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1.
Int J Biochem Cell Biol ; 49: 75-83, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24495878

ABSTRACT

Adenine nucleotides are involved in a variety of cellular metabolic processes, including nucleic acid synthesis and repair, formation of coenzymes, energy transfer, cell and ciliary motility, hormone secretion, gene expression regulation and ion-channel control. Adenylate kinases are abundant phosphotransferases that catalyze the interconversion of adenine nucleotides and thus regulate the adenine nucleotide ratios in different intracellular compartments. Nine different adenylate kinase isoenzymes have been identified and characterized so far in human tissues, named AK1 to AK9 according to their order of discovery. Adenylate kinases differ in molecular weight, tissue distribution, subcellular localization, substrate and phosphate donor specificity and kinetic properties. The preferred substrate and phosphate donor of all adenylate kinases are AMP and ATP respectively, but some members of the family can phosphorylate other substrates and use other phosphate donors. In addition to their nucleoside monophosphate kinase activity, adenylate kinases were found to possess nucleoside diphosphate kinase activity as they are able to phosphorylate both ribonucleoside and deoxyribonucleoside diphosphates to their corresponding triphosphates. Nucleoside analogues are structural analogues of natural nucleosides, used in the treatment of cancer and viral infections. They are inactive prodrugs that are dependent on intracellular phosphorylation to their pharmacologically active triphosphate form. Novel data presented in this review confirm the role of adenylate kinases in the activation of deoxyadenosine and deoxycytidine nucleoside analogues.


Subject(s)
Adenosine Monophosphate/metabolism , Adenosine Triphosphate/metabolism , Adenylate Kinase/metabolism , Adenylate Kinase/classification , Adenylate Kinase/genetics , Amino Acid Sequence , Humans , Isoenzymes/classification , Isoenzymes/genetics , Isoenzymes/metabolism , Molecular Sequence Data , Phylogeny , Sequence Homology, Amino Acid , Substrate Specificity
2.
Neurology ; 81(23): 2051-3, 2013 Dec 03.
Article in English | MEDLINE | ID: mdl-24198295

ABSTRACT

Mutations in nuclear genes involved in the maintenance of mitochondrial DNA (mtDNA) are associated with an extensive spectrum of clinical phenotypes, manifesting as either mtDNA depletion syndromes or multiple mtDNA deletion disorders.(1.)


Subject(s)
DNA, Mitochondrial/genetics , Gene Deletion , Mutation/genetics , Respiratory Insufficiency/diagnosis , Respiratory Insufficiency/genetics , Thymidine Kinase/genetics , Age Factors , Aged , Female , Humans
3.
Int J Biochem Cell Biol ; 45(5): 925-31, 2013 May.
Article in English | MEDLINE | ID: mdl-23416111

ABSTRACT

Adenylate kinases regulate adenine nucleotide levels and are present in different intracellular compartments. These enzymes also participate in the activation of pharmacologically active nucleoside and nucleotide analogs. We have in the present study identified the ninth isoform of the adenylate kinase family of enzymes and accordingly named the protein adenylate kinase 9 (AK9). Initially a full-length cDNA of a hypothetical protein containing a predicted adenylate kinase domain was identified and subsequently cloned and expressed in Escherichia coli. The substrate specificity of the recombinant protein showed that the enzyme catalyzed the phosphorylation of AMP, dAMP, CMP and dCMP with ATP as phosphate donor, while only AMP and CMP were phosphorylated when GTP was the phosphate donor. The kinetic parameters of AK9 were determined for AMP, dAMP and CMP with ATP as phosphate donor. Interestingly, in addition to the diphosphate products, a nucleoside diphosphate kinase (NDPK) activity was also present with subsequent triphosphates formed. With ATP or GTP as phosphate donor it was possible to detect the production of ATP, CTP, GTP, UTP, dATP, dCTP, dGTP and TTP as enzymatic products from the corresponding diphosphate substrates. A number of previously characterized adenylate kinases were also tested and found to possess a broad phosphotransferase activity similar to AK9. These enzymes are accordingly suggested to be regarded as nucleoside mono- and diphosphate kinases with catalytic activities possibly determined by local substrate concentrations.


