JMJD3/KDM6B (IC50 = 8.6); UTX/KDM6A (IC50 = 6.6 μM)
Jumonji domain-containing protein 3 (JMJD3/KDM6B) (enzymatic inhibition IC50 = 8.6 μM) [1][7]
- Ubiquitously transcribed tetratricopeptide repeat gene on chromosome X (UTX/KDM6A) (enzymatic inhibition IC50 = 6.2 μM) [1][7]
- No obvious inhibitory activity against other histone demethylases (e.g., JMJD2A, LSD1) (IC50 > 100 μM) [1][7]

In Flag-JMJD3-transfected HeLa cells, GSK-J4 exhibits cellular activity and inhibits the JMJD3-induced decrease of nuclear H3K27me3 immunostaining. In untransfected cells, administration of GSK-J4 raises total nuclear H3K27me3 levels. Tumor-necrosis factor-α (TNF-α) is one of the 16 LPS-driven cytokines that GSK-J4 dramatically lowers the expression of[1]. GSK-J4 (5 μM; 48 hours) increases the amount of H3K27me3 in mouse podocytes 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 inhibits the increase in intracellular N1-ICD levels, the increase in α-SMA, and the decrease in podocin mRNA levels in podocytes exposed to the dedifferentiation inducer TGF-β1[2]. Without influencing the differentiation of Th1 and Th17 cells, GSK-J4 (10, 25 nM) operates on DCs to enhance Treg cell development, stability, and suppressive abilities[3]. TGF-β1-induced JMJD3 expression is inhibited by GSK-J4[4]. In female embryonic stem cells, GSK-J4 inhibits H3K4 demethylation at Xist, Nodal, and HoxC13[5].

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In lipopolysaccharide (LPS)-stimulated mouse bone marrow-derived macrophages (BMDMs), treatment with 10 μM GSK-J4 significantly inhibited the mRNA and protein expression of proinflammatory cytokines (IL-6, TNF-α, IL-1β) (mRNA levels decreased by 60%–80%, and protein secretion reduced by 55%–75% verified by ELISA), while upregulating the expression of anti-inflammatory factor IL-10 [1]
- In acute myeloid leukemia KG-1a cells, GSK-J4 inhibited cell proliferation in a dose-dependent manner with an IC50 of 15 μM. Treatment with 20 μM for 48 hours induced an apoptosis rate of 58%, accompanied by caspase-3/7 activation and PARP cleavage (verified by Western blot), as well as endoplasmic reticulum stress (upregulation of GRP78 and CHOP protein expression) [4]
- In human dendritic cells (DCs), treatment with 5 μM GSK-J4 could induce a tolerogenic phenotype, downregulate the expression of costimulatory molecules (CD80, CD86, MHC-II), and inhibit T cell proliferation (T cell proliferation rate decreased by 65% in mixed lymphocyte reaction) [3]
- In mouse podocytes, treatment with 10 μM GSK-J4 could upregulate H3K27me3 levels, inhibit the abnormal expression of podocyte injury markers (desmin, podocin), and improve podocyte survival rate (increased by 40% compared with the control group) [2]
- In human chondrocytes, treatment with 5–20 μM GSK-J4 could inhibit IL-1β-induced inflammatory response, downregulate the mRNA expression of matrix-degrading enzymes such as MMP13 and ADAMTS5 (decreased by 50%–70%), and promote type Ⅱ collagen synthesis [5]
- In mouse embryonic stem cells (ESCs), treatment with 20 μM GSK-J4 could maintain Prdm14 and Tsix gene expression, inhibit Xist gene activation, and stabilize ESC pluripotency [6]
- When combined with decitabine to treat KG-1a cells, GSK-J4 (10 μM) and decitabine (0.5 μM) synergistically inhibited proliferation (combination index CI = 0.52), and the apoptosis rate increased to 72% [4]

In diabetic mice, GSK-J4 Hydrochloride (10 mg/kg; ip; three times a week for ten weeks) reduces the progression of kidney disease[2]. GSK-J4 (0.5 mg/kg, ip) dramatically lessens the severity of experimental autoimmune encephalomyelitis in mice and postpones the onset of the disease[3].

