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At least 343 records · Page 19Linked to original sources
PubMed1989

Highly specific inhibition of human immunodeficiency virus type 1 by a novel 6-substituted acyclouridine derivative.

A novel 6-substituted acyclouridine derivative, 1-[(2-hydroxy-ethoxy) methyl]-6-phenylthiothymine (HEPT), has proved to be a potent and selective inhibitor of human immunodeficiency virus type 1 (HIV-1) in vitro. HEPT inhibits HIV-1 replication in various T4 cell cultures as well as peripheral blood lymphocytes and macrophages. The 50% antiviral effective concentration for HIV-1 (HTLV-IIIB) in MT-4 cells is 7.0 microM, while the 50% cytotoxic concentration for mock-infected MT-4 cells is 740 microM. Although HEPT is inhibitory to various strains of HIV-1, it has no effect on the replication of other retroviruses including HIV type 2. In contrast with the dideoxynucleoside (i.e. azidothymidine) 5'-triphosphates, the triphosphate of HEPT does not interact with HIV-1 reverse transcriptase. The mechanism of action of HEPT remains subject of further study.

Antiviral Agents↗
PubMed1988

Investigations on the anti-HIV activity of 2',3'-dideoxyadenosine analogues with modifications in either the pentose or purine moiety. Potent and selective anti-HIV activity of 2,6-diaminopurine 2',3'-dideoxyriboside.

Several 2',3'-dideoxyadenosine analogues with modifications in either the ribose or purine moiety were evaluated for their inhibitory effects on the replication of human immunodeficiency virus (HIV) in MT-4 cell cultures. The 2',3'-dideoxyriboside of 2,6-diaminopurine (ddDAPR) inhibited HIV antigen expression and HIV-induced cytopathogenicity at a 50% effective dose of 2.4-3.8 microM, as compared to 3-6 microM for 2',3'-dideoxyadenosine (ddAdo), whereas 50% inhibition of MT-4 cell viability was noted only at a concentration of 477 and 889 microM, respectively. Both ddDAPR and ddAdo were only weakly inhibitory to the proliferation of a number of T-lymphoblast and T-lymphocyte cell lines, pointing to the selectivity of these compounds as anti-HIV agents. In contrast to ddAdo, ddDAPR was found to be a poor substrate for adenosine deaminase, which may be advantageous from a chemotherapeutic viewpoint. Substitution of an azido or fluoro group at the 2' and 3'-position of the ribose moiety in either "up" or "down" configurations resulted in a decrease of the anti-HIV potency and selectivity of ddAdo. In addition to ddDAPR other purine-modified ddAdo analogues, i.e. several pyrrolo[2,3-d]pyrimidine 2',3'-dideoxynucleosides, were investigated for their anti-HIV activity, but none of these derivatives proved as potent or selective as ddDAPR.

Adenosine Deaminase↗
PubMed1989

Regulation of purine deoxynucleoside phosphorylation by deoxycytidine kinase from human leukemic blast cells.

The kinetics and regulation of nucleoside phosphorylation by highly purified human deoxycytidine kinase from leukemic lymphoblasts were studied. The phosphorylation of purine nucleosides by this enzyme showed sensitivity to the endogenous inhibitors dCTP and UDP three times greater than the phosphorylation of dCyd. Examination of nucleotide pools in human T and B lymphoblasts disclosed that the levels of dCTP and UDP in these cells were sufficient to regulate kinase activity. The enhanced sensitivity of the kinase to dCTP and UDP was related to its reduced ability to interact with purine nucleosides. Comparison of the phosphorylation kinetics for pyrimidine and purine dideoxynucleosides used in antiviral therapy showed that the purine nucleosides were at least 50-fold less efficient as enzyme substrates. These results suggest that the phosphorylation of pharmacologically active purine nucleosides by deoxycytidine kinase is regulated by cellular nucleotide pools.

