Readout Technologies for Highly Miniaturized Kinase Assays Applicable to High-Throughput Screening in a 1536-Well Format

This article discusses the development of homogeneous, miniaturized assays for the identification of novel kinase inhibitors from very large compound collections. In particular, the suitability of time-resolved fluorescence resonance energy transfer (TR-RET) based on phospho-specific antibodies, an antibody-independent fluorescence polarization (FP) approach using metal-coated beads (IMAP ™ technology), and the determination of adenosine triphosphate consumption through chemilu-minescence is evaluated. These readouts are compared with regard to assay sensitivity, compound interference, reagent con-sumption, and performance in a 1536-well format, and practical considerations for their application in primary screening or in the identification of kinase substrates are discussed. All of the tested technologies were found to be suitable for miniatur-ized high-throughput screening (HTS) in principle, but each of them has distinct limitations and advantages. Therefore, the target-specific selection of the most appropriate readout technology is recommended to ensure maximal relevance of HTS campaigns. (Journal of Biomolecular Screening 2006:617-633) Key words: kinase, high-throughput screening (HTS), time-resolved fluorescence resonance energy transfer (TR-FRET), fluorescence polarization (FP), chemiluminescence, assay, IMAP © 2006 Society for Biomolecular Sciences www.sbsonline.org 61