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1.
Sci Rep ; 6: 25806, 2016 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-27194388

RESUMO

Human respiratory syncytial virus (hRSV) is a leading cause of acute lower respiratory tract infection in infants, elderly and immunocompromised individuals. To date, no specific antiviral drug is available to treat or prevent this disease. Here, we report that the Smoothened receptor (Smo) antagonist cyclopamine acts as a potent and selective inhibitor of in vitro and in vivo hRSV replication. Cyclopamine inhibits hRSV through a novel, Smo-independent mechanism. It specifically impairs the function of the hRSV RNA-dependent RNA polymerase complex notably by reducing expression levels of the viral anti-termination factor M2-1. The relevance of these findings is corroborated by the demonstration that a single R151K mutation in M2-1 is sufficient to confer virus resistance to cyclopamine in vitro and that cyclopamine is able to reduce virus titers in a mouse model of hRSV infection. The results of our study open a novel avenue for the development of future therapies against hRSV infection.


Assuntos
Vírus Sincicial Respiratório Humano/fisiologia , Transcrição Genética , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Alcaloides/farmacologia , Animais , Linhagem Celular , RNA Polimerases Dirigidas por DNA/metabolismo , Modelos Animais de Doenças , Farmacorresistência Viral/efeitos dos fármacos , Feminino , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Receptor Smoothened/metabolismo , Transcrição Genética/efeitos dos fármacos , Veratrum/química , Alcaloides de Veratrum/química , Alcaloides de Veratrum/farmacologia , Alcaloides de Veratrum/uso terapêutico , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
3.
Antimicrob Agents Chemother ; 55(5): 2004-10, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21343450

RESUMO

The evolution of the highly pathogenic H5N1 influenza virus produces genetic variations that can lead to changes in antiviral susceptibility and in receptor-binding specificity. In countries where the highly pathogenic H5N1 virus is endemic or causes regular epidemics, the surveillance of these changes is important for assessing the pandemic risk. In Cambodia between 2004 and 2010, there have been 26 outbreaks of highly pathogenic H5N1 influenza virus in poultry and 10 reported human cases, 8 of which were fatal. We have observed naturally occurring mutations in hemagglutinin (HA) and neuraminidase (NA) of Cambodian H5N1 viruses that were predicted to alter sensitivity to neuraminidase inhibitors (NAIs) and/or receptor-binding specificity. We tested H5N1 viruses isolated from poultry and humans between 2004 and 2010 for sensitivity to the NAIs oseltamivir (Tamiflu) and zanamivir (Relenza). All viruses were sensitive to both inhibitors; however, we identified a virus with a mildly decreased sensitivity to zanamivir and have predicted that a V149A mutation is responsible. We also identified a virus with a hemagglutinin A134V mutation, present in a subpopulation amplified directly from a human sample. Using reverse genetics, we verified that this mutation is adaptative for human α2,6-linked sialidase receptors. The importance of an ongoing surveillance of H5N1 antigenic variance and genetic drift that may alter receptor binding and sensitivities of H5N1 viruses to NAIs cannot be underestimated while avian influenza remains a pandemic threat.


Assuntos
Antivirais/farmacologia , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Virus da Influenza A Subtipo H5N1/enzimologia , Neuraminidase/antagonistas & inibidores , Animais , Linhagem Celular , Cães , Hemaglutininas/genética , Humanos , Neuraminidase/genética , Oseltamivir/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Suínos
4.
Curr Drug Targets ; 4(5): 389-408, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12816348

RESUMO

Influenza is a highly contagious, acute upper respiratory tract disease caused by influenza virus, a member of the Orthomyxoviridae family. The viral particles have two surface antigens, haemagglutinin and sialidase (neuraminidase) that extensively decorate the surface of the virus and have been implicated in viral attachment and fusion, and the release of virion progeny, respectively. The receptor for haemagglutinin is the terminal sialic acid residue of host cell surface sialyloligosaccharides, while sialidase catalyses the hydrolysis of terminal sialic acid residues from sialyloligosaccharides. Extensive crystallographic studies of both these proteins have revealed that the residues that interact with the sialic acid are strictly conserved. Therefore, these proteins make attractive targets for the design of drugs to halt the progression of the virus. Recent successful efforts in the search for new cures for influenza have led to the development of three clinically-useful anti-influenza drugs. All three are potent, selective inhibitors of influenza virus A and B sialidase. Strategies for the development of haemagglutinin inhibitors have also been devised.