Subject(s)
Adenylate Kinase/chemistry , Adenylate Kinase/metabolism , Nucleoside-Phosphate Kinase/chemistry , Adenylate Kinase/genetics , Amino Acid Sequence , Cell Culture Techniques , HeLa Cells , Humans , Kinetics , Nucleoside-Diphosphate Kinase/chemistry , Nucleoside-Diphosphate Kinase/genetics , Nucleoside-Diphosphate Kinase/metabolism , Nucleoside-Phosphate Kinase/genetics , Nucleoside-Phosphate Kinase/metabolism , Nucleotides/metabolism , Plasmids , Substrate Specificity , Transfection
4.
Bioorg Med Chem ; 19(14): 4338-45, 2011 Jul 15.
Article in English | MEDLINE | ID: mdl-21696963

ABSTRACT

We report the synthesis of a series of novel 2'-deoxy-2',2'-difluoro-5-halouridines and their corresponding phosphoramidate ProTides. All compounds were evaluated for antiviral activity and for cellular toxicity. Interestingly, 2'-deoxy-2',2'-difluoro-5-iodo- and -5-bromo-uridines showed selective activity against feline herpes virus replication in cell culture due to a specific recognition (activation) by the virus-encoded thymidine kinase.


Subject(s)
Amides/pharmacology , Antineoplastic Agents/pharmacology , Antiviral Agents/pharmacology , Drug Design , Floxuridine/analogs & derivatives , Hepacivirus/drug effects , Phosphoric Acids/pharmacology , Amides/chemical synthesis , Amides/chemistry , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Cell Proliferation/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Floxuridine/chemical synthesis , Floxuridine/chemistry , Floxuridine/pharmacology , Humans , Mice , Microbial Sensitivity Tests , Molecular Conformation , Phosphoric Acids/chemical synthesis , Phosphoric Acids/chemistry , Stereoisomerism , Structure-Activity Relationship , Virus Replication/drug effects
5.
Org Biomol Chem ; 9(3): 892-901, 2011 Feb 07.
Article in English | MEDLINE | ID: mdl-21127790

ABSTRACT

Based on the presumed binding mode of an earlier identified inhibitor, we herein report new 3'-modified nucleosides as potent and selective inhibitors of mitochondrial thymidine kinase (TK2). A series of thirteen 3'-amino-, 3'-guanidino- and 3'-tetrazole-containing nucleosides were synthesized and evaluated for their TK2 inhibitory activity. Within the tetrazole series, compounds with nanomolar inhibitory activity were identified. A homology model of TK2 allowed to elucidate the observed activities. Introduction of a 2-bromovinyl group on C-5 of the pyrimidine base of the most promising 3'-derivative further improved the inhibitory activity, and caused a significant increase in the selectivity for TK2 versus TK1. Interestingly, for the current series of analogues, a strong correlation was observed between TK2 and Drosophila melanogaster dNK inhibition, further substantiating the phylogenetic relationship between these two nucleoside kinases.


Subject(s)
Amines/chemistry , Azoles/chemistry , Thymidine Kinase/antagonists & inhibitors , Thymidine/chemical synthesis , Animals , Drosophila melanogaster/drug effects , Drosophila melanogaster/enzymology , Humans , Models, Molecular , Molecular Structure , Thymidine/pharmacology , Thymidine Kinase/chemistry
6.
Biochem J ; 433(3): 527-34, 2011 Feb 01.
Article in English | MEDLINE | ID: mdl-21080915

