ERβ Selectivity Research Articles

Novel estrogen receptor β/histone deacetylase dual-targeted near-infrared fluorescent probes as theranostic agents for imaging and treatment of prostate cancer

Estrogen receptor (ER) β and histone deacetylases (HDACs), when overexpressed, are associated closely with the occurrence and development of prostate cancer and are, therefore, considered important targets and biomarkers used in the clinical treatment of prostate cancer. The present study involved the design and synthesis of the first ERβ and HDAC dual-target near-infrared fluorescent probe with both imaging capacity and antitumor activity for prostate cancer. Both P1 and P2 probes exhibited excellent ERβ selectivity, with P1 being almost exclusively selective for ERβ compared to ERα. In addition, P1 exhibited good optical properties, such as strong near-infrared emission, large Stokes shift, and better anti-interference ability, along with excellent imaging ability for living cells. P1 also exhibited potent inhibitory activity against HDAC6 and DU-145 cells, with IC50 values of 52 nM and 0.96 μM, respectively. Further, P1 was applied successfully for the in vivo imaging of prostate cancer in a mouse model, and significant in vivo antitumor efficacy was achieved. The developed dual-target NIR fluorescent probe is expected to serve as an effective tool in the research on prostate cancer, leading to novel insights for the theranostic study of diseases related to ERβ and HDACs.

DDDR-12. ESTABLISHING A SAFE AND EFFICACIOUS ANTI-GLIOBLASTOMA THERAPY

Abstract Glioblastomas (GBM) are primary brain tumors that are among the most treatment-resistant human cancers with a median survival of only 14 months and a 5-year survival rate of less than 5%. The standard-of-care for GBM has remained unchanged for two decades and more than half of newly diagnosed patients do not see any therapeutic benefit with existing regimens. Due to a 25:1 failure ratio in developing new brain tumor treatments, the FDA has approved only four drugs against GBM in the past three decades. This failure in clinical development can be attributed primarily to severe toxic side effects, the inability of most compounds to cross the blood brain barrier (BBB) and use of pre-clinical models that do not represent the molecular heterogeneity of GBM. To fulfill this unmet need for curative treatments we are developing an anti-GBM therapy in a clinically relevant patient-derived in vivo model and selectively targeting estrogen receptor-beta (ERβ) using ent-28, a novel, highly selective and potent agonist of ERβ. ent-28 has 9000-fold higher selectivity for ERβ compared to estrogen, or 17β- estradiol; precluding non-specific binding of ERα. ent-28 inhibits the proliferation of GBM patient-derived adherent and stem cells but not of human astrocytes and neural progenitor cells in vitro. More critically, ent-28 is not toxic and does not have off- target side effects in immune-competent mice even after one year long chronic treatment. These data have enabled us to further establish that using an oral route of delivery, ent-28 crosses the BBB and significantly reduces or ablates intra-cranial, orthotopically implanted GBM patient-derived xenografts, resulting in increased survival of ent-28 treated mice. Pre-clinical data will be presented on safety, pharmacokinetics, metabolic stability, in vivo efficacy, mechanism of action and species rationalization for this novel, ERβ agonist that is being developed as an anti-GBM therapy.

The estrogen receptor modulatory activity and neuroprotective activity of novel prenylated flavonoids and their structure–activity relationship

AbstractPrenylated flavonoids are promising nutraceuticals with diverse bioactivities. However, publications on the mechanism of action and structure–activity relationship of prenylated flavonoids are limited. In the present work, six novel prenylated flavonoids (C8–C13) with diversity structure were prepared. Their estrogen receptor (ER) modulator activity and neuroprotective activity were investigated by cell assays. C8, C9, and C10 with 8‐C‐prenyl and 7‐OH showed agonistic activities toward ERα and ERβ, along with high ERβ selectivity. However, C11 and C12 were ERα‐selective agonists. The position and substituent moiety of prenylation, C‐ring configuration, and flavonoid skeleton were important for the ER modulating activities. Hydrogen bonds between 3′‐OH and Leu339 in ERβ, and 5′‐OH and Glu305 in ERβ might respond for the higher agonistic activity of C8 and C10, respectively. C8, C9, C11, and C12 showed more potency than 17β‐estradiol on neuroprotection in glutamate‐treated PC‐12 cells. C8 and C12 could reverse reactive oxygen species overproduction and reduction of Δψm induced by glutamate through increase of the expression of ERS1, SOD1, SOD2, CAT, and GPx4 and decrease of caspase‐3 expression, suggesting C8, and C9 might interact with ERα, promote antioxidant defense, mitigate oxidative stress, and improve mitochondrial function to exert the neuroprotection activities.

