Pyrimidine Nucleoside Kinase Research Articles

Potential application of thymidylate kinase in nucleoside analogue activation in Plasmodium falciparum

Plasmodium falciparum thymidylate kinase ( PfTMPK) shows a broad range of substrate tolerance when compared to the corresponding human enzyme. Besides 2′-deoxythymidine monophosphate (dTMP), PfTMPK can phosphorylate 3′-azido-2′,3′-dideoxythymidine monophosphate (AZTMP) very efficiently. In contrast, human thymidylate kinase (hTMPK) is 200 times less active towards AZTMP. We were interested to see if we could use PfTMPK to activate 3′-azido-2′,3′-deoxythymidine (AZT) derivatives as a strategy to treat malaria. P. falciparum lacks a pyrimidine nucleoside kinase which usually activates nucleoside and nucleoside analogues to the corresponding monophosphates. Therefore, several prodrug analogues of AZT and related nucleoside monophosphates were prepared and analysed for antiparasitic activity. The prodrugs showed an increase in activity over the parent nucleoside analogues, which showed no inhibition of parasite growth at the concentration tested. The evidence here reported provides a strategy that could be exploited for further anti-malarial design.

Hematopoiesis in the thymidine kinase 2 deficient mouse model of mitochondrial DNA depletion syndrome

Mitochondria are important for normal blood-cell development, and several diseases linked to mitochondrial DNA (mtDNA) show hematological manifestations. We recently generated a mouse strain deficient in expression of the mitochondrial pyrimidine nucleoside kinase thymidine kinase 2 (Tk2), showing that these mice exhibit progressive mtDNA depletion in multiple organs. We used this mouse strain as a model for mtDNA depletion syndromes to investigate the effects of mtDNA depletion on hematopoiesis. MtDNA levels in spleen from the Tk2-deficient mice were decreased 50%, but in contrast to all other investigated organs, both thymus and peripheral blood leukocytes showed normal mtDNA levels. Analysis of peripheral blood and cell populations in spleen, thymus, and bone marrow showed normal findings in the Tk2-deficient mice. The total rates of thymidine phosphorylation-which also include phosphorylation catalyzed by cytosolic Tk 1-in both spleen and thymus from wild-type mice were >50-fold higher than in liver, brain, and muscle. In summary, our data show that blood cells are less dependent on mitochondrial Tk2 compared with several other tissues and that these cells can synthesize deoxyribonucleotides required for mtDNA replication by alternative pathways such as phosphorylation of thymidine by cytosolic Tk1.

Mechanism of Action of (-)-(2R,4R)-1-(2-hydroxymethyl-1,3-dioxolan-4-yl)thymine as an anti-HIV agent

(-)-(2R,4R)-1-(2-Hydroxymethyl-1,3-dioxolan-4yl)thymine (DOT) is a thymidine analogue that has potent in vitro activity against wild-type and nucleoside reverse transcriptase inhibitor (NRTI)-resistant HIV. For nucleoside analogues to inhibit viral replication, they must be metabolized to the active triphosphate, which inhibits the viral reverse transcriptase (RT). Using purified enzymes, the kinetics of DOT phosphorylation, inhibition of wild-type and drug-resistant HIV-1 reverse transcriptase activity, and excision of DOT-5'-monophosphate (DOT-MP) from a chain-terminated primer were examined. DOT was phosphorylated by human thymidine kinase-1 (TK-1) but not by other pyrimidine nucleoside kinases, including the mitochondrial thymidine kinase (TK-2). Resistance to NRTIs involves decreased binding/incorporation and/or increased excision of the chain-terminating NRTI. RTs containing the D67N/K70R/T215Y/K219Q or T695-SS/T215Y mutations show enhanced removal of DOT-MP from terminated primer as well as approximately four-fold decreased binding/incorporation. The Q151M and K65R mutations appear to cause decreased inhibition by DOT-TP. However, both the K65R and Q151M mutations show decreased excision, which would confer greater stability on the terminated primer. These opposing mechanisms could offset the overall resistance profile and susceptibility. Little or no resistance was observed with the enzymes harbouring mutations resistant to lamivudine (M184V) and non-nucleoside RT inhibitors (K103N).

