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)

The rational design of antitumor and antiviral agents must ultimately take advantage of biochemical differences between normal host cells and transformed cells. The initial experiments must be performed with subcellular or cellular model systems. For the studies with arabinosyl nucleosides we have chosen those enzyme systems, synthesizing DNA and RNA; being precursor analogues, the different arabinosyl nucleosides have been added in the triphosphate state to the different DNA- and RNA polymerase assays. 1-beta-D-Arabinofuranosylcytosine-5'-triphosphate has been found to inhibit the RNA-dependent DNA polymerases (isolated from oncogenic RNA viruses) 200-fold more sensitively than viral and cellular DNA-dependent DNA polymerases. Recent results, showing that RNA-leukemia-virus-related sequences are present in DNA of some human leukemia patients might support the assumption that the efficacy of this antimetabolite in the treatment of acute leukemia is due to its, at least relative selective inhibitory activity on reverse transcriptase. 9-beta-D-Arabinofuranosyladenine-5'-triphosphate is a strong inhibitor of cellular DNA polymerases with the cytological consequence of an inhibition of cell proliferation. The clinical benefit of the compound in treatment of tumors is dependent on their levels of adenosine deaminase. The triphosphate of this compound is a 100-fold more sensitive inhibitor of the herpesvirus DNA polymerase compared to the cellular replicative DNA polymerase. In addition the analogue, incorporated into herpesvirus DNA, acts as chain terminator. These effects are the biochemical basis for the highly selective antiherpesvirus activity of this antimetabolite. The anomer 9-alpha-D-arabinofuranosyladenine-5'-triphosphate only inhibits cellular replicative DNA polymerase and has no effect on herpesvirus DNA polymerase. Consequently this agent acts only cytostatically and not antivirally. Concerning 1-beta-D-arabinofuranosyluracil and 1-beta-D-arabinofuranosylthymine no pronounced antitumor or antiviral effect is known.
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PMID:Rational design of arabinosyl nucleosides as antitumor and antiviral agents. 61 2

Of the dideoxynucleosides described to date, the purine analogues ddA and ddI have exhibited very favorable therapeutic ratios in vitro. ddI is presently undergoing extensive phase I-II clinical trials. Whereas the action of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) is usually to convert a given analogue of Ado to an inactive or less active form, ddI appears to retain the same biological activity as that of the parent ddA. An explanation for these observations was possible when we found that ddI (1) underwent only a slow cleavage to hypoxanthine through the action of PNP and (2) accumulated the same active antiviral metabolite (i.e., ddATP) as ddA in human lymphoid cells. The use of human lymphoid cells with deficiencies in cellular nucleoside kinases and of inhibitors of pathways of nucleotide metabolism have also revealed new aspects of dideoxypurine metabolism in human lymphoid cells, including the identification of a salvage pathway (phosphotransferase/5'-nucleotide pathway) by which ddA/ddI may be metabolized preferentially to the active nucleotide. The effectiveness of ddA and ddI as orally administered antiviral agents may be limited by their susceptibility to acid hydrolysis and the low efficiency for nucleotide conversion in human lymphoid cells. The presence of a fluorine atom in the arabinose configuration on C-2 confers resistance to solvolysis and renders the analogue less susceptible to enzymatic deamination and resistant to phosphorylytic cleavage by PNP. In addition, human lymphoid cells accumulated several fold higher levels of the putative active triphosphate, 2'-F-dd-ara-ATP, than those of ddA or ddI. This increased accumulation of the analogue triphosphate could be accounted for by a more direct conversion of 2'-F-dd-ara-A by a direct phosphorylation through dCyd kinase than ddA. Thus, a single substitution with fluorine at the 2' "up" position of the sugar moiety of ddA markedly improves several biochemical properties relating to dideoxynucleotide accumulation in human lymphoid cells. Whether there are significant alterations of other biochemical properties, such as the ability of the analogue triphosphate to interact with the target enzyme reverse transcriptase, has not yet been determined. Thus, a definitive resolution of the relative merit of ddA/ddI and its 2'-fluoro-arabinosyl analogue is not yet possible on the basis of the studies described here.
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PMID:Metabolism in human leukocytes of anti-HIV dideoxypurine nucleosides. 207 20

