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PubMed1995

Novel non-nucleoside inhibitors of HIV-1 reverse transcriptase. 3. Dipyrido[2,3-b:2',3'-e]diazepinones.

We have explored the potential of derivatives of the dipyrido[2,3-b:2',3'-e][1,4]diazepinone ring system as inhibitors of HIV-1 reverse transcriptase (RT). These compounds are isomeric to the potent RT inhibitor nevirapine and are available via a novel Smiles rearrangement on intermediates used for the synthesis of nevirapine analogs. Derivatives of this isomeric series are weaker inhibitors of RT than corresponding nevirapine analogs, although with appropriate substitution of the A- and C-pyridine rings activity can be improved.

Antiviral Agents↗
PubMed1999

Sequence analysis of the polymerase domain of HIV-1 reverse transcriptase in naive and zidovudine-treated individuals reveals a higher polymorphism in alpha-helices as compared with beta-strands.

We report a statistical analysis of genetic heterogeneity of the reverse transcriptase (RT)-coding region of human immunodeficiency virus type 1. Both newly determined sequences and sequences contained in the data banks have been examined. For the calculations, the viral samples and the regions within the RT molecule were divided in two groups. The viral samples were split into those from patients not subjected to antiretroviral therapy and those from patients treated with zidovudine (AZT, 3'-azido-3'-deoxythymidine) alone or in combination with other RT inhibitors. The RT-coding region was divided into segments encoding beta-strands and segments encoding alpha-helices. A significantly lower heterogeneity was observed in beta-strands relative to the alpha-helix coding segments. Application of the D test of Tajima has provided evidence of operation of negative (or purifying) selection in sequences from viruses of patients not subjected to antiretroviral treatment as well as in treated patients. In the group of untreated individuals, regions encoding beta-strands are subjected to stronger negative selection than those encoding alpha-helices. It is likely that the observed differences reflect stronger functional constraints in beta-strands than in alpha-helices of RT.

Adolescent↗
PubMedDate not supplied

Reverse transcriptase activity and Ty RNA are associated with virus-like particles in yeast.

The Ty element of yeast represents a class of eukaryotic transposons that show remarkable structural similarity to retroviral proviruses. Recently, these comparisons have been strengthened by a series of observations on the yeast Ty element: Ty transposes via an RNA intermediate; it contains a sequence (Fig. 1) which, when translated, is homologous to a conserved region found in all reverse transcriptases; a fusion protein encoded by Ty is produced by a frameshift event that is directly analogous to the production of Pr180gag-pol in a retrovirus such as Rous sarcoma virus. Here we identify the reverse transcriptase activity that, until now, has been presumed to mediate Ty transposition and show that it is sequestered in virus-like particles that also contain Ty RNA.

DNA Transposable Elements↗
PubMed2005

Quantification of the effects on viral DNA synthesis of reverse transcriptase mutations conferring human immunodeficiency virus type 1 resistance to nucleoside analogues.

Human immunodeficiency virus type I (HIV-1) reverse transcriptase (RT) resistance mutations reduce the susceptibility of the virus to nucleoside analogues but may also impair viral DNA synthesis. To further characterize the effect of nucleoside analogue resistance mutations on the efficiency and kinetics of HIV-1 DNA synthesis and to evaluate the impact of the depletion of deoxynucleoside triphosphates (dNTP) on this process, DNA synthesis was evaluated by allowing DNA synthesis to proceed with natural HIV-1 templates and primers, either within permeabilized viral particles or in newly infected cells, and quantifying the products by real-time PCR. Three recombinant viruses derived from three pNL4-3 molecular clones expressing mutations associated with resistance to zidovudine: a clone expressing RT mutation M184V, a clone expressing mutations M41L plus T215Y (M41L+T215Y), and clinical isolate BV34 (carrying seven resistance mutations). Following infection of P4 cells, the BV34 mutant, but not viruses expressing the M184V mutation or M41L+T215Y, exhibited a defect in DNA synthesis. Importantly, however, for mutants carrying the M184V mutation or M41L+T215Y mutations, a defect could be detected by using target cells in which dATP pools had been reduced by pretreatment with hydroxyurea. Based on these observations, we developed a recombinant-virus assay to assess the effects of hydroxyurea pretreatment on infectivity of viruses carrying plasma-derived RT sequences from patients with nucleoside resistance. Using this assay, we found that many, but not all, viruses carrying RT resistance mutations display an increased sensitivity to hydroxyurea, suggesting that the impact of RT resistance mutations on viral replication may be more profound in cell populations characterized by smaller dNTP pools.

