DNA Methyltransferases (DNMT) are crucial enzymes involved in epigenetic regulation of gene expression. Inhibitors of DNMT are commonly studied for the potential to serve as drugs in the treatment of cancers that are caused, in some part, by epigenetic abnormalities. Many of these studies focus on the impact of the inhibitor on total DNMT activity. The characterization of the mode of inhibition is frequently not addressed. In addition, the assays commonly used involve radioactive means, which are not always a viable option due to the risk of radioactivity. The primary goal of the project was to classify the mode of inhibition of two common inhibitors of methyltransferases, procainamide and hydralazine, using the E. <i>coli </i>CpG methyltransferase, M.SssI, as a model enzyme. A method for a non-radioactive assay was utilized to determine the mode of inhibition of these DNMT inhibitors. This assay uses agarose gel electrophoresis to determine if a specific restriction site on a plasmid of known sequence has been methylated, as methylation prevents restriction enzyme digestion. Comparing the density of cut DNA bands to uncut bands in the agarose gel allows for a determination of the rate of the methylation reaction at varying concentrations of DNA and inhibitor. Using a Dixon plot, the mode of inhibition and the ki can be determined simultaneously. Comparing the obtained Ki values to the known values will provide a means for evaluating the quality of data obtained by this approach to determine the mode of inhibition.

Classifying the mode of inhibition of DNA methyltransferase inhibitors

253rd National Meeting (2017)

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