Activators of AMPK: not just for type II diabetes.

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Medicinal Chemistry

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Activators of AMPK: not just for Type II diabetes

Recent discoveries of AMPK activators point to the large number of therapeutic candidates that can be transformed to successful designs of novel drugs. AMPK is a universal energy sensor and influences almost all physiological processes in the cells. Thus, regulation of the cellular energy metabolism can be achieved in selective tissues via the artificial activation of AMPK by small molecules. Recently, special attention has been given to direct activators of AMPK that are regulated by several nonspecific upstream factors. The direct activation of AMPK, by definition, should lead to more specific biological activities and as a result minimize possible side effects.

AMP-activated kinase (AMPK) is a member of a large family of highly conserved heterotrimeric serine/threonine kinases [1] . The main role of AMPK is to fine tune the cell’s energy needs by monitoring the availability of ATP and quickly responding to different environmental signals. Activated AMPK lowers the cell’s energy expenditure allowing the cell to undergo mild adaptations to accomplish permanent changes in body’s homeostasis [2–4] . AMPK’s structure has been extensively studied [5] . The enzyme is a heterotrimer complex composed of three subunits, a catalytic α- (63 kDa), two regulatory β-, and a γ-subunit. AMPK is encoded by seven different genes: α1, α2, β1, β 2, γ1, γ2 and γ3 [6] . The tentative possible combination of these genes creates 12 isoenzymes, which are differently distributed in the tissues of different species [7] . For example, the main AMPK isoform in human liver is α1β2γ1, however, in the dog and rat livers, the dominant AMPK isoforms are α1β1γ1 and α2β1γ1, respectively [7–9] . The crystalline structure of the whole mammalian AMPK complex (α1β2γ1) has been elucidated [10] and, in addition, the structures of several different subunits and their isoforms were successfully rendered. These structures are available as 3D images in the PDB [11] . In total, 18 descriptors in the

10.4155/FMC.14.74 © 2014 Future Science Ltd

Ilana Zaks1, Tamar Getter1 & Arie Gruzman*,1 Department of Chemistry, Faculty of Exact Sciences, Bar Ilan University, Ramat Gan, 5290002, Israel *Author for correspondence: Tel.: +972 3738 4597 Fax: +972 7384 053 [email protected] 1

PDB are relevant for ‘human AMPK’ [12–15] . Interactions between the AMPK complex’s different domains and subunits and interactions between the AMPK subunits and various ligands were studied using many in silico techniques, including molecular modeling, homology modeling and all-atom molecular dynamics simulations. In addition, much research was done on the structure-, ligandand fragment-based designs of potential AMPK activators and an impressive structural basis for developing AMPK activators and AMPK targeted drug discovery has been achieved [16] . As with many other kinases, AMPK activation is regulated by several different mechanisms: phosphorylation/dephosphorylation, allosteric regulation and myristoylation [17] . AMPK’s degree of activation is directly proportional to the degree of cellular stress stimuli, which deplete ATP resources. Moreover, certain physiological ATP-costly processes (e.g., proliferation) will also activate AMPK, further antagonizing such biochemical events [18] . The canonic AMPK activation occurs when AMP binds to the regulatory γ subunit’s CBS domain. This induces conformational changes in the whole AMPK complex and enhances the phosphorylation of Thr172 by upstream kinases in the catalytic α-subunit [19,20] . The ATP concentration-dependent dis-

Future Med. Chem. (2014) 6(11), 1325–1353

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Review Zaks, Getter & Gruzman sociation of the AMP molecule is followed by the binding of ATP instead of AMP to the CBS domain. ATP binding causes additional conformational changes in AMPK, triggering Thr172 dephosphorylation, usually by protein phosphatases 2A, resulting in loss of activity [21] . LKB1 is a major upstream kinase that phosphorylates the α-subunit’s Thr172 following activating of AMPK [22] . Another kinase that activates AMPK is CaMKKβ. Additionally, the AMPK complex has internal regulatory mechanisms such as an intrinsic Thr172-autophosphorylating ability, which allows for a baseline level of AMPK activity and the functioning of the autoinhibitory domain [23,24] . The body’s biggest consumer of ATP is the skeletal muscles. In this tissue, AMPK augments the rate of glucose uptake and upregulates the expression of genes encoding GLUT-4 and hexokinase II [25] . AMPK also exerts its catabolic function in skeletal muscles by enhancing lipid oxidation and by activating two major citric acid cycle enzymes: citrate synthase and succinate dehydrogenase [26,27] . The positive effect of AMPK on the rate of glucose uptake in skeletal muscles is independent of insulin’s action and this noninsulin-dependent blood glucose lowering effect is extremely important in regulating the blood glucose level in Type 2 diabetes mellitus (T2D) patients [28] . Thus, in insulin-resistant T2D patients, physical exercises (the physiological activation of AMPK) without any pharmacological intervention are able to reduce blood glucose levels to normal numbers [29] . Importantly, the physical exercises lead to wide changes in the energy metabolism in skeletal muscles and AMPKrelated effects are only part of this sophisticated orchestra. It was shown in several works that AMPK only partially regulates the contraction-stimulated glucose uptake and other AMPK-independent pathways that also contribute to massive increased glucose influx into working skeletal muscles [30–32] . AMPK activation in adipocytes causes massive fatty acid oxidation, a dramatic inhibition of lipogenic flux and the arrest of triglyceride, fatty acid and cholesterol synthesis [33] . Not surprisingly, AMPK also prevents the liberation of free fatty acids in adipocytes by inactivating the key regulatory enzyme, hormone-sensitive lipase, by phosphorylation [34] . AMPK is also involved in the crosstalk between fat tissue and skeletal muscles by regulating the expression of adiponectin, a physiKey term Indirect activators of AMPK: Small molecules that target upstream AMPK regulatory proteins or affect intracellular processes that do not directly activate AMPK. Usually, the reduction of ATP level is the main trigger for such a collateral effect.

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ological insulin sensitizer in skeletal muscles, which reduces the insulin resistance in skeletal muscles especially in obese T2D patients [35,36] . Besides skeletal muscles and adipocytes, the liver is the third major player that regulates blood glucose levels and energy metabolism in the body. Similar to adipose tissue, AMPK activation in the liver causes increased fatty acid oxidation, reduced lipogenesis, and blocks cholesterol and triglycerides biosynthesis [37] . Recent publications doubt the effect of AMPK on the liver gluconeogenesis [38–40] . Previously, it was widely accepted that this process is negatively regulated by AMPK [41]). However, Foretz et al. showed paradoxical data that an indirect activator of AMPK, metformin, inhibits hepatocyte glucose output despite the total absence of AMPK in hepatocytes [42] . Additionally, Miller et al. suggested a novel mechanism of metformin’s effect on hepatic gluconeogenesis, which involves being an antagonist to the physiological role of glucagon [38] . These reports are not surprising. It is of crucial importance for the function of any organism that such critical metabolic response will have multiple regulatory pathways [43] . Increased hepatic glucose output is one of the major problems in treating T2D [44] . Therefore, inhibition of liver gluconeogenesis by AMPK activators is an important characteristic for any future antidiabetic drug development for this class of compounds. AMPK activation in skeletal muscles, fat tissue and liver are beneficial for diabetic patients. However, in recent years this classical therapeutic application for AMPK activators was expanded to other therapeutic applications such as the development of anticancer, anti-inflammatory, antineurodegenerative, antiviral, hepatoprotective and platelet antiaggregation drugs (Figure 1) . In Table 1 & Figure 2A & B we present the biological data and chemical structures on non-direct AMPK activators. For the majority of these AMPK activators, the exact mode of action has yet to be elucidated. In many cases the activation of AMPK is only a secondary effect, resulting in activation of other regulatory or metabolic pathways. For example, any compound that inhibits the mitochondrial respiratory chain will also indirectly activate AMPK because of decreased production of ATP. Generally, many indirect AMPK activators act by modulating cellular ATP content [45] . Moreover, even well-known AMPK activators such as metformin and AICAR [45] are able to cause cellular biological effects in the total absence of AMPK. [46] . Additionally, the activation of upstream AMPK regulatory kinases (e.g., tumor suppressor LKB1) obviously will lead to the stimulation of many downstream targets, which only one of them is AMPK. Thus, it is very difficult to distinguish within these

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Review

Current and potential use of AMPK activators

x Reduction of myocardical damage

Anticancer proapoptotic effect

Amyloride degradation

Fat utilization

Anti-inflammatory effect

HCV infection inhibition

Anti-proliferative effect

x x

x

Inhibition of neointima formation

Inhibition of the IL-6 secretion from macrophages

x x

Inhibition of liver steatosis

x

Glucose Cholesterol Triglycerides Fatty acids

Liver production HO HO

OH O

HO

Antiaggregation effect

Inhibition of adipogenesis

H OH

HO

H H

Decreased level of blood lipids and glucose

Neuroprotective effect

Figure 1. Biological effects of AMPK activators. HCV: Hepatitis C virus.