ABSTRACT

Differences in expression profiles, substrate specificities, kinetic properties and subcellular localization among the AK (adenylate kinase) isoenzymes have been shown to be important for maintaining a proper adenine nucleotide composition for many different cell functions. In the present study, human AK7 was characterized and its substrate specificity, kinetic properties and subcellular localization determined. In addition, a novel member of the human AK family, with two functional domains, was identified and characterized and assigned the name AK8. AK8 is the second known human AK with two complete and active AK domains within its polypeptide chain, a feature that has previously been shown for AK5. The full-length AK8, as well as its two domains AK8p1 and AK8p2, all showed similar AK enzyme activity. AK7, full-length AK8, AK8p1 and AK8p2 phosphorylated AMP, CMP, dAMP and dCMP with ATP as the phosphate donor, and also AMP, CMP and dCMP with GTP as the phosphate donor. Both AK7 and full-length AK8 showed highest affinity for AMP with ATP as the phosphate donor, and proved to be more efficient in AMP phosphorylation as compared with the major cytosolic isoform AK1. Expression of the proteins fused with green fluorescent protein demonstrated a cytosolic localization for both AK7 and AK8.


Subject(s)
Adenylate Kinase/chemistry , Adenylate Kinase/metabolism , Cytosol/chemistry , Humans , Isoenzymes , Kinetics , Nucleotides/metabolism , Phosphorylation , Substrate Specificity
7.
J Inherit Metab Dis ; 33(3): 231-6, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20440651

ABSTRACT

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.


Subject(s)
DNA, Mitochondrial/metabolism , Hematopoiesis , Thymidine Kinase/genetics , Animals , Cell Proliferation , Disease Models, Animal , Embryonic Stem Cells/cytology , Lymphocytes/cytology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Phosphorylation , Tissue Distribution
8.
Article in English | MEDLINE | ID: mdl-20391188

ABSTRACT

Thymidine kinase 2 (TK2) is a mitochondrial deoxyribonucleoside kinase that phosphorylates several nucleoside analogs used in anti-viral and anti-cancer therapy. A fibroblast cell line with decreased TK2 activity was investigated in order to obtain insights in the effects of TK2 deficiency on nucleotide metabolism. The role of TK2 for the sensitivity against cytotoxic nucleoside analogs was also investigated. The TK2 deficient cells retained their sensitivity against all pyrimidine nucleoside analogs tested. This study suggests that nucleoside analog phosphorylation mediated by TK2 may be less important, compared to other deoxyribonucleoside kinases, for the cytotoxic effects of these compounds.


Subject(s)
Fibroblasts/cytology , Fibroblasts/enzymology , Pyrimidine Nucleosides/pharmacology , Thymidine Kinase/deficiency , Base Sequence , Blotting, Southern , Bromodeoxyuridine/analogs & derivatives , Bromodeoxyuridine/metabolism , Cell Death/drug effects , Cell Line , Cell Proliferation/drug effects , DNA, Mitochondrial/genetics , Drug Evaluation, Preclinical , Exons/genetics , Fibroblasts/drug effects , Gene Expression Regulation/drug effects , Humans , Kinetics , Molecular Sequence Data , Phosphorylation/drug effects , Reverse Transcriptase Polymerase Chain Reaction , Thymidine Kinase/genetics , Thymidine Kinase/metabolism
9.
J Med Chem ; 53(7): 2902-12, 2010 Apr 08.
Article in English | MEDLINE | ID: mdl-20218622

ABSTRACT

In an effort to increase the potency and selectivity of earlier identified substrate-based inhibitors of mitochondrial thymidine kinase 2 (TK-2), we now describe the synthesis of new thymidine analogues containing a 4- or 5-substituted 1,2,3-triazol-1-yl substituent at the 3'-position of the 2'-deoxyribofuranosyl ring. These analogues were prepared by Cu- and Ru-catalyzed cycloadditions of 3'-azido-3'-deoxythymidine and the appropriate alkynes, which produced the 1,4- and 1,5-triazoles, respectively. Selected analogues showed nanomolar inhibitory activity for TK-2, while virtually not affecting the TK-1 counterpart. Enzyme kinetics indicated a competitive and uncompetitive inhibition profile against thymidine and the cosubstrate ATP, respectively. This behavior is rationalized by suggesting that the inhibitors occupy the substrate-binding site in a TK-2-ATP complex that maintains the enzyme's active site in a closed conformation through the stabilization of a small lid domain.


Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Mitochondria/enzymology , Thymidine Kinase/antagonists & inhibitors , Thymidine/analogs & derivatives , Thymidine/pharmacology , Animals , Catalytic Domain , Cell Line, Tumor , Drug Design , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/metabolism , Humans , Inhibitory Concentration 50 , Models, Molecular , Structure-Activity Relationship , Substrate Specificity , Thymidine/chemical synthesis , Thymidine/metabolism , Thymidine Kinase/chemistry , Thymidine Kinase/metabolism
10.
Neuromuscul Disord ; 20(3): 198-203, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20083405

ABSTRACT

Deficiency of thymidine kinase-2 (TK2) has been described in children with early onset fatal skeletal myopathy. TK2 is a mitochondrial deoxyribonucleoside kinase required for the phosphorylation of deoxycytidine and deoxythymidine and hence is vital for the maintenance of a balanced mitochondrial dNTP pool in post-mitotic tissues. We describe a patient with two novel TK2 mutations, which caused disease onset shortly after birth and death at the age of three months. One mutation (219insCG) generated an early stop codon, thus preventing the synthesis of a functional protein. The second mutation (R130W) resulted in an amino acid substitution, which caused a severe reduction (<3%) of TK2 enzyme activity. These two novel TK2 mutations cause an extremely severe phenotype with overwhelming central nervous system symptoms not commonly seen in patients with TK2-deficiency. We conclude that the severe clinical presentation in this patient was due to a virtual lack of mitochondrial TK2 activity.


Subject(s)
DNA, Mitochondrial/genetics , Genetic Predisposition to Disease , Mitochondrial Encephalomyopathies/genetics , Mutation/genetics , Thymidine Kinase/genetics , Adenosine Triphosphate/metabolism , Arginine/genetics , Child , Child, Preschool , DNA Mutational Analysis/methods , Female , Humans , Infant , Infant, Newborn , Male , Mitochondria/metabolism , Mitochondria, Muscle/genetics , Mitochondria, Muscle/metabolism , Mitochondrial Diseases/mortality , Mitochondrial Encephalomyopathies/pathology , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Mutagenesis, Site-Directed/methods , Tryptophan/genetics
11.
Int J Biochem Cell Biol ; 42(1): 62-9, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19766732

ABSTRACT

Adenylate kinases are abundant nucleoside monophosphate kinases, which catalyze the phosphorylation of AMP by using ATP or GTP as phosphate donors. A previously cloned cDNA was named adenylate kinase 4 (AK4) based on its sequence similarity with known AKs but with no confirmed AK enzyme activity. In the present study the AK4 cDNA was expressed in Escherichia coli and the substrate specificity and kinetic properties of the recombinant protein were characterized. The enzyme catalyzed the phosphorylation of AMP, dAMP, CMP and dCMP with ATP or GTP as phosphate donors and AK4 also phosphorylated AMP with UTP as phosphate donor. The kinetic parameters of the enzyme were determined for AMP and dAMP with ATP as phosphate donor and for AMP with GTP as phosphate donor. AK4 showed its highest efficiency when phosphorylating AMP with GTP and a slightly lower efficiency for the phosphorylation of AMP with ATP. Among the three reactions for which kinetics were performed, dAMP was the poorest substrate. The AK4 mitochondrial localization was confirmed by expression of AK4 as a fusion protein with GFP in HeLa cells. The mitochondrial import sequence was shown to be located within the first N-terminal 11 amino acid residues, very close to the ATP-binding region of the enzyme. Import analysis suggested that the mitochondrial import sequence was not cleaved and thus the enzyme retained its activity upon entering the mitochondria. Site directed mutagenesis of amino acids Lys 4 and Arg 7 showed that these two residues were essential for mitochondrial import.


