Z-STILBENE AMPK ACTIVATOR COMPOUNDS, COMPOSITIONS, METHODS AND USES THEREOF

Abstract

The present invention relates to a compound having general formula I for use in the activation of AMPK. A composition comprising said compound for use in the activation of AMPK is also provided.

Claims

1. A method for use in the activation of AMPK comprising administering a compound having the general formula I, ##STR00014## wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are each independently selected from the group consisting of H; OH; OMe; O-glycoside; a sulfate; substituted and/or branched C1 to C20 alkyl; substituted and branched, C2 to C20 alkenyl; substituted and/or branched, C4 to C20 polyalkenyl; a derivative or analogue thereof; and a OCH3 group can cyclize with a neighboring OH group to form a methylene dioxy bridge.

2. Method according to claim 1 wherein said compound is a compound of general Formula I wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are each independently selected from the group consisting of H; CH3; OH; OMe; O-glycoside; and a sulfate.

3. Method according to claim 1 wherein said compound is a compound of general Formula I wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are each independently selected from the group consisting of H; OH; OMe; O-glycoside; a sulfate, and a derivative or analogue thereof.

4. Method according to claim 1 wherein said compound is a compound of general Formula I wherein R1, R2, R4, R5, R7, R8, and R9 are each independently selected from the group consisting of H; OH; OMe; O-glycoside; a sulfate; substituted and/or branched C1 to C20 alkyl; substituted and/or branched, C2 to C20 alkenyl; substituted and/or branched, C4 to C20 polyalkenyl; R3, R6, and R10 are H, or a derivative or analogue thereof; and a OCH3 group can cyclize with a neighboring OH group to form a methylene dioxy bridge.

5. Method according to claim 1 wherein said compound is a compound of general Formula I wherein R1, R2, R4, R5, R7, R8, and R9 are each independently selected from the group consisting of H; CH3; OH; OMe; O-glycoside; a sulfate; R3, R6, and R10 are H, or a derivative or analogue thereof.

6. A method according to claim 1, wherein said compound of general Formula I is selected from the group consisting of Pholidotol D, (Z)-Thunalbene, (Z)-3,3′-Dihydroxy-5-methoxystilbene, 3-[(1Z)-2-(3-Hydroxyphenyl)ethenyl]-5-methoxyphenol, Phenol, 3-[(1Z)-2-(3-hydroxyphenyl)ethenyl]-5-methoxy-; also known as CAS number 1006380-82-0.

7. A method according to claim 1 to treat or prevent a condition, disorder, or disease related to type 2 diabetes, non-alcoholic fatty liver disease and/or obesity.

8. A method according to claim 1, wherein the subject is a human.

9. A method according to claim 1, wherein the activation of AMPK is through a direct activation mechanism.

10. A method according to claim 1, wherein the activation of AMPK is in muscle, liver and/or kidney tissues.

11. A method according to claim 1, wherein the activation of AMPK is AMPK which comprises an α2 subunit, a β1 subunit, and a γ1 subunit.

12. A method according to claim 1, wherein the compound is obtained from a plant or plant extract.

13-14. (canceled)

15. A method according to claim 1, wherein the composition is a food, beverage, or dietary supplement.

16. A method according to claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier.

17-18. (canceled)

19. An in vitro method of activating AMPK, comprising contacting a compound having the general formula I, ##STR00015## wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are each independently selected from the group consisting of H; OH; OMe; O-glycoside; a sulfate; substituted and/or branched C1 to C20 alkyl; substituted and branched, C2 to C20 alkenyl; substituted and/or branched, C4 to C20 polyalkenyl; a derivative or analogue thereof; and a OCH3 group can cyclize with a neighboring OH group to form a methylene dioxy bridge with AMPK.

20. A method of treatment or prevention of a condition, disorder, or disease related to type 2 diabetes, non-alcoholic fatty liver disease and/or obesity comprising administration of a composition having the general formula I, ##STR00016## wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are each independently selected from the group consisting of H; OH; OMe; O-glycoside; a sulfate; substituted and/or branched C1 to C20 alkyl; substituted and branched, C2 to C20 alkenyl; substituted and/or branched, C4 to C20 polyalkenyl; a derivative or analogue thereof; and a OCH3 group can cyclize with a neighboring OH group to form a methylene dioxy bridge.

Description

BRIEF DESCRIPTION OF FIGURES

[0202] FIG. 1. Compound 1 Activation of Bacterially-Expressed AMPKα2β1γ1 Complexes.

[0203] Compound 1 is Pholidotol D, (Z)-Thunalbene, (Z)-3,3′-Dihydroxy-5-methoxystilbene, 3-[(1Z)-2-(3-Hydroxyphenyl)ethenyl]-5-methoxyphenol, Phenol, 3-[(1Z)-2-(3-hydroxyphenyl)ethenyl]-5-methoxy-; also known as CAS number 1006380-82-0.

