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PubMed2002

Inhibitors of HIV-1 reverse transcriptase and integrase: classical and emerging therapeutical approaches.

The rapid spread of the AIDS epidemic has stimulated the search for new agents able to arrest the replication of the causative virus, HIV. The best strategy for AIDS treatment involves a combination therapy using inhibitors of reverse transcriptase and protease. However, the emergence of HIV-1 strains resistant to these drugs and their cytotoxicity requires the synthesis and the biochemical and cellular characterization of new antiviral drugs, as well as the development of newer strategies and viral targets. In addition to reverse transcriptase and protease, other retroviral enzymes acting in the replicative cycle of HIV-1 are potential targets for chemotherapeutic intervention. Like all retroviruses, HIV-1 requires the integration of the proviral double-stranded DNA, arising from the reverse transcription step, into the host chromosome for its efficient replication, maintenance of a stably infected state and productive infection. DNA integration is carried out by integrase so this enzyme represents a key area in developing new anti-retroviral therapy. Another novel enzymatic target concerns the RNase H activity associated with the retroviral reverse transcriptase, since a functional RNase H is essential for retroviral replication. Inhibitors against HIV-1 integrase and RNase H having potential therapeutical propeties have not yet been described. We focus this review on the properties of inhibitors of reverse transcriptase and integrase. Some of these antiviral agents have been known for several years while others are emerging as new promising strategies based on the use of oligonucleotides with special emphasis on the SELEX approach, peptides and retrovirucides.

Acquired Immunodeficiency Syndrome↗
PubMed1976

Mechanism of interaction of avian myeloblastosis virus reverse transcriptase with avian myeloblastosis virus RNA.

The synthesis of DNA on avian myeloblastosis virus (AMV) RNA as the primer-template using AMV reverse transcriptase in vitro has been examined as a function of the concentrations of these components, as well as a function of the ionic strenth of the assay medium. The results are consistent with the hypothesis that two types of sites exist on the AMV RNA: inactive "dead-end" sites that merely bind the enzyme, and active binding sites that lead to DNA synthesis. Velocity sedimentation studies of reverse transcriptase reveal that the enzyme becomes a dimer (or oligomer) at low salt concentrations and it is at these concentrations that the two types of sites are evident on the RNA. At high salt concentration the enzyme, which exists primarily as a monomer, is inactive with AMV RNA, although it is active when poly(rA)dT10 is used as the primer-template. We have shown that inactive sites are not due to binding of the reverse transcriptase to nicked regions or to partially denatured RNA molecules. We deduce that inactive sites are those containing incorrect 4S primer molecules. These results are discussed in terms of the mechanism of the interaction of the reverse transcriptase with AMV RNA.

Avian Leukosis Virus↗
PubMed1998

4'-Acylated thymidine 5'-triphosphates: a tool to increase selectivity towards HIV-1 reverse transcriptase.

4'-Acylated thymidines represent a new class of DNA chain terminators, since they have been shown to act as post-incorporation chain-terminating nucleotides despite the presence of a free 3'-hydroxyl group. Here, we describe the action of the 4'-acetyl- (MeTTP) and 4'-propanoylthymidine 5'-triphosphate (EtTTP) on HIV-1 reverse transcriptase in RNA- and DNA-dependent DNA synthesis and on DNA synthesis catalyzed by the cellular DNA polymerases alpha, beta, delta and epsilon. MeTTP exhibits a high selectivity towards HIV-1 reverse transcriptase. By the use of the bulkier propanoyl group as the 4'-substituent of the nucleoside 5'-triphosphate, selectivity towards HIV-1 reverse transcriptase could be increased without affecting substrate efficiency. Thus, 4'-modifications may serve as a tool to increase selectivity towards HIV-1 reverse transcriptase.

Acylation↗
PubMed2004

Distinct effects of protease and reverse transcriptase inhibition in an immunological model of HIV-1 infection with impulsive drug effects.

