Complementary Assays Helping to Overcome Challenges for Identifying Neuraminidase Inhibitors

Richter, Martina; Schumann, Lilia; Walther, Elisabeth; Hoffmann, Anja; Braun, Heike; Grienke, Ulrike; Rollinger, Judith M.; Grafenstein, Susanne von; Liedl, Klaus R.; Kirchmair, Johannes; Wutzler, Peter; Sauerbrei, Andreas; Schmidtke, Michaela

doi:10.2217/fvl.14.97

Cited by 24 publications

(45 citation statements)

References 34 publications

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“…Also the Ruminococcus gnavus NA is more effectively inhibited by oseltamivir carboxylate than by zanamivir (IC 50 of 30 µM versus 11.89 mM) [ 42 ]. In contrast, we observed an increased inhibitory activity of zanamivir over oseltamivir carboxylate for bacterial VCNA (IC 50 of 52 µM versus 144 µM, Table 1 ), consistent with recent findings [ 44 ]. This highlights the difficulties in translating structure-based drug developments even between bacterial NAs of different origin, though NAs share a similar fold in their active sites [ 43 ].…”

Section: Discussionsupporting

confidence: 93%

Different Inhibitory Potencies of Oseltamivir Carboxylate, Zanamivir, and Several Tannins on Bacterial and Viral Neuraminidases as Assessed in a Cell-Free Fluorescence-Based Enzyme Inhibition Assay

2017

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Neuraminidase is a key enzyme in the life cycle of influenza viruses and is present in some bacterial pathogens. We here assess the inhibitory potency of plant tannins versus clinically used inhibitors on both a viral and a bacterial model neuraminidase by applying the 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (MUNANA)-based activity assay. A range of flavan-3-ols, ellagitannins and chemically defined proanthocyanidin fractions was evaluated in comparison to oseltamivir carboxylate and zanamivir for their inhibitory activities against viral influenza A (H1N1) and bacterial Vibrio cholerae neuraminidase (VCNA). Compared to the positive controls, all tested polyphenols displayed a weak inhibition of the viral enzyme but similar or even higher potency on the bacterial neuraminidase. Structure–activity relationship analyses revealed the presence of galloyl groups and the hydroxylation pattern of the flavan skeleton to be crucial for inhibitory activity. The combination of zanamivir and EPs® 7630 (root extract of Pelargonium sidoides) showed synergistic inhibitory effects on the bacterial neuraminidase. Co-crystal structures of VCNA with oseltamivir carboxylate and zanamivir provided insight into bacterial versus viral enzyme-inhibitor interactions. The current data clearly indicate that inhibitor potency strongly depends on the biological origin of the enzyme and that results are not readily transferable. The therapeutic relevance of our findings is briefly discussed.

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Section: Discussionsupporting

confidence: 93%

Different Inhibitory Potencies of Oseltamivir Carboxylate, Zanamivir, and Several Tannins on Bacterial and Viral Neuraminidases as Assessed in a Cell-Free Fluorescence-Based Enzyme Inhibition Assay

2017

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“…Neuraminidase activity of both NAs was further confirmed based on hemagglutination of NA-treated human erythrocytes in the presence of peanut lectin (HA assay) as described recently ( Richter et al, 2015 ; Walther et al, 2015 ).…”

Section: Methodsmentioning

confidence: 91%

“…Activity of 10-fold dilutions (1:1,000 to 1:100,000) of rNanA and rNanB was determined with a fluorescence-based NA assay (FL assay) using the substrate 2-(4-methylumbelliferyl)-α- D - N -acetylneuraminic acid sodium salt hydrate (MUNANA, Sigma–Aldrich GmbH) as published ( Richter et al, 2015 ). The activity of rNanB was additionally tested at pH 5.…”

Section: Methodsmentioning

confidence: 99%

“…In contrast, oseltamivir competitively inhibits NanA ( Gut et al, 2011 ; Walther et al, 2015 ). Two natural compounds, katsumadain A and the isoprenylated flavone artocarpin, also inhibited pneumococcal NA activity ( Richter et al, 2015 ; Walther et al, 2015 ). Hence, the dual acting NAIs (active against both viral and bacterial NA) might have the potential to combat the lethal synergism between influenza virus and secondary pneumococcal infection by targeting the interaction between both pathogens.…”

Section: Introductionmentioning

confidence: 98%

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Dual Acting Neuraminidase Inhibitors Open New Opportunities to Disrupt the Lethal Synergism between Streptococcus pneumoniae and Influenza Virus

Walther

Richter

et al. 2016

Front. Microbiol.

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Secondary infections with Streptococcus pneumoniae cause severe pneumonia and enhance lethality during influenza epidemics and pandemics. Structural and functional similarities with viral neuraminidase (NA) suggest that the highly prevalent pneumococcal NAs, NanA and NanB, might contribute to this lethal synergism by supporting viral replication and that dual acting NA inhibitors (NAIs) will disrupt it. To verify this hypothesis, NanA and NanB were expressed in E. coli. After confirming their activity in enzyme assays, in vitro models with influenza virus A/Jena/8178/09 (Jena/8178) and the recombinant NanA or NanB (rNanA and rNanB) were established in A549 and MDCK cells to mimic the role of these pneumococcal NAs during co-infection. Studies on the influence of both NAs on viral receptor expression, spread, and yield revealed a distinct effect of NanA and NanB on viral replication in these in vitro models. Both enzymes were able to support Jena/8178 replication at certain concentrations. This synergism was disrupted by the NAIs oseltamivir, DANA, katsumadain A, and artocarpin exerting an inhibitory effect on viral NA and NanA. Interestingly, katsumadain A and artocarpin inhibited rNanA and rNanB similarly. Zanamivir did not show activity. These results demonstrate a key role of pneumococcal NAs in the lethal synergism with influenza viruses and reveal opportunities for its effective disruption.

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“…Inhibition of viral NA was examined using a chemiluminescence‐based assay (NA‐star® kit; Tropix, Applied Biosystems, Darmstadt, Germany) as described in the literature with some modifications …”

Section: Methodsmentioning

confidence: 99%

Zanamivir Amidoxime- and N-Hydroxyguanidine-Based Prodrug Approaches to Tackle Poor Oral Bioavailability

Schade

Kotthaus

Riebling

et al. 2015

Journal of Pharmaceutical Sciences

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Complementary Assays Helping to Overcome Challenges for Identifying Neuraminidase Inhibitors

Cited by 24 publications

References 34 publications

Different Inhibitory Potencies of Oseltamivir Carboxylate, Zanamivir, and Several Tannins on Bacterial and Viral Neuraminidases as Assessed in a Cell-Free Fluorescence-Based Enzyme Inhibition Assay

Different Inhibitory Potencies of Oseltamivir Carboxylate, Zanamivir, and Several Tannins on Bacterial and Viral Neuraminidases as Assessed in a Cell-Free Fluorescence-Based Enzyme Inhibition Assay

Dual Acting Neuraminidase Inhibitors Open New Opportunities to Disrupt the Lethal Synergism between Streptococcus pneumoniae and Influenza Virus

Zanamivir Amidoxime- and N-Hydroxyguanidine-Based Prodrug Approaches to Tackle Poor Oral Bioavailability

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