Abstract
DNA methyltransferase 1 (DNMT1) is the enzyme most responsible for epigenetic modification of human DNA and the intended target of approved cancer drugs such as 5-aza-cytidine and 5-aza-2′-deoxycytidine. 5-aza nucleosides have complex mechanisms of action that require incorporation into DNA, and covalent trapping and proteolysis of DNMT isozymes. Direct DNMT inhibitors are needed to refine understanding of the role of specific DNMT isozymes in cancer etiology and, potentially, to improve cancer prevention and treatment. Here, we developed a high throughput pipeline for identification of direct DNMT1 inhibitors. The components of this screen include an activated form of DNMT1, a restriction enzyme-coupled fluorigenic assay performed in 384 well plates with a z-factor of 0.66, a counter screen against the restriction enzyme, a screen to eliminate DNA intercalators, and a differential scanning fluorimetry assay to validate direct binders. Using the Microsource Spectrum collection of 2320 compounds, this screen identified nine compounds with dose responses ranging from 300 nM to 11 µM, representing at least two different pharmacophores with DNMT1 inhibitory activity. Seven of nine inhibitors identified exhibited two to four-fold selectivity for DNMT1 versus DNMT3A.
Highlights
In eukaryotes, the most common DNA modification is methylation of the 5 carbon of cytosines, predominately in CpG dinucleotides
Cleavage of the product DNA releases the fluorophore from the quencher and generates fluorescence. Using this endonuclease-coupled DNA methylation assay, we showed that an N-terminal deletion of sequences up to and including the replication foci targeting sequence (RFTS) domain, the first 620 amino acids, results in an enzyme that is 640fold more active [27]
This de-repressed form of DNA methyltransferase 1 (DNMT1), with a kcat/Km of,106 M21 s21 [27], has sufficient catalytic power to allow for facile identification of inhibitors using the fluorogenic assay
Summary
The most common DNA modification is methylation of the 5 carbon of cytosines, predominately in CpG dinucleotides. Methylation patterns are established and maintained by a family of enzymes known as DNA methyltransferases (DNMTs). DNMT3A and DNMT3B, establish methylation patterns during germ cell and embryonic development. These proteins are aided by DNMT3L, a catalytically inactive isoform that forms complexes with DNMT3A and DNMT3B [1]. DNA methylation is an important epigenetic mark associated with gene repression that plays a critical role in development and differentiation, genome stability, genomic imprinting, X-chromosome inactivation and silencing of retrotransposons [4]. Though genetic changes associated with cancer cannot be corrected, epigenetic changes, such as DNA methylation, are dynamic and amenable to reversal. Epigenetic reprogramming, accomplished by pharmacological targeting of DNMTs, could be expected to result in restoration of a more differentiated and less proliferative state, and regression to a lower degree of drug resistance [13]
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