Deoxythymidine Phosphorylation Research Articles

Age-related metabolic changes limit efficacy of deoxynucleoside-based therapy in thymidine kinase 2-deficient mice

BackgroundThymidine kinase 2 (TK2) catalyses the phosphorylation of deoxythymidine (dThd) and deoxycytidine (dCtd) within mitochondria. TK2 deficiency leads to mtDNA depletion or accumulation of multiple deletions. In patients, TK2 mutations typically manifest as a rapidly progressive myopathy with infantile onset, leading to respiratory insufficiency and encephalopathy in the most severe clinical presentations. TK2-deficient mice develop the most severe form of the disease and die at average postnatal day 16. dThd+dCtd administration delayed disease progression and expanded lifespan of a knockin murine model of the disease. MethodsWe daily administered TK2 knockout mice (Tk2KO) from postnatal day 4 with equimolar doses of dThd+dCtd, dTMP+dCMP, dThd alone or dCtd alone. We monitored body weight and survival and studied different variables at 12 or 29 days of age. We determined metabolite levels in plasma and target tissues, mtDNA copy number in tissues, and the expression and activities of enzymes with a relevant role in mitochondrial dNTP anabolism or catabolism. FindingsdThd+dCtd treatment extended average lifespan of Tk2KO mice from 16 to 34 days, attenuated growth retardation, and rescued mtDNA depletion in skeletal muscle and other target tissues of 12-day-old mice, except in brain. However, the treatment was ineffective in 29-day-old mice that still died prematurely. Bioavailability of dThd and dCtd markedly decreased during mouse development. Activity of enzymes catabolizing dThd and dCtd increased with age in small intestine. Conversely, the activity of the anabolic enzymes decreased in target tissues during mouse development. We also found that administration of dThd alone had the same impact on survival to that of dThd+dCtd, whereas dCtd alone had no influence on lifespan. InterpretationdThd+dCtd treatment recruits alternative cytosolic salvage pathways for dNTP synthesis, suggesting that this therapy would be of benefit for any Tk2 mutation. dThd accounts for the therapeutic effect of the combined treatment in mice. During the first weeks after birth, mice experience marked tissue-specific metabolic regulations and ontogenetic changes in dNTP metabolism-related enzymes that limit therapeutic efficacy to early developmental stages. FundThis study was funded by grants from the Spanish Ministry of Industry, Economy and Competitiveness, the Spanish Instituto de Salud Carlos III, the Fundación Inocente, Inocente, AFM Téléthon and the Generalitat de Catalunya. The disclosed funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Novel 1,2,4-triazole and Purine Acyclic Cyclopropane Nucleoside Analogues: Synthesis, Antiviral and Cytostatic Activity Evaluations

Several published studies indicate that the acyclic guanine nucleoside analogues possessing bis(1,2-hydroxymethyl) substituted cyclopropane rings mimicking the sugar moiety are potent inhibitors of replication of several herpes viruses. Established synthetic methods and antiviral and cytostatic activity assays were used for the evaluation of new 1,2,4-triazole and purine acyclic nucleoside analogues. The synthesis of new types of acyclic nucleoside analogues which incorporate 1,2,4-triazole or purine moiety bound via flexible methylenic spacer to the bis(1,2-hydroxymethyl) cyclopropane ring. None of the new compounds showed pronounced antiviral activities at subtoxic concentrations on a broad panel of DNA and RNA viruses. Evaluation of their affinity for herpes simplex type 1 (HSV-1) and varicella-zoster virus-encoded thymidine kinases (VZV TK) also showed that none of the compounds was able to significantly inhibit 1 μM deoxythymidine phosphorylation by HSV-1 and VZV TK at 500 μM concentrations. The in vitro cytostatic activity evaluation results indicated a weak antiproliferative activity for all tested compounds. Only 6-pyrrolylpurine derivative bearing a carboxylic group substituted cyclopropane ring produced a rather slight inhibitory effect at higher micromolar concentrations on a breast carcinoma cell line (MCF-7) and no cytotoxic effect on human normal fibroblasts (WI 38). The lack of antiherpetic activity may be due to poor, if any, recognition of the compounds by virus-induced nucleoside kinases as an alternative substrate to become metabolically activated.

