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1.
Sci Adv ; 7(1)2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33523846

RESUMO

Here, we report the topology-matched design of heteromultivalent nanostructures as potent and broad-spectrum virus entry inhibitors based on the host cell membrane. Initially, we investigate the virus binding dynamics to validate the better binding performance of the heteromultivalent moieties as compared to homomultivalent ones. The heteromultivalent binding moieties are transferred to nanostructures with a bowl-like shape matching the viral spherical surface. Unlike the conventional homomultivalent inhibitors, the heteromultivalent ones exhibit a half maximal inhibitory concentration of 32.4 ± 13.7 µg/ml due to the synergistic multivalent effects and the topology-matched shape. At a dose without causing cellular toxicity, >99.99% reduction of virus propagation has been achieved. Since multiple binding sites have also been identified on the S protein of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), we envision that the use of heteromultivalent nanostructures may also be applied to develop a potent inhibitor to prevent coronavirus infection.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Vírus da Influenza A/efeitos dos fármacos , Influenza Humana/virologia , Nanopartículas/química , Neuraminidase/química , Animais , Antivirais/farmacologia , Sítios de Ligação , Membrana Celular/metabolismo , Cães , Membrana Eritrocítica/virologia , Humanos , Vírus da Influenza A/fisiologia , Células Madin Darby de Rim Canino , Ligação Proteica , Glicoproteína da Espícula de Coronavírus , Vírion , Ligação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos
2.
PLoS Pathog ; 16(8): e1008816, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32853241

RESUMO

Influenza A viruses (IAVs) cause seasonal epidemics and occasional pandemics. Most pandemics occurred upon adaptation of avian IAVs to humans. This adaptation includes a hallmark receptor-binding specificity switch of hemagglutinin (HA) from avian-type α2,3- to human-type α2,6-linked sialic acids. Complementary changes of the receptor-destroying neuraminidase (NA) are considered to restore the precarious, but poorly described, HA-NA-receptor balance required for virus fitness. In comparison to the detailed functional description of adaptive mutations in HA, little is known about the functional consequences of mutations in NA in relation to their effect on the HA-NA balance and host tropism. An understudied feature of NA is the presence of a second sialic acid-binding site (2SBS) in avian IAVs and absence of a 2SBS in human IAVs, which affects NA catalytic activity. Here we demonstrate that mutation of the 2SBS of avian IAV H5N1 disturbs the HA-NA balance. Passaging of a 2SBS-negative H5N1 virus on MDCK cells selected for progeny with a restored HA-NA balance. These viruses obtained mutations in NA that restored a functional 2SBS and/or in HA that reduced binding of avian-type receptors. Importantly, a particular HA mutation also resulted in increased binding of human-type receptors. Phylogenetic analyses of avian IAVs show that also in the field, mutations in the 2SBS precede mutations in HA that reduce binding of avian-type receptors and increase binding of human-type receptors. Thus, 2SBS mutations in NA can drive acquisition of mutations in HA that not only restore the HA-NA balance, but may also confer increased zoonotic potential.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/genética , Mutação , Neuraminidase/genética , Infecções por Orthomyxoviridae/virologia , Ácidos Siálicos/metabolismo , Replicação Viral , Substituição de Aminoácidos , Animais , Sítios de Ligação , Cães , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/isolamento & purificação , Células Madin Darby de Rim Canino , Neuraminidase/química , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/patologia , Ligação Proteica
3.
Exp Parasitol ; 216: 107943, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32598890