B-Lymphocytes↗
PubMed1993

Inhibition of mammalian DNA polymerase-associated 3' to 5' exonuclease activity by 5'-monophosphates of 3'-azido-3'-deoxythymidine and 3'-amino-3'-deoxythymidine.

3'-Azido-3'-deoxythymidine 5'-monophosphate (AZT-MP) has been hypothesized by us to possibly affect 3'-azido-3'-deoxythymidine (AZT) excision from host cell DNA. In the present study, AZT-MP inhibited 3' to 5' exonuclease activity of calf thymus DNA polymerase delta at pharmacological relevant intracellular concentrations. Other 2',3'-dideoxynucleoside-5'-monophosphate (ddN-MP) analogs, including 3'-amino-3'-deoxythymidine-5'-monophosphate (AMT-MP), were also assayed as potential inhibitors of 3' to 5' exonuclease activity. The monophosphate derivative of 3'-amino-3'-deoxythymidine (AMT), an in vivo toxic catabolite of AZT, was the most potent of the ddN-MP analogs tested, inhibiting this activity by more than 50% at 100 microM. These results suggest that inhibition of 3' to 5' exonuclease activities by AZT-MP and AMT-MP may increase steady-state levels of AZT in host DNA, accounting in part for the cell toxicity associated with this drug. The present study also raises the question of whether AZT-MP inhibition of this activity may lead to potential mutagenic effects due to inhibition of 3' to 5' exonuclease-mediated proofreading functions involved in DNA replication.

Animals↗
PubMed1995

Enhancement by hydroxyurea of the anti-human immunodeficiency virus type 1 potency of 2'-beta-fluoro-2',3'-dideoxyadenosine in peripheral blood mononuclear cells.

Ribonucleotide reductase inhibitors such as hydroxyurea (HU) and related compounds, at low, non-toxic doses, enhance the anti-human immunodeficiency virus type 1 (HIV-1) potency of both purine and pyrimidine 2',3'-dideoxynucleosides (ddNs) in human lymphocytes and macrophages. The most marked enhancement of inhibition of HIV-1 replication reported to date has been seen with the purine ddN 2',3'-dideoxyinosine (ddIno): a low level of HU (0.1 mM) permitted a 4.5-fold reduction in optimal ddIno dosage with no decrease in therapeutic effect or increase in toxicity. We report here even more marked enhancement by HU of the potency of the purine ddN 2'-beta-fluoro-2',3'-dideoxyadenosine (2'-beta-F-ddAdo), where the addition of 0.1 mM HU permitted a 7.1-fold reduction in the optimal dose of 2'-beta-F-ddAdo in the phytohemagglutinin-activated peripheral blood mononuclear cell HIV-1 test system.

Antiviral Agents↗
PubMed1988

Synthesis and evaluation of a series of 1-(3-alkyl-2,3-dideoxy-alpha,beta-D-erythro-pentofuranosyl)thymines.

A series of 1-(3-alkyl-2,3-dideoxy-alpha,beta-D-erythro-pentofuranosyl)thymines (3'-alkyl-3'-deoxythymidines) has been prepared from 5-O-(tert-butyldiphenylsilyl)-2,3-dideoxy-D-glycero-pent-2- enono-1,4-lactone ((S)-5-[(tert-butyldiphenylsilyl)oxymethyl]-2(5H)- furanone) by Michael addition of the appropriate organocopper reagent, followed by reduction of the lactone, acetylation of the resulting hemiacetal, and trimethylsilyl triflate-catalyzed coupling with 2,4-di-O-(trimethylsilyl)thymine. The protected nucleosides were desilylated by using tetrabutylammonium fluoride to give anomeric mixtures of the free nucleosides. The unsubstituted 2',3'-dideoxynucleoside analog was similarly prepared from 5-O-(tert-butyldiphenylsilyl)-2,3-dideoxy-D-glycero-pentono- 1,4-lactone ((S)5-[(tert-butyldiphenylsilyl)-oxymethyl]-dihydro-2(3H)-fu r anone).

