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Characterization of the 5-HT_2b Receptor in Evaluation of Aequorin Detection of Calcium Mobilization for Miniaturized GPCR High-Throughput Screening

HTS CoE, Pfizer Global Research and Development, Groton, Connecticut, mark.gilchrist{at}pfizer.com

Angela Cacace

Applied Genomics, Bristol-Myers Squibb Pharmaceuticals Research Institute, Wallingford, Connecticut

David G. Harden

Lead Discovery, Bristol-Myers Squibb Pharmaceuticals Research Institute, Wallingford, Connecticut

Fluorescent detection of calcium mobilization has been used successfully to identify modulators of G-protein—coupled receptors (GPCRs); however, inherent issues with fluorescence may limit its potential for high-throughput screening miniaturization. The data presented here demonstrate that the calcium-sensitive photoprotein aequorin (AequoScreen^TM), when compared with FLUO-4 in the same cellular background, allows for miniaturization of functional kinetic calcium flux assays, in which the rank order of potency and efficacy was maintained for a series of diverse small-molecule modulators. Small-volume (<10 µL) 384- and 1536-well aequorin assays were implemented by integration of acoustic dispensing (Echo 550^TM) and kinetic flash luminometry (CyBi Lumax^TM). The enhanced high signal-to-background ratios observed relative to fluorescence were readily manipulated by altering per-well cell densities and yielded acceptable screening statistics in miniaturized format for both agonist and antagonist screening scenarios. In addition, the authors demonstrate the feasibility of using agonist concentrations less than EC₅₀ in a miniaturized antagonist assay. These features, coupled with improved sample handling, should enhance sensitivity and provide the benefits of miniaturization including cost reduction and throughput gains. (Journal of Biomolecular Screening 2008:486-493)

Key Words: aequorin • G-protein—coupled receptors (GPCRs) • miniaturization • acoustic dispense

This version was published on July 1, 2008

Journal of Biomolecular Screening, Vol. 13, No. 6, 486-493 (2008)
DOI: 10.1177/1087057108319212


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