RESUMO

The search for novel therapeutic candidates against animal trypanosomiasis is an ongoing scientific endevour because of the negative impacts of the disease to the African livestock industry. In this study, the in vivo therapeutic potentials of phytol toward Trypanosoma congolense infection and the inhibitory effects on trypanosomal sialidase were investigated. Rats were infected with T. congolense and administered daily oral treatment of 50 and 100 mg/kg BW of phytol. Within the first 10 days of the treatment, no antitrypanosomal activity was recorded but a moderate trypanostatic activity was observed from day 17-day 21 pi. However, at 100 mg/kg BW, phytol demonstrated a significant (p < 0.05) ameliorative potentials toward T. congolense-induced host-associated pathological damages such as anaemia, hepatic and renal damages; and the data was comparable to diminazine aceturate. Moreover, the T. congolense caused a significant (p < 0.05) increase in free serum sialic acid level which was significantly (p < 0.05) prevented in the presence of phytol (100 mg/kg BW). In an in vitro analysis, phytol inhibited partially purified T. congolense sialidase using an uncompetitive inhibition pattern with inhibition binding constant of 261.24 µmol/mL. Subsequently, molecular docking revealed that the compound binds to homology modelled trypanosomal sialidase with a binding free energy of -6.7 kcal/mol which was mediated via a single hydrogen bond while Trp324 and Pro274 were the critical binding residues. We concluded that phytol has moderate trypanostatic activity but with a great potential in mitigating the host-associated cellular damages while the anaemia amelioration was mediated, in part, through the inhibition of sialidase.


Assuntos
Antiprotozoários/uso terapêutico , Inibidores Enzimáticos/farmacologia , Neuraminidase/antagonistas & inibidores , Fitol/uso terapêutico , Trypanosoma congolense/efeitos dos fármacos , Tripanossomíase Africana/veterinária , Animais , Antiprotozoários/farmacologia , Inibidores Enzimáticos/uso terapêutico , Gado , Doenças Negligenciadas/tratamento farmacológico , Doenças Negligenciadas/veterinária , Neuraminidase/química , Neuraminidase/isolamento & purificação , Fitol/farmacologia , Distribuição Aleatória , Ratos , Ratos Wistar , Trypanosoma congolense/enzimologia , Tripanossomíase Africana/tratamento farmacológico
4.
Gene ; 742: 144538, 2020 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-32184168

RESUMO

Lysosomal desialylation is the initial step in the degradation of sialo-glycopeptides that is essential for regenerating sialo-glycoconjugates. Neu1 sialidase is the enzyme responsible for the removal of sialic acid in the mammalian lysosome. Although Neu1 sialidases are conserved in fish similar to mammals, their physiological functions remain to be fully understood. Nile tilapia (Oreochromis niloticus) is known to possess two putative Neu1 sialidases (Neu1a and Neu1b) in the genome that may have arisen by gene duplication (specifically in cichlidae family members). This suggests that understanding the Neu1 sialidase in fish, particularly cichlids, could provide insights into the (novel) physiological functions of these genes. Moreover, characterization of the tilapia Neu1 sialidase is paramount to ensure clarity of the desialylation reaction performed by the fish sialidases (like the characterized tilapia sialidases Neu3 and Neu4). Therefore, this study focused on the characterization of the tilapia Neu1 sialidases. Neu1b exhibited narrow substrate specificity when compared with Neu1a, whereas the properties of these two Neu1 sialidases, such as cathepsin A-induced activation, optimal pH, and lysosomal localization, were conserved. Neu1a mRNA levels were detected in various tissues of tilapia as compared to the mRNA levels of Neu1b. Although the cloned construct of Neu1b contained an extra exon unlike tilapia Neu1a, the exon did not affect the enzymatic properties of Neu1b. This study suggests that tilapia Neu1a profiles were highly conserved with other vertebrate Neu1 isoforms, while Neu1b probably evolved independently in other members of the cichlidae family. Moreover, the expression of sialidase genes (neu1a, neu1b, neu3a, and neu4) were determined in various stages of tilapia embryogenesis using real-time PCR; sialidase gene expression is reported to be drastically and individually altered during embryogenesis in Japanese medaka (Oryzias latipes). The mRNA levels of neu1a drastically increased between 72 and 84 hpf and mildly decreased from 84 to 144 hpf. In contrast, the transcript levels of neu1b did not change between 84 and 144 hpf and the expression of neu3a gradually increased between 84 and 120 hpf and drastically decreased at 144 hpf. The highest level of the neu4 transcripts was detected at 84 hpf. These expression patterns were different from those in Japanese medaka, possibly due to the different developmental program found in the tilapia embryo accompanied with the unique profiles of the tilapia sialidases.


