Fluorescence- and luminescence-based methods for the determination of affinity and activity of neuropeptide Y2 receptor ligands

Ralf Ziemek; Albert Brennauer; Erich Schneider; Chiara Cabrele; Annette G Beck-Sickinger; Günther Bernhardt; Armin Buschauer

doi:10.1016/j.ejphar.2006.08.075

Fluorescence- and luminescence-based methods for the determination of affinity and activity of neuropeptide Y2 receptor ligands

Eur J Pharmacol. 2006 Dec 3;551(1-3):10-8. doi: 10.1016/j.ejphar.2006.08.075. Epub 2006 Sep 8.

Authors

Ralf Ziemek¹, Albert Brennauer, Erich Schneider, Chiara Cabrele, Annette G Beck-Sickinger, Günther Bernhardt, Armin Buschauer

Affiliation

¹ University of Regensburg, Institute of Pharmacy, Universitätsstr. 31, D-93040 Regensburg, Germany.

PMID: 17027743
DOI: 10.1016/j.ejphar.2006.08.075

Abstract

With respect to the discovery and characterization of neuropeptide Y(2) receptor ligands as pharmacological tools or potential drugs, fluorescence- and luminescence-based assays were developed to determine both the affinity and the activity of receptor agonists and antagonists. A flow cytometric binding assay is described for the hY(2) receptor stably expressed in CHO cells using cy5-labeled porcine neuropeptide Y and compared with a radioligand binding assay. Binding of the fluorescent ligand was visualized by confocal microscopy. Stable co-transfection with the chimeric G protein Gq(i5) enabled the establishment of a spectrofluorimetric fura-2 and a flow cytometric fluo-4 calcium assay. Further stable expression of apoaequorin targeted to the mitochondria allowed the establishment of an aequorin assay which could be performed in the 96-well format. The shape of the concentration-response curves of porcine neuropeptide Y in the presence of the Y(2)-selective receptor antagonist BIIE0246, characteristic of either competitive or insurmountable antagonism, depended on the period of incubation with the cells. Functional data of Y(2) receptor agonists and antagonists determined in the fluorescence- and luminescence-based assays were in good agreement.

Publication types

Comparative Study
Research Support, Non-U.S. Gov't

MeSH terms

Aequorin / drug effects
Aequorin / metabolism
Animals
Apoproteins / drug effects
Apoproteins / metabolism
Arginine / analogs & derivatives
Arginine / metabolism
Arginine / pharmacology
Benzazepines / metabolism
Benzazepines / pharmacology
Binding, Competitive
CHO Cells
Calcium Signaling* / drug effects
Cricetinae
Cricetulus
Dose-Response Relationship, Drug
Flow Cytometry / methods*
Fluorescent Dyes
GTP-Binding Protein alpha Subunits, Gq-G11 / drug effects
GTP-Binding Protein alpha Subunits, Gq-G11 / metabolism
Ligands
Microscopy, Confocal / methods*
Neuropeptide Y / analogs & derivatives
Neuropeptide Y / metabolism*
Neuropeptide Y / pharmacology
Peptide Fragments / metabolism
Peptide Fragments / pharmacology
Peptide YY / metabolism
Peptide YY / pharmacology
Protein Binding
Radioligand Assay
Receptors, Neuropeptide Y / drug effects
Receptors, Neuropeptide Y / metabolism*
Recombinant Fusion Proteins / drug effects
Recombinant Fusion Proteins / metabolism
Recombinant Proteins / drug effects
Recombinant Proteins / metabolism
Reproducibility of Results
Spectrometry, Fluorescence / methods*
Swine
Transfection

Substances

Apoproteins
Benzazepines
Fluorescent Dyes
Ligands
Neuropeptide Y
Peptide Fragments
Receptors, Neuropeptide Y
Recombinant Fusion Proteins
Recombinant Proteins
apoaequorin
neuropeptide Y (13-36)
neuropeptide Y2 receptor
Peptide YY
Aequorin
Arginine
GTP-Binding Protein alpha Subunits, Gq-G11
BIIE 0246