High-throughput synthesis optimization of sulfonamide NPY Y5 antagonists

A series of sulfonamide neuropeptide Y Y5 antagonists was optimized by preparation of sets of analogues using high-throughput synthesis and purification techniques. Testing of these compounds for their ability to bind to the human NPY Y5 receptor revealed separate SAR trends for sulfonamide amides versus sulfonamide ureas versus sulfonamide amines. By understanding these SAR trends, potent compounds were identified in all three series.     

Neuropeptide Y is neuroproliferative for post-natal hippocampal precursor cells

New neurones are produced in the adult hippocampus throughout life and are necessary for certain types of hippocampal learning. Little, however, is known about the control of hippocampal neurogenesis. We used primary hippocampal cultures from early post-natal rats and neuropeptide Y Y1 receptor knockout mice as well as selective neuropeptide Y receptor antagonists and agonists to demonstrate that neuropeptide Y is proliferative for nestin-positive, sphere-forming hippocampal precursor cells and beta-tubulin-positive neuroblasts and that the neuroproliferative effect of neuropeptide Y is mediated via its Y1 receptor. Immunohistochemistry confirmed Y1 receptor staining on both nestin-positive cells and beta-tubulin-positive cells in culture and short pulse 5-bromo-2-deoxyuridine studies demonstrated that neuropeptide Y has a proliferative effect on both cell types. These studies suggest that the proliferation of hippocampal neuroblasts and precursor cells is increased by neuropeptide Y and, therefore, that hippocampal learning and memory may be modulated by neuropeptide Y-releasing interneurones.     

8-Amino-5-nitro-6-phenoxyquinolines: Potential Non-peptidic Neuropeptide Y Receptor Ligands

The synthesis and pharmacological evaluation of analogues of PD 160170, a neuropeptide Y1 (NPY) receptor antagonist are reported. Phamacomodulation of this 8-amino-5-nitro-6-phenylsulfonylquinoline was carried out by replacing the sulfone moiety by oxygen. The corresponding ethers 11 - 16 were obtained by nucleophilic substitution of 8-acetamido-6-chloro-5-nitroquinoline 4 with phenols, followed by acidic hydrolysis of the intermediary amides 5 - 10. The test compounds 11 - 16 exerted no appreciable Y1 activity and they were also inactive in terms of Y5 receptor binding; their IC 50 values were >1 μM and 10 μM, respectively. The dramatic decrease in potency resulting from replacement of the sulfone function by an ether was confirmed by IP administration of 16 to ob / ob mice; after a 4-day administration, no decrease in food consumption or weight was observed.     

8-Amino-5-nitro-6-phenoxyquinolines: potential non-peptidic neuropeptide Y receptor ligands

The synthesis and pharmacological evaluation of analogues of PD 160170, a neuropeptide Y1 (NPY) receptor antagonist are reported. Phamacomodulation of this 8-amino-5-nitro-6-phenylsulfonylquinoline was carried out by replacing the sulfone moiety by oxygen. The corresponding ethers 11-16 were obtained by nucleophilic substitution of 8-acetamido-6-chloro-5-nitroquinoline 4 with phenols, followed by acidic hydrolysis of the intermediary amides 5-10. The test compounds 11-16 exerted no appreciable Y1 activity and they were also inactive in terms of Y5 receptor binding; their IC50 values were >1 microM and 10 microM, respectively. The dramatic decrease in potency resulting from replacement of the sulfone function by an ether was confirmed by IP administration of 16 to ob/ob mice; after a 4-day administration, no decrease in food consumption or weight was observed.     

Modeling of neuropeptide receptors Y1, Y4, Y5, and docking studies with neuropeptide antagonist

Neuropeptide Y (NPY), receptors belong to the G-protein coupled receptor superfamily. NPY mediates several physiological responses, such as blood pressure, food intake, sedation. These actions of NPY are mediated by six receptor subtypes denoted as Y1-Y5 and y6. Modeling of receptor subtypes and binding site identification is an important step in developing new therapeutic agents. We have attempted to model the three NPY receptor types, Y1, Y4, and Y5 using homology modeling and threading methods. The models are consistent with previously reported experimental evidence. To understand the interaction and selectivity of NPY analogues with different neuropeptide receptors, docking studies of two neuropeptide analogues (BVD10 and BVD15) with receptors Y1 and Y4 were carried out. Results of the docking studies indicated that the interaction of ligands BVD10 and BVD15 with Y1 and Y4 receptors are different. These results were evaluated for selectivity of peptide analogues BVD10 and BVD15 towards the receptors.     

