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

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 β-galactosidase (β-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 β-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 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 β-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 β-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 β-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.