DIRECT AMPK ACTIVATOR COMPOUNDS COMBINED WITH INDIRECT AMPK ACTIVATOR COMPOUNDS, COMPOSITIONS, METHODS AND USES THEREOF

Abstract

The present invention relates to a combinations of direct AMPK activators with indirect AMPK activators for use in activating AMPK. In particular, combinations of benzocoumarins of formula I which are direct AMPK activators with urolithins of formula VII which are indirect AMPK activators.

Claims

1. Combination of a direct AMPK activator compound, which binds directly to at least one alpha, beta or gamma subunit of AMPK; and an indirect AMPK activator compound, which does not bind directly to AMPK but alters the nucleotide status of the cell by lowering ATP in the cell and increasing AMP/ADP, to activate AMPK via the gamma-subunit; wherein the direct AMPK activator compound has the general formula I ##STR00019## wherein R1, R2, R3, R4, R5, R6, R7, and R8 are each independently selected from the group consisting of H; CH.sub.3; CH.sub.2OH; CHO; COOH; OH; OCH.sub.3; CO—(CH.sub.2).sub.2—CH.sub.3; O—CO—CH.sub.3; a halogen; O-glycoside; C-glycoside; acylated O-glycoside; acylated C-glycoside; sulfated O-glycoside; sulfated C-glycoside; and a sulfate; and the indirect AMPK activator compound has the general formula VII ##STR00020## wherein R1, R2, R3, and R4 are each independently selected from the group consisting of OH; OCH.sub.3; O-glycoside; C-glycoside; acylated O-glycoside; acylated C-glycoside; sulfated O-glycoside; sulfated C-glycoside; a sulfate; for use in the activation of AMPK.

2. Combination according to claim 1 for use in the activation of AMPK wherein said direct AMPK activator compound is a compound of Formula II ##STR00021## wherein R1, R2, R3, R4, and R5 are each independently selected from the group consisting of OH; OCH.sub.3; O-glycoside; C-glycoside; acylated O-glycoside; acylated C-glycoside; sulfated O-glycoside; sulfated C-glycoside; a halogen; a primary, secondary, or tertiary alcohol; a ketone; an aldehyde; a carboxylic acid; an ester; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; C1 to C20 alkyl; R6, and R7 are each independently H, OH; OCH.sub.3; O-glycoside; C-glycoside; acylated O-glycoside; acylated C-glycoside; sulfated O-glycoside; sulfated C-glycoside; a halogen; a primary, secondary, or tertiary alcohol; a ketone; an aldehyde; a carboxylic acid; an ester; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; a sulfate; and/or a derivative or analogue thereof, for use in the activation of AMPK.

3. Combination according to claim 1 for use in the activation of AMPK wherein said direct AMPK activator compound is a compound of Formula III ##STR00022## wherein R1, R2, R3, R4, and R5 are each independently selected from the group consisting of OH; OCH.sub.3; O-glycoside; C-glycoside; acylated O-glycoside; acylated C-glycoside; sulfated O-glycoside; sulfated C-glycoside; a halogen; a primary, secondary, or tertiary alcohol; a ketone; an aldehyde; a carboxylic acid; an ester; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; a sulfate; R6, and R7 are each independently H, OH; OCH.sub.3; O-glycoside; C-glycoside; acylated O-glycoside; acylated C-glycoside; sulfated O-glycoside; sulfated C-glycoside; a halogen; a primary, secondary, or tertiary alcohol; a ketone; an aldehyde; a carboxylic acid; an ester; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; a sulfate; and/or a derivative or analogue thereof, for use in the activation of AMPK.

