BIBP 3226, the first selective neuropeptide Y1 receptor antagonist: a review of its pharmacological properties.

Based on the assumption that the pharmacophoric groups interacting with the Y1 receptor are located in the C-terminal part of neuropeptide Y, low molecular weight compounds with high affinity and selectivity for the Y1 receptor were designed and synthesized. The prototype BIBP 3226 possesses affinity for the Y1 receptor in the nanomolar range. In addition, this compound is selective displaying rather low affinity for Y2, Y3, Y4 and a set of 60 other receptors. Both biochemical and pharmacological studies showed that BIBP 3226 behaves as a competitive antagonist. Using BIBP 3226 it was possible to investigate the role of NPY and/or Y1 receptors in blood pressure regulation. The interesting observation was that antagonism to Y1 receptors had no major influence on the basal blood pressure but attenuated stress induced hypertension. This strongly supports the hypothesis that NPY is mainly released during stress involving intense sympathetic nervous system activation. Moreover, BIBP 3226 can be used to characterize NPY receptor subtypes. For instance, we were able to show that presynaptic NPY receptors mediating catecholamine release do not solely belong to the Y2 subtype, but that presynaptic Y1 receptors also exist. In conclusion, BIBP 3226 has been shown to be an important tool for the elucidation of the physiological role of Y1 receptors in the cardiovascular system.

Subtype selectivity and antagonistic profile of the nonpeptide Y1 receptor antagonist BIBP 3226.

In the present study, the subtype specificity and species selectivity of the nonpeptide BIBP 3226, as well as its in vitro antagonism of neuropeptide Y (NPY)-mediated second messengers have been investigated. Radiolabeled NPY is potently displaced by BIBP 3226 [(R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenylmethyl]-D- arginine amide] on human Y1 receptor expressing Chinese hamster ovary-K1 cells (Ki = 0.47 +/- 0.07 nM). SK-N-MC human neuroblastoma cells (Ki = 5.1 +/- 0.5 nM) and the rat parietal cortex membranes (Ki = 6.8 +/- 0.7 nM). The interaction of BIBP 3226 with the Y1 receptor is stereoselective, because the (S)-enantiomer of the (R)-configured BIBP 3226 displays almost no affinity (Ki > 10,000 nM). In contrast, concentrations up to 10 microM BIBP 3226 do not displace [125I]NPY from the human Y2 receptor (neuroblastoma cell line SMS-KAN), the rabbit Y2 receptor (kidney) and the rat Y2 receptor (hippocampus). Functional antagonism could be shown for the human Y1 receptor: 0.1 microM BIBP 3226 antagonizes the NPY induced Ca++ mobilization (pKb = 7.5 +/- 0.17) as well as the NPY-mediated inhibition of cyclic AMP synthesis (pKb = 8.2 +/- 0.24) in SK-N-MC cells. In contrast, none of the formerly described putative antagonists PYX-2, [D-Trp32]NPY and benextramine could be characterized as high affinity Y1 receptor antagonists. The 18 amino acid NPY analog EXBP 68 Ile-Glu-Pro-Orn-Tyr-Arg-Leu-Arg-Tyr-NH2, cyclic (2,4'), (2',4')-diamide] displayed Y1-selective affinity with in vitro antagonistic properties (Ki = 0.33 +/- 0.04 nM and pKb = 8.4 +/- 0.07) in SK-N-MC cells. Therefore, BIBP 3226 is the first potent and subtype-selective nonpeptide Y1 receptor antagonist.

A rational approach for the development of reduced-size analogues of neuropeptide Y with high affinity to the Y1 receptor.

Four sets of centrally truncated analogues of neuropeptide Y have been synthesized. In each series the N-terminal part was constant, while the C-terminal segment was systematically varied in length. The C- and N-terminal parts were linked by 6-aminohexanoic acid. The affinity to the Y1 receptor was investigated on human neuroblastoma cells SK-N-MC. Significant differences were found between the series of peptides as well as within each set. Remarkably, the affinity did not solely depend on the length of the segment, and with increasing numbers of residues the IC50 values were not always decreased. With a given N-terminal segment, only one optimal length of the C-terminal segment was found, which suggests that it is not the amino acids themselves but their 3D arrangement and orientation that is important for high receptor affinity.

Labeling of neuropeptide Y receptors in SK-N-MC cells using the novel, nonpeptide Y1 receptor-selective antagonist [3H]BIBP3226.

