Abstract
The authors have developed a cell-based high-throughput screening (HTS)-compatible assay tomeasure EGFRdimerization using the InteraX TMenzyme complementation technology of Applied Biosystems. The cells contain 2 chimeric proteins with complementing deletionmutants of the beta galactosidase enzyme, each fused to the extracellular and transmembrane part of EGFR. On binding of EGF, EGF receptor dimerizes and an active beta galactosidase is built. The authors used this homogeneous 384-well assay to screen about 20,000 diverse compounds. From 2 independent primary screen runs 239 hits were identified. For run 1, amean S/Bratio of 4.26 and ameanZβ factor of 0.74were obtained, for run 2 amean S/Bratio of 3.88 and amean Zβ factor of 0.71 were obtained. After hit confirmation, repeated 4 times, 112 hits remainedwith a confirmation rate of 48.9%. Thirty of the 112 could be identified as cytotoxic. Fifty-one of the remaining 82 compounds could be shown to be inhibitors of the beta galactosidase enzymeitself. In summary, 31 compounds remained as potential EGFRdimerization or EGF stimulation inhibitors. The authors conclude that the InteraX TMsystemtechnology is HTS capable and can detect smallmolecule inhibitors capable of inhibiting protein-protein interactions.