A Radiolabeled Photoswitchable G Protein-Coupled Receptor Antagonist Enlightens Ligand Binding Kinetics Associated...

Photopharmacology offers powerful opportunities to control protein signaling using photoresponsive ligands. Despite the vast potential of photoswitchable ligands for spatiotemporal target protein control, research on ligand-protein binding kinetics of these ligands remains limited. Herein, we describe the discovery of the first radiolabeled photoswitchable ligand, [³H]VUF26063 ([³H]3f), to assess light-dependent ligand-protein binding kinetics in real time. The key compound (3f) is an arylazopyrazole-based antagonist targeting a prototypic family A G protein-coupled receptor (GPCR), the histamine H₃ receptor (H₃R), and enabled convenient radiolabeling via a growth vector on the pyrazole. Its photochemical properties, subnanomolar affinity of the trans isomer and a 50-fold decrease in affinity upon switching, allowed for reversible photochemical control of H₃R binding kinetics in real time. The kinetic binding data obtained with this radiolabeled ligand indicate that 3f isomerizes in the H₃R extended binding pocket upon illumination. Our results shed light on the binding kinetics of photoswitchable ligands and will have relevance beyond GPCRs as targets.