Application of the guanidine–acylguanidine bioisosteric approach to NPY Y2 receptor antagonists: bivalent, radiolabeled and fluorescent pharmacological tools
Neuropeptide Y (NPY), a 36 amino acid peptide, is widely distributed in the central and peripheral nervous system, where it acts as a neurotransmitter and exhibits a large number of physiological functions, including the regulation of blood pressure, control of food intake, anxiety, pain and hormone secretion. In humans, these effects are mediated by four receptor subtypes (Y1, Y2, Y4, Y5), all belonging to class A of the superfamily of G-protein coupled receptors (GPCRs). The Y2R is expressed, e. g., in sympathetic nerve endings, in the renal tubules and in distinct brain regions such as the hippocampus and the hypothalamus. It is discussed to be involved in several human diseases, for instance, epilepsy, obesity and cancer, and therefore regarded as an attractive target in drug design. However, antagonistic pharmacological tools for the elucidation of the receptor´s function in health and disease are still missing.
In 1999, the (S)-argininamide BIIE 0246, a C-terminal mimic of NPY, was reported the first highly potent and selective Y2R antagonist. NG-Acylation of BIIE 0246 was demonstrated to retain or even increase the Y2R affinity, thereby improving the pharmacokinetic properties. Thus, we prepared Nω-aminoacylated analogs as precursors towards radio- and fluorescence labeled as well as bivalent pharmacological tools according to the guanidine-acylguanidine bioisosteric approach. Such derivatives, bearing a free amino group, exhibited the highest Y2R affinities (Ki < 10 nM). Interestingly, masking the positive charge, e.g. by acylation, resulted in decreased affinities, presumably, due to the loss of an additional electrostatic interaction of the primary amine with the receptor.
Bivalent ligands were synthesized starting from various amine precursors derived from BIIE 0246 by acylation with different aliphatic dicarboxylic acids. Only minor differences in affinities (Ki = 61-300 nM) were observed for the majority of the compounds, irrespective of the diversity in length and chemical nature of the spacer. However, bivalent ligand 4.6, constructed from an amine precursor bearing an additional amino group in the linker, showed a binding affinity (Ki = 21 nM) and antagonistic activity (KB = 15 nM) in the same range as the monovalent antagonists, corroborating the affinity-enhancing effect of a positive charge in the linker.
Fluorescence ligands were synthesized from the amine precursors by acylation with succinimidyl esters or by ring transformation of pyrylium dyes. In terms of retaining affinity, the pyrylium dyes were superior to the bulky cyanine, hemicyanine and Bodipy dyes. Whereas the majority of the cyanine and hemicyanine labeled fluorescent ligands proved to be suitable for the detection of Y2Rs at the cell membrane by confocal microscopy, rapid cellular uptake was observed for most of the Py-1 and Py-5 coupled antagonists. Based on the results from binding studies and confocal microscopy, four fluorescent Y2R antagonists (5.4, 5.15-5.17) were chosen for flow cytometric binding studies. These compounds turned out to be applicable as labeled standard ligands in saturation and competition binding experiments. Interestingly, kinetic studies revealed a pseudo-irreversible binding of 5.16 at the Y2R. The endogenous ligand NPY was not able to displace the novel red-fluorescent Y2R antagonists 5.4 and 5.16 even at very high concentrations.
Acylation of the amine precursor (S)-3.48 with succinimidyl [2,3-3H]-propionate afforded the easily accessible, highly potent and selective tritiated Y2R antagonist [3H]-UR-PLN196 (A2: 43 nM, Ca2+ assay; KD: 43 nM, determined by kinetic studies), which was shown to be useful for the quantification of Y2R binding sites and for the radiochemical determination of Y2R binding affinities of small molecules. In addition, the novel radioligand was identified as an insurmountable antagonist against pNPY exhibiting pseudo-irreversible binding at the Y2R similar to the fluorescent labeled antagonist 5.16.
In conclusion, this work presents a straight-forward synthetic route towards labeled argininamide-type Y2R antagonists related to BIIE 0246 with improved physicochemical properties. These compounds are useful pharmacological tools for the detection of the Y2R in vitro and for detailed investigations of the antagonistic binding mode, respectively, as well as for the identification and characterization of small molecule Y2R antagonists. Moreover, these tracers are suitable to replace labeled NPY standard ligands in binding assays due to their beneficial properties in terms of Y2 receptor subtype selectivity, antagonistic mode of action, stability and kinetics
