Reducing costs while maintaining the highest readout quality is a precept of modern high-throughput screening. Given the trend toward nonradiometric screening platforms, this has been a big challenge for some kinase target classes. Common issues include lowsensitivity, susceptibility to nonspecific interference, or the need for costly reagents. In this study, the authors describe the feasibility ofminiaturization of a serine kinase assay using generic reagents in the Alpha Screen format. They have validated the robustness of this assay in the course of miniaturization from a 35-to 4.375-µ L final assay volume in 384-and 1536-well formats. Within this volume range, they consistently obtained Z• values above 0.5 and have investigated the suitability of these assay formats for measuring compound effects by testing a set of 25 previously identified active compounds. These active compounds were also reliably identified in the miniaturized assay formats. The results presented here show that the Alpha Screen technology permits robust and cost-efficient miniaturization of serine/threonine kinase assays.

The authors have developed a cell-based high-throughput screening (HTS)-compatible assay to measure EGFR dimerization using the InteraX enzyme complementation technology of Applied Biosystems. The cells contain 2 chimeric proteins with complementing deletion mutants 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, a mean S/B ratio of 4.26 and a mean Z' factor of 0.74 were obtained, for run 2 a mean S/B ratio of 3.88 and a mean Z' factor of 0.71 were obtained. After hit confirmation, repeated 4 times, 112 hits remained with 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 enzyme itself. In summary, 31 compounds remained as potential EGFR dimerization or EGF stimulation inhibitors. The authors conclude that the InteraX system technology is HTS capable and can detect small molecule inhibitors capable of inhibiting protein-protein interactions.

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