High molecular weight PEGylation of human pancreatic polypeptide at position 22 improves stability and reduces food intake in mice.

BACKGROUND AND PURPOSE: Human pancreatic polypeptide (hPP) is known to suppress appetite and food intake, thereby representing a potential therapeutic approach against obesity and associated metabolic disorders. The aim of this study was to improve hPP stability by covalent PEGylation with diverse molecular weight polyethylene glycols (PEGs) at two positions using promising lead structures while maintaining target activity. EXPERIMENTAL APPROACH: Modified peptides were synthesized by combined solid-phase and solution-phase peptide synthesis. Their potency was investigated in constitutively expressing human epithelial cells and isolated human colonic mucosa as well as receptor-transfected artificial cell lines. Human blood plasma and porcine liver homogenates were used to examine the in vitro stability of the analogues. The most promising variants were injected s.c. in C57BL/6JRj mice to monitor fasting-induced food intake and bioavailability. KEY RESULTS: In human epithelia and colonic mucosal preparations, activity of the modified hPP peptides depended on the core sequence and latency of the peptides was related to PEG size. Peptides modified with a 22 kDa PEG (PEG22) remained intact in blood plasma and on incubation with liver homogenates for more than 96 h. Finally, hPP2-36 , [K22 (PEG22)]hPP2-36 and [K22 (PEG22),Q34 ]hPP significantly reduced cumulative food intake in mice over 16 h after s.c. administration. CONCLUSIONS AND IMPLICATIONS: Modification with PEG22 at position 22 stabilizes hPP significantly while extending its biological activities and could be used in drug development prospectively.

Pancreatic polypeptide and its central Y4 receptors are essential for cued fear extinction and permanent suppression of fear.

BACKGROUND AND PURPOSE: Avoiding danger and finding food are closely related behaviours that are essential for surviving in a natural environment. Growing evidence supports an important role of gut-brain peptides in modulating energy homeostasis and emotional-affective behaviour. For instance, postprandial release of pancreatic polypeptide (PP) reduced food intake and altered stress-induced motor activity and anxiety by activating central Y4 receptors. EXPERIMENTAL APPROACH: We characterized [K(30) (PEG2)]hPP2-36 as long-acting Y4 receptor agonist and injected it peripherally into wildtype and Y4 receptor knockout (Y4KO) C57Bl/6NCrl mice to investigate the role of Y4 receptors in fear conditioning. Extinction and relapse after extinction was measured by spontaneous recovery and renewal. KEY RESULTS: The Y4KO mice showed impaired cued and context fear extinction without affecting acquisition, consolidation or recall of fear. Correspondingly, peripheral injection of [K(30) (PEG2)]hPP2-36 facilitated extinction learning upon fasting, an effect that was long-lasting and generalized. Furthermore, peripherally applied [K(30) (PEG2)]hPP2-36 before extinction inhibited the activation of orexin-expressing neurons in the lateral hypothalamus in WT, but not in Y4KO mice. CONCLUSIONS AND IMPLICATIONS: Our findings suggests suppression of excessive arousal as a possible mechanism for the extinction-promoting effect of central Y4 receptors and provide strong evidence that fear extinction requires integration of vegetative stimuli with cortical and subcortical information, a process crucially depending on Y4 receptors. Importantly, in the lateral hypothalamus two peptide systems, PP and orexin, interact to generate an emotional response adapted to the current homeostatic state. Detailed investigations of feeding-relevant genes may thus deliver multiple intervention points for treating anxiety-related disorders.

Functional characterization of the in vitro folded human y(1) receptor in lipid environment.

We describe the recombinant production of the human Y(1) receptor from inclusion bodies of E. coli cultures. The in vitro refolding was carried out in the presence of lipids from bovine brain extracts. Y(1) receptors in brain lipids compete for cellular receptors in competitive binding experiments.

Radiometal targeted tumor diagnosis and therapy with peptide hormones.

Radiometal labeled peptide hormones are promising tools for a new generation of radiopharmaceuticals, because their receptors frequently are overexpressed in many human tumors. Furthermore, peptide hormones are characterized by different advantages for clinical application, such as high tumor-to-background ratios as well as rapid blood clearance. Peptidic tumor targeting agents can be sub-divided into the following segments: peptide, spacer, bifunctional chelator and radioisotope. Here the biological and chemical properties of peptide hormones are summarized as well as their prerequisites for their use as tumor targeting agents. Additionally, promising bifunctional chelators and radioisotopes for radiometal labeling are reviewed. Some few special peptide hormones that have been pre-clinically or clinically investigated are furthermore presented, such as somatostatin, bombesin (BBS) / gastrin releasing peptide (GRP), vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY). In vitro and in vivo investigations of the binding affinity, selectivity, metabolic stability, bioavailability and biodistribution of radiolabeled peptide hormones could lead to potential peptide-based tumor targeting agents for tumor diagnosis and therapy.

