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.
authors.affiliation: BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark.
authors.forename: W
authors.initials: W
authors.lastname: Knecht
authors.forename: E
authors.initials: E
authors.lastname: Rozpedowska
authors.forename: C
authors.initials: C
authors.lastname: Le Breton
authors.forename: M
authors.initials: M
authors.lastname: Willer
authors.forename: Z
authors.initials: Z
authors.lastname: Gojkovic
authors.forename: M P B
authors.initials: MP
authors.lastname: Sandrini
authors.forename: T
authors.initials: T
authors.lastname: Joergensen
authors.forename: L
authors.initials: L
authors.lastname: Hasholt
authors.forename: B
authors.initials: B
authors.lastname: Munch-Petersen
authors.forename: J
authors.initials: J
authors.lastname: Piskur
mesh_terms: D000818:Animals; D000963:Antimetabolites; D000970:Antineoplastic Agents / Q000627:therapeutic use; D045744:Cell Line, Tumor; D017338:Cladribine / Q000627:therapeutic use; D003561:Cytarabine / Q000627:therapeutic use; D019020:Directed Molecular Evolution / Q000379:methods; D004331:Drosophila melanogaster / Q000201:enzymology*; D041722:Genes, Transgenic, Suicide; D015316:Genetic Therapy / Q000379:methods*; D005909:Glioblastoma / Q000628:therapy; D006801:Humans; D007928:Lethal Dose 50; D009154:Mutation; D009369:Neoplasms / Q000628:therapy*; D012516:Osteosarcoma / Q000628:therapy; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000378:metabolism; D011687:Purines / Q000378:metabolism; D013379:Substrate Specificity; D014161:Transduction, Genetic / Q000379:methods; D014740:Vidarabine / Q000031:analogs & derivatives / Q000627:therapeutic use
chemical_list: D000963:Antimetabolites; D000970:Antineoplastic Agents; D011687:Purines; D003561:Cytarabine; D017338:Cladribine; D017853:Phosphotransferases (Alcohol Group Acceptor); C020018:deoxyribonucleoside kinases; D014740:Vidarabine; C024352:fludarabine