AMPK; pan-AMPK

MK-8722 activates pAMPK complexes more powerfully and significantly than AMP. MK-8722 causes the phosphorylation of a number of additional known pAMPK targets in primary mouse hepatocytes, HepG2 cells, or primary human myocytes. The most potent off-target activity seen for MK-8722 is against the serotonin 5-HT2A receptor. [1]

In addition to reducing insulin resistance and hyperglycemia, pharmacological pan-AMPK activation by MK-8722 results in improvements in glucose homeostasis that are chronically sustainable. MK-8722 (30 mpk) acute treatment significantly reduces blood sugar and insulin levels. Chronic MK-8722 administration to mice also raises the level of Glut4 protein in the muscles. With increased cardiac and skeletal muscle glycogen, MK8722 causes cardiac hypertrophy in rhesus monkeys. [1]

AMPK activity assay and determination of activator EC50 values [1]
The enzymatic reaction was performed in three phases. In phase 1, the AMPK complex of interest was appropriately diluted (to twice the desired concentration in the final enzymatic reaction) in AMPK reaction buffer containing 20 mM HEPES, pH 7.3, 10 mM MgCl2, 6 mM DTT, 0.01% Brij 35, 200 mM ATP, and then phosphorylated/activated by addition of GST-tagged, truncated CaMKK2 (~20 nM final) (30) and incubation at room temperature for 30 min to yield pAMPK. For γ3 containing AMPK enzymes, the buffer used was 100mM Tris-HCl, pH 7.5, 100 mM KCl, 10 mM MgCl2, 6 mM DTT, 0.02% BSA and 200 uM ATP. In phase two, compound and pAMPK were pre-incubated by adding appropriately diluted AMPK activator (e.g., MK-8722 or AMP) in DMSO (1.2 mL total) to the reaction buffer containing pAMPK (15 mL per well; 384 well plates), the plate was vortexed briefly and then incubated at room temperature for 30 min. Finally, pAMPK reaction phase 3 was initiated by the addition of a fluorescein-labeled SAMS peptide, 5-FAM-HMRSAMSGLHLVKRR-COOH , in AMPK reaction buffer lacking ATP (15 mL; 1.5 mM final concentration peptide substrate). The plate was sealed and incubated at room temperature for 60 min, at which time the reaction was stopped by the addition of quench buffer consisting of 100 mM HEPES, pH 7.3, 0.015% Brij 35 and 40 mM EDTA. The reaction plate was centrifuged (2000 rpm for 4 min) and the supernatant read on the LabChip EZ reader using the following parameters: -550 downstream voltage, -2250 upstream voltage, -1.5 psi pressure, 40 s post-sample sip time, and 110 s final delay. The %product formed was calculated by taking the ratio of the substrate and product peak heights and multiplying by 100. EC50 and %activation parameters were calculated from %product vs. activator concentration plots using 4-parameter logistic curve fitting analysis. Selectivity of the MK-8722 was assessed in a broad array of pharmacologically relevant targets (120 Panlabs assays, including various enzymes, receptors, ion channels and transporters) at Eurofins.

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Synergistic effect of MK-8722 and AMP on AMPK activity [1]
For this study, pAMPK activity was determined by measuring the rate of ADP formation in the kinase reaction using an ADP-Glo Kinase Assay Kit. pAMPK12 (2 nM; generated using CAMKK2) was pre-incubated with saturating levels of activators, AMP at 25 mM and/or MK-8722 at 195 nM, in reaction buffer containing 100 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 100 mM KCl, I mM DTT, 0.015% Brij 35, and 0.02% BSA for 30 minutes at room temperature. The reactions were then initiated by addition of 100 mM of ATP and 10 mM peptide substrate (sequence: HMRSAMSGLH). The reactions were terminated as a function of incubation time, and ADP formation determined using the ADP-Glo™ Reagent per manufacturer instructions. Luminescence was measured using a SpectraMax M5 microplate reader. Data are from two independent studies.

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MK-8722 plasma protein binding [1]
Plasma protein binding to C57BL/6 mouse, Wistar-Han rat, rhesus macaque monkey and human plasma was determined by using a 96-well RED Equilibrium Dialyzer Plate. Equilibrium dialysis of plasma spiked with MK-8722 (10 mM) was performed against isotonic phosphate buffer (PBS). As MK-8722 is highly protein bound, diluted (10%) plasma was also included to estimate the binding of MK-8722 in undiluted (100%) plasma. At the end of the dialysis (4-6 hr), aliquots of the plasma and phosphate buffer were analyzed by LC-MS/MS to quantitate the MK-8722 in each compartment following protein precipitation using acetonitrile (see below).