Estrogen receptor β-targeted hypoxia-responsive near-infrared fluorescence probes for prostate cancer study

Aberrant expression of estrogen receptor β (ERβ) and tumor hypoxia have been observed in castration-resistant prostate cancer (CRPC); therefore, hypoxia-responsive labeling of ERβ will be beneficial for the early diagnosis and treatment of CRPC. Herein, we report the first ERβ-targeted hypoxia-responsive near-infrared fluorescent probes, which showed superior ERβ selectivity and favorable optical properties. These two probes exhibited excellent hypoxia responsiveness and specific mitochondrial ERβ imaging ability in CRPC cells. In addition, P1 displayed strong anti-interference ability and good tumor imaging capacity in vivo, contributing to effective diagnosis of CRPC. Mechanistic studies, including high resolution mass spectrometry (HRMS) and density functional theory (DFT) calculations, showed that the introduction of a nitro group quenched the probe fluorescence by inducing a PET effect, while in the hypoxic tumor microenvironment, reduction of the nitro group blocked the PET effect and turned on the probe fluorescence. These novel ERβ-targeted hypoxia-responsive near-infrared fluorescent probes may promote the study of prostate cancer.

Estrogen Receptor β-Targeted Near-Infrared Inherently Fluorescent Probe: A Potent Tool for Estrogen Receptor β Research.

Estrogen receptor β (ERβ) is associated with many diseases, and ERβ probes can help to reveal the complex role of ERβ and promote the development of ERβ-targeted therapy. Herein, we designed and synthesized the first ERβ-targeted near-infrared (NIR) inherently fluorescent probe P5, which showed the advantages of high ERβ selectivity, good optical properties, and excellent ERβ imaging capability in living cells. The probe was successfully utilized to explore ERβ motion characteristic, and for the first time, the diffusion coefficient of ERβ was obtained. Moreover, P5 was also successfully applied to the in vivo imaging of ERβ in the prostate cancer mice model. Therefore, this ERβ-targeted NIR probe might be employed as a potential tool for the research of ERβ and related diseases.

Abstract 1701: Estrogen receptor beta agonists as anti-tumor and immune-modulatory agents in malignant glioma

Abstract High grade, or malignant gliomas are Grade III and IV primary brain tumors that are among the most treatment-resistant human cancers. They have the worst prognosis of any central nervous system malignancy with a median survival of only 14 months and a 5-year survival rate of less than 5%. The standard-of-care (SOC) for high-grade glioma has remained unchanged for over a decade and more than half of newly diagnosed patients do not see any therapeutic benefit with existing regimens. Targeted immunotherapies have not improved the prognosis for glioma patients due to tumor-mediated mechanisms of immune evasion and suppression. Estrogen, or 17β- estradiol (E2), is a sex steroid hormone that inhibits development of gliomas in animal models and mediates its effects via Estrogen Receptor alpha (ERα), beta (ERβ), and the G-protein Coupled Estrogen Receptor (GPER). Early studies indicated that exogenous administration of estrogen mediates protection against glioma development in animal models, but estrogen therapy has adverse side effects due to non-specific targeting of Estrogen Receptors (ER) (α and β). In contrast to ERα, ERβ is a tumor-suppressor in gliomas, with lower expression correlating to increased tumor aggressiveness and worse prognosis. Thus, we have developed a novel, potent, and selective ERβ agonist, ent-28, and our data demonstrate its anti-glioma functions in patient cell lines in vitro, patient-derived xenografts, and orthotopic syngeneic models in vivo. While the more selective ERβ agonists have 200 to 1000-fold selectivity for ERβ, ent-28 has a 9000-fold beta-selectivity. Thus, the specificity of ent-28 for ERβ over ERα will allow us to minimize side-effects of non-selective ER-targeted therapy and maximize therapeutic benefit. ERβ agonists also have anti-inflammatory effects through inhibiting NF-κB activity and decreasing subsequent inflammatory cytokine secretion. Our preliminary gene set analyses indicate that immune signaling pathways are highly enriched in ent-28-treated patient cells compared to vehicle control, suggesting that ent-28 has immune-modulatory effects. To further elucidate the extrinsic effects of ERβ agonism on the tumor microenvironment (TME), we have assessed infiltration of immune subsets between ent-28 and vehicle-treated syngeneic tumor-bearing mice using immunohistochemistry (IHC). Here, we have demonstrated that ERβ agonists have anti-tumor activity in animal models of glioma and modulate the brain TME. Lack of immune infiltration and suppression within the glioma TME are a major barrier to immunotherapy responsiveness; thus, ERβ-mediated immune modulation may sensitize patients to immune-based therapies. Citation Format: Jordan Fredriksen Isaacs, Divya Ravi, Carmen L. del Genio, Zachary A. Shalit, Glenn C. Micalizio, Arti B. Gaur. Estrogen receptor beta agonists as anti-tumor and immune-modulatory agents in malignant glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1701.

