NPY Analogs Research Articles

Targeted modulation of gene expression through receptor-specific delivery of small interfering RNA peptide conjugates.

Small interfering RNA (siRNA) has emerged as a valuable tool to address RNA interference (RNAi) to modulate gene expression also in therapy. However, challenges such as inefficient cell targeting and rapid degradation in biological systems have limited its success. To address these issues, the development of a receptor-specific shuttle system represents a promising solution. [F7,P34]-NPY analogues were modified by solid-phase peptide synthesis, enabling non-covalent conjugation with siRNA. This modification yielded an efficient siRNA vehicle capable of binding and transporting its cargo into target cells without adversely affecting receptor activation or cell viability. Mass spectrometry and gel shift assays confirmed successful and stable siRNA binding under various conditions. Microscopy experiments further demonstrated the co-internalization of labeled peptides and siRNA in Hepa1c1 cells, highlighting the stability of the complex. In vitro quantitative RT-PCR experiments, targeting the TSC22D4 gene to normalize systemic glucose homeostasis and insulin resistance, revealed a functional peptide-based siRNA shuttle system with the ability to decrease mRNA expression to approximately 40%. These findings strengthen the potential of receptor-specific siRNA shuttle systems as efficient tools for gene therapy that offer a possibility for reducing side effects.

Controlling toxicity of Peptide-drug conjugates by different chemical linker structures.

The side effects of chemotherapy can be overcome by linking toxic agents to tumor-targeting peptides with cleavable linkers. Herein, this concept is demonstrated by addressing the human Y1 receptor (hY1 R), overexpressed in breast tumors, with analogues of the hY1 R-preferring [F(7) ,P(34) ]NPY. First, carboxytetramethylrhodamine was connected to [F(7) ,P(34) ]NPY by an amide, ester, disulfide, or enzymatic linkage. Live imaging revealed hY1 R-mediated delivery and allowed visualization of time-dependent intracellular release. Next, the fluorophore was replaced by the toxic agent methotrexate (MTX). In addition to linkage through the amide, ester, disulfide bond, or enzymatic cleavage site, a novel disulfide/ester linker was designed and coupled to [F(7) ,P(34) ]NPY by solid-phase peptide synthesis. Internalization studies showed hY1 R subtype selective uptake, and cell viability experiments demonstrated hY1 R-mediated toxicity that was clearly dependent on the linkage type. Fast release profiles for fluorophore-[F(7) ,P(34) ]NPY analogues correlated with high toxicities of MTX conjugates carrying the same linker types and emphasize the relevance of new structures connecting the toxophore and the carrier.

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

Selective NPY analogues are potent tools for tumour targeting. Their Y(1)-receptors are significantly over-expressed in human breast tumours, whereas normal breast tissue only expresses Y(2)-receptors. The endogenous peptide consists of 36 amino acids, whereas smaller peptides are preferred because of better labelling efficiencies. As Y(1)-receptor agonists enhance the tumour to background ratio compared to Y(1)-receptor antagonists, we were interested in the development of Y(1)-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 Y(1)-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 Y(1)-receptor internalization. For the first time, here we show the design and characterization of a small Y(1)-receptor selective agonist. This agonist might be a useful novel ligand for NPY-mediated tumour diagnostics and therapeutics.

Novel Chemically Modified Analogues of Neuropeptide Y for Tumor Targeting

The successful use of peptides as potential radiopharmaceuticals essentially requires the modification of the bioactive peptide hormones to introduce chelators for radiolabeling. In this study, four Y 1/Y 2 receptor-selective NPY analogues with different receptor subtype specificities have been investigated. For in vitro studies, the cold metal surrogate was used. Gallium and indium complexes were introduced by using 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid as bifunctional chelator. The peptides were synthesized by solid-phase peptide synthesis (SPPS), the chelator was coupled either at the N-terminus or at the N(epsilon) side chain of Lys(4) of the resin-bound peptide, and the labeling was performed in solution after cleavage. Competitive binding assays showed high binding affinity of the receptor-selective analogues at NPY receptor expressing cells. To test internalization of the novel peptide analogues and the metabolic stability in human blood plasma, the corresponding 5(6)-carboxyfluorescein (CF) analogues were prepared and investigated. One of the most promising analogues, the Y 1-receptor selective [Lys(DOTA)(4), Phe(7), Pro(34)]NPY was labeled with (111)In and injected into nude mice that bear MCF-7 breast cancer xenografts, and biodistribution studies were performed. In vitro and in vivo studies suggest that receptor-selective analogues of NPY have promising characteristics for future applications in nuclear medicine for breast tumor diagnosis and therapy.

