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Sample records for inhibitor-resistant viral potassium

  1. Selection of Inhibitor-Resistant Viral Potassium Channels Identifies a Selectivity Filter Site that Affects Barium and Amantadine Block

    Science.gov (United States)

    Fujiwara, Yuichiro; Arrigoni, Cristina; Domigan, Courtney; Ferrara, Giuseppina; Pantoja, Carlos; Thiel, Gerhard; Moroni, Anna; Minor, Daniel L.

    2009-01-01

    Background Understanding the interactions between ion channels and blockers remains an important goal that has implications for delineating the basic mechanisms of ion channel function and for the discovery and development of ion channel directed drugs. Methodology/Principal Findings We used genetic selection methods to probe the interaction of two ion channel blockers, barium and amantadine, with the miniature viral potassium channel Kcv. Selection for Kcv mutants that were resistant to either blocker identified a mutant bearing multiple changes that was resistant to both. Implementation of a PCR shuffling and backcrossing procedure uncovered that the blocker resistance could be attributed to a single change, T63S, at a position that is likely to form the binding site for the inner ion in the selectivity filter (site 4). A combination of electrophysiological and biochemical assays revealed a distinct difference in the ability of the mutant channel to interact with the blockers. Studies of the analogous mutation in the mammalian inward rectifier Kir2.1 show that the T→S mutation affects barium block as well as the stability of the conductive state. Comparison of the effects of similar barium resistant mutations in Kcv and Kir2.1 shows that neighboring amino acids in the Kcv selectivity filter affect blocker binding. Conclusions/Significance The data support the idea that permeant ions have an integral role in stabilizing potassium channel structure, suggest that both barium and amantadine act at a similar site, and demonstrate how genetic selections can be used to map blocker binding sites and reveal mechanistic features. PMID:19834614

  2. Potassium sorbate-A new aqueous copper corrosion inhibitor

    International Nuclear Information System (INIS)

    Abelev, Esta; Starosvetsky, David; Ein-Eli, Yair

    2007-01-01

    This work presents the novel nature of 2,4-hexadienoic acid potassium salt (potassium sorbate (KCH 3 CH=CHCH=CHCO 2 )) as an effective copper aqueous corrosion inhibitor. The influence of pH and potassium sorbate concentration on copper corrosion in aerated sulfate and chloride solutions is reported. Degree of copper protection was found to increase with an increase in potassium sorbate concentration; an optimum concentration of this inhibitor in sulfate solutions was found to be 10 g/L. Copper is highly resistant to corrosion attacks by chloride ions in the presence of potassium sorbate. X-ray photoelectron spectroscopy (XPS) studies suggest that copper protection is achieved via the formation of a mixed layer of cuprous oxide, cupric hydroxide and copper(II)-sorbate at the metal surface

  3. Rapid emergence of hepatitis C virus protease inhibitor resistance is expected

    Energy Technology Data Exchange (ETDEWEB)

    Rong, Libin [Los Alamos National Laboratory; Perelson, Alan S [Los Alamos National Laboratory; Ribeiro, Ruy M [Los Alamos National Laboratory

    2009-01-01

    Approximately 170 million people worldwide are infected with hepatitis C virus (HCV). Current therapy, consisting of pegylated interferon (PEG-IFN) and ribavirin (RBV), leads to sustained viral elimination in only about 45% of patients treated. Telaprevir (VX-950), a novel HCV NS3-4A serine protease inhibitor, has demonstrated substantial antiviral activity in patients with chronic hepatitis C genotype 1 infection. However, some patients experience viral breakthrough during dosing, with drug resistant variants being 5%-20% of the virus population as early as day 2 after treatment initiation. Why viral variants appear such a short time after the start of dosing is unclear, especially since this has not been seen with monotherapy for either human immunodeficiency virus or hepatitis B virus. Here, using a viral dynamic model, we explain why such rapid emergence of drug resistant variants is expected when potent HCV protease inhibitors are used as monotherapy. Surprisingly, our model also shows that such rapid emergence need not be the case with some potent HCV NS5B polymerase inhibitors. Examining the case of telaprevir therapy in detail, we show the model fits observed dynamics of both wild-type and drug-resistant variants during treatment, and supports combination therapy of direct antiviral drugs with PEG-IFN and/or RBV for hepatitis C.

  4. The prevalence of the pre-existing hepatitis C viral variants and the evolution of drug resistance in patients treated with the NS3-4a serine protease inhibitor telaprevir

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    Rong, Libin [Los Alamos National Laboratory; Ribeiro, Ruy M [Los Alamos National Laboratory; Perelson, Alan S [Los Alamos National Laboratory

    2008-01-01

    Telaprevir (VX-950), a novel hepatitis C virus (HCV) NS3-4A serine protease inhibitor, has demonstrated substantial antiviral activity in patients infected with HCV genotype 1. Some patients experience viral breakthrough, which has been shown to be associated with emergence of telaprevir-resistant HCV variants during treatment. The exact mechanisms underlying the rapid selection of drug resistant viral variants during dosing are not fully understood. In this paper, we develop a two-strain model to study the pre-treatment prevalence of the mutant virus and derive an analytical solution of the mutant frequency after administration of the protease inhibitor. Our analysis suggests that the rapid increase of the mutant frequency during therapy is not due to mutant growth but rather due to the rapid and profound loss of wild-type virus, which uncovers the pre-existing mutant variants. We examine the effects of backward mutation and hepatocyte proliferation on the pre-existence of the mutant virus and the competition between wild-type and drug resistant virus during therapy. We then extend the simple model to a general model with multiple viral strains. Mutations during therapy do not play a significant role in the dynamics of various viral strains, although they are capable of generating low levels of HCV variants that would otherwise be completely suppressed because of fitness disadvantages. Hepatocyte proliferation may not affect the pretreatment frequency of mutant variants, but is able to influence the quasispecies dynamics during therapy. It is the relative fitness of each mutant strain compared with wild-type that determines which strain(s) will dominate the virus population. The study provides a theoretical framework for exploring the prevalence of pre-existing mutant variants and the evolution of drug resistance during treatment with other protease inhibitors or HCV polymerase inhibitors.

  5. Nationwide experience of treatment with protease inhibitors in chronic hepatitis C patients in Denmark: identification of viral resistance mutations.

    Science.gov (United States)

    Sølund, Christina; Krarup, Henrik; Ramirez, Santseharay; Thielsen, Peter; Røge, Birgit T; Lunding, Suzanne; Barfod, Toke S; Madsen, Lone G; Tarp, Britta; Christensen, Peer B; Gerstoft, Jan; Laursen, Alex L; Bukh, Jens; Weis, Nina

    2014-01-01

    The first standard of care in treatment of chronic HCV genotype 1 infection involving directly acting antivirals was protease inhibitors telaprevir or boceprevir combined with pegylated-interferon and ribavirin (triple therapy). Phase III studies include highly selected patients. Thus, treatment response and development of viral resistance during triple therapy in a routine clinical setting needs to be determined. The aims of this study were to investigate treatment outcome and identify sequence variations after triple therapy in patients with chronic HCV genotype 1 infection in a routine clinical setting. 80 patients, who initiated and completed triple therapy in Denmark between May 2011 and November 2012, were included. Demographic data and treatment response were obtained from the Danish Database for Hepatitis B and C. Direct sequencing and clonal analysis of the RT-PCR amplified NS3 protease were performed in patients without cure following triple therapy. 38 (47%) of the patients achieved cure, 15 (19%) discontinued treatment due to adverse events and remained infected, and 27 (34%) experienced relapse or treatment failure of whom 15 of 21 analyzed patients had well-described protease inhibitor resistance variants detected. Most frequently detected protease variants were V36M and/or R155K, and V36M, in patients with genotype 1a and 1b infection, respectively. The cure rate after triple therapy in a routine clinical setting was 47%, which is substantially lower than in clinical trials. Resistance variants towards protease inhibitors were seen in 71% of patients failing therapy indicating that resistance could have an important role in treatment response.

  6. HIV protease drug resistance and its impact on inhibitor design.

    Science.gov (United States)

    Ala, P J; Rodgers, J D; Chang, C H

    1999-07-01

    The primary cause of resistance to the currently available HIV protease inhibitors is the accumulation of multiple mutations in the viral protease. So far more than 20 substitutions have been observed in the active site, dimer interface, surface loops and flaps of the homodimer. While many mutations reduce the protease's affinity for inhibitors, others appear to enhance its catalytic efficiency. This high degree of genetic flexibility has made the protease an elusive drug target. The design of the next generation of HIV protease inhibitors will be discussed in light of the current structural information.

  7. Fifteen years of HIV Protease Inhibitors: raising the barrier to resistance.

    Science.gov (United States)

    Wensing, Annemarie M J; van Maarseveen, Noortje M; Nijhuis, Monique

    2010-01-01

    HIV protease plays a crucial role in the viral life cycle and is essential for the generation of mature infectious virus particles. Detailed knowledge of the structure of HIV protease and its substrate has led to the design of specific HIV protease inhibitors. Unfortunately, resistance to all protease inhibitors (PIs) has been observed and the genetic basis of resistance has been well documented over the past 15 years. The arrival of the early PIs was a pivotal moment in the development of antiretroviral therapy. They made possible the dual class triple combination therapy that became known as HAART. However, the clinical utility of the first generation of PIs was limited by low bioavailability and high pill burdens, which ultimately reduced adherence and limited long-term viral inhibition. When therapy failure occurred multiple protease resistance mutations were observed, often resulting in broad class resistance. To combat PI-resistance development, second-generation approaches have been developed. The first advance was to increase the level of existing PIs in the plasma by boosting with ritonavir. The second was to develop novel PIs with high potency against the known PI-resistant HIV protease variants. Both approaches increased the number of protease mutations required for clinical resistance, thereby raising the genetic barrier. This review provides an overview of the history of protease inhibitor therapy, its current status and future perspectives. It forms part of a special issue of Antiviral Research marking the 25th anniversary of antiretroviral drug discovery and development, vol. 85, issue 1, 2010. Copyright 2009 Elsevier B.V. All rights reserved.

  8. Resistance Patterns Associated with HCV NS5A Inhibitors Provide Limited Insight into Drug Binding

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    Moheshwarnath Issur

    2014-11-01

    Full Text Available Direct-acting antivirals (DAAs have significantly improved the treatment of infection with the hepatitis C virus. A promising class of novel antiviral agents targets the HCV NS5A protein. The high potency and broad genotypic coverage are favorable properties. NS5A inhibitors are currently assessed in advanced clinical trials in combination with viral polymerase inhibitors and/or viral protease inhibitors. However, the clinical use of NS5A inhibitors is also associated with new challenges. HCV variants with decreased susceptibility to these drugs can emerge and compromise therapy. In this review, we discuss resistance patterns in NS5A with focus prevalence and implications for inhibitor binding.

  9. Stability of the resistance to the thiosemicarbazone derived from 5,6-dimethoxy-1-indanone, a non-nucleoside polymerase inhibitor of bovine viral diarrhea virus.

    Science.gov (United States)

    Castro, Eliana F; Campos, Rodolfo H; Cavallaro, Lucía V

    2014-01-01

    Bovine viral diarrhea virus (BVDV) is the prototype Pestivirus. BVDV infection is distributed worldwide and causes serious problems for the livestock industry. The thiosemicarbazone of 5,6-dimethoxy-1-indanone (TSC) is a non-nucleoside polymerase inhibitor (NNI) of BVDV. All TSC-resistant BVDV variants (BVDV-TSCr T1-5) present an N264D mutation in the NS5B gene (RdRp) whereas the variant BVDV-TSCr T1 also presents an NS5B A392E mutation. In the present study, we carried out twenty passages of BVDV-TSCr T1-5 in MDBK cells in the absence of TSC to evaluate the stability of the resistance. The viral populations obtained (BVDV R1-5) remained resistant to the antiviral compound and conserved the mutations in NS5B associated with this phenotype. Along the passages, BVDV R2, R3 and R5 presented a delay in the production of cytopathic effect that correlated with a decrease in cell apoptosis and intracellular accumulation of viral RNA. The complete genome sequences that encode for NS2 to NS5B, Npro and Erns were analyzed. Additional mutations were detected in the NS5B of BVDV R1, R3 and R4. In both BVDV R2 and R3, most of the mutations found were localized in NS5A, whereas in BVDV R5, the only mutation fixed was NS5A V177A. These results suggest that mutations in NS5A could alter BVDV cytopathogenicity. In conclusion, the stability of the resistance to TSC may be due to the fixation of different compensatory mutations in each BVDV-TSCr. During their replication in a TSC-free medium, some virus populations presented a kind of interaction with the host cell that resembled a persistent infection: decreased cytopathogenicity and viral genome synthesis. This is the first report on the stability of antiviral resistance and on the evolution of NNI-resistant BVDV variants. The results obtained for BVDV-TSCr could also be applied for other NNIs.

  10. A compensatory mutation provides resistance to disparate HIV fusion inhibitor peptides and enhances membrane fusion.

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    Matthew P Wood

    Full Text Available Fusion inhibitors are a class of antiretroviral drugs used to prevent entry of HIV into host cells. Many of the fusion inhibitors being developed, including the drug enfuvirtide, are peptides designed to competitively inhibit the viral fusion protein gp41. With the emergence of drug resistance, there is an increased need for effective and unique alternatives within this class of antivirals. One such alternative is a class of cyclic, cationic, antimicrobial peptides known as θ-defensins, which are produced by many non-human primates and exhibit broad-spectrum antiviral and antibacterial activity. Currently, the θ-defensin analog RC-101 is being developed as a microbicide due to its specific antiviral activity, lack of toxicity to cells and tissues, and safety in animals. Understanding potential RC-101 resistance, and how resistance to other fusion inhibitors affects RC-101 susceptibility, is critical for future development. In previous studies, we identified a mutant, R5-tropic virus that had evolved partial resistance to RC-101 during in vitro selection. Here, we report that a secondary mutation in gp41 was found to restore replicative fitness, membrane fusion, and the rate of viral entry, which were compromised by an initial mutation providing partial RC-101 resistance. Interestingly, we show that RC-101 is effective against two enfuvirtide-resistant mutants, demonstrating the clinical importance of RC-101 as a unique fusion inhibitor. These findings both expand our understanding of HIV drug-resistance to diverse peptide fusion inhibitors and emphasize the significance of compensatory gp41 mutations.

  11. Hepatitis C Virus Genotype 1 to 6 Protease Inhibitor Escape Variants: In Vitro Selection, Fitness, and Resistance Patterns in the Context of the Infectious Viral Life Cycle.

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    Serre, Stéphanie B N; Jensen, Sanne B; Ghanem, Lubna; Humes, Daryl G; Ramirez, Santseharay; Li, Yi-Ping; Krarup, Henrik; Bukh, Jens; Gottwein, Judith M

    2016-06-01

    Hepatitis C virus (HCV) NS3 protease inhibitors (PIs) are important components of novel HCV therapy regimens. Studies of PI resistance initially focused on genotype 1. Therefore, knowledge about the determinants of PI resistance for the highly prevalent genotypes 2 to 6 remains limited. Using Huh7.5 cell culture-infectious HCV recombinants with genotype 1 to 6 NS3 protease, we identified protease positions 54, 155, and 156 as hot spots for the selection of resistance substitutions under treatment with the first licensed PIs, telaprevir and boceprevir. Treatment of a genotype 2 isolate with the newer PIs vaniprevir, faldaprevir, simeprevir, grazoprevir, paritaprevir, and deldeprevir identified positions 156 and 168 as hot spots for resistance; the Y56H substitution emerged for three newer PIs. Substitution selection also depended on the specific recombinant. The substitutions identified conferred cross-resistance to several PIs; however, most substitutions selected under telaprevir or boceprevir treatment conferred less resistance to certain newer PIs. In a single-cycle production assay, across genotypes, PI treatment primarily decreased viral replication, which was rescued by PI resistance substitutions. The substitutions identified resulted in differential effects on viral fitness, depending on the original recombinant and the substitution. Across genotypes, fitness impairment induced by resistance substitutions was due primarily to decreased replication. Most combinations of substitutions that were identified increased resistance or fitness. Combinations of resistance substitutions with fitness-compensating substitutions either rescued replication or compensated for decreased replication by increasing assembly. This comprehensive study provides insight into the selection patterns and effects of PI resistance substitutions for HCV genotypes 1 to 6 in the context of the infectious viral life cycle, which is of interest for clinical and virological HCV research

  12. Stability of the resistance to the thiosemicarbazone derived from 5,6-Dimethoxy-1-indanone, a non-nucleoside polymerase inhibitor of bovine viral diarrhea virus

    OpenAIRE

    Castro, Eliana Florencia; Campos, Rodolfo Hector; Cavallaro, Lucía Vicenta

    2017-01-01

    Bovine viral diarrhea virus (BVDV) is the prototype Pestivirus. BVDV infection is distributed worldwide and causes serious problems for the livestock industry. The thiosemicarbazone of 5,6-dimethoxy-1-indanone (TSC) is a non-nucleoside polymerase inhibitor (NNI) of BVDV. All TSC-resistant BVDV variants (BVDV-TSCr T1–5) present an N264D mutation in the NS5B gene (RdRp) whereas the variant BVDV-TSCr T1 also presents an NS5B A392E mutation. In the present study, we carried out twenty passages of...

  13. Stability of the resistance to the thiosemicarbazone derived from 5,6-dimethoxy-1-indanone, a non-nucleoside polymerase inhibitor of bovine viral diarrhea virus.

    Directory of Open Access Journals (Sweden)

    Eliana F Castro

    Full Text Available Bovine viral diarrhea virus (BVDV is the prototype Pestivirus. BVDV infection is distributed worldwide and causes serious problems for the livestock industry. The thiosemicarbazone of 5,6-dimethoxy-1-indanone (TSC is a non-nucleoside polymerase inhibitor (NNI of BVDV. All TSC-resistant BVDV variants (BVDV-TSCr T1-5 present an N264D mutation in the NS5B gene (RdRp whereas the variant BVDV-TSCr T1 also presents an NS5B A392E mutation. In the present study, we carried out twenty passages of BVDV-TSCr T1-5 in MDBK cells in the absence of TSC to evaluate the stability of the resistance. The viral populations obtained (BVDV R1-5 remained resistant to the antiviral compound and conserved the mutations in NS5B associated with this phenotype. Along the passages, BVDV R2, R3 and R5 presented a delay in the production of cytopathic effect that correlated with a decrease in cell apoptosis and intracellular accumulation of viral RNA. The complete genome sequences that encode for NS2 to NS5B, Npro and Erns were analyzed. Additional mutations were detected in the NS5B of BVDV R1, R3 and R4. In both BVDV R2 and R3, most of the mutations found were localized in NS5A, whereas in BVDV R5, the only mutation fixed was NS5A V177A. These results suggest that mutations in NS5A could alter BVDV cytopathogenicity. In conclusion, the stability of the resistance to TSC may be due to the fixation of different compensatory mutations in each BVDV-TSCr. During their replication in a TSC-free medium, some virus populations presented a kind of interaction with the host cell that resembled a persistent infection: decreased cytopathogenicity and viral genome synthesis. This is the first report on the stability of antiviral resistance and on the evolution of NNI-resistant BVDV variants. The results obtained for BVDV-TSCr could also be applied for other NNIs.

  14. Cell-Specific Establishment of Poliovirus Resistance to an Inhibitor Targeting a Cellular Protein

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    Viktorova, Ekaterina G.; Nchoutmboube, Jules; Ford-Siltz, Lauren A.

    2015-01-01

    ABSTRACT It is hypothesized that targeting stable cellular factors involved in viral replication instead of virus-specific proteins may raise the barrier for development of resistant mutants, which is especially important for highly adaptable small (+)RNA viruses. However, contrary to this assumption, the accumulated evidence shows that these viruses easily generate mutants resistant to the inhibitors of cellular proteins at least in some systems. We investigated here the development of poliovirus resistance to brefeldin A (BFA), an inhibitor of the cellular protein GBF1, a guanine nucleotide exchange factor for the small cellular GTPase Arf1. We found that while resistant viruses can be easily selected in HeLa cells, they do not emerge in Vero cells, in spite that in the absence of the drug both cultures support robust virus replication. Our data show that the viral replication is much more resilient to BFA than functioning of the cellular secretory pathway, suggesting that the role of GBF1 in the viral replication is independent of its Arf activating function. We demonstrate that the level of recruitment of GBF1 to the replication complexes limits the establishment and expression of a BFA resistance phenotype in both HeLa and Vero cells. Moreover, the BFA resistance phenotype of poliovirus mutants is also cell type dependent in different cells of human origin and results in a fitness loss in the form of reduced efficiency of RNA replication in the absence of the drug. Thus, a rational approach to the development of host-targeting antivirals may overcome the superior adaptability of (+)RNA viruses. IMPORTANCE Compared to the number of viral diseases, the number of available vaccines is miniscule. For some viruses vaccine development has not been successful after multiple attempts, and for many others vaccination is not a viable option. Antiviral drugs are needed for clinical practice and public health emergencies. However, viruses are highly adaptable and can

  15. Hepatitis C Virus NS3/4A Protease Inhibitors Incorporating Flexible P2 Quinoxalines Target Drug Resistant Viral Variants.

    Science.gov (United States)

    Matthew, Ashley N; Zephyr, Jacqueto; Hill, Caitlin J; Jahangir, Muhammad; Newton, Alicia; Petropoulos, Christos J; Huang, Wei; Kurt-Yilmaz, Nese; Schiffer, Celia A; Ali, Akbar

    2017-07-13

    A substrate envelope-guided design strategy is reported for improving the resistance profile of HCV NS3/4A protease inhibitors. Analogues of 5172-mcP1P3 were designed by incorporating diverse quinoxalines at the P2 position that predominantly interact with the invariant catalytic triad of the protease. Exploration of structure-activity relationships showed that inhibitors with small hydrophobic substituents at the 3-position of P2 quinoxaline maintain better potency against drug resistant variants, likely due to reduced interactions with residues in the S2 subsite. In contrast, inhibitors with larger groups at this position were highly susceptible to mutations at Arg155, Ala156, and Asp168. Excitingly, several inhibitors exhibited exceptional potency profiles with EC 50 values ≤5 nM against major drug resistant HCV variants. These findings support that inhibitors designed to interact with evolutionarily constrained regions of the protease, while avoiding interactions with residues not essential for substrate recognition, are less likely to be susceptible to drug resistance.

  16. The molecular basis of drug resistance against hepatitis C virus NS3/4A protease inhibitors.

    Directory of Open Access Journals (Sweden)

    Keith P Romano

    Full Text Available Hepatitis C virus (HCV infects over 170 million people worldwide and is the leading cause of chronic liver diseases, including cirrhosis, liver failure, and liver cancer. Available antiviral therapies cause severe side effects and are effective only for a subset of patients, though treatment outcomes have recently been improved by the combination therapy now including boceprevir and telaprevir, which inhibit the viral NS3/4A protease. Despite extensive efforts to develop more potent next-generation protease inhibitors, however, the long-term efficacy of this drug class is challenged by the rapid emergence of resistance. Single-site mutations at protease residues R155, A156 and D168 confer resistance to nearly all inhibitors in clinical development. Thus, developing the next-generation of drugs that retain activity against a broader spectrum of resistant viral variants requires a comprehensive understanding of the molecular basis of drug resistance. In this study, 16 high-resolution crystal structures of four representative protease inhibitors--telaprevir, danoprevir, vaniprevir and MK-5172--in complex with the wild-type protease and three major drug-resistant variants R155K, A156T and D168A, reveal unique molecular underpinnings of resistance to each drug. The drugs exhibit differential susceptibilities to these protease variants in both enzymatic and antiviral assays. Telaprevir, danoprevir and vaniprevir interact directly with sites that confer resistance upon mutation, while MK-5172 interacts in a unique conformation with the catalytic triad. This novel mode of MK-5172 binding explains its retained potency against two multi-drug-resistant variants, R155K and D168A. These findings define the molecular basis of HCV N3/4A protease inhibitor resistance and provide potential strategies for designing robust therapies against this rapidly evolving virus.

  17. Identification of cellular and viral factors related to anti-hepatitis C virus activity of cyclophilin inhibitor.

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    Goto, Kaku; Watashi, Koichi; Inoue, Daisuke; Hijikata, Makoto; Shimotohno, Kunitada

    2009-10-01

    We have so far reported that an immunosuppressant cyclosporin A (CsA), a well-known cyclophilin (CyP) inhibitor (CPI), strongly suppressed hepatitis C virus (HCV) replication in cell culture, and that CyPB was a cellular cofactor for viral replication. To further investigate antiviral mechanisms of CPI, we here developed cells carrying CsA-resistant HCV replicons, by culturing the HCV subgenomic replicon cells for 4 weeks in the presence of CsA with G418. Transfection of total RNA from the isolated CsA-resistant cells to naïve Huh7 cells conferred CsA resistance, suggesting that the replicon RNA itself was responsible for the resistant phenotype. Of the identified amino acid mutations, D320E in NS5A conferred the CsA resistance. The replicon carrying the D320E mutation was sensitive to interferon-alpha, but was resistant to CsA and other CPIs including NIM811 and sanglifehrin A. Knockdown of individual CyP subtypes revealed CyP40, in addition to CyPA and CyPB, contributed to viral replication, and CsA-resistant replicons acquired independence from CyPA for efficient replication. These data provide important evidence on the mechanisms underlying the regulation of HCV replication by CyP and for designing novel and specific anti-HCV strategies with CPIs.

  18. Identification of a novel multiple kinase inhibitor with potent antiviral activity against influenza virus by reducing viral polymerase activity

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    Sasaki, Yutaka; Kakisaka, Michinori; Chutiwitoonchai, Nopporn [Viral Infectious Diseases Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Tajima, Shigeru [Department of Virology I, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo 162-8640 (Japan); Hikono, Hirokazu; Saito, Takehiko [Influenza and Prion Disease Research Center, National Institute of Animal Health, National Agriculture and Food Research Organization (NARO), 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856 (Japan); Aida, Yoko, E-mail: aida@riken.jp [Viral Infectious Diseases Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan)

    2014-07-18

    Highlights: • Screening of 50,000 compounds and subsequent lead optimization identified WV970. • WV970 has antiviral effects against influenza A, B and highly pathogenic viral strains. • WV970 inhibits viral genome replication and transcription. • A target database search suggests that WV970 may bind to a number of kinases. • KINOMEscan screening revealed that WV970 has inhibitory effects on 15 kinases. - Abstract: Neuraminidase inhibitors are the only currently available influenza treatment, although resistant viruses to these drugs have already been reported. Thus, new antiviral drugs with novel mechanisms of action are urgently required. In this study, we identified a novel antiviral compound, WV970, through cell-based screening of a 50,000 compound library and subsequent lead optimization. This compound exhibited potent antiviral activity with nanomolar IC{sub 50} values against both influenza A and B viruses but not non-influenza RNA viruses. Time-of-addition and indirect immunofluorescence assays indicated that WV970 acted at an early stage of the influenza life cycle, but likely after nuclear entry of viral ribonucleoprotein (vRNP). Further analyses of viral RNA expression and viral polymerase activity indicated that WV970 inhibited vRNP-mediated viral genome replication and transcription. Finally, structure-based virtual screening and comprehensive human kinome screening were used to demonstrate that WV970 acts as a multiple kinase inhibitor, many of which are associated with influenza virus replication. Collectively, these results strongly suggest that WV970 is a promising anti-influenza drug candidate and that several kinases associated with viral replication are promising drug targets.

  19. Identification of a novel multiple kinase inhibitor with potent antiviral activity against influenza virus by reducing viral polymerase activity

    International Nuclear Information System (INIS)

    Sasaki, Yutaka; Kakisaka, Michinori; Chutiwitoonchai, Nopporn; Tajima, Shigeru; Hikono, Hirokazu; Saito, Takehiko; Aida, Yoko

    2014-01-01

    Highlights: • Screening of 50,000 compounds and subsequent lead optimization identified WV970. • WV970 has antiviral effects against influenza A, B and highly pathogenic viral strains. • WV970 inhibits viral genome replication and transcription. • A target database search suggests that WV970 may bind to a number of kinases. • KINOMEscan screening revealed that WV970 has inhibitory effects on 15 kinases. - Abstract: Neuraminidase inhibitors are the only currently available influenza treatment, although resistant viruses to these drugs have already been reported. Thus, new antiviral drugs with novel mechanisms of action are urgently required. In this study, we identified a novel antiviral compound, WV970, through cell-based screening of a 50,000 compound library and subsequent lead optimization. This compound exhibited potent antiviral activity with nanomolar IC 50 values against both influenza A and B viruses but not non-influenza RNA viruses. Time-of-addition and indirect immunofluorescence assays indicated that WV970 acted at an early stage of the influenza life cycle, but likely after nuclear entry of viral ribonucleoprotein (vRNP). Further analyses of viral RNA expression and viral polymerase activity indicated that WV970 inhibited vRNP-mediated viral genome replication and transcription. Finally, structure-based virtual screening and comprehensive human kinome screening were used to demonstrate that WV970 acts as a multiple kinase inhibitor, many of which are associated with influenza virus replication. Collectively, these results strongly suggest that WV970 is a promising anti-influenza drug candidate and that several kinases associated with viral replication are promising drug targets

  20. SCREENING OF PROTEASE INHIBITORS RESISTANCE MUTATIONS IN HEPATITIS C VIRUS ISOLATES INFECTING ROMANIAN PATIENTS UNEXPOSED TO TRIPLE THERAPY.

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    Dinu, Sorin; Calistru, Petre-Iacob; Ceauşu, Emanoil; Târdeil, Graţiela; Oprişan, Gabriela

    2015-01-01

    Although the European recommendations include the use of new antiviral drugs for the treatment of hepatitis C, in Romania the current treatment remains interferon plus ribavirin. First generation viral protease inhibitors (i.e. boceprevir, telaprevir), which have raised the chances of obtaining viral clearance in up to 70% of infection cases produced by genotype 1 isolates, have not been introduced yet as standard treatment in our country. The success of these new antivirals is limited by the occurrence and selection of resistance mutations during therapy. We set-up a molecular study aiming to detect any resistance mutations to boceprevir and telaprevir harbored by hepatitis C isolates infecting Romanian patients naïve to viral protease inhibitors. Since these new antivirals are efficient and approved for genotype 1 infection, viral samples were genotyped following a protocol previously developed by our research group. We analyzed by both population sequencing and molecular cloning and sequencing the NS3 protease region of hepatitis C virus isolates infecting patients which were not previously exposed to boceprevir and telaprevir. All the analyzed samples were subtype 1b and resembled the samples collected in recent years from Romanian patients. Molecular cloning followed by sequencing showed great intra-host diversity, which is known to represent the source of isolates with different resistance phenotypes. Both population sequencing and molecular cloning followed by clone sequencing revealed two boceprevir resistance mutations (T54S and V55A), respectively, a telaprevir resistance mutation (T54S) in the sequences obtained from a patient with chronic hepatitis C. To our knowledge, this is the first study indicating the existence of pre-treatment resistance mutations to boceprevir and telaprevir in hepatitis C virus isolates infecting Romanian patients.

  1. Mutations in the Nonstructural Protein 3A Confer Resistance to the Novel Enterovirus Replication Inhibitor TTP-8307

    NARCIS (Netherlands)

    de Palma, Armando M.; Thibaut, Hendrik Jan; van der Linden, Lonneke; Lanke, Kjerstin; Heggermont, Ward; Ireland, Stephen; Andrews, Robert; Arimilli, Murty; Al-tel, Taleb H.; de Clercq, Erik; van Kuppeveld, Frank; Neyts, Johan

    2009-01-01

    A novel compound, TTP-8307, was identified as a potent inhibitor of the replication of several rhino- and enteroviruses. TTP-8307 inhibits viral RNA synthesis in a dose-dependent manner, without affecting polyprotein synthesis and/or processing. Drug-resistant variants of coxsackievirus B3 were all

  2. [Role of ATP-sensitive potassium channel activators in liver mitochondrial function in rats with different resistance to hypoxia].

    Science.gov (United States)

    Tkachenko, H M; Kurhaliuk, N M; Vovkanych, L S

    2003-01-01

    Effects of ATP-sensitive potassium (KATP) channels opener pinacidil (0.06 mg/kg) and inhibitor glibenclamide (1 mg/kg) in rats with different resistance to hypoxia on indices of ADP-stimulation of mitochondrial respiration by Chance, calcium capacity and processes of lipid peroxidation in liver has been investigated. We used next substrates of oxidation: 0.35 mM succinate, 1 mM alpha-ketoglutarate. Additional analyses contain the next inhibitors: mitochondrial fermentative complex I-10 mkM rotenone, succinate dehydrogenase 2 mM malonic acid. It was shown that effects of pinacidil induced the increasing of oxidative phosporylation efficacy and ATP synthesis together with lowering of calcium capacity in rats with low resistance to hypoxia. Effects of pinacidil were leveled by glibenclamide. These changes are connected with the increasing of respiratory rate, calcium overload and intensification of lipid peroxidation processes. A conclusion was made about protective effect of pinacidil on mitochondrial functioning by economization of oxygen-dependent processes, adaptive potentialities of organisms with low resistance to hypoxia being increased.

  3. A high throughput Cre–lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry

    Energy Technology Data Exchange (ETDEWEB)

    Esposito, Anthony M. [Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, Immunology Institute, New York, NY (United States); Cheung, Pamela [Integrated Screening Core, Icahn School of Medicine at Mount Sinai, New York, NY (United States); Swartz, Talia H.; Li, Hongru [Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, Immunology Institute, New York, NY (United States); Tsibane, Tshidi [Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY (United States); Durham, Natasha D. [Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, Immunology Institute, New York, NY (United States); Basler, Christopher F. [Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY (United States); Felsenfeld, Dan P. [Integrated Screening Core, Icahn School of Medicine at Mount Sinai, New York, NY (United States); Chen, Benjamin K., E-mail: benjamin.chen@mssm.edu [Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, Immunology Institute, New York, NY (United States)

    2016-03-15

    Enveloped virus entry occurs when viral and cellular membranes fuse releasing particle contents into the target cell. Human immunodeficiency virus (HIV) entry occurs by cell-free virus or virus transferred between infected and uninfected cells through structures called virological synapses. We developed a high-throughput cell-based assay to identify small molecule inhibitors of cell-free or virological synapse-mediated entry. An HIV clone carrying Cre recombinase as a Gag-internal gene fusion releases active Cre into cells upon viral entry activating a recombinatorial gene switch changing dsRed to GFP-expression. A screen of a 1998 known-biological profile small molecule library identified pharmacological HIV entry inhibitors that block both cell-free and cell-to-cell infection. Many top hits were noted as HIV inhibitors in prior studies, but not previously recognized as entry antagonists. Modest therapeutic indices for simvastatin and nigericin were observed in confirmatory HIV infection assays. This robust assay is adaptable to study HIV and heterologous viral pseudotypes. - Highlights: • Cre recombinase viral fusion assay screens cell-free or cell–cell entry inhibitors. • This Gag-iCre based assay is specific for the entry step of HIV replication. • Screened a library of known pharmacologic compounds for HIV fusion antagonists. • Many top hits were previously noted as HIV inhibitors, but here are classified as entry antagonists. Many top hits were previously noted as HIV inhibitors, but not as entry antagonists. • The assay is compatible with pseudotyping with HIV and heterologous viruses.

  4. A high throughput Cre–lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry

    International Nuclear Information System (INIS)

    Esposito, Anthony M.; Cheung, Pamela; Swartz, Talia H.; Li, Hongru; Tsibane, Tshidi; Durham, Natasha D.; Basler, Christopher F.; Felsenfeld, Dan P.; Chen, Benjamin K.

    2016-01-01

    Enveloped virus entry occurs when viral and cellular membranes fuse releasing particle contents into the target cell. Human immunodeficiency virus (HIV) entry occurs by cell-free virus or virus transferred between infected and uninfected cells through structures called virological synapses. We developed a high-throughput cell-based assay to identify small molecule inhibitors of cell-free or virological synapse-mediated entry. An HIV clone carrying Cre recombinase as a Gag-internal gene fusion releases active Cre into cells upon viral entry activating a recombinatorial gene switch changing dsRed to GFP-expression. A screen of a 1998 known-biological profile small molecule library identified pharmacological HIV entry inhibitors that block both cell-free and cell-to-cell infection. Many top hits were noted as HIV inhibitors in prior studies, but not previously recognized as entry antagonists. Modest therapeutic indices for simvastatin and nigericin were observed in confirmatory HIV infection assays. This robust assay is adaptable to study HIV and heterologous viral pseudotypes. - Highlights: • Cre recombinase viral fusion assay screens cell-free or cell–cell entry inhibitors. • This Gag-iCre based assay is specific for the entry step of HIV replication. • Screened a library of known pharmacologic compounds for HIV fusion antagonists. • Many top hits were previously noted as HIV inhibitors, but here are classified as entry antagonists. Many top hits were previously noted as HIV inhibitors, but not as entry antagonists. • The assay is compatible with pseudotyping with HIV and heterologous viruses.

  5. Escape from Human Immunodeficiency Virus Type 1 (HIV-1 Entry Inhibitors

    Directory of Open Access Journals (Sweden)

    Carol D. Weiss

    2012-12-01

    Full Text Available The human immunodeficiency virus (HIV enters cells through a series of molecular interactions between the HIV envelope protein and cellular receptors, thus providing many opportunities to block infection. Entry inhibitors are currently being used in the clinic, and many more are under development. Unfortunately, as is the case for other classes of antiretroviral drugs that target later steps in the viral life cycle, HIV can become resistant to entry inhibitors. In contrast to inhibitors that block viral enzymes in intracellular compartments, entry inhibitors interfere with the function of the highly variable envelope glycoprotein as it continuously adapts to changing immune pressure and available target cells in the extracellular environment. Consequently, pathways and mechanisms of resistance for entry inhibitors are varied and often involve mutations across the envelope gene. This review provides a broad overview of entry inhibitor resistance mechanisms that inform our understanding of HIV entry and the design of new inhibitors and vaccines.

  6. Antagonism of the Sodium-Potassium ATPase Impairs Chikungunya Virus Infection

    Directory of Open Access Journals (Sweden)

    Alison W. Ashbrook

    2016-05-01

    Full Text Available Chikungunya virus (CHIKV is a reemerging alphavirus that has caused epidemics of fever, arthralgia, and rash worldwide. There are currently no licensed vaccines or antiviral therapies available for the prevention or treatment of CHIKV disease. We conducted a high-throughput, chemical compound screen that identified digoxin, a cardiac glycoside that blocks the sodium-potassium ATPase, as a potent inhibitor of CHIKV infection. Treatment of human cells with digoxin or a related cardiac glycoside, ouabain, resulted in a dose-dependent decrease in infection by CHIKV. Inhibition by digoxin was cell type-specific, as digoxin treatment of either murine or mosquito cells did not diminish CHIKV infection. Digoxin displayed antiviral activity against other alphaviruses, including Ross River virus and Sindbis virus, as well as mammalian reovirus and vesicular stomatitis virus. The digoxin-mediated block to CHIKV and reovirus infection occurred at one or more postentry steps, as digoxin inhibition was not bypassed by fusion of CHIKV at the plasma membrane or infection with cell surface-penetrating reovirus entry intermediates. Selection of digoxin-resistant CHIKV variants identified multiple mutations in the nonstructural proteins required for replication complex formation and synthesis of viral RNA. These data suggest a role for the sodium-potassium ATPase in promoting postentry steps of CHIKV replication and provide rationale for modulation of this pathway as a broad-spectrum antiviral strategy.

  7. Resistance Analyses of Integrase Strand Transfer Inhibitors within Phase 3 Clinical Trials of Treatment-Naive Patients

    Directory of Open Access Journals (Sweden)

    Kirsten L. White

    2014-07-01

    Full Text Available The integrase (IN strand transfer inhibitors (INSTIs, raltegravir (RAL, elvitegravir (EVG and dolutegravir (DTG, comprise the newest drug class approved for the treatment of HIV-1 infection, which joins the existing classes of reverse transcriptase, protease and binding/entry inhibitors. The efficacy of first-line regimens has attained remarkably high levels, reaching undetectable viral loads in 90% of patients by Week 48; however, there remain patients who require a change in regimen due to adverse events, virologic failure with emergent resistance or other issues of patient management. Large, randomized clinical trials conducted in antiretroviral treatment-naive individuals are required for drug approval in this population in the US, EU and other countries, with the primary endpoint for virologic success at Week 48. However, there are differences in the definition of virologic failure and the evaluation of drug resistance among the trials. This review focuses on the methodology and tabulation of resistance to INSTIs in phase 3 clinical trials of first-line regimens and discusses case studies of resistance.

  8. Excitation block in a nerve fibre model owing to potassium-dependent changes in myelin resistance

    DEFF Research Database (Denmark)

    Brazhe, Alexey; Maksimov, G. V.; Mosekilde, Erik

    2011-01-01

    . Uptake of potassium leads to Schwann cell swelling and myelin restructuring that impacts the electrical properties of the myelin. In order to further understand the dynamic interaction that takes place between the myelin and the axon, we have modelled submyelin potassium accumulation and related changes...... in myelin resistance during prolonged high-frequency stimulation. We predict that potassium-mediated decrease in myelin resistance leads to a functional excitation block with various patterns of altered spike trains. The patterns are found to depend on stimulation frequency and amplitude and to range from...

  9. Prevalence of drug resistance and importance of viral load measurements in Honduran HIV-infected patients failing antiretroviral treatment.

    Science.gov (United States)

    Murillo, Wendy; de Rivera, I L; Parham, L; Jovel, E; Palou, E; Karlsson, A C; Albert, J

    2010-02-01

    The Honduran HIV/AIDS Program began to scale up access to HIV therapy in 2002. Up to May 2008, more than 6000 patients received combination antiretroviral therapy (cART). As HIV drug resistance is the major obstacle for effective treatment, the purpose of this study was to assess the prevalence of antiretroviral drug resistance in Honduran HIV-1-infected individuals. We collected samples from 138 individuals (97 adults and 41 children) on cART with virological, immunological or clinical signs of treatment failure. HIV-1 pol sequences were obtained using an in-house method. Resistance mutations were identified according to the 2007 International AIDS Society (IAS)-USA list and predicted susceptibility to cART was scored using the ANRS algorithm. Resistance mutations were detected in 112 patients (81%), 74% in adults and 98% in children. Triple-, dual- and single-class drug resistance was documented in 27%, 43% and 11% of the study subjects, respectively. Multiple logistic regression showed that resistance was independently associated with type of treatment failure [virological failure (odds ratio (OR) = 1) vs. immunological failure (OR = 0.11; 95% confidence interval (CI) 0.030-0.43) vs. clinical failure (OR = 0.037; 95% CI 0.0063-0.22)], route of transmission (OR = 42.8; 95% CI 3.73-491), and years on therapy (OR = 1.81; 95% CI 1.11-2.93). The prevalence of antiretroviral resistance was high in Honduran HIV-infected patients with signs of treatment failure. A majority of study subjects showed dual- or triple-class resistance to nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors and protease inhibitors. Virologically defined treatment failure was a strong predictor of resistance, indicating that viral load testing is needed to correctly identify patients with treatment failure attributable to resistance.

  10. Life cycle synchronization is a viral drug resistance mechanism.

    Directory of Open Access Journals (Sweden)

    Iulia A Neagu

    2018-02-01

    Full Text Available Viral infections are one of the major causes of death worldwide, with HIV infection alone resulting in over 1.2 million casualties per year. Antiviral drugs are now being administered for a variety of viral infections, including HIV, hepatitis B and C, and influenza. These therapies target a specific phase of the virus's life cycle, yet their ultimate success depends on a variety of factors, such as adherence to a prescribed regimen and the emergence of viral drug resistance. The epidemiology and evolution of drug resistance have been extensively characterized, and it is generally assumed that drug resistance arises from mutations that alter the virus's susceptibility to the direct action of the drug. In this paper, we consider the possibility that a virus population can evolve towards synchronizing its life cycle with the pattern of drug therapy. The periodicity of the drug treatment could then allow for a virus strain whose life cycle length is a multiple of the dosing interval to replicate only when the concentration of the drug is lowest. This process, referred to as "drug tolerance by synchronization", could allow the virus population to maximize its overall fitness without having to alter drug binding or complete its life cycle in the drug's presence. We use mathematical models and stochastic simulations to show that life cycle synchronization can indeed be a mechanism of viral drug tolerance. We show that this effect is more likely to occur when the variability in both viral life cycle and drug dose timing are low. More generally, we find that in the presence of periodic drug levels, time-averaged calculations of viral fitness do not accurately predict drug levels needed to eradicate infection, even if there is no synchronization. We derive an analytical expression for viral fitness that is sufficient to explain the drug-pattern-dependent survival of strains with any life cycle length. We discuss the implications of these findings for

  11. Convergent Akt activation drives acquired EGFR inhibitor resistance in lung cancer

    DEFF Research Database (Denmark)

    Jacobsen, Kirstine; Bertran-Alamillo, Jordi; Molina, Miguel Angel

    2017-01-01

    Non-small-cell lung cancer patients with activating epidermal growth factor receptor (EGFR) mutations typically benefit from EGFR tyrosine kinase inhibitor treatment. However, virtually all patients succumb to acquired EGFR tyrosine kinase inhibitor resistance that occurs via diverse mechanisms....... The diversity and unpredictability of EGFR tyrosine kinase inhibitor resistance mechanisms presents a challenge for developing new treatments to overcome EGFR tyrosine kinase inhibitor resistance. Here, we show that Akt activation is a convergent feature of acquired EGFR tyrosine kinase inhibitor resistance......, across a spectrum of diverse, established upstream resistance mechanisms. Combined treatment with an EGFR tyrosine kinase inhibitor and Akt inhibitor causes apoptosis and synergistic growth inhibition in multiple EGFR tyrosine kinase inhibitor-resistant non-small-cell lung cancer models. Moreover...

  12. Viral kinetics in patients with chronic hepatitis C treated with the serine protease inhibitor BILN 2061

    NARCIS (Netherlands)

    Herrmann, Eva; Zeuzem, Stefan; Sarrazin, Christoph; Hinrichsen, Holger; Benhamou, Yves; Manns, Michael P.; Reiser, Markus; Reesink, Henk; Calleja, José L.; Forns, Xavier; Steinmann, Gerhard G.; Nehmiz, Gerhard

    2006-01-01

    We analysed viral kinetics from a 2-day treatment with BILN 2061, a serine protease inhibitor of hepatitis C virus, in patients chronically infected with genotype 1 hepatitis C virus. The efficiency (E), describing inhibition of viral production, was above 99.45% in all patients with minor or

  13. Stem cell gene therapy for HIV: strategies to inhibit viral entry and replication.

    Science.gov (United States)

    DiGiusto, David L

    2015-03-01

    Since the demonstration of a cure of an HIV+ patient with an allogeneic stem cell transplant using naturally HIV-resistant cells, significant interest in creating similar autologous products has fueled the development of a variety of "cell engineering" approaches to stem cell therapy for HIV. Among the more well-studied strategies is the inhibition of viral entry through disruption of expression of viral co-receptors or through competitive inhibitors of viral fusion with the cell membrane. Preclinical evaluation of these approaches often starts in vitro but ultimately is tested in animal models prior to clinical implementation. In this review, we trace the development of several key approaches (meganucleases, short hairpin RNA (shRNA), and fusion inhibitors) to modification of hematopoietic stem cells designed to impart resistance to HIV to their T-cell and monocytic progeny. The basic evolution of technologies through in vitro and in vivo testing is discussed as well as the pros and cons of each approach and how the addition of postentry inhibitors may enhance the overall antiviral efficacy of these approaches.

  14. Proteasome inhibitors induce apoptosis and reduce viral replication in primary effusion lymphoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Saji, Chiaki [Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812 (Japan); Higashi, Chizuka; Niinaka, Yasufumi [Faculty of Medicine, University of Yamanashi, Chuoh-shi 409-3898 (Japan); Yamada, Koji [Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812 (Japan); Noguchi, Kohji [Faculty of Pharmacy, Keio University, 1-5-30 Shiba-koen, Minato-ku, Tokyo 105-8512 (Japan); Fujimuro, Masahiro, E-mail: fuji2@mb.kyoto-phu.ac.jp [Department of Cell Biology, Kyoto Pharmaceutical University, Misasagi-Shichonocho 1, Yamashinaku, Kyoto 607-8412 (Japan)

    2011-12-02

    Highlights: Black-Right-Pointing-Pointer Constitutive NF-{kappa}B signaling is essential for the survival and growth of PEL cells. Black-Right-Pointing-Pointer NF-{kappa}B signaling is upregulated by the proteasome-dependent degradation of I{kappa}B{alpha}. Black-Right-Pointing-Pointer Proteasome inhibitors suppress NF-{kappa}B signaling and induce apoptosis in PEL cells through stabilization of I{kappa}B{alpha}. Black-Right-Pointing-Pointer Proteasome inhibitors suppress viral replication in PEL cells during lytic KSHV infection. -- Abstract: Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by Kaposi's sarcoma-associated herpesvirus (KSHV). This study provides evidence that proteasomal activity is required for both survival of PEL cells stably harboring the KSHV genome and viral replication of KSHV. We evaluated the cytotoxic effects of proteasome inhibitors on PEL cells. The proteasome inhibitors MG132, lactacystin, and proteasome inhibitor I dramatically inhibited cell proliferation and induced apoptosis of PEL cells through the accumulation of p21 and p27. Furthermore, proteasome inhibitors induced the stabilization of NF-{kappa}B inhibitory molecule (I{kappa}B{alpha}) and suppressed the transcriptional activity of NF-{kappa}B in PEL cells. The NF-{kappa}B specific inhibitor BAY11-7082 also induced apoptosis in PEL cells. The constitutive activation of NF-{kappa}B signaling is essential for the survival and growth of B cell lymphoma cells, including PEL cells. NF-{kappa}B signaling is upregulated by proteasome-dependent degradation of I{kappa}B{alpha}. The suppression of NF-{kappa}B signaling by proteasome inhibitors may contribute to the induction of apoptosis in PEL cells. In addition, proteasome activity is required for KSHV replication in KSHV latently infected PEL cells. MG132 reduced the production of progeny virus from PEL cells at low concentrations, which do not affect PEL cell growth. These findings suggest that proteasome

  15. Unnecessary antiretroviral treatment switches and accumulation of HIV resistance mutations; two arguments for viral load monitoring in Africa.

    Science.gov (United States)

    Sigaloff, Kim C E; Hamers, Raph L; Wallis, Carole L; Kityo, Cissy; Siwale, Margaret; Ive, Prudence; Botes, Mariette E; Mandaliya, Kishor; Wellington, Maureen; Osibogun, Akin; Stevens, Wendy S; van Vugt, Michèle; de Wit, Tobias F Rinke

    2011-09-01

    This study aimed to investigate the consequences of using clinicoimmunological criteria to detect antiretroviral treatment (ART) failure and guide regimen switches in HIV-infected adults in sub-Saharan Africa. Frequencies of unnecessary switches, patterns of HIV drug resistance, and risk factors for the accumulation of nucleoside reverse transcriptase inhibitor (NRTI)-associated mutations were evaluated. Cross-sectional analysis of adults switching ART regimens at 13 clinical sites in 6 African countries was performed. Two types of failure identification were compared: diagnosis of clinicoimmunological failure without viral load testing (CIF only) or CIF with local targeted viral load testing (targeted VL). After study enrollment, reference HIV RNA and genotype were determined retrospectively. Logistic regression assessed factors associated with multiple thymidine analogue mutations (TAMs) and NRTI cross-resistance (≥2 TAMs or Q151M or K65R/K70E). Of 250 patients with CIF switching to second-line ART, targeted VL was performed in 186. Unnecessary switch at reference HIV RNA <1000 copies per milliliter occurred in 46.9% of CIF only patients versus 12.4% of patients with targeted VL (P < 0.001). NRTI cross-resistance was observed in 48.0% of 183 specimens available for genotypic analysis, comprising ≥2 TAMs (37.7%), K65R (7.1%), K70E (3.3%), or Q151M (3.3%). The presence of NRTI cross-resistance was associated with the duration of ART exposure and zidovudine use. Clinicoimmunological monitoring without viral load testing resulted in frequent unnecessary regimen switches. Prolonged treatment failure was indicated by extensive NRTI cross-resistance. Access to virological monitoring should be expanded to prevent inappropriate switches, enable early failure detection and preserve second-line treatment options in Africa.

  16. 4-Aminopyridine: a pan voltage-gated potassium channel inhibitor that enhances K7.4 currents and inhibits noradrenaline-mediated contraction of rat mesenteric small arteries

    DEFF Research Database (Denmark)

    Khammy, Makhala M; Kim, Sukhan; Bentzen, Bo H

    2018-01-01

    has not been systematically studied. The aim of this study was to investigate the pharmacological activity of 4-AP on Kv7.4 and Kv7.5 channels and characterize the effect of 4-AP on rat resistance arteries. EXPERIMENTAL APPROACH: Voltage clamp experiments were performed on Xenopus laevis oocytes......BACKGROUND AND PURPOSE: Kv7.4 and Kv7.5 channels are regulators of vascular tone. 4-Aminopyridine (4-AP) is considered a broad inhibitor of voltage-gated potassium (KV) channels, with little inhibitory effect on Kv7 family members at mmol concentrations. However, the effect of 4-AP on Kv7 channels...

  17. Mechanisms of Hepatitis C Viral Resistance to Direct Acting Antivirals.

    Science.gov (United States)

    Ahmed, Asma; Felmlee, Daniel J

    2015-12-18

    There has been a remarkable transformation in the treatment of chronic hepatitis C in recent years with the development of direct acting antiviral agents targeting virus encoded proteins important for viral replication including NS3/4A, NS5A and NS5B. These agents have shown high sustained viral response (SVR) rates of more than 90% in phase 2 and phase 3 clinical trials; however, this is slightly lower in real-life cohorts. Hepatitis C virus resistant variants are seen in most patients who do not achieve SVR due to selection and outgrowth of resistant hepatitis C virus variants within a given host. These resistance associated mutations depend on the class of direct-acting antiviral drugs used and also vary between hepatitis C virus genotypes and subtypes. The understanding of these mutations has a clear clinical implication in terms of choice and combination of drugs used. In this review, we describe mechanism of action of currently available drugs and summarize clinically relevant resistance data.

  18. Computational Analysis of Molecular Interaction Networks Underlying Change of HIV-1 Resistance to Selected Reverse Transcriptase Inhibitors.

    Science.gov (United States)

    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.

  19. Temperature-induced viral resistance in Emiliania huxleyi (Prymnesiophyceae).

    Science.gov (United States)

    Kendrick, B Jacob; DiTullio, Giacomo R; Cyronak, Tyler J; Fulton, James M; Van Mooy, Benjamin A S; Bidle, Kay D

    2014-01-01

    Annual Emiliania huxleyi blooms (along with other coccolithophorid species) play important roles in the global carbon and sulfur cycles. E. huxleyi blooms are routinely terminated by large, host-specific dsDNA viruses, (Emiliania huxleyi Viruses; EhVs), making these host-virus interactions a driving force behind their potential impact on global biogeochemical cycles. Given projected increases in sea surface temperature due to climate change, it is imperative to understand the effects of temperature on E. huxleyi's susceptibility to viral infection and its production of climatically active dimethylated sulfur species (DSS). Here we demonstrate that a 3°C increase in temperature induces EhV-resistant phenotypes in three E. huxleyi strains and that successful virus infection impacts DSS pool sizes. We also examined cellular polar lipids, given their documented roles in regulating host-virus interactions in this system, and propose that alterations to membrane-bound surface receptors are responsible for the observed temperature-induced resistance. Our findings have potential implications for global biogeochemical cycles in a warming climate and for deciphering the particular mechanism(s) by which some E. huxleyi strains exhibit viral resistance.

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

    DEFF Research Database (Denmark)

    Fox, Zoe; Phillips, Andrew; Cohen, Cal

    2008-01-01

    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...... 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.......BACKGROUND: 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...

  1. Ninety-nine is not enough: molecular characterization of inhibitor-resistant human immunodeficiency virus type 1 protease mutants with insertions in the flap region

    Czech Academy of Sciences Publication Activity Database

    Kožíšek, Milan; Grantz Šašková, Klára; Řezáčová, Pavlína; Brynda, Jiří; Maarseveen van, N. M.; De Jongh, D.; Boucher, Ch. A. B.; Kagan, R. M.; Nijhuis, M.; Konvalinka, Jan

    2008-01-01

    Roč. 82, č. 12 (2008), s. 5869-5878 ISSN 0022-538X R&D Projects: GA MŠk 1M0508; GA MZd NR8571 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520514 Keywords : HIV protease inhibitors * aspartic proteases * viral resistance * insertions Subject RIV: CE - Biochemistry Impact factor: 5.308, year: 2008

  2. Inhibition of post-transcriptional RNA processing by CDK inhibitors and its implication in anti-viral therapy.

    Directory of Open Access Journals (Sweden)

    Jitka Holcakova

    Full Text Available Cyclin-dependent kinases (CDKs are key regulators of the cell cycle and RNA polymerase II mediated transcription. Several pharmacological CDK inhibitors are currently in clinical trials as potential cancer therapeutics and some of them also exhibit antiviral effects. Olomoucine II and roscovitine, purine-based inhibitors of CDKs, were described as effective antiviral agents that inhibit replication of a broad range of wild type human viruses. Olomoucine II and roscovitine show high selectivity for CDK7 and CDK9, with important functions in the regulation of RNA polymerase II transcription. RNA polymerase II is necessary for viral transcription and following replication in cells. We analyzed the effect of inhibition of CDKs by olomoucine II on gene expression from viral promoters and compared its effect to widely-used roscovitine. We found that both roscovitine and olomoucine II blocked the phosphorylation of RNA polymerase II C-terminal domain. However the repression of genes regulated by viral promoters was strongly dependent on gene localization. Both roscovitine and olomoucine II inhibited expression only when the viral promoter was not integrated into chromosomal DNA. In contrast, treatment of cells with genome-integrated viral promoters increased their expression even though there was decreased phosphorylation of the C-terminal domain of RNA polymerase II. To define the mechanism responsible for decreased gene expression after pharmacological CDK inhibitor treatment, the level of mRNA transcription from extrachromosomal DNA was determined. Interestingly, our results showed that inhibition of RNA polymerase II C-terminal domain phosphorylation increased the number of transcribed mRNAs. However, some of these mRNAs were truncated and lacked polyadenylation, which resulted in decreased translation. These results suggest that phosphorylation of RNA polymerase II C-terminal domain is critical for linking transcription and posttrancriptional

  3. The S230R Integrase Substitution Associated with Viral Rebound during DTG Monotherapy Confers Low Levels INSTI Drug Resistance.

    Science.gov (United States)

    Pham, Hanh T; Labrie, Lydia; Wijting, Ingeborg E A; Hassounah, Said; Lok, Ka Yee; Portna, Inna; Goring, Mark; Han, Yingshan; Lungu, Cynthia; van der Ende, Marchina E; Brenner, Bluma G; Boucher, Charles A; Rijnders, Bart J A; van Kampen, Jeroen J A; Mesplède, Thibault; Wainberg, Mark A

    2018-03-29

    Dolutegravir (DTG) is an integrase strand-transfer inhibitor (INSTI) used for treatment of HIV-infected individuals. Due to its high genetic barrier to resistance, DTG has been clinically investigated as maintenance monotherapy to maintain viral suppression and to reduce complication and healthcare costs. Our study aims to explain the underlying mechanism related to the emergence of a S230R substitution in patients who experienced virological failure while using DTG monotherapy. We evaluated the effect of S230R substitution in regard to IN enzyme activity, viral infectivity, replicative capacity and susceptibility to different INSTIs by biochemical and cell-based assays. S230R substitution conferred 63% reduction in enzyme efficiency. The S230R virus was 1.29-fold less infectious than wildtype (WT), but could replicate in PM1 cells without significant delay. Resistance levels against DTG, CAB, RAL and EVG in tissue culture were 3.85-, 3.72-, 1.52-, and 1.21-fold, respectively. Our data indicate that the S230R substitution is comparable to the previously reported R263K in some respects. Virological failure under DTG monotherapy can occur through the development of such S230R or R263K mutations without the need for high levels DTG resistance.

  4. Excitation block in a nerve fibre model owing to potassium-dependent changes in myelin resistance.

    Science.gov (United States)

    Brazhe, A R; Maksimov, G V; Mosekilde, E; Sosnovtseva, O V

    2011-02-06

    The myelinated nerve fibre is formed by an axon and Schwann cells or oligodendrocytes that sheath the axon by winding around it in tight myelin layers. Repetitive stimulation of a fibre is known to result in accumulation of extracellular potassium ions, especially between the axon and the myelin. Uptake of potassium leads to Schwann cell swelling and myelin restructuring that impacts the electrical properties of the myelin. In order to further understand the dynamic interaction that takes place between the myelin and the axon, we have modelled submyelin potassium accumulation and related changes in myelin resistance during prolonged high-frequency stimulation. We predict that potassium-mediated decrease in myelin resistance leads to a functional excitation block with various patterns of altered spike trains. The patterns are found to depend on stimulation frequency and amplitude and to range from no block (less than 100 Hz) to a complete block (greater than 500 Hz). The transitional patterns include intermittent periodic block with interleaved spiking and non-spiking intervals of different relative duration as well as an unstable regime with chaotic switching between the spiking and non-spiking states. Intermittent conduction blocks are accompanied by oscillations of extracellular potassium. The mechanism of conductance block based on myelin restructuring complements the already known and modelled block via hyperpolarization mediated by the axonal sodium pump and potassium depolarization.

  5. Effect of potassium supply on drought resistance in sorghum: plant growth and macronutrient content

    International Nuclear Information System (INIS)

    Asgharipour, M.R.; Heidari, M.

    2011-01-01

    Nowadays, the main limiting natural resource is widely considered to be water. Therefore, research into crop management practices that enhance drought resistance and plant growth when water supply is limited has become increasingly essential. This study was conducted to evaluate the effect of potassium (K) nutritional status on the drought resistance of grain sorghum during 2009. Drought stress by reducing the yield components, especially the number of panicle per plant and one-hundred grain weight reduced grain yield and greatest yield (3499 kg ha/sup -1/) obtained at full irrigation. Potassium sulfate increased grain and biological yield by 28% and 22%, respectively compared to control through improving growth conditions. Drought stress increased the N content, while reduced water availability decreased the K and Na in plant. No K fertilized plants had the lowest leaf K and N and highest Na concentrations. Chlorophyll content increased significantly with increase in K supply and increased frequency of irrigation. Interaction effect of drought stress and potassium sulfate on all studied traits except chlorophyll content was significant and optimum soil K levels protects plants from drought. These observations indicate that adequate K nutrition can improve drought resistance of sorghum. (author)

  6. Gag mutations strongly contribute to HIV-1 resistance to protease inhibitors in highly drug-experienced patients besides compensating for fitness loss.

    Directory of Open Access Journals (Sweden)

    Elisabeth Dam

    2009-03-01

    Full Text Available Human immunodeficiency virus type 1 (HIV-1 resistance to protease inhibitors (PI results from mutations in the viral protease (PR that reduce PI binding but also decrease viral replicative capacity (RC. Additional mutations compensating for the RC loss subsequently accumulate within PR and in Gag substrate cleavage sites. We examined the respective contribution of mutations in PR and Gag to PI resistance and RC and their interdependence using a panel of HIV-1 molecular clones carrying different sequences from six patients who had failed multiple lines of treatment. Mutations in Gag strongly and directly contributed to PI resistance besides compensating for fitness loss. This effect was essentially carried by the C-terminal region of Gag (containing NC-SP2-p6 with little or no contribution from MA, CA, and SP1. The effect of Gag on resistance depended on the presence of cleavage site mutations A431V or I437V in NC-SP2-p6 and correlated with processing of the NC/SP2 cleavage site. By contrast, reverting the A431V or I437V mutation in these highly evolved sequences had little effect on RC. Mutations in the NC-SP2-p6 region of Gag can be dually selected as compensatory and as direct PI resistance mutations, with cleavage at the NC-SP2 site behaving as a rate-limiting step in PI resistance. Further compensatory mutations render viral RC independent of the A431V or I437V mutations while their effect on resistance persists.

  7. ERK mutations confer resistance to mitogen-activated protein kinase pathway inhibitors.

    Science.gov (United States)

    Goetz, Eva M; Ghandi, Mahmoud; Treacy, Daniel J; Wagle, Nikhil; Garraway, Levi A

    2014-12-01

    The use of targeted therapeutics directed against BRAF(V600)-mutant metastatic melanoma improves progression-free survival in many patients; however, acquired drug resistance remains a major medical challenge. By far, the most common clinical resistance mechanism involves reactivation of the MAPK (RAF/MEK/ERK) pathway by a variety of mechanisms. Thus, targeting ERK itself has emerged as an attractive therapeutic concept, and several ERK inhibitors have entered clinical trials. We sought to preemptively determine mutations in ERK1/2 that confer resistance to either ERK inhibitors or combined RAF/MEK inhibition in BRAF(V600)-mutant melanoma. Using a random mutagenesis screen, we identified multiple point mutations in ERK1 (MAPK3) and ERK2 (MAPK1) that could confer resistance to ERK or RAF/MEK inhibitors. ERK inhibitor-resistant alleles were sensitive to RAF/MEK inhibitors and vice versa, suggesting that the future development of alternating RAF/MEK and ERK inhibitor regimens might help circumvent resistance to these agents. ©2014 American Association for Cancer Research.

  8. Naturally occurring Vpr inhibitors from medicinal plants of Myanmar.

    Science.gov (United States)

    Win, Nwet Nwet; Ngwe, Hla; Abe, Ikuro; Morita, Hiroyuki

    2017-10-01

    Human immunodeficiency virus type-1 (HIV-1) is a lentiviral family member that encodes the retroviral Gag, Pol, and Env proteins, along with six additional accessory proteins, Tat, Rev, Vpu, Vif, Nef, and Vpr. The currently approved anti-HIV drugs target the Pol and Env encoded proteins. However, these drugs are only effective in reducing viral replication. Furthermore, the drugs' toxicities and the emergence of drug-resistant strains have become serious worldwide problems. Resistance eventually arises to all of the approved anti-HIV drugs, including the newly approved drugs that target HIV integrase (IN). Drug resistance likely emerges because of spontaneous mutations that occur during viral replication. Therefore, new drugs that effectively block other viral components must be developed to reduce the rate of resistance and suppress viral replication with little or no long-term toxicity. The accessory proteins may expand treatment options. Viral protein R (Vpr) is one of the promising drug targets among the HIV accessory proteins. However, the search for inhibitors continues in anti-HIV drug discovery. In this review, we summarize the naturally occurring compounds discovered from two Myanmar medicinal plants as well as their structure-activity relationships. A total of 49 secondary metabolites were isolated from Kaempferia pulchra rhizomes and Picrasama javanica bark, and the types of compounds were identified as isopimarane diterpenoids and picrasane quassinoids, respectively. Among the isolates, 7 diterpenoids and 15 quassinoids were found to be Vpr inhibitors lacking detectable toxicity, and their potencies varied according to their respective functionalities.

  9. Inhibitors of the Hepatitis C Virus Polymerase; Mode of Action and Resistance.

    Science.gov (United States)

    Eltahla, Auda A; Luciani, Fabio; White, Peter A; Lloyd, Andrew R; Bull, Rowena A

    2015-09-29

    The hepatitis C virus (HCV) is a pandemic human pathogen posing a substantial health and economic burden in both developing and developed countries. Controlling the spread of HCV through behavioural prevention strategies has met with limited success and vaccine development remains slow. The development of antiviral therapeutic agents has also been challenging, primarily due to the lack of efficient cell culture and animal models for all HCV genotypes, as well as the large genetic diversity between HCV strains. On the other hand, the use of interferon-α-based treatments in combination with the guanosine analogue, ribavirin, achieved limited success, and widespread use of these therapies has been hampered by prevalent side effects. For more than a decade, the HCV RNA-dependent RNA polymerase (RdRp) has been targeted for antiviral development, and direct-acting antivirals (DAA) have been identified which bind to one of at least six RdRp inhibitor-binding sites, and are now becoming a mainstay of highly effective and well tolerated antiviral treatment for HCV infection. Here we review the different classes of RdRp inhibitors and their mode of action against HCV. Furthermore, the mechanism of antiviral resistance to each class is described, including naturally occurring resistance-associated variants (RAVs) in different viral strains and genotypes. Finally, we review the impact of these RAVs on treatment outcomes with the newly developed regimens.

  10. Mechanisms of Hepatitis C Viral Resistance to Direct Acting Antivirals

    Directory of Open Access Journals (Sweden)

    Asma Ahmed

    2015-12-01

    Full Text Available There has been a remarkable transformation in the treatment of chronic hepatitis C in recent years with the development of direct acting antiviral agents targeting virus encoded proteins important for viral replication including NS3/4A, NS5A and NS5B. These agents have shown high sustained viral response (SVR rates of more than 90% in phase 2 and phase 3 clinical trials; however, this is slightly lower in real-life cohorts. Hepatitis C virus resistant variants are seen in most patients who do not achieve SVR due to selection and outgrowth of resistant hepatitis C virus variants within a given host. These resistance associated mutations depend on the class of direct-acting antiviral drugs used and also vary between hepatitis C virus genotypes and subtypes. The understanding of these mutations has a clear clinical implication in terms of choice and combination of drugs used. In this review, we describe mechanism of action of currently available drugs and summarize clinically relevant resistance data.

  11. Development of elvitegravir resistance and linkage of integrase inhibitor mutations with protease and reverse transcriptase resistance mutations.

    Directory of Open Access Journals (Sweden)

    Mark A Winters

    Full Text Available Failure of antiretroviral regimens containing elvitegravir (EVG and raltegravir (RAL can result in the appearance of integrase inhibitor (INI drug-resistance mutations (DRMs. While several INI DRMs have been identified, the evolution of EVG DRMs and the linkage of these DRMs with protease inhibitor (PI and reverse transcriptase inhibitor (RTI DRMs have not been studied at the clonal level. We examined the development of INI DRMs in 10 patients failing EVG-containing regimens over time, and the linkage of INI DRMs with PI and RTI DRMs in these patients plus 6 RAL-treated patients. A one-step RT-nested PCR protocol was used to generate a 2.7 kB amplicon that included the PR, RT, and IN coding region, and standard cloning and sequencing techniques were used to determine DRMs in 1,277 clones (mean 21 clones per time point. Results showed all patients had multiple PI, NRTI, and/or NNRTI DRMs at baseline, but no primary INI DRM. EVG-treated patients developed from 2 to 6 strains with different primary INI DRMs as early as 2 weeks after initiation of treatment, predominantly as single mutations. The prevalence of these strains fluctuated and new strains, and/or strains with new combinations of INI DRMs, developed over time. Final failure samples (weeks 14 to 48 typically showed a dominant strain with multiple mutations or N155H alone. Single N155H or multiple mutations were also observed in RAL-treated patients at virologic failure. All patient strains showed evidence of INI DRM co-located with single or multiple PI and/or RTI DRMs on the same viral strand. Our study shows that EVG treatment can select for a number of distinct INI-resistant strains whose prevalence fluctuates over time. Continued appearance of new INI DRMs after initial INI failure suggests a potent, highly dynamic selection of INI resistant strains that is unaffected by co-location with PI and RTI DRMs.

  12. PRACTICE OF USING VIRAL PROTEASE INHIBITORS IN CHILDREN WITH HIV INFECTION

    Directory of Open Access Journals (Sweden)

    V.B. Denisenko

    2010-01-01

    Full Text Available Selection of the most effective and safest high-active antiretroviral therapies is a critical issue faced by modern HIV medicine. Authors studied 28 children with HIV infection aged from 3 to 7 divided into two groups administered a combination of two HIV reverse transcriptase nucleoside inhibitors with viral protease nelfinavir inhibitors (n = 13 and lopinavir/ritonavir (n = 15. The subjects in both groups demonstrated a decreased frequency of HIV-associated symptoms and opportunistic infections, positive dynamics of immunological indicators, suppression of HIV replication. When lopinavir/ritonavir was administered, there was more even better dynamics in clinical, immunological and virologic parameters, which allows this medication to be recommended as a antiretroviral therapy for children. Key words: HIV infection, lopinavir/ritonavir, nelfinavir, children. (Pediatric Pharmacology. – 2010; 7(1:62-67

  13. Inhibition of herpes simplex virus type 1 entry by chloride channel inhibitors tamoxifen and NPPB

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Kai [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); College of Life Science and Technology, Jinan University, Guangzhou (China); Chen, Maoyun [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); College of pharmacy, Jinan University, Guangzhou (China); Xiang, Yangfei; Ma, Kaiqi [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); Jin, Fujun [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); College of pharmacy, Jinan University, Guangzhou (China); Wang, Xiao [School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006 (China); Wang, Xiaoyan; Wang, Shaoxiang [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); Wang, Yifei, E-mail: twang-yf@163.com [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China)

    2014-04-18

    Highlights: • We analyze the anti-HSV potential of chloride channel inhibitors. • Tamoxifen and NPPB show anti-HSV-1 and anti-ACV-resistant HSV-1 activities. • HSV-1 infection induces intracellular chloride concentration increasing. • Tamoxifen and NPPB inhibit HSV-1 early infection. • Tamoxifen and NPPB prevent the fusion process of HSV-1. - Abstract: Herpes simplex virus type 1 (HSV-1) infection is very common worldwide and can cause significant health problems from periodic skin and corneal lesions to encephalitis. Appearance of drug-resistant viruses in clinical therapy has made exploring novel antiviral agents emergent. Here we show that chloride channel inhibitors, including tamoxifen and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB), exhibited extensive antiviral activities toward HSV-1 and ACV-resistant HSV viruses. HSV-1 infection induced chloride ion influx while treatment with inhibitors reduced the increase of intracellular chloride ion concentration. Pretreatment or treatment of inhibitors at different time points during HSV-1 infection all suppressed viral RNA synthesis, protein expression and virus production. More detailed studies demonstrated that tamoxifen and NPPB acted as potent inhibitors of HSV-1 early entry step by preventing viral binding, penetration and nuclear translocation. Specifically the compounds appeared to affect viral fusion process by inhibiting virus binding to lipid rafts and interrupting calcium homeostasis. Taken together, the observation that tamoxifen and NPPB can block viral entry suggests a stronger potential for these compounds as well as other ion channel inhibitors in antiviral therapy against HSV-1, especially the compound tamoxifen is an immediately actionable drug that can be reused for treatment of HSV-1 infections.

  14. Inhibition of herpes simplex virus type 1 entry by chloride channel inhibitors tamoxifen and NPPB

    International Nuclear Information System (INIS)

    Zheng, Kai; Chen, Maoyun; Xiang, Yangfei; Ma, Kaiqi; Jin, Fujun; Wang, Xiao; Wang, Xiaoyan; Wang, Shaoxiang; Wang, Yifei

    2014-01-01

    Highlights: • We analyze the anti-HSV potential of chloride channel inhibitors. • Tamoxifen and NPPB show anti-HSV-1 and anti-ACV-resistant HSV-1 activities. • HSV-1 infection induces intracellular chloride concentration increasing. • Tamoxifen and NPPB inhibit HSV-1 early infection. • Tamoxifen and NPPB prevent the fusion process of HSV-1. - Abstract: Herpes simplex virus type 1 (HSV-1) infection is very common worldwide and can cause significant health problems from periodic skin and corneal lesions to encephalitis. Appearance of drug-resistant viruses in clinical therapy has made exploring novel antiviral agents emergent. Here we show that chloride channel inhibitors, including tamoxifen and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB), exhibited extensive antiviral activities toward HSV-1 and ACV-resistant HSV viruses. HSV-1 infection induced chloride ion influx while treatment with inhibitors reduced the increase of intracellular chloride ion concentration. Pretreatment or treatment of inhibitors at different time points during HSV-1 infection all suppressed viral RNA synthesis, protein expression and virus production. More detailed studies demonstrated that tamoxifen and NPPB acted as potent inhibitors of HSV-1 early entry step by preventing viral binding, penetration and nuclear translocation. Specifically the compounds appeared to affect viral fusion process by inhibiting virus binding to lipid rafts and interrupting calcium homeostasis. Taken together, the observation that tamoxifen and NPPB can block viral entry suggests a stronger potential for these compounds as well as other ion channel inhibitors in antiviral therapy against HSV-1, especially the compound tamoxifen is an immediately actionable drug that can be reused for treatment of HSV-1 infections

  15. Multi-step inhibition explains HIV-1 protease inhibitor pharmacodynamics and resistance

    Science.gov (United States)

    Rabi, S. Alireza; Laird, Gregory M.; Durand, Christine M.; Laskey, Sarah; Shan, Liang; Bailey, Justin R.; Chioma, Stanley; Moore, Richard D.; Siliciano, Robert F.

    2013-01-01

    HIV-1 protease inhibitors (PIs) are among the most effective antiretroviral drugs. They are characterized by highly cooperative dose-response curves that are not explained by current pharmacodynamic theory. An unresolved problem affecting the clinical use of PIs is that patients who fail PI-containing regimens often have virus that lacks protease mutations, in apparent violation of fundamental evolutionary theory. Here, we show that these unresolved issues can be explained through analysis of the effects of PIs on distinct steps in the viral life cycle. We found that PIs do not affect virion release from infected cells but block entry, reverse transcription, and post–reverse transcription steps. The overall dose-response curves could be reconstructed by combining the curves for each step using the Bliss independence principle, showing that independent inhibition of multiple distinct steps in the life cycle generates the highly cooperative dose-response curves that make these drugs uniquely effective. Approximately half of the inhibitory potential of PIs is manifest at the entry step, likely reflecting interactions between the uncleaved Gag and the cytoplasmic tail (CT) of the Env protein. Sequence changes in the CT alone, which are ignored in current clinical tests for PI resistance, conferred PI resistance, providing an explanation for PI failure without resistance. PMID:23979165

  16. Carbohydrate-Based Ice Recrystallization Inhibitors Increase Infectivity and Thermostability of Viral Vectors

    Science.gov (United States)

    Ghobadloo, Shahrokh M.; Balcerzak, Anna K.; Gargaun, Ana; Muharemagic, Darija; Mironov, Gleb G.; Capicciotti, Chantelle J.; Briard, Jennie G.; Ben, Robert N.; Berezovski, Maxim V.

