Homogenous Fluorescent Assays for Characterizing Small-Molecule Activators of AMP-Activated Protein Kinase (AMPK)

Abstract

AMP activated protein kinase (AMPK) is a key regulator of cellular metabolism. AMPK activity is modulated in part by binding of AMP to the γ-subunit of the kinase, which increases the activity of the catalytic α-subunit. Because increased AMPK activity in the liver and in skeletal muscle leads to increased fatty acid oxidation and decreased cholesterol and fatty acid biosynthesis, activators of AMPK are being sought for treatment of type-2 diabetes and other metabolic disorders. The unique mechanism of AMPK activation offers an opportunity to develop small molecules that directly upregulate AMPK activity, and there exists a need for simplified methods to identify and characterize small-molecules that show isoform-specific effects on AMPK. We have developed a suite of fluorescence-based assays to identify and characterize such compounds, and have used these to characterize and compare activity of recombinant AMPK α1β1γ1 and α2β1γ1 isoforms in response to small molecule activators and inhibitors.

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

“…The streptavidin donor beads have a b-ASP binding capacity of about 50 nM, a concentration that is two orders of magnitude below the Km of the reaction and leads to substrate exhaustion. We therefore set up a two-step assay ( Figure 2 a) to allow in the first step the kinase reaction to proceed at substrate concentrations (ATP: 100 μM; b-ASP peptide: 50 μM) above Km (Km ATP: 26–35 μM; Km of less optimal AMPK substrate peptide SAMS: 27 μM) [ 4 , 31 ], and then dilute the reaction 1000-fold to achieve a low concentration (50 nM) of total b-ASP (b-ASP + b- p ASP) that matches the streptavidin donor bead capacity during the subsequent (second step) Alphascreen reaction in 384 well plates. Because AMPK phosphorylates b-ASP more efficiently than the commonly used SAMS peptide substrate, it was important to establish conditions at which AMPK does not cause substrate exhaustion.…”

“…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. The net effect of these phosphorylation events is a cellular reprogramming to activate ATP-generating metabolic pathways, such as glucose and fatty acid mobilization, uptake, and catabolism, and the inhibition of ATP-consuming programs, such as the synthesis of macromolecular building blocks, growth, and proliferation [ 1 , 2 , 4 ].…”

“…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.…”