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:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Zidovudine triphosphate inhibits the hepatitis B virus (HBV) DNA polymerase (DNAp) in vitro. Serial measurements of serum HBV DNAp activity and HBV DNA were made in 14 consecutive male homosexual patients starting zidovudine for symptomatic HIV-1 infection. Median duration of treatment was 15 weeks (range 2-72). In the 13 patients with detectable DNAp/DNA pre-treatment, no significant change in either measure of viral replication was observed during the first 16 weeks of treatment compared with the 13 weeks prior to treatment. The lack of response may be due to the opposing effect of immunosuppression, or to a failure of in vivo activity.
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PMID:No effect of zidovudine on hepatitis B virus replication in homosexual men with symptomatic HIV-1 infection. 203 94

Several dideoxynucleosides, including 3'-azido-2',3'-dideoxythymidine (zidovudine, azidothymidine, AZT), 2',3'-dideoxycytidine (ddC), and 2',3'-dideoxyinosine (ddI), have been shown to be potent inhibitors of human immunodeficiency virus (HIV) replication in human T cells and macrophages. These compounds undergo anabolic phosphorylation within target cells to a 3'-triphosphate moiety; as triphosphates, they act at the level of HIV DNA polymerase (reverse transcriptase). AZT has been shown to reduce the morbidity and mortality of patients with severe HIV infection and to at least temporarily ameliorate certain cases of HIV-induced dementia. In phase 1 studies, ddC and ddI have been shown to induce immunologic and virologic improvements in patients with AIDS or related disorders; phase 2 studies of ddC and ddI are underway. The use of these drugs can be associated with toxicity. AZT can cause bone marrow toxicity or myositis with prolonged use, ddC can cause peripheral neuropathy at high doses, and ddI can cause sporadic pancreatitis and peripheral neuropathy at high doses. For each compound, however, a therapeutic window exists in which an anti-HIV effect can be attained without short-term toxicity in most patients. Dose-intensity appears to be an important determinant of the toxicity of dideoxynucleosides. Studies are underway to explore how the therapeutic profiles of these compounds may be enhanced by attention to scheduling or through the use of combination therapy.
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PMID:Initial clinical experience with dideoxynucleosides as single agents and in combination therapy. 207 27

The inhibition of HSV-1 DNA polymerase and HeLa DNA polymerases alpha and beta by diphosphoryl derivatives of acyclic phosphonylmethoxyalkyl nucleotide analogues was studied and compared with the inhibition by ACV-TP, araCTP, ddTTP and AZT-TP. In the series of phosphonylmethoxyethyl (PME-) derivatives of heterocyclic bases, the inhibitory effect of their diphosphates on HSV-1 DNA polymerase decreased in the order 2-amino-PMEApp (Ki = 0.03 microM) much greater than PMEGpp greater than PMEApp greater than PMETpp much greater than PMECpp much greater than n8z7PMEApp greater than PMEUpp. The diphosphate derivative of the antiherpes agent (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl) adenine (HPMPA) proved to be a relatively weak inhibitor of HSV-1 DNA polymerase (Ki = 1.4 microM). The inhibitors could be divided into three groups: (a) the diphosphoryl derivatives of acyclic nucleotide analogues (PME-type and HPMPA) and ACV-TP specifically inhibit HSV-1 DNA polymerase and DNA polymerase alpha and do not significantly inhibit DNA polymerase beta; (b) AZT-TP and ddTTP are effective only against DNA polymerase beta, and (c) araCTP inhibits all three enzymes. When dATP was omitted from the reaction mixture, the addition of HPMPApp stimulated DNA synthesis by HSV-1 DNA polymerase indicating that HPMPApp is an alternative substrate for in vitro DNA synthesis catalyzed by this enzyme.
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PMID:Inhibition of herpes simplex virus DNA polymerase by diphosphates of acyclic phosphonylmethoxyalkyl nucleotide analogues. 216 89

