Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.1.21 (thymidine kinase)
7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The efficacy of suicide herpes simplex virus-1 thymidine kinase (HSVtk)/ganciclovir (GCV) gene therapy is often limited by intrinsic resistance of tumor cells. Here we show that repair of GCV incorporated in DNA is a factor involved in GCV resistance. A protective role of DNA repair in GCV-induced cell killing is supported by the following findings: (a) GCV-exposed Chinese hamster ovary-HSVtk cells exhibited both reduced repair of GCV and cloning efficiency in the presence of a specific polymerase beta (beta-pol) inhibitor, prunasin; (b) DNA beta-pol-deficient mouse fibroblasts were more sensitive to the cytotoxic, apoptosis-inducing, and genotoxic (DNA breakage and chromosomal aberration-inducing) effects of GCV as compared with wild-type and beta-pol-complemented cell lines; (c) methoxyamine, an inhibitor of beta-pol-dependent short-patch base excision repair, sensitized wild-type and complemented beta-pol cells to GCV, whereas it had no effect on the sensitivity of beta-pol-null cells to GCV. Because methoxyamine-mediated sensitization of beta-pol wild-type and beta-pol-complemented cells to GCV did not reach the level of null cells, we suggest that both beta-pol-dependent short- and long-patch base excision repair are involved in protection of cells to GCV. Some implications for HSVtk/GCV gene therapy are being discussed.
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PMID:DNA polymerase beta mediates protection of mammalian cells against ganciclovir-induced cytotoxicity and DNA breakage. 1160 69

In vitro susceptibility assays of herpes simplex virus (HSV) do not necessarily correlate with treatment outcome. An HSV type 1 (HSV-1) isolate, N4, recovered from a patient who presented with herpes keratitis with localized immunosuppression, was characterized for susceptibility. Although the 50% inhibitory concentration (IC(50)) for this isolate was less than the accepted breakpoint for defining resistance to acyclovir (>2.0 microg/mL), the following lines of evidence suggest that the isolate was acyclovir resistant: (1) the clinical history confirmed that the infection was nonresponsive to acyclovir; (2) the in vitro susceptibility was similar to that of a thymidine kinase (TK)-negative, acyclovir-resistant virus SLU360; (3) the IC(50) of acyclovir was more than 10 times the IC(50) for an acyclovir-susceptible control strain; (4) plaque-purified clonal isolates were resistant to acyclovir (IC(50)s, >2.0 microg/mL); and (5) biochemical studies indicated that the HSV-1 N4 TK was partially impaired for acyclovir phosphorylation. Although residue changes were found in both the viral tk and pol coding regions of HSV-1 N4, characterization of a recombinant virus expressing the HSV-1 N4 polymerase suggested that the TK and Pol together conferred the acyclovir-resistance phenotype.
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PMID:Biochemical characterization of a virus isolate, recovered from a patient with herpes keratitis, that was clinically resistant to acyclovir. 1171 95

DNA polymerase beta (pol beta) is an ideal system for studying the role of its different amino acid residues in the fidelity of DNA synthesis. In this study, the T79S variant of pol beta was identified using an in vivo genetic screen. T79S is located in the N-terminal 8-kDa domain of pol beta and has no contact with either the DNA template or the incoming dNTP substrate. The T79S protein produced 8-fold more multiple mutations in the herpes simplex virus type 1-thymidine kinase assay than wild-type pol beta. Surprisingly, T79S is a misincorporation mutator only when using a 3'-recessed primer-template. In the presence of a single nucleotide-gapped DNA substrate, T79S displays an antimutator phenotype when catalyzing DNA synthesis opposite template C and has similar fidelity as wild type opposite templates A, G, or T. Threonine 79 is located directly between two helix-hairpin-helix motifs located within the 8-kDa and thumb domains of pol beta. As the pol beta enzyme closes into its active form, the helix-hairpin-helix motifs appear to assist in the production and stabilization of a 90 degrees bend of the DNA. The function of the bent DNA is to present the templating base to the incoming nucleotide substrate. We propose that Thr-79 is part of a hydrogen bonding network within the helix-hairpin-helix motifs that is important for positioning the DNA within the active site. We suggest that alteration of Thr-79 to Ser disrupts this hydrogen bonding network and results in an enzyme that is unable to bend the DNA into the proper geometry for accurate DNA synthesis.
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PMID:Threonine 79 is a hinge residue that governs the fidelity of DNA polymerase beta by helping to position the DNA within the active site. 1212 98

