AMPK Activation by "Compound 13"

MB7264 and “Compound 13” In vitro and vivo selective activation of

the AMPK 𝛼1 isoform

Jorge E Gómez-Galeno

A presentation based on:

A Potent and Selective AMPK Activator That Inhibits de Novo Lipogenesis

Jorge E. Gómez-Galeno, Qun Dang, Thanh H. Nguyen, Serge H. Boyer, Matthew P. Grote, Zhili Sun, Mingwei Chen, William A. Craigo, Paul D. van Poelje, Deidre A. MacKenna, Edward E. Cable, Paul A. Rolzin, Patricia D. Finn, Bert Chi, David L. Linemeyer, Scott J. Hecker, and Mark D. Erion

ACS Med. Chem. Lett., 2010, 1 (9), pp 478–482 DOI: 10.1021/ml100143q

AMP-activated Protein Kinase (AMPK)

✤ Widely distributed heterotrimeric ser/thr kinase:

✤ Catalytic alpha subunit, regulatory beta and gamma subunits

✤ Multiple isoforms (𝛼1, 𝛼2, 𝛽1, 𝛽2, 𝛾1, 𝛾2, 𝛾3)

✤ Metabolite sensor: sensitive to AMP/ATP ratio

✤ Activated during exercise, nutrient deprivation, hipoxia/ischemia

AMPK -- Metabolic Effects

✤ Expected effects of a liver targeted AMPK activator

Decreased fatty acid and cholesterol production

Decreased triglyceride accumulation

Increased fatty acid oxidation

http://www.huffingtonpost.com/paul-spector-md/exercise-in-a-bottle-nest_b_6221922.html

AMPK activators

N

NN

NNH2

O

OHOH

OPHOOH

O

O

NH2N

NNH2

O

OHOH

OR

R = H: AICAR R = PO3H2: ZMP

NH

S

HO

OH N

O

A-769662

MB7264

• No activity at 100µM against FBPase, 5’-nucleotidase, AMP deaminase, Adenosine Kinase, or Glycogen Phosphorylase

• No hits or cross-reactivity at 10 μM against 64 targets (MDS lead-profiling screen)

Liver AMPK Activation

Concentration (µM)

0.001 0.01 0.1 1 10 100

AM

PK

Act

ivat

ion

(V/V

0)

1.0

1.5

2.0

2.5

3.0

3.5

MB07264

AMP

EC50 = 7.6 nM

EC50 = 6.3 µM

Human

Liver AMPK Activation

Concentration (µM)

0.001 0.01 0.1 1 10 100

AM

PK

Act

ivat

ion

(V/V

0)

1.0

1.5

2.0

2.5

3.0

3.5

MB07264

AMP

EC50 = 7.6 nM

EC50 = 6.3 µM

Human

Concentration (µM)

0.001 0.01 0.1 1 10 100

AM

PK

Act

ivat

ion

(V/V

0)1

2

3

4

5

MB07264

AMP

EC50 = 20 nM

EC50 = 5.7 µM

Rat

Species-dependent Isoform Activity

Species α1 α2MB7264

Max Activation

Human >95% <5% 115%Rat 50% 50% 43%

Mouse 50% 50% 54%Guinea Pig 95% 5% 96%

MB7264

MB7264

O PO3Bn2N

O PNO

O

H2,Pd(OH)2/C

OO

OHChx2NHEthanol Chx2NH2

O

BnO2

O PO3R2O

HR = Et

R = H

R = CH2Ph

O PO3Bn2HON

XX = H

X = ClTMSBr, CH2Cl2

NNHChx

Chx OBn

BnO

Et3N, CH2Cl2

NH2OH-HCl

NaOAc, THF: H2O

NCS, DMF

MB7264 - properties

ONO

HO

PO3Et2ONO

HO

PO3H2

pKa 3.32, 3.93, 6.94

logD7.4 -4.3

pKa 3.23

Esterase Sensitive Prodrugs

O PO3H2

O

H O PO

H

OO

O

O2

I O

O

Et3N, CH3CN

(1) NH2OH-HCl, NaOAc

(2) NCS, DMF

O PHON

Cl

OO

O

O O PN O

O

O

O2

O

O2

O O

OO

Et3N, CH2Cl2

Intracellular concentration of MB7264*

Concentration Intracellular MB7264 (nmol/g)

100 µM < 55

10 µM

100 µM

254 ± 104

2151 ± 712O P

N OO

O

O2

O

O

O PNO

O

OO

OH

Chx2NH2 2

* Plated rat hepatocytes

Inhibition of de novo lipogenesis*

O PN O

O

O

O2

O

OR

R EC50 (nM) R EC50 (nM)

N/A > 10,000 OEt 42

t-Bu 100 O-(i-Pr) 27

i-Pr 20 OCH2Ph 609

Cyclopentyl 30 OC(CH3)2Et 390

* Plated rat hepatocytes

Inhibition of de novo lipogenesis*

O PN O

O

O

O2

O

OR

R In vivo DNL inhibition

R In vivo DNL inhibition

N/A ND OEt 73%

t-Bu 65% O-(i-Pr) 73%

i-Pr 78% OCH2Ph 34%

Cyclopentyl ND OC(CH3)2Et ND

*C57Bl/6 mice, 30 mg/kg ip

“Compound 13”

AMPK Activators - Summary

• MB07264 is a direct, selective, potent activator of AMPK that activates α1−containing complexes.

• In hepatocytes, a prodrug of MB07264 regulates several expected downstream targets: – Increases ACC phosphorylation

– Decreases malonyl CoA levels

– Inhibits de novo lipogenesis

• Prodrugs of MB7264 inhibit de novo lipogenesis in mice and rats (data not shown) and are preferentially distributed into liver tissues

Other studies on our MB7264 prodrug

http://www.huffingtonpost.com/paul-spector-md/exercise-in-a-bottle-nest_b_6221922.html

http://www.huffingtonpost.com/paul-spector-md/exercise-in-a-bottle-nest_b_6221922.html

http://www.huffingtonpost.com/paul-spector-md/exercise-in-a-bottle-nest_b_6221922.html

Studies confirming that: • MB7264 has 𝛼1 selectivity

and • its prodrugs inhibit liver de

novo lipogenesis

A prodrug of MB7264 in Oncology

Compound 13, an α1-selective small molecule activator of AMPK, potently inhibits melanoma cell proliferation Xueqing Hu, Fangzhen Jiang, Qi Bao, Huan Qian,

Quan Fang, Zheren Shao 

Tumor Biology, pp 1-8; First online: 14 August 2015 DOI: 10.1007/s13277-015-3854-8

Compound 13