Protective effect of neuropeptide Y2 receptor activation against methamphetamine-induced brain endothelial cell alterations.

Methamphetamine (METH) consumption is a health problem that leads to neurological and psychiatric disturbances. The cellular alterations behind these conditions have been extensively investigated and it is now well-established that METH causes cerebrovascular alterations being a key feature in drug-induced neuropathology. Although promising advances in understanding the blood-brain barrier (BBB) alterations induced by METH, there is still no available approach to counteract or diminish such effects. Interestingly, several studies show that neuropeptide Y (NPY) has an important protective role against METH-induced neuronal and glial toxicity, as well as behavioral deficits. Despite these beneficial effects of the NPY system, nothing is known about its role in brain endothelial cells under conditions of METH exposure. Thus, our aim was to unravel the effect of NPY and its receptors against METH-induced endothelial cell dysfunction. For that, we used a human brain microvascular endothelial cell line (hCMEC/D3) and our results demonstrate that endothelial cells express both NPY Y1 (Y1R) and Y2 (Y2R) receptors, but only Y2R is upregulated after METH exposure. Moreover, this drug of abuse induced endothelial cell death and elicited the production of reactive oxygen species (ROS) by these cells, which were prevented by the activation of Y2R. Additional, cell death and oxidative stress triggered by METH were dependent on the concentration of the drug. In sum, with the present study we identified for the first time the NPY system, and particularly the Y2R subtype, as a promising target to protect against METH-induced neurovascular dysfunction.

Neuropeptide Y and cannabinoids interaction in the amygdala after exposure to shock and reminders model of PTSD.

Modulation of cannabinoid and neuropeptide Y (NPY) receptors may offer therapeutic benefits for post-traumatic stress disorder (PTSD). In this study, we aimed to investigate the functional interaction between these systems in the basolateral amygdala (BLA) in a rat model of PTSD. Rats were exposed to the shock and reminders model of PTSD and tested for hyper arousal/PTSD- and depression-like behaviors 3 weeks later. Immediately after shock exposure rats were microinjected into the BLA with URB597, a selective inhibitor of fatty acid amide hydrolase (FAAH) that increases the levels of the endocannabinoid anandamide or with the NPY1 receptor agonist Leu31,Pro34-NPY (Leu). Intra-BLA URB597 prevented the shock/reminders-induced PTSD- behaviors (extinction, startle) and depression-behaviors (despair, social impairments). These preventing effects of URB597 on PTSD- and depression-like behaviors were shown to be mostly mediated by cannabinoid CB1 and NPY1 receptors, as they were blocked when URB597 was co-administered with a low dose of a CB1 or NPY1 receptor antagonist. Similarly, intra-BLA Leu prevented development of all the behaviors. Interestingly, a CB1 antagonist prevented the effects of Leu on despair and social behavior, but not the effects on extinction and startle. Moreover, exposure to shock and reminders upregulated CB1 and NPY1 receptors in the BLA and infralimbic prefrontal cortex and this upregulation was restored to normal with intra-BLA URB597 or Leu. The findings suggest that the functional interaction between the eCB and NPY1 systems is complex and provide a rationale for exploring novel therapeutic strategies that target the cannabinoid and NPY systems for stress-related diseases.

Carbaboranylation of Truncated C-Terminal Neuropeptide†Y Analogue Leads to Full hY1 Receptor Agonism.

The human Y1 receptor is overexpressed in breast tumour cells and is, therefore, a valuable target for site-selective drug delivery. The well-established hY1 R-selective ligand [Phe7,Pro34]NPY has been used to couple to drugs but its length of 36 amino acids also implies complex synthesis and high production costs. Therefore, shorter ligands are desirable. However, truncated versions of neuropeptide Y (NPY) usually result in reduced binding, antagonists, or only partial G-protein-biased agonists. Herein, we report on a nonamer peptide derived from the C terminus of NPY that is modified by a carbaborane, which is able to activate hY1 R fully in terms of G-protein activation but also arrestin recruitment and internalisation. We provide evidence that this unique behaviour is due to the bulky nature of the carbaborane cluster, which might address a specific second binding pocket in hY1 R that is crucial for arrestin recruitment. Thus, this study helps in deciphering ligand-induced onset of different pathways in hY1 R mediated signalling.

