Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019693 (HIV)
 170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Based on amino acid sequence information from purified mitochondrial thymidine kinase (TK2), a cDNA of 1930 bp was cloned, containing an open reading frame encoding 232 amino acid residues starting with the N-terminal sequence determined from the native human protein preparation. Northern blot analysis with the cDNA coding region demonstrated several TK2 mRNAs, with 2 and 4 kb forms present in many tissues. We also characterised N-terminally truncated (starting at position 18) human TK2 with pharmacologically important antiviral and cytostatic nucleoside analogues. Results were highly similar to those with the native TK2 preparation. The anti-leukaemic drug arabinosyl cytosine is phosphorylated. The antitumour drug difluorodeoxycytidine and its metabolite difluorodeoxyuridine are good substrates, with K(m) values of 66 and 29 microM, respectively, and a relative Vmax of 0.6 compared to that of thymidine. Negative cooperativity was found with thymidine and the anti-HIV drug 3'-azidothymidine, but the reaction followed Michaelis-Menten kinetics with deoxycytidine, arabinosyl cytosine, and arabinosyl thymine. The results demonstrate a broad substrate specificity and complex kinetics, and suggest a role for TK2 in the activation of chemotherapeutic nucleoside analogues.
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PMID:Human thymidine kinase 2: molecular cloning and characterisation of the enzyme activity with antiviral and cytostatic nucleoside substrates. 998 99

We have reported recently that a small element within the mouse histone H2a-coding region permits efficient cytoplasmic accumulation of intronless beta-globin cDNA transcripts. This sequence lowers the levels of spliced products from intron-containing constructs and can functionally replace Rev and the Rev-responsive element (RRE) in the nuclear export of unspliced HIV-1-related mRNAs. In work reported here, we further investigate the molecular mechanisms by which this element might work. We demonstrate here through both in vivo and in vitro assays that, in addition to promoting mRNA nuclear export, this element acts as a polyadenylation enhancer and as a potent inhibitor of splicing. Surprisingly, two other described intronless mRNA transport elements (from the herpes simplex virus thymidine kinase gene and hepatitis B virus) appear to function in a similar manner. These findings prompt us to suggest that a general feature of intronless mRNA transport elements might be a collection of phenotypes, including the inhibition of splicing and the enhancement of both polyadenylation and mRNA export.
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PMID:Intronless mRNA transport elements may affect multiple steps of pre-mRNA processing. 1007 34

A recombinant vaccinia virus (rvv) expressing, human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein, gp120, fused to a non-cleavable transmembrane protein, vvE13, elicited protection against a tumor cell line expressing HIV-1 full length envelope glycoprotein, gp160, in mice. Mice vaccinated with vvE13 exhibited a decreased incidence of tumor development and significantly smaller tumors in comparison to mice vaccinated with rvv gp160, vvE1, or a thymidine kinase minus (TK-) rvv, vSC11, or phosphate-buffered saline (PBS) injected controls. vvE13 and vvE1 also delayed tumor development, compared to vSC11 and PBS-injected controls; however, a statistical correlation could not be demonstrated due to the development of tumors in so few animals. Specificity toward HIV-1 envelope glycoprotein, was shown, since HIV-1 envelope-tumor prevention (incidence for vvE13 and size for vvE1 and vvE13 and delay for vvE1 and vvE13) was statistically superior with HIV-1 envelope expressing tumors compared to parenteral tumors. The vvE13 recombinant vaccinia virus expressing the HIV-1 envelope glycoprotein gp120 fused to a non-cleavable transmembrane protein elicits superior protection against tumors expressing the gp160 envelope glycoprotein, as compared to vvE1 expressing gp160.
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PMID:Enhanced HIV-1 envelope-tumor protection by a recombinant vaccinia virus expressing anchored HIV-1 gp120 lacking gp41. 1008 12

The thymidine kinase (TK) genes from herpes simplex virus (HSV) types 1 and 2 were recombined in vitro with a technique called DNA family shuffling. A high-throughput robotic screen identified chimeras with an enhanced ability to phosphorylate zidovudine (AZT). Improved clones were combined, reshuffled, and screened on increasingly lower concentrations of AZT. After four rounds of shuffling and screening, two clones were isolated that sensitize Escherichia coli to 32-fold less AZT compared with HSV-1 TK and 16,000-fold less than HSV-2 TK. Both clones are hybrids derived from several crossover events between the two parental genes and carry several additional amino acid substitutions not found in either parent, including active site mutations. Kinetic measurements show that the chimeric enzymes had acquired reduced K(M) for AZT as well as decreased specificity for thymidine. In agreement with the kinetic data, molecular modeling suggests that the active sites of both evolved enzymes better accommodate the azido group of AZT at the expense of thymidine. Despite the overall similarity of the two chimeric enzymes, each contains key contributions from different parents in positions influencing substrate affinity. Such mutants could be useful for anti-HIV gene therapy, and similar directed-evolution approaches could improve other enzyme-prodrug combinations.
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PMID:Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling. 1009 93

