UR-DEBa242: A Py-5-Labeled Fluorescent Multipurpose Probe for Investigations on the Histamine H₃ and H₄ Receptors

Comprehensively characterized fluorescent probes for the histamine H₃ receptor (H₃R) and especially for the H₄R orthologs [e.g., human (h) and mouse (m)] are highly needed as versatile complementary tools to radioligands. In view of fluorescent probes for BRET-based binding studies and for localizing the H₄R in live cells, we synthesized and biologically characterized Py-5-labeled histamine derivatives. The most notable compound was UR-DEBa242 (26, 1-[4-(1H-Imidazol-4-yl)butyl]-4-{(1E,3E)-4-[4-(dimethylamino)phenyl]buta-1,3-dienyl}-2,6-dimethylpyridinium hydrotrifluoroacetate trifluoroacetate), acting as a partial agonist at the hH₃R [pEC₅₀ (reporter gene) 8.77] and as an inverse agonist/antagonist at the h/mH₄Rs [pIC₅₀ (reporter gene) 8.76/7.08; pIC₅₀/pK_b (β-arrestin2) 7.81/7.30]. In confocal microscopy, 26 proved suitable for hH₄R localization and trafficking studies in live cells. BRET-based binding at the NLuc-hH_3,4Rs/mH₄R [pK_d 8.78/7.75/7.18, comparable to binding constants from radioligand binding/flow cytometry; fast association/dissociation (∼2 min)] revealed 26 as a useful molecular tool to determine hH_3,4Rs/mH₄R binding affinities of ligands binding to these receptors.