Assuntos
Ciclídeos/metabolismo , Proteínas de Peixes/metabolismo , Neuraminidase/metabolismo , Animais , Ciclídeos/genética , Ciclídeos/crescimento & desenvolvimento , Clonagem Molecular , Evolução Molecular , Feminino , Proteínas de Peixes/química , Proteínas de Peixes/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Neuraminidase/química , Neuraminidase/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Especificidade por Substrato/genética
5.
Arch Virol ; 165(4): 891-911, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32060794

RESUMO

Pandemics caused by influenza A virus (IAV) are responsible for the deaths of millions of humans around the world. One of these pandemics occurred in Mexico in 2009. Despite the impact of IAV on human health, there is no effective vaccine. Gene mutations and translocation of genome segments of different IAV subtypes infecting a single host cell make the development of a universal vaccine difficult. The design of immunogenic peptides using bioinformatics tools could be an interesting strategy to increase the success of vaccines. In this work, we used the predicted amino acid sequences of the neuraminidase (NA) and hemagglutinin (HA) proteins of different IAV subtypes to perform multiple alignments, epitope predictions, molecular dynamics simulations, and experimental validation. Peptide selection was based on the following criteria: promiscuity, protein surface exposure, and the degree of conservation among different medically relevant IAV strains. These peptides were tested using immunological assays to test their ability to induce production of antibodies against IAV. We immunized rabbits and mice and measured the levels of IgG and IgA antibodies in serum samples and nasal washes. Rabbit antibodies against the peptides P11 and P14 (both of which are hybrids of NA and HA) recognized HA from both group 1 (H1, H2, and H5) and group 2 (H3 and H7) IAV and also recognized the purified NA protein from the viral stock (influenza A Puerto Rico/916/34). IgG antibodies from rabbits immunized with P11 and P14 were capable of recognizing viral particles and inhibited virus hemagglutination. Additionally, intranasal immunization of mice with P11 and P14 induced specific IgG and IgA antibodies in serum and nasal mucosa, respectively. Interestingly, the IgG antibodies were found to have neutralizing capability. In conclusion, the peptides designed through in silico studies were validated in experimental assays.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Sequência de Aminoácidos , Animais , Biologia Computacional , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Imunização , Vírus da Influenza A/química , Vírus da Influenza A/genética , Vacinas contra Influenza/química , Vacinas contra Influenza/genética , Influenza Humana/imunologia , Influenza Humana/virologia , Camundongos , Camundongos Endogâmicos BALB C , Neuraminidase/química , Neuraminidase/genética , Neuraminidase/imunologia , Coelhos , Alinhamento de Sequência , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/imunologia
6.
Sci Rep ; 10(1): 2161, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034220

RESUMO

While molecular-targeted drugs have demonstrated strong therapeutic efficacy against diverse diseases such as cancer and infection, the appearance of drug resistance associated with genetic variations in individual patients or pathogens has severely limited their clinical efficacy. Therefore, precision medicine approaches based on the personal genomic background provide promising strategies to enhance the effectiveness of molecular-targeted therapies. However, identifying drug resistance mutations in individuals by combining DNA sequencing and in vitro analyses is generally time consuming and costly. In contrast, in silico computation of protein-drug binding free energies allows for the rapid prediction of drug sensitivity changes associated with specific genetic mutations. Although conventional alchemical free energy computation methods have been used to quantify mutation-induced drug sensitivity changes in some protein targets, these methods are often adversely affected by free energy convergence. In this paper, we demonstrate significant improvements in prediction performance and free energy convergence by employing an alchemical mutation protocol, MutationFEP, which directly estimates binding free energy differences associated with protein mutations in three types of a protein and drug system. The superior performance of MutationFEP appears to be attributable to its more-moderate perturbation scheme. Therefore, this study provides a deeper level of insight into computer-assisted precision medicine.


