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

作者：Marco Weisshaar, Robert M. Cox, Zachary P. Morehouse, Shiva K. Kyasa, Dan Yan, Phil Oberacker, Shuli Mao, Jennifer E. Golden, Anice C. Lowen, Michael G. Natchus, Richard K. Plemper · 发表于：Journal of Virology · 年份：2016 · DOI：10.1128/jvi.00898-16 · 被引用次数：27 · 研究领域：Respiratory viral infections research、Influenza Virus Research Studies、Virology and Viral Diseases

UNLABELLED: 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...