Effect of AMPK activators on cellular pACC and pAMPK [1]
HepG2 hepatoma cells and Hela cells were added (50,000 cells per well) to a 96-well plates and cultured overnight in DMEM containing 10% fetal bovine serum, Penicillin / Streptomycin and non-essential amino acids at 37 ºC in a 5% CO2 atmosphere. Freshly isolated mouse and rat primary hepatocytes (25,000 cells per well) were added to collagen I coated 96-well plates in IHAMC media containing 10% fetal bovine serum, Penicillin / Streptomycin and cultured overnight at 37 ºC in a 5% CO2 atmosphere. The following morning, the media was changed to DMEM without serum (99 mL per well) and cultured for an additional 2 hr. In studies examining the effect of Compound C, the cells were preincubated with or without Compound C at the indicated final concentration for 1 hr prior to AMPK activator addition. DMSO alone or AMPK activator (MK-8722, AICAR or ionomycin) in DMSO (1 mL, 1% DMSO final) was then added to yield the desired final concentration and culturing continued for 1 hr using MK-8722 and AICAR or for 5 min using ionomycin. Control studies demonstrated that DMSO control results did not change during incubation times from 5 to 60 min. Following the appropriate incubations, the media was then gently removed by suction and 60 uL of chilled cell lysis buffer containing PhosStop phosphatase inhibitors and complete protease...

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Effect of MK-8722 and insulin on 2-deoxy-D-glucose uptake in human skeletal muscle cells [1]
Primary human skeletal muscle cells and media were usedNon-differentiated muscle cells were plated at a density of 30,000 cells in 100 mL of growth medium in 96-well collagen-coated white/opaque plates. After 4-6 hr of culture at 37 ºC under a 5% CO2 atmosphere, the media was changed to basal media + 0.1% bovine serum albumin. DMSO with or without Compound C at a final concentration of 30 mM) was added and the culture incubated for 1 hr. At that point, DMSO with or without MK-8722 at a final concentration of 3 or 10 mM was added and the incubation continued overnight (2% final DMSO concentration). The next day, Humulin was added to a final concentration of 100 nM, followed by 1 hr incubation. The plate was then washed twice with PBS, then PBS/Ca+2/Mg+2 /0.1% BSA containing 100 mM 2-deoxy-D-glucose plus 0.5 mCi/mL [14C]-2-deoxy-D-glucose was added. Following a 10-min incubation, the plate was placed on ice, the media removed and the wells then washed three times with ice-cold PBS. The remaining cells were lysed using 50 mL cell lysis buffer for 1 hr at room temperature, 150 mL of Microscint 40 was added and radioactivity determined using a Microplate Scintillation and Luminescence Counter.

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MK-8722 effects on cellular ATP [1]
Mouse 3T3-L1 fibroblasts (passage #2-11) and rat hepatoma H4IIE cells were grown in monolayer. Differentiation of 3T3-L1 fibroblasts was initialed 5-7 days post-seeding in BioCoat T-150 flasks. Cells were incubated with differentiation media for 8-12 days (2 days plus IBMX, 2 days plus insulin, 4-8 days plus fetal bovine serum). Fully differentiated 3T3-L1 adipocytes were trypsinized and reseeded on a collagen-coated 96-well plate at a density of ~6-8 x 104 cells per well. Cells were starved for 60 min with starvation media (DMEM plus 25 mM D-glucose, 0.1 μM Dexamethasone and 1 μM insulin) and then treated with DMSO (1% final) with or without MK-8722 for 60 min. H4IIE cells were grown to ~90% confluency and treated with DMSO (1% final) with or without MK-8722 for 3 hr.

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MK-8722 cell permeability [1]
The cell permeability of MK-8722 was determined in LLC-PK1 cells cultured in 24-well transwell culture plates. Solutions containing [3H]-MK-8722 were prepared at a final concentration of 0.1, 0.5, or 1 μM in Hank’s Balanced Salt Solution containing 10 mM HEPES, pH7.4. These solutions were individually added to either the apical (A) or the basolateral (B) compartment of the culture plate, and buffer was added to the compartment opposite to that containing the compound. At t=3 hr, sample (50 mL) was taken out from both sides and counted for radioactivity.

Mice: Lean Housing Both C57BL/6 eDIO mice and C57BL/6 mice between the ages of 10 and 12 weeks are employed. 7-week-old db/db mice are employed. With free access to food and water and a constant temperature of 22°C, animals are kept on a 12/12-hour light-dark cycle. In a typical cage, four thin C57BL/6 mice are kept. eDIO mice are housed in individual cages. In a sizable rodent cage, eight db/db mice are kept. Before receiving compound treatments, C57BL/6 mice and db/db mice are kept on the regular rodent chow diet 7012 (5% dietary fat; 3.75 kcal/g). A diet high in fat (60 kcal%) is used to maintain eDIO mice. Use 10 mL/kg body weight when administering MK8722 orally in a standard vehicle or by the vehicle alone. By contrasting MK8722's effects with those of animals that had been given vehicles as treatments, various metabolic parameters are determined[1].

