| Size | Price | Stock | Qty |
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| 5mg |
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| Targets |
IC50: 60 nM (KDM6B)[2]
JMJD3 (KDM6B) and UTX (KDM6A). GSK-J1 lithium salt is a potent, selective, small-molecule inhibitor of the H3K27me3/me2 demethylases JMJD3 (KDM6B, Jumonji domain-containing protein 3) and UTX (KDM6A, ubiquitously transcribed tetratricopeptide repeat on chromosome X). These enzymes are Jumonji C (JmjC) domain-containing histone demethylases that remove the methyl groups from trimethylated and dimethylated lysine 27 on histone H3 (H3K27me3/me2), which is a repressive epigenetic mark associated with silenced genes. By binding to the active site of these demethylases and chelating the iron (Fe2+) cofactor, GSK-J1 inhibits their demethylase activity. This leads to an increase in H3K27me3 levels and thus enhanced repression of Polycomb target genes and other genes regulated by these demethylases. GSK-J1 is selective for the KDM6 family (KDM6A/B) over other JmjC demethylases (e.g., KDM4, KDM5). It is a valuable chemical probe for studying the role of H3K27 methylation in epigenetic regulation. |
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| ln Vitro |
GSK-J1 binds specifically to endogenous JMJD3 and is selective for H3K27 demethylases of the KDM6 subfamily. In an H3K27-dependent way, GSK-J1 suppresses human primary macrophages' ability to produce TNF-α[1]. At 1 mM α-ketoglutarate, GSK-J1 inhibits KDM5C's demethylase activity with 8.5 times more potency than KDM5B's, with IC50 values of 11 μM and 94 μM, respectively[3].
In vitro, GSK-J1 lithium salt is a potent inhibitor of KDM6B (JMJD3) with an IC50 of 60 nM, as measured by a biochemical demethylase assay using a peptide substrate and mass spectrometry detection. It also inhibits KDM6A (UTX) with similar potency (IC50 in the low nM range). In cell-based assays, treatment of human or mouse cells with GSK-J1 (0.1-10 uM) for 24-72 hours results in a dose-dependent increase in global H3K27me3 levels, as measured by Western blot (anti-H3K27me3 antibody) and by immunofluorescence. This effect is selective for H3K27me3; other histone marks (H3K4me3, H3K9me3, H3K36me3) are not significantly affected. GSK-J1 also reduces the expression of JMJD3 target genes, such as inflammatory cytokines (IL-6, IL-8, TNF-alpha) in macrophages stimulated with LPS, as measured by qPCR and ELISA. In cancer cell lines, GSK-J1 (1-10 uM) inhibits cell proliferation, induces cell cycle arrest, and promotes apoptosis. In primary mouse embryonic fibroblasts (MEFs), GSK-J1 promotes reprogramming to induced pluripotent stem cells (iPSCs) by enhancing the expression of pluripotency genes. GSK-J1 is not cytotoxic at concentrations up to 10 uM in most cell lines, as assessed by MTT assays. The lithium salt is more soluble than the free acid, facilitating cell culture studies. |
| ln Vivo |
In vivo, GSK-J1 lithium salt has been used in mouse models of inflammation, cancer, and other diseases. In a mouse model of lipopolysaccharide (LPS)-induced septic shock, intraperitoneal (i.p.) administration of GSK-J1 (10-50 mg/kg) reduces serum levels of pro-inflammatory cytokines (TNF-alpha, IL-6, IL-1beta) and improves survival, indicating anti-inflammatory effects mediated by increased H3K27me3 and suppression of inflammatory genes. In a mouse model of colitis (DSS-induced), GSK-J1 (10-30 mg/kg, i.p.) reduces disease activity index, colonic inflammation, and neutrophil infiltration. In cancer models, GSK-J1 (30-50 mg/kg, i.p., daily or every other day) reduces tumor growth in xenograft models of glioblastoma, breast cancer, and ovarian cancer. The compound is generally well-tolerated at doses up to 50 mg/kg, with no significant weight loss or organ toxicity reported. The lithium salt is used for formulation in saline or PBS. Because lithium itself has biological activity (e.g., used in bipolar disorder), control experiments should include lithium chloride alone to rule out effects of lithium ions. Typically, the concentration of lithium from GSK-J1 lithium salt is low (e.g., 0.1-1 mM in cell culture, 0.01-0.1 mmol/kg in vivo), which is lower than therapeutic lithium levels, but still should be controlled. GSK-J1 is a research tool; it is not an approved drug.