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.

Regulation of Anxiety‐like Behavior by Neuropeptide Y and Corticotropin Releasing Factor

Anxiety is an emotional response that may lead to many psychiatric disorders. The basolateral nucleus of the amygdala (BLA) is an important brain region for the regulation of the physiological and behavioral responses to stressors and appears to play an important role in the etiology of anxiety and panic disorder. Our laboratories have sought to understand the role of NPY and CRF in the BLA. We explored the actions of NPY and NPY analogs administered bilaterally into the BLA on anxiety-like behavior as assessed using the social interaction (SI) test. NPY and NPY analogs appear to produce anxiolytic-like effects via the Y1 and Y5 receptors while activation of the Y2 receptor produces anxiogenic-like effects. On the other hand, CRF and the related peptide, Urocortin, produce anxiogenic-like actions via the CRF1 receptor. In subsequent studies, we found that 3 day repeated “sub-threshold” administration of Ucn into the BLA results in a behavioral sensitivity to lactate in treated rats. Continued treatment of the rats for 5 days produced a persistent anxiety state that was observed for an additional 5 weeks without Ucn administration. Electrophysiological studies on the BLA of these animals suggest a profound loss of inhibitory input resulting in increased excitability. Therefore, the NPY and CRF systems play an important role in BLA excitability and may have an important role in the etiology of anxiety and panic disorder.

BODIPY-conjugated neuropeptide Y ligands: new fluorescent tools to tag Y1, Y2, Y4 and Y5 receptor subtypes.

N-terminal labelled fluorescent BODIPY-NPY peptide analogues were tested in Y1, Y2, Y4 and Y5 receptor-binding assays performed in rat brain membrane preparations and HEK293 cells expressing the rat Y1, Y2, Y4 and Y5 receptors. BODIPY TMR/FL-[Leu31, Pro34]NPY/PYY were able to compete for specific [125][Leu31, Pro34]PYY-binding sites with an affinity similar to that observed for the native peptide at the Y1 (Ki=1-6 nM), Y2 (Ki>1000 nM), Y4 (Ki=10 nM) and Y5 (Ki=1-4 nM) receptor subtypes. BODIPY FL-PYY(3-36) was able to compete for specific Y2 (Ki=10 nM) and Y5 (Ki=30 nM) binding sites, but had almost no affinity in Y1 and Y4 assays. BODIPY FL-hPP was able to compete with high affinity (Ki; 1 and 15 nM) only in Y4 and Y5 receptor-binding assays. BODIPY TMR-[cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP and BODIPY TMR-[hPP(1-17), Ala31, Aib32]NPY were potent competitors only on specific Y5-binding sites (Ki=0.1-0.6 nM). As expected, these fluorescent peptides inhibited forskolin-induced cAMP accumulation, demonstrating that they retained their agonist properties. When tested in confocal microscopy imaging, fluorescent Y1 and Y5 agonists internalized in a time-dependent manner in Y1 and Y5 transfected cells, respectively. These results demonstrate that BODIPY-conjugated NPY analogues retain their selectivity, affinity and agonist properties for the Y1, Y2, Y4 and Y5 receptor subtypes, respectively. Thus, they represent novel tools to study and visualize NPY receptors in living cells.

Neuropeptide Y inhibits spontaneous alpha-melanocyte-stimulating hormone (alpha-MSH) release via a Y(5) receptor and suppresses thyrotropin-releasing hormone-induced alpha-MSH secretion via a Y(1) receptor in frog melanotrope cells.

