The Neuropeptide Y Y1 Antagonist, 1229U91, A Potent Agonist for the Human Pancreatic Polypeptide-Preferring (NPY Y4) Receptor

Peptides, Vol. 19, No. 3, pp. 537–542, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/98 $19.00 1 .00

PII S0196-9781(97)00455-5

The Neuropeptide Y Y1 Antagonist, 1229U91, A Potent Agonist for the Human Pancreatic Polypeptide-Preferring (NPY Y4) Receptor DOUGLAS A. SCHOBER,* ANNE M. VAN ABBEMA,* DAVID L. SMILEY,† ROBERT F. BRUNS* AND DONALD R. GEHLERT*1 Lilly * Neuroscience and † Endocrine Research, Eli Lilly and Co., Lilly Corporate Center, Indianapolis, IN 46285 Received 8 September 1997; Accepted 27 October 1997 SCHOBER, D. A., A. M. VAN ABBEMA, D. L. SMILEY, R. F. BRUNS AND D. R. GEHLERT. The neuropeptide Y Y1 antagonist, 1229U91, a potent agonist for the human pancreatic polypeptide-preferring (NPY Y4) receptor. PEPTIDES 19(3) 537–542, 1998.—Recently, a novel high-affinity peptide antagonist, 1229U91, was published as a selective neuropeptide Y Y1 antagonist. The selectivity of 1229U91 was evaluated in the human NPY Y1 receptor containing cell line, SK-N-MC, and cells containing the cloned human NPY Y2, the pancreatic polypeptide-preferring (NPY Y4), and the NPY Y5 receptors. 1229U91 potently displaced [125I]peptide YY (PYY) binding to human NPY Y1 receptors (IC50 5 0.245 6 0.004 nM, n 5 4), but displayed little affinity for the human NPY Y2 and Y5 receptors (IC50 . 1000 nM). Interestingly, 1229U91 displaced [125I]-PYY with even greater affinity at the human NPY Y4 receptor (IC50 5 0.081 6 0.009 nM, n 5 4). Using a cyclic AMP accumulation assay, 1229U91 blocked NPY inhibition of forskolin-induced adenylate cyclase activity in NPY Y1 receptor containing SK-N-MC cells. In the human NPY Y4 receptor expressing cell line, 1229U91 did not block pancreatic polypeptide (PP) inhibition of forskolin stimulated adenylate cyclase. However, in the absence of PP, 1229U91 was able to inhibit forskolin stimulated cyclic AMP accumulation (IC50 5 7.16 6 2.8 nM, n 5 4). We conclude that 1229U91 binds non-selectively with high affinity to both human NPY Y1 and Y4 receptors. Furthermore, 1229U91 displays antagonist activity at the NPY Y1 receptor, while having agonist activity at the NPY Y4 receptor. © 1998 Elsevier Science Inc. Neuropeptide Y (NPY)

NPY receptor subtypes

Cyclic AMP

THE physiology controlling feeding behavior is complex and includes many factors that may be gastrointestinal, neural, and/or pancreatic in nature. These factors can either increase or decrease food intake. Neurotransmitters and modulators that increase food consumption include norepinephrine, galanin, opioids, growth hormone releasing factor and neuropeptide Y (NPY; Reference 20). NPY is a 36 amino acid peptide that was discovered in 1982 using an assay that detected C-terminally amidated peptides (25). Considered to be one of the most abundant peptides in the central nervous system (19), NPY along with PYY (24) and PP (15) form the PP-fold family of related peptides. When

NPY receptor agonists

injected into the brain of rats, NPY (5,23), PYY (23), and PP (5) produced a significant increase in food consumption and feeding behavior. The NPY Y1 receptor subtype was thought to mediate this behavior, because the putative NPY Y1 selective agonist, [Leu31,Pro34]-NPY, but not the NPY Y2 selective agonist, NPY (13–36), stimulated feeding (13). These data sparked an extensive search for NPY antagonists with hopes of developing agents to regulate feeding behaviors. Specifically, several peptide and non-peptide Y1 antagonists have emerged. Initially, receptor antagonists for NPY receptors were based on modified C-terminal fragments of NPY itself (26).

1 Requests for reprints should be addressed to Dr. Donald R. Gehlert, Lilly Neuroscience Research, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285.

