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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

AZT is an inhibitor of HIV reverse transcriptase which active form is AZT-triphosphate. Its clinical efficacy is limited by its toxicity and the emergence of mutant resistant viruses. This might be due to an unfficient cellular metabolism of AZT which leads to the intracellular accumulation of AZT monophosphate. We tested a possible enhancement of this metabolism with the HSV1 thymidine kinase, an enzyme that also possesses a good thymidilate kinase activity. We show that, compared to parental cells, the proportion of AZT triphosphate is increased 3 fold in HSV1-TK expressing cells, and that inhibition of HIV replication in these cells requires 3 to 10 fold less AZT. This observation forms the basis for a new gene therapy strategy named genetically controlled pharmacomodulation.
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PMID:[Genetically controlled pharmacomodulation: application to the treatment of HIV infection]. 788 39

The target protein (enzyme) with which antiviral agents interact determines their antiviral activity spectrum. Based on their activity spectrum, antiviral compounds could be divided into the following classes: (1) sulfated polysaccharides (i.e., dextran sulfate), which interact with the viral envelope glycoproteins and are inhibitory to a broad variety of enveloped viruses (i.e., retro-, herpes-, rhabdo-, and arenaviruses): (2) SAH hydrolase inhibitors (i.e., neplanocin A derivatives), which are particularly effective against poxvirus, (-)RNA viruses (paramyxovirus, rhabdovirus), and (+/-)RNA virus (reovirus); (3) OMP decarboxylase inhibitors (i.e., pyrazofurin) and CTP synthetase inhibitors (i.e., cyclopentenylcytosine), which are active against a broad range of DNA, (+)RNA, (-)RNA, and (+/-)RNA viruses; (4) IMP dehydrogenase inhibitors (i.e., ribavirin), which are also active against various (+)RNA and (-)RNA viruses and, in particular, ortho- and paramyxoviruses; (5) acyclic guanosine analogs (i.e., ganciclovir) and carbocyclic guanosine analogs (i.e., cyclobut-G), which are particularly active against herpesviruses (i.e., HSV-1, HSV-2, VZV, CMV); (6) thymidine analogs (i.e., BVDU, BVaraU), which are specifically active against HSV-1 and VZV because of their preferential phosphorylation by the virus-encoded thymidine kinase; (7) acyclic nucleoside phosphonates (i.e., HPMPA, HPMPC, PMEA, FPMPA), which, depending on the structure of the acyclic side chain, span an activity spectrum from DNA viruses (papova-, adeno-, herpes-, hepadna-, and poxvirus) to retroviruses (HIV); (8) dideoxynucleoside analogs (i.e., AZT, DDC), which act as chain terminators in the reverse transcriptase reaction and thus block the replication of retroviruses as well as hepadnaviruses; and (9) the TIBO, HEPT, and other TIBO-like compounds, which interact specifically with the reverse transcriptase of HIV-1 and thus block the replication of HIV-1, but not of HIV-2 or any other retrovirus.
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PMID:Antiviral agents: characteristic activity spectrum depending on the molecular target with which they interact. 843 May 18

The T-cell line Jurkat E6-1 was rendered resistant to zidovudine (AZT) in vitro by exposure to low but gradually increased concentrations of the drug. Biochemical pharmacology studies of [3H]AZT in the AZT-resistant T-cell lines showed a significant reduction of AZT phosphorylation to the mono-, di-, and triphosphate anabolites. Peripheral blood mononuclear cells (PBMCs) from pediatric patients with human immunodeficiency virus type 1 (HIV-1) infection showed a similar pattern of decreased AZT anabolism. Enzymatic studies with purified thymidine kinase (TK) preparations from these cell lines showed a gradual decline in Vmax related to their level of resistance to AZT. The Jurkat/AZT-20 and Jurkat/AZT-100 cells were studied in greater detail with reverse transcriptase/polymerase chain reaction (RT/PCR) cloned probes to determine possible molecular mechanisms of resistance to AZT. TK mRNA was significantly decreased (approximately 5- to 10-fold) in the AZT-resistant T-cell lines. Southern blot analyses indicated that there were no major rearrangements or deletions of the TK gene, but the 5' end of the gene in the AZT-resistant cells is highly methylated when compared to wild-type cells. No apparent differences were seen in thymidylate kinase (dTMPk) mRNA levels in the same T-cell lines. Thus the decreased expression of TK mRNA and resultant TK enzymatic activity is responsible for the observed reduction in the AZT anabolism in the resistant T-cell lines. Decreased T-cell TK activity could allow wild-type, AZT-sensitive HIV-1 to replicate in the presence of subinhibitory AZT triphosphate (AZT-TP) cellular concentrations enabling a genetic variant with drug resistance to emerge and outgrow the AZT-sensitive, wild-type virus.
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PMID:Development of zidovudine (AZT) resistance in Jurkat T cells is associated with decreased expression of the thymidine kinase (TK) gene and hypermethylation of the 5' end of human TK gene. 854 39

