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.     

Cloning and characterization of the guinea pig neuropeptide Y receptor Y5

The Y5 receptor has been postulated to be the main receptor mediating NPY-induced food intake in rats, based on its pharmacological profile and mRNA distribution. To further characterize this important receptor subtype, we isolated the Y5 gene in the guinea pig, a widely used laboratory animal in which all other known NPY receptors (Y1, Y2, Y4, y6) [2,13,33,37] have recently been cloned by our group. Our results show that the Y5 receptor is well conserved between species; guinea pig Y5 displays 96% overall amino acid sequence identity to human Y5, the highest identity reported for any non-primate NPY receptor orthologue, regardless of subtype. Thirteen of the twenty substitutions occur in the large third cytoplasmic loop. The identities between the guinea pig Y5 receptor and the dog, rat, and mouse Y5 receptors are 93%, 89%, and 89% respectively. When transiently expressed in EBNA cells, the guinea pig Y5 receptor showed a high binding affinity to iodinated porcine PYY with a dissociation constant of 0.41 nM. Competition experiments showed that the rank order of potency for NPY-analogues was PYY = NPY = NPY2-36 > gpPP > rPP > NPY 22-36. Thus the pharmacological profile of the guinea pig Y5 receptor agrees well with that reported for the Y5 receptor from other cloned species.     

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.     

A Y2 receptor mimetic aptamer directed against neuropeptide Y.

Neuropeptide Y (NPY) is a 36-amino acid neuropeptide that exerts its activity by at least five different receptor subtypes that belong to the family of G-protein-coupled receptors. We isolated an aptamer directed against NPY from a nuclease-resistant RNA library. Mapping experiments with N-terminally, C-terminally, and centrally truncated analogues of NPY revealed that the aptamer recognizes the C terminus of NPY. Individual replacement of the four arginine residues at positions 19, 25, 33, and 35 by l-alanine showed that arginine 33 is essential for binding. The aptamer does not recognize pancreatic polypeptide, a highly homologous Y4 receptor-specific peptide of the gut. Furthermore, the affinity of the aptamer to the Y5 receptor-selective agonist [Ala(31),Aib(32)]NPY and the Y1/Y5 receptor-binding peptide [Leu(31),Pro(34)]NPY was considerably reduced, whereas Y2 receptor-specific NPY mutants were bound well by the aptamer. Accordingly, the NPY epitope was recognized by the Y2 receptor, and the aptamer was highly similar. This Y2 receptor mimicking effect was further confirmed by competition binding studies. Whereas the aptamer competed with the Y2 receptor for binding of [(3)H]NPY with high affinity, a low affinity displacement of [(3)H]NPY was observed at the Y1 and the Y5 receptors. Consequently, competition at the Y2 receptor occurred with a considerably lower K(i) value compared with the Y1 and Y5 receptors. These results indicate that the aptamer mimics the binding of NPY to the Y2 receptor more closely than to the Y1 and Y5 receptors.     

Molecular recognition of a peptide mimic of the Lewis Y antigen by an anti-Lewis Y antibody

Peptides as mimics of carbohydrates display a distinct advantage in vaccine design because of ease of synthesis and their inherent T cell-dependent nature as immunogens. While peptides that mimic carbohydrates have been described, it is not clear how they do so. To further our insight into structural relationships between peptide-mimics and carbohydrate structures, we have analyzed a potential recognition scheme between the murine monoclonal antibody, B3, directed against the tumor-associated antigen Lewis Y oligosaccharide and a peptide identified from phage display screening with B3. The Lewis Y core antigen is a difucosylated structure consisting of four hexose units. The B3 antibody binds to the peptide sequence APWLYGPA in which the putative sequence APWLY is critical for binding to the antibody. Not having experimental structural information for B3, the crystal structure of another anti-Lewis Y antibody, BR96, solved in complex with a nonoate methyl ester Lewis Y tetrasaccharide, provides a molecular basis for LeY antigen recognition and specificity, and how this binding relates to peptide binding. As a guide to place the APWLY motif in the B3 combining site, a fragment library was searched for analogous compounds that have the potential to bind to B3. Our modeling study shows that the B3-peptide complex shares similar recognition features for the difucosylated type 2 lactoseries' structure. This analysis provides a molecular perspective for peptide mimicry of a carbohydrate epitope. © 1998 John Wiley & Sons, Ltd.     

