Tianzhu Guan

In vitro and in silico assessment of the structure-dependent binding of bisphenol analogues to glucocorticoid receptor

Widespread use of bisphenol A (BPA) and other bisphenol analogues has attracted increasing attention for their potential adverse effects. As environmental endocrine-disrupting compounds (EDCs), bisphenols (BPs) may activate a variety of nuclear receptors, including glucocorticoid receptor (GR). In this work, the binding of 11 BPs to GR was investigated by fluorescence polarization (FP) assay in combination with molecular dynamics simulations. The human glucocorticoid receptor was prepared as a soluble recombinant protein. A fluorescein-labeled dexamethasone derivative (Dex-fl) was employed as tracer. Competitive displacement of Dex-fl from GR by BPs showed that the binding affinities of bisphenol analogues were largely dependent on their characteristic functional groups. In order to further understand the relationship between BPs structures and their GR-mediated activities, molecular docking was utilized to explore the binding modes at the atomic level. The results confirmed that structural variations of bisphenol analogues contributed to different interactions of BPs with GR, potentially causing distinct toxic effects. Comparison of the calculated binding energies vs. experimental binding affinities yielded a good correlation (R2 = 0.8266), which might be helpful for the design of environmentally benign materials with reduced toxicities. In addition, the established FP assay based on GR exhibited the potential to offer an alternative to traditional methods for the detection of bisphenols.

Binding interactions of halogenated bisphenol A with mouse PPARα: In vitro investigation and molecular dynamics simulation

The binding of bisphenol A (BPA) and its halogenated derivatives (halogenated BPAs) to mouse peroxisome proliferator-activated receptor α ligand binding domain (mPPARα-LBD) was examined by a combination of in vitro investigation and in silico simulation. Fluorescence polarization (FP) assay showed that halogenated BPAs could bind to mPPARα-LBD* as the affinity ligands. The calculated electrostatic potential (ESP) illustrated the different charge distributions of halogenated BPAs with altered halogenation patterns. As electron-attracting substituents, halogens decrease the positive electrostatic potential and thereby have a significant influence on the electrostatic interactions of halogenated BPAs with mPPARα-LBD*. The docking results elucidated that hydrophobic and hydrogen-bonding interactions may also contribute to stabilize the binding of the halogenated BPAs to their receptor molecule. Comparison of the calculated binding energies with the experimentally determined affinities yielded a good correlation (R2=0.6659) that could provide a rational basis for designing environmentally benign chemicals with reduced toxicities. This work can potentially be used for preliminary screening of halogenated BPAs.

Estrogenicity of halogenated bisphenol A: in vitro and in silico investigations

The binding interactions of bisphenol A (BPA) and its halogenated derivatives (halogenated BPAs) to human estrogen receptor α ligand binding domain (hERα-LBD) was investigated using a combined in vitro and in silico approach. First, the recombinant hERα-LBD was prepared as a soluble protein in Escherichia coli BL21(DE3)pLysS. A native fluorescent phytoestrogen, coumestrol, was employed as tracer for the fluorescence polarization assay. The results of the in vitro binding assay showed that bisphenol compounds could bind to hERα-LBD as the affinity ligands. All the tested halogenated BPAs exhibited weaker receptor binding than BPA, which might be explained by the steric effect of substituents. Molecular docking studies elucidated that the halogenated BPAs adopted different conformations in the flexible hydrophobic ligand binding pocket (LBP), which is mainly dependent on their distinct halogenation patterns. The compounds with halogen substituents on the phenolic rings and on the bridging alkyl moiety acted as agonists and antagonists for hERα, respectively. Interestingly, all the compounds in the agonist conformation of hERα formed a hydrogen bond with His524, while the compounds in the antagonist conformation formed a hydrogen bond with Thr347. These docking results suggested a pivotal role of His524/Thr347 in maintaining the hERα structure in the biologically active agonist/antagonist conformation. Comparison of the calculated binding energies vs. experimental binding affinities yielded a good correlation, which might be applicable for the structure-based design of novel bisphenol compounds with reduced toxicities and for environmental risk assessment. In addition, based on hERα-LBD as a recognition element, the proposed fluorescence polarization assay may offer an alternative to chromatographic techniques for the multi-residue determination of bisphenol compounds.

