Isopoly-L-ornithine Derivatives of Thymine and Thymidine†

Abstract

L-Ornithine derivatives of thymine, and thymidine gave oligomers by solid phase elongation reactions. These oligomers 2 and 3, however, hardly interact with the complementary polynucleotide. Conformational studies of the oligomers with CD and NMR revealed that stable intramolecular hydrogen bonding was formed between thymine base and ornithine unit.

     

5-Bromovinyl 2′-Deoxyuridine Phosphorylation by Mitochondrial and Cytosolic Thymidine Kinase (TK2 and TK1) and Its Use in Selective Measurement of TK2 Activity in Crude Extracts

Mitochondrial thymidine kinase (TK2) is responsible for phosphorylation of thymidine and deoxycytidine and plays a crucial role in mitochondrial DNA precursor synthesis. TK2 is expressed in all tissues at low levels complicating accurate determinations, especially in tissues with high cytosolic thymidine kinase (TK1) activity. Recently, 5-bromovinyl 2 ′-deoxyuridine (BvdU) at 0.2 μ M was used to measure TK2 activity selectively. BvdU phosphorylation by pure human TK2 and TK1 was tested here, and the ratio of BvdU phosphorylation by TK2/TK1 was 91 at 0.2 μ M but was 500 at 2.5 μ M. Therefore, for reliable measurement of TK2 activity higher BvdU concentration should be used.     

The Synthesis of Carbocyclic 5′-Nor Thymidine and an Isomer as Oligonucleotide Monomers

Abstract

The chiral synthesis of (1S,3S,4S)-1-(3,4-dihydroxycyclopent-1-yl)-1H?thymine (carbocyclic 5′-nor thymidine, 4) has been achieved in 5 steps from (+)-(lR,4S)-4-hydroxy-2-cyclopenten-1-yl acetate (5) and N³?benzoylthymine. Compound 4 is viewed as a monomeric building block for poly-T-like oligomers.     

Thymidine Kinase 2 Deficiency-Induced mtDNA Depletion in Mouse Liver Leads to Defect β-Oxidation

Thymidine kinase 2 (TK2) deficiency in humans causes mitochondrial DNA (mtDNA) depletion syndrome. To study the molecular mechanisms underlying the disease and search for treatment options, we previously generated and described a TK2 deficient mouse strain (TK2^−/−) that progressively loses its mtDNA. The TK2^−/− mouse model displays symptoms similar to humans harboring TK2 deficient infantile fatal encephalomyopathy. Here, we have studied the TK2^−/− mouse model to clarify the pathological role of progressive mtDNA depletion in liver for the severe outcome of TK2 deficiency. We observed that a gradual depletion of mtDNA in the liver of the TK2^−/− mice was accompanied by increasingly hypertrophic mitochondria and accumulation of fat vesicles in the liver cells. The levels of cholesterol and nonesterified fatty acids were elevated and there was accumulation of long chain acylcarnitines in plasma of the TK2^−/− mice. In mice with hepatic mtDNA levels below 20%, the blood sugar and the ketone levels dropped. These mice also exhibited reduced mitochondrial β-oxidation due to decreased transport of long chain acylcarnitines into the mitochondria. The gradual loss of mtDNA in the liver of the TK2^−/− mice causes impaired mitochondrial function that leads to defect β-oxidation and, as a result, insufficient production of ketone bodies and glucose. This study provides insight into the mechanism of encephalomyopathy caused by TK2 deficiency-induced mtDNA depletion that may be used to explore novel therapeutic strategies.     

Phosphorylation of Isocarbostyril‐ and Difluorophenyl‐Nucleoside Thymidine Mimics by the Human Deoxynucleoside Kinases