Analgesic and anxiolytic effects of [Leu31,Pro34]-neuropeptide Y microinjected into the periaqueductal gray in rats.

Several reports have demonstrated that neuropeptide Y (NPY) is involved in food intake, epilepsy, circadian rhythms, drug seeking, pain and anxiety, and other physiological or pathological conditions. On the other hand, periaqueductal gray (PAG) is a key brain center for modulating pain, anxiety and fear. It is the main structure implicated in integrated defensive behaviors. One such behavior, tonic immobility (TI), resembles fear and is able to induce analgesia. After microinjection of [Leu31,Pro34]-Neuropeptide Y ([Leu31,Pro34]-NPY) into the PAG dorsal (D) or ventrolateral (VL) of adult male Wistar rats, the following parameters were assessed: i) the analgesic effect by means of the tail-flick test (TF), ii) the duration of TI as a passive defensive behavioral response and as an anxiety/fear model (considering both TF and TI as single behaviors), iii) TI-induced analgesia by the combination of TF/TI, and iv) the anxious-like state through the elevated plus maze (EPM), and defensive burying behavior (DBB). The results show that the microinjection of [Leu31,Pro34]-NPY into the PAG produced an analgesic effect (increasing the TF latency); overall decreased the TI duration, which might represent an important anti-fear effect. Moreover, [Leu31,Pro34]-NPY microinjected into the PAG allows for a TI-induced analgesic effect, as well as, a substantial anxiolytic effect (evidenced by the EPM and DBB models). Hence, [Leu31,Pro34]-NPY microinjected into the PAG, especially at 0.47nmol/0.5µL produces both analgesic and anxiolytic effects, in a higher magnitude within ventrolateral area.

Negative Effects of Acid Violet-17 and MBB Dual In Vitro on Different Ocular Cell Lines.

PURPOSE: Vital dyes have become a clinical standard during vitrectomy to visualize anatomical structures. It was the aim of this study to test the effect of two vital dyes (AV 17-M with 5% mannitol and MBB Dual) on different intraocular cells, to see whether in vitro test can be used as reliable preclinical testing tool. METHODS: Cell morphology was assessed via phase contrast pictures, cell viability via MTS assay and total cell amount via crystal violet staining. ARPE19 and 661W cells were chosen for toxicology testing at different exposure times (60 seconds, 15 minutes and 30 minutes). Vital dyes were completely removed after the staining period. RESULTS: Treatment with AV 17-M changed the morphology and the cell number at every time point investigated on ARPE19 and 661 W cells. ARPE19 cells treated with AV 17-M or MBB Dual displayed only a slight or no decrease in cell viability after the three different exposure times. AV-17 without medium to simulate a possible intraoperative use after fluid-air exchange showed a decrease in viability of 6%, 24% and 14%. A difference in cell density of 21%, 46% and 34% was noted after CV staining for AV 17-M, MBB Dual led to a decrease of 2%, 16% and 3% after 30 minutes compared to BSS. AV 17-M directly applied on 661W decreased viability significantly by 18% after 60 seconds, 33% after 15 minutes and 40% after 30 minutes. Cell density of 661W cells exposed relevant negative effects; after incubation of 60 seconds with AV 17-M, the cell amount was significantly lowered by 41% and MBB Dual by 12%. After 15 minutes, a loss of 48% cell amount was detected with AV 17-M and after 30 min 51%. MBB Dual led to 37% loss after 15 minutes and to 28% loss after 30 minutes. CONCLUSION: AV 17-M with 5% mannitol has a negative effect on different ocular cells.

NPY1 Receptors Participate in the Regulation of Myocardial Contractility in Rats.

Selective agonist (Leu(31)Pro(34)NPY) and blocker (BIBP-3226) of NPY1 receptors were used to determine the type of NPY receptors involved in myocardial contraction. Experiments with isometric contraction of myocardial strips from mature rats showed that the agonist produced the most potent effect in a concentration of 10-7 M. In this concentration, Leu(31)Pro(34)NPY showed the greatest positive inotropic effect on the contraction of the atria and ventricles. In contrast, selective blocker BIBP-3226 reduced the force of myocardial contractions. Pretreatment of myocardial strips with this blocker abolished the positive inotropic effect of Leu(31)Pro(34)NPY, which attested to important role of NPY1 receptors in myocardial contraction.

