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)

To design combination strategies for chronic hepatitis B therapy, we evaluated in vitro the inhibitory activity of 4 nucleoside analogs, (-)FTC, L-FMAU, DXG, and DAPD, in comparison with lamivudine (3TC) and PMEA. In a cell-free assay for the expression of wild-type duck hepatitis B virus (DHBV) reverse transcriptase, DAPD-TP was found to be the most active on viral minus strand DNA synthesis, including the priming reaction, followed by 3TC-TP, (-)FTC-TP, and DXG-TP, whereas L-FMAU-TP was a weak inhibitor. In cell culture experiments, important differences in drug concentration allowing a 50% inhibition of viral replication or polymerase activity (IC50s) were observed depending on the cell type used, showing that antiviral effect of nucleoside analogs may depend on their intracellular metabolism. IC50s obtained for wild-type DHBV replication in primary duck hepatocytes were much lower than with DHBV transfected LMH cells. IC50s were also significantly lower in the 2.2.1.5 and HepG2 cells compared with HBV transfected HuH7 cells. Moreover, L-FMAU inhibited preferentially HBV plus strand DNA synthesis in these cell lines. The antiviral effect of these inhibitors was also evaluated against 3TC-resistant mutants of the DHBV and HBV polymerases. These mutants were found to be cross resistant to (-)FTC. By contrast, the double DHBV polymerase mutant was sensitive to DXG-TP and DAPD-TP. Moreover, both purine analogs remained active against DHBV and HBV 3TC-resistant mutants in transfected LMH and HepG2 cells, respectively. In conclusion, the unique mechanism of action of these new inhibitors warrants further evaluation in experimental models to determine their capacity to delay or prevent the selection of drug resistant mutants.
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PMID:Inhibitory activity of dioxolane purine analogs on wild-type and lamivudine-resistant mutants of hepadnaviruses. 1219 65

Because prevention of heterosexual HIV transmission is not always possible, it is important to develop effective strategies of postexposure prophylaxis (PEP). Since in vivo comparison of drug potency is difficult, we developed an in vitro model with cells resembling primary targets during sexual transmission: monocyte-derived dendritic cells (MO-DCs), Langerhans cells (MO-LCs), and resting autologous CD4(+) T cells. Nucleoside and nonnucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs, respectively) were evaluated for their antiviral activity, when added immediately after infection or at a later time point. In parallel, their immune-suppressive effect was examined by measuring inhibition of mixed MO-DC/allogeneic CD4(+) T cell cultures. Most RTIs potently inhibited HIV replication, even if added 24 hr after infection (representing PEP). The sensitivity to antiretroviral drugs was similar in HIV-infected MO-DCs and MO-LCs, but decreased in cocultures with resting autologous CD4(+) T cells. The NNRTIs efavirenz and UC-781 as well as the NRTIs AZT, 3TC, and d4T showed a similar high potency in MO-DC plus autologous CD4(+) T cell cocultures as compared with CEM T cells, whereas their activity in phytohemagglutinin/interleukin 2 (PHA/IL-2)-activated CD4(+) T cells was lower. The dideoxynucleoside RTI abacavir as well as the phosphonates (R)-PMPA and PMEA were more active in infected MO-DCs as compared with either CEM T cells or PHA/IL-2 activated CD4(+) T cells. Infection in cocultures of MO-DCs and autologous CD4(+) T cells could be aborted in a proportion of the cultures, with high concentrations of PMEA and/or efavirenz, but not with AZT. Suppressive activity in mixed leukocyte cultures was observed only at very high concentrations of RTI. Our data suggest that cocultures of MO-DCs and autologous CD4(+) T cells can be used as a possible in vitro model to explore protocols for PEP after sexual HIV transmission.
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PMID:Activity of reverse transcriptase inhibitors in monocyte-derived dendritic cells: a possible in vitro model for postexposure prophylaxis of sexual HIV transmission. 1239 48