Assuntos
Resistência a Medicamentos , Simulação de Acoplamento Molecular/métodos , Mutação , Aldeído Redutase/antagonistas & inibidores , Aldeído Redutase/química , Aldeído Redutase/genética , Quinase do Linfoma Anaplásico/antagonistas & inibidores , Quinase do Linfoma Anaplásico/química , Quinase do Linfoma Anaplásico/genética , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Simulação de Acoplamento Molecular/normas , Neuraminidase/antagonistas & inibidores , Neuraminidase/química , Neuraminidase/genética , Sensibilidade e Especificidade
7.
Biochem Biophys Res Commun ; 523(2): 487-492, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-31889533

RESUMO

Bacterial sialidases are widely used to remove sialic acid (Sia) residues from glycans. Most of them cleave the glycosides of N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) under acidic pHs; however, currently available bacterial sialidases had no activity to the glycosides of deaminoneuraminic acid (Kdn). In this study, we found a novel sialidase from Sphingobacterium sp. strain HMA12 that could cleave any of the glycosides of Neu5Ac, Neu5Gc, and Kdn. It also had a broad linkage specificity, i.e., α2,3-, α2,6-, α2,8-, and α2,9-linkages, and the optimal pH at neutral ranges, pH 6.5-7.0. These properties are particularly important when sialidases are applied for in vivo digestion of the cell surface sialosides under physiological conditions. Interestingly, 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (Neu5Ac2en), which is a transition state analog-based inhibitor, competitively inhibited the enzyme-catalyzed reaction for Kdn as well as for Neu5Ac, suggesting that the active site is common to the Neu5Ac and Kdn residues. Taken together, this sialidase is versatile and useful for the in vivo research on sialo-glycoconjugates.


Assuntos
Glicosídeos/metabolismo , Neuraminidase/metabolismo , Ácidos Siálicos/metabolismo , Sphingobacterium/enzimologia , Motivos de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células CHO , Cricetulus , Concentração de Íons de Hidrogênio , Hidrólise , Ácido N-Acetilneuramínico/análogos & derivados , Ácido N-Acetilneuramínico/metabolismo , Ácido N-Acetilneuramínico/farmacologia , Ácidos Neuramínicos , Neuraminidase/antagonistas & inibidores , Neuraminidase/química , Neuraminidase/genética , Sphingobacterium/genética , Especificidade por Substrato , Temperatura
8.
Emerg Microbes Infect ; 9(1): 78-87, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31894728

RESUMO

The H7N9 influenza virus has been circulating in China for more than six years. The neuraminidase (NA) has gained great concern for the development of antiviral drugs, therapeutic antibodies, and new vaccines. In this study, we screened seven mouse monoclonal antibodies (mAbs) and compared their protective effects against H7N9 influenza virus. The epitope mapping from escape mutants showed that all the seven mAbs could bind to the head region of the N9 NA close to the enzyme activity sites, and four key sites of N9 NA were reported for the first time. The mAbs D3 and 7H2 could simultaneously inhibit the cleavage of the sialic acid of fetuin protein with large molecular weight and NA-XTD with small molecule weight in the NA inhibition experiment, prevent the formation of virus plaque at a low concentration, and effectively protect the mice from the challenge of the lethal dose of H7N9 virus.


Assuntos
Anticorpos Monoclonais/química , Subtipo H7N9 do Vírus da Influenza A/imunologia , Neuraminidase/antagonistas & inibidores , Neuraminidase/química , Infecções por Orthomyxoviridae/prevenção & controle , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Antígenos Virais , Domínio Catalítico , Linhagem Celular , Cães , Mapeamento de Epitopos , Humanos , Influenza Humana/tratamento farmacológico , Influenza Humana/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/tratamento farmacológico
9.
J Virol ; 94(5)2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31801857

RESUMO

To characterize bat influenza H18N11 virus, we propagated a reverse genetics-generated H18N11 virus in Madin-Darby canine kidney subclone II cells and detected two mammal-adapting mutations in the neuraminidase (NA)-like protein (NA-F144C and NA-T342A, N2 numbering) that increased the virus titers in three mammalian cell lines (i.e., Madin-Darby canine kidney, Madin-Darby canine kidney subclone II, and human lung adenocarcinoma [Calu-3] cells). In mice, wild-type H18N11 virus replicated only in the lungs of the infected animals, whereas the NA-T342A and NA-F144C/T342A mutant viruses were detected in the nasal turbinates, in addition to the lungs. Bat influenza viruses have not been tested for their virulence or organ tropism in ferrets. We detected wild-type and single mutant viruses each possessing NA-F144C or NA-T342A in the nasal turbinates of one or several infected ferrets, respectively. A mutant virus possessing both the NA-F144C and NA-T342A mutations was isolated from both the lung and the trachea, suggesting that it has a broader organ tropism than the wild-type virus. However, none of the H18N11 viruses caused symptoms in mice or ferrets. The NA-F144C/T342A double mutation did not substantially affect virion morphology or the release of virions from cells. Collectively, our data demonstrate that the propagation of bat influenza H18N11 virus in mammalian cells can result in mammal-adapting mutations that may increase the replicative ability and/or organ tropism of the virus; overall, however, these viruses did not replicate to high titers throughout the respiratory tract of mice and ferrets.IMPORTANCE Bats are reservoirs for several severe zoonotic pathogens. The genomes of influenza A viruses of the H17N10 and H18N11 subtypes have been identified in bats, but no live virus has been isolated. The characterization of artificially generated bat influenza H18N11 virus in mammalian cell lines and animal models revealed that this virus can acquire mammal-adapting mutations that may increase its zoonotic potential; however, the wild-type and mutant viruses did not replicate to high titers in all infected animals.


