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)

Passage of human immunodeficiency virus type 1 in the presence of increasing 2'-deoxy-3'-thiacytidine (3TC) concentrations results in high-level (> 100-fold) 3TC-resistant viruses. All 3TC-resistant viruses possess a substitution at the second codon (from a methionine into an isoleucine) at position 184 within the highly conserved motif (YMDD) of human immunodeficiency virus type 1 reverse transcriptase. 3TC-resistant viruses were cross-resistant to the (-) enantiomer of the fluorinated derivative of BCH-189 but remained susceptible to 2',3'-dideoxyinosine and 2',3'-dideoxycytidine. The susceptibilities of the 3TC-resistant viruses to the (+) enantiomers of BCH-189 and the fluorinated derivative of BCH-189 demonstrate an enantiomeric specificity for viruses selected under these conditions. Introduction of an isoleucine substitution at codon 184 into a background of two known 3'-azido-3'-deoxythymidine resistance mutations (amino acids 41 and 215) restored the susceptibility of this virus to 3'-azido-3'-deoxythymidine.
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PMID:High-level resistance to (-) enantiomeric 2'-deoxy-3'-thiacytidine in vitro is due to one amino acid substitution in the catalytic site of human immunodeficiency virus type 1 reverse transcriptase. 750 9

Combinations of antiretroviral drugs that prevent or delay the appearance of drug-resistant human immunodeficiency virus-type 1 (HIV-1) mutants are urgently required. Mutants resistant to 3'-azidothymidine (AZT, zidovudine) became phenotypically sensitive in vitro by mutation of residue 184 of viral reverse transcriptase to valine, which also induced resistance to (-)2'-deoxy-3'-thiacytidine (3TC). Furthermore, AZT-3TC coresistance was not observed during extensive in vitro selection with both drugs. In vivo AZT-3TC combination therapy resulted in a markedly greater decreased in serum HIV-1 RNA concentrations than treatment with AZT alone, even though valine-184 mutants rapidly emerged. Most samples assessed from the combination group remained AZT sensitive at 24 weeks of therapy, consistent with in vitro mutation studies.
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PMID:Potential mechanism for sustained antiretroviral efficacy of AZT-3TC combination therapy. 754 4

(-)-2'-deoxy-3'-thiacytidine (3TC) has been shown to be a potent, selective inhibitor of HIV replication in vitro, which requires phosphorylation to its 5'-triphosphate for antiviral activity. The intracellular concentration of 3TC 5'-triphosphate in phytohaemagglutinin (PHA)-stimulated peripheral blood lymphocytes (PBL) shows a linear dependence on the extracellular concentration of 3TC up to an extracellular 3TC concentration of 10 microM. At this extracellular concentration of 3TC, the resulting intracellular concentration of 3TC 5'-triphosphate is 5 microM. This value is similar to the inhibition constant (Ki) values for the competitive inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and human DNA polymerases (10-16 microM) by 3TC 5'-triphosphate. Since the concentration of 3TC producing 90% inhibition (IC90) of HIV replication in PBLs has been reported to be 76 nM, the antiviral activity of 3TC requires intracellular concentrations of 3TC 5'-triphosphate, which would result in very little inhibition of reverse transcriptase if its sole mode of action was competitive inhibition. This apparent discrepency may be explained by the ability of 3TC 5'-triphosphate to act as a substrate for reverse transcriptase. Primer extension assays have shown that 3TC 5'-triphosphate is a substrate for HIV-1 reverse transcriptase and DNA polymerase gamma, resulting in the incorporation of 3TC 5'-monophosphate into DNA. In the case of DNA polymerase gamma, the product of this reaction (i.e. double-stranded DNA with 3TC 5'-monophosphate incorporated at the 3'-terminus of the primer strand) is also a substrate for the 3'-5' exonuclease activity of this enzyme. This may explain the low levels of mitochondrial toxicity observed with 3TC.
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PMID:The intracellular phosphorylation of (-)-2'-deoxy-3'-thiacytidine (3TC) and the incorporation of 3TC 5'-monophosphate into DNA by HIV-1 reverse transcriptase and human DNA polymerase gamma. 757 60