N(ω)-Carbamoylation of the Argininamide Moiety: An Avenue to Insurmountable NPY Y1 Receptor Antagonists and a Radiolabeled Selective High-Affinity Molecular Tool ([(3)H]UR-MK299) with Extended Residence Time.

Analogues of the argininamide-type NPY Y1 receptor (Y1R) antagonist BIBP3226, bearing carbamoyl moieties at the guanidine group, revealed subnanomolar Ki values and caused depression of the maximal response to NPY (calcium assay) by up to 90% in a concentration- and time-dependent manner, suggesting insurmountable antagonism. To gain insight into the mechanism of binding of the synthesized compounds, a tritiated antagonist, (R)-N(α)-diphenylacetyl-N(ω)-[2-([2,3-(3)H]propionylamino)ethyl]aminocarbonyl-(4-hydroxybenzyl)arginin-amide ([(3)H]UR-MK299, [(3)H]38), was prepared. [(3)H]38 revealed a dissociation constant in the picomolar range (Kd 0.044 nM, SK-N-MC cells) and very high Y1R selectivity. Apart from superior affinity, a considerably lower target off-rate (t1/2 95 min) was characteristic of [(3)H]38 compared to that of the higher homologue containing a tetramethylene instead of an ethylene spacer (t1/2 3 min, Kd 2.0 nM). Y1R binding of [(3)H]38 was fully reversible and fully displaceable by nonpeptide antagonists and the agonist pNPY. Therefore, the insurmountable antagonism observed in the functional assay has to be attributed to the extended target-residence time, a phenomenon of relevance in drug research beyond the NPY receptor field.

Activation of Neuropeptide Y Receptors Modulates Retinal Ganglion Cell Physiology and Exerts Neuroprotective Actions In Vitro.

Neuropeptide Y (NPY) is expressed in mammalian retina but the location and potential modulatory effects of NPY receptor activation remain largely unknown. Retinal ganglion cell (RGC) death is a hallmark of several retinal degenerative diseases, particularly glaucoma. Using purified RGCs and ex vivo rat retinal preparations, we have measured RGC intracellular free calcium concentration ([Ca2+]i) and RGC spiking activity, respectively. We found that NPY attenuated the increase in the [Ca2+]i triggered by glutamate mainly via Y1 receptor activation. Moreover, (Leu31, Pro34)-NPY, a Y1/Y5 receptor agonist, increased the initial burst response of OFF-type RGCs, although no effect was observed on RGC spontaneous spiking activity. The Y1 receptor activation was also able to directly modulate RGC responses by attenuating the NMDA-induced increase in RGC spiking activity. These results suggest that Y1 receptor activation, at the level of inner or outer plexiform layers, leads to modulation of RGC receptive field properties. Using in vitro cultures of rat retinal explants exposed to NMDA, we found that NPY pretreatment prevented NMDA-induced cell death. However, in an animal model of retinal ischemia-reperfusion injury, pretreatment with NPY or (Leu31, Pro34)-NPY was not able to prevent apoptosis or rescue RGCs. In conclusion, we found modulatory effects of NPY application that for the first time were detected at the level of RGCs. However, further studies are needed to evaluate whether NPY neuroprotective actions detected in retinal explants can be translated into animal models of retinal degenerative diseases.

Synthesis and in vitro and in vivo evaluation of an (18)F-labeled neuropeptide Y analogue for imaging of breast cancer by PET.

