title: Resistance to Mutant Group 2 Influenza Virus Neuraminidases of an Oseltamivir-Zanamivir Hybrid Inhibitor.
abstract: Influenza virus neuraminidase (NA) drug resistance is one of the challenges to preparedness against epidemic and pandemic influenza virus infections. NA N1- and N2-containing influenza viruses are the primary cause of seasonal epidemics and past pandemics. The structural and functional basis underlying drug resistance of the influenza virus N1 NA is well characterized. Yet drug resistance of the N2 strain is not well understood. Here, we confirm that replacement of N2 E119 or I222 results in multidrug resistance, and when the replacements occur together, the sensitivity to NA inhibitors (NAI) is reduced severely. Using crystallographic studies, we showed that E119 replacement results in a loss of hydrogen bonding to oseltamivir and zanamivir, whereas I222 replacement results in a change in the hydrophobic environment that is critical for oseltamivir binding. Moreover, we found that MS-257, a zanamivir-oseltamivir hybrid inhibitor, is less susceptible to drug resistance. The binding mode of MS-257 shows that increased hydrogen bonding interactions between the inhibitor and NA active site anchor the inhibitor within the active site and allow adjustments in response to active-site modifications. Such stability is likely responsible for the observed reduced susceptibility to drug resistance. MS-257 serves as a next-generation anti-influenza virus drug candidate and serves also as a scaffold for further design of NAIs.
  
  
  OBJECTIVE
  Oseltamivir and zanamivir are the two major antiviral drugs available for the treatment of influenza virus infections. However, multidrug-resistant viruses have emerged in clinical cases, which pose a challenge for the development of new drugs. N1 and N2 subtypes exist in the viruses which cause seasonal epidemics and past pandemics. Although N1 drug resistance is well characterized, the molecular mechanisms underlying N2 drug resistance are unknown. A previous report showed that an N2 E119V/I222L dual mutant conferred drug resistance to seasonal influenza virus. Here, we confirm that these substitutions result in multidrug resistance and dramatically reduced sensitivity to NAI. We further elucidate the molecular mechanism underlying N2 drug resistance by solving crystal structures of the N2 E119V and I222L mutants and the dual mutant. Most importantly, we found that a novel oseltamivir-zanamivir hybrid inhibitor, MS-257, remains more effective against drug-resistant N2 and is a promising candidate as a next-generation anti-influenza virus drug.
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.|Center for Influenza Research and Early-warning, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.|University of Chinese Academy of Sciences, Beijing, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Gao
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
authors.forename: Jianxun
authors.initials: J
authors.lastname: Qi
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.|Center for Influenza Research and Early-warning, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
authors.forename: Yuhai
authors.initials: Y
authors.lastname: Bi
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
authors.forename: Lifeng
authors.initials: L
authors.lastname: Fu
authors.affiliation: Department of Chemistry, Simon Fraser University, Burnaby, BC, Canada.
authors.forename: Sankar
authors.initials: S
authors.lastname: Mohan
authors.affiliation: Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.
authors.forename: Yuhang
authors.initials: Y
authors.lastname: Chen
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
authors.forename: Xuebing
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Chemistry, Simon Fraser University, Burnaby, BC, Canada.
authors.forename: B Mario
authors.initials: BM
authors.lastname: Pinto
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Vavricka
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China tienpo@im.ac.cn gaof@im.ac.cn.
authors.forename: Po
authors.initials: P
authors.lastname: Tien
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China tienpo@im.ac.cn gaof@im.ac.cn.|National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China.|Research Network of Immunity and Health, Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing, China.|Center for Influenza Research and Early-warning, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
authors.forename: George F
authors.initials: GF
authors.lastname: Gao
mesh_terms: D019943:Amino Acid Substitution; D000998:Antiviral Agents / Q000494:pharmacology*; D024921:Drug Resistance, Multiple, Viral / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D009980:Influenza A virus / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation; D009439:Neuraminidase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics*; D053139:Oseltamivir / Q000494:pharmacology*; D014764:Viral Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics*; D053243:Zanamivir / Q000494:pharmacology*
chemical_list: D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D014764:Viral Proteins; D053139:Oseltamivir; D009439:Neuraminidase; D053243:Zanamivir