Aminopyrazoles with high affinity for the human neuropeptide Y5 receptor

1,3-Disubstituted-5-aminopyrazoles were prepared based on a lead compound found through high-throughput screening of our corporate compound library in an assay measuring affinity for the human neuropeptide Y5 receptor. The target compounds were prepared by cyclization of alpha-cyanoketones with appropriate hydrazines, followed by reduction and coupling to various sulfonamido-carboxylic acids. Several of these arylpyrazoles (e.g., 19 and 45) displayed high affinity for the human NPY Y5 receptor (<20nM IC(50)s).     

Modeling of Neuropeptide Receptors Y1, Y4, Y5, and Docking Studies with Neuropeptide Antagonist Analogues: Implications for Selectivity

Abstract Neuropeptide Y (NPY), receptors belong to the G-protein coupled receptor superfamily. NPY mediates several physiological responses, such as blood pressure, food intake, sedation. These actions of NPY are mediated by six receptor subtypes denoted as Y(1)-Y(5) and y(6). Modeling of receptor subtypes and binding site identification is an important step in developing new therapeutic agents. We have attempted to model the three NPY receptor types, Y1, Y4, and Y5 using homology modeling and threading methods. The models are consistent with previously reported experimental evidence. To understand the interaction and selectivity of NPY analogues with different neuropeptide receptors, docking studies of two neuropeptide analogues (BVD10 and BVD15) with receptors Y1 and Y4 were carried out. Results of the docking studies indicated that the interaction of ligands BVD10 and BVD15 with Y1 and Y4 receptors are different. These results were evaluated for selectivity of peptide analogues BVD10 and BVD15 towards the receptors.     

Modeling of Neuropeptide Receptors Y1, Y4, Y5, and Docking Studies with Neuropeptide Antagonist Analogues: Implications for Selectivity

Abstract

Neuropeptide Y (NPY), receptors belong to the G-protein coupled receptor superfamily. NPY mediates several physiological responses, such as blood pressure, food intake, sedation. These actions of NPY are mediated by six receptor subtypes denoted as Y₁-Y₅ and y₆. Modeling of receptor subtypes and binding site identification is an important step in developing new therapeutic agents. We have attempted to model the three NPY receptor types, Y1, Y4, and Y5 using homology modeling and threading methods. The models are consistent with previously reported experimental evidence. To understand the interaction and selectivity of NPY analogues with different neuropeptide receptors, docking studies of two neuropeptide analogues (BVD10 and BVD15) with receptors Y1 and Y4 were carried out. Results of the docking studies indicated that the interaction of ligands BVD10 and BVD15 with Y1 and Y4 receptors are different. These results were evaluated for selectivity of peptide analogues BVD10 and BVD15 towards the receptors.     

Neuropeptide Y stimulates DNA synthesis in human vascular smooth muscle cells through neuropeptide Y Y1 receptors

We investigated the mitogenic effect, measured as [3H]thymidine incorporation, of neuropeptide Y (NPY) on smooth muscle cells (SMCs) from human subcutaneous arteries (diameter: 0.4 mm). NPY stimulated DNA synthesis in a concentration-dependent manner, Emax 32 +/- 5% relative to control. The effect was potently antagonised by the NPY Y1 receptor antagonist BIBP3226 ((R)-N2-(diphenylacetyl)-N-[(4-hydroxy-phenyl)methyl]-D-arginine-a mide), indicating the effect to be mediated via the NPY Y1 receptor. Noradrenaline (NA) also induced mitogenesis, Emax 35 +/- 10% relative to control. When added together, NPY and NA potentiated the [3H]thymidine incorporation, Emax 109 +/- 38% relative to control. Also, this effect seems to be mediated by the NPY Y1 receptor, since BIBP3226 blocked the effect (44 +/- 9% relative to control). The mitogenic effect of NPY and NA, two important transmitters of the sympathetic nervous system, might have clinical consequences on conditions with elevated sympathetic nerve activity.     

Novel potent antagonists of human neuropeptide Y Y5 receptors. Part 2: substituted benzo[a]cycloheptene derivatives

Novel benzo[a]cycloheptene derivatives were prepared for the purpose of searching new neuropeptide Y-Y5 (NPY-Y5) receptor antagonists. The structure-activity relationships are described and compound 2o (FR226928) showed the most potent affinity for Y5 receptor of all we prepared and was found to have higher potency and better selectivity for Y5 over Y1 receptor affinities when compared with the known lead compound 1.     

Novel potent neuropeptide Y Y5 receptor antagonists: synthesis and structure-activity relationships of phenylpiperazine derivatives

A series of phenylpiperazine derivatives were synthesized and evaluated for their neuropeptide Y (NPY) Y5 receptor antagonistic activities. The benzindane portion of 2 was replaced by 1-phenylpiperazine, resulting in novel urea derivative 3f. Subsequent optimization of the phenylpiperazine template by substitution of the phenyl moiety resulted in a series of (2-methanesulfonamidephenyl)piperazine derivatives that showed potent binding affinity and antagonistic activity for the Y5 receptor.     

