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).
CypHer5 labelled anti -VSV-G antibody, 250 mg PA45407
IN Cell Analyzer 3000 25-8010-11
Granularity Analysis Module 63-0048-97
(for IN Cell Analyzer 3000)
Other materials required
MEM (Sigma) containing 10% (v/v) FBS, 200 µ g/ml G418, L-glutamine, non-essential amino acids
Assay medium: MEM (Sigma)
Agonist: Isoproterenol (Sigma)
Antagonist: Alprenolol (Sigma)
Fetal bovine serum (Gibco-BRL)
Geneticin G418 (Sigma)
Hoechst 33342 (Molecular Probes)
Non-essential amino acids (Sigma)
Phosphate buffered saline (PBS) solution
Poly-D-lysine, 5 mg (Sigma)
96-well assay plate(Packard Viewplate)
The 640 pharmacologically active small organic compounds were provided in 96-well format, organized by receptor class/activity into eight plates (80 compounds per plate, 2 mg/well).
3. Cholinergics/ion channel modulators
6. Signal transduction agents/opioids
8. Enzyme inhibitors/GABAergics
The β 2-adrenergic receptor agonist isoproteren ol (1 µ M) andantagonist alprenolol (1 µ M) were included as internal controls on each plate along with untreated cells for basal fluorescent levels.
Live-cell CypHer5 assay
1. Pre-coat 96-well plates with poly-D-lysine (recommended for HEK293 cells).
2. Seed cells at a density of 10–15 000 cells/well.
3. Incubate overnight at 37 ° C with 5% CO2.
4. Replace the culture medium with 100 µ l of fresh assay medium (MEM) containing 5 µ g/ml CypHer5 labelled anti-VSV-G antibody and 2.5 µ M Hoechst nuclear stain.
5. Add test compounds and controls to final assay concentrations of 40 µ g/ml and 1 µ M respectively.
6. Incubate at room temperature for 30 min.
7. Image on the IN Cell Analyzer 3000 with the following filter sets: 633 nm excitation and 695BP55 emission for CypHer5; 365 nm excitation and 450BP65 emission for Hoechst nuclear stain.
Quantitation of the receptor response can be achieved using the Granularity Analysis Module for the IN Cell Analyzer 3000, which identifies, counts, and analyzes grain-like structures within the cells. Figure 1 shows a scatter plot for β 2-adrenergic receptor expressing HEK cells, with and without isoproterenol stimulation. A signal:noise ratio of 10:1 and Z’ factor of 0.61 were obtained for this assay.
All eight LOPAC plates were imaged after incubation with control or test compounds. Granularity analysis results from the acquired images are shown in Figure 2 and summarized in Table 1.
Fourteen potential agonists were identified as showing an increase in CypHer5 fluorescence, indicating internalization of the β 2-adrenergic receptor. The majority (71 %) were from Plate 2: Adrenergics and histaminergics and included the β -adrenergic receptor agonists isoproterenol (three salts), epinephrine, salbutamol, and norepinephrine along with two α -adrenergic receptor agonists phenylephrine and iodoclonidine. Figure 3a shows receptor internalization responses for these agonists. The CypHer5 technology was effective in not only identifying the most active stimulants of β 2-adrenergic receptor internalization but also compounds with partial, low-affinity or non-specific action (3,6,7).
Putative agonists were also highlighted from three other plates. On Plate 4: Dopaminergics, the compound was identified as N-methyldopamine hydrochloride, also known as epinine hydrochloride or ibopamine. This is reported in the literature as a non-specific agonist for adrenergic receptors as well as a dopamine receptor agonist, with its affinity for dopaminergic > β 1 and β 2 adrenergic > α 1 and α 2 adrenergic receptors (4). On Plate 5: Glutaminergics, the compound was quisqualic acid, which has been shown to enhance norepinephrine release at excitatory amino acid receptors (5), suggesting it can lead indirectly to internalization of β 2-adrenergic receptors.
Figure 3b shows an example of the pattern of fluorescent grains obtained for the remaining two compounds on Plate 1: Adenosines/purinergics. They do not show the classic perinuclear clustering seen with internalization of β 2-adrenergic receptors into acidic endosomal vesicles and would appear to be false positives.
Agonist activation of cell surface receptors, which are used by the pharm aceutical industry as major drug targets, almost invariably results in the internalization of the receptor from the plasma membrane to the endosomal pathway within the cell. CypHer5 provides a generic approach to functional analysis and high-throughput primary and secondary screening of a broad range of cell surface receptors. CypHer5 and the IN Cell Analyzer 3000 are effective tools for the high-throughput screening of agonist-induced receptor internalization. They can also be used to identify the most active β 2-adrenergic receptor agonists present in a 640compound library along with compounds with potential agonist activity. The data presented support the use of CypHer5 in screening for new agonists of known receptors and potentially ligands of orphan receptors.
1. Milligan, G. Drug Discovery Today, 8 (13), 579–585 (2003).
2. Adie, E.J. et al. BioTechniques, 33, 1152 (2002).
3. Application Note: Pharmacological characterization of the β2-adrenergic
receptor with the pH sensitive dye, CypHer5, and IN Cell Analyzer 3000,
Amersham Biosciences, 18-1177-91, Edition AA (2003).
3. Pouleur et al. Clin. Cardiol., 18 (3 Suppl I), 117–21 (1995).
4. Pittaluga and Raiteri. J. Pharmacol. Exp. Ther,. 260 (1), 232–7 (1992).
5. Oakley RH et al. Journal of Biological Chemistry, 274 (45), 32248–32257 (1999).
6. Crider, J.Y. and Sharif, N.A. J. Ocul. Pharmacol. Ther., 18 (3), 221–30 (2002).
Note: The CypHer5 dye used in this application note was replaced in December 2003 by CypHer5E. This new pH-sensitive cyanine dye has the same mechanism of action as the previous compound but with the following enhanced features:
1. Higher extinction coeffic ient: can be used with cell lines that have lower receptor expression levels, enabling assays to be imaged on lower sensitivity instruments.
2. Increased sulfonation: Improved solubility, greater labeling efficiencies, and improved stability in solution.
More information comparing the performance of the two dyes can be found in the application note: The use of CypHer5E and the IN Cell Analyzer 1000 for live-cell receptor internalization studies (11-0008-22AA).
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