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.

Directed evolution of an orthogonal nucleoside analog kinase via fluorescence-activated cell sorting

“…The best variants of HSV–thymidine kinase (9,13) and Dm dNK (11,12,41) typically show <2-fold gains in k cat / K M for the NA. Furthermore, mutants selected by genetic complementation or cytotoxicity screening retain high activity for the native substrate.…”

Development of gene therapy in association with clinically used cytotoxic deoxynucleoside analogues

“…31,32 Sites involved in the substrate specificity have also been identified, and cells expressing some mutants are specifically more sensitive to purine derivatives than the wt Dm-dNK. [33][34][35] Finally, hybrid enzymes of Dm-dNK and human thymidine kinase 2 were shown to have higher activity on thymidine derivatives than Dm-dNK or TK2, but lower activity on fludarabine or gemcitabine. 36 Escherichia coli purine nucleoside phosphorylase Human PNP, a homotrimeric enzyme with a total molecular weight of about 100 kDa, is specific for 6-oxopurine nucleosides and has poor activity on 6-aminopurine nucleosides.…”

Bacterial Deoxyribonucleoside Kinases are Poor Suicide Genes in Mammalian Cells

“…The phosphorylated products were eluted from the paper disk and the quantity of the labeled monophosphate product determined in a scintillation counter. [8,15] …”