    2014-07-01

    The inability of vaccines to retain sufficient thermostability has been an obstacle to global vaccination programs. To address this major limitation, we utilized carbohydrate-based ice recrystallization inhibitors (IRIs) to eliminate the cold chain and stabilize the potency of Vaccinia virus (VV), Vesicular Stomatitis virus (VSV) and Herpes virus-1 (HSV-1). The impact of these IRIs was tested on the potency of the viral vectors using a plaque forming unit assay following room temperature storage, cryopreservation with successive freeze-thaw cycles and lyophilization. Viral potency after storage with all three conditions demonstrated that N-octyl-gluconamide (NOGlc) recovered the infectivity of shelf stored VV, 5.6 Log10 PFU mL-1 during 40 days, and HSV-1, 2.7 Log10 PFU mL-1 during 9 days. Carbon-linked antifreeze glycoprotein analogue ornithine-glycine-glycine-galactose (OGG-Gal) increases the recovery of VV and VSV more than 1 Log10 PFU mL-1 after 10 freeze-thaw cycles. In VSV, cryostorage with OGG-Gal maintains high infectivity and reduces temperature-induced aggregation of viral particles by 2 times that of the control. In total, OGG-Gal and NOGlc preserve virus potency during cryostorage. Remarkably, NOGlc has potential to eliminate the cold chain and permit room temperature storage of viral vectors.

  17. Canine osteosarcoma cells exhibit resistance to aurora kinase inhibitors.

    Science.gov (United States)

    Cannon, C M; Pozniak, J; Scott, M C; Ito, D; Gorden, B H; Graef, A J; Modiano, J F

    2015-03-01

    We evaluated the effect of Aurora kinase inhibitors AZD1152 and VX680 on canine osteosarcoma cells. Cytotoxicity was seen in all four cell lines; however, half-maximal inhibitory concentrations were significantly higher than in human leukaemia and canine lymphoma cells. AZD1152 reduced Aurora kinase B phosphorylation, indicating resistance was not because of failure of target recognition. Efflux mediated by ABCB1 and ABCG2 transporters is one known mechanism of resistance against these drugs and verapamil enhanced AZD1152-induced apoptosis; however, these transporters were only expressed by a small percentage of cells in each line and the effects of verapamil were modest, suggesting other mechanisms contribute to resistance. Our results indicate that canine osteosarcoma cells are resistant to Aurora kinase inhibitors and suggest that these compounds are unlikely to be useful as single agents for this disease. Further investigation of these resistance mechanisms and the potential utility of Aurora kinase inhibitors in multi-agent protocols is warranted. © 2013 Blackwell Publishing Ltd.

  18. Variability and resistance mutations in the hepatitis C virus NS3 protease in patients not treated with protease inhibitors

    Directory of Open Access Journals (Sweden)

    Luciana Bonome Zeminian

    2013-02-01

    Full Text Available The goal of treatment of chronic hepatitis C is to achieve a sustained virological response, which is defined as exhibiting undetectable hepatitis C virus (HCV RNA levels in serum following therapy for at least six months. However, the current treatment is only effective in 50% of patients infected with HCV genotype 1, the most prevalent genotype in Brazil. Inhibitors of the serine protease non-structural protein 3 (NS3 have therefore been developed to improve the responses of HCV-infected patients. However, the emergence of drug-resistant variants has been the major obstacle to therapeutic success. The goal of this study was to evaluate the presence of resistance mutations and genetic polymorphisms in the NS3 genomic region of HCV from 37 patients infected with HCV genotype 1 had not been treated with protease inhibitors. Plasma viral RNA was used to amplify and sequence the HCV NS3 gene. The results indicate that the catalytic triad is conserved. A large number of substitutions were observed in codons 153, 40 and 91; the resistant variants T54A, T54S, V55A, R155K and A156T were also detected. This study shows that resistance mutations and genetic polymorphisms are present in the NS3 region of HCV in patients who have not been treated with protease inhibitors, data that are important in determining the efficiency of this new class of drugs in Brazil.

  19. Detecting and treating breast cancer resistance to EGFR inhibitors

    Science.gov (United States)

    Moonlee, Sun-Young; Bissell, Mina J.; Furuta, Saori; Meier, Roland; Kenny, Paraic A.

    2016-04-05

    The application describes therapeutic compositions and methods for treating cancer. For example, therapeutic compositions and methods related to inhibition of FAM83A (family with sequence similarity 83) are provided. The application also describes methods for diagnosing cancer resistance to EGFR inhibitors. For example, a method of diagnosing cancer resistance to EGFR inhibitors by detecting increased FAM83A levels is described.

  20. Resistance to BET Inhibitor Leads to Alternative Therapeutic Vulnerabilities in Castration-Resistant Prostate Cancer.

    Science.gov (United States)

    Pawar, Aishwarya; Gollavilli, Paradesi Naidu; Wang, Shaomeng; Asangani, Irfan A

    2018-02-27

    BRD4 plays a major role in the transcription networks orchestrated by androgen receptor (AR) in castration-resistant prostate cancer (CRPC). Several BET inhibitors (BETi) that displace BRD4 from chromatin are being evaluated in clinical trials for CRPC. Here, we describe mechanisms of acquired resistance to BETi that are amenable to targeted therapies in CRPC. BETi-resistant CRPC cells displayed cross-resistance to a variety of BETi in the absence of gatekeeper mutations, exhibited reduced chromatin-bound BRD4, and were less sensitive to BRD4 degraders/knockdown, suggesting a BRD4-independent transcription program. Transcriptomic analysis revealed reactivation of AR signaling due to CDK9-mediated phosphorylation of AR, resulting in sensitivity to CDK9 inhibitors and enzalutamide. Additionally, increased DNA damage associated with PRC2-mediated transcriptional silencing of DDR genes was observed, leading to PARP inhibitor sensitivity. Collectively, our results identify the therapeutic limitation of BETi as a monotherapy; however, our BETi resistance data suggest unique opportunities for combination therapies in treating CRPC. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  1. Role of the K101E substitution in HIV-1 reverse transcriptase in resistance to rilpivirine and other nonnucleoside reverse transcriptase inhibitors.

    Science.gov (United States)

    Xu, Hong-Tao; Colby-Germinario, Susan P; Huang, Wei; Oliveira, Maureen; Han, Yingshan; Quan, Yudong; Petropoulos, Christos J; Wainberg, Mark A

    2013-11-01

    Resistance to the recently approved nonnucleoside reverse transcriptase inhibitor (NNRTI) rilpivirine (RPV) commonly involves substitutions at positions E138K and K101E in HIV-1 reverse transcriptase (RT), together with an M184I substitution that is associated with resistance to coutilized emtricitabine (FTC). Previous biochemical and virological studies have shown that compensatory interactions between substitutions E138K and M184I can restore enzyme processivity and the viral replication capacity. Structural modeling studies have also shown that disruption of the salt bridge between K101 and E138 can affect RPV binding. The current study was designed to investigate the impact of K101E, alone or in combination with E138K and/or M184I, on drug susceptibility, viral replication capacity, and enzyme function. We show here that K101E can be selected in cell culture by the NNRTIs etravirine (ETR), efavirenz (EFV), and dapivirine (DPV) as well as by RPV. Recombinant RT enzymes and viruses containing K101E, but not E138K, were highly resistant to nevirapine (NVP) and delavirdine (DLV) as well as ETR and RPV, but not EFV. The addition of K101E to E138K slightly enhanced ETR and RPV resistance compared to that obtained with E138K alone but restored susceptibility to NVP and DLV. The K101E substitution can compensate for deficits in viral replication capacity and enzyme processivity associated with M184I, while M184I can compensate for the diminished efficiency of DNA polymerization associated with K101E. The coexistence of K101E and E138K does not impair either viral replication or enzyme fitness. We conclude that K101E can play a significant role in resistance to RPV.

  2. Protease Inhibitors Drug Resistance Mutations in Turkish Patients with Chronic Hepatitis C.

    Science.gov (United States)

    Sargin Altunok, Elif; Sayan, Murat; Akhan, Sila; Aygen, Bilgehan; Yildiz, Orhan; Tekin Koruk, Suda; Mistik, Resit; Demirturk, Nese; Ural, Onur; Kose, Şükran; Aynioglu, Aynur; Korkmaz, Fatime; Ersoz, Gülden; Tuna, Nazan; Ayaz, Celal; Karakecili, Faruk; Keten, Derya; Inan, Dilara; Yazici, Saadet; Koculu, Safiye; Yildirmak, Taner

    2016-09-01

    Drug resistance development is an expected problem during treatment with protease inhibitors (PIs), this is largely due to the fact that Pls are low-genetic barrier drugs. Resistance-associated variants (RAVs) however may also occur naturally, and prior to treatment with Pls, the clinical impact of this basal resistance remains unknown. In Turkey, there is yet to be an investigation into the hepatitis C (HCV) drug associated resistance to oral antivirals. 178 antiviral-naïve patients infected with HCV genotype 1 were selected from 27 clinical centers of various geographical regions in Turkey and included in the current study. The basal NS3 Pls resistance mutations of these patients were analyzed. In 33 (18.5%) of the patients included in the study, at least one mutation pattern that can cause drug resistance was identified. The most frequently detected mutation pattern was T54S while R109K was the second most frequently detected. Following a more general examination of the patients studied, telaprevir (TVR) resistance in 27 patients (15.2%), boceprevir (BOC) resistance in 26 (14.6%) patients, simeprevir (SMV) resistance in 11 (6.2%) patients and faldaprevir resistance in 13 (7.3%) patients were detected. Our investigation also revealed that rebound developed in the presence of a Q80K mutation and amongst two V55A mutations following treatment with TVR, while no response to treatment was detected in a patient with a R55K mutation. We are of the opinion that drug resistance analyses can be beneficial and necessary in revealing which variants are responsible for pre-treatment natural resistance and which mutations are responsible for the viral breakthrough that may develop during the treatment. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  3. Intracellular mediators of potassium-induced aldosterone secretion

    International Nuclear Information System (INIS)

    Ganguly, A.; Chiou, S.; Davis, J.S.

    1990-01-01

    We have investigated the intracellular messengers of potassium in eliciting aldosterone secretion in calf adrenal glomerulosa cells since there were unresolved issues relating to the role of phosphoinositides, cAMP and protein kinases. We observed no evidence of hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) in 3 H-inositol labeled alf adrenal cells or increase of cAMP in response to potassium. Addition of calcium channel blocker, nitrendipine after stimulating adrenal glomerulosa cells with potassium, markedly inhibited aldosterone secretion. A calmodulin inhibitor (W-7) produced greater reduction of aldosterone secretion than an inhibitor of protein kinase C (H-7). These results suggest that a rise in cytosolic free calcium concentration through voltage-dependent calcium channel and calmodulin are the critical determinants of aldosterone secretion stimulated by potassium

  4. Innate resistance of New Zealand paua to abalone viral ganglioneuritis.

    Science.gov (United States)

    Corbeil, Serge; McColl, Kenneth A; Williams, Lynette M; Slater, Joanne; Crane, Mark St J

    2017-06-01

    The susceptibility of New Zealand paua (Haliotis iris) to infection by abalone herpesvirus (Haliotid herpesvirus 1; HaHV) and to the disease abalone viral ganglioneuritis (AVG) was determined. Infection challenges performed by intra-muscular injection and by immersion in infectious water containing HaHV demonstrated that New Zealand paua were highly resistant to infection by Haliotid herpesvirus 1 and were fully resistant to the disease AVG. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Potent host-directed small-molecule inhibitors of myxovirus RNA-dependent RNA-polymerases.

    Directory of Open Access Journals (Sweden)

    Stefanie A Krumm

    Full Text Available Therapeutic targeting of host cell factors required for virus replication rather than of pathogen components opens new perspectives to counteract virus infections. Anticipated advantages of this approach include a heightened barrier against the development of viral resistance and a broadened pathogen target spectrum. Myxoviruses are predominantly associated with acute disease and thus are particularly attractive for this approach since treatment time can be kept limited. To identify inhibitor candidates, we have analyzed hit compounds that emerged from a large-scale high-throughput screen for their ability to block replication of members of both the orthomyxovirus and paramyxovirus families. This has returned a compound class with broad anti-viral activity including potent inhibition of different influenza virus and paramyxovirus strains. After hit-to-lead chemistry, inhibitory concentrations are in the nanomolar range in the context of immortalized cell lines and human PBMCs. The compound shows high metabolic stability when exposed to human S-9 hepatocyte subcellular fractions. Antiviral activity is host-cell species specific and most pronounced in cells of higher mammalian origin, supporting a host-cell target. While the compound induces a temporary cell cycle arrest, host mRNA and protein biosynthesis are largely unaffected and treated cells maintain full metabolic activity. Viral replication is blocked at a post-entry step and resembles the inhibition profile of a known inhibitor of viral RNA-dependent RNA-polymerase (RdRp activity. Direct assessment of RdRp activity in the presence of the reagent reveals strong inhibition both in the context of viral infection and in reporter-based minireplicon assays. In toto, we have identified a compound class with broad viral target range that blocks host factors required for viral RdRp activity. Viral adaptation attempts did not induce resistance after prolonged exposure, in contrast to rapid

  6. Phosphatidic acid accumulation and catecholamine release in adrenal chromaffin cells: stimulation by high potassium and by nicotine, and effect of a diacylglycerol kinase inhibitor R 59 022.

    Science.gov (United States)

    Owen, P J; Jones, J A; Boarder, M R

    1991-09-01

    Using primary cultures of bovine adrenal chromaffin cells labelled with 32Pi, we show that stimulation with bradykinin, nicotine, or a depolarising concentration of potassium stimulates the accumulation of [32P]phosphatidic acid. The effects of nicotine and potassium are smaller than the effect of bradykinin, and are dependent entirely on extracellular calcium. The diacylglycerol kinase inhibitor R 59 022 attenuates the formation of phosphatidic acid by nicotine and depolarising concentrations of potassium. This inhibitor also blocks the nicotine and potassium stimulation of noradrenaline release from chromaffin cells. Using 45Ca2+ influx studies, we show that the nicotine-evoked calcium influx is also attenuated by R 59 022. These observations contrast with those in another report in which we showed that bradykinin stimulation of either [32P]phosphatidic acid accumulation or noradrenaline release is not affected by R 59 022. It is likely that the calcium influx produced by nicotine and depolarising potassium is blocked by R 59 022 by a mechanism that is independent of its ability to block diacylglycerol kinase. The nicotine- and potassium-stimulated [32P]phosphatidic acid accumulation is a consequence of this calcium influx and presumably reflects calcium activation of either phospholipase C or phospholipase D.

  7. [Determination of drug resistance mutations of NS3 inhibitors in chronic hepatitis C patients infected with genotype 1].

    Science.gov (United States)

    Şanlıdağ, Tamer; Sayan, Murat; Akçalı, Sinem; Kasap, Elmas; Buran, Tahir; Arıkan, Ayşe

    2017-04-01

    Direct-acting antiviral agents (DAA) such as NS3 protease inhibitors is the first class of drugs used for chronic hepatitis C (CHC) treatment. NS3 inhibitors (PI) with low genetic barrier have been approved to be used in the CHC genotype 1 infections, and in the treatment of compensated liver disease including cirrhosis together with pegile interferon and ribavirin. Consequently, the development of drug resistance during DAA treatment of CHC is a major problem. NS3 resistant variants can be detected before treatment as they can occurnaturally. The aim of this study was to investigate new and old generation NS3 inhibitors resistance mutations before DAA treatment in hepatitis C virus (HCV) that were isolated from CHC. The present study was conducted in 2015 and included 97 naive DAA patients infected with HCV genotype 1, who were diagnosed in Manisa and Kocaeli cities of Turkey. Magnetic particle based HCV RNA extraction and than RNA detection and quantification were performed using commercial real-time PCR assay QIASypmhony + Rotorgene Q/ArtusHCV QS-RGQ and COBAS Ampliprep/COBAS TaqMan HCV Tests. HCV NS3 viral protease genome region was amplified with PCR and mutation analysis was performed by Sanger dideoxy sequencing technique of NS3 protease codons (codon 32-185). HCV NS3 protease inhibitors; asunaprevir, boceprevir, faldaprevir, grazoprevir, pariteprevir, simeprevir and telaprevir were analysed for resistant mutations by Geno2pheno-HCV resistance tool. HCV was genotyped in all patients and 88 patients (n= 88/97, 91%) had genotype 1. Eight (n= 8/97, 8.2%) and 80 (n= 80/97, 82.4%) HCC patients were subgenotyped as 1a and 1b, respectively. Many aminoacid substitutions and resistance mutations were determined in 39/88 (44%) patients in the study group. Q80L, S122C/N, S138W were defined as potential substitutions (6/88 patients; 7%); R109K, R117C, S122G, I132V, I170V, N174S were described as potential resistance (34/88 patients; 39%); V36L, T54S, V55A, Q80H were

  8. Discovery of Dengue Virus NS4B Inhibitors

    Science.gov (United States)

    Wang, Qing-Yin; Dong, Hongping; Zou, Bin; Karuna, Ratna; Wan, Kah Fei; Zou, Jing; Susila, Agatha; Yip, Andy; Shan, Chao; Yeo, Kim Long; Xu, Haoying; Ding, Mei; Chan, Wai Ling; Gu, Feng; Seah, Peck Gee; Liu, Wei; Lakshminarayana, Suresh B.; Kang, CongBao; Lescar, Julien; Blasco, Francesca; Smith, Paul W.

    2015-01-01

    ABSTRACT The four serotypes of dengue virus (DENV-1 to -4) represent the most prevalent mosquito-borne viral pathogens in humans. No clinically approved vaccine or antiviral is currently available for DENV. Here we report a spiropyrazolopyridone compound that potently inhibits DENV both in vitro and in vivo. The inhibitor was identified through screening of a 1.8-million-compound library by using a DENV-2 replicon assay. The compound selectively inhibits DENV-2 and -3 (50% effective concentration [EC50], 10 to 80 nM) but not DENV-1 and -4 (EC50, >20 μM). Resistance analysis showed that a mutation at amino acid 63 of DENV-2 NS4B (a nonenzymatic transmembrane protein and a component of the viral replication complex) could confer resistance to compound inhibition. Genetic studies demonstrate that variations at amino acid 63 of viral NS4B are responsible for the selective inhibition of DENV-2 and -3. Medicinal chemistry improved the physicochemical properties of the initial “hit” (compound 1), leading to compound 14a, which has good in vivo pharmacokinetics. Treatment of DENV-2-infected AG129 mice with compound 14a suppressed viremia, even when the treatment started after viral infection. The results have proven the concept that inhibitors of NS4B could potentially be developed for clinical treatment of DENV infection. Compound 14a represents a potential preclinical candidate for treatment of DENV-2- and -3-infected patients. IMPORTANCE Dengue virus (DENV) threatens up to 2.5 billion people and is now spreading in many regions in the world where it was not previously endemic. While there are several promising vaccine candidates in clinical trials, approved vaccines or antivirals are not yet available. Here we describe the identification and characterization of a spiropyrazolopyridone as a novel inhibitor of DENV by targeting the viral NS4B protein. The compound potently inhibits two of the four serotypes of DENV (DENV-2 and -3) both in vitro and in vivo. Our

  9. Resistência de Eleusine indica aos inibidores de ACCase Eleusine indica resistance to ACCase inhibitors

    Directory of Open Access Journals (Sweden)

    R.A. Vidal

    2006-01-01

    Full Text Available Dentre as causas da ineficácia no controle de plantas daninhas destaca-se a resistência delas aos herbicidas. Os objetivos deste trabalho foram avaliar a suspeita de resistência de Eleusine indica a inibidores de acetil-CoA carboxilase (ACCase e investigar a ocorrência de resistência cruzada entre os inibidores de ACCase. Biótipo de Eleusine indica originado do Mato Grosso com suspeita de resistência aos herbicidas inibidores de ACCase foi avaliado em casa de vegetação na sua suscetibilidade para diversos produtos do grupo dos ariloxifenoxipropionatos e cicloexanodionas. Estudos de resposta à dose confirmaram que o biótipo era 18 vezes mais insensível ao sethoxydim do que biótipo suscetível nunca aspergido com herbicidas. Também se constatou resistência cruzada ao fenoxaprop, cyhalofop, propaquizafop e butroxydim. Não se observou resistência cruzada aos produtos fluazifop, haloxyfop, quizalofop e clethodim.Among the causes for weed control inefficacy, the worst one is resistance to herbicides. The objectives of this work were to evaluate an Eleusine indica biotype suspected of resistance to ACCase inhibitors and to investigate the occurrence of cross- resistance to several ACCase inhibitors. One biotype of Eleusine indica originated from Mato Grosso with suspected resistance to ACCase inhibitors was evaluated in a greenhouse in relation to its susceptibility to several products of the ariloxyphenoxypropionate and cyclohexanedione groups. Studies on dose response confirmed that the suspected biotype was 18 times more insensitive to sethoxydim than the susceptible biotype that had never been treated with herbicides. Cross-resistance was confirmed for fenoxaprop, cyhalofop, propaquizafop and butroxydim. No cross-resistance was observed with fluazifop, haloxyfop, quizalofop, and clethodim.

  10. Chemotherapeutics-resistance "arms" race: An update on mechanisms involved in resistance limiting EGFR inhibitors in lung cancer.

    Science.gov (United States)

    Singh, Pankaj Kumar; Silakari, Om

    2017-10-01

    Clinical reports suggest that EGFR-mutated lung cancer usually respond significantly towards small molecule tyrosine kinase inhibitors. Same studies also report the eventual development of acquired resistance within a median time interval of 9 to 14months. One of the major mechanisms involved in this acquired resistance was found to be a secondary point mutation at gate-keeper residue, EGFR T790M. However, there are other recent studies which disclose the role of few other novel key players such as, ZEB1, TOPK etc., in the development of tolerance towards the EGFR TKI's, along with other commonly known mechanisms, such as amplification of signalling pathways such as, c-MET, Erbb2, AXL, additional acquired secondary mutations (PIK3CA, BRAF), or phenotypic transformation (small cell or epithelial to mesenchymal transitions). Interestingly, a recent study showed development of resistance via another point mutation, C797S, in case of tumors which were previously resistant and were administered agents capable of overcoming T790M gatekeeper mutation based resistance. Thus, raising serious concern over the direction of drug development involving tyrosine kinases such as EGFR. Current approaches focussing on development of third generation inhibitors, dual inhibitors or inhibitors of HSP90 have shown significant activity but do not answer the long term question of resistance. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. The Second-Generation Maturation Inhibitor GSK3532795 Maintains Potent Activity Toward HIV Protease Inhibitor-Resistant Clinical Isolates.

    Science.gov (United States)

    Ray, Neelanjana; Li, Tianbo; Lin, Zeyu; Protack, Tricia; van Ham, Petronella Maria; Hwang, Carey; Krystal, Mark; Nijhuis, Monique; Lataillade, Max; Dicker, Ira

    2017-05-01

    Protease inhibitor (PI)-resistant HIV-1 isolates with primary substitutions in protease (PR) and secondary substitutions in Gag could potentially exhibit cross-resistance to maturation inhibitors. We evaluated the second-generation maturation inhibitor, GSK3532795, for activity toward clinical isolates with genotypic and phenotypic characteristics associated with PI resistance (longitudinal). Longitudinal clinical isolates from 15 PI-treated patients and 7 highly PI-resistant (nonlongitudinal) viruses containing major and minor PI resistance-associated mutations were evaluated for GSK3532795 sensitivity. Phenotypic sensitivity was determined using the PhenoSense Gag/PR assay (Monogram Biosciences) or in-house single- and multiple-cycle assays. Changes from baseline [CFB; ratio of post- to pre-treatment FC-IC50 (fold-change in IC50 versus wild-type virus)] Monogram (11 patients)] and 1.5 (1.0-2.2) [single-cycle (4 patients)]. The 2 post-PI treatment samples showing GSK3532795 CFB >3 (Monogram) were retested using single- and multiple-cycle assays. Neither sample had meaningful sensitivity changes in the multiple-cycle assay. Gag changes were not associated with an increased GSK3532795 CFB. GSK3532795 maintained antiviral activity against PI-resistant isolates with emergent PR and/or Gag mutations. This finding supports continued development of GSK3532795 in treatment-experienced patients with or without previous PI therapy.

  12. Effect of pinacidil on norepinephrine- and potassium-induced contractions and membrane potential in rat and human resistance vessels and in rat aorta

    International Nuclear Information System (INIS)

    Videbaek, L.M.; Aalkjaer, C.; Mulvany, M.J.

    1988-01-01

    The effect of pinacidil on contractile responses to norepinephrine, potassium, and membrane potential was examined in rat and human resistance vessels. In some experiments rat aorta was also used. Pinacidil (0.1-30 microM) caused a concentration-dependent relaxation of norepinephrine-induced contractions in all vessels studied. In the same concentration range, pinacidil had only little effect on potassium (125 mM) activated rat mesenteric and femoral resistance vessels. In denervated rat mesenteric resistance vessels, a depolarization with potassium (125 mM) before superimposing a norepinephrine tone markedly diminished the effect of pinacidil. In resting rat mesenteric resistance vessels, pinacidil (1-10 microM) caused a hyperpolarization of 10-15 mV. In rat aorta, pinacidil (10 microM) caused a significant (p less than 0.001) increase in 86 Rb+ efflux rate constant whereas 1 microM had no effect. The results of these experiments indicate that the vasodilating effect may be caused by a hyperpolarization of the vascular smooth muscle cell membrane

  13. Prevalence of polymorphisms with significant resistance to NS5A inhibitors in treatment-naive patients with hepatitis C virus genotypes 1a and 3a in Sweden.

    Science.gov (United States)

    Lindström, Ida; Kjellin, Midori; Palanisamy, Navaneethan; Bondeson, Kåre; Wesslén, Lars; Lannergard, Anders; Lennerstrand, Johan

    2015-08-01

    The future treatment of hepatitis C virus (HCV) infection will be combinations of direct-acting antivirals (DAAs) that not only target multiple viral targets, but are also effective against different HCV genotypes. Of the many drug targets in HCV, one promising target is the non-structural 5A protein (NS5A), against which inhibitors, namely daclatasvir, ledipasvir and ombitasvir, have shown potent efficacy. However, since HCV is known to have very high sequence diversity, development of resistance is a problem against but not limited to NS5A inhibitors (i.e. resistance also found against NS3-protease and NS5B non-nucleoside inhibitors), when used in suboptimal combinations. Furthermore, it has been shown that natural resistance against DAAs is present in treatment-naïve patients and such baseline resistance will potentially complicate future treatment strategies. A pan-genotypic population-sequencing method with degenerated primers targeting the NS5A region was developed. We have investigated the prevalence of baseline resistant variants in 127 treatment-naïve patients of HCV genotypes 1a, 1b, 2b and 3a. The method could successfully sequence more than 95% of genotype 1a, 1b and 3a samples. Interpretation of fold resistance data against the NS5A inhibitors was done with the help of earlier published phenotypic data. Baseline resistance variants associated with high resistance (1000-50,000-fold) was found in three patients: Q30H or Y93N in genotype 1a patients and further Y93H in a genotype 3a patient. Using this method, baseline resistance can be examined and the data could have a potential role in selecting the optimal and cost-efficient treatment for the patient.

  14. Structural Mechanism of the Pan-BCR-ABL Inhibitor Ponatinib (AP24534): Lessons for Overcoming Kinase Inhibitor Resistance

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Tianjun; Commodore, Lois; Huang, Wei-Sheng; Wang, Yihan; Thomas, Mathew; Keats, Jeff; Xu, Qihong; Rivera, Victor M.; Shakespeare, William C.; Clackson, Tim; Dalgarno, David C.; Zhu, Xiaotian (ARIAD)

    2012-01-20

    The BCR-ABL inhibitor imatinib has revolutionized the treatment of chronic myeloid leukemia. However, drug resistance caused by kinase domain mutations has necessitated the development of new mutation-resistant inhibitors, most recently against the T315I gatekeeper residue mutation. Ponatinib (AP24534) inhibits both native and mutant BCR-ABL, including T315I, acting as a pan-BCR-ABL inhibitor. Here, we undertook a combined crystallographic and structure-activity relationship analysis on ponatinib to understand this unique profile. While the ethynyl linker is a key inhibitor functionality that interacts with the gatekeeper, virtually all other components of ponatinib play an essential role in its T315I inhibitory activity. The extensive network of optimized molecular contacts found in the DFG-out binding mode leads to high potency and renders binding less susceptible to disruption by single point mutations. The inhibitory mechanism exemplified by ponatinib may have broad relevance to designing inhibitors against other kinases with mutated gatekeeper residues.

  15. Potential mechanisms of resistance to microtubule inhibitors.

    Science.gov (United States)

    Kavallaris, Maria; Annereau, Jean-Philippe; Barret, Jean-Marc

    2008-06-01

    Antimitotic drugs targeting the microtubules, such as the taxanes and vinca alkaloids, are widely used in the treatment of neoplastic diseases. Development of drug resistance over time, however, limits the efficacy of these agents and poses a clinical challenge to long-term improvement of patient outcomes. Understanding the mechanism(s) of drug resistance becomes paramount to allowing for alternative, if not improved, therapeutic options that might circumvent this challenge. Vinflunine, a novel microtubule inhibitor, has shown superior preclinical antitumor activity, and displays a different pattern of resistance, compared with other agents in the vinca alkaloid class.

  16. Establishment and characterization of arsenic trioxide resistant KB/ATO cells.

    Science.gov (United States)

    Zhang, Yun-Kai; Dai, Chunling; Yuan, Chun-Gang; Wu, Hsiang-Chun; Xiao, Zhijie; Lei, Zi-Ning; Yang, Dong-Hua; Le, X Chris; Fu, Liwu; Chen, Zhe-Sheng

    2017-09-01

    Arsenic trioxide (ATO) is used as a chemotherapeutic agent for the treatment of acute promyelocytic leukemia. However, increasing drug resistance is reducing its efficacy. Therefore, a better understanding of ATO resistance mechanism is required. In this study, we established an ATO-resistant human epidermoid carcinoma cell line, KB/ATO, from its parental KB-3-1 cells. In addition to ATO, KB/ATO cells also exhibited cross-resistance to other anticancer drugs such as cisplatin, antimony potassium tartrate, and 6-mercaptopurine. The arsenic accumulation in KB/ATO cells was significantly lower than that in KB-3-1 cells. Further analysis indicated that neither application of P-glycoprotein inhibitor, breast cancer resistant protein (BCRP) inhibitor, or multidrug resistance protein 1 (MRP1) inhibitor could eliminate ATO resistance. We found that the expression level of ABCB6 was increased in KB/ATO cells. In conclusion, ABCB6 could be an important factor for ATO resistance in KB/ATO cells. The ABCB6 level may serve as a predictive biomarker for the effectiveness of ATO therapy.

  17. Ropivacaine-Induced Contraction Is Attenuated by Both Endothelial Nitric Oxide and Voltage-Dependent Potassium Channels in Isolated Rat Aortae

    Directory of Open Access Journals (Sweden)

    Seong-Ho Ok

    2013-01-01

    Full Text Available This study investigated endothelium-derived vasodilators and potassium channels involved in the modulation of ropivacaine-induced contraction. In endothelium-intact rat aortae, ropivacaine concentration-response curves were generated in the presence or absence of the following inhibitors: the nonspecific nitric oxide synthase (NOS inhibitor Nω-nitro-L-arginine methyl ester (L-NAME, the neuronal NOS inhibitor Nω-propyl-L-arginine hydrochloride, the inducible NOS inhibitor 1400W dihydrochloride, the nitric oxide-sensitive guanylyl cyclase (GC inhibitor ODQ, the NOS and GC inhibitor methylene blue, the phosphoinositide-3 kinase inhibitor wortmannin, the cytochrome p450 epoxygenase inhibitor fluconazole, the voltage-dependent potassium channel inhibitor 4-aminopyridine (4-AP, the calcium-activated potassium channel inhibitor tetraethylammonium (TEA, the inward-rectifying potassium channel inhibitor barium chloride, and the ATP-sensitive potassium channel inhibitor glibenclamide. The effect of ropivacaine on endothelial nitric oxide synthase (eNOS phosphorylation in human umbilical vein endothelial cells was examined by western blotting. Ropivacaine-induced contraction was weaker in endothelium-intact aortae than in endothelium-denuded aortae. L-NAME, ODQ, and methylene blue enhanced ropivacaine-induced contraction, whereas wortmannin, Nω-propyl-L-arginine hydrochloride, 1400W dihydrochloride, and fluconazole had no effect. 4-AP and TEA enhanced ropivacaine-induced contraction; however, barium chloride and glibenclamide had no effect. eNOS phosphorylation was induced by ropivacaine. These results suggest that ropivacaine-induced contraction is attenuated primarily by both endothelial nitric oxide and voltage-dependent potassium channels.

  18. Inhibition of protease-inhibitor resistant hepatitis C virus replicons and infectious virus by intracellular intrabodies

    Science.gov (United States)

    Gal-Tanamy, Meital; Zemel, Romy; Bachmatov, Larissa; Jangra, Rohit K.; Shapira, Assaf; Villanueva, Rodrigo; Yi, MinKyung; Lemon, Stanley M.; Benhar, Itai; Tur-Kaspa, Ran

    2015-01-01

    Hepatitis C virus (HCV) infection is a common cause of chronic liver disease and a serious threat to human health. The HCV NS3/4A serine protease is necessary for viral replication and innate immune evasion, and represents a well-validated target for specific antiviral therapy. We previously reported the isolation of single-chain antibodies (scFvs) that inhibit NS3/4A protease activity in vitro. Expressed intracellularly (intrabodies), these scFvs blocked NS3-mediated proliferation of NS3-transfected cells. Here we show that anti-NS3 scFvs suppress HCV RNA replication when expressed intracellularly in Huh7 hepatoma cells bearing either subgenomic or genome-length HCV RNA replicons. The expression of intrabodies directed against NS3 inhibited the autonomous amplification of HCV replicons resistant to small molecule inhibitors of the NS3/4A protease, and replicons derived from different HCV genotypes. The combination of intrabodies and interferon-α had an additive inhibitory effect on RNA replication in the replicon model. Intrabody expression also inhibited production of infectious HCV in a cell culture system. The NS3 protease activity was inhibited by the intrabodies in NS3-expressing cells. In contrast, cell-free synthesis of HCV RNA by preformed replicase complexes was not inhibited by intrabodies, suggesting that the major mode of inhibition of viral replication is inhibition of NS3/4A protease activity and subsequent suppression of viral polyprotein processing. PMID:20705106

  19. APPLICATIONS OF POTASSIUM FERTILIZER AND Bacillus sp. BIOPESTICIDE FOR INCREASING TOMATO RESISTANCE TO BACTERIAL WILT DISEASE

    Directory of Open Access Journals (Sweden)

    Nur Prihatiningsih

    2011-02-01

    Full Text Available Bacterial wilt on tomato caused by Ralstonia solanacearum is a crucial disease, because it can reduce yield until 50%. The aims of this research were: 1 to find out biopesticide formula for Bacillus sp.growth, 2 to test Bacillus sp. against R. solanacearum in vitro, 3 to test potassium fertilizer combined with Bacillus sp. for enhancing tomato resistance to the bacterial wilt disease. The research was conducted in 2 steps i.e to test the persistence of Bacillus sp. in biopesticide formula, and to test the best combination of both potassium and the Bacillus sp. biopesticide. The results showed that Bacillus B298 was the best isolate in its persistence on the biopesticide formula of organic growth medium+CaCO3+CMC 1%+mannitol 1%, and in inhibiting R. solanacearum. The best biopesticide formula for the Bacillus sp. persistence was growth organic media+ CaCO3+CMC 1%+mannitol 1%. Bacillus sp. was able to increase tomato resistance to the bacterial wilt disease from the category of susceptible to be tolerant and becoming resistant.

  20. Structure-Based Design of Novel HIV-1 Protease Inhibitors to Combat Drug Resistance

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh,A.; Sridhar, P.; Leshchenko, S.; Hussain, A.; Li, J.; Kovalevsky, A.; Walters, D.; Wedelind, J.; Grum-Tokars, V.; et al.

    2006-01-01

    Structure-based design and synthesis of novel HIV protease inhibitors are described. The inhibitors are designed specifically to interact with the backbone of HIV protease active site to combat drug resistance. Inhibitor 3 has exhibited exceedingly potent enzyme inhibitory and antiviral potency. Furthermore, this inhibitor maintains impressive potency against a wide spectrum of HIV including a variety of multi-PI-resistant clinical strains. The inhibitors incorporated a stereochemically defined 5-hexahydrocyclopenta[b]furanyl urethane as the P2-ligand into the (R)-(hydroxyethylamino)sulfonamide isostere. Optically active (3aS,5R,6aR)-5-hydroxy-hexahydrocyclopenta[b]furan was prepared by an enzymatic asymmetrization of meso-diacetate with acetyl cholinesterase, radical cyclization, and Lewis acid-catalyzed anomeric reduction as the key steps. A protein-ligand X-ray crystal structure of inhibitor 3-bound HIV-1 protease (1.35 Angstroms resolution) revealed extensive interactions in the HIV protease active site including strong hydrogen bonding interactions with the backbone. This design strategy may lead to novel inhibitors that can combat drug resistance.

  1. Viral phenotype, antiretroviral resistance and clinical evolution in human immunodeficiency virus-infected children.

    Science.gov (United States)

    Mellado, M J; Cilleruelo, M J; Ortiz, M; Villota, J; García, M; Perez-Jurado, M L; Barreiro, G; Martín-Fontelos, P; Bernal, A

    1997-11-01

    The syncytium-inducing (SI) viral phenotype and the emergence of viral strains resistant to zidovudine have been described in persons infected with HIV, and in some cases they have been associated with poor prognosis. HIV isolates obtained from 37 HIV-infected children were analyzed to determine whether the SI viral phenotype and the mutation on the 215 position of the reverse transcriptase (M215) could be used as markers of disease progression. We performed peripheral blood coculture mononuclear cells, and we analyzed the induction of syncytia using the MT-2 cell line. The emergence of mutations on the 215 position was determined by PCR. We found a statistically significant association (P < 0.05) between SI viral phenotype and (1) recurrent serious bacterial infections, (2) absolute CD4+ cell counts <2 SD, (3) progression to AIDS and (4) death. Sixty percent of the children treated with zidovudine developed 215 mutant viral strains without statistically significant association with clinical or immunologic findings. The SI viral phenotype was statistically associated with the presence of the 215 mutation (P < 0.05). SI viral phenotype is a marker associated with a poor clinical and immunologic progression of the disease and it may facilitate the emergence of mutant strains in children treated with zidovudine.

  2. Pharyngitis - viral

    Science.gov (United States)

    ... throat is due to a viral infection. The antibiotics will not help. Using them to treat viral infections helps bacteria become resistant to antibiotics. With some sore throats (such as those caused ...

  3. Parallel screening of wild-type and drug-resistant targets for anti-resistance neuraminidase inhibitors.

    Directory of Open Access Journals (Sweden)

    Kai-Cheng Hsu

    Full Text Available Infection with influenza virus is a major public health problem, causing serious illness and death each year. Emergence of drug-resistant influenza virus strains limits the effectiveness of drug treatment. Importantly, a dual H275Y/I223R mutation detected in the pandemic influenza A 2009 virus strain results in multidrug resistance to current neuraminidase (NA drugs. Therefore, discovery of new agents for treating multiple drug-resistant (MDR influenza virus infections is important. Here, we propose a parallel screening strategy that simultaneously screens wild-type (WT and MDR NAs, and identifies inhibitors matching the subsite characteristics of both NA-binding sites. These may maintain their potency when drug-resistant mutations arise. Initially, we analyzed the subsite of the dual H275Y/I223R NA mutant. Analysis of the site-moiety maps of NA protein structures show that the mutant subsite has a relatively small volume and is highly polar compared with the WT subsite. Moreover, the mutant subsite has a high preference for forming hydrogen-bonding interactions with polar moieties. These changes may drive multidrug resistance. Using this strategy, we identified a new inhibitor, Remazol Brilliant Blue R (RB19, an anthraquinone dye, which inhibited WT NA and MDR NA with IC(50 values of 3.4 and 4.5 µM, respectively. RB19 comprises a rigid core scaffold and a flexible chain with a large polar moiety. The former interacts with highly conserved residues, decreasing the probability of resistance. The latter forms van der Waals contacts with the WT subsite and yields hydrogen bonds with the mutant subsite by switching the orientation of its flexible side chain. Both scaffolds of RB19 are good starting points for lead optimization. The results reveal a parallel screening strategy for identifying resistance mechanisms and discovering anti-resistance neuraminidase inhibitors. We believe that this strategy may be applied to other diseases with high

  4. Transcriptional changes associated with resistance to inhibitors of epidermal growth factor receptor revealed using metaanalysis

    International Nuclear Information System (INIS)

    Younis, Sidra; Javed, Qamar; Blumenberg, Miroslav

    2015-01-01

    EGFR is important in maintaining metabolic homeostasis in healthy cells, but in tumors it activates downstream signaling pathways, causing proliferation, angiogenesis, invasion and metastasis. Consequently, EGFR is targeted in cancers using reversible, irreversible or antibody inhibitors. Unfortunately, tumors develop inhibitor resistance by mutations or overexpressing EGFR, or its ligand, or activating secondary, EGFR-independent pathways. Here we present a global metaanalysis comparing transcriptional profiles from matched pairs of EGFR inhibitor-sensitive vs. -resistant cell lines, using 15 datasets comprising 274 microarrays. We also analyzed separately pairs of cell lines derived using reversible, irreversible or antibody inhibitors. The metaanalysis identifies commonalities in cell lines resistant to EGFR inhibitors: in sensitive cell lines, the ontological categories involving the ErbB receptors pathways, cell adhesion and lipid metabolism are overexpressed; however, resistance to EGFR inhibitors is associated with overexpression of genes for ErbB receptors-independent oncogenic pathways, regulation of cell motility, energy metabolism, immunity especially inflammatory cytokines biosynthesis, cell cycle and responses to exogenous and endogenous stimuli. Specifically in Gefitinib-resistant cell lines, the immunity-associated genes are overexpressed, whereas in Erlotinib-resistant ones so are the mitochondrial genes and processes. Unexpectedly, lines selected using EGFR-targeting antibodies overexpress different gene ontologies from ones selected using kinase inhibitors. Specifically, they have reduced expression of genes for proliferation, chemotaxis, immunity and angiogenesis. This metaanalysis suggests that ‘combination therapies’ can improve cancer treatment outcomes. Potentially, use of mitochondrial blockers with Erlotinib, immunity blockers with Gefitinib, tyrosine kinase inhibitors with antibody inhibitors, may have better chance of avoiding

  5. Acquired resistance to EGFR inhibitors: mechanisms and prevention strategies

    International Nuclear Information System (INIS)

    Viloria-Petit, Alicia M.; Kerbel, Robert S.

    2004-01-01

    Potent and specific, or relatively specific, inhibitors of epidermal growth factor receptor (EGFR) signaling, including monoclonal antibodies and small molecular weight compounds, have been successfully developed. Both types of agent have been found to have significant antitumor activity, especially when used in combination with radio- hormone- and chemotherapy in preclinical studies. Because of the potentiation of the conventional drug activity in these combination settings, inhibitors of EGFR signaling have often been referred to as sensitizers for chemotherapy or radiation, as well as drug resistance reversal agents. Phase II clinical trials in head-and-neck as well as lung cancer suggested this concept of chemosensitization might translate into the clinic, but this remains to be definitively proven in randomized, double-blind Phase III trials. Given the extensive preclinical literature on EGFR blocking drugs and the advanced clinical development of such agents, it is surprising that the possibility of development of acquired resistance to the EGFR inhibitors themselves, a common clinical problem with virtually all other currently used anticancer drugs, remains a largely unexplored subject of investigation. Here we summarize some of the possible mechanisms that can result in acquired resistance to EGFR-targeting drugs. Alternative combination therapies to circumvent and delay this problem are suggested

  6. Convergent Akt activation drives acquired EGFR inhibitor resistance in lung cancer

    DEFF Research Database (Denmark)

    Jacobsen, Kirstine; Bertran-Alamillo, Jordi; Molina, Miguel Angel

    2017-01-01

    Non-small-cell lung cancer patients with activating epidermal growth factor receptor (EGFR) mutations typically benefit from EGFR tyrosine kinase inhibitor treatment. However, virtually all patients succumb to acquired EGFR tyrosine kinase inhibitor resistance that occurs via diverse mechanisms....

  7. HIV-1 integrase inhibitor resistance among treatment naïve patients in the West of Scotland.

    Science.gov (United States)

    Bradley-Stewart, A; Urcia, C; MacLean, A; Aitken, C; Gunson, R

    2017-07-01

    Transmitted integrase inhibitor resistance is rare, with only a small number of cases reported world-wide to date. The aim of this study was to assess whether transmitted integrase inhibitor resistance has occurred in Scotland and if so, could there be a case for performing genotypic integrase resistance testing at baseline. The study population consisted of 106 treatment naïve, newly diagnosed, HIV positive patients. The patient samples were collected between October 2015 and March 2016 at the time of HIV diagnosis and prior to initiation of anti-retroviral therapy. The integrase region was amplified and sequenced. We detected integrase inhibitor resistance (T66I/T) at baseline in one patient sample. This is a non-polymorphic mutation seen in patients receiving elvitegravir which confers high-level resistance to elvitegravir and intermediate resistance to raltegravir. A further 10 patients had accessory mutations which have minimal or no effect on susceptibility to integrase inhibitors. Transmitted integrase inhibitor resistance remains rare. The results of the present study do not support performing integrase resistance testing at baseline. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  8. Human Immunodeficiency Virus Type 1 Protease and the Emergence of Drug Resistance

    DEFF Research Database (Denmark)

    Poulsen, Nina Rødtness

    in multi-drug-resistant PRs. Computational analysis of a vast number of inhibitor-resistant HIV-1 PR variants can broaden the knowledge of how and why the mutations arise, which would be a great advantage in the design on resistance-evading inhibitors. Here we present a diverse system to select...... in the virus life cycle has made it a major target for drug development and active site competitive inhibitors have been successful in the battle against HIV. Unfortunately, the massive drug pressure along with high-level replication and lack of proofreading by the viral reverse transcriptase have resulted...... for catalytically active HIV-1 PR in the presence of inhibitor. The system is based on the protein AraC, which regulates transcription of the araA, araB and araD genes necessary for arabinose catabolism in Escherichia coli, and its effectiveness was demonstrated by the isolation of both known and unknown inhibitor-resistant...

  9. Perturbation biology nominates upstream-downstream drug combinations in RAF inhibitor resistant melanoma cells.

    Science.gov (United States)

    Korkut, Anil; Wang, Weiqing; Demir, Emek; Aksoy, Bülent Arman; Jing, Xiaohong; Molinelli, Evan J; Babur, Özgün; Bemis, Debra L; Onur Sumer, Selcuk; Solit, David B; Pratilas, Christine A; Sander, Chris

    2015-08-18

    Resistance to targeted cancer therapies is an important clinical problem. The discovery of anti-resistance drug combinations is challenging as resistance can arise by diverse escape mechanisms. To address this challenge, we improved and applied the experimental-computational perturbation biology method. Using statistical inference, we build network models from high-throughput measurements of molecular and phenotypic responses to combinatorial targeted perturbations. The models are computationally executed to predict the effects of thousands of untested perturbations. In RAF-inhibitor resistant melanoma cells, we measured 143 proteomic/phenotypic entities under 89 perturbation conditions and predicted c-Myc as an effective therapeutic co-target with BRAF or MEK. Experiments using the BET bromodomain inhibitor JQ1 affecting the level of c-Myc protein and protein kinase inhibitors targeting the ERK pathway confirmed the prediction. In conclusion, we propose an anti-cancer strategy of co-targeting a specific upstream alteration and a general downstream point of vulnerability to prevent or overcome resistance to targeted drugs.

  10. Lack of integrase inhibitors associated resistance mutations among HIV-1C isolates.

    Science.gov (United States)

    Mulu, Andargachew; Maier, Melanie; Liebert, Uwe Gerd

    2015-12-01

    Although biochemical analysis of HIV-1 integrase enzyme suggested the use of integrase inhibitors (INIs) against HIV-1C, different viral subtypes may favor different mutational pathways potentially leading to varying levels of drug resistance. Thus, the aim of this study was to search for the occurrence and natural evolution of integrase polymorphisms and/or resistance mutations in HIV-1C Ethiopian clinical isolates prior to the introduction of INIs. Plasma samples from chronically infected drug naïve patients (N = 45), of whom the PR and RT sequence was determined previously, were used to generate population based sequences of HIV-1 integrase. HIV-1 subtype was determined using the REGA HIV-1 subtyping tool. Resistance mutations were interpreted according to the Stanford HIV drug resistance database ( http://hivdb.stanford.edu ) and the updated International Antiviral Society (IAS)-USA mutation lists. Moreover, rates of polymorphisms in the current isolates were compared with South African and global HIV-1C isolates. All subjects were infected with HIV-1C concordant to the protease (PR) and reverse transcriptase (RT) regions. Neither major resistance-associated IN mutations (T66I/A/K, E92Q/G, T97A, Y143HCR, S147G, Q148H/R/K, and N155H) nor silent mutations known to change the genetic barrier were observed. Moreover, the DDE-catalytic motif (D64G/D116G/E152 K) and signature HHCC zinc-binding motifs at codon 12, 16, 40 and 43 were found to be highly conserved. However, compared to other South African subtype C isolates, the rate of polymorphism was variable at various positions. Although the sample size is small, the findings suggest that this drug class could be effective in Ethiopia and other southern African countries where HIV-1C is predominantly circulating. The data will contribute to define the importance of integrase polymorphism and to improve resistance interpretation algorithms in HIV-1C isolates.

  11. Potent Allosteric Dengue Virus NS5 Polymerase Inhibitors: Mechanism of Action and Resistance Profiling.

    Directory of Open Access Journals (Sweden)

    Siew Pheng Lim

    2016-08-01

    Full Text Available Flaviviruses comprise major emerging pathogens such as dengue virus (DENV or Zika virus (ZIKV. The flavivirus RNA genome is replicated by the RNA-dependent-RNA polymerase (RdRp domain of non-structural protein 5 (NS5. This essential enzymatic activity renders the RdRp attractive for antiviral therapy. NS5 synthesizes viral RNA via a "de novo" initiation mechanism. Crystal structures of the flavivirus RdRp revealed a "closed" conformation reminiscent of a pre-initiation state, with a well ordered priming loop that extrudes from the thumb subdomain into the dsRNA exit tunnel, close to the "GDD" active site. To-date, no allosteric pockets have been identified for the RdRp, and compound screening campaigns did not yield suitable drug candidates. Using fragment-based screening via X-ray crystallography, we found a fragment that bound to a pocket of the apo-DENV RdRp close to its active site (termed "N pocket". Structure-guided improvements yielded DENV pan-serotype inhibitors of the RdRp de novo initiation activity with nano-molar potency that also impeded elongation activity at micro-molar concentrations. Inhibitors exhibited mixed inhibition kinetics with respect to competition with the RNA or GTP substrate. The best compounds have EC50 values of 1-2 μM against all four DENV serotypes in cell culture assays. Genome-sequencing of compound-resistant DENV replicons, identified amino acid changes that mapped to the N pocket. Since inhibitors bind at the thumb/palm interface of the RdRp, this class of compounds is proposed to hinder RdRp conformational changes during its transition from initiation to elongation. This is the first report of a class of pan-serotype and cell-active DENV RdRp inhibitors. Given the evolutionary conservation of residues lining the N pocket, these molecules offer insights to treat other serious conditions caused by flaviviruses.

  12. Systems biology analysis of mitogen activated protein kinase inhibitor resistance in malignant melanoma.

    Science.gov (United States)

    Zecena, Helma; Tveit, Daniel; Wang, Zi; Farhat, Ahmed; Panchal, Parvita; Liu, Jing; Singh, Simar J; Sanghera, Amandeep; Bainiwal, Ajay; Teo, Shuan Y; Meyskens, Frank L; Liu-Smith, Feng; Filipp, Fabian V

    2018-04-04

    Kinase inhibition in the mitogen activated protein kinase (MAPK) pathway is a standard therapy for cancer patients with activating BRAF mutations. However, the anti-tumorigenic effect and clinical benefit are only transient, and tumors are prone to treatment resistance and relapse. To elucidate mechanistic insights into drug resistance, we have established an in vitro cellular model of MAPK inhibitor resistance in malignant melanoma. The cellular model evolved in response to clinical dosage of the BRAF inhibitor, vemurafenib, PLX4032. We conducted transcriptomic expression profiling using RNA-Seq and RT-qPCR arrays. Pathways of melanogenesis, MAPK signaling, cell cycle, and metabolism were significantly enriched among the set of differentially expressed genes of vemurafenib-resistant cells vs control. The underlying mechanism of treatment resistance and pathway rewiring was uncovered to be based on non-genomic adaptation and validated in two distinct melanoma models, SK-MEL-28 and A375. Both cell lines have activating BRAF mutations and display metastatic potential. Downregulation of dual specific phosphatases, tumor suppressors, and negative MAPK regulators reengages mitogenic signaling. Upregulation of growth factors, cytokines, and cognate receptors triggers signaling pathways circumventing BRAF blockage. Further, changes in amino acid and one-carbon metabolism support cellular proliferation despite MAPK inhibitor treatment. In addition, treatment-resistant cells upregulate pigmentation and melanogenesis, pathways which partially overlap with MAPK signaling. Upstream regulator analysis discovered significant perturbation in oncogenic forkhead box and hypoxia inducible factor family transcription factors. The established cellular models offer mechanistic insight into cellular changes and therapeutic targets under inhibitor resistance in malignant melanoma. At a systems biology level, the MAPK pathway undergoes major rewiring while acquiring inhibitor resistance

  13. Inhibition of large conductance calcium-dependent potassium ...

    African Journals Online (AJOL)

    conductance, calcium and voltage- dependent potassium (BKCa) channels thereby promoting vasoconstriction. Our results show that the Rho-kinase inhibitor, Y-27632, induced concentration-dependent relaxation in rat mesenteric artery.

  14. Distinguishing HIV-1 drug resistance, accessory, and viral fitness mutations using conditional selection pressure analysis of treated versus untreated patient samples

    Directory of Open Access Journals (Sweden)

    Lee Christopher

    2006-05-01

    Full Text Available Abstract Background HIV can evolve drug resistance rapidly in response to new drug treatments, often through a combination of multiple mutations 123. It would be useful to develop automated analyses of HIV sequence polymorphism that are able to predict drug resistance mutations, and to distinguish different types of functional roles among such mutations, for example, those that directly cause drug resistance, versus those that play an accessory role. Detecting functional interactions between mutations is essential for this classification. We have adapted a well-known measure of evolutionary selection pressure (Ka/Ks and developed a conditional Ka/Ks approach to detect important interactions. Results We have applied this analysis to four independent HIV protease sequencing datasets: 50,000 clinical samples sequenced by Specialty Laboratories, Inc.; 1800 samples from patients treated with protease inhibitors; 2600 samples from untreated patients; 400 samples from untreated African patients. We have identified 428 mutation interactions in Specialty dataset with statistical significance and we were able to distinguish primary vs. accessory mutations for many well-studied examples. Amino acid interactions identified by conditional Ka/Ks matched 80 of 92 pair wise interactions found by a completely independent study of HIV protease (p-value for this match is significant: 10-70. Furthermore, Ka/Ks selection pressure results were highly reproducible among these independent datasets, both qualitatively and quantitatively, suggesting that they are detecting real drug-resistance and viral fitness mutations in the wild HIV-1 population. Conclusion Conditional Ka/Ks analysis can detect mutation interactions and distinguish primary vs. accessory mutations in HIV-1. Ka/Ks analysis of treated vs. untreated patient data can distinguish drug-resistance vs. viral fitness mutations. Verification of these results would require longitudinal studies. The result

  15. Overcoming resistance to beta-lactamase inhibitors: comparing sulbactam to novel inhibitors against clavulanate resistant SHV enzymes with substitutions at Ambler position 244.

    Science.gov (United States)

    Thomson, Jodi M; Distler, Anne M; Bonomo, Robert A

    2007-10-09

    Amino acid changes at Ambler position R244 in class A TEM and SHV beta-lactamases confer resistance to ampicillin/clavulanate, a beta-lactam/beta-lactamase inhibitor combination used to treat serious infections. To gain a deeper understanding of this resistance phenotype, we investigated the activities of sulbactam and two novel penem beta-lactamase inhibitors with sp2 hybridized C3 carboxylates and bicyclic R1 side chains against a library of SHV beta-lactamase variants at the 244 position. Compared to SHV-1 expressed in Escherichia coli, all 19 R244 variants exhibited increased susceptibility to ampicillin/sulbactam, an important difference compared to ampicillin/clavulanate. Kinetic analyses of SHV-1 and three SHV R244 (-S, -Q, and -L) variants revealed the Ki for sulbactam was significantly elevated for the R244 variants, but the partition ratios, kcat/kinact, were markedly reduced (13 000 --> inhibitors effectively restored ampicillin susceptibility in vitro. Compared to that of sulbactam, the kcat/kinact values of penems for SHV-1 and R244S were low (resistance can be overcome by designing penem inhibitors with strategic chemical properties that improve affinity and impair turnover.

  16. Hepatitis C virus protease inhibitor-resistance mutations: our experience and review.

    Science.gov (United States)

    Wu, Shuang; Kanda, Tatsuo; Nakamoto, Shingo; Imazeki, Fumio; Yokosuka, Osamu

    2013-12-21

    Direct-acting antiviral agents (DAAs) for hepatitis C virus (HCV) infection are one of the major advances in its medical treatment. The HCV protease inhibitors boceprevir and telaprevir were the first approved DAAs in the United States, Europe, and Japan. When combined with peginterferon plus ribavirin, these agents increase sustained virologic response rates to 70%-80% in treatment-naïve patients and previous-treatment relapsers with chronic HCV genotype 1 infection. Without peginterferon plus ribavirin, DAA mono-therapies increased DAA-resistance mutations. Several new DAAs for HCV are now in clinical development and are likely to be approved in the near future. However, it has been reported that the use of these drugs also led to the emergence of DAA-resistance mutations in certain cases. Furthermore, these mutations exhibit cross-resistance to multiple drugs. The prevalence of DAA-resistance mutations in HCV-infected patients who were not treated with DAAs is unknown, and it is as yet uncertain whether such variants are sensitive to DAAs. We performed a population sequence analysis to assess the frequency of such variants in the sera of HCV genotype 1-infected patients not treated with HCV protease inhibitors. Here, we reviewed the literature on resistance variants of HCV protease inhibitors in treatment naïve patients with chronic HCV genotype 1, as well as our experience.

  17. Resistance to pyridine-based inhibitor KF116 reveals an unexpected role of integrase in HIV-1 Gag-Pol polyprotein proteolytic processing.

    Science.gov (United States)

    Hoyte, Ashley C; Jamin, Augusta V; Koneru, Pratibha C; Kobe, Matthew J; Larue, Ross C; Fuchs, James R; Engelman, Alan N; Kvaratskhelia, Mamuka

    2017-12-01

    The pyridine-based multimerization selective HIV-1 integrase (IN) inhibitors (MINIs) are a distinct subclass of allosteric IN inhibitors. MINIs potently inhibit HIV-1 replication during virion maturation by inducing hyper- or aberrant IN multimerization but are largely ineffective during the early steps of viral replication. Here, we investigated the mechanism for the evolution of a triple IN substitution (T124N/V165I/T174I) that emerges in cell culture with a representative MINI, KF116. We show that HIV-1 NL4-3(IN T124N/V165I/T174I) confers marked (>2000-fold) resistance to KF116. Two IN substitutions (T124N/T174I) directly weaken inhibitor binding at the dimer interface of the catalytic core domain but at the same time markedly impair HIV-1 replication capacity. Unexpectedly, T124N/T174I IN substitutions inhibited proteolytic processing of HIV-1 polyproteins Gag and Gag-Pol, resulting in immature virions. Strikingly, the addition of the third IN substitution (V165I) restored polyprotein processing, virus particle maturation, and significant levels of replication capacity. These results reveal an unanticipated role of IN for polyprotein proteolytic processing during virion morphogenesis. The complex evolutionary pathway for the emergence of resistant viruses, which includes the need for the compensatory V165I IN substitution, highlights a relatively high genetic barrier exerted by MINI KF116. Additionally, we have solved the X-ray structure of the drug-resistant catalytic core domain protein, which provides means for rational development of second-generation MINIs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Resistance to Plum pox virus strain C in Arabidopsis thaliana and Chenopodium foetidum involves genome-linked viral protein and other viral determinants and might depend on compatibility with host translation initiation factors.

    Science.gov (United States)

    Calvo, María; Martínez-Turiño, Sandra; García, Juan Antonio

    2014-11-01

    Research performed on model herbaceous hosts has been useful to unravel the molecular mechanisms that control viral infections. The most common Plum pox virus (PPV) strains are able to infect Nicotiana species as well as Chenopodium and Arabidopsis species. However, isolates belonging to strain C (PPV-C) that have been adapted to Nicotiana spp. are not infectious either in Chenopodium foetidum or in Arabidopsis thaliana. In order to determine the mechanism underlying this interesting host-specific behavior, we have constructed chimerical clones derived from Nicotiana-adapted PPV isolates from the D and C strains, which differ in their capacity to infect A. thaliana and C. foetidum. With this approach, we have identified the nuclear inclusion a protein (VPg+Pro) as the major pathogenicity determinant that conditions resistance in the presence of additional secondary determinants, different for each host. Genome-linked viral protein (VPg) mutations similar to those involved in the breakdown of eIF4E-mediated resistance to other potyviruses allow some PPV chimeras to infect A. thaliana. These results point to defective interactions between a translation initiation factor and the viral VPg as the most probable cause of host-specific incompatibility, in which other viral factors also participate, and suggest that complex interactions between multiple viral proteins and translation initiation factors not only define resistance to potyviruses in particular varieties of susceptible hosts but also contribute to establish nonhost resistance.

  19. Schrödinger's Cheshire Cat: Are Haploid Emiliania huxleyi Cells Resistant to Viral Infection or Not?

    Science.gov (United States)

    Mordecai, Gideon J; Verret, Frederic; Highfield, Andrea; Schroeder, Declan C

    2017-03-18

    Emiliania huxleyi is the main calcite producer on Earth and is routinely infected by a virus (EhV); a double stranded DNA (dsDNA) virus belonging to the family Phycodnaviridae . E. huxleyi exhibits a haplodiploid life cycle; the calcified diploid stage is non-motile and forms extensive blooms. The haploid phase is a non-calcified biflagellated cell bearing organic scales. Haploid cells are thought to resist infection, through a process deemed the "Cheshire Cat" escape strategy; however, a recent study detected the presence of viral lipids in the same haploid strain. Here we report on the application of an E. huxleyi CCMP1516 EhV-86 combined tiling array (TA) that further confirms an EhV infection in the RCC1217 haploid strain, which grew without any signs of cell lysis. Reverse transcription polymerase chain reaction (RT-PCR) and PCR verified the presence of viral RNA in the haploid cells, yet indicated an absence of viral DNA, respectively. These infected cells are an alternative stage of the virus life cycle deemed the haplococcolithovirocell. In this instance, the host is both resistant to and infected by EhV, i.e., the viral transcriptome is present in haploid cells whilst there is no evidence of viral lysis. This superimposed state is reminiscent of Schrödinger's cat; of being simultaneously both dead and alive.

  20. Distinct apoptotic blocks mediate resistance to panHER inhibitors in HER2+ breast cancer cells.

    Science.gov (United States)

    Karakas, Bahriye; Ozmay, Yeliz; Basaga, Huveyda; Gul, Ozgur; Kutuk, Ozgur

    2018-05-04

    Despite the development of novel targeted therapies, de novo or acquired chemoresistance remains a significant factor for treatment failure in breast cancer therapeutics. Neratinib and dacomitinib are irreversible panHER inhibitors, which block their autophosphorylation and downstream signaling. Moreover, neratinib and dacomitinib have been shown to activate cell death in HER2-overexpressing cell lines. Here we showed that increased MCL1 and decreased BIM and PUMA mediated resistance to neratinib in ZR-75-30 and SKBR3 cells while increased BCL-XL and BCL-2 and decreased BIM and PUMA promoted neratinib resistance in BT474 cells. Cells were also cross-resistant to dacomitinib. BH3 profiles of HER2+ breast cancer cells efficiently predicted antiapoptotic protein dependence and development of resistance to panHER inhibitors. Reactivation of ERK1/2 was primarily responsible for acquired resistance in SKBR3 and ZR-75-30 cells. Adding specific ERK1/2 inhibitor SCH772984 to neratinib or dacomitinib led to increased apoptotic response in neratinib-resistant SKBR3 and ZR-75-30 cells, but we did not detect a similar response in neratinib-resistant BT474 cells. Accordingly, suppression of BCL-2/BCL-XL by ABT-737 was required in addition to ERK1/2 inhibition for neratinib- or dacomitinib-induced apoptosis in neratinib-resistant BT474 cells. Our results showed that different mitochondrial apoptotic blocks mediated acquired panHER inhibitor resistance in HER2+ breast cancer cell lines as well as highlighted the potential of BH3 profiling assay in prediction of panHER inhibitor resistance in breast cancer cells. Copyright © 2018 Elsevier B.V. All rights reserved.

  1. Perturbation biology nominates upstream–downstream drug combinations in RAF inhibitor resistant melanoma cells

    Science.gov (United States)

    Korkut, Anil; Wang, Weiqing; Demir, Emek; Aksoy, Bülent Arman; Jing, Xiaohong; Molinelli, Evan J; Babur, Özgün; Bemis, Debra L; Onur Sumer, Selcuk; Solit, David B; Pratilas, Christine A; Sander, Chris

    2015-01-01

    Resistance to targeted cancer therapies is an important clinical problem. The discovery of anti-resistance drug combinations is challenging as resistance can arise by diverse escape mechanisms. To address this challenge, we improved and applied the experimental-computational perturbation biology method. Using statistical inference, we build network models from high-throughput measurements of molecular and phenotypic responses to combinatorial targeted perturbations. The models are computationally executed to predict the effects of thousands of untested perturbations. In RAF-inhibitor resistant melanoma cells, we measured 143 proteomic/phenotypic entities under 89 perturbation conditions and predicted c-Myc as an effective therapeutic co-target with BRAF or MEK. Experiments using the BET bromodomain inhibitor JQ1 affecting the level of c-Myc protein and protein kinase inhibitors targeting the ERK pathway confirmed the prediction. In conclusion, we propose an anti-cancer strategy of co-targeting a specific upstream alteration and a general downstream point of vulnerability to prevent or overcome resistance to targeted drugs. DOI: http://dx.doi.org/10.7554/eLife.04640.001 PMID:26284497

  2. Multi-resistance strategy for viral diseases and short hairpin RNA verification method in pigs

    Directory of Open Access Journals (Sweden)

    Jong-nam Oh

    2018-04-01

    Full Text Available Objective Foot and mouth disease (FMD and porcine reproductive and respiratory syndrome (PRRS are major diseases that interrupt porcine production. Because they are viral diseases, vaccinations are of only limited effectiveness in preventing outbreaks. To establish an alternative multi-resistant strategy against FMD virus (FMDV and PRRS virus (PRRSV, the present study introduced two genetic modification techniques to porcine cells. Methods First, cluster of differentiation 163 (CD163, the PRRSV viral receptor, was edited with the clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 technique. The CD163 gene sequences of edited cells and control cells differed. Second, short hairpin RNA (shRNAs were integrated into the cells. The shRNAs, targeting the 3D gene of FMDV and the open reading frame 7 (ORF7 gene of PRRSV, were transferred into fibroblasts. We also developed an in vitro shRNA verification method with a target gene expression vector. Results shRNA activity was confirmed in vitro with vectors that expressed the 3D and ORF7 genes in the cells. Cells containing shRNAs showed lower transcript levels than cells with only the expression vectors. The shRNAs were integrated into CD163-edited cells to combine the two techniques, and the viral genes were suppressed in these cells. Conclusion We established a multi-resistant strategy against viral diseases and an in vitro shRNA verification method.

  3. A new pH-sensitive rectifying potassium channel in mitochondria from the embryonic rat hippocampus.

    Science.gov (United States)

    Kajma, Anna; Szewczyk, Adam

    2012-10-01

    Patch-clamp single-channel studies on mitochondria isolated from embryonic rat hippocampus revealed the presence of two different potassium ion channels: a large-conductance (288±4pS) calcium-activated potassium channel and second potassium channel with outwardly rectifying activity under symmetric conditions (150/150mM KCl). At positive voltages, this channel displayed a conductance of 67.84pS and a strong voltage dependence at holding potentials from -80mV to +80mV. The open probability was higher at positive than at negative voltages. Patch-clamp studies at the mitoplast-attached mode showed that the channel was not sensitive to activators and inhibitors of mitochondrial potassium channels but was regulated by pH. Moreover, we demonstrated that the channel activity was not affected by the application of lidocaine, an inhibitor of two-pore domain potassium channels, or by tertiapin, an inhibitor of inwardly rectifying potassium channels. In summary, based on the single-channel recordings, we characterised for the first time mitochondrial pH-sensitive ion channel that is selective for cations, permeable to potassium ions, displays voltage sensitivity and does not correspond to any previously described potassium ion channels in the inner mitochondrial membrane. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012). Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Taxane-Platin-Resistant Lung Cancers Co-develop Hypersensitivity to JumonjiC Demethylase Inhibitors

    Directory of Open Access Journals (Sweden)

    Maithili P. Dalvi

    2017-05-01

    Full Text Available Although non-small cell lung cancer (NSCLC patients benefit from standard taxane-platin chemotherapy, many relapse, developing drug resistance. We established preclinical taxane-platin-chemoresistance models and identified a 35-gene resistance signature, which was associated with poor recurrence-free survival in neoadjuvant-treated NSCLC patients and included upregulation of the JumonjiC lysine demethylase KDM3B. In fact, multi-drug-resistant cells progressively increased the expression of many JumonjiC demethylases, had altered histone methylation, and, importantly, showed hypersensitivity to JumonjiC inhibitors in vitro and in vivo. Increasing taxane-platin resistance in progressive cell line series was accompanied by progressive sensitization to JIB-04 and GSK-J4. These JumonjiC inhibitors partly reversed deregulated transcriptional programs, prevented the emergence of drug-tolerant colonies from chemo-naive cells, and synergized with standard chemotherapy in vitro and in vivo. Our findings reveal JumonjiC inhibitors as promising therapies for targeting taxane-platin-chemoresistant NSCLCs.

  5. [Computational prediction of human immunodeficiency resistance to reverse transcriptase inhibitors].

    Science.gov (United States)

    Tarasova, O A; Filimonov, D A; Poroikov, V V

    2017-10-01

    Human immunodeficiency virus (HIV) causes acquired immunodeficiency syndrome (AIDS) and leads to over one million of deaths annually. Highly active antiretroviral treatment (HAART) is a gold standard in the HIV/AIDS therapy. Nucleoside and non-nucleoside inhibitors of HIV reverse transcriptase (RT) are important component of HAART, but their effect depends on the HIV susceptibility/resistance. HIV resistance mainly occurs due to mutations leading to conformational changes in the three-dimensional structure of HIV RT. The aim of our work was to develop and test a computational method for prediction of HIV resistance associated with the mutations in HIV RT. Earlier we have developed a method for prediction of HIV type 1 (HIV-1) resistance; it is based on the usage of position-specific descriptors. These descriptors are generated using the particular amino acid residue and its position; the position of certain residue is determined in a multiple alignment. The training set consisted of more than 1900 sequences of HIV RT from the Stanford HIV Drug Resistance database; for these HIV RT variants experimental data on their resistance to ten inhibitors are presented. Balanced accuracy of prediction varies from 80% to 99% depending on the method of classification (support vector machine, Naive Bayes, random forest, convolutional neural networks) and the drug, resistance to which is obtained. Maximal balanced accuracy was obtained for prediction of resistance to zidovudine, stavudine, didanosine and efavirenz by the random forest classifier. Average accuracy of prediction is 89%.

  6. Efficacy and safety on tenofovire therapy in patients with hepatitis B viral infections resistent to lamivudin

    Directory of Open Access Journals (Sweden)

    Katanić N.

    2015-01-01

    Full Text Available Chronic viral hepatitis B (CHB still represents a significant world health problem despite obligatory and worldwide immunization against infections of viral hepatitis B. In some patients with chronic viral hepatitis B infections, in the natural course of the disease, progression towards cirhossis and hepatocellular carcinoma is primarily targeted by antiviral CHB therapy stopping further progression of the disease. Today on the market there exist two classes of pharmnaceutical drugs for treatment of CHB: a immunomodulatory therapy with conventional interferon alpha (INF and PEGylated interferon alpha-2a, b and oral antiviral therapy with nucleos( tide analogues. Lamivudine was for quite a period the only medicament available on our market for the treatment of HVB and in most of our patients led to the development of resistance. As of two years ago, a new oral analogue from the group of nucleotides is being registered in Serbia for market use: tenofovir disoproxil (TDF. In our work we have analysed 69 patients with chronic viral hepatitis B treated in the Clinic for Infectious and Tropical Diseases KCS Belgrade in the period between years 2012 and 2014. All patients involved in this reasearch were previously treated with LAM, and on subsequent development of resistance to LAM, TDF was used. TDF showed an excellent efficacy, a high resistance barrier and very few unwanted side effects over several years of treatment. Our experience with the use of this drug does not pertain to and acount for its long term use, in view of its brief availability on our market.

  7. Structure-based methods to predict mutational resistance to diarylpyrimidine non-nucleoside reverse transcriptase inhibitors.

    Science.gov (United States)

    Azeem, Syeda Maryam; Muwonge, Alecia N; Thakkar, Nehaben; Lam, Kristina W; Frey, Kathleen M

    2018-01-01

    Resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) is a leading cause of HIV treatment failure. Often included in antiviral therapy, NNRTIs are chemically diverse compounds that bind an allosteric pocket of enzyme target reverse transcriptase (RT). Several new NNRTIs incorporate flexibility in order to compensate for lost interactions with amino acid conferring mutations in RT. Unfortunately, even successful inhibitors such as diarylpyrimidine (DAPY) inhibitor rilpivirine are affected by mutations in RT that confer resistance. In order to aid drug design efforts, it would be efficient and cost effective to pre-evaluate NNRTI compounds in development using a structure-based computational approach. As proof of concept, we applied a residue scan and molecular dynamics strategy using RT crystal structures to predict mutations that confer resistance to DAPYs rilpivirine, etravirine, and investigational microbicide dapivirine. Our predictive values, changes in affinity and stability, are correlative with fold-resistance data for several RT mutants. Consistent with previous studies, mutation K101P is predicted to confer high-level resistance to DAPYs. These findings were further validated using structural analysis, molecular dynamics, and an enzymatic reverse transcription assay. Our results confirm that changes in affinity and stability for mutant complexes are predictive parameters of resistance as validated by experimental and clinical data. In future work, we believe that this computational approach may be useful to predict resistance mutations for inhibitors in development. Published by Elsevier Inc.

  8. Mutations in HIV-1 gag and pol Compensate for the Loss of Viral Fitness Caused by a Highly Mutated Protease

    Czech Academy of Sciences Publication Activity Database

    Kožíšek, Milan; Henke, S.; Grantz Šašková, Klára; Jacobs, G. B.; Schuch, A.; Buchholz, B.; Müller, V.; Kräusslich, H. G.; Řezáčová, Pavlína; Konvalinka, Jan; Bodem, J.

    2012-01-01

    Roč. 56, č. 8 (2012), s. 4320-4330 ISSN 0066-4804 R&D Projects: GA ČR GAP207/11/1798 Institutional research plan: CEZ:AV0Z40550506 Keywords : HIV protease * resistance * inhibitor * viral fitness * AG subtype Subject RIV: EE - Microbiology, Virology Impact factor: 4.565, year: 2012

  9. Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells

    International Nuclear Information System (INIS)

    Okabe, Seiichi; Tauchi, Tetsuzo; Tanaka, Yuko; Ohyashiki, Kazuma

    2013-01-01

    Highlights: •Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells okabe et al. •Imatinib or nilotinib resistance was involved Src family kinase. •The BCR-ABL point mutation (E334V) was highly resistant to imatinib or nilotinib. •Ponatinib was a powerful strategy against imatinib or nilotinib resistant Ph-positive cells. -- Abstract: Because a substantial number of patients with chronic myeloid leukemia acquire resistance to ABL tyrosine kinase inhibitors (TKIs), their management remains a challenge. Ponatinib, also known as AP24534, is an oral multi-targeted TKI. Ponatinib is currently being investigated in a pivotal phase 2 clinical trial. In the present study, we analyzed the molecular and functional consequences of ponatinib against imatinib- or nilotinib-resistant (R) K562 and Ba/F3 cells. The proliferation of imatinib- or nilotinib-resistant K562 cells did not decrease after treatment with imatinib or nilotinib. Src family kinase Lyn was activated. Point mutation Ba/F3 cells (E334 V) were also highly resistant to imatinib and nilotinib. Treatment with ponatinib for 72 h inhibited the growth of imatinib- and nilotinib-resistant cells. The phosphorylation of BCR-ABL, Lyn, and Crk-L was reduced. This study demonstrates that ponatinib has an anti-leukemia effect by reducing ABL and Lyn kinase activity and this information may be of therapeutic relevance

  10. Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Okabe, Seiichi, E-mail: okabe@tokyo-med.ac.jp; Tauchi, Tetsuzo; Tanaka, Yuko; Ohyashiki, Kazuma

    2013-06-07

    Highlights: •Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells okabe et al. •Imatinib or nilotinib resistance was involved Src family kinase. •The BCR-ABL point mutation (E334V) was highly resistant to imatinib or nilotinib. •Ponatinib was a powerful strategy against imatinib or nilotinib resistant Ph-positive cells. -- Abstract: Because a substantial number of patients with chronic myeloid leukemia acquire resistance to ABL tyrosine kinase inhibitors (TKIs), their management remains a challenge. Ponatinib, also known as AP24534, is an oral multi-targeted TKI. Ponatinib is currently being investigated in a pivotal phase 2 clinical trial. In the present study, we analyzed the molecular and functional consequences of ponatinib against imatinib- or nilotinib-resistant (R) K562 and Ba/F3 cells. The proliferation of imatinib- or nilotinib-resistant K562 cells did not decrease after treatment with imatinib or nilotinib. Src family kinase Lyn was activated. Point mutation Ba/F3 cells (E334 V) were also highly resistant to imatinib and nilotinib. Treatment with ponatinib for 72 h inhibited the growth of imatinib- and nilotinib-resistant cells. The phosphorylation of BCR-ABL, Lyn, and Crk-L was reduced. This study demonstrates that ponatinib has an anti-leukemia effect by reducing ABL and Lyn kinase activity and this information may be of therapeutic relevance.

