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1.
Eur J Med Chem ; 185: 111841, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31708183

RESUMO

Influenza A neuraminidase plays an indispensable role in the process of replication and transmission of influenza, so the neuraminidase inhibition can prevent the reproduction of the viruses therefore achieve the effect of treatment of influenza. However, drug resistance of neuraminidase inhibitors such as oseltamivir highlights the need to develop novel structural neuraminidase inhibitors. Here we explored a series of oseltamivir derivatives bearing pyridyl group. Among them, compound 23b exhibiting potent inhibitory activity against neuraminidase from H5N1 subtype was comparable to oseltamivir carboxylate. Cytopathic effect inhibition assay in MDCK cells indicated that compound 23b exerted powerful inhibitions on influenza viruses. And compound 23b were nontoxic to MDCK cells. Meanwhile, compound 23b showed high stability towards rat liver microsomes, human liver microsomes and human plasma. This research enriched the structural type of neuraminidase inhibitors.


Assuntos
Antivirais/farmacologia , Desenho de Drogas , Inibidores Enzimáticos/farmacologia , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Neuraminidase/antagonistas & inibidores , Oseltamivir/farmacologia , Animais , Antivirais/síntese química , Antivirais/química , Cães , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Virus da Influenza A Subtipo H5N1/enzimologia , Células Madin Darby de Rim Canino/efeitos dos fármacos , Células Madin Darby de Rim Canino/microbiologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Neuraminidase/metabolismo , Oseltamivir/síntese química , Oseltamivir/química , Relação Estrutura-Atividade
2.
BMC Complement Altern Med ; 19(1): 346, 2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31791311

RESUMO

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/metabolismo
3.
Nat Commun ; 10(1): 4816, 2019 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-31645552

RESUMO

Sialic acids are a family of related sugars that play essential roles in many biological events intimately linked to cellular recognition in both health and disease. Sialidases are therefore orchestrators of cellular biology and important therapeutic targets for viral infection. Here, we sought to define if uncharacterized sialidases would provide distinct paradigms in sialic acid biochemistry. We show that a recently discovered sialidase family, whose first member EnvSia156 was isolated from hot spring metagenomes, defines an unusual structural fold and active centre constellation, not previously described in sialidases. Consistent with an inverting mechanism, EnvSia156 reveals a His/Asp active center in which the His acts as a Brønsted acid and Asp as a Brønsted base in a single-displacement mechanism. A predominantly hydrophobic aglycone site facilitates accommodation of a variety of 2-linked sialosides; a versatility that offers the potential for glycan hydrolysis across a range of biological and technological platforms.


Assuntos
Domínio Catalítico , Neuraminidase/metabolismo , Ácidos Siálicos/metabolismo , Cristalografia por Raios X , Glicocálix/metabolismo , Neuraminidase/ultraestrutura , Estrutura Terciária de Proteína
4.
Mol Cell Biochem ; 462(1-2): 207-215, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31515676

RESUMO

The link between Neuraminidase 1 (Neu1) and cancer development has been highlighted in numerous studies. In an effort to understand the role of Neu1 in mammary carcinoma cells, we evaluated the effect of Neu1 on controlling cell proliferation and apoptosis, as well as regulating the expression of cadherins. By blocking the activity of Neu1 with oseltamivir phosphate or using siRNA to silence the Neu1 protein, we observed suppression of cell growth in MCF-7 and MDA-MB-231 cells. Enhanced cleaved caspase 3 expression was demonstrated in breast cancer cells treated with oseltamivir phosphate or in Neu1 knockdown mammary carcinoma cells. We also provided evidence of Neu1 reversing the epithelial-mesenchymal properties with associated changes to the respective cadherin family. Additional observations indicated that the phytochemical, honokiol downregulates the expression of Neu1. As a consequence of blocking Neu1, honokiol reduced the levels of sialic acid in the two subtypes of breast cancer. These findings provide evidence that Neu1 regulates cell growth and death, and facilitates cancer progression by modulating the expression levels of cadherins.


