In Silico and In Vitro multiple analysis approach for screening naturally derived ligands for red seabream aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AHR) is a key ligand-dependent transcription factor that mediates the toxic effects of compounds such as dioxin. Recently, natural ligands of AHR, including flavonoids, have been attracting physiological and toxicological attention as they have been reported to regulate major biological functions such as inflammation and anti-cancer by reducing the toxic effects of dioxin. Additionally, it is known that natural AHR ligands can accumulate in wildlife tissues, such as fish. However, studies in fish have investigated only a few ligands in experimental fish species, and the AHR response of marine fish to natural AHR ligands of various other structures has not been thoroughly investigated. To explore various natural AHR ligands in marine fish, which make up the most fish, it is necessary to develop new screening methods that consider the specificity of marine fish. In this study, we investigated the response of natural ligands by constructing in vitro and in silico experimental systems using red seabream as a model species. We attempted to develop a new predictive model to screen potential ligands that can induce transcriptional activation of red seabream AHR1 and AHR2 (rsAHR1 and rsAHR2). This was achieved through multiple analyses using in silico/ in vitro data and Tox21 big data. First, we constructed an in vitro reporter gene assay of rsAHR1 and rsAHR2 and measured the response of 10 representatives natural AHR ligands in COS-7 cells. The results showed that FICZ, Genistein, Daidzein, I3C, DIM, Quercetin and Baicalin induced the transcriptional activity of rsAHR1 and rsAHR2, while Resveratrol and Retinol did not induce the transcriptional activity of rsAHR isoforms. Comparing the EC50 values of the respective compounds in rsAHR1 and rsAHR2, FICZ, Genistein, and Daidzein exhibited similar isoform responses, but I3C, Baicalin, DIM and Quercetin show the isoform-specific responses. These results suggest that natural AHR ligands have specific profiling and transcriptional activity for each rsAHR isoform. In silico analysis, we constructed homology models of the ligand binding domains (LBDs) of rsAHR1 and rsAHR2 and calculated the docking energies (U_dock values) of natural ligands with measured in vitro transcriptional activity and dioxins reported in previous studies. The results showed a significant correlation (R2=0.74(rsAHR1), R2=0.83(rsAHR2)) between docking energy and transcriptional activity (EC50) value, suggesting that the homology model of rsAHR1 and rsAHR2 can be utilized to predict the potential transactivation of ligands. To broaden the applicability of the homology model to diverse compound structures and validate the correlation with transcriptional activity, we conducted additional analyses utilizing Tox21 big data. We calculated the docking energy values for 1860 chemicals in both rsAHR1 and rsAHR2, which were tested for transcriptional activation in Tox21 data against human AHR. By comparing the U_dock energy values between 775 active compounds and 1085 inactive compounds, a significant difference (p<0.001) was observed between the U_dock energy values in the two groups, suggesting that the U_dock value can be applied to distinguish the activation of compounds. Furthermore, we observed a significant correlation (R2=0.45) between the AC50 of Tox21 database and U_dock values of human AHR model. In conclusion, we calculated equations to translate the results of an in silico prediction model for ligand screening of rsAHR1 and rsAHR2 transactivation. This ligand screening model can be a powerful tool to quantitatively estimate AHR transactivation of major marine agents to which red seabream may be exposed. The study introduces a new screening approach for potential natural AHR ligands in marine fish, based on homology model-docking energy values of rsAHR1 and rsAHR2, with implications for future agonist development and applications bridging in silico and in vitro data.

Orthogonal assay for validation of Tox21 PPARγ data and applicability to in silico prediction model.

