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Sample records for non-nucleoside reverse transcriptase

  1. [Non-nucleoside reverse transcriptase inhibitors].

    PubMed

    Joly, V; Yeni, P

    2000-06-01

    The non-nucleoside reverse transcriptase inhibitors (NNRTIs) directly inhibit the HIV-1 reverse transcriptase (RT) by binding in a reversible and non-competitive manner to the enzyme. The currently available NNRTIs are nevirapine, delavirdine, and efavirenz; other compounds are under evaluation. NNRTIs are extensively metabolized in the liver through cytochrome P450, leading to pharmacokinetic interactions with compounds utilizing the same metabolic pathway, particularly PIs, whose plasma levels are altered in the presence of NNRTIs. NNRTIs are drugs with a low genetic barrier, i.e. a single mutation in RT genoma induces a high-level of phenotypic resistance, preventing the use of NNRTIs as monotherapy. In naive patients, several trials have shown the value of NNRTIs in combination with nucleosides and/or protease inhibitors. Small pilot studies have shown that NNRTIs may be useful as second-line therapy. However, due to the rapid emergence of resistant virus to these compounds in case of incomplete viral suppression, NNRTIs should not be added to current failing antiretroviral regimen. The most common side-effect reported with nevirapine and delavirdine is rash. The incidence of rash is rather similar under these two compounds, but severe rash is less frequent with delavirdine. The most common adverse reactions reported with efavirenz are central nervous system complaints such as dizziness. Rash is reported less frequently than with nevirapine or delavirdine, and is usually mild. NNRTIs resistance mutations are located in the amino acid residues aligning the NNRTI-binding "pocket" site. High-level resistance is often associated with a single point mutation which develops within this site (especially codon groups 100 - 108 and 181 - 190). Patients failing on one NNRTI are very likely to possess multiple NNRTI resistance mutations. NNRTIs should always be used as part of a potent antiretroviral therapy to insure suppression of viral replication, thus circumventing

  2. Optimization of 5-aryloxyimidazole non-nucleoside reverse transcriptase inhibitors.

    PubMed

    Jones, Lyn H; Allan, Gill; Corbau, Romuald; Hay, Duncan; Middleton, Donald S; Mowbray, Charles E; Newman, Sandra D; Perros, Manos; Randall, Amy; Vuong, Hannah; Webster, Rob; Westby, Mike; Williams, David

    2008-11-01

    A major problem associated with non-nucleoside reverse transcriptase inhibitors (NNRTIs) for the treatment of HIV is their vulnerability to mutations in the allosteric binding site of reverse transcriptase that can result in the development of a resistant virus. Herein we present the optimization of a series of 5-aryloxy imidazoles, which possess a balanced pharmacological profile against both wild-type enzyme and the clinically relevant mutations K103N and Y181C. Subtle structural changes were used to probe structure-activity relationships relating to both potency and metabolic stability, which led to an imidazole derivative with an impressive overall profile.

  3. Rilpivirine: a novel non-nucleoside reverse transcriptase inhibitor.

    PubMed

    Garvey, Lucy; Winston, Alan

    2009-07-01

    Combination antiretroviral therapy has transformed the prognosis and life expectancy of HIV-1 infected individuals in resource-rich settings. British guidelines currently recommend the non-nucleoside reverse transcriptase inhibitor (NNRTI) efavirenz as part of first-line treatment in therapy-naive HIV-1 infected individuals. However, efavirenz is limited by its low genetic barrier to the development of resistance, together with its potential for CNS toxicities. To overcome these obstacles, several 'next-generation' NNRTIs are in various stages of clinical development. Here, we review the journey of rilpivirine (also known as TMC278, Tibotec), from the discovery of the chemical compound, through successful Phase I and II development, to its current position of being studied in international Phase III trials for the treatment of therapy-naive HIV-1 infected subjects using a 25 mg daily dose. Pharmacokinetic findings and food and drug interactions are discussed, together with safety profile. Rilpivirine has demonstrated high antiviral activity (including against NNRTI-resistant isolates) in vitro, with similar rates of virological suppression in therapy-naive individuals at 96 weeks when compared to efavirenz. Rilpivirine seems to be well tolerated with less CNS disturbance than efavirenz, and has non-teratogenic potential; however, unfavorable interactions with acid suppressant medications will require heightened vigilance when rilpivirine is used in widespread clinical practice.

  4. Novel indole-3-sulfonamides as potent HIV non-nucleoside reverse transcriptase inhibitors (NNRTIs)

    SciTech Connect

    Zhao, Zhijian; Wolkenberg, Scott E.; Lu, Meiqing; Munshi, Vandna; Moyer, Gregory; Feng, Meizhen; Carella, Anthony V.; Ecto, Linda T.; Gabryelski, Lori J.; Lai, Ming-Tain; Prasad, Sridar G.; Yan, Youwei; McGaughey, Georgia B.; Miller, Michael D.; Lindsley, Craig W.; Hartman, George D.; Vacca, Joseph P.; Williams, Theresa M.

    2008-09-29

    This Letter describes the design, synthesis, and biological evaluation of novel 3-indole sulfonamides as potent non-nucleoside reverse transcriptase inhibitors (NNRTIs) with balanced profiles against common HIV RT mutants K103N and Y181C.

  5. Novel indazole non-nucleoside reverse transcriptase inhibitors using molecular hybridization based on crystallographic overlays.

    PubMed

    Jones, Lyn H; Allan, Gill; Barba, Oscar; Burt, Catherine; Corbau, Romuald; Dupont, Thomas; Knöchel, Thorsten; Irving, Steve; Middleton, Donald S; Mowbray, Charles E; Perros, Manos; Ringrose, Heather; Swain, Nigel A; Webster, Robert; Westby, Mike; Phillips, Chris

    2009-02-26

    A major problem associated with non-nucleoside reverse transcriptase inhibitors (NNRTIs) for the treatment of HIV is their lack of resilience to mutations in the reverse transcriptase (RT) enzyme. Using structural overlays of the known inhibitors efavirenz and capravirine complexed in RT as a starting point, and structure-based drug design techniques, we have created a novel series of indazole NNRTIs that possess excellent metabolic stability and mutant resilience.

  6. Crystal structures of HIV-1 reverse transcriptase complexes with thiocarbamate non-nucleoside inhibitors.

    PubMed

    Spallarossa, Andrea; Cesarini, Sara; Ranise, Angelo; Ponassi, Marco; Unge, Torsten; Bolognesi, Martino

    2008-01-25

    O-Phthalimidoethyl-N-arylthiocarbamates (TCs) have been recently identified as a new class of potent HIV-1 reverse transcriptase (RT) non-nucleoside inhibitors (NNRTIs), by means of computer-aided drug design techniques [Ranise A. Spallarossa, S. Cesarini, F. Bondavalli, S. Schenone, O. Bruno, G. Menozzi, P. Fossa, L. Mosti, M. La Colla, et al., Structure-based design, parallel synthesis, structure-activity relationship, and molecular modeling studies of thiocarbamates, new potent non-nucleoside HIV-1 reverse transcriptase inhibitor isosteres of phenethylthiazolylthiourea derivatives, J. Med. Chem. 48 (2005) 3858-3873]. To elucidate the atomic details of RT/TC interaction and validate an earlier TC docking model, the structures of three RT/TC complexes were determined at 2.8-3.0A resolution by X-ray crystallography. The conformations adopted by the enzyme-bound TCs were analyzed and compared with those of bioisosterically related NNRTIs.

  7. Crystal structures of HIV-1 reverse transcriptase complexes with thiocarbamate non-nucleoside inhibitors

    SciTech Connect

    Spallarossa, Andrea Cesarini, Sara; Ranise, Angelo; Ponassi, Marco; Unge, Torsten; Bolognesi, Martino

    2008-01-25

    O-Phthalimidoethyl-N-arylthiocarbamates (TCs) have been recently identified as a new class of potent HIV-1 reverse transcriptase (RT) non-nucleoside inhibitors (NNRTIs), by means of computer-aided drug design techniques [Ranise A. Spallarossa, S. Cesarini, F. Bondavalli, S. Schenone, O. Bruno, G. Menozzi, P. Fossa, L. Mosti, M. La Colla, et al., Structure-based design, parallel synthesis, structure-activity relationship, and molecular modeling studies of thiocarbamates, new potent non-nucleoside HIV-1 reverse transcriptase inhibitor isosteres of phenethylthiazolylthiourea derivatives, J. Med. Chem. 48 (2005) 3858-3873]. To elucidate the atomic details of RT/TC interaction and validate an earlier TC docking model, the structures of three RT/TC complexes were determined at 2.8-3.0 A resolution by X-ray crystallography. The conformations adopted by the enzyme-bound TCs were analyzed and compared with those of bioisosterically related NNRTIs.

  8. Novel theoretically designed HIV-1 non-nucleoside reverse transcriptase inhibitors derived from nevirapine.

    PubMed

    Liu, Jinfeng; He, Xiao; Zhang, John Z H

    2014-10-01

    A common problem with non-nucleoside reverse transcriptase inhibitors (NNRTIs) of HIV-1 is the emergence of mutations in the HIV-1 RT, in particular Lys103 → Asn (K103N) and Tyr181 → Cys (Y181C), which lead to resistance to this entire class of inhibitors. In this study, we theoretically designed two new non-nucleoside HIV-1 RT inhibitors, Mnev-1 and Mnev-2, derived from nevirapine, in order to reduce the resistance caused by those HIV-1 RT mutations. The binding modes of Mnev-1 and Mnev-2 with the wild-type HIV-1 RT and its mutants (K103N and Y181C) were suggested by molecular docking followed by 20-ns molecular dynamics (MD) simulations in explicit water of those binding complexes (HIV-1 RTs with the new inhibitors). A molecular mechanics/generalized Born surface area (MM/GBSA) calculation was carried out for multiple snapshots extracted from the MD trajectory to estimate the binding free energy. The results of the calculations show that each of the new inhibitors forms a stable hydrogen bond with His235 during the MD simulations, leading to tighter binding of the new inhibitors with their targets. In addition, the repulsive interaction with Cys181 in the Y181C-nevirapine complex is not present in the novel inhibitors. The binding affinities predicted using the MM/GBSA calculations indicate that the new inhibitors could be effective at bypassing the drug resistance of these HIV-1 RT mutants.

  9. Clinical perspective on drug-drug interactions with the non-nucleoside reverse transcriptase inhibitor rilpivirine.

    PubMed

    Crauwels, Herta; van Heeswijk, Rolf P G; Stevens, Marita; Buelens, Annemie; Vanveggel, Simon; Boven, Katia; Hoetelmans, Richard

    2013-01-01

    Rilpivirine (TMC278) is a non-nucleoside reverse transcriptase inhibitor approved in combination with other antiretrovirals for the treatment of HIV-1 infection in treatment-naive adults (Edurant(®) 25 mg once daily; Complera(®) [USA]/Eviplera(®) [EU] once daily single-tablet regimen). Rilpivirine should be administered with a meal to optimize bioavailability. Its solubility is pH dependent. Rilpivirine is primarily excreted via the feces with negligible renal elimination. Rilpivirine is predominantly metabolized by cytochrome P450 3A4. There is no clinically relevant effect of age, gender, bodyweight, race, estimated glomerular filtration rate, or hepatitis B/C coinfection status on rilpivirine pharmacokinetics in adults. Drug-drug interactions were investigated with cytochrome P450 3A substrates, inducers and inhibitors, drugs altering intragastric pH, antiretrovirals, and other often coadministered drugs. Rilpivirine 25 mg once daily does not have a clinically relevant effect on exposure of coadministered drugs. Coadministration with cytochrome P450 3A inhibitors (ketoconazole, ritonavir-boosted protease inhibitors, telaprevir) results in increased rilpivirine plasma concentrations, but these are not considered clinically relevant; no dose adjustments are required. Coadministration of rilpivirine with cytochrome P450 3A inducers (e.g. rifampin, rifabutin) or compounds increasing gastric pH (e.g. omeprazole, famotidine) results in decreased rilpivirine plasma concentrations, which may increase the risk of virologic failure and resistance development. Therefore, strong cytochrome P450 3A inducers and proton-pump inhibitors are contraindicated. Histamine-2 receptor antagonists and antacids can be coadministered with rilpivirine, provided doses are temporally separated. No dose adjustments are required when rilpivirine is coadministered with: acetaminophen, phosphodiesterase type 5 inhibitors (sildenafil, etc.), atorvastatin (and other statins), oral

  10. Non-nucleoside reverse transcriptase inhibitors: a review on pharmacokinetics, pharmacodynamics, safety and tolerability

    PubMed Central

    Usach, Iris; Melis, Virginia; Peris, José-Esteban

    2013-01-01

    Introduction Human immunodeficiency virus (HIV) type-1 non-nucleoside and nucleoside reverse transcriptase inhibitors (NNRTIs) are key drugs of highly active antiretroviral therapy (HAART) in the clinical management of acquired immune deficiency syndrome (AIDS)/HIV infection. Discussion First-generation NNRTIs, nevirapine (NVP), delavirdine (DLV) and efavirenz (EFV) are drugs with a low genetic barrier and poor resistance profile, which has led to the development of new generations of NNRTIs. Second-generation NNRTIs, etravirine (ETR) and rilpivirine (RPV) have been approved by the Food and Drug Administration and European Union, and the next generation of drugs is currently being clinically developed. This review describes recent clinical data, pharmacokinetics, metabolism, pharmacodynamics, safety and tolerability of commercialized NNRTIs, including the effects of sex, race and age differences on pharmacokinetics and safety. Moreover, it summarizes the characteristics of next-generation NNRTIs: lersivirine, GSK 2248761, RDEA806, BILR 355 BS, calanolide A, MK-4965, MK-1439 and MK-6186. Conclusions This review presents a wide description of NNRTIs, providing useful information for researchers interested in this field, both in clinical use and in research. PMID:24008177

  11. Diarylaniline Derivatives as a Distinct Class of HIV-1 Non-nucleoside Reverse Transcriptase Inhibitors

    PubMed Central

    Qin, Bingjie; Jiang, Xingkai; Lu, Hong; Tian, Xingtao; Barbault, Florent; Huang, Li; Qian, Keduo; Chen, Chin-Ho; Huang, Rong; Jiang, Shibo; Lee, Kuo-Hsiung; Xie, Lan

    2010-01-01

    By using structure-based drug design and isosteric replacement, diarylaniline and 1,5-diarylbenzene-1,2-diamine derivatives were synthesized and evaluated against wild type HIV-1 and drug-resistant viral strains, resulting in the discovery of diarylaniline derivatives as a distinct class of next-generation HIV-1 non-nucleoside reverse transcriptase inhibitor (NNRTI) agents. The most promising compound 37 showed significant EC50 values of 0.003-0.032 μM against HIV-1 wild-type strains and of 0.005-0.604 μM against several drug-resistant strains. Current results also revealed important structure-activity relationship (SAR) conclusions for diarylanilines and strongly support our hypothesis that an NH2 group on the central benzene ring ortho to the aniline moiety is crucial for interaction with K101 of the NNRTI binding site in HIV-1 RT, likely by forming H-bonds with K101. Furthermore, molecular modeling studies with molecular mechanism/general born surface area (MM/GBSA) technology demonstrated the rationality of our hypothesis. PMID:20527972

  12. Conformational analysis of nevirapine, a non-nucleoside HIV-1 reverse transcriptase inhibitor, based on quantum mechanical calculations

    NASA Astrophysics Data System (ADS)

    Hannongbua, Supa; Prasithichokekul, Sirikanok; Pungpo, Pornpan

    2001-11-01

    The structure and the conformational behavior of the HIV-1 reverse transcriptase inhibitor, 11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[3,2-b2',3'-e][1,4]diazepin-6-one (nevirapine), is investigated by semiempirical (MNDO, AM1 and PM3) method, ab initio at the HF/3-21G and HF/6-31G** levels and density functional theory at the B3LYP/6-31G** level. The fully optimized structure and rotational potential of the nitrogen and carbon bond in the cyclopropyl ring were examined in detail. A similar geometrical minimum is obtained from all methods which shows an almost identical structure to the geometry of the molecule in the complex structure with HIV-1 reverse transcriptase. To get some information on the structure in solution, NMR chemical shift calculations were also performed by a density functional theory at the B3LYP/6-31G** level, using GIAO approximation. The calculated 1H-NMR and 13C-NMR spectra for the energy minimum geometry agree well with the experimental results, which indicated that the geometry of nevirapine in solution is very similar to that of the molecule in the inhibition complex. Furthermore, the obtained results are compared to the conformational studies of other non-nucleoside reverse transcriptase inhibitors and reveal a common agreement of the non-nucleoside reverse transcriptase inhibitors. The specific butterfly-like shape and conformational flexibility within the side chain of the non-nucleoside reverse transcriptase inhibitors play an important role inducing conformational change of HIV-1 reverse transcriptase structure and are essential for the association at the inhibition pocket.

  13. Drug interaction profile for GSK2248761, a next generation non-nucleoside reverse transcriptase inhibitor

    PubMed Central

    Piscitelli, Steve; Kim, Joseph; Gould, Elizabeth; Lou, Yu; White, Scott; de Serres, Mark; Johnson, Mark; Zhou, Xiao-Jian; Pietropaolo, Keith; Mayers, Douglas

    2012-01-01

    AIM To evaluate potential drug interactions with antiretroviral therapies or supportive therapies for use in conjunction with the once daily, next generation non-nucleoside reverse transcriptase inhibitor GSK2248761 in patients with HIV-1 infection. METHODS A series of phase I drug interaction studies was conducted. RESULTS GSK2248761 was shown to be a weak CYP3A4 and CYP2D6 inhibitor in a clinical study with a probe cocktail. Mean plasma concentration–time profiles for atazanavir, tenofovir disoproxil fumarate/emtricitabine (TDF/FTC), darunavir (DRV, administered with ritonavir [RTV]), and drospirenone/ethinylestradiol were similar following co-administration of GSK2248761. Plasma raltegravir AUC(0,τ) and Cmax increased by 18% with no change in Cτ when raltegravir was co-administered with GSK2248761. Lopinavir (LPV) plasma AUC(0,τ), Cmax and Cτ decreased by 23%, 14% and 40%, respectively, following administration of lopinavir/ritonavir with GSK2248761. Atorvastatin, rosuvastatin and simvastatin AUC(0,∞) and Cmax increased following co-administration with GSK2248761, with the largest changes observed for simvastatin (3.7-fold and 4.3-fold). Changes in maximum and extent of GSK2248761 exposure were marginal after co-administration with atazanavir, TDF/FTC and raltegravir compared with GSK2248761 administered alone. Co-administration of GSK2248761 with DRV/RTV and LPV/RTV increased plasma GSK2248761 exposures by 1.25- to ≤2-fold compared with GSK2248761 administered alone, and increases in GSK2248761 exposure were higher following single dose co-administration of DRV/RTV or LPV/RTV compared with multiple doses. There were few drug-related AEs, and no treatment-related trends in blood chemistry, haematology, urinalysis, vital signs or ECG findings. CONCLUSIONS These studies indicate that GSK2248761 was safe and well tolerated in healthy adults treated in these studies at the doses and duration of therapy evaluated. PMID:22288567

  14. Differential responses of human hepatocytes to the non-nucleoside HIV-1 reverse transcriptase inhibitor nevirapine.

    PubMed

    Fang, Jia-Long; Beland, Frederick A

    2013-01-01

    Nevirapine is a non-nucleoside reverse transcriptase (RT) inhibitor used for the treatment of AIDS and the prevention of mother-to-child transmission of HIV-1. Despite its therapeutic benefits, treatment with nevirapine has been associated with significant incidences of liver and dermal toxicity. The present study examined the effects of nevirapine on cell growth and death in human hepatocyte HepG2 cells and THLE2 cells and the possible pathways involved in these effects. The concentrations of nevirapine inhibiting 50% cell growth were similar for both cell lines. Nevirapine (0-250 µM) treatment caused a slight increase in the amount of lactate dehydrogenase released into the medium. Apoptotic cell death did not contribute to the decrease in viable cells. Exposing of HepG2 cells to nevirapine caused G2/M phase arrest, and the activity of senescence-associated β-galactosidase was not altered. In THLE2 cells, the percentage of cells in G1/G0 phase was increased and cellular senescence was induced in a concentration-dependent manner. Endogenous non-telomeric RT activity was not detected in either cell line. Western blot analysis indicated lower levels of p53 and phospho-p53 (ser15) in HepG2 cells as compared to THLE2 cells; no significant changes in p53 or phospho-p53 (ser15) were noted with nevirapine treatment. These data demonstrate that nevirapine inhibits cell growth, induces cell cycle arrest at different phases, and has different effects on cellular senescence in HepG2 cells and THLE2 cells. The differential responses appear to be related to differences in the basal levels of p53 in the HepG2 cells and THLE2 cells.

  15. Drug interaction profile for GSK2248761, a next generation non-nucleoside reverse transcriptase inhibitor.

    PubMed

    Piscitelli, Steve; Kim, Joseph; Gould, Elizabeth; Lou, Yu; White, Scott; de Serres, Mark; Johnson, Mark; Zhou, Xiao-Jian; Pietropaolo, Keith; Mayers, Douglas

    2012-08-01

    To evaluate potential drug interactions with antiretroviral therapies or supportive therapies for use in conjunction with the once daily, next generation non-nucleoside reverse transcriptase inhibitor GSK2248761 in patients with HIV-1 infection. A series of phase I drug interaction studies was conducted. GSK2248761 was shown to be a weak CYP3A4 and CYP2D6 inhibitor in a clinical study with a probe cocktail. Mean plasma concentration-time profiles for atazanavir, tenofovir disoproxil fumarate/emtricitabine (TDF/FTC), darunavir (DRV, administered with ritonavir [RTV]), and drospirenone/ethinylestradiol were similar following co-administration of GSK2248761. Plasma raltegravir AUC(0,τ) and C(max) increased by 18% with no change in Cτ when raltegravir was co-administered with GSK2248761. Lopinavir (LPV) plasma AUC(0,τ), C(max) and Cτ decreased by 23%, 14% and 40%, respectively, following administration of lopinavir/ritonavir with GSK2248761. Atorvastatin, rosuvastatin and simvastatin AUC(0,∞) and C(max) increased following co-administration with GSK2248761, with the largest changes observed for simvastatin (3.7-fold and 4.3-fold). Changes in maximum and extent of GSK2248761 exposure were marginal after co-administration with atazanavir, TDF/FTC and raltegravir compared with GSK2248761 administered alone. Co-administration of GSK2248761 with DRV/RTV and LPV/RTV increased plasma GSK2248761 exposures by 1.25- to ≤2-fold compared with GSK2248761 administered alone, and increases in GSK2248761 exposure were higher following single dose co-administration of DRV/RTV or LPV/RTV compared with multiple doses. There were few drug-related AEs, and no treatment-related trends in blood chemistry, haematology, urinalysis, vital signs or ECG findings. These studies indicate that GSK2248761 was safe and well tolerated in healthy adults treated in these studies at the doses and duration of therapy evaluated. © 2012 ViiV Healthcare. British Journal of Clinical Pharmacology

  16. Structure-enhanced methods in the development of non-nucleoside inhibitors targeting HIV reverse transcriptase variants.

    PubMed

    Frey, Kathleen M

    2015-01-01

    Resistance continues to emerge as a leading cause for antiretroviral treatment failure. Several mutations in HIV reverse transcriptase (RT) confer resistance to non-nucleoside inhibitors (NNRTIs), vital components of antiretroviral combination therapies. Since the majority of mutations are located in the NNRTI binding pocket, crystal structures of RT variants in complex with NNRTIs have provided ideas for new drug design strategies. This article reviews the impact of RT crystal structures on the multidisciplinary design and development of new inhibitors with improved resistance profiles.

  17. Synthesis and biological evaluation of alkenyldiarylmethane HIV-1 non-nucleoside reverse transcriptase inhibitors that possess increased hydrolytic stability.

    PubMed

    Cullen, Matthew D; Deng, Bo-Liang; Hartman, Tracy L; Watson, Karen M; Buckheit, Robert W; Pannecouque, Christophe; Clercq, Erik De; Cushman, Mark

    2007-10-04

    Non-nucleoside inhibitors of HIV reverse transcriptase (NNRTIs), albeit not the mainstays of HIV/AIDS treatment, have become increasingly important in highly active antiretroviral therapy (HAART) due to their unique mechanism of action. Several years ago our group identified the alkenyldiarylmethanes (ADAMs) as a potent and novel class of NNRTIs; however, the most active compounds were found to be metabolically unstable. Subsequent work has led to the synthesis of 33 analogues, with improved metabolic profiles, through the replacement of labile esters with various heterocycles, nitriles, and thioesters. As a result, a number of hydrolytically stable NNRTIs were identified with anti-HIV activity in the nanomolar concentration range. Furthermore, an improved pharmacophore model has been developed based on the new ADAM series, in which a salicylic acid-derived aryl ring is oriented cis to the side chain and the aryl ring that is trans to the side chain contains a hydrogen bond acceptor site within the plane of the ring.

  18. Synthesis, anti-HIV activity, and metabolic stability of new alkenyldiarylmethane HIV-1 non-nucleoside reverse transcriptase inhibitors.

    PubMed

    Deng, Bo-Liang; Hartman, Tracy L; Buckheit, Robert W; Pannecouque, Christophe; De Clercq, Erik; Fanwick, Phillip E; Cushman, Mark

    2005-09-22

    Non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs) are part of the combination therapy currently used to treat HIV infection. Based on analogy with known HIV-1 NNRT inhibitors, 18 novel alkenyldiarylmethanes (ADAMs) containing 5-chloro-2-methoxyphenyl, 3-cyanophenyl, or 3-fluoro-5-trifluoromethylphenyl groups were synthesized and evaluated as HIV inhibitors. Their stabilities in rat plasma have also been investigated. Although introducing 5-chloro-2-methoxyphenyl or 3-fluoro-5-trifluoromethylphenyl groups into alkenyldiarylmethanes does not maintain the antiviral potency, the structural modification of alkenyldiarylmethanes with a 3-cyanophenyl substituent can be made without a large decrease in activity. The oxazolidinonyl group was introduced into the alkenyldiarylmethane framework and found to confer enhanced metabolic stability in rat plasma.

  19. Investigation of the alkenyldiarylmethane non-nucleoside reverse transcriptase inhibitors as potential cAMP phosphodiesterase-4B2 inhibitors.

    PubMed

    Cullen, Matthew D; Cheung, York-Fong; Houslay, Miles D; Hartman, Tracy L; Watson, Karen M; Buckheit, Robert W; Pannecouque, Christophe; De Clercq, Erik; Cushman, Mark

    2008-02-15

    The alkenyldiarylmethanes (ADAMs) are currently being investigated as non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTIs) of potential value in the treatment of HIV infection and AIDS. During the course of these studies, a number of ADAM analogues have been identified that protect HIV-infected cells from the cytopathic effects of the virus by an unknown, HIV-1 RT-independent mechanism. Since the phosphodiesterase 4 family is required for HIV infection, the effect of various ADAMs on the activity of PDE4B2 was investigated in an effort to determine if the ADAMs could possibly be targeting phosphodiesterases. Six compounds representative of the ADAM class were tested for inhibition of cAMP hydrolysis by PDE4B2 enzymatic activity. Four ADAMs were found to be weak inhibitors of PDE4B2 and two of them were inactive. The experimental results are consistent with an antiviral mechanism that does not include inhibition of PDE4 isoforms.

  20. HEPT derivatives as non-nucleoside inhibitors of HIV-1 reverse transcriptase: QSAR studies agree with the crystal structures

    NASA Astrophysics Data System (ADS)

    Gaudio, Anderson Coser; Montanari, Carlos Alberto

    2002-04-01

    The interest in the non-nucleoside inhibitors (NNIs) to the reverse transcriptase (RT) as anti-AIDS agents has grown in the last ten years. The compound 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) is the precursor of the most studied class of NNIs, from which hundreds of derivatives have been synthesized and tested. There are at least twelve QSAR studies about the HEPT derivatives as RT inhibitors. Most of the predictions derived by these studies are related to the nature of the active site near the substituents at positions N-1 and C-5, and at the C-6 phenyl ring. The validity of these models has been checked against the 3-D structure of HIV 1 RT-HEPT complexes available. Most of these predictions were confirmed at the molecular level.

  1. Design, Synthesis, and Evaluation of Diarylpyridines and Diarylanilines as Potent Non-nucleoside HIV-1 Reverse Transcriptase Inhibitors

    PubMed Central

    Tian, Xingtao; Qin, Bingjie; Wu, Zhiyuan; Wang, Xiaofeng; Lu, Hong; Morris-Natschke, Susan L.; Chen, Chin Ho; Jiang, Shibo; Lee, Kuo-Hsiung; Xie, Lan

    2010-01-01

    Based on the structures and activities of our previously identified non-nucleoside reverse transcriptase inhibitors (NNRTIs), we designed and synthesized two sets of derivatives, diarylpyridines (A) and diarylanilines (B), and tested their anti-HIV-1 activity against infection by HIV-1 NL4-3 and IIIB in TZM-bl and MT-2 cells, respectively. The results showed that most compounds exhibited potent anti-HIV-1 activity with low nanomolar EC50 values, and some of them, such as 13m, 14c, and 14e, displayed high potency with subnanomolar EC50 values, which were more potent than etravirine (TMC125, 1) in the same assays. Notably, these compounds were also highly effective against infection by multi-RTI-resistant strains, suggesting a high potential to further develop these compounds as a novel class of NNRTIs with improved antiviral efficacy and resistance profile. PMID:21049929

  2. Non-nucleoside reverse transcriptase inhibitors: perspectives on novel therapeutic compounds and strategies for the treatment of HIV infection.

    PubMed

    Buckheit, R W

    2001-08-01

    At present, the nucleoside reverse transcriptase (RT) inhibitors and protease inhibitors (PI) have dominated the therapeutic options for the treatment of human immunodeficiency virus (HIV) infection. From the initial monotherapeutic strategies, to the widely accepted multi-drug cocktails of today, the use of these two classes of compounds has successfully prolonged patient survival following infection with HIV. The efficacy of the multi-drug cocktails has delayed the onset of disease and generated hope that long-term therapy might allow the natural immune response to HIV infection to control both virus replication and pathogenesis within the context of an intact immune system despite the continuing presence of virus in various reservoirs within the body and the inability of these therapies to completely eradicate virus. However, the use of antiretroviral compounds for prolonged periods of time has also resulted in the appearance of significant drug-induced toxicity and metabolic abnormalities, as well as drug-induced variations in disease progression. Thus, continued research and development to identify new and improved antiretroviral agents will be a critical requirement in the foreseeable future. This ongoing research and development should also consider the challenges of defining more effective use of existing therapeutic agents, including the non-nucleoside reverse transcriptase inhibitors (NNRTIs).

  3. Dipyridodiazepinone analogs as human immunodeficiency virus type 1-specific non-nucleoside reverse transcriptase inhibitors: an overview.

    PubMed

    Lv, M; Xu, H

    2010-01-01

    According to World Health Organization (WHO)/Joint United Nations Programme on human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) (UNAIDS) Report in 2007, 33.2 million people are living with HIV, 2.5 million ones have been newly infected with HIV, and 2.1 million ones died from AIDS, including 330,000 children. Therefore, HIV/AIDS still remains a public health emergency and a leading cause of mortality worldwide. It is believed that reverse transcriptase (RT) is a crucial enzyme in the life cycle of HIV-1, and thereby RT has been the important drug target for antiretroviral (ARV) chemotherapy against AIDS. To our knowledge, dipyridodiazepinone analogs have been considered as one class of potential non-nucleoside reverse transcriptase inhibitors (NNRTIs), especially the structurally and chemically related nevirapine (Viramune(R)), which was the first NNRTI approved by the U. S. Food and Drug Administration (FDA) for the treatment of HIV-1 infection for adults in 1996 and for children in 1998. This review mainly highlights the progress of synthesis and structure-activity relationship (SAR) of dipyridodiazepinone analogs; in the meantime, the mechanism of action is also presented. It will pave the way for the design and development of novel dipyridodiazepinone analogs as NNRTIs in AIDS chemotherapy in the future.

  4. Exploring isoxazole and carboxamide derivatives as potential non-nucleoside reverse transcriptase inhibitors.

    PubMed

    Kurup, Sudheer S; Joshi, Kaustubh A

    2016-04-01

    Nonnucleoside reverse transciptase inhibitors (NNRTI) are a class of drug molecules with a specific target of HIV-1 reverse transcriptase (RT). In the present work, we evaluated a set of selected oxazole and carboxamide derivatives to identify potential pharmacophoric features using molecular docking approach. The docking approach employed has been validated by enrichment factor calculation at top 1% (EF1%). It shows a considerable improvement in EF1%value compared to earlier reported study carried out on specific dataset of ligands and decoys for RT, in the directory of useful decoys (DUD). The carboxamide derivatives show better activity as NNRT inhibitors than oxazole derivatives. From this study, four pharmacophoric groups including a triazine ring, an aniline substituent, a benzyl amide moiety and a trimethylphenoxy substituent have been recognized and used for designing new NNRT inhibitors. Newly designed molecules show significant enhancement in docking scores over the native ligand, parent and other training set molecules. In addition, some functional groups have also been identified to assist in improving the activity of these pharmacophores. Thus a nitrile group, an amide and fluoro substitution turn out to be an important requisite for NNRT potential inhibitors. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Rilpivirine resistance mutations in HIV patients failing non-nucleoside reverse transcriptase inhibitor-based therapies.

    PubMed

    Anta, Lourdes; Llibre, Josep M; Poveda, Eva; Blanco, José L; Alvarez, Marta; Pérez-Elías, María J; Aguilera, Antonio; Caballero, Estrella; Soriano, Vicente; de Mendoza, Carmen

    2013-01-02

    Rilpivirine (RPV) is the latest approved nonnucleoside reverse transcriptase inhibitor (NNRTI). It displays in-vitro activity extending over other NNRTI-resistant HIV strains. There is scarce information about the rate of RPV resistance-associated mutations (RAMs) in patients failing other NNRTIs. RPV RAMs were examined in plasma samples collected from HIV patients that had recently failed NNRTI-based regimens at 22 clinics in Spain. Resistance tests from a total of 1064 patients failing efavirenz (EFV) (54.5%), nevirapine (NVP) (40%) or etravirine (ETR) (5.5%) were examined. The prevalence of RPV RAMs was K101E (9.1%), K101P (1.4%), E138A (3.9%), E138G (0.3%), E138K (0.3%), E138Q (0.8%), V179L (0.2%), Y181C (21.8%), Y181I (0.5%), Y181V (0.2%), H221Y (8.3%), F227C (0.1%) and M230L (1.5%). K101E/M184I was seen in 1%. E138K/M184I were absent. Mutations L100I and V108I were significantly more frequent in patients failing EFV than NVP (7.9 vs. 0.2 and 12.2 vs. 7.3%, respectively). Conversely, Y181C, Y181I, V106A, H221Y and F227L were more prevalent following NVP than EFV failures. Using the Spanish resistance interpretation algorithm, 206 genotypes (19.3%) from patients failing NNRTI (NVP 52%, EFV 40.8% and ETR 7.8%) were considered as RPV resistant. In patients with ETR failure, cross-resistance to RPV was seen in 27.6%, mainly as result of Y181C (81.3%), V179I (43.8%), V90I (31.3%) and V108I (18.8%). RPV resistance is overall recognized in nearly 20% of patients failing other NNRTIs. It is more common following ETR (27.6%) or NVP (25%) failures than EFV (14.5%). E138 mutants are rarely seen in this context.

  6. Structure-Based Evaluation of Non-nucleoside Inhibitors with Improved Potency and Solubility That Target HIV Reverse Transcriptase Variants

    PubMed Central

    2015-01-01

    The development of novel non-nucleoside inhibitors (NNRTIs) with activity against variants of HIV reverse transcriptase (RT) is crucial for overcoming treatment failure. The NNRTIs bind in an allosteric pocket in RT ∼10 Å away from the active site. Earlier analogues of the catechol diether compound series have picomolar activity against HIV strains with wild-type RT but lose potency against variants with single Y181C and double K103N/Y181C mutations. As guided by structure-based and computational studies, removal of the 5-Cl substitution of compound 1 on the catechol aryl ring system led to a new analogue compound 2 that maintains greater potency against Y181C and K103N/Y181C variants and better solubility (510 μg/mL). Crystal structures were determined for wild-type, Y181C, and K103N/Y181C RT in complex with both compounds 1 and 2 to understand the structural basis for these findings. Comparison of the structures reveals that the Y181C mutation destabilizes the binding mode of compound 1 and disrupts the interactions with residues in the pocket. Compound 2 maintains the same conformation in wild-type and mutant structures, in addition to several interactions with the NNRTI binding pocket. Comparison of the six crystal structures will assist in the understanding of compound binding modes and future optimization of the catechol diether series. PMID:25700160

  7. Structure-based evaluation of non-nucleoside inhibitors with improved potency and solubility that target HIV reverse transcriptase variants.

    PubMed

    Frey, Kathleen M; Puleo, David E; Spasov, Krasimir A; Bollini, Mariella; Jorgensen, William L; Anderson, Karen S

    2015-03-26

    The development of novel non-nucleoside inhibitors (NNRTIs) with activity against variants of HIV reverse transcriptase (RT) is crucial for overcoming treatment failure. The NNRTIs bind in an allosteric pocket in RT ∼10 Å away from the active site. Earlier analogues of the catechol diether compound series have picomolar activity against HIV strains with wild-type RT but lose potency against variants with single Y181C and double K103N/Y181C mutations. As guided by structure-based and computational studies, removal of the 5-Cl substitution of compound 1 on the catechol aryl ring system led to a new analogue compound 2 that maintains greater potency against Y181C and K103N/Y181C variants and better solubility (510 μg/mL). Crystal structures were determined for wild-type, Y181C, and K103N/Y181C RT in complex with both compounds 1 and 2 to understand the structural basis for these findings. Comparison of the structures reveals that the Y181C mutation destabilizes the binding mode of compound 1 and disrupts the interactions with residues in the pocket. Compound 2 maintains the same conformation in wild-type and mutant structures, in addition to several interactions with the NNRTI binding pocket. Comparison of the six crystal structures will assist in the understanding of compound binding modes and future optimization of the catechol diether series.

  8. Resistance to the most recent protease and non-nucleoside reverse transcriptase inhibitors across HIV-1 non-B subtypes.

    PubMed

    Anta, Lourdes; Blanco, José L; Llibre, Josep M; García, Federico; Pérez-Elías, María J; Aguilera, Antonio; Pérez-Romero, Pilar; Caballero, Estrella; Vidal, Carmen; Cañizares, Angelina; Gutiérrez, Félix; Dalmau, David; Iribarren, José A; Soriano, Vicente; de Mendoza, Carmen

    2013-09-01

    Limited data are available on resistance to etravirine, rilpivirine, darunavir and tipranavir in patients infected with HIV-1 non-B subtypes, in which natural polymorphisms at certain positions could influence the barrier and/or pathways to drug resistance. FASTA format sequences from the reverse transcriptase and protease genes recorded within the Spanish Drug Resistance database (ResRIS) were examined. From 8272 genotypes derived from 5930 different HIV-1 patients included in ResRIS, 5276 genotypes had complete treatment information. Overall, 85% were from antiretroviral-experienced subjects and 7.5% belonged to HIV-1 non-B subtypes: CRF02_AG, C, F and G being the most prevalent variants. For etravirine, only G190A was more prevalent in B than non-B subtypes, whereas V90I and V179E were more frequent in non-B than B subtypes. For rilpivirine, V108I and Y188I were more frequent in B than non-B subtypes, whereas V90I was more prevalent in non-B subtypes. Despite these differences, the overall prevalence of resistance did not differ significantly when comparing etravirine or rilpivirine in B versus non-B subtypes (11.3% versus 7.4%, P = 0.13, and 10.5% versus 7.4%, P = 0.23, respectively). Despite more frequent natural polymorphisms in non-B than B subtypes at tipranavir resistance positions, the prevalence of tipranavir resistance was greater in B than non-B subtypes (11% versus 4.3%, P = 0.004), reflecting a greater antiretroviral exposure in the former. Darunavir resistance did not differ significantly when comparing B and non-B subtypes (5.8% versus 5.5%, P = 0.998). The rate of resistance to the most recently approved protease and non-nucleoside reverse transcriptase inhibitors is low in antiretroviral-experienced patients, regardless of the HIV-1 subtype.

  9. Etravirine and Rilpivirine Drug Resistance Among HIV-1 Subtype C Infected Children Failing Non-Nucleoside Reverse Transcriptase Inhibitor-Based Regimens in South India.

    PubMed

    Saravanan, Shanmugam; Kausalya, Bagavathi; Gomathi, Selvamurthi; Sivamalar, Sathasivam; Pachamuthu, Balakrishnan; Selvamuthu, Poongulali; Pradeep, Amrose; Sunil, Solomon; Mothi, Sarvode N; Smith, Davey M; Kantor, Rami

    2017-06-01

    We have analyzed reverse transcriptase (RT) region of HIV-1 pol gene from 97 HIV-infected children who were identified as failing first-line therapy that included first-generation non-nucleoside RT inhibitors (Nevirapine and Efavirenz) for at least 6 months. We found that 54% and 65% of the children had genotypically predicted resistance to second-generation non-nucleoside RT inhibitors drugs Etravirine (ETR) and Rilpivirine, respectively. These cross-resistance mutations may compromise future NNRTI-based regimens, especially in resource-limited settings. To complement these investigations, we also analyzed the sequences in Stanford database, Monogram weighted score, and DUET weighted score algorithms for ETR susceptibility and found almost perfect agreement between the three algorithms in predicting ETR susceptibility from genotypic data.

  10. Efavirenz Has the Highest Anti-Proliferative Effect of Non-Nucleoside Reverse Transcriptase Inhibitors against Pancreatic Cancer Cells.

    PubMed

    Hecht, Markus; Erber, Sonja; Harrer, Thomas; Klinker, Hartwig; Roth, Thomas; Parsch, Hans; Fiebig, Nora; Fietkau, Rainer; Distel, Luitpold V

    2015-01-01

    Cancer prevention and therapy in HIV-1-infected patients will play an important role in future. The non-nucleoside reverse transcriptase inhibitors (NNRTI) Efavirenz and Nevirapine are cytotoxic against cancer cells in vitro. As other NNRTIs have not been studied so far, all clinically used NNRTIs were tested and the in vitro toxic concentrations were compared to drug levels in patients to predict possible anti-cancer effects in vivo. Cytotoxicity was studied by Annexin-V-APC/7AAD staining and flow cytometry in the pancreatic cancer cell lines BxPC-3 and Panc-1 and confirmed by colony formation assays. The 50% effective cytotoxic concentrations (EC50) were calculated and compared to the blood levels in our patients and published data. The in vitro EC50 of the different drugs in the BxPC-3 pancreatic cancer cells were: Efavirenz 31.5 μmol/l (= 9944 ng/ml), Nevirapine 239 μmol/l (= 63,786 ng/ml), Etravirine 89.0 μmol/l (= 38,740 ng/ml), Lersivirine 543 μmol/l (= 168,523 ng/ml), Delavirdine 171 μmol/l (= 78,072 ng/ml), Rilpivirine 24.4 μmol/l (= 8941 ng/ml). As Efavirenz and Rilpivirine had the highest cytotoxic potential and Nevirapine is frequently used in HIV-1 positive patients, the results of these three drugs were further studied in Panc-1 pancreatic cancer cells and confirmed with colony formation assays. 205 patient blood levels of Efavirenz, 127 of Rilpivirine and 31 of Nevirapine were analyzed. The mean blood level of Efavirenz was 3587 ng/ml (range 162-15,363 ng/ml), of Rilpivirine 144 ng/ml (range 0-572 ng/ml) and of Nevirapine 4955 ng/ml (range 1856-8697 ng/ml). Blood levels from our patients and from published data had comparable Efavirenz levels to the in vitro toxic EC50 in about 1 to 5% of all patients. All studied NNRTIs were toxic against cancer cells. A low percentage of patients taking Efavirenz reached in vitro cytotoxic blood levels. It can be speculated that in HIV-1 positive patients having high Efavirenz blood levels pancreatic cancer

  11. Efavirenz Has the Highest Anti-Proliferative Effect of Non-Nucleoside Reverse Transcriptase Inhibitors against Pancreatic Cancer Cells

    PubMed Central

    Hecht, Markus; Erber, Sonja; Harrer, Thomas; Klinker, Hartwig; Roth, Thomas; Parsch, Hans; Fiebig, Nora; Fietkau, Rainer; Distel, Luitpold V.

    2015-01-01

    Background Cancer prevention and therapy in HIV-1-infected patients will play an important role in future. The non-nucleoside reverse transcriptase inhibitors (NNRTI) Efavirenz and Nevirapine are cytotoxic against cancer cells in vitro. As other NNRTIs have not been studied so far, all clinically used NNRTIs were tested and the in vitro toxic concentrations were compared to drug levels in patients to predict possible anti-cancer effects in vivo. Methods Cytotoxicity was studied by Annexin-V-APC/7AAD staining and flow cytometry in the pancreatic cancer cell lines BxPC-3 and Panc-1 and confirmed by colony formation assays. The 50% effective cytotoxic concentrations (EC50) were calculated and compared to the blood levels in our patients and published data. Results The in vitro EC50 of the different drugs in the BxPC-3 pancreatic cancer cells were: Efavirenz 31.5μmol/l (= 9944ng/ml), Nevirapine 239μmol/l (= 63786ng/ml), Etravirine 89.0μmol/l (= 38740ng/ml), Lersivirine 543μmol/l (= 168523ng/ml), Delavirdine 171μmol/l (= 78072ng/ml), Rilpivirine 24.4μmol/l (= 8941ng/ml). As Efavirenz and Rilpivirine had the highest cytotoxic potential and Nevirapine is frequently used in HIV-1 positive patients, the results of these three drugs were further studied in Panc-1 pancreatic cancer cells and confirmed with colony formation assays. 205 patient blood levels of Efavirenz, 127 of Rilpivirine and 31 of Nevirapine were analyzed. The mean blood level of Efavirenz was 3587ng/ml (range 162–15363ng/ml), of Rilpivirine 144ng/ml (range 0-572ng/ml) and of Nevirapine 4955ng/ml (range 1856–8697ng/ml). Blood levels from our patients and from published data had comparable Efavirenz levels to the in vitro toxic EC50 in about 1 to 5% of all patients. Conclusion All studied NNRTIs were toxic against cancer cells. A low percentage of patients taking Efavirenz reached in vitro cytotoxic blood levels. It can be speculated that in HIV-1 positive patients having high Efavirenz blood levels

  12. Systematic evaluation of methyl ester bioisosteres in the context of developing alkenyldiarylmethanes (ADAMs) as non-nucleoside reverse transcriptase inhibitors (NNRTIs) for anti-HIV-1 chemotherapy.

    PubMed

    Hoshi, Ayako; Sakamoto, Takeshi; Takayama, Jun; Xuan, Meiyan; Okazaki, Mari; Hartman, Tracy L; Buckheit, Robert W; Pannecouque, Christophe; Cushman, Mark

    2016-07-01

    The alkenyldiarylmethanes (ADAMs) are a class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) targeting HIV-1. Four chemically and metabolically stabilized ADAMs incorporating N-methoxyimidoyl halide replacements of the methyl esters of the lead compound were previously reported. In this study, twenty-five new ADAMs were synthesized in order to investigate the biological consequences of installing nine different methyl ester bioisosteres at three different locations. Attempts to define a universal rank order of methyl ester bioisosteres and discover the 'best' one in terms of inhibitory activity versus HIV-1 reverse transcriptase (RT) led to the realization that the potencies are critically dependent on the surrounding structure at each location, and therefore the definition of universal rank order is impossible. This investigation produced several new non-nucleoside reverse transcriptase inhibitors in which all three of the three methyl esters of the lead compound were replaced by methyl ester bioisosteres, resulting in compounds that are more potent as HIV-1 RT inhibitors and antiviral agents than the lead compound itself and are expected to also be more metabolically stable than the lead compound. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Synthesis, structure-activity relationship and molecular docking of cyclohexenone based analogous as potent non-nucleoside reverse-transcriptase inhibitors

    NASA Astrophysics Data System (ADS)

    Nazar, Muhammad Faizan; Abdullah, Muhammad Imran; Badshah, Amir; Mahmood, Asif; Rana, Usman Ali; Khan, Salah Ud-Din

    2015-04-01

    The chalcones core in compounds is advantageously chosen effective synthons, which offer exciting perspectives in biological and pharmacological research. The present study reports the successful development of eight new cyclohexenone based anti-reverse transcriptase analogous using rational drug design synthesis principles. These new cyclohexenone derivatives (CDs) were synthesized by following a convenient route of Robinson annulation, and the molecular structure of these CDs were later confirmed by various analytical techniques such as 1H NMR, 13C NMR, FT-IR, UV-Vis spectroscopy and mass spectrometry. All the synthesized compounds were screened theoretically and experimentally against reverse transcriptase (RT) and found potentially active reverse transcriptase (RT) inhibitors. Of the compounds studied, the compound 2FC4 showed high interaction with RT at non-nucleoside binding site, contributing high free binding energy (ΔG -8.01 Kcal) and IC50 (0.207 μg/ml), respectively. Further results revealed that the compounds bearing more halogen groups, with additional hydrophobic character, offered superior anti-reverse transcriptase activity as compared to rest of compounds. It is anticipate that the present study would be very useful for the selection of potential reverse transcriptase inhibitors featuring inclusive pharmacological profiles.

  14. Synthesis of alkenyldiarylmethanes (ADAMs) containing benzo[d]isoxazole and oxazolidin-2-one rings, a new series of potent non-nucleoside HIV-1 reverse transcriptase inhibitors.

    PubMed

    Deng, Bo-Liang; Zhao, Yujie; Hartman, Tracy L; Watson, Karen; Buckheit, Robert W; Pannecouque, Christophe; De Clercq, Erik; Cushman, Mark

    2009-03-01

    As a continuation of efforts to replace the metabolically labile methyl esters of lead alkenyldiarylmethanes (ADAMs) with stable bioisosteres, compounds bearing benzo[d]isoxazole and oxazolidine-2-one rings were designed and evaluated as a new series of potent HIV-1 non-nucleoside reverse transcriptase inhibitors with anti-HIV activity. All of the resulting ADAMs were found to inhibit HIV-1 RT with poly(rC) x oligo(dG) as the template primer. The most promising compound in this series was ADAM 3, with EC(50) values of 40 nM (vs HIV-1(RF)) and 20 nM (vs HIV-1(IIIB)). Compound 3 also inhibited HIV-1 reverse transcriptase with an IC(50) of 0.91 microM. ADAM 4 has an antiviral EC(50) of 0.6 microM in CEM-SS cells and a plasma half-life of 51.4 min.

  15. Synthesis of alkenyldiarylmethanes (ADAMs) containing benzo[d]isoxazole and oxazolidin-2-one rings, a new series of potent non-nucleoside HIV-1 reverse transcriptase inhibitors

    PubMed Central

    Deng, Bo-Liang; Zhao, Yujie; Hartman, Tracy L.; Watson, Karen; Buckheit, Robert W.; Pannecouque, Christophe; De Clercq, Erik; Cushman, Mark

    2009-01-01

    As a continuation of efforts to replace the metabolically labile methyl esters of the lead alkenyldiarylmethanes (ADAMs) with stable bioisosteres, compounds bearing benzo[d]isoxazole and oxazolidine-2-one rings were designed and evaluated as a new series of potent HIV-1 non-nucleoside reverse transcriptase inhibitors with anti-HIV activity. All of the resulting ADAMs were found to inhibit HIV-1 RT with poly(rC)·oligo(dG) as the template primer. The most promising compound in this series was ADAM 3, with EC50 values of 40 nM (vs HIV-1RF) and 20 nM (vs HIV-1IIIB). Compound 3 also inhibited HIV-1 reverse transcriptase with an IC50 of 0.91 μM. ADAM 4 has an antiviral EC50 of 0.6 μM in CEM-SS cells and a plasma half-life of 51.4 min. PMID:18952324

  16. Progress of bis(heteroaryl)piperazines (BHAPs) as non-nucleoside reverse transcriptase inhibitors (NNRTIs) against human immunodeficiency virus type 1 (HIV-1).

    PubMed

    Xu, Hui

    2010-01-01

    Since the first case of acquired immunodeficiency syndrome (AIDS) was reported in 1981, AIDS, as the global disease affecting 33.2 million people in 2007, has always been an unsolved problem worldwide. Reverse transcriptase (RT) is a crucial enzyme in the life cycle of human immunodeficiency virus type 1 (HIV-1), and thereby has been the prime drugs target for antiretroviral (ARV) therapy against AIDS. To date, two classes of RT inhibitors (RTIs), e.g., nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs), and a lot of compounds tested as RTIs have been described. To our knowledge, bis(heteroaryl)piperazines (BHAPs) have been considered as one class of promising NNRTIs, such as structurally and chemically related NNRTI delavirdine, which was approved by the U. S. Food and Drug Administration (FDA) for the treatment of HIV-1 infection in 1997. In this mini-review, we make attempts to report the progress of synthesis and structure-activity relationship (SAR) of BHAPs, in the meantime, the synergistic inhibition of HIV-1 replication by combining delavirdine with other HIV-1 inhibitors is also discussed. It will pave the way for the design and development of BHAPs as anti-HIV-1 agents in AIDS chemotherapy in the future.

  17. Discovery of Wild-type and Y181C Mutant Non-nucleoside HIV-1 Reverse Transcriptase Inhibitors Using Virtual Screening with Multiple Protein Structures

    PubMed Central

    Nichols, Sara E.; Domaoal, Robert A.; Thakur, Vinay V.; Tirado-Rives, Julian; Anderson, Karen S.; Jorgensen, William L.

    2009-01-01

    In order to discover non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs) that are effective against both wild-type (WT) virus and variants that encode the clinically troublesome Tyr181Cys (Y181C) RT mutation, virtual screening by docking was carried out using three RT structures and more than 2 million commercially available compounds. Two of the structures are for WT-virus with different conformations of Tyr181, while the third structure incorporates the Y181C modification. Eventually nine compounds were purchased and assayed. Three of the compounds show low-micromolar anti-viral activity towards either or both the wild-type and Y181C HIV-1 strains. The study illustrates a viable protocol to seek anti-HIV agents with enhanced resistance profiles. PMID:19374380

  18. Replacement of the metabolically labile methyl esters in the alkenyldiarylmethane series of non-nucleoside reverse transcriptase inhibitors with isoxazolone, isoxazole, oxazolone, or cyano substituents.

    PubMed

    Deng, Bo-Liang; Hartman, Tracy L; Buckheit, Robert W; Pannecouque, Christophe; De Clercq, Erik; Cushman, Mark

    2006-08-24

    The alkenyldiarylmethanes (ADAMs) are a unique class of non-nucleoside reverse transcriptase inhibitors that have potential value in the treatment of HIV/AIDS. However, the potential usefulness of the ADAMs is limited by the presence of metabolically labile methyl ester moieties. A series of novel ADAMs were therefore designed and synthesized in order to replace the metabolically labile methyl ester moieties of the existing ADAM lead compounds with hydrolytically stable, fused isoxazolone, isoxazole, oxazolone, or cyano substituents on the aromatic rings. The methyl ester and methoxy substituents on both of the aromatic rings in the parent compound 1 were successfully replaced with metabolically stable moieties with retention of anti-HIV activity and a general decrease in cytotoxicity.

  19. Synthesis, Anti-HIV Activity, and Metabolic Stability of New Alkenyldiarylmethane (ADAM) HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs)

    PubMed Central

    Deng, Bo-Liang; Hartman, Tracy L.; Buckheit, Robert W.; Pannecouque, Christophe; De Clercq, Erik; Fanwick, Phillip E.; Cushman, Mark

    2008-01-01

    Non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs) are part of the combination therapy currently used to treat HIV infection. Based on analogy with known HIV-1 NNRT inhibitors, eighteen novel alkenyldiarylmethanes (ADAMs) containing 5-chloro-2-methoxyphenyl, 3-cyanophenyl or 3-fluoro-5-trifluoromethylphenyl groups were synthesized and evaluated as HIV inhibitors. Their stabilities in rat plasma have also been investigated. Although introducing 5-chloro-2-methoxyphenyl, or 3-fluoro-5-trifluoromethylphenyl groups into alkenyldiarylmethanes does not maintain the antiviral potency, the structural modification of alkenyldiarylmethanes with a 3-cyanophenyl substituent can be made without a large decrease in activity. The oxazolidinonyl group was introduced into the alkenyldiarylmethane framework and found to confer enhanced metabolic stability in rat plasma. PMID:16162014

  20. Molecular design, synthesis and biological evaluation of BP-O-DAPY and O-DAPY derivatives as non-nucleoside HIV-1 reverse transcriptase inhibitors.

    PubMed

    Yang, Shiqiong; Pannecouque, Christophe; Daelemans, Dirk; Ma, Xiao-Dong; Liu, Yang; Chen, Fen-Er; De Clercq, Erik

    2013-07-01

    This paper reports the synthesis and antiviral evaluation of a series of non-nucleoside reverse transcriptase inhibitors (NNRTIs) that combine the peculiar structural features of diarylpyrimidine derivatives (DAPYs) and benzophenone derivatives (BPs). The DAPY derivatives bearing benzoyl or alkoxyl substitutes on the A-ring showed the inhibitory activity against wild-type HIV-1 at the cellular level within the range of EC50 values from micromolar to nanomolar. Among these compounds, 1u exhibited the most potent anti-HIV-1 activity (EC50 = 0.06 ± 0.01 μM, SI > 6260), which were about 1.8-fold more active than nevirapine (NVP) and delavirdine (DLV). In addition, the binding modes with HIV-1 RT and the preliminary SAR studies of these derivatives were also considered for further investigation.

  1. Synthesis and biological evaluation of piperidine-substituted triazine derivatives as HIV-1 non-nucleoside reverse transcriptase inhibitors.

    PubMed

    Chen, Xuwang; Zhan, Peng; Pannecouque, Christophe; Balzarini, Jan; De Clercq, Erik; Liu, Xinyong

    2012-05-01

    A novel series of piperidine-substituted triazine derivatives have been synthesized and evaluated for anti-HIV activities in MT-4 cells. Most compounds displayed extremely promising activity against wild-type HIV-1 with EC(50) values in low nanomolar concentration, better than that of Nevirapine, Delavirdine, Zidovudine and Dideoxycitidine, and higher potency towards the resistant mutant strain K103N/Y181C than that of Nevirapine and Delavirdine. Selected compounds were also assayed against reverse transcriptase with lower IC(50) values than that of Nevirapine. The structure-activity relationship (SAR) of these novel structural congeners was also discussed.

  2. Synthesis and anti-HIV activity of new alkenyldiarylmethane (ADAM) non-nucleoside reverse transcriptase inhibitors (NNRTIs) incorporating benzoxazolone and benzisoxazole rings.

    PubMed

    Deng, Bo-Liang; Cullen, Matthew D; Zhou, Zhigang; Hartman, Tracy L; Buckheit, Robert W; Pannecouque, Christophe; De Clercq, Erik; Fanwick, Phillip E; Cushman, Mark

    2006-04-01

    The HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) constitute a large and structurally diverse set of compounds, several of which are currently used in the treatment of AIDS. A series of novel alkenyldiarylmethanes (ADAMs) were designed and synthesized as part of an ongoing investigation to replace the metabolically labile methyl ester moieties found in the ADAM pharmacophore with stable modifications that retain the potent anti-HIV activity of the parent compounds. Unsurprisingly, the rat plasma half-lives of the new ADAMs were not improved when compared to the parent compounds, but all of the synthesized ADAMs inhibited the cytopathic effect of HIV-1 in cell culture. The most potent compound identified was (E)-5-[1-(3,7-dimethyl-2-oxo-2,3-dihydro-benzoxazol-5-yl)-5-methoxycarbonyl-pent-1-enyl]-2-methoxy-3-methylbenzoic acid methyl ester (7), which inhibited the cytopathic effects of both HIV-1(RF) and HIV-1(IIIB) strains in cell cultures with EC50 values of 30 and 90 nM, respectively, and inhibited HIV-1 reverse transcriptase with an IC50 of 20 nM.

  3. Synthesis, biological evaluation and molecular modeling of 4,6-diarylpyrimidines and diarylbenzenes as novel non-nucleosides HIV-1 reverse transcriptase inhibitors.

    PubMed

    Ribone, Sergio R; Leen, Volker; Madrid, Marcela; Dehaen, Wim; Daelemans, Dirk; Pannecouque, Christophe; Briñón, Margarita C

    2012-12-01

    A series of novel 4,6-diarylpyrimidines (4,6-DAPY) and diarylbenzenes (DABE) compounds were synthesized and evaluated as inhibitors of human immunodeficiency virus type-1 (HIV-1). Among them, the most potent HIV-1 inhibitors were 8b, 8d, 14b and 18 (EC(50) = 0.049, 0.381, 0.599 and 0.398 μM, respectively), with HIV-1 inhibitory activity improved or similar to nevirapine (NVP, EC(50) = 0.097 μM) and delavirdine (DEV, EC(50) = 0.55 μM). The other compounds displayed moderate activity (8c, EC(50) = 5.25 μM) or were inactive (8a and 14a) against HIV-1 replication. Molecular modeling studies were performed with the synthesized compounds in complex with the wild-type reverse transcriptase (RT). A correlation was found between the anti-HIV activity and the electrostatic energy of interaction with Lys101 residue. These findings enrich the SAR of these Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) families.

  4. Synthesis and biological evaluation of DAPY-DPEs hybrids as non-nucleoside inhibitors of HIV-1 reverse transcriptase.

    PubMed

    Wu, Hai-Qiu; Yao, Jin; He, Qiu-Qin; Chen, Wen-Xue; Chen, Fen-Er; Pannecouque, Christophe; De Clercq, Erik; Daelemans, Dirk

    2015-02-01

    A series of new DAPY-DPEs hybrids, combined the important pharmacophores of DAPYs and DPEs, has been synthesized and biologically evaluated for their anti-HIV activities against wild-type HIV-1 strain IIIB, double RT mutant (K103N+Y181C) strain RES056 and HIV-2 strain ROD in MT-4 cell cultures. Many promising candidates with potent inhibitory activity (wild-type) within the EC50 range from 0.16 to 0.013 μM were obtained. In particular, 3c, 3p, 3r and 3s displayed low nM level EC50 values (35, 13, 50 and 17 nM, respectively) and high selectivity (9342, 25131, 2890 and 11338, respectively), which were much more potent than NVP (EC50=0.31 μM, SI=48), 3TC (EC50=2.24 μM, SI>39), DDI (EC50=23.20 μM, SI>9) and DLV (EC50=0.65 μM, SI>67), and comparable to AZT (EC50=0.0071 μM, SI>13144) and EFV (EC50=0.0062 μM, SI>1014). The HIV-1 reverse transcriptase inhibitory assay confirmed that these DAPY-DPEs hybrids targeted HIV-1 RT. Molecular simulation was performed to investigate the potential binding mode of the newly synthesized compounds. And reasonable explanation for the activity results was discussed with docking method. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Structure-based virtual screening efforts against HIV-1 reverse transcriptase to introduce the new potent non-nucleoside reverse transcriptase inhibitor

    NASA Astrophysics Data System (ADS)

    Hosseini, Yaser; Mollica, Adriano; Mirzaie, Sako

    2016-12-01

    The human immunodeficiency virus (HIV) which is strictly related to the development of AIDS, is treated by a cocktail of drugs, but due its high propensity gain drug resistance, the rational development of new medicine is highly desired. Among the different mechanism of action we selected the reverse transcriptase (RT) inhibition, for our studies. With the aim to identify new chemical entities to be used for further rational drug design, a set of 3000 molecules from the Zinc Database have been screened by docking experiments using AutoDock Vina software. The best ranked compounds with respect of the crystallographic inhibitor MK-4965 resulted to be five compounds, and the best among them was further tested by molecular dynamics (MD) simulation. Our results indicate that comp1 has a stronger interaction with the subsite p66 of RT than MK-4965 and that both are able to stabilize specific conformational changes of the RT 3D structure, which may explain their activity as inhibitors. Therefore comp1 could be a good candidate for biological tests and further development.

  6. A randomized trial of Raltegravir replacement for protease inhibitor or non-nucleoside reverse transcriptase inhibitor in HIV-infected women with lipohypertrophy.

    PubMed

    Lake, Jordan E; McComsey, Grace A; Hulgan, Todd M; Wanke, Christine A; Mangili, Alexandra; Walmsley, Sharon L; Boger, M Sean; Turner, Ralph R; McCreath, Heather E; Currier, Judith S

    2012-09-01

    Lipohypertrophy in HIV-infected patients is associated with metabolic abnormalities. Raltegravir (RAL) is not known to induce fat changes or severe metabolic perturbations. HIV-infected women with central adiposity and HIV-1 RNA less than 50 copies per milliliter on non-nucleoside reverse transcriptase inhibitor (NNRTI)- or protease inhibitor (PI)-based antiretroviral therapy (ART) continued their nucleoside reverse transcriptase inhibitor (NRTI) backbone and were randomized to switch to open label RAL immediately or after 24 weeks. The primary end point was 24-week between-group change in computed tomography (CT)-quantified visceral adipose tissue (AT) volume. Fasting lipids, glucose, C-reactive protein (CRP), anthropometric measurements, and patient-reported quality of life assessments were also measured. Thirty-six subjects provided 80% power to detect a 10% between-group difference in visceral AT over 24 weeks. Thirty-seven of 39 enrolled subjects completed week 24. At entry, subjects were 75% black or Hispanic, and on 62% PI-based and 38% NNRTI-based regimens. The median age was 43 years, CD4 count 558 cells per microliter, and body mass index (BMI) 32 kg/m(2). After 24 weeks, no statistically significant changes in visceral or subcutaneous AT, anthropometrics, BMI, glucose, or CRP were observed. In subjects receiving RAL, significant improvements in total and LDL cholesterol (p=0.04), self-reported belly size (p=0.02) and composite body size (p=0.02) were observed. Body size changes correlated well with percent visceral AT change. No RAL-related adverse events occurred. Compared to continued PI or NNRTI, switch to RAL was associated with statistically significant 24-week improvements in total and LDL cholesterol but not AT volumes. Additional insights into AT and metabolic changes in women on RAL will be provided by 48-week follow-up of the immediate-switch arm.

  7. Mechanistic Study of Common Non-Nucleoside Reverse Transcriptase Inhibitor-Resistant Mutations with K103N and Y181C Substitutions

    PubMed Central

    Lai, Ming-Tain; Munshi, Vandna; Lu, Meiqing; Feng, MeiZhen; Hrin-Solt, Renee; McKenna, Philip M.; Hazuda, Daria J.; Miller, Michael D.

    2016-01-01

    Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are a mainstay of therapy for human immunodeficiency type 1 virus (HIV-1) infections. However, their effectiveness can be hampered by the emergence of resistant mutations. To aid in designing effective NNRTIs against the resistant mutants, it is important to understand the resistance mechanism of the mutations. Here, we investigate the mechanism of the two most prevalent NNRTI-associated mutations with K103N or Y181C substitution. Virus and reverse transcriptase (RT) with K103N/Y188F, K103A, or K103E substitutions and with Y181F, Y188F, or Y181F/Y188F substitutions were employed to study the resistance mechanism of the K103N and Y181C mutants, respectively. Results showed that the virus and RT with K103N/Y188F substitutions displayed similar resistance levels to the virus and RT with K103N substitution versus NNRTIs. Virus and RT containing Y181F, Y188F, or Y181F/Y188F substitution exhibited either enhanced or similar susceptibility to NNRTIs compared with the wild type (WT) virus. These results suggest that the hydrogen bond between N103 and Y188 may not play an important role in the resistance of the K103N variant to NNRTIs. Furthermore, the results from the studies with the Y181 or Y188 variant provide the direct evidence that aromatic π–π stacking plays a crucial role in the binding of NNRTIs to RT. PMID:27669286

  8. Mechanistic Study of Common Non-Nucleoside Reverse Transcriptase Inhibitor-Resistant Mutations with K103N and Y181C Substitutions.

    PubMed

    Lai, Ming-Tain; Munshi, Vandna; Lu, Meiqing; Feng, MeiZhen; Hrin-Solt, Renee; McKenna, Philip M; Hazuda, Daria J; Miller, Michael D

    2016-09-23

    Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are a mainstay of therapy for human immunodeficiency type 1 virus (HIV-1) infections. However, their effectiveness can be hampered by the emergence of resistant mutations. To aid in designing effective NNRTIs against the resistant mutants, it is important to understand the resistance mechanism of the mutations. Here, we investigate the mechanism of the two most prevalent NNRTI-associated mutations with K103N or Y181C substitution. Virus and reverse transcriptase (RT) with K103N/Y188F, K103A, or K103E substitutions and with Y181F, Y188F, or Y181F/Y188F substitutions were employed to study the resistance mechanism of the K103N and Y181C mutants, respectively. Results showed that the virus and RT with K103N/Y188F substitutions displayed similar resistance levels to the virus and RT with K103N substitution versus NNRTIs. Virus and RT containing Y181F, Y188F, or Y181F/Y188F substitution exhibited either enhanced or similar susceptibility to NNRTIs compared with the wild type (WT) virus. These results suggest that the hydrogen bond between N103 and Y188 may not play an important role in the resistance of the K103N variant to NNRTIs. Furthermore, the results from the studies with the Y181 or Y188 variant provide the direct evidence that aromatic π-π stacking plays a crucial role in the binding of NNRTIs to RT.

  9. A Randomized Trial of Raltegravir Replacement for Protease Inhibitor or Non-Nucleoside Reverse Transcriptase Inhibitor in HIV-Infected Women with Lipohypertrophy

    PubMed Central

    McComsey, Grace A.; Hulgan, Todd M.; Wanke, Christine A.; Mangili, Alexandra; Walmsley, Sharon L.; Boger, M. Sean; Turner, Ralph R.; McCreath, Heather E.; Currier, Judith S.

    2012-01-01

    Abstract Lipohypertrophy in HIV-infected patients is associated with metabolic abnormalities. Raltegravir (RAL) is not known to induce fat changes or severe metabolic perturbations. HIV-infected women with central adiposity and HIV-1 RNA less than 50 copies per milliliter on non-nucleoside reverse transcriptase inhibitor (NNRTI)- or protease inhibitor (PI)-based antiretroviral therapy (ART) continued their nucleoside reverse transcriptase inhibitor (NRTI) backbone and were randomized to switch to open label RAL immediately or after 24 weeks. The primary end point was 24-week between-group change in computed tomography (CT)-quantified visceral adipose tissue (AT) volume. Fasting lipids, glucose, C-reactive protein (CRP), anthropometric measurements, and patient-reported quality of life assessments were also measured. Thirty-six subjects provided 80% power to detect a 10% between-group difference in visceral AT over 24 weeks. Thirty-seven of 39 enrolled subjects completed week 24. At entry, subjects were 75% black or Hispanic, and on 62% PI-based and 38% NNRTI-based regimens. The median age was 43 years, CD4 count 558 cells per microliter, and body mass index (BMI) 32 kg/m2. After 24 weeks, no statistically significant changes in visceral or subcutaneous AT, anthropometrics, BMI, glucose, or CRP were observed. In subjects receiving RAL, significant improvements in total and LDL cholesterol (p=0.04), self-reported belly size (p=0.02) and composite body size (p=0.02) were observed. Body size changes correlated well with percent visceral AT change. No RAL-related adverse events occurred. Compared to continued PI or NNRTI, switch to RAL was associated with statistically significant 24-week improvements in total and LDL cholesterol but not AT volumes. Additional insights into AT and metabolic changes in women on RAL will be provided by 48-week follow-up of the immediate-switch arm. PMID:22823027

  10. Search for Non-nucleoside Inhibitors of HIV-1 Reverse Transcriptase using Chemical Similarity, Molecular Docking, and MM-GB/SA Scoring

    PubMed Central

    Barreiro, Gabriela; Guimarães, Cristiano R. W.; Tubert-Brohman, Ivan; Lyons, Theresa M.; Tirado-Rives, Julian; Jorgensen, William L.

    2008-01-01

    A virtual screening protocol has been applied to seek non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs) and its K103N mutant. First, a chemical similarity search on the Maybridge library was performed using known NNRTIs as reference structures. The top-ranked molecules obtained from this procedure plus 26 known NNRTIs were then docked into the binding sites of the wild-type reverse transcriptase (HIV-RT) and its K103N variant (K103N-RT) using Glide 3.5. The top-ranked 100 compounds from the docking for both proteins were post-scored with a procedure using molecular mechanics and continuum solvation (MM-GB/SA). The validity of the virtual screening protocol was supported by (i) testing of the MM-GB/SA procedure, (ii) agreement between predicted and crystallographic binding poses, (iii) recovery of known potent NNRTIs at the top of both rankings, and (iv) identification of top-scoring library compounds that are close in structure to recently reported NNRTI HTS-hits. However, purchase and assaying of selected top-scoring compounds from the library failed to yield active anti-HIV agents. Nevertheless, the highest-ranked database compound, S10087, was pursued as containing a potentially viable core. Subsequent synthesis and assaying of S10087 analogs proposed by further computational analysis yielded anti-HIV agents with EC50 values as low as 310 nM. Thus, with the aid of computational tools, it was possible to evolve a false positive into a true active. PMID:17949071

  11. Hybrids of [TSAO-T]-[foscarnet]: The first conjugate of foscarnet with a non-nucleoside reverse transcriptase inhibitor through a labile covalent ester bond.

    PubMed

    Velázquez, Sonsoles; Lobatón, Esther; De Clercq, Erik; Koontz, Dianna L; Mellors, John W; Balzarini, Jan; Camarasa, María-José

    2004-06-17

    This paper describes the first example of combination of non-nucleoside reverse transcriptase inhibitors such as TSAO derivatives and foscarnet (PFA) in a single molecule through a labile covalent ester bond. The essential criteria in the design of these hybrids [TSAO-T]-[PFA] was to explore if the conjugation of foscarnet with the highly lipophilic TSAO derivative may facilitate the penetration of the conjugates through the cell membrane and if the hybrids escape extracellular hydrolysis and regenerate the parent inhibitors intracellulary. Several [TSAO-T]-[PFA] conjugates proved markedly inhibitory to HIV-1. Some of them also showed potent activity against PFA-resistant HIV-1 strains but fewer had detectable inhibitory activity against TSAO-resistant HIV-1 strains. These results indicated a pivotal role of the TSAO component of the hybrid but not the PFA component in the activity of the conjugates. Moreover, stability studies of the [TSAO-T]-[PFA] conjugates demonstrated that the compounds were stable in PBS whereas some of the conjugates regenerated the parent inhibitors in extracts from CEM cells.

  12. Design and synthesis of a new series of modified CH-diarylpyrimidines as drug-resistant HIV non-nucleoside reverse transcriptase inhibitors.

    PubMed

    Meng, Ge; Liu, Yang; Zheng, Aqun; Chen, Fener; Chen, Wenxue; De Clercq, Erik; Pannecouque, Christophe; Balzarini, Jan

    2014-07-23

    This article reports the design, synthesis and antiviral evaluation of a new series of non-nucleoside reverse transcriptase inhibitors (NNRTIs). The basic skeleton of these target 18 molecules is diarylpyrimidine featuring a substituted amino group between the pyrimidine scaffold and the aryl wing. All of the new compounds have been characterized by spectra analysis. The entire target molecules were evaluated for their in vitro anti-HIV activity with controlling group of FDA approved drugs. Most of them showed good to potent activities against wild-type (WT) HIV-1 with IC50 values in the range of 0.0175-69.21 μM. 2-(4-Cyanophenylamino)-4-(2-cyanovinylphenylhydrazonomethyl)pyrimidine (1d) displayed potent anti-HIV-1 activity against WT HIV-1 with a selectivity index (SI) of 106367 and an IC50 value of 1.75 nM, which was 47 fold lower than that of AZT. Compound 1d also showed a broad-spectrum inhibitory activity, with an IC50 value of 5.33 μM and 5.05 μM against both HIV-1 double-mutated (K103N/Y181C) strain and HIV-2 strain, respectively. The preliminary structure-activity relationship (SAR) was also investigated. The binding modes with HIV-1 RT for both the wild type and mutant type have also been discussed.

  13. Development and Characterization of a Vaginal Film Containing Dapivirine, a Non- nucleoside Reverse Transcriptase Inhibitor (NNRTI), for prevention of HIV-1 sexual transmission

    PubMed Central

    Akil, Ayman; Parniak, Michael A.; Dezzuitti, Charlene S.; Moncla, Bernard J.; Cost, Marilyn R.; Li, Mingguang; Rohan, Lisa Cencia

    2012-01-01

    Dapivirine, a non-nucleoside reverse transcriptase inhibitor, is a potent and promising anti-HIV molecule. It is currently being investigated for use as a vaginal microbicide in two dosage forms, a semi-solid gel and a silicone elastomer ring. Quick-dissolving films are promising and attractive dosage forms that may provide an alternative platform for the vaginal delivery of microbicide drug candidates. Vaginal films may provide advantages such as discreet use, no product leakage during use, lack of requirement for an applicator for insertion, rapid drug release and minimal packaging and reduced wastage. Within this study the in vitro bioactivity of dapivirine as compared to the NNRTI UC781 was further established and a quick dissolve film was developed for vaginal application of dapivirine for prevention of HIV infection. The developed film was characterized with respect to its physical and chemical attributes including water content, mechanical strength, drug release profile, permeability, compatibility with lactobacilli and bioactivity. The anti-HIV activity of the formulated dapivirine film was confirmed in in vitro and ex vivo models. Importantly the physical and chemical properties of the film as well as its bioactivity were maintained for a period of 18 months. In conclusion, a vaginal film containing dapivirine was developed and characterized. The film was shown to prevent HIV-1 infection in vitro and ex vivo and have acceptable characteristics which make this film a promising candidate for testing as vaginal microbicide. PMID:22708075

  14. Viral resuppression and detection of drug resistance following interruption of a suppressive non-nucleoside reverse transcriptase inhibitor-based regimen.

    PubMed

    Fox, Zoe; Phillips, Andrew; Cohen, Cal; Neuhaus, Jacquie; Baxter, John; Emery, Sean; Hirschel, Bernard; Hullsiek, Kathy Huppler; Stephan, Christoph; Lundgren, Jens

    2008-11-12

    Interruption of a non-nucleoside reverse transcriptase inhibitor (NNRTI)-regimen is often necessary, but must be performed with caution because NNRTIs have a low genetic barrier to resistance. Limited data exist to guide clinical practice on the best interruption strategy to use. Patients in the drug-conservation arm of the Strategies for Management of Antiretroviral Therapy (SMART) trial who interrupted a fully suppressive NNRTI-regimen were evaluated. From 2003, SMART recommended interruption of an NNRTI by a staggered interruption, in which the NNRTI was stopped before the NRTIs, or by replacing the NNRTI with another drug before interruption. Simultaneous interruption of all antiretrovirals was discouraged. Resuppression rates 4-8 months after reinitiating NNRTI-therapy were assessed, as was the detection of drug-resistance mutations within 2 months of the treatment interruption in a subset (N = 141). Overall, 601/688 (87.4%) patients who restarted an NNRTI achieved viral resuppression. The adjusted odds ratio (95% confidence interval) for achieving resuppression was 1.94 (1.02-3.69) for patients with a staggered interruption and 3.64 (1.37-9.64) for those with a switched interruption compared with patients with a simultaneous interruption. At least one NNRTI-mutation was detected in the virus of 16.4% patients with simultaneous interruption, 12.5% patients with staggered interruption and 4.2% patients with switched interruption. Fewer patients with detectable mutations (i.e. 69.2%) achieved HIV-RNA of 400 copies/ml or less compared with those in whom no mutations were detected (i.e. 86.7%; P = 0.05). In patients who interrupt a suppressive NNRTI-regimen, the choice of interruption strategy may influence resuppression rates when restarting a similar regimen. NNRTI drug-resistance mutations were observed in a relatively high proportion of patients. These data provide additional support for a staggered or switched interruption strategy for NNRTI drugs.

  15. Structure-based drug design of non-nucleoside inhibitors for wild-type and drug-resistant HIV reverse transcriptase.

    PubMed

    Mao, C; Sudbeck, E A; Venkatachalam, T K; Uckun, F M

    2000-11-01

    The generation of anti-HIV agents using structure-based drug design methods has yielded a number of promising non-nucleoside inhibitors (NNIs) of HIV reverse transcriptase (RT). Recent successes in identifying potent NNIs are reviewed with an emphasis on the recent trend of utilizing a computer model of HIV RT to identify space in the NNI binding pocket that can be exploited by carefully chosen functional groups predicted to interact favorably with binding pocket residues. The NNI binding pocket model was used to design potent NNIs against both wild-type RT and drug-resistant RT mutants. Molecular modeling and score functions were used to analyze how drug-resistant mutations would change the RT binding pocket shape, volume, and chemical make-up, and how these changes could affect inhibitor binding. Modeling studies revealed that for an NNI of HIV RT to be active against RT mutants such as the especially problematic Y181C RT mutant, the following features are required: (a) the inhibitor should be highly potent against wild-type RT and therefore capable of tolerating a considerable activity loss against RT mutants (i.e. a picomolar-level inhibitor against wild-type RT may still be effective against RT mutants at nanomolar concentrations), (b) the inhibitor should maximize the occupancy in the Wing 2 region of the NNI binding site of RT, and (c) the inhibitor should contain functional groups that provide favorable chemical interactions with Wing 2 residues of wild-type as well as mutant RT. Our rationally designed NNI compounds HI-236, HI-240, HI-244, HI-253, HI-443, and HI-445 combine these three features and outperform other anti-HIV agents examined.

  16. Gastrointestinal-associated lymphoid tissue immune reconstitution in a randomized clinical trial of raltegravir versus non-nucleoside reverse transcriptase inhibitor-based regimens.