Assuntos
Apoptose , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Caderinas/metabolismo , Neuraminidase/metabolismo , Apoptose/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Lignanas/farmacologia , Ácido N-Acetilneuramínico/metabolismo
5.
Microbiology ; 165(11): 1181-1197, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31517596

RESUMO

Key to onset and progression of periodontitis is a complex relationship between oral bacteria and the host. The organisms most associated with severe periodontitis are the periodontal pathogens of the red complex: Tannerella forsythia, Treponema denticola and Porphyromonas gingivalis. These organisms express sialidases, which cleave sialic acid from host glycoproteins, and contribute to disease through various mechanisms. Here, we expressed and purified recombinant P. gingivalis sialidase SiaPG (PG_0352) and characterized its activity on a number of substrates, including host sialoglycoproteins and highlighting the inability to cleave diacetylated sialic acids - a phenomenon overcome by the NanS sialate-esterase from T. forsythia. Indeed SiaPG required NanS to maximize sialic acid harvesting from heavily O-acetylated substrates such as bovine salivary mucin, hinting at the possibility of interspecies cooperation in sialic acid release from host sources by these members of the oral microbiota. Activity of SiaPG and P. gingivalis was inhibited using the commercially available chemotherapeutic zanamivir, indicating its potential as a virulence inhibitor, which also inhibited sialic acid release from mucin, and was capable of inhibiting biofilm formation of P. gingivalis on oral glycoprotein sources. Zanamivir also inhibited attachment and invasion of oral epithelial cells by P. gingivalis and other periodontal pathogens, both in monospecies but also in multispecies infection experiments, indicating potential to suppress host-pathogen interactions of a mixed microbial community. This study broadens our understanding of the multifarious roles of bacterial sialidases in virulence, and indicates that their inhibition with chemotherapeutics could be a promising strategy for periodontitis therapy.


Assuntos
Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Neuraminidase/metabolismo , Porphyromonas gingivalis/enzimologia , Fatores de Virulência/metabolismo , Proteínas de Bactérias/genética , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Linhagem Celular , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Interações Microbianas , Mucinas/metabolismo , Mutação , Neuraminidase/genética , Polissacarídeos/metabolismo , Porphyromonas gingivalis/efeitos dos fármacos , Porphyromonas gingivalis/crescimento & desenvolvimento , Porphyromonas gingivalis/patogenicidade , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sialoglicoproteínas/metabolismo , Tannerella forsythia/enzimologia , Fatores de Virulência/genética , Zanamivir/farmacologia
6.
Cell Biochem Biophys ; 77(4): 319-333, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31559538

RESUMO

Influenza virus is known for its intermittent outbreaks affecting billions of people worldwide. Several neuraminidase inhibitors have been used in practice to overcome this situation. However, advent of new resistant mutants has limited its clinical utilization. In the recent years drug repurposing technique has attained the limelight as it is cost effective and reduces the time consumed for drug discovery. Here, we present multi-dimensional repurposing strategy that integrates the results of ligand-, energy-, receptor cavity, and shape-based pharmacophore algorithm to effectively identify novel drug candidate for influenza. The pharmacophore hypotheses were generated by utilizing the PHASE module of Schrödinger. The generated hypotheses such as AADP, AADDD, and DDRRNH, respectively, for ligand-, e-pharmacophore and receptor cavity based approach alongside shape of oseltamivir were successfully utilized to screen the DrugBank database. Subsequently, these models were evaluated for their differentiating ability using Enrichment calculation. Receiver operating curve and enrichment factors from the analysis indicate that the models possess better capability to screen actives from decoy set of molecules. Eventually, the hits retrieved from different hypotheses were subjected to molecular docking using Glide module of Schrödinger Suite. The results of different algorithms were then combined to eliminate false positive hits and to demonstrate reliable prediction performance than existing approaches. Of note, Pearson's correlation coefficients were calculated to examine the extent of correlation between the glide score and IC50 values. Further, the interaction profile, pharmacokinetic, and pharmacodynamics properties were analyzed for the hit compounds. The results from our analysis showed that alprostadil (DB00770) exhibits better binding affinity toward NA protein than the existing drug molecules. The biological activity of the hit was also predicted using PASS algorithm that renders the antiviral activity of the compound. Further, the results were validated using mutation analysis and molecular dynamic simulation studies. Indeed, this integrative filtering is able to exceed accuracy of other state-of-the-art methods for the drug discovery.


