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PubMed2000

Novel 1,5-diphenylpyrazole nonnucleoside HIV-1 reverse transcriptase inhibitors with enhanced activity versus the delavirdine-resistant P236L mutant: lead identification and SAR of 3- and 4-substituted derivatives.

Through computationally directed broad screening, a novel 1, 5-diphenylpyrazole (DPP) class of HIV-1 nonnucleoside reverse transcriptase inhibitors (NNRTIs) has been discovered. Compound 2 (PNU-32945) was found to have good activity versus wild-type (IC(50) = 2.3 microM) and delavirdine-resistant P236L (IC(50) = 1.1 microM) reverse transcriptase (RT). Also, PNU-32945 has an ED(50) for inhibition of viral replication in cell cultures of 0.1 microM and was shown to be noncytotoxic with a CC(50) > 10 microM. Structure-activity relationship studies on the 3- and 4-positions of PNU-32945 led to interesting selectivity and activity within the class. In particular, the 3-hydroxyethyl-4-ethyl congener 29 is a potent inhibitor of the P236L mutant (IC(50) = 0.65 microM), whereas it is essentially inactive versus the wild-type enzyme (IC(50) > 50 microM). Furthermore, this compound was significantly more active versus the P236L mutant than delavirdine. The synthesis and RT inhibitory activity of various 3- and 4-substituted analogues are discussed.

Anti-HIV Agents↗
PubMed2004

A prospective evaluation of the diagnostic and potential prognostic utility of urinary human telomerase reverse transcriptase mRNA in patients with bladder cancer.

Human telomerase reverse transcriptase (hTERT) mRNA expression has been considered a surrogate marker for telomerase activity based on its parallel detection in urological malignancies, including transitional cell carcinoma (TCC) of the bladder. The objective of this study was to prospectively evaluate the diagnostic performance of urine hTERT mRNA marker and urine cytology in the detection of bladder cancer. The multiplex hTERT/GAPDH (glyceraldehyde-3-phosphate dehydrogenase) reverse transcription polymerase chain reaction (RT-PCR) assay was employed to assess hTERT mRNA expression in urine sediments from 43 patients with clinically apparent TCC undergoing transurethral resection. Tumor grade and pathological stage were determined. The results of urine cytology were compared with urine hTERT mRNA expression. The control group consisted of 46 age-matched healthy volunteers without known urinary tract disease. The sensitivity of hTERT mRNA expression marker in the detection of bladder cancer was significantly better than urine cytology (95% versus 65%, p<0.001). The hTERT mRNA was detected with high sensitivity in both low and high grade tumors, and in superficial and invasive phenotypes. No correlation was seen between hTERT mRNA and the histopathological grade and stage. The specificity of urinary hTERT mRNA marker was 93.5%. The detection of hTERT mRNA expression in urine was a highly sensitive marker for the diagnosis of TCC of the bladder in this study. This urine-based marker shows promise as a non-invasive adjunct to cystoscopy in patients undergoing bladder tumor surveillance.

Aged↗
PubMed1996

Detectionof reverse transcriptase activity in live attenuated virus vaccines.

BACKGROUND: Safety considerations require that biological products for human use are free from any agent that might pose a potential health hazard. One method to detect the presence of retroviral particles is the reverse transcriptase (RT) assay. This assay is capable of detecting all infectious retrovirus particles, irrespective of genome or protein composition. Recently, a family of ultrasensitive RT tests, named product-enhanced reverse transcriptase (PERT) assays, has been designed with a detection limit that is 10(6) - 10(7) times lower than that of conventional RT tests. OBJECTIVES: To investigate with the PERT assay whether RT activity is detectable in live attenuated virus vaccines and to characterize eventual RT activities. STUDY DESIGN: A total of 12 different monovalent and one trivalent virus vaccines containing live attenuated viruses were tested for RT activity with the PERT assay and a conventional RT test. RT activities were investigated with respect to their susceptibility to RT inhibitors, association with physical particles, and their possible origin. RESULTS: One trivalent and five different monovalent vaccines contained RT activity when tested with the PERT assay, but were negative in a conventional RT assay. All lots tested of these vaccines showed RT activity. The activity in all vaccines was sensitive to AZT-triphosphate and ddTTP and at least part of it was associated with particles. Mg(2+)-dependent RT activity banded at a density of 1.14 g/ml. All positive vaccines were produced using chicken cells. CONCLUSIONS: The data indicate the systematic presence of partially particle-associated retroviral reverse transcriptase in attenuated live virus vaccines that are produced in chicken-derived cells. The identification and further characterization of these particles, as well as the elucidation of possible interactions with the human organism are imperative goals despite the fact that these vaccines have been safely used for many years.