[0204] Varying concentrations of compound 1 was incubated with phosphorylated purified AMPK α2β1γ1 for 30 mins. AMPK activity was determined using the HTRF KinEASE assay kit and results are presented as the ratio of 665/620 nm (STK1 phosphorylation).

[0205] FIG. 2. Compound 1 Increases the Phosphorylation of the AMPK Substrate, Acetyl-CoA Carboxylase (ACC), in U-2 OS Flp-In T-REx Mammalian Cells.

[0206] Compound 1 is Pholidotol D, (Z)-Thunalbene, (Z)-3,3′-Dihydroxy-5-methoxystilbene, 3-[(1Z)-2-(3-Hydroxyphenyl)ethenyl]-5-methoxyphenol, Phenol, 3-[(1Z)-2-(3-hydroxyphenyl)ethenyl]-5-methoxy-; also known as CAS number 1006380-82-0.

[0207] U-2 OS cells were treated with varying concentrations of compound 1 for 30 mins at 37 C. Phosphorylation of ACC was assessed using the HTRF Cisbio (pACC kit). Results are displayed as the average fold increase in activation relative to untreated cells.

EXAMPLES

Example 1

[0208] Compound 1 Activates Bacterially-Expressed AMPKα2β1γ1 Complexes.

[0209] The AMPKα2β1γ1 complex was expressed in bacteria and purified through the His-α subunit by nickel purification, purified through gel filtration and phosphorylated by incubation with CaMKKβ, and then further purified through a final gel filtration purification step. Phosphorylated purified AMPK was incubated with varying concentrations of ligand for 30 mins along with substrate and reagents from the HTRF-KinEASE Cisbio assay kit (STK S1 Kit). Phosphorylation of the substrate was measured by incubating with donor and acceptor antibodies for 2 h at room temperature as per the manufacturer's protocol (and Coulerie et al., (2016), see below) and phosphorylated peptide detected by performing HTRF. AMPK activity is displayed as the 665 nm/620 nm ratio.

[0210] FIG. 1 shows that compound 1 which is Pholidotol D, (Z)-Thunalbene, (Z)-3,3′-Dihydroxy-5-methoxystilbene, 3-[(1Z)-2-(3-Hydroxyphenyl)ethenyl]-5-methoxyphenol, Phenol, 3-[(1Z)-2-(3-hydroxyphenyl)ethenyl]-5-methoxy-; also known as CAS number 1006380-82-0; allosterically activates AMPKα2β1γ1 demonstrating that this compound is capable of directly activating AMPK.

[0211] Reference 1: Standardized LC×LC-ELSD Fractionation Procedure for the Identification of Minor Bioactives via the Enzymatic Screening of Natural Extracts. Coulerie P, Ratinaud Y, Moco S, Merminod L, Naranjo Pinta M, Boccard J, Bultot L, Deak M, Sakamoto K, Queiroz E F, Wolfender J L, Barron D. J Nat Prod. 2016 Nov. 23; 79(11):2856-2864. Epub 2016 Oct. 28.

Example 2

[0212] Compound 1 Increases the Phosphorylation of the AMPK Substrate, Acetyl-CoA Carboxylase (ACC), in U-2 OS Flp-In T-REx Mammalian Cells.

[0213] U-2 OS Flp-In T-REx cells were seeded at 50 K in a 96-well plate and left overnight at 37 C in DMEM GlutaMAX (Thermo Fisher Scientific) supplemented with 10% (vol/vol) FBS and 100 U/ml penicillin G, and 100 μg/ml streptomycin. Cells were treated for 30 mins with varying concentrations of compound 1 in media lacking FBS and then cells were lysed in 50 μl of Cisbio lysis buffer #1 supplemented with blocking solution as per the manufacturer's protocol (Cisbio). Cells were lysed for 30 mins at room temperature before 16 μl of lysate was incubated with 4 μl of the HTRF antibodies (1:40 dilution of the acceptor and donor (p)ACC antibodies, as per the manufacturers protocol). Lysates were incubated overnight with the antibodies before 665 nm/620 nm ratio was determined using a MolecularDevices i3 plate reader (with a HTRF cartridge add-on). The results are plotted as fold activation compared to the respective AMPK complex without any compound.

[0214] FIG. 2 shows that using the pACC HTRF assay kit (Cisbio), compound 1 which is Pholidotol D, (Z)-Thunalbene, (Z)-3,3′-Dihydroxy-5-methoxystilbene, 3-[(1Z)-2-(3-Hydroxyphenyl)ethenyl]-5-methoxyphenol, Phenol, 3-[(1Z)-2-(3-hydroxyphenyl)ethenyl]-5-methoxy-; also known as CAS number 1006380-82-0; increased the phosphorylation of the AMPK substrate, ACC, in a dose-dependent manner in U-2 OS Flp-In T-REx mammalian cells. Phosphorylation of ACC is widely used as a cellular indicator of AMPK activity. These data show that compound 1 is an AMPK activator in cells.