  11. Sulfonamide-Based Inhibitors of Aminoglycoside Acetyltransferase Eis Abolish Resistance to Kanamycin in Mycobacterium tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Garzan, Atefeh; Willby, Melisa J.; Green, Keith D.; Gajadeera, Chathurada S.; Hou, Caixia; Tsodikov, Oleg V.; Posey, James E.; Garneau-Tsodikova, Sylvie

    2016-12-08

    A two-drug combination therapy where one drug targets an offending cell and the other targets a resistance mechanism to the first drug is a time-tested, yet underexploited approach to combat or prevent drug resistance. By high-throughput screening, we identified a sulfonamide scaffold that served as a pharmacophore to generate inhibitors of Mycobacterium tuberculosis acetyltransferase Eis, whose upregulation causes resistance to the aminoglycoside (AG) antibiotic kanamycin A (KAN) in Mycobacterium tuberculosis. Rational systematic derivatization of this scaffold to maximize Eis inhibition and abolish the Eis-mediated KAN resistance of M. tuberculosis yielded several highly potent agents. A crystal structure of Eis in complex with one of the most potent inhibitors revealed that the inhibitor bound Eis in the AG-binding pocket held by a conformationally malleable region of Eis (residues 28–37) bearing key hydrophobic residues. These Eis inhibitors are promising leads for preclinical development of innovative AG combination therapies against resistant TB.

  12. Primary resistance to integrase strand-transfer inhibitors in Europe.

    NARCIS (Netherlands)

    M. Casadellá; P.M. van Ham (Petra); M. Noguera-Julian; A. van Kessel; C. Pou; L.M. Hofstra (Marije); G.A. Metcalf (Ginger A.); F. Garcia; D. Struck (Daniel); I. Alexiev (Ivailo); A.M. Bakken Kran; A.I.M. Hoepelman; L.G. Kostrikis (Leondios); S. Somogyi; K. Liitsola (Kirsi); M. Linka (Marek); C. Nielsen; D. Otelea (Dan); D. Paraskevis (Dimitrios); M. Poljak (Mario); E. Puchhammer-Stöckl (Elisabeth); D. Stanekova (Danica); M. Stanojevic (Maja); K. Van Laethem (Kristel); S. Zidovec Lepej (Snjezana); B. Clotet; C.A.B. Boucher (Charles); R. Paredes (Roger); A.M.J. Wensing (Annemarie)

    2015-01-01

    markdownabstractThe objective of this study was to define the natural genotypic variation of the HIV-1 integrase gene across Europe for epidemiological surveillance of integrase strand-transfer inhibitor (InSTI) resistance. This was a multicentre, cross-sectional study within the European SPREAD

  13. Primary resistance to integrase strand-transfer inhibitors in Europe

    NARCIS (Netherlands)

    Casadella, M.; van Ham, P. M.; Noguera-Julian, M.; van Kessel, A.; Pou, C.; Hofstra, L. M.; Santos, J. R.; Garcia, F.; Struck, D.; Alexiev, I.; Kran, A. M. Bakken; Hoepelman, A. I.; Kostrikis, L. G.; Somogyi, S.; Liitsola, K.; Linka, M.; Nielsen, C.; Otelea, D.; Paraskevis, D.; Poljak, M.; Puchhammer-Stoeckl, E.; Stanekova, D.; Stanojevic, M.; Van Laethem, K.; Lepej, S. Zidovec; Clotet, B.; Boucher, C. A. B.; Paredes, R.; Wensing, A. M. J.; Schuurman, R

    2015-01-01

    Objectives: The objective of this study was to define the natural genotypic variation of the HIV-1 integrase gene across Europe for epidemiological surveillance of integrase strand-transfer inhibitor (InSTI) resistance. Methods: This was a multicentre, cross-sectional study within the European

  14. The histone deacetylase inhibitor vorinostat (SAHA) increases the susceptibility of uninfected CD4+ T cells to HIV by increasing the kinetics and efficiency of postentry viral events.

    Science.gov (United States)

    Lucera, Mark B; Tilton, Carisa A; Mao, Hongxia; Dobrowolski, Curtis; Tabler, Caroline O; Haqqani, Aiman A; Karn, Jonathan; Tilton, John C

    2014-09-01

    Latently infected cells remain a primary barrier to eradication of HIV-1. Over the past decade, a better understanding of the molecular mechanisms by which latency is established and maintained has led to the discovery of a number of compounds that selectively reactivate latent proviruses without inducing polyclonal T cell activation. Recently, the histone deacetylase (HDAC) inhibitor vorinostat has been demonstrated to induce HIV transcription from latently infected cells when administered to patients. While vorinostat will be given in the context of antiretroviral therapy (ART), infection of new cells by induced virus remains a clinical concern. Here, we demonstrate that vorinostat significantly increases the susceptibility of CD4(+) T cells to infection by HIV in a dose- and time-dependent manner that is independent of receptor and coreceptor usage. Vorinostat does not enhance viral fusion with cells but rather enhances the kinetics and efficiency of postentry viral events, including reverse transcription, nuclear import, and integration, and enhances viral production in a spreading-infection assay. Selective inhibition of the cytoplasmic class IIb HDAC6 with tubacin recapitulated the effect of vorinostat. These findings reveal a previously unknown cytoplasmic effect of HDAC inhibitors promoting productive infection of CD4(+) T cells that is distinct from their well-characterized effects on nuclear histone acetylation and long-terminal-repeat (LTR) transcription. Our results indicate that careful monitoring of patients and ART intensification are warranted during vorinostat treatment and indicate that HDAC inhibitors that selectively target nuclear class I HDACs could reactivate latent HIV without increasing the susceptibility of uninfected cells to HIV. HDAC inhibitors, particularly vorinostat, are currently being investigated clinically as part of a "shock-and-kill" strategy to purge latent reservoirs of HIV. We demonstrate here that vorinostat increases the

  15. Schrödinger’s Cheshire Cat: Are Haploid Emiliania huxleyi Cells Resistant to Viral Infection or Not?

    Directory of Open Access Journals (Sweden)

    Gideon J. Mordecai

    2017-03-01

    Full Text Available Emiliania huxleyi is the main calcite producer on Earth and is routinely infected by a virus (EhV; a double stranded DNA (dsDNA virus belonging to the family Phycodnaviridae. E. huxleyi exhibits a haplodiploid life cycle; the calcified diploid stage is non-motile and forms extensive blooms. The haploid phase is a non-calcified biflagellated cell bearing organic scales. Haploid cells are thought to resist infection, through a process deemed the “Cheshire Cat” escape strategy; however, a recent study detected the presence of viral lipids in the same haploid strain. Here we report on the application of an E. huxleyi CCMP1516 EhV-86 combined tiling array (TA that further confirms an EhV infection in the RCC1217 haploid strain, which grew without any signs of cell lysis. Reverse transcription polymerase chain reaction (RT-PCR and PCR verified the presence of viral RNA in the haploid cells, yet indicated an absence of viral DNA, respectively. These infected cells are an alternative stage of the virus life cycle deemed the haplococcolithovirocell. In this instance, the host is both resistant to and infected by EhV, i.e., the viral transcriptome is present in haploid cells whilst there is no evidence of viral lysis. This superimposed state is reminiscent of Schrödinger’s cat; of being simultaneously both dead and alive.

  16. Schrödinger’s Cheshire Cat: Are Haploid Emiliania huxleyi Cells Resistant to Viral Infection or Not?

    Science.gov (United States)

    Mordecai, Gideon J.; Verret, Frederic; Highfield, Andrea; Schroeder, Declan C.

    2017-01-01

    Emiliania huxleyi is the main calcite producer on Earth and is routinely infected by a virus (EhV); a double stranded DNA (dsDNA) virus belonging to the family Phycodnaviridae. E. huxleyi exhibits a haplodiploid life cycle; the calcified diploid stage is non-motile and forms extensive blooms. The haploid phase is a non-calcified biflagellated cell bearing organic scales. Haploid cells are thought to resist infection, through a process deemed the “Cheshire Cat” escape strategy; however, a recent study detected the presence of viral lipids in the same haploid strain. Here we report on the application of an E. huxleyi CCMP1516 EhV-86 combined tiling array (TA) that further confirms an EhV infection in the RCC1217 haploid strain, which grew without any signs of cell lysis. Reverse transcription polymerase chain reaction (RT-PCR) and PCR verified the presence of viral RNA in the haploid cells, yet indicated an absence of viral DNA, respectively. These infected cells are an alternative stage of the virus life cycle deemed the haplococcolithovirocell. In this instance, the host is both resistant to and infected by EhV, i.e., the viral transcriptome is present in haploid cells whilst there is no evidence of viral lysis. This superimposed state is reminiscent of Schrödinger’s cat; of being simultaneously both dead and alive. PMID:28335465

  17. Early viral replication and induced or constitutive immunity in rainbow trout families with differential resistance to Infectious hematopoietic necrosis virus (IHNV)

    Science.gov (United States)

    Purcell, M.K.; LaPatra, S.E.; Woodson, J.C.; Kurath, G.; Winton, J.R.

    2010-01-01

    The main objective of this study was to assess correlates of innate resistance in rainbow trout full-sibling families that differ in susceptibility to Infectious hematopoietic necrosis virus (IHNV). As part of a commercial breeding program, full-sibling families were challenged with IHNV by waterborne exposure at the 1 g size to determine susceptibility to IHNV. Progeny from select families (N = 7 families) that varied in susceptibility (ranging from 32 to 90% cumulative percent mortality (CPM)) were challenged again at the 10 g size by intra-peritoneal injection and overall mortality, early viral replication and immune responses were evaluated. Mortality challenges included 20–40 fish per family while viral replication and immune response studies included 6 fish per family at each time point (24, 48 and 72 h post-infection (hpi)). CPM at the 1 g size was significantly correlated with CPM at the 10 g size, indicating that inherent resistance was a stable trait irrespective of size. In the larger fish, viral load was measured by quantitative reverse-transcriptase PCR in the anterior kidney and was a significant predictor of family disease outcome at 48 hpi. Type I interferon (IFN) transcript levels were significantly correlated with an individual's viral load at 48 and 72 hpi, while type II IFN gene expression was significantly correlated with an individual's viral load at 24 and 48 hpi. Mean family type I but not type II IFN gene expression was weakly associated with susceptibility at 72 hpi. There was no association between mean family susceptibility and the constitutive expression of a range of innate immune genes (e.g. type I and II IFN pathway genes, cytokine and viral recognition receptor genes). The majority of survivors from the challenge had detectable serum neutralizing antibody titers but no trend was observed among families. This result suggests that even the most resistant families experienced sufficient levels of viral replication to trigger specific

  18. Design, synthesis and antiviral evaluation of novel heteroarylcarbothioamide derivatives as dual inhibitors of HIV-1 reverse transcriptase-associated RNase H and RDDP functions.

    Science.gov (United States)

    Corona, Angela; Onnis, Valentina; Deplano, Alessandro; Bianco, Giulia; Demurtas, Monica; Distinto, Simona; Cheng, Yung-Chi; Alcaro, Stefano; Esposito, Francesca; Tramontano, Enzo

    2017-08-31

    In the continuous effort to identify new HIV-1 inhibitors endowed with innovative mechanisms, the dual inhibition of different viral functions would provide a significant advantage against drug-resistant variants. The HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H) is the only viral-encoded enzymatic activity that still lacks an efficient inhibitor. We synthesized a library of 3,5-diamino-N-aryl-1H-pyrazole-4-carbothioamide and 4-amino-5-benzoyl-N-phenyl-2-(substituted-amino)-1H-pyrrole-3-carbothioamide derivatives and tested them against RNase H activity. We identified the pyrazolecarbothioamide derivative A15, able to inhibit viral replication and both RNase H and RNA-dependent DNA polymerase (RDDP) RT-associated activities in the low micromolar range. Docking simulations hypothesized its binding to two RT pockets. Site-directed mutagenesis experiments showed that, with respect to wt RT, V108A substitution strongly reduced A15 IC50 values (12.6-fold for RNase H inhibition and 4.7-fold for RDDP), while substitution A502F caused a 9.0-fold increase in its IC50 value for RNase H, not affecting the RDDP inhibition, reinforcing the hypothesis of a dual-site inhibition. Moreover, A15 retained good inhibition potency against three non-nucleoside RT inhibitor (NNRTI)-resistant enzymes, confirming a mode of action unrelated to NNRTIs and suggesting its potential as a lead compound for development of new HIV-1 RT dual inhibitors active against drug-resistant viruses. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. PI3Kδ inhibitor idelalisib in combination with BTK inhibitor ONO/GS-4059 in diffuse large B cell lymphoma with acquired resistance to PI3Kδ and BTK inhibitors.

    Directory of Open Access Journals (Sweden)

    Anella Yahiaoui

    Full Text Available Activated B-cell-like diffuse large B-cell lymphoma relies on B-cell receptor signaling to drive proliferation and survival. Downstream of the B-cell receptor, the key signaling kinases Bruton's tyrosine kinase and phosphoinositide 3-kinase δ offer opportunities for therapeutic intervention by agents such as ibrutinib, ONO/GS-4059, and idelalisib. Combination therapy with such targeted agents could provide enhanced efficacy due to complimentary mechanisms of action. In this study, we describe both the additive interaction of and resistance mechanisms to idelalisib and ONO/GS-4059 in a model of activated B-cell-like diffuse large B-cell lymphoma. Significant tumor regression was observed with a combination of PI3Kδ and Bruton's tyrosine kinase inhibitors in the mouse TMD8 xenograft. Acquired resistance to idelalisib in the TMD8 cell line occurred by loss of phosphatase and tensin homolog and phosphoinositide 3-kinase pathway upregulation, but not by mutation of PIK3CD. Sensitivity to idelalisib could be restored by combining idelalisib and ONO/GS-4059. Further evaluation of targeted inhibitors revealed that the combination of idelalisib and the phosphoinositide-dependent kinase-1 inhibitor GSK2334470 or the AKT inhibitor MK-2206 could partially overcome resistance. Characterization of acquired Bruton's tyrosine kinase inhibitor resistance revealed a novel tumor necrosis factor alpha induced protein 3 mutation (TNFAIP3 Q143*, which led to a loss of A20 protein, and increased p-IκBα. The combination of idelalisib and ONO/GS-4059 partially restored sensitivity in this resistant line. Additionally, a mutation in Bruton's tyrosine kinase at C481F was identified as a mechanism of resistance. The combination activity observed with idelalisib and ONO/GS-4059, taken together with the ability to overcome resistance, could lead to a new therapeutic option in activated B-cell-like diffuse large B-cell lymphoma. A clinical trial is currently underway to

  20. Possible increase in insulin resistance and concealed glucose-coupled potassium-lowering mechanisms during acute coronary syndrome documented by covariance structure analysis.

    Science.gov (United States)

    Ito, Satoshi; Nagoshi, Tomohisa; Minai, Kosuke; Kashiwagi, Yusuke; Sekiyama, Hiroshi; Yoshii, Akira; Kimura, Haruka; Inoue, Yasunori; Ogawa, Kazuo; Tanaka, Toshikazu D; Ogawa, Takayuki; Kawai, Makoto; Yoshimura, Michihiro

    2017-01-01

    Although glucose-insulin-potassium (GIK) therapy ought to be beneficial for ischemic heart disease in general, variable outcomes in many clinical trials of GIK in acute coronary syndrome (ACS) had a controversial impact. This study was designed to examine whether "insulin resistance" is involved in ACS and to clarify other potential intrinsic compensatory mechanisms for GIK tolerance through highly statistical procedure. We compared the degree of insulin resistance during ACS attack and remission phase after treatment in individual patients (n = 104). During ACS, homeostasis model assessment of insulin resistance (HOMA-IR) values were significantly increased (Pcovariance structure analysis with a strong impact (β: 0.398, P = 0.015). Intriguingly, a higher incidence of myocardial infarction relative to unstable angina pectoris, as well as a longer hospitalization period were observed in patients with larger ΔK, indicating that ΔK also reflects disease severity of ACS. Insulin resistance most likely increases during ACS; however, ΔK was positively correlated with plasma glucose level, which overwhelmed insulin resistance condition. The present study with covariance structure analysis suggests that there are potential endogenous glucose-coupled potassium lowering mechanisms, other than insulin, regulating glucose metabolism during ACS.

  1. Hairpin RNA Targeting Multiple Viral Genes Confers Strong Resistance to Rice Black-Streaked Dwarf Virus

    Directory of Open Access Journals (Sweden)

    Fangquan Wang

    2016-05-01

    Full Text Available Rice black-streaked dwarf virus (RBSDV belongs to the genus Fijivirus in the family of Reoviridae and causes severe yield loss in rice-producing areas in Asia. RNA silencing, as a natural defence mechanism against plant viruses, has been successfully exploited for engineering virus resistance in plants, including rice. In this study, we generated transgenic rice lines harbouring a hairpin RNA (hpRNA construct targeting four RBSDV genes, S1, S2, S6 and S10, encoding the RNA-dependent RNA polymerase, the putative core protein, the RNA silencing suppressor and the outer capsid protein, respectively. Both field nursery and artificial inoculation assays of three generations of the transgenic lines showed that they had strong resistance to RBSDV infection. The RBSDV resistance in the segregating transgenic populations correlated perfectly with the presence of the hpRNA transgene. Furthermore, the hpRNA transgene was expressed in the highly resistant transgenic lines, giving rise to abundant levels of 21–24 nt small interfering RNA (siRNA. By small RNA deep sequencing, the RBSDV-resistant transgenic lines detected siRNAs from all four viral gene sequences in the hpRNA transgene, indicating that the whole chimeric fusion sequence can be efficiently processed by Dicer into siRNAs. Taken together, our results suggest that long hpRNA targeting multiple viral genes can be used to generate stable and durable virus resistance in rice, as well as other plant species.

  2. Hairpin RNA Targeting Multiple Viral Genes Confers Strong Resistance to Rice Black-Streaked Dwarf Virus.

    Science.gov (United States)

    Wang, Fangquan; Li, Wenqi; Zhu, Jinyan; Fan, Fangjun; Wang, Jun; Zhong, Weigong; Wang, Ming-Bo; Liu, Qing; Zhu, Qian-Hao; Zhou, Tong; Lan, Ying; Zhou, Yijun; Yang, Jie

    2016-05-11

    Rice black-streaked dwarf virus (RBSDV) belongs to the genus Fijivirus in the family of Reoviridae and causes severe yield loss in rice-producing areas in Asia. RNA silencing, as a natural defence mechanism against plant viruses, has been successfully exploited for engineering virus resistance in plants, including rice. In this study, we generated transgenic rice lines harbouring a hairpin RNA (hpRNA) construct targeting four RBSDV genes, S1, S2, S6 and S10, encoding the RNA-dependent RNA polymerase, the putative core protein, the RNA silencing suppressor and the outer capsid protein, respectively. Both field nursery and artificial inoculation assays of three generations of the transgenic lines showed that they had strong resistance to RBSDV infection. The RBSDV resistance in the segregating transgenic populations correlated perfectly with the presence of the hpRNA transgene. Furthermore, the hpRNA transgene was expressed in the highly resistant transgenic lines, giving rise to abundant levels of 21-24 nt small interfering RNA (siRNA). By small RNA deep sequencing, the RBSDV-resistant transgenic lines detected siRNAs from all four viral gene sequences in the hpRNA transgene, indicating that the whole chimeric fusion sequence can be efficiently processed by Dicer into siRNAs. Taken together, our results suggest that long hpRNA targeting multiple viral genes can be used to generate stable and durable virus resistance in rice, as well as other plant species.

  3. Identification of alpha interferon-induced envelope mutations of hepatitis C virus in vitro associated with increased viral fitness and interferon resistance

    DEFF Research Database (Denmark)

    Serre, Stéphanie B N; Krarup, Henrik B; Bukh, Jens

    2013-01-01

    Alpha interferon (IFN-α) is an essential component of innate antiviral immunity and of treatment regimens for chronic hepatitis C virus (HCV) infection. Resistance to IFN might be important for HCV persistence and failure of IFN-based therapies. Evidence for HCV genetic correlates of IFN resistance...... is limited. Experimental studies were hampered by lack of HCV culture systems. Using genotype (strain) 1a(H77) and 3a(S52) Core-NS2 JFH1-based recombinants, we aimed at identifying viral correlates of IFN-α resistance in vitro. Long-term culture with IFN-α2b in Huh7.5 cells resulted in viral spread...... with acquisition of putative escape mutations in HCV structural and nonstructural proteins. Reverse genetic studies showed that primarily amino acid changes I348T in 1a(H77) E1 and F345V/V414A in 3a(S52) E1/E2 increased viral fitness. Single-cycle assays revealed that I348T and F345V/V414A enhanced viral entry...

  4. Hepatitis C Virus NS3 Inhibitors: Current and Future Perspectives

    Directory of Open Access Journals (Sweden)

    Kazi Abdus Salam

    2013-01-01

    Full Text Available Currently, hepatitis C virus (HCV infection is considered a serious health-care problem all over the world. A good number of direct-acting antivirals (DAAs against HCV infection are in clinical progress including NS3-4A protease inhibitors, RNA-dependent RNA polymerase inhibitors, and NS5A inhibitors as well as host targeted inhibitors. Two NS3-4A protease inhibitors (telaprevir and boceprevir have been recently approved for the treatment of hepatitis C in combination with standard of care (pegylated interferon plus ribavirin. The new therapy has significantly improved sustained virologic response (SVR; however, the adverse effects associated with this therapy are still the main concern. In addition to the emergence of viral resistance, other targets must be continually developed. One such underdeveloped target is the helicase portion of the HCV NS3 protein. This review article summarizes our current understanding of HCV treatment, particularly with those of NS3 inhibitors.

  5. Bromodomain and Extra Terminal (BET Inhibitor Suppresses Macrophage-Driven Steroid-Resistant Exacerbations of Airway Hyper-Responsiveness and Inflammation.

    Directory of Open Access Journals (Sweden)

    Thi Hiep Nguyen

    Full Text Available Exacerbations of asthma are linked to significant decline in lung function and are often poorly controlled by corticosteroid treatment. Clinical investigations indicate that viral and bacterial infections play crucial roles in the onset of steroid-resistant inflammation and airways hyperresponsiveness (AHR that are hallmark features of exacerbations. We have previously shown that interferon γ (IFNγ and lipopolysaccharide (LPS cooperatively activate pulmonary macrophages and induce steroid-resistant airway inflammation and AHR in mouse models. Furthermore, we have established a mouse model of respiratory syncytial virus (RSV-induced exacerbation of asthma, which exhibits macrophage-dependent, steroid-resistant lung disease. Emerging evidence has demonstrated a key role for bromo- and extra-terminal (BET proteins in the regulation of inflammatory gene expression in macrophages. We hypothesised that BET proteins may be involved in the regulation of AHR and airway inflammation in our steroid-resistant exacerbation models.We investigated the effects of a BET inhibitor (I-BET-762 on the development of steroid-resistant AHR and airway inflammation in two mouse models. I-BET-762 administration decreased macrophage and neutrophil infiltration into the airways, and suppressed key inflammatory cytokines in both models. I-BET treatment also suppressed key inflammatory cytokines linked to the development of steroid-resistant inflammation such as monocyte chemoattractant protein 1 (MCP-1, keratinocyte-derived protein chemokine (KC, IFNγ, and interleukin 27 (IL-27. Attenuation of inflammation was associated with suppression of AHR.Our results suggest that BET proteins play an important role in the regulation of steroid-resistant exacerbations of airway inflammation and AHR. BET proteins may be potential targets for the development of future therapies to treat steroid-resistant inflammatory components of asthma.

  6. Neutralizing antibody and anti-retroviral drug sensitivities of HIV-1 isolates resistant to small molecule CCR5 inhibitors

    International Nuclear Information System (INIS)

    Pugach, Pavel; Ketas, Thomas J.; Michael, Elizabeth; Moore, John P.

    2008-01-01

    The small molecule CCR5 inhibitors are a new class of drugs for treating infection by human immunodeficiency virus type 1 (HIV-1). They act by binding to the CCR5 co-receptor and preventing its use during HIV-1-cell fusion. Escape mutants can be raised against CCR5 inhibitors in vitro and will arise when these drugs are used clinically. Here, we have assessed the responses of CCR5 inhibitor-resistant viruses to other anti-retroviral drugs that act by different mechanisms, and their sensitivities to neutralizing antibodies (NAbs). The rationale for the latter study is that the resistance pathway for CCR5 inhibitors involves changes in the HIV-1 envelope glycoproteins (Env), which are also targets for NAbs. The escape mutants CC101.19 and D1/85.16 were selected for resistance to AD101 and vicriviroc (VVC), respectively, from the primary R5 HIV-1 isolate CC1/85. Each escape mutant was cross-resistant to other small molecule CCR5 inhibitors (aplaviroc, maraviroc, VVC, AD101 and CMPD 167), but sensitive to protein ligands of CCR5: the modified chemokine PSC-RANTES and the humanized MAb PRO-140. The resistant viruses also retained wild-type sensitivity to the nucleoside reverse transcriptase inhibitor (RTI) zidovudine, the non-nucleoside RTI nevirapine, the protease inhibitor atazanavir and other attachment and fusion inhibitors that act independently of CCR5 (BMS-806, PRO-542 and enfuvirtide). Of note is that the escape mutants were more sensitive than the parental CC1/85 isolate to a subset of neutralizing monoclonal antibodies and to some sera from HIV-1-infected people, implying that sequence changes in Env that confer resistance to CCR5 inhibitors can increase the accessibility of some NAb epitopes. The need to preserve NAb resistance may therefore be a constraint upon how escape from CCR5 inhibitors occurs in vivo

  7. Antibacterial efficacy of Nisin, Pediocin 34 and Enterocin FH99 against Listeria monocytogenes and cross resistance of its bacteriocin resistant variants to common food preservatives.

    Science.gov (United States)

    Kaur, G; Singh, T P; Malik, R K

    2013-01-01

    Antilisterial efficiency of three bacteriocins, viz, Nisin, Pediocin 34 and Enterocin FH99 was tested individually and in combination against Listeria mononcytogenes ATCC 53135. A greater antibacterial effect was observed when the bacteriocins were combined in pairs, indicating that the use of more than one LAB bacteriocin in combination have a higher antibacterial action than when used individually. Variants of Listeria monocytogenes ATCC 53135 resistant to Nisin, Pediocin 34 and Enterocin FH99 were developed. Bacteriocin cross-resistance of wild type and their corresponding resistant variants were assessed and results showed that resistance to a bacteriocin may extend to other bacteriocins within the same class. Resistance to Pediocin 34 conferred cross resistance to Enterocin FH 99 but not to Nisin. Similarly resistance to Enterocin FH99 conferred cross resistance to Pediocin 34 but not to Nisin. Also, the sensitivity of Nisin, Pediocin 34 and Enterocin FH99 resistant variants of Listeria monocytogenes to low pH, salt, sodium nitrite, and potassium sorbate was assayed in broth and compared to the parental wild-type strain. The Nisin, Pediocin 34 and Enterocin FH99 resistant variants did not have intrinsic resistance to low pH, sodium chloride, potassium sorbate, or sodium nitrite. In no case were the bacteriocin resistant Listeria monocytogenes variants examined were more resistant to inhibitors than the parental strains.

  8. Antibacterial efficacy of Nisin, Pediocin 34 and Enterocin FH99 against Listeria monocytogenes and cross resistance of its bacteriocin resistant variants to common food preservatives

    Directory of Open Access Journals (Sweden)

    G. Kaur

    2013-01-01

    Full Text Available Antilisterial efficiency of three bacteriocins, viz, Nisin, Pediocin 34 and Enterocin FH99 was tested individually and in combination against Listeria mononcytogenes ATCC 53135. A greater antibacterial effect was observed when the bacteriocins were combined in pairs, indicating that the use of more than one LAB bacteriocin in combination have a higher antibacterial action than when used individually. Variants of Listeria monocytogenes ATCC 53135 resistant to Nisin, Pediocin 34 and Enterocin FH99 were developed. Bacteriocin cross-resistance of wild type and their corresponding resistant variants were assessed and results showed that resistance to a bacteriocin may extend to other bacteriocins within the same class. Resistance to Pediocin 34 conferred cross resistance to Enterocin FH 99 but not to Nisin. Similarly resistance to Enterocin FH99 conferred cross resistance to Pediocin 34 but not to Nisin. Also, the sensitivity of Nisin, Pediocin 34 and Enterocin FH99 resistant variants of Listeria monocytogenes to low pH, salt, sodium nitrite, and potassium sorbate was assayed in broth and compared to the parental wild-type strain. The Nisin, Pediocin 34 and Enterocin FH99 resistant variants did not have intrinsic resistance to low pH, sodium chloride, potassium sorbate, or sodium nitrite. In no case were the bacteriocin resistant Listeria monocytogenes variants examined were more resistant to inhibitors than the parental strains.

  9. A significant reduction in the frequency of HIV-1 drug resistance in Québec from 2001 to 2011 is associated with a decrease in the monitored viral load.

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    Hugues Charest

    Full Text Available BACKGROUND: HIV drug resistance represents a major threat for effective treatment. We assessed the trends in the frequency of drug resistance mutations and the monitored viral load (VL in treatment-naïve (TN and treatment-experienced (TE individuals infected with HIV-1 in Québec, Canada, between 2001 and 2011. METHODS AND FINDINGS: Resistance data were obtained from 4,105 and 5,086 genotypic tests performed on TN and TE patients, respectively. Concomitantly, 274,161 VL tests were carried out in the Province. Changes over time in drug resistance frequency and in different categories of VL were assessed using univariate logistic regression. Multiple logistic regression was used to evaluate associations between the rates of certain mutations and antiretroviral prescriptions. From 2001 to 2011, the proportion of undetectable VL test results continually increased, from 42.1% to 75.9%, while a significant decrease in the frequency of resistance mutations associated with protease inhibitors [PI (from 54% to 16%], nucleoside [NRTI (from 78% to 37% and non-nucleoside reverse transcriptase inhibitors [NNRTI (from 44% to 31%] was observed in TE patients. In TN individuals, the overall frequency of transmitted drug resistance was 13.1%. A multiple logistic regression analysis indicated that the introduction of co-formulated emtricitabine/tenofovir or emtricitabine/tenofovir/efavirenz was positively associated with the decrease of the frequency of the M184I/V mutations observed overtime (p = 0.0004. CONCLUSIONS: We observed a significant decrease in the frequency of drug resistance mutations in TE patients, concomitant with a decrease in the proportion of patients with detectable viremia. These findings may be related to both the increased potencies and adherence to therapy associated with newer antiretroviral regimens. Nevertheless, our data demonstrate that broad use of antiretrovirals does not increase the level of circulating drug resistant

  10. A novel small molecule inhibitor of hepatitis C virus entry.

    Directory of Open Access Journals (Sweden)

    Carl J Baldick

    Full Text Available Small molecule inhibitors of hepatitis C virus (HCV are being developed to complement or replace treatments with pegylated interferons and ribavirin, which have poor response rates and significant side effects. Resistance to these inhibitors emerges rapidly in the clinic, suggesting that successful therapy will involve combination therapy with multiple inhibitors of different targets. The entry process of HCV into hepatocytes represents another series of potential targets for therapeutic intervention, involving viral structural proteins that have not been extensively explored due to experimental limitations. To discover HCV entry inhibitors, we utilized HCV pseudoparticles (HCVpp incorporating E1-E2 envelope proteins from a genotype 1b clinical isolate. Screening of a small molecule library identified a potent HCV-specific triazine inhibitor, EI-1. A series of HCVpp with E1-E2 sequences from various HCV isolates was used to show activity against all genotype 1a and 1b HCVpp tested, with median EC50 values of 0.134 and 0.027 µM, respectively. Time-of-addition experiments demonstrated a block in HCVpp entry, downstream of initial attachment to the cell surface, and prior to or concomitant with bafilomycin inhibition of endosomal acidification. EI-1 was equally active against cell-culture adapted HCV (HCVcc, blocking both cell-free entry and cell-to-cell transmission of virus. HCVcc with high-level resistance to EI-1 was selected by sequential passage in the presence of inhibitor, and resistance was shown to be conferred by changes to residue 719 in the carboxy-terminal transmembrane anchor region of E2, implicating this envelope protein in EI-1 susceptibility. Combinations of EI-1 with interferon, or inhibitors of NS3 or NS5A, resulted in additive to synergistic activity. These results suggest that inhibitors of HCV entry could be added to replication inhibitors and interferons already in development.

  11. A targeted enzyme approach to sensitization of tyrosine kinase inhibitor-resistant breast cancer cells.

    Science.gov (United States)

    Giordano, Courtney R; Mueller, Kelly L; Terlecky, Laura J; Krentz, Kendra A; Bollig-Fischer, Aliccia; Terlecky, Stanley R; Boerner, Julie L

    2012-10-01

    Gefitinib is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) of potential use in patients with breast cancer. Unfortunately, in clinical studies, gefitinib is often ineffective indicating that resistance to EGFR inhibitors may be a common occurrence in cancer of the breast. EGFR has been shown to be overexpressed in breast cancer, and in particular remains hyperphosphorylated in cell lines such as MDA-MB-468 that are resistant to EGFR inhibitors. Here, we investigate the cause of this sustained phosphorylation and the molecular basis for the ineffectiveness of gefitinib. We show that reactive oxygen species (ROS), known to damage cellular macromolecules and to modulate signaling cascades in a variety of human diseases including cancers, appear to play a critical role in mediating EGFR TKI-resistance. Furthermore, elimination of these ROS through use of a cell-penetrating catalase derivative sensitizes the cells to gefitinib. These results suggest a new approach for the treatment of TKI-resistant breast cancer patients specifically, the targeting of ROS and attendant downstream oxidative stress and their effects on signaling cascades. Copyright © 2012. Published by Elsevier Inc.

  12. Anhedonia Predicts Poorer Recovery among Youth with Selective Serotonin Reuptake Inhibitor Treatment-Resistant Depression

    Science.gov (United States)

    McMakin, Dana L.; Olino, Thomas M.; Porta, Giovanna; Dietz, Laura J.; Emslie, Graham; Clarke, Gregory; Wagner, Karen Dineen; Asarnow, Joan R.; Ryan, Neal D.; Birmaher, Boris; Shamseddeen, Wael; Mayes, Taryn; Kennard, Betsy; Spirito, Anthony; Keller, Martin; Lynch, Frances L.; Dickerson, John F.; Brent, David A.

    2012-01-01

    Objective: To identify symptom dimensions of depression that predict recovery among selective serotonin reuptake inhibitor (SSRI) treatment-resistant adolescents undergoing second-step treatment. Method: The Treatment of Resistant Depression in Adolescents (TORDIA) trial included 334 SSRI treatment-resistant youth randomized to a medication…

  13. Virtual screening of the inhibitors targeting at the viral protein 40 of Ebola virus.

    Science.gov (United States)

    Karthick, V; Nagasundaram, N; Doss, C George Priya; Chakraborty, Chiranjib; Siva, R; Lu, Aiping; Zhang, Ge; Zhu, Hailong

    2016-02-17

    The Ebola virus is highly pathogenic and destructive to humans and other primates. The Ebola virus encodes viral protein 40 (VP40), which is highly expressed and regulates the assembly and release of viral particles in the host cell. Because VP40 plays a prominent role in the life cycle of the Ebola virus, it is considered as a key target for antiviral treatment. However, there is currently no FDA-approved drug for treating Ebola virus infection, resulting in an urgent need to develop effective antiviral inhibitors that display good safety profiles in a short duration. This study aimed to screen the effective lead candidate against Ebola infection. First, the lead molecules were filtered based on the docking score. Second, Lipinski rule of five and the other drug likeliness properties are predicted to assess the safety profile of the lead candidates. Finally, molecular dynamics simulations was performed to validate the lead compound. Our results revealed that emodin-8-beta-D-glucoside from the Traditional Chinese Medicine Database (TCMD) represents an active lead candidate that targets the Ebola virus by inhibiting the activity of VP40, and displays good pharmacokinetic properties. This report will considerably assist in the development of the competitive and robust antiviral agents against Ebola infection.

  14. Sensitization of multidrug-resistant human cancer cells to Hsp90 inhibitors by down-regulation of SIRT1

    Science.gov (United States)

    Kim, Hak-Bong; Lee, Su-Hoon; Um, Jee-Hyun; Oh, Won Keun; Kim, Dong-Wan; Kang, Chi-Dug; Kim, Sun-Hee

    2015-01-01

    The effectiveness of Hsp90 inhibitors as anticancer agents was limited in multidrug-resistant (MDR) human cancer cells due to induction of heat shock proteins (Hsps) such as Hsp70/Hsp27 and P-glycoprotein (P-gp)-mediated efflux. In the present study, we showed that resistance to Hsp90 inhibitors of MDR human cancer cells could be overcome with SIRT1 inhibition. SIRT1 knock-down or SIRT1 inhibitors (amurensin G and EX527) effectively suppressed the resistance to Hsp90 inhibitors (17-AAG and AUY922) in several MDR variants of human lymphoblastic leukemia and human breast cancer cell lines. SIRT1 inhibition down-regulated the expression of heat shock factor 1 (HSF1) and subsequently Hsps and facilitated Hsp90 multichaperone complex disruption via hyperacetylation of Hsp90/Hsp70. These findings were followed by acceleration of ubiquitin ligase CHIP-mediated mutant p53 (mut p53) degradation and subsequent down-regulation of P-gp in 17-AAG-treated MDR cancer cells expressing P-gp and mut p53 after inhibition of SIRT1. Therefore, combined treatment with Hsp90 inhibitor and SIRT1 inhibitor could be a more effective therapeutic approach for Hsp90 inhibitor-resistant MDR cells via down-regulation of HSF1/Hsps, mut p53 and P-gp. PMID:26416354

  15. Overcoming imatinib resistance using Src inhibitor CGP76030, Abl inhibitor nilotinib and Abl/Lyn inhibitor INNO-406 in newly established K562 variants with BCR-ABL gene amplification.

    Science.gov (United States)

    Morinaga, Koji; Yamauchi, Takahiro; Kimura, Shinya; Maekawa, Taira; Ueda, Takanori

    2008-06-01

    Because imatinib (IM) resistance in chronic myeloid leukemia is primarily caused by the re-establishment of Abl kinase, new inhibitors may be efficacious. We evaluated 3 new agents against 2 new K562 variants, IM-R1 and IM-R2 cells, which were developed having 7- and 27-fold greater IM resistance, respectively, than the parental K562 cells. Both variants possessed BCR-ABL gene amplification along with elevated levels of its transcript and protein. Greater BCR-ABL gene amplification was observed in IM-R2 cells than in IM-R1 cells, which was consistent with the higher mRNA and protein levels of Bcr-Abl, and ultimately correlated with the greater IM resistance in IM-R2 cells. No mutation in the Abl kinase domain was detected in either variant. Despite the absence of Lyn overexpression, the Src kinase inhibitor CGP76030 showed positive cooperability with IM in inhibiting cell growth of not only K562 cells but also these 2 variants. This might be because of the augmented inhibition of Erk1/2 phosphorylation. The new Abl kinase inhibitor nilotinib was 10-fold more potent than IM in inhibiting the growth of K562 cells. Nilotinib inhibited the growth of IM-R1 and IM-R2 cells as potently as K562 cells. The combination of nilotinib with CGP76030 showed little additivity, because the potency of nilotinib masked the efficacy of CGP76030. The new dual Abl/Lyn inhibitor INNO-406 (formerly NS-187) was slightly more potent than nilotinib in inhibiting the growth of all 3 cell lines. Because BCR-ABL gene amplification occurs in blast crisis, these inhibitors might overcome IM resistance in such patients' leukemia. (c) 2008 Wiley-Liss, Inc.

  16. The role of multikinase inhibitors target therapy in radioiodine-resistant differentiated thyroid cancer

    Directory of Open Access Journals (Sweden)

    P O Rumyantsev

    2015-06-01

    Full Text Available About 5-15% of patients with differentiated thyroid cancer (DTC primary or within follow-up have had distant metastases or inoperable tumor mass that are resistant to radioiodine therapy as well as dramatically deteriorate survival prognosis. Other treatment modalities (radiotherapy, chemotherapy etc. also ineffective. Certain expectances are associated with target therapy with multikinase inhibitors with are selectively blocking onco-kinase molecular pathways. This review is devoted to analysis of those multikinase inhibitors which have been implemented in patients with radioiodine DTC. Comparative analysis of two most perspective multikinase inhibitors (sorafenib and lenvatinib with evaluation of efficacy and adverse effects was conducted. Both of them successfully underwent 3 rd phase of clinical trial and were recommended as treatment of choice in progressive radioiodine-resistant DTC patients.

  17. Fluoxetine Is a Potent Inhibitor of Coxsackievirus Replication

    OpenAIRE

    Zuo, Jun; Quinn, Kevin K.; Kye, Steve; Cooper, Paige; Damoiseaux, Robert; Krogstad, Paul

    2012-01-01

    No antiviral drugs currently exist for the treatment of enterovirus infections, which are often severe and potentially life threatening. Molecular screening of small molecule libraries identified fluoxetine, a selective serotonin reuptake inhibitor, as a potent inhibitor of coxsackievirus replication. Fluoxetine did not interfere with either viral entry or translation of the viral genome. Instead, fluoxetine and its metabolite norfluoxetine markedly reduced the synthesis of viral RNA and prot...

  18. An effective HIV-1 integrase inhibitor screening platform: Rationality validation of drug screening, conformational mobility and molecular recognition analysis for PFV integrase complex with viral DNA.

    Science.gov (United States)

    Du, Wenyi; Zuo, Ke; Sun, Xin; Liu, Wei; Yan, Xiao; Liang, Li; Wan, Hua; Chen, Fengzheng; Hu, Jianping

    2017-11-01

    As an important target for the development of novel anti-AIDS drugs, HIV-1 integrase (IN) has been widely concerned. However, the lack of a complete accurate crystal structure of HIV-1 IN greatly blocks the discovery of novel inhibitors. In this work, an effective HIV-1 IN inhibitor screening platform, namely PFV IN, was filtered from all species of INs. Next, the 40.8% similarity with HIV-1 IN, as well as the high efficiency of virtual screening and the good agreement between calculated binding free energies and experimental ones all proved PFV IN is a promising screening platform for HIV-1 IN inhibitors. Then, the molecular recognition mechanism of PFV IN by its substrate viral DNA and six naphthyridine derivatives (NRDs) inhibitors was investigated through molecular docking, molecular dynamics simulations and water-mediated interactions analyses. The functional partition of NRDs IN inhibitors could be divided into hydrophobic and hydrophilic ones, and the Mg 2+ ions, water molecules and conserved DDE motif residues all interacted with the hydrophilic partition, while the bases in viral DNA and residues like Tyr212, Pro214 interacted with the hydrophobic one. Finally, the free energy landscape (FEL) and cluster analyses were performed to explore the molecular motion of PFV IN-DNA system. It is found that the association with NRDs inhibitors would obviously decrease the motion amplitude of PFV IN-DNA, which may be one of the most potential mechanisms of IN inhibitors. This work will provide a theoretical basis for the inhibitor design based on the structure of HIV-1 IN. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Prevalence of HIV-1 drug resistance in treated patients with viral load >50 copies/mL: a 2014 French nationwide study.

    Science.gov (United States)

    Assoumou, L; Charpentier, C; Recordon-Pinson, P; Grudé, M; Pallier, C; Morand-Joubert, L; Fafi-Kremer, S; Krivine, A; Montes, B; Ferré, V; Bouvier-Alias, M; Plantier, J-C; Izopet, J; Trabaud, M-A; Yerly, S; Dufayard, J; Alloui, C; Courdavault, L; Le Guillou-Guillemette, H; Maillard, A; Amiel, C; Vabret, A; Roussel, C; Vallet, S; Guinard, J; Mirand, A; Beby-Defaux, A; Barin, F; Allardet-Servent, A; Ait-Namane, R; Wirden, M; Delaugerre, C; Calvez, V; Chaix, M-L; Descamps, D; Reigadas, S

    2017-06-01

    Surveillance of HIV-1 resistance in treated patients with a detectable viral load (VL) is important to monitor, in order to assess the risk of spread of resistant viruses and to determine the proportion of patients who need new antiretroviral drugs with minimal cross-resistance. The HIV-1 protease and reverse transcriptase (RT) and integrase genes were sequenced in plasma samples from 782 consecutive patients on failing antiretroviral regimens, seen in 37 specialized centres in 2014. The genotyping results were interpreted using the ANRS v24 algorithm. Prevalence rates were compared with those obtained during a similar survey conducted in 2009. The protease and RT sequences were obtained in 566 patients, and the integrase sequence in 382 patients. Sequencing was successful in 60%, 78%, 78% and 87% of patients with VLs of 51-200, 201-500, 501-1000 and >1000 copies/mL, respectively. Resistance to at least one antiretroviral drug was detected in 56.3% of samples. Respectively, 3.9%, 8.7%, 1.5% and 3.4% of patients harboured viruses that were resistant to any NRTI, NNRTI, PI and integrase inhibitor (INI). Resistance rates were lower in 2014 than in 2009. Resistance was detected in 48.5% of samples from patients with a VL between 51 and 200 copies/mL. In France in 2014, 90.0% of patients in AIDS care centres were receiving antiretroviral drugs and 12.0% of them had VLs >50 copies/mL. Therefore, this study suggests that 6.7% of treated patients in France might transmit resistant strains. Resistance testing may be warranted in all treated patients with VL > 50 copies/mL. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  20. A Novel, Highly Selective Inhibitor of Pestivirus Replication That Targets the Viral RNA-Dependent RNA Polymerase

    Science.gov (United States)

    Paeshuyse, Jan; Leyssen, Pieter; Mabery, Eric; Boddeker, Nina; Vrancken, Robert; Froeyen, Matheus; Ansari, Israrul H.; Dutartre, Hélène; Rozenski, Jef; Gil, Laura H. V. G.; Letellier, Carine; Lanford, Robert; Canard, Bruno; Koenen, Frank; Kerkhofs, Pierre; Donis, Ruben O.; Herdewijn, Piet; Watson, Julia; De Clercq, Erik; Puerstinger, Gerhard; Neyts, Johan

    2006-01-01

    We report on the highly potent and selective antipestivirus activity of 5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine (BPIP). The 50% effective concentration (EC50) for inhibition of bovine viral diarrhea virus (BVDV)-induced cytopathic effect formation was 0.04 ± 0.01 μM. Comparable reduction of viral RNA synthesis (EC50 = 0.12 ± 0.02 μM) and production of infectious virus (EC50 = 0.074 ± 0.003 μM) were observed. The selectivity index (ratio of 50% cytostatic concentration/EC50) of BPIP was ∼2,000. BPIP was inactive against the hepatitis C virus subgenomic replicon and yellow fever virus but demonstrated weak activity against GB virus. Drug-resistant mutants were at least 300-fold less susceptible to BPIP than wild-type virus; showed cross-resistance to N-propyl-N-[2-(2H-1,2,4-triazino[5,6-b]indol-3-ylthio)ethyl]-1-propanamine (VP32947), and carried the F224S mutation in the viral RNA-dependent RNA polymerase (RdRp). When the F224S mutation was introduced into an infectious clone, the drug-resistant phenotype was obtained. BPIP did not inhibit the in vitro activity of recombinant BVDV RdRp, but did inhibit the activity of replication complexes (RCs). Computational docking revealed that F224 is located at the top of the finger domain of the polymerase. Docking of BPIP in the crystal structure of the BVDV RdRp revealed aromatic ring stacking, some hydrophobic contacts, and a hydrogen bond. Since two structurally unrelated compounds, i.e., BPIP and VP32947, target the same region of the BVDV RdRp, this position may be expected to be critical in the functioning of the polymerase or assembly of the RC. The potential of BPIP for the treatment of pestivirus and hepacivirus infections is discussed. PMID:16352539

  1. Evolution of inhibitor-resistant natural mutant forms of HIV-1 protease probed by pre-steady state kinetic analysis.

    Science.gov (United States)

    Zakharova, Maria Yu; Kuznetsova, Alexandra A; Kaliberda, Elena N; Dronina, Maria A; Kolesnikov, Alexander V; Kozyr, Arina V; Smirnov, Ivan V; Rumsh, Lev D; Fedorova, Olga S; Knorre, Dmitry G; Gabibov, Alexander G; Kuznetsov, Nikita A

    2017-11-01

    Pre-steady state kinetic analysis of mechanistic features of substrate binding and processing is crucial for insight into the evolution of inhibitor-resistant forms of HIV-1 protease. These data may provide a correct vector for rational drug design assuming possible intrinsic dynamic effects. These data should also give some clues to the molecular mechanism of protease action and resistance to inhibitors. Here we report pre-steady state kinetics of the interaction of wild type or mutant forms of HIV-1 protease with a FRET-labeled peptide. The three-stage "minimal" kinetic scheme with first and second reversible steps of substrate binding and with following irreversible peptide cleavage step adequately described experimental data. For the first time, a set of "elementary" kinetic parameters of wild type HIV-1 protease and its natural mutant inhibitor-resistant forms MDR-HM, ANAM-11 and prDRV4 were compared. Inhibitors of the first and second generation were used to estimate the inhibitory effects on HIV-1 protease activity. The resulting set of kinetic data supported that the mutant forms are kinetically unaffected by inhibitors of the first generation, proving their functional resistance to these compounds. The second generation inhibitor darunavir inhibited mutant forms MDR-HM and ANAM-11, but was ineffective against prDRV4. Our kinetic data revealed that these inhibitors induced different conformational changes in the enzyme and, thereby they have different mode of binding in the enzyme active site. These data confirmed hypothesis that the driving force of the inhibitor-resistance evolution is disruption of enzyme-inhibitor complex by changing of the contact network in the inhibitor binding site. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  2. Viral Marketing

    OpenAIRE

    Sorina Raula Gîrboveanu; Silvia Puiu

    2008-01-01

    With consumers showing increasing resistance to traditional forms of advertising such as TV or newspaper ads, marketers have turned to alternate strategies, including viral marketing. Viral marketing exploits existing social networks by encouraging customers to share product information with their friends.In our study we are able to directly observe the effectiveness of person to person word of mouth advertising for hundreds of thousands of products for the first time

  3. Classification of Breast Cancer Resistant Protein (BCRP) Inhibitors and Non-Inhibitors Using Machine Learning Approaches.

    Science.gov (United States)

    Belekar, Vilas; Lingineni, Karthik; Garg, Prabha

    2015-01-01

    The breast cancer resistant protein (BCRP) is an important transporter and its inhibitors play an important role in cancer treatment by improving the oral bioavailability as well as blood brain barrier (BBB) permeability of anticancer drugs. In this work, a computational model was developed to predict the compounds as BCRP inhibitors or non-inhibitors. Various machine learning approaches like, support vector machine (SVM), k-nearest neighbor (k-NN) and artificial neural network (ANN) were used to develop the models. The Matthews correlation coefficients (MCC) of developed models using ANN, k-NN and SVM are 0.67, 0.71 and 0.77, and prediction accuracies are 85.2%, 88.3% and 90.8% respectively. The developed models were tested with a test set of 99 compounds and further validated with external set of 98 compounds. Distribution plot analysis and various machine learning models were also developed based on druglikeness descriptors. Applicability domain is used to check the prediction reliability of the new molecules.

  4. Molecular Phylogenetics of Transmitted Drug Resistance in Newly Diagnosed HIV Type 1 Individuals in Denmark, a Nation-Wide Study

    DEFF Research Database (Denmark)

    Audelin, Anne Margrethe; Gerstoft, Jan; Obel, Niels

    2011-01-01

    Abstract Highly active antiretroviral treatment is compromised by viral resistance mutations. Transmitted drug resistance (TDR) is therefore monitored closely, but follow-up studies of these patients are limited. Virus from 1405 individuals diagnosed with HIV-1 in Denmark between 2001 and 2009...... without resistance mutations. We observed no difference in progression of the infection between individuals infected with TDR and individuals infected with wild-type HIV-1. The prevalence of TDR is low in Denmark and transmission of dual-drug-resistant HIV-1 is infrequent. The TDR isolates were shown...... resulting in a prevalence of 6.1%, with no changes over time. The main resistance mutations were nucleoside reverse transcriptase inhibitor (NRTI) mutation 215 revertants, as well as nonnucleoside reverse transcriptase inhibitor (NNRTI) mutation 103N/S and protease inhibitor (PI) mutations 90M and 85V...

  5. Molecular phylogenetics of transmitted drug resistance in newly diagnosed HIV Type 1 individuals in Denmark: a nation-wide study

    DEFF Research Database (Denmark)

    Audelin, Anne Margrethe; Gerstoft, Jan; Obel, Niels

    2011-01-01

    Abstract Highly active antiretroviral treatment is compromised by viral resistance mutations. Transmitted drug resistance (TDR) is therefore monitored closely, but follow-up studies of these patients are limited. Virus from 1405 individuals diagnosed with HIV-1 in Denmark between 2001 and 2009...... without resistance mutations. We observed no difference in progression of the infection between individuals infected with TDR and individuals infected with wild-type HIV-1. The prevalence of TDR is low in Denmark and transmission of dual-drug-resistant HIV-1 is infrequent. The TDR isolates were shown...... resulting in a prevalence of 6.1%, with no changes over time. The main resistance mutations were nucleoside reverse transcriptase inhibitor (NRTI) mutation 215 revertants, as well as nonnucleoside reverse transcriptase inhibitor (NNRTI) mutation 103N/S and protease inhibitor (PI) mutations 90M and 85V...

  6. Preclinical rationale for PI3K/Akt/mTOR pathway inhibitors as therapy for epidermal growth factor receptor inhibitor-resistant non-small-cell lung cancer.

    Science.gov (United States)

    Gadgeel, Shirish M; Wozniak, Antoinette

    2013-07-01

    Mutations in the epidermal growth factor receptor gene (EGFR) are frequently observed in non-small-cell lung cancer (NSCLC), occurring in about 40% to 60% of never-smokers and in about 17% of patients with adenocarcinomas. EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, have transformed therapy for patients with EGFR-mutant NSCLC and have proved superior to chemotherapy as first-line treatment for this patient group. Despite these benefits, there are currently 2 key challenges associated with EGFR inhibitor therapy for patients with NSCLC. First, only 85% to 90% of patients with the EGFR mutation derive clinical benefit from EGFR TKIs, with the remainder demonstrating innate resistance to therapy. Second, acquired resistance to EGFR TKIs inevitably occurs in patients who initially respond to therapy, with a median duration of response of about 10 months. Mutant EGFR activates various subcellular signaling cascades, including the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway, which demonstrates maintained activity in a variety of TKI-resistant cancers. Given the fundamental role of the PI3K/Akt/mTOR pathway in tumor oncogenesis, proliferation, and survival, PI3K pathway inhibitors have emerged as a possible solution to the problem of EGFR TKI resistance. However resistance to EGFR TKIs is associated with considerable heterogeneity and complexity. Preclinical experiments investigating these phenomena suggest that in some patients, PI3K inhibitors will have to be paired with other targeted agents if they are to be effective. This review discusses the preclinical data supporting PI3K/Akt/mTOR pathway inhibitor combinations in EGFR TKI-resistant NSCLC from the perspective of the various agents currently being investigated in clinical trials. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Efflux as a mechanism of antimicrobial drug resistance in clinical relevant microorganisms: the role of efflux inhibitors.

    Science.gov (United States)

    Willers, Clarissa; Wentzel, Johannes Frederik; du Plessis, Lissinda Hester; Gouws, Chrisna; Hamman, Josias Hendrik

    2017-01-01

    Microbial resistance against antibiotics is a serious threat to the effective treatment of infectious diseases. Several mechanisms exist through which microorganisms can develop resistance against antimicrobial drugs, of which the overexpression of genes to produce efflux pumps is a major concern. Several efflux transporters have been identified in microorganisms, which infer resistance against specific antibiotics and even multidrug resistance. Areas covered: This paper focuses on microbial resistance against antibiotics by means of the mechanism of efflux and gives a critical overview of studies conducted to overcome this problem by combining efflux pump inhibitors with antibiotics. Information was obtained from a literature search done with MEDLINE, Pubmed, Scopus, ScienceDirect, OneSearch and EBSCO host. Expert opinion: Efflux as a mechanism of multidrug resistance has presented a platform for improved efficacy against resistant microorganisms by co-administration of efflux pump inhibitors with antimicrobial agents. Although proof of concept has been shown for this approach with in vitro experiments, further research is needed to develop more potent inhibitors with low toxicity which is clinically effective.

  8. Hepatitis C Virus Resistance to Direct-Acting Antiviral Drugs in Interferon-Free Regimens.

    Science.gov (United States)

    Pawlotsky, Jean-Michel

    2016-07-01

    Treatment of hepatitis C virus (HCV) infection has progressed considerably with the approval of interferon-free, direct-acting antiviral (DAA)-based combination therapies. Although most treated patients achieve virological cure, HCV resistance to DAAs has an important role in the failure of interferon-free treatment regimens. The presence of viral variants resistant to NS5A inhibitors at baseline is associated with lower rates of virological cure in certain groups of patients, such as those with genotype 1a or 3 HCV, those with cirrhosis, and/or prior nonresponders to pegylated interferon-based regimens. DAA-resistant HCV is generally dominant at virological failure (most often relapse). Viruses resistant to NS3-4A protease inhibitors disappear from peripheral blood in a few weeks to months, whereas NS5A inhibitor-resistant viruses persist for years. Re-treatment options are available, but first-line treatment strategies should be optimized to efficiently prevent treatment failure due to HCV resistance. Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.

  9. Activity of dual SRC-ABL inhibitors highlights the role of BCR/ABL kinase dynamics in drug resistance

    Science.gov (United States)

    Azam, Mohammad; Nardi, Valentina; Shakespeare, William C.; Metcalf, Chester A.; Bohacek, Regine S.; Wang, Yihan; Sundaramoorthi, Raji; Sliz, Piotr; Veach, Darren R.; Bornmann, William G.; Clarkson, Bayard; Dalgarno, David C.; Sawyer, Tomi K.; Daley, George Q.

    2006-01-01

    Mutation in the ABL kinase domain is the principal mechanism of imatinib resistance in patients with chronic myelogenous leukemia. Many mutations favor active kinase conformations that preclude imatinib binding. Because the active forms of ABL and SRC resemble one another, we tested two dual SRC-ABL kinase inhibitors, AP23464 and PD166326, against 58 imatinib-resistant (IMR) BCR/ABL kinase variants. Both compounds potently inhibit most IMR variants, and in vitro drug selection demonstrates that active (AP23464) and open (PD166326) conformation-specific compounds are less susceptible to resistance than imatinib. Combinations of inhibitors suppressed essentially all resistance mutations, with the notable exception of T315I. Guided by mutagenesis studies and molecular modeling, we designed a series of AP23464 analogues to target T315I. The analogue AP23846 inhibited both native and T315I variants of BCR/ABL with submicromolar potency but showed nonspecific cellular toxicity. Our data illustrate how conformational dynamics of the ABL kinase accounts for the activity of dual SRC-ABL inhibitors against IMR-mutants and provides a rationale for combining conformation specific inhibitors to suppress resistance. PMID:16754879

  10. Evaluating Andrographolide as a Potent Inhibitor of NS3-4A Protease and Its Drug-Resistant Mutants Using In Silico Approaches

    Directory of Open Access Journals (Sweden)

    Vivek Chandramohan

    2015-01-01

    Full Text Available Current combination therapy of PEG-INF and ribavirin against the Hepatitis C Virus (HCV genotype-1 infections is ineffective in maintaining sustained viral response in 50% of the infection cases. New compounds in the form of protease inhibitors can complement the combination therapy. Asunaprevir is new to the drug regiment as the NS3-4A protease inhibitor, but it is susceptible to two mutations, namely, R155K and D168A in the protein. Thus, in our study, we sought to evaluate Andrographolide, a labdane-diterpenoid from the Andrographis paniculata plant as an effective compound for inhibiting the NS3-4A protease as well as its concomitant drug-resistant mutants by using molecular docking and dynamic simulations. Our study shows that Andrographolide has best docking scores of −15.0862, −15.2322, and −13.9072 compared to those of Asunaprevir −3.7159, −2.6431, and −5.4149 with wild-type R155K and D168A mutants, respectively. Also, as shown in the MD simulations, the compound was good in binding the target proteins and maintains strong bonds causing very less to negligible perturbation in the protein backbone structures. Our results validate the susceptibility of Asunaprevir to protein variants as seen from our docking studies and trajectory period analysis. Therefore, from our study, we hope to add one more option in the drug regiment to tackle drug resistance in HCV infections.

  11. A highly intensified ART regimen induces long-term viral suppression and restriction of the viral reservoir in a simian AIDS model.

    Directory of Open Access Journals (Sweden)

    Iart Luca Shytaj

    Full Text Available Stably suppressed viremia during ART is essential for establishing reliable simian models for HIV/AIDS. We tested the efficacy of a multidrug ART (highly intensified ART in a wide range of viremic conditions (10³-10⁷ viral RNA copies/mL in SIVmac251-infected rhesus macaques, and its impact on the viral reservoir. Eleven macaques in the pre-AIDS stage of the disease were treated with a multidrug combination (highly intensified ART consisting of two nucleosidic/nucleotidic reverse transcriptase inhibitors (emtricitabine and tenofovir, an integrase inhibitor (raltegravir, a protease inhibitor (ritonavir-boosted darunavir and the CCR5 blocker maraviroc. All animals stably displayed viral loads below the limit of detection of the assay (i.e. <40 RNA copies/mL after starting highly intensified ART. By increasing the sensitivity of the assay to 3 RNA copies/mL, viral load was still below the limit of detection in all subjects tested. Importantly, viral DNA resulted below the assay detection limit (<2 copies of DNA/5*10⁵ cells in PBMCs and rectal biopsies of all animals at the end of the follow-up, and in lymph node biopsies from the majority of the study subjects. Moreover, highly intensified ART decreased central/transitional memory, effector memory and activated (HLA-DR⁺ effector memory CD4⁺ T-cells in vivo, in line with the role of these subsets as the main cell subpopulations harbouring the virus. Finally, treatment with highly intensified ART at viral load rebound following suspension of a previous anti-reservoir therapy eventually improved the spontaneous containment of viral load following suspension of the second therapeutic cycle, thus leading to a persistent suppression of viremia in the absence of ART. In conclusion, we show, for the first time, complete suppression of viral load by highly intensified ART and a likely associated restriction of the viral reservoir in the macaque AIDS model, making it a useful platform for testing

  12. Drug Resistance to EGFR Inhibitors in Lung Cancer | Office of Cancer Genomics

    Science.gov (United States)

    The discovery of mutations in epidermal growth factor receptor (EGFR) has dramatically changed the treatment of patients with non-small-cell lung cancer (NSCLC), the leading cause of cancer deaths worldwide. EGFR-targeted therapies show considerable promise, but drug resistance has become a substantial issue. We reviewed the literature to provide an overview of the drug resistance to EGFR tyrosine kinase inhibitors (TKIs) in NSCLC. The mechanisms causing primary, acquired and persistent drug resistance to TKIs vary.

  13. A resurgence of β-lactamase inhibitor combinations effective against multidrug-resistant Gram-negative pathogens.

    Science.gov (United States)

    Bush, Karen

    2015-11-01

    β-Lactamase inhibitors (BLIs) have played an important role in combatting β-lactam resistance in Gram-negative bacteria, but their effectiveness has diminished with the evolution of diverse and deleterious varieties of β-lactamases. In this review, a new generation of BLIs and inhibitor combinations is presented, describing epidemiological information, pharmacodynamic studies, resistance identification and current clinical status. Novel serine BLIs of major interest include the non-β-lactams of the diazabicyclo[3.2.1]octanone (DBO) series. The DBOs avibactam, relebactam and RG6080 inhibit most class A and class C β-lactamases, with selected inhibition of class D enzymes by avibactam. The novel boronic acid inhibitor RPX7009 has a similar inhibitory profile. All of these inhibitors are being developed in combinations that are targeting primarily carbapenemase-producing Gram-negative pathogens. Two BLI combinations (ceftolozane/tazobactam and ceftazidime/avibactam) were recently approved by the US Food and Drug Administration (FDA) under the designation of a Qualified Infectious Disease Product (QIDP). Other inhibitor combinations that have at least completed phase 1 clinical trials are ceftaroline fosamil/avibactam, aztreonam/avibactam, imipenem/relebactam, meropenem/RPX7009 and cefepime/AAI101. Although effective inhibitor combinations are in development for the treatment of infections caused by Gram-negative bacteria with serine carbapenemases, better options are still necessary for pathogens that produce metallo-β-lactamases (MBLs). The aztreonam/avibactam combination demonstrates inhibitory activity against MBL-producing enteric bacteria owing to the stability of the monobactam to these enzymes, but resistance is still an issue for MBL-producing non-fermentative bacteria. Because all of the inhibitor combinations are being developed as parenteral drugs, an orally bioavailable combination would also be of interest. Copyright © 2015 Elsevier B.V. and the

  14. Kallistatin Ameliorates Influenza Virus Pathogenesis by Inhibition of Kallikrein-Related Peptidase 1-Mediated Cleavage of Viral Hemagglutinin

    Science.gov (United States)

    Leu, Chia-Hsing; Yang, Mei-Lin; Chung, Nai-Hui; Huang, Yen-Jang; Su, Yu-Chu; Chen, Yi-Cheng; Lin, Chia-Cheng; Shieh, Gia-Shing; Chang, Meng-Ya; Wang, Shainn-Wei; Chang, Yao; Chao, Julie; Chao, Lee

    2015-01-01

    Proteolytic cleavage of the hemagglutinin (HA) of influenza virus by host trypsin-like proteases is required for viral infectivity. Some serine proteases are capable of cleaving influenza virus HA, whereas some serine protease inhibitors (serpins) inhibit the HA cleavage in various cell types. Kallikrein-related peptidase 1 (KLK1, also known as tissue kallikrein) is a widely distributed serine protease. Kallistatin, a serpin synthesized mainly in the liver and rapidly secreted into the circulation, forms complexes with KLK1 and inhibits its activity. Here, we investigated the roles of KLK1 and kallistatin in influenza virus infection. We show that the levels of KLK1 increased, whereas those of kallistatin decreased, in the lungs of mice during influenza virus infection. KLK1 cleaved H1, H2, and H3 HA molecules and consequently enhanced viral production. In contrast, kallistatin inhibited KLK1-mediated HA cleavage and reduced viral production. Cells transduced with the kallistatin gene secreted kallistatin extracellularly, which rendered them more resistant to influenza virus infection. Furthermore, lentivirus-mediated kallistatin gene delivery protected mice against lethal influenza virus challenge by reducing the viral load, inflammation, and injury in the lung. Taking the data together, we determined that KLK1 and kallistatin contribute to the pathogenesis of influenza virus by affecting the cleavage of the HA peptide and inflammatory responses. This study provides a proof of principle for the potential therapeutic application of kallistatin or other KLK1 inhibitors for influenza. Since proteolytic activation also enhances the infectivity of some other viruses, kallistatin and other kallikrein inhibitors may be explored as antiviral agents against these viruses. PMID:26149981

  15. HDAC Inhibitors Disrupt Programmed Resistance to Apoptosis During Drosophila Development

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    Yunsik Kang

    2017-06-01

    Full Text Available We have previously shown that the ability to respond to apoptotic triggers is regulated during Drosophila development, effectively dividing the fly life cycle into stages that are either sensitive or resistant to apoptosis. Here, we show that the developmentally programmed resistance to apoptosis involves transcriptional repression of critical proapoptotic genes by histone deacetylases (HDACs. Administration of HDAC inhibitors (HDACi, like trichostatin A or suberoylanilide hydroxamic acid, increases expression of proapoptotic genes and is sufficient to sensitize otherwise resistant stages. Conversely, reducing levels of proapoptotic genes confers resistance to otherwise sensitive stages. Given that resistance to apoptosis is a hallmark of cancer cells, and that HDACi have been recently added to the repertoire of FDA-approved agents for cancer therapy, our results provide new insights for how HDACi help kill malignant cells and also raise concerns for their potential unintended effects on healthy cells.

  16. Raltegravir: first in class HIV integrase inhibitor

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    Zelalem Temesgen

    2008-06-01

    Full Text Available Zelalem Temesgen1, Dawd S Siraj21Mayo Clinic, Rochester, MN, USA; 2East Carolina University Greenville, NC, USAAbstract: On October 16, 2007, the US Food and Drug Administration (FDA approved raltegravir for treatment of human immunodeficiency virus (HIV-1 infection in combination with other antiretroviral agents in treatment-experienced adult patients who have evidence of viral replication and HIV-1 strains resistant to multiple antiretroviral agents. Raltegravir is first in a novel class of antiretroviral drugs known as integrase inhibitors. It has demonstrated potent anti HIV activity in both antiretroviral treatment-naïve and experienced patients. The most common adverse events reported with raltegravir during phase 2 and 3 clinical trials were diarrhea, nausea, and headache. Laboratory abnormalities include mild elevations in liver transaminases and creatine phosphokinase.Keywords: raltegravir, HIV, antiretroviral agents, integrase inhibitors

  17. Synthesis, antiviral evaluation and molecular docking studies of N4-aryl substituted/unsubstituted thiosemicarbazones derived from 1-indanones as potent anti-bovine viral diarrhea virus agents.

    Science.gov (United States)

    Soraires Santacruz, María C; Fabiani, Matías; Castro, Eliana F; Cavallaro, Lucía V; Finkielsztein, Liliana M

    2017-08-01

    A series of N 4 -arylsubstituted thiosemicarbazones derived from 1-indanones and a set of compounds lacking such substitution in the N 4 position of the thiosemicarbazone moiety were synthesized and evaluated for their anti-bovine viral diarrhea virus (BVDV) activity. Among these, derivatives 2 and 15 displayed high activity (EC 50 =2.7±0.4 and 0.7±0.1µM, respectively) as inhibitors of BVDV replication. Novel key structural features related to the anti-BVDV activity were identified by structure-activity relationship (SAR) analysis. In a previous study, the thiosemicarbazone of 5,6-dimethoxy-1-indanone (5,6-TSC) was characterized as a non-nucleoside inhibitor (NNI) of the BVDV RNA-dependent RNA polymerase. In the present work, cross-resistance assays were performed with the most active compounds. Such studies were carried out on 5,6-TSC resistant BVDV (BVDV-TSC r T1) carrying mutations in the viral polymerase. This BVDV mutant was also resistant to compound 15. Molecular docking studies and MM/PBSA calculations were performed to assess the most active derivatives at the 5,6-TSC viral polymerase binding site. The differences in the interaction pattern and the binding affinity of derivative 15 either to the wild type or BVDV-TSC r T1 polymerase were key factors to define the mode of action of this compound. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Genetically pyramiding protease-inhibitor genes for dual broad-spectrum resistance against insect and phytopathogens in transgenic tobacco.

    Science.gov (United States)

    Senthilkumar, Rajendran; Cheng, Chiu-Ping; Yeh, Kai-Wun

    2010-01-01

    Protease inhibitors provide a promising means of engineering plant resistance against attack by insects and pathogens. Sporamin (trypsin inhibitor) from sweet potato and CeCPI (phytocystatin) from taro were stacked in a binary vector, using pMSPOA (a modified sporamin promoter) to drive both genes. Transgenic tobacco lines of T0 and T1 generation with varied inhibitory activity against trypsin and papain showed resistance to both insects and phytopathogens. Larvae of Helicoverpa armigera that ingested tobacco leaves either died or showed delayed growth and development relative to control larvae. Transgenic tobacco-overexpressing the stacked genes also exhibited strong resistance against bacterial soft rot disease caused by Erwinia carotovora and damping-off disease caused by Pythium aphanidermatum. Thus, stacking protease-inhibitor genes, driven by the wound and pathogen responsive pMSPOA promoter, is an effective strategy for engineering crops to resistance against insects and phytopathogens.

  19. The structure of FIV reverse transcriptase and its implications for non-nucleoside inhibitor resistance.

    Directory of Open Access Journals (Sweden)

    Meytal Galilee

    2018-01-01

    Full Text Available Reverse transcriptase (RT is the target for the majority of anti-HIV-1 drugs. As with all anti-AIDS treatments, continued success of RT inhibitors is persistently disrupted by the occurrence of resistance mutations. To explore latent resistance mechanisms potentially accessible to therapeutically challenged HIV-1 viruses, we examined RT from the related feline immunodeficiency virus (FIV. FIV closely parallels HIV-1 in its replication and pathogenicity, however, is resistant to all non-nucleoside inhibitors (NNRTI. The intrinsic resistance of FIV RT is particularly interesting since FIV harbors the Y181 and Y188 sensitivity residues absent in both HIV-2 and SIV. Unlike RT from HIV-2 or SIV, previous efforts have failed to make FIV RT susceptible to NNRTIs concluding that the structure or flexibility of the feline enzyme must be profoundly different. We report the first crystal structure of FIV RT and, being the first structure of an RT from a non-primate lentivirus, enrich the structural and species repertoires available for RT. The structure demonstrates that while the NNRTI binding pocket is conserved, minor subtleties at the entryway can render the FIV RT pocket more restricted and unfavorable for effective NNRTI binding. Measuring NNRTI binding affinity to FIV RT shows that the "closed" pocket configuration inhibits NNRTI binding. Mutating the loop residues rimming the entryway of FIV RT pocket allows for NNRTI binding, however, it does not confer sensitivity to these inhibitors. This reveals a further layer of resistance caused by inherent FIV RT variances that could have enhanced the dissociation of bound inhibitors, or, perhaps, modulated protein plasticity to overcome inhibitory effects of bound NNRTIs. The more "closed" conformation of FIV RT pocket can provide a template for the development of innovative drugs that could unlock the constrained pocket, and the resilient mutant version of the enzyme can offer a fresh model for the study

  20. Sifuvirtide, a potent HIV fusion inhibitor peptide

    International Nuclear Information System (INIS)

    Wang, Rui-Rui; Yang, Liu-Meng; Wang, Yun-Hua; Pang, Wei; Tam, Siu-Cheung; Tien, Po; Zheng, Yong-Tang

    2009-01-01

    Enfuvirtide (ENF) is currently the only FDA approved HIV fusion inhibitor in clinical use. Searching for more drugs in this category with higher efficacy and lower toxicity seems to be a logical next step. In line with this objective, a synthetic peptide with 36 amino acid residues, called Sifuvirtide (SFT), was designed based on the crystal structure of gp41. In this study, we show that SFT is a potent anti-HIV agent with relatively low cytotoxicity. SFT was found to inhibit replication of all tested HIV strains. The effective concentrations that inhibited 50% viral replication (EC 50 ), as determined in all tested strains, were either comparable or lower than benchmark values derived from well-known anti-HIV drugs like ENF or AZT, while the cytotoxic concentrations causing 50% cell death (CC 50 ) were relatively high, rendering it an ideal anti-HIV agent. A GST-pull down assay was performed to confirm that SFT is a fusion inhibitor. Furthermore, the activity of SFT on other targets in the HIV life cycle was also investigated, and all assays showed negative results. To further understand the mechanism of action of HIV peptide inhibitors, resistant variants of HIV-1 IIIB were derived by serial virus passage in the presence of increasing doses of SFT or ENF. The results showed that there was cross-resistance between SFT and ENF. In conclusion, SFT is an ideal anti-HIV agent with high potency and low cytotoxicity, but may exhibit a certain extent of cross-resistance with ENF.

  1. Inhibition of bacterial multidrug resistance by celecoxib, a cyclooxygenase-2 inhibitor.

    Science.gov (United States)

    Kalle, Arunasree M; Rizvi, Arshad

    2011-01-01

    Multidrug resistance (MDR) is a major problem in the treatment of infectious diseases and cancer. Accumulating evidence suggests that the cyclooxygenase-2 (COX-2)-specific inhibitor celecoxib would not only inhibit COX-2 but also help in the reversal of drug resistance in cancers by inhibiting the MDR1 efflux pump. Here, we demonstrate that celecoxib increases the sensitivity of bacteria to the antibiotics ampicillin, kanamycin, chloramphenicol, and ciprofloxacin by accumulating the drugs inside the cell, thus reversing MDR in bacteria.

  2. [Bacterial efflux pumps - their role in antibiotic resistance and potential inhibitors].

    Science.gov (United States)

    Hricová, Kristýna; Kolář, Milan

    2014-12-01

    Efflux pumps capable of actively draining antibiotic agents from bacterial cells may be considered one of potential mechanisms of the development of antimicrobial resistance. The most important group of efflux pumps capable of removing several types of antibiotics include RND (resistance - nodulation - division) pumps. These are three proteins that cross the bacterial cell wall, allowing direct expulsion of the agent out from the bacterial cell. The most investigated efflux pumps are the AcrAB-TolC system in Escherichia coli and the MexAB-OprM system in Pseudomonas aeruginosa. Moreover, efflux pumps are able to export other than antibacterial agents such as disinfectants, thus decreasing their effectiveness. One potential approach to inactivation of an efflux pump is to use the so-called efflux pump inhibitors (EPIs). Potential inhibitors tested in vitro involve, for example, phenylalanyl-arginyl-b-naphthylamide (PAbN), carbonyl cyanide m-chlorophenylhydrazone (CCCP) or agents of the phenothiazine class.

  3. Alternative signaling pathways as potential therapeutic targets for overcoming EGFR and c-Met inhibitor resistance in non-small cell lung cancer.

    Directory of Open Access Journals (Sweden)

    Jason T Fong

    Full Text Available The use of tyrosine kinase inhibitors (TKIs against EGFR/c-Met in non-small cell lung cancer (NSCLC has been shown to be effective in increasing patient progression free survival (PFS, but their efficacy is limited due to the development of resistance and tumor recurrence. Therefore, understanding the molecular mechanisms underlying development of drug resistance in NSCLC is necessary for developing novel and effective therapeutic approaches to improve patient outcome. This study aims to understand the mechanism of EGFR/c-Met tyrosine kinase inhibitor (TKI resistance in NSCLC. H2170 and H358 cell lines were made resistant to SU11274, a c-Met inhibitor, and erlotinib, an EGFR inhibitor, through step-wise increases in TKI exposure. The IC50 concentrations of resistant lines exhibited a 4-5 and 11-22-fold increase for SU11274 and erlotinib, respectively, when compared to parental lines. Furthermore, mTOR and Wnt signaling was studied in both cell lines to determine their roles in mediating TKI resistance. We observed a 2-4-fold upregulation of mTOR signaling proteins and a 2- to 8-fold upregulation of Wnt signaling proteins in H2170 erlotinib and SU11274 resistant cells. H2170 and H358 cells were further treated with the mTOR inhibitor everolimus and the Wnt inhibitor XAV939. H358 resistant cells were inhibited by 95% by a triple combination of everolimus, erlotinib and SU11274 in comparison to 34% by a double combination of these drugs. Parental H2170 cells displayed no sensitivity to XAV939, while resistant cells were significantly inhibited (39% by XAV939 as a single agent, as well as in combination with SU11274 and erlotinib. Similar results were obtained with H358 resistant cells. This study suggests a novel molecular mechanism of drug resistance in lung cancer.

