We implemented an assay combining fluorescence and bright‐field microscopy to measure ligand binding to dopamine D3 receptors in live mammalian cells. For fluorescence intensity quantification from microscopy images, we developed a machine learning‐based user‐friendly software membrane tools. For the experiments, a novel fluorescent ligand NAPS‐Cy3B was synthesized. The subnanomolar affinity of NAPS‐Cy3B makes it a suitable ligand for the characterization of D3 receptors and its ligands in live HEK293 cells.

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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.

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Cyclic adenosine monophosphate (cAMP) is a second messenger of many G-protein-coupled receptors. The change in cellular cAMP level has widely been used to estimate the biological activity of various GPCR-specific agents. Förster resonance energy transfer (FRET) biosensors have been around for almost 10 years and became increasingly popular for cAMP detection. Ratiometric sensitized emission assay of a FRET biosensor can easily be implemented on fluorescence plate reader platforms. For such assays a considerable amount of cells expressing the desired biosensor is needed. A method to achieve sufficient and reproducible level of cAMP biosensor protein expression with the means of BacMam transduction system is the subject of this chapter.