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PubMed2002

Base pairing properties of 8-oxo-7,8-dihydroadenosine in cDNA synthesis by reverse transcriptases.

Incorporation of nucleotides opposite 8-oxo-7,8-dihydroadenosine (8-oxoA) in oligonucleotides with dNTPs by three reverse transcriptases (AMV-, MMRV-, RAV2-RT) in cDNA synthesis was studied. Guanine as well as thymine was incorporated preferentially by all reverse transcriptases. In the melting temperature experiment, 8-oxoA and 8-oxoA-Me formed base pairs with thymine and guanine with similar stabilities.

Autoradiography↗
PubMed2002

[Reversing malignant phenotypes of liver cancer cell lines with antisense gene to human telomerase reverse transcriptase].

OBJECTIVE: To explore the effect of antisense gene to human telomerase reverse transcriptase (hTRT) on reversing malignant phenotypes of liver cancer cell lines. METHODS: Sense and antisense eukaryotic expressing vector of hTRT gene was transfected into human liver cancer line HepG(2) with the DOTAP liposomal transfection method. Changes of cellular malignant phenotypes through proliferation capacity, telomerase activity, cloning formation in soft agar, invasive capacity in Borden's chamber model and tumorigenicity in nude mice were examined. RESULTS: Sense and antisense eukaryotic expressing vector was successfully transfected into HepG(2). The obtained transfectants termed HepG(2)-sense (HepG(2)-S) and HepG(2)-antisense (HepG(2)-AS) stably produced sense and antisense hTRT, respectively. HepG(2)-AS showed an obvious decrease in growth and telomerase activity. HepG(2)-AS penetrated cells through Matrigel were decreased significantly compared with HepG(2) and HepG(2)-S. Cloning efficiency in soft agar and tumorigenicity in nude mice was also markedly inhibited in HepG(2)-AS. CONCLUSIONS: Antisense gene to hTRT can significantly suppress cancer cell growth, partially reverse malignant phenotypes of HepG(2), which indicates that hTRT may be a new target gene for antisense gene therapy of liver cancer.

Animals↗
PubMed1996

Mutational studies of human immunodeficiency virus type 1 reverse transcriptase: the involvement of residues 183 and 184 in the fidelity of DNA synthesis.

The high error rates characteristic of human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) are a presumptive source of the viral hypermutability that impedes prevention and therapy of acquired immunodeficiency syndrome (AIDS). We have analyzed two mutants of HIV-1 RT by conducting a comparative study of the accuracy of DNA synthesis. Each mutant bears a single amino acid substitution adjacent to the two aspartic acid residues at positions 185 and 186 in the highly conserved DNA polymerase active site. The first mutant, Met 184-->Leu (M184L), displays a marked reduction in both misinsertion and mispair extension, suggesting a fidelity of DNA synthesis significantly higher than that of the wild-type HIV-1 RT. The second mutant, Tyr 183-->Phe (Y183F), shows a decrease in mispair extension with no significant change in misincorporation. Thus, the overall pattern of error-proneness of DNA synthesis is: wild-type HIV-1 RT > Y183F > M184L. Taken together, it is possible that residues 183 and 184 contribute to the low fidelity of DNA synthesis characteristic of the reverse transcriptases of HIV-1, HIV-2 and possibly, of other lentiviruses. Our observations may bear on the nature of potential mutations responsible for resistance to the nucleoside analogs used in chemotherapy of AIDS.

Base Sequence↗
PubMedDate not supplied

3D-quantitative structure-activity relationships of HEPT derivatives as HIV-1 reverse transcriptase inhibitors, based on Ab initio calculations.