Assuntos
Descoberta de Drogas , Reposicionamento de Medicamentos , Algoritmos , Alprostadil/química , Alprostadil/metabolismo , Alprostadil/uso terapêutico , Antivirais/química , Antivirais/metabolismo , Antivirais/uso terapêutico , Sítios de Ligação , Humanos , Influenza Humana/tratamento farmacológico , Influenza Humana/patologia , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Neuraminidase/química , Neuraminidase/genética , Neuraminidase/metabolismo , Oseltamivir/química , Oseltamivir/metabolismo , Ligação Proteica
7.
Emerg Microbes Infect ; 8(1): 1428-1437, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31560252

RESUMO

Switching of receptor binding preference has been widely considered as one of the necessary mutations for avian influenza viruses, enabling efficient transmissions between human hosts. By stably overexpressing B4GalNT2 gene in MDCK cells, surface α2,3-siallylactose receptors were modified without affecting α2,6-receptor expression. The cell line MDCK-B4GalNT2 was used as a tool to screen for α2,3-receptor requirements in a panel of influenza viruses with previously characterized glycan array data. Infection of viruses with α2,3-receptor binding capability was inhibited in MDCK-B4GalNT2 cells, with the exception of A/WSN/33 (WSN). Infection with the 2009 pandemic H1N1 strains, A/California/04/2009 (Cal04) and A/Hong Kong/415742/2009 (HK09), despite showing α2,6-receptor binding, was also found to be inhibited. Further investigation showed that viral inhibition was due to a reduction in viral entry rate and viral attachment. Recombinant WSN virus with the neuraminidase (NA) gene swapped to A/Puerto Rico/8/1934 (PR8) and Cal04 resulted in a significant viral inhibition in MDCK-B4GalNT2 cells. With oseltamivir, the NA active site was found to be important for the replication results of WSN, but not Cal04.


Assuntos
Vírus da Influenza A Subtipo H1N1/fisiologia , N-Acetilgalactosaminiltransferases/genética , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/metabolismo , Ligação Viral , Internalização do Vírus , Animais , Antivirais/farmacologia , Linhagem Celular , Cães , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/genética , Células Madin Darby de Rim Canino , N-Acetilgalactosaminiltransferases/metabolismo , Neuraminidase/genética , Oseltamivir/farmacologia , Replicação Viral/efeitos dos fármacos
8.
Eur J Med Chem ; 182: 111635, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31493744

RESUMO

The discovery of novel potent neuraminidase (NA) inhibitors remains an attractive approach for treating infectious diseases caused by influenza. In this study, we describe the design and synthesis of novel N-substituted oseltamivir derivatives for probing the 150-cavity which is nascent to the activity site of NA. NA inhibitory studies showed that new derivatives demonstrated the inhibitory activity with IC50 values at nM level against NA of a clinical influenza virus strain. Moreover, the in silico ADME predictions showed that the selected compounds had comparable properties with oseltamivir carboxylate, which demonstrated the druggablity of these derivatives. Furthermore, molecular docking studies showed that the most potent compound 6f and 10i could adopt different modes of binding interaction with NA, which may provide novel solutions for treating oseltamivir-resistant influenza. Based on the research results, we consider that compounds 6f and 10i have the potential for further studies as novel antiviral agents.


Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Simulação de Acoplamento Molecular , Neuraminidase/antagonistas & inibidores , Orthomyxoviridae/efeitos dos fármacos , Oseltamivir/farmacologia , Antivirais/síntese química , Antivirais/química , Simulação por Computador , Relação Dose-Resposta a Droga , Desenho de Drogas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Neuraminidase/metabolismo , Orthomyxoviridae/enzimologia , Oseltamivir/síntese química , Oseltamivir/química , Relação Estrutura-Atividade
9.
Int J Mol Sci ; 20(18)2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31509989

RESUMO

Altered cell surface glycosylation in congenital and acquired diseases has been shown to affect cell differentiation and cellular responses to external signals. Hence, it may have an important role in immune regulation; however, T cell surface glycosylation has not been studied in systemic lupus erythematosus (SLE), a prototype of autoimmune diseases. Analysis of the glycosylation of T cells from patients suffering from SLE was performed by lectin-binding assay, flow cytometry, and quantitative real-time PCR. The results showed that resting SLE T cells presented an activated-like phenotype in terms of their glycosylation pattern. Additionally, activated SLE T cells bound significantly less galectin-1 (Gal-1), an important immunoregulatory lectin, while other lectins bound similarly to the controls. Differential lectin binding, specifically Gal-1, to SLE T cells was explained by the increased gene expression ratio of sialyltransferases and neuraminidase 1 (NEU1), particularly by elevated ST6 beta-galactosamide alpha-2,6-sialyltranferase 1 (ST6GAL1)/NEU1 and ST3 beta-galactoside alpha-2,3-sialyltransferase 6 (ST3GAL6)/NEU1 ratios. These findings indicated an increased terminal sialylation. Indeed, neuraminidase treatment of cells resulted in the increase of Gal-1 binding. Altered T cell surface glycosylation may predispose the cells to resistance to the immunoregulatory effects of Gal-1, and may thus contribute to the pathomechanism of SLE.


Assuntos
Galectina 1/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Ativação Linfocitária , Linfócitos T/metabolismo , Adulto , Antígenos CD/genética , Antígenos CD/metabolismo , Feminino , Expressão Gênica , Glicosilação , Humanos , Lectinas/metabolismo , Lúpus Eritematoso Sistêmico/genética , Masculino , Pessoa de Meia-Idade , Neuraminidase/genética , Neuraminidase/metabolismo , Sialiltransferases/genética , Sialiltransferases/metabolismo , Propriedades de Superfície , Adulto Jovem
10.
BMC Womens Health ; 19(1): 109, 2019 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-31405377

RESUMO

BACKGROUND: This study aims to investigate the difference in vaginal microecology, local immunity and HPV infection among childbearing-age women with different degrees of cervical lesions. METHODS: A total of 432 patients were included in this study. Among these patients, 136 patients had LSIL, 263 patients had HSIL and 33 patients had CSCC. These patients were assigned as the research groups. In addition, 100 healthy females were enrolled and assigned as the control group. RESULTS: The microbiological indexes of vaginal secretions were evaluated. Furthermore, the concentrations of SIgA, IgG, IL-2 and IL-10 in vaginal lavage fluid, as well as the presence of HPV, mycoplasma and Chlamydia in cervical secretions, were detected. The results is that: (1) Differences in evaluation indexes of vaginal microecology among all research groups and the control group were statistically significant (P < 0.0001). As the degree of cervical lesions increased, the number of Lactobacillus decreased, and there was an increase in prevalence of bacterial imbalance, and the diversity, density and normal proportion of bacteria was reduced. Furthermore, the incidence of HPV, trichomonads, clue cell and Chlamydia infection increased. Moreover, the positive rate of H2O2 decreased, while the positive rates of SNa and GADP increased. (2) Differences in the ratio of IL-2 and IL-10 in the female genital tract among all research groups and the control group were statistically significant (P < 0.0001). CONCLUSIONS: As the degree of cervical lesions increased, IL-2 decreased, IL-10 increased and IL-2/IL-10 decreased, while SIgA and IgG were elevated. The reduction of dominant Lactobacillus in the vagina, impairment of H2O2 function, flora ratio imbalance, pathogen infections, reduction in IL-2/IL-10 ratio, and changes in SIgA and IgG levels could all be potential factors that influenced the pathogenicity of HPV infection and the occurrence and development of cervical lesions.


