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LIN28 inhibitor LI71 is a potent ,novel and cell-permeable LIN28 inhibitor, which abolishes LIN28-mediated oligouridylation with an IC50 of 7 uM. LIN28 inhibitor LI71 directly binds the cold shock domain (CSD) to suppress LIN28’s activity against let-7 in leukemia cells and embryonic stem cells.
| Targets |
LIN28 protein (specifically the Cold Shock Domain, CSD) – inhibits the LIN28:let-7 interaction and LIN28-mediated oligouridylation of let-7 precursors. IC50 in fluorescence polarization (FP) assay using LIN28 F73A: ~7 µM. IC50 in oligouridylation assay: ~27 µM. [1]
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| ln Vitro |
LI71 inhibits the binding between recombinant human LIN28 protein and FAM-labeled preE-let-7 RNA in a fluorescence polarization assay, with an IC50 of approximately 7 µM when using the LIN28 F73A mutant. [1]
LI71 inhibits LIN28-mediated oligouridylation of pre-let-7g by TUT4 (terminal uridylyltransferase 4) in a cell-free biochemical assay, with an IC50 of approximately 27 µM. It does not inhibit TUT4 catalytic activity in the absence of LIN28. [1] Saturation Transfer Difference (STD) NMR spectroscopy demonstrates that LI71 directly binds to the Cold Shock Domain (CSD) of LIN28, but not to the preE-let-7 RNA. The benzoic acid head group, cyclopentaquinoline body, and ethoxy tail of LI71 are in close contact with LIN28. Mutation of residue K102 in the CSD alters the STD signal, suggesting its involvement in LI71 binding. [1] Heteronuclear Single Quantum Coherence (HSQC) NMR titrations show minor intensity reductions for residues involved in RNA binding in both the CSD and ZKD of LIN28 upon addition of LI71, indicating interference with the LIN28:let-7 interaction. [1] Differential Scanning Fluorimetry (DSF) shows that LI71 destabilizes the LIN28:preE-let-7f complex, decreasing its melting temperature (Tm) from 63°C to 60°C. [1] Structure-activity relationship (SAR) studies using LI71 derivatives reveal that the carboxyl head group is critical for activity; modifications to this group abolish inhibition. Alterations to the ethoxy tail reduce activity. The enantiomer of LI71 shows moderately increased IC50 and retains binding ability, indicating the orientation of the carboxyphenyl group is important. [1] |
| Enzyme Assay |
LIN28-mediated oligouridylation assay: Recombinant full-length mouse LIN28A and truncated mouse TUT4 (residues 230–1424) were purified. Pre-let-7g RNA was transcribed in vitro and radiolabeled with [γ-³²P]ATP using T4 polynucleotide kinase. Mouse LIN28, mouse TUT4, and radiolabeled pre-let-7g were mixed and incubated for 40 minutes at 37°C in a buffer containing 20 mM Tris (pH 7.5), 5% glycerol, 6 mM MgCl₂, 6 mM DTT, 50 µM ZnCl₂, 40 mM KCl, and 200 µM UTP. Reactions were stopped by adding EDTA, SDS, and proteinase K, followed by incubation at 50°C for 30 minutes. Reaction products (pre-let-7g and oligouridylated pre-let-7g) were separated by denaturing PAGE and visualized. For inhibitor testing, assays were performed in the presence of 40 µM test compound or varying concentrations for dose-response. [1]
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| Cell Assay |
Dual luciferase reporter assay: HeLa cell lines stably expressing LIN28A or LIN28B (or GFP as control) were generated via retroviral transduction and selection. These cells were further lentivirally transduced with a pLenti construct containing a Renilla luciferase cassette with eight tandem let-7 recognition sites in its 3' UTR and a constitutively active Firefly luciferase cassette as an internal control. Stable polyclonal populations were selected with G418. Cells were treated with LI71 (up to 100 µM) or vehicle (DMSO) for 24 or 48 hours, then lysed. Dual luciferase activity was measured using a commercial assay kit. Renilla luciferase activity (normalized to Firefly) was used to assess let-7 activity, which is inversely correlated with LIN28 function. [1]