Pharmacology and quantitative structure-activity relationships of imidazolylpropylguanidines with mepyramine-like substructures as non-peptide neuropeptide Y Y1 receptor antagonists

The design of non-peptide, Y1-selective antagonists of neuropeptide Y (NPY) as pharmacological tools is in progress and is increasingly important as therapeutic applications are expected. Starting from the potent histamine H2 agonist and weak NPY Y1 antagonist arpromidine, 16 imidazolylpropylguanidine derivatives were synthesized and tested for Y1 antagonistic activity (inhibition of NPY-stimulated Ca2+ increase in human erythroleukemic cells), where the pheniramine-like moiety of arpromidine was replaced with 2-pyridylaminoalkyl, benzyl-(2-pyridyl)aminoalkyl, and phenyl-(2-pyridyl)alkylaminoalkyl partial structures derived from mepyramine. The pA2 values of the most active compounds are in the range of 6.2-6.5. Quantitative structure-activity relationships (QSAR) were investigated by fragment regression analysis. Results indicate that a tetramethylene spacer between the guanidino group and the amino nitrogen is optimal. For an at least moderate degree of Y1 antagonistic activity, a second benzyl or phenyl group must be present in addition to the 2-pyridyl ring. At this second group, hydrophobic substituents such as 3,4-di-CI and 4-Br further enhance Y1 antagonism. The most active derivative additionally bears a 5-Br substituent at the 2-pyridyl moiety. Structure-activity relationships suggest that the compounds might be able to partially imitate the role of NPY when interacting with Y1 receptors and thus behave as moderate non-peptide NPY Y1 antagonists.     

Conservation of human Y chromosome sequences among male great apes: Implications for the evolution of Y chromosomes

Nine newly described single-copy and lowcopy-number genomic DNA sequences isolated from a flow-sorted human Y chromosome library were mapped to regions of the human Y chromosome and were hybridized to Southern blots of male and female great ape genomic DNAs (Gorilla gorilla, Pan troglodytes, Pongo pygmaeus). Eight of the nine sequences mapped to the euchromatic Y long arm (Yq) in humans, and the ninth mapped to the short arm or pericentromeric region. All nine of the newly identified sequences and two additional human Yq sequences hybridized to restriction fragments in male but not female genomic DNA from the great apes, indicating Y chromosome localization. Seven of these 11 human Yq sequences hybridized to similarly-sized restriction endonuclease fragments in all the great ape species analyzed. The five human sequences that mapped to the most distal subregion of Yq (deletion of which region is associated with spermatogenic failure in humans) were hybridized to Southern blots generated by pulsed-field gel electrophoresis. These sequences define a region of approximately 1 Mb on human Yq in which HpaII tiny fragment (HTF) islands appear to be absent. The conservation of these human Yq sequences on great ape Y chromosomes indicates a greater stability in this region of the Y than has been previously described for most anonymous human Y chromosomal sequences. The stability of these sequences on great ape Y chromosomes seems remarkable given that this region of the Y does not undergo meiotic recombination and the sequences do not appear to encode genes for which positive selection might occur. Correspondence to: B. Steele Allen     

神经肽Y及其受体Y1、Y2对痛觉调制的研究进展

神经肽Y（neuropeptide Y,NPY）是一种由36个氨基酸残基组成的肽类激素,属胰多肽家族,广泛分布于中枢及外周神经组织的神经元中。NPY主要参与摄食行为、心血管活动、垂体分泌等生理功能的调节。NPY还参与了痛觉调制。NPY受体有Y1、Y2、Y3、Y4、Y5和Y6六种亚型。目前对Y1受体和Y2受体的研究较多,显示Y1受体和Y2受体参与痛觉调制。但现在对NPY在痛觉中的具体作用机制还不清楚。该文对NPY及其Y1受体、Y2受体在痛觉调制中的作用作一概述。     

