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

doi:10.1016/S0196-9781(97)00455-5

Peptides

Volume 19, Issue 3, 1998, Pages 537-542

https://doi.org/10.1016/S0196-9781(97)00455-5 Get rights and content

Abstract

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 = 0.245 ± 0.004 nM, n = 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 = 0.081 ± 0.009 nM, n = 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 = 7.16 ± 2.8 nM, n = 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.

Section snippets

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CHO cells containing stably expressed human NPY Y₂ [10], Y₄ [18], Y₅ (unpublished results) and a neuroepithelioma cell line expressing human NPY Y₁ receptors, SK-N-MC (ATCC, Rockville, MD), were washed once with phosphate-buffered saline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

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 = 0.245 ± 0.004 nM, n = 4) and BIBP3226 (IC₅₀ = 16.57 ± 1.12 nM, n = 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 both compounds had IC₅₀ values greater than 1000 nM.

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, 14and BIBP3226 7, 21exhibited 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

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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All three peptides signal via activation of G-protein-coupled receptors (GPCRs), of which four functional variants (Y1, Y2, Y4 and Y5) have been identified in humans (Herzog et al., 1993) (all of them acting in an inhibitory fashion through inhibition of cAMP production (Aakerlund et al., 1990)). While NPY and PYY have similar affinities for all four of the Y-receptors (highest affinity for Y2 followed by Y1 > Y5 and much lower for Y4), PP preferably binds to the Y4 receptors (Schober et al., 1998). While full-length NPY/PYY peptides are required for Y1 binding and activation, truncated versions such as NPY(aa3-36); ; ; or NPY(aa13-36); ; ; are able to bind Y2-receptors (Sheikh et al., 1989).
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This observation is supported by the fact that no significant shifts (<0.02 ppm) of the NH signals in the backbone were detected. Previous studies have shown that C-terminally derived NPY analogs are potent hY1R antagonists and equipotent hY4R agonists (Parker et al., 1998; Schober et al., 1998). After completing the hYR activation profile of the lead analog [Pro30, Nle31, Bpa32, Leu34]NPY 28–36 we observed a well preserved hY4R activity, although earlier binding studies revealed a negligible affinity.
Short selective neuropeptide Y (NPY) analogs are highly attractive because of their facile synthesis. Based on the reduced-size NPY analog [Pro³⁰, Nle³¹, Bpa³², Leu³⁴]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 Bpa³² 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 ¹H NMR spectroscopy revealed structural changes in the side chains of residues Pro³⁰ and Leu³⁴ 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.
GR 231118
2008, xPharm: The Comprehensive Pharmacology Reference
Self-regulation of agonist activity at the Y receptors
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Neuropeptide Y (NPY) is one of the most abundant neuropeptides, and is likely to be present at nanomolar levels over extended periods in the synaptic space of many forebrain areas. This might be linked to an evolved generalized toning activity through a number of other peptide receptors that use C-terminally amidated agonists (with LHRH and orexin receptors and GIR as examples). However, the Y1 and Y2 receptors (which constitute the bulk of Y receptors active in the neural matrix) possess subnanomolar affinities that, at saturating NPY levels, could produce excessive signaling, as well as receptor losses via repeated endocytosis. The related Y4 receptor shows an even higher agonist affinity, and faces the same problem in visceral and neural locations accessible to pancreatic polypeptide (PP). An examination of agonist peptide interaction with Y receptors shows that Y1 and Y4 receptors in particular (as located on either the intact cells, or on particulates derived from various cell types) develop a blockade dependent on ligand concentration, with the blocking ranks of [NPY] ≫ [peptide YY] (PYY) for the Y1, and [human PP] ⋙ [PYY-related Y4 agonist] for the Y4 receptor. This blockade is also echoed in a concentration-related reduction in biological activity of primary agonists (NPY and PP), resembling a partial agonism, and is influenced especially by the allosteric interactivity of agonists. With the Y2 receptor, the blocking by agonists is less pronounced, but the signaling by NPY-related peptides is apparently less than with PYY-related agonists. The extended occupancy and self-attenuation of primary agonist activity at Y receptors could represent an evolutionary solution contributing to a balancing of metabolic signaling, agonist clearance and receptor conservation.

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

Abstract

Section snippets

Receptor Binding Studies

Receptor Binding Pharmacology

Discussion

Acknowledgements

Analytical Biochem.

Peptides.

Analytical Biochem.

Eur. J. Pharmacol.

Neurochem. Int.

Physiol. Behav.

J. Biol. Chem.

Peptides.

J. Biol. Chem.

Peptides.

Nutrition.

Eur. J. Pharmacol.

Peptides.

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