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One such agent, PYX-2, was shown to block natural and NPY-induced carbohydrate feeding (17). Other peptide analogs such as, [D-Trp32]-NPY (3) and NPY (18 –36) (2) were characterized as competitive antagonists of NPY in rat hypothalamus and cardiac ventricular membranes, respectively. Nonpeptide compounds such as BIBP3226 (21), SR 120819A (22) and benextramine (8) have also emerged as putative NPY receptor antagonists. Recently, a novel peptide antagonist, 1229U91, was synthesized based on the C-terminus of NPY (6). This compound displaced [125I]NPY (6) and [125I]-PYY (14) with high affinity from the human NPY Y1 receptor containing cell line, SK-N-MC. Furthermore, the displacement was competitive in nature, and the compound was reported to be a pure antagonist at NPY receptors found in HEL cells (6). The selectivity of 1229U91, however, remains somewhat controversial. Radioligand binding studies in rat brain homogenates show 1229U91 displacing [3H]-NPY with 10 fold higher affinity than the Y1 containing SK-N-MC cells (6). These investigators concluded from their findings that 1229U91 did not appear selective for any particular NPY receptor subtype. In contrast, Kanatani et al. (14), reported that 1229U91 selectively inhibited [125I]-PYY binding using SK-N-MC and SK-N-BE2 cells. In the human NPY Y2 expressing cell line, SK-N-BE2, 1229U91 was 7000-fold less potent than in the SK-N-MC cells. Consequently, we evaluated the selectivity of 1229U91 and BIBP3226 using cell lines containing cloned human NPY Y2, Y4 and Y5 receptors stably expressed in Chinese Hamster Ovary (CHO) cells. Human NPY Y1 pharmacological studies were performed using the NPY Y1 containing SK-N-MC cells. In addition, cyclic AMP accumulation studies were performed to investigate the biological response of these agents in vitro with cells containing either the human NPY Y1 or Y4 receptors. METHOD Receptor Binding Studies CHO cells containing stably expressed human NPY Y2 (10), Y4 (18), Y5 (unpublished results) and a neuroepithelioma cell line expressing human NPY Y1 receptors, SK-N-MC (ATCC, Rockville, MD), were washed once with phosphate-buffered saline (PBS), scraped, and pelleted in fresh PBS. Binding assays were conducted as previously described (9) with isolated crude membrane homogenates. The cell pellets were resuspended using a Polytron homogenizer (Brinkmann, Westbury, NY) in 25 mM HEPES (pH 7.4) buffer containing 2.5 mM CaCl2, 1.0 mM MgCl2 and 2 g/l bacitracin. Incubations were performed at room temperature for 2 h in a final volume of 200 ml containing approximately 0.1 nM [125I]-PYY (SA 2200 Ci/mM, DuPontNEN, Boston, MA). Using a 96-well cell harvester (TOMTEC, Orange, CT), incubations were terminated by rapid filtration through GF/C filters (Wallac, Gaithersburg,

SCHOBER ET AL.

MD) presoaked in 0.3% polyethyleneimine (Sigma, St. Louis, MO). The filters were washed with 5 ml of ice-cold 50 mM Tris (pH 7.4) and rapidly dried at 60°C. The dried filters were treated with MeltiLex A melt-on scintillator sheets (Wallac), and the radioactivity retained on the filters counted using the Wallac 1205 Betaplate scintillation counter. Nonspecific binding was defined as the amount of radioactivity remaining on the filter after incubating in the presence of 1 mM human NPY (Peninsula, Belmont, CA) for the NPY Y1, Y2 and Y5 receptors, and 1 mM human PP (Bachem, King of Prussia, PA) for the NPY Y4 receptor. Various concentration of BIBP3226 (Peninsula) and 1229U91 (Eli Lilly and Company, Indianapolis, IN) were added to the displacement pharmacology studies to determine binding affinity. The results were analyzed using the Prism software package (Graphpad, San Diego, CA). Protein concentrations were measured using Coomassiet protein assay reagent (Pierce, Rockford, IL) with BSA for standards (4). Cyclic AMP Accumulation Assays The human NPY Y4 receptor cell line and the SK-N-MC cells were grown to confluency in 6-well plates with growth medium. The plates were placed in a humidified incubator at 37°C with 5% CO2. Once confluent, the cells were washed with 1 ml PBS, pre-labeled with growth medium containing 2 mCi/well [3H]adenine (ICN #27001) and returned to the incubator for 3 h. Cyclic AMP accumulation was then determined using the method of Alvarez and Daniels (1) with some modifications. The incubation buffer contained Tyrode’s balanced salts solution supplemented with 15 mM HEPES, 50 mM glucose, 100 mM rolipram (Schering AG, Berlin), 10 mM indolidan (Eli Lilly and Company), 10 mM phosphoramidon, and 12.6 mg bacitracin/100 ml (Sigma). The pH was adjusted to 7.4 with NaOH. Phenol red was added as an indicator of pH. An aliquot from a 2 mM forskolin stock in DMSO was added to the incubation buffer for a final concentration of 1 mM. Human NPY was dissolved at 100 mM in 0.1 N acetic acid. All compounds were dissolved and diluted in DMSO at a concentration 1003 the final concentration. A 10 ml aliquot was added to each polystyrene tube containing 1 ml incubation buffer. Using this procedure, each well contained 1% DMSO, except 1229U91 which was dissolved and diluted in water. Incubations were carried out at ambient temperature in a tissue culture hood. Pre-labeling medium was removed by aspiration, then each well was washed with 1 ml PBS. After washing, 1 ml of the incubation medium containing the test agents was added. After 10 min, the incubations were terminated by aspirating the incubation medium, washing with 1 ml PBS, then adding 0.5 ml of stop solution (70% ethanol with ;5000 dpm of [32P]cyclic AMP). The plates were then stored at 220°C