Structure-Activity Relationship of para-Carborane Selective Estrogen Receptor β Agonists.

Selective agonism of the estrogen receptor (ER) subtypes, ERα and ERβ, has historically been difficult to achieve due to the high degree of ligand-binding domain structural similarity. Multiple efforts have focused on the use of classical organic scaffolds to model 17β-estradiol geometry in the design of ERβ selective agonists, with several proceeding to various stages of clinical development. Carborane scaffolds offer many unique advantages including the potential for novel ligand/receptor interactions but remain relatively unexplored. We synthesized a series of para-carborane estrogen receptor agonists revealing an ERβ selective structure-activity relationship. We report ERβ agonists with low nanomolar potency, greater than 200-fold selectivity for ERβ over ERα, limited off-target activity against other nuclear receptors, and only sparse CYP450 inhibition at very high micromolar concentrations. The pharmacological properties of our para-carborane ERβ selective agonists measure favorably against clinically developed ERβ agonists and support further evaluation of carborane-based selective estrogen receptor modulators.

Novel daidzein molecules exhibited anti-prostate cancer activity through nuclear receptor ERβ modulation, in vitro and in vivo studies

Eight novel ERβ selective daidzein analogues (NCE1-8) were synthesized and their anti-cancer activity was evaluated by in vitro and in vivo methods. Cytotoxicity study, Receptor binding studies, Luciferase assay, cMYC & Cyclin D1 expression and Caspase 3, 8 & 9 activities were measured to ascertain the anticancer activity and mechanism. Uterotropic, anti-androgenic and anti-tumour activities were performed in rodents. The results revealed that NCEs produced anti-prostate cancer activity in DU145, LNCaP and PC3 cell lines and 50% more active than genistein. NCEs was significantly down-regulated cMYC & Cyclin D1 genes and elevated caspase 3 & 9 levels and did not show any difference in uterotropic, anti-androgenic activities. The tumour weight was also reduced. The NCE 1 and 2 have shown ERβ selectivity in receptor binding studies. Daidzein with methyl substitution at R or R1 position exhibited more ERβ selectivity and could be considered as lead molecules for anti-prostate cancer activity.

Structure-Activity Relationship of Phytoestrogen Analogs as ERα/β Agonists with Neuroprotective Activities.

A set of isoflavononid and flavonoid analogs was prepared and evaluated for estrogen receptor α (ERα) and ERβ transactivation and anti-neuroinflammatory activities. Structure-activity relationship (SAR) study of naturally occurring phytoestrogens, their metabolites, and related isoflavone analogs revealed the importance of the C-ring of isoflavonoids for ER activity and selectivity. Docking study suggested putative binding modes of daidzein 2 and dehydroequol 8 in the active site of ERα and ERβ, and provided an understanding of the promising activity and selectivity of dehydroequol 8. Among the tested compounds, equol 7 and dehydroequol 8 were the most potent ERα/β agonists with ERβ selectivity and neuroprotective activity. This study provides knowledge on the SAR of isoflavonoids for further development of potent and selective ER agonists with neuroprotective potential.

ER subtype selectivity of m-carborane-containing phenols: C-alkyl groups on the m-carborane cage enhance ERα selectivity.

Estrogen receptor (ER) exhibits two subtypes, ERα and ERβ, whose biological functions are quite different despite expression in the same tissues. We developed diiodo-m-carborane derivative 3a, which showed 14-fold selectivity for ERβ with high binding affinity toward ERβ. Interestingly, introduction of an alkyl group into the carbon atom of the m-carborane cage of 3a markedly enhanced the binding affinity toward ERα and decreased affinity toward ERβ. C-n-propyl derivative 3d showed 28-fold selectivity for ERα in an ER binding assay and promoted proliferation of MCF-7 breast cancer cells. Docking simulation studies suggest that the directions of the n-propyl group and the diiodo substituent introduced on the m-carborane cage play important roles for the control of ER subtype selectivity. As 3a and 3d showed ERβ and ERα selectivity with high binding affinity, respectively, these ligands may be useful as biological tools to aid in understanding the different roles of ER subtypes.