Assuntos
Antivirais/química , Inibidores Enzimáticos/química , Orthomyxoviridae/efeitos dos fármacos , Antivirais/administração & dosagem , Antivirais/farmacologia , Sítios de Ligação , Desenho de Fármacos , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Humanos , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Lipossomos , Micelas , Neuraminidase/antagonistas & inibidores , Neuraminidase/química , Relação Estrutura-Atividade
5.
J Med Chem ; 44(20): 3292-301, 2001 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-11563928

RESUMO

Rotaviruses cause severe gastroenteritis in infants and are estimated to be responsible for over 600 000 deaths annually, primarily in developing countries. The development of potential inhibitors of this virus is therefore of great interest, particularly since the safety and efficacy of rotaviral vaccines has recently been questioned. This study describes the synthesis of a variety of compounds that can be considered as mimetics of N-acetylneuraminic acid thioglycosides and the subsequent in vitro biological evaluation of these sialylmimetics as inhibitors of rotaviral infection. Our results show that readily accessible carbohydrate-based compounds have the potential to act as inhibitors of rotaviral replication in vitro, presumably through inhibition of the rotaviral adhesion process.


Assuntos
Antivirais/síntese química , Glicosídeos/síntese química , Ácido N-Acetilneuramínico/análogos & derivados , Ácido N-Acetilneuramínico/síntese química , Rotavirus/efeitos dos fármacos , Animais , Antivirais/química , Antivirais/farmacologia , Bovinos , Linhagem Celular , Glicosídeos/química , Glicosídeos/farmacologia , Humanos , Mimetismo Molecular , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/farmacologia , Testes de Neutralização , Relação Estrutura-Atividade
6.
Carbohydr Res ; 332(2): 133-9, 2001 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-11434370

RESUMO

The synthesis of 3-azido-3-deoxy, 3-amino-3-deoxy and 3-N-tert-butyloxycarbonyl-3-deoxy derivatives of 2-acetamido-2-deoxy-alpha,beta-D-mannose (N-acetyl-alpha,beta-D-mannosamine, ManNAc), is presented. The 3-azido-3-deoxy- and 3-N-tert-butyloxycarbonyl compounds were further characterised as their peracetates. A preliminary study has found that these C-3 nitrogen-substituted derivatives of ManNAc not to be substrates for Neu5Ac aldolase.


Assuntos
Hexosaminas/síntese química , Oxo-Ácido-Liases/metabolismo , Hexosaminas/metabolismo , Nitrogênio/química , Ressonância Magnética Nuclear Biomolecular , Especificidade por Substrato
7.
Bioorg Med Chem Lett ; 11(12): 1587-90, 2001 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-11412987

RESUMO

Glycomimetics are increasingly being recognised as powerful tools in the search for novel compounds that possess useful biological properties. This paper describes our preliminary efforts towards the development of novel mimetics of sialic acid thioglycosides. These sialylmimetics are readily prepared and have been shown, in some instances, to have biological properties similar to sialic acid thioglycosides.


Assuntos
Sialoglicoproteínas/síntese química , Animais , Humanos , Mimetismo Molecular , Sondas Moleculares , Sialoglicoproteínas/química , Tioglicosídeos/síntese química , Tioglicosídeos/química
8.
Protein Sci ; 10(4): 689-96, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11274459

RESUMO

2,3-didehydro-2-deoxy-N:-acetylneuraminic acid (DANA) is a transition state analog inhibitor of influenza virus neuraminidase (NA). Replacement of the hydroxyl at the C9 position in DANA and 4-amino-DANA with an amine group, with the intention of taking advantage of an increased electrostatic interaction with a conserved acidic group in the active site to improve inhibitor binding, significantly reduces the inhibitor activity of both compounds. The three-dimensional X-ray structure of the complexes of these ligands and NA was obtained to 1.4 A resolution and showed that both ligands bind isosterically to DANA. Analysis of the geometry of the ammonium at the C4 position indicates that Glu119 may be neutral when these ligands bind. A computational analysis of the binding energies indicates that the substitution is successful in increasing the energy of interaction; however, the gains that are made are not sufficient to overcome the energy that is required to desolvate that part of the ligand that comes in contact with the protein.


