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Research Area
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Description
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Cancer |
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Biological Activity
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Description
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A-769662 is a potent, reversible AMPK activator with EC50 of 0.8 μM and inhibits fatty acid synthesis with IC50 of 3.2 μM. |
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Targets
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AMPK |
Fatty acid synthesis |
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IC50 |
0.8 μM |
3.2 μM [1] |
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In Vitro
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A-769662 stimulates partially purified rat liver AMPK with EC50 with 0.8 μM. A-769662 activates AMPK purified from multiple tissues and species in a dose-responsive manner with modest variations in observed EC50s. EC50s determined for A-769662 using partially purified AMPK extracts from rat heart, rat muscle, or human embryonic kidney cells (HEKs) are 2.2 mM, 1.9 mM, or 1.1 mM, respectively. A 4 hours treatment of primary rat hepatocytes with A-769662 dose-dependently increases ACC phosphorylation, which correlated inhibition of fatty acid synthesis with IC50 of 3.2 μM. A-769662 also inhibits fatty acid sythesis in mouse hepatocytes with IC50 with 3.6 μM [1]A-769662 activates AMPK both allosterically and by inhibiting dephosphorylation of AMPK on Thr-172, similar to the effects of AMP. [2] A-769662 inhibits proteasomal function by an AMPK-independent mechanism. A-769662 affects the in vitro activity of purified 26S proteasomes but not the in vitro activity of purified 20S proteasomes. A-769662 has toxic effects on MEF cells. [3] A recent research shows A-769662 inhibited cell proliferation and DNA synthesis. [4] |
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In Vivo
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Short-term treatment of normal Sprague Dawley rats with A-769662 decreases liver malonyl CoA levels and the respiratory exchange ratio, VCO2/VO2, indicating an increased rate of whole-body fatty acid oxidation. Treatment of ob/ob mice with 30 mg/kg b.i.d. A-769662 decreases hepatic expression of PEPCK, G6Pase, and FAS, lowers plasma glucose by 40%, reduced body weight gain and significantly decreases both plasma and liver triglyceride levels. [1] |
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Clinical Trials
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Features
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Protocol
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Kinase Assay
[1]
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| 96-well AMPK assay |
AMPK activity is measured by monitoring phosphorylation of the SAMS peptide substrate (20 mM in standard assays and 100 mM in additivity assays) following a previously described protocol (Anderson et al., 2004). To determine whether A-769662-induced AMPK activation occurs in a reversible manner, AMP or A-769662 are preincubated with rat liver AMPK for 10 minutes at 20 times standard assay concentrations prior to dilution and measurement of AMPK activity. |
| Fatty Acid Synthesis Assay |
Primary rat hepatocytes are isolated and plated at 5 × 104 cells per well on BioCoat, collagen-coated, black-walled 96-well plates in DMEM supplemented with 10% FBS, 5 mM glucose, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 0.1 mM nonessential amino acids, 5 mg/ml ransferring, 100 nM dexamethasone, 100 nM insulin and 25 mg/ml gentamycin. After 4 hours medium is replaced with medium but without FBS and containing 100 nM triiodothyronine (T3). Following a 16 hours, 37 °C incubation, the incubation medium is removed and replaced with medium containing 14C acetate (2 mCi/ml) and AICAR or test A-769662 at the indicated concentrations. Cells are incubated 4 hours at 37 °C then the plates are rinsed with PBS. The final wash is replaced with Microscint20 and radioactivity incorporate into fatty acid monitored on a Wallac Microbeta plate reader. |
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Cell Assay
[3]
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| Cell Lines |
MEF cells |
| Concentrations |
300 μM |
| Incubation Time |
24 hours |
| Methods |
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. To determine the proportion of cells in each phase of the cell cycle, cells are harvested by trypsinization, collected by centrifugation, washed in PBS and fixed overnight in 80% ethanol at -20 °C. Subsequently, these fixed cells are centrifuged to remove the fixative and incubated for 20 minutes in the dark at room temperature in PBS containing 0.5 mg/mL RNase and 50 μg/mL propidium iodine. Flow cytometry analysis is performed as above. The proportion of cells in G1, S, and G2 is determined using the MODFIT program. Cell culture pictures are taken at the indicated times using a camera coupled to an inverted microscope with a 20 × objective. |
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Animal Study
[1]
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| Animal Models |
Sprague Dawley rats |
| Formulation |
A-769662 is dissolved in DMSO. |
| Doses |
30 mg/kg |
| Administration |
Administered via i.p. |
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References |
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[1] Cool B, et al, Cell Metab, 2006, 3(6), 403-416.
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[2] Sanders MJ, J Biol Chem, 2007, 282(45), 32539-32548.
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[3] Moreno D, et al, FEBS Lett, 2008, 583(17), 2650-2654.
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[4] Pevton KJ, et al, J Pharmacol Exp Ther, 2012, Jun 13.
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