The Experts below are selected from a list of 270 Experts worldwide ranked by ideXlab platform
Margaret E. Black - One of the best experts on this subject based on the ideXlab platform.
-
characterization of herpes simplex virus type 1 Thymidine Kinase mutants engineered for improved ganciclovir or acyclovir activity
Protein Science, 2009Co-Authors: Mark S Kokoris, Margaret E. BlackAbstract:Herpes Simplex Virus type 1 (HSV-1) Thymidine Kinase (TK) is currently the most widely used suicide agent for gene therapy of cancer. Tumor cells that express HSV-1 Thymidine Kinase are rendered sensitive to prodrugs due to preferential phosphorylation by this enzyme. Although ganciclovir (GCV) is the prodrug of choice for use with TK, this approach is limited in part by the toxicity of this prodrug. From a random mutagenesis library, seven Thymidine Kinase variants containing multiple amino acid substitutions were identified on the basis of activity towards ganciclovir and acyclovir based on negative selection in Escherichia coli. Using a novel affinity chromatography column, three mutant enzymes and the wild-type TK were purified to homogeneity and their kinetic parameters for Thymidine, ganciclovir, and acyclovir determined. With ganciclovir as the substrate, one mutant (mutant SR39) demonstrated a 14-fold decrease in Km compared to the wild-type enzyme. The most dramatic change is displayed by mutant SR26, with a 124-fold decrease in Km with acyclovir as the substrate. Such new “prodrug Kinases” could provide benefit to ablative gene therapy by now making it feasible to use the relatively nontoxic acyclovir at nanomolar concentrations or ganciclovir at lower, less immunosuppressive doses.
-
a guanylate Kinase hsv 1 Thymidine Kinase fusion protein enhances prodrug mediated cell killing
Gene Therapy, 2006Co-Authors: Candice Willmon, Elizabeth Krabbenhoft, Margaret E. BlackAbstract:A guanylate Kinase/HSV-1 Thymidine Kinase fusion protein enhances prodrug-mediated cell killing
-
optimizing prostate cancer suicide gene therapy using herpes simplex virus Thymidine Kinase active site variants
Human Gene Therapy, 2002Co-Authors: Allan J Pantuck, Margaret E. Black, Jamie Matherly, Amnon Zisman, David Nguyen, Frank Berger, Sanjiv S Gambhir, Arie S BelldegrunAbstract:The herpes simplex virus (HSV) Thymidine Kinase gene (tk) forms the basis of a widely used strategy for suicide gene therapy. A library of HSV Thymidine Kinase enzyme (TK) active site mutants having different affinities for guanosine analog prodrugs was developed. We sought to determine the optimal combination of tk variant and prodrug specifically for prostate cancer gene therapy, using in vitro and in vivo studies of adenovirally infected CL1, DU-145, and LNCaP tumor lines carrying wild-type tk, tk30, tk75, and sr39tk mutants expressed by a strong, constitutive cytomegalovirus promoter and treated with ganciclovir and acyclovir. In vitro experiments involving prostate cancer (CaP) cell line infection were carried out with a broad range of prodrug concentrations, and cell killing was determined by limiting dilution (colony-forming), MTT, and propidium iodide assays. In vivo studies based on CL1-GFP xenograft experiments were carried out to examine the ability of each TK variant to prevent tumor formation and to inhibit tumor growth and development of metastases in established orthotopic and subcutaneous tumors in SCID mice. Both in vitro and in vivo studies suggest improved killing with the sr39tk variant. Thus, the results suggest that the use of sr39tk in future trials of prostate cancer tk suicide gene therapy may be beneficial.
