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Screening for potential beta 2-adrenergic receptor antagonists using CypHer5E and IN Cell Analyzer 1000


Introduction
CypHer5, a pH sensitive dye, has shown utility in β2-adrenergic receptor agonist screening (1). CypHer5 has been used to obtain dose-response and rank-order potency data for both agonist and antagonist treatment of β2-adrenergic receptor expressing cells (2). For live-cell receptor internalization studies, an enhanced version of this dye, CypHer5E, has been developed (3). CypHer5E is a red-excited pH sensitive fluorogenic dye that displays minimal fluorescence at basic pH and maximal fluorescence at acidic pH. This characteristic makes it ideal for monitoring the translocation of cell surface receptors into the endosomal pathway. Such translocation is a key element of receptor trafficking and activation, particularly in G-protein coupled receptors (GPCRs) (4).

In this assay system, a CypHer5E-conjugated anti-VSV-G epitope antibody is incubated with a clonal stable HEK 293 cell line expressing N-terminally epitope tagged β2-adrenergic receptor. Upon agonist stimulation, a fluorescent signal is observed as the CypHer5E–antibody conjugate is internalized alongside the receptor into acidic endosomal vesicles.

This application note describes the validation of antagonist-format CypHer5E receptor internalization assays for high-throughput screening. A β2adrenergic receptor antagonist screen was performed using the LOPACcompound library, which consists of 640 well-characterized, pharmaceutically diverse compounds with known drug or drug-like activity. The data demonstrate that the β2-adrenergic receptor CypHer5E antagonist assay used in conjunction with IN Cell Analyzer 1000 was able to identify all specific β2-adrenergic receptor antagonists and additional structurally related compounds present in the LOPAC compound library. CypHer5E, consequently, can be used to screen for novel antagonists of known receptors and
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