Mapping of phosphorylation-dependent anti-tau monoclonal antibodies in immunoblots using human tau-constructs synthesized by native chemical ligation.

In Alzheimer's disease (AD) neurofibrillary tangles (NFT) are formed by hyperphosphorylated microtubule-associated tau protein. It is still a matter of controversy which phosphorylation sites are AD-specific and how these might be linked to the cause or progress of the disease. Whereas most research projects in this field rely on phosphorylation-dependent tau-specific monoclonal antibodies (mAbs), the phosphorylation patterns recognized by these mAbs are often not characterized in detail. Therefore, we synthesized unphosphorylated, two monophosphorylated (pThr231, pSer235), and the bisphosphorylated (pThr231+pSer235) tau226-240 peptides. The phosphopeptides were ligated via an N-terminal cysteine to the thioester-activated C-terminus of human aldo/keto reductase AKR1A1. After purification by preparative gel electrophoresis, the ligation products were analyzed by Western blotting and probed with phosphorylation-dependent anti-tau mAbs HPT-101, HPT-103, HPT-104, and HPT-110. The obtained specificities were very similar to the data obtained by ELISA, showing that ELISA-based epitope mapping studies are also valid for immunoblot analyses.

New [99mTc]bombesin analogues with improved biodistribution for targeting gastrin releasing-peptide receptor-positive tumors.

AIM: Bombesin (BBS) receptors are potential targets for diagnosis and therapy of breast and prostate tumors. To overcome the rapid degradation of natural BBS some modifications were introduced at positions 13 and 14. Additionally, a spacer was inserted between the chelator and the binding sequence in order to further improve the in vivo uptake. The analogues were labeled with the [(99m)Tc(CO)(3)]-core and tested. METHODS: Stability was analyzed in vitro in human plasma. Binding affinity and internalization were determined in vitro in prostate carcinoma PC-3 cells. Biodistribution studies and single photon emission computed tomography/X-ray computed tomography (SPECT/CT) imaging were performed in nude mice with PC-3 tumor xenografts. RESULTS: The changes introduced in the BBS(7-14) sequence substantially increased plasma stability. Affinity for gastrin releasing-peptide (GRP) receptors on PC-3 cells was comparable to that of the unmodified analogue with Kd<1 nM. The presence of a spacer in the molecule induced an increment in the in vivo uptake in pancreas and PC-3 xenografts (GRP receptor-positive tissues). The increase in pancreas and tumor uptake was higher when both spacer and stabilization are present in the same molecule. Moreover, in vivo uptake was highly specific. The tumor was clearly visualized by SPECT/CT. CONCLUSIONS: The modifications in the BBS(7-14) sequence led to a higher plasma stability while binding affinity remained unaffected. Stabilization resulted in improved biodistribution with better tumor to non-tumor ratios. However, the insertion of a spacer had a greater influence on the biodistribution. Analogues with both spacer and stabilization are the most promising radiopharmaceuticals for targeting GRP receptor-positive tumors.

Calcitonin-derived carrier peptides.

The recent discovery of carrier peptides offers new opportunities to translocate several bioactive molecules into the cytoplasm. Previous studies have shown that human calcitonin (hCT) and selected C-terminal sequences translocate in nasal epithelium. Moreover, the hCT(9-32) fragment was found to internalize efficiently a number of substances like fluorophores, nucleic acids or the enhanced green fluorescent protein (EGFP). In order to understand the uptake mechanism interactions of hCT(9-32) with membrane models of different lipid compositions have been investigated. From these studies it was possible to shed light on the conformational state of the peptide in the presence of membrane-like conditions. Further insight into the translocation mechanism was provided by fluorescence microscopy of truncated sequences of hCT that were shown to penetrate the plasma membrane and to distribute in a sectoral, punctuated pattern supporting an endocytotic internalization pathway as previously suggested.

PYY3-36 as an anti-obesity drug target.

