Readout technologies for highly miniaturized kinase assays applicable to high-throughput screening in a 1536-well format

被引：27

作者：

Klumpp, Martin ^{[1
]}

Boettcher, Andreas ^{[1
]}

Becker, Damaris ^{[1
]}

Meder, Gabriele ^{[1
]}

Blank, Jutta ^{[1
]}

Leder, Lukas ^{[1
]}

Forstner, Michael ^{[1
]}

Ottl, Johannes ^{[1
]}

Mayr, Lorenz M. ^{[1
]}

机构：

[1] Novartis Pharma AG, Novartis Inst Biomed Res Basel, CH-4002 Basel, Switzerland

来源：

JOURNAL OF BIOMOLECULAR SCREENING | 2006年 / 11卷 / 06期

关键词：

kinase; high-throughput screening (HTS); time-resolved fluorescence resonance energy transfer (TR-FRET); fluorescence polarization (FP); chemiluminescence; assay; IMAP;

D O I：

10.1177/1087057106288444

中图分类号：

Q5 [生物化学];

学科分类号：

071010 [生物化学与分子生物学]; 081704 [应用化学];

摘要：

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 (TM) 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.

引用

页码：617 / 633

页数：17

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