title: Readout technologies for highly miniaturized kinase assays applicable to high-throughput screening in a 1536-well format.
abstract: 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 chemiluminescence is evaluated. These readouts are compared with regard to assay sensitivity, compound interference, reagent consumption, 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 miniaturized 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.
authors.affiliation: Novartis Institute for Biomedical Research, Basel, Switzerland. martin.klumpp@novartis.com
authors.forename: Martin
authors.initials: M
authors.lastname: Klumpp
authors.forename: Andreas
authors.initials: A
authors.lastname: Boettcher
authors.forename: Damaris
authors.initials: D
authors.lastname: Becker
authors.forename: Gabriele
authors.initials: G
authors.lastname: Meder
authors.forename: Jutta
authors.initials: J
authors.lastname: Blank
authors.forename: Lukas
authors.initials: L
authors.lastname: Leder
authors.forename: Michael
authors.initials: M
authors.lastname: Forstner
authors.forename: Johannes
authors.initials: J
authors.lastname: Ottl
authors.forename: Lorenz M
authors.initials: LM
authors.lastname: Mayr
mesh_terms: D001681:Biological Assay / Q000379:methods; D004353:Drug Evaluation, Preclinical; D005454:Fluorescence Polarization / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D010316:Particle Size; D010455:Peptides / Q000737:chemistry; D011494:Protein Kinases / Q000032:analysis*; D013379:Substrate Specificity
chemical_list: D010455:Peptides; D011494:Protein Kinases