High-affinity neuropeptide Y receptor antagonists.

Abstract

Neuiropeptide Y (NPY) is one of the most abundant peptide transmitters in the mammalian brain. In the periphery it is costored and coreleased with norepinephrine from sympathetic nerve terminals. However, the physiological functions of this peptide remain unclear because of the absence of specific high-affinity receptor antagonists. Three potent NPY receptor antagonists were synthesized and tested for their biological activity in in vitro, ex vivo, and in vivo functional assays. We describe here the effects of these antagonists inhibiting specific radiolabeled NPY binding at Y1 and Y2 receptors and antagonizing the effects of NPY in human erythroleukmia cell intracellular calcium mobilization, perfusion pressure in the isolated rat kidney, and mean arterial blood pressure in anesthetized rats.Neuropeptide Y (NPY) is a 36-amino acid peptide with an N-terminal tyrosine and a C-terminal tyrosine amide, first isolated from porcine brain by Tatemoto et al. in 1982 (1). NPY has been found to be an abundant mammalian neuropeptide, widely distributed throughout the central and peripheral nervous systems (2-4). On the basis of the pharmacological effects observed in experimental animals after central or peripheral administration of NPY, the peptide has tentatively been implicated in the regulation of a wide variety of biological functions such as vascular tone, feeding behavior, mood, and hormone secretion among others (for a review see ref. 5). At least two NPY receptor subtypes have been described based on the relative affinity of different NPY agonists: NPY-Y1 receptors require essentially the full NPY sequence of amino acids (see Fig. 1) for activation and have high affinity for the analog [Leu31,Pro34]NPY, whereas NPY-Y2 receptors can be activated by NPY and the shorter C-terminal fragment, NPY13-36, but have low affinity for [Leu31,Pro34]NPY (6,7). A third subtype (NPY-Y3) that recognizes all three of the above peptides but is insensitive to the NPY homolog, peptide YY, has been proposed (8, 9). Direct demonstration of a physiological and pathophysiological role for NPY has been hampered by the lack of specific, high-affinity NPY receptor antagonists. Receptor antagonists based on modified Cterminal fragments of NPY (10) Peptide Synthesis. Peptides were synthesized by the solidphase method. Compound 2 was obtained by oxidation of the reduced monomer and purification of the dimer by HPLC. Compound 3 was synthesized by using standard solid-phase synthesis. Compound 4 was synthesized by coupling BOC-Lglutamic acid fluorenylmethyl ester and a-Boc 3-FmOC-Ldiamino propionic acid in position 8 and 6, respectively. Dimerization was achieved on the resin by treatment with piperidine followed by a coupling reagent. Detailed synthesis is described in the compounds' patent publication (15).Binding Assays.[3H]NPY binding to rat brain membranes was done as described (16) except that incubations were terminated by filtration on a Brandel cell harvester through a Whatman GF/B filter, previously soaked overnight in 0.3% po...

Modified, cyclic dodecapeptide analog of neuropeptide Y is the smallest full agonist at the human Y2 receptor

“…Analogs of Ac-25-36 with replacement of Asn 29, Leu a° or Ile 31 by helix reducing amino acids (Gly, Daa) have been found inactive [29] and large hydrophobic residues active [30]. In addition, molar Y2 receptor affinity was found for C-terminal analogs with replacement of Glu 34 by Leu and Thr ~2 by Tyr [31]. Our predictions have been confirmed in the present study since we have succeeded in developing a cyclic dodecapeptide exhibiting high receptor affinity and agonistic properties as strong as NPY itself.…”

Homodimerization of Neuropeptide Y Receptors Investigated by Fluorescence Resonance Energy Transfer in Living Cells

“…NPY modulates numerous physiological processes including regulation of cardiovascular (38) and renal functions, intestinal motility, memory (39), anxiety, seizures, feeding (40), circadian rhythm (41), and nociception. First speculations for homodimerization come from studies with recently reported truncated NPY analogues named ([P 30 ,C 31 (42,43) that bind after dimerization with a higher affinity to the hY 1 -receptor. Furthermore, the homodimeric, peptidergic GR231118 (Y 1 -antagonist, Y 4 agonist) obtains a higher affinity for the Y 1 -receptor than its monomeric form (42,43).…”

Novel analogues of neuropeptide Y with a preference for the Y1‐receptor

“…BIBP3226 [34], LY357897 [35] or BIBO 3304, a nonpeptide Y 1 -antagonist that is even capable of inhibiting NPY-stimulated food consumption in rats after ICV administration [30]. GW1229 (also known as 1229U91 or GR231118), a symmetrical dimeric peptide, has very high affinity for the Y 1 -receptor, but also binds to the Y 4 -receptor subtype and exhibits agonistic properties [36,37]. Until now, no selective agonist for the Y 1 -receptor has been published.…”