A human leukocyte antigen (HLA)-matched unrelated bone marrow transplantation (BMT) was performed in a 13-year-old patient with the congenital immunodeficiency syndrome, Wiskott-Aldrich syndrome. The patient had a history of acyclovir (ACV)-resistant (ACV(r)) herpes simplex virus type 1 (HSV-1) infections prior to BMT. After BMT, the skin lesions caused by HSV-1 relapsed on the face and genito-anal areas. Ganciclovir (GCV) therapy was initiated, but the mucocutaneous lesions worsened. An HSV-1 isolate recovered from the lesions during this episode was resistant to both ACV and GCV. The ACV(r) isolate was confirmed to have the same mutation in the viral thymidine kinase (TK) gene as that of the previously isolated ACV(r) isolates from the patient. After treatment switch to foscarnet (PFA), there was a satisfactory remission but not a complete recovery. Although the mucocutaneous lesions improved, a PFA-resistant (PFA(r)) HSV-1 was isolated 1 month after the start of PFA therapy. The PFA(r) HSV-1 isolate coded for the same mutation in the viral TK gene as the ACV(r) HSV-1 isolates. Furthermore, the PFA(r) isolate also expressed a mutated viral DNA polymerase (DNA pol) with an amino acid (Gly) substitution for Val at position 715. This is the first report on the clinical course of a BMT-associated ACV(r) HSV-1 infection that subsequently developed resistance to foscarnet as well.
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PMID:Bone marrow transplantation in a child with Wiskott-Aldrich syndrome latently infected with acyclovir-resistant (ACV(r)) herpes simplex virus type 1: emergence of foscarnet-resistant virus originating from the ACV(r) virus. 1221 Apr 36

Sequential herpes simplex virus type 1 (HSV-1) isolates were obtained from a paediatric haematopoietic stem cell transplant (HSCT) patient who received prolonged therapy with acyclovir (ACV) followed by foscarnet (PFA) and topical cidofovir (HPMPC) for severe persistent mucocutaneous HSV-1 infection. The isolates were retrospectively studied for drug resistance. The first resistant isolate associated with clinical failure of antiviral therapy emerged 44 days post-ACV treatment initiation. Susceptibility testing revealed an ACV-resistant HSV strain that demonstrated cross resistance to PFA in the absence of any previous PFA treatment. The observed cross resistance was conferred by a single amino acid substitution, Ser724Asn, in the HSV DNA polymerase (DNA pol) gene. During the subsequent course of ACV therapy, the ACV/PFA-cross-resistant isolates were replaced by ACV-resistant, PFA-sensitive isolates. These isolates carried no DNA pol mutations, but had an Arg163His substitution in the thymidine kinase gene. Upon subsequent switching of antiviral therapy from ACV to PFA, the original ACV/PFA-cross-resistant DNA pol mutant re-appeared. Our study shows the emergence of different drug-resistant HSV variants during ongoing, unchanged ACV therapy. Furthermore, a rapid re-selection of the original resistant variant was observed after switch. For optimal antiviral management of HSV infections in HSCT recipients, therapeutic decisions should be guided by drug susceptibility results whenever therapeutic failure is observed and/or when changes in antiviral treatment are considered.
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PMID:Sequential switching of DNA polymerase and thymidine kinase-mediated HSV-1 drug resistance in an immunocompromised child. 1504 May 41

