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This version was published on March 1, 2008
Journal of Biomolecular Screening, Vol. 13, No. 3, 218-228 (2008)
DOI: 10.1177/1087057108315036

Use of FLIPR Membrane Potential Dyes for Validation of High-Throughput Screening with the FLIPR and µARCS Technologies: Identification of Ion Channel Modulators Acting on the GABAA Receptor

Christoph Joesch

Biofocus DPI AG, Allschwil, Switzerland

Emelie Guevarra

Biofocus DPI AG, Allschwil, Switzerland

Serge P. Parel

Biofocus DPI AG, Allschwil, Switzerland

Andreas Bergner

Biofocus DPI AG, Allschwil, Switzerland

Peter Zbinden

Biofocus DPI AG, Allschwil, Switzerland

Daniel Konrad

bSys Biological Monitoring Systems GmbH, Witterswil, Switzerland

Hugo Albrecht

Biofocus DPI AG, Allschwil, Switzerland, hugo.albrecht{at}fhnw.ch, University of Applied Sciences Northwestern Switzerland, Muttenz, Switzerland

Fluorometric imaging plate reader (FLIPR) membrane potential dyes (FMP-Red-Dye and FMP-Blue-Dye) were evaluated for the detection of compounds acting either as positive allosteric modulators or agonists on the GABAA receptor (GABAAR). A stable HEK293 cell line with constitutive expression of the rat GABA AR {alpha}1, β2, and {gamma}2 genes was used to establish a functional high-throughput screening (HTS) assay based on measurement of the membrane potential change in living cells. The assay was validated with the FLIPR technology for identification of agonists and positive allosteric modulators using GABA and diazepam as model compounds. The FMP-Red-Dye showed better performance than the FMP-Blue-Dye, and the effects induced by GABA and diazepam were comparable to electrophysiology data. Subsequently, the assay was also validated with an ultra-HTS approach known as microarrayed compound screening (µARCS). The LOPAC library was used in a test screen for an initial assessment of the technology. Finally, the FLIPR and µARCS technologies were tested with a larger screening campaign. A focused library of 3520 putative positive modulators was tested with the FLIPR assay, and a diverse subset of 84,480 compounds was selected for screening with the µARCS technology. All hits were subjected to verification using the FLIPR technology, and confirmed hits were subsequently evaluated by EC50 determination. Finally, selected hits were further confirmed with electrophysiology testing. (Journal of Biomolecular Screening 2008:218-228)

Key Words: ion channel screening • FMP-Dye • FLIPR • GABAA receptor • µARCS


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