An electrochemical approach for detection of DNA methylation and assay of the methyltransferase activity

This work develops an electrochemical approach for rapid detection of the genomic DNA methylation level, assay of methyltransferase activity, and evaluation and screening of the inhibitors of methyltransferase. This method may be a help for the discovery of anticancer drugs.     

Determinants of cofactor binding to DNA methyltransferases: insights from a systematic series of structural variants of S-adenosylhomocysteine

S-Adenosylmethionine (AdoMet) is a commonly used cofactor, second only to ATP in the variety of reactions in which it participates. It is the methyl donor in the majority of methyl transfer reactions, including methylation of DNA, RNA, proteins and small molecules. Almost all structurally characterised methyltransferases share a conserved AdoMet-dependent methyltransferase fold, in which AdoMet is bound in the same orientation. Although potential interactions between the cofactor and methyltransferases have been inferred from crystal structures, there has not been a systematic study of the contributions of each functional group to binding. To explore the binding interaction we synthesised a series of seven analogues of the methyltransferase inhibitor S-adenosylhomocysteine (AdoHcy), each containing a single modification, and tested them for the ability to inhibit methylation by HhaI and HaeIII DNA methyltransferase. Comparison of the Ki values highlights the structural determinants for cofactor binding, and indicates which nucleoside and amino acid functional groups contribute significantly to AdoMet binding. An understanding of the binding of AdoHyc to methyltransferases will greatly assist the design of AdoMet inhibitors.     

脱氧核糖核酸甲基化分析方法在肿瘤诊断和治疗中的应用

脱氧核糖核酸( DNA)甲基化是表观遗传改变的主要作用方式,在基因表达调控、基因组印迹、胚胎发育、维持正常细胞功能等过程中起着极其重要的作用;异常甲基化可以导致肿瘤的发生、发展.因此,探讨甲基化形成与改变的可能机制,建立准确性好、灵敏度高、操作简单的DNA甲基化分析方法,可为某些肿瘤的早期诊断提供重要依据.本文综述了D...     

Chemoproteomics‐Enabled Discovery of a Potent and Selective Inhibitor of the DNA Repair Protein MGMT

We present a novel chemical scaffold for cysteine‐reactive covalent inhibitors. Chloromethyl triazoles (CMTs) are readily accessed in only two chemical steps, thus enabling the rapid optimization of the pharmacological properties of these inhibitors. We demonstrate the tunability of the CMTs towards a specific biological target by synthesizing AA‐CW236 as the first potent non‐pseudosubstrate inhibitor of the O⁶‐alkylguanine DNA methyltransferase (MGMT), a protein of major clinical significance for the treatment of several severe cancer forms. Using quantitative proteomics profiling techniques, we show that AA‐CW236 exhibits a high degree of selectivity towards MGMT. Finally, we validate the effectiveness of our MGMT inhibitor in combination with the DNA alkylating drug temozolomide in breast and colon cancer cells by fluorescence imaging and a cell‐viability assay. Our results may open a new avenue towards the development of a clinically approved MGMT inhibitor.     

Electrochemical strategy for sensing DNA methylation and DNA methyltransferase activity

The present work demonstrates a novel signal-off electrochemical method for the determination of DNA methylation and the assay of methyltransferase activity using the electroactive complex [Ru(NH₃)₆]³⁺ (RuHex) as a signal transducer. The assay exploits the electrostatic interactions between RuHex and DNA strands. Thiolated single strand DNA1 was firstly self-assembled on a gold electrode via Au–S bonding, followed by hybridization with single strand DNA2 to form double strand DNA containing specific recognition sequence of DNA adenine methylation MTase and methylation-responsive restriction endonuclease Dpn I. The double strand DNA may adsorb lots of electrochemical species ([Ru(NH₃)₆]³⁺) via the electrostatic interaction, thus resulting in a high electrochemical signal. In the presence of DNA adenine methylation methyltransferase and S-adenosyl-l-methionine, the formed double strand DNA was methylated by DNA adenine methylation methyltransferase, then the double strand DNA can be cleaved by methylation-responsive restriction endonuclease Dpn I, leading to the dissociation of a large amount of signaling probes from the electrode. As a result, the adsorption amount of RuHex reduced, resulting in a decrease in electrochemical signal. Thus, a sensitive electrochemical method for detection of DNA methylation is proposed. The proposed method yielded a linear response to concentration of Dam MTase ranging from 0.25 to 10 U mL⁻¹ with a detection limit of 0.18 U mL⁻¹ (S/N = 3), which might promise this method as a good candidate for monitoring DNA methylation in the future.     