In amphibians, the secretion of alpha-MSH by melanotrope cells is stimulated by TRH and inhibited by NPY. We have previously shown that NPY abrogates the stimulatory effect of TRH on alpha-MSH secretion. The aim of the present study was to characterize the receptor subtypes mediating the action of NPY and to investigate the intracellular mechanisms involved in the inhibitory effect of NPY on basal and TRH-induced alpha-MSH secretion. Y(1) and Y(5) receptor mRNAs were detected by RT-PCR and visualized by in situ hybridization histochemistry in the intermediate lobe of the pituitary. Various NPY analogs inhibited in a dose-dependent manner the spontaneous secretion of alpha-MSH from perifused frog neurointermediate lobes with the following order of potency porcine peptide YY (pPYY) > frog NPY (fNPY) > porcine NPY (pNPY)-2-36) > pNPY-(13-36) > [D-Trp(32)]pNPY > [Leu(31),Pro(34)]pNPY. The stimulatory effect of TRH (10(-8)6 M) on alpha-MSH release was inhibited by fNPY, pPYY, and [Leu(31),Pro(34)]pNPY, but not by pNPY-(13-36) and [D-Trp(32)]pNPY. These data indicate that the inhibitory effect of fNPY on spontaneous alpha-MSH release is preferentially mediated through Y(5) receptors, whereas the suppression of TRH-induced alpha-MSH secretion by fNPY probably involves Y(1) receptors. Pretreatment of neurointermediate lobes with pertussis toxin (PTX; 1 microg/ml; 12 h) did not abolish the inhibitory effect of fNPY on cAMP formation and spontaneous alpha-MSH release, but restored the stimulatory effect of TRH on alpha-MSH secretion, indicating that the adenylyl cyclase pathway is not involved in the action of fNPY on TRH-evoked alpha-MSH secretion. In the majority of melanotrope cells, TRH induces a sustained and biphasic increase in cytosolic Ca(2+) concentration. Preincubation of cultured cells with fNPY (10(-7) M) or omega-conotoxin GVIA (10(-7) M) suppressed the plateau phase of the Ca(2+) response induced by TRH. However, although fNPY abrogated TRH-evoked alpha-MSH secretion, omega-conotoxin did not, showing dissociation between the cytosolic Ca(2+) concentration increase and the secretory response. Collectively, these data indicate that in frog melanotrope cells NPY inhibits spontaneous alpha-MSH release and cAMP formation through activation of a Y(5) receptor coupled to PTX- insensitive G protein, whereas NPY suppresses the stimulatory effect of TRH on alpha-MSH secretion through a Y(1) receptor coupled to a PTX-sensitive G protein-coupled receptor.

Multiple NPY receptors Inhibit GABA(A) synaptic responses of rat medial parvocellular effector neurons in the hypothalamic paraventricular nucleus.

We have recently shown that NPY and alpha-melanocyte-stimulating hormone, which potently induce or inhibit feeding, respectively, have opposing modulatory actions on GABAergic synapses in the medial parvocellular region of the paraventricular hypothalamic nucleus (mpPVN). Because this action might underlie the effects of NPY on feeding, we have examined the pharmacology of NPY responses using electrophysiological recordings. Focal electrical stimulation within the PVN elicited a GABA(A) synaptic response in some mpPVN neurons, which was reversibly inhibited by NPY in a concentration-dependent manner (EC(50) = 28 nM). NPY did not alter the response to the GABA(A) agonist, muscimol. Agonist responses to NPY analogs were not consistent with a single NPY receptor subtype; the most subtype selective agonists were less effective than the more broadly selective ones. Antagonist blockade of individual receptor subtypes partly inhibited NPY action, while fully blocking effects of selective agonists. Combining Y1 and Y5 antagonists blocked actions of NPY entirely, but the Y2 antagonist also completely blocked actions of NPY in some neurons. NPY inhibits GABA(A) synaptic transmission onto mpPVN neurons, but this can be mediated by three different NPY receptors. Controversy regarding the receptor or receptor subtypes involved in NPY-mediated feeding may arise from the multiple NPY receptors present.

Studies of the human, rat, and guinea pig Y4 receptors using neuropeptide Y analogues and two distinct radioligands

The neuropeptide Y-family receptor Y4 differs extensively between human and rat in sequence, receptor binding, and anatomical distribution. We have investigated the differences in binding profile between the cloned human, rat, and guinea pig Y4 receptors using NPY analogues with single amino acid replacements or deletion of the central portion. The most striking result was the increase in affinity for the rat receptor, but not for human or guinea pig, when amino acid 34 was replaced with proline; [Ahx 8–20,Pro 34]NPY bound to the rat Y4 receptor with 20-fold higher affinity than [Ahx 8–20]NPY. Also, the rat Y4 tolerates alanine in position 34 since p[Ala 34]NPY bound with similar affinity as pNPY while the affinity for hY4 and gpY4 decreased about 50-fold. Alanine substitutions in position 33, 35, and 36 as well as the large loop-deletion, [Ahx 5–24]NPY, reduced the binding affinity to all three receptors more than 100-fold. NPY and PYY competed with 125I-hPP at Y4 receptors expressed in CHO cells according to a two-site model. This was investigated for gpY4 by saturation with either radiolabeled hPP or pPYY. The number of high-affinity binding-sites for 125I-pPYY was about 60% of the receptors recognized by 125I-hPP. Porcine [Ala 34]NPY and [Ahx 8–20]NPY bound to rY4 (but not to hY4 or gpY4) according to a two-site model. These results suggest that different full agonists can distinguish between different active conformations of the gpY4 receptor and that Y4 may display functional differences in vivo between human, guinea pig, and rat.

