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Purity: ≥98%
A-769662 (A769662; Thienopyridone analog A-769662 (A769662; A 769662) is a potent, alloesteric, and reversible AMPK (AMP-activated protein kinase) activator with anti-diabetic activity. It activates AMPK with an EC50 of 0.8 μM in cell-free assays, and shows little effect on GPPase/FBPase activity. The activity of AMPK isolated from rat muscle, human embryonic kidney cells (HEKs), or rat heart was activated by A-769662 with EC50 values of 1.1 mM, 1.9 mM, or 2.2 mM, respectively. With an IC50 value of 3.2 mM in primary rat hepatocytes, A-769662 inhibited the production of fatty acids. With an AMPK-independent mechanism, A769662 also inhibits the 26S proteasome.
| Targets |
AMPK (EC50 = 0.8 μM)
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| ln Vitro |
A-769662 is equally potent in activating the baculovirus expressed α1,β1,γ1 recombinant isoform of AMPK (EC50=0.7 μM). A-592107 exert dose-dependent AMPK activation with only minor variations in the observed EC50s in AMPK purified from various tissues and species. Rat heart, rat muscle, or human embryonic kidney cells (HEKs) were used to determine the EC50s for A-769662, and they were found to be 2.2 μM, 1.9 μM, or 1.1 μM, respectively[1]. A-769662 activates endogenous AMPK in LKB1-expressing (HEK293) and LKB1-deficient (CCL13) cells. A-769662 allosterically activates AMPK complexes that contain 1 and contain arginine 298 (R298G) substitutions. In the mutant 1-containing complexes, A-769662 inhibits dephosphorylation of Thr-172 to a similar extent as in the wild-type complexes[2]. Toxic effects of A769662 (300 M) on MEF cells. The proteasomal activity is reversibly inhibited by A769662[3].
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| ln Vivo |
A-769662 (30 mg/kg, i.p.) significantly reduced the respiratory exchange ratio (RER) in the SD rat. Malonyl CoA levels in the livers of animals treated with 30 mg/kg A-769662 (0.905 nmol/g) or 500 mg/kg metformin (0.574 nmol/g) are reduced by 33% and 58%, respectively. While the lower doses of A-769662 (3 and 10 mg/kg) had no effect on the diabetic ob/ob mice, the higher dose (30 mg/kg, b.i.d.) significantly lowers fed plasma glucose (30%–40% reduction)[1].
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| Enzyme Assay |
To assay glycogen phosphorylase b (GPb) activity, 1.5 μg/mL of rabbit GPb is added to a reaction mix containing 20 mM Na2HPO4 (pH 7.2), 2 mM MgSO4, 1 mM β-NADP (β-nicotinamide adenine dinucleotide phosphate), 1.4 U/mL G-6-PDH (Glucose-6-Phosphate-Dehydrogenase) and 3 U/mL PGM (phosphoglucomutase). The reaction is started by adding glycogen (final concentration 1 mg/mL) to the assay medium after adding AMP or test compounds at the designated concentrations. By measuring absorbance at 340 nm, GPb activity is evaluated following a 10-minute incubation period at 25°C.
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| Cell Assay |
Cell viability of MEF cells treated or not with A-769662 is performed as follows: cells are harvested by trypsinization and incubated with 0.5 mg/mL RNase and 50 μg/mL propidium iodine at room temperature in the dark; cell viability is analyzed by flow cytometry using a FACScanto flow cytometer, using an excitation laser at 488 nm and a propidium iodine fluorescence detection at 600 nm. Cells are harvested by trypsinization, collected by centrifugation, washed in PBS, and fixed overnight in 80% ethanol at -20 °C to ascertain the percentage of cells in each phase of the cell cycle. These fixed cells are then centrifuged to remove the fixative, and 20 minutes were spent incubating them in PBS containing 0.5 mg/mL RNase and 50 g/mL propidium iodine at room temperature in the dark. As stated above, flow cytometry analysis is carried out. The MODFIT program is used to calculate the percentage of G1, S, and G2 cells. At the designated times, cell culture images are captured using a camera connected to an inverted microscope with a 20 × objective.
