RESEARCH ARTICLE


A High Throughput Assay for Discovery of Small Molecules that Bind AMP-activated Protein Kinase (AMPK)



Sarah E Sinnett1, Jonathan Z Sexton2 , Jay E Brenman3, 4, *
1 Neurobiology Curriculum, University of North Carolina Chapel Hill (UNC);
2 Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University;
3 UNC Neuroscience Center;
4 Department of Cell Biology and Physiology, UNC


Article Metrics

CrossRef Citations:
0
Total Statistics:

Full-Text HTML Views: 1242
Abstract HTML Views: 524
PDF Downloads: 184
Total Views/Downloads: 1950
Unique Statistics:

Full-Text HTML Views: 371
Abstract HTML Views: 248
PDF Downloads: 105
Total Views/Downloads: 724



© Sinnett et al.; Licensee Bentham Open.

open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

* Address correspondence to this author at the Neuroscience Center and Department of Cell Biology and Physiology, UNC School of Medicine, Chapel Hill, NC 27599, USA, Tel: 919-843-3637, Fax: 919-966-9605, E-mail: brenman@med.unc.edu


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.

Keywords: : ADP, AMPK, canonical ATP-binding site, environment-sensitive fluorophore, high throughput assay, regulatory fragment.