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The aryl hydrocarbon receptor (AHR) mediates the toxic effects of dioxin-like compounds, such as 2,3,7,8-tetracholordibezno-p-dioxin (TCDD). Sequence and structural features of AHR can underlie species- and population-specific differences in its affinity for TCDD and other agonists. While most vertebrates possess at least one AHR that binds TCDD tightly, all characterized amphibian AHRs bind TCDD with very low affinity. Our previous analyses of AHRs from Xenopus laevis (a frog; order Anura) and Ambystoma mexicanum (a salamander; order Urodela) identified three key amino acid residues in the ligand binding domain (LBD) that underlie low affinity binding. In X. laevis AHR1β, these are A354, A370, and N325. In studying the AHR of the caecilian Gymnopis multiplicata, we sought to determine if AHRs from all three amphibian orders share the low affinity phenotype. G. multiplicata represents Order Apoda (caecilians), a clade of legless amphibians that diverged early from the class’s common lineage, prior to the split between frogs and salamanders. cDNA was isolated by RT-PCR from tissue provided by the Museum of Vertebrate Zoology at the University of California, Berkeley. The encoded protein (92.7 kDa) is monophyletic with vertebrate AHR1s, sharing 59% identity with X. laevis AHR1β and 63% identity with A. mexicanum AHR. An LBD homology model suggests that the basic architecture of the caecilian AHR closely resembles other species, and the LBD sequence includes the three signature residues of low TCDD affinity. We measured the TCDD responsiveness with transactivation assays employing pGudLuc6.1, a luciferase reporter construct driven by the mouse CYP1A1 enhancer region. For G. multiplicata AHR, the EC50 for reporter gene induction by TCDD was 28.46 nM, similar to X. laevis AHR1β (26.84 nM) and dramatically less than a chimeric frog AHR containing the mouse LBD (0.2 nM). Taken together, low TCDD responsiveness and sequence conservation with the frog and salamander AHRs suggest that this caecilian AHR also binds TCDD with low affinity. We predict G. multiplicata is similarly resistant to the toxic effects of TCDD and other xenobiotic AHR agonists. (NIH: R15ES011130, P42ES007381, R01ES007685)

The aryl hydrocarbon receptor (AHR) plays pleiotropic roles in the development and physiology of vertebrates in conjunction with xenobiotic and endogenous ligands. It is best known for mediating the toxic effects of dioxin-like pollutants such as 2,3,7,8-tetracholordibenzo-p-dioxin (TCDD). While most vertebrates possess at least one AHR that binds TCDD tightly, amphibian AHRs bind TCDD with very low affinity. Previous analyses of AHRs fromXenopus laevis(a frog; order Anura) andAmbystoma mexicanum(a salamander; order Urodela) identified three amino acid residues in the ligand-binding domain (LBD) that underlie low-affinity binding. InX. laevisAHR1β, these are A354, A370, and N325. Here we extend the analysis of amphibian AHRs to the caecilianGymnopis multiplicata, representing the remaining extant amphibian order, Apoda.G. multiplicataAHR groups with the monophyletic vertebrate AHR/AHR1 clade. The LBD includes all three signature residues of low TCDD affinity, and a structural homology model suggests that its architecture closely resembles those of other amphibians. In transactivation assays, the EC50 for reporter gene induction by TCDD was 17.17 nM, comparable toX. laevisAhR1β (26.23 nM) andAmbystomaAHR (34.09 nM) and dramatically higher than mouse AhR (0.13 nM), a trend generally reflected in direct measures of TCDD binding. These shared properties distinguish amphibian AHRs from the high-affinity proteins typical of both more ancient vertebrate groups (teleost fish) and those that appeared more recently (tetrapods). We suggest that AHRs with low TCDD affinity represent a basal characteristic that evolved in a common ancestor of all three extant amphibian groups.Research HighlightsA caecilian aryl hydrocarbon receptor exhibits low dioxin binding and sensitivity.The protein’s ligand-binding domain resembles frog and salamander AHRs in structure and function.AHR with low dioxin affinity likely evolved in a common ancestor of all three extant amphibian groups.Graphical Abstract

The Ah receptor (AhR) is a ligand-dependent transcription factor belonging to the basic helix-loop-helix Per-Arnt-Sim (bHLH-PAS) superfamily. Binding to and activation of the AhR by a variety of chemicals results in the induction of expression of diverse genes and production of a broad spectrum of biological and toxic effects. The AhR also plays important roles in several physiological responses, which has led it to become a novel target for the development of therapeutic drugs. Differences in the interactions of various ligands within the AhR ligand binding domain (LBD) may contribute to differential modulation of AhR functionality. We combined computational and experimental analyses to investigate the binding modes of a group of chemicals representative of major classes of AhR ligands. On the basis of a novel computational approach for molecular docking to the homology model of the AhR LBD that includes the receptor flexibility, we predicted specific residues within the AhR binding cavity that play a critical role in binding of three distinct groups of chemicals. The prediction was validated by site-directed mutagenesis and evaluation of the relative ligand binding affinities for the mutant AhRs. These results provide an avenue for understanding ligand modulation of the AhR functionality and for rational drug design.

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that modulates gene expression following its binding and activation by structurally diverse chemicals. Species differences in AhR functionality have been observed, with the mouse AhR (mAhR) and human AhR (hAhR) exhibiting significant differences in ligand binding, coactivator recruitment, gene expression and response. While the AhR agonist indirubin (IR) is a more potent activator of hAhR-dependent gene expression than the prototypical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), it is a significantly less potent activator of the mAhR. DNA binding analysis confirmed the greater potency/efficacy of IR in stimulating transformation/DNA binding of the hAhR in vitro and domain-swapping experiments demonstrated that the enhanced response to IR was primarily due to the hAhR ligand binding domain (LBD). Site-directed mutagenesis and functional analysis studies revealed that mutation of H326 and A349 in the mAhR LBD to the corresponding residues in the hAhR LBD significantly increased the potency of IR. Since these mutations had no significant effect on ligand binding, these residues likely contribute to an enhanced efficiency of transformation/DNA binding by IR-bound hAhR. Molecular docking to mAhR LBD homology models further elucidated the different roles of the A375V mutation in TCDD and IR binding, as revealed by [3H]TCDD competitive binding results. These results demonstrate the differential binding of structurally diverse ligands within the LBD of a given AhR and confirm that amino acid differences within the LBD of AhRs contribute to significant species differences in ligand response.

Natural products offer unexplored molecular frameworks for the development of chemical leads and innovative drugs. However, the structural complexity of natural products compared with synthetic drug-like molecules often limits the scaffold hopping potential of natural-product-inspired molecular design. Here we introduce a holistic molecular representation incorporating pharmacophore and shape patterns, which facilitates scaffold hopping from natural products to isofunctional synthetic compounds. This computational approach captures simultaneously the partial charge, atom distributions and molecular shape. In a prospective application, we use four natural cannabinoids as queries in a chemical database search for novel synthetic modulators of human cannabinoid receptors. Of the synthetic compounds selected by the new method, 35% are experimentally confirmed as active. These cannabinoid receptor modulators are structurally less complex than their respective natural product templates. The results of this study validate this holistic molecular representation for hit and lead finding in drug discovery., Communications Chemistry, 1 (1)

The Pregnane X Receptor (PXR) is a ligand-activated transcription factor belonging to the nuclear receptor family. PXR can bind diverse drugs and environmental toxicants with different binding modes, making it an intriguing target for drug discovery. Here we investigated the binding mechanism of the SR12813 ligand to elucidate the significant steps, from the ligand entrance pathway into the binding cavity, to the ligand-induced conformational changes, and to the exploration of its alternative binding geometries. We used the advanced Molecular Dynamics-based methods implemented in the BiKi suite and developed specific methodological approaches to overcome the complexity induced by the buried and flexible binding cavity. The adopted methods provided a full dynamic description of the binding event and allowed rationalization of the observed multiple binding modes. These results suggest that the same approach could be exploited for the study of other binding processes with similar characteristics.

The mediator of dioxin toxicity, aryl hydrocarbon receptor (AHR), has also important physiological functions. Selective AHR modulators (SAHRMs) share some effects of dioxins, except for their marked toxicity. We recently characterised toxicologically two novel SAHRMs, prodrugs IMA-08401 and IMA-07101 in rats, demonstrating that they are far less deleterious than the most toxic AHR-agonist, TCDD. Here, we analysed the in vitro toxicity and in silico AHR binding of the respective active, deacetylated metabolites, IMA-06201 (N-ethyl-N-phenyl-5-chloro-1,2-dihydro-4-hydroxy-1-methyl-2-oxo-quinoline-3-carboxamide) and IMA-06504 (N-(4-trifluoromethylphenyl)-1,2-dihydro-4-hydroxy-5-methoxy-1-methyl-2-oxo-quinoline-3-carboxamide). In H4IIE rat hepatoma cells, IMA-06201 and IMA-06504 induced CYP1A1 with comparable potencies and efficacies to those of TCDD. They had little effect on cell viability as assessed by LDH leakage and MTT reduction assays, and were not mutagenic in the Ames test, but IMA-06504 elicited a maximally 2.7-fold increase in micronuclei. Molecular docking simulations showed that similar to TCDD, they occupy the central region of AHR ligand binding cavity. Hence, while showing low to negligible in vitro toxicity, these novel SAHRMs bind to the AHR qualitatively in a similar fashion to TCDD, and appear comparably powerful AHR agonists. Combined with our earlier results demonstrating that they seem considerably less toxic in vivo than TCDD, these compounds are thus highly interesting new SAHRMs.

Molecular modeling has given important contributions to elucidation of the main stages in the AhR signal transduction pathway. Despite the lack of experimentally determined structures of the AhR functional domains, information derived from homologous systems has been exploited for modeling their structure and interactions. Homology models of the AhR PASB domain have provided information on the binding cavity and contributed to elucidate species-specific differences in ligand binding. Molecular Docking simulations of the ligand binding process have given insights into differences in binding of diverse agonists, antagonists, and selective AhR modulators, and their application to virtual screening of large databases of compounds have allowed identification of novel AhR ligands. Recently available structural information on protein–protein and protein-DNA complexes of other bHLH-PAS systems has opened the way for modeling the AhR:ARNT dimer structure and investigating the mechanisms of AhR transformation and DNA binding. Future research directions should include simulation of the protein dynamics to obtain a more reliable description of intermolecular interactions involved in signal transmission.