Pharmacological characterization of cloned chicken neuropeptide Y receptors Y1 and Y5

The neuropeptide Y (NPY) receptor subtypes Y1 and Y5 are involved in the regulation of feeding and several other physiological functions in mammals. To increase our understanding of the origin and mechanisms of the complex NPY system, we report here the cloning and pharmacological characterization of receptors Y1 and Y5 in the first non-mammal, chicken (Gallus gallus). The receptors display 80-83% and 64-72% amino acid sequence identity, respectively, with their mammalian orthologues. The three endogenous ligands NPY, peptide YY (PYY) and pancreatic polypeptide (PP) have similar affinities as in mammals, i.e. NPY and PYY have subnanomolar affinity for both receptors whereas chicken PP bound with nanomolar affinity to Y5 but not to Y1. A notable difference to mammalian receptor subtypes is that the Y1 antagonist SR120819A does not bind chicken Y1, whereas BIBP3226 does. The Y5 antagonist CGP71863A binds to the chicken Y5 receptor. Anatomically, both Y1 and Y5 have high mRNA expression levels in the infundibular nucleus which is the homologous structure of the hypothalamic arcuate nucleus in mammals. These results suggest that some of the selective Y1 and Y5 antagonists developed in mammals can be used to study appetite regulation in chicken.     

Distinct Changes in Peptide YY Binding to, and mRNA Levels of, Y1 and Y2 Receptors in the Rat Hippocampus Associated with Kindling Epileptogenesis

Abstract: Electrical kindling of the rat dorsal hippocampus induced significant changes in the binding of ¹²⁵I-peptide YY to Y1 and Y2 subtypes of neuropeptide Y receptors and in their mRNA levels in the area dentata as assessed by quantitative receptor autoradiography and in situ hybridization histochemistry. Binding to Y1 receptor sites decreased by 50% ( p < 0.05) in the molecular layer of the stimulated dentate gyrus, 2 days after preconvulsive stage 2 and 1 week or 1 month after generalized stage 5 seizures compared with sham-stimulated rats. Binding to Y2 receptor sites increased bilaterally by 36–87% ( p < 0.05) in the hilus at stage 2 and 1 week or 1 month after stage 5. No significant changes were observed after one afterdischarge or in the other hippocampal subfields or in the cortex. Y1 receptor mRNA signal decreased bilaterally by 50–64% ( p < 0.01) in the granule cell layer, 6 h but not 24 h after stages 2 and 5. The Y2 receptor mRNA signal was enhanced by 283% ( p < 0.01) in the stimulated granule cell layer 24 h after stage 2. At 6 and 24 h after stage 5, mRNA levels were increased both ipsilaterally (283 and 360%, respectively; p < 0.01) and contralaterally (190 and 260%, respectively; p < 0.05). No significant changes in level of either mRNA was found following one afterdischarge. These modifications, and the enhanced neuropeptide Y release previously shown in the hippocampus, suggest that kindling is associated with lasting changes in neuropeptide Y-mediated neurotransmission.     

Oligomerization of neuropeptide Y (NPY) Y2 receptors in CHO cells depends on functional pertussis toxin-sensitive G-proteins

Human neuropeptide Y Y2 receptors expressed in CHO cells are largely oligomeric, and upon solubilization are recovered by density gradient centrifugation as approximately 180 kDa complexes of receptor dimers and G-protein heterotrimers. A large fraction of the receptors is inactivated in the presence of pertussis toxin, in parallel with inactivation of Gi alpha subunits (with half-periods of about 4 h for both). This is accompanied by a very long-lasting loss of receptor dimers and of masked surface Y2 sites (an apparent receptor reserve pre-coupled mainly to Gi alpha subunit-containing G-proteins). However, surface Y2 receptors accessible to large peptide agonists are much less sensitive to the toxin. All surface Y2 receptors are rapidly blocked by Y2 antagonist BIIE0246, with a significant loss of the dimers, but with little change of basal Gi activity. However, both dimers and Y2 receptor compartmentalization are restored within 24 h after removal of the antagonist. In CHO cells, the maintenance and organization of Y2 receptors appear to critically depend on functional pertussis toxin-sensitive G-proteins.     

Pyrazolecarboxamide human neuropeptide Y5 receptor ligands with in vivo antifeedant activity

1-Aryl-3-carboxamido-5-alkylpyrazoles were prepared based on a hit found in high-throughput screening of our corporate compound library in an assay measuring affinity for the human neuropeptide Y5 receptor. 1-(3-Trifluoromethylphenyl)-3-[N-(5-quinolinyl)carboxamido]-5-methylpyrazole (31) bound to the human neuropeptide Y5 receptor with a 80 nM IC(50 )and was shown to inhibit cumulative food consumption 43.2% 2-6 h after ip dosing in a fasting-induced feeding model in rats.     