Acyclic nucleoside analogues as novel inhibitors of human mitochondrial thymidine kinase.

A series of acyclic nucleoside analogues of 5'-O-tritylthymidine have been synthesized and evaluated as potential human mitochondrial thymidine kinase (TK-2) inhibitors. In this series, the sugar moiety of the parent 5'-O-tritylthymidine has been replaced by aliphatic chains including (E)- and (Z)-butenol, butynol, or butanol. Among them the (Z)-butenyl derivative (10) showed an IC(50) against TK-2 of 1.5 microM, being 1 order of magnitude more potent than the parent 5'-O-tritylthymidine. This lead compound has been further modified by replacing the thymine base by other pyrimidine bases such as 5-iodouracil, 5-ethyluracil, 5-methylcytosine, 3-N-methylthymine, or 5,6-dihydrothymine, as well as by the purine base guanine. The trityl group has also been replaced by different aliphatic and aromatic acyl moieties including tert-butylacetyl, hexanoyl, decanoyl, and diphenylacetyl moieties. The evaluation of the compounds against TK-2 and the phylogenetically close HSV-1 TK has shown that the base moiety plays a crucial role in their interaction against these pyrimidine nucleoside kinases. Also, the presence of a lipophilic substituent, preferentially an aromatic moiety such as diphenylmethyl or triphenylmethyl, is required for efficient TK-2 inhibition. Whereas some compounds showed marked specificity for either TK-2 (i.e, the 5,6-dihydrothymine derivative, 26) or HSV-1 TK (i.e., the butynyl derivative, 11), some others, including the (Z)-and (E)-butenyl derivatives 10 and 12, showed significant inhibition against both enzymes. They also proved to be inhibitory against HSV-1 TK in intact human osteosarcoma cells that were transduced with the HSV-1 TK gene.

Selective abolishment of pyrimidine nucleoside kinase activity of herpes simplex virus type 1 thymidine kinase by mutation of alanine-167 to tyrosine.

Herpes simplex virus type 1 (HSV-1) encodes a thymidine kinase (TK) that markedly differs from mammalian nucleoside kinases in terms of substrate specificity. It recognizes both pyrimidine 2'-deoxynucleosides and a variety of purine nucleoside analogs. Based on a computer modeling study and in an attempt to modify this specificity, an HSV-1 TK mutant enzyme containing an alanine-to-tyrosine mutation at amino acid position 167 was constructed. Compared with wild-type HSV-1 TK, the purified mutant HSV-1 TK(A167Y) enzyme was heavily compromised in phosphorylating pyrimidine nucleosides such as (E)-5-(2-bromovinyl)-2'-deoxyuridine and the natural substrate dThd, whereas its ability to phosphorylate the purine nucleoside analogs ganciclovir (GCV) and lobucavir was only reduced approximately 2-fold. Moreover, a markedly decreased competition of natural pyrimidine nucleosides (i.e., thymidine) with purine nucleoside analogs for phosphorylation by HSV-1 TK(A167Y) was observed. Human osteosarcoma cells transduced with the wild-type HSV-1 TK gene were extremely sensitive to the cytostatic effects of antiherpetic pyrimidine [i.e., (E)-5-(2-bromovinyl)-2'-deoxyuridine] and purine (i.e., GCV) nucleoside analogs. Transduction with the HSV-1 TK(A167Y) gene sensitized the osteosarcoma cells to a variety of purine nucleoside analogs, whereas there was no measurable cytostatic activity of pyrimidine nucleoside analogs. The unique properties of the A167Y mutant HSV-1 TK may give this enzyme a therapeutic advantage in an in vivo setting due to the markedly reduced dThd competition with GCV for phosphorylation by the HSV-1 TK.

Cytidine Kinase: A Novel Pyrimidine Ribonucleoside Phosphorylating Enzyme in Escherzchza Coli

A previously undescribed pyrimidine ribonucleoside kinase that preferentially phosphorylates cytidine to yield its 5′-monophosphate form has been purified to near homogeneity from Escherichia coli.The enzyme was distinct from the known pyrimidine nucleoside kinase in the chromatographic profile, substrate specificity and molecular weight. * Telephone: +81-76-229-8828, Fax: +81-76-229-1352 †Present address: Kanazawa Gakuin College, 10 Sue-machi, Kanazawa 920-13, Japan. T.K. submitted this work as partial fulfillment of the requirement for Ph.D. at Kanazawa University.

