Shedding Light on the D1-Like Receptors: A Fluorescence-Based Toolbox for Visualization of the D1and D5Receptors

Abstract

AbstractDopamine D1-like receptors are the most abundant type of dopamine receptors in the central nervous system and, even after decades of discovery, still highly interesting for the study of neurological diseases. We herein describe the synthesis of a new set of fluorescent ligands, structurally derived from D1R antagonist SCH-23390 and labeled with two different fluorescent dyes, as tool compounds for the visualization of D1-like receptors. Pharmacological characterization in radioligand binding studies identified UR-NR435 (25) as a high-affinity ligand for D1-like receptors (pKi(D1R) = 8.34, pKi(D5R) = 7.62) with excellent selectivity towards D2-like receptors. Compound25proved to be a neutral antagonist at the D1R and D5R in a Gsheterotrimer dissociation assay, an important feature to avoid receptor internalization and degradation when working with whole cells. The neutral antagonist25displayed rapid association and complete dissociation to the D1R in kinetic binding studies using confocal microscopy verifying its applicability for fluorescence microscopy. Moreover, molecular brightness studies determined a single-digit nanomolar binding affinity of the ligand, which was in good agreement with radioligand binding data. For this reason, this fluorescent ligand is a useful tool for a sophisticated characterization of native D1receptors in a variety of experimental setups.