Assuntos
Antivirais/química , Cristalografia por Raios X/métodos , Inibidores Enzimáticos/química , Ácido N-Acetilneuramínico/análogos & derivados , Ácido N-Acetilneuramínico/química , Neuraminidase/antagonistas & inibidores , Orthomyxoviridae/enzimologia , Sítios de Ligação , Cristalografia por Raios X/instrumentação , Modelos Moleculares , Análise Numérica Assistida por Computador , Ácidos Siálicos/química , Solventes/química , Eletricidade Estática
9.
Biochem Biophys Res Commun ; 280(1): 104-9, 2001 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-11162485

RESUMO

We found that the hepatopancreas of oyster, Crassostrea virginica, contained a sialidase capable of releasing Neu5Gc from the novel polysialic acid chain (-->5-O(glycolyl)Neu5Gcalpha2-->)n more efficiently than from the conventional type of polysialic acid chains, (-->8Neu5Acalpha2-->)n, or (-->8Neu5Gcalpha2-->)n. We have partially purified this novel sialidase and compared its reactivity with that of microbial sialidases using four different sialic acid dimers, Neu5Gcalpha2-->5-O(glycolyl)Neu5Gc (Gg2), Neu5Acalpha2-->8Neu5Ac (A2), Neu5Gcalpha2-->8Neu5Gc (G2), and KDNalpha2-->8KDN (K2) as substrates. Hydrolysis was monitored by high performance anion-exchange chromatography with a CarboPac PA-100 column and pulsed amperometric detection, the method by which we can accurately quantitate both the substrate (sialiac acid dimers) and the product (sialic acid monomers). The oyster sialidase effectively hydrolyzed Gg2 and K2, whereas A2 and G2 were poor substrates. Neu5Ac2en but not KDN2en effectively inhibited the hydrolysis of Gg2 by the oyster sialidase. Likewise, the hydrolysis of K2 by the oyster sialidase was inhibited by a cognate inhibitor, KDN2en, but not by Neu5Ac2en. Using the new analytical method we found that Gg2 was hydrolyzed less efficiently than A2 but much more readily than G2 by Arthrobacter ureafaciens sialidase. This result was at variance with the previous report using the thiobarbituric acid method to detect the released free sialic acid [Kitazume, S., et al. (1994) Biochem. Biophys. Res. Commun. 205, 893-898]. In agreement with previous results, Gg2 was a poor substrate for Clostridium perfringens sialidase, while K2 was refractory to all microbial sialidases tested. Thus, the oyster sialidase is novel and distinct from microbial sialidases with regards to glycon- and linkage-specificity. This finding adds an example of the presence of diverse sialidases, in line with the diverse sialic acids and sialic acid linkages that exist in nature. The new sialidase should become useful for both structural and functional studies of sialoglycoconjugates.


Assuntos
Arthrobacter/enzimologia , Clostridium perfringens/enzimologia , Sistema Digestório/enzimologia , Neuraminidase/metabolismo , Oligossacarídeos/metabolismo , Vibrio cholerae/enzimologia , Animais , Cromatografia em Gel , Cromatografia por Troca Iônica , Dimerização , Eletroquímica , Cinética , Neuraminidase/isolamento & purificação , Ostreidae , Especificidade por Substrato
10.
Bioorg Med Chem ; 8(12): 2709-18, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11131162

RESUMO

The intimate involvement of carbohydrate-protein interactions in a number of important biological processes has prompted several research efforts towards developing new methods of investigating these glycobiological interactions. Biotinylated oligosaccharides are emerging as a new and powerful tool in this area of research, primarily due to their high affinity towards streptavidin and their ease of immobilization on matrices. Here we describe a novel synthetic approach towards biotinylated saccharides which incorporate a UV absorbing group into the final compounds. The synthetic strategy described is applicable to a variety of saccharides, with examples of biotinylated mono-, di-, and trisaccharides being prepared with overall high efficiency.


Assuntos
Metabolismo dos Carboidratos , Carboidratos/síntese química , Sondas Moleculares/síntese química , Sondas Moleculares/metabolismo , Proteínas/metabolismo , Biotina/química , Sequência de Carboidratos , Carboidratos/química , Sondas Moleculares/química , Dados de Sequência Molecular , Oligossacarídeos/síntese química , Oligossacarídeos/química , Oligossacarídeos/metabolismo
11.
Carbohydr Res ; 328(3): 287-92, 2000 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-11072835

RESUMO

An efficient new entry into N,N-dialkyl-S-glycosylsulfenamides is reported. The reaction of bis-activated alkyl halides in the presence of a secondary amine base with glycosylic S-acetyl derivatives (1-S-acetyl-1-thioaldoses or 2-S-acetyl-2-thioketoses) results in the formation of novel carbohydrate sulfonamides. These new carbohydrate-based sulfonamides may provide useful derivatives with biological activity, as well as provide reactive carbohydrate sulfonylating agents.


