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Thymidine kinase 1 (TK1) and deoxycytidine kinase (dCK) are required for the activation of thymidine and deoxycytidine analogs used in antiviral and anticancer therapies. Many anticancer drugs cause oxidative stress, and the rise of GSSG and other reactive oxygen species may lead to alteration in gene expression, protein, nucleic acids and lipid modifications. Here, we investigated the effects of oxidative stress and nucleoside analog on the expression and activity of TK1 and dCK. Treatment with GSSG resulted in glutathionylation of dCK and dGK but not TK1 and Dm-dNK, and glutathionylation led to increased dCK activity but decreased dGK activity. Treatment with hydrogen peroxide resulted in induction of TK1, however, the TK1 activity did not correlate with TK1 protein levels, indicating that TK1 protein was inactive. The cellular expression of dCK, however, was reduced but dCK activity was not affected at concentration ≤ 4 mM. Treatment with TFT or 5FdU resulted in downregulation of both TK1 and dCK. However, araC and dFdC treatment led to increased dCK protein but decreased dCK activity. In contrast, both TK1 protein and activity were upregulated after araC and dFdC treatment. Doxorubicin treatment led to upregulation of the TK1 but downregulation of dCK. In conclusion TK1 and dCK expression and activity are apparently affected by oxidative stress and treatment by nucleoside analogs. These results demonstrate the pharmacokinetic importance of characterizing the expression and activity of TK1 and dCK during chemotherapy with thymidine and deoxycytidine analogs in order to optimize their efficacy.

Background: Mycoplasma pneumoniae (Mpn) is a human pathogen that causes acute and chronic respiratory diseases and has been linked to many extrapulmonary diseases. Due to the lack of cell wall, Mpn is resistant to antibiotics targeting cell wall synthesis such as penicillin. During the last 10 years macrolide-resistant Mpn strains have been frequently reported in Asian countries and have been spreading to Europe and the United States. Therefore, new antibiotics are needed. In this study, 30 FDA-approved anticancer or antiviral drugs were screened for inhibitory effects on Mpn growth and selected analogs were further characterized by inhibition of target enzymes and metabolism of radiolabeled substrates., Results: Sixteen drugs showed varying inhibitory effects and seven showed strong inhibition of Mpn growth. The anticancer drug 6-thioguanine had a MIC (minimum inhibitory concentration required to cause 90% of growth inhibition) value of 0.20 μg ml(-1), whereas trifluorothymidine, gemcitabine and dipyridamole had MIC values of approximately 2 μg ml(-1). In wild type Mpn culture the presence of 6-thioguanine and dipyridamole strongly inhibited the uptake and metabolism of hypoxanthine and guanine while gemcitabine inhibited the uptake and metabolism of all nucleobases and thymidine. Trifluorothymidine and 5-fluorodeoxyuridine, however, stimulated the uptake and incorporation of radiolabeled thymidine and this stimulation was due to induction of thymidine kinase activity. Furthermore, Mpn hypoxanthine guanine phosphoribosyl transferase (HPRT) was cloned, expressed, and characterized. The 6-thioguanine, but not other purine analogs, strongly inhibited HPRT, which may in part explain the observed growth inhibition. Trifluorothymidine and 5-fluorodeoxyuridine were shown to be good substrates and inhibitors for thymidine kinase from human and Mycoplasma sources., Conclusion: We have shown that several anticancer and antiviral nucleoside and nucleobase analogs are potent inhibitors of Mpn growth and that the mechanism of inhibition are most likely due to inhibition of enzymes in the nucleotide biosynthesis pathway and nucleoside transporter. Our results suggest that enzymes in Mycoplasma nucleotide biosynthesis are potential targets for future design of antibiotics against Mycoplasma infection.

A library of purine and pyrimidine nucleosides modified with carborane or metallacarborane boron clusters at different locations, consisting of new molecules as well as already described compounds, was prepared. The compounds were tested as substrates for human deoxynucleoside kinases. Some conjugates, with modification attached to N3 of thymidine via a linker containing the triazole moiety, were efficiently phosphorylated by cytosolic thymidine kinase 1 and mitochondrial thymidine kinase 2. Higher phosphorylation levels were observed with thymidine kinase 1, the phosphorylation of nucleosides modified with metallacarboranes was observed for the first time.

