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:3.1.26.4 (RNase H)
2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

As human immunodeficiency virus type 1 (HIV-1) has become better understood, numerous drugs have been developed that act at virus-specific sites. These are challenging our ability to evaluate them thoroughly and rapidly. Zidovudine (AZT) remains the mainstay of anti-HIV-1 drugs. Recent controlled trials indicate it should be used early in infection (in those with CD4 cell counts less than 500/mm3) and in lower doses (500-600 mg/day). Prolonged AZT treatment in patients with AIDS, however, is often associated with viral resistance. Newer reverse transcriptase-inhibiting nucleoside derivatives are currently in phase II-III clinical trials. Other HIV-1 replicative sites under attack in clinical studies include binding and entry of virus, envelope protein glycosylation, and viral assembly and release. Agents that target HIV-1 proteinase, integrase, ribonuclease H, and products of regulatory genes such as tat are under development. Combination therapies that target different viral replicative sites likely will allow use of individual agents below their toxic concentrations and help prevent drug resistance. Innovative programs for expanded access to experimental drugs are needed that will permit expeditious clinical trials, optimize the gathering of useful information, and permit the widest access to promising treatments.
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PMID:Chemotherapy of human immunodeficiency virus infections: current practice and future prospects. 169 Dec 43

The effects of AZTMP and other nucleoside 5'-monophosphates on the RNA-dependent DNA polymerase and RNase H activities of a recombinant HIV reverse transcriptase have been investigated. Both activities are sensitive to inhibition by millimolar concentrations of AZTMP with MgCl2 as divalent cation activator. Substitution of Mn2+ for Mg2+ markedly potentiates the inhibition of RNase H activity by AZTMP, reducing the IC50 from 5 to 0.05 mM. In contrast, Mn2+ does not alter the sensitivity of the RNA-dependent DNA polymerase activity to inhibition by AZTMP. The inhibition of RNase H activity by AZTMP can be reversed by increasing concentrations of the substrate poly(A)/poly(dT), suggesting that AZTMP may compete with the substrate for binding at the active site of RNase H. Other nucleoside 5'-monophosphates do not inhibit RNase H in the presence of Mg2+. However, in the presence of Mn2+, deoxy- and dideoxynucleoside 5'-monophosphates that are complementary to the DNA strand of the heteroduplex substrate are somewhat inhibitory. The RNA-dependent DNA polymerase activity is a slightly inhibited by AZTMP and ddTMP in either Mg2+ or Mn2+, and substitution of Mn2+ for Mg2+ results in inhibition by ddAMP as well. Naturally occurring ribo- or deoxyribonucleoside 5'-monophosphates are not inhibitory at concentrations up to 5 mM. Since AZTTP inhibits the RNA-dependent DNA polymerase activity of HIV reverse transcriptase at nanomolar concentrations, it is unlikely that the inhibition of this activity by AZTMP plays a significant role in the antiviral effect of AZT. However, the inhibition of the RNase H activity by AZTMP, which can reach millimolar concentrations in vivo, may account for part of the sensitivity of the virus to AZT.
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PMID:Inhibition of the RNase H activity of HIV reverse transcriptase by azidothymidylate. 170 9

Poly(1-methyl-6-thioinosinic acid), or PMTI, is a single-stranded polyribonucleotide and is the first homopolyribonucleotide devoid of Watson-Crick hydrogen bonding sites to show potent human immunodeficiency virus (HIV) inhibition. PMTI was found to be active when evaluated against a variety of low passage clinical HIV isolates in fresh human peripheral blood cells, including T cell-tropic and monocyte-macrophage-tropic viruses, syncytium-inducing and non-syncytium-inducing viruses and viruses representative of the various HIV-1 clades (A through F). The compound was active against HIV-2, all nucleoside and non-nucleoside reverse transcriptase (RT) inhibitor drug-resistant virus isolates tested and interacted with AZT or ddl to synergistically inhibit HIV infection. In biochemical inhibition assays, PMTI was determined to be a potent inhibitor of HIV-1 and HIV-2 RT, including RTs with mutations that engender resistance to nucleoside and non-nucleoside RT inhibitors. PMTI inhibited both the polymerase and RNase H activities of HIV RT. PMTI did not inhibit HIV-1 protease or integrase. Cell-based mechanism of action assays indicated that PMTI also interfered with early events in the entry of HIV into target cells. Furthermore, PMTI inhibited the fusion of gp120-expressing and CD4-expressing cells, but at concentrations approximately 1 log10 greater than those that inhibited virus entry. These results suggest that the homopolyribonucleotide PMTI blocks HIV replication in human cells at its earliest stages by multiple mechanisms, inhibition of virus entry and inhibition of RT.
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PMID:PMTI, a broadly active unusual single-stranded polyribonucleotide, inhibits human immunodeficiency virus replication by multiple mechanisms. 1007 76

