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)

We have studied selected mutants of human immunodeficiency virus type 2 (HIV-2) reverse transcriptase (RT) in a cell-free system in order to assess whether the mutant proteins exhibit a reduction in the sensitivity to nucleoside analog inhibitors similar to that of HIV-1 RT. We have modified, by site-directed mutagenesis, several of those amino acid residues so that their equivalent substitutions in HIV-1 RT have led to the formation of HIV-1 RT variants with the highest degree of resistance to dideoxynucleoside triphosphates (i.e., Glu-89-->Gly, Leu-74-->Val, and Ser-215-->Tyr [which is comparable to the Thr-215-->Tyr mutation of HIV-1 RT] and the double mutations Glu-89-->Gly/Ser-215-->Tyr and and Leu-74-->Val/Ser-215-->Tyr). The similarity found between resistance of the newly generated HIV-2 RT mutants to nucleoside analogs and that of the comparable mutants of HIV-1 RT can support the notion that the overall protein folding around the DNA polymerase active site in HIV-2 RT is quite similar to that of HIV-1 RT.
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PMID:Resistance to nucleoside analogs of selective mutants of human immunodeficiency virus type 2 reverse transcriptase. 752 86

A number of structurally diverse compounds have been shown to be potent inhibitors of the DNA polymerase activity of human immunodeficiency virus (HIV-1) reverse transcriptase (RT). The compounds can be grouped into two broad classes: nucleoside analogs and nonnucleoside inhibitors. The nonnucleoside inhibitors are quite specific for the polymerase activity of HIV-1 RT; they do not affect the polymerase activity of HIV-2 RT or the ribonuclease H (RNase H) activity of either HIV-1 RT or HIV-2 RT. Structural, biochemical, and genetic analyses showed that this group of inhibitors binds in a hydrophobic pocket near the polymerase active site. Mutations in amino acids that line this hydrophobic pocket, for example at tyrosine 181, tyrosine 188, or lysine 103, lead to enzymes that are resistant to the nonnucleoside inhibitors. We have investigated the enzymatic properties of two mutants of HIV-1 RT in which residues 181 and 188 were replaced by the corresponding amino acids in HIV-2 RT (tyrosine 181-->isoleucine and tyrosine 188-->leucine). The two tyrosine mutants closely resemble the wild-type HIV-1 RT in almost all the catalytic functions tested, including the heat stability, sensitivity of the DNA polymerase activity to inhibition by deoxynucleoside analogs, inhibition by the zinc chelator o-phenanthroline, and the Km values calculated for the DNA polymerase activity. There is, however, a slight difference in the effect of orthophenanthroline on the RNase H activity. In addition, there is a subtle disparity in the fidelity of DNA synthesis (analyzed by a mispair extension assay), thus indicating that these mutant RTs are not likely to confer any selective advantages or disadvantages to the variant virions over wild-type virus.
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PMID:Enzymatic properties of two mutants of reverse transcriptase of human immunodeficiency virus type 1 (tyrosine 181-->isoleucine and tyrosine 188-->leucine), resistant to nonnucleoside inhibitors. 752 32

Genetic recombination between viral genomes has been shown to contribute to the generation of genetic diversity during retrovirus infections. The role of recombination in the development of human immunodeficiency virus type 1 (HIV-1) zidovudine resistance was investigated as a possible cause of the formation of the linked Leu-41/Tyr-215 resistance genotype. Zidovudine resistance is conferred by the presence of subsets of four or five amino acid substitutions in the HIV-1 reverse transcriptase. Zidovudine therapy of asymptomatic HIV-1-infected individuals results in the selection of drug-resistant variants that posses defined combinations of the five zidovudine resistance mutations. The linked Leu-41/Tyr-215 resistance genotype appears central to the continued development of high-level zidovudine resistance. By using genetically tagged mutant viruses, it was possible readily to select recombinant viruses from mixed infections of Leu-41 and Tyr-215 single mutants in the presence of zidovudine drup pressure. After three passages of a mixed infection in the presence of drug, 38% of clones screened were recombinant double mutants. In the absence of zidovudine selection, little change in the mixed virus populations was noted. No evidence of de novo generation of mutations at codons 41 and 215 was seen during any in vitro passage. This provides the first example of the role of retroviral recombination in the development of HIV-1 variants with increased drug resistance.
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PMID:Retroviral recombination can lead to linkage of reverse transcriptase mutations that confer increased zidovudine resistance. 752 34

