A Computational Approach to Evaluate the Androgenic Affinity of Iprodione, Procymidone, Vinclozolin and Their Metabolites

<div><p>Our research is aimed at devising and assessing a computational approach to evaluate the affinity of endocrine active substances (EASs) and their metabolites towards the ligand binding domain (LBD) of the androgen receptor (AR) in three distantly related species: human, rat, and zebrafish. We computed the affinity for all the selected molecules following a computational approach based on molecular modelling and docking. Three different classes of molecules with well-known endocrine activity (iprodione, procymidone, vinclozolin, and a selection of their metabolites) were evaluated. Our approach was demonstrated useful as the first step of chemical safety evaluation since ligand-target interaction is a necessary condition for exerting any biological effect. Moreover, a different sensitivity concerning AR LBD was computed for the tested species (rat being the least sensitive of the three). This evidence suggests that, in order not to over−/under-estimate the risks connected with the use of a chemical entity, further <i>in vitro</i> and/or <i>in vivo</i> tests should be carried out only after an accurate evaluation of the most suitable cellular system or animal species. The introduction of <i>in silico</i> approaches to evaluate hazard can accelerate discovery and innovation with a lower economic effort than with a fully wet strategy.</p></div

Global alignment of the selected AR LBD: the residues of the binding sites are highlighted with the following color-code: red for human AR, green for rat AR, and yellow for zebrafish AR.

<p>Global alignment of the selected AR LBD: the residues of the binding sites are highlighted with the following color-code: red for human AR, green for rat AR, and yellow for zebrafish AR.</p

Hazard evaluation pipeline for putative androgen disruptors.

<p>Step 1: database production; step 2: <i>in silico</i> binding assay; step 3 <i>in vitro</i> binding assay for the selected dataset; step 4: <i>in vitro</i> activity assays only for the high affinity molecules (positive hits); and identification of agonist (α = 1), partial agonist (1<α<0) and antagonist (α = 0) activity.</p

Molecular surface of the binding site and filling dummy atoms in the zebrafish AR LBD model, side (A) and top (B) view.

<p>Molecular surface of the binding site and filling dummy atoms in the zebrafish AR LBD model, side (A) and top (B) view.</p

Best pose of iprodione complexed with the AR LBD in each species.

<p>The ligand molecular surface is also rendered. A) Iprodione complexed with the human AR LBD, B) iprodione complexed with the rat AR LBD, and C) iprodione complexed with zebrafish AR LBD.</p

Box plot for all the calculated binding free energies.

<p>The yellow boxes represent the binding free energies of the vinclozolin and its metabolites for each species. The red boxes represent the binding free energies of iprodione and its metabolites for each species. The blue boxes represent the binding free energies of procymidone and its metabolites for each species. Outliers are marked as circles. Binding free energies of the endogenous hormones are marked with continuous lines.</p

Molecular docking validation dataset and RMSD values between the co-crystallized and the docked ligands.

<p>Molecular docking validation dataset and RMSD values between the co-crystallized and the docked ligands.</p

Experimental (from literature) and <i>in silico</i> (computed) dissociation constants for the three endogenous tested hormones.

<p>DS: Docking Score; ΔG-LM: binding free energy computed through low-mode molecular dynamics simulations; ΔG-MD: binding free energy computed through molecular docking; K_i: dissociation constant computed from molecular docking data.</p

AR LBD and binding site RMSD values, computed for α-carbons and for whole residues of the three selected receptors.

<p>AR LBD and binding site RMSD values, computed for α-carbons and for whole residues of the three selected receptors.</p

Experimental (from [27]) and <i>in silico</i> (computed) dissociation constants for the selected compounds with respect to rat AR binding site.

<p>ΔG-MD: binding free energy computed through molecular docking.</p