Comparative molecular field analysis (CoMFA) has been applied to a large set of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) analogues. The starting geometry of HEPT was obtained from crystallographic data of HEPT/HIV-1 reverse transcriptase (RT) complexes. The structures of 101 HEPT derivatives were considered and fully optimized by ab initio molecular orbital calculations at the HF/3-21G level. The best CoMFA model is satisfactory in both statistical significance and predictive ability. It shows excellent, high predictive ability as r2cv = 0.858. The derived model indicates the importance of steric contributions (64.4%) as well as electrostatic interactions for the HIV-1 RT inhibition. In addition, steric and electrostatic contour maps from this analysis agree well with the experimentally observed trend that there are steric interactions between the side chain of HEPT and an aromatic ring of Tyr181. It is concluded that a moderately sized group at C5 enhances contact with Tyr181 enough to push it into a position which renders the protein nonfunctional, but a smaller group has insufficient steric requirements to do this and a larger group renders the ligand too large for the cavity. The mutation-induced resistance of reverse transcriptase is explained by this analysis. The obtained results not only lead to a better understanding of structural requirements of this set of compounds for the inhibition but also enable the suggestions for new and more potent drugs.

Algorithms↗
PubMed2006

Stampidine as a novel nucleoside reverse transcriptase inhibit with potent anti-HIV activity.

Stavudine (STV, d4T, 2',3'-didehydro-3'-deoxythymidine, CAS 3056-17-5) is a standard anti-HIV drug. Stampidine (STAMP, DDE-113, HI-113, N-[p-(4-bromophenyl)-2',3'-didehydro-3'-deoxy-5'-thymidylyl]-L-alanine methyl ester, CAS 217178-62-6) is a novel aryl phosphate derivative of stavudine with more potent anti-HIV activity and more favorable pharmacodynamic features. The remarkable potency of stampidine against clinical HIV-1 isolates with NRTI- or NNRTI-resistance (NRTI, nucleoside reverse transcriptase inhibitor; NNRTI, non-nucleoside reverse transcriptase inhibitor) warrants the further development of this new anti-HIV agent.

Anti-HIV Agents↗
PubMed1995

The structure of unliganded reverse transcriptase from the human immunodeficiency virus type 1.

The crystal structure of the reverse transcriptase (RT) from the type 1 human immunodeficiency virus has been determined at 3.2-A resolution. Comparison with complexes between RT and the polymerase inhibitor Nevirapine [Kohlstaedt, L.A., Wang, J., Friedman, J.M., Rice, P.A. & Steitz, T.A. (1992) Science 256, 1783-1790] and between RT and an oligonucleotide [Jacobo-Molina, A., Ding, J., Nanni, R., Clark, A. D., Lu, X., Tantillo, C., Williams, R. L., Kamer, G., Ferris, A. L., Clark, P., Hizi, A., Hughes, S. H. & Arnold, E. (1993) Proc. Natl. Acad. Sci. USA 90, 6320-6324] reveals changes associated with ligand binding. The enzyme is a heterodimer (p66/p51), with domains labeled "fingers," "thumb," "palm," and "connection" in both subunits, and a ribonuclease H domain in the larger subunit only. The most striking difference between RT and both complex structures is the change in orientation of the p66 thumb (approximately 33 degrees rotation). Smaller shifts relative to the core of the molecule were also found in other domains, including the p66 fingers and palm, which contain the polymerase active site. Within the polymerase catalytic region itself, there are no rearrangements between RT and the RT/DNA complex. In RT/Nevirapine, the drug binds in the p66 palm near the polymerase active site, a region that is well-packed hydrophobic core in the unliganded enzyme. Room for the drug is provided by movement of a small beta-sheet within the palm domain of the Nevirapine complex. The rearrangement within the palm and thumb, as well as domain shifts relative to the enzyme core, may prevent correct placement of the oligonucleotide substrate when the drug is bound.

Binding Sites↗
PubMed1996

Reverse transcriptase inhibitors suppress telomerase function and induce senescence-like processes in cultured mouse fibroblasts.

Spontaneous transformation of mouse embryonic fibroblasts in the presence of the reverse transcriptase inhibitors azidothymidine and carbovir led to the formation of telomerase-free clones. After prolonged cultivation of fibroblasts in the presence of carbovir, resistant cells with a very high level of telomerase activity were obtained. Azidothymidine and carbovir, but not dideoxycytidine, induced senescence-like processes in cultures of immortal mouse fibroblasts. After long-term incubation, cell proliferation gradually decreased, their morphology becoming similar to that of the senescent ones. The process was reversible: after inhibitor removal, the cells, including the giant ones, entered mitoses. All these data suggest that reverse transcriptase inhibitors block telomerase function in mouse cells.

