Science.gov

Sample records for proline-based neuraminidase inhibitor

  1. Neuraminidase

    NASA Technical Reports Server (NTRS)

    2004-01-01

    The reproduction process of all strains of influenza are dependent on the same enzyme neuraminidase. Pharmaceutical companies have been developing drugs that can inhibit the function of neuraminidase hoping to create an effective weapon against the flu. Researchers from the pharmaceutical industry and from the Center for Macromolecular Crystallography have grown crystals of neuraminidase in space. These improved, space-grown crystals have provided information that have helped design drugs which form a stronger interaction with the enzyme. These drugs inhibit neuraminidase by attaching themselves to the enzyme. Since the drugs are less likely to detach from the enzyme, they are more effective, require smaller dosages, and have fewer side effects. Shown here is a segmented representation of the neuraminidase inhibitor compound sitting inside a cave-like contour of the neuraminidase enzyme surface. This cave-like formation present in every neuraminidase enzyme is the active site crucial to the flu's ability to infect. The space-grown crystals of neuraminidase have provided significant new details about the three-dimensional characteristics of this active site thus allowing researchers to design drugs that fit tighter into the site. Principal Investigator: Dr. Larry DeLucas

  2. Novel Potent Hepatitis C Virus NS3 Serine Protease Inhibitors Derived from Proline-Based Macrocycles

    SciTech Connect

    Chen, Kevin X.; Njoroge, F. George; Arasappan, Ashok; Venkatraman, Srikanth; Vibulbhan, Bancha; Yang, Weiying; Parekh, Tejal N.; Pichardo, John; Prongay, Andrew; Cheng, Kuo-Chi; Butkiewicz, Nancy; Yao, Nanhua; Madison, Vincent; Girijavallabhan, Viyyoor

    2008-06-30

    The hepatitis C virus (HCV) NS3 protease is essential for viral replication. It has been a target of choice for intensive drug discovery research. On the basis of an active pentapeptide inhibitor, 1, we envisioned that macrocyclization from the P2 proline to P3 capping could enhance binding to the backbone Ala156 residue and the S4 pocket. Thus, a number of P2 proline-based macrocyclic {alpha}-ketoamide inhibitors were prepared and investigated in an HCV NS3 serine protease continuous assay (K*{sub i}). The biological activity varied substantially depending on factors such as the ring size, number of amino acid residues, number of methyl substituents, type of heteroatom in the linker, P3 residue, and configuration at the proline C-4 center. The pentapeptide inhibitors were very potent, with the C-terminal acids and amides being the most active ones (24, K*{sub i} = 8 nM). The tetrapeptides and tripeptides were less potent. Sixteen- and seventeen-membered macrocyclic compounds were equally potent, while fifteen-membered analogues were slightly less active. gem-Dimethyl substituents at the linker improved the potency of all inhibitors (the best compound was 45, K*{sub i} = 6 nM). The combination of tert-leucine at P3 and dimethyl substituents at the linker in compound 47 realized a selectivity of 307 against human neutrophil elastase. Compound 45 had an IC{sub 50} of 130 nM in a cellular replicon assay, while IC{sub 50} for 24 was 400 nM. Several compounds had excellent subcutaneous AUC and bioavailability in rats. Although tripeptide compound 40 was 97% orally bioavailable, larger pentapeptides generally had low oral bioavailability. The X-ray crystal structure of compounds 24 and 45 bound to the protease demonstrated the close interaction of the macrocycle with the Ala156 methyl group and S4 pocket. The strategy of macrocyclization has been proved to be successful in improving potency (>20-fold greater than that of 1) and in structural depeptization.

  3. Peramivir Phosphonate Derivatives as Influenza Neuraminidase Inhibitors.

    PubMed

    Wang, Peng-Cheng; Fang, Jim-Min; Tsai, Keng-Chang; Wang, Shi-Yun; Huang, Wen-I; Tseng, Yin-Chen; Cheng, Yih-Shyun E; Cheng, Ting-Jen Rachel; Wong, Chi-Huey

    2016-06-01

    Peramivir is a potent neuraminidase (NA) inhibitor for treatment of influenza infection by intravenous administration. By replacing the carboxylate group in peramivir with a phosphonate group, phosphono-peramivir (6a), the dehydration and deoxy derivatives (7a and 8a) as well as their corresponding monoalkyl esters are prepared from a pivotal intermediate epoxide 12. Among these phosphonate compounds, the dehydration derivative 7a that has a relatively rigid cyclopentene core structure exhibits the strongest inhibitory activity (IC50 = 0.3-4.1 nM) against several NAs of wild-type human and avian influenza viruses (H1N1, H3N2, H5N1, and H7N9), although the phosphonate congener 6a is unexpectedly less active than peramivir. The inferior binding affinity of 6a is attributable to the deviated orientations of its phosphonic acid and 3-pentyl groups in the NA active site as inferred from the NMR, X-ray diffraction, and molecular modeling analyses. Compound 7a is active to the oseltamivir-resistant H275Y strains of H1N1 and H5N1 viruses (IC50 = 73-86 nM). The phosphonate monoalkyl esters (6b, 6c, 7b, 7c, 8b, and 8c) are better anti-influenza agents (EC50 = 19-89 nM) than their corresponding phosphonic acids (EC50 = 50-343 nM) in protection of cells from the viral infection. The phosphonate monoalkyl esters are stable in buffer solutions (pH 2.0-7.4) and rabbit serum; furthermore, the alkyl group is possibly tuned to attain the desired pharmacokinetic properties. PMID:27167096

  4. Widespread use of neuraminidase inhibitors in Japan.

    PubMed

    Sugaya, Norio

    2011-10-01

    Almost all patients with an influenza-like illness in Japan are now tested with rapid diagnostic tests, and when positive, they are treated with a neuraminidase inhibitor (NAI). Japan may have had the lowest case fatality rate for symptomatic illness (<0.001%, 198/20.7 million) in the H1N1/09 pandemic because of the universal implementation of early treatment with NAI. A study of 1,000 children hospitalized because of a H1N1/09 infection revealed that NAIs, primarily oseltamivir, had been used to treat 984 (98.4%) of the 1,000 patients. In 88.9% of the patients, treatment with NAIs was initiated within 48 h after the onset of illness. In addition to oseltamivir and zanamivir, the newly approved inhalant drug, laninamivir, and the newly approved intravenous drug, peramivir, were used in Japan during the 2010-2011 season. Neuropsychiatric disorders that were suspected of being adverse reactions to oseltamivir became a cause of concern in 2007. The Health, Labour and Welfare Ministry issued an emergency instruction to suspend the use of oseltamivir to treat patients between the ages of 10 and 19 years. However, according to the Vital Statistics data, the widespread use of oseltamivir has not caused an increase in deaths as a result of accidental falls or intentional jumps from buildings. Although oseltamivir is widely used in Japan, no outbreaks have been caused by oseltamivir-resistant viruses, and no serious illness caused by oseltamivir-resistant viruses has ever been reported.

  5. Neuraminidase Inhibitors from the Fruiting Body of Phellinus igniarius

    PubMed Central

    Kim, Ji-Yul; Kim, Dae-Won; Hwang, Byung Soon; Woo, E-Eum; Lee, Yoon-Ju; Jeong, Kyeong-Woon; Lee, In-Kyoung

    2016-01-01

    During our ongoing investigation of neuraminidase inhibitors from medicinal fungi, we found that the fruiting bodies of Phellinus igniarius exhibited significant inhibitory activity against neuraminidase from recombinant H3N2 influenza viruses. Two active compounds were isolated from the methanolic extract of P. igniarius through solvent partitioning and Sephadex LH-20 column chromatography. The active compounds were identified as phelligridins E and G on proton nuclear magnetic resonance (1H NMR) and electrospray ionization mass measurements. These compounds inhibited neuraminidases from recombinant rvH1N1, H3N2, and H5N1 influenza viruses, with IC50 values in the range of 0.7~8.1 µM. PMID:27433123

  6. Neuraminidase Inhibitors from the Fruiting Body of Phellinus igniarius.

    PubMed

    Kim, Ji-Yul; Kim, Dae-Won; Hwang, Byung Soon; Woo, E-Eum; Lee, Yoon-Ju; Jeong, Kyeong-Woon; Lee, In-Kyoung; Yun, Bong-Sik

    2016-06-01

    During our ongoing investigation of neuraminidase inhibitors from medicinal fungi, we found that the fruiting bodies of Phellinus igniarius exhibited significant inhibitory activity against neuraminidase from recombinant H3N2 influenza viruses. Two active compounds were isolated from the methanolic extract of P. igniarius through solvent partitioning and Sephadex LH-20 column chromatography. The active compounds were identified as phelligridins E and G on proton nuclear magnetic resonance ((1)H NMR) and electrospray ionization mass measurements. These compounds inhibited neuraminidases from recombinant rvH1N1, H3N2, and H5N1 influenza viruses, with IC50 values in the range of 0.7~8.1 µM. PMID:27433123

  7. Proline-Based Macrocyclic Inhibitors of the Hepatitis C Virus: Stereoselective Synthesis and Biological Activity

    SciTech Connect

    Chen, Kevin X.; Njoroge, F. George; Vibulbhan, Bancha; Prongay, Andrew; Pichardo, John; Madison, Vincent; Buevich, Alexei; Chan, Tze-Ming

    2008-06-30

    Macrocyclization through a Mitsunobu reaction was used to synthesize a 17-membered macrocycle. The bicyclic acetal core was prepared completely diastereoselectively. The macrocyclic peptidomimetic surrogate of the P2-P3 dipeptide moiety was designed to function as a hepatitis C virus (HCV) NS3 serine protease inhibitor, and the pentapeptide {alpha}-ketoamides derived from the macrocycle were shown to be potent HCV inhibitors.

  8. Exploring the chemical space of influenza neuraminidase inhibitors

    PubMed Central

    Anuwongcharoen, Nuttapat; Shoombuatong, Watshara; Tantimongcolwat, Tanawut; Prachayasittikul, Virapong

    2016-01-01

    The fight against the emergence of mutant influenza strains has led to the screening of an increasing number of compounds for inhibitory activity against influenza neuraminidase. This study explores the chemical space of neuraminidase inhibitors (NAIs), which provides an opportunity to obtain further molecular insights regarding the underlying basis of their bioactivity. In particular, a large set of 347 and 175 NAIs against influenza A and B, respectively, was compiled from the literature. Molecular and quantum chemical descriptors were obtained from low-energy conformational structures geometrically optimized at the PM6 level. The bioactivities of NAIs were classified as active or inactive according to their half maximum inhibitory concentration (IC50) value in which IC50 < 1µM and ≥ 10µM were defined as active and inactive compounds, respectively. Interpretable decision rules were derived from a quantitative structure–activity relationship (QSAR) model established using a set of substructure descriptors via decision tree analysis. Univariate analysis, feature importance analysis from decision tree modeling and molecular scaffold analysis were performed on both data sets for discriminating important structural features amongst active and inactive NAIs. Good predictive performance was achieved as deduced from accuracy and Matthews correlation coefficient values in excess of 81% and 0.58, respectively, for both influenza A and B NAIs. Furthermore, molecular docking was employed to investigate the binding modes and their moiety preferences of active NAIs against both influenza A and B neuraminidases. Moreover, novel NAIs with robust binding fitness towards influenza A and B neuraminidase were generated via combinatorial library enumeration and their binding fitness was on par or better than FDA-approved drugs. The results from this study are anticipated to be beneficial for guiding the rational drug design of novel NAIs for treating influenza infections. PMID

  9. Pandemic influenza vaccines and neuraminidase inhibitors: efficacy and side effects.

    PubMed

    Bijl, D

    2011-01-01

    At the time of the outbreak of the pandemic of New Influenza A (H1N1) pandemic influenza vaccines became available via an accelerated registration procedure. In 2005 large stocks of the neuraminidase inhibitor oseltamivir were built up in the Netherlands and other western countries. There was considerable doubt about the efficacy of this medicine. Initially reported positive effects of the drug were largely based on unpublished research, which was sponsored by the manufacturer and was partially written by ghostwriters. There now have been reports of rare and serious side effects. The first reports on the severity of the pandemic in Australia and New Zealand indicated a mild course.

  10. Using Support Vector Machine (SVM) for Classification of Selectivity of H1N1 Neuraminidase Inhibitors.

    PubMed

    Li, Yang; Kong, Yue; Zhang, Mengdi; Yan, Aixia; Liu, Zhenming

    2016-04-01

    Inhibition of the neuraminidase is one of the most promising strategies for preventing influenza virus spreading. 479 neuraminidase inhibitors are collected for dataset 1 and 208 neuraminidase inhibitors for A/P/8/34 are collected for dataset 2. Using support vector machine (SVM), four computational models were built to predict whether a compound is an active or weakly active inhibitor of neuraminidase. Each compound is represented by MASSC fingerprints and ADRIANA.Code descriptors. The predication accuracies for the test sets of all the models are over 78 %. Model 2B, which is the best model, obtains a prediction accuracy and a Matthews Correlation Coefficient (MCC) of 89.71 % and 0.81 on test set, respectively. The molecular polarizability, molecular shape, molecular size and hydrogen bonding are related to the activities of neuraminidase inhibitors. The models can be obtained from the authors. PMID:27491921

  11. Photolabeling of the alpha-neuraminidase/beta-galactosidase complex from human placenta with a photoreactive neuraminidase inhibitor

    SciTech Connect

    Warner, T.G.; Louie, A.; Potier, M. )

    1990-11-30

    Photolabeling of the alpha-neuraminidase/beta-galactosidase complex in human placenta was carried out using the radioactive photoprobe, 9-S-(4-azido-3,5-3H-2-nitrophenyl)-5-acetamido-2,6 anhydro-2,3,5,9- tetradeoxy-9- thio-D-glycero-D-galacto-non-2-enonic acid. Two intensely labeled bands at 61 and 46 kD were detected with autoradiography. Labeling of the 46 kD protein was blocked with the inclusion of the surfactant Triton X-100 in the photolysis mixture, indicating a nonspecific, hydrophobic interaction. The 61 kD protein was protected from labeling only when the neuraminidase inhibitor 2,3 dehydro N-acetyl neuraminic acid (1 mM) was present during photolysis. These results suggest that the neuraminidase activity resides among the proteins in the 61 kD molecular weight range comigrating with the lysosomal beta-galactosidase, under denaturing conditions.

  12. Integrating docking and molecular dynamics approaches for a series of proline-based 2,5-diketopiperazines as novel αβ-tubulin inhibitors.

    PubMed

    Fani, Najmeh; Bordbar, Abdol-Khalegh; Ghayeb, Yousef; Sepehri, Saghi

    2015-01-01

    In this work, docking tools were utilized in order to study the binding properties of more than five hundred of proline-based 2,5-diketopiperazine in the binding site of αβ-tubulin. Results revealed that 20 compounds among them showed lower binding energies in comparison with Tryprostatin-A, a well known tubulin inhibitor and therefore could be potential inhibitors of tubulin. However, the precise evaluation of binding poses represents the similar binding modes for all of these compounds and Tryprostatin-A. Finally, the best docked complex was subjected to a 25 ns molecular dynamics simulation to further validate the proposed binding mode of this compound.

  13. Influenza neuraminidase inhibitors: antiviral action and mechanisms of resistance

    PubMed Central

    McKimm‐Breschkin, Jennifer L.

    2012-01-01

    Please cite this paper as: McKimm‐Breschkin (2012) Influenza neuraminidase inhibitors: Antiviral action and mechanisms of resistance. Influenza and Other Respiratory Viruses 7(Suppl. 1), 25–36. There are two major classes of antivirals available for the treatment and prevention of influenza, the M2 inhibitors and the neuraminidase inhibitors (NAIs). The M2 inhibitors are cheap, but they are only effective against influenza A viruses, and resistance arises rapidly. The current influenza A H3N2 and pandemic A(H1N1)pdm09 viruses are already resistant to the M2 inhibitors as are many H5N1 viruses. There are four NAIs licensed in some parts of the world, zanamivir, oseltamivir, peramivir, and a long‐acting NAI, laninamivir. This review focuses on resistance to the NAIs. Because of differences in their chemistry and subtle differences in NA structures, resistance can be both NAI‐ and subtype specific. This results in different drug resistance profiles, for example, the H274Y mutation confers resistance to oseltamivir and peramivir, but not to zanamivir, and only in N1 NAs. Mutations at E119, D198, I222, R292, and N294 can also reduce NAI sensitivity. In the winter of 2007–2008, an oseltamivir‐resistant seasonal influenza A(H1N1) strain with an H274Y mutation emerged in the northern hemisphere and spread rapidly around the world. In contrast to earlier evidence of such resistant viruses being unfit, this mutant virus remained fully transmissible and pathogenic and became the major seasonal A(H1N1) virus globally within a year. This resistant A(H1N1) virus was displaced by the sensitive A(H1N1)pdm09 virus. Approximately 0·5–1·0% of community A(H1N1)pdm09 isolates are currently resistant to oseltamivir. It is now apparent that variation in non‐active site amino acids can affect the fitness of the enzyme and compensate for mutations that confer high‐level oseltamivir resistance resulting in minimal impact on enzyme function. PMID:23279894

  14. Screening of neuraminidase inhibitors from traditional Chinese medicines by integrating capillary electrophoresis with immobilized enzyme microreactor.

    PubMed

    Zhao, Haiyan; Chen, Zilin

    2014-05-01

    A simple and effective neuraminidase-immobilized capillary microreactor was fabricated by glutaraldehyde cross-linking technology for screening the neuraminidase inhibitors from traditional Chinese medicines. The substrate and product were separated by CE in short-end injection mode within 2 min. Dual-wavelength ultraviolet detection was employed to eliminate the interference from the screened compounds. The parameters relating to the separation efficiency and the activity of immobilized neuraminidase were systematically evaluated. The activity of the immobilized neuraminidase remained 90% after 30 days storage at 4°C. The immobilized NA microreactor could be continuously used for more than 200 runs. The Michaelis-Menten constant of neuraminidase was determined by the microreactor as 136.6 ± 10.8 μM. In addition, six in eighteen natural products were found as potent inhibitors and the inhibition potentials were ranked in the following order: bavachinin>bavachin>baicalein>baicalin>chrysin and vitexin. The half-maximal inhibitory concentrations were 59.52 ± 4.12, 65.28 ± 1.07, 44.79 ± 1.21 and 31.62 ± 2.04 for baicalein, baicalin, bavachin and bavachinin, respectively. The results demonstrated that the neuraminidase-immobilized capillary microreactor was a very effective tool for screening neuraminidase inhibitors from traditional Chinese medicines.

  15. Potential Drug-Like Inhibitors of Group 1 Influenza Neuraminidase Identified through Computer-Aided Drug Design

    PubMed Central

    Durrant, Jacob D.; McCammon, J. Andrew

    2010-01-01

    Pandemic (H1N1) influenza poses an imminent threat. Nations have stockpiled inhibitors of the influenza protein neuraminidase in hopes of protecting their citizens, but drug-resistant strains have already emerged, and novel therapeutics are urgently needed. In the current work, the computer program AutoGrow is used to generate novel predicted neuraminidase inhibitors. Given the great flexibility of the neuraminidase active site, protein dynamics are also incorporated into the computer-aided drug-design process. Several potential inhibitors are identified that are predicted to bind neuraminidase better than currently approved drugs. PMID:20427241

  16. Neuraminidase inhibitor R-125489 - A promising drug for treating influenza virus: Steered molecular dynamics approach

    SciTech Connect

    Mai, Binh Khanh; Li, Mai Suan

    2011-07-08

    Highlights: {yields} We study binding affinity of R-125489 and its prodrug CS-8958 to neuraminidase of pathogenic influenza viruses by molecular dynamics simulations. {yields} It is shown that, in agreement with experiments, R-125489 binds to neuraminidase more tightly than CS-8958. {yields} We predict that R-125489 can be used to treat not only wild-type but also tamiflu-resistant N294S, H274Y variants of A/H5N1 virus. {yields} The high correlation between theoretical and experimental data implies that SMD is a very promising tool for drug design. -- Abstract: Two neuraminidase inhibitors, oseltamivir and zanamivir, are important drug treatments for influenza. Oseltamivir-resistant mutants of the influenza virus A/H1N1 and A/H5N1 have emerged, necessitating the development of new long-acting antiviral agents. One such agent is a new neuraminidase inhibitor R-125489 and its prodrug CS-8958. An atomic level understanding of the nature of this antiviral agents binding is still missing. We address this gap in our knowledge by applying steered molecular dynamics (SMD) simulations to different subtypes of seasonal and highly pathogenic influenza viruses. We show that, in agreement with experiments, R-125489 binds to neuraminidase more tightly than CS-8958. Based on results obtained by SMD and the molecular mechanics-Poisson-Boltzmann surface area method, we predict that R-125489 can be used to treat not only wild-type but also tamiflu-resistant N294S, H274Y variants of A/H5N1 virus as its binding affinity does not vary much across these systems. The high correlation level between theoretically determined rupture forces and experimental data on binding energies for the large number of systems studied here implies that SMD is a promising tool for drug design.

  17. Reduced susceptibility to all neuraminidase inhibitors of influenza H1N1 viruses with haemagglutinin mutations and mutations in non-conserved residues of the neuraminidase

    PubMed Central

    McKimm-Breschkin, Jennifer L.; Williams, Janelle; Barrett, Susan; Jachno, Kim; McDonald, Mandy; Mohr, Peter G.; Saito, Takehiko; Tashiro, Masato

    2013-01-01

    Objectives We characterized human H1N1 influenza isolate A/Hokkaido/15/02, which has haemagglutinin and neuraminidase mutations that reduce drug susceptibility to oseltamivir, zanamivir and peramivir. Methods One wild-type and three mutant viruses were isolated by plaque purification. Viruses were tested in MUNANA-based enzyme assays, cell culture and receptor binding assays. Results Two viruses had a neuraminidase Y155H mutation that reduced susceptibility in the enzyme inhibition assay to all inhibitors by 30-fold to >100-fold. The Y155H mutation reduced plaque size and affected the stability, Km and pH activity profile of the enzyme. In contrast to previous mutants, this neuraminidase demonstrated a slower rate of inhibitor binding in the IC50 kinetics assay. One virus had both the Y155H mutation and a haemagglutinin D225G mutation that rescued the small-plaque phenotype of the Y155H virus and affected receptor binding and drug susceptibility in cell culture and binding assays. We also isolated a third mutant virus, with both neuraminidase V114I and haemagglutinin D225N mutations, which affected susceptibility in the enzyme inhibition assay and receptor binding, respectively, but to lesser extents than the Y155H and D225G mutations. Conclusions Neither Y155 nor V114 is conserved across neuraminidase subtypes. Furthermore, Y155 is not conserved even among avian and swine N1 viruses. Structurally, both residues reside far from the neuraminidase active site. D225 forms part of the receptor binding site of the haemagglutinin. We believe this is the first demonstration of a specific haemagglutinin mutation correlating with reduced drug susceptibility in plaque assays in both Madin Darby Canine Kidney and SIAT cells. PMID:23759505

  18. Structural basis for a class of nanomolar influenza A neuraminidase inhibitors

    NASA Astrophysics Data System (ADS)

    Kerry, Philip S.; Mohan, Sankar; Russell, Rupert J. M.; Bance, Nicole; Niikura, Masahiro; Pinto, B. Mario

    2013-10-01

    The influenza virus neuraminidase (NA) is essential for the virus life cycle. The rise of resistance mutations against current antiviral therapies has increased the need for the development of novel inhibitors. Recent efforts have targeted a cavity adjacent to the catalytic site (the 150-cavity) in addition to the primary catalytic subsite in order to increase specificity and reduce the likelihood of resistance. This study details structural and in vitro analyses of a class of inhibitors that bind uniquely in both subsites. Crystal structures of three inhibitors show occupation of the 150-cavity in two distinct and novel binding modes. We believe these are the first nanomolar inhibitors of NA to be characterized in this way. Furthermore, we show that one inhibitor, binding within the catalytic site, offers reduced susceptibility to known resistance mutations via increased flexibility of a pendant pentyloxy group and the ability to pivot about a strong hydrogen-bonding network.