The neuropeptide Y (NPY)/peptide YY (PYY) system has been implicated in the physiology of obesity for several decades. More recently ignited enormous interest in PYY3-36, an endogenous Y2-receptor agonist, as a promising anti-obesity compound. Despite this interest, there have been remarkably few subsequent reports reproducing or extending the initial findings, while at the same time studies finding no anti-obesity effects have surfaced. Out of 41 different rodent studies conducted (in 16 independent labs worldwide), 33 (83%) were unable to reproduce the reported effects and obtained no change or sometimes increased food intake, despite use of the same experimental conditions (i.e. adaptation protocols, routes of drug administration and doses, rodent strains, diets, drug vendors, light cycles, room temperatures). Among studies by authors in the original study, procedural caveats are reported under which positive effects may be obtained. Currently, data speak against a sustained decrease in food intake, body fat, or body weight gain following PYY3-36 administration and make the previously suggested role of the hypothalamic melanocortin system unlikely as is the existence of PYY deficiency in human obesity. We review the studies that are in the public domain which support or challenge PYY3-36 as a potential anti-obesity target.

Neuropeptide Y Y5 receptors inhibit kindling acquisition in rats.

Neuropeptide Y inhibits neuronal excitability and seizures in various experimental models. This peptide delays kindling epileptogenesis but the receptors involved in this action are unknown. We have studied the role of Y5 receptors in kindling using the selective antagonist GW438014A (IC50=210 nM), a small heterocycle molecule that crosses the blood-brain barrier, and the selective peptide agonist Ala31Aib34 NPY (IC50=6.0 nM). Intraperitoneal injection of GW438014A (10 mg/kg), 30 min before the beginning of a rapid-kindling protocol, significantly accelerated the rate of kindling acquisition as compared to vehicle-injected rats. Thus, the number of electrical stimuli required to reach stages 3 and 4-5 of kindling were reduced by 50% and 25%, respectively. The average afterdischarge duration in the stimulated hippocampus was prolonged by 2-fold. Conversely, kindling rate was delayed by intracerebroventricular administration of 24 nmol Ala31Aib32 NPY. Thus, the number of stimuli necessary to reach stages 2 and 3 of kindling was increased by 3- and 4-fold, respectively. During the stimulation protocol (40 stimuli) none of the rats treated with the Y5 agonist showed stages 4-5 seizures. Twenty-four hours after the last kindling stimulation, thus during the re-test session, Y5 agonist- or antagonist-treated rats had stages 4-5 seizures as their controls. In rats treated with both the antagonist and the agonist, kindling rate was similar to vehicle-injected rats. These data indicate that Y5 receptors mediate inhibitory effects of NPY in kindling and display anticonvulsant rather then antiepileptogenic effects upon agonist stimulation.

Biosynthesis of peptide hormones derived from precursor sequences.

The release of hormones is subject to a complex and finely tuned regulation system. The biosynthesis plays a key role by specifically converting the prohormone precursor into its biological active product(s). A family of mammalian proteases could be identified to be responsible for the endoproteolytic processing. These subtilisin/kexin-like prohormone convertases (PC) recognize their substrates at single or pairs of basic residues with a high substrate specificity. The so far known seven members include PC1/3, PC2, furin/PACE, PACE4, PC4, PC5/6 and PC7/SPC7/LPC/PC8. PC1/3 and PC2 are the most important enzymes for the processing of prohormones, whereas furin is the only one that causes lethality in knock-out models. Tissue-specific co-localization of the prohormone and the PC as well as distinct characteristics of both, like the secondary structures, determine the possible conversion processes. Identification of such determinants implies a great potential for the development of novel drug targets. To obtain sufficient amounts for the in vitro characterization of prohormones, chemical and recombinant synthesis methods have been developed. Application of expressed protein ligation lead to the semisynthesis of the first chemically modified analogs of a full-length proneurohormone (pro-neuropeptide Y). Structural analyses mainly on peptides of the pro-oxytocin/neurophysin system and on prosomatostatin highlighted the importance of flexible turn or loop structures adjacent to the cleavage site for the specific substrate-enzyme active site interaction. Prohormones and their processing show multiple functions. Therapeutic application including PC inhibitors is very promising for the treatment of disorders like cancer.

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.

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.

Novel peptide conjugates for tumor-specific chemotherapy.

