title: Application of beta-galactosidase enzyme complementation technology as a high throughput screening format for antagonists of the epidermal growth factor receptor.
abstract: We have applied enzyme complementation technology to develop a screen for antagonists of the epidermal growth factor (EGF) receptor. Chimeric proteins containing two weakly complementing deletion mutants of Escherichia coli beta-galactosidase (beta-gal), each fused to the EGF receptor extracellular and transmembrane domains, have been stably expressed in C2C12 cells. In this cell line, formation of active beta-gal is dependent on agonist-stimulated dimerization of the EGF receptor. We have developed a homogenous 384-well assay protocol and have applied this to characterize the pharmacology of the receptor and to develop a high throughput screen (HTS) for EGF receptor antagonists. The assay is tolerant to DMSO concentrations of up to 2% and, across 21 passages in culture, exhibits an EC(50) for EGF of 5.4 +/- 3.6 ng/ml (n = 11) and a Z' of 0.55 +/- 0.13 (n = 11). A random set of 1,280 compounds was screened in duplicate at 11 microM to examine the robustness of enzyme complementation technology and to characterize the false-positive hit rate in the assay. Using a cutoff of 40% inhibition of EGF-promoted beta-gal activity, the hit rate on day 1 was 2.5% and on day 2 was 1.9%. After retesting the active compounds, the hit rate was reduced to 0.4%, of which one of the compounds was identified as a beta-gal inhibitor and the remainder appeared to be nonspecific inhibitors in the assay. This technology is amenable to automated screen workstations, there are highly sensitive chemiluminescent and fluorescent beta-gal assay reagents amenable to detection in miniaturized plate formats, and the assay benefits from a low false-positive hit rate. Enzyme complementation technology may have wide application within the HTS environment for the detection of modulators of receptor activation or inhibitors of protein-protein interactions in mammalian cells.
authors.affiliation: Molecular Recognition, GlaxoSmithKline Medicines Research Centre, Stevenage, Hertfordshire, UK.
authors.forename: D L
authors.initials: DL
authors.lastname: Graham
authors.forename: N
authors.initials: N
authors.lastname: Bevan
authors.forename: P N
authors.initials: PN
authors.lastname: Lowe
authors.forename: M
authors.initials: M
authors.lastname: Palmer
authors.forename: S
authors.initials: S
authors.lastname: Rees
mesh_terms: D000818:Animals; D002460:Cell Line; D019281:Dimerization; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D066246:ErbB Receptors / Q000819:agonists / Q000037:antagonists & inhibitors* / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D005816:Genetic Complementation Test; D008163:Luminescent Measurements; D012680:Sensitivity and Specificity; D001616:beta-Galactosidase / Q000235:genetics*
chemical_list: D066246:ErbB Receptors; D001616:beta-Galactosidase; D004121:Dimethyl Sulfoxide