Design and synthesis of new non nucleoside inhibitors of DNMT3A.

DNA methylation, an epigenetic modification regulating gene expression, is a promising target in cancer. In an effort to identify new non nucleosidic inhibitors of DNA methyltransferases, the enzymes responsible for DNA methylation, we carried out a high-throughput screening of 66,000 chemical compounds based on an enzymatic assay against catalytic DNMT3A. A family of propiophenone derivatives was identified. After chemical optimization and structure activity relationship studies, a new inhibitor (33) was obtained with an EC50 of 2.1 µM against DNMT3A. The mechanism of inhibition of the compound was investigated as it forms a reactive Michael acceptor group in situ. Thereby, the Michael acceptor 20 was identified. This compound was further characterized for its biological activity in cancer cells.

Electrochemical assay for the signal-on detection of human DNA methyltransferase activity.

Strategies to detect human DNA methyltransferases are needed, given that aberrant methylation by these enzymes is associated with cancer initiation and progression. Here we describe a nonradioactive, antibody-free, electrochemical assay in which methyltransferase activity on DNA-modified electrodes confers protection from restriction for signal-on detection. We implement this assay with a multiplexed chip platform and show robust detection of both bacterial (SssI) and human (Dnmt1) methyltransferase activity. Essential to work with human methyltransferases, our unique assay design allows activity measurements on both unmethylated and hemimethylated DNA substrates. We validate this assay by comparison with a conventional radioactive method. The advantages of electrochemistry over radioactivity and fluorescence make this assay an accessible and promising new approach for the sensitive, label-free detection of human methyltransferase activity.