Machine-Learning-Enabled Virtual Screening for Inhibitors of Lysine-Specific Histone Demethylase 1.

A machine learning approach has been applied to virtual screening for lysine specific demethylase 1 (LSD1) inhibitors. LSD1 is an important anti-cancer target. Machine learning models to predict activity were constructed using Morgan molecular fingerprints. The dataset, consisting of 931 molecules with LSD1 inhibition activity, was obtained from the ChEMBL database. An evaluation of several candidate algorithms on the main dataset revealed that the support vector regressor gave the best model, with a coefficient of determination (R2) of 0.703. Virtual screening, using this model, identified five predicted potent inhibitors from the ZINC database comprising more than 300,000 molecules. The virtual screening recovered a known inhibitor, RN1, as well as four compounds where activity against LSD1 had not previously been suggested. Thus, we performed a machine-learning-enabled virtual screening of LSD1 inhibitors using only the structural information of the molecules.

A protocol for high-throughput screening of histone lysine demethylase 4 inhibitors using TR-FRET assay.

Identification of diverse chemotypes of selective KDM4 inhibitors is important for exploring and validating the roles of KDM4s in the pathogenesis of human disease and for developing therapies. Here, we report a protocol for high-throughput screening of KDM4 inhibitors using TR-FRET demethylation functional assay. We describe this protocol for screen of KDM4B inhibitors, which can be modified to screen inhibitors of other JmjC-domain-containing KDMs. For complete details on the use and execution of this protocol, please refer to Singh et al. (2021).

The metabesity factor HMG20A potentiates astrocyte survival and reactive astrogliosis preserving neuronal integrity.

Rationale: We recently demonstrated that the 'Metabesity' factor HMG20A regulates islet beta-cell functional maturity and adaptation to physiological stress such as pregnancy and pre-diabetes. HMG20A also dictates central nervous system (CNS) development via inhibition of the LSD1-CoREST complex but its expression pattern and function in adult brain remains unknown. Herein we sought to determine whether HMG20A is expressed in the adult CNS, specifically in hypothalamic astrocytes that are key in glucose homeostasis and whether similar to islets, HMG20A potentiates astrocyte function in response to environmental cues. Methods: HMG20A expression profile was assessed by quantitative PCR (QT-PCR), Western blotting and/or immunofluorescence in: 1) the hypothalamus of mice exposed or not to either a high-fat diet or a high-fat high-sucrose regimen, 2) human blood leukocytes and adipose tissue obtained from healthy or diabetic individuals and 3) primary mouse hypothalamic astrocytes exposed to either high glucose or palmitate. RNA-seq and cell metabolic parameters were performed on astrocytes treated or not with a siHMG20A. Astrocyte-mediated neuronal survival was evaluated using conditioned media from siHMG20A-treated astrocytes. The impact of ORY1001, an inhibitor of the LSD1-CoREST complex, on HMG20A expression, reactive astrogliosis and glucose metabolism was evaluated in vitro and in vivo in high-fat high-sucrose fed mice. Results: We show that Hmg20a is predominantly expressed in hypothalamic astrocytes, the main nutrient-sensing cell type of the brain. HMG20A expression was upregulated in diet-induced obesity and glucose intolerant mice, correlating with increased transcript levels of Gfap and Il1b indicative of inflammation and reactive astrogliosis. Hmg20a transcript levels were also increased in adipose tissue of obese non-diabetic individuals as compared to obese diabetic patients. HMG20A silencing in astrocytes resulted in repression of inflammatory, cholesterol biogenesis and epithelial-to-mesenchymal transition pathways which are hallmarks of reactive astrogliosis. Accordingly, HMG20A depleted astrocytes exhibited reduced mitochondrial bioenergetics and increased susceptibility to apoptosis. Neuron viability was also hindered in HMG20A-depleted astrocyte-derived conditioned media. ORY1001 treatment rescued expression of reactive astrogliosis-linked genes in HMG20A ablated astrocytes while enhancing cell surface area, GFAP intensity and STAT3 expression in healthy astrocytes, mimicking the effect of HMG20A. Furthermore, ORY1001 treatment protected against obesity-associated glucose intolerance in mice correlating with a regression of hypothalamic HMG20A expression, indicative of reactive astrogliosis attenuation with improved health status. Conclusion: HMG20A coordinates the astrocyte polarization state. Under physiological pressure such as obesity and insulin resistance that induces low grade inflammation, HMG20A expression is increased to induce reactive astrogliosis in an attempt to preserve the neuronal network and re-establish glucose homeostasis. Nonetheless, a chronic metabesity state or functional mutations will result in lower levels of HMG20A, failure to promote reactive astrogliosis and increase susceptibility of neurons to stress-induced apoptosis. Such effects could be reversed by ORY1001 treatment both in vitro and in vivo, paving the way for a new therapeutic approach for Type 2 Diabetes Mellitus.

