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)

Sequential isolates of human immunodeficiency virus type 1 (HIV-1) were obtained from patients with AIDS on short and long term treatment with zidovudine (3'-azido-3'-deoxythymidine; AZT). The isolates were tested for resistance to zidovudine by monitoring the inhibition of syncytium formation, HIV-1-specific immunofluorescence and p24 production in C8166 cells. The reverse transcriptase (RT) genes of zidovudine-sensitive (< 1 microM) and -resistant (10 to 15 microM) strains were amplified using the polymerase chain reaction and the products were sequenced directly. The predicted amino acid sequences of the RTs of zidovudine-sensitive and -resistant isolates showed 95 to 97% identity to the corresponding sequence of HIV-1SF2 which was used as a reference. Amino acid changes at positions 41, 67, 70, 215 and 219 which are known to be associated with zidovudine resistance were present in some, but not all isolates exhibiting zidovudine resistance in vitro. This indicates that mutations in the RT of HIV-1, other than those already identified, may be involved in conferring resistance to zidovudine.
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PMID:Sequence analysis of reverse transcriptase genes of zidovudine (AZT)-resistant and -sensitive human immunodeficiency virus type 1 strains. 767 40

Wild-type reverse transcriptase has evolved for the survival of human immunodeficiency virus type 1 (HIV-1) by natural selection. In contrast, therapy relying on inhibitors of reverse transcriptase by nucleosides like zidovudine (AZT) or dideoxyinosine (ddI), and by non-nucleosides like pyridinones or nevirapine, may exert different selection pressures on this enzyme. Therefore the acquisition of resistance to reverse transcriptase inhibitors by selection of mutations in the pol gene may require compromises in enzyme function that affect viral replication. As single mutations are unlikely to confer broad resistance when combinations of reverse transcriptase inhibitors are used, multiple mutations may occur that result in further compromises. Certain drug combinations may prevent the co-existence of adequate reverse transcription function and multi-drug resistance (MDR). Unlike bacterial or eukaryotic drug resistance, retroviral drug resistance is conferred only by mutations in its own genome and is limited by genome size. Combining drugs directed against the same essential viral protein may thus prevent HIV-1 MDR, whereas the conventional approach of targeting different HIV-1 proteins for combination therapy may not, because genomes with resistance mutations in different HIV-1 genes might recombine to develop MDR. Here we show that several mutations in the HIV-1 reverse transcriptase gene that confer resistance to inhibitors of this enzyme can attenuate viral replication. We tested whether combinations of mutations giving rise to single-agent resistance might further compromise or even abolish viral replication, and if multidrug-resistant viruses could be constructed. Certain combinations of mutations conferring resistance to AZT, ddI and pyridinone are incompatible with viral replication. These results indicate that evolutionary limitations exist to restrict development of MDR. Furthermore, a therapeutic strategy exploiting these limitations by using selected multidrug regimens directed against the same target may prevent development of MDR. This approach, which we call convergent combination therapy, eliminated HIV-1 replication and virus breakthrough in vitro, and may be applicable to other viral targets. Moreover, elimination of reverse transcription by convergent combination therapy may also limit MDR.
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PMID:Use of evolutionary limitations of HIV-1 multidrug resistance to optimize therapy. 833 79

Nuclear Overhauser effect experiments were performed at 500 MHz to determine the conformations of AZTTP and dTTP when bound to HIV-1 reverse transcriptase. The conformations of both ligands were found to be similar in the bound state. The orientation of the glycosidic angle is anti (chi = -120 degrees +/- 12 for AZTTP and -110 degrees +/- 12 for dTTP), gamma is +sc and the pucker of the 3'-azido-2',3'-dideoxy- and 2'-deoxyribose rings is predominantly C4' exo (P = 60 degrees +/- 10 for AZTTP and 55 degrees +/- 8 for dTTP). These results indicate that the unusual C4'endo/C3'exo pucker (P = 215 degrees) reported for the dideoxyribose ring of AZT in the solid state does not play a role in the interaction of HIV-1 reverse transcriptase with AZTTP.
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PMID:A comparison of the conformations of the 5'-triphosphates of zidovudine (AZT) and thymidine bound to HIV-1 reverse transcriptase. 768 61

