title: Detection of proton movement directly across viral membranes to identify novel influenza virus M2 inhibitors.
abstract: The influenza virus M2 protein is a well-validated yet underexploited proton-selective ion channel essential for influenza virus infectivity. Because M2 is a toxic viral ion channel, existing M2 inhibitors have been discovered through live virus inhibition or medicinal chemistry rather than M2-targeted high-throughput screening (HTS), and direct measurement of its activity has been limited to live cells or reconstituted lipid bilayers. Here, we describe a cell-free ion channel assay in which M2 ion channels are incorporated into virus-like particles (VLPs) and proton conductance is measured directly across the viral lipid bilayer, detecting changes in membrane potential, ion permeability, and ion channel function. Using this approach in high-throughput screening of over 100,000 compounds, we identified 19 M2-specific inhibitors, including two novel chemical scaffolds that inhibit both M2 function and influenza virus infectivity. Counterscreening for nonspecific disruption of viral bilayer ion permeability also identified a broad-spectrum antiviral compound that acts by disrupting the integrity of the viral membrane. In addition to its application to M2 and potentially other ion channels, this technology enables direct measurement of the electrochemical and biophysical characteristics of viral membranes.
authors.affiliation: Integral Molecular, Inc., Philadelphia, Pennsylvania, USA.
authors.forename: Chidananda
authors.initials: C
authors.lastname: Sulli
authors.forename: Soma S R
authors.initials: SS
authors.lastname: Banik
authors.forename: Justin
authors.initials: J
authors.lastname: Schilling
authors.forename: Allan
authors.initials: A
authors.lastname: Moser
authors.forename: Xiaoxiao
authors.initials: X
authors.lastname: Xiang
authors.forename: Riley
authors.initials: R
authors.lastname: Payne
authors.forename: Antony
authors.initials: A
authors.lastname: Wanless
authors.forename: Sharon H
authors.initials: SH
authors.lastname: Willis
authors.forename: Cheryl
authors.initials: C
authors.lastname: Paes
authors.forename: Joseph B
authors.initials: JB
authors.lastname: Rucker
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Doranz
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism / Q000821:virology*; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009980:Influenza A virus / Q000502:physiology*; D007251:Influenza, Human / Q000188:drug therapy / Q000473:pathology / Q000821:virology*; D007473:Ion Channels / Q000187:drug effects*; D008051:Lipid Bilayers / Q000378:metabolism; D011522:Protons; D054852:Small Molecule Libraries; D014763:Viral Matrix Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014771:Virion; D000086082:Viroporin Proteins
chemical_list: D000998:Antiviral Agents; D007473:Ion Channels; D008051:Lipid Bilayers; D011522:Protons; D054852:Small Molecule Libraries; D014763:Viral Matrix Proteins; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins