ABSTRACT
The field of epigenetics includes the study of C5 DNA methyltransferases (C5 DNA-MTases). Molecular studies on the inhibition of C5 DNA-MTases by 5-fluoro-2′-deoxcytidine and 5-aza-cytosine nucleosides showed that the reaction occurred by DNA base flipping. This universal mechanism was also shown for our novel DNA oligodeoxynucleotides containing the nucleobases of 2′-deoxy-5,6-dihydro-5-aza-cytidine (dDHAC) and 2′-deoxyzebularine (dZ). DNA oligodeoxynucleotides modified with dZ were as potent as DNA oligodeoxynucleotides containing 5-azacytosine. The weaker potency of zebularine as a drug was found to be related to its sluggish metabolism by dinucleotide reductase, RNR. Despite its lower potency, the stability of zebularine allowed the use of the drug for extended periods of time to produce heritable changes in demethylation and activation of tumor suppressor genes, such as p16. To overcome the low incorporation of zebularine into DNA, nucleotide prodrugs (ProTides) of dZ were developed to bypass the limiting RNR step. The dZ ProTides had a narrow specificity and required co-administration of thymidine. The use of a promiscuous kinase from Drosophila melanogaster (Dm-dNK) allowed incorporating dZ exclusively into DNA and unveiled an additional mechanism of zebularine.
