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

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.