Estrogen receptor alpha (ER) is a key biomarker for breast cancer, and the presence or absence of ER in breast and other hormone-dependent cancers decides treatment regimens and patient prognosis. ER is activated after ligand binding - typically by steroid. 2682 steroid compounds were used in a molecular docking study to identify novel ligands for ER and to predict compounds that may show anticancer activity. The effect of the most promising compounds was determined by a novel luciferase reporter assay. Two compounds, 7 and 12, showing ER inhibitory activity comparable to clinical inhibitors such as tamoxifen or fulvestrant were selected. We propose that the inhibitory effect of compounds 7 and 12 on ER is related to the presence of a double bond in their D-ring, which may protect against ER activation by reducing the electron density of the keto group, or may undergo metabolism leading to an active compound. Western blotting revealed that compound 12 decreased the level of ER in the breast cancer cell line MCF7, which was associated with reduced expression of both isoforms of the progesterone receptor, a well-known downstream target of ER. However, compound 12 has a different mechanism of action from fulvestrant. Furthermore, we found that compound 12 interferes with mitochondrial functions, probably by disrupting the electron transport chain, leading to induction of the intrinsic apoptotic pathway even in ER-negative breast cancer cells. In conclusion, the combination of computational and experimental methods shown here represents a rapid approach to determine the activity of compounds towards ER. Our data will not only contribute to research focused on the regulation of ER activity but may also be useful for the further development of novel steroid receptor-targeted drugs applicable in clinical practice.

Department of Experimental Biology Faculty of Science Palacký University Olomouc Šlechtitelů 27 78371 Olomouc Czech Republic

Department of Physical Chemistry Faculty of Science Palacký University Olomouc třída 17 listopadu 12 771 46 Olomouc Czech Republic

Institute of Organic Chemistry and Biochemistry AS CR Flemingovo náměstí 2 166 10 Praha 6 Czech Republic

Laboratory of Growth Regulators Faculty of Science of Palacký University and Institute of Experimental Botany of the Czech Academy of Sciences Šlechtitelů 27 783 71 Olomouc Czech Republic

Research Centre for Applied Molecular Oncology Masaryk Memorial Cancer Institute Žlutý kopec 7 656 53 Brno Czech Republic

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