Molecular docking studies can shed light into the molecular determinants of ligand binding. When no experimental structures are available, docking can also be applied to homology models. Given that the model quality, especially in the binding site, greatly affects the accuracy of docking predictions [1], development of strategies able to include protein flexibility [2] may help in the effective use of docking to homology models. In this work, ligand binding of structurally different ligands to the mouse Aryl hydrocarbon Receptor (mAhR) homology model is analyzed. Introduction AhR is a ligand-dependent transcription factor that responds to exogenous and endogenous chemicals with the induction of gene expression and production of diverse biological and toxic effects [3]. The mechanism is initiated by ligand binding to the AhR, which is present in the cytosol as a multiprotein complex, and the PAS-B domain acts as ligand binding domain (LBD). The most “classical” AhR ligands are the exogenous halogenated aromatic hydrocarbons (HAHs) (including the most toxic 2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD), polycyclic aromatic hydrocarbons (PAHs), and PAH-like chemicals; but also “non-classical” natural, endogenous and synthetic AhR ligands with diverse structure and physico-chemical characteristics have been identified [3]. Given that AhR shows different biological responses for different ligands, understanding the molecular determinants of binding could help in elucidating the mechanism of action associated to each ligand. Methods No experimental information is available for the AhR LBD but since last years the X-ray structures of many homologous PAS domains have been determined, in particular the Hypoxia-Inducible Factor 2α (HIF2α) shows nearly 30% of sequence identity with the mAhR LBD. 10 homology models of the mAhR LBD were developed using MODELLER, each one based on a different HIF2α template structure in order to describe the flexibility of the binding cavity. All the ligands and solvent molecules in the HIF2α internal cavity were maintained during the modeling and refinement stages. Docking was performed using Glide XP, that treats ligands as flexible. An ensemble docking approach based on ligand docking to the 10 LBD models was used to include receptor flexibility. Selected binding poses were subjected to Molecular Dynamics simulation (10ns–20ns). The binding free energy (ΔGbind) was calculated by the MM-GBSA approach implemented in AMBER and the most stabilizing contributions were obtained by per-residue decomposition analysis. Results & Conclusions The set of 12 ligands here analyzed is composed by both classical and non-classical AhR ligands with diverse structures and physico-chemical characteristics. Protein flexibility and plasticity were introduced in both the ensemble docking to multiple homology models and the post-docking MD simulations to obtain an accurate description of binding of such diverse chemicals within the binding cavity of the receptor. The ΔGbind analysis, performed to identify the most relevant residues for each pose stabilization, allowed to discriminate the molecular determinants for binding of the different ligands. Three main arrangements within the binding cavity were identified: hydrophobic molecules interact mainly with residues located at the bottom of the cavity (TCDD); others occupy the whole cavity and in some cases are stabilized by hydrogen bonds with residues in the middle of the cavity (FICZ); the smallest and polar ligands stay at the entrance of the cavity (leflunomide). These poses will be validated by a mutagenesis study focused on the key stabilizing residues predicted for each ligand by the computational protocol here presented 1. Bordogna A, Pandini A & Bonati L, J Comput Chem 32, 81 (2011) 2. Fan H, Irwin J & Webb B, J Chem Inf Model 2512 (2009) 3. Denison MS, Soshilov AA, He G, DeGroot DE & Zhao B, Toxicol Sci 124, 1 (2011)

Modern Molecular Modeling methods are at the core of mechanistic and computational toxicology, indeed these methods are able to analyze 3D molecular structures and their interactions in order to better understand the mechanism by which toxic compounds or drugs execute their action. Toxic effects of a molecule can be explained by the Adverse Outcome Pathway model (AOP); the molecular initiating event (MIE) can be the interaction of the molecule with a biological target, a protein for example. After the initial interaction many Key Events can link the MIE to the Adverse Outcome. It is useful to describe also the Key Event Relationship to assess the causal nature between two measurable biological events. Our focus of study is the Aryl hydrocarbon Receptor (AhR), a ligand-dependent transcription factor that responds to exogenous and endogenous chemicals with the induction of gene expression and production of diverse biological and toxic effects. Well known ligands of AhR are polychlorinated-dibenzodioxins (among them the 2,3,7,8-TCDD), dibenzofurans and biphenyls (PCB), and polycyclic aromatic hydrocarbons (PAH). The analysis of specific binding interactions of ligands within the AhR Ligand Binding Domain (LBD) would allow a deeper comprehension of the key molecular events regulating the mechanism of ligand-dependent and ligand-specific AhR activation. Aim of our current studies is to combine molecular modeling methods with experimental approaches to gain insights into the binding specificity of AhR ligands with different structures and properties