complex interactions between those that are specific to AMPK and those that are related to other biological effectors. Moreover, the majority of the AMPK activators have pleiotropic modes of action, making it difficult to point to AMPK activation as a specific pathway leading to a precise biological effect [47] . Several examples for the problematic interpretation of biological effects of nondirect AMPK activators are discussed below. This divergence is narrower in the use of direct AMPK activators. Diabetes, obesity & hyperlipidimia There is ongoing research in isolating and characterizing novel antidiabetic AMPK activators from traditional ethnic herbal medicines. Several such compounds were recently reported, among them: damulin B (1), betulinic acid (2), cyaniding-3-beta-glycoside (3), ursolic acid (4), baicalein (5), p-coumaric acid (6), gingerol (7), licochalcone A (8) and dantron (9) (Table 1 & Figure 2A) . Damulin B is a novel dammarane-type saponin that was isolated from Gynostemma pentaphyllum, a plant with a long history of use in traditional oriental medicine [83] . The structure of damu-


lin B is very similar to another well-known AMPK natural activator, ginsenoside Rh2 [84] . Both compounds have a four ring triterpenoidic moiety conjugated to a carbohydrate domain. Damulin increases β-oxidation and exhibits a stimulatory effect on the rate of glucose uptake by upregulating GLUT4 translocation to the plasma membrane in L6 myotubes [48] . The range of minimal active concentrations of the compound in different assays presented in the manuscript was between 3 and 37 μM and this is quite adequate for in vitro assays. However, additional structural–activity relationship experiments should be conducted for the sake of potentially decreasing the therapeutic concentration of the compound. Another compound that increases the rate of glucose uptake via the activation of AMPK is p-coumaric acid [85] . This compound was already known in the early 70s as an antioxidant, as are the majority of other phenols and polyphenols [86] . A p-coumaric acid can be found in hundreds of plants, even in common ones, such as tomatoes and carrots [87] . In rat L6 myotubes, p-coumaric acid not only increases the rate of glucose uptake but also positively upregulates lipid metabolism

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Ursolic acid

Baicalein

p-Coumaric acid

Gingerol

Licochalcone A

Danthron

(R)-5,7-dihydroxy-3(4-hydroxybenzyl)8-methoxy-6methylchroman-4-one

1,3-bis(3,5dichlorophenyl)urea (COH-SR4)

WS070117

4

5

6

7

8

9

10

11

12


SC

SC

SC

NC

NC

NC

NC

NC

NC

NC

NC

10.0

Triglyceride accumulation ↓

AMPK phosphorylation

L6 C2C12

HepG2

10 μM

Glucose uptake ↑

Lipid contents ↓

Lipid accumulation ↓ Expression of adipogenesisrelated transcription factors and lipogenic proteins ↓

B16-F0 A2058 Proliferation ↓ Hs600T Apoptosis

IAR-20

3T3-L1

2.0

–

Glucose uptake ↑

10.0

2.0

–

4.0

–

–

–

β-oxidation ↑ Glucose uptake ↑

–

Adipocytes differentiation ↓ 400.0

–

Fatty acid oxidation ↑

–

5.0

–

Glucose uptake ↑, β-oxidation ↑ Lipid accumulation ↓ Lipogenesis ↓

Dose (mg/kg)

In vitro Biological effect

HepG2 C2C12 Lipid synthesis ↓ Glucose consumption ↑

HepG2

L6

L6

–

3T3-L1

HepG2

HepG2

L6

Type of cells

2.5

10.0

0.1

5.0–10.0

150.0

12.5

–

20.0

1.0–5.0

40.0

12.0

MEC (μM)

LPS: Lipopolysuccharides; MEC: Minimal effective concentration; NC: Natural compounds; SC: Synthetic compounds; VSMC: Vascular smooth muscles cell.

Ampkinone

Cyanidin-3-O-βglucoside

3

13

Betulinic acid

2

NC

NC

1

Damulin B

Source

Structure Compound name number

Table 1. Biological activity of nondirect AMPK activators. In vivo

–

–

Intracellular lipid accumulation ↓

–

Biological effect

–

–

Triglyceride accumulation in liver ↓

–

–

–

Diet-induced Blood glucose ↓ obese mice Insulin ↓ Improved metabolic symptoms

High-fat diet Serum lipids ↓ hamsters

–

C57B mice Antineoplastic athymic nude effect mice

–

–

High fat-fed ICR mice

–

–

High-fat diet Serum lipids ↓ mice

–

–

High fat-fed ICR mice

–

Type of animals

[61]

[60]

[59]

[58]

[57]

[56]

[55]

[54]

[53]

[52]

[51]

[50]

[49]

[48]

Ref.

Review Zaks, Getter & Gruzman


1-(3,5-dimethoxy phenyl)-3-hydroxy4-(4-methoxyphenyl) azetidin-2-one

1-(4-(trifluoromethyl) phenyl)-3-(3,4,5trifluorophenyl)urea

(2R,3S)-1,2,3,4-tetra SC hydronaphthalene-2,3diyl bis (4-bromo-3,5- dihydroxybenzoate)

Demethoxycurcumin

Celastrol

Belinostat

Gambogic acid

7-(benzyloxy)-5,6dihydroxy-2-phenyl4H-chromen-4-one

Quercetin

15

16

17

18

19

20

21

22

23


–

1.0–8.0

2.5

10.0

0.3

5.0

10.0

1.0–3.0

0.003– 0.012

27.0

MEC (μM) AMPK phosphorylation

Biological effect

–

Several human cancer cell lines

U87

PANC-1

MCF-7

–

Not mentioned in the paper

–

–

Apoptosis cell growth ↓ Apoptosis

–

Apoptosis

–

–

Human Proliferation ↓ triplenegative breast cancer cells Apoptosis

–

MDA-MB-231 Proliferation ↓

LS174T

–

–

–

Dose (mg/kg)

Proliferation ↓

HuTu-80 Cytotoxicity Human SW48

L6

Type of cells

In vitro


NC

SC

NC

SC

NC

NC

SC

SC

(3R,4R)-3-(3,4SC dimethoxybenzyl)4-(4-((3,4dimethoxybenzyl) oxy)-3-methoxybenzyl) tetrahydrofuran

Source

14


Table 1. Biological activity of nondirect AMPK activators (cont.).

Mice with high cholesterolinduced neurotoxicity

–

–

–

–

–

–

–

–

–

Type of animals

In vivo

Neuro protective effect behavioral performance ↑

–

–

–

–

–

–

–

–

–

Biological effect

[71]

[70]

[69]

[68]

[67]

[66]

[65]

[64]

[63]

[62]

Ref.


Review

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1330


Methylene blue

KS370G

RSVA314 RSVA405

Nectandrin B

Sophocarpine

(Z)-N-(4-((3-((1methylcyclohexyl) methyl)-2,4dioxothiazolidin5-ylidene) methyl) phenyl)-4-nitro-3- (trifluoromethyl) benzenesulfonamide

26

27

28 and 29

30

31

32

SC

NC

NC

SC

SC

SC

5.0

2400.0

1.0–3.0

1.0

3.5

0.2–1.0

0.1 nM

25

MEC (μM)

– 20.0

–

Adipogenesis ↓ Proliferation ↓

Steatosis ↓

–

–

3.0

Nitric oxide ↓ Expression of inducible nitric oxide synthase and cyclooxygenase-2 ↓ Amyloide degradation

–


–

–

Glucose uptake ↑


Dose (mg/kg)

Biological effect

THP-1 human IL-6 production ↓ macrophages

Rat primary hepatocytes

VSMC

3T3-L1

HEK293, mice primary neurons

BV-2 rat primary cultured microglia

HT22

Primary neurons

Transfected by mutant SOD1 NSC34 motor neuron-like cells

Type of cells

In vitro


Latrepirdine

25

SC

SC

24

Reluzole

Source


Table 1. Biological activity of nondirect AMPK activators (cont.).

–

–

Mouse femoral artery injury model

–

–

LPS-treated mice

–

–

–

Type of animals

In vivo

–

–

Neointima formation ↓

-

–

Neuro inflammation ↓

–

–

–

Biological effect

[80]

[79]

[78]

[77]

[76]

[75]

[74]

[73]

[72]

Ref.




SC

8-hydroxy-2,2,14,14- tetramethylpenta decanedioic acid

2-octynoic

33

34

Human monocytederived macrophages

Type of cells

3.0–100.0 Primary rats hepatocytes HepG2

50.0

MEC (μM)

Dose (mg/kg) 30.0

30.0

Vascular inflammation ↓ Acute-phase cytokines ↓ Expression of adhesion molecules ↓

De novo lipid synthesis ↓

In vitro Biological effect


Source


Table 1. Biological activity of nondirect AMPK activators (cont.). In vivo Biological effect

Wistar rats Golden Syrian hamsters

Liver triglyceride ↓ Markers of oxidative metabolism ↓ Hepatic steatosis ↓ Plasma atherogenic lipoproteins ↓ β-oxidation ↑

C57BL/6 mice Homing of leukocytes into inflammation site ↓ Epididymal fat-pad mass ↓ Adipose tissue inflammation ↓

Type of animals

[82]

[81]

Ref.



