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.

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.

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.

Quantitative structure-activity relationship for estrogenic flavonoids from Psoralea corylifolia.

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 α ligand binding domain (hERα-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 hERα-LBD as affinity ligands, except for corylin. Then, molecular modeling was conducted to explore the binding modes between hERα-LBD and flavonoids. All the tested compounds fit into the hydrophobic binding pocket of hERα-LBD. The hydrophobic and hydrogen-bonding interactions are dominant forces to stabilize the flavonoids-hERα-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.

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.