    PubMed

    Asmuth, David M; Ma, Zhong-Min; Mann, Surinder; Knight, Thomas H; Yotter, Tammy; Albanese, Anthony; Melcher, Gregory P; Troia-Cancio, Paolo; Hayes, Timothy; Miller, Chris J; Pollard, Richard B

    2012-08-24

    To examine immune restoration in duodenal tissue and correlates of reduction of immune activation in chronic HIV-infected patients randomized to different treatment regimens. Randomized clinical trial (RCT) comparing raltegravir to a non-nucleoside reverse transcriptase inhibitor-based regimen, both with fixed-dose tenofovir difumerate/emtricitabine. Antiretroviral therapy (ART)-naive volunteers underwent upper endoscopy for duodenal biopsies before and after 9 months of therapy. Tissue was paraffin-embedded for immunohistochemistry or digested into single-cell suspensions for flow cytometry of lymphocyte subsets and activation phenotype. Plasma-soluble CD14 levels were measured as a surrogate for bacterial translocation. Sixteen HIV-positive and seven control individuals completed study procedures. Small increases in duodenal lamina propria CD4 T-cell numbers were observed, especially when viewed relative to populations in control volunteers, with no differences between treatment arms. The increase in CD4 T-cell percentage was due largely to declines in CD8 T-cell numbers, which were disproportionately increased compared to peripheral blood and controls. Patients randomized to the raltegravir arm had consistent declines in both sCD14 levels and CD8 T-cell numbers in the duodenal tissue lamina propria. This first RCT of lymphocyte population restoration in duodenal tissue demonstrates more modest increases in CD4 T-cell numbers during the first 9 months of therapy than when considering CD3/CD4 percentages only. Although reduced after 9 months of ART, disproportional increased CD8 populations persist in duodenal gastrointestinal-associated lymphoid tissue (GALT). Local rather than systemic antigenic stimulation appears to be driving expanded CD8 T lymphocytes in GALT. Factors other than viral-induced CD8 expansion may be contributing to this local immunologic response. © 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins

  17. The prevalence of transmitted resistance to first-generation non-nucleoside reverse transcriptase inhibitors and its potential economic impact in HIV-infected patients.

    PubMed

    Snedecor, Sonya J; Khachatryan, Alexandra; Nedrow, Katherine; Chambers, Richard; Li, Congyu; Haider, Seema; Stephens, Jennifer

    2013-01-01

    Non-nucleoside reverse transcriptase inhibitor (NNRTI)-based highly active antiretroviral therapy (HAART) including efavirenz is recommended as a 1(st)-line treatment choice in international HIV guidelines, and it is one of the most common components of initial therapy. Resistance to 1(st)-generation NNRTIs is found among treated and untreated HIV-infected individuals creating a subpopulation of HIV-infected individuals in whom efavirenz is not fully effective. This analysis reviewed published articles and conference abstracts to examine the prevalence of 1(st)-generation NNRTI resistance in Europe, the United States (US), and Canada and to identify published evidence of the economic consequences of resistance. The reported prevalence of NNRTI resistance was generally higher in US/Canada than in Europe and increased in both regions from their introduction in the late 1990s until the early 2000s. The most recent time-based trends suggest that NNRTI-resistance prevalence may be stable or decreasing. These estimates of resistance may be understated as resistance estimates using ultra-sensitive genotypic testing methods, which identify low-frequency mutations undetected by standard testing methods, showed increased prevalence of resistance by more than two-fold. No studies were identified that explicitly investigated the costs of drug resistance. Rather, most studies reported costs of treatment change, failure, or disease progression. Among those studies, annual HIV medical costs of those infected with HIV increased 1) as CD4 cells decreased, driven in part by hospitalization at lower CD4 cell counts; 2) for treatment changes, and 3) for each virologic failure. The possible erosion of efficacy or of therapy choices through resistance transmission or selection, even when present with low frequency, may become a barrier to the use of 1(st)-generation NNRTIs and the increased costs associated with regimen failure and disease progression underlie the importance of

  18. A nanoparticle-encapsulated non-nucleoside reverse-transcriptase inhibitor with enhanced anti-HIV-1 activity and prolonged circulation time in plasma.

    PubMed

    Li, Wen; Wang, Qian; Li, Yuan; Yu, Fei; Liu, Qi; Qin, Bingjie; Xie, Lan; Lu, Lu; Jiang, Shibo

    2015-01-01

    Non-nucleoside reverse-transcriptase inhibitors (NNRTIs), major components of highly active antiretroviral therapy (HAART), are effective in suppressing viral replication and preventing the progress of HIV-1 infection to AIDS. However, rapid blood clearance in vivo could significantly impair the efficiency of the anti-HIV-1 activity and result in multiple daily doses which might lead to poor patient compliance. Here we attempted to employ biodegradable organic nanoparticles (NPs) to encapsulate DAAN15h, a derivative of 4-substituted 1, 5-diarylaniline with potent anti-HIV activities. Nanoparticles encapsulating DAAN15h (NP-DAAN15h) displayed a spherical shape with a size of 97.01 ± 3.64 nm and zeta potential of -19.1 ± 3.78 mV, and they exhibited a sustained controlled release behavior in vitro. The cellular uptake of NPs on TZM-b1 cells, MT-2 cells and M7 cells, possibly through lipid raft-mediated and energydependent active transport processes, was significantly enhanced. NP-DAAN15h, which possessed no significant in vitro cytotoxicity, showed improved antiviral activity against laboratory-adapted and primary HIV-1 isolates with different subtypes and tropisms, including RT-resistant variants. NP-DAAN15h exhibited a significantly prolonged blood circulation time, decreased plasma elimination rate, and enhanced AUC(0-t). NP-DAAN15h, a nanoparticle-encapsulated NNRTI, exhibits enhanced cellular uptake, improved anti-HIV-1 efficacy and prolonged in vivo circulation time, suggesting good potential for further development as a new NNRTI formulation for clinical use.

  19. Efficacy of non-nucleoside reverse transcriptase inhibitor-based highly active antiretroviral therapy in Thai HIV-infected children aged two years or less.

    PubMed

    Puthanakit, Thanyawee; Aurpibul, Linda; Sirisanthana, Thira; Sirisanthana, Virat

    2009-03-01

    Twenty-six Thai HIV-infected children, aged 2 years or less were prospectively enrolled to receive non-nucleoside reverse transcription inhibitor-based highly active antiretroviral therapy (HAART). Twenty-two children (85%) had World Health Organization clinical stage 3 or 4. The median baseline CD4 cell percentage and plasma HIV RNA were 17% and 5.9 log 10 copies/mL, respectively. The median age at HAART initiation was 9.8 months (range, 1.5-24.0). One child died. The mean CD4 cell percentages at 24, 48, and 96 weeks of treatment were 26%, 31%, and 37%, respectively. The proportions of children with virologic suppression (<400 copies/mL) at week 24 and 48 were 14/26 (54%) and 19/26 (73%), respectively. Non-nucleoside reverse transcription inhibitor-based HAART is safe and effective in HIV-infected young children in a resource-limited setting.

  20. Synthesis and anti-HIV activity of new metabolically stable alkenyldiarylmethane non-nucleoside reverse transcriptase inhibitors incorporating N-methoxy imidoyl halide and 1,2,4-oxadiazole systems.

    PubMed

    Sakamoto, Takeshi; Cullen, Matthew D; Hartman, Tracy L; Watson, Karen M; Buckheit, Robert W; Pannecouque, Christophe; De Clercq, Erik; Cushman, Mark

    2007-07-12

    The alkenyldiarylmethanes (ADAMs) are a unique class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) that are capable of inhibiting HIV-1 reverse transcriptase (RT) through an allosteric mechanism. However, the potential usefulness of the ADAMs is limited by the presence of metabolically labile methyl ester moieties that are hydrolyzed by nonspecific esterases present in blood plasma, resulting in the formation of the inactive carboxylic acid metabolites. Therefore, to discover metabolically stable ADAMs, the design and synthesis of a new class of ADAMs with N-methoxy imidoyl halide and 1,2,4-oxadiazole systems were attempted. The resulting new ADAM 6 displayed enhanced metabolic stability in rat plasma (t1/2 = 61 h) along with the ability to inhibit HIV-1 reverse transcriptase and the cytopathic effect of HIV-1RF and HIV-1IIIB at submicromolar concentrations.

  1. Synthesis and Anti-HIV Activity of New Metabolically Stable Alkenyldiarylmethane (ADAM) Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) Incorporating N-Methoxy Imidoyl Halide and 1,2,4-Oxadiazole Systems

    PubMed Central

    Sakamoto, Takeshi; Cullen, Matthew D.; Hartman, Tracy L.; Watson, Karen M.; Buckheit, Robert W.; Pannecouque, Christophe; De Clercq, Erik; Cushman, Mark

    2008-01-01

    The alkenyldiarylmethanes (ADAMs) are a unique class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) that are capable of inhibiting HIV-1 reverse transcriptase (RT) through an allosteric mechanism. However, the potential usefulness of the ADAMs is limited by the presence of metabolically labile methyl ester moieties that are hydrolyzed by non-specific esterases present in blood plasma, resulting in the formation of the inactive carboxylic acid metabolites. Therefore, in order to discover metabolically stable ADAMs, the design and synthesis of a new class of ADAMs with N-methoxy imidoyl halide and 1,2,4-oxadiazole systems were attempted. The resulting new ADAM 6 displayed enhanced metabolic stability in rat plasma (t1/2 = 61 h) along with the ability to inhibit HIV-1 reverse transcriptase and the cytopathic effect of HIV-1RF and HIV-1IIIB at submicromolar concentrations. PMID:17579385

  2. Conformational landscape of the human immunodeficiency virus type 1 reverse transcriptase non-nucleoside inhibitor binding pocket: lessons for inhibitor design from a cluster analysis of many crystal structures.

    PubMed

    Paris, Kristina A; Haq, Omar; Felts, Anthony K; Das, Kalyan; Arnold, Eddy; Levy, Ronald M

    2009-10-22

    Clustering of 99 available X-ray crystal structures of HIV-1 reverse transcriptase (RT) at the flexible non-nucleoside inhibitor binding pocket (NNIBP) provides information about features of the conformational landscape for binding non-nucleoside inhibitors (NNRTIs), including effects of mutation and crystal forms. The ensemble of NNIBP conformations is separated into eight discrete clusters based primarily on the position of the functionally important primer grip, the displacement of which is believed to be one of the mechanisms of inhibition of RT. Two of these clusters are populated by structures in which the primer grip exhibits novel conformations that differ from the predominant cluster by over 4 A and are induced by the unique inhibitors capravirine and rilpivirine/TMC278. This work identifies a new conformation of the NNIBP that may be used to design NNRTIs. It can also be used to guide more complete exploration of the NNIBP free energy landscape using advanced sampling techniques.

  3. Potent dual anti-HIV and spermicidal activities of novel oxovanadium(V) complexes with thiourea non-nucleoside inhibitors of HIV-1 reverse transcriptase.

    PubMed

    D'Cruz, Osmond J; Dong, Yanhong; Uckun, Fatih M

    2003-03-07

    We have previously demonstrated that tetrahedral bis(cyclopentadienyl)vanadium(IV) complexes and square pyramidal oxovanadium(IV) complexes of vanadium are rapid and selective spermicidal agents at low micromolar concentrations. This study investigated the potential utility of oxovanadium in combination with thiourea non-nucleoside inhibitors (NNIs) of HIV-1 reverse transcriptase (RT) for the development of an effective dual-function anti-HIV spermicide. Two rationally designed substituted phenyl-ring containing pyridyl thiourea NNIs, N-[2-(2-chlorophenethyl)]-N(')-[2-(5-bromopyridyl)-thiourea) [1] and N-[2-(2-methoxyphenethyl)]-N(')-[2-(pyridyl)-thiourea [2] that exhibited subnanomolar IC(50) values against the drug-sensitive, drug-resistant, and multidrug-resistant strains of HIV-1, were complexed with oxovanadium. The oxovanadium-thiourea [OVT] NNIs, C(29)H(27)Br(2)Cl(2)N(6)O(2)S(2)V [3], and C(31)H(35)N(6)O(4)S(2)V [4], were synthesized by reacting VOSO(4), a V(IV) compound, with the corresponding deprotonated thiourea NNI compounds as ligands. Elemental analysis showed that each OVT-NNI used two thiourea molecules as ligands. The existence of the Vz.dbnd6;O bond (968cm(-1)) was confirmed by IR spectroscopy. No d-d bands were observed in the visible spectra of OVT-NNIs and their EPR spectra were featureless, indicating that the vanadium centers were oxidized to V(V). The new OVT-NNIs as well as their thiourea NNI ligands were evaluated for (i) anti-HIV activity using the cell-free recombinant RT inhibition assays, (ii) cellular HIV replication assays, (iii) spermicidal activity against human sperm by computer-assisted sperm analysis (CASA), and (iv) cytotoxicity against normal human female genital tract epithelial cell using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) dye-reduction assays. Similar to thiourea NNIs 1 and 2, the OVT-NNIs 3 and 4, exhibited potent anti-HIV activity with submicromolar IC(50[p24]) values (0.08 and 0.128 micro

  4. Molecular docking and 3D-QSAR studies on triazolinone and pyridazinone, non-nucleoside inhibitor of HIV-1 reverse transcriptase.

    PubMed

    Sivan, Sree Kanth; Manga, Vijjulatha

    2010-06-01

    Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are allosteric inhibitors of the HIV-1 reverse transcriptase. Recently a series of Triazolinone and Pyridazinone were reported as potent inhibitors of HIV-1 wild type reverse transcriptase. In the present study, docking and 3D quantitative structure activity relationship (3D QSAR) studies involving comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were performed on 31 molecules. Ligands were built and minimized using Tripos force field and applying Gasteiger-Hückel charges. These ligands were docked into protein active site using GLIDE 4.0. The docked poses were analyzed; the best docked poses were selected and aligned. CoMFA and CoMSIA fields were calculated using SYBYL6.9. The molecules were divided into training set and test set, a PLS analysis was performed and QSAR models were generated. The model showed good statistical reliability which is evident from the r2 nv, q2 loo and r2 pred values. The CoMFA model provides the most significant correlation of steric and electrostatic fields with biological activities. The CoMSIA model provides a correlation of steric, electrostatic, acceptor and hydrophobic fields with biological activities. The information rendered by 3D QSAR model initiated us to optimize the lead and design new potential inhibitors.

  5. From the traditional Chinese medicine plant Schisandra chinensis new scaffolds effective on HIV-1 reverse transcriptase resistant to non-nucleoside inhibitors.

    PubMed

    Xu, Lijia; Grandi, Nicole; Del Vecchio, Claudia; Mandas, Daniela; Corona, Angela; Piano, Dario; Esposito, Francesca; Parolin, Cristina; Tramontano, Enzo

    2015-04-01

    HIV-1 reverse transcriptase (RT) is still an extremely attractive pharmaceutical target for the identification of new inhibitors possibly active on drug resistant strains. Medicinal plants are a rich source of chemical diversity and can be used to identify novel scaffolds to be further developed by chemical modifications. We investigated the ability of the main lignans from Schisandra chinensis (Turcz.) Baill. fruits, commonly used in Traditional Chinese Medicine, to affect HIV-1 RT functions. We purified 6 lignans from Schisandra chinensis fruits and assayed their effects on HIV-1 RT and viral replication. Among the S. chinensis fruit lignans, Schisandrin B and Deoxyschizandrin selectively inhibited the HIV-1 RT-associated DNA polymerase activity. Structure activity relationship revealed the importance of cyclooctadiene ring substituents for efficacy. In addition, Schisandrin B was also able to impair HIV-1 RT drug resistant mutants and the early phases of viral replication. We identified Schisandrin B and Deoxyschizandrin as new scaffold for the further development of novel HIV-1 RT inhibitors.

  6. Introducing Catastrophe-QSAR. Application on Modeling Molecular Mechanisms of Pyridinone Derivative-Type HIV Non-Nucleoside Reverse Transcriptase Inhibitors

    PubMed Central

    Putz, Mihai V.; Lazea, Marius; Putz, Ana-Maria; Duda-Seiman, Corina

    2011-01-01

    The classical method of quantitative structure-activity relationships (QSAR) is enriched using non-linear models, as Thom’s polynomials allow either uni- or bi-variate structural parameters. In this context, catastrophe QSAR algorithms are applied to the anti-HIV-1 activity of pyridinone derivatives. This requires calculation of the so-called relative statistical power and of its minimum principle in various QSAR models. A new index, known as a statistical relative power, is constructed as an Euclidian measure for the combined ratio of the Pearson correlation to algebraic correlation, with normalized t-Student and the Fisher tests. First and second order inter-model paths are considered for mono-variate catastrophes, whereas for bi-variate catastrophes the direct minimum path is provided, allowing the QSAR models to be tested for predictive purposes. At this stage, the max-to-min hierarchies of the tested models allow the interaction mechanism to be identified using structural parameter succession and the typical catastrophes involved. Minimized differences between these catastrophe models in the common structurally influential domains that span both the trial and tested compounds identify the “optimal molecular structural domains” and the molecules with the best output with respect to the modeled activity, which in this case is human immunodeficiency virus type 1 HIV-1 inhibition. The best molecules are characterized by hydrophobic interactions with the HIV-1 p66 subunit protein, and they concur with those identified in other 3D-QSAR analyses. Moreover, the importance of aromatic ring stacking interactions for increasing the binding affinity of the inhibitor-reverse transcriptase ligand-substrate complex is highlighted. PMID:22272148

  7. Design, Synthesis and Biological Evaluation of 1-[(2-benzyloxyl/alkoxyl) methyl]-5-halo-6-aryluracils as Potent HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors with Improved Drug Resistance Profile

    PubMed Central

    Wang, Xiaowei; Zhang, Jianfang; Huang, Yang; Wang, Ruiping; Zhang, Liang; Qiao, Kang; Li, Li; Liu, Chang; Ouyang, Yabo; Xu, Weisi; Zhang, Zhili; Zhang, Liangren; Shao, Yiming; Jiang, Shibo; Ma, Liying; Liu, Junyi

    2012-01-01

    Since the emergence of drug-resistant mutants has limited the efficacy of non-nucleoside reverse transcriptase inhibitors (NNRTIs), it is essential to develop new antivirals with better drug-resistance and pharmacokinetic profiles. Here we designed and synthesized a series of 1-[(2-benzyloxyl/alkoxyl)methyl]-5-halo-6-aryluracils, the HEPT analogues, and evaluated their biological activity using Nevirapine and 18 (TNK-651) as reference compounds. Most of these compounds, especially 6b, 7b, 9b, 11b and 7c, exhibited highly potent anti-HIV-1 activity against both wild-type and NNRTI-resistant HIV-1 strains. The compound 7b, that had the highest selectivity index (SI = 38,215), is more potent than Nevirapine and 18. These results suggest that introduction of halogen at the C-5 position may contribute to the effectiveness of these compounds against RTI-resistant variants. In addition, m-substituents on the C-6 aromatic moiety could significantly enhance activity against NNRTI-resistant HIV-1 strains. These compounds can be further developed as next-generation NNRTIs with improved antiviral efficacy and drug-resistance profile. PMID:22283377

  8. Malaria in HIV-Infected Children Receiving HIV Protease-Inhibitor- Compared with Non-Nucleoside Reverse Transcriptase Inhibitor-Based Antiretroviral Therapy, IMPAACT P1068s, Substudy to P1060

    PubMed Central

    Hobbs, Charlotte V.; Gabriel, Erin E.; Kamthunzi, Portia; Tegha, Gerald; Tauzie, Jean; Petzold, Elizabeth; Barlow-Mosha, Linda; Chi, Benjamin H.; Li, Yonghua; Ilmet, Tiina; Kirmse, Brian; Neal, Jillian; Parikh, Sunil; Deygoo, Nagamah; Jean Philippe, Patrick; Mofenson, Lynne; Prescott, William; Chen, Jingyang; Musoke, Philippa; Palumbo, Paul; Duffy, Patrick E.; Borkowsky, William

    2016-01-01

    Background HIV and malaria geographically overlap. HIV protease inhibitors kill malaria parasites in vitro and in vivo, but further evaluation in clinical studies is needed. Methods Thirty-one children from Malawi aged 4–62 months were followed every 3 months and at intercurrent illness visits for ≤47 months (September 2009-December 2011). We compared malaria parasite carriage by blood smear microscopy (BS) and confirmed clinical malaria incidence (CCM, or positive BS with malaria symptoms) in children initiated on HIV antiretroviral therapy (ART) with zidovudine, lamivudine, and either nevirapine (NVP), a non-nucleoside reverse transcriptase inhibitor, or lopinavir-ritonavir (LPV-rtv), a protease inhibitor. Results We found an association between increased time to recurrent positive BS, but not CCM, when anti-malarial treatment and LPV-rtv based ART were used concurrently and when accounting for a LPV-rtv and antimalarial treatment interaction (adjusted HR 0.39; 95% CI (0.17,0.89); p = 0.03). Conclusions LPV-rtv in combination with malaria treatment was associated with lower risk of recurrent positive BS, but not CCM, in HIV-infected children. Larger, randomized studies are needed to confirm these findings which may permit ART optimization for malaria-endemic settings. Trial Registration ClinicalTrials.gov NCT00719602 PMID:27936233

  9. Selection and characterization of viruses resistant to the dual acting pyrimidinedione entry and non-nucleoside reverse transcriptase inhibitor IQP-0410.

    PubMed

    Buckheit, Robert W; Watson Buckheit, Karen; Sturdevant, Christa Buckheit; Buckheit, Robert W

    2013-11-01

    The 1-(2-hydroxyethoxymethyl)-6-(phenylthio)thymine (HEPT)-like compounds with homocyclic moieties at the N-1 of the pyrimidinedione, including the highly potent lead compound IQP-0410, inhibit HIV-1 at sub-nanomolar concentrations primarily through a typical non-nucleoside mechanism involving allosteric inhibition at the hydrophobic binding pocket of the HIV-1 RT. Like all NNRTIs, the pyrimidinediones have no activity against HIV-2 RT. The pyrimidinediones, however, also possess a second mode of action involving inhibition of virus entry at nanomolar concentrations which extends their range of action to include HIV-2. Entry inhibition occurs through recognition of a complex conformational binding site formed upon interaction of the virus with target cells, but does not involve direct inhibition of gp120-CD4 binding. In order to further explore the means by which the pyrimidinediones act, resistant strains of HIV-1 and HIV-2 were selected in cell culture and molecularly and biologically characterized. With HIV-1, three phases of resistance selection occurred which involve an initial appearance of single amino changes in the NNRTI binding pocket, followed by changes in the envelope glycoproteins gp120 and gp41, and subsequent multiple additional changes in the RT, resulting in high level resistance to IQP-0410. With HIV-2, resistance to entry inhibition was achieved with no resistance-engendering mutations detected in the HIV-2 RT. Detailed molecular and biological characterization of IQP-0410-resistant viruses was performed to define the resistance-engendering mutations present in the RT and envelope and to quantify cross-resistance to other HIV inhibitors. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. A Phase III Comparative Study of the Efficacy and Tolerability of Three Non-Nucleoside Reverse Transcriptase Inhibitor-Sparing Antiretroviral Regimens for Treatment-Naïve HIV-1-Infected Volunteers: A Randomized, Controlled Trial

    PubMed Central

    Lennox, Jeffrey L.; Landovitz, Raphael J.; Ribaudo, Heather J.; Ofotokun, Ighovwerha; Na, Lumine H.; Godfrey, Catherine; Kuritzkes, Daniel R.; Sagar, Manish; Brown, Todd T.; Cohn, Susan E.; McComsey, Grace A.; Aweeka, Francesca; Fichtenbaum, Carl J.; Presti, Rachel M.; Koletar, Susan L.; Haas, David W.; Patterson, Kristine B.; Benson, Constance A.; Baugh, Bryan P.; Leavitt, Randi Y.; Rooney, James F.; Seekins, Daniel; Currier, Judith S.

    2015-01-01

    Background Non-nucleoside reverse transcriptase (NNRTI) inhibitor-based antiretroviral therapy is not suitable for all treatment-naïve HIV-infected persons. Objective Perform a rigorous evaluation of three NNRTI-sparing initial antiretroviral regimens to demonstrate equivalence for virologic efficacy and tolerability. Design Phase-III, 1:1:1 randomized, open label, >96 week study. Setting Fifty-seven sites in United States and Puerto Rico. Patients Treatment naïve, ≥18 years, HIV-1 RNA >1000 copies/mL, no nucleoside reverse transcriptase or protease inhibitor resistance. Intervention Atazanavir 300 mg with ritonavir 100 mg, daily; or raltegravir 400 mg twice daily; or darunavir 800 mg with ritonavir 100 mg, daily; plus emtricitabine 200 mg + tenofovir disoproxil fumarate 300 mg daily. Measurements Virologic failure defined as confirmed HIV-1 RNA >1000 copies/mL between 16 and 24 weeks, or >200 copies/mL at or after 24 weeks; tolerability failure defined as discontinuation of atazanavir, raltegravir or darunavir for toxicity. A secondary endpoint was a combination of virologic efficacy and tolerability. Results Among 1,809 participants all pairwise comparisons of incidence of virologic failure over 96-weeks demonstrated equivalence within ±10%. Raltegravir and ritonavir-boosted darunavir were equivalent for tolerability, whereas ritonavir-boosted atazanavir resulted in a 12.7% and a 9.2% higher incidence of tolerability discontinuation than raltegravir and ritonavir-boosted darunavir respectively, primarily due to hyperbilirubinemia. For combined virologic efficacy and tolerability ritonavir-boosted darunavir was superior to ritonavir-boosted atazanavir, and raltegravir was superior to both protease inhibitors. Antiretroviral resistance at time of virologic failure was rare but more likely with raltegravir. Limitations Open label; ritonavir not provided Conclusions Over 2 years all three regimens attain high and equivalent rates of virologic control. Regimens

  11. Multicenter study of skin rashes and hepatotoxicity in antiretroviral-naïve HIV-positive patients receiving non-nucleoside reverse-transcriptase inhibitor plus nucleoside reverse-transcriptase inhibitors in Taiwan

    PubMed Central

    Wu, Pei-Ying; Cheng, Chien-Yu; Liu, Chun-Eng; Lee, Yi-Chien; Yang, Chia-Jui; Tsai, Mao-Song; Cheng, Shu-Hsing; Lin, Shih-Ping; Lin, De-Yu; Wang, Ning-Chi; Lee, Yi-Chieh; Sun, Hsin-Yun; Tang, Hung-Jen; Hung, Chien-Ching

    2017-01-01

    Objectives Two nucleos(t)ide reverse-transcriptase inhibitors (NRTIs) plus 1 non-NRTI (nNRTI) remain the preferred or alternative combination antiretroviral therapy (cART) for antiretroviral-naive HIV-positive patients in Taiwan. The three most commonly used nNRTIs are nevirapine (NVP), efavirenz (EFV) and rilpivirine (RPV). This study aimed to determine the incidences of hepatotoxicity and skin rashes within 4 weeks of initiation of cART containing 1 nNRTI plus 2 NRTIs. Methods Between June, 2012 and November, 2015, all antiretroviral-naive HIV-positive adult patients initiating nNRTI-containing cART at 8 designated hospitals for HIV care were included in this retrospective observational study. According to the national HIV treatment guidelines, patients were assessed at baseline, 2 and 4 weeks of cART initiation, and subsequently every 8 to 12 weeks. Plasma HIV RNA load, CD4 cell count and aminotransferases were determined. The toxicity grading scale of the Division of AIDS (DAIDS) 2014 was used for reporting clinical and laboratory adverse events. Results During the 3.5-year study period, 2,341 patients initiated nNRTI-containing cART: NVP in 629 patients, EFV 1,363 patients, and RPV 349 patients. Rash of any grade occurred in 14.1% (n = 331) of the patients. In multiple logistic regression analysis, baseline CD4 cell counts (per 100-cell/μl increase, adjusted odds ratio [AOR], 1.125; 95% confidence interval [95% CI], 1.031–1.228) and use of NVP (AOR, 2.443; 95% CI, 1.816–3.286) (compared with efavirenz) were independently associated with the development of skin rashes. Among the 1,455 patients (62.2%) with aminotransferase data both at baseline and week 4, 72 (4.9%) developed grade 2 or greater hepatotoxicity. In multiple logistic regression analysis, presence of antibody for hepatitis C virus (HCV) (AOR, 2.865; 95% CI, 1.439–5.704) or hepatitis B surface antigen (AOR, 2.397; 95% CI, 1.150–4.997), and development of skin rashes (AOR, 2.811; 95% CI, 1

  12. First-Line Antiretroviral Therapy With A Protease Inhibitor Versus Non-Nucleoside Reverse Transcriptase Inhibitor And Switch At Higher Versus Low Viral Load In Hiv-Infected Children: An Open-Label, Randomised Phase 2/3 Trial

    PubMed Central

    2011-01-01

    Background Randomised long-term comparisons between protease inhibitor(PI) and non-nucleoside reverse transcriptase inhibitor(NNRTI) first-line antiretroviral therapy(ART) and viral load(VL) switch criteria have never been undertaken in HIV-infected children. Methods PENPACT-1(ISRCTN73318385) assessed long-term effectiveness of ART-naïve children from Europe and North/South America initiating 2NRTIs+PI vs 2NRTIs+NNRTI, and switch to second-line at VL ≥1000c/ml vs ≥30000c/ml in a randomised open-label factorial design. The primary outcome was VL change between baseline and 4 years. Results 266 children were randomised(66 PI-1000, 65 PI-30000, 68 NNRTI-1000, 67 NNRTI-30000), and 263 analysed(3 NNRTI-30000 excluded); median age 6.5(IQR:2.8–12.9)years; mean(SD) CD4 18%(11); VL 5.1(0.8)log10c/ml. Median follow-up was 5.0(IQR:4.2–6.0)years; 188(71%) children were on first-line ART at trial end. For children starting second-line ART, median VLs at switch were 6720c/ml vs 35712c/ml in 1000 vs 30000; children in the 30000 group switched 41 weeks later, on average. At 4 years, mean VL reductions were −3.16 vs −3.31log10c/ml for PI vs NNRTI(difference −0.15log10c/ml,95%CI[−0.41,0.11];p=0.26), and −3.26 vs −3.20log10c/ml for 1000 vs 30000(difference 0.06log10c/ml,95%CI[−0.20,0.32];p=0.56); VL was <400c/ml in 82%PI vs 82%NNRTI, p=0.91 and 83%1000 vs 80%30000, p=0.42. Nine children with new CDC-C events, and 60 experiencing grade 3/4 adverse events were balanced across randomisations. PI resistance was uncommon and no increase in NRTI resistance occurred in PI-30000 compared to PI-1000. In contrast, NNRTI resistance was selected early (similar in 1000 and 30000), and ~10% more children accumulated NRTI mutations in NNRTI-30000 than NNRTI-1000. Conclusion There was no difference between initiating ART with PI or NNRTI-based regimens; both achieved good long-term virological outcomes. Delayed switching on NNRTI-based ART increases NRTI, but not NNRTI

  13. Long-term effectiveness of initiating non-nucleoside reverse transcriptase inhibitor- versus ritonavir-boosted protease inhibitor-based antiretroviral therapy: implications for first-line therapy choice in resource-limited settings

    PubMed Central

    Lima, Viviane D; Hull, Mark; McVea, David; Chau, William; Harrigan, P Richard; Montaner, Julio SG

    2016-01-01

    Introduction In many resource-limited settings, combination antiretroviral therapy (cART) failure is diagnosed clinically or immunologically. As such, there is a high likelihood that patients may stay on a virologically failing regimen for a substantial period of time. Here, we compared the long-term impact of initiating non-nucleoside reverse transcriptase inhibitor (NNRTI)- versus boosted protease inhibitor (bPI)-based cART in British Columbia (BC), Canada. Methods We followed prospectively 3925 ART-naïve patients who started NNRTIs (N=1963, 50%) or bPIs (N=1962; 50%) from 1 January 2000 until 30 June 2013 in BC. At six months, we assessed whether patients virologically failed therapy (a plasma viral load (pVL) >50 copies/mL), and we stratified them based on the pVL at the time of failure ≤500 versus >500 copies/mL. We then followed these patients for another six months and calculated their probability of achieving subsequent viral suppression (pVL <50 copies/mL twice consecutively) and of developing drug resistance. These probabilities were adjusted for fixed and time-varying factors, including cART adherence. Results At six months, virologic failure rates were 9.5 and 14.3 cases per 100 person-months for NNRTI and bPI initiators, respectively. NNRTI initiators who failed with a pVL ≤500 copies/mL had a 16% higher probability of achieving subsequent suppression at 12 months than bPI initiators (0.81 (25th–75th percentile 0.75–0.83) vs. 0.72 (0.61–0.75)). However, if failing NNRTI initiators had a pVL >500 copies/mL, they had a 20% lower probability of suppressing at 12 months than pVL-matched bPI initiators (0.37 (0.29–0.45) vs. 0.46 (0.38–0.54)). In terms of evolving HIV drug resistance, those who failed on NNRTI performed worse than bPI in all scenarios, especially if they failed with a viral load >500 copies/mL. Conclusions Our results show that patients who virologically failed at six months on NNRTI and continued on the same regimen had a

  14. Docking and 3-D QSAR studies on indolyl aryl sulfones. Binding mode exploration at the HIV-1 reverse transcriptase non-nucleoside binding site and design of highly active N-(2-hydroxyethyl)carboxamide and N-(2-hydroxyethyl)carbohydrazide derivatives.

    PubMed

    Ragno, Rino; Artico, Marino; De Martino, Gabriella; La Regina, Giuseppe; Coluccia, Antonio; Di Pasquali, Alessandra; Silvestri, Romano

    2005-01-13

    Three-dimensional quantitative structure-activity relationship (3-D QSAR) studies and docking simulations were developed on indolyl aryl sulfones (IASs), a class of novel HIV-1 non-nucleoside reverse transcriptase (RT) inhibitors (Silvestri, et al. J. Med. Chem. 2003, 46, 2482-2493) highly active against wild type and some clinically relevant resistant strains (Y181C, the double mutant K103N-Y181C, and the K103R-V179D-P225H strain, highly resistant to efavirenz). Predictive 3-D QSAR models using the combination of GRID and GOLPE programs were obtained using a receptor-based alignment by means of docking IASs into the non-nucleoside binding site (NNBS) of RT. The derived 3-D QSAR models showed conventional correlation (r(2)) and cross-validated (q(2)) coefficients values ranging from 0.79 to 0.93 and from 0.59 to 0.84, respectively. All described models were validated by an external test set compiled from previously reported pyrryl aryl sulfones (Artico, et al. J. Med. Chem. 1996, 39, 522-530). The most predictive 3-D QSAR model was then used to predict the activity of novel untested IASs. The synthesis of six designed derivatives (prediction set) allowed disclosure of new IASs endowed with high anti-HIV-1 activities.

  15. Molecular dynamics study of non-nucleoside reverse transcriptase inhibitor 4-[[4-[[4-[(E)-2-cyanoethenyl]-2,6-dimethylphenyl]amino]-2-pyrimidinyl]amino]benzonitrile (TMC278/rilpivirine) aggregates: correlation between amphiphilic properties of the drug and oral bioavailability

    PubMed Central

    Frenkel, Yulia Volovik; Gallicchio, Emilio; Das, Kalyan; Levy, Ronald M.; Arnold, Eddy

    2009-01-01

    The non-nucleoside reverse transcriptase inhibitor (NNRTI) TMC278/rilpivirine is an anti-AIDS therapeutic agent with high oral bioavailability despite its high hydrophobicity. Previous studies established a correlation between ability of the drug molecule to form stable, homogeneous populations of spherical nanoparticles (~100–120 nm in diameter) at low pH in surfactant-independent fashion, and good oral bioavailability. Here, we hypothesize that the drug is able to assume surfactant-like properties under physiologically relevant conditions, thus facilitating formation of nanostructuresin the absence of other surfactants. The results of all-atom molecular dynamics simulations indeed show that protonated drug molecules behave as surfactants at the water/aggregate interface while neutral drug molecules assist aggregate packing via conformational variability. Our simulation results suggest that amphiphilic behavior at low pH and intrinsic flexibility influence drug aggregation and are believed to play critical roles in the favorable oral bioavailability of hydrophobic drugs. PMID:19739675

  16. Development and in Vitro Evaluation of a Microbicide Gel Formulation for a Novel Non-Nucleoside Reverse Transcriptase Inhibitor Belonging to the N-Dihydroalkyloxybenzyloxopyrimidines (N-DABOs) Family.

    PubMed

    Tintori, Cristina; Brai, Annalaura; Dasso Lang, Maria Chiara; Deodato, Davide; Greco, Antonia Michela; Bizzarri, Bruno Mattia; Cascone, Lorena; Casian, Alexandru; Zamperini, Claudio; Dreassi, Elena; Crespan, Emmanuele; Maga, Giovanni; Vanham, Guido; Ceresola, Elisa; Canducci, Filippo; Ariën, Kevin K; Botta, Maurizio

    2016-03-24

    Preventing HIV transmission by the use of a vaginal microbicide is a topic of considerable interest in the fight against AIDS. Both a potent anti-HIV agent and an efficient formulation are required to develop a successful microbicide. In this regard, molecules able to inhibit the HIV replication before the integration of the viral DNA into the genetic material of the host cells, such as entry inhibitors or reverse transcriptase inhibitors (RTIs), are ideal candidates for prevention purpose. Among RTIs, S- and N-dihydroalkyloxybenzyloxopyrimidines (S-DABOs and N-DABOs) are interesting compounds active at nanomolar concentration against wild type of RT and with a very interesting activity against RT mutations. Herein, novel N-DABOs were synthesized and tested as anti-HIV agents. Furthermore, their mode of binding was studied by molecular modeling. At the same time, a vaginal microbicide gel formulation was developed and tested for one of the most promising candidates.

  17. Crystal structure of tert-butyldimethylsilyl-spiroaminooxathioledioxide-thymine (TSAO-T) in complex with HIV-1 reverse transcriptase (RT) redefines the elastic limits of the non-nucleoside inhibitor-binding pocket

    PubMed Central

    Das, Kalyan; Bauman, Joseph D.; Rim, Angela S.; Dharia, Chhaya; Clark, Arthur D.; Camarasa, María-José; Balzarini, Jan; Arnold, Eddy

    2012-01-01

    Tert-butyldimethylsilyl-spiroaminooxathioledioxide (TSAO) compounds have an embedded thymidine-analog backbone; however, TSAO compounds invoke non-nucleoside RT inhibitor (NNRTI) resistance mutations. Our crystal structure of RT:7 (TSAO-T) complex shows that 7 binds inside the NNRTI-binding pocket assuming a “dragon” shape, and interacts extensively with almost all the pocket residues. The structure also explains the structure-activity relationships and resistance data for TSAO compounds. The binding of 7 causes hyper-expansion of the pocket and significant rearrangement of RT subdomains. This non-optimal complex formation is apparently responsible (1) for the lower stability of a RT (p66/p51) dimer and (2) for the lower potency of 7 despite of its extensive interactions with RT. However, the HIV-1 RT:7 structure reveals novel design features, such as (1) interactions with the conserved Tyr183 from the YMDD-motif and (2) a possible way for an NNRTI to reach the polymerase active site that may be exploited in designing new NNRTIs. PMID:21446702

  18. Excretion and metabolism of lersivirine (5-{[3,5-diethyl-1-(2-hydroxyethyl)(3,5-14C2)-1H-pyrazol-4-yl]oxy}benzene-1,3-dicarbonitrile), a next-generation non-nucleoside reverse transcriptase inhibitor, after administration of [14C]Lersivirine to healthy volunteers.

    PubMed

    Vourvahis, Manoli; Gleave, Michelle; Nedderman, Angus N R; Hyland, Ruth; Gardner, Iain; Howard, Martin; Kempshall, Sarah; Collins, Claire; LaBadie, Robert

    2010-05-01

    Lersivirine [UK-453,061, 5-((3,5-diethyl-1-(2-hydroxyethyl)(3,5-14C2)-1H-pyrazol-4-yl)oxy)benzene-1,3-dicarbonitrile] is a next-generation non-nucleoside reverse transcriptase inhibitor, with a unique binding interaction within the reverse transcriptase binding pocket. Lersivirine has shown antiviral activity and is well tolerated in HIV-infected and healthy subjects. This open-label, Phase I study investigated the absorption, metabolism, and excretion of a single oral 500-mg dose of [14C]lersivirine (parent drug) and characterized the plasma, fecal, and urinary radioactivity of lersivirine and its metabolites in four healthy male volunteers. Plasma C(max) for total radioactivity and unchanged lersivirine typically occurred between 0.5 and 3 h postdose. The majority of radioactivity was excreted in urine (approximately 80%) with the remainder excreted in the feces (approximately 20%). The blood/plasma ratio of total drug-derived radioactivity [area under the plasma concentration-time profile from time zero extrapolated to infinite time (AUC(inf))] was 0.48, indicating that radioactive material was distributed predominantly into plasma. Lersivirine was extensively metabolized, primarily by UDP glucuronosyltransferase- and cytochrome P450-dependent pathways, with 22 metabolites being identified in this study. Analysis of precipitated plasma revealed that the lersivirine-glucuronide conjugate was the major circulating component (45% of total radioactivity), whereas unchanged lersivirine represented 13% of total plasma radioactivity. In vitro studies showed that UGT2B7 and CYP3A4 are responsible for the majority of lersivirine metabolism in humans.

  19. Structure-based design of N-[2-(1-piperidinylethyl)]-N'-[2-(5-bromopyridyl)]-thiourea and N-[2-(1-piperazinylethyl)]-N'-[2-(5-bromopyridyl)]-thiourea as potent non-nucleoside inhibitors of HIV-1 reverse transcriptase.

    PubMed

    Mao, C; Vig, R; Venkatachalam, T K; Sudbeck, E A; Uckun, F M

    1998-08-18

    A novel computer model of the HIV reverse transcriptase (RT) non-nucleoside inhibitor (NNI) binding pocket, which was generated using high resolution crystal structure information from 9 individual RT/NNI complexes, revealed previously unrecognized ligand derivatization sites for phenethylthiazolylthiourea (PETT) derivatives. Spatial gaps surrounding the pyridyl ring of the active PETT derivative trovirdine were discovered during modeling procedures. Docking studies using the computer-generated model of the binding pocket (composite binding pocket) suggested that the replacement of the planar pyridyl ring of trovirdine with a nonplanar piperidinyl or piperazinyl ring, which occupy larger volumes, would better fill the spacious Wing 2 region of the butterfly-shaped NNI binding pocket. The anti-HIV activity of the synthesized heterocyclic compounds N-[2-(1-piperidinylethyl)]-N'-[2-(5-bromopyridyl)]-thiourea and N-[2-(1-piperazinylethyl)]-N'-[2-(5-bromopyridyl)]-thiourea was examined in HTLVIIIB-infected peripheral blood mononuclear cells. Both compounds were more potent than trovirdine and abrogated HIV replication at nanomolar concentrations without any evidence of cytotoxicity.

  20. A reverse transcriptase ribozyme.

    PubMed

    Joyce, Gerald F; Samanta, Biswajit

    2017-09-26

    A highly evolved RNA polymerase ribozyme was found to also be capable of functioning as a reverse transcriptase, an activity that has never been demonstrated before for RNA. This activity is thought to have been crucial for the transition from RNA to DNA genomes during the early history of life on Earth, when it similarly could have arisen as a secondary function of an RNA-dependent RNA polymerase. The reverse transcriptase ribozyme can incorporate all four dNTPs and can generate products containing up to 32 deoxynucleotides. It is likely that this activity could be improved through evolution, ultimately enabling the synthesis of complete DNA genomes. DNA is much more stable compared to RNA and thus provides a larger and more secure repository for genetic information.

  1. Design of Annulated Pyrazoles As Inhibitors of HIV-1 Reverse Transcriptase

    SciTech Connect

    Sweeney, Z.K.; Harris, S.F.; Arora, N.; Javanbakht, H.; Li, Y.; Fretland, J.; Davidson, J.P.; Billedeau, J.R.; Gleason, S.; Hirschfeld, D.; Kennedy-Smith, J.J.; Mirzadegan, T.; Roetz, R.; Smith, M.; Sperry, S.; Suh, J.M.; Wu, J.; Tsing, S.; Villasenor, A.G.; Paul, A.; Su, G.

    2009-05-26

    Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are recommended components of preferred combination antiretroviral therapies used for the treatment of HIV. These regimens are extremely effective in suppressing virus replication. Structure-based optimization of diaryl ether inhibitors led to the discovery of a new series of pyrazolo[3,4-c]pyridazine NNRTIs that bind the reverse transcriptase enzyme of human immunodeficiency virus-1 (HIV-RT) in an expanded volume relative to most other inhibitors in this class. The binding mode maintains the {beta}13 and {beta}14 strands bearing Pro236 in a position similar to that in the unliganded reverse transcriptase structure, and the distribution of interactions creates the opportunity for substantial resilience to single point mutations. Several pyrazolopyridazine NNRTIs were found to be highly effective against wild-type and NNRTI-resistant viral strains in cell culture.

  2. Optimization of 2,4-Diarylanilines as Non-nucleoside HIV-1 Reverse Transcriptase Inhibitors

    PubMed Central

    Sun, Lian-Qi; Qin, Bingjie; Huang, Li; Qian, Keduo; Chen, Chin-Ho; Lee, Kuo-Hsiung; Xie, Lan

    2012-01-01

    The current optimization of 2,4-diarylaniline analogs (DAANs) on the central phenyl ring provided a series of new active DAAN derivatives 9a–9e, indicating an accessible modification approach that could improve anti-HIV potency against wild-type and resistant strains, aqueous solubility, and metabolic stability. A new compound 9e not only exhibited extremely high potency against wild-type virus (EC50 0.53 nM) and several resistant viral strains (EC50 0.36 – 3.9 nM), but also showed desirable aqueous solubility and metabolic stability, which were comparable or better than those of the anti-HIV-1 drug TMC278 (2). Thus, new compound 9e might be a potential drug candidate for further development of novel next-generation NNRTIs. PMID:22406117

  3. Discovery of diarylpyridine derivatives as novel non-nucleoside HIV-1 reverse transcriptase inhibitors

    PubMed Central

    Tian, Xingtao; Qin, Bingjie; Lu, Hong; Lai, Weihong; Jiang, Shibo; Lee, Kuo-Hsiung; Ho Chen, Chin; Xie, Lan

    2009-01-01

    Two series (4 and 5) of diarylpyridine derivatives were designed, synthesized, and evaluated for anti-HIV-1 activity. The most promising compound, 5e, inhibited HIV-1 IIIB, NL4-3, and RTMDR1 with low nanomolar EC50 values and selectivity indexes of >10,000. The results of this study indicate that diarylpyridine can be used as a novel scaffold to derive a new class of potent NNRTIs, active against both wild-type and drug resistant HIV-1 strains. PMID:19666220

  4. Design and synthesis of conformationally constrained inhibitors of non-nucleoside reverse transcriptase.

    PubMed

    Gomez, Robert; Jolly, Samson J; Williams, Theresa; Vacca, Joseph P; Torrent, Maricel; McGaughey, Georgia; Lai, Ming-Tain; Felock, Peter; Munshi, Vandna; Distefano, Daniel; Flynn, Jessica; Miller, Mike; Yan, Youwei; Reid, John; Sanchez, Rosa; Liang, Yuexia; Paton, Brenda; Wan, Bang-Lin; Anthony, Neville

    2011-11-24

    Highly active antiretroviral therapy (HAART) significantly reduces human immunodeficiency virus (HIV) viral load and has led to a dramatic decrease in acquired immunodeficiency syndrome (AIDS) related mortality. Despite this success, there remains a critical need for new HIV therapies to address the emergence of drug resistant viral strains. Next generation NNRTIs are sought that are effective against these mutant forms of the HIV virus. The bound conformations of our lead inhibitors, MK-1107 (1) and MK-4965 (2), were divergent about the oxymethylene linker, and each of these conformations was rigidified using two isomeric cyclic constraints. The constraint derived from the bioactive conformation of 2provided novel, highly potent NNRTIs that possess broad spectrum antiviral activity and good pharmacokinetic profiles. Systematic SAR led to the identification of indazole as the optimal conformational constraint to provide MK-6186 (3) and MK-7445 (6). Despite their reduced flexibility, these compounds had potency comparable to that of the corresponding acyclic ethers in both recombinant enzyme and cell based assays against both the wild-type and the clinically relevant mutant strains.

  5. Template definition by Tetrahymena telomerase reverse transcriptase.

    PubMed

    Miller, M C; Liu, J K; Collins, K

    2000-08-15

    The ribonucleoprotein enzyme telomerase extends chromosome ends by copying a specific template sequence within its integral RNA component. An active recombinant telomerase RNP is minimally composed of this RNA and the telomerase reverse transcriptase (TERT) protein, which contains sequence motifs conserved among viral reverse transcriptases (RTs), flanked by N- and C-terminal extensions specific to TERTs. We have used site-directed mutagenesis to explore the roles of Tetrahymena TERT in determining features of telomerase activity in general and in establishing the boundaries and use of an internal RNA template in specific. We identify a new ciliate-specific motif in the TERT N-terminus required for template definition. Moreover, several residues in reverse transcriptase motifs 1, 2, A and D are critical for specific aspects of internal template use. Our results indicate that the unique specificity of telomerase activity is conferred to a reverse transcriptase active site by TERT residues both within and beyond the RT motif region.

  6. Discovery and crystallography of bicyclic arylaminoazines as potent inhibitors of HIV-1 reverse transcriptase.

    PubMed

    Lee, Won-Gil; Frey, Kathleen M; Gallardo-Macias, Ricardo; Spasov, Krasimir A; Chan, Albert H; Anderson, Karen S; Jorgensen, William L

    2015-11-01

    Non-nucleoside inhibitors of HIV-1 reverse transcriptase (HIV-RT) are reported that incorporate a 7-indolizinylamino or 2-naphthylamino substituent on a pyrimidine or 1,3,5-triazine core. The most potent compounds show below 10 nanomolar activity towards wild-type HIV-1 and variants bearing Tyr181Cys and Lys103Asn/Tyr181Cys resistance mutations. The compounds also feature good aqueous solubility. Crystal structures for two complexes enhance the analysis of the structure-activity data.

  7. Substituted tetrahydroquinolines as potent allosteric inhibitors of reverse transcriptase and its key mutants

    SciTech Connect

    Su, Dai-Shi; Lim, John J.; Tinney, Elizabeth; Wan, Bang-Lin; Young, Mary Beth; Anderson, Kenneth D.; Rudd, Deanne; Munshi, Vandna; Bahnck, Carolyn; Felock, Peter J.; Lu, Meiqing; Lai, Ming-Tain; Touch, Sinoeun; Moyer, Gregory; DiStefano, Daniel J.; Flynn, Jessica A.; Liang, Yuexia; Sanchez, Rosa; Prasad, Sridhar; Yan, Youwei; Perlow-Poehnelt, Rebecca; Torrent, Maricel; Miller, Mike; Vacca, Joe P.; Williams, Theresa M.; Anthony, Neville J.; Merck

    2010-09-27

    Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are key elements of multidrug regimens, called HAART (Highly Active Antiretroviral Therapy), that are used to treat HIV-1 infections. Elucidation of the structure-activity relationships of the thiocarbamate moiety of the previous published lead compound 2 provided a series of novel tetrahydroquinoline derivatives as potent inhibitors of HIV-1 RT with nanomolar intrinsic activity on the WT and key mutant enzymes and potent antiviral activity in infected cells. The SAR optimization, mutation profiles, preparation of compounds, and pharmacokinetic profile of compounds are described.

  8. A novel mechanism for inhibition of HIV-1 reverse transcriptase.

    PubMed

    Skillman, A Geoffrey; Maurer, Karl W; Roe, Diana C; Stauber, Margaret J; Eargle, Dolan; Ewing, Todd J A; Muscate, Angelika; Davioud-Charvet, Elisabeth; Medaglia, Maxine V; Fisher, Robert J; Arnold, Edward; Gao, Hong Qiang; Buckheit, Robert; Boyer, Paul L; Hughes, Stephen H; Kuntz, Irwin D; Kenyon, George L

    2002-12-01

    The human immunodeficiency virus (HIV) epidemic is an important medical problem. Although combination drug regimens have produced dramatic decreases in viral load, current therapies do not provide a cure for HIV infection. We have used structure-based design and combinatorial medicinal chemistry to identify potent and selective HIV-1 reverse transcriptase (RT) inhibitors that may work by a mechanism distinct from that of current HIV drugs. The most potent of these compounds (compound 4, 2-naphthalenesulfonic acid, 4-hydroxy-7-[[[[5-hydroxy-6-[(4-cinnamylphenyl)azo]-7-sulfo-2-naphthalenyl]amino]carbonyl]amino]-3-[(4-cinnamylphenyl)azo], disodium salt) has an IC(50) of 90 nM for inhibition of polymerase chain extension, a K(d) of 40 nM for inhibition of DNA-RT binding, and an IC(50) of 25-100 nM for inhibition of RNaseH cleavage. The parent compound (1) was as effective against 10 nucleoside and non-nucleoside resistant HIV-1 RT mutants as it was against the wild-type enzyme. Compound 4 inhibited HIV-1 RT and murine leukemia virus (MLV) RT, but it did not inhibit T(4) DNA polymerase, T(7) DNA polymerase, or the Klenow fragment at concentrations up to 200 nM. Finally, compound 4 protected cells from HIV-1 infection at a concentration more than 40 times lower than the concentration at which it caused cellular toxicity.

  9. (PCG) Protein Crystal Growth HIV Reverse Transcriptase

    NASA Technical Reports Server (NTRS)

    1992-01-01

    HIV Reverse Transcriptase crystals grown during the USML-1 (STS-50) mission using Commercial Refrigerator/Incubator Module (CR/IM) at 4 degrees C and the Vapor Diffusion Apparatus (VDA). Reverse transcriptase is an enzyme responsible for copying the nucleic acid genome of the AIDS virus from RNA to DNA. Studies indicated that the space-grown crystals were larger and better ordered (beyond 4 angstroms) than were comparable Earth-grown crystals. Principal Investigators were Charles Bugg and Larry DeLucas.

  10. (PCG) Protein Crystal Growth HIV Reverse Transcriptase

    NASA Technical Reports Server (NTRS)

    1992-01-01

    HIV Reverse Transcriptase crystals grown during the USML-1 (STS-50) mission using Commercial Refrigerator/Incubator Module (CR/IM) at 4 degrees C and the Vapor Diffusion Apparatus (VDA). Reverse transcriptase is an enzyme responsible for copying the nucleic acid genome of the AIDS virus from RNA to DNA. Studies indicated that the space-grown crystals were larger and better ordered (beyond 4 angstroms) than were comparable Earth-grown crystals. Principal Investigators were Charles Bugg and Larry DeLucas.

  11. Reverse transcriptase: mediator of genomic plasticity.

    PubMed

    Brosius, J; Tiedge, H

    1995-01-01

    Reverse transcription has been an important mediator of genomic change. This influence dates back more than three billion years, when the RNA genome was converted into the DNA genome. While the current cellular role(s) of reverse transcriptase are not yet completely understood, it has become clear over the last few years that this enzyme is still responsible for generating significant genomic change and that its activities are one of the driving forces of evolution. Reverse transcriptase generates, for example, extra gene copies (retrogenes), using as a template mature messenger RNAs. Such retrogenes do not always end up as nonfunctional pseudogenes but form, after reinsertion into the genome, new unions with resident promoter elements that may alter the gene's temporal and/or spatial expression levels. More frequently, reverse transcriptase produces copies of nonmessenger RNAs, such as small nuclear or cytoplasmic RNAs. Extremely high copy numbers can be generated by this process. The resulting reinserted DNA copies are therefore referred to as short interspersed repetitive elements (SINEs). SINEs have long been considered selfish DNA, littering the genome via exponential propagation but not contributing to the host's fitness. Many SINEs, however, can give rise to novel genes encoding small RNAs, and are the migrant carriers of numerous control elements and sequence motifs that can equip resident genes with novel regulatory elements [Brosius J. and Gould S.J., Proc Natl Acad Sci USA 89, 10706-10710, 1992]. Retrosequences, such as SINEs and portions of retroelements (e.g., long terminal repeats, LTRs), are capable of donating sequence motifs for nucleosome positioning, DNA methylation, transcriptional enhancers and silencers, poly(A) addition sequences, determinants of RNA stability or transport, splice sites, and even amino acid codons for incorporation into open reading frames as novel protein domains. Retroposition can therefore be considered as a major

  12. Hepatotoxicity of nucleoside reverse transcriptase inhibitors.

    PubMed

    Montessori, Valentina; Harris, Marianne; Montaner, Julio S G

    2003-05-01

    Hepatotoxicity is an adverse effect of all available classes of antiretrovirals, including nucleoside reverse transcriptase inhibitors (NRTI). A syndrome of hepatic steatosis and lactic acidosis has been recognized as a rare, potentially fatal complication since the advent of NRTI monotherapy in the early 1990s. Today, NRTI remain the backbone of antiretroviral combination regimens, and, with the success of current treatment strategies, exposure to two or more of these agents may occur over a number of years. Hepatic steatosis and lactic acidosis are accordingly being observed more frequently, along with a more recently recognized syndrome of chronic hyperlactatemia. These as well as other adverse effects of NRTI are mediated by inhibition of human DNA polymerase gamma, resulting in mitochondrial dysfunction in the liver and other tissues. Early recognition and intervention are essential to avert serious outcomes.

  13. Synthesis and Anti-HIV-1 Evaluation of Some Novel MC-1220 Analogs as Non-Nucleoside Reverse Transcriptase Inhibitors.

    PubMed

    Loksha, Yasser M; Pedersen, Erik B; Loddo, Roberta; La Colla, Paolo

    2016-05-01

    Some novel MC-1220 analogs were synthesized by condensation of 4,6-dichloro-N-methylpyrimidin-2-amine derivatives (1a,b and 15) and/or 4-chloro-6-methoxy-N,N,5-trimethylpyrimidin-2-amine (2a) with the sodium salt of 2,6-difluorophenylacetonitrile followed by treatment with aqueous sodium hydroxide in methanol, alkylation, reduction, halogenation, and/or acidic hydrolysis. All synthesized compounds were evaluated for their activity against HIV-1. The most active compound in this study was compound 7, which showed activity against HIV-1 comparable to that of MC-1220. The only difference in structure between compound 7 and MC-1220 is a fluoro atom instead of a CH3 group.

  14. Chiral Indolylarylsulfone Non-Nucleoside Reverse Transcriptase Inhibitors as New Potent and Broad Spectrum Anti-HIV-1 Agents.

    PubMed

    Famiglini, Valeria; La Regina, Giuseppe; Coluccia, Antonio; Masci, Domiziana; Brancale, Andrea; Badia, Roger; Riveira-Muñoz, Eva; Esté, José A; Crespan, Emmanuele; Brambilla, Alessandro; Maga, Giovanni; Catalano, Myriam; Limatola, Cristina; Formica, Francesca Romana; Cirilli, Roberto; Novellino, Ettore; Silvestri, Romano

    2017-08-10

    We designed and synthesized a series of chiral indolyarylsulfones (IASs) as new HIV-1 NNRTIs. The new IASs 8-37 showed potent inhibition of the HIV-1 WT NL4-3 strain and of the mutant K103N, Y181C, Y188L, and K103N-Y181C HIV-1 strains. Six racemic mixtures, 8, 23-25, 31, and 33, were separated at semipreparative level into their pure enantiomers. The (R)-8 enantiomer bearing the chiral (α-methylbenzyl) was superior to the (S)-counterpart. IAS derivatives bearing the (S) alanine unit, (S)-23, (S,R)-25, (S)-31, and (S)-33, were remarkably more potent than the corresponding (R)-enantiomers. Compound 23 protected hippocampal neuronal cells from the excitotoxic insult, while efavirenz (EFV) did not contrast the neurotoxic effect of glutamate. The present results highlight the chiral IASs as new NNRTIs with improved resistance profile against the mutant HIV-1 strains and reduced neurotoxic effects.

  15. C-2-Aryl O-substituted HI-236 derivatives as non-nucleoside HIV-1 reverse-transcriptase inhibitors

    PubMed Central

    Hunter, Roger; Younis, Yassir; Muhanji, Clare I.; Curtin, Tanith-lea; Naidoo, Kevin J.; Petersen, Melissa; Bailey, Christopher M.; Basavapathruni, Aravind; Anderson, Karen S.

    2009-01-01

    Several novel thiourea derivatives of the NNRTI HI-236 substituted at the C-2 oxygen of the phenyl ring have been synthesized and evaluated for their inhibitory activity against HIV-1 (IIIB) replication in MT-2 cell cultures. The compounds were synthesized in order to fine-tune the activity of HI-236 as well as to gain insight into spatial characteristics in the pocket pertaining to the positional choice of tether in the design of [NRTI]-tether-[HI-236] bifunctional inhibitors. Two of the thiourea derivatives bearing a butynyl (6c) or hydroxyethyl tether (6n) were endowed with improved anti-HIV activity compared to HI-236. NNRTI activity was confirmed by a cell-free RT assay on six of the derivatives in which 6c returned an IC50 of 3.8 nM compared to 28 nM for HI-236, establishing it as an improved lead for HI-236. The structure-activity profile is discussed in terms of potential interactions in the NNRTI pocket as suggested by a docking model using AutoDock, which have a bearing on the bifunctional drug design. PMID:18996020

  16. Synthesis and biological evaluation of CHX-DAPYs as HIV-1 non-nucleoside reverse transcriptase inhibitors.

    PubMed

    Yan, Zi-Hong; Wu, Hai-Qiu; Chen, Wen-Xue; Wu, Yan; Piao, Hu-Ri; He, Qiu-Qin; Chen, Fen-Er; De Clercq, Erik; Pannecouque, Christophe

    2014-06-15

    A series of new diarylpyrimidines (DAPYs) characterized by a halogen atom on the methylene linker between wing I and the central pyrimidine ring was synthesized and evaluated for their anti-HIV activity in MT-4 cell cultures. The two most promising compounds 7f and 7g showed excellent activity against wild-type HIV-1 with low nanomolar EC50 values of 0.005 and 0.009 μM, respectively, which were comparable to or more potent than all the reference drugs zidovudine (AZT), lamivudine (3TC), nevirapine (NEV), efavirenz (EFV), delaviridine (DLV) and etravirine (ETV). In particular, 7g also displayed strong activity against the double mutant strain 103N + 181C with an EC50 value of 8.2 μM. The preliminary structure-activity relationship (SAR) and molecular docking analysis of this new series of CHX-DAPYs were also investigated.

  17. Design and synthesis of a new series of cyclopropylamino-linking diarylpyrimidines as HIV non-nucleoside reverse transcriptase inhibitors.

    PubMed

    Liu, Yang; Meng, Ge; Zheng, Aqun; Chen, Fener; Chen, Wenxue; De Clercq, Erik; Pannecouque, Christophe; Balzarini, Jan

    2014-10-01

    A new series of 29 diarylpyrimidine analogues featuring a cyclopropylamino group between the pyrimidine scaffold and the aryl wing have been synthesized. All of the new compounds have been characterized by spectra analysis. The target molecules were evaluated for their in vitro anti-HIV activity with FDA-approved drugs as references. Some of the compounds exhibited moderate to potent activities against wild-type HIV-1. The compound 4-((4-((cyclopropylamino)(2,5-difluorophenyl)methyl)pyrimidin-2-yl)amino)benzonitrile (1e) displayed potent anti-HIV-1 activity against WT HIV-1 with an IC50 of 0.099 μM and a selectivity index of 2302. The preliminary structure-activity relationship (SAR) of this new series of compounds was also investigated.

  18. Synthesis and biological evaluation of imidazole thioacetanilides as novel non-nucleoside HIV-1 reverse transcriptase inhibitors.

    PubMed

    Zhan, Peng; Liu, Xinyong; Zhu, Junjie; Fang, Zengjun; Li, Zhenyu; Pannecouque, Christophe; Clercq, Erik De

    2009-08-15

    A series of 2-(1-aryl-1H-imidazol-2-ylthio)acetamide [imidazole thioacetanilide (ITA)] derivatives were synthesized and evaluated as potent inhibitors of human immunodeficiency virus type-1 (HIV-1). Among them, the most potent HIV-1 inhibitors were 4a5 (EC(50)=0.18microM), and 4a2 (EC(50)=0.20microM), which were more effective than the lead compound L1 (EC(50)=2.053microM) and the reference drugs nevirapine and delavirdine. The preliminary structure-activity relationship (SAR) of the newly synthesized congeners is discussed.

  19. Physicochemical Property-Driven Optimization of Diarylaniline Compounds as Potent HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors

    PubMed Central

    Liu, Na; Qin, Bingjie; Sun, Lian-Qi; Yu, Fei; Lu, Lu; Jiang, Shibo; Lee, Kuo-Hsiung; Xie, Lan

    2014-01-01

    Using physicochemical property-driven optimization, twelve new diarylaniline compounds (DAANs) (7a–h, 11a–b and 12a–b) were designed and synthesized. Among them, compounds 12a–b not only showed high potency (EC50 0.96–4.92 nM) against both wild-type and drug-resistant viral strains with the lowest fold change (FC 0.91 and 5.13), but also displayed acceptable drug-like properties based on aqueous solubility and lipophilicity (LE > 0.3, LLE > 5, LELP < 10). The correlations between potency and physicochemical properties of these DAAN analogues are also described. Compounds 12a–b merit further development as potent clinical trial candidates against AIDS. PMID:25042339

  20. Intracellular Metabolism of Nucleoside/Nucleotide Analogues: a Bottleneck to Reach Active Drugs on HIV Reverse Transcriptase.

    PubMed

    Varga, Andrea; Lionne, Corinne; Roy, Béatrice

    2016-01-01

    To date, the most effective way to treat HIV is to use a highly active antiretroviral therapy (HAART) that combines three or more different drugs. The usual regimen consists of two nucleoside reverse transcriptase inhibitors and either a protease inhibitor, a non-nucleoside reverse transcriptase inhibitor, or an integrase strand transfer inhibitor. Due to the emerging resistance against the nucleoside analogues in use, there is a continuous need for the development of such therapeutic molecules with different structural features. In this review, we would like to summarize the state of knowledge of the antiretroviral nucleoside analogues intracellular metabolism. Indeed, these molecules have to be phosphorylated in the cell, a process that is often a bottleneck, to produce their pharmacologically active triphosphorylated forms. These forms can be used by the HIV reverse transcriptase. Because they lack a 3'-hydroxyl group, they block further extension of the viral DNA, and finally lead to early chain termination. Several kinases can act on the phosphorylation of these drugs; most of them have low nucleoside/nucleotide specificity. On the other hand, there are also nucleotidases in the cell, which can reverse the phosphorylation process, thus shifting the equilibrium from the active triphosphorylated state to the non-active (not-, mono- or di-phosphorylated) states of these analogues. Here, we would like to bring to the attention of the medicinal chemists that they have to take into account the limitation of the intracellular phosphorylation machinery when designing new nucleoside analogue drugs.

  1. Reverse transcriptase and intron number evolution

    PubMed Central

    Kuo, Alan; Grigoriev, Igor V.

    2014-01-01

    Background Introns are universal in eukaryotic genomes and play important roles in transcriptional regulation, mRNA export to the cytoplasm, nonsense-mediated decay as both a regulatory and a splicing quality control mechanism, R-loop avoidance, alternative splicing, chromatin structure, and evolution by exon-shuffling. Methods Sixteen complete fungal genomes were used 13 of which were sequenced and annotated by JGI. Ustilago maydis, Cryptococcus neoformans, and Coprinus cinereus (also named Coprinopsis cinerea) were from the Broad Institute. Gene models from JGI-annotated genomes were taken from the GeneCatalog track that contained the best representative gene models. Varying fractions of the GeneCatalog were manually curated by external users. For clarity, we used the JGI unique database identifier. Results The last common ancestor of eukaryotes (LECA) has an estimated 6.4 coding exons per gene (EPG) and evolved into the diverse eukaryotic life forms, which is recapitulated by the development of a stem cell. We found a parallel between the simulated reverse transcriptase (RT)-mediated intron loss and the comparative analysis of 16 fungal genomes that spanned a wide range of intron density. Although footprints of RT (RTF) were dynamic, relative intron location (RIL) to the 5'-end of mRNA faithfully traced RT-mediated intron loss and revealed 7.7 EPG for LECA. The mode of exon length distribution was conserved in simulated intron loss, which was exemplified by the shared mode of 75 nt between fungal and Chlamydomonas genomes. The dominant ancient exon length was corroborated by the average exon length of the most intron-rich genes in fungal genomes and consistent with ancient protein modules being ~25 aa. Combined with the conservation of a protein length of 400 aa, the earliest ancestor of eukaryotes could have 16 EPG. During earlier evolution, Ascomycota’s ancestor had significantly more 3'-biased RT-mediated intron loss that was followed by dramatic RTF loss

  2. Identification of a 3-aminoimidazo[1,2-a]pyridine inhibitor of HIV-1 reverse transcriptase

    PubMed Central

    2012-01-01

    Background Despite the effectiveness of highly active antiretroviral therapy (HAART), there remains an urgent need to develop new human immunodeficiency virus type 1 (HIV-1) inhibitors with better pharmacokinetic properties that are well tolerated, and that block common drug resistant virus strains. Methods Here we screened an in-house small molecule library for novel inhibitors of HIV-1 replication. Results An active compound containing a 3-aminoimidazo[1,2-a]pyridine scaffold was identified and quantitatively characterized as a non-nucleoside reverse transcriptase inhibitor (NNRTI). Conclusions The potency of this compound coupled with its inexpensive chemical synthesis and tractability for downstream SAR analysis make this inhibitor a suitable lead candidate for further development as an antiviral drug. PMID:23231773

  3. N-3 Hydroxylation of Pyrimidine-2,4-diones Yields Dual Inhibitors of HIV Reverse Transcriptase and Integrase

    PubMed Central

    2010-01-01

    A new molecular scaffold featuring an N-hydroxyimide functionality and capable of inhibiting both reverse transcriptase (RT) and integrase (IN) of human immunodeficiency virus (HIV) was rationally designed based on 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) non-nucleoside RT inhibitors (NNRTIs). The design involves a minimal 3-N hydroxylation of the pyrimidine ring of HEPT compound to yield a chelating triad which, along with the existing benzyl group, appeared to satisfy major structural requirements for IN binding. In the mean time, this chemical modification did not severely compromise the compound’s ability to inhibit RT. A preliminary structure−activity relationship (SAR) study reveals that this N-3 OH is essential for IN inhibition and that the benzyl group on N-1 side chain is more important for IN binding than the one on C-6. PMID:21499541

  4. Hierarchical database screenings for HIV-1 reverse transcriptase using a pharmacophore model, rigid docking, solvation docking, and MM-PB/SA.

    PubMed

    Wang, Junmei; Kang, Xinshan; Kuntz, Irwin D; Kollman, Peter A

    2005-04-07

    In this work, an efficient strategy was presented to search drug leads for human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) using hierarchical database screenings, which included a pharmacophore model, multiple-conformation rigid docking, solvation docking, and molecular mechanics-Poisson-Boltzmann/surface area (MM-PB/SA) sequentially. Encouraging results were achieved in searching a refined available chemical directory (ACD) database: the enrichment factor after the first three filters was estimated to be 25-fold; the hit rate for all the four filters was predicted to be 41% in a control test using 37 known HIV-1 non-nucleoside reverse transcriptase inhibitors; 10 out of 30 promising solvation-docking hits had MM-PB/SA binding free energies better than -6.8 kcal/mol and the best one, HIT15, had -17.0 kcal/mol. In conclusion, the hierarchical multiple-filter database searching strategy is an attractive strategy in drug lead exploration.

  5. Biochemical, inhibition and inhibitor resistance studies of xenotropic murine leukemia virus-related virus reverse transcriptase

    PubMed Central

    Ndongwe, Tanyaradzwa P.; Adedeji, Adeyemi O.; Michailidis, Eleftherios; Ong, Yee Tsuey; Hachiya, Atsuko; Marchand, Bruno; Ryan, Emily M.; Rai, Devendra K.; Kirby, Karen A.; Whatley, Angela S.; Burke, Donald H.; Johnson, Marc; Ding, Shilei; Zheng, Yi-Min; Liu, Shan-Lu; Kodama, Ei-Ichi; Delviks-Frankenberry, Krista A.; Pathak, Vinay K.; Mitsuya, Hiroaki; Parniak, Michael A.; Singh, Kamalendra; Sarafianos, Stefan G.

    2012-01-01

    We report key mechanistic differences between the reverse transcriptases (RT) of human immunodeficiency virus type-1 (HIV-1) and of xenotropic murine leukemia virus-related virus (XMRV), a gammaretrovirus that can infect human cells. Steady and pre-steady state kinetics demonstrated that XMRV RT is significantly less efficient in DNA synthesis and in unblocking chain-terminated primers. Surface plasmon resonance experiments showed that the gammaretroviral enzyme has a remarkably higher dissociation rate (koff) from DNA, which also results in lower processivity than HIV-1 RT. Transient kinetics of mismatch incorporation revealed that XMRV RT has higher fidelity than HIV-1 RT. We identified RNA aptamers that potently inhibit XMRV, but not HIV-1 RT. XMRV RT is highly susceptible to some nucleoside RT inhibitors, including Translocation Deficient RT inhibitors, but not to non-nucleoside RT inhibitors. We demonstrated that XMRV RT mutants K103R and Q190M, which are equivalent to HIV-1 mutants that are resistant to tenofovir (K65R) and AZT (Q151M), are also resistant to the respective drugs, suggesting that XMRV can acquire resistance to these compounds through the decreased incorporation mechanism reported in HIV-1. PMID:21908397

  6. Reverse transcriptase incorporation of 1,5-anhydrohexitol nucleotides

    PubMed Central

    Vastmans, Karen; Froeyen, Matheus; Kerremans, Luc; Pochet, Sylvie; Herdewijn, Piet

    2001-01-01

    Several reverse transcriptases were studied for their ability to accept anhydrohexitol triphosphates, having a conformationally restricted six-membered ring, as substrate for template-directed synthesis of HNA. It was found that AMV, M-MLV, M-MLV (H–), RAV2 and HIV-1 reverse transcriptases were able to recognise the anhydrohexitol triphosphate as substrate and to efficiently catalyse the incorporation of one non-natural anhydrohexitol nucleotide opposite a natural complementary nucleotide. However, only the dimeric enzymes, the RAV2 and HIV-1 reverse transcriptases, seemed to be able to further extend the primer with another anhydrohexitol building block. Subsequently, several HIV-1 mutants (4×AZT, 4×AZT/L100I, L74V, M184V and K65A) were likewise analysed, resulting in selection of K65A and, in particular, M184V as the most succesful mutant HIV-1 reverse transcriptases capable of elongating a DNA primer with several 1,5-anhydrohexitol adenines in an efficient way. Results of kinetic experiments in the presence of this enzyme revealed that incorporation of one anhydrohexitol nucleotide of adenine or thymine gave an increased (for 1,5-anhydrohexitol-ATP) and a slightly decreased (for 1,5-anhydrohexitol-TTP) Km value in comparison to that of their natural counterparts. However, no more than four analogues could be inserted under the experimental conditions required for selective incorporation. Investigation of incorporation of the altritol anhydrohexitol nucleotide of adenine in the presence of M184V and Vent (exo–) DNA polymerase proved that an adjacent hydroxyl group on C3 of 1,5-anhydrohexitol-ATP has a detrimental effect on the substrate activity of the six-ring analogue. These results could be rationalised based on the X-ray structure of HIV-1 reverse transcriptase. PMID:11470872

  7. The Reverse Transcriptase Encoded by LINE-1 Retrotransposons in the Genesis, Progression, and Therapy of Cancer

    PubMed Central

    Sciamanna, Ilaria; De Luca, Chiara; Spadafora, Corrado

    2016-01-01

    In higher eukaryotic genomes, Long Interspersed Nuclear Element 1 (LINE-1) retrotransposons represent a large family of repeated genomic elements. They transpose using a reverse transcriptase (RT), which they encode as part of the ORF2p product. RT inhibition in cancer cells, either via RNA interference-dependent silencing of active LINE-1 elements, or using RT inhibitory drugs, reduces cancer cell proliferation, promotes their differentiation and antagonizes tumor progression in animal models. Indeed, the non-nucleoside RT inhibitor efavirenz has recently been tested in a phase II clinical trial with metastatic prostate cancer patients. An in-depth analysis of ORF2p in a mouse model of breast cancer showed ORF2p to be precociously expressed in precancerous lesions and highly abundant in advanced cancer stages, while being barely detectable in normal breast tissue, providing a rationale for the finding that RT-expressing tumors are therapeutically sensitive to RT inhibitors. We summarize mechanistic and gene profiling studies indicating that abundant LINE-1-derived RT can “sequester” RNA substrates for reverse transcription in tumor cells, entailing the formation of RNA:DNA hybrid molecules and impairing the overall production of regulatory miRNAs, with a global impact on the cell transcriptome. Based on these data, LINE-1-ORF2 encoded RT has a tumor-promoting potential that is exerted at an epigenetic level. We propose a model whereby LINE1-RT drives a previously unrecognized global regulatory process, the deregulation of which drives cell transformation and tumorigenesis with possible implications for cancer cell heterogeneity. PMID:26904537

  8. Intravaginal ring delivery of the reverse transcriptase inhibitor TMC 120 as an HIV microbicide.

    PubMed

    Woolfson, A David; Malcolm, R Karl; Morrow, Ryan J; Toner, Clare F; McCullagh, Stephen D

    2006-11-15

    TMC 120 (Dapivirine) is a potent non-nucleoside reverse transcriptase inhibitor that is presently being developed as a vaginal HIV microbicide. To date, most vaginal microbicides under clinical investigation have been formulated as single-dose semi-solid gels, designed for application to the vagina before each act of intercourse. However, a clear rationale exists for providing long-term, controlled release of vaginal microbicides in order to afford continuous protection against heterosexually transmitted HIV infection and to improve user compliance. In this study we report on the incorporation of various pharmaceutical excipients into TMC 120 silicone, reservoir-type intravaginal rings (IVRs) in order to modify the controlled release characteristics of the microbicide. The results demonstrate that TMC 120 is released in zero-order fashion from the rings over a 28-day period and that release parameters could be modified by the inclusion of release-modifying excipients in the IVR. The hydrophobic liquid excipient isopropyl myristate had little effect on steady-state daily release rates, but did increase the magnitude and duration of burst release in proportion to excipient loading in the IVR. By comparison, the hydrophobic liquid poly(dimethylsiloxane) had little effect on TMC 120 release parameters. A hydrophilic excipient, lactose, had the surprising effect of decreasing TMC 120 burst release while increasing the apparent steady-state daily release in a concentration-dependent manner. Based on previous cell culture data and vaginal physiology, TMC120 is released from the various ring formulations in amounts potentially capable of maintaining a protective vaginal concentration. It is further predicted that the observed release rates may be maintained for at least a period of 1 year from a single ring device. TMC 120 release profiles and the mechanical properties of rings could be modified by the physicochemical nature of hydrophobic and hydrophilic excipients

  9. Homodimerization of the p51 Subunit of HIV-1 Reverse Transcriptase

    SciTech Connect

    Zheng, X.; Mueller, G; Cuneo, M; DeRose, E; London, R

    2010-01-01

    The dimerization of HIV reverse transcriptase (RT), required to obtain the active form of the enzyme, is influenced by mutations, non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleotide substrates, Mg ions, temperature, and specifically designed dimerization inhibitors. In this study, we have utilized nuclear magnetic resonance (NMR) spectroscopy of the [methyl-{sup 13}C]methionine-labeled enzyme and small-angle X-ray scattering (SAXS) to investigate how several of these factors influence the dimerization behavior of the p51 subunit. The {sup 1}H-{sup 13}C HSQC spectrum of p51 obtained at micromolar concentrations indicates that a significant fraction of the p51 adopts a 'p66-like' conformation. SAXS data obtained for p51 samples were used to determine the fractions of monomer and dimer in the sample and to evaluate the conformation of the fingers/thumb subdomain. All of the p51 monomer observed was found to adopt the compact, 'p51C' conformation observed for the p51 subunit in the RT heterodimer. The NMR and SAXS data indicate that the p51 homodimer adopts a structure that is similar to the p66/p51 heterodimer, with one p51C subunit and a second p51 subunit in an extended, 'p51E' conformation that resembles the p66 subunit of the heterodimer. The fractional dimer concentration and the fingers/thumb orientation are found to depend strongly on the experimental conditions and exhibit a qualitative dependence on nevirapine and ionic strength (KCl) that is similar to the behavior reported for the heterodimer and the p66 homodimer. The L289K mutation interferes with p51 homodimer formation as it does with formation of the heterodimer, despite its location far from the dimer interface. This effect is readily interpreted in terms of a conformational selection model, in which p51{sub L289K} has a much greater preference for the compact, p51C conformation. A reduced level of dimer formation then results from the reduced ratio of the p51E{sub L289K} to p51C

  10. The first demonstration of the existence of reverse transcriptases in bacteria.

    PubMed

    Inouye, Masayori

    2017-01-15

    It has been long thought that reverse transcriptases are unique to the eukaryotes. However, through our research on a peculiar single stranded DNA called msDNA in Myxococcus xanthus, it was predicted that its synthesis requires reverse transcriptases. Subsequently, Lim and Maas as well as our group demonstrated the existence of reverse transcriptases for the production of msDNA. In this review, I describe how the discovery of msDNA led to the discovery of reverse transcriptases in bacteria and discuss the evolutionary significance of the discovery of revise transcriptases in bacteria.

  11. Hybrid Ty1/HIV-1 elements used to detect inhibitors and monitor the activity of HIV-1 reverse transcriptase

    PubMed Central

    Nissley, Dwight V.; Boyer, Paul L.; Garfinkel, David J.; Hughes, Stephen H.; Strathern, Jeffrey N.

    1998-01-01

    We previously demonstrated that hybrid retrotransposons composed of the yeast Ty1 element and the reverse transcriptase (RT) of HIV-1 are active in the yeast Saccharomyces cerevisiae. The RT activity of these hybrid Ty1/HIV-1 (his3AI/AIDS RT; HART) elements can be monitored by using a simple genetic assay. HART element reverse transcription depends on both the polymerase and RNase H domains of HIV-1 RT. Here we demonstrate that the HART assay is sensitive to inhibitors of HIV-1 RT. (−)-(S)-8-Chloro-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione monohydrochloride (8 Cl-TIBO), a well characterized non-nucleoside RT inhibitor (NNRTI) of HIV-1 RT, blocks propagation of HART elements. HART elements that express NNRTI-resistant RT variants of HIV-1 are insensitive to 8 Cl-TIBO, demonstrating the specificity of inhibition in this assay. HART elements carrying NNRTI-resistant variants of HIV-1 RT can be used to identify compounds that are active against drug-resistant viruses. PMID:9811899

  12. Quantitative Structure activity Relationship Analysis of Pyridinone HIV-1 Reverse Transcriptase Inhibitors using the k Nearest Neighbor Method and QSAR-based Database Mining

    NASA Astrophysics Data System (ADS)

    Medina-Franco, Jose Luis; Golbraikh, Alexander; Oloff, Scott; Castillo, Rafael; Tropsha, Alexander

    2005-04-01

    We have developed quantitative structure-activity relationship (QSAR) models for 44 non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTIs) of the pyridinone derivative type. The k nearest neighbor ( kNN) variable selection approach was used. This method utilizes multiple descriptors such as molecular connectivity indices, which are derived from two-dimensional molecular topology. The modeling process entailed extensive validation including the randomization of the target property (Y-randomization) test and the division of the dataset into multiple training and test sets to establish the external predictive power of the training set models. QSAR models with high internal and external accuracy were generated, with leave-one-out cross-validated R 2 ( q 2) values ranging between 0.5 and 0.8 for the training sets and R 2 values exceeding 0.6 for the test sets. The best models with the highest internal and external predictive power were used to search the National Cancer Institute database. Derivatives of the pyrazolo[3,4- d]pyrimidine and phenothiazine type were identified as promising novel NNRTIs leads. Several candidates were docked into the binding pocket of nevirapine with the AutoDock (version 3.0) software. Docking results suggested that these types of compounds could be binding in the NNRTI binding site in a similar mode to a known non-nucleoside inhibitor nevirapine.