Assuntos
Quirópteros/virologia , Mutação , Neuraminidase/genética , Neuraminidase/metabolismo , Orthomyxoviridae/enzimologia , Orthomyxoviridae/genética , Replicação Viral/fisiologia , Animais , Linhagem Celular , Modelos Animais de Doenças , Feminino , Furões/virologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Neuraminidase/química , Orthomyxoviridae/crescimento & desenvolvimento , Infecções por Orthomyxoviridae/veterinária , Infecções por Orthomyxoviridae/virologia , Traqueia/virologia , Zoonoses/virologia
10.
Arch Virol ; 165(1): 201-206, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31745716

RESUMO

Neuraminidase (NA) thermostability of influenza A and B viruses isolated from birds, swine and humans was measured to evaluate its variability associated with host body temperature. The highest 50% inactivation temperature (IT50) was observed with H3N8 avian influenza virus (74 °C), and the lowest IT50 was observed with the seasonal human H3N2 virus (45.5 °C). The IT50 values of A(H1N1)pdm09 viruses 56.4-58.5 °C were statistically higher than that of the prepandemic strain A/Solomon Islands/03/06 (52.5 °C). An analysis of Ca2+ binding sites revealed the correspondence of amino acid changes to NA thermostability. This study demonstrates that changes in NA thermostability correspond to differences in host body temperature.


Assuntos
Influenzavirus A/enzimologia , Influenzavirus B/enzimologia , Neuraminidase/química , Animais , Aves/virologia , Temperatura Corporal , Estabilidade Enzimática , Humanos , Suínos , Termodinâmica , Proteínas Virais/química , Zoonoses/virologia
11.
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
12.
Cell Host Microbe ; 26(6): 729-738.e4, 2019 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-31757767

RESUMO

Influenza virus neuraminidase (NA) is a major target for small-molecule antiviral drugs. Antibodies targeting the NA surface antigen could also inhibit virus entry and egress to provide host protection. However, our understanding of the nature and range of target epitopes is limited because of a lack of human antibody structures with influenza neuraminidase. Here, we describe crystal and cryogenic electron microscopy (cryo-EM) structures of NAs from human-infecting avian H7N9 viruses in complex with five human anti-N9 antibodies, systematically defining several antigenic sites and antibody epitope footprints. These antibodies either fully or partially block the NA active site or bind to epitopes distant from the active site while still showing neuraminidase inhibition. The inhibition of antibodies to NAs was further analyzed by glycan array and solution-based NA activity assays. Together, these structural studies provide insights into protection by anti-NA antibodies and templates for the development of NA-based influenza virus vaccines and therapeutics.


Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Epitopos/ultraestrutura , Neuraminidase , Infecções por Orthomyxoviridae/tratamento farmacológico , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/ultraestrutura , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/ultraestrutura , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/ultraestrutura , Antivirais/imunologia , Microscopia Crioeletrônica , Epitopos/imunologia , Epitopos/metabolismo , Humanos , Subtipo H7N9 do Vírus da Influenza A/imunologia , Vacinas contra Influenza , Neuraminidase/química , Neuraminidase/ultraestrutura , Infecções por Orthomyxoviridae/prevenção & controle , Proteínas Virais/química , Proteínas Virais/ultraestrutura
13.
Molecules ; 24(20)2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31623198

RESUMO

Monosialotetrahexosylganglioside (GM1) has good activity on brain diseases and was developed to be a drug applied in clinics for neurological disorders and nerve injury. It is difficult to isolate GM1 in industry scale from the brains directly. In this work, a simple and highly efficient method with high yield was developed for the isolation, conversion, and purification of GM1 from a pig brain. Gangliosides (GLS) were first extracted by supercritical CO2 (SCE). The optimum extraction time of GLS by SCE was 4 h, and the ratio of entrainer to acetone powder from the pig brain was 3:1 (v/w). GM1 was then prepared from GLS by immobilized sialidase and purified by reverse-phase silica gel. Sodium alginate embedding was used for the immobilization of sialidase. Under the optimized method, the yield of high-purity GM1 was around 0.056%. This method has the potential to be applied in the production of GM1 in the industry.


Assuntos
Dióxido de Carbono/química , Enzimas Imobilizadas , Gangliosídeo G(M1)/química , Neuraminidase/química , Acetona/química , Animais , Encéfalo , Ácidos Graxos , Neuraminidase/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Especificidade por Substrato , Suínos
14.
Matrix Biol ; 84: 1-3, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31655291

RESUMO

This Thematic Minireview Series of Matrix Biology focused on elastin, from structure to disease celebrates the memory of Ladislas Robert, a pioneer in Matrix Biology in France and Europe. Since his first publication on elastin and elastases in 1957, the huge development in matrix biology led to major findings on elastic fibers and their component proteins including elastin architecture, the role of fibrillins and microfibril-binding proteins on elastin assembly, the effects of sequence variants of human tropoelastin on its assembly, structure and functions, the role of elastin peptides in health and diseases, the identification of neuraminidase-1 as a member of the elastin receptor complex, and the fate of elastic fibers upon aging, which are reviewed in this series. Two other reviews, focused on the design and use of elastin-like recombinamers as biomaterials, and on the circadian rhythms in skin and other elastic tissues, complete this series.


Assuntos
Elastina/genética , Elastina/metabolismo , Matriz Extracelular/metabolismo , Elastina/química , Variação Genética , História do Século XX , História do Século XXI , Humanos , Neuraminidase/química , Neuraminidase/genética , Neuraminidase/metabolismo , Tropoelastina/química , Tropoelastina/genética , Tropoelastina/metabolismo
15.
SAR QSAR Environ Res ; 30(12): 899-917, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31645133

RESUMO

Influenza A virus (IAV) has caused epidemic infections worldwide, with many strains resistant to inhibitors of a surface protein, neuraminidase (NA), due to point mutations on its structure. A novel NA inhibitor named peramivir was recently approved, but no exhaustive computational research regarding its binding affinity with wild-type and mutant NA has been conducted. In this study, a thorough investigation of IAV-NA PDB entries of 9 subtypes is described, providing a list of residues constituting the protein-ligand binding sites. The results of induced-fit docking approach point out key residues of wild-type NA participating in hydrogen bonds and/or ionic interactions with peramivir, among which Arg 368 is responsible for a peramivir-NA ionic interaction. Mutations on this residue greatly reduced the binding affinity of peramivir with NA, with 3 mutations R378Q, R378K and R378L (NA6) capable of deteriorating the docking performance of peramivir by over 50%. 200 compounds from 6-scaffolds were docked into these 3 mutant versions, revealing 18 compounds giving the most promising results. Among them, CMC-2012-7-1527-56 (benzoic acid scaffold, IC50 = 32 nM in inhibitory assays with IAV) is deemed the most potential inhibitor of mutant NA resisting both peramivir and zanamivir, and should be further investigated.


