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.

Heterodimerisation of G protein-coupled receptors: implications for drug design and ligand screening

“…After binding EGF, the EGF receptor dimerises and hence generates enzyme activity. The authors used this system to identify small molecules capable of inhibiting this dimerisation process [89]. Because compounds that specifically target GPCR heterodimers have the potential to achieve higher specificity with reduced side effects, define new drug targets or behave as modulators of GPCR activity in specific tissues, there is significant interest in the design of drugs that target GPCR heterodimers.…”

High-Throughput Screening Assays to Discover Small-Molecule Inhibitors of Protein Interactions

“…To date, it seems that PCA has not been used often to screen for inhibitors of protein interactions. In a proof-of-principle study, a -galactosidase complementation assay was used to screen for small-molecule inhibitors of EGFR dimerization [49]. A more frequent use of PCAs for drug screening can be anticipated in the future.…”