The pathogenesis of human immunodeficiency virus (HIV)-associated dementia has been linked to microglial responses after infection. We have recently confirmed expression of several ATP-dependent efflux transporters in microglia, namely, multidrug resistance protein 1 (MRP1) and P-glycoprotein (P-gp). In the present study, we investigated whether cultured rat microglia express two additional MRP family members, rMRP4 and rMRP5. Using reverse transcriptase-polymerase chain reaction, rMRP4 and rMRP5 mRNA was detected in primary cultures of microglia and in a rat microglia cell line, MLS-9. Western blot analysis further confirmed protein expression of the two MRP isoforms in MLS-9 cells. Bis(pivaloxymethyl)-9-(2-phosphonylmethoxyethyl)adenine [bis(POM)PMEA], a lipophilic ester prodrug of the well characterized MRP4 and 5 substrate 9-(2-phosphonylmethoxyethyl)adenine (PMEA), was chosen to examine transport characteristics in MLS-9. Using thin layer chromatography, we verified that more than 90% of radioactivity recovered in MLS-9 loaded with 1 microM [(3)H]bis(POM)PMEA for 1 h under ATP-depleting conditions was converted to PMEA. Efflux of PMEA by MLS-9 cell monolayers was ATP-dependent, glutathione-independent, and significantly inhibited by several MRP inhibitors (i.e., sulfinpyrazone, genistein, indomethacin, and probenecid) as well as the antiretroviral drug azidothymidine-monophosphate. Similar results were not observed in MRP1- or P-gp-overexpressing cell lines, suggesting that PMEA is not a substrate for either P-gp or MRP1. These studies provide further evidence that microglia express multiple subfamilies of ATP-binding cassette transporters (i.e., P-gp, MRP1, MRP4, and MRP5) that could restrict permeation of several different classes of antiretroviral drugs in a brain cellular target of HIV-1 infection.
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PMID:Multidrug resistance protein (MRP) 4- and MRP 5-mediated efflux of 9-(2-phosphonylmethoxyethyl)adenine by microglia. 1476 2

During chronic hepatitis B virus (HBV) infection, mutations in the precore (PC) or basal core promoter (BCP) region affecting HBV e antigen (HBeAg) expression occur commonly and represent the predominant virus species in patients with HBeAg-negative chronic hepatitis B. The PC mutation (G1896A+C1858T) creates a translational stop codon resulting in absent HBeAg expression, whereas BCP mutations (A1762T/G1764A) reduce HBeAg expression by transcriptional mechanisms. Treatment of chronic HBV infection with lamivudine (LMV) often selects drug-resistant strains with single (rtM204I) or double (rtL180M+rtM204V) point mutations in the YMDD motif of HBV reverse transcriptase. We cloned replication-competent HBV vectors (genotype A, adw2) combining mutations in the core (wild type [wt], PC, and BCP) and polymerase gene (wt, rtM204I, and rtL180M/M204V) and analyzed virus replication and drug sensitivity in vitro. Resistance to LMV (rtM204I/rtL180M+rtM204V) was accompanied by a reduced replication efficacy as evidenced by reduced pregenomic RNA, encapsidated progeny DNA, polymerase activity, and virion release. PC mutations alone did not alter virus replication but restored replication efficacy of the LMV-resistant mutants without affecting drug resistance. BCP mutants had higher replication capacities than did the wt, also in combination with LMV resistance mutations. All nine HBV constructs showed similar sensitivities to adefovir. In conclusion, BCP-PC mutations directly impact the replication capacity of LMV-resistant mutants. PC mutations compensated for replication inefficiency of LMV-resistant mutants, whereas BCP mutations increased viral replication levels to above the wt baseline values, even in LMV-resistant mutants, without affecting drug sensitivity in vitro. Adefovir may be an effective treatment when combinations of core and polymerase mutations occur.
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PMID:Basal core promoter and precore mutations in the hepatitis B virus genome enhance replication efficacy of Lamivudine-resistant mutants. 1528 Apr 61

Adefovir is classified as a nucleotide reverse transcriptase inhibitor because it acts by inhibiting hepatitis B virus DNA polymerase (reverse transcriptase) and causing DNA chain termination after its incorporation into the viral DNA. Adefovir dipivoxil is indicated for the treatment of chronic hepatitis B in adults with evidence of active viral replication and either evidence of persistent elevations in serum aminotransferases (alanine [ALT] or aspartate [AST]) or histologically active disease. It is useful in the treatment of patients with either hepatitis B e antigen-positive or -negative chronic hepatitis B. The recommended adefovir dipivoxil dose in the treatment of chronic hepatitis B in patients with adequate renal function is 10 mg once daily. Adefovir dipivoxil therapy can reduce viral load, improve ALT, and produce histologic improvement in patients with chronic hepatitis B. Improvements are generally seen within the first few weeks of therapy and have shown persistence up to at least 3 years with continued therapy. Therapy with adefovir dipivoxil is generally well tolerated. However, nephrotoxicity is a risk with adefovir therapy, especially in patients receiving higher doses (30-120 mg/d). Patients should have their renal function monitored closely throughout therapy and may require an adjustment in dose relative to changes in the creatinine clearance. Lactic acidosis and severe hepatomegaly with steatosis may also occur during therapy.
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PMID:Adefovir dipivoxil: focus on its use in the treatment of chronic hepatitis B. 1597 40

