Identification of DNMT1 Selective Antagonists Using a Novel Scintillation Proximity Assay

A novel scintillation proximity high throughput assay (SPA) to identify inhibitors of DNA methyltransferases was developed and used to screen over 180,000 compounds. The majority of the validated hits shared a quinone core and several were found to generate the reactive oxygen species, H 2 O 2 . Inhibition of the production of H 2 O 2 by the addition of catalase blocked the ability of this group of compounds to inhibit DNA methyltransferase (DNMT) activity. However, a related compound, SW155246, was identified that existed in an already reduced form of the quinone. This compound did not generate H 2 O 2 , and catalase did not block its ability to inhibit DNA methyltransferase. SW155246 showed a 30-fold preference for inhibition of human DNMT1 versus human or murine DNMT3A or -3B, inhibited global methylation in HeLa cells, and reactivated expression of the tumor suppressor gene RASSF1A in A549 cells. To our knowledge, this work represents the first description of selective chemical inhibitors of the DNMT1 enzyme. Background: DNA methylation contributes to the heritable regulation of gene expression. Results: Chemical inhibitors of DNA methyltransferase (DNMT) enzymes were identified. Conclusion: Several inhibitors nonspecifically inhibited multiple methyltransferases through generation of H 2 O 2 , but one compound showed selective inhibition of DNMT1 independent of the production of H 2 O 2 . Significance: Selective DNMT1 inhibitors will allow for more precise elucidation of the role of this enzyme in gene regulation.