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Screening for β2-adrenergic receptor agonists using the pH-sensitive dye,CypHer5, and the IN Cell Analyzer 3000


Introduction
High-throughput screening for potential targets is a major requirement in drug development and a key application is in the area of receptor activation, particularly G-protein coupled receptors (GPCRs) (1).

It is estimated that there are still 100–200 orphan GPCRs of potential therapeutic value requiring identification of natural or artificial ligands. The Sigma-RBI Library of Pharmacologically Active Compounds (LOPACTM) was used to demonstrate the capability of screening for potential agonists to the β 2-adrenergic receptor, a member of the Gs subfamily of GPCRs. This library consists of 640 well-characterized, pharmaceutically diverse compounds with known drug or drug-like activities. These compounds can be used to screen new drug targets for leads, guide secondary screening of large diverse libraries, characterize orphan receptors, and validate new high-throughput screening assays.

A clonal stable HEK293 cell line expressing the N-terminally VSV-G epitope-tagged β 2-adrenergic receptor was established using standard methods and assessed for receptor expression levels using radioligand binding studies (approximately 1.8 pmol/mg cell homogenate). The assay measuring β 2-adrenergic receptor internalization into acidic endosomal vesicles uses a red-excited pH-sensitive cyanine dye, CypHerTM5, which is effectively non-fluorescent at pH 7.4 and maximally fluorescent at pH 5.5 (2). An antibody to the N-terminal epitope-tag is labelled with CypHer5 and, on agonist stimulation, receptor internalization is detected as an increase in fluorescence in the cell. Dose response data and rank order potency were previously obtained for both agonist and antagonist treatment of β 2-adrenergic receptor-expressing cells (3).


Products used
CypHer5 labelled anti
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