Aryl urea derivatives of spiropiperidines as NPY Y5 receptor antagonists

Continuing medicinal chemistry studies to identify spiropiperidine-derived NPY Y5 receptor antagonists are described. Aryl urea derivatives of a variety of spiropiperidines were tested for their NPY Y5 receptor binding affinities. Of the spiropiperidines so far examined, spiro[3-oxoisobenzofurane-1(3H),4′-piperidine] was a useful scaffold for producing orally active NPY Y5 receptor antagonists. Oral administration of 5c significantly inhibited the Y5 agonist-induced food intake in rats with a minimum effective dose of 3 mg/kg. In addition, this compound was efficacious in decreasing body weight in diet-induced obese mice.     

Neuropeptide Y modulates functions of inflammatory cells in the rat: distinct role for Y1, Y2 and Y5 receptors

Neuropeptide Y (NPY) has been reported to be a potent anti-inflammatory peptide with ability to directly modulate activity of granulocytes and macrophages. The present study aimed to correlate the effects of NPY in vivo on lipopolysaccharide-induced air-pouch exudates cells and in vitro on peripheral blood leukocytes functions. The role of different Y receptors was examined using NPY-related peptides and antagonists with diverse subtype specificity and selectivity for Y receptors. Y1, Y2 and Y5 receptors were detected on air-pouch exudates cells (flow cytometry) and peripheral blood granulocytes (immunocitochemistry). NPY in vivo reduced inflammatory cells accumulation into the air pouch, and decreased their adherence and phagocytic capacity via Y2/Y5 and Y1/Y2 receptors, respectively. Quite the opposite, NPY in vitro potentiated adhesiveness and phagocytosis of peripheral blood granulocytes and monocytes by activating Y1 receptor. The differences between in vivo and in vitro effects of NPY on rat inflammatory cells functions are mostly due to dipeptidyl peptidase 4 activity. In addition, suppressive effect of NPY in vivo is highly dependent on the local microenvironment, peptide truncation and specific Y receptors interplay.     

Energy Metabolic Profile of Mice after Chronic Activation of Central NPY Y1, Y2, or Y5 Receptors**

Objective: Neuropeptide Y (NPY), a 36‐amino acid peptide with orexigenic properties, is expressed abundantly in the central nervous system and binds to several NPY receptor subtypes. This study examines the roles of the NPY Y1, Y2, and Y5 receptor(s) in energy homeostasis. Research Methods and Procedures: We administered intracerebroventricular NPY (3 μg/d) or selective peptide agonists for the Y1, Y2, and Y5 receptor subtypes to C57Bl/6 mice for 6 days by mini‐osmotic pumps to assess the role of each receptor subtype in NPY‐induced obesity. Energy expenditure (EE) and respiratory quotient (RQ) were studied using indirect calorimetry. Adiposity was measured by DXA scanning and fat pad dissection. Insulin sensitivity was tested by whole‐blood glucose measurement after an insulin challenge. Results: Central administration of the selective Y1 agonist, Y5 agonist, or NPY for 6 days in mice significantly increased body weight, adiposity, and RQ, with significant hyperphagia in the Y5 agonist‐ and NPY‐treated groups but not in the Y1 agonist‐treated group. The NPY, Y1, or Y5 agonist‐treated mice had little change in total EE during ad libitum and pair‐feeding conditions. Conversely, selective activation of the Y2 receptor reduced feeding and resulted in a significant, but transient, weight loss. Discussion: Central activation of both Y1 and Y5 receptors increases RQ and adiposity, whereas only Y5 receptor activation reduces energy expended per energy ingested. Selective activation of Y2 autoreceptors leads to hypophagia and transient weight loss, with little effect on total EE. Our study indicates that all three NPY receptor subtypes may play a role in regulating energy homeostasis in mice.     

Design, synthesis and SAR of a novel series of benzimidazoles as potent NPY Y5 antagonists

A novel class of benzimidazole NPY Y5 receptor antagonists was prepared exploiting a privileged spirocarbamate moiety. The structure-activity relationship of this series and efforts to achieve a profile suitable for further development and an appropriate pharmacokinetic profile in rat are described. Optimisation led to the identification of the brain penetrant, orally bioavailable Y5 antagonist 9b which significantly inhibited the food intake induced by a Y5 selective agonist with a minimal effective dose of 30mg/kg po.     