Journal Article↗
PubMed1999

Molecular cloning and characterization of AtTERT, a telomerase reverse transcriptase homolog in Arabidopsis thaliana.

On the basis of its predicted homology to human telomerase reverse transcriptase (hTERT), a cDNA for Arabidopsis thaliana TERT (AtTERT) has now been isolated from cultured cells. The cDNA contains an open reading frame of 3372 bp, encoding a protein with a predicted size of 131 kDa and isoelectric point of 9.9. The AtTERT protein contains the conserved reverse transcriptase motifs 1, 2 and A-E as well as the TERT-specific T motif. Reverse transcription-polymerase chain reaction analysis and an assay of telomerase activity revealed that both AtTERT mRNA and telomerase activity are abundant in shoot apical meristems but are not detectable in rosette leaves.

Amino Acid Sequence↗
PubMed2002

The molecular mechanism of multidrug resistance by the Q151M human immunodeficiency virus type 1 reverse transcriptase and its suppression using alpha-boranophosphate nucleotide analogues.

Nucleoside analogues are currently used to treat human immunodeficiency virus infections. The appearance of up to five substitutions (A62V, V75I, F77L, F116Y, and Q151M) in the viral reverse transcriptase promotes resistance to these drugs, and reduces efficiency of the antiretroviral chemotherapy. Using pre-steady state kinetics, we show that Q151M and A62V/V75I/F77L/F116Y/Q151M substitutions confer to reverse transcriptase (RT) the ability to discriminate an analogue relative to its natural counterpart, and have no effect on repair of the analogue-terminated DNA primer. Discrimination results from a selective decrease of the catalytic rate constant k(pol): 18-fold (from 7 to 0.3 s(-1)), 13-fold (from 1.9 to 0.14 s(-1)), and 12-fold (from 13 to 1 s(-1)) in the case of ddATP, ddCTP, and 3'-azido-3'-deoxythymidine 5'-triphosphate (AZTTP), respectively. The binding affinities of the triphosphate analogues for RT remain unchanged. Molecular modeling explains drug resistance by a selective loss of electrostatic interactions between the analogue and RT. Resistance was overcome using alpha-boranophosphate nucleotide analogues. Using A62V/V75I/F77L/F116Y/Q151M RT, k(pol) increases up to 70- and 13-fold using alpha-boranophosphate-ddATP and alpha-boranophosphate AZTTP, respectively. These results highlight the general capacity of such analogues to circumvent multidrug resistance when RT-mediated nucleotide resistance originates from the selective decrease of the catalytic rate constant k(pol).

Anti-HIV Agents↗
PubMed1991

Mutational analysis of two conserved sequence motifs in HIV-1 reverse transcriptase.

Two conserved sequence motifs, occurring in HIV-1 reverse transcriptase at residues 110-116 and 183-190, have been studied using site-directed mutagenesis of the cloned gene. In particular, aspartates at positions 185 and 186 have each been mutated to either asparagine or glutamate. The resulting mutant proteins were catalytically inactive but still able to bind the template-primer complex, poly rA-oligo dT. Other mutations in these regions resulted in reduced reverse trascriptase activity but the mutation of tyrosine-183 to serine caused a significant increase in the Km for dTTP and the Ki for inhibition by 3'-azidothymidine-triphosphate, 2',3'-dideoxythymidine-triphosphate and phosphonoformic acid.

Amino Acid Sequence↗
PubMed1994

Quantitative micro P30 and reverse transcriptase assays for Moloney murine leukemia virus.

An anti-P30 immunohistochemical and a reverse transcriptase assay for Moloney murine leukemia virus (MoMLV) are adapted to 96-well plates. The assay results are shown to be directly proportional to the number of infectious particles, and can therefore be used to estimate the infective titers of a virus preparation. The micro P30 assay yields a direct estimate of infectious centers, and the reverse transcriptase assay quantitates progeny from a single cycle of replication. The semi-automated nature of these assays is well suited to the analysis of a large number of samples and therefore permits the examination of the efficiency of the process of retroviral/MoMLV infection under varied times or conditions.

