3 research outputs found
Natural (dihydro)phenanthrene plant compounds are direct activators of AMPK through its allosteric drug and metabolite–binding site
AMP-activated protein kinase (AMPK) is a central energy sensor that coordinates the response to energy challenges to maintain cellular ATP levels. AMPK is a potential therapeutic target for treating metabolic disorders, and several direct synthetic activators of AMPK have been developed that show promise in preclinical models of type 2 diabetes. These compounds have been shown to regulate AMPK through binding to a novel allosteric drug and metabolite (ADaM)–binding site on AMPK, and it is possible that other molecules might similarly bind this site. Here, we performed a high-throughput screen with natural plant compounds to identify such direct allosteric activators of AMPK. We identified a natural plant dihydrophenathrene, Lusianthridin, which allosterically activates and protects AMPK from dephosphorylation by binding to the ADaM site. Similar to other ADaM site activators, Lusianthridin showed preferential activation of AMPKβ1-containing complexes in intact cells and was unable to activate an AMPKβ1 S108A mutant. Lusianthridin dose-dependently increased phosphorylation of acetyl-CoA carboxylase in mouse primary hepatocytes, which led to a corresponding decrease in de novo lipogenesis. This ability of Lusianthridin to inhibit lipogenesis was impaired in hepatocytes from β1 S108A knock-in mice and mice bearing a mutation at the AMPK phosphorylation site of acetyl-CoA carboxylase 1/2. Finally, we show that activation of AMPK by natural compounds extends to several analogs of Lusianthridin and the related chemical series, phenanthrenes. The emergence of natural plant compounds that regulate AMPK through the ADaM site raises the distinct possibility that other natural compounds share a common mechanism of regulation
Standardized LCĂ—LC-ELSD Fractionation Procedure for the Identification of Minor Bioactives via the Enzymatic Screening of Natural Extracts
To identify natural bioactive compounds
from complex mixtures such
as plant extracts, efficient fractionation for biological screening
is mandatory. In this context, a fully automated workflow based on
two-dimensional liquid chromatography (2D-LC Ă— LC) was developed,
allowing for the production of hundreds of semipure fractions per
extract. Moreover, the ELSD response was used for online sample weight
estimation and automated concentration normalization for subsequent
bioassays. To evaluate the efficiency of this protocol, an enzymatic
assay was developed using AMP-activated protein kinase (AMPK). The
activation of AMPK by nonactive extracts spiked with biochanin A,
a known AMPK activator, was enhanced greatly when the fractionation
workflow was applied compared to screening crude spiked extracts.
The performance of the workflow was further evaluated on a red clover
(<i>Trifolium pratense</i>) extract, which is a natural
source of biochanin A. In this case, while the crude extract or 1D
chromatography fractions failed to activate AMPK, semipure fractions
containing biochanin A were readily localized when produced by the
2D-LCĂ—LC-ELSD workflow. The automated fractionation methodology
presented demonstrated high efficiency for the detection of bioactive
compounds at low abundance in plant extracts for high-throughput screening.
This procedure can be used routinely to populate natural product libraries
for biological screening