Cytotoxic and antiretroviral activity of cycloSal-d4TMP derivatives were tested in a new AZT-resistant H9 cell subline (H9rAZT250). The results showed, that cycloSal-d4TMP derivatives overcame resistance of HIV-1 to d4T in H9rAZT250 cells, which exert decreased thymidine kinase (TK) gene expression.
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PMID:Effects of cycloSal-d4TMP derivatives in H9 cells with induced AZT resistance phenotype. 1043 12

The phosphoramidate triester prodrugs of anti-human HIV 2', 3'-dideoxynucleoside analogs (ddN) represent a convenient approach to bypass the first phosphorylation to ddN 5'-monophosphate (ddNMP), resulting in an improved formation of ddN 5'-triphosphate and, hence, higher antiviral efficacy. Although phosphoramidate derivatization markedly increases the anti-HIV activity of 2',3'-didehydro-2', 3'-dideoxythymidine (d4T) in both wild-type and thymidine kinase-deficient CEM cells, the concept is far less successful for the 3'-azido-2',3'-dideoxythymidine (AZT) triesters. We now investigated the metabolism of triester prodrugs of d4T and AZT using pure enzymes or different biological media. The efficiency of the first activation step, mediated by carboxylesterases, consists of the formation of the amino acyl ddNMP metabolite. The efficiency of this step was shown to be dependent on the amino acid, alkyl ester, and ddN moiety. Triesters that showed no conversion to the amino acyl ddNMP accumulated as the phenyl-containing intermediate and had poor, if any, anti-HIV activity. In contrast to the relative stability of the triesters in human serum, carboxylesterase-mediated cleavage of the prodrugs was found to be remarkably high in mouse serum. The subsequent conversion of the amino acyl ddNMP metabolite to ddNMP or ddN was highest in rat liver cytosolic enzyme preparations. Although L-alaninyl-d4TMP was efficiently converted to d4TMP, the main metabolite formed from L-alaninyl-AZTMP was the free nucleoside (AZT), thus explaining why d4T prodrugs, but not AZT prodrugs, retain anti-HIV activity in HIV-infected thymidine kinase-deficient cell cultures. The rat liver phosphoramidase responsible for the formation of ddNMP was shown to be distinct from creatine kinase, alkaline phosphatase, and phosphodiesterase.
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PMID:Characterization of the activation pathway of phosphoramidate triester prodrugs of stavudine and zidovudine. 1049 51

2',3'-Dideoxycytidine (ddC) and azidothymidine (AZT) inhibit HIV-1 replication and currently are used in AIDS therapy. Long-term use of the drugs is associated with the selection of drug-resistant HIV strains, thus limiting their effectiveness. Another mechanism, associated with their altered metabolism in host cells, also can cause "cellular" drug resistance. Human lymphocytic H9 cell lines (H9-ddC0.5w and H9-ddC5.0w) selected for ddC resistance by exposure to 0.5 and 5.0 microM ddC were found to be cross-resistant to AZT. Compared with controls, the thymidine kinase (TK) activities in H9-ddC0.5w and H9-ddC5.0w cells were 56.7 and 51.4% (with thymidine as a substrate) and 50.3 and 42% (with AZT as a substrate). Consequently the cellular incorporation of AZT and thymidine (24-hr incubation) also was reduced to 51.3 and 70.0% in H9-ddC0.5w cells and to 12.1 and 17.3% in H9-ddC5.0w cells. A 3-hr incubation with 25 microM AZT and ddC decreased their cellular incorporation to 50.5 and 76.15% in H9-ddC0.5w cells and to 12.95 and 47.8% in H9-ddC5.0w cells compared with H9 cells. Thus, the change in AZT accumulation did not correlate exactly with the decrease in TK activity and far exceeded the effect on ddC accumulation. Evidence is presented that ddC, in addition to deoxycytidine kinase, affected TK1 activity. The involvement of multidrug resistance proteins in the mechanism of the resistance was ruled out by the failure of trifluoperazine and verapamil to alter cellular accumulations of AZT, ddC, daunorubicin, and rhodamine-123. Development of cellular ddC and AZT cross-resistance may affect the therapeutic efficacy of these antiviral agents.
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PMID:Cross-resistance of dideoxycytidine-resistant cell lines to azidothymidine. 1053 51

The outbreak of HIV infection introduced a new phenomenon in varicella zoster virus (VZV) pathology, namely the long-standing wart-like skin lesions that are frequently associated with resistance to thymidine kinase (TK)-dependent antiviral agents. This paper reviews the clinical, histological, and molecular aspects and the therapeutic management of these verrucous lesions. The majority of lesions are characterized by chronically evolving, unique or multiple wart-like cutaneous lesions. The main histopathological features include hyperkeratosis, verruciform acanthosis and VZV-induced cytopathic changes with scant or absent cytolysis of infected keratinocytes. The mechanism that establishes the chronic nature of the lesions appears to be associated with a particular pattern of VZV gene expression exhibiting reduced or nondetectable gE and gB synthesis. Drug resistance to TK-dependent antiviral agents is a result of nonfunctional or deficient viral TK. This necessitates alternative therapeutic management using antiviral agents that target the viral DNA polymerase.
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PMID:Chronic verrucous varicella zoster virus skin lesions: clinical, histological, molecular and therapeutic aspects. 1056 18

