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 polypurine tract (PPT) from the HIV-1 genome is resistant to digestion by reverse transcriptase following (-)-strand synthesis and is used to prime (+)-strand synthesis during retroviral replication. We have determined the crystal structure of the asymmetric DNA/DNA analog16-mer duplex (CTTTTTAAAAGAAAAG/CTTTTCTTTTAAAAAG) comprising most of the "visible" portion of the RNA:DNA hybrid from the polypurine tract of HIV-1, which was previously reported in a complex with HIV-1 reverse transcriptase. Our 16-mer completely encompasses a 10-mer DNA duplex analog of the HIV-1 PPT. We report here a detailed analysis of our B' form 16-mer DNA structure, including three full pure A-tracts, as well as a comparative structural analysis with polypurine tract and other A-tract-containing nucleic acid structures. Our analysis reveals that the polypurine tract structures share structural features despite being different nucleic acid forms (i.e. DNA:DNA versus RNA:DNA). In addition, the previously reported A-tract-containing DNA molecules bound to topoisomerase I are remarkably similar to our polypurine tract 16-mer structure. On the basis of our analysis, we suggest that the specific topology of long pure A-tracts is remarkably comparable across a wide array of biological environments.
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PMID:Staying straight with A-tracts: a DNA analog of the HIV-1 polypurine tract. 1281 2

Human telomerase is a reverse transcriptase that is expressed in essentially all cancer cells, but not in the vast majority of normal somatic cells. Therefore, the specific inhibition of telomerase activity in tumors might have significant beneficial therapeutic effects. We have designed and evaluated oligonucleotide N3' --> P5' thio-phosphoramidates as telomerase template antagonists. In biochemical cell-free assays 11-13-mer thio-phosphoramidate oligonucleotides demonstrated sequence specific and dose dependent inhibition of telomerase with pico-molar IC50 values. Optimization of the oligonucleotide sequence and length resulted in the identification of a 13-mer-oligonucleotide thio-phosphoramidate GRN163 as a drug development candidate. In cell cultures GRN163 was able to inhibit telomerase activity in the absence of cationic lipid with approximately 1 microM IC50 values. Telomerase inhibition by GRN163 produced gradual telomere shortening, followed by cellular senescence and/or apoptosis of cancer derived cell lines.
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PMID:Oligonucleotide N3' --> P5' thio-phosphoramidate telomerase template antagonists as potential anticancer agents. 1456 32

Emtricitabine [(-)FTC; (-)-beta-L-2'-3'-dideoxy-5-fluoro-3'-thiacytidine] is an oxathiolane nucleoside analog recently approved by the Food and Drug Administration for the treatment of human immunodeficiency virus (HIV). Structurally, (-)FTC closely resembles lamivudine [(-)3TC] except that the former is 5-fluorinated on the cytosine ring. In HIV-1 reverse transcriptase (RT) enzymatic assays, the triphosphate of (-)FTC [(-)FTC-TP] was incorporated into both DNA-DNA and DNA-RNA primer-templates nearly 3- and 10-fold more efficiently than (-)3TC-TP. Animal studies and clinical trial studies have demonstrated a favorable safety profile for (-)FTC. However, a detailed study of the incorporation of (-)FTC-TP by human mitochondrial DNA polymerase gamma, a host enzyme associated with nucleoside toxicity, is required for complete understanding of the molecular mechanisms of inhibition and toxicity. We studied the incorporation of (-)FTC-TP and its enantiomer (+)FTC-TP into a DNA-DNA primer-template by recombinant human mitochondrial DNA polymerase in a pre-steady-state kinetic analysis. (-)FTC-TP was incorporated 2.9 x 10(5)-, 1.1 x 10(5)-, 1.6 x 10(3)-, 7.9 x 10(3)-, and 100-fold less efficiently than dCTP, ddCTP, (+)3TC-TP, (+)FTC-TP, and (-)3TC-TP, respectively. The rate of removal of (-)FTC-MP from the corresponding chain-terminated 24-mer DNA by polymerase gamma's 3'-->5' exonuclease activity was equal to the removal of (+)FTC-MP, 2-fold slower than the removal of (-)3TC-MP and (+)3TC-MP, and 4.6-fold slower than the excision of dCMP. These results demonstrate that there are clear differences between HIV-1 RT and polymerase gamma in terms of preferences for substrate structure.
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PMID:Relationship between antiviral activity and host toxicity: comparison of the incorporation efficiencies of 2',3'-dideoxy-5-fluoro-3'-thiacytidine-triphosphate analogs by human immunodeficiency virus type 1 reverse transcriptase and human mitochondrial DNA polymerase. 1504 33