  19. Design of multiligand inhibitors for the swine flu H1N1 neuraminidase binding site

    PubMed Central

    Narayanan, Manoj M; Nair, Chandrasekhar B; Sanjeeva, Shilpa K; Rao, PV Subba; Pullela, Phani K; Barrow, Colin J

    2013-01-01

    Viral neuraminidase inhibitors such as oseltamivir and zanamivir prevent early virus multiplication by blocking sialic acid cleavage on host cells. These drugs are effective for the treatment of a variety of influenza subtypes, including swine flu (H1N1). The binding site for these drugs is well established and they were designed based on computational docking studies. We show here that some common natural products have moderate inhibitory activity for H1N1 neuraminidase under docking studies. Significantly, docking studies using AutoDock for biligand and triligand forms of these compounds (camphor, menthol, and methyl salicylate linked via methylene bridges) indicate that they may bind in combination with high affinity to the H1N1 neuraminidase active site. These results also indicate that chemically linked biligands and triligands of these natural products could provide a new class of drug leads for the prevention and treatment of influenza. This study also highlights the need for a multiligand docking algorithm to understand better the mode of action of natural products, wherein multiple active ingredients are present. PMID:23983477

  20. In vitro neuraminidase inhibitory activity of four neuraminidase inhibitors against influenza virus isolates in the 2011-2012 season in Japan.

    PubMed

    Ikematsu, Hideyuki; Kawai, Naoki; Iwaki, Norio; Kashiwagi, Seizaburo

    2014-02-01

    The neuraminidase inhibitors oseltamivir phosphate (Tamiflu®), zanamivir (Relenza®), laninamivir octanoate (Inavir®), and peramivir (Rapiacta®) have been available for the treatment of influenza in Japan since 2010. To assess the extent of viral resistance, we measured the 50% inhibitory concentration (IC₅₀) of each drug for influenza virus isolates from the 2011-2012 influenza season. Specimens were obtained from patients prior to treatment. Viral isolation was done using Madine-Darby canine kidney cells, and the type and subtype of influenza A(H1N1)pdm09, A(H3N2), or influenza B were determined by RT-PCR using type- and subtype-specific primers. The IC₅₀ was determined by a neuraminidase inhibition assay using a fluorescent substrate. The lineage of influenza B virus was determined by direct sequencing of the hemagglutinin gene. Influenza A(H3N2) and influenza B viruses were isolated in 283 and 42 patients, respectively, while no influenza A(H1N1)pdm09 virus was isolated. No isolate showed an IC₅₀ value exceeding 50 nM for any of the neuraminidase inhibitors. IC50 values for A(H3N2) were similar between the 2010-2011 and 2011-2012 seasons. In contrast, the IC₅₀ values for influenza B viruses in the 2011-2012 season to the four drugs were significantly lower than those found in the 2010-2011 season. These results indicate that the currently epidemic influenza viruses are susceptible to all four neuraminidase inhibitors, with no trend for IC₅₀ values to increase in Japan at present.

  1. Surveillance of influenza isolates for susceptibility to neuraminidase inhibitors during the 2000-2002 influenza seasons.

    PubMed

    Mungall, Bruce A; Xu, Xiyan; Klimov, Alexander

    2004-07-01

    Neuraminidase (NA) inhibitors (NI) have recently been licensed for the prophylaxis and treatment of influenza virus infection in humans. This study has utilized a new chemiluminescent (CL) neuraminidase assay to routinely monitor more than a thousand influenza field isolates collected worldwide during the 2000-2002 seasons for susceptibility to both licensed NIs, zanamivir, and oseltamivir by determining the 50% inhibitory concentration (IC50). Our data demonstrated that influenza A viruses of the N2 subtype were less susceptible to zanamivir, but not oseltamivir, than those of the N1 subtype such that 41 of 45 confirmed H1N2 isolates could be reliably differentiated from H1N1 viruses based on their zanamivir susceptibility. Pre-titration of influenza A viruses appeared to have no effect on IC50 determined for either NI, while pre-titration of influenza B viruses significantly reduced oseltamivir IC50 and increased zanamivir IC50. Influenza B viruses were less susceptible to either compound than type A isolates. The CL assay is a rapid and reliable method for screening large numbers of influenza isolates for NI susceptibility. Reassortant viruses of the H1N2 subtype that started to circulate worldwide during the 2001-2002 season can be reliably separated from H1N1 viruses based on their zanamivir susceptibility, enabling large scale screening of H1 isolates for determining the prevalence of such reassortants.

  2. Virtual screening of Indonesian flavonoid as neuraminidase inhibitor of influenza a subtype H5N1

    NASA Astrophysics Data System (ADS)

    Parikesit, A. A.; Ardiansah, B.; Handayani, D. M.; Tambunan, U. S. F.; Kerami, D.

    2016-02-01

    Highly Pathogenic Avian Influenza (HPAI) H5N1 poses a significant threat to animal and human health worldwide. The number of H5N1 infection in Indonesia is the highest during 2005-2013, with a mortality rate up to 83%. A mutation that occurred in H5N1 strain made it resistant to commercial antiviral agents such as oseltamivir and zanamivir, so the more potent antiviral agent is needed. In this study, virtual screening of Indonesian flavonoid as neuraminidase inhibitor of H5N1 was conducted. Total 491 flavonoid compound obtained from HerbalDB were screened. Molecular docking was performed using MOE 2008.10. This research resulted in Guajavin B as the best ligand.

  3. Profiling and Characterization of Influenza Virus N1 Strains Potentially Resistant to Multiple Neuraminidase Inhibitors

    PubMed Central

    Baek, Yun Hee; Song, Min-Suk; Lee, Eun-Young; Kim, Young-il; Kim, Eun-Ha; Park, Su-Jin; Park, Kuk Jin; Kwon, Hyeok-il; Pascua, Philippe Noriel Q.; Lim, Gyo-Jin; Kim, Semi; Yoon, Sun-Woo; Kim, Myung Hee; Webby, Richard J.

    2014-01-01

    ABSTRACT Neuraminidase inhibitors (NAIs) have been widely used to control influenza virus infection, but their increased use could promote the global emergence of resistant variants. Although various mutations associated with NAI resistance have been identified, the amino acid substitutions that confer multidrug resistance with undiminished viral fitness remain poorly understood. We therefore screened a known mutation(s) that could confer multidrug resistance to the currently approved NAIs oseltamivir, zanamivir, and peramivir by assessing recombinant viruses with mutant NA-encoding genes (catalytic residues R152K and R292K, framework residues E119A/D/G, D198N, H274Y, and N294S) in the backbones of the 2009 pandemic H1N1 (pH1N1) and highly pathogenic avian influenza (HPAI) H5N1 viruses. Of the 14 single and double mutant viruses recovered in the backbone of pH1N1, four variants (E119D, E119A/D/G-H274Y) exhibited reduced inhibition by all of the NAIs and two variants (E119D and E119D-H274Y) retained the overall properties of gene stability, replicative efficiency, pathogenicity, and transmissibility in vitro and in vivo. Of the nine recombinant H5N1 viruses, four variants (E119D, E119A/D/G-H274Y) also showed reduced inhibition by all of the NAIs, though their overall viral fitness was impaired in vitro and/or in vivo. Thus, single mutations or certain combination of the established mutations could confer potential multidrug resistance on pH1N1 or HPAI H5N1 viruses. Our findings emphasize the urgency of developing alternative drugs against influenza virus infection. IMPORTANCE There has been a widespread emergence of influenza virus strains with reduced susceptibility to neuraminidase inhibitors (NAIs). We screened multidrug-resistant viruses by studying the viral fitness of neuraminidase mutants in vitro and in vivo. We found that recombinant E119D and E119A/D/G/-H274Y mutant viruses demonstrated reduced inhibition by all of the NAIs tested in both the backbone of

  4. Integrin-mediated cell migration is blocked by inhibitors of human neuraminidase.

    PubMed

    Jia, Feng; Howlader, Md Amran; Cairo, Christopher W

    2016-09-01

    Integrins are critical receptors in cell migration and adhesion. A number of mechanisms are known to regulate the function of integrins, including phosphorylation, conformational change, and cytoskeletal anchoring. We investigated whether native neuraminidase (Neu, or sialidase) enzymes which modify glycolipids could play a role in regulating integrin-mediated cell migration. Using a scratch assay, we found that exogenously added Neu3 and Neu4 activity altered rates of cell migration. We observed that Neu4 increased the rate of migration in two cell lines (HeLa, A549); while Neu3 only increased migration in HeLa cells. A bacterial neuraminidase was able to increase the rate of migration in HeLa, but not in A549 cells. Treatment of cells with complex gangliosides (GM1, GD1a, GD1b, and GT1b) resulted in decreased cell migration rates, while LacCer was able to increase rates of migration in both lines. Importantly, our results show that treatment of cells with inhibitors of native Neu enzymes had a dramatic effect on the rates of cell migration. The most potent compound tested targeted the human Neu4 isoenzyme, and was able to substantially reduce the rate of cell migration. We found that the lateral mobility of integrins was reduced by treatment of cells with Neu3, suggesting that Neu3 enzyme activity resulted in changes to integrin-co-receptor or integrin-cytoskeleton interactions. Finally, our results support the hypothesis that inhibitors of human Neu can be used to investigate mechanisms of cell migration and for the development of anti-adhesive therapies.

  5. Overexpression of the alpha-2,6-sialyltransferase in MDCK cells increases influenza virus sensitivity to neuraminidase inhibitors.

    PubMed

    Matrosovich, Mikhail; Matrosovich, Tatyana; Carr, Jackie; Roberts, Noel A; Klenk, Hans-Dieter

    2003-08-01

    No reliable cell culture assay is currently available for monitoring human influenza virus sensitivity to neuraminidase inhibitors (NAI). This can be explained by the observation that because of a low concentration of sialyl-alpha2,6-galactose (Sia[alpha2,6]Gal)-containing virus receptors in conventional cell lines, replication of human virus isolates shows little dependency on viral neuraminidase. To test whether overexpression of Sia(alpha2,6)Gal moieties in cultured cells could make them suitable for testing human influenza virus sensitivity to NAI, we stably transfected MDCK cells with cDNA of human 2,6-sialyltransferase (SIAT1). Transfected cells expressed twofold-higher amounts of 6-linked sialic acids and twofold-lower amounts of 3-linked sialic acids than parent MDCK cells as judged by staining with Sambucus nigra agglutinin and Maackia amurensis agglutinin, respectively. After transfection, binding of a clinical human influenza virus isolate was increased, whereas binding of its egg-adapted variant which preferentially bound 3-linked receptors was decreased. The sensitivity of human influenza A and B viruses to the neuraminidase inhibitor oseltamivir carboxylate was substantially improved in the SIAT1-transfected cell line and was consistent with their sensitivity in neuraminidase enzyme assay and with the hemagglutinin (HA) receptor-binding phenotype. MDCK cells stably transfected with SIAT1 may therefore be a suitable system for testing influenza virus sensitivity to NAI. PMID:12857911

  6. QSAR modeling and molecular interaction analysis of natural compounds as potent neuraminidase inhibitors.

    PubMed

    Sun, Jiaying; Mei, Hu

    2016-04-26

    Different QSAR models of 40 natural compounds as neuraminidase inhibitors (NIs) are developed to comprehend chemical-biological interactions and predict activities against neuraminidase (NA) from Clostridium perfringens. Based on the constitutional, topological and conformational descriptors, R(2) and Q(2) values of the obtained SRA model are 0.931 and 0.856. The R(2) and Q(2) values of the constructed HQSAR and almond models are 0.903 and 0.767, 0.904 and 0.511, respectively. Based on the pharmacophore alignment, R(2) and Q(2) values of the optimal CoMSIA model are 0.936 and 0.654. Moreover, Rtest(2) and Qext(2) of values of SRA, HQSAR, almond and CoMSIA models are 0.611 and 0.565, 0.753 and 0.750, 0.612 and 0.582, 0.582 and 0.571, respectively. So, QSAR models have good predictive capability. They can be further used to evaluate and screen new compounds. Moreover, hydrogen bonds and electrostatic factors have high contributions to activities. To understand molecular interactions between natural compounds and NA from Clostridium perfringens, molecular docking is investigated. The docking results elucidate that Arg266, Asp291, Asp328, Tyr485, Glu493, Arg555, Arg615 and Tyr655 are especially the key residues in the active site of 2bf6. Hydrogen bonds and electrostatics are key factors, which impact the interactions between NIs and NA. So, the influential factors of interactions between NIs and NA in the docking results are in agreement with the QSAR results. PMID:27008437

  7. Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, 2012-2013.

    PubMed

    Meijer, Adam; Rebelo-de-Andrade, Helena; Correia, Vanessa; Besselaar, Terry; Drager-Dayal, Renu; Fry, Alicia; Gregory, Vicky; Gubareva, Larisa; Kageyama, Tsutomu; Lackenby, Angie; Lo, Janice; Odagiri, Takato; Pereyaslov, Dmitriy; Siqueira, Marilda M; Takashita, Emi; Tashiro, Masato; Wang, Dayan; Wong, Sun; Zhang, Wenqing; Daniels, Rod S; Hurt, Aeron C

    2014-10-01

    Emergence of influenza viruses with reduced susceptibility to neuraminidase inhibitors (NAIs) is sporadic, often follows exposure to NAIs, but occasionally occurs in the absence of NAI pressure. The emergence and global spread in 2007/2008 of A(H1N1) influenza viruses showing clinical resistance to oseltamivir due to neuraminidase (NA) H275Y substitution, in the absence of drug pressure, warrants continued vigilance and monitoring for similar viruses. Four World Health Organization (WHO) Collaborating Centres for Reference and Research on Influenza and one WHO Collaborating Centre for the Surveillance, Epidemiology and Control of Influenza (WHO CCs) tested 11,387 viruses collected by WHO-recognized National Influenza Centres (NIC) between May 2012 and May 2013 to determine 50% inhibitory concentration (IC50) data for oseltamivir, zanamivir, peramivir and laninamivir. The data were evaluated using normalized IC50 fold-changes rather than raw IC50 data. Nearly 90% of the 11,387 viruses were from three WHO regions: Western Pacific, the Americas and Europe. Only 0.2% (n=27) showed highly reduced inhibition (HRI) against at least one of the four NAIs, usually oseltamivir, while 0.3% (n=39) showed reduced inhibition (RI). NA sequence data, available from the WHO CCs and from sequence databases (n=3661), were screened for amino acid substitutions associated with reduced NAI susceptibility. Those showing HRI were A(H1N1)pdm09 with NA H275Y (n=18), A(H3N2) with NA E119V (n=3) or NA R292K (n=1) and B/Victoria-lineage with NA H273Y (n=2); amino acid position numbering is A subtype and B type specific. Overall, approximately 99% of circulating viruses tested during the 2012-2013 period were sensitive to all four NAIs. Consequently, these drugs remain an appropriate choice for the treatment and prophylaxis of influenza virus infections.

  8. Synergistic Effect of Nitazoxanide with Neuraminidase Inhibitors against Influenza A Viruses In Vitro

    PubMed Central

    Belardo, Giuseppe; Cenciarelli, Orlando; La Frazia, Simone; Rossignol, Jean Francois

    2014-01-01

    The emergence of drug-resistant influenza A virus (IAV) strains represents a serious threat to global human health and underscores the need for novel approaches to anti-influenza chemotherapy. Combination therapy with drugs affecting different IAV targets represents an attractive option for influenza treatment. We have previously shown that the thiazolide anti-infective nitazoxanide (NTZ) inhibits H1N1 IAV replication by selectively blocking viral hemagglutinin maturation. Herein we investigate the anti-influenza activity of NTZ against a wide range of human and avian IAVs (H1N1, H3N2, H5N9, H7N1), including amantadine-resistant and oseltamivir-resistant strains, in vitro. We also investigate whether therapy with NTZ in combination with the neuraminidase inhibitors oseltamivir and zanamivir exerts synergistic, additive, or antagonistic antiviral effects against influenza viruses. NTZ was effective against all IAVs tested, with 50% inhibitory concentrations (IC50s) ranging from 0.9 to 3.2 μM, and selectivity indexes (SIs) ranging from >50 to >160, depending on the strain and the multiplicity of infection (MOI). Combination therapy studies were performed in cell culture-based assays using A/Puerto Rico/8/1934 (H1N1), A/WSN/1933 (H1N1), or avian A/chicken/Italy/9097/1997 (H5N9) IAVs; dose-effect analysis and synergism/antagonism quantification were performed using isobologram analysis according to the Chou-Talalay method. Combination index (CI) analysis indicated that NTZ and oseltamivir combination treatment was synergistic against A/Puerto Rico/8/1934 (H1N1) and A/WSN/1933 (H1N1) IAVs, with CI values ranging between 0.39 and 0.63, independently of the MOI used. Similar results were obtained when NTZ was administered in combination with zanamivir (CI = 0.3 to 0.48). NTZ-oseltamivir combination treatment was synergistic also against the avian A/chicken/Italy/9097/1997 (H5N9) IAV (CI = 0.18 to 0.31). Taken together, the results suggest that regimens that combine

  9. Characterization of Neuraminidase Inhibitors in Korean Papaver rhoeas Bee Pollen Contributing to Anti-Influenza Activities In Vitro.

    PubMed

    Lee, In-Kyoung; Hwang, Byung Soon; Kim, Dae-Won; Kim, Ji-Yul; Woo, E-Eum; Lee, Yoon-Ju; Choi, Hwa Jung; Yun, Bong-Sik

    2016-04-01

    The active constituents of Korean Papaver rhoeas bee pollen conferring neuraminidase inhibitory activities (H1N1, H3N2, and H5N1) were investigated. Six flavonoids and one alkaloid were isolated and characterized by nuclear magnetic resonance and mass spectrometry data. These included kaempferol-3-sophoroside (1), kaempferol-3-neohesperidoside (2), kaempferol-3-sambubioside (3), kaempferol-3-glucoside (4), quercetin-3-sophoroside (5), luteolin (6), and chelianthifoline (7). All compounds showed neuraminidase inhibitory activities with IC50 values ranging from 10.7 to 151.1 µM. The most potent neuraminidase inhibitor was luteolin, which was the dominant content in the ethyl acetate fraction. All tested compounds displayed noncompetitive inhibition of H3N2 neuraminidase. Furthermore, compounds 1-7 all reduced the severity of virally induced cytopathic effects as determined by the Madin-Darby canine kidney cell-based assay showing antiviral activity with IC50 values ranging from 10.7 to 33.4 µM (zanamivir: 58.3 µM). The active compounds were quantified by high-performance liquid chromatography, and the total amount of compounds 1-7 made up about 0.592 g/100 g bee pollen, contributing a rich resource of a natural antiviral product. PMID:26848705

  10. Prognosis of hospitalized patients with 2009 H1N1 influenza in Spain: influence of neuraminidase inhibitors

    PubMed Central

    Delgado-Rodríguez, Miguel; Castilla, Jesús; Godoy, Pere; Martín, Vicente; Soldevila, Nuria; Alonso, Jordi; Astray, Jenaro; Baricot, Maretva; Cantón, Rafael; Castro, Ady; Gónzález-Candelas, Fernando; Mayoral, José María; Quintana, José María; Pumarola, Tomás; Tamames, Sonia; Sáez, Marc; Domínguez, Angela

    2012-01-01

    Background The H1N1 influenza pandemic strain has been associated with a poor prognosis in hospitalized patients. The present report evaluates the factors influencing prognosis. Methods A total of 813 patients hospitalized with H1N1 influenza in 36 hospitals (nationwide) in Spain were analysed. Detailed histories of variables preceding hospital admission were obtained by interview, validating data on medications and vaccine with their attending physicians. Data on treatment and complications during hospital stay were recorded. As definition of poor outcome, the endpoints of death and admission to intensive care were combined; and as a further outcome, length of stay was used. Results The mean age was 38.5 years (SD 22.8 years). There were 10 deaths and 79 admissions to intensive care (combined, 88). The use of neuraminidase inhibitors was reported by 495 patients (60.9%). The variables significantly associated with a poor outcome were diabetes (OR = 2.21, 95% CI = 1.21–4.02), corticosteroid therapy (OR = 3.37, 95% CI = 1.39–8.20) and use of histamine-2 receptor antagonists (OR = 2.68, 95% CI = 1.14–6.36), while the use of neuraminidase inhibitors (OR = 0.57, 95% CI = 0.34–0.94) was protective. Neuraminidase inhibitors within the first 2 days after the influenza onset reduced hospital stay by a mean of 1.9 days (95% CI = 4.7–6.6). Conclusions The use of neuraminidase inhibitors decreases the length of hospital stay and admission to intensive care and/or death. PMID:22467633

  11. Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, 2014-2015.

    PubMed

    Hurt, Aeron C; Besselaar, Terry G; Daniels, Rod S; Ermetal, Burcu; Fry, Alicia; Gubareva, Larisa; Huang, Weijuan; Lackenby, Angie; Lee, Raphael T C; Lo, Janice; Maurer-Stroh, Sebastian; Nguyen, Ha T; Pereyaslov, Dmitriy; Rebelo-de-Andrade, Helena; Siqueira, Marilda M; Takashita, Emi; Tashiro, Masato; Tilmanis, Danielle; Wang, Dayan; Zhang, Wenqing; Meijer, Adam

    2016-08-01

    The World Health Organization (WHO) Collaborating Centres for Reference and Research on Influenza (WHO CCs) tested 13,312 viruses collected by WHO recognized National Influenza Centres between May 2014 and May 2015 to determine 50% inhibitory concentration (IC50) data for neuraminidase inhibitors (NAIs) oseltamivir, zanamivir, peramivir and laninamivir. Ninety-four per cent of the viruses tested by the WHO CCs were from three WHO regions: Western Pacific, the Americas and Europe. Approximately 0.5% (n = 68) of viruses showed either highly reduced inhibition (HRI) or reduced inhibition (RI) (n = 56) against at least one of the four NAIs. Of the twelve viruses with HRI, six were A(H1N1)pdm09 viruses, three were A(H3N2) viruses and three were B/Yamagata-lineage viruses. The overall frequency of viruses with RI or HRI by the NAIs was lower than that observed in 2013-14 (1.9%), but similar to the 2012-13 period (0.6%). Based on the current analysis, the NAIs remain an appropriate choice for the treatment and prophylaxis of influenza virus infections.

  12. The accuracy and timeliness of neuraminidase inhibitor dispensing data for predicting laboratory-confirmed influenza.

    PubMed

    Papenburg, J; Charland, K M; DE Serres, G; Buckeridge, D L

    2016-06-01

    Neuraminidase inhibitor (NI) dispensing has emerged as a possible automated data source for influenza surveillance. We aimed to evaluate its timeliness, correlation, and predictive accuracy in relation to influenza activity in Quebec, Canada, 2010-2013. Our secondary objective was to use the same metrics to compare NI dispensing to visits for influenza-like illness (ILI) in emergency departments (EDs). Provincial weekly counts of positive influenza laboratory tests were used as a reference measure for the level of influenza circulation. We applied ARIMA models to account for serial correlation. We computed cross-correlations to measure the strengths of association and lead-lag relationships between NI dispensing, ILI ED visits, and our reference indicator. Finally, using an ARIMA model, we evaluated the ability of NI dispensing and ILI ED visits to predict laboratory-confirmed influenza. NI dispensing was significantly correlated (R = 0·68) with influenza activity with no lag. The maximal correlation of ILI ED visits was not as strong (R = 0·50). Both NI dispensing and ILI ED visits were significant predictors of laboratory-confirmed influenza in a multivariable model; predictive potential was greatest when NI counts were lagged to precede laboratory surveillance by 2 weeks. We conclude that NI dispensing data provides timely and valuable information for influenza surveillance. PMID:26611607

  13. Peramivir: A Novel Intravenous Neuraminidase Inhibitor for Treatment of Acute Influenza Infections

    PubMed Central

    Alame, Malak M.; Massaad, Elie; Zaraket, Hassan

    2016-01-01

    Peramivir is a novel cyclopentane neuraminidase inhibitor of influenza virus. It was approved by the Food and Drug Administration in December 2014 for treatment of acute uncomplicated influenza in patients 18 years and older. For several months prior to approval, the drug was made clinically available under Emergency Use authorization during the 2009 H1N1 influenza pandemic. Peramivir is highly effective against human influenza A and B isolates as well as emerging influenza virus strains with pandemic potential. Clinical trials demonstrated that the drug is well-tolerated in adult and pediatric populations. Adverse events are generally mild to moderate and similar in frequency to patients receiving placebo. Common side effects include gastrointestinal disorders and decreased neutrophil counts but are self-limiting. Peramivir is administered as a single-dose via the intravenous route providing a valuable therapeutic alternative for critically ill patients or those unable to tolerate other administration routes. Successful clinical trials and post-marketing data in pediatric populations in Japan support the safety and efficacy of peramivir in this population where administration of other antivirals might not be feasible. PMID:27065996

  14. Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, 2013-2014.

