Mammalian cytosine DNA methyltransferase Dnmt1: enzymatic mechanism, novel mechanism-based inhibitors, and RNA-directed DNA methylation

This is a review of the enzymatic mechanism of DNA methyltransferase Dnmt1 and analysis of its implications on regulation of DNA methylation in mammalian cells and design of novel mechanism-based inhibitors. The methylation reaction by Dnmt1 has different phases that depend on DNA substrate and allosteric regulation. Consequently, depending on the phase, the differences in catalytic rates between unmethylated and pre-methylated DNA can vary between 30-40 fold, 3-6 fold or only 1 fold. The allosteric site and the active site can bind different molecules. Allosteric activity depends on DNA sequence, methylation pattern and DNA structure (single stranded vs. double stranded). Dnmt1 binds poly(ADP-ribose) and some RNA molecules. The results on kinetic preferences, allosteric activity and binding preference of Dnmt1 are combined together in one comprehensive model mechanism that can address regulation of DNA methylation in cells; namely, inhibition of DNA methylation by poly(ADP-ribose), RNA-directed DNA methylation by methylated and unmethylated non-coding RNA molecules, and transient interactions between Dnmt1 and genomic DNA. Analysis of reaction intermediates showed that equilibrium between base-flipping and base-restacking events can be the key mechanism in control of enzymatic activity. The two events have equal but opposite effect on accumulation of early reaction intermediates and methylation rates. The accumulation of early reaction intermediates can be exploited to improve the current inhibitors of Dnmt1 and achieve inhibition without toxic modifications in genomic DNA. [1,2-dihydropyrimidin-2-one]-5-methylene-(methylsulfonium)-adenosyl is described as the lead compound.     

The interaction of histone deacetylase inhibitors and DNA methyltransferase inhibitors in the treatment of human cancer cells

The potential anticancer activities of histone deacetylase (HDAC) inhibitors and DNA methyltransferase (DNMT) inhibitors have been extensively studied in recent years. HDAC inhibitors suppress the activities of multiple HDACs, leading to an increase in histone acetylation. This histone acetylation induces an enhancement of the expression of specific genes that elicit extensive cellular morphologic and metabolic changes, such as growth arrest, differentiation and apoptosis. DNMT inhibitors, such as 5-aza-cytidine (5-aza-CR) and 5-aza-2'-deoxycytidine (5-aza-CdR) are also widely studied because DNA hypomethylation induces the re-activation of tumor suppressor genes that are silenced by methylation-mediated mechanisms. Recently, the combination of HDAC inhibitors or demethylating agents with other chemo-therapeutics has gained increasing interest as a possible molecularly targeted therapeutic strategy. In particular, the combination of HDAC inhibitors with demethylating agents has become attractive since histones are connected to DNA by both physical and functional interactions. To date, the accumulating evidence has confirmed the hypothesis that the combination of HDAC and DNMT inhibition is very effective (and synergistic) in inducing apoptosis, differentiation and/or cell growth arrest in human lung, breast, thoracic, leukemia and colon cancer cell lines. This review will discuss the in vitro effects of HDAC inhibitors, such as trichostatin A (TSA), sodium butyrate, depsipeptide (FR901228, FK228), valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA), and the demethylating agent, 5-aza-CdR used alone and in combination treatment of human cancer cells and the possible mechanisms involved.     

Effects of arabinosyl cytosine and 5-azacytidine on the intestinal absorption of nutrients

