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.

The cross-linked pentapeptides (2R,7R)-diaminooctanedioyl-bis(Tyr-Arg-Leu-Arg-Tyr-amide) ((2R,7R)-BVD-74D, (2R,7R)-1) and octanedioyl-bis(Tyr-Arg-Leu-Arg-Tyr-amide) (2) as well as the pentapeptide Ac-Tyr-Arg-Leu-Arg-Tyr-amide (3) were previously described as neuropeptide Y Y4 receptor (Y4R) partial agonists. Here, we report on a series of analogues of (2R,7R)-1 and 2 in which Arg2, Leu3, or Arg4 were replaced by the respective aza-amino acids. The replacement of Arg2 in 3 with a carbamoylated arginine building block and the extension of the N-terminus by an additional arginine led to the high-affinity hexapeptide Ac-Arg-Tyr-Nω-[(4-aminobutyl)aminocarbonyl]Arg-Leu-Arg-Tyr-amide (35), which was used as a precursor for a d-amino acid scan. The target compounds were investigated for Y4R functional activity in assays with complementary readouts: aequorin Ca2+ and β-arrestin 1 or β-arrestin 2 assays. In contrast to the parent compounds, which are Y4R agonists, several ligands were able to suppress the effect elicited by the endogenous ligand pancreatic polypeptide and therefore represent a novel class of peptide Y4R antagonists.

Red-shifted azobenzene scaffolds have emerged as useful molecular photoswitches to expand potential applications of photopharmacological tool compounds. As one of them, tetra-ortho-fluoro azobenzene is well compatible for the design of visible-light-responsive systems, providing stable and bidirectional photoconversions and tissue-compatible characteristics. Using the unsubstituted azobenzene core and its tetra-ortho-fluorinated analogue, we have developed a set of uni- and bivalent photoswitchable toolbox derivatives of the highly potent muscarinic acetylcholine receptor agonist iperoxo. We investigated the impact of the substitution pattern on receptor activity and evaluated the different binding modes. Compounds 9b and 15b show excellent photochemical properties and biological activity as fluorination of the azobenzene core alters not only the photochromic behavior but also the pharmacological profile at the muscarinic M1 receptor. These findings demonstrate that incorporation of fluorinated azobenzenes not just may alter photophysical properties but can exhibit a considerably different biological profile that has to be carefully investigated.

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The neuropeptide Y (NPY) Y receptor (YR) is involved in energy homeostasis and considered a potential drug target for the treatment of obesity. Only a few molecular tools, i.e., radiolabeled and fluorescent ligands, for the investigation of the YR were reported. Previously, [Lys]hPP proved to be an appropriate full-length PP analog to prepare a fluorescent ligand by derivatization at the ε-amino group. To preclude oxidation upon long-term storage, we replaced the two methionine residues in [Lys]hPP by norleucine and prepared the corresponding [H]propionylated ([H]12) and cyanine labeled (13) peptides, which were characterized and compared with a set of reference compounds in binding (Y, Y, Y, and Y receptors) and functional (luciferase gene reporter, beta-arrestin-1,2) YR assays. Both molecular probes proved to be useful in radiochemical and flow cytometric saturation and competition YR binding experiments. Most strikingly, there was a different influence of the composition of buffer on equilibrium binding and kinetics: [H]12 affinity (K in Na-free buffer: 1.1 nM) clearly decreased with increasing sodium ion concentration, whereas dissociation and YR-mediated internalization of 13 (K in Na-free buffer: 10.8 nM) were strongly affected by the osmolarity of the buffer as demonstrated by confocal microscopy. Displacement of [H]12 and 13 revealed a tendency to higher apparent affinities for a set of reference peptides in hypotonic (Na-free) compared to isotonic buffers. The differences were negligible in the case of hPP but up to 270-fold in the case of GW1229 (GR231118). By contrast, no relevant influence of Na on YR affinity became obvious, when the radioligands [H]12 and [H]propionyl-pNPY were investigated in saturation binding and competition binding.