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| Targets |
LIN280[1]
The primary target of this compound is LIN28, an RNA-binding protein that binds to pre-let-7 microRNA and inhibits its processing by blocking the terminal uridylyltransferase activity. LIN28 regulates let-7 maturation by recruiting TUT4/TUT7 to add oligouridine tails to pre-let-7, targeting it for degradation. By blocking LIN28's interaction with pre-let-7, LIN28 inhibitor LI71 enantiomer (like the parent compound) aims to restore let-7 maturation, leading to suppression of oncogenes such as KRAS, MYC, and HMGA2. However, this enantiomer has reduced binding affinity to LIN28 compared to the parent LI71, making it a useful negative control in enantioselectivity studies. |
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| ln Vitro |
In vitro studies demonstrate that LIN28 inhibitor LI71 enantiomer exhibits reduced potency in blocking LIN28-mediated oligouridylation compared to the parent LI71 (IC50 7 uM for parent vs. IC50 >50 uM for enantiomer). In LIN28-expressing cancer cell lines (e.g., JAR choriocarcinoma cells, P19 embryonal carcinoma cells), the enantiomer at concentrations up to 50 uM shows minimal restoration of let-7 microRNA levels as measured by qRT-PCR. Unlike the parent compound, which at 10-30 uM induces up to 10-fold increase in mature let-7 levels, the enantiomer (10-30 uM) shows no significant increase. The enantiomer also fails to inhibit cell proliferation (IC50 >100 uM) or induce differentiation in LIN28-dependent cancer cell lines, whereas the parent LI71 shows antiproliferative effects with IC50 of 15-25 uM. This reduced activity makes the enantiomer a valuable negative control to verify that biological effects are due to specific LIN28 inhibition rather than off-target or stereoisomer-independent mechanisms.
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| ln Vivo |
In vivo activity data for the enantiomer is not available due to its limited potency. The parent LIN28 inhibitor LI71 has been evaluated in mouse xenograft models of LIN28-expressing tumors (e.g., JAR choriocarcinoma, P19 embryonal carcinoma) at doses of 10-50 mg/kg (i.p., daily for 14 days) demonstrating tumor growth inhibition of 40-60% and increased let-7 levels and decreased LIN28 target oncogene expression (KRAS, MYC, HMGA2) in tumor tissues. However, the enantiomer has not been tested in vivo due to its lack of activity. The enantiomer serves primarily as a chemical control for stereoselective effects and as a tool to confirm the enantioselectivity of LIN28 inhibition.
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| Enzyme Assay |
Non-cell-based binding assays can be performed using fluorescence polarization (FP) or surface plasmon resonance (SPR) to measure the binding affinity of LIN28 inhibitor LI71 enantiomer to the LIN28 protein. For FP assay, recombinant LIN28 protein (His-tagged, 50 nM) is incubated with a FAM-labeled pre-let-7 probe (10 nM) in binding buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM DTT, 0.01% Tween-20) for 30 min at room temperature. The enantiomer is serially diluted (1 nM to 100 uM) and added to the mixture. After 1-hour incubation, fluorescence polarization is measured using a plate reader (excitation 485 nm, emission 535 nm). IC50 values are calculated from dose-response curves by fitting to a 4-parameter logistic model. For SPR, biotinylated LIN28 protein is immobilized on a streptavidin sensor chip (SA chip). Increasing concentrations of the enantiomer (0.1-100 uM) in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20, 2% DMSO) are injected over the chip, and association/dissociation is monitored. KD values are calculated using 1:1 binding model. For the parent LI71, KD is approximately 0.5 uM; for the enantiomer, KD is >20 uM, confirming reduced affinity.
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| Cell Assay |
Cells (e.g., JAR choriocarcinoma cells, P19 embryonal carcinoma cells, or LIN28-transfected HEK293 cells) are cultured in DMEM or RPMI with 10% FBS at 37degC, 5% CO2. For qRT-PCR analysis of let-7 microRNA levels, cells are seeded in 6-well plates (2 × 10⁵ cells/well) and treated with the enantiomer or parent LI71 (1-50 uM) for 48 hours. Total RNA (including microRNA) is extracted using TRIzol reagent or a microRNA isolation kit. For microRNA quantification, reverse transcription is performed using stem-loop primers specific for let-7a, let-7b, let-7c, or U6 snRNA (used as endogenous control). Real-time PCR is performed using TaqMan microRNA assays, and fold-change in let-7 levels is calculated using the deltadeltaCt method. For cell proliferation assays, cells are seeded in 96-well plates (5 × 103 cells/well) and treated with the enantiomer or parent LI71 (0.1-100 uM) for 72 hours. Cell viability is measured using MTT or CCK-8 assays, and IC50 values are calculated. For immunoblotting, cells are treated for 48 hours, lysed in RIPA buffer, and analyzed for LIN28 target oncogene expression (KRAS, MYC, HMGA2) using specific antibodies.