Design, synthesis and biological evaluation of novel indone derivatives as selective ERβ modulators

To reduce the endometrial toxicity and improve the efficacy of current selective estrogen receptor modulators used in breast cancer treatment by enhancing ERβ selectivity, inspired by active resveratrol oligomer, a series of analogs (5a–f, 6a–b, 7a–d, 8a–d) were designed, synthesized and biologically evaluated. Among them, the chiral indone analog (2R,3R)-8a exhibited best antiproliferative activity against both breast cancer cell lines (MDA-MB-231 and MCF-7) and better safety profile on uterus than tamoxifen. Analog (2R,3R)-8a demonstrated good binding affinity and selectivity toward ERβ, which was further proved by both molecular docking and radiometric competitive binding assay. Other studies for (2R,3R)-8a also have been explored including cell cycle and apoptosis evaluation and in vitro metabolic stability studies. These results demonstrated that (2R,3R)-8a could be a promising lead compound for future exploration of selective ERβ anti-breast cancer agents.

The Development and Characterization of Estrogen Receptor Beta Agonists to Treat Cognitive Decline in Post‐Menopausal Women

Estrogens in the brain are critical in the protection of neural pathways, and in the enhancement of hippocampal memory consolidation. The estrogen receptor exists in two forms: alpha (ERα) and beta (ERβ), the latter of which is predominant in the hippocampus and a drug target for several cognitive disorders. Thus, we have developed a selective ERβ agonist to combat memory decline in post‐menopausal women affected by significantly lowered estrogen levels.The molecule reported is an A‐C estrogen that shows high potency and selectivity for ERβ. ERα agonist activity has been linked to increased risk of diseases such as breast cancer and stroke, among others. Selectivity for ERβ over ERα is therefore critical in the success of a drug molecule that acts like estrogen. TR‐FRET displacement assays were utilized to determine the most promising candidates from a library of lead molecules by detecting binding of the molecules to the ERβ ligand binding domain. ISP358‐2 was one of few potent molecules that displayed an EC50 < 30 nM, and also showed a 12‐fold selectivity of ERβ (EC50 = 24 ± 5 nM) over ERα (EC50 = 289 ± 92 nM). ERβ cell‐based assays further confirmed the molecule's potency, and ERα cell‐based potency showed ERβ selectivity. To verify our cell‐based assay results, ISP358‐2 was tested for selectivity in seven other nuclear hormone receptors in cell‐based assays. We saw no significant activity in any nuclear hormone receptor other than the estrogen receptor.We hypothesized that the selectivity observed was due to selective coactivator recruitment present only in the native full length ERβ. This hypothesis was supported by data obtained from the cell‐based transcriptional activation assay, which employs a full length and native estrogen receptor and most closely mimic the in vivo situation. The results obtained in this assay were profound in that ISP358‐2 showed an ERβ agonist potency of 31 ± 7 nm, and an ERα agonist potency of 20,400 ± 859 nM. This equates to a selectivity of ERβ of ~750 fold over ERα. These results suggest that the coactivator plays a role in the selectivity of ISP358‐2. In a TR‐FRET coactivator binding assay the results showed a 15 fold selectivity for ERβ (EC50 =191 ± 15 nM) over ERα (EC50 =2,940 ± 390 nM). However, the coactivator assay utilizes a PPARɣ derived peptide. Using docking studies, we showed that the brain‐relevant SRC‐1 peptide docks more favorably. Modeling studies also demonstrate that the conformational change is favorable for coactivator recruitment for ERβ when agonist ISP358‐2 is bound, but not for the agonist‐bound ERα.Support or Funding InformationThis work was supported by NIH grants R15GM118304 and R01DA038042.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Construction of benzofuranone library via a metal-free, one-pot intermolecular condensation, and their application as efficient estrogen receptor β modulators.

Facile synthesis of benzofuranone was achieved through a metal-free, one-pot intermolecular condensation between α-hydroxy aryl ketones and resorcinol derivatives. A library of 20 compounds with moderate to good overall yields was prepared. These compounds showed strong binding toward estrogen receptors along with good selectivity for ERβ (>190-fold over ERα). Anti-proliferative activity on DU-145, U-87, and MCF-7 cells gave inhibition IC50 values in the low μM range, which suggested the promising potential therapeutic applications of these new classes of benzofuranones.

