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 nucleotide 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 %.