3T3 Cells↗
PubMed2006

Indolyl aryl sulphones as HIV-1 non-nucleoside reverse transcriptase inhibitors: synthesis, biological evaluation and binding mode studies of new derivatives at indole-2-carboxamide.

New non-nucleoside reverse transcriptase inhibitors (NNRTIs) that are active against the commonly occurring mutations of HIV are urgently needed for the treatment of AIDS. We synthesized new NNRTIs of the indolyl aryl sulphone (IAS) family, which are endowed with high antiviral potency against HIV-1 wt (wild-type), and the Y181C and K103N-Y181C drug resistant mutant strains. Several new compounds were highly active in lymphocytes infected with primary isolates carrying the K103N-V1081-M184V and L1001-V1081 mutations. The design of new IASs was based on three-dimensional quantitative structure-activity relationship (3D QSAR) studies and docking simulations. A cross-docking study was also undertaken to gain some insights in to the binding mode of the newly synthesized IASs in the wt and mutated isoforms of reverse transcriptase.

Cell Line↗
PubMed2005

Efficacy and tolerability of a nucleoside reverse transcriptase inhibitor-sparing combination of lopinavir/ritonavir and efavirenz in HIV-1-infected patients.

BACKGROUND: Recommended antiretroviral regimens include a nucleoside reverse transcriptase inhibitor (NRTI) component. Class cross-resistance and mitochondrial toxicity are recognized as problems with this class of antiretrovirals. METHODS: In a pilot open-label study, 65 antiretroviral-naive and 21 experienced but nonnucleoside reverse transcriptase inhibitor-naive HIV-1-infected adults were given a combination of lopinavir/ritonavir (533.3/133.3 mg twice daily) and efavirenz (600 mg once daily) for 48 weeks. RESULTS: At baseline, the mean viral load was 4.84 log10 copies/mL and the mean CD4 count was 311 cells/mm. At week 24, the proportions of patients with a viral load <400 copies/mL were 78% and 93% using an intent-to-treat and on-treatment analysis, respectively. At week 48, proportions were 73% and 97%, respectively. Treatment discontinuation occurred in 21 patients during the 48-week period, with 33% of those attributable to drug-related adverse effects. A viral load >400 copies/mL at week 24 or 48 was associated with nonadherence in 3 patients and virologic failure in 1 patient. After an increase during the first 8 weeks, fasting lipid levels remained stable up to 48 weeks. CONCLUSION: The lopinavir/ritonavir-efavirenz combination is associated with a high rate of virologic response and should be compared with more classic NRTI-containing regimens in randomized and controlled clinical trials.

Adolescent↗
PubMed2001

Glutamic residue 438 within the protease-sensitive subdomain of HIV-1 reverse transcriptase is critical for heterodimer processing in viral particles.

The biological form of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a heterodimer consisting of two polypeptides, p66 and p51, which have identical N-termini. The p51 polypeptide is generated by action of viral protease cleaving the p66 polypeptide between residues Phe440 and Tyr441. Dimerization has been mostly studied using bacterially purified RT bearing amino acid changes in either subunit, but not in the context of HIV-1 particles. We introduced changes of conserved amino acid residues 430-438 into the protease-sensitive subdomain of the p66 subunit and analyzed the reverse transcriptase processing and function using purified variants and their corresponding HIV-1 recombinant clones. Our mutational analysis shows that the conserved Glu438 residue is critical for proper heterodimerization and function of virion-associated RT, but not of bacterially expressed RT. In contrast, the conserved Glu430, Glu432, and Pro433 residues are not important for dimerization of virion-associated RT. The network of interactions made by the Glu438 carboxyl group with neighboring residues is critical to protect the Phe440-Tyr441 from cleavage in the context of the p66/p51 heterodimer and may explain why the p66/p51 is not processed further to p51/p51.

Amino Acid Sequence↗
PubMed2000

Salvage therapy with abacavir plus a non-nucleoside reverse transcriptase inhibitor and a protease inhibitor in heavily pre-treated HIV-1 infected patients. Swiss HIV Cohort Study.