A microchip electrophoretic assay for DNA methyltransferase activity based on methylation‐sensitive endonuclease DpnⅡ

DNA methylation is a significant epigenetic modification and the methods for the detection of DNA methyltransferase (MTase) activity are important due to aberrant methylation closely related to the occurrence of cancer. In this study, a simple and rapid microchip electrophoresis (ME) coupled with LED‐induced fluorescence (LEDIF) method was presented for the detection of Dam MTase activity. This strategy was based on methylation‐sensitive endonuclease DpnⅡ which could recognize the same specific site 5′‐GATC‐3′ with Dam MTase in double‐stranded DNA (dsDNA). The adenines in the specific site could be methylated by Dam MTase, then the special site could not be digested by DpnⅡ. Both methylated dsDNA and unmethylated dsDNA could be analyzed by ME‐LEDIF after stained by SYBR gold. The results showed the fluorescence intensities of methylated dsDNA were directly proportional to Dam MTase activities in the range of 0.5–20 U/mL with a detection limit of 0.12 U/mL. Furthermore, the method could successfully be applied to evaluation experiments of Dam MTase inhibitors. The results confirmed the ME‐LEDIF method is a promising approach for inhibitors screening of DNA MTase and development of anticancer drugs     

Turn-on DNA damage sensors for the direct detection of 8-oxoguanine and photoproducts in native DNA

The integrity of the genetic information in all living organisms is constantly threatened by a variety of endogenous and environmental insults. To counter this risk, the DNA-damage response is employed for repairing lesions and maintaining genomic integrity. However, an aberrant DNA-damage response can potentially lead to genetic instability and mutagenesis, carcinogenesis, or cell death. To directly monitor DNA damage events in the context of native DNA, we have designed two new sensors utilizing genetically fragmented firefly luciferase (split luciferase). The sensors are comprised of a methyl-CpG binding domain (MBD) attached to one fragment of split luciferase for localizing the sensor to DNA (50-80% of the CpG dinucleotide sites in the genome are symmetrically methylated at cytosines), while a damage-recognition domain is attached to the complementary fragment of luciferase to probe adjacent nucleotides for lesions. Specifically, we utilized oxoguanine glycosylase 1 (OGG1) to detect 8-oxoguanine caused by exposure to reactive oxygen species and employed the damaged-DNA binding protein 2 (DDB2) for detection of pyrimidine dimer photoproducts induced by UVC light. These two sensors were optimized and validated using oligonucleotides, plasmids, and mammalian genomic DNA, as well as HeLa cells that were systematically exposed to a variety of environmental insults, demonstrating that this methodology utilizing MBD-directed DNA localization provides a simple, sensitive, and potentially general approach for the rapid profiling of specific chemical modifications associated with DNA damage and repair.     

Synthesis and Biological Activity of a Cytostatic Inhibitor of MLLr Leukemia Targeting the DOT1L Protein

Histone methyltransferase DOT1L catalyzes mono-, di- and trimethylation of histone 3 at lysine residue 79 (H3K79) and hypermethylation of H3K79 has been linked to the development of acute leukemias characterized by the MLL (mixed-lineage leukemia) rearrangements (MLLr cells). The inhibition of H3K79 methylation inhibits MLLr cells proliferation, and an inhibitor specific for DOT1L, pinometostat, was in clinical trials (Phase Ib/II). However, the compound showed poor pharmacological properties. Thus, there is a need to find new potent inhibitors of DOT1L for the treatment of rearranged leukemias. Here we present the design, synthesis, and biological evaluation of a small molecule that inhibits in the nM level the enzymatic activity of hDOT1L, H3K79 methylation in MLLr cells with comparable potency to pinometostat, associated with improved metabolic stability and a characteristic cytostatic effect.     

