Design, Synthesis and Pharmacological Characterization of Oligopeptidic and Non-Peptidic Neuropeptide Y Y4 Receptor Ligands

The 36-amino acid peptides neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) exert their biological effects via activation of four G protein-coupled receptors (Y1R, Y2R, Y4R and Y5R), contributing to the regulation of numerous (patho)physiological processes, such as feeding behavior, pain sensitivity, seizures and anxiety. Among the NPY receptors, the Y4R is unique, as it preferably binds PP over NPY and PYY. For a better understanding of the physiological role of the Y4R and to explore its relevance as a potential drug target, selective Y4R ligands are needed. Therefore, the aim of this work was the discovery of selective Y4R agonists and antagonists, including peptidic and non-peptidic ligands. The hexapeptide UR-KK236 (Ac-Arg-Tyr-Arg-Leu-Arg-Tyr-NH2, 2.1), a high affinity Y4R partial agonist (Ki = 3.4 nM), was used as lead structure for the development of new Y4R agonists and antagonists. N-terminal truncation of 2.1 by two amino acids resulted in a tetrapeptide (Arg-Leu-Arg-Tyr-NH2) (UR-AK32, 2.29) acting as Y4R partial agonist and showing as high Y4R affinity (Ki = 3.4 nM) as 2.1. Replacement of individual amino acids in 2.29 by unnatural amino acids such as naphthylalanine, β-homoleucine, 4-nitrophenylalanine and D-configured amino acids did not result in increased Y4R affinity. However, compound 2.29, representing the smallest high-affinity Y4R ligand to date, might serve as a lead structure for the development of non-peptidic Y4R ligands in future projects. Interestingly, replacement of Leu4 in 2.1 by Trp was identified as a key modification to turn Y4R agonism into antagonism (e.g. UR-AK1 (2.13), Arg-Tyr-Arg-Trp-Arg-Tyr-NH2). Compound 2.13, exhibiting a pKi of 7.57 (radioligand competition binding), behaved as an antagonist in Y4R -arrestin-1 and -arrestin-2 recruitment assays, as well as in a Y4R Ca2+ aequorin assay measuring G-protein activation (Kb values: 7.14, 6.87 and 7.32, respectively). The results from induced-fit docking studies, using homology models of the Y4R, suggested a different binding pose for the antagonist 2.13 compared to the agonists 2.1 and 2.29, being consistent with the experimental data. Moreover, based on the structures of 2.1, 2.13 and 2.29 and their key interactions with the Y4R, suggested by induced-fit docking studies, potential non-peptidic Y4R ligands with low molecular weight (< 503 g/mole) were designed. The virtual Y4R ligands were investigated by induced-fit docking using a Y4R homology model, and one series of compounds, containing a 3,5-bis(aminomethyl)benzoyl and a tricyclic 1H-pyrrolo[3,4-b]quinoline-1,3(2H)-dione moiety, was synthesized. Most of these compounds did not bind to the Y4R, but two compounds (UR-AK113, UR-AK118) displayed Y4R binding. The more potent ligand, UR-AK113 (4.32), containing two basic guanidine groups, exhibited a pKi value of 6.03 and showed agonistic behavior in a -arrestin 2 recruitment assay. In future approaches, 4.32 can potentially serve as a template for the synthesis of non-peptidic Y4R ligands showing higher Y4R affinity. Besides the discovery of the aforementioned peptidic Y4R antagonists, a major achievement of this work was the introduction of N-terminus-to-arginine side chain cyclized hexapeptides as a new class of Y4R (partial) agonists exhibiting high Y4R potency, affinity and selectivity. Among these compounds, UR-AK86c (cyclo[succinyl-Arg-Tyr-Arg(carb)]-Leu-Arg-Tyr-NH2, 5.18) and UR-AK95c (cyclo[succinyl-Arg-Trp-Arg(carb)]-Leu-Arg-Tyr-NH2, 5.24) (note: Arg(carb) stands for an amino-functionalized Nω-carbamoylated arginine) showed the highest Y4R affinities with pKi values > 10. Worth mentioning, the non-cyclized, linear precursor peptides of 5.18 and 5.24 exhibited considerably lower Y4R affinity (27-fold and 64-fold, respectively). Stability studies with 5.18 and 5.24 in human plasma at 37 °C revealed rather low plasma stabilities (half-lives of ca. 2 h) due to proteolytic cleavage within the linear C-terminal tripeptide sequence (amide bond between Leu4 and Arg5), which is crucial for Y4R binding. Therefore, structural optimizations of the cyclic peptides are required with respect to metabolic stability and in vivo applications. Notably, cyclic peptides such as 5.24, exhibiting even higher Y4R affinity than the endogenous ligand hPP, represent attractive lead structures for the synthesis of new Y4R radio- and fluorescent-ligands, and can possibly be used as tools to support the crystallization of the hY4R in its active state. In summary, the approaches pursued in this thesis afforded new useful Y4R ligands, which can serve as pharmacological tools to study the physiological role of the NPY Y4 receptor, or as lead structures for future ligand development including labeled molecular tools for the Y4R