title: New strategy for high throughput screening of anti-influenza virus M2 ion channel inhibitors.
abstract: Since the recent emergence of swine and avian flu, there has been a sense of urgency to discover new anti-influenza drugs. In this study, a stable cell line with M2 ion channel/ enhanced Green fluorescent protein (EGFP) co-expression was established in order to develop an EGFP-based high-throughput assay for screening M2 ion channel inhibitors. This assay directly monitors the proton conductivity of the M2 ion channel by measuring the fluorescence intensity of EGFP, which is dependent on and sensitive to pH. The ability of amantadine to inhibit the M2 ion channel was detected by this novel EGFP-based assay and then confirmed by a patch clamp recording assay. With this assay, (1S,2S,3S,5R)-(+)-3-Isopinocampheylamine and (1R,2R,3R,5S)-(-)-3-Isopinocampheylamine were identified to inhibit M2 ion channel with an antiviral profile similar to that of amantadine. This new technique will facilitate the discovery of pharmaceutical candidates and will aid in the development of new, potent, and safe anti-influenza agents.
authors.affiliation: State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, #190 Kai-Yuan Road, Guangzhou Science Park, China.
authors.forename: Chufang
authors.initials: C
authors.lastname: Li
authors.forename: Yan
authors.initials: Y
authors.lastname: Long
authors.forename: Zuoxian
authors.initials: Z
authors.lastname: Lin
authors.forename: Yanling
authors.initials: Y
authors.lastname: Jie
authors.forename: Yajuan
authors.initials: Y
authors.lastname: Xiao
authors.forename: Lingzhi
authors.initials: L
authors.lastname: Yang
authors.forename: Juanjuan
authors.initials: J
authors.lastname: Sun
authors.forename: Yizhong
authors.initials: Y
authors.lastname: Ren
authors.forename: Ling
authors.initials: L
authors.lastname: Chen
authors.forename: Zhiyuan
authors.initials: Z
authors.lastname: Li
mesh_terms: D019943:Amino Acid Substitution; D000998:Antiviral Agents / Q000494:pharmacology*; D053961:Azabicyclo Compounds / Q000494:pharmacology; D055808:Drug Discovery / Q000379:methods*; D024882:Drug Resistance, Viral; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D006863:Hydrogen-Ion Concentration; D053124:Influenza A Virus, H5N1 Subtype / Q000187:drug effects* / Q000378:metabolism; D007251:Influenza, Human / Q000188:drug therapy*; D008564:Membrane Potentials / Q000187:drug effects; D049990:Membrane Transport Modulators / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000086082:Viroporin Proteins
chemical_list: D000998:Antiviral Agents; D053961:Azabicyclo Compounds; D049452:Green Fluorescent Proteins; D049990:Membrane Transport Modulators; D050505:Mutant Proteins; D011994:Recombinant Proteins; D014763:Viral Matrix Proteins; C586712:3-isopinocampheylamine; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins; C485184:enhanced green fluorescent protein