Mutations in a conserved residue in the influenza virus neuraminidase active site decreases sensitivity to Neu5Ac2en-derived inhibitors

Abstract

The influenza virus neuraminidase (NA)-specific inhibitor zanamivir (4-guanidino-Neu5Ac2en) is effective in humans when administered topically within the respiratory tract. The search for compounds with altered pharmacological properties has led to the identification of a novel series of influenza virus NA inhibitors in which the triol group of zanamivir has been replaced by a hydrophobic group linked by a carboxamide at the 6 position (6-carboxamide). NWS/G70C variants generated in vitro, with decreased sensitivity to 6-carboxamide, contained hemagglutinin (HA) and/or NA mutations. HA mutants bound with a decreased efficiency to the cellular receptor and were cross-resistant to all the NA inhibitors tested. The NA mutation, an Arg-to-Lys mutation, was in a previously conserved site, Arg292, which forms part of a triarginyl cluster in the catalytic site. In enzyme assays, the NA was equally resistant to zanamivir and 4-amino-Neu5Ac2en but showed greater resistance to 6-carboxamide and was most resistant to a new carbocyclic NA inhibitor, GS4071, which also has a hydrophobic side chain at the 6 position. Consistent with enzyme assays, the lowest resistance in cell culture was seen to zanamivir, more resistance was seen to 6-carboxamide, and the greatest resistance was seen to GS4071. Substrate binding and enzyme activity were also decreased in the mutant, and consequently, virus replication in both plaque assays and liquid culture was compromised. Altered binding of the hydrophobic side chain at the 6 position or the triol group could account for the decreased binding of both the NA inhibitors and substrate.

FIG. 1

Structure of NA substrate and inhibitors used in the study. 1, Neu5Ac; 2, Neu5Ac2en; 3, 4-amino-Neu5Ac2en; 4, zanamivir (4-guanidino-Neu5Ac2en); 5, 6-carboxamide; 6, GS4071.

FIG. 2

Inhibition of the NWS/G70C parent and ir2 (Arg292Lys) NA variant enzyme activity in a MUNANA assay. (a) Neu5Ac2en; (b) 4-amino-Neu5Ac2en; (c) zanamivir; (d) 6-carboxamide; (e) GS4071 analog.

FIG. 3

Relative specific activity of the NWS/G70C parent and Arg292Lys NAs. Crystals of both were redissolved, and the activity was assessed in a 96-well MUNANA-based enzyme activity assay. The same dilutions were titrated in parallel in a capture ELISA, using the NC-10 monoclonal antibody to quantitate native NA protein.

FIG. 4

Effect of exogenous C. perfringens NA on the plaque morphology of the NWS/G70C parent and ir2 Arg292Lys NA mutant.

FIG. 5

Kinetics of replication of the NWS/G70C virus and the mutants. Cells were infected with an MOI of 0.1, and the yields were assayed by a TCID₅₀ assay. The effect of exogenous C. perfringens NA on the replication kinetics was also monitored for the ir2 mutant and G70C wild-type virus. See Table 1 for nomenclature of the variants.

FIG. 6

Kinetics of adsorption of the NWS/G70C virus and mutants. Approximately 100 PFU of virus was adsorbed onto MDCK cells for 15, 30, or 60 min before the inoculum was removed and overlay was added. The percent efficiency of adsorption was calculated by comparing the numbers of plaques for these times to numbers of plaques when virus was allowed to adsorb for 60 min at room temperature followed by 60 min at 37°C before the inoculum was removed.