Real Time Enzyme Inhibition Assays Provide Insights into Differences in Binding of Neuraminidase Inhibitors to Wild Type and Mutant Influenza Viruses
The influenza neuraminidase (NA) inhibitors zanamivir, oseltamivir and peramivir were all designed based on the knowledge that the transition state analogue of the cleaved sialic acid, 2-deoxy,2,3-dehydro N-acetyl neuraminic acid (DANA) was a weak inhibitor of NA. While DANA bound rapidly to the NA,...
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| Format: | Online |
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Public Library of Science
2011
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| Online Access: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3157426/ |
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pubmed-31574262011-08-19 Real Time Enzyme Inhibition Assays Provide Insights into Differences in Binding of Neuraminidase Inhibitors to Wild Type and Mutant Influenza Viruses Barrett, Susan Mohr, Peter G. Schmidt, Peter M. McKimm-Breschkin, Jennifer L. Research Article The influenza neuraminidase (NA) inhibitors zanamivir, oseltamivir and peramivir were all designed based on the knowledge that the transition state analogue of the cleaved sialic acid, 2-deoxy,2,3-dehydro N-acetyl neuraminic acid (DANA) was a weak inhibitor of NA. While DANA bound rapidly to the NA, modifications leading to the improved potency of these new inhibitors also conferred a time dependent or slow binding phenotype. Many mutations in the NA leading to decreased susceptibility result in loss of slow binding, hence this is a phenotypic marker of many but not all resistant NAs. We present here a simplified approach to determine whether an inhibitor is fast or slow binding by extending the endpoint fluorescent enzyme inhibition assay to a real time assay and monitoring the changes in IC50s with time. We carried out two reactions, one with a 30 min preincubation with inhibitor and the second without. The enzymatic reaction was started via addition of substrate and IC50s were calculated after each 10 min interval up to 60 min. Results showed that without preincubation IC50s for the wild type viruses started high and although they decreased continuously over the 60 min reaction time the final IC50s remained higher than for pre-incubated samples. These results indicate a slow equilibrium of association and dissociation and are consistent with slow binding of the inhibitors. In contrast, for viruses with decreased susceptibility, preincubation had minimal effect on the IC50s, consistent with fast binding. Therefore this modified assay provides additional phenotypic information about the rate of inhibitor binding in addition to the IC50, and critically demonstrates the differential effect of incubation times on the IC50 and K i values of wild type and mutant viruses for each of the inhibitors. Public Library of Science 2011-08-17 /pmc/articles/PMC3157426/ /pubmed/21858186 http://dx.doi.org/10.1371/journal.pone.0023627 Text en Barrett et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
| repository_type |
Open Access Journal |
| institution_category |
Foreign Institution |
| institution |
US National Center for Biotechnology Information |
| building |
NCBI PubMed |
| collection |
Online Access |
| language |
English |
| format |
Online |
| author |
Barrett, Susan Mohr, Peter G. Schmidt, Peter M. McKimm-Breschkin, Jennifer L. |
| spellingShingle |
Barrett, Susan Mohr, Peter G. Schmidt, Peter M. McKimm-Breschkin, Jennifer L. Real Time Enzyme Inhibition Assays Provide Insights into Differences in Binding of Neuraminidase Inhibitors to Wild Type and Mutant Influenza Viruses |
| author_facet |
Barrett, Susan Mohr, Peter G. Schmidt, Peter M. McKimm-Breschkin, Jennifer L. |
| author_sort |
Barrett, Susan |
| title |
Real Time Enzyme Inhibition Assays Provide Insights into Differences in Binding of Neuraminidase Inhibitors to Wild Type and Mutant Influenza Viruses |
| title_short |
Real Time Enzyme Inhibition Assays Provide Insights into Differences in Binding of Neuraminidase Inhibitors to Wild Type and Mutant Influenza Viruses |
| title_full |
Real Time Enzyme Inhibition Assays Provide Insights into Differences in Binding of Neuraminidase Inhibitors to Wild Type and Mutant Influenza Viruses |
| title_fullStr |
Real Time Enzyme Inhibition Assays Provide Insights into Differences in Binding of Neuraminidase Inhibitors to Wild Type and Mutant Influenza Viruses |
| title_full_unstemmed |
Real Time Enzyme Inhibition Assays Provide Insights into Differences in Binding of Neuraminidase Inhibitors to Wild Type and Mutant Influenza Viruses |
| title_sort |
real time enzyme inhibition assays provide insights into differences in binding of neuraminidase inhibitors to wild type and mutant influenza viruses |
| description |
The influenza neuraminidase (NA) inhibitors zanamivir, oseltamivir and peramivir were all designed based on the knowledge that the transition state analogue of the cleaved sialic acid, 2-deoxy,2,3-dehydro N-acetyl neuraminic acid (DANA) was a weak inhibitor of NA. While DANA bound rapidly to the NA, modifications leading to the improved potency of these new inhibitors also conferred a time dependent or slow binding phenotype. Many mutations in the NA leading to decreased susceptibility result in loss of slow binding, hence this is a phenotypic marker of many but not all resistant NAs. We present here a simplified approach to determine whether an inhibitor is fast or slow binding by extending the endpoint fluorescent enzyme inhibition assay to a real time assay and monitoring the changes in IC50s with time. We carried out two reactions, one with a 30 min preincubation with inhibitor and the second without. The enzymatic reaction was started via addition of substrate and IC50s were calculated after each 10 min interval up to 60 min. Results showed that without preincubation IC50s for the wild type viruses started high and although they decreased continuously over the 60 min reaction time the final IC50s remained higher than for pre-incubated samples. These results indicate a slow equilibrium of association and dissociation and are consistent with slow binding of the inhibitors. In contrast, for viruses with decreased susceptibility, preincubation had minimal effect on the IC50s, consistent with fast binding. Therefore this modified assay provides additional phenotypic information about the rate of inhibitor binding in addition to the IC50, and critically demonstrates the differential effect of incubation times on the IC50 and K
i values of wild type and mutant viruses for each of the inhibitors. |
| publisher |
Public Library of Science |
| publishDate |
2011 |
| url |
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3157426/ |
| _version_ |
1611471253538865152 |