Acetylation↗
PubMed1992

3'-Mercapto-2',3'-dideoxynucleotides are high effective terminators of DNA synthesis catalyzed by HIV reverse transcriptase.

Four 3'-mercapto-2',3'-dideoxynucleoside 5'-triphosphates (A, G, C and T) were tested as DNA chain terminator substrates for calf thymus alpha-DNA polymerase, E. coli DNA polymerase I Klenow fragment, terminal deoxynucleotidyl transferase and reverse transcriptases of AMV, HIV and MLV viruses. It was shown that the analogues selectively and irreversibly terminated DNA chain elongation by AMV and HIV reverse transcriptases and the terminal transferase. Other DNA polymerases tested did not use the nucleotide analogues as chain terminator substrate.

Base Sequence↗
PubMed1988

Characterization of human immunodeficiency virus gag/pol gene products expressed by recombinant vaccinia viruses.

Recombinant vaccinia viruses containing either the entire gag/pol gene or the reverse transcriptase (RT) domain of the human immunodeficiency virus (HIV) were constructed. In mammalian cells infected with the recombinant vaccinia virus containing the gag/pol gene, major and minor polypeptides of 55 and 41 kDa were made, but processed gag products (p24/p17/p15) were not detected. In addition, none of the products of the pol open-reading frame were seen. Both the 55- and 41-kDa gag proteins were post-translationally modified by addition of myristic acid residues in recombinant vaccinia-infected cells, and were immunoprecipitated by antiserum to p24 gag, as well as by antisera from HIV-infected patients. These results indicate that neither proteolytic processing nor other HIV proteins are required for myristilation, and suggest that the 55- and 41-kDa gag precursors share the same amino terminus as p17. Cells infected with a separate vaccinia recombinant containing a truncated piece of the gag/pol gene with added start and stop codons at the 5' and 3' ends of the RT reading frame synthesized a major 61-kDa and a minor 51-kDa protein product which reacted immunologically with both a monoclonal antibody to native HIV p66/51 and antisera from HIV-infected patients. These proteins were purified from recombinant vaccinia-infected mammalian cells, and their enzyme activity was found to be similar to that of authentic HIV RT. Cells infected with the vaccinia/RT vector contained approximately 200-fold more RT per milligram of protein than cells infected with HIV. Recombinant RT was inhibited by dideoxynucleoside triphosphates and should be useful in screening for specific inhibitors of this enzyme. Mice inoculated intradermally with 10(8) plaque-forming units of the vaccinia/RT vector developed specific antibodies to the p66/51 proteins of HIV, but anti-HIV antibodies were not detected in mice inoculated with the vaccinia/gag vector.

Antigens, Viral↗
PubMed1995

Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.

Severe and prolonged impairment of mitochondrial beta-oxidation leads to microvesicular steatosis, and, in severe forms, to liver failure, coma and death. Impairment of mitochondrial beta-oxidation may be either genetic or acquired, and different causes may add their effects to inhibit beta-oxidation severely and trigger the syndrome. Drugs and some endogenous compounds can sequester coenzyme A and/or inhibit mitochondrial beta-oxidation enzymes (aspirin, valproic acid, tetracyclines, several 2-arylpropionate anti-inflammatory drugs, amineptine and tianeptine); they may inhibit both mitochondrial beta-oxidation and oxidative phosphorylation (endogenous bile acids, amiodarone, perhexiline and diethylaminoethoxyhexestrol), or they may impair mitochondrial DNA transcription (interferon-alpha), or decrease mitochondrial DNA replication (dideoxynucleoside analogues), while other compounds (ethanol, female sex hormones) act through a combination of different mechanisms. Any investigational molecule should be screened for such effects.

Animals↗
PubMed1992

Different pattern of activity of inhibitors of the human immunodeficiency virus in lymphocytes and monocyte/macrophages.

