Histone deacetylase inhibitor assay based on fluorescence resonance energy transfer
Histone deacetylase inhibitor assay based on fluorescence resonance energy transfer
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DOI：
10.1016/j.ab.2006.12.019
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发表时间：
2007-03-01
影响因子：
2.9
通讯作者：
Meyer-Almes, Franz-Josef
中科院分区：
文献类型：
--
作者：
Riester, Daniel;Hildmann, Christian;Meyer-Almes, Franz-Josef
组蛋白脱乙酰酶(HDACs)是真核细胞中转录调控基因表达的重要酶。此外，近年来，HDAC作为恶性肿瘤化疗干预的关键靶点占据了重要地位。然而，这些新化疗药物的开发进展在很大程度上取决于是否存在适合于抑制剂筛选的生物检测方法。在这里，我们提出了第一个非同位素竞争结合分析HDACs。该测定原理已经用来自Bordeella/Alcaliges种FB188的HDAC同源物FB188组蛋白去乙酰化酶样氨基水解酶进行了验证。该分析基于一种新的荧光HDAC抑制剂，该抑制剂在与酶结合时显示与色氨酸的荧光共振能量转移。在与其他HDAC抑制剂竞争的情况下，荧光抑制剂的置换伴随着荧光共振能量转移的减少。该方法非常适合于抑制剂结合的动力学研究和HDAC抑制剂的鉴定，例如，在药物发现中的高通量抑制剂筛选的背景下。(C)2006 Elsevier Inc.保留所有权利。
Histone deacetylases (HDACs) are important enzymes for the transcriptional regulation of gene expression in eukaryotic cells. Furthermore, in recent years HDACs occupied a major position as key targets for chemotherapeutic intervention in malignant diseases. However, progress in the development of these new chemotherapeutics is largely dependent on the existence of bioassays well-suited to inhibitor screening. Herein, we present the first nonisotopic competition binding assay for HDACs. The assay principle has been demonstrated using the well-established HDAC homolog FB188 histone deacetylase-like amidohydrolase from Bordetella/Alcaligenes species FB188. The assay is based on a new fluorescent HDAC inhibitor that shows fluorescence resonance energy transfer with tryptophans upon binding to the enzyme. In a competition situation with other HDAC inhibitors the displacement of the fluorescent inhibitor is accompanied by a decrease of fluorescence resonance energy transfer. The assay is well suited to kinetic studies of inhibitor binding and to HDAC inhibitor identification, e.g., in the context of high-throughput inhibitor screening in drug discovery. (c) 2006 Elsevier Inc. All rights reserved.