Assuntos
Alcanos/síntese química , Sulfonamidas/síntese química , Compostos de Enxofre/síntese química , Alcanos/química , Estrutura Molecular , Sulfonamidas/química
12.
Carbohydr Res ; 328(4): 445-8, 2000 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-11093699

RESUMO

A concise route to novel mimetics of Kdn2en, based on delta4-uronic acids, from D-glucurono-6,3-lactone is presented. Uronic acid-based mimetics in which an aliphatic ether (O-glycoside), a thioether (S-glycoside), or acetamide takes the place of the natural C-6 glycerol sidechain of the sialic acid were synthesized from the key intermediate, methyl 2,3,4-tri-O-acetyl-alpha-D-glucopyranosyluronate bromide.


Assuntos
Mimetismo Molecular , Ácidos Siálicos/química , Ácidos Urônicos/síntese química , Glucuronatos/química , Estrutura Molecular
13.
Anal Biochem ; 285(1): 21-32, 2000 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-10998260

RESUMO

Rat liver Golgi and Saccharomyces cerevisiae-expressed CMP-Neu5Ac transport protein were reconstituted in phosphatidylcholine liposomes and transport of CMP-Neu5Ac into these proteoliposomes was determined. The separation of transported substrate from free substrate was performed using Multiscreen minicolumns loaded with Sephadex G-50 resin (fine). The CMP-Neu5Ac transport characteristics of the rat liver Golgi and S. cerevisiae-expressed transporters, determined using this separation system, were very similar to those previously reported. Inhibition studies, utilizing the above procedure, revealed that the main structural features required for recognition of glycosyl nucleosides by the rat liver Golgi CMP-Neu5Ac transport protein were the nature of the nucleoside base and the anomeric configuration of the associated carbohydrate. In general, pyrimidine-based glycosyl nucleosides were found to inhibit transport to a far greater extent than purine-based glycosyl nucleosides, an observation that is in good agreement with previous reports. These results indicate that the reconstitution procedure, in conjunction with Multiscreen minicolumns, is an effective high-throughput method for the determination of CMP-Neu5Ac transport.


Assuntos
Proteínas de Transporte/análise , Ácido N-Acetilneuramínico Citidina Monofosfato/metabolismo , Complexo de Golgi/metabolismo , Fígado/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Cromatografia em Gel , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Lipossomos , Nucleosídeos/metabolismo , Nucleotídeos/metabolismo , Ratos
14.
Bioorg Med Chem ; 8(3): 657-64, 2000 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10732983

RESUMO

Several C-9 modified N-acetylneuraminic acid derivatives have been synthesised and evaluated as substrates of N-acetylneuraminic acid aldolase. Simple C-9 acyl or ether modified derivatives of N-acetylneuraminic acid were found to be accepted as substrates by the enzyme, albeit being transformed more slowly than Neu5Ac itself. 1H NMR spectroscopy was used to evaluate the extent of the enzyme catalysed transformation of these compounds. Interestingly, the chain-extended Neu5Ac derivative 16 is not a substrate for N-acetylneuraminate lyase and behaves as an inhibitor of the enzyme.


Assuntos
Ácido N-Acetilneuramínico/química , Oxo-Ácido-Liases/metabolismo , Proteínas de Bactérias , Sítios de Ligação , Escherichia coli/enzimologia , Cinética , Membranas Artificiais , Sondas Moleculares , Ácido N-Acetilneuramínico/análogos & derivados , Ácido N-Acetilneuramínico/síntese química , Oxo-Ácido-Liases/antagonistas & inibidores , Oxo-Ácido-Liases/química , Especificidade por Substrato
15.
J Med Microbiol ; 49(2): 121-6, 2000 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10670562

