Dukorn, Stefanie and Littmann, Timo and Keller, Max and Kuhn, Kilian and Cabrele, Chiara and Baumeister, Paul and Bernhardt, Guenther and Buschauer, Armin (2017) Fluorescence- and Radiolabeling of [Lys(4),Nle(17,30)]hPP Yields Molecular Tools for the NPY Y-4 Receptor. BIOCONJUGATE CHEMISTRY, 28 (4). pp. 1291-1304. ISSN 1043-1802,
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The neuropeptide Y (NPY) Y-4 receptor (Y4R) is involved in energy homeostasis and considered a potential drug target for the treatment of obesity. Only a few molecular tools, i.e., radiolabeled and fluorescent ligands, for the investigation of the Y4R were reported. Previously, [Lys(4)]hPP proved to be an appropriate full-length PP analog to prepare a fluorescent ligand by derivatization at the epsilon-amino group. To preclude oxidation upon long-term storage, we replaced the two methionine residues in [Lys(4)]hPP by norleucine and prepared the corresponding [H-3]propionylated ([H-3]12) and cyanine labeled (13) peptides, which were characterized and compared with a set of reference compounds in binding (Y-1, Y-2, Y-4, and Y-5 receptors) and functional (luciferase gene reporter, beta-arrestin-1,2) Y4R assays. Both molecular probes proved to be useful in radiochemical and flow cytometric saturation and competition Y4R binding experiments. Most strikingly, there was a different influence of the composition of buffer on equilibrium binding and kinetics: [H-3]12 affinity (K-d in Na+-free buffer: 1.1 nM) clearly decreased with increasing sodium ion concentration, whereas dissociation and Y4R-mediated internalization of 13 (K-d in Nat-free buffer: 10.8 nM) were strongly affected by the osmolarity of the buffer as demonstrated by confocal microscopy. Displacement of [H-3]12 and 13 revealed a tendency to higher apparent affinities for a set of reference peptides in hypotonic (Na+-free) compared to isotonic buffers. The differences were negligible in the case of hPP but up to 270-fold in the case of GW1229 (GR231118). By contrast, no relevant influence of Na+ on Y5R affinity became obvious, when the radioligands [H]12 and [H-3]propionyl-pNPY were investigated in saturation binding and competition binding.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | PANCREATIC-POLYPEPTIDE RECEPTORS; HIGH-AFFINITY RADIOLIGAND; NEUROPEPTIDE-Y; FUNCTIONAL EXPRESSION; AGONIST BINDING; HIGHLY POTENT; LIGANDS; ANALOGS; RAT; ANTAGONISTS; |
| Subjects: | 500 Science > 540 Chemistry & allied sciences |
| Divisions: | Chemistry and Pharmacy > Institute of Pharmacy Chemistry and Pharmacy > Institute of Pharmacy > Pharmaceutical/Medicinal Chemistry II (Prof. Buschauer) |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 14 Dec 2018 13:11 |
| Last Modified: | 20 Feb 2019 07:22 |
| URI: | https://pred.uni-regensburg.de/id/eprint/1109 |
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