A beta-lactamase-dependent Gal4-estrogen receptor beta transactivation assay for the ultra-high throughput screening of estrogen receptor beta agonists in a 3456-well format.
Estrogen action is mediated via two estrogen receptor (ER) subtypes, ERalpha and ERbeta. Selective ER modulators with balanced high affinity for ERalpha and ERbeta have been developed as therapeutics for the treatment of a variety of diseases, including hormone-responsive breast cancer and osteoporosis. Recent data based primarily on the evaluation of ER-knockout mice have revealed that ERalpha and ERbeta may regulate separate and distinct biological processes. The identification of ERbeta specific ligands could further enhance our understanding of ERbeta biology. In addition, compounds targeting ERbeta may prove useful as therapeutic agents with activity profiles distinguishable from that of estradiol. To discover novel selective ligands for ERbeta, we developed and characterized a cell-based Gal4-ERbeta beta-lactamase reporter gene assay (GERTA) in CHO cells for the ligand-induced activation of the human ERbeta. The sensitivity and selectivity of this assay were found to be comparable to those of an ER ligand-binding assay. The assay was optimized for screening in an ultra high throughput 3456-well nanoplate format and was successfully used to screen a large compound collection for ERbeta agonists. Compounds identified in a primary screen were tested in an in vitro ligand-binding assay to characterize further the selectivity and potency for ERbeta.
Peekhaus
Norbert T
NT
Department of Atherosclerosis and Endocrinology, Merck Research Laboratories, Rahway, NJ 07065, USA. norbert.peekhaus@merck.com
Ferrer
Marc
M
Chang
Tina
T
Kornienko
Oleg
O
Schneeweis
Jonathan E
JE
Smith
Todd S
TS
Hoffman
Ira
I
Mitnaul
Lyndon J
LJ
Chin
Jayne
J
Fischer
Paul A
PA
Blizzard
Tim A
TA
Birzin
Elizabeth T
ET
Chan
Wanda
W
Inglese
James
J
Strulovici
Berta
B
Rohrer
Susan P
SP
Schaeffer
James M
JM
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000378:metabolism / Q000494:pharmacology; D047629:Estrogen Receptor beta; D005822:Genetic Vectors; D006801:Humans; D036103:Nanotechnology / Q000379:methods*; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D011960:Receptors, Estrogen / Q000819:agonists* / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000502:physiology*; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
D047629:Estrogen Receptor beta; D011960:Receptors, Estrogen; D014157:Transcription Factors; D004958:Estradiol; D001618:beta-Lactamases