Assuntos
Antivirais/química , Ciclopentanos/química , Inibidores Enzimáticos/química , Guanidinas/química , Neuraminidase/química , Proteínas Virais/química , Ácidos Carbocíclicos , Sítios de Ligação , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Mutação , Neuraminidase/antagonistas & inibidores , Neuraminidase/genética , Relação Quantitativa Estrutura-Atividade , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genética
16.
Artigo em Inglês | MEDLINE | ID: mdl-31649895

RESUMO

There is an unmet public health need for a universal influenza vaccine (UIV) to provide broad and durable protection from influenza virus infections. The identification of broadly protective antibodies and cross-reactive T cells directed to influenza viral targets present a promising prospect for the development of a UIV. Multiple targets for cross-protection have been identified in the stalk and head of hemagglutinin (HA) to develop a UIV. Recently, neuraminidase (NA) has received significant attention as a critical component for increasing the breadth of protection. The HA stalk-based approaches have shown promising results of broader protection in animal studies, and their feasibility in humans are being evaluated in clinical trials. Mucosal immune responses and cross-reactive T cell immunity across influenza A and B viruses intrinsic to live attenuated influenza vaccine (LAIV) have emerged as essential features to be incorporated into a UIV. Complementing the weakness of the stand-alone approaches, prime-boost vaccination combining HA stalk, and LAIV is under clinical evaluation, with the aim to increase the efficacy and broaden the spectrum of protection. Preexisting immunity in humans established by prior exposure to influenza viruses may affect the hierarchy and magnitude of immune responses elicited by an influenza vaccine, limiting the interpretation of preclinical data based on naive animals, necessitating human challenge studies. A consensus is yet to be achieved on the spectrum of protection, efficacy, target population, and duration of protection to define a "universal" vaccine. This review discusses the recent advancements in the development of UIVs, rationales behind cross-protection and vaccine designs, and challenges faced in obtaining balanced protection potency, a wide spectrum of protection, and safety relevant to UIVs.


Assuntos
Proteção Cruzada/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Anticorpos Antivirais/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Reações Cruzadas/imunologia , Epitopos de Linfócito T/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Vacinas contra Influenza/normas , Influenza Humana/imunologia , Influenza Humana/virologia , Neuraminidase/química , Neuraminidase/imunologia , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/normas , Vacinologia/métodos , Vacinologia/normas , Proteínas Virais/química , Proteínas Virais/imunologia
17.
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
18.
Eur J Histochem ; 63(3)2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31505925

RESUMO

In mammals, the alveolarization process develops predominantly after birth. Airway cells display a complex assemblage of glycans on their surface. These glycans, particularly terminal glycan extensions, are important effective carriers of information that change during the differentiation process. Nevertheless, few systematic data are reported about the cell surface sugar residue content during post-natal lung development. In the present work, we aimed to identify and semi-quantify N-acetylgalactosamine (GalNAc)/galactose (Gal) residues on the bronchioloalveolar cell surface in rat lung sections from 1-, 4-, 8- day old and adult animals and link these data with the lung glycocalyx composition. Horseradish peroxidase-conjugated lectin from Glycine max (soybean agglutinin, SBA) was used, and light microscopy methodologies were performed. SBA labelling intensity was studied before and after sialidase pre-treatment, at one-, four- and eight-day-old animals and adult animals. For semi-quantitative evaluation of SBA binding intensity, two investigators performed the analysis independently, blinded to the type of experiment. Reactivity of the lectin was assessed in bronchiolar and respiratory portion/alveolar epithelial cell surfaces. We evidenced a stronger positive reaction when lung sections were pre-treated with neuraminidase before incubation with the lectin in one- and four-day-old animals and adult animals. These results were not so manifest in eight-day-old animals. This binding pattern, generally points towards the presence of terminal but mainly sub-terminal GalNAc/Gal residues probably capped by sialic acids on the rat bronchiolar/respiratory tract epithelial cells. As this glycan extension is common in O- and N-glycans, our results suggest that these glycan classes can be present in bronchioloalveolar cells immediately after birth and exist during the postnatal period. The results observed in eight-day-old rat lung sections may be due to the dramatic lung morphologic changes and the possible underlying biological mechanisms that occur during this age-moment.


