UMLS:C0019693 - FACTA Search

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Query: UMLS:C0019693 (HIV)
170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of hydroxyurea (HU), an inhibitor of ribonucleotide reductase, on the replication of human immunodeficiency virus type 1 (HIV-1) in activated peripheral blood mononuclear cells were studied. The inhibition of HIV-1 replication by HU alone was dose dependent, with a 90% inhibitory concentration of 0.4 mM, a plasma concentration tolerated by patients with oncological diseases. HU at lower concentrations (< 0.1 mM) was found to potentiate the antiviral activity of 2',3'-dideoxyinosine (ddl), 3'-azido-2',3'- dideoxythymidine, and 2',3'-dideoxycytidine against HIV-1, with the potentiation being ddl greater than 3'-azido-2',3'- dideoxythymidine = 2',3'-dideoxycytidine. In the presence of 0.1 mM HU, the 90% inhibitory concentration of ddl was reduced by 6-fold in activated peripheral blood mononuclear cells. The potentiating effect of HU on ddl action was time dependent, with the greatest inhibition of HIV-1 growth being seen when HU was present during and after virus adsorption, i.e., apparently coinciding with the time of proviral DNA synthesis. A brief incubation of activated cells with HU and ddl at low concentrations before virus exposure reduced p24 production by > 50%. Analyses using high performance liquid chromatography and enzymatic assays suggested that the greater degree of potentiation by HU of the action of ddl, compared with the other dideoxynucleosides, is due to the more effective inhibition by HU of dATP synthesis, compared with the synthesis of the other deoxynucleoside triphosphates (dGTP, dTTP, and dCTP). The present study suggests that, for appropriate agents, pharmacological reduction of deoxynucleoside triphosphate levels represents a potential therapeutic approach for inhibition of HIV-1 replication.
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PMID:Anti-human immunodeficiency virus type 1 activity of hydroxyurea in combination with 2',3'-dideoxynucleosides. 796 58

A new class of very potent and selective non-nucleoside inhibitors of HIV reverse transcriptase (RT) has recently been identified. The prototype compound trovirdine (LY 300046 HCl) and one analogue, MSC-127, have been studied with respect to inhibition of wild-type HIV-1 RT and RT with various mutations known to give rise to resistance to other non-nucleoside RT inhibitors, namely Leu100-->Ile (Ile100), Glu138-->Arg (Arg138), Tyr181-->Cys (Cys181) and Tyr188-->His (His188). The inhibition of HIV-1 RT by trovirdine and MSC-127 was reversible and template dependent. Trovirdine inhibited HIV-1 RT with an IC50 of 0.007 microM when employing heteropolymeric primer/template (oligo-DNA/ribosomal RNA) and dGTP as substrate. Enzyme kinetic studies showed that inhibition of RT by trovirdine was non-competitive with regard to deoxynucleoside triphosphates and uncompetitive with respect to varied primer/template under steady-state conditions. The amino acid changes Leu100, Tyr181 and Tyr188 gave rise to 25-, 147- and 12-fold decrease in inhibition by trovirdine. Enzyme-kinetic studies on trovirdine have been carried out using various RT mutants and compared to the properties of the earlier reported non-nucleoside RT inhibitors 9-Cl-TIBO, nevirapine and L-697,661.
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PMID:Inhibition of human immunodeficiency virus type 1 wild-type and mutant reverse transcriptases by the phenyl ethyl thiazolyl thiourea derivatives trovirdine and MSC-127. 866 92

In the search for effective antiviral agents, we have found 4-(2, 6-dichlorophenyl)-1, 2, 5-thiadiazol-3-yl-N-methyl, N-ethylcarbamate (RD4-2025) to be a highly potent and selective inhibitor of human immunodeficiency virus type 1 (HIV-1) in vitro. The 50% effective concentration of RD4-2025 for HIV-1-induced cytopathic effect in MT-4 cells was 37 nM, yet no antiviral activity was observed against HIV-2. In HIV-1 reverse transcriptase (RT) assays, RD4-2025 inhibited both RNA-dependent and DNA-dependent DNA polymerase activities of a recombinant HIV-1 RT with 50% inhibitory concentrations of 0.11 and 3.5 microM, respectively. However, the compound did not affect the activity of human DNA polymerase alpha. Kinetic studies revealed that the inhibition was noncompetitive with respect to dGTP as the substrate and poly(C)/(dG) 12-18 as the template/primer. These results were in accordance with those of nonnucleoside RT inhibitors (NNRTIs), such as R89439 (an alpha-anilinophenylacetamide derivative) and nevirapine, indicating that RD4-2025 also belongs to the family of NNRTIs.
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PMID:Inhibitory effect of 4-(2, 6-dichlorophenyl)-1, 2, 5-thiadiazol-3-yl-N-methyl, N-ethylcarbamate on replication of human immunodeficiency virus type 1 and the mechanism of action. 879 26