-
enhancement of tumor ablation by a selected hsv 1 Thymidine Kinase mutant
Gene Therapy, 1999Co-Authors: Mark S Kokoris, P Sabo, Elinor T Adman, Margaret E. BlackAbstract:With the advent of gene therapy, herpes simplex virus type I (HSV-1) Thymidine Kinase (TK) has garnered much interest as a suicide gene for cancer ablation. As a means to improve the overall efficacy of the prodrug-gene activation approach, as well as to reduce ganciclovir-mediated toxicity, a large library of mutant Thymidine Kinases was generated and screened for the ability to enhance in vitro cell sensitivity to the prodrugs, ganciclovir (GCV) and acyclovir (ACV). Enzyme kinetics of one Thymidine Kinase mutant from this library that contains six amino acid substitutions at or near the active site reveals a distinct mechanism for providing enhanced prodrug-mediated killing in mammalian cells. In in vitro rat C6 cell prodrug sensitivity assays the TK mutant (mutant 30) achieves nanomolar IC50 values with GCV and ACV, in contrast to IC50values of 30 μM and >100 μM, respectively, for wild-type TK. In a mouse xenograft tumor model, growth of mutant 30 expressing tumors is restricted by ganciclovir at a dose at least 10- fold lower than one that impedes growth of wild-type TK-expressing tumors. Furthermore, in the presence of GCV a substantial bystander effect is observable when only 20% of the tumor cells express mutant 30 whereas no restriction in tumor growth is seen in tumors bearing the wild-type TK under the same conditions. The enhanced sensitization to prodrugs conferred by mutant 30 is apparently due to a 35-fold increase in Thymidine Km which results in reduced competition between prodrug and Thymidine at the active site. This provides mutant 30 a substantial kinetic advantage despite very high Kms for both ganciclovir and acyclovir. Molecular modeling of the mutations within the active site suggests that a tyrosine substitution at alanine 168 (A168) alters Thymidine and prodrug interactions by causing catalytically important residues to move. The use of mutant 30 in place of the wild-type TK should provide a more effective gene therapy of cancer.
-
creation of drug specific herpes simplex virus type 1 Thymidine Kinase mutants for gene therapy
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Margaret E. Black, Terry G Newcomb, Heather Marie P Wilson, Lawrence A LoebAbstract:Abstract Herpes simplex virus type 1 (HSV-1) Thymidine Kinase is currently used as a suicide agent in the gene therapy of cancer. This therapy is based on the preferential phosphorylation of nucleoside analogs by tumor cells expressing HSV-1 Thymidine Kinase. However, the use of HSV-1 Thymidine Kinase is limited in part by the toxicity of the nucleoside analogs. We have used random sequence mutagenesis to create new HSV-1 Thymidine Kinases that, compared with wild-type Thymidine Kinase, render cells much more sensitive to specific nucleoside analogs. A segment of the HSV-1 Thymidine Kinase gene at the putative nucleoside binding site was substituted with random nucleotide sequences. Mutant enzymes that demonstrate preferential phosphorylation of the nucleoside analogs, ganciclovir or acyclovir, were selected from more than one million Escherichia coli transformants. Among the 426 active mutants we have isolated, 26 demonstrated enhanced sensitivity to ganciclovir, and 54 were more sensitive to acyclovir. Only 6 mutant enzymes displayed sensitivity to both ganciclovir and acyclovir when expressed in E. coli. Analysis of 3 drug-sensitive enzymes demonstrated that 1 produced stable mammalian cell transfectants that are 43-fold more sensitive to ganciclovir and 20-fold more sensitive to acyclovir.
Orly Elpeleg - One of the best experts on this subject based on the ideXlab platform.
-
mitochondrial deoxyribonucleoside triphosphate pools in Thymidine Kinase 2 deficiency
Biochemical and Biophysical Research Communications, 2003Co-Authors: Ann Saada, Orly Elpeleg, Hanna Mandel, Efrat Benshalom, Rivka Zyslin, Chaya MillerAbstract:Deficiency of mitochondrial Thymidine Kinase (TK2) is associated with mitochondrial DNA (mtDNA) depletion and manifests by severe skeletal myopathy in infancy. In order to elucidate the pathophysiology of this condition, mitochondrial deoxyribonucleoside triphosphate (dNTP) pools were determined in patients' fibroblasts. Despite normal mtDNA content and cytochrome c oxidase (COX) activity, mitochondrial dNTP pools were imbalanced. Specifically, deoxyThymidine triphosphate (dTTP) content was markedly decreased, resulting in reduced dTTP:deoxycytidine triphosphate ratio. These findings underline the importance of balanced mitochondrial dNTP pools for mtDNA synthesis and may serve as the basis for future therapeutic interventions.