Assuntos
Acetilgalactosamina/metabolismo , Brônquios/citologia , Células Epiteliais/metabolismo , Galactose/metabolismo , Alvéolos Pulmonares/citologia , Animais , Brônquios/crescimento & desenvolvimento , Feminino , Histocitoquímica/métodos , Peroxidase do Rábano Silvestre/química , Neuraminidase/química , Lectinas de Plantas/química , Gravidez , Alvéolos Pulmonares/crescimento & desenvolvimento , Ratos Wistar , Proteínas de Soja/química
19.
Nat Microbiol ; 4(12): 2298-2309, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31527796

RESUMO

Major histocompatibility complex class II (MHC-II) molecules of multiple species function as cell-entry receptors for the haemagglutinin-like H18 protein of the bat H18N11 influenza A virus, enabling tropism of the viruses in a potentially broad range of vertebrates. However, the function of the neuraminidase-like N11 protein is unknown because it is dispensable for viral infection or the release of H18-pseudotyped viruses. Here, we show that infection of mammalian cells with wild-type H18N11 leads to the emergence of mutant viruses that lack the N11 ectodomain and acquired mutations in H18. An infectious clone of one such mutant virus, designated rP11, appeared to be genetically stable in mice and replicated to higher titres in mice and cell culture compared with wild-type H18N11. In ferrets, rP11 antigen and RNA were detected at low levels in various tissues, including the tonsils, whereas the wild-type virus was not. In Neotropical Jamaican fruit bats, wild-type H18N11 was found in intestinal Peyer's patches and was shed to high concentrations in rectal samples, resulting in viral transmission to naive contact bats. Notably, rP11 also replicated efficiently in bats; however, only restored full-length N11 viruses were transmissible. Our findings suggest that wild-type H18N11 replicates poorly in mice and ferrets and that N11 is a determinant for viral transmission in bats.


Assuntos
Quirópteros/virologia , Vírus da Influenza A/fisiologia , Infecções por Orthomyxoviridae/transmissão , Infecções por Orthomyxoviridae/veterinária , Infecções por Orthomyxoviridae/virologia , Orthomyxoviridae/fisiologia , Animais , Linhagem Celular , Furões/virologia , Células HEK293 , Especificidade de Hospedeiro , Humanos , Vírus da Influenza A/patogenicidade , Mamíferos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Neuraminidase/química , Neuraminidase/genética , Orthomyxoviridae/genética , Orthomyxoviridae/patogenicidade , Receptor de Interferon alfa e beta/genética , Receptores de Interferon/genética , Replicação Viral
20.
Nat Microbiol ; 4(12): 2216-2225, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31406333

RESUMO

A(H3N2) virus predominated recent influenza seasons, which has resulted in the rigorous investigation of haemagglutinin, but whether neuraminidase (NA) has undergone antigenic change and contributed to the predominance of A(H3N2) virus is unknown. Here, we show that the NA of the circulating A(H3N2) viruses has experienced significant antigenic drift since 2016 compared with the A/Hong Kong/4801/2014 vaccine strain. This antigenic drift was mainly caused by amino acid mutations at NA residues 245, 247 (S245N/S247T; introducing an N-linked glycosylation site at residue 245) and 468. As a result, the binding of the NA of A(H3N2) virus by some human monoclonal antibodies, including those that have broad reactivity to the NA of the 1957 A(H2N2) and 1968 A(H3N2) reference pandemic viruses as well as contemporary A(H3N2) strains, was reduced or abolished. This antigenic drift also reduced NA-antibody-based protection against in vivo virus challenge. X-ray crystallography showed that the glycosylation site at residue 245 is within a conserved epitope that overlaps the NA active site, explaining why it impacts antibody binding. Our findings suggest that NA antigenic drift impacts protection against influenza virus infection, thus highlighting the importance of including NA antigenicity for consideration in the optimization of influenza vaccines.


Assuntos
Vírus da Influenza A Subtipo H3N2/enzimologia , Vírus da Influenza A Subtipo H3N2/imunologia , Vacinas contra Influenza/imunologia , Neuraminidase/química , Neuraminidase/imunologia , Animais , Anticorpos Monoclonais , Antígenos Virais/genética , Antígenos Virais/imunologia , Domínio Catalítico , Cristalografia por Raios X , Modelos Animais de Doenças , Genes Virais/genética , Glicosilação , Hong Kong , Humanos , Imunogenicidade da Vacina , Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/prevenção & controle , Camundongos , Modelos Moleculares , Mutação , Neuraminidase/genética , Infecções por Orthomyxoviridae/imunologia , Conformação Proteica , Análise de Sequência de Proteína , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/imunologia
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