Characterization of a novel, high‐affinity and selective fluorescent antagonist for the 5HT1A receptor

Abstract

The use of fluorescent ligands in G‐protein‐coupled receptor pharmacology to study ligand‐receptor interactions is now a viable alternative to the use of traditional radioligands, as it is non‐invasive, allows the study of interactions in real‐time and in single cells and is environmentally friendly. We have characterized a new, fluorescent antagonist acting selectively at the 5HT1A receptor. Using Chinese Hamster Ovary (CHO) cells stably expressing the 5‐HT1A receptor and a cyclic AMP response element‐coupled reporter gene (human secreted placental alkaline phosphatase (SPAP)), we calculated the log KB value of the ligand from the SPAP assay as −8.49 ± 0.14 (n=3). The ligand showed no significant affinity at closely‐related 5‐HT receptors. Confocal imaging on the ImageXpress Ultra confocal plate reader revealed clear membrane binding at concentrations consistent with the affinity value from the functional assay and a concentration‐dependent displacement of this binding by an unlabeled competitor, NAN‐190. These data show that this molecule is a high‐affinity, selective fluorescent antagonist at the 5HT1A receptor and thus can be used to probe the pharmacology and kinetics of ligand‐receptor interactions at this receptor.This research was supported by the award to AAF of a Nottingham Advanced Research Fellowship and a Marie Curie International Incoming Fellowship.