A number of 2',3'-dideoxynucleosides have been shown to inhibit the in vitro infectivity and cytopathic effect of the human immunodeficiency virus (HIV). These compounds, as their 5'-triphosphates, inhibit viral reverse transcriptase by competing with the natural substrate at the same binding site on the enzyme. Dideoxynucleoside triphosphates can also be incorporated into growing DNA chains which then blocks further DNA elongation because they lack the 3'-hydroxyl group required for further polymerization. Among these nucleosides, 2', 3'-dideoxyadenosine 2',3'-dideoxyadenosine (ddA) and 2',3'-dideoxyinosine (ddI) show promising in vitro activity. Because adenosine is rapidly converted to inosine by adenosine deaminase, the in vivo conversion of ddA to ddI was studied to determine suitability of measuring plasma levels of ddI and to assess the bioavailability and pharmacokinetics of ddA. This report describes and compares the pharmacokinetics of ddA and ddI in the mouse.
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PMID:Comparative pharmacokinetics of new anti-HIV agents: 2',3'-dideoxyadenosine and 2',3'-dideoxyinosine. 249 44

The 2',3'-dideoxynucleosides (ddNs) are currently undergoing clinical evaluation as antiretroviral agents in HIV-infected individuals. When phosphorylated, the ddNs (ddNTPs) function as chain-terminating substrate analogues with reverse transcriptase, thereby inhibiting HIV replication. These nucleoside analogues can also inhibit, by chain-terminating additions, the primitive lymphoid DNA polymerase, terminal deoxynucleotidyl transferase (TdT). To determine the effect of possible intracellular chain-terminating additions of ddNMPs by TdT, we exposed a series of TdT-positive and TdT-negative cell lines to 2',3'-dideoxyadenosine (ddA), a representative ddN. At ddA concentrations 25-fold higher than required for inhibition of HIV replication, progressive dose-related cytotoxicity was observed in the TdT-positive cell lines. This was accentuated by the adenosine deaminase inhibitor Coformycin (CF), presumably by enhancing the intracellular generation of ddATP from ddA. A central role of TdT in mediating the ddA/CF cytotoxicity was suggested by studies in a pre-B-cell line rendered TdT positive by infection with a TdT cDNA-containing retroviral vector. After a 48-hour continuous exposure period to 250 mumol/L ddA and 30 mumol/L CF, 30% cell death was observed in the TdT-negative parental line, whereas 90% cell death was observed in the TdT-positive daughter line. Exposure of fresh TdT-positive leukemic cells to ddA/CF for 72 hours ex vivo resulted in cytotoxicity (six cases of acute lymphocytic leukemia [ALL]) while not affecting TdT-negative acute leukemic cells (six cases). We conclude that ddA/CF selectively damages TdT-positive cells, presumably by chain-terminating additions of ddAMP, and that this may have therapeutic relevance in TdT-positive malignant disease.
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PMID:2',3'-Dideoxyadenosine is selectively toxic for TdT-positive cells. 283 1

Peripheral blood lymphocytes from a patient with adenosine deaminase (ADA) deficiency were transduced in vitro with a replication-defective retroviral vector containing a human ADA-cDNA. Eighteen months after the last of a series of infusions of autologous retroviral vector-treated cells, vector sequences were detectable in DNA isolated from peripheral blood mononuclear cells (PBMCs), with an average copy number approaching one per cell. Increased ADA enzyme activity reaching approximately one-quarter normal levels was found in this population of cells. Other evidence of long-term retroviral vector expression in vivo included neomycin phosphotransferase (NPT) activity and demonstration of persistent vector mRNA by reverse transcriptase polymerase chain reaction (RT-PCR). No evidence of spontaneous reversion of either mutant endogenous ADA allele was found.
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PMID:Molecular analysis of T lymphocyte-directed gene therapy for adenosine deaminase deficiency: long-term expression in vivo of genes introduced with a retroviral vector. 877 14

In 1991 we reported gene transduction into autologous long-term repopulating marrow cells in dogs using amphotropic helper-free retrovirus vectors containing the bacterial neomycin phosphotransferase gene (neo) and the human adenosine deaminase gene (ADA). Two of the dogs are still alive and healthy now more than 5 years after transplantation of transduced autologous marrow cells. In one of the surviving dogs, polymerase chain reaction (PCR) analysis showed the neo and ADA genes to be present in peripheral blood granulocytes and lymphocytes up to the present time. The estimated percentage of neo-positive cells ranged from < 0.001% to 0.1%. ADA mRNA expression was detected by reverse transcriptase PCR (RT-PCR) in granulocytes 63 months after transplantation. The other surviving dog failed to show either persistence or expression of the transduced genes after 50 months. Three additional dogs have been transplanted according to the same transduction protocols and with the same retrovirus vectors, and persistence of the transduced neo gene has been documented in peripheral blood myeloid and lymphoid cells along with G418-resistant colony-forming unit-granulocyte/macrophage (CFU-GM) for now more than 2 years. These findings represent the longest follow-up of retrovirus-mediated gene transduction in any animal species. Long-term transduction efficiency, though, has remained low and will need to be improved for therapeutic application to be possible.
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PMID:Long-term persistence of canine hematopoietic cells genetically marked by retrovirus vectors. 882 72