The current progress in antiviral therapy is related to our better understanding of the viral multiplication, with potential targets for specific antiviral action at each step of the multiplication cycle inside the infected cell. Amantadine and Rimantadine are anti-influenza A drugs interfering with the penetration and the release of the virus. Most of the other antiviral drugs which are clinically available have the same target in common, namely the viral DNA polymerase. This holds true for modified nucleosides such as Acycloguanosine (Acyclovir), DHPG, Adenine-Arabinoside, Azidothymidine as well as pyrophosphate derivatives such as phosphonoformic acid. Unfortunately the antiviral chemotherapy must confront 3 obstacles: 1) a possible interference with the normal cellular metabolism, leading to residual cytotoxic side effects; 2) the genetic variability of the viruses, producing drug-resistant mutants and 3) the inability of any antiviral chemotherapeutic agent known to date to eradicate latent viral infection. A new approach of the control of latent infection is suggested with anti sense oligonucleotides of hybridons.
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PMID:Perspectives in antiviral chemotherapy. 221 May 92

The thymidine analog 3'-azido-3'-deoxythymidine (BW A509U, azidothymidine) can inhibit human immunodeficiency virus (HIV) replication effectively in the 50-500 nM range [Mitsuya, H., Weinhold, K. J., Furman, P. A., St. Clair, M. H., Nusinoff-Lehrman, S., Gallo, R. C., Bolognesi, D., Barry, D. W. & Broder, S. (1985) Proc. Natl. Acad. Sci. USA 82, 7096-7100]. In contrast, inhibition of the growth of uninfected human fibroblasts and lymphocytes has been observed only at concentrations above 1 mM. The nature of this selectivity was investigated. Azidothymidine anabolism to the 5'-mono-, di-, and -triphosphate derivatives was similar in uninfected and HIV-infected cells. The level of azidothymidine monophosphate was high, whereas the levels of the di- and triphosphate were low (less than or equal to 5 microM and less than or equal to 2 microM, respectively). Cytosolic thymidine kinase (EC 2.7.1.21) was responsible for phosphorylation of azidothymidine to its monophosphate. Purified thymidine kinase catalyzed the phosphorylations of thymidine and azidothymidine with apparent Km values of 2.9 microM and 3.0 microM. The maximal rate of phosphorylation with azidothymidine was equal to 60% of the rate with thymidine. Phosphorylation of azidothymidine monophosphate to the diphosphate also appeared to be catalyzed by a host-cell enzyme, thymidylate kinase (EC 2.7.4.9). The apparent Km value for azidothymidine monophosphate was 2-fold greater than the value for dTMP (8.6 microM vs. 4.1 microM), but the maximal phosphorylation rate was only 0.3% of the dTMP rate. These kinetic constants were consistent with the anabolism results and indicated that azidothymidine monophosphate is an alternative-substrate inhibitor of thymidylate kinase. This conclusion was reflected in the observation that cells incubated with azidothymidine had reduced intracellular levels of dTTP. IC50 (concentration of inhibitor that inhibits enzyme activity 50%) values were determined for azidothymidine triphosphate with HIV reverse transcriptase and with immortalized human lymphocyte (H9 cell) DNA polymerase alpha. Azidothymidine triphosphate competed about 100-fold better for the HIV reverse transcriptase than for the cellular DNA polymerase alpha. The results reported here suggest that azidothymidine is nonselectively phosphorylated but that the triphosphate derivative efficiently and selectively binds to the HIV reverse transcriptase. Incorporation of azidothymidylate into a growing DNA strand should terminate DNA elongation and thus inhibit DNA synthesis.
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PMID:Phosphorylation of 3'-azido-3'-deoxythymidine and selective interaction of the 5'-triphosphate with human immunodeficiency virus reverse transcriptase. 243 Feb 86

