Design,Synthesis,and Pharmacology of Fluorescently Labeled Analogs of Serotonin: Application to Screening of the 5-HT 2C Receptor

Abstract

Novel fluorescent derivatives of serotonin have been synthesized and used as tracers for the development of a 5-HT_2C fluorescence polarization assay. Serotonin analogs that feature a fluorescent probe attached through an ether linkage at the tryptamine 5-position have high affinity for the 5-HT_2C receptor, and affinity is dependent on both linker length and pendent dye. These variables have been optimized to generate Cy3B derivative 5a, which has 10-fold higher 5-HT_2C affinity relative to serotonin (K_d = 0.23 nM). In receptor activation experiments, 5a acts as a full agonist of 5-HT_2C. Upon binding to 5-HT_2C cell membranes, 5a shows a robust increase in fluorescence polarization (FP) signal. In an FP binding assay using 5a as a tracer ligand, K_i values for known 5-HT_2C agonists and antagonists showed excellent agreement with K_i values from radioligand binding (r ² = 0.93). The FP ligand assay is suitable for high-throughput drug screening applications with respect to speed of analysis, displaceable signal, precision, and sensitivity to various reagents. A 384-well-based high-throughput assay that is rapid, economical, and predictive of test compounds' ability to bind to the 5-HT_2C receptor has been compiled and validated. (Journal of Biomolecular Screening 2009:360-370)

Keywords

serotonin fluorescence polarization high-throughput screening radioligand binding 5-HT2C

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