The (-) enantiomers of 2',3'-dideoxy-5-fluoro-3'-thiacytidine [(-)-FTC] and 2',3'-dideoxy-3'-thiacytidine [(-)-BCH-189] were recently shown to inhibit selectively human immunodeficiency viruses (HIV) and hepatitis B virus in vitro. In the current study, the potential for HIV type 1 (HIV-1) resistance to these compounds was evaluated by serial passage of the virus in human peripheral blood mononuclear cells and MT-2 cells in the presence of increasing drug concentrations. Highly drug-resistant HIV-1 variants dominated the replicating virus population after two or more cycles of infection. The resistant variants were cross-resistant to (-)-FTC, (-)-BCH-189, and their (+) congeners but remained susceptible to 2',3'-dideoxycytidine, 3'-azido-3'-deoxythymidine, 3'-fluoro-3'-deoxythymidine, 2',3'-dideoxyinosine, phosphonoformate, the TIBO compound R82150, and the bis(heteroaryl)piperazine derivative U-87201E. Reverse transcriptase derived from drug-resistant viral particles was 15- to 50-fold less susceptible to the 5'-triphosphates of FTC and BCH-189 compared with enzyme from parental drug-susceptible virus. DNA sequence analysis of the reverse transcriptase gene amplified from resistant viruses consistently identified mutations at codon 184 from Met (ATG) to Val (GTG or GTA) or Ile (ATA). Sequence analysis of amplified reverse transcriptase from a patient who had received (-)-BCH-189 therapy for 4 months demonstrated a mixture of the Met-184-to-Val (GTG) mutation and the parental genotype, indicating that the Met-184 mutation can occur in vivo.
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PMID:Characterization of human immunodeficiency viruses resistant to oxathiolane-cytosine nucleosides. 768 16

Resistant variants of human immunodeficiency virus type 1 (HIV-1) have been selected by limited passage in MT4 cells of both wild-type and 3'-azido-3'-deoxythymidine (AZT, zidovudine)-resistant strains with the nucleoside analogues (-)-2'-deoxy-3'-thiacytidine (3TC) and (-)-2'-deoxy-5-fluoro-3'-thiacytidine (FTC). Virus variants selected independently were crossresistant to both inhibitors. This rapid in vitro selection of resistant virus has not previously been seen with nucleoside analogues but is reminiscent of that observed with the nonnucleoside reverse transcriptase inhibitors. However, passage of wild-type virus with a combination of AZT and FTC appreciably delayed emergence of FTC-resistant virus. DNA sequence analysis of the reverse transcriptase coding region from FTC-resistant virus revealed changes at codon 184 in the highly conserved Tyr, Met, Asp, Asp (YMDD) region. When the mutation Met184-->Val was introduced into the infectious clone HXB2, this change alone accounted for the resistance (> 1000-fold) seen with both 3TC and FTC, and for a 5- to 15-fold reduction in sensitivity to their (+) enantiomers. It had no effect on susceptibility to AZT or nevirapine and minimal effect on susceptibility to 2',3'-dideoxyinosine and 2',3'-dideoxycytidine. To determine the influence of this mutation in a background of mutations conferring resistance to AZT and nonnucleoside reverse transcriptase inhibitors, a series of HIV-1 variants were created by site-directed mutagenesis. All mutants with Met184-->Val were cross-resistant to 3TC and FTC. The Met184-->Val mutation did not influence nevirapine resistance, but resistance to AZT was suppressed. Similar suppression of AZT resistance was seen with Tyr181-->Cys. Interestingly, when both Met184-->Val and Tyr181-->Cys substitutions were present, highly resistant virus reverted to complete AZT sensitivity. Assessment of the interactive effects of multiple drug-resistance mutations may help to establish a rationale for using these drugs in the future therapy of HIV disease.
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PMID:Rapid in vitro selection of human immunodeficiency virus type 1 resistant to 3'-thiacytidine inhibitors due to a mutation in the YMDD region of reverse transcriptase. 768 7