A new 32-mer 2',5'-oligoribonucleotide of 1-methyl-6-thioinosinic acid (10) has been synthesized. The design of this unique oligoribonucleotide is based on the reported HIV inhibitory activities of both 2',5'-oligonucleotides and the 3',5'-oligoribonucleotides containing the 1-methyl-6-mercaptopurine base. Tm and CD studies of 10 revealed that it has no organized secondary structure, presumably due to the rigidity of the molecule. The synthesized oligomer, 10, showed a potent inhibitory effect on HIV-1RF and significantly inhibited HIV-1 reverse transcriptase.
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PMID:An unusual "senseless" 2',5'-oligoribonucleotide with potent anti-HIV activity. 1511 22

The chemokine receptors CXCR4 and CCR5 are used as the main co-receptors by the T-cell-tropic (CXCR4-dependent, X4) and macrophage-tropic (CCR5-dependent, R5) HIV-1 strains, respectively, for entering their target cells. The natural ligands for CXCR4, the CXC-chemokine SDF-1 and CCR5, the CC-chemokines RANTES, MIP-1alpha and MIP-1beta are described to inhibit viral entry. In this review we focus on chemokine receptor/HIV co-receptor inhibitors. Modified chemokines such as Met-RANTES and AOP-RANTES showed antiviral activity against R5 viruses. Several low-molecular weight CCR5 antagonists have been described (such as TAK-779 and SCH-C) with potent antiviral activity. The latter compound is also orally available and is able to decrease R5 viral load levels in HIV-infected subjects. Several peptidic compounds, such as T22 (an 18-mer), T134 (a 14-mer), ALX40-4C (a 9-mer) and CGP 64222 (also a 9-mer) have anti-HIV activity and have been identified as CXCR4 antagonists. Also, the HIV-1 Tat protein has been described as a "natural" CXCR4 antagonist with anti-HIV-1 activity. The most potent and specific CXCR4 antagonists are the bicyclam derivatives, which also potently inhibit X4 HIV replication. AMD3100 has proved to be a highly specific CXCR4 antagonist, which consistently blocks X4 viral replication in any target cell-type evaluated so far. AMD3100 was selected as the clinical drug candidate, which, after initial phase I (safety) studies, had proceeded to phase II (efficacy) trials. The compound dose-dependently inhibited X4 viruses after 10 days of continuous infusion of the drug. Recently, the orally bioavailable CXCR4 antagonist, AMD070, is presented as a candidate HIV drug. We believe that chemokine receptor antagonists will become important new antiviral drugs to combat AIDS. Both (CXCR4 and CCR5) chemokine receptor inhibitors will be needed in combination to inhibit viral replication and even in combinations of antiviral drugs that also target other aspects of the HIV replication cycle, such as reverse transcriptase and protease, to obtain optimum therapeutic effects.
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PMID:HIV co-receptors as targets for antiviral therapy. 1513 47