Anti-HIV Agents↗
PubMed2002

Potency of nonnucleoside reverse transcriptase inhibitors (NNRTIs) used in combination with other human immunodeficiency virus NNRTIs, NRTIs, or protease inhibitors.

Efavirenz and a series of related quinazolinone nonnucleoside inhibitors of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) were evaluated in a series of two-drug combinations with several nucleoside RT inhibitors (NRTIs), nonnucleoside RT inhibitors (NNRTIs), and protease inhibitors (PIs). These combinations were tested in an established HIV-1 RT enzyme assay and a cell-based yield reduction assay with HIV-1 (replicative form [RF])-infected MT-2 cells. Synergy, additivity, and antagonism were determined in the two different assay systems by the method of Chou and Talalay (T.-C. Chou and P. Talalay, Adv. Enzyme Reg. 22:27-55, 1984). Efavirenz, DPC082, DPC083, DPC961, and DPC963 used in combination with the NRTIs zidovudine and lamivudine acted synergistically to inhibit RT activity in the HIV-1 RT enzyme assay and additively to slightly synergistically to inhibit HIV-1 (RF) replication in the yield reduction assay. The five NNRTIs in combination with the PI nelfinavir acted additively in the yield reduction assay to inhibit HIV-1 replication. Interestingly, efavirenz in combination with a second NNRTI acted additively to inhibit HIV-1 RT function in the enzyme assay, while it acted antagonistically to inhibit HIV-1 (RF) replication in the yield reduction assay. These data suggest that antiretroviral combination regimens containing multiple NNTRIs should be given thorough consideration before being used.

Alkynes↗
PubMed2005

In vitro HIV type 1 reverse transcriptase inhibitory activities of Thai medicinal plants and Canna indica L. rhizomes.

Water and 80% ethanol extracts of 20 Thai medicinal plants used to treat AIDS were tested for their HIV type 1 reverse transcriptase inhibitory activity. The water extracts of Ipomoea carnea subsp. fistulosa (aerial parts), Vitex glabrata (branch), Vitex trifolia (aerial part), Vitex negundo (aerial part), Canna indica (rhizome), and Justicia gendarussa (aerial part) showed HIV-1 RT inhibition ratio (% IR) higher than 90% at a 200 microg/ml concentration. The water extract of Canna indica rhizomes (IC(50) 22.56 microg/ml) was selected for further study, i.e. for its HIV-1 RT inhibition activity and the purification and characterization of the active proteins. Proteins in water extract were fractionated by ammonium sulfate precipitation and separated by sodium dodecyl sulfate acrylamide gel electrophoresis (SDS-PAGE), yielding two proteins, Cip31 (31 kDa) and Cip14 (14 kDa) with IC50 of 17.41 and 19.25 microg/ml and isoelectric point (pI) of 3.5 and 6.35, respectively. Both proteins showed significant HIV-1 RT inhibition.

HIV Reverse Transcriptase↗
PubMed2006

Compounds from rose (Rosa rugosa) flowers with human immunodeficiency virus type 1 reverse transcriptase inhibitory activity.