  4. The HDAC inhibitor SB939 overcomes resistance to BCR-ABL kinase Inhibitors conferred by the BIM deletion polymorphism in chronic myeloid leukemia.

    Directory of Open Access Journals (Sweden)

    Muhammad Rauzan

    Full Text Available Chronic myeloid leukemia (CML treatment has been improved by tyrosine kinase inhibitors (TKIs such as imatinib mesylate (IM but various factors can cause TKI resistance in patients with CML. One factor which contributes to TKI resistance is a germline intronic deletion polymorphism in the BCL2-like 11 (BIM gene which impairs the expression of pro-apoptotic splice isoforms of BIM. SB939 (pracinostat is a hydroxamic acid based HDAC inhibitor with favorable pharmacokinetic, physicochemical and pharmaceutical properties, and we investigated if this drug could overcome BIM deletion polymorphism-induced TKI resistance. We found that SB939 corrects BIM pre-mRNA splicing in CML cells with the BIM deletion polymorphism, and induces apoptotic cell death in CML cell lines and primary cells with the BIM deletion polymorphism. More importantly, SB939 both decreases the viability of CML cell lines and primary CML progenitors with the BIM deletion and restores TKI-sensitivity. Our results demonstrate that SB939 overcomes BIM deletion polymorphism-induced TKI resistance, and suggest that SB939 may be useful in treating CML patients with BIM deletion-associated TKI resistance.

  5. N348I in the connection domain of HIV-1 reverse transcriptase confers zidovudine and nevirapine resistance.

    Directory of Open Access Journals (Sweden)

    Soo-Huey Yap

    2007-12-01

    Full Text Available The catalytically active 66-kDa subunit of the human immunodeficiency virus type 1 (HIV-1 reverse transcriptase (RT consists of DNA polymerase, connection, and ribonuclease H (RNase H domains. Almost all known RT inhibitor resistance mutations identified to date map to the polymerase domain of the enzyme. However, the connection and RNase H domains are not routinely analysed in clinical samples and none of the genotyping assays available for patient management sequence the entire RT coding region. The British Columbia Centre for Excellence in HIV/AIDS (the Centre genotypes clinical isolates up to codon 400 in RT, and our retrospective statistical analyses of the Centre's database have identified an N348I mutation in the RT connection domain in treatment-experienced individuals. The objective of this multidisciplinary study was to establish the in vivo relevance of this mutation and its role in drug resistance.The prevalence of N348I in clinical isolates, the time taken for it to emerge under selective drug pressure, and its association with changes in viral load, specific drug treatment, and known drug resistance mutations was analysed from genotypes, viral loads, and treatment histories from the Centre's database. N348I increased in prevalence from below 1% in 368 treatment-naïve individuals to 12.1% in 1,009 treatment-experienced patients (p = 7.7 x 10(-12. N348I appeared early in therapy and was highly associated with thymidine analogue mutations (TAMs M41L and T215Y/F (p < 0.001, the lamivudine resistance mutations M184V/I (p < 0.001, and non-nucleoside RTI (NNRTI resistance mutations K103N and Y181C/I (p < 0.001. The association with TAMs and NNRTI resistance mutations was consistent with the selection of N348I in patients treated with regimens that included both zidovudine and nevirapine (odds ratio 2.62, 95% confidence interval 1.43-4.81. The appearance of N348I was associated with a significant increase in viral load (p < 0.001, which

  6. A novel HDAC inhibitor, CG200745, inhibits pancreatic cancer cell growth and overcomes gemcitabine resistance.

    Science.gov (United States)

    Lee, Hee Seung; Park, Soo Been; Kim, Sun A; Kwon, Sool Ki; Cha, Hyunju; Lee, Do Young; Ro, Seonggu; Cho, Joong Myung; Song, Si Young

    2017-01-30

    Pancreatic cancer is predominantly lethal, and is primarily treated using gemcitabine, with increasing resistance. Therefore, novel agents that increase tumor sensitivity to gemcitabine are needed. Histone deacetylase (HDAC) inhibitors are emerging therapeutic agents, since HDAC plays an important role in cancer initiation and progression. We evaluated the antitumor effect of a novel HDAC inhibitor, CG200745, combined with gemcitabine/erlotinib on pancreatic cancer cells and gemcitabine-resistant pancreatic cancer cells. Three pancreatic cancer-cell lines were used to evaluate the antitumor effect of CG200745 combined with gemcitabine/erlotinib. CG200745 induced the expression of apoptotic proteins (PARP and caspase-3) and increased the levels of acetylated histone H3. CG200745 with gemcitabine/erlotinib showed significant growth inhibition and synergistic antitumor effects in vitro. In vivo, gemcitabine/erlotinib and CG200745 reduced tumor size up to 50%. CG200745 enhanced the sensitivity of gemcitabine-resistant pancreatic cancer cells to gemcitabine, and decreased the level of ATP-binding cassette-transporter genes, especially multidrug resistance protein 3 (MRP3) and MRP4. The novel HDAC inhibitor, CG200745, with gemcitabine/erlotinib had a synergistic anti-tumor effect on pancreatic cancer cells. CG200745 significantly improved pancreatic cancer sensitivity to gemcitabine, with a prominent antitumor effect on gemcitabine-resistant pancreatic cancer cells. Therefore, improved clinical outcome is expected in the future.

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

    Directory of Open Access Journals (Sweden)

    Lucianna Helene Santos

    2015-11-01

    Full Text Available 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.

  8. THE INFLUENCE OF POTASSIUM DICHROMATE Cr (VI ADMINISTRATION DURATION ON GLOBULAR RESISTANCE IN FEMALE RATS

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    LETIŢIA STANA

    2009-05-01

    Full Text Available The „in vivo” experiment has had as aim the study of different Cr(VI doses administration on globular resistance in female rats related to administration duration. Study was carried out on 56 female rats divided in 8 groups, 6 experimental and 2 control that received potassium dichromate in drinking water in doses of 25 ppm, 50 ppm and 75ppm Cr(VI, for 3 months, respectively, 6 months. Decrease of globular resistance (in terms of haemolysis degree in hypotonic solutions at increasing dose (up to 0.8% NaCl at 75 ppm dose in all experimental groups, in direct relation with the duration of administration was registered. Control groups were in physiological limits. The results of the present study revealed the affecting of erythrocyte membrane in function of administration duration and chromium intake level, because of oxidative lesions produced by it.

  9. Transcriptome analysis of the Spodoptera frugiperda ascovirus in vivo provides insights into how its apoptosis inhibitors and caspase promote increased synthesis of viral vesicles and virion progeny.

    Science.gov (United States)

    Zaghloul, Heba; Hice, Robert; Arensburger, Peter; Federici, Brian A

    2017-09-27

    Ascoviruses are ds DNA viruses that attack caterpillars and differ from all other viruses by inducing nuclear lysis followed by cleavage of host cells into numerous anucleate vesicles in which virus replication continues as these grow in the blood. Ascoviruses are also unusual in that most encode apoptosis inhibitors and caspase or caspase-like proteins. A robust cell line to study the novel molecular biology of ascovirus replication in vitro is lacking. Therefore, we used strand-specific RNA-Seq to study transcription in vivo in third instars of Spodoptera frugiperda infected with the Spodoptera frugiperda ascovirus, a member of the type species, Spodoptera frugiperda ascovirus (SfAV-1a), sampling transcripts at different time points after infection. We targeted transcription of two types of SfAV-1a genes; first, 44 core genes that occur in several ascovirus species, and second, 26 genes predicted in silico to have metabolic functions likely involved in synthesizing viral vesicle membranes. Gene cluster analysis showed differences in temporal expression of SfAV-1a genes, enabling their assignment to three temporal classes; early, late and very late. Inhibitors of apoptosis (IAP-like proteins; ORF016, ORF025 and ORF074) were expressed early, whereas its caspase (ORF073) was expressed very late, which correlated with apoptotic events leading to viral vesicle formation. Expression analysis revealed that a Diedel gene homolog (ORF121), the only known "virokine," was highly expressed, implying this ascovirus protein helps evade innate host immunity. Lastly, single-nucleotide resolution of RNA-Seq data revealed 15 bicistronic and tricistronic messages along the genome, an unusual occurrence for large ds DNA viruses. IMPORTANCE Unlike all other DNA viruses, ascoviruses code for an executioner caspase, apparently involved in a novel cytopathology in which viral replication induces nuclear lysis followed by cell cleavage yielding numerous large anucleate viral vesicles

  10. Microenvironment-Mediated Mechanisms of Resistance to HER2 Inhibitors Differ between HER2+ Breast Cancer Subtypes.

    Science.gov (United States)

    Watson, Spencer S; Dane, Mark; Chin, Koei; Tatarova, Zuzana; Liu, Moqing; Liby, Tiera; Thompson, Wallace; Smith, Rebecca; Nederlof, Michel; Bucher, Elmar; Kilburn, David; Whitman, Matthew; Sudar, Damir; Mills, Gordon B; Heiser, Laura M; Jonas, Oliver; Gray, Joe W; Korkola, James E

    2018-03-28

    Extrinsic signals are implicated in breast cancer resistance to HER2-targeted tyrosine kinase inhibitors (TKIs). To examine how microenvironmental signals influence resistance, we monitored TKI-treated breast cancer cell lines grown on microenvironment microarrays composed of printed extracellular matrix proteins supplemented with soluble proteins. We tested ∼2,500 combinations of 56 soluble and 46 matrix microenvironmental proteins on basal-like HER2+ (HER2E) or luminal-like HER2+ (L-HER2+) cells treated with the TKIs lapatinib or neratinib. In HER2E cells, hepatocyte growth factor, a ligand for MET, induced resistance that could be reversed with crizotinib, an inhibitor of MET. In L-HER2+ cells, neuregulin1-β1 (NRG1β), a ligand for HER3, induced resistance that could be reversed with pertuzumab, an inhibitor of HER2-HER3 heterodimerization. The subtype-specific responses were also observed in 3D cultures and murine xenografts. These results, along with bioinformatic pathway analysis and siRNA knockdown experiments, suggest different mechanisms of resistance specific to each HER2+ subtype: MET signaling for HER2E and HER2-HER3 heterodimerization for L-HER2+ cells. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  11. V3-independent competitive resistance of a dual-X4 HIV-1 to the CXCR4 inhibitor AMD3100.

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    Yosuke Maeda

    Full Text Available A CXCR4 inhibitor-resistant HIV-1 was isolated from a dual-X4 HIV-1 in vitro. The resistant variant displayed competitive resistance to the CXCR4 inhibitor AMD3100, indicating that the resistant variant had a higher affinity for CXCR4 than that of the wild-type HIV-1. Amino acid sequence analyses revealed that the resistant variant harbored amino acid substitutions in the V2, C2, and C4 regions, but no remarkable changes in the V3 loop. Site-directed mutagenesis confirmed that the changes in the C2 and C4 regions were principally involved in the reduced sensitivity to AMD3100. Furthermore, the change in the C4 region was associated with increased sensitivity to soluble CD4, and profoundly enhanced the entry efficiency of the virus. Therefore, it is likely that the resistant variant acquired the higher affinity for CD4/CXCR4 by the changes in non-V3 regions. Taken together, a CXCR4 inhibitor-resistant HIV-1 can evolve using a non-V3 pathway.

  12. Improving plasma resistance and lowering roughness in an ArF photoresist by adding a chemical reaction inhibitor

    International Nuclear Information System (INIS)

    Jinnai, Butsurin; Uesugi, Takuji; Koyama, Koji; Samukawa, Seiji; Kato, Keisuke; Yasuda, Atsushi; Maeda, Shinichi; Momose, Hikaru

    2010-01-01

    Major challenges associated with 193 nm lithography using an ArF photoresist are low plasma resistance and roughness formation in the ArF photoresist during plasma processes. We have previously found decisive factors affecting the plasma resistance and roughness formation in an ArF photoresist: plasma resistance is determined by UV/VUV radiation, and roughness formation is dominated by chemical reactions. In this study, based on our findings on the interaction between plasma radiation species and ArF photoresist polymers, we proposed an ArF photoresist with a chemical reaction inhibitor, which can trap reactive species from the plasma, and characterized the performances of the resultant ArF photoresist through neutral beam experiments. Hindered amine light stabilizers, i.e. 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy (HO-TEMPO), were used as the chemical reaction inhibitor. Etching rates of the ArF photoresist films were not dependent on the HO-TEMPO content in the irradiations without chemical reactions or under UV/VUV radiation. However, in the irradiation with chemical reactions, the etching rates of the ArF photoresist films decreased as the HO-TEMPO content increased. In addition, the surface roughness decreased with the increase in the additive amount of chemical reaction inhibitor. According to FTIR analysis, a chemical reaction inhibitor can inhibit the chemical reactions in ArF photoresist films through plasma radicals. These results indicate that a chemical reaction inhibitor is effective against chemical reactions, resulting in improved plasma resistance and less roughness in an ArF photoresist. These results also support our suggested mechanism of plasma resistance and roughness formation in an ArF photoresist.

  13. Molecular characterization of hepatitis C virus for determination of subtypes and detection of resistance mutations to protease inhibitors in a group of intravenous drug users co-infected with HIV.

    Science.gov (United States)

    Silva, Tânia; Cortes Martins, Helena; Coutinho, Rodrigo; Leitão, Emília; Silva, Rui; Pádua, Elizabeth

    2015-09-01

    Modifications in therapeutic regimens for the treatment of hepatitis C virus (HCV) have been observed since the approval of viral protease inhibitors (PI), and the selection of natural drug-resistant variants has been also reported. Thus, it becomes crucial to be aware of consequences of new therapeutic approaches and make available tools for monitoring the infection. The study aimed to apply an "in-house" method for amplification and sequencing of the NS3 region which is the target of PI, and allowing simultaneously the classification of viral subtypes and identification of resistance mutations. Forty-seven samples collected from HIV injecting drug users and drug naive for HCV protease inhibitors were tested for anti-HCV antibodies, 93.6% of them had a positive result and in 70.5% was determined HCV active infection. High frequency of subtype 1a (46.2%), followed by an equal proportion of subtypes 3a, 4a, and 4d (15.4%) was obtained. Two potential recombinants, RF1_2k/1b (3.8%) and 2q/2k (3.8%) were identified. Substitutions V36L/P, T54A, I72L/N/T/V, Q80K/G, S122R/T, D168Q, and I170L/V were observed in 65.4% of the samples. The T54A and Q80K mutations, and the combination V36L + T54A were also identified. Polymorphisms were observed exclusively associated with specific genotypes, particularly, I72L and D168Q with genotype 3, and S122T with genotype 4. The V36L substitution was identified in 92.8% of sequences of non-genotype 1 denoting that this amino acid substitution is a natural polymorphism associated with non-genotype 1 strains. Although no major PI resistance mutations were detected, a more extensive study is needed to evaluate the impact of mutations identified in efficacy of PI treatment. © 2015 Wiley Periodicals, Inc.

  14. Crystal structure of the sodium-potassium pump (Na+,K+-ATPase) with bound potassium and ouabain

    OpenAIRE

    Ogawa, Haruo; Shinoda, Takehiro; Cornelius, Flemming; Toyoshima, Chikashi

    2009-01-01

    The sodium-potassium pump (Na+,K+-ATPase) is responsible for establishing Na+ and K+ concentration gradients across the plasma membrane and therefore plays an essential role in, for instance, generating action potentials. Cardiac glycosides, prescribed for congestive heart failure for more than 2 centuries, are efficient inhibitors of this ATPase. Here we describe a crystal structure of Na+,K+-ATPase with bound ouabain, a representative cardiac glycoside, at 2.8 Å resolution in a state analog...

  15. Presence of viral RNA and proteins in exosomes from the cellular clones resistant to Rift Valley Fever Virus infection.

    Directory of Open Access Journals (Sweden)

    Noor eAhsan

    2016-02-01

    Full Text Available Rift Valley Fever Virus (RVFV is a RNA virus that belongs to the genus Phlebovirus, family Bunyaviridae. It infects humans and livestock and causes Rift Valley fever. RVFV is considered an agricultural pathogen by the USDA, as it can cause up to 100% abortion in cattle and extensive death of newborns. In addition, it is designated as Category A pathogen by the CDC and the NIAID. In some human cases of RVFV infection, the virus causes fever, ocular damage, liver damage, hemorrhagic fever, and death. There are currently limited options for vaccine candidates, which include the MP-12 and clone 13 versions of RVFV. Viral infections often deregulate multiple cellular pathways that contribute to replication and host pathology. We have previously shown that latent HIV-1 and HTLV-1 infected cells secrete exosomes that contain short viral RNAs, limited number of genomic RNAs, and viral proteins. These exosomes largely target neighboring cells and activate the NF-кB pathway, leading to cell proliferation and overall better viral replication. In this manuscript, we studied the effects of exosome formation from RVFV infected cells and their function on recipient cells. We initially infected cells, isolated resistant clones, and further purified using dilution cloning. We then characterized these cells as resistant to new RVFV infection, but sensitive to other viral infections, including Venezuelan Equine Encephalitis Virus (VEEV. These clones contained normal markers (i.e. CD63 for exosomes and were able to activate the TLR pathway in recipient reporter cells. Interestingly, the exosome rich preparations, much like their host cell, contained viral RNA (L, M, and S genome. The RNAs were detected using qRT-PCR in both parental and exosomal preparations as well as in CD63 immunoprecipitates. Viral proteins such as N and a modified form of NSs were present in some of these exosomes. Finally, treatment of recipient cells (T- cells and monocytic cells showed

  16. The Design of New HIV-IN Tethered Bifunctional Inhibitors using Multiple Microdomain Targeted Docking.

    Science.gov (United States)

    Ciubotaru, Mihai; Musat, Mihaela Georgiana; Surleac, Marius; Ionita, Elena; Petrescu, Andrei Jose; Abele, Edgars; Abele, Ramona

    2018-04-05

    Currently used antiretroviral HIV therapy drugs exclusively target critical groups in the enzymes essential for the viral life cycle. Increased mutagenesis of their genes, changes these viral enzymes which once mutated can evade therapeutic targeting, effects which confer drug resistance. To circumvent this, our review addresses a strategy to design and derive HIV-Integrase (HIV-IN) inhibitors which simultaneously target two IN functional domains, rendering it inactive even if the enzyme accumulates many mutations. First we review the enzymatic role of IN to insert the copied viral DNA into a chromosome of the host T lymphocyte, highlighting its main functional and structural features to be subjected to inhibitory action. From a functional and structural perspective we present all classes of HIV-IN inhibitors with their most representative candidates. For each chosen compound we also explain its mechanism of IN inhibition. We use the recently resolved cryo EM IN tetramer intasome DNA complex [1] onto which we dock various reference IN inhibitory chemical scaffolds such as to target adjacent functional IN domains. Pairing compounds with complementary activity, which dock in the vicinity of a IN structural microdomain, we design bifunctional new drugs which may not only be more resilient to IN mutations but also may be more potent inhibitors than their original counterparts. In the end of our review we propose synthesis pathways to link such paired compounds with enhanced synergistic IN inhibitory effects. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  17. Chitosan as an effective inhibitor of multidrug resistant Acinetobacter baumannii.

    Science.gov (United States)

    Costa, E M; Silva, S; Vicente, S; Veiga, M; Tavaria, F; Pintado, M M

    2017-12-15

    Over the last two decades worldwide levels of antibiotic resistance have risen leading to the appearance of multidrug resistant microorganisms. Acinetobacter baumannii is a known skin pathogen which has emerged as a major cause of nosocomial outbreaks due to its capacity to colonize indwelling medical devices and natural antibiotic resistance. With chitosan being an effective antimicrobial agent against antibiotic resistant microorganisms, the aim of this work was to access its potential as an alternative to traditional antimicrobials in the management of A. baumannii growth. What the results showed was that both chitosan MW's tested were active upon A. baumannii's planktonic and sessile growth. For planktonic growth MICs and MBCs were obtained at relatively low concentrations (0.5-2mg/mL) while for sessile growth chitosan proved to be an effective inhibitor of A. baumannii's adhesion and biofilm formation. Considering these results chitosan shows a high potential for control of A. baumannii infections. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Virologic failure of protease inhibitor-based second-line antiretroviral therapy without resistance in a large HIV treatment program in South Africa.

    Directory of Open Access Journals (Sweden)

    Julie H Levison

    Full Text Available We investigated the prevalence of wild-type virus (no major drug resistance and drug resistance mutations at second-line antiretroviral treatment (ART failure in a large HIV treatment program in South Africa.HIV-infected patients ≥ 15 years of age who had failed protease inhibitor (PI-based second-line ART (2 consecutive HIV RNA tests >1000 copies/ml on lopinavir/ritonavir, didanosine, and zidovudine were identified retrospectively. Patients with virologic failure were continued on second-line ART. Genotypic testing for drug resistance was performed on frozen plasma samples obtained closest to and after the date of laboratory confirmed second-line ART failure. Of 322 HIV-infected patients on second-line ART, 43 were adults with confirmed virologic failure, and 33 had available plasma for viral sequencing. HIV-1 RNA subtype C predominated (n = 32, 97%. Mean duration on ART (SD prior to initiation of second-line ART was 23 (17 months, and time from second-line ART initiation to failure was 10 (9 months. Plasma samples were obtained 7(9 months from confirmed failure. At second-line failure, 22 patients (67% had wild-type virus. There was no major resistance to PIs found. Eleven of 33 patients had a second plasma sample taken 8 (5.5 months after the first. Median HIV-1 RNA and the genotypic resistance profile were unchanged.Most patients who failed second-line ART had wild-type virus. We did not observe evolution of resistance despite continuation of PI-based ART after failure. Interventions that successfully improve adherence could allow patients to continue to benefit from second-line ART therapy even after initial failure.

  19. High potency inhibition of hERG potassium channels by the sodium–calcium exchange inhibitor KB-R7943

    Science.gov (United States)

    Cheng, Hongwei; Zhang, Yihong; Du, Chunyun; Dempsey, Christopher E; Hancox, Jules C

    2012-01-01

    BACKGROUND AND PURPOSE KB-R7943 is an isothiourea derivative that is used widely as a pharmacological inhibitor of sodium–calcium exchange (NCX) in experiments on cardiac and other tissue types. This study investigated KB-R7943 inhibition of hERG (human ether-à-go-go-related gene) K+ channels that underpin the cardiac rapid delayed rectifier potassium current, IKr. EXPERIMENTAL APPROACH Whole-cell patch-clamp measurements were made of hERG current (IhERG) carried by wild-type or mutant hERG channels and of native rabbit ventricular IKr. Docking simulations utilized a hERG homology model built on a MthK-based template. KEY RESULTS KB-R7943 inhibited both IhERG and native IKr rapidly on membrane depolarization with IC50 values of ∼89 and ∼120 nM, respectively, for current tails at −40 mV following depolarizing voltage commands to +20 mV. Marked IhERG inhibition also occurred under ventricular action potential voltage clamp. IhERG inhibition by KB-R7943 exhibited both time- and voltage-dependence but showed no preference for inactivated over activated channels. Results of alanine mutagenesis and docking simulations indicate that KB-R7943 can bind to a pocket formed of the side chains of aromatic residues Y652 and F656, with the compound's nitrobenzyl group orientated towards the cytoplasmic side of the channel pore. The structurally related NCX inhibitor SN-6 also inhibited IhERG, but with a markedly reduced potency. CONCLUSIONS AND IMPLICATIONS KB-R7943 inhibits IhERG/IKr with a potency that exceeds that reported previously for acute cardiac NCX inhibition. Our results also support the feasibility of benzyloxyphenyl-containing NCX inhibitors with reduced potential, in comparison with KB-R7943, to inhibit hERG. PMID:21950687

  20. Emerging Viral Diseases of Tomato Crops

    NARCIS (Netherlands)

    Hanssen, I.M.; Lapidot, M.; Thomma, B.P.H.J.

    2010-01-01

    Viral diseases are an important limiting factor in many crop production systems. Because antiviral products are not available, control strategies rely on genetic resistance or hygienic measures to prevent viral diseases, or on eradication of diseased crops to control such diseases. Increasing

  1. Effects of sequence changes in the HIV-1 gp41 fusion peptide on CCR5 inhibitor resistance

    International Nuclear Information System (INIS)

    Anastassopoulou, Cleo G.; Ketas, Thomas J.; Sanders, Rogier W.; Johan Klasse, Per; Moore, John P.

    2012-01-01

    A rare pathway of HIV-1 resistance to small molecule CCR5 inhibitors such as Vicriviroc (VCV) involves changes solely in the gp41 fusion peptide (FP). Here, we show that the G516V change is critical to VCV resistance in PBMC and TZM-bl cells, although it must be accompanied by either M518V or F519I to have a substantial impact. Modeling VCV inhibition data from the two cell types indicated that G516V allows both double mutants to use VCV-CCR5 complexes for entry. The model further identified F519I as an independent determinant of preference for the unoccupied, high-VCV affinity form of CCR5. From inhibitor-free reversion cultures, we also identified a substitution in the inner domain of gp120, T244A, which appears to counter the resistance phenotype created by the FP substitutions. Examining the interplay of these changes will enhance our understanding of Env complex interactions that influence both HIV-1 entry and resistance to CCR5 inhibitors.

  2. The importance of potassium in resistance to crown rot disease in alfalfa

    Science.gov (United States)

    Nitrogen, phosphate, and potassium (potash, K2O) are the three most important nutrients in alfalfa growth and development. Nitrogen fertilization is not required because alfalfa has a high rate of biological nitrogen fixation. Phosphorus and potassium are frequently applied as fertilizer, but potass...

  3. Potassium acceptor doping of ZnO crystals

    Directory of Open Access Journals (Sweden)

    Narendra S. Parmar

    2015-05-01

    Full Text Available ZnO bulk single crystals were doped with potassium by diffusion at 950°C. Positron annihilation spectroscopy confirms the filling of zinc vacancies and a different trapping center for positrons. Secondary ion mass spectroscopy measurements show the diffusion of potassium up to 10 μm with concentration ∼1 × 1016 cm−3. IR measurements show a local vibrational mode (LVM at 3226 cm−1, at a temperature of 9 K, in a potassium doped sample that was subsequently hydrogenated. The LVM is attributed to an O–H bond-stretching mode adjacent to a potassium acceptor. When deuterium substitutes for hydrogen, a peak is observed at 2378 cm−1. The O-H peak is much broader than the O-D peak, perhaps due to an unusually low vibrational lifetime. The isotopic frequency ratio is similar to values found in other hydrogen complexes. Potassium doping increases the resistivity up to 3 orders of magnitude at room temperature. The doped sample has a donor level at 0.30 eV.

  4. Potassium acceptor doping of ZnO crystals

    Science.gov (United States)

    Parmar, Narendra S.; Corolewski, Caleb D.; McCluskey, Matthew D.; Lynn, K. G.

    2015-05-01

    ZnO bulk single crystals were doped with potassium by diffusion at 950°C. Positron annihilation spectroscopy confirms the filling of zinc vacancies and a different trapping center for positrons. Secondary ion mass spectroscopy measurements show the diffusion of potassium up to 10 μm with concentration ˜1 × 1016 cm-3. IR measurements show a local vibrational mode (LVM) at 3226 cm-1, at a temperature of 9 K, in a potassium doped sample that was subsequently hydrogenated. The LVM is attributed to an O-H bond-stretching mode adjacent to a potassium acceptor. When deuterium substitutes for hydrogen, a peak is observed at 2378 cm-1. The O-H peak is much broader than the O-D peak, perhaps due to an unusually low vibrational lifetime. The isotopic frequency ratio is similar to values found in other hydrogen complexes. Potassium doping increases the resistivity up to 3 orders of magnitude at room temperature. The doped sample has a donor level at 0.30 eV.

  5. Potassium acceptor doping of ZnO crystals

    Energy Technology Data Exchange (ETDEWEB)

    Parmar, Narendra S., E-mail: nparmar@wsu.edu; Lynn, K. G. [Center for Materials Research, Washington State University, Pullman, Washington 99164-2711 (United States); Department of Physics and Astronomy, Washington State University, Pullman, Washington 99164-2814 (United States); Corolewski, Caleb D.; McCluskey, Matthew D. [Department of Physics and Astronomy, Washington State University, Pullman, Washington 99164-2814 (United States)

    2015-05-15

    ZnO bulk single crystals were doped with potassium by diffusion at 950°C. Positron annihilation spectroscopy confirms the filling of zinc vacancies and a different trapping center for positrons. Secondary ion mass spectroscopy measurements show the diffusion of potassium up to 10 μm with concentration ∼1 × 10{sup 16} cm{sup −3}. IR measurements show a local vibrational mode (LVM) at 3226 cm{sup −1}, at a temperature of 9 K, in a potassium doped sample that was subsequently hydrogenated. The LVM is attributed to an O–H bond-stretching mode adjacent to a potassium acceptor. When deuterium substitutes for hydrogen, a peak is observed at 2378 cm{sup −1}. The O-H peak is much broader than the O-D peak, perhaps due to an unusually low vibrational lifetime. The isotopic frequency ratio is similar to values found in other hydrogen complexes. Potassium doping increases the resistivity up to 3 orders of magnitude at room temperature. The doped sample has a donor level at 0.30 eV.

  6. ON012380: A Non-ATP Competitive Inhibitor of BCR-ABL for the Therapy of Imatinib-Resistant CMLs

    Science.gov (United States)

    2010-05-01

    imatinib resistance with a novel ABL kinase inhibitor. Science. 2004; 305(5682):399-401 3. Weisberg E, Manley PW, Breitenstein W, Bruggen J, Cowan-Jacob... Manley PW, Cowan-Jacob SW, Hochhaus A, Griffin JD. Second generation inhibitors of BCR-ABL for the treatment of imatinib-resistant chronic myeloid...araldehyde (5) (10 mmol), glacial acetic acid (5 mL), and a catalytic amount (100 lL ) of benzyl amine was re- fluxed for 5–8 h. After completion of

  7. Oncogene Mimicry as a Mechanism of Primary Resistance to BRAF Inhibitors

    Directory of Open Access Journals (Sweden)

    Martin L. Sos

    2014-08-01

    Full Text Available Despite the development of potent RAF/mitogen-activated protein kinase (MAPK pathway inhibitors, only a fraction of BRAF-mutant patients benefit from treatment with these drugs. Using a combined chemogenomics and chemoproteomics approach, we identify drug-induced RAS-RAF-MEK complex formation in a subset of BRAF-mutant cancer cells characterized by primary resistance to vemurafenib. In these cells, autocrine interleukin-6 (IL-6 secretion may contribute to the primary resistance phenotype via induction of JAK/STAT3 and MAPK signaling. In a subset of cell lines, combined IL-6/MAPK inhibition is able to overcome primary resistance to BRAF-targeted therapy. Overall, we show that the signaling plasticity exerted by primary resistant BRAF-mutant cells is achieved by their ability to mimic signaling features of oncogenic RAS, a strategy that we term “oncogene mimicry.” This model may guide future strategies for overcoming primary resistance observed in these tumors.

  8. Targeted Morphoproteomic Profiling of Ewing's Sarcoma Treated with Insulin-Like Growth Factor 1 Receptor (IGF1R) Inhibitors: Response/Resistance Signatures

    Science.gov (United States)

    Subbiah, Vivek; Naing, Aung; Brown, Robert E.; Chen, Helen; Doyle, Laurence; LoRusso, Patricia; Benjamin, Robert; Anderson, Pete; Kurzrock, Razelle

    2011-01-01

    Background Insulin-like growth factor 1 receptor (IGF1R) targeted therapies have resulted in responses in a small number of patients with advanced metastatic Ewing's sarcoma. We performed morphoproteomic profiling to better understand response/resistance mechanisms of Ewing's sarcoma to IGF1R inhibitor-based therapy. Methodology/Principal Findings This pilot study assessed two patients with advanced Ewing's sarcoma treated with IGF1R antibody alone followed by combined IGF1R inhibitor plus mammalian target of rapamycin (mTOR) inhibitor treatment once resistance to single-agent IGF1R inhibitor developed. Immunohistochemical probes were applied to detect p-mTOR (Ser2448), p-Akt (Ser473), p-ERK1/2 (Thr202/Tyr204), nestin, and p-STAT3 (Tyr 705) in the original and recurrent tumor. The initial remarkable radiographic responses to IGF1R-antibody therapy was followed by resistance and then response to combined IGF1R plus mTOR inhibitor therapy in both patients, and then resistance to the combination regimen in one patient. In patient 1, upregulation of p-Akt and p-mTOR in the tumor that relapsed after initial response to IGF1R antibody might explain the resistance that developed, and the subsequent response to combined IGF1R plus mTOR inhibitor therapy. In patient 2, upregulation of mTOR was seen in the primary tumor, perhaps explaining the initial response to the IGF1R and mTOR inhibitor combination, while the resistant tumor that emerged showed activation of the ERK pathway as well. Conclusion/Significance Morphoproteomic analysis revealed that the mTOR pathway was activated in these two patients with advanced Ewing's sarcoma who showed response to combined IGF1R and mTOR inhibition, and the ERK pathway in the patient in whom resistance to this combination emerged. Our pilot results suggests that morphoproteomic assessment of signaling pathway activation in Ewing's sarcoma merits further investigation as a guide to understanding response and resistance signatures. PMID

  9. Inhibiting and healing effects of potassium permanganate for silane films

    Energy Technology Data Exchange (ETDEWEB)

    She, Zuxin; Li, Qing, E-mail: liqingswu@yeah.net; Wang, Shaoyin; Luo, Fei; Chen, Funan; Li, Longqin

    2013-07-31

    In this study, the inhibiting and healing effects of potassium permanganate for silane films were investigated, and the optimal mole ratio of MnO{sub 4}{sup −}/Cl{sup −} was also obtained. The inhibiting process and healing mechanism were studied by electrochemical measurements and scanning electron microcopy coupled with energy dispersive spectroscopy. Results demonstrated that the introduction of potassium permanganate to electrolyte could stop the development of corrosion process and the optimal inhibiting mole ratio of MnO{sub 4}{sup −}/Cl{sup −} is 2 × 10{sup −1} with a protective efficiency about 99.24%. According to its high protective efficiency and the nice results of long-term immersion test, potassium permanganate as an inhibitor could prolong the lifetime of silane films and expand its scope of application. - Highlights: • Healing sol–gel film was obtained by adding KMnO{sub 4} into electrolyte. • An optimal inhibitor mole ratio of MnO{sub 4}{sup −}/Cl{sup −} for Si sol–gel was 2 × 10{sup −1}. • The best protective efficiency was approximately 99.24%. • The inhibiting effect may be due to production of insoluble manganese hydroxide/oxide.

  10. A focused fragment library targeting the antibiotic resistance enzyme - Oxacillinase-48: Synthesis, structural evaluation and inhibitor design.

    Science.gov (United States)

    Akhter, Sundus; Lund, Bjarte Aarmo; Ismael, Aya; Langer, Manuel; Isaksson, Johan; Christopeit, Tony; Leiros, Hanna-Kirsti S; Bayer, Annette

    2018-02-10

    β-Lactam antibiotics are of utmost importance when treating bacterial infections in the medical community. However, currently their utility is threatened by the emergence and spread of β-lactam resistance. The most prevalent resistance mechanism to β-lactam antibiotics is expression of β-lactamase enzymes. One way to overcome resistance caused by β-lactamases, is the development of β-lactamase inhibitors and today several β-lactamase inhibitors e.g. avibactam, are approved in the clinic. Our focus is the oxacillinase-48 (OXA-48), an enzyme reported to spread rapidly across the world and commonly identified in Escherichia coli and Klebsiella pneumoniae. To guide inhibitor design, we used diversely substituted 3-aryl and 3-heteroaryl benzoic acids to probe the active site of OXA-48 for useful enzyme-inhibitor interactions. In the presented study, a focused fragment library containing 49 3-substituted benzoic acid derivatives were synthesised and biochemically characterized. Based on crystallographic data from 33 fragment-enzyme complexes, the fragments could be classified into R 1 or R 2 binders by their overall binding conformation in relation to the binding of the R 1 and R 2 side groups of imipenem. Moreover, binding interactions attractive for future inhibitor design were found and their usefulness explored by the rational design and evaluation of merged inhibitors from orthogonally binding fragments. The best inhibitors among the resulting 3,5-disubstituted benzoic acids showed inhibitory potential in the low micromolar range (IC 50  = 2.9 μM). For these inhibitors, the complex X-ray structures revealed non-covalent binding to Arg250, Arg214 and Tyr211 in the active site and the interactions observed with the mono-substituted fragments were also identified in the merged structures. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  11. The Second-Generation Maturation Inhibitor GSK3532795 Maintains Potent Activity Toward HIV Protease Inhibitor–Resistant Clinical Isolates

    Science.gov (United States)

    Ray, Neelanjana; Li, Tianbo; Lin, Zeyu; Protack, Tricia; van Ham, Petronella Maria; Hwang, Carey; Krystal, Mark; Nijhuis, Monique; Lataillade, Max

    2017-01-01

    Background: Protease inhibitor (PI)-resistant HIV-1 isolates with primary substitutions in protease (PR) and secondary substitutions in Gag could potentially exhibit cross-resistance to maturation inhibitors. We evaluated the second-generation maturation inhibitor, GSK3532795, for activity toward clinical isolates with genotypic and phenotypic characteristics associated with PI resistance (longitudinal). Methods: Longitudinal clinical isolates from 15 PI-treated patients and 7 highly PI-resistant (nonlongitudinal) viruses containing major and minor PI resistance-associated mutations were evaluated for GSK3532795 sensitivity. Phenotypic sensitivity was determined using the PhenoSense Gag/PR assay (Monogram Biosciences) or in-house single- and multiple-cycle assays. Changes from baseline [CFB; ratio of post- to pre-treatment FC-IC50 (fold-change in IC50 versus wild-type virus)] Monogram (11 patients)] and 1.5 (1.0–2.2) [single-cycle (4 patients)]. The 2 post-PI treatment samples showing GSK3532795 CFB >3 (Monogram) were retested using single- and multiple-cycle assays. Neither sample had meaningful sensitivity changes in the multiple-cycle assay. Gag changes were not associated with an increased GSK3532795 CFB. Conclusions: GSK3532795 maintained antiviral activity against PI-resistant isolates with emergent PR and/or Gag mutations. This finding supports continued development of GSK3532795 in treatment-experienced patients with or without previous PI therapy. PMID:28234686

  12. Preclinical and Clinical Resistance Profile of EDP-239, a Novel Hepatitis C Virus NS5A Inhibitor.

    Science.gov (United States)

    Owens, Christopher M; Brasher, Bradley B; Polemeropoulos, Alex; Rhodin, Michael H J; McAllister, Nicole; Wong, Kelly A; Jones, Christopher T; Jiang, Lijuan; Lin, Kai; Or, Yat Sun

    2016-10-01

    EDP-239, a potent and selective hepatitis C virus (HCV) nonstructural protein 5A (NS5A) inhibitor developed for the treatment of HCV infection, has been investigated in vitro and in vivo This study sought to characterize genotypic changes in the HCV NS5A sequence of genotype 1 (GT1) replicons and to compare those changes to GT1 viral RNA mutations isolated from clinical trial patients. Resistance selection experiments in vitro using a subgenomic replicon identified resistance-associated mutations (RAMs) at GT1a NS5A amino acid positions 24, 28, 30, 31, and 93 that confer various degrees of resistance to EDP-239. Key RAMs were similarly identified in GT1b NS5A at amino acid positions 31 and 93. Mutations F36L in GT1a and A92V in GT1b do not confer resistance to EDP-239 individually but were found to enhance the resistance of GT1a K24R and GT1b Y93H. RAMs were identified in GT1 patients at baseline or after dosing with EDP-239 that were similar to those detected in vitro Baseline RAMs identified at NS5A position 93 in GT1, or positions 28 or 30 in GT1a only, correlated with a reduced treatment response. RAMs at additional positions were also detected and may have contributed to reduced EDP-239 efficacy. The most common GT1a and GT1b RAMs found to persist up to weeks 12, 24, or 48 were those at NS5A positions 28, 30, 31, 58 (GT1a only), and 93. Those RAMs persisting at the highest frequencies up to weeks 24 or 48 were L31M and Q30H/R for GT1a and L31M and Y93H for GT1b. (This study has been registered at ClinicalTrials.gov under identifier NCT01856426.). Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  13. HIV-1 protease-substrate coevolution in nelfinavir resistance.

    Science.gov (United States)

    Kolli, Madhavi; Ozen, Ayşegül; Kurt-Yilmaz, Nese; Schiffer, Celia A

    2014-07-01

    Resistance to various human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. The virus accumulates mutations within the protease (PR) that render the PIs less potent. Occasionally, Gag sequences also coevolve with mutations at PR cleavage sites contributing to drug resistance. In this study, we investigated the structural basis of coevolution of the p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations by determining crystal structures of wild-type and NFV-resistant HIV-1 protease in complex with p1-p6 substrate peptide variants with L449F and/or S451N. Alterations of residue 30's interaction with the substrate are compensated by the coevolving L449F and S451N cleavage site mutations. This interdependency in the PR-p1-p6 interactions enhances intermolecular contacts and reinforces the overall fit of the substrate within the substrate envelope, likely enabling coevolution to sustain substrate recognition and cleavage in the presence of PR resistance mutations. Resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. Mutations in HIV-1 protease selected under the pressure of protease inhibitors render the inhibitors less potent. Occasionally, Gag sequences also mutate and coevolve with protease, contributing to maintenance of viral fitness and to drug resistance. In this study, we investigated the structural basis of coevolution at the Gag p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations. Our structural analysis reveals the interdependency of protease-substrate interactions and how coevolution may restore substrate recognition and cleavage in the presence of protease drug resistance mutations. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  14. Use of resistant ACCase mutants to screen for novel inhibitors against resistant and susceptible forms of ACCase from grass weeds.

    Science.gov (United States)

    Shukla, Amit; Nycholat, Corwin; Subramanian, Mani V; Anderson, Richard J; Devine, Malcolm D

    2004-08-11

    The aryloxyphenoxypropionic acid (AOPP) and cyclohexanedione (CHD) herbicides inhibit the first committed enzyme in fatty acid biosynthesis, acetyl CoA carboxylase (ACCase). The frequent use of AOPP and CHD herbicides has resulted in the development of resistance to these herbicides in many grass weed species. New herbicides that inhibit both the susceptible and resistant forms of ACCase in grass weeds would have obvious commercial appeal. In the present study, an attempt was made to identify molecules that target both the herbicide-sensitive and -resistant forms of ACCase. Seven experimental compounds, either CHD-like or AOPP-CHD hybrids, were synthesized and assayed against previously characterized susceptible and resistant forms of ACCase. All seven compounds inhibited ACCase from sensitive biotypes of Setaria viridis and Eleusine indica (I50 values from 6.4 to >100 microM) but were not particularly potent compared to some commercialized herbicides (I50 values of 0.08-5.6 microM). In almost all cases, the I50 values for each compound assayed against the resistant ACCases were higher than those against the corresponding sensitive ACCase, indicating reduced binding to the resistant ACCases. One compound, a CHD analogue, was almost equally effective against the resistant and susceptible ACCases, although it was not a very potent ACCase inhibitor per se (I50 of 51 and 76 microM against susceptible ACCase from S. viridis and E. indica, respectively). The AOPP-CHD hybrid molecules also inhibited some of the resistant ACCases, with I50 values ranging from 6.4 to 50 microM. These compounds may be good leads for developing ACCase inhibitors that target a wider range of ACCase isoforms, including those found in AOPP- and CHD-resistant weed biotypes.

  15. The effect of a beta-lactamase inhibitor peptide on bacterial membrane structure and integrity: a comparative study.

    Science.gov (United States)

    Alaybeyoglu, Begum; Uluocak, Bilge Gedik; Akbulut, Berna Sariyar; Ozkirimli, Elif

    2017-05-01

    Co-administration of beta-lactam antibiotics and beta-lactamase inhibitors has been a favored treatment strategy against beta-lactamase-mediated bacterial antibiotic resistance, but the emergence of beta-lactamases resistant to current inhibitors necessitates the discovery of novel non-beta-lactam inhibitors. Peptides derived from the Ala46-Tyr51 region of the beta-lactamase inhibitor protein are considered as potent inhibitors of beta-lactamase; unfortunately, peptide delivery into the cell limits their potential. The properties of cell-penetrating peptides could guide the design of beta-lactamase inhibitory peptides. Here, our goal is to modify the peptide with the sequence RRGHYY that possesses beta-lactamase inhibitory activity under in vitro conditions. Inspired by the work on the cell-penetrating peptide pVEC, our approach involved the addition of the N-terminal hydrophobic residues, LLIIL, from pVEC to the inhibitor peptide to build a chimera. These residues have been reported to be critical in the uptake of pVEC. We tested the potential of RRGHYY and its chimeric derivative as a beta-lactamase inhibitory peptide on Escherichia coli cells and compared the results with the action of the antimicrobial peptide melittin, the beta-lactam antibiotic ampicillin, and the beta-lactamase inhibitor potassium clavulanate to get mechanistic details on their action. Our results show that the addition of LLIIL to the N-terminus of the beta-lactamase inhibitory peptide RRGHYY increases its membrane permeabilizing potential. Interestingly, the addition of this short stretch of hydrophobic residues also modified the inhibitory peptide such that it acquired antimicrobial property. We propose that addition of the hydrophobic LLIIL residues to the peptide N-terminus offers a promising strategy to design novel antimicrobial peptides in the battle against antibiotic resistance. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European

  16. Counteracting quasispecies adaptability: extinction of a ribavirin-resistant virus mutant by an alternative mutagenic treatment.

    Directory of Open Access Journals (Sweden)

    Celia Perales

    Full Text Available BACKGROUND: Lethal mutagenesis, or virus extinction promoted by mutagen-induced elevation of mutation rates of viruses, may meet with the problem of selection of mutagen-resistant variants, as extensively documented for standard, non-mutagenic antiviral inhibitors. Previously, we characterized a mutant of foot-and-mouth disease virus that included in its RNA-dependent RNA polymerase replacement M296I that decreased the sensitivity of the virus to the mutagenic nucleoside analogue ribavirin. METHODOLOGY AND PRINCIPAL FINDINGS: Replacement M296I in the viral polymerase impedes the extinction of the mutant foot-and-mouth disease virus by elevated concentrations of ribavirin. In contrast, wild type virus was extinguished by the same ribavirin treatment and, interestingly, no mutants resistant to ribavirin were selected from the wild type populations. Decreases of infectivity and viral load of the ribavirin-resistant M296I mutant were attained with a combination of the mutagen 5-fluorouracil and the non-mutagenic inhibitor guanidine hydrocloride. However, extinction was achieved with a sequential treatment, first with ribavirin, and then with a minimal dose of 5-fluorouracil in combination with guanidine hydrochloride. Both, wild type and ribavirin-resistant mutant M296I exhibited equal sensitivity to this combination, indicating that replacement M296I in the polymerase did not confer a significant cross-resistance to 5-fluorouracil. We discuss these results in relation to antiviral designs based on lethal mutagenesis. CONCLUSIONS: (i When dominant in the population, a mutation that confers partial resistance to a mutagenic agent can jeopardize virus extinction by elevated doses of the same mutagen. (ii A wild type virus, subjected to identical high mutagenic treatment, need not select a mutagen-resistant variant, and the population can be extinguished. (iii Extinction of the mutagen-resistant variant can be achieved by a sequential treatment of a

  17. Derivation of a JC virus-resistant human glial cell line: implications for the identification of host cell factors that determine viral tropism

    International Nuclear Information System (INIS)

    Gee, Gretchen V.; Manley, Kate; Atwood, Walter J.

    2003-01-01

    JC virus (JCV) is a common human polyomavirus that infects 70-80% of the population worldwide. In immunosuppressed individuals, JCV infects oligodendrocytes and causes a fatal demyelinating disease known as progressive multifocal leukoencephalopathy (PML). The tropism of JCV is restricted to oligodendrocytes, astrocytes, and B lymphocytes. Several mechanisms may contribute to the restricted tropism of JCV, including the presence or absence of cell-type-specific transcription and replication factors and the presence or absence of cell-type-specific receptors. We have established a system to investigate cellular factors that influence viral tropism by selecting JCV-resistant cells from a susceptible glial cell line (SVG-A). SVG-A cells were subjected to several rounds of viral infection using JC virus (M1/SVEΔ). A population of resistant cells emerged (SVGR2) that were refractory to infection with the Mad-4 strain of JCV, the hybrid virus M1/SVEΔ, as well as to the related polyomavirus SV40. SVGR2 cells were as susceptible as the SVG-A cells to infection with an unrelated amphotropic retrovirus. The stage at which these cells are resistant to infection was investigated and the block appears to be at early viral gene transcription. This system should ultimately allow us to identify glial specific factors that influence the tropism of JCV

  18. Tenofovir-based regimens associated with less drug resistance in HIV-1-infected Nigerians failing first-line antiretroviral therapy.

    Science.gov (United States)

    Etiebet, Mary-Ann A; Shepherd, James; Nowak, Rebecca G; Charurat, Man; Chang, Harry; Ajayi, Samuel; Elegba, Olufunmilayo; Ndembi, Nicaise; Abimiku, Alashle; Carr, Jean K; Eyzaguirre, Lindsay M; Blattner, William A

    2013-02-20

    In resource-limited settings, HIV-1 drug resistance testing to guide antiretroviral therapy (ART) selection is unavailable. We retrospectively conducted genotypic analysis on archived samples from Nigerian patients who received targeted viral load testing to confirm treatment failure and report their drug resistance mutation patterns. Stored plasma from 349 adult patients on non-nucleoside reverse transcriptase inhibitor (NNRTI) regimens was assayed for HIV-1 RNA viral load, and samples with more than 1000 copies/ml were sequenced in the pol gene. Analysis for resistance mutations utilized the IAS-US 2011 Drug Resistance Mutation list. One hundred and seventy-five samples were genotyped; the majority of the subtypes were G (42.9%) and CRF02_AG (33.7%). Patients were on ART for a median of 27 months. 90% had the M184V/I mutation, 62% had at least one thymidine analog mutation, and 14% had the K65R mutation. 97% had an NNRTI resistance mutation and 47% had at least two etravirine-associated mutations. In multivariate analysis tenofovir-based regimens were less likely to have at least three nucleoside reverse transcriptase inhibitor (NRTI) mutations after adjusting for subtype, previous ART, CD4, and HIV viral load [P < 0.001, odds ratio (OR) 0.04]. 70% of patients on tenofovir-based regimens had at least two susceptible NRTIs to include in a second-line regimen compared with 40% on zidovudine-based regimens (P = 0.04, OR = 3.4). At recognition of treatment failure, patients on tenofovir-based first-line regimens had fewer NRTI drug-resistant mutations and more active NRTI drugs available for second-line regimens. These findings can inform strategies for ART regimen sequencing to optimize long-term HIV treatment outcomes in low-resource settings.

  19. Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus.

    Directory of Open Access Journals (Sweden)

    Anna Lundin

    2014-05-01

    Full Text Available Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs, a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6, a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV, and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.

  20. A novel HDAC inhibitor, CG200745, inhibits pancreatic cancer cell growth and overcomes gemcitabine resistance

    OpenAIRE

    Lee, Hee Seung; Park, Soo Been; Kim, Sun A; Kwon, Sool Ki; Cha, Hyunju; Lee, Do Young; Ro, Seonggu; Cho, Joong Myung; Song, Si Young

    2017-01-01

    Pancreatic cancer is predominantly lethal, and is primarily treated using gemcitabine, with increasing resistance. Therefore, novel agents that increase tumor sensitivity to gemcitabine are needed. Histone deacetylase (HDAC) inhibitors are emerging therapeutic agents, since HDAC plays an important role in cancer initiation and progression. We evaluated the antitumor effect of a novel HDAC inhibitor, CG200745, combined with gemcitabine/erlotinib on pancreatic cancer cells and gemcitabine-resis...

  1. High-throughput screening using pseudotyped lentiviral particles: a strategy for the identification of HIV-1 inhibitors in a cell-based assay.

    Science.gov (United States)

    Garcia, Jean-Michel; Gao, Anhui; He, Pei-Lan; Choi, Joyce; Tang, Wei; Bruzzone, Roberto; Schwartz, Olivier; Naya, Hugo; Nan, Fa-Jun; Li, Jia; Altmeyer, Ralf; Zuo, Jian-Ping

    2009-03-01

    Two decades after its discovery the human immunodeficiency virus (HIV) is still spreading worldwide and killing millions. There are 25 drugs formally approved for HIV currently on the market, but side effects as well as the emergence of HIV strains showing single or multiple resistances to current drug-therapy are causes for concern. Furthermore, these drugs target only 4 steps of the viral cycle, hence the urgent need for new drugs and also new targets. In order to tackle this problem, we have devised a cell-based assay using lentiviral particles to look for post-entry inhibitors of HIV-1. We report here the assay development, validation as well as confirmation of the hits using both wild-type and drug-resistant HIV-1 viruses. The screening was performed on an original library, rich in natural compounds and pure molecules from Traditional Chinese Medicine pharmacopoeia, which had never been screened for anti-HIV activity. The identified hits belong to four chemical sub-families that appear to be all non-nucleoside reverse transcriptase inhibitors (NNRTIs). Secondary tests with live viruses showed that there was good agreement with pseudotyped particles, confirming the validity of this approach for high-throughput drug screens. This assay will be a useful tool that can be easily adapted to screen for inhibitors of viral entry.

  2. Impact of potassium bromate and potassium iodate in a pound cake system.

    Science.gov (United States)

    Wilderjans, Edith; Lagrain, Bert; Brijs, Kristof; Delcour, Jan A

    2010-05-26

    This study investigates the impact of the oxidants potassium bromate and potassium iodate (8, 16, 32, 64, and 128 micromol/g dry matter of egg white protein) on pound cake making. The impact of the oxidants on egg white characteristics was studied in a model system. Differential scanning calorimetry showed that the oxidants caused egg white to denature later. During heating in a rapid visco analyzer, the oxidants caused the free sulfhydryl (SH) group levels to decrease more intensively and over a smaller temperature range. The oxidants made the proteins more resistant to decreases in protein extractability in sodium dodecyl sulfate containing buffer during cake recipe mixing and less resistant to such decreases during cake baking. We assume that, during baking, the degree to which SH/disulfide exchange and SH oxidation can occur depends on the properties of the protein at the onset of the process. In our view, the prevention of extractability loss during mixing increased the availability of SH groups and caused more such loss during baking. During cooling, all cakes baked with added oxidants showed less collapse. On the basis of the presented data, we put forward that only those protein reactions that occur during baking contribute to the formation of a network that supports final cake structure and prevents collapse.

  3. Antiviral drug resistance and helicase-primase inhibitors of herpes simplex virus.

    Science.gov (United States)

    Field, Hugh J; Biswas, Subhajit

    2011-02-01

    A new class of chemical inhibitors has been discovered that interferes with the process of herpesvirus DNA replication. To date, the majority of useful herpesvirus antivirals are nucleoside analogues that block herpesvirus DNA replication by targeting the DNA polymerase. The new helicase-primase inhibitors (HPI) target a different enzyme complex that is also essential for herpesvirus DNA replication. This review will place the HPI in the context of previous work on the nucleoside analogues. Several promising highly potent HPI will be described with a particular focus on the identification of drug-resistance mutations. Several HPI have good pharmacological profiles and are now at the outset of phase II clinical trials. Provided there are no safety issues to stop their progress, this new class of compound will be a major advance in the herpesvirus antiviral field. Furthermore, HPI are likely to have a major impact on the therapy and prevention of herpes simplex virus and varicella zoster in both immunocompetent and immunocompromised patients alone or in combination with current nucleoside analogues. The possibility of acquired drug-resistance to HPI will then become an issue of great practical importance. Copyright © 2010 Elsevier Ltd. All rights reserved.

  4. Potassium silicate and calcium silicate on the resistance of soybean to Phakopsora pachyrhizi infection

    Directory of Open Access Journals (Sweden)

    Maria Fernanda Cruz

    2013-01-01

    Full Text Available The control of Asian Soybean Rust (ASR, caused by Phakopsora pachyrhizi, has been difficult due to the aggressiveness of the pathogen and the lack of resistant cultivars. The objective of this study was to evaluate the effects of spray of potassium silicate (PS and soil amendment with calcium silicate (CS on soybean resistance to ASR. The PS solution was sprayed to leaves 24 hours prior to fungal inoculation while CS was amended to the soil at thirty-five days before sowing. The infection process of P. pachyrhizi was investigated by scanning electron microscopy. The uredia on leaves of plants sprayed with PS were smaller and more compact than those observed on the leaves of plants grown in soil amended with CS or in soil non-amended with CS (control treatment. On leaves of plants from the control treatment, uredia produced many urediniospores at 9 days after inoculation, and the ASR severity was 15, 8 and 9%, respectively, for plants from control, PS and CS treatments. In conclusion, the spray of PS contributed to reduce the number of uredia per cm² of leaf area and both PS spray and CS resulted in lower ASR symptoms.

  5. Random Plant Viral Variants Attain Temporal Advantages During Systemic Infections and in Turn Resist other Variants of the Same Virus.

    Science.gov (United States)

    Zhang, Xiao-Feng; Guo, Jiangbo; Zhang, Xiuchun; Meulia, Tea; Paul, Pierce; Madden, Laurence V; Li, Dawei; Qu, Feng

    2015-10-20

    Infection of plants with viruses containing multiple variants frequently leads to dominance by a few random variants in the systemically infected leaves (SLs), for which a plausible explanation is lacking. We show here that SL dominance by a given viral variant is adequately explained by its fortuitous lead in systemic spread, coupled with its resistance to superinfection by other variants. We analyzed the fate of a multi-variant turnip crinkle virus (TCV) population in Arabidopsis and N. benthamiana plants. Both wild-type and RNA silencing-defective plants displayed a similar pattern of random dominance by a few variant genotypes, thus discounting a prominent role for RNA silencing. When introduced to plants sequentially as two subpopulations, a twelve-hour head-start was sufficient for the first set to dominate. Finally, SLs of TCV-infected plants became highly resistant to secondary invasions of another TCV variant. We propose that random distribution of variant foci on inoculated leaves allows different variants to lead systemic movement in different plants. The leading variants then colonize large areas of SLs, and resist the superinfection of lagging variants in the same areas. In conclusion, superinfection resistance is the primary driver of random enrichment of viral variants in systemically infected plants.

  6. Resistance analysis and characterization of NITD008 as an adenosine analog inhibitor against hepatitis C virus.

    Science.gov (United States)

    Qing, Jie; Luo, Rui; Wang, Yaxin; Nong, Junxiu; Wu, Ming; Shao, Yan; Tang, Ruoyi; Yu, Xi; Yin, Zheng; Sun, Yuna

    2016-02-01

    Hepatitis disease caused by hepatitis C virus (HCV) is a severe threat to global public health, affecting approximately 3% of the world's population. Sofosbuvir (PSI-7977), a uridine nucleotide analog inhibitor targeting the HCV NS5B polymerase, was approved by FDA at the end of 2013 and represents a key step towards a new era in the management of HCV infection. Previous study identified NITD008, an adenosine nucleoside analog, as the specific inhibitor against dengue virus and showed good antiviral effect on other flaviviruses or enteroviruses. In this report, we systematically analyzed the anti-HCV profile of NITD008, which was discovered to effectively suppress the replication of different strains of HCV in human hepatoma cells with a low nanomolar activity. On genotype 2a virus, or 2a, 1a, and 1b replicon cells, EC50 values were 8.7 nM, 93.3 nM, 60.0 nM and 67.2 nM, and selective index values were >2298.9, >214.4, >333.3, >298.5 respectively. We demonstrated that resistance to NITD008 was conferred by mutation in NS5B (S282T) in the HCV infectious virus genotype 2a (JFH-1). Then, we compared the resistant profiles of NITD008 and PSI-7977, and found that the folds change of EC50 of NITD008 to full replicon cells containing mutation S282T was much bigger than PSI-7977(folds 76.50 vs. 4.52). Analysis of NITD008 cross-resistance against previously reported NS5B drug-selected mutations showed that the resistance pattern of NITD008 was not completely similar to PSI-7977, and meanwhile, S282T resistant mutation to NITD008 emerge more easily in cell culture than PSI-7977. Interestingly, NITD008 displayed significant synergistic effects with the NS5B polymerase inhibitor PSI-7977, however, only additive effects with alpha interferon (IFNα-2b), ribavirin, and an NS3 protease inhibitor. These results verify that NITD008 is an effective analog inhibitor against hepatitis C virus and a good research tool as a supplement to other types of nucleoside analogs. Copyright

  7. CRISPR/Cas9-mediated viral interference in plants

    KAUST Repository

    Ali, Zahir; Abulfaraj, Aala A.; Idris, Ali; Ali, Shawkat; Tashkandi, Manal; Mahfouz, Magdy M.

    2015-01-01

    These data establish the efficacy of the CRISPR/Cas9 system for viral interference in plants, thereby extending the utility of this technology and opening the possibility of producing plants resistant to multiple viral infections.

  8. Inhibition of bovine viral diarrhea virus RNA synthesis by thiosemicarbazone derived from 5,6-dimethoxy-1-indanone.

    Science.gov (United States)

    Castro, Eliana F; Fabian, Lucas E; Caputto, María E; Gagey, Dolores; Finkielsztein, Liliana M; Moltrasio, Graciela Y; Moglioni, Albertina G; Campos, Rodolfo H; Cavallaro, Lucía V

    2011-06-01

    In the present work, we described the activity of the thiosemicarbazone derived from 5,6-dimethoxy-1-indanone (TSC), which we previously characterized as a new compound that inhibits bovine viral diarrhea virus (BVDV) infection. We showed that TSC acts at a point of time that coincides with the onset of viral RNA synthesis and that it inhibits the activity of BVDV replication complexes (RCs). Moreover, we have selected five BVDV mutants that turned out to be highly resistant to TSC but still susceptible to ribavirin (RBV). Four of these resistant mutants carried an N264D mutation in the viral RNA-dependent RNA polymerase (RdRp). The remaining mutant showed an A392E mutation within the same protein. Some of these mutants replicated slower than the wild-type (wt) virus in the absence of TSC, whereas others showed a partial reversion to the wt phenotype over several passages in the absence of the compound. The docking of TSC in the crystal structure of the BVDV RdRp revealed a close contact between the indane ring of the compound and several residues within the fingers domain of the enzyme, some hydrophobic contacts, and hydrogen bonds with the thiosemicarbazone group. Finally, in the mutated RdRp from resistant BVDV, these interactions with TSC could not be achieved. Interestingly, TSC inhibited BVDV replication in cell culture synergistically with RBV. In conclusion, TSC emerges as a new nonnucleoside inhibitor of BVDV RdRp that is synergistic with RBV, a feature that turns it into a potential compound to be evaluated against hepatitis C virus (HCV).

  9. Discovery and characterization of a potent and selective inhibitor of Aedes aegypti inward rectifier potassium channels.

    Directory of Open Access Journals (Sweden)

    Rene Raphemot

    Full Text Available Vector-borne diseases such as dengue fever and malaria, which are transmitted by infected female mosquitoes, affect nearly half of the world's population. The emergence of insecticide-resistant mosquito populations is reducing the effectiveness of conventional insecticides and threatening current vector control strategies, which has created an urgent need to identify new molecular targets against which novel classes of insecticides can be developed. We previously demonstrated that small molecule inhibitors of mammalian Kir channels represent promising chemicals for new mosquitocide development. In this study, high-throughput screening of approximately 30,000 chemically diverse small-molecules was employed to discover potent and selective inhibitors of Aedes aegypti Kir1 (AeKir1 channels heterologously expressed in HEK293 cells. Of 283 confirmed screening 'hits', the small-molecule inhibitor VU625 was selected for lead optimization and in vivo studies based on its potency and selectivity toward AeKir1, and tractability for medicinal chemistry. In patch clamp electrophysiology experiments of HEK293 cells, VU625 inhibits AeKir1 with an IC50 value of 96.8 nM, making VU625 the most potent inhibitor of AeKir1 described to date. Furthermore, electrophysiology experiments in Xenopus oocytes revealed that VU625 is a weak inhibitor of AeKir2B. Surprisingly, injection of VU625 failed to elicit significant effects on mosquito behavior, urine excretion, or survival. However, when co-injected with probenecid, VU625 inhibited the excretory capacity of mosquitoes and was toxic, suggesting that the compound is a substrate of organic anion and/or ATP-binding cassette (ABC transporters. The dose-toxicity relationship of VU625 (when co-injected with probenecid is biphasic, which is consistent with the molecule inhibiting both AeKir1 and AeKir2B with different potencies. This study demonstrates proof-of-concept that potent and highly selective inhibitors of mosquito

  10. Discovery of dapivirine, a nonnucleoside HIV-1 reverse transcriptase inhibitor, as a broad-spectrum antiviral against both influenza A and B viruses.

    Science.gov (United States)

    Hu, Yanmei; Zhang, Jiantao; Musharrafieh, Rami Ghassan; Ma, Chunlong; Hau, Raymond; Wang, Jun

    2017-09-01

    The emergence of multidrug-resistant influenza viruses poses a persistent threat to public health. The current prophylaxis and therapeutic interventions for influenza virus infection have limited efficacy due to the continuous antigenic drift and antigenic shift of influenza viruses. As part of our ongoing effort to develop the next generation of influenza antivirals with broad-spectrum antiviral activity and a high genetic barrier to drug resistance, in this study we report the discovery of dapivirine, an FDA-approved HIV nonnucleoside reverse transcriptase inhibitor, as a broad-spectrum antiviral against multiple strains of influenza A and B viruses with low micromolar efficacy. Mechanistic studies revealed that dapivirine inhibits the nuclear entry of viral ribonucleoproteins at the early stage of viral replication. As a result, viral RNA and protein synthesis were inhibited. Furthermore, dapivirine has a high in vitro genetic barrier to drug resistance, and its antiviral activity is synergistic with oseltamivir carboxylate. In summary, the in vitro antiviral results of dapivirine suggest it is a promising candidate for the development of the next generation of dual influenza and HIV antivirals. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Effect of neuraminidase inhibitor-resistant mutations on pathogenicity of clade 2.2 A/Turkey/15/06 (H5N1) influenza virus in ferrets.

    Science.gov (United States)

    Ilyushina, Natalia A; Seiler, Jon P; Rehg, Jerold E; Webster, Robert G; Govorkova, Elena A

    2010-05-27

    The acquisition of neuraminidase (NA) inhibitor resistance by H5N1 influenza viruses has serious clinical implications, as this class of drugs can be an essential component of pandemic control measures. The continuous evolution of the highly pathogenic H5N1 influenza viruses results in the emergence of natural NA gene variations whose impact on viral fitness and NA inhibitor susceptibility are poorly defined. We generated seven genetically stable recombinant clade 2.2 A/Turkey/15/06-like (H5N1) influenza viruses carrying NA mutations located either in the framework residues (E119A, H274Y, N294S) or in close proximity to the NA enzyme active site (V116A, I117V, K150N, Y252H). NA enzyme inhibition assays showed that NA mutations at positions 116, 117, 274, and 294 reduced susceptibility to oseltamivir carboxylate (IC(50)s increased 5- to 940-fold). Importantly, the E119A NA mutation (previously reported to confer resistance in the N2 NA subtype) was stable in the clade 2.2 H5N1 virus background and induced cross-resistance to oseltamivir carboxylate and zanamivir. We demonstrated that Y252H NA mutation contributed for decreased susceptibility of clade 2.2 H5N1 viruses to oseltamivir carboxylate as compared to clade 1 viruses. The enzyme kinetic parameters (V(max), K(m) and K(i)) of the avian-like N1 NA glycoproteins were highly consistent with their IC(50) values. None of the recombinant H5N1 viruses had attenuated virulence in ferrets inoculated with 10(6) EID(50) dose. Most infected ferrets showed mild clinical disease signs that differed in duration. However, H5N1 viruses carrying the E119A or the N294S NA mutation were lethal to 1 of 3 inoculated animals and were associated with significantly higher virus titers (Pinfluenza drugs that target different virus/host factors and can limit the emergence of resistance.

  12. Synergism between demethylation inhibitor fungicides or gibberellin inhibitor plant growth regulators and bifenthrin in a pyrethroid-resistant population of Listronotus maculicollis (Coleoptera: Curculionidae).

    Science.gov (United States)

    Ramoutar, D; Cowles, R S; Requintina, E; Alm, S R

    2010-10-01

    In 2007-2008, the "annual bluegrass weevil," Listronotus maculicollis Kirby (Coleoptera: Curculionidae), a serious pest of Poa annua L. (Poales: Poaceae) on U.S. golf courses, was shown to be resistant to two pyrethroids, bifenthrin and lambda-cyhalothrin. In 2008, we showed that bifenthrin resistance was principally mediated by oxidase detoxification (cytochrome P450 [P450]). P450s can be inhibited by demethylation inhibitor fungicides and gibberellin inhibitor plant growth regulators, both of which are commonly used on golf courses. We tested these compounds for synergistic activity with bifenthin against a pyrethroid-resistant population of L. maculicollis. The LD50 value for bifenthrin was significantly reduced from 87 ng per insect (without synergists) to 9.6-40 ng per insect after exposure to the fungicides fenarimol, fenpropimorph, prochloraz, propiconazole, and pyrifenox and the plant growth regulators flurprimidol, paclobutrazol, and trinexapac-ethyl. Simulated field exposure with formulated products registered for use on turf revealed enhanced mortality when adult weevils were exposed to bifenthrin (25% mortality, presented alone) combined with field dosages of propiconizole, fenarimol, flurprimidol, or trinexapac-ethyl (range, 49-70% mortality).

  13. The influence of TAP1 and TAP2 gene polymorphisms on TAP function and its inhibition by viral immune evasion proteins.

    Science.gov (United States)

    Praest, P; Luteijn, R D; Brak-Boer, I G J; Lanfermeijer, J; Hoelen, H; Ijgosse, L; Costa, A I; Gorham, R D; Lebbink, R J; Wiertz, E J H J

    2018-06-04

    Herpesviruses encode numerous immune evasion molecules that interfere with the immune system, particularly with certain stages in the MHC class I antigen presentation pathway. In this pathway, the transporter associated with antigen processing (TAP) is a frequent target of viral immune evasion strategies. This ER-resident transporter is composed of the proteins TAP1 and TAP2, and plays a crucial role in the loading of viral peptides onto MHC class I molecules. Several variants of TAP1 and TAP2 occur in the human population, some of which are linked to autoimmune disorders and susceptibility to infections. Here, we assessed the influence of naturally occurring TAP variants on peptide transport and MHC class I expression. In addition, we tested the inhibitory capacity of three viral immune evasion proteins, the TAP inhibitors US6 from human cytomegalovirus, ICP47 from herpes simplex virus type 1 and BNLF2a from Epstein-Barr virus, for a series of TAP1 and TAP2 variants. Our results suggest that these TAP polymorphisms have no or limited effect on peptide transport or MHC class I expression. Furthermore, our study indicates that the herpesvirus-encoded TAP inhibitors target a broad spectrum of TAP variants; inhibition of TAP is not affected by the naturally occurring polymorphisms of TAP tested in this study. Our findings suggest that the long-term coevolution of herpesviruses and their host did not result in selection of inhibitor-resistant TAP variants in the human population. Copyright © 2018. Published by Elsevier Ltd.

  14. Inward rectifier potassium (Kir2.1) channels as end-stage boosters of endothelium-dependent vasodilators.

    Science.gov (United States)

    Sonkusare, Swapnil K; Dalsgaard, Thomas; Bonev, Adrian D; Nelson, Mark T

    2016-06-15

    Increase in endothelial cell (EC) calcium activates calcium-sensitive intermediate and small conductance potassium (IK and SK) channels, thereby causing hyperpolarization and endothelium-dependent vasodilatation. Endothelial cells express inward rectifier potassium (Kir) channels, but their role in endothelium-dependent vasodilatation is not clear. In the mesenteric arteries, only ECs, but not smooth muscle cells, displayed Kir currents that were predominantly mediated by the Kir2.1 isoform. Endothelium-dependent vasodilatations in response to muscarinic receptor, TRPV4 (transient receptor potential vanilloid 4) channel and IK/SK channel agonists were highly attenuated by Kir channel inhibitors and by Kir2.1 channel knockdown. These results point to EC Kir channels as amplifiers of vasodilatation in response to increases in EC calcium and IK/SK channel activation and suggest that EC Kir channels could be targeted to treat endothelial dysfunction, which is a hallmark of vascular disorders. Endothelium-dependent vasodilators, such as acetylcholine, increase intracellular Ca(2+) through activation of transient receptor potential vanilloid 4 (TRPV4) channels in the plasma membrane and inositol trisphosphate receptors in the endoplasmic reticulum, leading to stimulation of Ca(2+) -sensitive intermediate and small conductance K(+) (IK and SK, respectively) channels. Although strong inward rectifier K(+) (Kir) channels have been reported in the native endothelial cells (ECs) their role in EC-dependent vasodilatation is not clear. Here, we test the idea that Kir channels boost the EC-dependent vasodilatation of resistance-sized arteries. We show that ECs, but not smooth muscle cells, of small mesenteric arteries have Kir currents, which are substantially reduced in EC-specific Kir2.1 knockdown (EC-Kir2.1(-/-) ) mice. Elevation of extracellular K(+) to 14 mm caused vasodilatation of pressurized arteries, which was prevented by endothelial denudation and Kir channel

  15. A Biochemical/Biophysical Assay Dyad for HTS-Compatible Triaging of Inhibitors of the HIV-1 Nef/Hck SH3 Interaction

    KAUST Repository

    Breuer, Sebastian

    2013-07-26

    The current treatment regimens for HIV include over 20 anti-retrovirals. However, adverse drug effects and the emergence of drug resistance necessitates the continued improvement of the existing drug classes as well as the development of novel drugs that target as yet therapeutically unexploited viral and cellular pathways. Here we demonstrate a strategy for the discovery of protein-protein interaction inhibitors of the viral pathogenicity factor HIV-1 Nef and its interaction with the host factor SH3. A combination of a time-resolved fluorescence resonance energy resonance energy transfer-based assay and a label-free resonant waveguide grating-based assay was optimized for high-throughput screening formats.

  16. A Biochemical/Biophysical Assay Dyad for HTS-Compatible Triaging of Inhibitors of the HIV-1 Nef/Hck SH3 Interaction

    KAUST Repository

    Breuer, Sebastian; Espinola, Sheryll; Morelli, Xavier; Torbett, Bruce E; Arold, Stefan T.; Engels, Ingo H

    2013-01-01

    The current treatment regimens for HIV include over 20 anti-retrovirals. However, adverse drug effects and the emergence of drug resistance necessitates the continued improvement of the existing drug classes as well as the development of novel drugs that target as yet therapeutically unexploited viral and cellular pathways. Here we demonstrate a strategy for the discovery of protein-protein interaction inhibitors of the viral pathogenicity factor HIV-1 Nef and its interaction with the host factor SH3. A combination of a time-resolved fluorescence resonance energy resonance energy transfer-based assay and a label-free resonant waveguide grating-based assay was optimized for high-throughput screening formats.

  17. Resistance and cross-resistance profile of the diaryltriazine NNRTI and candidate microbicide UAMC01398.

    Science.gov (United States)

    Ariën, Kevin K; Venkatraj, Muthusamy; Michiels, Johan; Joossens, Jurgen; Vereecken, Katleen; Van der Veken, Pieter; Heeres, Jan; De Winter, Hans; Heyndrickx, Leo; Augustyns, Koen; Vanham, Guido

    2016-05-01

    The resistance development, cross-resistance to other NNRTIs and the impact of resistance on viral replicative fitness were studied for the new and potent NNRTI UAMC01398. Resistance was selected by dose escalation and by single high-dose selection against a comprehensive panel of NNRTIs used as therapeutics and NNRTIs under investigation for pre-exposure prophylaxis of sexual HIV transmission. A panel of 27 site-directed mutants with single mutations or combinations of mutations involved in reverse transcriptase (RT) inhibitor-mediated resistance was developed and used to confirm resistance to UAMC01398. Cross-resistance to other NNRTIs was assessed, as well as susceptibility of UAMC01398-resistant HIV to diarylpyrimidine-resistant viruses. Finally, the impact of UAMC01398 resistance on HIV replicative fitness was studied. We showed that UAMC01398 has potent activity against dapivirine-resistant HIV, that at least four mutations in the RT are required in concert for resistance and that the resistance profile is similar to rilpivirine, both genotypically and phenotypically. Resistance development to UAMC01398 is associated with a severe fitness cost. These data, together with the enhanced safety profile and good solubility in aqueous gels, make UAMC01398 an excellent candidate for HIV topical prevention. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  18. Discovery of Novel Bovine Viral Diarrhea Inhibitors Using Structure-Based Virtual Screening on the Envelope Protein E2

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    Mariela Bollini

    2018-03-01

    Full Text Available Bovine viral diarrhea virus (BVDV is a member of the genus Pestivirus within the family Flaviviridae. BVDV causes both acute and persistent infections in cattle, leading to substantial financial losses to the livestock industry each year. The global prevalence of persistent BVDV infection and the lack of a highly effective antiviral therapy have spurred intensive efforts to discover and develop novel anti-BVDV therapies in the pharmaceutical industry. Antiviral targeting of virus envelope proteins is an effective strategy for therapeutic intervention of viral infections. We performed prospective small-molecule high-throughput docking to identify molecules that likely bind to the region delimited by domains I and II of the envelope protein E2 of BVDV. Several structurally different compounds were purchased or synthesized, and assayed for antiviral activity against BVDV. Five of the selected compounds were active displaying IC50 values in the low- to mid-micromolar range. For these compounds, their possible binding determinants were characterized by molecular dynamics simulations. A common pattern of interactions between active molecules and aminoacid residues in the binding site in E2 was observed. These findings could offer a better understanding of the interaction of BVDV E2 with these inhibitors, as well as benefit the discovery of novel and more potent BVDV antivirals.

  19. Discovery of novel bovine viral diarrhea inhibitors using structure-based virtual screening on the envelope protein E2

    Science.gov (United States)

    Bollini, Mariela; Leal, Emilse S.; Adler, Natalia S.; Aucar, María G.; Fernández, Gabriela A.; Pascual, María J.; Merwaiss, Fernando; Alvarez, Diego E.; Cavasotto, Claudio N.

