Cell lines expressing recombinant aequorin and a G-protein coupled receptor for functional screening ( ceptor ORL-1 Orexin recepto...)

Cell lines expressing recombinant aequorin and a G-protein coupled receptor for functional screening

ceptor ORL-1

Orexin receptors 1 and 2 Purinergic P2Y2

Serotonin receptors : 5-HT_1B, 5-HT_2B

TSH-Receptor

This system can be used to detect either agonists or antagonists. Surmountable antagonists as well as non-surmountable antagonists are detected.

With a throughput of up to 1000 assays per hour, it is suitable for large-scale drug screening.

EXAMPLES OF RESULTS

This functional screening assay has been tested with several kinds of G-protein coupled receptors. EC₅₀ values are similar to affinity values obtain by radioligand binding and/or other functional assays.

Human chemokine CCR5 receptor:

A CHO cell line expressing the chemokine CCR5 receptor, the G_α16 coupling protein and apoaequorin was established. Cells were prepared as described above and were injected onto a series of dilutions of known ligands placed in the wells of a 96-well plate. The emitted light was immediately recorded for 30 seconds. After reading the first well, cells were injected into the next well and emitted light was recorded, etc. For each plate, a series of curves representing the intensity of the emitted light as a function of time for each well was displayed (figure 2). The intensity of the emitted light was integrated over 30 s using the MicroLumat Winglow software, yielding for each well one value representative of the emitted light, and hence of the stimulation of the CCR5 receptor by the agonist present in the well. These values can be plotted against the logarithm of the ligand concentration to generate a doseresponse curve as shown in figure 3. These allow the determination of half-maximal response doses (EC₅₀) for each ligand. For the generation of these data, 288 measurements were performed in less than 3 hours
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