Assessing histone demethylase inhibitors in cells: lessons learned.
Assessing histone demethylase inhibitors in cells: lessons learned.
复制标题
DOI：
10.1186/s13072-017-0116-6
复制
发表时间：
2017
影响因子：
3.9
通讯作者：
Müller S
中科院分区：
文献类型：
--
作者：
Hatch SB;Yapp C;Montenegro RC;Savitsky P;Gamble V;Tumber A;Ruda GF;Bavetsias V;Fedorov O;Atrash B;Raynaud F;Lanigan R;Carmichael L;Tomlin K;Burke R;Westaway SM;Brown JA;Prinjha RK;Martinez ED;Oppermann U;Schofield CJ;Bountra C;Kawamura A;Blagg J;Brennan PE;Rossanese O;Müller S
组蛋白赖氨酸去甲基化酶（kdm）由于其在染色质组织中的调节作用以及与癌症和精神障碍等疾病的密切关联而成为药物靶点。首个针对KDM1的KDM抑制剂已经进入临床试验，并且正在努力开发针对该靶标类的有效、选择性和细胞活性“探针”分子。评估KDM抑制剂在细胞中的特异性作用及其细胞选择性的稳健细胞分析是开发高质量抑制剂的先决条件。在这里，我们描述了使用高含量的细胞免疫荧光测定作为一种方法来证明靶参与细胞。开发了一组kdm的Jumonji C亚家族的检测，以涵盖JmjC系统发育树的所有主要分支。这些试验比较了化合物对野生型KDM蛋白和催化失活KDM蛋白的活性，其中参与活性位点铁配位的残基发生突变，使酶活性失活。这些突变体对于评估KDM抑制剂的特定作用和揭示对组蛋白甲基化状态的间接影响至关重要。报道的检测方法利用了异位表达的去甲基化酶，我们证明了它们用于分析几种最近发现的KDM抑制剂及其结构匹配的非活性对照。生成的数据与评估内源性KDM抑制的分析结果相吻合，并证实了在分离酶的生化分析中观察到的选择性。我们发现细胞渗透性和与2-氧葡萄糖酸盐的竞争都会影响生化活性向细胞抑制的转化。基于高含量的免疫荧光分析已经建立了8个KDM成员的2-氧戊二酸依赖加氧酶，覆盖了JmjC-KDM系统发育树的所有主要分支。使用全长、野生型和催化无活性突变的异位表达蛋白，以及结构匹配的无活性对照化合物，可以检测由于化合物毒性导致组蛋白甲基化变化的非特异性效应。开发的检测方法提供了一种组蛋白赖氨酸去甲基化酶家族范围的工具，用于评估KDM抑制剂的细胞活性和靶向疗效。此外，所提供的数据可能为进一步的研究提供信息，以评估组蛋白赖氨酸甲基化抑制剂的细胞活性。本文的在线版本（doi:10.1186/s13072-017- 016 -6）包含补充材料，可供授权用户使用。
Histone lysine demethylases (KDMs) are of interest as drug targets due to their regulatory roles in chromatin organization and their tight associations with diseases including cancer and mental disorders. The first KDM inhibitors for KDM1 have entered clinical trials, and efforts are ongoing to develop potent, selective and cell-active ‘probe’ molecules for this target class. Robust cellular assays to assess the specific engagement of KDM inhibitors in cells as well as their cellular selectivity are a prerequisite for the development of high-quality inhibitors. Here we describe the use of a high-content cellular immunofluorescence assay as a method for demonstrating target engagement in cells. A panel of assays for the Jumonji C subfamily of KDMs was developed to encompass all major branches of the JmjC phylogenetic tree. These assays compare compound activity against wild-type KDM proteins to a catalytically inactive version of the KDM, in which residues involved in the active-site iron coordination are mutated to inactivate the enzyme activity. These mutants are critical for assessing the specific effect of KDM inhibitors and for revealing indirect effects on histone methylation status. The reported assays make use of ectopically expressed demethylases, and we demonstrate their use to profile several recently identified classes of KDM inhibitors and their structurally matched inactive controls. The generated data correlate well with assay results assessing endogenous KDM inhibition and confirm the selectivity observed in biochemical assays with isolated enzymes. We find that both cellular permeability and competition with 2-oxoglutarate affect the translation of biochemical activity to cellular inhibition. High-content-based immunofluorescence assays have been established for eight KDM members of the 2-oxoglutarate-dependent oxygenases covering all major branches of the JmjC-KDM phylogenetic tree. The usage of both full-length, wild-type and catalytically inactive mutant ectopically expressed protein, as well as structure-matched inactive control compounds, allowed for detection of nonspecific effects causing changes in histone methylation as a result of compound toxicity. The developed assays offer a histone lysine demethylase family-wide tool for assessing KDM inhibitors for cell activity and on-target efficacy. In addition, the presented data may inform further studies to assess the cell-based activity of histone lysine methylation inhibitors. The online version of this article (doi:10.1186/s13072-017-0116-6) contains supplementary material, which is available to authorized users.