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)

[2',5'-Bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro- 5"-(4"-amino-1",2"-oxathiole-2", 2"-dioxide)thymine (TSAO-T) is a representative of a novel class of nucleoside analogues that are endowed with a potent and specific activity against human immunodeficiency virus (HIV) type 1 and are targeted at the HIV-1 reverse transcriptase (RT). Inhibition of HIV-1 RT by TSAO-T was reversible and noncompetitive with respect to dGTP as the substrate and poly(C).oligo(dG) as the template/primer. In contrast with the nonnucleoside derivatives tetrahydroimidazo-[4,5,1-jk][1,4]- benzodiazepin-2(1H)-thione (TIBO) (R-82150), nevirapine (BI-RG-587) and the HEPT derivative I-HEPU-SdM, TSAO-T was not inhibitory to HIV-1 RT in the presence of other homopolymeric template/primers. It did not interfere with the DNA-dependent DNA polymerase function of HIV-1 RT, HIV-2 RT, herpes simplex virus type 1 DNA polymerase, or Taq polymerase. However, TSAO-T proved inhibitory to the HIV-1 RT reaction primed by Escherichia coli 16S/23S rRNA, irrespective of the nature of the radiolabeled 2'-deoxynucleotide 5'-triphosphate (dNTP) used. TSAO-T does not act as a DNA chain terminator. It interacts with HIV-1 RT at a nonsubstrate (dNTP)-binding site.
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PMID:Kinetics of inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase by the novel HIV-1-specific nucleoside analogue [2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5 "- (4"-amino-1",2"-oxathiole-2",2"-dioxide)thymine (TSAO-T). 137 14

Recently, several classes of compounds have been shown to be extremely selective inhibitors of human immunodeficiency virus type 1 (HIV-1) replication in vitro. These include the tetrahydro-imidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-one and -thione (TIBO), 1-(2-hydroxyethoxymethyl)-6-(phenylthio)-thymine (HEPT), dipyridodiazepinone, pyridinone and bis(heteroaryl)piperazine derivatives. The hallmark of these new antiviral compounds is a specific interaction with reverse transcriptase (RT) of HIV-1. They are inactive against HIV-2 and any other viruses tested. Here we describe that, in addition to the HIV-1 strains, two simian immunodeficiency virus (SIV) strains from African green monkeys (SIVagm3 and SIVagmTYO-1) are also sensitive to the TIBO class of compounds. TIBO and HEPT derivatives block the replication of SIVagm in cell culture at micromolar concentrations. Kinetics of inhibition of SIVagm RT by TIBO are competitive with respect to the natural substrate (dGTP). Amino acid alignments and site-directed mutagenesis point to the critical role of amino acid residues Y181 and Y188 in the sensitivity of HIV-1 RT and SIVagm RT to inhibition by the TIBO derivatives. Antiviral efficacy studies with this range of compounds and using sensitive SIV strains are now feasible in monkeys.
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PMID:Differential inhibitory effects of TIBO derivatives on different strains of simian immunodeficiency virus. 137 81

Carbovir (the carbocyclic analog of 2'-3'-didehydro-2',3'-dideoxyguanosine) is a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) replication. Assays were developed to assess the mechanism of inhibition by the 5'-triphosphate of carbovir of HIV-1 reverse transcriptase using either RNA or DNA templates that contain all four natural nucleotides. Carbovir-TP was a potent inhibitor of HIV-1 reverse transcriptase using either template with Ki values similar to that observed by AZT-TP, ddGTP, and ddTTP. The kinetic constants for incorporation of these nucleotide analogs into DNA by HIV-1 reverse transcriptase using either template were similar to the values seen for their respective natural nucleotides. In addition, the incorporation of either carbovir-TP or AZT-TP in the presence of dGTP or dTTP, respectively, indicated that the mechanism of inhibition by these two nucleotide analogs was due to their incorporation into the DNA resulting in chain termination. Carbovir-TP was not a potent inhibitor of DNA polymerase alpha, beta, or gamma, or DNA primase. Given the potent activity of carbovir-TP against HIV-1 reverse transcriptase and its lack of activity against human DNA polymerases, we believe that further evaluation of this compound as a potential drug for the treatment of HIV-1 infection is warranted.
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PMID:Mechanism of inhibition of human immunodeficiency virus type 1 reverse transcriptase and human DNA polymerases alpha, beta, and gamma by the 5'-triphosphates of carbovir, 3'-azido-3'-deoxythymidine, 2',3'-dideoxyguanosine and 3'-deoxythymidine. A novel RNA template for the evaluation of antiretroviral drugs. 170 54