    2018-03-01

    Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus within the family Flaviviridae. BVDV causes both acute and persistent infections in cattle, leading to substantial financial losses to the livestock industry each year. The global prevalence of persistent BVDV infection and the lack of a highly effective antiviral therapy have spurred intensive efforts to discover and develop novel anti-BVDV therapies in the pharmaceutical industry. Antiviral targeting of virus envelope proteins is an effective strategy for therapeutic intervention of viral infections. We performed prospective small-molecule high-throughput docking to identify molecules that likely bind to the region delimited by domains I and II of the envelope protein E2 of BVDV. Several structurally different compounds were purchased or synthesized, and assayed for antiviral activity against BVDV. Five of the selected compounds were active displaying IC50 values in the low- to mid-micromolar range. For these compounds, their possible binding determinants were characterized by molecular dynamics simulations. A common pattern of interactions between active molecules and aminoacid residues in the binding site in E2 was observed. These findings could offer a better understanding of the interaction of BVDV E2 with these inhibitors, as well as benefit the discovery of novel and more potent BVDV antivirals.

  20. Novel mechanisms to inhibit HIV reservoir seeding using Jak inhibitors.

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    Christina Gavegnano

    2017-12-01

    Full Text Available Despite advances in the treatment of HIV infection with ART, elucidating strategies to overcome HIV persistence, including blockade of viral reservoir establishment, maintenance, and expansion, remains a challenge. T cell homeostasis is a major driver of HIV persistence. Cytokines involved in regulating homeostasis of memory T cells, the major hub of the HIV reservoir, trigger the Jak-STAT pathway. We evaluated the ability of tofacitinib and ruxolitinib, two FDA-approved Jak inhibitors, to block seeding and maintenance of the HIV reservoir in vitro. We provide direct demonstration for involvement of the Jak-STAT pathway in HIV persistence in vivo, ex vivo, and in vitro; pSTAT5 strongly correlates with increased levels of integrated viral DNA in vivo, and in vitro Jak inhibitors reduce the frequency of CD4+ T cells harboring integrated HIV DNA. We show that Jak inhibitors block viral production from infected cells, inhibit γ-C receptor cytokine (IL-15-induced viral reactivation from latent stores thereby preventing transmission of infectious particles to bystander activated T cells. These results show that dysregulation of the Jak-STAT pathway is associated with viral persistence in vivo, and that Jak inhibitors target key events downstream of γ-C cytokine (IL-2, IL-7 and IL-15 ligation to their receptors, impacting the magnitude of the HIV reservoir in all memory CD4 T cell subsets in vitro and ex vivo. Jak inhibitors represent a therapeutic modality to prevent key events of T cell activation that regulate HIV persistence and together, specific, potent blockade of these events may be integrated to future curative strategies.

  1. Corrosion of a hot potassium carbonate CO/sub 2/ removal plant

    International Nuclear Information System (INIS)

    Johnson, J.J.

    1987-01-01

    After ten years of successful operation, a hot potassium carbonate CO/sub 2/ removal plant experienced severe corrosion to the 2'' (50 mm) thick carbon steel absorber process vessel over a fourteen month period. This corrosive attack resulted in complete penetration on three separate occasions. Although the cause of this corrosion is still uncertain, it appears to be the result of decreasing strength of the vanadium pentoxide inhibitor, due to increasing concentrations of hydrogen sulfide in the feed gas. After extensive research, Chevron believes that stainless steel metallurgy or replacement of the hot potassium carbonate process are the only reliable long-term solutions

  2. Effect of neuraminidase inhibitor-resistant mutations on pathogenicity of clade 2.2 A/Turkey/15/06 (H5N1 influenza virus in ferrets.

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    Natalia A Ilyushina

    2010-05-01

    Full Text Available The acquisition of neuraminidase (NA inhibitor resistance by H5N1 influenza viruses has serious clinical implications, as this class of drugs can be an essential component of pandemic control measures. The continuous evolution of the highly pathogenic H5N1 influenza viruses results in the emergence of natural NA gene variations whose impact on viral fitness and NA inhibitor susceptibility are poorly defined. We generated seven genetically stable recombinant clade 2.2 A/Turkey/15/06-like (H5N1 influenza viruses carrying NA mutations located either in the framework residues (E119A, H274Y, N294S or in close proximity to the NA enzyme active site (V116A, I117V, K150N, Y252H. NA enzyme inhibition assays showed that NA mutations at positions 116, 117, 274, and 294 reduced susceptibility to oseltamivir carboxylate (IC(50s increased 5- to 940-fold. Importantly, the E119A NA mutation (previously reported to confer resistance in the N2 NA subtype was stable in the clade 2.2 H5N1 virus background and induced cross-resistance to oseltamivir carboxylate and zanamivir. We demonstrated that Y252H NA mutation contributed for decreased susceptibility of clade 2.2 H5N1 viruses to oseltamivir carboxylate as compared to clade 1 viruses. The enzyme kinetic parameters (V(max, K(m and K(i of the avian-like N1 NA glycoproteins were highly consistent with their IC(50 values. None of the recombinant H5N1 viruses had attenuated virulence in ferrets inoculated with 10(6 EID(50 dose. Most infected ferrets showed mild clinical disease signs that differed in duration. However, H5N1 viruses carrying the E119A or the N294S NA mutation were lethal to 1 of 3 inoculated animals and were associated with significantly higher virus titers (P<0.01 and inflammation in the lungs compared to the wild-type virus. Our results suggest that highly pathogenic H5N1 variants carrying mutations within the NA active site that decrease susceptibility to NA inhibitors may possess increased

  3. Pharmacologic inhibition of COX-1 and COX-2 in influenza A viral infection in mice.

    Directory of Open Access Journals (Sweden)

    Michelle A Carey

    Full Text Available BACKGROUND: We previously demonstrated that cyclooxygenase (COX-1 deficiency results in greater morbidity and inflammation, whereas COX-2 deficiency leads to reduced morbidity, inflammation and mortality in influenza infected mice. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the effects of COX-1 and COX-2 inhibitors in influenza A viral infection. Mice were given a COX-1 inhibitor (SC-560, a COX-2 inhibitor (celecoxib or no inhibitor beginning 2 weeks prior to influenza A viral infection (200 PFU and throughout the course of the experiment. Body weight and temperature were measured daily as indicators of morbidity. Animals were sacrificed on days 1 and 4 post-infection and bronchoalveolar lavage (BAL fluid was collected or daily mortality was recorded up to 2 weeks post-infection. Treatment with SC-560 significantly increased mortality and was associated with profound hypothermia and greater weight loss compared to celecoxib or control groups. On day 4 of infection, BAL fluid cells were modestly elevated in celecoxib treated mice compared to SC-560 or control groups. Viral titres were similar between treatment groups. Levels of TNF-alpha and G-CSF were significantly attenuated in the SC-560 and celecoxib groups versus control and IL-6 levels were significantly lower in BAL fluid of celecoxib treated mice versus control and versus the SC-560 group. The chemokine KC was significantly lower in SC-560 group versus control. CONCLUSIONS/SIGNIFICANCE: Treatment with a COX-1 inhibitor during influenza A viral infection is detrimental to the host whereas inhibition of COX-2 does not significantly modulate disease severity. COX-1 plays a critical role in controlling the thermoregulatory response to influenza A viral infection in mice.

  4. HIV Viral Load

    Science.gov (United States)

    ... PF4 Antibody Hepatitis A Testing Hepatitis B Testing Hepatitis C Testing HER2/neu Herpes Testing High-sensitivity C-reactive Protein (hs-CRP) Histamine Histone Antibody HIV Antibody and HIV Antigen (p24) HIV Antiretroviral Drug Resistance Testing, Genotypic HIV Viral Load HLA Testing HLA- ...

  5. Diagnosis, antiretroviral therapy, and emergence of resistance to antiretroviral agents in HIV-2 infection: a review

    Directory of Open Access Journals (Sweden)

    Maia Hightower

    Full Text Available Human immunodeficiency virus type 1 (HIV-1 and type 2 (HIV-2 are the causative agents of AIDS. HIV-2 is prevalent at moderate to high rates in West African countries, such as Senegal, Guinea, Gambia, and Cape Verde. Diagnosis of HIV-2 is made with a positive HIV-1/HIV-2 ELISA or simple/rapid assay, followed by one or two confirmatory tests specific for HIV-2. Following CD4+ T cell counts, HIV-2 viral burden and clinical signs and symptoms of immunodeficiency are beneficial in monitoring HIV-2 disease progression. Although non-nucleoside reverse transcriptase inhibitors are ineffective in treating HIV-2, nucleoside reverse transcriptase inhibitors and protease inhibitors can be effective in dual and triple antiretroviral regimens. Their use can decrease HIV-2 viral load, increase CD4+ T cell counts and improve AIDS-related symptoms. HIV-2 resistance to various nucleoside reverse transcriptase inhibitors and protease inhibitors, including zidovudine, lamivudine, ritonavir and indinavir, has been identified in some HIV-2 infected patients on antiretroviral therapy. The knowledge of HIV-2 peculiarities, when compared to HIV-1, is crucial to helping diagnose and guide the clinician in the choice of the initial antiretroviral regimen and for monitoring therapy success.

  6. Systematic review of influenza resistance to the neuraminidase inhibitors

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    Boivin Guy

    2011-05-01

    Full Text Available Abstract Background Antivirals play a critical role in the prevention and the management of influenza. One class of antivirals, neuraminidase inhibitors (NAIs, is effective against all human influenza viruses. Currently there are two NAI drugs which are licensed worldwide: oseltamivir (Tamiflu® and zanamivir (Relenza®; and two drugs which have received recent approval in Japan: peramivir and laninamivir. Until recently, the prevalence of antiviral resistance has been relatively low. However, almost all seasonal H1N1 strains that circulated in 2008-09 were resistant to oseltamivir whereas about 1% of tested 2009 pandemic H1N1 viruses were found to be resistant to oseltamivir. To date, no studies have demonstrated widespread resistance to zanamivir. It seems likely that the literature on antiviral resistance associated with oseltamivir as well as zanamivir is now sufficiently comprehensive to warrant a systematic review. The primary objectives were to systematically review the literature to determine the incidence of resistance to oseltamivir, zanamivir, and peramivir in different population groups as well as assess the clinical consequences of antiviral resistance. Methods We searched MEDLINE and EMBASE without language restrictions in September 2010 to identify studies reporting incidence of resistance to oseltamivir, zanamivir, and peramivir. We used forest plots and meta-analysis of incidence of antiviral resistance associated with the three NAIs. Subgroup analyses were done across a number of population groups. Meta-analysis was also performed to evaluate associations between antiviral resistance and clinical complications and symptoms. Results We identified 19 studies reporting incidence of antiviral resistance. Meta-analysis of 15 studies yielded a pooled incidence rate for oseltamivir resistance of 2.6% (95%CI 0.7% to 5.5%. The incidence rate for all zanamivir resistance studies was 0%. Only one study measured incidence of antiviral

  7. Accumulation of MxB/Mx2-resistant HIV-1 Capsid Variants During Expansion of the HIV-1 Epidemic in Human Populations.

    Science.gov (United States)

    Wei, Wei; Guo, Haoran; Ma, Min; Markham, Richard; Yu, Xiao-Fang

    2016-06-01

    Recent studies have identified human myxovirus resistance protein 2 (MxB or Mx2) as an interferon induced inhibitor of HIV-1 replication. However, whether HIV-1 can overcome MxB restriction without compromise of viral fitness has been undefined. Here, we have discovered that naturally occurring capsid (CA) variants can render HIV-1 resistant to the activity of MxB without losing viral infectivity or the ability to escape from interferon induction. Moreover, these MxB resistant HIV-1 variants do not lose MxB recognition. Surprisingly, MxB resistant CA variants are most commonly found in the Clade C HIV-1 that is the most rapidly expanding Clade throughout the world. Accumulation of MxB resistant mutations is also observed during HIV-1 spreading in human populations. These findings support a potential role for MxB as a selective force during HIV-1 transmission and evolution. Copyright © 2016. Published by Elsevier B.V.

  8. Overcoming chemotherapy drug resistance by targeting inhibitors of apoptosis proteins (IAPs).

    Science.gov (United States)

    Rathore, Rama; McCallum, Jennifer E; Varghese, Elizabeth; Florea, Ana-Maria; Büsselberg, Dietrich

    2017-07-01

    Inhibitors of apoptosis (IAPs) are a family of proteins that play a significant role in the control of programmed cell death (PCD). PCD is essential to maintain healthy cell turnover within tissue but also to fight disease or infection. Uninhibited, IAPs can suppress apoptosis and promote cell cycle progression. Therefore, it is unsurprising that cancer cells demonstrate significantly elevated expression levels of IAPs, resulting in improved cell survival, enhanced tumor growth and subsequent metastasis. Therapies to target IAPs in cancer has garnered substantial scientific interest and as resistance to anti-cancer agents becomes more prevalent, targeting IAPs has become an increasingly attractive strategy to re-sensitize cancer cells to chemotherapies, antibody based-therapies and TRAIL therapy. Antagonism strategies to modulate the actions of XIAP, cIAP1/2 and survivin are the central focus of current research and this review highlights advances within this field with particular emphasis upon the development and specificity of second mitochondria-derived activator of caspase (SMAC) mimetics (synthetic analogs of endogenously expressed inhibitors of IAPs SMAC/DIABLO). While we highlight the potential of SMAC mimetics as effective single agent or combinatory therapies to treat cancer we also discuss the likely clinical implications of resistance to SMAC mimetic therapy, occasionally observed in cancer cell lines.

  9. Hepatitis C Virus: Viral Quasispecies and Genotypes.

    Science.gov (United States)

    Tsukiyama-Kohara, Kyoko; Kohara, Michinori

    2017-12-22

    Hepatitis C virus (HCV) mainly replicates in the cytoplasm, where it easily establishes persistent infection, resulting in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Due to its high rate of mutation, HCV forms viral quasispecies, categorized based on the highly variable regions in the envelope protein and nonstructural 5A protein. HCV possesses seven major genotypes, among which genotype 1 is the most prevalent globally. The distribution of HCV genotypes varies based on geography, and each genotype has a different sensitivity to interferon treatment. Recently-developed direct-acting antivirals (DAAs), which target viral proteases or polymerases, mediate drastically better antiviral effects than previous therapeutics. Although treatment with DAAs has led to the development of drug-resistant HCV mutants, the most recently approved DAAs show improved pan-genomic activity, with a higher barrier to viral resistance.

  10. Recessive Resistance to Plant Viruses: Potential Resistance Genes Beyond Translation Initiation Factors

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    Masayoshi Hashimoto

    2016-10-01

    Full Text Available The ability of plant viruses to propagate their genomes in host cells depends on many host factors. In the absence of an agrochemical that specifically targets plant viral infection cycles, one of the most effective methods for controlling viral diseases in plants is taking advantage of the host plant’s resistance machinery. Recessive resistance is conferred by a recessive gene mutation that encodes a host factor critical for viral infection. It is a branch of the resistance machinery and, as an inherited characteristic, is very durable. Moreover, recessive resistance may be acquired by a deficiency in a negative regulator of plant defense responses, possibly due to the autoactivation of defense signaling. Eukaryotic translation initiation factor (eIF 4E and eIF4G and their isoforms are the most widely exploited recessive resistance genes in several crop species, and they are effective against a subset of viral species. However, the establishment of efficient, recessive resistance-type antiviral control strategies against a wider range of plant viral diseases requires genetic resources other than eIF4Es. In this review, we focus on recent advances related to antiviral recessive resistance genes evaluated in model plants and several crop species. We also address the roles of next-generation sequencing and genome editing technologies in improving plant genetic resources for recessive resistance-based antiviral breeding in various crop species.

  11. Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant.

    Science.gov (United States)

    Duan, X; Li, X; Xue, Q; Abo-el-Saad, M; Xu, D; Wu, R

    1996-04-01

    We introduced the potato proteinase inhibitor II (PINII) gene (pin2) into several Japonica rice varieties, and regenerated a large number of transgenic rice plants. Wound-inducible expression of the pin2 gene driven by its own promoter, together with the first intron of the rice actin 1 gene (act1), resulted in high-level accumulation of the PINII protein in the transgenic plants. The introduced pin2 gene was stably inherited in the second, third, and fourth generations, as shown by molecular analyses. Based on data from the molecular analyses, several homozygous transgenic lines were obtained. Bioassay for insect resistance with the fifth-generation transgenic rice plants showed that transgenic rice plants had increased resistance to a major rice insect pest, pink stem borer (Sesamia inferens). Thus, introduction of an insecticidal proteinase inhibitor gene into cereal plants can be used as a general strategy for control of insect pests.

  12. Inhibition of influenza virus infection and hemagglutinin cleavage by the protease inhibitor HAI-2

    Energy Technology Data Exchange (ETDEWEB)

    Hamilton, Brian S.; Chung, Changik; Cyphers, Soreen Y.; Rinaldi, Vera D.; Marcano, Valerie C.; Whittaker, Gary R., E-mail: grw7@cornell.edu

    2014-07-25

    Highlights: • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza HA cleavage activation. • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza virus infection. • Comparative analysis of HAI-2 for vesicular stomatitis virus and human parainfluenza virus type-1. • Analysis of the activity of HAI-2 in a mouse model of influenza. - Abstract: Influenza virus remains a significant concern to public health, with the continued potential for a high fatality pandemic. Vaccination and antiviral therapeutics are effective measures to circumvent influenza virus infection, however, multiple strains have emerged that are resistant to the antiviral therapeutics currently on the market. With this considered, investigation of alternative antiviral therapeutics is being conducted. One such approach is to inhibit cleavage activation of the influenza virus hemagglutinin (HA), which is an essential step in the viral replication cycle that permits viral-endosome fusion. Therefore, targeting trypsin-like, host proteases responsible for HA cleavage in vivo may prove to be an effective therapeutic. Hepatocyte growth factor activator inhibitor 2 (HAI-2) is naturally expressed in the respiratory tract and is a potent inhibitor of trypsin-like serine proteases, some of which have been determined to cleave HA. In this study, we demonstrate that HAI-2 is an effective inhibitor of cleavage of HA from the human-adapted H1 and H3 subtypes. HAI-2 inhibited influenza virus H1N1 infection in cell culture, and HAI-2 administration showed protection in a mouse model of influenza. HAI-2 has the potential to be an effective, alternative antiviral therapeutic for influenza.

  13. Inhibition of influenza virus infection and hemagglutinin cleavage by the protease inhibitor HAI-2

    International Nuclear Information System (INIS)

    Hamilton, Brian S.; Chung, Changik; Cyphers, Soreen Y.; Rinaldi, Vera D.; Marcano, Valerie C.; Whittaker, Gary R.

    2014-01-01

    Highlights: • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza HA cleavage activation. • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza virus infection. • Comparative analysis of HAI-2 for vesicular stomatitis virus and human parainfluenza virus type-1. • Analysis of the activity of HAI-2 in a mouse model of influenza. - Abstract: Influenza virus remains a significant concern to public health, with the continued potential for a high fatality pandemic. Vaccination and antiviral therapeutics are effective measures to circumvent influenza virus infection, however, multiple strains have emerged that are resistant to the antiviral therapeutics currently on the market. With this considered, investigation of alternative antiviral therapeutics is being conducted. One such approach is to inhibit cleavage activation of the influenza virus hemagglutinin (HA), which is an essential step in the viral replication cycle that permits viral-endosome fusion. Therefore, targeting trypsin-like, host proteases responsible for HA cleavage in vivo may prove to be an effective therapeutic. Hepatocyte growth factor activator inhibitor 2 (HAI-2) is naturally expressed in the respiratory tract and is a potent inhibitor of trypsin-like serine proteases, some of which have been determined to cleave HA. In this study, we demonstrate that HAI-2 is an effective inhibitor of cleavage of HA from the human-adapted H1 and H3 subtypes. HAI-2 inhibited influenza virus H1N1 infection in cell culture, and HAI-2 administration showed protection in a mouse model of influenza. HAI-2 has the potential to be an effective, alternative antiviral therapeutic for influenza

  14. Src mediates cigarette smoke-induced resistance to tyrosine kinase inhibitors in NSCLC cells.

    Science.gov (United States)

    Filosto, Simone; Baston, David S; Chung, Samuel; Becker, Cathleen R; Goldkorn, Tzipora

    2013-08-01

    The EGF receptor (EGFR) is a proto-oncogene commonly dysregulated in several cancers including non-small cell lung carcinoma (NSCLC) and, thus, is targeted for treatment using tyrosine kinase inhibitors (TKI) such as erlotinib. However, despite the efficacy observed in patients with NSCLC harboring oncogenic variants of the EGFR, general ineffectiveness of TKIs in patients with NSCLC who are current and former smokers necessitates identification of novel mechanisms to overcome this phenomenon. Previously, we showed that NSCLC cells harboring either wild-type (WT) EGFR or oncogenic mutant (MT) L858R EGFR become resistant to the effects of TKIs when exposed to cigarette smoke, evidenced by their autophosphorylation and prolonged downstream signaling. Here, we present Src as a target mediating cigarette smoke-induced resistance to TKIs in both WT EGFR- and L858R MT EGFR-expressing NSCLC cells. First, we show that cigarette smoke exposure of A549 cells leads to time-dependent activation of Src, which then abnormally binds to the WT EGFR causing TKI resistance, contrasting previous observations of constitutive binding between inactive Src and TKI-sensitive L858R MT EGFR. Next, we show that Src inhibition restores TKI sensitivity in cigarette smoke-exposed NSCLC cells, preventing EGFR autophosphorylation in the presence of erlotinib. Furthermore, we show that overexpression of a dominant-negative Src (Y527F/K295R) restores TKI sensitivity to A549 exposed to cigarette smoke. Importantly, the TKI resistance that emerges even in cigarette smoke-exposed L858R EGFR-expressing NSCLC cells could be eliminated with Src inhibition. Together, these findings offer new rationale for using Src inhibitors for treating TKI-resistant NSCLC commonly observed in smokers.

  15. Cancer Cell Resistance to Aurora Kinase Inhibitors: Identification of Novel Targets for Cancer Therapy

    Czech Academy of Sciences Publication Activity Database

    Hrabáková, Rita; Kollaredy, M.; Tylečková, Jiřina; Halada, Petr; Hajdúch, M.; Gadher, S. J.; Kovářová, Hana

    2013-01-01

    Roč. 12, č. 1 (2013), s. 455-469 ISSN 1535-3893 R&D Projects: GA MŠk LC07017 Institutional support: RVO:67985904 ; RVO:61388971 Keywords : Aurora kinase inhibitors * resistance * p53 * apoptosis Subject RIV: CE - Biochemistry Impact factor: 5.001, year: 2013

  16. Integrase-independent HIV-1 infection is augmented under conditions of DNA damage and produces a viral reservoir

    International Nuclear Information System (INIS)

    Ebina, Hirotaka; Kanemura, Yuka; Suzuki, Yasutsugu; Urata, Kozue; Misawa, Naoko; Koyanagi, Yoshio

    2012-01-01

    HIV-1 possesses a viral protein, integrase (IN), which is necessary for its efficient integration in target cells. However, it has been reported that an IN-defective HIV strain is still capable of integration. Here, we assessed the ability of wild type (WT) HIV-1 to establish infection in the presence of IN inhibitors. We observed a low, yet clear infection of inhibitor-incubated cells infected with WT HIV which was identical to cells infected with IN-deficient HIV, D64A. Furthermore, the IN-independent integration could be enhanced by the pretreatment of cells with DNA-damaging agents suggesting that integration is mediated by a DNA repair system. Moreover, significantly faster viral replication kinetics with augmented viral DNA integration was observed after infection in irradiated cells treated with IN inhibitor compared to nonirradiated cells. Altogether, our results suggest that HIV DNA has integration potential in the presence of an IN inhibitor and may serve as a virus reservoir.

  17. Integrase-independent HIV-1 infection is augmented under conditions of DNA damage and produces a viral reservoir

    Energy Technology Data Exchange (ETDEWEB)

    Ebina, Hirotaka, E-mail: hebina@virus.kyoto-u.ac.jp; Kanemura, Yuka; Suzuki, Yasutsugu; Urata, Kozue; Misawa, Naoko; Koyanagi, Yoshio

    2012-05-25

    HIV-1 possesses a viral protein, integrase (IN), which is necessary for its efficient integration in target cells. However, it has been reported that an IN-defective HIV strain is still capable of integration. Here, we assessed the ability of wild type (WT) HIV-1 to establish infection in the presence of IN inhibitors. We observed a low, yet clear infection of inhibitor-incubated cells infected with WT HIV which was identical to cells infected with IN-deficient HIV, D64A. Furthermore, the IN-independent integration could be enhanced by the pretreatment of cells with DNA-damaging agents suggesting that integration is mediated by a DNA repair system. Moreover, significantly faster viral replication kinetics with augmented viral DNA integration was observed after infection in irradiated cells treated with IN inhibitor compared to nonirradiated cells. Altogether, our results suggest that HIV DNA has integration potential in the presence of an IN inhibitor and may serve as a virus reservoir.

  18. Conjugation Inhibitors and Their Potential Use to Prevent Dissemination of Antibiotic Resistance Genes in Bacteria

    Directory of Open Access Journals (Sweden)

    Elena Cabezón

    2017-11-01

    Full Text Available Antibiotic resistance has become one of the most challenging problems in health care. Bacteria conjugation is one of the main mechanisms whereby bacteria become resistant to antibiotics. Therefore, the search for specific conjugation inhibitors (COINs is of interest in the fight against the spread of antibiotic resistances in a variety of laboratory and natural environments. Several compounds, discovered as COINs, are promising candidates in the fight against plasmid dissemination. In this review, we survey the effectiveness and toxicity of the most relevant compounds. Particular emphasis has been placed on unsaturated fatty acid derivatives, as they have been shown to be efficient in preventing plasmid invasiveness in bacterial populations. Biochemical and structural studies have provided insights concerning their potential molecular targets and inhibitory mechanisms. These findings open a new avenue in the search of new and more effective synthetic inhibitors. In this pursuit, the use of structure-based drug design methods will be of great importance for the screening of ligands and binding sites of putative targets.

  19. Inhibition of Bovine Viral Diarrhea Virus RNA Synthesis by Thiosemicarbazone Derived from 5,6-Dimethoxy-1-Indanone▿

    Science.gov (United States)

    Castro, Eliana F.; Fabian, Lucas E.; Caputto, María E.; Gagey, Dolores; Finkielsztein, Liliana M.; Moltrasio, Graciela Y.; Moglioni, Albertina G.; Campos, Rodolfo H.; Cavallaro, Lucía V.

    2011-01-01

    In the present work, we described the activity of the thiosemicarbazone derived from 5,6-dimethoxy-1-indanone (TSC), which we previously characterized as a new compound that inhibits bovine viral diarrhea virus (BVDV) infection. We showed that TSC acts at a point of time that coincides with the onset of viral RNA synthesis and that it inhibits the activity of BVDV replication complexes (RCs). Moreover, we have selected five BVDV mutants that turned out to be highly resistant to TSC but still susceptible to ribavirin (RBV). Four of these resistant mutants carried an N264D mutation in the viral RNA-dependent RNA polymerase (RdRp). The remaining mutant showed an A392E mutation within the same protein. Some of these mutants replicated slower than the wild-type (wt) virus in the absence of TSC, whereas others showed a partial reversion to the wt phenotype over several passages in the absence of the compound. The docking of TSC in the crystal structure of the BVDV RdRp revealed a close contact between the indane ring of the compound and several residues within the fingers domain of the enzyme, some hydrophobic contacts, and hydrogen bonds with the thiosemicarbazone group. Finally, in the mutated RdRp from resistant BVDV, these interactions with TSC could not be achieved. Interestingly, TSC inhibited BVDV replication in cell culture synergistically with RBV. In conclusion, TSC emerges as a new nonnucleoside inhibitor of BVDV RdRp that is synergistic with RBV, a feature that turns it into a potential compound to be evaluated against hepatitis C virus (HCV). PMID:21430053

  20. Activity of a potent hepatitis C virus polymerase inhibitor in the chimpanzee model.

    Science.gov (United States)

    Chen, Chih-Ming; He, Yupeng; Lu, Liangjun; Lim, Hock Ben; Tripathi, Rakesh L; Middleton, Tim; Hernandez, Lisa E; Beno, David W A; Long, Michelle A; Kati, Warren M; Bosse, Todd D; Larson, Daniel P; Wagner, Rolf; Lanford, Robert E; Kohlbrenner, William E; Kempf, Dale J; Pilot-Matias, Tami J; Molla, Akhteruzzaman

    2007-12-01

    A-837093 is a potent and specific nonnucleoside inhibitor of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase. It possesses nanomolar potencies in both enzymatic and replicon-based cell culture assays. In rats and dogs this compound demonstrated an oral plasma half-life of greater than 7 h, and its bioavailability was >60%. In monkeys it had a half-life of 1.9 h and 15% bioavailability. Its antiviral efficacy was evaluated in two chimpanzees infected with HCV in a proof-of-concept study. The design included oral dosing of 30 mg per kg of body weight twice a day for 14 days, followed by a 14-day posttreatment observation. Maximum viral load reductions of 1.4 and 2.5 log(10) copies RNA/ml for genotype 1a- and 1b-infected chimpanzees, respectively, were observed within 2 days after the initiation of treatment. After this initial drop in the viral load, a rebound of plasma HCV RNA was observed in the genotype 1b-infected chimpanzee, while the genotype 1a-infected chimpanzee experienced a partial rebound that lasted throughout the treatment period. Clonal analysis of NS5B gene sequences derived from the plasma of A-837093-treated chimpanzees revealed the presence of several mutations associated with resistance to A-837093, including Y448H, G554D, and D559G in the genotype 1a-infected chimpanzee and C316Y and G554D in the genotype 1b-infected chimpanzee. The identification of resistance-associated mutations in both chimpanzees is consistent with the findings of in vitro selection studies, in which many of the same mutations were selected. These findings validate the antiviral efficacy and resistance development of benzothiadiazine HCV polymerase inhibitors in vivo.

  1. Hepatitis C Virus: Viral Quasispecies and Genotypes

    Directory of Open Access Journals (Sweden)

    Kyoko Tsukiyama-Kohara

    2017-12-01

    Full Text Available Hepatitis C virus (HCV mainly replicates in the cytoplasm, where it easily establishes persistent infection, resulting in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Due to its high rate of mutation, HCV forms viral quasispecies, categorized based on the highly variable regions in the envelope protein and nonstructural 5A protein. HCV possesses seven major genotypes, among which genotype 1 is the most prevalent globally. The distribution of HCV genotypes varies based on geography, and each genotype has a different sensitivity to interferon treatment. Recently-developed direct-acting antivirals (DAAs, which target viral proteases or polymerases, mediate drastically better antiviral effects than previous therapeutics. Although treatment with DAAs has led to the development of drug-resistant HCV mutants, the most recently approved DAAs show improved pan-genomic activity, with a higher barrier to viral resistance.

  2. Activity of second-generation ALK inhibitors against crizotinib-resistant mutants in an NPM-ALK model compared to EML4-ALK

    International Nuclear Information System (INIS)

    Fontana, Diletta; Ceccon, Monica; Gambacorti-Passerini, Carlo; Mologni, Luca

    2015-01-01

    Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor involved in both solid and hematological tumors. About 80% of ALK-positive anaplastic large-cell lymphoma (ALCL) cases are characterized by the t(2;5)(p23;q35) translocation, encoding for the aberrant fusion protein nucleophosmin (NPM)-ALK, whereas 5% of non-small-cell lung cancer (NSCLC) patients carry the inv(2)(p21;p23) rearrangement, encoding for the echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion. The ALK/c-MET/ROS inhibitor crizotinib successfully improved the treatment of ALK-driven diseases. However, several cases of resistance appeared in NSCLC patients, and ALK amino acid substitutions were identified as a leading cause of resistance to crizotinib. Second-generation ALK inhibitors have been developed in order to overcome crizotinib resistance. In this work, we profiled in vitro the activity of crizotinib, AP26113, ASP3026, alectinib, and ceritinib against six mutated forms of ALK associated with clinical resistance to crizotinib (C1156Y, L1196M, L1152R, G1202R, G1269A, and S1206Y) and provide a classification of mutants according to their level of sensitivity/resistance to the drugs. Since the biological activity of ALK mutations extends beyond the specific type of fusion, both NPM-ALK- and EML4-ALK-positive cellular models were used. Our data revealed that most mutants may be targeted by using different inhibitors. One relevant exception is represented by the G1202R substitution, which was highly resistant to all drugs (>10-fold increased IC 50 compared to wild type) and may represent the most challenging mutation to overcome. These results provide a prediction of cross-resistance of known crizotinib-resistant mutations against all second-generation tyrosine kinase inhibitors (TKIs) clinically available, and therefore could be a useful tool to help clinicians in the management of crizotinib-resistance cases

  3. The multi-targeted kinase inhibitor sorafenib inhibits enterovirus 71 replication by regulating IRES-dependent translation of viral proteins.

    Science.gov (United States)

    Gao, Meng; Duan, Hao; Liu, Jing; Zhang, Hao; Wang, Xin; Zhu, Meng; Guo, Jitao; Zhao, Zhenlong; Meng, Lirong; Peng, Yihong

    2014-06-01

    The activation of ERK and p38 signal cascade in host cells has been demonstrated to be essential for picornavirus enterovirus 71 (EV71) replication and up-regulation of virus-induced cyclooxygenase-2 (COX-2)/prostaglandins E2 (PGE2) expression. The aim of this study was to examine the effects of sorafenib, a clinically approved anti-cancer multi-targeted kinase inhibitor, on the propagation and pathogenesis of EV71, with a view to its possible mechanism and potential use in the design of therapy regimes for Hand foot and mouth disease (HFMD) patients with life threatening neurological complications. In this study, non-toxic concentrations of sorafenib were shown to inhibit the yield of infectious progeny EV71 (clinical BC08 strain) by about 90% in three different cell types. A similar inhibitory effect of sorafenib was observed on the synthesis of both viral genomic RNA and the VP1 protein. Interestingly, sorafenib exerted obvious inhibition of the EV71 internal ribosomal entry site (IRES)-mediated translation, the first step in picornavirus replication, by linking it to a firefly luciferase reporter gene. Sorafenib was also able to prevent both EV71-induced CPE and the activation of ERK and p38, which contributes to up-regulation COX-2/PGE2 expression induced by the virus. Overall, this study shows that sorafenib strongly inhibits EV71 replication at least in part by regulating viral IRES-dependent translation of viral proteins, indicating a novel potential strategy for the treatment of HFMD patients with severe neurological complications. To our knowledge, this is the first report that investigates the mechanism by which sorafenib inhibits EV71 replication. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Rational Design of Novel Allosteric Dihydrofolate Reductase Inhibitors Showing Antibacterial Effects on Drug-Resistant Escherichia coli Escape Variants.

    Science.gov (United States)

    Srinivasan, Bharath; Rodrigues, João V; Tonddast-Navaei, Sam; Shakhnovich, Eugene; Skolnick, Jeffrey

    2017-07-21

    In drug discovery, systematic variations of substituents on a common scaffold and bioisosteric replacements are often used to generate diversity and obtain molecules with better biological effects. However, this could saturate the small-molecule diversity pool resulting in drug resistance. On the other hand, conventional drug discovery relies on targeting known pockets on protein surfaces leading to drug resistance by mutations of critical pocket residues. Here, we present a two-pronged strategy of designing novel drugs that target unique pockets on a protein's surface to overcome the above problems. Dihydrofolate reductase, DHFR, is a critical enzyme involved in thymidine and purine nucleotide biosynthesis. Several classes of compounds that are structural analogues of the substrate dihydrofolate have been explored for their antifolate activity. Here, we describe 10 novel small-molecule inhibitors of Escherichia coli DHFR, EcDHFR, belonging to the stilbenoid, deoxybenzoin, and chalcone family of compounds discovered by a combination of pocket-based virtual ligand screening and systematic scaffold hopping. These inhibitors show a unique uncompetitive or noncompetitive inhibition mechanism, distinct from those reported for all known inhibitors of DHFR, indicative of binding to a unique pocket distinct from either substrate or cofactor-binding pockets. Furthermore, we demonstrate that rescue mutants of EcDHFR, with reduced affinity to all known classes of DHFR inhibitors, are inhibited at the same concentration as the wild-type. These compounds also exhibit antibacterial activity against E. coli harboring the drug-resistant variant of DHFR. This discovery is the first report on a novel class of inhibitors targeting a unique pocket on EcDHFR.

  5. The Potent ALK Inhibitor Brigatinib (AP26113) Overcomes Mechanisms of Resistance to First- and Second-Generation ALK Inhibitors in Preclinical Models.

    Science.gov (United States)

    Zhang, Sen; Anjum, Rana; Squillace, Rachel; Nadworny, Sara; Zhou, Tianjun; Keats, Jeff; Ning, Yaoyu; Wardwell, Scott D; Miller, David; Song, Youngchul; Eichinger, Lindsey; Moran, Lauren; Huang, Wei-Sheng; Liu, Shuangying; Zou, Dong; Wang, Yihan; Mohemmad, Qurish; Jang, Hyun Gyung; Ye, Emily; Narasimhan, Narayana; Wang, Frank; Miret, Juan; Zhu, Xiaotian; Clackson, Tim; Dalgarno, David; Shakespeare, William C; Rivera, Victor M

    2016-11-15

    Non-small cell lung cancers (NSCLCs) harboring ALK gene rearrangements (ALK + ) typically become resistant to the first-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI) crizotinib through development of secondary resistance mutations in ALK or disease progression in the brain. Mutations that confer resistance to second-generation ALK TKIs ceritinib and alectinib have also been identified. Here, we report the structure and first comprehensive preclinical evaluation of the next-generation ALK TKI brigatinib. A kinase screen was performed to evaluate the selectivity profile of brigatinib. The cellular and in vivo activities of ALK TKIs were compared using engineered and cancer-derived cell lines. The brigatinib-ALK co-structure was determined. Brigatinib potently inhibits ALK and ROS1, with a high degree of selectivity over more than 250 kinases. Across a panel of ALK + cell lines, brigatinib inhibited native ALK (IC 50 , 10 nmol/L) with 12-fold greater potency than crizotinib. Superior efficacy of brigatinib was also observed in mice with ALK + tumors implanted subcutaneously or intracranially. Brigatinib maintained substantial activity against all 17 secondary ALK mutants tested in cellular assays and exhibited a superior inhibitory profile compared with crizotinib, ceritinib, and alectinib at clinically achievable concentrations. Brigatinib was the only TKI to maintain substantial activity against the most recalcitrant ALK resistance mutation, G1202R. The unique, potent, and pan-ALK mutant activity of brigatinib could be rationalized by structural analyses. Brigatinib is a highly potent and selective ALK inhibitor. These findings provide the molecular basis for the promising activity being observed in ALK + , crizotinib-resistant patients with NSCLC being treated with brigatinib in clinical trials. Clin Cancer Res; 22(22); 5527-38. ©2016 AACR. ©2016 American Association for Cancer Research.

  6. Consideration of Viral Resistance for Optimization of Direct Antiviral Therapy of Hepatitis C Virus Genotype 1-Infected Patients.

    Science.gov (United States)

    Dietz, Julia; Susser, Simone; Berkowski, Caterina; Perner, Dany; Zeuzem, Stefan; Sarrazin, Christoph

    2015-01-01

    Different highly effective interferon-free treatment options for chronic hepatitis C virus (HCV) infection are currently available. Pre-existence of resistance associated variants (RAVs) to direct antiviral agents (DAAs) reduces sustained virologic response (SVR) rates by 3-53% in hepatitis C virus (HCV) genotype 1 infected patients depending on different predictors and the DAA regimen used. Frequencies of single and combined resistance to NS3, NS5A and NS5B inhibitors and consequences for the applicability of different treatment regimens are unknown. Parallel population based sequencing of HCV NS3, NS5A and NS5B genes in 312 treatment-naïve Caucasian HCV genotype 1 infected patients showed the presence of major resistant variants in 20.5% (NS3), 11.9% (NS5A), and 22.1% (NS5B) with important differences for HCV subtypes. In NS3, Q80K was observed in 34.7% and 2.1% of subtype 1a and 1b patients, respectively while other RAVs to second generation protease inhibitors were detected rarely (1.4%). Within NS5A RAVs were observed in 7.1% of subtype 1a and 17.6% in subtype 1b infected patients. RAVs to non-nucleoside NS5B inhibitors were observed in 3.5% and 44.4% of subtype 1a and 1b patients, respectively. Considering all three DAA targets all subtype 1a and 98.6% of subtype 1b infected patients were wildtype for at least one interferon free DAA regimen currently available. In conclusion, baseline resistance testing allows the selection of at least one RAVs-free treatment option for nearly all patients enabling a potentially cost- and efficacy-optimized treatment of chronic hepatitis C.

  7. Consideration of Viral Resistance for Optimization of Direct Antiviral Therapy of Hepatitis C Virus Genotype 1-Infected Patients.

    Directory of Open Access Journals (Sweden)

    Julia Dietz

    Full Text Available Different highly effective interferon-free treatment options for chronic hepatitis C virus (HCV infection are currently available. Pre-existence of resistance associated variants (RAVs to direct antiviral agents (DAAs reduces sustained virologic response (SVR rates by 3-53% in hepatitis C virus (HCV genotype 1 infected patients depending on different predictors and the DAA regimen used. Frequencies of single and combined resistance to NS3, NS5A and NS5B inhibitors and consequences for the applicability of different treatment regimens are unknown. Parallel population based sequencing of HCV NS3, NS5A and NS5B genes in 312 treatment-naïve Caucasian HCV genotype 1 infected patients showed the presence of major resistant variants in 20.5% (NS3, 11.9% (NS5A, and 22.1% (NS5B with important differences for HCV subtypes. In NS3, Q80K was observed in 34.7% and 2.1% of subtype 1a and 1b patients, respectively while other RAVs to second generation protease inhibitors were detected rarely (1.4%. Within NS5A RAVs were observed in 7.1% of subtype 1a and 17.6% in subtype 1b infected patients. RAVs to non-nucleoside NS5B inhibitors were observed in 3.5% and 44.4% of subtype 1a and 1b patients, respectively. Considering all three DAA targets all subtype 1a and 98.6% of subtype 1b infected patients were wildtype for at least one interferon free DAA regimen currently available. In conclusion, baseline resistance testing allows the selection of at least one RAVs-free treatment option for nearly all patients enabling a potentially cost- and efficacy-optimized treatment of chronic hepatitis C.

  8. Differential effect of EGFR inhibitors on tamoxifen-resistant breast cancer cells.

    Science.gov (United States)

    Kim, Sangmin; Lee, Jeongmin; Oh, Soo Jin; Nam, Seok Jin; Lee, Jeong Eon

    2015-09-01

    Although tamoxifen is the most common and effective therapy for treatment of estrogen receptor-α (ER-α) breast cancer patients, resistance of endocrine therapy occurs, either de novo or acquired during therapy. Here, we investigated the clinical value of epidermal growth factor receptor (EGFR) in tamoxifen-resistant (TamR) patients and the differential effect of EGFR inhibitors, neratinib and gefitinib, on TamR breast cancer cell model. The morphology of TamR MCF7 cells showed mesenchymal phenotypes and did not induce cell death by tamoxifen treatment compared with tamoxifen‑sensitive (TamS) MCF7 cells. In addition, mesenchymal marker proteins, including N-cadherin (N-cad), fibronectin (FN), and Slug, significantly increased in TamR cells. In contrast, ER-α and E-cadherin (E-cad) were greatly decreased. We also found that the levels of EGFR and HER2 expression were increased in TamR cells. Furthermore, we observed that EGFR expression was directly involved with poor prognosis of tamoxifen-treated breast cancer patients using the GSE1378 date set. Thus, we treated TamR and TamS cells with EGFR inhibitors, neratinib and gefitinib, respectively. Interestingly, neratinib induced apoptotic cell death of TamR but not gefitinib. Cleaved PARP-1 expression was also increased by neratinib treatment in TamR cells. Therefore, we suggest that neratinib may be a potential therapeutic drug for treating TamR breast cancer.

  9. A serine palmitoyltransferase inhibitor blocks hepatitis C virus replication in human hepatocytes.

    Science.gov (United States)

    Katsume, Asao; Tokunaga, Yuko; Hirata, Yuichi; Munakata, Tsubasa; Saito, Makoto; Hayashi, Hitohisa; Okamoto, Koichi; Ohmori, Yusuke; Kusanagi, Isamu; Fujiwara, Shinya; Tsukuda, Takuo; Aoki, Yuko; Klumpp, Klaus; Tsukiyama-Kohara, Kyoko; El-Gohary, Ahmed; Sudoh, Masayuki; Kohara, Michinori

    2013-10-01

    Host cell lipid rafts form a scaffold required for replication of hepatitis C virus (HCV). Serine palmitoyltransferases (SPTs) produce sphingolipids, which are essential components of the lipid rafts that associate with HCV nonstructural proteins. Prevention of the de novo synthesis of sphingolipids by an SPT inhibitor disrupts the HCV replication complex and thereby inhibits HCV replication. We investigated the ability of the SPT inhibitor NA808 to prevent HCV replication in cells and mice. We tested the ability of NA808 to inhibit SPT's enzymatic activity in FLR3-1 replicon cells. We used a replicon system to select for HCV variants that became resistant to NA808 at concentrations 4- to 6-fold the 50% inhibitory concentration, after 14 rounds of cell passage. We assessed the ability of NA808 or telaprevir to inhibit replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in mice with humanized livers (transplanted with human hepatocytes). NA808 was injected intravenously, with or without pegylated interferon alfa-2a and HCV polymerase and/or protease inhibitors. NA808 prevented HCV replication via noncompetitive inhibition of SPT; no resistance mutations developed. NA808 prevented replication of all HCV genotypes tested in mice with humanized livers. Intravenous NA808 significantly reduced viral load in the mice and had synergistic effects with pegylated interferon alfa-2a and HCV polymerase and protease inhibitors. The SPT inhibitor NA808 prevents replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in cultured hepatocytes and in mice with humanized livers. It might be developed for treatment of HCV infection or used in combination with pegylated interferon alfa-2a or HCV polymerase or protease inhibitors. Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.

  10. Selective inhibition of influenza virus protein synthesis by inhibitors of DNA function

    International Nuclear Information System (INIS)

    Minor, P.D.; Dimmock, N.J.

    1977-01-01

    Various known inhibitors of cellular DNA function were shown to inhibit cellular RNA synthesis and influenza (fowl plague) virus multiplication. The drugs were investigated for their effect upon the synthesis of influenza virus proteins. According to this effect they could be classified with previously studied compounds as follows: Group I (ethidium bromide, proflavine, and N-nitroquinoline-N-oxide) inhibited both viral and cellular protein synthesis; Group II (nogalomycin, daunomycin and α-amanitin) inhibited viral but not cellular protein synthesis, and all viral proteins were inhibited coordinately; Group III (mithramycin, echinomycin, and actinomycin D) inhibited all viral but not cellular protein synthesis at high concentrations, but at a lower critical concentration inhibited the synthesis of viral haemagglutinin, neuraminidase, and M protein preferentially; Group IV(uv irradiation and camptothecin) inhibited the synthesis of viral haemagglutinin, neuraminidase, and M protein, but not other viral proteins, even at high doses. The mode of action of these inhibitors is discussed in relation to the mechanism of the nuclear events upon which influenza virus multiplication is dependent

  11. Engineering cotton (Gossypium hirsutum L.) for resistance to cotton leaf curl disease using viral truncated AC1 DNA sequences.

    Science.gov (United States)

    Hashmi, Jamil A; Zafar, Yusuf; Arshad, Muhammad; Mansoor, Shahid; Asad, Shaheen

    2011-04-01

    Several important biological processes are performed by distinct functional domains found on replication-associated protein (Rep) encoded by AC1 of geminiviruses. Two truncated forms of replicase (tAC1) gene, capable of expressing only the N-terminal 669 bp (5'AC1) and C-terminal 783 bp (3'AC1) nucleotides cloned under transcriptional control of the CaMV35S were introduced into cotton (Gossypium hirsutum L.) using LBA4404 strain of Agrobacterium tumefaciens to make use of an interference strategy for impairing cotton leaf curl virus (CLCuV) infection in transgenic cotton. Compared with nontransformed control, we observed that transgenic cotton plants overexpressing either N-terminal (5'AC1) or C-terminal (3'AC1) sequences confer resistance to CLCuV by inhibiting replication of viral genomic and β satellite DNA components. Molecular analysis by Northern blot hybridization revealed high transgene expression in early and late growth stages associated with inhibition of CLCuV replication. Of the eight T(1) transgenic lines tested, six had delayed and minor symptoms as compared to nontransformed control lines which developed disease symptoms after 2-3 weeks of whitefly-mediated viral delivery. Virus biological assay and growth of T(2) plants proved that transgenic cotton plants overexpressing 5'- and 3'AC1 displayed high resistance level up to 72, 81%, respectively, as compared to non-transformed control plants following inoculation with viruliferous whiteflies giving significantly high cotton seed yield. Progeny analysis of these plants by polymerase chain reaction (PCR), Southern blotting and virus biological assay showed stable transgene, integration, inheritance and cotton leaf curl disease (CLCuD) resistance in two of the eight transgenic lines having single or two transgene insertions. Transgenic cotton expressing partial AC1 gene of CLCuV can be used as virus resistance source in cotton breeding programs aiming to improve virus resistance in cotton crop.

  12. Effect of Neuraminidase Inhibitor–Resistant Mutations on Pathogenicity of Clade 2.2 A/Turkey/15/06 (H5N1) Influenza Virus in Ferrets

    OpenAIRE

    Ilyushina, Natalia A.; Seiler, Jon P.; Rehg, Jerold E.; Webster, Robert G.; Govorkova, Elena A.

    2010-01-01

    The acquisition of neuraminidase (NA) inhibitor resistance by H5N1 influenza viruses has serious clinical implications, as this class of drugs can be an essential component of pandemic control measures. The continuous evolution of the highly pathogenic H5N1 influenza viruses results in the emergence of natural NA gene variations whose impact on viral fitness and NA inhibitor susceptibility are poorly defined. We generated seven genetically stable recombinant clade 2.2 A/Turkey/15/06-like (H5N...

  13. Small-molecule inhibition of HIV pre-mRNA splicing as a novel antiretroviral therapy to overcome drug resistance.

    Directory of Open Access Journals (Sweden)

    Nadia Bakkour

    2007-10-01

    Full Text Available The development of multidrug-resistant viruses compromises antiretroviral therapy efficacy and limits therapeutic options. Therefore, it is an ongoing task to identify new targets for antiretroviral therapy and to develop new drugs. Here, we show that an indole derivative (IDC16 that interferes with exonic splicing enhancer activity of the SR protein splicing factor SF2/ASF suppresses the production of key viral proteins, thereby compromising subsequent synthesis of full-length HIV-1 pre-mRNA and assembly of infectious particles. IDC16 inhibits replication of macrophage- and T cell-tropic laboratory strains, clinical isolates, and strains with high-level resistance to inhibitors of viral protease and reverse transcriptase. Importantly, drug treatment of primary blood cells did not alter splicing profiles of endogenous genes involved in cell cycle transition and apoptosis. Thus, human splicing factors represent novel and promising drug targets for the development of antiretroviral therapies, particularly for the inhibition of multidrug-resistant viruses.

  14. [A method for genetic transformation of maize for resistance to viral diseases].

    Science.gov (United States)

    Valdez, Marta; Madriz, Kenneth; Ramírez, Pilar

    2004-09-01

    A system for the genetic transformation of maize was developed for two Costa Rican varieties: CR-7 and Diamantes 8843, that can allow the subsequent transfer of viral-derived genes in order to confer resistance to the disease caused by maize rayado fino virus (MRFV). The method is based on particle bombardment of organogenic calli derived from shoot tips. On the other hand, the molecular construction pRFcp-bar, containing the coat protein gene of MRFV and the marker gene bar, was elaborated. For the visual selection of the transformed material was used also the plasmid pDM803 that contains the reporter gene uidA (GUS). The results indicate that devices evaluated: the PIG ("Particle Inflow Gun") and the Bio-Rad are both enough efficient to transfer foreign genes to the genome of the maize.

  15. CO2 Capture by Absorption with Potassium Carbonate

    Energy Technology Data Exchange (ETDEWEB)

    Rochelle, Gary T; Seibert, Frank; Closmann, Fred; Cullinane, Tim; Davis, Jason; Goff, George; Hilliard, Marcus; McLees, John; Plaza, Jorge M; Sexton, Andrew; Wagener, David Van; Zu, Qing; Veawab, Amornvadee; Nainar, Manjula

    2007-08-31

    The objective of this work is to improve the process for CO{sub 2} capture by alkanolamine absorption/stripping by developing an alternative solvent, aqueous K{sub 2}CO3 promoted by piperazine (PZ). Pilot plant testing was performed in a 16.8-inch ID absorber and stripper with recirculation of air and CO{sub 2}. Three solvents (7 m MEA, 5 m K{sup +}/2.5 m PZ, and 6.4 m K{sup +}/1.6 m PZ) were tested in four campaigns with three different absorber packings. Pilot plant testing established that 5 m K{sup +}/2.5 m PZ requires two times less packing than 7 m MEA and three times less packing than 6.4 m K{sup +}/1.6 m PZ. A rigorous model of the thermodynamics and mass transfer was developed in the RateSep{trademark} block of AspenPlus{reg_sign}. The double matrix stripper reduces energy consumption by 5 to 15%. The best K{sup +}/PZ solvent, 4 m K{sup +}/4 m PZ, and the best process configuration, double matrix stripper with a double intercooled absorber, requires equivalent work of 40 kJ/mole CO{sub 2} to produce CO{sub 2} at 10 MPa. Inhibitor A is effective at reducing oxidative degradation over a wide range of metal concentrations and solvent types. Piperazine is resistant to oxidative degradation catalyzed by dissolved iron, but it oxidizes at rates comparable to monoethanolamine (MEA) in the presence of dissolved copper. The thermal degradation of MEA becomes significant at 120 C, but loaded piperazine solutions appear to be resistant to thermal degradation up to 135 C. The vapor pressure of PZ over typical lean solution at 40 C will be less than 25 ppm, which is less than the 40 ppm expected for MEA. Significant problems with foaming were encountered and alleviated by antifoamants in the pilot plant campaigns with K{sup +}/PZ. Potassium sulfate is not very soluble in 4 m K{sup +}/4 m PZ, so SO{sub 2} absorption and oxidation to sulfate in the bottom of the absorber may require operation with a slurry of potassium sulfate solids.

  16. Maintenance of serum potassium with sodium zirconium cyclosilicate (ZS-9) in heart failure patients : results from a phase 3 randomized, double-blind, placebo-controlled trial

    NARCIS (Netherlands)

    Anker, Stefan D.; Kosiborod, Mikhail; Zannad, Faiez; Pina, Ileana L.; McCullough, Peter A.; Filippatos, Gerasimos; van der Meer, Peter; Ponikowski, Piotr; Rasmussen, Henrik S.; Lavin, Philip T.; Singh, Bhupinder; Yang, Alex; Deedwania, Prakash

    2015-01-01

    AimsHyperkalaemia in heart failure patients limits use of cardioprotective renin-angiotensin-aldosterone system inhibitors (RAASi). Sodium zirconium cyclosilicate (ZS-9) is a selective potassium ion trap, whose mechanism of action may allow for potassium binding in the upper gastrointestinal tract

  17. Characterization of Dengue Virus Resistance to Brequinar in Cell Culture▿

    Science.gov (United States)

    Qing, Min; Zou, Gang; Wang, Qing-Yin; Xu, Hao Ying; Dong, Hongping; Yuan, Zhiming; Shi, Pei-Yong

    2010-01-01

    Brequinar is an inhibitor of dihydroorotate dehydrogenase, an enzyme that is required for de novo pyrimidine biosynthesis. Here we report that brequinar has activity against a broad spectrum of viruses. The compound not only inhibits flaviviruses (dengue virus, West Nile virus, yellow fever virus, and Powassan virus) but also suppresses a plus-strand RNA alphavirus (Western equine encephalitis virus) and a negative-strand RNA rhabdovirus (vesicular stomatitis virus). Using dengue virus serotype 2 (DENV-2) as a model, we found that brequinar suppressed the viral infection cycle mainly at the step of RNA synthesis. Supplementing the culture medium with pyrimidines (cytidine or uridine) but not purines (adenine or guanine) could be used to reverse the inhibitory effect of the compound. Continuous culturing of DENV-2 in the presence of brequinar generated viruses that were partially resistant to the inhibitor. Sequencing of the resistant viruses revealed two amino acid mutations: one mutation (M260V) located at a helix in the domain II of the viral envelope protein and another mutation (E802Q) located at the priming loop of the nonstructural protein 5 (NS5) polymerase domain. Functional analysis of the mutations suggests that the NS5 mutation exerts resistance through enhancement of polymerase activity. The envelope protein mutation reduced the efficiency of virion assembly/release; however, the mutant virus became less sensitive to brequinar inhibition at the step of virion assembly/release. Taken together, the results indicate that (i) brequinar blocks DENV RNA synthesis through depletion of intracellular pyrimidine pools and (ii) the compound may also exert its antiviral activity through inhibition of virion assembly/release. PMID:20606073

  18. [Immunotherapy for refractory viral infections].

    Science.gov (United States)

    Morio, Tomohiro; Fujita, Yuriko; Takahashi, Satoshi

    Various antiviral agents have been developed, which are sometimes associated with toxicity, development of virus-resistant strain, and high cost. Virus-specific T-cell (VST) therapy provides an alternative curative therapy that can be effective for a prolonged time without eliciting drug resistance. VSTs can be directly separated using several types of capture devices and can be obtained by stimulating peripheral blood mononuclear cells with viral antigens (virus, protein, or peptide) loaded on antigen-presenting cells (APC). APC can be transduced with virus-antigen coding plasmid or pulsed with overlapping peptides. VST therapy has been studied in drug non-responsive viral infections after hematopoietic cell transplantation (HCT). Several previous studies have demonstrated the efficacy of VST therapy without significant severe GVHD. In addition, VSTs from a third-party donor have been prepared and administered for post-HCT viral infection. Although target viruses of VSTs include herpes virus species and polyomavirus species, a wide variety of pathogens, such as papillomavirus, intracellular bacteria, and fungi, can be treated by pathogen-specific T-cells. Perhaps, these specific T-cells could be used for opportunistic infections in other immunocompromised hosts in the near future.

  19. Anti-cancer effect of HIV-1 viral protein R on doxorubicin resistant neuroblastoma.

    Directory of Open Access Journals (Sweden)

    Richard Y Zhao

    Full Text Available Several unique biological features of HIV-1 Vpr make it a potentially powerful agent for anti-cancer therapy. First, Vpr inhibits cell proliferation by induction of cell cycle G2 arrest. Second, it induces apoptosis through multiple mechanisms, which could be significant as it may be able to overcome apoptotic resistance exhibited by many cancerous cells, and, finally, Vpr selectively kills fast growing cells in a p53-independent manner. To demonstrate the potential utility of Vpr as an anti-cancer agent, we carried out proof-of-concept studies in vitro and in vivo. Results of our preliminary studies demonstrated that Vpr induces cell cycle G2 arrest and apoptosis in a variety of cancer types. Moreover, the same Vpr effects could also be detected in some cancer cells that are resistant to anti-cancer drugs such as doxorubicin (DOX. To further illustrate the potential value of Vpr in tumor growth inhibition, we adopted a DOX-resistant neuroblastoma model by injecting SK-N-SH cells into C57BL/6N and C57BL/6J-scid/scid mice. We hypothesized that Vpr is able to block cell proliferation and induce apoptosis regardless of the drug resistance status of the tumors. Indeed, production of Vpr via adenoviral delivery to neuroblastoma cells caused G2 arrest and apoptosis in both drug naïve and DOX-resistant cells. In addition, pre-infection or intratumoral injection of vpr-expressing adenoviral particles into neuroblastoma tumors in SCID mice markedly inhibited tumor growth. Therefore, Vpr could possibly be used as a supplemental viral therapeutic agent for selective inhibition of tumor growth in anti-cancer therapy especially when other therapies stop working.

  20. Oncogenic Receptor Tyrosine Kinases Directly Phosphorylate Focal Adhesion Kinase (FAK) as a Resistance Mechanism to FAK-Kinase Inhibitors.

    Science.gov (United States)

    Marlowe, Timothy A; Lenzo, Felicia L; Figel, Sheila A; Grapes, Abigail T; Cance, William G

    2016-12-01

    Focal adhesion kinase (FAK) is a major drug target in cancer and current inhibitors targeted to the ATP-binding pocket of the kinase domain have entered clinical trials. However, preliminary results have shown limited single-agent efficacy in patients. Despite these unfavorable data, the molecular mechanisms that drive intrinsic and acquired resistance to FAK-kinase inhibitors are largely unknown. We have demonstrated that receptor tyrosine kinases (RTK) can directly bypass FAK-kinase inhibition in cancer cells through phosphorylation of FAK's critical tyrosine 397 (Y397). We also showed that HER2 forms a direct protein-protein interaction with the FAK-FERM-F1 lobe, promoting direct phosphorylation of Y397. In addition, FAK-kinase inhibition induced two forms of compensatory RTK reprogramming: (i) the rapid phosphorylation and activation of RTK signaling pathways in RTK High cells and (ii) the long-term acquisition of RTKs novel to the parental cell line in RTK Low cells. Finally, HER2 +: cancer cells displayed resistance to FAK-kinase inhibition in 3D growth assays using a HER2 isogenic system and HER2 + cancer cell lines. Our data indicate a novel drug resistance mechanism to FAK-kinase inhibitors whereby HER2 and other RTKs can rescue and maintain FAK activation (pY397) even in the presence of FAK-kinase inhibition. These data may have important ramifications for existing clinical trials of FAK inhibitors and suggest that individual tumor stratification by RTK expression would be important to predict patient response to FAK-kinase inhibitors. Mol Cancer Ther; 15(12); 3028-39. ©2016 AACR. ©2016 American Association for Cancer Research.

  1. Effectiveness of Ritonavir-Boosted Protease Inhibitor Monotherapy in Clinical Practice Even with Previous Virological Failures to Protease Inhibitor-Based Regimens.

    Directory of Open Access Journals (Sweden)

    Luis F López-Cortés

    Full Text Available Significant controversy still exists about ritonavir-boosted protease inhibitor monotherapy (mtPI/rtv as a simplification strategy that is used up to now to treat patients that have not experienced previous virological failure (VF while on protease inhibitor (PI -based regimens. We have evaluated the effectiveness of two mtPI/rtv regimens in an actual clinical practice setting, including patients that had experienced previous VF with PI-based regimens.This retrospective study analyzed 1060 HIV-infected patients with undetectable viremia that were switched to lopinavir/ritonavir or darunavir/ritonavir monotherapy. In cases in which the patient had previously experienced VF while on a PI-based regimen, the lack of major HIV protease resistance mutations to lopinavir or darunavir, respectively, was mandatory. The primary endpoint of this study was the percentage of participants with virological suppression after 96 weeks according to intention-to-treat analysis (non-complete/missing = failure.A total of 1060 patients were analyzed, including 205 with previous VF while on PI-based regimens, 90 of whom were on complex therapies due to extensive resistance. The rates of treatment effectiveness (intention-to-treat analysis and virological efficacy (on-treatment analysis at week 96 were 79.3% (CI95, 76.8-81.8 and 91.5% (CI95, 89.6-93.4, respectively. No relationships were found between VF and earlier VF while on PI-based regimens, the presence of major or minor protease resistance mutations, the previous time on viral suppression, CD4+ T-cell nadir, and HCV-coinfection. Genotypic resistance tests were available in 49 out of the 74 patients with VFs and only four patients presented new major protease resistance mutations.Switching to mtPI/rtv achieves sustained virological control in most patients, even in those with previous VF on PI-based regimens as long as no major resistance mutations are present for the administered drug.

  2. Hepatitis A virus-encoded miRNAs attenuate the accumulation of viral genomic RNAs in infected cells.

    Science.gov (United States)

    Shi, Jiandong; Sun, Jing; Wu, Meini; Hu, Ningzhu; Hu, Yunzhang

    2016-06-01

    The establishment of persistent infection with hepatitis A virus (HAV) is the common result of most HAV/cell culture systems. Previous observations show that the synthesis of viral RNAs is reduced during infection. However, the underlying mechanism is poorly understood. We characterized three HAV-encoded miRNAs in our previous study. In this study, we aim to investigate the impact of these miRNAs on the accumulation of viral RNAs. The results indicated that the synthesis of viral genomic RNAs was dramatically reduced (more than 75 % reduction, P viral miRNA mimics. Conversely, they were significantly increased (more than 3.3-fold addition, P viral miRNA inhibitors. The luciferase reporter assay of miRNA targets showed that viral miRNAs were fully complementary to specific sites of the viral plus or minus strand RNA and strongly inhibited their expressions. Further data showed that the relative abundance of viral genomic RNA fragments that contain miRNA targets was also dramatically reduced (more than 80 % reduction, P viral miRNAs were overexpressed with miRNA mimics. In contrast, they were significantly increased (approximately 2-fold addition, P viral miRNAs were inhibited with miRNA inhibitors. In conclusion, these data suggest a possible mechanism for the reduction of viral RNA synthesis during HAV infection. Thus, we propose that it is likely that RNA virus-derived miRNA could serve as a self-mediated feedback regulator during infection.

  3. Roles of the Picornaviral 3C Proteinase in the Viral Life Cycle and Host Cells

    Directory of Open Access Journals (Sweden)

    Di Sun

    2016-03-01

    Full Text Available The Picornaviridae family comprises a large group of non-enveloped viruses that have a major impact on human and veterinary health. The viral genome contains one open reading frame encoding a single polyprotein that can be processed by viral proteinases. The crucial 3C proteinases (3Cpros of picornaviruses share similar spatial structures and it is becoming apparent that 3Cpro plays a significant role in the viral life cycle and virus host interaction. Importantly, the proteinase and RNA-binding activity of 3Cpro are involved in viral polyprotein processing and the initiation of viral RNA synthesis. In addition, 3Cpro can induce the cleavage of certain cellular factors required for transcription, translation and nucleocytoplasmic trafficking to modulate cell physiology for viral replication. Due to interactions between 3Cpro and these essential factors, 3Cpro is also involved in viral pathogenesis to support efficient infection. Furthermore, based on the structural conservation, the development of irreversible inhibitors and discovery of non-covalent inhibitors for 3Cpro are ongoing and a better understanding of the roles played by 3Cpro may provide insights into the development of potential antiviral treatments. In this review, the current knowledge regarding the structural features, multiple functions in the viral life cycle, pathogen host interaction, and development of antiviral compounds for 3Cpro is summarized.

  4. Basic thermal–mechanical properties and thermal shock, fatigue resistance of swaged + rolled potassium doped tungsten

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xiaoxin; Yan, Qingzhi, E-mail: qzyan@ustb.edu.cn; Lang, Shaoting; Xia, Min; Ge, Changchun

    2014-09-15

    Highlights: • The potassium doped tungsten grade was achieved via swaging + rolling process. • The cracking threshold of the W–K alloy was in the range of 0.44–0.66 GW/m{sup 2}. • Recrystallization occurred at 0.66–1.1 GW/m{sup 2} during the thermal shock tests. • No cracks emerged during the thermal fatigue tests (0.44 GW/m{sup 2}, 1000 cycles). • Recrystallization occurred after 1000 cycles during the thermal fatigue tests. - Abstract: The potassium doped tungsten (W–K) grade was achieved via swaging + rolling process. The swaged + rolled W–K alloy exhibited acceptable thermal conductivity of 159.1 W/m K and ductile-to-brittle transition temperature of about 873 K while inferior mechanical properties attributed to the coarse pores and small deformation degree. Then the thermal shock, fatigue resistance of the W–K grade were characterized by an electron beam facility. Thermal shock tests were conducted at absorbed power densities varied from 0.22 to 1.1 GW/m{sup 2} in a step of 0.22 GW/m{sup 2}. The cracking threshold was in the range of 0.44–0.66 GW/m{sup 2}. Furthermore, recrystallization occurred in the subsurface of the specimens tested at 0.66–1.1 GW/m{sup 2} basing on the analysis of microhardness and microstructure. Thermal fatigue tests were performed at 0.44 GW/m{sup 2} up to 1000 cycles and no cracks emerged throughout the tests. Moreover, recrystallization occurred after 1000 cycles.

  5. Low Potassium (Hypokalemia)

    Science.gov (United States)

    Symptoms Low potassium (hypokalemia) By Mayo Clinic Staff Low potassium (hypokalemia) refers to a lower than normal potassium level ... 2 millimoles per liter (mmol/L). A very low potassium level (less than 2.5 mmol/L) ...

  6. Inhibitor specificity of recombinant and endogenous caspase-9.

    Science.gov (United States)

    Ryan, Ciara A; Stennicke, Henning R; Nava, Victor E; Burch, Jennifer B; Hardwick, J Marie; Salvesen, Guy S

    2002-01-01

    Apoptosis triggered through the intrinsic pathway by radiation and anti-neoplastic drugs is initiated by the activation of caspase-9. To elucidate control mechanisms in this pathway we used a range of synthetic and natural reagents. The inhibitory potency of acetyl-Asp-Glu-Val-Asp-aldehyde ('Ac-DEVD-CHO'), benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone ('Z-VAD-FMK') and the endogenous caspase inhibitor X-chromosome-linked inhibitor of apoptosis protein ('XIAP') against recombinant caspase-9 were predictive of the efficacy of these compounds in a cell-free system. However, the viral proteins CrmA and p35, although potent inhibitors of recombinant caspase-9, had almost no ability to block caspase-9 in this system. These findings were also mirrored in cell expression studies. We hypothesize that the viral inhibitors CrmA and p35 are excluded from reacting productively with the natural form of active caspase-9 in vivo, making the potency of inhibitors highly context-dependent. This is supported by survival data from a mouse model of apoptosis driven by Sindbis virus expressing either p35 or a catalytic mutant of caspase-9. These results consolidate previous findings that CrmA is a potent inhibitor of caspase-9 in vitro, yet fails to block caspase-9-mediated cell death. PMID:12067274

  7. Study on the Efficacy of Some Current Herbicides for Control of Resistant and Susceptible Canarygrass (Phalaris spp. Biotypes to Acetyl CoA Carboxylase (ACCase Inhibitors

    Directory of Open Access Journals (Sweden)

    e Zand

    2011-02-01

    Full Text Available Abstract Two separate greenhouse experiments were conducted in the greenhouse facilities of the Iranian Plant Protection Research Institute, Tehran, to study the efficacy of some herbicides to control of resistant and susceptible P. minor and P. paradoxa biotypes. In each experiment, resistant and susceptible biotypes were treated separately by 19 herbicide treatments. Treatments included 10 ACCase inhibitors, 6 Acetolactate Synthase (ALS inhibitors, prosulfocarb, flamprop-M-isopropyl, isoproturon plus diflufenican and a non-sprayed control. To evaluate the effects of treatments, different characteristics including percent damage based on EWRC scores at 15 and 30 days after spraying, percentage of survived plants after spraying relative to before spraying, and percentage of dry weight and wet weight of individual plants relative to control were studied. Results showed that the susceptible biotypes of P. minor were best controlled by clodinafop propargyl and pinoxaden at 450 ml/ha while pinoxaden at 450 ml/ha and cycloxydim were best options for control of the resistant biotype. Among ALS inhibitors, iodosulfuron plus mesosulfuron could control susceptible and resistant biotypes of P. minor very effectively and semi-satisfactory, respectively. Iodosulfuron plus mesosulfuron and sulfosulfuron plus metsulfuron could remarkably reduce the wet weight of individual plants compared to control so that the plants were not damaging any more. Among other herbicides, isoproturon plus diflufenican could control the susceptible and resistant biotypes semi-satisfactory and very effectively, respectively. Keywords: Herbicide resistance, ACCase inhibitors, ALS inhibitors

  8. Effect of potassium-salt muds on gamma ray, and spontaneous potential measurements

    International Nuclear Information System (INIS)

    Cox, J.W.; Raymer, L.L.

    1976-01-01

    Interpretations of the gamma ray and Spontaneous Potential curves generally assume the presence of sodium chloride as the dominant salt in both the formation water and the mud filtrate. However, potassium-salt muds are increasingly being used by the oil industry. The potassium cation is significantly different from the sodium cation in its radioactive and electrochemical properties. Natural potassium contains a radioactive isotope which emits gamma rays. Thus, the presence of potassium salts in the mud system may contribute to Gamma-Ray tool response. Since the Gamma Ray is used quantitatively in many geological sequences as an indicator of clay content, a way to correct for the effect of potassium in the mud column is desirable. Correction methods and charts based on laboratory measurements and field observations are presented. The effect of temperature on the resistivity of potassium muds is also briefly discussed. From data available, it appears to be similar to that for NaCl muds. On the bases of field observations and laboratory work, the electrochemical properties of potassium-chloride and potassium-carbonate muds and mud filtrates are discussed. Activity relationships are proposed, and the influence of these salts on the SP component potentials--namely, the liquid-junction, membrane, and bi-ionic potentials--is described. Several field examples are presented

  9. Modulation of HIV-1 Gag NC/p1 cleavage efficiency affects protease inhibitor resistance and viral replicative capacity

    Czech Academy of Sciences Publication Activity Database

    Maarseveen van, N. M.; Andersson, Dan; Lepšík, Martin; Fun, A.; Schipper, P. J.; Jong de, D.; Boucher, Ch. A. B.; Nijhuis, M.

    2012-01-01

    Roč. 9, č. 29 (2012), s. 1-7 ISSN 1742-4690 EU Projects: European Commission(XE) 37693 - HIV PI RESISTANCE Grant - others:Dutch AIDS Fund(XE) 2006028; (NWO) VIDI(XE) 91796349 Institutional research plan: CEZ:AV0Z40550506 Keywords : HIV-1 * protease * Gag * resistance * cleavage Subject RIV: CE - Biochemistry Impact factor: 5.657, year: 2012

  10. Cholesterol biosynthesis inhibitor RO 48-8071 suppresses growth of hormone-dependent and castration-resistant prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Liang Y

    2016-05-01

    Full Text Available Yayun Liang,1 Benford Mafuvadze,1 Johannes D Aebi,2 Salman M Hyder1 1Dalton Cardiovascular Research Center and Department of Biomedical Sciences, University of Missouri-Columbia, Columbia, MO, USA; 2Medicinal Chemistry, Roche Pharma Research and Early Development (pRED, Roche Innovation Center Basel, F Hoffmann-La Roche Ltd., Basel, Switzerland Abstract: Standard treatment for primary prostate cancer includes systemic exposure to chemotherapeutic drugs that target androgen receptor or antihormone therapy (chemical castration; however, drug-resistant cancer cells generally emerge during treatment, limiting the continued use of systemic chemotherapy. Patients are then treated with more toxic standard therapies. Therefore, there is an urgent need for novel and more effective treatments for prostate cancer. The cholesterol biosynthetic pathway is an attractive therapeutic target for treating endocrine-dependent cancers because cholesterol is an essential structural and functional component of cell membranes as well as the metabolic precursor of endogenous steroid hormones. In this study, we have examined the effects of RO 48-8071 (4'-[6-(allylmethylaminohexyloxy]-4-bromo-2'-fluorobenzophenone fumarate; Roche Pharmaceuticals internal reference: RO0488071 (RO, which is an inhibitor of 2, 3-oxidosqualene cyclase (a key enzyme in the cholesterol biosynthetic pathway, on prostate cancer cells. Exposure of both hormone-dependent and castration-resistant human prostate cancer cells to RO reduced prostate cancer cell viability and induced apoptosis in vitro. RO treatment reduced androgen receptor protein expression in hormone-dependent prostate cancer cells and increased estrogen receptor β (ERβ protein expression in both hormone-dependent and castration-resistant prostate cancer cell lines. Combining RO with an ERβ agonist increased its ability to reduce castration-resistant prostate cancer cell viability. In addition, RO effectively suppressed the

  11. Urocortin2 prolongs action potential duration and modulates potassium currents in guinea pig myocytes and HEK293 cells.

    Science.gov (United States)

    Yang, Li-Zhen; Zhu, Yi-Chun

    2015-07-05

    We previously reported that activation of corticotropin releasing factor receptor type 2 by urocortin2 up-regulates both L-type Ca(2+) channels and intracellular Ca(2+) concentration in ventricular myocytes and plays an important role in cardiac contractility and arrhythmogenesis. This study goal was to further test the hypothesis that urocortin2 may modulate action potentials as well as rapidly and slowly activating delayed rectifier potassium currents. With whole cell patch-clamp techniques, action potentials and slowly activating delayed rectifier potassium currents were recorded in isolated guinea pig ventricular myocytes, respectively. And rapidly activating delayed rectifier potassium currents were tested in hERG-HEK293 cells. Urocortin2 produced a time- and concentration-dependent prolongation of action potential duration. The EC50 values of action potential duration and action potential duration at 90% of repolarization were 14.73 and 24.3nM respectively. The prolongation of action potential duration of urocortin2 was almost completely or partly abolished by H-89 (protein kinase A inhibitor) or KB-R7943 (Na(+)/Ca(2+) exchange inhibitor) pretreatment respectively. And urocortin2 caused reduction of rapidly activating delayed rectifier potassium currents in hERG-HEK293 cells. In addition, urocortin2 slowed the rate of slowly activating delayed rectifier potassium channel activation, and rightward shifted the threshold of slowly activating delayed rectifier potassium currents to more positive potentials. Urocortin2 prolonged action potential duration via activation of protein kinase A and Na(+)/ Ca(2+) exchange in isolated guinea pig ventricular myocytes in a time- and concentration- dependent manner. In hERG-HEK293 cells, urocortin2 reduced rapidly activating delayed rectifier potassium current density which may contribute to action potential duration prolongation. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Natural coagulation inhibitors and active protein c resistance in preeclampsia

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    Cengiz Demir

    2010-01-01

    Full Text Available INTRODUCTION: The etiology of preeclampsia is not fully established. A few studies have shown a relationship between natural coagulation inhibitors and preeclampsia. OBJECTIVES: The purpose of this study was to investigate the status of natural coagulation inhibitors and active protein C resistance (APC-R in preeclampsia. PATIENTS AND METHODS: We studied 70 women with preeclampsia recruited consecutively and 70 healthy pregnant and 70 nonpregnant women as controls. Plasma protein C (PC, free protein S (fPS, antithrombin III (ATIII and APC-R were evaluated. RESULTS: ATIII values were found to be significantly lower in preeclamptic patients than in the control groups (p< 0.001. Nevertheless, there was no significant difference between the healthy pregnant and nonpregnant women groups (p=0.141. The fPS values of the preeclamptic and healthy pregnant groups were lower than that of the nonpregnant group (p< 0.001, and the fPS value of the preeclamptic pregnant women was lower than that of healthy pregnant women (p<0.001. The PC value of the preeclamptic pregnant women was lower than that of the control groups (p< 0.001. The PC value of the healthy pregnant women was lower than that of the nonpregnant women (p< 0.001. The mean APC activity values were lower in the preeclamptic patients than that of the control groups (p< 0.001, p< 0.001. The APC-R positivity rates of the preeclamptic groups were higher than that of the control groups (p<0.001. CONCLUSIONS: This study demonstrated that ATIII, fPS, PC values and APC resistance were lower and APC-R positivity was higher in preeclamptic women than in normal pregnant and nonpregnant women.