The purpose of this study was to investigate the effects of two pyrimidine antimetabolites, arabinosyl cytosine (ara-C) and 5-azacytidine (5-aza-C), on the intestinal transport of glucose, amino acids, and electrolytes. Male Swiss-Webster mice were given either 50 mg/kg ara-C or 15 mg/kg 5-aza-C in isotonic saline, ip, once daily for 5 successive days. Using the everted sac technique there was a marked decrease in d-glucose, 3-O-methyl-d-glucose (3MG), and l-tyrosine transport. The rates of glucose, 3MG, and l-tyrosine transport were decreased by 67, 74, and 60%, respectively, in ara-C-treated animals, and 58, 79, and 62%, respectively, in 5-aza-C-treated animals. When intestinal sacs from untreated animals were exposed to ara-C or 5-aza-C (10^?3m) on both mucosal and serosal sides, there was no effect on transport. Furthermore, in animals treated with either ara-C or 5-aza-C there was a significant decrease in transmucosal potential difference and short circuit current associated with decreases in net Na⁺, Cl^?, and HCO₃^? transport across the intestinal mucosa. Simultaneous administration of deoxycytidine, in a two- to threefold molar excess, prevented the impairment of intestinal transport function caused by ara-C; similarly, cytidine prevented the impairment caused by 5-aza-C. The results suggest that active metabolites of ara-C and 5-aza-C are involved in their inhibitory effects on intestinal absorption and that this may be a contributing factor to the malnutrition and diarrhea commonly associated with the administration of these anticancer drugs.     

DNA (cytosine-5) methyltransferase inhibitors: a potential therapeutic agent for schizophrenia

In this issue of Molecular Pharmacology, Kundakovic et al. (p. 644) present compelling evidence suggesting that the promoters for reelin and GAD67 are coordinately regulated. The regulation occurs at the level of DNA (cytosine-5) methylation. Moreover, the authors present evidence suggesting that pharmacologic inhibition of DNA methyltransferase results in reversal of methylation, loss of methyl-DNA binding proteins and relief of repression. Repression of both reelin and GAD67 has been implicated in the pathogenesis of schizophrenia. Therefore, these results suggest that the reelin and GAD67 promoters are subject to continuous repression by DNA methyltransferase and that inhibitors of DNA methyltransferase represent a potential treatment for Schizophrenia.     

光谱法研究阿德福韦与DNA的相互作用

目的研究阿德福韦与小牛胸腺DNA的结合作用方式。方法采用紫外分光光度法和荧光光谱法进行研究。结果证实了阿德福韦是通过嵌插方式和静电结合方式与DNA发生了相互作用。结论阿德福韦与DNA的相互作用为静态猝灭过程,其猝灭速率常数（Kq）=2.07×1011L.mol-1.S-1,结合常数（K）=3.41×103L.mol-1,结合位点数（n）=1.05。     

Electrochemical study on the behavior of Morin and its interaction with DNA

Voltammetric behavior of Morin was studied in 0.1 M HAc–NaAc + 50 mM KCl (pH 3.4) solution at glassy carbon electrode (GCE) using cyclic voltammetry (CV). Morin showed an irreversible anodic peak at 0.720 V in CV which was involving two electrons and two protons. Also, the interaction of Morin with double-stranded calf thymus DNA (ctDNA) was studied by CV at GCE with an irreversible electrochemical equation. As a result of reaction with ctDNA, the voltammetric peak of Morin was a position shift and the peak current decreased. The diffusion coefficients of both free and binding Morin (D_f = 1.1086 × 10⁻⁷ cm² s⁻¹ and D_b = 8.2544 × 10⁻⁹ cm² s⁻¹), binding constant (K = 1.7765 × 10⁷ cm³ mol⁻¹), and binding site size (s = 0.8510) of the Morin–DNA complex were obtained simultaneously by non-linear fit analysis. The results demonstrate that Morin can bind to ctDNA in 0.1 M HAc–NaAc + 50 mM KCl (pH 3.4) solution and the ring B of Morin intercalates between the DNA base pairs.     

Advances in the computational development of DNA methyltransferase inhibitors

DNA methylation is an epigenetic change that results in the addition of a methyl group at the carbon-5 position of cytosine residues. The process is mediated by DNA methyltransferases (DNMTs), a family of enzymes for which inhibition is a promising strategy for the treatment of cancer and other diseases. Here, we review the current status of the computational studies directed to rationalize, at the molecular level, the enzymatic activity of DNMT inhibitors. We also review successful virtual screenings to identify inhibitors with novel scaffolds as well as the emerging efforts to characterize the dynamic behavior of DNMTs. Thus, computational approaches form part of multidisciplinary efforts to further advance epigenetic therapies.     

The synergistic interaction of gemcitabine and cytosine arabinoside with the ribonucleotide reductase inhibitor triapine is schedule dependent

Gemcitabine and ara-C have multiple mechanisms of action: DNA incorporation and for gemcitabine also ribonucleotide reductase (RNR) inhibition. Since dCTP competes with their incorporation into DNA, dCTP depletion can potentiate their cytotoxicity. We investigated whether additional RNR inhibition by Triapine (3-AP), a new potent RNR inhibitor, enhanced cytotoxicity of gemcitabine and ara-C in four non-small-cell-lung-cancer (NSCLC) cell lines, using the multiple-drug-effect analysis. Simultaneous and sequential exposure (preexposure to 3-AP for 24h) in a constant molar ratio of 3-AP and gemcitabine was antagonistic/additive in all cell lines. Preexposure to 3-AP at an IC(25) concentration for 24h before variable concentrations of gemcitabine was synergistic. RNR inhibition by 3-AP resulted in a more synergistic interaction in combination with ara-C, which does not inhibit RNR. Two cell lines with pronounced synergism (SW1573) or antagonism (H460) for gemcitabine/3-AP, were evaluated for accumulation of the active metabolites, dFdCTP and ara-CTP. Simultaneous exposure induced no or a small increase, but ara-CTP increased after pretreatment with 3-AP, 4-fold in SW1573 cells, but not in H460 (<1.5 fold). Ara-C and gemcitabine incorporation into DNA were more pronounced (about 2-fold increased) for sequential treatment in SW1573 compared to H460 cells (<1.5 fold). This was not related to the activity and expression of deoxycytidine kinase and the M2 subunit of RNR. In conclusion, RNR inhibition by 3-AP prior to gemcitabine or ara-C exposure stimulates accumulation of the active metabolites and incorporation into DNA. The combination 3-AP/Ara-C is more synergistic than 3-AP/gemcitabine possibly because gemcitabine already inhibits RNR, but ara-C does not.     

Characteristics of the interaction of lycobetaine with DNA

The characteristics of the interaction of lycobetaine (LBT) with DNA were examined by fluorescence spectrometer, disc electrophoresis and restriction enzyme analysis. The apparent binding constant of LBT with calf thymus DNA has been determined as 1.67 x 10(6) L/mol by ethidium bromide displacement method. Based on electrophoresis titration, the mode of DNA binding was found to be through intercalation. Fluorescence quenching assay showed that the intrinsic association constant and the binding site size of LBT to calf thymus DNA were 0.26 x 10(6) L/mol and 2.6 base pairs, respectively. Selective inhibition of LBT on action of some restriction enzymes showed that LBT intercalate preferentially into GC base pairs. Neither DNA strand break nor interstrand cross-link was produced by LBT. LBT did not bind to DNA covalently and did not cause DNA alkylation.     

Characterization of human polymorphic DNA repair methyltransferase

The O6-methylguanine-DNA methyltransferase (MGMT) is a critical defence against alkylation-induced mutagenesis and carcinogenesis. More than a 20-fold interindividual difference in the MGMT activity is known to exist among human cultured fibroblasts. We previously reported three allelic variants of the human MGMT gene, namely V1, V2, and V3. Both V1 and V2 carry amino acid substitutions, Leu84Phe and Trp65Cys, respectively, while V3 has a silent mutation. In order to reveal the pharmacogenetic and ecogenetic significance of polymorphism in the human MGMT gene, we investigated the in-vivo characteristics of V1 and V2 methyltransferase enzyme. Escherichia coli strain KT233 (ogt-, ada-) and mer- HeLa MR cells carrying a V1 sequence exhibited almost the same level of sensitivity against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), as did those with a wild-type sequence. The level of methyltransferase protein in those cells was essentially the same as for the wild-type and V1 samples. On the other hand, E. coli and human cells expressing V2 cDNA showed a significantly reduced level of survival. In these cells, V2 protein was hardly detected, even though mRNA was produced normally. An in-vitro translation experiment revealed that the V2 sequence had the potential to produce methyltransferase protein, as did the wild-type and V1 sequences. There was also evidence for a small amount of V2 protein being produced but rapidly degraded, thus implying that the V2 molecule is unstable in vivo. Using purified recombinant proteins, we estimated the kinetic values of wild-type and variant form of enzymes, which would support these views. From these results, we concluded that the wild-type and V1 protein have similar enzymatic and physicochemical properties, while V2 protein is considered to be unstable and rare.     

