Multisubstrate Deoxyribonucleoside Kinase Research Articles

Tanshinone IIA enhances bystander cell killing of cancer cells expressing Drosophila melanogaster deoxyribonucleoside kinase in nuclei and mitochondria.

Heterologous expression of the Drosophila melanogaster multi-substrate deoxyribonucleoside kinase (Dm-dNK) increases the sensitivity of cancer cells to several cytotoxic nucleoside analogs. Thus, it may be used as a suicide gene in combined gene/chemotherapy treatment of cancer. To further characterize this potential suicide gene, we constructed two retroviral vectors that enabled the expression of Dm-dNK in cancer cells. One vector harbored the wild‑type enzyme that localized to the nucleus. The other vector harbored a mitochondrial localized mutant enzyme that was constructed by deleting the nuclear localization signal and fusing it to a mitochondrial import signal of cytochrome c oxidase. A thymidine kinase-deficient osteosarcoma cell line was transduced with the recombinant viruses. The sensitivity and bystander cell killing in the presence of pyrimidine nucleoside analogs (E)-5-(2-bromovinyl)‑2'‑deoxyuridine and 1-β-D-arabinofuranosylthymine were investigated. TanshinoneIIA is a constituent of Danshen; a traditional Chinese medicine used in the treatment of cardiovascular diseases. This study also looked at the influence of TanshinoneIIA on the bystander effect and the underlying mechanisms. We showed that sensitivity of the osteosarcoma cell line to the nucleoside analogs and the efficiency of bystander cell killing were independent of the subcellular localization of Dm-dNK. The enhanced effect of tanshinone IIA on the bystander effect was related to the increased expression of Cx43 and Cx26.

Synergistic antitumor effect of adenovirus armed with Drosophila melanogaster deoxyribonucleoside kinase and nucleoside analogs for human breast carcinoma in vitro and in vivo.

BackgroundSuicide gene therapy in cancer can selectively kill tumors without damaging normal tissues. Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK), an original suicide kinase, makes use of the carcinomatous suicide gene therapy for broader substrate specificity and a higher catalytic rate.MethodsTo enhance the anti-tumor efficacy of Dm-dNK and maintain its substrate specificity and safety control in the meantime, the conditionally replicative gene–viral system, ZD55–dNK (which contains the selective replication adenovirus, ZD55, encoded with Dm-dNK), was investigated in pushing a deeper development of this strategy. Selective replication, cell killing efficacy, and cytotoxicity, in combination with chemotherapy, were applied to two breast cell lines (MDA231 and MCF7 cells), two normal cell lines (WI38 and MRC5 cells), and the MCF7 xenograft model in vivo.ResultsThe preclinical study showed that ZD55–dNK, combined with 2′,2′-difluorodeoxycytidine (DFDC), synergistically inhibited adenovirus replication in vitro but maintained specifically cancer cell killing efficacy. ZD55–dNK also greatly improved the antineoplastic effect in vitro and in breast cancer xenograft in vivo.ConclusionThe concomitant use of ZD55–dNK and DFDC is possibly a novel and promising approach to breast cancer treatment, and further investigation on the safe control of excessive virus replication and the efficacy of this approach in humans is warranted.

Potent antitumoral effects of targeted promoter-driven oncolytic adenovirus armed with Dm-dNK for breast cancer in vitro and in vivo

Currently, no curative treatments are available for late-stage metastatic or recurrent breast cancer, because the cancer tolerates both chemotherapy and endocrine therapy. In this study, we investigated the feasibility of a dual-regulated oncolytic adenoviral vector with a novel suicide gene to treat breast cancer. Following targeted gene virotherapy of conditionally replicating adenoviruses (CRAds), the novel suicide gene of multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-DNK) was inserted into the double-regulated oncolytic adenovirus SG500 to ensure more safety and enhanced antitumor activity against breast cancer both in vitro and in vivo. Selective replication, cell-killing efficacy, and cytotoxicity, combined with chemotherapeutics were investigated in several breast cell lines (MDA-MB-231 and MCF-7), normal cells (WI-38 and MRC-5), and human (MDA-MB-231) tumor models in vivo. The double-regulated SG500-dNK had high cell-killing activity in breast cancer. Replication was similar to wild-type in breast cells and was attenuated in normal cells. SG500-dNK combined with the chemotherapeutics (E)-5-(2-bromovinyl)-2′-deoxyuridine (Bvdu) and 2′,2′-difluoro-deoxycytidine (dFdC) resulted in synergistically enhanced cell killing and greatly improved antitumor efficacy in vitro or in breast xenografts in vivo. These data suggest that the novel oncolytic variant SG500-dNK is a promising candidate for targeting breast tumors specifically when combined with chemotherapeutics.

