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Development of Homogeneous High-Affinity Agonist Binding Assays for 5-HT 2 Receptor Subtypes

Development of Homogeneous High-Affinity Agonist Binding Assays for 5-HT 2 Receptor Subtypes


The serotonin (5-hydroxytryptamine) 5-HT 2 receptor subfamily consists of three members, 5-HT 2A , 5-HT 2B , and 5-HT 2 C. These receptors share high homology in their amino acid sequence, have similar signaling pathways, and have been indicated to play important roles in feeding, anxiety, aggression, sexual behavior, mood, and pain. Subtype-selective agonists and antagonists have been explored as drugs for hypertension, Parkinson's disease, sleep disorders, anxiety, depression, schizophrenia, and obesity. In this study, we report the development of homogeneous agonist binding assays in a scintillation proximity assay (SPA) format to determine the high-affinity binding state of agonist compounds for the human 5-HT 2C , 5-HT 2A , and 5-HT 2B receptors. The 5-HT 2 agonist 1-(4- 125 Iiodo-2,5-dimethoxyphenyl)-2-aminopropane ( 125 IDOI) was used to label the high-affinity sites for the 5-HT 2A and 5-HT 2C receptors. The high-affinity sites for the 5-HT 2B receptor were labeled with 3 Hlysergic acid diethylamide. Total receptor expression was determined with the 5-HT 2 antagonist 3 Hmesulergine for the 5-HT 2B and 5-HT 2C receptors, and 3 Hketanserin for the 5-HT 2A receptor. The agonist high-affinity binding sites accounted for 2.3% (5-HT 2C receptor), 4.0% (5-HT 2A receptor), and 22% (5-HT 2B receptor) of the total receptor population. Competition binding studies using known agonists indicated high Z′ values of the agonist binding assays in SPA format ( Z ′ > 0.70). The K i values of 5-HT, ( R )(-)DOI, and VER-3323 for the 5-HT 2A , 5-HT 2B , and 5-HT 2C receptors by SPA format were equivalent to published data determined by filtration binding assays. These results indicate that agonist binding assays in SPA format can be easily adapted to a highthroughput assay to screen for selective 5-HT 2C receptor agonists, as well as for selectivity profiling of the compounds.
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