Review

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OH OH

OH HO HO

H

HO HO

O O

HO Cyanidin-3-O-beta-glucoside (3)

O HO

H

OH

O

OH

H

O

HO

p-Coumaric acid (6)

Baicalein (5)

HO

HO O

OH

OH

O

O

O

OH

HN Cl

OH

Cl

Cl

WS070117 (12)

1,3-bis(3,5-dichlorophenyl)urea (11)

N

N

N

Cl

O

N

N

O

O

O

O

O

O O

O

O

O

O

O OH

O

O

Ampkinone (13)

F3C

N

O

O

O O

O

F

O

F F

O (3R,4R)-3-(3,4-dimethoxybenzyl)4-(4-((3,4-dimethoxybenzyl)oxy)3-methoxybenzyl)tetrahydrofuran (14)

H N

H N

OH

O O

O

7-(benzyloxy)-5,6-dihydroxy-2-phenyl-4H-chromen-4-one (10)

Licochalcone A (8) H N

O

HO

O

OH

Dantron (9)

H N

Gingerol (7)

O

Ursolic acid (4)

HO

HO

O

HO COOH

OH

HO

Damulin B (1)

OH

OH

O

H Betulinic acid (2)

HO OH

O OH

HO

O

O

O

H

O

HO

OH

H

HO

+

O

1-(4-(trifluoromethyl)phenyl)-3-(3,4,5trifluorophenyl)urea (16)

1-(3,5-dimethoxyphenyl)-3-hydroxy -4-(4-methoxyphenyl)azetidin-2-one (15)

Figure 2A. Chemical structures of nondirect AMPK activators.

by promoting fatty acid β-oxidation and decreasing oleic acid-induced triglyceride accumulation [53] . All these effects were obtained at a concentration of 12 μM. These results suggest that p-coumaric acid has the potential to prevent or lower insulin resistance and

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T2D by modulating glucose and lipid metabolism. However, in comparison to other phenol-based AMPK activators, p-coumaric acid does not have any advanced properties, such as better bioavailability, effectiveness or potency. Moreover, these effects of p-coumaric acid



OH

O

Br O

Review

O

O

OH

OH

HO

O

OH O

Demethoxycurcumine (18)

H

O

O OH

O

HO

Celastrol (19)

Br OH

OH

O

(2R,3S)-1,2,3,4-tetrahydronaphthalene-2,3-diyl bis(4-bromo-3,5-dihydroxybenzoate) (17)

O

O

O HO

O

O

O

OH H

O N H OH

F F

O N H

S

OH

O

Belinostat (20)

O

Gambogic acid (21)

S

F

OH

O

Reluzole (24)

+ N

S

N

OH

HO

Cl

– O

Methylene blue (26)

OH

R-5,7-dihydroxy-3-(4-hydroxybenzyl)8-methoxy-6-methylchroman-4-one (22)

N

NH2

N H

O

HO

N

N H

O

OH

HO

N

OH O

Latrepirdine (25)

HO

Quercetin (23)

N N

H N

OH

N

O

OH

O

O HO

RSVA405 (29)

Nectandrin B (30)

O

OH

F RSVA314 (28)

F

H

F

S OH

O O

H H

N

N

O2N

O HO

O

H N N

N

HO

KS370G (27)

OH

8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid (33)

H N

O

O S

O

H

Sophocarpine (31)

N

O

O

OH (Z)-N-(4-((3-((1-methylcyclohexyl)methyl)-2,4-dioxothiazolidin-5-ylidene) methyl)phenyl)-4-nitro-3-(trifluoromethyl)benzenesulfonamide (32)

2-Octynoic acid (34)

Figure 2B. Chemical structures of nondirect AMPK activators.

can be related to its well-known antioxidant properties. The augmentation in the rate of glucose uptake and upregulation of lipid metabolism can be achieved independently from the AMPK activation as reported for resveratrol [88] . Moreover, many other biological


effects of p-coumaric acid, such as decreasing the risk of stomach cancer [89] , reducing the myocardial infarct size in rats [90] , inhibition of melanogenesis [91] and prevention of hepatotoxicity in ethanol-induced mice [92] , are AMPK independent.


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Review Zaks, Getter & Gruzman Another phenolic molecule that was reported to be an AMPK activator is gingerol, which is the major pungent phenol component in ginger [54] . This plant has been used in oriental medicine for treating several human diseases, including T2D [93] . Several recent studies have shown that gingerol reduces blood glucose levels in diabetic animal models and augments glucose uptake in vitro [94,95] . It was shown that gingerol’s antidiabetic mechanism of action may be related to AMPK-dependent stimulation of glucose uptake in skeletal muscles, since the compound increases the rate of glucose uptake in L6 myotubes, as described by Li et al. [96] . The same group also researched whether the effect of gingerol on the rate of glucose uptake in L6 is isoform specific. They selectively knocked down AMPKα1 or AMPKα2 by transfecting corresponding siRNAs. The results showed that gingerol stimulates glucose uptake only in the AMPKα2 knockdown cells. Although, the active concentration of gingerol in these experiments was very high (150 μM), these data show that the AMPK-dependent effect of gingerol on the rate of glucose uptake is mediated by the AMPKα1 isoform. Such results are important in further AMPK isoform-specific drug design. Designing isoforms of AMPK-specific compounds is the most comprehensively used approach in the field. AMPK also regulates lipid metabolism and, therefore, it is not surprising that this AMPK property was targeted as a potential therapeutic target for both diabetes and hyperlipidemia disorders. Several natural structures of AMPK activators that exhibit beneficial effects predominantly on lipid homeostasis, especially in hepatocytes (in vitro) and in liver (in vivo), were recently reported. For example, betulinic acid, a pentacyclic triterpene isolated from many plants, in a concentration of 40 μM was shown to decrease the accumulation of lipids in HepG2 cells and in rat primary hepatocytes. Moreover, an identical effect of the compound on lipid accumulation was observed in vivo. Quan et al. reported that mice fed a diet containing high amounts of lipids following the administration of betulinic acid in concentrations of 5 mg/kg, showed a significant reduction in hepatic steatosis [49] . Betulinic acid is also known as a potential anticancer agent, although the correlation between the activation of AMPK and its antiproliferative effect has not yet been reported [97] . One of the mechanisms of its anticancer effect is related to the induction of mitochondrial outer membrane permeabilization, which obviously can lead to mithochondrial dysfunction and, as a result, to a decrease in ATP production and further activation of AMPK [98] . Additionally, betulinic acid affects several AMPK-independent intracellular pathways (induction of Bcl-2 proteins, modulation of NF-kB activity, inhi-

1334


bition of T-cell cytokines and inducing tissue-damaging reactive oxygen species), which might be also involved in lipid metabolism regulation [99] . All these data support the idea that reduction of hepatic steatosis can be mediated by several AMPK-independent regulatory pathways. Another natural AMPK activator studied by this research group is licocachalcone A, a major phenolic compound of the glycyrrhiza plant [55] . Using the same mouse model, they showed that 10 mg/kg of licocachalcone dramatically decreased the accumulation of triglycerides in the mice livers and in concentrations of 5 μM, the compound exhibited similar inhibitory effects on triglyceride storage in HepG2 human hepatocellular liver carcinoma cells. Such impressive in vivo effects using a relatively low dose, makes this tested compound a very good antilipidemic drug candidate. Future investigation of the pharmacokinetic properties and possible toxic effects of licocachalcone A will better assess its potential as a new drug. Another naturally occurring compound is cyanidin-3-O-βglucoside, which shows a calmodulin kinase kinase (CaKK)-dependent AMPK activation in HepG2 cells. As a result of AMPK activation, the rate of fatty acid oxidation dramatically increased in these cells. It is important to mention that this effect was obtained at a 1 μM concentration, which is very reasonable for in vitro assays and gives hope for suitable in vivo effects [50] . However, it is also important to mention that activating CaKK leads to other non-AMP-related biological effects. Thus, the potential use of cyanidin-3-Oβ-glucoside as a selective antihyperlipidemic drug is limited by its nonspecific mode of action. Obesity is one of the major contributing factors towards developing T2D. Thus, inhibiting differentiation of 3T3-L1 preadipocytes to mature adipocytes by ursolic acid (a naturally occurring pentacyclic triterpenoid) is an interesting finding that may eventually lead to the development of new antiobesity drugs. Treatment with 10 μM of ursolic acid for 6 days reduced adipogenesis in differentiated 3T3-L1 cells by approximately 30%. This effect was mediated by the activation of LKB1, the upstream canonic regulator of AMPK activity [51] . Another well-known naturally occurring compound, baicalein has shown beneficial effects in mice with metabolic syndrome induced by a high-fat diet [52] . The control mice developed obesity, dyslipidemia, fatty liver and insulin resistance. However, the mice treated with baicalein (400 mg/kg, indeed very high dose) showed no such symptoms. Baicalein’s effect on the metabolic syndrome was found to be mediated by a specific AMPKα2-dependent activation in hepatocytes, which led to the lipid-lowering effect.