  13. Docking study of HIV-1 reverse transcriptase with phytochemicals

    PubMed Central

    Seal, Abhik; Aykkal, Riju; Babu, Rosana O; Ghosh, Mriganka

    2011-01-01

    Natural products are important sources of drug discovery. In this context groups of different set of phytochemicals were taken and docked into the different cavities of the Reverse transcriptase (PDB ID: 1REV) of Human immunodeficiency virus (HIV) and results were discussed. Natural compounds such as Curcumin, Geranin, Gallotannin, Tiliroside, Kaempferol-3-o-glucoside and Trachelogenin were found to very effective according to its binding energy and ligand efficiency score. Those compounds also were found to have no adverse effect as carcinogenicity and mutagenicity and favorable drug likeness score. Hence, considering the facts those compounds could use effectively for HIV-1 drug discovery. PMID:21423889

  14. Oxidative base damage in RNA detected by reverse transcriptase.

    PubMed Central

    Rhee, Y; Valentine, M R; Termini, J

    1995-01-01

    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. Images PMID:7545285

  15. Identification and characterization of sea squirt telomerase reverse transcriptase.

    PubMed

    Li, Yang; Yates, Joel A; Chen, Julian J-L

    2007-10-01

    Telomerase is essential for maintaining telomere length and chromosome stability in most eukaryotic organisms. The telomerase ribonucleoprotein complex consists of two essential components, the catalytic telomerase reverse transcriptase protein (TERT) and the intrinsic telomerase RNA. The sea squirts, as urochordates, occupy a key position in the phylogenetic tree of the chordates: they diverged from the other chordates just before the lineage of vertebrates, and thus provide special insight into the origin and evolution of vertebrate genes. Here, we report the cloning and characterization of TERT genes from two sea squirts, Ciona intestinalis and Ciona savignyi. The C. intestinalis TERT (CinTERT) gene encodes 907 amino acids and consists of 17 exons, which are similar to vertebrate TERT genes. The C. savignyi TERT (CsaTERT) gene encodes 843 amino acids, but surprisingly does not contain any introns. Both Ciona TERTs contain all of the reverse transcriptase (RT) motifs (1, 2, A, B, C, D, and E) that are typically present in telomerase and viral RTs. Interestingly, the alignment of Ciona and vertebrate TERT sequences reveals a previously unknown motif, named motif 3, located between motifs 2 and A. The Ciona TERT gene is expressed in all tissues analyzed except the brain and heart. This is the first report of the TERT gene in invertebrate chordates.

  16. Discovery of Novel Inhibitors of HIV-1 Reverse Transcriptase Through Virtual Screening of Experimental and Theoretical Ensembles

    PubMed Central

    Ivetac, Anthony; Swift, Sara E.; Boyer, Paul L.; Diaz, Arturo; Naughton, John; Young, John A. T.; Hughes, Stephen H.; McCammon, J. Andrew

    2014-01-01

    Non-nucleoside Reverse Transcriptase Inhibitors (NNRTIs) are potent anti-HIV chemotherapeutics. Although there are FDA-approved NNRTIs, challenges such as the development of resistance have limited their utility. Here we describe the identification of novel NNRTIs through a combination of computational and experimental approaches. Based on the known plasticity of the NNRTI binding pocket (NNIBP), we adopted an ensemble-based virtual screening strategy: coupling receptor conformations from 10 x-ray crystal structures with 120 snapshots from a total of 480 ns of Molecular Dynamics (MD) trajectories. A screening library of 2,864 National Cancer Institute (NCI) compounds was built and docked against the ensembles in a hierarchical fashion. 16 diverse compounds were tested for their ability to block HIV infection in human tissue cultures using a luciferase-based reporter assay. 3 promising compounds were further characterized, using a HIV-1 RT based polymerase assay, to determine the specific mechanism of inhibition. We found that 2 of the 3 compounds inhibited the polymerase activity of RT (with potency similar to the positive control, the FDA-approved drug nevirapine). Through a computational approach, we were able to discover 2 compounds which inhibit HIV replication and block the activity of RT, thus offering the potential for optimization into mature inhibitors. PMID:24405985

  17. Efficient Discovery of Potent Anti-HIV Agents Targeting the Tyr181Cys Variant of HIV Reverse Transcriptase

    PubMed Central

    Jorgensen, William L.; Bollini, Mariela; Thakur, Vinay V.; Domaoal, Robert A.; Spasov, Krasimir A.; Anderson, Karen S.

    2011-01-01

    Non-nucleoside inhibitors of HIV reverse transcriptase (NNRTIs) are being pursued with guidance from molecular modeling including free energy perturbation (FEP) calculations for protein-inhibitor binding affinities. The previously reported pyrimidinylphenylamine 1 and its chloro analog 2 are potent anti-HIV agents; they inhibit replication of wild-type HIV-1 in infected human T-cells with EC50 values of 2 and 10 nM. However, they show no activity against viral strains containing the Tyr181Cys (Y181C) mutation in HIV-RT. Modeling indicates that the problem is likely associated with extensive interaction between the dimethylallyloxy substituent and Tyr181. As an alternative, a phenoxy group is computed to be oriented in a manner diminishing the contact with Tyr181. However, this replacement leads to a roughly 1000-fold loss of activity for 3 (2.5 μM). The present report details the efficient, computationally-driven evolution of 3 to novel NNRTIs with sub-10 nM potency towards both wild-type HIV-1 and Y181C-containing variants. The critical contributors were FEP substituent scans for the phenoxy and pyrimidine rings and recognition of potential benefits of addition of a cyanovinyl group to the phenoxy ring. PMID:21853995

  18. HIV-1 Reverse Transcriptase Structure with RNase H Inhibitor dihydroxy benzoyl naphthyl Hydrazone Bound at a Novel Site

    SciTech Connect

    Himmel,D.; Sarafianos, S.; Dharmasena, S.; Hossain, M.; McCoy-Simandle, K.; Ilina, T.; Clark, A.; Knight, J.; Julias, J.; et al.

    2007-01-01

    The rapid emergence of drug-resistant variants of human immunodeficiency virus, type 1 (HIV-1), has limited the efficacy of anti-acquired immune deficiency syndrome (AIDS) treatments, and new lead compounds that target novel binding sites are needed. We have determined the 3.15 {angstrom} resolution crystal structure of HIV-1 reverse transcriptase (RT) complexed with dihydroxy benzoyl naphthyl hydrazone (DHBNH), an HIV-1 RT RNase H (RNH) inhibitor (RNHI). DHBNH is effective against a variety of drug-resistant HIV-1 RT mutants. While DHBNH has little effect on most aspects of RT-catalyzed DNA synthesis, at relatively high concentrations it does inhibit the initiation of RNA-primed DNA synthesis. Although primarily an RNHI, DHBNH binds >50 {angstrom} away from the RNH active site, at a novel site near both the polymerase active site and the non-nucleoside RT inhibitor (NNRTI) binding pocket. When DHBNH binds, both Tyr181 and Tyr188 remain in the conformations seen in unliganded HIV-1 RT. DHBNH interacts with conserved residues (Asp186, Trp229) and has substantial interactions with the backbones of several less well-conserved residues. On the basis of this structure, we designed substituted DHBNH derivatives that interact with the NNRTI-binding pocket. These compounds inhibit both the polymerase and RNH activities of RT.

  19. Inhibition of HIV-1 Reverse Transcriptase Dimerization by Small Molecules.

    PubMed

    Tintori, Cristina; Corona, Angela; Esposito, Francesca; Brai, Annalaura; Grandi, Nicole; Ceresola, Elisa Rita; Clementi, Massimo; Canducci, Filippo; Tramontano, Enzo; Botta, Maurizio

    2016-04-15

    Because HIV-1 reverse transcriptase is an enzyme whose catalytic activity depends on its heterodimeric structure, this system could be a target for inhibitors that perturb the interactions between the protein subunits, p51 and p66. We previously demonstrated that the small molecule MAS0 reduced the association of the two RT subunits and simultaneously inhibited both the polymerase and ribonuclease H activities. In this study, some analogues of MAS0 were rationally selected by docking studies and evaluated in vitro for their ability to disrupt dimeric assembly. Two inhibitors were identified with improved activity compared to MAS0. This study lays the basis for the rational design of more potent inhibitors of RT dimerization.

  20. Asymmetric conformational maturation of HIV-1 reverse transcriptase.

    PubMed

    Zheng, Xunhai; Perera, Lalith; Mueller, Geoffrey A; DeRose, Eugene F; London, Robert E

    2015-06-03

    HIV-1 reverse transcriptase utilizes a metamorphic polymerase domain that is able to adopt two alternate structures that fulfill catalytic and structural roles, thereby minimizing its coding requirements. This ambiguity introduces folding challenges that are met by a complex maturation process. We have investigated this conformational maturation using NMR studies of methyl-labeled RT for the slower processes in combination with molecular dynamics simulations for rapid processes. Starting from an inactive conformation, the p66 precursor undergoes a unimolecular isomerization to a structure similar to its active form, exposing a large hydrophobic surface that facilitates initial homodimer formation. The resulting p66/p66' homodimer exists as a conformational heterodimer, after which a series of conformational adjustments on different time scales can be observed. Formation of the inter-subunit RH:thumb' interface occurs at an early stage, while maturation of the connection' and unfolding of the RH' domains are linked and occur on a much slower time scale.

  1. Transcriptional Regulation of Telomerase Reverse Transcriptase (TERT) by MYC

    PubMed Central

    Khattar, Ekta; Tergaonkar, Vinay

    2017-01-01

    Telomerase elongates telomeres and is crucial for maintaining genomic stability. While stem cells and cancer cells display high telomerase activity, normal somatic cells lack telomerase activity primarily due to transcriptional repression of telomerase reverse transcriptase (TERT), the catalytic component of telomerase. Transcription factor binding, chromatin status as well as epigenetic modifications at the TERT promoter regulates TERT transcription. Myc is an important transcriptional regulator of TERT that directly controls its expression by promoter binding and associating with other transcription factors. In this review, we discuss the current understanding of the molecular mechanisms behind regulation of TERT transcription by Myc. We also discuss future perspectives in investigating the regulation of Myc at TERT promoter during cancer development. PMID:28184371

  2. Telomerase reverse transcriptase (TERT) promoter mutations in Korean melanoma patients.

    PubMed

    Roh, Mi Ryung; Park, Kyu-Hyun; Chung, Kee Yang; Shin, Sang Joon; Rha, Sun Young; Tsao, Hensin

    2017-01-01

    Telomerase reverse transcriptase (TERT) is the reverse transcriptase component of the telomeric complex, which synthesizes terminal DNA to protect chromosomal ends and to maintain genomic integrity. In melanoma, mutation in TERT promoter region is a common event and theses promoter variants have been shown to be associated with increased gene expression, decreased telomere length and poorer outcome. In this study, we determined the frequency of TERT promoter mutation in 88 Korean primary melanoma patients and aimed to see the association of TERT promoter mutation status to other major molecular features, such as BRAF, NRAS, KIT mutations and correlate with clinicopathological features. In our study, acral melanoma (n=46, 52.3%) was the most common type. Overall, TERT promoter mutation was observed in 15 cases (17%) with ten c. -124C>T altertions and five c. -146C>T alterations. None of our samples showed CC>TT mutation which is considered pathognomonic of UV induction. Among the 46 acral melanoma patients, 5 patients (10.9%) harbored TERT promoter mutation. Tumors with TERT promoter mutation showed significantly greater Breslow thickness compared to WT tumors (P=0.039). A combined analysis for the presence of TERT promoter and BRAF mutations showed that patients with both TERT promoter and BRAF mutation showed decreased survival compared with those with only TERT promoter mutation, only BRAF mutation, or without mutations in either TERT promoter or BRAF (P=0.035). Our data provides additional evidence that UV-induced TERT promoter mutation frequencies vary depending on melanoma subtype, but preserves its prognostic value.

  3. Giant Reverse Transcriptase-Encoding Transposable Elements at Telomeres.

    PubMed

    Arkhipova, Irina R; Yushenova, Irina A; Rodriguez, Fernando

    2017-09-01

    Transposable elements are omnipresent in eukaryotic genomes and have a profound impact on chromosome structure, function and evolution. Their structural and functional diversity is thought to be reasonably well-understood, especially in retroelements, which transpose via an RNA intermediate copied into cDNA by the element-encoded reverse transcriptase, and are characterized by a compact structure. Here, we report a novel type of expandable eukaryotic retroelements, which we call Terminons. These elements can attach to G-rich telomeric repeat overhangs at the chromosome ends, in a process apparently facilitated by complementary C-rich repeats at the 3'-end of the RNA template immediately adjacent to a hammerhead ribozyme motif. Terminon units, which can exceed 40 kb in length, display an unusually complex and diverse structure, and can form very long chains, with host genes often captured between units. As the principal polymerizing component, Terminons contain Athena reverse transcriptases previously described in bdelloid rotifers and belonging to the enigmatic group of Penelope-like elements, but can additionally accumulate multiple cooriented ORFs, including DEDDy 3'-exonucleases, GDSL esterases/lipases, GIY-YIG-like endonucleases, rolling-circle replication initiator (Rep) proteins, and putatively structural ORFs with coiled-coil motifs and transmembrane domains. The extraordinary length and complexity of Terminons and the high degree of interfamily variability in their ORF content challenge the current views on the structural organization of eukaryotic retroelements, and highlight their possible connections with the viral world and the implications for the elevated frequency of gene transfer. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  4. Telomerase reverse transcriptase (TERT) promoter mutations in Korean melanoma patients

    PubMed Central

    Roh, Mi Ryung; Park, Kyu-Hyun; Chung, Kee Yang; Shin, Sang Joon; Rha, Sun Young; Tsao, Hensin

    2017-01-01

    Telomerase reverse transcriptase (TERT) is the reverse transcriptase component of the telomeric complex, which synthesizes terminal DNA to protect chromosomal ends and to maintain genomic integrity. In melanoma, mutation in TERT promoter region is a common event and theses promoter variants have been shown to be associated with increased gene expression, decreased telomere length and poorer outcome. In this study, we determined the frequency of TERT promoter mutation in 88 Korean primary melanoma patients and aimed to see the association of TERT promoter mutation status to other major molecular features, such as BRAF, NRAS, KIT mutations and correlate with clinicopathological features. In our study, acral melanoma (n=46, 52.3%) was the most common type. Overall, TERT promoter mutation was observed in 15 cases (17%) with ten c. -124C>T altertions and five c. -146C>T alterations. None of our samples showed CC>TT mutation which is considered pathognomonic of UV induction. Among the 46 acral melanoma patients, 5 patients (10.9%) harbored TERT promoter mutation. Tumors with TERT promoter mutation showed significantly greater Breslow thickness compared to WT tumors (P=0.039). A combined analysis for the presence of TERT promoter and BRAF mutations showed that patients with both TERT promoter and BRAF mutation showed decreased survival compared with those with only TERT promoter mutation, only BRAF mutation, or without mutations in either TERT promoter or BRAF (P=0.035). Our data provides additional evidence that UV-induced TERT promoter mutation frequencies vary depending on melanoma subtype, but preserves its prognostic value. PMID:28123854

  5. Interaction of HIV-1 reverse transcriptase ribonuclease H with an acylhydrazone inhibitor.

    PubMed

    Gong, Qingguo; Menon, Lakshmi; Ilina, Tatiana; Miller, Lena G; Ahn, Jinwoo; Parniak, Michael A; Ishima, Rieko

    2011-01-01

    HIV-1 reverse transcriptase is a bifunctional enzyme, having both DNA polymerase (RNA- and DNA-dependent) and ribonuclease H activities. HIV-1 reverse transcriptase has been an exceptionally important target for antiretroviral therapeutic development, and nearly half of the current clinically used antiretrovirals target reverse transcriptase DNA polymerase. However, no inhibitors of reverse transcriptase ribonuclease H are on the market or in preclinical development. Several drug-like small molecule inhibitors of reverse transcriptase ribonuclease H have been described, but little structural information is available about the interactions between reverse transcriptase ribonuclease H and inhibitors that exhibit antiviral activity. In this report, we describe NMR studies of the interaction of a new ribonuclease H inhibitor, BHMP07, with a catalytically active HIV-1 reverse transcriptase ribonuclease H domain fragment. We carried out solution NMR experiments to identify the interaction interface of BHMP07 with the ribonuclease H domain fragment. Chemical shift changes of backbone amide signals at different BHMP07 concentrations clearly demonstrate that BHMP07 mainly recognizes the substrate handle region in the ribonuclease H fragment. Using ribonuclease H inhibition assays and reverse transcriptase mutants, the binding specificity of BHMP07 was compared with another inhibitor, dihydroxy benzoyl naphthyl hydrazone. Our results provide a structural characterization of the ribonuclease H inhibitor interaction and are likely to be useful for further improvements of the inhibitors.

  6. [Predictive factors of clinically significant drug-drug interactions among regimens based on protease inhibitors, non-nucleoside reverse transcriptase inhibitors and raltegravir].

    PubMed

    Cervero, Miguel; Torres, Rafael; Jusdado, Juan José; Pastor, Susana; Agud, Jose Luis

    2016-04-15

    To determine the prevalence and types of clinically significant drug-drug interactions (CSDI) in the drug regimens of HIV-infected patients receiving antiretroviral treatment. retrospective review of database. Centre: Hospital Universitario Severo Ochoa, Infectious Unit. one hundred and forty-two participants followed by one of the authors were selected from January 1985 to December 2014. from their outpatient medical records we reviewed information from the last available visit of the participants, in relation to HIV infection, comorbidities, demographics and the drugs that they were receiving; both antiretroviral drugs and drugs not related to HIV infection. We defined CSDI from the information sheet and/or database on antiretroviral drug interactions of the University of Liverpool (http://www.hiv-druginteractions.org) and we developed a diagnostic tool to predict the possibility of CSDI. By multivariate logistic regression analysis and by estimating the diagnostic performance curve obtained, we identified a quick tool to predict the existence of drug interactions. Of 142 patients, 39 (29.11%) had some type of CSDI and in 11.2% 2 or more interactions were detected. In only one patient the combination of drugs was contraindicated (this patient was receiving darunavir/r and quetiapine). In multivariate analyses, predictors of CSDI were regimen type (PI or NNRTI) and the use of 3 or more non-antiretroviral drugs (AUC 0.886, 95% CI 0.828 to 0.944; P=.0001). The risk was 18.55 times in those receiving NNRTI and 27,95 times in those receiving IP compared to those taking raltegravir. Drug interactions, including those defined as clinically significant, are common in HIV-infected patients treated with antiretroviral drugs, and the risk is greater in IP-based regimens. Raltegravir-based prescribing, especially in patients who receive at least 3 non-HIV drugs could avoid interactions. Copyright © 2016 Elsevier España, S.L.U. All rights reserved.

  7. Recovery from lipodystrophy in HIV-infected children after substitution of stavudine with zidovudine in a non-nucleoside reverse transcriptase inhibitor-based antiretroviral therapy.

    PubMed

    Aurpibul, Linda; Puthanakit, Thanyawee; Taejaroenkul, Sineenart; Sirisanthana, Thira; Sirisanthana, Virat

    2012-04-01

    Substitution of stavudine with zidovudine may lead to recovery from lipodystrophy (LD) in HIV-infected children. We prospectively followed HIV-infected children enrolled in an earlier LD study conducted between 2002 and 2004 at Chiang Mai University Hospital in northern Thailand. In 2006, stavudine was substituted with zidovudine. All children were evaluated by a clinical LD checklist modified from that of the European Pediatric LD study group together with waist/hip measurement at baseline and 24, 48, 72, and 96 weeks after substitution. The waist-to-hip ratios were converted to age- and sex-adjusted z scores based on normal ranges in healthy Thai children. Forty-five lipodystrophic children with 36 episodes of lipohypertrophy and 22 episodes of lipoatrophy were enrolled. By weeks 48 and 96 after substitution, 40% and 47% of lipohypertrophy resolved, whereas 59% and 73% of lipoatrophy resolved, respectively. The rate of resolution of lipoatrophy was higher than that of lipohypertrophy at 48 weeks after substitution and thereafter. Ninety-six weeks after changing to zidovudine therapy, 8 children still had LD (1 with both lipoatrophy and lipohypertrophy, 7 with lipohypertrophy). No clinically significant hematologic adverse event was observed. Substitution of stavudine with zidovudine resulted in decreased severity or resolution of LD among HIV-infected children and adolescents.

  8. Hybrid chemistry. Part 4: Discovery of etravirine-VRX-480773 hybrids as potent HIV-1 non-nucleoside reverse transcriptase inhibitors.

    PubMed

    Wan, Zheng-Yong; Tao, Yuan; Wang, Ya-Feng; Mao, Tian-Qi; Yin, Hong; Chen, Fen-Er; Piao, Hu-Ri; De Clercq, Erik; Daelemans, Dirk; Pannecouque, Christophe

    2015-08-01

    A novel series of etravirine-VRX-480773 hybrids were designed using structure-guided molecular hybridization strategy and fusing the pharmacophore templates of etravirine and VRX-480773. The anti-HIV-1 activity and cytotoxicity was evaluated in MT-4 cell cultures. The most active hybrid compound in this series, N-(2-chlorophenyl)-2-((4-(4-cyano-2,6-dimethylphenoxy)pyrimidin-2-yl)thio)acetamide 3d (EC50=0.24 , SI>1225), was more potent than delavirdine (EC50=0.66 μM, SI>67) in the anti-HIV-1 in vitro cellular assay. Studies of structure-activity relationships established a correlation between anti-HIV activity and the substitution pattern of the acetanilide group.

  9. Conformation depends on 4D-QSAR analysis using EC-GA method: pharmacophore identification and bioactivity prediction of TIBOs as non-nucleoside reverse transcriptase inhibitors.

    PubMed

    Akyüz, Lalehan; Sarıpınar, Emin

    2013-08-01

    The electron conformational and genetic algorithm methods (EC-GA) were integrated for the identification of the pharmacophore group and predicting the anti HIV-1 activity of tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepinone (TIBO) derivatives. To reveal the pharmacophore group, each conformation of all compounds was arranged by electron conformational matrices of congruity. Multiple comparisons of these matrices, within given tolerances for high active and low active TIBO derivatives, allow the identification of the pharmacophore group that refers to the electron conformational submatrix of activity. The effects of conformations, internal and external validation were investigated by four different models based on an ensemble of conformers and a single conformer, both with and without a test set. Model 1 using an ensemble of conformers for the training (39 compounds) and test sets (13 compounds), obtained by the optimum seven parameters, gave satisfactory results (R²(training) = 0.878, R²(test)= 0.910, q² = 0.840, q²(ext1) = 0.926 and q²(ext2) = 0.900).

  10. 2D, 3D-QSAR and docking studies of 1,2,3-thiadiazole thioacetanilides analogues as potent HIV-1 non-nucleoside reverse transcriptase inhibitors

    PubMed Central

    2012-01-01

    Background The discovery of clinically relevant inhibitors of HIV-RT for antiviral therapy has proven to be a challenging task. To identify novel and potent HIV-RT inhibitors, the quantitative structure–activity relationship (QSAR) approach became very useful and largely widespread technique forligand-based drug design. Methods We perform the two- and three-dimensional (2D and 3D) QSAR studies of a series of 1,2,3-thiadiazole thioacetanilides analogues to elucidate the structural properties required for HIV-RT inhibitory activity. Results The 2D-QSAR studies were performed using multiple linear regression method, giving r2 = 0.97 and q2 = 0.94. The 3D-QSAR studies were performed using the stepwise variable selection k-nearest neighbor molecular field analysis approach; a leave-one-out cross-validated correlation coefficient q2 = 0.89 and a non-cross-validated correlation coefficient r2 = 0.97 were obtained. Docking analysis suggests that the new series have comparable binding affinity with the standard compounds. Conclusions This approach showed that hydrophobic and electrostatic effects dominantly determine binding affinities which will further useful for development of new NNRTIs. PMID:22691718

  11. Design, Synthesis, and Preclinical Evaluations of Novel 4-Substituted 1,5-Diarylanilines as Potent HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitor (NNRTI) Drug Candidates

    PubMed Central

    Sun, Lian-Qi; Zhu, Lei; Qian, Keduo; Qin, Bingjie; Huang, Li; Chen, Chin Ho; Lee, Kuo-Hsiung; Xie, Lan

    2012-01-01

    Twenty-one new 4-substituted diarylaniline compounds (DAANs) (Scheme 2, series 13, 14, and 15) were designed, synthesized, and evaluated against wild-type and drug resistant HIV-1 viral strains. As a result, approximately a dozen new DAANs showed high potency with low nano- to sub-nanomolar EC50 values ranging from 0.2 to 10 nM. The three most promising compounds 14e, 14h, and 15h exhibited high potency against wild-type and drug-resistant viral strains with EC50 values at the sub-nanomolar level (0.29–0.87 nM), and were comparable to or more potent than the new NNRTI drug riplivirine (2) in the same assays. Drug-like physicochemical property assessments revealed that the most active DAANs (EC50 <10 nM) have better aqueous solubility (>1–90 μg/mL at pH 7.4 and pH 2) and metabolic stability in vitro than 2, as well as desirable log P values (<5) and polar surface area (PSA) (<140 Å2). These promising results warrant further development of this novel compound class as potential potent anti-AIDS clinical trial candidates. PMID:22856541

  12. Reverse transcriptase activity of an intron encoded polypeptide.

    PubMed Central

    Fassbender, S; Brühl, K H; Ciriacy, M; Kück, U

    1994-01-01

    A number of group II introns from eukaryotic organelles and prokaryotes contain open reading frames for polypeptides with homology to retroviral reverse transcriptases (RTs). We have used the yeast transposon (Ty) system to express ORFs for RTs from eukaryotic organelles. This includes the mitochondrial coxI intron i1 from the fungus Podospora anserina, the plastid petD intron from the alga Scenedesmus obliquus and the mitochondrial RTL gene from the alga Chlamydomonas reinhardtii. The ORFs were fused with the TYA ORF from the yeast retrotransposon Ty to produce virus-like particles in the recipient strains with detectable amounts of the RT-like polypeptides. Analysis of the heterologous gene products revealed biochemical evidence that the P. anserina intron encodes an RNA-directed DNA polymerase with properties typically found for RTs of viral or retrotransposable origin. In vitro assays showed that the intron encoded RT is sensitive to RT inhibitors such as N-ethylmaleimide and dideoxythymidine triphosphate but is insensitive against the DNA polymerase inhibitor aphidicolin. The direct biochemical evidence provided here supports the idea that intron encoded RTs are involved in intron transposition events. Images PMID:7514530

  13. Involvement of Telomerase Reverse Transcriptase in Heterochromatin Maintenance

    PubMed Central

    Maida, Yoshiko; Yasukawa, Mami; Okamoto, Naoko; Ohka, Seii; Kinoshita, Keita; Totoki, Yasushi; Ito, Takashi K.; Minamino, Tohru; Nakamura, Hiromi; Yamaguchi, Satoko; Shibata, Tatsuhiro

    2014-01-01

    In the fission yeast Schizosaccharomyces pombe, centromeric heterochromatin is maintained by an RNA-directed RNA polymerase complex (RDRC) and the RNA-induced transcriptional silencing (RITS) complex in a manner that depends on the generation of short interfering RNA. In association with the telomerase RNA component (TERC), the telomerase reverse transcriptase (TERT) forms telomerase and counteracts telomere attrition, and without TERC, TERT has been implicated in the regulation of heterochromatin at locations distinct from telomeres. Here, we describe a complex composed of human TERT (hTERT), Brahma-related gene 1 (BRG1), and nucleostemin (NS) that contributes to heterochromatin maintenance at centromeres and transposons. This complex produced double-stranded RNAs homologous to centromeric alpha-satellite (alphoid) repeat elements and transposons that were processed into small interfering RNAs targeted to these heterochromatic regions. These small interfering RNAs promoted heterochromatin assembly and mitotic progression in a manner dependent on the RNA interference machinery. These observations implicate the hTERT/BRG1/NS (TBN) complex in heterochromatin assembly at particular sites in the mammalian genome. PMID:24550003

  14. Human Specific Regulation of the Telomerase Reverse Transcriptase Gene

    PubMed Central

    Zhang, Fan; Cheng, De; Wang, Shuwen; Zhu, Jiyue

    2016-01-01

    Telomerase, regulated primarily by the transcription of its catalytic subunit telomerase reverse transcriptase (TERT), is critical for controlling cell proliferation and tissue homeostasis by maintaining telomere length. Although there is a high conservation between human and mouse TERT genes, the regulation of their transcription is significantly different in these two species. Whereas mTERT expression is widely detected in adult mice, hTERT is expressed at extremely low levels in most adult human tissues and cells. As a result, mice do not exhibit telomere-mediated replicative aging, but telomere shortening is a critical factor of human aging and its stabilization is essential for cancer development in humans. The chromatin environment and epigenetic modifications of the hTERT locus, the binding of transcriptional factors to its promoter, and recruitment of nucleosome modifying complexes all play essential roles in restricting its transcription in different cell types. In this review, we will discuss recent progress in understanding the molecular mechanisms of TERT regulation in human and mouse tissues and cells, and during cancer development. PMID:27367732

  15. A widespread class of reverse transcriptase-related cellular genes.

    PubMed

    Gladyshev, Eugene A; Arkhipova, Irina R

    2011-12-20

    Reverse transcriptases (RTs) polymerize DNA on RNA templates. They fall into several structurally related but distinct classes and form an assemblage of RT-like enzymes that, in addition to RTs, also includes certain viral RNA-dependent RNA polymerases (RdRP) synthesizing RNA on RNA templates. It is generally believed that most RT-like enzymes originate from retrotransposons or viruses and have no specific function in the host cell, with telomerases being the only notable exception. Here we report on the discovery and properties of a unique class of RT-related cellular genes collectively named rvt. We present evidence that rvts are not components of retrotransposons or viruses, but single-copy genes with a characteristic domain structure that may contain introns in evolutionarily conserved positions, occur in syntenic regions, and evolve under purifying selection. These genes can be found in all major taxonomic groups including protists, fungi, animals, plants, and even bacteria, although they exhibit patchy phylogenetic distribution in each kingdom. We also show that the RVT protein purified from one of its natural hosts, Neurospora crassa, exists in a multimeric form and has the ability to polymerize NTPs as well as dNTPs in vitro, with a strong preference for NTPs, using Mn(2+) as a cofactor. The existence of a previously unknown class of single-copy RT-related genes calls for reevaluation of the current views on evolution and functional roles of RNA-dependent polymerases in living cells.

  16. International perspectives on antiretroviral resistance. Nonnucleoside reverse transcriptase inhibitor resistance.

    PubMed

    Deeks, S G

    2001-03-01

    Although understanding of nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance is less clearly established than that of other classes of antiretroviral drugs, certain facts have been established. The treatment-associated genetic mutation profiles of the available NNRTIs have been mapped, and resistance has been found to develop rapidly after initiation of NNRTI therapy. Despite the chemical diversity of the NNRTIs, cross-resistance among agents of this class is nearly universal. Although the viral replicative capacity ("fitness") of NNRTI-induced viral variants has not been extensively studied, available data suggest that NNRTI-selected mutations confer little damage to viral fitness, and thus a single point mutation produces a strain that is both resistant and fit. Furthermore, with continued therapy, viral evolution persists, creating species with greater numbers of mutations and higher level phenotypic resistance. Taken together, these facts suggest that continued use of NNRTIs after emergence of resistance will produce variants of complex mutational patterns that limit future treatment options, and, therefore, strong consideration should be given to discontinuing NNRTIs after virologic failure is confirmed. This article describes the scientific literature establishing the efficacy and limitations of NNRTI therapy and attempts to define a role for this class of drug in the long-term treatment of HIV-1 disease.

  17. The p66 Immature Precursor of HIV-1 Reverse Transcriptase

    PubMed Central

    Sharaf, Naima G.; Poliner, Eric; Slack, Ryan L.; Christen, Martin T.; Byeon, In-Ja L.; Parniak, Michael A.; Gronenborn, Angela M.; Ishima, Rieko

    2015-01-01

    In contrast to the wealth of structural data available for the mature p66/p51 heterodimeric human immunodeficiency virus type 1 reverse transcriptase (RT), the structure of the homodimeric p66 precursor remains unknown. In all X-ray structures of mature RT, free or complexed, the processing site in the p66 subunit, for generating the p51 subunit, is sequestered into a β-strand within the folded ribonuclease H (RNH) domain and is not readily accessible to proteolysis, rendering it difficult to propose a simple and straightforward mechanism of the maturation step. Here, we investigated, by solution NMR, the conformation of the RT p66 homodimer. Our data demonstrate that the RNH and Thumb domains in the p66 homodimer are folded and possess conformations very similar to those in mature RT. This finding suggests that maturation models which invoke a complete or predominantly unfolded RNH domain are unlikely. The present study lays the foundation for further in-depth mechanistic investigations at the atomic level. PMID:24771554

  18. [Studies on telomerase reverse transcriptase components and liver cancer].

    PubMed

    Cai, J J; Guo, X L

    2016-07-20

    Telomeres are DNA tandem repeats at the ends of chromosomes in eukaryotic cells and play important roles in maintaining the stability and integrity of chromosomes. Telomeres are gradually shortened with cell proliferation, and when they are shortened to a certain length, the cells experience senescence and apoptosis. However, a small number of cells can maintain the length of telomeres and restore their function through related mechanisms (activation of telomerase or other mechanisms), and some cells may even be immortalized. Therefore, telomere and telomerase are thought to be closely associated with tumor development and progression. It has been confirmed that telomerase activation is an early event in the development of primary liver cancer, especially the important component of telomerase telomerase reverse transcriptase (TERT), which plays an important role in this process. Here this article reviews the latest research advances in the function and regulation mechanisms of telomerase and the role of TERT in the development, progression, and treatment of primary liver cancer, especially hepatocellular carcinoma, so as to provide a molecular genetic basis for intervention of liver cancer and related targeted therapy.

  19. Effects of HIV-1 reverse transcriptase connection subdomain mutations on polypurine tract removal and initiation of (+)-strand DNA synthesis

    PubMed Central

    Betancor, Gilberto; Álvarez, Mar; Marcelli, Barbara; Andrés, Cristina; Martínez, Miguel A.; Menéndez-Arias, Luis

    2015-01-01

    HIV-1 reverse transcriptase (RT) connection subdomain mutations at positions 348, 369 and 376 have been associated with resistance to non-nucleoside RT inhibitors (NNRTIs). N348I may interfere with the initiation of (+)-strand DNA synthesis by reducing polypurine tract (PPT) removal in the presence of nevirapine. The effect of NNRTIs on the RNase H-mediated cleavage of PPT-containing template-primers has been studied with wild-type HIV-1 RT and mutants N348I, T369I, T369V, T376S and N348I/T369I. In the presence of NNRTIs, all RTs were able to stimulate PPT cleavage after primer elongation. The enhancing effects of nevirapine and efavirenz were reduced in RTs carrying mutation N348I, and specially N348I/T369I. However, those mutations had no effect on rilpivirine-mediated cleavage. Prior to elongation, the PPT remains resilient to cleavage, although efavirenz and rilpivirine facilitate RNase H-mediated trimming of its 3′-end. The integrity of the 3′-end is essential for the initiation of (+)-strand DNA synthesis. In the presence of dNTPs, rilpivirine was the most effective inhibitor of (+)-strand DNA synthesis blocking nucleotide incorporation and preventing usage of available PPT primers. The N348I/T369I RT showed reduced ability to generate short RNA products revealing a cleavage window defect. Its lower RNase H activity could be attributed to enhanced rigidity compared to the wild-type enzyme. PMID:25662223

  20. Telomerase reverse transcriptase moonlights: Therapeutic targets beyond telomerase.

    PubMed

    Maida, Yoshiko; Masutomi, Kenkichi

    2015-11-01

    Telomeres, the repetitive sequences at chromosomal ends, protect intact chromosomes. Telomeres progressively shorten through successive rounds of cell divisions, and critically shortened telomeres trigger senescence and apoptosis. The enzyme that elongates telomeres and maintains their structure is known as telomerase. The catalytic subunit of this enzyme (telomerase reverse transcriptase [TERT]) is expressed at a high level in malignant cells, but at a very low level in normal cells. Although telomerase activity was long believed to be the only function of TERT, emerging evidence indicates that TERT plays roles beyond telomeres. For example, TERT contributes to stem cell maintenance and cell reprogramming processes in a manner independent of its canonical function. Even some types of splice variants that lack the telomerase catalytic domains exhibit the functions in a manner that does not depend on telomerase activity. We recently demonstrated that the RNA-dependent RNA polymerase (RdRP) activity of TERT is involved in regulation of gene silencing and heterochromatic transcription. Moreover, TERT RdRP activity is mediated by a newly identified complex, distinct from the authentic telomerase complex, that plays a role in cancer stem cells in a telomere maintenance independent manner. TERT has attracted interest as a molecular target for anticancer treatment, but previous efforts aimed at developing novel therapeutic strategies focused only on the canonical function of TERT. However, accumulating evidence about the non-canonical functions of TERT led us to speculate that the functions other than telomerase might be therapeutic targets as well. In this review, we discuss the non-canonical functions of TERT and their potential applications for anticancer treatment. © 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

  1. Novel aptamer inhibitors of human immunodeficiency virus reverse transcriptase.

    PubMed

    DeStefano, Jeffrey J; Nair, Gauri R

    2008-06-01

    Primer-template-based double-stranded nucleic acids capable of binding human immunodeficiency virus reverse transcriptase (HIV-RT) with high affinity were used as starting material to develop small single-stranded loop-back DNA aptamers. The original primer-templates were selected using a SELEX (Systematic Evolution of Ligands by EXponential enrichment) approach and consisted of 46- and 50-nt primer and template strands, respectively. The major determinant of the approximately 10-fold tighter binding in selected sequences relative to control primer-templates was a run of 6.8 G residues at the 3' primer end. Sixty, thirty-seven, twenty-seven, and twenty-two nucleotide loop-back single-stranded versions that retained the base pairs near the 3' primer terminus were constructed. Both the 60- and 37-nt versions retained high affinity for RT with K(d) values of approximately 0.44 nM and 0.66 nM, respectively. Random sequence primer-templates of the same length had K(d)s of approximately 20 nM and approximately 161 nM. The shorter 27- and 22-nt aptamers bound with reduced affinity. Several modifications of the 37-nt aptamer were also tested including changes to the terminal 3' G nucleotide and internal bases in the G run, replacement of specific nucleotides with phosphothioates, and alterations to the 5' overhang. Optimal binding required a 4- to 5-nt overhang, and internal changes within the G run had a pronounced negative effect on binding. Phosphothioate nucleotides or the presence of a 3' dideoxy G residue did not alter affinity. The 37-nt aptamer was a potent inhibitor of HIV-RT in vitro and functioned by blocking binding of other primer-templates.

  2. Long-lasting protection of activity of nucleoside reverse transcriptase inhibitors and protease inhibitors (PIs) by boosted PI containing regimens.

    PubMed

    Scherrer, Alexandra U; Böni, Jürg; Yerly, Sabine; Klimkait, Thomas; Aubert, Vincent; Furrer, Hansjakob; Calmy, Alexandra; Cavassini, Matthias; Elzi, Luigia; Vernazza, Pietro L; Bernasconi, Enos; Ledergerber, Bruno; Günthard, Huldrych F

    2012-01-01

    The accumulation of mutations after long-lasting exposure to a failing combination antiretroviral therapy (cART) is problematic and severely reduces the options for further successful treatments. We studied patients from the Swiss HIV Cohort Study who failed cART with nucleoside reverse transcriptase inhibitors (NRTIs) and either a ritonavir-boosted PI (PI/r) or a non-nucleoside reverse transcriptase inhibitor (NNRTI). The loss of genotypic activity <3, 3-6, >6 months after virological failure was analyzed with Stanford algorithm. Risk factors associated with early emergence of drug resistance mutations (<6 months after failure) were identified with multivariable logistic regression. Ninety-nine genotypic resistance tests from PI/r-treated and 129 from NNRTI-treated patients were analyzed. The risk of losing the activity of ≥1 NRTIs was lower among PI/r- compared to NNRTI-treated individuals <3, 3-6, and >6 months after failure: 8.8% vs. 38.2% (p = 0.009), 7.1% vs. 46.9% (p<0.001) and 18.9% vs. 60.9% (p<0.001). The percentages of patients who have lost PI/r activity were 2.9%, 3.6% and 5.4% <3, 3-6, >6 months after failure compared to 41.2%, 49.0% and 63.0% of those who have lost NNRTI activity (all p<0.001). The risk to accumulate an early NRTI mutation was strongly associated with NNRTI-containing cART (adjusted odds ratio: 13.3 (95% CI: 4.1-42.8), p<0.001). The loss of activity of PIs and NRTIs was low among patients treated with PI/r, even after long-lasting exposure to a failing cART. Thus, more options remain for second-line therapy. This finding is potentially of high relevance, in particular for settings with poor or lacking virological monitoring.

  3. Docking, molecular dynamics and quantitative structure-activity relationship studies for HEPTs and DABOs as HIV-1 reverse transcriptase inhibitors.

    PubMed

    Mao, Yating; Li, Yan; Hao, Ming; Zhang, Shuwei; Ai, Chunzhi

    2012-05-01

    As a key component in combination therapy for acquired immunodeficiency syndrome (AIDS), non-nucleoside reverse transcriptase inhibitors (NNRTIs) have been proven to be an essential way in stopping HIV-1 replication. In the present work, in silico studies were conducted on a series of 119 NNRTIs, including 1-(2-hydroxyethoxymethyl)-6-(phenylthio)thymine (HEPT) and dihydroalkoxybenzyloxopyrimidine (DABO) derivatives by using the comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis (CoMSIA), docking simulations and molecular dynamics (MD). The statistical results of the optimal model, the ligand-based CoMSIA one (Q(2) = 0.48, R(ncv)(2) =0.847, R(pre)(2) = 0.745) validates its satisfactory predictive capacity both internally and externally. The contour maps, docking and MD results correlate well with each other, drawing conclusions as follows: 1) Compounds with bulky substituents in position-6 of ring A, hydrophobic groups around position- 1, 2, 6 are preferable to the biological activities; 2) Two hydrogen bonds between RT inhibitor and the Tyr 318, Lys 101 residues, respectively, and a π-π bond between the inhibitor and Trp 188 are formed and crucial to the orientation of the active conformation of the molecules; 3) The binding pocket is essentially hydrophobic, which are determined by residues such as Trp 229, Tyr 318, Val 179, Tyr 188 and Val 108, and hydrophobic substituents may bring an improvement to the biological activity; 4) DABO and HEPT derivatives have different structures but take a similar mechanism to inhibit RT. The potency difference between two isomers in HEPTs can be explained by the distinct locations of the 6-naphthylmethyl substituent and the reasons are explained in details. All these results could be employed to alter the structural scaffold in order to develop new HIV-1 RT inhibitors that have an improved biological property. To the best of our knowledge, this is the first report on 3D

  4. Regulation of the Telomerase Reverse Transcriptase Subunit through Epigenetic Mechanisms

    PubMed Central

    Lewis, Kayla A.; Tollefsbol, Trygve O.

    2016-01-01

    Chromosome-shortening is characteristic of normal cells, and is known as the end replication problem. Telomerase is the enzyme responsible for extending the ends of the chromosomes in de novo synthesis, and occurs in germ cells as well as most malignant cancers. There are three subunits of telomerase: human telomerase RNA (hTERC), human telomerase associated protein (hTEP1), or dyskerin, and human telomerase reverse transcriptase (hTERT). hTERC and hTEP1 are constitutively expressed, so the enzymatic activity of telomerase is dependent on the transcription of hTERT. DNA methylation, histone methylation, and histone acetylation are basic epigenetic regulations involved in the expression of hTERT. Non-coding RNA can also serve as a form of epigenetic control of hTERT. This epigenetic-based regulation of hTERT is important in providing a mechanism for reversibility of hTERT control in various biological states. These include embryonic down-regulation of hTERT contributing to aging and the upregulation of hTERT playing a critical role in over 90% of cancers. Normal human somatic cells have a non-methylated/hypomethylated CpG island within the hTERT promoter region, while telomerase-positive cells paradoxically have at least a partially methylated promoter region that is opposite to the normal roles of DNA methylation. Histone acetylation of H3K9 within the promoter region is associated with an open chromatin state such that transcription machinery has the space to form. Histone methylation of hTERT has varied control of the gene, however. Mono- and dimethylation of H3K9 within the promoter region indicate silent euchromatin, while a trimethylated H3K9 enhances gene transcription. Non-coding RNAs can target epigenetic-modifying enzymes, as well as transcription factors involved in the control of hTERT. An epigenetics diet that can affect the epigenome of cancer cells is a recent fascination that has received much attention. By combining portions of this diet with

  5. Reverse transcriptase-related proteins in telomeres and in certain chromosomal loci of Rhynchosciara (Diptera: Sciaridae).

    PubMed

    Gorab, Eduardo

    2003-04-01

    The localization of reverse transcriptase-related proteins in polytene chromosomes of dipterans was investigated using previously characterized antibodies to a recombinant polypeptide containing conserved motifs of insect reverse transcriptases. The immunoreactions were carried out with polytene chromosome squashes of eight sciarids, one chironomid and three Drosophila species. Telomeric staining was regularly observed on chromosomes of the sciarid Rhynchosciara americana under normal growth conditions. Five of eight chromosomal tips were labelled except for the heterochromatic ends that are occasionally found associated forming a chromocentre in the salivary gland. Reverse transcriptase-related proteins were detected at chromosomal tips of young larvae and remained bound to the telomeres throughout larval development. As in salivary gland chromosomes, five non-telocentric ends of the chromosomes from Malpighian tubules of R. americana appeared clearly stained with anti-reverse transcriptase. The occurrence of telomeric reverse transcriptase in R. americana correlates with the presence of RNA in addition to an unusual enrichment with homopolymeric dA/dT DNA associated with the telomeric heterochromatin. The antibodies also reacted with a few interstitial sites in chromosomes of four Rhynchosciara species, one band overlapping the histone gene locus of three species in the americana -like group. The results provide evidence for a reverse transcriptase-related protein as a constitutive component in telomeres of R. americana and also in certain interstitial loci of Rhynchosciara species in which RNA was immunologically detected in the form of RNA:DNA hybrids.

  6. HIV-1 Protease, Reverse Transcriptase, and Integrase Variation

    PubMed Central

    Sankaran, Kris; Varghese, Vici; Winters, Mark A.; Hurt, Christopher B.; Eron, Joseph J.; Parkin, Neil; Holmes, Susan P.; Holodniy, Mark; Shafer, Robert W.

    2016-01-01

    ABSTRACT HIV-1 protease (PR), reverse transcriptase (RT), and integrase (IN) variability presents a challenge to laboratories performing genotypic resistance testing. This challenge will grow with increased sequencing of samples enriched for proviral DNA such as dried blood spots and increased use of next-generation sequencing (NGS) to detect low-abundance HIV-1 variants. We analyzed PR and RT sequences from >100,000 individuals and IN sequences from >10,000 individuals to characterize variation at each amino acid position, identify mutations indicating APOBEC-mediated G-to-A editing, and identify mutations resulting from selective drug pressure. Forty-seven percent of PR, 37% of RT, and 34% of IN positions had one or more amino acid variants with a prevalence of ≥1%. Seventy percent of PR, 60% of RT, and 60% of IN positions had one or more variants with a prevalence of ≥0.1%. Overall 201 PR, 636 RT, and 346 IN variants had a prevalence of ≥0.1%. The median intersubtype prevalence ratios were 2.9-, 2.1-, and 1.9-fold for these PR, RT, and IN variants, respectively. Only 5.0% of PR, 3.7% of RT, and 2.0% of IN variants had a median intersubtype prevalence ratio of ≥10-fold. Variants at lower prevalences were more likely to differ biochemically and to be part of an electrophoretic mixture compared to high-prevalence variants. There were 209 mutations indicative of APOBEC-mediated G-to-A editing and 326 mutations nonpolymorphic treatment selected. Identification of viruses with a high number of APOBEC-associated mutations will facilitate the quality control of dried blood spot sequencing. Identifying sequences with a high proportion of rare mutations will facilitate the quality control of NGS. IMPORTANCE Most antiretroviral drugs target three HIV-1 proteins: PR, RT, and IN. These proteins are highly variable: many different amino acids can be present at the same position in viruses from different individuals. Some of the amino acid variants cause drug

  7. HIV-1 Protease, Reverse Transcriptase, and Integrase Variation.

    PubMed

    Rhee, Soo-Yon; Sankaran, Kris; Varghese, Vici; Winters, Mark A; Hurt, Christopher B; Eron, Joseph J; Parkin, Neil; Holmes, Susan P; Holodniy, Mark; Shafer, Robert W

    2016-07-01

    HIV-1 protease (PR), reverse transcriptase (RT), and integrase (IN) variability presents a challenge to laboratories performing genotypic resistance testing. This challenge will grow with increased sequencing of samples enriched for proviral DNA such as dried blood spots and increased use of next-generation sequencing (NGS) to detect low-abundance HIV-1 variants. We analyzed PR and RT sequences from >100,000 individuals and IN sequences from >10,000 individuals to characterize variation at each amino acid position, identify mutations indicating APOBEC-mediated G-to-A editing, and identify mutations resulting from selective drug pressure. Forty-seven percent of PR, 37% of RT, and 34% of IN positions had one or more amino acid variants with a prevalence of ≥1%. Seventy percent of PR, 60% of RT, and 60% of IN positions had one or more variants with a prevalence of ≥0.1%. Overall 201 PR, 636 RT, and 346 IN variants had a prevalence of ≥0.1%. The median intersubtype prevalence ratios were 2.9-, 2.1-, and 1.9-fold for these PR, RT, and IN variants, respectively. Only 5.0% of PR, 3.7% of RT, and 2.0% of IN variants had a median intersubtype prevalence ratio of ≥10-fold. Variants at lower prevalences were more likely to differ biochemically and to be part of an electrophoretic mixture compared to high-prevalence variants. There were 209 mutations indicative of APOBEC-mediated G-to-A editing and 326 mutations nonpolymorphic treatment selected. Identification of viruses with a high number of APOBEC-associated mutations will facilitate the quality control of dried blood spot sequencing. Identifying sequences with a high proportion of rare mutations will facilitate the quality control of NGS. Most antiretroviral drugs target three HIV-1 proteins: PR, RT, and IN. These proteins are highly variable: many different amino acids can be present at the same position in viruses from different individuals. Some of the amino acid variants cause drug resistance and occur mainly

  8. Vaginal microbicide film combinations of two reverse transcriptase inhibitors, EFdA and CSIC, for the prevention of HIV-1 sexual transmission

    PubMed Central

    Zhang, Wei; Hu, Minlu; Shi, Yuan; Gong, Tiantian; Dezzutti, Charlene S.; Moncla, Bernard; Sarafianos, Stefan G.; Parniak, Michael A.; Rohan, Lisa C.

    2015-01-01

    Purpose EFdA is a potent nucleoside reverse transcriptase inhibitor (NRTI) with activity against a wide spectrum of wild-type and drug resistant HIV-1 variants. CSIC is a tight-binding non-nucleoside reverse transcriptase inhibitor (NNRTI) with demonstrated anti-HIV properties important for use in topical prevention of HIV transmission. The objective of this study was to develop and characterize film-formulated EFdA and CSIC for use as a female-controlled vaginal microbicide to prevent sexual transmission of HIV. Methods Assessments of EFdA- and CSIC-loaded films included physicochemical characteristics, in vitro cytotoxicity, epithelia integrity studies, compatibility with the normal vaginal Lactobacillus flora and anti-HIV bioactivity evaluations. Results No significant change in physicochemical properties or biological activity of the combination films were noted during 3 months storage. In vitro cytotoxicity and bioactivity testing showed that 50% cytotoxic concentration (CC50) of either EFdA or CSIC was several orders of magnitude higher than the 50% effective concentration (EC50) values. Film-formulated EFdA and CSIC combination showed additive inhibitory activity against wild type and drug-resistant variants of HIV. Epithelial integrity studies demonstrated that the combination vaginal film had a much lower toxicity to HEC-1A monolayers compared to that of VCF®, a commercial vaginal film product containing nonoxynol-9. Polarized ectocervical explants showed films with drug alone or in combination were effective at preventing HIV infection. Conclusions Our data suggest that vaginal microbicide films containing a combination of the NRTI EFdA and the NNRTI CSIC have potential to prevent HIV-1 sexual transmission. PMID:25794967

  9. Two proteins with reverse transcriptase activities associated with hepatitis B virus-like particles

    SciTech Connect

    Bavand, M.R.; Laub, O. )

    1988-02-01

    Recent studies suggest that hepatitis B virus (HBV), despite being a DNA virus, replicates via an RNA intermediate. The HBV life cycle is therefore a permuted version of the RNA retroviral life cycle. Sequence homology between retroviral reverse transcriptase and the putative HBV polymerase gene product suggests the presence of an HBV reverse transcriptase. As yet, there has been no direct evidence that reverse transcriptase activity is present in the viral particle. The authors used activity gel analysis to detect the in situ catalytic activities of DNA polymerases after sodium dodecyl sulfate-polyacrylamide gel electrophorsis. These studies demonstrated that HBV-like particles secreted by a differentiated human hepatoma cell line tranfected with genomic HBV DNA contain two major polymerase activities which migrate as {approximately}90- and {approximately}70-kilodalton (kDa) proteins. This demonstrated, for the first time, that HBV-like particles contain a novel DNA polymerase-reverse transcriptase activity. Furthermore, they propose that the 70-kDa reverse transcriptase may be produced by proteolytic self-cleavage of the 90-kDa precursor protein.

  10. Oral Cyclosporin A Inhibits CD4 T cell P-glycoprotein Activity in HIV-Infected Adults Initiating Treatment with Nucleoside Reverse Transcriptase Inhibitors

    PubMed Central

    Hulgan, Todd; Donahue, John P.; Smeaton, Laura; Pu, Minya; Wang, Hongying; Lederman, Michael M.; Smith, Kimberly; Valdez, Hernan; Pilcher, Christopher; Haas, David W.

    2010-01-01

    Purpose P-glycoprotein limits tissue penetration of many antiretroviral drugs. We characterized effects of the P-glycoprotein substrate cyclosporin A on T cell P-glycoprotein activity in HIV-infected AIDS Clinical Trials Group study A5138 participants. Methods We studied P-glycoprotein activity on CD4 and CD8 T cells in 16 participants randomized to receive oral cyclosporin A (n=9) or not (n=7) during initiation antiretroviral therapy (ART) that did not include protease or non-nucleoside reverse transcriptase inhibitors. Results CD4 T cell P-glycoprotein activity decreased by a median of 8 percentage points with cyclosporin A/ART (difference between cyclosporin A/ART versus ART only P=0.001). Plasma trough cyclosporin A concentrations correlated with change in P-glycoprotein activity in several T cell subsets. Conclusions Oral cyclosporin A can inhibit peripheral blood CD4 T cell P-glycoprotein activity. Targeted P-glycoprotein inhibition might enhance delivery of ART to T cells. PMID:19779705

  11. Inhibition of viral reverse transcriptase and human sperm DNA polymerase by anti-sperm antibodies.

    PubMed Central

    Witkin, S S; Higgins, P J; Bendich, A

    1978-01-01

    The IgG fraction of serum from a rabbit immunized with detergent-prepared human sperm nuclei inhibited the DNA polymerase activities in human sperm and seminal fluid as well as the partially purified reverse transcriptase of the baboon endogenous type-C retrovirus (BEV). The analogous enzymes from lysates of oncogenic type-C viruses was unaffected. IgG from the serum of individual partners from infertile marriages similarly inhibited both purified BEV reverse transcriptase and human sperm DNA polymerase, but not a DNA polymerase isolated from human prostatic fluid. The data suggest that BEV reverse transcriptase and the human sperm DNA polymerase are antigenically related. Furthermore, the sperm appears to be auto-antigenic and the antibodies thus formed may be capable of interfering with reproductive success. PMID:82498

  12. Viral fitness: relation to drug resistance mutations and mechanisms involved: nucleoside reverse transcriptase inhibitor mutations.

    PubMed

    Weber, Jan; Henry, Kenneth R; Arts, Eric J; Quiñones-Mateu, Miguel E

    2007-03-01

    Nucleoside and nucleotide reverse transcriptase inhibitors constitute the backbone of highly active antiretroviral therapy in the treatment of HIV-1 infection. One of the major obstacles in achieving the long-term efficacy of anti-HIV-1 therapy is the development of resistance. The advent of resistance mutations is usually accompanied by a change in viral replicative fitness. This review focuses on the most common nucleoside reverse transcriptase inhibitor-associated mutations and their effects on HIV-1 replicative fitness. Recent studies have explained the two main mechanisms of resistance to nucleoside reverse transcriptase inhibitors and their role in HIV-1 replicative fitness. The first involves mutations directly interfering with binding or incorporation and seems to impact replicative fitness more adversely than the second mechanism, which involves enhanced excision of the newly incorporated analogue. Further studies have helped explain the antagonistic effects between amino acid substitutions, K65R, L74V, M184V, and thymidine analogue mutations, showing how viral replicative fitness influences the evolution of thymidine analogue resistance pathways. Nucleoside reverse transcriptase inhibitor resistance mutations impact HIV-1 replicative fitness to a lesser extent than protease resistance mutations. The monitoring of viral replicative fitness may help in the management of HIV-1 infection in highly antiretroviral-experienced individuals.

  13. Comparison of antigen assay and reverse transcriptase assay for detecting human immunodeficiency virus in culture.

    PubMed Central

    Feorino, P; Forrester, B; Schable, C; Warfield, D; Schochetman, G

    1987-01-01

    We compared an antigen capture assay (Abbott Laboratories, North Chicago, Ill.) with a reverse transcriptase assay to identify and quantify human immunodeficiency virus (HIV) in culture. In direct comparisons of serial dilutions of lymphadenopathy-associated virus type 1, the antigen assay was 100-fold more sensitive than the reverse transcriptase assay in detecting the virus. The antigen assay reacted strongly with 60 different HIV isolates but did not cross-react with human T-cell lymphotropic virus type I, human T-cell lymphotropic virus type II, cytomegalovirus, varicella-zoster virus, herpes simplex virus type 1, Epstein-Barr virus, adenovirus type 5, or poliovirus type 1 or with extracts from four different control human cell lines and eight different phytohemagglutinin-stimulated normal human lymphocytes. Peripheral blood lymphocyte samples from 50 individuals were evaluated by both the antigen assay and the reverse transcriptase assay. The cells from the 34 seropositive individuals were all positive by the antigen assay (range, 3 to 9 days; average time, 5.9 days) and the reverse transcriptase assay (range, 7 to 16 days; average time, 9.6 days). Cells from the 16 seronegative individuals were negative by both assays. These results indicate that the antigen assay is an important addition to the monitoring of HIV production in the lymphocytes of infected patients. PMID:2448334

  14. Detection of multiple, novel reverse transcriptase coding sequences in human nucleic acids: relation to primate retroviruses

    SciTech Connect

    Shih, A.; Misra, R.; Rush, M.G.

    1989-01-01

    A variety of chemically synthesized oligonucleotides designed on the basis of amino acid and/or nucleotide sequence data were used to detect a large number of novel reverse transcriptase coding sequences in human and mouse DNAs. Procedures involving Southern blotting, library screening, and the polymerase chain reaction were all used to detect such sequences; the polymerase chain reaction was the most rapid and productive approach. In the polymerase chain reaction, oligonucleotide mixtures based on consensus sequence homologies to reverse transcriptase coding sequences and unique oligonucleotides containing perfect homology to the coding sequences of human T-cell leukemia virus types I and II were both effective in amplifying reverse transcriptase-related DNA. It is shown that human DNA contains a wide spectrum of retrovirus-related reverse transcriptase coding sequences, including some that are clearly related to human T-cell leukemia virus types I and II, some that are related to the L-1 family of long interspersed nucleotide sequences, and others that are related to previously described human endogenous proviral DNAs. In addition, human T-cell leukemia virus type I-related sequences appear to be transcribed in both normal human T cells and in a cell line derived from a human teratocarcinoma.

  15. Synthesis and evaluation of novel quinolinones as HIV-1 reverse transcriptase inhibitors.

    PubMed

    Patel, M; McHugh, R J; Cordova, B C; Klabe, R M; Bacheler, L T; Erickson-Viitanen, S; Rodgers, J D

    2001-07-23

    A series of 4,4-disubstituted quinolinones was prepared and evaluated as HIV-1 reverse transcriptase inhibitors. The C-3 substituted compound 9h displayed improved antiviral activity against clinically significant single (K103N) and double (K103N/L100I) mutant viruses.

  16. Thiazolobenzimidazole: biological and biochemical anti-retroviral activity of a new nonnucleoside reverse transcriptase inhibitor.

    PubMed

    Buckheit, R W; Hollingshead, M G; Germany-Decker, J; White, E L; McMahon, J B; Allen, L B; Ross, L J; Decker, W D; Westbrook, L; Shannon, W M

    1993-07-01

    Thiazolobenzimidazole (NSC 625487) was a highly potent inhibitor of human immunodeficiency virus-induced cell killing and viral replication in a variety of human cell lines, as well as fresh human peripheral blood lymphocytes and macrophages. The compound was active against a panel of biologically diverse laboratory and clinical strains of HIV-1, including the AZT-resistant strain G910-6. However, the agent was inactive against HIV-2 and a pyridinone-resistant strain (A17) of HIV-1, a strain which is cross-resistant to several structurally diverse members of a common pharmacologic class of nonnucleoside reverse transcriptase inhibitors. The compound selectively inhibited HIV-1 reverse transcriptase but not HIV-2 reverse transcriptase. Combinations of thiazolobenzimidazole with either AZT or ddI synergistically inhibited HIV-1 induced cell killing in vitro. Thiazolobenzimidazole also inhibited the replication of the Rauscher murine leukemia retrovirus. Thus, thiazolobenzimidazole is a new active anti-HIV-1 chemotype and may represent a subclass of nonnucleoside reverse transcriptase inhibitors with an enhanced range of anti-retroviral activity.

  17. Soft shell clams Mya arenaria with disseminated neoplasia demonstrate reverse transcriptase activity

    USGS Publications Warehouse

    House, M.L.; Kim, C.H.; Reno, P.W.

    1998-01-01

    Disseminated neoplasia (DN), a proliferative cell disorder of the circulatory system of bivalves, was first reported in oysters in 1969. Since that time, the disease has been determined to be transmissible through water-borne exposure, but the etiological agent has not been unequivocally identified. In order to determine if a viral agent, possibly a retrovirus, could be the causative agent of DN, transmission experiments were performed, using both a cell-free filtrate and a sucrose gradient-purified preparation of a cell-free filtrate of DN positive materials. Additionally, a PCR-enhanced reverse transcriptase assay was used to determine if reverse transcriptase was present in tissues or hemolymph from DN positive soft shell clams Mya arenaria. DN was transmitted to healthy clams by injection with whole DN cells, but not with cell-free flitrates prepared from either tissues from DN positive clams, or DN cells. The cell-free preparations from DN-positive tissues and hemolymph having high levels of DN cells in circulation exhibited positive reactions in the PCR-enhanced reverse transcriptase assay. Cell-free preparations of hemolymph from clams having low levels of DN (<0.1% of cells abnormal), hemocytes from normal soft shell clams, and normal soft shell clam tissues did not produce a positive reaction in the PCR enhanced reverse transcriptase assay.

  18. Impact of reverse transcriptase resistance on the efficacy of TMC125 (etravirine) with two nucleoside reverse transcriptase inhibitors in protease inhibitor-naïve, nonnucleoside reverse transcriptase inhibitor-experienced patients: study TMC125-C227.

    PubMed

    Ruxrungtham, K; Pedro, R J; Latiff, G H; Conradie, F; Domingo, P; Lupo, S; Pumpradit, W; Vingerhoets, J H; Peeters, M; Peeters, I; Kakuda, T N; De Smedt, G; Woodfall, B

    2008-11-01

    TMC125-C227, an exploratory phase II, randomized, controlled, open-label trial, compared the efficacy and safety of TMC125 (etravirine) with an investigator-selected protease inhibitor (PI) in nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant, protease inhibitor-naïve, HIV-1-infected patients. Patients were randomized to TMC125 800 mg twice a day (bid) (phase II formulation; n=59) or the control PI (n=57), plus two nucleoside reverse transcriptase inhibitors (NRTIs). In an unplanned interim analysis, patients receiving TMC125 demonstrated suboptimal virological responses relative to the control PI. Therefore, trial enrolment was stopped prematurely and TMC125 treatment discontinued after a median of 14.3 weeks. In this first-line NNRTI-failure population, baseline NRTI and NNRTI resistance was high and reduced virological responses were observed relative to the control PI. No statistically significant relationship was observed between TMC125 exposure and virological response at week 12. TMC125 was better tolerated than a boosted PI for gastrointestinal-, lipid- and liver-related events. In a PI-naïve population, with baseline NRTI and NNRTI resistance and NRTI recycling, TMC125 was not as effective as first use of a PI. Therefore the use of TMC125 plus NRTIs alone may not be optimal in PI-naïve patients with first-line virological failure on an NNRTI-based regimen. Baseline two-class resistance, rather than pharmacokinetics or other factors, was the most likely reason for suboptimal responses.

  19. N348I in HIV-1 reverse transcriptase counteracts the synergy between zidovudine and nevirapine.

    PubMed

    Yap, Soo Huey; Herman, Brian D; Radzio, Jessica; Sluis-Cremer, Nicolas; Tachedjian, Gilda

    2012-10-01

    The efficacy of regimens that include both zidovudine and nevirapine can be explained by the synergistic interactions between these drugs. N348I in HIV-1 reverse transcriptase confers decreased susceptibility to zidovudine and nevirapine. Here, we demonstrate that N348I reverses the synergistic inhibition of HIV-1 by zidovudine and nevirapine. Also, the efficiency of zidovudine-monophosphate excision in the presence of nevirapine is greater for N348I HIV-1 reverse transcriptase compared with the wild-type enzyme. These data help explain the frequent selection of N348I in regimens that contain zidovudine and nevirapine, and suggest that the selection of N348I should be monitored in resource-limited settings where these drugs are routinely used.

  20. Homologous recombination promoted by reverse transcriptase during copying of two distinct RNA templates.

    PubMed Central

    Negroni, M; Ricchetti, M; Nouvel, P; Buc, H

    1995-01-01

    Retroviruses are known to mutate at high rates. An important source of genetic variability is recombination taking place during reverse transcription of internal regions of the two genomic RNAs. We have designed an in vitro model system, involving genetic markers carried on two RNA templates, to allow a search for individual recombination events and to score their frequency of occurrence. We show that Moloney murine leukemia virus reverse transcriptase alone promotes homologous recombination efficiently. While RNA concentration has little effect on recombination frequency, there is a clear correlation between the amount of reverse transcriptase used in the assay and the extent of recombination observed. Under conditions mimicking the in vivo situation, a rate compatible with ex vivo estimates has been obtained. PMID:7542781

  1. The group II intron maturase: a reverse transcriptase and splicing factor go hand in hand.

    PubMed

    Zhao, Chen; Pyle, Anna Marie

    2017-05-18

    The splicing of group II introns in vivo requires the assistance of a multifunctional intron encoded protein (IEP, or maturase). Each IEP is also a reverse-transcriptase enzyme that enables group II introns to behave as mobile genetic elements. During splicing or retro-transposition, each group II intron forms a tight, specific complex with its own encoded IEP, resulting in a highly reactive holoenzyme. This review focuses on the structural basis for IEP function, as revealed by recent crystal structures of an IEP reverse transcriptase domain and cryo-EM structures of an IEP-intron complex. These structures explain how the same IEP scaffold is utilized for intron recognition, splicing and reverse transcription, while providing a physical basis for understanding the evolutionary transformation of the IEP into the eukaryotic splicing factor Prp8. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. A polymorphism at position 400 in the connection subdomain of HIV-1 reverse transcriptase affects sensitivity to NNRTIs and RNaseH activity.

    PubMed

    Wright, David W; Deuzing, Ilona P; Flandre, Philippe; van den Eede, Peter; Govaert, Micheline; Setiawan, Laurentia; Coveney, Peter V; Marcelin, Anne-Geneviève; Calvez, Vincent; Boucher, Charles A B; Beerens, Nancy

    2013-01-01

    Reverse transcriptase (RT) plays an essential role in HIV-1 replication, and inhibition of this enzyme is a key component of HIV-treatment. However, the use of RT inhibitors can lead to the emergence of drug-resistant variants. Until recently, most clinically relevant resistance mutations were found in the polymerase domain of RT. Lately, an increasing number of resistance mutations has been identified in the connection and RNaseH domain. To further explore the role of these domains we analyzed the complete RT sequence of HIV-1 subtype B patients failing therapy. Position A/T400 in the connection subdomain is polymorphic, but the proportion of T400 increases from 41% in naïve patients to 72% in patients failing therapy. Previous studies suggested a role for threonine in conferring resistance to nucleoside RT inhibitors. Here we report that T400 also mediates resistance to non-nucleoside RT inhibitors. The susceptibility to NVP and EFV was reduced 5-fold and 2-fold, respectively, in the wild-type subtype B NL4.3 background. We show that substitution A400T reduces the RNaseH activity. The changes in enzyme activity are remarkable given the distance to both the polymerase and RNaseH active sites. Molecular dynamics simulations were performed, which provide a novel atomistic mechanism for the reduction in RNaseH activity induced by T400. Substitution A400T was found to change the conformation of the RNaseH primer grip region. Formation of an additional hydrogen bond between residue T400 and E396 may play a role in this structural change. The slower degradation of the viral RNA genome may provide more time for dissociation of the bound NNRTI from the stalled RT-template/primer complex, after which reverse transcription can resume.

  3. A Polymorphism at Position 400 in the Connection Subdomain of HIV-1 Reverse Transcriptase Affects Sensitivity to NNRTIs and RNaseH Activity

    PubMed Central

    Flandre, Philippe; van den Eede, Peter; Govaert, Micheline; Setiawan, Laurentia; Coveney, Peter V.; Marcelin, Anne-Geneviève; Calvez, Vincent; Boucher, Charles A. B.; Beerens, Nancy

    2013-01-01

    Reverse transcriptase (RT) plays an essential role in HIV-1 replication, and inhibition of this enzyme is a key component of HIV-treatment. However, the use of RT inhibitors can lead to the emergence of drug-resistant variants. Until recently, most clinically relevant resistance mutations were found in the polymerase domain of RT. Lately, an increasing number of resistance mutations has been identified in the connection and RNaseH domain. To further explore the role of these domains we analyzed the complete RT sequence of HIV-1 subtype B patients failing therapy. Position A/T400 in the connection subdomain is polymorphic, but the proportion of T400 increases from 41% in naïve patients to 72% in patients failing therapy. Previous studies suggested a role for threonine in conferring resistance to nucleoside RT inhibitors. Here we report that T400 also mediates resistance to non-nucleoside RT inhibitors. The susceptibility to NVP and EFV was reduced 5-fold and 2-fold, respectively, in the wild-type subtype B NL4.3 background. We show that substitution A400T reduces the RNaseH activity. The changes in enzyme activity are remarkable given the distance to both the polymerase and RNaseH active sites. Molecular dynamics simulations were performed, which provide a novel atomistic mechanism for the reduction in RNaseH activity induced by T400. Substitution A400T was found to change the conformation of the RNaseH primer grip region. Formation of an additional hydrogen bond between residue T400 and E396 may play a role in this structural change. The slower degradation of the viral RNA genome may provide more time for dissociation of the bound NNRTI from the stalled RT-template/primer complex, after which reverse transcription can resume. PMID:24098331

  4. Inhibition of Reverse Transcriptase Activity Increases Stability of the HIV-1 Core

    PubMed Central

    Yang, Yang; Fricke, Thomas

    2013-01-01

    Previous studies showed that HIV-1 reverse transcription occurs during or before uncoating, linking mechanistically reverse transcription with uncoating. Here we show that inhibition of reverse transcriptase (RT) during HIV-1 infection by pharmacologic or genetic means increased the stability of the HIV-1 core during infection. Interestingly, HIV-1 particles with increased core stability were resistant to the core-destabilizing effects of rhesus TRIM5α (TRIM5αrh). Collectively, this work implies that the surface of the HIV-1 core is dynamic and changes upon the ongoing processes within the core. PMID:23077298

  5. Structural basis for activation of α-boranophosphate nucleotide analogues targeting drug-resistant reverse transcriptase

    PubMed Central

    Meyer, Philippe; Schneider, Benoît; Sarfati, Simon; Deville-Bonne, Dominique; Guerreiro, Catherine; Boretto, Joëlle; Janin, Joël; Véron, Michel; Canard, Bruno

    2000-01-01

    AIDS chemotherapy is limited by inadequate intracellular concentrations of the active triphosphate form of nucleoside analogues, leading to incomplete inhibition of viral replication and the appearance of drug-resistant virus. Drug activation by nucleoside diphosphate kinase and inhibition of HIV-1 reverse transcriptase were studied comparatively. We synthesized analogues with a borano (BH3–) group on the α-phosphate, and found that they are substrates for both enzymes. X-ray structures of complexes with nucleotide diphosphate kinase provided a structural basis for their activation. The complex with d4T triphosphate displayed an intramolecular CH…O bond contributing to catalysis, and the Rp diastereoisomer of thymidine α-boranotriphosphate bound like a normal substrate. Using α-(Rp)-boranophosphate derivatives of the clinically relevant compounds AZT and d4T, the presence of the α-borano group improved both phosphorylation by nucleotide diphosphate kinase and inhibition of reverse transcription. Moreover, repair of blocked DNA chains by pyrophosphorolysis was reduced significantly in variant reverse transcriptases bearing substitutions found in drug-resistant viruses. Thus, the α-borano modification of analogues targeting reverse transcriptase may be of generic value in fighting viral drug resistance. PMID:10899107

  6. Structural basis for activation of alpha-boranophosphate nucleotide analogues targeting drug-resistant reverse transcriptase.

    PubMed

    Meyer, P; Schneider, B; Sarfati, S; Deville-Bonne, D; Guerreiro, C; Boretto, J; Janin, J; Véron, M; Canard, B

    2000-07-17

    AIDS chemotherapy is limited by inadequate intracellular concentrations of the active triphosphate form of nucleoside analogues, leading to incomplete inhibition of viral replication and the appearance of drug-resistant virus. Drug activation by nucleoside diphosphate kinase and inhibition of HIV-1 reverse transcriptase were studied comparatively. We synthesized analogues with a borano (BH(3)(-)) group on the alpha-phosphate, and found that they are substrates for both enzymes. X-ray structures of complexes with nucleotide diphosphate kinase provided a structural basis for their activation. The complex with d4T triphosphate displayed an intramolecular CH.O bond contributing to catalysis, and the R(p) diastereoisomer of thymidine alpha-boranotriphosphate bound like a normal substrate. Using alpha-(R(p))-boranophosphate derivatives of the clinically relevant compounds AZT and d4T, the presence of the alpha-borano group improved both phosphorylation by nucleotide diphosphate kinase and inhibition of reverse transcription. Moreover, repair of blocked DNA chains by pyrophosphorolysis was reduced significantly in variant reverse transcriptases bearing substitutions found in drug-resistant viruses. Thus, the alpha-borano modification of analogues targeting reverse transcriptase may be of generic value in fighting viral drug resistance.

  7. Polyurethane intravaginal ring for controlled delivery of dapivirine, a nonnucleoside reverse transcriptase inhibitor of HIV-1.

    PubMed

    Gupta, Kavita M; Pearce, Serena M; Poursaid, Azadeh E; Aliyar, Hyder A; Tresco, Patrick A; Mitchnik, Mark A; Kiser, Patrick F

    2008-10-01

    Women-controlled methods for prevention of male-to-female sexual transmission of HIV-1 are urgently needed. Providing inhibitory concentrations of HIV-1 reverse transcriptase inhibitors to impede the replication of the virus in the female genital tissue offers a mechanism for prophylaxis of HIV-1. To this end, an intravaginal ring device that can provide long duration delivery of dapivirine, a nonnucleoside reverse transcriptase inhibitor of HIV-1, was developed utilizing a medical-grade polyether urethane. Monolithic intravaginal rings were fabricated and sustained release with cumulative flux linear with time was demonstrated under sink conditions for a period of 30 days. The release rate was directly proportional to the amount of drug loaded. Another release study conducted for a week utilizing liposome dispersions as sink conditions, to mimic the partitioning of dapivirine into vaginal tissue, also demonstrated release rates constant with time. These results qualify polyether urethanes for development of intravaginal rings for sustained delivery of microbicidal agents.

  8. Inhibition of reverse transcriptase by tyrosinase generated quinones related to levodopa and dopamine.

    PubMed

    Wick, M M; Fitzgerald, G

    1981-12-01

    Several derivatives of levodopa have been shown to be potent inhibitors of the sulfhydryl enzyme, RNA dependent DNA polymerase, reverse transcriptase (RT). In the presence of the polyphenol oxidase, tyrosinase, the inhibitory values were between 10(-6) M and 10(-5) M. Structure-activity studies revealed that active oxidation or reduction was necessary for this potent inhibitory response. Spectrophotometric analysis showed that the presence of both the quinone and quinol was required for maximum inhibitory activity. These data suggest that the common intermediate of oxidation of quinols or reduction of quinones (i.e., semiquinone) is the active species. The use of tyrosinase provides a convenient model for the detection of the actual inhibitory interaction of a free-radical (semiquinone) with a biologically important macromolecule, reverse transcriptase.

  9. Developing novel nonnucleoside HIV-1 reverse transcriptase inhibitors: beyond the butterfly.

    PubMed

    Basavapathruni, Aravind; Anderson, Karen S

    2006-01-01

    To date three nonnucleoside reverse transcriptase inhibitors (NNRTIs) have been approved by the U.S. Food and Drug Administration for the treatment of human immunodeficiency virus type 1 infection. A limiting factor in the effectiveness of these agents is the development of resistance, manifested by amino acid substitutions within the virally encoded reverse transcriptase (RT). Understanding the mechanism of action of these agents and how resistance develops have broadened the field of NNRTI research to elucidate structural and biochemical features of inhibition in hopes of creating better inhibitors. In this review, the history of NNRTIs will preface the many studies characterizing inhibition and the development of a new paradigm for understanding the molecular mechanism of drug resistance to NNRTIs. Combination therapies including nonnucleoside inhibitors will be discussed, concluding with remarks on potential new inhibitors.

  10. Crystal structures of HIV-1 nonnucleoside reverse transcriptase inhibitors: N-benzyl-4-methyl-benzimidazoles

    NASA Astrophysics Data System (ADS)

    Ziółkowska, Natasza E.; Michejda, Christopher J.; Bujacz, Grzegorz D.

    2009-07-01

    HIV-1 nonnucleoside reverse transcriptase inhibitors are potentially specific and effective drugs in AIDS therapy. The presence of two aromatic systems with an angled orientation in the molecule of the inhibitor is crucial for interactions with HIV-1 RT. The inhibitor drives like a wedge into the cluster of aromatic residues of RT HIV-1 and restrains the enzyme in a conformation that blocks the chemical step of nucleotide incorporation. Structural studies provide useful information for designing new, more active inhibitors. The crystal structures of four NNRTIs are presented here. The investigated compounds are derivatives of N-benzyl-4-methyl-benzimidazole with various aliphatic and aromatic substituents at carbon 2 positions and a 2,6-dihalogeno-substituted N-benzyl moiety. Structural data reported here show that the conformation of the investigated compounds is relatively rigid. Such feature is important for the nonnucleoside inhibitor binding to HIV-1 reverse transcriptase.

  11. Novel Structure of Ty3 Reverse Transcriptase | Center for Cancer Research

    Cancer.gov

    Retrotransposons are mobile genetic elements that self amplify via a single-stranded RNA intermediate, which is converted to double-stranded DNA by an encoded reverse transcriptase (RT) with both DNA polymerase (pol) and ribonuclease H (RNase) activities. Categorized by whether they contain flanking long terminal repeat (LTR) sequences, retrotransposons play a critical role in the architecture of eukaryotic genomes and are the evolutionary origin of retroviruses, including human immunodeficiency virus (HIV).

  12. Incidence of cancer in children perinatally exposed to nucleoside reverse transcriptase inhibitors.

    PubMed

    Benhammou, Valérie; Warszawski, Josiane; Bellec, Stéphanie; Doz, François; André, Nicolas; Lacour, Brigitte; Levine, Martine; Bavoux, Françoise; Tubiana, Roland; Mandelbrot, Laurent; Clavel, Jacqueline; Blanche, Stéphane

    2008-10-18

    Long-term studies of tolerance to perinatal exposure to antiretroviral nucleoside reverse transcriptase inhibitors are required, in view of the potential genotoxicity of some of these molecules. To evaluate the incidence of cancers in uninfected children born to HIV-infected mothers. Cancers were detected in a nationwide prospective cohort of children born to HIV-infected mothers by standardized questionnaire during the prospective follow-up period of 2 years; thereafter, they were detected by spontaneous pharmacovigilance declaration and by crosschecking data with the national registries of childhood cancer. Standardized incidence ratio for incidence comparisons with general population. Ten cases of cancer were detected among the 9127 exposed HIV-uninfected children (median age: 5.4 years, 53 052 person-years of follow-up). The overall incidence did not differ significantly from that expected for the general population: 10 cases observed versus 8.9 and 9.6 expected depending on whether 1990-1999 or 2000-2004 national rates were used as reference [standardized incidence ratio of 1.1 (0.3-1.5) and 1.0 (0.5-1.9)]. Five cases of central nervous system cancer were observed (standardized incidence ratio of 3.1 [1.0-7.2] P = 0.05 and 2.4 [0.8-5.6], P = 0.12). The relative risk of cancer for children exposed to didanosine-lamivudine combination was higher than that for zidovudine monotherapy [hazard ratio: 13.6 (2.5-73.9)]. This study did not evidence an overall increase in cancer risk in nucleoside reverse transcriptase inhibitor exposed children until 5 years of age. Results suggesting associations with specific nucleoside reverse transcriptase inhibitor combinations need further investigations. A longer surveillance, including differential analysis of the different cancer sites and various nucleoside reverse transcriptase inhibitors administered is warranted.

  13. Inhibitory effect of aqueous dandelion extract on HIV-1 replication and reverse transcriptase activity

    PubMed Central

    2011-01-01

    Background Acquired immunodeficiency syndrome (AIDS), which is caused by the human immunodeficiency virus (HIV), is an immunosuppressive disease that results in life-threatening opportunistic infections. The general problems in current therapy include the constant emergence of drug-resistant HIV strains, adverse side effects and the unavailability of treatments in developing countries. Natural products from herbs with the abilities to inhibit HIV-1 life cycle at different stages, have served as excellent sources of new anti-HIV-1 drugs. In this study, we aimed to investigate the anti-HIV-1 activity of aqueous dandelion extract. Methods The pseudotyped HIV-1 virus has been utilized to explore the anti-HIV-1 activity of dandelion, the level of HIV-1 replication was assessed by the percentage of GFP-positive cells. The inhibitory effect of the dandelion extract on reverse transcriptase activity was assessed by the reverse transcriptase assay kit. Results Compared to control values obtained from cells infected without treatment, the level of HIV-1 replication and reverse transcriptase activity were decreased in a dose-dependent manner. The data suggest that dandelion extract has a potent inhibitory activity against HIV-1 replication and reverse transcriptase activity. The identification of HIV-1 antiviral compounds from Taraxacum officinale should be pursued. Conclusions The dandelion extract showed strong activity against HIV-1 RT and inhibited both the HIV-1 vector and the hybrid-MoMuLV/MoMuSV retrovirus replication. These findings provide additional support for the potential therapeutic efficacy of Taraxacum officinale. Extracts from this plant may be regarded as another starting point for the development of an antiretroviral therapy with fewer side effects. PMID:22078030

  14. Crystal Structures of HIV-1 Reverse Transcriptase with Picomolar Inhibitors Reveal Key Interactions for Drug Design

    PubMed Central

    Frey, Kathleen M.; Bollini, Mariela; Mislak, Andrea C.; Cisneros, José A.; Gallardo-Macias, Ricardo; Jorgensen, William L.; Anderson, Karen S.

    2012-01-01

    X-ray crystal structures at 2.9 Å resolution are reported for complexes of catechol diethers 1 and 2 with HIV-1 reverse transcriptase. The results help elucidate the structural origins of the extreme antiviral activity of the compounds. The possibility of halogen bonding between the inhibitors and Pro95 is addressed. Structural analysis reveals key interactions with conserved residues P95 and W229 of importance for design of inhibitors with high potency and favorable resistance profiles. PMID:23163887

  15. Reverse transcriptase activity in tissues of the soft shell clam Mya arenaria affected with haemic neoplasia.

    PubMed

    AboElkhair, M; Synard, S; Siah, A; Pariseau, J; Davidson, J; Johnson, G; Greenwood, S J; Casey, J W; Berthe, F C J; Cepica, A

    2009-10-01

    Since all retroviruses possess reverse transcriptase (RT) enzyme, reverse transcriptase activity has been the main supportive evidence of retroviral etiology of haemic neoplasia (HN) in soft shell clams, Mya arenaria. The objective of the present study was to search for a putative retrovirus in various tissues of diseased clams following quantification of RT activity (biochemical indicator of retroviral infection). The clams were assessed by flow cytometry (FCM) for diagnosis of HN. RT activity was quantified by TaqMan-product enhanced reverse transcriptase (TM-PERT) assay in four different organs, gonad, gills, digestive gland, and mantle, at various stages of HN. The digestive gland, the organ with the highest RT activity, and haemocytes, the target cell of HN, were assessed by EM for presence of retroviruses. All organs were assessed by histology. The results of this study demonstrated that although all organs of healthy clams have some background RT activity, the activity observed in most of organs of diseased clams was significantly increased (p<0.05). An association was observed between the degree of neoplastic cell infiltration and the level of RT activity. Digestive gland showed the highest and most consistent RT activity in both healthy and diseased clams. No evidence for the existence of a retrovirus like particle was found by positive staining EM. The presence of RT activity without indications of retroviral particles in digestive gland and haemocytes suggests a probable endogenous source of RT.

  16. Interpreting Reverse Transcriptase Termination and Mutation Events for Greater Insight into the Chemical Probing of RNA.

    PubMed

    Sexton, Alec N; Wang, Peter Y; Rutenberg-Schoenberg, Michael; Simon, Matthew D

    2017-09-05

    Chemical probing has the power to provide insight into RNA conformation in vivo and in vitro, but interpreting the results depends on methods to detect the chemically modified nucleotides. Traditionally, the presence of modified bases was inferred from their ability to halt reverse transcriptase during primer extension and the locations of termination sites observed by electrophoresis or sequencing. More recently, modification-induced mutations have been used as a readout for chemical probing data. Given the variable propensity for mismatch incorporation and read-through with different reverse transcriptases, we examined how termination and mutation events compare to each other in the same chemical probing experiments. We found that mutations and terminations induced by dimethyl sulfate probing are both specific for methylated bases, but these two measures have surprisingly little correlation and represent largely nonoverlapping indicators of chemical modification data. We also show that specific biases for modified bases depend partly on local sequence context and that different reverse transcriptases show different biases toward reading a modification as a stop or a mutation. These results support approaches that incorporate analysis of both termination and mutation events into RNA probing experiments.

  17. Human telomerase acts as a hTR-independent reverse transcriptase in mitochondria.

    PubMed

    Sharma, Nilesh K; Reyes, Aurelio; Green, Paula; Caron, Matthieu J; Bonini, Marcelo G; Gordon, Donna M; Holt, Ian J; Santos, Janine Hertzog

    2012-01-01

    Human telomerase reverse transcriptase (hTERT) is localized to mitochondria, as well as the nucleus, but details about its biology and function in the organelle remain largely unknown. Here we show, using multiple approaches, that mammalian TERT is mitochondrial, co-purifying with mitochondrial nucleoids and tRNAs. We demonstrate the canonical nuclear RNA [human telomerase RNA (hTR)] is not present in human mitochondria and not required for the mitochondrial effects of telomerase, which nevertheless rely on reverse transcriptase (RT) activity. Using RNA immunoprecipitations from whole cell and in organello, we show that hTERT binds various mitochondrial RNAs, suggesting that RT activity in the organelle is reconstituted with mitochondrial RNAs. In support of this conclusion, TERT drives first strand cDNA synthesis in vitro in the absence of hTR. Finally, we demonstrate that absence of hTERT specifically in mitochondria with maintenance of its nuclear function negatively impacts the organelle. Our data indicate that mitochondrial hTERT works as a hTR-independent reverse transcriptase, and highlight that nuclear and mitochondrial telomerases have different cellular functions. The implications of these findings to both the mitochondrial and telomerase fields are discussed.

  18. Human telomerase acts as a hTR-independent reverse transcriptase in mitochondria

    PubMed Central

    Sharma, Nilesh K.; Reyes, Aurelio; Green, Paula; Caron, Matthieu J.; Bonini, Marcelo G.; Gordon, Donna M.; Holt, Ian J.; Santos, Janine Hertzog

    2012-01-01

    Human telomerase reverse transcriptase (hTERT) is localized to mitochondria, as well as the nucleus, but details about its biology and function in the organelle remain largely unknown. Here we show, using multiple approaches, that mammalian TERT is mitochondrial, co-purifying with mitochondrial nucleoids and tRNAs. We demonstrate the canonical nuclear RNA [human telomerase RNA (hTR)] is not present in human mitochondria and not required for the mitochondrial effects of telomerase, which nevertheless rely on reverse transcriptase (RT) activity. Using RNA immunoprecipitations from whole cell and in organello, we show that hTERT binds various mitochondrial RNAs, suggesting that RT activity in the organelle is reconstituted with mitochondrial RNAs. In support of this conclusion, TERT drives first strand cDNA synthesis in vitro in the absence of hTR. Finally, we demonstrate that absence of hTERT specifically in mitochondria with maintenance of its nuclear function negatively impacts the organelle. Our data indicate that mitochondrial hTERT works as a hTR-independent reverse transcriptase, and highlight that nuclear and mitochondrial telomerases have different cellular functions. The implications of these findings to both the mitochondrial and telomerase fields are discussed. PMID:21937513

  19. Cross-sectional study of virological failure and multinucleoside reverse transcriptase inhibitor resistance at 12 months of antiretroviral therapy in Western India

    PubMed Central

    Karade, Santosh K.; Ghate, Manisha V.; Chaturbhuj, Devidas N.; Kadam, Dileep B.; Shankar, Subramanian; Gaikwad, Nitin; Gurav, Shraddha; Joshi, Rajneesh; Sane, Suvarna S.; Kulkarni, Smita S.; Kurle, Swarali N.; Paranjape, Ramesh S.; Rewari, Bharat B.; Gangakhedkar, Raman R.

    2016-01-01

    Abstract The free antiretroviral therapy (ART) program in India has scaled up to register second largest number of people living with HIV/AIDS across the globe. To assess the effectiveness of current first-line regimen we estimated virological suppression on completion of 1 year of ART. The study describes the correlates of virological failure (VF) and multinucleoside reverse transcriptase inhibitor (NRTI) drug resistance mutations (DRMs). In this cross-sectional study conducted between June and August 2014, consecutive adults from 4 State sponsored ART clinics of western India were recruited for plasma viral load screening at 12 ± 2 months of ART initiation. Individuals with plasma viral load >1000 copies/mL were selected for HIV drug resistance (HIVDR) genotyping. Logistic regression analyses were performed to assess factors associated with VF and multi-NRTI resistance mutations. Criteria adopted for multi-NRTI resistance mutation were either presence of K65R or 3 or more thymidine analog mutations (TAMs) or presence of M184V along with 2 TAMs. Of the 844 study participants, virological suppression at 1 year was achieved in 87.7% of individuals. Factors significantly associated with VF (P < 0.005) were 12 months CD4 count of ≤100 cells/μL (adjusted OR −7.11), low reported adherence (adjusted OR −4.44), and those living without any partner (adjusted OR −1.98). In patients with VF, the prevalence of non-nucleoside reverse transcriptase inhibitor (NNRTI) DRM (78.75%) were higher as compared to NRTI (58.75%). Multi-NRTI DRMs were present in 32.5% of sequences and were significantly associated with CD4 count of ≤100 cells/μL at baseline (adjusted OR −13.00) and TDF-based failing regimen (adjusted OR −20.43). Additionally, low reported adherence was negatively associated with multi-NRTI resistance (adjusted OR −0.11, P = 0.015). K65R mutation was significantly associated with tenofovir (TDF)-based failing regimen (P < 0

  20. SAMHD1 Has Differential Impact on the Efficacies of HIV Nucleoside Reverse Transcriptase Inhibitors

    PubMed Central

    Huber, Andrew D.; Michailidis, Eleftherios; Schultz, Megan L.; Ong, Yee T.; Bloch, Nicolin; Puray-Chavez, Maritza N.; Leslie, Maxwell D.; Ji, Juan; Lucas, Anthony D.; Kirby, Karen A.; Landau, Nathaniel R.

    2014-01-01

    Sterile alpha motif- and histidine/aspartic acid domain-containing protein 1 (SAMHD1) limits HIV-1 replication by hydrolyzing deoxynucleoside triphosphates (dNTPs) necessary for reverse transcription. Nucleoside reverse transcriptase inhibitors (NRTIs) are components of anti-HIV therapies. We report here that SAMHD1 cleaves NRTI triphosphates (TPs) at significantly lower rates than dNTPs and that SAMHD1 depletion from monocytic cells affects the susceptibility of HIV-1 infections to NRTIs in complex ways that depend not only on the relative changes in dNTP and NRTI-TP concentrations but also on the NRTI activation pathways. PMID:24867973

  1. Elevated Human telomerase reverse transcriptase gene expression in blood cells associated with chronic and arsenic exposure in Inner Mongolia, China

    EPA Science Inventory

    BACKGROUND: Arsenic exposure is associated with human cancer. Telomerase containing the catalytic subunit, human telomerase reverse transcriptase (hTERT), can extend telomeres of chromosomes, delay senescence and promoting cell proliferation leading to tumorigenesis. OBJECTIVE:...

  2. Elevated Human telomerase reverse transcriptase gene expression in blood cells associated with chronic and arsenic exposure in Inner Mongolia, China

    EPA Science Inventory

    BACKGROUND: Arsenic exposure is associated with human cancer. Telomerase containing the catalytic subunit, human telomerase reverse transcriptase (hTERT), can extend telomeres of chromosomes, delay senescence and promoting cell proliferation leading to tumorigenesis. OBJECTIVE:...

  3. Enzyme engineering through evolution: thermostable recombinant group II intron reverse transcriptases provide new tools for RNA research and biotechnology.

    PubMed

    Collins, Kathleen; Nilsen, Timothy W

    2013-08-01

    Current investigation of RNA transcriptomes relies heavily on the use of retroviral reverse transcriptases. It is well known that these enzymes have many limitations because of their intrinsic properties. This commentary highlights the recent biochemical characterization of a new family of reverse transcriptases, those encoded by group II intron retrohoming elements. The novel properties of these enzymes endow them with the potential to revolutionize how we approach RNA analyses.

  4. Computational drug design strategies applied to the modelling of human immunodeficiency virus-1 reverse transcriptase inhibitors

    PubMed Central

    Santos, Lucianna Helene; Ferreira, Rafaela Salgado; Caffarena, Ernesto Raúl

    2015-01-01

    Reverse transcriptase (RT) is a multifunctional enzyme in the human immunodeficiency virus (HIV)-1 life cycle and represents a primary target for drug discovery efforts against HIV-1 infection. Two classes of RT inhibitors, the nucleoside RT inhibitors (NRTIs) and the nonnucleoside transcriptase inhibitors are prominently used in the highly active antiretroviral therapy in combination with other anti-HIV drugs. However, the rapid emergence of drug-resistant viral strains has limited the successful rate of the anti-HIV agents. Computational methods are a significant part of the drug design process and indispensable to study drug resistance. In this review, recent advances in computer-aided drug design for the rational design of new compounds against HIV-1 RT using methods such as molecular docking, molecular dynamics, free energy calculations, quantitative structure-activity relationships, pharmacophore modelling and absorption, distribution, metabolism, excretion and toxicity prediction are discussed. Successful applications of these methodologies are also highlighted. PMID:26560977

  5. Computational drug design strategies applied to the modelling of human immunodeficiency virus-1 reverse transcriptase inhibitors.

    PubMed

    Santos, Lucianna Helene; Ferreira, Rafaela Salgado; Caffarena, Ernesto Raúl

    2015-11-01

    Reverse transcriptase (RT) is a multifunctional enzyme in the human immunodeficiency virus (HIV)-1 life cycle and represents a primary target for drug discovery efforts against HIV-1 infection. Two classes of RT inhibitors, the nucleoside RT inhibitors (NRTIs) and the nonnucleoside transcriptase inhibitors are prominently used in the highly active antiretroviral therapy in combination with other anti-HIV drugs. However, the rapid emergence of drug-resistant viral strains has limited the successful rate of the anti-HIV agents. Computational methods are a significant part of the drug design process and indispensable to study drug resistance. In this review, recent advances in computer-aided drug design for the rational design of new compounds against HIV-1 RT using methods such as molecular docking, molecular dynamics, free energy calculations, quantitative structure-activity relationships, pharmacophore modelling and absorption, distribution, metabolism, excretion and toxicity prediction are discussed. Successful applications of these methodologies are also highlighted.

  6. Analysis of transmitted drug resistance in Spain in the years 2007-2010 documents a decline in mutations to the non-nucleoside drug class.

    PubMed

    Monge, S; Guillot, V; Alvarez, M; Peña, A; Viciana, P; García-Bujalance, S; Pérez Elias, M J; Iribarren, J A; Gutiérrez, F; Itziar Casado, M; Garcia, F

    2012-11-01

    We have studied transmitted drug resistance (TDR) in 1.864 antiretroviral-naïve patients entering CoRIS (Spain) during 2007-2010. An overall 8.58% TDR was observed (3.92%, nucleoside reverse transcriptase inhibitors (NRTIs); 3.86%, non-nucleoside reverse transcriptase inhibitors (NNRTIs); 2.31%, protease inhibitors), with a significant decreasing trend over time for NNRTIs (5.53%, 2007; 2.45%, 2010; p for trend = 0.044). Non-B subtype prevalence was 15.93%, with a significant increase (11.95%, 2007; 18.14%, 2010; p for trend = 0.018), mainly related to immigration. Having no formal education increased the risk of TDR to NNRTIs (OR, 7.26), and carrying a non-B subtype reduced the risk of TDR to NRTIs (OR, 0.27). These findings may have important implications for treatment guidelines and laboratory testing recommendations. © 2012 The Authors. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.

  7. HuR interacts with human immunodeficiency virus type 1 reverse transcriptase, and modulates reverse transcription in infected cells

    PubMed Central

    Lemay, Julie; Maidou-Peindara, Priscilla; Bader, Thomas; Ennifar, Eric; Rain, Jean-Christophe; Benarous, Richard; Liu, Lang Xia

    2008-01-01

    Reverse transcription of the genetic material of human immunodeficiency virus type 1 (HIV-1) is a critical step in the replication cycle of this virus. This process, catalyzed by reverse transcriptase (RT), is well characterized at the biochemical level. However, in infected cells, reverse transcription occurs in a multiprotein complex – the reverse transcription complex (RTC) – consisting of viral genomic RNA associated with viral proteins (including RT) and, presumably, as yet uncharacterized cellular proteins. Very little is known about the cellular proteins interacting with the RTC, and with reverse transcriptase in particular. We report here that HIV-1 reverse transcription is affected by the levels of a nucleocytoplasmic shuttling protein – the RNA-binding protein HuR. A direct protein-protein interaction between RT and HuR was observed in a yeast two-hybrid screen and confirmed in vitro by homogenous time-resolved fluorescence (HTRF). We mapped the domain interacting with HuR to the RNAse H domain of RT, and the binding domain for RT to the C-terminus of HuR, partially overlapping the third RRM RNA-binding domain of HuR. HuR silencing with specific siRNAs greatly impaired early and late steps of reverse transcription, significantly inhibiting HIV-1 infection. Moreover, by mutagenesis and immunoprecipitation studies, we could not detect the binding of HuR to the viral RNA. These results suggest that HuR may be involved in and may modulate the reverse transcription reaction of HIV-1, by an as yet unknown mechanism involving a protein-protein interaction with HIV-1 RT. PMID:18544151

  8. Antiviral Activity of MK-4965, a Novel Nonnucleoside Reverse Transcriptase Inhibitor▿

    PubMed Central

    Lai, Ming-Tain; Munshi, Vandna; Touch, Sinoeun; Tynebor, Robert M.; Tucker, Thomas J.; McKenna, Philip M.; Williams, Theresa M.; DiStefano, Daniel J.; Hazuda, Daria J.; Miller, Michael D.

    2009-01-01

    Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are the mainstays of therapy for the treatment of human immunodeficiency virus type 1 (HIV-1) infections. However, the effectiveness of NNRTIs can be hampered by the development of resistance mutations which confer cross-resistance to drugs in the same class. Extensive efforts have been made to identify new NNRTIs that can suppress the replication of the prevalent NNRTI-resistant viruses. MK-4965 is a novel NNRTI that possesses both diaryl ether and indazole moieties. The compound displays potency at subnanomolar concentrations against wild-type (WT), K103N, and Y181C reverse transcriptase (RT) in biochemical assays. MK-4965 is also highly potent against the WT virus and two most prevalent NNRTI-resistant viruses (viruses that harbor the K103N or the Y181C mutation), against which it had 95% effective concentrations (EC95s) of <30 nM in the presence of 10% fetal bovine serum. The antiviral EC95 of MK-4965 was reduced approximately four- to sixfold when it was tested in 50% human serum. Moreover, MK-4965 was evaluated with a panel of 15 viruses with NNRTI resistance-associated mutations and showed a superior mutant profile to that of efavirenz but not to that of etravirine. MK-4965 was similarly effective against various HIV-1 subtypes and viruses containing nucleoside reverse transcriptase inhibitor or protease inhibitor resistance-conferring mutations. A two-drug combination study showed that the antiviral activity of MK-4965 was nonantagonistic with each of the 18 FDA-licensed drugs tested vice versa in the present study. Taken together, these in vitro data show that MK-4965 possesses the desired properties for further development as a new NNRTI for the treatment of HIV-1 infection. PMID:19289522

  9. Direct CRISPR spacer acquisition from RNA by a natural reverse-transcriptase-Cas1 fusion protein

    PubMed Central

    Sidote, David J.; Markham, Laura M.; Sanchez-Amat, Antonio; Bhaya, Devaki; Lambowitz, Alan M.; Fire, Andrew Z.

    2016-01-01

    CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) systems mediate adaptive immunity in diverse prokaryotes. CRISPR-associated Cas1 and Cas2 proteins have been shown to enable adaptation to new threats in Type I and II CRISPR systems by the acquisition of short segments of DNA (“spacers”) from invasive elements. In several Type III CRISPR systems, Cas1 is naturally fused to a reverse transcriptase (RT). In the marine bacterium Marinomonas mediterranea (MMB-1), we show that an RT-Cas1 fusion enables the acquisition of RNA spacers in vivo in an RT-dependent manner. In vitro, the MMB-1 RT-Cas1 and Cas2 proteins catalyze ligation of RNA segments into the CRISPR array, followed by reverse transcription. These observations outline a host-mediated mechanism for reverse information flow from RNA to DNA. PMID:26917774

  10. Reverse transcriptase and endonuclease activities encoded by Penelope-like retroelements

    PubMed Central

    Pyatkov, Konstantin I.; Arkhipova, Irina R.; Malkova, Natalia V.; Finnegan, David J.; Evgen'ev, Michael B.

    2004-01-01

    Penelope-like elements are a class of retroelement that have now been identified in >50 species belonging to at least 10 animal phyla. The Penelope element isolated from Drosophila virilis is the only transpositionally active representative of this class isolated so far. The single ORF of Penelope and its relatives contains regions homologous to a reverse transcriptase of atypical structure and to the GIY-YIG, or Uri, an endonuclease (EN) domain not previously found in retroelements. We have expressed the single ORF of Penelope in a baculovirus expression system and have shown that it encodes a polyprotein with reverse transcriptase activity that requires divalent cations (Mn2+ and Mg2+). We have also expressed and purified the EN domain in Escherichia coli and have demonstrated that it has EN activity in vitro. Mutations in the conserved residues of the EN catalytic module abolish its nicking activity, whereas the DNA-binding properties of the mutant proteins remain unaffected. Only one strand of the target sequence is cleaved, and there is a certain degree of cleavage specificity. We propose that the Penelope EN cleaves the target DNA during transposition, generating a primer for reverse transcription. Our results show that an active Uri EN has been adopted by a retrotransposon. PMID:15465912

  11. Modeling of Plasmodium falciparum Telomerase Reverse Transcriptase Ternary Complex: Repurposing of Nucleoside Analog Inhibitors.

    PubMed

    Mohanty, Pallavi; Gupta, Akanksha; Bhatnagar, Sonika

    2015-12-01

    The Plasmodium falciparum telomerase reverse transcriptase (PfTERT) is a ribonucleoprotein that assists the maintenance of the telomeric ends of chromosomes by reverse transcription of its own RNA subunit. It represents an attractive therapeutic target for eradication of the plasmodial parasite at the asexual liver stage. Automated modeling using MUSTER and knowledge-based techniques were used to obtain a three-dimensional model of the active site of reverse transcriptase domain of PfTERT, which is responsible for catalyzing the addition of incoming dNTPs to the growing DNA strand in presence of divalent magnesium ions. Further, the ternary complex of the active site of PfTERT bound to a DNA-RNA duplex was also modeled using Haddock server and represents the functional form of the enzyme. Initially, established nucleoside analog inhibitors of PfTERT, AZTTP, and ddGTP were docked in the modeled binding site of the PfTERT ternary complex using AutoDock v4.2. Subsequently, docking studies were carried out with 14 approved nucleoside analog inhibitors. Docking studies predicted that floxuridine, gemcitabine, stavudine, and vidarabine have high affinity for the PfTERT ternary complex. Further analysis on the basis of known side effects led us to propose repositioning of vidarabine as a suitable drug candidate for inhibition of PfTERT.

  12. Copy-choice recombination by reverse transcriptases: Reshuffling of genetic markers mediated by RNA chaperones

    PubMed Central

    Negroni, Matteo; Buc, Henri

    2000-01-01

    Copy-choice recombination efficiently reshuffles genetic markers in retroviruses. In vivo, the folding of the genomic RNA is controlled by the nucleocapsid protein (NC). We show that binding of NC onto the acceptor RNA molecule is sufficient to enhance recombination, providing evidence for a mechanism where the structure of the acceptor template determines the template switch. NC as well as another RNA chaperone (StpA) converts recombination into a widespread process no longer restricted to rare hot spots, an effect maximized when both the NC and the reverse transcriptase come from HIV-1. These data suggest that RNA chaperones confer a higher genetic flexibility to retroviruses. PMID:10829081

  13. Reversible immortalization of sheep fetal fibroblast cells by tetracycline-inducible expression of human telomerase reverse transcriptase.

    PubMed

    Zhang, Na; Li, Jianwei; Zhong, Xia; An, Xiaorong; Hou, Jian

    2016-08-01

    To achieve reversible immortalization of cells, we design a modified tetracycline-inducible expression (Tet-on) system to conditionally regulate the ectopic expression of human telomerase reverse transcriptase (hTERT) in primary cells. The hTERT gene, hygromycin-resistant gene and all essential elements for achieving tetracycline induction were combined into a single plasmid vector. Sheep fetal fibroblast cells were transfected with the vector and four putative immortalized cell clones were obtained via induction of hTERT expression by doxycycline. These immortalized cells maintained a normal karyotype and showed no transformed phenotype after 250 days continuous culture. When hTERT expression was switched off by withdrawal of doxycycline, the immortalized cells reverted to a normal proliferative state and eventually senesced after limited divisions. This single-plasmid based Tet-on inducible hTERT expression system can be applied for reversible immortalization of animal cells.

  14. Combinations of mutations in the connection domain of human immunodeficiency virus type 1 reverse transcriptase: assessing the impact on nucleoside and nonnucleoside reverse transcriptase inhibitor resistance.

    PubMed

    Gupta, Soumi; Fransen, Signe; Paxinos, Ellen E; Stawiski, Eric; Huang, Wei; Petropoulos, Christos J

    2010-05-01

    Recent reports have described the effect of mutations in the connection and RNase H domains of reverse transcriptase (RT) on nucleoside and nonnucleoside reverse transcriptase inhibitor (NRTI and NNRTI, respectively) resistance in the presence of thymidine analog resistance mutations (TAMs) and NNRTI mutations (J. H. Brehm, D. Koontz, J. D. Meteer, V. Pathak, N. Sluis-Cremer, and J. W. Mellors, J. Virol. 81:7852-7859, 2007; K. A. Delviks-Frankenberry, G. N. Nikolenko, R. Barr, and V. K. Pathak, J. Virol. 81:6837-6845, 2007; G. N. Nikolenko, K. A. Delviks-Frankenberry, S. Palmer, F. Maldarelli, M. J. Fivash, Jr., J. M. Coffin, and V. K. Pathak, Proc. Natl. Acad. Sci. U. S. A. 104:317-322, 2007; G. N. Nikolenko, S. Palmer, F. Maldarelli, J. W. Mellors, J. M. Coffin, and V. K. Pathak, Proc. Natl. Acad. Sci. U. S. A. 102:2093-2098, 2005; and S. H. Yap, C. W. Sheen, J. Fahey, M. Zanin, D. Tyssen, V. D. Lima, B. Wynhoven, M. Kuiper, N. Sluis-Cremer, P. R. Harrigan, and G. Tachedjian, PLoS Med. 4:e335, 2007). In the present study, novel mutations in the connection domain of RT (T369I/V), first identified in patient-derived viruses, were characterized, and their effects on NNRTI and NNRTI susceptibility were determined. Furthermore, the effect of N348I on NRTI and NNRTI resistance was confirmed. HIV-1 with either N348I or T369I/V demonstrated reduced susceptibility to nevirapine (NVP), efavirenz (EFV), delaviridine (DLV), and zidovudine (ZDV) compared to wild-type HIV-1. However, HIV-1 with T369I and N348I demonstrated 10- to 60-fold resistance to these same drugs. In clinical samples, these two connection domain RT mutations were predominantly observed in viruses containing TAMs and NNRTI mutations and did not alter the susceptible-resistant classifications of these samples. Introduction of T369I, N348I, or T369I/N348I also reduced replication capacity (RC). These observations suggest that it may be of scientific interest to test these mutations against new NNRTI

  15. Prevalence of transmitted nucleoside analogue-resistant HIV-1 strains and pre-existing mutations in pol reverse transcriptase and protease region: outcome after treatment in recently infected individuals.

    PubMed

    Balotta, C; Berlusconi, A; Pan, A; Violin, M; Riva, C; Colombo, M C; Gori, A; Papagno, L; Corvasce, S; Mazzucchelli, R; Facchi, G; Velleca, R; Saporetti, G; Galli, M; Rusconi, S; Moroni, M

    2000-03-01

    We retrospectively studied 38 Italian recently HIV-1-infected subjects who seroconverted from 1994 to 1997 to investigate: (i) the prevalence of nucleoside reverse transcriptase inhibitors (NRTI)-related mutations at primary infection; (ii) the proportion of naturally occurring mutations in reverse transcriptase (RT) and protease regions of patients naive for non-nucleoside RT inhibitors (NNRTIs) and protease inhibitors (PIs); (iii) the drug-susceptibility to NRTIs and PIs in subjects with NRTI- and/or PI-related mutations; and (iv) the outcome of seroconverters treated with various NRTIs or NRTI/PI regimens. Baseline HIV-1 plasma viraemia and absolute CD4 count at baseline could not be used to distinguish patients with NRTI- and/or PI-related pre-existing mutations from those with wild-type virus (P = 0.693 and P = 0.542, respectively). The frequency of zidovudine-related mutations was 21% in the study period. The response to treatment was not significantly different in subjects with or without genotypic zidovudine-related mutations at primary infection (P = 0.744 for HIV-1 RNA and P = 0.102 for CD4 cells). Some natural variation (2.6%) was present within regions 98-108 and 179-190 of RT involved in NNRTI resistance. The high natural polymorphism in the protease region present in our patients was similar to that reported by others. In our study some PI-associated substitutions, thought to be compensatory in protease enzymatic function, could confer intermediate to high PI-resistance. As discrepancies between genotypic and phenotypic results may exist in recent seroconverters, our data suggest that the role of transmitted NRTI- and PI-resistant variants remain to be fully elucidated in vivo.

  16. Pyrroloaryls and pyrroloheteroaryls: Inhibitors of the HIV fusion/attachment, reverse transcriptase and integrase.

    PubMed

    Patel, Rahul V; Park, Se Won

    2015-09-01

    Heterocyclic compounds execute a very important role in drug design and discovery. This article provides the basic milestones of the research for pyrroloaryl and pyrroloheteroaryl based components targeting HIV viral replication cycle. Anti-HIV activity is elaborated for several classes of pyrrolo-compounds as pyrrolopyridines, pyrrolopyrimidines, pyrrolopyridazines, pyrrolobenzodiazepinones, pyrrolobenzothiazepines, pyrrolobenzoxazepinones, pyrrolophenanthridines, pyrroloquinoxalines, pyrrolotriazines, pyrroloquinolines, pyrrolopyrazinones, pyrrolothiatriazines, arylthiopyrroles and pyrrolopyrazolones targeting two essential HIV enzymes, reverse transcriptase and integrase as well as attachment/fusion of HIV virons to the host CD-4 cell. Such attempts were resulted in a discovery of highly potent anti-HIV agents suitable for clinical trials, for example, BMS-378806, BMS-585248, BMS-626529, BMS-663068, BMS-488043 and BMS-663749, etc. as anti-HIV attachment agents, triciribine, QX432, BI-1 and BI-2 as HIV RT inhibitors which are in preclinical or clinical development. Mechanism of action of compounds presented in this article towards the suppression of HIV attachment/fusion as well as against the activities of HIV enzymes reverse transcriptase and integrase has been discussed. Relationships of new compounds' molecular framework and HIV viral target has been overviewed in order to facilitate further construction of promising anti-HIV agents in future drug discovery process.

  17. High-throughput sequencing of human plasma RNA by using thermostable group II intron reverse transcriptases

    PubMed Central

    Qin, Yidan; Yao, Jun; Wu, Douglas C.; Nottingham, Ryan M.; Mohr, Sabine; Hunicke-Smith, Scott; Lambowitz, Alan M.

    2016-01-01

    Next-generation RNA-sequencing (RNA-seq) has revolutionized transcriptome profiling, gene expression analysis, and RNA-based diagnostics. Here, we developed a new RNA-seq method that exploits thermostable group II intron reverse transcriptases (TGIRTs) and used it to profile human plasma RNAs. TGIRTs have higher thermostability, processivity, and fidelity than conventional reverse transcriptases, plus a novel template-switching activity that can efficiently attach RNA-seq adapters to target RNA sequences without RNA ligation. The new TGIRT-seq method enabled construction of RNA-seq libraries from <1 ng of plasma RNA in <5 h. TGIRT-seq of RNA in 1-mL plasma samples from a healthy individual revealed RNA fragments mapping to a diverse population of protein-coding gene and long ncRNAs, which are enriched in intron and antisense sequences, as well as nearly all known classes of small ncRNAs, some of which have never before been seen in plasma. Surprisingly, many of the small ncRNA species were present as full-length transcripts, suggesting that they are protected from plasma RNases in ribonucleoprotein (RNP) complexes and/or exosomes. This TGIRT-seq method is readily adaptable for profiling of whole-cell, exosomal, and miRNAs, and for related procedures, such as HITS-CLIP and ribosome profiling. PMID:26554030

  18. High-throughput sequencing of human plasma RNA by using thermostable group II intron reverse transcriptases.

    PubMed

    Qin, Yidan; Yao, Jun; Wu, Douglas C; Nottingham, Ryan M; Mohr, Sabine; Hunicke-Smith, Scott; Lambowitz, Alan M

    2016-01-01

    Next-generation RNA-sequencing (RNA-seq) has revolutionized transcriptome profiling, gene expression analysis, and RNA-based diagnostics. Here, we developed a new RNA-seq method that exploits thermostable group II intron reverse transcriptases (TGIRTs) and used it to profile human plasma RNAs. TGIRTs have higher thermostability, processivity, and fidelity than conventional reverse transcriptases, plus a novel template-switching activity that can efficiently attach RNA-seq adapters to target RNA sequences without RNA ligation. The new TGIRT-seq method enabled construction of RNA-seq libraries from <1 ng of plasma RNA in <5 h. TGIRT-seq of RNA in 1-mL plasma samples from a healthy individual revealed RNA fragments mapping to a diverse population of protein-coding gene and long ncRNAs, which are enriched in intron and antisense sequences, as well as nearly all known classes of small ncRNAs, some of which have never before been seen in plasma. Surprisingly, many of the small ncRNA species were present as full-length transcripts, suggesting that they are protected from plasma RNases in ribonucleoprotein (RNP) complexes and/or exosomes. This TGIRT-seq method is readily adaptable for profiling of whole-cell, exosomal, and miRNAs, and for related procedures, such as HITS-CLIP and ribosome profiling. © 2015 Qin et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  19. Transcription Regulation of the Human Telomerase Reverse Transcriptase (hTERT) Gene

    PubMed Central

    Ramlee, Muhammad Khairul; Wang, Jing; Toh, Wei Xun; Li, Shang

    2016-01-01

    Embryonic stem cells and induced pluripotent stem cells have the ability to maintain their telomere length via expression of an enzymatic complex called telomerase. Similarly, more than 85%–90% of cancer cells are found to upregulate the expression of telomerase, conferring them with the potential to proliferate indefinitely. Telomerase Reverse Transcriptase (TERT), the catalytic subunit of telomerase holoenzyme, is the rate-limiting factor in reconstituting telomerase activity in vivo. To date, the expression and function of the human Telomerase Reverse Transcriptase (hTERT) gene are known to be regulated at various molecular levels (including genetic, mRNA, protein and subcellular localization) by a number of diverse factors. Among these means of regulation, transcription modulation is the most important, as evident in its tight regulation in cancer cell survival as well as pluripotent stem cell maintenance and differentiation. Here, we discuss how hTERT gene transcription is regulated, mainly focusing on the contribution of trans-acting factors such as transcription factors and epigenetic modifiers, as well as genetic alterations in hTERT proximal promoter. PMID:27548225

  20. [Research progress of dual inhibitors targeting HIV-1 reverse transcriptase and integrase].

    PubMed

    Liu, Hong; Zhan, Peng; Liu, Xin-Yong

    2013-04-01

    Both reverse transcriptase (RT) and integrase (IN) play crucial roles in the life cycle of HIV-1, which are also key targets in the area of anti-HIV drug research. Reverse transcriptase inhibitors are involved in the most employed drugs used to treat AIDS patients and HIV-infected people, while one of the integrase inhibitors has already been approved by US FDA to appear on the market. Great achievement has been made in the research on both, separately. Recently, much more attention of medicinal chemistry researchers has been attracted to the strategies of multi-target drugs. Compounds with excellent potency against both HIV RT and IN, evidently defined as dual inhibitors targeting both enzymes, have been obtained through considerable significant exploration, which can be classified into two categories according to different strategies. Combinatorial chemistry approach together with high throughput screening methods and multi-target-based virtual screening strategy have been useful tools for identifying selective anti-HIV compounds for long times; Rational drug design based on pharmacophore combination has also led to remarkable results. In this paper, latest progress of both categories in the discovery and structural modification will be covered, with a view to contribute to the career of anti-HIV research.

  1. Structure of a Group II Intron Complexed with its Reverse Transcriptase

    PubMed Central

    Qu, Guosheng; Kaushal, Prem Singh; Wang, Jia; Shigematsu, Hideki; Piazza, Carol Lyn; Agrawal, Rajendra Kumar; Belfort, Marlene; Wang, Hong-Wei

    2016-01-01

    Bacterial group II introns are large catalytic RNAs related to nuclear spliceosomal introns and eukaryotic retrotransposons. They self-splice to yield mature RNA, and integrate into DNA as retroelements. A fully active group II intron forms a ribonucleoprotein complex comprising the intron ribozyme and an intron-encoded protein, with multiple activities including reverse transcriptase. This activity is responsible for copying the intron RNA into the DNA target. Here we report cryo-EM structures of an endogenously spliced Lactococcus lactis group IIA intron in its ribonucleoprotein complex form at 3.8 Å resolution and in its protein-depleted form at 4.5 Å resolution, revealing functional coordination of the intron RNA with the protein. Remarkably, the protein structure reveals a close relationship of the reverse transcriptase catalytic domain to telomerase, whereas the active center for splicing resembles the spliceosomal Prp8 protein. These extraordinary similarities hint at intricate ancestral relationships and provide new insights into splicing and retromobility. PMID:27136327

  2. Regulation of the reverse transcriptase of human immunodeficiency virus type 1 by dNTPs.

    PubMed Central

    West, A B; Roberts, T M; Kolodner, R D

    1992-01-01

    Reverse transcriptase (RNA-directed DNA polymerase, EC 2.7.7.49) of human immunodeficiency virus type 1 has been examined with respect to the steady-state kinetics of polymerization of dNTPs into product DNA. With dNTPs as variable substrate, the kinetics of polymerization deviated from standard Michaelis-Menten kinetics. Substrate inhibition was observed at high substrate concentrations and negative cooperativity was seen at lower substrate concentrations. Examination of incorporation of substrate dNMPs in the presence of nucleotides not complementing the template demonstrated that dNTPs may act as noncompetitive inhibitors, as well as substrate. The Ki of the enzyme for dNTPs was 104 microM. A working model is presented that accounts for the substrate inhibition. In this model, the reverse transcriptase is a multisubunit holoenzyme, where noncompetitive inhibition is mediated by one subunit binding nucleotide and down-regulating the enzymatically active 64-kDa subunit. With additional assumptions, this model can accommodate the negative cooperativity observed. Images PMID:1384060

  3. Parameterization of AZT-A widely used nucleoside inhibitor of HIV-1 reverse transcriptase

    NASA Astrophysics Data System (ADS)

    Carvalho, Alexandra T. P.; Fernandes, Pedro A.; Ramos, Maria J.

    Seven nucleoside reverse transcriptase (RT) inhibitors are currently used in the clinical treatment of acquired immunodeficiency syndrome (AIDS). These substrate analogues block DNA synthesis by the viral enzyme RT. However, the emergence of resistant variants of RT allied to their long-term toxicity requires the design of new and better RT inhibitors, with long-term in vivo efficacy. In this work we used density functional theory (DFT) calculations to develop a set of molecular mechanics (MM) parameters committed to the AMBER force field for one of the most used in the clinic nucleoside reverse transcriptase inhibitors (NRTIs): zidovudine (AZT). These parameters were tested by comparing the optimized geometries of AZT at both the DFT and MM levels of theory. The ability of the new parameters to reproduce the torsional energy of the azide group was also verified by scanning the surface in MM with the new parameters and comparing the results with the same potential energy surface (PES) at the DFT level. Finally, the parameters were validated through classical MD simulations of AZT in aqueous environment.

  4. A novel motif in telomerase reverse transcriptase regulates telomere repeat addition rate and processivity

    PubMed Central

    Xie, Mingyi; Podlevsky, Joshua D.; Qi, Xiaodong; Bley, Christopher J.; Chen, Julian J.-L.

    2010-01-01

    Telomerase is a specialized reverse transcriptase that adds telomeric DNA repeats onto chromosome termini. Here, we characterize a new telomerase-specific motif, called motif 3, in the catalytic domain of telomerase reverse transcriptase, that is crucial for telomerase function and evolutionally conserved between vertebrates and ciliates. Comprehensive mutagenesis of motif 3 identified mutations that remarkably increase the rate or alter the processivity of telomere repeat addition. Notably, the rate and processivity of repeat addition are affected independently by separate motif 3 mutations. The processive telomerase action relies upon a template translocation mechanism whereby the RNA template and the telomeric DNA strand separate and realign between each repeat synthesis. By analyzing the mutant telomerases reconstituted in vitro and in cells, we show that the hyperactive mutants exhibit higher repeat addition rates and faster enzyme turnovers, suggesting higher rates of strand-separation during template translocation. In addition, the strong correlation between the processivity of the motif 3 mutants and their ability to use an 8 nt DNA primer, suggests that motif 3 facilitates realignment between the telomeric DNA and the template RNA following strand-separation. These findings support motif 3 as a key determinant for telomerase activity and processivity. PMID:20044353

  5. Fidelity of classwide-resistant HIV-2 reverse transcriptase and differential contribution of K65R to the accuracy of HIV-1 and HIV-2 reverse transcriptases

    PubMed Central

    Álvarez, Mar; Sebastián-Martín, Alba; García-Marquina, Guillermo; Menéndez-Arias, Luis

    2017-01-01

    Nucleoside reverse transcriptase (RT) inhibitors constitute the backbone of current therapies against human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2, respectively). However, mutational pathways leading to the development of nucleoside analogue resistance are different in both types of HIV. In HIV-2, resistance to all approved nucleoside analogues is conferred by the combination of RT substitutions K65R, Q151M and M184V. Nucleotide incorporation kinetic analyses of mutant and wild-type (WT) HIV-2 RTs show that the triple-mutant has decreased catalytic efficiency due to the presence of M184V. Although similar effects were previously reported for equivalent mutations in HIV-1 RT, the HIV-2 enzymes were catalytically less efficient. Interestingly, in highly divergent HIV-1 RTs, K65R confers several-fold increased accuracy of DNA synthesis. We have determined the intrinsic fidelity of DNA synthesis of WT HIV-2 RT and mutants K65R and K65R/Q151M/M184V. Our results show that those changes in HIV-2 RT have a relatively small impact on nucleotide selectivity. Furthermore, we found that there were less than two-fold differences in error rates obtained with forward mutation assays using mutant and WT HIV-2 RTs. A different conformation of the β3-β4 hairpin loop in HIV-1 and HIV-2 RTs could probably explain the differential effects of K65R. PMID:28333133

  6. Fidelity of classwide-resistant HIV-2 reverse transcriptase and differential contribution of K65R to the accuracy of HIV-1 and HIV-2 reverse transcriptases.

    PubMed

    Álvarez, Mar; Sebastián-Martín, Alba; García-Marquina, Guillermo; Menéndez-Arias, Luis

    2017-03-23

    Nucleoside reverse transcriptase (RT) inhibitors constitute the backbone of current therapies against human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2, respectively). However, mutational pathways leading to the development of nucleoside analogue resistance are different in both types of HIV. In HIV-2, resistance to all approved nucleoside analogues is conferred by the combination of RT substitutions K65R, Q151M and M184V. Nucleotide incorporation kinetic analyses of mutant and wild-type (WT) HIV-2 RTs show that the triple-mutant has decreased catalytic efficiency due to the presence of M184V. Although similar effects were previously reported for equivalent mutations in HIV-1 RT, the HIV-2 enzymes were catalytically less efficient. Interestingly, in highly divergent HIV-1 RTs, K65R confers several-fold increased accuracy of DNA synthesis. We have determined the intrinsic fidelity of DNA synthesis of WT HIV-2 RT and mutants K65R and K65R/Q151M/M184V. Our results show that those changes in HIV-2 RT have a relatively small impact on nucleotide selectivity. Furthermore, we found that there were less than two-fold differences in error rates obtained with forward mutation assays using mutant and WT HIV-2 RTs. A different conformation of the β3-β4 hairpin loop in HIV-1 and HIV-2 RTs could probably explain the differential effects of K65R.

  7. Structure of a group II intron in complex with its reverse transcriptase.

    PubMed

    Qu, Guosheng; Kaushal, Prem Singh; Wang, Jia; Shigematsu, Hideki; Piazza, Carol Lyn; Agrawal, Rajendra Kumar; Belfort, Marlene; Wang, Hong-Wei

    2016-06-01

    Bacterial group II introns are large catalytic RNAs related to nuclear spliceosomal introns and eukaryotic retrotransposons. They self-splice, yielding mature RNA, and integrate into DNA as retroelements. A fully active group II intron forms a ribonucleoprotein complex comprising the intron ribozyme and an intron-encoded protein that performs multiple activities including reverse transcription, in which intron RNA is copied into the DNA target. Here we report cryo-EM structures of an endogenously spliced Lactococcus lactis group IIA intron in its ribonucleoprotein complex form at 3.8-Å resolution and in its protein-depleted form at 4.5-Å resolution, revealing functional coordination of the intron RNA with the protein. Remarkably, the protein structure reveals a close relationship between the reverse transcriptase catalytic domain and telomerase, whereas the active splicing center resembles the spliceosomal Prp8 protein. These extraordinary similarities hint at intricate ancestral relationships and provide new insights into splicing and retromobility.

  8. Expression of soluble, enzymatically active, human immunodeficiency virus reverse transcriptase in Escherichia coli and analysis of mutants.

    PubMed Central

    Hizi, A; McGill, C; Hughes, S H

    1988-01-01

    We have constructed a plasmid that, when introduced into Escherichia coli, induces the synthesis of large quantities of a protein with an apparent molecular mass of 66 kDa that differs from human immunodeficiency virus (HIV) RNA-dependent DNA polymerase (deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase or reverse transcriptase, EC 2.7.7.49) only in that it has two additional amino-terminal amino acids. This protein is soluble in E. coli extracts, is active in reverse transcriptase assays, and shows inhibition profiles with dideoxy-TTP and dideoxy-GTP that are indistinguishable from the viral enzyme. The deletion of 23 amino-terminal or carboxyl-terminal amino acids or the insertion of 5 amino acids at position 143 substantially decreases the polymerizing activity of the HIV reverse transcriptase made in E. coli. The properties of a 51-kDa reverse transcriptase-related protein made in E. coli suggests that the p51 found in the virion probably does not have substantial polymerizing activity. The full-length HIV reverse transcriptase and the various mutant proteins produced in E. coli should be quite useful for structural and biochemical analyses as well as for the production of antibodies. Images PMID:2448794

  9. Interactions of Murine Leukemia Virus Core Components: Characterization of Reverse Transcriptase Packaged in the Absence of 70S Genomic RNA

    PubMed Central

    Gerwin, Brenda I.; Levin, Judith G.

    1977-01-01

    Virions produced by cells in the presence of actinomycin D (Act D virions) contain reverse transcriptase but are deficient in 70S genomic RNA. To assess the role of genomic RNA in encapsidation of a functional reverse transcriptase and to study the interaction of the enzyme and its template in the cores of intact virions, the reverse transcriptase enzymes of normal and Act D virions were compared. The enzymes were indistinguishable by column chromatography, sedimentation velocity, or template/primer preferences. In addition, these enzymes showed equal sensitivity to inactivation by antibodies directed against Rauscher murine leukemia virus DNA polymerase. The enzymes from Act D and normal virions had similar thermal decay rates and were both protected against heat denaturation by natural and synthetic template/primers. By these criteria, the DNA polymerase molecules synthesized and assembled into virions in the absence of genomic RNA are identical to those packaged under normal conditions. Additional studies designed to measure protection of reverse transcriptase by genomic RNA were carried out by comparing the thermal lability of the enzyme in intact Act D and normal virions. The thermal decay rate of reverse transcriptase in Act D virions was identical to that in control virions. In contrast to the lability of the virion-associated enzyme, however, genomic RNA in control virions was stable to heat treatment. PMID:72160

  10. Interactions between avian myeloblastosis reverse transcriptase and tRNATrp. Mapping of complexed tRNA with chemicals and nucleases.

    PubMed

    Garret, M; Romby, P; Giegé, R; Litvak, S

    1984-03-12

    The interactions between beef tRNATrp with avian myeloblastosis reverse transcriptase have been studied by statistical chemical modifications of phosphate (ethylnitrosourea) and cytidine (dimethyl sulfate) residues, as well as by digestion of complexed tRNA by Cobra venom nuclease and Neurospora crassa endonuclease. Results with nucleases and chemicals show that reverse transcriptase interacts preferentially with the D arm, the anticodon stem and the T psi stem. All these regions are located in the outside of the L-shaped structure of tRNA. This domain of interaction is different to that reported previously in the complex of beef tRNA with the cognate aminoacyl-tRNA synthetase (M. Garret et al.; Eur. J. Biochem. In press). Avian reverse transcriptase destabilizes the region of tRNA where most of the tertiary interactions maintaining the structure of tRNA are located.

  11. Interactions between avian myeloblastosis reverse transcriptase and tRNATrp. Mapping of complexed tRNA with chemicals and nucleases.

    PubMed Central

    Garret, M; Romby, P; Giegé, R; Litvak, S

    1984-01-01

    The interactions between beef tRNATrp with avian myeloblastosis reverse transcriptase have been studied by statistical chemical modifications of phosphate (ethylnitrosourea) and cytidine (dimethyl sulfate) residues, as well as by digestion of complexed tRNA by Cobra venom nuclease and Neurospora crassa endonuclease. Results with nucleases and chemicals show that reverse transcriptase interacts preferentially with the D arm, the anticodon stem and the T psi stem. All these regions are located in the outside of the L-shaped structure of tRNA. This domain of interaction is different to that reported previously in the complex of beef tRNA with the cognate aminoacyl-tRNA synthetase (M. Garret et al.; Eur. J. Biochem. In press). Avian reverse transcriptase destabilizes the region of tRNA where most of the tertiary interactions maintaining the structure of tRNA are located. Images PMID:6200830

  12. RNA-seq of human reference RNA samples using a thermostable group II intron reverse transcriptase.

    PubMed

    Nottingham, Ryan M; Wu, Douglas C; Qin, Yidan; Yao, Jun; Hunicke-Smith, Scott; Lambowitz, Alan M

    2016-04-01

    Next-generation RNA sequencing (RNA-seq) has revolutionized our ability to analyze transcriptomes. Current RNA-seq methods are highly reproducible, but each has biases resulting from different modes of RNA sample preparation, reverse transcription, and adapter addition, leading to variability between methods. Moreover, the transcriptome cannot be profiled comprehensively because highly structured RNAs, such as tRNAs and snoRNAs, are refractory to conventional RNA-seq methods. Recently, we developed a new method for strand-specific RNA-seq using thermostable group II intron reverse transcriptases (TGIRTs). TGIRT enzymes have higher processivity and fidelity than conventional retroviral reverse transcriptases plus a novel template-switching activity that enables RNA-seq adapter addition during cDNA synthesis without using RNA ligase. Here, we obtained TGIRT-seq data sets for well-characterized human RNA reference samples and compared them to previous data sets obtained for these RNAs by the Illumina TruSeq v2 and v3 methods. We find that TGIRT-seq recapitulates the relative abundance of human transcripts and RNA spike-ins in ribo-depleted, fragmented RNA samples comparably to non-strand-specific TruSeq v2 and better than strand-specific TruSeq v3. Moreover, TGIRT-seq is more strand specific than TruSeq v3 and eliminates sampling biases from random hexamer priming, which are inherent to TruSeq. The TGIRT-seq data sets also show more uniform 5' to 3' gene coverage and identify more splice junctions, particularly near the 5' ends of mRNAs, than do the TruSeq data sets. Finally, TGIRT-seq enables the simultaneous profiling of mRNAs and lncRNAs in the same RNA-seq experiment as structured small ncRNAs, including tRNAs, which are essentially absent with TruSeq.

  13. Similarities between long interspersed element-1 (LINE-1) reverse transcriptase and telomerase

    PubMed Central

    Kopera, Huira C.; Moldovan, John B.; Morrish, Tammy A.; Moran, John V.

    2011-01-01

    Long interspersed element-1 (LINE-1 or L1) retrotransposons encode two proteins (ORF1p and ORF2p) that contain activities required for conventional retrotransposition by a mechanism termed target-site primed reverse transcription. Previous experiments in XRCC4 or DNA protein kinase catalytic subunit-deficient CHO cell lines, which are defective for the nonhomologous end-joining DNA repair pathway, revealed an alternative endonuclease-independent (ENi) pathway for L1 retrotransposition. Interestingly, some ENi retrotransposition events in DNA protein kinase catalytic subunit-deficient cells are targeted to dysfunctional telomeres. Here we used an in vitro assay to detect L1 reverse transcriptase activity to demonstrate that wild-type or endonuclease-defective L1 ribonucleoprotein particles can use oligonucleotide adapters that mimic telomeric ends as primers to initiate the reverse transcription of L1 mRNA. Importantly, these ribonucleoprotein particles also contain a nuclease activity that can process the oligonucleotide adapters before the initiation of reverse transcription. Finally, we demonstrate that ORF1p is not strictly required for ENi retrotransposition at dysfunctional telomeres. Thus, these data further highlight similarities between the mechanism of ENi L1 retrotransposition and telomerase. PMID:21940498

  14. Characterization of Moloney murine leukaemia virus/avian myeloblastosis virus chimeric reverse transcriptases.

    PubMed

    Yasukawa, Kiyoshi; Mizuno, Masaki; Inouye, Kuniyo

    2009-03-01

    Reverse transcriptases (RTs) from Moloney murine leukaemia virus (MMLV) and avian myeloblastosis virus (AMV) contain all the fingers, palm, thumb, connection and RNase H domains. The fingers, palm and thumb domains are thought to be involved in the reverse transcription activity, and the RNase H domain is in the RNase H activity. In this study, we characterized four chimeric RTs which comprise one of the fingers, palm, thumb and RNase H domains originated from AMV RT and the other three and connection domains originated from MMLV RT. Unexpectedly, all chimeric RTs exhibited the same characteristics: their specific reverse transcription activities decreased to less than 0.1% of that of MMLV RT, while their specific RNase H activities were approximately 20% of that of MMLV RT. The decreases in the two activities of the chimeric RTs were ascribed to the decreases in k(cat). Based on that the reverse transcription activity of MMLV RT was impaired by substituting its RNase H domain with that from AMV RT, we propose that in MMLV RT, there might be an interaction between the fingers/palm/thumb domain and the RNase H domain.

  15. Similarities between long interspersed element-1 (LINE-1) reverse transcriptase and telomerase.

    PubMed

    Kopera, Huira C; Moldovan, John B; Morrish, Tammy A; Garcia-Perez, Jose Luis; Moran, John V

    2011-12-20

    Long interspersed element-1 (LINE-1 or L1) retrotransposons encode two proteins (ORF1p and ORF2p) that contain activities required for conventional retrotransposition by a mechanism termed target-site primed reverse transcription. Previous experiments in XRCC4 or DNA protein kinase catalytic subunit-deficient CHO cell lines, which are defective for the nonhomologous end-joining DNA repair pathway, revealed an alternative endonuclease-independent (ENi) pathway for L1 retrotransposition. Interestingly, some ENi retrotransposition events in DNA protein kinase catalytic subunit-deficient cells are targeted to dysfunctional telomeres. Here we used an in vitro assay to detect L1 reverse transcriptase activity to demonstrate that wild-type or endonuclease-defective L1 ribonucleoprotein particles can use oligonucleotide adapters that mimic telomeric ends as primers to initiate the reverse transcription of L1 mRNA. Importantly, these ribonucleoprotein particles also contain a nuclease activity that can process the oligonucleotide adapters before the initiation of reverse transcription. Finally, we demonstrate that ORF1p is not strictly required for ENi retrotransposition at dysfunctional telomeres. Thus, these data further highlight similarities between the mechanism of ENi L1 retrotransposition and telomerase.

  16. Human immunodeficiency virus fitness in vivo: calculations based on a single zidovudine resistance mutation at codon 215 of reverse transcriptase.

    PubMed

    Goudsmit, J; De Ronde, A; Ho, D D; Perelson, A S

    1996-08-01

    We monitored a subject newly infected with a zidovudine-resistant human immunodeficiency virus type 1 strain and found that in the absence of drug, the viral population with the resistance-conferring tyrosine (TAC) codon 215 of reverse transcriptase was gradually replaced. By using standard formulas to model the effects of selection at a single locus in an asexual haploid population, the relative fitness gain of the viral population with a single mutation at codon 215 creating a serine (TCC) was calculated. We concluded that a viral population with a serine at reverse transcriptase codon 215 conferring zidovudine sensitivity was between 0.4 and 2.3% more fit.

  17. A reverse transcriptase PCR technique for the detection and viability assessment of Kluyveromyces marxianus in yoghurt.

    PubMed

    Mayoral, María Belén; Martin, Rosario; Hernández, Pablo E; González, Isabel; García, Teresa

    2006-09-01

    A fast and sensitive reverse transcriptase PCR (RT-PCR) method was developed for the detection of viable Kluyveromyces marxianus in yoghurt. Yeast-specific primers were used with the RT-PCR to evaluate the suitability of 18S rRNA as a target for the detection of viable yeasts in pure culture and yoghurt. The RT-PCR assay was able to detect down to 10(2) CFU ml(-1) in yoghurt samples contaminated with viable yeast cells. Application of the RT-PCR method to commercial yoghurt samples demonstrated the utility of this technique for detection of low concentrations of viable yeast cells in naturally contaminated dairy products. The 18S rRNA molecule is an appropriate target for cell viability assessment because of its limited persistence after cell death and the resultant high level of sensitivity of the assay.

  18. Endogenous Telomerase Reverse Transcriptase N-Terminal Tagging Affects Human Telomerase Function at Telomeres In Vivo

    PubMed Central

    Chiba, Kunitoshi; Vogan, Jacob M.; Wu, Robert A.; Gill, Manraj S.; Zhang, Xiaozhu; Collins, Kathleen

    2016-01-01

    ABSTRACT Telomerase action at telomeres is essential for the immortal phenotype of stem cells and the aberrant proliferative potential of cancer cells. Insufficient telomere maintenance can cause stem cell and tissue failure syndromes, while increased telomerase levels are associated with tumorigenesis. Both pathologies can arise from only small perturbation of telomerase function. To analyze telomerase at its low endogenous expression level, we genetically engineered human pluripotent stem cells (hPSCs) to express various N-terminal fusion proteins of the telomerase reverse transcriptase from its endogenous locus. Using this approach, we found that these modifications can perturb telomerase function in hPSCs and cancer cells, resulting in telomere length defects. Biochemical analysis suggests that this defect is multileveled, including changes in expression and activity. These findings highlight the unknown complexity of telomerase structural requirements for expression and function in vivo. PMID:27872149

  19. The telomerase reverse transcriptase subunit from the dimorphic fungus Ustilago maydis.

    PubMed

    Bautista-España, Dolores; Anastacio-Marcelino, Estela; Horta-Valerdi, Guillermo; Celestino-Montes, Antonio; Kojic, Milorad; Negrete-Abascal, Erasmo; Reyes-Cervantes, Hortensia; Vázquez-Cruz, Candelario; Guzmán, Plinio; Sánchez-Alonso, Patricia

    2014-01-01

    In this study, we investigated the reverse transcriptase subunit of telomerase in the dimorphic fungus Ustilago maydis. This protein (Trt1) contains 1371 amino acids and all of the characteristic TERT motifs. Mutants created by disrupting trt1 had senescent traits, such as delayed growth, low replicative potential, and reduced survival, that were reminiscent of the traits observed in est2 budding yeast mutants. Telomerase activity was observed in wild-type fungus sporidia but not those of the disruption mutant. The introduction of a self-replicating plasmid expressing Trt1 into the mutant strain restored growth proficiency and replicative potential. Analyses of trt1 crosses in planta suggested that Trt1 is necessary for teliospore formation in homozygous disrupted diploids and that telomerase is haploinsufficient in heterozygous diploids. Additionally, terminal restriction fragment analysis in the progeny hinted at alternative survival mechanisms similar to those of budding yeast.

  20. Cancer-Specific Telomerase Reverse Transcriptase (TERT) Promoter Mutations: Biological and Clinical Implications

    PubMed Central

    Liu, Tiantian; Yuan, Xiaotian; Xu, Dawei

    2016-01-01

    The accumulated evidence has pointed to a key role of telomerase in carcinogenesis. As a RNA-dependent DNA polymerase, telomerase synthesizes telomeric DNA at the end of linear chromosomes, and attenuates or prevents telomere erosion associated with cell divisions. By lengthening telomeres, telomerase extends cellular life-span or even induces immortalization. Consistent with its functional activity, telomerase is silent in most human normal somatic cells while active only in germ-line, stem and other highly proliferative cells. In contrast, telomerase activation widely occurs in human cancer and the enzymatic activity is detectable in up to 90% of malignancies. Recently, hotspot point mutations in the regulatory region of the telomerase reverse transcriptase (TERT) gene, encoding the core catalytic component of telomerase, was identified as a novel mechanism to activate telomerase in cancer. This review discusses the cancer-specific TERT promoter mutations and potential biological and clinical significances. PMID:27438857

  1. The Telomerase Reverse Transcriptase Subunit from the Dimorphic Fungus Ustilago maydis

    PubMed Central

    Horta-Valerdi, Guillermo; Celestino-Montes, Antonio; Kojic, Milorad; Negrete-Abascal, Erasmo; Reyes-Cervantes, Hortensia; Vázquez-Cruz, Candelario; Guzmán, Plinio; Sánchez-Alonso, Patricia

    2014-01-01

    In this study, we investigated the reverse transcriptase subunit of telomerase in the dimorphic fungus Ustilago maydis. This protein (Trt1) contains 1371 amino acids and all of the characteristic TERT motifs. Mutants created by disrupting trt1 had senescent traits, such as delayed growth, low replicative potential, and reduced survival, that were reminiscent of the traits observed in est2 budding yeast mutants. Telomerase activity was observed in wild-type fungus sporidia but not those of the disruption mutant. The introduction of a self-replicating plasmid expressing Trt1 into the mutant strain restored growth proficiency and replicative potential. Analyses of trt1 crosses in planta suggested that Trt1 is necessary for teliospore formation in homozygous disrupted diploids and that telomerase is haploinsufficient in heterozygous diploids. Additionally, terminal restriction fragment analysis in the progeny hinted at alternative survival mechanisms similar to those of budding yeast. PMID:25299159

  2. Single-molecule imaging of telomerase reverse transcriptase in human telomerase holoenzyme and minimal RNP complexes.

    PubMed

    Wu, Robert Alexander; Dagdas, Yavuz S; Yilmaz, S Tunc; Yildiz, Ahmet; Collins, Kathleen

    2015-10-12

    Telomerase synthesizes chromosome-capping telomeric repeats using an active site in telomerase reverse transcriptase (TERT) and an integral RNA subunit template. The fundamental question of whether human telomerase catalytic activity requires cooperation across two TERT subunits remains under debate. In this study, we describe new approaches of subunit labeling for single-molecule imaging, applied to determine the TERT content of complexes assembled in cells or cell extract. Surprisingly, telomerase reconstitutions yielded heterogeneous DNA-bound TERT monomer and dimer complexes in relative amounts that varied with assembly and purification method. Among the complexes, cellular holoenzyme and minimal recombinant enzyme monomeric for TERT had catalytic activity. Dimerization was suppressed by removing a TERT domain linker with atypical sequence bias, which did not inhibit cellular or minimal enzyme assembly or activity. Overall, this work defines human telomerase DNA binding and synthesis properties at single-molecule level and establishes conserved telomerase subunit architecture from single-celled organisms to humans.

  3. Endogenous Telomerase Reverse Transcriptase N-Terminal Tagging Affects Human Telomerase Function at Telomeres In Vivo.

    PubMed

    Chiba, Kunitoshi; Vogan, Jacob M; Wu, Robert A; Gill, Manraj S; Zhang, Xiaozhu; Collins, Kathleen; Hockemeyer, Dirk

    2017-02-01

    Telomerase action at telomeres is essential for the immortal phenotype of stem cells and the aberrant proliferative potential of cancer cells. Insufficient telomere maintenance can cause stem cell and tissue failure syndromes, while increased telomerase levels are associated with tumorigenesis. Both pathologies can arise from only small perturbation of telomerase function. To analyze telomerase at its low endogenous expression level, we genetically engineered human pluripotent stem cells (hPSCs) to express various N-terminal fusion proteins of the telomerase reverse transcriptase from its endogenous locus. Using this approach, we found that these modifications can perturb telomerase function in hPSCs and cancer cells, resulting in telomere length defects. Biochemical analysis suggests that this defect is multileveled, including changes in expression and activity. These findings highlight the unknown complexity of telomerase structural requirements for expression and function in vivo. Copyright © 2017 American Society for Microbiology.

  4. Theoretical investigation on nevirapine and HIV-1 reverse transcriptase binding site interaction, based on ONIOM method

    NASA Astrophysics Data System (ADS)

    Kuno, Mayuso; Hannongbua, Supa; Morokuma, Keiji

    2003-10-01

    The ONIOM method was applied to the interaction of nevirapine with the HIV-1 reverse transcriptase binding site. The isolated complex of pyridine (part of nevirapine) and methyl phenol (part of Tyr181) was found at the MP2/6-31+G(d) level to have stacking interaction with 8.8 kcal/mol binding energy. Optimization of nevirapine and Tyr181 geometry in the pocket of 16 amino acid residues at the ONIOM3(MP2/6-31G(d):HF/3-21G:PM3) level gave the complex structure with weak hydrogen bonding but without stacking interaction. The binding energy of 8.9 kcal/mol comes almost entirely from the interaction of nevirapine with amino acid residues other than Tyr181.

  5. Potent NLRP3 Inflammasome Activation by the HIV Reverse Transcriptase Inhibitor Abacavir.

    PubMed

    Toksoy, Atiye; Sennefelder, Helga; Adam, Christian; Hofmann, Sonja; Trautmann, Axel; Goebeler, Matthias; Schmidt, Marc

    2017-02-17

    There is experimental and clinical evidence that some exanthematous allergic drug hypersensitivity reactions are mediated by drug-specific T cells. We hypothesized that the capacity of certain drugs to directly stimulate the innate immune system may contribute to generate drug-specific T cells. Here we analyzed whether abacavir, an HIV-1 reverse transcriptase inhibitor often inducing severe delayed-type drug hypersensitivity, can trigger innate immune activation that may contribute to its allergic potential. We show that abacavir fails to generate direct innate immune activation in human monocytes but potently triggers IL-1β release upon pro-inflammatory priming with phorbol ester or Toll-like receptor stimulation. IL-1β processing and secretion were sensitive to Caspase-1 inhibition, NLRP3 knockdown, and K(+) efflux inhibition and were not observed with other non-allergenic nucleoside reverse transcriptase inhibitors, identifying abacavir as a specific inflammasome activator. It further correlated with dose-dependent mitochondrial reactive oxygen species production and cytotoxicity, indicating that inflammasome activation resulted from mitochondrial damage. However, both NLRP3 depletion and inhibition of K(+) efflux mitigated abacavir-induced mitochondrial reactive oxygen species production and cytotoxicity, suggesting that these processes were secondary to NLRP3 activation. Instead, depletion of cardiolipin synthase 1 abolished abacavir-induced IL-1β secretion, suggesting that mitochondrial cardiolipin release may trigger abacavir-induced inflammasome activation. Our data identify abacavir as a novel inflammasome-stimulating drug allergen. They implicate a potential contribution of innate immune activation to medication-induced delayed-type hypersensitivity, which may stimulate new concepts for treatment and prevention of drug allergies.

  6. Topical nonnucleoside reverse transcriptase inhibitor MC 1220 partially prevents vaginal RT-SHIV infection of macaques.

    PubMed

    Stolte-Leeb, Nicole; Loddo, Roberta; Antimisiaris, Sophia; Schultheiss, Tina; Sauermann, Ulrike; Franz, Monika; Mourtas, Spyridon; Parsy, Christophe; Storer, Richard; La Colla, Paolo; Stahl-Hennig, Christiane

    2011-09-01

    The availability of an effective vaginal microbicide would be a major step toward containment of HIV transmission as well as allowing women self-protection against HIV infection. Here we evaluated the efficacy of vaginal application of the potent nonnucleoside reverse transcriptase inhibitor (NNRTI) MC 1220 against vaginal challenge of macaques with RT-SHIV, a chimeric simian immunodeficiency virus (SIV) containing the reverse transcriptase (RT) gene of HIV-1. Challenge infection of monkeys with RT-SHIV currently represents the only nonhuman primate model available to test the anti-HIV-1 effects of NNRTIs. Two different gel formulations containing different MC 1220 concentrations were evaluated for efficacy in female rhesus macaques exposed to RT-SHIV. Five groups of five animals each were treated with two different gel compositions containing no drug, 0.1% or 0.5% MC 1220, followed by vaginal RT-SHIV challenge 30 min later. One animal in each group treated with the low concentration of MC 1220 as well as one control animal remained uninfected after vaginal challenge. By contrast, three of the animals receiving 0.5% MC 1220 remained uninfected, suggesting a threshold of the drug. Despite being negative for plasma viral RNA and absence of seroconversion, almost all uninfected animals exhibited SIV-specific T cells, either in the periphery or in lymph nodes draining the portal of virus entry. Our results make MC 1220 a promising compound for further development as a topical microbicide and warrant additional testing with improved formulation, long-lasting vaginal delivery systems, or even combinations with other inhibitors.

  7. A Non-Canonical Function of Zebrafish Telomerase Reverse Transcriptase Is Required for Developmental Hematopoiesis

    PubMed Central

    Koshimizu, Eriko; Hanai, Jun-ichi; Raftopoulou, Christina; Murphey, Ryan D.; Bayliss, Peter E.; Imai, Yoichi; Burns, Caroline Erter; Masutomi, Kenkichi; Gagos, Sarantis; Zon, Leonard I.; Roberts, Thomas M.; Kishi, Shuji

    2008-01-01

    Although it is clear that telomerase expression is crucial for the maintenance of telomere homeostasis, there is increasing evidence that the TERT protein can have physiological roles that are independent of this central function. To further examine the role of telomerase during vertebrate development, the zebrafish telomerase reverse transcriptase (zTERT) was functionally characterized. Upon zTERT knockdown, zebrafish embryos show reduced telomerase activity and are viable, but develop pancytopenia resulting from aberrant hematopoiesis. The blood cell counts in TERT-depleted zebrafish embryos are markedly decreased and hematopoietic cell differentiation is impaired, whereas other somatic lineages remain morphologically unaffected. Although both primitive and definitive hematopoiesis is disrupted by zTERT knockdown, the telomere lengths are not significantly altered throughout early development. Induced p53 deficiency, as well as overexpression of the anti-apoptotic proteins Bcl-2 and E1B-19K, significantly relieves the decreased blood cells numbers caused by zTERT knockdown, but not the impaired blood cell differentiation. Surprisingly, only the reverse transcriptase motifs of zTERT are crucial, but the telomerase RNA-binding domain of zTERT is not required, for rescuing complete hematopoiesis. This is therefore the first demonstration of a non-canonical catalytic activity of TERT, which is different from “authentic” telomerase activity, is required for during vertebrate hematopoiesis. On the other hand, zTERT deficiency induced a defect in hematopoiesis through a potent and specific effect on the gene expression of key regulators in the absence of telomere dysfunction. These results suggest that TERT non-canonically functions in hematopoietic cell differentiation and survival in vertebrates, independently of its role in telomere homeostasis. The data also provide insights into a non-canonical pathway by which TERT functions to modulate specification of

  8. A Novel Molecular Mechanism of Dual Resistance to Nucleoside and Nonnucleoside Reverse Transcriptase Inhibitors ▿

    PubMed Central

    Nikolenko, Galina N.; Delviks-Frankenberry, Krista A.; Pathak, Vinay K.

    2010-01-01

    Recently, mutations in the connection subdomain (CN) and RNase H domain of HIV-1 reverse transcriptase (RT) were observed to exhibit dual resistance to nucleoside and nonnucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs). To elucidate the mechanism by which CN and RH mutations confer resistance to NNRTIs, we hypothesized that these mutations reduce RNase H cleavage and provide more time for the NNRTI to dissociate from the RT, resulting in the resumption of DNA synthesis and enhanced NNRTI resistance. We observed that the effect of the reduction in RNase H cleavage on NNRTI resistance is dependent upon the affinity of each NNRTI to the RT and further influenced by the presence of NNRTI-binding pocket (BP) mutants. D549N, Q475A, and Y501A mutants, which reduce RNase H cleavage, enhance resistance to nevirapine (NVP) and delavirdine (DLV), but not to efavirenz (EFV) and etravirine (ETR), consistent with their increase in affinity for RT. Combining the D549N mutant with NNRTI BP mutants further increases NNRTI resistance from 3- to 30-fold, supporting the role of NNRTI-RT affinity in our NNRTI resistance model. We also demonstrated that CNs from treatment-experienced patients, previously reported to enhance NRTI resistance, also reduce RNase H cleavage and enhance NNRTI resistance in the context of the patient RT pol domain or a wild-type pol domain. Together, these results confirm key predictions of our NNRTI resistance model and provide support for a unifying mechanism by which CN and RH mutations can exhibit dual NRTI and NNRTI resistance. PMID:20219933

  9. Energetics of mutation-induced changes in potency of lersivirine against HIV-1 reverse transcriptase.

    PubMed

    Kar, Parimal; Knecht, Volker

    2012-06-07

    Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are key components of highly active antiretroviral therapy for the treatment of HIV-1. A common problem with the first generation NNRTIs is the emergence of mutations in the HIV-1 reverse transcriptase (RT), in particular, K103N and Y181C, which lead to resistance to the entire class of inhibitor. Here we have evaluated the relative affinity of the newly designed NNRTI lersivirine (LRV) against drug-resistant mutations in HIV-1 RT using the molecular mechanics generalized Born surface area (MM-GBSA) method. Eight single and one double mutant variants of RT are considered. Our results are in good agreement with experimental results and yield insights into the mechanisms underlying mutation-induced changes in the potency of LRV against RT. The strongest (54-fold) increase in the dissociation constant is found for the mutant F227C, originating from reduced electrostatic and van der Waals interactions between LRV and RT as well as a higher energetic penalty from the desolvation of polar groups. For the mutants K103N and Y181C only a moderate (2-fold) increase in the dissociation constant is found, due to a balance of opposite changes in the polar solvation as well as the electrostatic and van der Waals interactions between LRV and RT. The dissociation constant is decreased for the Y188C and G190A (2-fold), the M184V (5-fold), and the Y188C/Y188C mutant (10-fold), due to stronger electrostatic interactions between LRV and RT. Our results thus suggest that LRV is a highly potent and selective NNRTI, with excellent efficacy against NNRTI-resistant viruses, which is in agreement with experimental observations.

  10. Antiviral Activity of GW678248, a Novel Benzophenone Nonnucleoside Reverse Transcriptase Inhibitor

    PubMed Central

    Ferris, Robert G.; Hazen, Richard J.; Roberts, Grace B.; St. Clair, Marty H.; Chan, Joseph H.; Romines, Karen R.; Freeman, George A.; Tidwell, Jeffrey H.; Schaller, Lee T.; Cowan, Jill R.; Short, Steven A.; Weaver, Kurt L.; Selleseth, Dean W.; Moniri, Kelly R.; Boone, Lawrence R.

    2005-01-01

    The compound GW678248 is a novel benzophenone nonnucleoside reverse transcriptase inhibitor (NNRTI). Preclinical assessment of GW678248 indicates that this compound potently inhibits wild-type (WT) and mutant human immunodeficiency virus type 1 (HIV-1) reverse transcriptase in biochemical assays, with 50% inhibitory concentrations (IC50s) between 0.8 and 6.8 nM. In HeLa CD4 MAGI cell culture virus replication assays, GW678248 has an IC50 of ≤21 nM against HIV-1 isogenic strains with single or double mutations known to be associated with NNRTI resistance, including L100I, K101E, K103N, V106A/I/M, V108I, E138K, Y181C, Y188C, Y188L, G190A/E, P225H, and P236L and various combinations. An IC50 of 86 nM was obtained with a mutant virus having V106I, E138K, and P236L mutations that resulted from serial passage of WT virus in the presence of GW678248. The presence of 45 mg/ml human serum albumin plus 1 mg/ml α-1 acid glycoprotein increased the IC50 approximately sevenfold. Cytotoxicity studies with GW678248 indicate that the 50% cytotoxicity concentration is greater than the level of compound solubility and provides a selectivity index of >2,500-fold for WT, Y181C, or K103N HIV-1. This compound exhibits excellent preclinical antiviral properties and, as a prodrug designated GW695634, is being developed as a new generation of NNRTI for the treatment of HIV-1 in combination with other antiretroviral agents. PMID:16189079

  11. Rapid and Simultaneous Detection of Major Drug Resistance Mutations in Reverse Transcriptase Gene for HIV-1 CRF01_AE, CRF07_BC and Subtype B in China Using Sequenom MassARRAY® System.

    PubMed

    Cheung, Ka-Wai; Peng, Qiaoli; He, Liufen; Cai, Kanru; Jiang, Qiang; Zhou, Boping; To, Sabrina Wai-Chi; Yam, Wing-Cheong; Liu, Li; Chen, Zhiwei; Wang, Hui

    2016-01-01

    The development of a rapid, high-throughput and cost-effective HIV-1 drug resistance (HIV-DR) testing system is a challenge for areas consisting different HIV-1 strains. In this study, we established a broadly reactive multiplex assay that could simultaneously detect major drug resistance mutations at 8 loci, which are associated with resistance to commonly used nucleoside reverse transcriptase inhibitors (NRTIs) and Non-nucleoside reverse transcriptase inhibitors (NNRTIs), in specimens of HIV-1 CRF01_AE, CRF07_BC and subtype B, the three major circulating strains in China, using the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) provided by Sequenom MassARRAY® system. To establish the assay, pol gene fragments were prepared from the plasma viral RNA of 159 patients by nested PCR and the presence of wild type and mutant alleles at the 8 loci were analyzed by MALDI-TOF MS. In terms of loci, the detection rate of the alleles was greater than 97% for M41L, K65R, M184V and G190A, 91.2% for K101E/Q/P, 91.2% for T215F/Y, 89.9% for K103N/S and 80.5% for L210W. In terms of individuals, 80% of the alleles were detected in 95.4% CRF01_AE patients, 100% CRF07_BC patients and 83.3% subtype B patients. Importantly, the MALDI-TOF MS results were concordant to the drug resistance profiles of patients obtained from conventional sequencing analysis after excluded the failed detections. Using plasmid templates, the assay was estimated to be sensitive to detect drug resistant variants at level about 20% of the circulating viral population. The capability of this assay to detect mixed viral populations was further verified by two different patient specimens. In conclusion, this study evaluated the use of Sequenom MassARRAY® system for high-throughput detection of HIV-DR mutations towards the commonly used reverse transcriptase inhibitors in China.

  12. Rapid and Simultaneous Detection of Major Drug Resistance Mutations in Reverse Transcriptase Gene for HIV-1 CRF01_AE, CRF07_BC and Subtype B in China Using Sequenom MassARRAY® System

    PubMed Central

    He, Liufen; Cai, Kanru; Jiang, Qiang; Zhou, Boping; To, Sabrina Wai-Chi; Yam, Wing-Cheong; Liu, Li; Chen, Zhiwei; Wang, Hui

    2016-01-01

    The development of a rapid, high-throughput and cost-effective HIV-1 drug resistance (HIV-DR) testing system is a challenge for areas consisting different HIV-1 strains. In this study, we established a broadly reactive multiplex assay that could simultaneously detect major drug resistance mutations at 8 loci, which are associated with resistance to commonly used nucleoside reverse transcriptase inhibitors (NRTIs) and Non-nucleoside reverse transcriptase inhibitors (NNRTIs), in specimens of HIV-1 CRF01_AE, CRF07_BC and subtype B, the three major circulating strains in China, using the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) provided by Sequenom MassARRAY® system. To establish the assay, pol gene fragments were prepared from the plasma viral RNA of 159 patients by nested PCR and the presence of wild type and mutant alleles at the 8 loci were analyzed by MALDI-TOF MS. In terms of loci, the detection rate of the alleles was greater than 97% for M41L, K65R, M184V and G190A, 91.2% for K101E/Q/P, 91.2% for T215F/Y, 89.9% for K103N/S and 80.5% for L210W. In terms of individuals, 80% of the alleles were detected in 95.4% CRF01_AE patients, 100% CRF07_BC patients and 83.3% subtype B patients. Importantly, the MALDI-TOF MS results were concordant to the drug resistance profiles of patients obtained from conventional sequencing analysis after excluded the failed detections. Using plasmid templates, the assay was estimated to be sensitive to detect drug resistant variants at level about 20% of the circulating viral population. The capability of this assay to detect mixed viral populations was further verified by two different patient specimens. In conclusion, this study evaluated the use of Sequenom MassARRAY® system for high-throughput detection of HIV-DR mutations towards the commonly used reverse transcriptase inhibitors in China. PMID:27092551

  13. Emergent HIV-1 Drug Resistance Mutations Were Not Present at Low-Frequency at Baseline in Non-Nucleoside Reverse Transcriptase Inhibitor-Treated Subjects in the STaR Study.

    PubMed

    Porter, Danielle P; Daeumer, Martin; Thielen, Alexander; Chang, Silvia; Martin, Ross; Cohen, Cal; Miller, Michael D; White, Kirsten L

    2015-12-07

    At Week 96 of the Single-Tablet Regimen (STaR) study, more treatment-naïve subjects that received rilpivirine/emtricitabine/tenofovir DF (RPV/FTC/TDF) developed resistance mutations compared to those treated with efavirenz (EFV)/FTC/TDF by population sequencing. Furthermore, more RPV/FTC/TDF-treated subjects with baseline HIV-1 RNA >100,000 copies/mL developed resistance compared to subjects with baseline HIV-1 RNA ≤100,000 copies/mL. Here, deep sequencing was utilized to assess the presence of pre-existing low-frequency variants in subjects with and without resistance development in the STaR study. Deep sequencing (Illumina MiSeq) was performed on baseline and virologic failure samples for all subjects analyzed for resistance by population sequencing during the clinical study (n = 33), as well as baseline samples from control subjects with virologic response (n = 118). Primary NRTI or NNRTI drug resistance mutations present at low frequency (≥2% to 20%) were detected in 6.6% of baseline samples by deep sequencing, all of which occurred in control subjects. Deep sequencing results were generally consistent with population sequencing but detected additional primary NNRTI and NRTI resistance mutations at virologic failure in seven samples. HIV-1 drug resistance mutations emerging while on RPV/FTC/TDF or EFV/FTC/TDF treatment were not present at low frequency at baseline in the STaR study.

  14. Emergent HIV-1 Drug Resistance Mutations Were Not Present at Low-Frequency at Baseline in Non-Nucleoside Reverse Transcriptase Inhibitor-Treated Subjects in the STaR Study

    PubMed Central

    Porter, Danielle P.; Daeumer, Martin; Thielen, Alexander; Chang, Silvia; Martin, Ross; Cohen, Cal; Miller, Michael D.; White, Kirsten L.

    2015-01-01

    At Week 96 of the Single-Tablet Regimen (STaR) study, more treatment-naïve subjects that received rilpivirine/emtricitabine/tenofovir DF (RPV/FTC/TDF) developed resistance mutations compared to those treated with efavirenz (EFV)/FTC/TDF by population sequencing. Furthermore, more RPV/FTC/TDF-treated subjects with baseline HIV-1 RNA >100,000 copies/mL developed resistance compared to subjects with baseline HIV-1 RNA ≤100,000 copies/mL. Here, deep sequencing was utilized to assess the presence of pre-existing low-frequency variants in subjects with and without resistance development in the STaR study. Deep sequencing (Illumina MiSeq) was performed on baseline and virologic failure samples for all subjects analyzed for resistance by population sequencing during the clinical study (n = 33), as well as baseline samples from control subjects with virologic response (n = 118). Primary NRTI or NNRTI drug resistance mutations present at low frequency (≥2% to 20%) were detected in 6.6% of baseline samples by deep sequencing, all of which occurred in control subjects. Deep sequencing results were generally consistent with population sequencing but detected additional primary NNRTI and NRTI resistance mutations at virologic failure in seven samples. HIV-1 drug resistance mutations emerging while on RPV/FTC/TDF or EFV/FTC/TDF treatment were not present at low frequency at baseline in the STaR study. PMID:26690199

  15. Sensitive detection of the K103N non-nucleoside reverse transcriptase inhibitor resistance mutation in treatment-naïve HIV-1 infected individuals by rolling circle amplification.

    PubMed

    Wang, Bin; Dwyer, Dominic E; Chew, Choo Beng; Kol, Chenda; He, Zhong Ping; Joshi, Hemal; Steain, Megan C; Cunningham, Anthony L; Saksena, Nitin K

    2009-10-01

    Primary or transmitted antiretroviral drug resistance mutations pose a significant obstacle for optimizing antiviral treatment. When present at low-levels, resistance mutations are less likely to be detected by standard genotyping assays. This study utilizes a novel rolling circle amplification (RCA) method using padlock probes to achieve the sensitive, specific and low-level detection of the NNRTI resistance K103N from 59 HIV+ treatment-naïve patients from Beijing, China. Using standard genotyping methods, primary drug resistance mutations to either protease or RT inhibitors were found in 25% (15/59) of patients attending hospital clinics in Beijing. Among these 15 patients with antiretroviral (ARV) resistance mutations, standard sequence-based genotyping revealed that most (10/15) had the 103N. Using a highly sensitive RCA assay, 5 more patients among the 59 treatment-naïve cohort were found to have the 103N, but at low-levels, leading to an overall rate of 103N at 25.4% (15/59) in this population. The high prevalence of the 103N suggests that baseline resistance testing should be performed before treatment in this population. Importantly, the new RCA technology allows large-scale, sensitive detection of drug resistance mutations, including detection of minority populations with minimal equipment requirement.

  16. Synthesis and biological evaluation of novel 5-alkyl-2-arylthio-6-((3,4-dihydroquinolin-1(2H)-yl)methyl)pyrimidin-4(3H)-ones as potent non-nucleoside HIV-1 reverse transcriptase inhibitors.

    PubMed

    Zhang, Jing; Zhan, Peng; Wu, Jingde; Li, Zhenyu; Jiang, Yan; Ge, Weiying; Pannecouque, Christophe; De Clercq, Erik; Liu, Xinyong

    2011-07-15

    A series of novel S-DABO analogues of 5-alkyl-2-arylthio-6-((3,4-dihydroquinolin-1(2H)-yl)methyl)pyrimidin-4(3H)-ones were synthesized and evaluated as inhibitors of human immunodeficiency virus type-1 (HIV-1). Among them, the most potent HIV-1 inhibitors were compounds 6c1,6c6, and 6b1 (EC(50)=0.24 ± 0.05, 0.38 ± 0.13, 0.39 ± 0.05 μM, respectively), which possess improved or similar HIV-1 inhibitory activity compared with nevirapine (NVP) (EC(50)=0.21 μM) and delavirdine (DLV) (EC(50)=0.32 μM). None of these compounds were active against HIV-2 replication. Furthermore, enzyme inhibitory assays were performed with selected derivatives against HIV-1 wtRT, confirming that the main target of these compounds is the HIV-1 RT and these new S-DABOs are acting as NNRTIs. The preliminary structure-activity relationship (SAR) of these new congeners is discussed briefly and rationalized by docking studies.

  17. SINGLE-MOLECULE STUDY OF DNA POLYMERIZATION ACTIVITY OF HIV-1 REVERSE TRANSCRIPTASE ON DNA TEMPLATES

    PubMed Central

    Kim, Sangjin; Schroeder, Charles M.; Xie, X. Sunney

    2009-01-01

    Human Immunodeficiency Virus-1 reverse transcriptase (HIV-1 RT) is a multifunctional polymerase responsible for reverse transcription of the HIV genome, including DNA replication on both RNA and DNA templates. During reverse transcription in vivo, HIV-1 RT replicates through various secondary structures on RNA and single-stranded DNA templates without the need for a nucleic acid unwinding protein, such as a helicase. In order to understand the mechanism of polymerization through secondary structures, we investigated the DNA polymerization activity of HIV-1 RT on long single-stranded DNA templates using a multiplexed single-molecule DNA flow-stretching assay. We observed that HIV-1 RT performs fast primer extension DNA synthesis on single-stranded regions of DNA (18.7 nt/s) and switches its activity to slow strand displacement synthesis at DNA hairpin locations (2.3 nt/s). Furthermore, we found that the rate of strand displacement synthesis is dependent on the GC content in hairpin stems and template stretching force. This indicates that the strand displacement synthesis occurs through a mechanism that is neither completely active nor passive, i.e. the opening of the DNA hairpin is driven by a combination of free energy released during dNTP hydrolysis and thermal fraying of base pairs. Our experimental observations provide new insight into the interchanging modes of DNA replication by HIV-1 RT on long single-stranded DNA templates. PMID:19968999

  18. Single-molecule study of DNA polymerization activity of HIV-1 reverse transcriptase on DNA templates.

    PubMed

    Kim, Sangjin; Schroeder, Charles M; Xie, X Sunney

    2010-02-05

    HIV-1 RT (human immunodeficiency virus-1 reverse transcriptase) is a multifunctional polymerase responsible for reverse transcription of the HIV genome, including DNA replication on both RNA and DNA templates. During reverse transcription in vivo, HIV-1 RT replicates through various secondary structures on RNA and single-stranded DNA (ssDNA) templates without the need for a nucleic acid unwinding protein, such as a helicase. In order to understand the mechanism of polymerization through secondary structures, we investigated the DNA polymerization activity of HIV-1 RT on long ssDNA templates using a multiplexed single-molecule DNA flow-stretching assay. We observed that HIV-1 RT performs fast primer extension DNA synthesis on single-stranded regions of DNA (18.7 nt/s) and switches its activity to slow strand displacement synthesis at DNA hairpin locations (2.3 nt/s). Furthermore, we found that the rate of strand displacement synthesis is dependent on the GC content in hairpin stems and template stretching force. This indicates that the strand displacement synthesis occurs through a mechanism that is neither completely active nor passive: that is, the opening of the DNA hairpin is driven by a combination of free energy released during dNTP (deoxyribonucleotide triphosphate) hydrolysis and thermal fraying of base pairs. Our experimental observations provide new insight into the interchanging modes of DNA replication by HIV-1 RT on long ssDNA templates.

  19. AZT resistance of simian foamy virus reverse transcriptase is based on the excision of AZTMP in the presence of ATP

    PubMed Central

    Hartl, Maximilian J.; Kretzschmar, Benedikt; Frohn, Anne; Nowrouzi, Ali; Rethwilm, Axel; Wöhrl, Birgitta M.

    2008-01-01

    Azidothymidine (AZT, zidovudine) is one of the few nucleoside inhibitors known to inhibit foamy virus replication. We have shown previously that up to four mutations in the reverse transcriptase gene of simian foamy virus from macaque (SFVmac) are necessary to confer high resistance against AZT. To characterize the mechanism of AZT resistance we expressed two recombinant reverse transcriptases of highly AZT-resistant SFVmac in Escherichia coli harboring three (K211I, S345T, E350K) or four mutations (K211I, I224T, S345T, E350K) in the reverse transcriptase gene. Our analyses show that the polymerization activity of these mutants is impaired. In contrast to the AZT-resistant reverse transcriptase of HIV-1, the AZT resistant enzymes of SFVmac reveal differences in their kinetic properties. The SFVmac enzymes exhibit lower specific activities on poly(rA)/oligo(dT) and higher KM-values for polymerization but no change in KD-values for DNA/DNA or RNA/DNA substrates. The AZT resistance of the mutant enzymes is based on the excision of the incorporated inhibitor in the presence of ATP. The additional amino acid change of the quadruple mutant appears to be important for regaining polymerization efficiency. PMID:18096624

  20. Validation of a real-time reverse transcriptase-PCR assay for the detection of H7 avian influenza virus

    USDA-ARS?s Scientific Manuscript database

    A subtype specific H7 real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay developed by the Southeast Poultry Research Laboratory (SEPRL) for the detection of H7 in North and South American wild aquatic birds and poultry was validated as a collaborative effort by the SEPRL and Na...

  1. Use of propidium monoazide in reverse transcriptase PCR to distinguish between infectious and noninfectious enteric viruses in water samples

    EPA Science Inventory

    Human enteric viruses can be present in untreated and inadequately treated drinking water. Molecular methods, such as the reverse transcriptase PCR (RT-PCR), can detect viral genomes in a few hours, but they cannot distinguish between infectious and noninfectious viruses. Since o...

  2. The Need for Development of New HIV-1 Reverse Transcriptase and Integrase Inhibitors in the Aftermath of Antiviral Drug Resistance

    PubMed Central

    Wainberg, Mark A.

    2012-01-01

    The use of highly active antiretroviral therapy (HAART) involves combinations of drugs to achieve maximal virological response and reduce the potential for the emergence of antiviral resistance. There are two broad classes of reverse transcriptase inhibitors, the nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs). Since the first classes of such compounds were developed, viral resistance against them has necessitated the continuous development of novel compounds within each class. This paper considers the NRTIs and NNRTIs currently in both preclinical and clinical development or approved for second line therapy and describes the patterns of resistance associated with their use, as well as the underlying mechanisms that have been described. Due to reasons of both affordability and availability, some reverse transcriptase inhibitors with low genetic barrier are more commonly used in resource-limited settings. Their use results to the emergence of specific patterns of antiviral resistance and so may require specific actions to preserve therapeutic options for patients in such settings. More recently, the advent of integrase strand transfer inhibitors represents another major step forward toward control of HIV infection, but these compounds are also susceptible to problems of HIV drug resistance. PMID:24278679

  3. Use of propidium monoazide in reverse transcriptase PCR to distinguish between infectious and noninfectious enteric viruses in water samples

    EPA Science Inventory

    Human enteric viruses can be present in untreated and inadequately treated drinking water. Molecular methods, such as the reverse transcriptase PCR (RT-PCR), can detect viral genomes in a few hours, but they cannot distinguish between infectious and noninfectious viruses. Since o...

  4. Comparison of reverse transcriptase PCR, reverse transcriptase loop-mediated isothermal amplification, and culture-based assays for Salmonella detection from pork processing environments.

    PubMed

    Techathuvanan, Chayapa; Draughon, Frances Ann; D'Souza, Doris Helen

    2011-02-01

    Novel rapid Salmonella detection assays without the need for sophisticated equipment or labor remain in high demand. Real-time reverse transcriptase PCR (RT-PCR) assays, though rapid and sensitive, require expensive thermocyclers, while a novel RT loop-mediated isothermal amplification (RT-LAMP) method requires only a simple water bath. Our objective was to compare the detection sensitivity of Salmonella Typhimurium from the pork processing environment by RT-LAMP, RT-PCR, and culture-based assays. Carcass and surface swabs and carcass rinses were obtained from a local processing plant. Autoclaved carcass rinses (500 ml) were spiked with Salmonella Typhimurium and filtered. Filters were placed in stomacher bags containing tetrathionate broth (TTB) and analyzed with or without 10-h enrichment at 37 °C. Natural swabs were stomached with buffered peptone water, and natural carcass rinses were filtered, preenriched, and further enriched in TTB. Serially-diluted enriched samples were enumerated by spread plating on xylose lysine Tergitol 4 agar. RNA was extracted from 5 ml of enriched TTB with TRIzol. RT-LAMP assay using previously described invA primers was conducted at 62 °C for 90 min in a water bath with visual detection and by gel electrophoresis. SYBR Green I-based-real-time RT-PCR was carried out with invA primers followed by melt temperature analysis. The results of RT-LAMP detection for spiked carcass rinses were comparable to those of RT-PCR and cultural plating, with detection limits of 1 log CFU/ml, although they were obtained significantly faster, within 24 h including preenrichment and enrichment. RT-LAMP showed 4 of 12 rinse samples positive, while RT-PCR showed 1 of 12 rinse samples positive. For swabs, 6 of 27 samples positive by RT-LAMP and 5 of 27 by RT-PCR were obtained. This 1-day RT-LAMP assay shows promise for routine Salmonella screening by the pork industry.

  5. Dolutegravir Plus Two Nucleoside Reverse Transcriptase Inhibitors versus Efavirenz Plus Two Nucleoside Reverse Transcriptase Inhibitors As Initial Antiretroviral Therapy for People with HIV: A Systematic Review.

    PubMed

    Rutherford, George W; Horvath, Hacsi

    2016-01-01

    Dolutegravir (DTG) is a once-daily unboosted second-generation integrase-inhibitor that along with two nucleoside reverse transcriptase inhibitors is one of several regimens recommended by the United States, United Kingdom and European Union for first-line antiretroviral treatment of people with HIV infection. Our objective was to review the evidence for the efficacy and safety of DTG-based first-line regimens compared to efavirenz (EFV)-based regimens. We conducted a systematic review. We comprehensively searched a range of databases as well as conference abstracts and a trials registry. We used Cochrane methods in screening and data collection and assessed each study's risk of bias with the Cochrane tool. We meta-analyzed data using a fixed-effects model. We used GRADE to assess evidence quality. From 492 search results, we identified two randomized controlled trials, reported in five peer-reviewed articles and one conference abstract. One trial tested two DTG-based regimens (DTG + abacavir (ABC) + lamivudine (3TC) or DTG + tenofovir + emtricitabine) against an EFV-based regimen (EFV+ ABC+3TC). The other trial tested DTG+ABC+3TC against EFV+ABC+3TC. In meta-analysis, DTG-containing regimens were superior to EFV-containing regimens at 48 weeks and at 96 weeks (RR = 1.10, 95% CI 1.04-1.16; and RR = 1.12, 95% CI 1.04-1.21, respectively). In one trial, the DTG-containing regimen was superior at 144 weeks (RR = 1.13, 95% CI 1.02-1.24). DTG-containing regimens were superior in reducing treatment discontinuation compared to those containing EFV at 96 weeks and at 144 weeks (RR = 0.27, 95% CI 0.15-0.50; and RR = 0.28, 95% CI 0.16-0.48, respectively). Risk of serious adverse events was similar in each regimen at 96 weeks (RR = 1.15, 95% CI 0.80-1.63) and 144 weeks (RR = 0.93, 95% CI 0.68-1.29). Risk of bias was moderate overall, as was GRADE evidence quality. DTG-based regimens should be considered in future World Health Organization guidelines for initial HIV treatment.

  6. Dolutegravir Plus Two Nucleoside Reverse Transcriptase Inhibitors versus Efavirenz Plus Two Nucleoside Reverse Transcriptase Inhibitors As Initial Antiretroviral Therapy for People with HIV: A Systematic Review

    PubMed Central

    Rutherford, George W.; Horvath, Hacsi

    2016-01-01

    Background Dolutegravir (DTG) is a once-daily unboosted second-generation integrase-inhibitor that along with two nucleoside reverse transcriptase inhibitors is one of several regimens recommended by the United States, United Kingdom and European Union for first-line antiretroviral treatment of people with HIV infection. Our objective was to review the evidence for the efficacy and safety of DTG-based first-line regimens compared to efavirenz (EFV)-based regimens. Methods We conducted a systematic review. We comprehensively searched a range of databases as well as conference abstracts and a trials registry. We used Cochrane methods in screening and data collection and assessed each study’s risk of bias with the Cochrane tool. We meta-analyzed data using a fixed-effects model. We used GRADE to assess evidence quality. Results From 492 search results, we identified two randomized controlled trials, reported in five peer-reviewed articles and one conference abstract. One trial tested two DTG-based regimens (DTG + abacavir (ABC) + lamivudine (3TC) or DTG + tenofovir + emtricitabine) against an EFV-based regimen (EFV+ ABC+3TC). The other trial tested DTG+ABC+3TC against EFV+ABC+3TC. In meta-analysis, DTG-containing regimens were superior to EFV-containing regimens at 48 weeks and at 96 weeks (RR = 1.10, 95% CI 1.04–1.16; and RR = 1.12, 95% CI 1.04–1.21, respectively). In one trial, the DTG-containing regimen was superior at 144 weeks (RR = 1.13, 95% CI 1.02–1.24). DTG-containing regimens were superior in reducing treatment discontinuation compared to those containing EFV at 96 weeks and at 144 weeks (RR = 0.27, 95% CI 0.15–0.50; and RR = 0.28, 95% CI 0.16–0.48, respectively). Risk of serious adverse events was similar in each regimen at 96 weeks (RR = 1.15, 95% CI 0.80–1.63) and 144 weeks (RR = 0.93, 95% CI 0.68–1.29). Risk of bias was moderate overall, as was GRADE evidence quality. Conclusions DTG-based regimens should be considered in future World

  7. Sargassum fusiforme fraction is a potent and specific inhibitor of HIV-1 fusion and reverse transcriptase.

    PubMed

    Paskaleva, Elena E; Lin, Xudong; Duus, Karen; McSharry, James J; Veille, Jean-Claude L; Thornber, Carol; Liu, Yanze; Lee, David Yu-Wei; Canki, Mario

    2008-01-15

    Sargassum fusiforme (Harvey) Setchell has been shown to be a highly effective inhibitor of HIV-1 infection. To identify its mechanism of action, we performed bioactivity-guided fractionation on Sargassum fusiforme mixture. Here, we report isolation of a bioactive fraction SP4-2 (S. fusiforme), which at 8 mug/ml inhibited HIV-1 infection by 86.9%, with IC50 value of 3.7 mug. That represents 230-fold enhancement of antiretroviral potency as compared to the whole extract. Inhibition was mediated against both CXCR4 (X4) and CCR5 (R5) tropic HIV-1. Specifically, 10 mug/ml SP4-2 blocked HIV-1 fusion and entry by 53%. This effect was reversed by interaction of SP4-2 with sCD4, suggesting that S. fusiforme inhibits HIV-1 infection by blocking CD4 receptor, which also explained observed inhibition of both X4 and R5-tropic HIV-1. SP4-2 also inhibited HIV-1 replication after virus entry, by directly inhibiting HIV-1 reverse transcriptase (RT) in a dose dependent manner by up to 79%. We conclude that the SP4-2 fraction contains at least two distinct and biologically active molecules, one that inhibits HIV-1 fusion by interacting with CD4 receptor, and another that directly inhibits HIV-1 RT. We propose that S. fusiforme is a lead candidate for anti-HIV-1 drug development.

  8. Sargassum fusiforme fraction is a potent and specific inhibitor of HIV-1 fusion and reverse transcriptase

    PubMed Central

    Paskaleva, Elena E; Lin, Xudong; Duus, Karen; McSharry, James J; Veille, Jean-Claude L; Thornber, Carol; Liu, Yanze; Lee, David Yu-Wei; Canki, Mario

    2008-01-01

    Sargassum fusiforme (Harvey) Setchell has been shown to be a highly effective inhibitor of HIV-1 infection. To identify its mechanism of action, we performed bioactivity-guided fractionation on Sargassum fusiforme mixture. Here, we report isolation of a bioactive fraction SP4-2 (S. fusiforme), which at 8 μg/ml inhibited HIV-1 infection by 86.9%, with IC50 value of 3.7 μg. That represents 230-fold enhancement of antiretroviral potency as compared to the whole extract. Inhibition was mediated against both CXCR4 (X4) and CCR5 (R5) tropic HIV-1. Specifically, 10 μg/ml SP4-2 blocked HIV-1 fusion and entry by 53%. This effect was reversed by interaction of SP4-2 with sCD4, suggesting that S. fusiforme inhibits HIV-1 infection by blocking CD4 receptor, which also explained observed inhibition of both X4 and R5-tropic HIV-1. SP4-2 also inhibited HIV-1 replication after virus entry, by directly inhibiting HIV-1 reverse transcriptase (RT) in a dose dependent manner by up to 79%. We conclude that the SP4-2 fraction contains at least two distinct and biologically active molecules, one that inhibits HIV-1 fusion by interacting with CD4 receptor, and another that directly inhibits HIV-1 RT. We propose that S. fusiforme is a lead candidate for anti-HIV-1 drug development. PMID:18197976

  9. TNFα is involved in neuropathic pain induced by nucleoside reverse transcriptase inhibitor in rats.

    PubMed

    Zheng, Xuexing; Ouyang, Handong; Liu, Shue; Mata, Marina; Fink, David J; Hao, Shuanglin

    2011-11-01

    In patients with HIV/AIDS, neuropathic pain is a common neurological complication. Infection with the HIV itself may lead to neuropathic pain, and painful symptoms are enhanced when patients are treated with nucleoside reverse transcriptase inhibitors (NRTIs). The mechanisms by which NRTIs contribute to the development of neuropathic pain are not known. In the current studies, we tested the role of TNFα in antiretroviral drug-induced neuropathic pain. We administered 2',3'-dideoxycytidine (ddC, one of the NRTIs) systemically to induce mechanical allodynia. We found that ddC induced overexpression of both mRNA and proteins of GFAP and TNFα in the spinal dorsal horn. TNFα was colocalized with GFAP in the spinal dorsal horn and with NeuN in the DRG. Knockdown of TNFα with siRNA blocked the mechanical allodynia induced by ddC. Intrathecal administration of glial inhibitor or recombinant TNF soluble receptor, reversed mechanical allodynia induced by ddC. These results suggest that TNFα is involved in NRTI-induced neuropathic pain. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction.

    PubMed

    Zhu, Y Y; Machleder, E M; Chenchik, A; Li, R; Siebert, P D

    2001-04-01

    Here, we describe a fast, simple method for constructing full-length cDNA libraries using SMART technology. This novel procedure uses the template-switching activity of Moloney murine leukemia virus (MMLV) reverse transcriptase to synthesize and anchor first-strand cDNA in one step. Following reverse transcription, three cycles of PCR are performed using a modified oligo(dT) primer and an anchor primer to enrich the cDNA population for full-length sequences. Starting with 1 microgram human skeletal muscle poly(A)+ RNA, a cDNA library was constructed that contained 3 x 10(6) independent clones with an average insert size of 2 kb. Sequence analysis of 172 randomly selected clones showed that 77% of cDNA clones corresponding to known genes contained intact open reading frames. The average length of complete open reading frames was 2.4 kb. Furthermore, 86% of the full-length clones retained longer 5' UTR sequences than the longest 5' end deposited in the GenBank database. cDNA libraries generated using this method will be useful for accelerating the collection of mRNA 5' end sequence information, which is currently very limited in GenBank.

  11. Epigenetic perturbation driving asleep telomerase reverse transcriptase: Possible therapeutic avenues in carcinoma.

    PubMed

    Kumar, Ajay; Nilednu, Pritish; Kumar, Azad; Sharma, Nilesh Kumar

    2017-03-01

    In the last decade, implications of human telomerase reverse transcriptase (hTERT), a component of ribonucleoprotein telomerase in aging, senescence, and stem cell are highly evident. Besides, the activation of hTERT is also being documented several cancer types including carcinoma. The awakening of telomerase during carcinoma initiation and development is being seen with different perspectives including genetic and epigenetic tools and events. In view of several tumor progenitors genes (also referred as epigenetic mediators), telomerase is placed as key enzyme to achieve the carcinoma phenotype and sustain during the progression. It is true that swaying of telomerase in carcinoma could be facilitated with dedicated set of epigenetic modulators and modifiers players. These epigenetic alterations are heritable, potentially reversible, and seen as the epigenetic signature of carcinoma. Several papers converge to suggest that DNA methylation, histone modification, and small non-coding RNAs are the widely appreciated epigenetic changes towards hTERT modulation. In this review, we summarize the contribution of epigenetic factors in the telomerase activation and discuss potential avenues to achieve therapeutic intervention in carcinoma.

  12. RNase H activity associated with reverse transcriptase from feline immunodeficiency virus.

    PubMed Central

    Cronn, R C; Whitmer, J D; North, T W

    1992-01-01

    Reverse transcription of retroviral genomes requires the action of an RNase H for template switching and primer generation. In this report, we compare enzymatic properties of the RNase H associated with the reverse transcriptase (RT) from feline immunodeficiency virus (FIV) and that from human immunodeficiency virus (HIV). Both enzymes displayed substrate preference for poly[3H](rG) . poly(dC) hybird over poly[3H](rA) . poly(dT) and cation preference for Mg2+ over Mn2+. Activity of the FIV RNase H upon poly(rG) . poly(dC) produced hydrolysis products from 1 to 6 nucleotides in length, similar to that reported for HIV. Dextran sulfates were effective inhibitors of both the FIV and HIV RNase H and RT activities. Nearly identical inhibition constants (0.12 nM) were obtained for all enzyme activities with dextran sulfate 500,000, while different inhibition constants were observed with dextran sulfate 8,000. Our results suggest that FIV and HIV RTs contain a conserved region that is sensitive to the larger dextran sulfate and that dextran sulfate 8,000 may interact at a different site or by a different mechanism. Images PMID:1370549

  13. Activation of anti-reverse transcriptase nucleotide analogs by nucleoside diphosphate kinase: improvement by alpha-boranophosphate substitution.

    PubMed

    Schneider, B; Meyer, P; Sarfati, S; Mulard, L; Guerreiro, C; Boretto, J; Janin, J; Véron, M; Deville-Bonne, D; Canard, B

    2001-01-01

    Nucleoside activation by nucleoside diphosphate kinase and inhibition of HIV-1 reverse transcriptase were studied comparatively for a new class of nucleoside analogs with a borano (BH3-) or a thio (SH) group on the alpha-phosphate. Both the alpha-Rp-borano derivatives of AZT and d4T improved phosphorylation by NDP kinase, inhibition of reverse transcription as well as stability of alpha-borano nonophosphate derivatives in terminated viral DNA chain.

  14. Lipid metabolism and lipodystrophy in HIV-1-infected patients: the role played by nonnucleoside reverse transcriptase inhibitors.

    PubMed

    Sension, Michael; Deckx, Henri

    2015-01-01

    Dyslipidemia and lipodystrophy represent significant healthcare concerns in HIV-infected patients due to their association with diabetes mellitus and increased cardiovascular disease risk. Since the lipid effects of the nonnucleoside reverse transcriptase inhibitors are not well characterized, we systematically summarized the effects of nonnucleoside reverse transcriptase inhibitor treatment on dyslipidemia and lipodystrophy in HIV-1 infection. As with other classes of antiretroviral agents, the nonnucleoside reverse transcriptase inhibitors are associated with lipid changes, although individual agents exhibit differing effects on lipid profiles. Comparative trials have shown that the risk for hypertriglyceridemia is lower with efavirenz than with the use of ritonavir-boosted lopinavir, but there is a greater likelihood of hypercholesterolemia compared to ritonavir-boosted atazanavir. Data also suggest that efavirenz results in greater increases in plasma lipid levels than integrase inhibitors and CC-chemokine-receptor-5 antagonists. Lipid disturbances are less frequent with the newer nonnucleoside reverse transcriptase inhibitors than with efavirenz. However, in most cases, no change in the total:high-density lipoprotein-cholesterol ratio was seen between the efavirenz and comparator groups. Switching from efavirenz to etravirine or rilpivirine, or the integrase inhibitors raltegravir or elvitegravir, resulted in significant reductions in lipid levels. There appears to be minimal potential for efavirenz or rilpivirine to result in development of lipodystrophy. Overall, nonnucleoside reverse transcriptase inhibitors have a smaller impact on plasma lipids than ritonavir-boosted protease inhibitors, with the newer agents exhibiting more favorable lipid profiles than efavirenz. When considering antiretroviral regimens, awareness of the different lipid effect profiles of the third agent is important, without forgetting the critical contribution of the background

  15. Identification of Non-nucleoside Human Ribonucleotide Reductase Modulators

    DOE PAGES

    Ahmad, Md. Faiz; Huff, Sarah E.; Pink, John; ...

    2015-10-21

    Ribonucleotide reductase (RR) catalyzes the rate-limiting step of dNTP synthesis and is an established cancer target. Drugs targeting RR are mainly nucleoside in nature. In this study, we sought to identify non-nucleoside small-molecule inhibitors of RR. Using virtual screening, binding affinity, inhibition, and cell toxicity, we have discovered a class of small molecules that alter the equilibrium of inactive hexamers of RR, leading to its inhibition. Several unique chemical categories, including a phthalimide derivative, show micromolar IC50s and KDs while demonstrating cytotoxicity. A crystal structure of an active phthalimide binding at the targeted interface supports the noncompetitive mode of inhibitionmore » determined by kinetic studies. Furthermore, the phthalimide shifts the equilibrium from dimer to hexamer. Finally, together, these data identify several novel non-nucleoside inhibitors of human RR which act by stabilizing the inactive form of the enzyme.« less

  16. Identification of Non-nucleoside Human Ribonucleotide Reductase Modulators

    SciTech Connect

    Ahmad, Md. Faiz; Huff, Sarah E.; Pink, John; Alam, Intekhab; Zhang, Andrew; Perry, Kay; Harris, Michael E.; Misko, Tessianna; Porwal, Suheel K.; Oleinick, Nancy L.; Miyagi, Masaru; Viswanathan, Rajesh; Dealwis, Chris Godfrey

    2015-10-21

    Ribonucleotide reductase (RR) catalyzes the rate-limiting step of dNTP synthesis and is an established cancer target. Drugs targeting RR are mainly nucleoside in nature. In this study, we sought to identify non-nucleoside small-molecule inhibitors of RR. Using virtual screening, binding affinity, inhibition, and cell toxicity, we have discovered a class of small molecules that alter the equilibrium of inactive hexamers of RR, leading to its inhibition. Several unique chemical categories, including a phthalimide derivative, show micromolar IC50s and KDs while demonstrating cytotoxicity. A crystal structure of an active phthalimide binding at the targeted interface supports the noncompetitive mode of inhibition determined by kinetic studies. Furthermore, the phthalimide shifts the equilibrium from dimer to hexamer. Finally, together, these data identify several novel non-nucleoside inhibitors of human RR which act by stabilizing the inactive form of the enzyme.

  17. Highly efficient inhibition of human immunodeficiency virus type 1 reverse transcriptase by aptamers functionalized gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Shiang, Yen-Chun; Ou, Chung-Mao; Chen, Shih-Ju; Ou, Ting-Yu; Lin, Han-Jia; Huang, Chih-Ching; Chang, Huan-Tsung

    2013-03-01

    We have developed aptamer (Apt)-conjugated gold nanoparticles (Apt-Au NPs, 13 nm in diameter) as highly effective inhibitors for human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). Two Apts, RT1t49 (Aptpol) and ODN 93 (AptRH), which recognize the polymerase and RNase H regions of HIV-1 RT, are used to conjugate Au NPs to prepare Aptpol-Au NPs and AptRH-Au NPs, respectively. In addition to DNA sequence, the surface density of the aptamers on Au NPs (nApt-Au NPs; n is the number of aptamer molecules on each Au NP) and the linker length number (Tm; m is the base number of the deoxythymidine linker) between the aptamer and Au NPs play important roles in determining their inhibition activity. A HIV-lentiviral vector-based antiviral assay has been applied to determine the inhibitory effect of aptamers or Apt-Au NPs on the early stages of their replication cycle. The nuclease-stable G-quadruplex structure of 40AptRH-T45-Au NPs shows inhibitory efficiency in the retroviral replication cycle with a decreasing infectivity (40.2%).We have developed aptamer (Apt)-conjugated gold nanoparticles (Apt-Au NPs, 13 nm in diameter) as highly effective inhibitors for human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). Two Apts, RT1t49 (Aptpol) and ODN 93 (AptRH), which recognize the polymerase and RNase H regions of HIV-1 RT, are used to conjugate Au NPs to prepare Aptpol-Au NPs and AptRH-Au NPs, respectively. In addition to DNA sequence, the surface density of the aptamers on Au NPs (nApt-Au NPs; n is the number of aptamer molecules on each Au NP) and the linker length number (Tm; m is the base number of the deoxythymidine linker) between the aptamer and Au NPs play important roles in determining their inhibition activity. A HIV-lentiviral vector-based antiviral assay has been applied to determine the inhibitory effect of aptamers or Apt-Au NPs on the early stages of their replication cycle. The nuclease-stable G-quadruplex structure of 40AptRH-T45

  18. AZT resistance alters enzymatic properties and creates an ATP-binding site in SFVmac reverse transcriptase.

    PubMed

    Schneider, Anna; Schweimer, Kristian; Rösch, Paul; Wöhrl, Birgitta M

    2015-02-22

    The replication of simian foamy virus from macaques can be inhibited by the nucleoside reverse transcriptase inhibitor azidothymidine (AZT, zidovudine). Four substitutions in the protease-reverse transcriptase (PR-RT) protein (K211I, I224T, S345T, E350K) are necessary to obtain highly AZT resistant and fully replication competent virus. AZT resistance is based on the excision of the incorporated AZTMP in the presence of ATP. I224T is a polymorphism which is not essential for AZT resistance per se, but is important for regaining efficient replication of the resistant virus. We constructed PR-RT enzymes harboring one to four amino acid substitutions to analyze them biochemically and to determine their ability to remove the incorporated AZTMP. S345T is the only single substitution variant exhibiting significant AZTMP excision activity. Although K211I alone showed no AZTMP excision activity, excision efficiency doubled when K211I was present in combination with S345T and E350K. K211I also decreased nucleotide binding affinity and increased fidelity. NMR titration experiments revealed that a truncated version of the highly AZT resistant mt4 variant, comprising only the fingers-palm subdomains was able to bind ATP with a KD-value of ca. 7.6 mM, whereas no ATP binding could be detected in the corresponding wild type protein. We could show by NMR spectroscopy that S345T is responsible for ATP binding, probably by making a tryptophan residue accessible. Although AZT resistance in SFVmac is based on excision of the incorporated AZTMP like in HIV-1, the functions of the resistance substitutions in SFVmac PR-RT appear to be different. No mutation resulting in an aromatic residue like F/Y215 in HIV, which is responsible for π-π-stacking interactions with ATP, is present in SFVmac. Instead, S345T is responsible for creating an ATP binding site, probably by making an already existing tryptophan more accessible, which in turn can interact with ATP. This is in contrast to HIV-1

  19. Coevolutionary Analysis Identifies Protein–Protein Interaction Sites between HIV-1 Reverse Transcriptase and Integrase

    PubMed Central

    Hetti Arachchilage, Madara; Piontkivska, Helen

    2016-01-01

    The replication of human immunodeficiency virus-1 (HIV-1) requires reverse transcription of the viral RNA genome and integration of newly synthesized pro-viral DNA into the host genome. This is mediated by the viral proteins reverse transcriptase (RT) and integrase (IN). The formation and stabilization of the pre-integration complex (PIC), which is an essential step for reverse transcription, nuclear import, chromatin targeting, and subsequent integration, involves direct and indirect modes of interaction between RT and IN proteins. While epitope-based treatments targeting IN–viral DNA and IN–RT complexes appear to be a promising combination for an anti-HIV treatment, the mechanisms of IN-RT interactions within the PIC are not well understood due to the transient nature of the protein complex and the intrinsic flexibility of its components. Here, we identify potentially interacting regions between the IN and RT proteins within the PIC through the coevolutionary analysis of amino acid sequences of the two proteins. Our results show that specific regions in the two proteins have strong coevolutionary signatures, suggesting that these regions either experience direct and prolonged interactions between them that require high affinity and/or specificity or that the regions are involved in interactions mediated by dynamic conformational changes and, hence, may involve both direct and indirect interactions. Other regions were found to exhibit weak, but positive correlations, implying interactions that are likely transient and/or have low affinity. We identified a series of specific regions of potential interactions between the IN and RT proteins (e.g., specific peptide regions within the C-terminal domain of IN were identified as potentially interacting with the Connection domain of RT). Coevolutionary analysis can serve as an important step in predicting potential interactions, thus informing experimental studies. These studies can be integrated with structural data

  20. Efficient N-tailing of blunt DNA ends by Moloney murine leukemia virus reverse transcriptase

    PubMed Central

    Ohtsubo, Yoshiyuki; Nagata, Yuji; Tsuda, Masataka

    2017-01-01

    Moloney murine leukemia virus reverse transcriptase (MMLV-RT) is a widely used enzyme for cDNA synthesis. Here we show that MMLV-RT has a strong template-independent polymerase activity using blunt DNA ends as substrate that generates 3′ overhangs of A, C, G, or T. Nucleotides were appended efficiently in the order A > G > T > C, and tail lengths varied from 4 to 5, 2 to 7, 2 to 4, and 2 to 3 for A, C, G, and T, respectively. The activity was so strong that nearly all our test DNA ends were appended with at least one A, C, G, or T. The N-tailing activity of MMLV-RT was enhanced in the presence of Mn2+, and the G-, C-, and T-tailing activities were further enhanced by dCMP, dGMP, and dAMP, respectively. This is the first report of an enzymatic activity that almost thoroughly appends two or more As, or one or more Cs, Gs, or Ts to the 3′ end of double-stranded DNA, which would enable exhaustive analysis of DNA samples. The N-tailing activity of MMLV-RT is potentially useful in many biotechnological applications. PMID:28150748

  1. A novel ribonuclease with potent HIV-1 reverse transcriptase inhibitory activity from cultured mushroom Schizophyllum commune.

    PubMed

    Zhao, Yong-Chang; Zhang, Guo-Qing; Ng, Tzi-Bun; Wang, He-Xiang

    2011-10-01

    A 20-kDa ribonuclease (RNase) was purified from fresh fruiting bodies of cultured Schizophyllum commune mushrooms. The RNase was not adsorbed on Affi-gel blue gel but adsorbed on DEAE-cellulose and CM-cellulose. It exhibited maximal RNase activity at pH 6.0 and 70°C. It demonstrated the highest ribonucleolytic activity toward poly (U) (379.5 μ/mg), the second highest activity toward poly (C) (244.7 μ/mg), less activity toward poly (A) (167.4 μ/mg), and much weaker activity toward poly (G) (114.5 μ/mg). The RNase inhibited HIV-1 reverse transcriptase with an IC(50) of 65 μM. No effect on [(3)H-methyl]-thymidine uptake by lymphoma MBL2 cells and leukemia L1210 cells was observed at 100 μM concentration of the RNase. A comparison of RNases from S. commune and Volvariella volvacea revealed that they demonstrated some similarities in N-terminal amino acid sequence, optimum pH and polyhomoribonucleotide specificity. However, some differences in chromatographic behavior and molecular mass were observed.

  2. A reverse transcriptase-polymerase chain reaction assay for detecting Highlands J virus.

    PubMed

    Whitehouse, C A; Guibeau, A; McGuire, D; Takeda, T; Mather, T N

    2001-01-01

    Highlands J (HJ) virus is an arbovirus frequently recovered at high rates in mosquitoes collected in the eastern United States. HJ virus is primarily a veterinary pathogen causing disease in domestic birds including turkeys, chickens, and partridges. It has an enzootic cycle similar to eastern equine encephalitis (EEE) virus and is often used as an indicator species in EEE surveillance programs. Current immunologic techniques to identify HJ virus are often inefficient and can involve cross-reactivity of antibodies. Therefore, we developed a molecular-based assay by a reverse transcriptase (RT)-polymerase chain reaction (PCR) technique. Primers were constructed from conserved sequences of the E1 coding region from 19 strains of HJ virus. PCR amplifications from serial dilutions of HJ virus-infected Vero cell culture supernatants indicated that this assay could detect viral RNA at concentrations of 10 plaque-forming units per reaction. Extracted RNAs from western equine encephalitis, EEE, LaCrosse, and Jamestown Canyon viruses were not detected with this assay. RNA extracted directly from the brain tissue of a dead house sparrow and from a pool of Culiseta mosquitoes yielded a PCR product of the expected size. The RT-PCR technique developed was both sensitive and specific for detecting HJ virus from infected cell culture supernatants, bird brain tissues, and mosquitoes. This new assay will permit rapid and accurate diagnosis of HJ virus, both enhancing surveillance activities for EEE transmission risk and monitoring infections in domestic poultry and wild birds.

  3. Reverse transcriptase directs viral evolution in a deep ocean methane seep

    NASA Astrophysics Data System (ADS)

    Paul, B. G.; Bagby, S. C.

    2013-12-01

    Deep ocean methane seeps are sites of intense microbial activity, with complex communities fueled by aerobic and anaerobic methanotrophy. Methane consumption in these communities has a substantial impact on the global carbon cycle, yet little is known about their evolutionary history or their likely evolutionary trajectories in a warming ocean. As in other marine systems, viral predation and virally mediated horizontal gene transfer are expected to be major drivers of evolutionary change in these communities; however, the host cells' resistance to cultivation has impeded direct study of the viral population. We conducted a metagenomic study of viruses in the anoxic sediments of a deep methane seep in the Santa Monica Basin in the Southern California Bight. We retrieved 1660 partial viral genomes, tentatively assigning 1232 to bacterial hosts and 428 to archaea. One abundant viral genome, likely hosted by Clostridia species present in the sediment, was found to encode a diversity-generating retroelement (DGR), a module for reverse transcriptase-mediated directed mutagenesis of a distal tail fiber protein. While DGRs have previously been described in the viruses of human pathogens, where diversification of viral tail fibers permits infection of a range of host cell types, to our knowledge this is the first description of such an element in a marine virus. By providing a mechanism for massively broadening potential host range, the presence of DGRs in these systems may have a major impact on the prevalence of virally mediated horizontal gene transfer, and even on the phylogenetic distances across which genes are moved.

  4. Universal reverse-transcriptase real-time PCR for infectious hematopoietic necrosis virus (IHNV).

    PubMed

    Purcell, Maureen K; Thompson, Rachel L; Garver, Kyle A; Hawley, Laura M; Batts, William N; Sprague, Laura; Sampson, Corie; Winton, James R

    2013-10-11

    Infectious hematopoietic necrosis virus (IHNV) is an acute pathogen of salmonid fishes in North America, Europe and Asia and is reportable to the World Organization for Animal Health (OIE). Phylogenetic analysis has identified 5 major virus genogroups of IHNV worldwide, designated U, M, L, E and J; multiple subtypes also exist within those genogroups. Here, we report the development and validation of a universal IHNV reverse-transcriptase real-time PCR (RT-rPCR) assay targeting the IHNV nucleocapsid (N) gene. Properties of diagnostic sensitivity (DSe) and specificity (DSp) were defined using laboratory-challenged steelhead trout Oncorhynchus mykiss, and the new assay was compared to the OIE-accepted conventional PCR test and virus isolation in cell culture. The IHNV N gene RT-rPCR had 100% DSp and DSe and a higher estimated diagnostic odds ratio (DOR) than virus culture or conventional PCR. The RT-rPCR assay was highly repeatable within a laboratory and highly reproducible between laboratories. Field testing of the assay was conducted on a random sample of juvenile steelhead collected from a hatchery raceway experiencing an IHN epizootic. The RT-rPCR detected a greater number of positive samples than cell culture and there was 40% agreement between the 2 tests. Overall, the RT-rPCR assay was highly sensitive, specific, repeatable and reproducible and is suitable for use in a diagnostic setting.

  5. Telomerase reverse-transcriptase homozygous mutations in autosomal recessive dyskeratosis congenita and Hoyeraal-Hreidarsson syndrome

    PubMed Central

    Marrone, Anna; Walne, Amanda; Tamary, Hannah; Masunari, Yuka; Kirwan, Michael; Beswick, Richard; Vulliamy, Tom; Dokal, Inderjeet

    2010-01-01

    Dyskeratosis congenita (DC) is a multisystem bone marrow failure syndrome characterized by a triad of mucocutaneous abnormalities and an increased predisposition to malignancy. X-linked DC is due to mutations in DKC1, while heterozygous mutations in TERC (telomerase RNA component) and TERT (telomerase reverse transcriptase) have been found in autosomal dominant DC. Many patients with DC remain uncharacterized, particularly families displaying autosomal recessive (AR) inheritance. We have now identified novel homozygous TERT mutations in 2 unrelated consanguineous families, where the index cases presented with classical DC or the more severe variant, Hoyeraal-Hreidarsson (HH) syndrome. These TERT mutations resulted in reduced telomerase activity and extremely short telomeres. As these mutations are homozygous, these patients are predicted to have significantly reduced telomerase activity in vivo. Interestingly, in contrast to patients with heterozygous TERT mutations or hemizygous DKC1 mutations, these 2 homozygous TERT patients were observed to have higher-than-expected TERC levels compared with controls. Collectively, the findings from this study demonstrate that homozygous TERT mutations, resulting in a pure but severe telomerase deficiency, produce a phenotype of classical AR-DC and its severe variant, the HH syndrome. PMID:17785587

  6. Structural Maturation of HIV-1 Reverse Transcriptase-A Metamorphic Solution to Genomic Instability.

    PubMed

    London, Robert E

    2016-09-27

    Human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT)-a critical enzyme of the viral life cycle-undergoes a complex maturation process, required so that a pair of p66 precursor proteins can develop conformationally along different pathways, one evolving to form active polymerase and ribonuclease H (RH) domains, while the second forms a non-functional polymerase and a proteolyzed RH domain. These parallel maturation pathways rely on the structural ambiguity of a metamorphic polymerase domain, for which the sequence-structure relationship is not unique. Recent nuclear magnetic resonance (NMR) studies utilizing selective labeling techniques, and structural characterization of the p66 monomer precursor have provided important insights into the details of this maturation pathway, revealing many aspects of the three major steps involved: (1) domain rearrangement; (2) dimerization; and (3) subunit-selective RH domain proteolysis. This review summarizes the major structural changes that occur during the maturation process. We also highlight how mutations, often viewed within the context of the mature RT heterodimer, can exert a major influence on maturation and dimerization. It is further suggested that several steps in the RT maturation pathway may provide attractive targets for drug development.

  7. AIDS virus reverse transcriptase defined by high level expression in Escherichia coli.

    PubMed Central

    Larder, B; Purifoy, D; Powell, K; Darby, G

    1987-01-01

    The causative agent of AIDS the human immunodeficiency virus (HIV) encodes as part of its pol gene a reverse transcriptase (RT) which has a key role in the replication of the virus and thus constitutes an ideal target for antiviral chemotherapy. The purified HIV RT from virus particles consists of two related polypeptides of 66 and 51 kd mol. wt and similar polypeptides are found on expression of the complete HIV pol gene using prokaryotic systems. Here we describe the expression of the 66-kd protein in Escherichia coli and demonstrate that this polypeptide alone has authentic RT activity. Thus, a central HIV pol gene segment encodes and is sufficient for high levels of RT activity. The RT has been purified from E. coli extracts using a purification procedure involving two chromotography steps resulting in an enzyme preparation near homogeneity. Deletion of the C-terminal region of the RT thought to encode the RNase H domain resulted in loss of polymerase activity. Images Fig. 2. Fig. 4. PMID:2446866

  8. Efficient N-tailing of blunt DNA ends by Moloney murine leukemia virus reverse transcriptase.

    PubMed

    Ohtsubo, Yoshiyuki; Nagata, Yuji; Tsuda, Masataka

    2017-02-02

    Moloney murine leukemia virus reverse transcriptase (MMLV-RT) is a widely used enzyme for cDNA synthesis. Here we show that MMLV-RT has a strong template-independent polymerase activity using blunt DNA ends as substrate that generates 3' overhangs of A, C, G, or T. Nucleotides were appended efficiently in the order A > G > T > C, and tail lengths varied from 4 to 5, 2 to 7, 2 to 4, and 2 to 3 for A, C, G, and T, respectively. The activity was so strong that nearly all our test DNA ends were appended with at least one A, C, G, or T. The N-tailing activity of MMLV-RT was enhanced in the presence of Mn(2+), and the G-, C-, and T-tailing activities were further enhanced by dCMP, dGMP, and dAMP, respectively. This is the first report of an enzymatic activity that almost thoroughly appends two or more As, or one or more Cs, Gs, or Ts to the 3' end of double-stranded DNA, which would enable exhaustive analysis of DNA samples. The N-tailing activity of MMLV-RT is potentially useful in many biotechnological applications.

  9. Regulatory roles of LINE-1-encoded reverse transcriptase in cancer onset and progression

    PubMed Central

    Sciamanna, Ilaria; Gualtieri, Alberto; Piazza, Pier Vincenzo; Spadafora, Corrado

    2014-01-01

    LINE-1 retrotransposons encode the reverse transcriptase (RT) enzyme, required for their own mobility, the expression of which is inhibited in differentiated tissues while being active in tumors. Experimental evidence indicate that the inhibition of LINE-1-derived RT restores differentiation in cancer cells, inhibits tumor progression and yields globally reprogrammed transcription profiles. Newly emerging data suggest that LINE-1-encoded RT modulates the biogenesis of miRNAs, by governing the balance between the production of regulatory double-stranded RNAs and RNA:DNA hybrid molecules, with a direct impact on global gene expression. Abnormally high RT activity unbalances the transcriptome in cancer cells, while RT inhibition restores ‘normal’ miRNA profiles and their regulatory networks. This RT-dependent mechanism can target the myriad of transcripts - both coding and non-coding, sense and antisense - in eukaryotic transcriptomes, with a profound impact on cell fates. LINE-1-encoded RT emerges therefore as a key regulator of a previously unrecognized mechanism in tumorigenesis PMID:25478632

  10. Prolonged expansion of human nucleus pulposus cells expressing human telomerase reverse transcriptase mediated by lentiviral vector.

    PubMed

    Wu, Jianhong; Wang, Deli; Ruan, Dike; He, Qing; Zhang, Yan; Wang, Chaofeng; Xin, Hongkui; Xu, Cheng; Liu, Yue

    2014-01-01

    Human degenerative disc disease (DDD) is characterized by progressive loss of human nucleus pulposus (HNP) cells and extracellular matrix, in which the massive deposition are secreted by HNP cells. Cell therapy to supplement HNP cells to degenerated discs has been thought to be a promising strategy to treat DDD. However, obtaining a large quality of fully functional HNP cells has been severely hampered by limited proliferation capacity of HNP cells in vitro. Previous studies have used lipofectamine or recombinant adeno-associated viral (rAAV) vectors to deliver human telomerase reverse transcriptase (hTERT) into ovine or HNP cells to prolong the activity of nucleus pulposus cells with limited success. Here we developed a lentiviral vector bearing both hTERT and a gene encoding green fluorescence protein (L-hTERT/EGFP). This vector efficiently mediated both hTERT and EGFP into freshly isolated HNP cells. The expressions of both transgenes in L-hTERT/EGFP transduced HNP cells were detected up to day 210 post viral infection, which was twice as long as rAAV vector did. Furthermore, we observed restored telomerase activity, maintained telomere length, delayed cell senescence, and increased cell proliferation rate in those L-hTERT/EGFP transduced HNP cells. Our study suggests that lentiviral vector might be a useful gene delivery vehicle for HNP cell therapy to treat DDD.

  11. The (5Z)-5-Pentacosenoic and 5-Pentacosynoic Acids Inhibit the HIV-1 Reverse Transcriptase.

    PubMed

    Moreira, Lizabeth Giménez; Orellano, Elsie A; Rosado, Karolyna; Guido, Rafael V C; Andricopulo, Adriano D; Soto, Gabriela Ortiz; Rodríguez, José W; Sanabria-Ríos, David J; Carballeira, Néstor M

    2015-10-01

    The natural fatty acids (5Z)-5-pentacosenoic and (9Z)-9-pentacosenoic acids were synthesized for the first time in eight steps starting from either 4-bromo-1-butanol or 8-bromo-1-butanol and in 20-58% overall yields, while the novel fatty acids 5-pentacosynoic and 9-pentacosynoic acids were also synthesized in six steps and in 34-43% overall yields. The ∆(5) acids displayed the best IC50's (24-38 µM) against the HIV-1 reverse transcriptase (RT) enzyme, comparable to nervonic acid (IC50 = 12 µM). The ∆(9) acids were not as effective towards HIV-RT with the (9Z)-9-pentacosenoic acid displaying an IC50 = 54 µM and the 9-pentacosynoic acid not inhibiting the enzyme at all. Fatty acid chain length and position of the unsaturation was important for the observed inhibition. None of the synthesized fatty acids were toxic (IC50 > 500 µM) towards peripheral blood mononuclear cells. Molecular modeling studies indicated the structural determinants underlying the biological activity of the most potent compounds. These results provide new insights into the structural requirements that must be present in fatty acids so as to enhance their inhibitory potential towards HIV-RT.

  12. Telomerase reverse transcriptase promoter mutations in hepatitis B virus-associated hepatocellular carcinoma

    PubMed Central

    Yang, Xunjun; Guo, Xiuchan; Chen, Yao; Chen, Guorong; Ma, Yin; Huang, Kate; Zhang, Yuning; Zhao, Qiongya; Winkler, Cheryl A.; An, Ping; Lyu, Jianxin

    2016-01-01

    Telomerase reverse transcriptase (TERT) promoter mutations are among the most frequent noncoding somatic mutations in multiple cancers, including hepatocellular carcinoma (HCC). The clinical and pathological implications of TERT promoter mutations in hepatitis B virus (HBV)-associated HCC have not been resolved. To investigate TERT promoter mutations, protein expression, and their clinical-pathological implications, we sequenced the TERT promoter region for hotspot mutations in HCC tissues and performed immunostaining for TERT protein expression from HBV-associated HCC in Chinese patients. Of 276 HCC tumor DNA samples sequenced, 85 (31%) carried TERT promoter mutations. TERT promoter mutations were more frequent in those with low α-fetoprotein (AFP) serum levels (p = 0.03), advanced age (p = 0.04), and in those lacking HCC family history (p = 0.02), but were not correlated with HCC stages and grades. TERT protein levels were higher in HCC (n = 28) compared to normal liver tissues (n = 8) (p =0.001), but did not differ between mutated and non-mutated tumor tissues. In conclusion, TERT promoter mutations are common somatic mutations in HCC of Han Chinese with HBV infection. Detection of TERT promoter mutations in those with low levels of AFP may aid diagnosis of HCC with atypical presentation. PMID:27056898

  13. High fidelity simian immunodeficiency virus reverse transcriptase mutants have impaired replication in vitro and in vivo.

    PubMed

    Lloyd, Sarah B; Lichtfuss, Marit; Amarasena, Thakshila H; Alcantara, Sheilajen; De Rose, Robert; Tachedjian, Gilda; Alinejad-Rokny, Hamid; Venturi, Vanessa; Davenport, Miles P; Winnall, Wendy R; Kent, Stephen J

    2016-05-01

    The low fidelity of HIV replication facilitates immune and drug escape. Some reverse transcriptase (RT) inhibitor drug-resistance mutations increase RT fidelity in biochemical assays but their effect during viral replication is unclear. We investigated the effect of RT mutations K65R, Q151N and V148I on SIV replication and fidelity in vitro, along with SIV replication in pigtailed macaques. SIVmac239-K65R and SIVmac239-V148I viruses had reduced replication capacity compared to wild-type SIVmac239. Direct virus competition assays demonstrated a rank order of wild-type>K65R>V148I mutants in terms of viral fitness. In single round in vitro-replication assays, SIVmac239-K65R demonstrated significantly higher fidelity than wild-type, and rapidly reverted to wild-type following infection of macaques. In contrast, SIVmac239-Q151N was replication incompetent in vitro and in pigtailed macaques. Thus, we showed that RT mutants, and specifically the common K65R drug-resistance mutation, had impaired replication capacity and higher fidelity. These results have implications for the pathogenesis of drug-resistant HIV.

  14. Efavirenz a nonnucleoside reverse transcriptase inhibitor of first-generation: Approaches based on its medicinal chemistry.

    PubMed

    Bastos, Mônica M; Costa, Carolina C P; Bezerra, Talitha C; da Silva, Fernando de C; Boechat, Núbia

    2016-01-27

    Acquired immunodeficiency syndrome (AIDS) is a disease caused by human immunodeficiency virus (HIV) that affects individuals on all continents. In 1987, the antiretroviral therapy began increasing survival rates and improving the quality of life for patients. Efavirenz (EFV) is a drug widely used in the treatment of HIV-AIDS since 1998. Belonging to a class of nonnucleoside reverse transcriptase inhibitors (NNRTI), it directly blocks the action of the enzyme and consequently the multiplication of the virus. Although EFV has provided excellent results in reducing viral load, cases of resistance associated with adverse effects have led to the search to find new analogs of this drug. Although many researchers are involved in this quest, curiously there is still no clinical substitute for EFV. To develop a second-generation version of EFV, it is essential understand the structure-activity relationships of the derivative compounds. Thus, the aims of the present review are to compare EFV and its derivatives using medicinal chemistry and to describe the main synthetic routes.

  15. Derivation and characterization of goat fetal fibroblast cells induced with human telomerase reverse transcriptase.

    PubMed

    Xie, Ying; Zhao, Xiaoe; Jia, Hongxiang; Ma, Baohua

    2013-01-01

    Fetal fibroblast cells (FFCs) are often used as donor cells for somatic cell nuclear transfer (SCNT) because they are easy to culture and suitable for genetic manipulation. However, through genetic modification process, which required FFCs to be cultured in vitro for several passages, cells tended to age very rapidly and became inappropriate for SCNT. Human telomerase reverse transcriptase (hTERT) possessed the activity of human telomerase and maintains telomere in dividing cells; therefore, hTERT can be transfected into somatic cells to extend their lifespan. In this study, we transfected a Xinong Saanen Dairy Goat FFC line with hTERT. Then, we tested several characteristics of transfected cells, including growth curve, expression and activity of hTERT, tumorigenicity, and expression of oct4 and nanog. The result showed that hTERT could significantly extend the lifespan of transfected cells in vitro. hTERT mRNA was expressed in hTERT-transfected cells. Moreover, hTERT-transfected cells presented enhanced telomerase activity and longer telomere than untransfected cells at the same passage. On the other hand, hTERT-transfected cells can maintain normal karyotype even after several times of subculture in vitro. After inoculation of hTERT-transfected cells in nude mouse, none of them developed tumors on the vaccination site. Interestingly, transfection of hTERT can improve expression of nanog and oct4 in Xinong Saanen Dairy Goat FFCs, especially in low generation after transfection, but with increasing subculture, this effect gradually weakened.

  16. Reverse transcriptase activity from human embryonal carcinoma cells NTera2D1.

    PubMed Central

    Deragon, J M; Sinnett, D; Labuda, D

    1990-01-01

    We have identified an RNA-dependent DNA polymerase activity in the microsomal fraction of human pluri-potential embryonal carcinoma cells NTera2D1, which are known to express the full length coding strand of the genomic Line-1 (L1) elements. This activity was classified as a reverse transcriptase (RT) based on its utilization of an RT specific synthetic poly(Cm) template in the presence of Mn2+ ions. Treatment of the cell by ultraviolet irradiation (200 erg/mm2) which resulted in a 2- to 3-fold enhancement of the RT activity, was required for the reproducible detection of the activity throughout the entire purification procedure. More than a 100-fold enrichment in RT activity was obtained by centrifugation in a glycerol step gradient and a linear sucrose density gradient followed by Sephacryl S-1000 gel filtration. These experiments demonstrated that the RT activity was associated with a macromolecular complex having the characteristics of a viral-like particle with a major protein component of 37 kd. The presence of L1 mRNA in RT-containing fractions suggests that the activity identified could originate from L1 elements and/or be involved in the mechanism of retroposition. Images Fig. 2. Fig. 3. Fig. 4. PMID:1698616

  17. Distinguishing Functional Amino Acid Covariation from Background Linkage Disequilibrium in HIV Protease and Reverse Transcriptase

    PubMed Central

    Wang, Qi; Lee, Christopher

    2007-01-01

    Correlated amino acid mutation analysis has been widely used to infer functional interactions between different sites in a protein. However, this analysis can be confounded by important phylogenetic effects broadly classifiable as background linkage disequilibrium (BLD). We have systematically separated the covariation induced by selective interactions between amino acids from background LD, using synonymous (S) vs. amino acid (A) mutations. Covariation between two amino acid mutations, (A,A), can be affected by selective interactions between amino acids, whereas covariation within (A,S) pairs or (S,S) pairs cannot. Our analysis of the pol gene — including the protease and the reverse transcriptase genes — in HIV reveals that (A,A) covariation levels are enormously higher than for either (A,S) or (S,S), and thus cannot be attributed to phylogenetic effects. The magnitude of these effects suggests that a large portion of (A,A) covariation in the HIV pol gene results from selective interactions. Inspection of the most prominent (A,A) interactions in the HIV pol gene showed that they are known sites of independently identified drug resistance mutations, and physically cluster around the drug binding site. Moreover, the specific set of (A,A) interaction pairs was reproducible in different drug treatment studies, and vanished in untreated HIV samples. The (S,S) covariation curves measured a low but detectable level of background LD in HIV. PMID:17726544

  18. Reverse transcriptase genes are highly abundant and transcriptionally active in marine plankton assemblages

    PubMed Central

    Lescot, Magali; Hingamp, Pascal; Kojima, Kenji K; Villar, Emilie; Romac, Sarah; Veluchamy, Alaguraj; Boccara, Martine; Jaillon, Olivier; Iudicone, Daniele; Bowler, Chris; Wincker, Patrick; Claverie, Jean-Michel; Ogata, Hiroyuki

    2016-01-01

    Genes encoding reverse transcriptases (RTs) are found in most eukaryotes, often as a component of retrotransposons, as well as in retroviruses and in prokaryotic retroelements. We investigated the abundance, classification and transcriptional status of RTs based on Tara Oceans marine metagenomes and metatranscriptomes encompassing a wide organism size range. Our analyses revealed that RTs predominate large-size fraction metagenomes (>5 μm), where they reached a maximum of 13.5% of the total gene abundance. Metagenomic RTs were widely distributed across the phylogeny of known RTs, but many belonged to previously uncharacterized clades. Metatranscriptomic RTs showed distinct abundance patterns across samples compared with metagenomic RTs. The relative abundances of viral and bacterial RTs among identified RT sequences were higher in metatranscriptomes than in metagenomes and these sequences were detected in all metatranscriptome size fractions. Overall, these observations suggest an active proliferation of various RT-assisted elements, which could be involved in genome evolution or adaptive processes of plankton assemblage. PMID:26613339

  19. Telomerase reverse transcriptase promotes cancer cell proliferation by augmenting tRNA expression

    PubMed Central

    Khattar, Ekta; Kumar, Pavanish; Liu, Chia Yi; Akıncılar, Semih Can; Raju, Anandhkumar; Lakshmanan, Manikandan; Maury, Julien Jean Pierre; Qiang, Yu; Li, Shang; Tan, Ern Yu; Hui, Kam M.; Loh, Yuin Han

    2016-01-01

    Transcriptional reactivation of telomerase reverse transcriptase (TERT) reconstitutes telomerase activity in the majority of human cancers. Here, we found that ectopic TERT expression increases cell proliferation, while acute reductions in TERT levels lead to a dramatic loss of proliferation without any change in telomere length, suggesting that the effects of TERT could be telomere independent. We observed that TERT determines the growth rate of cancer cells by directly regulating global protein synthesis independently of its catalytic activity. Genome-wide TERT binding across 5 cancer cell lines and 2 embryonic stem cell lines revealed that endogenous TERT, driven by mutant promoters or oncogenes, directly associates with the RNA polymerase III (pol III) subunit RPC32 and enhances its recruitment to chromatin, resulting in increased RNA pol III occupancy and tRNA expression in cancers. TERT-deficient mice displayed marked delays in polyomavirus middle T oncogene–induced (PyMT-induced) mammary tumorigenesis, increased survival, and reductions in tRNA levels. Ectopic expression of either RPC32 or TERT restored tRNA levels and proliferation defects in TERT-depleted cells. Finally, we determined that levels of TERT and tRNA correlated in breast and liver cancer samples. Together, these data suggest the existence of a unifying mechanism by which TERT enhances translation in cells to regulate cancer cell proliferation. PMID:27643433

  20. [Application of fluorescent real-time reverse transcriptase-polymerase chain reaction in detecting influenza viruses].

    PubMed

    Cheng, Xiao-wen; Zhou, Li; Zhao, Jin; Fang, Shi-song; Yu, Lei; Ye, Bao-ying; He, Jian-fan; Lu, Xing; Zhang, Zai-qing; Yang, Hong

    2004-09-01

    To apply fluorescent real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in detecting influenza viruses. A total of 207 oral swab samples were obtained in 16 collections from SARS patients and suspected influenza outbreak cases. They were subjected to influenza virus detection by fluorescent real-time RT-PCR, MDCK cell culture, and hemagglutinin inhibition assay. Out of 207 samples, 79 (38.16%) were positive for influenza viruses when tested by fluorescent real-time PCR, and 62 (29.95%) positive when tested by MDCK cell culture. There was a statistically significant difference between them (chi square=8.64, P less than 0.005). From 104 cases in 9 collections dual serum samples were obtainable. When tested with hemagglutinin inhibition assay, 64 cases (61.54%) showed a 4-fold increase against H3N2 antigen. This study showed that fluorescent real-time PCR is a reliable, sensitive, and fast method for detecting influenza viruses.

  1. Real time measurements of elongation by a reverse transcriptase using surface plasmon resonance.

    PubMed Central

    Buckle, M; Williams, R M; Negroni, M; Buc, H

    1996-01-01

    A rapid direct assay for polymerase-induced elongation along a given template is an obligate requirement for understanding the processivity of polymerization and the mode of action of drugs and inhibitors on this process. Surface plasmon resonance can be used to follow the association and the dissociation rates of a given reverse transcriptase on DNA.RNA and DNA.DNA hybrids immobilized on a biotin-streptavidin surface. The addition of nucleotides complementary to the template strand produces an increase in the local mass, as deduced from an increase in the measured signal, due to elongation of the primer strand that allows an estimation of both the extent and rate of the polymerization process. The terminator drug 3'-deoxy-3'-azidothymidine triphosphate completely abolishes the increase in signal as would be expected from an inhibition of elongation. This technique provides a sensitive assay for the affinities of different polymerases for specific templates and for the effects of terminators of the elongation process. PMID:8570654

  2. Structure-activity relationship studies on a novel family of specific HIV-1 reverse transcriptase inhibitors.

    PubMed

    Bonache, María-Cruz; Chamorro, Cristina; Lobatón, Esther; De Clercq, Erik; Balzarini, Jan; Velázquez, Sonsoles; Camarasa, María-José; San-Félix, Ana

    2003-09-01

    We have previously reported the discovery and preliminary structure-activity relationships of a new class of specific HIV-1 reverse transcriptase (RT) inhibitors whose prototype compound is the 1-[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3-N-[(carboxy) methyl]-thymine. In an attempt to increase the inhibitory efficacy against HIV-1 RT of this new class of nucleosides, and to further explore the structural features required for anti-HIV-1 activity, different types of modifications have been carried out on the prototype compound. These include substitution of the tert-butyldimethylsilyl groups by other liphophilic groups, replacement of the carboxy group at the N-3 position of the nucleobase by other functional groups, change in the length of the spacer between the thymine and the carboxylic acid residue and substitution of the thymine moiety by other pyrimidine (uracil, 5-ethyluracil) or purine (hypoxanthine) nucleobases. In addition, the most salient structural features of this new class of HIV-1-specific nucleosides have been incorporated into classical HIV RT nucleoside inhibitors such as ddl, AZT, d4T. Our studies demonstrate that both the carboxymethyl moiety at the nucleobase and tert-butyldimethylsilyl groups at the sugar are important structural components since deletion of either of them is detrimental to the antiviral activity.

  3. HIV-1 protease inhibits its homologous reverse transcriptase by protein-protein interaction.

    PubMed Central

    Böttcher, M; Grosse, F

    1997-01-01

    The reading frame of the HIV-1 pol gene, encoding protease (PR) and reverse transcriptase (RT), including RNase H as well as integrase, was fused to the bacterialbeta-galactosidase gene and overexpressed in Escherichia coli cells. The resulting fusion protein was cleaved autocatalytically leading to PR, RT and integrase. Immunoprecipitations of bacterial crude extracts with anti-RT antibodies precipitated both RT and PR. Co-precipitation of PR and RT was also observed with anti-PR antibodies, strongly suggesting a physical interaction between fully processed RT and PR within the bacterial cell. Physical interactions were confirmed with purified components by means of an ELISA assay. Furthermore, purified PR inhibited the DNA synthesis activity of purified RT, while its RNase H activity remained unaffected. The type of inhibition was uncompetitive with respect to poly(rA).oligo(dT); the inhibition constant was 50-100 nM. A possible physiological significance of this type of interaction is discussed. PMID:9108151

  4. Amphiphilic cationic nanogels as brain-targeted carriers for activated nucleoside reverse transcriptase inhibitors

    PubMed Central

    Warren, G; Makarov, E; Lu, Y; Senanayake, T; Rivera, K; Gorantla, S; Poluektova, LY; Vinogradov, SV

    2015-01-01

    Progress in AIDS treatment shifted emphasis towards limiting adverse effects of antiviral drugs while improving the treatment of hard-to-reach viral reservoirs. Many therapeutic nucleoside reverse transcriptase inhibitors (NRTI) have a limited access to the central nervous system (CNS). Increased NRTI levels induced various complications during the therapy, including neurotoxicity, due to the NRTI toxicity to mitochondria. Here, we describe an innovative design of biodegradable cationic cholesterol-ε-polylysine nanogel carriers for delivery of triphosphorylated NRTIs that demonstrated high anti-HIV activity along with low neurotoxicity, warranting minimal side effects following systemic administration. Efficient CNS targeting was achieved by nanogel modification with brain-specific peptide vectors. Novel dual and triple-drug nanoformulations, analogous to therapeutic NRTI cocktails, displayed equal or higher antiviral activity in HIV-infected macrophages compared to free drugs. Our results suggest potential alternative approach to HIV-1 treatment focused on the effective nanodrug delivery to viral reservoirs in the CNS and reduced neurotoxicity. PMID:25559020

  5. Two highly antigenic sites in the human immunodeficiency virus type 1 reverse transcriptase.

    PubMed Central

    Björling, E; Boucher, C A; Samuelsson, A; Wolfs, T F; Utter, G; Norrby, E; Chiodi, F

    1993-01-01

    Antibodies to human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) are found in the serum of the majority of infected individuals, and inhibition of RT polymerase activity by HIV-1-positive sera can be demonstrated in vitro. The binding sites of human antibodies on the protein have not yet been identified. We synthesized overlapping peptides covering the entire RT protein of HIV-1 and used them in an enzyme-linked immunosorbent assay system to map the reactivities of HIV-1 and HIV-2 antibody-positive sera. Two highly antigenic regions were identified by both HIV serotypes. One region was found in the central part of the RT protein (amino acids 261 to 280) and another was found at the carboxy terminus in the RNase H portion of RT (amino acids 517 to 536). Comparison of the serological results with the crystal structure of the RT revealed that the antigenic region in the RNase H portion is located at the surface of the protein. The other antibody-binding site (amino acids 261 to 280) was located in the "thumb" region of the polymerase domain of RT. Polyclonal antibodies to either of the antibody-binding sites do not affect the polymerase activity of the RT protein. PMID:7681439

  6. Universal reverse-transcriptase real-time PCR for infectious hematopoietic necrosis virus (IHNV)

    USGS Publications Warehouse

    Purcell, Maureen K.; Thompson, Rachel L.; Garver, Kyle A.; Hawley, Laura M.; Batts, William N.; Sprague, Laura; Sampson, Corie; Winton, James R.

    2013-01-01

    Infectious hematopoietic necrosis virus (IHNV) is an acute pathogen of salmonid fishes in North America, Europe and Asia and is reportable to the World Organization for Animal Health (OIE). Phylogenetic analysis has identified 5 major virus genogroups of IHNV worldwide, designated U, M, L, E and J; multiple subtypes also exist within those genogroups. Here, we report the development and validation of a universal IHNV reverse-transcriptase real-time PCR (RT-rPCR) assay targeting the IHNV nucleocapsid (N) gene. Properties of diagnostic sensitivity (DSe) and specificity (DSp) were defined using laboratory-challenged steelhead trout Oncorhynchus mykiss, and the new assay was compared to the OIE-accepted conventional PCR test and virus isolation in cell culture. The IHNV N gene RT-rPCR had 100% DSp and DSe and a higher estimated diagnostic odds ratio (DOR) than virus culture or conventional PCR. The RT-rPCR assay was highly repeatable within a laboratory and highly reproducible between laboratories. Field testing of the assay was conducted on a random sample of juvenile steelhead collected from a hatchery raceway experiencing an IHN epizootic. The RT-rPCR detected a greater number of positive samples than cell culture and there was 40% agreement between the 2 tests. Overall, the RT-rPCR assay was highly sensitive, specific, repeatable and reproducible and is suitable for use in a diagnostic setting.

  7. The methylation status and expression of human telomerase reverse transcriptase is significantly high in oral carcinogenesis.

    PubMed

    Haraguchi, Kazuya; Yada, Naomi; Sato, Shinobu; Habu, Manabu; Hayakawa, Mana; Takahashi, Osamu; Sasaguri, Masaaki; Takenaka, Shigeori; Yoshioka, Izumi; Matsuo, Kou; Tominaga, Kazuhiro

    2017-09-01

    Telomerase activity is present in most cancers and is tightly regulated by the expression of human telomerase reverse transcriptase (hTERT). Hypermethylation in the promoter region of hTERT contributes to the regulation of hTERT expression. In this study, we investigated the methylation and expression of hTERT in oral squamous cell carcinoma (OSCC), oral leukoplakia, and normal oral mucosa. Furthermore, we investigated the significance of hTERT to the clinicopathological findings of OSCC. 35 OSCC, 50 oral leukoplakia (epithelial dysplasia n = 25, squamous cell hyperplasia n = 25), and 10 normal oral mucosa samples were investigated through methylation-specific PCR. Immunohistochemistry was analyzed in 35 OSCC, 50 oral leukoplakia, and 4 normal oral mucosa samples. The methylation and expression of hTERT increased from normal oral mucosa to oral leukoplakia to OSCC. In OSCC, all samples were methylated. However, partial methylation (20%) or unmethylation (80%), but never complete methylation, was observed in normal oral mucosa. Additionally, hTERT expression correlated with cervical lymph node metastasis. These results suggested that the methylation and expression of hTERT is high in oral carcinogenesis and may play an important role in oral cancer. hTERT expression may also be predictive of cervical lymph node metastasis. © 2017 APMIS. Published by John Wiley & Sons Ltd.

  8. Single-molecule imaging of telomerase reverse transcriptase in human telomerase holoenzyme and minimal RNP complexes

    PubMed Central

    Wu, Robert Alexander; Dagdas, Yavuz S; Yilmaz, S Tunc; Yildiz, Ahmet; Collins, Kathleen

    2015-01-01

    Telomerase synthesizes chromosome-capping telomeric repeats using an active site in telomerase reverse transcriptase (TERT) and an integral RNA subunit template. The fundamental question of whether human telomerase catalytic activity requires cooperation across two TERT subunits remains under debate. In this study, we describe new approaches of subunit labeling for single-molecule imaging, applied to determine the TERT content of complexes assembled in cells or cell extract. Surprisingly, telomerase reconstitutions yielded heterogeneous DNA-bound TERT monomer and dimer complexes in relative amounts that varied with assembly and purification method. Among the complexes, cellular holoenzyme and minimal recombinant enzyme monomeric for TERT had catalytic activity. Dimerization was suppressed by removing a TERT domain linker with atypical sequence bias, which did not inhibit cellular or minimal enzyme assembly or activity. Overall, this work defines human telomerase DNA binding and synthesis properties at single-molecule level and establishes conserved telomerase subunit architecture from single-celled organisms to humans. DOI: http://dx.doi.org/10.7554/eLife.08363.001 PMID:26457608

  9. NFAT5 regulates transcription of the mouse telomerase reverse transcriptase gene

    SciTech Connect

    Fujiki, Tsukasa; Udono, Miyako; Kotake, Yojiro; Yamashita, Makiko; Shirahata, Sanetaka; Katakura, Yoshinori

    2010-12-10

    We aimed to clarify the transcription-regulation mechanisms of the mouse telomerase reverse transcriptase gene (mTERT). First, we searched for the promoter region required for transcriptional activation of mTERT and identified an enhancer cis-element (named mTERT-EE) located between - 200 and - 179 bp of the mouse TERT gene (mTERT). EMSA results suggested that nuclear factor of activated T cells (NFAT) member proteins bind to mTERT-EE. We then identified NFAT5 as the factor binding to mTERT-EE and found that it activates the transcription of the mTERT core promoter. The results that siRNA directed against NFAT5 significantly reduced mTERT expression and mTERT core promoter activity and that the expressions of NFAT5 and mTERT were well correlated in various mouse tissues except liver suggest that NFAT5 dominantly and directly regulates mTERT expression. To clarify their functionality further, we investigated the effect of hypertonic stress, a known stimulus affecting the expression and transcriptional activity of NFAT5, on mTERT expression. The result indicated that hypertonic stress activates mTERT transcription via the activation and recruitment of NFAT5 to the mTERT promoter. These results provide useful information about the transcription-regulation mechanisms of mTERT.

  10. Human telomerase reverse transcriptase regulation by DNA methylation, transcription factor binding and alternative splicing (Review).

    PubMed

    Avin, Brittany A; Umbricht, Christopher B; Zeiger, Martha A

    2016-12-01

    The catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT), plays an essential role in telomere maintenance to oppose cellular senescence and, is highly regulated in normal and cancerous cells. Regulation of hTERT occurs through multiple avenues, including a unique pattern of CpG promoter methylation and alternative splicing. Promoter methylation affects the binding of transcription factors, resulting in changes in expression of the gene. In addition to expression level changes, changes in promoter binding can affect alternative splicing in a cotranscriptional manner. The alternative splicing of hTERT results in either the full length transcript which can form the active telomerase complex with hTR, or numerous inactive isoforms. Both regulation strategies are exploited in cancer to activate telomerase, however, the exact mechanism is unknown. Therefore, unraveling the link between promoter methylation status and alternative splicing for hTERT could expose yet another level of hTERT regulation. In an attempt to provide insight into the cellular control of active telomerase in cancer, this review will discuss our current perspective on CpG methylation of the hTERT promoter region, summarize the different forms of alternatively spliced variants, and examine examples of transcription factor binding that affects splicing.

  11. Crystal Engineering of HIV-1 Reverse Transcriptase for structure-Based Drug Design

    SciTech Connect

    Bauman,J.; Das, K.; Ho, W.; Baweja, M.; Himmel, D.; Clark, A.; Oren, D.; Shatkin, A.; Arnold, E.

    2008-01-01

    HIV-1 reverse transcriptase (RT) is a primary target for anti-AIDS drugs. Structures of HIV-1 RT, usually determined at {approx}2.5-3.0 Angstroms resolution, are important for understanding enzyme function and mechanisms of drug resistance in addition to being helpful in the design of RT inhibitors. Despite hundreds of attempts, it was not possible to obtain the structure of a complex of HIV-1 RT with TMC278, a nonnucleoside RT inhibitor (NNRTI) in advanced clinical trials. A systematic and iterative protein crystal engineering approach was developed to optimize RT for obtaining crystals in complexes with TMC278 and other NNRTIs that diffract X-rays to 1.8 Angstroms resolution. Another form of engineered RT was optimized to produce a high-resolution apo-RT crystal form, reported here at 1.85 Angstroms resolution, with a distinct RT conformation. Engineered RTs were mutagenized using a new, flexible and cost effective method called methylated overlap-extension ligation independent cloning. Our analysis suggests that reducing the solvent content, increasing lattice contacts, and stabilizing the internal low-energy conformations of RT are critical for the growth of crystals that diffract to high resolution. The new RTs enable rapid crystallization and yield high-resolution structures that are useful in designing/developing new anti-AIDS drugs.

