Mechanisms of substrate selectivity for Bacillus anthracis thymidylate kinase

Bacillus anthracis is well known in connection with biological warfare. The search for new drug targets and antibiotics is highly motivated because of upcoming multiresistant strains. Thymidylate kinase is an ideal target since this enzyme is at the junction of the de novo and salvage synthesis of dTTP, an essential precursor for DNA synthesis. Here the expression and characterization of thymidylate kinase from B. anthracis (Ba-TMPK) is presented. The enzyme phosphorylated deoxythymidine-5'-monophosphate (dTMP) efficiently with K (m) and V (max) values of 33 microM and 48 micromol mg(-1) min(-1), respectively. The efficiency of deoxyuridine-5'-monophosphate phosphorylation was approximately 10% of that of dTMP. Several dTMP analogs were tested, and D-FMAUMP (2'-fluoroarabinosyl-5-methyldeoxyuridine-5'-monophosphate) was selectively phosphorylated with an efficiency of 172% of that of D-dTMP, but L-FMAUMP was a poor substrate as were 5-fluorodeoxyuridine-5'-monophosphate (5FdUMP) and 2',3'-dideoxy-2',3'-didehydrothymidine-5'-monophosphate (d4TMP). No activity could be detected with 3'-azidothymidine-5'-monophosphate (AZTMP). The corresponding nucleosides known as efficient anticancer and antiviral compounds were also tested, and d-FMAU was a strong inhibitor with an IC(50) value of 10 microM, while other nucleosides--L-FMAU, dThd, 5-FdUrd, d4T, and AZT, and 2'-arabinosylthymidine--were poor inhibitors. A structure model was built for Ba-TMPK based on the Staphylococcus aureus TMPK structure. Docking with various substrates suggested mechanisms explaining the differences in substrate selectivity of the human and the bacterial TMPKs. These results may serve as a start point for development of new antibacterial agents.     

Selective assays for thymidine kinase 1 and 2 and deoxycytidine kinase and their activities in extracts from human cells and tissues.

Human cells salvage pyrimidine deoxyribonucleosides via 5'-phosphorylation which is also the route of activation of many chemotherapeutically used nucleoside analogs. Key enzymes in this metabolism are the cytosolic thymidine kinase (TK1), the mitochondrial thymidine kinase (TK2) and the cytosolic deoxycytidine kinase (dCK). These enzymes are expressed differently in different tissues and cell cycle phases, and they display overlapping substrate specificities. Thymidine is phosphorylated by both thymidine kinases, and deoxycytidine is phosphorylated by both dCK and TK2. The enzymes also phosphorylate nucleoside analogs with very different efficiencies. Here we present specific radiochemical assays for the three kinase activities utilizing analogs as substrates that are by more than 90 percent phosphorylated solely by one of the kinases; i.e. 3'-azido-2',3'-dideoxythymidine (AZT) as substrate for TK1, 1-beta-D-arabinofuranosylthymidine (AraT) for TK2 and 2-chlorodeoxyadenosine (CdA) for dCK. We determined the fraction of the total deoxycytidine and thymidine phosphorylating activity that was provided by each of the three enzymes in different human cells and tissues, such as resting and proliferating lymphocytes, lymphocytic cells of leukemia patients (chronic lymphocytic, chronic myeloic and hairy cell leukemia), muscle, brain and gastrointestinal tissue. The detailed knowledge of the pyrimidine deoxyribonucleoside kinase activities and substrate specificities are of importance for studies on chemotherapeutically active nucleoside analogs, and the assays and data presented here should be valuable tools in that research.     

Cloning and characterization of the multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster.

A Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) was reported to phosphorylate all four natural deoxyribonucleosides as well as several nucleoside analogs (Munch-Petersen, B., Piskur, J., and Sondergaard, L. (1998) J. Biol. Chem. 273, 3926-3931). The broad substrate specificity of this enzyme together with a high catalytic rate makes it unique among the nucleoside kinases. We have in the present study cloned the Dm-dNK cDNA, expressed the 29-kDa protein in Escherichia coli, and characterized the recombinant enzyme for the phosphorylation of nucleosides and clinically important nucleoside analogs. The recombinant enzyme preferentially phosphorylated the pyrimidine nucleosides dThd, dCyd, and dUrd, but phosphorylation of the purine nucleosides dAdo and dGuo was also efficiently catalyzed. Dm-dNK is closely related to human and herpes simplex virus deoxyribonucleoside kinases. The highest level of sequence similarity was noted with human mitochondrial thymidine kinase 2, and these enzymes also share many substrates. The cDNA cloning and characterization of Dm-dNK will be the basis for studies on the use of this multisubstrate nucleoside kinase as a suicide gene in combined gene/chemotherapy of cancer.     

Cloning,expression, and purification of His-tagged rat mevalonate kinase

Mevalonate kinase catalyzes the phosphorylation of mevalonic acid to form mevalonate 5-phosphate, which plays a key role in regulating cholesterol biosynthesis in animal cells. Deficiency of mevalonate kinase activity in the human body has been linked to mevalonic aciduria and hyperimmunoglobulinemia D/periodic fever syndrome (HIDS). We cloned the gene of rat mevalonate kinase into a bacterial expression vector pLM1 with six continuous histidine codons attached to the 5(') of the gene. The cloned gene was overexpressed in Escherichia coli and the soluble protein was purified with a nickel HiTrap chelating metal affinity column in 90% yield to apparent homogeneity. The purified rat mevalonate kinase had a dimeric structure composed of identical subunits. Based on SDS-PAGE, the subunit was 42 kDa. The specific activity of the purified His-tagged rat mevalonate kinase was 32.7 micromol/min/mg and the optimal pH was found to be 7.0-8.0 in phosphate buffer. The Michaelis constant K(M) was 35 microM for (RS)-mevalonate and 953 microM for ATP, respectively. The V(max) was determined to be 38.7 micromol/min/mg. The overexpression of rat mevalonate kinase in E. coli and one-step purification of the highly active rat mevalonate kinase will facilitate further our investigation of this enzyme through site-directed mutagenesis and enzyme-catalyzed reactions with substrate analogs.     

Phosphorylation of the yeast choline kinase by protein kinase C. Identification of Ser25 and Ser30 as major sites of phosphorylation

The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent and dependent on the concentrations of choline kinase (K(m) = 27 microg/ml) and ATP (K(m) = 15 microM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSSQRRHS (V5max/K(m) = 17.5 mm(-1) micromol min(-1) mg(-1)) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway, whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Although the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHSLTRQ) containing Ser30 was a substrate (V(max)/K(m) = 3.0 mm(-1) micromol min(-1) mg(-1)) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C.     

Carbocyclic thymidine derivatives efficiently inhibit Plasmodium falciparum thymidylate kinase (PfTMK)

During the course of our research into new anti-malaria drugs, Plasmodium falciparum thymidylate kinase (PfTMK) has emerged as an important drug target because of its unique substrate specificity. Compared with human thymidylate kinase (HsTMK), PfTMK shows broader substrate specificity, which includes both purine and pyrimidine nucleotides. PfTMK accepts both 2'-deoxyguanosine monophosphate (dGMP) and thymidine monosphosphate (TMP) as substrates. We have evaluated the inhibitory activity of seven carbocyclic thymidine analogs and report the first structure-activity relationship for these inhibitors against PfTMK. The 2',3' dideoxycarbocyclic derivative of thymidine showed the most potent inhibition of the enzyme. The K(i)(dTMP) and K(i)(dGMP) values were 20 and 7 μM respectively. Thus, further modifications of carbocyclic thymidine analogs represent a good strategy for developing more powerful thymidylate kinase inhibitors.     

Sodium-dependent nucleoside transport in choroid plexus from rabbit. Evidence for a single transporter for purine and pyrimidine nucleosides.