To identify interactions a nucleoside analog library (NAL) consisting of 45 FDA-approved nucleoside analogs was screened against 23 enzymes of the human nucleotide metabolism using a thermal shift assay. The method was validated with deoxycytidine kinase; eight interactions known from the literature were detected and five additional interactions were revealed after the addition of ATP, the second substrate. The NAL screening gave relatively few significant hits, supporting a low rate of "off target effects." However, unexpected ligands were identified for two catabolic enzymes guanine deaminase (GDA) and uridine phosphorylase 1 (UPP1). An acyclic guanosine prodrug analog, valaciclovir, was shown to stabilize GDA to the same degree as the natural substrate, guanine, with a ΔT(agg) around 7°C. Aciclovir, penciclovir, ganciclovir, thioguanine and mercaptopurine were also identified as ligands for GDA. The crystal structure of GDA with valaciclovir bound in the active site was determined, revealing the binding of the long unbranched chain of valaciclovir in the active site of the enzyme. Several ligands were identified for UPP1: vidarabine, an antiviral nucleoside analog, as well as trifluridine, idoxuridine, floxuridine, zidovudine, telbivudine, fluorouracil and thioguanine caused concentration-dependent stabilization of UPP1. A kinetic study of UPP1 with vidarabine revealed that vidarabine was a mixed-type competitive inhibitor with the natural substrate uridine. The unexpected ligands identified for UPP1 and GDA imply further metabolic consequences for these nucleoside analogs, which could also serve as a starting point for future drug design.

Bacillus anthracis, which causes anthrax, has attracted attention because of its potential use as a biological weapon. The risk of multidrug resistance against B. anthracis increases the need for antibiotics with new molecular targets. Nucleoside analogs are well-known antiviral and anticancer prodrugs, and thymidine kinase catalyzes the rate-limiting step in the activation of pyrimidine nucleoside analogs used in chemotherapy. The thymidine kinase gene from B. anthracis Sterne strain (34F2) (Ba-TK) was cloned and expressed in E. coli, and the product was purified and characterized regarding its substrate specificity. Ba-TK phosphorylated pyrimidine nucleosides and all natural nucleoside triphosphates served as phosphate donors. Size exclusion chromatography indicated a dimeric form of Ba-TK, regardless of the presence of ATP. Thymidine was the most efficient substrate with a low K(m) value (0.6 microM) and a V(max) of 3.3 micromol dTMP mg(-1) min(-1), but deoxyuridine (K(m)=4.2 microM, V(max)=4.1 micromol dUMP mg(-1) min(-1)) was also a good substrate. Several pyrimidine analogs were also tested and analogs with 5-position modifications showed higher activities compared to analogs with 3'- and N3-position modifications. Deoxyuridine analogs were the most potent inhibitors of B. anthracis growth in vitro. These results may be used to guide future development of nucleoside analogs against B. anthracis.

Ureaplasma urealyticum (U. urealyticum), belonging to the class Mollicutes, is a human pathogen colonizing the urogenital tract and causes among other things respiratory diseases in premature infants. We have studied the salvage of pyrimidine deoxynucleosides in U. urealyticum and cloned a key salvage enzyme, thymidine kinase (TK) from U. urealyticum. Recombinant Uu-TK was expressed in E. coli, purified and characterized with regards to substrate specificity and feedback inhibition. Uu-TK efficiently phosphorylated thymidine (dThd) and deoxyuridine (dUrd) as well as a number of pyrimidine nucleoside analogues. All natural ribonucleoside/deoxyribonucleoside triphosphates, except dTTP, served as phosphate donors, while dTTP was a feedback inhibitor. The level of Uu-TK activity in U. urealyticum extracts increased upon addition of dUrd to the growth medium. Fluoropyrimidine nucleosides inhibited U. urealyticum and M. pneumoniae growth and this inhibitory effect could be reversed by addition of dThd, dUrd or deoxytetrahydrouridine to the growth medium. Thus, the mechanism of inhibition was most likely the depletion of dTTP, either via a blocked thymidine kinase reaction and/or thymidylate synthesis step and these metabolic reactions should be suitable targets for antimycoplasma chemotherapy.