BACKGROUND: Highly active antiretroviral therapy (HAART) may fail, especially in pre-treated patients. OBJECTIVE: To examine retrospectively whether heavily pre-treated patients not responding to HAART at least once respond to a salvage therapy with abacavir, a nucleoside reverse transcriptase inhibitor (NRTI) plus a non-nucleoside analogue reverse transcriptase inhibitor (NNRTI) and one or two protease inhibitors (PI). PATIENTS: We retrospectively identified and analysed patients followed in the Swiss HIV Cohort Study with > 1000 HIV RNA copies/ml on HAART, naive to abacavir who were switched to abacavir plus one NNRTI (efavirenz or nevirapine) and one or two PI which had not been used in the previous HAART. RESULTS: Of 23 identified HIV-infected patients with four (median) therapy changes before salvage, 10 patients (43%) achieved a decrease of plasma HIV RNA > 0.5 log10 at 6 months of therapy. After 6 months only two patients had an HIV-1 RNA < 500 copies/ml, one of them < 50 copies/ml. Seven patients increased their CD4 cell counts by > 30% above baseline. Three patients, all with CD4 cell counts < 100 x 10(6)/l before salvage therapy had a > 30% decline in CD4 cell count. An extended number of resistance-associated mutations was found in almost all patients at baseline. One patient had two new AIDS-defining events. Five patients (22%) discontinued treatment because of side-effects, mainly occurrence of a rash. CONCLUSION: Salvage therapy in intensively pre-treated patients has a low virological success rate despite usage of abacavir and NNRTI. Nevertheless, this did not correlate with immunological and clinical course. This study emphasizes the difficulty of second-line treatment in HIV-1 infection and stresses the need for new compounds.

Adult↗
PubMed1993

Human immunodeficiency virus reverse transcriptase. Expression in Escherichia coli, purification, and characterization of a functionally and structurally asymmetric dimeric polymerase.

Human immunodeficiency virus (HIV) reverse transcriptase isolated from viral particles contains two subunits, p51 and p66. We have produced both subunits in separate Escherichia coli strains using expression vectors. Stop codons were placed immediately after the codon for the carboxyl-terminal residue of the mature processed p51 and p66 subunits found in viral particles. Insertion of a methionine in front of the HIV protease cleavage site in the recombinant protein enabled synthesis of both subunits with the natural amino-terminal proline, since E. coli methionine aminopeptidase cleaves a Met-Pro amino-terminal linkage. That this occurred to an extent greater than 95% was confirmed by sequencing the purified subunits. Examination of the activities of the individual p51 and p66 subunits on a variety of templates and under solution conditions optimized for each subunit revealed a significant catalytic activity for the natural p51 subunit. This result contrasts to results reported earlier for many recombinant forms without the natural amino and/or carboxyl termini. As expected from earlier work, the optimal homopolymeric template for the p66 subunit was poly(rA). For the p51 subunit, poly(dC) was found to be the optimal template; its activity is 2- to 4-fold greater than p66 on poly(dC). The p51 subunit is 13- to 50-fold less active on poly(rC). These findings are discussed in the context of our earlier hypothesis (McHenry, C. S. (1989) in Molecular Biology of Chromosome Function (Adolph, K., ed) Chap. 5, Springer-Verlag, New York) that the HIV reverse transcriptase might be functionally asymmetric with distinct plus- and minus-strand polymerases.

Base Sequence↗
PubMed1999

A new class of anti-HIV agents: synthesis and activity of conjugates of HIV protease inhibitors with a reverse transcriptase inhibitor.

Conjugates of HIV protease inhibitors with a reverse transcriptase inhibitor were synthesized, which expressed excellent antiviral activity compared with that of the individual components. The remarkable antiviral activity of the conjugated compounds may be due to their penetration into the cell and later splitting into two different classes of anti-HIV agents.

Anti-HIV Agents↗
PubMed2006

Design of nevirapine derivatives insensitive to the K103N and Y181C HIV-1 reverse transcriptase mutants.

Nevirapine (Viramune) belongs to the first generation of non-nucleoside reverse transcriptase inhibitors (NNRTIs). Its efficiency is limited by drug resistant mutations, such as K103N and Y181C, so, the aim of this work was to design novel nevirapine analogues insensitive to the K103N and Y181C HIV-1 RT. 360 Nevirapine derivatives were designed using a combinatorial library design approach and these compounds were docked into the binding pocket of mutant HIV-1 RT enzyme structures, using the GOLD program. 124 Compounds having a GoldScore higher than that of nevirapine (55.00 and 52.00 for K103N and Y181C mutants, respectively) were first retrieved and submitted to a topological analysis with the SILVER program. Consequently, 31 compounds presenting a significant percentage of the surfaces buried upon binding (>80%) and exhibiting hydrogen bonds to either N103 or C181 residues of the HIV-RT were selected. To ensure that these compounds had hydrogen bonding interaction to either N103 or C181 residues, their interaction energies were estimated by quantum chemical calculations (QCCs). Finally, QCCs represent an alternative method for performing post docking procedure.

