Title : Discovery of novel bacterial genes encoding the enzymes acting on modified uracil/uridine derivatives and their use for gene therapy in cancer treatment
Abstract:
Modified nucleotides are present in several RNA species in all Domains of Life. While the biosynthetic pathways of these nucleotides were well studied in recent years, much less attention was drawn to the degradation of different RNAs and the return of modified nucleotides or their constitutents into the metabolism. Using an Escherichia coli uracil auxotroph strain, we screened the metagenomic libraries for genes, which would allow the conversion of either 2-thiouracil, isocytosine’ or 2’-O-methyluridine into uracil and thereby lead to the growth on a defined synthetic medium. We have demonstrated that Domain of Unknown Function 523 (DUF523) containing protein is involved in the conversion of 2-thiouracil into uracil in vivo. We have also purified several recombinant isocytosine deaminases and a nucleoside hydrolase and demonstrated their enzymatic activities in vitro. These enzymes are also capable of converting the potential prodrugs 5-fluoroisocytosine, 5-fluorouridine, 5-fluoro-2’-O-methyluridine, and 5-fluoro-2’-deoxyuridine into a well-known anticancer drug 5-fluorouracil. The human glioblastoma U87MG and colorectal adenocarcinoma Caco-2 cell lines were transfected with the recoded isocytosine deaminase genes, and their citotoxicity together with 5-fluoroisoicytosine was demonstrated. The therapeutic potential of the isocytosine deaminase/5-fluoroisocytosine pair has been demonstrated in vivo, where the co-injection of the isocytosine deaminase-encoding mesenchymal stem cells and 5-fluoroisocytosine have been shown to increase longevity of tumorized mice by 50%.
Audience take away:
• In this presentation, the audience will learn that our environment is a source of the previously undescribed enzymatic activities. It will be emphasized that the enzymes acting on the analogs of modified nucleosides and/or heterocyclic bases my act as the non-toxic prodrug activators converting them into the active drugs. The preclinical studies demonstrating relevance of the proposed gene/prodrug pair for the cancer treatment will be presented and the advantages of the proposed prodrug activation system over the existing ones will be discussed.
