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the bound ANS is competed with agonists and antagonists (Fig. 11). The ANS fluorogenic assay is a homogeneous assay adaptable to HTS. The disadvantages of this assay are that it requires relatively large amount of protein and that ANS binds to hydrophobic pockets not necessarily in the active binding site. Even when the screening of a compound library is done with this assay, the hits have to be confirmed by another suitable method.

fmat receptor-ligand-binding assays. FMAT is fluorescence-based, homogeneous cell and bead based nonradioactive assay (described in Chaps. 3 and 4). FMAT can be used for G-protein coupled receptors and other membrane receptors on intact whole cell with peptide/protein ligands. Ligand labeled with Cy5 dye is coated on the bead surface and incubated with cells in a 96-, 384-, or 1536-well plate. The binding of fluorescent ligand to the whole cell receptor on the plate is measured in mm2 without interference from background fluorescence. Different size beads can be labeled with different CY5-dye-labeled ligands for different receptors, and the cells expressing these receptors are incubated with these beads. The fluorescence associated with each receptor-bound fluorescent ligand can be measured, thus enabling a screen for more than one receptor binding (multiplexing). Other receptors tested by FMAT include substance P, Neuropeptide Y, galanin, neurokinin A, bradykinin, somatostatin, angiotensin, and nuclear receptors [45].

Figure 11 A fluoregenic assay using anilinonaphthalene sulfonic acid (ANS) for an orphan nuclear receptor. ANS in aqueous solution is nonfluorescent, and when it binds to protein it yields fluorescence. (A) Dose-dependent response to receptor concentration. Fluorescence signal increased with increasing ANS and receptor concentrations. (B) Competition binding of ANS by agonists. The known agonists competed ANS binding to the receptor, but some of them competed only partially, whereas they competed fully in a gel-filtration binding assay with a 3H-agonist, suggesting that ANS also binds to other hydrophobic pockets in addition to the agonist binding pocket.

Figure 11 A fluoregenic assay using anilinonaphthalene sulfonic acid (ANS) for an orphan nuclear receptor. ANS in aqueous solution is nonfluorescent, and when it binds to protein it yields fluorescence. (A) Dose-dependent response to receptor concentration. Fluorescence signal increased with increasing ANS and receptor concentrations. (B) Competition binding of ANS by agonists. The known agonists competed ANS binding to the receptor, but some of them competed only partially, whereas they competed fully in a gel-filtration binding assay with a 3H-agonist, suggesting that ANS also binds to other hydrophobic pockets in addition to the agonist binding pocket.

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