IOX1 activity as sepsis therapy and an antibiotic against multidrug-resistant bacteria.

Sepsis is caused by organ dysfunction initiated by an unrestrained host immune response to infection. The emergence of antibiotic-resistant bacteria has rapidly increased in the last decades and has stimulated a firm research platform to combat infections caused by antibiotic-resistant bacteria that cannot be eradicated with conventional antibiotics. Strategies like epigenetic regulators such as lysine demethylase (Kdm) has received attention as a new target. Thus, we sought to investigate the epigenetic mechanisms in sepsis pathophysiology with the aim of discovering new concepts for treatment. A transcriptome analysis of dendritic cells during their inflammatory state identified Kdm as a critical molecule in sepsis regulation. Next, 8-hydroxyquinoline-5-carboxylic acid (IOX1) ability to control endotoxemia induced by Lipopolysaccharide and bacterial sepsis was demonstrated. IOX1 has been shown to regulate endotoxemia and sepsis caused by Escherichia coli and carbapenem-resistant Acinetobacter baumannii and has also contributed to the suppression of multidrug-resistant bacterial growth through the inhibition of DNA Gyrase. These findings show that IOX1 could be a component agent against bacterial sepsis by functioning as a broad-spectrum antibiotic with dual effects.

Target separation and antitumor metastasis activity of sesquiterpene-based lysine-specific demethylase 1 inhibitors from zedoary turmeric oil.

Lysine-specific histone demethylase 1 (LSD1) was the first histone demethylase identified in epigenetics and has recently emerged as an attractive therapeutic target for treating tumors. To date, almost all reported LSD1 inhibitors have been chemosynthesized; however, natural products possess pharmacological and biological activity and can be sources for drug development. Here, we established a target separation countercurrent chromatography technique to isolate LSD1 inhibitors from zedoary turmeric oil. Four sesquiterpene-based LSD1 inhibitors were efficiently obtained with an inhibition ratio equal to or less than that of the positive control drug. Compound 2 showed the most potent inhibitory activity, with a half-maximal inhibitory concentration of 3.97 µM, and was further tested to determine its ability to inhibit LSD1 and its antitumor metastatic effects in MDA-MB-231 cells. These four compounds are the first sesquiterpene-based natural LSD1 inhibitors to be characterized. Our findings provide a new molecular framework for studying LSD1 inhibitors and offer a template for designing more sesquiterpene-based LSD1 inhibitors with potential antitumor activity.

Raloxifene, identified as a novel LSD1 inhibitor, suppresses the migration of renal cell carcinoma.

Aim: As an important epigenetic modulator, histone lysine-specific demethylase 1 (LSD1) has been proved to be associated with the progression of renal cell carcinoma (RCC). Discovering novel LSD1 inhibitors offers therapeutic potential for RCC treatment. Methods & Results: We identified raloxifene as a novel LSD1 inhibitor (IC50 = 2.08 µM) through small compound library screening. Molecular docking indicated raloxifene might bind LSD1 in the flavin adenine dinucleotide (FAD) binding cavity in a reversible manner. Cell viability and migration assays showed raloxifene could suppress the proliferation and migration of RCC cells bearing overexpressed LSD1. Conclusion: Our findings indicated that LSD1 might be a promising therapeutic target for RCC and that raloxifene could serve as a lead compound for further anti-RCC metastasis drug discovery.

Capsaicin: A "hot" KDM1A/LSD1 inhibitor from peppers.

Capsaicinoids are plant secondary metabolites, and capsaicin is the main principal that responsible to the pungency of chili peppers, with widely application as food additive. In our study, capsaicin was characterized as lysine specific demethylase 1A (KDM1A/LSD1) inhibitor with IC50 of 0.6 ± 0.0421 µM in biochemical level, and can bind KDM1A recombinant directly and reversibly. Further cellular study confirmed that capsaicin can bind and inhibit KDM1A in gastric cancer cell line BGC-823 and further inhibit cell invasion and migration by reversing epithelial-mesenchymal transition (EMT). In sum, our findings identified KDM1A as a target of capsaicin and reveals capsaicin as a modifier of histone methylation for the first time, which may provide a new skeleton for further optimization of KDM1A inhibitor.

