A spectrophotometric assay for histone deacetylase 8
A spectrophotometric assay for histone deacetylase 8
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DOI：
10.1016/j.ab.2007.08.031
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发表时间：
2008-01-01
影响因子：
2.9
通讯作者：
Zheng, Weiping
中科院分区：
文献类型：
--
作者：
Fatkins, David G.;Zheng, Weiping
人们一直在积极探索经典蛋白质去乙酰化酶的抑制剂，以开发新一代的癌症治疗方法。因此，开发一种用于经典酶催化反应的新型便捷检测平台可能会促进药物研发进程。我们之前的研究表明，在由经典酶组蛋白去乙酰化酶8（HDAC8）催化的、源自人p53肿瘤抑制蛋白C末端的肽模板上的反应中，N - ε - 硫代乙酰 - 赖氨酸对N - ε - 乙酰 - 赖氨酸具有功能模拟作用。基于此，我们通过用埃尔曼试剂5,5'-二硫代双（2 - 硝基苯甲酸）对酶促脱硫乙酰化产生的硫代乙酸酯进行定量，开发了一种分光光度法HDAC8检测方法。我们进一步证明，这种新的检测方法对HDAC8相对于HDAC1和2以及对存在于HeLa细胞核提取物中的其他经典蛋白质去乙酰化酶具有选择性，因此它不仅可能适用于筛选HDAC8选择性抑制剂，而且还适用于在（病理）生理条件下选择性地评估HDAC8的活性。（C）2007爱思唯尔公司。保留所有权利。
Inhibitors for the classical protein deacetylase enzymes have been actively pursued to develop the next generation of cancer therapy. Developing a novel convenient assay platform for the classical enzyme-catalyzed reactions could thus facilitate the drug discovery process. Based on our previous studies demonstrating the functional mimicry of N-epsilon-thioacetyl-lysine for N-epsilon-acetyl-lysine in the reaction catalyzed by the classical enzyme histone deacetylase 8 (HDAC8) on a peptide template derived from the C terminus of the human p53 tumor suppressor protein, we have developed a spectrophotometric HDAC8 assay via quantifying thioacetate produced from the enzymatic dethioacetylation with Ellman's reagent 5,5'-dithiobis(2-nitrobenzoate). We further demonstrated that this novel assay was selective for HDAC8 versus HDAC1 and 2 and for other classical protein deacetylase enzymes present in the HeLa nuclear extracts, thus making it potentially suitable not only for screening HDAC8-selective inhibitors but also for selectively assessing HDAC8 activity under (patho)physiological conditions. (C) 2007 Elsevier Inc. All rights reserved.