Recent progress in the identification of adenosine monophosphate-activated protein kinase (AMPK) activators

doi:10.1016/j.bmcl.2016.09.065

Bioorganic & Medicinal Chemistry Letters

Volume 26, Issue 21, 1 November 2016, Pages 5139-5148

https://doi.org/10.1016/j.bmcl.2016.09.065 Get rights and content

Abstract

Adenosine monophosphate-activated protein kinase (AMPK), a serine/threonine heterotrimeric protein kinase, is a critical regulator of cellular and whole body energy homeostasis. There are twelve known AMPK isoforms that are differentially expressed in tissues and species. Dysregulation of AMPK signaling is associated with a multitude of human pathologies. Hence isoform-selective activators of AMPK are actively being sought for the treatment of cardiovascular and metabolic diseases. The present review summarizes the status of direct AMPK activators from the patent and published literature.

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Acknowledgements

We thank Russell Miller for providing a graphic and for helpful comments and Francis Rajamohan for providing a figure.

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Citation Excerpt :
We have recently reported our own direct AMPK activator, an analogue of the benzimidazole series.13 These molecules bind at the interface of the glycogen-binding domain of the β-subunit and the α-subunit resulting in a conformational change and activation.14,15 In addition, the pan-AMPK activator O304 developed by the group at Betagenon protects Thr172 from dephosphorylation has shown some efficacy in a Phase II clinical study.16
Using an in-cell AMPK activation assay, we have developed structure–activity relationships around a hit pyridine dicarboxamide 5 that resulted in 40 (R419). A particular focus was to retain the on-target potency while also improving microsomal stability and reducing off-target activities, including hERG inhibition. We were able to show that removing a tertiary amino group from the piperazine unit of hit compound 5 improved microsomal stability while hERG inhibition was improved by modifying the substitution of the central core pyridine ring. The SAR resulted in 40, which continues to maintain on-target potency. Compound 40 was able to activate AMPK in vivo after oral administration and showed efficacy in animal models investigating activation of AMPK as a therapy for glucose control (both db/db and DIO mouse models).
Natural (dihydro)phenanthrene plant compounds are direct activators of AMPK through its allosteric drug and metabolite–binding site
2022, Journal of Biological Chemistry
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.
Amarogentin, a secoiridoid glycoside, activates AMP- activated protein kinase (AMPK)to exert beneficial vasculo-metabolic effects
2019, Biochimica et Biophysica Acta - General Subjects
Citation Excerpt :
Among the direct activators, A769662, 991 and salicyclate activate AMPK upon binding to the allosteric site ADaM [31,47,48]. However, their limited potency and oral bioavailability has led to modifications in their core structure as seen for PF06409577, which was taken up to phase I clinical trial before being withdrawn [47]. In this study we identified a natural Gentiana derived secoiridoid glycoside, Amarogentin (AG), that acts as a direct activator of AMPK.
AMP-activated protein kinase (AMPK) is a drug target for treatment of metabolic and cardiovascular complications. Extracts of Gentianaceace plants exhibit anti-diabetic and anti-atherosclerotic effects, however, whether their phyto-constitutents activate AMPK remains to be determined.
Molecular docking of Gentiana lutea constituents was performed with crystal structure of human α₂β₁γ₁ trimeric AMPK (PDB ID: 4CFE). Binding of Amarogentin (AG) to α₂ subunit was confirmed through isothermal titration calorimetry (ITC) and in vitro kinase assays were performed. L6 myotube, HUH7 and endothelial cell cultures were employed to validate in silico and in vitro observations. Lipid lowering and anti-atherosclerotic effects were confirmed in streptozotocin induced diabetic mice via biochemical measurements and through heamatoxylin and eosin, Masson's trichrome and Oil Red O staining.
AG interacts with the α₂ subunit of AMPK and activates the trimeric kinase with an EC₅₀ value of 277 pM. In cell culture experiments, AG induced phosphorylation of AMPK as well as its downstream targets, acetyl-coA-carboxylase (ACC) and endothelial nitric oxide synthase (eNOS). Additionally, it enhanced glucose uptake in myotubes and blocked TNF-α induced endothelial inflammation. Oral supplementation of AG significantly attenuated diabetes-mediated neointimal thickening, and collagen and lipid deposition in the aorta. It also improved circulating levels of lipids and liver function in diabetic mice.
In conclusion, AG exerts beneficial vasculo-metabolic effects by activating AMPK.
Amarogentin, a naturally occurring secoiridoid glycoside, is a promising lead for design and synthesis of novel drugs for treatment and management of dyslipidemia and cardiovascular diseases.
Chemical genetic screen identifies Gapex-5/GAPVD1 and STBD1 as novel AMPK substrates
