Identification and Characterization of Influenza Virus Entry Inhibitors through Dual Myxovirus High-Throughput Screening

• Type of Material: Article
• Authors: Marco Weisshaar, Georgia State University; Robert Cox, Georgia State University; Zachary Morehouse, Georgia State University; Shiva Kumar Kyasa, Emory University; Dan Yan, Emory University; Phil Oberacker, Georgia State University; Shuli Mao, Emory University; Jennifer E. Golden, University of Wisconsin; Anice Lowen, Emory University; Michael Natchus, Emory University; Richard Plemper, Emory University
• Language: English
• Date: 2016-08-15
• Publisher: American Society for Microbiology
• Publication Version: Final Published Version
• Copyright Statement: © 2016, American Society for Microbiology. All Rights Reserved.
• Final Published Version (URL): http://dx.doi.org/10.1128/JVI.00898-16
• Title of Journal or Parent Work: Journal of Virology
• ISSN: 0022-538X
• Volume: 90
• Issue: 16
• Start Page: 7368
• End Page: 7387
• Grant/Funding Information: This work was supported in part by Public Health Service grants AI119196, AI011002, and HD079327 from the NIH/NIAID and NIH/NICHD (to R.K.P.).
• Keywords: RESPIRATORY SYNCYTIAL VIRUS; CONFORMATIONAL-CHANGE; POLYMERASE INHIBITOR; Life Sciences & Biomedicine; REDUCED SENSITIVITY; UNITED-STATES; IN-VIVO; A VIRUS; MEMBRANE-FUSION; 1ST-IN-CLASS MEDICINES; NEURAMINIDASE INHIBITORS; Science & Technology; Virology
• Research Categories: Biology, Virology; Biology, Microbiology; Health Sciences, Immunology

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.