| Size | Price | Stock | Qty |
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| Targets |
3F8 targets glycogen synthase kinase 3 beta (GSK3β). It is an ATP-competitive inhibitor of GSK3β. In the presence of 10 μM ATP, the IC₅₀ of 3F8 is 34 nM; in the presence of 100 μM ATP, the IC₅₀ increases to 304 nM. In a panel of 22 representative kinases, 5 μM 3F8 inhibited GSK3β activity by 91%, demonstrating selectivity. [1]
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| ln Vitro |
In a β-catenin-TCF reporter assay using 293T cells, treatment with 4 μM 3F8 increased luciferase activity approximately 15-fold compared to control, indicating that 3F8 upregulates the Wnt pathway. [1]
Computational docking analysis showed that 3F8 fits into the ATP pocket of human GSK3β with a predicted binding free energy of -9.9 kcal/mol, suggesting it is an ATP-competitive inhibitor. [1] In an in vitro GSK3β activity assay, 3F8 inhibited human GSK3β in an ATP-competitive manner. The IC₅₀ was 34 nM at 10 μM ATP and 304 nM at 100 μM ATP. Under the same conditions, 3F8 had a lower IC₅₀ than SB216763, a commonly used GSK3 inhibitor. [1] In a selectivity screen against 22 representative mammalian kinases, 5 μM 3F8 inhibited GSK3β activity by 91%, while showing much lower inhibition against other kinases, confirming its selectivity for GSK3. [1] |
| ln Vivo |
In zebrafish embryos, 3F8 treatment induces a dose-dependent “no-eyes” phenotype (loss of eyes and forebrain), resembling typical Wnt overexpression. The effective concentration (Cₑ) to cause the no-eyes phenotype is 7.5 μM. The ratio Cₑ/IC₅₀ for 3F8 is 221, which is lower than that of other tested GSK3 inhibitors (SB216763, GSK-3b inhibitor IX, GSK-3b inhibitor XII, and GSK-3 inhibitor XV, TWS119), implying that 3F8 is more efficient in vivo, likely due to better absorption and/or stability. [1]
Using 3F8, the timing of forebrain determination was studied: embryos treated with 3.75 μM 3F8 at the 2-cell stage all showed the no-eyes phenotype; the required concentration gradually increased to 11.25 μM until the shield stage; after gastrulation (2-somite stage), 3F8 no longer affected brain development even at 75 μM. [1] When 3F8 was removed at 2 days post-fertilization by washing with fish water, 22% of “no-eyes” embryos developed a forebrain and eyes by 5 dpf, indicating that committed forebrain progenitor cells remain and can regenerate. [1] Whole mount in situ hybridization showed that expression of forebrain markers pax2a and dlx2a was sensitive to 3F8 treatment; at doses that ablate both genes, no forebrain regeneration was observed. [1] |
| Enzyme Assay |
GSK3β activity assay (ELISA-based): Recombinant human GSK3β was added to reactions with or without inhibitors (3F8, its analogs, or SB216763). Reaction buffer contained 40 mM HEPES (pH 7.2), 5 mM MgCl₂, 5 mM EDTA, 50 μg/mL heparin, and ATP at either 10, 30, or 100 μM. The substrate was recombinant human TAU-441. Control reactions were set up without ATP, without TAU-441, or without GSK3β. Reaction mixtures were incubated at 30°C for 1 hour. The amount of phosphorylated TAU-441 was measured using a human Tau [pS396] ELISA kit. Absorbance at 450 nm was read. Activity curves were fitted using a four-parameter algorithm. [1]
Kinase selectivity profiling: A panel of 22 representative mammalian kinases was tested for inhibition by 3F8 at a concentration of 5 μM in 1% DMSO using a commercial kinase profiling service. [1] |
| Cell Assay |
β-catenin-TCF reporter assay (superTOPflash): 293T cells grown in 24-well plates were transfected with 200 ng of superTOPflash reporter plasmid and 20 ng of RLTK (Renilla luciferase control) plasmid. Twenty-four hours after transfection, 3F8 was added to cells at indicated concentrations for 18 hours. Cell extracts were then prepared and sequentially assayed for firefly and Renilla luciferase activities using a dual-luciferase reporter assay system. Firefly luciferase readings were normalized against Renilla luciferase. [1]
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| Animal Protocol |
Zebrafish embryo chemical screening: Wild-type AB strain zebrafish embryos were arrayed into 96-well plates (6 embryos per well) in 200 μL fresh fish water containing 1× antibiotic-antimycotic solution. 3F8 was added to embryos at approximately 10 μM using automated robots before the shield stage. Embryos were allowed to grow in the compound solution for up to 7 days. The “no-eyes” phenotype was examined visually under a dissection microscope. Stock solutions of 3F8 were made in DMSO, and working solutions were diluted from DMSO stocks into fish water. [1]