The aqueous extracts and ethanol precipitates of aqueous extracts of 18 medicinal herbs traditionally used in China were screened for their ability to inhibit human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) in-vitro. Among the samples screened at a concentration of 500 microg mL-1, dried rose (Rosa rugosa) flowers showed the strongest inhibition. The ethanol precipitate of the aqueous extract of R. rugosa was processed and two components (P1 and P2) were obtained after ion exchange chromatography on DEAE-cellulose. Then, P1-a (Mr 150 kDa) and P1-b (Mr 8 kDa) were isolated from P1 by gel filtration on Sephadex G-200. They inhibited the activity of HIV-1 RT with an IC50 of 158 nM and 148.16 microg mL-1 (18.5 microM), respectively. Further structural analyses revealed that P1-a was a polysaccharide-peptide complex, and P1-b was a polymer consisting of acteoside and acteoside derivatives identified by Fourier transform infrared spectroscopy, nuclear magnetic resonance, assays of carbohydrate and protein contents and high-performance liquid chromatography electrospray ionization mass spectrometry.

Chromatography, Gel↗
PubMed2006

Dimerization inhibitors of HIV-1 reverse transcriptase, protease and integrase: a single mode of inhibition for the three HIV enzymes?

The genome of human immunodeficiency virus type 1 (HIV-1) encodes 15 distinct proteins, three of which provide essential enzymatic functions: a reverse transcriptase (RT), an integrase (IN), and a protease (PR). Since these enzymes are all homodimers, pseudohomodimers or multimers, disruption of protein-protein interactions in these retroviral enzymes may constitute an alternative way to achieve HIV-1 inhibition. A growing number of dimerization inhibitors for these enzymes is being reported. This mini review summarizes some approaches that have been followed for the development of compounds that inhibit those three enzymes by interfering with the dimerization interfaces between the enzyme subunits.

Anti-HIV Agents↗
PubMed1995

Oxidative base damage in RNA detected by reverse transcriptase.

Oxidative base damage in DNA and metabolic defects in the recognition and removal of such damage play important roles in mutagenesis and human disease. The extent to which cellular RNA is a substrate for oxidative damage and the possible biological consequences of RNA base oxidation, however, remain largely unexplored. Since oxidatively modified RNA may contribute to the high mutability of retroviral genomic DNA, we have been interested in developing methods for the sequence specific detection of such damage. We show here that a primer extension assay using AMV reverse transcriptase (RT) can be used to reveal oxidatively damaged sites in RNA. This finding extends the currently known range of RNA modifications detectable with AMV reverse transcriptase. Analogous assays using DNA polymerases to detect base damage in DNA substrates appear to be restricted to lesions at thymine. Oxidative base damage in the absence of any detectable chain breaks was produced by dye photosensitization of RNA. Six out of 20 dyes examined were capable of producing RT detectable lesions. RT stops were seen predominantly at purines, although many pyrimidine sites were also detected. Dye specific photofootprints revealed by RT analysis suggests differential dye binding to the RNA substrate. Some of the photoreactive dyes described here may have potential utility in RNA structural analysis, particularly in the identification of stem-loop regions in complex RNAs.

Base Sequence↗
PubMed1989

Investigation of the stereochemical course of DNA synthesis catalysed by human immunodeficiency virus type 1 reverse transcriptase.

The Sp-isomer of thymidine 5'-O-(1-thiotriphosphate) is used as substrate by HIV-1 reverse transcriptase. The absolute configuration of the internucleotide linkage in the oligonucleotide product was identified by 31p NMR spectroscopy to be the Rp-isomer, indicating that incorporation of dTTP alpha S into the oligonucleotide proceeded with inversion of configuration at the alpha-phosphorus. The mechanistic implications of these observations are discussed.

Catalysis↗
PubMed1999

Histochemical localization of reverse transcriptase in polytene chromosomes of chironomids.

The localization of a reverse transcriptase-related protein in salivary gland polytene chromosomes was investigated by immunohistochemistry in two species of Chironomus. The antibodies used were raised against a recombinant protein containing phylogenetically conserved motifs of reverse transcriptases and derived from an abundant non-LTR element previously identified in Chironomus. Immunoreactive protein was found in some telomeres, in a centromeric region, in a few interstitial bands and in Balbiani ring 3. The telomeric signal was probably dependent on transcription and increased dramatically when telomeric heat shock puffs were induced. A correlation with transcription was also seen in Balbiani ring 3, the immunobinding of which disappeared after inhibition of transcription with actinomycin D.

