Drosophila deoxyribonucleoside kinase mutants with enhanced ability to phosphorylate purine analogs

W. Knecht, E. Rozpedowska, C. Le Breton, M. Willer, Z. Gojkovic, M. P. B. Sandrini, T. Jørgensen, L. Hasholt, Birgitte Munch-Petersen, J. Piskur

    Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

    Resumé

    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.
    OriginalsprogEngelsk
    TidsskriftGene Therapy (Basingstoke)
    Vol/bind14
    Udgave nummer17
    Sider (fra-til)1278-1286
    Antal sider9
    ISSN0969-7128
    DOI
    StatusUdgivet - 2007

    Citer dette

    Knecht, W., Rozpedowska, E., Le Breton, C., Willer, M., Gojkovic, Z., Sandrini, M. P. B., ... Piskur, J. (2007). Drosophila deoxyribonucleoside kinase mutants with enhanced ability to phosphorylate purine analogs. Gene Therapy (Basingstoke), 14(17), 1278-1286. https://doi.org/10.1038/sj.gt.3302982
    Knecht, W. ; Rozpedowska, E. ; Le Breton, C. ; Willer, M. ; Gojkovic, Z. ; Sandrini, M. P. B. ; Jørgensen, T. ; Hasholt, L. ; Munch-Petersen, Birgitte ; Piskur, J. / Drosophila deoxyribonucleoside kinase mutants with enhanced ability to phosphorylate purine analogs. I: Gene Therapy (Basingstoke). 2007 ; Bind 14, Nr. 17. s. 1278-1286.
    @article{583030e0a3df11dcaf2d000ea68e967b,
    title = "Drosophila deoxyribonucleoside kinase mutants with enhanced ability to phosphorylate purine analogs",
    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.",
    author = "W. Knecht and E. Rozpedowska and {Le Breton}, C. and M. Willer and Z. Gojkovic and Sandrini, {M. P. B.} and T. J{\o}rgensen and L. Hasholt and Birgitte Munch-Petersen and J. Piskur",
    year = "2007",
    doi = "10.1038/sj.gt.3302982",
    language = "English",
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    Knecht, W, Rozpedowska, E, Le Breton, C, Willer, M, Gojkovic, Z, Sandrini, MPB, Jørgensen, T, Hasholt, L, Munch-Petersen, B & Piskur, J 2007, 'Drosophila deoxyribonucleoside kinase mutants with enhanced ability to phosphorylate purine analogs' Gene Therapy (Basingstoke), bind 14, nr. 17, s. 1278-1286. https://doi.org/10.1038/sj.gt.3302982

    Drosophila deoxyribonucleoside kinase mutants with enhanced ability to phosphorylate purine analogs. / Knecht, W.; Rozpedowska, E.; Le Breton, C.; Willer, M.; Gojkovic, Z.; Sandrini, M. P. B.; Jørgensen, T.; Hasholt, L.; Munch-Petersen, Birgitte; Piskur, J.

    I: Gene Therapy (Basingstoke), Bind 14, Nr. 17, 2007, s. 1278-1286.

    Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

    TY - JOUR

    T1 - Drosophila deoxyribonucleoside kinase mutants with enhanced ability to phosphorylate purine analogs

    AU - Knecht, W.

    AU - Rozpedowska, E.

    AU - Le Breton, C.

    AU - Willer, M.

    AU - Gojkovic, Z.

    AU - Sandrini, M. P. B.

    AU - Jørgensen, T.

    AU - Hasholt, L.

    AU - Munch-Petersen, Birgitte

    AU - Piskur, J.

    PY - 2007

    Y1 - 2007

    N2 - 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.

    AB - 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.

    U2 - 10.1038/sj.gt.3302982

    DO - 10.1038/sj.gt.3302982

    M3 - Journal article

    VL - 14

    SP - 1278

    EP - 1286

    JO - Gene Therapy

    JF - Gene Therapy

    SN - 0969-7128

    IS - 17

    ER -