Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027819 (neuroblastoma)
 27,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Truncated, branched, and/or cyclic neuropeptide Y (NPY) analogues were tested for their ability to bind to the neuroblastoma cells, SK-N-MC (Y1 receptor) and SK-N-BE(2) (Y2 receptor). The design of such analogues was inspired by models of NPY based on the crystal structure of avian pancreatic polypeptide. The minimum length of the backbone was investigated using the following truncated analogues [binding affinity (nM) for Y1 and Y2 receptor subtypes respectively are given in parentheses]: des-AA10-17[D-Ala9]NPY (100, 0.9), des-AA7-23[D-Ala6]NPY (> 1000, 1.2), des-AA4-26[D-Ala3]NPY (> 1000, 120), cyclo(7,20)-des-AA10-17[Glu7,D- Ala9,D-Dpr20]NPY (100, nd), cyclo(2,27)-des-AA7-23[Glu2,D-Ala6,D-Dpr27]NPY (> 1000, 3.6), cyclo(2,30)- des-AA7-23[Glu2,D-Ala6,-D-Dpr30]NPY (> 1000, nd), cyclo(1,30)-des-AA4-26[Glu1,D-Ala3,D-Dpr30]NPY (> 1000, > 1000). A new family of branched NPY analogues corresponding to the partial deletion of the polyproline helix with conservation of the N-terminus was also examined: des-AA7-23[(Ac-NPY14-22)-epsilon-D-Lys6]NPY (> 1000, 2.1), des-AA7-23[(Ac-NPY7-22)-epsilon-D-Lys6]NPY (> 1000, 5.1), des-AA7-23-[(Ac-LEALEG-NPY14-22)-epsilon-D-Lys6]NPY (> 1000, 4.8). Finally, the role played by the flexible tail (residues 32-36) was studied with the following cyclic analogues: cyclo(30,34)-[Lys30,Glu34]NPY18-36 (> 1000, 360), cyclo(30,34)-[Orn30,Gly34]NPY18-36 (> 1000, 950), cyclo(30,34)-[Dpr30,Glu34]NPY18-36 (> 1000, 590), cyclo(33,36)-[Lys33,Glu36]NPY (> 1000, > 1000), cyclo(33,36)-[Lys33,Glu36]NPY18-36 (> 1000, > 1000). These results suggest that the Y1 receptor is highly discriminatory since deletion of residues 10-17, shown to have little effect on Y2 binding affinity, reduces Y1 affinity 50-fold. Bridging sites and constructs have been identified that may serve as useful leads in the design of more potent and selective analogues. We have identified two positions (9 and 6) where the introduction of a D amino acid is not detrimental to binding affinity. Whether this modification leads to the stabilization of a yet unidentified turn compatible with high Y2 receptor affinity will have to be determined by spectroscopic methods. Finally, stabilizing a putative alpha-helical conformation of the C-terminal heptapeptide of NPY18-36 has a deleterious effect on the Y1 and Y2 receptors.
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PMID:Truncated, branched, and/or cyclic analogues of neuropeptide Y: importance of the pancreatic peptide fold in the design of specific Y2 receptor ligands. 143 76

