Non-nucleoside DNA Methyltransferase Research Articles

Nucleoside and Non-Nucleoside DNA Methyltransferase 1 Inhibitors in Epigenetic and Combination Therapies in Cancer: A Scoping Review

Nucleoside and Non-Nucleoside DNA Methyltransferase 1 Inhibitors in Epigenetic and Combination Therapies in Cancer: A Scoping Review

The methylome of Biomphalaria glabrata and other mollusks: enduring modification of epigenetic landscape and phenotypic traits by a new DNA methylation inhibitor

Background5-Methylcytosine (5mC) is an important epigenetic mark in eukaryotes. Little information about its role exists for invertebrates. To investigate the contribution of 5mC to phenotypic variation in invertebrates, alteration of methylation patterns needs to be produced. Here, we apply new non-nucleoside DNA methyltransferase inhibitors (DNMTi) to introduce aleatory changes into the methylome of mollusk species.ResultsFlavanone inhibitor Flv1 was efficient in reducing 5mC in the freshwater snails Biomphalaria glabrata and Physa acuta, and to a lesser degree, probably due to lower stability in sea water, in the oyster Crassostrea gigas. Flv1 has no toxic effects and significantly decreased the 5mC level in the treated B. glabrata and in its offspring. Drug treatment triggers significant variation in the shell height in both generations. A reduced representation bisulfite-sequencing method called epiGBS corroborates hypomethylation effect of Flv1 in both B. glabrata generations and identifies seven Differential Methylated Regions (DMR) out of 32 found both in Flv1-exposed snails and its progeny, from which 5 were hypomethylated, demonstrating a multigenerational effect. By targeted bisulfite sequencing, we confirmed hypomethylation in a locus and show that it is associated with reduced gene expression.ConclusionsFlv1 is a new and efficient DNMTi that can be used to induce transient and heritable modifications of the epigenetic landscape and phenotypic traits in mollusks, a phylum of the invertebrates in which epigenetics is understudied.

Maintaining barrier function of infected gingival epithelial cells by inhibition of DNA methylation.

Infection and inflammation induce epigenetic changes that alter gene expression. In periodontal disease, inflammation, and microbial dysbiosis occur, which can lead to compromised barrier function of the gingival epithelia. Here, we tested the hypotheses that infection of cultured human gingival epithelial (HGEp) cells with Porphyromonas gingivalis disrupts barrier function by inducing epigenetic alterations and that these effects can be blocked by inhibitors of DNA methylation. Primary HGEp cells were infected with P. gingivalis either in the presence or absence of the non-nucleoside DNA methyltransferase (DNMT) inhibitors RG108, (-) epigallocatechin-3-gallate (EGCG), or curcumin. Barrier function was assessed as transepithelial electrical resistance (TEER). DNA methylation and mRNA abundance were quantified for genes encoding components of three cell-cell junction complexes, CDH1, PKP2, and TJP1. Cell morphology and the abundance of cell-cell junction proteins were evaluated by confocal microscopy. Compared to non-infected cells, P. gingivalis infection decreased TEER (P<0.0001) of HGEp cells; increased methylation of the CDH1, PKP2, and TJP1 (P<0.0001); and reduced their expression (mRNA abundance) (P<0.005). Pretreatment with DNMT inhibitors prevented these infection-induced changes in HGEp cells, as well as the altered morphology associated with infection. Pathogenic infection induced changes in DNA methylation and impaired the barrier function of cultured primary gingival epithelial cells, which suggests a mechanism for systemic consequences of periodontal disease. Inhibition of these events by non-nucleoside DNMT inhibitors represents a potential strategy to treat periodontal disease.

DNA Methylation May be Involved in the Analgesic Effect of Hyperbaric Oxygen via Regulating FUNDC1.

