Shedding Light on the D₁ -Like Receptors: A Fluorescence-Based Toolbox for Visualization of the D₁ and D₅ Receptors

Dopamine 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 D₁ R 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 (pK_i (D₁ R)=8.34, pK_i (D₅ R)=7.62) with excellent selectivity towards D₂ -like receptors. Compound 25 proved to be a neutral antagonist at the D₁ R and D₅ R in a G_s heterotrimer dissociation assay, an important feature to avoid receptor internalization and degradation when working with whole cells. The neutral antagonist 25 displayed rapid association and complete dissociation to the D₁ R 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 D₁ receptors in a variety of experimental setups.