title: A point mutation in influenza B neuraminidase confers resistance to peramivir and loss of slow binding.
abstract: The influenza neuraminidase (NA) inhibitors peramivir, oseltamivir, and zanamivir are potent inhibitors of NAs from both influenza A and B strains. In general, these inhibitors are slow, tight binders of NA, exhibiting time-dependent inhibition. A mutant of influenza virus B/Yamagata/16/88 which was resistant to peramivir was generated by passage of the virus in tissue culture, in the presence of increasing concentrations (0.1-120 microM over 15 passages) of the compound. Whereas the wild type (WT) virus was inhibited by peramivir with an EC(50) value of 0.10 microM, virus isolated at passages 3 and 15 displayed EC(50) values of 10 and >50 microM, respectively. Passage 3 virus contained 3 hemagglutinin (HA) mutations, but no NA mutation. Passage 15 (P15R) virus contained an additional 3 HA mutations, plus the NA mutation His273Tyr. The mechanism of inhibition of WT and P15R NA by peramivir was examined in enzyme assays. The WT and P15R NAs displayed IC(50) values of 8.4+/-0.4 and 127+/-16 nM, respectively, for peramivir. Peramivir inhibited the WT enzyme in a time-dependent fashion, with a K(i) value of 0.066+/-0.002nM. In contrast, the P15R enzyme did not display the property of slow binding and was inhibited competitively with a K(i) value of 4.69+/-0.44nM. Molecular modeling suggested that His273 was relatively distant from peramivir (>5A) in the NA active site, but that Tyr273 introduced a repulsive interaction between the enzyme and inhibitor, which may have been responsible for peramivir resistance.
authors.affiliation: Johnson & Johnson Pharmaceutical Research & Development, L.L.C., Raritan, NJ 08869, USA. ebaum@prdus.jnj.com
authors.forename: Ellen Z
authors.initials: EZ
authors.lastname: Baum
authors.forename: Pamela C
authors.initials: PC
authors.lastname: Wagaman
authors.forename: Linh
authors.initials: L
authors.lastname: Ly
authors.forename: Ignatius
authors.initials: I
authors.lastname: Turchi
authors.forename: Jianhua
authors.initials: J
authors.lastname: Le
authors.forename: Doris
authors.initials: D
authors.lastname: Bucher
authors.forename: Karen
authors.initials: K
authors.lastname: Bush
mesh_terms: D000146:Acids, Carbocyclic; D000998:Antiviral Agents / Q000378:metabolism / Q000494:pharmacology*; D002478:Cells, Cultured; D003517:Cyclopentanes / Q000378:metabolism / Q000494:pharmacology*; D024882:Drug Resistance, Viral; D006146:Guanidines; D006388:Hemagglutinins / Q000737:chemistry; D006801:Humans; D009981:Influenza B virus / Q000201:enzymology* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009439:Neuraminidase / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics* / Q000502:physiology*; D011485:Protein Binding; D010948:Viral Plaque Assay
chemical_list: D000146:Acids, Carbocyclic; D000998:Antiviral Agents; D003517:Cyclopentanes; D006146:Guanidines; D006388:Hemagglutinins; D009439:Neuraminidase; C414210:peramivir