4(S)-(6-Amino-9H-purin-9-yl)tetrahydro-2(S)-furanmethanol (IsoddA) is the most antivirally active member of a novel class of optically active isomeric dideoxynucleosides in which the base has been transposed from the natural 1' position to the 2' position and the absolute configuration is (S,S). IsoddA was active against human immunodeficiency virus type 1 (HIV-1) (strain IIIB), HIV-2 (strain ZY), and HIV-1 clinical isolates. Combinations of the compound with zidovudine (3'-azido-3'-deoxythymidine), 2',3'-dideoxyinosine, or 5-fluoro-2'-deoxy-3'-thiacytidine showed synergistic inhibition of HIV. A moderate reduction of activity was observed with clinical isolates resistant to zidovudine. An IsoddA-resistant virus (eightfold-increased 50% inhibitory concentration) was selected in vitro by repeated passage of HIV-1 (HXB2) in the presence of increasing concentrations of IsoddA. The reverse transcriptase-coding region of the mutant virus contained a single base change resulting in a change at codon 184 from Met to Val. IsoddA was also active against hepatitis B virus (HBV) in vitro; however, it lacked substantial selective activity in an in vivo HBV model. IsoddA was inefficiently phosphorylated in CEM cells; however, the half-life of the triphosphate was 9.4 h, and IsoddATP was a potent inhibitor of HIV-1 reverse transcriptase, with a Ki of 16 nM. The cytotoxicity 50% inhibitory concentrations of IsoddA were greater than 100 microM for CEM, MOLT-4, IM9, and the HepG2-derived HBV-infected 2.2.15 (subclone P5A) cell lines but were 12 and 11 microM for human granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Antiviral, metabolic, and pharmacokinetic properties of the isomeric dideoxynucleoside 4(S)-(6-amino-9H-purin-9-yl)tetrahydro-2(S)-furanmethanol. 854 Jul 5

The human immunodeficiency virus (HIV) type 1 reverse transcriptase (RT) is an important target for therapeutic intervention and for HIV-1-specific cytotoxic T lymphocytes (CTL). An HLA-A2-restricted CTL epitope containing the sequence YMDD, which is highly conserved among human and animal retroviruses and essential for function of the RNA-dependent DNA polymerase, is identified. The drug resistance mutation at RT amino acid 184 (M184V), associated with (-)-2'-deoxy-3'-thiacytidine (lamivudine), (-)-2'-deoxy-5-fluoro-3'-thiacytidine (FTC), and dideoxyinosine resistance, is located within this epitope and abolishes recognition by an established CTL response. This study demonstrates that the CTL response may target functionally relevant regions of the RT protein and suggests drug therapy may select for viral variants with altered susceptibility to established cellular immune responses.
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PMID:Recognition of the highly conserved YMDD region in the human immunodeficiency virus type 1 reverse transcriptase by HLA-A2-restricted cytotoxic T lymphocytes from an asymptomatic long-term nonprogressor. 856 16

The carbocyclic nucleoside 1592U89 is a selective inhibitor of the human immunodeficiency virus (HIV), targeting the reverse transcriptase (RT). In vitro selection studies were undertaken to generate resistant variants with both HIV type 1 (HIV-1) wild-type strain HIV-1(HXB2) and 3'-azido-3'-deoxythymidine (AZT)-resistant strain HIV-1(RTMC). At least two or three mutations in RT were required to produce a 10-fold reduction in susceptibility. The first RT mutation selected was at codon 184, methionine (M) to valine (V), for HIV-1(HXB2) and HIV-1(RTMC), conferring two- and fivefold resistance, respectively. Two additional mutations were selected with HIV-1(HXB2), either leucine (L) 74 to V and lysine (K) 65 to arginine (R) (first-passage series) or L74 to V and tyrosine (Y) 115 to phenylalanine (F) (second-passage series). Cloned variants, obtained from the 1592U89 selection, were either double RT mutants 65R/184V and 74V/184V or triple RT mutant 74V/115Y/184V. Molecular clones were constructed with single, double, and triple combinations of these mutations for resistance analysis with different RT inhibitors. Each individual mutation conferred only low-level resistance (two- to fourfold) to 1592U89 in the HXB2 background. Double mutants containing the 184V mutation and triple mutants showed slightly greater levels of resistance to 1592U89 (7- to 11-fold). Some of the 1592U89-resistant variants were cross-resistant with 2',3'-dideoxycytidine, 2',3'-dideoxyinosine, and (-)-2'-deoxy-3'-thiacytidine, but none were resistant to 2',3'-didehydro-3'-deoxythymidine or AZT.
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PMID:Combination of mutations in human immunodeficiency virus type 1 reverse transcriptase required for resistance to the carbocyclic nucleoside 1592U89. 914 75

