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)

A new class of membrane-active ether lipid (EL) analogs of platelet-activating factor were studied for in vitro anti-HIV-1 activity. Human T-cell (CEM-ss) monolayers or suspension cultures were used to determine effects of structural modifications of Type A phosphorus-containing and Type B nonphosphorus EL analogs on (a) the inhibitory concentration50 (IC50) for HIV-1 syncytial plaque formation and cell growth, and, (b) virus budding at the cell plasma membrane. Results indicate that representative Type A and Type B EL inhibit HIV-1 but not herpes simplex virus type 2 plaque formation when added before or up to 2 days after viral infection. Anti-HIV-1 activity does not involve direct inactivation of virus infectivity. Type A EL (IC50 range = 0.2-1.4 microM) with alkyoxy, alkylthio, or alkyamido substitution at glycerol position 1 and ethoxy or methoxy substitution at position 2, and Type B compounds (IC50 range = 0.33-0.63 microM) with an inverse choline or nitrogen heterocyclic substitution at position 3 have selective activity against HIV-1-infected T-cells. EL treatment of HIV-1-infected cells is associated with subsequent release of reverse transcriptase activity, but infectious virus production is inhibited with time after infection. Electron microscopic examination of HIV-1-infected and EL-treated cells revealed absence of detectable budding virus at the plasma membrane but presence of intracytoplasmic vacuolar virus particles. In summary, these data suggest that EL analogs are a novel class of agents that induce defective intracytoplasmic vacuolar HIV-1 formation in T-cells. Being membrane interactive, EL are ideally suited for combination chemotherapy with DNA-interactive anti-HIV nucleoside analogs.
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PMID:Novel membrane-interactive ether lipid analogs that inhibit infectious HIV-1 production and induce defective virus formation. 169 29

The water-soluble ammonium salt of 3'-azido-5'-(O-ethoxycarbonylphosphinyl)-3'-deoxythymidine (ECP-AZT), the prototype of a novel class of compounds incorporating two active antiretroviral agents, in this case 3'-azido-3'-deoxythymidine (AZT) and phosphonoformic acid (PFA), within the same structure, was synthesized and tested as an inhibitor of the replication of human immunodeficiency virus type 1 (HIV-1) in Jurkat cells, a CD4+ human T-lymphocyte cell line. The corresponding 5'-(O-methoxycarbonylphosphinyl) derivative (MCP-AZT) was also prepared. The rationale for the synthesis of ECP-AZT and MCP-AZT was that they may be cleaved intracellularly to AZT and PFA via hydrolysis of the phosphate ester bond or to AZT 5'-monophosphate by oxidative cleavage of the carbon-phosphorus bond. ECP-AZT was found to block viral replication at a 50% inhibitory concentration (IC50) of ca. 10(-6) M as measured by reverse transcriptase (RT) activity in supernatants from cultures of infected cells. Little or no inhibition of cell growth was observed at this concentration, and there was less than 20% inhibition of cell growth at 10(-4) M. AZT itself was a more potent inhibitor of HIV-1 replication than ECP-AZT, but was also more cytotoxic. The antiviral selectivity of ECP-AZT, defined as the ratio IC50 (virus inhibition)/IC50(cell growth inhibition), was in the range considered to be therapeutic for anti-AIDS nucleosides.
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PMID:Inhibition of human immunodeficiency virus type 1 replication by phosphonoformate esters of 3'-azido-3'-deoxythymidine. 222 76

The Sp-isomer of thymidine 5'-O-(1-thiotriphosphate) is used as substrate by HIV-1 reverse transcriptase. The absolute configuration of the internucleotide linkage in the oligonucleotide product was identified by 31p NMR spectroscopy to be the Rp-isomer, indicating that incorporation of dTTP alpha S into the oligonucleotide proceeded with inversion of configuration at the alpha-phosphorus. The mechanistic implications of these observations are discussed.
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PMID:Investigation of the stereochemical course of DNA synthesis catalysed by human immunodeficiency virus type 1 reverse transcriptase. 247 23