3T3 Cells↗
PubMed1981

Evidence for two forms of reverse transcriptase in human placenta of a patient with breast cancer. Purification and biochemical characterization of the enzymes.

Two DNA polymerases with properties of viral RNA-directed DNA polymerase were found in the placenta of a patient with breast cancer. Both enzyme activities were purified by column-chromatographic procedures or by preparative isoelectric focusing. The most distinguishing feature of the two enzymes is their specificity to transcribe (rA)n . (dT)12 or (rC)n . (dG)18. The two enzymes differ with respect to their elution profiles from the phosphocellulose column, isoelectric point, molecular weight, bivalent-cation requirements and thermal stability. Serological analysis of the (rA)n . (dT)12-activated enzyme showed that this enzyme is immunologically not related to DNA polymerase-gamma, or to any of the reverse transcriptases purified from retroviruses of avian, murine and subprimate origin. However, the activity of this enzyme was neutralized by antibodies to reverse transcriptase purified from human spleen of a patient with myelofibrosis [Chandra & Steel (1977) Biochem. J. 167, 513-524]. Attempts to purify reverse transcriptase of normal human placenta were repeatedly unsuccessful. Once the crude homogenate of normal placenta was freed from endogenous nucleic acids, no (rC)n . (dG)18-dependent activity cold be detected.U

Breast Neoplasms↗
PubMed2000

Interaction of p55 reverse transcriptase from the Saccharomyces cerevisiae retrotransposon Ty3 with conformationally distinct nucleic acid duplexes.

The 55-kDa reverse transcriptase (RT) domain of the Ty3 POL3 open reading frame was purified and evaluated on conformationally distinct nucleic acid duplexes. Purified enzyme migrated as a monomer by size exclusion chromatography. Enzymatic footprinting indicate Ty3 RT protects template nucleotides +7 through -21 and primer nucleotides -1 through -24. Contrary to previous data with retroviral enzymes, a 4-base pair region of the template-primer duplex remained nuclease accessible. The C-terminal portion of Ty3 RT encodes a functional RNase H domain, although the hydrolysis profile suggests an increased spatial separation between the catalytic centers. Despite conservation of catalytically important residues in the RNase H domain, Fe(2+) fails to replace Mg(2+) in the RNase H catalytic center for localized generation of hydroxyl radicals, again suggesting this domain may be structurally distinct from its retroviral counterparts. RNase H specificity was investigated using a model system challenging the enzyme to select the polypurine tract primer from within an RNA/DNA hybrid, extend this into (+) DNA, and excise the primer from nascent DNA. Purified RT catalyzed each of these three steps but was almost inactive on a non-polypurine tract RNA primer. Our studies provide the first detailed characterization of the enzymatic activities of a retrotransposon reverse transcriptase.

Base Sequence↗
PubMed1994

Design of structure-based reverse transcriptase inhibitors.

Based on the crystallographic structure of the active site in the reverse transcriptase (RT) of human immunodeficiency virus (HIV), a group of hydrophobic polyadenylic acid (5') derivatives were designed and synthesized as inhibitors of the enzyme. These compounds were found to inhibit all six of the RTs tested, with IC50 = 10(-11)-10(-8) M, but did not inhibit either RNA polymerase II (even at 10(-5) M) or DNA polymerase I up to 10(-6) M inhibitor concentration. The underivatized poly(A) did not inhibit any of the RTs tested under the same conditions. In aqueous solutions of purified HIV-1 RT, poly-2'-O-(2,4-dinitrophenyl)-oligo(A) was found to inhibit the enzyme reversibly and compete with the primer-template poly(A)-(dT)12, whereas poly-2'-O-(3-fluoro-4,6-dinitrophenyl)-poly(A) was found to inactivate HIV-1 RT irreversibly by covalent labeling. A comparison of physicochemical properties of the hybrids poly(A)-poly(dT) and dinitrophenyl-poly(A)-poly(dT) shows that the hydrophobic dinitrophenyl groups stabilize double helical structures. These inhibitors were also found to be effective in keeping susceptible lymphocytes viable in the presence of HIV-1 (wild type). The effective inhibitor concentrations (EC50) were found to be 0.2-2.6 microgram/ml. No toxic effect on the host cells was found even at 100-1000-fold higher inhibitor concentrations.

