ML324

ML324 is a potent and cell-permeable JMJD2 demethylase inhibitor (IC50 = 920 nM). [1]
Jumonji-domain-containing proteins (JMJD) is the largest class of N ε -methyl lysine demethylase, an enzyme that demethylates the tri-methylated H3K9. JMJD takes part in gene transcription regulation. [1]
ML324 acted as an antiviral agent that effectively inhibited HSV and hCMV IE gene expression in HFF and MRC-5 cells, resulted in suppression of HSV plaques formation and inhibition of HSV infection spread. [1]
ML324 also blocked HSV-1 reactivation and inhibited the formation of HSV plaque in mouse ganglia explant model of latently infected mice. [1]
References:
1.Rai G, Kawamura A, Tumber A, Liang Y, Vogel JL, Arbuckle JH, Rose NR,
Dexheimer TS, Foley TL, King ON, Quinn A, Mott BT, Schofield CJ, Oppermann U,Jadhav A, Simeonov A, Kristie TM, Maloney DJ. Discovery of ML324, a JMJD2
demethylase inhibitor with demonstrated antiviral activity. 2012 Dec 17 [updated 2013 Sep 16]. Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.

Inhibition of the histone demethylase KDM4B leads to activation of KDM1A, attenuates bacterial-induced pro-inflammatory cytokine release, and reduces osteoclastogenesis.[Pubmed:29927684]

Epigenetics. 2018;13(5):557-572.

Periodontal disease (PD) afflicts 46% of Americans with no effective adjunctive therapies available. While most pharmacotherapy for PD targets bacteria, the host immune response is responsible for driving tissue damage and bone loss in severe disease. Herein, we establish that the histone demethylase KDM4B is a potential drug target for the treatment of PD. Immunohistochemical staining of diseased periodontal epithelium revealed an increased abundance of KDM4B that correlates with inflammation. In murine calvarial sections exposed to Aggregatibacter actinomycetemcomitans lipopolysaccharide (Aa-LPS), immunohistochemical staining revealed a significant increase in KDM4B protein expression. The 8-hydroxyquinoline ML324 is known to inhibit the related demethylase KDM4E in vitro, but has not been evaluated against any other targets. Our studies indicate that ML324 also inhibits KDM4B (IC50: 4.9 muM), and decreases the pro-inflammatory cytokine response to an Aa-LPS challenge in vitro. Our results suggest that KDM4B inhibition-induced immunosuppression works indirectly, requiring new protein synthesis. In addition, fluorescence-stained macrophages exhibited a significant decrease in global monomethyl histone 3 lysine 4 (H3K4me) levels following an Aa-LPS challenge that was prevented by KDM4B inhibition, suggesting this effect is produced through KDM1A-mediated demethylation of H3K4. Finally, ML324 inhibition of KDM4B in osteoclast progenitors produced a significant reduction in Aa-LPS-induced osteoclastogenesis. These data link histone methylation with host immune response to bacterial pathogens in PD, and suggest a previously unreported, alternative mechanism for epigenetic control of the host inflammatory environment. As such, KDM4B represents a new therapeutic target for treating hyper-inflammatory diseases that result in bone destruction.

Inhibition of KDM4A activity as a strategy to suppress interleukin-6 production and attenuate colitis induction.[Pubmed:28511912]

Clin Immunol. 2017 Jul;180:120-127.

4-Chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) functions as a hapten and fluoresces upon binding to proteins. Therefore, fluorescence visualization of hapten-proteins is a feature of the colitis induced by NBD-Cl. Using this colitis model, we located activated fibroblasts in the vicinity of hapten-proteins upon colitis induction and observed interleukin (IL)-6 production in the activated fibroblasts. We screened herbal ingredients using primary fibroblasts stimulated with tumor necrosis factor alpha (TNF-alpha) and found the suppressive action of Atractylodin on IL-6 production. Under TNF-alpha stimulation, Atractylodin induced the tri-methylation of histone H3 at lysine residue 9, which impaired the binding between NF-kappaB and the IL-6 promoter on the genomic DNA. Atractylodin inhibited KDM4A but not KDM6A activity. Atractylodin administration attenuated colitis induction. The KDM4A inhibitor ML324 showed similar actions on IL-6 production and colitis induction. We propose the inhibition of KDM4A activity as a strategy to suppress IL-6 production and attenuate colitis induction.