The binding of tritium-labelled BIBP3226, N2-(diphenylacetyl)-N-[(4-hydroxy-phenyl)methyl]-D-arginine amide, to human neuroblastoma SK-N-MC cells was investigated. [3H]BIBP3226 reversibly binds to neuropeptide Y receptors of the Y1 subtype expressed in SK-N-MC cells with a KD of 2.1 +/- 0.3 nM (mean +/- S.E.M., n = 3) and a Bmax of 58,400 +/- 1100 sites/cell. Non-specific binding did not exceed 30% of the total radioactivity bound at KD. In competition experiments [3H]BIBP3226 is concentration-dependently displaced by neuropeptide Y and its peptide analogues with an affinity pattern neuropeptide Y = [Leu31, Pro34]neuropeptide Y >> neuropeptide Y-(18-36). This rank order of potencies is consistent with the interaction of [3H]BIBP3226 with neuropeptide Y receptors of the Y1 subtype. Therefore, [3H]BIBP3226 can be used as selective ligand to study neuropeptide Y Y1 receptors.

The first highly potent and selective non-peptide neuropeptide Y Y1 receptor antagonist: BIBP3226.

The design and subsequent in vitro and in vivo biological characterisation of the first potent and selective non-peptide neuropeptide Y Y1 receptor antagonist, BIBP3226 ((R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-argininami de) is reported. BIBP3226 displaced 125I-labelled neuropeptide Y with high affinity (Ki = 7 nM) from the human neuropeptide Y Y1 receptor and proved to be highly selective. BIBP3226 displayed potent antagonistic properties both in in vitro and in vivo models and thus represents the first selective non-peptide neuropeptide Y Y1 receptor antagonist.

Complete L-alanine scan of neuropeptide Y reveals ligands binding to Y1 and Y2 receptors with distinguished conformations.

The synthesis of more than fifty 36-residue oligopeptide analogs of neuropeptide Y (NPY) and their affinity to human Y1 and Y2 receptors is described. Each amino acid of the natural sequence was replaced by L-alanine, the four alanine residues at position 12, 14, 18 and 23 were replaced by glycine. Additional residues were exchanged to closely related ones in order to characterize the prerequisites for binding. A combination of automated single and multiple peptide synthesis using fluoren-9-ylmethoxycarbonyl/tert-butoxy strategy was applied. The purified peptides were characterized by electrospray mass spectrometry, analytical HPLC and amino acid analysis. Binding was investigated by displacement of 125I-labelled neuropeptide Y from human neuroblastoma cell lines SK-N-MC and SMS-KAN. Whereas Pro2 and the integrity of the neuropeptide Y loop is important for the binding to the Y1 receptor, exchanges within the C-terminal helix affect the affinity to the Y2 receptor. The C-terminal pentapeptide amide is important for both receptors and probably represents the binding site. However, Arg33 and Arg35 may not be exchanged by L-alanine in the Y1 system, whereas Arg35 and Tyr36 are the most susceptible residues in the Y2 system. In order to distinguish between conformational effects and direct hormone/receptor interaction via the side chains of neuropeptide Y, circular dichroic studies of the alanine-containing peptides were performed and structure affinity relationships are discussed. Comparing the affinities of the neuropeptide Y analogs to Y1 and Y2 receptors significant differences were found for the two binding sites, which suggests a different active conformation of neuropeptide Y at the two subtypes of receptors. Using molecular dynamics calculations, two distinct conformations were identified which are in good agreement with the data obtained by structure/affinity investigations.

Characterization of muscarinic receptors in guinea-pig uterus.

To characterize the muscarinic receptor present in guinea-pig uterus smooth muscle the affinities of a series of 27 muscarinic receptor antagonists for M1 (rat cortex), M2 (rat heart), M3 (rat submandibular gland), m4 (transfected in CHO cells) and muscarinic binding sites in guinea-pig uterus smooth muscle were determined in radioligand binding studies. In addition, functional experiments were performed to assess pKB values of the antagonist for muscarinic receptors in guinea-pig atrium and uterus. The results obtained are consistent with the presence of M2 receptors in the uterus through which the functional contractile response is mediated. Correlation coefficients of 0.98, 0.91 and 0.91 were calculated for the following linear regressions: pKi uterus vs. pKi M2, pKB uterus vs. pKi M2 and pKB uterus vs. pKB atrium. This study also revealed that the compounds dicyclomine, DAU 5884, DAU 6202 as well as AQ-RA 721 could distinguish m4 from M2 sites and are therefore important tools to characterize muscarinic receptor subtypes. In addition, DAU 5884 and DAU 6202 have been identified as highly potent M1 selective antagonists.