Ziemek, Ralf and Schneider, Erich and Kraus, Anja and Cabrele, Chiara and Beck-Sickinger, Annette G. and Bernhardt, Günther and Buschauer, Armin (2007) Determination of affinity and activity of ligands at the human neuropeptide Y Y4 receptor by flow cytometry and aequorin luminescence. Journal of Receptors and Signal Transduction 27 (4), pp. 217-233.
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Fluorescence-labeled neuropeptide Y (NPY) has been used in flow cytometric binding assays for the determination of affinity constants of NPY Y1, Y2, and Y5 receptor ligands. Because the binding of fluorescent NPY is insufficient for competition studies at the human Y4 receptor (hY4R), we replaced Glu-4 in hPP with Lys for the derivatization with cyanine-5. Because cy5-[K(4)]hPP has high affinity (Kd 5.6 nM) to the hY4R, it was used as a probe in a flow cytometric binding assay. Specific binding of cy5-[K(4)]hPP to hY4R was visualized by confocal microscopy. The hY(4)R, the chimeric G protein G(qi5) and mitochondrially targeted apoaequorin were stably coexpressed in CHO cells. Aequorin luminescence was quantified in a microplate reader and by a CCD camera. By application of these methods 3-cyclohexyl-N-[(3-1H-imidazol-4-ylpropylamino)(imino)methyl]propanamide (UR-AK49) was discovered as the first nonpeptidic Y4R antagonist (pKi 4.17), a lead to be optimized in terms of potency and selectivity.
|Institutions:|| Chemistry and Pharmacy > Institut für Organische Chemie > Arbeitskreis Dr. Chiara Cabrele|
Chemistry and Pharmacy > Institute of Pharmacy > Pharmaceutical/Medicinal Chemistry II (Prof. Buschauer)
|Projects:||GRK 760, Graduiertenkolleg Medizinische Chemie|
|Keywords:||NPY Y4 receptor; Flow cytometry; Chimeric G protein; Aequorin; Nonpeptide NPY Y4 receptor antagonist|
|Subjects:||500 Science > 570 Life sciences|
600 Technology > 610 Medical sciences Medicine
|Refereed:||Yes, this version has been refereed|
|Created at the University of Regensburg:||Yes|
|Owner:||Prof. Armin Buschauer|
|Deposited On:||21 Feb 2008 08:05|
|Last Modified:||05 Aug 2009 13:42|