| Size | Price | Stock | Qty |
|---|---|---|---|
| 5mg |
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| 10mg |
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| 100mg | |||
| Other Sizes |
| Targets |
EphA receptors
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| ln Vitro |
Among the 21 phenolics screened by an ELISA-binding assay, just urolithin C, urolithin D, and ellagic acid succeeded to inhibit the EphA2-ephrin-A1 binding. Urolithin D, the most active, was a competitive and reversible antagonist of EphA receptors able to discriminate between EphA and EphB receptors, showing intra-classes selectivity. Molecular modeling and structure-activity relationships shed light on the binding mode and selective activity of urolithin D. This catabolite blocked EphA2 phosphorylation mediated by ephrin-A1, while lacking cytotoxicity and anti-proliferative effects, and was inactive on the EphA2 kinase assay.
Conclusion: The mechanisms behind the cancer preventive properties of foods rich in flavan-3-ols and caffeoylquinic acids are not associated with metabolic pathways directly linked to the Eph-ephrin system. However, the ellagitannin-derived colonic metabolite urolithin D was able to exert remarkable and selective EphA-ephrin-A inhibition, which might impact on prostate cancer prevention [1].
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| Enzyme Assay |
EphA2 kinase activity [1]
The kinase activity of the EphA2 kinase domain was evaluated with the “EphA2 kinase assay/inhibitor assay kit” following manufacturer instructions. EphA2 kinase activity is not inhibited by Urolithin D/UroD [1] The observed inhibition of the EphA2 phosphorylation could also be linked to a direct inhibition of the kinase domain. To rule out this hypothesis, 30 μM of UroD were incubated with EphA2 kinase domain, a substrate peptide, and appropriate reagents. Under these conditions, 100 nM of dasatinib, used as reference compound, completely blocked EphA2 kinase activity whereas UroD was inactive, thus confirming its action as protein-protein inhibitor (Fig. 8). EGFR kinase activity [1] In order to partially assess the specific interaction of Urolithin D/UroD with Eph-receptors, we performed a functional assay on another tyrosine-kinase receptor (EGFR). UroD 30 μM was completely inactive when tested against the phosphorylation of EGF receptors induced by EGF in PC3 cells whereas the EGFR kinase inhibitor gefitinib (10 μM), used as reference compound, completely abolished response to EGF (Fig. 9). |
| Cell Assay |
Phosphorylation of EphA2 and EGFR in cells [1]
EphA2-, and EGFR-phosphorylation was measured in cell lysates using DuoSet® IC Sandwich ELISA following manufacturer's protocol. Briefly, 96-well ELISA high binding plates were incubated overnight with 100 μL/well of the specific capture antibody diluted in sterile PBS at the proper working concentrations. Next day, the wells were washed and blocked for 1 h and 100 μL/well of lysates (10 μg/well) were added for 2 h. Then, wells were incubated with the specific detection antibody and the phosphorylation level was revealed utilizing a standard HRP format and tetra-methylbenzidine through a colorimetric reaction read at 450 nm. Each step was performed at room temperature and followed by the washing of each well. LDH and MTT assays [1] Cytotoxicity was evaluated with CytoTox 96® Non-Radioactive Cytotoxicity Assay, following the manufacturer's protocol. Cells were seeded in 96-well plates (100 μL/well) at a density of 105 cells/mL and the day after treated with compounds or lysis buffer for 2 or 15 h. After incubation, released LDH in culture supernatants was measured using a 30-min coupled enzymatic assay, which results in the conversion of a tetrazolium salt (INT) into a red formazan product. The amount of colour formed is proportional to the number of lysed cells and quantified by an ELISA plate reader at 492 nm. The results were expressed as the ratio between absorbance of the cells treated with the compounds and the cells treated with lysis buffer. Cell viability was evaluated using the MTT colorimetric assay. Cells were seeded in 96-well plates at a density of 105cells/mL and the day after treated with compounds or 1% DMSO for 15 or 72 h. MTT was added at the final concentration of 1 mg/mL and incubated for 2 h. The resulting formazan crystals were solubilized with DMSO 100 μL/well. The absorbance was measured at 550 nm using an ELISA plate reader and the results were expressed as the ratio between absorbance of the cell treated with the compounds and untreated cells. |
| References | |
| Additional Infomation |
