title: Microfluidic Mobility Shift Profiling of Lysine Acetyltransferases Enables Screening and Mechanistic Analysis of Cellular Acetylation Inhibitors.
abstract: 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.
authors.affiliation: Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Frederick, Maryland 21702, United States.
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Sorum
authors.affiliation: Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Frederick, Maryland 21702, United States.
authors.forename: Jonathan H
authors.initials: JH
authors.lastname: Shrimp
authors.affiliation: Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Frederick, Maryland 21702, United States.
authors.forename: Allison M
authors.initials: AM
authors.lastname: Roberts
authors.affiliation: Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Frederick, Maryland 21702, United States.
authors.forename: David C
authors.initials: DC
authors.lastname: Montgomery
authors.affiliation: Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Frederick, Maryland 21702, United States.
authors.forename: Neil K
authors.initials: NK
authors.lastname: Tiwari
authors.affiliation: Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health , Rockville, Maryland 20850, United States.
authors.forename: Madhu
authors.initials: M
authors.lastname: Lal-Nag
authors.affiliation: Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health , Rockville, Maryland 20850, United States.
authors.forename: Anton
authors.initials: A
authors.lastname: Simeonov
authors.affiliation: Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health , Rockville, Maryland 20850, United States.
authors.forename: Ajit
authors.initials: A
authors.lastname: Jadhav
authors.affiliation: Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Frederick, Maryland 21702, United States.
authors.forename: Jordan L
authors.initials: JL
authors.lastname: Meier
mesh_terms: D000107:Acetylation / Q000187:drug effects; D000070837:Lysine Acetyltransferases / Q000235:genetics / Q000378:metabolism*; D046210:Microfluidic Analytical Techniques; D054852:Small Molecule Libraries
chemical_list: D054852:Small Molecule Libraries; D000070837:Lysine Acetyltransferases