In vitro and in silico perspectives on estrogenicity of tanshinones from Salvia miltiorrhiza

This work aims to investigate the structure-activity relationship for binding and activation of human estrogen receptor α ligand binding domain (hERα-LBD) with tanshinones by a combination of in vitro and in silico approaches. The recombinant hERα-LBD was expressed in E. coli strain. The direct binding interactions of tanshinones with hERα-LBD and their ERα agonistic potency were investigated by fluorescence polarization (FP) and reporter gene assays, respectively. FP assay suggested that the tested tanshinones can bind to hERα-LBD as affinity ligands. Tanshinones acted as agonists of hERα as demonstrated by transactivation of estrogen response element (ERE) in transiently transfected MCF-7 cells and by molecular docking of these compounds into the hydrophobic binding pocket of hERα-LBD. Interestingly, comparison of the calculated binding energies versus Connolly solvent-excluded volume and experimental binding affinities showed a good correlation. This work may provide insight into chemical and pharmacological characterization of novel bioactive compounds from Salvia miltiorrhiza.

Estrogenic properties of coumarins and meroterpene from the fruits of Cullen corylifolium: Experimental and computational studies

Coumarins and meroterpene from the fruits of Cullen corylifolium were evaluated for their ability to bind and activate human estrogen receptor α (hERα) by a combination of in vitro studies and molecular dynamics simulations. The recombinant hERα ligand binding domain (hERα-LBD) was produced in BL21 (DE3)pLysS and the fluorescence polarization (FP) assay was performed to determine the binding affinities of coumarins and meroterpene with receptor protein. These compounds displayed distinct binding potency toward hERα-LBD, generally increased with their increasing molecular length and Connolly solvent-excluded volume (CSEV). In an estrogen response element-luciferase (ERE-Luc) reporter gene assay, coumarins and meroterpene acted as agonists of human estrogen receptor α. Subsequently, molecular docking was conducted to elucidate the molecular mechanism behind their agonistic activities. Coumarins and meroterpene adopted an agonist conformation within the cavity of hERα-LBD. The hydrophobic and hydrogen-bonding interactions were dominant forces to stabilize their binding. The structure-activity relationship analysis suggested that the presence of hydroxyl groups and prenyl group were crucial for possessing estrogenic activities. Comparison of the calculated binding energies with the determined binding affinities yielded a good correlation (R2 = 0.9727). In conclusion, molecular modeling techniques can potentially be applied for in silico screening of selective estrogen receptor modulators (SERMs) from undescribed compounds.

Quantitative structure-activity relationship for estrogenic flavonoids from Psoralea corylifolia

Graphical abstract Figure. No Caption available. HighlightsThe structure‐activity relationship for estrogenic flavonoids was investigated.Flavonoids exhibited dose‐dependent binding to human estrogen receptor &agr;.Hydrophobic and hydrogen‐bonding interactions are dominant forces to stabilize the binding.Molecular docking showed potential for predicting affinities of undescribed compounds. ABSTRACT A combination of in vitro and in silico approaches was employed to investigate the estrogenic activities of flavonoid compounds from Psoralea corylifolia. In order to develop fluorescence polarization (FP) assay for flavonoids, a soluble recombinant protein human estrogen receptor &agr; ligand binding domain (hER&agr;‐LBD) was produced in Escherichia coli strain. The competition binding experiment was performed by using coumestrol (CS) as a tracer. The result of FP assay suggested that the tested flavonoids can bind to hER&agr;‐LBD as affinity ligands, except for corylin. Then, molecular modeling was conducted to explore the binding modes between hER&agr;‐LBD and flavonoids. All the tested compounds fit into the hydrophobic binding pocket of hER&agr;‐LBD. The hydrophobic and hydrogen‐bonding interactions are dominant forces to stabilize the flavonoids‐hER&agr;‐LBD binding. It can be speculated from molecular docking study that the hydroxyl groups and prenyl group are essential for flavonoid compounds to possess estrogenic activities. Both methylation of hydroxyl group and cyclization of prenyl group significantly diminish the estrogenic potency of flavonoids. Furthermore, quantitative structure‐activity relationship (QSAR) analysis was performed by the calculated binding energies of flavonoids coupled with their determined binding affinities. Comparison between the docking scores and the pIC50 values yields an R‐squared value of 0.9722, indicating that the estrogenic potency of flavonoids is structure‐dependent. In conclusion, molecular docking can potentially be applied for predicting the receptor‐binding properties of undescribed compounds based on their molecular structure.

