In the shadow of SARS-CoV-2, influenza seems to be an innocent virus, although new zoonotic influenza viruses evolved by mutations may lead to severe pandemics. According to WHO, there is an urgent need for better antiviral drugs. Blocking viral hemagglutinin with multivalent N-acetylneuraminic acid derivatives is a promising approach to prevent influenza infection. Moreover, dual inhibition of both hemagglutinin and neuraminidase may result in a more powerful effect. Since both viral glycoproteins can bind to neuraminic acid, we have prepared three series of amphiphilic self-assembling 2-thio-neuraminic acid derivatives constituting aggregates in aqueous medium to take advantage of their multivalent effect. One of the series was prepared by the azide-alkyne click reaction, and the other two by the thio-click reaction to yield neuraminic acid derivatives containing lipophilic tails of different sizes and an enzymatically stable thioglycosidic bond. Two of the three bis-octyl derivatives produced proved to be active against influenza viruses, while all three octyl derivatives bound to hemagglutinin and neuraminidase from H1N1 and H3N2 influenza types.
- MeSH
- chřipka lidská * farmakoterapie MeSH
- hemaglutininové glykoproteiny viru chřipky metabolismus MeSH
- hemaglutininy farmakologie MeSH
- kyselina N-acetylneuraminová farmakologie metabolismus MeSH
- kyseliny neuraminové MeSH
- lidé MeSH
- neuraminidasa metabolismus MeSH
- virus chřipky A, podtyp H1N1 * MeSH
- virus chřipky A, podtyp H3N2 MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Influenza neuraminidase is responsible for the escape of new viral particles from the infected cell surface. Several neuraminidase inhibitors are used clinically to treat patients or stockpiled for emergencies. However, the increasing development of viral resistance against approved inhibitors has underscored the need for the development of new antivirals effective against resistant influenza strains. A facile, sensitive, and inexpensive screening method would help achieve this goal. Recently, we described a multiwell plate-based DNA-linked inhibitor antibody assay (DIANA). This highly sensitive method can quantify femtomolar concentrations of enzymes. DIANA also has been applied to high-throughput enzyme inhibitor screening, allowing the evaluation of inhibition constants from a single inhibitor concentration. Here, we report the design, synthesis, and structural characterization of a tamiphosphor derivative linked to a reporter DNA oligonucleotide for the development of a DIANA-type assay to screen potential influenza neuraminidase inhibitors. The neuraminidase is first captured by an immobilized antibody, and the test compound competes for binding to the enzyme with the oligo-linked detection probe, which is then quantified by qPCR. We validated this novel assay by comparing it with the standard fluorometric assay and demonstrated its usefulness for sensitive neuraminidase detection as well as high-throughput screening of potential new neuraminidase inhibitors.
- MeSH
- antivirové látky chemie farmakologie MeSH
- chřipka lidská farmakoterapie enzymologie virologie MeSH
- DNA chemie MeSH
- inhibitory enzymů chemie farmakologie MeSH
- kyseliny fosforité chemie MeSH
- lidé MeSH
- neuraminidasa antagonisté a inhibitory metabolismus MeSH
- oseltamivir analogy a deriváty chemie MeSH
- preklinické hodnocení léčiv metody MeSH
- reprodukovatelnost výsledků MeSH
- virové proteiny antagonisté a inhibitory metabolismus MeSH
- virus chřipky A účinky léků enzymologie fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Influenza virus causes severe respiratory infections that are responsible for up to half a million deaths worldwide each year. Two inhibitors targeting viral neuraminidase have been approved to date (oseltamivir, zanamivir). However, the rapid development of antiviral drug resistance and the efficient transmission of resistant viruses among humans represent serious threats to public health. The approved influenza neuraminidase inhibitors have (oxa)cyclohexene scaffolds designed to mimic the oxonium transition state during enzymatic cleavage of sialic acid. Their active forms contain a carboxylate that interacts with three arginine residues in the enzyme active site. Recently, the phosphonate group was successfully used as an isostere of the carboxylate in oseltamivir, and the resulting compound, tamiphosphor, was identified as a highly active neuraminidase inhibitor. However, the structure of the complex of this promising inhibitor with neuraminidase has not yet been reported. Here, we analyzed the interaction of a set of oseltamivir and tamiphosphor derivatives with neuraminidase from the A/California/07/2009 (H1N1) influenza virus. We thermodynamically characterized the binding of oseltamivir carboxylate or tamiphosphor to the neuraminidase catalytic domain by protein microcalorimetry, and we determined crystal structure of the catalytic domain in complex with tamiphosphor at 1.8 Å resolution. This structural information should aid rational design of the next generation of neuraminidase inhibitors.
