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.     

Identification of neuropeptide Y cleavage products in human blood to improve metabolic stability

Regulatory, receptor-binding peptides are considered as the agents of choice for diagnostic imaging and therapy of cancers, because their receptors are overexpressed in various human cancer cells. It has been recently indicated that there is a putative role of NPY in breast tumors. The expression of the two best-investigated NPY receptor subtypes, Y1 and Y2, in breast tissue shows predominant occurrence of the Y1 receptor subtype in tumors, whereas Y2 receptors are found in nonproliferative tissue. To investigate the usefulness of NPY analogs for tumor diagnosis and therapy, we investigated the metabolic stability of receptor-selective NPY analogs in human blood plasma. NPY analogs were synthesized by Fmoc/t-Bu solid-phase strategy. Prior to the cleavage of peptides from the resin, they were labeled with 5(6)-carboxyfluorescein (CF) either at the N-terminus or at the side chain of Lys4. For the metabolic stability study, the digestion of peptides was monitored by HPLC and the cleavage products were identified by MALDI-ToF mass spectrometry. The data showed that full-length [Phe7, Pro34]NPY analogs, which show high binding affinity to Y1 receptors are enzymatically more stable than centrally truncated analogs, which show high binding affinity to Y2 receptors. Furthermore, the N-terminally CF-labeled Y1 and Y2 receptor-selective peptides were found to be enzymatically more resistant than their counterparts containing the CF label at Lys4 side chain.     

The first selective agonist for the neuropeptide YY5 receptor increases food intake in rats

The first Y(5) receptor-selective analog of neuropeptide Y (NPY), [Ala(31),Aib(32)]NPY, has been developed and biologically characterized. Using competition binding assays on cell lines that express different Y receptors, we determined the affinity of this analog to be 6 nm at the human Y(5) receptor, >500 nm at the Y(1) and Y(2) receptors, and >1000 nm at the Y(4) receptor. Activity studies performed in vitro using a cAMP enzyme immunoassay, and in vivo using food intake studies in rats, showed that the peptide acted as an agonist. Further peptides obtained by the combination of the Ala(31)-Aib(32) motif with chimeric peptides containing segments of NPY and pancreatic polypeptide displayed the same selectivity and even higher affinity (up to 0.2 nm) for the Y(5) receptor. In vivo administration of the new Y(5) receptor-selective agonists significantly stimulated feeding in rats. The NMR solution structures of NPY and [Ala(31),Aib(32)]NPY showed a different conformation in the C-terminal region, where the alpha-helix of NPY was substituted by a more flexible, 3(10)-helical turn structure.     

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

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.     

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.     

In vitro and in vivo evaluation of 64Cu-labeled DOTA-linker-bombesin(7-14) analogues containing different amino acid linker moieties

The gastrin-releasing peptide receptor (GRPR) is overexpressed on a variety of tumor types and has been targeted with radiolabeled peptides for detection and therapy of these cancers. Analogues of the 14 amino acid bombesin (BN) peptide have been radiolabeled with both gamma- and positron-emitting radionuclides for detection of GRPR-expressing tumors. We have previously evaluated BN analogues radiolabeled with the positron-emitter, copper-64 (64Cu), that contained various aliphatic linkers placed between the BN peptide and the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator. These studies showed that the analogues could be used for positron-emission tomographic (PET) imaging of GRPR-positive tumors in mice but clinical translation would be hindered by significant uptake in background tissues. Therefore, the purpose of this study was to determine if the use of amino acid linkers placed between the DOTA chelate and the BN peptide would reduce nontarget tissue uptake, while maintaining good prostate tumor uptake. The linkers studied utilized three amino acid combinations of glycine (G), serine (S), or glutamic acid (E). In vitro assays in PC-3 cells showed that the glutamic acid-containing linkers had poor binding and internalization, while the other analogues had IC50 values <100 nM and good internalization. In vivo, these same analogues demonstrated tumor-specific uptake and good imaging characteristics that were comparable to, or better than the previously reported 64Cu-labeled DOTA-BN analogues. Overall, this study shows that BN analogues containing amino acid linkers can be used for the PET imaging of GRPR-expressing prostate cancer and that these linkers lead to lower background tissue uptake.     