The overall goal of this study was to determine the mechanisms by which nucleosides are transported in choroid plexus. Choroid plexus tissue slices obtained from rabbit brain were depleted of ATP with 2,4-dinitrophenol. Uridine and thymidine accumulated in the slices against a concentration gradient in the presence of an inwardly directed Na+ gradient. The Na(+)-driven uptake of uridine and thymidine was saturable with Km values of 18.1 +/- 2.0 and 13.0 +/- 2.3 microM and Vmax values of 5.5 +/- 0.3 and 1.0 +/- 0.2 nmol/g/s, respectively. Na(+)-driven uridine uptake was inhibited by naturally occurring ribo- and deoxyribonucleosides (adenosine, cytidine, and thymidine) but not by synthetic nucleoside analogs (dideoxyadenosine, dideoxycytidine, cytidine arabinoside, and 3'-azidothymidine). Both purine (guanosine, inosine, formycin B) and pyrimidine nucleosides (uridine and cytidine) were potent inhibitors of Na(+)-thymidine transport with IC50 values ranging between 5 and 23 microM. Formycin B competitively inhibited Na(+)-thymidine uptake and thymidine trans-stimulated formycin B uptake. These data suggest that both purine and pyrimidine nucleosides are substrates of the same system. The stoichiometric coupling ratios between Na+ and the nucleosides, guanosine, uridine, and thymidine, were 1.87 +/- 0.10, 1.99 +/- 0.35, and 2.07 +/- 0.09, respectively. The system differs from Na(+)-nucleoside co-transport systems in other tissues which are generally selective for either purine or pyrimidine nucleosides and which have stoichiometric ratios of 1. This study represents the first direct demonstration of a unique Na(+)-nucleoside co-transport system in choroid plexus.     

Improved yields for baculovirus-mediated expression of human His(6)-PDK1 and His(6)-PKBbeta/Akt2 and characterization of phospho-specific isoforms for design of inhibitors that stabilize inactive conformations

PDK1 and PKB/Akt have a pleckstrin homology (PH) domain at the C-terminus and N-terminus, respectively, which stabilizes an unphosphorylated, autoinhibited conformation. Binding of the PH domain to a phospholipid second messenger causes relief of autoinhibition, which results in kinase phosphorylation and activation. Baculovirus-mediated expression in Sf9 insect cells of both His(6)-PDK1 and His(6)-PKBbeta/Akt2 were optimized, which significantly improved the yields (5-fold) of the affinity purified enzymes over previously reported values. Isoelectric focusing (IEF) and Western analyses indicated that the apparent V(max)=192+/-13 U/mg and K(m) (PDK-Tide)=55+/-10 microM of purified His(6)-PDK1 results from a mixture of at least three different phospho-specific isoforms (pI values of 6.8, 6.5, and 6.4). A purely unphosphorylated isoform of His(6)-PDK1 (pI=6.8) was generated by treatment with lambda protein phosphatase (lambdaPP), which decreased V(max) to 2.4+/-0.4 U/mg and increased K(m) (PDK-Tide) to 217+/-61 microM. Isoelectric focusing and Western analyses indicated that the apparent V(max)=0.21+/-0.03 U/mg and K(m) (Crosstide)=87+/-30 microM of purified His(6)-PKBbeta/Akt2 results from a mixture of the enzyme monophosphorylated either at Ser-474 ( approximately 90%) or at Thr-309 ( approximately 10%). A purely unphosphorylated isoform of His(6)-PKBbeta/Akt2 (pI=6.4) was generated by treatment with lambdaPP, which decreased V(max) approximately 2-fold. The optimization of high-level production and detailed characterization of purified and lambdaPP-treated His(6)-PDK1 and His(6)-PKBbeta/Akt2 will facilitate detailed structural and kinetic studies aimed at understanding the mechanism of second messenger-induced activation.     