    PubMed

    Takashita, Emi; Meijer, Adam; Lackenby, Angie; Gubareva, Larisa; Rebelo-de-Andrade, Helena; Besselaar, Terry; Fry, Alicia; Gregory, Vicky; Leang, Sook-Kwan; Huang, Weijuan; Lo, Janice; Pereyaslov, Dmitriy; Siqueira, Marilda M; Wang, Dayan; Mak, Gannon C; Zhang, Wenqing; Daniels, Rod S; Hurt, Aeron C; Tashiro, Masato

    2015-05-01

    Four World Health Organization (WHO) Collaborating Centres for Reference and Research on Influenza and one WHO Collaborating Centre for the Surveillance, Epidemiology and Control of Influenza (WHO CCs) tested 10,641 viruses collected by WHO-recognized National Influenza Centres between May 2013 and May 2014 to determine 50% inhibitory concentration (IC50) data for neuraminidase inhibitors (NAIs) oseltamivir, zanamivir, peramivir and laninamivir. In addition, neuraminidase (NA) sequence data, available from the WHO CCs and from sequence databases (n=3206), were screened for amino acid substitutions associated with reduced NAI susceptibility. Ninety-five per cent of the viruses tested by the WHO CCs were from three WHO regions: Western Pacific, the Americas and Europe. Approximately 2% (n=172) showed highly reduced inhibition (HRI) against at least one of the four NAIs, commonly oseltamivir, while 0.3% (n=32) showed reduced inhibition (RI). Those showing HRI were A(H1N1)pdm09 with NA H275Y (n=169), A(H3N2) with NA E119V (n=1), B/Victoria-lineage with NA E117G (n=1) and B/Yamagata-lineage with NA H273Y (n=1); amino acid position numbering is A subtype and B type specific. Although approximately 98% of circulating viruses tested during the 2013-2014 period were sensitive to all four NAIs, a large community cluster of A(H1N1)pdm09 viruses with the NA H275Y substitution from patients with no previous exposure to antivirals was detected in Hokkaido, Japan. Significant numbers of A(H1N1)pdm09 NA H275Y viruses were also detected in China and the United States: phylogenetic analyses showed that the Chinese viruses were similar to those from Japan, while the United States viruses clustered separately from those of the Hokkaido outbreak, indicative of multiple resistance-emergence events. Consequently, global surveillance of influenza antiviral susceptibility should be continued from a public health perspective.

  15. Influenza neuraminidase

    PubMed Central

    Air, Gillian M.

    2011-01-01

    Please cite this paper as: Air. (2012) Influenza neuraminidase. Influenza and Other Respiratory Viruses 6(4), 245–256. Influenza neuraminidase is the target of two licensed antivirals that have been very successful, with several more in development. However, neuraminidase has been largely ignored as a vaccine target despite evidence that inclusion of neuraminidase in the subunit vaccine gives increased protection. This article describes current knowledge on the structure, enzyme activity, and antigenic significance of neuraminidase. PMID:22085243

  16. Structure Optimization of Neuraminidase Inhibitors as Potential Anti-Influenza (H1N1Inhibitors) Agents Using QSAR and Molecular Docking Studies

    PubMed Central

    Inamdar, Poonam; Bhandari, Shashikant; Sonawane, Bhagyashri; Hole, Asha; Jadhav, Chintamani

    2014-01-01

    The urgent need of neuraminidase inhibitors (NI) has provided an impetus for understanding the structure requisite at molecular level. Our search for selective inhibitors of neuraminidase has led to the identification of pharmacophoric requirements at various positions around acyl thiourea pharmacophore. The main objective of present study is to develop selective NI, with least toxicity and drug like ADMET properties. Electronic, Steric requirements were defined using kohnone nearest neighbour- molecular field analysis (kNN-MFA) model of 3D-QSAR studies. Results generated by QSAR studies showed that model has good internal as well as external predictivity. Such defined requirements were used to generate new chemical entities which exhibit higher promising predicted activities. To check selective binding of designed NCE’s docking studies were carried out using the crystal structure of the neuraminidase enzyme having co-crystallized ligand Oseltamivir. Thus, molecular modelling provided a good platform to optimize the acyl thiourea pharmacophore for designing its derivatives having potent anti-viral activity. PMID:24734056

  17. Structure-guided discovery of potent and dual-acting human parainfluenza virus haemagglutinin-neuraminidase inhibitors.

    PubMed

    Guillon, Patrice; Dirr, Larissa; El-Deeb, Ibrahim M; Winger, Moritz; Bailly, Benjamin; Haselhorst, Thomas; Dyason, Jeffrey C; von Itzstein, Mark

    2014-01-01

    Human parainfluenza viruses (hPIVs) cause upper and lower respiratory tract disease in children that results in a significant number of hospitalizations and impacts health systems worldwide. To date, neither antiviral drugs nor vaccines are approved for clinical use against parainfluenza virus, which reinforces the urgent need for new therapeutic discovery strategies. Here we use a multidisciplinary approach to develop potent inhibitors that target a structural feature within the hPIV type 3 haemagglutinin-neuraminidase (hPIV-3 HN). These dual-acting designer inhibitors represent the most potent designer compounds and efficiently block both hPIV cell entry and virion progeny release. We also define the binding mode of these inhibitors in the presence of whole-inactivated hPIV and recombinantly expressed hPIV-3 HN by Saturation Transfer Difference NMR spectroscopy. Collectively, our study provides an antiviral preclinical candidate and a new direction towards the discovery of potential anti-parainfluenza drugs. PMID:25327774

  18. Synthesis of C-4-modified zanamivir analogs as neuraminidase inhibitors and their anti-AIV activities.

    PubMed

    Ye, Deju; Shin, Woo-Jin; Li, Ning; Tang, Wei; Feng, Enguang; Li, Jian; He, Pei-Lan; Zuo, Jian-Ping; Kim, Hanjo; Nam, Ky-Youb; Zhu, Weiliang; Seong, Baik-Lin; No, Kyoung Tai; Jiang, Hualiang; Liu, Hong

    2012-08-01

    With the introduction of bioisosteres of the guanidinium group together with scaffold hopping, 35 zanamivir analogs with C-4-modification were synthesized, and their inhibitory activities against both group-1 and group-2 neuraminidase (H5N1 and H3N2) were determined. Compound D26 exerts the most potency, with IC(50) values of 0.58 and 2.72 μM against N2 and N1, respectively. Further preliminary anti-avian influenza virus (AIV, H5N1) activities against infected MDCK cells were evaluated, and D5 exerts ∼58% protective against AIV infection, which was comparable to zanamivir (∼67%). In a rat pharmacokinetic study, compound D5 showed an increased plasma half-life (t(1/2)) compared to zanamivir following either intravenous or oral administration. This study may represent a new start point for the future development of improved anti-AIV agents.

  19. Neuraminidase Ribbon Diagram

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Ribbons is a program developed at UAB used worldwide to graphically depict complicated protein structures in a simplified format. The program uses sophisticated computer systems to understand the implications of protein structures. The Influenza virus remains a major causative agent for a large number of deaths among the elderly and young children and huge economic losses due to illness. Finding a cure will have a general impact both on the basic research of viral pathologists of fast evolving infectious agents and clinical treatment of influenza virus infection. The reproduction process of all strains of influenza are dependent on the same enzyme neuraminidase. Shown here is a segmented representation of the neuraminidase inhibitor compound sitting inside a cave-like contour of the neuraminidase enzyme surface. This cave-like formation present in every neuraminidase enzyme is the active site crucial to the flu's ability to infect. The space-grown crystals of neuraminidase have provided significant new details about the three-dimensional characteristics of this active site thus allowing researchers to design drugs that fit tighter into the site. Principal Investigator: Dr. Larry DeLucas

  20. Unique Determinants of Neuraminidase Inhibitor Resistance among N3, N7, and N9 Avian Influenza Viruses

    PubMed Central

    Song, Min-Suk; Marathe, Bindumadhav M.; Kumar, Gyanendra; Wong, Sook-San; Rubrum, Adam; Zanin, Mark; Choi, Young-Ki; Webster, Robert G.; Govorkova, Elena A.

    2015-01-01

    ABSTRACT 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. IMPORTANCE 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

  1. Multiple Influenza A (H3N2) Mutations Conferring Resistance to Neuraminidase Inhibitors in a Bone Marrow Transplant Recipient

    PubMed Central

    Eshaghi, Alireza; Shalhoub, Sarah; Rosenfeld, Paul; Li, Aimin; Higgins, Rachel R.; Stogios, Peter J.; Savchenko, Alexei; Bastien, Nathalie; Li, Yan; Rotstein, Coleman

    2014-01-01

    Immunocompromised patients are predisposed to infections caused by influenza virus. Influenza virus may produce considerable morbidity, including protracted illness and prolonged viral shedding in these patients, thus prompting higher doses and prolonged courses of antiviral therapy. This approach may promote the emergence of resistant strains. Characterization of neuraminidase (NA) inhibitor (NAI)-resistant strains of influenza A virus is essential for documenting causes of resistance. In this study, using quantitative real-time PCR along with conventional Sanger sequencing, we identified an NAI-resistant strain of influenza A (H3N2) virus in an immunocompromised patient. In-depth analysis by deep gene sequencing revealed that various known markers of antiviral resistance, including transient R292K and Q136K substitutions and a sustained E119K (N2 numbering) substitution in the NA protein emerged during prolonged antiviral therapy. In addition, a combination of a 4-amino-acid deletion at residues 245 to 248 (Δ245-248) accompanied by the E119V substitution occurred, causing resistance to or reduced inhibition by NAIs (oseltamivir, zanamivir, and peramivir). Resistant variants within a pool of viral quasispecies arose during combined antiviral treatment. More research is needed to understand the interplay of drug resistance mutations, viral fitness, and transmission. PMID:25246391

  2. Identification of Suitable Natural Inhibitor against Influenza A (H1N1) Neuraminidase Protein by Molecular Docking

    PubMed Central

    Sahoo, Maheswata; Jena, Lingaraja; Rath, Surya Narayan

    2016-01-01

    The influenza A (H1N1) virus, also known as swine flu is a leading cause of morbidity and mortality since 2009. There is a need to explore novel anti-viral drugs for overcoming the epidemics. Traditionally, different plant extracts of garlic, ginger, kalmegh, ajwain, green tea, turmeric, menthe, tulsi, etc. have been used as hopeful source of prevention and treatment of human influenza. The H1N1 virus contains an important glycoprotein, known as neuraminidase (NA) that is mainly responsible for initiation of viral infection and is essential for the life cycle of H1N1. It is responsible for sialic acid cleavage from glycans of the infected cell. We employed amino acid sequence of H1N1 NA to predict the tertiary structure using Phyre2 server and validated using ProCheck, ProSA, ProQ, and ERRAT server. Further, the modelled structure was docked with thirteen natural compounds of plant origin using AutoDock4.2. Most of the natural compounds showed effective inhibitory activity against H1N1 NA in binding condition. This study also highlights interaction of these natural inhibitors with amino residues of NA protein. Furthermore, among 13 natural compounds, theaflavin, found in green tea, was observed to inhibit H1N1 NA proteins strongly supported by lowest docking energy. Hence, it may be of interest to consider theaflavin for further in vitro and in vivo evaluation. PMID:27729839

  3. Dual Acting Neuraminidase Inhibitors Open New Opportunities to Disrupt the Lethal Synergism between Streptococcus pneumoniae and Influenza Virus

    PubMed Central

    Walther, Elisabeth; Xu, Zhongli; Richter, Martina; Kirchmair, Johannes; Grienke, Ulrike; Rollinger, Judith M.; Krumbholz, Andi; Saluz, Hans P.; Pfister, Wolfgang; Sauerbrei, Andreas; Schmidtke, Michaela

    2016-01-01

    Secondary infections with Streptococcus pneumoniae cause severe pneumonia and enhance lethality during influenza epidemics and pandemics. Structural and functional similarities with viral neuraminidase (NA) suggest that the highly prevalent pneumococcal NAs, NanA and NanB, might contribute to this lethal synergism by supporting viral replication and that dual acting NA inhibitors (NAIs) will disrupt it. To verify this hypothesis, NanA and NanB were expressed in E. coli. After confirming their activity in enzyme assays, in vitro models with influenza virus A/Jena/8178/09 (Jena/8178) and the recombinant NanA or NanB (rNanA and rNanB) were established in A549 and MDCK cells to mimic the role of these pneumococcal NAs during co-infection. Studies on the influence of both NAs on viral receptor expression, spread, and yield revealed a distinct effect of NanA and NanB on viral replication in these in vitro models. Both enzymes were able to support Jena/8178 replication at certain concentrations. This synergism was disrupted by the NAIs oseltamivir, DANA, katsumadain A, and artocarpin exerting an inhibitory effect on viral NA and NanA. Interestingly, katsumadain A and artocarpin inhibited rNanA and rNanB similarly. Zanamivir did not show activity. These results demonstrate a key role of pneumococcal NAs in the lethal synergism with influenza viruses and reveal opportunities for its effective disruption. PMID:27047471

  4. In Vitro Characterization of A-315675, a Highly Potent Inhibitor of A and B Strain Influenza Virus Neuraminidases and Influenza Virus Replication

    PubMed Central

    Kati, Warren M.; Montgomery, Debra; Carrick, Robert; Gubareva, Larisa; Maring, Clarence; McDaniel, Keith; Steffy, Kevin; Molla, Akhteruzzaman; Hayden, Frederick; Kempf, Dale; Kohlbrenner, William

    2002-01-01

    A-315675 is a novel, pyrrolidine-based compound that was evaluated in this study for its ability to inhibit A and B strain influenza virus neuraminidases in enzyme assays and influenza virus replication in cell culture. A-315675 effectively inhibited influenza A N1, N2, and N9 and B strain neuraminidases with inhibitor constant (Ki) values between 0.024 and 0.31 nM. These values were comparable to or lower than the Ki values measured for oseltamivir carboxylate (GS4071), zanamivir, and BCX-1812, except for the N1 enzymes that were found to be the most sensitive to BCX-1812. The time-dependent inhibition of neuraminidase catalytic activity observed with A-315675 is likely due to its very low rate of dissociation from the active site of neuraminidase. The half times for dissociation of A-315675 from B/Memphis/3/89 and A/Tokyo/3/67 (H3N2) influenza virus neuraminidases of 10 to 12 h are significantly slower than the half times measured for oseltamivir carboxylate (33 to 60 min). A-315675 inhibited the replication of several laboratory strains of influenza virus in cell culture with potencies that were comparable or superior to those for oseltamivir carboxylate and BCX-1812, except for the A/H1N1 viruses that were found to be two- to fourfold more susceptible to BCX-1812. A-315675 and oseltamivir carboxylate exhibited comparable potencies against a panel of A/H1N1 and A/H3N2 influenza virus clinical isolates, but A-315675 was found to be significantly more potent than oseltamivir carboxylate against the B strain isolates. The favorable in vitro results relative to other clinically effective agents provide strong support for the further investigation of A-315675 as a potential therapy for influenza virus infections. PMID:11897583

  5. Effectiveness of neuraminidase inhibitors in preventing hospitalization during the H1N1 influenza pandemic in British Columbia, Canada

    PubMed Central

    Marra, Fawziah; Chong, Mei; Henry, Bonnie; Patrick, David M.; Kendall, Perry

    2014-01-01

    Objectives In British Columbia (BC), Canada, neuraminidase inhibitors (NIs) were publicly funded during the 2009 A(H1N1)pdm09 pandemic for treatment of high-risk patients and/or anyone with moderate-to-severe illness. We assessed antiviral effectiveness (AVE) against hospitalization in that context. Methods A population-based cohort study was conducted using linked administrative data. The cohort included all individuals living in BC during the study period (1 September to 31 December 2009) with a diagnostic code consistent with influenza or pandemic H1N1. The main study period pertained to the second-wave A(H1N1)pdm09 circulation (1 October to 31 December 2009), with sensitivity analyses around the more specific pandemic peak (18 October to 7 November). Exposure was defined by same-day NI prescription. The main outcome was all-cause hospitalization within 14 days of the outpatient influenza diagnosis. Cox proportional hazards models assessed AVE with 1 : 1 propensity-score matching and covariate adjustment. Results After matching, there were 304/58 061 NI-exposed and 345/58 061 unexposed patients hospitalized during the main study period. The very young [<6 months (35.0; 95% CI 16.7–73.4)], the old [65–79 years (13.7; 95% CI 10.1–18.6)] and the very old [≥80 years (38.7; 95% CI 26.6–56.5)] had the highest hospitalization rate per 1000 patients overall. Fully adjusted AVE against all-cause hospitalization during the main study period was 16% (95% CI 2%–28%), similar to the pandemic peak (15%; 95% CI −4%–30%). Conclusions The use of NIs was associated with modest protection against hospitalization during the 2009 pandemic, but appeared underutilized in affected age groups with the highest hospitalization risk. PMID:24346762

  6. Anti-influenza neuraminidase inhibitor oseltamivir phosphate induces canine mammary cancer cell aggressiveness.

    PubMed

    de Oliveira, Joana T; Santos, Ana L; Gomes, Catarina; Barros, Rita; Ribeiro, Cláudia; Mendes, Nuno; de Matos, Augusto J; Vasconcelos, M Helena; Oliveira, Maria José; Reis, Celso A; Gärtner, Fátima

    2015-01-01

    Oseltamivir phosphate is a widely used anti-influenza sialidase inhibitor. Sialylation, governed by sialyltransferases and sialidases, is strongly implicated in the oncogenesis and progression of breast cancer. In this study we evaluated the biological behavior of canine mammary tumor cells upon oseltamivir phosphate treatment (a sialidase inhibitor) in vitro and in vivo. Our in vitro results showed that oseltamivir phosphate impairs sialidase activity leading to increased sialylation in CMA07 and CMT-U27 canine mammary cancer cells. Surprisingly, oseltamivir phosphate stimulated, CMT-U27 cell migration and invasion capacity in vitro, in a dose-dependent manner. CMT-U27 tumors xenograft of oseltamivir phosphate-treated nude mice showed increased sialylation, namely α2,6 terminal structures and SLe(x) expression. Remarkably, a trend towards increased lung metastases was observed in oseltamivir phosphate-treated nude mice. Taken together, our findings revealed that oseltamivir impairs canine mammary cancer cell sialidase activity, altering the sialylation pattern of canine mammary tumors, and leading, surprisingly, to in vitro and in vivo increased mammary tumor aggressiveness. PMID:25850034

  7. Anti-Influenza Neuraminidase Inhibitor Oseltamivir Phosphate Induces Canine Mammary Cancer Cell Aggressiveness

    PubMed Central

    de Oliveira, Joana T.; Santos, Ana L.; Gomes, Catarina; Barros, Rita; Ribeiro, Cláudia; Mendes, Nuno; de Matos, Augusto J.; Vasconcelos, M. Helena; Oliveira, Maria José; Reis, Celso A.; Gärtner, Fátima

    2015-01-01

    Oseltamivir phosphate is a widely used anti-influenza sialidase inhibitor. Sialylation, governed by sialyltransferases and sialidases, is strongly implicated in the oncogenesis and progression of breast cancer. In this study we evaluated the biological behavior of canine mammary tumor cells upon oseltamivir phosphate treatment (a sialidase inhibitor) in vitro and in vivo. Our in vitro results showed that oseltamivir phosphate impairs sialidase activity leading to increased sialylation in CMA07 and CMT-U27 canine mammary cancer cells. Surprisingly, oseltamivir phosphate stimulated, CMT-U27 cell migration and invasion capacity in vitro, in a dose-dependent manner. CMT-U27 tumors xenograft of oseltamivir phosphate-treated nude mice showed increased sialylation, namely α2,6 terminal structures and SLe(x) expression. Remarkably, a trend towards increased lung metastases was observed in oseltamivir phosphate-treated nude mice. Taken together, our findings revealed that oseltamivir impairs canine mammary cancer cell sialidase activity, altering the sialylation pattern of canine mammary tumors, and leading, surprisingly, to in vitro and in vivo increased mammary tumor aggressiveness. PMID:25850034

  8. Synthesis and evaluation of influenza A viral neuraminidase candidate inhibitors based on a bicyclo[3.1.0]hexane scaffold.

    PubMed

    Colombo, Cinzia; Pinto, B Mario; Bernardi, Anna; Bennet, Andrew J

    2016-07-01

    This manuscript describes a novel class of derivatives based on a bicyclo[3.1.0]hexane scaffold, proposed as mimics of sialic acid in a distorted boat conformation that is on the catalytic pathway of neuraminidases (sialidases). A general synthetic route for these constrained-ring molecules was developed using a photochemical reaction followed by a Johnson-Corey-Chaykovsky cyclopropanation. Functionalization with the goal of occupying the 150-cavity was also exploited. Inhibition assays demonstrated low micromolar inhibition against both group-1 (H5N1) and group-2 (H9N2) influenza neuraminidase subtypes, indicating good affinity for the alpha and beta sialic acid mimics and 150-cavity-targeted derivatives. These results provide a validation of a bicyclo[3.1.0]hexane scaffold as a mimic of a distorted sialic acid bound in the neuraminidase active site during catalysis. PMID:27305457

  9. The Value of Neuraminidase Inhibitors for the Prevention and Treatment of Seasonal Influenza: A Systematic Review of Systematic Reviews

    PubMed Central

    Michiels, Barbara; Van Puyenbroeck, Karolien; Verhoeven, Veronique; Vermeire, Etienne; Coenen, Samuel

    2013-01-01

    Controversy has arisen regarding the effectiveness of neuraminidase inhibitors (NIs), especially against influenza-related complications. A literature search was performed to critically assess the evidence collected by the available systematic reviews (SRs) regarding the benefits and disadvantages of NIs (oseltamivir, zanamivir) compared to placebos in healthy and at-risk individuals of all ages for prophylaxis and treatment of seasonal influenza. A SR was done using the Cochrane Database of Systematic Reviews, Health Technology Assessment Database, Database of Abstracts of Reviews of Effects, and Medline (January 2006–July 2012). Two reviewers selected SRs based on randomized clinical trials, which were restricted to intention-to-treat results, and they assessed review (AMSTAR) and study quality indicators (GRADE). The SRs included (N = 9) were of high quality. The efficacy of NIs in prophylaxis ranged from 64% (16–85) to 92% (37–99); the absolute risk reduction ranged from 1.2% to 12.1% (GRADE moderate to low). Clinically relevant treatment benefits of NIs were small in healthy adults and children suffering from influenza-like illness (GRADE high to moderate). Oseltamivir reduced antibiotic usage in healthy adults according to one SR, but this was not confirmed by other reviews (GRADE low). Zanamivir showed a preventive effect on antibiotic usage in children (95% (77–99);GRADE moderate) and on the occurrence of bronchitis in at-risk individuals (59% (30–76);GRADE moderate). No evidence was available on the treatment benefits of NIs in elderly and at-risk groups and their effects on hospitalization and mortality. In oseltamivir trials, nausea, vomiting and diarrhea were significant side-effects. For zanamivir trials, no adverse effects have been reported. The combination of diagnostic uncertainty, the risk for virus strain resistance, possible side effects and financial cost outweigh the small benefits of oseltamivir or zanamivir for the prophylaxis

  10. Neuraminidase assays.

    PubMed

    Aymard, M; Ferraris, O; Gerentes, L; Jolly, J; Kessler, N

    2003-01-01

    Anti-neuraminidase (NA) antibodies (Ab) play a role in protection against influenza and in combination with anti-HA Ab they increase the protection in mice. To control the NA content of vaccines, which should improve vaccine standardisation and may benefit vaccine efficacy, a series of questions must be addressed: 1) The antigenic characterization of NA in vaccine strains and seed lots is based on the measurement of the enzymatic (E) activity using fetuin as substrate. The antigenic profile is established by inhibiting the E activity with post infectious ferret antisera. Overnight incubation ensures sensitivity, and fetuin substrate gives specificity by detection of variant specific antibodies. Several difficulties have to be overcome, such as the low level of E activity in MDCK grown viruses, and the lability of N1. 2) The NA protein content of the vaccines (in bulk or final product) can be measured by an ELISA capture test but the lability of the NA proteins at 4 degrees C must be checked. 3) The anti NA Ab response can be measured using a neuraminidase inhibition test. --The steric hindrance by HI antibodies does not exceed a titre of 20 in human sera. --Triton treatment of viruses reduces the steric hindrance in polyclonal sera and monoclonal antibodies but unmasks epitopes. 4) The correlations between neuraminidase inhibition, neutralization and protection, has been established in the mouse model, but remains to be shown in humans. 5) The use of a small fluorescent (MUN) or chemiluminescent (NA-STAR) substrate can be used for the rapid differentiation of N1 from N2 and NB, but not for the titration of protective NI antibodies.