The thymidine mimics isocarbostyril nucleosides and difluorophenyl nucleosides were tested as deoxynucleoside kinase substrates using recombinant human cytosolic thymidine kinase (TK1) and deoxycytidine kinase (dCK), and mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK). The isocarbostyril nucleoside compound 1‐(2‐deoxy‐β‐D‐ribofuranosyl)‐isocarbostyril (EN1) was a poor substrate with all the enzymes. The phosphorylation rates of EN1 with TK1 and TK2 were < 1% relative to Thd, where as the phosphorylation rates for EN1 were 1.4% and 1.1% with dCK and dGK relative to dCyd and dGuo, respectively. The analogue 1‐(2‐deoxy‐β‐D‐ribofuranosyl)‐7‐iodoisocarbostyril (EN2) showed poor relative‐phosphorylation efficiencies (k _cat /K _m ) with both TK1 and dGK, but not with TK2. The k _cat /K _m value for EN2 with TK2 was 12.6% relative to that for Thd. Of the difluorophenyl nucleosides, 5‐(1′‐(2′‐deoxy‐β‐D‐ribofuranosyl))‐2,4‐difluorotoluene (JW1) and 1‐(1′‐(2′‐deoxy‐β‐D‐ribofuranosyl))‐2,4‐difluoro‐5‐iodobenzene (JW2) were substrates for TK1 with phosphorylation efficiencies of about 5% relative to that for Thd. Both analogues were considerably more efficient substrates for TK2, with k _cat /K _m values of 45% relative to that for Thd. 2,5‐Difluoro‐4‐[1‐(2‐deoxy‐β‐L‐ribofuranosyl)]‐aniline (JW5), a L‐nucleoside mimic, was phosphorylated up to 15% as efficiently as deoxycytidine by dCK. These data provide a possible explanation for the previously reported lack of cytotoxicity of the isocarbostyril‐ and difluorophenyl nucleosides, but potential mitochondrial effects of EN2, JW1 and JW2 should be further investigated.     

P1-(Thymidine 5′-)P1-amino-triphosphate: Synthesis,Hydrolysis and Reaction with Cytidine in Alkali1

Abstract

The title compound 1 is prepared from thymidine 5′-phos-phorodiamidate (2) and inorganic pyrophosphate (3) in anhydrous DMF, at 30–32°C. The products of alkaline hydrolysis of 1, at room temperature, are: thymidine 5′-phosphoramidate (4), thymidine 3′-phosphoramidate (8) and thymidine (9) as well as 3 and inorganic trimetaphosphate (10). In 1 N NH₄OH, 1 reacts with cytidine (15) to form cytidylyl-/2^T(3′)-5′/-thymidine (16) and a mixture of cytidine 2′,3′-cyclic phosphate (17) and 9.     

Role of Platelet Derived Endothelial Cell Growth Factor/Thymidine Phosphorylase in Fluoropyrimidine Sensitivity and Potential Role of Deoxyribose‐1‐Phosphate

Thymidine phosphorylase (TP) catalyzes the phosphorolytic cleavage of thymidine (TdR) to thymine and deoxyribose‐1‐phosphate (dR‐1‐P). TP, which is overexpressed in a wide variety of solid tumors, is involved in the activation and inactivation of fluoropyrimidines. We investigated the role of TP in 5′‐deoxy‐5‐fluorouridine (5′DFUR), 5‐fluorouracil (5FU) and trifluorothymidine (TFT) sensitivity. TP had no effect on TFT while it activated 5′DFUR and to a lesser extent 5FU. In order to provide an explanation for this difference in activation of 5′DFUR and 5FU, we studied the role of the 5FU co‐substrate, dR‐1‐P, needed for its activation.     

Thymidine Phosphorylase is Noncompetitively Inhibited by 5′-O-Trityl-Inosine (KIN59) and Related Compounds

We found that 5′-O-trityl-inosine (KIN59) inhibits recombinant bacterial (E. coli) and human thymidine phosphorylase (TPase) with an IC₅₀ of 44 μM and 67 μM, respectively. In contrast to previously described TPase inhibitors, KIN59 does not compete with thymidine (dThd) at the pyrimidine nucleoside-binding site or with inorganic phosphate (Pi) at the phosphate-binding site of the enzyme. These findings are strongly suggestive for the presence of an allosteric binding site at the enzyme. TPase is identical to the angiogenic protein platelet-derived endothelial cell growth factor (PD-ECGF). As such, PD-ECGF stimulates angiogenesis in the chick chorioallantoic membrane (CAM) assay. This angiogenic response was completely inhibited by KIN59. Inosine did not inhibit the enzyme or the angiogenic effect of TPase, confirming that the 5′-O-trityl group in KIN59 is essential for the observed effect. Our observations indicate that allosteric sites in TPase may regulate its biological activity.     

