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PubMed2004

Kinetics of nucleotide incorporation opposite DNA bulky guanine N2 adducts by processive bacteriophage T7 DNA polymerase (exonuclease-) and HIV-1 reverse transcriptase.

Six oligonucleotides with carcinogen derivatives bound at the N2 atom of deoxyguanosine were prepared, including adducts derived from butadiene, acrolein, crotonaldehyde, and styrene, and examined for effects on the replicative enzymes bacteriophage DNA polymerase T7- (T7-) and HIV-1 reverse transcriptase for comparison with previous work on smaller DNA adducts. All of these adducts strongly blocked dCTP incorporation opposite the adducts. dATP was preferentially incorporated opposite the acrolein and crotonaldehyde adducts, and dTTP incorporation was preferred at the butadiene- and styrene-derived adducts. Steady-state kinetic analysis indicated that the reduced catalytic efficiency with adducted DNA involved both an increased Km and attenuated kcat. Fluorescence estimates of Kd and pre-steady-state kinetic measurements of koff showed no significantly decreased affinity of T7- with the adducted oligonucleotides or the dNTP. Pre-steady-state kinetics showed no burst phase kinetics for dNTP incorporation with any of the modified oligonucleotides. These results indicate that phosphodiester bond formation or a conformational change of the enzyme.DNA complex is rate-limiting instead of the step involving release of the oligonucleotide. Thio elemental effects for dNTP incorporation were generally relatively small but variable, indicating that the presence of adducts may sometimes make phosphodiester bond formation rate-limiting but not always.

Bacteriophage T7↗
PubMed1999

HIV-1 reverse transcriptase is phosphorylated in vitro and in a cellular system.

Phosphorylation modulates the activity of many proteins that interact with nucleic acids including DNA and RNA polymerases. The HIV-1 reverse transcriptase (RT) is essential during the replicative cycle of the HIV-1 virus. HIV-1 RT has several potential sites for phosphorylation that could regulate its activities. In this work, the phosphorylation of HIV-1 RT is examined in vitro and in vivo, to evaluate any role for this modification in regulating RT metabolism. Recombinant unphosphorylated HIV-1 RT heterodimer expressed in bacteria can be phosphorylated in vitro by several purified mammalian protein kinases. Seven kinases were tested, and five of these enzymes phosphorylated HIV-1 RT. Using an insect baculovirus expression system, the 66 kDa HIV-1 RT was also phosphorylated in vivo. However, HIV-1 RT immunoprecipitated from H9-lymphoma cells infected with HIV-1 showed negligible phosphorylation. Our results indicate that purified HIV-1 RT can be phosphorylated by several mammalian protein kinases in vitro and during expression in baculovirus infected insect cells.

Amino Acid Sequence↗
PubMed2005

Screening of selected wood-damaging fungi for the HIV-1 reverse transcriptase inhibitors.

Extracts obtained using methanol and dichloromethane from 57 species of wood damaging fungi were investigated for their ability to inhibit HIV-1 reverse transcriptase activity in vitro using a non-radioactive assay. Sixty-three samples were tested all together; some species were represented more than one isolate. Thirteen methanolic extracts exhibited more than 40% inhibition and two among them inhibited the enzyme by more than 80%. All extracts obtained with dichloromethane were inferior to methanolic extracts in their inhibitory activity. The most active fungal species discovered in the first screening were Laetiporus sulphureus and Poria monticola, followed by Poria vaillanti and Chondrostereum purpureum. In the second screening, Laetiporus sulphureus was selected for detailed examination and different isolates were tested. Preliminary findings confirmed the presence of an acidic compound with the amino group in the most active fraction.

Ascomycota↗
PubMed2002

Insertion of a small peptide of six amino acids into the beta7-beta8 loop of the p51 subunit of HIV-1 reverse transcriptase perturbs the heterodimer and affects its activities.

BACKGROUND: HIV-1 RT is a heterodimeric enzyme, comprising of the p66 and p51 subunits. Earlier, we have shown that the beta7-beta8 loop of p51 is a key structural element for RT dimerization (Pandey et al., Biochemistry 40: 9505, 2001). Deletion or alanine substitution of four amino acid residues of this loop in the p51 subunit severely impaired DNA binding and catalytic activities of the enzyme. To further examine the role of this loop in HIV-1 RT, we have increased its size such that the six amino acids loop sequences are repeated in tandem and examined its impact on the dimerization process and catalytic function of the enzyme. RESULTS: The polymerase and the RNase H activities of HIV-1 RT carrying insertion in the beta7-beta8 loop of both the subunits (p66INS/p51INS) were severely impaired with substantial loss of DNA binding ability. These enzymatic activities were restored when the mutant p66INS subunit was dimerized with the wild type p51. Glycerol gradient sedimentation analysis revealed that the mutant p51INS subunit was unable to form stable dimer either with the wild type p66 or mutant p66INS. Furthermore, the p66INS/p66INS mutant sedimented as a monomeric species, suggesting its inability to form stable homodimer. CONCLUSION: The data presented herein indicates that any perturbation in the beta7-beta8 loop of the p51 subunit of HIV-1 RT affects the dimerization process resulting in substantial loss of DNA binding ability and catalytic function of the enzyme.

Amino Acids↗
PubMedDate not supplied

Metabolism of 3-[2-(benzoxazol-2-yl)ethyl]-5-ethyl-6-methylpyridin-2 (1H)-one (L-696,229), an HIV-1 reverse transcriptase inhibitor, by rat liver slices and in humans.

Healthy subjects were administered single oral doses of 800 mg or 400 mg 3-[2-(benzoxazol-2-yl)ethyl]-5-ethyl-6-methylpyridin-2(1H)-o ne (L-696,229), a nonnucleoside inhibitor of the human immunodeficiency virus-type 1 (HIV-1) reverse transcriptase (RT). Plasma or urine samples were collected over a period of 48 hr. Pooled plasma (0.5-6 hr) and urine (0-24 hr) samples were analyzed by HPLC-UV and HIV-1 RT inhibition assay using poly rC.dG as a template primer. The parent compound and several common metabolites were detected in both samples. The metabolic profiles were also similar to those obtained from a rat liver slice incubation with [3H]L-696,229. The in vitro metabolites were identified by NMR and MS as 5 alpha-hydroxyethyl- (major), 5,6-dihydrodiol-, 6'-hydroxy-, 6-hydroxymethyl-, and 5-vinyl analogs, and a benzoxazole ring hydrolysis product. Most of the significant metabolites in human plasma and urine were found to be identical to the in vitro metabolites, as established by HPLC-UV and MS. Hydrolysis of the plasma and urine with beta-glucuronidase/sulfatase indicated the presence of significant amounts of conjugates of the parent compound and 5 alpha-hydroxyethyl metabolite. Most of the other primary metabolites were also present in conjugated forms, albeit in small quantities. In addition, two secondary metabolites were isolated and identified from the hydrolyzed urine as 5-acetyl-6'-hydroxy- and 5 alpha-hydroxyethyl-6-hydroxymethyl- analogs.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗
PubMed2005

Mechanism of action of a novel viral mutagenic covert nucleotide: molecular interactions with HIV-1 reverse transcriptase and host cell DNA polymerases.

A novel non-chain terminating nucleoside analog anti-HIV inhibitor, KP-1212 has been designed to form base pairs with multiple bases that may lead to mutagenesis in the HIV-1 viral genome. After multiple replication cycles, the accumulation of mutations surpasses a crucial threshold beyond which the virus can no longer replicate. HIV-1 reverse transcriptase (RT) incorporates the KP-1212 monophosphate into the genome during viral replication after metabolic activation of the KP-1212 nucleoside to the triphosphate. The propensity for forming alternate base pairs with the KP-1212 nucleotide leads to mismatched nucleotides and the subsequent misincorporation is the basis for the inhibitory activity. The results showed that HIV-1 RT and human mitochondrial DNA polymerase (Pol gamma) incorporated KP-1212-TP with a significant level of efficiency, whereas mouse DNA polymerase beta (Pol beta) did not. Misincorporation studies suggest that both HIV-1 RT and Pol gamma may cause mutations at significantly high rates. These in vitro data confirm the mechanistic basis of KP-1212 as a viral mutagen but suggest that there may be a potential for toxicity to the mitochondria.

Anti-HIV Agents↗
PubMed2003

Inhibition of HIV-1 reverse transcriptase by RNA aptamers in Escherichia coli.

A better understanding of aptamer function in bacteria would help to establish simple model systems for screening RNA-protein interactions within an intracellular context. Escherichia coli DNA polymerase I mutants (Pol I(ts)) fail to grow at 37 degrees C unless an exogenous DNA polymerase such as HIV-1 reverse transcriptase (RT) is expressed within the cell. Here, we show that four RNA aptamers that inhibit HIV-1 RT in vitro block complementation by HIV-1 RT when expressed in vivo. No other essential functions are impaired by aptamer expression at either temperature. Intracellular aptamer RNA concentrations from induced cultures were measured to range from 76 to 180 nM, which is comparable with exogenously expressed HIV-1 RT levels in these cells. RT polymerase activity was reduced to background levels in cell-free extracts prepared from cultures expressing both HIV-1 RT and the 70.28 aptamer, compared with extracts from cultures expressing HIV-1 RT alone. Intracellularly expressed RNA aptamers can thus be used to generate conditional null mutants in bacteria by titrating an essential protein.

Escherichia coli↗
PubMed1989

Human immunodeficiency virus reverse transcriptase expressed in transformed yeast cells. Biochemical properties and interactions with bovine tRNALys.

Human immunodeficiency virus (HIV) reverse transcriptase has been purified from yeast transformed by an autoreplicating plasmid containing the retroviral DNA polymerase gene. The previously described purification procedure for the yeast-expressed reverse transcriptase [Barr, P.J., Power, M.D., Chun Ting Lee-Ng, Gibson, H. & Luciw, P. (1987) Bio/Technology 5, 486-489] has been substantially modified, leading to an increased yield and a higher degree of purity. Several biochemical properties of the enzyme are described (template specificity, effect of DNA synthesis inhibitors); interestingly, HIV reverse transcriptase is highly resistant to N-ethylmaleimide. A complex between the human retroviral enzyme and the bovine tRNALys was shown, using a direct approach, by glycerol gradient centrifugation, as well as by the protective and specific effect of the tRNALys against enzyme inactivation by thermal denaturation and trypsin digestion. A competitive type of inhibition of HIV reverse transcriptase by tRNALys, but not by tRNAVal, is observed when viral RNA or activated DNA are used as templates.

Animals↗
PubMed2004

Flexible docking of pyridinone derivatives into the non-nucleoside inhibitor binding site of HIV-1 reverse transcriptase.

Potent non-nucleoside reverse transcriptase inhibitors (NNRTIs) of the pyridinone derivative type were docked into nine NNRTIs binding pockets of HIV-1 reverse transcriptase (RT) structures. The docking results indicate that pyridinone analogues adopt a butterfly conformation and share the same binding mode as the crystal inhibitors in the pocket geometries of nevirapine, 1051U91, 9-Cl-TIBO, Cl-alpha-APA, efavirenz, UC-781, and S-1153. The results are in agreement with the data concerning mutational and structure-activity relationships available for pyridinone analogues and aid in the understanding, at the molecular level, of the biological response of published hybrid pyridinone molecules. Strategies to design further pyridinone derivatives active against RT containing mutations are discussed.

Anti-HIV Agents↗
PubMed1999

Mechanistic studies show that (-)-FTC-TP is a better inhibitor of HIV-1 reverse transcriptase than 3TC-TP.

Of all of the nucleoside inhibitors approved by the FDA for treatment of AIDS, (-)-beta-2',3'-dideoxy-3'-thiacytidine (3TC, lamivudine) is the only one with the unnatural (-)-beta-L configuration. The fluorinated derivative (-)-beta-2', 3'-dideoxy-5-fluoro-3'-thiacytidine [(-)-FTC] and its triphosphate form have also been reported to have excellent antiretroviral activity against HIV-1 reverse transcriptase (RT). Preliminary results of clinical trials suggest that (-)-FTC is 6- to 10-fold more potent than 3TC. However, the molecular mechanism for the observed enhanced clinical potency of (-)-FTC to inhibit viral replication is not understood. The present mechanistic studies used a transient kinetic approach and were designed to compare the incorporation of 3TC-TP and (-)-FTC-TP into DNA by HIV-1 RT and illuminate key features that may play a role in the differential potency. Here we show that (-)-FTC-TP is incorporated 10-fold more efficiently than 3TC-TP during HIV-1 RT-catalyzed RNA-dependent DNA synthesis. The enhanced incorporation efficiency of (-)-FTC-TP may be a key mechanistic feature that, in part, is responsible for the enhanced potency of (-)-FTC observed in ongoing clinical trials.

Base Sequence↗
PubMed1998

Cytotoxic T-lymphocyte responses to HIV-1 reverse transcriptase (review).

Cytotoxic T lymphocytes (CTL) play an important role in the control of human immunodeficiency virus (HIV) infection. CTL responses have been demonstrated for most of the HIV gene products, predominantly gag, pol, and env-encoded proteins, and also for the regulatory proteins Nef, Tat, Vif, or Rev. The HIV-1 reverse transcriptase (RT), which derives from expression of the pol gene, is an important target of cellular immune responses in infected individuals. More than 40 different peptides containing RT-specific CTL epitopes have been identified. The most conserved and frequently detected are located in the 'fingers' and 'palm' subdomains of the enzyme, but other epitopes have been found in the 'thumb' and 'connection' subdomains as well as in the RNase H domain. Studies on the sequence variability and functional role of amino acids forming CTL epitopes are relevant for addressing important questions relative to viral escape from immmune control and the future design of anti-AIDS vaccines.