Multiple Y receptors mediate pancreatic polypeptide responses in mouse colon mucosa

A functional study has been performed to characterise the Y receptors responsible for NPY, PYY and PP-stimulated responses in mouse colonic mucosal preparations. Electrogenic ion secretion was stimulated with VIP following which NPY, PYY and PP analogues were, to varying degrees, inhibitory. PYY(3-36), hPP, Gln 23hPP and rPP were effective but less potent than full length PYY, NPY or their Pro 34-substituted analogues, while the Y 5 agonist Ala 31, Aib 32hNPY was the least active peptide tested. The Y 1 antagonists, BIBP3226 and BIBO3304 virtually abolished Pro 34PYY and PYY responses while PYY(3-36) responses were selectively inhibited by the Y 2 antagonist, BIIE0246. A combination of BIBO3304 and BIIE0246 also partially attenuated hPP responses, leaving residual effects that were most probably Y 4-mediated. Thus we conclude that Y 1, Y 2 and Y 4 receptors attenuate ion secretion in mouse colon.

NPY receptor subtypes involved in the contraction of the proximal colon of the rat

Experiments were designed to determine the receptor subtype(s) involved in the contraction of the rat proximal colon to NPY. In this tissue, mRNA of Y 2 and Y 4 NPY receptor subtypes were highly expressed, whereas Y 5 mRNA levels were very low and Y 1 mRNA levels were intermediate. NPY analogues induced contractions with the following order of potency: rPP>hPP=PYY=NPY=[Leu 31, Pro 34]NPY>NPY (2–36)=[ d-Trp 32]NPY>NPY (13–36). Responses to NPY, PYY and NPY (13–36) were not or partially affected by tetrodotoxin, in contrast to the responses to [Leu 31,Pro 34]NPY, rPP, hPP and [ d-Trp 32]NPY which were fully blocked. Atropine did not inhibit the contractions to NPY, PYY and [Leu 31,Pro 34]NPY but significantly affected those to NPY (13–36), [ d-Trp 32]NPY, rPP and hPP. The specific Y 1 receptor antagonist BIBP 3226 was ineffective but JCF 104 and JCF 105 (two compounds with preferential affinity toward the hY 5 receptor versus the hY 1 or hY 2 receptor) abolished the contractions provoked by the NPY analogues. These results suggest that NPY activates three receptor subtypes, a Y 2 subtype possibly by a direct action on the smooth muscle cells, as well as a Y 4 and a Y 5 (or `Y 5-like') subtype which, respectively, release acetylcholine and an unknown neurotransmitter.

Is there really an NPY Y3 receptor?

NPY is an abundant neuropeptide that is widely distributed throughout the central and peripheral nervous systems. Based on pharmacological and cloning data, there are believed to be six different types of NPY receptors. The Y3 receptor is the only one of the six that has not been cloned or well characterized. Y3 receptors have been shown to be important in the regulation of visceral afferents within the nucleus tractus solitarius (NTS). In the present experiments, we have compared the effects of NPY and various analogs on Ba 2+ currents in acutely dissociated neurons from the NTS and the neighboring area postrema (AP). No neurons from either NTS or AP responded to [ d-Trp 32]NPY suggesting that these areas lack Y5 receptors. However, we found a group of NTS neurons that only responded to NPY and not PYY or any other analogs. This agonist profile corresponds to that described for NPY Y3 receptors. No AP neurons showed this type of agonist profile. Other neurons responded to a variety of NPY analogs indicating the presence of Y1, Y2, and Y4 receptors in both nuclei.

Neuropeptide Y receptors involved in calcium channel regulation in PC12 cells

Our laboratory has previously used NGF-differentiated PC12 cells as a sympathetic neuronal model to investigate the effects of NPY on catecholamine synthesis and release. We have additionally used these cells to demonstrate the NPY-induced inhibition of Ca 2+ channels which was suggested by those studies. In the present work, multiple NPY, PYY, and PP analogs are utilized to further define the receptor subtypes involved in this Ca 2+ channel modulation. We find that in PC12 cells NPY and PP modulate Ca 2+ channels through Y1, Y2, Y3, and Y4 receptors. In addition, we show that these receptors are differentially coupled to N, L, and non-N, non-L Ca 2+ channel subtypes. The results of the present study in combination with our previous investigations demonstrate an intriguing and complex role for NPY and PP in the modulation of sympathetic neurotransmission.