The TIBO, HEPT, nevirapine, pyridinone, BHAP, TSAO, and alpha-APA derivatives, although belonging to structurally diverging classes of molecules, share remarkable common features. They are specifically active against the reverse transcriptase of HIV-1 (TIBO and HEPT also, to a certain extent, against the reverse transcriptase of SIVagm strains), but not against the reverse transcriptases of HIV-2 or any other retroviruses. Nor are they active against any of the cellular DNA polymerases. These HIV-1-specific RT inhibitors seem to interact with a specific target site (YQYMDDLY) at positions 181-188, which is distinct from, but functionally and spatially related to, the substrate (dNTP) binding site. The tyrosine residues Y181 and Y188 play a crucial role in the interaction of TIBO and its congeners with their target site. The HIV-1-specific RT inhibitors have proven to inhibit the replication of various HIV-1 strains, including AZT-resistant HIV-1 strains, in different cell culture systems, including peripheral blood lymphocytes and monocyte/macrophages. In vitro they exhibit selectivity indexes of up to 5 orders of magnitude, which means that they are inhibitory to virus replication in cell culture at concentrations that are up to 100,000 times lower than the concentrations at which they are toxic to the host cells. As a rule, the HIV-1-specific RT inhibitors are orally bioavailable, as has been demonstrated with the TIBO and HEPT derivatives, nevirapine, pyridinones, and the alpha-APA derivatives in rats, dogs, monkeys, and humans. They sustain plasma drug levels that are well above the concentration required to inhibit virus replication in cell culture. Clinical studies have been undertaken with TIBO R82913, nevirapine, and pyridinones, and others (i.e., alpha-APA R89439) will soon follow. The problem of virus-drug resistance, which seems to readily emerge in vitro, will have to be addressed in the in vivo studies.
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PMID:HIV-1-specific RT inhibitors: highly selective inhibitors of human immunodeficiency virus type 1 that are specifically targeted at the viral reverse transcriptase. 768 60

Triciribine (TCN) and its 5'-monophosphate (TCN-P) are novel tricyclic compounds with known antitumor activity; TCN-P is currently in phase II human clinical trials. We now report that these compounds have potent and selective activity against HIV-1 and HIV-2. Using a syncytial plaque assay, TCN and TCN-P were active against HIV-1 at 0.01-0.02 microM and had differential selectivities of 2250 and 1900, respectively, compared to 1850 for AZT. In contrast, TCN and TCN-P had minimal selectivity against human cytomegalovirus (50 and 27, respectively). TCN and TCN-P markedly inhibited HIV-1-induced p24 core antigen production, reverse transcriptase, and infectious virus production in a dose-dependent manner using HIV-1 acutely infected CEM-SS, H9, and persistently infected H9IIIB and U1 cells. In acutely infected PBL cells, TCN and TCN-P inhibited reverse transcriptase and infectious virus production but not p24 core antigen production. Using a microtiter XTT assay, TCN and TCN-P were active against a panel of HIV-1 and HIV-2 strains at IC50 values ranging from 0.02 to 0.46 microM. Evaluation of matched pairs of predrug and postdrug therapy HIV-1 isolates established that AZT-resistant and TIBO-resistant variants of HIV-1 were sensitive to TCN or TCN-P. Furthermore, unlike AZT and other fraudulent nucleosides, neither TCN, TCN-P, nor TCN-TP inhibited the viral reverse transcriptase. Thus, even though triciribine is a nucleoside chemically, it does not act biologically by classic nucleoside modalities but rather by a unique mechanism yet to be elucidated.
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PMID:Activity of triciribine and triciribine-5'-monophosphate against human immunodeficiency virus types 1 and 2. 768 12

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

Carbovir (CBV) [the (--)-enantiomer of the carbocyclic analog of 2',3'-dideoxy-2',3'-didehydroguanosine] is a potent inhibitor of human immunodeficiency virus type 1 (HIV) replication in vitro. We have characterized the metabolism of CBV and its effect on cellular metabolism in an effort to better understand its mechanism of action. CBV was primarily metabolized to the 5'-triphosphate of CBV (CBV-TP) to concentrations sufficient to inhibit HIV reverse transcriptase. Infection of CEM cells with HIV did not affect the metabolism of CBV. In CEM cells, there was no evidence of the degradation of CBV by purine nucleoside phosphorylase. The half-life of CBV-TP in CEM cells was 2.5 h, similar to that of the 5'-triphosphate of zidovudine (AZT). However, unlike the levels of the 5'-triphosphate of AZT, CBV-TP levels declined without evidence of a plateau. CBV did not affect the metabolism of AZT, and AZT did not affect the metabolism of CBV. A small amount of CBV was incorporated into DNA in intact CEM cells, and this incorporation was increased by incubation with mycophenolic acid, an inhibitor of IMP dehydrogenase. CBV specifically inhibited the incorporation of nucleic acid precursors into DNA but had no effect on the incorporation of radiolabeled precursors into RNA or protein. CBV did not decrease the level of TTP, dGTP, dCTP, or dATP. These results suggested that the cytotoxicity of CBV was due to the inhibition of DNA synthesis. Further studies are necessary to identify the target(s) responsible for growth inhibition.
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PMID:Metabolism of carbovir, a potent inhibitor of human immunodeficiency virus type 1, and its effects on cellular metabolism. 768 93