Imaging of Y1R expression in breast cancer is still a challenging task. Herein, we report a suitable (18)F-labeled high-molecular-weight glycopeptide for imaging of peripheral neuropeptide Y (NPY) Y1 receptor (Y1R)-positive tumors by preclinical small-animal positron emission tomography (PET). The Y1R-preferring NPY [F(7),P(34)]NPY analogue was functionalized with an alkyne-bearing propargylglycine (Pra) in position 4. The corresponding fluoroglycosylated (FGlc) peptide analogue [Pra(4)(FGlc),F(7),P(34)]NPY and its (18)F-labeled analogue were synthesized by click chemistry-based fluoroglycosylation. The radiosynthesis was performed by (18)F-fluoroglycosylation starting from the 2-triflate of the ß-mannosylazide and the alkyne peptide [Pra(4),F(7),P(34)]NPY. The radiosynthesis of the(18)F-labeled analogue was optimized using a minimum amount of peptide precursor (40 nmol), proceeding with an overall radiochemical yield of 20-25% (nondecay corrected) in a total synthesis time of 75 min with specific activities of 40-70 GBq/µmol. In comparison to NPY and [F(7),P(34)]NPY, in vitro Y1R and Y2R activation studies with the cold [Pra(4)(FGlc),F(7),P(34)]NPY on stably transfected COS-7 cells displayed a high potency for the induction of Y1R-specific inositol accumulation (pEC50 = 8.5 ± 0.1), whereas the potency at Y2R was significantly decreased. Internalization studies on stably transfected HEK293 cells confirmed a strong glycopeptide-mediated Y1R internalization and a substantial Y1R subtype selectivity over Y2R. In vitro autoradiography with Y1R-positive MCF-7 tumor tissue slices indicated high specific binding of the (18)F-labeled glycopeptide, when binding was reduced by 95% ([Pra(4),F(7),P(34)]NPY) and by 86% (BIBP3226 Y1R antagonist) in competition studies. Biodistribution and small-animal PET studies on MCF-7 breast tumor-bearing nude mice revealed radiotracer uptake in the MCF-7 tumor of 1.8%ID/g at 20 min p.i. and 0.7%ID/g at 120 min p.i. (n = 3-4), increasing tumor-to-blood ratios from 1.2 to 2.4, and a tumor retention of 76 ± 4% (n = 4; 45-90 min p.i.). PET imaging studies with MCF-7 tumor-bearing nude mice demonstrated uptake of the (18)F-labeled glycopeptide in the tumor region at 60 min p.i., whereas only negligible tumor uptake was observed in animals injected with a nonbinding (18)F-labeled glycopeptide pendant as a measure of nonspecific binding. In conclusion, PET imaging experiments with the (18)F-labeled NPY glycopeptide revealed Y1R-specific binding uptake in MCF-7 tumors in vivo together with decreased kidney uptake compared to DOTA-derivatives of this peptide. We consider this glycopeptide to be a potent lead peptide for the design of improved (18)F-glycopeptides with shorter amino acid sequences that would further facilitate PET imaging studies of Y1R-positive breast tumors.

Receptor-mediated uptake of boron-rich neuropeptide y analogues for boron neutron capture therapy.

Peptidic ligands selectively targeting distinct G protein-coupled receptors that are highly expressed in tumor tissue represent a promising approach in drug delivery. Receptor-preferring analogues of neuropeptide Y (NPY) bind and activate the human Y1 receptor subtype (hY1 receptor), which is found in 90% of breast cancer tissue and in all breast-cancer-derived metastases. Herein, novel highly boron-loaded Y1 -receptor-preferring peptide analogues are described as smart shuttle systems for carbaboranes as (10) B-containing moieties. Various positions in the peptide were screened for their susceptibility to carbaborane modification, and the most promising positions were chosen to create a multi-carbaborane peptide containing 30 boron atoms per peptide with excellent activation and internalization patterns at the hY1 receptor. Boron uptake studies by inductively coupled plasma mass spectrometry revealed successful uptake of the multi-carbaborane peptide into hY1 -receptor-expressing cells, exceeding the required amount of 10(9) boron atoms per cell. This result demonstrates that the NPY/hY receptor system can act as an effective transport system for boron-containing moieties.

Galanin receptor 2-neuropeptide Y Y1 receptor interactions in the amygdala lead to increased anxiolytic actions.

Galanin (GAL) and neuropeptide Y (NPY) are neuropeptides involved in behaviors associated with anxiety. Both neuropeptides interact in several central functions. However, the potential behavioral and cellular interactions between them in anxiety are unknown. GAL was found to act through GAL receptor 2 (GALR2) to enhance NPYY1 receptor (NPYY1R)-mediated anxiolytic behaviors in rats. Using receptor autoradiography, c-Fos expression and in situ proximity ligation assay, the medial paracapsular intercalated nuclei of the amygdala were determined to be a key area in the interaction probably involving the formation of GALR2/NPYY1R heteroreceptor complexes. In cell cultures costimulation of GALR2 and NPYY1R induced changes in the functions of these receptors. The changes involved a potentiation of the decrease in the phosphorylation of CREB induced by NPYY1R and a delay in the internalization of NPYY1R. These results indicate that GALR2/NPYY1R interactions can provide a novel integrative amygdaloid mechanism in anxiety.

