Fluorescent ligands for dopamine D2/D3 receptors

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.

A Versatile Sub-Nanomolar Fluorescent Ligand Enables NanoBRET Binding Studies and Single-Molecule Microscopy at the Histamine H3 Receptor

“…Unfortunately, the capacity of the unpublished NLuc-receptor constructs to bind their endogenous ligands could not be thoroughly controlled given the lack of validated fluorescent ligands. Instead, it was only presumed based on sufficient surface expression of these constructs and based on the experience that N-terminal insertion of the NLuc usually does not affect the binding ability of class A GPCRs. ,− Receptor cell surface expression was confirmed by an ELISA using a NanoLuc antibody (Cat. No.…”

Fluorescent Ligands Targeting the Intracellular Allosteric Binding Site of the Chemokine Receptor CCR2

“…For the establishment of our NanoBRET binding assay, we referred to a recently published protocol. 32 The fluorescent ligands (8−16) were dissolved in DMSO (1 mM) and further diluted to varying concentrations in assay buffer (50 mM Na 2 HPO 4 , 50 mM KH 2 PO 4 , pH 7.4, 1 mg/mL saponin, 5% FBS), and 5 μL of these dilutions were pipetted to a 384-well plate. To determine total binding, 5 μL of assay buffer was added to the corresponding wells, while 5 μL of a 4 solution in assay buffer (final assay concentration: 10 μM) was used to determine nonspecific binding.…”

“…For the establishment of our NanoBRET binding assay, we referred to a recently published protocol . The fluorescent ligands ( 8 – 16 ) were dissolved in DMSO (1 mM) and further diluted to varying concentrations in assay buffer (50 mM Na 2 HPO 4 , 50 mM KH 2 PO 4 , pH 7.4, 1 mg/mL saponin, 5% FBS), and 5 μL of these dilutions were pipetted to a 384-well plate.…”

Optimizing the Expression of Human Dopamine Receptors in Escherichia coli

“…To test this approach, we used a fluorescent ligand NMP130, which was recently developed for the D 3 receptor and used for analyses in human embryonic kidney (HEK) 293 cells [32]. Radioligand competition assays revealed an almost 20-fold higher affinity of the ligand NMP130 for the D 3 receptor (0.76 nM) than for D 2S (15 nM) [32]. Thus, we tested the ligand with E. coli cells expressing D 3 .…”

“…cells [32]. Radioligand competition assays revealed an almost 20-fold higher affinity of the ligand NMP130 for the D3 receptor (0.76 nM) than for D2S (15 nM) [32]. Thus, we tested the ligand with E. coli cells expressing D3.…”