title: Selective ligands of estrogen receptor β discovered using pharmacophore mapping and structure-based virtual screening.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Chen
authors.affiliation: Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Dang
authors.initials: D
authors.lastname: Wu
authors.affiliation: Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Han-ping
authors.initials: HP
authors.lastname: Bian
authors.affiliation: Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Guang-lin
authors.initials: GL
authors.lastname: Kuang
authors.affiliation: Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Jiang
authors.affiliation: Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Wei-hua
authors.initials: WH
authors.lastname: Li
authors.affiliation: Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Gui-xia
authors.initials: GX
authors.lastname: Liu
authors.affiliation: Department of Gynecology, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011, China.
authors.forename: Shi-en
authors.initials: SE
authors.lastname: Zou
authors.affiliation: Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Huang
authors.affiliation: Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Tang
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism; D016466:CHO Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003412:Cricetulus; D019925:Cyclin A / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D047629:Estrogen Receptor beta / Q000819:agonists* / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005260:Female; D006801:Humans; D008024:Ligands; D061986:MCF-7 Cells; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D019925:Cyclin A; D019927:Cyclin E; D047629:Estrogen Receptor beta; D008024:Ligands; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2