A novel cyclic analog of neuropeptide Y specific for the Y2 receptor.

The low-molecular-mass, cyclic analog of neuropeptide Y, [Ahx5-24, gamma-Glu2-epsilon-Lys30] NPY (YESK-Ahx-RHYINKITRQRY; Ahx, 6-aminohexanoic acid; NPY, neuropeptide Y), was synthesized and investigated for receptor binding, inhibition of forskolin-stimulated cAMP accumulation, inhibition of electrically stimulated rat vas deferens contractions and ability to increase blood pressure. Like the linear peptide [Ahx5-24] NPY (YPSK-Ahx-RHYINLITRQRY), the more rigid, cyclic analog showed good correlation between receptor binding to rabbit kidney membranes and biological activity in the vas deferens assay. Binding of this peptide to a new Y2-receptor-expressing cell line was slightly reduced, compared to the linear peptide [Ahx5-24] NPY, however inhibition of cAMP accumulation was even more efficient. Unlike the linear peptide [Ahx5-24] NPY, the cyclic analog did not induce a blood pressure increase in rats. Reduced binding to Y1 receptor-expressing SK-N-MC cells, as well as the loss of capability of signal transduction, suggest that only Y2-mediated activity is preserved after cyclization. The selectivity of the cyclic compound for Y2 subtypes of NPY receptors with respect to inhibition of cAMP accumulation is more than fortyfold increased, as compared to the linear NPY-(13-36) peptide, which has been used to determine Y2 selectivity so far.     

Discovery of substituted 2,4,4-triarylimidazoline derivatives as potent and selective neuropeptide Y Y5 receptor antagonists

Novel imidazoline derivatives were discovered to be potent neuropeptide Y Y5 receptor antagonists. High-throughput screening of Merck sample collections against the human Y5 receptor resulted in the identification of 2,4,4-triphenylimidazoline (1), which had an IC₅₀ of 54 nM. Subsequent optimization led to the identification of several potent derivatives.     

Cloned neuropeptide Y (NPY) Y1 and pancreatic polypeptide Y4 receptors expressed in Chinese hamster ovary cells show considerable agonist-driven internalization, in contrast to the NPY Y2 receptor.

Guinea-pig neuropeptide Y1 and rat pancreatic polypeptide Y4 receptors expressed in Chinese hamster ovary cells were internalized rapidly upon attachment of selective peptide agonists. The Y1 and Y2, but not the Y4, receptor also internalized the nonselective neuropeptide Y receptor agonist, human/rat neuropeptide Y. The internalization of guinea-pig neuropeptide Y2 receptor expressed in Chinese hamster ovary cells was small at 37 degrees C, and essentially absent at or below 15 degrees C, possibly in connection to the large molecular size of the receptor-ligand complexes (up to 400 kDa for the internalized fraction). The rate of intake was strongly temperature dependent, with essentially no internalization at 6 degrees C for any receptor. Internalized receptors were largely associated with light, endosome-like particulates. Sucrose dose-dependently decreased the internalization rate for all receptors, while affecting ligand attachment to cell membrane sites much less. Internalization of the Y1 and the Y4 receptors could be blocked, and that of the Y2 receptor significantly inhibited, by phenylarsine oxide, which also unmasked spare cell-surface receptors especially abundant for the Y2 subtype. The restoration of Y1 and Y4 receptors after agonist peptide pretreatment was decreased significantly by cycloheximide and monensin. Thus, in Chinese hamster ovary cells the Y1 and Y4 receptors have much larger subcellular dynamics than the Y2 receptor. This differential could also hold in organismic systems, and is comparable with the known differences in internalization of angiotensin, bradykinin, somatostatin and opioid receptor subtypes.     

D-Trp(34)] neuropeptide Y is a potent and selective neuropeptide Y Y(5) receptor agonist with dramatic effects on food intake

The neuropeptide Y (NPY) Y(5) receptor has been proposed to mediate several physiological effects of NPY, including the potent orexigenic activity of the peptide. However, the lack of selective NPY Y(5) receptor ligands limits the characterization of the physiological roles of this receptor. Screening of several analogs of NPY revealed that [D-Trp(34)]NPY is a potent and selective NPY Y(5) receptor agonist. Unlike the prototype selective NPY Y(5) receptor agonist [D-Trp(32)]NPY, [D-Trp(34)]NPY markedly increases food intake in rats, an effect that is blocked by the selective NPY Y(5) receptor antagonist CGP 71683A. These data demonstrate that [D-Trp(34)]NPY is a useful tool for studies aimed at determining the physiological roles of the NPY Y(5) receptor.