Antitumor effects of oncolytic adenovirus armed with Drosophila melanogaster deoxyribonucleoside kinase in colorectal cancer

Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK) was applied as a cancer gene therapeutic approach. To improve the antitumor effect of Dm-dNK, a novel suicide gene system based on an oncolytic adenovirus vector was developed to produce therapeutic effects towards colorectal cancer cells. We constructed an oncolytic adenoviral vector (ZD55), which was designed by deletion of the E1B-55 kDa gene for selective replication in tumor cells, containing suicide gene (Dm-dNK) driven by a cytomegalovirus promoter. We analysed the expression and activity of Dm-dNK in colorectal cancer cells (HCT-116 and SW620) via reverse transcription (RT)-PCR and enzyme assay. We assessed selective cytotoxic effects of Dm-dNK with the presence of the analogs (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), difluorodeoxycytidine (dFdC) or 1-β-D-arabinofuranosylthymine (ara-T) by MTT and FACS; the variation of oncolytic adenovirus was detected by titer assay and western blot analysis. Our data showed that ZD55-Dm-dNK mediated high expression of Dm-dNK in HCT-116 and SW620 cancer cell lines and low levels of expression in WI-38 and MRC-5 normal cells, strong cytotoxicity was observed only in tumor cells after ZD55-Dm-dNK infection combined with nucleoside analogs (NA). When ZD55-Dm-dNK was combined with BVDU or dFdC, it produced a synergistic inhibitive effect of adenovirus replication while maintaining specific cancer cell killing activity. The results suggest that the novel oncolytic virus ZD55-Dm-dNK, in combination with NA, has potential as an efficient selective antitumoral agent and may produce synergistic effects in safe control of adenovirus, which is a new promising therapeutic for colorectal cancer.

The multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster as a therapeutic suicide gene of breast cancer cells

The multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) was investigated for its broader substrate specificity and higher catalytic rate as a suicide gene in a combined gene/chemotherapy of cancer. To evaluate the effects of nucleoside analog phosphorylation by Dm-dNK in vitro and in vivo, we generated a replication-deficient retroviral vector expressing Dm-dNK to transduce human breast cancer cells MCF7 (ER+) and MDA-MB-231 (ER-). We further determined the enzymatic activity and the sensitivity of the nontransduced and Dm-dNK-transduced 231/dNK and MCF7/dNK cells to the pyrimidine nucleoside analogs araC and araT. The data obtained show that Dm-dNK is enzymatically active and its overexpression in the nuclei of breast cancer cells results in an increased sensitivity to the nucleoside analogs araC and araT in vitro. Furthermore, subcutaneously transplanted 231/dNK cells were significantly inhibited after araC treatment, whereas nontransduced cancer cells continued to grow and develop in vivo. These results suggest that the Dm-dNK/nucleoside analog system could be a novel therapeutic strategy for treating breast cancer and improving anti-tumor efficacy, as well as for optimizing approaches for suicide gene therapy.

Synergistic therapeutic effect in gastric cancer cells produced by oncolytic adenovirus encoding Drosophila melanogaster deoxyribonucleoside kinase

Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK), a novel suicide kinase, was applied as a cancer gene therapeutic approach. To improve the antitumor effect of Dm-dNK and its substrate with the selectivity and safety control in consideration, the conditionally replicative gene-viral system ZD55-dNK, which includes the replicationselective adenovirus ZD55 armed with the Dm-dNK, was used to further explore the potential of this approach. When ZD55-dNK was combined with BVDU it produced a synergistic inhibitive effect of adenovirus replication in vitro while maintaining specific cancer cell killing activity. From a clinical standpoint, this approach is promising for its considerably low toxic side effects.