One of the major problems in cancer chemotherapy are the severe side effects that limit the dose of the anticancer drugs because of their unselectivity for tumor versus normal cells. In the present work, we show that coupling of anthracyclines to peptides is a promising approach to obtain selectivity. The peptide-drug conjugate was designed to bind to specific receptors expressed on the tumor cells with subsequent internalization of the ligand-receptor complex. Neuropeptide Y (NPY), a 36-amino acid peptide of the pancreatic polypeptide family, was chosen as model peptide because NPY receptors are overexpressed in a number of neuroblastoma tumors and the thereof derived cell lines. Daunorubicin and doxorubicin, two widely used antineoplastic agents in tumor therapy, were covalently linked to NPY via two spacers that differ in stability: an acid-sensitive hydrazone bond at the 13-keto position of daunorubicin and a stable amide bond at the 3'-amino position of daunorubicin and doxorubicin. Receptor binding of these three conjugates ([C(15)]-NPY-Dauno-HYD, [C(15)]-NPY-Dauno-MBS, and [C(15)]-NPY-Doxo-MBS) was determined at the human neuroblastoma cell line SK-N-MC, which selectively expresses the NPY Y(1) receptor subtype, and cytotoxic activity was evaluated using a XTT-based colorimetric cellular cytotoxicity assay. The different conjugates were able to bind to the receptor with affinities ranging from 25 to 51 nM, but only the compound containing the acid-sensitive bond ([C(15)]-NPY-Dauno-HYD) showed cytotoxic activity comparable to the free daunorubicin. This cytotoxicity is Y(1) receptor-mediated as shown in blocking studies with BIBP 3226, because tumor cells that do not express NPY receptors were sensitive to free daunorubicin, but not to the peptide-drug conjugate. The intracellular distribution was investigated by confocal laser scanning microscopy. We found evidence that the active conjugate [C(15)]-NPY-Dauno-HYD releases daunorubicin, which is localized close to the nucleus, whereas the inactive conjugate [C(15)]-NPY-Dauno-MBS is distributed distantly from the nucleus and does not seem to release the drug within the cell.

Y-receptor affinity modulation by the design of pancreatic polypeptide/neuropeptide Y chimera led to Y(5)-receptor ligands with picomolar affinity.

Neuropeptide Y (NPY) and pancreatic polypeptide (PP) bind to the Y-receptors with very different affinities: NPY has high affinity for the receptors Y(1), Y(2) and Y(5), while PP binds only to Y(4)-receptor with picomolar affinity. By exchanging of specific amino acid positions between the two peptides, we developed 38 full-length PP/NPY chimeras with binding properties that are completely different from those of the two native ligands. Pig NPY (pNPY) analogs containing the segment 19-23 from human PP (hPP) bound to the Y-receptors with much lower affinity than NPY itself. The affinity of the hPP analog containing the pNPY segments 1-7 and 19-23 was comparable to that of pNPY at the Y(1)- and Y(5)-receptor subtypes, and to that of hPP at the Y(4)-receptor. Furthermore, the presence of the segments 1-7 from chicken PP (cPP) and 19-23 from pNPY within the hPP sequence led to a ligand with IC(50) of 40 pM at the Y(5)-receptor. This is the most potent Y(5)-receptor ligand known so far, with 15-fold higher affinity than NPY.

Fluorescence-labeled octapeptides as substrates for histone deacetylase.

The determination of histone deacetylase (HDAC) activity and the screening of potential inhibitors is gaining increasing importance due to the involvement of HDAC in transcription regulation. The level of histone acetylation can be modulated by HDAC inhibitors resulting in differentiation and/or apoptosis in cancer cells. We have previously reported the development of a nonisotopic assay for HDAC using a fluorescent derivative of epsilon-acetyl lysine. Here we report fluorescein-labeled octapeptides which are substrates for HDAC that bear closer resemblance to the native substrate. HPLC with fluorescence detection is successfully applied to the analysis of the time- and site-dependent deacetylation. LC-MS analyses are used to confirm the findings. The observed selectivity toward one of two possible deacetylation sites might result from steric hindrance by the label but the methodology presented here could be applied to similar larger peptides which might be improved tools to characterize HDAC site selectivity in vitro.

Peptides as carrier for tumor diagnosis and treatment.

The specific binding of peptides to their receptors can be used to meet the key requirement in tumor targeting: selective addressing of neoplasm. Because of their small size, peptides exhibit faster blood clearance and higher target-to-background ratios compared to macromolecular compounds. In radiopharmacy, these advantages have been attended, and radiolabelled receptor-binding peptides have emerged as a new class of radiopharmaceuticals. Over the last years, nuclear medicine has evaluated various peptides for tumor scintigraphy. The challenge is to label bioactive peptides without affecting their receptor binding properties. Size, plasma protein binding, lipophilicity and sensitivity to proteolysis are to be considered, as well as biodistribution, metabolism and excretion characteristics. The variety of experiences gained in the development of peptide analogues and radiolabelling methods, and latest results from in vitro, in vivo and clinical studies will be presented. The tumor receptor-targeting approach with peptides can be extended to cancer chemotherapy. One of the major problems in classic chemotherapy is the non-specific toxicity of most anticancer agents against normal cells. Coupling cytotoxic drugs to macromolecular carriers has been shown to be a promising approach for efficient drug targeting. In the past few years, peptides were introduced as carriers. Different conjugates, composed of a peptide carrier and a cytotoxic moiety, have been investigated so far. Anticancer drugs were coupled to analogues of luteinizing hormone-releasing hormone, bombesin, somatostatin and neuropeptide Y. Suitable candidates maintained their binding affinity and could preserve the cytotoxic activity in vitro and in vivo, resulting in a peptide-mediated selective chemotherapy.