  11. In vitro neuraminidase inhibitory concentration (IC50) of four neuraminidase inhibitors against clinical isolates of the influenza viruses circulating in the 2010-2011 to 2014-2015 Japanese influenza seasons.

    PubMed

    Ikematsu, Hideyuki; Kawai, Naoki; Iwaki, Norio; Kashiwagi, Seizaburo

    2016-09-01

    To assess the extent of viral resistance to the four neuraminidase inhibitors (NAIs), we measured their 50% inhibitory concentration (IC50) for influenza virus isolates from the 2014-2015 influenza season for comparison with those circulating in the 2010-2011 to 2013-2014 influenza seasons. Viral isolation was done with specimens obtained prior to treatment, and the type and subtype of influenza was determined by RT-PCR using type- and subtype-specific primers. The IC50 was determined by a neuraminidase inhibition assay using a fluorescent substrate. IC50 was measured for 200 influenza A(H3N2) and 19 influenza B in the 2014-2015 season, and no virus with highly reduced sensitivity to the four NAIs was detected. The ratios of the geometric means of the A(H3N2) IC50s of 2014-2015 to those of the 2010-2011, 2011-2012, 2012-2013, and 2013-2014 seasons ranged from 0.72 to 1.05, 0.82 to 1.22, 0.69 to 1.00, and 0.70 to 1.03, respectively. The ratios of the geometric mean of the B IC50s to the previous four seasons ranged from 0.59 to 1.28, 0.66 to 1.34, 0.84 to 1.21, and 1.06 to 1.47, respectively. There was no trend in the change of the IC50s for A(H3N2) or B. Significant differences were found in some seasons, but the differences in the IC50s were all less than two fold. These results show change in the geometric mean IC50 by season but with no trend, which indicates that the influence of viral mutation on the effectiveness of these NAIs was minute for A(H3N2) and B over the past five seasons.

  12. Real time enzyme inhibition assays provide insights into differences in binding of neuraminidase inhibitors to wild type and mutant influenza viruses.

    PubMed

    Barrett, Susan; Mohr, Peter G; Schmidt, Peter M; McKimm-Breschkin, Jennifer L

    2011-01-01

    The influenza neuraminidase (NA) inhibitors zanamivir, oseltamivir and peramivir were all designed based on the knowledge that the transition state analogue of the cleaved sialic acid, 2-deoxy,2,3-dehydro N-acetyl neuraminic acid (DANA) was a weak inhibitor of NA. While DANA bound rapidly to the NA, modifications leading to the improved potency of these new inhibitors also conferred a time dependent or slow binding phenotype. Many mutations in the NA leading to decreased susceptibility result in loss of slow binding, hence this is a phenotypic marker of many but not all resistant NAs. We present here a simplified approach to determine whether an inhibitor is fast or slow binding by extending the endpoint fluorescent enzyme inhibition assay to a real time assay and monitoring the changes in IC(50)s with time. We carried out two reactions, one with a 30 min preincubation with inhibitor and the second without. The enzymatic reaction was started via addition of substrate and IC(50)s were calculated after each 10 min interval up to 60 min. Results showed that without preincubation IC(50)s for the wild type viruses started high and although they decreased continuously over the 60 min reaction time the final IC(50)s remained higher than for pre-incubated samples. These results indicate a slow equilibrium of association and dissociation and are consistent with slow binding of the inhibitors. In contrast, for viruses with decreased susceptibility, preincubation had minimal effect on the IC(50)s, consistent with fast binding. Therefore this modified assay provides additional phenotypic information about the rate of inhibitor binding in addition to the IC(50), and critically demonstrates the differential effect of incubation times on the IC(50) and K(i) values of wild type and mutant viruses for each of the inhibitors.

  13. Computational modeling and validation studies of 3-D structure of neuraminidase protein of H1N1 influenza A virus and subsequent in silico elucidation of piceid analogues as its potent inhibitors.

    PubMed

    Gupta, Chhedi Lal; Akhtar, Salman; Bajpaib, Preeti; Kandpal, K N; Desai, G S; Tiwari, Ashok K

    2013-01-01

    Emergence of the drug resistant variants of the Influenza A virus in the recent years has aroused a great need for the development of novel neuraminidase inhibitors for controlling the pandemic. The neuraminidase (NA) protein of the influenza virus has been the most potential target for the anti-influenza. However, in the absence of any experimental structure of the drug targeting NA protein of H1N1 influenza A virus as zanamivir and oseltamivir, the comprehensive study of the interaction of the drug molecules with the target protein has been missing. Hence in this study a computational 3-D structure of neuraminidase of H1N1 influenza A virus has been developed using homology modeling technique, and the same was validated for its reliability by ProSA web server in term of energy profile & Z scores and PROCHECK program followed by Ramachandran plot. Further, the developed 3-D model had been employed for docking studies with the class of compounds as Piceid and its analogs. In this context, two novel compounds (ChemBank ID 2110359 and 3075417) were found to be more potent inhibitors of neuraminidase than control drugs as zanamivir and oseltamivir in terms of their robust binding energies, strong inhibition constant (Ki) and better hydrogen bond interactions between the protein-ligand complex. The interaction of these compounds with NA protein has been significantly studied at the molecular level.

  14. Novel Genotyping and Quantitative Analysis of Neuraminidase Inhibitor Resistance-Associated Mutations in Influenza A Viruses by Single-Nucleotide Polymorphism Analysis▿§

    PubMed Central

    Duan, Susu; Boltz, David A.; Li, Jiang; Oshansky, Christine M.; Marjuki, Henju; Barman, Subrata; Webby, Richard J.; Webster, Robert G.; Govorkova, Elena A.

    2011-01-01

    Neuraminidase (NA) inhibitors are among the first line of defense against influenza virus infection. With the increased worldwide use of the drugs, antiviral susceptibility surveillance is increasingly important for effective clinical management and for public health epidemiology. Effective monitoring requires effective resistance detection methods. We have developed and validated a novel genotyping method for rapid detection of established NA inhibitor resistance markers in influenza viruses by single nucleotide polymorphism (SNP) analysis. The multi- or monoplex SNP analysis based on single nucleotide extension assays was developed to detect NA mutations H275Y and I223R/V in pandemic H1N1 viruses, H275Y in seasonal H1N1 viruses, E119V and R292K in seasonal H3N2 viruses, and H275Y and N295S in H5N1 viruses. The SNP analysis demonstrated high sensitivity for low-content NA amplicons (0.1 to 1 ng/μl) and showed 100% accordant results against a panel of defined clinical isolates. The monoplex assays for the H275Y NA mutation allowed precise and accurate quantification of the proportions of wild-type and mutant genotypes in virus mixtures (5% to 10% discrimination), with results comparable to those of pyrosequencing. The SNP analysis revealed the lower growth fitness of an H275Y mutant compared to the wild-type pandemic H1N1 virus by quantitatively genotyping progeny viruses grown in normal human bronchial epithelial cells. This novel method offers high-throughput screening capacity, relatively low costs, and the wide availability of the necessary equipment, and thus it could provide a much-needed approach for genotypic screening of NA inhibitor resistance in influenza viruses. PMID:21730113

  15. The Neuraminidase Inhibitor Oseltamivir Is Effective Against A/Anhui/1/2013 (H7N9) Influenza Virus in a Mouse Model of Acute Respiratory Distress Syndrome

    PubMed Central

    Baranovich, Tatiana; Burnham, Andrew J.; Marathe, Bindumadhav M.; Armstrong, Jianling; Guan, Yi; Shu, Yuelong; Peiris, Joseph Malik Sriyal; Webby, Richard J.; Webster, Robert G.; Govorkova, Elena A.

    2014-01-01

    Background. High mortality and uncertainty about the effectiveness of neuraminidase inhibitors (NAIs) in humans infected with influenza A(H7N9) viruses are public health concerns. Methods. Susceptibility of N9 viruses to NAIs was determined in a fluorescence-based assay. The NAI oseltamivir (5, 20, or 80 mg/kg/day) was administered to BALB/c mice twice daily starting 24, 48, or 72 hours after A/Anhui/1/2013 (H7N9) virus challenge. Results. All 12 avian N9 and 3 human H7N9 influenza viruses tested were susceptible to NAIs. Without prior adaptation, A/Anhui/1/2013 (H7N9) caused lethal infection in mice that was restricted to the respiratory tract and resulted in pulmonary edema and acute lung injury with hyaline membrane formation, leading to decreased oxygenation, all characteristics of human acute respiratory distress syndrome. Oseltamivir at 20 and 80 mg/kg protected 80% and 88% of mice when initiated after 24 hours, and the efficacy decreased to 70% and 60%, respectively, when treatment was delayed by 48 hours. Emergence of oseltamivir-resistant variants was not detected. Conclusions. H7N9 viruses are comparable to currently circulating influenza A viruses in susceptibility to NAIs. Based on these animal studies, early treatment is associated with improved outcomes. PMID:24133191

  16. Penetration of GS4071, a novel influenza neuraminidase inhibitor, into rat bronchoalveolar lining fluid following oral administration of the prodrug GS4104.

    PubMed Central

    Eisenberg, E J; Bidgood, A; Cundy, K C

    1997-01-01

    GS4071 is a novel potent inhibitor of influenza neuraminidase (Ki < 1 nM) with low (< 5%) oral bioavailability in animals. An ethyl ester prodrug of GS4071, GS4104, has exhibited good oral bioavailability in rat, mouse, and dog models and is currently being developed for the treatment of influenza A and B virus infections. Since influenza virus replicates primarily in the surface epithelial cells of the respiratory tract, the ability of the prodrug to deliver GS4071 to the bronchoalveolar lining fluid (BALF) following an oral dose of GS4104 should be an important indicator of its potential efficacy. In the present study, we determined the concentration-time profiles of GS4071 in the BALF and plasma of rats following oral administration of GS4104. The BALF was sampled by bronchoalveolar lavage with endogenous urea as a dilution marker. The concentration of GS4071 in BALF reached a peak at 2 h (1 h after the plasma peak) and declined at a slower rate than plasma levels, suggesting slow clearance of drug from the lung acini. The ratios of the area-under-the-curve (AUC) values of GS4071 in BALF to those in plasma were 1.05 for AUC from 0 to 6 h (AUC(0-6)) and 1.51 for AUC(0-infinity), indicating significant penetration of the parent drug into the lower respiratory tracts of rats following oral administration of the prodrug. No unchanged GS4104 was detected in BALF. PMID:9303391

  17. Post-marketing safety evaluation of the intravenous anti-influenza neuraminidase inhibitor peramivir: A drug-use investigation in patients with high risk factors.

    PubMed

    Komeda, Takuji; Ishii, Shingo; Itoh, Yumiko; Sanekata, Masaki; Yoshikawa, Takayoshi; Shimada, Jingoro

    2016-10-01

    Peramivir, the only injectable anti-influenza neuraminidase inhibitor medically available in Japan at present, is considered first-line treatment in patients with high risk factors for influenza exacerbation. We conducted a drug-use investigation of peramivir in inpatients with high risk factors (old age, pregnancy, and underlying disease such as chronic respiratory disease) from January 2010 to March 2013. Data of 772 patients from 124 facilities across Japan were collected; peramivir's safety in 770 patients and effectiveness in 688 patients were examined. In total, 412 adverse events were observed in 219 patients (28.4%). Of these, 155 events were adverse drug reactions (ADRs) observed in 98 patients (12.7%). Major ADRs (≥2%) were increased aspartate aminotransferase (5.1%), increased alanine aminotransferase (3.8%) and decreased white blood cell count (2.5%). Fourteen serious ADRs were observed in 12 patients (1.6%). All serious ADRs were resolved or improved except for two events for which outcomes were unknown. Multivariate analyses revealed that ADR incidences were significantly associated with these four backgrounds of patients: medical history, no influenza vaccination, renal impairment and other infection(s). With regard to its effectiveness, the median time to alleviation of both influenza symptoms and fever was 3 days, including the first day of administration, which was the same as in other previous surveillance studies. This surveillance study indicated the safety of peramivir in the treatment of influenza inpatients with high risk factors under routine clinical settings. PMID:27497712

  18. Competitive Fitness of Influenza B Viruses with Neuraminidase Inhibitor-Resistant Substitutions in a Coinfection Model of the Human Airway Epithelium

    PubMed Central

    Burnham, Andrew J.; Armstrong, Jianling; Lowen, Anice C.; Webster, Robert G.

    2015-01-01

    ABSTRACT Influenza A and B viruses are human pathogens that are regarded to cause almost equally significant disease burdens. Neuraminidase (NA) inhibitors (NAIs) are the only class of drugs available to treat influenza A and B virus infections, so the development of NAI-resistant viruses with superior fitness is a public health concern. The fitness of NAI-resistant influenza B viruses has not been widely studied. Here we examined the replicative capacity and relative fitness in normal human bronchial epithelial (NHBE) cells of recombinant influenza B/Yamanashi/166/1998 viruses containing a single amino acid substitution in NA generated by reverse genetics (rg) that is associated with NAI resistance. The replication in NHBE cells of viruses with reduced inhibition by oseltamivir (recombinant virus with the E119A mutation generated by reverse genetics [rg-E119A], rg-D198E, rg-I222T, rg-H274Y, rg-N294S, and rg-R371K, N2 numbering) or zanamivir (rg-E119A and rg-R371K) failed to be inhibited by the presence of the respective NAI. In a fluorescence-based assay, detection of rg-E119A was easily masked by the presence of NAI-susceptible virus. We coinfected NHBE cells with NAI-susceptible and -resistant viruses and used next-generation deep sequencing to reveal the order of relative fitness compared to that of recombinant wild-type (WT) virus generated by reverse genetics (rg-WT): rg-H274Y > rg-WT > rg-I222T > rg-N294S > rg-D198E > rg-E119A ≫ rg-R371K. Based on the lack of attenuated replication of rg-E119A in NHBE cells in the presence of oseltamivir or zanamivir and the fitness advantage of rg-H274Y over rg-WT, we emphasize the importance of these substitutions in the NA glycoprotein. Human infections with influenza B viruses carrying the E119A or H274Y substitution could limit the therapeutic options for those infected; the emergence of such viruses should be closely monitored. IMPORTANCE Influenza B viruses are important human respiratory pathogens contributing to a

  19. In vitro activity of favipiravir and neuraminidase inhibitor combinations against oseltamivir-sensitive and oseltamivir-resistant pandemic influenza A (H1N1) virus.

    PubMed

    Tarbet, E Bart; Vollmer, Almut H; Hurst, Brett L; Barnard, Dale L; Furuta, Yousuke; Smee, Donald F

    2014-06-01

    Few anti-influenza drugs are licensed in the United States for the prevention and therapy of influenza A and B virus infections. This shortage, coupled with continuously emerging drug resistance, as detected through a global surveillance network, seriously limits our anti-influenza armamentarium. Combination therapy appears to offer several advantages over traditional monotherapy in not only delaying development of resistance but also potentially enhancing single antiviral activity. In the present study, we evaluated the antiviral drug susceptibilities of fourteen pandemic influenza A (H1N1) virus isolates in MDCK cells. In addition, we evaluated favipiravir (T-705), an investigational drug with a broad antiviral spectrum and a unique mode of action, alone and in dual combination with the neuraminidase inhibitors (NAIs) oseltamivir, peramivir, or zanamivir, against oseltamivir-sensitive pandemic influenza A/California/07/2009 (H1N1) and oseltamivir-resistant A/Hong Kong/2369/2009 (H1N1) virus. Mean inhibitory values showed that the tested virus isolates remained sensitive to commonly used antiviral drugs, with the exception of the Hong Kong virus isolate. Drug dose-response curves confirmed complete drug resistance to oseltamivir, partial sensitivity to peramivir, and retained susceptibility to zanamivir and favipiravir against the A/Hong Kong/2369/2009 virus. Three-dimensional analysis of drug interactions using the MacSynergy(TM) II program indicated an overall synergistic interaction when favipiravir was combined with the NAIs against the oseltamivir-sensitive influenza virus, and an additive effect against the oseltamivir-resistant virus. Although the clinical relevance of these drug combinations remains to be evaluated, results obtained from this study support the use of combination therapy with favipiravir and NAIs for treatment of human influenza virus infections.

  20. Neuraminidase Inhibitors and Hospital Mortality in British Patients with H1N1 Influenza A: A Re-Analysis of Observational Data

    PubMed Central

    Wolkewitz, Martin; Schumacher, Martin

    2016-01-01

    Background Observational studies claimed reducing effects of neuraminidase inhibitors (NI) on hospital mortality in patients with H1N1 influenza A. It has been criticized that such findings are prone to common and serious survival biases. Methods With observational data from the FLU-CIN study group, multi-state and dynamic prediction models have been used to avoid such biases. The data included 1391 patients with confirmed pandemic influenza A/H1N1 infection collected during 2009-2010 in the UK. Due to their close relationship, the main outcome measures were hospital death and length of hospital stay. Findings There is no direct effect of NI on the hospital death rate; the hazard ratio (HR) of NI was 1.03 (95%-CI: 0.64–1.66). The discharge rate is increased for NI patients (HR = 1.89 (95%-CI: 1.65–2.16)) indicating that NI-treated patients stay shorter in hospital than NI-untreated patients, on average 3.10 days (95%-CI: 2.07–4.14). We also showed that the initiation timing of NI treatment (≤ 2 days versus > 2 days after onset) made no difference on the effects on the hospital death and discharge hazards. The hazard ratios remain stable after adjusting for potential confounders measured at admission (such as comorbidities and influenza-related clinical symptoms). Conclusions The potential beneficial effect of NI on hospitalized patients in the UK is rather a reduction of the length of hospital stay than a reduction of the mortality rate. There seems to be no confounding by indication and no differences if NI is given early or late. Different effects could be present in other populations (such as non-hospitalized individuals) or countries. Careful interpretation of the effect on length of hospital stay is needed due to potentially different discharge policies of NI-treated and NI-untreated patients. PMID:27583403

  1. Fitness Costs for Influenza B Viruses Carrying Neuraminidase Inhibitor-Resistant Substitutions: Underscoring the Importance of E119A and H274Y

    PubMed Central

    Burnham, Andrew J.; Baranovich, Tatiana; Marathe, Bindumadhav M.; Armstrong, Jianling; Webster, Robert G.

    2014-01-01

    Influenza B viruses cause annual outbreaks of respiratory illness in humans and are increasingly recognized as a major cause of influenza-associated pediatric mortality. Neuraminidase (NA) inhibitors (NAIs) are the only available therapy for patients infected with influenza B viruses, and the potential emergence of NAI-resistant viruses is a public health concern. The NA substitutions located within the enzyme active site could not only reduce NAI susceptibility of influenza B virus but also affect virus fitness. In this study, we investigated the effect of single NA substitutions on the fitness of influenza B/Yamanashi/166/1998 viruses (Yamagata lineage). We generated recombinant viruses containing either wild-type (WT) NA or NA with a substitution in the catalytic (R371K) or framework (E119A, D198E, D198Y, I222T, H274Y, and N294S) residues. We assessed NAI susceptibility, NA biochemical properties, NA protein expression, and virus replication in vitro and in differentiated normal human bronchial epithelial (NHBE) cells. Our results showed that four NA substitutions (D198E, I222T, H274Y, and N294S) conferred reduced inhibition by oseltamivir and three (E119A, D198Y, and R371K) conferred highly reduced inhibition by oseltamivir, zanamivir, and peramivir. All NA substitutions, except for D198Y and R371K, were genetically stable after seven passages in MDCK cells. Cell surface NA protein expression was significantly increased by H274Y and N294S substitutions. Viruses with the E119A, I222T, H274Y, or N294S substitution were not attenuated in replication efficiency in vitro or in NHBE cells. Overall, viruses with the E119A or H274Y NA substitution possess fitness comparable to NAI-susceptible virus, and the acquisition of these substitutions by influenza B viruses should be closely monitored. PMID:24566185

  2. Supply of Neuraminidase Inhibitors Related to Reduced Influenza A (H1N1) Mortality during the 2009–2010 H1N1 Pandemic: An Ecological Study

    PubMed Central

    Hubbard, Roderick J.; Li, Jiabai; Meyer, Alison E.; Stephens, Peter; Mounts, Anthony W.; Rolfes, Melissa A.; Penn, Charles R.

    2012-01-01

    Background The influenza A (H1N1) pandemic swept across the globe from April 2009 to August 2010 affecting millions. Many WHO Member States relied on antiviral drugs, specifically neuraminidase inhibitors (NAIs) oseltamivir and zanamivir, to treat influenza patients in critical condition. Such drugs have been found to be effective in reducing severity and duration of influenza illness, and likely reduced morbidity during the pandemic. However, it is less clear whether NAIs used during the pandemic reduced H1N1 mortality. Methods Country-level data on supply of oseltamivir and zanamivir were used to predict H1N1 mortality (per 100,000 people) from July 2009 to August 2010 in forty-two WHO Member States. Poisson regression was used to model the association between NAI supply and H1N1 mortality, with adjustment for economic, demographic, and health-related confounders. Results After adjustment for potential confounders, each 10% increase in kilograms of oseltamivir, per 100,000 people, was associated with a 1.6% reduction in H1N1 mortality over the pandemic period (relative rate (RR) = 0.84 per log increase in oseltamivir supply). While the supply of zanamivir was considerably less than that of oseltamivir in each Member State, each 10% increase in kilogram of active zanamivir, per 100,000, was associated with a 0.3% reduction in H1N1 mortality (RR = 0.97 per log increase). Conclusion While there are limitations to the ecologic nature of these data, this analysis offers evidence of a protective relationship between antiviral drug supply and influenza mortality and supports a role for influenza antiviral use in future pandemics. PMID:22984431

  3. Competitive Fitness of Influenza B Viruses Possessing E119A and H274Y Neuraminidase Inhibitor Resistance-Associated Substitutions in Ferrets.

    PubMed

    Pascua, Philippe Noriel Q; Marathe, Bindumadhav M; Burnham, Andrew J; Vogel, Peter; Webby, Richard J; Webster, Robert G; Govorkova, Elena A

    2016-01-01

    Neuraminidase (NA) inhibitors (NAIs) are the only antiviral drugs recommended for influenza treatment and prophylaxis. Although NAI-resistant influenza B viruses that could pose a threat to public health have been reported in the field, their fitness is poorly understood. We evaluated in ferrets the pathogenicity and relative fitness of reverse genetics (rg)-generated influenza B/Yamanashi/166/1998-like viruses containing E119A or H274Y NA substitutions (N2 numbering). Ferrets inoculated with NAI-susceptible rg-wild-type (rg-WT) or NAI-resistant (rg-E119A or rg-H274Y) viruses developed mild infections. Growth of rg-E119A virus in the nasal cavities was delayed, but the high titers at 3 days post-inoculation (dpi) were comparable to those of the rg-WT and rg-H274Y viruses (3.6-4.1 log10TCID50/mL). No virus persisted beyond 5 dpi and replication did not extend to the trachea or lungs. Positive virus antigen-staining of the nasal turbinate epithelium was intermittent with the rg-WT and rg-H274Y viruses; whereas antigen-staining for the rg-E119A virus was more diffuse. Virus populations in ferrets coinoculated with NAI-susceptible and -resistant viruses (1:1 mixture) remained heterogeneous at 5 dpi but were predominantly rg-WT (>70%). Although the E119A substitution was associated with delayed replication in ferrets, the H274Y substitution did not measurably affect viral growth properties. These data suggest that rg-H274Y has undiminished fitness in single virus inoculations, but neither rg-E119A nor rg-H274Y gained a fitness advantage over rg-WT in direct competition experiments without antiviral drug pressure. Taken together, our data suggest the following order of relative fitness in a ferret animal model: rg-WT > rg-H274Y > rg-E119A. PMID:27466813

  4. Competitive Fitness of Influenza B Viruses Possessing E119A and H274Y Neuraminidase Inhibitor Resistance–Associated Substitutions in Ferrets

    PubMed Central

    Pascua, Philippe Noriel Q.; Marathe, Bindumadhav M.; Burnham, Andrew J.; Vogel, Peter; Webby, Richard J.; Webster, Robert G.; Govorkova, Elena A.