Pharmacological studies indicate that peptide YY (PYY) and neuropeptide Y interact with multiple binding sites, categorized as Y1 and Y2 subtypes. In order to identify and structurally characterize the Y1 and Y2 receptors we covalently cross-linked [125I-Tyr36]PYY to its receptors. The Y2 receptor in rat hippocampus and rabbit kidney membranes was affinity labeled using different homo- and heterobifunctional cross-linking reagents. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography resulted in a major labeled protein band of Mr = 50,000 in both hippocampal and kidney membranes, which was unaffected by reducing agents. The Y1 receptor was analyzed in membranes from the MC-IXC human neuroblastoma cell line. Autoradiography revealed two labeled bands at Mr = 70,000 and 45,000. As the intensity of the Mr = 45,000 band was reduced by protease inhibitors, it is likely that this band is a degradation product of the larger band. Labeling of these proteins was obtained only when N-5-azido-2-nitrobenzoyloxysuccinimide was employed for cross-linking followed by exposure to UV light. Labeling of the two cross-linked bands was unaffected by reducing agents. The binding of radiolabeled PYY and the intensity of the cross-linked bands, for both the Y1 and Y2 receptors, were inhibited similarly in a dose-dependent manner by increasing concentrations of unlabeled PYY. When exposed to agarose-coupled lectins, the detergent-solubilized Y1 receptor-hormone complex was completely adsorbed by wheat germ agglutinin and partially by ricin communis II. The cross-linked Y2 receptor was almost totally adsorbed by wheat germ agglutinin-agarose and partially adsorbed by concanavalin A. The adsorptions were in all cases blocked by the appropriate hapten sugar. These results indicate that the Y1 receptor is a glycoprotein with a Mr = 70,000 binding subunit, whereas the Y2 receptor is a glycoprotein with a Mr = 50,000 binding subunit. These results provide evidence that the Y1 and Y2 subtypes of neuropeptide Y and PYY receptors, previously characterized pharmacologically, are structurally distinct glycoproteins, not disulfide-linked to other subunits.
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PMID:Structural characterization of Y1 and Y2 receptors for neuropeptide Y and peptide YY by affinity cross-linking. 215 75

Neuropeptide Y (NPY), a 36-residue polypeptide produced abundantly in both nervous and peripheral tissues, appears to play a significant role in the regulation of diverse biological processes, including feeding behavior and cardiovascular and psychotropic functions. The actions of NPY are mediated through effective binding to specific receptors of which two, designated Y1 and Y2, have been well characterized. A shortened cyclic analogue of NPY, des-AA10-17-cyclo-7/21[Cys7,21]NPY, was shown to retain high affinity for both human neuroblastoma SK-N-MC and SK-N-BE2 cell types (expressing Y1 and Y2 receptors, respectively). Increasing the size of the ring (des-AA10-17-cyclo-2/27[Cys2,27]NPY) in the present study produced a high-affinity analogue (Ki = 3.0 vs 0.3 nM for NPY) that bound exclusively to Y2 receptors. Using the feedback from structure-activity relationships, we also describe the optimization of specific substitutions and bridging arrangements leading to the production of other truncated, high-affinity Y1 selective analogues which bind, as does NPY itself, in the low-nanomolar range. Of greatest significance, des-AA10-17-cyclo-7/21[Cys7,21,Pro34]NPY (11) was found to possess agonistic properties with an affinity comparable to that of the native NPY molecule when tested for its ability to inhibit norepinephrine-stimulated cAMP release in SK-N-MC human neuroblastoma cells. Compound 11 also caused an increase in blood pressure in anesthetized rats. However, in two central nervous system models of Y1 receptor function, stimulation of feeding and anxiolytic activity, this analogue was inactive, which suggests the presence of a new subclass of receptors. In summary, the present results demonstrate that residues 10-17 of NPY are not directly involved in either Y1 or Y2 receptor recognition or activation. This suggests that the selectivity of NPY receptors is highly dependent on subtle conformational changes such as the substitution of residue 34 to a proline or the introduction of intramolecular constraints. Additionally, we have produced an analogue of NPY that selectively activates peripheral NPY Y1 receptors.
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PMID:Y1 and Y2 receptor selective neuropeptide Y analogues: evidence for a Y1 receptor subclass. 747 86

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.
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PMID:Subtype selectivity and antagonistic profile of the nonpeptide Y1 receptor antagonist BIBP 3226. 756 43

