Quantitative Analysis of Histone Deacetylase-1 Selective Histone Modifications by Differential Mass Spectrometry
Quantitative Analysis of Histone Deacetylase-1 Selective Histone Modifications by Differential Mass Spectrometry
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DOI：
10.1021/pr800510p
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发表时间：
2008-12-01
影响因子：
4.4
通讯作者：
Hendrickson, Ronald C.
中科院分区：
文献类型：
--
作者：
Lee, Anita Y. H.;Paweletz, Cloud P.;Hendrickson, Ronald C.
1类和2类组蛋白脱乙酰酶（HDAC）酶的抑制剂已在人体临床试验中显示出抗肿瘤活性。最近，人们对开发亚型选择性HDAC抑制剂产生了兴趣，这些抑制剂旨在保留抗癌活性，同时减少潜在的副作用。考虑到HDAC 1在肿瘤增殖和存活中的作用，已经开始努力选择性靶向HDAC 1。HDAC 1特异性抑制剂的开发将需要鉴定与体外和体内HDAC 1抑制密切相关的HDAC 1选择性药效学标志物。HDAC靶点接合的现有组蛋白标记物是使用泛HDAC抑制剂开发的，并且不一定代表同种型特异性抑制剂的稳健读数。因此，我们启动了一种蛋白质组学方法来鉴定HDAC 1抑制的读数。该方法涉及使用差示质谱法（dMS）通过分析用各种HDAC抑制剂处理的富含组蛋白的细胞级分来鉴定组蛋白的翻译后变化。在这项研究中，我们分析了从HCT 116人结肠癌细胞系中分离的组蛋白，这些细胞系已经用对HDAC 1、HDAM和3以及HDAC 1、3和6酶具有特异性的多种化学类别的化合物处理过。在两个独立的实验中，我们确定了24个与HDAC 1抑制相关的特征。在由HDAC 1选择性抑制剂调节的肽中，有来自组蛋白H2 B的Ac-H2 B-K5和来自组蛋白H3的Ac-H3-K18。使用针对特定组蛋白乙酰基-赖氨酸残基的市售抗体来确认这些肽也提供了体内和体外HDAC 1选择性抑制剂的药效学读数。这些结果显示了dMS在指导鉴定特异性读数以帮助开发HDAC选择性抑制剂方面的效用。
Inhibitors of class 1 and class 2 histone deacetylase (HDAC) enzymes have shown antitumor activity in human clinical trials. More recently, there has been interest in developing subtype-selective HDAC inhibitors designed to retain anticancer activity while reducing potential side effects. Efforts have been initiated to selectively target HDAC1 given its role in tumor proliferation and survival. The development of HDAC1-specific inhibitors will require the identification of HDAC1-selective pharmacodynamic markers that correlate closely with HDAC1-inhibition in vitro and in vivo. Existing histone markers of HDAC target engagement were developed using pan-HDAC inhibitors and do not necessarily represent robust readouts for isoform-specific inhibitors. Therefore, we have initiated a proteomic approach to identify readouts for HDAC1 inhibition. This approach involves the use of differential mass spectrometry (dMS) to identify post-translational changes in histones by profiling histone-enriched cellular fractions treated with various HDAC inhibitors. In this study, we profiled histones isolated from the HCT116 human colon cancer cell line that have been treated with compounds from multiple chemical classes that are specific for HDAC1; HDAM and 3; and HDAC1, 3, and 6 enzymes. In two independent experiments, we identified 24 features that correlated with HDAC1-inhibition. Among the peptides modulated by HDAC1-selective inhibitors were Ac-H2B-K5 from histone H2B, and Ac-H3-K18 from histone H3. Commercially available antibodies to specific histone acetyl-lysine residues were used to confirm that these peptides also provide pharmacodynamic readouts for HDAC1-selective inhibitors in vivo and in vitro. These results show the utility of dMS in guiding the identification of specific readouts to aid in the development of HDAC-selective inhibitors.