Four 25-mer oligonucleotides containing m4T(T*) with the 3'-->5' sequences A-T*-A, A-T*-C, G-T*-G and G-T*-C, together with four oligonucleotides containing the same sequences with unmodified T, were studied for the effect of the immediate 5' and 3' bases flanking m4T on in vitro replication. Under enzyme-limiting conditions, all m4T-containing oligomers showed a varying sequence-dependent blockage at the base 3' to m4T. However, with time and high concentrations of dNTP's, full replication beyond m4T did occur with Klenow fragment (Kf), although substantial blockage remained, particularly in the case when the sequence was 3'-A-T*-A-5'. The extent of blockage at the base 3' to m4T, compared to complete replication using Kf, followed the order A-T*-A > A-T*-C >> G-T*-C > or = G-T*-G. Two other DNA polymerases, HIV-RT and Sequenase, which, unlike Kf, do not have 3' proofreading activity differ, agreed with data obtained with Kf. We conclude that the extent of complete replication of m4T-containing oligomers is primarily a function of the 3' flanking base with a lesser effect of the 5' base and requires specific dGTP insertion opposite m4T. These data suggest that a 5' G . C pair favors the formation of the following T*-G and this latter step is rate-limiting. However, once a T*-G pair is formed, rapid elongation to full length transcripts occurs.
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PMID:Replication of O4-methylthymine-containing oligonucleotides: effect of 3' and 5' flanking bases on formation and extension of O4-methylthymine . guanine basepairs. 884 83

The enzyme dUTP pyrophosphatase (dUTPase, EC 3.6.1.23) is essential for cellular DNA replication and cell viability by virtue of its role in reducing the availability of dUTP as a substrate for DNA polymerases. Several members of the onco- and lentivirus families of retroviruses encode dUTPases and mutant strains of these viruses defective in this enzyme exhibit suboptimal replication kinetics. Among the lentiviruses there exists a surprising phylogenetic discontinuity in the distribution of dUTPase genes: non-primate viruses (EIAV, CAEV, FIV, visna) contain such genes whereas the primate viruses (HIVs, SIVs) do not. The reason for this difference is unknown. We suggest the following explanation: (1) the nuclear and mitochondrial compartmentalization of the mammalian dUTPase, combined with the cytoplasmic location of ribonucleotide reductase, leads to the net synthesis of dUTP, together with dCTP, dGTP and dATP in the cytoplasm; (2) this combination of dNTPs serves as a "toxic cocktail" for viral replication by virtue of its ability to promote the synthesis of uracil-substituted DNA; (3) many viruses have adapted to this challenge by encoding dUTPases that are free of normal cellular regulatory constraints; and (4) the fortuitous expression of a dUTPase encoded by one or more human endogenous retroviruses (HERVs) has led to the evolutionary loss of the putative ancestral dUTPase gene of primate lentiviruses. Thus, we propose that efficient replication of HIV in humans depends upon expression of a dUTPase encoded by an endogenous retrovirus. If this proposal is correct, then the entry of HIV into target cells is necessary, but not sufficient, for replication of the virus in humans.
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PMID:HIV and human endogenous retroviruses: an hypothesis with therapeutic implications. 910 94

