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Drosophila deoxyribonucleoside kinase mutants with enhanced ability to phosphorylate purine analogs

Gene Therapy volume 14, pages 1278–1286 (2007)Cite this article

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.

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Abbreviations

AraA:

vidarabine, 9-β-D-arabinofuranosyladenine

AraC, cytarabine:

1-β-D-arabinofuranosylcytosine

AZT:

azidothymidine

CdA:

cladribine, 2-chloro 2′-deoxyadenosine

dAdo:

deoxyadenosine

dCyd:

deoxycytidine

ddA:

dideoxyadenosine

ddC:

(zalcitabine) 2′,3′-dideoxycytidine

dGuo:

deoxyguanosine

dN:

deoxyribonucleoside

dNK:

deoxyribnonucleoside kinase

dNMP:

deoxyribonucleoside monophosphate

Dm-dNK:

Drosophila melanogaster multisubstrate deoxyribonucleoside kinase

dTTP:

thymidine triphosphate

dUrd:

deoxyuridine

F-AraA:

(fludarabine) 9-β-D-arabinofuranosyl-2-fluoroadenine

Thd:

thymidine

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Acknowledgements

We thank the Danish and Swedish Research Councils, the Danish Cancer Society, Swedish Cancer Fonden and the Meyer Foundation (Copenhagen) for their financial support.

Author information

Author notes

  1. W Knecht

    Present address: 6Current address: AstraZeneca R&D Mölndal, Mölndal SE-43183, Sweden.,

  2. M P B Sandrini

    Present address: 7Current address: Department of Molecular Biology, University of Copenhagen, Universitetsparken 13, Copenhagen DK-2100, Denmark.,

Authors and Affiliations

  1. BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark

    W Knecht, C Le Breton, M P B Sandrini, T Joergensen & J Piskur

  2. Cell and Organism Biology, Lund University, Lund, Sweden

    E Rozpedowska, M P B Sandrini & J Piskur

  3. ZGene A/S, Agern Allé 7, Hørsholm, Denmark

    M Willer & Z Gojkovic

  4. Department of Medical Biochemistry and Genetics, Section of Neurogenetics, University of Copenhagen, Copenhagen, Denmark

    L Hasholt

  5. Department of Science, Systems and Models, Roskilde University, Roskilde, Denmark

    B Munch-Petersen

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  1. W Knecht
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Correspondence to J Piskur.

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Knecht, W., Rozpedowska, E., Le Breton, C. et al. Drosophila deoxyribonucleoside kinase mutants with enhanced ability to phosphorylate purine analogs. Gene Ther 14, 1278–1286 (2007). https://doi.org/10.1038/sj.gt.3302982

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  • DOI: https://doi.org/10.1038/sj.gt.3302982

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