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. (Journal of Biomolecular Screening 2006:617-633)

Targeting Ceramide Metabolism with a Potent and Specific Ceramide Kinase Inhibitor

“…We developed a homogeneous CerK assay for high-throughput screening based on a luminescent reaction measuring ATP consumption (Koresawa and Okabe, 2004;Klumpp et al, 2006). After miniaturization of this assay to a 1536-well plate format, we screened over a million compounds for inhibition of CerK.…”

Multiplexing Terbium- and Europium-Based TR-FRET Readouts to Increase Kinase Assay Capacity

“…We have used this strategy to independently monitor multiple kinase reactions from within a single assay well, which may be useful in generating a "firstpass" look at compound specificity when screening compound libraries and also may be compatible with other commonly used kinase assay formats. 10,11 Because both Tb-and Eu-based TR-FRET assays across many target classes have been well described in the literature, we expect that this strategy can be easily adapted to a range of targets. In addition, applications can be envisioned in which the second assay performed in the well may be a control to improve confidence in the results from the primary assay, and studies toward such applications are under way in our laboratories.…”

Development of a CERT START Domain–Ceramide HTRF Binding Assay and Application to Pharmacological Studies and Screening

“…12 Moreover, the fluorescence background produced by chemical samples, buffers, or proteins, generally presenting lifetimes in the nanosecond range, could be eliminated using time-resolved methodology (TR-FRET). Homogeneous time-resolved fluorescence (HTRF 13 ), an homogeneous-phase TR-FRET technology, has been developed for high-throughput screening (HTS) 14 and was already successfully applied to screen diverse compound libraries. 15,16 Here, TR-FRET was applied for the first time to monitor a protein-lipid, namely, START-Cer, interaction.…”