Identification of compounds by high-content screening that induce cytoplasmic to nuclear localization of a fluorescent estrogen receptor α chimera and exhibit agonist or antagonist activity in vitro.
We have completed a robust high-content imaging screen for novel estrogen receptor α (ERα) agonists and antagonists by quantitation of cytoplasmic to nuclear translocation of an estrogen receptor chimera in 384-well plates. The screen was very robust, with Z' values >0.7 and coefficients of variation (CV) <5%. The screen utilized a stably transfected green fluorescent protein-tagged glucocorticoid/estrogen receptor (GFP-GRER) chimera, which consisted of the N-terminus of the glucocorticoid receptor fused to the human ERα ligand binding domain. The GFP-GRER exhibited cytoplasmic localization in the absence of ERα ligands and translocated to the nucleus in response to stimulation with ERα agonists and antagonists. The BD Pathway 435 imaging system was used for image acquisition, analysis of translocation dynamics, and cytotoxicity measurements. We screened 224,891 samples from our synthetic, pure natural product libraries, prefractionated natural product extracts library, and crude natural product extracts library, which produced a 0.003% hit rate. In addition to identifying several known ER ligands, five compounds were discovered that elicited significant activity in the screen. Transactivation potential studies demonstrated that two hit compounds behave as agonists, while three compounds elicited antagonist activity in MCF-7 cells.
1SAIC-Frederick, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, USA.
Angie B
AB
Dull
Anuja A
AA
George
Ekaterina I
EI
Goncharova
Jason R
JR
Evans
Antony
A
Wamiru
Laura K
LK
Cartner
Gordon L
GL
Hager
James B
JB
McMahon
D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D047628:Estrogen Receptor alpha / Q000819:agonists / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D008024:Ligands; D061986:MCF-7 Cells; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology
C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D008024:Ligands; D011993:Recombinant Fusion Proteins; D054852:Small Molecule Libraries; D049452:Green Fluorescent Proteins
cell-based assay; cytotoxicity; estrogen receptor; high-content screening; nuclear translocation