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)

Drug susceptibility and mutations in the reverse transcriptase (RT) gene were analyzed with 167 virus isolates from 38 patients treated with nevirapine, a potent nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) RT. Resistant isolates emerged quickly and uniformly in all patients administered nevirapine either as monotherapy or in combination with zidovudine (AZT). Resistance developed as early as 1 week, indicating rapid turnover of the virus population. The development of resistance was associated with the loss of antiviral drug activity as measured by CD4 lymphocyte counts and levels of HIV p24 antigen and RNA in serum. In addition to mutations at amino acid residues 103, 106, and 181 that had been identified by selection in cell culture, mutations at residues 108, 188, and 190 were also found in the patient isolates. Sequences from patient clones documented cocirculating mixtures of populations of different mutants. The most common mutation with monotherapy, tyrosine to cysteine at residue 181, was prevented from emerging by coadministration of AZT, which resulted in the selection of alternative mutations. The observations documented that, under selective drug pressure, the circulating virus population can change rapidly, and many alternative mutants can emerge, often in complex mixtures. The addition of a second RT inhibitor, AZT, significantly altered the pattern of mutations in the circulating population of HIV.
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PMID:Nevirapine resistance mutations of human immunodeficiency virus type 1 selected during therapy. 750

Thiazolo-iso-indolinone derivatives with high specificity toward the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) were identified. The most potent compound, BM +51.0836, inhibited HIV-1 RT at a 50% inhibitory concentration of 90 nM in vitro. In cell culture assays, similar 50% inhibitory concentrations were obtained with high specificity for HIV-1. These substances were equally active against a zidovudine-resistant isolate. No antiviral effect was observed with an HIV-2 isolate. HIV-1 isolates resistant to the thiazolo-iso-indolinones were generated in cell culture, and the nucleotide sequences of the respective RT genes were analyzed subsequently. Comparison of the deduced amino acid sequences with the wild-type sequence showed an amino acid change at position 181 (Tyr to Cys). Substitutions of amino acid Lys-101 and Lys-103 as well as Tyr-181 and/or Tyr-188 by site-directed mutagenesis led to resistance against the thiazolo-iso-indolinones. A chimeric HIV-2 RT, substituted with amino acids at positions 179 to 190 from HIV-1, acquired only partial susceptibility to BM +51.0836.
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PMID:Viral resistance to the thiazolo-iso-indolinones, a new class of nonnucleoside inhibitors of human immunodeficiency virus type 1 reverse transcriptase. 750 44

High level resistance to 3'-azido-3'-deoxythymidine (AZT, zidovudine or Retrovir) is conferred by the presence of four or five mutations (Met-41-->Leu; Asp-67-->Asn; Lys-70-->Arg; Thr-215-->Tyr or Phe; Lys-219-->Gln) in the human immunodeficiency virus (HIV) reverse transcriptase. The order of appearance of these five mutations in asymptomatic patients during therapy has been studied. This has enabled us to propose a model for the acquisition of zidovudine resistance mutations during the treatment of high-risk asymptomatic HIV-infected individuals. A consistent acquisition pattern of mutations at codons 41, 70 and 215 was observed in 17 individuals. Complex mixtures of HIV species containing different combinations of single and linked double resistance mutations were present early in zidovudine therapy in isolates from two patients studied in detail. From these mixtures the linked Leu-41/Tyr-215 genotype outgrew all others initially. The development of each new virus population is likely to be mediated primarily by the increase in the level of drug resistance rather than changes in the growth kinetics of the virus. This leads us to conclude that one major driving force in the outgrowth of different mutant viruses is the selective advantage conferred by higher levels of drug resistance.
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PMID:Zidovudine treatment results in the selection of human immunodeficiency virus type 1 variants whose genotypes confer increasing levels of drug resistance. 750 70

