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J Virol, March 1998, p. 2456-2462, Vol. 72, No. 3
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

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

Jennifer L. McKimm-Breschkin,1,* Anjali Sahasrabudhe,1 Tony J. Blick,1 Mandy McDonald,1 Peter M. Colman,1 Graham J. Hart,2 Richard C. Bethell,2 and Joseph N. Varghese1

Biomolecular Research Institute, Parkville, Australia,1 and Enzyme Pharmacology Unit, Glaxo Wellcome Research and Development Ltd., Stevenage SG1 2NY, United Kingdom2

Received 13 October 1997/Accepted 24 November 1997

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led to the identification of a novel series of influenza virus NA inhibitors in which the triol group of zanamivir has been replaced by a hydrophobic group linked by a carboxamide at the 6 position (6-carboxamide).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.

* Corresponding author. Mailing address: Biomolecular Research Institute, 343 Royal Parade, Parkville, 3052 Australia. Phone: 61 3 9662 7200. Fax: 61 3 9662 7101. E-mail: jenny.mckimm-breschkin{at}bioresi.com.au.




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Copyright © 1998 by the American Society for Microbiology. All rights reserved.