-
mtdna depletion myopathy elucidation of the tissue specificity in the mitochondrial Thymidine Kinase tk2 deficiency
Molecular Genetics and Metabolism, 2003Co-Authors: Ann Saada, Orly Elpeleg, Avraham ShaagAbstract:Decreased mitochondrial Thymidine Kinase (TK2) activity is associated with mitochondrial DNA (mtDNA) depletion and respiratory chain dysfunction and is manifested by isolated, fatal skeletal myopathy. Other tissues such as liver, brain, heart, and skin remain unaffected throughout the patients' life. In order to elucidate the mechanism of tissue specificity in the disease we have investigated the expression of the mitochondrial deoxynucleotide carrier, the mtDNA content and the activity of TK2 in mitochondria of various tissues. Our results suggest that low basal TK2 activity combined with a high requirement for mitochondrial encoded proteins in muscle predispose this tissue to the devastating effect of TK2 deficiency.
-
mutant mitochondrial Thymidine Kinase in mitochondrial dna depletion myopathy
Nature Genetics, 2001Co-Authors: Ann Saada, Avraham Shaag, Yoram Nevo, Hanna Mandel, Orly ElpelegAbstract:The mitochondrial deoxyribonucleotide (dNTP) pool is separated from the cytosolic pool because the mitochondria inner membrane is impermeable to charged molecules. The mitochondrial pool is maintained by either import of cytosolic dNTPs through dedicated transporters1,2 or by salvaging deoxynucleosides within the mitochondria; apparently, enzymes of the de novo dNTP synthesis pathway are not present in the mitochondria. In non-replicating cells, where cytosolic dNTP synthesis is down-regulated, mtDNA synthesis depends solely on the mitochondrial salvage pathway enzymes, the deoxyribonucleosides Kinases. Two of the four human deoxyribonucleoside Kinases, deoxyguanosine Kinase (dGK) and Thymidine Kinase-2 (TK2), are expressed in mitochondria3,4,5,6. Human dGK efficiently phosphorylates deoxyguanosine and deoxyadenosine, whereas TK2 phosphorylates deoxyThymidine, deoxycytidine and deoxyuridine. Here we identify two mutations in TK2, histidine 90 to asparagine and isoleucine 181 to asparagine, in four individuals who developed devastating myopathy and depletion of muscular mitochondrial DNA in infancy. In these individuals, the activity of TK2 in muscle mitochondria is reduced to 14–45% of the mean value in healthy control individuals. Mutations in TK2 represent a new etiology for mitochondrial DNA depletion, underscoring the importance of the mitochondrial dNTP pool in the pathogenesis of mitochondrial depletion.
Staffan Eriksson - One of the best experts on this subject based on the ideXlab platform.