Background Neuropathic pain (NP) is a type of chronic pain which lacks predictable, effective, and safe therapeutic options. We investigated the role of hyperbaric oxygen (HBO) in expression of FUN14 domain-containing 1 (FUNDC1), which is associated with DNA methylation. Methods We randomly divided rats into four groups: sham operation (S), S + HBO, chronic constriction injury (CCI), and CCI + HBO. Lumbar (L)4 and L5 dorsal root ganglia (DRGs) were used to assess expression of DNA methyltransferase (DNMT)1, DNMT3a, and DNMT3b by western blotting and RT-PCR. Pain-related behaviors were evaluated using mechanical withdrawal threshold and thermal withdrawal latency analysis. Western blotting was also used to assess expression of FUNDC1, BCL2, and adenovirus E1B19 kDa-interacting protein 3-like (NIX) and BCL2 and adenovirus E1B19 kDa-interacting protein3 (BNIP3). And we also examined the changes of FUNDC1 with immunofluorescence. Nonnucleoside DNA methyltransferase inhibitor RG108 was administered prior to CCI. The pain-related behavior and western blotting changes were examined in all groups. Results DNMT3a expression was higher on day 14 after CCI. HBO downregulated DNMT3a mRNA and protein expression, but not those of DNMT1 and DNMT3b. HBO increased pain-related behavior significantly, while it was down-regulated by RG108. In HBO groups, FUNDC1, NIX, and BNIP3 expression was upregulated more significantly than in the CCI group. In addition, FUNDC1 protein colocalized with NeuN and rarely with glutamine synthetase. However, expression was reduced when RG108 was administered. Immunofluorescence showed that FUNDC1 was upregulated after HBO treatment. Conclusion Our findings suggest that DNA methylation is involved in the analgesic effect of HBO via the regulation of FUNDC1.

Synthesis of novel 3-halo-3-nitroflavanones and their activities as DNA methyltransferase inhibitors in cancer cells

The implication of DNA methylation in cancer is today clearly established. Despite that nucleoside analogues are currently used for leukaemia treatment, their low stability in physiological conditions and their lack of selectivity arise the need for the identification of non-nucleoside DNA methyltransferase inhibitors. Here, we describe the synthesis and pharmacological characterisation of a novel class of DNA methyltransferase inhibitors: the 3-halo-3-nitroflavanones. We showed that 3-bromo-3-nitroflavanones 3b and 4a have a micromolar DNMT inhibition and an increased potency in a cell reporter model. Interestingly they are significantly more stable than the reference compounds and induce a low cytotoxicity, supporting them as new candidates for the development of non-cytotoxic cell-reprogramming epi-drugs for anticancer treatment.

Optimizing treatment of DNA methyltransferase inhibitor RG108 on porcine fibroblasts for somatic cell nuclear transfer.

Aberration in DNA methylation is believed to be one of the major causes of abnormal gene expression and inefficiency of somatic cell nuclear transfer (SCNT). RG108, a non-nucleoside DNA methyltransferase (DNMT) inhibitor, has been reported to facilitate somatic nuclear reprogramming and improved blastocyst formation. The aim of this study was to investigate interaction effect of RG108 treatment time (24-72hr) and concentrations (0.05-50µM) on donor cells, and further to optimize the treatment for porcine SCNT. Our results showed that RG108 treatment resulted in time-dependent decrease of genome-wide DNA methylation on foetal fibroblasts, which only happened after 72-hr treatment in our experiments, and no interaction effect between treatment time and concentration. Remarkable decrease of methylation in imprinted gene H19 and increased apoptosis was observed in 5 and 50µM RG108-treated cells. Furthermore, the blastocyst rates of SCNT embryos were increased as the fibroblasts treated with RG108 at 5 and 50µM, and additional treatment during cultivation of SCNT embryos would not provide any advantage for blastocyst formation. In conclusion, the RG108 treatment of 72hr and 5μM would be optimized time and concentration for porcine foetal fibroblasts to improve the SCNT embryonic development. In addition, combined treatment of RG108 on donor cells and SCNT embryos would not be beneficial for embryonic development.

In-silico investigations into natural products as nonnucleoside DNA methyltransferase 1 inhibitors for treating epi-mutation in gastric cancer

Purpose: To explore in silico methods to search for the best reported non-nucleoside DNA methyltransferase 1 (DNMT1) inhibitor of epimutation in gastric cancer.Methods: A dataset of reported non-nucleoside DNMT1 inhibitors was used to target the active site of crystallized DNMT1 protein. Molecular docking simulations were carried out using AutoDock 4.2.6 l. The results were analyzed using Discovery studio visualizer.Results: In silico analysis of known natural non-nucleoside DNMT1 inhibitors gave genistein as the top ranked compound with ΔG of -6.39 Kcal/mol. Further, the results indicated that epigallocatechin gallate and curcumin are poor non-nucleoside DNMT1 inhibitors, as the in silico data suggest that they failed to bind to the catalytic site of DNMT1.Conclusion: The results indicate that genistein is the top rated compound for DNMT1 inhibition. Previous in vitro and in vivo work by other researchers seem to validate the findings of the study.Keywords: Epi-mutation, DNA methyltransferase, Non-nucleoside, DNMT1 inhibitor, Docking

Non-Nucleoside DNA Methyltransferase Inhibition Attenuates Pressure Overload Induced Cardiac Hypertrophy in Rats

Non-Nucleoside DNA Methyltransferase Inhibition Attenuates Pressure Overload Induced Cardiac Hypertrophy in Rats

Structural optimization of non-nucleoside DNA methyltransferase inhibitor as anti-cancer agent.