1229U91, A POTENT AGONIST FOR THE HUMAN NPY Y4 RECEPTOR

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FIG. 1. Displacement of 100 pM [125I]-PYY binding to human NPY Y1, Y2, Y4 and Y5 receptor cell membrane homogenates by various concentrations of 1229U91 (Œ) and BIBP3226 (■). Specific binding is expressed as a percentage of maximal specific binding. Specific binding represents approximately 80, 90, 70, and 80% for the NPY Y1, Y2, Y4 and Y5 receptors, respectively. The curves were best fit to a one site model and IC50 values for each inhibitor determined using Prism software. A, 1229U91 and BIBP3226 had IC50 values of 0.245 6 0.004 nM and 16.57 6 1.12 nM, respectively, in SK-N-MC cells containing the human NPY Y1 receptor. B, These same compounds had IC50 values . 1000 nM in CHO cells expressing the human NPY Y2 receptor. C, In the Y4 containing cell line, 1229U91 displaced with high affinity (IC50 5 0.081 6 0.009 nM), while BIBP3226 was inactive. D, At the human NPY Y5, these compounds had IC50 values . 1000 nM. Each value represents an average of 4 (n 5 4) determinations performed in duplicate (mean 6 SEM).

overnight for later isolation of [3H]cyclic AMP by alumina column chromatography. Alumina columns were prepared by adding 1.3 g of dry Alcoa chromatographic alumina, F-20, 80-200 mesh (Schoofs Inc.) to each polypropylene Econo-Column (BioRad, Hercules, CA). The alumina was measured out using a RCBS Uniflow adjustable gunpowder measure with stand (Omark Industries, Oro, CA). Twenty ml aliquots from each well were taken for measurement of total [3H]adenine incorporation. The remaining contents of each well were applied onto the dry alumina columns, which were placed over a tray of 20 ml scintillation vials for collection. After 15 min, the columns were eluted with 4 ml of 100 mM ammonium acetate. Ten ml of Ready Protein Plus scintillation fluid (Beckman Instruments Inc., Fullerton, CA) was

added to each vial, capped and shaken. Vials were counted for 10 min for both [32P] (to measure recovery) and [3H]. The results were analyzed using the Prism software package (Graphpad, San Diego, CA). RESULTS Receptor Binding Pharmacology The affinity of 1229U91 and BIBP3226 to displace 100 pM [125I]-PYY binding to NPY Y1, Y2, Y4 and Y5 receptor expressing cell line homogenates was examined (Fig. 1). Both 1229U91 (IC50 5 0.245 6 0.004 nM, n 5 4) and BIBP3226 (IC50 5 16.57 6 1.12 nM, n 5 4) were effective inhibitors of [125I]-PYY binding to the human NPY Y1 receptor. In the human NPY Y2 and Y5 containing cells, 1229U91 was only slightly more potent than BIBP3226, but