4. Combination according to claim 1 for use in the activation of AMPK wherein said direct AMPK activator compound is a compound of Formula IV ##STR00023## wherein R1, R2, and R3 are each independently selected from the group consisting of OH; OCH.sub.3; O-glycoside; C-glycoside; acylated O-glycoside; acylated C-glycoside; sulfated O-glycoside; sulfated C-glycoside; a halogen; a primary, secondary, or tertiary alcohol; a ketone; an aldehyde; a carboxylic acid; an ester; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; a sulfate; R4, and R5 are each independently selected from the group consisting of H, OH; OCH.sub.3; O-glycoside; C-glycoside; acylated O-glycoside; acylated C-glycoside; sulfated O-glycoside; sulfated C-glycoside; a halogen; a primary, secondary, or tertiary alcohol; a ketone; an aldehyde; a carboxylic acid; an ester; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; a sulfate; and/or a derivative or analogue thereof, for use in the activation of AMPK.

5. Combination according to claim 1 for use in the activation of AMPK wherein said direct AMPK activator compound is a compound of Formula V ##STR00024## wherein R1, R2, R3, and R4 are each independently selected from the group consisting of OH; OCH.sub.3; O-glycoside; C-glycoside; acylated O-glycoside; acylated C-glycoside; sulfated O-glycoside; sulfated C-glycoside; a halogen; a primary, secondary, or tertiary alcohol; a ketone; an aldehyde; a carboxylic acid; an ester; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; a sulfate; R5 and R6 are each independently H; OH; OCH.sub.3; O-glycoside; C-glycoside; acylated O-glycoside; acylated C-glycoside; sulfated O-glycoside; sulfated C-glycoside; a halogen; a primary, secondary, or tertiary alcohol; a ketone; an aldehyde; a carboxylic acid; an ester; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; a sulfate; and/or a derivative or analogue thereof, for use in the activation of AMPK.

6. Combination according to claim 1 for use in the activation of AMPK, wherein said direct AMPK activator compound is 3,10-Dihydroxy-8-methoxy-6H-benzo[c]chromen-6-one; 6H-Dibenzo[b,d]pyran-6-one, 3,10-Dihydroxy-8-methoxy; 3,10-Dihydroxy-8-methoxy-6H-dibenzo[b,d]pyran-6-one.

7. Combination according to claim 1 for the activation of AMPK wherein the indirect AMPK activator is a compound of Formula VII selected from the group consisting of: ##STR00025##

8. Combination according to claim 1 for the activation of AMPK wherein the indirect AMPK activator is Urolithin B.

9. Combination according to claim 1 for the activation of AMPK wherein the direct AMPK activator is selected from the group consisting of 3,10-Dihydroxy-8-methoxy-6H-benzo[c]chromen-6-one; 6H-Dibenzo[b,d]pyran-6-one, 3,10-Dihydroxy-8-methoxy; 3,10-Dihydroxy-8-methoxy-6H-dibenzo[b, d]pyran-6-one and the indirect AMPK activator is Urolithin B.

10. A method according to claim 1 for the activation of AMPK to treat or prevent a condition, disorder, or disease selected from the group consisting of cardiometabolic health, obesity, type 2 diabetes, non-alcoholic fatty liver disease, cardiovascular disease, and/or cancer comprising administering to a subject in need of same a combination of a direct AMPK activator compound, which binds directly to at least one alpha, beta or gamma subunit of AMPK; and an indirect AMPK activator compound, which does not bind directly to AMPK but alters the nucleotide status of the cell by lowering ATP in the cell and increasing AMP/ADP, to activate AMPK via the gamma-subunit wherein the direct AMPK activator compound has the general formula I ##STR00026## wherein R1, R2, R3, R4, R5, R6, R7, and R8 are each independently selected from the group consisting of H; CH, CH.sub.2OH; CHO; COOH; OH; OCH.sub.3; CO—(CH.sub.2).sub.2—CH.sub.3; O—CO—CH.sub.3, a halogen; O-glycoside; C-glycoside; acylated O-glycoside; acylated C-glycoside; sulfated O-glycoside; sulfated C-glycoside; and a sulfate; and the indirect AMPK activator compound has the general formula VII ##STR00027## wherein R1, R2, R3, and R4 are each independently selected from the group consisting of OH; OCH.sub.3; O-glycoside; C-glycoside; acylated O-glycoside; acylated C-glycoside; sulfated O-glycoside; sulfated C-glycoside; a sulfate; for use in the activation of AMPK.