Inhibition of DNA methyltransferase 1 (DNMT1) can reverse the malignant behavior of cancer cells by restoring expression of aberrantly silenced genes that are required for differentiation, senescence, and apoptosis. Clinically used DNMT1 inhibitors decitabine and azacitidine inhibit their target by covalent trapping after incorporation into DNA as azacytidine analogs. These nucleoside compounds are prone to rapid enzymatic inactivation in blood, posing challenges to the development of purely epigenetic dosing schedules. Non-nucleoside compounds that suppress expression or function of DNMT1 may overcome this problem. Using a high-throughput PCR-based site specific chromatin condensation assay, we identified a compound that reactivated Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A) in myeloma cells and suppressed expression of DNMT1 from a library of 5120 chemically diverse small molecules. Lead optimization was performed to generate 26 new analogs with lung cancer proliferation and DNMT1 expression as activity readout. Two of the new derivatives showed 2 fold improvement of growth inhibiting potency and also decreased DNMT1 protein levels in lung cancer cells.

Abstract 2946: Effects of two novel quinoline-based non-nucleoside DNA methyltransferase inhibitors against bone sarcomas

Abstract Introduction. Identification of new drugs against sarcomas still represents an urgent clinical need due to the rarity of these diseases. Aberrant DNA methylation of gene promoter regions is recognized to be highly involved in cancer and efforts have been made to synthesize non-nucleoside compounds that can effectively modulate gene expression, with bearable side effects. Two novel DNMT inhibitors (DNMTi), MC3353 and MC3343, were tested in Ewing sarcoma (EWS) and osteosarcoma (OS) cell lines to determine their effects on proliferation, apoptosis/necrosis induction, and cell differentiation. Methods and Results. We evaluated specificity on DNMTs inhibition of these compounds together with anticancer activities (cell growth inhibition, cell cycle perturbations, effects on programmed cell death) in a panel of EWS and OS cell lines. Cellular response to DNMTi treatment was assessed also at the molecular level by evaluation of key regulators of cell cycle and apoptosis. Effects on neural differentiation were assessed by β-III tubulin and heavy neurofilament modulation in Ewing sarcoma cells. Concurrently, effects on osteoblastic differentiation were carried out on Saos-2 OS cell line and evaluated by RT-PCR. Both inhibitors determined a significant inhibition of DNMTs activity. In vitro, MC3343 was found to slow cell proliferation by increasing the percentage of cells in G1 or G2/M phases, while MC3353 compound was unable to modulate cell cycle but induced an increase in cell death indicating a cytotoxic rather than a cytostatic effect. Modulation of key regulators of cell cycle and evaluation of PARP cleavage confirmed these results. Both DNMTi induced cell differentiation. In EWS, treatment significantly modulated the expression of neural markers (positivity to β-III tubulin; NF-H; neurite outgrowth). In OS cells, DNMTi increased both matrix mineralization and expression of genes specifically related to osteoblastogenesis. Conclusions. Non-nucleoside DNMTi may represent a possible new therapeutic approach to bone sarcoma. Grants from: Association for Cancer Research (IG2013_14049; to KS); 5 per mille contributions to Rizzoli Institute. FIRB RBFR10ZJQT, IIT-Sapienza Project, and FP7 Projects BLUEPRINT/282510 and A-ParaDDise/602080 to AM. CC is a recipient of a fellowship from the Associazione Onlus “il Pensatore: Matteo Amitrano” Citation Format: Maria Cristina Manara, Sergio Valente, Camilla Cristalli, Cristina Baricordi, Clemens Zwergel, Paola B Arimondo, Piero Picci, Antonello Mai, Katia Scotlandi. Effects of two novel quinoline-based non-nucleoside DNA methyltransferase inhibitors against bone sarcomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2946. doi:10.1158/1538-7445.AM2015-2946

