Microfluidic Mobility Shift Profiling of Lysine Acetyltransferases Enables Screening and Mechanistic Analysis of Cellular Acetylation Inhibitors.
Microfluidic Mobility Shift Profiling of Lysine Acetyltransferases Enables Screening and Mechanistic Analysis of Cellular Acetylation Inhibitors.
复制标题
赖氨酸乙酰转移酶的微流体迁移率变化分析可实现细胞乙酰化抑制剂的筛选和机理分析。
DOI：
10.1021/acschembio.5b00709
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发表时间：
2016
影响因子：
4
通讯作者：
Meier,JordanL
中科院分区：
文献类型：
--
作者：
Sorum,AlexanderW;Shrimp,JonathanH;Roberts,AllisonM;Montgomery,DavidC;Tiwari,NeilK;Lal-Nag,Madhu;Simeonov,Anton;Jadhav,Ajit;Meier,JordanL
赖氨酸乙酰转移酶(KATS)在包括癌症在内的许多疾病中是信号的关键调节因子。确定KATS在疾病中的靶向功能的一个主要挑战是缺乏具有良好特性的、细胞活性的KAT抑制剂。为了应对这一挑战，我们在这里报告了一个用于发现和表征小分子Kat抑制剂的微流控迁移率转换平台。开发了四种Kat酶(p300、CREBBP、Morf和Gcn5)的新型荧光肽底物。酶催化的乙酰化改变了微流控芯片中这些多肽的电泳迁移率，从而可以方便和直接地监测Kat的活性。一个Pilot Screen被用来展示微流控迁移率变化图谱用于识别已知的和新的Kat活性调节剂的效用。KAT活性的实时动力学监测显示，用于细胞研究的天然产物KAT抑制剂藤黄酚表现出时间依赖性和洗涤剂敏感的抑制作用，与基于聚集的机制一致。相反，细胞通透性双底物抑制剂TAT-CoA表现出有效的和时间无关的KAT抑制作用，突出了它作为KAT活性细胞抑制剂的潜在用途。这些研究将微流控迁移率变化图谱定义为发现和表征KAT活性小分子抑制剂的强大平台，并为KAT抑制剂在细胞环境中的应用提供了潜在重要的机制见解。
Lysine acetyltransferases (KATs) are critical regulators of signaling in many diseases, including cancer. A major challenge in establishing the targetable functions of KATs in disease is a lack of well-characterized, cell-active KAT inhibitors. To confront this challenge, here we report a microfluidic mobility shift platform for the discovery and characterization of small molecule KAT inhibitors. Novel fluorescent peptide substrates were developed for four well-known KAT enzymes (p300, Crebbp, Morf, and Gcn5). Enzyme-catalyzed acetylation alters the electrophoretic mobility of these peptides in a microfluidic chip, allowing facile and direct monitoring of KAT activity. A pilot screen was used to demonstrate the utility of microfluidic mobility shift profiling to identify known and novel modulators of KAT activity. Real-time kinetic monitoring of KAT activity revealed that garcinol, a natural product KAT inhibitor used in cellular studies, exhibits time-dependent and detergent-sensitive inhibition, consistent with an aggregation-based mechanism. In contrast, the cell-permeable bisubstrate inhibitor Tat-CoA exhibited potent and time-independent KAT inhibition, highlighting its potential utility as a cellular inhibitor of KAT activity. These studies define microfluidic mobility shift profiling as a powerful platform for the discovery and characterization of small molecule inhibitors of KAT activity, and provide mechanistic insights potentially important for the application of KAT inhibitors in cellular contexts.