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.

Past strategies and future directions for identifying AMP-activated protein kinase (AMPK) modulators

“…A recently developed high-throughput assay aims to detect small molecules that bind AMPK through displacement of a protein-sensitive fluorescent probe shown to bind the AMPK regulatory region (Fig. 4) (S. Sinnett, J. Sexton, & J. Brenman, 2013).…”

“…Equipped with these new insights, researchers were then able to design a high-throughput screening assay using MANT-ADP, a protein-sensitive fluorescent probe that detects competitive binding of ADP to the regulatory fragment and full-length AMPK (Fig. 4) (S. Sinnett, et al, 2013).…”

“…Understandably, the moderate to high expression levels of α 1 , β 1 , and γ 1 in many tissues may motivate researchers to use purified α 1 β 1 γ 1 in protein-based assays. Indeed, the MANT-ADP binding assay described earlier was validated and optimized in the presence of α 1 β 1 γ 1 (Neumann, Woods, Carling, Wallimann, & Schlattner, 2003; S. Sinnett, et al, 2013).…”

A Highly Sensitive Non-Radioactive Activity Assay for AMP-Activated Protein Kinase (AMPK)

“…AMP-Activated Protein Kinase (AMPK) is a three-subunit protein kinase that functions as central cellular energy sensor and regulator of energy homeostasis in eukaryotes [ 1 , 2 , 3 , 4 ]. AMPK detects cellular energy states as ratios of AMP, ADP, and ATP (adenylate energy charge [ 5 ] ([ATP]+0.5x[ADP])/([ATP]+[ADP]+[AMP])) [ 6 ] by competitive binding of all three adenine nucleotides to three separate sites in its γ-subunit [ 6 , 7 , 8 ]. Energy stress, i.e., high ratios of AMP, and ADP, to ATP, strongly activate the AMPK kinase activity by multiple mechanisms [ 9 , 10 , 11 , 12 , 13 ], resulting in the phosphorylation of numerous metabolic and regulatory proteins in the cell.…”

“…Consequently, development of therapeutic AMPK activity modulators is pursued by many pharmaceutical companies. Direct AMPK activation is determined by various kinase assays, including relatively low throughput radioactive [ 16 , 17 ] and HPLC-based [ 18 ] kinase assays, as well as high throughput fluorescence-based assays [ 4 , 7 , 19 ], which have relatively small signal-to-noise ratios and suffer from fluorescence interference by a substantial fraction of screening compounds. Here, we present a sensitive two-step kinase assay that is amenable to high throughput screening, has an exceptional (ca.…”