Biosynthesis and scavenging of pyrimidines by pathogenic mycobacteria

Mycobacterium microti incorporated a wide range of exogenously supplied pyrimidines into its nucleic acids. M. avium incorporated a relatively narrow range of pyrimidines but both M. avium and M. microti when recovered after growth in vivo incorporated a slightly wider range of pyrimidines than the same strains grown in vitro. M. microti and M. leprae could not take up uridine nucleotides directly but could utilize the pyrimidines by hydrolysing them to uridine and then taking up the uridine. Pyrimidine biosynthesis, judged by the ability to incorporate carbon from CO2 or aspartate into pyrimidines was readily detected in non-growing suspensions of M. microti and M. avium harvested from Dubos medium, which does not contain pyrimidines. The biosynthetic activity was diminished in mycobacteria grown in vivo when there is likely to be a source of pyrimidines which they might use. Relative activities for pyrimidine biosynthesis de novo in M. microti were 100 for cells isolated from Dubos medium, 6 for cells isolated from Dubos medium containing the pyrimidine cytidine and 11 from cells recovered after growth in mice. In contrast, relative activities for a scavenging reaction, uracil incorporation, were 100, 71 and 59, respectively. Three key enzymes in the pathway of pyrimidine biosynthesis de novo were detected in M. microti and M. avium. Two, dihydroorotate synthase and orotate phosphoribosyltransferase appeared to be constitutive in M. microti and M. avium. Aspartate transcarbamoylase activity was higher in these mycobacteria grown in vivo than in Dubos medium but it was repressed in M. microti or M. avium grown in Dubos medium in the presence of 50 microM-pyrimidine. Aspartate transcarbamoylase was strongly inhibited by the feedback inhibitors ATP, CTP and UTP. Enzymes for scavenging pyrimidines were detected at low specific activities in all mycobacteria studied. Activities of phosphoribosyltransferases, enzymes that convert bases directly to nucleotides, were not related to the ability of intact mycobacteria to take up pyrimidine bases while activities of pyrimidine nucleoside kinases were generally related to the ability of intact mycobacteria to take up nucleosides. Phosphoribosyltransferase activity for uracil, cytosine, orotic acid and--in organisms grown in Dubos medium with 50 microM-uridine-thymine, as well as kinases for uridine, deoxyuridine, cytidine and thymidine were detected in M. microti. However, M. avium only contained uracil and orotate phosphoribosyltransferase, uridine, cytidine and thymidine kinase, and additionally deoxyuridine kinase when grown axenically with 50 microM-uracil, reflecting its more limited abilities in pyrimidine scavenging.

Rat cytomegalovirus induces cellular purine and pyrimidine nucleoside kinases in rat embryo fibroblasts and TK− rat-2 cells. Correlations with the antiviral activity of acyclovir

Rat cytomegalovirus (RCMV) induces a cytosol thymidine kinase (TK) in G0-phase rat embryo fibroblasts (REF), but not in a TK deficient rat cell line (R-2), though virus titers in both cell types reached comparable levels. The results indicate that TK is neither virus-coded nor is required for a productive infection in R-2 cells. A deoxycytidine kinase (dCK) is induced in either growing or RCMV-infected REF and R-2 cells, suggesting that dCK is essential for both host-cell and viral DNA synthesis. A deoxyguanosine kinase (dGK) is detectable in low concentrations in either growing or G0-phase REF and R-2 cells suggesting that this enzyme is cell-cycle independent. In contrast, RCMV induces high persisting levels of dGK, particularly in R-2 cells, indicating that this enzyme is of crucial importance for viral DNA synthesis. By comparison of thermostabilities and electrophoretic mobilities (Rf for TK, dCK and dGK were 0.12; 0.97; and 0.54, respectively) the enzymes were found to be substrate specific but of cellular origin. In contrast to TK and dCK, only dGK is inhibited by Acyclovir (Ki = 320 microM). It is suggested that RCMV inducable dGK is an important enzyme determining the in vitro anti-CMV activity of Acyclovir.