Forebrain determination timing study: Zebrafish embryos were treated with various concentrations of 3F8 (e.g., 3.75 μM, 11.25 μM, up to 75 μM) at different developmental stages (2-cell, shield stage, 2-somite stage). Treated embryos were observed for brain and eye formation. [1] Regeneration experiment: After 2 days of 3F8 treatment at a concentration that induced the no-eyes phenotype, embryos were washed with fish water to remove the compound and then allowed to develop until 5 days post-fertilization. The percentage of embryos that regenerated forebrain and eyes was quantified. [1] |
| Toxicity/Toxicokinetics |
In zebrafish embryos, 3F8 treatment at concentrations that induce the no-eyes phenotype caused mild heart edema and hypertrophy but otherwise normal body shape and appearance compared to DMSO-treated controls. Treatment of transgenic zebrafish with GFP-labeled internal organs (blood vessels, liver, pancreas) showed no visible necrosis or abnormality, suggesting that 3F8 does not have obvious non-specific developmental toxicity in zebrafish. [1]
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| References | |
| Additional Infomation |
3F8 (5-ethyl-7,8-dimethoxy-1H-pyrrolo[3,4-c]-isoquinoline-1,3-(2H)-dione) is a novel small-molecule GSK3 inhibitor identified from a screen of a 4000-compound chemical library (DIVERSet™) using the zebrafish “no-eyes” phenotype as an assay. This phenotype reflects ectopic activation of the Wnt signaling pathway. 3F8 is more potent than SB216763, a commonly used GSK3 inhibitor, under the same in vitro assay conditions. A derivative, 3F8.1 (7,8-dimethoxy-5-propyl-1H-pyrrolo[3,4-c]isoquinoline-1,3-(2H)-dione), showed similar activity. Based on the structure of 3F8, new analogs (6a and 6b) were synthesized; they inhibited GSK3β with IC₅₀ values of 270 nM and 92 nM, respectively. 3F8 has a Cₑ/IC₅₀ ratio of 221, indicating good in vivo efficiency. The compound can be used as a tool for studying GSK3 activity and Wnt signaling, and as a potential lead for drug discovery targeting GSK3-related diseases including type II diabetes, Alzheimer‘s disease, and cancers. [1]
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| Molecular Formula |
C15H14N2O4
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|---|---|
| Molecular Weight |
286.29
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| Exact Mass |
286.095
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| CAS # |
159109-11-2
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| PubChem CID |
23829003
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| Appearance |
Typically exists as solid at room temperature
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| LogP |
1.708
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
21
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| Complexity |
441
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C(C1C2=C(C=C(OC)C(OC)=C2)C2C(=O)NC(=O)C=2N=1)C
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| InChi Key |
ULVWJFBHQIXEPE-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C15H14N2O4/c1-4-9-7-5-10(20-2)11(21-3)6-8(7)12-13(16-9)15(19)17-14(12)18/h5-6H,4H2,1-3H3,(H,17,18,19)
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| Chemical Name |
5-Ethyl-7,8-dimethoxy-pyrrolo[3,4-c]isoquinoline-1,3-dione
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| Synonyms |
3F 8 3F83F-8
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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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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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 | 3.4930 mL | 17.4648 mL | 34.9296 mL | |
| 5 mM | 0.6986 mL | 3.4930 mL | 6.9859 mL | |
| 10 mM | 0.3493 mL | 1.7465 mL | 3.4930 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.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT01183416 | COMPLETEDWITH RESULTS | Drug: 3F8 monoclonal antibody and 13-cis-Retinoic Acid | Neuroblastoma | Memorial Sloan Kettering Cancer Center | 2010-08 | Phase 2 |
| NCT01183429 | COMPLETEDWITH RESULTS | Drug: 3F8 and 13-cis-retinoic acid | Neuroblastoma | Memorial Sloan Kettering Cancer Center | 2010-08-12 | Phase 2 |
| NCT01183884 | TERMINATEDWITH RESULTS | Biological: 3F8/GM-CSF Immunotherapy Plus 13-Cis-Retinoic Acid |
Neuroblastoma | Memorial Sloan Kettering Cancer Center | 2010-08 | Phase 2 |
| NCT01183897 | COMPLETEDWITH RESULTS | Biological: 3F8/GM-CSF Immunotherapy Plus 13-Cis-Retinoic |
Neuroblastoma | Memorial Sloan Kettering Cancer Center | 2010-08-12 | Phase 2 |
| NCT00450307 | COMPLETED | Biological: monoclonal antibody 3F8 Biological: sargramostim |
Neuroblastoma | Memorial Sloan Kettering Cancer Center | 2005-06 | Phase 1 |
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