Precise separation of lysine-specific demethylase 1 inhibitors from Corydalis yanhusuo using multi-mode counter-current chromatography guided by virtual screening.

It is significant to precisely isolate potential active compounds from medicinal herbs containing multiple compounds. Herein, a new strategy for precise separation of lysine-specific demethylase 1 (LSD1) inhibitors from the rhizome of Corydalis yanhusuo (RCY) using counter-current chromatography (CCC) guided by molecular docking and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis was established. First, representative alkaloids from RCY were docked with LSD1 for screening active skeleton compounds. Simultaneously, the crude extract of RCY was preliminarily separated via pH-zone refining CCC. Subsequently, guided by LC-MS/MS analysis of the fragmentation pathways, three potential active fractions were obtained, followed by further online-storage and recycling CCC separation. Finally, three high-purity target quaternary alkaloids compound 3 (dehydrocorydaline), 7 (coptisine), and 8 (columbamine) were successfully isolated as a new class of potential natural LSD1 inhibitors by only one CCC instrument with multiple modes. Compound 3, with the highest LSD1 inhibition ratio of 2.44 µM, was tested for its ability to inhibit tumor invasion and metastasis in U2OS cells. Therefore, the CCC separation guided by virtual screening is a promising method for the targeted isolation of enzyme inhibitors from medicinal herbs.

Selective lysine-specific demethylase 1 inhibitor, NCL1, could cause testicular toxicity via the regulation of apoptosis.

BACKGROUND: Recent studies have shown that epigenetic alterations, such as those involving lysine-specific demethylase 1 (LSD1), lead to oncogenic activation and highlight such alterations as therapeutic targets. However, studies evaluating the effect of LSD1 inhibitors on male fertility are lacking. OBJECTIVES: We analyzed the potential toxicity of a new selective LSD1 inhibitor, N-[(1S)-3-[3-(trans-2-aminocyclopropyl)phenoxy]-1-(benzylcarbamoyl)propyl] benzamide (NCL1), in testes. MATERIALS AND METHODS: Human testicular samples were immunohistochemically analyzed. Six-week-old male C57BL/6J mice were injected intraperitoneally with dimethyl sulfoxide vehicle (n = 15), or 1.0 (n = 15) or 3.0 (n = 15) mg/kg NCL1 biweekly. After five weeks, toxicity and gene expression were analyzed in testicular samples by ingenuity pathway analysis (IPA) using RNA sequence data and quantitative reverse transcriptase (qRT)-PCR; hormonal damage was analyzed in blood samples. NCL1 treated GC-1, TM3, and TM4 cell lines were analyzed by cell viability, chromatin immunoprecipitation, flow cytometry, and Western blot assays. RESULTS: LSD1 was mainly expressed in human Sertoli and germ cells, with LSD1 levels significantly decreased in a progressive meiosis-dependent manner; germ cells showed similar expression patterns in normal spermatogenesis and early/late maturation arrest. Histological examination revealed significantly increased levels of abnormal seminiferous tubules in 3.0 mg/kg NCL1-treated mice compared to control, with increased cellular detachment, sloughing, vacuolization, eosinophilic changes, and TUNEL-positive cells. IPA and qRT-PCR revealed NCL1 treatment down-regulated LSD1 activity. NCL1 also reduced total serum testosterone levels. Western blots of mouse testicular samples revealed NCL1 induced a marked elevation in cleaved caspases 3, 7, and 8, and connexin 43 proteins. NCL1 treatment significantly reduced GC-1, but not TM3 and TM4, cell viability in a dose-dependent manner. In flow cytometry analysis, NCL1 induced apoptosis in GC-1 cells. CONCLUSIONS: High-dose NCL1 treatment targeting LSD1 caused dysfunctional spermatogenesis and induced caspase-dependent apoptosis. This suggests the LSD1 inhibitor may cause testicular toxicity via the regulation of apoptosis.

Natural protoberberine alkaloids, identified as potent selective LSD1 inhibitors, induce AML cell differentiation.

