Identification of aryl hydrocarbon receptor allosteric antagonists from clinically approved drugs.

The human aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor, plays a pivotal role in a diverse array of pathways in biological and pathophysiological events. This position AhR as a promising target for both carcinogenesis and antitumor strategies. In this study we utilized computational modeling to screen and identify FDA-approved drugs binding to the allosteric site between α2 of bHLH and PAS-A domains of AhR, with the aim of inhibiting its canonical pathway activity. Our findings indicated that nilotinib effectively fits into the allosteric pocket and forms interactions with crucial residues F82, Y76, and Y137. Binding free energy value of nilotinib is the lowest among top hits and maintains stable within its pocket throughout entire (MD) simulations time. Nilotinib has also substantial interactions with F295 and Q383 when it binds to orthosteric site and activate AhR. Surprisingly, it does not influence AhR nuclear translocation in the presence of AhR agonists; instead, it hinders the formation of the functional AhR-ARNT-DNA heterodimer assembly, preventing the upregulation of regulated enzymes like CYP1A1. Importantly, nilotinib exhibits a dual impact on AhR, modulating AhR activity via the PAS-B domain and working as a noncompetitive allosteric antagonist capable of blocking the canonical AhR signaling pathway in the presence of potent AhR agonists. These findings open a new avenue for the repositioning of nilotinib beyond its current application in diverse diseases mediated via AhR.

Discovery of 4'-trifluoromethylchalcones as novel, potent and selective hCYP1B1 inhibitors without concomitant AhR activation.

Human cytochrome P450 1B1 (hCYP1B1), an extrahepatic cytochrome P450 enzyme over-expressed in various tumors, has been validated as a promising target for preventing and treating cancers. Herein, two series of chalcone derivatives were synthesized to discover potent hCYP1B1 inhibitors without AhR agonist effect. Structure-activity relationship (SAR) studies demonstrated that 4'-trifluoromethyl on the B-ring strongly enhanced the anti-hCYP1B1 effects, identifying A9 as a promising lead compound. Further SAR analysis on A9 derivatives (modified A-ring of 4'-trifluoromethylchalcone) showed that introducing 2-methoxyl improved the anti-hCYP1B1 effect and selectivity, while introducing a methoxyl at the C-4 site was beneficial for avoiding AhR activation. Ultimately, five 4'-trifluoromethyl chalcones were identified as potent hCYP1B1 inhibitors (IC50 < 10 nM), while B18 exhibits the most potent anti-hCYP1B1 effect (IC50 = 3.6 nM), suitable metabolic stability and good cell-permeability. B18 also acted as an AhR antagonist and could down-regulate hCYP1B1 in living systems. Mechanistic studies showed that B18 potently inhibited hCYP1B1 in a competitive inhibition manner (Ki = 3.92 nM), while docking simulations revealed that B18 could tightly bind to the catalytic cavity of hCYP1B1 mainly via hydrophobic and hydrogen-bonding interactions. Furthermore, B18 could potently inhibit hCYP1B1 in living cells and showed remarkable anti-migration ability on MFC-7 cells. Taken together, this study deciphered the SARs of chalcones as hCYP1B1 inhibitors and provided several potent hCYP1B1 inhibitors as promising candidates for the development of more efficacious anti-migration agents.

Molecular dynamics simulations depict structural motions of the whole human aryl hydrocarbon receptor influencing its binding of ligands and HSP90.

The aryl hydrocarbon receptor (AhR) has broad biological functions when its ligands activate it; the non-binding interactions with AhR have not been fully elucidated due to the absence of a complete tridimensional (3D) structure. Therefore, utilization of the whole 3D structure from Homo sapiens AhR by in silico studies will allow us to better study and analyze the binding mode of its full and partial agonists, and antagonists, as well as its interaction with the HSP90 chaperone. The 3D AhR structure was obtained from I-TASSER and subjected to molecular dynamics (MD) simulations to obtain different structural conformations and determine the most populated AhR conformer by clustering analyses. The AhR-3D structures selected from MD simulations and those from clustering analyses were used to achieve docking studies with some of its ligands and protein-protein docking with HSP90. Once the AhR-3D structure was built, its Ramachandran maps and energy showed a well-qualified 3D model. MD simulations showed that the per-Arnt-Sim homology (PAS) PAS A, PAS B, and Q domains underwent conformational changes, identifying the conformation when agonists were binding also, and HSP90 was binding near the PAS A, PAS B, and Q domains. However, when antagonists are binding, HSP90 does not bind near the PAS A, PAS B, and Q domains. These studies show that the complex agonist-AhR-HSP90 can be formed, but this complex is not formed when an antagonist is binding. Knowing the conformations when the ligands bind to AHR and the behavior of HSP90 allows for an understanding of its activity.Communicated by Ramaswamy H. Sarma.

