Discovery of Environment-Sensitive Fluorescent Ligands of β-Adrenergic Receptors for Cell Imaging and NanoBRET Assay.

By fusing several environment-sensitive fluorophores to the pharmacophore mirabegron, a series of new fluorescent ligands for β-adrenergic receptors (β-ARs) were produced with a turn-on mechanism and high binding affinity to β-ARs efficiently. Compound L5 with the pyridinium moiety possessed the most favorable combination of properties after systematic comparison and optimization, including high affinity and acceptable cytotoxicity, remarkable fluorescence enhancement (up to 30-fold) upon binding with β-ARs, and feasible visualizing ability of β-ARs in living cells under no-wash conditions. Furthermore, a NanoLuc-based bioluminescence resonance energy transfer (NanoBRET) binding assay based on compound L5 was developed and may be used in high-throughput screening (HTS) in the drug discovery of β-ARs due to its unique fluorescence spectroscopic features. Overall, as the first environment-sensitive fluorescent ligand, molecule L5 could be a useful tool for understanding the pharmacology of β-ARs.