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 H-3 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 hH(3)R [pEC(50) (reporter gene) 8.77] and as an inverse agonist/antagonist at the h/mH(4)Rs [pIC(50) (reporter gene) 8.76/7.08; pIC(50)/pK(b) (beta-arrestin2) 7.81/7.30]. In confocal microscopy, 26 proved suitable for hH(4)R localization and trafficking studies in live cells. BRET-based binding at the NLuc-hH(3,4)Rs/mH(4)R [pK(d) 8.78/7.75/7.18, comparable to binding constants from radioligand binding/flow cytometry; fast association/dissociation (similar to 2 min)] revealed 26 as a useful molecular tool to determine hH(3,4)Rs/mH(4)R binding affinities of ligands binding to these receptors.