  13. High-throughput screening identifies Ceefourin 1 and Ceefourin 2 as highly selective inhibitors of multidrug resistance protein 4 (MRP4).

    Science.gov (United States)

    Cheung, Leanna; Flemming, Claudia L; Watt, Fujiko; Masada, Nanako; Yu, Denise M T; Huynh, Tony; Conseil, Gwenaëlle; Tivnan, Amanda; Polinsky, Alexander; Gudkov, Andrei V; Munoz, Marcia A; Vishvanath, Anasuya; Cooper, Dermot M F; Henderson, Michelle J; Cole, Susan P C; Fletcher, Jamie I; Haber, Michelle; Norris, Murray D

    2014-09-01

    Multidrug resistance protein 4 (MRP4/ABCC4), a member of the ATP-binding cassette (ABC) transporter superfamily, is an organic anion transporter capable of effluxing a wide range of physiologically important signalling molecules and drugs. MRP4 has been proposed to contribute to numerous functions in both health and disease; however, in most cases these links remain to be unequivocally established. A major limitation to understanding the physiological and pharmacological roles of MRP4 has been the absence of specific small molecule inhibitors, with the majority of established inhibitors also targeting other ABC transporter family members, or inhibiting the production, function or degradation of important MRP4 substrates. We therefore set out to identify more selective and well tolerated inhibitors of MRP4 that might be used to study the many proposed functions of this transporter. Using high-throughput screening, we identified two chemically distinct small molecules, Ceefourin 1 and Ceefourin 2, that inhibit transport of a broad range of MRP4 substrates, yet are highly selective for MRP4 over other ABC transporters, including P-glycoprotein (P-gp), ABCG2 (Breast Cancer Resistance Protein; BCRP) and MRP1 (multidrug resistance protein 1; ABCC1). Both compounds are more potent MRP4 inhibitors in cellular assays than the most widely used inhibitor, MK-571, requiring lower concentrations to effect a comparable level of inhibition. Furthermore, Ceefourin 1 and Ceefourin 2 have low cellular toxicity, and high microsomal and acid stability. These newly identified inhibitors should be of great value for efforts to better understand the biological roles of MRP4, and may represent classes of compounds with therapeutic application. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Reverse transcriptase inhibitors as microbicides.

    Science.gov (United States)

    Lewi, Paul; Heeres, Jan; Ariën, Kevin; Venkatraj, Muthusamy; Joossens, Jurgen; Van der Veken, Pieter; Augustyns, Koen; Vanham, Guido

    2012-01-01

    The CAPRISA 004 study in South Africa has accelerated the development of vaginal and rectal microbicides containing antiretrovirals that target specific enzymes in the reproduction cycle of HIV, especially reverse transcriptase inhibitors (RTI). In this review we discuss the potential relevance of HIV-1 RTIs as microbicides, focusing in the nucleotide RTI tenofovir and six classes of nonnucleoside RTIs (including dapivirine, UC781, urea and thiourea PETTs, DABOs and a pyrimidinedione). Although tenofovir and dapivirine appear to be most advanced in clinical trials as potential microbicides, several issues remain unresolved, e.g., the importance of nonhuman primates as a "gatekeeper" for clinical trials, the emergence and spread of drug-resistant mutants, the combination of microbicides that target different phases of viral reproduction and the accessibility to microbicides in low-income countries. Thus, here we discuss the latest research on RTI as microbicides in the light of the continuing spread of the HIV pandemic from the point of view of medicinal chemistry, virological, and pharmaceutical studies.

  15. Competitive fitness of influenza B viruses with neuraminidase inhibitor-resistant substitutions in a coinfection model of the human airway epithelium.

    Science.gov (United States)

    Burnham, Andrew J; Armstrong, Jianling; Lowen, Anice C; Webster, Robert G; Govorkova, Elena A

    2015-04-01

    of seasonal influenza virus infections worldwide. The development of resistance to a single class of available antivirals, the neuraminidase (NA) inhibitors (NAIs), is a public health concern. Amino acid substitutions in the NA glycoprotein of influenza B virus not only can confer antiviral resistance but also can alter viral fitness. Here we used normal human bronchial epithelial (NHBE) cells, a model of the human upper respiratory tract, to examine the replicative capacities and fitness of NAI-resistant influenza B viruses. We show that virus with an E119A NA substitution can replicate efficiently in NHBE cells in the presence of oseltamivir or zanamivir and that virus with the H274Y NA substitution has a relative fitness greater than that of the wild-type NAI-susceptible virus. This study is the first to use NHBE cells to determine the fitness of NAI-resistant influenza B viruses. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  16. Boronic acid-modified lipid nanocapsules: a novel platform for the highly efficient inhibition of hepatitis C viral entry

    Science.gov (United States)

    Khanal, Manakamana; Barras, Alexandre; Vausselin, Thibaut; Fénéant, Lucie; Boukherroub, Rabah; Siriwardena, Aloysius; Dubuisson, Jean; Szunerits, Sabine

    2015-01-01

    The search for viral entry inhibitors that selectively target viral envelope glycoproteins has attracted increasing interest in recent years. Amongst the handful of molecules reported to show activity as hepatitis C virus (HCV) entry inhibitors are a variety of glycan-binding proteins including the lectins, cyanovirin-N (CV-N) and griffithsin. We recently demonstrated that boronic acid-modified nanoparticles are able to reduce HCV entry through a similar mechanism to that of lectins. A major obstacle to any further development of these nanostructures as viral entry inhibitors is their only moderate maximal inhibition potential. In the present study, we report that lipid nanocapsules (LNCs), surface-functionalized with amphiphilic boronic acid (BA) through their post-insertion into the semi-rigid shell of the LNCs, are indeed far superior as HCV entry inhibitors when compared with previously reported nanostructures. These 2nd generation particles (BA-LNCs) are shown to prevent HCV infection in the micromolar range (IC50 = 5.4 μM of BA moieties), whereas the corresponding BA monomers show no significant effects even at the highest analyzed concentration (20 μM). The new BA-LNCs are the most promising boronolectin-based HCV entry inhibitors reported to date and are thus observed to show great promise in the development of a pseudolectin-based therapeutic agent.The search for viral entry inhibitors that selectively target viral envelope glycoproteins has attracted increasing interest in recent years. Amongst the handful of molecules reported to show activity as hepatitis C virus (HCV) entry inhibitors are a variety of glycan-binding proteins including the lectins, cyanovirin-N (CV-N) and griffithsin. We recently demonstrated that boronic acid-modified nanoparticles are able to reduce HCV entry through a similar mechanism to that of lectins. A major obstacle to any further development of these nanostructures as viral entry inhibitors is their only moderate maximal

  17. Hsp90 inhibitors reduce influenza virus replication in cell culture

    International Nuclear Information System (INIS)

    Chase, Geoffrey; Deng, Tao; Fodor, Ervin; Leung, B.W.; Mayer, Daniel; Schwemmle, Martin; Brownlee, George

    2008-01-01

    The viral RNA polymerase complex of influenza A virus consists of three subunits PB1, PB2 and PA. Recently, the cellular chaperone Hsp90 was shown to play a role in nuclear import and assembly of the trimeric polymerase complex by binding to PB1 and PB2. Here we show that Hsp90 inhibitors, geldanamycin or its derivative 17-AAG, delay the growth of influenza virus in cell culture resulting in a 1-2 log reduction in viral titre early in infection. We suggest that this is caused by the reduced half-life of PB1 and PB2 and inhibition of nuclear import of PB1 and PA which lead to reduction in viral RNP assembly. Hsp90 inhibitors may represent a new class of antiviral compounds against influenza viruses

  18. Efeitos da suplementação de potássio via sal de cozinha sobre a pressão arterial e a resistência à insulina em pacientes obesos hipertensos em uso de diuréticos Effects of potassium supplementation by salt on arterial blood pressure and insulin resistance in hypertensive obese patients on diuretic therapy

    Directory of Open Access Journals (Sweden)

    Maria Alice de Gouveia Pereira

    2005-02-01

    Full Text Available OBJETIVO: Avaliar os efeitos da suplementação de potássio, por intermédio do sal de cozinha contendo cloreto de potássio, associada à dieta hipocalórica e à atividade física aeróbica, sobre a pressão arterial e índices de resistência à insulina em pacientes hipertensos com obesidade abdominal. MÉTODOS: Estudo prospectivo duplo-cego, randomizado, em 22 pacientes hipertensos com excesso de peso (índice de massa corporal >27kg/m² e controle insatisfatório da pressão arterial durante o uso de diuréticos pressão arterial sistólica >140 e 90 e OBJECTIVE: The objectives of the present study were to evaluate the effects of oral potassium supplementation, associated with a hypocaloric diet and aerobic exercises, on plasma potassium levels, blood pressure and insulin resistance, in centrally obese, not well controlled hypertensive patients on diuretic therapy; waist to hip ratio >0.85 in women, and >0.95 in men; systolic blood pressure >140mmHg and 90mmHg and <105mmHg. METHODS: This was a prospective double-blind randomized study including 22 patients divided in 2 groups: sodium chloride n=10, and potassium chloride n=12. For 12 weeks, each group received cooking salt containing, either 100% sodium chloride, or 50% sodium chloride and 50% potassium chloride. All patients were submitted to a hypocaloric diet and advised to increase their physical activity; a 40-minute walk three times a week. Before and after the study period, all patients were submitted to determinations of body mass index, body composition, waist circumference, sodium and potassium urinary excretions, sodium and potassium serum levels, 24h ambulatory blood pressure monitoring, oral glucose tolerance test with serum insulin measurements at fasting and 120 minutes after glucose load, and serum lipid profile. RESULTS: In both groups, no changes were observed in serum sodium and potassium levels, in blood glucose and insulin levels, insulin resistance indexes and serum

  19. Ganciclovir-Resistant Cytomegalovirus Infection in a Kidney Transplant Recipient Successfully Treated with Foscarnet and Everolimus.

    Science.gov (United States)

    Menghi, Viola; Comai, Giorgia; Baraldi, Olga; Liviano D'Arcangelo, Giovanni; Lazzarotto, Tiziana; La Manna, Gaetano

    2016-01-01

    Cytomegalovirus (CMV) infection remains a major cause of morbidity, graft failure, and death in kidney transplant recipients. We describe a case of a 53-year-old CMV-seronegative man who underwent renal transplant from a CMV-positive donor and who developed ganciclovir- (GCV-) resistant CMV infection. Foscarnet was started while immunosuppressive therapy was modified with the introduction of everolimus minimizing tacrolimus dosage. Only two weeks after the start of this treatment regimen was the patient's viral load negative. At two-year follow-up the patient has no clinical or laboratory signs of CMV infection and a good and stable renal function or graft survival. In our case, administration of an mTOR inhibitor combined with foscarnet led to rapid and persistent viral clearance without compromising short- and medium-term graft function. This combination therapy supports the need for the kidney transplant community to individualize a target therapy for each type of GCV-resistant CMV infection.

  20. Effects of plant urease inhibitor on crop nutrition and soil characters

    International Nuclear Information System (INIS)

    Wang Zhengyin; Xu Weihong; Huang Yun; Yuan Lujiang; Jia Zhongyuan; Zhou Jun; Ding Shuying

    2002-01-01

    A pot experiment was conducted to investigate the effects of 15 N-urea and 4 kinds of plant materials (P 1 , P 2 , P 3 and P 4 ) as urease inhibitor on sorghum and rice nutrition and soil characters. The results indicated that the growth, above-ground parts and roots weight of rice and sorghum were respectively promoted by 4 plant urease inhibitors and P 1 with little change of chl.a/chl.b ratios in these treatments. The content of amino acid in rice leaf and utilization rate of nitrogen by rice were enhanced by 12.9%-25.1% and 5.2%-7.7% respectively, and the utilization rate of nitrogen by sorghum was improved by urease inhibitor treatments (except P 1 ). Plant urease inhibitor could obviously increase the apparent utilization rate of nitrogen by 4.3%-19.2% for two crops and improve phosphorus and potassium uptake by rice plant but decrease phosphorus and potassium uptake by sorghum plant. The contents of soil alkali-hydrolyzable nitrogen were increased by plant urease inhibitor under two cultivated condition. The inhibition time of plant urease inhibitor to soil urease was short and it disappeared as 36 days of rice growth under flooded condition, while the activities of soil urease were decreased by 10.6%-18.3% at 48 days of sorghum growth in upland soil

  1. In Vitro Antiviral Activity and Resistance Profile of the Next-Generation Hepatitis C Virus NS5A Inhibitor Pibrentasvir.

    Science.gov (United States)

    Ng, Teresa I; Krishnan, Preethi; Pilot-Matias, Tami; Kati, Warren; Schnell, Gretja; Beyer, Jill; Reisch, Thomas; Lu, Liangjun; Dekhtyar, Tatyana; Irvin, Michelle; Tripathi, Rakesh; Maring, Clarence; Randolph, John T; Wagner, Rolf; Collins, Christine

    2017-05-01

    Pibrentasvir (ABT-530) is a novel and pan-genotypic hepatitis C virus (HCV) NS5A inhibitor with 50% effective concentration (EC 50 ) values ranging from 1.4 to 5.0 pM against HCV replicons containing NS5A from genotypes 1 to 6. Pibrentasvir demonstrated similar activity against a panel of chimeric replicons containing HCV NS5A of genotypes 1 to 6 from clinical samples. Resistance selection studies were conducted using HCV replicon cells with NS5A from genotype 1a, 1b, 2a, 2b, 3a, 4a, 5a, or 6a at a concentration of pibrentasvir that was 10- or 100-fold over its EC 50 for the respective replicon. With pibrentasvir at 10-fold over the respective EC 50 , only a small number of colonies (0.00015 to 0.0065% of input cells) with resistance-associated amino acid substitutions were selected in replicons containing genotype 1a, 2a, or 3a NS5A, and no viable colonies were selected in replicons containing NS5A from other genotypes. With pibrentasvir at 100-fold over the respective EC 50 , very few colonies (0.0002% of input cells) were selected by pibrentasvir in genotype 1a replicon cells while no colonies were selected in other replicons. Pibrentasvir is active against common resistance-conferring substitutions in HCV genotypes 1 to 6 that were identified for other NS5A inhibitors, including those at key amino acid positions 28, 30, 31, or 93. The combination of pibrentasvir with HCV inhibitors of other classes produced synergistic inhibition of HCV replication. In summary, pibrentasvir is a next-generation HCV NS5A inhibitor with potent and pan-genotypic activity, and it maintains activity against common amino acid substitutions of HCV genotypes 1 to 6 that are known to confer resistance to currently approved NS5A inhibitors. Copyright © 2017 Ng et al.

  2. Full Viral Suppression, Low-Level Viremia, and Quantifiable Plasma HIV-RNA at the End of Pregnancy in HIV-Infected Women on Antiretroviral Treatment.

    Science.gov (United States)

    Baroncelli, Silvia; Pirillo, Maria F; Tamburrini, Enrica; Guaraldi, Giovanni; Pinnetti, Carmela; Degli Antoni, Anna; Galluzzo, Clementina M; Stentarelli, Chiara; Amici, Roberta; Floridia, Marco

    2015-07-01

    There is limited information on full viral suppression and low-level HIV-RNA viremia in HIV-infected women at the end of pregnancy. We investigated HIV-RNA levels close to delivery in women on antiretroviral treatment in order to define rates of complete suppression, low-level viremia, and quantifiable HIV-RNA, exploring as potential determinants some clinical and viroimmunological variables. Plasma samples from a national study in Italy, collected between 2003 and 2012, were used. According to plasma HIV-RNA levels, three groups were defined: full suppression (target not detected), low-level viremia (target detected but HIV-RNA (≥37 copies/ml). Multivariable logistic regression was used to define determinants of full viral suppression and of quantifiable HIV-RNA. Among 107 women evaluated at a median gestational age of 35 weeks, 90 (84.1%) had HIV-RNA HIV-RNA was 109 copies/ml (IQR 46-251), with only one case showing resistance (mutation M184V; rate: 9.1%). In multivariable analyses, women with higher baseline HIV-RNA levels and with hepatitis C virus (HCV) coinfection were significantly more likely to have quantifiable HIV-RNA in late pregnancy. Full viral suppression was significantly more likely with nonnucleoside reverse transcriptase inhibitor (NNRTI)-based regimens and significantly less likely with higher HIV-RNA in early pregnancy. No cases of HIV transmission occurred. In conclusion, HIV-infected pregnant women showed a high rate of viral suppression and a low resistance rate before delivery. In most cases no target HIV-RNA was detected in plasma, suggesting a low risk of subsequent virological rebound and development of resistance. Women with high levels of HIV-RNA in early pregnancy and those who have concomitant HCV infection should be considered at higher risk of having quantifiable HIV-RNA at the end of pregnancy.

  3. SGLT2-inhibitor and DPP-4 inhibitor improve brain function via attenuating mitochondrial dysfunction, insulin resistance, inflammation, and apoptosis in HFD-induced obese rats.

    Science.gov (United States)

    Sa-Nguanmoo, Piangkwan; Tanajak, Pongpan; Kerdphoo, Sasiwan; Jaiwongkam, Thidarat; Pratchayasakul, Wasana; Chattipakorn, Nipon; Chattipakorn, Siriporn C

    2017-10-15

    Dipeptidyl peptidase-4 inhibitor (vildagliptin) has been shown to exert beneficial effects on insulin sensitivity and neuroprotection in obese-insulin resistance. Recent studies demonstrated the neuroprotection of the sodium-glucose co-transporter 2 inhibitor (dapagliflozin) in diabetes. However, the comparative effects of both drugs and a combination of two drugs on metabolic dysfunction and brain dysfunction impaired by the obese-insulin resistance have never been investigated. Forty male Wistar rats were divided into two groups, and received either a normal-diet (ND, n=8) or a high-fat diet (HFD, n=32) for 16weeks. At week 13, the HFD-fed rats were divided into four subgroups (n=8/subgroup) to receive either a vehicle, vildagliptin (3mg/kg/day) dapagliflozin (1mg/kg/day) or combined drugs for four weeks. ND rats were given a vehicle for four weeks. Metabolic parameters and brain function were investigated. The results demonstrated that HFD rats developed obese-insulin resistance and cognitive decline. Dapagliflozin had greater efficacy on improved peripheral insulin sensitivity and reduced weight gain than vildagliptin. Single therapy resulted in equally improved brain mitochondrial function, insulin signaling, apoptosis and prevented cognitive decline. However, only dapagliflozin improved hippocampal synaptic plasticity. A combination of the drugs had greater efficacy in improving brain insulin sensitivity and reducing brain oxidative stress than the single drug therapy. These findings suggested that dapagliflozin and vildagliptin equally prevented cognitive decline in the obese-insulin resistance, possibly through some similar mechanisms. Dapagliflozin had greater efficacy than vildagliptin for preserving synaptic plasticity, thus combined drugs could be the best therapeutic approach for neuroprotection in the obese-insulin resistance. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Short- and long-term inhibition of cardiac inward-rectifier potassium channel current by an antiarrhythmic drug bepridil.

    Science.gov (United States)

    Ma, Fangfang; Takanari, Hiroki; Masuda, Kimiko; Morishima, Masaki; Ono, Katsushige

    2016-07-01

    Bepridil is an effective antiarrhythmic drug on supraventricular and ventricular arrhythmias, and inhibitor of calmodulin. Recent investigations have been elucidating that bepridil exerts antiarrhythmic effects through its acute and chronic application for patients. The aim of this study was to identify the efficacy and the potential mechanism of bepridil on the inward-rectifier potassium channel in neonatal rat cardiomyocytes in acute- and long-term conditions. Bepridil inhibited inward-rectifier potassium current (I K1) as a short-term effect with IC50 of 17 μM. Bepridil also reduced I K1 of neonatal cardiomyocytes when applied for 24 h in the culture medium with IC50 of 2.7 μM. Both a calmodulin inhibitor (W-7) and an inhibitor of calmodulin-kinase II (KN93) reduced I K1 when applied for 24 h as a long-term effect in the same fashion, suggesting that the long-term application of bepridil inhibits I K1 more potently than that of the short-term application through the inhibition of calmodulin kinase II pathway in cardiomyocytes.

  5. Identification of a methylated oligoribonucleotide as a potent inhibitor of HIV-1 reverse transcription complex.

    Science.gov (United States)

    Grigorov, Boyan; Bocquin, Anne; Gabus, Caroline; Avilov, Sergey; Mély, Yves; Agopian, Audrey; Divita, Gilles; Gottikh, Marina; Witvrouw, Myriam; Darlix, Jean-Luc

    2011-07-01

    Upon HIV-1 infection of a target cell, the viral reverse transcriptase (RT) copies the genomic RNA to synthesize the viral DNA. The genomic RNA is within the incoming HIV-1 core where it is coated by molecules of nucleocapsid (NC) protein that chaperones the reverse transcription process. Indeed, the RT chaperoning properties of NC extend from the initiation of cDNA synthesis to completion of the viral DNA. New and effective drugs against HIV-1 continue to be required, which prompted us to search for compounds aimed at inhibiting NC protein. Here, we report that the NC chaperoning activity is extensively inhibited in vitro by small methylated oligoribonucleotides (mODN). These mODNs were delivered intracellularly using a cell-penetrating-peptide and found to impede HIV-1 replication in primary human cells at nanomolar concentrations. Extensive analysis showed that viral cDNA synthesis was severely impaired by mODNs. Partially resistant viruses with mutations in NC and RT emerged after months of passaging in cell culture. A HIV-1 molecular clone (NL4.3) bearing these mutations was found to replicate at high concentrations of mODN, albeit with a reduced fitness. Small, methylated ODNs such as mODN-11 appear to be a new type of highly potent inhibitor of HIV-1.

  6. Overcoming multidrug resistance in Dox-resistant neuroblastoma cell lines via treatment with HPMA copolymer conjugates containing anthracyclines and P-gp inhibitors

    Czech Academy of Sciences Publication Activity Database

    Koziolová, Eva; Janoušková, Olga; Cuchalová, Lucie; Hvězdová, Zuzana; Hraběta, J.; Eckschlager, T.; Sivák, Ladislav; Ulbrich, Karel; Etrych, Tomáš; Šubr, Vladimír

    2016-01-01

    Roč. 233, 10 July (2016), s. 136-146 ISSN 0168-3659 R&D Projects: GA ČR(CZ) GAP301/12/1254; GA MŠk(CZ) LO1507 Institutional support: RVO:61389013 ; RVO:61388971 Keywords : N-(2-hydroxypropyl)methacrylamide copolymers * multidrug resistance * P-glycoprotein inhibitors Subject RIV: CD - Macromolecular Chemistry ; FD - Oncology ; Hematology (MBU-M) Impact factor: 7.786, year: 2016

  7. Low-abundance HIV drug-resistant viral variants in treatment-experienced persons correlate with historical antiretroviral use.

    Science.gov (United States)

    Le, Thuy; Chiarella, Jennifer; Simen, Birgitte B; Hanczaruk, Bozena; Egholm, Michael; Landry, Marie L; Dieckhaus, Kevin; Rosen, Marc I; Kozal, Michael J

    2009-06-29

    It is largely unknown how frequently low-abundance HIV drug-resistant variants at levels under limit of detection of conventional genotyping (<20% of quasi-species) are present in antiretroviral-experienced persons experiencing virologic failure. Further, the clinical implications of low-abundance drug-resistant variants at time of virologic failure are unknown. Plasma samples from 22 antiretroviral-experienced subjects collected at time of virologic failure (viral load 1380 to 304,000 copies/mL) were obtained from a specimen bank (from 2004-2007). The prevalence and profile of drug-resistant mutations were determined using Sanger sequencing and ultra-deep pyrosequencing. Genotypes were interpreted using Stanford HIV database algorithm. Antiretroviral treatment histories were obtained by chart review and correlated with drug-resistant mutations. Low-abundance drug-resistant mutations were detected in all 22 subjects by deep sequencing and only in 3 subjects by Sanger sequencing. In total they accounted for 90 of 247 mutations (36%) detected by deep sequencing; the majority of these (95%) were not detected by standard genotyping. A mean of 4 additional mutations per subject were detected by deep sequencing (p<0.0001, 95%CI: 2.85-5.53). The additional low-abundance drug-resistant mutations increased a subject's genotypic resistance to one or more antiretrovirals in 17 of 22 subjects (77%). When correlated with subjects' antiretroviral treatment histories, the additional low-abundance drug-resistant mutations correlated with the failing antiretroviral drugs in 21% subjects and correlated with historical antiretroviral use in 79% subjects (OR, 13.73; 95% CI, 2.5-74.3, p = 0.0016). Low-abundance HIV drug-resistant mutations in antiretroviral-experienced subjects at time of virologic failure can increase a subject's overall burden of resistance, yet commonly go unrecognized by conventional genotyping. The majority of unrecognized resistant mutations correlate with

  8. Low-abundance HIV drug-resistant viral variants in treatment-experienced persons correlate with historical antiretroviral use.

    Directory of Open Access Journals (Sweden)

    Thuy Le

    Full Text Available BACKGROUND: It is largely unknown how frequently low-abundance HIV drug-resistant variants at levels under limit of detection of conventional genotyping (<20% of quasi-species are present in antiretroviral-experienced persons experiencing virologic failure. Further, the clinical implications of low-abundance drug-resistant variants at time of virologic failure are unknown. METHODOLOGY/PRINCIPAL FINDINGS: Plasma samples from 22 antiretroviral-experienced subjects collected at time of virologic failure (viral load 1380 to 304,000 copies/mL were obtained from a specimen bank (from 2004-2007. The prevalence and profile of drug-resistant mutations were determined using Sanger sequencing and ultra-deep pyrosequencing. Genotypes were interpreted using Stanford HIV database algorithm. Antiretroviral treatment histories were obtained by chart review and correlated with drug-resistant mutations. Low-abundance drug-resistant mutations were detected in all 22 subjects by deep sequencing and only in 3 subjects by Sanger sequencing. In total they accounted for 90 of 247 mutations (36% detected by deep sequencing; the majority of these (95% were not detected by standard genotyping. A mean of 4 additional mutations per subject were detected by deep sequencing (p<0.0001, 95%CI: 2.85-5.53. The additional low-abundance drug-resistant mutations increased a subject's genotypic resistance to one or more antiretrovirals in 17 of 22 subjects (77%. When correlated with subjects' antiretroviral treatment histories, the additional low-abundance drug-resistant mutations correlated with the failing antiretroviral drugs in 21% subjects and correlated with historical antiretroviral use in 79% subjects (OR, 13.73; 95% CI, 2.5-74.3, p = 0.0016. CONCLUSIONS/SIGNIFICANCE: Low-abundance HIV drug-resistant mutations in antiretroviral-experienced subjects at time of virologic failure can increase a subject's overall burden of resistance, yet commonly go unrecognized by conventional

  9. Effect of Lopinavir and Nevirapine Concentrations on Viral Outcomes in Protease Inhibitor-experienced HIV-infected Children.

    Science.gov (United States)

    Moholisa, Retsilisitsoe R; Schomaker, Michael; Kuhn, Louise; Castel, Sandra; Wiesner, Lubbe; Coovadia, Ashraf; Strehlau, Renate; Patel, Faeezah; Pinillos, Francoise; Abrams, Elaine J; Maartens, Gary; McIlleron, Helen

    2016-12-01

    Adequate exposure to antiretroviral drugs is necessary to achieve and sustain viral suppression. However, the target antiretroviral concentrations associated with long-term viral suppression have not been adequately defined in children. We assessed the relationship between plasma lopinavir or nevirapine concentrations and the risk of subsequent viremia in children initially suppressed on antiretroviral therapy. After an induction phase of antiretroviral treatment, 195 children with viral suppression (viral load ≤400 copies/mL) were randomized to continue a lopinavir/ritonavir-based regimen or to switch to a nevirapine-based regimen (together with lamivudine and stavudine). Viral load and lopinavir or nevirapine concentrations were measured at clinic visits 4, 8, 12, 16, 20, 24, 36, 52, 64 and 76 weeks post randomization. Cox multiple failure event models were used to estimate the effects of drug concentrations on the hazard of viremia (viral load >50 copies/mL) RESULTS:: At randomization, the median (interquartile range) age, CD4 T-Lymphocyte percentage, weight-for-age and weight-for-height z scores were 19 (16-24) months, 29% (23-37), -0.6 (-1.3 to 0.2) and -3.2 (-4.1 to -2.1), respectively. The proportion of children with viral load 51-400 copies/mL at randomization was 43%. The hazard of subsequent viremia during follow-up was increased for lopinavir concentrations <1 versus ≥1 mg/L [adjusted hazard ratio 0.62 (95% confidence interval, 0.40-0.94)] and for children with viral loads 51-400 copies/mL at randomization. Nevirapine concentrations were not significantly associated with subsequent viremia. Plasma lopinavir concentrations predicted viral outcomes in children receiving lopinavir-based antiretroviral therapy. Our findings support a minimum target concentration of ≥1 mg/L of lopinavir to ensure sustained viral suppression.

  10. Inhibitin: a specific inhibitor of sodium/sodium exchange in erythrocytes.

    OpenAIRE

    Morgan, K; Brown, R C; Spurlock, G; Southgate, K; Mir, M A

    1986-01-01

    An inhibitor of ouabain-insensitive sodium/sodium exchange in erythrocytes has been isolated from leukemic promyelocytes. To explore the specific effects of this inhibitor, named inhibitin, sodium transport experiments were carried out in human erythrocytes. Inhibitin reduced ouabain-insensitive bidirectional sodium transport. It did not change net sodium fluxes, had no significant effect on rubidium influx, and did not inhibit sodium-potassium-ATPase activity. The inhibitory effect of inhibi...

  11. Basic thermal-mechanical properties and thermal shock, fatigue resistance of swaged + rolled potassium doped tungsten

    Science.gov (United States)

    Zhang, Xiaoxin; Yan, Qingzhi; Lang, Shaoting; Xia, Min; Ge, Changchun

    2014-09-01

    The potassium doped tungsten (W-K) grade was achieved via swaging + rolling process. The swaged + rolled W-K alloy exhibited acceptable thermal conductivity of 159.1 W/m K and ductile-to-brittle transition temperature of about 873 K while inferior mechanical properties attributed to the coarse pores and small deformation degree. Then the thermal shock, fatigue resistance of the W-K grade were characterized by an electron beam facility. Thermal shock tests were conducted at absorbed power densities varied from 0.22 to 1.1 GW/m2 in a step of 0.22 GW/m2. The cracking threshold was in the range of 0.44-0.66 GW/m2. Furthermore, recrystallization occurred in the subsurface of the specimens tested at 0.66-1.1 GW/m2 basing on the analysis of microhardness and microstructure. Thermal fatigue tests were performed at 0.44 GW/m2 up to 1000 cycles and no cracks emerged throughout the tests. Moreover, recrystallization occurred after 1000 cycles.

  12. Serum Potassium Levels and Outcome in Acute Heart Failure (Data from the PROTECT and COACH Trials).

    Science.gov (United States)

    Tromp, Jasper; Ter Maaten, Jozine M; Damman, Kevin; O'Connor, Christopher M; Metra, Marco; Dittrich, Howard C; Ponikowski, Piotr; Teerlink, John R; Cotter, Gad; Davison, Beth; Cleland, John G F; Givertz, Michael M; Bloomfield, Daniel M; van der Wal, Martje H L; Jaarsma, Tiny; van Veldhuisen, Dirk J; Hillege, Hans L; Voors, Adriaan A; van der Meer, Peter

    2017-01-15

    Serum potassium is routinely measured at admission for acute heart failure (AHF), but information on association with clinical variables and prognosis is limited. Potassium measurements at admission were available in 1,867 patients with AHF in the original cohort of 2,033 patients included in the Patients Hospitalized with acute heart failure and Volume Overload to Assess Treatment Effect on Congestion and Renal FuncTion trial. Patients were grouped according to low potassium (5.0 mEq/l) levels. Results were verified in a validation cohort of 1,023 patients. Mean age of patients was 71 ± 11 years, and 66% were men. Low potassium was present in 115 patients (6%), normal potassium in 1,576 (84%), and high potassium in 176 (9%). Potassium levels increased during hospitalization (0.18 ± 0.69 mEq/l). Patients with high potassium more often used angiotensin-converting enzyme inhibitors and mineralocorticoid receptor antagonists before admission, had impaired baseline renal function and a better diuretic response (p = 0.005), independent of mineralocorticoid receptor antagonist usage. During 180-day follow-up, a total of 330 patients (18%) died. Potassium levels at admission showed a univariate linear association with mortality (hazard ratio [log] 2.36, 95% confidence interval 1.07 to 5.23; p = 0.034) but not after multivariate adjustment. Changes of potassium levels during hospitalization or potassium levels at discharge were not associated with outcome after multivariate analysis. Results in the validation cohort were similar to the index cohort. In conclusion, high potassium levels at admission are associated with an impaired renal function but a better diuretic response. Changes in potassium levels are common, and overall levels increase during hospitalization. In conclusion, potassium levels at admission or its change during hospitalization are not associated with mortality after multivariate adjustment. Copyright © 2016 The Authors. Published by Elsevier Inc

  13. High Rates of Baseline Drug Resistance and Virologic Failure Among ART-naive HIV-infected Children in Mali.

    Science.gov (United States)

    Crowell, Claudia S; Maiga, Almoustapha I; Sylla, Mariam; Taiwo, Babafemi; Kone, Niaboula; Oron, Assaf P; Murphy, Robert L; Marcelin, Anne-Geneviève; Traore, Ban; Fofana, Djeneba B; Peytavin, Gilles; Chadwick, Ellen G

    2017-11-01

    Limited data exist on drug resistance and antiretroviral treatment (ART) outcomes in HIV-1-infected children in West Africa. We determined the prevalence of baseline resistance and correlates of virologic failure (VF) in a cohort of ART-naive HIV-1-infected children baseline (before ART) and at 6 months. Resistance was defined according to the Stanford HIV Genotypic Resistance database. VF was defined as viral load ≥1000 copies/mL after 6 months of ART. Logistic regression was used to evaluate factors associated with VF or death >1 month after enrollment. Post hoc, antiretroviral concentrations were assayed on baseline samples of participants with baseline resistance. One-hundred twenty children with a median age 2.6 years (interquartile range: 1.6-5.0) were included. Eighty-eight percent reported no prevention of mother-to-child transmission exposure. At baseline, 27 (23%), 4 (3%) and none had non-nucleoside reverse transcriptase inhibitor (NNRTI), nucleoside reverse transcriptase inhibitor or protease inhibitor resistance, respectively. Thirty-nine (33%) developed VF and 4 died >1 month post-ART initiation. In multivariable analyses, poor adherence [odds ratio (OR): 6.1, P = 0.001], baseline NNRTI resistance among children receiving NNRTI-based ART (OR: 22.9, P baseline NNRTI resistance (OR: 5.8, P = 0.018) were significantly associated with VF/death. Ten (38%) with baseline resistance had detectable levels of nevirapine or efavirenz at baseline; 7 were currently breastfeeding, but only 2 reported maternal antiretroviral use. Baseline NNRTI resistance was common in children without reported NNRTI exposure and was associated with increased risk of treatment failure. Detectable NNRTI concentrations were present despite few reports of maternal/infant antiretroviral use.

  14. Overcoming Resistance of Cancer Cells to PARP-1 Inhibitors with Three Different Drug Combinations.

    Directory of Open Access Journals (Sweden)

    Michal Yalon

    Full Text Available Inhibitors of poly[ADP-ribose] polymerase 1 (PARPis show promise for treatment of cancers which lack capacity for homologous recombination repair (HRR. However, new therapeutic strategies are required in order to overcome innate and acquired resistance to these drugs and thus expand the array of cancers that could benefit from them. We show that human cancer cell lines which respond poorly to ABT-888 (a PARPi, become sensitive to it when co-treated with vorinostat (a histone deacetylase inhibitor (HDACi. Vorinostat also sensitized PARPis insensitive cancer cell lines to 6-thioguanine (6-TG-a drug that targets PARPis sensitive cells. The sensitizing effect of vorinostat was associated with increased phosphorylation of eukaryotic initiation factor (eIF 2α which in and of itself increases the sensitivity of cancer cells to ABT-888. Importantly, these drug combinations did not affect survival of normal fibroblasts and breast cells, and significantly increased the inhibition of xenograft tumor growth relative to each drug alone, without affecting the mice weight or their liver and kidney function. Our results show that combination of vorinostat and ABT-888 could potentially prove useful for treatment of cancer with innate resistance to PARPis due to active HRR machinery, while the combination of vorinostat and 6-TG could potentially overcome innate or acquired resistance to PARPis due to secondary or reversal BRCA mutations, to decreased PARP-1 level or to increased expression of multiple drug resistant proteins. Importantly, drugs which increase phosphorylation of eIF2α may mimic the sensitizing effect of vorinostat on cellular response to PARPis or to 6-TG, without activating all of its downstream effectors.

  15. Protease inhibitor associated mutations compromise the efficacy of therapy in human immunodeficiency virus – 1 (HIV-1 infected pediatric patients: a cross-sectional study

    Directory of Open Access Journals (Sweden)

    Petrova Anna

    2007-07-01

    Full Text Available Abstract Background Although the introduction of combined therapy with reverse transcriptase and protease inhibitors has resulted in considerable decrease in HIV related mortality; it has also induced the development of multiple drug-resistant HIV-1 variants. The few studies on HIV-1 mutagenesis in HIV infected children have not evaluated the impact of HIV-1 mutations on the clinical, virological and immunological presentation of HIV disease that is fundamental to optimizing the treatment regimens for these patients. Results A cross sectional study was conducted to evaluate the impact of treatment regimens and resistance mutation patterns on the clinical, virological, and immunological presentation of HIV disease in 41 children (25 male and 16 female at the Robert Wood Johnson Pediatric AIDS Program in New Brunswick, New Jersey. The study participants were symptomatic and had preceding treatment history with combined ARV regimens including protease inhibitors (PIs, nucleoside reverse transcriptase inhibitors (NRTIs and non-nucleoside reverse transcriptase inhibitors (NNRTIs. Fifteen (36.6% children were treated with NRTI+NNRTI+ PI, 6 (14.6% with NRTI+NNRTIs, 13 (31.7% with NRTI+PIs, and the remaining 7 (17.1% received NRTIs only. Combined ARV regimens did not significantly influence the incidence of NRTI and NNRTI associated mutations. The duration of ARV therapy and the child's age had no significant impact on the ARV related mutations. The clinico-immunological presentation of the HIV disease was not associated with ARV treatment regimens or number of resistance mutations. However, primary mutations in the protease (PR gene increased the likelihood of plasma viral load (PVL ≥ 10,000 copies/mL irrespective of the child's age, duration of ARV therapy, presence of NRTI and NNRTI mutation. Viremia ≥ 10,000 copies/mL was recorded in almost all the children with primary mutations in the PR region (n = 12/13, 92.3% as compared with only 50.0% (n

  16. Deciphering mechanisms of drug sensitivity and resistance to Selective Inhibitor of Nuclear Export (SINE) compounds

    International Nuclear Information System (INIS)

    Crochiere, Marsha; Kashyap, Trinayan; Kalid, Ori; Shechter, Sharon; Klebanov, Boris; Senapedis, William; Saint-Martin, Jean-Richard; Landesman, Yosef

    2015-01-01

    Exportin 1 (XPO1) is a well-characterized nuclear export protein whose expression is up-regulated in many types of cancers and functions to transport key tumor suppressor proteins (TSPs) from the nucleus. Karyopharm Therapeutics has developed a series of small-molecule Selective Inhibitor of Nuclear Export (SINE) compounds, which have been shown to block XPO1 function both in vitro and in vivo. The drug candidate, selinexor (KPT-330), is currently in Phase-II/IIb clinical trials for treatment of both hematologic and solid tumors. The present study sought to decipher the mechanisms that render cells either sensitive or resistant to treatment with SINE compounds, represented by KPT-185, an early analogue of KPT-330. Using the human fibrosarcoma HT1080 cell line, resistance to SINE was acquired over a period of 10 months of constant incubation with increasing concentration of KPT-185. Cell viability was assayed by MTT. Immunofluorescence was used to compare nuclear export of TSPs. Fluorescence activated cell sorting (FACS), quantitative polymerase chain reaction (qPCR), and immunoblots were used to measure effects on cell cycle, gene expression, and cell death. RNA from naïve and drug treated parental and resistant cells was analyzed by Affymetrix microarrays. Treatment of HT1080 cells with gradually increasing concentrations of SINE resulted in > 100 fold decrease in sensitivity to SINE cytotoxicity. Resistant cells displayed prolonged cell cycle, reduced nuclear accumulation of TSPs, and similar changes in protein expression compared to parental cells, however the magnitude of the protein expression changes were more significant in parental cells. Microarray analyses comparing parental to resistant cells indicate that a number of key signaling pathways were altered in resistant cells including expression changes in genes involved in adhesion, apoptosis, and inflammation. While the patterns of changes in transcription following drug treatment are similar in parental

  17. Elucidating the Interdependence of Drug Resistance from Combinations of Mutations.

    Science.gov (United States)

    Ragland, Debra A; Whitfield, Troy W; Lee, Sook-Kyung; Swanstrom, Ronald; Zeldovich, Konstantin B; Kurt-Yilmaz, Nese; Schiffer, Celia A

    2017-11-14

    HIV-1 protease is responsible for the cleavage of 12 nonhomologous sites within the Gag and Gag-Pro-Pol polyproteins in the viral genome. Under the selective pressure of protease inhibition, the virus evolves mutations within (primary) and outside of (secondary) the active site, allowing the protease to process substrates while simultaneously countering inhibition. The primary protease mutations impede inhibitor binding directly, while the secondary mutations are considered accessory mutations that compensate for a loss in fitness. However, the role of secondary mutations in conferring drug resistance remains a largely unresolved topic. We have shown previously that mutations distal to the active site are able to perturb binding of darunavir (DRV) via the protein's internal hydrogen-bonding network. In this study, we show that mutations distal to the active site, regardless of context, can play an interdependent role in drug resistance. Applying eigenvalue decomposition to collections of hydrogen bonding and van der Waals interactions from a series of molecular dynamics simulations of 15 diverse HIV-1 protease variants, we identify sites in the protease where amino acid substitutions lead to perturbations in nonbonded interactions with DRV and/or the hydrogen-bonding network of the protease itself. While primary mutations are known to drive resistance in HIV-1 protease, these findings delineate the significant contributions of accessory mutations to resistance. Identifying the variable positions in the protease that have the greatest impact on drug resistance may aid in future structure-based design of inhibitors.

  18. Contribution of HIV minority variants to drug resistance in an integrase strand transfer inhibitor-based therapy

    Czech Academy of Sciences Publication Activity Database

    Weber, Jan; Gibson, R. M.; Meyer, A. M.; Winner, D.; Robertson, D. L.; Miller, M. D.; Quinones-Mateu, M. E.

    2013-01-01

    Roč. 18, Suppl. 1 (2013), A66-A66 ISSN 1359-6535. [International Workshop on HIV & Hepatitis Virus Drug Resistance Curative Strategies. 04.06.2013-08.06.2013, Toronto] Institutional support: RVO:61388963 Keywords : HIV minority variants * integrase inhibitor * replicative fitness Subject RIV: CE - Biochemistry

  19. Potassium fluorotitanate preparation

    International Nuclear Information System (INIS)

    Perillo, Patricia; Ares, Osvaldo; Botbol, Jose.

    1989-01-01

    In order to determine the best conditions for potassium fluotitanate preparation as intermediate step in the electrolytic production of metalic titanium, the effects of a number of experimental variables have been studied. This method is a process of sintering titanium dioxide with potassium fluosilicate and potassium chloride, followed by leaching with boiling water and further crystallization by cooling the solution. An overall yield of 90% has been attained under the following conditions: working temperature: 750 deg C; heating time for sintering: 3 hours; molar ratio: titanium dioxide: potassium fluosilicate: potassium chloride: 1 : 2 : 0.4; number of leachings: 6. (Author) [es

  20. The prototype HIV-1 maturation inhibitor, bevirimat, binds to the CA-SP1 cleavage site in immature Gag particles

    Directory of Open Access Journals (Sweden)

    Nguyen Albert T

    2011-12-01

    Full Text Available Abstract Background Bevirimat, the prototype Human Immunodeficiency Virus type 1 (HIV-1 maturation inhibitor, is highly potent in cell culture and efficacious in HIV-1 infected patients. In contrast to inhibitors that target the active site of the viral protease, bevirimat specifically inhibits a single cleavage event, the final processing step for the Gag precursor where p25 (CA-SP1 is cleaved to p24 (CA and SP1. Results In this study, photoaffinity analogs of bevirimat and mass spectrometry were employed to map the binding site of bevirimat to Gag within immature virus-like particles. Bevirimat analogs were found to crosslink to sequences overlapping, or proximal to, the CA-SP1 cleavage site, consistent with previous biochemical data on the effect of bevirimat on Gag processing and with genetic data from resistance mutations, in a region predicted by NMR and mutational studies to have α-helical character. Unexpectedly, a second region of interaction was found within the Major Homology Region (MHR. Extensive prior genetic evidence suggests that the MHR is critical for virus assembly. Conclusions This is the first demonstration of a direct interaction between the maturation inhibitor, bevirimat, and its target, Gag. Information gained from this study sheds light on the mechanisms by which the virus develops resistance to this class of drug and may aid in the design of next-generation maturation inhibitors.

  1. A new class of HIV-1 protease inhibitor: the crystallographic structure, inhibition and chemical synthesis of an aminimide peptide isostere.

    Science.gov (United States)

    Rutenber, E E; McPhee, F; Kaplan, A P; Gallion, S L; Hogan, J C; Craik, C S; Stroud, R M

    1996-09-01

    The essential role of HIV-1 protease (HIV-1 PR) in the viral life cycle makes it an attractive target for the development of substrate-based inhibitors that may find efficacy as anti-AIDS drugs. However, resistance has arisen to potent peptidomimetic drugs necessitating the further development of novel chemical backbones for diversity based chemistry focused on probing the active site for inhibitor interactions and binding modes that evade protease resistance. AQ148 is a potent inhibitor of HIV-1 PR and represents a new class of transition state analogues incorporating an aminimide peptide isostere. A 3-D crystallographic structure of AQ148, a tetrapeptide isostere, has been determined in complex with its target HIV-1 PR to a resolution of 2.5 A and used to evaluate the specific structural determinants of AQ148 potency and to correlate structure-activity relationships within the class of related compounds. AQ148 is a competitive inhibitor of HIV-1 PR with a Ki value of 137 nM. Twenty-nine derivatives have been synthesized and chemical modifications have been made at the P1, P2, P1', and P2' sites. The atomic resolution structure of AQ148 bound to HIV-1 PR reveals both an inhibitor binding mode that closely resembles that of other peptidomimetic inhibitors and specific protein/inhibitor interactions that correlate with structure-activity relationships. The structure provides the basis for the design, synthesis and evaluation of the next generation of hydroxyethyl aminimide inhibitors. The aminimide peptide isostere is a scaffold with favorable biological properties well suited to both the combinatorial methods of peptidomimesis and the rational design of potent and specific substrate-based analogues.

  2. Regulation of voltage-gated potassium channels attenuates resistance of side-population cells to gefitinib in the human lung cancer cell line NCI-H460.

    Science.gov (United States)

    Choi, Seon Young; Kim, Hang-Rae; Ryu, Pan Dong; Lee, So Yeong

    2017-02-21

    Side-population (SP) cells that exclude anti-cancer drugs have been found in various tumor cell lines. Moreover, SP cells have a higher proliferative potential and drug resistance than main population cells (Non-SP cells). Also, several ion channels are responsible for the drug resistance and proliferation of SP cells in cancer. To confirm the expression and function of voltage-gated potassium (Kv) channels of SP cells, these cells, as well as highly expressed ATP-binding cassette (ABC) transporters and stemness genes, were isolated from a gefitinib-resistant human lung adenocarcinoma cell line (NCI-H460), using Hoechst 33342 efflux. In the present study, we found that mRNA expression of Kv channels in SP cells was different compared to Non-SP cells, and the resistance of SP cells to gefitinib was weakened with a combination treatment of gefitinib and Kv channel blockers or a Kv7 opener, compared to single-treatment gefitinib, through inhibition of the Ras-Raf signaling pathway. The findings indicate that Kv channels in SP cells could be new targets for reducing the resistance to gefitinib.

  3. Biochemical and muscle studies in patients with acute onset post-viral fatigue syndrome.

    OpenAIRE

    Preedy, V R; Smith, D G; Salisbury, J R; Peters, T J

    1993-01-01

    AIMS--To investigate in detail various biochemical and pathophysiological indices of muscle pathology in acute onset post-viral fatigue syndrome (PVFS). METHODS--Twenty three patients with PVFS (of mean duration 4.6 years) were subjected to needle biopsy for histomorphometry and total RNA contents. Plasma analysis included serology and creatine kinase activities. Indices of whole body mass were also measured--namely, whole body potassium content and plasma carnosinase activities. RESULTS--Abo...

  4. AP24534, a Pan-BCR-ABL Inhibitor for Chronic Myeloid Leukemia, Potently Inhibits the T315I Mutant and Overcomes Mutation-Based Resistance

    Science.gov (United States)

    O’Hare, Thomas; Shakespeare, William C.; Zhu, Xiaotian; Eide, Christopher A.; Rivera, Victor M.; Wang, Frank; Adrian, Lauren T.; Zhou, Tianjun; Huang, Wei-Sheng; Xu, Qihong; Metcalf, Chester A.; Tyner, Jeffrey W.; Loriaux, Marc M.; Corbin, Amie S.; Wardwell, Scott; Ning, Yaoyu; Keats, Jeffrey A.; Wang, Yihan; Sundaramoorthi, Raji; Thomas, Mathew; Zhou, Dong; Snodgrass, Joseph; Commodore, Lois; Sawyer, Tomi K.; Dalgarno, David C.; Deininger, Michael W.N.; Druker, Brian J.; Clackson, Tim

    2009-01-01

    SUMMARY Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABLT315I mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and pre-clinical evaluation of AP24534, a potent, orally available multi-targeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABLT315I-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML. PMID:19878872

  5. AP24534, a Pan-BCR-ABL Inhibitor for Chronic Myeloid Leukemia, Potently Inhibits the T315I Mutant and Overcomes Mutation-Based Resistance

    Energy Technology Data Exchange (ETDEWEB)

    O’Hare, Thomas; Shakespeare, William C.; Zhu, Xiaotian; Eide, Christopher A.; Rivera, Victor M.; Wang, Frank; Adrian, Lauren T.; Zhou, Tianjun; Huang, Wei-Sheng; Xu, Qihong; Metcalf, III, Chester A.; Tyner, Jeffrey W.; Loriaux, Marc M.; Corbin, Amie S.; Wardwell, Scott; Ning, Yaoyu; Keats, Jeffrey A.; Wang, Yihan; Sundaramoorthi, Raji; Thomas, Mathew; Zhou, Dong; Snodgrass, Joseph; Commodore, Lois; Sawyer, Tomi K.; Dalgarno, David C.; Deininger, Michael W.N.; Druker, Brian J.; Clackson, Tim; (OHSU- Cancer Instit.); (ARIAD)

    2010-09-07

    Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABL{sup T315I} mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and preclinical evaluation of AP24534, a potent, orally available multitargeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABL{sup T315I}-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML.

  6. Mechanisms of Acquired Resistance to ALK Inhibitors and the Rationale for Treating ALK-positive Lung Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Isozaki, Hideko [Department of Clinical Pharmaceutics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama 700-8558 (Japan); Takigawa, Nagio, E-mail: ntakigaw@gmail.com [Department of General Internal Medicine 4, Kawasaki Medical School, Okayama 700-8505 (Japan); Kiura, Katsuyuki [Department of Allergy and Respiratory Medicine, Okayama University Hospital, Okayama 700-8558 (Japan)

    2015-04-30

    The discovery of an echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene led to improved clinical outcomes in patients with lung cancer after the development of the first ALK-targeting agent, crizotinib. Some second-generation ALK tyrosine kinase inhibitors (TKIs), which might be more potent than crizotinib or effective on crizotinib-resistant patients, have been developed. Although these ALK-TKIs show an excellent response initially, most patients eventually acquire resistance. Therefore, careful consideration of the resistance mechanisms might lead to superior therapeutic strategies. Here, we summarize the history of ALK-TKIs and their underlying resistance mechanisms in both the preclinical and clinical settings. In addition, we discuss potential future treatment strategies in ALK-TKI-naïve and -resistant patients with lung cancer harboring the EML4-ALK fusion gene.

  7. Tissue transglutaminase (TG2) is involved in the resistance of cancer cells to the histone deacetylase (HDAC) inhibitor vorinostat.

    Science.gov (United States)

    Carbone, Carmine; Di Gennaro, Elena; Piro, Geny; Milone, Maria Rita; Pucci, Biagio; Caraglia, Michele; Budillon, Alfredo

    2017-03-01

    Vorinostat demonstrated preclinical and clinical efficacy in human cancers and is the first histone deacetylase inhibitor (HDACi) approved for cancer treatment. Tissue transglutaminase (TG2) is a multifunctional enzyme that catalyzes a Ca 2+ dependent transamidating reaction resulting in covalent cross-links between proteins. TG2 acts also as G-protein in trans-membrane signaling and as a cell surface adhesion mediator. TG2 up-regulation has been demonstrated in several cancers and its expression levels correlate with resistance to chemotherapy and metastatic potential. We demonstrated that the anti-proliferative effect of the HDACi vorinostat is paralleled by the induction of TG2 mRNA and protein expression in cancer cells but not in ex vivo treated peripheral blood lymphocytes. This effect was also shared by other pan-HDACi and resulted in increased TG2 transamidating activity. Notably, high TG2 basal levels in a panel of cancer cell lines correlated with lower vorinostat antiproliferative activity. Notably, in TG2-knockdown cancer cells vorinostat anti-proliferative and pro-apoptotic effects were enhanced, whereas in TG2-full-length transfected cells were impaired, suggesting that TG2 could represent a mechanism of intrinsic or acquired resistance to vorinostat. In fact, co-treatment of tumor cells with inhibitors of TG2 transamidating activity potentiated the antitumor effect of vorinostat. Moreover, vorinostat-resistant MCF7 cells selected by stepwise increasing concentrations of the drug, significantly overexpressed TG2 protein compared to parental cells, and co-treatment of these cells with TG2 inhibitors reversed vorinostat-resistance. Taken together, our data demonstrated that TG2 is involved in the resistance of cancer cells to vorinostat, as well as to other HDACi.

  8. Acquired resistance to HSP90 inhibitor 17-AAG and increased metastatic potential are associated with MUC1 expression in colon carcinoma cells.

    Science.gov (United States)

    Liu, Xin; Ban, Li-Li; Luo, Gang; Li, Zhi-Yao; Li, Yun-Feng; Zhou, Yong-Chun; Wang, Xi-Cai; Jin, Cong-Guo; Ye, Jia-Gui; Ma, Ding-Ding; Xie, Qing; Huang, You-Guang

    2016-06-01

    Heat shock protein 90 (HSP90) is a molecular chaperone required for the stability and function of many proteins. The chaperoning of oncoproteins by HSP90 enhances the survival, growth, and invasive potential of cancer cells. HSP90 inhibitors are promising new anticancer agents, in which the benzoquinone ansamycin 17-allylamino-17-demethoxygeldanamycin (17-AAG) is currently in clinical evaluation. However, the implications of acquired resistance to this class of drug remain largely unexplored. In the present study, we have generated isogenic human colon cancer cell lines that are resistant to 17-AAG by continued culturing in the compound. Cross-resistance was found with another HSP90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin. The resistant cells showed obvious morphology changes with a metastatic phenotype and significant increases in migration and adhesion to collagens. Western blotting analysis of epithelial-mesenchymal transition molecular markers found that expression of E-cadherin downregulated, whereas expression of N-cadherin and β-catenin upregulated in the resistant cells. Mucin 1 (MUC1) has been reported to mediate metastasis as well as chemical resistance in many cancers. Here, we found that MUC1 expression was significantly elevated in the acquired drug resistance cells. 17-AAG treatment could decrease MUC1 more in parental cells than in acquired 17-AAG-resistant cells. Further study found that knockdown of MUC1 expression by small interfering RNA could obviously re-sensitize the resistant cells to 17-AAG treatment, and decrease the cell migration and adhesion. These were coupled with a downregulation in N-cadherin and β-catenin. The results indicate that HSP90 inhibitor therapies in colon carcinomas could generate resistance and increase metastatic potential that might mediated by upregulation of MUC1 expression. Findings from this study further our understanding of the potential clinical effects of HSP90-directed therapies in

  9. NK Cell Receptor/H2-Dk–Dependent Host Resistance to Viral Infection Is Quantitatively Modulated by H2 q Inhibitory Signals

    Science.gov (United States)

    Fodil-Cornu, Nassima; Loredo-Osti, J. Concepción; Vidal, Silvia M.

    2011-01-01

    The cytomegalovirus resistance locus Cmv3 has been linked to an epistatic interaction between two loci: a Natural Killer (NK) cell receptor gene and the major histocompatibility complex class I (MHC-I) locus. To demonstrate the interaction between Cmv3 and H2k, we generated double congenic mice between MA/My and BALB.K mice and an F2 cross between FVB/N (H-2q) and BALB.K (H2k) mice, two strains susceptible to mouse cytomegalovirus (MCMV). Only mice expressing H2k in conjunction with Cmv3MA/My or Cmv3FVB were resistant to MCMV infection. Subsequently, an F3 cross was carried out between transgenic FVB/H2-Dk and MHC-I deficient mice in which only the progeny expressing Cmv3FVB and a single H2-Dk class-I molecule completely controlled MCMV viral loads. This phenotype was shown to be NK cell–dependent and associated with subsequent NK cell proliferation. Finally, we demonstrated that a number of H2q alleles influence the expression level of H2q molecules, but not intrinsic functional properties of NK cells; viral loads, however, were quantitatively proportional to the number of H2q alleles. Our results support a model in which H-2q molecules convey Ly49-dependent inhibitory signals that interfere with the action of H2-Dk on NK cell activation against MCMV infection. Thus, the integration of activating and inhibitory signals emanating from various MHC-I/NK cell receptor interactions regulates NK cell–mediated control of viral load. PMID:21533075

  10. Novel Inhibitors of Staphyloxanthin Virulence Factor in Comparison with Linezolid and Vancomycin versus Methicillin-Resistant, Linezolid-Resistant, and Vancomycin-Intermediate Staphylococcus aureus Infections in Vivo.

    Science.gov (United States)

    Ni, Shuaishuai; Wei, Hanwen; Li, Baoli; Chen, Feifei; Liu, Yifu; Chen, Wenhua; Xu, Yixiang; Qiu, Xiaoxia; Li, Xiaokang; Lu, Yanli; Liu, Wenwen; Hu, Linhao; Lin, Dazheng; Wang, Manjiong; Zheng, Xinyu; Mao, Fei; Zhu, Jin; Lan, Lefu; Li, Jian

    2017-10-12

    Our previous work ( Wang et al. J. Med. Chem. 2016 , 59 , 4831 - 4848 ) revealed that effective benzocycloalkane-derived staphyloxanthin inhibitors against methicillin-resistant Staphylococcus aureus (S. aureus) infections were accompanied by poor water solubility and high hERG inhibition and dosages (preadministration). In this study, 92 chroman and coumaran derivatives as novel inhibitors have been addressed for overcoming deficiencies above. Derivatives 69 and 105 displayed excellent pigment inhibitory activities and low hERG inhibition, along with improvement of solubility by salt type selection. The broad and significantly potent antibacterial spectra of 69 and 105 were displayed first with normal administration in the livers and hearts in mice against pigmented S. aureus Newman, Mu50 (vancomycin-intermediate S. aureus), and NRS271 (linezolid-resistant S. aureus), compared with linezolid and vancomycin. In summary, both 69 and 105 have the potential to be developed as good antibacterial candidates targeting virulence factors.

  11. Histone deacetylase inhibitors improve the replication of oncolytic herpes simplex virus in breast cancer cells.

    Directory of Open Access Journals (Sweden)

    James J Cody

    Full Text Available New therapies are needed for metastatic breast cancer patients. Oncolytic herpes simplex virus (oHSV is an exciting therapy being developed for use against aggressive tumors and established metastases. Although oHSV have been demonstrated safe in clinical trials, a lack of sufficient potency has slowed the clinical application of this approach. We utilized histone deacetylase (HDAC inhibitors, which have been noted to impair the innate antiviral response and improve gene transcription from viral vectors, to enhance the replication of oHSV in breast cancer cells. A panel of chemically diverse HDAC inhibitors were tested at three different doses (LD50 for their ability to modulate the replication of oHSV in breast cancer cells. Several of the tested HDAC inhibitors enhanced oHSV replication at low multiplicity of infection (MOI following pre-treatment of the metastatic breast cancer cell line MDA-MB-231 and the oHSV-resistant cell line 4T1, but not in the normal breast epithelial cell line MCF10A. Inhibitors of class I HDACs, including pan-selective compounds, were more effective for increasing oHSV replication compared to inhibitors that selectively target class II HDACs. These studies demonstrate that select HDAC inhibitors increase oHSV replication in breast cancer cells and provides support for pre-clinical evaluation of this combination strategy.

  12. Antiretroviral-treated HIV-1 patients can harbour resistant viruses in CSF despite an undetectable viral load in plasma.

    Science.gov (United States)

    Soulie, Cathia; Grudé, Maxime; Descamps, Diane; Amiel, Corinne; Morand-Joubert, Laurence; Raymond, Stéphanie; Pallier, Coralie; Bellecave, Pantxika; Reigadas, Sandrine; Trabaud, Mary-Anne; Delaugerre, Constance; Montes, Brigitte; Barin, Francis; Ferré, Virginie; Jeulin, Hélène; Alloui, Chakib; Yerly, Sabine; Signori-Schmuck, Anne; Guigon, Aurélie; Fafi-Kremer, Samira; Haïm-Boukobza, Stéphanie; Mirand, Audrey; Maillard, Anne; Vallet, Sophie; Roussel, Catherine; Assoumou, Lambert; Calvez, Vincent; Flandre, Philippe; Marcelin, Anne-Geneviève

    2017-08-01

    HIV therapy reduces the CSF HIV RNA viral load (VL) and prevents disorders related to HIV encephalitis. However, these brain disorders may persist in some cases. A large population of antiretroviral-treated patients who had a VL > 1.7 log 10 copies/mL in CSF with detectable or undetectable VL in plasma associated with cognitive impairment was studied, in order to characterize discriminatory factors of these two patient populations. Blood and CSF samples were collected at the time of neurological disorders for 227 patients in 22 centres in France and 1 centre in Switzerland. Genotypic HIV resistance tests were performed on CSF. The genotypic susceptibility score was calculated according to the last Agence Nationale de Recherche sur le Sida et les hépatites virales Action Coordonnée 11 (ANRS AC11) genotype interpretation algorithm. Among the 227 studied patients with VL > 1.7 log 10 copies/mL in CSF, 195 had VL detectable in plasma [median (IQR) HIV RNA was 3.7 (2.7-4.7) log 10 copies/mL] and 32 had discordant VL in plasma (VL plasma compared with patients with plasma VL > 1.7 log 10 copies/mL. Resistance to antiretrovirals was observed in CSF for the two groups of patients. Fourteen percent of this population of patients with cognitive impairment and detectable VL in CSF had well controlled VL in plasma. Thus, it is important to explore CSF HIV (VL and genotype) even if the HIV VL is controlled in plasma because HIV resistance may be observed. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  13. Histological transformation after acquired resistance to epidermal growth factor tyrosine kinase inhibitors.

    Science.gov (United States)

    Shao, Yi; Zhong, Dian-Sheng

    2018-04-01

    Non-small-cell lung cancer patients with sensitive epidermal growth factor receptor mutations generally respond well to tyrosine kinase inhibitors (TKIs). However, acquired resistance will eventually develop place after 8-16 months. Several mechanisms contribute to the resistance including T790M mutation, c-Met amplification, epithelial mesenchymal transformation and PIK3CA mutation; however, histological transformation is a rare mechanism. The patterns and mechanisms underlying histological transformation need to be explored. We searched PubMed, EMBASE and search engines Google Scholar, Medical Matrix for literature related to histological transformation. Case reports, cases series, and clinical and basic medical research articles were reviewed. Sixty-one articles were included in this review. Cases of transformation to small-cell lung cancer, squamous cell carcinoma, large-cell neuroendocrine carcinoma and sarcoma after TKI resistance have all been reported. As the clinical course differed dramatically between cases, a new treatment scheme needs to be recruited. The mechanisms underlying histological transformation have not been fully elucidated and probably relate to cancer stem cells, driver genetic alterations under selective pressure or the heterogeneity of the tumor. When TKI resistance develops, we recommend that patients undergo a second biopsy to determine the reason, guide the next treatment and predict the prognosis.

  14. A novel inhibitor of dengue virus replication that targets the capsid protein.

    Science.gov (United States)

    Byrd, Chelsea M; Dai, Dongcheng; Grosenbach, Douglas W; Berhanu, Aklile; Jones, Kevin F; Cardwell, Kara B; Schneider, Christine; Wineinger, Kristin A; Page, Jessica M; Harver, Chris; Stavale, Eric; Tyavanagimatt, Shanthakumar; Stone, Melialani A; Bartenschlager, Ralf; Scaturro, Pietro; Hruby, Dennis E; Jordan, Robert

    2013-01-01

    Dengue viruses (DENV) infect 50 to 100 million people worldwide per year, of which 500,000 develop severe life-threatening disease. This mosquito-borne illness is endemic in most tropical and subtropical countries and has spread significantly over the last decade. While there are several promising vaccine candidates in clinical trials, there are currently no approved vaccines or therapeutics available for treatment of dengue infection. Here, we describe a novel small-molecule compound, ST-148, that is a potent inhibitor of all four serotypes of DENV in vitro. ST-148 significantly reduced viremia and viral load in vital organs and tended to lower cytokine levels in the plasma in a nonlethal model of DENV infection in AG129 mice. Compound resistance mapped to the DENV capsid (C) gene, and a direct interaction of ST-148 with C protein is suggested by alterations of the intrinsic fluorescence of the protein in the presence of compound. Thus, ST-148 appears to interact with the DENV C protein and inhibits a distinct step(s) of the viral replication cycle.

  15. Natural Polymorphisms Conferring Resistance to HCV Protease and Polymerase Inhibitors in Treatment-Naïve HIV/HCV Co-Infected Patients in China.

    Directory of Open Access Journals (Sweden)

    Kali Zhou

    Full Text Available The advent of direct-acting agents (DAAs has improved treatment of HCV in HIV co-infection, but may be limited by primary drug resistance. This study reports the prevalence of natural polymorphisms conferring resistance to NS3/4A protease inhibitors and NS5B polymerase inhibitors in treatment-naïve HIV/HCV co-infected individuals in China.Population based NS3/4A sequencing was completed for 778 treatment-naïve HIV/HCV co-infected patients from twelve provinces. NS3 sequences were amplified by nested PCR using in-house primers for genotypes 1-6. NS5B sequencing was completed for genotyping in 350 sequences. Resistance-associated variants (RAVs were identified in positions associated with HCV resistance.Overall, 72.8% (566/778 of all HCV sequences had at least one RAV associated with HCV NS3/4A protease inhibitor resistance. Variants were found in 3.6% (7/193 of genotype 1, 100% (23/23 of genotype 2, 100% (237/237 of genotype 3 and 92% (299/325 of genotype 6 sequences. The Q80K variant was present in 98.4% of genotype 6a sequences. High-level RAVs were rare, occurring in only 0.8% of patients. 93% (64/69 patients with genotype 1b also carried the C316N variant associated with NS5B low-level resistance.The low frequency of high-level RAVs associated with primary HCV DAA resistance among all genotypes in HIV/HCV co-infected patients is encouraging. Further phenotypic studies and clinical research are needed.

  16. Neuraminidase inhibitor resistance in influenza: assessing the danger of its generation and spread.

    Directory of Open Access Journals (Sweden)

    Andreas Handel

    2007-12-01

    Full Text Available Neuraminidase Inhibitors (NI are currently the most effective drugs against influenza. Recent cases of NI resistance are a cause for concern. To assess the danger of NI resistance, a number of studies have reported the fraction of treated patients from which resistant strains could be isolated. Unfortunately, those results strongly depend on the details of the experimental protocol. Additionally, knowing the fraction of patients harboring resistance is not too useful by itself. Instead, we want to know how likely it is that an infected patient can generate a resistant infection in a secondary host, and how likely it is that the resistant strain subsequently spreads. While estimates for these parameters can often be obtained from epidemiological data, such data is lacking for NI resistance in influenza. Here, we use an approach that does not rely on epidemiological data. Instead, we combine data from influenza infections of human volunteers with a mathematical framework that allows estimation of the parameters that govern the initial generation and subsequent spread of resistance. We show how these parameters are influenced by changes in drug efficacy, timing of treatment, fitness of the resistant strain, and details of virus and immune system dynamics. Our study provides estimates for parameters that can be directly used in mathematical and computational models to study how NI usage might lead to the emergence and spread of resistance in the population. We find that the initial generation of resistant cases is most likely lower than the fraction of resistant cases reported. However, we also show that the results depend strongly on the details of the within-host dynamics of influenza infections, and most importantly, the role the immune system plays. Better knowledge of the quantitative dynamics of the immune response during influenza infections will be crucial to further improve the results.

  17. Effect of phosphodiesterase inhibitors on human arteries in vitro

    NARCIS (Netherlands)

    Vroom, M. B.; Pfaffendorf, M.; van Wezel, H. B.; van Zwieten, P. A.

    1996-01-01

    In the present study, we investigated if the relaxant effects of phosphodiesterase (PDE) III inhibitors on human vessels could be inhibited by a nitric oxide synthase blocker, L-NAME, or by a blocker of ATP-sensitive potassium channels (KATP), glibenclamide. The experiments were performed using an

  18. A translational study of resistance emergence using sequential direct-acting antiviral agents for hepatitis C using ultra-deep sequencing.

    Science.gov (United States)

    Abe, Hiromi; Hayes, C Nelson; Hiraga, Nobuhiko; Imamura, Michio; Tsuge, Masataka; Miki, Daiki; Takahashi, Shoichi; Ochi, Hidenori; Chayama, Kazuaki

    2013-09-01

    Direct-acting antiviral agents (DAAs) against hepatitis C virus (HCV) have recently been developed and are ultimately hoped to replace interferon-based therapy. However, DAA monotherapy results in rapid emergence of resistant strains and DAAs must be used in combinations that present a high genetic barrier to resistance, although viral kinetics of multidrug-resistant strains remain poorly characterized. The aim of this study is to track the emergence and fitness of resistance using combinations of telaprevir and NS5A or NS5B inhibitors with genotype 1b clones. HCV-infected chimeric mice were treated with DAAs, and resistance was monitored using direct and ultra-deep sequencing. Combination therapy with telaprevir and BMS-788329 (NS5A inhibitor) reduced serum HCV RNA to undetectable levels. The presence of an NS3-V36A telaprevir resistance mutation resulted in poor response to telaprevir monotherapy but showed significant HCV reduction when telaprevir was combined with BMS-788329. However, a BMS-788329-resistant strain emerged at low frequency. Infection with a BMS-788329-resistant NS5A-L31V mutation rapidly resulted in gain of an additional NS5A-Y93A mutation that conferred telaprevir resistance during combination therapy. Infection with dual NS5AL31V/NS5AY93H mutations resulted in poor response to combination therapy and development of telaprevir resistance. Although HCV RNA became undetectable soon after the beginning of combination therapy with BMS-788329 and BMS-821095 (NS5B inhibitor), rebound with emergence of resistance against all three drugs occurred. Triple resistance also occurred following infection with the NS3V36A/NS5AL31V/NS5AY93H triple mutation. Resistant strains easily develop from cloned virus strains. Sequential use of DAAs should be avoided to prevent emergence of multidrug-resistant strains.

  19. A kinase inhibitor screen identifies Mcl-1 and Aurora kinase A as novel treatment targets in antiestrogen-resistant breast cancer cells

    DEFF Research Database (Denmark)

    Thrane, S; Pedersen, A M; Thomsen, M B H

    2015-01-01

    Antiestrogen resistance is a major problem in breast cancer treatment. Therefore, the search for new therapeutic targets and biomarkers for antiestrogen resistance is crucial. In this study, we performed a kinase inhibitor screen on antiestrogen responsive MCF-7 cells and a panel of MCF-7-derived...

  20. Combined effects of EGFR tyrosine kinase inhibitors and vATPase inhibitors in NSCLC cells

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Hyeon-Ok [KIRAMS Radiation Biobank, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Hong, Sung-Eun [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Kim, Chang Soon [Department of Microbiological Engineering, Kon-Kuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, 143–701 (Korea, Republic of); Park, Jin-Ah; Kim, Jin-Hee; Kim, Ji-Young; Kim, Bora [KIRAMS Radiation Biobank, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Chang, Yoon Hwan; Hong, Seok-Il; Hong, Young Jun [Department of Laboratory Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Park, In-Chul, E-mail: parkic@kirams.re.kr [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Lee, Jin Kyung, E-mail: jklee@kirams.re.kr [KIRAMS Radiation Biobank, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Department of Laboratory Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of)

    2015-08-15

    Despite excellent initial clinical responses of non-small cell lung cancer (NSCLC) patients to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), many patients eventually develop resistance. According to a recent report, vacuolar H + ATPase (vATPase) is overexpressed and is associated with chemotherapy drug resistance in NSCLC. We investigated the combined effects of EGFR TKIs and vATPase inhibitors and their underlying mechanisms in the regulation of NSCLC cell death. We found that combined treatment with EGFR TKIs (erlotinib, gefitinib, or lapatinib) and vATPase inhibitors (bafilomycin A1 or concanamycin A) enhanced synergistic cell death compared to treatments with each drug alone. Treatment with bafilomycin A1 or concanamycin A led to the induction of Bnip3 expression in an Hif-1α dependent manner. Knock-down of Hif-1α or Bnip3 by siRNA further enhanced cell death induced by bafilomycin A1, suggesting that Hif-1α/Bnip3 induction promoted resistance to cell death induced by the vATPase inhibitors. EGFR TKIs suppressed Hif-1α and Bnip3 expression induced by the vATPase inhibitors, suggesting that they enhanced the sensitivity of the cells to these inhibitors by decreasing Hif-1α/Bnip3 expression. Taken together, we conclude that EGFR TKIs enhance the sensitivity of NSCLC cells to vATPase inhibitors by decreasing Hif-1α/Bnip3 expression. We suggest that combined treatment with EGFR TKIs and vATPase inhibitors is potentially effective for the treatment of NSCLC. - Highlights: • Co-treatment with EGFR TKIs and vATPase inhibitors induces synergistic cell death • EGFR TKIs enhance cell sensitivity to vATPase inhibitors via Hif-1α downregulation • Co-treatment of these inhibitors is potentially effective for the treatment of NSCLC.

  1. Potassium toxicity at low serum potassium levels with refeeding syndrome.

    Science.gov (United States)

    Vemula, Praveen; Abela, Oliver G; Narisetty, Keerthy; Rhine, David; Abela, George S

    2015-01-01

    Refeeding syndrome is a life-threatening condition occurring in severely malnourished patients after initiating feeding. Severe hypophosphatemia with reduced adenosine triphosphate production has been implicated, but little data are available regarding electrolyte abnormalities. In this case, we report electrocardiographic changes consistent with hyperkalemia during potassium replacement after a serum level increase from 1.9 to 2.9 mEq/L. This was reversed by lowering serum potassium back to 2.0 mEq/L. In conclusion, the patient with prolonged malnutrition became adapted to low potassium levels and developed potassium toxicity with replacement. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Double-blind, randomized, placebo-controlled trial of the use of topical 10% potassium hydroxide solution in the treatment of molluscum contagiosum.

    Science.gov (United States)

    Short, Katherine A; Fuller, L Claire; Higgins, Elisabeth M

    2006-01-01

    Molluscum contagiosum is a common viral infection of the skin that frequently affects children. Lesions take between 6 and 18 months to resolve spontaneously and are a source of great embarrassment to both caretakers and children, often affecting attendance at school and limiting social activity. Treatment options to date have been poorly tolerated by children but recent studies have suggested that potassium hydroxide may be beneficial. This double-blind, randomized, placebo-controlled study compared 10% potassium hydroxide with placebo (normal saline). Twenty patients, aged 2 to 12 years, were recruited. Parents applied a solution twice daily to lesional skin until signs of inflammation appeared. Children were examined by the same observer on days 0, 15, 30, 60, and 90. Seventy percent of children receiving topical potassium hydroxide cleared, compared with 20% in the placebo group. Further dosing studies are required to identify whether weaker concentrations of potassium hydroxide are as efficacious, with less irritancy.