The Therapeutic Potential of Urolithin A for Cancer Treatment and Prevention.

BACKGROUND: Urolithin A is the metabolite of natural polyphenol ellagic acid and ellagitannins generated by gut microbiota. Urolithin A is better absorbed in the gastrointestinal tract than its parent substances. Thus, the variable effects of ellagitannin-reach food (like pomegranate fruit, walnuts, tea, and others) on people's health might be linked with the differences in individual microbiota content. Urolithin A possesses various anti-inflammatory and anti-cancer effects, as shown by in vivo and in vitro studies. OBJECTIVES: In the current review, we consider anti-inflammatory and direct anti-cancer urolithin A effects as well as their molecular mechanisms, which might be the basement of clinical trials, estimating urolithin A anti-cancer effects. CONCLUSION: Urolithin A attenuated the pro-inflammatory factors production (IL-6, IL-1ß, NOS2 and others) in vitro studies. Oral urolithin A treatment caused prominent anti-cancer and anti-inflammatory action in various in vivo studies, including colitis rat model, carrageenan-induced paw edema mice model, models of pancreatic cancer, and models of obesity. The main molecular mechanisms of these effects might be the modulation of aryl hydrocarbon receptors, which antagonism may lead to decreasing of chronic inflammation. Other primary targets of urolithin A might be the processes of protein phosphorylation (for instance, it decreases the phosphorylation of protein kinase B) and p53 stabilization. Anti-inflammatory effects of urolithin A can be reached in physiologically relevant concentrations. This might be of vital importance for preventing immune suppression associated with chronic inflammation in cancer. Considering the favorable urolithin A safety profile, it is a promising compound for cancer treatment and prevention.

AhR Antagonist Promotes Differentiation of Papillary Thyroid Cancer via Regulating circSH2B3/miR-4640-5P/IGF2BP2 Axis.

Abnormally high expression of aryl hydrocarbon receptor (AhR) has been implicated in dedifferentiation of radioiodine-refractory papillary thyroid cancer (RR-PTC). This study aimed to evaluate the differentiation effect of AhR antagonist in PTC, and to explore the potential mechanism of it. Results showed that AhR antagonists promoted differentiation of PTC, as shown as increase in 125I uptake and Na/I symporter (NIS) expression level. CircRNA microarray in K1 cells treated with StemRegenin 1(SR1) revealed that hsa_circ_0006741 (circSH2B3) was down-regulated in SR1 treated K1 cells. Downregulation of circSH2B3 increased 125I uptake and NIS expression levels. CircSH2B3 acted as an endogenous sponge of hsa-miR-4640-5p and modulated IGF2BP2 expression. IGF2BP2 overexpression induced dedifferentiation of PTC, while silencing IGF2BP2 accelerated differentiation of PTC cells. Rescue studies showed that the dedifferentiation activity of AhR was modulated by the circSH2B3/miR-4640-5p/IGF2BP2 axis. Our findings confirmed for the first time that AhR antagonists promote differentiation of PTC via inhibiting the circSH2B3/miR-4640-5p/IGF2BP2 axis, offering a novel therapeutic approach and a potential marker for differentiation of PTC.

Elucidating the aryl hydrocarbon receptor antagonism from a chemical-structural perspective.

The aryl hydrocarbon receptor (AhR) plays an important role in several biological processes such as reproduction, immunity and homoeostasis. However, little is known on the chemical-structural and physicochemical features that influence the activity of AhR antagonistic modulators. In the present report, in vitro AhR antagonistic activity evaluations, based on a chemical-activated luciferase gene expression (AhR-CALUX) bioassay, and an extensive literature review were performed with the aim of constructing a structurally diverse database of contaminants and potentially toxic chemicals. Subsequently, QSAR models based on Linear Discriminant Analysis and Logistic Regression, as well as two toxicophoric hypotheses were proposed to model the AhR antagonistic activity of the built dataset. The QSAR models were rigorously validated yielding satisfactory performance for all classification parameters. Likewise, the toxicophoric hypotheses were validated using a diverse set of 350 decoys, demonstrating adequate robustness and predictive power. Chemical interpretations of both the QSAR and toxicophoric models suggested that hydrophobic constraints, the presence of aromatic rings and electron-acceptor moieties are critical for the AhR antagonism. Therefore, it is hoped that the deductions obtained in the present study will contribute to elucidate further on the structural and physicochemical factors influencing the AhR antagonistic activity of chemical compounds.

Urolithin A Is a Dietary Microbiota-Derived Human Aryl Hydrocarbon Receptor Antagonist.