To identify interactions a nucleoside analog library (NAL) consisting of 45 FDA-approved nucleoside analogs was screened against 23 enzymes of the human nucleotide metabolism using a thermal shift assay. The method was validated with deoxycytidine kinase; eight interactions known from the literature were detected and five additional interactions were revealed after the addition of ATP, the second substrate. The NAL screening gave relatively few significant hits, supporting a low rate of "off target effects." However, unexpected ligands were identified for two catabolic enzymes guanine deaminase (GDA) and uridine phosphorylase 1 (UPP1). An acyclic guanosine prodrug analog, valaciclovir, was shown to stabilize GDA to the same degree as the natural substrate, guanine, with a ΔTagg around 7°C. Aciclovir, penciclovir, ganciclovir, thioguanine and mercaptopurine were also identified as ligands for GDA. The crystal structure of GDA with valaciclovir bound in the active site was determined, revealing the binding of the long unbranched chain of valaciclovir in the active site of the enzyme. Several ligands were identified for UPP1: vidarabine, an antiviral nucleoside analog, as well as trifluridine, idoxuridine, floxuridine, zidovudine, telbivudine, fluorouracil and thioguanine caused concentration-dependent stabilization of UPP1. A kinetic study of UPP1 with vidarabine revealed that vidarabine was a mixed-type competitive inhibitor with the natural substrate uridine. The unexpected ligands identified for UPP1 and GDA imply further metabolic consequences for these nucleoside analogs, which could also serve as a starting point for future drug design. [ABSTRACT FROM AUTHOR]

Abstract: SUCLA2-related mitochondrial DNA (mtDNA) depletion syndrome is a result of mutations in the β subunit of the ADP-dependent isoform of the Krebs cycle succinyl-CoA synthase (SCS). The mechanism of tissue specificity and mtDNA depletion is elusive but complementation by the GDP-dependent isoform encoded by SUCLG2, and the association with mitochondrial nucleoside diphosphate kinase (NDPK), is a plausible link. We have investigated this relationship by studying SUCLA2 deficient fibroblasts derived from patients and detected normal mtDNA content and normal NDPK activity. However, knockdown of SUCLG2 by shRNA in both patient and control fibroblasts resulted in a significant decrease in mtDNA amount, decreased NDPK and cytochrome c oxidase activities, and a marked growth impairment. This suggests that, SUCLG2, to a higher degree than SUCLA2, is crucial for mtDNA maintenance and that mitochondrial NDPK is involved. Although results pertain to a cell culture system, the findings might explain the pathomechanism and tissue specificity in mtDNA depletion caused by defective SUCLA2. [Copyright &y& Elsevier]

Thymidine kinase (TK) is the key enzyme in salvaging thymidine to produce thymidine monophosphate. Owing to its ability to phosphorylate nucleoside analogue prodrugs, TK has gained attention as a rate-limiting drug activator. We describe the structures of two bacterial TKs, one from the pathogen Bacillus anthracis in complex with the substrate dT, and the second from the food-poison-associated Bacillus cereus in complex with the feedback inhibitor dTTP. Interestingly, in contrast with previous structures of TK in complex with dTTP, in this study dTTP occupies the phosphate donor site and not the phosphate acceptor site. This results in several conformational changes compared with TK structures described previously. One of the differences is the way tetramers are formed. Unlike B. anthracis TK, B. cereus TK shows a loose tetramer. Moreover, the lasso-domain is in open conformation in B. cereus TK without any substrate in the active site, whereas in B. anthracis TK the loop conformation is closed and thymidine occupies the active site. Another conformational difference lies within a region of 20 residues that we refer to as phosphate-binding β-hairpin. The phosphate-binding β-hairpin seems to be a flexible region of the enzyme which becomes ordered upon formation of hydrogen bonds to the α-phosphate of the phosphate donor, dTTP. In addition to descriptions of the different conformations that TK may adopt during the course of reaction, the oligomeric state of the enzyme is investigated. [ABSTRACT FROM AUTHOR]

Thymidine kinases have been found in most organisms, from viruses and bacteria to mammals. Ureaplasma urealyticum ( parvum), which belongs to the class of cell-wall-lacking Mollicutes, has no de novo synthesis of DNA precursors and therefore has to rely on the salvage pathway. Thus, thymidine kinase ( Uu-TK) is the key enzyme in dTTP synthesis. Recently the 3D structure of Uu-TK was determined in a feedback inhibitor complex, demonstrating that a lasso-like loop binds the thymidine moiety of the feedback inhibitor by hydrogen bonding to main-chain atoms. Here the structure with the substrate deoxythymidine is presented. The substrate binds similarly to the deoxythymidine part of the feedback inhibitor, and the lasso-like loop binds the base and deoxyribose moieties as in the complex determined previously. The catalytic base, Glu97, has a different position in the substrate complex from that in the complex with the feedback inhibitor, having moved in closer to the 5′-OH of the substrate to form a hydrogen bond. The phosphorylation of and inhibition by several nucleoside analogues were investigated and are discussed in the light of the substrate binding pocket, in comparison with human TK1. Kinetic differences between Uu-TK and human TK1 were observed that may be explained by structural differences. The tight interaction with the substrate allows minor substitutions at the 3 and 5 positions of the base, only fluorine substitutions at the 2′-Ara position, but larger substitutions at the 3′ position of the deoxyribose. [ABSTRACT FROM AUTHOR]

