Betagenon o304

1

AMPK activator O304

Peripheral Vascular Disease, Type 2 Diabetes & Cancer

AMP-activated protein kinase

(AMPK)

2

- Master regulator of energy charge at

cellular and organism level

- Validated target in energy balance disorders

- Activated by ATP depletion

Identified in cellular screen designed to

identify compounds which activates AMPK

without reducing cellular ATP

3

O304

4

Mechanism of Action

O304

O304 increases p-T172 AMPK

and the downstream target p-S79 AC

in normal cells

5

AMPK

P

T172

AMPK P

S79

ACC

p-T172 AMPKa

AMPKa

p-S79 ACC

ACC

V.C. 2.5

O304 (µM)

10 5

0,0

1,0

2,0

3,0

4,0

V.C. 2.5 5 10

Arb

. U

nits

p-T172 AMPK

O304 (µM)

0,0

1,0

2,0

3,0

4,0

V.C. 2.5 5 10

Arb

. U

nits

p-S79 ACC

O304 (µM)

O304 activates AMPK and increases

cellular ATP

6

Dose and time dependent

1h

p-T172 AMPK

AMPK

2.5 5 10 V.C.

O304 (µM)

p-T172 AMPK

AMPK

8h

2.5 5 10 V.C.

O304 (µM)

p-172 AMPK

AMPK

16h

2.5 5 10 V.C.

O304 (µM)

ATP/prot

1,0 1,0 1,0 1,1

0,0

0,5

1,0

1,5

2,0

2,5

Arb

. U

nits

V.C. 2.5 5 10

O304 (µM)

ATP/prot

1,0 1,0 1,1 1,3

0,0

0,5

1,0

1,5

2,0

2,5

2.5 5 10

O304 (µM)

V.C.

1,0

1,2 1,4

1,8

0,0

0,5

1,0

1,5

2,0

2,5 ATP/prot

2.5 5 10

O304 (µM)

V.C.

Both O304 and Metformin activates AMPK

7

0,0

1,0

2,0

3,0

4,0

V.C. 2.5 5 10 1 2 5

Re

lative

p-T

17

2 A

MP

K

le

ve

l

p-T172 AMPK/AMPK

Metformin (mM)

O304 (M)

V.C. 2.5 5 10 1 2 5

O304 (M)

AMPK

ACC

p-T172 AMPK

p-S79 ACC

Metformin (mM)

HUVEC

0,0

0,5

1,0

1,5

V.C. 2.5 5 10 1 2 5

Rela

tive A

TP

/pro

tein

levels

ATP/prot

Metformin (mM)

O304 (M)

O304 increases-Metformin decreases

cellular ATP

8

Rate of dephosphorylation of p-T172 AMPK

key regulatory mechanism

- Main upstream kinase LKB1 is constitutively active

- AMP and ADP inhibit dephosphorylation of p-T172 AMPK

- AMP but not ADP allosterically activates AMPK

- Phosphorylation of T172 AMPK is required for activity

- AMPK activated by ATP depletion

Garcia-Haro et. al., 2010

Protein phosphatases PP1 and PP2C

dephosphorylate p-T172 AMPK

9

O304 suppresses the dephosphorylation of

human recombinant p-T172 AMPK

by PP2C (and PP1)

Developed ELISA to monitor the dephosphorylation of p-T172 AMPK

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AMPK111

0,0

0,2

0,4

0,6

0,8

1,0

1,2

p-T

172 A

MP

K

A

rb.