  3. Unique chloride-sensing properties of WNK4 permit the distal nephron to modulate potassium homeostasis.

    Science.gov (United States)

    Terker, Andrew S; Zhang, Chong; Erspamer, Kayla J; Gamba, Gerardo; Yang, Chao-Ling; Ellison, David H

    2016-01-01

    Dietary potassium deficiency activates thiazide-sensitive sodium chloride cotransport along the distal nephron. This may explain, in part, the hypertension and cardiovascular mortality observed in individuals who consume a low-potassium diet. Recent data suggest that plasma potassium affects the distal nephron directly by influencing intracellular chloride, an inhibitor of the with-no-lysine kinase (WNK)-Ste20p-related proline- and alanine-rich kinase (SPAK) pathway. As previous studies used extreme dietary manipulations, we sought to determine whether the relationship between potassium and NaCl cotransporter (NCC) is physiologically relevant and clarify the mechanisms involved. We report that modest changes in both dietary and plasma potassium affect NCC in vivo. Kinase assay studies showed that chloride inhibits WNK4 kinase activity at lower concentrations than it inhibits activity of WNK1 or WNK3. Also, chloride inhibited WNK4 within the range of distal cell chloride concentration. Mutation of a previously identified WNK chloride-binding motif converted WNK4 effects on SPAK from inhibitory to stimulatory in mammalian cells. Disruption of this motif in WNKs 1, 3, and 4 had different effects on NCC, consistent with the three WNKs having different chloride sensitivities. Thus, potassium effects on NCC are graded within the physiological range, which explains how unique chloride-sensing properties of WNK4 enable it to mediate effects of potassium on NCC in vivo. Copyright © 2015 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  4. Everolimus downregulates estrogen receptor and induces autophagy in aromatase inhibitor-resistant breast cancer cells

    International Nuclear Information System (INIS)

    Lui, Asona; New, Jacob; Ogony, Joshua; Thomas, Sufi; Lewis-Wambi, Joan

    2016-01-01

    mTOR inhibition of aromatase inhibitor (AI)-resistant breast cancer is currently under evaluation in the clinic. Everolimus/RAD001 (Afinitor®) has had limited efficacy as a solo agent but is projected to become part of combination therapy for AI-resistant breast cancer. This study was conducted to investigate the anti-proliferative and resistance mechanisms of everolimus in AI-resistant breast cancer cells. In this study we utilized two AI-resistant breast cancer cell lines, MCF-7:5C and MCF-7:2A, which were clonally derived from estrogen receptor positive (ER+) MCF-7 breast cancer cells following long-term estrogen deprivation. Cell viability assay, colony formation assay, cell cycle analysis and soft agar anchorage-independent growth assay were used to determine the efficacy of everolimus in inhibiting the proliferation and tumor forming potential of MCF-7, MCF-7:5C, MCF-7:2A and MCF10A cells. Confocal microscopy and transmission electron microscopy were used to evaluate LC3-II production and autophagosome formation, while ERE-luciferase reporter, Western blot, and RT-PCR analyses were used to assess ER expression and transcriptional activity. Everolimus inhibited the proliferation of MCF-7:5C and MCF-7:2A cells with relatively equal efficiency to parental MCF-7 breast cancer cells. The inhibitory effect of everolimus was due to G1 arrest as a result of downregulation of cyclin D1 and p21. Everolimus also dramatically reduced estrogen receptor (ER) expression (mRNA and protein) and transcriptional activity in addition to the ER chaperone, heat shock protein 90 protein (HSP90). Everolimus restored 4-hydroxy-tamoxifen (4OHT) sensitivity in MCF-7:5C cells and enhanced 4OHT sensitivity in MCF-7 and MCF-7:2A cells. Notably, we found that autophagy is one method of everolimus insensitivity in MCF-7 breast cancer cell lines. This study provides additional insight into the mechanism(s) of action of everolimus that can be used to enhance the utility of mTOR inhibitors as

  5. Development and evaluation of a phenotypic assay monitoring resistance formation to protease inhibitors in HIV-1-infected patients.

    Science.gov (United States)

    Gehringer, Heike; Von der Helm, Klaus; Seelmeir, Sigrid; Weissbrich, Benedikt; Eberle, Josef; Nitschko, Hans

    2003-05-01

    A novel phenotypic assay, based on recombinant expression of the HIV-1-protease was developed and evaluated; it monitors the formation of resistance to protease inhibitors. The HIV-1 protease-encoding region from the blood sample of patients was amplified, ligated into the expression vector pBD2, and recombinantly expressed in Escherichia coli TG1 cells. The resulting recombinant enzyme was purified by a newly developed one-step acid extraction protocol. The protease activity was determined in presence of five selected HIV protease inhibitors and the 50% inhibitory concentration (IC(50)) to the respective protease inhibitors determined. The degree of resistance was expressed in terms of x-fold increase in IC(50) compared to the IC(50) value of an HIV-1 wild type protease preparation. The established test system showed a reproducible recombinant expression of each individual patients' HIV-1 protease population. Samples of nine clinically well characterised HIV-1-infected patients with varying degrees of resistance were analysed. There was a good correlation between clinical parameters and the results obtained by this phenotypic assay. For the majority of patients a blind genotypic analysis of the patients' protease domain revealed a fair correlation to the results of the phenotypic assay. In a minority of patients our phenotypic results diverged from the genotypic ones. This novel phenotypic assay can be carried out within 8-10 days, and offers a significant advantage in time to the current employed phenotypic tests.

  6. The allosteric HIV-1 integrase inhibitor BI-D affects virion maturation but does not influence packaging of a functional RNA genome

    NARCIS (Netherlands)

    van Bel, Nikki; van der Velden, Yme; Bonnard, Damien; Le Rouzic, Erwann; Das, Atze T.; Benarous, Richard; Berkhout, Ben

    2014-01-01

    The viral integrase (IN) is an essential protein for HIV-1 replication. IN inserts the viral dsDNA into the host chromosome, thereby aided by the cellular co-factor LEDGF/p75. Recently a new class of integrase inhibitors was described: allosteric IN inhibitors (ALLINIs). Although designed to

  7. The emerging profile of cross-resistance among the nonnucleoside HIV-1 reverse transcriptase inhibitors.

    Science.gov (United States)

    Sluis-Cremer, Nicolas

    2014-07-31

    Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are widely used to treat HIV-1-infected individuals; indeed most first-line antiretroviral therapies typically include one NNRTI in combination with two nucleoside analogs. In 2008, the next-generation NNRTI etravirine was approved for the treatment of HIV-infected antiretroviral therapy-experienced individuals, including those with prior NNRTI exposure. NNRTIs are also increasingly being included in strategies to prevent HIV-1 infection. For example: (1) nevirapine is used to prevent mother-to-child transmission; (2) the ASPIRE (MTN 020) study will test whether a vaginal ring containing dapivirine can prevent HIV-1 infection in women; (3) a microbicide gel formulation containing the urea-PETT derivative MIV-150 is in a phase I study to evaluate safety, pharmacokinetics, pharmacodynamics and acceptability; and (4) a long acting rilpivirine formulation is under-development for pre-exposure prophylaxis. Given their widespread use, particularly in resource-limited settings, as well as their low genetic barriers to resistance, there are concerns about overlapping resistance between the different NNRTIs. Consequently, a better understanding of the resistance and cross-resistance profiles among the NNRTI class is important for predicting response to treatment, and surveillance of transmitted drug-resistance.

  8. An orally available, small-molecule polymerase inhibitor shows efficacy against a lethal morbillivirus infection in a large animal model.

    Science.gov (United States)

    Krumm, Stefanie A; Yan, Dan; Hovingh, Elise S; Evers, Taylor J; Enkirch, Theresa; Reddy, G Prabhakar; Sun, Aiming; Saindane, Manohar T; Arrendale, Richard F; Painter, George; Liotta, Dennis C; Natchus, Michael G; von Messling, Veronika; Plemper, Richard K

    2014-04-16

    Measles virus is a highly infectious morbillivirus responsible for major morbidity and mortality in unvaccinated humans. The related, zoonotic canine distemper virus (CDV) induces morbillivirus disease in ferrets with 100% lethality. We report an orally available, shelf-stable pan-morbillivirus inhibitor that targets the viral RNA polymerase. Prophylactic oral treatment of ferrets infected intranasally with a lethal CDV dose reduced viremia and prolonged survival. Ferrets infected with the same dose of virus that received post-infection treatment at the onset of viremia showed low-grade viral loads, remained asymptomatic, and recovered from infection, whereas control animals succumbed to the disease. Animals that recovered also mounted a robust immune response and were protected against rechallenge with a lethal CDV dose. Drug-resistant viral recombinants were generated and found to be attenuated and transmission-impaired compared to the genetic parent virus. These findings may pioneer a path toward an effective morbillivirus therapy that could aid measles eradication by synergizing with vaccination to close gaps in herd immunity due to vaccine refusal.

  9. CyclinG1 Amplification Enhances Aurora Kinase Inhibitor-Induced Polyploid Resistance and Inhibition of Bcl-2 Pathway Reverses the Resistance

    Directory of Open Access Journals (Sweden)

    Wenfeng Zhang

    2017-08-01

    Full Text Available Background/Aims: CyclinG1 (CycG1 is frequently overexpressed in solid tumors and overexpression of CycG1 promotes cell survival upon paclitaxel exposure by inducing polyploidy. Whether and how CycG1 regulates polyploidization caused by small molecular targeted inhibitors remains unclear. Methods: Immunohistochemistry and immunoblotting were utilized to examine protein expression. Cell proliferation was measured by ATPlite assay, and cell cycle distribution and apoptosis were measured by flow cytometry and/or DNA fragmentation assays. Results: Overexpression of CycG1 in breast cancer cells caused apoptosis-resistant polyploidy upon treatment with Aurora kinase inhibitor, ZM447439 (ZM. Addition of ABT-263, a small-molecule BH3 mimetic, to ZM, produced a synergistic loss of cell viability with greater sustained tumor growth inhibition in breast cancer cell lines. Decrease of Mcl-1 and increase of NOXA caused by ZM treatment, were responsible for the synergy. Furthermore, CycG1 was highly expressed in Triple-Negative-Breast-Cancer patients treated with paclitaxel and was paralleled by decreased cell survival. Conclusion: CycG1 is a crucial factor in ZM-induced polyploidy resistance, and ABT-263/ZM combination hold therapeutic utility in the CycG1-amplified subset of breast cancer and CycG1, thus, is a promising target in breast cancer.

  10. Transmitted drug resistance in the CFAR network of integrated clinical systems cohort: prevalence and effects on pre-therapy CD4 and viral load.

    Directory of Open Access Journals (Sweden)

    Art F Y Poon

    Full Text Available Human immunodeficiency virus type 1 (HIV-1 genomes often carry one or more mutations associated with drug resistance upon transmission into a therapy-naïve individual. We assessed the prevalence and clinical significance of transmitted drug resistance (TDR in chronically-infected therapy-naïve patients enrolled in a multi-center cohort in North America. Pre-therapy clinical significance was quantified by plasma viral load (pVL and CD4+ cell count (CD4 at baseline. Naïve bulk sequences of HIV-1 protease and reverse transcriptase (RT were screened for resistance mutations as defined by the World Health Organization surveillance list. The overall prevalence of TDR was 14.2%. We used a Bayesian network to identify co-transmission of TDR mutations in clusters associated with specific drugs or drug classes. Aggregate effects of mutations by drug class were estimated by fitting linear models of pVL and CD4 on weighted sums over TDR mutations according to the Stanford HIV Database algorithm. Transmitted resistance to both classes of reverse transcriptase inhibitors was significantly associated with lower CD4, but had opposing effects on pVL. In contrast, position-specific analyses of TDR mutations revealed substantial effects on CD4 and pVL at several residue positions that were being masked in the aggregate analyses, and significant interaction effects as well. Residue positions in RT with predominant effects on CD4 or pVL (D67 and M184 were re-evaluated in causal models using an inverse probability-weighting scheme to address the problem of confounding by other mutations and demographic or risk factors. We found that causal effect estimates of mutations M184V/I (-1.7 log₁₀pVL and D67N/G (-2.1[³√CD4] and 0.4 log₁₀pVL were compensated by K103N/S and K219Q/E/N/R. As TDR becomes an increasing dilemma in this modern era of highly-active antiretroviral therapy, these results have immediate significance for the clinical management of HIV-1

  11. A single amino acid substitution in the core protein of West Nile virus increases resistance to acidotropic compounds.

    Directory of Open Access Journals (Sweden)

    Miguel A Martín-Acebes

    Full Text Available West Nile virus (WNV is a worldwide distributed mosquito-borne flavivirus that naturally cycles between birds and mosquitoes, although it can infect multiple vertebrate hosts including horses and humans. This virus is responsible for recurrent epidemics of febrile illness and encephalitis, and has recently become a global concern. WNV requires to transit through intracellular acidic compartments at two different steps to complete its infectious cycle. These include fusion between the viral envelope and the membrane of endosomes during viral entry, and virus maturation in the trans-Golgi network. In this study, we followed a genetic approach to study the connections between viral components and acidic pH. A WNV mutant with increased resistance to the acidotropic compound NH4Cl, which blocks organelle acidification and inhibits WNV infection, was selected. Nucleotide sequencing revealed that this mutant displayed a single amino acid substitution (Lys 3 to Glu on the highly basic internal capsid or core (C protein. The functional role of this replacement was confirmed by its introduction into a WNV infectious clone. This single amino acid substitution also increased resistance to other acidification inhibitor (concanamycin A and induced a reduction of the neurovirulence in mice. Interestingly, a naturally occurring accompanying mutation found on prM protein abolished the resistant phenotype, supporting the idea of a genetic crosstalk between the internal C protein and the external glycoproteins of the virion. The findings here reported unveil a non-previously assessed connection between the C viral protein and the acidic pH necessary for entry and proper exit of flaviviruses.

  12. A single amino acid substitution in the core protein of West Nile virus increases resistance to acidotropic compounds.

    Science.gov (United States)

    Martín-Acebes, Miguel A; Blázquez, Ana-Belén; de Oya, Nereida Jiménez; Escribano-Romero, Estela; Shi, Pei-Yong; Saiz, Juan-Carlos

    2013-01-01

    West Nile virus (WNV) is a worldwide distributed mosquito-borne flavivirus that naturally cycles between birds and mosquitoes, although it can infect multiple vertebrate hosts including horses and humans. This virus is responsible for recurrent epidemics of febrile illness and encephalitis, and has recently become a global concern. WNV requires to transit through intracellular acidic compartments at two different steps to complete its infectious cycle. These include fusion between the viral envelope and the membrane of endosomes during viral entry, and virus maturation in the trans-Golgi network. In this study, we followed a genetic approach to study the connections between viral components and acidic pH. A WNV mutant with increased resistance to the acidotropic compound NH4Cl, which blocks organelle acidification and inhibits WNV infection, was selected. Nucleotide sequencing revealed that this mutant displayed a single amino acid substitution (Lys 3 to Glu) on the highly basic internal capsid or core (C) protein. The functional role of this replacement was confirmed by its introduction into a WNV infectious clone. This single amino acid substitution also increased resistance to other acidification inhibitor (concanamycin A) and induced a reduction of the neurovirulence in mice. Interestingly, a naturally occurring accompanying mutation found on prM protein abolished the resistant phenotype, supporting the idea of a genetic crosstalk between the internal C protein and the external glycoproteins of the virion. The findings here reported unveil a non-previously assessed connection between the C viral protein and the acidic pH necessary for entry and proper exit of flaviviruses.

  13. Allosteric small-molecule kinase inhibitors

    DEFF Research Database (Denmark)

    Wu, Peng; Clausen, Mads Hartvig; Nielsen, Thomas E.

    2015-01-01

    current barriers of kinase inhibitors, including poor selectivity and emergence of drug resistance. In spite of the small number of identified allosteric inhibitors in comparison with that of inhibitors targeting the ATP pocket, encouraging results, such as the FDA-approval of the first small...

  14. Integrase inhibitors in late pregnancy and rapid HIV viral load reduction.

    Science.gov (United States)

    Rahangdale, Lisa; Cates, Jordan; Potter, JoNell; Badell, Martina L; Seidman, Dominika; Miller, Emilly S; Coleman, Jenell S; Lazenby, Gweneth B; Levison, Judy; Short, William R; Yawetz, Sigal; Ciaranello, Andrea; Livingston, Elizabeth; Duthely, Lunthita; Rimawi, Bassam H; Anderson, Jean R; Stringer, Elizabeth M

    2016-03-01

    Minimizing time to HIV viral suppression is critical in pregnancy. Integrase strand transfer inhibitors (INSTIs), like raltegravir, are known to rapidly suppress plasma HIV RNA in nonpregnant adults. There are limited data in pregnant women. We describe time to clinically relevant reduction in HIV RNA in pregnant women using INSTI-containing and non-INSTI-containing antiretroviral therapy (ART) options. We conducted a retrospective cohort study of pregnant HIV-infected women in the United States from 2009 through 2015. We included women who initiated ART, intensified their regimen, or switched to a new regimen due to detectable viremia (HIV RNA >40 copies/mL) at ≥20 weeks gestation. Among women with a baseline HIV RNA permitting 1-log reduction, we estimated time to 1-log RNA reduction using the Kaplan-Meier estimator comparing women starting/adding an INSTI in their regimen vs other ART. To compare groups with similar follow-up time, we also conducted a subgroup analysis limited to women with ≤14 days between baseline and follow-up RNA data. This study describes 101 HIV-infected pregnant women from 11 US clinics. In all, 75% (76/101) of women were not taking ART at baseline; 24 were taking non-INSTI containing ART, and 1 received zidovudine monotherapy. In all, 39% (39/101) of women started an INSTI-containing regimen or added an INSTI to their ART regimen. Among 90 women with a baseline HIV RNA permitting 1-log reduction, the median time to 1-log RNA reduction was 8 days (interquartile range [IQR], 7-14) in the INSTI group vs 35 days (IQR, 20-53) in the non-INSTI ART group (P pregnancy. Inclusion of an INSTI may play a role in optimal reduction of HIV RNA for HIV-infected pregnant women presenting late to care or failing initial therapy. Larger studies are urgently needed to assess the safety and effectiveness of this approach. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Alternative alkali resistant deNO{sub x} technologies. Appendix 1

    Energy Technology Data Exchange (ETDEWEB)

    Putluru, S.S.R.; Degn Jensen, A.

    2011-07-01

    The increased use of biomass as fuel has created some new challenges to establish SCR flue gas treatment technology. One of these challenges comes from biomass complex chemical composition, which includes potassium shown to have a negative impact on the SCR catalyst. Studies have shown that potassium deactivates SCR catalyst and reduces its ability to reduce NO to N{sub 2}. An attempt was made to protect the SCR catalyst from alkali poisoning by the imposition of a coating on the catalyst surface. Various compounds were coated on a commercial catalyst supplied by Haldor Topsoee A/S and tested for alkali poisoning resistance. These materials were broadly divided as metal oxides, zeolites and other materials. The coated catalysts were exposed to potassium chloride aerosols at 350 deg. C for 650-1200 h. SCR activity, SEM and EDX measurements were performed to analyze the coated catalysts resistance to potassium poisoning. Coated catalysts (Mg, Mg containing compounds and Zeolites) showed appreciable alkali resistivity compared to the uncoated reference catalyst. Coated catalysts showed high potassium concentration at the surface of the coating and low potassium concentration across the cross section when compared to the uncoated reference catalyst. Thus, it is assumed that the coating layer accumulates the potassium at the surface and prevents to penetrate through the catalyst. The overall assessment is that it is possible to protect an SCR catalyst from potassium poisoning by the imposition of coating layer. (Author)

  16. Viral pneumonias: Typical and atypical findings

    International Nuclear Information System (INIS)

    Westhoff-Bleck, M.; Bleck, J.S.; Schirg, E.

    1987-01-01

    The clinical and radiological features of viral pneumonias are summarized and discussed. Although viral infections of the lung belong to atypical pneumonias they demonstrate not always the radiographic pattern of an interstitial pneumonia. Characteristic radiographic findings are quite rare. In most cases the microbial etiology cannot be predicted from chest radiographs. The appearance varies depending on the virulence of the organism and the resistence of the host. In this regard knowledge of epidemiological data as well as patients condition and underlying disease is of utmost importance. Differentiation between community- and hospital-acquired infection may be very helpful. (orig.) [de

  17. Effects of low energy helium plasma irradiation on potassium doped tungsten

    Energy Technology Data Exchange (ETDEWEB)

    Shu, Xiaoyan [Key Laboratory of Radiation Physics and Technology of Ministry of Education, Institute of Nuclear Science and Technology, Sichuan University, Chengdu (China); Key Subject Laboratory of National Defense for Radioactive Waste and Environmental Security, Southwest University of Science and Technology, Mianyang (China); Huang, Bo [Key Laboratory of Radiation Physics and Technology of Ministry of Education, Institute of Nuclear Science and Technology, Sichuan University, Chengdu (China); Liu, Dongping; Fan, Hongyu [School of Physics and Materials Engineering, Dalian Nationalities University, Dalian (China); Liu, Ning [Key Laboratory of Radiation Physics and Technology of Ministry of Education, Institute of Nuclear Science and Technology, Sichuan University, Chengdu (China); Tang, Jun, E-mail: tangjun@scu.edu.cn [Key Laboratory of Radiation Physics and Technology of Ministry of Education, Institute of Nuclear Science and Technology, Sichuan University, Chengdu (China)

    2017-04-15

    Effects of helium plasma irradiation on spark plasma sintering (SPS) W-K, pure W and traditionally sintered commercial W-K have been studied, concerning the density, grain size and potassium content as the influence factors. Pinholes are formed under 120 eV He ions at 600 °C and 1 × 10{sup 23} m{sup −2} fluence on the surface of all samples. It is found that SPS-sintered W-K shows the best irradiation resistance among the present samples, and SPS-sintered pure W exhibits higher irradiation tolerance than commercial W-K. Different He-plasma tolerance was observed among the SPS-sintered W-K samples due to varied potassium content and grain size. In addition, the microstructure evolution under helium irradiation, the growth-migration of helium bubbles and their interactions of potassium bubbles have also been discussed.

  18. Expansion of Viral Load Testing and the Potential Impact on HIV Drug Resistance.

    Science.gov (United States)

    Raizes, Elliot; Hader, Shannon; Birx, Deborah

    2017-12-01

    The US President's Emergency Plan for AIDS Relief (PEPFAR) supports aggressive scale-up of antiretroviral therapy (ART) in high-burden countries and across all genders and populations at risk toward global human immunodeficiency virus (HIV) epidemic control. PEPFAR recognizes the risk of HIV drug resistance (HIVDR) as a consequence of aggressive ART scale-up and is actively promoting 3 key steps to mitigate the impact of HIVDR: (1) routine access to routine viral load monitoring in all settings; (2) optimization of ART regimens; and (3) routine collection and analysis of HIVDR data to monitor the success of mitigation strategies. The transition to dolutegravir-based regimens in PEPFAR-supported countries and the continuous evolution of HIVDR surveillance strategies are essential elements of PEPFAR implementation. Published by Oxford University Press for the Infectious Diseases Society of America 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  19. NK cell receptor/H2-Dk-dependent host resistance to viral infection is quantitatively modulated by H2q inhibitory signals.

    Science.gov (United States)

    Fodil-Cornu, Nassima; Loredo-Osti, J Concepción; Vidal, Silvia M

    2011-04-01

    The cytomegalovirus resistance locus Cmv3 has been linked to an epistatic interaction between two loci: a Natural Killer (NK) cell receptor gene and the major histocompatibility complex class I (MHC-I) locus. To demonstrate the interaction between Cmv3 and H2(k), we generated double congenic mice between MA/My and BALB.K mice and an F(2) cross between FVB/N (H-2(q)) and BALB.K (H2(k)) mice, two strains susceptible to mouse cytomegalovirus (MCMV). Only mice expressing H2(k) in conjunction with Cmv3(MA/My) or Cmv3(FVB) were resistant to MCMV infection. Subsequently, an F(3) cross was carried out between transgenic FVB/H2-D(k) and MHC-I deficient mice in which only the progeny expressing Cmv3(FVB) and a single H2-D(k) class-I molecule completely controlled MCMV viral loads. This phenotype was shown to be NK cell-dependent and associated with subsequent NK cell proliferation. Finally, we demonstrated that a number of H2(q) alleles influence the expression level of H2(q) molecules, but not intrinsic functional properties of NK cells; viral loads, however, were quantitatively proportional to the number of H2(q) alleles. Our results support a model in which H-2(q) molecules convey Ly49-dependent inhibitory signals that interfere with the action of H2-D(k) on NK cell activation against MCMV infection. Thus, the integration of activating and inhibitory signals emanating from various MHC-I/NK cell receptor interactions regulates NK cell-mediated control of viral load.

  20. Application of virus-like particles (VLP) to NMR characterization of viral membrane protein interactions

    Energy Technology Data Exchange (ETDEWEB)

    Antanasijevic, Aleksandar; Kingsley, Carolyn [University of Illinois at Chicago, Department of Biochemistry and Molecular Genetics (United States); Basu, Arnab; Bowlin, Terry L. [Microbiotix Inc. (United States); Rong, Lijun [University of Illinois at Chicago, Department of Microbiology and Immunology (United States); Caffrey, Michael, E-mail: caffrey@uic.edu [University of Illinois at Chicago, Department of Biochemistry and Molecular Genetics (United States)

    2016-03-15

    The membrane proteins of viruses play critical roles in the virus life cycle and are attractive targets for therapeutic intervention. Virus-like particles (VLP) present the possibility to study the biochemical and biophysical properties of viral membrane proteins in their native environment. Specifically, the VLP constructs contain the entire protein sequence and are comprised of native membrane components including lipids, cholesterol, carbohydrates and cellular proteins. In this study we prepare VLP containing full-length hemagglutinin (HA) or neuraminidase (NA) from influenza and characterize their interactions with small molecule inhibitors. Using HA-VLP, we first show that VLP samples prepared using the standard sucrose gradient purification scheme contain significant amounts of serum proteins, which exhibit high potential for non-specific interactions, thereby complicating NMR studies of ligand-target interactions. We then show that the serum contaminants may be largely removed with the addition of a gel filtration chromatography step. Next, using HA-VLP we demonstrate that WaterLOGSY NMR is significantly more sensitive than Saturation Transfer Difference (STD) NMR for the study of ligand interactions with membrane bound targets. In addition, we compare the ligand orientation to HA embedded in VLP with that of recombinant HA by STD NMR. In a subsequent step, using NA-VLP we characterize the kinetic and binding properties of substrate analogs and inhibitors of NA, including study of the H274Y-NA mutant, which leads to wide spread resistance to current influenza antivirals. In summary, our work suggests that VLP have high potential to become standard tools in biochemical and biophysical studies of viral membrane proteins, particularly when VLP are highly purified and combined with control VLP containing native membrane proteins.

  1. The anti-apoptotic BAG3 protein is involved in BRAF inhibitor resistance in melanoma cells.

    Science.gov (United States)

    Guerriero, Luana; Palmieri, Giuseppe; De Marco, Margot; Cossu, Antonio; Remondelli, Paolo; Capunzo, Mario; Turco, Maria Caterina; Rosati, Alessandra

    2017-10-06

    BAG3 protein, a member of BAG family of co-chaperones, has a pro-survival role in several tumour types. BAG3 anti-apoptotic properties rely on its characteristic to bind several intracellular partners, thereby modulating crucial events such as apoptosis, differentiation, cell motility, and autophagy. In human melanomas, BAG3 positivity is correlated with the aggressiveness of the tumour cells and can sustain IKK-γ levels, allowing a sustained activation of NF-κB. Furthermore, BAG3 is able to modulate BRAFV600E levels and activity in thyroid carcinomas. BRAFV600E is the most frequent mutation detected in malignant melanomas and is targeted by Vemurafenib, a specific inhibitor found to be effective in the treatment of advanced melanoma. However, patients with BRAF-mutated melanoma may result insensitive ab initio or, mostly, develop acquired resistance to the treatment with this molecule. Here we show that BAG3 down-modulation interferes with BRAF levels in melanoma cells and sensitizes them to Vemurafenib treatment. Furthermore, the down-modulation of BAG3 protein in an in vitro model of acquired resistance to Vemurafenib can induce sensitization to the BRAFV600E specific inhibition by interfering with BRAF pathway through reduction of ERK phosphorylation, but also on parallel survival pathways. Future studies on BAG3 molecular interactions with key proteins responsible of acquired BRAF inhibitor resistance may represent a promising field for novel multi-drugs treatment design.

  2. Targeting the Warburg effect with a novel glucose transporter inhibitor to overcome gemcitabine resistance in pancreatic cancer cells

    Science.gov (United States)

    Lai, I-Lu; Chou, Chih-Chien; Lai, Po-Ting; Fang, Chun-Sheng; Shirley, Lawrence A.; Yan, Ribai; Mo, Xiaokui; Bloomston, Mark; Kulp, Samuel K.; Bekaii-Saab, Tanios; Chen, Ching-Shih

    2014-01-01

    Gemcitabine resistance remains a significant clinical challenge. Here, we used a novel glucose transporter (Glut) inhibitor, CG-5, as a proof-of-concept compound to investigate the therapeutic utility of targeting the Warburg effect to overcome gemcitabine resistance in pancreatic cancer. The effects of gemcitabine and/or CG-5 on viability, survival, glucose uptake and DNA damage were evaluated in gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer cell lines. Mechanistic studies were conducted to determine the molecular basis of gemcitabine resistance and the mechanism of CG-5-induced sensitization to gemcitabine. The effects of CG-5 on gemcitabine sensitivity were investigated in a xenograft tumor model of gemcitabine-resistant pancreatic cancer. In contrast to gemcitabine-sensitive pancreatic cancer cells, the resistant Panc-1 and Panc-1GemR cells responded to gemcitabine by increasing the expression of ribonucleotide reductase M2 catalytic subunit (RRM2) through E2F1-mediated transcriptional activation. Acting as a pan-Glut inhibitor, CG-5 abrogated this gemcitabine-induced upregulation of RRM2 through decreased E2F1 expression, thereby enhancing gemcitabine-induced DNA damage and inhibition of cell survival. This CG-5-induced inhibition of E2F1 expression was mediated by the induction of a previously unreported E2F1-targeted microRNA, miR-520f. The addition of oral CG-5 to gemcitabine therapy caused greater suppression of Panc-1GemR xenograft tumor growth in vivo than either drug alone. Glut inhibition may be an effective strategy to enhance gemcitabine activity for the treatment of pancreatic cancer. PMID:24879635

  3. Novel benzoxazole inhibitor of dengue virus replication that targets the NS3 helicase.

    Science.gov (United States)

    Byrd, Chelsea M; Grosenbach, Douglas W; Berhanu, Aklile; Dai, Dongcheng; Jones, Kevin F; Cardwell, Kara B; Schneider, Christine; Yang, Guang; Tyavanagimatt, Shanthakumar; Harver, Chris; Wineinger, Kristin A; Page, Jessica; Stavale, Eric; Stone, Melialani A; Fuller, Kathleen P; Lovejoy, Candace; Leeds, Janet M; Hruby, Dennis E; Jordan, Robert

    2013-04-01

    Dengue virus (DENV) is the predominant mosquito-borne viral pathogen that infects humans with an estimated 50 to 100 million infections per year worldwide. Over the past 50 years, the incidence of dengue disease has increased dramatically and the virus is now endemic in more than 100 countries. Moreover, multiple serotypes of DENV are now found in the same geographic region, increasing the likelihood of more severe forms of disease. Despite extensive research, there are still no approved vaccines or therapeutics commercially available to treat DENV infection. Here we report the results of a high-throughput screen of a chemical compound library using a whole-virus assay that identified a novel small-molecule inhibitor of DENV, ST-610, that potently and selectively inhibits all four serotypes of DENV replication in vitro. Sequence analysis of drug-resistant virus isolates has identified a single point mutation, A263T, in the NS3 helicase domain that confers resistance to this compound. ST-610 inhibits DENV NS3 helicase RNA unwinding activity in a molecular-beacon-based helicase assay but does not inhibit nucleoside triphosphatase activity based on a malachite green ATPase assay. ST-610 is nonmutagenic, is well tolerated (nontoxic) in mice, and has shown efficacy in a sublethal murine model of DENV infection with the ability to significantly reduce viremia and viral load compared to vehicle controls.

  4. Genetic basis of Spodoptera frugiperda (Lepidoptera: Noctuidae) resistance to the chitin synthesis inhibitor lufenuron.

    Science.gov (United States)

    do Nascimento, Antonio Rogério Bezerra; Farias, Juliano Ricardo; Bernardi, Daniel; Horikoshi, Renato Jun; Omoto, Celso

    2016-04-01

    An understanding of the genetic basis of insect resistance to insecticides is important for the establishment of insect resistance management (IRM) strategies. In this study we evaluated the inheritance pattern of resistance to the chitin synthesis inhibitor lufenuron in Spodoptera frugiperda. The LC50 values (95% CI) were 0.23 µg lufenuron mL(-1) water (ppm) (0.18-0.28) for the susceptible strain (SUS) and 210.6 µg mL(-1) (175.90-258.10) for the lufenuron-resistant strain (LUF-R), based on diet-overlay bioassay. The resistance ratio was ≈ 915-fold. The LC50 values for reciprocal crosses were 4.89 µg mL(-1) (3.79-5.97) for female LUF-R and male SUS and 5.74 µg mL(-1) (4.70-6.91) for female SUS and male LUF-R, indicating that the inheritance of S. frugiperda resistance to lufenuron is an autosomal, incompletely recessive trait. Backcrosses of the progeny of reciprocal crosses with the parental LUF-R showed a polygenic effect. The estimated minimum number of independent segregations was in the 11.02 range, indicating that resistance to lufenuron is associated with multiple genes in S. frugiperda. Based on genetic crosses, the inheritance pattern of lufenuron resistance in S. frugiperda was autosomal, incompletely recessive and polygenic. Implications of this finding to IRM are discussed in this paper. © 2015 Society of Chemical Industry.

  5. Handling of potassium

    International Nuclear Information System (INIS)

    Schwarz, N.; Komurka, M.

    1983-03-01

    As a result for the Fast Breeder Development extensive experience is available worldwide with respect to Sodium technology. Due to the extension of the research program to topping cycles with Potassium as the working medium, test facilities with Potassium have been designed and operated in the Institute of Reactor Safety. The different chemical properties of Sodium and Potassium give rise in new safety concepts and operating procedures. The handling problems of Potassium are described in the light of theoretical properties and own experiences. Selected literature on main safety and operating problems complete this report. (Author) [de

  6. The inhibitory effects of potassium chloride versus potassium silicate application on 137Cs uptake by rice

    International Nuclear Information System (INIS)

    Fujimura, Shigeto; Yoshioka, Kunio; Ota, Takeshi; Ishikawa, Tetsuya; Sato, Makoto; Satou, Mutsuto

    2016-01-01

    After the accident at the Fukushima Dai-ichi Nuclear Power Plant owned by the Tokyo Electric Power Company on 11 March 2011, potassium fertilizer was applied to agricultural fields in the southern Tohoku and northern Kanto regions of Japan to reduce the uptake of radiocesium by crops. In this study, we examined the effects of two types of potassium fertilizers, potassium chloride (a readily available potassium fertilizer) and potassium silicate (a slow-release potassium fertilizer), as well as a split application of potassium, on the accumulation of 137 Cs by rice plants in two pot experiments. The 137 Cs concentrations in the brown rice and in the above-ground plants were significantly lower after potassium chloride application than after potassium silicate application. The potassium ion (K + ) concentrations in soil solutions sampled 9 and 21 d after transplanting were significantly higher for the potassium chloride application than for the potassium silicate application. The K + concentrations in soil solutions observed in the application of potassium silicate were similar to those in the treatment when no potassium was applied. This finding indicates that the application of potassium silicate did not sufficiently increase the available K + for rice plants in the soil, which led to a greater uptake of 137 Cs after the potassium silicate application than after the application of potassium chloride. The 137 Cs concentration in brown rice was higher in the split application of potassium fertilizer with the second application at the full heading stage than that without split application and the split application with the second application before heading. - Highlights: • Potassium application reduced 137 Cs uptake by rice grown in pot experiments. • Readily available K fertilizer more effectively decreased brown rice 137 Cs concentration. • Potassium should be applied before heading to reduce brown rice 137 Cs concentration.

  7. Evaluation of candidate biomarkers to predict cancer cell sensitivity or resistance to PARP-1 inhibitor treatment

    DEFF Research Database (Denmark)

    Oplustilova, L.; Wolanin, K.; Bartkova, J.

    2012-01-01

    combinations with camptothecin or ionizing radiation. Furthermore, monitoring pARsylation and Rad51 foci formation as surrogate markers for PARP activity and HR, respectively, supported their candidacy for biomarkers of PARP-1i responses. As to resistance mechanisms, we confrmed the role of the multidrug......(ADp-ribose) polymerase-1 (PARP-1), an enzyme critical for repair pathways alternative to HR. While promising, treatment with PARP-1 inhibitors (PARP-1i) faces some hurdles, including (1) acquired resistance, (2) search for other sensitizing, non-BRCA1/2 cancer defects and (3) lack of biomarkers to predict response...

  8. Rilpivirine: a new non-nucleoside reverse transcriptase inhibitor.

    Science.gov (United States)

    Sharma, Mamta; Saravolatz, Louis D

    2013-02-01

    Rilpivirine is a new non-nucleoside reverse transcriptase inhibitor (NNRTI) that is approved for HIV-1 treatment-naive adult patients in combination with other antiretroviral agents. The recommended dose is a 25 mg tablet once daily taken orally with a meal. Due to cytochrome P450 3A4 enzyme induction or gastric pH increase, rilpivirine cannot be coadministered with a number of other drugs (anticonvulsants, rifabutin, rifampicin, rifapentine, proton pump inhibitors, systemic dexamethasone and St John's wort). Rilpivirine should be used with caution when coadministered with a drug with a known risk for torsade de pointes. Rilpivirine has a better tolerability than a comparative NNRTI, efavirenz, in clinical trials, with fewer central nervous system adverse effects, rashes, lipid abnormalities and discontinuation rates. Virological failure occurs more commonly with higher baseline viral loads (>100,000 copies/mL) and lower baseline CD4 counts (<50 cells/mm(3)). Seventeen NNRTI mutations have been associated with decreased susceptibility to rilpivirine: K101E/P, E138A/G/K/Q/R, V179L, Y181C/I/V, H221Y, F227C, M230I/L, Y188L and the combination L100I + K103N. Resistance to rilpivirine largely excludes future use of the NNRTI class.

  9. In Vitro Antiviral Activity and Resistance Profile Characterization of the Hepatitis C Virus NS5A Inhibitor Ledipasvir.

    Science.gov (United States)

    Cheng, Guofeng; Tian, Yang; Doehle, Brian; Peng, Betty; Corsa, Amoreena; Lee, Yu-Jen; Gong, Ruoyu; Yu, Mei; Han, Bin; Xu, Simin; Dvory-Sobol, Hadas; Perron, Michel; Xu, Yili; Mo, Hongmei; Pagratis, Nikos; Link, John O; Delaney, William

    2016-01-11

    Ledipasvir (LDV; GS-5885), a component of Harvoni (a fixed-dose combination of LDV with sofosbuvir [SOF]), is approved to treat chronic hepatitis C virus (HCV) infection. Here, we report key preclinical antiviral properties of LDV, including in vitro potency, in vitro resistance profile, and activity in combination with other anti-HCV agents. LDV has picomolar antiviral activity against genotype 1a and genotype 1b replicons with 50% effective concentration (EC50) values of 0.031 nM and 0.004 nM, respectively. LDV is also active against HCV genotypes 4a, 4d, 5a, and 6a with EC50 values of 0.11 to 1.1 nM. LDV has relatively less in vitro antiviral activity against genotypes 2a, 2b, 3a, and 6e, with EC50 values of 16 to 530 nM. In vitro resistance selection with LDV identified the single Y93H and Q30E resistance-associated variants (RAVs) in the NS5A gene; these RAVs were also observed in patients after a 3-day monotherapy treatment. In vitro antiviral combination studies indicate that LDV has additive to moderately synergistic antiviral activity when combined with other classes of HCV direct-acting antiviral (DAA) agents, including NS3/4A protease inhibitors and the nucleotide NS5B polymerase inhibitor SOF. Furthermore, LDV is active against known NS3 protease and NS5B polymerase inhibitor RAVs with EC50 values equivalent to those for the wild type. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  10. Effect of Functional Bread Rich in Potassium, γ-Aminobutyric Acid and Angiotensin-Converting Enzyme Inhibitors on Blood Pressure, Glucose Metabolism and Endothelial Function

    Science.gov (United States)

    Becerra-Tomás, Nerea; Guasch-Ferré, Marta; Quilez, Joan; Merino, Jordi; Ferré, Raimon; Díaz-López, Andrés; Bulló, Mònica; Hernández-Alonso, Pablo; Palau-Galindo, Antoni; Salas-Salvadó, Jordi

    2015-01-01

    Abstract Because it has been suggested that food rich in γ-aminobutyric acid (GABA) or angiotensin-converting enzyme inhibitor (ACEI) peptides have beneficial effects on blood pressure (BP) and other cardiovascular risk factors, we tested the effects of low-sodium bread, but rich in potassium, GABA, and ACEI peptides on 24-hour BP, glucose metabolism, and endothelial function. A randomized, double-blind, crossover trial was conducted in 30 patients with pre or mild-to-moderate hypertension, comparing three 4-week nutritional interventions separated by 2-week washout periods. Patients were randomly assigned to consume 120 g/day of 1 of the 3 types of bread for each nutritional intervention: conventional wheat bread (CB), low-sodium wheat bread enriched in potassium (LSB), and low-sodium wheat bread rich in potassium, GABA, and ACEI peptides (LSB + G). For each period, 24-hour BP measurements, in vivo endothelial function, and biochemical samples were obtained. After LSB + G consumption, 24-hour ambulatory BP underwent a nonsignificant greater reduction than after the consumption of CB and LSB (0.26 mm Hg in systolic BP and −0.63 mm Hg in diastolic BP for CB; −0.71 mm Hg in systolic BP and −1.08 mm Hg in diastolic BP for LSB; and −0.75 mm Hg in systolic BP and −2.12 mm Hg in diastolic BP for LSB + G, respectively). Diastolic BP at rest decreased significantly during the LSB + G intervention, although there were no significant differences in changes between interventions. There were no significant differences between interventions in terms of changes in in vivo endothelial function, glucose metabolism, and peripheral inflammatory parameters. Compared with the consumption of CB or LSB, no greater beneficial effects on 24-hour BP, endothelial function, or glucose metabolism were demonstrated after the consumption of LSB + G in a population with pre or mild-to-moderate hypertension. Further studies are warranted to clarify the

  11. The inhibitory effects of potassium chloride versus potassium silicate application on (137)Cs uptake by rice.

    Science.gov (United States)

    Fujimura, Shigeto; Yoshioka, Kunio; Ota, Takeshi; Ishikawa, Tetsuya; Sato, Makoto; Satou, Mutsuto

    2016-03-01

    After the accident at the Fukushima Dai-ichi Nuclear Power Plant owned by the Tokyo Electric Power Company on 11 March 2011, potassium fertilizer was applied to agricultural fields in the southern Tohoku and northern Kanto regions of Japan to reduce the uptake of radiocesium by crops. In this study, we examined the effects of two types of potassium fertilizers, potassium chloride (a readily available potassium fertilizer) and potassium silicate (a slow-release potassium fertilizer), as well as a split application of potassium, on the accumulation of (137)Cs by rice plants in two pot experiments. The (137)Cs concentrations in the brown rice and in the above-ground plants were significantly lower after potassium chloride application than after potassium silicate application. The potassium ion (K(+)) concentrations in soil solutions sampled 9 and 21 d after transplanting were significantly higher for the potassium chloride application than for the potassium silicate application. The K(+) concentrations in soil solutions observed in the application of potassium silicate were similar to those in the treatment when no potassium was applied. This finding indicates that the application of potassium silicate did not sufficiently increase the available K(+) for rice plants in the soil, which led to a greater uptake of (137)Cs after the potassium silicate application than after the application of potassium chloride. The (137)Cs concentration in brown rice was higher in the split application of potassium fertilizer with the second application at the full heading stage than that without split application and the split application with the second application before heading. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. HIV-1 drug resistance prevalence, drug susceptibility and variant characterization in the Jacobi Medical Center paediatric cohort, Bronx, NY, USA.

    Science.gov (United States)

    de Mulder, M; York, V A; Wiznia, A A; Michaud, H A; Nixon, D F; Holguin, A; Rosenberg, M G

    2014-03-01

    With the advent of combined antiretroviral therapy (cART), perinatally HIV-infected children are surviving into adolescence and beyond. However, drug resistance mutations (DRMs) compromise viral control, affecting the long-term effectiveness of ART. The aims of this study were to detect and identify DRMs in a HIV-1 infected paediatric cohort. Paired plasma and dried blood spots (DBSs) specimens were obtained from HIV-1 perinatally infected patients attending the Jacobi Medical Center, New York, USA. Clinical, virological and immunological data for these patients were analysed. HIV-1 pol sequences were generated from samples to identify DRMs according to the International AIDS Society (IAS) 2011 list. Forty-seven perinatally infected patients were selected, with a median age of 17.7 years, of whom 97.4% were carrying subtype B. They had a mean viral load of 3143 HIV-1 RNA copies/mL and a mean CD4 count of 486 cells/μL at the time of sampling. Nineteen patients (40.4%) had achieved undetectable viraemia (40.5% had a CD4 count of > 500 cells/μL. Most of the patients (97.9%) had received cART, including protease inhibitor (PI)-based regimens in 59.6% of cases. The DRM prevalence was 54.1, 27.6 and 27.0% for nucleoside reverse transcriptase inhibitors (NRTIs), PIs and nonnucleoside reverse transcriptase inhibitors (NNRTIs), respectively. Almost two-thirds (64.9%) of the patients harboured DRMs to at least one drug class and 5.4% were triple resistant. The mean nucleotide similarity between plasma and DBS sequences was 97.9%. Identical DRM profiles were present in 60% of plasma-DBS paired sequences. A total of 30 DRMs were detected in plasma and 26 in DBSs, with 23 present in both. Although more perinatally HIV-1-infected children are reaching adulthood as a result of advances in cART, our study cohort presented a high prevalence of resistant viruses, especially viruses resistant to NRTIs. DBS specimens can be used for DRM detection. © 2013 British HIV Association.

  13. Influence of antihypertensive therapy, sodium intake and the concentration of potassium in plasma on concentration of aldosterone and plasma renin activity

    Directory of Open Access Journals (Sweden)

    Lalić Tijana

    2013-01-01

    Full Text Available Introduction: Primary aldosteronism (PA is a group of disorders which are characterized by inadequate and non-suppressible production of aldosterone. The prevalence of PA is increasing in hypertensive population. The golden standard of screening for primary aldosteronism, determination of aldosterone/plasma renin activity (ARR, is influenced by numerous exogenous and endogenous factors. Testing cannot always be conducted under optimal conditions. Objective: To determine influence of antihypertensive drugs and concentrations of potassium and sodium in blood and urine on values of aldosterone and plasma renin activity. Methods: In this retrospective study, we analyzed medical reports of patients admitted to Department of thyroid gland disease in the period from 2009 to 2011, with increased risk for primary aldosteronism. Body weight and height, sodium and potassium in serum and urine, plasma aldosterone concentrations and plasma renin activity, data on medicines and comorbidity were analyzed in all patients. In processing data, statistical methods descriptive analysis, Student T test and univariate linear regression were applied. Result: Of 137 patients, there were more patients with resistant hypertension (53,28% than with adrenal tumors (46,72%. Most patients used calcium channel blockers. Treatment with alpha blockers and calcium channel blockers does not influence ARR. Beta blockers and ACE inhibitors can influence ARR and diuretics and vasodilatators have definite influence. Diabetes mellitus can have higher risk of false negative results. Urine sodium excretion is significantly correlated with plasma aldosteron and serum potassium. Plasma aldosteron and PRA are significantly correlated with concentrations of electrolites in urine. Conclusion: Increased prevalence of primary aldosteronism necessitates need for accurate and better diagnostics.

  14. The Inhibition Effect of Potassium Iodide on the Corrosion of Pure Iron in Sulphuric Acid

    Directory of Open Access Journals (Sweden)

    Tarik Attar

    2014-01-01

    Full Text Available The use of inorganic inhibitors as an alternative to organic compounds is based on the possibility of degradation of organic compounds with time and temperature. The inhibition effect of potassium iodide on the corrosion of pure iron in 0.5 M H2SO4 has been studied by weight loss. It has been observed from the results that the inhibition efficiency (IE% of KI increases from 82.17% to 97.51% with the increase in inhibitor concentration from 1·10−4 to 2·10−3 M. The apparent activation energy (Ea and the equilibrium constant of adsorption (Kads were calculated. The adsorption of the inhibitor on the pure iron surface is in agreement with Langmuir adsorption isotherm.

  15. BAG3-dependent expression of Mcl-1 confers resistance of mutant KRAS colon cancer cells to the HSP90 inhibitor AUY922.

    Science.gov (United States)

    Wang, Chun Yan; Guo, Su Tang; Croft, Amanda; Yan, Xu Guang; Jin, Lei; Zhang, Xu Dong; Jiang, Chen Chen

    2018-02-01

    Past studies have shown that mutant KRAS colon cancer cells are susceptible to apoptosis induced by the HSP90 inhibitor AUY922. Nevertheless, intrinsic and acquired resistance remains an obstacle for the potential application of the inhibitor in the treatment of the disease. Here we report that Mcl-1 is important for survival of colon cancer cells in the presence of AUY922. Mcl-1 was upregulated in mutant KRAS colon cancer cells selected for resistance to AUY922-induced apoptosis. This was due to its increased stability mediated by Bcl-2-associated athanogene domain 3 (BAG3), which was also increased in resistant colon cancer cells by heat shock factor 1 (HSF1) as a result of chronic endoplasmic reticulum (ER) stress. Functional investigations demonstrated that inhibition of Mcl-1, BAG3, or HSF1 triggered apoptosis in resistant colon cancer cells, and rendered AUY922-naïve colon cancer cells more sensitive to the inhibitor. Together, these results identify that the HSF1-BAG3-Mcl-1 signal axis is critical for protection of mutant KRAS colon cancer cells from AUY922-induced apoptosis, with potential implications for targeting HSF1/BAG3/Mcl-1 to improve the efficacy of AUY922 in the treatment of colon cancer. © 2017 Wiley Periodicals, Inc.

  16. The inhibitor of volume-regulated anion channels DCPIB activates TREK potassium channels in cultured astrocytes

    Czech Academy of Sciences Publication Activity Database

    Minieri, L.; Pivoňková, Helena; Caprini, M.; Harantová, Lenka; Anděrová, Miroslava; Ferroni, S.

    2013-01-01

    Roč. 168, č. 5 (2013), s. 1240-1254 ISSN 0007-1188 R&D Projects: GA ČR GAP303/10/1338 Institutional support: RVO:68378041 Keywords : two-pore-domain potassium channels * patch clamp * neuroprotection Subject RIV: FH - Neurology Impact factor: 4.990, year: 2013

  17. Tyrosine and aurora kinase inhibitors diminish transport function of multidrug resistance-associated protein (MRP 4 and breast cancer resistance protein (BCRP

    Directory of Open Access Journals (Sweden)

    Rhiannon N. Hardwick

    2016-12-01

    Full Text Available Tyrosine and aurora kinases are important effectors in signal transduction pathways that are often involved in aberrant cancer cell growth. Tyrosine (TKI and aurora (AKI kinase inhibitors are anti-cancer agents specifically designed to target such signaling pathways through TKI/AKI binding to the ATP-binding pocket of kinases thereby leading to diminished kinase activity. Some TKIs have been identified as inhibitors of ATP-binding cassette (ABC transporters such as P-glycoprotein and breast cancer resistance protein (BCRP, which are commonly upregulated in malignant cells. TKI/AKIs have been investigated as ABC transporter inhibitors in order to facilitate the accumulation of concomitantly administered chemo-therapeutics within cancer cells. However, ABC transporters are prominently expressed in the liver and other eliminating organs, and their inhibition has been linked to intracellular accumulation of drugs, altered disposition, and toxicity. The potential for TKIs/AKIs to inhibit other important hepatic efflux transporters, particularly multidrug resistance-associated proteins (MRPs, remains unknown. The aim of the current study was to compare the inhibitory potency of 20 selected TKI/AKIs against MRP4 and BCRP through the use of inverted membrane vesicle assays. Relative IC50 values were estimated by determining TKI/AKI inhibition of MRP4-mediated [3H]-dehydroepiandrosterone sulfate uptake and BCRP-mediated [3H]-estrone sulfate uptake. To provide insight to the clinical relevance of TKI/AKI inhibition of ABC efflux transporters, the ratio of the steady-state maximum total plasma concentration (Css to the IC50 for each compound was calculated with Css/IC50 ratio >0.1 deemed potentially clinically relevant. Such analysis identified several potentially clinically relevant inhibitors of MRP4: alisertib, danusertib, erlotinib, lapatinib, neratinib, nilotinib, pazopanib, sorafenib, and tozasertib. The potentially clinically relevant inhibition of

  18. Selection of HIV resistance associated with antiretroviral therapy initiated due to pregnancy and suspended postpartum.

    Science.gov (United States)

    Ellis, Giovanina M; Huang, Sharon; Hitti, Jane; Frenkel, Lisa M

    2011-11-01

    Compare the risk of HIV drug resistance in women stopping suppressive nelfinavir (NFV)-based or Nevirapine (NVP)-based antiretroviral therapy (ART) after pregnancy. Specimens collected after stopping ART were tested for drug resistance by an oligonucleotide ligation assay and consensus sequencing. When postpartum drug resistance was detected, specimens obtained at study entry and during ART were evaluated. Sixteen of 38 women with ART-induced suppression of viral replication suspended ART postpartum. Resistance mutations were detected in 75% who stopped NFV-ART and in 50% who stopped NVP-ART. M184V, associated with Lamivudine resistance, was more frequent among those randomized to NFV-ART compared with NVP-ART (6 of 8 versus 1 of 8; P = 0.04), and nonnucleoside reverse transcriptase inhibitor resistance was detected in 4 of 8 stopping NVP-ART. HIV drug resistance was frequently observed among women who stopped suppressive NVP-ART or NFV-ART postpartum. This suggests that NFV-ART may have suboptimal potency, that staggering discontinuation of NVP-ART may be warranted, and/or ART adherence may be lax in women who choose to stop ART postpartum.

  19. High HIV-1 Diversity and Prevalence of Transmitted Drug Resistance Among Antiretroviral-Naive HIV-Infected Pregnant Women from Rio de Janeiro, Brazil.

    Science.gov (United States)

    Delatorre, Edson; Silva-de-Jesus, Carlos; Couto-Fernandez, José Carlos; Pilotto, Jose H; Morgado, Mariza G

    2017-01-01

    Antiretroviral (ARV) resistance mutations in human immunodeficiency virus type 1 (HIV-1) infection may reduce the efficacy of prophylactic therapy to prevent mother-to-child transmission (PMTCT) and future treatment options. This study evaluated the diversity and the prevalence of transmitted drug resistance (TDR) in protease (PR) and reverse transcriptase (RT) regions of HIV-1 pol gene among 87 ARV-naive HIV-1-infected pregnant women from Rio de Janeiro, Brazil, between 2012 and 2015. The viral diversity comprised HIV-1 subtypes B (67.8%), F1 (17.2%), and C (4.6%); the circulating recombinant forms 12_BF (2.3%), 28/29_BF, 39_BF, 02_AG (1.1% each) and unique recombinants forms (4.5%). The overall prevalence of any TDR was 17.2%, of which 5.7% for nucleoside RT inhibitors, 5.7% for non-nucleoside RT inhibitors, and 8% for PR inhibitors. The TDR prevalence found in this population may affect the virological outcome of the standard PMTCT ARV-regimens, reinforcing the importance of continuous monitoring.

  20. Oral potassium supplementation in surgical patients.

    Science.gov (United States)

    Hainsworth, Alison J; Gatenby, Piers A

    2008-08-01

    Hospital inpatients are frequently hypokalaemic. Low plasma potassium levels may cause life threatening complications, such as cardiac arrhythmias. Potassium supplementation may be administered parenterally or enterally. Oral potassium supplements have been associated with oesophageal ulceration, strictures and gastritis. An alternative to potassium salt tablets or solution is dietary modification with potassium rich food stuffs, which has been proven to be a safe and effective method for potassium supplementation. The potassium content of one medium banana is equivalent to a 12 mmol potassium salt tablet. Potassium supplementation by dietary modification has been shown to be equally efficacious to oral potassium salt supplementation and is preferred by the majority of patients. Subsequently, it is our practice to replace potassium using dietary modification, particularly in surgical patients having undergone oesophagogastrectomy or in those with peptic ulcer disease.

  1. Substituted 2,6-bis(benzimidazol-2-yl)pyridines: a novel chemical class of pestivirus inhibitors that targets a hot spot for inhibition of pestivirus replication in the RNA-dependent RNA polymerase.

    Science.gov (United States)

    Musiu, Simone; Pürstinger, Gerhard; Stallinger, Sylvia; Vrancken, Robert; Haegeman, Andy; Koenen, Frank; Leyssen, Pieter; Froeyen, Mathy; Neyts, Johan; Paeshuyse, Jan

    2014-06-01

    2,6-Bis(benzimidazol-2-yl)pyridine (BBP/CSFA-0) was identified in a CPE-based screening as a selective inhibitor of the in vitro bovine viral diarrhea virus (BVDV) replication. The EC50-values for the inhibition of BVDV-induced cytopathic (CPE) effect, viral RNA synthesis and the production of infectious virus were 0.3±0.1μM, 0.05±0.01μM and 0.3±0.04μM, respectively. Furthermore, BBP/CSFA-0 inhibits the in vitro replication of the classical swine fever virus (CSFV) with an EC50 of 0.33±0.25μM. BBP/CSFA-0 proved in vitro inactive against the hepatitis C virus, that belongs like BVDV and CSFV to the family of Flaviviridae. Modification of the substituents on the two 1H-benzimidazole groups of BBP resulted in analogues equipotent in anti-BVDV activity (EC50=0.7±0.1μM), devoid of cytotoxicity (S.I.=142). BBP resistant BVDV was selected for and was found to carry the I261M mutation in the viral RNA-dependent RNA polymerase (RdRp). Likewise, BBP-resistant CSFV was selected for; this variant carries either an I261N or a P262A mutation in NS5B. Molecular modeling revealed that I261 and P262 are located in a small cavity near the fingertip domain of the pestivirus polymerase. BBP-resistant BVDV and CSFV proved to be cross-resistant to earlier reported pestivirus inhibitors (BPIP, AG110 and LZ37) that are known to target the same region of the RdRp. BBP did not inhibit the in vitro activity of recombinant BVDV RdRp but inhibited the activity of BVDV replication complexes (RCs). BBP interacts likely with the fingertip of the pestivirus RdRp at the same position as BPIP, AG110 and LZ37. This indicates that this region is a "hot spot" for inhibition of pestivirus replication. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Comparative study of 5% and 2.5% potassium hydroxide solution for molluscum contagiosum in children.

    Science.gov (United States)

    Uçmak, Derya; Akkurt, Meltem Zeynep; Kacar, Seval Dogruk; Sula, Bilal; Arica, Mustafa

    2014-03-01

    Molluscum contagiosum (MC) is a pediatric viral infection that is fairly contagious. Although various treatment methods are available, the presence of facial lesions limits options of therapy. We aimed to test an alternative treatment consisting of application of two different concentrations of potassium hydroxide (of KOH 5% solution and of KOH 2.5% solution) aqueous solution. In this study we evaluated the effectiveness and side-effects of daily applications of potassium hydroxide (KOH) aqueous solution at 2.5% and 5% concentrations, twice daily in 29 children with MC. Out of a total of 29 patients with molluscum contagiosum included in the study, 13 patients in the 2.5% KOH group and 12 patients in the KOH 5% group completed the study. Families were instructed to apply potassium hydroxide twice a day. The assessment of response and side-effects were performed on days 0, 15, 30, 45 and 60 (visits were numbered 1, 2, 3, 4 and 5, respectively) and one month after. We had a total of 11 (44%) patients who completely recovered after the fifth visit. While eight (66.7%) of these 11 patients were in the 5% treatment group, three (23.1%) patients were in the 2.5% treatment group, and there was a statistically meaningful difference (p Potassium hydroxide solution at a concentration of 5% was more effective than 2.5% in our patients. The treatment was well-tolerated on the face with the advantage of administration of lower concentrations. This study suggests potassium hydroxide may be a more preferable mode of treatment for molluscum contagiosum lesions on the face.

  3. Review The Emerging Profile of Cross-Resistance among the Nonnucleoside HIV-1 Reverse Transcriptase Inhibitors

    Directory of Open Access Journals (Sweden)

    Nicolas Sluis-Cremer

    2014-07-01

    Full Text Available Nonnucleoside reverse transcriptase inhibitors (NNRTIs are widely used to treat HIV-1-infected individuals; indeed most first-line antiretroviral therapies typically include one NNRTI in combination with two nucleoside analogs. In 2008, the next-generation NNRTI etravirine was approved for the treatment of HIV-infected antiretroviral therapy-experienced individuals, including those with prior NNRTI exposure. NNRTIs are also increasingly being included in strategies to prevent HIV-1 infection. For example: (1 nevirapine is used to prevent mother-to-child transmission; (2 the ASPIRE (MTN 020 study will test whether a vaginal ring containing dapivirine can prevent HIV-1 infection in women; (3 a microbicide gel formulation containing the urea-PETT derivative MIV-150 is in a phase I study to evaluate safety, pharmacokinetics, pharmacodynamics and acceptability; and (4 a long acting rilpivirine formulation is under-development for pre-exposure prophylaxis. Given their widespread use, particularly in resource-limited settings, as well as their low genetic barriers to resistance, there are concerns about overlapping resistance between the different NNRTIs. Consequently, a better understanding of the resistance and cross-resistance profiles among the NNRTI class is important for predicting response to treatment, and surveillance of transmitted drug-resistance.

  4. Inhibition of cornifins and up-regulation of protease inhibitors in cervicovaginal lavage imparts resistance to heterosexual HIV transmission

    Directory of Open Access Journals (Sweden)

    Sushama Rokade

    2017-12-01

    Full Text Available HIV-exposed seronegative individuals (HESNs are persons who remain seronegative despite repeated exposure to HIV, suggesting an in vivo resistance mechanism to HIV. Elucidation of endogenous factors responsible for this phenomenon may aid in the development of new classes of microbicides and therapeutics. The genital mucosal secretions of both men and women are known to contain a spectrum of antimicrobials and immune mediators that may contribute to resistance against HIV-1. Existence of HIV serodiscordant couples is a testimony to mucosal factors in the genital tract that prevent sexual transmission of the virus. We attempted to map such mucosal factors in female genital secretions of the serodiscordant couples in comparison with HIV infected and healthy participants using quantitative proteomics. The cervico vaginal lavage (CVL samples were collected from three groups of study participants (HIV infected, n=30; Un-infected Controls, n=10; Serodiscordant, n=24. Abundant proteins, albumin and globulins were removed from the pooled samples using multiple affinity removal spin cartridge (Agilent to enhance the sensitivity of iTRAQ proteomics analysis. Initial analysis identified a total of 135 proteins and associated 497 peptide matches. Serodiscordant females showed significantly down regulated levels of Cornifin A, B and C, Neutrophil gelatinase, myeloperoxidase and eosinophil peroxidase. Cornifins are cross-linked envelope protein of keratinocytes and are upregulated during inflammation. Downregulation of oxidative stress inducing enzymes and cornifins suggests immune-quiescence in serodiscordant females. CVL of these women showed significantly upregulated levels of Mucin 5B, S100A7, Alpha-2-macroglobulin, Cystatin A (protease inhibitor, Lacto-transferrin, SLPI (anti-leukoproteinase inhibitor and SERPIN G1 (protease inhibitor.  Significantly elevated levels of Cystatin B and Elafin in the CVL of serodiscordant females were confirmed by ELISA

  5. In vitro effects of beta-lactams combined with beta-lactamase inhibitors against methicillin-resistant Staphylococcus aureus.

    Science.gov (United States)

    Kobayashi, S; Arai, S; Hayashi, S; Sakaguchi, T

    1989-01-01

    The effects of combinations of beta-lactams with two beta-lactamase inhibitors, sulbactam and clavulanic acid, were determined in vitro against 22 clinical isolates of methicillin-resistant Staphylococcus aureus. Combinations of cefpirome, cefotaxime, and cefazolin with sulbactam (10 micrograms/ml) showed synergistic effects against more than 70% of the strains. Combinations of methicillin and penicillin G with sulbactam also showed synergistic effects against 50 and 68% of the strains, respectively, while cefotiam, moxalactam, flomoxef, and cefmetazole in combination with sulbactam showed such effects against only 40% or fewer. Clavulanic acid was synergistic only when combined with penicillin G, the effect probably being due to the beta-lactamase inhibition by the inhibitor. Sulbactam did not improve the antimicrobial activities of the beta-lactams against methicillin-susceptible S. aureus strains. At 42 degrees C the MICs of cefotaxime, methicillin, and flomoxef alone were markedly decreased from the values at 35 degrees C, and no synergy between these beta-lactams and sulbactam appeared. The resistance to penicillin G was not inhibited by incubation at 42 degrees C, and combinations of penicillin G with sulbactam and clavulanic acid showed synergy. The amounts of beta-lactamase produced were not related to the decreases in the MICs of the beta-lactams, except for penicillin G combined with sulbactam. Clavulanic acid showed slightly stronger beta-lactamase-inhibiting activity than sulbactam did. These results suggest that the synergy between sulbactam and the beta-lactams, except for penicillin G, may not be due to beta-lactamase inhibition but to suppression of the methicillin-resistant S. aureus-specific resistance based on other factors. PMID:2786369

  6. Anti-influenza drugs: the development of sialidase inhibitors.

    Science.gov (United States)

    von Itzstein, Mark; Thomson, Robin

    2009-01-01

    Viruses, particularly those that are harmful to humans, are the 'silent terrorists' of the twenty-first century. Well over four million humans die per annum as a result of viral infections alone. The scourge of influenza virus has plagued mankind throughout the ages. The fact that new viral strains emerge on a regular basis, particularly out of Asia, establishes a continual socio-economic threat to mankind. The arrival of the highly pathogenic avian influenza H5N1 heightened the threat of a potential human pandemic to the point where many countries have put in place 'preparedness plans' to defend against such an outcome. The discovery of the first designer influenza virus sialidase inhibitor and anti-influenza drug Relenza, and subsequently Tamiflu, has now inspired a number of continuing efforts towards the discovery of next generation anti-influenza drugs. Such drugs may act as 'first-line-of-defence' against the spread of influenza infection and buy time for necessary vaccine development particularly in a human pandemic setting. Furthermore, the fact that influenza virus can develop resistance to therapeutics makes these continuing efforts extremely important. An overview of the role of the virus-associated glycoprotein sialidase (neuraminidase) and some of the most recent developments towards the discovery of anti-influenza drugs based on the inhibition of influenza virus sialidase is provided in this chapter.

  7. Mechanisms of Acquired Drug Resistance to the HDAC6 Selective Inhibitor Ricolinostat Reveals Rational Drug-Drug Combination with Ibrutinib.

    Science.gov (United States)

    Amengual, Jennifer E; Prabhu, Sathyen A; Lombardo, Maximilian; Zullo, Kelly; Johannet, Paul M; Gonzalez, Yulissa; Scotto, Luigi; Serrano, Xavier Jirau; Wei, Ying; Duong, Jimmy; Nandakumar, Renu; Cremers, Serge; Verma, Akanksha; Elemento, Olivier; O'Connor, Owen A

    2017-06-15

    Purpose: Pan-class I/II histone deacetylase (HDAC) inhibitors are effective treatments for select lymphomas. Isoform-selective HDAC inhibitors are emerging as potentially more targeted agents. ACY-1215 (ricolinostat) is a first-in-class selective HDAC6 inhibitor. To better understand the discrete function of HDAC6 and its role in lymphoma, we developed a lymphoma cell line resistant to ACY-1215. Experimental Design: The diffuse large B-cell lymphoma cell line OCI-Ly10 was exposed to increasing concentrations of ACY-1215 over an extended period of time, leading to the development of a resistant cell line. Gene expression profiling (GEP) was performed to investigate differentially expressed genes. Combination studies of ACY-1215 and ibrutinib were performed in cell lines, primary human lymphoma tissue, and a xenograft mouse model. Results: Systematic incremental increases in drug exposure led to the development of distinct resistant cell lines with IC 50 values 10- to 20-fold greater than that for parental lines. GEP revealed upregulation of MAPK10, HELIOS, HDAC9, and FYN, as well as downregulation of SH3BP5 and LCK. Gene-set enrichment analysis (GSEA) revealed modulation of the BTK pathway. Ibrutinib was found to be synergistic with ACY-1215 in cell lines as well as in 3 primary patient samples of lymphoma. In vivo confirmation of antitumor synergy was demonstrated with a xenograft of DLBCL. Conclusions: The development of this ACY-1215-resistant cell line has provided valuable insights into the mechanistic role of HDAC6 in lymphoma and offered a novel method to identify rational synergistic drug combinations. Translation of these findings to the clinic is underway. Clin Cancer Res; 23(12); 3084-96. ©2016 AACR . ©2016 American Association for Cancer Research.

  8. Anti-viral properties and mode of action of standardized Echinacea purpurea extract against highly pathogenic avian Influenza virus (H5N1, H7N7 and swine-origin H1N1 (S-OIV

    Directory of Open Access Journals (Sweden)

    Schoop Roland

    2009-11-01

    Full Text Available Abstract Background Influenza virus (IV infections are a major threat to human welfare and animal health worldwide. Anti-viral therapy includes vaccines and a few anti-viral drugs. However vaccines are not always available in time, as demonstrated by the emergence of the new 2009 H1N1-type pandemic strain of swine origin (S-OIV in April 2009, and the acquisition of resistance to neuraminidase inhibitors such as Tamiflu® (oseltamivir is a potential problem. Therefore the prospects for the control of IV by existing anti-viral drugs are limited. As an alternative approach to the common anti-virals we studied in more detail a commercial standardized extract of the widely used herb Echinacea purpurea (Echinaforce®, EF in order to elucidate the nature of its anti-IV activity. Results Human H1N1-type IV, highly pathogenic avian IV (HPAIV of the H5- and H7-types, as well as swine origin IV (S-OIV, H1N1, were all inactivated in cell culture assays by the EF preparation at concentrations ranging from the recommended dose for oral consumption to several orders of magnitude lower. Detailed studies with the H5N1 HPAIV strain indicated that direct contact between EF and virus was required, prior to infection, in order to obtain maximum inhibition in virus replication. Hemagglutination assays showed that the extract inhibited the receptor binding activity of the virus, suggesting that the extract interferes with the viral entry into cells. In sequential passage studies under treatment in cell culture with the H5N1 virus no EF-resistant variants emerged, in contrast to Tamiflu®, which produced resistant viruses upon passaging. Furthermore, the Tamiflu®-resistant virus was just as susceptible to EF as the wild type virus. Conclusion As a result of these investigations, we believe that this standard Echinacea preparation, used at the recommended dose for oral consumption, could be a useful, readily available and affordable addition to existing control options

  9. Inhibition of p70S6K1 Activation by Pdcd4 Overcomes the Resistance to an IGF-1R/IR Inhibitor in Colon Carcinoma Cells.

    Science.gov (United States)

    Zhang, Yan; Wang, Qing; Chen, Li; Yang, Hsin-Sheng

    2015-03-01

    Agents targeting insulin-like growth factor 1 receptor (IGF-1R) are being actively examined in clinical trials. Although there has been some initial success of single-agent targeting IGF-1R, attempts in later studies failed because of resistance. This study aimed to understand the effects of programmed cell death 4 (Pdcd4) on the chemosensitivity of the IGF-1R inhibitor OSI-906 in colorectal cancer cells and the mechanism underlying this impact. Using OSI-906-resistant and -sensitive colorectal cancer cells, we found that the Pdcd4 level directly correlates with cell chemosensitivity to OSI-906. In addition, tumors derived from Pdcd4 knockdown cells resist the growth inhibitory effect of OSI-906 in a colorectal cancer xenograft mouse model. Moreover, Pdcd4 enhances the antiproliferative effect of OSI-906 in resistant cells through suppression of p70S6K1 activation. Knockdown of p70S6K1, but not p70S6K2, significantly increases the chemosensitivity of OSI-906 in cultured colorectal cancer cells. Furthermore, the combination of OSI-906 and PF-4708671, a p70S6K1 inhibitor, efficiently suppresses the growth of OSI-906-resistant colon tumor cells in vitro and in vivo. Taken together, activation of p70S6K1 that is inhibited by Pdcd4 is essential for resistance to the IGF-1R inhibitor in colon tumor cells, and the combinational treatment of OSI-906 and PF-4708671 results in enhanced antiproliferation effects in colorectal cancer cells in vitro and in vivo, providing a novel venue to overcome the resistance to the IGF-1R inhibitor in treating colorectal cancer. ©2015 American Association for Cancer Research.

  10. Mechanistic Characterization of GS-9190 (Tegobuvir), a Novel Nonnucleoside Inhibitor of Hepatitis C Virus NS5B Polymerase▿

    Science.gov (United States)

    Shih, I-hung; Vliegen, Inge; Peng, Betty; Yang, Huiling; Hebner, Christy; Paeshuyse, Jan; Pürstinger, Gerhard; Fenaux, Martijn; Tian, Yang; Mabery, Eric; Qi, Xiaoping; Bahador, Gina; Paulson, Matthew; Lehman, Laura S.; Bondy, Steven; Tse, Winston; Reiser, Hans; Lee, William A.; Schmitz, Uli; Neyts, Johan; Zhong, Weidong

    2011-01-01

    GS-9190 (Tegobuvir) is a novel imidazopyridine inhibitor of hepatitis C virus (HCV) RNA replication in vitro and has demonstrated potent antiviral activity in patients chronically infected with genotype 1 (GT1) HCV. GS-9190 exhibits reduced activity against GT2a (JFH1) subgenomic replicons and GT2a (J6/JFH1) infectious virus, suggesting that the compound's mechanism of action involves a genotype-specific viral component. To further investigate the GS-9190 mechanism of action, we utilized the susceptibility differences between GT1b and GT2a by constructing a series of replicon chimeras where combinations of 1b and 2a nonstructural proteins were encoded within the same replicon. The antiviral activities of GS-9190 against the chimeric replicons were reduced to levels comparable to that of the wild-type GT2a replicon in chimeras expressing GT2a NS5B. GT1b replicons in which the β-hairpin region (amino acids 435 to 455) was replaced by the corresponding sequence of GT2a were markedly less susceptible to GS-9190, indicating the importance of the thumb subdomain of the polymerase in this effect. Resistance selection in GT1b replicon cells identified several mutations in NS5B (C316Y, Y448H, Y452H, and C445F) that contributed to the drug resistance phenotype. Reintroduction of these mutations into wild-type replicons conferred resistance to GS-9190, with the number of NS5B mutations correlating with the degree of resistance. Analysis of GS-9190 cross-resistance against previously reported NS5B drug-selected mutations showed that the resistance pattern of GS-9190 is different from other nonnucleoside inhibitors. Collectively, these data demonstrate that GS-9190 represents a novel class of nonnucleoside polymerase inhibitors that interact with NS5B likely through involvement of the β-hairpin in the thumb subdomain. PMID:21746939

  11. Assessment of FIV-C infection of cats as a function of treatment with the protease inhibitor, TL-3

    Directory of Open Access Journals (Sweden)

    de Rozières Sohela

    2004-11-01

    Full Text Available Abstract Background The protease inhibitor, TL-3, demonstrated broad efficacy in vitro against FIV, HIV and SIV (simian immunodeficiency virus, and exhibited very strong protective effects on early neurologic alterations in the CNS of FIV-PPR infected cats. In this study, we analyzed TL-3 efficacy using a highly pathogenic FIV-C isolate, which causes a severe acute phase immunodeficiency syndrome, with high early mortality rates. Results Twenty cats were infected with uncloned FIV-C and half were treated with TL-3 while the other half were left untreated. Two uninfected cats were used as controls. The general health and the immunological and virological status of the animals was monitored for eight weeks following infection. All infected animals became viremic independent of TL-3 treatment and seven of 20 FIV-C infected animals developed severe immunodepletive disease in conjunction with significantly (p ≤ 0.05 higher viral RNA loads as compared to asymptomatic animals. A marked and progressive increase in CD8+ T lymphocytes in animals surviving acute phase infection was noted, which was not evident in symptomatic animals (p ≤ 0.05. Average viral loads were lower in TL-3 treated animals and of the 6 animals requiring euthanasia, four were from the untreated cohort. At eight weeks post infection, half of the TL-3 treated animals and only one of six untreated animals had viral loads below detection limits. Analysis of protease genes in TL-3 treated animals with higher than average viral loads revealed sequence variations relative to wild type protease. In particular, one mutant, D105G, imparted 5-fold resistance against TL-3 relative to wild type protease. Conclusions The findings indicate that the protease inhibitor, TL-3, when administered orally as a monotherapy, did not prevent viremia in cats infected with high dose FIV-C. However, the modest lowering of viral loads with TL-3 treatment, the greater survival rate in symptomatic animals of

  12. KV7 potassium channels

    DEFF Research Database (Denmark)

    Stott, Jennifer B; Jepps, Thomas Andrew; Greenwood, Iain A

    2014-01-01

    Potassium channels are key regulators of smooth muscle tone, with increases in activity resulting in hyperpolarisation of the cell membrane, which acts to oppose vasoconstriction. Several potassium channels exist within smooth muscle, but the KV7 family of voltage-gated potassium channels have been...

  13. Potent nonnucleoside reverse transcriptase inhibitors target HIV-1 Gag-Pol.

    Directory of Open Access Journals (Sweden)

    Anna Figueiredo

    2006-11-01

    Full Text Available Nonnucleoside reverse transcriptase inhibitors (NNRTIs target HIV-1 reverse transcriptase (RT by binding to a pocket in RT that is close to, but distinct, from the DNA polymerase active site and prevent the synthesis of viral cDNA. NNRTIs, in particular, those that are potent inhibitors of RT polymerase activity, can also act as chemical enhancers of the enzyme's inter-subunit interactions. However, the consequences of this chemical enhancement effect on HIV-1 replication are not understood. Here, we show that the potent NNRTIs efavirenz, TMC120, and TMC125, but not nevirapine or delavirdine, inhibit the late stages of HIV-1 replication. These potent NNRTIs enhanced the intracellular processing of Gag and Gag-Pol polyproteins, and this was associated with a decrease in viral particle production from HIV-1-transfected cells. The increased polyprotein processing is consistent with premature activation of the HIV-1 protease by NNRTI-enhanced Gag-Pol multimerization through the embedded RT sequence. These findings support the view that Gag-Pol multimerization is an important step in viral assembly and demonstrate that regulation of Gag-Pol/Gag-Pol interactions is a novel target for small molecule inhibitors of HIV-1 production. Furthermore, these drugs can serve as useful probes to further understand processes involved in HIV-1 particle assembly and maturation.

  14. Interaction of the EGFR inhibitors gefitinib, vandetanib, pelitinib and neratinib with the ABCG2 multidrug transporter: implications for the emergence and reversal of cancer drug resistance.