RESUMO

The potential of motile Aeromonas species to cause human gastrointestinal infections has been recognised recently. Considerable worldwide epidemiological, microbiological and clinical investigations have shown that some strains of the different motile aeromonads are of increasing enteropathogenic significance, especially in children, the elderly and in immunocompromised individuals. Some of the diarrhoeal symptoms of Aeromonas-associated gastro-enteritis have been attributed to enterotoxins. In this study, 15 Aeromonas isolates from clinical and non-clinical sources, representing the three motile aeromonads commonly associated with gastro-enteritis (A. caviae, A. hydrophila and A. veronii biovar sobria), were tested for their ability to cause fluid accumulation in infant mice by the suckling mouse technique. Eight isolates were found to produce enterotoxin. Of these, an A. veronii biovar sobria strain (AS15), isolated from lamb kidney, was found to produce the highest enterotoxin score. An enterotoxin of c. 40 kDa produced by A. veronii biovar sobria AS15 was purified by Sephacryl S-100 gel filtration and high-performance liquid chromatography. This enterotoxin caused marked fluid accumulation in infant mice by the suckling mouse technique. The purified enterotoxin cross-reacted with cholera toxin antibodies and was readily inactivated by heating at 56 degrees C for 10 min. The production of a 'cholera-like' enterotoxin by Aeromonas isolates from samples of animal origin suggests that these organisms could be of public health significance in food products.


Assuntos
Aeromonas/metabolismo , Enterotoxinas , Gastroenterite/microbiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Aeromonas/classificação , Aeromonas/isolamento & purificação , Animais , Animais Lactentes , Anticorpos Antibacterianos/imunologia , Toxina da Cólera/imunologia , Reações Cruzadas , Enterotoxinas/biossíntese , Enterotoxinas/imunologia , Enterotoxinas/isolamento & purificação , Enterotoxinas/toxicidade , Congelamento , Infecções por Bactérias Gram-Negativas/veterinária , Temperatura Alta , Humanos , Rim/microbiologia , Camundongos , Ovinos/microbiologia , Doenças dos Ovinos/microbiologia
16.
Bioorg Med Chem Lett ; 10(24): 2791-4, 2000 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-11133093

RESUMO

1H NMR spectroscopy has been used to investigate the transfer of sialic acid from sialic acid donor molecules to acceptor molecules using the trans-sialidase from Typanosoma cruzi. It is clearly demonstrated that NMR spectroscopy is an efficient and powerful means of monitoring the trans-sialidase promoted transfer of sialic acid from donor to acceptor.


Assuntos
Glicoproteínas/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/metabolismo , Ressonância Magnética Nuclear Biomolecular , Sialiltransferases/metabolismo , Trypanosoma cruzi/enzimologia , Amino Açúcares/química , Amino Açúcares/metabolismo , Animais , Doença de Chagas/enzimologia , Doença de Chagas/etiologia , Doença de Chagas/parasitologia , Glicoproteínas/química , Lactose/análogos & derivados , Lactose/química , Lactose/metabolismo , Ácido N-Acetilneuramínico/química , Neuraminidase/química , Prótons , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Sialiltransferases/química
17.
Glycoconj J ; 16(1): 13-7, 1999 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10580646

RESUMO

The synthesis of thiosialosides as potential biological probes for investigations involving the use of sialic acid-recognising proteins has been reinvestigated. It has been found that the most efficient method for the preparation of thiosialosides free from any 2,3-didehydro sialic acid contaminants involves an intermediate HPLC purification of thiosialosides as their methyl esters. Subsequent methyl ester hydrolysis provides thiosialosides (eg. 6 and 14) which are suitable for studies involving the use of sialic acid-recognising proteins.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Ácido N-Acetilneuramínico/química , Tioglicosídeos/química , Sensibilidade e Especificidade , Ácidos Siálicos/química
20.
Carbohydr Res ; 322(3-4): 201-8, 1999 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-10637984

RESUMO

A novel N-acetylneuraminic acid analogue, 2-S-(5'-aminopentyl) 5-acetamido-3,5-dideoxy-2-thio-D-glycero-alpha-D-galacto-2- nonulopyranosidonic acid, as well as the thiosialoside 2-S-(2'-aminoethyl) 5-acetamido-3,5-dideoxy-2-thio-D-glycero-alpha-D-galacto-2- nonulopyranosidonic acid, have been synthesised and successfully coupled to CNBr-activated Sepharose 4B through the terminal amino group. The resultant affinity resins have proved efficient in purifying a number of sialic acid-recognising proteins such as Vibrio cholerae sialidase, sialidase-L from leech, trans-sialidase from Trypanosoma cruzi, and sialyltransferases from rat liver, all in high yield.


Assuntos
Ácido N-Acetilneuramínico/análogos & derivados , Proteínas/isolamento & purificação , Animais , Cálcio/metabolismo , Cromatografia de Afinidade/métodos , Brometo de Cianogênio , Sanguessugas , Ligantes , Fígado/enzimologia , Neuraminidase/metabolismo , Ratos , Proteínas Recombinantes/metabolismo , Sefarose , Sialiltransferases/metabolismo , Trypanosoma cruzi
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