Ciossek Thomas, Julius Heiko, Wieland Heike, Maier Thomas, Beckers Thomas
Therapeutic Area Oncology, ALTANA Pharma-a member of the Nycomed Group, Byk-Gulden Str. 2, 78467 Konstanz, Germany.
Anal Biochem. 2008 Jan 1;372(1):72-81. doi: 10.1016/j.ab.2007.07.024. Epub 2007 Aug 2.
Most cellular assays that quantify the efficacy of histone deacetylase (HDAC) inhibitors measure hyperacetylation of core histone 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. IC(50) values derived from concentration-effect curves correlated well with EC(50) values derived from a cellomics array scan histone 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.
大多数用于量化组蛋白去乙酰化酶（HDAC）抑制剂功效的细胞分析方法都是检测核心组蛋白H3和H4的高乙酰化状态。在此，我们描述了一种新方法，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。进入HeLa宫颈癌细胞或K562慢性髓性白血病细胞后，会形成去乙酰化产物Boc-K-AMC，细胞裂解后，该产物会被胰蛋白酶切割，最终释放出荧光团AMC。使用该分析方法测定了知名HDAC抑制剂辛二酰苯胺异羟肟酸、LBH589、曲古抑菌素A和MS275的细胞活性。从浓度效应曲线得出的IC(50)值与从细胞组学阵列扫描组蛋白H3高乙酰化分析得出的EC(50)值高度相关。细胞HDAC活性分析已适用于均相形式，完全兼容机器人筛选。在Tecan Genesis Freedom工作站上生成的浓度效应曲线具有高度可重复性，信噪比为5.7，Z'因子为0.88，表明该分析方法非常稳健。最后，在一次中通量筛选实验中对一个聚焦于HDAC抑制剂的文库进行了分析。细胞HDAC活性的抑制与HeLa细胞的细胞毒性和组蛋白H3高乙酰化以及生化分析中对人重组HDAC1的抑制密切相关。因此，通过使用Boc-K(Ac)-AMC作为细胞可渗透的HDAC底物，可以以简单且均相的方式在完整细胞中测量包括含HDAC1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。
