A fluorogenic histone deacetylase assay well suited for high-throughput activity screening
A fluorogenic histone deacetylase assay well suited for high-throughput activity screening
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DOI：
10.1016/s1074-5521(02)00305-8
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发表时间：
2003-01-01
影响因子：
--
通讯作者：
Schwienhorst, A
中科院分区：
文献类型：
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作者：
Wegener, D;Wirsching, F;Schwienhorst, A
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。最近的研究结果表明，HDAC可能是恶性疾病化疗干预的关键靶点。因此，一种简便而灵敏的HDAC活性荧光检测方法将加快HDAC在转录调控和体外筛选药物发现方面的研究。在本研究中，我们合成了一种新型的HDACs荧光底物，它的C端带有一个E-乙酰化的赖氨酰基和一个相邻的MCA基团。乙酰化赖氨酰部分去乙酰化后，分子成为胰酶的底物，在检测的后续步骤中释放高荧光的AMC分子。荧光强度与脱乙酰基底物分子的量成正比，即HDAC活性。非同位素、均一化验非常适合于高通量的HDAC抑制剂筛选。
Histone deacetylases (HDACs) are important enzymes for the transcriptional regulation of gene expression in eukaryotic cells. Recent findings suggest that HDACs could be key targets for chemotherapeutic intervention in malignant diseases. A convenient and sensitive fluorogenic assay for HDAC activity would therefore expedite studies of HDAC in transcriptional regulation and in vitro screening for drug discovery. In this study, novel fluorogenic substrates of HDACs were synthesized with an E-acetylated lysyl moiety and an adjacent MCA moiety at the C terminus of the peptide chain. Upon deacetylation of the acetylated lysyl moiety, molecules became substrates for trypsin, which released highly fluorescent AMC molecules in a subsequent step of the assay. The fluorescence increased in direct proportion to the amount of deacetylated substrate molecules, i.e., HDAC activity. The nonisotopic, homogeneous assay is well suited for high-throughput HDAC inhibitor screening.