IS DNA TOPOISOMERASE INVOLVED IN THE UV EXCISION REPAIR PROCESS? NEW EVIDENCE FROM STUDIES WITH DNA INTERCALATING AND NON-INTERCALATING ANTITUMOR AGENTS

Abstract— The effects of selected DNA intercalating and non-intercalating drugs on the UV excision repair process in human fibroblasts have been examined. 9-Amino acridine, acridine orange, quinacrine, doxorubicin (adriamycin), ethidium bromide and actinomycin-D all inhibited the removal of pyrimidine dimers from cellular DNA by inhibiting the incision process as monitored by the nick translation assay and by an endonuclease-sensitive site assay. These agents also partially inhibited incision by the M. luteus endonuclease in an in vitro system. This is the only class of compounds tested to date that appears to block this early step of repair in mammalian cells. The DNA topoisomerase inhibitors, m -amsacrine and VP-16 (etoposide) and the bacterial gyrase inhibitors nalidixic acid and oxolinic acid were shown not to inhibit UV repair. As shown previously, however, novobiocin does block dimer removal and we show here that it is a potent inhibitor of the M. luteus UV endonuclease. While it has recently been demonstrated that many DNA intercalating agents block the strand-passing activity of DNA topoisomerase II giving rise to protein associated DNA strand breaks, the finding that the specific inhibitors of topoisomerase, m -AMSA and VP-16, do not inhibit repair, even though they block this strand passing activity, strongly suggests that inhibition of DNA topoisomerase is not associated with inhibition of DNA repair.     

The Bioluminescence Resonance Energy Transfer from Firefly Luciferase to a Synthetic Dye and its Application for the Rapid Homogeneous Immunoassay of Progesterone

The sensitive BRET system for the homogeneous immunoassay of a low‐molecular weight antigen was developed using progesterone as an example. Two thermostable mutants of the Luciola mingrelica firefly luciferase (Luc)—the “red” mutant with λ_max.em = 590 nm (RedLuc) and the “green” mutant with λ_max.em = 550 nm (GreenLuc)—were tested as the donors. The water‐soluble Alexa Fluor 610× (AF) dye was selected as the acceptor because its two absorption maxima, located at 550 and 610 nm, are close to the bioluminescence maxima of the GreenLuc and RedLuc, respectively. The methods for the synthesis of the luciferase–progesterone (Luc–Pg) conjugate and the conjugate of the dye and the polyclonal antiprogesterone antibody (AF–Ab) were developed. Both conjugates retained their functional properties, had high antigen–antibody binding activity, and demonstrated a high BRET signal. The homogeneous immunoassay system based on the BRET from the firefly luciferase to the synthetic dye was established to assay progesterone as a model antigen. Optimization of the assay conditions, the composition of the reaction mixture, and the concentrations of the donor and the acceptor made it possible to reach the minimum detectable progesterone concentration of 0.5 ng mL^?1.     