3'-Azido-3'-deoxythymidine (AZT), a thymidine analogue widely used in the treatment of AIDS patients and for prevention of the onset of AIDS in HIV-seropositive individuals, causes tumors in mice exposed as adults or in utero. The purpose of this study was to investigate the potential mechanisms of AZT mutagenicity and carcinogenicity by quantifying the incorporation of AZT into cellular DNA, measuring AZT-induced thymidine kinase (TK) mutant frequencies (Mfs), and determining the percentage of loss of heterozygosity (LOH) in spontaneous or AZT-induced TK mutants in the human lymphoblastoid cell line, TK6. Cells were exposed to 300 microM AZT for 0, 1, 3, or 6 days, or to 0, 33, 100, 300, or 900 microM AZT for 3 days (n = 5 flasks/group). The effects of exposure concentration on incorporation of AZT into cellular DNA were evaluated by an AZT radioimmunoassay, and the effects of duration and concentration of AZT exposure on the TK Mfs were assessed by a cell-cloning assay. AZT was incorporated into DNA in a dose-related manner at concentrations up to 300 microM, above which no further increase was observed. TK Mf increased with the extended duration and with incremental concentrations of AZT exposure. There was a positive correlation (P = 0.036, coefficient = 0.903) between AZT-DNA incorporation and AZT-induced TK Mfs, suggesting that AZT incorporation into cellular DNA has a direct role in the genotoxicity of AZT. Southern blot analyses indicated that 84% (6.2 x 10(-6)/7.4 x 10(-6)) of AZT-induced mutants were attributable to LOH, consistent with the known mechanism of AZT as a DNA chain terminator. Considering the importance of LOH in human carcinogenesis, AZT-induced LOH warrants further study.
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PMID:Relationships between DNA incorporation, mutant frequency, and loss of heterozygosity at the TK locus in human lymphoblastoid cells exposed to 3'-azido-3'-deoxythymidine. 1077 14

Didanosine (ddI) is used in the treatment of HIV-1 infection alone and in combination with azidothymidine (AZT). When combined with AZT, patients exhibit improved patterns of surrogate markers after sequential combination regimens of ddI and AZT compared to either drug monotherapy. We have investigated the biochemical mechanism(s) of this synergistic drug combination in human PBMC cells and in human T-cell lines sensitive and resistant to AZT due to lack of thymidine kinase (TK). DdI is preferentially activated to its triphosphate anabolite, ddATP, at 3:1 ratio in human T-lymphocytes compared to monocytes from the same individual. There are no apparent differences in the intracellular concentrations of ddATP in Jurkat/0 and Jurkat/AZT-10, an AZT resistant human T-cell line, when ddI is administered alone or in combination with AZT, hence there appears to be a case of collateral sensitivity. Intracellular increases of AZTTP concentrations in patient's PBMC cells have been determined clinically after AZT alone and in a combination regimen with ddI. A stochastic biochemical model has been developed that estimates the velocity of HIV-RT under uninhibited and inhibited conditions by the active anabolites, AZTTP and ddATP. This model provides a rational explanation for the greater inhibition of HIV-RT in the presence of both inhibitors, AZTTP and ddATP, as compared to the presence of either anabolite triphosphate alone. Expanding this model to describe the inhibition of HIV-RT in the presence of three competitive inhibitors, AZTTP, ddATP and 3TCTP demonstrated that the presence of these HIV-RT inhibitors resulted in an even greater inhibition of this viral enzyme necessary for HIV integration and replication. Hence, a more effective inhibition of HIV-RT enzyme is achieved by the combination of the three drugs, AZT, ddl and 3TC. In an effort to verify this model with experimental data the kinetics of HIV-RT were studied in the absence and after inhibition by AZTTP or ddATP alone, both AZTTP + ddATP or AZTTP + ddATP + 3TCTP. Treatment of HIV-RT with high concentrations of these triphosphate inhibitors, as high as 3Kis, inhibited this enzyme to greater than 90% of untreated control. However, a small percentage of residual HIV-RT, 6%, was uninhibited even after exposure to 3Ki concentrations of each inhibitor. These studies strongly suggested that: 1) AZT plus ddI or AZT plus ddI plus 3TC are synergistic at the active anabolite level against HIV-RT; 2) the combination of the three nucleoside analog drugs (AZT, ddI 3TC) is needed for more effective inhibition of HIV-RT; 3) that the combination of the triphosphates at concentrations much greater than those pharnacologically achieved in T-Cells or PBMC under treatment conditions did not inhibit completely HIV-RT. Hence, the three nucleoside HIV-RT inhibitors must be combined with other classes of antiviral drugs or T-cell specific inhibitor drugs.
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PMID:Pharmacodynamic studies (PD) of didanosine (ddI) alone and in combination with azidothymidine (AZT) in human T-cells; a stochastic biochemical approach to antiretroviral nucleoside drug combination in inhibiting HIV-reverse transcriptase (RT). 1090 70


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