Abstract
Fluorescent ligands are versatile tools for the study of G protein-coupled receptors. Depending on the fluorophore, they can be used for a range of different applications, including fluorescence microscopy and bioluminescence or fluorescence resonance energy transfer (BRET or FRET) assays. Starting from phenylpiperazines and indanylamines, privileged scaffolds for dopamine D2-like receptors, we developed dansyl-labeled fluorescent ligands that are well accommodated in the binding pockets of D2 and D3 receptors. These receptors are the target proteins for the therapy for several neurologic and psychiatric disorders, including Parkinson’s disease and schizophrenia. The dansyl-labeled ligands exhibit binding affinities up to 0.44 nM and 0.29 nM at D2R and D3R, respectively. When the dansyl label was exchanged for sterically more demanding xanthene or cyanine dyes, fluorescent ligands 10a-c retained excellent binding properties and, as expected from their indanylamine pharmacophore, acted as agonists at D2R. While the Cy3B-labeled ligand 10b was used to visualize D2R and D3R on the surface of living cells by total internal reflection microscopy, ligand 10a comprising a rhodamine label showed excellent properties in a NanoBRET binding assay at D3R.
Highlights
Dopamine receptors belong to the large family of G protein-coupled receptors (GPCRs)
For an initial set of fluorescent ligands, we focused on the incorporation of a dansyl label, a naphthalene-derived fluorophore emitting green light
Since we have previously found that the binding pockets of D 2R and D 3R can accommodate flexible linkers[14] and sterically more demanding substituents in their extended binding pocket[36,37], installation of the dansyl fluorophore was envisioned through sulfonamide-formation either directly with the primary benzylamine or through addition of a short 1,3-diaminopropane spacer to the benzoic acid precursors
Summary
Dopamine receptors belong to the large family of G protein-coupled receptors (GPCRs). Whereas numerous assay technologies, such as enzyme fragment complementation, fluorescence or bioluminescence resonance energy transfer (FRET or BRET), proximity-based assays and even label-free methods have been employed for the determination of functional activity at G PCRs8, the characterization of a ligand’s affinity towards a given receptor is mostly based on radioligand competition This binding assay has been proven useful due to the high chemical similarity of unlabeled and labeled probe molecules, the highly specific detection of radioactive labels, and the relatively easy assay format[9]. We have developed Cy3B-labeled fluorescent ligands with an N-propylamino-5-hydroxytetraline or phenylpiperazine substructure for TIRF microscopy studies of D 2R and D3R homodimerization in living c ells[14] Taking advantage of this expertise, we describe the design, molecular docking, chemical synthesis and pharmacological characterization of novel fluorescent ligands targeting the D2-like receptor subfamily. Our novel ligands can be used to label D2R and D3R in living cells (TIRF microscopy) or in a newly established D 3R-NanoBRET ligand binding assay that will open up the path for future drug discovery campaigns
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