MS Binding Assays with UNC0642 as reporter ligand for the MB327 binding site of the nicotinic acetylcholine receptor.

Intoxications with organophosphorus compounds (OPCs) based chemical warfare agents and insecticides may result in a detrimental overstimulation of muscarinic and nicotinic acetylcholine receptors evolving into a cholinergic crisis leading to death due to respiratory failure. In the case of the nicotinic acetylcholine receptor (nAChR), overstimulation leads to a desensitization of the receptor, which cannot be pharmacologically treated so far. Still, compounds interacting with the MB327 binding site of the nAChR like the bispyridinium salt MB327 have been found to re-establish the functional activity of the desensitized receptor. Only recently, a series of quinazoline derivatives with UNC0642 as one of the most prominent representatives has been identified to address the MB327 binding site of the nAChR, as well. In this study, UNC0642 has been utilized as a reporter ligand to establish new Binding Assays for this target. These assays follow the concept of MS Binding Assays for which by assessing the amount of bound reporter ligand by mass spectrometry no radiolabeled material is required. According to the results of the performed MS Binding Assays comprising saturation and competition experiments it can be concluded, that UNC0642 used as a reporter ligand addresses the MB327 binding site of the Torpedo-nAChR. This is further supported by the outcome of ex vivo studies carried out with poisoned rat diaphragm muscles as well as by in silico studies predicting the binding mode of UNC0646, an analog of UNC0642 with the highest binding affinity, in the recently proposed binding site of MB327 (MB327-PAM-1). With UNC0642 addressing the MB327 binding site of the Torpedo-nAChR, this and related quinazoline derivatives represent a promising starting point for the development of novel ligands of the nAChR as antidotes for the treatment of intoxications with organophosphorus compounds. Further, the new MS Binding Assays are a potent alternative to established assays and of particular value, as they do not require the use of radiolabeled material and are based on a commercially available compound as reporter ligand, UNC0642, exhibiting one of the highest binding affinities for the MB327 binding site known so far.

A Review of Advanced Cutaneous Melanoma Therapies and Their Mechanisms, from Immunotherapies to Lysine Histone Methyl Transferase Inhibitors.

Advanced cutaneous melanoma is considered to be the most aggressive type of skin cancer and has variable rates of treatment response. Currently, there are some classes of immunotherapy and target therapies for its treatment. Immunotherapy can inhibit tumor growth and its recurrence by triggering the host's immune system, whereas targeted therapy inhibits specific molecules or signaling pathways. However, melanoma responses to these treatments are highly heterogeneous, and patients can develop resistance. Epigenomics (DNA/histone modifications) contribute to cancer initiation and progression. Epigenetic alterations are divided into four levels of gene expression regulation: DNA methylation, histone modification, chromatin remodeling, and non-coding RNA regulation. Deregulation of lysine methyltransferase enzymes is associated with tumor initiation, invasion, development of metastases, changes in the immune microenvironment, and drug resistance. The study of lysine histone methyltransferase (KMT) and nicotinamide N-methyltransferase (NNMT) inhibitors is important for understanding cancer epigenetic mechanisms and biological processes. In addition to immunotherapy and target therapy, the research and development of KMT and NNMT inhibitors is ongoing. Many studies are exploring the therapeutic implications and possible side effects of these compounds, in addition to their adjuvant potential to the approved current therapies. Importantly, as with any drug development, safety, efficacy, and specificity are crucial considerations when developing methyltransferase inhibitors for clinical applications. Thus, this review article presents the recently available therapies and those in development for advanced cutaneous melanoma therapy.

The histone methyltransferase G9a mediates stress-regulated alcohol drinking.