-
3 1 2 3 triazol 1 yl 3 deoxyThymidine analogs as substrates for human and ureaplasma parvum Thymidine Kinase for structure activity investigations
Bioorganic & Medicinal Chemistry, 2010Co-Authors: Liya Wang, Luigi A Agrofoglio, Dominique Devillebonne, Tamara R Mcbrayer, Steven J Coats, Raymond F Schinazi, Staffan ErikssonAbstract:Abstract The pathogenic mycoplasma Ureaplasma parvum (Up) causes opportunistic infections and relies on salvage of nucleosides for DNA synthesis and Up Thymidine Kinase (UpTK) provides the necessary Thymidine nucleotides. The anti-HIV compound 3-azido-3′-deoxyThymidine (AZT) is a good substrate for TK. Methods for a rapid and efficient synthesis of new 3′-α-[1,2,3]triazol-3′-deoxyThymidine analogs from AZT under Huisgen conditions are described. Thirteen 3′-analogues were tested with human cytosolic Thymidine Kinase (hTK1) and UpTK. The new analogs showed higher efficiencies (Km/Vmax values) in all cases with UpTK than with hTK1. Still, hTK1 was preferentially inhibited by 9 out of 10 tested analogs. Structural models of UpTK and hTK1 were constructed and used to explain the kinetic results. Two different binding modes of the nucleosides within the active sites of both enzymes were suggested with one predominating in the bacterial enzyme and the other in hTK1. These results will aid future development of anti-mycoplasma nucleosides.
-
Structures of Thymidine Kinase 1 of human and mycoplasmic origin.
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Martin Welin, Staffan Eriksson, Liya Wang, Birgitte Munch-petersen, U. Kosinska, Nils-egil Mikkelsen, Cecilia Carnrot, Chunying Zhu, Hans EklundAbstract:Cytosolic Thymidine Kinase 1, TK1, is a well known cell-cycle-regulated enzyme of importance in nucleotide metabolism as well as an activator of antiviral and anticancer drugs such as 3′-azido-3′-deoxyThymidine (AZT). We have now determined the structures of the TK1 family, the human and Ureaplasma urealyticum enzymes, in complex with the feedback inhibitor dTTP. The TK1s have a tetrameric structure in which each subunit contains an α/β-domain that is similar to ATPase domains of members of the RecA structural family and a domain containing a structural zinc. The zinc ion connects β-structures at the root of a β-ribbon that forms a stem that widens to a lasso-type loop. The Thymidine of dTTP is hydrogen-bonded to main-chain atoms predominantly coming from the lasso loop. This binding is in contrast to other deoxyribonucleoside Kinases where specific interactions occur with side chains. The TK1 structure differs fundamentally from the structures of the other deoxyribonucleoside Kinases, indicating a different evolutionary origin.
-
sensitive nonradiometric method for determining Thymidine Kinase 1 activity
Clinical Chemistry, 2004Co-Authors: Anders B. Öhrvik, Maria Lindh, Roland Einarsson, Jacques Grassi, Staffan ErikssonAbstract:Background: Thymidine Kinase 1 (TK1) is a cytoplasmic enzyme, produced only in the S-phase of proliferating cells, that has potential as a tumor marker. Specific determination of TK1 in serum is difficult, in part because of differences in the physical properties of serum TK1 compared with cytoplasmic TK1. Methods: The first step in the new assay was phosphorylation of 3′-azido-2′,3′-deoxyThymidine (AZT) to AZT 5′-monophosphate (AZTMP) by TK1 present in patient material. The AZTMP formed was measured in a competitive immunoassay with specific anti-AZTMP antibodies and AZTMP-labeled peroxidase. Results were compared with those of a TK radioenzyme assay (REA) for 78 samples from patients suffering from hematologic diseases. Results: The detection limit was 78 μIU/L, and within-run CVs <20% were seen for samples with TK1 down to 130 μIU/L. Cross-determination of the mitochondrial isoenzyme TK2 activity was <0.1%. Between-assay imprecision (CV) was 3.5–7.4%, and the within-assay imprecision was 4.1–9.1%. In studies of recovery and linearity on dilution, measured values ranged from 84% to 115% of expected at concentrations of 0.26–10.4 mIU/L. Results of the new assay (mIU/L) = 0.109 × TK REA (U/L) + 0.092. Heterophilic antibodies did not interfere in the assay. The upper 95th percentile, in 100 healthy individuals, was 0.94 mIU/L, and the median value was 0.43 mIU/L. Conclusion: The TK1 enzyme-labeled immunoassay uses a stable substrate, is precise, appears to be accurate, and is resistant to interferences. It may provide a practical tool in the management of hematologic malignancies.