We present an immunological model that considers the dynamics of CD4+ T cells interacting with free virions, reverse transcriptase inhibiting drugs and protease inhibiting drugs. We divide the T cells into multiple classes and use impulsive differential equations to describe the drug activity. As expected, we find that insufficient dosing of either drug corresponds to high viral load and a large population of infectious T cells. The model further predicts that, in the absence of physiological limits on tolerable drug concentrations, sufficiently frequent dosing with the reverse transcriptase inhibitor alone could theoretically maintain the CD4+ T cell count arbitrarily close to the T cell count in the uninfected immune system. However, for frequent dosing of the protease inhibitor alone, the limiting T cell populations may not be enough to maintain the immune system. Furthermore, frequent dosing of both drugs has the same net effect on the T cell population as frequent dosing of the reverse transcriptase inhibitor only. Thus, the two drug classes can have fundamentally different effects on the long-term dynamics and the reverse transcriptase inhibitor, in particular, plays a crucial role in maintaining the immune system. We also provide estimates for the dosing intervals of each drug that could theoretically sustain the T cell population at a desired level.

CD4 Lymphocyte Count↗
PubMed1989

Spermine stimulates RNA-dependent reverse transcriptase activity.

The polyamine spermine can not replace Mg2+ in the reactions catalyzed by avian myeloblastosis virus reverse transcriptase. In the presence of suboptimal Mg2+ concentrations, spermine enhanced the reverse transcriptase activities with viral 70 S RNA and synthetic polyriboadenylic acid as templates, about 4-fold and 2-fold, respectively. A similar effect occurred in the polyriboadenylic acid-directed reaction using Mn2+ as divalent cation. On the other hand, no stimulation by spermine was observed with activated DNA and polydeoxyadenylic acid as template in the reactions catalyzed by reverse transcriptase.

Avian Leukosis Virus↗
PubMed1999

Crystallographic analysis of the binding modes of thiazoloisoindolinone non-nucleoside inhibitors to HIV-1 reverse transcriptase and comparison with modeling studies.

We have determined the crystal structures of thiazoloisoindolinone non-nucleoside inhibitors in complex with HIV-1 reverse transcriptase to high-resolution limits of 2.7 A (BM +21.1326) and 2. 52 A (BM +50.0934). We find that the binding modes of this series of inhibitors closely resemble that of "two-ring" non-nucleoside reverse transcriptase inhibitors. The structures allow rationalization of stereochemical requirements, structure-activity data, and drug resistance data. Comparisons with our previous structures suggest modifications to the inhibitors that might improve resilience to drug-resistant mutant forms of reverse transcriptase. Comparison with earlier modeling studies reveals that the predicted overlap of thiazoloisoindolinones with TIBO was largely correct, while that with nevirapine was significantly different.

Crystallography, X-Ray↗
PubMed1989

Infectious potential of human immunodeficiency virus type 1 reverse transcriptase mutants with altered inhibitor sensitivity.

There is considerable interest in the potential of human immunodeficiency virus type 1 (HIV-1) to develop drug resistance, especially as 3'-azido-3'-deoxythymidine (Retrovir) is now in widespread clinical use to treat people with AIDS and AIDS-related complex (ARC). To address this possibility, mutations in the HIV reverse transcriptase [deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase (RNA-directed), EC 2.7.7.49] gene have been introduced by site-directed mutagenesis of cloned constructs in Escherichia coli. Analysis of the recombinant mutant reverse transcriptase from a number of these constructs revealed enzymes that maintained enzyme activity but had a reduced ability to recognize inhibitors such as azidothymidine triphosphate. To assess the infectivity of these mutants, several constructs of proviral HIV clones with mutant reverse transcriptase genes have been made and used to transfect T cells. All five mutants tested have lower infectious potential, suggesting considerable levels of reverse transcriptase activity are required for efficient virus replication. Viable virus recovered from two clones showed decreased sensitivity to the antiviral compound phosphonoformate, thus demonstrating the potential for drug-resistant HIV to replicate. However, although the reverse transcriptase from these mutant viruses showed decreased sensitivity to azidothymidine triphosphate, paradoxically these viruses were hypersensitive to azidothymidine when tested in culture.

Amino Acid Sequence↗
PubMed2003

HIV resistance to nevirapine and other non-nucleoside reverse transcriptase inhibitors.

Nevirapine and other members of the non-nucleoside reverse transcriptase inhibitor (NNRTI) family of anti-HIV-1 drugs are essential components of antiretroviral treatment regimens. Unfortunately, drug resistance has become an important issue with respect to all therapeutic targets in HIV-1. This paper summarizes current knowledge about the mutations in the reverse transcriptase gene of HIV-1 that are responsible for drug resistance and the mechanisms whereby drug resistance develops.

Drug Resistance, Viral↗
PubMed1999

Combination therapy with efavirenz, nelfinavir, and nucleoside reverse-transcriptase inhibitors in children infected with human immunodeficiency virus type 1. Pediatric AIDS Clinical Trials Group 382 Team.

