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Design, Synthesis, and Pharmacology of Fluorescently Labeled Analogs of Serotonin: Application to Screening of the 5-HT_2C Receptor

Department of Cardiovascular, Metabolic, and Endocrine Diseases, Pfizer Global Research and Development, Groton, Connecticut, peter.cornelius{at}pfizer.com

Eunsun Lee

Department of Cardiovascular, Metabolic, and Endocrine Diseases, Pfizer Global Research and Development, Groton, Connecticut

Wen Lin

Department of Exploratory Medicinal Sciences, Pfizer Global Research and Development, Groton, Connecticut

Ruduan Wang

Department of Cardiovascular, Metabolic, and Endocrine Diseases, Pfizer Global Research and Development, Groton, Connecticut

Wendy Werner

Department of Cardiovascular, Metabolic, and Endocrine Diseases, Pfizer Global Research and Development, Groton, Connecticut

Janice A. Brown

Department of Cardiovascular, Metabolic, and Endocrine Diseases, Pfizer Global Research and Development, Groton, Connecticut

Frank Stuhmeier

Primary Pharmacology Group, Sandwich, Kent, UK, Pfizer Global Research and Development, Groton, Connecticut

James G. Boyd

Department of Exploratory Medicinal Sciences, Pfizer Global Research and Development, Groton, Connecticut

Kim McClure

Department of Cardiovascular, Metabolic, and Endocrine Diseases, Pfizer Global Research and Development, Groton, Connecticut

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 (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-HT_2C receptor has been compiled and validated. (Journal of Biomolecular Screening 2009:360-370)

Key Words: serotonin • fluorescence polarization • high-throughput screening • radioligand binding • 5-HT2C

Journal of Biomolecular Screening, Vol. 14, No. 4, 360-370 (2009)
DOI: 10.1177/1087057109331804


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