Thirty-one herpes simplex virus type one (HSV-1) isolates from 12 haematopoietic stem cell transplant recipients with persistent HSV infections despite acyclovir (ACV) prophylaxis or treatment, were genotypically and phenotypically characterized. The relationship between drug susceptibility of the isolates and mutations in thymidine kinase (TK) and DNA polymerase (DNA pol) genes was examined. In all 12 patients, HSV infections were due to ACV-resistant, foscarnet-sensitive viruses. Out of 31 isolates examined, 23 were resistant and eight were sensitive to ACV; eight patients carried viruses with frameshift mutations in the TK gene (due to addition or deletion of single nucleotides in homopolymeric repeats). These mutations were found at codon 61 (G deletion, one patient), 146 (G insertion, five patients) and 153 or 185 (C deletion, one patient each). In four patients, viruses were selected during ACV therapy that contained novel amino acid substitutions in the TK gene (H58R, G129D, A189V, R216H, R220C). Their possible role in ACV resistance was further confirmed phenotypically and by the absence of any resistance-associated mutations in the DNA pol gene. These substitutions were located in ATP- or nucleoside-binding sites or in conserved regions of the TK gene. In addition, a single mutation, Q570R, in the delta-region C of the DNA pol gene, was identified in an isolate from a single patient with resistance to ACV. Our study confirms and expands previous data on genotypic changes associated with ACV resistance of HSV-1 clinical isolates.
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PMID:Genotypic and phenotypic characterization of acyclovir-resistant herpes simplex viruses isolated from haematopoietic stem cell transplant recipients. 1545 88

Low levels of gene delivery in vivo using replication-defective retroviral vectors have severely limited their application for clinical protocols. To overcome this problem, we describe here a semi-replication-competent retrovirus (s-RCR) in which the gag-pol and envelope (VSV-G, vesicular stomatitis virus G protein) genes were split into two vectors. This system offers potential advantages over both replication-defective vectors, in terms of efficiency of in vivo spread through a tumor, and all-in-one replication-competent vectors in terms of the payload of therapeutic genes that can be carried. We achieved a viral titer of s-RCR viruses approximately 70-fold higher than VSV-G pseudotyped, replication-defective vectors. In addition, s-RCR vectors induced tumor killing by the cytotoxicity of VSV-G during viral spread. Inclusion of the herpes simplex virus thymidine kinase (HSVtk30) gene into vectors significantly improved tumor killing activity followed by ganciclovir (GCV) treatment in vitro under conditions of low-level viral replication. However, at high levels of viral spread, VSV-G-mediated cytotoxicity predominated. Xenografts of human fibrosarcoma HT1080 cells, preinfected by semi-replicative green fluorescent protein vectors (semi-GFP), were completely non-tumorigenic in nude mice. Implantation of cells preinfected by semi-replicative TK30 vectors (semi-TK30) mixed with parental HT1080 cells at a ratio of 1:1 efficiently prevented tumor growth in mice treated by GCV. Direct intratumoral injection of HT1080 tumors growing in nude mice, or B16 murine melanoma in immunocompetent mice, with semi-TK30 viruses significantly prolonged survival. Injection of autologous cells (B16) producing semi-TK30 vector into B16 tumors prolonged survival only in mice treated with GCV but not with phosphate-buffered saline (PBS). In contrast, when xenogeneic cells (293T) producing semi-TK30 vectors were injected into B16 tumors, an optimal survival advantage was obtained in mice treated with PBS rather than GCV. These data indicate that complex interactions exist between direct cytotoxicity of VSV-G and HSVtk expression when placed in the context of additional immune parameters, which combine to determine the efficacy of the therapy. Taken together, our data suggest that s-RCR vectors have some potential advantages for development to deliver genes into tumors for cancer treatment but that a combination of factors will impact on the decision as to whether the s-RCR strategy is worth developing to full clinical trials.
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PMID:VSV-G pseudotyped, MuLV-based, semi-replication-competent retrovirus for cancer treatment. 1672 95