Biophysical characterization of vaccinia virus thymidine kinase substrate utilization

To provide information for the development of new antiviral compounds that inhibit orthopoxviruses, further characterization of the kinetics and thermodynamics that underlie substrate utilization reactions of vaccinia virus thymidine kinase (VVTK) has been undertaken. The kinetics of 2′deoxythymidine phosphorylation by VVTK and the thermodynamics of complex formation between VVTK and the substrate 2′ deoxythymidine were determined using spectroscopic and calorimetric techniques. These studies demonstrated that kinetic parameters for 2′ deoxythymidine phosphorylation by VVTK were 25 μM and 0.2 s −1 for K m and k cat, respectively. The enthalpy change, Δ H, for the enzyme catalyzed reaction is −18.1 kcal/mol. Thermodynamic studies for the formation of the enzyme substrate complex demonstrated a binding affinity ( K a) of 4 × 10 4 M −1, an enthalpy change for binding (Δ H) of −17.4 kcal/mol, and a reaction stoichiometry of two molecules of substrate binding to each enzyme tetramer. Kinetic and thermodynamic data were in agreement ( K a ≈ 1/ K m) and showed similarities to literature values reported for herpes simplex virus thymidine kinase (HSV-TK) and human thymidine kinase 1 (hTK1) with respect to k cat but not with respect to K m. The K m value found for VVTK in this study is nearly two orders of magnitude larger than the values reported for the hTK1 and the HSV TK enzymes.

The Study of Coupling Agents for Phosphorylation of 3′-Azido-3′-deoxythymidine with [32P]Orthophosphoric Acid

The kinetics of 3'-azido-3'-deoxythymidine phosphorylation with [32P]orthophosphoric acid was studied in the presence of various coupling agents. The most effective method with the use of BrCN provided the isolation of the target 3'-azido-3'-deoxythymidine 5'-[32P]monophosphate in 46% yield and a high specific radioactivity (>100 Ci/mmol).

The Crystal Structure of Mycobacterium tuberculosis Thymidylate Kinase in Complex with 3‘-Azidodeoxythymidine Monophosphate Suggests a Mechanism for Competitive Inhibition,

Tuberculosis (TB) is the primary cause of mortality among infectious diseases. Mycobacterium tuberculosis thymidylate kinase (TMPK(Mtub)) catalyzes the ATP-dependent phosphorylation of deoxythymidine 5'-monophosphate (dTMP). Essential to DNA replication, this enzyme represents a promising target for developing new drugs against TB, because the configuration of its active site is unique within the TMPK family. Indeed, it has been proposed that, as opposed to other TMPKs, catalysis by TMPK(Mtub) necessitates the transient binding of a magnesium ion coordinating the phosphate acceptor. Moreover, 3'-azidodeoxythymidine monophosphate (AZTMP) is a competitive inhibitor of TMPK(Mtub), whereas it is a substrate for human and other TMPKs. Here, the crystal structures of TMPK(Mtub) in complex with deoxythymidine (dT) and AZTMP were determined to 2.1 and 2.0 A resolution, respectively, and suggest a mechanism for inhibition. The azido group of AZTMP perturbs the induced-fit mechanism normally adopted by the enzyme. Magnesium is prevented from binding, and the resulting electrostatic environment precludes phosphoryl transfer from occurring. Our data provide a model for drug development against tuberculosis.

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.

Human Immunodeficiency Virus Resistance to AZT in MOLT4/8 Cells is Associated with a Lack of AZT Phosphorylation and is Bypassed by AZT-Monophosphate SATE Prodrugs

Human T lymphoid MOLT4/8 cells were grown continuously for more than 2 years in a medium containing 3′-azido-2′,3′-dideoxythymidine (zidovudine; AZT) at a concentration of 250 μM. These cells, designated MOLT-4/8rAZT250, were used to test the cytotoxic and antiviral activity of AZT. Intracellular accumulation of AZT, expression of the multidrug resistance 1 (MDR-1) gene, thymidine kinase (TK) gene and activity of the TK enzyme in cellular extracts were measured. The results showed that both the cytotoxic and antiviral activity of AZT were significantly lower in MOLT4/8rAZT250 than in MOLT4/8 cells; concentrations required to inhibit 50% production of the p24 human immunodeficiency virus type 1 (HIV-1) antigen of two laboratory strains were at least 100-fold higher in resistant cells. The MDR-1 gene was not expressed in the resistant cells. TK mRNA expression was significantly lower in the resistant than in the sensitive cells. TK enzymatic activity for deoxythymidine phosphorylation was impaired in MOLT4/8rAZT250 cells compared to the sensitive cells. AZT was phosphorylated only in the sensitive cells whereas no phosphorylation of AZT was found in the resistant cells. We tested whether several AZT-monophosphate triesters, which bypass cellular TK, could overcome resistance to the cytotoxic and antiviral activity of AZT. The bis( t-butylSATE) phosphotriester derivative of AZT showed comparable cytotoxic and antiviral activity in sensitive and resistant cells. The results demonstrated that MOLT4/8rAZT250 cells exert resistance to the anti-HIV activity of the drug mainly owing to the lack of AZT phosphorylation and that resistance may be bypassed by using AZT-monophosphate SATE prodrugs.

ChemInform Abstract: SYNTHESIS AND BIOLOGICAL ACTIVITIES OF SOME URONIC ACIDS, URONATES, URONAMIDES, AND URONONITRILES OF PYRIMIDINE NUCLEOSIDES

Abstract35 Pyrimidin‐Nucleosidanaloga (I) werden in 21 ‐ 98%iger Ausbeute dargestellt und als WachstumJnhibitoren von Sar‐ coma 180‐Zellen, HerpesSimplex Virus (Typ 1) und Streptococcus faecium getestet.