Using guanine nucleotides, pertussis toxin, and specific antisera against the COOH-terminals of the alpha-subunits of Gi1/2, Gi3, and G(o), the binding and biological response of the Y2 receptor (Y2R) for peptide YY (PYY) was probed in SMS-KAN neuroblastoma cells. The specific binding of radiolabeled PYY exhibited a single apparent dissociation constant, KD = 76 pM for intact cells and KD = 906 pM for permeabilized cells. However, other data suggested existence of multiple receptor affinity states. A shift in KD and a decrease in apparent number of binding sites (Bmax) was observed in permeabilized cells when incubated with guanine nucleotides. By contrast, in membrane preparations guanine nucleotides induced only a decrease in Bmax. In intact cells, agonist exposure inhibited the intracellular accumulation of forskolin-stimulated cyclic AMP by 80% (IC50 = 420 nM) compared with 94% inhibition (IC50 = 380 nM) in permeabilized cells. In permeabilized cells, preincubation with antisera against alpha i1/2 and alpha i3 blocked the functional response of PYY, with anti-alpha i3 being the most potent; whereas anti-alpha o failed to affect the cyclic AMP levels. These results suggest that permeabilized SMS-KAN cells serve as a good model system for analysis of Y2R binding kinetics and functional response and that the Y2R interacts directly with several different GiS (but not G(o)).
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PMID:Coupling of the human Y2 receptor for neuropeptide Y and peptide YY to guanine nucleotide inhibitory proteins in permeabilized SMS-KAN cells. 783 57

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.
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PMID:Complete L-alanine scan of neuropeptide Y reveals ligands binding to Y1 and Y2 receptors with distinguished conformations. 795 31

We have carried out functional and in vitro studies on a novel analog of neuropeptide Y which shows selectivity for the prejunctional or neuropeptide Y Y2 receptor. In anaesthetised rats N-acetyl [Leu28,Leu31]neuropeptide Y-(24-36) attenuates cardiac vagal action (a prejunctional or neuropeptide Y Y2 action) and has no significant pressor effects (postjunctional or neuropeptide Y Y1 action). In the human neuroblastoma cell line (SMS-KAN) which expresses and endogenous Y2-like neuropeptide Y receptor, N-acetyl [Leu28,Leu31]neuropeptide Y-(24-36) competes with peptide YY for binding sites with an IC50 of 0.5 +/- 0.1 nM. In contrast in a fibroblast Chinese hamster ovary cell line which expresses the cloned human neuropeptide Y Y1 receptor and is used to study changes in cytosolic calcium evoked by (a neuropeptide Y Y1 effect), N-acetyl [Leu28,Leu31]neuropeptide Y-(24-36) showed no activity even at high concentrations. The steric structure for this novel compound has been determined using proton nuclear magnetic resonance (NMR) spectroscopy and it is consistent with the C-terminal end of published structures of neuropeptide Y. We suggest acetylation and amino acid substitutions stabilise the molecule and allow it to bind only to the neuropeptide Y Y2 receptor.
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PMID:A novel neuropeptide Y analog, N-acetyl [Leu28,Leu31]neuropeptide Y-(24-36), with functional specificity for the presynaptic (Y2) receptor. 808 64

In an effort to gain insight into the bioactive conformation of neuropeptide Y upon interaction with its receptors, all single-point D-amino acid substituted NPY analogues were prepared, and their Y1 and Y2 receptor binding affinities were evaluated using the human neuroblastoma cell lines, SK-N-MC and SK-N-BE2, respectively. Solid-phase synthesis (Boc strategy) followed by preparative HPLC purification produced analogues of high purity that were characterized by RP-HPLC, AAA, LSIMS, CZE, and optical rotation. Of the 37 isomers (a naturally occurring glycine at position 9 was replaced by Ala and D-Ala), Y1 receptor binding was most perturbed by chiral inversion of residues at the C-terminus (residues 20, 27, 29-35, Ki > or = 300 nM). Substitutions at residues 2-5, 28, and 36 had Ki values ranging from 40 to 260 nM. Substitutions at all other positions yielded analogues with affinities ranging from 1.5 to 20 nM. Binding affinities to the Y2 class of receptors all measured in the low or sub-nanomolar concentrations, with the exception of C-terminally modified isomers (residues 30-35). Only [D-Arg33]- and [D-Gln34]NPY displayed no measurable binding affinity to Y2 receptors at the highest concentration tested (1000 nM). Representative analogues were selected on the basis of their binding affinities and position in the sequence for structural analysis using circular dichroism (CD) spectroscopy. Of the nine peptide evaluated ([D-Pro5]-, [Ala9]-, [D-Glu10]-, [D-Asp11]-, [D-Ala18]-, [D-Tyr20]-, [D-Tyr27]-, and [D-Arg33]NPY), only [D-Tyr27]NPY expressed a definitive correlation between loss of binding affinity and disruption of secondary structure by having the propensity to form beta-sheets at the expense of alpha-helical content. It was concluded that although the incorporation of a single D-amino acid within the sequence of NPY may confer a conformational perturbation, the receptor interaction was only affected when certain critical residues were modified, findings that provide a basis for the identification of the binding pharmacophore of NPY.
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PMID:Neuropeptide Y: Y1 and Y2 affinities of the complete series of analogues with single D-residue substitutions. 825 9

