real time enzyme inhibition assays provide insights into differences in binding of neuraminidase inhibitors to wild type and mutant influenza viruses实时酶抑制试验提供见解不同的绑定的野生型和突变体流感病毒神经氨酸酶抑制剂.pdfVIP

Real Time Enzyme Inhibition Assays Provide Insights into Differences in Binding of Neuraminidase Inhibitors to Wild Type and Mutant Influenza Viruses 1 2 1 1 Susan Barrett , Peter G. Mohr , Peter M. Schmidt , Jennifer L. McKimm-Breschkin * 1 CSIRO Materials Science and Engineering, Parkville, Australia, 2 CSIRO Australian Animal Health Laboratory, Geelong, Australia Abstract 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 con