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Drosophila deoxyribonucleoside kinase mutants with enhanced ability to phosphorylate purine analogs

Knecht, W.; Rozpedowska, Elzbieta LU ; Le Breton, C.; Willer, M.; Gojkovic, Z.; Sandrini, Michael LU ; Joergensen, T.; Hasholt, L.; Munch-Petersen, B. and Piskur, Jure LU (2007) In Gene Therapy 14(17). p.1278-1286
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... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
purines, nucleoside analogs, directed evolution, deoxyribonucleoside kinase, cancer gene therapy
in
Gene Therapy
volume
14
issue
17
pages
1278 - 1286
publisher
Nature Publishing Group
external identifiers
  • wos:000248882800004
  • scopus:34548150268
ISSN
0969-7128
DOI
10.1038/sj.gt.3302982
language
English
LU publication?
yes
id
efcc76bd-ae89-45ff-8b94-ab96ce69e523 (old id 691942)
date added to LUP
2007-12-12 08:36:31
date last changed
2017-06-18 03:43:13
@article{efcc76bd-ae89-45ff-8b94-ab96ce69e523,
  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.},
  author       = {Knecht, W. and Rozpedowska, Elzbieta and Le Breton, C. and Willer, M. and Gojkovic, Z. and Sandrini, Michael and Joergensen, T. and Hasholt, L. and Munch-Petersen, B. and Piskur, Jure},
  issn         = {0969-7128},
  keyword      = {purines,nucleoside analogs,directed evolution,deoxyribonucleoside kinase,cancer gene therapy},
  language     = {eng},
  number       = {17},
  pages        = {1278--1286},
  publisher    = {Nature Publishing Group},
  series       = {Gene Therapy},
  title        = {Drosophila deoxyribonucleoside kinase mutants with enhanced ability to phosphorylate purine analogs},
  url          = {http://dx.doi.org/10.1038/sj.gt.3302982},
  volume       = {14},
  year         = {2007},
}