Inophyllums are novel non-nucleoside inhibitors of human immunodeficiency virus (HIV) type 1 reverse transcriptase identified through an enzyme screening program and isolated from the plant Calophyllum inophyllum. The kinetics of reverse transcriptase inhibition by inophyllum B were characterized using recombinant purified enzyme, a heteropolymeric RNA template, and a scintillation proximity assay. Preincubation of inhibitor with the enzyme-template-primer complex for 11 min was required for maximal inhibition of reverse transcriptase to occur, suggesting that inophyllum B had a slow on-rate and that template-primer must bind to reverse transcriptase prior to inhibitor binding. Inhibition of reverse transcriptase by inophyllums was shown to be reversible. When thymidine triphosphate was the variable substrate, inophyllum B inhibited reverse transcriptase noncompetitively with a Ki of 42 nM. Enzyme inhibition with respect to template-primer was uncompetitive with a Ki of 26 nM. Reverse transcriptase enzymes containing point mutations in which tyrosine 181 was changed to either cysteine or isoleucine exhibited marginal resistance to inophyllums but were resistant to (+)-(5S)-4,5,6,7-tetrahydro-9-chloro-5-methyl-6- (3-methyl-2-butenyl)-imidazo[4,5,1-j,k][1,4]benzodiazepin-2-(1H)-t hione (TIBO R82913). A mutant enzyme in which tyrosine 188 was changed to leucine was cross-resistant to both inophyllum B and TIBO R82913, as was HIV type 2 reverse transcriptase. These studies suggest that inophyllum B and TIBO R82913 bind to distinct but overlapping sites. Inhibition of avian myeloblastosis virus reverse transcriptase and Moloney murine leukemia virus reverse transcriptase by inophyllum B was detectible, suggesting that these inhibitors may be more promiscuous than other previously described non-nucleoside inhibitors. Inophyllums were active against HIV type 1 in cell culture with IC50 values of approximately 1.5 microM. These studies imply that the inophyllums have a novel mechanism of interaction with reverse transcriptase and as such could conceivably play a role in combination therapy.
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PMID:Kinetic and mutational analysis of human immunodeficiency virus type 1 reverse transcriptase inhibition by inophyllums, a novel class of non-nucleoside inhibitors. 750

Mutation in the human immunodeficiency virus type 1 reverse transcriptase (RT) at codon 215 has been shown to play a significant role in resistance to zidovudine (AZT). Substitution of threonine with tyrosine or phenylalanine alone confers decreased susceptibility to the inhibitor. In this study we constructed a panel of 10 viruses with different amino acids at this codon, including 7 novel mutants, and assessed their susceptibilities to AZT. The majority of the new mutants were AZT sensitive, whereas the Thr-215-->Trp mutant was partially resistant (threefold less susceptible). A combination of the Thr-215-->Trp with the other AZT resistance mutations Lys-70-->Arg and Met-41-->Leu gave additive resistance. The Thr-215-->Phe virus was less AZT resistant than the Thr-215-->Tyr mutant, both on its own and when each was combined with the Met-41-->Leu mutant. These observations confirm the general hypothesis that increased bulk of the amino acid side chains at this position confers decreased AZT sensitivity. A leucine-to-valine substitution at codon 74 has previously been found to confer dideoxynucleoside resistance. We constructed mutants with five novel amino acid substitutions (Ala, Gly, Glu, Met, and Asp) at codon 74. Of these, only one (that with the Met substitution) retained enough RT activity to yield viable virus. It thus appears that there are severe structure-function constraints on the amino acid side chains at this position in the enzyme. The activities of the Leu-74-->Ala and Leu-74-->Met RT enzymes expressed in Escherichia coli appeared to have reduced susceptibility to ddGTP compared with the wild-type enzyme. The mutants described in this work may prove useful for correlation with structural studies of the human immunodeficiency virus type 1 RT.
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PMID:Mutagenic study of codons 74 and 215 of the human immunodeficiency virus type 1 reverse transcriptase, which are significant in nucleoside analog resistance. 751 65

The dipyridodiazepinone Nevirapine is a potent and highly specific inhibitor of the reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1). It is a member of an important class of nonnucleoside drugs that appear to share part or all of the same binding site on the enzyme but are susceptible to a variety of spontaneous drug-resistance mutations. The co-crystal-structure of HIV-1 RT and Nevirapine has been solved previously at 3.5-A resolution and now is partially refined against data extending to 2.9-A spacing. The drug is bound in a hydrophobic pocket and in contact with some 38 protein atoms from the p66 palm and thumb subdomains. Most, but not all, nonnucleoside drug-resistance mutations map to residues in close contact with Nevirapine. The major effects of these mutations are to introduce steric clashes with the drug molecule or to remove favorable protein-drug contacts. Additionally, four residues (Phe-227, Trp-229, Leu-234, and Tyr-319) in contact with Nevirapine have not been selected as sites of drug-resistance mutations, implying that there may be limitations on the number and types of resistance mutations that yield viable virus. Strategies of inhibitor design that target interactions with these conserved residues may yield drugs that are less vulnerable to escape mutations.
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PMID:Structure of the binding site for nonnucleoside inhibitors of the reverse transcriptase of human immunodeficiency virus type 1. 751 27

