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Application of ß-Galactosidase Enzyme Complementation Technology as a High Throughput Screening Format for Antagonists of the Epidermal Growth Factor Receptor

Molecular Recognition, GlaxoSmithKline Medicines Research Centre, Stevenage, Hertfordshire, UK

Nicola Bevan

Molecular Screening, GlaxoSmithKline Medicines Research Centre, Stevenage, Hertfordshire, UK

Peter N. Lowe

Molecular Recognition, GlaxoSmithKline Medicines Research Centre, Stevenage, Hertfordshire, UK

Michelle Palmer

Applied Biosystems, Advanced Discovery Sciences, Bedford, MA

Stephen Rees

Molecular Screening, GlaxoSmithKline Medicines Research Centre, Stevenage, Hertfordshire, UK

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 ß-galactosidase (ß-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 ß-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 EC₅₀ 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 µM 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 ß-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 ß-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 ß-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.

Journal of Biomolecular Screening, Vol. 6, No. 6, 401-411 (2001)
DOI: 10.1177/108705710100600606


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