Development and Characterization of a Highly Selective Turn-On Fluorescent Ligand for β₃-Adrenergic Receptor

For the precise visualization of GPCR, subtype selectivity of turn-on fluorescent ligands is of major relevance. Although there are many thriving β-adrenergic receptors (β-ARs) probes, none of them are selective to the β₃-subtype, which severely limits the development of β₃-AR investigations. Using a polyethylene glycol (PEG) chain to conjugate the Py-5 fluorophore with mirabegron, we present here a highly selective fluorescent ligand, H2, for β₃-AR. It was established by the radioligand and NanoLuc-based bioluminescence resonance energy transfer (NanoBRET) binding experiments that molecule H2 has a substantially higher affinity for β₃-AR than the other two subtypes (1/3, 45-fold; 2/3, 16-fold). More crucially, when molecule H2 was incubated with β₃-AR, the turn-on fluorescent signals could be quickly released. The subsequent investigations, which included cell imaging, tissue imaging, and flow-cytometry analysis, proved that molecule H2 may make it possible to quickly and accurately fluorescently identify β₃-AR at different levels. We offer a prospective fluorescent turn-on ligand with exceptional selectivity for β₃-AR as a result of our combined efforts.