Electron paramagnetic resonance backbone dynamics studies on spin-labelled neuropeptide Y analogues.

Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system of mammalians. NPY acts by binding to at least five G-protein coupled receptors (GPCRs) which have been named Y1, Y2, Y4, Y5 and Y6. Three spin-labelled NPY analogues containing the nitroxide group of the amino acid TOAC (2.2.6.6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) as a paramagnetic probe were synthesized by solid-phase peptide synthesis. Synthetic problems owing to the sensitivity of nitroxide towards acidic and reducing conditions have been overcome by using a cleavage cocktail that contains anisole and cresol scavengers. Concerning the receptor binding preferences, the analogues [TOAC34]-pNPY and [Ala31, TOAC32]-pNPY showed a marked selectivity for the Y5 receptor, while [TOAC2]-pNPY maintained a significant binding also to the Y2 receptor subtype. The modifications of the native peptide structure caused by the introduction of TOAC were examined by circular dichroism. In order to determine the rotational correlation time of the spin probes, electron paramagnetic resonance measurements were performed in solution and in the presence of liposomes. This allowed us to evaluate the backbone dynamics of the different parts of the NPY molecule in the free and membrane bound states. The results of these studies showed that NPY Interacts with liposomes by using the C-terminal alpha-helix while the N-terminal tail retains a flexibility that is comparable to that of the peptide in solution as already shown by NMR studies on DPC micelles. Furthermore, we demonstrated that TOAC-labelllng is a valuable tool to investigate changes in the backbone conformation and dynamics. This may be of major importance for peptides and small proteins when they bind to cell membranes.     

PYY preference is a common characteristic of neuropeptide Y receptors expressed in human, rat, and mouse gastrointestinal epithelia

This investigation describes the relative potencies of four peptide agonists, namely, peptide YY (PYY), [Leu3l,Pro34]PYY (Pro34pYY), neuropeptide Y (NPY), and [Leu31,Pro34]NPY (Pro34NPY), as antisecretory agents in human, rat, and mouse gastrointestinal preparations. The inhibition of agonist responses by the Y1-receptor antagonist BIBP 3226 was also tested in each preparation. An unexpectedly pronounced preference for PYY and Pro34PYY was observed in functional studies of two human epithelial lines stably transfected with the rat Y1 receptor (Y1-7 and C1Y1-6). NPY and Pro34NPY were at least an order of magnitude less effective than PYY in these functional studies but were only marginally less potent in displacement binding studies using membrane preparations of the same clonal lines. The orders of agonist potency obtained in Y1-7 and C1Y1-6 epithelia were compared with those obtained from a single human colonic adenocarcinoma cell line (Colony-6, which constitutively expresses Y1 receptors) and also from mucosal preparations of rat and mouse descending colon. Similar peptide orders of potency were obtained in rat and mouse colonic mucosae and Colony-6 epithelia, all of which exhibited PYY preference (although less pronounced than with Y1-7 and C1Y1-6 epithelia) and significant sensitivity to the Y1 receptor antagonist, BIBP 3226. We have compared the pharmacology of these five mammalian epithelial preparations and provide cautionary evidence against the reliance upon agonist concentration-response relationships alone, in the characterization of NPY receptor types.     

A neuropeptide Y receptor Y1-subfamily gene from an agnathan, the European river lamprey. A potential ancestral gene.

We report here the isolation and functional expression of a neuropeptide Y (NPY) receptor from the river lamprey, Lampetra fluviatilis. The receptor displays approximately 50% amino-acid sequence identity to all previously cloned Y1-subfamily receptors including Y1, Y4, and y6 and the teleost subtypes Ya, Yb and Yc. Phylogenetic analyses point to a closer relationship with Y4 and Ya/b/c suggesting that the lamprey receptor could possibly represent a pro-orthologue of some or all of those gnathostome receptors. Our results support the notion that the Y1 subfamily increased in number by genome or large-scale chromosome duplications, one of which may have taken place prior to the divergence of lampreys and gnathostomes whereas the second duplication probably occurred in the gnathostome lineage after this split. Functional expression of the lamprey receptor in a cell line facilitated specific binding of the three endogenous lamprey peptides NPY, peptide YY and peptide MY with picomolar affinities. Binding studies with a large panel of NPY analogues revealed indiscriminate binding properties similar to those of another nonselective Y1-subfamily receptor, zebrafish Ya. RT-PCR detected receptor mRNA in the central nervous system as well as in several peripheral organs suggesting diverse functions. This lamprey receptor is evolutionarily the most distant NPY receptor that clearly belongs to the Y1 subfamily as defined in mammals, which shows that subtypes Y2 and Y5 arose even earlier in evolution.     

