Development of a universal radioactive DNA methyltransferase inhibition test for high-throughput screening and mechanistic studies

DNA methylation is an important epigenetic mark in eukaryotes, and aberrant pattern of this modification is involved in numerous diseases such as cancers. Interestingly, DNA methylation is reversible and thus is considered a promising therapeutic target. Therefore, there is a need for identifying ne...

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Bibliographic Details
Published in:Nucleic acids research 2013-10, Vol.41 (19), p.e185-e185
Main Authors: Gros, Christina, Chauvigné, Laura, Poulet, Anaïs, Menon, Yoann, Ausseil, Frédéric, Dufau, Isabelle, Arimondo, Paola B
Format: Article
Language:English
Subjects:
Cell Extracts
Cell Line
Dimethyl Sulfoxide
DNA (Cytosine-5-)-Methyltransferase 1
DNA (Cytosine-5-)-Methyltransferases - antagonists & inhibitors
DNA Methylation - drug effects
Enzyme Inhibitors - pharmacology
High-Throughput Screening Assays - methods
Humans
Methods Online
Solvents
Tritium
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Summary:DNA methylation is an important epigenetic mark in eukaryotes, and aberrant pattern of this modification is involved in numerous diseases such as cancers. Interestingly, DNA methylation is reversible and thus is considered a promising therapeutic target. Therefore, there is a need for identifying new small inhibitors of C5 DNA methyltransferases (DNMTs). Despite the development of numerous in vitro DNMT assays, there is a lack of reliable tests suitable for high-throughput screening, which can also give insights into inhibitor mechanisms of action. We developed a new test based on scintillation proximity assay meeting these requirements. After optimizing our assay on human DNMT1 and calibrating it with two known inhibitors, we carried out S-Adenosyl-l-Methionine and DNA competition studies on three inhibitors and were able to determine each mechanism of action. Finally, we showed that our test was applicable to 3 other methyltransferases sources: human DNMT3A, bacterial M.SssI and cellular extracts as well.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkt753