A homogeneous single-label quenching resonance energy transfer assay for a δ-opioid receptor-ligand using intact cells

Analyst. 2013 Sep 7;138(17):4907-14. doi: 10.1039/c3an00736g. Epub 2013 Jun 26.

Abstract

This study, a homogeneous assay system for delta opioid receptor binding ligands has been developed using Quenching Resonance Energy Transfer (QRET). The QRET system allows receptor-ligand binding assays on intact cells using a single-label approach and a nonspecific quenching mechanism. Binding of antagonists or agonists to the receptor can be defined using a europium(III) labeled ligand. In the presence of the unlabeled ligand the labeled ligand is displaced and remains in solution. The non-bound labeled ligand is not protected by the target receptor, and the luminescence signal is quenched. For this objective, a Eu(III) labeled peptide molecule with three different linkers (AX0, AX1 and AX2) was designed. Peptides were evaluated using the homogeneous QRET technique, radioligand binding assays and the heterogeneous time-resolved luminescence (TRL) technique. Using the Eu-AX0 peptide and the QRET method, a panel of opioid compounds (naltrexone, naltrindole, SCN-80, DPDPE and DAMGO) was tested to prove the assay performance. The signal-to-background ratio for the tested opioid ligand ranged from 3.3 to 12.0. The QRET method showed prominent performance also in high DMSO concentrations. QRET is a homogenous and a non-radioactive detection system for screening and this is the first attempt to utilize peptide ligands in the QRET concept.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Count
  • Dimethyl Sulfoxide / chemistry
  • Europium / chemistry
  • Fluorescence Resonance Energy Transfer*
  • HEK293 Cells
  • Humans
  • Ligands
  • Models, Molecular
  • Oligopeptides / chemistry
  • Oligopeptides / metabolism
  • Protein Conformation
  • Receptors, Opioid, delta / chemistry
  • Receptors, Opioid, delta / metabolism*
  • Staining and Labeling*

Substances

  • Ligands
  • Oligopeptides
  • Receptors, Opioid, delta
  • Europium
  • Dimethyl Sulfoxide