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| Targets |
7-Methoxyisoflavone is an isoflavone derivative and an activator of adenosine monophosphate-activated protein kinase (AMPK). Serum-induced reduction in AMPK phosphorylation is avoided when serum-starved cells are fed with 10% FBS in the presence of 7-methoxyisoflavone [1].
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| ln Vitro |
7-Methoxyisoflavone is an isoflavone derivative and an activator of adenosine monophosphate-activated protein kinase (AMPK). Serum-induced reduction in AMPK phosphorylation is avoided when serum-starved cells are fed with 10% FBS in the presence of 7-methoxyisoflavone [1].
In an in vitro kinase assay using recombinant human AMPK (α1β1γ1), 7-Methoxyisoflavone at a concentration of 40 µM resulted in a relative AMPK activity of 108% (the highest among 37 tested isoflavones, tied with 7-Hydroxyisoflavone), indicating it acts as an AMPK activator. The activity is measured relative to 100% phosphorylation control. [1] Western blot analysis was performed on serum-starved 3T3L1 preadipocytes to confirm AMPK activation. Serum starvation for 24 hours increased phosphorylation of AMPK at Thr172. Subsequent treatment with 10% FBS for 30 minutes decreased this phosphorylation. However, co-treatment with 10% FBS and 40 µM 7-Methoxyisoflavone prevented the serum-induced decrease in AMPK phosphorylation, confirming its AMPK-activating effect in cells. [1] In silico docking studies were performed to elucidate the molecular binding mode of 7-Methoxyisoflavone to AMPK. Using the human AMPK structure (PDB: 4cfe), flexible docking was performed. The analysis indicated that the 7-Methoxyisoflavone-AMPK complex involves hydrophobic interactions with Ile46 (from the α2 subunit) and forms three hydrogen bonds with Lys29 (α2), Arg83 (β1), and Asn111 (β1). The binding energy for the complexes ranged from -15.89 to -12.77 kcal/mol. [1] |
| Enzyme Assay |
An in vitro kinase assay for AMPK was performed using a commercial kit. Briefly, recombinant human AMPK (α1β1γ1) at 0.49 nM was incubated with 50 µM ATP and a peptide substrate (AlaMetAlaArgAlaAlaSerAlaAlaAlaLeuAlaArgArgArg). Test compounds, including 7-Methoxyisoflavone, were tested at a concentration of 40 µM. The kinase reaction allows for phosphorylation of the peptide substrate. The assay utilizes a fluorescence resonance energy transfer (FRET) principle, where phosphorylation of the substrate peptide disrupts FRET between coumarin and fluorescein. Fluorescence signals were measured with excitation at 400 nm and emissions at 445 nm (coumarin) and 520 nm (fluorescein). The degree of phosphorylation (and thus AMPK activity in the presence of the compound) was calculated relative to a 100% phosphorylated control. All experiments were performed in triplicate. [1]
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| Cell Assay |
AMPK Phosphorylation Western Blot in 3T3L1 Cells: 3T3L1 preadipocytes were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells were serum-starved for 24 hours to induce AMPK activation (increased p-AMPK Thr172). Subsequently, cells were treated with 10% FBS in the absence or presence of 40 µM 7-Methoxyisoflavone for 30 minutes. After treatment, cells were harvested and lysed in a buffer containing HEPES, Triton X-100, glycerol, NaCl, leupeptin, and phenylmethylsulfonyl fluoride. Cell lysates were subjected to electrophoresis and immunoblotting. Phosphorylated AMPK (Thr172) was detected using a specific primary antibody and an enhanced chemiluminescence detection system. [1]
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| References | |
| Additional Infomation |
7-Methoxy isoflavone is a methoxy isoflavone, that is, an isoflavone in which the 7-position is substituted with a methoxy group.
7-Methoxy isoflavone is a synthetic isoflavone derivative characterized by the 7-position of the isoflavone core structure being substituted with a methoxy group. [1] This study tested 37 isoflavones in a cell-free system and found that 7-methoxy isoflavone is one of the most potent activators of AMPK. [1] Its mechanism of action is thought to be the direct binding to and activation of the AMPK heterotrimeric complex. Computer docking results showed that it binds to an interface involving α and β subunits that differs from the binding site of known potent inhibitors (astrosporins) but shares some residues with known activators (benzimidazole 992). [1] This study concludes that findings regarding the structure-activity relationship and binding mode of 7-methoxy isoflavone contribute to the design of new AMPK modulators. [1] |
| Molecular Formula |
C16H12O3
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| Molecular Weight |
252.26468
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| Exact Mass |
252.079
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| CAS # |
1621-56-3
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| PubChem CID |
638006
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| Appearance |
White to off-white solid powder
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| Density |
1.24g/cm3
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| Boiling Point |
421.2ºC at 760 mmHg
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| Flash Point |
200.3ºC
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| Vapour Pressure |
2.66E-07mmHg at 25°C
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| Index of Refraction |
1.614
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| LogP |
3.468
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
19
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| Complexity |
368
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
IECSQLKWZBEUGA-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C16H12O3/c1-18-12-7-8-13-15(9-12)19-10-14(16(13)17)11-5-3-2-4-6-11/h2-10H,1H3
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| Chemical Name |
7-methoxy-3-phenylchromen-4-one
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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 : ~25 mg/mL (~99.10 mM)
H2O : < 0.1 mg/mL |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (9.91 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 (9.91 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.9642 mL | 19.8208 mL | 39.6416 mL | |
| 5 mM | 0.7928 mL | 3.9642 mL | 7.9283 mL | |
| 10 mM | 0.3964 mL | 1.9821 mL | 3.9642 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.
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