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AMP-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis, acting as a sensor of energy and nutrient status. As such, AMPK is considered a promising drug target for treatment of medical conditions particularly associated with metabolic dysfunctions. To better understand the downstream effectors and physiological consequences of AMPK activation, we have employed a chemical genetic screen in mouse primary hepatocytes in an attempt to identify novel AMPK targets. Treatment of hepatocytes with a potent and specific AMPK activator 991 resulted in identification of 65 proteins phosphorylated upon AMPK activation, which are involved in a variety of cellular processes such as lipid/glycogen metabolism, vesicle trafficking, and cytoskeleton organisation. Further characterisation and validation using mass spectrometry followed by immunoblotting analysis with phosphorylation site-specific antibodies identified AMPK-dependent phosphorylation of Gapex-5 (also known as GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1)) on Ser902 in hepatocytes and starch-binding domain 1 (STBD1) on Ser175 in multiple cells/tissues. As new promising roles of AMPK as a key metabolic regulator continue to emerge, the substrates we identified could provide new mechanistic and therapeutic insights into AMPK-activating drugs in the liver.
Comparison of lactate and β-hydroxybutyrate in the treatment of concanavalin-A induced hepatitis
2018, International Immunopharmacology
Citation Excerpt :
AMPK is activated in conditions of cellular stress associated with reduced ATP and increased AMP levels [37]. Upon activation, AMPK works to activate pathways that generate ATP like catabolic processes and inhibit pathways that consume ATP like anabolic processes [38]. The elevated expression of p-AMPK in the livers of Con A-intoxicated mice points to the cellular stress from which the hepatocytes suffer.
Simple metabolites released during physical exercise and fasting like lactate (Lac) and β-hydroxybutyrate (BHB) have recently been shown to possess anti-inflammatory properties. However, the effects of these metabolites in immune mediated hepatitis are still unknown. Accordingly, we investigated the role of Lac, BHB and their combination on experimentally induced hepatic inflammation. Adult male mice were administered concanavalin A (Con A, 15 mg/kg, intravenous) for 12 h. In the treatment groups, mice were treated 1 h after Con A-intoxication with Lac (500 mg/kg, intraperitoneal), BHB (300 mg/kg, intraperitoneal) and their combination. The results demonstrated that Lac and BHB, especially when combined together, alleviated Con A-induced hepatocellular injury (ALT, AST and LDH) and necrosis (hematoxylin-eosin and electron microscopy). These beneficial effects correlated with attenuating Con A-induced elevation in hepatic oxidative stress parameters (MDA and NOx). Mechanistically, administration of Lac and BHB led to inhibition of Con A-induced phosphorylation of JNK and AMPK proteins in the liver to the same extent. These effects were concordant with curbing Con A-mediated overexpression of the pro-inflammatory cytokines TNF-α, IL-6 and IL-12 and activation of the transcription factor NF-κB. The marked anti-inflammatory properties of combining Lac and BHB were attributed to their cooperation in repressing immune cells (monocytes and neutrophils) infiltration to the liver. Unlike BHB, Lac administration markedly induced the reparative STAT3 and ERK phosphorylation in the livers of Con A-intoxicated mice at the early time point. In conclusion, the simultaneous use of Lac and BHB might be an auspicious strategy for limiting immune mediated hepatitis.
AMPK activation promotes gastroprotection through mutual interaction with the gaseous mediators H <inf>2</inf> S, NO, and CO
2018, Nitric Oxide - Biology and Chemistry
Citation Excerpt :
This enzyme exists as a heterotrimeric complex of 3 subunits (α, β, and γ). The α-subunit contains the catalytic domain and has two isoforms, α1 and α2, that are phosphorylated at Thr172 upon enzyme activation [14]. AMPK activation increases NO and CO by stimulation of NO synthase (eNOS) and heme oxygenase-1 (HO-1), respectively [15,16].
Activation of 5′ adenosine monophosphate-activated protein kinase (AMPK) stimulates production of the gaseous mediators nitric oxide (NO) and carbon monoxide (CO), which are involved in mucosal defense and gastroprotection. As AMPK itself has gastroprotective effects against several gastric ulcer etiologies, in the present study, we aimed to elucidate whether AMPK may also prevent ethanol-induced injury and play a key role in the associated gastroprotection mediated by hydrogen sulfide (H₂S), NO, and CO. Mice were pretreated with AICAR (20 mg/kg, an AMPK activator) alone or with 50% ethanol. Other groups were pretreated with respective gaseous mediator inhibitors PAG, l-NAME, or ZnPP IX 30 min prior to AICAR, or with gaseous mediator donors NaHS, Lawesson's reagent and l-cysteine (H₂S), SNP, l-Arginine (NO), Hemin, or CORM-2 (CO) 30 min prior to ethanol with or without compound C (10 mg/kg, a non-selective AMPK inhibitor). H₂S, nitrate/nitrite (NO₃⁻/NO₂⁻), bilirubin levels, GSH and MDA concentration were evaluated in the gastric mucosa. The gastric mucosa was also collected for histopathological analysis and AMPK expression assessment by immunohistochemistry. Pretreatment with AICAR attenuated the ethanol-induced injury and increased H₂S and bilirubin levels but not NO₃⁻/NO₂⁻ levels in the gastric mucosa. In addition, inhibition of H₂S, NO, or CO synthesis exacerbated the ethanol-induced gastric damage and inhibited the gastroprotection by AICAR. Pretreatment with compound C reversed the gastroprotective effect of NaHS, Lawesson's reagent, l-cysteine, SNP, l-Arginine, CORM-2, or Hemin. Compound C also reversed the effect of NaHS on H₂S production, SNP on NO₃⁻/NO₂⁻ levels, and Hemin on bilirubin levels. Immunohistochemistry revealed that AMPK is present at basal levels mainly in the gastric mucosa cells, and was increased by pretreatment with NaHS, SNP, and CORM-2. In conclusion, our findings indicate that AMPK activation exerts gastroprotection against ethanol-induced gastric damage and mutually interacts with H₂S, NO, or CO to facilitate this process.