An additional AMPK activator from traditional Chinese medicine sources with potential antidiabetic properties is (R)-5,7-dihydroxy-3-(4-hydroxybenzyl)8-methoxy-6-methylchroman-4-one (10). This homoisoflavonoid effectively activates AMPK and its downstream effector, acetyl-CoA carboxylase (ACC) in IAR-20 rat liver epithelial cells in concentrations of 10 μM [57] . In parallel with the search for novel naturally occurring compounds that can act as antidiabetic drugs via AMPK activation, there are ongoing efforts to create novel synthetic AMPK activators for T2D/obesity treatments. We would like to highlight four recent examples of such molecules: (11), WS070117 (12), ampkinone (13) and (3R,4R)-3-(3,4-dimethoxybenzyl)4-(4-((3,4-dimethoxybenzyl)oxy)-3-methoxybenzyl) tetrahydrofuran (14). Inhibition of adipocyte differentiation to adult fat storing cells has been targeted by several obesity treatment strategies for the development of antiobesity and antidiabetic drugs. It was reported that COH-SR4, an aromatic urea derivative, inhibits adipocyte differentiation in 3T3-L1 cells [59] . The effect mainly occurs at the early phase of differentiation through the inhibition of mitotic clonal expansion and cell cycle arrest at the G1/S phase transition. COHSR4 significantly decreases lipid accumulation and downregulates the expression of many important adipogenesis-related transcription factors. The compound indirectly activates AMPK and two of its downstream regulatory proteins, raptor and tuberous sclerosis protein 2. The critical involvement of AMPK in adiposities differentiation was shown by a selective knockdown of AMPKα1/α2, which abrogates COH-SR4’s effect on adipogenesis and lipid accumulation in 3T3-L1 cells. Activation of AMPK was also achieved by using the synthetic adenosine derivative WS070117. This molecule contains the polyacetylated inosine moiety and has shown lipid lowering effects in vivo, as was reported by Zeqin et al. [60] . It is also important to mention that WS070117 is active in relatively low doses of only 2 mg/kg per day. This dose was sufficient to reduce serum levels of triglycerides, low density lipoproteins and total cholesterol. In addition, hepatic cholesterol and triglyceride contents decreased dramatically. In HepG2 as expected, WS070117 reduced lipid contents and activated AMPK. The effective concentration of the compound was also in the low range of approximately 1 μM. The excellent ability of phosphorylated AMPK to block the de novo hepatic lipogenesis is well known [100] and potent compounds like WS070117, which are able to mediate this effect via AMPK, can certainly be considered to be good candidates for future drug development. However, WS070117’s adenosinelike structure should be modified or masked because


Review

of the already known side effects of adenosine-based compounds in humans [101] . Oh et al. recently reported that ampkinone, a novel synthetic AMPK activator, shows antidiabetic and antiobesity effects in vivo [61] . Interestingly, the compound induced AMPK phosphorylation via activation of both upstream kinases, LKB1 and CaMKK. Ampkinone augments the rate of glucose uptake in both L6 and C2C12 myotubes and at a dose of 10 μg/kg per day and significantly reduces blood glucose levels in obese mice. Shen et al. reported an interesting structure–activity relationship study of arctigenin, which resulted in the design and synthesis of (3R,4R)-3-(3,4dimethoxybenzyl) -4- (4- ((3,4-dimethoxybenzyl) oxy)-3-methoxybenzyl)tetrahydrofuran (14). This molecule effectively activates AMPK in L6 myoblasts. According to this study, the 2-(3,4-dimethoxyphenyl) ethyl ether moiety was critical for inducing AMPK phosphorylation [62] . Metformine is still considered the primary AMPK activator type antidiabetic drug, although, in many cell cultures the effect of metformine on glucose metabolism is independent from AMPK activation [42,102–104] . Ongoing attempts to find naturally occurring molecules and in designing molecules with better pharmacokinetic and pharmacodynamic properties than metformin continues. Developing an ‘exercise pill’ is still a concept that many research groups in academia and industry are trying to materialize. AMPK activation in T2D patients is an excellent alternative to the not so effective insulin-dependent glucose uptake upregulation in skeletal muscles, the main process that downregulates the blood glucose level. A relatively new approach is to use AMPK activators as potential antiobesity therapeutic agents. However, AMPK activation in the brain, especially in the areas that regulate food intake, leads to increased appetite [105] . Thus, any potential antiobesity AMPK activators should have chemical structures that will not allow them to penetrate the blood–brain barrier (BBB). Otherwise, a very effective therapeutic approach against obesity, namely appetite reduction, will be replaced by a highly potent obesity risk factor, namely increased appetite. In this scenario, the peripheral positive effect of antiobese AMPK activators will be neutralized by increased food intake stimuli. All of the above compounds activate AMPK indirectly. Together with the activation of AMPK, the compounds also affect additional cellular regulatory mechanisms. This fact may explain the relatively high effective concentrations of several of the above described compounds. Additionally, such nonselective modes of action may lead to a broad spectrum of side effects. Moreover, the planned effect (AMPK activa-


1335

Review Zaks, Getter & Gruzman tion) occurs in all tissues and not only in the relevant tissues for glucose and lipid homeostasis regulation such as skeletal muscles, fat, liver and pancreas. The activation of AMPK in other tissues may be problematic, although there are big discrepancies in the data about benefits of the activation of AMPK in β-cells [106] . We therefore surmise that the therapeutic use of indirect activators of AMPK for future treatment of the described metabolic disorders will be limited. Conversely, direct tissue-specific (predominantly skeletal muscles and liver) AMPK isoform activators have huge therapeutic potential. Cancer The potential use of AMPK activators as anticancer agents is based on data published by Evans et al. in 2005 [107] . The authors showed that metformin, significantly lowered the risk of cancer (up to 30%) in T2D patients. This statistical study was later backed by biochemical evidences that indeed showed that AMPK plays an essential role in cancer transformation, progression and even in drug resistance to chemotherapy [108,109] . This finding is not surprising considering the general character of AMPK’s function, which is to block anabolic processes and allow catabolic processes. These actions obviously lead to an antiproliferative effect resulting in tumor suppression. In addition to the previously discussed molecular mechanisms that inhibit cellular ATP use for anabolic purposes (cell proliferation), there are additional regulatory pathways that are specific for oncogenic processes that AMPK controls. For example, AMPK directly regulates the main tumor suppressive factor p53 [110] and antagonizes the functions of the cell division regulator, mTOR [111] . However, a significant number of publications have raised doubts about AMPK’s role in cancer biogenesis and progression. First of all, AMPK is physiologically activated under hypoxia and protects cells in this stressful condition. However, hypoxia is the normal environment for a majority of cancer cells. Therefore, on the one hand, AMPK activation suppresses cancer progression because of its anabolic characteristic, but on the other hand, it protects ischemic cancer cells and supplies them with energy for survival via upregulated glycolysis (the Warburg effect). Moreover, in several human cancers, AMPK expression is significantly elevated and it does not interfere with the tumor’s progression [112–115] . Additionally, Shackelford et al. in their recent report presented data that the AMPK activator, phenformin, is most active in LKB1 compromised tumors [116] . Therefore, the results from the phenformin study might be explained by activation of unrelated AMPK pathways. Taken together, these data indicate that under certain conditions and in specific types of cancers, the

1336


antitumor activity of AMPK mimetics results from the balance between AMPK’s antiproliferative and antianabolic effects [117,118] . Moreover, in certain instances, both types of these AMPK effects were shown to be identically unregulated at the same time in same tumor [119] . Therefore, based on a general characteristic of AMPK actions, AMPK activators cannot be used as universal anticancer drugs as once thought. Despite this, several naturally occurring and synthetic AMPK activators were recently identified and tested for their possible antiproliferative properties in specific types of cancer. Some of them have biological effects in the nanomolar concentration range and are nontoxic to normal cells. For example, a series of novel 1,4-diaryl-2-azetidinones (15) that activate AMPK have shown strong antiproliferative activity, cell cycle inhibitory effects, and selective apoptosis induction in human duodenal and colon cancer cell lines [63] . It is important to mention that together with AMPK activation there is a strong possibility that additional mechanisms contribute to these biological effects. It is known that polyaromatic β-lactams derivatives exhibit cancer cell cytotoxicity by inducing DNA damage, inhibiting DNA replication and activating apoptosis [120,121] . Diaryl-2-azetidinones were shown to inhibit tubulin polymerization following the stimulation of the apoptotic pathway by activating the caspase-3 and PARP proteins. Therefore, AMPK activation may be only a secondary event of the massive cell stress caused by DNA damage, for example, and not the direct effect of the diaryl-2-azetidinones [122] . Nevertheless, even after only in vivo studies, the potential anticancer activity of 1,4-diaryl-2-azetidinones should be evaluated. If indeed the tested compounds selectively inhibit the proliferation of colon cancer, the therapeutic impact might be enormous due to the current lack of effective anticolon cancer chemotherapeutic agents [123] . Proliferation of human colon adenocarcinoma cells was inhibited by another synthetic AMPK activator: 1-(4-(trifluoromethyl)phenyl)-3-(3,4,5-trifluorophenyl)urea (16) [64] . In this case, the active concentration of the compound was 3 μM and not 12 nM as in diaryl-2-azetidinones. Such high in vitro effective concentration makes the potential use of the compound as an anticancer drug almost impossible. However, this is the first report of a novel family of AMPK activators, namely fluorinated N,N’-diarylureas and by using known medicinal chemistry tools for optimization of biological active molecules, it is possible to reduce the active in vitro concentration to the nanomole range. It is interesting to note that the structure of the aromatic urea AMPK activators (described in the diabetes and obesity sections of this review) are comparable to the fluorinated N,N’-diarylureas and in addition to the



