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

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 for potential ligands of non-G-protein coupled receptors (5) and orphan receptors.


Other materials required
LOPAC compound library (Sigma)

HEK293 cell line expressing VSV-G epitope tagged β2-adrenergic receptor

Culture medium:
MEM (Sigma)
10% (v/v) FBS (Sigma)
200 µg/ml Geneticin 418 (Sigma)
L-glutamine (Gibco-BRL)
Non-essential amino acids (Sigma)

Assay medium: MEM, phenol red free (Invitrogen)

Agonist: isoproterenol (Sigma)

Antagonist: alprenolol (Sigma)

Hoechst33342 (Molecular Probes)

Poly-D-lysine, 5 mg (Sigma)

96-well assay plate (Greiner Bio-One Ltd.)

LOPAC compounds and plate configuration
1 LOPAC’s 640 pharmacologically active compounds were organized by receptor class/activity into eight 96-well plates (80 compounds per plate at 1 mM) as follows:

Plate 1: adenosines/purinergics

Plate 2: adrenergics/histaminergics

Plate 3: cholinergics/ion channel modulators

Plate 4: dopaminergics

Plate 5: glutaminergics

Plate 6: signal transduction agents/opioids

Plate 7: serotonergics

Plate 8: enzyme inhibitors/GABAergics

The compounds were further diluted in assay medium to give final well concentrations of 100 nM. This value was determined during a pre-screen library titration study to be the optimum screen concentration for the LOPAC compounds in 0.01% DMSO. Agonist and antagonist controls were placed symmetrically in columns 1 and 12 of each plate. Twelve replicate wells contained a known agonist, isoproterenol, and four replicate wells contai ned a known antagonist, alprenolol, each diluted in 0.01% DMSO. DMSO at ≤1% was demonstrated to have no detectable adverse effect on the cells or the assay (data not shown).

Live-cell CypHer5E antagonist assay
1. Pre-coat 96-well plates with poly-D-lysine, recommended for HEK293 cells.

2. Seed cells at a density of 8000 cells/well in 100 µl of culture medium.

3. Incubate ~72 h in a humidified environment at 37 ºC and 5% CO2.

4. Equilibrate the plate to room temperature (22–25 ºC) for 5 min.

5. Add 60 µl of fresh assay medium (MEM) containing 2–2.5 µg/ml CypHer5E labelled anti-VSV-G epitope antibody and 5 µM Hoechst nuclear stain.

6. Incubate at room temperature for 10 min.

7. Add 20 µl assay medium containing control or LOPAC test compound to a final assay concentration of 100 nM.

8. Incubate at room temperature for 10 min.

9. Add 20 µl assay medium containing agonist to a final assay concentration of 100 nM and incubate at 37 ºC for 30 min.

10. Image on IN Cell Analyzer 1000 (exposure time 300 ms/field, 620 nm excitation, and 700 nm emission for CypHer5E; exposure time 500 ms/field, 360 nm excitation, and 535 nm emission for Hoechst).

11. Analyze the images using the Granularity Analysis Module, following the instructions in the user guide

Representative IN Cell Analyzer 1000 images show internalization of CypHer5E labeled antibody into acidic endosomes in an agonist-only control well (Fig 1a) or a test well treated with another known agonist (Fig 1c). No cell-associated CypHer5E fluorescence is detected in the presence of antagonist (Fig 1b and 1d). The Granularity Analysis Module for IN Cell Analyzer 1000 enables scientists to quantify the juxta-n uclear clustering of CypHer5E fluorescent granules by identifying, counting, and analyzing the grain-like CypHer5E fluorescent structures adjacent to the nucleus of each cell.

Data from each test compound well were normalized to agonistonly controls on the same assay plate. Plate quality was assessed using the Z` coefficient (6) calculated from controls on each plate. Seven of the eight plates had a Z` = 0.5, as shown in Figure 2. No false positives were detected on plate 1, despite a slightly lower Z` of < 0.3.

A scatterplot of normalized data for the entire screen is shown in Figure 3. The plot also displays the agonist and antagonist control responses for each plate. Hit compounds were defined as those having greater than 90% inhibition of CypHer5E internalization into acidic endosomes.

Using this cut-off criterion for hits, the screen identified a total of eight hit compounds, including all seven known 2-adrenoreceptor antagonists in the 640-compound library (Table 1). The eighth hit compound, S(-)-pindolol, is a structural isomer of ()-pindobind, one of the seven known antagonists. It, too, has been reported to have antagonist activity (7). Under the conditions reported here, the screen was highly specific for 2-adrenoceptor antagonists; no 1-adrenoceptor antagonists in the library were detected as false positives.

Screen quality was assessed by both Z` factor (6) and signal-tonoise ratio (S:N)* assessments (8). The Z` factor and S:N for the screen were 0.3 and 6.6, respectively. These values reflect the small library size and the relatively high proportion of active compounds.

The CypHer5E antagonist assay described here was able to identify all specific 2-adrenergic receptor antagonists and additional structurally related compounds present in the 640-compound LOPAC library

CypHer5E assays can be effectively deployed in a high-throughput format on IN Cell Analyzer 1000 to identify potential antagonists to the 2-adrenergic receptor

CypHer5E technology can identify compounds that affect receptor trafficking and can be a useful screen for novel antagonists of known receptors and for ligands of non-G protein coupled receptors and orphan receptors

1. Application Note: Screening for 2-adrenergic receptor agonists using the pH-sensitive dye, CypHer 5, and IN Cell Analyzer 3000, Amersham Biosciences, 11-0002-81.

2. Application note: Pharmacological characterization of 2-adrenergic receptor with pH-sensitive dye, CypHer5, and IN Cell Analyzer 3000, Amersham Biosciences, 11-0002-82.

3. Application note: The use of CypHer5E and the IN Cell Analyzer 1000 for live-cell receptor internalization studies, Amersham Biosciences, 11-1114-02.

4. Milligan, G. High-content assays for ligand regulation of G-protein coupled receptors. Drug Discovery Today 8, 579585 (2003).

5. Application note: The use of CypHer5 for receptor internalization studies in both a range of GPCRs and a non-GPCR, Amersham Biosciences, 11-0002-80.

6. Zhang et al. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. Journal of Biomolecular Screening 4 (2), 6773 (1999).

7. Horinouchi, T. and Koike, K. Functional properties of atypical -adrenoceptors on the guinea pig duodenum. European Journal of Pharmacology 416, 153163 (2001).

8. Roquemore, E. P. et al. Is Z factor the best assessment for the quality of cellular assays delivering higher content? Poster presented at 9th Annual Conference of the Society for Biomolecular Screening, Portland, Oregon, USA (2003).

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