Characteristics of deoxythymidine transport and deoxythymidine kinase in 3T3 cells

The structural requirements for deoxythymidine transport are specific. Structural alteration of deoxythymidine at the 2′- and 5′-positions results in compounds which do not compete for deoxythymidine transport. Similarly, naturally occurring nucleosides do not have a demonstrable affinity for the deoxythymidine transport site. An exception is an apparent tolerance for replacement of the 5-methyl group by certain substituents. The structural requirements for deoxythymidine kinase and the transport process are similar for 5-bromodeoxyuridine, 5-fluorodeoxyuridine, and deoxyuridine. However, 5-aminodeoxyuridine inhibits deoxythymidine transport but not deoxythymidine kinase, whereas 2′,5′-dideoxythymidine inhibits deoxythymidine kinase but has no effect on deoxythymidine transport. These differences indicate that deoxythymidine kinase is dissociated from the transport process. Confirmatory evidence for this conclusion is provided by the finding that intracellular deoxythymidine has the highest specific activity of all intracellular deoxythymidine-containing compounds after exposure to radioactive deoxythymidine for 1 min. These data show that extracellular deoxythymidine is transported into the cell as deoxythymidine. Deoxythymidine is rapidly converted to dTMP intracellularly by deoxythymidine kinase. Accumulation of dTMP in the presence of 2,4-dinitrophenol confirms previous studies that intracellular phosphorylation of deoxythymidine is very rapid even when ATP production is limited.

The metabolism of deoxyribonucleosides in Lactobacillus acidophilus: Regulation of deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine kinase activities by nucleotides

1. 1. The presence of deoxycytidine kinase, as well as of deoxyadenosine, deoxyguanosine and thymidine kinase activities, is reported in Lactobacillus acidophilus. 2. 2. Phosphorylation of each deoxynucleoside is inhibited most effectively by its homoogous deoxynucleoside triphosphate. Besides inhibiting deoxycytidine kinase, dCTP also inhibits deoxyguanosine kinase activity, but stimulates deoxyadenosine and deoxythymidine phosphorylation. 3. 3. The inhibition of deoxycytidine, deoxyguanosine or deoxyadenosine phosphorylation by the corresponding triphosphate is reversed by UTP and dUTP, but not by dTTP which augmented the inhibition in certain cases. The inhibition of thymidine kinase by dTTP was readily reversed by dCTP. 4. 4. Preliminary purification of the bacterial deoxycytidine kinase is described, and its properties are compared with those of the enzyme from calf thymus. The pH curve is biphasic, with optima at pH 7.6 and 10.5. The mol. wt., as estimated by gel filtration, is only about 35 000. The enzyme is slightly activated by mercurial reagents, apparently without loss of regulatory properties. Initial kinetic experiments yielded linear double-reciprocal plots when either deoxycytidine or ATP-Mg 2− was varied. The end-product inhibitor dCTP apparently is competitive; K i dCTP, 5.0·10 −5 M.

Properties of deoxythymidine kinase partially purified from noninfected and virus-infected mouse fibroblast cells

Deoxythymidine kinase, partially purified from noninfected LM mouse fibroblast cells, was rapidly inactivated when incubated in the absence of substrates at 38°C. The half-life of the enzyme was 29–43 minutes whether prepared from LM cells in the logarithmic phase of growth, the phase of negative growth acceleration, the stationary phase, or from LM cells which had been incubated for 24 hours with 50 μg deoxythymidine per milliliter prior to harvest. The deoxythymidine kinase induced in LM (TK −) cells by herpes simplex virus was also very unstable. In contrast, deoxythymidine kinase prepared from vaccinia-infected LM or LM (TK −) cells was considerably more stable with a half-life greater than 300 minutes. The addition of ATP (15 m M), dTTP (0.025 m M), or deoxythymidine (0.05 to 0.1 m M) significantly protected the LM cell enzyme against thermal inactivation. However, charcoal treatment of the vaccinia-induced enzyme did not decrease the stability of that enzyme. Immunological differences and differences in apparent Michaelis constants for deoxyuridine were also observed between the vaccinia-induced deoxythymidine kinase and the enzyme from noninfected LM cells. However, the temperature characteristics of the reactions, calculated from the V m values at 38° and 30°, were similar, and these enzymes were nearly equal in susceptibility to inhibition by dTTP. With either enzyme, the phosphorylation of deoxyuridine was competitively inhibited by fluorodeoxyuridine, bromodeoxyuridine, iododeoxyuridine, or deoxythymidine. The phosphorylation of deoxythymidine was inhibited by low concentrations of bromodeoxyuridine or iododeoxyuridine, and by high concentrations of deoxyuridine or fluorodeoxyuridine.