Real Time Enzyme Inhibition Assays Provide Insights into Differences in Binding of Neuraminidase Inhibitors to Wild Type and Mutant Influenza Viruses

被引:37
作者
Barrett, Susan [1 ]
Mohr, Peter G. [2 ]
Schmidt, Peter M. [1 ]
McKimm-Breschkin, Jennifer L. [1 ]
机构
[1] CSIRO Mat Sci & Engn, Parkville, Vic, Australia
[2] CSIRO Australian Anim Hlth Lab, Geelong, Vic, Australia
来源
PLOS ONE | 2011年 / 6卷 / 08期
基金
澳大利亚国家健康与医学研究理事会;
关键词
SLOW-BINDING; ACTIVE-SITE; B VIRUSES; OSELTAMIVIR; SENSITIVITY; RESISTANCE; PERAMIVIR; SIALIDASE; SUSCEPTIBILITY; MUTATIONS;
D O I
10.1371/journal.pone.0023627
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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 IC(50)s 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 IC(50)s were calculated after each 10 min interval up to 60 min. Results showed that without preincubation IC(50)s for the wild type viruses started high and although they decreased continuously over the 60 min reaction time the final IC(50)s 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 IC(50)s, consistent with fast binding. Therefore this modified assay provides additional phenotypic information about the rate of inhibitor binding in addition to the IC(50), and critically demonstrates the differential effect of incubation times on the IC(50) and K(i) values of wild type and mutant viruses for each of the inhibitors.
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页数:9
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