We investigated the inhibitory effects of aurochloric acid (AuCl4H) on reverse transcriptase (RT) derived from avian myeloblastosis virus and DNA polymerase alpha (pol. alpha) purified from HeLa S3 cells. The activities of RT, pol. alpha and E. coli DNA polymerase I (pol. I) with dTTP as the substrate were inhibited 50% at AuCl4H concentrations of 18 microM, 43 microM and 230 microM, respectively. AuCl4H inhibited RT activity competitively with respect to the substrate, dTTP, and uncompetitively with the template/primer, (rA)n(dT)12-18. In assays with dGTP as the substrate, 50% inhibitions of RT, pol. alpha and pol. I activities were observed at AuCl4H concentrations of 100 microM, 450 microM and 580 microM, respectively. AuCl4H inhibited RT activity uncompetitively with respect to the substrate, dGTP, and noncompetitively with the template/primer, (rC)n(dG)12-18. AuCl4H at concentrations causing more than 50% inhibition of RT activity had little inhibitory effect on the colony-forming ability of HeLa cells or their syntheses of DNA, RNA and protein.
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PMID:Inhibition of avian myeloblastosis virus reverse transcriptase by aurochloric acid. 170 21

Human immunodeficiency virus 1 (HIV-1) reverse transcriptase has been found to conduct error-prone synthesis on DNA and RNA templates. We find here that tolerance of an A:G mispair with poly(rA) as template is particularly strong, such that extensive poly(dG) synthesis is conducted. This type of extensive misincorporation is not observed with several reference DNA polymerases. Surprisingly, HIV reverse transcriptase processivity and kcat for dGMP misincorporation and normal dTMP incorporation are about the same. However, the Km value for dGTP in poly(dG) synthesis is approximately 1000-fold higher than the Km for dTTP in poly(dT) synthesis. Comparison of thermodynamic parameters for dGMP misincorporation and normal dNMP incorporation indicates a lower energy of activation for dGMP misincorporation than for normal dNMP incorporation. Entropy of activation (delta S*) for normal dTMP incorporation is positive (approximately 10 cal/kmol), whereas delta S* for dGMP misincorporation is negative (-36 cal/kmol). Since differences in delta S* are usually considered to reflect differences in solvation for the transition state complex, these results are consistent with the interpretation that the active site of HIV reverse transcriptase is flexible enough to misincorporate dGMP without the usual dispersion of water molecules.
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PMID:Thermodynamics of A:G mismatch poly(dG) synthesis by human immunodeficiency virus 1 reverse transcriptase. 170 95

A novel dipyridodiazepinone, 6,11-dihydro-11-cyclopropyl-4-methyldipyrido[2,3-b:2',3'-e]- [1,4]diazepin-6-one (BI-RG-587), is a selective noncompetitive inhibitor of HIV-1 reverse transcriptase (RT-1). An azido photoaffinity analogue of BI-RG-587 was synthesized and found to irreversibly inhibit the enzyme upon UV irradiation. BI-RG-587 and close structural analogues competitively protected RT-1 from inactivation by the photoaffinity label. A thiobenzimidazolone (TIBO) derivative, a nonnucleoside inhibitor of RT-1, also protected the enzyme from photoinactivation, which suggests a common binding site for these compounds. Substrates dGTP, template-primer, and tRNA afforded no protection from enzyme inactivation. A tritiated photoaffinity probe was found to stoichiometrically and selectively label p66 such that 1 mol of probe inactivates 1 mol of RT-1.
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PMID:A novel dipyridodiazepinone inhibitor of HIV-1 reverse transcriptase acts through a nonsubstrate binding site. 170 36