Natural protoberberine alkaloids were first identified and characterized as potent, selective and cellular active lysine specific demethylase 1 (LSD1) inhibitors. Due to our study, isoquinoline-based tetracyclic scaffold was identified as the key structural element for their anti-LSD1 activity, subtle changes of substituents attached to the core structure led to dramatic changes of the activity. Among these protoberberine alkaloids, epiberberine potently inhibited LSD1 (IC50 = 0.14 ± 0.01 µM) and was highly selective to LSD1 over MAO-A/B. Furthermore, epiberberine could induce the expression of CD86, CD11b and CD14 in THP-1 and HL-60 cells, confirming its cellular activity of inducing acute myeloid leukemia (AML) cells differentiation. Moreover, epiberberine prolonged the survival of THP-1 cells bearing mice and inhibited the growth of AML cells in vivo without obvious global toxicity. These findings give the potential application of epiberberine in AML treatment, and the isoquinoline-based tetracyclic scaffold could be used for further development of LSD1 inhibitors.

Inhibition of lysine-specific histone demethylase 1A results in meiotic aberration during oocyte maturation in vitro in goats.

Histone methylation is associated with oocyte maturation in several species and is also expected in goat oocytes, while the mechanism is still unclear. Therefore, single-cell RNA sequencing (scRNA-seq) was performed on goat germinal vesicle (GV) and metaphase II (MII) oocytes, and the functions of lysine-specific histone demethylase 1A (LSD1), one of the differentially expressed genes (DEGs) were investigated during in vitro maturation (IVM) of goat oocytes. Through scRNA-seq, 4516 DEGs were identified from GV oocytes and MII oocytes in goats, among which there were 16 histone methyltransferase and demethylase DEGs (including LSD1). The functions of LSD1 during IVM of goat oocytes were investigated through its inhibitor, GSK-LSD1. We found that the first polar body extrusion rate of goat oocytes significantly reduced with an increase in GSK-LSD1 concentration supplemented into IVM medium (0 µM: 58.84 ± 0.95%; 2.5 µM: 52.14 ± 0.51%, P < 0.01; 50 µM: 41.22 ± 0.42%, P < 0.001; 100 µM: 29.78 ± 1.78%, P < 0.001). Moreover, compared with the control group, the level of H3K4me2 methylation and p-H2AX in goat oocytes significantly increased (P < 0.001 and P < 0.01, respectively) upon 50-µM GSK-LSD1 treatment for 12 h. Furthermore, abnormalities in spindle assembly (25.94 ± 1.02% vs. 71.15 ± 3.32%; P < 0.01) and chromosome alignment (22.93 ± 1.11% vs. 76.03 ± 3.25%; P < 0.01) were observed, and cytoskeletal organization (15.31 ± 1.60% vs. 67.50 ± 3.09%; P < 0.001) was disrupted upon treatment with 50-µM GSK-LSD1 for 12 h, which compared with that in the control group. Additionally, the ratio of BCL2:BAX significantly higher (P < 0.01) in oocytes with 50-µM GSK-LSD1 treatment than that in control group. Collectively, these results indicate the important role of LSD1 in meiotic maturation of goat oocytes. Our data not only clarify dynamic changes in mRNA during oocyte maturation but also provide a theoretical basis and technical means for further studies of meiotic maturation of goat oocytes.

Design, synthesis and biological evaluation of curcumin analogues as novel LSD1 inhibitors.

Histone lysine-specific demethylase 1 (LSD1) was the first discovered histone demethylase. Inactivating LSD1 or downregulating its expression inhibits cancer-cell development, and thus, it is an attractive molecular target for the development of novel cancer therapeutics. In this study, we worked on the structural optimization of natural products and identified 30 novel LSD1 inhibitors. Utilizing a structure-based drug design strategy, we designed and synthesized a series of curcumin analogues that were shown to be potent LSD1 inhibitors in the enzyme assay. Compound WB07 displayed the most potent LSD1 inhibitory activity, with an IC50 value of 0.8 µM. Moreover, WA20 showed an anticlonogenic effect on A549 cells with an IC50 value of 4.4 µM. Molecular docking simulations were also carried out, and the results indicated that the inhibitors bound to the protein active site located around the key residues of Asp555 and Asp556. These findings suggested that compounds WA20 and WB07 are the first curcumin analogue-based LSD1 inhibitors with remarkable A549 suppressive activity, providing a novel scaffold for the development of LSD1 inhibitors.

Deferiprone: Pan-selective Histone Lysine Demethylase Inhibition Activity and Structure Activity Relationship Study.