Human immunodeficiency virus type 1 (HIV-1) recombinant reverse transcriptase (RT) containing lysine (Lys) instead of glutamic acid (Glu) at position 138 proved fully resistant to the inhibitory effect of TSAO derivatives, but retained marked sensitivity to all other HIV-1-specific inhibitors investigated. In contrast, 181 Tyr-->Cys mutated RT lost sensitivity to all HIV-1-specific inhibitors. There was a close correlation between the sensitivity/resistance pattern of HIV-1-specific inhibitors against mutated (138 Glu-->Lys) recombinant HIV-1 RT and mutant virus strains selected for resistance against TSAO-m3T in cell culture and proven to contain the 138-Lys mutation as the sole mutation within the amino acid 50-270 region of their RT.
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PMID:Sensitivity of (138 Glu-->Lys) mutated human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) to HIV-1-specific RT inhibitors. 751 68

To evaluate the potential that multiply resistant human immunodeficiency virus type 1 variants may arise during combination nucleoside and nonnucleoside reverse transcriptase inhibitor therapy, we constructed a series of mutant reverse transcriptase enzymes and viruses that coexpressed various combinations of resistance-associated amino acid substitutions. Substitutions at residues 100 (Leu-->Ile) and 181 (Tyr-->Cys), which mediate resistance to the nonnucleosides, suppressed resistance to 3'-azido-3'-deoxythymidine (AZT) when coexpressed with AZT-specific substitutions. However, a number of viral variants that exhibited significantly reduced susceptibilities to both classes of inhibitors were constructed.
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PMID:Susceptibilities of human immunodeficiency virus type 1 enzyme and viral variants expressing multiple resistance-engendering amino acid substitutions to reserve transcriptase inhibitors. 752 28

Two mutants of HIV-1 reverse transcriptase (RT), Tyr-188-->His and Glu-138-->Arg have been prepared and their catalytic properties and sensitivities to inhibitors studied. As compared to wild type RT, a reduction in catalytic efficiency and turn over number was observed, especially for the Tyr-188-->His mutant. The non-nucleoside inhibitors nevirapine, L-697,661 and 9-Cl-TIBO caused a mixed type of inhibition of RT (Arg-138) with respect to substrate, and with the exception of a non-competitive inhibition by nevirapine, also a mixed type of inhibition of RT (His-188). Foscarnet (PFA) caused a non-competitive type of inhibition of RT (Arg-138) and a mixed inhibition of RT (His-188). The inhibition by ddG-TP was competitive with both mutant RTs. Inhibition by nevirapine gave IC50 values of 0.15, 0.23 and 0.72 microM; by 9-Cl-TIBO of 0.20, 2.50 and 10.3 microM; by L-697,661 of 0.064, 0.28 and 0.60 microM; by ddGTP of 0.13, 0.14 and 0.02 microM; by PFA of 17.0, 48.0 and 15.0 microM for RT wt, RT (Arg-138) and RT (His-188), respectively.
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PMID:Enzymatic properties and sensitivity to inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase with Glu-138-->Arg and Tyr-188-->His mutations. 752 39

Hepadnaviruses employ a unique mechanism for the initiation of RNA-directed DNA synthesis. Initially, four bases (5'-GTAA-3') are added to a tyrosine residue of the viral polymerase by reverse transcription of a bulge sequence in epsilon, a stem-loop structure which functions as the packaging signal for pregenomic RNA. This protein-DNA complex acts as the primer for minus-strand elongation from the 3' sequence, DR1. To understand this process in greater detail, we investigated whether the protein-mediated priming of viral DNA synthesis is affected by nucleotide analogs. By using cell-free expression of duck hepatitis B virus (DHBV) reverse transcriptase (G.-H. Wang and C. Seeger, Cell 71:663-670, 1992), the 5'-triphosphate of the thymidine analog fialuridine (FIAU) was shown to inhibit the incorporation of radiolabeled TMP into primer DNA in a dose-dependent manner. Inhibition by the 5'-triphosphate of FIAU (FIAU-TP) was nearly complete at a concentration of 10 microM. The dideoxynucleotide analogs ddGTP, ddTTP, and 3'-azidodeoxythymidine triphosphate, known inhibitors of DHBV endogenous DNA polymerase, did not affect substantially the synthesis of primer DNA. Alternate substrate analysis suggested that FIAU is incorporated efficiently into nascent primer DNA as an analog of thymidine. Using site-directed mutagenesis to construct a mutant RNA template yielding a primer with the sequence 5'-GTAC-3', we demonstrated that FIAU-TP inhibited the incorporation of TMP, had no effect on that of dAMP, and decreased markedly the incorporation of dCMP. These results show that the synthesis of full-length DHBV primer DNA is inhibited by FIAU-TP but not by the dideoxynucleotide analogs that we tested. The significance of these findings as they relate to HBV DNA replication is discussed.
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PMID:Priming of duck hepatitis B virus reverse transcription in vitro: premature termination of primer DNA induced by the 5'-triphosphate of fialuridine. 752 86


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