Characterization and expression analysis of two distinct neuropeptide Ya paralogues in Jian carp (Cyprinus carpio var. Jian).

Two distinct neuropeptide Ya paralogues (jlNPYa1 and jlNPYa2) were cloned and characterized in Jian carp (Cyprinus carpio var. Jian), with a highly conserved organization encoded by four exons and three introns. The cDNAs for jlNPYa1 and jlNPYa2 were 693 and 730 bp in size, respectively. jlNPYa1 and jlNPYa2 both encoded a 96-amino acid protein, which shared 97.9 % identity. Phylogenetic tree showed that it has two NPYa genes, called jlNPYa1 and jlNPYa2, that presumably resulted from the tetraploidization event in the carp lineage. Analysis of expression profiles of jlNPYa1 and jlNPYa2 showed that the two NPY genes had a broad tissue distribution but expressed primarily in the forebrain, hypothalamus, testis and liver. The expression pattern was different in juvenile and adult (female and male) Jian carp. In juvenile, the highest expression level of jlNPYa1 and jlNPYa2 was detected in the testis. In adult, it was detected in the forebrain. In female hypothalamus, the expression level of jlNPYa1 was significantly higher than that of jlNPYa2. However, the opposite was true in male hypothalamus. The differing distribution patterns of the two NPY genes suggested that jlNPYa1 and jlNPYa2 might play different roles in Jian carp.

Neuropeptide Y in the central nucleus of amygdala regulates the anxiolytic effect of agmatine in rats.

In the present study, modulation of anxiolytic action of agmatine by neuropeptide Y (NPY) in the central nucleus of amygdala (CeA) is evaluated employing Vogel's conflict test (VCT) in rats. The intra-CeA administration of agmatine (0.6 and 1.2µmol/rat), NPY (10 and 20pmol/rat) or NPY Y1/Y5 receptors agonist [Leu(31), Pro(34)]-NPY (30 and 60pmol/rat) significantly increased the number of punished drinking licks following 15min of treatment. Combination treatment of subeffective dose of NPY (5pmol/rat) or [Leu(31), Pro(34)]-NPY (15pmol/rat) and agmatine (0.3µmol/rat) produced synergistic anxiolytic-like effect. However, intra-CeA administration of selective NPY Y1 receptor antagonist, BIBP3226 (0.25 and 0.5mmol/rat) produced anxiogenic effect. In separate set of experiment, pretreatment with BIBP3226 (0.12mmol/rat) reversed the anxiolytic effect of agmatine (0.6µmol/rat). Furthermore, we evaluated the effect of intraperitoneal injection of agmatine (40mg/kg) on NPY-immunoreactivity in the nucleus accumbens shell (AcbSh), lateral part of bed nucleus of stria terminalis (BNSTl) and CeA. While agmatine treatment significantly decreased the fibers density in BNSTl, increase was noticed in AcbSh. In addition, agmatine reduced NPY-immunoreactive cells in the AcbSh and CeA. Immunohistochemical data suggest the enhanced transmission of NPY from the AcbSh and CeA. Taken together, this study suggests that agmatine produced anxiolytic effect which might be regulated via modulation of NPYergic system particularly in the CeA.

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.

Possible involvement of neuropeptide Y Y1 receptors in antidepressant like effect of agmatine in rats.