Phytoestrogenic Activity of Blackcurrant Anthocyanins Is Partially Mediated through Estrogen Receptor Beta.

Phytoestrogens are plant compounds with estrogenic effects found in many foods. We have previously reported phytoestrogen activity of blackcurrant anthocyanins (cyanidin-3-glucoside, cyanidin-3-rutinoside, delphinidin-3-glucoside, and delphinidin-3-rutinoside) via the estrogen receptor (ER)α. In this study, we investigated the participation of ERβ in the phytoestrogen activity of these anthocyanins. Blackcurrant anthocyanin induced ERβ-mediated transcriptional activity, and the IC50 of ERβ was lower than that of ERα, indicating that blackcurrant anthocyanins have a higher binding affinity to ERβ. In silico docking analysis of cyanidin and delphinidin, the core portions of the compound that fits within the ligand-binding pocket of ERβ, showed that similarly to 17β-estradiol, hydrogen bonds formed with the ERβ residues Glu305, Arg346, and His475. No fitting placement of glucoside or rutinoside sugar chains within the ligand-binding pocket of ERβ-estradiol complex was detected. However, as the conformation of helices 3 and 12 in ERβ varies depending on the ligand, we suggest that the surrounding structure, including these helices, adopts a conformation capable of accommodating glucoside or rutinoside. Comparison of ERα and ERβ docking structures revealed that the selectivity for ERβ is higher than that for ERα, similar to genistein. These results show that blackcurrant anthocyanins exert phytoestrogen activity via ERβ.

Design and synthesis of iodocarborane-containing ligands with high affinity and selectivity toward ERβ

The selectivity and the binding affinity of previously reported carborane-containing ligands 2 and 3 toward ERβ remains to be optimized. To improve their biological profiles, a series of iodinated carboranyl phenol derivatives (4–6) were designed and synthesized as prospective ERβ-selective ligands with high affinity. Several iodinated carboranyl phenols showed high relative binding affinity (RBA) values for both ERs, and especially for ERβ, due to suitable hydrophobic interactions of the iodine atoms with the hydrophobic amino acid residues of the ERβ ligand-binding domains. Among these derivatives, 9,10-diiodo-m-carborane 5f exhibited a more than 100% increase of the RBA values toward ERβ, a 14-fold increased selectivity for ERβ over ERα, and ER-agonistic activity in MCF-7 cell proliferation assays.

Synthesis and structure–activity relationships of 1-benzylindane derivatives as selective agonists for estrogen receptor beta

The estrogen receptor beta (ERβ) selective agonist is considered a promising candidate for the treatment of estrogen deficiency symptoms in ERβ-expressing tissues, without the risk of breast cancer, and multiple classes of compounds have been reported as ERβ selective agonists. Among them, 6-6 bicyclic ring-containing structures (e.g., isoflavone phytoestrogens) are regarded as one of the cyclized analogues of isobutestrol 5b, and suggest that other cyclized scaffolds comprising 5-6 bicyclic rings could also act as selective ERβ ligands. In this study, we evaluated the selective ERβ agonistic activity of 1-(4-hydroxybenzyl)indan-5-ol 7a and studied structure–activity relationship (SAR) of its derivatives. Some functional groups improved the properties of 7a; introduction of a nitrile group on the indane-1-position resulted in higher selectivity for ERβ (12a), and further substitution with a fluoro or a methyl group to the pendant phenyl ring was also preferable (12b, d, and e). Subsequent chiral resolution of 12a identified that R-12a has a superior profile over S-12a. This is comparable to diarylpropionitrile (DPN) 5c, one of the promising selective ERβ agonists and indicates that this indane-based scaffold has the potential to provide better ERβ agonistic probes.

Molecular Docking Reveals Binding Features of Estrogen Receptor Beta Selective Ligands.