Monocyte/macrophages (M/M) are important targets for HIV in the body, and represent the majority of cells infected by the virus in some body compartments such as the central nervous system (CNS). M/M can be different from T-lymphocytes in terms of surface antigens, cell replication and drug metabolism. Thus, we evaluated, in M/M and in T-lymphocytes, the pattern of viral inhibition induced by various anti-HIV drugs, and assessed some of the mechanisms of action related to such antiviral activity. Inhibitors of HIV binding on CD4 receptors have similar activity in M/M and T-lymphocytes, while AZT and other dideoxynucleosides (ddN) are in general more active against HIV in M/M than in T-lymphocytes. This phenomenon can be related to the increased ratio in M/M of ddN-triphosphate/deoxynucleoside-triphosphate, and can at least in part explain the ability of zidovudine and didanosine in improving neurological dysfunctions in AIDS patients. Moreover, the antiviral activity of AZT (but not of other ddN- or HIV-binding inhibitors) is potently enhanced by cytokines like granulocyte-macrophage colony stimulating factor (GM-CSF) in M/M, while anti-HIV activity of TIBO compounds in M/M is not down-modulated by GM-CSF and other cytokines. Finally, non-toxic concentrations of adriamycin, an anticancer drug reported to be active against DNA viruses, can inhibit HIV replication in M/M (but not in T-lymphocytes). Taken together, these results suggest that M/M are selective targets for HIV with peculiarities different from those of T-lymphocytes. Thus, promising anti-HIV compounds should be evaluated both in T-cells and in M/M before reaching clinical trials. This may help in selecting drugs with good chances of being effective in patients with HIV-related disease.

Adenine↗
PubMed1992

Use of a standardized cell culture assay to assess activities of nucleoside analogs against hepatitis B virus replication.

A cell culture system for the evaluation of compounds which inhibit HBV replication (Korba and Milman, Antiviral Res. 15:217, 1991) has been developed into a standardized assay. Toxicity of test compounds was assessed by the uptake of neutral red dye under culture and treatment conditions which were identical to those used for the antiviral assays. A total of 667 separate cultures of 2.2.15 cells were evaluated for this study. In 86 untreated cell cultures, representing 15 experiments over a 24-month period, the levels of extracellular HBV virion DNA and intracellular HBV DNA forms were found to vary by less than 2.5-fold overall. Virion DNA in serum and intracellular viral DNA replication intermediates [RI] are the two most reliable and commonly followed markers of hepadnavirus replication in patients and experimental animals. In these assays, levels of extracellular HBV virion DNA and intracellular HBV RI were well correlated in 2.2.15 cells. Less correlation was observed between the levels of HBV virion DNA and the 3.2-kb episomal HBV genomes present in the cells. A threshold level of 22-37 intracellular replicating HBV genomes appeared to be required before virions were detected in the culture medium. The activities of several 2'-substituted and 3'-substituted deoxynucleoside analogs against HBV replication were compared using this standardized assay. Dideoxycytosine [ddC] and dideoxyguanosine [ddG] were the most selective 2',3'-dideoxynucleosides against HBV in 2.2.15 cells. Substitution of fluorine at the 2' position abolished the antiviral activity of ddC, but enhanced the selective antiviral activities of dideoxythymidine and dideoxyuracil. Several 2'-fluorinated pyrimidine arabinosyl furanosides, reported to be potent (but toxic) inhibitors of hepadnaviruses in vivo, demonstrated relatively low selective antiviral activities in 2.2.15 cells. The current data base allows for validation of any given set of test evaluations through statistical analysis of both the positive and the negative treatment controls present in each experiment; thus, relevant comparisons of the selectivity of anti-HBV activities for different compounds examined in future experiments can be made.

Antiviral Agents↗
PubMed1993

Influence of template primary structure on 3'-azido-3'-deoxythymidine triphosphate incorporation into DNA.