The template mRNA was extracted from Schistosoma japonicum. The first strand of cDNA was synthesized by AMV-reverse transcriptase. The second strand cDNA was first digested by RNase H to remove mRNA and was then synthesized by AMV-reverse transcriptase, T4-DNA polymerase. Sizing of cDNA was applied on a NACS column to remove small fragments of less than 1 kb. Homopolymeric tailing of vector (pUC18) was done with dGTP and DNA terminal transferase and tailing of the cDNA with dCTP was carried out under the same conditions. After annealing, the plasmids with cDNA were transformed into E. coli MC1061. The efficiency of cloning was about 10(4)/micrograms mRNA with 30% of the transformants having the inserts of cDNA (Figs. 1-2).
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PMID:[Construction of a cDNA library of Schistosoma japonicum]. 171 36

Substrate inhibition was observed with the heterodimeric (p66/p51) and the homodimeric (p66/p66, p51/p51) forms of human immunodeficiency virus type 1 reverse transcriptase (RNA-dependent DNA polymerase, EC 2.7.7.49). An apparent Ki value of 195 +/- 37 microM was determined for dTTP using the bacterial cloned and expressed heterodimer. Similar values were obtained with the homodimeric and the virus-encoded enzymes. When poly-(rC).p(dG)10 was used as template-primer, dGTP exhibited substrate inhibition with an apparent Ki value of 189 +/- 32 microM. Substrate inhibition was not observed with dTTP when DNA.DNA template-primers were used. Hill coefficients for substrate binding determined in the presence of saturating concentrations of template-primer were equal to 1.0, suggesting that substrate inhibition of the heterodimer is not the result of an allosteric mechanism involving the p51 subunit. Furthermore, UV crosslinking experiments with [gamma-32P]dTTP showed crosslinking only to the p66 subunit. Substrate inhibition was not as pronounced with other retroviral reverse transcriptases as it was with human immunodeficiency type 1 reverse transcriptase.
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PMID:Substrate inhibition of the human immunodeficiency virus type 1 reverse transcriptase. 171 79

N3-Methyl derivative of 3'-azido-3'-deoxythymidine 5'-triphosphate (Me-AZTTP) showed a potent inhibitory effect on HIV-1 reverse transcriptase using MS2 phage RNA as the template. The inhibition mechanism of MeAZTTP was noncompetitive with respect to any of the template MS2 RNA, dATP and dCTP. On the other hand, MeAZTTP showed a mixed-type inhibition with respect to dGTP and dTTP. These results indicate that MeAZTTP competes not only with dTTP but also with dGTP.
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PMID:Inhibitory effect of N3-methyl derivative of 3'-azido-3'-deoxythymidine 5'-triphosphate on the activity of HIV-1 reverse transcriptase. 172 10

2',3'-dideoxyguanosine 5'-triphosphate (ddGTP) was found to be an efficient substrate for DNA polymerase beta when activated DNA was used as the template.primer. Under the optimized reaction conditions with activated DNA, the rate of the incorporation of ddGTP into DNA was almost equal to that of the corresponding normal substrate dGTP. The Km value for ddGTP (1.8 microM) was smaller than that for dGTP (7.8 microM). In contrast, ddGTP was not utilized as a substrate for DNA polymerase gamma with any of the activated DNA and (dC)n.(dG)12-18 as the template primer. Other DNA polymerases such as DNA polymerase alpha, E coli DNA polymerase I and retroviral reverse transcriptase could poorly utilize ddGTP as a substrate. Some of the kinetic properties of DNA polymerase beta revealed toward ddGTP are also described. Since DNA polymerase beta plays a role in DNA repair, the present results predict possible appearance of cytotoxicity or clinical side effect(s) of 2',3'-dideoxyguanosine (ddG), known as a potent inhibitor of human immunodeficiency virus, when ddG is administered to the patients with acquired immune deficiency syndrome (AIDS) or AIDS-related complex.
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PMID:Differential utilization of 2',3'-dideoxyguanosine 5'-triphosphate as a substrate for various DNA polymerases. 193 1


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