Tetrahydro-imidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-one and -thione (TIBO) derivatives were shown to specifically block human immunodeficiency virus type 1 (HIV-1) replication through a unique interaction with the HIV-1 reverse transcriptase (RT). Through further modification of the lead compounds and structure-activity relationship analysis several new TIBO derivatives that show high potency, selectivity, and specificity against HIV-1 have been obtained. A new TIBO derivative, R86183, inhibits the replication of HIV-1, but not HIV-2, in a variety of CD4+ T-cell lines and peripheral blood lymphocytes, at a concentration of 0.3 to 30 nM, which is at least 4 orders of magnitude lower than the 50% cytotoxic concentration. Whereas an HIV-1 strain containing the Leu-100-->Ile mutation in the RT gene is about 400-fold less susceptible, R86183 still inhibits the replication of an HIV-1 strain containing the Tyr-181-->Cys RT mutation by 50% at a concentration of 130 nM. R86183 inhibits the poly(C).oligo(dG)12-18-directed HIV-1 RT reaction by 50% at a concentration of 57 nM. The antiviral activity of 22 TIBO derivatives in cell culture correlated well with their activity against HIV-1 RT. No such correlation was found for their cytotoxicity. The combination of R86183 with either zidovudine or didanosine resulted in a synergistic inhibition of HIV-1 (strain IIIB) replication. Combination of R86183 with the protease inhibitor Ro31-8959 was found to be additive. Also described is a dilution protocol circumventing overestimation and underestimation of antiviral activity due to adherence to plastic surfaces.
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PMID:New tetrahydroimidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-one and -thione derivatives are potent inhibitors of human immunodeficiency virus type 1 replication and are synergistic with 2',3'-dideoxynucleoside analogs. 753 37

Three structural analogs of 5-ethyl-1-benzyloxymethyl-6-(phenylthio)uracil (E-BPU) inhibited human immunodeficiency virus type 1 (HIV-1) replication without cytotoxicity in vitro and were more potent than azidothymidine and were as potent as E-BPU. The target of these compounds is HIV-1 reverse transcriptase. Reverse transcriptases resistant to nevirapine (tyrosine at position 181 to cysteine) and TIBO R82150 (leucine at position 100 to isoleucine) are cross resistant to E-BPU analogs. Nevirapine- or TIBO R82150-resistant HIV-1 were cross resistant to E-BPU analogs but were inhibited at concentrations 11- to 135-fold lower than the cytotoxic doses.
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PMID:Action of uracil analogs on human immunodeficiency virus type 1 and its reverse transcriptase. 753 30

Five structurally related thiophene and furane analogues of the oxathiin carboxanilide derivative NSC 615985 (UC84) (designated UC10, UC68, UC81, UC42, and UC16) were identified as potent inhibitors of HIV-1 replication in cell culture and HIV-1 reverse transcriptase activity. These compounds were markedly active against a series of mutant HIV-1 strains, containing the Leu-100-->Ile, Val-106-->Ala, Glu-138-->Lys, or Tyr-181-->Cys mutations in their reverse transcriptase. However, the thiocarboxanilide derivatives selected for mutations at amino acid positions 100 (Leu-->Ile), 101 (Lys-->Ile/Glu), 103 (Lys-->Thr/Asp) and 141 (Gly-->Glu) in the HIV-1 reverse transcriptase. The compounds completely suppressed HIV-1 replication and prevented the emergence of resistant virus strains when used at 1.3-6.6 microM--that is, 10- to 25-fold lower than the concentration required for nevirapine and bis(heteroaryl)piperazine (BHAP) U90152 to do so. If UC42 was combined with the [2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5"-(4"-amino-1",2"- oxathiole-2",2"-dioxide)]-beta-D-pentofuranosyl (TSAO) derivative of N3-methylthymine (TSAO-m3T), virus breakthrough could be prevented for a much longer time, and at much lower concentrations, than if the compounds were used individually. Virus breakthrough could be suppressed for even longer, and at lower drug concentrations, if BHAP was added to the combination of UC42 with TSAO-m3T, which points to the feasibility of two- or three-drug combinations in preventing virus breakthrough and resistance development.
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PMID:Suppression of the breakthrough of human immunodeficiency virus type 1 (HIV-1) in cell culture by thiocarboxanilide derivatives when used individually or in combination with other HIV-1-specific inhibitors (i.e., TSAO derivatives). 753 17

A series of 23 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine derivatives that were highly potent inhibitors of wild-type human immunodeficiency virus type 1 strain IIIB (HIV-1/IIIB) replication in CEM cells were evaluated against a panel of HIV-1 mutant strains containing the replacement of leucine by isoleucine at position 100 (100-Leu-->Ile), 103-Lys-->Asn, 106-Val-->Ala, 138-Glu-->Lys, 181-Tyr-->Cys, 181-Tyr-->Ile, or 188-Tyr-->His in their reverse transcriptase (RT). A different structure-antiviral activity relationship was found, depending on the nature of the mutated amino acid in the HIV-1 RT. The results show that 5-ethyl-1-ethoxymethyl-6-(3,5-dimethylbenzyl)uracil, 5-ethyl-1-ethoxymethyl-6-(3,5-dimethylphenylthio)uracil, and 5-ethyl-1-ethoxymethyl-6-(3,5-dimethylphenylthio)-2-thiouracil remain active against the majority of viruses containing single mutations which confer resistance to nonnucleoside RT inhibitors.
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PMID:Differential activities of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine derivatives against different human immunodeficiency virus type 1 mutant strains. 754 Mar 84

