A homogeneous cellular histone deacetylase assay suitable for compound profiling and robotic screening
A homogeneous cellular histone deacetylase assay suitable for compound profiling and robotic screening
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DOI：
10.1016/j.ab.2007.07.024
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发表时间：
2008-01-01
影响因子：
2.9
通讯作者：
Beckers, Thomas
中科院分区：
文献类型：
--
作者：
Ciossek, Thomas;Julius, Heiko;Beckers, Thomas
大多数量化组蛋白去乙酰化酶（HDAC）抑制剂功效的细胞检测是测量核心组蛋白H3和H4的过度乙酰化。在此我们描述一种新方法，即使用底物Boc - K(Ac) - 7 - 氨基 - 4 - 甲基香豆素（AMC）直接测量细胞HDAC酶活性。在穿透HeLa宫颈癌细胞或K562慢性髓性白血病细胞后，形成去乙酰化产物Boc - K - AMC，细胞裂解后，该产物被胰蛋白酶切割，最终释放出荧光团AMC。使用该检测方法测定了辛二酰苯胺异羟肟酸（suberoylanilide hydroxamic acid）、LBH589、曲古抑菌素A（trichostatin A）和MS275作为已知HDAC抑制剂的细胞效力。从浓度 - 效应曲线得出的IC50值与从细胞组学阵列扫描组蛋白H3过度乙酰化检测得出的EC50值具有良好的相关性。细胞HDAC活性检测被调整为均相形式，与机器人筛选完全兼容。在Tecan Genesis Freedom工作站上生成的浓度 - 效应曲线具有高度重现性，信噪比为5.7，Z'因子为0.88，表明该检测非常稳健。最后，在一个中等通量的筛选活动中对一个以HDAC抑制剂为重点的文库进行了分析。细胞HDAC活性的抑制与HeLa细胞的细胞毒性和组蛋白H3过度乙酰化以及在生化检测中对人重组HDAC1的抑制具有良好的相关性。因此，通过使用Boc - K(Ac) - AMC作为一种细胞可渗透的HDAC底物，包括含HDAC1的复合物在内的各种蛋白质赖氨酸特异性去乙酰化酶的活性能够以一种简单且均相的方式在完整细胞中进行测量。（C）2007爱思唯尔公司。保留所有权利。
Most cellular assays that quantify the efficacy of histone deacetylase (HDAC) inhibitors measure hyperacetylation of core historic proteins H3 and H4. Here we describe a new approach, directly measuring cellular HDAC enzymatic activity using the substrate Boc-K(Ac)-7-amino-4-methylcoumarin (AMC). After penetration into HeLa cervical carcinoma or K562 chronic myeloid leukemia cells, the deacetylated product Boc-K-AMC is formed which, after cell lysis, is cleaved by trypsin, finally releasing the fluorophor AMC. The cellular potency of suberoylanilide hydroxamic acid, LBH589, trichostatin A, and MS275 as well-known HDAC inhibitors was determined using this assay. IC50 values derived from concentration-effect curves correlated well with EC50 values derived from a cellomics array scan historic H3 hyperacetylation assay. The cellular HDAC activity assay was adapted to a homogeneous format, fully compatible with robotic screening. Concentration-effect curves generated on a Tecan Genesis Freedom workstation were highly reproducible with a signal-to-noise ratio of 5.7 and a Z' factor of 0.88, indicating a very robust assay. Finally, a HDAC-inhibitor focused library was profiled in a medium-throughput screening campaign. Inhibition of cellular HDAC activity correlated well with cytotoxicity and histone H3 hyperacetylation in HeLa cells and with inhibition of human recombinant HDAC1 in a biochemical assay. Thus, by using Boc-K(Ac)-AMC as a cell-permeable HDAC substrate, the activity of various protein lysine-specific deacetylases including HDAC1-containing complexes is measurable in intact cells in a simple and homogeneous manner. (C) 2007 Elsevier Inc. All rights reserved.