Methodology and Problems of Protein‐Ligand Docking: Case Study of Dihydroorotate Dehydrogenase,Thymidine Kinase,and Phosphodiesterase 4

Abstract

The docking methodology was applied to three different therapeutically interesting enzymes: human dihydroorotate dehydrogenase (DHODH), Herpes simplex virus type I thymidine kinase (HSV1 TK) and human phosphodiesterase 4 (PDE4). Programs FlexX, AutoDock and DOCK where used. The three targets represent three distinct cases. For DHODH and HSV1 TK, the binding modes of substrate and inhibitors within the active site are known, while the binding orientation of cAMP within PDE4 has been solely hypothesized. Active site of DHODH is mainly hydrophobic and the binding mode of the inhibitor brequinar was used as a template for evaluating the docking strategies. The presence of cofactors revealed to be crucial for the definition of the docking site. The HSV1 TK active site is small and polar and contains crystal water molecules and ATP. Docking of thymidine and aciclovir (ACV) within the active site was analyzed by keeping or removing water molecules. It showed the crucial role of water in predicting the binding of pyrimidines and purines. The crystal structure of PDE4 contains magnesium and zinc cations as well as catalytic water molecule but no ligand. Several docking experiments of cAMP and rolipram were performed and the results showed clear‐cut dependence between the ligand orientation and the presence of metals in the active site. All three cases show specific problems of the docking methodology, depending on the character of the active site.     

Synthesis of 5-Halogeno-6-amino-2′-deoxyurldines and their Analogs as Potential Inhibitors of Thymidine Phosphorylase

ABSTRACT

5-Halogeno-6-amino-2′-deoxyuridines were synthesized from 2′-deoxyuridine as potential thymidine phosphorylase (ThdPase) inhibitors. Among the compounds synthesized, 5-bromo-6-amino-2′-deoxyuridine (6) and 5-iodo-6-amino-2′-deoxyuridine (9) were found to inhibit ThdPase activity with IC50 values of 1.3 μM and 6.5 μM, respectively. In vitro cell culture studies showed that compound (6) can significantly enhance the cytotoxic effects of 5-fluoro-2′-deoxyuridine against a human colon cancer HCT-8 cell line.     

Synthesis and Antiviral Evaluation of 3′-Substituted Thymidine Analogues Derived from 3′-Amino-3′-deoxythymidine

Abstract

To assess the structure-activity relationship for antiviral activity, a series of 3′-deoxy-3′-N-functionalized thymidine analogues were synthesized. Several of these thymidine analogues show moderate in vitro activity against HIV-1 and HIV-2.     

The Effect of α-Interferon on Thymidine,Uridine, and Leucine Uptake and Ultrastructure of Peripheral Blood Mononuclear Cells from Multiple Myeloma Patients

The effect of IFN α-2b on thymidine, uridine, and leucine uptake was examined on peripheral blood mononuclear cells (PBMC) of healthy donors and 15 patients with multiple myeloma (MM). In addition, the surface ultrastructure of the cells incubated without or with IFN α-2b was examined with a scanning electron microscope. The results showed that IFN had no effect on thymidine, uridine, or leucine uptake of unstimulated MM and control PBMC. On the other hand IFN inhibited thymidine, and uridine uptake of PWM-stimulated MM PBMC, but had no effect on healthy donor stimulated PBMC. 1FN inhibited also thymidine and uridine uptake in PHA-stimulated healthy donors and MM patients′ PBMC. The cellular surface ultrastructure of MM lymphocytes incubated with 100 u/ml IFN showed disappearance of the microvilli and formation of cellular pits, whereas in healthy donor lymphocytes IFN caused flattening of microvilli.     

Temperament and character in cross-cultural comparisons between Swedish and Iranian people and Iranian refugees in Sweden--personality in transition?

