Development of homogeneous high-affinity agonist binding assays for 5-HT2 receptor subtypes

Area of Science:

• Pharmacology
• Neuroscience
• Biochemistry

Background:

• The serotonin (5-hydroxytryptamine) 5-HT2 receptor subfamily (5-HT2A, 5-HT2B, 5-HT2C) plays crucial roles in mood, anxiety, and neurological disorders.
• Selective agonists and antagonists targeting these receptors are investigated for therapeutic applications in conditions like depression, schizophrenia, and obesity.

Purpose of the Study:

• To develop homogeneous agonist binding assays in a scintillation proximity assay (SPA) format.
• To determine the high-affinity binding state of agonist compounds for human 5-HT2A, 5-HT2B, and 5-HT2C receptors.

Main Methods:

• Utilized [125I]DOI for 5-HT2A and 5-HT2C receptors, and [3H]lysergic acid diethylamide for 5-HT2B receptors to label high-affinity sites.
• Determined total receptor expression using [3H]mesulergine (5-HT2B, 5-HT2C) and [3H]ketanserin (5-HT2A).
• Performed competition binding studies with known agonists to assess assay performance and validate results against filtration binding assays.

Main Results:

• Agonist high-affinity binding sites represented 2.3% (5-HT2C), 4.0% (5-HT2A), and 22% (5-HT2B) of the total receptor population.
• Agonist binding assays in SPA format demonstrated high Z' values (Z' > 0.70), indicating reliability.
• SPA-determined Ki values for 5-HT, (R)(-)DOI, and VER-3323 were consistent with published filtration assay data.

Conclusions:

• Agonist binding assays in SPA format are adaptable for high-throughput screening.
• These assays are valuable for identifying selective 5-HT2C receptor agonists.
• The developed SPA assays facilitate selectivity profiling of compounds targeting the 5-HT2 receptor subfamily.