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

Jan 28, 2006Assay and drug development technologies

Creating uniform tests to measure strong binding of activating molecules to 5-HT2 receptor types

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Abstract

Agonist binding assays reveal that 22% of the total receptor population corresponds to the high-affinity sites of the 5-HT2B receptor.

Three serotonin receptors, 5-HT2A, 5-HT2B, and 5-HT2C, share high similarities in structure and are involved in various physiological processes.
High-affinity binding sites for the 5-HT2A and 5-HT2C receptors were labeled using a specific agonist, while the 5-HT2B receptor sites were labeled with a different compound.
The proportion of agonist high-affinity binding sites is 2.3% for the 5-HT2C receptor, 4.0% for the 5-HT2A receptor, and 22% for the 5-HT2B receptor.
Validation studies showed high Z' values (> 0.70) for the agonist binding assays, indicating robust assay performance.
Ki values obtained for several compounds at the 5-HT2 receptors matched previously published data, supporting the reliability of the SPA format.

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The serotonin (5-hydroxytryptamine) 5-HT2 receptor subfamily consists of three members, 5-HT2A, 5-HT2B, and 5-HT2C. 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-HT2C, 5-HT2A, and 5-HT2B receptors. The 5-HT2 agonist 1-(4- [125I]iodo-2,5-dimethoxyphenyl)-2-aminopropane ([125I]DOI) was used to label the high-affinity sites for the 5-HT2A and 5-HT2C receptors. The high-affinity sites for the 5-HT2B receptor were labeled with [3H]lysergic acid diethylamide. Total receptor expression was determined with the 5-HT2 antagonist [3H]mesulergine for the 5-HT2B and 5-HT2C receptors, and [3H]ketanserin for the 5-HT2A receptor. The agonist high-affinity binding sites accounted for 2.3% (5-HT(2C) receptor), 4.0% (5-HT2A receptor), and 22% (5-HT2B 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 Ki values of 5-HT, (R)(-)DOI, and VER-3323 for the 5-HT2A, 5-HT2B, and 5-HT2C 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 high throughput assay to screen for selective 5-HT2C receptor agonists, as well as for selectivity profiling of the compounds.

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

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