Salvage of pyrimidine nucleosides by Trichomonas vaginalis

Trichomonas vaginalis is incapable of de novo pyrimidine biosynthesis because it cannot incorporate bicarbonate, aspartate or orotate into its pyrimidine nucleotides or nucleic acids. The organism can salvage exogenous cytidine > uridine > uracil and thymidine, and incorporate them into the nucleotide pool. A portion of cytidine is converted to CMP, CDP and CTP by cytidine phosphotransferase and nucleotide kinases. Some cytidine and most of uracil are, however, converted first to uridine by cytidine deaminase and uridine phosphorylase respectively; uridine is then incorporated into UMP, UDP and UTP by uridine phosphotransferase and nucleotide kinases. The two phosphotransferases, found mainly in the non-sedimentable fraction of T. vaginalis, provide the main avenue of pyrimidine salvage. No significant levels of pyrimidine phosphoribosyl transferase or nucleoside kinases can be detected in the extract. T. vaginalis has no appreciable dihydrofolate reductase or thymidylate synthetase; it grows normally in millimolar concentrations of methotrexate, pyrimethamine, or trimethoprim, and cannot incorporate labels from exogenous uracil or uridine into DNA. It has an enzyme thymidine phosphotransferase in the sedimentable fraction which converts thymidine to TMP. Thymidine salvage in T. vaginalis is thus totally isolated from the rest of the pyrimidine salvage.

Capacity of deoxycytidine to selectively antagonize cytotoxicity of 5-halogenated analogs of deoxycytidine without loss of antiherpetic activity.

Enzyme kinetic studies from this laboratory (M. Dobersen and S. Greer, Biochemistry 17:920-928, 1978) suggested that deoxycytidine could antagonize the toxicity of 5-halogenated analogs of deoxycytidine without interfering with their antiviral activity. Antagonism by deoxycytidine of the toxicity of 5-chlorodeoxycytidine without impairing its anti-herpes simplex virus type 2 activity is demonstrated in the present studies. Tetrahydrouridine, an inhibitor of cytidine deaminase, was utilized. The high Km for deoxycytidine (0.6 mM) with respect to the herpes pyrimidine nucleoside kinase as compared with the low Km for 5-chlorodeoxycytidine (1.1 microM) accounts for the absence of antagonism of the antiviral activity. The high Km for 5-chlorodeoxycytidine (56 microM) as compared with the low Km of deoxycytidine (2 microM) with respect to mammalian deoxycytidine kinase accounts, in great part, for the antagonism of toxicity. In addition, antagonism of toxicity by deoxycytidine is the result of factors other than the kinetic parameters of nucleoside kinases, as indicated by its antagonism of the cytotoxicity of 5-chlorodeoxyuridine. This may be attributed to replenishment of low dCTP pools, diminished because of effector inhibition of ribonucleoside diphosphate reductase by Cl-dUTP. Resistance of the herpes-encoded enzymes to effector control may also play a role in the selective antagonism. Cell culture studies with high concentrations of tetrahydrouridine and 2'-deoxytetrahydrouridine suggest that competition by deoxycytidine for deaminases may not play a major role. The fact that deoxycytidine antagonizes the toxicity of chlorodeoxyuridine also argues against competition for the deaminases as a major reason for its effect. Limited studies with a topical herpes simplex virus type 2 infection system indicate heightened efficacy of 5-chlorodeoxycytidine (and tetrahydrouridine) when deoxycytidine is coadministered. The concepts of selective antagonism of a chemotherapeutic agent derived from these studies may be applied to other approaches that extent beyond viral chemotherapy.

Comparison of pyrimidine and purine nucleoside secretion and nucleoside kinase expression in resident and elicited peritoneal macrophages.

Thymidine was found in culture supernatants from both normal peritoneal macrophages and macrophages elicited in vivo with LPS under conditions in which there was no detectable TK activity. Abrogation of thymidine secretion was observed only when elicited macrophages were cultured in the presence of macrophage growth factor (MGF), whereas resident macrophages continued secreting under similar conditions. Similarly, TK activity was induced with MGF only in elicited, but not in resident, macrophages; LPS added in vitro failed to render resident macrophages susceptible to stimulation by MGF. Tests for adenosine and deoxyadenosine in macrophage supernatants proved that these nucleosides were not secreted by these cells. Although AK was consistently expressed in both resident and elicited macrophages, no significant dAK levels could be detected in either cell population. In contrast to TK, both purine nucleoside kinases could not be modulated or induced with MGF. The data indicate different reactivity to MGF by resident and elicited macrophages and a difference in pyrimidine vs purine nucleoside secretion as well as nucleoside kinase expression by these cells.