  12. Optimization of the reverse transcriptase polymerase chain reaction for the detection of circulating prostate cells

    PubMed Central

    McIntyre, I G; Spreckley, K; Clarke, R B; Anderson, E; Clarke, N W; George, N J R

    2000-01-01

    The reverse transcriptase polymerase chain reaction (RT-PCR) is a sensitive technique that can detect prostate-specific messenger RNA in circulating blood. Many authors have studied the potential of RT-PCR as a staging technique in prostate cancer (PC). Clinical sensitivity and in some cases specificity has been disappointing. Few authors have been able to correlate RT-PCR result with patient stage. We have compared the results of using two different RT-PCR protocols with different sensitivities on blood samples from prostate cancer patients. An 80-amplification-cycle nested primer RT-PCR assay had a detection limit of 10 prostate cells and a 50-cycle RT-PCR could detect 20 cells in 5 ml blood. The 80-cycle assay detected prostate mRNA in four of 10 female samples, whereas the 50-cycle assay detected it in none. There was little difference in the assays’ ability to detect prostate mRNA in advanced PC patients. The 50-cycle assay could differentiate between hormone-escaped, stable hormone-treated and untreated localized PC patients, whereas the 80-cycle assay could not. Each blood sample must be assayed several times with RT-PCR to avoid false-negative results and, if this is done, assay specificity can be increased with little effect on clinical sensitivity. © 2000 Cancer Research Campaign PMID:10993644

  13. Reverse transcriptase genes are highly abundant and transcriptionally active in marine plankton assemblages.

    PubMed

    Lescot, Magali; Hingamp, Pascal; Kojima, Kenji K; Villar, Emilie; Romac, Sarah; Veluchamy, Alaguraj; Boccara, Martine; Jaillon, Olivier; Iudicone, Daniele; Bowler, Chris; Wincker, Patrick; Claverie, Jean-Michel; Ogata, Hiroyuki

    2016-05-01

    Genes encoding reverse transcriptases (RTs) are found in most eukaryotes, often as a component of retrotransposons, as well as in retroviruses and in prokaryotic retroelements. We investigated the abundance, classification and transcriptional status of RTs based on Tara Oceans marine metagenomes and metatranscriptomes encompassing a wide organism size range. Our analyses revealed that RTs predominate large-size fraction metagenomes (>5 μm), where they reached a maximum of 13.5% of the total gene abundance. Metagenomic RTs were widely distributed across the phylogeny of known RTs, but many belonged to previously uncharacterized clades. Metatranscriptomic RTs showed distinct abundance patterns across samples compared with metagenomic RTs. The relative abundances of viral and bacterial RTs among identified RT sequences were higher in metatranscriptomes than in metagenomes and these sequences were detected in all metatranscriptome size fractions. Overall, these observations suggest an active proliferation of various RT-assisted elements, which could be involved in genome evolution or adaptive processes of plankton assemblage.

  14. Structural Basis of the Allosteric Inhibitor Interaction on the HIV-1 Reverse Transcriptase RNase H domain

    PubMed Central

    Christen, Martin T.; Menon, Lakshmi; Myshakina, Nataliya A.; Ahn, Jinwoo; Parniak, Michael A.; Ishima, Rieko

    2012-01-01

    HIV-1 reverse transcriptase (RT) has been an attractive target for the development of antiretroviral agents. Although this enzyme is bi-functional, having both DNA polymerase and ribonuclease H (RNH) activities, there is no clinically approved inhibitor of the RNH activity. Here, we characterize the structural basis and molecular interaction of an allosteric site inhibitor, BHMP07, with the wild type (WT) RNH fragment. Solution NMR experiments for inhibitor titration on WT RNH showed relatively wide chemical shift perturbations, suggesting a long-range conformational effect on the inhibitor interaction. Comparisons of the inhibitor-induced NMR chemical-shift changes of RNH with those of RNH dimer, in the presence and absence of Mg2+, were performed to determine and verify the interaction site. The NMR results, with assistance of molecular docking, indicate that BHMP07 preferentially binds to a site that is located between the RNH active site and the region encompassing helices B and D (the “substrate-handle region”). The interaction site is consistent with the previous proposed site, identified using a chimeric RNH (p15-EC) [Gong, el (2011) Chem. Biol. Drug Des. 77, 39-47], but with slight differences that reflect the characteristics of the amino acid sequences in p15-EC compared to the WT RNH. PMID:22846652

  15. Sequence Quality Analysis Tool for HIV Type 1 Protease and Reverse Transcriptase

    PubMed Central

    DeLong, Allison K.; Wu, Mingham; Bennett, Diane; Parkin, Neil; Wu, Zhijin; Hogan, Joseph W.

    2012-01-01

    Abstract Access to antiretroviral therapy is increasing globally and drug resistance evolution is anticipated. Currently, protease (PR) and reverse transcriptase (RT) sequence generation is increasing, including the use of in-house sequencing assays, and quality assessment prior to sequence analysis is essential. We created a computational HIV PR/RT Sequence Quality Analysis Tool (SQUAT) that runs in the R statistical environment. Sequence quality thresholds are calculated from a large dataset (46,802 PR and 44,432 RT sequences) from the published literature (http://hivdb.Stanford.edu). Nucleic acid sequences are read into SQUAT, identified, aligned, and translated. Nucleic acid sequences are flagged if with >five 1–2-base insertions; >one 3-base insertion; >one deletion; >six PR or >18 RT ambiguous bases; >three consecutive PR or >four RT nucleic acid mutations; >zero stop codons; >three PR or >six RT ambiguous amino acids; >three consecutive PR or >four RT amino acid mutations; >zero unique amino acids; or <0.5% or >15% genetic distance from another submitted sequence. Thresholds are user modifiable. SQUAT output includes a summary report with detailed comments for troubleshooting of flagged sequences, histograms of pairwise genetic distances, neighbor joining phylogenetic trees, and aligned nucleic and amino acid sequences. SQUAT is a stand-alone, free, web-independent tool to ensure use of high-quality HIV PR/RT sequences in interpretation and reporting of drug resistance, while increasing awareness and expertise and facilitating troubleshooting of potentially problematic sequences. PMID:21916749

  16. HIV-1 Subtype C Reverse Transcriptase and Protease Genotypes in Zimbabwean Patients Failing Antiretroviral Therapy

    PubMed Central

    KANTOR, RAMI; ZIJENAH, LYNN S.; SHAFER, ROBERT W.; MUTETWA, SOLOMON; JOHNSTON, ELIZABETH; LLOYD, ROBERT; VON LIEVEN, ANDREA; ISRAELSKI, DENNIS; KATZENSTEIN, DAVID A.

    2008-01-01

    HIV-1 drug resistance mutations have been identified and characterized mostly in subtype B HIV-1 infection. The extent to which antiretroviral drugs select for drug resistance mutations in non-subtype B HIV-1 is not known. We obtained HIV-1 reverse transcriptase (RT) and protease sequences from 21 Zimbabwean patients failing antiretroviral drug therapy. We compared these sequences with 56 published RT and protease subtype C sequences from untreated patients, 990 RT and 1140 protease subtype B sequences from treated patients, and 340 RT and 907 protease subtype B sequences from untreated patients and identified four mutation categories of subtype C HIV-1. Seventeen of the 21 patients (81%) had known drug resistance mutations. Mutations at 15 RT and 11 protease positions were more common in subtype C isolates than in subtype B isolates. HIV-1 subtype C-infected individuals receiving antiretroviral therapy develop many of the known subtype B drug resistance mutations. Comparison of subtype C RT and protease sequences with a large database of subtype B sequences identified subtype C-specific polymorphisms and candidate drug resistance mutations. PMID:12512512

  17. Crystal engineering of HIV-1 reverse transcriptase for structure-based drug design.

    PubMed

    Bauman, Joseph D; Das, Kalyan; Ho, William C; Baweja, Mukta; Himmel, Daniel M; Clark, Arthur D; Oren, Deena A; Boyer, Paul L; Hughes, Stephen H; Shatkin, Aaron J; Arnold, Eddy

    2008-09-01

    HIV-1 reverse transcriptase (RT) is a primary target for anti-AIDS drugs. Structures of HIV-1 RT, usually determined at approximately 2.5-3.0 A resolution, are important for understanding enzyme function and mechanisms of drug resistance in addition to being helpful in the design of RT inhibitors. Despite hundreds of attempts, it was not possible to obtain the structure of a complex of HIV-1 RT with TMC278, a nonnucleoside RT inhibitor (NNRTI) in advanced clinical trials. A systematic and iterative protein crystal engineering approach was developed to optimize RT for obtaining crystals in complexes with TMC278 and other NNRTIs that diffract X-rays to 1.8 A resolution. Another form of engineered RT was optimized to produce a high-resolution apo-RT crystal form, reported here at 1.85 A resolution, with a distinct RT conformation. Engineered RTs were mutagenized using a new, flexible and cost effective method called methylated overlap-extension ligation independent cloning. Our analysis suggests that reducing the solvent content, increasing lattice contacts, and stabilizing the internal low-energy conformations of RT are critical for the growth of crystals that diffract to high resolution. The new RTs enable rapid crystallization and yield high-resolution structures that are useful in designing/developing new anti-AIDS drugs.

  18. Design and synthesis of novel distamycin-modified nucleoside analogues as HIV-1 reverse transcriptase inhibitors.

    PubMed

    Li, Chao; Ma, Chunying; Zhang, Jin; Qian, Ning; Ding, Jingjing; Qiao, Renzhong; Zhao, Yufen

    2014-02-01

    Design and synthesis of nucleoside analogues have persistently attracted extensive interest because of their potential application in the field of antiviral therapy, and its study also receives additional impetus for improvement in the ProTide technology. Previous studies have made great strides in the design and discovery of monophosphorylated nucleoside analogues as potential kinase-independent antiretrovirals. In this work, a series of nucleoside phosphoramidates modified by distamycin analogues was synthesized and evaluated as nucleoside reverse transcriptase inhibitors (NRTIs) in HIV-1-infected MT-4 and CEM cells, including variations in nucleoside, alkyl moiety, and the structure of distamycin analogues. These compounds exhibited modest potency with the EC50 value in the range of 1.3- to 6.5-fold lower than their corresponding parent drugs in MT-4 cells, which may be attributed to increasing intracellular availability due to the existence of distamycin analogue with favorable hydrophilic-lipophilic equilibrium. Meanwhile, the length of distamycin analogue was considered and assessed as an important factor that could affect antiviral activity and cytotoxicity. Enzymatic and metabolic stability studies have been performed in order to better understand the antiviral behavior of these compounds. The present work revealed the compounds to have a favorable and selective anti-HIV-1 activity in MT-4 and CEM cells, and helped to develop strategies for design and synthesis of effective monophosphorylated nucleoside analogues, which may be applied to antiretroviral research as NRTIs. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Typing and Subtyping Influenza Virus Using DNA Microarrays and Multiplex Reverse Transcriptase PCR

    PubMed Central

    Li, Jiping; Chen, Shu; Evans, David H.

    2001-01-01

    A model DNA microarray has been prepared and shown to facilitate typing and subtyping of human influenza A and B viruses. Reverse transcriptase PCR was used to prepare cDNAs encoding ∼500-bp influenza virus gene fragments, which were then cloned, sequenced, reamplified, and spotted to form a glass-bound microarray. These target DNAs included multiple fragments of the hemagglutinin, neuraminidase, and matrix protein genes. Cy3- or Cy5-labeled fluorescent probes were then hybridized to these target DNAs, and the arrays were scanned to determine the probe binding site(s). The hybridization pattern agreed perfectly with the known grid location of each target, and the signal-to-background ratio varied from 5 to 30. No cross-hybridization could be detected beyond that expected from the limited degree of sequence overlap between different probes and targets. At least 100 to 150 bp of homology was required for hybridization under the conditions used in this study. Combinations of Cy3- and Cy5-labeled DNAs can also be hybridized to the same chip, permitting further differentiation of amplified molecules in complex mixtures. In a more realistic test of the technology, several sets of multiplex PCR primers that collectively target influenza A and B virus strains were identified and were used to type and subtype several previously unsequenced influenza virus isolates. The results show that DNA microarray technology provides a useful supplement to PCR-based diagnostic methods. PMID:11158130

  20. A real-time reverse transcriptase polymerase chain reaction for detection and quantification of Vesiculovirus

    PubMed Central

    Tolardo, Aline Lavado; de Souza, William Marciel; Romeiro, Marilia Farignoli; Vieira, Luiz Carlos; Luna, Luciano Kleber de Souza; Henriques, Dyana Alves; de Araujo, Jansen; Siqueira, Carlos Eduardo Hassegawa; Colombo, Tatiana Elias; Aquino, Victor Hugo; da Fonseca, Benedito Antonio Lopes; Bronzoni, Roberta Vieira de Morais; Nogueira, Maurício Lacerda; Durigon, Edison Luiz; Figueiredo, Luiz Tadeu Moraes

    2016-01-01

    Vesiculoviruses (VSV) are zoonotic viruses that cause vesicular stomatitis disease in cattle, horses and pigs, as well as sporadic human cases of acute febrile illness. Therefore, diagnosis of VSV infections by reliable laboratory techniques is important to allow a proper case management and implementation of strategies for the containment of virus spread. We show here a sensitive and reproducible real-time reverse transcriptase polymerase chain reaction (RT-PCR) for detection and quantification of VSV. The assay was evaluated with arthropods and serum samples obtained from horses, cattle and patients with acute febrile disease. The real-time RT-PCR amplified the Piry, Carajas, Alagoas and Indiana Vesiculovirus at a melting temperature 81.02 ± 0.8ºC, and the sensitivity of assay was estimated in 10 RNA copies/mL to the Piry Vesiculovirus. The viral genome has been detected in samples of horses and cattle, but not detected in human sera or arthropods. Thus, this assay allows a preliminary differential diagnosis of VSV infections. PMID:27276185

  1. Structural investigation of HIV-1 nonnucleoside reverse transcriptase inhibitors: 2-Aryl-substituted benzimidazoles

    NASA Astrophysics Data System (ADS)

    Ziółkowska, Natasza E.; Michejda, Christopher J.; Bujacz, Grzegorz D.

    2009-11-01

    Acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV) is one of the most destructive epidemics in history. Inhibitors of HIV enzymes are the main targets to develop drugs against that disease. Nonnucleoside reverse transcriptase inhibitors of HIV-1 (NNRTIs) are potentially effective and nontoxic. Structural studies provide information necessary to design more active compounds. The crystal structures of four NNRTI derivatives of 2-aryl-substituted N-benzyl-benzimidazole are presented here. Analysis of the geometrical parameters shows that the structures of the investigated inhibitors are rigid. The important geometrical parameter is the dihedral angle between the planes of the π-electron systems of the benzymidazole and benzyl moieties. The values of these dihedral angles are in a narrow range for all investigated inhibitors. There is no significant difference between the structure of the free inhibitor and the inhibitor in the complex with RT HIV-1. X-ray structures of the investigated inhibitors are a good basis for modeling enzyme-inhibitor interactions in rational drug design.

  2. Snapshot of the equilibrium dynamics of a drug bound to HIV-1 reverse transcriptase

    NASA Astrophysics Data System (ADS)

    Kuroda, Daniel G.; Bauman, Joseph D.; Challa, J. Reddy; Patel, Disha; Troxler, Thomas; Das, Kalyan; Arnold, Eddy; Hochstrasser, Robin M.

    2013-03-01

    The anti-AIDS drug rilpivirine undergoes conformational changes to bind HIV-1 reverse transcriptase (RT), which is an essential enzyme for the replication of HIV. These changes allow it to retain potency against mutations that otherwise would render the enzyme resistant. Here we report that water molecules play an essential role in this binding process. Femtosecond experiments and theory expose the molecular level dynamics of rilpivirine bound to HIV-1 RT. Two nitrile substituents, one on each arm of the drug, are used as vibrational probes of the structural dynamics within the binding pocket. Two-dimensional vibrational echo spectroscopy reveals that one nitrile group is unexpectedly hydrogen-bonded to a mobile water molecule, not identified in previous X-ray structures. Ultrafast nitrile-water dynamics are confirmed by simulations. A higher (1.51 Å) resolution X-ray structure also reveals a water-drug interaction network. Maintenance of a crucial anchoring hydrogen bond may help retain the potency of rilpivirine against pocket mutations despite the structural variations they cause.

  3. Pharmacogenetics of nucleoside reverse-transcriptase inhibitor-associated peripheral neuropathy

    PubMed Central

    Kallianpur, Asha R; Hulgan, Todd

    2009-01-01

    Peripheral neuropathy is an important complication of antiretroviral therapy. Nucleoside reverse-transcriptase inhibitor (NRTI)-associated mitochondrial dysfunction, inflammation and nutritional factors are implicated in its pathogenesis. Pharmacogenetic and genomic studies investigating NRTI neurotoxicity have only recently become possible via the linkage of HIV clinical studies to large DNA repositories. Preliminary case–control studies using these resources suggest that host mitochondrial DNA haplogroup polymorphisms in the hemochromatosis gene and proinflammatory cytokine genes may influence the risk of peripheral neuropathy during antiretroviral therapy. These putative risk factors await confirmation in other HIV-infected populations but they have strong biological plausibility. Work to identify underlying mechanisms for these associations is ongoing. Large-scale studies incorporating clearly defined and validated methods of neuropathy assessment and the use of novel laboratory models of NRTI-associated neuropathy to clarify its pathophysiology are now needed. Such investigations may facilitate the development of more effective strategies to predict, prevent and ameliorate this debilitating treatment toxicity in diverse clinical settings. PMID:19374518

  4. Robust Suppression of HIV Replication by Intracellularly Expressed Reverse Transcriptase Aptamers Is Independent of Ribozyme Processing

    PubMed Central

    Lange, Margaret J; Sharma, Tarun K; Whatley, Angela S; Landon, Linda A; Tempesta, Michael A; Johnson, Marc C; Burke, Donald H

    2012-01-01

    RNA aptamers that bind human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) also inhibit viral replication, making them attractive as therapeutic candidates and potential tools for dissecting viral pathogenesis. However, it is not well understood how aptamer-expression context and cellular RNA pathways govern aptamer accumulation and net antiviral bioactivity. Using a previously-described expression cassette in which aptamers were flanked by two “minimal core” hammerhead ribozymes, we observed only weak suppression of pseudotyped HIV. To evaluate the importance of the minimal ribozymes, we replaced them with extended, tertiary-stabilized hammerhead ribozymes with enhanced self-cleavage activity, in addition to noncleaving ribozymes with active site mutations. Both the active and inactive versions of the extended hammerhead ribozymes increased inhibition of pseudotyped virus, indicating that processing is not necessary for bioactivity. Clonal stable cell lines expressing aptamers from these modified constructs strongly suppressed infectious virus, and were more effective than minimal ribozymes at high viral multiplicity of infection (MOI). Tertiary stabilization greatly increased aptamer accumulation in viral and subcellular compartments, again regardless of self-cleavage capability. We therefore propose that the increased accumulation is responsible for increased suppression, that the bioactive form of the aptamer is one of the uncleaved or partially cleaved transcripts, and that tertiary stabilization increases transcript stability by reducing exonuclease degradation. PMID:22948672

  5. Regulatory roles of LINE-1-encoded reverse transcriptase in cancer onset and progression.

    PubMed

    Sciamanna, Ilaria; Gualtieri, Alberto; Piazza, Pier Francesco; Spadafora, Corrado

    2014-09-30

    LINE-1 retrotransposons encode the reverse transcriptase (RT) enzyme, required for their own mobility, the expression of which is inhibited in differentiated tissues while being active in tumors. Experimental evidence indicate that the inhibition of LINE-1-derived RT restores differentiation in cancer cells, inhibits tumor progression and yields globally reprogrammed transcription profiles. Newly emerging data suggest that LINE-1-encoded RT modulates the biogenesis of miRNAs, by governing the balance between the production of regulatory double-stranded RNAs and RNA:DNA hybrid molecules, with a direct impact on global gene expression. Abnormally high RT activity unbalances the transcriptome in cancer cells, while RT inhibition restores "normal" miRNA profiles and their regulatory networks. This RT-dependent mechanism can target the myriad of transcripts - both coding and non-coding, sense and antisense - in eukaryotic transcriptomes, with a profound impact on cell fates. LINE-1-encoded RT emerges therefore as a key regulator of a previously unrecognized mechanism in tumorigenesis.

  6. Human Telomerase Reverse Transcriptase (hTERT) Positively Regulates 26S Proteasome Activity.

    PubMed

    Im, Eunju; Yoon, Jong Bok; Lee, Han-Woong; Chung, Kwang Chul

    2017-08-01

    Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase, an RNA-dependent DNA polymerase that elongates telomeric DNA. hTERT displays several extra-telomeric functions that are independent of its telomere-regulatory function, including tumor progression, and neuronal cell death regulation. In this study, we evaluated these additional hTERT non-telomeric functions. We determined that hTERT interacts with several 19S and 20S proteasome subunits. The 19S regulatory particle and 20S core particle are part of 26S proteasome complex, which plays a central role in ubiquitin-dependent proteolysis. In addition, hTERT positively regulated 26S proteasome activity independent of its enzymatic activity. Moreover, hTERT enhanced subunit interactions, which may underlie hTERT's ability of hTERT to stimulate the 26S proteasome. Furthermore, hTERT displayed cytoprotective effect against ER stress via the activation of 26S proteasome in acute myeloid leukemia cells. Our data suggest that hTERT acts as a novel chaperone to promote 26S proteasome assembly and maintenance. J. Cell. Physiol. 232: 2083-2093, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  7. Ultraviolet-induced transformation of keratinocytes: possible involvement of long interspersed element-1 reverse transcriptase.

    PubMed

    Banerjee, Gautam; Gupta, Nishma; Tiwari, Jyoti; Raman, Govindarajan

    2005-02-01

    The normal human keratinocyte cell line, HaCaT, was transformed using multiple doses of ultraviolet (UV)A+B (UVA, 150-200 mJ/cm(2) and UVB, 15-20 mJ/cm(2) x 6). Malignant transformation was confirmed by upregulation of Cyclin D1 (mRNA) and formation of colonies on soft agar. To identify the genes involved in this transformation process, we have done rapid amplification of polymorphic DNA using RNA from unexposed and multiple-exposed cells. Six percent PAGE showed several differentially regulated genes in exposed cells compared with unexposed cells. Total 19 genes were identified, cloned and sequenced. Three of these 19 cloned genes showed 99% homology at both DNA and protein levels to a stretch of 540 bp (180 aa) of long interspersed element (LINE)-1 reverse transcriptase (RT) open reading frame (ORF-2). Colonies from soft agar showed upregulation of this gene compared with non-colonized (lawn on soft agar) cells as detected by RT-PCR. This data implicates LINE-1 RT (ORF-2) in UV-induced malignancy and can possibly be used as a marker for the diagnosis of UV-induced skin cancer.

  8. Domain structure and three-dimensional model of a group II intron-encoded reverse transcriptase

    PubMed Central

    BLOCKER, FORREST J.H.; MOHR, GEORG; CONLAN, LORI H.; QI, LI; BELFORT, MARLENE; LAMBOWITZ, ALAN M.

    2005-01-01

    Group II intron-encoded proteins (IEPs) have both reverse transcriptase (RT) activity, which functions in intron mobility, and maturase activity, which promotes RNA splicing by stabilizing the catalytically active RNA structure. The LtrA protein encoded by the Lactococcus lactis Ll.LtrB group II intron contains an N-terminal RT domain, with conserved sequence motifs RT1 to 7 found in the fingers and palm of retroviral RTs; domain X, associated with maturase activity; and C-terminal DNA-binding and DNA endonuclease domains. Here, partial proteolysis of LtrA with trypsin and Arg-C shows major cleavage sites in RT1, and between the RT and X domains. Group II intron and related non-LTR retroelement RTs contain an N-terminal extension and several insertions relative to retroviral RTs, some with conserved features implying functional importance. Sequence alignments, secondary-structure predictions, and hydrophobicity profiles suggest that domain X is related structurally to the thumb of retroviral RTs. Three-dimensional models of LtrA constructed by “threading” the aligned sequence on X-ray crystal structures of HIV-1 RT (1) account for the proteolytic cleavage sites; (2) suggest a template–primer binding track analogous to that of HIV-1 RT; and (3) show that conserved regions in splicing-competent LtrA variants include regions of the RT and X (thumb) domains in and around the template–primer binding track, distal regions of the fingers, and patches on the protein’s back surface. These regions potentially comprise an extended RNA-binding surface that interacts with different regions of the intron for RNA splicing and reverse transcription. PMID:15574519

  9. Poly(A) polymerase modification and reverse transcriptase PCR amplification of environmental RNA.

    PubMed

    Botero, Lina M; D'Imperio, Seth; Burr, Mark; McDermott, Timothy R; Young, Mark; Hassett, Daniel J

    2005-03-01

    We describe a combination of two established techniques for a novel application for constructing full-length cDNA clone libraries from environmental RNA. The cDNA was cloned without the use of prescribed primers that target specific genes, and the procedure did not involve random priming. Purified RNA was first modified by addition of a poly(A) tail and then was amplified by using a commercially available reverse transcriptase PCR (RT-PCR) cDNA synthesis kit. To demonstrate the feasibility of this approach, a cDNA clone library was constructed from size-fractionated RNA (targeting 16S rRNA) purified from a geothermally heated soil in Yellowstone National Park in Wyoming. The resulting cDNA library contained clones representing Bacteria and Eukarya taxa and several mRNAs. There was no exact clone match between this library and a separate cDNA library generated from an RT-PCR performed with unmodified rRNA and Bacteria-specific forward and universal reverse primers that were designed from cultivated organisms; however, both libraries contained representatives of the Firmicutes and the alpha-Proteobacteria. Unexpectedly, there were no Archaea clones in the library generated from poly(A)-modified RNA. Additional RT-PCRs performed with universal and Archaea-biased primers and unmodified RNA demonstrated the presence of novel Archaea in the soil. Experiments with pure cultures of Sulfolobus solfataricus and Halobacterium halobium revealed that some Archaea rRNA may not be a suitable substrate for the poly(A) tail modification step. The protocol described here demonstrates the feasibility of directly accessing prokaryote RNA (rRNA and/or mRNA) in environmental samples, but the results also illustrate potentially important problems.

  10. Poly(A) Polymerase Modification and Reverse Transcriptase PCR Amplification of Environmental RNA

    PubMed Central

    Botero, Lina M.; D'Imperio, Seth; Burr, Mark; McDermott, Timothy R.; Young, Mark; Hassett, Daniel J.

    2005-01-01

    We describe a combination of two established techniques for a novel application for constructing full-length cDNA clone libraries from environmental RNA. The cDNA was cloned without the use of prescribed primers that target specific genes, and the procedure did not involve random priming. Purified RNA was first modified by addition of a poly(A) tail and then was amplified by using a commercially available reverse transcriptase PCR (RT-PCR) cDNA synthesis kit. To demonstrate the feasibility of this approach, a cDNA clone library was constructed from size-fractionated RNA (targeting 16S rRNA) purified from a geothermally heated soil in Yellowstone National Park in Wyoming. The resulting cDNA library contained clones representing Bacteria and Eukarya taxa and several mRNAs. There was no exact clone match between this library and a separate cDNA library generated from an RT-PCR performed with unmodified rRNA and Bacteria-specific forward and universal reverse primers that were designed from cultivated organisms; however, both libraries contained representatives of the Firmicutes and the α-Proteobacteria. Unexpectedly, there were no Archaea clones in the library generated from poly(A)-modified RNA. Additional RT-PCRs performed with universal and Archaea-biased primers and unmodified RNA demonstrated the presence of novel Archaea in the soil. Experiments with pure cultures of Sulfolobus solfataricus and Halobacterium halobium revealed that some Archaea rRNA may not be a suitable substrate for the poly(A) tail modification step. The protocol described here demonstrates the feasibility of directly accessing prokaryote RNA (rRNA and/or mRNA) in environmental samples, but the results also illustrate potentially important problems. PMID:15746328

  11. Solution structure of a reverse transcriptase recognition site of a LINE RNA from zebrafish.

    PubMed

    Otsu, Maina; Kajikawa, Masaki; Okada, Norihiro; Kawai, Gota

    2017-10-01

    Long interspersed nuclear element (LINE) is known to be transposed by reverse transcription using its RNA transcript. Recognition of the 3' stem-loop of LINE RNA by its reverse transcriptase (RT) is an important step of the retrotransposition. Our previous study revealed that the second G residue (G8) in the GGAUA loop of a 17mer LINE RNA from eel, UnaL2-17, is recognized by its RT and the U residue (U10) in the same loop is required to maintain the loop structure (Baba S, Kajikawa M, Okada N, Kawai G. Solution structure of an RNA stem-loop derived from the 3' conserved region of eel LINE UnaL2. RNA 2004;10:1380-1387). ZfL2-2, a LINE from zebrafish, has the same 3' stem-loop with UnaL2 and ZfL2-1 has similar but distinct 3' stem-loop with an insertion which can form an additional stem-loop. Here, we determined the solution structure of the 34mer RT recognition site of the LINE RNA (ZfL2-1-34). It was found that ZfL2-1-34 forms a hairpin with an internal loop, the tertiary structure of which is superimposed with that of ZfL2-2. It is noted that A10 and the inserted stem-loop, starting with A12, in ZfL2-1-34 located at the positions corresponding to those of G8 and U10, respectively, in UnaL2-17. These results strongly suggest that the two LINEs share the similar recognition mechanism and the A10 in ZfL2-1-34 is the determinant recognized by its RT. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  12. The reverse transcriptase encoded by LINE-1 retrotransposons in the genesis, progression and therapy of cancer

    NASA Astrophysics Data System (ADS)

    Sciamanna, Ilaria; De Luca, Chiara; Spadafora, Corrado

    2016-02-01

    In higher eukaryotic genomes, Long Interspersed Nuclear Element 1 (LINE-1) retrotransposons represent a large family of repeated genomic elements. They transpose using a reverse transcriptase (RT), which they encode as part of the ORF2p product. RT inhibition in cancer cells, either via RNA interference-dependent silencing of active LINE-1 elements, or using RT inhibitory drugs, reduces cancer cell proliferation, promotes their differentiation and antagonizes tumor progression in animal models. Indeed, the nonnucleoside RT inhibitor efavirenz has recently been tested in a phase II clinical trial with metastatic prostate cancer patients. An in-depth analysis of ORF2p in a mouse model of breast cancer showed ORF2p to be precociously expressed in precancerous lesions and highly abundant in advanced cancer stages, while being barely detectable in normal breast tissue, providing a rationale for the finding that RT-expressing tumours are therapeutically sensitive to RT inhibitors. We summarise mechanistic and gene profiling studies indicating that highly abundant LINE-1-derived RT can “sequester” RNA substrates for reverse transcription in tumor cells, entailing the formation of RNA:DNA hybrid molecules and impairing the overall production of regulatory miRNAs, with a global impact on the cell transcriptome. Based on these data, LINE-1-ORF2 encoded RT has a tumor-promoting potential that is exerted at an epigenetic level. We propose a model whereby LINE1-RT drives a previously unrecognized global regulatory process, the deregulation of which drives cell transformation and tumorigenesis and possibly implicated in cancer cell heterogeneity.

  13. Human telomerase reverse transcriptase expression in Diff-Quik-stained FNA samples from thyroid nodules.

    PubMed

    Siddiqui, M T; Greene, K L; Clark, D P; Xydas, S; Udelsman, R; Smallridge, R C; Zeiger, M A; Saji, M

    2001-06-01

    Fine-needle aspiration (FNA) is a highly sensitive method in the differential diagnosis of thyroid nodules. However, 10% of thyroid FNAs are indeterminate for cancer, and thus additional markers may be useful diagnostically. The authors have demonstrated previously that human telomerase reverse transcriptase (hTERT) gene expression is useful in the distinction of benign lesions from malignant lesions. They therefore wondered whether the detection of hTERT gene expression was feasible using archival slides. To establish an experimental system, ribonucleic acid was extracted from human anaplastic thyroid carcinoma cell line (ARO) in cytologic specimens, and reverse transcription-polymerase chain reaction (RT-PCR) for hTERT expression was performed. RT-PCR analysis for hTERT gene detection was then performed using 58 Diff-Quik-stained archival FNA samples collected retrospectively. RT-PCR for human thyroglobulin (hTg) or beta-actin gene expression served as a positive control. Successful PCR results were obtained from 48 of the 58 cases. All 10 slides in which no RT-PCR products were noted were older than 3 years. hTERT gene expression was demonstrated in FNAs from two of seven cases (29%) of hyperplastic nodule, one of one case (100%) of Hashimoto's thyroiditis, three of eight cases (38%) of follicular adenoma, three of eight cases (38%) of Hürthle cell adenoma, three of four cases (75%) of follicular carcinoma, two of two cases (100%) of Hürthle cell carcinoma, and 11 of 18 cases (61%) of papillary carcinoma. All but one of the available 33 corresponding frozen samples exhibited the same RT-PCR results. This study demonstrates that Diff-Quik-stained thyroid FNA specimens less than 3 years old can be used for the detection of hTERT gene expression by RT-PCR. This test, along with careful cytopathologic examination, may improve our ability to differentiate benign lesions from malignant lesions in indeterminate FNA samples from thyroid nodules.

  14. Detection and genotyping of human rotavirus VP4 and VP7 genes by reverse transcriptase PCR and reverse hybridization.

    PubMed

    van Doorn, Leen-Jan; Kleter, Bernhard; Hoefnagel, Evert; Stainier, Isabelle; Poliszczak, Annick; Colau, Brigitte; Quint, Wim

    2009-09-01

    Rotavirus infections can be diagnosed in stool samples by serological and molecular methods. We developed a novel reverse transcriptase PCR (RT-PCR) method for the amplification of rotavirus RNA and a reverse hybridization assay on a strip to detect amplimers and identify the specific G and P genotypes present in human stool specimens. An additional aim was to permit specific identification of the rotavirus G1P[8] strain, used in the Rotarix vaccine. Novel broad-spectrum PCR primers were developed for both VP4 and VP7, permitting the amplification of a wide range of rotavirus genotypes. Primer sets comprise mixtures of defined primer sequences. For the identification of G and P genotypes, two reverse hybridization strip assays were developed. Both the VP4 and the VP7 strip contain universal probes for the detection of VP4 and VP7 sequences, irrespective of the G or P genotype. The VP4 strip contains type-specific probes for P[4], P[6], P[8], P[9], and P[10]. The VP7 strip contains type-specific probes for G1, G2, G3, G4, G5, G6, G8, and G9. In addition, probes to distinguish between wild-type G1 and G1 vaccine strain sequences were present. Testing by analysis of multiple reference strains confirmed that both RT-PCR methods allowed the detection of a broad spectrum of genotypes. RT-PCR for VP7 was more sensitive than RT-PCR for VP4, but all samples identified as positive for rotavirus antigen by an enzyme-linked immunosorbent assay (ELISA) were also positive for both VP4 and VP7. The high specificity of the reverse hybridization method was confirmed by sequence analysis as well as by type-specific PCR, and the vaccine strain could also be specifically identified. The reverse hybridization method permits accurate identification of mixed infections with different genotypes. Rotavirus genotypes for which no type-specific probes were present on the strip were adequately identified by the universal detection probes. The assay was formally validated by analyses of

  15. Commercial reverse transcriptase as source of false-positive strand-specific RNA detection in human cells.

    PubMed

    Moison, Celine; Arimondo, Paola B; Guieysse-Peugeot, Anne-Laure

    2011-10-01

    Recently, an increasing number of studies describe the existence of non-coding RNAs (ncRNAs) involved in gene expression modulation. Since the observation that antisense ncRNAs are implicated in human disorders, there is more and more interest in ncRNAs. A commonly used technique to investigate the expression of an antisense ncRNAs is strand-specific reverse transcription coupled with polymerase chain reaction (RT-PCR). The advantage of this accurate technique is that it does not require any special equipment or expertise. The disadvantage is that it can lead easily to false-positive results. We applied strand-specific RT-PCR to investigate the presence of antisense ncRNA associated to Retinoic Acid Receptor Beta 2 (RARβ2) in different human tumoral cell lines. By performing this technique, we observed false-positive detection of ncRNA. For accurate interpretation of the results in RT-PCR experiments, we introduced a «No primer» control that reveals non-specific cDNA synthesis. Moreover, we report the presence of non-specific cDNA amplification with five of the most frequently used reverse transcriptase in absence of added primers. We found that the choice of the reverse transcriptase as well as the conditions of the reaction (RT temperature and PCR cycle number) are important parameters to choose as the different reverse transcriptases do not display the same cDNA synthesis background. This previously observed phenomenon was reported to originate from the «self-priming» of RNA template. Here, we report rather the presence of RNA contaminants associated with one of the reverse transcriptase studied that might contribute to non-specific cDNA synthesis.

  16. Heterologous induction of Ty1 retrotransposition: Reverse transcriptase plays a key role in initiation of the retrotransposition cycle

    SciTech Connect

    Reznik, N.L.; Kidgotko, O.V.; Zolotova, L.I.

    1995-12-01

    A new method was developed to study the mechanism of initiation of the retrotransposition cycle: retrotransposons of Drosophila melanogaster, gypsy, copia, and 17.6 were expressed in yeast under the control of strong yeast promoters. Expression of retrotransposons induced formation of viruslike particles (VLPs) associated with full-length Ty1 RNA and DNA sequences. This phenomenon was termed heterologous induction. When the gene for reverse transcriptase of human immunodeficiency virus (HIV) was expressed in yeast, the same results were obtained. These data allowed us to assume that the excess of active reverse transcriptase plays the key role in induction of transposition. Possible mechanisms of induction of Ty1 transposition by homologous and heterologous elements are discussed. 34 refs., 5 figs.

  17. Human mammaglobin: a superior marker for reverse-transcriptase PCR in detecting circulating tumor cells in breast cancer patients.

    PubMed

    Li, GuangLiang; Zhang, Jing; Jin, KeTao; He, KuiFeng; Wang, HaoHao; Lu, HaiQi; Teng, LiSong

    2011-04-01

    Breast cancer is the most frequent cancer in women in the USA and the second most common cause of death in females who develop cancer. Recently, the detection of circulating tumor cells has emerged as a promising tool for monitoring the progression of clinically occult micrometastases in breast cancer patients. Sensitive molecular techniques, primarily based upon the reverse-transcriptase PCR, using various molecules as markers, have been developed to detect circulating tumor cells. Among those molecules, human mammaglobin mRNA has been found to be the most specific marker for the hematogenous spread of breast cancer cells. In this article, we review the current knowledge regarding the use of reverse-transcriptase PCR for detecting human mammaglobin mRNA as a biomarker for circulating tumor cells in breast cancer patients, and evaluate the clinical implications of human mammaglobin since it was first isolated in 1996.

  18. Structure of HIV-1 nonnucleoside reverse transcriptase inhibitors derivatives of N-benzyl-benzimidazole with different substituents in position 4

    NASA Astrophysics Data System (ADS)

    Ziółkowska, Natasza E.; Michejda, Christopher J.; Bujacz, Grzegorz D.

    2010-01-01

    The constant development of new drugs against HIV-1 is necessary due to global expansion of AIDS and HIV-1 drug resistance. Nonnucleoside reverse transcriptase inhibitors of HIV-1 (NNRTIs) are potentially effective and nontoxic drugs in AIDS therapy. The crystal structures of six nonnucleoside inhibitors of HIV-1 reverse transcriptase (RT) derivatives of N-benzyl-benzimidazole are reported here. The investigated compounds belong to the group of so called "butterfly like" inhibitors with characteristic two π-electron moieties with an angled orientation. The structural data show the influence of the substituents of the benzimidazole ring on the geometry of the molecule and correlation between the structure of the inhibitor and its biological activity.

  19. Novel Codon Insert in HIV Type 1 Clade B Reverse Transcriptase Associated with Low-Level Viremia During Antiretroviral Therapy

    PubMed Central

    Gianella, Sara; Vazquez, Homero; Ignacio, Caroline; Zweig, Adam C.; Richman, Douglas D.; Smith, Davey M.

    2014-01-01

    Abstract We investigated the pol genotype in two phylogenetically and epidemiologically linked partners, who were both experiencing persistent low-level viremia during antiretroviral therapy. In one partner we identified a new residue insertion between codon 248 and 249 of the HIV-1 RNA reverse transcriptase (RT) coding region (HXB2 numbering). We then investigated the potential impact of identified mutations in RT and antiretroviral binding affinity using a novel computational approach. PMID:24020934

  20. Detection of BCR-ABL Fusion mRNA Using Reverse Transcriptase Loop-mediated Isothermal Amplification

    SciTech Connect

    Dugan, L C; Hall, S; Kohlgruber, A; Urbin, S; Torres, C; Wilson, P

    2011-12-08

    RT-PCR is commonly used for the detection of Bcr-Abl fusion transcripts in patients diagnosed with chronic myelogenous leukemia, CML. Two fusion transcripts predominate in CML, Br-Abl e13a2 and e14a2. They have developed reverse transcriptase isothermal loop-mediated amplification (RT-LAMP) assays to detect these two fusion transcripts along with the normal Bcr transcript.

  1. RNA dependent DNA replication fidelity of HIV-1 reverse transcriptase: evidence of discrimination between DNA and RNA substrates.

    PubMed

    Kerr, S G; Anderson, K S

    1997-11-18

    The RNA dependent DNA replication fidelity of HIV-1 reverse transcriptase has been investigated using pre-steady-state kinetics under single turnover conditions. In contrast to previous estimates of low replication fidelity of HIV-1 reverse transcriptase, the present study finds the enzyme to be more highly discriminating when an RNA/DNA template-primer is employed as compared with the corresponding DNA/DNA template-primer. The basis of this selectivity is due to extremely slow polymerization kinetics for incorporation of an incorrect deoxynucleotide. The maximum rates for misincorporation (kpol) of dGTP, dCTP, and dTTP opposite a template uridine were 0.2, 0.03, and 0.003 s-1, respectively. The equilibrium dissociation constants (Kd) for the incorrect nucleotide opposite a template uridine were 1.0, 1.1, and 0.7 mM for dGTP, dCTP, and dTTP, respectively. These kinetic values provide fidelity estimates of 26 000 for discrimination against dGTP, 176 000 for dCTP, and 1 x 10(6) for dTTP misincorporation at this position. Similar observations were obtained when incorrect nucleotide misincorporation was examined opposite a template adenine. Thus in a direct comparison of RNA/DNA and DNA/DNA template-primer substrates, HIV-1 RT exhibits approximately a 10-60-fold increase in fidelity. This study augments our current understanding of the similarities and differences of catalytic activity of HIV-1 reverse transcriptase using RNA and DNA substrates. Moreover, these studies lend further support for a model for nucleotide incorporation by HIV-1 reverse transcriptase involving a two-step binding mechanism governed by a rate-limiting conformational change for correct incorporation.

  2. Novel codon insert in HIV type 1 clade B reverse transcriptase associated with low-level viremia during antiretroviral therapy.

    PubMed

    Chaillon, Antoine; Gianella, Sara; Vazquez, Homero; Ignacio, Caroline; Zweig, Adam C; Richman, Douglas D; Smith, Davey M

    2014-02-01

    We investigated the pol genotype in two phylogenetically and epidemiologically linked partners, who were both experiencing persistent low-level viremia during antiretroviral therapy. In one partner we identified a new residue insertion between codon 248 and 249 of the HIV-1 RNA reverse transcriptase (RT) coding region (HXB2 numbering). We then investigated the potential impact of identified mutations in RT and antiretroviral binding affinity using a novel computational approach.

  3. Genetic identification of potential RNA-binding regions in a group II intron-encoded reverse transcriptase

    PubMed Central

    Gu, Shan-Qing; Cui, Xiaoxia; Mou, Sijiong; Mohr, Sabine; Yao, Jun; Lambowitz, Alan M.

    2010-01-01

    Mobile group II introns encode a reverse transcriptase that binds the intron RNA to promote RNA splicing and intron mobility, the latter via reverse splicing of the excised intron into DNA sites, followed by reverse transcription. Previous work showed that the Lactococcus lactis Ll.LtrB intron reverse transcriptase, denoted LtrA protein, binds with high affinity to DIVa, a stem–loop structure at the beginning of the LtrA open reading frame and makes additional contacts with intron core regions that stabilize the active RNA structure for forward and reverse splicing. LtrA's binding to DIVa down-regulates its translation and is critical for initiation of reverse transcription. Here, by using high-throughput unigenic evolution analysis with a genetic assay in which LtrA binding to DIVa down-regulates translation of GFP, we identified regions at LtrA's N terminus that are required for DIVa binding. Then, by similar analysis with a reciprocal genetic assay, we confirmed that residual splicing of a mutant intron lacking DIVa does not require these N-terminal regions, but does require other reverse transcriptase (RT) and X/thumb domain regions that bind the intron core. We also show that N-terminal fragments of LtrA by themselves bind specifically to DIVa in vivo and in vitro. Our results suggest a model in which the N terminus of nascent LtrA binds DIVa of the intron RNA that encoded it and nucleates further interactions with core regions that promote RNP assembly for RNA splicing and intron mobility. Features of this model may be relevant to evolutionarily related non-long-terminal-repeat (non-LTR)-retrotransposon RTs. PMID:20179150

  4. Interaction of aurintricarboxylic acid (ATA) with four nucleic acid binding proteins DNase I, RNase A, reverse transcriptase and Taq polymerase

    NASA Astrophysics Data System (ADS)

    Ghosh, Utpal; Giri, Kalyan; Bhattacharyya, Nitai P.

    2009-12-01

    In the investigation of interaction of aurintricarboxylic acid (ATA) with four biologically important proteins we observed inhibition of enzymatic activity of DNase I, RNase A, M-MLV reverse transcriptase and Taq polymerase by ATA in vitro assay. As the telomerase reverse transcriptase (TERT) is the main catalytic subunit of telomerase holoenzyme, we also monitored effect of ATA on telomerase activity in vivo and observed dose-dependent inhibition of telomerase activity in Chinese hamster V79 cells treated with ATA. Direct association of ATA with DNase I ( Kd = 9.019 μM)), RNase A ( Kd = 2.33 μM) reverse transcriptase ( Kd = 0.255 μM) and Taq polymerase ( Kd = 81.97 μM) was further shown by tryptophan fluorescence quenching studies. Such association altered the three-dimensional conformation of DNase I, RNase A and Taq polymerase as detected by circular dichroism. We propose ATA inhibits enzymatic activity of the four proteins through interfering with DNA or RNA binding to the respective proteins either competitively or allosterically, i.e. by perturbing three-dimensional structure of enzymes.

  5. Antimycobacterial and HIV-1 Reverse Transcriptase Activity of Julianaceae and Clusiaceae Plant Species from Mexico

    PubMed Central

    Gómez-Cansino, Rocio; Espitia-Pinzón, Clara Inés; Campos-Lara, María Guadalupe; Guzmán-Gutiérrez, Silvia Laura; Segura-Salinas, Erika; Echeverría-Valencia, Gabriela; Torras-Claveria, Laura; Cuevas-Figueroa, Xochitl Marisol; Reyes-Chilpa, Ricardo

    2015-01-01

    The extracts of 14 Julianaceae and 5 Clusiaceae species growing in Mexico were tested in vitro (50 µg/mL) against Mycobacterium tuberculosis H37Rv and HIV reverse transcriptase (HIV-RT). The Julianaceae bark and leaf extracts inhibited M. tuberculosis (>84.67%) and HIV-RT (<49.89%). The Clusiaceae leaves extracts also inhibited both targets (>58.3% and >67.6%), respectively. The IC50 values for six selected extracts and their cytotoxicity (50 µg/mL) to human macrophages were then determined. Amphipterygium glaucum, A. molle, and A. simplicifolium fairly inhibited M. tuberculosis with IC50 of 1.87–2.35 µg/mL; but their IC50 against HIV-RT was 59.25–97.83 µg/mL. Calophyllum brasiliense, Vismia baccifera, and Vismia mexicana effect on M. tuberculosis was noteworthy (IC50 3.02–3.64 µg/mL) and also inhibited RT-HIV (IC50 26.24–35.17 µg/mL). These 6 extracts (50 µg/mL) presented low toxicity to macrophages (<23.8%). The HPLC profiles of A. glaucum, A. molle, and A. simplicifolium indicated that their antimycobacterial activity cannot be related to masticadienonic, 3α, or 3β-hydromasticadienonic acids, suggesting that other compounds may be responsible for the observed activity or this might be a synergy result. The anti-HIV-RT and antimycobacterial activities induced by C. brasiliense can be attributed to the content of calanolides A, B, as well as soulatrolide. PMID:25983849

  6. Transgenic rat model of childhood-onset dermatitis by overexpressing telomerase reverse transcriptase (TERT).

    PubMed

    Kaneko, Ryosuke; Sato, Atsuko; Hamada, Shun; Yagi, Takeshi; Ohsawa, Ichiro; Ohtsuki, Mamitaro; Kobayashi, Eiji; Hirabayashi, Masumi; Murakami, Takashi

    2016-08-01

    Childhood-onset dermatitis is one of the most common skin disorders in children. Although various mouse models that mirror aspects of dermatitis have become available, there is still a need for an animal model that develops dermatitis in childhood and is more suitable for performing tissue transplantation experiments. There is emerging evidence that peripheral blood T lymphocytes from patients with dermatitis have significantly increased telomerase activity. Here, we developed telomerase reverse transcriptase (TERT)-expressing transgenic (Tg) rats that spontaneously developed eczematous skin inflammation in childhood. Newborn TERT-Tg rats developed visible dermatitis in 56 % of cases, and the skin lesions microscopically showed spongiosis and acanthosis with infiltration of lymphocytes, eosinophils and mast cells. TERT-Tg rats with dermatitis exhibited increased CD4 (2.5-fold) and CD8 (fivefold) T cell numbers compared with dermatitis-free TERT-Tg rats. Stronger TERT activity was observed in the peripheral lymphocytes of dermatitis-positive TERT-Tg rats than those of dermatitis-free TERT-Tg rats. RT-PCR analysis revealed that IL-4 was markedly elevated in the spleen of dermatitis-positive TERT-Tg rats, and that interferon-gamma was increased in the dermatitis lesions. Moreover, skin grafting of TERT-Tg rats with dermatitis onto T cell-deficient nude rats demonstrated that the inflamed skin lesions could not be maintained. Taken together, the results suggest that TERT activation in T lymphocytes is one of the potential predisposing factors for dermatitis. Moreover, our results demonstrated that the TERT-Tg rats mirror aspects of human childhood-onset dermatitis and that these animals represent a potential animal model system for studying childhood-onset dermatitis.

  7. Cloning and molecular characterization of telomerase reverse transcriptase (TERT) and telomere length analysis of Peromyscus leucopus

    PubMed Central

    Zhao, Xin; Ueda, Yasutaka; Kajigaya, Sachiko; Alaks, Glen; Desierto, Marie J; Townsley, Danielle M.; Dumitriu, Bogdan; Chen, Jichun; Lacy, Robert C.; Young, Neal S.

    2015-01-01

    Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase complex that regulates telomerase activity to maintain telomere length for all animals with linear chromosomes. As the Mus musculus (MM) laboratory mouse has very long telomeres compared to humans, a potential alternative animal model for telomere research is the Peromyscus leucopus (PL) mouse that has telomere lengths close to the human range and has the wild counterparts for comparison. We report the full TERT coding sequence (pTERT) from PL mice to use in the telomere research. Comparative analysis with eight other mammalian TERTs revealed a pTERT protein considerably homologous to other TERTs and preserved all TERT specific-sequence signatures, yet with some distinctive features. pTERT displayed the highest nucleotide and amino acid sequence homology with hamster TERT. Unlike human but similar to MM mice, pTERT expression was detected in various adult somatic tissues of PL mice, with the highest expression in testes. Four different captive stocks of PL mice and wild-captured PL mice each displayed group-specific average telomere lengths, with the longest and shortest telomeres in inbred and outbred stock mice, respectively. pTERT showed considerable numbers of synonymous and nonsynonymous mutations. A pTERT proximal promoter region cloned was homologous among PL and MM mice and rat, but with species-specific features. From PL mice, we further cloned and characterized ribosomal protein, large, P0 (pRPLP0) to use as an internal control for various assays. Peromyscus mice have been extensively used for various studies, including human diseases, for which pTERT and pRPLP0 would be useful tools. PMID:25962353

  8. Cloning and molecular characterization of telomerase reverse transcriptase (TERT) and telomere length analysis of Peromyscus leucopus.

    PubMed

    Zhao, Xin; Ueda, Yasutaka; Kajigaya, Sachiko; Alaks, Glen; Desierto, Marie J; Townsley, Danielle M; Dumitriu, Bogdan; Chen, Jichun; Lacy, Robert C; Young, Neal S

    2015-08-15

    Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase complex that regulates telomerase activity to maintain telomere length for all animals with linear chromosomes. As the Mus musculus (MM) laboratory mouse has very long telomeres compared to humans, a potential alternative animal model for telomere research is the Peromyscus leucopus (PL) mouse that has telomere lengths close to the human range and has the wild counterparts for comparison. We report the full TERT coding sequence (pTERT) from PL mice to use in the telomere research. Comparative analysis with eight other mammalian TERTs revealed a pTERT protein considerably homologous to other TERTs and preserved all TERT specific-sequence signatures, yet with some distinctive features. pTERT displayed the highest nucleotide and amino acid sequence homology with hamster TERT. Unlike human but similar to MM mice, pTERT expression was detected in various adult somatic tissues of PL mice, with the highest expression in testes. Four different captive stocks of PL mice and wild-captured PL mice each displayed group-specific average telomere lengths, with the longest and shortest telomeres in inbred and outbred stock mice, respectively. pTERT showed considerable numbers of synonymous and nonsynonymous mutations. A pTERT proximal promoter region cloned was homologous among PL and MM mice and rat, but with species-specific features. From PL mice, we further cloned and characterized ribosomal protein, large, P0 (pRPLP0) to use as an internal control for various assays. Peromyscus mice have been extensively used for various studies, including human diseases, for which pTERT and pRPLP0 would be useful tools.

  9. Extended spectrum of HIV-1 reverse transcriptase mutations in patients receiving multiple nucleoside analog inhibitors

    PubMed Central

    Gonzales, Matthew J.; Wu, Thomas D.; Taylor, Jonathan; Belitskaya, Ilana; Kantor, Rami; Israelski, Dennis; Chou, Sunwen; Zolopa, Andrew R.; Fessel, W. Jeffrey; Shafer, Robert W.

    2008-01-01

    Objective To characterize reverse transcriptase (RT) mutations by their association with extent of nucleoside RT inhibitor (NRTI) therapy. To identify mutational clusters in RT sequences from persons receiving multiple NRTI. Design A total of 1210 RT sequences from persons with known antiretroviral therapy were analyzed: 641 new sequences were performed at Stanford University Hospital; 569 were previously published. Methods Chi-square tests and logistic regression were done to identify associations between mutations and NRTI therapy. Correlation studies were done to identify mutational clusters. The Benjamini-Hochberg procedure was used to correct for multiple comparisons. Results Mutations at 26 positions were significantly associated with NRTI including 17 known resistance mutations (positions 41, 44, 62, 65, 67, 69, 70, 74, 75, 77, 116, 118, 151, 184, 210, 215, 219) and nine previously unreported mutations (positions 20, 39, 43, 203, 208, 218, 221, 223, 228). The nine new mutations correlated linearly with number of NRTI; 777 out of 817 (95%) instances occurred with known drug resistance mutations. Positions 203, 208, 218, 221, 223, and 228 were conserved in untreated persons; positions 20, 39, and 43 were polymorphic. Most NRTI-associated mutations clustered into three groups: (i) 62, 65, 75, 77, 115, 116, 151; (ii) 41, 43, 44, 118, 208, 210, 215, 223; (iii) 67, 69, 70, 218, 219, 228. Conclusions Mutations at nine previously unreported positions are associated with NRTI therapy. These mutations are probably accessory because they occur almost exclusively with known drug resistance mutations. Most NRTI mutations group into one of three clusters, although several (e.g., M184V) occur in multiple mutational contexts. PMID:12660525

  10. Telomerase Reverse Transcriptase Has an Extratelomeric Function in Somatic Cell Reprogramming*

    PubMed Central

    Kinoshita, Taisuke; Nagamatsu, Go; Saito, Shigeru; Takubo, Keiyo; Horimoto, Katsuhisa; Suda, Toshio

    2014-01-01

    Reactivation of the endogenous telomerase reverse transcriptase (TERT) catalytic subunit and telomere elongation occur during the reprogramming of somatic cells to induced pluripotent stem (iPS) cells. However, the role of TERT in the reprogramming process is unclear. To clarify its function, the reprogramming process was examined in TERT-KO somatic cells. To exclude the effect of telomere elongation, tail-tip fibroblasts (TTFs) from first generation TERT-KO mice were used. Although iPS cells were successfully generated from TERT-KO TTFs, the efficiency of reprogramming these cells was markedly lower than that of WT TTFs. The gene expression profiles of iPS cells induced from TERT-KO TTFs were similar to those of WT iPS cells and ES cells, and TERT-KO iPS cells formed teratomas that differentiated into all three germ layers. These data indicate that TERT plays an extratelomeric role in the reprogramming process, but its function is dispensable. However, TERT-KO iPS cells showed transient defects in growth and teratoma formation during continuous growth. In addition, TERT-KO iPS cells developed chromosome fusions that accumulated with increasing passage numbers, consistent with the fact that TERT is essential for the maintenance of genome structure and stability in iPS cells. In a rescue experiment, an enzymatically inactive mutant of TERT (D702A) had a positive effect on somatic cell reprogramming of TERT-KO TTFs, which confirmed the extratelomeric role of TERT in this process. PMID:24733392

  11. A uniquely prevalent nonnucleoside reverse transcriptase inhibitor resistance mutation in Russian subtype A HIV-1 viruses

    PubMed Central

    Kolomeets, Anna N.; Varghese, Vici; Lemey, Philippe; Bobkova, Marina R.; Shafer, Robert W.

    2015-01-01

    Background The subtype A variant in the Former Soviet Union (AFSU) causes most of Russia’s HIV-1 infections. However, the spectrum of drug-resistance mutations (DRMs) in antiretroviral experienced patients with this variant has not been studied. Methods Between 2010 and 2013, genotypic resistance testing was performed on plasma samples from 366 antiretroviral-experienced patients in Siberia. Results Three-hundred patients (82%) had subtype AFSU and 55 (15%) had CRF02_AG viruses. The pattern of DRMs was consistent with patient antiretroviral history with one exception. G190S was the most common nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance mutation, occurring in 55 (33%) subtype AFSU viruses from 167 NNRTI-experienced patients compared with none of 37 CRF02_AG viruses from NNRTI-experienced patients (P < 0.001). The next most common subtype AFSU NNRTI-resistance mutation, K103N, occurred in 25 (15%) viruses. Wild-type glycine (G) at position 190 is encoded by GGC in more than 99% of published AFSU strains. By contrast, G190 is encoded by GGA or GGG in 97% of other subtypes and in subtype A strains outside of the FSU. Therefore, G190S results from a single G→A transition: G (GGC) → S (AGC) almost exclusively in subtype AFSU viruses. Conclusion The predisposition of subtype AFSU to G190S is concerning because G→A is the most common HIV-1 mutation and because G190S causes higher levels of nevirapine and efavirenz resistance than K103N. This study exemplifies the need for characterizing the genetic mechanisms of resistance in diverse populations and warrants studies to verify that NRTI/NNRTI regimens are as efficacious in treating subtype AFSU as viruses belonging to other subtypes. PMID:25259833

  12. Structural Integrity of the Ribonuclease H domain in HIV-1 Reverse Transcriptase

    PubMed Central

    Slack, Ryan L.; Spiriti, Justin; Ahn, Jinwoo; Parniak, Michael A.; Zuckerman, Daniel M.; Ishima, Rieko

    2015-01-01

    The mature form of reverse transcriptase (RT) is a heterodimer comprising the intact 66-kDa subunit (p66) and a smaller 51-kDa subunit (p51) that is generated by removal of most of the RNase H (RNH) domain from a p66 subunit by proteolytic cleavage between residues 440/441. Viral infectivity is eliminated by mutations such as F440A and E438N in the proteolytic cleavage sequence, while normal processing and virus infectivity are restored by a compensatory mutation, T477A, that is located more than 10 Å away from the processing site. The molecular basis for this compensatory effect has remained unclear. We therefore investigated structural characteristics of RNH mutants using computational and experimental approaches. Our Nuclear Magnetic Resonance and Differential Scanning Fluorimetry results show that both F440A and E438N mutations disrupt RNH folding. Addition of the T477A mutation restores correct folding of the RNH domain despite the presence of the F440A or E438N mutations. Molecular dynamics simulations suggest that the T477A mutation affects the processing site by altering relative orientations of secondary structure elements. Predictions of sequence tolerance suggest that phenylalanine and tyrosine are structurally preferred at residues 440 and 441, respectively, which are the P1 and P1’ substrate residues known to require bulky side chains for substrate specificity. Interestingly, our study demonstrates that the processing site residues, which are critical for protease substrate specificity and must be exposed to the solvent for efficient processing, also function to maintain proper RNH folding in the p66/p51 heterodimer. PMID:26061827

  13. QSAR Modeling Using Large-Scale Databases: Case Study for HIV-1 Reverse Transcriptase Inhibitors.

    PubMed

    Tarasova, Olga A; Urusova, Aleksandra F; Filimonov, Dmitry A; Nicklaus, Marc C; Zakharov, Alexey V; Poroikov, Vladimir V

    2015-07-27

    Large-scale databases are important sources of training sets for various QSAR modeling approaches. Generally, these databases contain information extracted from different sources. This variety of sources can produce inconsistency in the data, defined as sometimes widely diverging activity results for the same compound against the same target. Because such inconsistency can reduce the accuracy of predictive models built from these data, we are addressing the question of how best to use data from publicly and commercially accessible databases to create accurate and predictive QSAR models. We investigate the suitability of commercially and publicly available databases to QSAR modeling of antiviral activity (HIV-1 reverse transcriptase (RT) inhibition). We present several methods for the creation of modeling (i.e., training and test) sets from two, either commercially or freely available, databases: Thomson Reuters Integrity and ChEMBL. We found that the typical predictivities of QSAR models obtained using these different modeling set compilation methods differ significantly from each other. The best results were obtained using training sets compiled for compounds tested using only one method and material (i.e., a specific type of biological assay). Compound sets aggregated by target only typically yielded poorly predictive models. We discuss the possibility of "mix-and-matching" assay data across aggregating databases such as ChEMBL and Integrity and their current severe limitations for this purpose. One of them is the general lack of complete and semantic/computer-parsable descriptions of assay methodology carried by these databases that would allow one to determine mix-and-matchability of result sets at the assay level.

  14. CHIP promotes human telomerase reverse transcriptase degradation and negatively regulates telomerase activity.

    PubMed

    Lee, Ji Hoon; Khadka, Prabhat; Baek, Seung Han; Chung, In Kwon

    2010-12-31

    The maintenance of eukaryotic telomeres requires telomerase, which is minimally composed of a telomerase reverse transcriptase (TERT) and an associated RNA component. Telomerase activity is tightly regulated by expression of human (h) TERT at both the transcriptional and post-translational levels. The Hsp90 and p23 molecular chaperones have been shown to associate with hTERT for the assembly of active telomerase. Here, we show that CHIP (C terminus of Hsc70-interacting protein) physically associates with hTERT in the cytoplasm and regulates the cellular abundance of hTERT through a ubiquitin-mediated degradation. Overexpression of CHIP prevents nuclear translocation of hTERT and promotes hTERT degradation in the cytoplasm, thereby inhibiting telomerase activity. In contrast, knockdown of endogenous CHIP results in the stabilization of cytoplasmic hTERT. However, it does not affect the level of nuclear hTERT and has no effect on telomerase activity and telomere length. We further show that the binding of CHIP and Hsp70 to hTERT inhibits nuclear translocation of hTERT by dissociating p23. However, Hsp90 binding to hTERT was not affected by CHIP overexpression. These results suggest that CHIP can remodel the hTERT-chaperone complexes. Finally, the amount of hTERT associated with CHIP peaks in G(2)/M phases but decreases during S phase, suggesting a cell cycle-dependent regulation of hTERT. Our data suggest that CHIP represents a new pathway for modulating telomerase activity in cancer.

  15. Biophysical and enzymatic properties of the simian and prototype foamy virus reverse transcriptases

    PubMed Central

    2010-01-01

    Background The foamy virus Pol protein is translated independently from Gag using a separate mRNA. Thus, in contrast to orthoretroviruses no Gag-Pol precursor protein is synthesized. Only the integrase domain is cleaved off from Pol resulting in a mature reverse transcriptase harboring the protease domain at the N-terminus (PR-RT). Although the homology between the PR-RTs from simian foamy virus from macaques (SFVmac) and the prototype foamy virus (PFV), probably originating from chimpanzee, exceeds 90%, several differences in the biophysical and biochemical properties of the two enzymes have been reported (i.e. SFVmac develops resistance to the nucleoside inhibitor azidothymidine (AZT) whereas PFV remains AZT sensitive even if the resistance mutations from SFVmac PR-RT are introduced into the PFV PR-RT gene). Moreover, contradictory data on the monomer/dimer status of the foamy virus protease have been published. Results We set out to purify and directly compare the monomer/dimer status and the enzymatic behavior of the two wild type PR-RT enzymes from SFVmac and PFV in order to get a better understanding of the protein and enzyme functions. We determined kinetic parameters for the two enzymes, and we show that PFV PR-RT is also a monomeric protein. Conclusions Our data show that the PR-RTs from SFV and PFV are monomeric proteins with similar biochemical and biophysical properties that are in some aspects comparable with MLV RT, but differ from those of HIV-1 RT. These differences might be due to the different conditions the viruses are confronted with in dividing and non-dividing cells. PMID:20113504

  16. Rapid diagnosis of Argentine hemorrhagic fever by reverse transcriptase PCR-based assay.

    PubMed Central

    Lozano, M E; Enría, D; Maiztegui, J I; Grau, O; Romanowski, V

    1995-01-01

    Argentine hemorrhagic fever (AHF) is an endemo-epidemic disease caused by Junín virus. This report demonstrates that a reverse transcriptase (RT) PCR-based assay developed in our laboratory to detect Junín virus in whole blood samples is sensitive and specific. The experiments were conducted in a double-blinded manner using 94 clinical samples collected in the area in which AHF is endemic. The RT-PCR-based assay was compared with traditional methodologies, including enzyme-linked immunosorbent assay, plaque neutralization tests, and occasionally viral isolation. The calculated parameters for RT-PCR diagnosis, with seroconversion as the "gold standard," were 98% sensitivity and 76% specificity. It is noteworthy that 94% of the patients with putative false-positive results (RT-PCR positive and no seroconversion detected) exhibited febrile syndromes of undefined etiology. These results could be interpreted to mean that most of those patients with febrile syndromes were actually infected with Junín virus but did not develop a detectable immune response. Furthermore, 8 laboratory-fabricated samples and 25 blood samples of patients outside the area in which AHF is endemic tested in a similar way were disclosed correctly (100% match). The RT-PCR assay is the only laboratory test available currently for the early and rapid diagnosis of AHF. It is sensitive enough to detect the low viremia found during the period in which immune plasma therapy can be used effectively, reducing mortality rates from 30% to less than 1%. PMID:7542268

  17. Resistance mutations against dolutegravir in HIV integrase impair the emergence of resistance against reverse transcriptase inhibitors.

    PubMed

    Oliveira, Maureen; Mesplède, Thibault; Quashie, Peter K; Moïsi, Daniela; Wainberg, Mark A

    2014-03-27

    Among 1222 antiretroviral-naive patients who received dolutegravir (DTG) as part of first-line therapy, none has developed resistance against this compound after 48-96 weeks of follow-up. Moreover, only four occurrences of virological failure with resistance mutations have been documented in previously drug-experienced patients who received DTG as a first time integrase inhibitor as a component of a second-line regimen. The R263K integrase resistance mutation was observed in two of these individuals who received suboptimal background regimens. We have previously selected mutations at position R263K, G118R, H51Y, and E138K as being associated with low-level resistance to DTG. Now, we sought to investigate the facility with which resistance on the part of R263K-containing viruses might develop. We tested the ability of DTG-resistant viruses containing either the R263K or G118R and/or H51Y mutations to develop further resistance against several reverse transcriptase inhibitors during in-vitro selection experiments. Our results show that DTG-resistant viruses are impaired in their ability to acquire further resistance to each of nevirapine and lamivudine as a consequence of their relative inability to develop resistance mutations associated with these two compounds. Our findings provide an explanation for the fact that no individual has yet progressed to virological failure with resistance mutations associated with dolutegravir in clinical trials in which patients received dolutegravir together with an optimized background regimen.

  18. Antimycobacterial and HIV-1 Reverse Transcriptase Activity of Julianaceae and Clusiaceae Plant Species from Mexico.

    PubMed

    Gómez-Cansino, Rocio; Espitia-Pinzón, Clara Inés; Campos-Lara, María Guadalupe; Guzmán-Gutiérrez, Silvia Laura; Segura-Salinas, Erika; Echeverría-Valencia, Gabriela; Torras-Claveria, Laura; Cuevas-Figueroa, Xochitl Marisol; Reyes-Chilpa, Ricardo

    2015-01-01

    The extracts of 14 Julianaceae and 5 Clusiaceae species growing in Mexico were tested in vitro (50 µg/mL) against Mycobacterium tuberculosis H37Rv and HIV reverse transcriptase (HIV-RT). The Julianaceae bark and leaf extracts inhibited M. tuberculosis (>84.67%) and HIV-RT (<49.89%). The Clusiaceae leaves extracts also inhibited both targets (>58.3% and >67.6%), respectively. The IC50 values for six selected extracts and their cytotoxicity (50 µg/mL) to human macrophages were then determined. Amphipterygium glaucum, A. molle, and A. simplicifolium fairly inhibited M. tuberculosis with IC50 of 1.87-2.35 µg/mL; but their IC50 against HIV-RT was 59.25-97.83 µg/mL. Calophyllum brasiliense, Vismia baccifera, and Vismia mexicana effect on M. tuberculosis was noteworthy (IC50 3.02-3.64 µg/mL) and also inhibited RT-HIV (IC50 26.24-35.17 µg/mL). These 6 extracts (50 µg/mL) presented low toxicity to macrophages (<23.8%). The HPLC profiles of A. glaucum, A. molle, and A. simplicifolium indicated that their antimycobacterial activity cannot be related to masticadienonic, 3α, or 3β-hydromasticadienonic acids, suggesting that other compounds may be responsible for the observed activity or this might be a synergy result. The anti-HIV-RT and antimycobacterial activities induced by C. brasiliense can be attributed to the content of calanolides A, B, as well as soulatrolide.

  19. Derivatives of mesoxalic acid block translocation of HIV-1 reverse transcriptase.

    PubMed

    Bernatchez, Jean A; Paul, Rakesh; Tchesnokov, Egor P; Ngure, Marianne; Beilhartz, Greg L; Berghuis, Albert M; Lavoie, Rico; Li, Lianhai; Auger, Anick; Melnyk, Roman A; Grobler, Jay A; Miller, Michael D; Hazuda, Daria J; Hecht, Sidney M; Götte, Matthias

    2015-01-16

    The pyrophosphate mimic and broad spectrum antiviral phosphonoformic acid (PFA, foscarnet) was shown to freeze the pre-translocational state of the reverse transcriptase (RT) complex of the human immunodeficiency virus type 1 (HIV-1). However, PFA lacks a specificity domain, which is seen as a major reason for toxic side effects associated with the clinical use of this drug. Here, we studied the mechanism of inhibition of HIV-1 RT by the 4-chlorophenylhydrazone of mesoxalic acid (CPHM) and demonstrate that this compound also blocks RT translocation. Hot spots for inhibition with PFA or CPHM occur at template positions with a bias toward pre-translocation. Mutations at active site residue Asp-185 compromise binding of both compounds. Moreover, divalent metal ions are required for the formation of ternary complexes with either of the two compounds. However, CPHM contains both an anchor domain that likely interacts with the catalytic metal ions and a specificity domain. Thus, although the inhibitor binding sites may partly overlap, they are not identical. The K65R mutation in HIV-1 RT, which reduces affinity to PFA, increases affinity to CPHM. Details with respect to the binding sites of the two inhibitors are provided on the basis of mutagenesis studies, structure-activity relationship analyses with newly designed CPHM derivatives, and in silico docking experiments. Together, these findings validate the pre-translocated complex of HIV-1 RT as a specific target for the development of novel classes of RT inhibitors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Prospective multi-institutional study of reverse transcriptase polymerase chain reaction for molecular staging of melanoma.

    PubMed

    Scoggins, Charles R; Ross, Merrick I; Reintgen, Douglas S; Noyes, R Dirk; Goydos, James S; Beitsch, Peter D; Urist, Marshall M; Ariyan, Stephan; Davidson, B Scott; Sussman, Jeffrey J; Edwards, Michael J; Martin, Robert C G; Lewis, Angela M; Stromberg, Arnold J; Conrad, Andrew J; Hagendoorn, Lee; Albrecht, Jeffrey; McMasters, Kelly M

    2006-06-20

    To evaluate the prognostic significance of molecular staging using reverse transcriptase polymerase chain reaction (RT-PCR) in detecting occult melanoma cells in sentinel lymph nodes (SLNs) and circulating bloodstream. In this multicenter study, eligibility criteria included patient age 18 to 71 years, invasive melanoma > or = 1.0 mm Breslow thickness, and no clinical evidence of metastasis. SLN biopsy and wide excision of the primary tumor were performed. SLNs were examined by serial-section histopathology and S-100 immunohistochemistry. A portion of each SLN was frozen for RT-PCR. In addition, RT-PCR was performed on peripheral-blood mononuclear cells (PBMCs). RT-PCR analysis was performed using four markers: tyrosinase, MART1, MAGE3, and GP-100. Disease-free survival (DFS), distant-DFS (DDFS), and overall survival (OS) were analyzed. A total of 1,446 patients with histologically negative SLNs underwent RT-PCR analysis. At a median follow-up of 30 months, there was no difference in DFS, DDFS, or OS between the RT-PCR-positive (n = 620) and RT-PCR-negative (n = 826) patients. Analysis of PBMC from 820 patients revealed significant differences in DFS and DDFS, but not OS, for patients with detection of more than one RT-PCR marker in peripheral blood. In this large, prospective, multi-institutional study, RT-PCR analysis on SLNs and PBMCs provides no additional prognostic information beyond standard histopathologic analysis of SLNs. Detection of more than one marker in PBMC is associated with a worse prognosis. RT-PCR remains investigational and should not be used to direct adjuvant therapy at this time.

  1. Identification of mechanistically distinct inhibitors of HIV-1 reverse transcriptase through fragment screening

    PubMed Central

    La, Jennifer; Latham, Catherine F.; Tinetti, Ricky N.; Johnson, Adam; Tyssen, David; Huber, Kelly D.; Sluis-Cremer, Nicolas; Simpson, Jamie S.; Headey, Stephen J.; Chalmers, David K.; Tachedjian, Gilda

    2015-01-01

    Fragment-based screening methods can be used to discover novel active site or allosteric inhibitors for therapeutic intervention. Using saturation transfer difference (STD) NMR and in vitro activity assays, we have identified fragment-sized inhibitors of HIV-1 reverse transcriptase (RT) with distinct chemical scaffolds and mechanisms compared to nonnucleoside RT inhibitors (NNRTIs) and nucleoside/nucleotide RT inhibitors (NRTIs). Three compounds were found to inhibit RNA- and DNA-dependent DNA polymerase activity of HIV-1 RT in the micromolar range while retaining potency against RT variants carrying one of three major NNRTI resistance mutations: K103N, Y181C, or G190A. These compounds also inhibit Moloney murine leukemia virus RT but not the Klenow fragment of Escherichia coli DNA polymerase I. Steady-state kinetic analyses demonstrate that one of these fragments is a competitive inhibitor of HIV-1 RT with respect to deoxyribonucleoside triphosphate (dNTP) substrate, whereas a second compound is a competitive inhibitor of RT polymerase activity with respect to the DNA template/primer (T/P), and consequently also inhibits RNase H activity. The dNTP competing RT inhibitor retains activity against the NRTI-resistant mutants K65R and M184V, demonstrating a drug resistance profile distinct from the nucleotide competing RT inhibitors indolopyridone-1 (INDOPY-1) and 4-dimethylamino-6-vinylpyrimidine-1 (DAVP-1). In antiviral assays, the T/P competing compound inhibits HIV-1 replication at a step consistent with an RT inhibitor. Screening of additional structurally related compounds to the three fragments led to the discovery of molecules with improved potency against HIV-1 RT. These fragment inhibitors represent previously unidentified scaffolds for development of novel drugs for HIV-1 prevention or treatment. PMID:26038551

  2. Sublimation characterization and vapor pressure estimation of an HIV nonnucleoside reverse transcriptase inhibitor using thermogravimetric analysis.

    PubMed

    Xie, Minli; Ziemba, Theresa M; Maurin, Michael B

    2003-01-01

    The purpose of this research is to investigate the sublimation process of DPC 963, a second-generation nonnucleoside reverse transcriptase inhibitor for HIV-1 retrovirus, and to better understand the effect of sublimation during active pharmaceutical ingredient (API) manufacture and formulation development, especially the drying processes. Sublimation of DPC 963 at 150 degrees C and above was determined by thermogravimetric analysis-Fourier transform infrared (TGA-FTIR). The rates of sublimation at different temperatures were measured using isothermal TGA. Condensed material was collected and analyzed by differential scanning calorimetry (DSC), x-ray powder diffraction (XRPD), and infrared (IR) spectrometry. Benzoic acid was used as a reference standard to derive a linear logarithmic relationship between sublimation/evaporation rate and vapor pressure specific to the TGA system used in this study. Sublimation and evaporation of DPC 963 were found to follow apparent zero-order kinetics. Using the Eyring equation, the enthalpy and entropy of the sublimation and evaporation processes were obtained. The enthalpies of sublimation and evaporation were found to be 29 and 22 kcal/mol, respectively. The condensed material from the vapor phase was found to exist in 2 physical forms, amorphous and crystalline. Using benzoic acid as a reference standard, vapor pressure of DPC 963 at different temperatures was calculated using the linear logarithmic relationship obtained. DPC 963 undergoes sublimation at appreciable rates at 150 degrees C and above but this is not likely to pose a serious issue during the manufacturing process. Vapor pressure estimation using thermogravimetric analysis provided sufficient accuracy to be used as a fast, simple, and safe alternative to the traditional methods of vapor pressure determination.

  3. Tissue-specific expression of telomerase reverse transcriptase gene variants in Nicotiana tabacum.

    PubMed

    Jurečková, Jana Fišerová; Sýkorová, Eva; Hafidh, Said; Honys, David; Fajkus, Jiří; Fojtová, Miloslava

    2017-03-01

    In tobacco, three sequence variants of the TERT gene have been described. We revealed unbalanced levels of TERT variant transcripts in vegetative tobacco tissues and enhanced TERT transcription and telomerase activity in reproductive tissues. Telomerase is a ribonucleoprotein complex responsible for the maintenance of telomeres, structures delimiting ends of linear eukaryotic chromosomes. In the Nicotiana tabacum (tobacco) allotetraploid plant, three sequence variants (paralogs) of the gene coding for the telomerase reverse transcriptase subunit (TERT) have been described, two of them derived from the maternal N. sylvestris genome (TERT_Cs, TERT_D) and one originated from the N. tomentosiformis paternal genome (TERT_Ct). In this work, we analyzed the transcription of TERT variants in correlation with telomerase activity in tobacco tissues. High and approximately comparable levels of TERT_Ct and TERT_Cs transcripts were detected in seedlings, roots, flower buds and leaves, while the transcript of the TERT_D variant was markedly underrepresented. Similarly, in N. sylvestris tissues, TERT_Cs transcript significantly predominated. A specific pattern of TERT transcripts was found in samples of tobacco pollen with the TERT_Cs variant clearly dominating particularly at the early stage of pollen development. Detailed analysis of TERT_C variants representation in functionally distinct fractions of pollen transcriptome revealed their prevalence in large ribonucleoprotein particles encompassing translationally silent mRNA; only a minority of TERT_Ct and TERT_Cs transcripts were localized in actively translated polysomes. Histones of the TERT_C chromatin were decorated predominantly with the euchromatin-specific epigenetic modification in both telomerase-positive and telomerase-negative tobacco tissues. We conclude that the existence and transcription pattern of tobacco TERT paralogs represents an interesting phenomenon and our results indicate its functional significance

  4. Nucleoside reverse transcriptase inhibitors induce a mitophagy-associated endothelial cytotoxicity that is reversed by coenzyme Q10 cotreatment.

    PubMed

    Xue, Stephen Y; Hebert, Valeria Y; Hayes, Danicia M; Robinson, Corie N; Glover, Mitzi; Dugas, Tammy R

    2013-08-01

    Cardiovascular complications have been documented in HIV-1 infected populations, and antiretroviral therapy may play a role. Nucleoside reverse transcriptase inhibitors (NRTIs) are antiretrovirals known to induce mitochondrial damage in endothelial cells, culminating in endothelial dysfunction, an initiating event in atherogenesis. Though the mechanism for NRTI-induced endothelial toxicity is not yet clear, our prior work suggested that a mitochondrial oxidative stress may be involved. To further delineate the mechanism of toxicity, endothelial cells were treated with NRTIs of varying subclasses, and the level of reactive oxygen species (ROS) and mitochondrial function were assessed. To test whether rescue of mitochondrial electron transport attenuated NRTI-induced endothelial cytotoxicity, in some cases, cells were cotreated with the electron transport cofactor coenzyme Q10 (Q10). At 4-6h, NRTIs increased levels of ROS but decreased the activities of electron transport chain complexes I-IV, levels of ATP and the NAD/NADH ratio. Moreover, nitric oxide levels were decreased, whereas endothelin-1 release was increased. Q10 abolished NRTI-induced mitochondria injury and effects on endothelial agonist production. Interestingly, in cells treated with NRTIs only, markers for mitochondrial toxicity returned to baseline levels by 18-24h, suggesting a compensatory mechanism for clearing damaged mitochondria. Using confocal microscopy, with confirmation utilizing the autophagy and mitophagy markers LC-3 and Nix, respectively, we observed autophagy of mitochondria at 8-10h after treatment. Q10 prevented NRTI-mediated increase in LC-3. These findings suggest that NRTI-induced mitophagy may be involved in NRTI-induced endothelial dysfunction and that this damage likely results from oxidant injury. Further, Q10 supplementation could potentially prevent NRTI-induced endothelial dysfunction.

  5. Murine leukemia virus pol gene products: analysis with antisera generated against reverse transcriptase and endonuclease fusion proteins expressed in Escherichia coli

    SciTech Connect

    Hu, S.C.; Court, D.L.; Zweig, M.; Levin, J.G.

    1986-10-01

    The organization of the murine leukemia virus (MuLV) pol gene was investigated by expressing molecular clones containing AKR MuLV reverse transcriptase or endonuclease or both gene segments in Escherichia coli and generating specific antisera against the expressed bacterial proteins. Reaction of these antisera with detergent-disrupted virus precipitated and 80-kilodalton (kDa) protein, the MuLV reverse transcriptase, and a 46-kDa protein which we believe is the viral endonuclease. A third (50-kDa) protein, related to reverse transcriptase, was also precipitated. Bacterial extracts of clones expressing reverse transcriptase and endonuclease sequences competed with the viral 80- and 46-kDa proteins, respectively. These results demonstrate that the antisera are specific for viral reverse transcriptase and endonuclease. Immunoprecipitation of AKR MuLV with antisera prepared against a bacterial protein containing only endonuclease sequences led to the observation that reverse transcriptase and endonuclease can be associated as a complex involving a disulfide bond(s).