The "bystander effect" refers to the death of unmodified tumor cells when in contact with ganciclovir (GCV)-exposed, herpes simplex virus-thymidine kinase (HSV-TK)-modified tumor cells. Although the exact mechanism or mechanisms involved in mediating the bystander effect in vivo are unknown, our findings suggest that an intact host immune system is required for the phenomenon to occur. The present study was designed to establish the effect of HSV-TK-modified tumor cells and GCV on the tumor and its microenvironment in vivo. In sublethally irradiated and immunodeficient Balb/c mice, the bystander effect was observed to be diminished or abrogated. Histopathologic examination of the tumor mass from immunocompetent mice demonstrated centralized hemorrhagic tumor necrosis (38%) after inoculation of the HSV-TK-modified tumor cells and GCV in tumor-bearing mice compared with the control mice (5%), indicating that cytokines such as tumor necrosis factor-alpha (TNF-alpha) were being released locally. This hypothesis was underscored using reverse transcriptase polymerase chain reaction (RT-PCR), by the demonstration of cytokine mRNA expression in mice treated with HSV-TK-expressing tumors and GCV. Semiquantitative PCR analysis for TNF-alpha using PCR-MIMIC on tumor samples from mice treated on days 1 and 4 showed a two-fold increase in the level on mRNA expression. Also, immunohistochemical staining for TNF-alpha showed that mononuclear inflammatory cells infiltrating the tumor were its source. Finally, characterization of tumor-infiltrating lymphocytes (TIL) in experimental animals demonstrated a two- to three-fold increase in the number of macrophages and T cells compared with control animals. These results demonstrate that, in vivo, the bystander effect is mediated in part by an antitumor response through the release of cytokines. Further, the cytokine milieu and tumor microenvironment can be modulated following injection of HSV-TK cells and GCV to enhance the host immune response, which is of potential use in clinical trials.
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PMID:In vivo analysis of the 'bystander effect': a cytokine cascade. 864 34

The anti-human immunodeficiency virus (anti-HIV) agent 2',3'-didehydro-3'-deoxythymidine (D4T), like other 2',3'-dideoxynucleosides, requires conversion to its 5'-triphosphate to exert its pharmacological effect. Although D4T-triphosphate is unusually potent as an inhibitor of HIV-1 reverse transcriptase, the phosphorylation of the drug at low dose levels is inefficient because of its low affinity as an alternate substrate for the initial phosphorylation enzyme thymidine kinase. Because thymidine kinase is under feedback regulatory control by the physiological deoxynucleoside-5'-triphosphate dTTP, we examined the effect on D4T phosphorylation and thus, potentially, on its antiviral activity, of a variety of agents that lower intracellular dTTP pools. We found that agents that inhibit the de novo pyrimidine biosynthetic pathway have the ability to increase D4T phosphorylation, the most effective being two inhibitors of thymidylate formation, methotrexate and 5-fluoro-2'-deoxyuridine, compounds that block the enzymes dihydrofolate reductase and thymidylate synthetase, respectively. Because HIV itself lacks the capacity to synthesize dTTP and the other deoxynucleoside triphosphates essential for viral replication, combinations of D4T with modulatory agents that deplete host-cell dTTP, unlike conventional anti-HIV drug monotherapy directed solely at viral enzymes, have the ability to inhibit replication of mutant HIV strains as well as of wild-type virus.
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PMID:2',3'-Didehydro-3'-deoxythymidine: regulation of its metabolic activation by modulators of thymidine-5'-triphosphate biosynthesis. 870 Jan 8

Acyclovir is an effective drug for the treatment of HSV and VZV infections, which after phosphorylation to the triphosphate, inhibits viral DNA polymerase. Acyclovir has low oral bioavailability, therefore prodrugs have been developed, and the L-valyl ester, valaciclovir, recently has been licensed for the treatment of shingles. Ganciclovir is used against CMV, and famciclovir, a lipophilic prodrug of penciclovir, is marketed for shingles. The acyclic nucleoside phosphonates are active against thymidine kinase-resistant viral strains. Promising analogs are PMEA (in clinical trial for the treatment of AIDS) and (S)-HPMPC (good in vivo activity against HSV, VZV, CMV, and EBV). Oligonucleotides incorporating acyclic nucleosides at the 3'-and 5'-ends, or constituted of amino acyclic nucleosides, are resistant to cleavage by nucleases and may be useful in antisense and/or antigene therapy. HEPT is active against HIV-1: It binds in a hydrophic pocket on reverse transcriptase, rather than in the polymerase active site. Some acyclic nucleosides are potent inhibitors of purine and pyrimidine nucleoside phosphorylase. These compounds may have a therapeutic niche in combination therapy with antiviral and anticancer nucleosides, and in the treatment of diseases involving the T-cell.
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PMID:Acyclic nucleosides as antiviral compounds. 873 25

