title: A High Throughput Assay for Discovery of Small Molecules that Bind AMP-activated Protein Kinase (AMPK).
abstract: AMPK is a conserved heterotrimeric serine-threonine kinase that regulates anabolic and catabolic pathways in eukaryotes. Its central role in cellular and whole body metabolism makes AMPK a commonly proposed therapeutic target for illnesses characterized by abnormal energy regulation, including cancer and diabetes. Many AMPK modulators, however, produce AMPK-independent effects. To identify drugs that modulate AMPK activity independent of the canonical ATP-binding pocket found throughout the kinome, we designed a robust fluorescence-based high throughput screening assay biased toward the identification of molecules that bind the regulatory region of AMPK through displacement of MANT-ADP, a fluorescent ADP analog. Automated pin tools were used to rapidly transfer small molecules to a low volume assay mixture on 384-well plates. Prior to assay validation, we completed a full assay optimization to maximize the signal-to-background and reduce variability for robust detection of small molecules displacing MANT-ADP. After validation, we screened 13,120 molecules and identified 3 positive hits that dose-dependently inhibited the protein-bound signal of MANT-ADP in the presence of both full-length AMPK and the truncated "regulatory fragment" of AMPK, which is missing the kinase active site. The average Z'-factor for the screen was 0.55 and the compound confirmation rate was 60%. Thus, this fluorescence-based assay may be paired with in vitro kinase assays and cell-based assays to help identify molecules that selectively regulate AMPK with fewer off-target effects on other kinases.
authors.affiliation: Neurobiology Curriculum, University of North Carolina Chapel Hill (UNC);
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Sinnett
authors.affiliation: Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University;
authors.forename: Jonathan Z
authors.initials: JZ
authors.lastname: Sexton
authors.affiliation: UNC Neuroscience Center; ; Department of Cell Biology and Physiology, UNC.
authors.forename: Jay E
authors.initials: JE
authors.lastname: Brenman
keywords: ADP; AMPK; canonical ATP-binding site; environment-sensitive fluorophore; high throughput assay; regulatory fragment.