Agmatine and neuropeptide Y (NPY) are widely distributed in central nervous system and critically involved in modulation of depressive behavior in experimental animals. However their mutual interaction, if any, in regulation of depression remain largely unexplored. In the present study we explored the possible interaction between agmatine and neuropeptide Y in regulation of depression like behavior in forced swim test. We found that acute intracerebroventricular (i.c.v.) administration of agmatine (20-40µg/rat), NPY (5 and 10µg/rat) and NPY Y1 receptor agonist, [Leu(31), Pro(34)]-NPY (0.4 and 0.8ng/rat) dose dependently decreased immobility time in forced swim test indicating their antidepressant like effects. In combination studies, the antidepressant like effect of agmatine (10µg/rat) was significantly potentiated by NPY (1 and 5µg/rat, icv) or [Leu(31), Pro(34)]-NPY (0.2 and 0.4ng/rat, icv) pretreatment. Conversely, pretreatment of animals with NPY Y1 receptor antagonist, BIBP3226 (0.1ng/rat, i.c.v.) completely blocked the antidepressant like effect of agmatine (20-40µg/rat) and its synergistic effect with NPY (1µg/rat, icv) or [Leu(31), Pro(34)]-NPY (0.2ng/rat, icv). The results of the present study showed that, agmatine exerts antidepressant like effects via NPYergic system possibly mediated by the NPY Y1 receptor subtypes and suggest that interaction between agmatine and neuropeptide Y may be relevant to generate the therapeutic strategies for the treatment of depression.

NPY Y1 receptors differentially modulate GABAA and NMDA receptors via divergent signal-transduction pathways to reduce excitability of amygdala neurons.

Neuropeptide Y (NPY) administration into the basolateral amygdala (BLA) decreases anxiety-like behavior, mediated in part through the Y1 receptor (Y1R) isoform. Activation of Y1Rs results in G-protein-mediated reduction of cAMP levels, which results in reduced excitability of amygdala projection neurons. Understanding the mechanisms linking decreased cAMP levels to reduced excitability in amygdala neurons is important for identifying novel anxiolytic targets. We studied the intracellular mechanisms of activation of Y1Rs on synaptic transmission in the BLA. Activating Y1Rs by [Leu(31),Pro(34)]-NPY (L-P NPY) reduced the amplitude of evoked NMDA-mediated excitatory postsynaptic currents (eEPSCs), without affecting AMPA-mediated eEPSCs, but conversely increased the amplitude of GABAA-mediated evoked inhibitory postsynaptic currents (eIPSCs). Both effects were abolished by the Y1R antagonist, PD160170. Intracellular GDP-ß-S, or pre-treatment with either forskolin or 8Br-cAMP, eliminated the effects of L-P NPY on both NMDA- and GABAA-mediated currents. Thus, both the NMDA and GABAA effects of Y1R activation in the BLA are G-protein-mediated and cAMP-dependent. Pipette inclusion of protein kinase A (PKA) catalytic subunit blocked the effect of L-P NPY on GABAA-mediated eIPSCs, but not on NMDA-mediated eEPSCs. Conversely, activating the exchange protein activated by cAMP (Epac) with 8CPT-2Me-cAMP blocked the effect of L-P NPY on NMDA-mediated eEPSCs, but not on GABAA-mediated eIPSCs. Thus, NPY regulates amygdala excitability via two signal-transduction events, with reduced PKA activity enhancing GABAA-mediated eIPSCs and Epac deactivation reducing NMDA-mediated eEPSCs. This multipathway regulation of NMDA- and GABAA-mediated currents may be important for NPY plasticity and stress resilience in the amygdala.

Manipulating Y receptor subtype activation of short neuropeptide Y analogs by introducing carbaboranes.

Short selective neuropeptide Y (NPY) analogs are highly attractive because of their facile synthesis. Based on the reduced-size NPY analog [Pro(30), Nle(31), Bpa(32), Leu(34)]NPY 28-36 position 32 was identified as a key position to alter the preferential activation pattern of the human neuropeptide Y receptors (hYRs). By replacing benzoylphenylalanine (Bpa) by a biphenylalanine (Bip) the photostability was first improved while the biological activity was maintained. SAR-studies showed that both aromatic rings have a high influence on the preferential hYR subtype activation. Interestingly, replacement of Bpa(32) by a strongly hydrophobic moiety changed the hYR subtype preference of the analog. Whereas the parent compound is able to activate the human neuropeptide Y1 receptor (hY1R) subtype, the introduction of an N(ε)-ortho-carbaboranyl propionic acid modified lysine resulted in a loss of activity at the hY1R but in an increased activity at both the hY2R and the hY4R. However, subsequent receptor internalization studies with this novel analog revealed that receptor internalization can neither be triggered at the hY2R nor at the hY4R suggesting a biased ligand. Surprisingly, investigations by (1)H NMR spectroscopy revealed structural changes in the side chains of residues Pro(30) and Leu(34) which nicely correlates with the shift from hY1R/hY4R to hY2R/hY4R activation preference. Thus, position 32 has been identified to switch the bioactive conformation and subsequently influences receptor subtype activation behavior.