Resistance to zidovudine (3'-azido-3'-deoxythymidine, AZT) by the human immunodeficiency virus, type 1, requires multiple amino acid substitutions such as D67N/K70R/T215F/K219Q in the viral reverse transcriptase (RT). In this background of AZT resistance, additional "suppressive" substitutions such as Y181C restore sensitivity to AZT. In order to characterize the mechanism of this AZT resistance suppression, the Y181C substitution was introduced into both wild-type and AZT-resistant reverse transcriptase. The introduction of the Y181C substitution suppresses the increased repair (or unblocking) of the AZTMP-terminated primer provided by the AZT resistance substitutions in RT using either DNA or RNA templates, independently from the RT RNase H activity. Contrary to wild-type RT, the low level of unblocking activity is not due to inhibition by the next correct nucleotide binding to the RT/AZTMP-terminated primer complex. When Y181C is added to the AZT resistance substitutions, ATP binds with less affinity to the AZTMP-terminated primer-RT binary complex. These results provide an insight into one possible molecular mechanism of re-sensitization of AZT-resistant viruses by suppressive substitutions.
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PMID:The Y181C substitution in 3'-azido-3'-deoxythymidine-resistant human immunodeficiency virus, type 1, reverse transcriptase suppresses the ATP-mediated repair of the 3'-azido-3'-deoxythymidine 5'-monophosphate-terminated primer. 1290 45

Despite the key role played by the RNase H of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) in viral proliferation, only a few inhibitors of RNase H have been reported. Using in vitro combinatorial selection methods and the RNase H domain of the HIV RT, we have selected double-stranded DNA thioaptamers (aptamers with selected thiophosphate backbone substitutions) that inhibit RNase H activity and viral replication. The selected thioaptamer sequences had a very high proportion of G residues. The consensus sequence for the selected thioaptamers showed G clusters separated by single residues at the 5'-end of the sequence. Gel electrophoresis mobility shift assays and nuclear magnetic resonance spectroscopy showed that the selected thioaptamer binds to the isolated RNase H domain, but did not bind to a structurally similar RNase H from Escherichia coli. The lead thioaptamer, R12-2, showed specific binding to HIV-1 RT with a binding constant (K(d)) of 70 nM. The thioaptamer inhibited the RNase H activity of intact HIV-1 RT. In cell culture, transfection of thioaptamer R12-2 (0.5 microg/mL) markedly inhibited viral production and exhibited a dose response of inhibition with R12-2 concentrations ranging from 0.03 to 2.0 microg/mL (IC(50) < 100 nM). Inhibition was also seen across a wide range of virus inoculum, ranging from a multiplicity of infection (moi) of 0.0005 to 0.05, with a reduction of the level of virus production by more than 50% at high moi. Suppression of virus was comparable to that seen with AZT when moi <or= 0.005.
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PMID:Combinatorial selection, inhibition, and antiviral activity of DNA thioaptamers targeting the RNase H domain of HIV-1 reverse transcriptase. 1604 16

Metal ions are essential for DNA polymerase and RNase H activities of HIV-1 reverse transcriptase (RT). RT studies are routinely performed at 6-8 mM Mg2+, despite the fact that the in vivo concentration might be as low as 0.2 mM. We studied the influence of MgCl2 and ATP, which likely binds a significant fraction of the magnesium pool in vivo, on the DNA polymerase and RNase H activities of HIV-1 RT, its inhibition by nucleoside RT inhibitors (NRTIs) and primer unblocking by AZT-resistant RT. At low Mg2+ concentration, reverse transcription of a natural template strongly increased despite a dramatically reduced intrinsic polymerase activity under such conditions. Low Mg2+ concentrations affected the RNA stability and indirectly decreased its degradation by the RNase H activity. The reduced RNA degradation prevented premature dissociation of the template and primer strands that otherwise generated dead-end DNA products. In addition, low Mg2+ dramatically decreased the incorporation of NRTIs into DNA and increased nucleotide excision by AZT-resistant RT. The latter effect is also most likely owing to the diminished cleavage of the RNA template. Thus, differences in the free Mg2+ concentration between different cell types or during the cell cycle might strongly affect HIV-1 replication and its inhibition.
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PMID:Mg2+ dependency of HIV-1 reverse transcription, inhibition by nucleoside analogues and resistance. 1639 22

Inhibitors of the excision reaction catalysed by HIV-1 RT (reverse transcriptase) represent a promising approach in the fight against HIV, because these molecules would interfere with the main mechanism of resistance of this enzyme towards chain-terminating nucleotides. Only a limited number of compounds have been demonstrated to inhibit this reaction to date, including NNRTIs (non-nucleoside RT inhibitors) and certain pyrophosphate analogues. We have found previously that 2GP (2-O-galloylpunicalin), an antiviral compound extracted from the leaves of Terminalia triflora, was able to inhibit both the RT and the RNase H activities of HIV-1 RT without affecting cell proliferation or viability. In the present study, we show that 2GP also inhibited the ATP- and PP(i)-dependent phosphorolysis catalysed by wild-type and AZT (3'-azido-3'-deoxythymidine)-resistant enzymes at sub-micromolar concentrations. Kinetic and direct-binding analysis showed that 2GP was a non-competitive inhibitor against the nucleotide substrate, whereas it competed with the binding of RT to the template-primer (K(d)=85 nM). As expected from its mechanism of action, 2GP was active against mutations conferring resistance to NNRTIs and AZT. The combination of AZT with 2GP was highly synergistic when tested in the presence of pyrophosphate, indicating that the inhibition of RT-catalysed phosphorolysis was responsible for the synergy found. Although other RT inhibitors that compete with the template-primer have been described, this is the first demonstration that these compounds can be used to block the excision of chain terminating nucleotides, providing a rationale for their combination with nucleoside analogues.
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PMID:A new strategy to inhibit the excision reaction catalysed by HIV-1 reverse transcriptase: compounds that compete with the template-primer. 1735 25