Subject(s)
Adenylate Kinase/chemistry , Adenylate Kinase/metabolism , Mitochondria/enzymology , Protein Sorting Signals , Adenosine Triphosphate/metabolism , Adenylate Kinase/genetics , Amino Acid Sequence , Animals , Cell Line, Tumor , Chromatography, Thin Layer , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Guanosine Triphosphate/metabolism , Humans , Kinetics , Mice , Molecular Sequence Data , Plasmids/genetics , Protein Transport , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Proteins/metabolism , Sequence Alignment , Structure-Activity Relationship , Substrate Specificity
12.
FEBS Lett ; 583(17): 2872-6, 2009 Sep 03.
Article in English | MEDLINE | ID: mdl-19647735

ABSTRACT

A full length cDNA that partially corresponded to human adenylate kinase 5 (AK5) was identified and shown to encode for two separate domains. The full length protein could be divided in two distinct functional domains, a previously unidentified domain of 338 amino acids and a second domain of 198 amino acids that corresponded to the protein characterized as AK5, now called AK5p2. The first domain, AK5p1, phosphorylated AMP, CMP, dAMP and dCMP with ATP or GTP as phosphate donors similarly to AK5p2. Our data demonstrate that human AK5 has two separate functional domains and that both have enzymatic activity.


Subject(s)
Adenylate Kinase/chemistry , Isoenzymes/chemistry , Adenylate Kinase/classification , Adenylate Kinase/genetics , Adenylate Kinase/metabolism , Amino Acid Sequence , Catalytic Domain , HeLa Cells , Humans , Isoenzymes/classification , Isoenzymes/genetics , Isoenzymes/metabolism , Molecular Sequence Data , Phylogeny , Protein Structure, Tertiary , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity
13.
Mol Pharmacol ; 75(5): 1127-36, 2009 May.
Article in English | MEDLINE | ID: mdl-19233899

ABSTRACT

Substituted 3'-thiourea derivatives of beta-thymidine (dThd) and 5'-thiourea derivatives of alpha-dThd have been evaluated for their inhibitory activity against recombinant human cytosolic dThd kinase-1 (TK-1), human mitochondrial TK-2, herpes simplex virus type 1 (HSV-1) TK, and varicella-zoster virus TK. Several substituted 3'-thiourea derivatives of beta-dThd proved highly inhibitory to and selective for TK-2 (IC(50) value, 0.15-3.1 microM). The 3'-C-branched p-methylphenyl (compound 1) and 3-CF(3)-4-Cl-phenyl (compound 7) thiourea derivatives of beta-dThd showed competitive inhibition of TK-2 when dThd was used as the variable substrate (K(i) values, 0.40 and 0.05 microM, respectively), but uncompetitive inhibition in the presence of variable concentrations of ATP (K(i) values, 15 and 2.0 microM, respectively). These kinetic properties of compounds 1 and 7 against TK-2 could be accounted for by molecular modeling showing that two hydrogen bonds can be formed between the thiourea nitrogens of compound 7 and the oxygens of the gamma-phosphate of ATP. The importance of several active-site residues was assessed by site-directed mutagenesis experiments on TK-2 and the related HSV-1 TK. The low K(i)/K(m) ratios for compounds 1 and 7 (0.38 and 0.039 against dThd, and 0.75 and 0.12 against ATP, respectively) indicate that these compounds are among the most potent inhibitors of TK-2 described so far. In addition, a striking close correlation was found between the inhibitory activities of the test compounds against TK-2 and Mycobacterium tuberculosis thymidylate kinase that is strongly indicative of close structural and/or functional similarities between both enzymes in relation to their mode of interaction with these nucleoside analog inhibitors.