A Molecular Tool Targeting the Base-Flipping Activity of Human UHRF1

During DNA replication, ubiquitin-like, containing PHD and RING fingers domains 1 (UHRF1) plays key roles in the inheritance of methylation patterns to daughter strands by recognizing through its SET and RING-associated domain (SRA) the methylated CpGs and recruiting DNA methyltransferase 1 (DNMT1). Herein, our goal is to identify UHRF1 inhibitors targeting the 5′-methylcytosine (5mC) binding pocket of the SRA domain to prevent the recognition and flipping of 5mC and determine the molecular and cellular consequences of this inhibition. For this, we used a multidisciplinary strategy combining virtual screening and molecular modeling with biophysical assays in solution and cells. We identified an anthraquinone compound able to bind to the 5mC binding pocket and inhibit the base-flipping process in the low micromolar range. We also showed in cells that this hit impaired the UHRF1/DNMT1 interaction and decreased the overall methylation of DNA, highlighting the critical role of base flipping for DNMT1 recruitment and providing the first proof of concept of the druggability of the 5mC binding pocket. The selected anthraquinone appears thus as a key tool to investigate the role of UHRF1 in the inheritance of methylation patterns, as well as a starting point for hit-to-lead optimizations.     

Rapid,single-step nucleic acid detection

A rapid detection method for nucleic acid based on bioluminescence resonance energy transfer (BRET) from the luminescence donor Renilla luciferase to an acceptor quantum dot upon oligonucleotide probe hybridization has been developed. Utilizing a competitive assay, we detected the target nucleic acid by correlating the BRET signal with the amount of target present in the sample. This method allows for the detection of as little as 4 pmol (20 nM) of nucleic acid in a single-step, homogeneous format both in vitro in a buffer matrix as well as in a cellular matrix. Using this method, one may perform nucleic acid detection in as little as 30 min, showing much improvement over time-consuming blotting methods and solid-phase methods which require multiple wash steps to remove unbound probe. This is the first report on the use of quantum dots as a BRET acceptor in the development of a nucleic acid hybridization assay. An erratum to this article can be found at     

Coupling hybridization chain reaction with DNAzyme recycling for enzyme-free and dual amplified sensitive fluorescent detection of methyltransferase activity

Aberrant DNA methylation originated from changes in DNA methyltransferase activity can lead to many genetic diseases and tumor types, and the monitoring of methyltransferase activity is thus of great importance in disease diagnosis and drug screening. In this work, by combing hybridization chain reaction (HCR) and metal ion-dependent DNAzyme recycling, we have developed a convenient enzyme-free signal amplification strategy for highly sensitive detection of DNA adenine methyltransferase (Dam MTase) activity and its inhibitors. The Dam MTase-induced methylation and subsequent cleavage of the methylated hairpin DNA probes by DpnI endonuclease lead to the release of ssDNA triggers for HCR formation of many Mg²⁺-dependent DNAzymes, in which the fluorescently quenched substrate sequences are catalytically and cyclically cleaved by Mg²⁺ to generate remarkably amplified fluorescent signals for highly sensitive detection of Dam MTase at 7.23 × 10⁻⁴ U/mL. In addition, the inhibition of different drugs to Dam MTase activity can also be evaluated with the developed method. With the advantages of simplicity and significant signal amplification over other common methods, the demonstrated biosensing approach thus offers great potential for highly sensitive detection of various methyltransferases and provides a convenient platform for drug screening for therapeutic applications.     

Firefly Luciferase‐based Fusion Proteins and their Applications in Bioanalysis

Firefly luciferase is widely used in molecular biology and bioanalytical systems as a reporter molecule due to the high quantum yield of the bioluminescence, availability of stable mutant forms of the enzyme with prescribed spectral characteristics and abundance of bacterial expression systems suitable for production of recombinant proteins in limitless quantities. In this review, we described fusion proteins of luciferase with biotin‐binding domain and streptavidin, with proteins A and G, antibodies, with DNA‐ and RNA‐binding proteins, as well as fusion proteins designed for BRET systems. The firefly luciferase‐based fusion proteins are represented as an effective tool for the development of different bioanalytical systems such as (1) systems in which luciferase is attached to the surface of the target and the bioluminescence signal is detected from the specific complexes formed; (2) BRET‐based systems, in which the specific interaction induces changes in the bioluminescence spectrum; and (3) systems that use modified or split luciferases, in which the luciferase activity changes under the action of the analyte. All these systems have wide application in biochemical analysis of physiologically important compounds, for the detection of pathogenic bacteria and viruses, for evaluation of protein–protein interactions, assaying of metabolites involved in cell communication and cell signaling.     

