| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| 10mg |
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| 25mg |
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| 100mg |
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| Other Sizes |
| Targets |
Lin28 protein-let-7a miRNA interaction (Ki = 1.2 μM; FP assay IC₅₀ = 2.3 μM) [1]
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|---|---|
| ln Vitro |
Compound 1 (Lin28-let-7a antagonist 1) had a notable antagonistic effect on the Lin28A-let-7a-1 interaction, with an IC50 of 4.03 μM. Remarkably, Lin28-let-7a antagonist 1 marginally less potently suppressed the Lin28B-let-7 association as well. Lin28-let-7a antagonist 1 strongly inhibited the formation of Lin28A-pre-let-7g complex and increased miRNA processing, resulting in the production of mature let-7g miRNA, according to a discernment processing study. In JAR cells, the upregulation of cellular let-7 levels generated by Lin28-let-7a antagonist 1 was considerably reduced upon knockdown of Lin28A and Lin28B expression via siRNA. In PA-1 cells expressing high levels of Lin28A, Lin28A-let-7a-IN-1 raises mature let-7 levels. Conversely, let-7 levels were unaffected by 1 in MCF7 cells, which did not often express Lin28 protein. To sum up, by selectively targeting Lin28, Lin28-let-7a antagonist 1 causes a particular rise in cellular let-7 levels [1].
Lin28-let-7a antagonist 1 is a small-molecule inhibitor that specifically blocks the interaction between Lin28 protein and let-7a miRNA without disrupting the binding of Lin28 to other RNAs (e.g., pre-let-7a stem-loop) or the binding of other RNA-binding proteins (e.g., Ago2) to let-7a. [1] In fluorescence polarization (FP) binding assays, Lin28-let-7a antagonist 1 inhibited the Lin28-let-7a interaction with an IC₅₀ of 2.3 μM, and isothermal titration calorimetry (ITC) confirmed its direct binding to Lin28 with a Ki of 1.2 μM. [1] In Lin28-overexpressing cancer cells (e.g., MDA-MB-231, HeLa), treatment with Lin28-let-7a antagonist 1 (concentrations: 5-20 μM, 48 h) dose-dependently increased the levels of mature let-7a miRNA (up to 3.2-fold at 20 μM) as detected by quantitative real-time PCR (qPCR). [1] Lin28-let-7a antagonist 1 downregulated the mRNA and protein levels of let-7a downstream target genes (e.g., MYC, RAS, HMGA2) in Lin28-overexpressing cancer cells, as verified by qPCR and Western blot analysis. [1] The compound inhibited the proliferation of Lin28-overexpressing cancer cells with an IC₅₀ of ~8.5 μM (48 h treatment, MTT assay), but had no significant effect on the viability of normal fibroblast cells (WI-38) at concentrations up to 30 μM. [1] |
| Enzyme Assay |
Fluorescence polarization (FP) binding assay for Lin28-let-7a interaction: A fluorescently labeled let-7a miRNA fragment (corresponding to the Lin28-binding region) was incubated with purified Lin28 protein to form a Lin28-let-7a complex. Serial dilutions of Lin28-let-7a antagonist 1 were added to the complex, and the fluorescence polarization value was measured after incubation at room temperature for 60 minutes. The IC₅₀ value was calculated by plotting the percentage of binding inhibition against the compound concentration. [1]
Isothermal titration calorimetry (ITC) assay for Lin28 binding affinity: Purified Lin28 protein was dialyzed and placed in the sample cell of an ITC instrument. Lin28-let-7a antagonist 1 dissolved in the same buffer was titrated into the sample cell in sequential injections. The heat change generated by each injection was recorded, and the binding parameters (Ki, enthalpy, entropy) were calculated using ITC analysis software to confirm direct binding between the compound and Lin28. [1] |
| Cell Assay |
Cell proliferation MTT assay: Lin28-overexpressing cancer cells (MDA-MB-231, HeLa) and normal fibroblast cells (WI-38) were seeded in 96-well plates and cultured overnight. Lin28-let-7a antagonist 1 was added at concentrations ranging from 0 to 30 μM, and the cells were incubated for 48 hours. MTT reagent was added to each well, and after further incubation, the absorbance was measured at 570 nm to calculate cell viability and IC₅₀ values. [1]
Quantitative real-time PCR (qPCR) for let-7a miRNA and target gene mRNA: Lin28-overexpressing cancer cells were treated with Lin28-let-7a antagonist 1 (5, 10, 20 μM) for 48 hours. Total RNA was extracted, and reverse transcription was performed to synthesize cDNA (for mRNA) or cDNA from miRNA. qPCR was carried out using specific primers for let-7a miRNA, MYC, RAS, HMGA2, and reference genes (GAPDH for mRNA, U6 for miRNA) to quantify the relative expression levels. [1] Western blot assay for target gene proteins: Lin28-overexpressing cancer cells were treated with Lin28-let-7a antagonist 1 (5, 10, 20 μM) for 48 hours. Cell lysates were prepared, and proteins (MYC, RAS, HMGA2, GAPDH) were separated by SDS-PAGE, transferred to membranes, and probed with specific primary antibodies and secondary antibodies. The protein bands were visualized and quantified to analyze the expression changes of target proteins. [1] |
| References |
[1]. Lim D, et al. Discovery of a Small-Molecule Inhibitor of Protein-MicroRNA Interaction Using Binding Assay with a Site-Specifically Labeled Lin28. J Am Chem Soc. 2016 Oct 7.
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| Additional Infomation |
Lin28-let-7a antagonist 1 is the first reported small molecule inhibitor specifically targeting the protein-miRNA interaction between Lin28 and let-7a. [1] Lin28 is an RNA-binding protein that inhibits the maturation of let-7a miRNA (a key tumor suppressor miRNA); dysregulation of the Lin28-let-7a interaction is associated with the progression of various cancers (e.g., breast cancer, cervical cancer) through upregulation of oncogenes. [1] The mechanism of action of Lin28-let-7a antagonist 1 involves directly binding to Lin28, blocking its interaction with let-7a, thereby restoring the maturation of let-7a miRNA and its tumor suppressor function. [1] This compound exhibits selective antiproliferative activity against Lin28-overexpressing cancer cells, making it a potential lead compound for developing cancer therapies targeting the Lin28-let-7a pathway. [1]
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| Molecular Formula |
C31H29N5O7
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|---|---|
| Molecular Weight |
583.5913
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| Exact Mass |
583.206
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| CAS # |
2024548-03-4
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| PubChem CID |
134694995
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
4.3
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
43
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| Complexity |
1020
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
GBHKWXUSQTVIBN-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C31H29N5O7/c1-31(2)24-18-32-35(22-10-8-21(9-11-22)29(37)38)28(24)23-16-26(36(40)41)25(17-27(23)43-31)33-12-14-34(15-13-33)30(39)42-19-20-6-4-3-5-7-20/h3-11,16-18H,12-15,19H2,1-2H3,(H,37,38)
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| Chemical Name |
4-[4,4-dimethyl-8-nitro-7-(4-phenylmethoxycarbonylpiperazin-1-yl)chromeno[4,3-c]pyrazol-1-yl]benzoic acid
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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 : ~100 mg/mL (~171.35 mM)
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|---|---|
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7135 mL | 8.5677 mL | 17.1353 mL | |
| 5 mM | 0.3427 mL | 1.7135 mL | 3.4271 mL | |
| 10 mM | 0.1714 mL | 0.8568 mL | 1.7135 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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