The development of novel drugs active against multi-drug resistant (MDR) HIV-1 strains is urgently required. HIV protease inhibitors and reverse transcriptase inhibitors constitute two categories of important drugs, which have greatly improved the clinical treatment of HIV-infected patients by their cocktail use designated as highly active anti-retroviral therapy (HAART). By combinatorial chemistry involving substructure units contained in known HIV protease inhibitors, we found effective protease inhibitors, TYA5 and TYB5, which showed potent anti-HIV activity even against MDR strains. Selection of drug-resistant viruses is also decreased when these new agents are tested in vitro. Subsequently, introduction of an (E)-alkene dipeptide isostere into TYB5 led to the development of a pure non-peptide protease inhibitor, TYB1. We have also studied the development of effective inhibitors for blocking HIV-entry into host cells based on recent discovery of an HIV entry mechanism involving the viral usage of chemokine receptors as coreceptors, CXCR4 and CCR5. We developed highly selective CXCR4 antagonists, T22 and T140 (18-mer and 14-mer peptides, respectively), which strongly suppress T-cell line-tropic HIV-1 (X4-HIV-1) entry through their specific binding to CXCR4. Recently, molecular-size reduction of T140 yielded low molecular weight CXCR4 antagonists, which might be more useful leads to drug-like structures. In this review, we discuss the development of two types of anti-HIV agents, protease inhibitors and CXCR4 antagonists, which would improve clinical AIDS chemotherapy.
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PMID:Two orthogonal approaches to overcome multi-drug resistant HIV-1s: development of protease inhibitors and entry inhibitors based on CXCR4 antagonists. 1518 Apr 58

2-Naphthalenesulfonic acid (4-hydroxy-7-[[[[5-hydroxy-6-[(4 cinnamylphenyl)azo]-7-sulfo-2-naphthalenyl]amino]-carbonyl]amino]-3-[(4-cinnamylphenyl)]azo (KM-1)) is a novel non-nucleoside reverse transcriptase inhibitor (NNRTI) that was designed to bind at an unconventional site on human immunodeficiency virus type 1 reverse transcriptase (RT) (Skillman, A. G., Maurer, K. W., Roe, D. C., Stauber, M. J., Eargle, D., Ewing, T. J., Muscate, A., Davioud-Charvet, E., Medaglia, M. V., Fisher, R. J., Arnold, E., Gao, H. Q., Buckheit, R., Boyer, P. L., Hughes, S. H., Kuntz, I. D., and Kenyon, G. L. (2002) Bioorg. Chem. 30, 443-458). We have investigated the mechanism by which KM-1 inhibits wild-type human immunodeficiency virus type 1 RT by using pre-steady state kinetic methods to examine the effect of KM-1 on the parameters governing the single nucleotide incorporation catalyzed by RT. Analysis of the pre-steady-state burst phase of dATP incorporation showed that KM-1 decreased the amplitude of the reaction as previously shown for other NNRTIs, because of the slow equilibration of the inhibitor with RT. In the ternary enzyme-DNA-KM-1 complex (E-DNA-I), incorporation of the next nucleotide onto the primer is blocked. However, unlike conventional NNRTIs, the inhibitory effect was caused primarily by weakening the DNA binding affinity and displacing DNA from the enzyme. Wild-type RT binds a 25/45-mer DNA duplex with an apparent K(d) of 3 nm, which was increased to 400 nm upon saturation with KM-1. Likewise, the apparent K(d) for KM-1 binding to RT increased at higher DNA concentrations. We therefore conclude that KM-1 represents a new class of inhibitor distinct from nevirapine and related NNRTIs. KM-1 can bind to RT in both the absence and presence of DNA but weakens the affinity for DNA 140-fold so that it favors DNA dissociation. The data suggest that KM-1 distorts RT conformation and misaligns DNA at the active site.
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PMID:Novel mechanism of inhibition of HIV-1 reverse transcriptase by a new non-nucleoside analog, KM-1. 1523 30