Hepatitis B viruses establish a chronic, productive, and noncytopathic infection of hepatocytes. Viral products are produced by transcription from multiple copies (5-50) of covalently closed circular (ccc) viral DNA. This cccDNA does not replicate, but can be replaced by DNA precursors that are synthesized in the cytoplasm. The present study was carried out to determine if long-term treatment with an inhibitor of viral DNA synthesis would lead to loss of virus products, including cccDNA, from the liver of woodchucks chronically infected with woodchuck hepatitis virus. Viral DNA synthesis was inhibited with the nucleoside analog, lamivudine (2'-deoxy-3'-thiacytidine). Lamivudine treatment produced a slow but progressive decline in viral titers in serum, to about 0.3% or less of the initial level. However, even after maintenance of drug therapy for 3-12 months, > 95% of the hepatocytes in most animals were still infected. Significant declines in the percentage of infected hepatocytes and of intrahepatic cccDNA levels were observed in only three woodchucks, two in the group receiving lamivudine and one in the placebo control group. Moreover, virus titers eventually rose in woodchucks receiving lamivudine, suggesting that drug-resistant viruses began to spread through the liver starting at least as early as 9-12 months of treatment. Three types of mutation that may be associated with drug resistance were found at this time, in a region upstream of the YMDD motif in the active site of the viral reverse transcriptase. The YMDD motif itself remained unchanged. Not unexpectedly, the lamivudine therapy did not have a impact on development of liver cancer.
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PMID:Lamivudine therapy of WHV-infected woodchucks. 961 64

Cirrhosis and hepatocellular carcinoma occur as long-term complications of chronic hepatitis B virus (HBV) infection. Antiviral therapy is potentially a successful approach for the treatment of patients with HBV infection, which includes the nucleoside analog, lamivudine [(-)2'-deoxy-3'-thiacytidine, 3TC]. Although resistance to lamivudine therapy has been reported in several HBV-infected patients, the pattern of resistance-associated mutations in HBV has not been fully characterized. We report a DNA sequence database that includes a 500-base pair region of the HBV polymerase gene from 20 patients with clinical manifestations of lamivudine resistance. Analysis of the database reveals two patterns of amino acid substitutions in the tyrosine, methionine, aspartate, aspartate (YMDD) nucleotide-binding locus of the HBV polymerase. HBV DNA from the sera of patients in Group I exhibits a substitution of valine for methionine at residue 552, accompanied by a substitution of methionine for leucine at residue 528. Patients in Group II had only an isoleucine-for-methionine substitution at position 552. Reconstruction of these mutations in an HBV replication-competent plasmid was performed in a transient transfection cell assay to determine the function/relevance of these mutations to lamivudine resistance. Both Group I and Group II mutations resulted in a substantial decrease in sensitivity to lamivudine treatment (> 10,000-fold shift in IC50 over wild-type [wt] IC50), strongly indicating that these mutations were involved in resistance to lamivudine. A hypothetical model of the HBV reverse transcriptase has been generated for further study of the role of these mutations in lamivudine resistance.
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PMID:Identification and characterization of mutations in hepatitis B virus resistant to lamivudine. Lamivudine Clinical Investigation Group. 962 Mar 41


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