An in vitro nucleoside analog screening method for cancer gene therapy

Suicide genes that sensitize cells to drugs that are normally nontoxic at therapeutic levels represent an important approach in human gene therapy research. We have developed an in vitro screening assay to assess the modulation of nucleoside analogs after transfection of a vector expressing the herpes simplex virus thymidine kinase gene (HSV-TK). The thymidine kinase gene enhances nucleoside phosphorylation to nucleotides that kill cells by blocking DNA elongation. Cells lines used are 3T3-NIH fibroblasts (parental cells) and 3T3-TKc3 (HSV-TK gene-transfected 3T3-NIH). Two types of analysis are performed: a cytotoxicity assay, the neutral red uptake assay to assess the IC₅₀ on the two cell lines, and an HPLC analysis coupled to a radiochemical flow detector to evaluate metabolic profiles after incubation of cells with tritiated analogs. Results show that cells expressing the HSV-TK gene are more sensitive than the parent cells to the effect of acyclovir or ganciclovir, the reference purine analog drugs, and also to the effect of pyrimidine analogs, bromodeoxyuridine, bromovinyldeoxyuridine, and ethyldeoxyuridine. Promising nucleoside analogs for gene therapy that can be achieved by HSV-TK could be evaluated using this model.Abbreviations ACV acyclovir - ACV-MP acyclovir monophosphate - ACV-DP acyclovir diphosphate - ACV-TP acyclovir triphosphate - BDU bromodeoxyuridine - BVDU bromovinyldeoxyuridine - EDU ethyldeoxyuridine - FDU fluorodeoxyuridine - GCV ganciclovir - HSV-TK herpes simplex virus thymidine kinase gene - IDU iododeoxyuridine - NA nucleoside analog     

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.     

Flavonoids as inhibitors of human carbonyl reductase 1

Human carbonyl reductase 1 (CBR1), that is one of the enzymes responsible for the reduced efficiency of treatments by the antineoplastic agents anthracyclines, was functionally expressed in Saccharomyces cerevisiae. CBR1 was purified and kinetically characterised using daunorubicin as substrate. CBR1-catalysed reduction of daunorubicin followed an apparent Michaelis-Menten kinetics with K(M)=85.2+/-26.7microM and V(max)=3490+/-220micromol/(mingprotein). The type of inhibition for the flavonoid compound rutin was determined by studying initial reaction rates in the presence of rutin. The inhibition kinetics was found to follow an apparent mixed inhibition with K(ic)=1.8+/-1.2microM and K(iu)=2.8+/-1.6microM. IC50-values were also determined for a set of flavonoids in order to identify essential structure for inhibition activity. Computational docking experiments of the four best inhibitors to the catalytic site of CBR1 showed that the flavonoid skeleton structure was the binding part of the molecule. The presence of a sugar moiety in 1 and 2, or a sugar mimicking part in 9, directed the orientation of the flavonoid so that the sugars were pointing outwards, giving rise to a stabilising effect to the binding. Finally, additional binding epitopes that interacted with various parts of the flavonoid ligand were identified and could potentially be targeted for further improvement of inhibition activity. These included; hydrogen-binding sites surrounding Ser139 and Cys226, Met234 and Tyr193 or Trp229; aromatic-aromatic interaction with Tyr193, Trp229 or NADPH; van der Waals interactions with Ile140.     

Molecularly imprinted polymer of 5-methyluridine for solid-phase extraction of pyrimidine nucleoside cancer markers in urine

Normal and modified urinary nucleosides represent potential biomarkers for cancer diagnosis. To selectively extract modified nucleosides, we developed a molecularly imprinted polymer (MIP) of 5-methyluridine as selective material for molecularly imprinted solid-phase extraction (MISPE). The MIPs were obtained from vinyl-phenylboronate ester derivative of the template, acrylamide and pentaerythritol triacrylate co-polymer, and were tested in batch and cartridge experiments with aqueous samples. Our results indicated that the imprinted polymer was selective for pyrimidine nucleosides with a K(d) and a B(max) of 46 microM and 18 micromol/g, respectively. Finally, a MISPE of the most common pyrimidine nucleoside cancer markers in urine sample was realized.     

Genetic analysis of nucleoside transport in Leishmania donovani.