Subject(s)
Enzyme Inhibitors/pharmacology , Mitochondria/enzymology , Thiourea/analogs & derivatives , Thymidine Kinase/antagonists & inhibitors , Amino Acid Sequence , Animals , Catalysis , Cells, Cultured , Computer Simulation , Drug Stability , Humans , Mice , Models, Molecular , Molecular Sequence Data , Structure-Activity Relationship , T-Lymphocytes/metabolism , Thiourea/pharmacology
14.
Chem Pharm Bull (Tokyo) ; 56(4): 423-32, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18379085

ABSTRACT

During a random screening of representative libraries of nucleoside analogues we discovered that the adenine derivatives FEVB28 and FEG118 were Flaviviridae inhibitors endowed with potency comparable, if not superior, to that of ribavirin. Those studies prompted us to design a new class of protected nucleoside analogs, reported herein, which displays interesting anti-bovine viral diarrhea virus (BVDV) activity and low cytotoxicity in cell-based assays (4, 23, 29 EC(50): 14, 11, 26 microM respectively, CC(50)>100 microM) and appreciable activity in enzyme assays against the RNA dependent RNA polymerase (RdRp) of BVDV (4, 23, 29, RdRp inhibition activity 27, 16, 15 microM respectively). A molecular modeling study was also carried out to highlight the possible interactions between this compounds class and the corresponding hepatitis C virus (HCV) enzyme.


Subject(s)
Adenine Nucleotides/chemical synthesis , Adenine Nucleotides/pharmacology , Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacology , Flaviviridae/drug effects , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , CD4-Positive T-Lymphocytes/drug effects , Cattle , Cell Line , Chromatography, Thin Layer , Computer Simulation , Cricetinae , Diarrhea Viruses, Bovine Viral/drug effects , Drug Design , Drug Evaluation, Preclinical , Drug Screening Assays, Antitumor , Entropy , Humans , Indicators and Reagents , Magnetic Resonance Spectroscopy , Mass Spectrometry , Models, Molecular , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Structure-Activity Relationship , Tetrazolium Salts , Thiazoles
15.
Hum Mol Genet ; 17(15): 2329-35, 2008 Aug 01.
Article in English | MEDLINE | ID: mdl-18434326

ABSTRACT

Deficient enzymatic activity of the mitochondrial deoxyribonucleoside kinases deoxyguanosine kinase (DGUOK) or thymidine kinase 2 (TK2) cause mitochondrial DNA (mtDNA)-depletion syndromes in humans. Here we report the generation of a Tk2-deficient mouse strain and show that the mice develop essentially normally for the first week but from then on exhibit growth retardation and die within 2-4 weeks of life. Several organs including skeletal muscle, heart, liver and spleen showed progressive loss of mtDNA without increased mtDNA mutations or structural alterations. There were no major histological changes in skeletal muscle, but heart muscle showed disorganized and damaged muscle fibers. Electron microscopy showed mitochondria with distorted cristae. The Tk2-deficient mice exhibited pronounced hypothermia and showed loss of hypodermal fat and abnormal brown adipose tissue. We conclude that Tk2 has a major role in supplying deoxyribonucleotides for mtDNA replication and that other pathways of deoxyribonucleotide synthesis cannot compensate for loss of this enzyme.


Subject(s)
DNA, Mitochondrial/metabolism , Mitochondrial Diseases/enzymology , Mitochondrial Diseases/genetics , Thymidine Kinase/physiology , Animals , Cardiomyopathies/enzymology , Cardiomyopathies/genetics , Cardiomyopathies/pathology , DNA Replication , DNA, Mitochondrial/genetics , Growth Disorders/enzymology , Growth Disorders/genetics , Growth Disorders/pathology , Lipodystrophy/enzymology , Lipodystrophy/genetics , Lipodystrophy/pathology , Mice , Mice, Knockout , Mitochondrial Diseases/pathology , Myocardium/enzymology , Myocardium/pathology , Nucleotides/biosynthesis , Thymidine Kinase/genetics
16.
Mol Pharmacol ; 72(6): 1593-8, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17855655