Computational and experimental characterization of estrogenic activities of 20(S, R)-protopanaxadiol and 20(S, R)-protopanaxatriol

Background As the main metabolites of ginsenosides, 20(S, R)-protopanaxadiol [PPD(S, R)] and 20(S, R)-protopanaxatriol [PPT(S, R)] are the structural basis response to a series of pharmacological effects of their parent components. Although the estrogenicity of several ginsenosides has been confirmed, however, the underlying mechanisms of their estrogenic effects are still largely unclear. In this work, PPD(S, R) and PPT(S, R) were assessed for their ability to bind and activate human estrogen receptor α (hERα) by a combination of in vitro and in silico analysis. Methods The recombinant hERα ligand-binding domain (hERα-LBD) was expressed in E. coli strain. The direct binding interactions of ginsenosides with hERα-LBD and their ERα agonistic potency were investigated by fluorescence polarization and reporter gene assays, respectively. Then, molecular dynamics simulations were carried out to simulate the binding modes between ginsenosides and hERα-LBD to reveal the structural basis for their agonist activities toward receptor. Results Fluorescence polarization assay revealed that PPD(S, R) and PPT(S, R) could bind to hERα-LBD with moderate affinities. In the dual luciferase reporter assay using transiently transfected MCF-7 cells, PPD(S, R) and PPT(S, R) acted as agonists of hERα. Molecular docking results showed that these ginsenosides adopted an agonist conformation in the flexible hydrophobic ligand-binding pocket. The stereostructure of C-20 hydroxyl group and the presence of C-6 hydroxyl group exerted significant influence on the hydrogen bond network and steric hindrance, respectively. Conclusion This work may provide insight into the chemical and pharmacological screening of novel therapeutic agents from ginsenosides.

Estrogen receptor-based multi-residue screening of bisphenol compounds in urine

Human exposure to bisphenol compounds (BPs) has been implicated in the development of several chronic diseases. Instead of exploiting the traditional methods for determination of BPs, this work confirms that the human estrogen receptor α ligand binding domain (hERα‐LBD) is a powerful recognition element that can be used to monitor multi‐residue of BPs in urine samples by fluorescence polarization (FP) assay. Test parameters were optimized for the best performance. Under the optimal conditions, the IC50 values of BPs are in the range of 0.04–1.61 μg mL−1. Recovery experiments were then performed to assess the accuracy and precision of the established method. The results detected by FP assay show good agreements with that of liquid chromatography–tandem mass spectrometry method with a fit of R2 = 0.9372 and 0.9640 for BPE and BPAP, respectively. A computational methodology, ligand‐based pharmacophore model, was also employed to further explore the broad‐specific of tested compounds. It was found that the two hydrogen bond acceptor features and one hydrophobic aliphatic feature were essential for the corresponding cross‐reactivity results from the FP assay. All these results suggest that the established method can be successfully applied to monitor the occurrence of BPs in urine.

Estrogen receptor-based fluorescence polarization assay for bisphenol analogues and molecular modeling study of their complexation mechanism

A fluorescence polarization (FP) assay based on estrogen receptor was developed for the determination of bisphenol compounds (BPs). The human estrogen receptor α ligand binding domain (hERα-LBD) and coumestrol were employed as recognition element and fluorescent probe, respectively. Competitive displacement of tracer from receptor suggested that BPs exhibited dose-dependent binding to hERα-LBD. In order to elucidate the structural basis for the interaction between BPs and hERα-LBD, molecular dynamics simulations were performed to explore their complexation mechanism. The docked bisphenol compounds adopted agonist/antagonist conformations with varying positions and orientations in the hydrophobic binding pocket, depending on their structural characteristics of bridging moieties. Interestingly, the calculated binding energies were generally correlated with the experimentally measured affinities, indicating a potential advantage of the molecular modeling approach in predicting the binding potencies of putative ligands. Considering that the real samples may contain more than one BP, the established FP assay can potentially be used as a pre-screening method to determine the total amounts of bisphenol compounds.

Fluorescence polarization assay for the simultaneous determination of bisphenol A, bisphenol F and their diglycidyl ethers in canned tuna

ABSTRACT This work developed a fluorescence polarization (FP) assay for simultaneous monitoring BPA, BPF, BADGE, and BFDGE in canned tuna. The assay was based on the competitive binding between bisphenol analogues (BPs) and dexamethasone fluorescein (Dex-fl) for mouse peroxisome proliferator-activated receptor α ligand binding domain (mPPARα-LBD*). The soluble form of mPPARα-LBD* was expressed in Escherichia coli strain Rosetta (DE3), as a recognition element on detection of BPs. Under optimized conditions, four bisphenol analytes were detected at concentrations corresponding to the specific migration limits (SMLs) as required by the European Union. The FP assay showed IC50 values of 2.80, 6.51, 1.72, and 4.28 mg L−1 with a limit of detection of 0.35, 0.08, 0.10, and 0.49 mg L−1 for BPA, BPF, BADGE, and BFDGE, respectively. The analysis of spiked canned tuna samples showed the acceptable recoveries ranged from 87.9% to 77.3%, with variation coefficients ranging from 9.0% to 15.8%. With the high sensitivity and wide-range affinities to BPs, the developed assay based on mPPARα-LBD* exhibited the potential to be a screening assay for fast detection of BPs in canned tuna.