Animals↗
PubMed2000

Comparison of a point mutation assay with a line probe assay for the detection of the major mutations in the HIV-1 reverse transcriptase gene associated with reduced susceptibility to nucleoside analogues.

This study compares the performance of a line probe assay (LiPA) for the detection of the major mutations associated with reduced sensitivity to nucleoside analogues with a well characterised point mutation assay (PMA). Plasma samples obtained from patients in a trial of four reverse transcriptase inhibitors (MRC Quattro Trial) were tested by both LiPA and PMA at baseline, 32nd and 64th weeks for the presence of drug resistance associated mutations in the reverse transcriptase (RT) gene. HIV-1 RNA was extracted from plasma by the Boom method and amplified by RT-PCR prior to being tested by LiPA or PMA. Assay discrepancies were further investigated by sequencing of the RT gene. Of 275 samples available from 98 trial subjects, 246 samples were successfully amplified by PCR and analysed by LiPA and PMA for six mutations. Of the 1476 individual codons analysed, LiPA successfully assayed 1444 (97.8%) and PMA gave a result with 1418 (96.1%). LiPA failed to give a result for 32 codons from 22 samples and PMA failed with 58 codons from 38 samples. Gross differences between the two assays, in which one scored a codon as wild-type only and the other as mutant only or vice versa, occurred at 28 codons analysed (1.9%) representing 26 samples from 20 subjects. Sequencing of 22 of the 26 samples confirmed the LiPA result in nine cases, the PMA result in 11 and detected a novel variant at codon 215 in four cases. The PMA and LiPA approach to the detection of the major mutations that are genotypically associated with reduced sensitivity to nucleoside analogues can correctly detect mutations in 97% of the cases.

Acetamides↗
PubMed1999

Structure-based design of non-nucleoside reverse transcriptase inhibitors of drug-resistant human immunodeficiency virus.

A computer model of reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1) was used to design thiourea compounds that were predicted to inhibit RT. The RT model was used to approximate how changes in binding pocket shape, volume and chemical properties resulting from residue mutations would affect inhibitor binding. Our lead compound, N-[2-(2,5-dimethoxyphenylethyl)]-N'-[2-(5-bromopyridyl)]-thi ourea (HI-236) was tested against clinically observed non-nucleoside inhibitor (NNI)-resistant mutated strains of HIV. HI-236 was more potent than trovirdine, MKC-442 and zidovudine against the drug-sensitive HIV-1 strain IIIB, 50-100 times more effective than delavirdine or nevirapine and twice as effective as our recently reported lead compound N-[2-(2-fluorophenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (HI-240) against the NNI-resistant Y181C mutant HIV-1 strain A17. HI-236 was highly effective against the multidrug-resistant HIV-1 strain RT-MDR containing multiple mutations involving the RT residues 74V, 41L, 106A and 215Y. In general, thiourea compounds such as HI-236 and HI-240 showed better inhibition of drug-resistant strains of HIV-1 than thioalkylbenzyl-pyrimidine compounds such as HI-280 and HI-281. The improved activity of thioureas against RT mutants is consistent with a structural analysis of the NNI binding pocket model of RT. The activity of HI-236 against RT-MDR was superior to that of other anti-HIV agents tested, in the following order, from high to low activity; HI-236 (IC50 5 nM), HI-240 (IC50 6 nM), trovirdine (IC50 20 nM), zidovudine (IC50 150 nM), MKC-442 (IC50 300 nM), delavirdine (IC50 400 nM) and nevirapine (IC50 5 microM).