We have investigated anaerobic respiration of the archaeal model organism Halobacterium sp. strain NRC-1 by using phenotypic and genetic analysis, bioinformatics, and transcriptome analysis. NRC-1 was found to grow on either dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO) as the sole terminal electron acceptor, with a doubling time of 1 day. An operon, dmsREABCD, encoding a putative regulatory protein, DmsR, a molybdopterin oxidoreductase of the DMSO reductase family (DmsEABC), and a molecular chaperone (DmsD) was identified by bioinformatics and confirmed as a transcriptional unit by reverse transcriptase PCR analysis. dmsR, dmsA, and dmsD in-frame deletion mutants were individually constructed. Phenotypic analysis demonstrated that dmsR, dmsA, and dmsD are required for anaerobic respiration on DMSO and TMAO. The requirement for dmsR, whose predicted product contains a DNA-binding domain similar to that of the Bat family of activators (COG3413), indicated that it functions as an activator. A cysteine-rich domain was found in the dmsR gene, which may be involved in oxygen sensing. Microarray analysis using a whole-genome 60-mer oligonucleotide array showed that the dms operon is induced during anaerobic respiration. Comparison of dmsR+ and DeltadmsR strains by use of microarrays showed that the induction of the dmsEABCD operon is dependent on a functional dmsR gene, consistent with its action as a transcriptional activator. Our results clearly establish the genes required for anaerobic respiration using DMSO and TMAO in an archaeon for the first time.
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PMID:Genomic analysis of anaerobic respiration in the archaeon Halobacterium sp. strain NRC-1: dimethyl sulfoxide and trimethylamine N-oxide as terminal electron acceptors. 1571 36

We have previously reported the potent in vitro HIV-1 anti-reverse transcriptase activity of a 35-mer of 4-thio-deoxyuridylate [(s(4)dU)(35)]. In efforts to define its activity in a more physiological system, studies were carried out to determine the stage of viral infection that this compound mediates its anti-viral effect. Results of the studies reported herein show that (s(4)dU)(35) is nontoxic and is capable of inhibiting both single and multi-drug resistant HIV strains (IC(50): 0.8-25.4 microg/ml) in vitro. Besides its previously reported anti-RT activity, (s(4)dU)(35) mediated its antiviral action by preventing virus attachment (IC(50): 0.002-0.003 microg/ml), and was stable in vitro and slowly degraded by DNAses. Competition studies and fluorescence resonance energy transfer (FRET) experiments indicated that (s(4)dU)(35) preferentially binds to CD4 receptors, but not to CD48. Confocal laser scanning microscopy (CLSM) studies showed that (s(4)dU)(35) did not penetrate into the cells and colocalized with cell surface thioredoxin. Our studies identify (s(4)dU)(35) as a potential novel HIV entry inhibitor that may have utility as either a systemic antiretroviral or as a preventing agent for HIV transmission.
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PMID:Potent inhibition of HIV-1 entry by (s4dU)35. 1578 Aug 71

A fluorescence polarization (FP) microplate assay suitable for screening compounds against the ribonuclease H (RNase H) activity of HIV-1 reverse transcriptase has been developed. This homogeneous assay uses a hybrid 18-mer DNA/RNA duplex substrate composed of an RNA oligonucleotide labeled with 6-carboxytetramethyl rhodamine at the 3' end that is annealed to a complementary unlabeled DNA strand. The labeled RNA/DNA duplex demonstrated Michaelis-Menten kinetics with a Km value of 9.6+/-2.8 nM. Substrate cleavage by RNase H to produce small RNA fragments (1-4 mer) resulted in a large change in the measured FP value. This FP assay was amenable to kinetics protocols as well as stopped endpoint measurements. When using the latter for conducting robotics runs, Z' values greater than 0.8 typically were observed. The stopped endpoint FP assay was used successfully in a high-throughput screening campaign to screen 1.8 million compounds for RNase H inhibition.
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PMID:A fluorescence polarization assay for screening inhibitors against the ribonuclease H activity of HIV-1 reverse transcriptase. 1652 35


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