The histamine H 3 receptor (H 3 R) is considered an attractive drug target for various neurological diseases. We here report the synthesis of UR-NR266, a novel fluorescent H 3 R ligand. Broad pharmacological characterization revealed UR-NR266 as a sub-nanomolar compound at the H 3 R 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 H 3 R ligands in NanoBRET competition binding using UR-NR266 highlights its value as a versatile pharmacological tool to analyze future H 3 R 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 H 3 R 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.

The ability of G protein-coupled receptors (GPCRs) to form dimers, and particularly heterodimers, offers potential for targeted therapeutics with improved selectivity. However, studying dimer pharmacology is challenging, because of signaling cross-talk or because dimerization may often be transient in nature. Here we develop a system to isolate the pharmacology of precisely defined GPCR dimers, trapped by bimolecular fluorescence complementation (BiFC). Specific effects of agonist activation on such dimers are quantified using automated imaging and analysis of their internalization, controlled for by simultaneous assessment of endocytosis of one coexpressed protomer population. We applied this BiFC system to study example neuropeptide Y (NPY) Y1 receptor dimers. Incorporation of binding-site or phosphorylation-site mutations into just one protomer of a Y1/Y1 BiFC homodimer had no impact on efficient NPY-stimulated endocytosis, demonstrating that single-site agonist occupancy, and one phosphorylated monomer within this dimer, was sufficient. For two Y1 receptor heterodimer combinations (with the Y4 receptor or b2-adrenoceptor), agonist and antagonist pharmacology was explained by independent actions on the respective orthosteric binding sites. However, Y1/Y5 receptor BiFC dimers, compared with the constituent subtypes, were characterized by reduced potency and efficacy of Y5-selective peptide agonists, the inactivity of Y1-selective antagonists, and a change from surmountable to nonsurmountable antagonism for three unrelated Y5 antagonists. Thus, allosteric interactions between Y1 and Y5 receptors modify the pharmacology of the heterodimer, with implications for potential antiobesity agents that target centrally coexpressed Y1 and Y5 receptors to suppress appetite.

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The G-protein-coupled Y 4 -receptor (Y 4 R) and its endogenous ligand, pancreatic polypeptide (PP), suppress appetite in response to food intake and, thus, are attractive drug targets for body-weight control. The Cterminus of human PP (hPP), T 32 -R 33 -P 34 -R 35 -Y 36 -NH 2 , penetrates deep into the binding pocket with its tyrosine-amide and di-arginine motif. Here, we present two C-terminally amidated α,γ-hexapeptides (1a/b) with sequence Ac-R 31 -γ-CBAA 32 -R 33 -L 34 -R 35 -Y 36 -NH 2 , where γ-CBAA is the (1R,2S,3R)configured 2-(aminomethyl)-3-phenylcyclobutanecarboxyl moiety (1a) or its mirror image (1b). Both peptides bind the Y 4 R (K i of 1a/b: 0.66/12 nM) and act as partial agonists (intrinsic activity of 1a/b: 50/39%). Their induced-fit binding poses in the Y 4 R pocket are unique and build ligand− receptor contacts distinct from those of the C-terminus of the endogenous ligand hPP. We conclude that energetically favorable interactions, although they do not match those of the native ligand hPP, still guarantee high binding affinity (with 1a rivaling hPP) but not the maximum receptor activation.