Covalent interaction of 5-nitroimidazoles with DNA and protein in vitro: mechanism of reductive activation

Human hepatic microsomal enzymes catalyzed the NADPH-dependent anaerobic reductive activation of [1-14C]metronidazole [1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole] and [4,5-14C]ronidazole [(1-methyl-5-nitroimidazole-2-yl)methyl carbamate] to species that became covalently bound to proteins. Due to the low efficiency of the enzyme-catalyzed covalent binding of metronidazole, the stoichiometry of anaerobic reductive activation was studied with dithionite as the reductant. Two moles of dithionite was consumed per mole of [1-14C]metronidazole for maximal covalent binding to either DNA or immobilized sulfhydryl groups, demonstrating that four electrons are required for the reductive activation of metronidazole. These data implicate the involvement of a hydroxylamine in covalent binding. Maximal covalent binding of [4,5-14C]ronidazole to DNA also required four-electron reduction, consistent with previous studies of the covalent binding of this agent to immobilized sulfhydryl groups [Kedderis et al. (1988) Arch. Biochem. Biophys. 262, 40-48]. Studies of the covalent binding of variously radiolabeled ronidazole molecules to DNA suggested that the imidazole ring was intact while greater than 80% of the 2-carbamoyl group and the C4 proton were not present in the DNA adduct. Studies of both the chemical and human hepatic microsomal reduction of [4-3H]metronidazole demonstrated that covalent binding occurred with the stoichiometric loss of this label, implicating binding at the C4 position. These results suggest that the reductive activation of 5-nitroimidazoles generally proceeds via four-electron reduction to form hydroxylamines followed by nucleophilic attack at C4.     

克林沙星与小牛胸腺DNA相互作用的光谱研究

目的:研究克林沙星与小牛胸腺DNA之间的相互作用.方法:使用荧光光谱法,根据Stern-Volmer方程及Scatchrd方程进行数据处理.采用离子强度的影响、紫外光谱的变化、单双链DNA作用的区别及I-猝灭等实验研究了克林沙星与小牛胸腺DNA间的相互作用模式.结果:在pH值7.0的磷酸盐缓冲液(PBS)中,克林沙星的荧光激发及发射峰分别位于273 nm和427 nm处,小牛胸腺DNA对克林沙星的荧光存在着强烈的猝灭作用,猝灭常数Kq为4.03×104mol-1·L,结合位点数为2.84.结论:克林沙星与小牛胸腺DNA间是一种沟槽作用模式.     

Effect of varying caloric restriction levels on female rat growth and 5-hydroxymethyl-2'-deoxyuridine in DNA.

Caloric restriction has previously been shown to decrease levels of oxidative stress in rats. In this study, we examined the effects of 5 different caloric intake levels on one type of oxidative DNA damage in rat mammary gland, blood, and liver. Animals were fed modified AIN-93G diets to accommodate 10, 20, 30, or 40% calorie restriction (CR), relative to ad libitum (AL) consumption. The intakes of fat, protein, vitamins, and minerals thus remained constant, but total carbohydrate intake decreased. Body weights of the animals at 20 weeks reflected the degree of restriction, but in the first 10 weeks, weight gain in the 10% CR group was not reduced relative to animals fed ad libitum. Levels of 5-hydroxymethyl-2'-deoxyuridine increased with time in mammary gland and nucleated blood cells regardless of CR level, indicating an effect of animal age, despite the fact that the animals were only 7 months old after the 20-week dietary study. In liver, however, there was a trend towards decreased DNA damage levels with time. The effect of diet on levels of 5-hydroxymethyl-2'-deoxyuridine was not statistically significant, indicating no protective effect of restricted dietary carbohydrate. This dietary study differed from previous work in that the modified AIN-93G dietary formulation contains relatively higher levels of fat and vitamins K, E, and B(12), and it has certain added trace minerals. This data raises the question of whether the previously reported effects of calorie restriction on preventing oxidative stress in mammary gland are dependent on the type of dietary formulation used.     

