title: Identification and Characterization of Influenza Virus Entry Inhibitors through Dual Myxovirus High-Throughput Screening.
abstract: Influenza A virus (IAV) infections cause major morbidity and mortality, generating an urgent need for novel antiviral therapeutics. We recently established a dual myxovirus high-throughput screening protocol that combines a fully replication-competent IAV-WSN strain and a respiratory syncytial virus reporter strain for the simultaneous identification of IAV-specific, paramyxovirus-specific, and broad-spectrum inhibitors. In the present study, this protocol was applied to a screening campaign to assess a diverse chemical library with over 142,000 entries. Focusing on IAV-specific hits, we obtained a hit rate of 0.03% after cytotoxicity testing and counterscreening. Three chemically distinct hit classes with nanomolar potency and favorable cytotoxicity profiles were selected. Time-of-addition, minigenome, and viral entry studies demonstrated that these classes block hemagglutinin (HA)-mediated membrane fusion. Antiviral activity extends to an isolate from the 2009 pandemic and, in one case, another group 1 subtype. Target identification through biolayer interferometry confirmed binding of all hit compounds to HA. Resistance profiling revealed two distinct escape mechanisms: primary resistance, associated with reduced compound binding, and secondary resistance, associated with unaltered binding. Secondary resistance was mediated, unusually, through two different pairs of cooperative mutations, each combining a mutation eliminating the membrane-proximal stalk N-glycan with a membrane-distal change in HA1 or HA2. Chemical synthesis of an analog library combined with in silico docking extracted a docking pose for the hit classes. Chemical interrogation spotlights IAV HA as a major druggable target for small-molecule inhibition. Our study identifies novel chemical scaffolds with high developmental potential, outlines diverse routes of IAV escape from entry inhibition, and establishes a path toward structure-aided lead development.
  
  
  
  This study is one of the first to apply a fully replication-competent third-generation IAV reporter strain to a large-scale high-throughput screen (HTS) drug discovery campaign, allowing multicycle infection and screening in physiologically relevant human respiratory cells. A large number of potential druggable targets was thus chemically interrogated, but mechanistic characterization, positive target identification, and resistance profiling demonstrated that three chemically promising and structurally distinct hit classes selected for further analysis all block HA-mediated membrane fusion. Viral escape from inhibition could be achieved through primary and secondary resistance mechanisms. In silico docking predicted compound binding to a microdomain located at the membrane-distal site of the prefusion HA stalk that was also previously suggested as a target site for chemically unrelated HA inhibitors. This study identifies an unexpected chemodominance of the HA stalk microdomain for small-molecule inhibitors in IAV inhibitor screening campaigns and highlights a novel mechanism of cooperative resistance to IAV entry blockers.
authors.affiliation: Institute for Biomedical Sciences, Georgia State University, Atlanta, GA, USA.
authors.forename: Marco
authors.initials: M
authors.lastname: Weisshaar
authors.affiliation: Institute for Biomedical Sciences, Georgia State University, Atlanta, GA, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Cox
authors.affiliation: Institute for Biomedical Sciences, Georgia State University, Atlanta, GA, USA.
authors.forename: Zachary
authors.initials: Z
authors.lastname: Morehouse
authors.affiliation: Emory Institute for Drug Development, Emory University, Atlanta, GA, USA.
authors.forename: Shiva K
authors.initials: SK
authors.lastname: Kyasa
authors.affiliation: Institute for Biomedical Sciences, Georgia State University, Atlanta, GA, USA.
authors.forename: Dan
authors.initials: D
authors.lastname: Yan
authors.affiliation: Institute for Biomedical Sciences, Georgia State University, Atlanta, GA, USA.
authors.forename: Phil
authors.initials: P
authors.lastname: Oberacker
authors.affiliation: Emory Institute for Drug Development, Emory University, Atlanta, GA, USA.
authors.forename: Shuli
authors.initials: S
authors.lastname: Mao
authors.affiliation: Department of Pharmacology, University of Wisconsin, Madison, WI, USA.
authors.forename: Jennifer E
authors.identifier: 0000-0002-6813-3710
authors.initials: JE
authors.lastname: Golden
authors.affiliation: Department of Microbiology & Immunology, Emory University School of Medicine, Atlanta, GA, USA.
authors.forename: Anice C
authors.initials: AC
authors.lastname: Lowen
authors.affiliation: Emory Institute for Drug Development, Emory University, Atlanta, GA, USA.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Natchus
authors.affiliation: Institute for Biomedical Sciences, Georgia State University, Atlanta, GA, USA rplemper@gsu.edu.
authors.forename: Richard K
authors.initials: RK
authors.lastname: Plemper
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology* / Q000633:toxicity; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D024882:Drug Resistance, Viral; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006801:Humans; D009980:Influenza A virus / Q000187:drug effects* / Q000502:physiology*; D009154:Mutation; D011485:Protein Binding; D053586:Virus Internalization / Q000187:drug effects*
chemical_list: D000998:Antiviral Agents; D019267:Hemagglutinin Glycoproteins, Influenza Virus