To further elucidate the minimum bioactive conformation of neuropeptide Y (NPY), a series of truncated and conformationally constrained analogues has been prepared. The synthesis and purification of these peptides was achieved using routine laboratory strategies and techniques. Parent molecules consisted of the native NPY N-terminal 1-4 and C-terminal 25-36 segments, having the residue 5-24 core replaced by either a single flexible omega-aminoalkanoic acid, or a more rigid Pro-Gly or Pro-DAla sequence which was expected to constrain a putative turn, and allow the N- and C-termini to align. Cross-linking between residues 2 and 27 through lactamization using side-chain length and chirality suggested by computer simulations, resulted in cyclo-(2/27)-des-AA7-24[Glu2,Gly6,DDpr27]NPY that exhibited very high affinity (Ki = 0.3 versus 0.3 nM for NPY) for the Y2 receptor using SK-N-BE2 human neuroblastoma cells, yet very low affinity for the Y1 receptor using SK-N-MC human neuroblastoma cells (Ki = 130 versus 2.0 nM for NPY). The added constraint resulting from bridging in this analogue as well as in others suggested that the combination of the deletion of residues 5-24 and the introduction of an internal ring produced exclusive selectivity for the Y2 receptor with little or no loss of affinity. The tolerance of structural recognition was further demonstrated as a second ring was introduced which was expected to constrain the amphiphilic alpha-helix, resulting in the full Y2 agonist dicyclo (2/27,28/32)-des-AA7-24 [Glu2,32,DAla6,DDpr27,Lys28]NPY. Improvement of Y1 binding activity was achieved only by including more residues (des-AA10-17) in the central PP-fold region, while allowing limited flexibility of the termini. Although the length of the bridge seemed to have little effect on binding potency, changes in the location of and chirality at the bridgehead resulted in analogues with different binding affinities. Combination of optimum structural modifications resulted in cyclo-(7/21)-des-AA10-18[Cys7,21]NPY, an analogue shortened by 25% but retaining comparable binding properties to that of native NPY at Y1 and Y2 receptor types (Ki = 5.1 and 1.3 nM, respectively).
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PMID:Defining structural requirements for neuropeptide Y receptors using truncated and conformationally restricted analogues. 842 66

In order to stabilize the C-terminal dodecapeptide of neuropeptide Y (NPY) we replaced Leu28 and Thr32 by Lys and Glu, respectively, and subsequently linked these residues by lactamization. This peptide analog of NPY shows a more than 100-fold increase in affinity compared to the C-terminal linear dodecapeptide in receptor binding studies performed at human neuroblastoma cells SMS-KAN, which exclusively express the Y2 receptor subtype. Signal transduction was investigated by measuring Ca2+ current inhibition in human SH-SY5Y cells and cyclic [Lys28-Glu32] NPY Ac-25-36 and NPY were shown to be equipotent in this assay. Thus, this molecule is the smallest Y2 receptor selective full agonist of NPY. Using 2D-NMR experiments and molecular modelling techniques, the structures of the linear and cyclic peptides have been investigated and significant differences have been found, which may explain the improvement in biological activity. Thus, a model of the bioactive conformation of NPY at the human Y2 receptor is suggested.
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PMID:Modified, cyclic dodecapeptide analog of neuropeptide Y is the smallest full agonist at the human Y2 receptor. 884 57


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