In the present study, templates containing a specific segment of the G gamma-globin gene were constructed and incorporation of 3'-azido-3'-deoxythymidine 5'-triphosphate (AZT-TP) or 2',3'-dideoxythymidine 5'-triphosphate (ddTTP) into these templates was compared to that observed in M13 bacteriophage DNA. Investigations on the intrinsic fidelity of T7 DNA polymerase in reactions with AZT-TP and without deoxythymidine 5'-triphosphate, resulted in DNA synthesis beyond the first T site, suggesting that other normal deoxynucleotides misincorporated at these T sites. Modified T7 DNA polymerase incorporated AZT-TP into T sites of elongating DNA strands. Chain termination at noncomplementary sites was also observed with AZT-TP when a genomic DNA template was used and interestingly, this phenomenon was not detected in the presence of a M13 DNA template. These DNA template-dependent effects were not detected with either ddTTP, 2',3'-dideoxycytidine 5'-triphosphate, or 2',3'-didehydro-2',3'-dideoxythymidine 5'-triphosphate (D4T-TP). A variation in the extent of chain termination at T sites was observed with D4T-TP suggesting that each 2',3'-dideoxynucleoside may exhibit unique chain termination patterns along with the template sequence.

Antiviral Agents↗
PubMed1995

Inhibition of visna virus replication and cytopathic effect in sheep choroid plexus cell cultures by selected anti-HIV agents.

Several anti-HIV agents were tested against visna virus replication and cytopathic effect (CPE) in sheep choroid plexus cell cultures. Sulphated polysaccharides (i.e., dextran sulphate, pentosan polysulphate and heparin) and plant lectins, which inhibit viral adsorption and fusion, were found to be 10- to 40-fold less active against visna virus than against HIV. Bicyclam derivatives were at least 250-fold less active against visna virus and the highly HIV-1 specific TIBO derivatives were without a significant inhibitory effect on visna virus at subtoxic concentrations. In contrast, several 2',3'-dideoxynucleosides and acyclic nucleoside phosphonate analogues, which inhibit reverse transcription, were found to be very effective inhibitors of visna virus replication and viral CPE in cell culture.

Animals↗
PubMed1986

Nucleoside 5'-triphosphates modified at sugar residues as substrates for calf thymus terminal deoxynucleotidyl transferase and for AMV reverse transcriptase.

Terminal deoxynucleotidyl transferase from calf thymus and RNA-directed DNA polymerase (reverse transcriptase) from the avian myeloblastosis virus catalyze the incorporation of 3'-amino-2',3'-dideoxynucleoside 5'-triphosphates, as well as some of their 3'-derivatives, 3'-amino-3'-deoxyarabinonucleoside 5'-triphosphates and some other nucleoside 5'-triphosphates modified at sugar residues. After incorporation of the appropriate 5'-mononucleotide residue into the DNA, further chain elongation is blocked. This finding opens up a possibility for selective inhibition of DNA synthesis catalyzed by a certain enzyme.

Animals↗
PubMed1986

Nucleoside 5'-triphosphates with modified sugars as substrates for DNA polymerases.

A number of nucleoside 5'-triphosphate analogs were tested with Escherichia coli DNA polymerase I and Klenow fragment of the enzyme, bacteriophage T4 DNA polymerase and calf thymus DNA polymerase alpha. It was shown that 3'-amino-2',3'-dideoxynucleoside 5'-triphosphates as well as a number of 3'-derivatives of dTTP(3'NH2) are able to terminate DNA synthesis catalyzed by each enzyme if the reaction is performed in the absence of natural substrates. ddNTP and dNTP(3'F) were found to be inactive with DNA polymerase alpha only, but araNTP(3'NH2) was inactive with E. coli DNA polymerase I. dTTP(3'N3), dGTP(3'N'3), dCTP(3'N3), araNTP(3'N3) and (alpha-thio)dTTP(3'F) were unable to inhibit any of the above-mentioned DNA polymerases, in contrast to reverse transcriptase, accessible to the most nucleotide analogs tested.