11. Method according to claim 10 for the activation of AMPK, wherein the subject is a human.

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

13. Method according to claim 1, wherein the compounds are obtained from a plant or plant extract.

14. (canceled)

15. Combination according to claim 1 wherein said combination is formulated as a food, beverage, or dietary supplement.

16. Combination according to claim 1 wherein said combination is formulated as a pharmaceutical product.

17. (canceled)

18. A method of treatment of a condition, disorder, or disease related to cardiometabolic health, obesity, type 2 diabetes, non-alcoholic fatty liver disease, cardiovascular disease, and/or cancer comprising administration of the combination of a direct AMPK activator compound, which binds directly to at least one alpha, beta or gamma subunit of AMPK, and an indirect AMPK activator compound, which does not bind directly to AMPK but alters the nucleotide status of the cell by lowering ATP in the cell and increasing AMP/ADP, to activate AMPK via the gamma-subunit; wherein the direct AMPK activator compound has the general formula I ##STR00028## wherein R1, R2, R3, R4, R5, R6, R7, and R8 are each independently selected from the group consisting of H; CH.sub.3; CH.sub.2OH, CHO; COOH; OH; OCH.sub.3; CO—(CH.sub.2).sub.2—CH.sub.3; O—CO—CH.sub.3; a halogen; O-glycoside; C-glycoside; acylated O-glycoside; acylated C-glycoside; sulfated O-glycoside; sulfated C-glycoside; and a sulfate; and the indirect AMPK activator compound has the general formula VII ##STR00029## wherein R1, R2, R3, and R4 are each independently selected from the group consisting of OH; OCH.sub.3; O-glycoside; C-glycoside; acylated O-glycoside; acylated C-glycoside; sulfated O-glycoside; sulfated C-glycoside; a sulfate; for use in the activation of AMPK to a human in need of same.

Description

BRIEF DESCRIPTION OF FIGURES

[1048] FIG. 1. Compound 1 increases the phosphorylation of the AMPK substrate, acetyl-CoA carboxylase (ACC), in U-2 OS Flp-In T-REx mammalian cells.

[1049] 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 ratio 665/620 nm (±SEM) of 3 independent experiments.

[1050] Compound 1: indirect activator (Urolithin B) 3-hydroxy-6H-benzo[c]chromen-6-one; CAS: 1139-83-9

[1051] FIG. 2. Compound 2 improves the dose-response curve of Compound 1, for activation of AMPK in cells.

[1052] U-2 OS cells were treated with varying concentrations of compound 1 for 30 mins at 37 C in the presence or absence of 11 μM of compound 2. Phosphorylation of ACC was assessed using the HTRF Cisbio (pACC kit). Results are displayed as the ratio 665/620 nm (±SEM) of 3 independent experiments.

[1053] Compound 1: indirect activator (Urolithin B) 3-hydroxy-6H-benzo[c]chromen-6-one; CAS: 1139-83-9

[1054] Compound 2: direct activator (Benzocoumarin) 3,10-Dihydroxy-8-methoxy-6H-benzo[c]chromen-6-one; 6H-Dibenzo[b,d]pyran-6-one, 3,10-Dihydroxy-8-methoxy; 3,10-Dihydroxy-8-methoxy-6H-dibenzo[b,d]pyran-6-one

[1055] FIG. 3. Compound 2 does not improve the dose-response curve of Compound 1 in cells stably expressing an ADaM-binding site AMPK mutant (β1 S108A).

[1056] The β1 WT or β1 S108A mutant were stably expressed in AMPKβ1β2 double knockout cells and were treated with varying concentrations of compound 1 for 30 mins at 37 C in the presence or absence of 11 μM of compound 2. Phosphorylation of ACC was assessed using the HTRF Cisbio (pACC kit). Results are displayed as the average fold increase in activation (±SEM) of 3 independent experiments.