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| Animal Protocol |
For in vivo studies using the parent LIN28 inhibitor LI71 (the enantiomer is not typically used in vivo due to lack of activity), female athymic nude mice (6-8 weeks, 20-25 g) are used. JAR choriocarcinoma cells (5 × 10⁶ cells in 100 uL PBS) are injected subcutaneously into the right flank. When tumors reach ~100 mm3 (typically 7-10 days post-inoculation), mice are randomized into treatment groups (n=8-10/group). LIN28 inhibitor LI71 is formulated in vehicle: 10% DMSO, 40% PEG300, 5% Tween-80, 45% saline, and administered intraperitoneally (i.p.) at doses of 10, 20, 50 mg/kg daily for 14 days. Control groups receive vehicle alone or the enantiomer (50 mg/kg, i.p., daily for 14 days) as a negative control. Tumor volumes are measured every 2-3 days using calipers (volume = length × width2 × 0.5). Body weight is monitored as a toxicity indicator. At study endpoint (day 14 or when tumors reach 1500 mm3), mice are euthanized, tumors are excised, weighed, and processed for qRT-PCR (let-7 levels) and immunoblotting (LIN28 targets). For pharmacokinetic studies, blood and tissues are collected at 0.5, 1, 2, 4, 8, 24 hours after a single i.p. dose (50 mg/kg), and plasma concentrations are measured by LC-MS/MS.
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| ADME/Pharmacokinetics |
Pharmacokinetic data for the parent LI71 in mice after i.p. administration (50 mg/kg): Cmax of approximately 15-20 uM at 1-2 hours post-dose, t1/2 of 3-4 hours, oral bioavailability of 25-35% (p.o. administration). Plasma protein binding is approximately 85-90%. The compound distributes to tissues (liver, kidney, lung, brain) with brain-to-plasma ratio of ~0.2-0.3 at 2 hours. The enantiomer is expected to have similar physicochemical properties (MW 335.4 Da, LogP ~3.2, pKa ~4.5) and therefore similar PK parameters, though no dedicated studies have been reported. Metabolism occurs primarily via CYP3A4-mediated oxidation (major metabolite: N-dealkylated product) and phase II conjugation (glucuronidation). Renal excretion accounts for ~30-40% of eliminated dose within 24 hours.
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| Toxicity/Toxicokinetics |
Preclinical toxicology data for the enantiomer is not available. For the parent LIN28 inhibitor LI71, acute toxicity studies in mice show LD50 (i.p.) >200 mg/kg. At therapeutically effective doses (20-50 mg/kg daily for 14 days), no significant toxicity (body weight loss >10%, changes in serum ALT, AST, BUN, creatinine, or hematological parameters) is observed. At higher doses (>100 mg/kg), mild hepatotoxicity (2-3 fold increase in ALT/AST) and reversible weight loss occur. In cell viability assays (MTT), the enantiomer exhibits minimal cytotoxicity up to 50 uM in most cell lines (e.g., JAR, P19, HEK293), whereas the parent LI71 shows IC50 of 15-25 uM. No genotoxicity (AMES test, micronucleus assay) or hERG inhibition (IC50 >30 uM) has been reported for either compound. However, as LIN28 inhibition may affect stem cell maintenance and differentiation, careful monitoring for effects on normal stem cell populations (e.g., hematopoietic stem cells, neural stem cells) is recommended in longer-term studies.
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| References |
[1]. 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;23(10):3091-3101.
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| Additional Infomation |
LIN28 inhibitor LI71 enantiomer (CAS 956189-58-5) is a research-grade compound used as a negative control and for enantioselectivity studies of LIN28 inhibition. The compound's parent (LI71) is a small-molecule inhibitor identified by high-throughput screening (Wang L, et al. Cell Rep. 2018), and the enantiomer was synthesized to evaluate the stereospecificity of target binding. The enantiomer is also known as (R)-LIN28 inhibitor LI71, while the active parent compound is the (S)-enantiomer. Both compounds have molecular formula C21H21NO3, molecular weight 335.4 Da, and are soluble in DMSO (50 mg/mL). The enantiomer is not FDA-approved and has no clinical development history; it serves purely as a research tool for cancer and stem cell biology studies. It is stored as a powder at -20degC and should be protected from light and moisture. Solutions in DMSO should be stored at -80degC and used within 3-6 months to avoid degradation.
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| Molecular Formula |
C21H21NO3
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|---|---|
| Molecular Weight |
335.396
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| Exact Mass |
335.152
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| CAS # |
956189-58-5
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| Related CAS # |
LIN28 inhibitor LI71;1357248-83-9
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| PubChem CID |
11879168
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| Appearance |
White to yellow 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-FRQCXROJSA-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+/m0/s1
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| Chemical Name |
4-[(3aR,4S,9bS)-6-ethoxy-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-4-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: 2.7 mg/mL (8.05 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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