Estrogen receptors exist as two subtypes ERα and ERβ, which are characterized by various distributions in human tissues and diverse transcription regulation. Ligands capable of selective ERβ activation show positive effects in treatment of such diseases as certain cancers, endometriosis, inflammatory diseases, and assist in maintaining cardiovascular and nervous system health. Thus far, there are no pharmaceutical drugs available acting on this target. In order to provide new treatment for such diseases, a new generation of selective estrogen receptor modulators is required. This remains an unsolved task due to several difficulties. It is known that minor modifications of ER agonists can influence the selectivity of their binding. The majority of designed ligands acting on ER possess chiral centers thus exist as stereoisomers. Unfortunately, not every spatial isomer is individually considered in experimental research. The molecular docking was applied to investigate the structural basis of diverse selectivity and binding affinity of selected estrogen receptor β agonists. Docking simulations revealed that terminal aromatic rings positioned in the A- and D-ring regions are a factor that determines binding affinity of ERβ agonists. This positioning can be ascribed to the presence of two terminal hydroxyl groups, a rigid linker, and the introduction of aliphatic substituents. The side substituents of underlined molecular scaffold should adopt inside characterized cavities I and II in order to provide selectivity. The bulkiness, attachment to linker and stereochemistry of the substituents affect ERβ selectivity. These molecular features should be considered during search and design of new improved ERβ agonists.

Design and synthesis of carborane-containing estrogen receptor-beta (ERβ)-selective ligands.

Candidates for highly selective estrogen receptor-beta (ERβ) ligands (6a-c, 7a-c, 8a and 8b) were designed and synthesized based on carborane-containing ER ligands 1 and 2 as lead compounds. Among them, p-carboranylcyclohexanol derivatives 8a and 8b exhibited high ERβ selectivity in competitive binding assay: for example, 8a showed 56-fold selectivity for ERβ over ERα. Docking studies of 8a and 8b with the ERα and ERβ ligand-binding domains (LBDs) suggested that the p-carborane cage of the ligands is located close to key amino acid residues that influence ER-subtype selectivity, that is, Leu384 in the ERα LBD and Met336 in the ERβ LBD. The p-carborane cage in 8a and 8b appears to play a crucial role in the increased ERβ selectivity.

Highly selective salicylketoxime-based estrogen receptor β agonists display antiproliferative activities in a glioma model.

Estrogen receptor β (ERβ) selective agonists are considered potential therapeutic agents for a variety of pathological conditions, including several types of cancer. Their development is particularly challenging, since differences in the ligand binding cavities of the two ER subtypes α and β are minimal. We have carried out a rational design of new salicylketoxime derivatives which display unprecedentedly high levels of ERβ selectivity for this class of compounds, both in binding affinity and in cell-based functional assays. An endogenous gene expression assay was used to further characterize the pharmacological action of these compounds. Finally, these ERβ-selective agonists were found to inhibit proliferation of a glioma cell line in vitro. Most importantly, one of these compounds also proved to be active in an in vivo xenograft model of human glioma, thus demonstrating the high potential of this type of compounds against this devastating disease.

Selective ligands of estrogen receptor β discovered using pharmacophore mapping and structure-based virtual screening.

Aim:To discover novel ligands of estrogen receptor (ER) β using pharmacophore mapping and structure-based screening.Methods:A computer-aided strategy combining pharmacophore mapping and structure-based screening was used to screen the Maybridge and Enamine databases. Yeast two-hybrid (Y2H) assay was used to detect the activity and selectivity of the chosen compounds. The transcriptional activities of the chosen compounds were demonstrated with luciferase reporter assays. The anti-proliferative effects of ER antagonists against MCF-7 and MDA-MB-231 breast cancer cells were examined using MTT assay, and the mechanisms of action were analyzed with flow cytometry analysis and Western blotting.Results:Through in silico screen, 95 compounds were chosen for testing in Y2H assay, which led to 20 potent ligands, including 10 agonists, 8 antagonists and 2 partial agonists with EC50 or IC50 values at μmol/L. Furthermore, 6 agonists exhibited absolute selectivity for ERβ, and 3 agonists showed higher selectivity for ERβ. The agonists 1g and 1h (10, 25, and 50 μmol/L) dose-dependently increased ER transcriptional activities, whereas the antagonists 2a and 2d (10, 25, and 50 μmol/L) caused dose-dependent inhibition on the activities. The antagonists and partial agonists at 100 μmol/L suppressed the proliferation of ERα positive MCF-7 cells and ERβ positive MDA-MB-231 cells, but were more effective against MDA-MB-231 cells. Treatment of MDA-MB-231 cells with antagonists 2a and 2d (25 and 50 μmol/L) dose-dependently increased the population of cells in the S phase. Both 2a and 2d treatment dose-dependently decreased the expression levels of cyclin A and CDK2. Meanwhile, the downregulation of cyclin E was only caused by 2d, while 2a treatment did not cause significant changes in the protein levels of cyclin E.Conclusion:The selective ligands discovered in this study are promising drug candidates to be used as molecular probes to explore the differences between ERα and ERβ.