Determination of affinity and activity of ligands at the human neuropeptide Y Y4 receptor by flow cytometry and aequorin luminescence

Fluorescence-labeled neuropeptide Y (NPY) has been used in flow cytometric binding assays for the determination of affinity constants of NPY Y1, Y2, and Y5 receptor ligands. Because the binding of fluorescent NPY is insufficient for competition studies at the human Y4 receptor (hY4R), we replaced Glu-4 in hPP with Lys for the derivatization with cyanine-5. Because cy5-[K(4)]hPP has high affinity (Kd 5.6 nM) to the hY4R, it was used as a probe in a flow cytometric binding assay. Specific binding of cy5-[K(4)]hPP to hY4R was visualized by confocal microscopy. The hY(4)R, the chimeric G protein G(qi5) and mitochondrially targeted apoaequorin were stably coexpressed in CHO cells. Aequorin luminescence was quantified in a microplate reader and by a CCD camera. By application of these methods 3-cyclohexyl-N-[(3-1H-imidazol-4-ylpropylamino)(imino)methyl]propanamide (UR-AK49) was discovered as the first nonpeptidic Y4R antagonist (pKi 4.17), a lead to be optimized in terms of potency and selectivity.     

99mTc-labeled neuropeptide Y analogues as potential tumor imaging agents.

The possible use of neuropeptide Y (NPY) as a novel radiopeptide has been investigated. NPY is a 36-amino acid peptide of the pancreatic polypeptide family, which is expressed in the peripheral and central nervous system, and is one of the most abundant neuropeptides in the brain. Its receptors are produced in a number of neuroblastoma and the thereof derived cell lines. As structure-activity relationships of NPY are well-known, we could assume where a radionuclide might be introduced without affecting receptor affinity. We applied the novel [99mTc(OH2)3(CO)3]+ aqua complex and PADA (2-picolylamine-N,N-diacetic acid) as bifunctional chelating agent. The peptides were synthesized by solid-phase peptide synthesis, and PADA was coupled to the side chain of Lys4 of the resin-bound peptide. Upon postlabeling of [K4(PADA)]-NPY, 99mTc(CO)3 did not only bind to the desired PADA, but presumably as well to the His in position 26. Since the replacement of His26 by Ala only slightly decreased binding affinity, [K4(PADA),A26]-NPY was specifically postlabeled, and the 185Re surrogate maintained high binding affinity. Furthermore, the prelabeling approach has been applied for the centrally truncated analogue [Ahx5-24]-NPY, which is highly selective for the Y2 receptor. The resulting Ac-[Ahx5-24,K4(99mTc(CO)3-PADA)]-NPY was produced with a yield of only 16%. Therefore, postlabeling was applied for the short analogue as well, again substituting His26 by Ala. Competitive binding assays using (185)Re as a surrogate for 99mTc showed high binding affinity of Ac-[Ahx5-24,K4(185Re(CO)3-PADA),A26]-NPY. Internalization studies with the corresponding 99mTc-labeled analogue revealed receptor-mediated internalization. Furthermore, biodistribution studies were performed in mice, and stability was tested in human plasma. Our centrally truncated analogue revealed a 6-fold increased stability compared to the natural peptide NPY. We conclude that Ac-[Ahx5-24,K4(99mTc(CO)3-PADA),A26]-NPY has promising characteristics for future applications in nuclear medicine.     