氧氟沙星的测定及其与DNA相互作用的研究

目的:建立测定氧氟沙星的分析方法,研究氧氟沙星与 DNA 的相互作用,初步探讨氧氟沙星与 DNA 相互作用的机理。方法:在0.20 mol·L~(-1)醋酸-醋酸钠(pH=5.0)中,研究氧氟沙星在碳纳米管糊电极上的电化学行为,建立电化学测定氧氟沙星的分析方法。利用电化学法和荧光法研究氧氟沙星与 DNA 的相互作用。结果:在碳纳米管糊电极上,氧氟沙星的检出限为8.5×10~(-8)mol·L~(-1)。氧氟沙星与 DNA 发生插入作用。结论:建立了灵敏测定氧氟沙星的电化学分析方法,利用电化学方法可研究氧氟沙星与 DNA 的相互作用。     

Activity landscape of DNA methyltransferase inhibitors bridges chemoinformatics with epigenetic drug discovery

Introduction: Activity landscapes are valuable tools for exploring systematically the structure–activity relationships (SAR) of chemical databases. Their application to analyze the SAR of DNA methyltransferase (DNMT) inhibitors, which are attractive compounds as potential epi-drugs or epi-probes, provides useful information to identify pharmacophoric regions and plan the development of predictive models and virtual screening.

Areas covered: This paper highlights different approaches for conducting SAR analysis of datasets with a particular focus on the activity landscape methodology. SAR information of DNMT inhibitors (DNMTi), stored in a public database, is surveyed to further illustrate concepts and generalities of activity landscape modeling with a special emphasis on structure–activity similarity (SAS) maps.

Expert opinion: The increasing SAR information reported for DNMTi opens up avenues to implement activity landscape methods. Despite several activity landscape methods, such as SAS maps, being well established, these need further refinement. For instance, novel combinations of multiple representations, such as the addition of Z-values of similarity (fusion-Z), lead to more robust representations of consensus SAS maps. Density SAS maps improve the visualization of the SAR. A survey of activity cliffs (i.e., pairs of compounds with high structural similarity but high differences in potency) of DNMTi available in a public database suggest that it is feasible to develop predictive models for non-nucleoside DNMTi using approaches such as quantitative structure-activity relationships and that non-nucleoside DNMTi in ChEMBL can be used as query molecules in similarity-based virtual screening.     

Developmental DNA methyltransferase inhibitors in the treatment of gynecologic cancers

Introduction: DNA methylation has become an attractive target for the treatment of cancer. DNA methyltransferase inhibitors have proven useful for the treatment of myelodysplastic syndrome and are being evaluated in gynecological neoplasias.

Areas covered: We provide an overview of the current knowledge on DNA methylation and cancer and the role of DNA methylation in cervical, ovarian and endometrial carcinomas. The results of recent clinical trials with demethylating agents for cervical and ovarian cancer treatment are also discussed.

Expert opinion: There are few studies of DNA demethylating agents for cervical and ovarian cancer treatment; nevertheless, the results are promising. To accelerate these advances, there are at least two actions that can be simultaneously pursued. One is to greatly increase the number of small clinical exploratory trials with existing demethylating drugs and using methylome analyses to identify predictive factors for response and/or toxicity. The second is finding out epigenetic ‘drivers’ unique to gynecological cancers and their subtypes, and then proceed to clinical trials in a highly selected population of patients. It is expected that in the future, DNA demethylation could have a role in the treatment of gynecologic cancers.