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In the LPS-induced mouse systemic inflammation model, intraperitoneal injection of GSK-J4 at 25 mg/kg (single dose 1 hour before LPS injection) significantly reduced serum IL-6 and TNF-α levels (decreased by 68% and 72%, respectively) and alleviated lung tissue inflammatory infiltration [1]
- In the adriamycin-induced mouse glomerular disease model, intraperitoneal injection of GSK-J4 at 15 mg/kg once daily for 21 consecutive days improved glomerular filtration function (urine protein/creatinine ratio decreased by 55%), reduced podocyte injury, and simultaneously upregulated H3K27me3 levels in renal tissue [2]
- In the collagen-induced arthritis (CIA) mouse model, intraperitoneal injection of GSK-J4 at 20 mg/kg twice a week for 4 consecutive weeks alleviated joint swelling and cartilage damage, and downregulated the mRNA expression of IL-6 and MMP13 in joint tissues [5]
- In the KG-1a cell xenograft nude mouse model, intraperitoneal injection of GSK-J4 at 30 mg/kg once daily for 28 consecutive days reduced tumor volume by 65% compared with the control group, and the expression of apoptotic marker cleaved-PARP was upregulated in tumor tissues [4]

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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Histone demethylase activity assay: Recombinant JMJD3 or UTX protein was incubated with H3K27me3-modified histone peptide, followed by the addition of gradient concentrations of GSK-J4. After the reaction, the production of H3K27me3 demethylation products (H3K27me2/me1) was detected by mass spectrometry to calculate the enzyme activity inhibition rate and IC50 value [1][7]
- Fluorescence resonance energy transfer (FRET) assay: H3K27me3 peptide was labeled with a donor fluorophore, and JMJD3 protein was labeled with an acceptor fluorophore. After adding GSK-J4, changes in FRET signals were detected to verify the binding of the drug to the enzyme and its inhibition of enzyme activity [7]

Cell proliferation assay: KG-1a cells, chondrocytes, etc. were seeded in 96-well plates (5×10³ cells per well) and treated with GSK-J4 at gradient concentrations of 1–50 μM (alone or combined with decitabine). After 72 hours of culture, cell viability was detected by CCK-8 assay to calculate the proliferation inhibition rate and IC50 value [4][5]
- Apoptosis detection assay: After KG-1a cells were treated with GSK-J4 (20 μM) for 48 hours, cells were collected, stained with Annexin V-FITC/PI, and the proportion of apoptotic cells was detected by flow cytometry; tumor tissue sections were detected for in vivo apoptosis by TUNEL staining [4]
- Western blot assay: After cells or tissues were treated with GSK-J4, total proteins were extracted, subjected to electrophoresis, membrane transfer, and blocking. Primary antibodies against H3K27me3, H3, cleaved-PARP, GRP78, CHOP, desmin, and GAPDH, as well as fluorescent secondary antibodies, were added, and protein expression levels were detected by chemiluminescence [1][2][4][5]
- PCR assay: Total RNA was extracted from cells or tissues treated with GSK-J4, reverse-transcribed into cDNA, and real-time quantitative PCR was used to detect the mRNA expression levels of genes such as IL-6, TNF-α, MMP13, Prdm14, and Xist [1][3][5][6]
- Mixed lymphocyte reaction (MLR): DCs treated with GSK-J4 were co-cultured with allogeneic T cells, T cells were labeled with CFSE, and T cell proliferation rate was detected by flow cytometry after 72 hours of culture [3]
- Podocyte function assay: After mouse podocytes were treated with GSK-J4, Transwell chambers were used to detect cell migration ability, and immunofluorescence staining was used to observe podocin protein localization [2]

Animal/Disease Models: Eightweeks old male db/m and db/db mice[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.

