A Time-Resolved FRET Cell-Based Binding Assay for the Apelin Receptor

ChemMedChem. 2017 Jun 21;12(12):925-931. doi: 10.1002/cmdc.201700106. Epub 2017 Apr 20.

Abstract

Analogues of apelin-13 carrying diverse spacers and an ad hoc DY647-derived fluorophore were designed and synthesized by chemoselective acylation of α-hydrazinopeptides. The resulting probes retain very high affinity and efficacy for both the wild-type and SNAP-tagged apelin receptor (ApelinR). They give a time-resolved FRET (TR-FRET) signal with rare-earth lanthanides used as donor fluorophores grafted onto the SNAP-tagged receptor. This specific signal allowed the validation of a binding assay with a high signal-to-noise ratio. In such an assay, the most potent sub-nanomolar fluorescent probe was found to be competitively displaced by the endogenous apelin peptides with binding constants similar to those obtained in a classical radioligand assay. We have thus validated the first TR-FRET cell-based binding assay for ApelinR with potential high-throughput screening applications.

Keywords: G protein-coupled receptors; TR-FRET assays; apelin; chemical ligation; fluorescent probes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apelin Receptors
  • Binding Sites / drug effects
  • Dose-Response Relationship, Drug
  • Fluorescence Resonance Energy Transfer*
  • HEK293 Cells
  • High-Throughput Screening Assays
  • Humans
  • Lanthanoid Series Elements / chemistry
  • Lanthanoid Series Elements / pharmacology*
  • Ligands
  • Molecular Structure
  • Organometallic Compounds / chemical synthesis
  • Organometallic Compounds / chemistry
  • Organometallic Compounds / pharmacology*
  • Receptors, G-Protein-Coupled / agonists*
  • Structure-Activity Relationship
  • Time Factors

Substances

  • APLNR protein, human
  • Apelin Receptors
  • Lanthanoid Series Elements
  • Ligands
  • Organometallic Compounds
  • Receptors, G-Protein-Coupled