Differential contributions of de novo and maintenance DNA methyltransferases to object memory processing in the rat hippocampus and perirhinal cortex – a double dissociation

Epigenetic mechanisms are increasingly acknowledged as major players in memory formation. Specifically, DNA methylation is necessary for the formation of long-term memory in various brain regions, including the hippocampus (HPC); however, its role in the perirhinal cortex (PRh), a structure critical for object memory, has not been characterized. Moreover, the mnemonic effects of selective DNA methyltransferase (DNMT) inhibition have not yet been investigated systematically, despite distinct roles for de novo (DNMT3a, 3b) and maintenance (DNMT1) methyltransferases. Consequently, we assessed the effects of various DNMT inhibitors within the HPC and PRh of rats using the object-in-place paradigm, which requires both brain regions. The non-nucleoside DNA methyltransferase inhibitor RG-108 impaired long-term object-in-place memory in both regions. Furthermore, intracranial administration of Accell short-interference RNA sequences to inhibit the expression of individual DNMTs implicated DNMT3a and DNMT1 in the HPC and PRh effects, respectively. mRNA expression analyses revealed a complementary pattern of results, as only de novo DNMT3a and DNMT3b mRNA was upregulated in the HPC (dentate gyrus) following object-in-place learning, whereas DNMT1 mRNA was selectively upregulated in the PRh. These results reinforce the established functional double dissociation between the HPC and PRh and imply the operation of different epigenetic mechanisms in brain regions dedicated to long-term memory processing for different types of information.

Identifying novel selective non-nucleoside DNA methyltransferase 1 inhibitors through docking-based virtual screening.

The DNA methyltransferases (DNMTs) found in mammals include DNMT1, DNMT3A, and DNMT3B and are attractive targets in cancer chemotherapy. DNMT1 was the first among the DNMTs to be characterized, and it is responsible for maintaining DNA methylation patterns. A number of DNMT inhibitors have been reported, but most of them are nucleoside analogs that can lead to toxic side effects and lack specificity. By combining docking-based virtual screening with biochemical analyses, we identified a novel compound, DC_05. DC_05 is a non-nucleoside DNMT1 inhibitor with low micromolar IC50 values and significant selectivity toward other AdoMet-dependent protein methyltransferases. Through a process of similarity-based analog searching, compounds DC_501 and DC_517 were found to be more potent than DC_05. These three potent compounds significantly inhibited cancer cell proliferation. The structure-activity relationship (SAR) and binding modes of these inhibitors were also analyzed to assist in the future development of more potent and more specific DNMT1 inhibitors.

Anti-neoplastic properties of hydralazine in prostate cancer.

Prostate cancer (PCa) is a major cause of cancer-related morbidity and mortality worldwide. Although early disease is often efficiently managed therapeutically, available options for advanced disease are mostly ineffective. Aberrant DNA methylation associated with gene-silencing of cancer-related genes is a common feature of PCa. Therefore, DNA methylation inhibitors might constitute an attractive alternative therapy. Herein, we evaluated the anti-cancer properties of hydralazine, a non-nucleoside DNA methyltransferases (DNMT) inhibitor, in PCa cell lines. In vitro assays showed that hydralazine exposure led to a significant dose and time dependent growth inhibition, increased apoptotic rate and decreased invasiveness. Furthermore, it also induced cell cycle arrest and DNA damage. These phenotypic effects were particularly prominent in DU145 cells. Following hydralazine exposure, decreased levels of DNMT1, DNMT3a and DNMT3b mRNA and DNMT1 protein were depicted. Moreover, a significant decrease in GSTP1, BCL2 and CCND2 promoter methylation levels, with concomitant transcript re-expression, was also observed. Interestingly, hydralazine restored androgen receptor expression, with upregulation of its target p21 in DU145 cell line. Protein array analysis suggested that blockage of EGF receptor signaling pathway is likely to be the main mechanism of hydralazine action in DU145 cells. Our data demonstrate that hydralazine attenuated the malignant phenotype of PCa cells, and might constitute a useful therapeutic tool.

Design, synthesis, inhibitory activity, and binding mode study of novel DNA methyltransferase 1 inhibitors

To identify novel non-nucleoside DNA methyltransferase (DNMT) inhibitors, we designed and synthesized a series of maleimide derivatives. Among this series, compounds 5– 8 were found to be more potent DNMT1 inhibitors than RG108, a DNMT1 inhibitor reported previously by Siedlecki et al. The binding mode analysis of compound 5 is also reported.