Reverse transcriptase was purified from human immunodeficiency virus (HIV). It utilized the artificial primer-template poly(rA)-oligo(dT)12-18 more efficiently than activated calf thymus DNA, poly(rI)-oligo(dC)12-18, poly(rC)-oligo(dG)12-18, or poly(rCm)-oligo(dG)12-18. Maximum activity was observed at pH 7.0 to 7.6 in the presence of 5 mM MgCl2 and 100 mM KCl. 3'-Azido-3'-deoxythymidine triphosphate competed with dTTP for binding to HIV reverse transcriptase. Different kinetic constants were obtained with different primer-templates. Km and Ki values of 2.8 and 0.04 microM, respectively, were obtained with poly(rA)-oligo(dT)12-18. The corresponding values were 1.2 and 0.3 microM, respectively, with activated calf thymus DNA and 0.3 and 0.01 microM, respectively, with extracted virus and native template. Inhibition of the host cell DNA polymerases alpha and beta was considerably weaker. The Km and Ki values obtained with activated calf thymus DNA as the primer-template were 2.4 and 230 microM, respectively, for DNA polymerase alpha and 6.0 and 73 microM, respectively, for DNA polymerase beta. 3'-Azido-3'-deoxythymidine triphosphate could also serve as an alternate substrate for HIV reverse transcriptase. The resulting incorporation of 3'-azido-3'-deoxythymidine triphosphate into poly(rA)-oligo(dT)12-18 caused chain termination and premature deceleration of the reaction. The terminated primer could not be elongated when incubated with dTTP and HIV reverse transcriptase.
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PMID:3'-Azido-3'-deoxythymidine triphosphate as an inhibitor and substrate of purified human immunodeficiency virus reverse transcriptase. 244 66

The reverse transcriptase from human immunodeficiency virus type 1 was purified from the virus to near homogeneity. The enzyme was shown to possess both RNA-dependent and DNA-dependent DNA-synthesizing activity. Activated DNA as a heteropolymeric substrate was used as efficiently as was the homopolymeric substrate poly(rA)-oligo(dT). The Michaelis-Menten constants were determined for each of the four nucleotides needed to elongate a natural template primer. Azidothymidine triphosphate, a well-known inhibitor of the enzyme, inhibited the enzyme competitively with respect to dTTP and noncompetitively with respect to the other nucleotides. Azidothymidine triphosphate acted as an efficient inhibitor of cellular DNA polymerase gamma, whereas other enzymes of eucaryotic DNA metabolism, namely, DNA polymerase alpha-primase and DNA polymerase beta, were not inhibited. This finding may explain why some acquired immunodeficiency syndrome patients suffer side effects during azidothymidine therapy.
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PMID:Azidothymidine triphosphate is an inhibitor of both human immunodeficiency virus type 1 reverse transcriptase and DNA polymerase gamma. 248 2

Combinations of 3'-azido-3'-deoxythymidine and phosphonoformate produced a moderate synergistic inhibitory effect against human immunodeficiency virus type 1 in vitro at concentrations that are easily achieved in humans. The synergistic effect was more pronounced with increasing concentrations and was not secondary to toxic effects of the drugs. 3'-Azido-3'-deoxythymidine neither inhibited the replication of human cytomegalovirus in human embryonic lung fibroblasts nor interfered with the anticytomegalovirus effect of phosphonoformate. By using partially purified reverse transcriptase of human immunodeficiency virus type 1 and human cytomegalovirus DNA polymerase, various combinations of 3'-azido-3'-deoxythymidine-5'-triphosphate and phosphonoformate produced strong indications of additive interactions. The synergistic interactions in infected cells and the additive effects observed at the reverse transcriptase level indicate that mechanisms other than the reverse transcriptase may be of importance for the inhibition of human immunodeficiency virus replication by these two compounds. A concomitant treatment of cytomegalovirus infections, such as cytomegalovirus retinitis, with phosphonoformate in patients with acquired immunodeficiency syndrome receiving 3'-azido-3'-deoxythymidine may be appropriate, and this combination may also be useful in controlling human immunodeficiency virus infection.
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PMID:Combinations of 3'-azido-3'-deoxythymidine (zidovudine) and phosphonoformate (foscarnet) against human immunodeficiency virus type 1 and cytomegalovirus replication in vitro. 254 87