  6. In Vitro Selection of HIV-1 CRF08_BC Variants Resistant to Reverse Transcriptase Inhibitors

    PubMed Central

    Wu, Hao; Zhang, Xiao-Min; Zhang, Hao-Jie; Zhang, Qiwei; Chen, Zhiwei; Huang, Jian-Dong

    2015-01-01

    Abstract Human immunodeficiency virus type 1 (HIV-1) circulating recombinant form 08_BC (CRF08_BC), carrying the recombinant reverse transcriptase (RT) gene from subtypes B and C, has recently become highly prevalent in Southern China. As the number of patients increases, it is important to characterize the drug resistance mutations of CRF08_BC, especially against widely used antiretrovirals. In this study, clinically isolated virus (2007CNGX-HK), confirmed to be CRF08_BC with its sequence deposited in GenBank (KF312642), was propagated in human peripheral blood mononuclear cells (PBMCs) with increasing concentrations of nevirapine (NVP), efavirenz (EFV), or lamivudine (3TC). Three different resistance patterns led by initial mutations of Y181C, E138G, and Y188C were detected after the selection with NVP. Initial mutations, in combination with other previously reported substitutions (K20R, D67N, V90I, K101R/E, V106I/A, V108I, F116L, E138R, A139V, V189I, G190A, D218E, E203K, H221Y, F227L, N348I, and T369I) or novel mutations (V8I, S134N, C162Y, L228I, Y232H, E396G, and D404N), developed during NVP selection. EFV-associated variations contained two initial mutations (L100I and Y188C) and three other mutations (V106L, F116Y, and A139V). Phenotypic analyses showed that E138R, Y181C, and G190A contributed high-level resistance to NVP, while L100I and V106L significantly reduced virus susceptibility to EFV. Y188C was 20-fold less sensitive to both NVP and EFV. As expected, M184I alone, or with V90I or D67N, decreased 3TC susceptibility by over 1,000-fold. Although the mutation profile obtained in culture may be different from the patients, these results may still provide useful information to monitor and optimize the antiretroviral regimens. PMID:25482475

  7. Tempol protects cardiomyocytes from nucleoside reverse transcriptase inhibitor-induced mitochondrial toxicity.

    PubMed

    Liu, Yongmin; Shim, Eunwoo; Nguyen, Phuonggiang; Gibbons, Alexander T; Mitchell, James B; Poirier, Miriam C

    2014-05-01

    Nucleoside reverse transcriptase inhibitors (NRTIs), essential components of combinational therapies used for treatment of human immunodeficiency virus-1, damage heart mitochondria. Here, we have shown mitochondrial compromise in H9c2 rat cardiomyocytes exposed for 16 passages (P) to the NRTIs zidovudine (AZT, 50μM) and didanosine (ddI, 50μM), and we have demonstrated protection from mitochondrial compromise in cells treated with 200μM 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine (Tempol) or 200μM 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethylpiperidine (Tempol-H), along with AZT/ddI, for 16P. Exposure to AZT/ddI caused a moderate growth inhibition at P3, P5, P7, and P13, which was not altered by addition of Tempol or Tempol-H. Mitochondrial oxidative phosphorylation capacity was determined as uncoupled oxygen consumption rate (OCR) by Seahorse XF24 Analyzer. At P5, P7, and P13, AZT/ddI-exposed cells showed an OCR reduction of 8.8-57.2% in AZT/ddI-exposed cells, compared with unexposed cells. Addition of Tempol or Tempol-H, along with AZT/ddI, resulted in OCR levels increased by about 300% above the values seen with AZT/ddI alone. The Seahorse data were further supported by electron microscopy (EM) studies in which P16 cells exposed to AZT/ddI/Tempol had less mitochondrial pathology than P16 cells exposed to AZT/ddI. Western blots of P5 cells showed that Tempol and Tempol-H upregulated expression of mitochondrial uncoupling protein-2 (UCP-2). However, Complex I activity that was reduced by AZT/ddI, was not restored in the presence of AZT/ddI/Tempol. Superoxide levels were increased in the presence of AZT/ddI and significantly decreased in cells exposed to AZT/3TC/Tempol at P3, P7, and P10. In conclusion, Tempol protects against NRTI-induced mitochondrial compromise, and UCP-2 plays a role through mild uncoupling.

  8. Tempol Protects Cardiomyocytes from Nucleoside Reverse Transcriptase Inhibitor-Induced Mitochondrial Toxicity

    PubMed Central

    Liu, Yongmin; Shim, Eunwoo; Nguyen, Phuonggiang; Gibbons, Alexander T.; Mitchell, James B.; Poirier, Miriam C.

    2014-01-01

    Nucleoside reverse transcriptase inhibitors (NRTIs), essential components of combinational therapies used for treatment of human immunodeficiency virus-1, damage heart mitochondria. Here, we have shown mitochondrial compromise in H9c2 rat cardiomyocytes exposed for 16 passages (P) to the NRTIs zidovudine (AZT, 50μM) and didanosine (ddI, 50μM), and we have demonstrated protection from mitochondrial compromise in cells treated with 200μM 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine (Tempol) or 200μM 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethylpiperidine (Tempol-H), along with AZT/ddI, for 16P. Exposure to AZT/ddI caused a moderate growth inhibition at P3, P5, P7, and P13, which was not altered by addition of Tempol or Tempol-H. Mitochondrial oxidative phosphorylation capacity was determined as uncoupled oxygen consumption rate (OCR) by Seahorse XF24 Analyzer. At P5, P7, and P13, AZT/ddI-exposed cells showed an OCR reduction of 8.8–57.2% in AZT/ddI-exposed cells, compared with unexposed cells. Addition of Tempol or Tempol-H, along with AZT/ddI, resulted in OCR levels increased by about 300% above the values seen with AZT/ddI alone. The Seahorse data were further supported by electron microscopy (EM) studies in which P16 cells exposed to AZT/ddI/Tempol had less mitochondrial pathology than P16 cells exposed to AZT/ddI. Western blots of P5 cells showed that Tempol and Tempol-H upregulated expression of mitochondrial uncoupling protein-2 (UCP-2). However, Complex I activity that was reduced by AZT/ddI, was not restored in the presence of AZT/ddI/Tempol. Superoxide levels were increased in the presence of AZT/ddI and significantly decreased in cells exposed to AZT/3TC/Tempol at P3, P7, and P10. In conclusion, Tempol protects against NRTI-induced mitochondrial compromise, and UCP-2 plays a role through mild uncoupling. PMID:24591154

  9. Telomerase Reverse Transcriptase Locus Polymorphisms and Cancer Risk: A Field Synopsis and Meta-Analysis

    PubMed Central

    Verdi, Daunia; Pooley, Karen A.; Landi, Maria T.; Egan, Kathleen M.; Baird, Duncan M.; Prescott, Jennifer; De Vivo, Immaculata; Nitti, Donato

    2012-01-01

    Background Several recent studies have provided evidence that polymorphisms in the telomerase reverse transcriptase (TERT) gene sequence are associated with cancer development, but a comprehensive synopsis is not available. We conducted a systematic review and meta-analysis of the available molecular epidemiology data regarding the association between TERT locus polymorphisms and predisposition to cancer. Methods A systematic review of the English literature was conducted by searching PubMed, Embase, Cancerlit, Google Scholar, and ISI Web of Knowledge databases for studies on associations between TERT locus polymorphisms and cancer risk. Random-effects meta-analysis was performed to pool per-allele odds ratios for TERT locus polymorphisms and risk of cancer, and between-study heterogeneity and potential bias sources (eg, publication and chasing bias) were assessed. Because the TERT locus includes the cleft lip and palate transmembrane 1-like (CLPTM1L) gene, which is in linkage disequilibrium with TERT, CLPTM1L polymorphisms were also analyzed. Cumulative evidence for polymorphisms with statistically significant associations was graded as “strong,” “moderate,” and “weak” according to the Venice criteria. The joint population attributable risk was calculated for polymorphisms with strong evidence of association. Results Eighty-five studies enrolling 490 901 subjects and reporting on 494 allelic contrasts were retrieved. Data were available on 67 TERT locus polymorphisms and 24 tumor types, for a total of 221 unique combinations of polymorphisms and cancer types. Upon meta-analysis, a statistically significant association with the risk of any cancer type was found for 22 polymorphisms. Strong, moderate, and weak cumulative evidence for association with at least one tumor type was demonstrated for 11, 9, and 14 polymorphisms, respectively. For lung cancer, which was the most studied tumor type, the estimated joint population attributable risk for three

  10. Hepatitis B virus reverse transcriptase mutations in treatment Naïve chronic hepatitis B patients.

    PubMed

    Singla, Bhupesh; Chakraborti, Anuradha; Sharma, Bal Krishan; Kapil, Shweta; Chawla, Yogesh K; Arora, Sunil K; Das, Ashim; Dhiman, Radha K; Duseja, Ajay

    2013-07-01

    Mutations in the reverse transcriptase (RT) region of the hepatitis B virus (HBV) genome lead to decreased susceptibility to nucleos(t)ide analogs approved for treatment of HBV infection. The aim of this study was to detect and analyze pre-existing HBV RT mutations in treatment naïve patients with chronic hepatitis B. Seventy one chronic HBV treatment naïve patients were enrolled from January 2009 to June 2011. HBV RT sequence analysis was done by using direct bidirectional sequencing of semi-nested PCR products. HBV genotypes were determined by multiplex PCR. Genotype D was found in 64 patients (90.1%) followed by genotype C and A which were present in 5 (7.0%) and 2 (2.8%) patients respectively. The results of the RT sequence analysis showed mutations in 34 (47.9%) patients. The rtH248N mutation was the most common mutation, accounting for 47.1% patients. Other common mutations included rtD263E/S, rtM129L, rtF122L/V/I, rtS135Y/H, rtQ149K, rtL91I, rtH126R, rtC256S/G, rtY257W, rtS259T and rtE271D, which were present in 26.5% (9/34), 29.4% (10/34), 20.6% (7/34), 20.6% (7/34), 20.6% (7/34), 17.6% (6/34), 14.7% (5/34), 14.7% (5/34), 11.8% (4/34), 11.8% (4/34) and 11.8% (4/34) patients respectively. The known primary drug resistance mutations were found in 3 (8.8%) patients. The present study shows the presence of RT amino acid substitutions in treatment-naïve patients with chronic hepatitis B, which may decrease susceptibility to available oral antiviral drugs. On the basis of the finding of this study, genotypic testing is recommended before the start of therapy in naïve patients, so that suitable antiviral drugs can be prescribed.

  11. Subunit-specific mutational analysis of residue N348 in HIV-1 reverse transcriptase.

    PubMed

    Radzio, Jessica; Sluis-Cremer, Nicolas

    2011-08-22

    N348I in HIV-1 reverse transcriptase (RT) confers resistance to zidovudine (AZT) and nevirapine. Biochemical studies demonstrated that N348I indirectly increases AZT resistance by decreasing the frequency of secondary ribonuclease H (RNase H) cleavages that reduce the RNA/DNA duplex length of the template/primer (T/P) and diminish the efficiency of AZT-monophosphate (MP) excision. By contrast, there is some discrepancy in the literature in regard to the mechanisms associated with nevirapine resistance: one study suggested that it is due to decreased inhibitor binding while others suggest that it may be related to the decreased RNase H cleavage phenotype. From a structural perspective, N348 in both subunits of RT resides distal to the enzyme's active sites, to the T/P binding tract and to the nevirapine-binding pocket. As such, the structural mechanisms associated with the resistance phenotypes are not known. Using a novel modelled structure of RT in complex with an RNA/DNA T/P, we identified a putative interaction between the β14-β15 loop in the p51 subunit of RT and the RNA template. Substitution of the asparagine at codon 348 in the p51 subunit with either isoleucine or leucine abrogated the observed protein-RNA interaction, thus, providing a possible explanation for the decreased RNase H phenotype. By contrast, alanine or glutamine substitutions exerted no effect. To validate this model, we introduced the N348I, N348L, N348A and N348Q mutations into RT and purified enzymes that contained subunit-specific mutations. N348I and N348L significantly decreased the frequency of secondary RNase H cleavages and increased the enzyme's ability to excise AZT-MP. As predicted by the modelling, this phenotype was due to the mutation in the p51 subunit of RT. By contrast, the N348A and N348Q RTs exhibited RNase H cleavage profiles and AZT-MP excision activities similar to the wild-type enzyme. All N348 mutant RTs exhibited decreased nevirapine susceptibility, although the N

  12. Antisense oligodeoxynucleotide against human telomerase reverse transcriptase inhibits the proliferation of Eca-109 esophageal carcinoma cells

    PubMed Central

    FAN, XIANG-KUI; YAN, RUI-HUA; LI, BAO-JIANG; CHEN, XIANG-MING; WEI, LIN; WANG, ZHOU

    2014-01-01

    Previous studies have demonstrated that the growth of tumor cells may be inhibited by antisense oligonucleotides (ASODNs) targeted against human telomerase (hTR) or human telomerase reverse transcriptase (hTERT), resulting in antitumor activity in a wide variety of tumors. However, few studies have investigated the effect of hTERT gene-targeted ASODNs on telomerase activity and cell proliferation in human esophageal cancer. In the present study, an MTT assay was used to determine the growth inhibition rate of Eca-109 cells treated with a hTERT-targeted phosphorothioate-ASODN (PS-ASODN). An inverted microscope was used to observe the morphologic changes of the cells following treatment with 5 μM PS-ASODN for 10 days. Telomerase activity was detected using the silver staining semi-quantitative telomeric repeat amplification protocol (TRAP) assay. Following treatment with the PS-ASODN (1–5 μmol/l), the proliferation of the Eca-109 cells was inhibited. The differences in inhibition rate between the PS-ASODN and blank control groups were statistically significant (P<0.05) when the concentration of the PS-ASODN was ≥2 μmol/l, whereas no statistically significant difference was identified between the non-specific-ASODN and blank control groups. The inhibition rate increased gradually as the concentration of the PS-ASODN increased and with time, suggesting that the PS-ASODN inhibited the growth of Eca-109 cells in a concentration-dependent, time-dependent and sequence-specific manner. The growth rate of the cells incubated with the PS-ASODN was reduced compared with that of the control cells. Cells treated with the PS-ASODN became round, suspended and reduced in size. The PS-ASODN was also found to inhibit telomerase activity. The ability of the PS-ASODN to inhibit the telomerase activity and cell proliferation of the Eca-109 cell line suggests that ASODNs have the potential to be novel therapeutic agents for the treatment of esophageal cancer. PMID:25187833

  13. Semiquantification of circulating hepatocellular carcinoma cells by reverse transcriptase polymerase chain reaction.

    PubMed Central

    Wong, I. H.; Leung, T.; Ho, S.; Lau, W. Y.; Chan, M.; Johnson, P. J.

    1997-01-01

    Hepatocellular carcinoma (HCC) is one of the most common and rapidly fatal malignancies worldwide. Treatment options are severely limited by the frequent presence of metastases. If hepatocyte-specific mRNAs are detected in the circulation, it is possible to infer the presence of circulating, presumably malignant, liver cells. If these can be quantified, it is possible to predict the likelihood of haematogenous metastasis. In this investigation, we have attempted to gain an index of the mass of circulating HCC cells (with reference to the number of hepatoblastoma cells) by measuring the amounts of PCR products for albumin (alb) mRNA and alpha-fetoprotein (afp) mRNA by reverse transcriptase polymerase chain reaction (RT-PCR) and Southern blot analysis. For calibration, total RNA from 1-10(6) HepG2 cells was mixed with total RNA from 10(6) normal peripheral mononuclear cells. A linear relationship was demonstrated between the amount of alb- or afp PCR product and the level of HepG2 total RNA spiked. The assay is sensitive down to a detection level of one HepG2 cell. Alb mRNA was detected in 50% of 18 normal subjects and afp mRNA in only two normal subjects. The alb mRNA cut-off level for the normal was exceeded by seven normal subjects and 34 out of 64 HCC patients, and that for afp mRNA was exceeded by six HCC patients but none of the normal subjects. The level of alb mRNA detected was not linearly proportional to the amount of afp mRNA detected in peripheral blood of the same patients, suggesting heterogeneous expression of alb and afp genes in different circulating tumour cells. In addition, no significant linear association between the levels of afp mRNA and serum AFP was observed. Semiquantification of both mRNA markers for HCC cell detection may prove useful in prediction of metastases. Images Figure 1 Figure 2 Figure 5 Figure 6 PMID:9303362

  14. Anti-HIV efficacy and biodistribution of nucleoside reverse transcriptase inhibitors delivered as squalenoylated prodrug nanoassemblies.

    PubMed

    Hillaireau, Hervé; Dereuddre-Bosquet, Nathalie; Skanji, Rym; Bekkara-Aounallah, Fawzia; Caron, Joachim; Lepêtre, Sinda; Argote, Sébastien; Bauduin, Laurent; Yousfi, Rahima; Rogez-Kreuz, Christine; Desmaële, Didier; Rousseau, Bernard; Gref, Ruxandra; Andrieux, Karine; Clayette, Pascal; Couvreur, Patrick

    2013-07-01

    Due to their hydrophilic nature, most nucleoside reverse transcriptase inhibitors (NRTIs) display a variable bioavailability after oral administration and a poor control over their biodistribution, thus hampering their access to HIV sanctuaries. The limited cellular uptake and activation in the triphosphate form of NRTIs further restrict their efficacy and favour the emergence of viral resistance. We have shown that the conjugation of squalene (sq) to the nucleoside analogues dideoxycytidine (ddC) and didanosine (ddI) leads to amphiphilic prodrugs (ddC-sq and ddI-sq) that spontaneously self-organize in water as stable nanoassemblies of 100-300 nm. These nanoassemblies can also be formulated with polyethylene glycol coupled to either cholesterol (Chol-PEG) or squalene (sq-PEG). When incubated with peripheral blood mononuclear cells (PBMCs) in vitro infected with HIV, the NRTI-sq prodrugs enhanced the antiviral efficacy of the parent NRTIs, with a 2- to 3-fold decrease of the 50% effective doses and a nearly 2-fold increase of the selectivity index. This was also the case with HIV-1 strains resistant to ddC and/or ddI. The enhanced antiviral activity of ddI-sq was correlated with an up to 5-fold increase in the intracellular concentration of the corresponding pharmacologically active metabolite ddA-TP. The ddI-sq prodrug was further investigated in vivo by the oral route, the preferred route of administration of NRTIs. Pharmacokinetics studies performed on rats showed that the prodrug maintained low amounts of free ddI in the plasma. Administration of (3)H-ddI-sq led to radioactivity levels higher in the plasma and relevant organs in HIV infection as compared to administration of free (3)H-ddI. Taken together, these results show the potential of the squalenoylated prodrugs of NRTIs to enhance their absorption and improve their biodistribution, but also to enhance their intracellular delivery and antiviral efficacy towards HIV-infected cells.

  15. Viral resistance to human immunodeficiency virus type 1-specific pyridinone reverse transcriptase inhibitors.

    PubMed Central

    Nunberg, J H; Schleif, W A; Boots, E J; O'Brien, J A; Quintero, J C; Hoffman, J M; Emini, E A; Goldman, M E

    1991-01-01

    Human immunodeficiency virus type 1 (HIV-1)-specific pyridinone reverse transcriptase (RT) inhibitors prevent HIV-1 replication in cell culture (M. E. Goldman, J. H. Nunberg, J. A. O'Brien, J.C. Quintero, W. A. Schleif, K. F. Freund, S. L. Gaul, W. S. Saari, J. S. Wai, J. M. Hoffman, P. S. Anderson, D. J. Hupe, E. A. Emini, and A. M. Stern, Proc. Natl. Acad. Sci. USA 88:6863-6867, 1991). In contrast to nucleoside analog inhibitors, such as AZT, which need to be converted to triphosphates by host cells, these compounds act directly to inhibit RT via a mechanism which is noncompetitive with respect to deoxynucleoside triphosphates. As one approach to define the mechanism of action of pyridinone inhibitors, we isolated resistant mutants of HIV-1 in cell culture. Serial passage in the presence of inhibitor yielded virus which was 1,000-fold resistant to compounds of this class. Bacterially expressed RTs molecularly cloned from resistant viruses were also resistant. The resistant RT genes encoded two amino acid changes, K-103 to N and Y-181 to C, each of which contributed partial resistance. The mutation at amino acid 181 lies adjacent to the conserved YG/MDD motif found in most DNA and RNA polymerases. The mutation at amino acid 103 lies within a region of RT which may be involved in PPi binding. The resistant viruses, although sensitive to nucleoside analogs, were cross-resistant to the structurally unrelated RT inhibitors TIBO R82150 (R. Pauwels, K. Andries, J. Desmyter, D. Schols, M. J. Kukla, H. J. Breslin, A. Raeymaeckers, J. Van Gelder, R. Woestenborghs, J. Heykanti, K. Schellekens, M. A. C. Janssen, E. De Clercq, and P. A. J. Janssen, Nature [London] 343:470-474, 1990) and BI-RG-587 (V. J. Merluzzi, K. D. Hargrave, M. Labadia, K. Grozinger, M. Skoog, J. C. Wu, C.-K. Shih, K. Eckner, S. Hattox, J. Adams, A. S. Rosenthal, R. Faanes, R. J. Eckner, R. A. Koup, and J. L. Sullivan, Science 250:1411-1413, 1990). Thus, these nonnucleoside analog inhibitors may share a

  16. Sequence and structure based models of HIV-1 protease and reverse transcriptase drug resistance

    PubMed Central

    2013-01-01

    Background Successful management of chronic human immunodeficiency virus type 1 (HIV-1) infection with a cocktail of antiretroviral medications can be negatively affected by the presence of drug resistant mutations in the viral targets. These targets include the HIV-1 protease (PR) and reverse transcriptase (RT) proteins, for which a number of inhibitors are available on the market and routinely prescribed. Protein mutational patterns are associated with varying degrees of resistance to their respective inhibitors, with extremes that can range from continued susceptibility to cross-resistance across all drugs. Results Here we implement statistical learning algorithms to develop structure- and sequence-based models for systematically predicting the effects of mutations in the PR and RT proteins on resistance to each of eight and eleven inhibitors, respectively. Employing a four-body statistical potential, mutant proteins are represented as feature vectors whose components quantify relative environmental perturbations at amino acid residue positions in the respective target structures upon mutation. Two approaches are implemented in developing sequence-based models, based on use of either relative frequencies or counts of n-grams, to generate vectors for representing mutant proteins. To the best of our knowledge, this is the first reported study on structure- and sequence-based predictive models of HIV-1 PR and RT drug resistance developed by implementing a four-body statistical potential and n-grams, respectively, to generate mutant attribute vectors. Performance of the learning methods is evaluated on the basis of tenfold cross-validation, using previously assayed and publicly available in vitro data relating mutational patterns in the targets to quantified inhibitor susceptibility changes. Conclusion Overall performance results are competitive with those of a previously published study utilizing a sequence-based strategy, while our structure- and sequence

  17. Isolation and characterization of reverse transcriptase fragments of LTR retrotransposons from the genome of Chenopodium quinoa (Amaranthaceae).

    PubMed

    Kolano, Bozena; Bednara, Edyta; Weiss-Schneeweiss, Hanna

    2013-10-01

    High heterogeneity was observed among conserved domains of reverse transcriptase ( rt ) isolated from quinoa. Only one Ty1- copia rt was highly amplified. Reverse transcriptase sequences were located predominantly in pericentromeric region of quinoa chromosomes. The heterogeneity, genomic abundance, and chromosomal distribution of reverse transcriptase (rt)-coding fragments of Ty1-copia and Ty3-gypsy long terminal repeat retrotransposons were analyzed in the Chenopodium quinoa genome. Conserved domains of the rt gene were amplified and characterized using degenerate oligonucleotide primer pairs. Sequence analyses indicated that half of Ty1-copia rt (51 %) and 39 % of Ty3-gypsy rt fragments contained intact reading frames. High heterogeneity among rt sequences was observed for both Ty1-copia and Ty3-gypsy rt amplicons, with Ty1-copia more heterogeneous than Ty3-gypsy. Most of the isolated rt fragments were present in quinoa genome in low copy numbers, with only one highly amplified Ty1-copia rt sequence family. The gypsy-like RNase H fragments co-amplified with Ty1-copia-degenerate primers were shown to be highly amplified in the quinoa genome indicating either higher abundance of some gypsy families of which rt domains could not be amplified, or independent evolution of this gypsy-region in quinoa. Both Ty1-copia and Ty3-gypsy retrotransposons were preferentially located in pericentromeric heterochromatin of quinoa chromosomes. Phylogenetic analyses of newly amplified rt fragments together with well-characterized retrotransposon families from other organisms allowed identification of major lineages of retroelements in the genome of quinoa and provided preliminary insight into their evolutionary dynamics.

  18. Prediction of response to preoperative chemoradiotherapy in rectal cancer by using reverse transcriptase polymerase chain reaction analysis of four genes.

    PubMed

    Watanabe, Toshiaki; Kobunai, Takashi; Akiyoshi, Takashi; Matsuda, Keiji; Ishihara, Soichiro; Nozawa, Keijiro

    2014-01-01

    Patients with rectal cancer exhibit a wide spectrum of responses to chemoradiotherapy. Several gene expression signatures have been reported to predict the response to chemoradiotherapy in rectal cancer, but the lack of practical assays has restricted the clinical use of this technique. We aimed to identify a set of discriminating genes that can be used for the clinical prediction of response to chemoradiotherapy in rectal cancer. This study is a retrospective analysis of tumor samples in a single institute. Sixty-two patients who underwent preoperative chemoradiotherapy were studied. Gene expression was initially studied in 46 training samples by microarray analysis, and the association between gene expression and response to chemoradiotherapy was evaluated. Quantitative reverse transcriptase polymerase chain reaction was performed to validate the microarray expression levels of the discriminating genes. We developed a gene expression model for the prediction of response to chemoradiotherapy based on the reverse transcriptase polymerase chain reaction findings and validated it by using 16 independent test samples. We identified 24 discriminating probes with expression levels that differed significantly between responders and nonresponders. Among 18 genes identified by Gene Symbol, real-time reverse transcriptase polymerase chain reaction showed significant differences in the expression of 16 genes between responders and nonresponders. We constructed a predictive model by using different sets of these 16 genes, and the highest accuracy rate (89.1%) was obtained by using LRRIQ3, FRMD3, SAMD5, and TMC7. The predictive accuracy rate of this 4-gene signature in the independent set of 16 patients was 81.3%. Validation in a different and large cohort of patients is necessary. The 4-gene signature identified in this study is closely associated with response to chemoradiotherapy in rectal cancer.

  19. APOBEC3G is a single-stranded DNA cytidine deaminase and functions independently of HIV reverse transcriptase

    PubMed Central

    Suspène, Rodolphe; Sommer, Peter; Henry, Michel; Ferris, Stéphane; Guétard, Denise; Pochet, Sylvie; Chester, Ann; Navaratnam, Naveenan; Wain-Hobson, Simon; Vartanian, Jean-Pierre

    2004-01-01

    In the absence of the viral vif gene, human immunodeficiency virus (HIV) may be restricted by the APOBEC3G gene on chromosome 22. The role of the HIV Vif protein is to exclude host cell APOBEC3G from the budding virion. As APOBEC3G shows sequence homology to cytidine deaminases, it is presumed that in the absence of Vif, cytidine residues in the cDNA are deaminated yielding uracil. It is not known if additional proteins mediate APOBEC3G function or if deamination occurs in concert with reverse transcription. This report describes an in vitro assay showing that Baculovirus derived APOBEC3G alone extensively deaminates cDNA independently of reverse transcriptase. It reproduces the dinucleotide context typical of G → A hypermutants derived from a Δvif virus. By using an RNaseH– form of reverse transcriptase, it was shown that the cDNA has to be free of its RNA template to allow deamination. APOBEC3G deamination of dC or dCTP was not detected. In short, APOBEC3G is a single-stranded DNA cytidine deaminase capable of restricting retroviral replication. PMID:15121899

  20. Treatment-limiting toxicities associated withnucleoside analogue reverse transcriptase inhibitor therapy: A prospective, observational study**

    PubMed Central

    Palacios, Rosario; Santos, Jesús; Camino, Xavier; Arazo, Piedad; Torres Perea, Rafael; Echevarrfa, Santiago; Ribera, Esteban; Sánchez de la Rosa, Rainel; Moreno Guillen, Santiago

    2005-01-01

    Background: The Recover Study is an ongoing, prospective study designed 10 to assess toxicity associated with the use of nucleoside analogue reverse transcriptase inhibitors (NRTIs) (stavudine, zidovudine, lamivudine, didanosine, abacavir) in HIV-1-infected patients receiving highly active antiretroviral therapy (HAART) in routine clinical practice. This project is being conducted at 120 HIV units at teaching hospitals across Spain. Objective: The aim of this study was to identify the most common treatment-limiting 10 moderate to severe clinical and laboratory adverse effects (AEs), and the individual NRTIs involved in the development of these effects, in HIV-1-infected patients receiving HAART who discontinued use of an NRTI in the Recover Study. Methods: Patients eligible for participation in the Recover Study are aged10 ≥18 years; have virologically documented HIV-1 infection; have sustained viral suppression (viral load <200 cells/mL or stable, heavily experienced [ie, have received ≥3 antiretroviral regimens] patients with viral load <5000 cells/mL) for ≥6 months; are receiving HAART; are undergoing active follow-up; and have developed 2:1 NRTI-associated AE that, in the opinion of a study investigator and under the conditions of routine clinical practice, justified discontinuation of treatment with the offending drug (principal AE/offending NRTI). The present study included patients recruited for the Recover Study between September 2002 and May 2003. Results: A total of 1391 patients were enrolled (966 men, 425 women; mean 1 age, 42 years [range, 18–67 years]). Five hundred six patients (36.4%) had been diagnosed with AIDS. The mean duration of treatment with the offending NRTI was 74 months (range, 6–156 months). Seven hundred nine patients (51.0%) were receiving fourth-line (or more) therapy. Eight hundred twenty-one patients (59.0%) were receiving nonnucleoside analogues, and 552 patients (39.7%), protease inhibitors, as components of their HAART

  1. Human telomerase reverse transcriptase binds to a pre-organized hTR in vivo exposing its template

    PubMed Central

    Zemora, Georgeta; Handl, Stefan; Waldsich, Christina

    2016-01-01

    Telomerase is a specialized reverse transcriptase that is responsible for telomere length maintenance. As in other organisms, the minimal components required for an active human telomerase are the template-providing telomerase RNA (hTR) and the enzymatic entity telomerase reverse transcriptase (hTERT). Here, we explored the structure of hTR and the hTERT-induced conformational changes within hTR in living cells. By employing an in vivo DMS chemical probing technique, we showed that the pseudoknot and associated triple helical scaffold form stably in vivo independently of hTERT. In fact, the dimethyl-sulfate (DMS) modification pattern suggests that hTR alone is capable of adopting a conformation that is suited to interact with hTERT. However, in the absence of hTERT the template region of hTR is only weakly accessible to DMS-modifications. The predominant change after binding of hTERT to hTR is the exposure of the template region. PMID:26481359

  2. Lersivirine, a Nonnucleoside Reverse Transcriptase Inhibitor with Activity against Drug-Resistant Human Immunodeficiency Virus Type 1▿ ‡

    PubMed Central

    Corbau, Romuald; Mori, Julie; Phillips, Chris; Fishburn, Lesley; Martin, Alex; Mowbray, Charles; Panton, Wendy; Smith-Burchnell, Caroline; Thornberry, Adele; Ringrose, Heather; Knöchel, Thorsten; Irving, Steve; Westby, Mike; Wood, Anthony; Perros, Manos

    2010-01-01

    The nonnucleoside reverse transcriptase inhibitors (NNRTIs) are key components of highly active antiretroviral therapy (HAART) for the treatment of human immunodeficiency virus type 1 (HIV-1). A major problem with the first approved NNRTIs was the emergence of mutations in the HIV-1 reverse transcriptase (RT), in particular K103N and Y181C, which led to resistance to the entire class. We adopted an iterative strategy to synthesize and test small molecule inhibitors from a chemical series of pyrazoles against wild-type (wt) RT and the most prevalent NNRTI-resistant mutants. The emerging candidate, lersivirine (UK-453,061), binds the RT enzyme in a novel way (resulting in a unique resistance profile), inhibits over 60% of viruses bearing key RT mutations, with 50% effective concentrations (EC50s) within 10-fold of those for wt viruses, and has excellent selectivity against a range of human targets. Altogether lersivirine is a highly potent and selective NNRTI, with excellent efficacy against NNRTI-resistant viruses. PMID:20660667

  3. Lersivirine, a nonnucleoside reverse transcriptase inhibitor with activity against drug-resistant human immunodeficiency virus type 1.

    PubMed

    Corbau, Romuald; Mori, Julie; Phillips, Chris; Fishburn, Lesley; Martin, Alex; Mowbray, Charles; Panton, Wendy; Smith-Burchnell, Caroline; Thornberry, Adele; Ringrose, Heather; Knöchel, Thorsten; Irving, Steve; Westby, Mike; Wood, Anthony; Perros, Manos

    2010-10-01

    The nonnucleoside reverse transcriptase inhibitors (NNRTIs) are key components of highly active antiretroviral therapy (HAART) for the treatment of human immunodeficiency virus type 1 (HIV-1). A major problem with the first approved NNRTIs was the emergence of mutations in the HIV-1 reverse transcriptase (RT), in particular K103N and Y181C, which led to resistance to the entire class. We adopted an iterative strategy to synthesize and test small molecule inhibitors from a chemical series of pyrazoles against wild-type (wt) RT and the most prevalent NNRTI-resistant mutants. The emerging candidate, lersivirine (UK-453,061), binds the RT enzyme in a novel way (resulting in a unique resistance profile), inhibits over 60% of viruses bearing key RT mutations, with 50% effective concentrations (EC(50)s) within 10-fold of those for wt viruses, and has excellent selectivity against a range of human targets. Altogether lersivirine is a highly potent and selective NNRTI, with excellent efficacy against NNRTI-resistant viruses.

  4. The major reverse-transcriptase-incompetent splice variant of the human telomerase protein inhibits telomerase activity but protects from apoptosis

    PubMed Central

    Listerman, Imke; Sun, Jie; Gazzaniga, Francesca S.; Lukas, Jason L.; Blackburn, Elizabeth H.

    2013-01-01

    hTERT (TERT), the catalytic protein subunit of telomerase, is subjected to numerous alternative splicing events, but the regulation and function of these splice variants is obscure. Full-length hTERT includes conserved domains that encode reverse transcriptase activity, RNA binding and other functions. The major splice variant termed α+β− or β-deletion is highly expressed in stem and cancer cells, where it codes for a truncated protein lacking most of the reverse transcriptase domain but retaining the known RNA binding motifs. In a breast cancer cell panel, we found that β-deletion was the hTERT transcript that was most highly expressed. Splicing of this transcript was controlled by the splice regulators SRSF11, HNRNPH2 and HNRNPL and the β-deletion transcript variant was associated with polyribosomes in cells. When ectopically overexpressed, β-deletion protein competed for binding to hTR (TERC) RNA, thereby inhibiting endogenous telomerase activity. Overexpressed β-deletion protein localized to the nucleus and mitochondria and it protected breast cancer cells from cisplatin-induced apoptosis. Our results reveal that a major hTERT splice variant can confer a growth advantage to cancer cells independent of telomere maintenance, suggesting hTERT makes multiple contributions to cancer pathophysiology. PMID:23610451

  5. SYBR Green II Dye-Based Real-Time Assay for Measuring Inhibitor Activity Against HIV-1 Reverse Transcriptase.

    PubMed

    Kokkula, Chakradhar; Palanisamy, Navaneethan; Ericstam, Malin; Lennerstrand, Johan

    2016-10-01

    There are arrays of in vitro assays to quantify the activity of HIV-1 reverse transcriptase (HIV-1 RT). These assays utilize either chemically customized/labelled nucleotides, or TaqMan probes, or radiolabeled nucleotides/primers. Although several real-time PCR assays exist commercially for measuring the RT activity, which are usually used for quantifying the viral titres, these assays are not optimized for measuring the inhibitory concentrations (IC50) of HIV-1 RT inhibitors. Moreover, a recently established inorganic pyrophosphate-coupled enzyme assay cannot be employed for studying nonphosphorylated nucleoside reverse transcriptase inhibitors (NRTIs). In the present study, we have developed a novel one-step assay with native nucleotide substrates and SYBR Green II dye to determine IC50 values of triphosphorylated NRTIs against HIV-1 RT. Using exact batches of wild-type and mutant RT, and triphosphorylated NRTIs, we showed that our method gave IC50 values for inhibitors similar to that of an earlier published colorimetric assay with BrdUTP substrate (CABS). Our assay should be suitable for high-throughput screening of antiretroviral drugs and could also be suitable for studying drug resistance profiles. Additionally, we also used our assay to study inhibition by AZT in its nonphosphorylated form by supplementing the reaction mixture with necessary kinases and ATP.

  6. Human telomerase reverse transcriptase binds to a pre-organized hTR in vivo exposing its template.

    PubMed

    Zemora, Georgeta; Handl, Stefan; Waldsich, Christina

    2016-01-08

    Telomerase is a specialized reverse transcriptase that is responsible for telomere length maintenance. As in other organisms, the minimal components required for an active human telomerase are the template-providing telomerase RNA (hTR) and the enzymatic entity telomerase reverse transcriptase (hTERT). Here, we explored the structure of hTR and the hTERT-induced conformational changes within hTR in living cells. By employing an in vivo DMS chemical probing technique, we showed that the pseudoknot and associated triple helical scaffold form stably in vivo independently of hTERT. In fact, the dimethyl-sulfate (DMS) modification pattern suggests that hTR alone is capable of adopting a conformation that is suited to interact with hTERT. However, in the absence of hTERT the template region of hTR is only weakly accessible to DMS-modifications. The predominant change after binding of hTERT to hTR is the exposure of the template region.

  7. The C-terminal extension of human telomerase reverse transcriptase is necessary for high affinity binding to telomeric DNA.

    PubMed

    Tomlinson, Christopher G; Holien, Jessica K; Mathias, Jordan A T; Parker, Michael W; Bryan, Tracy M

    2016-01-01

    The ribonucleoprotein enzyme telomerase maintains telomeres and is essential for cellular immortality in most cancers. Insight into the telomerase mechanism can be gained from short telomere syndromes, in which mutation of telomerase components manifests in telomere dysfunction. We carried out detailed kinetic analyses and molecular modelling of a disease-associated mutant in the C-terminal extension of the reverse transcriptase subunit of human telomerase. The kinetic analyses revealed that the mutation substantially impacts the affinity of telomerase for telomeric DNA, but the magnitude of this impact varies for primers with different 3' ends. Molecular dynamics simulations corroborate this finding, revealing that the mutation results in greater movement of a nearby loop, impacting the DNA-RNA helix differentially with different DNA primers. Thus, the data indicate that this region is the location of one of the enzyme conformational changes responsible for the long-standing observation that off-rates of telomerase vary with telomeric 3' end sequence. Our data provide a molecular basis for a disease-associated telomerase mutation, and the first direct evidence for a role of the C-terminal extension in DNA binding affinity, a function analogous to the "thumb" domain of retroviral reverse transcriptases. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  8. Sustained high proportion of zidovudine-resistant HIV variants despite prolonged substitution of zidovudine by other nucleoside reverse transcriptase inhibitors.

    PubMed

    Bélec, Laurent; Legoff, Jérôme; Si-Mohamed, Ali; Andréoletti, Laurent; Mbopi-Kéou, François-Xavier; Kolberg, Janice; Matta, Mathieu; Detmer, Jill; Piketty, Christophe; Kazatchkine, Michel D

    2002-09-01

    The consequences of zidovudine (ZDV) replacement by other nucleoside reverse transcriptase inhibitors on the expression of resistance mutations at codons 215 and 41 of the reverse transcriptase (RT) gene was investigated prospectively in 66 patients harboring mutant genotypes who were changed to an effective two- or three-drug combination antiretroviral regimen. Quantitation of mutant (MUT) viral populations at codon 215 by means of RT-PCR with differential hybridization of amplicons specific for MUT and wild (WT) variants revealed no difference in the proportion of 215 MUT variants prior to (93.5 +/- 2.4%) and 12 to 20 months after (96.9 +/- 1.9%) ZDV replacement, independently of a therapeutic change for stavudine. The fitness of the variants harboring the ZDV-resistant MUT 215 genotype following drug withdrawal was calculated to be 96 to 99% of that of the variants harboring the WT 215 genotype. The apparent stability of ZDV-resistant variants in the study population may have two main complementary explanations: persistent selective pressure secondary to partial cross-resistance due to the new regimens given after the therapeutic alteration and suppression of viral replication after the therapeutic alteration that could have hampered the replacement of less fit variants by fitter variants. These findings indicate that, at least within 15 months following discontinuation of ZDV, an effective antiretroviral therapy is insufficient to allow for ZDV-resistant strains to disappear, and thus to allow for the safe re-introduction of the drug.

  9. APOBEC3DE Inhibits LINE-1 Retrotransposition by Interacting with ORF1p and Influencing LINE Reverse Transcriptase Activity.

    PubMed

    Liang, Weizi; Xu, Jiwei; Yuan, Wensu; Song, Xuan; Zhang, Jianyong; Wei, Wei; Yu, Xiao-Fang; Yang, Ying

    2016-01-01

    Human long interspersed elements 1 (LINE-1 or L1) is the only autonomous non-LTR retroelement in humans and has been associated with genome instability, inherited genetic diseases, and the development of cancer. Certain human APOBEC3 family proteins are known to have LINE-1 restriction activity. The mechanisms by which APOBEC3 affects LINE-1 retrotransposition are not all well characterized; here, we confirm that both A3B and A3DE have a strong ability to inhibit LINE-1 retrotransposition. A3DE interacts with LINE-1 ORF1p to target LINE-1 ribonucleoprotein particles in an RNA-dependent manner. Moreover, A3DE binds to LINE-1 RNA and ORF1 protein in cell culture system. Fluorescence microscopy demonstrated that A3DE co-localizes with ORF1p in cytoplasm. Furthermore, A3DE inhibits LINE-1 reverse transcriptase activity in LINE-1 ribonucleoprotein particles in a cytidine deaminase-independent manner. In contrast, A3B has less inhibitory effects on LINE-1 reverse transcriptase activity despite its strong inhibition of LINE-1 retrotransposition. This study demonstrates that different A3 proteins have been evolved to inhibit LINE-1 activity through distinct mechanisms.

  10. Identification of a putative binding site for [2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide)thymine (TSAO) derivatives at the p51-p66 interface of HIV-1 reverse transcriptase.

    PubMed

    Rodríguez-Barrios, F; Pérez, C; Lobatón, E; Velázquez, S; Chamorro, C; San-Félix, A; Pérez-Pérez, M J; Camarasa, M J; Pelemans, H; Balzarini, J; Gago, F

    2001-06-07

    A binding site for TSAO-m(3)T at the interface between the p66 and p51 subunits of HIV-1 reverse transcriptase (RT) and distinct from that of "classical" HIV-1 non-nucleoside inhibitors is proposed. The feasibility of the binding mode was assessed by carrying out nanosecond molecular dynamics simulations for the complexes of TSAO-m(3)T with reduced models of both the wild-type enzyme and a more sensitive R172A mutant. The molecular model is in agreement with a previous proposal, with known structure-activity and mutagenesis data for this unique class of inhibitors, and also with recent biochemical evidence indicating that TSAO analogues can affect enzyme dimerization. The relative importance of residues involved in dimer formation and TSAO-RT complex stabilization was assessed by a combination of surface area accessibility, molecular mechanics, and continuum electrostatics calculations. A structure-based modification introduced into the lead compound yielded a new derivative with improved antiviral activity.

  11. An unusual mechanism of self-primed reverse transcription requires the RNase H domain of reverse transcriptase to cleave an RNA duplex.

    PubMed

    Levin, H L

    1996-10-01

    The reverse transcription of retroviruses and long terminal repeat-containing retrotransposons requires that tRNA species serve as primers. We recently reported that the long terminal repeat-containing retrotransposon Tf1 is a unique exception in that reverse transcription is independent of tRNA and is instead initiated by a self-priming mechanism. The first 11 bases of the Tf1 transcript fold back and anneal to the primer binding site in a process that results in the priming of minus-strand strong-stop DNA. Data presented here demonstrate that a cleavage occurs between the 11th and 12th bases of the transcript, resulting in the generation of the primer. Mutagenesis experiments presented here indicate that the RNase H domain of the Tf1 reverse transcriptase is required for the cleavage reaction, suggesting that this RNase H may have the novel ability to cleave double-stranded RNA at the end of a duplexed region.

  12. Comparative Study of different msDNA (multicopy single-stranded DNA) structures and phylogenetic comparison of reverse transcriptases (RTs): evidence for vertical inheritance.

    PubMed

    Das, Rasel; Shimamoto, Tadashi; Hosen, Sultan Mohammad Zahid; Arifuzzaman, Mohammad

    2011-01-01

    The multi-copy single-stranded DNA (msDNA) is yielded by the action of reverse transcriptase of retro-element in a wide range of pathogenic bacteria. Upon this phenomenon, it has been shown that msDNA is only produced by Eubacteria because many Eubacteria species contained reverse transcriptase in their special retro-element. We have screened around 111 Archaea at KEGG (Kyoto Encyclopedia of Genes and Genomes) database available at genome net server and observed three Methanosarcina species (M.acetivorans, M.barkeri and M.mazei), which also contained reverse transcriptase in their genome sequences. This observation of reverse transcriptase in Archaea raises questions regarding the origin of this enzyme. The evolutionary relationship between these two domains of life (Eubacteria and Archaea) hinges upon the phenomenon of retrons. Interestingly, the evolutionary trees based on the reverse transcriptases (RTs) and 16S ribosomal RNAs point out that all the Eubacteria RTs were descended from Archaea RTs during their evolutionary times. In addition, we also have shown some significant structural features among the newly identified msDNA-Yf79 in Yersinia frederiksenii with other of its related msDNAs (msDNA-St85, msDNA-Vc95, msDNA-Vp96, msDNA-Ec78 and msDNA-Ec83) from pathogenic bacteria. Together the degree of sequence conservation among these msDNAs, the evolutionary trees and the distribution of these ret (reverse transcriptase) genes suggest a possible evolutionary scenario. The single common ancestor of the organisms of Eubacteria and Archaea subgroups probably achieved this ret gene during their evolution through the vertical descent rather than the horizontal transformations followed by integration into this organism genome by a mechanism related to phage recognition and/or transposition.

  13. Structure of the HIV-1 reverse transcriptase Q151M mutant: insights into the inhibitor resistance of HIV-1 reverse transcriptase and the structure of the nucleotide-binding pocket of Hepatitis B virus polymerase

    SciTech Connect

    Nakamura, Akiyoshi; Tamura, Noriko; Yasutake, Yoshiaki

    2015-10-23

    The structure of the HIV-1 reverse transcriptase Q151M mutant was determined at a resolution of 2.6 Å in space group P321. Hepatitis B virus polymerase (HBV Pol) is an important target for anti-HBV drug development; however, its low solubility and stability in vitro has hindered detailed structural studies. Certain nucleotide reverse transcriptase (RT) inhibitors (NRTIs) such as tenofovir and lamivudine can inhibit both HBV Pol and Human immunodeficiency virus 1 (HIV-1) RT, leading to speculation on structural and mechanistic analogies between the deoxynucleotide triphosphate (dNTP)-binding sites of these enzymes. The Q151M mutation in HIV-1 RT, located at the dNTP-binding site, confers resistance to various NRTIs, while maintaining sensitivity to tenofovir and lamivudine. The residue corresponding to Gln151 is strictly conserved as a methionine in HBV Pol. Therefore, the structure of the dNTP-binding pocket of the HIV-1 RT Q151M mutant may reflect that of HBV Pol. Here, the crystal structure of HIV-1 RT Q151M, determined at 2.6 Å resolution, in a new crystal form with space group P321 is presented. Although the structure of HIV-1 RT Q151M superimposes well onto that of HIV-1 RT in a closed conformation, a slight movement of the β-strands (β2–β3) that partially create the dNTP-binding pocket was observed. This movement might be caused by the introduction of the bulky thioether group of Met151. The structure also highlighted the possibility that the hydrogen-bonding network among amino acids and NRTIs is rearranged by the Q151M mutation, leading to a difference in the affinity of NRTIs for HIV-1 RT and HBV Pol.

  14. Development of a series of 3-hydroxyquinolin-2(1H)-ones as selective inhibitors of HIV-1 reverse transcriptase associated RNase H activity.

    PubMed

    Suchaud, Virginie; Bailly, Fabrice; Lion, Cédric; Tramontano, Enzo; Esposito, Francesca; Corona, Angela; Christ, Frauke; Debyser, Zeger; Cotelle, Philippe

    2012-06-15

    We report herein the synthesis of a series of 3-hydroxyquinolin-2(1H)-one derivatives. Esters and amide groups were introduced at position 4 of the basis scaffold and some modulations of the benzenic moiety were performed. Most compounds presented selective inhibitory properties in the 10-20 μM range against HIV-1 reverse transcriptase associated ribonuclease H activity, without affecting the integrase and reverse transcriptase DNA polymerase activities. Unfortunately all tested compounds exhibited high cellular cytotoxicity in cell culture which limited their applications as antiviral agents.

  15. Inhibition of HIV-1 reverse transcriptase, toxicological and chemical profile of Calophyllum brasiliense extracts from Chiapas, Mexico.

    PubMed

    César, García-Zebadúa Julio; Alfonso, Magos-Guerrero Gil; Marius, Mumbrú-Massip; Elizabeth, Estrada-Muñoz; Angel, Contreras-Barrios Miguel; Maira, Huerta-Reyes; Guadalupe, Campos-Lara María; Manuel, Jiménez-Estrada; Ricardo, Reyes-Chilpa

    2011-10-01

    Calophyllum species are sources of calanolides, which inhibit human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). The hexane extract of the leaves from C. brasiliense collected in Soconusco, State of Chiapas, Mexico, analyzed by HPLC showed to contain apetalic acid, calanolides B, and C. It showed potent anti-HIV-1 RT inhibition (IC(50)=20.2 μg/ml), but was not toxic in mice (LD(50)=1.99 g/kg). The histological study of the mice treated at the highest dose revealed no alteration on hepatocytes, and an increase in the number of spleen megakaryocytes. These results suggest this extract is suitable to continue studies for developing a phytodrug against HIV-1.

  16. Structure of HIV-1 Reverse Transcriptase with the Inhibitor β-thujaplicinol Bound at the RNase H Active Site

    PubMed Central

    Himmel, Daniel M.; Maegley, Karen A.; Pauly, Tom A.; Bauman, Joseph D.; Das, Kalyan; Dharia, Chhaya; Clark, Arthur D.; Ryan, Kevin; Hickey, Michael J.; Love, Robert A.; Hughes, Stephen H.; Bergqvist, Simon; Arnold, Eddy

    2012-01-01

    Summary Novel inhibitors are needed to counteract the rapid emergence of drug-resistant HIV variants. HIV-1 reverse transcriptase (RT) has both DNA polymerase and RNase H (RNH) enzymatic activities, but approved drugs that inhibit RT target the polymerase. Inhibitors that act against new targets, like RNH, would be effective against all of the current drug-resistant variants. Here, we present 2.80 Å and 2.04 Å resolution crystal structures of an RNH inhibitor, β-thujaplicinol, bound at the RNH active site of both HIV-1 RT and an isolated RNH domain. β-thujaplicinol chelates two divalent metal ions at the RNH active site. We provide biochemical evidence that β-thujaplicinol is a slow-binding RNH inhibitor with non-competitive kinetics and suggest that it forms a tropylium ion that interacts favorably with RT and the RNA:DNA substrate. PMID:20004166

  17. An immortalized goat mammary epithelial cell line induced with human telomerase reverse transcriptase (hTERT) gene transfer.

    PubMed

    He, Y L; Wu, Y H; He, X N; Liu, F J; He, X Y; Zhang, Y

    2009-06-01

    Although mammary epithelial cell lines can provide a rapid and reliable indicator of gene expression efficiency of transgenic animals, their short lifespan greatly limits this application. To provide stable and long lifespan cells, goat mammary epithelial cells (GMECs) were transduced with pLNCX2-hTERT by retrovirus-mediated gene transfer. Transduced GMECs were evaluated by reverse transcriptase polymerase chain reaction (RT-PCR), proliferation assays, karyotype analysis, telomerase activity assay, western blotting, soft agar assay, and injection into nude mice. Non-transduced GMECs were used as a control. The hTERT-GMECs had higher telomerase activity and extended proliferative lifespan compared to non-transfected GMECs; even after Passage 50, hTERT-GMECs had a near diploid complement of chromosomes. Furthermore, they did not gain the anchorage-independent growth property and were not associated with a malignant phenotype in vitro or in vivo.

  18. The 'helix clamp' in HIV-1 reverse transcriptase: a new nucleic acid binding motif common in nucleic acid polymerases.

    PubMed Central

    Hermann, T; Meier, T; Götte, M; Heumann, H

    1994-01-01

    Amino acid sequences homologous to 259KLVGKL (X)16KLLR284 of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) are conserved in several nucleotide polymerizing enzymes. This amino acid motif has been identified in the crystal structure model as an element of the enzyme's nucleic acid binding apparatus. It is part of the helix-turn-helix structure, alpha H-turn-alpha I, within the 'thumb' region of HIV-1 RT. The motif grasps the complexed nucleic acid at one side. Molecular modeling studies on HIV-1 RT in complex with a nucleic acid fragment suggest that the motif has binding function in the p66 subunit as well as in the p51 subunit, acting as a kind of 'helix clamp'. Given its wide distribution within the nucleic acid polymerases, the helix clamp motif is assumed to be a structure of general significance for nucleic acid binding. Images PMID:7527138

  19. Mutations in the Primer Grip of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Impair Proviral DNA Synthesis and Virion Maturation

    PubMed Central

    Yu, Qiang; Ottmann, Michele; Pechoux, Christine; Le Grice, Stuart; Darlix, Jean-Luc

    1998-01-01

    This report describes the effects of mutating highly conserved residues in the primer grip domain of human immunodeficiency virus type 1 reverse transcriptase (RT) on virus formation and infectivity. Among a series of RT mutant viruses, three (M230A, L234D, and W239A) were found to be noninfectious or very poorly infectious. Our data indicate that these mutations in RT caused severe defects in proviral DNA synthesis. Interestingly, assembly and maturation of mutant virus M230A were similar to those of the wild type, while mutants L234D and W239A showed impaired maturation. The immature morphology of RT mutants L234D and W239A is due at least in part to premature cleavage of the gag-pol precursor, prior to virion budding, indicating that intracellular stability of Pr160gag-pol is of key importance during virus assembly. PMID:9696874

  20. Particular interaction between efavirenz and the HIV-1 reverse transcriptase binding site as explained by the ONIOM2 method

    NASA Astrophysics Data System (ADS)

    Nunrium, Peerapol; Kuno, Mayuso; Saen-oon, Suwipa; Hannongbua, Supa

    2005-03-01

    Particular interaction between efavirenz and the HIV-1 reverse transcriptase binding site was investigated, based on the B3LYP/6-31G(d,p) and ONIOM2 methods. The interaction between efavirenz and Lys101 was found to be the strongest interaction, typically, -11.29 kcal/mol. The stability of this complex system leads to the foundation of the estimated binding energy of approximately -22.66 kcal/mol. Moreover, two hydrogen bonds between benzoxazin-2-one, and the backbone carbonyl oxygen and the backbone amino hydrogen of Lys101 were observed. These hydrogen bond interactions play an important role in the bound efavirenz/HIV-1 RT complex.

  1. Development of a multiplex real-time reverse transcriptase-polymerase chain reaction for equine infectious anemia virus (EIAV).

    PubMed

    Cook, R Frank; Cook, S J; Li, F Li; Montelaro, R C; Issel, C J

    2002-08-01

    A single-tube reverse transcriptase-polymerase chain reaction (RT-PCR) using a fluorogenic real-time PCR detection method is described for the quantitation of equine infectious anemia virus (EIAV) RNA in the plasma of equids. To compensate for variations inherent in sample preparation a multiplex real-time RT-PCR system was developed that permitted the simultaneous calculation of the nucleic acid recovery rate along with the copy number of viral RNA molecules. Detection of EIAV RNA was linear from 10(9) to 10(1) molecules with intra- and inter-assay variability of less than 1% at 10(8), 10(6), 10(4) and 10(2) molecules.

  2. Enhanced detection of RNA by MMLV reverse transcriptase coupled with thermostable DNA polymerase and DNA/RNA helicase.

    PubMed

    Okano, Hiroyuki; Katano, Yuta; Baba, Misato; Fujiwara, Ayako; Hidese, Ryota; Fujiwara, Shinsuke; Yanagihara, Itaru; Hayashi, Tsukasa; Kojima, Kenji; Takita, Teisuke; Yasukawa, Kiyoshi

    2017-01-01

    Detection of mRNA is a valuable method for monitoring the specific gene expression. In this study, we devised a novel cDNA synthesis method using three enzymes, the genetically engineered thermostable variant of reverse transcriptase (RT), MM4 (E286R/E302K/L435R/D524A) from Moloney murine leukemia virus (MMLV), the genetically engineered variant of family A DNA polymerase with RT activity, K4polL329A from thermophilic Thermotoga petrophila K4, and the DNA/RNA helicase Tk-EshA from a hyperthermophilic archaeon Thermococcus kodakarensis. By optimizing assay conditions for three enzymes using Taguchi's method, 100 to 1000-fold higher sensitivity was achieved for cDNA synthesis than conventional assay condition using only RT. Our results suggest that DNA polymerase with RT activity and DNA/RNA helicase are useful to increase the sensitivity of cDNA synthesis.

  3. Cytosolic RNA:DNA Duplexes Generated by Endogenous Reverse Transcriptase Activity as Autonomous Inducers of Skin Inflammation in Psoriasis

    PubMed Central

    Griez, Anthony; Guilhou, Jean-Jacques; Girard, Céline; Nagot, Nicolas; Van de Perre, Philippe; Dujols, Pierre

    2017-01-01

    Psoriasis is a chronic skin disease of unknown ætiology. Recent studies suggested that a large amount of cytosolic DNA (cyDNA) in keratinocytes is breaking keratinocytes DNA tolerance and promotes self-sustained inflammation in the psoriatic lesion. We investigated the origin of this cyDNA. We show that, amongst all the possible DNA structures, the cyDNA could be present as RNA:DNA duplexes in keratinocytes. We further show that endogenous reverse transcriptase activities generate such duplexes and consequently activate the production of Th1-inflammatory cytokines. These observations open a new research avenue related to endogenous retroelements for the aetiology of psoriasis and probably of other human chronic inflammatory diseases. PMID:28095445

  4. Design and synthesis of α-carboxy nucleoside phosphonate analogues and evaluation as HIV-1 reverse transcriptase-targeting agents.

    PubMed

    Keane, Sarah J; Ford, Alan; Mullins, Nicholas D; Maguire, Nuala M; Legigan, Thibaut; Balzarini, Jan; Maguire, Anita R

    2015-03-06

    The synthesis of the first series of a new class of nucleoside phosphonate analogues is described. Addition of a carboxyl group at the α position of carbocyclic nucleoside phosphonate analogues leads to a novel class of potent HIV reverse transcriptase (RT) inhibitors, α-carboxy nucleoside phosphonates (α-CNPs). Key steps in the synthesis of the compounds are Rh-catalyzed O-H insertion and Pd-catalyzed allylation reactions. In cell-free assays, the final products are markedly inhibitory against HIV RT and do not require phosphorylation to exhibit anti-RT activity, which indicates that the α-carboxyphosphonate function is efficiently recognized by HIV RT as a triphosphate entity, an unprecedented property of nucleoside monophosph(on)ates.

  5. [Telomerase reverse transcriptase (TERT) promoter mutations in the tumors of human endocrine organs: Biological and prognostic value].

    PubMed

    Selivanova, L S; Volganova, K S; Abrosimov, A Y U

    2016-01-01

    The analysis of the data available in the literature has shown that telomerase reverse transcriptase TERT promoter may serve as promising markers of malignancy, aggressive disease course, and poor prognosis for malignant tumors of endocrine organs. Considering the established association of mutations with tumors having a poor prognosis (high-grade and anaplastic carcinoma of the thyroid), it is reasonable to perform prognostic-value investigations in a group of low-grade thyroid carcinomas that may occasionally recur and may be resistant to radioactive iodine therapy, i.e. can demonstrate a poor course and prognosis. TERT promoter mutations may be a specific marker of the clinically aggressive forms of adrenocortical carcinoma, but the determination of its diagnostic value calls for additional investigations that will have the larger number cases and establish the association with clinical features and survival rates.

  6. Prospective application of reverse transcriptase polymerase chain reaction for diagnosing influenza infections in respiratory samples from a children's hospital.

    PubMed Central

    Claas, E C; van Milaan, A J; Sprenger, M J; Ruiten-Stuiver, M; Arron, G I; Rothbarth, P H; Masurel, N

    1993-01-01

    A prospective clinical evaluation of the reverse transcriptase polymerase chain reaction (RNA PCR) for detection of influenza viruses was carried out with specimens from 342 patients of a children's hospital in The Netherlands. The RNA PCR, carried out directly on the specimens without an organic extraction, showed a sensitivity and specificity which are superior to those of direct immunofluorescence and comparable to those of cell culture combined with immunofluorescence (culture/IF). Negative results can be obtained within 2 days by the RNA PCR but may take up to 14 days by culture/IF. Because culturing is the standard technique for the detection of respiratory viruses, at this moment there are no strong arguments to replace culture/IF with RNA PCR for the detection of influenza A virus. Images PMID:8370755

  7. Leptin upregulates telomerase activity and transcription of human telomerase reverse transcriptase in MCF-7 breast cancer cells

    SciTech Connect

    Ren, He; Zhao, Tiansuo; Wang, Xiuchao; Gao, Chuntao; Wang, Jian; Yu, Ming; Hao, Jihui

    2010-03-26

    The aim was to analyze the mechanism of leptin-induced activity of telomerase in MCF-7 breast cancer cells. We found that leptin activated telomerase in a dose-dependent manner; leptin upregulated the expression of Human Telomerase Reverse Transcriptase (hTERT) at mRNA and protein levels; blockade of signal transducer and activator of transcription 3 (STAT3) phosphorylation significantly counteracted leptin-induced hTERT transcription and protein expression; chromatin immunoprecipitation analysis showed that leptin enhanced the binding of STAT3 to the hTERT promoter. This study uncovers a new mechanism of the proliferative effect of leptin on breast cancer cells and provides a new explanation of obesity-related breast cancer.

  8. Enzymatic Pre-treatment of Wastewater to Minimize Recovery by Reverse Transcriptase PCR of RNA from Inactive Bacteriophages.

    PubMed

    Unnithan, Veena V; Unc, Adrian; Smith, Geoffrey B

    2015-07-01

    Quantitative viral risk assessments for wastewaters are notoriously difficult. The often considered quantitative reverse transcriptase PCR reflects poorly on virus infectivity rates leading to inaccurate risk interpretations. Various techniques focused on the degradation of the nucleic acids of non-infective viruses were previously employed. We comparatively assessed the effectiveness of such enzymatic treatments for MS2 bacteriophage in treated wastewaters. The single use of RNase A at an appropriate concentration may be as effective as the combination of RNase followed by Proteinase K and more rapid. While all tested enzymatic treatments minimized recovery of RNA (>95 %) in the absence of infective MS2, none completely eliminated the signal recovery. Selection of any enzymatic protocol for minimizing recovery of RNA from degraded, non-infective viruses should balance the methods efficacy with its expediency.

  9. Efavirenz or nevirapine in three-drug combination therapy with two nucleoside or nucleotide-reverse transcriptase inhibitors for initial treatment of HIV infection in antiretroviral-naïve individuals

    PubMed Central

    Mbuagbaw, Lawrence; Mursleen, Sara; Irlam, James H; Spaulding, Alicen B; Rutherford, George W; Siegfried, Nandi

    2016-01-01

    Background The advent of highly active antiretroviral therapy (ART) has reduced the morbidity and mortality due to HIV infection. The World Health Organization (WHO) ART guidelines focus on three classes of antiretroviral drugs, namely nucleoside or nucleotide reverse transcriptase inhibitors (NRTI), non-nucleoside reverse transcriptase inhibitors (NNRTI) and protease inhibitors. Two of the most common medications given as first-line treatment are the NNRTIs, efavirenz (EFV) and nevirapine (NVP). It is unclear which NNRTI is more efficacious for initial therapy. This systematic review was first published in 2010. Objectives To determine which non-nucleoside reverse transcriptase inhibitor, either EFV or NVP, is more effective in suppressing viral load when given in combination with two nucleoside reverse transcriptase inhibitors as part of initial antiretroviral therapy for HIV infection in adults and children. Search methods We attempted to identify all relevant studies, regardless of language or publication status, in electronic databases and conference proceedings up to 12 August 2016. We searched MEDLINE, Embase, the Cochrane Central Register of Controlled Trials (CENTRAL), the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP) and ClinicalTrials.gov to 12 August 2016. We searched LILACS (Latin American and Caribbean Health Sciences Literature) and the Web of Science from 1996 to 12 August 2016. We checked the National Library of Medicine (NLM) Gateway from 1996 to 2009, as it was no longer available after 2009. Selection criteria We included all randomized controlled trials (RCTs) that compared EFV to NVP in people with HIV without prior exposure to ART, irrespective of the dosage or NRTI's given in combination. The primary outcome of interest was virological success. Other primary outcomes included mortality, clinical progression to AIDS, severe adverse events, and discontinuation of therapy for any reason. Secondary

  10. Impact of HIV-1 reverse transcriptase polymorphism at codons 211 and 228 on virological response to didanosine.

    PubMed

    Marcelin, Anne-Genevieve; Flandre, Philippe; Furco, Andre; Wirden, Marc; Molina, Jean-Michel; Calvez, Vincent

    2006-01-01

    To determine the potential impact of reverse transcriptase (RT) mutations, other than those currently known to confer nucleoside reverse transcriptase inhibitors (NRTIs) resistance, on the virological response to didanosine (ddl). In the placebo-controlled Jaguar trial, 168 patients were randomly assigned to receive ddl (n=111) or placebo (n=57) in addition to their currently failing regimen for 4 weeks. The virological response was a reduction of HIV-1 RNA from baseline to week 4. In an univariate analysis, we investigated the impact on the virological response to ddl of all the mutations in the RT gene (codons 21-236), except those known to confer NRTI resistance. Using the removing procedure, with a test for trend (Jonckheere's test), a new potential score was calculated incorporating all potential mutations associated to the week 4 virological response. Two RT polymorphisms were associated with a reduced virological response to ddl, R211A/D/G/K/S and L228H/M/R, and one with a better virological response: F214L. A mutation score (M41L+D67N+T69D-K70R +L74V-M 1 84V/I+T21 5Y/F+ K219Q/E+ R211A/D/G/K/S+ L228H/M/R), including two RT polymorphisms not previously described to be associated with ddl resistance (211 and 228) and RT mutations previously described, was associated with a continuum of virological response and increased the predictability of virological response to ddl. RT polymorphisms should be taken into account to define algorithms able to correctly define resistance to NRTIs and more specifically ddl.

  11. LRE2, an active human L1 element, has low level transcriptional activity and extremely low reverse transcriptase activity

    SciTech Connect

    Holmes, S.E.; Dombroski, B.A.; Sassaman, D.M.

    1994-09-01

    Previously, we found a 2 kb insertion containing a rearranged L1 element plus a unique sequence component (USC) within exon 48 of the dystrophin gene of a patient with muscular dystrophy. We used the USC to clone the precursor of this insertion, the second known {open_quotes}active{close_quotes} human L1 element. The locus LRE2 (L1 Retrotransposable Element 2) has an allele derived from the patient which matches the insertion sequence exactly. LRE2 has a perfect 13-15 bp target site duplication, 2 open reading frames (ORFs), and an unusual 21 bp truncation of the 5{prime} end in a region known to be important for L1 transcription. The truncated LRE2 promoter has about 20% of the transcriptional activity of a previously studied L1 promoter after transfection into NTera2D1 cells of a construct in which the L1 promoter drives the expression of a lacZ gene. In addition, the reverse transcriptase (RT) encoded by LRE2 is active in an in vivo pseudogene assay in yeast and an in vitro assay. However, in both assays the RT of LRE2 is 1-5% as active as that of LRE1. These data demonstrate that multiple {open_quotes}active{close_quotes} L1 elements exist in the human genome, and that active elements can have highly variable rates of transcription and reverse transcriptase activity. That the RT of LRE2 has extremely low activity suggests the possibility that retrotransposition of an L1 element may in some cases involve an RT encoded by another L1 element.

  12. Nanogel-Conjugated Reverse Transcriptase Inhibitors and Their Combinations as Novel Antiviral Agents with Increased Efficacy against HIV-1 Infection.

    PubMed

    Senanayake, T H; Gorantla, S; Makarov, E; Lu, Y; Warren, G; Vinogradov, S V

    2015-12-07

    Nucleoside reverse transcriptase inhibitors (NRTIs) are an integral part of the current antiretroviral therapy (ART), which dramatically reduced the mortality from AIDS and turned the disease from lethal to chronic. The further steps in curing the HIV-1 infection must include more effective targeting of infected cells and virus sanctuaries inside the body and modification of drugs and treatment schedules to reduce common complications of the long-term treatment and increase patient compliancy. Here, we describe novel NRTI prodrugs synthesized from cholesteryl-ε-polylysine (CEPL) nanogels by conjugation with NRTI 5'-succinate derivatives (sNRTI). Biodegradability, small particle size, and high NRTI loading (30% by weight) of these conjugates; extended drug release, which would allow a weekly administration schedule; high therapeutic index (>1000) with a lower toxicity compared to NRTIs; and efficient accumulation in macrophages known as carriers for HIV-1 infection are among the most attractive properties of new nanodrugs. Nanogel conjugates of zidovudine (AZT), lamivudine (3TC), and abacavir (ABC) have been investigated individually and in formulations similar to clinical NRTI cocktails. Nanodrug formulations demonstrated 10-fold suppression of reverse transcriptase activity (EC90) in HIV-infected macrophages at 2-10, 2-4, and 1-2 μM drug levels, respectively, for single nanodrugs and dual and triple nanodrug cocktails. Nanogel conjugate of lamivudine was the most effective single nanodrug (EC90 2 μM). Nanodrugs showed a more favorable pharmacokinetics compared to free NRTIs. Infrequent iv injections of PEGylated CEPL-sAZT alone could efficiently suppress HIV-1 RT activity to background level in humanized mouse (hu-PBL) HIV model.

  13. The G-Patch Domain of Mason-Pfizer Monkey Virus Is a Part of Reverse Transcriptase

    PubMed Central

    Křížová, Ivana; Hadravová, Romana; Štokrová, Jitka; Günterová, Jana; Doležal, Michal; Ruml, Tomáš

    2012-01-01

    Mason-Pfizer monkey virus (M-PMV), like some other betaretroviruses, encodes a G-patch domain (GPD). This glycine-rich domain, which has been predicted to be an RNA binding module, is invariably localized at the 3′ end of the pro gene upstream of the pro-pol ribosomal frameshift sequence of genomic RNAs of betaretroviruses. Following two ribosomal frameshift events and the translation of viral mRNA, the GPD is present in both Gag-Pro and Gag-Pro-Pol polyproteins. During the maturation of the Gag-Pro polyprotein, the GPD transiently remains a C-terminal part of the protease (PR), from which it is then detached by PR itself. The destiny of the Gag-Pro-Pol-encoded GPD remains to be determined. The function of the GPD in the retroviral life cycle is unknown. To elucidate the role of the GPD in the M-PMV replication cycle, alanine-scanning mutational analysis of its most highly conserved residues was performed. A series of individual mutations as well as the deletion of the entire GPD had no effect on M-PMV assembly, polyprotein processing, and RNA incorporation. However, a reduction of the reverse transcriptase (RT) activity, resulting in a drop in M-PMV infectivity, was determined for all GPD mutants. Immunoprecipitation experiments suggested that the GPD is a part of RT and participates in its function. These data indicate that the M-PMV GPD functions as a part of reverse transcriptase rather than protease. PMID:22171253

  14. Integration of maternal genome into the neonate genome through breast milk mRNA transcripts and reverse transcriptase.

    PubMed

    Irmak, M Kemal; Oztas, Yesim; Oztas, Emin

    2012-06-07

    Human milk samples contain microvesicles similar to the retroviruses. These microvesicles contain mRNA transcripts and possess reverse transcriptase activity. They contain about 14,000 transcripts representing the milk transcriptome. Microvesicles are also enriched with proteins related to "caveolar-mediated endocytosis signaling" pathway. It has recently been reported that microvesicles could be transferred to other cells by endocytosis and their RNA content can be translated and be functional in their new location. A significant percentage of the mammalian genome appears to be the product of reverse transcription, containing sequences whose characteristics point to RNA as a template precursor. These are mobile elements that move by way of transposition and are called retrotransposons. We thought that retrotransposons may stem from about 14,000 transcriptome of breast milk microvesicles, and reviewed the literature.The enhanced acceptance of maternal allografts in children who were breast-fed and tolerance to the maternal MHC antigens after breastfeeding may stem from RNAs of the breast milk microvesicles that can be taken up by the breastfed infant and receiving maternal genomic information. We conclude that milk microvesicles may transfer genetic signals from mother to neonate during breastfeeding. Moreover, transfer of wild type RNA from a healthy wet-nurse to the suckling neonate through the milk microvesicles and its subsequent reverse transcription and integration into the neonate genome could result in permanent correction of the clinical manifestations in genetic diseases.

  15. Role of Murine Leukemia Virus Reverse Transcriptase Deoxyribonucleoside Triphosphate-Binding Site in Retroviral Replication and In Vivo Fidelity

    PubMed Central

    Halvas, Elias K.; Svarovskaia, Evguenia S.; Pathak, Vinay K.

    2000-01-01

    Retroviral populations exhibit a high evolutionary potential, giving rise to extensive genetic variation. Error-prone DNA synthesis catalyzed by reverse transcriptase (RT) generates variation in retroviral populations. Structural features within RTs are likely to contribute to the high rate of errors that occur during reverse transcription. We sought to determine whether amino acids within murine leukemia virus (MLV) RT that contact the deoxyribonucleoside triphosphate (dNTP) substrate are important for in vivo fidelity of reverse transcription. We utilized the previously described ANGIE P encapsidating cell line, which expresses the amphotropic MLV envelope and a retroviral vector (pGA-1). pGA-1 expresses the bacterial β-galactosidase gene (lacZ), which serves as a reporter of mutations. Extensive mutagenesis was performed on residues likely to interact with the dNTP substrate, and the effects of these mutations on the fidelity of reverse transcription were determined. As expected, most substitution mutations of amino acids that directly interact with the dNTP substrate significantly reduced viral titers (>10,000-fold), indicating that these residues played a critical role in catalysis and viral replication. However, the D153A and A154S substitutions, which are predicted to affect the interactions with the triphosphate, resulted in statistically significant increases in the mutation rate. In addition, the conservative substitution F155W, which may affect interactions with the base and the ribose, increased the mutation rate 2.8-fold. Substitutions of residues in the vicinity of the dNTP-binding site also resulted in statistically significant decreases in fidelity (1.3- to 2.4-fold). These results suggest that mutations of residues that contact the substrate dNTP can affect viral replication as well as alter the fidelity of reverse transcription. PMID:11044079

  16. A quantitative reverse transcriptase polymerase chain reaction-based assay to detect carcinoma cells in peripheral blood.

    PubMed Central

    Helfrich, W.; ten Poele, R.; Meersma, G. J.; Mulder, N. H.; de Vries, E. G.; de Leij, L.; Smit, E. F.

    1997-01-01

    The presence of tumour cells in the circulation may predict disease recurrence and metastasis. To improve on existing methods of cytological or immunocytological detection, we have developed a sensitive and quantitative technique for the detection of carcinoma cells in blood, using the reverse transcriptase polymerase chain reaction (RT-PCR) identifying transcripts of the pancarcinoma-associated tumour marker EGP-2 (KSA or 17-1A antigen). The amount of EGP2 mRNA was quantified using an internal recombinant competitor RNA standard with known concentration and which is both reversely transcribed and co-amplified in the same reaction, allowing for a reliable assessment of the initial amount of EGP2 mRNA in the sample. Calibration studies, seeding blood with MCF-7 breast carcinoma cells, showed that the assay can detect ten tumour cells among 1.0 x 10(6) leucocytes. The PCR assay revealed that normal bone marrow expresses low levels of EGP2 mRNA, although immunocytochemistry with the anti-EGP2 MAb MOC31 could not identify any positively stained cell. Analyses using this RT-PCR assay may prove to have applications to the assessment of circulating tumour cells in clinical samples. Images Figure 3 Figure 4 PMID:9218728

  17. Direct detection of infectious bursal disease virus from clinical samples by in situ reverse transcriptase-linked polymerase chain reaction.

    PubMed

    Cardoso, Tereza C; Rosa, Ana C G; Astolphi, Rafael D; Vincente, Rafael M; Novais, Juliana B; Hirata, Karina Y; Luvizotto, Maria Cecilia R

    2008-08-01

    The presence of the very virulent (vv) Brazilian strain of infectious bursal disease virus (IBDV) was determined in the bursa of Fabricius, thymus and liver of 2-week-old broilers from a flock with a higher than expected mortality. For this purpose, a direct in situ reverse transcriptase (RT)-linked polymerase chain reaction (PCR) method was developed using specific primers for vvIBDV. Unlabelled forward and reverse biotinylated oligonucleotides were used for RT-PCR in a one-step method and the respective products were revealed by a direct enzymatic reaction. The results were compared with those obtained by standard RT-PCR using general primers for IBDV and virus isolation. The virus isolation, RT-PCR and in situ RT-PCR revealed positive results on the bursa of Fabricius in 86%, 80% and 100%, respectively. The in situ RT-PCR detected vvIBDV in all tested thymus and liver samples, whereas the standard RT-PCR detected virus in 80% and 90% of the samples, respectively. After three consecutive passages on chicken embryonated eggs, IBDV was isolated from 64% of the thymus samples and 30% of the liver samples. In the present study, no classical or antigenic variants of IBDV were detected. The developed in situ RT-PCR assay was able to detect the very virulent strain of IBDV with a higher sensitivity than the conventional RT-PCR and virus isolation.

  18. Efavirenz or nevirapine in three-drug combination therapy with two nucleoside or nucleotide-reverse transcriptase inhibitors for initial treatment of HIV infection in antiretroviral-naïve individuals.

    PubMed

    Mbuagbaw, Lawrence; Mursleen, Sara; Irlam, James H; Spaulding, Alicen B; Rutherford, George W; Siegfried, Nandi

    2016-12-10

    The advent of highly active antiretroviral therapy (ART) has reduced the morbidity and mortality due to HIV infection. The World Health Organization (WHO) ART guidelines focus on three classes of antiretroviral drugs, namely nucleoside or nucleotide reverse transcriptase inhibitors (NRTI), non-nucleoside reverse transcriptase inhibitors (NNRTI) and protease inhibitors. Two of the most common medications given as first-line treatment are the NNRTIs, efavirenz (EFV) and nevirapine (NVP). It is unclear which NNRTI is more efficacious for initial therapy. This systematic review was first published in 2010. To determine which non-nucleoside reverse transcriptase inhibitor, either EFV or NVP, is more effective in suppressing viral load when given in combination with two nucleoside reverse transcriptase inhibitors as part of initial antiretroviral therapy for HIV infection in adults and children. We attempted to identify all relevant studies, regardless of language or publication status, in electronic databases and conference proceedings up to 12 August 2016. We searched MEDLINE, Embase, the Cochrane Central Register of Controlled Trials (CENTRAL), the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP) and ClinicalTrials.gov to 12 August 2016. We searched LILACS (Latin American and Caribbean Health Sciences Literature) and the Web of Science from 1996 to 12 August 2016. We checked the National Library of Medicine (NLM) Gateway from 1996 to 2009, as it was no longer available after 2009. We included all randomized controlled trials (RCTs) that compared EFV to NVP in people with HIV without prior exposure to ART, irrespective of the dosage or NRTI's given in combination.The primary outcome of interest was virological success. Other primary outcomes included mortality, clinical progression to AIDS, severe adverse events, and discontinuation of therapy for any reason. Secondary outcomes were change in CD4 count, treatment failure

  19. Premature strand transfer by the HIV-1 reverse transcriptase during strong-stop DNA synthesis.

    PubMed Central

    Klaver, B; Berkhout, B

    1994-01-01

    Reverse transcription of retroviral genomes starts near the 5' end of the viral RNA by use of an associated tRNA primer. According to the current model of reverse transcription, the initial cDNA product, termed minus-strand strong-stop DNA, 'jumps' to a repeated sequence (R region) at the 3' end of the RNA template. The human retroviruses have relatively long R regions (97-247 nucleotides) when compared to murine and avian viruses (16-68 nucleotides). This suggests that the full complement of the R region is not required for strand transfer and that partial cDNA copies of the 5' R can prematurely jump to the 3' R. To test this hypothesis, we generated mutants of the human immunodeficiency virus with R region changes and analyzed whether 5' or 3' R sequences were inherited by the progeny. We found that in most cases, 5' R-encoded sequences are dominant, which is consistent with the model of reverse transcription. Using a selection protocol, however, we were also able to identify progeny viruses with R sequences derived from the original 3' R element. These results suggest that partial strong stop cDNAs can be transferred with R region homologies much shorter than 97 nucleotides. Images PMID:7510065

  20. Phylogenetic Insights into the Functional Relationship between Primate Lentiviral Reverse Transcriptase and Accessory Proteins Vpx/Vpr

    PubMed Central

    Sakai, Yosuke; Doi, Naoya; Miyazaki, Yasuyuki; Adachi, Akio; Nomaguchi, Masako

    2016-01-01

    The efficiency of reverse transcription to synthesize viral DNA in infected cells greatly influences replication kinetics of retroviruses. However, viral replication in non-dividing cells such as resting T cells and terminally differentiated macrophages is potently and kinetically restricted by a host antiviral factor designated SAMHD1 (sterile alpha motif and HD-domain containing protein 1). SAMHD1 reduces cellular deoxynucleoside triphosphate (dNTP) pools and affects viral reverse transcription step. Human immunodeficiency virus type 2 (HIV-2) and some simian immunodeficiency viruses (SIVs) have Vpx or Vpr to efficiently degrade SAMHD1. Interestingly, the reverse transcriptase (RT) derived from HIV-1 that encodes no anti-SAMHD1 proteins has been previously demonstrated to uniquely exhibit a high enzymatic activity. It is thus not irrational to assume that some viruses may have acquired or lost the specific RT property to better adapt themselves to the low dNTP environments confronted in non-dividing cells. This adaptation process may probably be correlated with the SAMHD1-antagonizing ability by viruses. In this report, we asked whether such adaptive events can be inferable from Vpx/Vpr and RT phylogenetic trees overlaid with SAMHD1-degrading capacity of Vpx/Vpr and with kinetic characteristics of RT. Resultant two trees showed substantially similar clustering patterns, and therefore suggested that the properties of RT and Vpx/Vpr can be linked. In other words, HIV/SIVs may possess their own RT proteins to adequately react to various dNTP circumstances in target cells. PMID:27803699

  1. Suicide Inhibitors of Reverse Transcriptase in the Therapy of AIDS and Other Retroviruses

    DTIC Science & Technology

    1988-07-01

    8217Max murn 2LO wOr’YI) A rapid assay procedure for the enzyme has been developed based upon dot blotting acid washing using DE filter paper dises...Procedures for Reverse Transcriptose A rapid assay procedure far the enzyme has been developed based upon dot blotting acid washing using DE filter...inhibition of HTLV III/HIV replication in H-9 cells (15). Other drugs including the dideoxycytidines 5 which are also based upon -nhibition of RT by chain

  2. A protein with antiproliferative, antifungal and HIV-1 reverse transcriptase inhibitory activities from caper (Capparis spinosa) seeds.