Units

AMPK

PP2C

0,0

0,2

0,4

0,6

0,8

1,0

1,2

AMPK211

AMPK

PP2C 2.5 5 10

O304 (M)

2.5 5 10

O304 (M)

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Rat liver AMPK

p-T172 AMPK

PP2C AMPK

10M 150M

O304 ADP

0,0

0,2

0,4

0,6

0,8

1,0

1,2

Rela

tive

p-1

72

AM

PK

a l

eve

l

Human recombinant AMPK

p-T172 AMPK

0,0

0,2

0,4

0,6

0,8

1,0

1,2

Rela

tive

p-1

72

AM

PK

le

ve

l

PP2C AMPK

10M 150M

O304 ADP

O304 suppresses the dephosphorylation of

p-T172 AMPK by PP2C of partially

purified rat liver AMPK

O304 does not inhibit PP2C or PP1

12

0

10000

20000

30000

40000

50000

RFU

PP1

PP1 -PP1 2.5 5 10

O304 (M)

0

10000

20000

30000

40000

50000

RF

U

PP2C

PP2C -PP2C 2.5 5 10

O304 (M)

O304 does not activate AMPK in

LKB1-deficient HeLa cells

AMPK P T172

AMPK LKB1 X

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ACC

AMPK

p-S79 ACC

p-T172 AMPK

V.C. 2.5 5 10 1

Ionomycin O304(M) (M)

Ca2+ Ionophore

O304 requires LKB1

Activation of AMPK only in physiologically relevant cells!

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AMPK

P T172

AMPK

P T172

AMPK

P T172

LKB1

PP2C/PP1

AMPK

P T172 O304 X

O304 suppresses the dephosphorylation of p-T172 AMPK

by PP2C and PP1 without inhibiting their activity

Mechanism of action of O304

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On-target

AMPK activity declines with aging and obesity

Mice fed very high fat diet (vHFD)

Animal model

High oral availability of O304

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Single dose in rat

0

1000

2000

3000

4000

O304 20mg/kg O304 40mg/kg

Arb

. U

nits

AUC

0

20

40

60

80

100

120

140

0 20 40 60

O304 µ

M

Time (h)

PK

O304 40 mg/kg

O304 20 mg/kg

O304 activates AMPK in liver

of mice fed vHFD

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**

0

0,5

1

1,5

2

V.C. O304 20mg/kg

Rela

tive

pT

17

2-A

MP

K le

ve

l

p-T172 AMPK / AMPK

V.C. O304 20mg/kg

p-T172 AMPK

AMPK

Mice were treated with O304 at 20mg/kg and fed vHFD for 13 weeks

Chronic AMPK activation

regulates expression of target genes

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Hardie, et. al., Chemistry&Biology 19, 2012

O304 suppresses the expression of

ACC, FAS, and SCD-1and reduces fatty liver

in mice fed vHFD

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Mice were fed vHFD for 3 weeks and thereafter O304 at 40mg/kg/day and vHFD for 10 weeks

FASN

SCD-1

-Actin

ACC

V.C. O304 40mg/kg

0

20

40

60

80

100

120

VC O304

To

tal lip

ids (

mg/g

liv

er)

*

0

5

10

15

20

25

30

35

40

VC O304

Trig

lycerd

es (

mg/g

liv

er)

*

AMPK

Vascular system

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Multiple proposed beneficial effects of

AMPK activation on vasculature

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Maintains endothelial redox balance

Relaxes blood vessels / reduces hypertension

Suppresses smooth muscle proliferation

Promotes re-endothelization / vascular repair

Reduces cardiac hypertrophy and ischema / reperfusion damage

Suppresses platelet activation

Increases angiogenesis/arteriogenesis and microvascular perfusion

22

AMPK-Endurance-Vascular function

- AICAR increases endurance in normal sedentary mice

- Loss of AMPK in muscle causes severe reduction in

endurance due to reduced capillary density in muscle

- AICAR increases microvascular perfusion in muscle

- Aged normal sedentary mice

- Genetically obese mice

Animal models-Endurance

O304 increases significantly endurance

of normal sedentary 14 month old mice

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V.C.

O304

V.C.