Alanine↗
PubMed1992

Inhibition of human immunodeficiency virus type 1 reverse transcriptase by 3'-blocked oligonucleotide primers.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) (EC 2.7.7.49) with a high specific activity has been purified from the overexpressing Escherichia coli strain DH5 alpha [pJS3.7]. Steady-state kinetics of DNA synthesis catalysed by RT were analysed on polyriboadenylate 20-mer of (3'-5')deoxythymidylate [poly(rA).(dT)20] and polyribouridylate 20-mer of (3'-5')-deoxyadenylate [poly(rU).(dA)20] homopolymeric template-primers. Km values of 40 and 140 nM (3'-OH ends) and kcat values of 4 and 0.14 sec-1 were determined for the two different substrates. Oligonucleotide primers (dA)20 and (dT)20 were elongated in a terminal transferase-catalysed reaction (EC 2.7.7.31) with ddATP, 3'-dATP (cordycepin), 2',3'-epoxy-ATP and arabino-ATP; and ddTTP, 3'-azido-TTP, 3'-dUTP, 3'-F-dTTP and rUTP, respectively. The resulting oligonucleotides were hybridized to their complementary templates and the inhibitory potential of these compounds towards DNA synthesis started from unchanged primers was measured. Oligonucleotides with unextendable 3'-groups were shown to act as strong inhibitors of DNA synthesis catalysed by HIV-1 RT. In particular, poly(rA).(dT)20-[ddTMP] and poly(rU).(dA)20-[3'-dAMP] were potent competitive inhibitors, displaying Ki values of about 6 and 12 nM, respectively. Also 3'-azido-, and 3'-fluoro-terminated oligonucleotides showed competitive inhibition with inhibition constants in the range of 20-35 nM. In contrast, 2',3'-epoxy-terminated (dA)21 displayed a mixed-type inhibition with a Ki value of 67 nM. Arabino-terminated (dA)21 was found to be an uncompetitive inhibitor of HIV-1 RT with an inhibition constant of 318 nM. Arabino-terminated primers did not act as strict chain terminators because they could be elongated by HIV-1 RT. This study provides information on the structure-activity relationship of modified 3'-termini of primer molecules which might be exploited as inhibitors of HIV in the future.

Cloning, Molecular↗
PubMed2005

Analysis of human immunodeficiency virus type 1 reverse transcriptase subunit structure/function in the context of infectious virions and human target cells.

The reverse transcriptase (RT) of all retroviruses is required for synthesis of the viral DNA genome. The human immunodeficiency virus type 1 (HIV-1) RT exists as a heterodimer made up of 51-kDa and 66-kDa subunits. The crystal structure and in vitro biochemical analyses indicate that the p66 subunit of RT is primarily responsible for the enzyme's polymerase and RNase H activities. Since both the p51 and p66 subunits are generated from the same coding region, as part of the Pr160(Gag-Pol) precursor protein, there are inherent limitations for studying subunit-specific function with intact provirus in a virologically relevant context. Our lab has recently described a novel system for studying the RT heterodimer (p51/p66) wherein a LTR-vpr-p51-IRES-p66 expression cassette provided in trans to an RT-deleted HIV-1 genome allows precise molecular analysis of the RT heterodimer. In this report, we describe in detail the specific approaches, alternative strategies, and pitfalls that may affect the application of this novel assay for analyzing RT subunit structure/function in infectious virions and human target cells. The ability to study HIV-1 RT subunit structure/function in a physiologically relevant context will advance our understanding of both RT and the process of reverse transcription. The study of antiretroviral drugs in a subunit-specific virologic context should provide new insights into drug resistance and viral fitness. Finally, we anticipate that this approach will help elucidate determinants that mediate p51-p66 subunit interactions, which is essential for structure-based drug design targeting RT heterodimerization.

Blotting, Western↗
PubMed2006

Update on primary HIV-1 resistance in Argentina: emergence of mutations conferring high-level resistance to nonnucleoside reverse transcriptase inhibitors in drug-naive patients.