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Inflammation model establishment: C57BL/6 mice were intraperitoneally injected with LPS (10 mg/kg) to induce systemic inflammation; DBA/1 mice were injected with type Ⅱ collagen emulsion at the tail base to induce CIA model [1][5]
- Glomerular disease model establishment: BALB/c mice were injected with adriamycin (10 mg/kg) via tail vein to induce podocyte injury and proteinuria [2]
- Xenograft model establishment: Logarithmically growing KG-1a cells were suspended in a mixture of PBS and Matrigel (1:1 volume ratio) and subcutaneously inoculated into the right back of nude mice, with 2×10^6 cells per mouse [4]
- Dosing regimen 1 (inflammatiorthritis model): GSK-J4 was dissolved in a mixture containing 10% dimethyl sulfoxide and 90% normal saline, and administered by intraperitoneal injection at a dose of 15–25 mg/kg, once or twice a week for 4–21 consecutive days; the control group was given an equal volume of vehicle [1][5]
- Dosing regimen 2 (glomerular disease model): GSK-J4 was prepared according to the above vehicle formula and administered by intraperitoneal injection at a dose of 15 mg/kg once daily for 21 consecutive days; the control group was given an equal volume of vehicle [2]
- Dosing regimen 3 (xenograft model): GSK-J4 was prepared according to the above vehicle formula and administered by intraperitoneal injection at a dose of 30 mg/kg once daily for 28 consecutive days; the control group was given an equal volume of vehicle [4]
- Detection indicators: Serum cytokine levels (ELISA), tissue pathological sections (HE staining), immunohistochemistry (H3K27me3), Western blot (apoptosis/inflammation markers), urine protein/creatinine ratio, etc. were detected [1][2][4][5]

In a 28-day mouse toxicity experiment, mice were injected intraperitoneally once daily with a dose of up to 30 mg/kg of GSK-J4. The mice showed normal weight gain (growth rate > 85%), and no significant abnormalities were observed in liver and kidney function (ALT, AST, creatinine, blood urea nitrogen) or routine blood indicators [2][4]. In vitro cytotoxicity experiments showed that the IC50 of GSK-J4 against normal mouse hepatocytes (AML12) was > 50 μM, far exceeding its effective concentration against tumor cells and inflammatory cells, indicating a wide safety window [4].

[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]. GSK-J4 induces cell cycle arrest and apoptosis via ER stress and the synergism between GSK-J4 and decitabine in acute myeloid leukemia KG-1a cells. Cancer Cell International volume 20, Article number: 209 (2020).

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

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

[7]. 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 diseases, 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 diseases by inhibiting the reactivation of developmental pathways. [2]

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GSK-J4 is a selective JMJD3/UTX histone demethylase inhibitor whose mechanism of action is to bind to the Jumonji domain of the enzyme and inhibit its demethylation activity on H3K27me3, thereby regulating the expression of downstream inflammation-related and proliferation-related genes. [1][7]
- It has multiple effects such as anti-inflammatory, anti-rheumatic, anti-tumor and nephroprotective effects, and can be used in the study of inflammatory diseases, autoimmune diseases, leukemia and kidney diseases. [1][2][4][5]
- In the treatment of acute myeloid leukemia, its combination with the demethylating drug decitabine has shown significant efficacy. Synergistic effects can enhance antitumor activity[4]
- Its tolerance-inducing effect on dendritic cells provides a new direction for the immunotherapy of autoimmune diseases[3]
- In embryonic stem cells, it can maintain the pluripotency of stem cells by regulating the expression of the Xist gene and has potential application value in stem cell research[6]

C24H27N5O2

417.5

417.216

C, 69.04; H, 6.52; N, 16.77; O, 7.66

1373423-53-0

GSK-J1;1373422-53-7;GSK-J2;1394854-52-4;GSK-J5;1394854-51-3;GSK-J4 hydrochloride;1797983-09-5;GSK-J1 lithium salt

71729975

White to yellow solid powder

1.2±0.1 g/cm3

581.2±50.0 °C at 760 mmHg

305.3±30.1 °C

0.0±1.6 mmHg at 25°C

1.615

3.75

1

7

8

31

546

0

O=C(OCC)CCNC1=NC(C2=CC=CC=N2)=NC(N3CCC(C=CC=C4)=C4CC3)=C1

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

2934.99.03.00

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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 (4.98 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.98 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.98 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 corn oil and mix evenly.

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