Animals↗
PubMed1988

The influence of a double-stranded hindrance on DNA synthesis performed by DNA polymerase alpha, T4 DNA polymerase, DNA polymerase I (Klenow fragment) and AMV reverse transcriptase.

The influence of a double-stranded region on DNA synthesis performed by a series of DNA polymerases on a single-stranded template was studied. Two types of double-stranded hindrances were employed: a stable hairpin formed by the template alone and a region formed by the template and an extraneous oligonucleotide complementary to the template. While T4 and calf thymus alpha DNA polymerases are strongly arrested at the beginning of either of the two double-stranded hindrances, the Klenow fragment of E. coli DNA polymerase I and avian myeloblastosis virus reverse transcriptase can pass through the double-stranded regions. The DNA chain-elongation rate seems to be undisturbed in the case of reverse transcriptase but greatly reduced for DNA polymerase I.

Animals↗
PubMed1987

Conformational properties of 3'-azido-3'deoxy-thymidine (AZT), an inhibitor of HIV reverse transcriptase.

The low-energy conformations of 3'-azido-3'-deoxy-thymidine, (AZT), an inhibitor of retroviral reverse transcriptase, have been studied by molecular mechanics techniques. A force-field has been developed for the azido group by quantum-mechanical methods, and used in the analysis. The global low-energy structure of AZT has C3'-endo sugar pucker, an anti glycosidic angle, and a g+ C4'-C5' conformation. It is concluded that the AZT molecule has conformational properties that are very similar to those of standard deoxypyrimidines.

Antiviral Agents↗
PubMed2001

Trifluoromethyl-containing 3-alkoxymethyl- and 3-aryloxymethyl-2-pyridinones are potent inhibitors of HIV-1 non-nucleoside reverse transcriptase.

3-Alkoxymethyl- and 3-aryloxymethyl-2-pyridinones were synthesized and evaluated for activity as non-nucleoside reverse transcriptase inhibitors (NNRTIs) of HIV-1. It was found that several compounds were potent inhibitors of HIV-1 with the most potent compound 24 exhibiting an IC90 = 32 nM. Compound 24 also possessed a potent resistance profile as demonstrated by submicromolar IC90s against several clinically meaningful mutant virus strains.

Anti-HIV Agents↗
PubMed1993

A mutation in reverse transcriptase of bis(heteroaryl)piperazine-resistant human immunodeficiency virus type 1 that confers increased sensitivity to other nonnucleoside inhibitors.

Several nonnucleoside inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been described, including Nevirapine, thiobenzimidazolone (TIBO) derivatives, pyridinone derivatives such as L-697,661, and the bis(heteroaryl)piperazines (BHAPs). HIV-1 resistant to L-697,661 or Nevirapine emerges rapidly in infected patients treated with these drugs, and the resistance is caused primarily by substitutions at amino acids 181 and 103 of RT that also confer cross resistance to the other nonnucleoside inhibitors. We describe derivation and characterization of two BHAP-resistant HIV-1 variants that differ from this pattern of cross resistance. With both variants, HIV-1 resistance to BHAP RT inhibitors was caused by a RT mutation that results in a proline-to-leucine substitution at amino acid 236 (P236L). Rather than conferring cross resistance to other RT inhibitors, this substitution sensitized RT 7- to 10-fold to Nevirapine, TIBO R82913, and L-697,661 without influencing sensitivity to nucleoside analogue RT inhibitors. This sensitization caused by P236L was also observed in cell culture with BHAP-resistant HIV-1. The effects of the P236L RT substitution suggest that emergence of BHAP-resistant virus in vivo could produce a viral population sensitized to inhibition by these other nonnucleoside RT inhibitors.