Drosophila melanogaster deoxyribonucleoside kinase activates gemcitabine

Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK) can additionally sensitize human cancer cell lines towards the anti-cancer drug gemcitabine. We show that this property is based on the Dm-dNK ability to efficiently phosphorylate gemcitabine. The 2.2Å resolution structure of Dm-dNK in complex with gemcitabine shows that the residues Tyr70 and Arg105 play a crucial role in the firm positioning of gemcitabine by extra interactions made by the fluoride atoms. This explains why gemcitabine is a good substrate for Dm-dNK.

A multisubstrate deoxyribonucleoside kinase from plants

Deoxyribonucleoside kinases catalyze the rate limiting step during the salvage of deoxyribonucleosides and convert them into the corresponding monophosphate compounds. We have identified and characterized a unique multisubstrate deoxyribonucleoside kinase from plants. The phylogenetic relationship and biochemical properties suggest that this deoxyribonucleoside kinase represents a living fossil resembling the progenitor of the modern animal deoxycytidine, deoxyguanosine and thymidine 2 kinases. The broad substrate specificity makes this enzyme an interesting candidate to be evaluated as a suicide gene in anti-cancer therapy.

Structural studies of nucleoside analog and feedback inhibitor binding to Drosophila melanogaster multisubstrate deoxyribonucleoside kinase

The Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (dNK; EC 2.7.1.145) has a high turnover rate and a wide substrate range that makes it a very good candidate for gene therapy. This concept is based on introducing a suicide gene into malignant cells in order to activate a prodrug that eventually may kill the cell. To be able to optimize the function of dNK, it is vital to have structural information of dNK complexes. In this study we present crystal structures of dNK complexed with four different nucleoside analogs (floxuridine, brivudine, zidovudine and zalcitabine) and relate them to the binding of substrate and feedback inhibitors. dCTP and dGTP bind with the base in the substrate site, similarly to the binding of the feedback inhibitor dTTP. All nucleoside analogs investigated bound in a manner similar to that of the pyrimidine substrates, with many interactions in common. In contrast, the base of dGTP adopted a syn-conformation to adapt to the available space of the active site.

Mitochondrial Expression of theDrosophila melanogasterMultisubstrate Deoxyribonucleoside Kinase

The multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) is studied as a candidate suicide gene for applications in combined gene/chemotherapy of cancer. We have created an engineered Dm-dNK nucleoside kinase that is targeted to the mitochondrial matrix. The enzyme was expressed in a thymidine kinase 1-deficient osteosarcoma cell line, and the sensitivity of the cells to cytotoxic nucleoside analogs was determined when the enzyme was targeted to either the nucleus or the mitochondrial matrix. Although the total deoxythymidine (dThd) phosphorylation activity was similar in cells expressing Dm-dNK in the nucleus or in the mitochondria, the cells expressing the enzyme in the mitochondria showed higher sensitivity to the antiproliferative activity of several pyrimidine nucleoside analogs, such as (E)-5-(2-bromovinyl)-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, and 5-fluoro-2'-deoxyuridine. Labeling studies using [3H]dThd showed that the cells expressing the mitochondrial enzyme had an increased incorporation of [3H]dThd into DNA, shown to be due to a higher [3H]dTTP specific activity of the total dTTP pool in the cells in which Dm-dNK was targeted to the mitochondria. The difference in the specific activity of the dTTP pool is a result of different contributions of the de novo and the salvage pathways for the dTTP synthesis in transduced cells. In summary, these findings suggest that mitochondrial targeting of Dm-dNK facilitates nucleoside and nucleoside analog phosphorylation and could be used as a strategy to enhance the efficacy of nucleoside analog phosphorylation and concomitantly their cytostatic potential.

Enhanced toxicity of purine nucleoside analogs in cells expressing Drosophila melanogaster nucleoside kinase mutants

The multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) is investigated for possible use as a suicide gene in combined gene/chemotherapy of cancer. The enzyme has broader substrate specificity and higher catalytic rate compared to herpes simplex type 1 thymidine kinase and other known dNKs. Although the enzyme has broad substrate specificity, it has a preference for pyrimidine nucleosides and nucleoside analogs. We have evaluated the substrate specificity and kinetic properties of Dm-dNK proteins containing M88R, V84A+M88R or V84A+M88R+A110D mutations in the amino-acid sequence. These engineered enzymes showed a relative increase in phosphorylation of purine nucleoside analogs such as ganciclovir, 9-beta-D-arabinofuranosylguanine and 2',2'-difluorodeoxyguanosine compared to the wild-type enzyme. The mutant enzymes were expressed in an osteosarcoma thymidine kinase-deficient cell line and the sensitivity of the cell line to nucleoside analogs was determined. The cells expressing the M88R mutant enzyme showed the highest increased sensitivity to purine nucleoside analogs with 8- to 80-fold decreased inhibition constant IC(50) compared to untransduced control cells or cells expressing the wild-type nucleoside kinase. In summary, our data show that enzyme engineering can be used to shift the substrate specificity of the Dm-dNK to selectively increase the sensitivity of cells expressing the enzyme to purine nucleoside analogs.

New ultrasensitive 32P-postlabelling method for the analysis of 3,N4-etheno-2′-deoxycytidine in human urine

Etheno–DNA adducts are generated from exogenous carcinogens such as vinyl chloride and urethane and also from endogenous lipid peroxidation products such as trans-4-hydroxy-2-nonenal (HNE). The present authors and others have established that 1,N6-ethenodeoxyadenosine (εdA) and 3,N4-ethenodeoxycytidine (εdC) are present in human urine and could be explored as biomarkers for monitoring whole-body oxidative stress. The present study reports on a new ultrasensitive 32P-postlabelling/thin-layer chromatography (TLC) method for the analysis of εdC as deoxynucleoside in human urine. The urine samples were purified and enriched on a solid-phase silica C-18 column followed by a semi-preparative reverse-phase high-performance liquid chromatography. The purified sample was labelled with a multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster (Dm-dNK) in the presence of 5′-bromo-2′-deoxyuridine (BrdU) as internal standard. The absolute sensitivity of the method was 0.1 fmol εdC detectable in 500 µl of human urine. The analysis of human urine samples from 15 healthy volunteers revealed a mean εdC level of 2.49±1.76 (SD) fmol µmol−1 creatinine (range 0.66–6.42). By this non-invasive method, εdC in human urine could be explored as a biomarker for oxidative stress-related human diseases.

The Drosophila melanogaster UMP-CMP kinase cDNA encodes an N-terminal mitochondrial import signal

Drosophila melanogaster cells express a multi-substrate deoxyribonucleoside kinase that phosphorylates both purine and pyrimidine deoxyribonucleosides. The subsequent phosphorylation step is catalyzed by nucleoside monophosphate kinases. We have cloned and characterized the D. melanogaster UMP-CMP kinase ( Dm.UMP-CMP kinase) to further study the nucleotide metabolizing enzymes in these cells. The kinase, encoded by the dak1 gene, was ≈60% similar to the human UMP-CMP kinase and predominantly phosphorylated CMP, dCMP, and UMP. Expression analysis showed that the Dm.UMP-CMP kinase mRNA was constitutively expressed throughout the Drosophila development. The open-reading frame of the Dm.UMP-CMP kinase cDNA was extended in the 5 ′-region compared to UMP-CMP kinases of other species. The extended region encoded an N-terminal sequence with properties characteristic of a mitochondrial import signal. Expression of the enzyme in fusion with the green fluorescent protein verified that the N-terminal signal targeted the enzyme to mitochondria. This is the first time a mitochondrial pyrimidine nucleoside monophosphate kinase has been cloned from any organism and we discuss the implication of this finding for deoxyribonucleoside salvage in both Drosophila and other organisms.

Active site mutants of Drosophila melanogaster multisubstrate deoxyribonucleoside kinase.

The multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) is sequence-related to three human deoxyribonucleoside kinases and to herpes simplex virus type-1 thymidine kinase. Dm-dNK phosphorylates both purine and pyrimidine deoxyribonucleosides and nucleoside analogues although it has a preference for pyrimidine nucleosides. We performed site-directed mutagenesis on residues that, based on structural data, are involved in substrate recognition. The aim was to increase the phosphorylation efficiency of purine nucleoside substrates to create an improved enzyme to be used in suicide gene therapy. A Q81N mutation showed a relative increase in deoxyguanosine phosphorylation compared with the wild-type enzyme although the efficiency of deoxythymidine phosphorylation was 10-fold lower for the mutant. In addition to residue Q81 the function of amino acids N28, I29 and F114 was investigated by different substitutions. All of the mutated enzymes showed decreased efficiency of thymidine phosphorylation in comparison with the wild-type enzyme supporting their importance for substrate binding and/or catalysis as proposed by the recently solved structure of Dm-dNK.