Urolithin D is a hydroxycoumarin. It has been reported that pomegranate (Punica granatum) contains urolithin D, and relevant data are available. In summary, the results of this study indicate that the mechanism behind the reported anticancer properties of foods rich in flavan-3-ol and caffeoylquinic acid is not related to metabolic pathways directly linked to the Eph-ephrin system. Instead, urolithin D (UroD), a colonic metabolite derived from ellagitannins, selectively inhibits the EphA-ephrin-A complex, which may have implications for prostate cancer prevention. This selective characteristic is particularly noteworthy because intestinal cells express the Eph-ephrin system, which regulates epithelial cell renewal by modulating cell migration patterns along the crypt-villi axis by regulating the activity of stem cells at the base of the Lieberkühn crypts.52 It is noteworthy that this process is mediated by the interaction of the EphB receptor with ephrin-B. The lack of EphB receptor activity in UroD may help avoid interfering with intestinal lamina renewal. However, since the large intestine is the site with the highest concentration of UroD in human tissues, further research is needed on the potential multiple roles of this ellagitannin metabolite in cancer prevention to study the ability of UroD to inhibit the migration of colon cancer cells. [1]
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| Molecular Formula |
C13H8O6
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|---|---|
| Molecular Weight |
260.20
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| Exact Mass |
260.032
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| CAS # |
131086-98-1
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| Related CAS # |
Urolithin D; 131086-98-1
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| PubChem CID |
5482042
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| Appearance |
White to off-white solid powder
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| Density |
1.8±0.1 g/cm3
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| Boiling Point |
634.1±55.0 °C at 760 mmHg
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| Flash Point |
253.0±25.0 °C
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| Vapour Pressure |
0.0±1.9 mmHg at 25°C
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| Index of Refraction |
1.801
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| Source |
Originated from microorganisms: Human gut Enterocloster species
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| LogP |
1.07
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
0
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| Heavy Atom Count |
19
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| Complexity |
372
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1=CC(=C(C2=C1C3=CC(=C(C=C3C(=O)O2)O)O)O)O
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| InChi Key |
NEZDQSKPNPRYAW-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C13H8O6/c14-8-2-1-5-6-3-9(15)10(16)4-7(6)13(18)19-12(5)11(8)17/h1-4,14-17H
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| Chemical Name |
3,4,8,9-tetrahydroxybenzo[c]chromen-6-one
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| Synonyms |
Urolithin D; 131086-98-1; 3,4,8,9-Tetrahydroxybenzo[c]chromen-6-one; CHEMBL6338; 3,4,8,9-Tetrahydroxy-6H-benzo[c]chromen-6-one; 6H-Dibenzo(b,d)pyran-6-one, 3,4,8,9-tetrahydroxy-; 6H-Dibenzo[b,d]pyran-6-one, 3,4,8,9-tetrahydroxy-; Urolithin-D;
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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 Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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: ~125 mg/mL (~480.4 mM; with ultrasonication)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (7.99 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween-80 + 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 DMSO stock solution (20.8 mg/mL) to 400 μL of PEG300 and mix well; then add 50 μL of Tween-80 and mix well; finally add 450 μL of physiological saline and 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: ≥ 2.08 mg/mL (7.99 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 DMSO stock solution (20.8 mg/mL) to 900 μL of 20% SBE-β-CD saline and mix well. 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. Solubility in Formulation 3: ≥ 2.08 mg/mL (7.99 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 DMSO stock solution (20.8 mg/mL) to 900 μL of corn oil and mix well. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.8432 mL | 19.2160 mL | 38.4320 mL | |
| 5 mM | 0.7686 mL | 3.8432 mL | 7.6864 mL | |
| 10 mM | 0.3843 mL | 1.9216 mL | 3.8432 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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