inhibitory effect on adipocyte differentiation in 3T3L1 cells [60] , the aromatic urea AMPK activators also show strong in vitro antiproliferative and proapoptotic effects in melanoma cells. The compound 11 was used in a mouse melanoma model and the compound exhibits a significant antiplastic effect at a dose of 4 μg/kg per day [58] . Altogether, this data provides an interesting observation that a halogenated diaromatic urea scaffold might be used to design and synthesize selective AMPK activators, which will be effective against specific types of cancer cells. Several AMPK activators have shown noteworthy antiproliferative effects in human breast cancer cells in vitro, including a synthetic derivative of epigallocatechin gallate, (2R,3S)-1,2,3,4-tetrahydronaphthalene2,3-diylbis(4-bromo-3,5-dihydroxybenzoate) (17) and two naturally occurring molecules, demethoxycurcumin (18) and celastrol (19). Di Chen et al. reported that compound 17 was more biologically active than the parent compound, the well-known AMPK activator, epigallocatechin gallate [65] . Activation of AMPK by this epigallocatechin gallate analog results in the inhibition of human breast cancer cell proliferation, upregulation of the cyclin-dependent kinase inhibitor p21, downregulation of the mTOR pathway and growth suppression of the stem cell population in these cells. It is important to mention that the antiproliferative effect of gallic acid derivatives can be attributed to other biochemical pathways that they affect, for example, promotion of antioxidant activity, inhibition of NF-κB and AP-1, regulation of the cell cycle, inhibition of receptor tyrosine kinase pathways, control of epigenetic modifications, and modulation of the immune system [124] . However, all these pathways are regulated by AMPK as well. Therefore, the activation of AMPK might be the key event in potential anticancer use of gallate analogs. This important mechanism of action should be tested especially due to their selective antiproliferative activity against breast cancer, which makes potential use of these novel gallate derivatives extremely important. Celastrol, a pentacydic triterpenoid with strong antioxidant properties, shows antiproliferative activity and induces apoptosis in breast cancer cells in the nanomolar range of concentrations. Celastrol strongly induces AMPK’s function in the cells resulting in the increased activity of the p53. The AMPK inhibitor, compound C, completely blocks p53 phosphorylation that was induced by celastrol. These data indicate that the apoptotic effect of celastrol on breast cancer cells at least partially is mediated by AMPK [67] . It is known that celastrol is an inhibitor of tumor growth (e.g., melanoma). Celastrol triggers caspase-dependent and -independent cell death and suppresses the PI3K/


Review

AKT signaling pathway, which is associated with cell proliferation, survival and metastasis in cancer cells [125,126] . Additionally, celastrol induces cell cycle arrest at the G1 phase in human myeloma cells via modulation of the NF-κB pathway [127] . Recently, Aggarwal et al. summarized the role of celastrol in prevention and treatment of colorectal cancer. Their review mentions that in addition to the phosphorylation of AMPK by celastrol, 24 additional proteins are also upregulated [128] . Therefore, it is very difficult to pinpoint the exact role of AMPK in such a multivalent anticancer effect of celastrol. A very aggressive type of breast malignancy with a poor prognosis and ineffective treatment options, even during the early stages of the disease, is the “triplenegative” breast cancer [129] . This type of breast cancer is characterized by the lack of expression of the estrogen receptor, progesterone receptor and human epidermal growth factor receptor-2 [130] . Currently, there are no effective drugs to treat this type of breast cancer. Therefore, results about an effective in vitro proliferation inhibition of triple-negative breast cancer cells by an AMPK activator might be a remarkable first step for a potential treatment. Recently, it was reported that demethoxycurcumin in concentrations of about 5 μM blocked the activity of the eukaryotic initiation factor 4E-binding protein-1 signaling pathway and mRNA translation via mTOR in ‘triple-negative’ breast cancer cells via AMPK activation [66] . Moreover, AMPK activation causes a decrease in the activity and/or expression of several lipogenic enzymes, such as fatty acid synthase and ACC. Very recent reports have shown that AMPK activators inhibit the growth and viability of various types of cancer cells. For example, belinostat (20), an experimental anticancer drug candidate designed as a histone deacetylase inhibitor, activates one of the upstream regulators of AMPK, TAK-1 (TGF-β activated kinase 1), which then phosphorylates AMPK, thus inhibiting the proliferation rate of pancreatic cancer cells in vitro [68] . Again, it is very difficult to attribute the antiproliferative properties of belinostat exclusively to AMPK stimulation because activation of TAK-1 can lead to intracellular effects via its other downstream targets such as p38, JNK-MAPK and NF-κb [131] . Gambogic acid (21), a naturally occurring compound isolated from gamboges, shows remarkable growth inhibition and induces apoptosis in cultured U87 glioma cells [69] . This is an important observation because of the very limited arsenal of chemiotherapeutic agents against cancers of the CNS. Additionally, the chemical structure of gambogic acid seems to be lipophilic enough to penetrate the BBB. Nevertheless, its more lipophilic amide derivative (without the negative


1337

1338

A769662

PT-1

2-((benzo[d]thiazol2-ylmethyl)thio)6-ethoxybenzo[d] thiazole

(E)-3-((3-((4chlorophenyl) (phenyl)methylene)2-oxoindolin-1-yl) methyl)benzoic acid

2-chloro-7-hydroxy1-(2’-hydroxy-3’methoxy-[1,1’biphenyl]-4-yl)6-phenyl-1Hpyrrolo[3,2-b] pyridin-5(4H)-one

38


39

40

41

42

SC

SC

SC

SC

SC

NC

Salicylic acid

37

NC

SC

Coldycepin mono phosphate

Metformin

35

Source

36

Compound name

Structure number

–

β1 subunit, allosteric inhibition of Thr172 dephosphorylation

5.0 5.0

10.0

7.6

α1 subunit?

α2β1γ1

α1β1γ1

50.0

40.0

100.0

100.0

α1 subunit

500.0

3.000

β1 subunit, allosteric inhibition of Thr172 dephosphorylation

0.0001

15.00

γ subunit? γ subunit?

MEC (μM)

–


AMPK activation

Biological effect

AMPK

L6 myotubes

L6 myotubes

L6 myotubes

Rat platelets

HepG2

Rat platelets

Primary mice hepatocytes

AMPK activation

Glucose uptake ↑

Glucose uptake ↑

30.0

50.0

30.0

–

–

Aggregation ↓ Glucose uptake ↑

–

–

Aggregation ↓ Triglyceride contents ↓

30.0

–

6.0

250.0

–

Dose (mg\kg)


Isolated mice Glucose soleus muscle uptake ↑

–

HEK-293 Primary mice hepatocytes

AMPK

Type of cells

In vitro/ex vivo Interaction with AMPK

Table 2. Biological effects of direct AMPK activators.

–

–

–

ob/ob mice

Wistar rats

Blood glucose level ↓

Improved glucose tolerance

[183]

[182]

[181]

[181]

[179]

[180]

[179]

[175]

Fat utilization ↑ –

[178]

[177]

[176]

[175]

[174]

Ref.

–

Delay in the development of ischemic contracture Myocardial damage ↓

Fat utilization ↑

–

KKAy Blood glucose level ↓ diabetic mice

–

–

–

–

Mice

–

Mice

Mice

–

Type of Biological effect animals

In vivo



[12]

45

1000 γ subunit SC AICAR (ZMP)

[185]

– – – AMPK 20.0 α1β1γ1 α1β2γ1 NC


44

Sanguinarine

AMPK activation

[184]

Ref. In vivo

De novo hepatic lipogenesis ↓ Mice 30.0 De novo lipogenesis ↓ Rat primary hepatocytes Human and rat AMPK 10.0 (((5-(5-oxo-4,5SC dihydroisoxazol3-yl)furan-2-yl) phosphoryl)bis(oxy)) bis(methylene) bis(2,2dimethylpropanoate) 43

Type of cells MEC (μM) Interaction with AMPK

Source Compound name Structure number

Table 2. Biological effects of direct AMPK activators (cont.).

In vitro/ex vivo

Biological effect

Dose (mg\kg)

Type of Biological effect animals


Review

charge of the carboxylic acid moiety) is also active and has better chances to reach the brain [132] . The selectivity of the AMPK activators’ antiproliferative or apoptotic effects in cancer cells is not yet fully understood. The question of why certain compounds can inhibit cancer cell proliferation of a specific type of cancer is still an open issue. Some AMPK activators show broad antiproliferative effects. For example, compound 22, the lipophilic baicalein derivative, inhibits the proliferation rate of several types of human cancer cells, including epidermoid carcinoma, ovarian adenocarcinoma, prostate carcinoma and cervical carcinoma. This effect was obtained in the 1.0–8.0-μM range [70] . Despite tremendous strides in understanding of the role of AMPK in cancer progression, it is still too early to conclude if targeting AMPK is a suitable goal for developing novel anticancer therapy. Several clinical trails with metformin, are underway and the results are not yet publicly available. No doubt, any AMPK activators that are more potent then metformin in any tested AMPK-related cellular assays, and work in the nanomolar concentration range, should be tested in animal cancer models and after that in human clinical trials. The excellent antidiabetic effect of metformin is achieved at very high doses (∼1.5 g per day). Therefore, it will be very problematic to increase the dose of metformin for anticancer therapy if needed without experiencing side effects. Thus, more potent available AMPK activators, in effective therapeutic low anticancer doses, should be researched. A recently published article by Liu et al., doubts the involvement of AMPK activation in the anticancer effects of metformin and AICAR. The authors showed that both AMPK activators indeed inhibit proliferation (in vitro and in vivo), but through AMPK-independent mechanisms [133] . Moreover, both compounds work on different pathways. Metformin directly inhibits mTOR, whereas AICAR causes an arrest of the cell cycle inducing proteasomal degradation of the G2M phosphatase cdc25c. Additional research is needed for a more comprehensive understanding of AMPK’s role in the antiproliferative effects of AMPK activators. If indeed, the role of AMPK in proliferation inhibition will be shown, future anticancer therapies based on AMPK activation should go in two directions: investigation of specific types of cancer that are sensitive to such therapy and combination therapies with other anticancer drugs that might reveal a potent and effective synergistic combination of an AMPK activator and a known anticancer drugs. Neuroprotective therapy Recently, the amount of publications about the potential therapeutic use of AMPK activators in the CNS


