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...

Peptide analogs of neuropeptide Y (NPY) with a Tyr-32 and Leu-34 replacement resulted in the decapeptide TyrIleAsnLeuIleTyrArgLeuArgTyr-NH2 (9; Table 1) and a 3700-fold improvement in affinity at Y2 (rat brain; IC50 = 8.2 +/- 3 nM) receptors when compared to the native NPY(27-36) C-terminal fragment. In addition, compound 9 was an agonist at Y1 (human erythroleukemia (HEL) cell; ED50 = 8.8 +/- 0.5 nM) receptors with potency comparable to that of NPY(1-36) (ED50 = 5 nM). Molecular dynamics and 1H-NMR were used to propose a solution structure of decapeptide 9 and for subsequent analog design. The replacement of Leu with Pro at position 4 of decapeptide 9 afforded an antagonist of NPY in HEL cells (18, TyrIleAsnProIleTyrArgLeuArgTyr-NH2; IC50 = 100 +/- 5 nM). Deletion of the N-terminal tyrosine of 18 resulted in a 10-fold improvement in antagonistic activity with a parallel 4-fold decrease in Y2 affinity. This potent antagonist may provide further insight into the physiological role(s) for NPY in the mammalian and peripheral nervous system.

Traditional approaches to discovery of selective estrogen receptor modulators (SERMs) have relied on ER binding and cell-based estrogen response element-driven assays to identify compounds that are osteoprotective but nonproliferative in breast and uterine tissues. To discover new classes of potential SERMs, we have employed a cell-free microsphere-based binding assay to rapidly characterize ERalpha interactions with conformation-sensing cofactor or phage display peptides. Peptide profiles of constrained triarenes were compared to known proliferative and nonproliferative ER ligands to discover potent quinoline-based ligands with minimal Ishikawa cell stimulation.

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