A Potent Cell-active Allosteric Inhibitor of Murine DNA Cytosine C5 Methyltransferase

Flynn, James E.; Fang, Jing‐Yuan; Mikovits, Judy A.; Reich, Norbert O.

doi:10.1074/jbc.m209839200

Cited by 32 publications

(65 citation statements)

References 52 publications

(87 reference statements)

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“…The molecular mechanism of the allosteric activation of Dnmt1 is unknown, although the CXXC domain [52] and residues 284-287 of murine Dnmt1 [107] have been implicated in the process. In addition, an inhibitory effect of unmethylated substrates has been demonstrated in several studies, [106,108,109] suggesting that binding of an unmethylated DNA to the N-terminal part of Dnmt1 leads to repression of the enzymatic activity on hemimethylated DNA. The binding site for this substrate inhibition effect might be localised in the first 501 amino acids of Dnmt1.…”

Section: Enzymatic Properties Of Dnmt1mentioning

confidence: 95%

Structure and Function of Mammalian DNA Methyltransferases

2010

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DNA methylation plays an important role in epigenetic signalling, having an impact on gene regulation, chromatin structure, development and disease. Here, we review the structures and functions of the mammalian DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b, including their domain structures, catalytic mechanisms, localisation, regulation, post-translational modifications and interaction with chromatin and other proteins, summarising data obtained in genetic, cell biology and enzymatic studies. We focus on the question of how the molecular and enzymatic properties of these enzymes are connected to the dynamics of DNA methylation patterns and to the roles the enzymes play in the processes of de novo and maintenance DNA methylation. Recent enzymatic and genome-wide methylome data have led to a new model of genomic DNA methylation patterns based on the preservation of average levels of DNA methylation in certain regions, rather than the methylation states of individual CG sites.

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Section: Enzymatic Properties Of Dnmt1mentioning

confidence: 95%

Structure and Function of Mammalian DNA Methyltransferases

2010

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“…In this study, we have done a comparative benchmark analysis of compounds that have been previously reported to inhibit DNA methyltransferase activity in cancer cell lines (see Table 2 for a summary of results). This group of drugs did not include antisense oligonucleotides that function via DNA methyltransferase mRNA degradation (20,35) or compounds that have been shown to inhibit DNA methyltransferases indirectly, either through their chemical reactivity (36) or through the inhibition of signaling pathways associated with DNA methylation (37).…”

Section: Discussionmentioning

confidence: 99%

Functional Diversity of DNA Methyltransferase Inhibitors in Human Cancer Cell Lines

et al. 2006

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DNA methyltransferase inhibitors represent promising new drugs for cancer therapies. The first of these compounds (5-azacytidine, Vidaza) has recently been approved as an antitumor agent, and others are presently in various stages of their preclinical or clinical development. Most of the archetypal inhibitors have been established and characterized in different experimental systems, which has thus far precluded their direct comparison. We have now established defined experimental conditions that allowed a comparative analysis of the six most widely known DNA methyltransferase inhibitors: 5-azacytidine (5-aza-CR), 5-aza-2V-deoxycytidine (5-aza-CdR), zebularine, procaine, (À)-epigallocatechin-3-gallate (EGCG), and RG108. Of these, 5-aza-CR, 5-aza-CdR, zebularine, and EGCG were found to exhibit significant cytotoxicity in human cancer cell lines. 5-aza-CdR and EGCG were also found to be genotoxic, as evidenced by the induction of micronuclei. In addition, 5-aza-CR, 5-aza-CdR, zebularine, and RG108 caused concentration-dependent demethylation of genomic DNA, whereas procaine and EGCG failed to induce significant effects. Finally, the experiments in cancer cell lines were complemented by a cell-free in vitro assay with purified recombinant DNA methyltransferase, which indicated that RG108 is the only drug capable of direct enzyme inhibition. These results show a substantial diversity in the molecular activities of DNA methyltransferase inhibitors and provide valuable insights into the developmental potential of individual drugs. (Cancer Res 2006; 66(5): 2794-800)

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“…DNMT1 is subject to strong allosteric substrate inhibition by regions of unmethylated DNA [54,55]. Interestingly, DNMT1 is also activated by nearby methylated cytosines on the complementary DNA strand [56][57][58][59][60].…”

Section: Dna Methyltransferasementioning

confidence: 99%

The biological significance of substrate inhibition: A mechanism with diverse functions

2010

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Many enzymes are inhibited by their own substrates, leading to velocity curves that rise to a maximum and then descend as the substrate concentration increases. Substrate inhibition is often regarded as a biochemical oddity and experimental annoyance. We show, using several case studies, that substrate inhibition often has important biological functions. In each case we discuss, the biological significance is different. Substrate inhibition of tyrosine hydroxylase results in a steady synthesis of dopamine despite large fluctuations in tyrosine due to meals. Substrate inhibition of acetylcholinesterase enhances the neural signal and allows rapid signal termination. Substrate inhibition of phosphofructokinase ensures that resources are not devoted to manufacturing ATP when it is plentiful. In folate metabolism, substrate inhibition maintains reactions rates in the face of substantial folate deprivation. Substrate inhibition of DNA methyltransferase serves to faithfully copy DNA methylation patterns when cells divide while preventing de novo methylation of methyl-free promoter regions. Keywords:.b iological function; enzyme kinetics; substrate inhibitionThe kinetics of an enzymatic reaction are typically studied by varying the concentration of substrate and plotting the rate of product formation as a function of substrate concentration. In the conventional case this yields a typical hyperbolic Michaelis-Menten curve, and a linear reciprocal LineweaverBurk plot, from which the kinetic constants of the enzyme can be calculated. A surprisingly large number of enzymes do not behave in this conventional way. Instead, their velocity curves rise to a maximum and then decline as the substrate concentration goes up. This phenomenon is referred to as substrate inhibition, and it is estimated that it occurs in some 20% of enzymes [1]. A partial list of enzymes that show substrate inhibition appears in Box 1.Substrate inhibition is often interpreted as an abnormality that comes from using artificially high substrate concentration in a laboratory setting. In a review article on the mechanisms of substrate inhibition in 1994, Kuehl [2] commented that ''although recognized early on as an almost universal phenomenon, it has nevertheless met an almost universal disinterest. Probably the main reason for this neglect is that the majority of enzymologists and many authorities in the field regard substrate inhibition as being almost always a nonphysiological phenomenon.'' There are several reasons for suspecting that substrate inhibition is not a pathological phenomenon, but a biologically relevant regulatory mechanism. First, in many cases normal substrate concentrations are to the right of the velocity maximum, which indicates that these enzymes typically operate under substrate inhibition. Second, many enzymes have specialized sites where a second substrate molecule can bind and act as an allosteric inhibitor. For those enzymes, substrate inhibition is clearly a specially evolved property. Third, evidence is accumulating that substr...

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A Potent Cell-active Allosteric Inhibitor of Murine DNA Cytosine C5 Methyltransferase

Cited by 32 publications

References 52 publications

Structure and Function of Mammalian DNA Methyltransferases

Structure and Function of Mammalian DNA Methyltransferases

Functional Diversity of DNA Methyltransferase Inhibitors in Human Cancer Cell Lines

The biological significance of substrate inhibition: A mechanism with diverse functions

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