JMJD3/KDM6B (IC50 = 8.6); UTX/KDM6A (IC50 = 6.6 μM)
GSK-J4 is a prodrug of the selective histone H3K27 demethylase inhibitor GSK-J1. It targets the KDM6 subfamily of jumonji (JMJ) histone demethylases, specifically JMJD3 and UTX, which demethylate H3K27me3. GSK-J1 (the active form) shows an IC₅₀ of 60 nM against JMJD3 in an AlphaScreen assay, and selectively inhibits JMJD3 and UTX over other JMJ family demethylases and a panel of 100 protein kinases (no significant inhibition at 30 µM) [1]

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].

---

GSK-J4 (the ethyl ester prodrug of GSK-J1) is rapidly hydrolyzed by macrophage esterases to release the active acid GSK-J1 intracellularly. In human primary macrophages, GSK-J4 (30 µM) significantly reduced the expression of 16 out of 34 LPS-induced cytokines, including TNF-α, as assessed by PCR array. GSK-J4 inhibited TNF-α protein production in a dose-dependent manner with an IC₅₀ of 9 µM, while its inactive isomer GSK-J5 had no effect. GSK-J4 (25 µM) preserved nuclear H3K27me3 staining in Flag-JMJD3-transfected HeLa cells, whereas GSK-J5 did not. GSK-J4 also blocked TNF-α production in macrophages derived from rheumatoid arthritis patients [1]

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].

---

In an adriamycin-induced nephrotoxicity model in BALB/c mice (a model of focal segmental glomerulosclerosis), administration of GSK-J4 (10 mg/kg, i.p., thrice weekly for 10 days) markedly attenuated the rise in albuminuria. The treatment also increased renal H3K27me3 content by approximately 4-fold, as shown by immunoblotting of kidney homogenates, and increased podocyte nuclear H3K27me3 levels, as detected by dual immunofluorescence microscopy. GSK-J4 treatment limited glomerular Jagged-1 upregulation and loss of the podocyte protein nephrin. In a diabetic nephropathy model using db/db mice (obese, type 2 diabetic), treatment with GSK-J4 (10 mg/kg, i.p., thrice weekly for 10 weeks) attenuated the development of glomerular injury. Specifically, it diminished albuminuria and the accumulation of matrix within the glomerular mesangium, attenuated podocyte foot process effacement (measured by transmission electron microscopy), and reduced glomerular upregulation of α-SMA and Jagged-1. In a late-intervention study in the adriamycin model, initiating GSK-J4 treatment (10 mg/kg, i.p., on days 4, 7, and 11) 4 days after adriamycin injection (when podocyte H3K27me3 levels were already reduced and albuminuria was established) prevented further podocyte H3K27me3 loss and halted the progression of albuminuria over the subsequent 8 days. A similar delayed intervention in a subtotal nephrectomy (SNx) model also abated the increases in glomerular Jagged-1 content and albuminuria [2]

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]

---

JMJD3 inhibitory activity was measured using an AlphaScreen assay. The assay detects demethylation of a biotinylated H3K27me3 peptide by recombinant JMJD3, using an antibody that recognizes the demethylated product. The IC₅₀ value for GSK-J1 was determined to be 60 nM in this assay. Selectivity against other JMJ demethylases was assessed using thermal shift assays, mass spectrometry-based demethylation assays, and antibody-based assays. For thermal shift, a significant stabilization (>1 °C) was observed only for UTX and JMJD3, indicating selective binding of GSK-J1 to the KDM6 subfamily [1]

For intracellular compound quantification, human primary macrophages were treated with 30 µM of GSK-J1, GSK-J2, GSK-J4, or GSK-J5 for 1 hour. Cells were lysed, and the concentrations of GSK-J1 and GSK-J2 in the lysates were measured. The ester prodrugs GSK-J4 and GSK-J5 delivered higher intracellular levels of the active acids compared to direct administration of the acids. For cytokine analysis, human primary macrophages from healthy donors were pretreated with compounds (e.g., 30 µM GSK-J4 or GSK-J5) for 1 hour, then stimulated with LPS for 2-6 hours. TNF-α protein in supernatants was quantified by ELISA, and mRNA expression of cytokines was analyzed by quantitative PCR array. For H3K27me3 immunostaining, HeLa cells were transfected with Flag-JMJD3, treated with 25 µM GSK-J4 or GSK-J5, fixed, and stained with antibodies against Flag and H3K27me3. Images were acquired by fluorescence microscopy. For siRNA knockdown, human primary macrophages were transfected with scrambled, JMJD3-specific, or UTX-specific siRNA. After 48 hours, cells were treated with compounds and stimulated with LPS, and TNF-α production was measured [1]

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.