Nondenaturing gel electrophoresis was used to study the nucleotide substrate-induced conformational change in reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). Dead-end complex was formed between HIV-1 RT, dideoxynucleotide chain-terminated primer, and DNA template in the presence of deoxynucleotide triphosphate (dNTP) complementary to the next position on the template. Complexes which form in the absence of the next complementary dNTP were disrupted by adding excess poly(rA)/oligo(dT) or heparin just prior to electrophoresis. Dead-end complex formation by noncomplementary dNTP's or ribonucleotides was at least 2000-fold less efficient than with the complementary nucleotide. When dA was the next nucleotide on the template, analogues of dTTP supported dead-end complex formation with increased apparent Kd (dTTP < dideoxy-TTP approximately alpha-thio-dTTP < dUTP < 3'-azidothymidine triphosphate). A similar relationship was observed for dGTP analogues across from dC on the template (dGTP < dideoxy-GTP < alpha-thio-dGTP << dITP < dideoxy-ITP). The optimal length of the primer/template duplex region for dead-end complex formation was between 20 and 32 base pairs. Primer-template with a mismatched primer terminus did not support dead-end complex formation, and primer terminated with 3'-azidothymidine formed dead-end complex with 25-fold elevated apparent Kd. By contrast, dead-end complex formation on primer terminated with dideoxy-IMP base paired with dC on the template was more efficient than on primer terminated with dideoxy-GMP. Implications for the mechanisms of discrimination between nucleotide analogues by HIV-1 RT are discussed.
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PMID:Nucleotide-induced stable complex formation by HIV-1 reverse transcriptase. 915 15

The RNA dependent DNA replication fidelity of HIV-1 reverse transcriptase has been investigated using pre-steady-state kinetics under single turnover conditions. In contrast to previous estimates of low replication fidelity of HIV-1 reverse transcriptase, the present study finds the enzyme to be more highly discriminating when an RNA/DNA template-primer is employed as compared with the corresponding DNA/DNA template-primer. The basis of this selectivity is due to extremely slow polymerization kinetics for incorporation of an incorrect deoxynucleotide. The maximum rates for misincorporation (kpol) of dGTP, dCTP, and dTTP opposite a template uridine were 0.2, 0.03, and 0.003 s-1, respectively. The equilibrium dissociation constants (Kd) for the incorrect nucleotide opposite a template uridine were 1.0, 1.1, and 0.7 mM for dGTP, dCTP, and dTTP, respectively. These kinetic values provide fidelity estimates of 26 000 for discrimination against dGTP, 176 000 for dCTP, and 1 x 10(6) for dTTP misincorporation at this position. Similar observations were obtained when incorrect nucleotide misincorporation was examined opposite a template adenine. Thus in a direct comparison of RNA/DNA and DNA/DNA template-primer substrates, HIV-1 RT exhibits approximately a 10-60-fold increase in fidelity. This study augments our current understanding of the similarities and differences of catalytic activity of HIV-1 reverse transcriptase using RNA and DNA substrates. Moreover, these studies lend further support for a model for nucleotide incorporation by HIV-1 reverse transcriptase involving a two-step binding mechanism governed by a rate-limiting conformational change for correct incorporation.
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PMID:RNA dependent DNA replication fidelity of HIV-1 reverse transcriptase: evidence of discrimination between DNA and RNA substrates. 936 77

The ribonucleotide reductase inhibitor hydroxyurea exhibits potent synergism, even at low, non-cytotoxic concentrations, with the anti-HIV-1 dideoxynucleoside 2',3'-dideoxyinosine, bringing about failure of HIV DNA synthesis and, thus, of HIV replication. To elucidate the incompletely defined role of hydroxyurea in the hydroxyurea/dideoxyinosine interaction and, in particular, to identify the reasons for the unusual selective inhibitory action of the combination on retroviral rather than on cellular DNA synthesis, we prepared specific cDNA probes to determine the effects of low-level hydroxyurea on mammalian cell ribonucleotide reductase M1 and M2 subunit mRNA, while simultaneously quantitating the effects of the drug on cell cycle and on deoxynucleoside triphosphate pools. While dTTP, dCTP, and dGTP pools changed little or even increased in the presence of low-level hydroxyurea, there took place a rapid and specific inhibition of M2-subunit-catalyzed generation of dATP, with consequent slowing of cellular DNA synthesis and prolongation of S phase. However, the latter effect, in turn, resulted in increased M2 subunit mRNA transcription (a process blocked in Go/G1-phase cells, with full-length functional M2 transcripts being generated only during S phase) and, hence, in a return to normal levels of dATP and to a normal rate of cellular DNA synthesis. Because of this self-regulating mechanism, hydroxyurea-induced host-cell toxicity was obviated under conditions where HIV DNA synthesis, a process sensitive to both dATP depletion and the chain-terminating properties of the other inhibitory component of the combination (ddATP derived from dideoxyinosine), was unable to recover.
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PMID:Role of the M2 subunit of ribonucleotide reductase in regulation by hydroxyurea of the activity of the anti-HIV-1 agent 2',3'-dideoxyinosine. 969 94

