A surface plasmon resonance assay for measurement of neuraminidase inhibition, sensitivity of wild-type influenza neuraminidase and its H274Y mutant to the antiviral drugs zanamivir and oseltamivir.
Antiviral resistance is currently monitored by a labelled enzymatic assay, which can give inconsistent results because of the short half-life of the labelled product, and variations in assay conditions. In this paper, we describe a competitive surface plasmon resonance (SPR) inhibition assay for measuring the sensitivities of wild-type neuraminidase (WT NA) and the H274Y (histidine 274 tyrosine) NA mutant to antiviral drugs. The two NA isoforms were expressed in High-five™ (Trichoplusia ni) insect cells. A spacer molecule (1,6-hexanediamine (HDA)) was conjugated to the 7-hydroxyl group of zanamivir, and the construct (HDA-zanamivir) was immobilized onto a SPR sensor chip to obtain a final immobilization response of 431 response units. The immobilized HDA-zanamivir comprised a bio-specific ligand for the WT and mutant proteins. The effects of the natural substrate (sialic acid) and two inhibitors (zanamivir and oseltamivir) on NA binding to the immobilized ligand were studied. The processed SPR data was analysed to determine 50% inhibitory concentrations (IC50-spr ), using a log dose-response curve fit. Although both NA isoforms had almost identical IC50-spr values for sialic acid (WT = 5.5 nM; H274Y mutant = 3.25 nM) and zanamivir (WT = 2.16 nM; H274Y mutant = 2.42 nM), there were significant differences between the IC50-spr values obtained for the WT (7.7 nM) and H274Y mutant (256 nM) NA in the presence of oseltamivir, indicating that oseltamivir has a reduced affinity for the H274Y mutant. The SPR inhibition assay strategy presented in this work could be applied for the rapid screening of newly emerging variants of NA for their sensitivity to antiviral drugs.
Biomolecular Interaction Centre, University of Canterbury, Christchurch, New Zealand.|Chemical and Process Engineering, University of Canterbury, Private Bag 4800, 8140, Christchurch, Canterbury, New Zealand.
Balaji
B
Somasundaram
Biomolecular Interaction Centre, University of Canterbury, Christchurch, New Zealand.|Chemical and Process Engineering, University of Canterbury, Private Bag 4800, 8140, Christchurch, Canterbury, New Zealand.
Conan J
CJ
Fee
Biomolecular Interaction Centre, University of Canterbury, Christchurch, New Zealand.|Chemical and Process Engineering, University of Canterbury, Private Bag 4800, 8140, Christchurch, Canterbury, New Zealand.
Rayleen
R
Fredericks
Biomolecular Interaction Centre, University of Canterbury, Christchurch, New Zealand.|Department of Chemistry, University of Canterbury, Private Bag 4800, 8140, Christchurch, New Zealand.
Andrew J A
AJ
Watson
Biomolecular Interaction Centre, University of Canterbury, Christchurch, New Zealand.|Department of Chemistry, University of Canterbury, Private Bag 4800, 8140, Christchurch, New Zealand.
Antony J
AJ
Fairbanks
National Centre for Biosecurity and Infectious Disease (NCBID), Institute of Environmental Science and Research (ESR), 66 Ward Street, 5018, Upper Hutt, New Zealand.
Richard J
RJ
Hall
D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology*; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000378:metabolism*; D006801:Humans; D007251:Influenza, Human / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000821:virology; D020128:Inhibitory Concentration 50; D007313:Insecta / Q000166:cytology; D009154:Mutation; D009439:Neuraminidase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D053139:Oseltamivir / Q000737:chemistry / Q000494:pharmacology*; D020349:Surface Plasmon Resonance; D053243:Zanamivir / Q000737:chemistry / Q000494:pharmacology*
D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D053139:Oseltamivir; D009439:Neuraminidase; D053243:Zanamivir
antiviral drugs; drug resistance; influenza neuraminidase; inhibition assay; surface plasmon resonance