhibition, the expression levels of proinflammatory cytokines (TNF-α, IL-1ß, IL-8, and IL-10) and the inflammation-related protein COX-2 were downregulated by CST. CONCLUSIONS: CST inhibited IAV replication by downregulating the NF-κB signal transduction pathway. CST may be a potential agent or supplement against IAV infection.