---

For the adriamycin nephrotoxicity model in BALB/c mice, eight-week-old male BALB/c mice received a single tail-vein injection of adriamycin (10 mg/kg in PBS) or PBS (vehicle). Mice were then randomly allocated to receive GSK-J4 (10 mg/kg) or vehicle (0.1% DMSO in PBS) by intraperitoneal (i.p.) injection thrice weekly for 10 days (total of 5 injections). For the late-intervention study, mice received adriamycin as above, and four days later, they were stratified by albuminuria level to receive GSK-J4 (10 mg/kg) or vehicle by i.p. injection on days 4, 7, and 11. For the diabetic nephropathy model, eight-week-old male db/m (non-diabetic control) and db/db (diabetic) mice were randomly allocated to receive GSK-J4 (10 mg/kg in 0.1% DMSO in PBS) or vehicle alone by thrice-weekly i.p. injections for 10 weeks. For the subtotal nephrectomy (SNx) delayed intervention model, C57BL/6 mice underwent SNx surgery. Six weeks later, they were randomized to receive GSK-J4 (10 mg/kg, thrice weekly i.p.) or vehicle for an additional 4 weeks. Urine was collected using metabolic cages for albumin and creatinine measurement [2]

GSK-J4 is an ethyl ester prodrug designed to improve its cell permeability, superior to the polar carboxylic acid GSK-J1. In human primary macrophages, after administration of 30 µM GSK-J4 for 1 hour, the intracellular GSK-J1 concentration reached 11.8 ± 0.6 µM, while direct administration of 30 µM GSK-J1 only resulted in an intracellular GSK-J1 concentration of 1.6 ± 1.6 µM. This confirms that the prodrug can be efficiently hydrolyzed into active acid by esterases in the cell[1].

Under the experimental conditions used, human primary macrophages were treated with GSK-J4 at concentrations up to 30 µM, and no cytotoxicity was observed (assessed by cytotoxicity assays). [1]

[1]. A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response. Nature. 2012 Aug 16;488(7411):404-8.

[2]. Shifts in podocyte histone H3K27me3 regulate mouse and human glomerular disease. J Clin Invest. 2018 Jan 2;128(1):483-499.

[3]. 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]. H3K27me3 demethylases regulate in vitro chondrogenesis and chondrocyte activity in osteoarthritis. Arthritis Res Ther. 2016 Jul 7;18(1):158.

[5]. Histone demethylation maintains Prdm14 and Tsix expression and represses xIst in embryonic stem cells. PLoS One. 2015 May 20;10(5):e0125626.

[6]. Inhibition of demethylases by GSK-J1/J4. Nature. 2014 Oct 2;514(7520):E1-2.

Histone modifications control cell fate determination during normal development and lead to cell dedifferentiation during disease. This study aimed to investigate the extent to which dynamic changes in histones affect the differentiation phenotype of adult glomerular podocytes, which are normally in a quiescent state. To this end, we investigated the consequences of altering the balance of the inhibitory histone H3 lysine trimethylation at position 27 (H3K27me3) labeling in podocytes. Doxorubicin nephrotoxicity and partial nephrectomy (SNx) studies showed that the absence of the histone methyltransferase EZH2 in podocytes reduced H3K27me3 levels and made mice more susceptible to glomerular disease. H3K27me3 was enriched in the promoter region of the Notch ligand Jag1 in podocytes, and relieving the inhibition of Jag1 by inhibiting or knocking down EZH2 promoted podocyte dedifferentiation. Conversely, inhibition of the Jumonji C-domain-containing demethylases Jmjd3 and UTX increased H3K27me3 levels in podocytes and alleviated glomerular disease induced by doxorubicin nephrotoxicity, nephrectomy (SNx), and diabetes. Glomerular podocytes from patients with focal segmental glomerulosclerosis or diabetic nephropathy showed decreased H3K27me3 content and increased UTX content. Similar to human disease, inhibition of Jmjd3 and UTX slowed the progression of nephropathy in mice with established glomerular damage and reduced H3K27me3 levels. These findings together suggest that surface-stable chromatin modifications can be dynamically regulated in quiescent cells and that epigenetic reprogramming can improve the prognosis of glomerular disease by inhibiting the reactivation of developmental pathways. [2]

---

GSK-J4 is an ethyl ester prodrug of GSK-J1, which is the first selective catalytic site inhibitor of H3K27me3-specific demethylases JMJD3 and UTX (KDM6 subfamily). GSK-J1 was discovered through a structure-directed approach based on the cocrystal structure of JMJD3 with substrates and cofactors. GSK-J4 was used in experiments to inhibit intracellular JMJD3/UTX demethylase activity. Its inactive regioisomer GSK-J5 was used as an important negative control. This study showed that JMJD3 and UTX-mediated H3K27 demethylation is essential for the activation of pro-inflammatory genes (such as TNF-α) in human primary macrophages. Inhibition with GSK-J4 blocked the loss of the LPS-induced TNFA transcription start site H3K27me3 and the recruitment of RNA polymerase II, but did not affect the upstream NF-κB signaling pathway. This suggests that targeting KDM6 demethylases may have the potential to regulate inflammatory responses [1].

C24H27N5O2.HCL

453.96

453.193

C, 63.50; H, 6.22; Cl, 7.81; N, 15.43; O, 7.05

1797983-09-5

GSK-J4;1373423-53-0

71729974

Light yellow to yellow solids at room temperature

2

7

8

32

546

0

O(CC)C(CCNC1=CC(=NC(C2=CC=CC=N2)=N1)N1CCC2=CC=CC=C2CC1)=O.Cl

WBKCKEHGXNWYMO-UHFFFAOYSA-N

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)

ethyl 3-((2-(pyridin-2-yl)-6-(1,2,4,5-tetrahydro-3H-benzo[d]azepin-3-yl)pyrimidin-4-yl)amino)propanoate hydrochloride

2934.99.9001

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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

---

 (Please use freshly prepared in vivo formulations for optimal results.)