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

1. Marco Weisshaar^a,
2. Robert Cox^a,
3. Zachary Morehouse^a,
4. Shiva Kumar Kyasa^b,
5. Dan Yan^a,
6. Phil Oberacker^a,
7. Shuli Mao^b,
8. Jennifer E. Golden^c,
9. Anice C. Lowen^d,
10. Michael G. Natchus^b and
11. Richard K. Plemper^a

1. ^aInstitute for Biomedical Sciences, Georgia State University, Atlanta, GA, USA
2. ^bEmory Institute for Drug Development, Emory University, Atlanta, GA, USA
3. ^cDepartment of Pharmacology, University of Wisconsin, Madison, WI, USA
4. ^dDepartment of Microbiology & Immunology, Emory University School of Medicine, Atlanta, GA, USA

ABSTRACT

Influenza A virus (IAV) infections induce serious morbidity and mortality, generating an urgent necessity 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 along with over 142,000 entries. Focusing on IAV-individual hits, we obtained a strike price of 0.03% after cytotoxicity testing and counterscreening. 3 chemically distinct strike classes along 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, yet another group 1 subtype. Target identification through biolayer interferometry confirmed binding of every one of strike compounds to HA. Resistance profiling revealed two distinct escape mechanisms: primary resistance, associated along with low compound binding, and secondary resistance, associated along with unaltered binding. Secondary resistance was mediated, unusually, through two various pairs of cooperative mutations, each combining a mutation eliminating the membrane-proximal stalk N-glycan along with a membrane-distal adjustment in HA1 or HA2. Chemical synthesis of an analog library combined along with in silico docking extracted a docking pose for the strike classes. Chemical interrogation spotlights IAV HA as a serious druggable target for small-molecule inhibition. Our study identifies novel chemical scaffolds along with higher developmental potential, outlines diverse routes of IAV escape from entry inhibition, and establishes a road toward structure-aided lead development.

IMPORTANCE This study is among the very first to use 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 lot of potential druggable targets was thus chemically interrogated, however mechanistic characterization, positive target identification, and resistance profiling demonstrated that 3 chemically promising and structurally distinct strike classes decided on for further analysis every one of block HA-mediated membrane fusion. Viral escape from inhibition could be attained 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 additionally 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.

FOOTNOTES

• • Received 7 Might 2016.
  • Accepted 26 Might 2016.
  • Accepted manuscript posted online 1 June 2016.
• Address correspondence to Richard K. Plemper, rplemper{at}gsu.edu.

• Citation Weisshaar M, Cox R, Morehouse Z, Kumar Kyasa S, Yan D, Oberacker P, Mao S, Golden JE, Lowen AC, Natchus MG, Plemper RK. 2016. Identification and characterization of influenza virus entry inhibitors through dual myxovirus high-throughput screening. J Virol 90:7368–7387. doi:10.1128/JVI.00898-16.

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• Copyright © 2016, American Society for Microbiology. every one of Rights Reserved.

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