First selective agonist of the neuropeptide Y1‐receptor with reduced size

Selective NPY analogues are potent tools for tumour targeting. Their Y₁‐receptors are significantly over‐expressed in human breast tumours, whereas normal breast tissue only expresses Y₂‐receptors. The endogenous peptide consists of 36 amino acids, whereas smaller peptides are preferred because of better labelling efficiencies. As Y₁‐receptor agonists enhance the tumour to background ratio compared to Y₁‐receptor antagonists, we were interested in the development of Y₁‐receptor selective agonists. We designed 19 peptides containing the C‐terminus of NPY (28–36) with several modifications. By using competition receptor binding affinity assays, we identified three NPY analogues with high Y₁‐receptor affinity and selectivity. Metabolic stability studies in human blood plasma of the N‐terminally 5(6)‐carboxyfluorescein (CF) labelled peptides resulted in half‐lives of several hours. Furthermore, the degradation pattern revealed proteolytic degradation of the peptides by amino peptidases. The most promising peptide was further investigated in receptor activation and internalization studies. Signal transduction assays revealed clear agonistic properties, which could be confirmed by microscopy studies that showed clear Y₁‐receptor internalization. For the first time, here we show the design and characterization of a small Y₁‐receptor selective agonist. This agonist might be a useful novel ligand for NPY‐mediated tumour diagnostics and therapeutics. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Highly potent and small neuropeptide Y agonist obtained by linking NPY 1-4 via spacer to alpha-helical NPY 25-36

Analogues of neuropeptide Y (NPY) containing small N- and C-terminal segments linked via flexible spacer arms were found to exhibit receptor binding affinity constants almost as high as NPY as well as post- and presynaptic NPY-agonistic activities. One of the most active analogues contains N-terminal NPY segment 1-4 linked via epsilon-aminocaproic acid (Aca) to the C-terminal partially alpha-helical peptide amide segment 25-36. NPY 1-4-Aca-25-36 is the first highly potent NPY agonist, which is of considerably reduced size in comparison to the native hormone. The analogues are accessible by solid-phase synthesis using Fmoc strategy.     

乳腺癌荷瘤鼠99mTc-DOTANOC显像与生长抑素受体表达水平的相关性研究

目的:探讨乳腺癌荷瘤鼠模型中肿瘤组织中的生长押素受体(SSTR)的表达水平与99mTc-DOTANOC显像的相关性研究.方法:配体交换法标记99mTc-DOTANOC,通过尾静脉注射乳腺癌荷瘤鼠模型,行99mTc-DOTANOC显像,勾画ROI计算肿瘤与对侧正常组织(T/NT)的放射性比值并测定肿瘤及主要脏器单位组织的放射性摄取百分值(%ID/g),采用逆转录聚合酶反应(RT-PCR)检测肿瘤组织中各SSTR亚型mRNA的表达水平,对SSTR亚型表达水平与T/NT放射性摄取比值进行相关性研究.结果:99mTc-DOTANOC乳腺癌荷瘤鼠模型显像示肿瘤部位有较高的放射性浓聚,与对侧正常组织T/NT比值较高,4h达到2.41±0.21;99mTc-DOTANOC荷瘤鼠体内生物分布示药物在肿瘤部位有较高的摄取;RT-PCR示乳腺癌组织中有着丰富的SSTR表达,SSTR3和SSTR2亚型表达水平较高,两者mRNA的表达水平与荷瘤鼠显像T/NT比值呈正相关(两者分别r=0.94,r=81,P<0.05).结论:乳腺癌细胞株MDA-MB-435 高表达SSTR2和SSTR3,其中SSTR3和SSTR2 mRNA表达水平与肿瘤组织对99mTc-DOTANOC的摄取呈正相关.第三代生长抑素类似物99mTc-DOTANOC受体显像对乳腺癌有较好的影像诊断价值.     

Biophysical methods to study ligand-receptor interactions of neuropeptide Y

Neuropeptide Y (NPY) is a 36 amino acids peptide amide that was isolated for the first time almost 20 years ago from porcine brain. NPY displays a multiplicity of physiological effects that are transmitted by at least six G-protein coupled receptors (GPCRs) named Y(1), Y(2), Y(3), Y(4), Y(5), and y(6). Because of the difficulty in obtaining high-resolution crystallographic structures from GPCRs that all belong to seven transmembrane helices proteins, a variety of biophysical methods have been applied in order to characterize the interaction of ligand and receptor. In this review article we present the most relevant outcomes of the studies performed in this field by our group and others. The use of photoaffinity labeling allowed the molecular characterization of the Y(2) receptor. The concerted application of molecular modeling and mutagenesis studies led to a model for the interaction of the natural agonist and nonpeptide antagonists with the Y(1) receptor. The three-dimensional (3D) structure and dynamics of micelle-bound NPY and their implications for receptor selection have been studied by NMR. The characterization of the tertiary and quaternary structure of the NPY dimer in solution at millimolar concentrations has been performed by NMR and extended to physiologically relevant concentrations by fluorescence resonance energy transfer (FRET) experiments performed with fluorescence-labeled analogues.     

