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 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.
Klumpp
Martin
M
Novartis Institute for Biomedical Research, Basel, Switzerland. martin.klumpp@novartis.com
Boettcher
Andreas
A
Becker
Damaris
D
Meder
Gabriele
G
Blank
Jutta
J
Leder
Lukas
L
Forstner
Michael
M
Ottl
Johannes
J
Mayr
Lorenz M
LM
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
D010455:Peptides; D011494:Protein Kinases