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 AMPKß1-containing complexes in intact cells and was unable to activate an AMPKß1 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 ß1 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.

Coordination of GPR40 and Ketogenesis Signaling by Medium Chain Fatty Acids Regulates Beta Cell Function.

Diabetes prevalence increases with age, and ß-cell dysfunction contributes to the incidence of the disease. Dietary lipids have been recognized as contributory factors in the development and progression of the disease. Unlike long chain triglycerides, medium chain triglycerides (MCT) increase fat burning in animal and human subjects as well as serum C-peptide in type 2 diabetes patients. We evaluated the beneficial effects of MCT on ß-cells in vivo and in vitro. MCT improved glycemia in aged rats via ß-cell function assessed by measuring insulin secretion and content. In ß-cells, medium chain fatty acid (MCFA)-C10 activated fatty acid receptor 1 FFAR1/GPR40, while MCFA-C8 induced mitochondrial ketogenesis and the C8:C10 mixture improved ß cell function. We showed that GPR40 signaling positively impacts ketone body production in ß-cells, and chronic treatment with ß-hydroxybutyrate (BHB) improves ß-cell function. We also showed that BHB and MCFA help ß-cells recover from lipotoxic stress by improving mitochondrial function and increasing the expression of genes involved in ß-cell function and insulin biogenesis, such as Glut2, MafA, and NeuroD1 in primary human islets. MCFA offers a therapeutic advantage in the preservation of ß-cell function as part of a preventative strategy against diabetes in at risk populations.

A novel ATP-synthase-independent mechanism coupling mitochondrial activation to exocytosis in insulin-secreting cells.

Pancreatic ß-cells sense glucose, promoting insulin secretion. Glucose sensing requires the sequential stimulation of glycolysis, mitochondrial metabolism and Ca2+ entry. To elucidate how mitochondrial activation in ß-cells contributes to insulin secretion, we compared the effects of glucose and the mitochondrial substrate methylsuccinate in the INS-1E insulin-secreting cell line at the respective concentrations at which they maximally activate mitochondrial respiration. Both substrates induced insulin secretion with distinct respiratory profiles, mitochondrial hyperpolarization, NADH production and ATP-to-ADP ratios. In contrast to glucose, methylsuccinate failed to induce large [Ca2+] rises and exocytosis proceeded largely independently of mitochondrial ATP synthesis. Both glucose- and methylsuccinate-induced secretion was blocked by diazoxide, indicating that Ca2+ is required for exocytosis. Dynamic assessment of the redox state of mitochondrial thiols revealed a less marked reduction in response to methylsuccinate than with glucose. Our results demonstrate that insulin exocytosis can be promoted by two distinct mechanisms one of which is dependent on mitochondrial ATP synthesis and large Ca2+ transients, and one of which is independent of mitochondrial ATP synthesis and relies on small Ca2+ signals. We propose that the combined effects of Ca2+ and redox reactions can trigger insulin secretion by these two mechanisms.

Standardized LC×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 × 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 (Trifolium pratense) 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-LC×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.

Chemokine receptor antagonist development.

This chapter describes assays that focus on the characterization of compounds identified in high--throughput screening campaigns, and the subsequent medicinal chemistry programs. They cover methods to determine potency in buffer, the effect of whole blood on the compounds' activity and finally the pharmacokinetic (PK)/pharmacodynamic (PD) -relationship of the compounds in a rodent species.

Discovery of a novel series of CXCR3 antagonists.

The discovery of a novel series of CXCR3 antagonists is described. Starting from an HTS positive, iterative optimization gave potent compounds (IC(50) 15 nM in a chemotaxis assay). The strategy employed to improve the metabolic stability of these derivatives is described.