O304

0

300

600

900

1200

V.C. O304

(m)

Before treatment

*

0

300

600

900

1200

V.C. O304

(m)

After 30 doses

Distribution of mice exhibiting

different running distance

Before treatment

After 30 doses

Mice were treated with O304 at 20 mg/kg/day for 30 days

0

300

600

900

1200

1500

1800

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

(m)

n=14

n=14

0

300

600

900

1200

1500

1800

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

(m)

n=14

n=14

Significantly lower lactate levels

at exhaustion in normal 14 month old mice

treated with O304

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p=0.013

*

0,0

1,0

2,0

3,0

4,0

5,0

Rest Exhaustion

Lacta

te m

mol/L

V.C. O304

Blood lactate

Mice were treated with O304 at 20 mg/kg/day for 30 days

O304 increases significantly endurance

in Ob/Ob mice

0

100

200

300

400

V.C. O304

Running distance (m

) *

25

Mice were treated with O304 at 20mg/kg/day for 15 days

Increase in endurance induced by O304

in aged mice persists after 1 week but not after

2 weeks of washout

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Cardiovascular effect?

Laser Doppler Images (LDI) of left hind paw

O304 increases peripheral blood

perfusion in 14 months old mice

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8 w. old control mice

14 months old mice V.C.

14 months old mice O304 20 mg/kg/day for 25 days

28

Distribution of individual mice

with different perfusion of left hind paw

0

3

6

9

12

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Max p

erf

usio

n A

rb.

units

8w old

O304

V.C.

p=0.0096

n=10

vs p=0.036

n=13

vs

p=0.098

n=16

vs

n=16

n=16

n=16

O304 - Vascular effects

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Increases expression of Vegf and nNOS

Increases peripheral blood flow

Increases no. of -SMA+ blood vessels in Soleus muscle

Increases cardiac stroke volume

Reduces blood pressure

Increases bleeding time/reduces platelet activation

Reduces ET-1 and TSP-1 levels

Type 2 Diabetes

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Insulin resistance and β-cell dysfunction

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AMPK-Type 2 Diabetes

- Ensures normal glucose sensing in β-cells

- Reduces fat and insulin resistance in liver

- Promotes glucose uptake in muscle

32

- Glucose-stimulated insulin secretion is impaired by

AMPKα2 knockout in β-cells

- Promotes trafficking of KATP channels which couple

glucose metabolism to insulin secretion in β-cells

- Facilitates the decrease in insulin secretion in response

to glucose deprivation

AMPK ensures normal glucose sensing in β-cells

O304 stimulates GSIS

33

Perturbed -cell function in Diabetes

Type 2 Diabetics (~70%) have deposits

of islet amyloid polypeptide (IAPP) in -cells

Rodent IAPP is not amyloidogenic;

IAPP amyloidogenesis in diabetes not well studied

Transgenic overexpression of amyloidogenic

human IAPP in mouse β-cells

Animal model

Macroautophagy eliminates aggregated

proteins in mammalian cells

- AMPK induces autophagy by multiple pathways

including suppression of mTOR signaling

- Exercise induces AMPK and autophagy in muscle

and in pancreatic -cells

34

O304 induces autophagy in -cells

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Induction of insulin resistance by high fat diet

in mice with perturbed β-cell function due to

overexpression of hIAPP in β-cells

Animal model of Type 2 Diabetes

Studies performed in collaboration with

Prof. H. Edlund Umea University

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O304 - Type 2 Diabetes

O304 ameliorates both insulin resistance and

β-cell dysfunction - hallmarks of type 2 diabetes

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Increased plasma levels of IGF-1 are

correlated to increased Diabetes mortality

O304 reduces diet-induced increase

in plasma levels of IGF-1

AMPK - Cancer

- Stabilizes p53, p21WAF1,p27CIP1 and causes cell cycle arrest

- Inhibits the synthesis of fatty acids, triglycerides, cholesterol,

glycogen, ribosomal RNA and proteins, and thus cell growth

- Inhibits mTORC1 and translation of many proteins required

for rapid cell growth

- Exhibits 'anti-Warburg' effect - Promotes oxidative metabolism

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O304 - Tumour cells

- Inhibits mTORC1 signaling

- Suppresses cell growth and induces

cell cycle arrest

- Suppresses lipid synthesis

- Reduces diet-induced hyperinsulinemia

and increased plasma levels of IGF-1

40

Clinical development of O304

In Phase I clinical trials

Future Indications:

Type 2 Diabetes & Vascular complications

Peripheral vascular disease

Cancer