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| Enzyme Assay |
For non-cellular binding assays, a biochemical demethylase assay using a synthetic H3K27me3 peptide and recombinant KDM6B protein is standard. Incubate KDM6B (0.1-1 uM) with H3K27me3 peptide (1-10 uM) in reaction buffer (50 mM HEPES pH 7.5, 50 uM (NH4)2Fe(SO4)2, 100 uM alpha-ketoglutarate, 2 mM ascorbic acid, 1 mM DTT) for 30-60 min at 37degC. GSK-J1 lithium salt is dissolved in DMSO or water and added at varying concentrations (0.01-10000 nM) during the reaction. Terminate the reaction by adding 0.5% TFA. Desalt the sample and analyze by MALDI-TOF mass spectrometry to detect the demethylation product (H3K27me2 or H3K27me1). Quantify the ratio of trimethylated to dimethylated/monomethylated peaks. IC50 is determined by fitting dose-response curves. For a fluorescence-based assay, use a formaldehyde dehydrogenase-coupled assay or a formaldehyde detection kit (since demethylation produces formaldehyde). This is less common. For a tritium release assay, use 3H-labeled methyl groups on H3K27me3 peptide, incubate with enzyme, and measure 3H released into the supernatant. IC50 is calculated. For selectivity profiling, test the compound against other KDM family members (KDM4A, KDM5A, etc.) using similar assays. GSK-J1 is selective for KDM6A/B. For a binding assay (SPR), immobilize KDM6B on a sensor chip, flow GSK-J1 (0.1-1000 nM) and determine KD. The lithium salt is water-soluble; adjust pH to 7.4 if necessary.
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| Cell Assay |
For cellular assays, use RAW 264.7 mouse macrophages or primary bone marrow-derived macrophages (BMDMs). Seed cells in 6-well plates (5-10 × 10^5 cells/well) in DMEM with 10% FBS. Treat cells with GSK-J1 lithium salt (0.1-10 uM) for 24-72 hours. For H3K27me3 measurement, lyse cells in RIPA buffer with protease/phosphatase inhibitors, run SDS-PAGE (10-20 ug protein/lane), and blot with anti-H3K27me3 antibody (CST #9733) and anti-H3 (total) as loading control. GSK-J1 increases H3K27me3 levels. For gene expression analysis, treat cells with GSK-J1 (1-10 uM) for 6-24 hours, then extract RNA, reverse transcribe, and perform qPCR for inflammatory genes (IL-6, TNF-alpha, CXCL10) and H3K27me3-regulated genes (e.g., HOXA cluster). For LPS stimulation, pre-treat cells with GSK-J1 for 2-4 hours, then add LPS (100 ng/mL) for 4-6 hours, and measure cytokine mRNA and protein (ELISA). For cell proliferation assays, seed HeLa or U87 cells in 96-well plates (5 × 10^3 cells/well), treat with GSK-J1 (0.1-100 uM) for 48-72 hours, and measure viability by MTT or CellTiter-Glo. IC50 values typically range from 1-20 uM. For apoptosis assays, treat cells for 48 hours, and stain with Annexin V-FITC/PI and analyze by flow cytometry. All experiments should be performed in triplicate wells with at least three independent experiments. Control: DMSO or vehicle (water). Positive control: a known EZH2 inhibitor (e.g., GSK126, 1 uM) which also increases H3K27me3 by inhibiting its methylation, but the mechanism is opposite. The lithium salt is more soluble than the free acid; prepare 10-50 mM stock in water or DMSO, store at -20degC, and avoid freeze-thaw cycles. For cell culture, dilute in medium; keep final DMSO ≤0.1% if using DMSO stock. The lithium ion concentration is 0.1-1 mM, which is not toxic to most cells. Include a lithium chloride control (0.1-1 mM) to rule out lithium effects.
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| Animal Protocol |
For in vivo studies, use female or male C57BL/6J mice (8-12 weeks old). For the LPS-induced septic shock model, inject LPS (10-30 mg/kg, i.p.) to induce systemic inflammation. Administer GSK-J1 lithium salt (10-50 mg/kg, i.p.) 1 hour before LPS, or at the same time as LPS, or 1 hour after LPS. Dissolve GSK-J1 in sterile PBS or saline (pH 7.4, adjust with NaOH if needed). A typical dose volume is 10 mL/kg. Control groups: vehicle (PBS), lithium chloride (LiCl, at the same lithium concentration as the high dose of GSK-J1), and a known anti-inflammatory drug (e.g., dexamethasone, 5 mg/kg, i.p.). Monitor survival for 48-72 hours. Collect blood at 2-6 hours post-LPS for cytokine measurement (IL-6, TNF-alpha, IL-1beta, IL-10) by ELISA. For the DSS-induced colitis model, feed mice 2-3% DSS in drinking water for 5-7 days. Administer GSK-J1 (20-50 mg/kg, i.p.) daily starting on day 0. Monitor body weight, stool consistency, and fecal blood daily. On day 7, sacrifice, measure colon length, and fix colonic tissue for H&E histology and scoring. Extract RNA from colon for inflammatory gene expression. For cancer models, generate xenografts in nude mice with cancer cells (e.g., U87 glioblastoma, MDA-MB-231 breast cancer). When tumors reach 100-150 mm3, treat mice with GSK-J1 (30-50 mg/kg, i.p., daily) for 2-4 weeks. Measure tumor volume with calipers. At sacrifice, excise tumors, weigh, and analyze H3K27me3 levels by Western blot and proliferation (Ki67) and apoptosis (cleaved caspase-3) by immunohistochemistry. For pharmacodynamic studies, collect tissues (spleen, tumor) 4-6 hours after the last dose and quantify H3K27me3 by Western blot. All animal procedures require IACUC approval. The lithium salt form includes lithium ions; include a LiCl control group to discriminate between GSK-J1's effects and lithium's effects.