-
molecular characterization of Thymidine Kinase from ureaplasma urealyticum nucleoside analogues as potent inhibitors of mycoplasma growth
Molecular Microbiology, 2003Co-Authors: Cecilia Carnrot, Staffan Eriksson, Rahma Wehelie, Goran Bolske, Liya WangAbstract:Ureaplasma urealyticum (U. urealyticum), belonging to the class Mollicutes, is a human pathogen colonizing the urogenital tract and causes among other things respiratory diseases in premature infants. We have studied the salvage of pyrimidine deoxynucleosides in U. urealyticum and cloned a key salvage enzyme, Thymidine Kinase (TK) from U. urealyticum. Recombinant Uu-TK was expressed in E. coli, purified and characterized with regards to substrate specificity and feedback inhibition. Uu-TK efficiently phosphorylated Thymidine (dThd) and deoxyuridine (dUrd) as well as a number of pyrimidine nucleoside analogues. All natural ribonucleoside/deoxyribonucleoside triphosphates, except dTTP, served as phosphate donors, while dTTP was a feedback inhibitor. The level of Uu-TK activity in U. urealyticum extracts increased upon addition of dUrd to the growth medium. Fluoropyrimidine nucleosides inhibited U. urealyticum and M. pneumoniae growth and this inhibitory effect could be reversed by addition of dThd, dUrd or deoxytetrahydrouridine to the growth medium. Thus, the mechanism of inhibition was most likely the depletion of dTTP, either via a blocked Thymidine Kinase reaction and/or thymidylate synthesis step and these metabolic reactions should be suitable targets for antimycoplasma chemotherapy.
-
selective assays for Thymidine Kinase 1 and 2 and deoxycytidine Kinase and their activities in extracts from human cells and tissues
Biochemical and Biophysical Research Communications, 1992Co-Authors: Elias S J Arner, Tatjana Spasokoukotskaja, Staffan ErikssonAbstract:Abstract 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-β-D-arabinofura-anosylThymidine (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.
Caterina Garone - One of the best experts on this subject based on the ideXlab platform.
-
deoxycytidine and deoxyThymidine treatment for Thymidine Kinase 2 deficiency
Annals of Neurology, 2017Co-Authors: Carlos Lopezgomez, Caterina Garone, Beatriz Garciadiaz, Saba Tadesse, Rebecca J Levy, Maria J Sanchezquintero, Marti Juanolafalgarona, Emanuele BarcaAbstract:Objective Thymidine Kinase 2 (TK2), a critical enzyme in the mitochondrial pyrimidine salvage pathway, is essential for mitochondrial DNA (mtDNA) maintenance. Mutations in the nuclear gene, TK2, cause TK2 deficiency, which manifests predominantly in children as myopathy with mtDNA depletion. Molecular bypass therapy with the TK2 products, deoxycytidine monophosphate (dCMP) and deoxyThymidine monophosphate (dTMP), prolongs the life span of Tk2-deficient (Tk2–/–) mice by 2- to 3-fold. Because we observed rapid catabolism of the deoxynucleoside monophosphates to deoxyThymidine (dT) and deoxycytidine (dC), we hypothesized that: (1) deoxynucleosides might be the major active agents and (2) inhibition of deoxycytidine deamination might enhance dTMP+dCMP therapy. Methods To test these hypotheses, we assessed two therapies in Tk2–/– mice: (1) dT+dC and (2) coadministration of the deaminase inhibitor, tetrahydrouridine (THU), with dTMP+dCMP. Results We observed that dC+dT delayed disease onset, prolonged life span of Tk2-deficient mice and restored mtDNA copy number as well as respiratory chain enzyme activities and levels. In contrast, dCMP+dTMP+THU therapy decreased life span of Tk2–/– animals compared to dCMP+dTMP. Interpretation Our studies demonstrate that deoxynucleoside substrate enhancement is a novel therapy, which may ameliorate TK2 deficiency in patients. Ann Neurol 2017;81:641–652