    PubMed

    Lam, Sze-Kwan; Ng, Tzi-Bun

    2009-05-01

    A protein exhibiting an N-terminal amino acid sequence with some similarity to imidazoleglycerol phosphate synthase was purified from fresh Capparis spinosa melon seeds. The purification protocol entailed anion exchange chromatography on DEAE-cellulose, cation exchange chromatography on SP-Sepharose, and finally gel filtration by fast protein liquid chromatography on Superdex 75. The protein was adsorbed using 20 mM Tris-HCl buffer (pH 7.4) and desorbed using 1 M NaCl in the starting buffer from the DEAE-cellulose column and SP-Sepharose column. The protein demonstrated a molecular mass of 38 kDa in gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that it was monomeric. The protein inhibited proliferation of hepatoma HepG2 cells, colon cancer HT29 cells and breast cancer MCF-7 cells with an IC(50) of about 1, 40 and 60 microM, respectively. It inhibited HIV-1 reverse transcriptase with IC(50) of 0.23 microM. It inhibited mycelial growth in the fungus, Valsa mali. It did not exhibit hemagglutinating, ribonuclease, mitogenic or protease inhibitory activities.

  3. Combining Telomerase Reverse Transcriptase Genetic Variant rs2736100 with Epidemiologic Factors in the Prediction of Lung Cancer Susceptibility

    PubMed Central

    Wang, Xu; Ma, Kewei; Chi, Lumei; Cui, Jiuwei; Jin, Lina; Hu, Ji-Fan; Li, Wei

    2016-01-01

    Genetic variants from a considerable number of susceptibility loci have been identified in association with cancer risk, but their interaction with epidemiologic factors in lung cancer remains to be defined. We sought to establish a forecasting model for identifying individuals with high-risk of lung cancer by combing gene single-nucleotide polymorphisms with epidemiologic factors. Genotyping and clinical data from 500 lung cancer cases and 500 controls were used for developing the logistic regression model. We found that lung cancer was associated with telomerase reverse transcriptase (TERT) rs2736100 single-nucleotide polymorphism. The TERT rs2736100 model was still significantly associated with lung cancer risk when combined with environmental and lifestyle factors, including lower education, lower BMI, COPD history, heavy cigarettes smoking, heavy cooking emission, and dietary factors (over-consumption of meat and deficiency in fish/shrimp, vegetables, dairy products, and soybean products). These data suggest that combining TERT SNP and epidemiologic factors may be a useful approach to discriminate high and low-risk individuals for lung cancer. PMID:27162544

  4. Combining Telomerase Reverse Transcriptase Genetic Variant rs2736100 with Epidemiologic Factors in the Prediction of Lung Cancer Susceptibility.

    PubMed

    Wang, Xu; Ma, Kewei; Chi, Lumei; Cui, Jiuwei; Jin, Lina; Hu, Ji-Fan; Li, Wei

    2016-01-01

    Genetic variants from a considerable number of susceptibility loci have been identified in association with cancer risk, but their interaction with epidemiologic factors in lung cancer remains to be defined. We sought to establish a forecasting model for identifying individuals with high-risk of lung cancer by combing gene single-nucleotide polymorphisms with epidemiologic factors. Genotyping and clinical data from 500 lung cancer cases and 500 controls were used for developing the logistic regression model. We found that lung cancer was associated with telomerase reverse transcriptase (TERT) rs2736100 single-nucleotide polymorphism. The TERT rs2736100 model was still significantly associated with lung cancer risk when combined with environmental and lifestyle factors, including lower education, lower BMI, COPD history, heavy cigarettes smoking, heavy cooking emission, and dietary factors (over-consumption of meat and deficiency in fish/shrimp, vegetables, dairy products, and soybean products). These data suggest that combining TERT SNP and epidemiologic factors may be a useful approach to discriminate high and low-risk individuals for lung cancer.

  5. Crystal structures of the reverse transcriptase-associated ribonuclease H domain of xenotropic murine leukemia-virus related virus

    SciTech Connect

    Zhou, Dongwen; Chung, Suhman; Miller, Maria; Le Grice, Stuart F.J.; Wlodawer, Alexander

    2012-06-19

    The ribonuclease H (RNase H) domain of retroviral reverse transcriptase (RT) plays a critical role in the life cycle by degrading the RNA strands of DNA/RNA hybrids. In addition, RNase H activity is required to precisely remove the RNA primers from nascent (-) and (+) strand DNA. We report here three crystal structures of the RNase H domain of xenotropic murine leukemia virus-related virus (XMRV) RT, namely (i) the previously identified construct from which helix C was deleted, (ii) the intact domain, and (iii) the intact domain complexed with an active site {alpha}-hydroxytropolone inhibitor. Enzymatic assays showed that the intact RNase H domain retained catalytic activity, whereas the variant lacking helix C was only marginally active, corroborating the importance of this helix for enzymatic activity. Modeling of the enzyme-substrate complex elucidated the essential role of helix C in binding a DNA/RNA hybrid and its likely mode of recognition. The crystal structure of the RNase H domain complexed with {beta}-thujaplicinol clearly showed that coordination by two divalent cations mediates recognition of the inhibitor.

  6. Single Active Site Mutation Causes Serious Resistance of HIV Reverse Transcriptase to Lamivudine: Insight from Multiple Molecular Dynamics Simulations.

    PubMed

    Moonsamy, Suri; Bhakat, Soumendranath; Walker, Ross C; Soliman, Mahmoud E S

    2016-03-01

    Molecular dynamics simulations, binding free energy calculations, principle component analysis (PCA), and residue interaction network analysis were employed in order to investigate the molecular mechanism of M184I single mutation which played pivotal role in making the HIV-1 reverse transcriptase (RT) totally resistant to lamivudine. Results showed that single mutations at residue 184 of RT caused (1) distortion of the orientation of lamivudine in the active site due to the steric conflict between the oxathiolane ring of lamivudine and the side chain of beta-branched amino acids Ile at position 184 which, in turn, perturbs inhibitor binding, (2) decrease in the binding affinity by (~8 kcal/mol) when compared to the wild-type, (3) variation in the overall enzyme motion as evident from the PCA for both systems, and (4) distortion of the hydrogen bonding network and atomic interactions with the inhibitor. The comprehensive analysis presented in this report can provide useful information for understanding the drug resistance mechanism against lamivudine. The results can also provide some potential clues for further design of novel inhibitors that are less susceptible to drug resistance.

  7. The brown algae Pl.LSU/2 group II intron-encoded protein has functional reverse transcriptase and maturase activities.

    PubMed

    Zerbato, Madeleine; Holic, Nathalie; Moniot-Frin, Sophie; Ingrao, Dina; Galy, Anne; Perea, Javier

    2013-01-01

    Group II introns are self-splicing mobile elements found in prokaryotes and eukaryotic organelles. These introns propagate by homing into precise genomic locations, following assembly of a ribonucleoprotein complex containing the intron-encoded protein (IEP) and the spliced intron RNA. Engineered group II introns are now commonly used tools for targeted genomic modifications in prokaryotes but not in eukaryotes. We speculate that the catalytic activation of currently known group II introns is limited in eukaryotic cells. The brown algae Pylaiella littoralis Pl.LSU/2 group II intron is uniquely capable of in vitro ribozyme activity at physiological level of magnesium but this intron remains poorly characterized. We purified and characterized recombinant Pl.LSU/2 IEP. Unlike most IEPs, Pl.LSU/2 IEP displayed a reverse transcriptase activity without intronic RNA. The Pl.LSU/2 intron could be engineered to splice accurately in Saccharomyces cerevisiae and splicing efficiency was increased by the maturase activity of the IEP. However, spliced transcripts were not expressed. Furthermore, intron splicing was not detected in human cells. While further tool development is needed, these data provide the first functional characterization of the PI.LSU/2 IEP and the first evidence that the Pl.LSU/2 group II intron splicing occurs in vivo in eukaryotes in an IEP-dependent manner.

  8. A Laccase with Antiproliferative and HIV-I Reverse Transcriptase Inhibitory Activities from the Mycorrhizal Fungus Agaricus placomyces

    PubMed Central

    Sun, Jian; Chen, Qing-Jun; Cao, Qing-Qin; Wu, Ying-Ying; Xu, Li-Jing; Zhu, Meng-Juan; Ng, Tzi-Bun; Wang, He-Xiang; Zhang, Guo-Qing

    2012-01-01

    A novel 68 kDa laccase was purified from the mycorrhizal fungus Agaricus placomyces by utilizing a procedure that comprised three successive steps of ion exchange chromatography and gel filtration as the final step. The monomeric enzyme exhibited the N-terminal amino acid sequence of DVIGPQAQVTLANQD, which showed only a low extent of homology to sequences of other fungal laccases. The optimal temperature for A. placomyces laccase was 30°C, and optimal pH values for laccase activity towards the substrates 2,7′-azinobis[3-ethylbenzothiazolone-6-sulfonic acid] diammonium salt (ABTS) and hydroquinone were 5.2 and 6.8, respectively. The laccase displayed, at 30°C and pH 5.2, Km values of 0.392 mM towards hydroquinone and 0.775 mM towards ABTS. It potently suppressed proliferation of MCF 7 human breast cancer cells and Hep G2 hepatoma cells and inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity with an IC50 of 1.8 μM, 1.7 μM, and 1.25 μM, respectively, signifying that it is an antipathogenic protein. PMID:23093860

  9. Ab initio molecular dynamics studies on HIV-1 reverse transcriptase triphosphate binding site: implications for nucleoside-analog drug resistance.

    PubMed Central

    Alber, F.; Carloni, P.

    2000-01-01

    Quantum-chemical methods are used to shed light on the functional role of residues involved in the resistance of HIV-1 reverse transcriptase against nucleoside-analog drugs. Ab initio molecular dynamics simulations are carried out for models representing the adduct between the triphosphate substrate and the nucleoside binding site. The triphosphate is considered either deprotonated or protonated at the gamma-position. Although the protonated form already experiences large rearrangements in the ps time scale, the fully deprotonated state exhibits a previously unrecognized low-barrier hydrogen bond between Lys65 and gamma-phosphate. Absence of this interaction in Lys65-->Arg HIV-1 RT might play a prominent role in the resistance of this mutant for nucleoside analogs (Gu Z et al., 1994b, Antimicrob Agents Chemother 38:275-281; Zhang D et al., 1994, Antimicrob Agents Chemother 38:282-287). Water molecules present in the active site, not detected in the X-ray structure, form a complex H-bond network. Among these waters, one may be crucial for substrate recognition as it bridges Gln151 and Arg72 with the beta-phosphate. Absence of this stabilizing interaction in Gln151-->Met HIV-1 RT mutant may be a key factor for the known drug resistance of this mutant toward dideoxy-type drugs and AZT (Shirasaka T et al., 1995, Proc Natl Acad Sci USA 92:2398-2402: Iversen AK et al., 1996, J Virol 70:1086-1090). PMID:11206075

  10. CEBP factors regulate telomerase reverse transcriptase promoter activity in whey acidic protein-T mice during mammary carcinogenesis.

    PubMed

    Kumar, Mukesh; Witt, Britta; Knippschild, Uwe; Koch, Sylvia; Meena, Jitendra K; Heinlein, Christina; Weise, Julia M; Krepulat, Frauke; Kuchenbauer, Florian; Iben, Sebastian; Rudolph, Karl-Lenhard; Deppert, Wolfgang; Günes, Cagatay

    2013-05-01

    Telomerase is activated in the majority of invasive breast cancers, but the time point of telomerase activation during mammary carcinogenesis is not clear. We have recently presented a transgenic mouse model to study human telomerase reverse transcriptase (TERT) gene expression in vivo (hTERTp-lacZ). In the present study, hTERTp-lacZxWAP-T bitransgenic mice were generated to analyze the mechanisms responsible for human and mouse TERT upregulation during tumor progression in vivo. We found that telomerase activity and TERT expression were consistently upregulated in SV40-induced invasive mammary tumors compared to normal and hyperplastic tissues and ductal carcinoma in situ (DCIS). Human and mouse TERT genes are regulated similarly in the breast tissue, involving the CEBP transcription factors. Loss of CEBP-α and induction of CEBP-β expression correlated well with the activation of TERT expression in mouse mammary tumors. Transfection of CEBP-α into human or murine cells resulted in TERT repression, whereas knockdown of CEBP-α in primary human mammary epithelial cells resulted in reactivation of endogenous TERT expression and telomerase activity. Conversely, ectopic expression of CEBP-β activated endogenous TERT gene expression. Moreover, ChIP and EMSA experiments revealed binding of CEBP-α and CEBP-β to human TERT-promoter. This is the first evidence indicating that CEBP-α and CEBP-β are involved in TERT gene regulation during carcinogenesis. Copyright © 2012 UICC.

  11. Evaluation of reverse transcriptase polymerase chain reaction for the detection of eastern equine encephalomyelitis virus during vector surveillance.

    PubMed

    Monroy, A M; Scott, T W; Webb, B A

    1996-05-01

    A reverse transcriptase polymerase chain reaction (RT-PCR) assay was evaluated for the detection of eastern equine encephalomyelitis virus (EEEV). EEEV was detected by amplification of a 416-bp PCR product from within the E2 gene. Internal restriction endonuclease digestion and hybridizations to EEEV RNA demonstrated that the PCR product was amplified from EEEV. PCR amplifications from serial dilutions of an EEEV isolate identified by a neutralization test and titered by an infectious assay in cell culture indicated that this RT-PCR assay detected viral RNA at concentrations below 1 plaque forming unit(PFU) per reaction. The performance of the PCR assay in detection of EEEV was compared with an infectious assay detection procedure (IA/IFA) as part of the New Jersey 1993 vector surveillance program. During 1993, 7,007 field-collected Culiseta melanura (Coquillett) were assayed in 522 pools by both RT-PCR and IA/IFA. EEEV was detected in 95 pools by RT-PCR and 17 pools by IA/IFA; all IA/IFA positive pools were also positive by RT-PCR. During the 1993 field season, RT-PCR consistently detected virus at enzootic foci earlier that IA/IFA and in greater numbers of mosquito pools. The data indicated that viral RNA may be present earlier and in more mosquitoes than indicated by IA/IFA.

  12. Anticancer DNA vaccine based on human telomerase reverse transcriptase generates a strong and specific T cell immune response.

    PubMed

    Thalmensi, Jessie; Pliquet, Elodie; Liard, Christelle; Escande, Marie; Bestetti, Thomas; Julithe, Marion; Kostrzak, Anna; Pailhes-Jimenez, Anne-Sophie; Bourges, Emanuèle; Loustau, Maria; Caumartin, Julien; Lachgar, Abderrahim; Huet, Thierry; Wain-Hobson, Simon; Langlade-Demoyen, Pierre

    2016-03-01

    Human telomerase reverse transcriptase (hTERT) is overexpressed in more than 85% of human cancers regardless of their cellular origin. As immunological tolerance to hTERT can be overcome not only spontaneously but also by vaccination, it represents a relevant universal tumor associated antigen (TAA). Indeed, hTERT specific cytotoxic T lymphocyte (CTL) precursors are present within the peripheral T-cell repertoire. Consequently, hTERT vaccine represents an attractive candidate for antitumor immunotherapy. Here, an optimized DNA plasmid encoding an inactivated form of hTERT, named INVAC-1, was designed in order to trigger cellular immunity against tumors. Intradermal injection of INVAC-1 followed by electrogene transfer (EGT) in a variety of mouse models elicited broad hTERT specific cellular immune responses including high CD4(+) Th1 effector and memory CD8(+) T‑cells. Furthermore, therapeutic INVAC‑1 immunization in a HLA-A2 spontaneous and aggressive mouse sarcoma model slows tumor growth and increases survival rate of 50% of tumor-bearing mice. These results emphasize that INVAC-1 based immunotherapy represents a relevant cancer vaccine candidate.

  13. Anticancer DNA vaccine based on human telomerase reverse transcriptase generates a strong and specific T cell immune response

    PubMed Central

    Thalmensi, Jessie; Pliquet, Elodie; Liard, Christelle; Escande, Marie; Bestetti, Thomas; Julithe, Marion; Kostrzak, Anna; Pailhes-Jimenez, Anne-Sophie; Bourges, Emanuèle; Loustau, Maria; Caumartin, Julien; Lachgar, Abderrahim; Huet, Thierry; Wain-Hobson, Simon; Langlade-Demoyen, Pierre

    2016-01-01

    ABSTRACT Human telomerase reverse transcriptase (hTERT) is overexpressed in more than 85% of human cancers regardless of their cellular origin. As immunological tolerance to hTERT can be overcome not only spontaneously but also by vaccination, it represents a relevant universal tumor associated antigen (TAA). Indeed, hTERT specific cytotoxic T lymphocyte (CTL) precursors are present within the peripheral T-cell repertoire. Consequently, hTERT vaccine represents an attractive candidate for antitumor immunotherapy. Here, an optimized DNA plasmid encoding an inactivated form of hTERT, named INVAC-1, was designed in order to trigger cellular immunity against tumors. Intradermal injection of INVAC-1 followed by electrogene transfer (EGT) in a variety of mouse models elicited broad hTERT specific cellular immune responses including high CD4+ Th1 effector and memory CD8+ T‑cells. Furthermore, therapeutic INVAC‑1 immunization in a HLA-A2 spontaneous and aggressive mouse sarcoma model slows tumor growth and increases survival rate of 50% of tumor-bearing mice. These results emphasize that INVAC-1 based immunotherapy represents a relevant cancer vaccine candidate. PMID:27141336

  14. A Novel Laccase with Potent Antiproliferative and HIV-1 Reverse Transcriptase Inhibitory Activities from Mycelia of Mushroom Coprinus comatus

    PubMed Central

    Zhao, Shuang; Rong, Cheng-Bo; Kong, Chang; Liu, Yu; Xu, Feng; Miao, Qian-Jiang; Wang, Shou-Xian; Wang, He-Xiang

    2014-01-01

    A novel laccase was isolated and purified from fermentation mycelia of mushroom Coprinus comatus with an isolation procedure including three ion-exchange chromatography steps on DEAE-cellulose, CM-cellulose, and Q-Sepharose and one gel-filtration step by fast protein liquid chromatography on Superdex 75. The purified enzyme was a monomeric protein with a molecular weight of 64 kDa. It possessed a unique N-terminal amino acid sequence of AIGPVADLKV, which has considerably high sequence similarity with that of other fungal laccases, but is different from that of C. comatus laccases reported. The enzyme manifested an optimal pH value of 2.0 and an optimal temperature of 60°C using 2,2′-azinobis(3-ethylbenzothiazolone-6-sulfonic acid) diammonium salt (ABTS) as the substrate. The laccase displayed, at pH 2.0 and 37°C, Km values of 1.59 mM towards ABTS. It potently suppressed proliferation of tumor cell lines HepG2 and MCF7, and inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) with an IC50 value of 3.46 μM, 4.95 μM, and 5.85 μM, respectively, signifying that it is an antipathogenic protein. PMID:25540778

  15. Culture and Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Proven Mycobacterium Tuberculosis Endophthalmitis: A Case Series.

    PubMed

    Rishi, Ekta; Rishi, Pukhraj; Therese, K Lily; Ramasubban, Gayathri; Biswas, Jyotirmay; Sharma, Tarun; Bhende, Pramod; Susvar, Pradeep; Agarwal, Mamta; George, Amala Elizabeth; Delhiwala, Kushal; Sharma, Vishal Rajan

    2016-09-06

    To report early confirmation of Mycobacterium tuberculosis (MTB) endophthalmitis by detection of 85B mRNA in vitreous by a reverse transcriptase polymerase chain reaction (RT-PCR) technique. Retrospective, interventional case series of 5 patients with MTB endogenous endophthalmitis. Vitreous aspirate was subjected to Ziehl-Neelsen (ZN) staining, BACTEC MicroMGIT culture, RT-PCR targeting the 85B gene, real-time PCR targeting the IS6110 region, and nested PCR targeting the MPB64 gene and IS6110 region. Correlation between detection of MTB RNA, culture positivity, and ZN staining was studied. Five patients with endophthalmitis with no history of tuberculosis revealed acid-fast bacilli on ZN staining of vitreous. RT-PCR detected 85B RNA within 24 h. Culture for MTB was positive in 3/5 patients after 1 month. None of the eyes recovered any useful vision. RT-PCR can detect viable MTB RNA and provide evidence of active infection much earlier than culture.

  16. Unfolding the HIV-1 reverse transcriptase RNase H domain – how to lose a molecular tug-of-war

    SciTech Connect

    Zheng, Xunhai; Pedersen, Lars C.; Gabel, Scott A.; Mueller, Geoffrey A.; DeRose, Eugene F.; London, Robert E.

    2016-01-14

    Formation of the mature HIV-1 reverse transcriptase (RT) p66/p51 heterodimer requires subunit-specific processing of the p66/p66' homodimer precursor. Since the ribonuclease H (RH) domain contains an occult cleavage site located near its center, cleavage must occur either prior to folding or subsequent to unfolding. Recent NMR studies have identified a slow, subunit-specific RH domain unfolding process proposed to result from a residue tug-of-war between the polymerase and RH domains on the functionally inactive, p66' subunit. Here, we describe a structural comparison of the isolated RH domain with a domain swapped RH dimer that reveals several intrinsically destabilizing characteristics of the isolated domain that facilitate excursions of Tyr427 from its binding pocket and separation of helices B and D. These studies provide independent support for the subunit-selective RH domain unfolding pathway in which instability of the Tyr427 binding pocket facilitates its release followed by domain transfer, acting as a trigger for further RH domain destabilization and subsequent unfolding. As further support for this pathway, NMR studies demonstrate that addition of an RH active site-directed isoquinolone ligand retards the subunit-selective RH' domain unfolding behavior of the p66/p66' homodimer. As a result, this study demonstrates the feasibility of directly targeting RT maturation with therapeutics.

  17. Unfolding the HIV-1 reverse transcriptase RNase H domain – how to lose a molecular tug-of-war

    DOE PAGES

    Zheng, Xunhai; Pedersen, Lars C.; Gabel, Scott A.; ...

    2016-01-14

    Formation of the mature HIV-1 reverse transcriptase (RT) p66/p51 heterodimer requires subunit-specific processing of the p66/p66' homodimer precursor. Since the ribonuclease H (RH) domain contains an occult cleavage site located near its center, cleavage must occur either prior to folding or subsequent to unfolding. Recent NMR studies have identified a slow, subunit-specific RH domain unfolding process proposed to result from a residue tug-of-war between the polymerase and RH domains on the functionally inactive, p66' subunit. Here, we describe a structural comparison of the isolated RH domain with a domain swapped RH dimer that reveals several intrinsically destabilizing characteristics of themore » isolated domain that facilitate excursions of Tyr427 from its binding pocket and separation of helices B and D. These studies provide independent support for the subunit-selective RH domain unfolding pathway in which instability of the Tyr427 binding pocket facilitates its release followed by domain transfer, acting as a trigger for further RH domain destabilization and subsequent unfolding. As further support for this pathway, NMR studies demonstrate that addition of an RH active site-directed isoquinolone ligand retards the subunit-selective RH' domain unfolding behavior of the p66/p66' homodimer. As a result, this study demonstrates the feasibility of directly targeting RT maturation with therapeutics.« less

  18. Unfolding the HIV-1 reverse transcriptase RNase H domain – how to lose a molecular tug-of-war

    PubMed Central

    Zheng, Xunhai; Pedersen, Lars C.; Gabel, Scott A.; Mueller, Geoffrey A.; DeRose, Eugene F.; London, Robert E.

    2016-01-01

    Formation of the mature HIV-1 reverse transcriptase (RT) p66/p51 heterodimer requires subunit-specific processing of the p66/p66′ homodimer precursor. Since the ribonuclease H (RH) domain contains an occult cleavage site located near its center, cleavage must occur either prior to folding or subsequent to unfolding. Recent NMR studies have identified a slow, subunit-specific RH domain unfolding process proposed to result from a residue tug-of-war between the polymerase and RH domains on the functionally inactive, p66′ subunit. Here, we describe a structural comparison of the isolated RH domain with a domain swapped RH dimer that reveals several intrinsically destabilizing characteristics of the isolated domain that facilitate excursions of Tyr427 from its binding pocket and separation of helices B and D. These studies provide independent support for the subunit-selective RH domain unfolding pathway in which instability of the Tyr427 binding pocket facilitates its release followed by domain transfer, acting as a trigger for further RH domain destabilization and subsequent unfolding. As further support for this pathway, NMR studies demonstrate that addition of an RH active site-directed isoquinolone ligand retards the subunit-selective RH′ domain unfolding behavior of the p66/p66′ homodimer. This study demonstrates the feasibility of directly targeting RT maturation with therapeutics. PMID:26773054

  19. Telomerase Reverse Transcriptase Is Required for the Localization of Telomerase RNA to Cajal Bodies and Telomeres in Human Cancer Cells

    PubMed Central

    Tomlinson, Rebecca L.; Abreu, Eladio B.; Ziegler, Tania; Ly, Hinh; Counter, Christopher M.

    2008-01-01

    Telomere maintenance by telomerase is critical for the unlimited division potential of most human cancer cells. The two essential components of human telomerase, telomerase RNA (hTR) and telomerase reverse transcriptase (hTERT), are recruited from distinct subnuclear sites to telomeres during S phase. Throughout the remainder of the cell cycle hTR is found primarily in Cajal bodies. The localization of hTR to Cajal bodies and telomeres is specific to cancer cells where telomerase is active and is not observed in primary cells. Here we show that the trafficking of hTR to both telomeres and Cajal bodies depends on hTERT. RNA interference–mediated depletion of hTERT in cancer cells leads to loss of hTR from both Cajal bodies and telomeres without affecting hTR levels. In addition, expression of hTERT in telomerase-negative cells (including primary and ALT cancer cell lines) induces hTR to localize to both sites. Factors that did not stimulate hTR localization in our experiments include increased hTR RNA levels and Cajal body numbers, and expression of SV40 large T antigen and oncogenic Ras. Our findings suggest that the trafficking of telomerase to Cajal bodies and telomeres in cancer cells correlates with and depends on the assembly of the enzyme. PMID:18562689

  20. 2´,3´-Dialdehyde of ATP, ADP, and adenosine inhibit HIV-1 reverse transcriptase and HIV-1 replication.

    PubMed

    Schachter, Julieta; Valadao, Ana Luiza Chaves; Aguiar, Renato Santana; Barreto-de-Souza, Victor; Rossi, Atila Duque; Arantes, Pablo Ricardo; Verli, Hugo; Quintana, Paula Gabriela; Heise, Norton; Tanuri, Amilcar; Bou-Habib, Dumith Chequer; Persechini, Pedro Muanis

    2014-01-01

    The 2´3´-dialdehyde of ATP or oxidized ATP (oATP) is a compound known for specifically making covalent bonds with the nucleotide-binding site of several ATP-binding enzymes and receptors. We investigated the effects of oATP and other oxidized purines on HIV-1 infection and we found that this compound inhibits HIV-1 and SIV infection by blocking early steps of virus replication. oATP, oxidized ADP (oADP), and oxidized Adenosine (oADO) impact the natural activity of endogenous reverse transcriptase enzyme (RT) in cell free virus particles and are able to inhibit viral replication in different cell types when added to the cell cultures either before or after infection. We used UFLC-UV to show that both oADO and oATP can be detected in the cell after being added in the extracellular medium. oATP also suppresses RT activity and replication of the HIV-1 resistant variants M184V and T215Y. We conclude that oATP, oADP and oADO display anti HIV-1 activity that is at in least in part due to inhibitory activity on HIV-1 RT.

  1. IMMORTALIZATION OF HUMAN AND RHESUS MACAQUE PRIMARY ANTIGEN-SPECIFIC T CELLS BY RETROVIRALLY TRANSDUCED TELOMERASE REVERSE TRANSCRIPTASE

    PubMed Central

    Barsov, Eugene V.

    2011-01-01

    Human and rhesus macaque primary antigen-specific T cells derived from infected or immunized individuals or animals are a valuable material with which to study cellular immune responses against pathogens and tumors. Antigen-specific T cells can be expanded in vitro but have a finite proliferative life span. After a limited period in culture, primary T cells undergo replicative senescence and stop dividing. This restricts their applicability to short term experiments and complicates their use in adoptive immunotherapy. The proliferative life span of primary human and rhesus macaque T cells can be considerably extended by ectopically expressed human telomerase reverse transcriptase (TERT). Antigen-specific T cells transduced with TERT-expressing retroviral vectors can proliferate and expand in culture for long periods of time while maintaining their primary T cell characteristics including antigen-specific responses. Thus, TERT-immortalized T cells are an important and valuable resource for studying T cell immune responses and, potentially, for adoptive immunotherapy. PMID:22048804

  2. Exclusion of exon 2 is a common mRNA splice variant of primate telomerase reverse transcriptases.

    PubMed

    Withers, Johanna B; Ashvetiya, Tamara; Beemon, Karen L

    2012-01-01

    Telomeric sequences are added by an enzyme called telomerase that is made of two components: a catalytic protein called telomerase reverse transcriptase (TERT) and an integral RNA template (TR). Telomerase expression is tightly regulated at each step of gene expression, including alternative splicing of TERT mRNA. While over a dozen different alternative splicing events have been reported for human TERT mRNA, these were all in the 3' half of the coding region. We were interested in examining splicing of the 5' half of hTERT mRNA, especially since exon 2 is unusually large (1.3 kb). Internal mammalian exons are usually short, typically only 50 to 300 nucleotides, and most long internal exons are alternatively processed. We used quantitative RT-PCR and high-throughput sequencing data to examine the variety and quantity of mRNA species generated from the hTERT locus. We determined that there are approximately 20-40 molecules of hTERT mRNA per cell in the A431 human cell line. In addition, we describe an abundant, alternatively-spliced mRNA variant that excludes TERT exon 2 and was seen in other primates. This variant causes a frameshift and results in translation termination in exon 3, generating a 12 kDa polypeptide.

  3. A novel lectin with highly potent antiproliferative and HIV-1 reverse transcriptase inhibitory activities from cicada (Cicada flammata).

    PubMed

    Ye, Xiu Juan; Ng, Tzi Bun

    2010-05-01

    A dimeric lectin with a molecular weight of 60 kDa and high hemagglutinating activity was isolated from dried cicadas. It was adsorbed on Q-Sepharose and unadsorbed on Affi-Gel Blue gel. Its hemagglutinating activity was stable up to 55 degrees C and between pH 2 and 13. The activity was inhibited by glucuronic acid and raffinose, K(+) ions, and Mg(2+) ions. Cicada lectin potently inhibited proliferation of HepG2 hepatoma and MCF 7 breast cancer cells, with an IC(50) value of 0.76 and 0.49 microM, respectively. It potently inhibited HIV-1 reverse transcriptase activity with an IC(50) of 0.36 microM but was devoid of mitogenic activity on spleen cells. Its N-terminal sequence exhibited slight similarity to a conserved hypothetical protein from Culex quinquefasciatus and a gene product from transcript GH19834-RA of Drosophila grimshawi, but there was no resemblance to lectins from other insects, including Drosophila, Sarcophaga, Glossina, and Aedes species.

  4. Deletion of the telomerase reverse transcriptase gene and haploinsufficiency of telomere maintenance in Cri du chat syndrome.

    PubMed

    Zhang, Anju; Zheng, Chengyun; Hou, Mi; Lindvall, Charlotta; Li, Ke-Jun; Erlandsson, Fredrik; Björkholm, Magnus; Gruber, Astrid; Blennow, Elisabeth; Xu, Dawei

    2003-04-01

    Cri du chat syndrome (CdCS) results from loss of the distal portion of chromosome 5p, where the telomerase reverse transcriptase (hTERT) gene is localized (5p15.33). hTERT is the rate-limiting component for telomerase activity that is essential for telomere-length maintenance and sustained cell proliferation. Here, we show that a concomitant deletion of the hTERT allele occurs in all 10 patients with CdCS whom we examined. Induction of hTERT mRNA in proliferating lymphocytes derived from five of seven patients was lower than that in unaffected control individuals (P<.05). The patient lymphocytes exhibited shorter telomeres than age-matched unaffected individuals (P<.0001). A reduction in replicative life span and a high rate of chromosome fusions were observed in cultured patient fibroblasts. Reconstitution of telomerase activity by ectopic expression of hTERT extended the telomere length, increased the population doublings, and prevented the end-to-end fusion of chromosomes. We conclude that hTERT is limiting and haploinsufficient for telomere maintenance in humans in vivo. Accordingly, the hTERT deletion may be one genetic element contributing to the phenotypic changes in CdCS.

  5. Mutations in the HIV-1 reverse transcriptase tryptophan repeat motif affect virion maturation and Gag-Pol packaging.

    PubMed

    Chiang, Chien-Cheng; Tseng, Ying-Tzu; Huang, Kuo-Jung; Pan, Yen-Yu; Wang, Chin-Tien

    2012-01-20

    Our goal was to determine the contribution of HIV-1 reverse transcriptase tryptophan repeat motif residues to virion maturation. With the exception of W402A, we found none of the single substitution mutations exerted major impacts on virus assembly or processing. However, all mutants except for W410A exhibited significant decreases in virus-associated RT, presumably a result of unstable RT mutant degradation. Mutations W398A, W401A and W406A decreased the enhancement effect of efavirenz on PR-mediated Gag processing efficiency, which is in agreement with their destabilizing RT effects. Furthermore, combined double or triple W398, W401 and W406 mutations significantly affected virus processing and Gag-Pol packaging. Further analyses suggest that inefficient PR-mediated Gag cleavage partly accounts for the virion processing defect. Our results support the idea that in addition to playing a role in RT heterodimer stabilization, the RT Trp repeat motif in the Gag-Pol context is also involved in PR activation via Gag-Pol/Gag-Pol interaction. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. Free Energy-Based Virtual Screening and Optimization of RNase H Inhibitors of HIV-1 Reverse Transcriptase

    PubMed Central

    2016-01-01

    We report the results of a binding free energy-based virtual screening campaign of a library of 77 α-hydroxytropolone derivatives against the challenging RNase H active site of the reverse transcriptase (RT) enzyme of human immunodeficiency virus-1. Multiple protonation states, rotamer states, and binding modalities of each compound were individually evaluated. The work involved more than 300 individual absolute alchemical binding free energy parallel molecular dynamics calculations and over 1 million CPU hours on national computing clusters and a local campus computational grid. The thermodynamic and structural measures obtained in this work rationalize a series of characteristics of this system useful for guiding future synthetic and biochemical efforts. The free energy model identified key ligand-dependent entropic and conformational reorganization processes difficult to capture using standard docking and scoring approaches. Binding free energy-based optimization of the lead compounds emerging from the virtual screen has yielded four compounds with very favorable binding properties, which will be the subject of further experimental investigations. This work is one of the few reported applications of advanced-binding free energy models to large-scale virtual screening and optimization projects. It further demonstrates that, with suitable algorithms and automation, advanced-binding free energy models can have a useful role in early-stage drug-discovery programs. PMID:27713931

  7. A Laccase with HIV-1 Reverse Transcriptase Inhibitory Activity from the Broth of Mycelial Culture of the Mushroom Lentinus tigrinus

    PubMed Central

    Xu, LiJing; Wang, HeXiang; Ng, TziBun

    2012-01-01

    A 59 kDa laccase with inhibitory activity against HIV-1 reverse transcriptase (IC50 = 2.4 μM) was isolated from the broth of mycelial culture of the mushroom Lentinus tigrinus. The isolation procedure involved ion exchange chromatography on DEAE-cellulose and CM-cellulose, and gel filtration by fast protein liquid chromatography on Superdex 75. The laccase was adsorbed on both types of ion exchangers. About 95-fold purification was achieved with a 25.9% yield of the enzyme. The procedure resulted in a specific enzyme activity of 76.6 U/mg. Its N-terminal amino acid sequence was GIPDLHDLTV, which showed little similarity to other mushroom laccase and other Lentinus tigrinus strain laccase. Its characteristics were different from previously reported laccase of other Lentinus tigrinus strain. Maximal laccase activity was observed at a pH of 4 and at a temperature of 60°C, respectively. This study yielded the information about the potentially exploitable activities of Lentinus tigrinus laccase. PMID:22536022

  8. Association of telomerase reverse transcriptase promoter mutations with clinicopathological features and prognosis of thyroid cancer: a meta-analysis

    PubMed Central

    Su, Xingyun; Jiang, Xiaoxia; Wang, Weibin; Wang, Haiyong; Xu, Xin; Lin, Aihui; Teng, Xiaodong; Wu, Huiling; Teng, Lisong

    2016-01-01

    The clinicopathological and prognostic significance of telomerase reverse transcriptase (TERT) promoter mutations have been widely investigated in thyroid cancer; however, the results are still discrepant. Systematic searches were performed in PubMed, Web of Science, Scopus, Ovid, and the Cochran Library databases for relevant articles prior to April 2016. Mutation rates were synthesized by R statistical software. The odds ratio or standardized mean difference with 95% confidence interval was pooled by Stata. A total of 22 studies with 4,907 cases were included in this meta-analysis. TERT promoter mutations tended to present in aggressive histological types including poorly differentiated thyroid cancer (33.37%), anaplastic thyroid cancer (38.69%), and tall-cell variant papillary thyroid cancer (30.23%). These promoter mutations were likely to exist in older patients and males and were well associated with larger tumor size, extrathyroidal extension, vascular invasion, lymph node metastasis, distant metastasis, advanced tumor stage, disease recurrence/persistence, and mortality. In addition, TERT promoter mutations (especially C228T) tended to coexist with BRAFV600E mutation, which indicated more aggressive tumor behavior. Therefore, TERT promoter mutations may be promising biomarkers for early diagnosis, risk stratification, prognostic prediction, and management of thyroid cancer. PMID:27956840

  9. The Brown Algae Pl.LSU/2 Group II Intron-Encoded Protein Has Functional Reverse Transcriptase and Maturase Activities

    PubMed Central

    Zerbato, Madeleine; Holic, Nathalie; Moniot-Frin, Sophie; Ingrao, Dina; Galy, Anne; Perea, Javier

    2013-01-01

    Group II introns are self-splicing mobile elements found in prokaryotes and eukaryotic organelles. These introns propagate by homing into precise genomic locations, following assembly of a ribonucleoprotein complex containing the intron-encoded protein (IEP) and the spliced intron RNA. Engineered group II introns are now commonly used tools for targeted genomic modifications in prokaryotes but not in eukaryotes. We speculate that the catalytic activation of currently known group II introns is limited in eukaryotic cells. The brown algae Pylaiella littoralis Pl.LSU/2 group II intron is uniquely capable of in vitro ribozyme activity at physiological level of magnesium but this intron remains poorly characterized. We purified and characterized recombinant Pl.LSU/2 IEP. Unlike most IEPs, Pl.LSU/2 IEP displayed a reverse transcriptase activity without intronic RNA. The Pl.LSU/2 intron could be engineered to splice accurately in Saccharomyces cerevisiae and splicing efficiency was increased by the maturase activity of the IEP. However, spliced transcripts were not expressed. Furthermore, intron splicing was not detected in human cells. While further tool development is needed, these data provide the first functional characterization of the PI.LSU/2 IEP and the first evidence that the Pl.LSU/2 group II intron splicing occurs in vivo in eukaryotes in an IEP-dependent manner. PMID:23505475

  10. High Sequence Conservation of Human Immunodeficiency Virus Type 1 Reverse Transcriptase under Drug Pressure despite the Continuous Appearance of Mutations

    PubMed Central

    Ceccherini-Silberstein, Francesca; Gago, Federico; Santoro, Maria; Gori, Caterina; Svicher, Valentina; Rodríguez-Barrios, Fátima; d'Arrigo, Roberta; Ciccozzi, Massimo; Bertoli, Ada; Monforte, Antonella d'Arminio; Balzarini, Jan; Antinori, Andrea; Perno, Carlo-Federico

    2005-01-01

    To define the extent of sequence conservation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in vivo, the first 320 amino acids of RT obtained from 2,236 plasma-derived samples from a well-defined cohort of 1,704 HIV-1-infected individuals (457 drug naïve and 1,247 drug treated) were analyzed and examined in structural terms. In naïve patients, 233 out of these 320 residues (73%) were conserved (<1% variability). The majority of invariant amino acids clustered into defined regions comprising between 5 and 29 consecutive residues. Of the nine longest invariant regions identified, some contained residues and domains critical for enzyme stability and function. In patients treated with RT inhibitors, despite profound drug pressure and the appearance of mutations primarily associated with resistance, 202 amino acids (63%) remained highly conserved and appeared mostly distributed in regions of variable length. This finding suggests that participation of consecutive residues in structural domains is strictly required for cooperative functions and sustainability of HIV-1 RT activity. Besides confirming the conservation of amino acids that are already known to be important for catalytic activity, stability of the heterodimer interface, and/or primer/template binding, the other 62 new invariable residues are now identified and mapped onto the three-dimensional structure of the enzyme. This new knowledge could be of help in the structure-based design of novel resistance-evading drugs. PMID:16051864

  11. Structure of HIV-1 reverse transcriptase bound to a novel 38-mer hairpin template-primer DNA aptamer.

    PubMed

    Miller, Matthew T; Tuske, Steve; Das, Kalyan; DeStefano, Jeffrey J; Arnold, Eddy

    2016-01-01

    The development of a modified DNA aptamer that binds HIV-1 reverse transcriptase (RT) with ultra-high affinity has enabled the X-ray structure determination of an HIV-1 RT-DNA complex to 2.3 Å resolution without the need for an antibody Fab fragment or RT-DNA cross-linking. The 38-mer hairpin-DNA aptamer has a 15 base-pair duplex, a three-deoxythymidine hairpin loop, and a five-nucleotide 5'-overhang. The aptamer binds RT in a template-primer configuration with the 3'-end positioned at the polymerase active site and has 2'-O-methyl modifications at the second and fourth duplex template nucleotides that interact with the p66 fingers and palm subdomains. This structure represents the highest resolution RT-nucleic acid structure to date. The RT-aptamer complex is catalytically active and can serve as a platform for studying fundamental RT mechanisms and for development of anti-HIV inhibitors through fragment screening and other approaches. Additionally, the structure allows for a detailed look at a unique aptamer design and provides the molecular basis for its remarkably high affinity for RT. © 2015 The Protein Society.

  12. Quantitative structure-activity relationships and comparative molecular field analysis of TIBO derivatised HIV-1 reverse transcriptase inhibitors

    NASA Astrophysics Data System (ADS)

    Hannongbua, Supa; Pungpo, Pornpan; Limtrakul, Jumras; Wolschann, Peter

    1999-11-01

    Quantitative structure-activity relationships (QSAR) and Comparative Molecular Field Analysis (CoMFA) have been applied in order to explain the structural requirements of HIV-1 reverse transcriptase (HIV-1 RT) inhibitory activity of TIBO derivatives on the MT-4 cells. The best QSAR model is satisfactory in both statistical significance and predictive ability. The derived structural descriptors indicate the importance of electronic contributions toward the HIV-1 RT inhibition of this class of compounds. However, it could not reveal any hydrophobic influence because of high collinearity between C2 and log P variables. In order to cope with steric interaction in the correlation, 3D-QSAR was performed using CoMFA. The obtained CoMFA model shows high predictive ability, r2 cv=0.771, and clearly demonstrates its potential in the steric feature of the molecules through contour maps, explaining a majority (81.8%) of the variance in the data. Consequently, these results can be useful in identifying the structural requirements of TIBO derivatives and helpful for better understanding the HIV-1 RT inhibition. Eventually, they provide a beneficial basis to design new and more potent inhibitors of HIV-1 RT.

  13. A critical role of nicotinamide phosphoribosyltransferase in human telomerase reverse transcriptase induction by resveratrol in aortic smooth muscle cells

    PubMed Central

    Huang, Peixin; Riordan, Sean M.; Heruth, Daniel P.; Grigoryev, Dmitry N.; Zhang, Li Qin; Ye, Shui Qing

    2015-01-01

    Aging is the predominant risk factor for cardiovascular diseases and contributes to a considerably more severe outcome in patients with acute myocardial infarction. Resveratrol, a polyphenol found in red wine, is a caloric restriction mimetic with potential anti-aging properties which has emerged as a beneficial nutraceutical for patients with cardiovascular disease. Although resveratrol is widely consumed as a nutritional supplement, its mechanism of action remains to be elucidated fully. Here, we report that resveratrol activates human nicotinamide phosphoribosyltransferase (NAMPT), SIRT4 and telomerase reverse transcriptase (hTERT) in human aortic smooth muscle cells. Similar observations were obtained in resveratrol treated C57BL/6J mouse heart and liver tissues. Resverotrol can also augment telomerase activity in both human pulmonary microvascular endothelial cells and A549 cells. Blocking NAMPT and SIRT4 expression prevents induction of hTERT in human aortic smooth muscle cells while overexpression of NAMPT elevates the telomerase activity induced by resveratrol in A549 cells. Together, these results identify a NAMPT-SIRT4-hTERT axis as a novel mechanism by which resveratrol may affect the anti-aging process in human aortic smooth muscle cells, mouse hearts and other cells. These findings enrich our understanding of the positive effects of resveratrol in human cardiovascular diseases. PMID:25926556

  14. Natural product-inspired esters and amides of ferulic and caffeic acid as dual inhibitors of HIV-1 reverse transcriptase.

    PubMed

    Sonar, Vijay P; Corona, Angela; Distinto, Simona; Maccioni, Elias; Meleddu, Rita; Fois, Benedetta; Floris, Costantino; Malpure, Nilesh V; Alcaro, Stefano; Tramontano, Enzo; Cottiglia, Filippo

    2017-04-21

    Using an HIV-1 Reverse Transcriptase (RT)-associated RNase H inhibition assay as lead, bioguided fractionation of the dichloromethane extract of the Ocimum sanctum leaves led to the isolation of five triterpenes (1-5) along with three 3-methoxy-4-hydroxy phenyl derivatives (6-8). The structure of this isolates were determined by 1D and 2D NMR experiments as well as ESI-MS. Tetradecyl ferulate (8) showed an interesting RNase H IC50 value of 12.4 μM and due to the synthetic accessibility of this secondary metabolite, a structure-activity relationship study was carried out. A series of esters and amides of ferulic and caffeic acids were synthesized and, among all, the most active was N-oleylcaffeamide displaying a strong inhibitory activity towards both RT-associated functions, ribonuclease H and DNA polymerase. Molecular modeling studies together with Yonetani-Theorell analysis, demonstrated that N-oleylcaffeamide is able to bind both two allosteric site located one close to the NNRTI binding pocket and the other close to RNase H catalytic site.

  15. Development of an RNA extraction protocol for detection of waterborne viruses by reverse transcriptase quantitative PCR (RT-qPCR).

    PubMed

    Jothikumar, N; Sobsey, M D; Cromeans, T L

    2010-10-01

    RNA extraction from environmental samples yields frequently an RNA preparation containing inhibitors of molecular reactions. Commercial RNA extraction kits commonly permit extraction of only 0.1-0.2 ml sample volume. An RNA extraction buffer (RNAX buffer) was formulated for the extraction of viral RNA from 4.0 ml using a silica column based protocol. To evaluate the RNAX buffer based protocol, we used hepatitis A virus (HAV) and coxsackievirus B3 (CVB3) to monitor the RNA extraction efficiency from environmental samples. For evaluation of viral RNA recovery from water concentrates which were prepared from river and pond water by PEG concentration, serial ten fold dilutions of two waterborne viruses were added to the water concentrates for evaluation by quantitative detection. Quantitative recovery of HAV and CVB3 was determined by reverse transcriptase quantitative real-time PCR (RT-qPCR). The extracted RNA was compatible with RT-qPCR and sensitivity of detection of 0.8PFU per reaction was found with RNAX buffer and the developed protocol. This level of sensitivity was obtained using viral RNA extracted from 4.0 ml of an inoculated water sample concentrate. The RNAX buffer developed in this study could be applicable to the detection of other pathogens in water and food.

  16. Validation of a real-time reverse transcriptase-PCR assay for the detection of H7 avian influenza virus

    USGS Publications Warehouse

    Pedersen, J.; Killian, M.L.; Hines, N.; Senne, D.; Panigrahy, B.; Ip, H.S.; Spackman, Erica

    2010-01-01

    This report describes the validation of an avian influenza virus (AIV) H7 subtype-specific real-time reverse transcriptasePCR (rRT-PCR) assay developed at the Southeast Poultry Research Laboratory (SEPRL) for the detection of H7 AI in North and South American wild aquatic birds and poultry. The validation was a collaborative effort by the SEPRL and the National Veterinary Services Laboratories. The 2008 H7 rRT-PCR assay detects 101 50% embryo infectious doses per reaction, or 103104 copies of transcribed H7 RNA. Diagnostic sensitivity and specificity were estimated to be 97.5% and 82.4%, respectively; the assay was shown to be specific for H7 AI when tested with >270 wild birds and poultry viruses. Following validation, the 2008 H7 rRT-PCR procedure was adopted as an official U.S. Department of Agriculture procedure for the detection of H7 AIV. The 2008 H7 assay replaced the previously used (2002) assay, which does not detect H7 viruses currently circulating in wild birds in North and South America. ?? 2010 American Association of Avian Pathologists.

  17. Complicated RNA splicing of chicken telomerase reverse transcriptase revealed by profiling cells both positive and negative for telomerase activity.

    PubMed

    Chang, Hong; Delany, Mary E

    2006-09-01

    Telomerase reverse transcriptase (TERT) is an essential component of the telomerase ribonucleoprotein complex which maintains telomeres. The objective of this study was to investigate chicken TERT (cTERT) alternative RNA splicing profiles of samples varying for telomerase activity and immortalization parameters. These included systems both in vivo (gastrula embryo, embryo and adult liver) and in vitro (chicken embryo fibroblasts (CEFs) and DT40 cells). Nineteen cTERT variants were discovered, which were generated through exon skipping, intron retention, and alternative usage of splice donor and acceptor sites. Three variants were predicted to introduce in-frame mutations, whereas the others were predicted to have premature termination codons. The number of cTERT variants detected ranged from 10 in adult liver to 13 in CEFs. One variant (V4) was found in all samples and was predicted to generate a truncated protein lacking telomerase catalytic activity. Interestingly, the standard TERT expected from the full-length transcript was expressed not only in telomerase-positive, but also in telomerase-negative samples. The complicated expression profiles of cTERT in various cell systems suggest that sophisticated regulatory pathways are involved in cTERT pre-mRNA editing. Further, these results support the body of increasing evidence that alternative splicing of TERT, both in human and chicken, contributes to telomerase activity regulation.

  18. National survey of the prevalence and conditions of selection of HIV-1 reverse transcriptase K70E mutation.

    PubMed

    Delaugerre, C; Flandre, P; Marcelin, A G; Descamps, D; Tamalet, C; Cottalorda, J; Schneider, V; Yerly, S; LeGoff, J; Morand-Joubert, L; Chaix, M L; Costagliola, D; Calvez, V

    2008-05-01

    Tenofovir disoproxil fumarate (TDF) has become an important component of HIV combination therapy because of its potency and once-daily dosing. Key mutation associated with resistance to TDF is a K65R in the reverse transcriptase (RT) gene. According to occurrence of K70E mutation after failure to TDF regimen, this mutation was recently reported as a mutation associated with TDF resistance in most resistance genotypic algorithms. The aim of this study was to analyze, retrospectively, the prevalence and conditions of selection of HIV-1 RT K70E mutation from a national clinical survey. Absence of selection of K70E in 850 HIV-1-infected naive patients suggests its role in NRTI drug resistance. Prevalence of K70E RT was low (99/41601, 0.24%) in patients treated between 1999 and 2005. Conversely with K65R mutation, thymidine analog mutations (TAMs) can be concomitantly observed with K70E mutation but its frequency decreased as the number of TAM increases. Concomitant association of K65R and K70E was possible but infrequent (11%). At the time of K70E selection, 60% of patients had received or received TDF-containing regimen and one-third received exclusive NRTI regimen. In conclusion, the K70E mutation could be an alternative pathway of TDF resistance, but as the K65R mutation, other NRTI as ABC, ddI, and 3TC could be also associated with the K70E selection.

  19. Impact of HIV-1 Subtype and Antiretroviral Therapy on Protease and Reverse Transcriptase Genotype: Results of a Global Collaboration

    PubMed Central

    Kantor, Rami; Katzenstein, David A; Efron, Brad; Carvalho, Ana Patricia; Wynhoven, Brian; Cane, Patricia; Clarke, John; Sirivichayakul, Sunee; Soares, Marcelo A; Snoeck, Joke; Pillay, Candice; Rudich, Hagit; Rodrigues, Rosangela; Holguin, Africa; Ariyoshi, Koya; Bouzas, Maria Belen; Cahn, Pedro; Sugiura, Wataru; Soriano, Vincent; Brigido, Luis F; Grossman, Zehava; Morris, Lynn; Vandamme, Anne-Mieke; Tanuri, Amilcar; Phanuphak, Praphan; Weber, Jonathan N; Pillay, Deenan; Harrigan, P. Richard; Camacho, Ricardo; Schapiro, Jonathan M; Shafer, Robert W

    2005-01-01

    Background The genetic differences among HIV-1 subtypes may be critical to clinical management and drug resistance surveillance as antiretroviral treatment is expanded to regions of the world where diverse non-subtype-B viruses predominate. Methods and Findings To assess the impact of HIV-1 subtype and antiretroviral treatment on the distribution of mutations in protease and reverse transcriptase, a binomial response model using subtype and treatment as explanatory variables was used to analyze a large compiled dataset of non-subtype-B HIV-1 sequences. Non-subtype-B sequences from 3,686 persons with well characterized antiretroviral treatment histories were analyzed in comparison to subtype B sequences from 4,769 persons. The non-subtype-B sequences included 461 with subtype A, 1,185 with C, 331 with D, 245 with F, 293 with G, 513 with CRF01_AE, and 618 with CRF02_AG. Each of the 55 known subtype B drug-resistance mutations occurred in at least one non-B isolate, and 44 (80%) of these mutations were significantly associated with antiretroviral treatment in at least one non-B subtype. Conversely, of 67 mutations found to be associated with antiretroviral therapy in at least one non-B subtype, 61 were also associated with antiretroviral therapy in subtype B isolates. Conclusion Global surveillance and genotypic assessment of drug resistance should focus primarily on the known subtype B drug-resistance mutations. PMID:15839752

  20. Molecular Docking Studies of Marine Diterpenes as Inhibitors of Wild-Type and Mutants HIV-1 Reverse Transcriptase

    PubMed Central

    Miceli, Leonardo A.; Teixeira, Valéria L.; Castro, Helena C.; Rodrigues, Carlos R.; Mello, Juliana F. R.; Albuquerque, Magaly G.; Cabral, Lucio M.; de Brito, Monique A.; de Souza, Alessandra M. T.

    2013-01-01

    AIDS is a pandemic responsible for more than 35 million deaths. The emergence of resistant mutations due to drug use is the biggest cause of treatment failure. Marine organisms are sources of different molecules, some of which offer promising HIV-1 reverse transcriptase (RT) inhibitory activity, such as the diterpenes dolabelladienotriol (THD, IC50 = 16.5 µM), (6R)-6-hydroxydichotoma-3,14-diene-1,17-dial (HDD, IC50 = 10 µM) and (6R)-6-acetoxydichotoma-3,14-diene-1,17-dial (ADD, IC50 = 35 µM), isolated from a brown algae of the genus Dictyota, showing low toxicity. In this work, we evaluated the structure-activity relationship (SAR) of THD, HDD and ADD as anti HIV-1 RT, using a molecular modeling approach. The analyses of stereoelectronic parameters revealed a direct relationship between activity and HOMO (Highest Occupied Molecular Orbital)-LUMO (Lowest Unoccupied Molecular Orbital) gap (ELUMO–EHOMO), where antiviral profile increases with larger HOMO-LUMO gap values. We also performed molecular docking studies of THD into HIV-1 RT wild-type and 12 different mutants, which showed a seahorse conformation, hydrophobic interactions and hydrogen bonds with important residues of the binding pocket. Based on in vitro experiments and docking studies, we demonstrated that mutations have little influence in positioning and interactions of THD. Following a rational drug design, we suggest a modification of THD to improve its biological activity. PMID:24172210

  1. Expanded-Spectrum Nonnucleoside Reverse Transcriptase Inhibitors Inhibit Clinically Relevant Mutant Variants of Human Immunodeficiency Virus Type 1

    PubMed Central

    Corbett, Jeffrey W.; Ko, Soo S.; Rodgers, James D.; Jeffrey, Susan; Bacheler, Lee T.; Klabe, Ronald M.; Diamond, Sharon; Lai, Chii-Ming; Rabel, Shelley R.; Saye, Jo Anne; Adams, Stephen P.; Trainor, George L.; Anderson, Paul S.; Erickson-Viitanen, Susan K.

    1999-01-01

    A research program targeted toward the identification of expanded-spectrum nonnucleoside reverse transcriptase inhibitors which possess increased potency toward K103N-containing mutant human immunodeficiency virus (HIV) and which maintain pharmacokinetics consistent with once-a-day dosing has resulted in the identification of the 4-cyclopropylalkynyl-4-trifluoromethyl-3,4-dihydro-2(1H)quinazolinones DPC 961 and DPC 963 and the 4-cyclopropylalkenyl-4-trifluoromethyl-3,4-dihydro-2(1H)quinazolinones DPC 082 and DPC 083 for clinical development. DPC 961, DPC 963, DPC 082, and DPC 083 all exhibit low-nanomolar potency toward wild-type virus, K103N and L100I single-mutation variants, and many multiply amino acid-substituted HIV type 1 mutants. This high degree of potency is combined with a high degree of oral bioavailability, as demonstrated in rhesus monkeys and chimpanzees, and with plasma serum protein binding that can result in significant free levels of drug. PMID:10582878

  2. Free Energy-Based Virtual Screening and Optimization of RNase H Inhibitors of HIV-1 Reverse Transcriptase.

    PubMed

    Zhang, Baofeng; D'Erasmo, Michael P; Murelli, Ryan P; Gallicchio, Emilio

    2016-09-30

    We report the results of a binding free energy-based virtual screening campaign of a library of 77 α-hydroxytropolone derivatives against the challenging RNase H active site of the reverse transcriptase (RT) enzyme of human immunodeficiency virus-1. Multiple protonation states, rotamer states, and binding modalities of each compound were individually evaluated. The work involved more than 300 individual absolute alchemical binding free energy parallel molecular dynamics calculations and over 1 million CPU hours on national computing clusters and a local campus computational grid. The thermodynamic and structural measures obtained in this work rationalize a series of characteristics of this system useful for guiding future synthetic and biochemical efforts. The free energy model identified key ligand-dependent entropic and conformational reorganization processes difficult to capture using standard docking and scoring approaches. Binding free energy-based optimization of the lead compounds emerging from the virtual screen has yielded four compounds with very favorable binding properties, which will be the subject of further experimental investigations. This work is one of the few reported applications of advanced-binding free energy models to large-scale virtual screening and optimization projects. It further demonstrates that, with suitable algorithms and automation, advanced-binding free energy models can have a useful role in early-stage drug-discovery programs.

  3. Mechanisms associated with HIV-1 resistance to acyclovir by the V75I mutation in reverse transcriptase.

    PubMed

    Tchesnokov, Egor P; Obikhod, Aleksandr; Massud, Ivana; Lisco, Andrea; Vanpouille, Christophe; Brichacek, Beda; Balzarini, Jan; McGuigan, Christopher; Derudas, Marco; Margolis, Leonid; Schinazi, Raymond F; Götte, Matthias

    2009-08-07

    It has recently been demonstrated that the anti-herpetic drug acyclovir (ACV) also displays antiviral activity against the human immunodeficiency virus type 1 (HIV-1). The triphosphate form of ACV is accepted by HIV-1 reverse transcriptase (RT), and subsequent incorporation leads to classical chain termination. Like all approved nucleoside analogue RT inhibitors (NRTIs), the selective pressure of ACV is associated with the emergence of resistance. The V75I mutation in HIV-1 RT appears to be dominant in this regard. By itself, this mutation is usually not associated with resistance to currently approved NRTIs. Here we studied the underlying biochemical mechanism. We demonstrate that V75I is also selected under the selective pressure of a monophosphorylated prodrug that was designed to bypass the bottleneck in drug activation to the triphosphate form (ACV-TP). Pre-steady-state kinetics reveal that V75I discriminates against the inhibitor at the level of catalysis, whereas binding of the inhibitor remains largely unaffected. The incorporated ACV-monophosphate (ACV-MP) is vulnerable to excision in the presence of the pyrophosphate donor ATP. V75I compromises binding of the next nucleotide that can otherwise provide a certain degree of protection from excision. Collectively, the results of this study suggest that ACV is sensitive to two different resistance pathways, which warrants further investigation regarding the detailed resistance profile of ACV. Such studies will be crucial in assessing the potential clinical utility of ACV and its derivatives in combination with established NRTIs.

  4. Telomerase reverse transcriptase is required for the localization of telomerase RNA to cajal bodies and telomeres in human cancer cells.

    PubMed

    Tomlinson, Rebecca L; Abreu, Eladio B; Ziegler, Tania; Ly, Hinh; Counter, Christopher M; Terns, Rebecca M; Terns, Michael P

    2008-09-01

    Telomere maintenance by telomerase is critical for the unlimited division potential of most human cancer cells. The two essential components of human telomerase, telomerase RNA (hTR) and telomerase reverse transcriptase (hTERT), are recruited from distinct subnuclear sites to telomeres during S phase. Throughout the remainder of the cell cycle hTR is found primarily in Cajal bodies. The localization of hTR to Cajal bodies and telomeres is specific to cancer cells where telomerase is active and is not observed in primary cells. Here we show that the trafficking of hTR to both telomeres and Cajal bodies depends on hTERT. RNA interference-mediated depletion of hTERT in cancer cells leads to loss of hTR from both Cajal bodies and telomeres without affecting hTR levels. In addition, expression of hTERT in telomerase-negative cells (including primary and ALT cancer cell lines) induces hTR to localize to both sites. Factors that did not stimulate hTR localization in our experiments include increased hTR RNA levels and Cajal body numbers, and expression of SV40 large T antigen and oncogenic Ras. Our findings suggest that the trafficking of telomerase to Cajal bodies and telomeres in cancer cells correlates with and depends on the assembly of the enzyme.

  5. Synthesis of the (5Z)-5-Pentacosenoic and 5-Pentacosynoic Acids as Novel HIV-1 Reverse Transcriptase Inhibitors

    PubMed Central

    Moreira, Lizabeth Giménez; Orellano, Elsie A.; Rosado, Karolyna; Guido, Rafael V. C.; Andricopulo, Adriano D.; Soto, Gabriela O.; Rodríguez, José W.; Sanabria-Ríos, David J.; Carballeira, Néstor M.

    2016-01-01

    The natural fatty acids (5Z)-5-pentacosenoic and (9Z)-9-pentacosenoic acids were synthesized for the first time in eight steps starting from either 4-bromo-1-butanol or 8-bromo-1-butanol and in 20-58% overall yields, while the novel fatty acids 5-pentacosynoic and 9-pentacosynoic acids were also synthesized in six steps and in 34-43% overall yields. The Δ5 acids displayed the best IC50’s (24-38 µM) against the HIV-1 reverse transcriptase (RT) enzyme, comparable to nervonic acid (IC50 = 12 µM). The Δ9 acids were not as effective towards HIV-RT with the (9Z)-9-pentacosenoic acid displaying an IC50 = 54 µM. Fatty acid chain length and position of the unsaturation was critical for the observed inhibition. Molecular modeling studies indicated the structural determinants underlying the biological activity of the most potent compounds. These results provide new insights into the structural requirements that must be present in fatty acids so as to enhance their inhibitory potential towards HIV-RT. PMID:26345647

  6. Computational Analysis of Molecular Interaction Networks Underlying Change of HIV-1 Resistance to Selected Reverse Transcriptase Inhibitors.

    PubMed

    Kierczak, Marcin; Dramiński, Michał; Koronacki, Jacek; Komorowski, Jan

    2010-12-12

    Despite more than two decades of research, HIV resistance to drugs remains a serious obstacle in developing efficient AIDS treatments. Several computational methods have been developed to predict resistance level from the sequence of viral proteins such as reverse transcriptase (RT) or protease. These methods, while powerful and accurate, give very little insight into the molecular interactions that underly acquisition of drug resistance/hypersusceptibility. Here, we attempt at filling this gap by using our Monte Carlo feature selection and interdependency discovery method (MCFS-ID) to elucidate molecular interaction networks that characterize viral strains with altered drug resistance levels. We analyzed a number of HIV-1 RT sequences annotated with drug resistance level using the MCFS-ID method. This let us expound interdependency networks that characterize change of drug resistance to six selected RT inhibitors: Abacavir, Lamivudine, Stavudine, Zidovudine, Tenofovir and Nevirapine. The networks consider interdependencies at the level of physicochemical properties of mutating amino acids, eg,: polarity. We mapped each network on the 3D structure of RT in attempt to understand the molecular meaning of interacting pairs. The discovered interactions describe several known drug resistance mechanisms and, importantly, some previously unidentified ones. Our approach can be easily applied to a whole range of problems from the domain of protein engineering. A portable Java implementation of our MCFS-ID method is freely available for academic users and can be obtained at: http://www.ipipan.eu/staff/m.draminski/software.htm.

  7. Activation of the human nuclear xenobiotic receptor PXR by the reverse transcriptase-targeted anti-HIV drug PNU-142721

    SciTech Connect

    Cheng, Yuan; Redinbo, Matthew R.

    2012-10-09

    The human pregnane X receptor (PXR) is a member of the nuclear receptor superfamily of ligand-regulated transcription factors. PXR responds to a structurally diverse variety of endogenous and xenobiotic compounds, and coordinates the expression of genes central to the metabolism and excretion of potentially harmful chemicals, including human therapeutics. The reverse transcriptase inhibitor PNU-142721 has been designed to treat human immunodeficiency virus (HIV) infection. Although this compound has anti-HIV activity, it was established using cell-based assays that PNU-142721 is an efficacious PXR agonist. We present here the 2.8 {angstrom} resolution crystal structure of the human PXR ligand-binding domain in complex with PNU-142721. PXR employs one hydrogen bond and fourteen van der Waals contacts to interact with the ligand, but allows two loops adjacent to the ligand-binding pocket to remain disordered in the structure. These observations highlight the role structural flexibility plays in PXR's promiscuous responses to xenobiotics. The crystal structure also explains why PNU-173575, a thiomethyl metabolite of PNU-142721, exhibits enhanced PXR activation relative to the unmodified compound and why PNU-142721 can also activate rat PXR. Taken together, the results presented here elucidate the structural basis for PXR activation by PNU-142721 and related chemicals.

  8. International Collaborative Study To Compare Reverse Transcriptase PCR Assays for Detection and Genotyping of Noroviruses

    PubMed Central

    Vinjé, Jan; Vennema, Harry; Maunula, Leena; von Bonsdorff, Carl-Henrik; Hoehne, Marina; Schreier, Eckart; Richards, Alison; Green, Jon; Brown, David; Beard, Suzanne S.; Monroe, Stephan S.; de Bruin, Erwin; Svensson, Lennart; Koopmans, Marion P. G.

    2003-01-01

    To allow more rapid and internationally standardized assessment of the spread of noroviruses (previously called Norwalk-like viruses [NLVs]) as important food-borne pathogens, harmonization of methods for their detection is needed. Diagnosis of NLVs in clinical diagnostic laboratories is usually performed by reverse transciptase PCR (RT-PCR) assays. In the present study, the performance of five different RT-PCR assays for the detection of NLVs was evaluated in an international collaborative study by five laboratories in five countries with a coded panel of 91 fecal specimens. The assays were tested for their sensitivity, detection limit, and ease of standardization. In total, NLVs could be detected by at least one RT-PCR assay in 69 (84%) of the samples that originally tested positive. Sensitivity ranged from 52 to 73% overall and from 54 to 100% and 58 to 85% for genogroup I and II viruses, respectively. In all, 64% of the false-negative results were obtained with a set of diluted stools (n = 20) that may have lost quality upon storage. Sensitivity was improved when these samples were excluded from analysis. No one single assay stood out as the best, although the p1 assay demonstrated the most satisfactory overall performance. To promote comparability of data, this assay will be recommended for newly starting groups in future collaborative studies. PMID:12682125

  9. A tumor-promoting mechanism mediated by retrotransposon-encoded reverse transcriptase is active in human transformed cell lines

    PubMed Central

    Sciamanna, Ilaria; Gualtieri, Alberto; Cossetti, Cristina; Osimo, Emanuele Felice; Ferracin, Manuela; Macchia, Gianfranco; Aricò, Eleonora; Prosseda, Gianni; Vitullo, Patrizia; Misteli, Tom; Spadafora, Corrado

    2013-01-01

    LINE-1 elements make up the most abundant retrotransposon family in the human genome. Full-length LINE-1 elements encode a reverse transcriptase (RT) activity required for their own retrotranpsosition as well as that of non-autonomous Alu elements. LINE-1 are poorly expressed in normal cells and abundantly in cancer cells. Decreasing RT activity in cancer cells, by either LINE-1-specific RNA interference, or by RT inhibitory drugs, was previously found to reduce proliferation and promote differentiation and to antagonize tumor growth in animal models. Here we have investigated how RT exerts these global regulatory functions. We report that the RT inhibitor efavirenz (EFV) selectively downregulates proliferation of transformed cell lines, while exerting only mild effects on non-transformed cells; this differential sensitivity matches a differential RT abundance, which is high in the former and undetectable in the latter. Using CsCl density gradients, we selectively identify Alu and LINE-1 containing DNA:RNA hybrid molecules in cancer but not in normal cells. Remarkably, hybrid molecules fail to form in tumor cells treated with EFV under the same conditions that repress proliferation and induce the reprogramming of expression profiles of coding genes, microRNAs (miRNAs) and ultraconserved regions (UCRs). The RT-sensitive miRNAs and UCRs are significantly associated with Alu sequences. The results suggest that LINE-1-encoded RT governs the balance between single-stranded and double-stranded RNA production. In cancer cells the abundant RT reverse-transcribes retroelement-derived mRNAs forming RNA:DNA hybrids. We propose that this impairs the formation of double-stranded RNAs and the ensuing production of small regulatory RNAs, with a direct impact on gene expression. RT inhibition restores the ‘normal’ small RNA profile and the regulatory networks that depend on them. Thus, the retrotransposon-encoded RT drives a previously unrecognized mechanism crucial to the

  10. Quantitative reverse transcriptase-polymerase chain reaction measured expression of MDR1 and MRP in primary breast carcinoma.

    PubMed

    Dexter, D W; Reddy, R K; Geles, K G; Bansal, S; Myint, M A; Rogakto, A; Leighton, J C; Goldstein, L J

    1998-06-01

    To evaluate the clinical significance of drug resistance mechanisms in breast cancer, we examined the expression of MDR1 and MRP in primary breast carcinoma and normal adjacent tissue using a highly quantitative and reproducible reverse transcription-PCR assay. Expression of both genes was observed in all specimens examined, both tumor (n = 74) and normal adjacent tissue (n = 55). The expression of MDR1, however, was low, with the level of expression being 25 times less than the drug-resistant control cell line KB 8-5. Immunohistochemical analysis of P-glycoprotein corroborated the PCR results; only 6% (2 of 31) were positive for JSB1 staining, and 0 of 32 were positive for for UIC2. MRP expression did not exceed control cell line levels, and immunohistochemistry detected moderate levels of expression. MDR1 expression was independent of grade, stage, tumor size, nodal status, metastasis, and estrogen receptor and progesterone receptor status. There was, however, a significant correlation of MDR1 expression with age and histology. Approximately twice the expression of MDR1 was observed in the < 50 age group compared to the > 50 age group, and lobular carcinoma had 4 times the expression of MDR1 of other histological types. MRP expression was independent of all other clinical parameters. Thus, these results show that although MDR1 expression is detectable in primary breast carcinoma by PCR, this expression as measured by quantitative reverse transcriptase-PCR is extremely low. The significance of these low levels is yet to be determined. MDR1 expression was higher in < 50 age group and lobular carcinoma, which may contribute to poor prognosis associated with young age and lobular histology.

  11. A tumor-promoting mechanism mediated by retrotransposon-encoded reverse transcriptase is active in human transformed cell lines.

    PubMed

    Sciamanna, Ilaria; Gualtieri, Alberto; Cossetti, Cristina; Osimo, Emanuele Felice; Ferracin, Manuela; Macchia, Gianfranco; Aricò, Eleonora; Prosseda, Gianni; Vitullo, Patrizia; Misteli, Tom; Spadafora, Corrado

    2013-12-01

    LINE-1 elements make up the most abundant retrotransposon family in the human genome. Full-length LINE-1 elements encode a reverse transcriptase (RT) activity required for their own retrotranpsosition as well as that of non-autonomous Alu elements. LINE-1 are poorly expressed in normal cells and abundantly in cancer cells. Decreasing RT activity in cancer cells, by either LINE-1-specific RNA interference, or by RT inhibitory drugs, was previously found to reduce proliferation and promote differentiation and to antagonize tumor growth in animal models. Here we have investigated how RT exerts these global regulatory functions. We report that the RT inhibitor efavirenz (EFV) selectively downregulates proliferation of transformed cell lines, while exerting only mild effects on non-transformed cells; this differential sensitivity matches a differential RT abundance, which is high in the former and undetectable in the latter. Using CsCl density gradients, we selectively identify Alu and LINE-1 containing DNA:RNA hybrid molecules in cancer but not in normal cells. Remarkably, hybrid molecules fail to form in tumor cells treated with EFV under the same conditions that repress proliferation and induce the reprogramming of expression profiles of coding genes, microRNAs (miRNAs) and ultraconserved regions (UCRs). The RT-sensitive miRNAs and UCRs are significantly associated with Alu sequences. The results suggest that LINE-1-encoded RT governs the balance between single-stranded and double-stranded RNA production. In cancer cells the abundant RT reverse-transcribes retroelement-derived mRNAs forming RNA:DNA hybrids. We propose that this impairs the formation of double-stranded RNAs and the ensuing production of small regulatory RNAs, with a direct impact on gene expression. RT inhibition restores the 'normal' small RNA profile and the regulatory networks that depend on them. Thus, the retrotransposon-encoded RT drives a previously unrecognized mechanism crucial to the

  12. Inhibition of LINE-1 retrotransposon-encoded reverse transcriptase modulates the expression of cell differentiation genes in breast cancer cells.

    PubMed

    Patnala, Radhika; Lee, Sung-Hun; Dahlstrom, Jane E; Ohms, Stephen; Chen, Long; Dheen, S Thameem; Rangasamy, Danny

    2014-01-01

    Long Interspersed Elements (L1 elements) are biologically active retrotransposons that are capable of autonomous replication using their own reverse transcriptase (RT) enzyme. Expression of the normally repressed RT has been implicated in cancer cell growth. However, at present, little is known about the expression of L1-encoded RT activity or the molecular changes that are associated with RT activity in the development of breast cancer. Here, we report that RT activity is widespread in breast cancer cells. The expression of RT protein decreased markedly in breast cancer cells after treatment with the antiretroviral drug, efavirenz. While the majority of cells showed a significant reduction in proliferation, inhibition of RT was also accompanied by cell-specific differences in morphology. MCF7 cells displayed elongated microtubule extensions that adhered tightly to their substrate, while a large fraction of the T47D cells that we studied formed long filopodia projections. These morphological changes were reversible upon cessation of RT inhibition, confirming their dependence on RT activity. We also carried out gene expression profiling with microarrays and determined the genes that were differentially expressed during the process of cellular differentiation. Genes involved in proliferation, cell migration, and invasive activity were repressed in RT-inhibited cells. Concomitantly, genes involved in cell projection, formation of vacuolar membranes, and cell-to-cell junctions were significantly upregulated in RT-inhibited cells. qRT-PCR examination of the mRNA expression of these genes in additional cell lines yielded close correlation between their differential expression and the degree of cellular differentiation. Our study demonstrates that the inhibition of L1-encoded RT can reduce the rate of proliferation and promote differentiation of breast cancer cells. Together, these results provide a direct functional link between the expression of L1 retrotransposons and

  13. Preformulation studies of EFdA, a novel nucleoside reverse transcriptase inhibitor for HIV prevention

    PubMed Central

    Zhang, Wei; Parniak, Michael A.; Mitsuya, Hiroaki; Sarafianos, Stefan G.; Graebing, Phillip W.; Rohan, Lisa C.

    2014-01-01

    4′-Ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is a novel nucleoside analog of great interest because of its superior activity against wild-type and multidrug-resistant HIV-1 strains, and favorable safety profiles in vitro and in vivo. The aim of this work was to provide preformulation information of EFdA important for delivery system development. A simple, accurate and specific reverse-phase high performance liquid chromatographic (RP-HPLC) method with UV detection was developed for quantification of EFdA. In addition, physicochemical characterizations including pH solubility profile, octanol/water partition coefficient (Log Po/w), DSC analysis, field emission scanning electron microscopy, and stability studies under various conditions were conducted. EFdA existed in planar or flake shape, with a melting point of ~130 °C, and had a pH dependent solubility. The log Po/w value of EFdA was −1.19. The compound was stable upon exposure to pH levels from 3 to 9 and showed good stability at elevated temperature (65 °C). In vitro cytotoxicity assessments were performed in two different epithelial cell lines. In cell-based studies, the EFdA selectivity index (50% cytotoxic concentration [CC50] values/50% effective concentration [EC50]) was found to be greater than 1 × 103. Permeability studies using cell- and tissue-based models showed that EFdA had an apparent permeability coefficient (Papp) <1 × 10−6cm/s and that the paracelluar pathway was the dominant transport route for EFdA. Overall, EFdA possesses favorable characteristics for further formulation development. PMID:23841536

  14. Identification of bacteria enduring endodontic treatment procedures by a combined reverse transcriptase-polymerase chain reaction and reverse-capture checkerboard approach.

    PubMed

    Rôças, Isabela N; Siqueira, José F

    2010-01-01

    This study identified the bacterial taxa enduring endodontic treatment procedures by using a combined 16S ribosomal RNA-based reverse-transcriptase polymerase chain reaction (RT-PCR) and reverse-capture checkerboard hybridization approach. Samples were taken from infected canals of 15 teeth with apical periodontitis before treatment (S1), after chemomechanical preparation with NaOCl as the irrigant (S2), and after interappointment medication with a calcium hydroxide paste (S3). Bacterial presence was first screened by a DNA-based single PCR assay. RNA extracts were subjected to RT-PCR, and the resulting products were surveyed for the presence of 28 targeted taxa by using the checkerboard method. Bacteria were found in all S1 samples. Detectable levels of bacterial ribosomal RNA, used as an indicator of viability, were observed in 60% of the cases after chemomechanical preparation and 53% after intracanal medication. The most prevalent taxa in S1 were Olsenella uli (67%), Pyramidobacter piscolens (60%), Streptococcus species (53%), and Bacteroidetes clone X083 (53%). Streptococcus species (47%), Fusobacterium nucleatum (40%), and O. uli (33%) prevailed in S2, whereas Streptococcus species (47%), Propionibacterium acnes (27%), and O. uli (27%) were the most frequent taxa in S3. The present study with a combined molecular approach revealed that bacterial diversity was overall markedly reduced by treatment procedures. Although bacterial taxa more frequently identified in post-treatment samples emerge as potential risk factors for persistent disease, this remains to be determined by longitudinal studies.

  15. Human Immunodeficiency Virus Reverse Transcriptase and Protease Sequence Database: an expanded data model integrating natural language text and sequence analysis programs.

    PubMed

    Kantor, R; Machekano, R; Gonzales, M J; Dupnik, K; Schapiro, J M; Shafer, R W

    2001-01-01

    The HIV Reverse Transcriptase and Protease Sequence Database is an on-line relational database that catalogs evolutionary and drug-related sequence variation in the human immunodeficiency virus (HIV) reverse transcriptase (RT) and protease enzymes, the molecular targets of anti-HIV therapy (http://hivdb.stanford.edu). The database contains a compilation of nearly all published HIV RT and protease sequences, including submissions from International Collaboration databases and sequences published in journal articles. Sequences are linked to data about the source of the sequence sample and the antiretroviral drug treatment history of the individual from whom the isolate was obtained. During the past year 3500 sequences have been added and the data model has been expanded to include drug susceptibility data on sequenced isolates. Database content has also been integrated with didactic text and the output of two sequence analysis programs.

  16. Specific detection of RT activity in culture supernantants of retrovirus-producing cells, using synthetic DNA as competitor in polymerase enhanced reverse transcriptase assay.

    PubMed

    Voisset, C; Tönjes, R R; Breyton, P; Mandrand, B; Paranhos-Baccalà, G

    2001-05-01

    The polymerase enhanced reverse transcriptase (PERT) assay is a highly sensitive assay for the detection of reverse transcriptase (RT) activity in culture supernatants of retrovirus-producing cells. However, some cellular DNA-dependent DNA polymerases exhibit RT-like activities in this assay. A synthetic DNA competitor which suppresses the RT-like activities of cellular DNA-dependent DNA polymerases was used in a modified PERT assay technique for specific detection of RT activity in culture supernatants of retrovirus-producing cells. We determined the optimum condition of the assay and evaluated its specificity. This improved PERT assay is easy to perform and is able to detect minute amounts of purified RT, as well as RT in crude cell lysates and concentrated culture supernatants.

  17. Structural studies of series HIV-1 nonnucleoside reverse transcriptase inhibitors 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)-benzimidazoles with different 4-substituents

    NASA Astrophysics Data System (ADS)

    Ziółkowska, Natasza E.; Michejda, Christopher J.; Bujacz, Grzegorz D.

    2010-03-01

    Over the past 10 years, several anti-viral drugs have become available to fight the HIV infection. Antiretroviral treatment reduces the mortality of AIDS. Nonnucleoside inhibitors of HIV-1 reverse transcriptase are specific and potentially nontoxic drugs against AIDS. The crystal structures of five nonnucleoside inhibitors of HIV-1 reverse transcriptase are presented here. The structural parameters, especially those describing