Constrained (l-)-S-adenosyl-l-homocysteine (SAH) analogues as DNA methyltransferase inhibitors

Potent SAH analogues with constrained homocysteine units have been designed and synthesized as inhibitors of human DNMT enzymes. The five membered (2S,4S)-4-mercaptopyrrolidine-2-carboxylic acid, in 1a, was a good replacement for homocysteine, while the corresponding six-member counterpart was less active. Further optimization of 1a, changed the selectivity profile of these inhibitors. A Chloro substituent at the 2-position of 1a, compound 1d, retained potency against DNMT1, while N⁶ alkylation, compound 7a, conserved DNMT3b2 activity. The concomitant substitutions of 1a at both 2- and N⁶ positions reduced activity against both enzymes.     

Structure of human DNMT2, an enigmatic DNA methyltransferase homolog that displays denaturant-resistant binding to DNA

DNMT2 is a human protein that displays strong sequence similarities to DNA (cytosine-5)-methyltransferases (m(5)C MTases) of both prokaryotes and eukaryotes. DNMT2 contains all 10 sequence motifs that are conserved among m(5)C MTases, including the consensus S:-adenosyl-L-methionine-binding motifs and the active site ProCys dipeptide. DNMT2 has close homologs in plants, insects and Schizosaccharomyces pombe, but no related sequence can be found in the genomes of Saccharomyces cerevisiae or Caenorhabditis elegans. The crystal structure of a deletion mutant of DNMT2 complexed with S-adenosyl-L-homocysteine (AdoHcy) has been determined at 1.8 A resolution. The structure of the large domain that contains the sequence motifs involved in catalysis is remarkably similar to that of M.HHAI, a confirmed bacterial m(5)C MTase, and the smaller target recognition domains of DNMT2 and M.HHAI are also closely related in overall structure. The small domain of DNMT2 contains three short helices that are not present in M.HHAI. DNMT2 binds AdoHcy in the same conformation as confirmed m(5)C MTases and, while DNMT2 shares all sequence and structural features with m(5)C MTases, it has failed to demonstrate detectable transmethylase activity. We show here that homologs of DNMT2, which are present in some organisms that are not known to methylate their genomes, contain a specific target-recognizing sequence motif including an invariant CysPheThr tripeptide. DNMT2 binds DNA to form a denaturant-resistant complex in vitro. While the biological function of DNMT2 is not yet known, the strong binding to DNA suggests that DNMT2 may mark specific sequences in the genome by binding to DNA through the specific target-recognizing motif.     

Identification and sequencing of the gene encoding DNA methyltransferase 3 (DNMT3) from sea cucumber,Apostichopus japonicus

Molecular Biology Reports - The sea cucumber Apostichopus japonicus is well known as a traditional tonic food and as a commercially important cultured aquatic species. This species produces...     

Recognition of unusual DNA structures by human DNA (cytosine-5)methyltransferase

The symmetry of the responses of the human DNA (cytosine-5)methyltransferase to alternative placements of 5-methylcytosine in model oligodeoxynucleotide duplexes containing unusual structures has been examined. The results of these experiments more clearly define the DNA recognition specificity of the enzyme. A simple three-nucleotide recognition motif within the CG dinucleotide pair can be identified in each enzymatically methylated duplex. The data can be summarized by numbering the four nucleotides in the dinucleotide pair thus: 1 4/2 3. With reference to this numbering scheme, position 1 can be occupied by cytosine or 5-methylcytosine; position 2 can be occupied by guanosine or inosine; position 3, the site of enzymatic methylation, can be occupied only by cytosine; and position 4 can be occupied by guanosine, inosine, O6-methylguanosine, cytosine, adenosine, an abasic site, or the 3' hydroxyl group at the end of a gapped molecule. Replacing the guanosine normally found at position 4 with any of the moieties introduces unusual (non-Watson-Crick) pairing at position 3 and generally enhances methylation of the cytosine at that site. The exceptional facility of the enzyme in actively methylating unusual DNA structures suggests that the evolution of the DNA methyltransferase, and perhaps DNA methylation itself, may be linked to the biological occurrence of unusual DNA structures.     

Molecular dynamics simulations of human DNA methyltransferase 3B with selective inhibitor nanaomycin A

DNA methyltransferases (DNMTs) are involved in epigenetic regulation of the genome and are promising targets for therapeutic intervention in cancer and other diseases. Until now, very limited information is available concerning the molecular dynamics of DNMTs. The natural product nanaomycin A is the first selective inhibitor of DNMT3B that induce genomic demethylation. Herein we report long (>100 ns) molecular dynamics simulations for human DNMT3B bound to nanaomycin A with and without the presence of the cofactor S-adenosyl-l-methionine (SAM). We concluded that SAM favors the binding of nanaomycin A to DNMT3B. Key interactions of nanaomycin A with DNMT3B involve long lasting interactions with Arg731, Arg733, Arg832, and the catalytic Cys651. Results further support the previous hypothesis that nanaomycin A has key interactions with amino acid residues involved in the mechanism of methylation. This work represents one of the first molecular dynamics studies of DNMT3B. Results of this work shed light on the structure and binding recognition process of a key epigenetic enzyme with a small molecule inhibitor.     