[1]. Science. 2017 Aug 4;357(6350):507-511.

5'-Adenosine monophosphate-activated protein kinase (AMPK) is a master regulator of energy homeostasis in eukaryotes. Despite three decades of investigation, the biological roles of AMPK and its potential as a drug target remain incompletely understood, largely because of a lack of optimized pharmacological tools. We developed MK-8722, a potent, direct, allosteric activator of all 12 mammalian AMPK complexes. In rodents and rhesus monkeys, MK-8722-mediated AMPK activation in skeletal muscle induced robust, durable, insulin-independent glucose uptake and glycogen synthesis, with resultant improvements in glycemia and no evidence of hypoglycemia. These effects translated across species, including diabetic rhesus monkeys, but manifested with concomitant cardiac hypertrophy and increased cardiac glycogen without apparent functional sequelae.[1]

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Our results show that systemic, pharmacological pan-AMPK activation by MK-8722 leads to chronically sustainable improvements in glucose homeostasis, including the amelioration of insulin resistance and hyperglycemia. No evidence of hypoglycemia was found in any study using MK-8722, including high-dose 6- to 8-month studies in normoglycemic rats and rhesus monkeys. These effects appear to be mediated predominantly by insulin-independent skeletal muscle glucose uptake. Glucose lowering without a requirement for increased insulin levels should reduce the demand on the pancreatic β cell for insulin and, as a consequence, may increase the durability of antihyperglycemic action. This profile is both unique and highly desirable for patients with T2DM, differing from all currently available therapeutic agents. On the other hand, MK-8722 treatment induced reversible cardiac hypertrophy that was not associated with notable functional sequelae or ECG abnormalities in the time frames studied. Our data suggest that this hypertrophy is not due to the observed cardiac glycogen accumulation. Rather, the effect is reminiscent of the physiological cardiac hypertrophy observed in elite athletes. It is well established that exercise can activate cardiac AMPK (29). Whether this effect is tolerable in humans having the pathophysiology of metabolic syndrome, obesity, and overt diabetes remains to be determined. While this manuscript was in the final stages of review, Cokorinos et al. reported that a close analog of MK-8722 (PF-739) was able to activate skeletal muscle AMPK and lower glucose in preclinical species independent of hepatic AMPK activation. [1]

C24H20CLN3O4

449.886304855347

449.114

C, 64.07; H, 4.48; Cl, 7.88; N, 9.34; O, 14.22

1394371-71-1

1394371-71-1

89558344

White to off-white solid powder

3.8

2

6

4

32

645

4

C1[C@H]([C@@H]2[C@H](O1)[C@@H](CO2)OC3=NC4=NC(=C(C=C4N3)Cl)C5=CC=C(C=C5)C6=CC=CC=C6)O

XQMNBTZLYOOAGA-UGESXGAOSA-N

InChI=1S/C24H20ClN3O4/c25-19-16(15-8-6-14(7-9-15)13-4-2-1-3-5-13)10-26-23-20(19)27-24(28-23)32-18-12-31-21-17(29)11-30-22(18)21/h1-10,17-18,21-22,29H,11-12H2,(H,26,27,28)/t17-,18-,21-,22-/m1/s1

(3R,3aR,6R,6aR)-6-[[6-chloro-5-(4-phenylphenyl)-1H-imidazo[4,5-b]pyridin-2-yl]oxy]-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-3-ol

MK8722; MK 8722; 1394371-71-1; CHEMBL4167177; (3R,3aR,6R,6aR)-6-[[6-chloro-5-(4-phenylphenyl)-1H-imidazo[4,5-b]pyridin-2-yl]oxy]-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-3-ol; 1,4:3,6-Dianhydro-2-O-(5-[1,1'-biphenyl]-4-yl-6-chloro-3H-imidazo[4,5-b]pyridin-2-yl)-D-mannitol; (3R,3aR,6R,6aR)-6-[(5-{[1,1'-biphenyl]-4-yl}-6-chloro-3H-imidazo[4,5-b]pyridin-2-yl)oxy]-hexahydrofuro[3,2-b]furan-3-ol; RW3ZG69SHT; MK-8722

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~90 mg/mL (~115.1 mM)
Ethanol: ~2 mg/mL (~4.5 mM)

Solubility in Formulation 1: 2.08 mg/mL (4.62 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.62 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.62 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)