Recent studies have identified a role for mutations in the connection and RNase H domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) resistance to nucleoside analog RT inhibitors (NRTI). To provide insight into the biochemical mechanism(s) involved, we investigated the effect of the G333D mutation in the connection domain of RT on resistance to zidovudine (AZT) and lamivudine (3TC) in enzymes that contain both M184V and thymidine analog mutations (TAMs; M41L, L210W, and T215Y). Our results from steady-state kinetic, pre-steady-state kinetic, and thermodynamic analyses indicate that G333D facilitates dual resistance to AZT and 3TC in two ways. First, in combination with M184V, G333D increased the ability of HIV-1 RT to effectively discriminate between the normal substrate dCTP and 3TC-triphosphate. Second, G333D enhanced the ability of RT containing TAMs and M184V to bind template/primer terminated by AZT-monophosphate (AZT-MP), thereby restoring ATP-mediated excision of AZT-MP under steady-state assay conditions. This study is the first to elucidate a molecular mechanism whereby a mutation in the connection domain of RT can affect NRTI susceptibility at the enzyme level.
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PMID:Mechanisms by which the G333D mutation in human immunodeficiency virus type 1 Reverse transcriptase facilitates dual resistance to zidovudine and lamivudine. 1796 7

Previous biochemical studies have demonstrated that synergy between non-nucleoside reverse transcriptase (RT) inhibitors (NNRTI) and nucleoside RT inhibitors (NRTIs) is due to inhibition by the NNRTI of the rate at which HIV-1 RT facilitates ATP-mediated excision of NRTIs from chain-terminated template/primers (T/P). However, these studies did not take into account the possible effects of NNRTI on the ribonuclease H (RNase H) activity of RT, despite recent evidence that suggests an important role for this activity in the NRTI excision phenotype. Accordingly, in this study, we compared the ability of efavirenz to inhibit the incorporation and excision of zidovudine (AZT) by HIV-1 RT using DNA/DNA and RNA/DNA T/Ps that were identical in sequence. Whereas IC(50) values for the inhibition of AZT-triphosphate incorporation by efavirenz were essentially similar for both DNA/DNA and RNA/DNA T/P, a 19-fold difference in IC(50) was observed between the AZT-monophosphate excision reactions, the RNA/DNA T/P substrate being significantly more sensitive to inhibition. Analysis of the RNase H cleavage events generated during ATP-mediated excision reactions demonstrated that efavirenz dramatically increased the rate of appearance of a secondary cleavage product that decreased the T/P duplex length to only 10 nucleotides. Studies designed to delineate the relationship between T/P duplex length and efficiency of AZT excision demonstrated that RT could not efficiently unblock chain-terminated T/P if the RNA/DNA duplex length was less than 12 nucleotides. Taken together, these results highlight an important role for RNase H activity in the NRTI excision phenotype and in the mechanism of synergy between NNRTI and NRTI.
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PMID:Efavirenz accelerates HIV-1 reverse transcriptase ribonuclease H cleavage, leading to diminished zidovudine excision. 1802 10

We previously proposed that mutations in the connection subdomain (cn) of HIV-1 reverse transcriptase increase AZT resistance by altering the balance between nucleotide excision and template RNA degradation. To test the predictions of this model, we analyzed the effects of previously identified cn mutations in combination with thymidine analog mutations (D67N, K70R, T215Y, and K219Q) on in vitro RNase H activity and AZT monophosphate (AZTMP) excision. We found that cn mutations G335C/D, N348I, A360I/V, V365I, and A376S decreased primary and secondary RNase H cleavages. The patient-derived cns increased ATP- and PPi-mediated AZTMP excision on an RNA template compared with a DNA template. One of 5 cns caused an increase in ATP-mediated AZTMP excision on a DNA template, whereas three cns showed a higher ratio of ATP- to PPi-mediated excision, indicating that some cn mutations also affect excision on a DNA substrate. Overall, the results strongly support the model that cn mutations increase AZT resistance by reducing template RNA degradation, thereby providing additional time for RT to excise AZTMP.
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PMID:HIV-1 reverse transcriptase connection subdomain mutations reduce template RNA degradation and enhance AZT excision. 1866 7


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