Microfluidic Mobility Shift Profiling of Lysine Acetyltransferases Enables Screening and Mechanistic Analysis of Cellular Acetylation Inhibitors
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- 作者：Alexander W. Sorum ; Jonathan H. Shrimp ; Allison M. Roberts ; David C. Montgomery ; Neil K. Tiwari ; Madhu Lal-Nag ; Anton Simeonov ; Ajit Jadhav ; Jordan L. Meier
- 刊名：ACS Chemical Biology
- 出版年：2016
- 出版时间：March 18, 2016
- 年：2016
- 卷：11
- 期：3
- 页码：734-741
- 全文大小：458K
- ISSN：1554-8937
文摘
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.