NPY receptors in human cancer: a review of current knowledge

Many peptide hormone receptors are over-expressed in human cancer, permitting an in vivo targeting of tumors for diagnostic and therapeutic purposes. NPY receptors are novel and promising candidates in this field. Using in vitro receptor autoradiography, Y1 and Y2 receptors have been found to be expressed in breast carcinomas, adrenal gland and related tumors, renal cell carcinomas, and ovarian cancers in both tumor cells and tumor-associated blood vessels. Pathophysiologically, tumoral NPY receptors may be activated by endogenous NPY released from intratumoral nerve fibers or tumor cells themselves, and mediate NPY effects on tumor cell proliferation and tumoral blood supply. Clinically, tumoral NPY receptors may be targeted with NPY analogs coupled with adequate radionuclides or cytotoxic agents for a scintigraphic tumor imaging and/or tumor therapy.     

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.     

Receptor autoradiography as mean to explore the possible functional relevance of neuropeptides: focus on new agonists and antagonists to study natriuretic peptides, neuropeptide Y and calcitonin gene-related peptides

Over the past 20 years, receptor autoradiography has proven most useful to provide clues as to the role of various families of peptides expressed in the brain. Early on, we used this method to investigate the possible roles of various brain peptides. Natriuretic peptide (NP), neuropeptide Y (NPY) and calcitonin (CT) peptide families are widely distributed in the peripheral and central nervous system and induced multiple biological effects by activating plasma membrane receptor proteins. The NP family includes atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). The NPY family is composed of at least three peptides NPY, peptide YY (PYY) and the pancreatic polypeptides (PPs). The CT family includes CT, calcitonin gene-related peptide (CGRP), amylin (AMY), adrenomedullin (AM) and two newly isolated peptides, intermedin and calcitonin receptor-stimulating peptide (CRSP). Using quantitative receptor autoradiography as well as selective agonists and antagonists for each peptide family, in vivo and in vitro assays revealed complex pharmacological responses and radioligand binding profile. The existence of heterogeneous populations of NP, NPY and CT/CGRP receptors has been confirmed by cloning. Three NP receptors have been cloned. One is a single-transmembrane clearance receptor (NPR-C) while the other two known as CG-A (or NPR-A) and CG-B (or NPR-B) are coupled to guanylate cyclase. Five NPY receptors have been cloned designated as Y(1), Y(2), Y(4), Y(5) and y(6). All NPY receptors belong to the seven-transmembrane G-protein coupled receptors family (GPCRs; subfamily type I). CGRP, AMY and AM receptors are complexes which include a GPCR (the CT receptor or CTR and calcitonin receptor-like receptor or CRLR) and a single-transmembrane domain protein known as receptor-activity-modifying-proteins (RAMPs) as well as an intracellular protein named receptor-component-protein (RCP). We review here tools that are currently available in order to target each NP, NPY and CT/CGRP receptor subtype and establish their respective pathophysiological relevance.     

Modular synthesis of non-peptidic bivalent NPY Y1 receptor antagonists

According to a 'bivalent ligand approach' to increase the affinity of the potent argininamide-type NPY Y(1) receptor antagonist BIBP-3226, dimeric ligands were synthesized in which two molecules of the parent compound were linked by different spacers via N(G)-acylation at the guanidino groups. A synthetic route for the preparation of the title compounds was developed, which includes a copper(I)-catalyzed azide alkyne cycloaddition as the key step. Three bivalent analogues of BIBP-3226 were prepared showing nanomolar antagonistic activity and binding affinity to the NPY Y(1) receptor (calcium assay on HEL cells, radioligand binding assay on SK-N-MC cells), but these ligands were not superior to the parent compound and there was no correlation with the length or the chemical nature of the spacer. A trivalent BIBP-3226 derivate showed, surprisingly, no affinity to the NPY Y(1) receptor at all.