Synthesis of a ligand–quencher conjugate for the ligand binding study of the aryl hydrocarbon receptor using a FRET assay

Abstract

The aryl hydrocarbon receptor (AhR) is a transcription factor that induces the adaptive responses upon binding to a wide range of exogenous chemicals in cells. The traditional method for studying AhR ligands is to determine their effect on transcriptional activities (e.g., luciferase expression) in cultured cells. In this paper, we sought to investigate the feasibility of studying the ligand binding with the AhR using a Forster resonance energy transfer (FRET) assay—a novel approach. Two conjugates (βNFQ and 8) containing β-naphthoflavone and DABCYL (a quencher) with different linkers were therefore synthesized for evaluation. The luciferase expression and green fluorescent protein (GFP) expression assays showed that βNFQ is a partial AhR agonist. Simultaneous incubation of cultured cells containing expressed GFP-AhR fusion protein with βNFQ and a known AhR ligand 3-methylchloranthrene reduced the fluorescence of GFP-AhR to a lesser extent, suggesting that the reduction in fluorescence resulted from the competitive binding of ligands to GFP-AhR. Although the fluorescent quenching by βNFQ is modest, our results suggest that FRET could be a valuable tool for identifying AhR ligands with the use of a more efficient ligand–quencher.