Enzymes of the purine salvage pathway play an important role in altering the in vivo pharmacokinetics of 2',3'-dideoxypurine nucleosides. This study examines the pharmacokinetics of enzyme-resistant 2'-beta-fluoro analogues of 2',3'-dideoxyinosine (ddI) and 2',3'-dideoxyadenosine (ddA). 2'-beta-Fluoro-2',3'-dideoxyinosine (F-ddI) is an acid-stable analogue of ddI that is highly resistant to purine nucleoside phosphorylase, the principal enzyme in ddI metabolism. 2'-beta-Fluoro-2',3'-dideoxyadenosine (F-ddA), an acid-stable and purine nucleoside phosphorylase-resistant analogue of ddA, is converted in vivo to F-ddI by adenosine deaminase (ADA) but is a much poorer substrate for this enzyme than is ddA. Both F-ddA and F-ddI have been shown to have activity against human immunodeficiency virus in vitro, and F-ddA has been selected by the National Cancer Institute for clinical trials as a new human immunodeficiency virus reverse transcriptase inhibitor. The pharmacokinetics of F-ddI and ddI were compared at equivalent doses in chronically catheterized rats. Because ddI and F-ddI are isosteres having nearly identical lipophilicity, this comparison is likely to reflect primarily metabolic differences. The clearance of F-ddI was substantially reduced, in comparison with that of ddI (27.3 ml/min/kg vs. 90.9 ml/min/kg), resulting in higher systemic concentrations at steady state and prolonged retention of F-ddI after termination of infusions, consistent with a significant metabolic component in the clearance of ddI. Concentrations of F-ddA and F-ddI during and after infusions of F-ddA were determined in both untreated and 2'-deoxycoformycin-pretreated rats. In untreated rats, F-ddA was rapidly eliminated from plasma, with a total clearance of 68.5 ml/kg/min. Metabolic clearance of F-ddA to F-ddI accounted for 58% of this value (bioconversion t1/2 = 9.8 +/- 1.9 min). Pretreatment with 2'-deoxycoformycin, an ADA inhibitor, reduced the clearance of F-ddA to 23.8 ml/min/kg, leading to 2.9 +/- 0.4-fold higher steady-state plasma concentrations of F-ddA, in agreement with a 2.5-fold enhancement predicted by a compartmental model assuming complete ADA inhibition.
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PMID:Role of altered metabolism in dideoxynucleoside pharmacokinetics. Studies of 2'-beta-fluoro-2',3'-dideoxyinosine and 2'-beta-fluoro-2',3'-dideoxyadenosine in rats. 889 19

The anabolism of 1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, a selective inhibitor of human immunodeficiency virus (HIV), was characterized in human T-lymphoblastoid CD4+ CEM cells. 1592U89 was ultimately anabolized to the triphosphate (TP) of the guanine analog (-)-carbovir (CBV), a potent inhibitor of HIV reverse transcriptase. However, less than 2% of intracellular 1592U89 was converted to CBV, an amount insufficient to account for the CBV-TP levels observed. 1592U89 was anabolized to its 5'-monophosphate (MP) by the recently characterized enzyme adenosine phosphotransferase, but neither its diphosphate (DP) nor its TP was detected. The MP, DP, and TP of CBV were found in cells incubated with either 1592U89 or CBV, with CBV-TP being the major phosphorylated species. We confirmed that CBV is phosphorylated by 5'-nucleotidase and that mycophenolic acid increased the formation of CBV-TP from CBV 75-fold. However, mycophenolic acid did not stimulate 1592U89 anabolism to CBV-TP. The adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) did not inhibit CBV-TP formation from CBV or 1592U89, whereas the adenylate deaminase inhibitor 2'-deoxycoformycin selectively inhibited 1592U89 anabolism to CBV-TP and reversed the antiviral activity of 1592U89. 1592U89-MP was not a substrate for adenylate deaminase but was a substrate for a distinct cytosolic deaminase that was inhibited by 2'-deoxycoformycin-5'-MP. Thus, 1592U89 is phosphorylated by adenosine phosphotransferase to 1592U89-MP, which is converted by a novel cytosolic enzyme to CBV-MP. CBV-MP is then further phosphorylated to CBV-TP by cellular kinases. This unique activation pathway enables 1592U89 to overcome the pharmacokinetic and toxicological deficiencies of CBV while maintaining potent and selective anti-HIV activity.
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PMID:Unique intracellular activation of the potent anti-human immunodeficiency virus agent 1592U89. 914 76