Antiviral Agents↗
PubMed1998

Line probe assay for detection of human immunodeficiency virus type 1 mutations conferring resistance to nucleoside inhibitors of reverse transcriptase: comparison with sequence analysis.

We compared the line probe assay (LiPA) to sequence analysis for the detection of mutations conferring resistance to nucleoside inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Plasma samples from 40 patients who had received zidovudine, dideoxyinosine, and dideoxycytosine, alone or in combination, and who were enrolled in the ALTIS 2 clinical trial (lamivudine [3TC] plus stavudine) were tested at enrollment and at week 24. RT PCR products from plasma were used for LiPA, and DNA was used for sequence analysis. LiPA gave uninterpretable results for 8.5% of the analyzed codons corresponding to 63 samples, mainly for codons 41, 69, and 70. Several minor discrepancies between the two methods occurred, mainly due to the ability of LiPA to detect mixed populations while sequence analyses detect a single homogeneous population. LiPA is suitable for detecting mixed populations and easy to implement in clinical laboratories and might be useful for epidemiological surveys of primary HIV-1 resistance.

Anti-HIV Agents↗
PubMed2002

Pre-existing distortions in nucleic acid structure aid polypurine tract selection by HIV-1 reverse transcriptase.

Precise cleavage at the polypurine tract (PPT)/U3 junction by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase RNase H is critical for generating a correct viral DNA end for subsequent integration. Using potassium permanganate (KMnO(4)) modification, we have identified a significant distortion in the nucleic acid structure at the HIV-1 PPT/U3 junction in the absence of trans-acting factors. Unusually high reactivity of template thymine +1 is detected when the PPT primer is extended by DNA or RNA at its 3' terminus. Chemical footprinting suggests that the extent of base unstacking in the wild-type species is comparable when the +1 A:T base pair is replaced by a C:T mismatch. However, reactivity of this template base is diminished after alterations to upstream (rA)(4):(dT)(4) or (rG)(6):(dC)(6) tracts. Importantly, there is a correlation between the structural deformation at base pair +1 and precise cleavage at the PPT/U3 junction by HIV-1 reverse transcriptase/RNase H. KMnO(4) modification also revealed unusually high reactivity for one of two (dT)(4):(rA)(4) duplexes upstream of the PPT/U3 junction, suggesting a significant structural distortion within the PPT itself in the absence of the retroviral polymerase. Structural abnormalities in this region are not only essential for resistance of the PPT to hydrolysis but also significantly impact the conformation of the PPT/U3 junction. Our data collectively suggest that the entire PPT sequence contributes to the structural distortion at the PPT/U3 junction, potentially providing a mechanism for its selective processing.

Base Pair Mismatch↗
PubMed2006

Arylthiopyrrole (AThP) derivatives as non-nucleoside HIV-1 reverse transcriptase inhibitors: synthesis, structure-activity relationships, and docking studies (part 2).

Arylthio isopropyl pyridinylmethylpyrrolemethanols (AThPs) have been recently reported as a new class of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) inhibitors acting at the non-nucleoside binding site (NNBS) of this enzyme. Docking experiments of the potent inhibitors 4k (IC(50) = 0.24 microM, SI = 167) and 5e (IC(50) = 0.11 microM, SI > 1667) of wild-type RT prompted the synthesis and biological evaluation of novel AThP derivatives featuring a number of polar groups in position 3 of the pyrrole ring and larger and more hydrophobic alicyclic substituents in place of the isopropyl group at position 4. Among the compounds synthesized and tested in cell-based assays against HIV-1 infected cells, 19b was the most active, with EC(50) = 0.007 microM, CC(50) = 114.5 microm, and SI = 16357. This compound and its precursor 18b retained interesting activities against clinically relevant drug-resistant RT forms carrying K103N, Y181I, and L100I mutations. Docking calculations of 10, 14, 18b, and 19b were also performed to investigate their binding mode into the RT NNBS and to rationalize both structure-activity relationship and resistance data.

Binding Sites↗

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