Cyclic analogs of galanin and neuropeptide Y by hydrocarbon stapling.

Hydrocarbon stapling is an effective strategy to stabilize the helical conformation of bioactive peptides. Here we describe application of stapling to anticonvulsant neuropeptides, galanin (GAL) and neuropeptide Y (NPY), that are implicated in modulating seizures in the brain. Dicarba bridges were rationally introduced into minimized analogs of GAL and NPY resulting in increased α-helical content, in vitro metabolic stability and n-octanol/water partitioning coefficient (logD). The stapled analogs retained agonist activities towards their respective receptors and suppressed seizures in a mouse model of epilepsy.

Incorporation of monodisperse oligoethyleneglycol amino acids into anticonvulsant analogues of galanin and neuropeptide y provides peripherally acting analgesics.

Delivery of neuropeptides into the central and/or peripheral nervous systems supports development of novel neurotherapeutics for the treatment of pain, epilepsy and other neurological diseases. Our previous work showed that the combination of lipidization and cationization applied to anticonvulsant neuropeptides galanin (GAL) and neuropeptide Y (NPY) improved their penetration across the blood-brain barrier yielding potent antiepileptic lead compounds, such as Gal-B2 (NAX 5055) or NPY-B2. To dissect peripheral and central actions of anticonvulsant neuropeptides, we rationally designed, synthesized and characterized GAL and NPY analogues containing monodisperse (discrete) oligoethyleneglycol-lysine (dPEG-Lys). The dPEGylated analogues Gal-B2-dPEG(24), Gal-R2-dPEG(24) and NPY-dPEG(24) displayed analgesic activities following systemic administration, while avoiding penetration into the brain. Gal-B2-dPEG(24) was synthesized by a stepwise deprotection of orthogonal 4-methoxytrityl and allyloxycarbonyl groups, and subsequent on-resin conjugations of dPEG(24) and palmitic acids, respectively. All the dPEGylated analogues exhibited substantially decreased hydrophobicity (expressed as logD values), increased in vitro serum stabilities and pronounced analgesia in the formalin and carrageenan inflammatory pain assays following systemic administration, while lacking apparent antiseizure activities. These results suggest that discrete PEGylation of neuropeptides offers an attractive strategy for developing neurotherapeutics with restricted penetration into the central nervous system.

Central nervous system neuropeptide Y signaling via the Y1 receptor partially dissociates feeding behavior from lipoprotein metabolism in lean rats.

Elevated plasma triglyceride (TG) levels contribute to an atherogenic dyslipidemia that is associated with obesity, diabetes, and metabolic syndrome. Numerous models of obesity are characterized by increased central nervous system (CNS) neuropeptide Y (NPY) tone that contributes to excess food intake and obesity. Previously, we demonstrated that intracerebroventricular (icv) administration of NPY in lean fasted rats also elevates hepatic production of very low-density lipoprotein (VLDL)-TG. Thus, we hypothesize that elevated CNS NPY action contributes to not only the pathogenesis of obesity but also dyslipidemia. Here, we sought to determine whether the effects of NPY on feeding and/or obesity are dissociable from effects on hepatic VLDL-TG secretion. Pair-fed, icv NPY-treated, chow-fed Long-Evans rats develop hypertriglyceridemia in the absence of increased food intake and body fat accumulation compared with vehicle-treated controls. We then modulated CNS NPY signaling by icv injection of selective NPY receptor agonists and found that Y1, Y2, Y4, and Y5 receptor agonists all induced hyperphagia in lean, ad libitum chow-fed Long-Evans rats, with the Y2 receptor agonist having the most pronounced effect. Next, we found that at equipotent doses for food intake NPY Y1 receptor agonist had the most robust effect on VLDL-TG secretion, a Y2 receptor agonist had a modest effect, and no effect was observed for Y4 and Y5 receptor agonists. These findings, using selective agonists, suggest the possibility that the effect of CNS NPY signaling on hepatic VLDL-TG secretion may be relatively dissociable from effects on feeding behavior via the Y1 receptor.