Modeling of the aryl hydrocarbon receptor (AhR) ligand binding domain and its utility in virtual ligand screening to predict new AhR ligands

Abstract

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor; the AhR Per-AhR/Arnt-Sim (PAS) domain binds ligands. We developed homology models of the AhR PAS domain to characterize previously observed intra- and interspecies differences in ligand binding using molecular docking. In silico structure-based virtual ligand screening using our model resulted in the identification of pinocembrin and 5-hydroxy-7-methoxyflavone, which promoted nuclear translocation and transcriptional activation of AhR and AhR-dependent induction of endogenous target genes.

Figure 1

(A) Interspecies multiple sequence alignment of the AhR-LBD performed with the program ClustalW. (B) Evolutionary tree of different species.

Figure 2

(A) Homology model of the mouse AhR-LBD with the protein backbone displayed as ribbon and colored by secondary structure (ICM v3.5-1n, Molsoft). The residues are displayed as sticks and colored by atom type with the carbon atoms in white. The binding pocket volume is colored in green and was calculated using the program ICM Pocket Finder (ICM v3.5-1n, Molsoft). (B–D) TCDD docking orientation into mouse (B), human (C), zebrafish isoform 2 (D) AhR-LBD binding pocket. The protein backbone is displayed as ribbon and colored by secondary structure. The residues are displayed as sticks and colored by atom type with the carbon atoms in white. TCDD is displayed as sticks and colored by atom type (carbon atoms in yellow). (E) mouse AhR-LBD binding pocket volume is colored in green and was calculated using the program ICM Pocket Finder. The protein backbone is displayed as ribbon and colored by secondary structure. The residues are displayed as sticks and colored by atom type with the carbon atoms in orange (wild type) and white (A375L mutant). (F–G) binding pocket region identified by ICM Pocket Finder of the zebrafish AhR-LBD of isoform 2 (F) and isoform 1a (G) colored in translucent blue and dark blue respectively. The protein backbone is displayed as ribbon and colored by secondary structure. Residues are displayed as sticks and colored by atom type with carbon atoms in white. Docked TCDD is displayed as sticks and colored in red.

Figure 3

ITE (A, C) and FICZ (B, D) docking orientation respectively into mouse (A, B), human (C, D) AhR-LBD homology model. The protein backbone is displayed as ribbon and colored by secondary structure. The residues are displayed as sticks and colored by atom type with the carbon atoms in white. FICZ and ITE are displayed as sticks and colored by atom type with carbon atoms in yellow. H-bonds are represented by white dashed lines.

Figure 4

(A) Mouse Hepa1 cells transfected with the AhR response element (AhRE)/ xenobiotic response element (XRE)-luciferase reporter were treated with compounds at the indicated concentrations for 6 hours and assayed for reporter gene activity. (B) Docking orientation of 5-hydroxy-7-methoxyflavone and pinocembrin into mouse AhR-LBD binding pocket (ICM v3.5-1n, Molsoft). The protein backbone is displayed as ribbon and colored by secondary structure. The residues are displayed as sticks and colored by atom type with the carbon atoms in green. The ligands are displayed as sticks, colored by atom type with carbon atoms in yellow. H-bonds are represented as black dashed lines. (C) Western blot showing the expression of AhR in the mouse Hepa1 derivative cell line C12, which has a significantly decreased abundance of AhR, and the C12+AhR, wildtype AhR re-expressing cells. The blot shows 30 µg of whole cell lysate from each line (upper panel). Semi-quantitative RT-PCR for AhR target genes following exposure to vehicle (0.1% DMSO), TCDD (1 nM), 5-hydroxy-7-methoxyflavone (20 µM) and pinocembrin (20 µM) for 18 hours. GAPDH expression control was used to indicate similar amount of RNA usage (lower panel). CYP1A1 (Cytochrome p450 1A1), and NADPH-QO (NADPH quinone-oxidoreductase). PCR cycle numbers are indicated.

Figure 5

Nuclear localization of AhR in Hepa1 cells. Cells were treated with the indicated compounds for 60 minutes, fixed and then immunostained with AhR followed by fluoresceinisothiocyanate(FITC)-conjugated secondary antibody. Cells were stained by 4,6-diamino-2-phenylindole (DAPI) to visualize nucleus and were imaged on a Zeiss Axiovert S100TV microscope.