Novel Method for Quantifying AhR-Ligand Binding Affinities Using Microscale Thermophoresis.

Anne Stinn,Pedro Moura-Alves,Michael Kolbe,Stefan H E Kaufmann,Jens Furkert

doi:10.3390/bios11030060

Abstract

The aryl hydrocarbon receptor (AhR) is a highly conserved cellular sensor of a variety of environmental pollutants and dietary-, cell- and microbiota-derived metabolites with important roles in fundamental biological processes. Deregulation of the AhR pathway is implicated in several diseases, including autoimmune diseases and cancer, rendering AhR a promising target for drug development and host-directed therapy. The pharmacological intervention of AhR processes requires detailed information about the ligand binding properties to allow specific targeting of a particular signaling process without affecting the remaining. Here, we present a novel microscale thermophoresis-based approach to monitoring the binding of purified recombinant human AhR to its natural ligands in a cell-free system. This approach facilitates a precise identification and characterization of unknown AhR ligands and represents a screening strategy for the discovery of potential selective AhR modulators.

Highlights

Biomolecular interactions like protein-protein or protein-ligand interactions play an important role in almost all physiological processes
All compounds were obtained from commercial sources, solubilized in dimethyl sulfoxide (DMSO), and stored protected from light. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was obtained from LGC Standard. [3H]TCDD diluted in ethanol was from Hartmann Analytic. 1-Hydroxyphenazine (1-HP) was purchased from TCI Europe N.V., 6-Formylindolo [3,2-b] carbazole (FICZ) and 2-Methyl-2H-pyrazole-3-carboxylic acid (2-methyl-4-o-tolylazo-phenyl)-amide (CH-223191) from Sigma–Aldrich. 1-HP, CH-223191 and FICZ were kept at RT, 4 ◦C or −20 ◦C, respectively
Besides the apparent sensitivity towards amendments in the protocol, some studies stress the fact that the radioligand binding assay largely suffers from the insolubility of TCDD and other aryl hydrocarbon receptor (AhR) ligands in water [26,27]

Summary

Introduction

Biomolecular interactions like protein-protein or protein-ligand interactions play an important role in almost all physiological processes. Quantitative binding studies can be performed by a number of biophysical approaches, such as surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) [2,3]. These techniques are often limited either due to the immobilization of one of the interaction partners that may interfere with binding (SPR) or due to high sample consumptions (ITC), respectively. Originally identified as dioxin receptor, extensive research in the last years has demonstrated that the receptor is a Biosensors 2021, 11, 60.

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