The Sp diastereomer of thymidine 5'-O-(1-thiotriphosphate) was polymerized by avian myeloblastosis virus reverse transcriptase using poly(A) . d(pT)10 as template-primer. Degradation of the template poly(A) by alkaline hydrolysis and isolation by gel chromatography gave a single-stranded poly(d(p(S)T)), a polymer of thymidine 5'-phosphorothioate. To determine the configuration of the phosphorothioate internucleotide linkage, this material was degraded by snake venom phosphodiesterase. Comparison of the rates of degradation by snake venom phosphodiesterase of poly(d(p(S)T)) prepared by reverse transcriptase and DNA polymerase I showed them to be very similar. Since it has been established earlier than the latter enzyme produces polymers with phosphorothioate linkages of the Rp configuration (Burgers, P. M. J., and Eckstein, F. (1979) J. Biol. Chem. 254, 6889-6893), it is concluded that the polymer produces by reverse transcriptase has the same stereochemistry. Further proof for this assignment comes from comparison by 31P nmr of this polymer with the diastereomers of synthetic 5'-O-thymidyl 3'-O-thymidyl phosphorothioate. The chemical shift observed for the polymer was identical with that of the Rp isomer of 5'-O-thymidyl 3'-O-thymidyl phosphorothioate. Avian myeloblastosis virus reverse transcriptase therefore polymerizes deoxynucleoside 5'-triphosphates with inversion of configuration at the alpha-phosphorus. This result indicates that direct nucleophilic attack by the 3-hydroxyl group of the growing polymer on the alpha-phosphoryl group occurs without formation of a covalent enzyme intermediate.
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PMID:Stereochemical course of polymerization catalyzed by avian myeloblastosis virus reverse transcriptase. 617 37

Analysis of controlled studies performed by the Polycythemia Vera Study Group (P.V.S.G.) and the European Organization for Research in Treatment of Cancer (E.O.R.T.C.) indicate that busulphan (Myleran) (BU) is the treatment of choice for polycythemia vera (PV). BU is particularly effective as compared to aspirin and dipyridamole (Persantine) or radioactive phosphorus (32P) in preventing the thrombotic and atherosclerotic complications of PV. In contradistinction to chlorambucil (CM), BU is not associated with an unacceptable increase in the incidence of leukemia. The pharmacology of BU remains unclear, but certainly it cannot be considered a classic alkylating agent. BU suppresses the activity of the reverse transcriptase-like RNA dependent DNA polymerase in the platelets of these patients. A clearer understanding of the role of BU in the treatment of the myeloproliferative disorders will provide important insights into the etiology and pathogenesis not only of preneoplastic states, but also thrombosis and atherosclerosis.
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PMID:Busulphan: effect on platelet RNA dependent DNA polymerase--implications in the treatment of polycythemia vera, thrombosis and atherosclerosis. 618 58

We present a full-coordinate model of residues 1-319 of the polymerase domain of HIV-I reverse transcriptase. This model was constructed from the x-ray crystallographic structure of Jacobo-Molina et al. (Jacobo-Molina et al., P.N.A.S. USA 90, 6320-6324 (1993)) which is currently available to the degree of C-coordinates. The backbone and side-chain atoms were constructed using the MAXSPROUT suite of programs (L. Holm and C. Sander, J. Mol. Biol. 218, 183-194 (1991)) and refined through molecular modeling. A seven base pair A-form dsDNA was positioned in the nucleic acid binding cleft to represent the template-primer complex. The orientation of the template-primer complex in the nucleic acid binding cleft was guided by the positions of phosphorus atoms in the crystal structure.
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PMID:A full-coordinate model of the polymerase domain of HIV-1 reverse transcriptase and its interaction with a nucleic acid substrate. 753 52

Heteropolyoxotungstates of the Keggin class containing different heteroatoms were tested for inhibition of two strains of human immunodeficiency virus 1 (HIV-1); they exhibited varying antiviral activity. Compounds containing boron were inactive, only one of those containing phosphorus showed selective anti-viral activity, whereas all silicon-containing compounds exhibited significant anti-viral activity in C8166 cells infected with the IIIB strain. Their effectiveness was some 10-fold higher in JM cells with selectivity indices of about 2000. The silicotungstates were effective inhibitors of HIV reverse transcriptase, showing greater inhibition with RNA/DNA template primers than with DNA/DNA template.primer. Kinetic analysis demonstrated that they inhibit the enzyme by different mechanisms, as, of the four compounds examined, two competed with template.primer and two competed with deoxynucleoside triphosphate. Inhibition of DNA polymerase activity by these compounds was compared using polymerases from different sources, including human; although not necessarily most specific for HIV-1 reverse transcriptase, they did not inhibit all DNA polymerases to a similar degree.
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PMID:Anti-(human immunodeficiency virus) activity of polyoxotungstates and their inhibition of human immunodeficiency virus reverse transcriptase. 753 11

