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 beta-adrenergic receptors (beta-ARs) were produced with a turn-on mechanism and high binding affinity to beta-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 beta-ARs, and feasible visualizing ability of beta-ARs in living cells under no-wash conditions. Furthermore, a NanoLuc-based bioluminescence resonance energy transfer (NanoBRET) binding assay based on compound 1,5 was developed and may be used in high-throughput screening (HTS) in the drug discovery of beta-ARs due to its unique fluorescence spectroscopic features. Overall, as the first environment-sensitive fluorescent ligand, molecule 1,5 could be a useful tool for understanding the pharmacology of beta-ARs.

Automated summary

A series of new fluorescent ligands with high binding affinity and visualizing ability for beta-adrenergic receptors (beta-ARs) have been successfully developed. Compound L5 showed the most favorable combination of properties, including high affinity, remarkable fluorescence enhancement upon binding, and the ability to visualize beta-ARs in living cells without wash steps. Additionally, a NanoLuc-based binding assay using compound 1,5 has been developed for high-throughput screening of beta-ARs.