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| Animal Protocol |
After acclimatization, lean and ob/ob mice are distributed randomly to the various treatment groups based on body weight and fed glucose levels (tail snip) at 8 AM. A subset of the animals representing each treatment group (n = 10 lean ob/+ and n = 10 ob/ob littermates) also has baseline plasma insulin samples taken. Ob/ob and lean littermate studies were conducted in two different ways: first, for a short period of time (5 days), and then for a longer period of time (14 days), in order to assess effectiveness and characterize the body weight change seen in the earlier study more thoroughly. Treatment groups for the 5 day study are as follows: ob/ob vehicle (0.2% hydroxypropyl methylcellulose [HPMC], i.p., b.i.d.), A-592107 (10 or 100 mg/kg, i.p., b.i.d.), A-769662 (3 or 30 mg/kg, i.p., b.i.d.), AICAR (375 mg/kg, s.c., b.i.d.), or metformin (450 mg/kg, p.o., q.d., with vehicle in PM), and lean littermates treated with vehicle (i.p., b.i.d.). Treatment groups for the 14 day ob/ob and lean littermate study are as follows: ob/ob vehicle (0.2% HPMC, i.p., b.i.d.), A-769662 (3, 10, or 30 mg/kg, i.p., b.i.d.), or metformin, and lean littermates treated with vehicle or 30 mg/kg of A-769662 (i.p., b.i.d.).
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| References |
| Molecular Formula |
C20H12N2O3S
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|---|---|
| Molecular Weight |
360.3859
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| Exact Mass |
360.05686
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| Elemental Analysis |
C, 66.65; H, 3.36; N, 7.77; O, 13.32; S, 8.90
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| CAS # |
844499-71-4
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| Related CAS # |
844499-71-4
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| Appearance |
Solid powder
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| SMILES |
N#CC1=C(O)C(C(C2=CC=C(C3=CC=CC=C3O)C=C2)=CS4)=C4NC1=O
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| InChi Key |
CTESJDQKVOEUOY-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C20H12N2O3S/c21-9-14-18(24)17-15(10-26-20(17)22-19(14)25)12-7-5-11(6-8-12)13-3-1-2-4-16(13)23/h1-8,10,23H,(H2,22,24,25)
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| Chemical Name |
4-hydroxy-3-[4-(2-hydroxyphenyl)phenyl]-6-oxo-7H-thieno[2,3-b]pyridine-5-carbonitrile
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| Synonyms |
A-769662; A 769662; A769662
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: ~72 mg/mL (~199.8 mM)
Water: <1 mg/mL Ethanol: <1 mg/mL |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.94 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 25.0 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (6.94 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 25.0 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (6.94 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 1% DMSO+30% polyethylene glycol+1% Tween 80: 30mg/mL |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.7748 mL | 13.8739 mL | 27.7477 mL | |
| 5 mM | 0.5550 mL | 2.7748 mL | 5.5495 mL | |
| 10 mM | 0.2775 mL | 1.3874 mL | 2.7748 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
A769662 inhibits proteasomal function by an AMPK-independent mechanism.FEBS Lett.2008 Jul 23;582(17):2650-4. |
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Inhibition of proteasomal activity by A769662 is reversible.FEBS Lett.2008 Jul 23;582(17):2650-4. |
A769662 affects the in vitro activity of purified 26S proteasomes but not the in vitro activity of purified 20S proteasomes.FEBS Lett.2008 Jul 23;582(17):2650-4. |
A769662 has toxic effects on MEF cells.FEBS Lett.2008 Jul 23;582(17):2650-4. |
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A-769662 allosterically activates AMPK and protects against dephosphorylation of Thr-172.J Biol Chem.2007 Nov 9;282(45):32539-48 |
A-769662 activates endogenous AMPK in LKB1-expressing (HEK293) and LKB1-deficient (CCL13) cells.J Biol Chem.2007 Nov 9;282(45):32539-48 |
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