AIDS Vaccines↗
PubMed1996

All-atom models for the non-nucleoside binding site of HIV-1 reverse transcriptase complexed with inhibitors: a 3D QSAR approach.

Several molecular modeling techniques were used to generate an all-atom molecular model of a receptor binding site starting only from Ca atom coordinates. The model consists of 48 noncontiguous residues of the non-nucleoside binding site of HIV-1 reverse transcriptase and was generated using a congeneric series of nevirapine analogs as structural probes. On the basis of the receptor-ligand atom contacts, the program HINT was used to develop a 3D quantitative structure activity relationship that predicted the rank order of binding affinities for the series of inhibitors. Electronic profiles of the ligands in their docked conformations were characterized using electrostatic potential maps and frontier orbital calculations. These results led to the development of a 3D stereoelectronic pharmacophore which was used to construct 3D queries for database searches. A search of the National Cancer Institute's open database identified a lead compound that exhibited moderate antiviral activity.

Antiviral Agents↗
PubMed2004

Mutations in HIV-1 reverse transcriptase potentially associated with hypersusceptibility to nonnucleoside reverse-transcriptase inhibitors: effect on response to efavirenz-based therapy in an urban observational cohort.

BACKGROUND: Hypersusceptibility to nonnucleoside reverse-transcriptase inhibitors (NNRTIs) was described in association with reverse-transcriptase (RT) mutations conferring resistance to nucleoside reverse-transcriptase inhibitors (NRTIs). We evaluated the effect of RT mutations associated with hypersusceptibility to NNRTIs on the response to efavirenz-based therapy. METHODS: We analyzed an observational database of patients for whom highly active antiretroviral therapy failed and who received genotypic resistance testing-guided therapy, either efavirenz or protease inhibitor (PI) based. Study end points were achievement of virus load <80 copies/mL, achievement of virus load <80 copies/mL without rebound to >500 copies/mL, and changes in CD4 cell counts. RESULTS: The baseline RT mutations M41L, M184V, L210W, and T215Y and the M41L/T215Y and M41L/T215Y/M184V combinations were associated with virological suppression for efavirenz-treated patients, whereas, for PI-treated patients, only the M184V mutation was associated with virological suppression, and the L210W mutation showed a negative correlation; no correlation was found between any mutation and virological response without rebound. CONCLUSIONS: The M41L, M184V, L210W, and T215Y mutations were associated with a better, although transient, virological outcome in patients treated with efavirenz-based regimens.

Adult↗
PubMed2003

Probing the molecular mechanisms of AZT drug resistance mediated by HIV-1 reverse transcriptase using a transient kinetic analysis.

Several hypotheses have been proposed to explain the development of resistance to the anti-HIV drug AZT. Clinical findings show that AZT resistance mutations in HIV-1 reverse transcriptase (RT) not only reduce susceptibility to thymidine analogues but may also confer multi-dideoxynucleoside resistance. In this report, we describe transient kinetic studies establishing the biochemical effects of AZT resistance mutations in HIV-1 RT on the incorporation and removal of natural and unnatural deoxynucleotides. While the physiological role remains to be elucidated, the largest biochemical difference between wild-type and AZT resistant HIV-1 RT manifested itself during ATP-mediated deoxynucleotide removal. Enhanced removal resulted from an increase in the maximum rate of chain terminator excision, suggesting that mutated residues play a role in the optimal alignment of substrates for ATP-mediated removal. The efficiency of pyrophosphorolysis was not increased by the presence of AZT resistance mutations. However, a 2-fold decrease in the extent of inhibition caused by the next correct nucleotide during pyrophosphorolytic cleavage of a D4TMP chain-terminated primer may illustrate how this mutant can utilize pyrophosphate to enhance resistance. The inability of RT to catalyze removal of a chain terminator from an RNA-RNA primer-template may show how slight changes in selectivity against AZTMP incorporation during the initiation of DNA synthesis can contribute to high-level resistance. Taken together, these results suggest that multiple modes of resistance may be conferred by these mutations. Structure-activity studies of chain terminator removal suggest that analogues that form tight interactions with residues in the RT active site may be more prone to resistance mechanisms mediated by removal.