A simple radioisotopic assay for nucleoside kinases employing alumina for separation of nucleosides and nucleotides

A rapid, simple, and sensitive radiochemical assay for the measurement of purine or pyrimidine nucleoside kinases (EC 2.7.1.-) is described. The substrate (thymidine, deoxyuridine, deoxycytidine, deoxyguanosine, deoxyadenosine, uridine, cytidine, and adenosine) is separated from the product (the respective 5′-nucleotide) on neutral alumina columns which retain the nucleotides but not the nucleosides. The nucleotides are recovered by elution with 0.4 m sodium phosphate buffer, pH 7.6.

Herpes simplex virus type 2 induced pyrimidine nucleoside kinase: enzymic basis for the selective antiherpetic effect of 5-halogenated analogs of deoxycytidine

Herpes simplex virus type 2 induced pyrimidine nucleoside kinase: enzymic basis for the selective antiherpetic effect of 5-halogenated analogs of deoxycytidine

PEI-cellulose thin-layer chromatography: A highly versatile system for separating purine and pyrimidine nucleotides from nucleosides and free bases

A simple, rapid, and highly versatile thin-layer system is described for separating purine and pyrimidine nucleotides from their respective nucleosides and free bases. This system, which employs anion-exchange chromatography on PEI-cellulose, retains both ribonucleotides and deoxyribonucleotides at the origin and permits ribonucleosides, deoxyribonucleosides, and free bases to migrate away. Distilled water serves as the developing solvent. The ability to retain nucleotides at the origin makes PEI-cellulose ideally suited for use in the assay of nucleotide synthesizing enzymes. These enzymes include purine phosphoribosyl-transferases, purine nucleoside kinases, and pyrimidine nucleoside kinases.

Influence of glucosamine on the uptake of nucleosides by chick fibroblasts and on the incorporation into RNA

As a consequence of the reduction of the UTP pool by glucosamine the velocity of the uptake of uridine into the cells is drastically increased. Since the same effect is observed for the uptake of cytidine but not of adenosine, it is concluded that UTP (or a derivative of it) acts in vivo by negative feedback on the pyrimidine nucleoside kinase or a common transport system. After increasing the UTP or CTP pools the velocity of the uptake of both pyrimidine nucleosides is markedly reduced. Therefore, also CTP should regulate the uptake of pyrimidine nucleosides by negative feedback. Furthermore, glucosamine interferes with the conversion of UTP to CTP.

The appearance of pyrimidine nucleoside and deoxynucleoside kinase activities in peanut axes

The appearance of pyrimidine nucleoside and deoxynucleoside kinase activities in peanut axes was followed during the first 96 hr of germination. The activation or synthesis of these enzymes preceded increases in RNA, DNA and fresh weight. The incorporation of DNA precursors into DNA occurred only after the formation of the pyrimidine deoxynucleoside kinases.

Enzymes of uracil metabolism in the Ehrlich ascites tumour and mammalian liver

The content of enzymes involved in uracil metabolism (pyrimidine nucleoside phosphorylase, pyrimidine nucleoside kinase and enzymes of uracil catabolism) in extracts from rat liver, mouse liver and the Ehrlich ascites tumour was studied. Much higher levels of enzymes involved in anabolic reactions were found in ascites tumour extracts than in liver, whereas the enzymes of uracil catabolism which were present in rat and mouse liver were absent from the tumour. Especially the relatively high concentration of pyrimidine nucleoside kinase in the tumour extracts probably explains the better utilization of free uracil for polynucleotide synthesis in this tissue as compared to rat liver. Pyrimidine nucleoside kinase needed a high concentration of uridine ( ca. 0.01 M) for optimal synthesis of nucleosides, while a considerably lower concentration of cytidine (less than 0.0055 M) was sufficient. This partly explains the much better utilization of cytidine for polynucleotide synthesis as compared to uridine.