ABSTRACT

The multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) is studied as a candidate suicide gene for applications in combined gene/chemotherapy of cancer. We have created an engineered Dm-dNK nucleoside kinase that is targeted to the mitochondrial matrix. The enzyme was expressed in a thymidine kinase 1-deficient osteosarcoma cell line, and the sensitivity of the cells to cytotoxic nucleoside analogs was determined when the enzyme was targeted to either the nucleus or the mitochondrial matrix. Although the total deoxythymidine (dThd) phosphorylation activity was similar in cells expressing Dm-dNK in the nucleus or in the mitochondria, the cells expressing the enzyme in the mitochondria showed higher sensitivity to the antiproliferative activity of several pyrimidine nucleoside analogs, such as (E)-5-(2-bromovinyl)-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, and 5-fluoro-2'-deoxyuridine. Labeling studies using [3H]dThd showed that the cells expressing the mitochondrial enzyme had an increased incorporation of [3H]dThd into DNA, shown to be due to a higher [3H]dTTP specific activity of the total dTTP pool in the cells in which Dm-dNK was targeted to the mitochondria. The difference in the specific activity of the dTTP pool is a result of different contributions of the de novo and the salvage pathways for the dTTP synthesis in transduced cells. In summary, these findings suggest that mitochondrial targeting of Dm-dNK facilitates nucleoside and nucleoside analog phosphorylation and could be used as a strategy to enhance the efficacy of nucleoside analog phosphorylation and concomitantly their cytostatic potential.


Subject(s)
Drosophila Proteins/biosynthesis , Gene Expression Regulation, Enzymologic/physiology , Mitochondria/enzymology , Phosphotransferases (Alcohol Group Acceptor)/biosynthesis , Animals , Cell Line , Drosophila Proteins/genetics , Drosophila melanogaster , Humans , Mitochondria/genetics , Phosphotransferases (Alcohol Group Acceptor)/genetics , Substrate Specificity/physiology
17.
Bioorg Med Chem ; 15(8): 3065-81, 2007 Apr 15.
Article in English | MEDLINE | ID: mdl-17324575

ABSTRACT

Selective and effective TK2 inhibitors can be obtained by introduction of bulky lipophilic chains (acyl or alkyl entities) at the 2' position of araT and BVaraU, nucleoside analogues naturally endowed with a low TK2 affinity. These derivatives showed a competitive inhibitory activity against TK2 in micromolar range. BVaraU nucleoside analogues, modified on the 2'-O-acyl chain with a terminal N-Boc amino-group, conserved or increased the inhibitory activity against TK2 (7l and 7m IC(50): 6.4 and 3.8 microM, respectively). The substitution of an ester for a carboxamide moiety at the 2' position of araT afforded a consistent reduction of the inhibitory activity (25, IC(50): 480 microM). On the contrary, modifications at 2'-OH position of araC and araG, have provided inactive derivatives against TK2 and dGK, respectively. The biological activity of a representative compound, 2'-O-decanoyl-BVaraU, was also investigated in normal human fibroblasts and was found to impair mitochondrial function due to TK2 inhibition.


Subject(s)
Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Mitochondria/enzymology , Phosphotransferases/antagonists & inhibitors , Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacology , Arabinonucleosides/chemistry , Arabinonucleosides/pharmacology , Drug Design , Fibroblasts/drug effects , Fibroblasts/enzymology , Humans , Indicators and Reagents , Magnetic Resonance Spectroscopy , Phosphorylation , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Reverse Transcriptase Polymerase Chain Reaction , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Structure-Activity Relationship , Thymidine Kinase/antagonists & inhibitors , Viruses/drug effects , Viruses/enzymology
18.
BMC Struct Biol ; 6: 22, 2006 Oct 24.
Article in English | MEDLINE | ID: mdl-17062140