The epigenetic enzyme G9a is a histone methyltransferase that dimethylates lysine 9 on histone H3 (H3K9me2), and in the adult nucleus accumbens (NAc), G9a regulates multiple behaviors associated with substance use disorder. We show here that chronic intermittent ethanol (CIE) exposure in male mice reduced both G9a and H3K9me2 levels in the adult NAc, but not dorsal striatum. Viral-mediated reduction of G9a in the NAc had no effects on baseline volitional ethanol drinking or escalated alcohol drinking produced by CIE exposure; however, NAc G9a was required for stress-regulated changes in ethanol drinking, including potentiated alcohol drinking produced by activation of the kappa-opioid receptor. In addition, we observed that chronic systemic administration of a G9a inhibitor, UNC0642, also blocked stress-potentiated alcohol drinking. Together, our findings suggest that chronic alcohol use, similar to other abused substances, produces a NAc-selective reduction in G9a levels that serves to limit stress-regulated alcohol drinking. Moreover, our findings suggest that pharmacological inhibition of G9a might provide a novel therapeutic approach to treat stress-induced alcohol drinking, which is a major trigger of relapse in individuals suffering from AUD.

EHMT2 promotes the pathogenesis of hepatocellular carcinoma by epigenetically silencing APC expression.

BACKGROUND: Hepatocellular carcinoma (HCC), the second leading cause of cancer death worldwide, alone accounts for over half (466,100) of new cancer cases and 422,100 deaths based on the average year incidence rates of 2009 to 2011 in China. Due to unclear and complex underlying mechanisms for HCC development, effective therapy for HCC is still unavailable. The Wnt-ß-catenin pathway is a critical contributor of HCC pathogenesis: 40-70% of HCCs from patients harbor the nuclear accumulation of ß-catenin protein. However, the mechanisms for ß-catenin activation are not fully understood. METHODS: The deletion of EHMT2 in Hep3B and Huh1 cells was achieved by transiently transfecting cells with pX459 plasmids, which carry EHMT2 specific small guide RNA (sgRNA) sequences for Cas9 protein. All experiments were performed in triplicate and repeated more than three times. RESULTS: In the present study, we observed that EHMT2 (but not EHMT1) mRNA and protein levels were significantly elevated in HCC compared with normal controls. Next, the results of Ki67 staining, as well as MTT, soft-agar and xenograft assays, in wild-type and EHMT2-/- Hep3B and Huh1 cancer stem cells collectively revealed that the elevation of EHMT2 expression is required for the tumorigenesis of HCC. Meanwhile, we found that elevated EHMT2 expression contributes to the activation of Wnt-ß-catenin signaling: deletion of EHMT2 in Hep3B or Huh1 cells promoted the cytoplasmic localization of ß-catenin and restrained the expression of Wnt-ß-catenin signaling targets such as Myc, CCND1, MMP-7, etc. We demonstrated that EMHT2 directly mediates the H3K9me2 methylation of the APC promoter to epigenetically silence its expression. More intriguingly, our findings also showed that UNC0642, a specific inhibitor of EHMT2, exhibits anti-tumorigenesis effects in HCC both in vitro and in vivo, which were largely abolished by deletion of EHMT2 or overexpression of APC in Hep3B and Huh1 cells. CONCLUSION: Altogether, our observations emphasize that the EHMT2-APC axis is a critical contributor to Wnt-ß-catenin pathway activation in HCC, and UNC0642 may be a potential candidate for target drug treatment of HCC.

RNA-seq and miRNA-seq data from pharmacological inhibition of the G9a/GLP histone methyltransferase complex with UNC0642 in SAMP8 mice.

Growing evidence demonstrates the epigenetic modulation as a key event in Alzheimer's disease (AD) pathology. Furthermore, recent data suggests that the epigenetic regulation by the methyltransferase G9a is a crucial mechanism involved in learning and memory formation. Taking this into account, we hereby provide genomics data from pharmacological intervention with UNC0642, a potent and selective G9a/GLP in SAMP8 mice, a model of Alzheimer's disease (AD). We have generated novel RNA-seq and miRNA-seq data for three groups, healthy SAMR1, SAMP8 control and SAMP8 treated with UNC0642 (5 mg/Kg). Thus, the new data can be used to find miRNA regulation, and the mRNA's modified in AD under G9a/GLP inhibition.