Mitochondrial toxicity limits nucleoside reverse transcriptase inhibitors (NRTIs) for acquired immune deficiency syndrome. NRTI triphosphates, the active moieties, inhibit human immunodeficiency virus reverse transcriptase and eukaryotic mitochondrial DNA polymerase pol-gamma. NRTI phosphorylation seems to correlate with mitochondrial toxicity, but experimental evidence is lacking. Transgenic mice (TGs) with cardiac overexpression of thymidine kinase isoforms (mitochondrial TK2 and cytoplasmic TK1) were used to study NRTI mitochondrial toxicity. Echocardiography and nuclear magnetic resonance imaging defined cardiac performance and structure. TK gene copy and enzyme activity, mitochondrial (mt) DNA and polypeptide abundance, succinate dehydrogenase and cytochrome oxidase histochemistry, and electron microscopy correlated with transgenesis, mitochondrial structure, and biogenesis. Antiretroviral combinations simulated therapy. Untreated hTK1 or TK2 TGs exhibited normal left ventricle mass. In TK2 TGs, cardiac TK2 gene copy doubled, activity increased 300-fold, and mtDNA abundance doubled. Abundance of the 17-kd subunit of complex I, succinate dehydrogenase histochemical activity, and cristae density increased. NRTIs increased left ventricle mass 20% in TK2 TGs. TK activity increased 3 logs in hTK1 TGs, but no cardiac phenotype resulted. NRTIs abrogated functional effects of transgenically increased TK2 activity but had no effect on TK2 mtDNA abundance. Thus, NRTI mitochondrial phosphorylation by TK2 is integral to clinical NRTI mitochondrial toxicity.
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PMID:Targeted transgenic overexpression of mitochondrial thymidine kinase (TK2) alters mitochondrial DNA (mtDNA) and mitochondrial polypeptide abundance: transgenic TK2, mtDNA, and antiretrovirals. 1732 72

Growth inhibition of the DNA virus vaccinia (VACV) by NO is known to occur at the level of DNA synthesis. This inhibition is partially reversed by addition of deoxyribonucleosides, suggesting that NO or NO-related species inhibit viral ribonucleotide reductase (RR). However, the effect of NO on VACV-encoded RR or other DNA-synthesizing enzymes has not been demonstrated. In order to study the effects of NO on VACV-encoded RR, DNA polymerase (DNA pol) and thymidine kinase (TK), we generated a VACV recombinant expressing murine macrophage iNOS under control of a VACV early/late promoter p7.5. Using this recombinant, we demonstrate that expression of iNOS and the resulting production of NO inhibit activity of the viral RR, but not of viral DNA pol and TK. This NO-mediated inhibition of viral RR occurred around the same time as the increase of ADP levels, while it preceded the block in VACV DNA synthesis and the decrease of ATP levels. In addition, we tested the effects of DPTA/NONOate on the growth of different VACV mutants. Fold-inhibition of the growth of VACV deletion mutant for TK was comparable to that of wild-type VACV. VACV containing amplification of the gene for the small subunit of RR appeared to be least sensitive to DPTA/NONOate, while VACV deletion mutant for the large subunit of RR was most sensitive. The results provide a direct evidence for NO-mediated inhibition of VACV-encoded RR.
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PMID:The effect of nitric oxide on vaccinia virus-encoded ribonucleotide reductase. 1895 91

Herpes simplex viruses (HSV) type 1 and type 2 are responsible for recurrent orolabial and genital infections. The standard therapy for the management of HSV infections includes acyclovir (ACV) and penciclovir (PCV) with their respective prodrugs valacyclovir and famciclovir. These compounds are phosphorylated by the viral thymidine kinase (TK) and then by cellular kinases. The triphosphate forms selectively inhibit the viral DNA polymerase (DNA pol) activity. Drug-resistant HSV isolates are frequently recovered from immunocompromised patients but rarely found in immunocompetent subjects. The gold standard phenotypic method for evaluating the susceptibility of HSV isolates to antiviral drugs is the plaque reduction assay. Plaque autoradiography allows the associated phenotype to be distinguished (TK-wild-type, TK-negative, TK-low-producer, or TK-altered viruses or mixtures of wild-type and mutant viruses). Genotypic characterization of drug-resistant isolates can reveal mutations located in the viral TK and/or in the DNA pol genes. Recombinant HSV mutants can be generated to analyze the contribution of each specific mutation with regard to the drug resistance phenotype. Most ACV-resistant mutants exhibit some reduction in their capacity to establish latency and to reactivate, as well as in their degree of neurovirulence in animal models of HSV infection. For instance, TK-negative HSV mutants establish latency with a lower efficiency than wild-type strains and reactivate poorly. DNA pol HSV mutants exhibit different degrees of attenuation of neurovirulence. The management of ACV- or PCV-resistant HSV infections includes the use of the pyrophosphate analogue foscarnet and the nucleotide analogue cidofovir. There is a need to develop new antiherpetic compounds with different mechanisms of action.
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PMID:Resistance of herpes simplex viruses to nucleoside analogues: mechanisms, prevalence, and management. 2107 29


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