    2016-01-01

    Neuraminidase (NA) inhibitors (NAIs) are the only antiviral drugs recommended for influenza treatment and prophylaxis. Although NAI-resistant influenza B viruses that could pose a threat to public health have been reported in the field, their fitness is poorly understood. We evaluated in ferrets the pathogenicity and relative fitness of reverse genetics (rg)–generated influenza B/Yamanashi/166/1998-like viruses containing E119A or H274Y NA substitutions (N2 numbering). Ferrets inoculated with NAI-susceptible rg–wild-type (rg-WT) or NAI-resistant (rg-E119A or rg-H274Y) viruses developed mild infections. Growth of rg-E119A virus in the nasal cavities was delayed, but the high titers at 3 days post-inoculation (dpi) were comparable to those of the rg-WT and rg-H274Y viruses (3.6–4.1 log10TCID50/mL). No virus persisted beyond 5 dpi and replication did not extend to the trachea or lungs. Positive virus antigen-staining of the nasal turbinate epithelium was intermittent with the rg-WT and rg-H274Y viruses; whereas antigen-staining for the rg-E119A virus was more diffuse. Virus populations in ferrets coinoculated with NAI-susceptible and -resistant viruses (1:1 mixture) remained heterogeneous at 5 dpi but were predominantly rg-WT (>70%). Although the E119A substitution was associated with delayed replication in ferrets, the H274Y substitution did not measurably affect viral growth properties. These data suggest that rg-H274Y has undiminished fitness in single virus inoculations, but neither rg-E119A nor rg-H274Y gained a fitness advantage over rg-WT in direct competition experiments without antiviral drug pressure. Taken together, our data suggest the following order of relative fitness in a ferret animal model: rg-WT > rg-H274Y > rg-E119A. PMID:27466813

  5. Influenza A viruses of swine circulating in the United States during 2009–2014 are susceptible to neuraminidase inhibitors but show lineage-dependent resistance to adamantanes

    PubMed Central

    Baranovich, Tatiana; Bahl, Justin; Marathe, Bindumadhav M.; Culhane, Marie; Stigger-Rosser, Evelyn; Darnell, Daniel; Kaplan, Bryan S.; Lowe, James F.; Webby, Richard J.; Govorkova, Elena A.

    2015-01-01

    Antiviral drug susceptibility is one of the evaluation criteria of pandemic potential posed by an influenza virus. Influenza A viruses of swine (IAV-S) can play an important role in generating novel variants, yet limited information is available on the drug resistance profiles of IAV-S circulating in the U.S. Phenotypic analysis of the IAV-S isolated in the U.S. (2009–2011) (n=105) revealed normal inhibition by the neuraminidase (NA) inhibitors (NAIs) oseltamivir, zanamivir, and peramivir. Screening NA sequences from IAV-S collected in the U.S. since 1930 showed 0.03% (1/3396) sequences with clinically relevant H274Y-NA substitution. Phenotypic analysis of IAV-S isolated in the U.S. (2009–2011) confirmed amantadine resistance caused by the S31N-M2 and revealed an intermediate level of resistance caused by the I27T-M2. The majority (96.7%, 589/609) of IAV-S with the I27T-M2 in the influenza database were isolated from pigs in the U.S. The frequency of amantadine-resistant markers among IAV-S in the U.S. was high (71%), and their distribution was M-lineage dependent. All IAV-S of the Eurasian avian M lineage were amantadine-resistant and possessed either a single S31N-M2 substitution (78%, 585/747) or its combination with the V27A-M2 (22%, 162/747). The I27T-M2 substitution accounted for 43% (429/993) of amantadine resistance in classic swine M lineage. Phylogenetic analysis showed that both S31N-M2 and I27T-M2 emerged stochastically but appeared to be fixed in the U.S. IAV-S population. This study defines a drug-susceptibility profile, identifies the frequency of drug-resistant markers, and establishes a phylogenetic approach for continued antiviral-susceptibility monitoring of IAV-S in the U.S. PMID:25701593

  6. Neuraminidase inhibitor susceptibility profile of pandemic and seasonal influenza viruses during the 2009-2010 and 2010-2011 influenza seasons in Japan.

    PubMed

    Dapat, Clyde; Kondo, Hiroki; Dapat, Isolde C; Baranovich, Tatiana; Suzuki, Yasushi; Shobugawa, Yugo; Saito, Kousuke; Saito, Reiko; Suzuki, Hiroshi

    2013-09-01

    Two new influenza virus neuraminidase inhibitors (NAIs), peramivir and laninamivir, were approved in 2010 which resulted to four NAIs that were used during the 2010-2011 influenza season in Japan. This study aims to monitor the susceptibility of influenza virus isolates in 2009-2010 and 2010-2011 influenza seasons in Japan to the four NAIs using the fluorescence-based 50% inhibitory concentration (IC₅₀) method. Outliers were identified using box-and-whisker plot analysis and full NA gene sequencing was performed to determine the mutations that are associated with reduction of susceptibility to NAIs. A total of 117 influenza A(H1N1)pdm09, 59 A(H3N2), and 18 type B viruses were tested before NAI treatment and eight A(H1N1)pdm09 and 1 type B viruses were examined from patients after NAI treatment in the two seasons. NA inhibition assay showed type A influenza viruses were more susceptible to NAIs than type B viruses. The peramivir and laninamivir IC₅₀ values of both type A and B viruses were significantly lower than the oseltamivir and zanamivir IC₅₀ values. Among influenza A(H1N1)pdm09 viruses, the prevalence of H274Y viruses increased from 0% in the 2009-2010 season to 3% in the 2010-2011 season. These H274Y viruses were resistant to oseltamivir and peramivir with 200-300 fold increase in IC₅₀ values but remained sensitive to zanamivir and laninamivir. Other mutations in NA, such as I222T and M241I were identified among the outliers. Among influenza A(H3N2) viruses, two outliers were identified with D151G and T148I mutations, which exhibited a reduction in susceptibility to oseltamivir and zanamivir, respectively. Among type B viruses, no outliers were identified to the four NAIs. For paired samples that were collected before and after drug treatment, three (3/11; 27.3%) H274Y viruses were identified among A(H1N1)pdm09 viruses after oseltamivir treatment but no outliers were found in the laninamivir-treatment group (n=3). Despite widespread use of

  7. Neuraminidase in Bacteroides fragilis.

    PubMed Central

    Berg, J O; Lindqvist, L; Andersson, G; Nord, C E

    1983-01-01

    A neuraminidase from Bacteroides fragilis was purified 542-fold by isoelectric focusing, adsorption chromatography on Affi-Gel 202, and gel filtration chromatography on Sephadex G-200. On isoelectric focusing the neuraminidase was resolved into three differently charged fractions with pI values of 6.8, 7.1, and 7.4. The major component of pI 7.1 was used for further purification. The purified enzyme had optimal activity at pH 6.4 with N-acetylneuraminlactose as the substrate. Its molecular weight, determined by Sephadex G-200 gel filtration chromatography, was 92,000. The neuraminidase hydrolyzed terminal neuraminic acid residues from N-acetylneuraminlactose, fetuin, bovine submaxillary mucin, and porcine stomach lining mucin. A new method for the detection of neuraminidase activity is described which is based on rocket affinoelectrophoresis. It utilizes the differences in the interaction of sialylated and desialylated mucin with Helix pomatia lectin, enzymatic activity being detected by formation of affinorockets after incubation of the neuraminidase with bovine submaxillary mucin. PMID:6614909

  8. Crystal Structures of Respiratory Pathogen Neuraminidases

    SciTech Connect

    Hsiao, Y.; Parker, D; Ratner, A; Prince, A; Tong, L

    2009-01-01

    Currently there is pressing need to develop novel therapeutic agents for the treatment of infections by the human respiratory pathogens Pseudomonas aeruginosa and Streptococcus pneumoniae. The neuraminidases of these pathogens are important for host colonization in animal models of infection and are attractive targets for drug discovery. To aid in the development of inhibitors against these neuraminidases, we have determined the crystal structures of the P. aeruginosa enzyme NanPs and S. pneumoniae enzyme NanA at 1.6 and 1.7 {angstrom} resolution, respectively. In situ proteolysis with trypsin was essential for the crystallization of our recombinant NanA. The active site regions of the two enzymes are strikingly different. NanA contains a deep pocket that is similar to that in canonical neuraminidases, while the NanPs active site is much more open. The comparative studies suggest that NanPs may not be a classical neuraminidase, and may have distinct natural substrates and physiological functions. This work represents an important step in the development of drugs to prevent respiratory tract colonization by these two pathogens.

  9. Supply of neuraminidase inhibitors related to reduced influenza A (H1N1) mortality during the 2009-2010 H1N1 pandemic: summary of an ecological study.

    PubMed

    Miller, Paula E; Rambachan, Aksharananda; Hubbard, Roderick J; Li, Jiabai; Meyer, Alison E; Stephens, Peter; Mounts, Anthony W; Rolfes, Melissa A; Penn, Charles R

    2013-09-01

    When the influenza A (H1N1) pandemic spread across the globe from April 2009 to August 2010, many WHO Member States used antiviral drugs, specifically neuraminidase inhibitors (NAIs) oseltamivir and zanamivir, to treat influenza patients in critical condition. Antivirals have been found to be effective in reducing severity and duration of influenza illness, and likely reduce morbidity; however, it is unclear whether NAIs used during the pandemic reduced H1N1 mortality. To assess the association between antivirals and influenza mortality, at an ecologic level, country-level data on supply of oseltamivir and zanamivir were compared to laboratory-confirmed H1N1 deaths (per 100 000 people) from July 2009 to August 2010 in 42 WHO Member States. From this analysis, it was found that each 10% increase in kilograms of oseltamivir, per 100 000 people, was associated with a 1·6% reduction in H1N1 mortality over the pandemic period [relative rate (RR) = 0·84 per log increase in oseltamivir supply]. Each 10% increase in kilogram of active zanamivir, per 100 000, was associated with a 0·3% reduction in H1N1 mortality (RR = 0·97 per log increase). While limitations exist in the inference that can be drawn from an ecologic evaluation, this analysis offers evidence of a protective relationship between antiviral drug supply and influenza mortality and supports a role for influenza antiviral use in future pandemics. This article summarises the original study described previously, which can be accessed through the following citation: Miller PE, Rambachan A, Hubbard RJ, Li J, Meyer AE, et al. (2012) Supply of Neuraminidase Inhibitors Related to Reduced Influenza A (H1N1) Mortality during the 2009-2010 H1N1 Pandemic: An Ecological Study. PLoS ONE 7(9): e43491.

  10. Proline-based Phosphoramidite Reagents for the Reductive Ligation of S-Nitrosothiols

    PubMed Central

    Xian, Ming

    2015-01-01

    S-Nitrosothiols have many biological implications but are rarely used in organic synthesis. In this work we report the development of proline-based phosphoramidite substrates that can effectively convert S-nitrosothiols to proline-based sulfenamides through a reductive ligation process. A unique property of this method is that the phosphine oxide moiety on the ligation products can be readily removed under acidic conditions. In conjugation with the facile preparation of S-nitrosothiols (RSNO) from the corresponding thiols (RSH), this method provides a new way to prepare proline-based sulfenamides from simple thiol starting materials. PMID:26758493

  11. Toll-like receptor 9 ligand D-type oligodeoxynucleotide D35 as a broad inhibitor for influenza A virus replication that is associated with suppression of neuraminidase activity.

    PubMed

    Yamada, Hiroshi; Nagase, Satoshi; Takahashi, Kazuo; Sakoda, Yoshihiro; Kida, Hiroshi; Okamoto, Shigefumi

    2016-05-01

    The most effective drugs available to treat influenza are neuraminidase (NA) inhibitors, which provide important additional measures for the control of influenza virus infections. However, since the emergence of NA inhibitor-resistant viruses may compromise the clinical utility of this class of anti-influenza agents, it is very important to develop new anti-influenza agents which target a different region in NA responsible for its sensitivity from that for NA inhibitors and could be used to treat NA inhibitors-resistant isolates. The oligodeoxynucleotide D35, multimerized and aggregated, suppressed replication of influenza A viruses except A/WSN/33 (WSN). The suppressive viral replication by D35 depended on G-terad and multimer formation. The range of the suppressive viral replication at the late stage, including virus assembly and release from infected cells, was much larger than that at the initial stage, viral attachment and entry. D35 suppressed NA activity of influenza A viruses. Furthermore, replacing the NA gene of A/Puerto Rico/8/34 (PR8), in which viral replication was inhibited by D35 at the late stage, with the NA gene from WSN, in which viral replication was not inhibited, eliminated the D35-dependent suppression. D35 showed an additive anti-influenza effect with oseltamivir. It was also effective in vivo. These results suggest that the influenza virus NA mainly contributes to the D35-suppressible virus release from infected cells at the late stage. In addition, because administration of D35 into the virus-infected mice suppressed viral replication and weight loss, clinical application of D35 could be considered. PMID:26923882

  12. Antipneumococcal activity of neuraminidase inhibiting artocarpin.

    PubMed

    Walther, E; Richter, M; Xu, Z; Kramer, C; von Grafenstein, S; Kirchmair, J; Grienke, U; Rollinger, J M; Liedl, K R; Slevogt, H; Sauerbrei, A; Saluz, H P; Pfister, W; Schmidtke, M

    2015-05-01

    Streptococcus (S.) pneumoniae is a major cause of secondary bacterial pneumonia during influenza epidemics. Neuraminidase (NA) is a virulence factor of both pneumococci and influenza viruses. Bacterial neuraminidases (NAs) are structurally related to viral NA and susceptible to oseltamivir, an inhibitor designed to target viral NA. This prompted us to evaluate the antipneumococcal potential of two NA inhibiting natural compounds, the diarylheptanoid katsumadain A and the isoprenylated flavone artocarpin. Chemiluminescence, fluorescence-, and hemagglutination-based enzyme assays were applied to determine the inhibitory efficiency (IC(50) value) of the tested compounds towards pneumococcal NAs. The mechanism of inhibition was studied via enzyme kinetics with recombinant NanA NA. Unlike oseltamivir, which competes with the natural substrate of NA, artocarpin exhibits a mixed-type inhibition with a Ki value of 9.70 μM. Remarkably, artocarpin was the only NA inhibitor (NAI) for which an inhibitory effect on pneumococcal growth (MIC: 0.99-5.75 μM) and biofilm formation (MBIC: 1.15-2.97 μM) was observable. In addition, we discovered that the bactericidal effect of artocarpin can reduce the viability of pneumococci by a factor of >1000, without obvious harm to lung epithelial cells. This renders artocarpin a promising natural product for further investigations.

  13. Antipneumococcal activity of neuraminidase inhibiting artocarpin.

    PubMed

    Walther, E; Richter, M; Xu, Z; Kramer, C; von Grafenstein, S; Kirchmair, J; Grienke, U; Rollinger, J M; Liedl, K R; Slevogt, H; Sauerbrei, A; Saluz, H P; Pfister, W; Schmidtke, M

    2015-05-01

    Streptococcus (S.) pneumoniae is a major cause of secondary bacterial pneumonia during influenza epidemics. Neuraminidase (NA) is a virulence factor of both pneumococci and influenza viruses. Bacterial neuraminidases (NAs) are structurally related to viral NA and susceptible to oseltamivir, an inhibitor designed to target viral NA. This prompted us to evaluate the antipneumococcal potential of two NA inhibiting natural compounds, the diarylheptanoid katsumadain A and the isoprenylated flavone artocarpin. Chemiluminescence, fluorescence-, and hemagglutination-based enzyme assays were applied to determine the inhibitory efficiency (IC(50) value) of the tested compounds towards pneumococcal NAs. The mechanism of inhibition was studied via enzyme kinetics with recombinant NanA NA. Unlike oseltamivir, which competes with the natural substrate of NA, artocarpin exhibits a mixed-type inhibition with a Ki value of 9.70 μM. Remarkably, artocarpin was the only NA inhibitor (NAI) for which an inhibitory effect on pneumococcal growth (MIC: 0.99-5.75 μM) and biofilm formation (MBIC: 1.15-2.97 μM) was observable. In addition, we discovered that the bactericidal effect of artocarpin can reduce the viability of pneumococci by a factor of >1000, without obvious harm to lung epithelial cells. This renders artocarpin a promising natural product for further investigations. PMID:25592264

  14. Kinetic, thermodynamic and structural analysis of tamiphosphor binding to neuraminidase of H1N1 (2009) pandemic influenza.

    PubMed

    Albiñana, Carlos Berenguer; Machara, Aleš; Řezáčová, Pavlína; Pachl, Petr; Konvalinka, Jan; Kožíšek, Milan

    2016-10-01

    Influenza virus causes severe respiratory infections that are responsible for up to half a million deaths worldwide each year. Two inhibitors targeting viral neuraminidase have been approved to date (oseltamivir, zanamivir). However, the rapid development of antiviral drug resistance and the efficient transmission of resistant viruses among humans represent serious threats to public health. The approved influenza neuraminidase inhibitors have (oxa)cyclohexene scaffolds designed to mimic the oxonium transition state during enzymatic cleavage of sialic acid. Their active forms contain a carboxylate that interacts with three arginine residues in the enzyme active site. Recently, the phosphonate group was successfully used as an isostere of the carboxylate in oseltamivir, and the resulting compound, tamiphosphor, was identified as a highly active neuraminidase inhibitor. However, the structure of the complex of this promising inhibitor with neuraminidase has not yet been reported. Here, we analyzed the interaction of a set of oseltamivir and tamiphosphor derivatives with neuraminidase from the A/California/07/2009 (H1N1) influenza virus. We thermodynamically characterized the binding of oseltamivir carboxylate or tamiphosphor to the neuraminidase catalytic domain by protein microcalorimetry, and we determined crystal structure of the catalytic domain in complex with tamiphosphor at 1.8 Å resolution. This structural information should aid rational design of the next generation of neuraminidase inhibitors.

  15. A virtual screening approach for identifying plants with anti H5N1 neuraminidase activity.

    PubMed

    Ikram, Nur Kusaira Khairul; Durrant, Jacob D; Muchtaridi, Muchtaridi; Zalaludin, Ayunni Salihah; Purwitasari, Neny; Mohamed, Nornisah; Rahim, Aisyah Saad Abdul; Lam, Chan Kit; Normi, Yahaya M; Rahman, Noorsaadah Abd; Amaro, Rommie E; Wahab, Habibah A

    2015-02-23

    Recent outbreaks of highly pathogenic and occasional drug-resistant influenza strains have highlighted the need to develop novel anti-influenza therapeutics. Here, we report computational and experimental efforts to identify influenza neuraminidase inhibitors from among the 3000 natural compounds in the Malaysian-Plants Natural-Product (NADI) database. These 3000 compounds were first docked into the neuraminidase active site. The five plants with the largest number of top predicted ligands were selected for experimental evaluation. Twelve specific compounds isolated from these five plants were shown to inhibit neuraminidase, including two compounds with IC50 values less than 92 μM. Furthermore, four of the 12 isolated compounds had also been identified in the top 100 compounds from the virtual screen. Together, these results suggest an effective new approach for identifying bioactive plant species that will further the identification of new pharmacologically active compounds from diverse natural-product resources. PMID:25555059

  16. A Virtual Screening Approach For Identifying Plants with Anti H5N1 Neuraminidase Activity

    PubMed Central

    2016-01-01

    Recent outbreaks of highly pathogenic and occasional drug-resistant influenza strains have highlighted the need to develop novel anti-influenza therapeutics. Here, we report computational and experimental efforts to identify influenza neuraminidase inhibitors from among the 3000 natural compounds in the Malaysian-Plants Natural-Product (NADI) database. These 3000 compounds were first docked into the neuraminidase active site. The five plants with the largest number of top predicted ligands were selected for experimental evaluation. Twelve specific compounds isolated from these five plants were shown to inhibit neuraminidase, including two compounds with IC50 values less than 92 μM. Furthermore, four of the 12 isolated compounds had also been identified in the top 100 compounds from the virtual screen. Together, these results suggest an effective new approach for identifying bioactive plant species that will further the identification of new pharmacologically active compounds from diverse natural-product resources. PMID:25555059

  17. Combining Molecular Docking and Molecular Dynamics to Predict the Binding Modes of Flavonoid Derivatives with the Neuraminidase of the 2009 H1N1 Influenza A Virus

    PubMed Central

    Lu, Shih-Jen; Chong, Fok-Ching

    2012-01-01

    Control of flavonoid derivatives inhibitors release through the inhibition of neuraminidase has been identified as a potential target for the treatment of H1N1 influenza disease. We have employed molecular dynamics simulation techniques to optimize the 2009 H1N1 influenza neuraminidase X-ray crystal structure. Molecular docking of the compounds revealed the possible binding mode. Our molecular dynamics simulations combined with the solvated interaction energies technique was applied to predict the docking models of the inhibitors in the binding pocket of the H1N1 influenza neuraminidase. In the simulations, the correlation of the predicted and experimental binding free energies of all 20 flavonoid derivatives inhibitors is satisfactory, as indicated by R2 = 0.75. PMID:22605992

  18. Detection of influenza virus neuraminidase-specific antibodies by an enzyme-linked immunosorbent assay.

    PubMed

    Khan, M W; Gallagher, M; Bucher, D; Cerini, C P; Kilbourne, E D

    1982-07-01

    An enzyme-linked immunosorbent assay was developed for the titration of antibodies in human sera to influenza virus neuraminidase, employing partially purified N1 neuraminidase. Specificity of the test was demonstrated, and the test was more sensitive than either the conventional neuraminidase inhibition or plaque size reduction tests in detecting anti-neuraminidase antibody.

  19. Inhibitors

    MedlinePlus

    ... Community Counts Blood Safety Inhibitors Articles & Key Findings Free Materials Videos Starting the Conversation Playing it Safe A Look at Hemophilia Joint Range of Motion My Story Links to Other Websites ...

  20. Neuraminidase of Influenza A Virus Binds Lysosome-Associated Membrane Proteins Directly and Induces Lysosome Rupture

    PubMed Central

    Ju, Xiangwu; Yan, Yiwu; Liu, Qiang; Li, Ning; Sheng, Miaomiao; Zhang, Lifang; Li, Xiao; Liang, Zhu; Huang, Fengming; Liu, Kangtai; Zhao, Yan; Zhang, Yanxu; Zou, Zhen; Du, Jianchao; Zhong, Ying; Zhou, Huandi; Yang, Peng; Lu, Huijun; Tian, Mingyao; Li, Dangsheng; Zhang, Jianming

    2015-01-01

    ABSTRACT As a recycling center, lysosomes are filled with numerous acid hydrolase enzymes that break down waste materials and invading pathogens. Recently, lysosomal cell death has been defined as “lysosomal membrane permeabilization and the consequent leakage of lysosome contents into cytosol.” Here, we show that the neuraminidase (NA) of H5N1 influenza A virus markedly deglycosylates and degrades lysosome-associated membrane proteins (LAMPs; the most abundant membrane proteins of lysosome), which induces lysosomal rupture, and finally leads to cell death of alveolar epithelial carcinoma A549 cells and human tracheal epithelial cells. The NA inhibitors peramivir and zanamivir could effectively block the deglycosylation of LAMPs, inhibit the virus cell entry, and prevent cell death induced by the H5N1 influenza virus. The NA of seasonal H1N1 virus, however, does not share these characteristics. Our findings not only reveal a novel role of NA in the early stage of the H5N1 influenza virus life cycle but also elucidate the molecular mechanism of lysosomal rupture crucial for influenza virus induced cell death. IMPORTANCE The integrity of lysosomes is vital for maintaining cell homeostasis, cellular defense and clearance of invading pathogens. This study shows that the H5N1 influenza virus could induce lysosomal rupture through deglycosylating lysosome-associated membrane proteins (LAMPs) mediated by the neuraminidase activity of NA protein. NA inhibitors such as peramivir and zanamivir could inhibit the deglycosylation of LAMPs and protect lysosomes, which also further interferes with the H5N1 influenza virus infection at early stage of life cycle. This work is significant because it presents new concepts for NA's function, as well as for influenza inhibitors' mechanism of action, and could partially explain the high mortality and high viral load after H5N1 virus infection in human beings and why NA inhibitors have more potent therapeutic effects for lethal avian

  1. Neuraminidase enhances in vitro expansion of human erythroid progenitors.

    PubMed

    Bodivit, Gwellaouen; Chadebech, Philippe; Vigon, Isabelle; Yacia, Azzedine; Roziers, Nicolas Burin des; Pirenne, France; Fichelson, Serge

    2016-06-01

    In spite of recent key improvements, in vitro mass production of erythrocytes from human stem cells is still limited by difficulties in obtaining sufficient numbers of erythroid progenitors. In fact, such progenitors are as scarce in the bone marrow as in peripheral blood. We used a two-step culture model of human cord blood-derived erythroid progenitors in the presence or absence of high-purity neuraminidase, in a serum-free, defined culture medium. Granulocytic and megakaryocytic progenitor cell expansions were also studied. We show that significant enhancement of erythroid cell generation is obtained when CD34(+) human hematopoietic progenitors are cultured in the presence of neuraminidase. Interestingly, in so doing, expanded red cell progenitors remained erythropoietin-dependent for further expansion and survival, and cells thus generated displayed a normal phenotype. Moreover, the activity of neuraminidase on these cells can be reversed by simple cell washing. Finally, growth of cells of the other myeloid lineages (granulocytes and megakaryocytes) is either decreased or unchanged in the presence of neuraminidase. This specific feature of neuraminidase, that of stimulation of human red cell progenitor proliferation, provides a safe technique for producing greater numbers of in vitro-generated red blood cells for both basic research and transfusion use. PMID:27478075

  2. Photodegradation and inhibition of drug-resistant influenza virus neuraminidase using anthraquinone-sialic acid hybrids.