The kinetics of 8-oxo-7,8-dihydroguanosine triphosphate (8-oxo-dGTP) incorporation into DNA by Escherichia coli polymerases I exo- (KF-) and II exo- (Pol II-), HIV-1 RT reverse transcriptase (HIV-1 RT), and bacteriophage T7 exo- (T7(-)) were examined to determine the misincorporation potential for 8-oxo-dGTP and to investigate the role of base pairing symmetry in DNA polymerase fidelity. 8-Oxo-dGTP was found to be a poor substrate for the four polymerases, with insertion efficiencies >10(4)-fold lower than for dGTP incorporation. Insertion efficiencies of 8-oxo-dGTP were also consistently lower than for incorporation of dNTPs opposite template 8-oxo-G, previously studied in this laboratory. In steady-state reactions, T7(-) had a high preference for 8-oxo-dGTP insertion opposite A (97%) and HIV-1 RT, KF-, and Pol II- preferred to insert 8-oxo-dGTP opposite C. Misinsertion frequencies for 8-oxo-dGTP also varied considerably from frequencies of misinsertion at template 8-oxo-G adducts for Pol II-, HIV-1 RT, and T7(-). Pre-steady-state incorporation of 8-oxo-dGTP opposite C (but not opposite A) by HIV-1 RT, KF-, and Pol II- displayed biphasic curves, with rates of initial incorporation 2- to 11-fold lower than normal dGTP incorporation. Although extension past template 8-oxo-G adducts had previously been shown to occur preferentially for the mispair, extension past primer 8-oxo-G:template A or C pairs was variable. The low and comparable estimated Kd values for dGTP and 8-oxo-dGTP binding to HIV-1 RT alone or HIV-1 RT.DNA complexes indicated that the initial binding was nonselective and had high affinity. The large difference (>3 orders of magnitude) in kinetic Kdapp values for 8-oxo-dGTP and dGTP binding to HIV-1 RT.DNA indicates that there are contributions to the kinetically determined Kdapp (such as conformational change and/or phosphodiester bond formation) which may be involved in the selection against 8-oxo-dGTP. The differences in binding (Kdapp), incorporation, and extension kinetics of 8-oxo-dGTP compared to normal dNTP incorporation at template 8-oxo-G adducts indicate that polymerase fidelity does not depend solely upon the overall geometry of Watson-Crick base pairs and reflects the asymmetry of the enzyme active site.
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PMID:Steady-state and pre-steady-state kinetic analysis of 8-oxo-7,8-dihydroguanosine triphosphate incorporation and extension by replicative and repair DNA polymerases. 974 38

We have used 8-O-dGTP, a mutagenic nucleotide generated by oxidative metabolism, to probe the misincorporation potential of HIV-1 reverse transcriptase (RT) during DNA synthesis templated by the same nucleotide sequence as either RNA or DNA. With either template, 8-O-dGMP was misincorporated opposite template A, yielding characteristic A-->C transversions. The error rate with DNA was similar to that with RNA, suggesting that base misincorporation by the RT during first-strand and second-strand replication may contribute equally to the HIV-1 base substitution mutation rate. The rate of 8-O-dGMP misincorporation differed by more than 10-fold among the 20 adenines in the M13mp2 template where A-->C transversions can be detected. The transversion distribution was similar with the two templates, indicating that the effects of flanking nucleotides on misincorporation rates were similar. This is consistent with structural and biochemical data suggesting that HIV-1 RT binds RNA x DNA and DNA x DNA template-primers in the same orientation. The similarities in error rates and distribution further indicate that, despite differences in the structures of free RNA x DNA and DNA x DNA duplexes (e.g., minor groove dimensions), the polymerase active site that assembles upon substrate binding establishes a similar degree of nucleotide selectivity with both types of template-primers. Comparison of the RT error distribution to that observed with two Pol I family DNA polymerases and a Pol alpha family polymerase revealed common hot and cold spots for misincorporation. This suggests that the local nucleotide sequence influences the nucleotide selectivity of four polymerases in a similar manner, despite their differences in structure, biochemical properties, and functions.
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PMID:The base substitution fidelity of HIV-1 reverse transcriptase on DNA and RNA templates probed with 8-oxo-deoxyguanosine triphosphate. 1052

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