Genetic dissection of nucleoside transport in Leishmania donovani indicates that the insect vector form of these parasites possesses two biochemically distinct nucleoside transport systems. The first transports inosine, guanosine, and formycin B, and the second transports pyrimidine nucleosides and the adenosine analogs, formycin A and tubercidin. Adenosine is transported by both systems. A mutant, FBD5, isolated by virtue of its resistance to growth inhibition by 5 microM formycin B, cannot efficiently transport inosine, guanosine, or formycin B. This cell line is also cross-resistant to growth inhibition by a spectrum of cytotoxic analogs of inosine and guanosine. A second parasite mutant, TUBA5, isolated for its resistance to 20 microM tubercidin, cannot take up from the culture medium radiolabeled tubercidin, formycin A, uridine, cytidine, or thymidine. Both the FBD5 and the TUBA5 cell lines have about a 50% reduced capacity to take up adenosine, indicating that adenosine is transported by both systems. A tubercidin-resistant clonal derivative of FBD5, FBD5-TUB, has acquired the combined biochemical phenotype of each single mutant. The wild-type and mutant cell lines transport purine bases and uracil with equal efficiency. Mutational analysis of the relative growth sensitivities to cytotoxic nucleoside analogs and the selective capacities to take up exogenous radiolabeled nucleosides from the culture medium have enabled us to define genetically the multiplicity and substrate specificities of the nucleoside transport systems in L. donovani promastigotes.     

Nucleoside and nucleobase transporters of primary human cardiac microvascular endothelial cells: characterization of a novel nucleobase transporter

Levels of cardiovascular active metabolites, like adenosine, are regulated by nucleoside transporters of endothelial cells. We characterized the nucleoside and nucleobase transport capabilities of primary human cardiac microvascular endothelial cells (hMVECs). hMVECs accumulated 2-[3H]chloroadenosine via the nitrobenzylmercaptopurine riboside-sensitive equilibrative nucleoside transporter 1 (ENT1) at a V(max) of 3.4 +/- 1 pmol.microl(-1).s(-1), with no contribution from the nitrobenzylmercaptopurine riboside-insensitive ENT2. Inhibition of 2-chloroadenosine uptake by ENT1 blockers produced monophasic inhibition curves, which are also compatible with minimal ENT2 expression. The nucleobase [3H]hypoxanthine was accumulated within hMVECs (K(m) = 96 +/- 37 microM; V(max) = 1.6 +/- 0.3 pmol.microl(-1).s(-1)) despite the lack of a known nucleobase transport system. This novel transporter was dipyridamole-insensitive but could be inhibited by adenine (K(i) = 19 +/- 7 microM) and other purine nucleobases, including chemotherapeutic analogs. A variety of other cell types also expressed the nucleobase transporter, including the nucleoside transporter-deficient PK(15) cell line (PK15NTD). Further characterization of [3H]hypoxanthine uptake in the PK15NTD cells showed no dependence on Na(+) or H(+). PK15NTD cells expressing human ENT2 accumulated 4.5-fold more [3H]hypoxanthine in the presence of the ENT2 inhibitor dipyridamole than did PK15NTD cells or hMVECs, suggesting trapping of ENT2-permeable metabolites. Understanding the nucleoside and nucleobase transporter profiles in the vasculature will allow for further study into their roles in pathophysiological conditions such as hypoxia or ischemia.     

Human CYP1A1 variants lead to differential eicosapentaenoic acid metabolite patterns

To answer the question whether the most common allelic variants of human CYP1A1, namely CYP1A1.1 (wild type), CYP1A1.2 (Ile462Val), and CYP1A1.4 (Thr461Asn), differ in their catalytic activity towards eicosapentaenoic acid (EPA), in vitro enzymatic assays were performed in reconstituted CYP1A1 systems. All CYP1A1 variants catalyzed EPA epoxygenation and hydroxylation to 17(R),18(S)-epoxyeicosatetraenoic acid (17(R),18(S)-EETeTr) and 19-OH-EPA, yet with varying catalytic efficiency and distinct regiospecificity. CYP1A1.1 and CYP1A1.4 formed 17(R),18(S)-EETeTr as main product (K(m)=53 and 50 microM; V(max)=0.60 and 0.50 pmol/min/pmol; V(max)/K(m)=0.11 and 0.10 microM(-1)min(-1), respectively), followed by 19-OH-EPA (K(m)=76 and 93 microM; V(max)=0.37 and 0.37 pmol/min/pmol; V(max)/K(m)=0.005 and 0.004 microM(-1)min(-1), respectively). The variant CYP1A1.2 produced almost equal amounts of both metabolites, but its catalytic efficiency for hydroxylation was five times higher (K(m)=66 microM; V(max)=1.7 pmol/min/pmol; V(max)/K(m)=0.026 microM(-1)min(-1)) and that for epoxygenation was twice higher (K(m)=66 microM; V(max)=1.5 pmol/min/pmol; V(max)/K(m)=0.023 microM(-1)min(-1)) than those of the wild-type enzyme. Thus, the Ile462Val polymorphism in human CYP1A1 affects EPA metabolism and may contribute to interindividual variance in the local production of physiologically active fatty acid metabolites in the cardiovascular system and other extrahepatic tissues, where CYP1A1 is expressed or induced by polycyclic aromatic hydrocarbons and other xenobiotics.     