Chronic hepatitis B is a common disease and approximately 20% of infected patients with compensated cirrhosis will decompensate over 5 years. If untreated, the survival of decompensated cirrhosis is poor (15% at 5 years). The extent of hepatitis B virus (HBV) replication, as assessed by serum HBV-DNA level, is a strong predictor of the risk of disease progression and hepatocellular carcinoma. This provides a rationale for antiviral therapy to arrest progression of liver disease. Lamivudine is a pyrimidine analogue that inhibits HBV-DNA reverse transcriptase. It decreases HBV replication, normalises alanine aminotransferase levels and reduces hepatic inflammation and fibrosis in patients with chronic hepatitis B. This article will focus on the use of lamivudine in patients with HBV-cirrhosis. In patients with compensated HBV-cirrhosis, a randomised, placebo-controlled trial has shown that lamivudine significantly reduced the rate of disease progression and hepatocellular carcinoma development over a 3-year period. In patients with decompensated cirrhosis, treatment with lamivudine can produce spectacular improvements of liver function, but the improvement is slow and a clinical benefit is usually not observed until after at least 3-6 months of treatment. A major drawback of lamivudine treatment is the development of resistance, observed in 15-20% of patients after 1 year and up to 70% after 5 years of continued treatment. Thus, patients with HBV-cirrhosis treated with lamivudine should have regular monitoring of serum HBV-DNA levels and prompt institution of additional antiviral therapy if viral breakthrough is observed. Adefovir, tenofovir and entecavir have demonstrated efficacy in patients with lamivudine resistance. In patients with decompensated cirrhosis, in whom the development of resistance can be fatal, combination therapy (such as lamivudine plus adefovir) may prove more effective than monotherapy and this issue needs further study.
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PMID:Lamivudine treatment in patients with chronic hepatitis B and cirrhosis. 1692 9

Emergence of a lamivudine (LAM)-resistant hepatitis B virus (HBV) with amino acid substitutions in the YMDD motif is a well-documented problem during long-term LAM therapy. Entecavir (ETV) is a new drug approved for treatment of HBV infection with or without LAM-resistant mutants. This report describes an ETV-resistant strain of HBV, which emerged after prolonged ETV therapy in a patient who did not respond to LAM therapy. Direct sequence analysis of the ETV-resistant strain showed appearance of amino acid substitution rtS202G in the reverse transcriptase (RT) domain, together with rtL180M + M204V substitution that had developed at the emergence of LAM-resistant mutant. In vitro analysis demonstrated that the rtL180M + M204V + S202G mutant strain displayed a 200-fold and a 5-fold reduction in susceptibility to ETV compared with the wild- type and the rtL180M + M204V mutant strain, respectively. Adefovir was effective against the ETV-resistant strain both in vitro and during the clinical course. In conclusion, this study showed that virological and biochemical breakthrough due to ETV could occur in patients infected with LAM-resistant HBV and confirmed that the addition of rtS202G substitution to the rtL180M + M204V mutant strain is responsible for ETV resistance and we could treat the resistant mutant successfully.
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PMID:Successful treatment of an entecavir-resistant hepatitis B virus variant. 1793 65

Of the multitude of reverse transcriptase inhibitors and protease inhibitors that have been pursued for the treatment of HIV infections, nine compounds (viz. zidovudine, didanosine, zalcitabine, stavudine, lamivudine, saquinavir, ritonavir, indinavir and nevirapine) have been approved and several others (i.e. adefovir dipivoxyl [bis(POM)-PMEA], PMPA, bis(POC)-PMPA, 1592U89, delavirdine, loviride, MKC-442, nelfinavir and VX-478) are under clinical development. All these compounds can select for mutations in the reverse transcriptase or protease that confer various degrees of resistance or diminished susceptibility to the compounds. Both the reverse transcriptase and protease are able to accumulate multiple mutations in their genome, thus engendering high-level resistance. To avoid drug resistance from emerging it is recommended to use from the beginning combinations of the different drugs at sufficiently high (that is maximal tolerated) doses. If installed as soon as possible after infection, when it has become evident that the virus is replicating, these drug combinations may achieve a pronounced and sustained virus suppression. This should be reflected by a dramatic reduction of viral load in both the plasma and lymphnodes. With the most effective drug combination regimens, the viral load may even fall under the threshold of detection, and this may clinically translate into an arrest or prevention of progression to AIDS.
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PMID:Development of resistance of human immunodeficiency virus (HIV) to anti-HIV agents: how to prevent the problem? 1861 16

