Development of novel cellular histone-binding and chromatin-displacement assays for bromodomain drug discovery.
Development of novel cellular histone-binding and chromatin-displacement assays for bromodomain drug discovery.
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DOI：
10.1186/s13072-015-0026-4
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发表时间：
2015
影响因子：
3.9
通讯作者：
Andersen JN
中科院分区：
文献类型：
--
作者：
Zhan Y;Kost-Alimova M;Shi X;Leo E;Bardenhagen JP;Shepard HE;Appikonda S;Vangamudi B;Zhao S;Tieu TN;Jiang S;Heffernan TP;Marszalek JR;Toniatti C;Draetta G;Tyler J;Barton M;Jones P;Palmer WS;Geck Do MK;Andersen JN
“读取”组蛋白密码的蛋白质是表观遗传控制的核心元素，而结合乙酰基-赖氨酸基序的溴结构域越来越被认为是疾病状态的潜在介质。值得注意的是，第一种基于BET溴结构域的疗法已经进入临床试验，并且对剖析其他含溴结构域蛋白在人类疾病中的治疗相关性存在广泛的兴趣。通常，药物开发通过高通量筛选来促进和加速，其中测定需要是灵敏的、稳健的、具有成本效益的和可扩展的。然而，对于溴结构域，它缺乏催化活性，否则可以监测（使用经典酶学），基于细胞的，药物靶点接合测定的发展一直具有挑战性。因此，与其他蛋白质家族（例如，组蛋白脱乙酰酶和甲基转移酶）。在这里，我们提出了一套新的染色质和组蛋白结合试验使用AlphaLISA，原位细胞提取和荧光为基础的，高内容的成像。首先，使用TRIM 24作为实例，从生物化学肽竞争测定法修改同质的基于珠粒的AlphaScreen技术以测量TRIM 24布罗莫结构域与细胞中内源性组蛋白H3的结合（AlphaLISA）。第二，开发了一种目标不可知的高通量成像平台，以量化化学探针从染色质/核结构解离内源性蛋白质的能力。在保持总体核形态的同时，该程序从具有通过免疫荧光（IF）或荧光蛋白显微镜观察到的药物靶向置换的细胞中提取可溶性、非染色质结合的蛋白质。这些试验的药理学评价交叉验证了其实用性、灵敏度和耐用性。最后，使用遗传学和药理学方法，我们剖析了TRIM 24、BRD 4、ATAD 2和SMARCA 2对染色质结合的结构域贡献，说明了原位细胞提取平台的多功能性/实用性。总之，我们已经开发了两种新的互补的和基于细胞的药物-靶标接合测定，扩展了用于溴结构域工具化合物开发的药效学测定的库。这些测定已经通过成功的TRIM 24布罗莫结构域抑制剂程序进行了验证，其中使用基于细胞的测定优化微摩尔先导分子（IACS-6558）以产生第一个单位数纳摩尔TRIM 24抑制剂（IACS-9571）。总之，本文所述的测定平台准备加速新型化学探针的发现和开发，以实现基于表观遗传学的疗法的前景。本文的在线版本（doi：10.1186/s13072-015-0026-4）包含补充材料，可供授权用户使用。
Proteins that ‘read’ the histone code are central elements in epigenetic control and bromodomains, which bind acetyl-lysine motifs, are increasingly recognized as potential mediators of disease states. Notably, the first BET bromodomain-based therapies have entered clinical trials and there is a broad interest in dissecting the therapeutic relevance of other bromodomain-containing proteins in human disease. Typically, drug development is facilitated and expedited by high-throughput screening, where assays need to be sensitive, robust, cost-effective and scalable. However, for bromodomains, which lack catalytic activity that otherwise can be monitored (using classical enzymology), the development of cell-based, drug-target engagement assays has been challenging. Consequently, cell biochemical assays have lagged behind compared to other protein families (e.g., histone deacetylases and methyltransferases). Here, we present a suite of novel chromatin and histone-binding assays using AlphaLISA, in situ cell extraction and fluorescence-based, high-content imaging. First, using TRIM24 as an example, the homogenous, bead-based AlphaScreen technology was modified from a biochemical peptide-competition assay to measure binding of the TRIM24 bromodomain to endogenous histone H3 in cells (AlphaLISA). Second, a target agnostic, high-throughput imaging platform was developed to quantify the ability of chemical probes to dissociate endogenous proteins from chromatin/nuclear structures. While overall nuclear morphology is maintained, the procedure extracts soluble, non-chromatin-bound proteins from cells with drug-target displacement visualized by immunofluorescence (IF) or microscopy of fluorescent proteins. Pharmacological evaluation of these assays cross-validated their utility, sensitivity and robustness. Finally, using genetic and pharmacological approaches, we dissect domain contribution of TRIM24, BRD4, ATAD2 and SMARCA2 to chromatin binding illustrating the versatility/utility of the in situ cell extraction platform. In summary, we have developed two novel complementary and cell-based drug-target engagement assays, expanding the repertoire of pharmacodynamic assays for bromodomain tool compound development. These assays have been validated through a successful TRIM24 bromodomain inhibitor program, where a micromolar lead molecule (IACS-6558) was optimized using cell-based assays to yield the first single-digit nanomolar TRIM24 inhibitor (IACS-9571). Altogether, the assay platforms described herein are poised to accelerate the discovery and development of novel chemical probes to deliver on the promise of epigenetic-based therapies. The online version of this article (doi:10.1186/s13072-015-0026-4) contains supplementary material, which is available to authorized users.