Direct delivery of the herpes simplex virus thymidine kinase (HSVtk) gene, in combination with the prodrug ganciclovir (GC), has been used for the treatment of localised, inoperable tumours. Several groups have shown that when rodent tumours are ablated in vivo with suicide genes, anti-tumour immunity can also be generated. Hence, this approach may also be useful in treating disseminated disease. Here we have studied the mechanisms associated with this anti-tumour immunity. In B16 HSVtk+ tumours being killed in vivo with GC treatment, we observed the induction of a pronounced intratumoural infiltrate of macrophages, CD4+ and CD8+ T cells. In addition, using reverse transcriptase polymerase chain reaction, expression of interleukin (IL)-2, IL-12, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and granulocyte/macrophage colony-stimulating factor (GM-CSF) but not IL-4, IL-6 or IL-10, was observed, a profile of cytokine expression which resembles that of a Th1 immune response. To complement these findings, we also investigated the mechanisms by which expression of HSVtk leads to cell death. Our data show that B16/HSVtk+ cells die predominantly by necrosis, rather than apoptosis, on exposure to GC, a process which may be associated with the generation of anti-tumour inflammatory responses. From these data we propose a model for the induction of anti-tumour immunity using suicide genes and discuss the development of improved vectors for gene therapy to augment these effects in vivo.
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PMID:Generation of an anti-tumour immune response in a non-immunogenic tumour: HSVtk killing in vivo stimulates a mononuclear cell infiltrate and a Th1-like profile of intratumoural cytokine expression. 913 53

ZF5, which we have cloned as a repressor on the mouse c-myc promoter, is a zinc finger protein containing Kruppel-type zinc finger and ZiN/POZ domains. In a reverse transcriptase PCR assay using mouse skeletal muscle RNA, we identified a 827 bp PCR product including the zinc finger domain of ZF5 and the acidic domain of VP16. The presence of the VP16 acidic domain induced the reduction of DNA-binding activity of the zinc finger domain. In addition, the inhibitory effect of the VP16 acidic domain was demonstrated on the human immunodeficiency virus (HIV) promoter, but there was no effect on the thymidine kinase (TK) promoter.
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PMID:Detection of mouse skeletal muscle-specific product, which includes ZF5 zinc fingers and a VP16 acidic domain, by reverse transcriptase PCR. 922 18

Latent infections of neurons by herpes simplex virus form reservoirs of recurrent viral infections that resist cure. In latently infected neurons, viral gene expression is severely repressed; only the latency-associated transcripts (LATs) are expressed abundantly. Using sensitive reverse transcriptase PCR assays, we analyzed the effects of a deletion mutation in the LAT locus on viral gene expression in latently infected mouse trigeminal ganglia. The deletion mutation, which reduced expression of the major LATs 10(5)-fold, resulted in a approximately 5-fold increase in accumulation of transcripts from the immediate-early gene encoding ICP4, an essential transactivator of viral gene expression. The LAT deletion also resulted in a >10-fold increase in the accumulation of transcripts from the early gene encoding thymidine kinase, whose expression during productive infection stringently depends on ICP4, and positively affected the correlation of the levels of these transcripts with the levels of ICP4 transcripts. We also detected transcripts antisense to ICP4 RNA, which were in substantial excess to ICP4 transcripts in ganglia latently infected with wild-type virus. In contrast to its effects on productive-cycle transcripts, the LAT deletion reduced the accumulation of these antisense transcripts approximately 15-fold. Thus, a viral function associated with the LAT locus represses the accumulation of transcripts from at least two productive-cycle genes in latently infected mouse ganglia. We discuss possible mechanisms and consequences of this repression.
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PMID:A viral function represses accumulation of transcripts from productive-cycle genes in mouse ganglia latently infected with herpes simplex virus. 922 77

We have developed murine leukemia virus (MLV)-based self-inactivating and self-activating vectors to show that the previously demonstrated high-frequency direct repeat deletions are not unique to spleen necrosis virus (SNV) or the neomycin drug resistance gene. Retroviral vectors pKD-HTTK and pKD-HTpTK containing direct repeats composed of segments of the herpes simplex virus type 1 thymidine kinase (HTK) gene were constructed; in pKD-HTpTK, the direct repeat flanked the MLV packaging signal. The generation of hypoxanthine-aminopterin-thymidine-resistant colonies after one cycle of retroviral replication demonstrated functional reconstitution of the HTK gene. Quantitative Southern analysis indicated that direct repeat deletions occurred in 57 and 91% of the KD-HTTK and KD-HTpTK proviruses, respectively. These results demonstrate that (i) deletion of direct repeats occurs at similar high frequencies in SNV and MLV vectors, (ii) MLV psi can be efficiently deleted by using direct repeats, (iii) suicide genes can be functionally reconstituted during reverse transcription, and (iv) the psi region may be a hot spot for reverse transcriptase template switching events.
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PMID:Psi- vectors: murine leukemia virus-based self-inactivating and self-activating retroviral vectors. 922 21


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