qPCR for microRNA expression: K562 leukemia cells (endogenously expressing LIN28B) or mouse embryonic stem cells (mESCs; Lin28a/Lin28b double-knockout cells expressing exogenous wild-type LIN28A) were treated with 100 µM LI71 or DMSO for 48 hours. Total RNA was isolated using TRIzol. cDNA was synthesized using a microRNA-specific reverse transcription system. Mature and precursor let-7 microRNA species were quantified by quantitative PCR (qPCR) using microRNA-specific primer assays, with U6 small nuclear RNA as an endogenous control. [1] Cell viability/apoptosis assay: HeLa cells or K562 cells treated with LI71 (up to 100 µM) for 48 hours were harvested and stained with Annexin V-APC, followed by analysis using flow cytometry to assess apoptosis. [1] |
| Toxicity/Toxicokinetics |
Cytotoxicity: Annexin V staining and flow cytometry assessment showed that treatment with LI71 at concentrations up to 100 µM for 48 hours did not significantly increase apoptosis in HeLa or K562 cells. [1]
This study indicated that LI71 "has good solubility in solution and low cytotoxicity even at high micromolar concentrations." [1] |
| References |
Wang L, et al. Small-Molecule Inhibitors Disrupt let-7 Oligouridylation and Release the Selective Blockade of let-7 Processing by LIN28. Cell Rep. 2018 Jun 5;23(10):3091-3101.
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| Additional Infomation |
LI71 is a small molecule inhibitor discovered through high-throughput screening of 101,017 compounds using fluorescence polarization. [1] It shares a benzoic acid moiety with another lead compound, LI20. [1] LI71's mechanism of action involves direct binding to the cold shock domain (CSD) of LIN28, competitively binding to RNA, thereby disrupting LIN28-mediated let-7 microRNA maturation arrest. [1] It can restore the level of mature let-7 in LIN28-expressing cancer cells (K562) and embryonic stem cells. [1] The authors believe that although LI71 has relatively low potency (micromolar levels), it holds promise as a candidate for future drug chemistry optimization due to its unique mechanism of action, good solubility, and low cytotoxicity. [1]
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| Molecular Formula |
C21H21NO3
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|---|---|
| Molecular Weight |
335.3963
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| Exact Mass |
335.15
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| Elemental Analysis |
C, 75.20; H, 6.31; N, 4.18; O, 14.31
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| CAS # |
1357248-83-9
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| Related CAS # |
LIN28 inhibitor LI71 enantiomer;956189-58-5
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| PubChem CID |
6541811
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| Appearance |
Solid powder
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| LogP |
4.2
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
25
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| Complexity |
511
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| Defined Atom Stereocenter Count |
3
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| SMILES |
CCOC1=CC=CC2=C1N[C@H]([C@@H]3[C@H]2C=CC3)C4=CC=C(C=C4)C(=O)O
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| InChi Key |
QWJMABCFVYELBB-GJYPPUQNSA-N
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| InChi Code |
InChI=1S/C21H21NO3/c1-2-25-18-8-4-7-17-15-5-3-6-16(15)19(22-20(17)18)13-9-11-14(12-10-13)21(23)24/h3-5,7-12,15-16,19,22H,2,6H2,1H3,(H,23,24)/t15-,16+,19+/m1/s1
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| Chemical Name |
4-[(3aS,4R,9bR)-6-ethoxy-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-4-yl]benzoic acid
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| Synonyms |
LIN28 inhibitor LI71; L-I71; L I71;
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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 (~298.15 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 | 2.9815 mL | 14.9076 mL | 29.8151 mL | |
| 5 mM | 0.5963 mL | 2.9815 mL | 5.9630 mL | |
| 10 mM | 0.2982 mL | 1.4908 mL | 2.9815 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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