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Purity: ≥98%
GSK J4 (GSK-J4) is a novel, cell permeable, and potent prodrug of GSK J1 with anti-inflammatory effects. It is the first selective and dual inhibitor of H3K27 histone demethylase (KDM) JMJD3 and UTX with IC50 of 60 nM in a cell-free assay and is inactive against a panel of demethylases of the JMJ family. GSK-J4 is used to probe the consequences of demethylation of H3K27me3. GSK-J4 inhibits the lipopolysaccharide-induced production of cytokines, including pro-inflammatory tumour necrosis factor (TNF). GSK-J4 (0.5 mg/kg, i.p.) significantly reduces the severity and delays the onset of the disease of the mouse model of experimental autoimmune encephalomyelitis.
| Targets |
JMJD3/KDM6B (IC50 = 8.6); UTX/KDM6A (IC50 = 6.6 μM)
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| ln Vitro |
In Flag-JMJD3-transfected HeLa cells, GSK-J4 hydrochloride exhibits cellular activity by averting JMJD3-induced decrease of nuclear H3K27me3 immunostaining. In untransfected cells, GSK-J4 administration raised the levels of total nuclear H3K27me3. Tumor necrosis factor-α (TNF-α) is one of the 16 LPS-driven cytokines that GSK-J4 dramatically lowers the expression of [1]. In mouse podocytes, GSK-J4 hydrochloride increased the amount of H3K27me3 by more than three times. In cultured podocytes, GSK-J4 lowers the levels of Jagged-1 protein and mRNA while raising H3K27me3. Similarly, pretreatment with GSK-J4 inhibited the increase in intracellular N1-ICD levels, the increase in α-SMA, and the decrease in podocyte protein mRNA levels when podocytes were subjected to the dedifferentiation inducer TGF-β1 [2]. Hydrochloride GSK-J4 While having little effect on Th1 and Th17 cell differentiation, hydrochloride (10, 25 nM) operates on DC to enhance Treg cell differentiation, stability, and inhibitory capacity [3]. Hydrochloride GSK-J4 TGF-β1-induced JMJD3 expression is inhibited by hydrochloride [4]. Hydrochloride GSK-J4 In female embryonic stem cells, hydrochloride suppresses the H3K4 demethylation of Xist, Nodal, and HoxC13 [5].
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| ln Vivo |
Hydrochloride GSK-J4 In diabetic mice, hydrochloride (10 mg/kg; intraperitoneal injection; three times a week for 10 weeks) is administered to prevent renal damage [2]. Hydrochloride GSK-J4 In a mouse model, hydrochloride (0.5 mg/kg, ip) considerably lessens the severity and postpones the development of experimental autoimmune encephalomyelitis [3].
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| Enzyme Assay |
The jumonji (JMJ) family of histone demethylases are Fe2+- and α-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes. These enzymes demethylate lysine residues in histones in a methylation-state and sequence-specific context. Considerable effort has been devoted to gaining a mechanistic understanding of the roles of histone lysine demethylases in eukaryotic transcription, genome integrity and epigenetic inheritance, as well as in development, physiology and disease. However, because of the absence of any selective inhibitors, the relevance of the demethylase activity of JMJ enzymes in regulating cellular responses remains poorly understood. Here we present a structure-guided small-molecule and chemoproteomics approach to elucidating the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX). The liganded structures of human and mouse JMJD3 provide novel insight into the specificity determinants for cofactor, substrate and inhibitor recognition by the KDM6 subfamily of demethylases. We exploited these structural features to generate the first small-molecule catalytic site inhibitor that is selective for the H3K27me3-specific JMJ subfamily. We demonstrate that this inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. Our results resolve the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provide encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family.[1]
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| Animal Protocol |
Animal/Disease Models: Eightweeks old male db/m and db/db mice on a BKS background[2]
Doses: 10 mg/kg Route of Administration: ip; thrice-weekly for 10 weeks Experimental Results: Attenuated the development of kidney disease in diabetic mice. |
| References |
[1]. Kruidenier L, et al. A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response. Nature. 2012 Aug 16;488(7411):404-8.
