Development of Homogeneous High-Affinity Agonist Binding Assays for 5-HT₂Receptor Subtypes

Abstract

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.

Structure–Activity Relationships of Phenylalkylamines as Agonist Ligands for 5‐HT2A Receptors

“…Binding affinities can be determined by radioligand competition assays using rat brain homogenate or cloned human receptors. [106] Several antagonist and agonist radioligands, with known binding properties, are available for competition assays at the 5-HT 2A receptor. [107][108][109] Agonists and antagonists display different affinities for various receptor states, [110] an observation accounted for by receptor theories.…”

Radiosynthesis and Evaluation of 11C-CIMBI-5 as a 5-HT2A Receptor Agonist Radioligand for PET

“…Briefly, rats were injected in the tail vein with 11 C-CIMBI-5 (3.9 6 3.5 MBq/kg; specific radioactivity, 30.9 GBq/mmol). The rats were decapitated at 5 (n 5 2), 15 (n 5 2), 30 (n 5 4), 45 (n 5 2), and 60 min (n 5 4); the brains were quickly removed, placed on ice, and dissected into frontal cortex 5 1,381 6 231 D 2 1,600 6 333 5-HT 7 1,670 6 125 5-HT 5A 2,200 6 385 D 1 3,718 6 365 5-HT 1B 3,742 6 553 Norepinephrine transporter 4,574 6 270 Dopamine transporter 5,031 6 343 D 5 7,872 6 933 (first 3 mm of the brain) and cerebellum. Blood from the trunk was collected immediately, and plasma was isolated by centrifugation (1,500 rpm, 10 min).…”

“…However, whereas 5-HT 2A antagonists bind to the total pool of receptors, 5-HT 2A agonists bind only to the high-affinity state of the receptor (6,7). Thus, a 5-HT 2A receptor agonist ligand holds promise for the selective mapping of 5-HT 2A receptors in their functional state; therefore, alterations in agonist binding measured in vivo with PET may be more relevant for assessing dysfunction in the 5-HT 2A receptor system in specific patient or population groups.…”

New Thoughts on Thought Disorders in Parkinson's Disease: Review of Current Research Strategies and Challenges

“…Much of the serotonin effect of these atypical antipsychotics relates to drug interactions with the 5HT-2a receptors, which in the central nervous system can be found in the cortex, basal ganglia (caudate and putamen), and hippocampus [102]. Interestingly, 5HT-2a antagonists or inverse agonists including clozapine also have demonstrated improvement in parkinsonian motor function in primates and humans [103, 104].…”