ABSTRACT
Transduced deoxyribonucleoside kinases (dNK) can be used to kill recipient cells in combination with nucleoside prodrugs. The Drosophila melanogaster multisubstrate dNK (Dm-dNK) displays a superior turnover rate and has a great plasticity regarding its substrates. We used directed evolution to create Dm-dNK mutants with increased specificity for several nucleoside analogs (NAs) used as anticancer or antiviral drugs. Four mutants were characterized for the ability to sensitize Escherichia coli toward analogs and for their substrate specificity and kinetic parameters. The mutants had a reduced ability to phosphorylate pyrimidines, while the ability to phosphorylate purine analogs was relatively similar to the wild-type enzyme. We selected two mutants, for expression in the osteosarcoma 143B, the glioblastoma U-87M-G and the breast cancer MCF7 cell lines. The sensitivities of the transduced cell lines in the presence of the NAs fludarabine (F-AraA), cladribine (CdA), vidarabine and cytarabine were compared to the parental cell lines. The sensitivity of 143B cells was increased by 470-fold in the presence of CdA and of U-87M-G cells by 435-fold in the presence of F-AraA. We also show that a choice of the selection and screening system plays a crucial role when optimizing suicide genes by directed evolution.
Subject(s)
Antimetabolites , Drosophila melanogaster/enzymology , Genetic Therapy/methods , Mutation , Neoplasms/therapy , Phosphotransferases (Alcohol Group Acceptor)/genetics , Animals , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Cladribine/therapeutic use , Cytarabine/therapeutic use , Directed Molecular Evolution/methods , Genes, Transgenic, Suicide , Glioblastoma/therapy , Humans , Lethal Dose 50 , Osteosarcoma/therapy , Phosphorylation , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Purines/metabolism , Substrate Specificity , Transduction, Genetic/methods , Vidarabine/analogs & derivatives , Vidarabine/therapeutic useABSTRACT
Thymidine kinases (TKs) appear to be almost ubiquitous and are found in nearly all prokaryotes, eukaryotes, and several viruses. They are the key enzymes in thymidine salvage and activation of several anti-cancer and antiviral drugs. We show that bacterial TKs can be subdivided into 2 groups. The TKs from Gram-positive bacteria are more closely related to the eukaryotic TK1 enzymes than are TKs from Gram-negative bacteria.
Subject(s)
Bacteria/enzymology , Gene Expression Regulation, Bacterial , Phosphotransferases (Alcohol Group Acceptor)/biosynthesis , Thymidine Kinase/biosynthesis , Amino Acid Sequence , Evolution, Molecular , Humans , Models, Molecular , Molecular Sequence Data , Nucleosides/chemistry , Open Reading Frames , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Phylogeny , Pyrimidines/chemistry , Sequence Homology, Amino Acid , Species Specificity , Thymidine Kinase/chemistryABSTRACT
Twenty-six fully sequenced archaeal genomes were searched for genes coding for putative deoxyribonucleoside kinases (dNKs). We identified only 5 human-like thymidine kinase 1 genes (TK1s) and none for non-TK1 kinases. Four TK1s were identified in the Euryarchaea and one was found in the Crenarchaea, while none was found in Nanoarchaeum. The identified TK1s have high identity to Gram-positive bacteria TK1s. The TK1s from archaea, Gram-positive bacteria and eukaryotes share the same common ancestor, while the TK1s from Gram-negative bacteria belong to a less-related subgroup. It seems that a functional deoxyribonucleoside salvage pathway is not crucial for the archaeal cell.
