Drosophila deoxyribonucleoside kinase mutants with enhanced ability to phosphorylate purine analogs

Wolfgang Knecht, E. Rozpedowska, C. Le Breton, Mette Willer, Zoran Gojkovic, M.P.B. Sandrini, Tina Jørgensen, L. Hasholt, B. Munch-Petersen, Jure Piskur

    Research output: Contribution to journalJournal articleResearchpeer-review

    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 435fold 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.
    Original languageEnglish
    JournalGene Therapy
    Volume14
    Issue number17
    Pages (from-to)1278-1286
    ISSN0969-7128
    DOIs
    Publication statusPublished - 2007

    Keywords

    • nucleoside analogs
    • cancer gene therapy
    • directed evolution
    • deoxyribonucleoside kinase
    • purines

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