[2]. Majumder S, et al. Shifts in podocyte histone H3K27me3 regulate mouse and human glomerular disease. J Clin Invest. 2018 Jan 2;128(1):483-499. [3]. Donas C, et al. The histone demethylase inhibitor GSK-J4 limits inflammation through the induction of a tolerogenic phenotype on DCs. J Autoimmun. 2016 Dec;75:105-117. [4]. Yapp C, et al. H3K27me3 demethylases regulate in vitro chondrogenesis and chondrocyte activity in osteoarthritis. Arthritis Res Ther. 2016 Jul 7;18(1):158 [5]. Kamikawa YF, et al. Histone demethylation maintains Prdm14 and Tsix expression and represses xIst in embryonic stem cells. PLoS One. 2015 May 20;10(5):e0125626 [6]. Heinemann B, et al. Inhibition of demethylases by GSK-J1/J4. Nature. 2014 Oct 2;514(7520):E1-2 |
| Additional Infomation |
Histone protein modifications control fate determination during normal development and dedifferentiation during disease. Here, we set out to determine the extent to which dynamic changes to histones affect the differentiated phenotype of ordinarily quiescent adult glomerular podocytes. To do this, we examined the consequences of shifting the balance of the repressive histone H3 lysine 27 trimethylation (H3K27me3) mark in podocytes. Adriamycin nephrotoxicity and subtotal nephrectomy (SNx) studies indicated that deletion of the histone methylating enzyme EZH2 from podocytes decreased H3K27me3 levels and sensitized mice to glomerular disease. H3K27me3 was enriched at the promoter region of the Notch ligand Jag1 in podocytes, and derepression of Jag1 by EZH2 inhibition or knockdown facilitated podocyte dedifferentiation. Conversely, inhibition of the Jumonji C domain-containing demethylases Jmjd3 and UTX increased the H3K27me3 content of podocytes and attenuated glomerular disease in adriamycin nephrotoxicity, SNx, and diabetes. Podocytes in glomeruli from humans with focal segmental glomerulosclerosis or diabetic nephropathy exhibited diminished H3K27me3 and heightened UTX content. Analogous to human disease, inhibition of Jmjd3 and UTX abated nephropathy progression in mice with established glomerular injury and reduced H3K27me3 levels. Together, these findings indicate that ostensibly stable chromatin modifications can be dynamically regulated in quiescent cells and that epigenetic reprogramming can improve outcomes in glomerular disease by repressing the reactivation of developmental pathways.[2]
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| Molecular Formula |
C24H27N5O2.HCL
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| Molecular Weight |
453.96
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| Exact Mass |
453.193146
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| Elemental Analysis |
C, 63.50; H, 6.22; Cl, 7.81; N, 15.43; O, 7.05
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| CAS # |
1797983-09-5
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| Related CAS # |
GSK-J4;1373423-53-0
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| PubChem CID |
71729974
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| Appearance |
Light yellow to yellow solids at room temperature
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| tPSA |
80.2Ų
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| SMILES |
O(CC)C(CCNC1=CC(=NC(C2=CC=CC=N2)=N1)N1CCC2=CC=CC=C2CC1)=O.Cl
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| InChi Key |
WBKCKEHGXNWYMO-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C24H27N5O2/c1-2-31-23(30)10-14-26-21-17-22(28-24(27-21)20-9-5-6-13-25-20)29-15-11-18-7-3-4-8-19(18)12-16-29/h3-9,13,17H,2,10-12,14-16H2,1H3,(H,26,27,28)
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| Chemical Name |
ethyl 3-((2-(pyridin-2-yl)-6-(1,2,4,5-tetrahydro-3H-benzo[d]azepin-3-yl)pyrimidin-4-yl)amino)propanoate hydrochloride
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| Synonyms |
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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. |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.58 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.08 mg/mL (4.58 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: 2% DMSO+dd H2O:10mg/mL |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2028 mL | 11.0142 mL | 22.0284 mL | |
| 5 mM | 0.4406 mL | 2.2028 mL | 4.4057 mL | |
| 10 mM | 0.2203 mL | 1.1014 mL | 2.2028 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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