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Digest paperRecent progress in the identification of adenosine monophosphate-activated protein kinase (AMPK) activators

Abstract

Graphical abstract

Section snippets

Acknowledgements

Curr. Opin. Cell Biol.

Trends Endocrinol. Metab.

Metabolism

Trends Endocrinol. Metab.

Expert Opin. Ther. Pat.

Structure

Cell Metab.

Mol. Cell. Endocrinol.

J. Biol. Chem.

J. Lipid Res.

J. Biol. Chem.

Cell Metab.

Trends Pharmacol. Sci.

Cancer Lett.

Cell Metab.

Cell. Signal.

Kidney Int.

Vascul. Pharmacol.

Trends Endocrinol. Metab.

Cell Metab.

Cell Metab.

Protein Expr. Purif.

J. Biol. Chem.

FEBS Lett.

Chem. Biol.

Bioorg. Med. Chem. Lett.

FEBS Lett.

Chem. Biol.

J. Biol. Chem.

Chem. Biol.

Physiol. Rev.

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Nature

Cell Res.

Nat. Commun.

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Nature

Nature

Science

Biochem. J.

Diabetes

J. Physiol.

Exp. Physiol.

Diabetologia

Science

FASEB J.

J. Clin. Invest.

Digest paper
Recent progress in the identification of adenosine monophosphate-activated protein kinase (AMPK) activators