Abnormal overexpression of G9a in melanoma cells promotes cancer progression via upregulation of the Notch1 signaling pathway.

Malignant melanoma is a type of very dangerous skin cancer. Histone modifiers usually become dysregulated during the process of carcinoma development, thus there is potential for a histone modifier inhibitor as a useful drug for cancer therapy. There is a multitude of evidence regarding the role of G9a, a histone methyltransferase (HMTase), in tumorigenesis. In this study, we first showed that G9a was significantly upregulated in melanoma patients. Using the TCGA database, we found a significantly higher expression of G9a in primary melanoma samples (n = 461) compared to normal skin samples (n = 551). Next, we knocked down G9a in human M14 and A375 melanoma cell lines in vitro via small interfering RNA (siRNA). This resulted in a significant decrease in cell viability, migration and invasion, and an increase in cell apoptosis. UNC0642 is a small molecule inhibitor of G9a that demonstrates minimal cell toxicity and good in vivo pharmacokinetic characteristics. We investigated the role of UNC0642 in melanoma cells, and detected its anti-cancer effects in vitro and in vivo. Next, we treated cells with UNC0642, and observed a significant decrease in cell viability in M14 and A375 cell lines. Furthermore, treatment with UNC0642 resulted in increased apoptosis. In immunocompetent mice bearing A375 engrafts, treatment with UNC0642 inhibited tumor growth. Results of Western blot analysis revealed that administration of UNC0642 or silencing of G9a expression by siRNA reduced Notch1 expression significantly and decreased the level of Hes1 in A375. All in all, the data from our study demonstrates potential of G9a as a therapeutic target in the treatment of melanoma.

Inhibition of G9a by a small molecule inhibitor, UNC0642, induces apoptosis of human bladder cancer cells.

Urinary bladder cancer (UBC) is characterized by frequent recurrence and metastasis despite the standard chemotherapy with gemcitabine and cisplatin combination. Histone modifiers are often dysregulated in cancer development, thus they can serve as an excellent drug targets for cancer therapy. Here, we investigated whether G9a, one of the histone H3 methyltransferases, was associated with UBC development. We first analyzed clinical data from public databases and found that G9a was significantly overexpressed in UBC patients. The TCGA Provisional dataset showed that the average expression level of G9a in primary UBC samples (n = 408) was 1.6-fold as much as that in normal bladder samples (n = 19; P < 0.001). Then we used small interfering RNA to knockdown G9a in human UBC T24 and J82 cell lines in vitro, and observed that the cell viability was significantly decreased and cell apoptosis induced. Next, we choosed UNC0642, a small molecule inhibitor targeting G9a, with low cytotoxicity, and excellent in vivo pharmacokinetic properties, to test its anticancer effects against UBC cells in vitro and in vivo. Treatment with UNC0642 dose-dependently decreased the viability of T24, J82, and 5637 cells with the IC50 values of 9.85 ± 0.41, 13.15 ± 1.72, and 9.57 ± 0.37 µM, respectively. Furthermore, treatment with UNC0642 (1-20 µM) dose-dependently decreased the levels of histone H3K9me2, the downstream target of G9a, and increased apoptosis in T24 and J82 cells. In nude mice bearing J82 engrafts, administration of UNC0642 (5 mg/kg, every other day, i.p., for 6 times) exerted significant suppressive effect on tumor growth without loss of mouse body weight. Moreover, administration of UNC0642 significantly reduced Ki67 expression and increased the level of cleaved Caspase 3 and BIM protein in J82 xenografts evidenced by immunohistochemistry and western blot analysis, respectively. Taken together, our data demonstrated that G9a may be a promising therapeutic target for UBC, and an epigenetics-based therapy by UNC0642 is suggested.