title: Identification of Novel Fusion Inhibitors of Influenza A Virus by Chemical Genetics.
abstract: A previous screening of more than 50,000 compounds led to the identification of a pool of bioactive small molecules with inhibitory effect on the influenza A virus. One of these compounds, now widely known as nucleozin, is a small molecule that targets the influenza A virus nucleoprotein. Here we identify and characterize two structurally different novel fusion inhibitors of the influenza A virus group 1 hemagglutinin (HA), FA-583 and FA-617, with low nanomolar activities. Escape mutants that are highly resistant to each of these compounds were generated, and both were found to carry mutations localized in close proximity to the B-loop of the hemagglutinin 2 protein, which plays a crucial role in the virion-host cell fusion process. Recombinant virus, generated through reverse genetics, confirmed the resistance phenotype. In addition, the proposed binding pockets predicted by molecular docking studies are in accordance with the resistance-bearing mutation sites. We show through mechanistic studies that FA-583 and FA-617 act as fusion inhibitors by prohibiting the low-pH-induced conformational change of hemagglutinin. Our study has offered concrete biological and mechanistic explorations for the strategic development of novel fusion inhibitors of influenza A viruses.
  
  
  OBJECTIVE
  Here we report two structurally distinctive novel fusion inhibitors of influenza A virus that act by interfering with the structural change of HA at acidic pH, a process necessary for successful entry of the virus. Mutational and molecular docking studies have identified their binding pockets situated in close proximity to the B-loop region of hemagglutinin 2. The reduced sensitivity of FA-583- or FA-617-associated mutants to another compound suggests a close proximity and even partial overlap of their binding sites on hemagglutinin. Amino acid sequence alignments and crystal structure analyses of group 1 and group 2 hemagglutinins have shed light on the possible binding mode of these two compounds. This report offers new lead compounds for the design of fusion inhibitors for influenza A viruses and further shows that analysis by forward chemical genetics is a highly effective approach for the identification of novel compounds that can perturb the infectivity of viruses and to probe new druggable targets or druggable domains in various viruses.
authors.affiliation: Department of Microbiology, HKU Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong.
authors.forename: Kin Kui
authors.initials: KK
authors.lastname: Lai
authors.affiliation: Department of Microbiology, HKU Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong.
authors.forename: Nam Nam
authors.initials: NN
authors.lastname: Cheung
authors.affiliation: Department of Microbiology, HKU Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong.
authors.forename: Fang
authors.initials: F
authors.lastname: Yang
authors.affiliation: Department of Microbiology, HKU Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong.
authors.forename: Jun
authors.initials: J
authors.lastname: Dai
authors.affiliation: AIDS Institute, HKU Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong.
authors.forename: Li
authors.initials: L
authors.lastname: Liu
authors.affiliation: Department of Microbiology, HKU Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong AIDS Institute, HKU Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong.
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Department of Microbiology, HKU Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong State Key Laboratory for Emerging Infectious Disease and the Research Centre of Infection and Immunology, HKU Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong.
authors.forename: Kong Hung
authors.initials: KH
authors.lastname: Sze
authors.affiliation: Department of Microbiology, HKU Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong State Key Laboratory for Emerging Infectious Disease and the Research Centre of Infection and Immunology, HKU Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong.
authors.forename: Honglin
authors.initials: H
authors.lastname: Chen
authors.affiliation: Department of Microbiology, HKU Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong State Key Laboratory for Emerging Infectious Disease and the Research Centre of Infection and Immunology, HKU Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong kyyuen@hku.hk rytkao@hku.hk.
authors.forename: Kwok-Yung
authors.initials: KY
authors.lastname: Yuen
authors.affiliation: Department of Microbiology, HKU Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong State Key Laboratory for Emerging Infectious Disease and the Research Centre of Infection and Immunology, HKU Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong kyyuen@hku.hk rytkao@hku.hk.
authors.forename: Richard Yi Tsun
authors.initials: RY
authors.lastname: Kao
mesh_terms: D000818:Animals; D002460:Cell Line; D024882:Drug Resistance, Viral; D006389:Hemagglutinins, Viral / Q000235:genetics; D006801:Humans; D009980:Influenza A virus / Q000187:drug effects* / Q000235:genetics; D062105:Molecular Docking Simulation; D009154:Mutation; D059386:Reverse Genetics; D065147:Viral Fusion Protein Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology*
chemical_list: D006389:Hemagglutinins, Viral; D065147:Viral Fusion Protein Inhibitors; C452559:hemagglutinin HA-2 fusogenic peptide, Influenza virus