Adenosine Triphosphate↗
PubMed1990

Rapid purification of homodimer and heterodimer HIV-1 reverse transcriptase by metal chelate affinity chromatography.

We have modified an Escherichia coli vector expressing 66-kDa HIV-1 reverse transcriptase (p66) so that it simultaneously expresses this and the pol-coded protease. The twin expression cassette yields high quantities of both reverse transcriptase and protease; however, under these conditions, 50% of the over-expressed p66 reverse transcriptase is processed, resulting in accumulation of large quantities of p66/p51 enzyme. Furthermore, addition of a poly(histidine) affinity label at the amino terminus of the reverse-transcriptase-coding sequence (His-p66) permits a simple, rapid purification of milligram quantities of either p66 or p66/p51 enzyme from a crude lysate by metal chelate affinity chromatography. Purified His-p66 and His-p66/His-p51 reverse transcriptase exhibit both reverse transcriptase and RNase H activity. Purification by metal chelate chromatography of a p66/p51 enzyme wherein only the p66 component is labelled strengthens the argument for the existence of a heterodimer.

Chelating Agents↗
PubMed2002

Destabilization of the HIV-1 reverse transcriptase dimer upon interaction with N-acyl hydrazone inhibitors.

N-(4-tert-butylbenzoyl)-2-hydroxy-1-naphthaldehyde hydrazone (BBNH) inhibits both the DNA polymerase and ribonuclease H (RNase H) activities of the human immunodeficiency virus type 1 reverse transcriptase. In this study, we show that BBNH binding impacts on the stability of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) heterodimer. The Gibbs free energy of dimer dissociation of HIV-1 RT is decreased in the presence of increasing concentrations of BBNH, resulting in a loss in stability of 3.8 kcal mol(-1). To evaluate whether this observed phenomenon was mediated by BBNH binding to one or more sites in RT, we synthesized a variety of BBNH analogs and identified (4-t-butylbenzoyl)-2-hydroxy-1-salicylyl hydrazone (BBSH) and (4,N,N-dimethylaminobenzoyl)-2-hydroxy-1-naphthyl hydrazone as specific inhibitors of RT DNA polymerase or RT RNase H activity, respectively. Interestingly, only BBSH provided significant destabilization of the HIV-1 RT dimer. The identification of these specific inhibitors, in combination with other biochemical data, suggests a model in which two molecules of BBNH bind per RT heterodimer. In this regard, only the binding of hydrazone molecules in the DNA polymerase domain activity elicits the observed destabilization of the HIV-1 RT heterodimer.

DNA-Directed DNA Polymerase↗
PubMed2001

A highly prevalent polymorphism at codon 72 of HIV-1 reverse transcriptase in Argentina prevents hybridization reaction at codon 74 in the LiPA genotyping test.

The aim of this study was to compare the performance of two commercial drug-resistance HIV-1genotyping kits: LiPA (Innogenetics, Belgium) and TruGene (Visible Genetics, Canada). Samples from 103 HIV-1 infected individuals from Argentina were studied. The average correlation between the two methods was 92.81%. More codons could be analysed by TruGene than by LiPA (610/618 and 541/618 codons, respectively). The sequences of the samples and the LiPA probes were aligned showing that a silent mutation at codon 72 (AGA-->AGG) of the HIV-1 reverse transcriptase, which was not recognised by the LiPA probes, was present in 35/36 non-reactive samples for position 74. The overall prevalence of this polymorphism in the population studied was 39.81%. When sequences from different parts of the world were analysed 189/3395 (5.63%) carried the mutation at codon 72, while 23/133 (17.29%) of the sequences from Latin America (excluding Argentina) had this mutation. In both cases, the prevalence of this polymorphism in the Argentinean population was significantly higher. This highlights the importance of carrying out studies on the performance of genotyping kits outside the United States and Europe, especially in areas where non-B HIV-1 subtypes are prevalent.

Argentina↗
PubMed2004

QSAR modelling of HIV-1 reverse transcriptase inhibition by benzoxazinones using a combination of P_VSA and pharmacophore feature descriptors.

In pursuit of better anti-HIV drugs, a quantitative structure-activity relationship analysis using a novel set of 2D descriptors was performed on a series of HIV-1 reverse transcriptase inhibitory benzoxazinones. The QSAR models derived from the above mentioned descriptors were found to be statistically significant and exhibited superior predictive power. The results of the study justify the application of the descriptors for exploring the binding mode of the benzoxazinones to the enzyme.

Combinatorial Chemistry Techniques↗