    Science.gov (United States)

    Hegedüs, Csilla; Truta-Feles, Krisztina; Antalffy, Géza; Várady, György; Német, Katalin; Ozvegy-Laczka, Csilla; Kéri, György; Orfi, László; Szakács, Gergely; Settleman, Jeffrey; Váradi, András; Sarkadi, Balázs

    2012-08-01

    Human ABCG2 is a plasma membrane glycoprotein that provides physiological protection against xenobiotics. ABCG2 also significantly influences biodistribution of drugs through pharmacological tissue barriers and confers multidrug resistance to cancer cells. Moreover, ABCG2 is the molecular determinant of the side population that is characteristically enriched in normal and cancer stem cells. Numerous tumors depend on unregulated EGFR signaling, thus inhibition of this receptor by small molecular weight inhibitors such as gefitinib, and the novel second generation agents vandetanib, pelitinib and neratinib, is a promising therapeutic option. In the present study, we provide detailed biochemical characterization regarding the interaction of these EGFR inhibitors with ABCG2. We show that ABCG2 confers resistance to gefitinib and pelitinib, whereas the intracellular action of vandetanib and neratinib is unaltered by the presence of the transporter. At higher concentrations, however, all these EGFR inhibitors inhibit ABCG2 function, thereby promoting accumulation of ABCG2 substrate drugs. We also report enhanced expression of ABCG2 in gefitinib-resistant non-small cell lung cancer cells, suggesting potential clinical relevance of ABCG2 in acquired drug resistance. Since ABCG2 has important impact on both the pharmacological properties and anti-cancer efficiencies of drugs, our results regarding the novel EGFR inhibitors should provide useful information about their therapeutic applicability against ABCG2-expressing cancer cells depending on EGFR signaling. In addition, the finding that these EGFR inhibitors efficiently block ABCG2 function may help to design novel drug-combination therapeutic strategies. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Primary resistance to integrase strand transfer inhibitors in patients infected with diverse HIV-1 subtypes in sub-Saharan Africa

    NARCIS (Netherlands)

    Inzaule, Seth C.; Hamers, Raph L.; Noguera-Julian, Marc; Casadellà, Maria; Parera, Mariona; Rinke de Wit, Tobias F.; Paredes, Roger

    2018-01-01

    To investigate the prevalence and patterns of major and accessory resistance mutations associated with integrase strand transfer inhibitors (INSTIs), across diverse HIV-1 subtypes in sub-Saharan Africa. pol gene sequences were obtained using Illumina next-generation sequencing from 425 INSTI-naive

  16. In Vitro Contractile Response of Rabbit Myometrium to BKCa and KATP Potassium Channel Openers

    Directory of Open Access Journals (Sweden)

    Soňa Fraňová

    2009-01-01

    Full Text Available The aim of the study was to evaluate the participation of ligand-sensitive potassium large conductance calcium-activated channels (BKCa and ATP-sensitive potassium channels in uterine smooth muscle reactivity during different stages of the experimentally induced proliferatory and secretory phase in the sexual cycle in ovariectomised rabbits in vitro. The myometrial reactivity to oxytocin (10-6 mol l-1 was investigated by an in vitro method in female rabbits 14 days after ovariectomy treated with 17β-estradiol - 1 mg/kg/day i.m. for 7 days, or with a combination of progesterone 2 mg/kg/day s.c. for 7 days and 17β-estradiol - 0.2 mg/ kg/day (day 3–7. The strips of myometrial smooth muscle were incubated with a specific opener (NS 1619 and an antagonist (TEA of potassium large conductance calcium-activated channel, or with a specific opener (pinacidil and an antagonist (glybenclamide of ATP-sensitive potassium channels before the administration of oxytocin. NS1619 produced more potent inhibition of the oxytocin-induced contraction during the gestagen dominance (experimental secretory phase than the one observed during the oestrogen dominance (experimental proliferatory phase. TEA antagonized the NS1619 induced inhibition of the myometrial contraction. In the matter of KATP potassium channels, after the administration of pinacidil we observed a similar situation in the changes of myometrial contractility. Pinacidil produced more pronounced inhibition of oxytocin-induced contraction during the secretory phase, and its effect was abolished by the selective inhibitor glybenclamide. Our experimental results indicate that both potassium large conductance calcium-activated channels and ATP-sensitive potassium channels significantly participate in the regulation of myometrial oxytocin-induced contractions and the activity of these channels is probably influenced by the levels of oestrogens and gestagens.

  17. Structural and Functional Insights into Foamy Viral Integrase

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    Cha-Gyun Shin

    2013-07-01

    Full Text Available Successful integration of retroviral DNA into the host chromosome is an essential step for viral replication. The process is mediated by virally encoded integrase (IN and orchestrated by 3'-end processing and the strand transfer reaction. In vitro reaction conditions, such as substrate specificity, cofactor usage, and cellular binding partners for such reactions by the three distinct domains of prototype foamy viral integrase (PFV-IN have been described well in several reports. Recent studies on the three‑dimensional structure of the interacting complexes between PFV-IN and DNA, cofactors, binding partners, or inhibitors have explored the mechanistic details of such interactions and shown its utilization as an important target to develop anti-retroviral drugs. The presence of a potent, non-transferable nuclear localization signal in the PFV C-terminal domain extends its use as a model for investigating cellular trafficking of large molecular complexes through the nuclear pore complex and also to identify novel cellular targets for such trafficking. This review focuses on recent advancements in the structural analysis and in vitro functional aspects of PFV-IN.

  18. Chronic potassium depletion increases adrenal progesterone production that is necessary for efficient renal retention of potassium.

    Science.gov (United States)

    Elabida, Boutaïna; Edwards, Aurélie; Salhi, Amel; Azroyan, Anie; Fodstad, Heidi; Meneton, Pierre; Doucet, Alain; Bloch-Faure, May; Crambert, Gilles

    2011-08-01

    Modern dietary habits are characterized by high-sodium and low-potassium intakes, each of which was correlated with a higher risk for hypertension. In this study, we examined whether long-term variations in the intake of sodium and potassium induce lasting changes in the plasma concentration of circulating steroids by developing a mathematical model of steroidogenesis in mice. One finding of this model was that mice increase their plasma progesterone levels specifically in response to potassium depletion. This prediction was confirmed by measurements in both male mice and men. Further investigation showed that progesterone regulates renal potassium handling both in males and females under potassium restriction, independent of its role in reproduction. The increase in progesterone production by male mice was time dependent and correlated with decreased urinary potassium content. The progesterone-dependent ability to efficiently retain potassium was because of an RU486 (a progesterone receptor antagonist)-sensitive stimulation of the colonic hydrogen, potassium-ATPase (known as the non-gastric or hydrogen, potassium-ATPase type 2) in the kidney. Thus, in males, a specific progesterone concentration profile induced by chronic potassium restriction regulates potassium balance.

  19. Intrinsic resistance to tyrosine kinase inhibitors is associated with poor clinical outcome in metastatic renal cell carcinoma

    International Nuclear Information System (INIS)

    Busch, Jonas; Grünwald, Viktor; Seidel, Christoph; Weikert, Steffen; Wolff, Ingmar; Kempkensteffen, Carsten; Weinkauf, Lisa; Hinz, Stefan; Magheli, Ahmed; Miller, Kurt

    2011-01-01

    Data on sequential therapy in patients with metastatic renal cell carcinoma (mRCC) and intrinsic resistance to receptor tyrosine kinase inhibitor (rTKI) treatment remains vague. We retrospectively studied treatment characteristics and outcome of mRCC patients refractory to first rTKI therapy. Thirty-five mRCC patients (male, 18; female, 11) with primary resistance to first rTKI therapy (sunitinib, n = 28; sorafenib, n = 7) and a median treatment interval of 2.4 months (1 - 4.6) were identified. In 22 patients, progressive disease (PD) was determined by a new metastatic lesion. Of these, 16 patients received subsequent therapy with 12 patients remaining refractory and 4 patients achieving disease stabilization. In 13 patients continuous growth of existing metastatic lesions determined PD. Of these, 9 received sequential therapy with 6 achieving disease stabilization. Altogether, 25 patients were treated sequentially (rTKI: n = 15; mTOR-inhibitor: n = 10) and achieved a median PFS of 3.2 months (range, 1-16.6). Fifteen patients failed to respond to either line of therapy. Disease control was not associated with type of subsequent therapy. Median OS was 14.9 months (CI: 5.5-24.4). Intrinsic resistance to rTKI is associated with a low chance of response to sequential therapy and a poor prognosis in mRCC patients

  20. HIV genotype resistance testing in antiretroviral (ART) exposed Indian children--a need of the hour.

    Science.gov (United States)

    Shah, Ira; Parikh, Shefali

    2013-04-01

    Development of drug resistance in HIV infected children with treatment failure is a major impediment to selection of appropriate therapy. HIV genotype resistance assays predict drug resistance on the basis of mutations in the viral genome. However, their clinical utility, especially in a resource limited setting is still a subject of debate. The authors report two cases in which both the children suffered from treatment failure of various antiretroviral therapy regimes. In both the cases, Genotype Resistance Testing (GRT) prompted a radical change from proposed failure therapy as per existing guidelines. GRT was specifically important for the selection of a new dual Nucleoside reverse transcriptase inhibitors (NRTI) component of failure regimen by identifying TAMS and M184V mutations in the HIV genome. These case reports highlight the importance of GRT in children failing multiple antiretroviral regimes; and emphasizes the need to recognize situations where GRT is absolutely essential to guide appropriate therapy, even in a resource limited setting.

  1. Resistance to EGF receptor inhibitors in glioblastoma mediated by phosphorylation of the PTEN tumor suppressor at tyrosine 240.

    Science.gov (United States)

    Fenton, Tim R; Nathanson, David; Ponte de Albuquerque, Claudio; Kuga, Daisuke; Iwanami, Akio; Dang, Julie; Yang, Huijun; Tanaka, Kazuhiro; Oba-Shinjo, Sueli Mieko; Uno, Miyuki; Inda, Maria del Mar; Wykosky, Jill; Bachoo, Robert M; James, C David; DePinho, Ronald A; Vandenberg, Scott R; Zhou, Huilin; Marie, Suely K N; Mischel, Paul S; Cavenee, Webster K; Furnari, Frank B

    2012-08-28

    Glioblastoma multiforme (GBM) is the most aggressive of the astrocytic malignancies and the most common intracranial tumor in adults. Although the epidermal growth factor receptor (EGFR) is overexpressed and/or mutated in at least 50% of GBM cases and is required for tumor maintenance in animal models, EGFR inhibitors have thus far failed to deliver significant responses in GBM patients. One inherent resistance mechanism in GBM is the coactivation of multiple receptor tyrosine kinases, which generates redundancy in activation of phosphoinositide-3'-kinase (PI3K) signaling. Here we demonstrate that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is frequently phosphorylated at a conserved tyrosine residue, Y240, in GBM clinical samples. Phosphorylation of Y240 is associated with shortened overall survival and resistance to EGFR inhibitor therapy in GBM patients and plays an active role in mediating resistance to EGFR inhibition in vitro. Y240 phosphorylation can be mediated by both fibroblast growth factor receptors and SRC family kinases (SFKs) but does not affect the ability of PTEN to antagonize PI3K signaling. These findings show that, in addition to genetic loss and mutation of PTEN, its modulation by tyrosine phosphorylation has important implications for the development and treatment of GBM.

  2. HIV drug resistance following a decade of the free antiretroviral therapy programme in India: A review.

    Science.gov (United States)

    Karade, Santosh; Chaturbhuj, Devidas N; Sen, Sourav; Joshi, Rajneesh K; Kulkarni, Smita S; Shankar, Subramanian; Gangakhedkar, Raman R

    2018-01-01

    The objective of this review was to assess the burden of HIV drug resistance mutations (DRM) in Indian adults exposed to first-line antiretroviral therapy (ART) as per national guidelines. An advanced search of the published literature on HIV drug resistance in India was performed in the PubMed and Scopus databases. Data pertaining to age, sex, CD4 count, viral load, and prevalence of nucleoside reverse transcriptase inhibitor (NRTI)/non-nucleoside reverse transcriptase inhibitor (NNRTI) DRM were extracted from each publication. Year-wise Indian HIV-1 reverse transcriptase (RT) sequences were retrieved from the Los Alamos HIV database and mutation analyses were performed. A time trend analysis of the proportion of sequences showing NRTI resistance mutations among individuals exposed to first-line ART was conducted. Overall, 23 studies (1046 unique RT sequences) were identified indicating a prevalence of drug resistance to NRTI and NNRTI. The proportion of RT sequences with any DRM, any NRTI DRM, and any NNRTI DRM was 78.39%, 68.83%, and 73.13%, respectively. The temporal trend analysis of individual DRM from sequences retrieved during 2004-2014 indicated a rising trend in K65R mutations (p=0.013). Although the overall burden of resistance against first-line ART agents remained steady over the study decade, periodic monitoring is essential. There is the need to develop an HIV-1 subtype C-specific resistance database in India. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  3. Discovery of 2,4-diarylaminopyrimidines bearing a resorcinol motif as novel ALK inhibitors to overcome the G1202R resistant mutation.

    Science.gov (United States)

    Geng, Kaijun; Xia, Zongjun; Ji, Yinchun; Zhang, Ruisi Ruthy; Sun, Deqiao; Ai, Jing; Song, Zilan; Geng, Meiyu; Zhang, Ao

    2018-01-20

    To address drug resistance caused by ALK kinase mutations, especially the most refractory and predominant mutation G1202R for the second-generation ALK inhibitor, a series of new diarylaminopyrimidine analogues were designed by incorporating a resorcinol moiety (A-ring) to interact the ALK kinase domain where the G1202R is located. Compound 12d turns out as the most potent with IC 50 values of 1.7, 3.5, and 1.8 nM against ALK wild type, gatekeeper mutant L1196M, and the G1202R mutant, respectively. More importantly, compound 12d has excellent inhibitory effects against the proliferation of BaF3 cells specifically expressing ALK wild type, gatekeeper L1196M, and the most challenging mutant G1202R, with IC 50 values all less than 1.5 nM. Collectively, compound 12d is worthy of further investigation as a new more potent third-generation ALK inhibitor to circumvent drug resistance of both the first-generation and the second-generation inhibitors. Copyright © 2017. Published by Elsevier Masson SAS.

  4. Hepatitis C Variability, Patterns of Resistance, and Impact on Therapy

    Directory of Open Access Journals (Sweden)

    Cristina Simona Strahotin

    2012-01-01

    Full Text Available Hepatitis C (HCV, a leading cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma, is the most common indication for liver transplantation in the United States. Although annual incidence of infection has declined since the 1980s, aging of the currently infected population is expected to result in an increase in HCV burden. HCV is prone to develop resistance to antiviral drugs, and despite considerable efforts to understand the virus for effective treatments, our knowledge remains incomplete. This paper reviews HCV resistance mechanisms, the traditional treatment with and the new standard of care for hepatitis C treatment. Although these new treatments remain PEG-IFN-α- and ribavirin-based, they add one of the newly FDA approved direct antiviral agents, telaprevir or boceprevir. This new “triple therapy” has resulted in greater viral cure rates, although treatment failure remains a possibility. The future may belong to nucleoside/nucleotide analogues, non-nucleoside RNA-dependent RNA polymerase inhibitors, or cyclophilin inhibitors, and the treatment of HCV may ultimately parallel that of HIV. However, research should focus not only on effective treatments, but also on the development of a HCV vaccine, as this may prove to be the most cost-effective method of eradicating this disease.

  5. Apoptosis-related molecular differences for response to tyrosin kinase inhibitors in drug-sensitive and drug-resistant human bladder cancer cells

    Directory of Open Access Journals (Sweden)

    Jixia Li

    2013-01-01

    Full Text Available Context: The epidermal growth factor receptor (EGFR family is reportedly overexpressed in bladder cancer, and tyrosine kinaseinhibitors (TKIs have been suggested as treatment. Gefitinib is a selective inhibitor of the EGFR and lapatinib is a dual inhibitor of both the EGFR and HER2 (human EGFR type 2 receptor. Both compounds compete with the binding of adenosine triphosphate (ATP to the tyrosine kinase domain of the respective receptors to inhibit receptor autophosphorylation causing suppression of signal transduction. Unfortunately, resistance to these inhibitors is a major clinical problem. Aims: To compare the apoptosis signaling pathway(s induced by gefitinib and lapatinib, in UM-UC-5 (drug-sensitive and UM-UC-14 (drug-resistant bladder cancer cells and to identify molecular differences that might be useful predictors of their efficacy. Materials and Methods: Cell proliferation, cell cycle and apoptosis assay were used to detect the effect of TKIs on UM-UC-5 and UM-UC-14 cells. Molecular differences for response to TKIs were examined by protein array. Results: TKIs strongly inhibited cell proliferation and induced cell cycle G1 arrest and apoptosis in UM-UC-5 cells. Most notable apoptosis molecular differences included decreased claspin, trail, and survivin by TKIs in the sensitive cells. In contrast, TKIs had no effect on resistant cells. Conclusions: Claspin, trail, and survivin might be used to determine the sensitivity of bladder cancers to TKIs.

  6. Penicillin V Potassium

    Science.gov (United States)

    Penicillin V potassium is used to treat certain infections caused by bacteria such as pneumonia and other ... heart valves and other symptoms) from coming back. Penicillin V potassium is in a class of medications ...

  7. The mTOR inhibitor, everolimus (RAD001), overcomes resistance to imatinib in quiescent Ph-positive acute lymphoblastic leukemia cells

    International Nuclear Information System (INIS)

    Kuwatsuka, Y; Minami, M; Minami, Y; Sugimoto, K; Hayakawa, F; Miyata, Y; Abe, A; Goff, D J; Kiyoi, H; Naoe, T

    2011-01-01

    In Ph-positive (Ph + ) leukemia, the quiescent cell state is one of the reasons for resistance to the BCR-ABL-kinase inhibitor, imatinib. In order to examine the mechanisms of resistance due to quiescence and the effect of the mammalian target of rapamycin inhibitor, everolimus, for such a resistant population, we used Ph + acute lymphoblastic leukemia patient cells serially xenotransplanted into NOD/SCID/IL2rγ null (NOG) mice. Spleen cells from leukemic mice showed a higher percentage of slow-cycling G 0 cells in the CD34 + CD38 − population compared with the CD34 + CD38 + and CD34 − populations. After ex vivo imatinib treatment, more residual cells were observed in the CD34 + CD38 − population than in the other populations. Although slow-cycling G 0 cells were insensitive to imatinib in spite of BCR-ABL and CrkL dephosphorylation, combination treatment with everolimus induced substantial cell death, including that of the CD34 + CD38 − population, with p70-S6 K dephosphorylation and decrease of MCL-1 expression. The leukemic non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mouse system with the in vivo combination treatment with imatinib and everolimus showed a decrease of tumor burden including CD34 + cells. These results imply that treatment with everolimus can overcome resistance to imatinib in Ph + leukemia due to quiescence

  8. Evaluation of the potassium adsorption capacity of a potassium adsorption filter during rapid blood transfusion.

    Science.gov (United States)

    Matsuura, H; Akatsuka, Y; Muramatsu, C; Isogai, S; Sugiura, Y; Arakawa, S; Murayama, M; Kurahashi, M; Takasuga, H; Oshige, T; Yuba, T; Mizuta, S; Emi, N

    2015-05-01

    The concentration of extracellular potassium in red blood cell concentrates (RCCs) increases during storage, leading to risk of hyperkalemia. A potassium adsorption filter (PAF) can eliminate the potassium at normal blood transfusion. This study aimed to investigate the potassium adsorption capacity of a PAF during rapid blood transfusion. We tested several different potassium concentrations under a rapid transfusion condition using a pressure bag. The adsorption rates of the 70-mEq/l model were 76·8%. The PAF showed good potassium adsorption capacity, suggesting that this filter may provide a convenient method to prevent hyperkalemia during rapid blood transfusion. © 2015 International Society of Blood Transfusion.

  9. Successive increases in the resistance of Drosophila to viral infection through a transposon insertion followed by a Duplication.

    Directory of Open Access Journals (Sweden)

    Michael M Magwire

    2011-10-01

    Full Text Available To understand the molecular basis of how hosts evolve resistance to their parasites, we have investigated the genes that cause variation in the susceptibility of Drosophila melanogaster to viral infection. Using a host-specific pathogen of D. melanogaster called the sigma virus (Rhabdoviridae, we mapped a major-effect polymorphism to a region containing two paralogous genes called CHKov1 and CHKov2. In a panel of inbred fly lines, we found that a transposable element insertion in the protein coding sequence of CHKov1 is associated with increased resistance to infection. Previous research has shown that this insertion results in a truncated messenger RNA that encodes a far shorter protein than the susceptible allele. This resistant allele has rapidly increased in frequency under directional selection and is now the commonest form of the gene in natural populations. Using genetic mapping and site-specific recombination, we identified a third genotype with considerably greater resistance that is currently rare in the wild. In these flies there have been two duplications, resulting in three copies of both the truncated allele of CHKov1 and CHKov2 (one of which is also truncated. Remarkably, the truncated allele of CHKov1 has previously been found to confer resistance to organophosphate insecticides. As estimates of the age of this allele predate the use of insecticides, it is likely that this allele initially functioned as a defence against viruses and fortuitously "pre-adapted" flies to insecticides. These results demonstrate that strong selection by parasites for increased host resistance can result in major genetic changes and rapid shifts in allele frequencies; and, contrary to the prevailing view that resistance to pathogens can be a costly trait to evolve, the pleiotropic effects of these changes can have unexpected benefits.

  10. Potassium maldistribution revisited

    African Journals Online (AJOL)

    Background:This study investigated maldistribution of concentrated 15% potassium chloride after injection into .... and latter experiments referred to for example as “Control 1” ..... be further investigated as a reliable, simple method of potassium.

  11. Detection of potassium deficiency on palm oil tree (Elaeis guineensis (jacq)) by laser induced fluorescence

    International Nuclear Information System (INIS)

    Diomande, K.; Konate, A.; Krou Adjo, V.; Soro, A.; Ebby, N.; Ballo, K.

    1998-02-01

    The potassium is the main nutrient element which plays a significant role on oil palm tree (Elaeis guineensis (jacq)) production and its resistance to the dry season. One can observe 30% decrease of the production in case of potassium deficiency. The potassium nutrition control of an oil palm tree field is a very important activity and leads to the fertilization policy. The Laser Induced Fluorescence (L.I.F.) is a fast and simple method compared to the classical one, ''Diagnostic Foliaire'', usually used in agronomy. We used the L.I.F. method to detect the oil palm tree stress caused by potassium deficiency, analysing the fluorescence spectrum of the chlorophyll a. We proved that the intensity ratio of the fluorescence spectrum R=F690/F73S is superior to 0.5 when the tree is under stress and its value is around 0.4 in case of intact tree. (author)

  12. In vitro cross-resistance profile of nucleoside reverse transcriptase inhibitor (NRTI) BMS-986001 against known NRTI resistance mutations.

    Science.gov (United States)

    Li, Zhufang; Terry, Brian; Olds, William; Protack, Tricia; Deminie, Carol; Minassian, Beatrice; Nowicka-Sans, Beata; Sun, Yongnian; Dicker, Ira; Hwang, Carey; Lataillade, Max; Hanna, George J; Krystal, Mark

    2013-11-01

    BMS-986001 is a novel HIV nucleoside reverse transcriptase inhibitor (NRTI). To date, little is known about its resistance profile. In order to examine the cross-resistance profile of BMS-986001 to NRTI mutations, a replicating virus system was used to examine specific amino acid mutations known to confer resistance to various NRTIs. In addition, reverse transcriptases from 19 clinical isolates with various NRTI mutations were examined in the Monogram PhenoSense HIV assay. In the site-directed mutagenesis studies, a virus containing a K65R substitution exhibited a 0.4-fold change in 50% effective concentration (EC50) versus the wild type, while the majority of viruses with the Q151M constellation (without M184V) exhibited changes in EC50 versus wild type of 0.23- to 0.48-fold. Susceptibility to BMS-986001 was also maintained in an L74V-containing virus (0.7-fold change), while an M184V-only-containing virus induced a 2- to 3-fold decrease in susceptibility. Increasing numbers of thymidine analog mutation pattern 1 (TAM-1) pathway mutations correlated with decreases in susceptibility to BMS-986001, while viruses with TAM-2 pathway mutations exhibited a 5- to 8-fold decrease in susceptibility, regardless of the number of TAMs. A 22-fold decrease in susceptibility to BMS-986001 was observed in a site-directed mutant containing the T69 insertion complex. Common non-NRTI (NNRTI) mutations had little impact on susceptibility to BMS-986001. The results from the site-directed mutants correlated well with the more complicated genotypes found in NRTI-resistant clinical isolates. Data from clinical studies are needed to determine the clinically relevant resistance cutoff values for BMS-986001.

  13. Ethanol from lignocellulose - Fermentation inhibitors, detoxification and genetic engineering of Saccharomyces cerevisiae for enhanced resistance

    Energy Technology Data Exchange (ETDEWEB)

    Larsson, Simona

    2000-07-01

    Ethanol can be produced from lignocellulose by first hydrolysing the material to sugars, and then fermenting the hydrolysate with the yeast Saccharomyces cerevisiae. Hydrolysis using dilute sulphuric acid has advantages over other methods, however, compounds which inhibit fermentation are generated during this kind of hydrolysis. The inhibitory effect of aliphatic acids, furans, and phenolic compounds was investigated. The generation of inhibitors during hydrolysis was studied using Norway spruce as raw material. It was concluded that the decrease in the fermentability coincided with increasing harshness of the hydrolysis conditions. The decrease in fermentability was not correlated solely to the content of aliphatic acids or furan derivatives. To increase the fermentability, detoxification is often employed. Twelve detoxification methods were compared with respect to the chemical composition of the hydrolysate and the fermentability after treatment. The most efficient detoxification methods were anion-exchange at pH 10.0, overliming and enzymatic detoxification with the phenol-oxidase laccase. Detailed analyses of ion exchange revealed that anion exchange and unspecific hydrophobic interactions greatly contributed to the detoxification effect, while cation exchange did not. The comparison of detoxification methods also showed that phenolic compounds are very important fermentation inhibitors, as their selective removal with laccase had a major positive effect on the fermentability. Selected compounds; aliphatic acids, furans and phenolic compounds, were characterised with respect to their inhibitory effect on ethanolic fermentation by S. cerevisiae. When aliphatic acids or furans were compared, the inhibitory effects were found to be in the same range, but the phenolic compounds displayed widely different inhibitory effects. The possibility of genetically engineering S. cerevisiae to achieve increased inhibitor resistance was explored by heterologous expression of

  14. 21 CFR 184.1619 - Potassium carbonate.

    Science.gov (United States)

    2010-04-01

    ... solution of potassium hydroxide with excess carbon dioxide to produce potassium carbonate; (3) By treating a solution of potassium hydroxide with carbon dioxide to produce potassium bicarbonate, which is... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Potassium carbonate. 184.1619 Section 184.1619 Food...

  15. Synergy against drug-resistant HIV-1 with the microbicide antiretrovirals, dapivirine and tenofovir, in combination.

    Science.gov (United States)

    Schader, Susan M; Colby-Germinario, Susan P; Schachter, Jordana R; Xu, Hongtao; Wainberg, Mark A

    2011-08-24

    To evaluate the candidate antiretroviral microbicide compounds, dapivirine (DAP) and tenofovir (TFV), alone and in combination against the transmission of wild-type and nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV-1 from different subtypes. We determined single-drug efficacy of the RTIs, DAP and TFV, against subtype B and non-B wild-type and NNRTI-resistant HIV-1 in vitro. To assess breadth of activity, compounds were tested alone and in combination against wild-type and NNRTI-resistant subtype C primary HIV-1 isolates and complimentary clonal HIV-1 from subtypes B, C and CRF02_AG to control for viral variation. Early infection was quantified by counting light units emitted from TZM-bl cells less than 48-h postinfection. Combination ratios were based on drug inhibitory concentrations (IC(50)s) and combined effects were determined by calculating combination indices. Both candidate microbicide antiretrovirals demonstrated potent anti-NNRTI-resistant HIV-1 activity in vitro, albeit the combination protected better than the single-drug treatments. Of particular interest, the DAP with TFV combination exhibited synergy (50% combination index, CI(50) = 0.567) against subtype C NNRTI-resistant HIV-1, whereas additivity (CI(50) = 0.987) was observed against the wild-type counterpart from the same patient. The effect was not compounded by the presence of subdominant viral fractions, as experiments using complimentary clonal subtype C wild-type (CI(50) = 0.968) and NNRTI-resistant (CI(50) = 0.672) HIV-1, in lieu of the patient quasispecies, gave similar results. This study supports the notion that antiretroviral drug combinations may retain antiviral activity against some drug-resistant HIV-1 despite subtype classification and quasispecies diversity.

  16. Pore Polarity and Charge Determine Differential Block of Kir1.1 and Kir7.1 Potassium Channels by Small-Molecule Inhibitor VU590.

    Science.gov (United States)

    Kharade, Sujay V; Sheehan, Jonathan H; Figueroa, Eric E; Meiler, Jens; Denton, Jerod S

    2017-09-01

    VU590 was the first publicly disclosed, submicromolar-affinity (IC 50 = 0.2 μ M), small-molecule inhibitor of the inward rectifier potassium (Kir) channel and diuretic target, Kir1.1. VU590 also inhibits Kir7.1 (IC 50 ∼ 8 μ M), and has been used to reveal new roles for Kir7.1 in regulation of myometrial contractility and melanocortin signaling. Here, we employed molecular modeling, mutagenesis, and patch clamp electrophysiology to elucidate the molecular mechanisms underlying VU590 inhibition of Kir1.1 and Kir7.1. Block of both channels is voltage- and K + -dependent, suggesting the VU590 binding site is located within the pore. Mutagenesis analysis in Kir1.1 revealed that asparagine 171 (N171) is the only pore-lining residue required for high-affinity block, and that substituting negatively charged residues (N171D, N171E) at this position dramatically weakens block. In contrast, substituting a negatively charged residue at the equivalent position in Kir7.1 enhances block by VU590, suggesting the VU590 binding mode is different. Interestingly, mutations of threonine 153 (T153) in Kir7.1 that reduce constrained polarity at this site (T153C, T153V, T153S) make wild-type and binding-site mutants (E149Q, A150S) more sensitive to block by VU590. The Kir7.1-T153C mutation enhances block by the structurally unrelated inhibitor VU714 but not by a higher-affinity analog ML418, suggesting that the polar side chain of T153 creates a barrier to low-affinity ligands that interact with E149 and A150. Reverse mutations in Kir1.1 suggest that this mechanism is conserved in other Kir channels. This study reveals a previously unappreciated role of membrane pore polarity in determination of Kir channel inhibitor pharmacology. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  17. Double stranded viral RNA induces inflammation and insulin resistance in skeletal muscle from pregnant women in vitro.

    Science.gov (United States)

    Lappas, Martha

    2015-05-01

    Maternal peripheral insulin resistance and increased inflammation are two features of pregnancies complicated by pre-existing maternal obesity and gestational diabetes mellitus (GDM). There is now increasing evidence that activation of Toll-like receptor (TLR) signalling pathways by viral products may play a role in the pathophysiology of diabetes. Thus, the aim of this study was to assess the effect of the TLR3 ligand and viral dsRNA analogue polyinosinic polycytidilic acid (poly(I:C)) on inflammation and the insulin signalling pathway in skeletal muscle from pregnant women. Human skeletal muscle tissue explants were performed to determine the effect of poly(I:C) on the expression and secretion of markers of inflammation, and the insulin signalling pathway and glucose uptake. Poly(I:C) significantly increased the expression of a number of inflammatory markers in skeletal muscle from pregnant women. Specifically, there was an increase in the expression and/or secretion of the pro-inflammatory cytokines TNF-α, and IL-6 and the pro-inflammatory chemokines IL-8 and MCP-1. These effect of poly(I:C) appear to mediated via a number of signalling molecules including the pro-inflammatory transcription factor NF-κB, and the serine threonine kinases GSK3 and AMPKα. Additionally, poly(I:C) decreased insulin stimulated GLUT-4 expression and glucose uptake in skeletal muscle from pregnant women. The in vitro data presented in this study suggests that viral infection may contribute to the pathophysiology of pregnancies complicated by pre-existing maternal obesity and/or GDM. It should be noted that the in vitro studies cannot be directly used to infer the same outcomes in the intact subject. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Phase 1 study of INNO-406, a dual Abl/Lyn kinase inhibitor, in Philadelphia chromosome-positive leukemias after imatinib resistance or intolerance.

    Science.gov (United States)

    Kantarjian, Hagop; le Coutre, Phillipp; Cortes, Jorge; Pinilla-Ibarz, Javier; Nagler, Arnon; Hochhaus, Andreas; Kimura, Shinya; Ottmann, Oliver

    2010-06-01

    : INNO-406, a dual v-abl Abelson murine leukemia viral oncogene homolog (Abl)/v-yes-1 Yamaguchi sarcoma viral-related oncogene homolog (Lyn) tyrosine kinase inhibitor (TKI), has demonstrated specific Lyn kinase inhibitory activity with no or limited activity against other sarcoma (Src) family member kinases. Several breakpoint cluster region (Bcr)-Abl kinase domain mutations are sensitive to INNO-406 in vitro, including mutations that involve a phenylalanine-to-leucine or phenylalanine-to-valine substitution at codon 317 (F317L and F317V, respectively). In the current study, the authors evaluated the use of INNO-406 in patients with Philadelphia (Ph) chromosome-positive chronic myelogenous leukemia (CML) or acute lymphocytic leukemia (ALL) after imatinib resistance or intolerance. : A dose-escalation study was conducted at a starting dose of oral INNO-406 30 mg once daily. Cohorts of at least 3 patients were treated at each dose level until the maximum tolerated dose (MTD) was reached. Twice-daily dosing also was evaluated. Therapy was allowed to continue for a maximum of 24 months. : INNO-406 was administered to 56 patients with imatinib resistance (n = 40) or intolerance (n = 16). Other previous treatments included nilotinib (n = 20 patients), dasatinib (n = 26 patients), and dasatinib/nilotinib (n = 9 patients). Common mutations at the time of study entry included a tyrosine-to-histidine substitution at codon 253 (Y253H) (n = 6 patients), a glycine-to-glutamic acid substitution at codon 250 (G250E) (n = 4 patients), a threonine-to-isoleucine substitution at codon 315 (T315I) (n = 4 patients), and F317L (n = 3 patients). Of 31 patients with CML in chronic phase who received INNO-406, the major cytogenetic response rate was 19%. No responses were observed in patients who had CML in accelerated phase, CML in blastic phase, or Ph-positive ALL. The dose-limiting toxicities (DLTs) at an INNO-406 dose of 480 mg twice daily were liver function abnormalities and

  19. A Duo of Potassium-Responsive Histidine Kinases Govern the Multicellular Destiny of Bacillus subtilis.

    Science.gov (United States)

    Grau, Roberto R; de Oña, Paula; Kunert, Maritta; Leñini, Cecilia; Gallegos-Monterrosa, Ramses; Mhatre, Eisha; Vileta, Darío; Donato, Verónica; Hölscher, Theresa; Boland, Wilhelm; Kuipers, Oscar P; Kovács, Ákos T

    2015-07-07

    Multicellular biofilm formation and surface motility are bacterial behaviors considered mutually exclusive. However, the basic decision to move over or stay attached to a surface is poorly understood. Here, we discover that in Bacillus subtilis, the key root biofilm-controlling transcription factor Spo0A~Pi (phosphorylated Spo0A) governs the flagellum-independent mechanism of social sliding motility. A Spo0A-deficient strain was totally unable to slide and colonize plant roots, evidencing the important role that sliding might play in natural settings. Microarray experiments plus subsequent genetic characterization showed that the machineries of sliding and biofilm formation share the same main components (i.e., surfactin, the hydrophobin BslA, exopolysaccharide, and de novo-formed fatty acids). Sliding proficiency was transduced by the Spo0A-phosphorelay histidine kinases KinB and KinC. We discovered that potassium, a previously known inhibitor of KinC-dependent biofilm formation, is the specific sliding-activating signal through a thus-far-unnoticed cytosolic domain of KinB, which resembles the selectivity filter sequence of potassium channels. The differential expression of the Spo0A~Pi reporter abrB gene and the different levels of the constitutively active form of Spo0A, Sad67, in Δspo0A cells grown in optimized media that simultaneously stimulate motile and sessile behaviors uncover the spatiotemporal response of KinB and KinC to potassium and the gradual increase in Spo0A~Pi that orchestrates the sequential activation of sliding, followed by sessile biofilm formation and finally sporulation in the same population. Overall, these results provide insights into how multicellular behaviors formerly believed to be antagonistic are coordinately activated in benefit of the bacterium and its interaction with the host. Alternation between motile and sessile behaviors is central to bacterial adaptation, survival, and colonization. However, how is the collective

  20. Potassium supplements for oral diarrhoea regimens.

    Science.gov (United States)

    Clements, M L; Levine, M M; Black, R E; Hughes, T P; Rust, J; Tome, F C

    1980-10-18

    A study is proposed for supplementing potassium loss from diarrhea in rehydration therapies with fresh fruit and other naturally potassium-rich foods. Bananas contain .1 mol of potassium per gm. Freshly squeezed lemon or orange juices were tested for potassium and sodium content and found to have very low potassium concentration. Therefore, the banana was chosen for an upcoming study that will determine if infants and children suffering from diarrhea can ingest the amounts of the fruit necessary to elevate the potassium level sufficiently. Bananas as the potassium source are thought to be well-accepted in developing areas.

  1. The Effects of Different Tillage Methods on Available Soil Potassium Measured by Various Extractors in a Soil with High Specific Surface Area

    Directory of Open Access Journals (Sweden)

    M. Hosseini

    2016-02-01

    Full Text Available Introduction: The effects of any tillage method on soil properties, depends on location (soil, water and air and the number of (years their implementation. Soil compaction reduces yield through increased soil mechanical resistance against root growth and lower water and nutrient use efficiency (Gamda et al. 18 & Ishagh et al 23. Soil surface and sub surface compaction both reduce yield due to limited root growth and plant potassium uptake (Doulan et al. 14. Sabt et al. (50 reported that in the study area, which the lands are mostly illite clay (high specific surface area with sufficient nitrogen, soil potassium is the most important limiting factor for the growth of wheat.Considering the point that loess soils in Golestan Province have a high specific surface area,they can provide potassium for plants to produce crop, but for a higher production, potassium fertilizers should be used. Previous studies indicated that production of wheat is limited due to potassium deficiency (4, 49, 54 and 57. In these soils with a high specific surface area, the speed of movement of potassium from the soil solution is low, and doing solimits wheat yield.In loess soils containing high illite and high specific surface area (eg, soilsin the series of Rahmat Abad of Gorgan, ammonium acetate measured potassium on exchange and solution surfaces, which is highly correlated with grain yield (54 . There is a high correlation between grain yield with overload of potassium and Na TPB extraction (57. The aim of this study was to absorb potassium (limiting factor for plant growth with different tillage systemsat different depths. International recommendations towards reducing the depth and intensity of tillage (from minimum tillage to no-tillage in order to reduce erosion and oxidation of organic substances plays an important role in determining the amount of greenhouse gases. If potassium absorption does not reduceafter reducing tillage intensity,low or no-tillage methods

  2. Drug resistance prevalence in human immunodeficiency virus type 1 infected pediatric populations in Honduras and El Salvador during 1989-2009.

    Science.gov (United States)

    Holguín, Africa; Erazo, Karen; Escobar, Gustavo; de Mulder, Miguel; Yebra, Gonzalo; Martín, Leticia; Jovel, Luis Enrique; Castaneda, Luis; Pérez, Elsy

    2011-05-01

    Emergence of viral resistance is a major obstacle for antiretroviral treatment (ART) effectiveness. Human immunodeficiency virus type-1 (HIV-1) variants and drug-resistance mutations were identified in naive and antiretroviral drug-experienced children with virologic failure, in Honduras and El Salvador. Dried blood spots (DBS) from 80 individuals (54 from Honduras, 26 from El Salvador) infected during their childhood between 1989 and 2009 were collected in 2009. The HIV pol region was amplified and sequenced to identify antiretroviral-resistant mutations according to the 2009 International AIDS Society. The genotypic drug resistance interpretation was performed using the Stanford algorithm. HIV-1 variants were characterized by phylogenetic analysis and subtyping tools. HIV-1 protease and reverse transcription sequences were obtained from DBS specimens in 71 and 66 patients, respectively, of the 80 patients. All children were native Central Americans carrying subtype B, with a mean age of 9 years, most were male (65%), perinatally infected (96%), with moderate/severe AIDS symptoms (70%), and receiving first line ART at the time of sequencing (65%). Diagnostic delay was frequently observed. Infected children from Honduras presented longer ART experience and clinical outcomes, and more frequent severe symptoms. Resistant variants infected 1 of 11 naive children from El Salvador but none of the perinatally infected naive children from Honduras. Resistance was higher among ART-exposed individuals in both countries and similar for protease inhibitors (16%), nucleoside reverse transcription inhibitors (44%-52%), and nonnucleoside reverse-transcription inhibitors (66.7%). One in 10 pretreated children in each country was infected with resistant viruses to the 3 drug families. Our data support the need for continued surveillance of resistance patterns using DBS at national levels among naive and pretreated children to optimize the ART regimens.

  3. Potassium adsorption ratios as an indicator for the fate of agricultural potassium in groundwater

    NARCIS (Netherlands)

    Griffioen, J.

    2001-01-01

    Fertilization of agricultural land in groundwater infiltration areas often causes deterioration of groundwater quality. In addition to nitrogen and phosphorous, potassium deserves attention. The fate of potassium in the subsurface is controlled mainly by cation-exchange. Use of the Potassium

  4. The Trend of Antibiotic Resistance in Shigellosis and the Diagnostic Value of Erythrocyte Sedimentation Rate for Its Differentiation from Viral Gastroenteritides in Aliasghar Children Hospital, Tehran (1996-2006

    Directory of Open Access Journals (Sweden)

    A.R. Nateghian

    2007-07-01

    Full Text Available Background and objectives As a common infectious disease in childhood, shigellosis has a variety of seasonal and microbiologic patterns and there is a concerning trend of resistance in various parts of the world and even a single country. Inappropriate empiric therapy for any acute episode of gastroenteritis, which might be based on lab studies, could potentially lead to even further resistance. In this report, the erythrocyte sedimentation rate was studied in patients with acute gastroenteritis. Methods Medical records of 117 patients were reviewed in this study. The mean and standard deviation of some of the acute phase inflammatory indicators were compared in two groups of shigellosis and viral gastroenteritides using independent samples t test. Results The mean age of the patients was 49 months, and almost half of them were male. Shigella sonnei was the commonest species isolated from these patients. Shigellosis was found to be more frequent during August to October. After 2001, resistance to ampicillin and cotrimoxazole was observed in 75% and 90% of isolates respectively. A few cases were resistant to third generation of cephalosporins (< 3%. In shigellosis, the erythrocyte sedimentation rate was significantly higher than viral gastroenteritis. Conclusion Empiric use of cotrimoxazole is no longer recommended in children suspicious of contracting shigellosis in Tehran. To avoid unnecessary prescription of antibiotics for acute gastroenteritis, a normal result of erythrocyte sedimentation rate might be helpful.Keywords: Gastroenteritis, Shigella, Bacterial Antibiotic Resistance, Antibiotics, Erythrocyte Sedimentation Rate

  5. Unique Determinants of Neuraminidase Inhibitor Resistance among N3, N7, and N9 Avian Influenza Viruses.

    Science.gov (United States)

    Song, Min-Suk; Marathe, Bindumadhav M; Kumar, Gyanendra; Wong, Sook-San; Rubrum, Adam; Zanin, Mark; Choi, Young-Ki; Webster, Robert G; Govorkova, Elena A; Webby, Richard J

    2015-11-01

    Human infections with avian influenza viruses are a serious public health concern. The neuraminidase (NA) inhibitors (NAIs) are the frontline anti-influenza drugs and are the major option for treatment of newly emerging influenza. Therefore, it is essential to identify the molecular markers of NAI resistance among specific NA subtypes of avian influenza viruses to help guide clinical management. NAI-resistant substitutions in NA subtypes other than N1 and N2 have been poorly studied. Here, we identified NA amino acid substitutions associated with NAI resistance among influenza viruses of N3, N7, and N9 subtypes which have been associated with zoonotic transmission. We applied random mutagenesis and generated recombinant influenza viruses carrying single or double NA substitution(s) with seven internal genes from A/Puerto Rico/8/1934 (H1N1) virus. In a fluorescence-based NA inhibition assay, we identified three categories of NA substitutions associated with reduced inhibition by NAIs (oseltamivir, zanamivir, and peramivir): (i) novel subtype-specific substitutions in or near the enzyme catalytic site (R152W, A246T, and D293N, N2 numbering), (ii) subtype-independent substitutions (E119G/V and/or D and R292K), and (iii) substitutions previously reported in other subtypes (Q136K, I222M, and E276D). Our data show that although some markers of resistance are present across NA subtypes, other subtype-specific markers can only be determined empirically. The number of humans infected with avian influenza viruses is increasing, raising concerns of the emergence of avian influenza viruses resistant to neuraminidase (NA) inhibitors (NAIs). Since most studies have focused on NAI-resistance in human influenza viruses, we investigated the molecular changes in NA that could confer NAI resistance in avian viruses grown in immortalized monolayer cells, especially those of the N3, N7, and N9 subtypes, which have caused human infections. We identified not only numerous NAI-resistant

  6. Potassium in milk and milk products

    International Nuclear Information System (INIS)

    Sombrito, E.Z.; Nuguid, Z.F.S.; Tangonan, M.C.

    1989-01-01

    The amount of potassium in imported processed milk was determined by gamma spectral analysis. The results show that the potassium content of diluted infant formula milk is closest to the reported mean concentration of potassium in human milk while other milk types have potassium values similar to the potassium content of cow milk. (Auth.). 2 figs., 5 refs

  7. Manejo da Planta Daninha Brachiaria plantaginea Resistente aos herbicidas Inibidores da ACCase Management of the Weed Brachiaria plantaginea Resistant to ACCase Inhibitor Herbicides

    Directory of Open Access Journals (Sweden)

    P.J. Christoffoleti

    2001-04-01

    study a population of alexandergrass (Brachiaria plantaginea resistant to ACCase inhibitor herbicides (ariloxyphenoxypropionic and cyclohexanodiones, usually sprayed on soybean under the conservation tillage system. Two experiments were conducted, one under field conditions, comparing the efficacy of non-selective herbicides to ACCase inhibitors, and another under greenhouse conditions, using ACCase inhibitors with nitrogenous additives in the spray solution. Resistant seeds were collected from a site of suspected resistant population, and compared to a population of alexandergrass that had never been sprayed with ACCase inhibitors, the susceptible population. The experiment with non-selective herbicides was conducted under field conditions, but plants were confined to pots of 50 L capacity, avoiding the dissemination of the seeds to adjacent areas. It was then concluded that resistant plants did not show multiple resistance to non selective herbicides with alternative mechanisms of action, with glyphosate, paraquat, paraquat + diuron, MSMA and glufosinate alternative herbicides being possible to be used to control the weed during the winter to manage populations of alexandergrass resistant to ACCase inhibitor herbicides. The additive experiment was conducted under greenhouse conditions in pots, using seeds from the same populations used in the non selective experiment. The herbicides tested were ACCase inhibitors, and the additive treatments were ammonium sulfate and urea. It was then concluded that the additives did not enhance ACCase inhibitor herbicide efficacy in neither of the alexandergrass populations.

  8. High levels of X-linked Inhibitor-of-Apoptosis Protein (XIAP) are indicative of radio chemotherapy resistance in rectal cancer

    International Nuclear Information System (INIS)

    Flanagan, L.; Kehoe, J.; Fay, J.; Bacon, O.; Lindner, A.U.; Kay, E.W.; Deasy, J.; McNamara, D.A.; Prehn, J.H.M.

    2015-01-01

    The mainstay of treatment in rectal cancer is neoadjuvant radio chemotherapy prior to surgery, in an attempt to downstage the tumour, allowing for more complete removal during surgery. In 40 % of cases however, this neoadjuvant radio chemotherapy fails to achieve tumour regression, partly due insufficient apoptosis signaling. X-linked Inhibitor of Apoptosis Protein (XIAP) is an anti-apoptotic protein that has been reported to contribute to disease progression and chemotherapy resistance. We obtained rectal biopsy normal and matched tumour tissue from 29 rectal cancer patients with varying degrees of tumour regression, and using Western blot, examined anti-apoptotic XIAP and pro-apoptotic Smac protein levels in these tissues, with the aim to examine whether disturbed XIAP/Smac levels may be an indicator of neoadjuvant radio chemotherapy resistance. Expression of inhibitor of apoptosis proteins cIAP-1 and cIAP-2 was also examined. We found that levels of XIAP increased in accordance with the degree of radio chemotherapy resistance of the tissue. Levels of this protein were also significantly higher in tumour tissue, compared to matched normal tissue in highly resistant tissue. In contrast, Smac protein levels did not increase with radio chemotherapy resistance, and the protein was similarly expressed in normal and tumour tissue, indicating a shift in the balance of these proteins. Post treatment surgical resection tissue was available for 8 patients. When we compared matched tissue pre- and post- radio chemotherapy we found that XIAP levels increased significantly during treatment in both normal and tumour tissue, while Smac levels did not change. cIAP-1 and cIAP-2 levels were not differentially expressed in varying degrees of radio chemotherapy resistance, and neoadjuvant therapy did not alter expression of these proteins. These data indicate that disturbance of the XIAP/Smac balance may be a driver of radio chemotherapy resistance, and hence high levels of XIAP may

  9. Comparison of ALS functionality and plant growth in ALS-inhibitor susceptible and resistant Myosoton aquaticum L.

    Science.gov (United States)

    Liu, Weitang; Bai, Shuang; Jia, Sisi; Guo, Wenlei; Zhang, Lele; Li, Wei; Wang, Jinxin

    2017-10-01

    Herbicide target-site resistance mutations may cause pleiotropic effects on plant ecology and physiology. The effect of several known (Pro197Ser, Pro197Leu Pro197Ala, and Pro197Glu) target-site resistance mutations of the ALS gene on both ALS functionality and plant vegetative growth of weed Myosoton aquaticum L. (water chickweed) have been investigated here. The enzyme kinetics of ALS from four purified water chickweed populations that each homozygous for the specific target-site resistance-endowing mutations were characterized and the effect of these mutations on plant growth was assessed via relative growth rate (RGR) analysis. Plants homozygous for Pro197Ser and Pro197Leu exhibited higher extractable ALS activity than susceptible (S) plants, while all ALS mutations with no negative change in ALS kinetics. The Pro197Leu mutation increased ALS sensitivity to isoleucine and valine, and Pro197Glu mutation slightly increased ALS sensitivity to isoleucine. RGR results indicated that none of these ALS resistance mutations impose negative pleiotropic effects on relative growth rate. However, resistant (R) seeds had a lowed germination rate than S seeds. This study provides baseline information on ALS functionality and plant growth characteristics associated with ALS inhibitor resistance-endowing mutations in water chickweed. Copyright © 2017. Published by Elsevier Inc.

  10. A Rapid Screening Assay Identifies Monotherapy with Interferon-ß and Combination Therapies with Nucleoside Analogs as Effective Inhibitors of Ebola Virus.

    Directory of Open Access Journals (Sweden)

    Stephen D S McCarthy

    2016-01-01

    Full Text Available To date there are no approved antiviral drugs for the treatment of Ebola virus disease (EVD. While a number of candidate drugs have shown limited efficacy in vitro and/or in non-human primate studies, differences in experimental methodologies make it difficult to compare their therapeutic effectiveness. Using an in vitro model of Ebola Zaire replication with transcription-competent virus like particles (trVLPs, requiring only level 2 biosafety containment, we compared the activities of the type I interferons (IFNs IFN-α and IFN-ß, a panel of viral polymerase inhibitors (lamivudine (3TC, zidovudine (AZT tenofovir (TFV, favipiravir (FPV, the active metabolite of brincidofovir, cidofovir (CDF, and the estrogen receptor modulator, toremifene (TOR, in inhibiting viral replication in dose-response and time course studies. We also tested 28 two- and 56 three-drug combinations against Ebola replication. IFN-α and IFN-ß inhibited viral replication 24 hours post-infection (IC50 0.038μM and 0.016μM, respectively. 3TC, AZT and TFV inhibited Ebola replication when used alone (50-62% or in combination (87%. They exhibited lower IC50 (0.98-6.2μM compared with FPV (36.8μM, when administered 24 hours post-infection. Unexpectedly, CDF had a narrow therapeutic window (6.25-25μM. When dosed >50μM, CDF treatment enhanced viral infection. IFN-ß exhibited strong synergy with 3TC (97.3% inhibition or in triple combination with 3TC and AZT (95.8% inhibition. This study demonstrates that IFNs and viral polymerase inhibitors may have utility in EVD. We identified several 2 and 3 drug combinations with strong anti-Ebola activity, confirmed in studies using fully infectious ZEBOV, providing a rationale for testing combination therapies in animal models of lethal Ebola challenge. These studies open up new possibilities for novel therapeutic options, in particular combination therapies, which could prevent and treat Ebola infection and potentially reduce drug

  11. Effect of polacrilin potassium as disintegrant on bioavailability of diclofenac potassium in tablets : a technical note.

    Science.gov (United States)

    Bele, Mrudula H; Derle, Diliprao V

    2012-09-01

    Polacrilin potassium is an ion exchange resin used in oral pharmaceutical formulations as a tablet disintegrant. It is a weakly acidic cation exchange resin. Chemically, it is a partial potassium salt of a copolymer of methacrylic acid with divinyl benzene. It ionizes to an anionic polymer chain and potassium cations. It was hypothesized that polacrilin potassium may be able to improve the permeability of anionic drugs according to the Donnan membrane phenomenon. The effect of polacrilin potassium on the permeability of diclofenac potassium, used as a model anionic drug, was tested in vitro using diffusion cells and in vivo by monitoring serum levels in rats. The amount of drug permeated across a dialysis membrane in vitro was significantly more in the presence of polacrilin potassium. Significant improvement was found in the extent of drug absorption in vivo. It could be concluded that polacrilin potassium may be used as a high-functionality excipient for improving the bioavailability of anionic drugs having poor gastrointestinal permeability.

  12. 21 CFR 184.1625 - Potassium citrate.

    Science.gov (United States)

    2010-04-01

    ... acid with potassium hydroxide or potassium carbonate. It occurs as transparent crystals or a white... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Potassium citrate. 184.1625 Section 184.1625 Food... Specific Substances Affirmed as GRAS § 184.1625 Potassium citrate. (a) Potassium citrate (C6H5K3O7·H2O, CAS...

  13. Roles of circulating WNT-signaling proteins and WNT-inhibitors in human adiposity, insulin resistance, insulin secretion, and inflammation.

    Science.gov (United States)

    Almario, R U; Karakas, S E

    2015-02-01

    Wingless-type MMTV integration site family member (WNT) signaling and WNT-inhibitors have been implicated in regulation of adipogenesis, insulin resistance, pancreatic function, and inflammation. Our goal was to determine serum proteins involved in WNT signaling (WNT5 and WISP2) and WNT inhibition (SFRP4 and SFRP5) as they relate to obesity, serum adipokines, insulin resistance, insulin secretion, and inflammation in humans. Study population comprised 57 insulin resistant women with polycystic ovary syndrome (PCOS) and 27 reference women. In a cross-sectional study, blood samples were obtained at fasting, during oral, and frequently sampled intravenous glucose tolerance tests. Serum WNT5, WISP2, and SFRP4 concentrations did not differ between PCOS vs. reference women. Serum WNT5 correlated inversely with weight both in PCOS and reference women, and correlated directly with insulin response during oral glucose tolerance test in PCOS women. Serum WISP2 correlated directly with fatty acid binding protein 4. Serum SFRP5 did not differ between obese (n=32) vs. nonobese (n=25) PCOS women, but reference women had lower SFRP5 (pPCOS groups). Serum SFRP5 correlated inversely with IL-1β, TNF-α, cholesterol, and apoprotein B. These findings demonstrated that WNT5 correlated inversely with adiposity and directly with insulin response, and the WNT-inhibitor SFRP5 may be anti-inflammatory. Better understanding of the role of WNT signaling in obesity, insulin resistance, insulin secretion, lipoprotein metabolism, and inflammation is important for prevention and treatment of metabolic syndrome, diabetes and cardiovascular disease. © Georg Thieme Verlag KG Stuttgart · New York.

  14. CONFERENCE REPORT ANTIRETROVIRAL RESISTANCE

    African Journals Online (AJOL)

    2004-08-02

    Aug 2, 2004 ... development of new agents with potential clinical utility for treating resistant ... data on the emergence of resistance among Thai women given zidovudine ... had achieved full virological suppression (viral load. < 50 copies/ml).

  15. Diversity in viral anti-PKR mechanisms: a remarkable case of evolutionary convergence.

    Directory of Open Access Journals (Sweden)

    Elena Domingo-Gil

    Full Text Available Most viruses express during infection products that prevent or neutralize the effect of the host dsRNA activated protein kinase (PKR. Translation of Sindbis virus (SINV mRNA escapes to PKR activation and eIF2 phosphorylation in infected cells by a mechanism that requires a stem loop structure in viral 26S mRNA termed DLP to initiate translation in the absence of functional eIF2. Unlike the rest of viruses tested, we found that Alphavirus infection allowed a strong PKR activation and eIF2α phosphorylation in vitro and in infected animals so that the presence of DLP structure in mRNA was critical for translation and replication of SINV. Interestingly, infection of MEFs with some viruses that express PKR inhibitors prevented eIF2α phosphorylation after superinfection with SINV, suggesting that viral anti-PKR mechanisms could be exchangeable. Thus, translation of SINV mutant lacking the DLP structure (ΔDLP in 26S mRNA was partially rescued in cells expressing vaccinia virus (VV E3 protein, a known inhibitor of PKR. This case of heterotypic complementation among evolutionary distant viruses confirmed experimentally a remarkable case of convergent evolution in viral anti-PKR mechanisms. Our data reinforce the critical role of PKR in regulating virus-host interaction and reveal the versatility of viruses to find different solutions to solve the same conflict.

  16. Struggle for space: viral extinction through competition for cells.

    Science.gov (United States)

    Cuesta, José A; Aguirre, Jacobo; Capitán, José A; Manrubia, Susanna C

    2011-01-14

    The design of protocols to suppress the propagation of viral infections is an enduring enterprise, especially hindered by limited knowledge of the mechanisms leading to viral extinction. Here we report on infection extinction due to intraspecific competition to infect susceptible hosts. Beneficial mutations increase the production of viral progeny, while the host cell may develop defenses against infection. For an unlimited number of host cells, a feedback runaway coevolution between host resistance and progeny production occurs. However, physical space limits the advantage that the virus obtains from increasing offspring numbers; thus, infection clearance may result from an increase in host defenses beyond a finite threshold. Our results might be relevant to devise improved control strategies in environments with mobility constraints or different geometrical properties.

  17. A Rapid Phenotypic Whole Cell Screening Approach for the Identification of Small Molecule Inhibitors that Counter Beta-lactamase Resistance in Pseudomonas aeruginosa

    Science.gov (United States)

    Collia, Deanna; Bannister, Thomas D.; Tan, Hao; Jin, Shouguang; Langaee, Taimour; Shumate, Justin; Scampavia, Louis; Spicer, Timothy P.

    2017-01-01

    Pseudomonas aeruginosa is an opportunistic human pathogen which is prevalent in hospitals and continues to develop resistance to multiple classes of antibiotics. Historically, β-lactam antibiotics have been the first line of therapeutic defense. However, the emergence of multidrug-resistant (MDR) strains of P. aeruginosa, such as AmpC β-lactamase overproducing mutants, limits the effectiveness of current antibiotics. Among AmpC hyper producing clinical isolates, inactivation of AmpG, which is essential for the expression of AmpC, increases bacterial sensitivity to β-lactam antibiotics. We hypothesize that inhibition of AmpG activity will enhance the efficacy of β-lactams against P. aeruginosa. Here, using a highly drug resistant AmpC inducible laboratory strain PAO1, we describe an ultra-high throughput whole cell turbidity assay designed to identify small molecule inhibitors of the AmpG. We screened 645K compounds to identify compounds with the ability to inhibit bacterial growth in the presence of Cefoxitin; an AmpC inducer, and identified 2,663 inhibitors which were also tested in the absence of Cefoxitin to determine AmpG specificity. The Z′ and S:B were robust at 0.87 ± 0.05 and 2.2 ± 0.2, respectively. Through a series of secondary and tertiary studies, including a novel luciferase based counterscreen, we ultimately identified 8 potential AmpG specific inhibitors. PMID:28850797

  18. The mTOR inhibitor, everolimus (RAD001), overcomes resistance to imatinib in quiescent Ph-positive acute lymphoblastic leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Kuwatsuka, Y; Minami, M; Minami, Y; Sugimoto, K; Hayakawa, F; Miyata, Y; Abe, A [Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya (Japan); Goff, D J [Moores Cancer Center, University of California San Diego School of Medicine, La Jolla, CA (United States); Kiyoi, H [Department of Infectious Diseases, Nagoya University Hospital, Nagoya (Japan); Naoe, T [Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya (Japan)

    2011-05-01

    In Ph-positive (Ph{sup +}) leukemia, the quiescent cell state is one of the reasons for resistance to the BCR-ABL-kinase inhibitor, imatinib. In order to examine the mechanisms of resistance due to quiescence and the effect of the mammalian target of rapamycin inhibitor, everolimus, for such a resistant population, we used Ph{sup +} acute lymphoblastic leukemia patient cells serially xenotransplanted into NOD/SCID/IL2rγ{sup null} (NOG) mice. Spleen cells from leukemic mice showed a higher percentage of slow-cycling G{sub 0} cells in the CD34{sup +}CD38{sup −} population compared with the CD34{sup +}CD38{sup +} and CD34{sup −} populations. After ex vivo imatinib treatment, more residual cells were observed in the CD34{sup +}CD38{sup −} population than in the other populations. Although slow-cycling G{sub 0} cells were insensitive to imatinib in spite of BCR-ABL and CrkL dephosphorylation, combination treatment with everolimus induced substantial cell death, including that of the CD34{sup +}CD38{sup −} population, with p70-S6 K dephosphorylation and decrease of MCL-1 expression. The leukemic non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mouse system with the in vivo combination treatment with imatinib and everolimus showed a decrease of tumor burden including CD34{sup +} cells. These results imply that treatment with everolimus can overcome resistance to imatinib in Ph{sup +} leukemia due to quiescence.

  19. Potassium and Your CKD Diet

    Science.gov (United States)

    ... vegetable in your diet, leach them before using. Leaching is a process by which some potassium can be pulled out ... out of my favorite high-potassium vegetables? The process of leaching will help pull potassium out of some high- ...

  20. Surface engineering of titanium with potassium hydroxide and its effects on the growth behavior of mesenchymal stem cells.

    Science.gov (United States)

    Cai, Kaiyong; Lai, Min; Yang, Weihu; Hu, Ran; Xin, Renlong; Liu, Qing; Sung, K L Paul

    2010-06-01

    To improve the corrosion resistance and biological performance of commercially pure titanium (cp-Ti) substrates, potassium hydroxide was employed to modify the surfaces of titanium substrates, followed by biomimetic deposition of apatite on the substrates in a simulated body fluid. The morphologies of native and treated titanium substrates were characterized by field emission scanning electron microscopy (FE-SEM). Treatment with potassium hydroxide led to the formation of intermediate layers of potassium titanate on the surfaces of titanium substrates, while apatite was subsequently deposited onto the intermediate layer. The formation of potassium titanate and apatite was confirmed by thin-film X-ray diffraction and FE-SEM equipped with energy dispersive spectroscopy, respectively. Electrochemical impedance spectroscopy showed that the formed potassium titanate layer improved the corrosion-resistance properties of titanium substrates. The influence of modified titanium substrates on the biological behavior of mesenchymal stem cells (MSCs), including osteogenic differentiation, was investigated in vitro. Compared with cp-Ti substrates, MSCs cultured onto alkali- and heat-treated titanium substrates and apatite-deposited titanium substrates displayed significantly higher (P<0.05 or P<0.01) proliferation and differentiation levels of alkaline phosphatase and osteocalcin in 7 and 14day cultures, respectively. More importantly, our results suggest that the modified titanium substrates have great potential for inducing MSCs to differentiate into osteoblasts. The approach presented here may be exploited to fabricate titanium-based implants. Copyright 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  1. Obtaining of potassium dicyan-argentate

    International Nuclear Information System (INIS)

    Sattarova, M.A.; Solojenkin, P.M.

    1997-01-01

    This work is devoted to obtaining of potassium dicyan-argentate. By means of exchange reaction between silver nitrate and potassium cyanide the potassium dicyan-argentate was synthesized. The analysis of obtained samples was carried out by means of titration and potentiometry.

  2. Extrarenal potassium adaptation: role of skeletal muscle

    International Nuclear Information System (INIS)

    Blachley, J.D.; Crider, B.P.; Johnson, J.H.

    1986-01-01

    Following the ingestion of a high-potassium-content diet for only a few days, the plasma potassium of rats rises only modestly in response to a previously lethal dose of potassium salts. This acquired tolerance, termed potassium adaptation, is principally the result of increased capacity to excrete potassium into the urine. However, a substantial portion of the acute potassium dose is not immediately excreted and is apparently translocated into cells. Previous studies have failed to show an increase in the content of potassium of a variety of tissues from such animals. Using 86 Rb as a potassium analogue, we have shown that the skeletal muscle of potassium-adapted rats takes up significantly greater amounts of potassium in vivo in response to an acute challenge than does that of control animals. Furthermore, the same animals exhibit greater efflux of 86 Rb following the termination of the acute infusion. We have also shown that the Na+-K+-ATPase activity and ouabain-binding capacity of skeletal muscle microsomes are increased by the process of potassium adaptation. We conclude that skeletal muscle is an important participant in potassium adaptation and acts to temporarily buffer acute increases in the extracellular concentration of potassium

  3. Subgroup and resistance analyses of raltegravir for resistant HIV-1 infection

    DEFF Research Database (Denmark)

    Cooper, David A; Steigbigel, Roy T; Gatell, Jose M

    2008-01-01

    BACKGROUND: We evaluated the efficacy of raltegravir and the development of viral resistance in two identical trials involving patients who were infected with human immunodeficiency virus type 1 (HIV-1) with triple-class drug resistance and in whom antiretroviral therapy had failed. METHODS: We c...

  4. Suppressive Effects of the Site 1 Protease (S1P) Inhibitor, PF-429242, on Dengue Virus Propagation.

    Science.gov (United States)

    Uchida, Leo; Urata, Shuzo; Ulanday, Gianne Eduard L; Takamatsu, Yuki; Yasuda, Jiro; Morita, Kouichi; Hayasaka, Daisuke

    2016-02-10

    Dengue virus (DENV) infection causes one of the most widespread mosquito-borne diseases in the world. Despite the great need, effective vaccines and practical antiviral therapies are still under development. Intracellular lipid levels are regulated by sterol regulatory elements-binding proteins (SREBPs), which are activated by serine protease, site 1 protease (S1P). Small compound PF-429242 is known as a S1P inhibitor and the antivirus effects have been reported in some viruses. In this study, we examined the anti-DENV effects of PF-429242 using all four serotypes of DENV by several primate-derived cell lines. Moreover, emergence of drug-resistant DENV mutants was assessed by sequential passages with the drug. DENV dependency on intracellular lipids during their infection was also evaluated by adding extracellular lipids. The addition of PF-429242 showed suppression of viral propagation in all DENV serotypes. We showed that drug-resistant DENV mutants are unlikely to emerge after five times sequential passages through treatment with PF-429242. Although the levels of intracellular cholesterol and lipid droplets were reduced by PF-429242, viral propagations were not recovered by addition of exogenous cholesterol or fatty acids, indicating that the reduction of LD and cholesterol caused by PF-429242 treatment is not related to its mechanism of action against DENV propagation. Our results suggest that PF-429242 is a promising candidate for an anti-DENV agent.

  5. Structural studies of series HIV-1 nonnucleoside reverse transcriptase inhibitors 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)-benzimidazoles with different 4-substituents

    Science.gov (United States)

    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 the angular orientation of the π-electron systems and influencing biological activity, were determined for all of the investigated inhibitors. The chemical character and orientation of the substituent at C4 position of the benzimidazole moiety substantially influences the anti-viral activity. The structural data of the investigated inhibitors is a good basis for modeling enzyme-inhibitor interactions for structure-assisted drug design.

  6. Alpha-momorcharin enhances Tobacco mosaic virus resistance in tobaccoNN by manipulating jasmonic acid-salicylic acid crosstalk.

    Science.gov (United States)

    Yang, Ting; Zhu, Li-Sha; Meng, Yao; Lv, Rui; Zhou, Zhuo; Zhu, Lin; Lin, Hong-Hui; Xi, De-Hui

    2018-04-01

    Alpha-momorcharin (α-MMC) is a type-I ribosome inactivating protein (RIP) with a molecular weight of 29 kDa found in plants. This protein has been shown to be effective against a broad range of human viruses and also has anti-tumor activities. However, the mechanism by which α-MMC induces plant defense responses and regulates the N gene to promote resistance to the Tobacco mosaic virus (TMV) is still not clear. By using pharmacological and infection experiments, we found that α-MMC enhances TMV resistance of tobacco plants containing the N gene (tobacco NN ). Our results showed that plants pretreated with 0.5 mg/ml α-MMC could relieve TMV-induced oxidative damage, had enhanced the expression of the N gene and increased biosynthesis of jasmonic acid (JA) and salicylic acid (SA). Moreover, transcription of JA and SA signaling pathway genes were increased, and their expression persisted for a longer period of time in plants pretreated with α-MMC compared with those pretreated with water. Importantly, exogenous application of 1-Aminobenzotriazole (ABT, SA inhibitor) and ibuprofen (JA inhibitor) reduced α-MMC induced plant resistance under viral infection. Thus, our results revealed that α-MMC enhances TMV resistance of tobacco NN plants by manipulating JA-SA crosstalk. Copyright © 2017 Elsevier GmbH. All rights reserved.

  7. Drug resistance to inhibitors of the human double minute-2 E3 ligase is mediated by point mutations of p53, but can be overcome with the p53 targeting agent RITA.

    Science.gov (United States)

    Jones, Richard J; Bjorklund, Chad C; Baladandayuthapani, Veerabhadran; Kuhn, Deborah J; Orlowski, Robert Z

    2012-10-01

    The human double minute (HDM)-2 E3 ubiquitin ligase plays a key role in p53 turnover and has been validated preclinically as a target in multiple myeloma (MM) and mantle cell lymphoma (MCL). HDM-2 inhibitors are entering clinical trials, and we therefore sought to understand potential mechanisms of resistance in lymphoid models. Wild-type p53 H929 MM and Granta-519 MCL cells resistant to MI-63 or Nutlin were generated by exposing them to increasing drug concentrations. MI-63-resistant H929 and Granta-519 cells were resistant to Nutlin, whereas Nutlin-resistant cells displayed cross-resistance to MI-63. These cells also showed cross-resistance to bortezomib, doxorubicin, cisplatin, and melphalan, but remained sensitive to the small molecule inhibitor RITA (reactivation of p53 and induction of tumor cell apoptosis). HDM-2 inhibitor-resistant cells harbored increased p53 levels, but neither genotoxic nor nongenotoxic approaches to activate p53 induced HDM-2 or p21. Resequencing revealed wild-type HDM-2, but mutations were found in the p53 DNA binding and dimerization domains. In resistant cells, RITA induced a G(2)-M arrest, upregulation of p53 targets HDM-2, PUMA, and NOXA, and PARP cleavage. Combination regimens with RITA and MI-63 resulted in enhanced cell death compared with RITA alone. These findings support the possibility that p53 mutation could be a primary mechanism of acquired resistance to HDM-2 inhibitors in MCL and MM. Furthermore, they suggest that simultaneous restoration of p53 function and HDM-2 inhibition is a rational strategy for clinical translation.

  8. Eleusine indica resistance to Accase inhibitors

    OpenAIRE

    Vidal, Ribas Antonio; Portes, Emerson da Silva; Lamego, Fabiane Pinto; Trezzi, Michelangelo Muzell

    2006-01-01

    Dentre as causas da ineficácia no controle de plantas daninhas destaca-se a resistência delas aos herbicidas. Os objetivos deste trabalho foram avaliar a suspeita de resistência de Eleusine indica a inibidores de acetil-CoA carboxilase (ACCase) e investigar a ocorrência de resistência cruzada entre os inibidores de ACCase. Biótipo de Eleusine indica originado do Mato Grosso com suspeita de resistência aos herbicidas inibidores de ACCase foi avaliado em casa de vegetação na sua suscetibilidade...

  9. Mutations Conferring Resistance to Viral DNA Polymerase Inhibitors in Camelpox Virus Give Different Drug-Susceptibility Profiles in Vaccinia Virus

    Czech Academy of Sciences Publication Activity Database

    Duraffour, S.; Andrei, G.; Topalis, D.; Krečmerová, Marcela; Crance, J. M.; Garin, D.; Snoeck, R.

    2012-01-01

    Roč. 86, č. 13 (2012), s. 7310-7325 ISSN 0022-538X Institutional support: RVO:61388963 Keywords : camelpox virus * CMLV * vaccinia virus VACV * acyclic nucleoside phosphonates * HPMPDAP * cidofovir * drug resistance Subject RIV: CC - Organic Chemistry Impact factor: 5.076, year: 2012

  10. VEGF attenuated increase of outward delayed-rectifier potassium currents in hippocampal neurons induced by focal ischemia via PI3-K pathway.

    Science.gov (United States)

    Wu, K W; Yang, P; Li, S S; Liu, C W; Sun, F Y

    2015-07-09

    We recently indicated that the vascular endothelial growth factor (VEGF) protects neurons against hypoxic death via enhancement of tyrosine phosphorylation of Kv1.2, an isoform of the delayed-rectifier potassium channels through activation of the phosphatidylinositol 3-kinase (PI3-K) signaling pathway. The present study investigated whether VEGF could attenuate ischemia-induced increase of the potassium currents in the hippocampal pyramidal neurons of rats after ischemic injury. Adult male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion (MCAO) to induce brain ischemia. The whole-cell patch-clamp technique was used to record the potassium currents of hippocampal neurons in brain slices from the ischemically injured brains of the rats 24h after MCAO. We detected that transient MCAO caused a significant increase of voltage-gated potassium currents (Kv) and outward delayed-rectifier potassium currents (IK), but not outward transient potassium currents (IA), in the ipsilateral hippocampus compared with the sham. Moreover, we found that VEGF could acutely, reversibly and voltage-dependently inhibit the ischemia-induced IK increase. This inhibitory effect of VEGF could be completely abolished by wortmannin, an inhibitor of PI3-K. Our data indicate that VEGF attenuates the ischemia-induced increase of IK via activation of the PI3-K signaling pathway. Published by Elsevier Ltd.

  11. 21 CFR 184.1631 - Potassium hydroxide.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Potassium hydroxide. 184.1631 Section 184.1631 Food... Specific Substances Affirmed as GRAS § 184.1631 Potassium hydroxide. (a) Potassium hydroxide (KOH, CAS Reg... pellets, flakes, sticks, lumps, and powders. Potassium hydroxide is obtained commercially from the...

  12. Hepatitis C virus cell-cell transmission and resistance to direct-acting antiviral agents

    DEFF Research Database (Denmark)

    Xiao, Fei; Fofana, Isabel; Heydmann, Laura

    2014-01-01

    Hepatitis C virus (HCV) is transmitted between hepatocytes via classical cell entry but also uses direct cell-cell transfer to infect neighboring hepatocytes. Viral cell-cell transmission has been shown to play an important role in viral persistence allowing evasion from neutralizing antibodies....... In contrast, the role of HCV cell-cell transmission for antiviral resistance is unknown. Aiming to address this question we investigated the phenotype of HCV strains exhibiting resistance to direct-acting antivirals (DAAs) in state-of-the-art model systems for cell-cell transmission and spread. Using HCV...... genotype 2 as a model virus, we show that cell-cell transmission is the main route of viral spread of DAA-resistant HCV. Cell-cell transmission of DAA-resistant viruses results in viral persistence and thus hampers viral eradication. We also show that blocking cell-cell transmission using host...

  13. Insect symbiotic bacteria harbour viral pathogens for transovarial transmission.

    Science.gov (United States)

    Jia, Dongsheng; Mao, Qianzhuo; Chen, Yong; Liu, Yuyan; Chen, Qian; Wu, Wei; Zhang, Xiaofeng; Chen, Hongyan; Li, Yi; Wei, Taiyun

    2017-03-06

    Many insects, including mosquitoes, planthoppers, aphids and leafhoppers, are the hosts of bacterial symbionts and the vectors for transmitting viral pathogens 1-3 . In general, symbiotic bacteria can indirectly affect viral transmission by enhancing immunity and resistance to viruses in insects 3-5 . Whether symbiotic bacteria can directly interact with the virus and mediate its transmission has been unknown. Here, we show that an insect symbiotic bacterium directly harbours a viral pathogen and mediates its transovarial transmission to offspring. We observe rice dwarf virus (a plant reovirus) binding to the envelopes of the bacterium Sulcia, a common obligate symbiont of leafhoppers 6-8 , allowing the virus to exploit the ancient oocyte entry path of Sulcia in rice leafhopper vectors. Such virus-bacterium binding is mediated by the specific interaction of the viral capsid protein and the Sulcia outer membrane protein. Treatment with antibiotics or antibodies against Sulcia outer membrane protein interferes with this interaction and strongly prevents viral transmission to insect offspring. This newly discovered virus-bacterium interaction represents the first evidence that a viral pathogen can directly exploit a symbiotic bacterium for its transmission. We believe that such a model of virus-bacterium communication is a common phenomenon in nature.

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

    Science.gov (United States)

    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.

  15. Synthesis of poly[N-(2-hydroxypropyl)methacrylamide] conjugates of inhibitors of the ABC transporter that overcome multidrug resistance in doxorubicin-resistant P388 cells in vitro

    Czech Academy of Sciences Publication Activity Database

    Šubr, Vladimír; Sivák, Ladislav; Koziolová, Eva; Braunová, Alena; Pechar, Michal; Strohalm, Jiří; Kabešová, Martina; Říhová, Blanka; Ulbrich, Karel; Kovář, Marek

    2014-01-01

    Roč. 15, č. 8 (2014), s. 3030-3043 ISSN 1525-7797 R&D Projects: GA ČR GAP301/12/1254; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61389013 ; RVO:61388971 Keywords : N-(2-hydroxypropyl)methacrylamide copolymers * multidrug resistance * P-glycoprotein inhibitors Subject RIV: CD - Macromolecular Chemistry; FD - Oncology ; Hematology (MBU-M) Impact factor: 5.750, year: 2014

  16. 21 CFR 184.1622 - Potassium chloride.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Potassium chloride. 184.1622 Section 184.1622 Food... Specific Substances Affirmed as GRAS § 184.1622 Potassium chloride. (a) Potassium chloride (KCl, CAS Reg... levels not to exceed current good manufacturing practice. Potassium chloride may be used in infant...