We have utilized UV-induced cross-linking of [methyl-3H]dTTP to identify the nucleotide binding site on heterodimeric HIV-1 reverse transcriptase (RT). RT was derivatized by irradiating a solution containing [methyl-3H]dTTP and purified recombinant RT for 10 min. The UV-induced cross-linking reaction between dTTP and RT is linear with time of UV exposure up to 10 min, and it has been determined previously that dTTP cross-linking is half-maximal at 90 microM [Cheng, N., Painter, G. R., & Furmann, P.A. (1991) Biochem. Biophys. Res. Commun. 174, 785-789]. Under these reaction conditions, only the 66-kDa subunit of the 66-kDa/51-kDa RT heterodimer was labeled with dTTP. The [methyl-3H]dTTP-labeled RT was fragmented with trypsin and endoproteinase Asp-N, and peptides were purified on reversed phase HPLC. The peptide covalently linked to [methyl-3H]dTTP was subjected to amino acid sequence analysis. The sequencing data localized the nucleotide binding site of RT to Lys-73 in the vicinity of several mutation sites linked to antiviral drug resistance. Since most effective anti-AIDS compounds are inhibitors of RT, information about its dNTP binding site may make it possible to understand the basis for the antiviral activity of nucleoside analogs such as AZT, ddI, and ddC. This information may also be useful for a more rationally based design of anti-HIV agents.
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PMID:Identification of the nucleotide binding site of HIV-1 reverse transcriptase using dTTP as a photoaffinity label. 768 65

Specific mutations in the human immunodeficiency virus type 1 (HIV-1) pol gene that cause zidovudine (3'-azido-2',3'-dideoxythymidine; AZT) and didanosine (2',3'-dideoxyinosine; ddI) resistance were studied. The 50% inhibitory concentrations (IC50s) of nucleosides for cloned viruses containing these mutations were compared with the IC50s of the corresponding triphosphate analogs for mutant recombinant-expressed reverse transcriptases (RTs). Changes in ddATP inhibition of RNA-dependent DNA polymerase activity fully accounted for the ddI resistance of the virus caused by a Leu-74-->Val substitution in RT, including an augmentation by the AZT-selected substitutions Thr-215-->Tyr and Lys-219-->Gln in RT. In contrast, the AZT-selected substitutions studied did not cause as great a change in the IC50 of AZT-triphosphate (AZT-TP) for polymerase as they did in the IC50 of AZT for mutant virus. In addition, the mutation at codon 74 suppressed AZT resistance in the virus caused by the mutations at codons 215 and 219 but did not suppress the AZT-TP resistance of enzyme containing these same mutations in RT. The mutation at codon 74 was found in clinical isolates whether or not the patient had received AZT prior to starting ddI therapy. AZT resistance coexisted with ddI resistance following acquisition of Leu-74-->Val in three clinical isolates, indicating that the suppressive effect of Val-74 on the AZT resistance of the virus does not occur in all genetic contexts. When this suppression of AZT resistance was seen in the virus, Val-74 did not appear to cause mutually exclusive changes in AZT-TP and ddATP binding to RT in vitro. The results of the in vitro experiments and characterization of clinical isolates suggest that there are differences in the functional effects of these AZT and ddI resistance mutations.
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PMID:pol mutations conferring zidovudine and didanosine resistance with different effects in vitro yield multiply resistant human immunodeficiency virus type 1 isolates in vivo. 768 22

The reverse transcriptase enzyme of human immunodeficiency virus type 1 (HIV-1) is the target for many inhibitors. Amino-acid substitutions in functional regions of the enzyme that abolish reverse transcriptase activity also prevent HIV-1 replication. But selection pressure by drugs such as AZT (3'-azido-3'deoxythymidine, zidovudine), ddI (2',3'-dideoxyinosine) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) causes outgrowth of resistant variants due to non-lethal mutations in the enzyme. Reports of synergy and lack of cross-resistance between reverse transcriptase inhibitors (refs 7, 9, 10, 12-14, 17, 18, 20, 21), plus the reversal of AZT resistance by mutations induced by ddI and NNRTIs, have indicated that specific drug combinations directed at reverse transcriptase might curtail resistance. Chow et al. extended this concept in a report that specific multiple combinations of resistance mutations in the reverse transcriptase can significantly impair HIV-1 replication. They concluded that evolutionary limitations may exist to prevent the emergence of multidrug resistance to inhibitors of reverse transcriptase. We report here that HIV-1 co-resistant to AZT, ddI and the NNRTI nevirapine can be readily selected in cell culture starting with dual AZT- and ddI-resistant virus. We found no evidence for 'replication incompatible' combinations of resistance mutations, although a mutation (M184-->V) conferring oxathiolane-cytosine nucleoside resistance in reverse transcriptase completely suppressed AZT resistance in a triple-resistant background. These in vitro observations suggest that triple drug combination therapy might ultimately result in co-resistant HIV-1, although they do not preclude assessment of such combinations for treatment of HIV-1 disease.
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PMID:Convergent combination therapy can select viable multidrug-resistant HIV-1 in vitro. 769 2


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