Here we present a survey including 52 drug-naive recently HIV-1-infected subjects from Buenos Aires City and province (79%) and 3 other regions in Argentina (21%). Recent infections were established from previous negative serology (32/52), indeterminate Western blot (12/52), or acute retroviral syndrome after high-risk HIV exposure (8/52) within 9 months before genotyping (median time, 4.2 months). Genotyping was performed from plasma by sequencing both protease and reverse transcriptase. Phylogenetic analysis combined with bootscanning resulted in 21 subtype B sequences and 31 B/F recombinants (RecBF). On protease, minor resistance-related mutations were found in both subtype B and RecBF with low frequencies. The substitution L89M, recently suggested as a resistance-related mutation in some subtype F viruses, was observed in 1 RecBF. On reverse transcriptase, major resistance-related mutations were found in 4 of 52 (7.7%) patients from different health centers: M41L (subtype B) and K103N+/-P225H (1 RecBF and 2 subtype B). The greater than 5% resistance threshold found indicates a need for sentinel resistance surveillances calling for an update in the current resistance testing guidelines in Argentina.

Argentina↗
PubMed1991

Human immunodeficiency virus reverse transcriptase T helper epitopes identified in mice and humans: correlation with a cytotoxic T cell epitope.

T cell immunity may be critical to development of a vaccine for human immunodeficiency virus (HIV-1). T helper epitopes were identified in three predominantly conserved regions in the HIV-1 reverse transcriptase by using reverse transcriptase-immunized mice of five major histocompatibility complex haplotypes. One peptide (residues 38-52) that stimulated H-2k T cells also contained an epitope recognized by cytotoxic T cells from the same mice and from HIV-infected patients. Such concordance between helper and cytotoxic T lymphocyte epitopes, observed in four cases, may be important in vaccine development. Peptide 36-52 was recognized by interleukin-2-producing peripheral blood T cells from 9 of 17 HIV-seropositive humans studied, of multiple human leukocyte antigen-DR and -DQ types. The broad recognition of this peptide by both helper and cytotoxic T cells substantiates its potential importance in a vaccine.

Adult↗
PubMed2004

The cellular pharmacology of nucleoside- and nucleotide-analogue reverse-transcriptase inhibitors and its relationship to clinical toxicities.

Nucleoside- and nucleotide-analogue reverse-transcriptase inhibitors (NRTIs) require intracellular phosphorylation for anti-human immunodeficiency virus (HIV) activity and toxicity. Long-term toxicities associated with NRTIs may be related to overactivation of this process. In vitro experiments have shown increased rates of NRTI and endogenous nucleoside phosphorylation to be associated with cellular activation. Patients with advanced HIV disease often have overexpression of cytokines, which corresponds to an elevated cellular activation state. These patients also have higher rates of NRTI phosphorylation and NRTI toxicity, suggesting an interaction between a proinflammatory biological state, NRTI phosphorylation, and toxicity. Studies suggest that women may have higher rates of NRTI phosphorylation than do men, as well as increased risk for NRTI-induced toxicity. Future research is needed to understand the NRTI activation process and improve the long-term toxicity profile of NRTIs. Such research should include comparisons of NRTI phosphorylation according to sex and cellular activation state (i.e., elevated vs. low).

Antiviral Agents↗
PubMed2002

Synthesis of 1-(3-phthalimido-2-oxobutyl)-4-substituted- phenylpiperazines and their anti-HIV reverse transcriptase activity.

AIM: Synthesis of 1-(3-phthalimido-2-oxobutyl)-4-substituted- phenylpiperazines (5-15). METHODS: The starting material nitrogen mustard hydrochloride (16), reacted with the corresponding substituted anilines to afford piperazine hydrochlorides (17-27), which were then coupled with 1-bromo-3-phthalimidobutan-2-one (4) to give the target compounds. RESULTS: Eleven target compounds (5-15) were synthesized, which were characterized by 1HNMR, IR and elemental analysis. CONCLUSION: Anti-HIV-1 RT using HIV reverse transcriptase P-66 protein test showed that compounds 11, 14, 10 and 13 possessed inhibitory effects against HIV-1 reverse transcriptase (RT), with IC50 29.80, 35.20, 43.77 and 63.76 mumol.L-1, respectively.

HIV Reverse Transcriptase↗