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FIG. 2. Forskolin stimulated [3H]-cyclic AMP accumulation in the SK-N-MC cells (A) and in CHO cells expressing the human Y4 receptor (C). [3H]cyclic AMP accumulation for agonist response curves are expressed as a percentage achieved by stimulating with 1 mm forskolin. Antagonist response curves are expressed as the percent blockade of 1 nM NPY or PP, which was the concentration that sub-maximally inhibited 1 mm forskolin stimulated [3H]cyclic AMP in the NPY Y1 (B) and NPY Y4 (D) containing cells, respectively. Each value represents an average of two to four (n 5 2-4) determinations performed in triplicate. The results were analyzed using the Prism software package (Graphpad, San Diego, CA).

both compounds had IC50 values greater than 1000 nM. Interestingly, 1229U91 was a very potent displacer of [125I]PYY in the human NPY Y4 receptor cell line. In fact, 1229U91 (IC50 5 0.081 6 0.009 nM, n 5 4) was slightly more potent at this receptor than the human NPY Y1 receptor. In contrast, the affinity of BIBP3226 for the human NPY Y4 receptor was just as poor as the human NPY Y2 and Y5 receptor (IC50 . 1000 nM). Cyclic AMP Accumulation Assays Receptor binding data demonstrated that both BIBP3226 and 1229U91 compounds have high affinity for the human NPY Y1 receptor subtype. In cyclic AMP accumulation assays performed with SK-N-MC cells, these compounds exhibited antagonist properties. Both BIBP3226 (IC50 5 24.6 6 4.9 nM, n 5 2) and 1229U91 (IC50 5 5.27 6 1.63 nM, n 5 3) prevented the inhibition of forskolin induced

cyclic AMP accumulation by 1 nM NPY in the this cell line (Fig. 2b). Although these values are less potent than those seen in the receptor binding experiments, the rank order of potency was similar. One plausible explanation for these differences could be that unlike the radioligand binding assays which are performed under equilibrium conditions, the cyclic AMP accumulation assays are incubated with the agonist for only 10 min. There was no detectable effect on forskolin stimulated cyclic AMP accumulation for 1229U91 (Fig. 2a) and BIBP3226 (not shown) when tested in the absence of 1 nM NPY. Furthermore, NPY (IC50 5 0.348 6 0.043 nM, n 5 4) potently inhibited 1 mM forskolin stimulated adenylate cyclase (Fig. 2a). Therefore, in these experiments, both BIBP3226 and 1229U91 were antagonists at the human NPY Y1 receptor. In light of the ability of 1229U91 to displace [125I]-PYY in the NPY Y4 receptor cell line, we tested the effects of this

1229U91, A POTENT AGONIST FOR THE HUMAN NPY Y4 RECEPTOR

peptide in cyclic AMP accumulation assays. Human PP alone was a potent inhibitor of 1 mM forskolin stimulated cyclic AMP accumulation (IC50 5 0.188 6 0.012 nM, n 5 3) in this cell line (Fig. 2c). However, neither BIBP3226 (Fig. 2d) nor 1229U91 (not shown) could inhibit 1 nM human PP effects on forskolin stimulated cyclase activity at the NPY Y4 receptor. Therefore, neither compound appeared to be an antagonist at this receptor. Interestingly, in the absence of human PP, 1229U91 (IC50 5 7.16 6 2.8 nM, n 5 4) potently inhibited forskolin stimulated cyclic AMP accumulation (Fig. 2c). A similar level of inhibition was achieved with 1229U91 when compared to human PP, suggesting it was a full agonist. Therefore, 1229U91 is a relatively potent, full agonist at the human NPY Y4 receptor, but with somewhat lower potency than human PP. DISCUSSION In this paper, we examined the receptor selectivity of two putative NPY receptor antagonists. We evaluated the affinity of 1229U91 and BIBP3226 for human NPY Y1, Y2, Y4 and Y5 receptor containing cells labeled with 100 pM [125I]PYY. In several studies, 1229U91 (6,12,14) and BIBP3226 (7,21) exhibited high affinity for the NPY Y1 receptor subtype. These findings were substantiated further when we compared the pharmacology of 1229U91 and BIBP3226 in the NPY Y1 containing cell line, SK-N-MC. At the NPY Y2 receptor, BIBP3226 was inactive up to 1 mM, while differing activities have been reported for 1229U91. The affinity of 1229U91 for the NPY Y2 receptor subtype has been evaluated using either SK-N-BE2 cells, (12,14), rat brain membranes (6), or a cloned NPY Y2 containing CHO cell line (present study). We found that 1229U91 did not substantially displace [125I]-PYY to the cloned human Y2 receptor, similar to findings using the SK-N-BE2 cell line (12,14). Therefore, 1229U91, like BIBP3226, can pharmacologically distinguish the NPY Y1 receptor from the NPY Y2 and Y5 subtype. In contrast to these findings, Daniels et al. (6) reported that 1229U91 was not selective, since it displaced [125I]-NPY from rat brain homogenates with very high affinity. However, it has been shown previously using homogenate and autoradiographic methods that the rat brain contains both the NPY Y1 and Y2 receptor subtypes (9). Therefore, to pharmacologically distinguish the receptor selectivity, cell lines containing either cloned or naturally expressed receptor subtypes should be utilized. The affinity of 1229U91 and BIBP3226 was evaluated using a CHO cell line stably transfected with the human NPY Y4 receptor. We discovered that 1229U91 was a potent inhibitor of [125I]PYY binding (11), while BIBP3226 was inactive. We speculated that the NPY Y4 receptor was similar to the NPY Y1 receptor because the amino- and carboxyl-terminal regions of the native peptides participate in the binding (11). Both BIBP3226 (21) and 1229U91 (6) were designed based on the C-terminal portion of NPY. Therefore, additional re-