Receptor binding profiles of NPY analogues and fragments in different tissues and cell lines.

To investigate receptor selectivity and possible species selectivity of a number of NPY analogues and fragments, receptor binding studies were performed using cell lines and membranes of several species. NPY displays 4-25-fold higher affinity for the Y2 receptor than for the Y1 receptor. The affinity of [Leu31,Pro34]NPY is 7-60-fold higher for the Y1 receptor when compared with the Y2 subtype. Species selectivity within the Y2 receptors is demonstrated by PYY(3-36), NPY(2-36), NPY(22-36), and NPY(26-36). It is shown that NPY(22-36) is species selective for the human Y2 subtype (K1 of 0.3 nM) compared with the rabbit and rat Y2 receptor (K1 of 2 and 10 nM, respectively). PYY(3-36) displays highest affinity for the human and rabbit Y2 subtype (K1 of 0.03 and 0.17 nM). The screening of NPY analogues and fragments revealed that highest affinity for the human Y2 receptor is shown by NPY(2-36) and PYY(3-36). In addition, PYY(3-36) and NPY(2-36) are not only subtype selective, but also species selective.

Activation of neuropeptide Y 1 and neuropeptide Y 2 receptors by substituted and truncated neuropeptide Y analogs: identification of signal epitopes

Neuropeptide Y (NPY-(1–36)) acts on Y 1 and Y 2 receptors at the sympathetic neuroeffector junction. Various truncated NPY analogs were tested in the isolated guinea-pig caval vein where NPY is a vasoconstrictor (Y 1 receptors) and in isolated rat vas deferens, by monitoring the suppression of electrically evoked contractions (Y 2 receptors). The aim of this study was to define which parts of the NPY-(1–36) molecule were required to activate these receptors. NPY-(1–36), [Pro 34]NPY and [Glu 16, Ser 18, Ala 22, Leu 28,31] NPY (ESALL-NPY), the latter being an analog with increased α-helicity in the 14–31 region, evoked vasoconstriction with similar potency and efficacy. Cyclic as well as linear NPY analogs having the 4 to 7 N-terminal amino acid residues linked to the 9 to 19 C-terminal residues by an 8-aminooctanoic acid (Aoc) residue were 25–50 times less potent than NPY-(1–36) itself. In the cyclic analogs, a disulfide bond was introduced to bring the N- and C-termini close together. Linear Aoc 2–27-NPY was virtually inactive. The Y 1 receptor needs an intact N-terminal end of NPY in order to become fully activated. The requirements for the C-terminus are less stringent, since substitutions in this part of the molecule resulted in fully active analogs. The central portion of the molecule may impose steric constraints on the N- and C-terminal ends, thereby facilitating Y 1 receptor activation, but it does not seem to be essential for receptor recognition. NPY-(2–36) and NPY-(5–36) were only slightly less potent than the parent molecule in suppressing electrically evoked twitches in the vas deferens. ESALL-NPY was virtually equipotent with NPY-(1–36). [Aoc 8–17]NPY and [Aoc 5–24]NPY and the corresponding cyclic analogs effectively suppressed the stimulated twitches, but were about 10 times less potent than NPY-(1–36). [Aoc 2–27]NPY was much less potent than the parent molecule. Substitution with proline in position 34 resulted in a marked loss of efficacy and potency. The results show tha extensive N-terminal or central truncation does not greatly impair Y 2 receptor recognition, whereas NPY analogs with proline in position 34 are poorly recognized. Hence, the Y 2 receptor requires an intact NPY C-terminus, whereas demands for the N-terminal and mid-molecule parts are less stringent.

Specific [ 3H]propionyl-neuropeptide Y (NPY) binding in rabbit aortic membranes: Comparisons with binding in rat brain and biological responses in rat vas deferens

The binding of biologically active [3H]propionyl-NPY to rabbit aortic membranes was specific and saturable. Scatchard analysis indicated a single class of binding sites with a Kd of 1.1 nM. The rank order of potencies for displacement of [3H]propionyl-NPY binding by NPY analogs in the aorta correlated with their potencies in displacing binding in brain and their activity in inhibiting contractions of the field-stimulated rat vas deferens. However, differences were noted in the absolute or relative potencies of other related polypeptides both in regards to aorta compared to brain NPY binding and NPY binding compared to activity in the vas deferens. Collectively, the results support proposals for heterogeneity of NPY receptors.