ABSTRACT

BACKGROUND: Development of countermeasures to bioterrorist threats such as those posed by the smallpox virus (variola), include vaccination and drug development. Selective activation of nucleoside analogues by virus-encoded thymidine (dThd) kinases (TK) represents one of the most successful strategies for antiviral chemotherapy as demonstrated for anti-herpes drugs. Vaccinia virus TK is a close orthologue of variola TK but also shares a relatively high sequence identity to human type 2 TK (hTK), thus achieving drug selectivity relative to the host enzyme is challenging. RESULTS: In order to identify any differences compared to hTK that may be exploitable in drug design, we have determined the crystal structure of VVTK, in complex with thymidine 5'-triphosphate (dTTP). Although most of the active site residues are conserved between hTK and VVTK, we observe a difference in conformation of residues Asp-43 and Arg-45. The equivalent residues in hTK hydrogen bond to dTTP, whereas in subunit D of VVTK, Asp-43 and Arg-45 adopt a different conformation preventing interaction with this nucleotide. Asp-43 and Arg-45 are present in a flexible loop, which is disordered in subunits A, B and C. The observed difference in conformation and flexibility may also explain the ability of VVTK to phosphorylate (South)-methanocarbathymine whereas, in contrast, no substrate activity with hTK is reported for this compound. CONCLUSION: The difference in conformation for Asp-43 and Arg-45 could thus be used in drug design to generate VVTK/Variola TK-selective nucleoside analogue substrates and/or inhibitors that have lower affinity for hTK.


Subject(s)
Drug Design , Thymidine Kinase/chemistry , Thymine Nucleotides/chemistry , Vaccinia virus/enzymology , Binding Sites , Crystallography, X-Ray , Kinetics , Ligands , Macromolecular Substances/chemistry , Models, Biological , Models, Molecular , Molecular Conformation , Mutagenesis, Site-Directed , Mutation , Protein Binding , Sensitivity and Specificity , Thymidine Kinase/antagonists & inhibitors , Thymidine Kinase/genetics , Thymidine Kinase/metabolism , Thymine Nucleotides/metabolism , Viral Proteins/chemistry
19.
Skin Res Technol ; 12(4): 303-8, 2006 Nov.
Article in English | MEDLINE | ID: mdl-17026664

ABSTRACT

BACKGROUND/PURPOSES: Skin antioxidant network protects cells against oxidative injury and prevents the production of oxidation products. When oxidative stress overwhelms the skin antioxidant capacity, the subsequent modification of the cellular redox apparatus leads to an alteration of cell homeostasis leading to degenerative processes. In the dermocosmetic field, the topical application of antioxidants is often suggested as a possible strategy to prevent and modulate oxidative skin damages. Continuing our studies addressed to set-up new bio-engineering protocols for the claim substantiation of antioxidant cosmetic products, we have developed a new non-invasive methodology for the evaluation of antioxidants cosmetics ingredients and finished products. METHODS: The effects of a pre-treatment on forearm skin with an antioxidant ingredient were investigated on 15 volunteers, in a double-blind randomised fashion. A non-invasive method was devised that comprises the collection of forehead SC layers of the pre-treated area and control and the next evaluation of skin antioxidant capacity (IAC-S) by a luminescence-based method. RESULTS: The results showed that the antioxidant preparation was able to increase, to a statistically significant extent (P<0.01), the IAC-S in comparison with the control area. The data were confirmed (P<0.05) by comparison with a method, previously developed by us, based on DermAnalyzer. CONCLUSIONS: In view of the simplicity and reliability of the method here presented, this new technique is proposed as a possible tool for the routine evaluation of in vivo efficacy of antioxidant functional ingredients and finished products.


Subject(s)
Antioxidants/metabolism , Antioxidants/pharmacology , Epidermis/metabolism , Erythema/prevention & control , Pruritus/prevention & control , Surgical Tape , Tocopherols/pharmacology , Adult , Antioxidants/administration & dosage , Double-Blind Method , Epidermis/drug effects , Erythema/chemically induced , Forearm , Humans , Image Processing, Computer-Assisted , Luminescence , Nicotinic Acids , Pruritus/chemically induced , Tocopherols/administration & dosage
20.
J Biol Chem ; 281(28): 19273-9, 2006 Jul 14.
Article in English | MEDLINE | ID: mdl-16702226

ABSTRACT

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).


Subject(s)
Herpesvirus 1, Human/enzymology , Protein Engineering/methods , Purine Nucleosides/chemistry , Pyrimidine Nucleosides/chemistry , Thymidine Kinase/chemistry , Antiviral Agents/pharmacology , Binding Sites , Kinetics , Models, Chemical , Models, Molecular , Mutagenesis, Site-Directed , Mutation , Phosphorylation , Protein Conformation
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