Design, Synthesis, and Pharmacology of Fluorescently Labeled Analogs of Serotonin: Application to Screening of the 5-HT2C Receptor

Novel fluorescent derivatives of serotonin have been synthesized and used as tracers for the development of a 5-HT2c 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-HT2c receptor, and affinity is dependent on both linker length and pendent dye. These variables have been optimized to generate uCy3B derivative 5a, which has 10-fold higher 5-HT2c affinity relative to serotonin (Kd = 0.23 nM). In receptor activation experiments, 5a acts as a full agonist of 5-HT2c. Upon binding to 5-HT2c cell membranes, 5a shows a robust increase in fluorescence polarization (FP) signal. In an FP binding assay using 5a as a tracer ligand, Ki values for known 5-HT2c agonists and antagonists showed excellent agreement with Ki values from radioligand binding (r2 = 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-HT2c receptor has been compiled and validated.