Mutations in a Conserved Residue in the Influenza Virus Neuraminidase Active Site Decreases Sensitivity to Neu5Ac2en-Derived Inhibitors

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led to the identification of a novel series of influenza virus NA inhibitors in which the triol group of zanamivir has been replaced by a hydrophobic group linked by a carboxamide at the 6 position (6-carboxamide).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.Influenza virus possesses two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). HA is responsible for recognition of the cell surface receptor, and NA is thought to be responsible for the elution of progeny virions from infected cells, and from each other by cleavage of terminal sialic acid residues (Neu5Ac). The potential of NA as a target for antiviral therapy was investigated many years ago, when Meindl and Tuppy (13) first synthesized the unsaturated sialic acid analog Neu5Ac2en, which inhibited influenza virus replication in vitro but not in vivo (16, 17). Based on the knowledge of the three-dimensional structure of NA complexed with Neu5Ac (23), a derivative of Neu5Ac2en with a substitution of a guanidinium group at the 4 position, 4-guanidino-Neu5Ac2en (zanamivir), has been synthesized and has been shown to have potent antiviral activity both in vitro and in vivo when administered topically within the respiratory tract (7, 25, 27). 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 was replaced with a hydrophobic group linked by a carboxamide at the 6 position (21). An essential aspect of drug development is determining if and how resistant variants may arise after prolonged exposure to the inhibitor. We and others have reported the generation of variants with decreased sensitivity to zanamivir as a result of mutations in either NA (1, 3, 4, 12, 22) or HA (3, 11). We were interested in determining whether we could also isolate variants to the 6-carboxamide derivative of zanamivir by in vitro passaging in the presence of the inhibitor.