BACKGROUND: Consistent long-term viral suppression has been difficult to achieve in children with human immunodeficiency virus type 1 (HIV-1) infection. We tested the safety and antiviral efficacy of a novel combination consisting of efavirenz, nelfinavir, and one or more nucleoside reverse-transcriptase inhibitors in 57 children previously treated with only nucleoside reverse-transcriptase inhibitors. METHODS: The children were monitored for 48 weeks after the initiation of therapy. We assessed plasma concentrations of efavirenz and nelfinavir, plasma HIV-1 RNA levels, and lymphocyte subpopulations. RESULTS: At base line, the 57 HIV-1-infected children (age range, 3.8 to 16.8 years) had a median of 699 CD4 cells per cubic millimeter and 10,000 copies of HIV-1 RNA per milliliter of plasma. The most common treatment-related effects of at least moderate severity were rash (in 30 percent of children), diarrhea (in 18 percent), neutropenia (in 12 percent), and biochemical abnormalities (in 12 percent). Serious side effects were uncommon. The mean values for the area under the curve for efavirenz and nelfinavir corresponded to expected values. In an intention-to-treat analysis, 76 percent of children had plasma HIV-1 RNA levels of less than 400 copies per milliliter after 48 weeks of therapy and 63 percent had levels of less than 50 copies per milliliter. A high plasma HIV-1 RNA level at base line significantly decreased the likelihood that plasma levels of HIV-1 RNA would become undetectable during treatment. CONCLUSIONS: In HIV-1-infected children who were previously treated with nucleoside reverse-transcriptase inhibitors, the combination of efavirenz, nelfinavir, and nucleoside reverse-transcriptase inhibitors was generally well tolerated and had a potent and sustained antiviral effect.

Adolescent↗
PubMed1999

HIV reverse transcriptase inhibitors of natural origin.

Inhibitors of HIV reverse transcriptase (RT) are important drugs for the treatment of acquired immuno-deficiency syndrome (AIDS). One approach to identify novel inhibitors of HIV-1-RT is the screening of natural compounds. Many natural products have been shown to be active as RT inhibitors. These compounds belong to a wide range of different structural classes, e.g., coumarins, flavonoids, tannins, alkaloids, lignans, terpenes, naphtho- and anthraquinones, and polysaccharides. The life forms from which the bioactive compounds were isolated are as equally diverse and comprise terrestrial and marine plants, micro-organisms, and marine animals. From the most extensive screening effort, carried out by the NCl, calanolide A, isolated from the terrestrial plant Calophyllum lanigerum (Guttiferae), has been discovered as the most interesting natural RT inhibitor. The promise of this natural product probably relates to a novel mechanism of action. The current review describes natural products from various sources that are able to inhibit HIV-RT.

Acquired Immunodeficiency Syndrome↗
PubMed2004

Nucleoside-analog resistance mutations in HIV-1 reverse transcriptase and their influence on polymerase fidelity and viral mutation rates.

Nucleoside-analog inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) were the first drugs used against the virus. It is long known that monotherapy with these and other drugs leads to the rapid development of viral resistance and it is being increasingly appreciated that a significant percentage of individuals receiving highly active antiretroviral therapy (HAART) also develop resistance. Considering the fact that RT is responsible both for optimal rate of replication and an accurate copying of the viral genome, the consequence of drug-resistance mutations in RT to the biochemistry of this enzyme and to the biology of the virus are critically important. The biochemistry of HIV-1 reverse transcriptase variants harboring nucleoside-analog resistance mutations has been studied extensively. In this review, we describe a number of studies into the polymerase fidelity of nucleoside-analog resistant HIV-1 reverse transcriptase as well as the mutation rate of HIV-1 harboring these mutations.

Antiretroviral Therapy, Highly Active↗
PubMed2004

In situ human telomerase reverse transcriptase expression pattern in normal and neoplastic ovarian tissues.