High-throughput screening data from the Tox21 database is used for prioritizing hazardous chemicals and building in silico-based toxicity prediction models. One of the Tox21 dataset, peroxisome proliferator-activated receptor-gamma (PPARγ), a nuclear receptor superfamily, identified various endpoints in HEK293 cells. PPARγ mediates various toxic effects when its receptors are activated or inhibited by ligands such as thiazolidinedione and GW9662. In this study, an orthogonal assay was constructed to verify the effectiveness of the Tox21 PPARγ data, and the effect of highly reliable data on in silico model construction was investigated. The orthogonal assay was a reporter gene assay based on the PPARγ ligand binding domain in CV-1 cells. Only 39% of agonists and 55% of antagonists had similar responses in CV-1 and HEK293 cells. Thus, the effectiveness of Tox21 data on PPARγ may vary depending on the cell line. However, in silico PLS-DA analysis with only high-reliability data (i.e., the same response in both cell lines), yielded more accurate prediction of the activity of potential chemical ligands, despite the small number of samples. Thus, obtaining reliable chemical screening data for PPARγ through orthogonal analysis, even for only limited chemicals, supports the construction of highly predictive in silico models with improved screening efficiency.

Multi-step structure-activity relationship screening efficiently predicts diverse PPARγ antagonists.

In discovering the potential antagonist of peroxisome proliferator-activated receptor gamma (PPARγ), the structure-activity relationship (SAR) is a useful in silico method. However, it is difficult for conventional SAR approaches to predict the activities of antagonists owing to the large structural diversity of antagonistic compounds. This study provides evidence that multi-step SAR screening is applicable for predicting PPARγ antagonists by combining different complementary methodologies. We constructed three models: read-across-like SAR, docking-simulation-interpreting SAR, and deep-learning-based SAR. To provide user-customized prediction results, our multi-step SAR screening model combined the three SAR models in a stepwise manner, which subdivided them according to potential levels of the PPARγ antagonist. The read-across-like SAR, which considered specific antagonist scaffolds, revealed the highest positive predictive value (PPV). The docking-simulation-interpreting SAR, which considered the molecular surface features, revealed high statistics for the PPV and the true-positive rate (TPR). The deep-learning-based SAR showed the highest TPR at the last classification step. This multi-step SAR screening covered the antagonists of high reliability provided by a read-across-like SAR, as well as the antagonists of diverse scaffolds provided by docking-simulation-interpreting SAR and deep-learning-based SAR. Therefore, to predict PPARγ antagonists, multi-step SAR screening could be as a useful tool.

The AHR1-ARNT1 dimerization pair is a major regulator of the response to natural ligands, but not to TCDD, in the chicken.

The activation of the aryl hydrocarbon receptor (AHR) occurs through the binding of dioxin-like compounds (DLCs) or natural ligands. In this pathway, the AHR-ARNT (AHR nuclear translocator) heterodimer serves to regulate critical physiological functions, such as immune responses and the metabolism of xenobiotics. Birds have three AHR isoforms (AHR1, AHR1ß, and AHR2) and two ARNT isoforms (ARNT1 and ARNT2). However, how AHR and ARNT dimerization pair in birds regulates the AHR signaling pathway in an isoform-specific manner remains unknown. In this study, we initially sought to clarify the major chicken AHR-ARNT (ckAHR-ckARNT) pairs by estimating the mRNA tissue distributions of various ckAHR and ckARNT isoforms. Our results indicated that the ckAHR1-ckARNT1 represented the major dimerization pair in most tissues except the brain. We then measured the transactivation potencies of various ckAHR-ckARNT pairs by natural ligands and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), in in vitro reporter gene assays using COS-7 and LMH cell lines. Our results from the in vitro assays demonstrated that the ckAHR1-ckARNT1 pair was strongly activated by the five natural ligands, namely, 6-formylindolo [3,2-b]carbazole, L-kynurenin, kynurenic acid, indoxyl-3-sulfate, and 1,3,7-tribromodibenzo-p-dioxin, but not by TCDD. In in silico ligand docking simulations with ckAHR1 homology models, all the natural ligands showed a interaction pattern that was distinct from that observed with anthropogenic DLCs, including TCDD. In conclusion, our findings indicate that the ckAHR1-ckARNT1 may be the most important dimerization pair in most tissues for regulating the physiological functions driven by natural ligands, although it was less reactive to TCDD.