[1057] Compound 1: indirect activator (Urolithin B) 3-hydroxy-6H-benzo[c]chromen-6-one; CAS: 1139-83-9

[1058] Compound 2: direct activator (Benzocoumarin) 3,10-Dihydroxy-8-methoxy-6H-benzo[c]chromen-6-one; 6H-Dibenzo[b,d]pyran-6-one, 3,10-Dihydroxy-8-methoxy; 3,10-Dihydroxy-8-methoxy-6H-dibenzo[b,d]pyran-6-one

[1059] FIG. 4. Compound 2 does not improve the dose-response curve of compound 1 in cells stably expressing the β2 isoform.

[1060] U-2 OS cells AMPKβ1β2 double knockout cells stably expressing AMPK β2 were treated with varying concentrations of compound 1 for 30 mins at 37 C in the presence or absence of 11 μM of compound 2. Phosphorylation of ACC was assessed using the HTRF Cisbio (pACC kit). Results are displayed as the average fold increase in activation (±SEM) of 3 independent experiments.

[1061] Compound 1: indirect activator (Urolithin B) 3-hydroxy-6H-benzo[c]chromen-6-one; CAS: 1139-83-9

[1062] Compound 2: direct activator (Benzocoumarin) 3,10-Dihydroxy-8-methoxy-6H-benzo[c]chromen-6-one; 6H-Dibenzo[b,d]pyran-6-one, 3,10-Dihydroxy-8-methoxy; 3,10-Dihydroxy-8-methoxy-6H-dibenzo[b,d]pyran-6-one

EXAMPLES

Example 1

[1063] Compound 1 Increases the Phosphorylation of the AMPK Substrate, Acetyl-CoA Carboxylase (ACC), in U-2 OS Flp-In T-REx Mammalian Cells.

[1064] Natural compounds typically activate AMPK almost exclusively through their ability to interfere with ATP production of the cell, typically by inhibiting mitochondrial respiration. As a consequence, this perturbs the adenine nucleotide levels within the cell and leads to activation of AMPK through AMP and ADP binding to the AMPK γ subunit. This mechanism of AMPK activation has been termed “indirect” due to the fact that natural compounds do not directly bind to AMPK to achieve activation. In contrast, AMPK can be “directly” activated by binding of small molecules to the allosteric drug and metabolite (ADaM) binding site formed at the interface between the AMPK α subunit kinase domain and the AMPK β subunit.

[1065] We have now identified a new benzocoumarin compound, compound 2, which directly binds to the ADaM-site and activates AMPK. We achieved synergistic activation of AMPK by treating cells with a combination of an indirect benzocoumarin activator (compound 1) and this newly identified direct benzocoumarin activator (compound 2).

[1066] First, we monitored the activation of AMPK within cells using the indirect activator, compound 1. 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 the 665 nm/620 nm ratio was determined using a MolecularDevices i3 plate reader (with a HTRF cartridge add-on).

[1067] FIG. 1 shows that using the pACC HTRF assay kit (Cisbio), Compound 1 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 extensively used as a cellular read-out of AMPK activity.

Example 2

[1068] Compound 2 Improves the Dose-Response Curve of Compound 1 for the Phosphorylation of the AMPK Substrate, Acetyl-CoA Carboxylase (ACC), in U-2 OS Flp-In T-REx Mammalian Cells.

[1069] To investigate whether treatment of compound 1 with our new identified direct benzocoumarin AMPK activator, compound 2, could improve the activation of AMPK, cells were treated for 30 mins with varying concentrations of compound 1 in the presence or absence of 11 μM compound 2. Cells were cultured and analysed according to example 1.

