title: Design, synthesis, and pharmacology of fluorescently labeled analogs of serotonin: application to screening of the 5-HT2C receptor.
abstract: 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 Cy3B 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.
authors.affiliation: Department of Cardiovascular, Metabolic, and Endocrine Diseases, Pfizer Global Research and Development, Groton, Connecticut 06340, USA. peter.cornelius@pfizer.com
authors.forename: Peter
authors.initials: P
authors.lastname: Cornelius
authors.forename: Eunsun
authors.initials: E
authors.lastname: Lee
authors.forename: Wen
authors.initials: W
authors.lastname: Lin
authors.forename: Ruduan
authors.initials: R
authors.lastname: Wang
authors.forename: Wendy
authors.initials: W
authors.lastname: Werner
authors.forename: Janice A
authors.initials: JA
authors.lastname: Brown
authors.forename: Frank
authors.initials: F
authors.lastname: Stuhmeier
authors.forename: James G
authors.initials: JG
authors.lastname: Boyd
authors.forename: Kim
authors.initials: K
authors.lastname: McClure
mesh_terms: D000818:Animals; D001681:Biological Assay; D015195:Drug Design; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D051379:Mice; D041681:NIH 3T3 Cells; D044405:Receptor, Serotonin, 5-HT2C / Q000032:analysis* / Q000378:metabolism; D012015:Reference Standards; D015203:Reproducibility of Results; D012701:Serotonin / Q000031:analogs & derivatives*; D013194:Staining and Labeling; D013329:Structure-Activity Relationship
chemical_list: D005456:Fluorescent Dyes; D044405:Receptor, Serotonin, 5-HT2C; D012701:Serotonin