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A Time-Resolved FRET Cell-Based Binding Assay for the Apelin Receptor
Christel Valencia1, Céline Dujet2, Jean-François Margathe3
1Université de Strasbourg, CNRS, Plateforme de Chimie Biologie Intégrative, PCBIS, UMS3286, Labex MEDALIS, 6700, Strasbourg, France.
Chemmedchem
|April 5, 2017
Summary
Researchers developed novel fluorescent probes for the apelin receptor (ApelinR). These probes enable a new time-resolved Förster resonance energy transfer (TR-FRET) assay for drug discovery.
Area of Science:
- Biochemistry
- Pharmacology
- Molecular Biology
Background:
- The apelin receptor (ApelinR) plays a crucial role in cardiovascular and metabolic functions.
- Developing robust assays for ApelinR is essential for identifying novel therapeutic agents.
Purpose of the Study:
- To design and synthesize novel fluorescent probes for ApelinR.
- To validate a new time-resolved Förster resonance energy transfer (TR-FRET) cell-based binding assay for ApelinR.
Main Methods:
- Synthesis of apelin-13 analogues with diverse spacers and a DY647 fluorophore via chemoselective acylation.
- Utilizing SNAP-tagged ApelinR and rare-earth lanthanides for TR-FRET signal generation.
- Validation of the binding assay using competitive displacement with endogenous apelin peptides.
Main Results:
- Synthesized probes demonstrated high affinity and efficacy for both wild-type and SNAP-tagged ApelinR.
- A validated TR-FRET assay with a high signal-to-noise ratio was established.
- The assay successfully quantified binding affinities comparable to radioligand assays.
Conclusions:
- The first TR-FRET cell-based binding assay for ApelinR was successfully validated.
- This assay shows potential for high-throughput screening applications in drug discovery.
- Novel fluorescent probes offer a valuable tool for studying ApelinR pharmacology.