Mosquito has a single multisubstrate deoxyribonucleoside kinase characterized by unique substrate specificity

In mammals four deoxyribonucleoside kinases, with a relatively restricted specificity, catalyze the phosphorylation of the four natural deoxyribonucleosides. When cultured mosquito cells, originating from the malaria vector Anopheles gambiae, were examined for deoxyribonucleoside kinase activities, only a single enzyme was isolated. Subsequently, the corresponding gene was cloned and over-expressed. While the mosquito kinase (Ag-dNK) phosphorylated all four natural deoxyribonucleosides, it displayed an unexpectedly higher relative efficiency for the phosphorylation of purine versus pyrimidine deoxyribonucleosides than the fruit fly multisubstrate deoxyribonucleoside kinase (EC 2.7.1.145). In addition, Ag-dNK could also phosphorylate some medically interesting nucleoside analogs, like stavudine (D4T), 2-chloro-deoxyadenosine (CdA) and 5-bromo-vinyl-deoxyuridine (BVDU). Although the biological significance of multisubstrate deoxyribonucleoside kinases and their diversity among insects remains unclear, the observed variation provides a whole range of applications, as species specific and highly selective targets for insecticides, they have a potential to be used in the enzymatic production of various (di-)(deoxy-)ribonucleoside monophosphates, and as suicide genes in gene therapy.

A few amino acid substitutions can convert deoxyribonucleoside kinase specificity from pyrimidines to purines.

In mammals, the four native deoxyribonucleosides are phosphorylated to the corresponding monophosphates by four deoxyribonucleoside kinases, which have specialized substrate specificities. These four enzymes are likely to originate from a common progenitor kinase. Insects appear to have only one multisubstrate deoxyribonucleoside kinase (dNK, EC 2.7.1.145), which prefers pyrimidine nucleosides, but can also phosphorylate purine substrates. When the structures of the human deoxyguanosine kinase (dGK, EC 2.7.1.113) and the dNK from Drosophila melanogaster were compared, a limited number of amino acid residues were identified and proposed to be responsible for the substrate specificity. Three of these key residues in Drosophila dNK were then mutagenized and the mutant enzymes were characterized regarding their ability to phosphorylate native deoxyribonucleosides and nucleoside analogs. The mutations converted the dNK substrate specificity from predominantly pyrimidine specific into purine specific. A similar scenario could have been followed during the evolution of kinases. Upon gene duplication of the progenitor kinase, only a limited number of single amino acid changes has taken place in each copy and resulted in substrate-specialized enzymes.

Nucleoside Analog Cytotoxicity and Bystander Cell Killing of Cancer Cells Expressing Drosophila melanogaster Deoxyribonucleoside Kinase in the Nucleus or Cytosol

We have recently shown that the overexpression of Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK) in cancer cell lines increases the cells' sensitivity to several cytotoxic nucleoside analogs and the enzyme may accordingly be used as a suicide gene in combined gene/chemotherapy treatment of cancer. To further characterize the enzyme for possible use as a suicide gene, we constructed a replication-deficient retroviral vector that expressed either the wild-type enzyme that localizes to the cell nucleus or a mutant (arg247ser) that localizes to the cytosol. A thymidine kinase-deficient osteosarcoma cell line was transduced with the recombinant virus and we compared the sensitivity and bystander cell killing when the cell lines were incubated with the pyrimidine nucleoside analogs (E)-5-(2-bromovinyl)-2′-deoxyuridine and 1-β-d-arabinofuranosylthymine. In summary, we showed that the cells' sensitivity and the efficiency of bystander cell killing were not dependent on whether Dm-dNK was located in the nucleus or cytosol.

Bystander effects of cancer cell lines transduced with the multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster and synergistic enhancement by hydroxyurea.

The multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) can be expressed in human cells with retained enzymatic activity. The cells expressing Dm-dNK exhibit increased sensitivity to several cytotoxic nucleoside analogs. In this study, we further evaluated Dm-dNK as a potential novel suicide gene in combination with (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) as the prodrug. We used two human cancer cell lines transduced with a retrovirus encoding the Dm-dNK cDNA and investigated whether the cells expressing the enzyme can induce cell death of untransduced cells, a phenomenon known as the "bystander effect". A bystander effect was observed in a thymidine kinase-deficient human osteosarcoma cell line but not in the MIA PaCa-2 human pancreatic adenocarcinoma cell line. The cytotoxicity of BVDU increased in both cell lines when the compound was used in combination with subtoxic concentrations of hydroxyurea. Hydroxyurea also enhanced the bystander effect in the osteosarcoma cells, but not in the MIA PaCa-2 cells, treated with BVDU. These findings indicate that BVDU phosphorylated by Dm-dNK in transduced cancer cells may also induce bystander cell death in certain cell lines.

Retroviral Transduction of Cancer Cell Lines with the Gene Encoding Drosophila melanogaster Multisubstrate Deoxyribonucleoside Kinase

Nucleoside kinases from several species are investigated as "suicide genes" for treatment of malignant tumors by combined gene/chemotherapy. We have recently cloned a multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK), and we have shown that the enzyme phosphorylates cytotoxic pyrimidine and purine nucleoside analogs. The broad substrate specificity of the enzyme, as well as its very high catalytic rate, makes it a unique member of the nucleoside kinase enzyme family. In the present study, we evaluated Dm-dNK as a suicide gene by constructing a replication-deficient retroviral vector that expresses the enzyme. The human pancreatic adenocarcinoma cell line MIA PaCa-2 and a thymidine kinase-deficient osteosarcoma cell line were transduced with the recombinant virus. We showed that Dm-dNK can be expressed in human cells, that the enzyme retained its enzymatic activity, and that it is localized in the cell nuclei due to a nuclear localization signal in its C-terminal region. The cells expressing Dm-dNK exhibited increased sensitivity to several cytotoxic nucleoside analogs, such as 1-beta-d-arabinofuranosylcytosine, 1-beta-d-arabinofuranosylthymine, (E)-5-(2-bromovinyl)-2'-deoxyuridine, 2-chloro-2'-deoxyadenosine, and 2',2'-difluorodeoxycytidine. These findings suggest that Dm-dNK may be used as a suicide gene in combined gene/chemotherapy of cancer.

Identification of residues involved in the specificity and regulation of the highly efficient multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster

In contrast to all known deoxyribonucleoside kinases, a single highly efficient deoxyribonucleoside kinase from Drosophila melanogaster (Dm-dNK) is able to phosphorylate all precursor nucleosides for DNA synthesis. Dm-dNK was mutated in vitro by high-frequency random mutagenesis, expressed in the thymidine kinase-deficient Escherichia coli strain KY895 and clones were selected for sensitivity to the nucleoside analogs 1-beta-d-arabinofuranosylcytosine (AraC, Cytarabine), 3′-azido-2′,3′-dideoxythymidine (AZT, Zidovudine, Retrovir®), 2′,3′-dideoxyadenosine (ddA) and 2′,3′-dideoxycytidine (ddC, Zalcitabine, Hivid®). Thirteen mutants with increased sensitivity compared to the wild-type Dm-dNK were isolated from a relatively small pool of less than 10,000 clones. Eight mutant Dm-dNKs increased the sensitivity of KY895 to more than one analog, and two of these mutants even to all four nucleoside analogs. Surprisingly, the mutations did not map to the five regions which are highly conserved among deoxyribonucleoside kinases. The molecular background of improved sensitivity was characterized for the double-mutant MuD (N45D, N64D), where the LD100 value of transformed KY895 decreased 316-fold for AZT and more than 11-fold for ddC when compared to wild-type Dm-dNK. Purified recombinant MuD displayed higher Km values for the native substrates than wild-type Dm-dNK and the Vmax values were substantially lower. On the other hand, the Km and Vmax values for AZT and the Km value for ddC were nearly unchanged between MuD and wild-type Dm-dNK. Additionally, a decrease in feedback inhibition of MuD by thymidine triphosphate (TTP) was found. This study demonstrates how high-frequency mutagenesis combined with a parallel selection for desired properties provides an insight into the structure-function relationships of the multisubstrate kinase from D. melanogaster. At the same time these mutant enzymes exhibit properties useful in biotechnological and medical applications.