Deferiprone (DFP) is a hydroxypyridinone-derived iron chelator currently in clinical use for iron chelation therapy. DFP has also been known to elicit antiproliferative activities, yet the mechanism of this effect has remained elusive. We herein report that DFP chelates the Fe2+ ion at the active sites of selected iron-dependent histone lysine demethylases (KDMs), resulting in pan inhibition of a subfamily of KDMs. Specifically, DFP inhibits the demethylase activities of six KDMs - 2A, 2B, 5C, 6A, 7A and 7B - with low micromolar IC50s while considerably less active or inactive against eleven KDMs - 1A, 3A, 3B, 4A-E, 5A, 5B and 6B. The KDM that is most sensitive to DFP, KDM6A, has an IC50 that is between 7- and 70-fold lower than the iron binding equivalence concentrations at which DFP inhibits ribonucleotide reductase (RNR) activities and/or reduces the labile intracellular zinc ion pool. In breast cancer cell lines, DFP potently inhibits the demethylation of H3K4me3 and H3K27me3, two chromatin posttranslational marks that are subject to removal by several KDM subfamilies which are inhibited by DFP in cell-free assay. These data strongly suggest that DFP derives its anti-proliferative activity largely from the inhibition of a sub-set of KDMs. The docked poses adopted by DFP at the KDM active sites enabled identification of new DFP-based KDM inhibitors which are more cytotoxic to cancer cell lines. We also found that a cohort of these agents inhibited HP1-mediated gene silencing and one lead compound potently inhibited breast tumor growth in murine xenograft models. Overall, this study identified a new chemical scaffold capable of inhibiting KDM enzymes, globally changing histone modification profiles, and with specific anti-tumor activities.

Inhibition of histone lysine-specific demethylase 1 elicits breast tumor immunity and enhances antitumor efficacy of immune checkpoint blockade.

Immunotherapy strategies have been emerging as powerful weapons against cancer. Early clinical trials reveal that overall response to immunotherapy is low in breast cancer patients, suggesting that effective strategies to overcome resistance to immunotherapy are urgently needed. In this study, we investigated whether epigenetic reprograming by modulating histone methylation could enhance effector T lymphocyte trafficking and improve therapeutic efficacy of immune checkpoint blockade in breast cancer with focus on triple-negative breast cancer (TNBC) subtype. In silico analysis of The Cancer Genome Atlas (TCGA) data shows that expression of histone lysine-specific demethylase 1 (LSD1) is inversely associated with the levels of cytotoxic T cell-attracting chemokines (C-C motif chemokine ligand 5 (CCL5), C-X-C motif chemokine ligand 9 and 10 (CXCL9, CXCL10)) and programmed death-ligand 1 (PD-L1) in clinical TNBC specimens. Tiling chromatin immunoprecipitation study showed that re-expression of chemokines by LSD1 inhibition is associated with increased H3K4me2 levels at proximal promoter regions. Rescue experiments using concurrent treatment with small interfering RNA or inhibitor of chemokine receptors blocked LSD1 inhibitor-enhanced CD8+ T cell migration, indicating a critical role of key T cell chemokines in LSD1-mediated CD8+ lymphocyte trafficking to the tumor microenvironment. In mice bearing TNBC xenograft tumors, anti-PD-1 antibody alone failed to elicit obvious therapeutic effect. However, combining LSD1 inhibitors with PD-1 antibody significantly suppressed tumor growth and pulmonary metastasis, which was associated with reduced Ki-67 level and augmented CD8+ T cell infiltration in xenograft tumors. Overall, these results suggest that LSD1 inhibition may be an effective adjuvant treatment with immunotherapy as a novel management strategy for poorly immunogenic breast tumors.

Oral administration of the LSD1 inhibitor ORY-3001 increases fetal hemoglobin in sickle cell mice and baboons.

Increased levels of fetal hemoglobin (HbF) lessen the severity of symptoms and increase the life span of patients with sickle cell disease (SCD). More effective strategies to increase HbF are needed because the current standard of care, hydroxyurea, is not effective in a significant proportion of patients. Treatment of the millions of patients projected worldwide would best be accomplished with an orally administered drug therapy that increased HbF. LSD1 is a component of corepressor complexes that repress γ-globin gene expression and are a therapeutic target for HbF reactivation. We have shown that subcutaneous administration of RN-1, a pharmacological LSD1 inhibitor, increased γ-globin expression in SCD mice and baboons, which are widely acknowledged as the best animal model in which to test the activity of HbF-inducing drugs. The objective of this investigation was to test the effect of oral administration of a new LSD1 inhibitor, ORY-3001. Oral administration of ORY-3001 to SCD mice (n = 3 groups) increased γ-globin expression, Fetal Hemoglobin (HbF)-containing (F) cells, and F reticulocytes (retics). In normal baboons (n = 7 experiments) treated with ORY-3001, increased F retics, γ-globin chain synthesis, and γ-globin mRNA were observed. Experiments in anemic baboons (n = 2) showed that ORY-3001 increased F retics (PA8695, predose = 24%, postdose = 66.8%; PA8698: predose = 13%, postdose = 93.6%), γ-globin chain synthesis (PA8695: predose = 0.07 γ/γ+ß, postdose = 0.20 γ/γ+ß; PA8698: predose = 0.02 γ/γ+ß, postdose = 0.44 γ/γ+ß), and γ-globin mRNA (PA8695: predose = 0.06 γ/γ+ß, postdose = 0.18 γ/γ+ß; PA8698: predose = 0.03 γ/γ+ß, postdose = 0.33 γ/γ+ß). We conclude that oral administration of ORY-3001 increases F retics, γ-globin chain synthesis, and γ-globin mRNA in baboons and SCD mice, supporting further efforts toward the development of this drug for SCD therapy.