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search will be necessary to determine the structure-activity requirements of 1229U91 at the NPY Y4 receptor. The functional properties of BIBP3226 and 1229U91 were also investigated in this study using cyclic AMP accumulation assays. These data are consistent with previous studies describing the antagonist properties and potencies of 1229U91 (6,12,14) and BIBP3226 (7,21) in NPY Y1 receptor containing cells. Although both BIBP3226 and 1229U91 blocked NPY inhibition of forskolin stimulated cyclic AMP accumulation in the SK-N-MC cells, 1229U91 was about 10-fold more potent. In the cell line expressing the human NPY Y4 receptor, BIBP3226 neither inhibited cyclic AMP accumulation alone, nor blocked the inhibition caused by 1 nM human PP. These results are consistent with the low affinity of BIBP3226 for the human NPY Y4 receptor. Interestingly, 1229U91 behaved as a full agonist in the cell line containing the human NPY Y4 receptor. However, 1229U91 was approximately 2 log orders less potent than the endogenous ligand, human PP. Therefore, 1229U91 is not only a potent antagonist for the human NPY Y1 receptor, but an equipotent agonist for the human NPY Y4 receptor. The physiological implications for these findings may substantially influence the use of this agent to evaluate mechanisms involved in ingestive behavior. The physiology behind feeding behavior is multi-faceted and may have many regulatory factors that can either increase or decrease food consumption (20). NPY is only one such factor that has a pronounced effect on ingestive behavior. Centrally administered NPY can dramatically increases food intake and ingestive behavior (5). Historically, feeding behavior was thought to be mediated through a NPY Y1-like receptor subtype. This was based on studies where the putative NPY Y1 selective agonist, [Leu31,Pro34]NPY strongly elicited a robust feeding response (13,16). In addition, recent evidence further supports the role that a NPY Y1-like receptor may regulate feeding behavior. This was based on a study where the putative NPY Y1 selective antagonist, 1229U91, produced a statistically significant reduction in the feeding response induced by NPY (14). However, other receptor subtypes for the PP-fold family may also play an important role in modulating feeding behavior. This is not surprising that an innate behavior, such as feeding, would have multiple regulatory factors. For instance, PP, a member of the PP-fold family, also has been reported to stimulate feeding behavior when injected ICV (5). Therefore, when evaluating the potential efficacy of agents to regulate ingestive behavior, consideration for receptor selectivity becomes even greater. We can see this complexity even with 1229U91. Both NPY and PP can stimulate food consumption, yet 1229U91 has been shown to inhibit feeding (14) even though it displays agonist properties for the NPY Y4 receptor. In summary, using either cloned or endogenously expressed receptor containing cell lines, 1229U91 displayed

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antagonist properties for the human NPY Y1, but agonist properties for the human NPY Y4 receptors. Therefore, the potential use of 1229U91 for studying the physiological effects of NPY Y1 receptors in vivo raises concerns about its specificity when evaluating its role in studying feeding behavior.

ACKNOWLEDGEMENTS The authors wish to thank Lisa Beavers, Dwayne Johnson and Robert Gadski (Lilly Research Labs) for supplying the CHO cells transfected with the human NPY Y2, the pancreatic polypeptide-preferring (NPY Y4), and Y5 receptors. Furthermore, we like to thank Susan Gackenheimer for data on BIBP3226 at the human NPY Y5 receptor.

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