1339

Review Zaks, Getter & Gruzman has dramatically increased. Neurons and glial cells are exceedingly active but do not have sufficient capacity for nutrient and energy storage and, as a result, they are extremely sensitive to intracellular ATP level fluctuations [134] . Accordingly, we would expect that the energy stabilization functions of AMPK play a neuroprotective role in different neurological diseases. However, AMPK’s function in brain pathologies is not so obvious. In all the classical energy restricted situations such as ischemia, hypoxia and glucose deprivation, AMPK is indeed activated [135–137] . In strokes it was shown that the overactivation of AMPK worsens the disease symptoms and prognosis and, conversely, AMPK inhibition was found in this case to be neuroprotective [138] . However, in neurodegenerative diseases, such as Huntington’s and Alzheimer’s, it seems that activation of AMPK has more neuroprotective properties than neurotoxic ones [139–147] . Several comprehensive reports showed opposite results, indicating that AMPK activation leads to neuronal apoptosis in a murine HT22 hippocampal cell line and in a mouse Huntington disease model (R6/2) [148,149] . These contradictory results regarding AMPK’s role in neuron survival under restricted energy metabolism requires additional research to clarify the potential therapeutic benefits of AMPK stimulation in the brain. It was suggested that this discrepancy is simply due to the time of incubation and/or administration of the AMPK activators [150] . When the AMPK activators are given for short periods of time, AMPK activation in neurons are beneficial. However, long-term administration is detrimental to neuronal survival [151] . Nevertheless, as a potential target, the protective functions of AMPK in neurodegenerative diseases recently gained the attention of medicinal chemists. Successful treatments of neurodegenerative diseases usually depend on long-term neuronal survivability, despite the disease. Sometimes, there is a fine line between neuronal death and persistence (or even regeneration following recovery of the brain functions) and even small bursts of energy could change the destiny of these neurons from degeneration to survival [152] . Several reports about the beneficial effects of AMPK activators in neurons were recently published. Lu et al. described that quercetin (23), a natural multifunctional flavonoid, activates AMPK and protects the mouse brain against cholesterol-induced neurotoxicity [71] . Quercetin, also activates AMPK in cancer cells, causing apoptosis in these cells [153] . However, as numerously mentioned in this review, it is very difficult (especially in vivo) to conclude that activation of AMPK is the main cause for the neuroprotective effect of such polypotent antioxidant compounds such as quercetin. Additionally, the potential use of the com-

1340


pound as a neuroprotective agent is limited due to the high effective doses in humans [154] . An interesting phenomenon related to AMPK activation in an in vitro ALS model was observed. Riluzole (24), the only US FDA-approved drug for ALS treatment, activates AMPK in a dose- and time-dependent manner [72] . The mechanism of action of reluzole is not clear, although in the scientific literature, its anti-ALS effect is attributed to its antiglutamatergic activity [155] . Interestingly, glutamate excitation decreases ATP levels in neurons and in response, AMPK is activated. It was reported that even negligible restrictions of glucose availability dramatically increases neuronal vulnerability to glutamate [156,157] . Therefore, neurons will either successfully recover if they have adequate amounts of ATP or if not, will go towards the apoptotic pathway [158] . Thus, the data that show that reluzole leads to neuroprotective effect in neurons via activate AMPK points out the possibility that activation of AMPK is one of the biological effects of reluzole that helps neurons fight glutamate toxicity. Additionally, a well-known drug, latrepirdine (25) (H2 receptor antagonist), was recently found to have neuroprotective properties at (sub)nanomolar concentrations of only 0.1 nM. Latrepirdine activates AMPK and protects neurons against glutamate toxicity and, as a result, the compound reduces neuronal excitability [73] . Having such a low effective dose and that latrepirdine passes the BBB and has a known pharmacokinetic profile makes latrepirdine an ideal contender for future detailed investigation of its effect in different neuronal pathologies. Another report describes how methylene blue (26) via AMPK activation protects neurons from serum deprivation-induced apoptosis [74] . In contrast with the described above compound, methylene blue is not a suitable drug candidate because of high human toxicity (the compound was used to treat malaria in the 30 s) and low selectivity [75] . Thus, this in vitro study is still interesting from the mechanistic point of view. In addition, solid evidence was provided showing that together with neurons, AMPK activation protects another important player in normal brain physiology, glia cells [159] . In these cells, AMPK also mediates an antiapoptotic effect. Lu et al. showed that the AMPK activator phenylethyl amide, a derivative of caffeic acid (KS370G) (27), significantly inhibits the release of nitric oxide and the expression of inducible nitric oxide synthase and cyclooxygenase-2 in glial cells and protects them from apoptosis. This is a very interesting observation because in many neurological disorders and pathological conditions, the glia cells together with the neurons are damaged and a compound with potential glial protective properties should be added to the arsenal of neurological diseases treatment agents [160] .



H2N

H N

N NH

N

NH2 HO

NH

N

O

Metformin (35)

O OH

N N

O

OH

NH S A769662 (38)

Salicylate (37)

OH

N

HO

OH

Review

Cordycepin (36) H N

O

N

O

S

NO2

OH

Cl

HO

Cl

O O

OH

O

O O

PT-1 (39)

N

OH

N

NH

Cl 2-chloro-7-hydroxy-1-)2´-hydroxy-3´methoxy-[1,1´-biphenyl]-4-yl)-6-phenyl-1Hpyrrolo[3,2-b]pyridin-5(4H)-one (42)

(E)-3-((3-((4-chlorophenyl)(phenyl)methylene) -2-oxoindolin-1-yl)methyl)benzoic acid (41) N S

O

HO

O

S

+N

O

2(benzo[d]thiazol-2-ylmethylthio)6-ethoxybenzo[d]thiazole (40)

N

O

O

P

O N

O

HO O

O O

O

H 2N

S

N

NH2 N

OH AICAR (45)

O

O

Sanguinarine (44)

O

O O O

(((5-(5-oxo-4,5-dihydroisoxazol-3-yl)furan-2-yl) phosphoryl)bis(oxy))bis(methylene) bis(2,2-dimethylpropanoate) (43)

Figure 3. Chemical structures of direct activators of AMPK.

Amyloid-β peptide degradation remains one of the most important targets for the possible treatment of Alzheimer’s disease [161] . It was shown that a wellknown natural polyphenol compound, resveratrol, decreases amyloid-β levels by inducing its proteolytic clearance in cell lines and in primary neurons [162–164] and lowers amyloid deposition in Alzheimer’s disease mice models [163,165–166] . Based on these observations, a library screening of synthetic resveratrol analogs was preformed [76] . The research team successfully found several compounds that inhibit amyloid-β accumulation in vitro, which were 40-times more potent than the parent molecule. Two of these molecules, RSVA314 (28) and RSVA405 (29), were further characterized and were found to activate one of the upstream AMPK regulators, CaMKKβ, following the phosphorylation of AMPK, which leads to increased autophagy and


degradation of amyloid-β by the lysosomal proteolitic system. Interestingly, RSVA314 has shown inhibition of adipogenesis in adipocytes also via AMPK activation [77] . It is important to mention that activation of CaMKKβ leads to the activation of two other signaling proteins beside AMPK, CaMKI and CaMKIV [167] , and the inhibition of amyloid-β accumulation can be a result of the AMPK-independent effect of CaMKK activation. Further research should be done to clarify if indeed this promising antiamyloid accumulation effect is indeed mediated specifically by AMPK. Taken together, all these findings indicate that it is still premature to expect AMPK activators to be used for neurological disease treatments in the near future. The proapoptotic properties of AMPK activators, even if they are only observed after long-term use or in extreme situations, cast a shadow on the unquestionable


1341


N

HO

S

N

S

S (38)

α

OH

N

γ (37) Cl

O O

OH

(39)

H 2N N HO O

N

N

N

HO

OH

NH O

O

NH2

N

N

HO

H N

N

O

H2N

(36)

+N

OH

S

NO2

O

O

β

(40) H N

NH

AMPK

O

S

O

O

OH

N

NH2

(45)

OH

NH (35)

O

O O

OH HO

(44)

O O O

N

O

P

N O

O

O

O

O

Cl

NH (42)

Cl

O

O O

(43)

O

OH

N (41)

Figure 4. Unit specific binding of direct AMPK activators.

beneficial neuroprotective effects of such compounds. Much additional work is needed for exact target validation. For example, the identification and characterization of known and still unknown downstream