Characteristics of Na(+)-dependent intestinal nucleoside transport in the pig

Since the capacity of nucleic acid digestion and absorption appears to be comparatively high in the pig, we investigated the properties of transport of (3)H-labelled nucleosides across the porcine intestinal brush border membrane (BBM) using BBM vesicles isolated from the small intestine of slaughter pigs. In the presence of a transmembrane Na(+) gradient, uridine, thymidine and guanosine transiently accumulated in the vesicular lumen beyond the equilibrium (60 min) value suggesting the presence of Na(+)/nucleoside cotransporters in the BBM. The findings of inhibitory studies are consistent with the presence of two Na(+)-dependent nucleoside transporters with overlapping substrate specificity, one for pyrimidine nucleosides (N2) and one for purine nucleosides (N1). Guanosine appeared to be a specific substrate for N1, while this applies to thymidine for N2. Transport of thymidine and guanosine were also inhibited by 2 mmol/l D-glucose and alpha-methyl-D-glucoside. The maximal transport capacity (V(max)) for Na(+)-dependent thymidine and guanosine transport were much higher than reported for other monogastric species. Unlike in other species tested, there was no proximal-to-distal gradient, neither in nucleoside transport activity nor in the inhibition of nucleoside transport by monosaccharides in the porcine small intestine. The high intestinal nucleoside transport activity may contribute to the high digestive capacity for nucleic acids in the pig.     

Identification and characterisation of high affinity nucleoside and nucleobase transporters in Toxoplasma gondii

The protozoan parasite Toxoplasma gondii depends upon salvaging the purines that it requires. We have re-analysed purine transport in T. gondii and identified novel nucleoside and nucleobase transporters. The latter transports hypoxanthine (TgNBT1; K(m)=0.91+/-0.19 microM) and is inhibited by guanine and xanthine: it is the first high affinity nucleobase transporter to be identified in an apicomplexan parasite. The previously reported nucleoside transporter, TgAT1, is low affinity with K(m) values of 105 and 134 microM for adenosine and inosine, respectively. We have now identified a second nucleoside transporter, TgAT2, which is high affinity and inhibited by adenosine, inosine, guanosine, uridine and thymidine (K(m) values 0.28-1.5 microM) as well as cytidine (K(i)=32 microM). TgAT2 also recognises several nucleoside analogues with therapeutic potential. We have investigated the basis for the broad specificity of TgAT2 and found that hydrogen bonds are formed with the 3' and 5' hydroxyl groups and that the base groups are bound through H-bonds with either N3 of the purine ring or N(3)H of the pyrimidine ring, and most probably pi-pi-stacking as well. The identification of these high affinity purine nucleobase and nucleoside transporters reconciles for the first time the low abundance of free nucleosides and nucleobases in the intracellular environment with the efficient purine salvage carried out by T. gondii.     

Metabolism of 4'-azidothymidine. A compound with potent and selective activity against the human immunodeficiency virus.