Telomerase is a ribonuclear protein reverse transcriptase that maintains telomere length in eukaryotic cells. Activation of telomerase has been implicated in human cellular immortalization and carcinogenesis. Telomerase activity in ovarian neoplasm has been studied using polymerase chain reaction (PCR)-based methods and shown to be correlated with malignancy. However, we believe those results must be interpreted with caution because such studies used a heterogeneous mix of cells, including normal cell type known to express telomerase when activated. The present study used in situ hybridization that allows determination of the type of cells expressing telomerase, as well as the intensity of that expression, in ovarian neoplasms. A total of 75 specimens were studied. Epithelial telomerase reverse transcriptase mRNA expression was detected in 28 of 31 epithelial ovarian carcinomas, 1 of 1 malignant granulosa cell tumor, 7 of 9 serous borderline ovarian tumors, 11 of 11 mucinous borderline ovarian tumors, 4 of 5 serous cystadenofibromas, 2 of 4 serous cystadenomas, 8 of 8 mucinous cystadenomas, and 0 of 6 normal ovaries except the corpus luteum. Telomerase expression is heterogeneously found in both benign and malignant epithelial tissues. We conclude that human telomerase reverse transcriptase mRNA expression does not seem to be a reliable marker for clinical use in differentiating between benign and malignant tumors.

Corpus Luteum↗
PubMedDate not supplied

Reverse transcriptase PCR and staging prostate cancer.

OBJECTIVE: To present a brief review of the diagnostic dilemma of staging prostate cancer and how a novel diagnostic technique, the reverse transcriptase polymerase chain reaction, is being used as an aid to better stage and manage the disease. DATA SOURCE: Research articles about prostate cancer and the reverse transcriptase polymerase chain reaction published in the last 5 years, as well as data gathered by the authors. STUDY SELECTION: Performed by the authors. DATA EXTRACTION: Performed by the authors. DATA SYNTHESIS: Prostate cancer is the most common cancer among men in the U.S. A wide variety of methods are used for the diagnosis; however, accurate staging of the disease to determine the most effective treatment is a problem. Because metastatic prostate cancer is routinely understaged, the reverse transcriptase polymerase chain reaction to identify prostate cancer cells in the circulatory system is becoming an important diagnostic aid for staging and monitoring the disease. It is analytically and clinically sensitive as well as specific. CONCLUSION: The reverse transcriptase polymerase chain reaction is a highly accurate aid in staging and monitoring prostate cancer. Its prognostic value, particularly when a small number of prostate cancer cells are detected in the circulatory system requires further long-term follow-up studies.

DNA, Neoplasm↗
PubMedDate not supplied

Prevalence of nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance-associated mutations and polymorphisms in NNRTI-naïve HIV-infected patients.

BACKGROUND: The efficacy of treatment containing nonnucleoside reverse transcriptase inhibitors (NNRTIs) could be compromised in NNRTI-naïve patients already harboring a virus resistant to NNRTIs. On the contrary, hypersusceptibility to NNRTIs in patients having failed nucleoside reverse transcriptase inhibitor (NRTI)-containing regimens has been described and has been associated with improved outcome. METHOD: We assessed the prevalence of NNRTI resistance-associated mutations or polymorphisms in 146 antiretroviral-naïve patients and in 181 HIV-infected patients who were given an NNRTI-based regimen. We phenotypically evaluated the NNRTI susceptibility of 41 strains presenting with amino acid substitutions at positions involved in NNRTI resistance. RESULTS: In the 268 genotypically analyzable samples, the overall prevalence of NNRTI resistance-associated mutations was 2% (6/268 patients). The prevalence of strains with amino acid substitutions at reverse transcriptase (RT) gene positions (A98, K101, K103, V106, V108, V179) involved in NNRTI resistance was 15%. Hypersusceptibility to NNRTI was rare (2%, 1/41) in those samples. RT substitutions at positions involved in NNRTI resistance were not associated with a significantly worse virologic outcome in NNRTI-treated patients. Our understanding of small shifts in IC50 values (higher or lower) toward NNRTI is very limited. The significance of many RT mutations on NNRTI susceptibility is not clear. CONCLUSION: In contrast to resistance mutations, RT substitutions at positions involved in NNRTI resistance are frequent. They are not associated with a worse virologic outcome or with decreased phenotypic susceptibility to NNRTIs. It may be prudent not to rule out the use of NNRTIs in patients with small shifts in IC50 values or poorly understood mutations.

Adult↗
PubMed2002

Template-competitive inhibitors of HIV-1 reverse transcriptase: design, synthesis and inhibitory activity.