Assuntos
Infecções por Chlamydia/epidemiologia , Infecções por Papillomavirus/epidemiologia , Lesões Intraepiteliais Escamosas Cervicais/epidemiologia , Vagina/imunologia , Vagina/microbiologia , Adulto , Líquidos Corporais/metabolismo , China/epidemiologia , Chlamydia trachomatis/isolamento & purificação , Coagulase/metabolismo , Feminino , Humanos , Peróxido de Hidrogênio/metabolismo , Imunoglobulina A/metabolismo , Imunoglobulina G/metabolismo , Interleucina-10/metabolismo , Interleucina-2/metabolismo , Mycoplasma/isolamento & purificação , Neuraminidase/metabolismo , Papillomaviridae/isolamento & purificação , Esfregaço Vaginal , Adulto Jovem
11.
Artigo em Inglês | MEDLINE | ID: mdl-31297339

RESUMO

The most frequent form of hemolytic-uremic syndrome (HUS) is associated with infections caused by Shiga-like toxin-producing Enterohaemorrhagic Escherichia coli (STEC). In rarer cases HUS can be triggered by Streptococcus pneumoniae. While production of Shiga-like toxins explains STEC-HUS, the mechanisms of pneumococcal HUS are less well-known. S. pneumoniae produces neuraminidases with activity against cell surface sialic acids that are critical for factor H-mediated complement regulation on cells and platelets. The aim of this study was to find out whether S. pneumoniae neuraminidase NanA could trigger complement activation and hemolysis in whole blood. We studied clinical S. pneumoniae isolates and two laboratory strains, a wild-type strain expressing NanA, and a NanA deletion mutant for their ability to remove sialic acids from various human cells and platelets. Red blood cell lysis and activation of complement was measured ex vivo by incubating whole blood with bacterial culture supernatants. We show here that NanA expressing S. pneumoniae strains and isolates are able to remove sialic acids from cells, and platelets. Removal of sialic acids by NanA increased complement activity in whole blood, while absence of NanA blocked complement triggering and hemolytic activity indicating that removal of sialic acids by NanA could potentially trigger pHUS.


Assuntos
Neuraminidase/sangue , Neuraminidase/metabolismo , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/metabolismo , Proteínas de Bactérias/genética , Plaquetas/metabolismo , Proteínas do Sistema Complemento/efeitos dos fármacos , Eritrócitos , Células HEK293 , Hemólise , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , Inflamação , Neuraminidase/genética , Neuraminidase/farmacologia , Infecções Pneumocócicas/microbiologia , Deleção de Sequência , Ácidos Siálicos
12.
Virology ; 534: 80-86, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31220651

RESUMO

The immunopathological mechanisms as well as the role played by influenza A virus infection of human leukocytes and induction of apoptosis have not been fully elucidated. We confirm here that the percentage of cells that are infected is less than the percent of apoptotic cells. Depletion of monocytes/macrophages and depletion of cells expressing influenza neuraminidase from the cultures after exposure to virus decreased lymphocyte apoptosis. Treatment of virus-exposed leukocyte cultures with anti-neuraminidase antibodies but not with anti-hemagglutinin antibodies, reduced lymphocyte production of active caspase-3 and induction of apoptosis. Different strains of virus induced different levels of apoptosis. Variations in induction of apoptosis correlated with production and expression of viral neuraminidase by infected leukocytes. The data suggest that cell surface expression of neuraminidase plays an important role in the induction of apoptosis in human lymphocytes. The benefit, or cost, to the host of lymphocyte apoptosis warrants continued investigation.