    PubMed

    Aoki, Yusuke; Tanimoto, Shuho; Takahashi, Daisuke; Toshima, Kazunobu

    2013-02-11

    The anthraquinone-sialic acid hybrids designed effectively degraded not only non-drug-resistant neuraminidase but also drug-resistant neuraminidase, which is an important target of anti-influenza therapy. Degradation was achieved using long-wavelength UV radiation in the absence of any additives and under neutral conditions. Moreover, the hybrids efficiently inhibited neuraminidase activities upon photo-irradiation. PMID:23282898

  3. Sequence and crystallization of influenza virus B/Beijing/1/87 neuraminidase

    SciTech Connect

    Burmeister, W.P.; Daniels, R.S.; Dayan, S.; Gagnon, J.; Cusack, S.; Ruigrok, R.W. )

    1991-01-01

    Influenza B/Beijing/1/87 neuraminidase heads were isolated from virus via trypsin digestion and characterized by PAGE, N-terminal sequencing, electron microscopy, and enzyme activity. The heads were crystallized into two crystal forms; tetragonal plates, like other neuraminidase crystals described before, that diffract to medium resolution (3 A) and a new form consisting of trigonal prisms or needles that diffract to high resolution (at least 2 A). The gene segment coding for neuraminidase was sequenced and compared with the neuraminidase sequence of B/Lee/40. The deduced amino acid sequences for neuraminidase showed only a 7% difference, whereas those for the NB proteins differed by 20%.

  4. A Newcastle disease virus hemagglutinin-neuraminidase mutation that diminished hemagglutination and neuraminidase activity did not change infectivity and pathogenecity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infection by Newcastle disease virus (NDV), a member of the Paramyxoviridae family, requires recognition and attachment of the virions to host cell receptors containing sialic acid residues. Recognition of sialic acid-containing proteins is mediated by the hemagglutinin-neuraminidase (HN) protein of...

  5. Neuroinflammation induced by intracerebroventricular injection of microbial neuraminidase.

    PubMed

    Granados-Durán, Pablo; López-Ávalos, María D; Grondona, Jesús M; Gómez-Roldán, María Del Carmen; Cifuentes, Manuel; Pérez-Martín, Margarita; Alvarez, Martina; Rodríguez de Fonseca, Fernando; Fernández-Llebrez, Pedro

    2015-01-01

    In the present paper, we describe the facts that took place in the rat brain after a single injection of the enzyme neuraminidase from Clostridium perfringens into the right lateral ventricle. After injection, it diffused through the cerebrospinal fluid of the ipsilateral ventricle and the third ventricle, and about 400 μm into the periventricular brain parenchyma. The expression of ICAM1 in the endothelial cells of the periventricular vessels, IBA1 in microglia, and GFAP in astrocytes notably increased in the regions reached by the injected neuraminidase. The subependymal microglia and the ventricular macrophages begun to express IL1β and some appeared to cross the ependymal layer. After about 4 h of the injection, leukocytes migrated from large venules of the affected choroid plexus, the meninges and the local subependyma, and infiltrated the brain. The invading cells arrived orderly: first neutrophils, then macrophage-monocytes, and last CD8α-positive T-lymphocytes and B-lymphocytes. Leukocytes in the ventricles and the perivascular zones penetrated the brain parenchyma passing through the ependyma and the glia limitans. Thus, it is likely that a great part of the damage produced by microorganism invading the brain may be due to their neuraminidase content.

  6. Neuroinflammation Induced by Intracerebroventricular Injection of Microbial Neuraminidase

    PubMed Central

    Granados-Durán, Pablo; López-Ávalos, María D.; Grondona, Jesús M.; Gómez-Roldán, María del Carmen; Cifuentes, Manuel; Pérez-Martín, Margarita; Alvarez, Martina; Rodríguez de Fonseca, Fernando; Fernández-Llebrez, Pedro

    2015-01-01

    In the present paper, we describe the facts that took place in the rat brain after a single injection of the enzyme neuraminidase from Clostridium perfringens into the right lateral ventricle. After injection, it diffused through the cerebrospinal fluid of the ipsilateral ventricle and the third ventricle, and about 400 μm into the periventricular brain parenchyma. The expression of ICAM1 in the endothelial cells of the periventricular vessels, IBA1 in microglia, and GFAP in astrocytes notably increased in the regions reached by the injected neuraminidase. The subependymal microglia and the ventricular macrophages begun to express IL1β and some appeared to cross the ependymal layer. After about 4 h of the injection, leukocytes migrated from large venules of the affected choroid plexus, the meninges and the local subependyma, and infiltrated the brain. The invading cells arrived orderly: first neutrophils, then macrophage-monocytes, and last CD8α-positive T-lymphocytes and B-lymphocytes. Leukocytes in the ventricles and the perivascular zones penetrated the brain parenchyma passing through the ependyma and the glia limitans. Thus, it is likely that a great part of the damage produced by microorganism invading the brain may be due to their neuraminidase content. PMID:25853134

  7. Molecular docking of potential inhibitors for influenza H7N9.

    PubMed

    Liu, Zekun; Zhao, Junpeng; Li, Weichen; Wang, Xinkun; Xu, Jingxuan; Xie, Jin; Tao, Ke; Shen, Li; Zhang, Ran

    2015-01-01

    As a new strain of virus emerged in 2013, avian influenza A (H7N9) virus is a threat to the public health, due to its high lethality and pathogenicity. Furthermore, H7N9 has already generated various mutations such as neuraminidase R294K mutation which could make the anti-influenza oseltamivir less effective or ineffective. In this regard, it is urgent to develop new effective anti-H7N9 drug. In this study, we used the general H7N9 neuraminidase and oseltamivir-resistant influenza virus neuraminidase as the acceptors and employed the small molecules including quercetin, chlorogenic acid, baicalein, and oleanolic acid as the donors to perform the molecular docking for exploring the binding abilities between these small molecules and neuraminidase. The results showed that quercetin, chlorogenic acid, oleanolic acid, and baicalein present oseltamivir-comparable high binding potentials with neuraminidase. Further analyses showed that R294K mutation in neuraminidase could remarkably decrease the binding energies for oseltamivir, while other small molecules showed stable binding abilities with mutated neuraminidase. Taken together, the molecular docking studies identified four potential inhibitors for neuraminidase of H7N9, which might be effective for the drug-resistant mutants.

  8. Understanding the cross-resistance of oseltamivir to H1N1 and H5N1 influenza A neuraminidase mutations using multidimensional computational analyses

    PubMed Central

    Singh, Ashona; Soliman, Mahmoud E

    2015-01-01

    This study embarks on a comprehensive description of the conformational contributions to resistance of neuraminidase (N1) in H1N1 and H5N1 to oseltamivir, using comparative multiple molecular dynamic simulations. The available data with regard to elucidation of the mechanism of resistance as a result of mutations in H1N1 and H5N1 neuraminidases is not well established. Enhanced post-dynamic analysis, such as principal component analysis, solvent accessible surface area, free binding energy calculations, and radius of gyration were performed to gain a precise insight into the binding mode and origin of resistance of oseltamivir in H1N1 and H5N1 mutants. Three significant features reflecting resistance in the presence of mutations H274Y and I222K, of the protein complexed with the inhibitor are: reduced flexibility of the α-carbon backbone; an improved ΔEele of ~15 (kcal/mol) for H1N1 coupled with an increase in ΔGsol (~13 kcal/mol) from wild-type to mutation; a low binding affinity in comparison with the wild-type of ~2 (kcal/mol) and ~7 (kcal/mol) with respect to each mutation for the H5N1 systems; and reduced hydrophobicity of the overall surface structure due to an impaired hydrogen bonding network. We believe the results of this study will ultimately provide a useful insight into the structural landscape of neuraminidase-associated binding of oseltamivir. Furthermore, the results can be used in the design and development of potent inhibitors of neuraminidases. PMID:26257512

  9. The Mechanism by which 146-N-Glycan Affects the Active Site of Neuraminidase.

    PubMed

    Liu, Pi; Wang, Zhonghua; Zhang, Lijie; Li, Dongmei; Lin, Jianping

    2015-01-01

    One of the most conserved glycosylation sites of neuraminidase (NA) is 146-N-glycan. This site is adjacent to the 150-cavity of NA, which is found within the active site and thought to be a target for rational drug development against the antiviral resistance of influenza. Here, through a total of 2.4 μs molecular dynamics (MD) simulations, we demonstrated that 146-N-glycan can stabilize the conformation of the 150-loop that controls the volume of the 150-cavity. Moreover, with 146-N-glycan, our simulation result was more consistent with crystal structures of NAs than simulations conducted without glycans. Cluster analysis of the MD trajectories showed that 146-N-glycan adopted three distinct conformations: monomer-bridged, dimer-bridged and standing. Of these conformations, the dimer-bridged 146-N-glycan was the most stable one and contributed to stabilization of the 150-loop conformation. Furthermore, our simulation revealed that various standing conformations of 146-N-glycan could block the entrance of the binding pocket. This result was consistent with experimental data and explained the relatively low activity of inhibitors with flexible substituents toward the 150-cavity. Together, our results lead us to hypothesize that rigid and hydrophobic substituents could serve as better inhibitors targeting the 150-cavity. PMID:26267136

  10. Structure prediction and analysis of neuraminidase sequence variants.

    PubMed

    Thayer, Kelly M

    2016-07-01

    Analyzing protein structure has become an integral aspect of understanding systems of biochemical import. The laboratory experiment endeavors to introduce protein folding to ascertain structures of proteins for which the structure is unavailable, as well as to critically evaluate the quality of the prediction obtained. The model system used is the highly mutable influenza virus protein neuraminidase, which is the key target in the development of therapeutics. In light of recent pandemics, understanding how mutations confer drug resistance, which translates at the molecular level to understanding how different sequence variants differ, constitutes an area of great interest because of the ramifications in public health. This lab targets upper level undergraduate biochemistry students, and aims to introduce tools to be used to explore protein folding and protein visualization in the context of the neuraminidase case study. Students proceed to critically evaluate the folded models by comparison with crystallographic structures. When validity is established, they fold a neuraminidase sequence for which a structure is not available. Through structural alignment and visual inspection of the 150 loop, students gain molecular insight into two possible conformations of the protein, which are actively being studied. Folding the third chosen sequence mimics a true research environment in allowing students to generate a structure from a sequence for which a structure was not previously available, and to assess whether their particular variant has an open or closed loop. From this vantage, they are then challenged to speculate about the connection between loop conformation and drug susceptibility. © 2016 by The International Union of Biochemistry and Molecular Biology, 44(4):361-376, 2016. PMID:26900942

  11. Evaluation of Three Influenza Neuraminidase Inhibition Assays for Use in a Public Health Laboratory Setting During the 2011–2012 Influenza Season

    PubMed Central

    Murtaugh, William; Mahaman, Lalla; Healey, Benjamin; Peters, Heather; Anderson, Barbara; Tran, Mandy; Ziese, Marci

    2013-01-01

    Objectives We evaluated the implementation of three commericially available neuraminidase inhibition assays in a public health laboratory (PHL) setting. We also described the drug susceptibility patterns of human influenza A and B circulating in Maryland during the 2011–2012 influenza season. Methods From January to May 2012, 169 influenza virus isolates were tested for phenotypic susceptibility to oseltamivir, zanamivir, and peramivir using NA-FluorTM, NA-Star®, and NA-XTDTM concurrently. A 50% neuraminidase inhibitory concentration (IC50) value was calculated to determine drug susceptibility. We used the standard deviation based on the median absolute deviation of the median analysis to determine the potential for reduced drug susceptibility. We evaluated each assay for the use of resources in high- and low-volume testing scenarios. Results One of the 25 2009 influenza A (H1N1) pandemic isolates tested was resistant to oseltamivir and peramivir, and sensitive to zanamivir, on all three platforms. Eighty-two influenza A (H3N2) and 62 B isolates were sensitive to all three drugs in all three assays. For a low-volume scenario, NA-Star and NA-XTD took 120 minutes to complete, while NA-Fluor required 300 minutes to complete. The lowest relative cost favored NA-Star. In a high-volume scenario, NA-Fluor had the highest throughput. Reagent use was most efficient when maximizing throughput. Cost efficiency from low- to high-volume testing improved the most for NA-Star. Conclusions Our evaluation showed that both chemiluminescent and fluorescent neuraminidase inhibition assays can be successfully implemented in a PHL setting to screen circulating influenza strains for neuraminidase inhibitor resistance. For improved PHL influenza surveillance, it may be essential to develop guidelines for phenotypic drug-resistance testing that take into consideration a PHL's workload and available resources. PMID:23997307

  12. Virulence factors of influenza A viruses: WSN virus neuraminidase required for plaque production in MDBK cells.

    PubMed

    Schulman, J L; Palese, P

    1977-10-01

    The genetic basis for the distinctive capacity of influenza A/WSN/33 (H0N1) virus (WSN virus) to produce plaques on bovine kidney (MDBK) cells was found to be related to virus neuraminidase. Recombinant viruses that derived only the neuraminidase of WSN virus were capable of producing plaques, whereas recombinant viruses identical to WSN except for neuraminidase did not produce plaques. With viruses that do not contain WSN neuraminidase, infectivity of virus yields from MDBK cells was increased approximately 1,000-fold after in vitro treatment with trypsin. In contrast, no significant increase in infectivity was observed after trypsin treatment of viruses containing WSN neuraminidase. In addition, polyacrylamide gel analysis of proteins of WSN virus obtained after infection of MDBK cells demonstrated that hemagglutinin was present in the cleaved form (HA1 + HA2), whereas only uncleaved hemagglutinin was obtained with a recombinant virus that derived all of its genes from WSN virus except its neuraminidase. These data are in accord with the hypothesis that neuraminidase may facilitate production of infectious particles by removing sialic acid residues and exposing appropriate cleavage sites on hemagglutinin.

  13. Neuraminidase inhibitory activities of quaternary isoquinoline alkaloids from Corydalis turtschaninovii rhizome.

    PubMed

    Kim, Jang Hoon; Ryu, Young Bae; Lee, Woo Song; Kim, Young Ho

    2014-11-01

    Clostridium perfringens is a Gram-positive spore-forming bacterium that causes food poisoning. The neuraminidase (NA) protein of C. perfringens plays a pivotal role in bacterial proliferation and is considered a novel antibacterial drug target. Based on screens for novel NA inhibitors, a 95% EtOH extract of Corydalis turtschaninovii rhizome showed NA inhibitory activity (68% at 30 μg/ml), which resulted in the isolation of 10 isoquinoline alkaloids; namely, palmatine (1), berberine (2), coptisine (3), pseudodehydrocorydaline (4), jatrorrhizine (5), dehydrocorybulbine (6), pseudocoptisine (7), glaucine (8), corydaline (9) and tetrahydrocoptisine (10). Interestingly, seven quaternary isoquinoline alkaloids 1-7 (IC50 = 12.8 ± 1.5 to 65.2 ± 4.5 μM) showed stronger NA inhibitory activity than the tertiary alkaloids 8-10. In addition, highly active compounds 1 and 2 showed reversible non-competitive behavior based on a kinetic study. Molecular docking simulations using the Autodock 4.2 software increased our understanding of receptor-ligand binding of these compounds. In addition, we demonstrated that compounds 1 and 2 suppressed bacterial growth.

  14. Hydration of ligands of influenza virus neuraminidase studied by the fragment molecular orbital method.

    PubMed

    Tokuda, Kana; Watanabe, Chiduru; Okiyama, Yoshio; Mochizuki, Yuji; Fukuzawa, Kaori; Komeiji, Yuto

    2016-09-01

    The fragment molecular orbital (FMO) method was applied to quantum chemical calculations of neuramic acid, the natural substrate of the influenza virus neuraminidase, and two of its competitive inhibitors, Oseltamivir (Tamiful(®)) and Zanamivir (Relenza(®)), to investigate their hydrated structures and energetics. Each of the three ligands was immersed in an explicit water solvent, geometry-optimized by classical MM and QM/MM methods, and subjected to FMO calculations with 2-, 3-, and 4-body corrections under several fragmentation options. The important findings were that QM/MM optimization was preferable to obtain reliable hydrated structures of the ligands, that the 3-body correction was important for quantitative evaluation of the solvation energy, and that the dehydration effect was most remarkable near the hydrophobic sections of the ligands. In addition, the hydration energy calculated by the explicit solvent was compared with the hydration free energy calculated by the implicit solvent via the Poisson-Boltzmann equation, and the two showed a fairly good correlation. These findings will serve as useful information for rapid drug design. PMID:27611645

  15. Neuraminidase 1 is a Negative Regulator of Lysosomal Exocytosis

    PubMed Central

    Yogalingam, Gouri; Bonten, Erik J.; van de Vlekkert, Diantha; Hu, Huimin; Moshiach, Simon; Connell, Samuel A.; d’Azzo, Alessandra

    2009-01-01

    SUMMARY Lysosomal exocytosis is a Ca2+-regulated mechanism that involves proteins responsible for cytoskeletal attachment and fusion of lysosomes with the plasma membrane. However, whether luminal lysosomal enzymes contribute to this process remains unknown. Here we show that neuraminidase Neu1 negatively regulates lysosomal exocytosis in hematopoietic cells by processing the sialic acids on the lysosomal membrane protein Lamp-1. In macrophages from Neu1-deficient mice, a model of the disease sialidosis, and in patients’ fibroblasts, oversialylated Lamp-1 enhances lysosomal exocytosis. Silencing of Lamp-1 reverts this phenotype by interfering with the docking of lysosomes at the plasma membrane. In Neu1-/- mice the excessive exocytosis of serine proteases in the bone niche leads to inactivation of extracellular serpins, premature degradation of VCAM-1, and loss of bone marrow retention. Our findings uncover an unexpected mechanism influencing lysosomal exocytosis and argue that exacerbations of this process form the basis for certain genetic diseases. PMID:18606142

  16. Interface dynamics explain assembly dependency of influenza neuraminidase catalytic activity

    PubMed Central

    von Grafenstein, Susanne; Wallnoefer, Hannes G.; Kirchmair, Johannes; Fuchs, Julian E.; Huber, Roland G.; Schmidtke, Michaela; Sauerbrei, Andreas; Rollinger, Judith M.; Liedl, Klaus R.

    2015-01-01

    Influenza virus neuraminidase (iNA) is a homotetrameric surface protein of the influenza virus and an established target for antiviral drugs. In contrast to neuraminidases (NAs) of other biological systems (non-iNAs), enzymatic activity of iNA is only observed in a quaternary assembly and iNA needs the tetramerization to mediate enzymatic activity. Obviously, differences on a molecular level between iNA and non-iNAs are responsible for this intriguing observation. Comparison between protein structures and multiple sequence alignment allow the identification of differences in amino acid composition in crucial regions of the enzyme, such as next to the conserved D151 and the 150-loop. These differences in amino acid sequence and protein tetramerization are likely to alter the dynamics of the system. Therefore, we performed molecular dynamics simulations to investigate differences in the molecular flexibility of monomers, dimers, and tetramers of iNAs of subtype N1 (avian 2004, pandemic 1918 and pandemic 2009 iNA) and as comparison the non-iNA monomer from Clostridium perfringens. We show that conformational transitions of iNA are crucially influenced by its assembly state. The protein–protein interface induces a complex hydrogen-bonding network between the 110-helix and the 150-loop, which consequently stabilizes the structural arrangement of the binding site. Therefore, we claim that these altered dynamics are responsible for the dependence of iNA’s catalytic activity on the tetrameric assembly. Only the tetramerization-induced balance between stabilization and altered local flexibility in the binding site provides the appropriate arrangement of key residues for iNA’s catalytic activity. PMID:24279589

  17. Bioluminescence-based neuraminidase inhibition assay for monitoring influenza virus drug susceptibility in clinical specimens.

    PubMed

    Marjuki, Henju; Mishin, Vasiliy P; Sleeman, Katrina; Okomo-Adhiambo, Margaret; Sheu, Tiffany G; Guo, Lizheng; Xu, Xiyan; Gubareva, Larisa V

    2013-11-01

    The QFlu prototype bioluminescence-based neuraminidase (NA) inhibition (NI) assay kit was designed to detect NA inhibitor (NAI)-resistant influenza viruses at point of care. Here, we evaluated its suitability for drug susceptibility assessment at a surveillance laboratory. A comprehensive panel of reference viruses (n = 14) and a set of 90 seasonal influenza virus A and B isolates were included for testing with oseltamivir and/or zanamivir in the QFlu assay using the manufacturer-recommended protocol and a modified version attuned to surveillance requirements. The 50% inhibitory concentrations (IC50s) generated were compared with those of NI assays currently used for monitoring influenza drug susceptibility, the fluorescent (FL) and chemiluminescent (CL) assays. To provide proof of principle, clinical specimens (n = 235) confirmed by real-time reverse transcription (RT)-PCR to contain influenza virus A(H1N1)pdm09 and prescreened for the oseltamivir resistance marker H275Y using pyrosequencing were subsequently tested in the QFlu assay. All three NI assays were able to discriminate the reference NA variants and their matching wild-type viruses based on the difference in their IC50s. Unless the antigenic types were first identified, certain NA variants (e.g., H3N2 with E119V) could be detected among seasonal viruses using the FL assays only. Notably, the QFlu assay identified oseltamivir-resistant A(H1N1)pdm09 viruses carrying the H275Y marker directly in clinical specimens, which is not feasible with the other two phenotypic assays, which required prior virus culturing in cells. Furthermore, The QFlu assay allows detection of the influenza virus A and B isolates carrying established and potential NA inhibitor resistance markers and may become a useful tool for monitoring drug resistance in clinical specimens. PMID:23917311

  18. Combining crystallographic information and an aspherical-atom data bank in the evaluation of the electrostatic interaction energy in an enzyme–substrate complex: influenza neuraminidase inhibition

    SciTech Connect

    Dominiak, Paulina M.; Volkov, Anatoliy; Dominiak, Adam P.; Jarzembska, Katarzyna N.; Coppens, Philip

    2009-05-01

    The electrostatic component of the enzyme/inhibitor interaction of a wide range influenza neuraminidases and inhibitors has been analyzed using transferable aspherical-atom densities from a recently compiled databank. Results are subdivided into the contributions of individual active-site residues and different functional groups of the inhibitors, and the effect of the Arg292→Lys mutation is considered. Although electrostatic interactions contribute only a part of the interaction energies between macromolecules, unlike dispersion forces they are highly directional and therefore dominate the nature of molecular packing in crystals and in biological complexes and contribute significantly to differences in inhibition strength among related enzyme inhibitors. In the reported study, a wide range of complexes of influenza neuraminidases with inhibitor molecules (sialic acid derivatives and others) have been analyzed using charge densities from a transferable aspherical-atom data bank. The strongest interactions of the residues are with the acidic group at the C2 position of the inhibitor (∼−300 kJ mol{sup −1} for —COO{sup −} in non-aromatic inhibitors, ∼−120–210 kJ mol{sup −1} for —COO{sup −} in aromatic inhibitors and ∼−450 kJ mol{sup −1} for —PO{sub 3}{sup 2−}) and with the amino and guanidine groups at C4 (∼−250 kJ mol{sup −1}). Other groups contribute less than ∼100 kJ mol{sup −1}. Residues Glu119, Asp151, Glu227, Glu276 and Arg371 show the largest variation in electrostatic energies of interaction with different groups of inhibitors, which points to their important role in the inhibitor recognition. The Arg292→Lys mutation reduces the electrostatic interactions of the enzyme with the acidic group at C2 for all inhibitors that have been studied (SIA, DAN, 4AM, ZMR, G20, G28, G39 and BCZ), but enhances the interactions with the glycerol group at C6 for inhibitors that contain it. This is in agreement with the lower level

  19. [The effects of complex platinum compounds on the neuraminidase activity of the Sendai virus].

