Abstract
Nucleoside analogues (NA) are prodrugs that are phosphorylated by deoxyribonucleoside kinases (dNKs) as the first step towards a compound toxic to the cell. During the last 20 years, research around dNKs has gone into new organisms other than mammals and viruses. Newly discovered dNKs have been tested as enzymes for suicide gene therapy. The tomato thymidine kinase 1 (ToTK1) is a dNK that has been selected for its in vitro kinetic properties and then successfully been tested in vivo for the treatment of malignant glioma. We present the selection of two improved variants of ToTK1 generated by random protein engineering for suicide gene therapy with the NA azidothymidine (AZT). We describe their selection, recombinant production and a subsequent kinetic and biochemical characterization. Their improved performance in killing of E. coli KY895 is accompanied by an increase in specificity for the NA AZT over the natural substrate thymidine as well as a decrease in inhibition by dTTP, the end product of the nucleoside salvage pathway for thymidine. The understanding of the enzymatic properties improving the variants efficacy is instrumental to further develop dNKs for use in suicide gene therapy.
Highlights
Deoxyribonucleoside triphosphates for DNA synthesis are provided via de novo and salvage pathways
The two tomato thymidine kinase 1 (ToTK1) variants characterized in detail showed that improved performance in cell killing of E. coli KY895 is accompanied by an increase in specificity for the Nucleoside analogues (NA) AZT over the natural substrate dThd as well as a decrease in inhibition of the kinase activity by dTTP, the end product of the nucleoside salvage pathway for dThd
As previously done for DmdNK random mutagenesis was done by error prone PCR using Deoxyribonucleoside triphosphates (dNTP) analogs [28,29]
Summary
Deoxyribonucleoside triphosphates (dNTP) for DNA synthesis are provided via de novo and salvage pathways. The first dNK used in suicide gene therapy was a viral thymidine kinase from herpes simplex virus (HSV-TK) used in combination with ganciclovir (GCV) as prodrug This system can be regarded as the prototype model for gene-directed enzyme prodrug therapy [11,12]. The two ToTK1 variants characterized in detail showed that improved performance in cell killing of E. coli KY895 is accompanied by an increase in specificity for the NA AZT over the natural substrate dThd as well as a decrease in inhibition of the kinase activity by dTTP, the end product of the nucleoside salvage pathway for dThd. Our results show possibilities and molecular mechanisms important to create new variants of TK1s with increased NA efficacy that can be tested in suicide gene therapy
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