KDM4B histone demethylase and G9a regulate expression of vascular adhesion proteins in cerebral microvessels.[Pubmed:28327608]

Sci Rep. 2017 Mar 22;7:45005.

Intercellular adhesion molecule 1 (ICAM1) mediates the adhesion and transmigration of leukocytes across the endothelium, promoting inflammation. We investigated the epigenetic mechanism regulating ICAM1 expression. The pro-inflammatory cytokine TNF-alpha dramatically increased ICAM1 mRNA and protein levels in human brain microvascular endothelial cells and mouse brain microvessels. Chromatin immunoprecipitation revealed that TNF-alpha reduced methylation of histone H3 at lysines 9 and 27 (H3K9 and H3K27), well-known residues involved in gene suppression. Inhibition of G9a and EZH2, histone methyltransferases responsible for methylation at H3K9 and H3K27, respectively as well as G9a overexpression demonstrated the involvement of G9a in TNF-alpha-induced ICAM1 expression and leukocyte adhesion and transmigration. A specific role for KDM4B, a histone demethylase targeting H3K9me2, in TNF-alpha-induced ICAM1 upregulation was validated with siRNA. Moreover, treating mice with a KDM4 inhibitor ML324 blocked TNF-alpha-mediated neutrophil adhesion. Similarly, TNF-alpha-induced VCAM1 expression was suppressed by G9a overexpression and KDM4B knockdown. Collectively, we demonstrated that modification of H3K9me2 by G9a and KDM4B regulates expression of vascular adhesion molecules, and that depletion of these proteins or KDM4B reduces inflammation-induced leukocyte extravasation. Thus, blocking ICAM1 or KDM4B could offer a novel therapeutic opportunity treating brain diseases.

Histone Lysine Demethylases of JMJD2 or KDM4 Family are Important Epigenetic Regulators in Reward Circuitry in the Etiopathology of Depression.[Pubmed:27711046]

Neuropsychopharmacology. 2017 Mar;42(4):854-863.

Major depressive disorder (MDD) is debilitating mental illness and is one of the leading contributors to global burden of disease, but unfortunately newer and better drugs are not forthcoming. The reason is lack of complete understanding of molecular mechanisms underlying the development of this disorder. Recent research shows dysregulation in epigenetic regulatory mechanisms, particularly the transcriptionally repressive di- and tri-methylation of histone 3 lysine 9 (H3K9me2/me3) in nucleus accumbens (NAc), a critical region of the reward pathway involved in the development of anhedonia, the hallmark of depression. However, the role of histone lysine demethylases, which can remove methylation from H3K9, in particular Jumonji domain containing demethylases 2 or Jmjd2 family, has not been studied. Using social defeat stress-induced mouse model of depression, this study uncovered that transcripts of most of the Jmjd2 members were unchanged after 5 days of defeat during the onset of depression, but were downregulated after 10 days of defeat in full-blown depression. Blocking the Jumonji domain containing demethylases by chronic administration of inhibitors dimethyloxalylglycine (DMOG) and ML324 resulted in depression-like phenotype even in absence of stress exposure, which was associated with an increase in transcriptionally repressive epigenetic marks H3K9me2/me3 in NAc, causing altered neuroplastic changes as reported in NAc in depression models. Thus, we report for the first time that Jmjd2 class demethylases are critical epigenetic regulators involved in etiopathology of depression and related disorders and activation of these demethylases can be a good strategy in the treatment of MDD and related psychiatric disorders.