The transition between dependence on maternal transcripts and proteins inherited in the oocyte and embryonic gene expression in the human preimplantation embryo occurs at the four- to eight-cell stage. Recently, studies using reverse transcriptase polymerase chain reaction (RT-PCR) have detected paternal transcripts for the Y-linked genes, ZFY and SRY, and the myotonic dystrophy associated protein kinase gene, DK, as early as the late pronucleate one-cell stage. However, expression at the protein level has not been demonstrated and its function at these early stages is unknown. Using coding sequence polymorphisms to distinguish maternal and paternal transcripts, we have examined the transcription of two ubiquitously expressed genes: X-linked glucose-6-phosphate dehydrogenase (G6PD) and adenosine deaminase (ADA). Both G6PD and ADA are housekeeping genes with TATA-less promoters which, because of their roles in metabolism and ubiquitous expression, may provide a more reliable indication of the timing of activation of the embryonic genome. They also each have biallelic polymorphisms with a high heterozygosity ratio which can be detected by restriction digestion. Couples undergoing in vitro fertilization (IVF) were screened for these polymorphisms. Individual spare oocytes and embryos at different stages of preimplantation development were analyzed by RT-PCR and appropriate restriction digestion in those cases in which the male partner carried a different allele to the female partner. In addition, since only female embryos inherit the paternal allele of X-linked G6PD, cDNA was also analyzed for ZFX/ZFY transcripts to identify the sex of each embryo. One hundred and twenty three individual oocytes and embryos were analyzed by RT-PCR and restriction digestion to detect the paternal transcripts from the polymorphic alleles. Maternal transcripts for G6PD, ADA, and ZFX were detected in all oocytes and embryos and at all stages. Following restriction digestion, paternal G6PD and ZFY transcripts were first detected at the four-cell stage and paternal ADA transcripts in an embryo at the three-cell stage coinciding with the onset of dependency on transcription from the embryonic genome. This approach should be widely applicable to other genes since similar polymorphisms exist in the coding regions of many genes.
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PMID:Paternal transcripts for glucose-6-phosphate dehydrogenase and adenosine deaminase are first detectable in the human preimplantation embryo at the three- to four-cell stage. 936 38

The present study describes a new method for microassay of the activity of 5'-nucleotidase (5'-ND) and adenosine deaminase (ADA) in the microdissected nephron segments. The nephron segments including glomeruli, proximal convoluted and straight tubules (PCT and PST), cortical and medullary thick ascending limbs, and cortical and medullary collecting ducts were microdissected. 5'-ND and ADA in the nondenatured lysate of 20-mm microdissected tubules and 20 glomeruli were separated by agarose gel electrophoresis and by isoelectric focusing, respectively. The gels were incubated with specific substrates and staining dyes to exhibit the dephosphorylation by 5'-ND or deamination by ADA. The enzyme activity was estimated by measuring the intensity of the reaction bands on the gels. The 5'-ND activity was detected in all microdissected tubular segments and glomeruli. Among these nephron segments, PCT and PST exhibited the greatest enzyme activity, averaging 1142 and 939 mU/mg tissue protein, respectively. The activity of ADA was also detected in all tubular segments and glomeruli. However, the greatest activity of this enzyme was found in the glomeruli (649.8 mU/mg protein). Using reverse transcriptase-polymerase chain reaction technique, we verified the presence of mRNA of 5'-ND and ADA in all microdissected tubular segments and glomeruli. Based on these results, we conclude that 5'-ND and ADA are present in all nephron segments studied, but the activity of these enzymes is nonuniformly expressed along the nephron. This microassay is a highly specific, sensitive, and reliable method for the segmental analysis of adenosine metabolism in the kidney.
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PMID:Microassay of 5'-nucleotidase and adenosine deaminase activity in microdissected nephron segments. 988 22


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