Bioactivity-guided cut countercurrent chromatography for isolation of lysine-specific demethylase 1 inhibitors from Scutellaria baicalensis Georgi.

Countercurrent chromatography (CCC) has gradually become a widely used method for preparative separation of bioactive natural molecules. These molecules generally contain distinct scaffolds and characteristics that cannot be readily isolated from plants. While one-dimensional CCC is typically used for the initial purification with insufficiently resolved peaks after locating bioactive components, two-dimensional (2D) or multi-dimensional CCC strategies are employed to improve the resolution of peaks. However, these methods usually present certain disadvantages, such as complicated procedures and increased time consumption, experimental costs, and equipment requirements. Here, a bioactivity-guided cut CCC strategy was established to isolate lysine-specific demethylase 1 (LSD1) inhibitors from Scutellaria baicalensis Georgi. Gradient-elution CCC coupled with real-time detection of LSD1 inhibition by the collected fractions was developed. Next, an online-storage recycling CCC mode was designed to enable the active fractions to be stored in coils, and these active fractions were further separated to obtain pure compounds by using sequential recycling elution. In this strategy, active fractions are first identified, and then pure LSD1 inhibitors are isolated in the 2D CCC mode through continuous separation on a single instrument. By using our bioactivity-guided cut CCC strategy, we successfully isolated six natural LSD1 inhibitors from S. baicalensis Georgi, five of which were identified for the first time as natural LSD1 inhibitors.

An Overview on Screening Methods for Lysine Specific Demethylase 1 (LSD1) Inhibitors.

BACKGROUND: In the past few years, great of attention has been paid to the identification and characterization of selective and potent inhibitors of the first identified histone demethylase LSD1, which may erase mono- and di-methylated histone 3 lysine 4 and 9. As the aberrant overexpression of LSD1 is involved in various pathological processes, especially cancer, obtaining selective and potent LSD1 inhibitors has emerged as a crucial issue in medicinal chemistry research. METHOD: Until now, several LSD1 inhibitor screening models have been established, including enzyme coupled assay, LC-MS based assay, and FRET based assay. Nevertheless, due to some special instrument requirement and additional costs of LC-MS and FRET, the enzyme coupled assay is the most widely applied method for LSD1 inhibitor screening. RESULT: We summarized and compared several reported in vitro LSD1 inhibitor screening models. Each of them has distinct advantages and disadvantages, and none of these methods is perfect. In order to exclude the false positive results, at least one additional method should be applied to screen LSD1 inhibitors.

Inhibition of KDM4A activity as a strategy to suppress interleukin-6 production and attenuate colitis induction.

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 α (TNF-α) and found the suppressive action of Atractylodin on IL-6 production. Under TNF-α stimulation, Atractylodin induced the tri-methylation of histone H3 at lysine residue 9, which impaired the binding between NF-κB 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.

Epigenetic therapies by targeting aberrant histone methylome in AML: molecular mechanisms, current preclinical and clinical development.

While the current epigenetic drug development is still largely restricted to target DNA methylome, emerging evidence indicates that histone methylome is indeed another major epigenetic determinant for gene expression and frequently deregulated in acute myeloid leukaemia (AML). The recent advances in dissecting the molecular regulation and targeting histone methylome in AML together with the success in developing lead compounds specific to key histone methylation-modifying enzymes have revealed new opportunities for effective leukaemia treatment. In this article, we will review the emerging functions of histone methyltransferases and histone demethylases in AML, especially MLL-rearranged leukaemia. We will also examine recent preclinical and clinical studies that show significant promises of targeting these histone methylation-modifying enzymes for AML treatment.