[1070] FIG. 2 shows that using the pACC HTRF assay kit (Cisbio), compound 1 increases the phosphorylation of the AMPK substrate, ACC, in a dose-dependent manner in U-2 OS Flp-In T-REx mammalian cells. Surprisingly, in cells treated with compound 1 in the presence of a fixed concentration of compound 2 showed a left-shift in the dose-response curve. This suggests that there is an improved ability of compound 1 to activate AMPK when compound 2 is present. This synergistic activation of AMPK was achieved when using two AMPK activators with differing mechanisms of action, namely, one directly and the other indirectly activating AMPK.

Example 3

[1071] Compound 2 does not Improve the Dose-Response Curve of Compound 1 in Cells Expressing AMPK Complexes Containing a Mutation at the Allosteric Drug and Metabolite (ADaM) Site in Cells (S108A).

[1072] We investigated whether the ability of compound 2 to improve the activation profile of compound 1, was dependent on the ability of compound 2 to bind to the ADaM-site of AMPK. Interference of the regulation at the ADaM-site can be induced by either introducing a mutation at site serine 108 within the β subunit of the ADaM-site, or by switching the β isoform from β1 to β2. Activation of the β2 subunit isoform weakens or abolishes the ability of ADaM-site activators to stimulate AMPK.

[1073] AMPKβ1/β2 double knockout U-2 OS Flp-In™ T-Rex™ cell lines were generated by Horizon Discovery (Cambridge, UK). Cells were genotyped and analysed by Western blotting to confirm that there was a complete knockout of AMPKβ1/β2. We took these AMPKβ1/β2 double knockout cells, and re-introduced the expression of human β1 wild-type (WT) or a β1 Serine 108 to alanine mutation (S108A). This was achieved using the Flp-In™ system (Invitrogen) present in this cell line and stable cells expressing β1 WT or a β1 S108A mutant were generated according to the manufacturers' protocols. Re-expression of the β1 subunit was confirmed by Western blot analysis.

[1074] Cells stably expressing β1 WT or a β1 S108A mutant were treated with varying concentrations of Compound 1 in the presence or absence of a fixed concentration (11 μM) of compound 2. Cells lysates were subjected to the pACC HTRF (Cisbio) assay to determine the level of phosphorylation of the AMPK substrate, ACC, in cell lysates, as in example 1. As shown in FIG. 3, Compound 1 was able to dose-dependently increase pACC in cells stably expressing the β1 WT subunit. Compound 2 was capable of causing a left-shift and improvement in the ability of compound 1 to activate AMPK in β1 WT cells. In contrast, Compound 2 was not able to cause a left-shift and improvement in the dose-response curve of compound 1 in cells expressing the β1 S108A mutant. This suggests that the ability of compound 2 to improve the activation of compound 1, is mediated by its ability to bind to the ADaM-site of AMPK.

Example 4

[1075] In Cells, Compound 2 does not Improve the Dose-Response of Compound 1 to Activate AMPK Complexes Containing the β2 Isoform Subunit.

[1076] We took AMPKβ1/β2 double knockout cells, and re-introduced the expression of the human β2 WT isoform. Re-expression of the β2 subunit was confirmed by Western blot analysis and were shown to be expressed to a similar extent. Cells stably expressing β2 WT were treated with varying concentrations of Compound 1 in the absence or presence of a fixed concentration (11 μM) of compound 2. Cell lysates were subjected to the pACC HTRF (Cisbio) assay to determine the level of phosphorylation of the AMPK substrate, ACC, in cell lysates, as in Example 1. As shown in FIG. 4, Compound 2 did not improve the dose-response curve of compound 1 in cells expressing the β2 WT isoform. Direct activation AMPK by binding to the ADaM site was impaired or abolished in β2-containing complexes in vitro and in cells. The inability of compound 2 to improve the activation of AMPK by compound 1, was consistent with the knowledge we have generated showing the compound 2 binds to the ADaM-site of AMPK. Taken together, we showed that in cells, Compound 2 binding to the ADaM-pocket, and indirect activation of AMPK (compound 1) result in synergistic activation of AMPK. Low doses of compound 1 in combination with compound 2 could activate AMPK. This demonstrated the advantage of combining two AMPK activators with different modes of action.