Discovery of potent and selective small molecule NPY Y5 receptor antagonists

The discovery of a new class of sulfonamide NPY Y5 receptor antagonists is described. Optimization of this series led to the identification of compounds with high affinity for the hY5 subtype and excellent selectivity over the other NPY receptor subtypes. The SAR for this series was examined and a model for understanding the ligand-receptor interactions was developed.     

ICV NPY Y1 receptor agonist but not Y5 agonist induces torpor-like hypothermia in cold-acclimated Siberian hamsters

The reduced metabolism derived from daily torpor enables numerous small mammals, including Siberian hamsters, to survive periods of energetic challenge. Little is known of the neural mechanisms underlying the initiation and expression of torpor. Hypothalamic neuropeptide Y (NPY) contributes to surviving energetic challenges by both increasing food ingestion and reducing metabolic expenditure. Intracerebroventricular injections of NPY in cold-acclimated Siberian hamsters induce torpor-like hypothermia comparable to natural torpor. Multiple NPY receptor subtypes have been identified, and the Y1 receptor and Y5 receptor both contribute to the orexigenic effect of NPY. The purpose of this research was to compare and contrast the effects of Y1 receptor activation by a specific Y1 agonist ([D-Arg25]-NPY) or Y5 receptor activation by a specific Y5 agonist ([D-Trp34]-NPY) on body temperature and subsequent food intake in cold-acclimated Siberian hamsters. Intracerebroventricular injections of Y1 agonist produced torporlike hypothermia closely resembling that induced by intracerebroventricular NPY. The intracerebroventricular Y5 agonist infrequently produced hypothermia reaching criterion for torpor and that failed to resemble either NPY-induced or natural torpor. Combined injections of Y1 and Y5 agonists resulted in hypothermia comparable to Y5 agonist treatments alone, negating the mimicry of NPY treatment seen with Y1 agonist alone. Prior treatment with Y1 agonist or Y5 agonist surprisingly had lingering effects on NPY-induced torpor expression, Y1 agonist enhanced and Y5 agonist inhibited the effect of NPY. The ability of NPY to induce torporlike hypothermia, especially its initiation, most likely involves activation of the NPY Y1 receptor subtype.     

Ligands of the neuropeptide Y Y2 receptor

Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian brain and exerts a variety of physiological processes in humans via four different receptor subtypes Y1, Y2, Y4 and Y5. Y2 receptor is the most abundant Y subtype receptor in the central nervous system and implicated with food intake, bone formation, affective disorders, alcohol and drugs of abuse, epilepsy, pain, and cancer. The lack of small molecule non-peptidic Y2 receptor modulators suitable as in vivo pharmacological tools hampered the progress to uncover the precise pharmacological role of Y2. Only in recent years, several potent, selective and non-peptidic Y2 antagonists have been discovered providing the tools to validate Y2 receptor as a therapeutic target. This Letter reviews Y2 receptor modulators mainly non-peptidic antagonists and their structure–activity relationships.     

Novel analogues of neuropeptide Y with a preference for the Y1-receptor.

Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian brain and acts in humans via at least three receptor subtypes: Y1, Y2, and Y5. Whereas selective agonists and antagonists are known for the Y2- and Y5-receptors, the Y1-receptor still lacks a highly selective agonist. This work presents the first NPY-based analogues with Y1-receptor preference and agonistic properties. Furthermore, the importance of specific amino acids of NPY for binding to the Y-receptor subtypes is presented. Amongst the analogues tested, [Phe7,Pro34]pNPY (where pNPY is porcine neuropeptide Y) showed the most significant Y1-receptor preference (> 1 : 3000-fold), with subnanomolar affinity to the Y1-receptor, and Ki values of approximately 30 nM for the Y2- and Y5-subtype, respectively. Variations of position 6, especially [Arg6,Pro34]pNPY and variations within positions 20-23 of NPY were found to result in further analogues with significant Y1-receptor preference (1 : 400-1 : 2000). In contrast, cyclo S-S [Cys20,Cys24]pNPY was found to be a highly selective ligand at the Y2-receptor, binding only threefold less efficiently than NPY. Analogues containing variations of positions 31 and 32 showed highly reduced affinity to the Y1-receptor, while binding to the Y5-receptor was affected less. Inhibition of cAMP-accumulation of selected peptides with replacements within position 20-23 of NPY showed preserved agonistic properties. The NPY analogues tested give insights into ligand-receptor interaction of NPY at the Y1-, Y2- and Y5-receptor and contribute to our understanding of subtype selectivity. Furthermore, the Y1-receptor-preferring peptides are novel tools that will provide insight into the physiological role of the Y1-receptor.     

