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. 2018 Mar;93(3):197-207.
doi: 10.1124/mol.117.108696. Epub 2018 Jan 2.

A Computational-Based Approach to Identify Estrogen Receptor α/ β Heterodimer Selective Ligands

Carlos G Coriano  1 , Fabao Liu  1 , Chelsie K Sievers  1 , Muxuan Liang  1 , Yidan Wang  1 , Yoongho Lim  1 , Menggang Yu  1 , Wei Xu  2
Affiliations

A Computational-Based Approach to Identify Estrogen Receptor α/ β Heterodimer Selective Ligands

Carlos G Coriano et al. Mol Pharmacol. 2018 Mar.

Abstract

The biologic effects of estrogens are transduced by two estrogen receptors (ERs), ERα and ERβ, which function in dimer forms. The ERα/α homodimer promotes and the ERβ/β inhibits estrogen-dependent growth of mammary epithelial cells; the functions of ERα/β heterodimers remain elusive. Using compounds that promote ERα/β heterodimerization, we have previously shown that ERα/β heterodimers appeared to inhibit tumor cell growth and migration in vitro. Further dissection of ERα/β heterodimer functions was hampered by the lack of ERα/β heterodimer-specific ligands. Herein, we report a multistep workflow to identify the selective ERα/β heterodimer-inducing compound. Phytoestrogenic compounds were first screened for ER transcriptional activity using reporter assays and ER dimerization preference using a bioluminescence resonance energy transfer assay. The top hits were subjected to in silico modeling to identify the pharmacophore that confers ERα/β heterodimer specificity. The pharmacophore encompassing seven features that are potentially important for the formation of the ERα/β heterodimer was retrieved and subsequently used for virtual screening of large chemical libraries. Four chemical compounds were identified that selectively induce ERα/β heterodimers over their respective homodimers. Such ligands will become unique tools to reveal the functional insights of ERα/β heterodimers.

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Figures

Fig. 1.
Fig. 1.
Transcriptional and ligand-binding assays of 37 flavonoid compounds. (A) T47D-KBLuc transcriptional assays showing ERE-luciferase reporter activity of 13 out of 37 flavonoid compounds from four different subclasses, revealed 13 phytoestrogenic compounds able to transcriptionally activate ER in a dose-dependent manner. The red line represents a 2-fold cutoff for positive hits. RLU, relative luciferase unit, normalized to β-gal control. Data are shown as mean ± S.D. (B) Relative ligand-binding affinity of 12 compounds to ERα or ERβ.
Fig. 2.
Fig. 2.
BRET assays in HEK293 cells show dimer selectivity of different flavonoid subclasses. (A–C) Fold change of BRET ratios when cells were treated with indicated compounds: (A) ERα/α, (B) ERβ/β, and (C) ERα/β (10 nM E2 was used as a positive control). Each compound represents an individual experiment; those that induced dimer interaction at a threshold value of P < 0.05 were considered statistically significant. Fold change is relative to the negative control DMSO. Data are shown as mean ± S.D. of three biologic replicates. *Indicates compounds that significantly induced dimerization as determined by two-way analysis of variance. (D) Western blot analysis of Flag-tagged ERβ in MCF7-Flag-ERβ cells. (E and F) Relative ATP6V0E1 and BAG1 mRNAs levels in MCF7-Flag-ERβ cells treated with indicated compounds. (G) Compound 29-induced recruitment of ERβ to the BAG1 and ATP6V0E1 promoters in MCF7-Flag-ERβ cells shown by ChIP assays. (H) The enrichment of ERα on the BAG1 and ATP6V0E1 promoters in MCF7-Flag-ERβ cells after compound 29 treatment shown by ChIP assays. *Indicates statistically significant P < 0.05, **P < 0.01.
Fig. 3.
Fig. 3.
Generation and selection of a pharmacophore hypothesis model of ERα/β heterodimer-inducing ligands. A ligand-based pharmacophore hypothesis was generated using GALAHAD. (A) Structures and bioactivity values of the training set chemicals used to generate ligand-based pharmacophore. The structures of the six lead compounds (cosmosiin, two isoflavones, four flavanones, and a flavone) identified from the cell-based assays. (B) Plot of the different criteria used to select the best model. Plot of the energy, sterics, Mol_QRY and H_Bond values for GALAHAD models with selected four ligands that contribute to the consensus feature. (A) Sterics vs. energy (B) Pharmacophore similarity vs. energy (C) Pharmacophore similarity vs. sterics. The open circle represents the ideal best scoring for each condition. The red diamond represents model 6. (C) Selected pharmacophore hypothesis GALAHAD model. GALAHAD assumes pharmacophore/shape and alignments from sets of ligand molecules, to generate a pharmacophore hypothesis that can be used for a 3D search query. GALAHAD models were derived by using the ligands in the training set, which contains seven features identified by GALAHAD represented by blue, green, and purple spheres. The three hydrophobes are centered in the benzopyran and phenyl rings. The three acceptor atoms are in green and a donor atom is in purple.
Fig. 4.
Fig. 4.
3D search query of two commercially available databases, the Chembridge and the Maybridge databases, which together have over a million chemicals, resulted in a refined hit list of 167 compounds. (A) Represents a schematic of the 3D virtual screening of the ChemBridge and Maybridge databases. (B) Dose-response data of BRET assays in HEK293 cells, illustrating dimerization profile of selected hits. Data are shown as mean ± S.D. of three biologic replicates. Data are normalized to DMSO control. (C) Measurement of compound binding to ERα and ERβ using in vitro fluorescence polarization competition binding assays.
Fig. 5.
Fig. 5.
Mutant ERα and ERβ LBDs reveal ERα as the dominant heterodimeric partner in the presence of selective ERα/β heterodimer compounds. (A) Heterodimerization of the wild-type ERα and ERβ. (B) Mutation in the ERβ LBD does not affect heterodimerization with ERα. (C) Heterodimerization of mutant ERα with mutant ERβ. (D) No dimerization is observed between mutant ERα and wild-type ERβ. Data are shown as mean ± S.D. * Indicates statistically significant <0.05.
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References

    1. Anstead GM, Carlson KE, Katzenellenbogen JA. (1997) The estradiol pharmacophore: ligand structure-estrogen receptor binding affinity relationships and a model for the receptor binding site. Steroids 62:268–303. - PubMed
    1. Brzozowski AM, Pike AC, Dauter Z, Hubbard RE, Bonn T, Engström O, Ohman L, Greene GL, Gustafsson JA, Carlquist M. (1997) Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389:753–758. - PubMed
    1. Caballero J. (2010) 3D-QSAR (CoMFA and CoMSIA) and pharmacophore (GALAHAD) studies on the differential inhibition of aldose reductase by flavonoid compounds. J Mol Graph Model 29:363–371. - PubMed
    1. Cowley SM, Hoare S, Mosselman S, Parker MG. (1997) Estrogen receptors α and β form heterodimers on DNA. J Biol Chem 272:19858–19862. - PubMed
    1. Grober OMV, Mutarelli M, Giurato G, Ravo M, Cicatiello L, De Filippo MR, Ferraro L, Nassa G, Papa MF, Paris O, et al. (2011) Global analysis of estrogen receptor beta binding to breast cancer cell genome reveals an extensive interplay with estrogen receptor alpha for target gene regulation. BMC Genomics 12:36. - PMC - PubMed

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