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

The histamine H receptor (HR) is considered an attractive drug target for various neurological diseases. We here report the synthesis of UR-NR266, a novel fluorescent HR ligand. Broad pharmacological characterization revealed UR-NR266 as a sub-nanomolar compound at the HR with an exceptional selectivity profile within the histamine receptor family. The presented neutral antagonist showed fast association to its target and complete dissociation in kinetic binding studies. Detailed characterization of standard HR ligands in NanoBRET competition binding using UR-NR266 highlights its value as a versatile pharmacological tool to analyze future HR ligands. The low nonspecific binding observed in all experiments could also be verified in TIRF and confocal microscopy. This fluorescent probe allows the highly specific analysis of native HR in various assays ranging from optical high throughput technologies to biophysical analyses and single-molecule studies in its natural environment. An off-target screening at 14 receptors revealed UR-NR266 as a selective compound.