DNA↗
PubMed1991

Experimental studies of the hematologic and immune system toxicity of nucleoside derivatives used against HIV infection.

Adverse effects stemming from the therapeutic use of dideoxynucleoside derivatives continue to occur in patients with AIDS or AIDS-related complex. For example, the continued use of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-didoxycytidine (ddC), both of which confer clinical benefits in AIDS patients, may be complicated by anemia, neutropenia and thrombocytopenia and ddC also causes peripheral neuropathy. Subsequently, the National Toxicology Program (NTP) has undertaken efforts to define and characterize the toxicities associated with currently employed and potential AIDS therapeutics in experimental animals, with particular emphasis on the hematopoietic and immune systems. In addition to AZT and ddC, 2',3'-dideoxyadenosine (ddA), 2',3'-dideoxyinosine (ddI) and 2',3'-didehydrodideoxythymidine (d4T) have been examined. The present studies describe: (1) the development of a poorly regenerative macrocytic anemia in mice exposed to AZT or ddC. This anemia demonstrates a rapid and progressive time course of toxicity and reversibility after cessation of treatment; (2) the selective suppression of erythroid progenitor cells in mice exposed to d4T without concomitant effects on myeloid stem cells. Myelotoxicity appears to show metabolism-dependent strain-specificity and is more evident following in vitro exposure than in vivo exposure; and (3) the immunosuppressive effects following subchronic (30-day) exposure. Of the nucleoside derivatives studied, only ddA and ddI altered immune function and these changes were confined to suppression of antibody responses. It can be concluded that the overall similarities in the hematopoietic and immune system effects between rodents and humans indicate that such animal toxicology studies provide important information relevant to the toxicity of these drugs.

Animals↗
PubMed1987

Application of high-performance liquid chromatography and thermospray high-performance liquid chromatography-mass spectrometry to the analysis and identification of 2',3'-dideoxyadenosine and its metabolite in biological media.

Reversed-phase high-performance liquid chromatographic (HPLC) procedures are described for determining the stability of 2',3'-dideoxyadenosine (DDA) in biological fluids at therapeutic dosages. The validated methodology uses both direct injection and solid-phase extraction techniques. Deamination of DDA to 2',3'-dideoxyinosine (DDI) in plasma by adenosine deaminase was monitored by HPLC, and the identification of DDI verified by thermospray HPLC-mass spectrometry. This methodology should prove useful in future studies concerning the stability and metabolism of dideoxynucleosides.

Adenosine Deaminase↗
PubMed1994

Determination of 2'-deoxy-3'-thiacytidine (3TC) in human urine by liquid chromatography: direct injection with column switching.

3TC (GR109714X) is a cytidine dideoxynucleoside analogue which has been shown to have in vitro activity against a variety of strains of HIV-1 and is currently being investigated in clinical trials as a treatment for HIV infection. An HPLC method for the determination of 3TC in human urine has been developed and validated. The method allows direct injection of urine (10 microliters) using HPLC column switching followed by UV detection. On-line extraction is performed using a Spherisorb-SCX (5 microns, 20 x 4.0 mm) eluted with deionized water at 1 ml min-1. 3TC is retained while the bulk of urine constituents are eluted to waste. The SCX column is then backflushed to a BDS-Hypersil-C18 (5 microns, 250 x 4.6 mm) and eluted with 100 mM acetate pH 4.5-methanol (95:5, v/v) for final separation. 3TC is detected by UV absorbance at lambda = 285. The quantitation range of the assay was 0.5-500 micrograms ml-1. The method has demonstrated sufficient ruggedness to be used in support of 3TC clinical trials. Application to other cytidine analogues including DDC has been demonstrated.

Chromatography, High Pressure Liquid↗