Determination of affinity and activity of ligands at the human neuropeptide Y Y4 receptor by flow cytometry and aequorin luminescence.
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.
Ziemek
Ralf
R
Institut für Pharmazie, Universität Regensburg, Regensburg, Germany.
Schneider
Erich
E
Kraus
Anja
A
Cabrele
Chiara
C
Beck-Sickinger
Annette G
AG
Bernhardt
Günther
G
Buschauer
Armin
A
D000331:Aequorin / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D049449:Luminescence; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D011485:Protein Binding; D017476:Receptors, Neuropeptide Y / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D017476:Receptors, Neuropeptide Y; D011993:Recombinant Fusion Proteins; D000331:Aequorin; C109491:neuropeptide Y4 receptor