DNA Methyl Transferases


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Development of inhibitors of DNMTs

Epigenetics is defined as the study of changes that occur on chromosomes without affecting the sequence of DNA and that lead to stable and heritable phenotypes. These changes consist mainly in the installation of epigenetic marks (methyl, acetyl, ubiquityl and sumoyl goups) on the histone tails (lysine and arginine) or on the DNA. These transformations are performed by epigenetic enzymes called writers. The epigenetic marks and then read by proteins called readers to generate a cell response. When the marks are no longer needed, they are removed by enzymes called erasers. These epigenetic processes control the level of compaction of DNA thus enabling (euchromatin) or repressing (heterochromatin) gene transcription. The delicate and complex orchestration of all epigenetic processes permit the regulation of cell processes including cell differentiation.

The methylation of DNA is an epignetic process that leads to the installation of a methyl group CH3 at the C5 position of cytosine on DNA, mainly in regions called CpG islands. This modification is repressive since it leads to the silencing of methylated genes by preventing the binding of transcription factors. The recruitment of MBDs (Methyl Binding Domain proteins) also contributes to the silencing of methylated genes.

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Although it is less dynamic than the methylation of histone tails, the methylation of DNA is nonetheless a reversible process. Enzymes called TET perform the demethylation of DNA by oxidizing the CH3 group into the corresponding aldehyde CHO and then the carboxylic acid CO2H. The balance between methylation and demethylation processes is crucial for the maintenance of cellular functions and plays a particularly important role in cell differentiation.

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DNA methyltransferases DNMTs are enzymes that belong to the class of epigenetic writers. These enzymes catalyze the transfer of a CH3 group from SAM (S-adenosylmethionine) to the C5 position of cytosine to produce C5-Me-cytosine on DNA along with SAH (S-adenosylhomocysteine). DNMT3A and DNMT3B are responsible for de novo methylation by installing the first methyl groups on unmethylated DNA during the development of the embryo. DNMT3L (DNMT3-like) lacks certain catalytic domains and is thus catalytically inactive. However, this protein plays a crucial role by modulating the activity of DNMT3A and DNMT3B. Lastly, DNMT1 shows a greater selectivity towards hemi-methylated DNA and is thus in charge of maintaining the DNA methylation patterns during cell division. The DNA methylation mechanism begins by the attack of the DNMT's cysteine to the C6 position of cytosine to generate an amino-enamine-like structure that attacks the methyl group of SAM through its C5 carbon. The removal of the hydrogen atom in C5 re-establishes the double bond, producing C5-Me-cytosine, and regenerates the catalytic DNMT's cysteine.

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An overexpression of DNMTs along with a dysregulation of DNA methylation patterns has been observed in various types of cancers, leading to a global hypomethylation of DNA and a local hypermethylation and silencing of certain genes such as the tumour supressor genes (TSGs). The inhibition of DNMTs leads to the demethylation and reactivation of epigenetically silenced genes by preventing the maintenance of the methylation patterns, thus demonstrating the value of DNMTs as drug targets in drug discovery.

Vidaza and dacogen are the only commercially available drugs that target DNMTs. These pharmaceutics do not inhibit directly DNMTs. Instead, these compounds are first phosphorylated and then incorporated in the DNA where they trap the DNMTs' catalytic cysteine, leading to the degradation of the enzymes. However, due to their mechanism of action, these drugs show considerable cytotoxicity. In addition, because of their nucleosidic structures, these therapeutic agents possess low half-lives as well as poor pharmacokinetic profiles.

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Few natural products have demonstrated a capacity to inhibit DNMTs and reactivate epigenetically silenced genes including TSGs. However, these compounds often operate through an indirect mode of action or a non specific or unknown mechanism. In addition, they often present chemical features that are undesirable in medicinal chemistry. Therefore, there exist a considerable need for small non-nucleoside DNMT inhibitors that possess adequate biopharmaceutical properties.

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Our group made its entry in the field of DNMTs in 2013 by exploring the SAR and activity of NSC319745, a compound that was reported as a potential inhibitor of DNMTs. The resynthesis of this compound indicated that when pure, this molecule does show inhibitory activity against DNMTs. SAR studies led to the identification of AK–I–85, a compound that inhibits DNMT3A with an IC50 of 36uM.

XRay NSC106084 et 14778.tiff

Afterwards, our group developed a synthetic route to access NSC106084 and NSC14778 and demonstrated that these compounds, when pure do not inhibit DNMTs. The nature of these compounds was confirmed by X-ray crystallography. Our work on DNMT inhibitors is important because it emphasizes the challenges associated with the discovery of inhibitors for these epigenetic enzymes. It also shows the tendency of assays to lead to false positives, the necessity of confirming the activity of hits with resynthesized materials, and the variability between assays.

Our group is currently working on the development of new DNMT inhibitors. To reach this goal, we have established collaborations with leaders across the world in the fields of biochemistry, NMR and computational chemistry. Our approach is based on various techniques such as resynthesis of hits from the literature, docking, NMR screening and rational design.