Urolithins (e.g., UroA and B) are gut microbiota-derived metabolites of the natural polyphenol ellagic acid. Urolithins are associated with various health benefits, including attenuation of inflammatory signaling, anti-cancer effects and repression of lipid accumulation. The molecular mechanisms underlying the beneficial effects of urolithins remain unclear. We hypothesize that some of the human health benefits of urolithins are mediated through the aryl hydrocarbon receptor (AHR). Utilizing a cell-based reporter system, we tested urolithins for the capacity to modulate AHR activity. Cytochrome P450 1A1 (CYP1A1) mRNA levels were assessed by real-time quantitative polymerase chain reaction. Competitive ligand binding assays were performed to determine whether UroA is a direct ligand for the AHR. Subcellular AHR protein levels were examined utilizing immunoblotting analysis. AHR expression was repressed in Caco-2 cells by siRNA transfection to investigate AHR-dependency. UroA and B were able to antagonize 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced AHR-mediated transcriptional activity. Furthermore, UroA and B attenuated TCDD-mediated stimulation of CYP1A1 mRNA levels. In addition, competitive ligand binding assays characterized UroA as a direct AHR ligand. Consistent with other AHR antagonists, UroA failed to induce AHR retention in the nucleus. AHR is necessary for UroA-mediated attenuation of cytokine-induced interleukin 6 (IL6) and prostaglandin-endoperoxide synthase 2 (PTGS2) expression in Caco-2 cells. Here we identified UroA as the first dietary-derived human selective AHR antagonist produced by the gut microbiota through multi-step metabolism. Furthermore, previously reported anti-inflammatory activity of UroA may at least in part be mediated through AHR.

Towards Resolving the Pro- and Anti-Tumor Effects of the Aryl Hydrocarbon Receptor.

We have postulated that the aryl hydrocarbon receptor (AHR) drives the later, more lethal stages of some cancers when chronically activated by endogenous ligands. However, other studies have suggested that, under some circumstances, the AHR can oppose tumor aggression. Resolving this apparent contradiction is critical to the design of AHR-targeted cancer therapeutics. Molecular (siRNA, shRNA, AHR repressor, CRISPR-Cas9) and pharmacological (AHR inhibitors) approaches were used to confirm the hypothesis that AHR inhibition reduces human cancer cell invasion (irregular colony growth in 3D Matrigel cultures and Boyden chambers), migration (scratch wound assay) and metastasis (human cancer cell xenografts in zebrafish). Furthermore, these assays were used for a head-to-head comparison between AHR antagonists and agonists. AHR inhibition or knockdown/knockout consistently reduced human ER−/PR−/Her2− and inflammatory breast cancer cell invasion, migration, and metastasis. This was associated with a decrease in invasion-associated genes (e.g., Fibronectin, VCAM1, Thrombospondin, MMP1) and an increase in CDH1/E-cadherin, previously associated with decreased tumor aggression. Paradoxically, AHR agonists (2,3,7,8-tetrachlorodibenzo-p-dioxin and/or 3,3′-diindolylmethane) similarly inhibited irregular colony formation in Matrigel and blocked metastasis in vivo but accelerated migration. These data demonstrate the complexity of modulating AHR activity in cancer while suggesting that AHR inhibitors, and, under some circumstances, AHR agonists, may be useful as cancer therapeutics.

In vivo effects of the pure aryl hydrocarbon receptor antagonist GNF-351 after oral administration are limited to the gastrointestinal tract.

BACKGROUND AND PURPOSE: GNF-351 is a potent aryl hydrocarbon receptor (AHR) antagonist that inhibits dioxin response element-dependent and independent activities. Here, the absorption, metabolism and in vivo AHR antagonist activity of GNF-351 were investigated. EXPERIMENTAL APPROACH: LC-MS metabolomics was used to analyse GNF-351 metabolism in vitro and in vivo. Recombinant drug-metabolizing enzymes were employed to determine the enzymes involved in GNF-351 metabolism. Analysis of target AHR genes was performed to investigate the inhibitory effects of GNF-351 towards AHR activation. KEY RESULTS: Several phase I metabolites were generated after GNF-351 was incubated with microsomes from human or mouse liver and intestine, including two oxidized GNF-351 and one tri-demethylated GNF-351. Poor absorption from the intestine resulted in no detectable levels of GNF-351 in mouse serum (0-6 h) and urine (24 h) and almost all GNF-351 was found in the faeces after 24 h. Analysis of faeces further revealed all the in vitro phase I metabolites. Novel metabolites were detected, including one di-oxidized GNF-351, two oxidized and tri-demethylated GNF-351, one dehydrogenated product of oxidized GNF-351, and one sulfation product of di-oxidized GNF-351. Cytochromes P450 were demonstrated to be the major enzymes involved in metabolism of GNF-351. After oral administration to mice, GNF-351 readily inhibited ß-naphthoflavone-induced AHR activation in ileum and colon, but not that in the liver. CONCLUSION AND IMPLICATIONS: While poor absorption and extensive metabolism after oral administration limited the in vivo effects of the pure AHR antagonist GNF-351 in liver, it could be used to inhibit AHR activation in intestine and colon.