-
Research Article Deoxypyrimidine monophosphate bypass therapy for Thymidine Kinase 2 deficiency
2016Co-Authors: Caterina Garone, Valentina Emmanuele, Luis C Lopez, Saba Tadesse, Kurenai Tanji, Catarina M Quinzii, Beatriz Garcia-diaz, O Akman, Michio HiranoAbstract:Autosomal recessive mutations in the Thymidine Kinase 2 gene (TK2) cause mitochondrial DNA depletion, multiple deletions, or both due to loss of TK2 enzyme activity and ensuing unbalanced deoxynucleotide triphosphate (dNTP) pools. To bypass Tk2 defi-ciency, we administered deoxycytidine and deoxyThymidine mono-phosphates (dCMP+dTMP) to the Tk2 H126N (Tk2/) knock-in mouse model from postnatal day 4, when mutant mice are pheno-typically normal, but biochemically affected. Assessment of 13-day-old Tk2/ mice treated with dCMP+dTMP 200 mg/kg/day each (Tk2/200dCMP/dTMP) demonstrated that in mutant animals, the compounds raise dTTP concentrations, increase levels of mtDNA, ameliorate defects of mitochondrial respiratory chain enzymes, and significantly prolong their lifespan (34 days with treatment versus 13 days untreated). A second trial of dCMP+dTMP each at 400 mg/kg/day showed even greater pheno-typic and biochemical improvements. In conclusion, dCMP/dTMP supplementation is the first effective pharmacologic treatment for Tk2 deficiency
-
deoxypyrimidine monophosphate bypass therapy for Thymidine Kinase 2 deficiency
Embo Molecular Medicine, 2014Co-Authors: Caterina Garone, Beatriz Garciadiaz, Valentina Emmanuele, Luis C Lopez, Saba Tadesse, Hasan O Akman, Kurenai Tanji, Catarina M QuinziiAbstract:Autosomal recessive mutations in the Thymidine Kinase 2 gene (TK2) cause mitochondrial DNA depletion, multiple deletions, or both due to loss of TK2 enzyme activity and ensuing unbalanced deoxynucleotide triphosphate (dNTP) pools. To bypass Tk2 deficiency, we administered deoxycytidine and deoxyThymidine monophosphates (dCMP+dTMP) to the Tk2 H126N (Tk2 � /� ) knock-in mouse model from postnatal day 4, when mutant mice are phenotypically normal, but biochemically affected. Assessment of 13-day-old Tk2 � /� mice treated with dCMP+dTMP 200 mg/kg/day each (Tk2 � /� 200dCMP/dTMP ) demonstrated that in mutant animals, the compounds raise dTTP concentrations, increase levels of mtDNA, ameliorate defects of mitochondrial respiratory chain enzymes, and significantly prolong their lifespan (34 days with treatment versus 13 days untreated). A second trial of dCMP+dTMP each at 400 mg/kg/day showed even greater phenotypic and biochemical improvements. In conclusion, dCMP/dTMP supplementation is the first effective pharmacologic treatment for Tk2 deficiency.
Robert W Overell - One of the best experts on this subject based on the ideXlab platform.
-
dominant positive and negative selection using a hygromycin phosphotransferase Thymidine Kinase fusion gene
Molecular and Cellular Biology, 1991Co-Authors: Stephen D Lupton, Laurie L Brunton, Victoria A Kalberg, Robert W OverellAbstract:The hygromycin phosphotransferase gene was fused in-frame with the herpes simplex virus type 1 Thymidine Kinase gene. The resulting fusion gene (termed HyTK) confers hygromycin B resistance for dominant positive selection and ganciclovir sensitivity for negative selection and provides a means by which these selectable phenotypes may be expressed and regulated as a single genetic entity.