The aim of the study was a cross-cultural comparison of personality traits between individuals from two very different cultures and refugees who resettled several years before from one to the other. Four hundred forty four Swedish individuals of the normal population; and 100 Iranian refugees in Sweden, and a group of 335 individuals from Tehran, capital of Iran, were investigated by means of the Temperament and Character Inventory, a questionnaire to assess temperament and character Iranians are those that are most frequently correctly classified followed by the Swedish based on temperament scores by means of a Discriminance analyses. Iranian refugees in Sweden were classified to about 50 per cent as Swedish and to slightly more then one-third as Iranians. Especially concerning character, 4 per cent only could be correctly classified as refugees. The results give some perspective on the adaptation process and personality changes in refugees several years after resettlement in another country with a complete different culture.     

New Efficient Synthesis of Thymidine Cyclic 3′, 5′- Phosphorofluoridate and Its Sulfur Analogue via the Phosphoroamidite Route

Abstract

We describe the convenient synthesis of thymidine cyclic 3′, 5′-phosphorofluoridate 6, which is superior to that previously reported. Our procedure is based on a sequence of reactions utilizing 3 as the key substrate. Similar sequence of reaction leads to the sulfur analogues of 6 the thymidine cyclic 3′, 5′-phosphorofluoridothioate 7.     

Dual localization of β-glucuronidase and acid phosphatase in lysosomes and in microsomes

Summary The dual localization of certain hydrolases in lysosomes and in endoplasmic reticulum as studied in enzyme staining reactions is now supported by cytobiochemical studies on mouse liver and kidney -glucuronidase and acid phosphatase. Use was made of the renal -glucuronidase response to endogenous androgen for both studies. Accordingly, sucrose homogenates were prepared of liver and kidney of male BALB/C mice previously injected with gonadotrophin along with control animals receiving saline instead. The homogenates were subjected to differential ultracentrifugation yielding six fractions. These were characterized as to their organelle composition by measurements of marker enzymes and by observations with the electron microscope. In all subcellular fractions, -glucuronidase was uniformly increased 5 to 8 times over the corresponding control value and, in fractions rich in lysosomes, this enzyme was easily released by alternate freezing and thawing. On the other hand, the microsomal -glucuronidase and acid phosphatase enzymes were not liberated by freezing and thawing nor were they after treatment with 0.1 % Triton X-100 and by employing other reagents and conditions which are known to release lysosomal enzymes. In contrast to microsomal acid phosphatase, microsomal -glucuronidase activity could be liberated by treatment with hyaluronidase. This soluble -glucuronidase showed the same optimum pH, Michaelis Constant and heat inactivation behavior as the lysosomal -glucuronidase prepared by freezing and thawing treatment. These observations define two populations of microsomal vesicles each identifiable by an individual membrane-associated acid hydrolase. One of these -glucuronidase, increases in specific activity in the animal on androgens and is released by hyaluronidase and the other, acid phosphatase, does not respond to androgen and is not released by hyaluronidase. There would appear to be a variety of mechanisms by which hydrolases enter into association with the membranes of the endoplasmic reticulum and from there, a variety of routes to the lysosomes. A comment is made concerning the question of acid phosphatases and -glucuronidase as enzyme markers for lysosomes in mouse kidney.Aided in part by Research Grant, P-106, of the American Cancer Society, Inc., New York, and by U.S.P.H.S. Grant CA-07538 and by a Research Career Award, CA-K6-18453 to William H. Fishman.     

Protection by zinc against UVA— and UVB-induced cellular and genomic damage in vivo and in vitro

For many years, zinc salts have been used both topically and orally to treat minor burns and abrasions as well as to enhance wound repair in man and animals. In this study we describe the protective effects of zinc against UV-induced genotoxicity in vitro and against sunburn cell formation in mouse skin in vivo. Cultured skin cells from neonatal mice showed a dramatic increase in the number of micronuclei as a result of UVA and UVB irradiation. Inclusion of zinc at 5 μg/mL in the medium significantly reduced the frequency of micronuclei and of micronucleated cells. In hairless mice, topical application of zinc chloride for 5 consecutive days or a single application 2 h prior to UV exposure reduced the number of sunburn cells in the epidermis as did application of zinc 1 h after exposure. Application 2 h after irradiation also tended to have a protective effect, although there was a large variation between animals. It is proposed that an influx of zinc can protect epidermal cells against some of the more delayed effects of UV-induced damage.