On the synthesis of orexin A: a novel one-step procedure to obtain peptides with two intramolecular disulphide bonds.

An efficient strategy for the synthesis of orexin A, a recently discovered neuropeptide with two intramolecular disulphide bonds, was developed. Four different methods for the synthesis of peptides containing two disulphide bonds were compared and optimized with respect to reaction time, purity of the crude peptide and yield of the purified peptide. A new one-step cyclization method in solution is presented for fast, easy and high yield synthesis of orexin A, based on iodine oxidation in acetic acid/water and S-acetamidomethyl (S-Acm) and S-trityl (S-Trt) for side-chain protection of cysteine. Disulphide formation without selective side-chain protection leads to the formation of different mono- and bicyclic configurations of orexin A. These data stress the requirement of selective cysteine side-chain protection in the synthesis of orexin A.

Monitoring of the internalization of neuropeptide Y on neuroblastoma cell line SK-N-MC.

Neuropeptide Y (NPY) is an important neuromodulator in the central and peripheral nervous system. The peptide acts through different NPY receptor subtypes (Y1-Y5, y6) that belong to the family of G protein-coupled receptors. In general, cellular responses to prolonged exposure to agonists of G protein-coupled receptors are attenuated, often through internalization of the receptors and their bound ligands. In this study, a fluorescent labeled NPY derivative was synthesized and characterized to investigate the internalization of NPY in the human neuroblastoma cell line SK-N-MC. Internalization was proven by binding experiments and subsequent acidic washing as well as by direct visualization by means of confocal laser scanning microscopy. Approximately 20-30% of the fluorescent labeled NPY and a tritium-marked NPY were resistant to acid removal of cell surface-bound ligands indicating internalization. Extracellular fluorescent labeled NPY was found to be distributed heterogeneously in a clustered pattern, which suggests that the ligand-receptor complex is collected in pits and caveolae followed by endocytosis.

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.

Molecular characterization of the ligand-receptor interaction of the neuropeptide Y family.

Neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) belong to the NPY hormone family and activate a class of receptors called the Y-receptors, and also belong to the large superfamily of the G-protein coupled receptors. Structure-affinity and structure-activity relationship studies of peptide analogs, combined with studies based on site-directed mutagenesis and anti-receptor antibodies, have given insight into the individual characterization of each receptor subtype relative to its interaction with the ligand, as well as to its biological function. A number of selective antagonists at the Y1-receptor are available whose structures resemble that of the C-terminus of NPY. Some of these compounds, like BIBP3226, BIBO3304 and GW1229, have recently been used for in vivo investigations of the NPY-induced increase in food intake. Y2-receptor selective agonists are the analog cyclo-(28/32)-Ac-[Lys28-Glu32]-(25-36)-pNPY and the TASP molecule containing two units of the NPY segment 21-36. Now the first antagonist with nanomolar affinity for the Y2-receptor is also known, BIIE0246. So far, the native peptide PP has been shown to be the most potent ligand at the Y4-receptor. However, by the design of PP/NPY chimera, some analogs have been found that bind not only to the Y4-, but also to the Y5-receptor with subnanomolar affinities, and are as potent as NPY at the Y1-receptor. For the characterization of the Y5-receptor in vitro and in vivo, a new class of highly selective agonists is now available. This consists of analogs of NPY and of PP/NPY chimera which all contain the motif Ala31-Aib32. This motif has been shown to induce a 3(10)-helical turn in the region 28-31 of NPY and is suggested to be the key motif for high Y5-receptor selectivity. The results of feeding experiments in rats treated with the first highly specific Y5-receptor agonists support the hypothesis that this receptor plays a role in the NPY-induced stimulation of food intake. In conclusion, the selective compounds for the different Y receptor subtypes known so far are promising tools for a better understanding of the physiological properties of the hormones of the NPY family and related receptors.