- MeSH
- antivirové látky chemie farmakologie MeSH
- chřipka lidská virologie MeSH
- inhibitory enzymů chemie farmakologie MeSH
- katalytická doména MeSH
- kinetika MeSH
- kyseliny fosforité metabolismus terapeutické užití MeSH
- lidé MeSH
- neuraminidasa antagonisté a inhibitory metabolismus MeSH
- oseltamivir analogy a deriváty metabolismus terapeutické užití MeSH
- pandemie MeSH
- termodynamika MeSH
- vazba proteinů MeSH
- virus chřipky A, podtyp H1N1 enzymologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Hemojuvelin (HJV) is one of essential components for expression of hepcidin, a hormone which regulates iron transport. HJV is mainly expressed in muscle and liver, and processing of HJV in both tissues is similar. However, hepcidin is expressed in liver but not in muscle and the role of the muscle HJV is yet to be established. Our preliminary analyses of mouse tissue HJV showed that the apparent molecular masses of HJV peptides are different in liver (50 kDa monomer and 35 and 20 kDa heterodimer fragments) and in muscle (55 kDa monomer and a 34 kDa possible large fragment of heterodimer). One possible explanation is glycosylation which could lead to difference in molecular mass. RESULTS: We investigated glycosylation of HJV in both liver and muscle tissue from mice. PNGase F treatment revealed that the HJV large fragments of liver and muscle were digested to peptides with similar masses, 30 and 31 kDa, respectively, and the liver 20 kDa small fragment of heterodimer was digested to 16 kDa, while the 50 kDa liver and 55 kDa muscle monomers were reduced to 42 and 48 kDa, respectively. Endo H treatment produced distinct digestion profiles of the large fragment: a small fraction of the 35 kDa peptide was reduced to 33 kDa in liver, while the majority of the 34 kDa peptide was digested to 33 kDa and a very small fraction to 31 kDa in muscle. In addition, liver HJV was found to be neuraminidase-sensitive but its muscle counterpart was neuraminidase-resistant. CONCLUSIONS: Our results indicate that different oligosaccharides are attached to liver and muscle HJV peptides, which may contribute to different functions of HJV in the two tissues.
- MeSH
- extracelulární prostor metabolismus MeSH
- genový knockout MeSH
- glykopeptidasa metabolismus MeSH
- glykosylace MeSH
- játra cytologie metabolismus MeSH
- membránové proteiny nedostatek genetika izolace a purifikace metabolismus MeSH
- myši MeSH
- neuraminidasa metabolismus MeSH
- orgánová specificita MeSH
- svaly cytologie metabolismus MeSH
- transport proteinů MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- degenerace nervu MeSH
- dospělí MeSH
- fibroblasty enzymologie MeSH
- játra patologie MeSH
- lidé MeSH
- mladiství MeSH
- myoklonus komplikace MeSH
- neuraminidasa metabolismus nedostatek MeSH
- oligosacharidy moč MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
- MeSH
- alfa-glukosidasy chemie MeSH
- isoelektrická fokusace MeSH
- krysa rodu rattus MeSH
- mikroklky enzymologie MeSH
- morčata MeSH
- neuraminidasa metabolismus MeSH
- rozpustnost MeSH
- techniky in vitro MeSH
- tenké střevo enzymologie ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- morčata MeSH
- zvířata MeSH
- Publikační typ
- srovnávací studie MeSH
- MeSH
- časové faktory MeSH
- daktinomycin farmakologie MeSH
- druhová specificita MeSH
- glykoproteiny MeSH
- hemaglutininy virové metabolismus MeSH
- kultivační techniky MeSH
- kultivované buňky enzymologie MeSH
- kuřecí embryo MeSH
- kyseliny neuraminové metabolismus MeSH
- myši MeSH
- neuraminidasa metabolismus MeSH
- Orthomyxoviridae imunologie patogenita MeSH
- vaječný bílek MeSH
- virová hemaglutinace MeSH
- virus newcastleské nemoci imunologie patogenita MeSH
- zvířata MeSH
- Check Tag
- kuřecí embryo MeSH
- myši MeSH
- zvířata MeSH