The acyclic pyrimidine nucleoside phosphonate (ANP) phosphonylmethoxyethoxydiaminopyrimidine (PMEO-DAPym) differs from other ANPs in that the aliphatic alkyloxy linker is bound to the C-6 of the 2,4-diaminopyrimidine base through an ether bond, instead of the traditional alkyl linkage to the N-1 or N-9 of the pyrimidine or purine base. In this study, we have analyzed the molecular interactions between PMEO-DAPym-diphosphate (PMEO-DAPym-pp) and the active sites of wild-type (WT) and drug-resistant HIV-1 reverse transcriptase (RT). Pre-steady-state kinetic analyses revealed that PMEO-DAPym-pp is a good substrate for WT HIV-1 RT: its catalytic efficiency of incorporation (k(pol)/K(d)) is only 2- to 3-fold less than that of the corresponding prototype purine nucleotide analogs PMEA-pp or (R)PMPA-pp. HIV-1 RT recognizes PMEO-DAPym-pp as a purine base instead of a pyrimidine base and incorporates it opposite to thymine (in DNA) or uracil (in RNA). Molecular modeling demonstrates that PMEO-DAPym-pp fits into the active site of HIV-1 RT without significant perturbation of key amino acid residues and mimics an open incomplete purine ring that allows the canonical Watson-Crick base pairing to be maintained. PMEO-DAPym-pp is incorporated more efficiently than (R)PMPA-pp by mutant K65R HIV-1 RT and is not as efficiently excised as (R)PMPA by HIV-1 RT containing thymidine analog mutations. Overall, the data revealed that PMEO- DAPym represents the prototype compound of a novel class of pyrimidine acyclic nucleoside phosphonates that are recognized as a purine nucleotide and should form the rational basis for the design and development of novel purine nucleo(s)(t)ide mimetics as potential antiviral or antimetabolic agents.
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PMID:The acyclic 2,4-diaminopyrimidine nucleoside phosphonate acts as a purine mimetic in HIV-1 reverse transcriptase DNA polymerization. 2016 90

Prolonged treatment of chronic hepatitis B (CHB) with nucleoside analogues (NAs) almost invariably engenders viral resistance, and sequential NAs monotherapy can promote multi-drug resistance. This study aimed to investigate the molecular characteristics and the mutation profile of multi-drug resistant hepatitis B virus (HBV). The complete genome of HBV isolated from a multi-drug refractory patient was amplified and cloned, and 22 clones were selected for sequencing. The homology of the full-length genome between clones ranged from 98.7% to 99.9%. A precore stop codon mutation of G1896A and basic core promoter (BCP) mutations A1762T/G1764A were detected in a majority of clones. A phylogenetic analysis showed that all clones were classified as subgenotype B2. Three mutations in the surface (S) antigen region, sC76Y, sP120T and sI195M, were detected in 100%, 100% and 77.3% of the clones, respectively. In the core (C) antigen region, a mutation at codon 135 (cP135Q) was detected in 100% of clones. Lamivudine (LAM)-resistant mutations, rtL180M and rtM204V/I were detected in 86.4% of clones. Adefovir (ADV) or entecavir (ETV)-resistant mutations were not detected. Several novel mutations, such as rtT128N, rtA222T, rtS256G, rtL271M, rtS332R, and rtN/T337D, were present in a majority of clones. Furthermore, six pairs of mutations in the overlapping reverse transcriptase (RT) gene and S gene were detected. In conclusion, the complex HBV mutation profile detected in the multi-drug refractory patient highlights the problems associated with the ongoing selection of mutations, including further compensatory mutations as well as potential cross-resistance mutations.
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PMID:Characterization of hepatitis virus B isolated from a multi-drug refractory patient. 2097 Apr 66


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