3-Aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase, is a stimulator, not an inhibitor, of DNA repair

An inhibitor of poly(ADP-ribose) synthesis, 3-aminobenzamide (3AB), at low concentrations (0.01-0.1 mM) was found to reduce strand-break frequencies and increase repair replication in human lymphoid cells damaged by methyl methanesulfonate. A concentration of 0.1 mM 3AB was adequate to produce a maximum effect on strand-break frequencies and repair replication. This evidence, together with our previous measurements, demonstrates that 3AB cannot be regarded as an inhibitor of DNA repair; rather, it actually accelerates the ligation of DNA repair patches. Previous considerations of 3AB as a repair inhibitor may have derived from the use of excessive concentrations above 1 mM that may have stimulated additional damage and from the use of ethyl alcohol as a solvent for 3AB. Interpretations of the role of single-strand breaks and poly(ADP-ribose) in DNA repair, differentiation, and gene activity may need reevaluation because they have frequently been based on an erroneous notion of 3AB as a repair inhibitor, when its mode of action is, in fact, more complex.     

Effects of prenatal Poly I:C exposure on global histone deacetylase (HDAC) and DNA methyltransferase (DNMT) activity in the mouse brain

The aim of our study was to investigate the brain-specific epigenetic effects on global enzymatic histone deacetylase (HDAC) and DNA methyltransferase (DNMT) activity after prenatal exposure to maternal immune challenge by polyinosinic:polycytidylic acid (Poly I:C) at gestational day (GD) 17 in C57BL/6JRccHsd mouse offspring. Pregnant mice were randomly divided into 2 groups, receiving either 5 mg/kg Poly I:C or phosphate buffered saline (PBS) intravenously at GD 17. Subsequently, the effects on whole brain enzymatic HDAC and DNMT activity and the protein levels of various HDAC isoforms were assessed in the offspring. Overall, a significant sex × treatment interaction effect was observed after prenatal exposure to maternal immune challenge by Poly I:C, indicative of increased global HDAC activity particularly in female offspring from mothers injected with Poly I:C when compared to controls. Results on the levels of specific HDAC isoforms suggested that neither differences in the levels of HDAC1, HDAC2, HDAC3, HDAC4 or HDAC6 could explain the increased global HDAC activity observed in female Poly I:C offspring. In conclusion, we show that Poly I:C administration to pregnant mice alters global brain HDAC, but not DNMT activity in adult offspring, whereas it is still unclear which specific HDAC(s) mediate(s) this effect. These results indicate the necessity for further research on the epigenetic effects of Poly I:C.     

Methylation of slipped duplexes, snapbacks and cruciforms by human DNA(cytosine-5)methyltransferase.

When human DNA(cytosine-5)methyltransferase was used to methylate a series of snapback oligodeoxy-nucleotides of differing stem lengths, each containing a centrally located CG dinucleotide recognition site, the enzyme required a minimum of 22 base pairs in the stem for maximum activity. Extrahelical cytosines in slipped duplexes that were 30 base pairs in length acted as effective methyl acceptors and were more rapidly methylated than cytosines that were Watson-Crick paired. Duplexes containing hairpins of CCG repeats in cruciform structures in which the enzyme recognition sequence was disrupted by a C.C mispair were also more rapidly methylated than control Watson-Crick-paired duplexes. Since enzymes have higher affinities for their transition states than for their substrates, the results with extrahelical and mispaired cytosines suggest that these structures can be viewed as analogs of the transition state intermediates produced during catalysis by methyltransferases.     

DNA methyltransferase inhibitor assay system based on the HBx-induced DNA methylation of E-cadherin

We here report a simple assay system for DNA methyltransferase (DNMT) inhibitors based on the HBx-induced DNA methylation of E-cadherin. A stable cell line named G1 was generated by co-transfecting E-cadherin luciferase reporter and HBx-expression plasmid into HepG2 cells. Treatment of G1 cells with DNMT inhibitors, 5-azacytidine, 5-aza-2′-deoxycytidine, and procainamaid, dose-dependently inhibited DNA methylation of E-cadherin promoter in the reporter, resulting in up-regulation of luciferase levels and its enzyme activity. Treatment with all-trans retinoic acid that is known to inhibit DNMT expression, also induced similar effects. Our system can be useful for development of epi-drugs targeting DNA methylation in malignancies.     

Human uric acid transporter 1 (hURAT1): an inhibitor structure-activity relationship (SAR) study

The current study describes the chemical synthesis of a series of (2-ethylbenzofuran-3-yl)(substituted-phenyl)methanone compounds and their subsequent in vitro testing via oocytes expressing hURAT1. The experimental data support the notion that a potent hURAT1 inhibitor requires an anion (i.e., a formal negative charge) to interact with the positively charged hURAT1 binding pocket. An anion appears to be a primary requirement in order to be a hURAT1 substrate (i.e., urate) or inhibitor. We discuss the inhibitor structure-activity relationship and how electronically donating or withdrawing groups attached to the B-ring can decrease or increase inhibitory potency, respectively.