Development of a quantitative, cell-based, high-content screening assay for epidermal growth factor receptor modulators.
OBJECTIVE
To develop a robust, cell-based, high-content screening (HCS) assay based on receptor internalization for the identification of novel modulators of the epidermal growth factor receptor (EGFR).


METHODS
Agonist-induced receptor internalization is part of the signaling cascade of EGFR. Fluorescent-tagged epidermal growth factor (EGF) was used to visualize the internalized receptor-ligand complex. The fluorescent intracellular spots were detected and measured with an ArrayScan HCS reader. Compounds that can competitively bind to EGFR or interfere with EGFR internalization process would result in a reduced number and intensity of intracellular fluorescent spots. This assay was validated, optimized, and applied to a large-scale screening of a library containing 48,000 synthetic compounds.


RESULTS
The competition between fluorescent EGF and unlabeled EGF reveals the IC(50) of unlabeled EGF is approximately 0.2 nmol/L, which is comparable with other published reports. Thirteen compounds with a relatively high degree of interference with EGFR internalization were identified. One of the compounds was proven to be agonist of the EGFR since it induced phosphorylation of the receptor and extracellular signal-regulated protein kinase (ERK).


CONCLUSIONS
This automated, objective, and easy-to-use assay provided abundant information, quantitative results, and demonstrated the potential use of HCS methods in searching membrane receptor modulators.
Wang
Jue
J
The National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Xie
Xin
X
D001667:Binding, Competitive / Q000187:drug effects; D015195:Drug Design; D004353:Drug Evaluation, Preclinical; D004705:Endocytosis / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000819:agonists* / Q000037:antagonists & inhibitors* / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects; D015262:Xenobiotics / Q000737:chemistry / Q000494:pharmacology*
D015262:Xenobiotics; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D048049:Extracellular Signal-Regulated MAP Kinases