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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

¹ Pfizer Global Research and Development
² Bristol-Myers Squibb Pharmaceuticals Research Institute

^* To whom correspondence should be addressed. E-mail: David.Harden{at}bms.com.

	Abstract

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 EC<Sub>50</Sub> 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 XXXX:xx-xx)

First published on June 19, 2008, doi:10.1177/1087057108319212

Journal of Biomolecular Screening 2008;13:486.

A more recent version of this article appeared on July 1, 2008


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