    PubMed

    Repanovici, R; Călinoiu, A; Iliescu, R; Löber, G; Popa, L M

    1989-01-01

    The effect of di- and tetravalent cis-diaminoplatinum chlorides on Sendai virus envelop HN glycoprotein was investigated. The partial inhibition of neuraminidase activity was greater in the case of the divalent platinum complex derivative. PMID:2556835

  20. Novel binding patterns between ganoderic acids and neuraminidase: Insights from docking, molecular dynamics and MM/PBSA studies.

    PubMed

    Yang, Zhiwei; Wu, Fei; Yuan, Xiaohui; Zhang, Lei; Zhang, Shengli

    2016-04-01

    Recently, ganoderic acids (GAs) give rise to the attractive candidates of novel neuraminidase (NA) inhibitors. However, there is still no evident conclusion about their binding patterns. To this end, docking, molecular dynamics and MM/PBSA methods were combined to study the binding profiles of GAs with the N1 protein and familiar H274Y and N294S mutations (A/Vietnam/1203/04 stain). It was found that the binding affinities of ganoderic acid DM and Z (ΔGbind, -16.83 and -10.99 kcal mol(-1)) are comparable to that of current commercial drug oseltamivir (-23.62 kcal mol(-1)). Electrostatic interaction is the main driving force, and should be one important factor to evaluate the binding quality and rational design of NA inhibitors. The 150-loop residues Asp151 and Arg152 played an important role in the binding processes. Further analysis revealed that ganoderic acid DM is a potential source of anti-influenza ingredient, with novel binding pattern and advantage over oseltamivir. It had steric hindrance on the 150 cavity of N1 protein, and exerted activities across the H274Y and N294S mutations. This work also pointed out how to effectively design dual-site NA inhibitors and reinforce their affinities. These findings should prove valuable for the in-depth understanding of interactions between NA and GAs, and warrant the experimental aspects to design novel anti-influenza drugs. PMID:26905206

  1. Influenza virus neuraminidase (NA): a target for antivirals and vaccines.

    PubMed

    Jagadesh, Anitha; Salam, Abdul Ajees Abdul; Mudgal, Piya Paul; Arunkumar, Govindakarnavar

    2016-08-01

    Influenza, the most common infectious disease, poses a great threat to human health because of its highly contagious nature and fast transmissibility, often leading to high morbidity and mortality. Effective vaccination strategies may aid in the prevention and control of recurring epidemics and pandemics associated with this infectious disease. However, antigenic shifts and drifts are major concerns with influenza virus, requiring effective global monitoring and updating of vaccines. Current vaccines are standardized primarily based on the amount of hemagglutinin, a major surface antigen, which chiefly constitutes these preparations along with the varying amounts of neuraminidase (NA). Anti-influenza drugs targeting the active site of NA have been in use for more than a decade now. However, NA has not been approved as an effective antigenic component of the influenza vaccine because of standardization issues. Although some studies have suggested that NA antibodies are able to reduce the severity of the disease and induce a long-term and cross-protective immunity, a few major scientific issues need to be addressed prior to launching NA-based vaccines. Interestingly, an increasing number of studies have shown NA to be a promising target for future influenza vaccines. This review is an attempt to consolidate studies that reflect the strength of NA as a suitable vaccine target. The studies discussed in this article highlight NA as a potential influenza vaccine candidate and support taking the process of developing NA vaccines to the next stage. PMID:27255748

  2. On the agglutinogens of red cells developed with proteolytic enzymes and neuraminidase.

    PubMed

    Sagisaka, K; Takahashi, K

    1976-10-01

    It has been known that the agglutinability of human red cells is changed or enhanced by treatments with proteolytic enzymes or neuraminidase. In this paper, the serological properties of agglutinogens developed by proteolytic enzymes (bromelin, ficin, papain, trypsin and pronase) and neuraminidase are investigated by using antisera to trypsin- and neuraminidase-treated red cells. The adsorptions of the antiserum to trypsinized red cells with the cells treated with each of the proteolytic enzymes showed that the agglutinogens uncovered by bromelin, ficin and papain were different from those by pronase and trypsin. It was demonstrated that pronase was the most effective enzyme to uncover the agglutinogen located on deeper site of red cell membrane. This was confirmed by the agglutination with the test cells treated twice with two kinds of the enzymes. The reactions of the antiserum to neuraminidase-treated red cells treated with six kinds of the enzymes indicated that the agglutinogens developed by neuraminidase resembled those by bromelin, ficin and papain more than those by trypsin and pronase. PMID:982434

  3. Neuraminidase Activity and Resistance of 2009 Pandemic H1N1 Influenza Virus to Antiviral Activity in Bronchoalveolar Fluid

    PubMed Central

    Ruangrung, Kanyarat; Suptawiwat, Ornpreya; Maneechotesuwan, Kittipong; Boonarkart, Chompunuch; Chakritbudsabong, Warunya; Assawabhumi, Jirawatna; Bhattarakosol, Parvapan; Uiprasertkul, Mongkol; Puthavathana, Pilaipan; Wiriyarat, Witthawat; Jongkaewwattana, Anan

    2016-01-01

    ABSTRACT Human bronchoalveolar fluid is known to have anti-influenza activity. It is believed to be a frontline innate defense against the virus. Several antiviral factors, including surfactant protein D, are believed to contribute to the activity. The 2009 pandemic H1N1 influenza virus was previously shown to be less sensitive to surfactant protein D. Nevertheless, whether different influenza virus strains have different sensitivities to the overall anti-influenza activity of human bronchoalveolar fluid was not known. We compared the sensitivities of 2009 pandemic H1N1, seasonal H1N1, and seasonal H3N2 influenza virus strains to inhibition by human bronchoalveolar lavage (BAL) fluid. The pandemic and seasonal H1N1 strains showed lower sensitivity to human BAL fluid than the H3N2 strains. The BAL fluid anti-influenza activity could be enhanced by oseltamivir, indicating that the viral neuraminidase (NA) activity could provide resistance to the antiviral defense. In accordance with this finding, the BAL fluid anti-influenza activity was found to be sensitive to sialidase. The oseltamivir resistance mutation H275Y rendered the pandemic H1N1 virus but not the seasonal H1N1 virus more sensitive to BAL fluid. Since only the seasonal H1N1 but not the pandemic H1N1 had compensatory mutations that allowed oseltamivir-resistant strains to maintain NA enzymatic activity and transmission fitness, the resistance to BAL fluid of the drug-resistant seasonal H1N1 virus might play a role in viral fitness. IMPORTANCE Human airway secretion contains anti-influenza activity. Different influenza strains may vary in their susceptibilities to this antiviral activity. Here we show that the 2009 pandemic and seasonal H1N1 influenza viruses were less sensitive to human bronchoalveolar lavage (BAL) fluid than H3N2 seasonal influenza virus. The resistance to the pulmonary innate antiviral activity of the pandemic virus was determined by its neuraminidase (NA) gene, and it was shown that the

  4. [Contribution to the antigenic study of influenza viruses in animals. I.--Neuraminidase of the equine influenza viruses (author's transl)].

    PubMed

    Fontaine, M; Aymard-Henry, M

    1975-01-01

    From the Revised Nomenclature of WHO, the fowl influenza virus A/Duck/Ukraine/63 (Hav7 Neq2) has the same neuraminidase as the equine virus A/equi 2/Miami/63 (Heq2 Neq2); the A/Chicken Germany "N"/49 virus has the same neuraminidase as the equine virus A/equi 1/Prague/56. A comparative study of the antigenic specificities confirms that the Neq2 neuraminidases are closely connected, whatever their animal origin, and that the fowl strain Hav7 Neq2 can be used for the titration of anti Neq2 antibodies in the serums of animals immunized with the equine virus Heq2 Neq2. The Neqi neuraminidases of various animal origins are connected, but the neuraminidase of the fowl strain Hav2 Neqi is slightly inhibited by the anti Neq1 antibodies of animals immunized with the Heq1 Neq1 virus: to titrate the anti Neq1 antibodies of equine origin, the H72 Neq1 recombinant should therefore be used. The antigenic characterization of the different equine influenza strains isolated since 1967 by the study of their neuraminidase has been completed: The various neuraminidases, like the hemagglutinins of the various strains belonging to the sub-type A equi2 are closely connected; a minor antigenic variation, concerning the two surface antigens, seems to exist between the strain A equi 1/Prague/56 and the strain of the same subtype isolated in 1973.

  5. Anomeric specificity and protein-substrate interactions support the 3D model for the hemagglutinin-neuraminidase from sendai virus.

    PubMed

    Bellini, T; Pasti, C; Manfrinato, M C; Tomasi, M; Dallocchio, F

    1999-08-27

    The 3D structure of paramyxovirus hemagglutinin-neuraminidase has not yet been resolved; however, a theoretical model has been built by using influenza virus and bacterial neuraminidases as template [V. C. Epa (1997) Proteins Struct. Funct. Gen. 29, 264-281]. Two common features of the catalytic mechanism of the neuraminidases of known 3D structure are the anomeric specificity and the involvement of a tyrosine residue in the stabilization of the transition state. These key features have been investigated on the water-soluble ectodomain of the hemagglutinin-neuraminidase from Sendai virus (cHN). The anomeric specificity of the hydrolysis of the substrate by cHN has been investigated by NMR spectroscopy. The immediate product of the reaction was the alpha-anomer, meaning that cHN belongs between glycohydrolases retaining anomeric configuration like influenza virus neuraminidase. Measurements of the UV difference spectrum upon binding of the substrate analogue 2,3-dehydro 2-deossi N-acetyl neuraminic acid indicate the ionization of a tyrosine residue and decreased polarity in the environment of a tryptophan residue. Functional significance of the spectral data was derived from the known structure of influenza neuraminidase, where a tyrosinate ion is involved in the stabilization of the transition-state carbonium ion, and a tryptophan residue is involved in the binding of the acetyl moiety of the substrate. The data give experimental support to the 3D model of paramyxovirus neuraminidase.

  6. Structural characterization of a protective epitope spanning A(H1N1)pdm09 influenza virus neuraminidase monomers

    PubMed Central

    Wan, Hongquan; Yang, Hua; Shore, David A.; Garten, Rebecca J.; Couzens, Laura; Gao, Jin; Jiang, Lianlian; Carney, Paul J.; Villanueva, Julie; Stevens, James; Eichelberger, Maryna C.

    2015-01-01

    A(H1N1)pdm09 influenza A viruses predominated in the 2013–2014 USA influenza season, and although most of these viruses remain sensitive to Food and Drug Administration-approved neuraminidase (NA) inhibitors, alternative therapies are needed. Here we show that monoclonal antibody CD6, selected for binding to the NA of the prototypic A(H1N1)pdm09 virus, A/California/07/2009, protects mice against lethal virus challenge. The crystal structure of NA in complex with CD6 Fab reveals a unique epitope, where the heavy-chain complementarity determining regions (HCDRs) 1 and 2 bind one NA monomer, the light-chain CDR2 binds the neighbouring monomer, whereas HCDR3 interacts with both monomers. This 30-amino-acid epitope spans the lateral face of an NA dimer and is conserved among circulating A(H1N1)pdm09 viruses. These results suggest that the large, lateral CD6 epitope may be an effective target of antibodies selected for development as therapeutic agents against circulating H1N1 influenza viruses. PMID:25668439

  7. Comparison of neuraminidases of the same subtype but from different species using a new method of titration.

    PubMed

    Fiszon, B; Hannoun, C

    1990-01-01

    Neuraminidase is one of the two surface glycoproteins of influenza virions. In order to compare neuraminidases of the same subtype but isolated from different species (man, birds, pig), a new and simple method was adapted and optimized using peanut hemagglutinin. Results were very similar to those obtained with the classical method recommended by the WHO, using fetuin as a substrate. The technique was used to examine the relationship between animal and human neuraminidases belonging to serotypes N1 and N2. The results confirm the possible role of ducks as a reservoir for influenza viruses and the eventuality of interspecific exchanges.

  8. Molecular distribution of amino acid substitutions on neuraminidase from the 2009 (H1N1) human influenza pandemic virus.

    PubMed

    Quiliano, Miguelmiguel; Valdivia-Olarte, Hugo; Olivares, Carlos; Requena, David; Gutiérrez, Andrés H; Reyes-Loyola, Paola; Tolentino-Lopez, Luis E; Sheen, Patricia; Briz, Verónica; Muñoz-Fernández, Maria A; Correa-Basurto, José; Zimic, Mirko

    2013-01-01

    The pandemic influenza AH1N1 (2009) caused an outbreak of human infection that spread to the world. Neuraminidase (NA) is an antigenic surface glycoprotein, which is essential to the influenza infection process, and is the target of anti-flu drugs oseltamivir and zanamivir. Currently, NA inhibitors are the pillar pharmacological strategy against seasonal and global influenza. Although mutations observed after NA-inhibitor treatment are characterized by changes in conserved amino acids of the enzyme catalytic site, it is possible that specific amino acid substitutions (AASs) distant from the active site such as H274Y, could confer oseltamivir or zanamivir resistance. To better understand the molecular distribution pattern of NA AASs, we analyzed NA AASs from all available reported pandemic AH1N1 NA sequences, including those reported from America, Africa, Asia, Europe, Oceania, and specifically from Mexico. The molecular distributions of the AASs were obtained at the secondary structure domain level for both the active and catalytic sites, and compared between geographic regions. Our results showed that NA AASs from America, Asia, Europe, Oceania and Mexico followed similar molecular distribution patterns. The compiled data of this study showed that highly conserved amino acids from the NA active site and catalytic site are indeed being affected by mutations. The reported NA AASs follow a similar molecular distribution pattern worldwide. Although most AASs are distributed distantly from the active site, this study shows the emergence of mutations affecting the previously conserved active and catalytic site. A significant number of unique AASs were reported simultaneously on different continents.

  9. Molecular distribution of amino acid substitutions on neuraminidase from the 2009 (H1N1) human influenza pandemic virus

    PubMed Central

    Quiliano, MiguelMiguel; Valdivia-Olarte, Hugo; Olivares, Carlos; Requena, David; Gutiérrez, Andrés H; Reyes-Loyola, Paola; Tolentino-Lopez, Luis E; Sheen, Patricia; Briz, Verónica; Muñoz-Fernández, Maria A; Correa-Basurto, José; Zimic, Mirko

    2013-01-01

    The pandemic influenza AH1N1 (2009) caused an outbreak of human infection that spread to the world. Neuraminidase (NA) is an antigenic surface glycoprotein, which is essential to the influenza infection process, and is the target of anti-flu drugs oseltamivir and zanamivir. Currently, NA inhibitors are the pillar pharmacological strategy against seasonal and global influenza. Although mutations observed after NA-inhibitor treatment are characterized by changes in conserved amino acids of the enzyme catalytic site, it is possible that specific amino acid substitutions (AASs) distant from the active site such as H274Y, could confer oseltamivir or zanamivir resistance. To better understand the molecular distribution pattern of NA AASs, we analyzed NA AASs from all available reported pandemic AH1N1 NA sequences, including those reported from America, Africa, Asia, Europe, Oceania, and specifically from Mexico. The molecular distributions of the AASs were obtained at the secondary structure domain level for both the active and catalytic sites, and compared between geographic regions. Our results showed that NA AASs from America, Asia, Europe, Oceania and Mexico followed similar molecular distribution patterns. The compiled data of this study showed that highly conserved amino acids from the NA active site and catalytic site are indeed being affected by mutations. The reported NA AASs follow a similar molecular distribution pattern worldwide. Although most AASs are distributed distantly from the active site, this study shows the emergence of mutations affecting the previously conserved active and catalytic site. A significant number of unique AASs were reported simultaneously on different continents. PMID:23930018

  10. Microsecond Molecular Dynamics Simulations of Influenza Neuraminidase Suggest a Mechanism for the Increased Virulence of Stalk-Deletion Mutants

    PubMed Central

    2016-01-01

    Deletions in the stalk of the influenza neuraminidase (NA) surface protein are associated with increased virulence, but the mechanisms responsible for this enhanced virulence are unclear. Here we use microsecond molecular dynamics simulations to explore the effect of stalk deletion on enzymatic activity, contrasting NA proteins from the A/swine/Shandong/N1/2009 strain both with and without a stalk deletion. By modeling and simulating neuraminidase apo glycoproteins embedded in complex-mixture lipid bilayers, we show that the geometry and dynamics of the neuraminidase enzymatic pocket may differ depending on stalk length, with possible repercussions on the binding of the endogenous sialylated-oligosaccharide receptors. We also use these simulations to predict previously unrecognized druggable “hotspots” on the neuraminidase surface that may prove useful for future efforts aimed at structure-based drug design. PMID:27141956

  11. Characterizing Loop Dynamics and Ligand Recognition in Human- and Avian-Type Influenza Neuraminidases via Generalized Born Molecular Dynamics and End-Point Free Energy Calculations

    SciTech Connect

    Amaro, Rommie E; Cheng, Xiaolin; Ivanov, Ivaylo N; Xu, Dong; McCammon, Jonathan

    2009-01-01

    The comparative dynamics and inhibitor binding free energies of group-1 and group-2 pathogenic influenza A subtype neuraminidase (NA) enzymes are of fundamental biological interest and relevant to structure-based drug design studies for antiviral compounds. In this work, we present seven generalized Born molecular dynamics simulations of avian (N1)- and human (N9)-type NAs in order to probe the comparative flexibility of the two subtypes, both with and without the inhibitor oseltamivir bound. The enhanced sampling obtained through the implicit solvent treatment suggests several provocative insights into the dynamics of the two subtypes, including that the group-2 enzymes may exhibit similar motion in the 430-binding site regions but different 150-loop motion. End-point free energy calculations elucidate the contributions to inhibitor binding free energies and suggest that entropic considerations cannot be neglected when comparing across the subtypes. We anticipate the findings presented here will have broad implications for the development of novel antiviral compounds against both seasonal and pandemic influenza strains.

  12. Abrogation of neuraminidase reduces biofilm formation, capsule biosynthesis, and virulence of Porphyromonas gingivalis.

    PubMed

    Li, Chen; Kurniyati; Hu, Bo; Bian, Jiang; Sun, Jianlan; Zhang, Weiyan; Liu, Jun; Pan, Yaping; Li, Chunhao

    2012-01-01

    The oral bacterium Porphyromonas gingivalis is a key etiological agent of human periodontitis, a prevalent chronic disease that affects up to 80% of the adult population worldwide. P. gingivalis exhibits neuraminidase activity. However, the enzyme responsible for this activity, its biochemical features, and its role in the physiology and virulence of P. gingivalis remain elusive. In this report, we found that P. gingivalis encodes a neuraminidase, PG0352 (SiaPg). Transcriptional analysis showed that PG0352 is monocistronic and is regulated by a sigma70-like promoter. Biochemical analyses demonstrated that SiaPg is an exo-α-neuraminidase that cleaves glycosidic-linked sialic acids. Cryoelectron microscopy and tomography analyses revealed that the PG0352 deletion mutant (ΔPG352) failed to produce an intact capsule layer. Compared to the wild type, in vitro studies showed that ΔPG352 formed less biofilm and was less resistant to killing by the host complement. In vivo studies showed that while the wild type caused a spreading type of infection that affected multiple organs and all infected mice were killed, ΔPG352 only caused localized infection and all animals survived. Taken together, these results demonstrate that SiaPg is an important virulence factor that contributes to the biofilm formation, capsule biosynthesis, and pathogenicity of P. gingivalis, and it can potentially serve as a new target for developing therapeutic agents against P. gingivalis infection.

  13. High-throughput neuraminidase substrate specificity study of human and avian influenza A viruses

    PubMed Central

    Li, Yanhong; Cao, Hongzhi; Dao, Nguyet; Luo, Zheng; Yu, Hai; Chen, Yi; Baumgarth, Nicole; Cardona, Carol; Chen, Xi

    2011-01-01

    Despite the importance of neuraminidase (NA) activity in effective infection by influenza A viruses, limited information exists about the differences of substrate preferences of viral neuraminidases from different hosts or from different strains. Using a high-throughput screening format and a library of twenty α2–3- or α2–6-linked para-nitrophenol-tagged sialylgalactosides, substrate specificity of NAs on thirty-seven strains of human and avian influenza A viruses was studied using intact viral particles. Neuraminidases of all viruses tested cleaved both α2–3- and α2–6-linked sialosides but preferred α2–3-linked ones and the activity was dependent on the terminal sialic acid structure. In contrast to NAs of other subtypes of influenza A viruses which did not cleave 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn) or 5-deoxy Kdn (5d–Kdn), NAs of all N7 subtype viruses tested had noticeable hydrolytic activities on α2–3-linked sialosides containing Kdn or 5d–Kdn. Additionally, group 1 NAs showed efficient activity in cleaving N-azidoacetylneuraminic acid from α2 –3-linked sialoside. PMID:21501853

  14. Abrogation of Neuraminidase Reduces Biofilm Formation, Capsule Biosynthesis, and Virulence of Porphyromonas gingivalis

    PubMed Central

    Li, Chen; Kurniyati; Hu, Bo; Bian, Jiang; Sun, Jianlan; Zhang, Weiyan; Liu, Jun; Pan, Yaping

    2012-01-01

    The oral bacterium Porphyromonas gingivalis is a key etiological agent of human periodontitis, a prevalent chronic disease that affects up to 80% of the adult population worldwide. P. gingivalis exhibits neuraminidase activity. However, the enzyme responsible for this activity, its biochemical features, and its role in the physiology and virulence of P. gingivalis remain elusive. In this report, we found that P. gingivalis encodes a neuraminidase, PG0352 (SiaPg). Transcriptional analysis showed that PG0352 is monocistronic and is regulated by a sigma70-like promoter. Biochemical analyses demonstrated that SiaPg is an exo-α-neuraminidase that cleaves glycosidic-linked sialic acids. Cryoelectron microscopy and tomography analyses revealed that the PG0352 deletion mutant (ΔPG352) failed to produce an intact capsule layer. Compared to the wild type, in vitro studies showed that ΔPG352 formed less biofilm and was less resistant to killing by the host complement. In vivo studies showed that while the wild type caused a spreading type of infection that affected multiple organs and all infected mice were killed, ΔPG352 only caused localized infection and all animals survived. Taken together, these results demonstrate that SiaPg is an important virulence factor that contributes to the biofilm formation, capsule biosynthesis, and pathogenicity of P. gingivalis, and it can potentially serve as a new target for developing therapeutic agents against P. gingivalis infection. PMID:22025518

  15. A surface plasmon resonance assay for measurement of neuraminidase inhibition, sensitivity of wild-type influenza neuraminidase and its H274Y mutant to the antiviral drugs zanamivir and oseltamivir.

    PubMed

    Somasundaram, Balaji; Fee, Conan J; Fredericks, Rayleen; Watson, Andrew J A; Fairbanks, Antony J; Hall, Richard J

    2015-09-01

    Antiviral resistance is currently monitored by a labelled enzymatic assay, which can give inconsistent results because of the short half-life of the labelled product, and variations in assay conditions. In this paper, we describe a competitive surface plasmon resonance (SPR) inhibition assay for measuring the sensitivities of wild-type neuraminidase (WT NA) and the H274Y (histidine 274 tyrosine) NA mutant to antiviral drugs. The two NA isoforms were expressed in High-five™ (Trichoplusia ni) insect cells. A spacer molecule (1,6-hexanediamine (HDA)) was conjugated to the 7-hydroxyl group of zanamivir, and the construct (HDA-zanamivir) was immobilized onto a SPR sensor chip to obtain a final immobilization response of 431 response units. The immobilized HDA-zanamivir comprised a bio-specific ligand for the WT and mutant proteins. The effects of the natural substrate (sialic acid) and two inhibitors (zanamivir and oseltamivir) on NA binding to the immobilized ligand were studied. The processed SPR data was analysed to determine 50% inhibitory concentrations (IC50-spr ), using a log dose-response curve fit. Although both NA isoforms had almost identical IC50-spr values for sialic acid (WT = 5.5 nM; H274Y mutant = 3.25 nM) and zanamivir (WT = 2.16 nM; H274Y mutant = 2.42 nM), there were significant differences between the IC50-spr values obtained for the WT (7.7 nM) and H274Y mutant (256 nM) NA in the presence of oseltamivir, indicating that oseltamivir has a reduced affinity for the H274Y mutant. The SPR inhibition assay strategy presented in this work could be applied for the rapid screening of newly emerging variants of NA for their sensitivity to antiviral drugs.