In the present study, we investigated the effect of fluoropyrimidines on the growth of Ureaplasma urealyticum. Addition of fluoropyrimidines strongly inhibited bacterial growth. Growth inhibition by these analogues could be reversed by addition of either thymidine or deoxyuridine, suggesting inhibition of thymidylate biosynthesis as the mechanism in operation. [ABSTRACT FROM AUTHOR]

Mycoplasmas are unable to synthesize purine and pyrimidine bases de novo. Therefore, salvage of existing nucleosides and bases is essential for their survival. Four mycoplasma species were studied with regard to their ability to phosphorylate deoxynucleosides. High levels of thymidine kinase (TK), deoxycytidine kinase (dCK), deoxyguanosine kinase (dGK) and deoxyadenosine kinase (dAK) activities were detected in extracts from Mycoplasma pneumoniae, Mycoplasma mycoides subsp. mycoides SC (M. mymySC), Acholeplasma laidlawii (A. laidlawii) and Mycoplasma arginini (M. arginini). Nucleoside phosphotransferase activities were found at high levels in A. laidlawii and low levels in M. arginini. Pyrophosphate-dependent deoxynucleoside kinase activities were detected mainly in A. laidlawii and M. mymySC extracts. Two open reading frames were identified in the M. mymySC genome; one showed 25% sequence identity to human dGK and the other one had about 26% sequence identity to human TK1. The M. mymySC dGK-like enzyme was cloned, expressed in Escherichia coli and affinity-purified. This enzyme phosphorylated dAdo, dGuo and dCyd, and the highest catalytic rate was with dAdo as substrate. Therefore, we suggest that this enzyme should be named deoxyadenosine kinase. The physiological role of mycoplasma dAK and TK may be to support the unusually large dATP and dTTP pools required for replication of mycoplasma genomes. [ABSTRACT FROM AUTHOR]

Mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) catalyze the initial rate limiting phosphorylation of deoxynucleosides and are essential enzymes for mitochondrial function. Chemotherapy using nucleoside analogs is often associated with mitochondrial toxicities. Here we showed that incubation of U2OS cells with didanosine (ddI, 2',3'-dideoxyinosine), a purine nucleoside analog used in the highly active antiretroviral therapy (HAART), led to selective degradation of both mitochondrial TK2 and dGK while the cytosolic deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1) were not affected. Addition of guanosine to the ddI-treated cells prevented the degradation of mitochondrial TK2 and dGK. The levels of intracellular reactive oxygen species and protein oxidation in ddI-treated and control cells were also measured. The results suggest that down-regulation of mitochondrial TK2 and dGK may be a mechanism of mitochondrial toxicity caused by antiviral and anticancer nucleoside analogs. [ABSTRACT FROM AUTHOR]

Abstract: The pathogenic mycoplasma Ureaplasma parvum (Up) causes opportunistic infections and relies on salvage of nucleosides for DNA synthesis and Up thymidine kinase (UpTK) provides the necessary thymidine nucleotides. The anti-HIV compound 3´-azido-3′-deoxythymidine (AZT) is a good substrate for TK. Methods for a rapid and efficient synthesis of new 3′-α-[1,2,3]triazol-3′-deoxythymidine analogs from AZT under Huisgen conditions are described. Thirteen 3′-analogues were tested with human cytosolic thymidine kinase (hTK1) and UpTK. The new analogs showed higher efficiencies (K m/V max values) in all cases with UpTK than with hTK1. Still, hTK1 was preferentially inhibited by 9 out of 10 tested analogs. Structural models of UpTK and hTK1 were constructed and used to explain the kinetic results. Two different binding modes of the nucleosides within the active sites of both enzymes were suggested with one predominating in the bacterial enzyme and the other in hTK1. These results will aid future development of anti-mycoplasma nucleosides. [Copyright &y& Elsevier]