Assuntos
Apoptose , Membrana Celular/virologia , Vírus da Influenza A/enzimologia , Influenza Humana/virologia , Linfócitos/citologia , Neuraminidase/metabolismo , Proteínas Virais/metabolismo , Animais , Caspase 3/genética , Caspase 3/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/genética , Influenza Humana/enzimologia , Influenza Humana/genética , Influenza Humana/fisiopatologia , Linfócitos/virologia , Neuraminidase/genética , Proteínas Virais/genética
13.
PLoS Pathog ; 15(6): e1007860, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31181126

RESUMO

Influenza A virus (IAV) neuraminidase (NA) receptor-destroying activity and hemagglutinin (HA) receptor-binding affinity need to be balanced with the host receptor repertoire for optimal viral fitness. NAs of avian, but not human viruses, contain a functional 2nd sialic acid (SIA)-binding site (2SBS) adjacent to the catalytic site, which contributes to sialidase activity against multivalent substrates. The receptor-binding specificity and potentially crucial contribution of the 2SBS to the HA-NA balance of virus particles is, however, poorly characterized. Here, we elucidated the receptor-binding specificity of the 2SBS of N2 NA and established an important role for this site in the virion HA-NA-receptor balance. NAs of H2N2/1957 pandemic virus with or without a functional 2SBS and viruses containing this NA were analysed. Avian-like N2, with a restored 2SBS due to an amino acid substitution at position 367, was more active than human N2 on multivalent substrates containing α2,3-linked SIAs, corresponding with the pronounced binding-specificity of avian-like N2 for these receptors. When introduced into human viruses, avian-like N2 gave rise to altered plaque morphology and decreased replication compared to human N2. An opposite replication phenotype was observed when N2 was combined with avian-like HA. Specific bio-layer interferometry assays revealed a clear effect of the 2SBS on the dynamic interaction of virus particles with receptors. The absence or presence of a functional 2SBS affected virion-receptor binding and receptor cleavage required for particle movement on a receptor-coated surface and subsequent NA-dependent self-elution. The contribution of the 2SBS to virus-receptor interactions depended on the receptor-binding properties of HA and the identity of the receptors used. We conclude that the 2SBS is an important and underappreciated determinant of the HA-NA-receptor balance. The rapid loss of a functional 2SBS in pandemic viruses may have served to balance the novel host receptor-repertoire and altered receptor-binding properties of the corresponding HA protein.


Assuntos
Vírus da Influenza A Subtipo H2N2 , Vírus da Influenza A Subtipo H3N2 , Neuraminidase , Receptores Virais , Proteínas Virais , Vírion , Animais , Sítios de Ligação , Cães , Humanos , Vírus da Influenza A Subtipo H2N2/química , Vírus da Influenza A Subtipo H2N2/genética , Vírus da Influenza A Subtipo H2N2/metabolismo , Vírus da Influenza A Subtipo H3N2/química , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/metabolismo , Células Madin Darby de Rim Canino , Ácido N-Acetilneuramínico/genética , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/química , Neuraminidase/genética , Neuraminidase/metabolismo , Receptores Virais/química , Receptores Virais/genética , Receptores Virais/metabolismo , Células Vero , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vírion/química , Vírion/genética , Vírion/metabolismo
14.
Molecules ; 24(11)2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31185617

RESUMO

A series of NH2-sulfonyl oseltamivir analogues were designed, synthesized, and their inhibitory activities against neuraminidase from H5N1 subtype evaluated. The results indicated that the IC50 value of compound 4a, an oseltamivir analogue via methyl sulfonylation of C5-NH2, was 3.50 µM. Molecular docking simulations suggested that 4a retained most of the interactions formed by oseltamivir carboxylate moieties and formed an additional hydrogen bond with the methylsulfonyl group. Meanwhile, 4a showed high stability towards human liver microsomes. More importantly, 4a without basic moieties is not a zwitterion as reported on the general structure of neuraminidase inhibitors. This research will provide valuable reference for the research of new types of neuraminidase inhibitors.


Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Neuraminidase/antagonistas & inibidores , Oseltamivir/análogos & derivados , Oseltamivir/síntese química , Antivirais/química , Inibidores Enzimáticos/química , Humanos , Concentração Inibidora 50 , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Neuraminidase/metabolismo , Oseltamivir/química , Oseltamivir/farmacologia
15.
J Chem Phys ; 150(22): 220901, 2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-31202243

RESUMO

As molecular scientists have made progress in their ability to engineer nanoscale molecular structure, we face new challenges in our ability to engineer molecular dynamics (MD) and flexibility. Dynamics at the molecular scale differs from the familiar mechanics of everyday objects because it involves a complicated, highly correlated, and three-dimensional many-body dynamical choreography which is often nonintuitive even for highly trained researchers. We recently described how interactive molecular dynamics in virtual reality (iMD-VR) can help to meet this challenge, enabling researchers to manipulate real-time MD simulations of flexible structures in 3D. In this article, we outline various efforts to extend immersive technologies to the molecular sciences, and we introduce "Narupa," a flexible, open-source, multiperson iMD-VR software framework which enables groups of researchers to simultaneously cohabit real-time simulation environments to interactively visualize and manipulate the dynamics of molecular structures with atomic-level precision. We outline several application domains where iMD-VR is facilitating research, communication, and creative approaches within the molecular sciences, including training machines to learn potential energy functions, biomolecular conformational sampling, protein-ligand binding, reaction discovery using "on-the-fly" quantum chemistry, and transport dynamics in materials. We touch on iMD-VR's various cognitive and perceptual affordances and outline how these provide research insight for molecular systems. By synergistically combining human spatial reasoning and design insight with computational automation, technologies such as iMD-VR have the potential to improve our ability to understand, engineer, and communicate microscopic dynamical behavior, offering the potential to usher in a new paradigm for engineering molecules and nano-architectures.


Assuntos
Simulação de Dinâmica Molecular , Software , Realidade Virtual , Benzamidinas/metabolismo , Ciclofilina A/química , Humanos , Subtipo H7N9 do Vírus da Influenza A/enzimologia , Relações Interpessoais , Ligantes , Neuraminidase/metabolismo , Compostos Orgânicos/química , Oseltamivir/metabolismo , Ligação Proteica , Conformação Proteica , Teoria Quântica , Tripsina/metabolismo
16.
J Sep Sci ; 42(16): 2621-2627, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31166086

RESUMO

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/farmacologia
17.
ACS Chem Biol ; 14(6): 1195-1204, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31120724

RESUMO

Sialidases are widely distributed in nature and are involved in many physiological and pathological processes. Sialidases are expressed and work in various tissues and organelles. Clarification of the localization of sialidases is very helpful as a way to understand their functions. We previously developed a novel fluorogenic probe for sialidases, BTP3-Neu5Ac, that visualized the localization of sialidase activity in live cells and tissues by precipitating the hydrophobic fluorescent compound; however, for the purpose of accurate fluorescence imaging of sialidase-expressing cells or the distribution of intracellular sialidase activity, BTP3-Neu5Ac was inadequate in imaging performance. We report the design and development of a sialidase imaging probe that improves the sensitivity and accuracy of in situ fluorescence imaging performance as well as increases the hydrophobicity by attaching linear unsaturated hydrocarbon chains into the hydrophobic fluorescent compound of BTP3-Neu5Ac. The newly developed probe showed low diffusivity and high brightness for fluorescence imaging, and it enabled sensitive and highly accurate imaging of viral sialidase in virus-infected cells and sialidase-expressing cells as well as mammalian sialidase in the rat brain. The probe also enabled the fluorescence imaging of intracellular viral sialidase in live-virus-infected cells. The newly developed probe is expected to be a useful tool that will contribute to the progress of research on sialidases in various fields such as research on viruses and brains.


Assuntos
Corantes Fluorescentes/química , Vírus da Influenza A/enzimologia , Vírus da Influenza B/enzimologia , Neuraminidase/metabolismo , Animais , Encéfalo/enzimologia , Células COS , Linhagem Celular , Cães , Hidrocarbonetos/química , Células Madin Darby de Rim Canino , Masculino , Mamíferos , Imagem Óptica/métodos , Ratos , Ratos Wistar
18.
Vet Res ; 50(1): 37, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118100

RESUMO

The fusogenically activated F and HN proteins of virulent NDV induce complete autophagic flux in DF-1 and A549 cells. However, the effect of both glycoproteins on mitochondria remains elusive. Here, we found that F and HN cooperation increases mitochondrial biogenesis but does not cause the mitochondria damage. We observed that both glycoproteins change the morphological characteristics and spatial distribution of intracellular mitochondria. F and HN cooperate cooperatively to induce ER stress and UPRmt. Our preliminary data suggested that F and HN cooperatively disturb mitochondrial fusion-fission homeostasis to enhance mitochondrial biogenesis, and eventually meet the energy demand of syncytium formation.