4'-Azidothymidine (ADRT) is a novel nucleoside analog, that selectively inhibits human immunodeficiency virus replication in human lymphocytes. Unlike the dideoxyribonucleoside analogs and 3'-azido-2',3'-dideoxythymidine (AZT), ADRT retains the 3'-hydroxy group. The pathways of ADRT metabolism were elucidated by determining: (i) the kinetics of the interactions of ADRT and its metabolites with enzymes of thymidine metabolic pathways, (ii) the pool sizes of phosphorylated metabolites, and (iii) the nature of ADRT incorporation into human DNA. ADRT is not a substrate for thymidine phosphorylase, but is metabolized by kinases. Thymidine kinase phosphorylates ADRT to ADRT monophosphate (ADRT-MP). For this enzyme, ADRT has a Ki value of 5.2 microM, in comparison to a Km value of 0.7 microM for thymidine. The Km value of ADRT toward thymidine kinase is 8.3 microM and the rate of ADRT phosphorylation is 1.4% that of thymidine phosphorylation. ADRT-MP has a low affinity toward thymidylate kinase (a Ki value of 28.9 microM versus a Km value of 0.56 microM for thymidylate), and toward thymidylate synthase (a Ki value of 180 microM versus a Km value of 8 microM for deoxyuridylate). The results suggest that ADRT can be activated effectively by cellular kinases without significant interference of normal thymidine metabolism. In cultured human lymphocytes (A3.01, H9, and U937 cells), ADRT was phosphorylated efficiently to ADRT 5'-triphosphate (ADRT-TP), which is the major metabolite of ADRT. The intracellular concentrations of ADRT-TP ranged from 1 to 3.3 microM after 24 h of incubation with 2 microM of ADRT and the half-life of ADRT-TP varied from 3 to 6 h. Although ADRT-TP is a poor competitive inhibitor against dTTP toward DNA polymerases alpha and beta with Ki values of 62.5 and 150 microM, respectively. ADRT-MP was found to be internally incorporated into cellular DNA. The extent of ADRT-MP substitution for dTMP in DNA was 1 in 6979 for A3.01 cells incubated with 2.9 microM ADRT for 24 h. Internal incorporation of ADRT-MP contrasts with the mechanism of other 2',3'-dideoxynucleoside analogs (i.e. AZT, ddC, ddI, d4T...), which are DNA chain terminators. This finding indicates that a 3'-deoxy structure in a nucleoside analog is not a prerequisite for anti-human immunodeficiency virus activity.     

Engineering of a single conserved amino acid residue of herpes simplex virus type 1 thymidine kinase allows a predominant shift from pyrimidine to purine nucleoside phosphorylation

Studies of herpes simplex virus type 1 (HSV-1) thymidine (dThd) kinase (TK) crystal structures show that purine and pyrimidine bases occupy distinct positions in the active site but approximately the same geometric plane. The presence of a bulky side chain, such as tyrosine at position 167, would not be sterically favorable for pyrimidine or pyrimidine nucleoside analogue binding, whereas purine nucleoside analogues would be less affected because they are located further away from the phenylalanine side chain. Site-directed mutagenesis of the conserved Ala-167 and Ala-168 residues in HSV-1 TK resulted in a wide variety of differential affinities and catalytic activities in the presence of the natural substrate dThd and the purine nucleoside analogue drug ganciclovir (GCV), depending on the nature of the amino acid mutation. A168H- and A167F-mutated HSV-1 TK enzymes turned out to have a virtually complete knock-out of dThd kinase activity (at least approximately 4-5 orders of magnitude lower) presumably due to a steric clash between the mutated amino acid and the dThd ring. In contrast, a full preservation of the GCV (and other purine nucleoside analogues) kinase activity was achieved for A168H TK. The enzyme mutants also markedly lost their binding capacity for dThd and showed a substantially diminished feedback inhibition by thymidine 5'-triphosphate. The side chain size at position 168 seems to play a less important role regarding GCV or dThd selectivity than at position 167. Instead, the nitrogen-containing side chains from A168H and A168K seem necessary for efficient ligand discrimination. This explains why A168H-mutated HSV-1 TK fully preserves its GCV kinase activity (Vmax/Km 4-fold higher than wild-type HSV-1 TK), although still showing a severely compromised dThd kinase activity (Vmax/Km 3-4 orders of magnitude lower than wild-type HSV-1 TK).