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ACS Chem Biol. 2016 Mar 18;11(3):734-41. doi: 10.1021/acschembio.5b00709. Epub 2015 Oct 14.

Microfluidic Mobility Shift Profiling of Lysine Acetyltransferases Enables Screening and Mechanistic Analysis of Cellular Acetylation Inhibitors.

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Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Frederick, Maryland 21702, United States.
Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health , Rockville, Maryland 20850, United States.


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.

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