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AMPK effectors that are involved in mediating the therapeutic outcomes, specifically in the brain, need to be elucidated. All of the AMPK activators that might be used as neuroprotective drugs in the future should



have excellent BBB penetration characteristics. This is not a trivial matter and is very chemically challenging. New horizons In this review, we have already discussed the antiproliferative effects of AMP activators in cancer cells. A novel application for this effect is to slow the proliferative rate of vascular smooth muscle cells by using nectadrin B, a naturally occurring diphenol AMPK activator [168] . Nectadrin B (30) suppresses cell proliferation induced by PDGF. The effect of nectadrin B was positively correlated with AMPK-mediated induction of p53 and p21, which is initiated by Rb phosphorylation and E2F1 expression following significant downregulation of proliferation of vascular smooth muscle cells [78] . Additionally, Hien et al. reported that together with the activation of AMPK, Nectandrin B inhibits TNF-α-induced monocytoid THP-1 cell adhesion to ECV 304 human endothelial cells [169] . The compound also suppresses TNFα-induced proteins and mRNA expression of two cell adhesion molecules, VCAM-1 and ICAM-1. In addition, expression of cyclooxygenase-2 and inducible nitric oxide synthase were diminished by the tested compound. Dominant-negative expression of mutated AMPK in endothelium cells only partially blocks the biological effect of nectandrin B. Again, these results show the difficulties in distinguishing the activation of AMPK and other biological effects of the compound in its antiarteriosclerotic effect. In vitro results were supported by in vivo experiments in a mouse femoral artery injury model. Nectadrin B in concentrations of 20 μg/kg successfully inhibited neointimal formation. Such biological activity opens an exciting possibility for effective prevention of arteriosclerosis because the generation of neointima in vesicles is one of the first steps in developing atherosclerotic plaques [170] . Another naturally occurring AMPK activator, sophocarpine (31), has been reported as an effective molecule for treating nonalcoholic steatohepatitis in rats and for modulating adipocytokine synthesis [79] . The authors isolated primary rat hepatocytes and incubated them with toxic doses of oleic acid for 24 h to induce steatotisis. The cells were then treated with sophocarpine for 72 h. The treatment significantly improved the steatosis, decreased leptin expression and increased adiponectin expression. These data suggest that a pharmacological agent that is able to activate AMPK in hepatocytes can be used to treat steatosis, a disease that has no effective therapy beside liver transplantation, especially in its advanced stages. Several scientific reports disclose the anti-inflammatory function of AMPK. This is predominantly caused by inhibiting the production of inflammatory cyto-


Review

kines, especially IL-6, in macrophages [171,172] . Based on this observation, Guh et al. screened a library of small organic molecules and identified a potent activator of AMPK, which suppresses the production of IL-6 in THP-1 human macrophages. The compound 32 has some structural homology to PT-1 (an AMPK autoinhibition blocker) and is active in concentrations of 5 μM [80] . The anti-inflammatory properties of another synthetic AMPK activator, the saturated fatty acid analog ETC-1002 (33), were investigated in primary human monocyte-derived macrophages and in mice models of inflammation [81] . It is important to mention that the blood lipid-lowering effect of ETC-1002 has been intensively studied and, recently, the compound went to clinical trials and was found to lower LDL and beneficially modulate other cardiometabolic risk factors [82] . Monocytes treated with ETC-1002 were found to have upregulated levels of phosphorylated AMPK, reduced activity of MAPKs, and decreased production of proinflammatory cytokines and chemokines. The silencing of LKB1 in macrophages has led to ETC-1002’s loss of activity. In this case, the data clearly indicate that the effect of ETC-1002 is mediated by an upstream AMPK regulator and does not directly affect AMPK. In vivo, ETC-1002 suppresses thioglycollate-induced accumulation of leukocytes in the mouse peritoneal cavity and diminishes the expression of macrophagespecific inflammatory marker 4F/80. These results were positively correlated with decreased epididymal fat-pad mass and IL-6 levels. In addition, ETC-1002 also inhibits de novo lipid synthesis in hepatocytes and more importantly reduces liver triglycerides, markers of oxidative stress, hepatic steatosis, plasma atherogenic lipoproteins and enhanced β-oxidation in animal models. Taken together, these results show that ETC-1002 may be used as or be the basis for developing modern drugs with multiple therapeutic targets that treat patients with inflammations that are attributed to insulin resistance and vascular complications of metabolic syndrome. Moreover, the anti-inflammatory effects of this AMPK activator bring new possibilities for treating autoimmune diseases. Finally, very intriguing results were published about the effect of another fatty acid analog, 2-octynoic acid (2-OA) (34), on the prevention of human hepatitis C virus (HCV) infections in human cells [173] . 2-OA affects the lipid accumulation in HCV replicon cells and virus-infected hepatocytes. 2-OA specifically suppresses HCV RNA replication and the virus production with no cytotoxicity to the host cells. The study demonstrated that 2-OA activates AMPK and its downstream regulated protein, acetyl-CoA carboxylase. 2-OA’s effect is via the phosphorylation of AMPK. An inhibitor of AMPK


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Review Zaks, Getter & Gruzman (compound C) and a knockdown of AMPK were introduced to the cells, and, as a result, the antiviral effect of 2-OA was eliminated. Here, this solid data indicate that the biological effect of the compound is indeed mediated by activation of AMPK. It will be interesting to see if AMPK plays a similar role in hepatic infections caused by different viruses and viral infections in different tissues. Such information can bring new therapeutic strategies for developing novel antiviral drugs. Taking together, these new applications for AMPK activators again show that AMPK is involved in regulation of a diverse spectrum of cellular functions. The selectivity, in terms of activation of the tissue specific AMPK isoforms, should help to produce a more focused AMPK activator. These examples also show that AMPK activators can be used to develop potential drugs against almost any target event where energy metabolism plays a critical role. Direct activators In this chapter we will summarize the recent achievements in medicinal chemistry of direct AMPK activators that show promising biological results and can be used as prototypes for developing more specific drugs, despite the polyvalent functions of AMPK. Table 2 & Figure 3 summarize the information about the structure and biological activity of the direct activators of AMPK. Figure 4 explains the sites of AMPK where direct activators bind. Metformin (35) is known as an indirect AMPK activator. However, recent evidence indicates that metformin directly interacts with AMPK. A series of experiments have shown that metformin indeed binds to AMPK with a reasonable affinity (K D = 13.2 ± 4.37 μM), which is not surprising given metformin’s average daily dose of 1.6 g. The ‘wet’ tests were conducted after in silico prediction docking experiments showed that metformin may interact with the γ subunit of AMPK. Subsequent fluorescence spectrum and ForteBio assays indicate that metformin indeed strongly binds to the γ subunit of AMPK and such interactions also result in a decrease of the α-helicity of the γ-subunit determined by CD spectra [174] . It is important to note that AICAR, the prodrug of the ZMP that interacts directly with AMPK, also binds to the γ-subunit [12] . In silico docking measurements were also used to explain the biological effect of the naturally occurring Key term Direct activators of AMPK: Small molecules that selectively target AMPK subunits, causing specific biological effects in selected tissues.

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compound, cordycepin (36) [186] . An analogy with AICAR’s proposed mechanism of action was based on the conversion of cordycepin to its monophosphate derivative, which acts as an AMP analog that binds to the γ-subunit and activates AMPK. The modeling calculation supports this hypothesis and provides the structural basis that there is a high level of the probability that cordycepin monophosphate binds to AMPK with a high affinity. Further in vitro experiments reveal that cordycepin monophosphate functions as an AMP mimietic and activates AMPK in the nanomolar concentration range. It is interesting to note that the parent compound, cordycepin, is not active in the AMPK in vitro assay [175] . This shows that for AMPK activation, the cordycepin molecule has to be phosphorylated. This can also explain the dozens of cordycepin’s other biological activities reported so far [187] . It is also possible that cordycepin without the phosphate group affects other intracellular signaling pathways and its phosphorylated form interacts with AMPK as we previously described. Salicylic acid (37) and its derivatives/prodrugs, aspirin and salsalate have been broadly used from ancient times until the present. Salicylates and other COX-1/2 inhibitors have shown antihyperglycemic effect [188–190] , but because of high doses that cause severe side effects, their structures were never properly studied for designing novel antidiabetic drugs. Recently, an interesting discovery was made about salicylate’s mechanism of action [176] . Salicylate binds directly to the β1 subunit of AMPK, causing an allosteric change in the AMPK complex, which inhibits the dephosphorylation of Thr172. This constant Thr172 phosphorylation state of the enzyme keeps it active. This is a nice example of the endless surprises in science that new mechanisms of action are discovered for a drug that was first introduced to the market over 100 years ago. It is interesting to note that salicylate binds to the exact moiety in the β1-subunit of AMPK that A769662 (38), another direct activator of the enzyme, binds. A769662 and salicylate do not have any structure similarity beside the shared phenol moiety. A769662, a thienopyridone derivative, was discovered in 2006 [177] . It was shown by Xiao et al. that the compound binds between the kinase domain and the module that recognizes the carbohydrates, stabilizing the contact surface between these two compartments. The authors also suggest that the shape of the activator-binding cavity implies the presence of another molecule (most likely an endogenuse ligand not yet unidentified) that regulates the physiological activity of AMPK. [191] . As published in the original paper, A769662 directly stimulates the activity of purified rat liver AMPK at a concentration of 0.8 μM, and in ob/ob mice. A 30 mg/kg daily dose of this compound lowered



