Peptidic Neuropeptide Y Y4 Receptor Agonists: Synthesis, Pharmacology and Stability

The neuropeptide Y (NPY) Y4 receptor (Y4R) represents a potential therapeutic target with respect to the treatment of diet-related diseases such as obesity due to its involvement in regulating food intake and gastrointestinal tract motility, among others. Within the family of NPY receptors, the Y4 subtype is one of the least studied. There have been several efforts to develop Y4R ligands, largely with the aim of obtaining potential therapeutics. Given the lack of high-affinity molecular probes that allow for the study of these ligands in vitro under conditions that are physiologically relevant, i.e. using sodium-containing buffers, the new Y4R radioligand 3H2.5 (3HUR-JG102) was characterized in a sodium-containing and, for comparison, also in a sodium-free buffer. The findings from this study showed that 3H2.5 is suitable for Y4R ligand binding studies under physiological-like conditions (sodium-containing buffer). 3H2.5 was also used in autoradiographic Y4R binding studies using tissues of rat brain and rat intestines, to assess its suitability for this technique. This study revealed low to moderate expression of Y4R in certain brain regions, including the hypothalamus, midbrain, medulla, and brain stem. Also, Y4R binding of 3H2.5 was detected in rat intestines, known to express Y4R. These findings suggested that this radioligand is suitable for autoradiographic Y4R binding studies at cryosections of organs. Another aspect of this thesis focused on identifying the proteolytic enzyme(s) and susceptible amide bond(s) associated with the proteolytic degradation of the cyclic hexapeptide 2.4 (UR-AK86C) (half-life in human plasma ca. 2 h), a potential lead compound for drug development. It also focused on developing plasma-stable analogs of 2.4 with improved stability in human plasma. By Nα-methylation of the proteolytically susceptible amide bond, replacement of the respective amino acids, or elongating the peptide backbone, 13 analogs of 2.4 were synthesized. The study revealed that angiotensin-converting enzyme (ACE), which cleaves the Leu4-Arg5 peptide bond in 2.4, is responsible for the degradation of this peptide in human plasma. Concerning the development of plasma-stable analogs of 2.4, eight peptides with significantly improved plasma stabilities (half-live ≥ 9 h) were obtained. However, the structural changes also led to significant decreases in their Y4R binding affinities and agonism (up to 4 log units) compared to 2.4. Out of these analogs, 4.9b (UR-AG27), containing a cyclopentyl-Gly in position 4 instead of Leu, emerged as a potential lead structure candidate with significantly improved plasma stability (half-life ca. 19.5 h) while exhibiting partial Y4R agonism (cAMP assay: pEC50 = 7.60, Emax = 93%) and moderately high Y4R binding affinity (pKi = 8.58). New analogs of 2.4 and 4.9b were also synthesized, with the guanidine groups in the peptides being replaced by weakly basic methyl-carbamoylguanidine moieties to reduce their positive net charge at pH 7.4, hence potentially increasing their penetration across biological membranes. While the analogs of 2.4 (5.8b (UR-AG13), 5.9b (UR-AG11), and 5.10b (UR-AG15)) showed increased lipophilicity but decreased Y4R binding affinities (up to 1.5 log units), the analog of 4.9b (5.13b (UR-AG41)) exhibited enhanced lipophilicity along with a Y4R binding affinity and agonism similar to 4.9b. Translocation studies of 4.9b and 5.13b using HBEC-5i cells revealed that these analogs were substantially proteolytically degraded by cellular enzymes within 24 h of incubation, producing degradation products similar to those found after their incubation (24 h) in human plasma. Further studies indicated that ACE and its variants were not responsible for their degradation, suggesting a need for additional investigations. Lastly, part of this thesis focused on elucidating the effect of sodium on the NPY receptor (YR) binding profiles (Y1R, Y2R, Y4R, Y5R) of the endogenous agonists NPY, peptide YY, and pancreatic polypeptide, as well as of selected YR antagonists. The study showed that the presence of sodium generally reduces the binding of agonists to YRs while increasing the binding of antagonists. Notably, the effect of sodium on YR binding was dependent on the receptor subtype and the type of agonist or antagonist studied