Combined inhibition of HDAC and DNMT1 induces p85α/MEKmediated cell cycle arrest by dual target inhibitor 208 in U937 cells

Herein we reported an efficient dual DNMT and HDAC inhibitor 208 with great antiproliferative activity against U937 cells. Further studies revealed 208 affected the whole proteome profile and could induce G1 cell cycle arrest and apoptosis in U937 cells through upregulating CDK inhibitor p16 and downregulating cyclin-dependent kinases and their activators.     

Homology modeling,docking and structure-based pharmacophore of inhibitors of DNA methyltransferase

DNA methyltransferase 1 (DNMT1) is an emerging epigenetic target for the treatment of cancer and other diseases. To date, several inhibitors from different structural classes have been published. In this work, we report a comprehensive molecular modeling study of 14 established DNTM1 inhibitors with a herein developed homology model of the catalytic domain of human DNTM1. The geometry of the homology model was in agreement with the proposed mechanism of DNA methylation. Docking results revealed that all inhibitors studied in this work have hydrogen bond interactions with a glutamic acid and arginine residues that play a central role in the mechanism of cytosine DNA methylation. The binding models of compounds such as curcumin and parthenolide suggest that these natural products are covalent blockers of the catalytic site. A pharmacophore model was also developed for all DNMT1 inhibitors considered in this work using the most favorable binding conformations and energetic terms of the docked poses. To the best of our knowledge, this is the first pharmacophore model proposed for compounds with inhibitory activity of DNMT1. The results presented in this work represent a conceptual advance for understanding the protein–ligand interactions and mechanism of action of DNMT1 inhibitors. The insights obtained in this work can be used for the structure-based design and virtual screening for novel inhibitors targeting DNMT1.     

Notl-Msell methylation-sensitive amplied fragment length polymorhism for DNA methylation analysis of human cancers

We have applied a methylation-sensitive restriction endonuclease, NotI, to the existing amplified fragment length polymorphism (AFLP) method and developed NotI-MseI methylation-sensitive-AFLP (MS-AFLP). NotI-MseI MS-AFLP allows the analysis of DNA methylation alterations at the NotI sites scattered over the genome. Hypermethylation and hypomethylation are visualized by the decrease and increase in the band intensity of DNA fingerprints. Identification of consistent changes can be facilitated through parallel electrophoresis of multiple samples. DNA fragments exhibiting alterations can be cloned from fingerprint bands by amplification of gel-eluted DNA with the same pair of primers used for radioactive fingerprint presentation. Fluorescent NotI-MseI MS-AFLP offers a safer method of studying the alterations in DNA methylation, and may be applied to the hybridization of DNA microarrays in the future. Using NotI-MseI MS-AFLP, we observed frequent hypomethylation of a satellite DNA repeat sequence in a majority of breast tumors.     

From Natural Products to Drugs for Epimutation Computer-Aided Drug Design

The epimutational event, i.e., ectopic methylation in tumor suppressor genes, can lead to gene silencing, thus promoting prognosis of cancer. The progression of DNA methylation is a cycle of demethylation, de novo methylation, and maintenance methylation. The enzyme responsible for maintenance of methylation status is DNA methyltransferase 1 (DNMT1), the continuous activity of which is required to maintain the pattern of epimutation; thus, its inhibition is a promising strategy for the treatment of cancer. To the best of our knowledge, this study is the first to focus on the recently developed crystal structure of the catalytic site of DNMT1. Here in this study, we have used the crystal structure for the development of non-nucleoside DNMT1 inhibitors using virtual screening (VS), absorption, distribution, metabolism, elimination/toxicology analysis, and molecular docking studies. In this study, VS was carried out on 48,531 natural products to create a subset of lead-like natural products. Three of them were found to form hydrogen bonds with the catalytic site of the DNMT1 (Cys 1226). Thus, this study adumbrates potential lead compounds for treatment of epimutation.