We report the design, synthesis and activity studies on a novel class of template-competitive reverse transcriptase inhibitors (TCRTIs). The TCRTIs are 1,N(6)-etheno analogues of a series of dATP-based template-competitive DNA polymerase inhibitors synthesized in our laboratory (Moore, B. M.; Jalluri, R.; Doughty, M.B. Biochemistry 1996, 35, 11634). Thus, nucleotides 2-(4-azidophenacyl)thio-1,N(6)-etheno-2'-deoxyadenosine 5'-triphosphate 1, the tetrafluoro analogue 2-(4-azido-2,3,5,6-tetrafluorophenacyl)thio-1,N(6)-etheno-2'-deoxyadenosine 5'-triphosphate 2 and its analogues were synthesized by alkylation of 2-thio-1,N(6)-etheno-2'-deoxyadenosine 5'-monophosphate with the corresponding chloro- or bromo-alkyl halides and converted to the triphosphate. Kinetically, nucleotides 1 and 2 are both competitive inhibitors of reverse transcriptase versus template/primer with K(i)'s of 8.0 and 7.4 microM, respectively, and non-competitive inhibitors versus TTP with K(i)'s of 15 and 10 microM, respectively. Nucleotide 3, which differs from 1 only in that it lacks the etheno group, non-complementary nucleotide triphosphates, and related monophosphates and nucleosides, are completely inactive as inhibitors of reverse transcriptase at concentrations up to 1 mM. Photoinactivation of RT by 1 was both time- and concentration-dependent, and protected by template/primer but not by dNTPs. The concentration-dependent inactivation data gave a K(D,app) of 17.2 microM and maximum inactivation of 90%, and radiolabeled [beta, gamma-32P]-1 photoincorporated specifically and covalently into the p66 subunit of RT. Thus the photoinactivation data support our main conclusion from the kinetic data that this class of RT inhibitors are non-substrate and template-competitive.

Binding, Competitive↗
PubMed1993

Error-prone polymerization by HIV-1 reverse transcriptase. Contribution of template-primer misalignment, miscoding, and termination probability to mutational hot spots.

We have observed previously that DNA template-directed polymerization by the type 1 human immunodeficiency virus reverse transcriptase is error-prone for single-nucleotide substitution, addition and deletion errors at homopolymeric sequences. We have also noted strong termination of processive synthesis at these positions (Bebenek, K., Abbotts, J., Roberts, J. D., Wilson, S. H., and Kunkel, T. A. (1989) J. Biol. Chem. 264, 16948-16956). Here we have tested three models to explain errors at these hot spots: template-primer misalignment for deletion errors, and dislocation and direct miscoding for substitution errors. The approach involves introducing single-nucleotide changes within or flanking the homopolymeric hot spots and examining the effects that these changes have on human immunodeficiency virus type 1 (HIV-1) reverse transcriptase error rate, error specificity, and termination probability. The results obtained suggest that single-nucleotide deletion errors in homopolymeric runs result from template-primer misalignment and that both direct miscoding and template-primer dislocation contribute to the base substitution hot spots. The data also suggest that base substitution errors at one position can be templated by the preceding nucleotide or either of the next two nucleotides. Frameshift error rates at homopolymeric sites were affected by changes in the sequences flanking the runs, including single-nucleotide differences in the single-stranded template strand and in the double-stranded primer region as many as six nucleotides distant from the hot spot. Both increases and decreases in frameshift fidelity were observed, and most of these correlated with concomitant increases or decreases in the probability that HIV-1 reverse transcriptase terminated processive synthesis within the run. These data provide further support for a relationship between the frameshift fidelity and the processivity of DNA-dependent DNA synthesis by HIV-1 reverse transcriptase.

Base Sequence↗
PubMed1990

Expression, purification, and crystallization of the HIV-1 reverse transcriptase (RT).

The HIV-1 pol gene proteins (protease, reverse transcriptase, and endonuclease) were expressed in Escherichia coli N4830-1 by the use of the inducible expression vector pWS60 into which the pol gene was inserted. The p66/p51 heterodimer of reverse transcriptase (RT) was isolated in a highly pure and active form. Crystals of the p66/p51 heterodimer were obtained by the vapor diffusion hanging drop technique. The present crystal quality is still not adequate for high resolution X-ray investigation.

Crystallization↗
PubMedDate not supplied

[Reverse transcriptase and its biological role].

Elements with open-reading translation frames homologous to retroviral RNA-dependent DNA polymerases (reverse transcriptases) were found in eukaryotic cell genomes. Expression of endogenous reverse transcriptases in prokaryotic and eukaryotic cells is confirmed directly and indirectly. Evolution and function of reverse transcriptases are analyzed, and their phylogenetic and ontogenetic role is evaluated.

Biological Evolution↗