title: Evaluation of the InteraX system technology in a high-throughput screening environment.
abstract: 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.
authors.affiliation: Department of Integrated Lead Discovery, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany. frank.buettner@bc.boehringer-ingelheim.com
authors.forename: Frank H
authors.initials: FH
authors.lastname: Büttner
authors.forename: Renate
authors.initials: R
authors.lastname: Kumpf
authors.forename: Susanne
authors.initials: S
authors.lastname: Menzel
authors.forename: Dominique
authors.initials: D
authors.lastname: Reulle
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Valler
mesh_terms: D000818:Animals; D001331:Automation; D001665:Binding Sites; D002460:Cell Line; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods*; D004798:Enzymes / Q000737:chemistry*; D066246:ErbB Receptors / Q000737:chemistry; D051379:Mice; D008954:Models, Biological; D011485:Protein Binding; D015203:Reproducibility of Results; D012984:Software; D013672:Technology / Q000379:methods; D001616:beta-Galactosidase / Q000737:chemistry
chemical_list: D004798:Enzymes; D066246:ErbB Receptors; D001616:beta-Galactosidase