plasma glucose levels by 40%, reducing bodyweight gain and significantly decreasing both plasma and liver triglyceride levels. These results provide a new impetus to develop direct AMPK activators for the treatment of T2D. Since then, several interesting discoveries related to A769662 were made. For example, it was reported that A769662 augments the rate of the glucose uptake in isolated skeletal muscles [178] . However, the physiological relevance of the results is questionable, since A769662 induces its positive effect on the rate of glucose uptake through a PI3K-dependent pathway that is usually activated by insulin [192] . In another study, relatively high concentrations (100 μM) of A769662 showed an in vitro antiaggregation effect in rat platelets [179] . A report by Kim et al. showed an in vivo inhibitory effect of A-769662 on the development of ischemic contracture and a reduction of myocardial damage [193] . Interestingly, there are no reports that this oldest known direct AMPK activator causes an antiproliferative effect. This information supports the hypothesis that the anticancer effects of some AMPK activators are not related to the ability to activate AMPK itself. The catalytic α1 subunit of AMPK has in its structure the autoinhibitory domain, which is activated by ATP. Chen et al. shows that the autoinhibitory domain binds to the hinge region of its own kinase domain, which allows the interaction with both amino-terminal and carboxy-terminal lobes. This restricts the mobility that is critical for the kinase action parts of the α1 subunit and as result, AMPK is inactivated [194] . This regulatory mechanism makes possible the development of new types of small compounds that activate AMPK by blocking the autoinhibition domain. This elegant approach to liberate AMPK from its own autoinhibition was used by Pang et al. as a basis to develop novel antidiabetic drugs [180] . Out of several tested compounds, a thiazolidin derivative, named PT1 (39) was able to bind to this specific sequence. Although the screening of the compound library was conducted on the α1 subunit by itself, in further experiments it was shown that PT1 activates the physiological relevant heterotrimer of AMPK (α1β1γ1) by interrupting AMPK’s autoinhibition. In silico molecular modeling and mutagenesis experiments revealed that PT1 might interact with the autoinhibitory domain and directly block the autoinhibitory activity of AMPK [195] . In addition, the compound was tested in L6 myotubes where the activation of AMPK led to the phosphorylation of ACC. Moreover, PT1 significantly reduces the cell lipid content in the HepG2 cells. Several other research groups reported that PT1 shows AMPK-related biological activity. Liu et al. described the antiaggregation effects of the compound on rat platelets [179] and Meltzer-Mats et al. showed that PT1 increases the rate of glucose uptake


Review

in hyperglycemic-like conditions in L6 myotubes [181] . This research group designed a pharmacophore model based on PT1’s structure and used it to synthesize several benzo[d]thiazole thioethers. One molecule (40), which was generated out of the pharmacophore, directly activates AMPK in a cell-free kinase assay. In addition, the compound increases the rate of glucose uptake in L6 myotubes. Moreover, compound 40 was active in KKAy diabetic mice, where it decreased the blood glucose values until almost normoglycemic levels. Interestingly, despite a very low structural similarity between PT1 and compound 40, in silico calculations were conducted based on the binding mode and identified that PT-1 and compound 40 have high binding scores and most probably interact within the same area of AMPK. An additional synthesized direct activator based on the PT1 structure was recently described by Yu et al. [182] . The design of the most active compound was based on the alkene oxindole scaffold (41). The molecule showed direct activation of AMPK, via interaction with the α2 AMPK subunit. This indicates that PT1 and its analogs are not α1 subunit-specific molecules. This molecule was tested in diet-induced obesity and diabetes mouse models. Improved glucose tolerance and decreased insulin resistance were observed at the end of the treatment. Orally active direct AMPK activators were designed and synthesized by Mirguet et al. [183] . A new pyridone scaffold-based molecule shows oral activity in a reasonable therapeutic dose. In addition, the lead drug candidate (42), exhibits selectivity towards activation of the α1β1γ1 AMPK complex and the pharmacokinetic properties of the lead compound are very encouraging in terms of the future drugability of the molecule. Phosphonic acid derivatives (43) with the ability to directly activate AMPK were recently identified by screening a targeted library of AMP mimetics [184] . A furan-based phosphonic acid molecule has a very slight similarity to AMP, the cell’s natural activator of AMPK. Despite not being similar to AMP, this molecule was able to directly activate rat and human AMPK with extremely high potency. The EC50 was determined in the in vitro kinase assay to be 6 nM. Despite having an excellent in vitro antilipogenic effect, the compound is metabolically unstable. This prevents the original compound to show biological effects at such low concentrations in vivo. However, ester-based lipophilic prodrugs of the parent compound are active in vivo and inhibit de novo lipogenesis in animal models of hyperlipidemia. Interestingly, even though it has high potency, this tested compound is only a partial activator of the rat liver AMPK, and this partial activation is sufficient to fully inhibit de novo lipogenesis in rat hepatocytes. These data opens new directions for designing and synthesizing a


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Review Zaks, Getter & Gruzman full inhibitor based on the furan-phosphonic acid scaffold. Such a molecule might be the ultimate activator of AMPK in the liver, the main site of metformin’s AMPK activation, which works only at very high doses. The final direct activator of AMPK that was recently reported is the naturally occurring molecule sanguinarine (44) [185] . Sanguinarine directly activates AMPK through the interaction with AMPK in the cleft between the β and γ subunits, similar to AMP. The physiological relevance of AMPK activation by sanguinarine was shown in cell-based assays, which confirm that the downstream acetyl-CoA carboxylase (ACC) is phosphorylated after sanguinarine-induced AMPK activation. Sanguinarine exclusively interacts and activates α1γ1-containing AMPK complexes and selectivity for β1- and β2-containing AMPK complexes was not observed. The molecule equally activates both types of β-subunits in a similar fashion. This compound, as many naturally obtained AMPK activators, exhibits a broad spectrum of biological effects of which the majority are not related to AMPK activation [196] . Thus, the physiological relevance of these results should be properly investigated. Conclusion & future perspective There is a concerted effort to design drugs around AMPK as a target for the treatment of several severe human diseases. The most significant problem in targeting AMPK, a complex involved in almost all biochemical regulator processes, is to achieve selectivity of certain biological effects in a specific tissue. Discovering selective molecules depends on our ability to produce highly isoform specific AMPK activators or alternatively, to synthesize selective modulators of downstream AMPK targets. However, if we follow the last option, we will be digressing from the idea of using the direct activation of AMPK as a target for drug design. Much work

is needed in the field of molecular and cellular biology to identify the exact selective pathways in which AMPK is involved. We also need to elucidate the type of AMPK isoform that is involved in specific biochemical processes and structure biologists have yet to obtain all crystal structures of different AMPK complexes composed of different isoforms of the subunits. Such breakthroughs will provide a strong molecular basis for computer-aided drug design approaches that have the power to develop new drugs. In terms of specificity, the direct AMPK activators bring new levels of complexity to the expected selectivity in the endless orchestra of AMPK-induced effects. This specific approach of designing direct and maybe even covalent activators of AMPK is important in creating future drugs that are based on AMPK activation. The specific pattern of distribution of AMPK isoforms in different tissues and species should be utilized for developing future novel AMPK activators and for interpreting the in vivo results of such molecules. Ideal AMPK activators should be tissue specific, in terms of their ability to selectively activate certain AMPK isoform. In our opinion, the direct activators of specific isoforms of AMPK is the group of compounds that has the greatest structural potential to generate new drugs against cancer, diabetes and all other diseases discussed herein. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Executive summary • AMPK has a specific structure, variety of functions, and a specific role in different tissues. • Indirect AMPK activators with antidiabetic, antiobesity and antihyperlipidemic activity have recently been reported, as natural and synthetic compounds. • Indirect AMPK activators as potential novel anticancer therapeutics have also recently been reported. Some AMPK activators have antiproliferative activity and the antiproliferative effect’s multipotential character of indirect AMPK activators might be mediated by AMPK-independent pathways. • The use of the indirect AMPK activators as a potential neuroprotective agents is possible. • Review of the new biological activities of indirect AMPK activators is condacted: –– Description of possible antiarteriosclerotic applications of AMPK activators; –– Discussion about potential liver protective properties of AMPK activators; –– Analysis of potential anti-inflammatory activity of AMPK activators. • The direct AMPK activators and their biological effects are discussed in this article, including analysis of the properties of γ subunit binding AMPK activators, α subunit binding AMPK activators as potential drug prototypes, biological properties of the β subunit binding AMPK activators, and specific AMPK isoforms binders as the promising potential drug candidates. • Future perspectives of the use of AMPK activators as potential drugs are listed in this article.

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•

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•

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AMPK Activators as a Drug for Diabetes, Cancer and Cardiovascular Disease.

AMPK activation: a therapeutic target for type 2 diabetes?

Adults Are Not Just Enormous Children: Type 2 Diabetes Mellitus in Adults With Congenital Heart Disease.

Type II diabetes: search for primary defects.

AMPK activators: mechanisms of action and physiological activities.

Structural basis of AMPK regulation by small molecule activators.

Development of Potent Adenosine Monophosphate Activated Protein Kinase (AMPK) Activators.

Discovery of Pyridones As Oral AMPK Direct Activators.

Ranaviruses: not just for frogs.

Hyperproinsulinemia in type II diabetes.

B-type natriuretic Peptide: not just a heart throb.

Lessons from Nature: Sources and Strategies for Developing AMPK Activators for Cancer Chemotherapeutics.

Assessment of whole-genome regression for type II diabetes.

Naltrexone: Not Just for Opioids Anymore.

Writing is not just for other people!

Natural Nrf2 activators in diabetes.

Wheel running not just for lab mice.

Not just for cars: Lean methodology.

Not just pathophysiology.

Not just a fracture.

Structure, not just function.

Combination therapy with insulin and sulfonylureas for type II diabetes.

Not just hot air.

Lipoproteins and diuretics in type II diabetes.