Foscarnet (phosphonoformic acid) is a pyrophosphate analog that inhibits the replication of human immunodeficiency virus type 1 (HIV-1) in vitro and in patients with AIDS. HIV-1 resistance to foscarnet has not been reported despite long-term foscarnet therapy of AIDS patients with cytomegalovirus disease. We therefore attempted to select foscarnet-resistant HIV-1 in vitro by serial endpoint passage of virus in 400 microM foscarnet. After 13 cycles of passage in MT-2 cells, virus exhibiting > or = 8.5-fold foscarnet resistance was isolated. The reverse transcriptase (RT) from resistant virions exhibited a similar level of foscarnet resistance in enzyme inhibition assays (approximately 10-fold resistance). Foscarnet-resistant virus showed increased susceptibility to 3'-azido-3'-deoxythymidine (90-fold) and to the HIV-1-specific RT inhibitors TIBO R82150 (30-fold) and nevirapine (20-fold). DNA sequence analysis of RT clones from resistant virus revealed the coexistence of two mutations in all clones: Gln-161 to Leu (CAA to CTA) and His-208 to Tyr (CAT to TAT). Sequence analysis of six clinical HIV-1 isolates showing reduced susceptibility to foscarnet revealed the Tyr-208 mutation in two, the Leu-161 mutation in one, and a Trp-88-to-Ser or -Gly mutation in four isolates. Site-specific mutagenesis and production of mutant recombinant viruses demonstrated that the Leu-161, Ser-88, and Tyr-208 mutations reduced HIV-1 susceptibility to foscarnet 10.5-, 4.3-, and 2.4-fold, respectively, in MT-2 cells. In the crystal structure of HIV-1 RT, the Gln-161 residue lies in the alpha E helix beneath the putative deoxynucleoside triphosphate (dNTP) binding site. The Gln-161-to-Leu mutation may affect the structure of the dNTP binding site and its affinity for foscarnet. The location of the Trp-88 residue in the Beta5a strand of HIV-1 RT suggest that the Ser-88 mutation affects template-primer binding, as do several mutations that affect RT susceptibility to nucleoside analogs.
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PMID:Novel mutations in reverse transcriptase of human immunodeficiency virus type 1 reduce susceptibility to foscarnet in laboratory and clinical isolates. 754 60

The replacement of either Tyr-181 or Tyr-188 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) by the corresponding HIV-2 RT amino acids Ile-181 or Leu-188 is known to result in active mutant enzymes (Y181I; Y188L) with virtual loss of sensitivity towards three structural classes of nonnucleoside RT inhibitors; L-697,661, nevirapine, and TIBO R82913. The bisheteroarylpiperazine (BHAP) U-90152S, a highly specific inhibitor (IC50, 0.29 +/- 0.01 microM) of HIV-1 RT, inhibited the recombinant Y181I and Y188L HIV-1 RT mutants with IC50 values of 3.6 +/- 0.15 microM and 0.71 +/- 0.02 microM, respectively. Construction and in vitro analysis of double mutants Y181I/Y188L and Y181C/Y188L of HIV-1 RT showed > 150-fold resistance to U-90152S. An HIV-2 RT mutant containing amino acids 176-190 from HIV-1 RT acquired full sensitivity to U-90152S (IC50, 0.26 +/- 0.01 microM). It is concluded that simultaneous mutations at Tyr-181 and Tyr-188 of HIV-1 RT promotes resistance to U-90152S.
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PMID:Simultaneous mutations at Tyr-181 and Tyr-188 in HIV-1 reverse transcriptase prevents inhibition of RNA-dependent DNA polymerase activity by the bisheteroarylpiperazine (BHAP) U-90152s. 754 2

Initial studies suggested that major histocompatibility complex class I-restricted viral epitopes could be predicted by the presence of particular residues termed anchors. However, recent studies showed that nonanchor positions of the epitopes are also significant for class I binding and recognition by cytotoxic T lymphocytes (CTLs). We investigated if changing nonanchor amino acids could increase class I affinity, complex stability, and T-cell recognition of a natural viral epitope. This concept was tested by using the HLA-A 0201-restricted human immunodeficiency virus type 1 epitope from reverse transcriptase (pol). Position 1 (P1) amino acid substitutions were emphasized because P1 alterations may not alter the T-cell receptor interaction. The peptide with the P1 substitution of tyrosine for isoleucine (I1Y) showed a binding affinity for HLA-A 0201 similar to that of the wild-type pol peptide in a cell lysate assembly assay. Surprisingly, I1Y significantly increased the HLA-A 0201-peptide complex stability at the cell surface. I1Y sensitized HLA-A 0201-expressing target cells for wild-type pol-specific CTL lysis as well as wild-type pol. Peripheral blood lymphocytes from three HLA-A2 HIV-seropositive individuals were stimulated in vitro with I1Y and wild-type pol. I1Y stimulated a higher wild-type pol-specific CTL response than wild-type pol in all three donors. Thus, I1Y may be an "improved" epitope for use as a CTL-based human immunodeficiency virus vaccine component. The design of improved epitopes has important ramifications for prophylaxis and therapeutic vaccine development.
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PMID:Amino-terminal alteration of the HLA-A*0201-restricted human immunodeficiency virus pol peptide increases complex stability and in vitro immunogenicity. 754 95


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