Assuntos
Retículo Endoplasmático/virologia , Hemaglutininas/metabolismo , Mitocôndrias/metabolismo , Neuraminidase/metabolismo , Doença de Newcastle/metabolismo , Vírus da Doença de Newcastle/metabolismo , Resposta a Proteínas não Dobradas , Células A549/metabolismo , Células A549/virologia , Animais , Western Blotting , Retículo Endoplasmático/metabolismo , Homeostase , Humanos , Mitocôndrias/virologia
19.
Carbohydr Res ; 478: 33-45, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31054381

RESUMO

Trypanosoma cruzi trans-sialidase (TcTS) is a cell surface protein that participates in the adhesion and invasion mechanisms of the parasite into the host cells, making it an attractive target for inhibitors design. In order to contribute to the knowledge of the interaction between TcTS and their acceptor substrates, we designed and synthesized a library of 20 benzyl lactosides substituted in C-6 of the glucose residue with a series of 1,2,3-triazole derivatives containing different aromatic substituents in the C-4 position. The library was prepared by alkyne-azide cycloaddition reaction catalyzed by Cu(I) ("click chemistry") between a benzyl ß-lactoside functionalized with an azide group in the C-6 position and a series of 2-propargyl phenyl ethers. Herein we analyzed the chromatographic behavior on high performance anion exchange chromatography (HPAEC) of the triazoyl-lactose derivatives and their activity as acceptors of TcTS and inhibitors of the sialylation of N-acetyllactosamine. The triazoyl derivatives were obtained with excellent yields and all of them behaved as moderate alternative substrates. The presence of bulky hydrophobic substituents dramatically increased the retention times in HPAEC but did not affect significantly their acceptor properties toward TcTS.


Assuntos
Amino Açúcares/antagonistas & inibidores , Glicoproteínas/metabolismo , Glicosídeos/farmacologia , Neuraminidase/metabolismo , Trypanosoma cruzi/enzimologia , Amino Açúcares/metabolismo , Configuração de Carboidratos , Glicoproteínas/química , Glicosídeos/síntese química , Glicosídeos/química , Interações Hidrofóbicas e Hidrofílicas , Neuraminidase/química , Especificidade por Substrato
20.
Carbohydr Res ; 479: 48-58, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31132642

RESUMO

Cells are covered by a complex array of carbohydrates. Among them, sialosides are of key importance in intracellular adhesion, recognition and signaling. The need for structurally diverse sialosides impelled the search for efficient synthetic methods since their isolation from natural sources is a difficult task. The enzymatic approach obviates the need of a chemical synthesis for protecting or participating groups in the substrates. The trans-sialidase of Trypanosoma cruzi (TcTS) is highly stereospecific for the transfer of sialic acid from an α-sialylglycoside donor to a terminal ß-galactopyranosyl unit in the acceptor substrate to form the α-Neu5Ac-(2 → 3)-ß-D-Galp motif. The enzyme was cloned and easily available glycoproteins, e.g. fetuin, may be used as donors of sialic acid, constituting strong points for the scalability of TcTS-catalyzed reactions. This review outlines the preparative use of TcTS for the sialylation of oligosaccharides. A detailed description of the substrates used as sialic acid donors, the acceptor substrates and the methods employed to monitor the reaction is included.


Assuntos
Glicoproteínas/metabolismo , Ácido N-Acetilneuramínico/química , Neuraminidase/metabolismo , Oligossacarídeos/química , Oligossacarídeos/síntese química , Trypanosoma cruzi/enzimologia , Técnicas de Química Sintética
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