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

Comprehensively characterized fluorescent probes for the histamine H receptor (HR) and especially for the HR 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 HR in live cells, we synthesized and biologically characterized Py-5-labeled histamine derivatives. The most notable compound was UR-DEBa242 (, 1-[4-(1-Imidazol-4-yl)butyl]-4-{(1,3)-4-[4-(dimethylamino)phenyl]buta-1,3-dienyl}-2,6-dimethylpyridinium hydrotrifluoroacetate trifluoroacetate), acting as a partial agonist at the hHR [pEC (reporter gene) 8.77] and as an inverse agonist/antagonist at the h/mHRs [pIC (reporter gene) 8.76/7.08; pIC/p (β-arrestin2) 7.81/7.30]. In confocal microscopy, proved suitable for hHR localization and trafficking studies in live cells. BRET-based binding at the NLuc-hHRs/mHR [p 8.78/7.75/7.18, comparable to binding constants from radioligand binding/flow cytometry; fast association/dissociation (∼2 min)] revealed as a useful molecular tool to determine hHRs/mHR binding affinities of ligands binding to these receptors.