A novel membrane-soluble prodrug of the 5'-monophosphate derivative of 3TC containing a phenyl group and the methyl ester of L-alanine linked to the phosphorus through a phosphoramidate bond with the primary amino moiety (designated Cf 1109) was prepared. The 3TC prodrug proved less potent an inhibitor of HIV-1 and HIV-2 replication in CEM cell cultures than 3TC, but lost only 20-fold antiviral potency in 2'-deoxycytidine kinase-deficient CEM/dCK- cells compared with a more than 2,000-fold decrease of activity of 3TC. In contrast, 3TC and Cf 1109 proved equally highly effective in inhibiting HBV release in supernatants of HBV-transfected Hep G2 2.2.15 cell cultures (50% effective concentration approximately 0.02 microM). Both compounds easily selected for highly resistant HIV-1 strains at a comparable speed of breakthrough. The mutant viruses contained an 184-Ile and/or 184-Val amino acid change in their reverse transcriptase. Our data are suggestive for a relatively poor delivery of 3TC-MP in the intact CEM cells but a remarkably high delivery of 3TC and/or 3TC-MP in the intact Hep G2 2.2.15 cells.
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PMID:Anti-HIV and anti-HBV activity and resistance profile of 2',3'-dideoxy-3'-thiacytidine (3TC) and its arylphosphoramidate derivative CF 1109. 875 70

Chondrocytes isolated from normal adult human articular cartilage were infected with a retroviral vector encoding a temperature-sensitive mutant of the simian virus 40 large tumor antigen and a linked geneticin (G418)-resistance marker. G418-resistant colonies were then isolated, ring-cloned, and expanded in serum-containing media. Several immortalized chondrocyte cell lines were established from the clones that survived, some of which have been maintained in continuous culture for over 2 years. Despite serial subcultures and maintenance as monolayers, these cells retain expression of markers specific for cells of the lineage, namely type II collagen and aggrecan, detected immunocytochemically. We also examined the phenotype of three of these immortalized cell lines (designated HAC [human articular chondrocyte]) using a pellet culture system, and in this report, we present evidence that a prototype of these lines (HAC-F cells) expresses markers normally associated with hypertrophic chondrocytes. When HAC-F cells were cultivated in centrifuge tubes, for periods of up to 63 days, at 39 degrees C with mild and intermittent centrifugation they continued to express both lineage markers; total type II collagen/pellet remained stable, whereas there was a temporal decrease in cartilage-specific glycosaminoglycans content. In addition, in the presence of ascorbate but in the absence of a phosphate donor or inorganic phosphate supplement, the cells also begin to express a hypertrophic phenotype characterized by type X collagen synthesis and extensive mineralization of the extracellular matrix in late stage cultures. The mRNA encoding type X collagen was detected in the cell pellets by reverse transcriptase polymerase chain reaction as early as day 2, and anti-type X collagen immunoreactivity was subsequently localized in the matrix. The mineral was characterized by energy-dispersive X-ray microanalysis as containing calcium (Ca) and phosphorus (P) with a Ca:P peak height ratio close to that of mineralized bone tissue. The unexpected phenotype of this human chondrocyte cell line provides an interesting opportunity for studying chondrocyte maturation in vitro.
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PMID:Expression of type X collagen and matrix calcification in three-dimensional cultures of immortalized temperature-sensitive chondrocytes derived from adult human articular cartilage. 952 44

(R)-9-(2-Phosphonylmethoxypropyl)adenine (PMPA) is an acyclic nucleoside phosphonate that has been shown to be effective in the treatment of AIDS although it has a shorter separation between the adenine and phosphorus than dideoxy-AMP and dAMP. By using pre-steady state kinetic methods, we examined the incorporation of the diphosphate of PMPA, 2',3'-dideoxyadenosine 5'-triphosphate (ddATP), and dATP catalyzed by wild-type human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, an exonuclease-deficient T7 DNA polymerase (T7 exo-), and wild-type rat DNA polymerase beta in order to evaluate the selectivity of PMPA as an antiviral inhibitor. With a DNA/DNA or DNA/RNA 22/43-mer duplex, the diphosphate of PMPA (PMPApp) is as effective as ddATP in reactions catalyzed by HIV-1 reverse transcriptase in that both analogs have similar substrate specificity constants (kp/Kd) which are only 5-fold lower than dATP. In contrast, PMPApp is a much weaker inhibitor of the reaction catalyzed by T7 exo- (with the DNA/DNA 22/43-mer duplex) in that PMPApp has a 5 x 10(-4)-fold lower kp/Kd than ddATP and dATP. The lower kp/Kd of PMPApp is due to a 1000-2000-fold lower incorporation rate (kp) and a 35-45-fold lower binding constant (Kd). Similarly, PMPApp is 800-fold less inhibitory toward polymerase beta with the DNA/DNA 22/43-mer duplex, whereas in studies with a single nucleotide gapped DNA (22-20/43-mer) PMPApp is 13-fold less inhibitory than ddATP. Although parallel studies will need to be performed using appropriate human polymerases, these results begin to define the mechanistic basis for the reported lower toxicity of PMPA in the treatment of AIDS.
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PMID:Selective inhibition of HIV-1 reverse transcriptase by an antiviral inhibitor, (R)-9-(2-Phosphonylmethoxypropyl)adenine. 976 48


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