Anti-HIV Agents↗
PubMed1997

Resistance to nevirapine of HIV-1 reverse transcriptase mutants: loss of stabilizing interactions and thermodynamic or steric barriers are induced by different single amino acid substitutions.

The kinetic parameters governing the inhibition by Nevirapine of the RNA-dependent DNA synthesis catalyzed by HIV-1 reverse transcriptase have been determined by steady-state kinetic analysis with the wild-type enzyme and with mutant reverse transcriptases containing the single amino acid substitutions L100I, K103N, V106A, V179D, Y181I and Y188L. While the mutant V179D was inhibited by Nevirapine as the wild-type enzyme, all the other mutations displayed a 17 to 90-fold reduced sensitivity to the drug in the order: Y181I<(i.e. less sensitive) Y188L < V106A < L100I < K103N < wild-type. Determination of the rate constants for Nevirapine binding (kon) and dissociation (koff) for the mutant and wild-type enzymes showed that mutations L100I and V106A increased the koff values by 12 and 8.5-fold, respectively, without significantly affecting the kon, whereas mutation K103N decreased the kon 5-fold without increasing the koff. Mutations Y181I and Y188L, on the other hand, conferred resistance to Nevirapine affecting both koff and kon values. In addition, mutations L100I and Y181I reduced the catalytic potential of HIV-1 RT. Thus, Nevirapine resistance could arise from a combination of loss of stabilizing interactions and emergence of steric and thermodynamic barriers for drug binding, depending on the particular amino acid substitution involved.

Anti-HIV Agents↗
PubMed2005

Upstream stimulatory factor (USF) as a transcriptional suppressor of human telomerase reverse transcriptase (hTERT) in oral cancer cells.

Telomerase activity is suppressed in normal human somatic tissues but is activated in cancer cells and immortal cell lines. The reverse transcriptase (RT) subunit human telomerase reverse transcriptase (hTERT) is the key regulator of telomerase activity. The hTERT promoter contains E-box elements and may allow upstream stimulatory factor (USF), a basic helix-loop-helix (bHLH) leucine zipper family proteins, to bind and regulate the expression. In this study, we investigated whether and how USF effect on hTERT. Through luciferase reporter assays, we found that both USF1 and USF2 possess a comparable effect on the inhibition of hTERT expression. Immunoprecipitation (IP) and immunoblotting (IB) analysis reveal that the suppression of hTERT by USF was not through the interaction of USF with c-myc or mad, nor disturbed the cellular protein levels of those. In gel mobility shift and chromatin immunoprecipitation (CHIP) assays, we found that the USF suppression is through direct binding at the E-box site of hTERT promoter and rendering the effect actively. Analysis on clinical normal and tumor tissues reveal that the expression of USF1 and USF2 was lower in the tumor tissues, correlated with hTERT expression and telomerase activity. Taking together, our results demonstrate that USF is a negative transcriptional repressor for hTERT in oral cancer cells. It is possible that USF lose the inhibitory effect on hTERT expression leading to telomerase reactivation and oral carcinogenesis.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed1997

Gliotoxicity, reverse transcriptase activity and retroviral RNA in monocyte/macrophage culture supernatants from patients with multiple sclerosis.

In investigating a possible link between a novel retroviral agent (provisionally called MSRV), recently characterised in multiple sclerosis (MS), and the neuropathology of MS, it was found that there was a significant correlation between gliotoxicity and reverse transcriptase activity in monocyte/macrophage culture supernatants (MMCS) unique to MS patients. MMCS from healthy controls and patients with other neurological diseases did not display either gliotoxicity or reverse transcriptase activity. The observed gliotoxic effect was an initial, intermediate filament network disorganization and subsequent cell death which was specific to astrocytes and oligodendrocytes. The reverse transcriptase activity and MSRV-specific RNA were observed during the first 2 weeks of culture in MMCS from patients with active MS. The further elucidation of the molecular form(s) of this gliotoxic factor and its original source may be crucial in elucidating important etiopathogenic mechanisms in MS.

Animals↗