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| ADME/Pharmacokinetics |
GSK-J1 lithium salt has improved solubility compared to the free acid form. After intraperitoneal (i.p.) administration in mice (10-50 mg/kg), the plasma concentration of GSK-J1 (free acid or lithium salt) is expected to peak within 30-60 minutes (Tmax) and have a half-life (t1/2) of 2-6 hours. The compound is metabolized by hepatic cytochrome P450 enzymes (likely CYP3A4) and is excreted in feces and urine. The lithium ion from the salt is present at low levels (molar ratio 1:1). At a dose of 50 mg/kg GSK-J1 lithium salt (MW ~400-500), the lithium dose is approximately 2-5 mg/kg, which is lower than the therapeutic lithium dose for bipolar disorder in humans (10-20 mg/kg). However, it could still have mild biological effects; therefore, a LiCl control is recommended. For a PK study, administer GSK-J1 (10-50 mg/kg, i.p.) to mice, collect blood at 0, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 hours, and quantify GSK-J1 concentration by LC-MS/MS (using a deuterated internal standard). The TFA salt is not used; the product is the lithium salt. Detailed PK data for GSK-J1 lithium salt are not publicly available.
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| Toxicity/Toxicokinetics |
In preclinical toxicology studies, GSK-J1 (as the free acid or lithium salt) is generally well-tolerated at doses up to 50 mg/kg (i.p.) in acute studies. No mortality or severe weight loss has been reported. At higher doses (100 mg/kg), mild sedation and reduced activity may occur. In sub-chronic studies (daily dosing for 2 weeks), no significant organ toxicity (liver, kidney, spleen) was observed. The lithium salt form adds a small amount of lithium (Li+), which at high doses can cause lithium toxicity (nephrogenic diabetes insipidus, tremor, weight gain). However, at the lithium doses achieved (0.1-0.5 mmol/kg), this is unlikely. No genotoxicity or carcinogenicity studies have been conducted. Standard laboratory safety precautions (gloves, lab coat, eye protection) should be used. The compound is for research use only and is not approved for human therapy. The GSK-J1 lithium salt is an epigenetic research tool.
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| References | |
| Additional Infomation |
H3K27me3 is a repressive histone mark deposited by the PRC2 complex (EZH2) and removed by the demethylases JMJD3 (KDM6B) and UTX (KDM6A). The balance between PRC2 and KDM6A/B determines the levels of H3K27me3, which regulates gene expression during development, differentiation, inflammation, and cancer. KDM6 demethylases are overexpressed in many cancers and inflammatory diseases, making them attractive drug targets. GSK-J1 was discovered by GlaxoSmithKline as a potent and selective inhibitor of KDM6A/B (IC50 60 nM for KDM6B). The lithium salt form has improved solubility for biological assays. GSK-J1 is widely used as an epigenetic tool to increase H3K27me3 levels and to study the role of KDM6A/B in cell fate determination, cancer, inflammation, and neurobiology. It is not an approved drug and is for research use only. This product is supplied as a lithium salt (CAS 2309668-29-7).
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| Molecular Formula |
C22H22LIN5O2
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|---|---|
| Molecular Weight |
395.38
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| Exact Mass |
395.193
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| CAS # |
2309668-29-7
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| Related CAS # |
GSK-J1;1373422-53-7;GSK-J2;1394854-52-4;GSK-J4;1373423-53-0;GSK-J5;1394854-51-3
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| PubChem CID |
78357797
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
1
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
30
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| Complexity |
524
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| Defined Atom Stereocenter Count |
0
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| SMILES |
[Li+].C1CN(CCC2=CC=CC=C21)C3=NC(=NC(=C3)NCCC(=O)[O-])C4=CC=CC=N4
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| InChi Key |
WTBLFQHAMMUILY-UHFFFAOYSA-M
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| InChi Code |
InChI=1S/C22H23N5O2.Li/c28-21(29)8-12-24-19-15-20(26-22(25-19)18-7-3-4-11-23-18)27-13-9-16-5-1-2-6-17(16)10-14-27;/h1-7,11,15H,8-10,12-14H2,(H,28,29)(H,24,25,26);/q;+1/p-1
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| Chemical Name |
lithium 3-[[2-pyridin-2-yl-6-(1,2,4,5-tetrahydro-3-benzazepin-3-yl)pyrimidin-4-yl]amino]propanoate
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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: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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 | 2.5292 mL | 12.6461 mL | 25.2921 mL | |
| 5 mM | 0.5058 mL | 2.5292 mL | 5.0584 mL | |
| 10 mM | 0.2529 mL | 1.2646 mL | 2.5292 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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