Neuropeptide Y (NPY) receptors belong to the G protein-coupled receptor (GPCR) superfamily and play important roles in food intake, anxiety and cancer regulation1,2. The NPY/Y receptor system has emerged as one of the most complex networks with three peptide ligands (NPY, peptide YY and pancreatic polypeptide) binding to four receptors in mammals, namely Y1, Y2, Y4 and Y5 receptors, with different affinity and selectivity3. NPY is the most powerful stimulant of food intake and this effect is primarily mediated by Y1 receptor (Y1R)4. A number of peptides and small-molecule compounds have been characterized as Y1R antagonists and have shown clinical potential in the treatment of obesity4, tumor1 and bone loss5. However, their clinical usage has been hampered by low potency and selectivity, poor brain penetration ability or lack of oral bioavailability6. Here we report crystal structures of the human Y1R bound to two selective antagonists UR-MK299 and BMS-193885 at 2.7 and 3.0 Å resolution, respectively. The structures combined with mutagenesis studies reveal binding modes of Y1R to several structurally diverse antagonists and determinants of ligand selectivity. The Y1R structure and molecular docking of the endogenous agonist NPY, together with nuclear magnetic resonance (NMR), photo-crosslinking and functional studies, provide insights into the binding behavior of the agonist and for the first time determine the interaction of its N terminus with the receptor. These insights into Y1R can enable structure-based drug discovery targeting NPY receptors.

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Currently employed histamine H2 receptor (H2R) radioligands possess several drawbacks, for example, high non-specificity, insurmountable binding, or short half-life. We report the synthesis and the chemical and pharmacological characterization of the highly stable carbamoylguanidine-type radioligand [3H]­UR-KAT479 ([3H]23), a subtype selective histamine H2 receptor G protein-biased agonist. [3H]23 was characterized by saturation, kinetic, and competition binding assays at the human, guinea pig, and mouse H2 receptors (co-)­expressed in HEK293­(T) cells. [3H]23 reversibly bound to the respective H2Rs with moderate to high affinity (human/guinea pig/mouse K d: 24/28/94 nM). In order to investigate the applicability of carbamoylguanidine-type ligands in animal studies elucidating the role of the H2R in the brain, we performed a preliminary partitioning experiment in the whole human/mouse blood, which indicated a low binding of [3H]23 to red blood cells. These properties turn [3H]23 into a powerful tool for the determination of binding affinities and demonstrate the promising pharmacokinetic profile of carbamoylguanidine-type ligands.

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G protein activation represents an early key event in the complex GPCR signal transduction process and is usually studied by label-dependent methods targeting specific molecular events. However, the constrained environment of such “invasive” techniques could interfere with biological processes. Although histamine receptors (HRs) represent (evolving) drug targets, their signal transduction is not fully understood. To address this issue, we established a non-invasive dynamic mass redistribution (DMR) assay for the human H1–4Rs expressed in HEK cells, showing excellent signal-to-background ratios above 100 for histamine (HIS) and higher than 24 for inverse agonists with pEC50 values consistent with literature. Taking advantage of the integrative nature of the DMR assay, the involvement of endogenous Gαq/11, Gαs, Gα12/13 and Gβγ proteins was explored, pursuing a two-pronged approach, namely that of classical pharmacology (G protein modulators) and that of molecular biology (Gα knock-out HEK cells). We showed that signal transduction of hH1–4Rs occurred mainly, but not exclusively, via their canonical Gα proteins. For example, in addition to Gαi/o, the Gαq/11 protein was proven to contribute to the DMR response of hH3,4Rs. Moreover, the Gα12/13 was identified to be involved in the hH2R mediated signaling pathway. These results are considered as a basis for future investigations on the (patho)physiological role and the pharmacological potential of H1–4Rs.