We previously demonstrated that 3'-azido-3'-deoxythymidine (AZT) inhibits growth proliferation of human bone marrow progenitor cells in vitro [Antimicrob. Agents Chemother. 31:452-454 (1987)]. The present study evaluates the effect of toxic concentrations of AZT on possible sites of toxicity in human bone marrow cells. Exposure of cells over a 6-hr period to AZT concentrations between 0.5 and 50 microM resulted in a decreased incorporation of tritiated deoxyguanosine into DNA. Unchanged AZT and its phosphorylated metabolites accumulated within cells after exposure to 10 microM [3H]AZT. 3'-Azido-3'-deoxythymidine-5'-monophosphate was the predominant metabolite, reaching a concentration of 49.2 +/- 14.1 pmol/10(6) cells after 48 hr, and a continuous increase was observed in all phosphorylated derivative levels between 2 and 48 hr of incubation. Using a highly sensitive and specific DNA polymerase assay, endogenous deoxyribonucleotide pool size(s) were analyzed for 48 hr after incubation of cells with a pharmacologically relevant concentration of 10 microM AZT. After a 6-hr exposure, 2'-deoxycytidine-5'-triphosphate and 2'-deoxythymidine-5'-triphosphate pools represented approximately 86 and 70% of the control values; levels returned to normal after 24 hr and remained subsequently unchanged. Nucleic acids of human bone marrow cells exposed for 24 hr to 10 microM [3H]AZT were purified and analyzed by cesium sulfate density gradient. No radioactivity was detected in the RNA region, whereas a significant amount was associated with the DNA region. Hydrolysis of radiolabeled DNA and subsequent analysis by high performance liquid chromatography demonstrated specific incorporation of AZT into DNA. In additional studies, the amount of AZT incorporated into DNA was correlated with the initial extracellular AZT concentration. In particular, a significant relationship (p less than 0.0001) between the level of AZT incorporated into DNA and the inhibition of clonal growth was observed at concentrations of AZT between 1 and 25 microM (IC50 and IC85 for human bone marrow cells). In summary, these studies demonstrate that AZT is incorporated into DNA of human bone marrow cells and suggest that incorporation of AZT into DNA may be one mechanism responsible for AZT-induced bone marrow toxicity. In contrast, imbalance of deoxyribonucleotide pools by AZT appears unlikely to be associated with inhibition of DNA synthesis and toxicity in human bone marrow cells.
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PMID:Cellular pharmacology of 3'-azido-3'-deoxythymidine with evidence of incorporation into DNA of human bone marrow cells. 274 33

The thymidine analog 3'-azido-3'-deoxythymidine (BW A509U; azidothymidine [AZT]) had potent bactericidal activity against many members of the family Enterobacteriaceae, including strains of Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, Shigella flexneri, and Enterobacter aerogenes. AZT also had bactericidal activity against Vibrio cholerae and the fish pathogen Vibrio anguillarum. AZT had no activity against Pseudomonas aeruginosa, gram-positive bacteria, anaerobic bacteria, Mycobacterium tuberculosis, nontuberculosis mycobacteria, or most fungal pathogens. Several lines of evidence indicated that AZT must be activated to the nucleotide level to inhibit cellular metabolism: AZT was a substrate for E. coli thymidine kinase; spontaneously arising AZT-resistant mutants of E. coli ML-30 and S. typhimurium were deficient in thymidine kinase; and intact E. coli ML-30 cells converted [3H]AZT to its mono-, di-, and triphosphate metabolites. Of the phosphorylated metabolites, AZT-5'-triphosphate was the most potent inhibitor of replicative DNA synthesis in toluene-permeabilized E. coli pol A mutant cells. AZT-treated E. coli cultures grown in minimal medium contained highly elongated cells consistent with the inhibition of DNA synthesis. AZT-triphosphate was a specific DNA chain terminator in the in vitro DNA polymerization reaction catalyzed by the Klenow fragment of E. coli DNA polymerase I. Thus, DNA chain termination may explain the lethal properties of this compound against susceptible microorganisms.
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PMID:Antibacterial activity and mechanism of action of 3'-azido-3'-deoxythymidine (BW A509U). 355 32


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