Isolation and characterization of Y chromosome DNA probes.

A sorted, cloned Y chromosome phage library was screened for unique Y chromosome sequences. Of the thousands of plaques screened, 13 did not hybridize to radiolabeled 46,XX total chromosomal DNA. Three plaques were characterized further. Clone Y1 hybridized to multiple restriction enzyme fragments in both male and female DNA with more intense bands in male DNA. Clone Y2, also found in female and male DNA, is probably located in the pseudosutosomal region because extra copies of either the X or Y chromosomes increased Y2 restriction enzyme fragment intensity in total cellular DNA. Clone Y5 was male specific in three of four restriction enzyme digests although in the fourth a light hybridizing band was observed in both male and female DNA. Clone Y5 was sublocalized to band Yq 11.22 by hybridization to a panel of cellular DNA from patients with Y chromosome rearrangements. Clone Y5 can be used to test for retention of the proximally long arm Y suggested to cause gonadal cancer in carrier females. The long series of GA repeats in Y5, anticipated to be polymorphic, may provide a sensitive means to follow Y chromosome variation in human populations.     

Y chromosomal DNA of Drosophila hydei

Six recombinant DNA clones are described, which are derived from the Y chromosome of Drosophila hydei. They reveal characteristic features of Y chromosomal DNA sequences. Three of the cloned inserts are Y-specific and are members of the same family of repeated sequences associated with the lampbrush loop-forming fertility gene "nooses" in the short arm of the Y chromosome. The other three cloned sequences are members of three different families of repeated sequences, but display a small amount of homology to one another and to the family of the nooses sequences. These three cloned sequences are found preferentially in the Y chromosome, but also in other chromosomal positions. The Y chromosomal copies are located in the short arm of the Y chromosome. The other copies are found in autosomal kinetochore-associated heterochromatin or, for one of the cloned sequences, in one band of the giant chromosome 4, in addition to the kinetochore heterochromatin.     

Y1 and Y5 receptors are both required for the regulation of food intake and energy homeostasis in mice

Neuropeptide Y (NPY) acting in the hypothalamus is one of the most powerful orexigenic agents known. Of the five known Y receptors, hypothalamic Y1 and Y5 have been most strongly implicated in mediating hyperphagic effects. However, knockout of individual Y1 or Y5 receptors induces late-onset obesity--and Y5 receptor knockout also induces hyperphagia, possibly due to redundancy in functions of these genes. Here we show that food intake in mice requires the combined actions of both Y1 and Y5 receptors. Germline Y1Y5 ablation in Y1Y5(-/-) mice results in hypophagia, an effect that is at least partially mediated by the hypothalamus, since mice with adult-onset Y1Y5 receptor dual ablation targeted to the paraventricular nucleus (PVN) of the hypothalamus (Y1Y5(Hyp/Hyp)) also exhibit reduced spontaneous or fasting-induced food intake when fed a high fat diet. Interestingly, despite hypophagia, mice with germline or hypothalamus-specific Y1Y5 deficiency exhibited increased body weight and/or increased adiposity, possibly due to compensatory responses to gene deletion, such as the decreased energy expenditure observed in male Y1Y5(-/-) animals relative to wildtype values. While Y1 and Y5 receptors expressed in other hypothalamic areas besides the PVN--such as the dorsomedial nucleus and the ventromedial hypothalamus--cannot be excluded from having a role in the regulation of food intake, these studies demonstrate the pivotal, combined role of both Y1 and Y5 receptors in the mediation of food intake.