  16. Novel hemagglutinin-based influenza virus inhibitors

    PubMed Central

    Shen, Xintian; Zhang, Xuanxuan

    2013-01-01

    Influenza virus has caused seasonal epidemics and worldwide pandemics, which caused tremendous loss of human lives and socioeconomics. Nowadays, only two classes of anti-influenza drugs, M2 ion channel inhibitors and neuraminidase inhibitors respectively, are used for prophylaxis and treatment of influenza virus infection. Unfortunately, influenza virus strains resistant to one or all of those drugs emerge frequently. Hemagglutinin (HA), the glycoprotein in influenza virus envelope, plays a critical role in viral binding, fusion and entry processes. Therefore, HA is a promising target for developing anti-influenza drugs, which block the initial entry step of viral life cycle. Here we reviewed recent understanding of conformational changes of HA in protein folding and fusion processes, and the discovery of HA-based influenza entry inhibitors, which may provide more choices for preventing and controlling potential pandemics caused by multi-resistant influenza viruses. PMID:23977436

  17. Single-Domain Antibodies Targeting Neuraminidase Protect against an H5N1 Influenza Virus Challenge

    PubMed Central

    Cardoso, Francisco Miguel; Ibañez, Lorena Itatí; Van den Hoecke, Silvie; De Baets, Sarah; Smet, Anouk; Roose, Kenny; Schepens, Bert; Descamps, Francis J.; Fiers, Walter; Muyldermans, Serge

    2014-01-01

    ABSTRACT Influenza virus neuraminidase (NA) is an interesting target of small-molecule antiviral drugs. We isolated a set of H5N1 NA-specific single-domain antibodies (N1-VHHm) and evaluated their in vitro and in vivo antiviral potential. Two of them inhibited the NA activity and in vitro replication of clade 1 and 2 H5N1 viruses. We then generated bivalent derivatives of N1-VHHm by two methods. First, we made N1-VHHb by genetically joining two N1-VHHm moieties with a flexible linker. Second, bivalent N1-VHH-Fc proteins were obtained by genetic fusion of the N1-VHHm moiety with the crystallizable region of mouse IgG2a (Fc). The in vitro antiviral potency against H5N1 of both bivalent N1-VHHb formats was 30- to 240-fold higher than that of their monovalent counterparts, with 50% inhibitory concentrations in the low nanomolar range. Moreover, single-dose prophylactic treatment with bivalent N1-VHHb or N1-VHH-Fc protected BALB/c mice against a lethal challenge with H5N1 virus, including an oseltamivir-resistant H5N1 variant. Surprisingly, an N1-VHH-Fc fusion without in vitro NA-inhibitory or antiviral activity also protected mice against an H5N1 challenge. Virus escape selection experiments indicated that one amino acid residue close to the catalytic site is required for N1-VHHm binding. We conclude that single-domain antibodies directed against influenza virus NA protect against H5N1 virus infection, and when engineered with a conventional Fc domain, they can do so in the absence of detectable NA-inhibitory activity. IMPORTANCE Highly pathogenic H5N1 viruses are a zoonotic threat. Outbreaks of avian influenza caused by these viruses occur in many parts of the world and are associated with tremendous economic loss, and these viruses can cause very severe disease in humans. In such cases, small-molecule inhibitors of the viral NA are among the few treatment options for patients. However, treatment with such drugs often results in the emergence of resistant viruses

  18. Epistatic interactions between neuraminidase mutations facilitated the emergence of the oseltamivir-resistant H1N1 influenza viruses

    PubMed Central

    Duan, Susu; Govorkova, Elena A.; Bahl, Justin; Zaraket, Hassan; Baranovich, Tatiana; Seiler, Patrick; Prevost, Kristi; Webster, Robert G.; Webby, Richard J.

    2014-01-01

    Oseltamivir-resistant H1N1 influenza viruses carrying the H275Y neuraminidase mutation predominated worldwide during the 2007–2009 seasons. While several neuraminidase substitutions were found to be necessary to counteract the adverse effects of H275Y, the order and impact of evolutionary events involved remain elusive. Here, we reconstruct H1N1 neuraminidase phylogeny during 1999–2009, estimate the timing and order of crucial amino acid changes, and evaluate their impact on the biological outcome of the H275Y mutation. Of the twelve neuraminidase substitutions that occurred during 1999–2009, five (chronologically, V234M, R222Q, K329E, D344N, H275Y, and D354G) are necessary for maintaining full neuraminidase function in the presence of the H275Y mutation by altering protein accumulation or enzyme affinity/activity. The sequential emergence and cumulative effects of these mutations clearly illustrate a role for epistasis in shaping the emergence and subsequent evolution of a drug-resistant virus population, which can be useful in understanding emergence of novel viral phenotypes of influenza. PMID:25297528

  19. Epistatic interactions between neuraminidase mutations facilitated the emergence of the oseltamivir-resistant H1N1 influenza viruses.

    PubMed

    Duan, Susu; Govorkova, Elena A; Bahl, Justin; Zaraket, Hassan; Baranovich, Tatiana; Seiler, Patrick; Prevost, Kristi; Webster, Robert G; Webby, Richard J

    2014-01-01

    Oseltamivir-resistant H1N1 influenza viruses carrying the H275Y neuraminidase mutation predominated worldwide during the 2007-2009 seasons. Although several neuraminidase substitutions were found to be necessary to counteract the adverse effects of H275Y, the order and impact of evolutionary events involved remain elusive. Here we reconstruct H1N1 neuraminidase phylogeny during 1999-2009, estimate the timing and order of crucial amino acid changes and evaluate their impact on the biological outcome of the H275Y mutation. Of the 12 neuraminidase substitutions that occurred during 1999-2009, 5 (chronologically, V234M, R222Q, K329E, D344N, H275Y and D354G) are necessary for maintaining full neuraminidase function in the presence of the H275Y mutation by altering protein accumulation or enzyme affinity/activity. The sequential emergence and cumulative effects of these mutations clearly illustrate a role for epistasis in shaping the emergence and subsequent evolution of a drug-resistant virus population, which can be useful in understanding emergence of novel viral phenotypes of influenza.

  20. Isolation of a biologically active soluble form of the hemagglutinin-neuraminidase protein of Sendai virus.

    PubMed Central

    Thompson, S D; Laver, W G; Murti, K G; Portner, A

    1988-01-01

    As a first step in establishing the three-dimensional structure of the Sendai virus hemagglutinin-neuraminidase (HN), we have isolated and characterized a potentially crystallizable form of the molecule. The sequence of HN, a surface glycoprotein, predicts a protein with an uncharged hydrophobic region near the amino terminus which is responsible for anchorage in the viral envelope. To avoid rosette formation (aggregation), which would preclude crystallization, this hydrophobic tail was removed from a membrane-free form of HN by proteolytic digestion. This digestion resulted in a single product with a molecular weight of about 10,000 less than native HN. N-terminal amino acid sequence analysis of cleaved HN (C-HN) indicated a single cleavage site at amino acid residue 131, resulting in a product consisting of the carboxyl-terminal 444 amino acids of HN. Functional analyses revealed that C-HN retained full neuraminidase activity and was able to bind erythrocytes, indicating that the N-terminal 131 residues were not necessary for these biological activities. Furthermore, this cleavage product retained the antigenic structure of intact HN, since monoclonal antibodies still bound to C-HN in enzyme-linked immunosorbent assay and Western (immuno-) blot analysis. Viewed by electron microscopy, the dimeric and tetrameric forms of intact HN form rosettes while C-HN maintains the oligomeric structure but no longer aggregates. Furthermore, the electron micrographs revealed a C-HN tetramer strikingly similar to the influenza virus neuraminidase in both size and gross structural features. Images PMID:2846877

  1. Immunobiological properties of influenza A (H7N9) hemagglutinin and neuraminidase proteins.

    PubMed

    Jiang, Li; Changsom, Don; Lerdsamran, Hatairat; Wiriyarat, Witthawat; Masamae, Wanibtisam; Noisumdaeng, Pirom; Jongkaewwattana, Anan; Puthavathana, Pilaipan

    2016-10-01

    Recombinant vaccinia viruses harboring the complete hemagglutinin (HA) or neuraminidase (NA) genes from the influenza A/Anhui/1/2013 (H7N9) virus were constructed (rVac-H7 HA and rVac-N9 NA viruses). The HA and NA proteins were expressed in the cytoplasm and on the plasma membrane of thymidine-kinase-negative (TK(-)) cells infected with these recombinant viruses. Only one form of the HA protein was expressed in infected TK(-) cells, with a molecular weight (MW) of 75 kDa, but three forms were found when the culture medium was supplemented with trypsin (MWs of 75, 50 and 27 kDa), which was similar to what was found in Madin-Darby canine kidney (MDCK) cells infected with reverse genetic (rg) influenza viruses carrying HA genes of H7N9 virus origin. One form of hyperglycosylated NA protein with a MW of 75 kDa was produced in rVac-N9-NA-virus-infected TK(-) or MDCK cells. The MW decreased to 55 kDa after deglycosylation. The hyperglycosylated recombinant NA protein demonstrated sialidase activity in a fetuin-based neuraminidase assay. The rVac-H7 HA and rVac-N9 NA viruses elicited significantly higher anti-HA and anti-NA antibody titers in BALB/c mice that were immunized once than in ICR mice. The anti-HA and anti-NA antibodies showed activity against homosubtypic HA or NA, but not against heterosubtypic HA or NA, as determined by hemagglutination-inhibition and microneutralization assays for anti-HA antibodies and neuraminidase-inhibition and replication-inhibition assays for anti-NA antibodies. Taken together, our data demonstrated immunobiological properties of recombinant HA and NA proteins that might be useful for vaccine development. PMID:27406044

  2. Immunologic response to influenza virus neuraminidase is influenced by prior experience with the associated viral hemagglutinin. III. Reduced generation of neuraminidase-specific helper T cells in hemagglutinin-primed mice.

    PubMed

    Johansson, B E; Moran, T M; Bona, C A; Kilbourne, E D

    1987-09-15

    In BALB/c mice primed by influenza virus infection to H3 hemagglutinin and N2 neuraminidase, presentation of N2 in association with a heterosubtypic (H7) hemagglutinin results in production of a greater amount of N2 antibody than is found with homologous (H3N2) reimmunization. Titration of primed helper T cell (Th) activity by adoptive transfer of purified T cells to athymic mice given H6N2 vaccine demonstrates a lesser number of N2-specific Th cells in mice subjected to homologous reimmunization. We conclude that Th cells participate in the mediation of intermolecular (intravirionic) antigenic competition between influenza virus hemagglutinin and neuraminidase.

  3. Flavonol dimers from callus cultures of Dysosma versipellis and their in vitro neuraminidase inhibitory activities.

    PubMed

    Chen, Ridao; Duan, Ruigang; Wei, Yannan; Zou, Jianhua; Li, Junwei; Liu, Xiaoyue; Wang, Haiyan; Guo, Ying; Li, Qiuhong; Dai, Jungui

    2015-12-01

    A chemical investigation of callus cultures of Dysosma versipellis led to the isolation of five new flavonol dimers, dysoverines A-E (1-5), together with 12 known compounds (6-17). The structures of new compounds were determined by the extensive spectroscopic data analyses. The biosynthetic pathway of the new compounds was proposed to involve O-methylation, prenylation, and Diels-Alder cycloaddition, which successively occurred in cultured plant cells. Compounds 1-17 exhibited in vitro neuraminidase inhibitory activities with the IC50 values of 31.0-93.9μM. PMID:26481138

  4. Fusion mutants of Newcastle disease virus selected with monoclonal antibodies to the hemagglutinin-neuraminidase.

    PubMed Central

    Iorio, R M; Glickman, R L

    1992-01-01

    The Australia-Victoria (AV) isolate of Newcastle disease virus (NDV) induces fusion from within but not fusion from without. L1, a neuraminidase (NA)-deficient virus derived from AV, has the opposite fusion phenotype from the wild-type virus. It fails to induce the former mode of fusion, but has gained a limited ability to promote the latter. Monoclonal antibodies to antigenic site 23 on the hemagglutinin-neuraminidase (HN) glycoprotein have previously been shown to select variants of the AV isolate that have altered NA activity or receptor-binding affinity. By using an antibody to this site, variants of L1 have been selected. Three of the variants have gained an increased affinity for sialic acid-containing receptors, as evidenced by the resistance of their hemagglutinating activity to the presence of reduced amounts of sialic acid on the surface of chicken erythrocytes. All four variants still have very low levels of NA activity, comparable to that of the parent virus, L1. The alteration in receptor-binding affinity results in a decreased potential for elution from cellular receptors and correlates with an increased ability to promote both modes of fusion. A single amino acid substitution in the HN protein of each variant, responsible for its escape from neutralization, has been identified. These studies identify two HN residues, 193 and 203, at which monoclonal antibody-selected substitution influences the receptor recognition properties of NDV and may influence its ability to promote syncytium formation. Images PMID:1404607

  5. Quantitative Comparison of Human Parainfluenza Virus Hemagglutinin-Neuraminidase Receptor Binding and Receptor Cleavage

    PubMed Central

    Tappert, Mary M.; Porterfield, J. Zachary; Mehta-D'Souza, Padmaja; Gulati, Shelly

    2013-01-01

    The human parainfluenza virus (hPIV) hemagglutinin-neuraminidase (HN) protein binds (H) oligosaccharide receptors that contain N-acetylneuraminic acid (Neu5Ac) and cleaves (N) Neu5Ac from these oligosaccharides. In order to determine if one of HN′s two functions is predominant, we measured the affinity of H for its ligands by a solid-phase binding assay with two glycoprotein substrates and by surface plasmon resonance with three monovalent glycans. We compared the dissociation constant (Kd) values from these experiments with previously determined Michaelis-Menten constants (Kms) for the enzyme activity. We found that glycoprotein substrates and monovalent glycans containing Neu5Acα2-3Galβ1-4GlcNAc bind HN with Kd values in the 10 to 100 μM range. Km values for HN were previously determined to be on the order of 1 mM (M. M. Tappert, D. F. Smith, and G. M. Air, J. Virol. 85:12146–12159, 2011). A Km value greater than the Kd value indicates that cleavage occurs faster than the dissociation of binding and will dominate under N-permissive conditions. We propose, therefore, that HN is a neuraminidase that can hold its substrate long enough to act as a binding protein. The N activity can therefore regulate binding by reducing virus-receptor interactions when the concentration of receptor is high. PMID:23740997

  6. Computational Assay of H7N9 Influenza Neuraminidase Reveals R292K Mutation Reduces Drug Binding Affinity

    NASA Astrophysics Data System (ADS)

    Woods, Christopher J.; Malaisree, Maturos; Long, Ben; McIntosh-Smith, Simon; Mulholland, Adrian J.

    2013-12-01

    The emergence of a novel H7N9 avian influenza that infects humans is a serious cause for concern. Of the genome sequences of H7N9 neuraminidase available, one contains a substitution of arginine to lysine at position 292, suggesting a potential for reduced drug binding efficacy. We have performed molecular dynamics simulations of oseltamivir, zanamivir and peramivir bound to H7N9, H7N9-R292K, and a structurally related H11N9 neuraminidase. They show that H7N9 neuraminidase is structurally homologous to H11N9, binding the drugs in identical modes. The simulations reveal that the R292K mutation disrupts drug binding in H7N9 in a comparable manner to that observed experimentally for H11N9-R292K. Absolute binding free energy calculations with the WaterSwap method confirm a reduction in binding affinity. This indicates that the efficacy of antiviral drugs against H7N9-R292K will be reduced. Simulations can assist in predicting disruption of binding caused by mutations in neuraminidase, thereby providing a computational `assay.'

  7. Sequence diversity of NanA manifests in distinct enzyme kinetics and inhibitor susceptibility

    NASA Astrophysics Data System (ADS)

    Xu, Zhongli; von Grafenstein, Susanne; Walther, Elisabeth; Fuchs, Julian E.; Liedl, Klaus R.; Sauerbrei, Andreas; Schmidtke, Michaela

    2016-04-01

    Streptococcus pneumoniae is the leading pathogen causing bacterial pneumonia and meningitis. Its surface-associated virulence factor neuraminidase A (NanA) promotes the bacterial colonization by removing the terminal sialyl residues from glycoconjugates on eukaryotic cell surface. The predominant role of NanA in the pathogenesis of pneumococci renders it an attractive target for therapeutic intervention. Despite the highly conserved activity of NanA, our alignment of the 11 NanAs revealed the evolutionary diversity of this enzyme. The amino acid substitutions we identified, particularly those in the lectin domain and in the insertion domain next to the catalytic centre triggered our special interest. We synthesised the representative NanAs and the mutagenized derivatives from E. coli for enzyme kinetics study and neuraminidase inhibitor susceptibility test. Via molecular docking we got a deeper insight into the differences between the two major variants of NanA and their influence on the ligand-target interactions. In addition, our molecular dynamics simulations revealed a prominent intrinsic flexibility of the linker between the active site and the insertion domain, which influences the inhibitor binding. Our findings for the first time associated the primary sequence diversity of NanA with the biochemical properties of the enzyme and with the inhibitory efficiency of neuraminidase inhibitors.

  8. Sequence diversity of NanA manifests in distinct enzyme kinetics and inhibitor susceptibility

    PubMed Central

    Xu, Zhongli; von Grafenstein, Susanne; Walther, Elisabeth; Fuchs, Julian E.; Liedl, Klaus R.; Sauerbrei, Andreas; Schmidtke, Michaela

    2016-01-01

    Streptococcus pneumoniae is the leading pathogen causing bacterial pneumonia and meningitis. Its surface-associated virulence factor neuraminidase A (NanA) promotes the bacterial colonization by removing the terminal sialyl residues from glycoconjugates on eukaryotic cell surface. The predominant role of NanA in the pathogenesis of pneumococci renders it an attractive target for therapeutic intervention. Despite the highly conserved activity of NanA, our alignment of the 11 NanAs revealed the evolutionary diversity of this enzyme. The amino acid substitutions we identified, particularly those in the lectin domain and in the insertion domain next to the catalytic centre triggered our special interest. We synthesised the representative NanAs and the mutagenized derivatives from E. coli for enzyme kinetics study and neuraminidase inhibitor susceptibility test. Via molecular docking we got a deeper insight into the differences between the two major variants of NanA and their influence on the ligand-target interactions. In addition, our molecular dynamics simulations revealed a prominent intrinsic flexibility of the linker between the active site and the insertion domain, which influences the inhibitor binding. Our findings for the first time associated the primary sequence diversity of NanA with the biochemical properties of the enzyme and with the inhibitory efficiency of neuraminidase inhibitors. PMID:27125351

  9. Hemagglutination and the closest distance of approach of normal, neuraminidase- and papain-treated erythrocytes.

    PubMed

    van Oss, C J; Absolom, D R

    1984-01-01

    By means of potential energy versus distance diagrams, derived from electrophoretic and surface tension data, the minimum distances of approach of normal (NOR) and of neuraminidase-treated (NEU) and papain-treated (PAP) human erythrocytes could be determined. The minimum distances between the actual cell membranes of two opposing red cells are: 184 A (NOR), 111 A (NEU), and 113 A (PAP), which agrees well with the fact that anti-D (Rho) antibodies of the IgG-class (which have a maximum distance of approximately 120 A between the two antibody-active sites) can hemagglutinate NEU and PAP cells, but are incapable of hemagglutinating normal D (Rho)-positive erythrocytes. PMID:6431697

  10. Hemagglutination and the closest distance of approach of normal, neuraminidase- and papain-treated erythrocytes.

    PubMed

    van Oss, C J; Absolom, D R

    1984-01-01

    By means of potential energy versus distance diagrams, derived from electrophoretic and surface tension data, the minimum distances of approach of normal (NOR) and of neuraminidase-treated (NEU) and papain-treated (PAP) human erythrocytes could be determined. The minimum distances between the actual cell membranes of two opposing red cells are: 184 A (NOR), 111 A (NEU), and 113 A (PAP), which agrees well with the fact that anti-D (Rho) antibodies of the IgG-class (which have a maximum distance of approximately 120 A between the two antibody-active sites) can hemagglutinate NEU and PAP cells, but are incapable of hemagglutinating normal D (Rho)-positive erythrocytes.

  11. Radiation inactivation analysis of influenza virus reveals different target sizes for fusion, leakage, and neuraminidase activities

    SciTech Connect

    Gibson, S.; Jung, C.Y.; Takahashi, M.; Lenard, J.

    1986-10-07

    The size of the functional units responsible for several activities carried out by the influenza virus envelope glycoproteins was determined by radiation inactivation analysis. Neuraminidase activity, which resides in the glycoprotein NA, was inactivated exponentially with an increasing radiation dose, yielding a target size of 94 +/- 5 kilodaltons (kDa), in reasonable agreement with that of the disulfide-bonded dimer (120 kDa). All the other activities studied are properties of the HA glycoprotein and were normalized to the known molecular weight of the neuraminidase dimer. Virus-induced fusion activity was measured by two phospholipid dilution assays: relief of energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dipalmitoyl-L-alpha- phosphatidylethanolamine (N-NBD-PE) and N-(lissamine rhodamine B sulfonyl)-dioleoyl-L-alpha-phosphatidylethanolamine (N-Rh-PE) in target liposomes and relief of self-quenching of N-Rh-PE in target liposomes. Radiation inactivation of fusion activity proceeded exponentially with radiation dose, yielding normalized target sizes of 68 +/- 6 kDa by assay i and 70 +/- 4 kDa by assay ii. These values are close to the molecular weight of a single disulfide-bonded (HA1 + HA2) unit (75 kDa), the monomer of the HA trimer. A single monomer is thus inactivated by each radiation event, and each monomer (or some part of it) constitutes a minimal functional unit capable of mediating fusion. Virus-induced leakage of calcein from target liposomes and virus-induced leakage of hemoglobin from erythrocytes (hemolysis) both showed more complex inactivation behavior: a pronounced shoulder was present in both inactivation curves, followed by a steep drop in activity at higher radiation levels.

  12. [Analysis of the Genetic Evolution of Neuraminidases of Influenza A Subtype N9 Viruses].

    PubMed

    Wan, Yong; Tang, Hua

    2015-03-01

    This study analyzed the genetic evolution of neuraminidases (NAs) of influenza A subtype N9 viruses with the aim of determining the genetic origin of the novel avian A/H7N9 influenza virus. The NA sequences of influenza A subtype N9 viruses available from NCBI were used to construct a phylogenetic tree using the programs ClustalX 2.0 and MEGA 6.0. This analysis indicated that the novel avian A/H7N9 influenza virus is located in the modern Eurasian phylogenetic cluster. This cluster was then further analyzed by estimating the overall rate of evolutionary change and the selective pressure at the nucleotide level using the program BEAST 2.1.2 and the web interface Datamonkey, and by generating an amino acid sequence entropy plot using Bioedit software. In this cluster, the mean rate of nucleotide substitutions in NA was found to be 3.8354 x 10(-3) and the mean ratio of non-synonymous (dN) to synonymous (dS) substitutions per site (dN/dS) was 0.140413. A particularly high level of amino acid mutation entropy was identified at nucleotides 16, 19, 40, 53, 81, 84, 112, 256, 335, 359, and 401. This genetic evolution analysis suggests that the nucleotide substitutions that characterize the novel avian A/H7N9 influenza virus neuraminidase are likely to result from the overall genetic evolution of influenza A subtype N9 virus NAs, and not from selective stress. Phylogenetic analysis suggests that the influenza A virus (A/duck/Siberia/700/1996(H11N9)) isolated in 1996 appears to be the common ancestor of the more recent influenza A subtype N9 viruses NAs.

  13. [A mechanism for limiting reproduction influenza A virus reassortants with incomplete functional compatibility of hemagglutinin and neuraminidase gene products].

    PubMed

    Kropotkina, E A; Rudneva, I A; Rusanova, O S; Varich, N L; Kadoshnikov, Iu P; Gambarian, A S; Kaverin, N V

    1998-01-01

    The mechanism of decrease in the level of virus accumulation in reassortants with hemagglutinin (HA) and neuraminidase (NA) genes from different parents is studied. The reassortant viruses and their passage variants do not differ by the rate of virus protein production or their stability in infected cells. Electron microscopy and titration of infectious virus in culture fluid and cell-associated virus showed that the variants selected by serial passages accumulated mainly in the culture fluid, whereas the initial reassortant virions were predominantly cell-associated. These data suggest that incomplete removal of sialic acid residues by viral neuraminidase N1 in some reassortants results in re-attachment of virions to the infected cells and thus impairs the virus dissemination, which may be regarded as a reassortant-limiting factor probably significant for virus evolution.

  14. Analysis of parainfluenza virus-5 hemagglutinin-neuraminidase protein mutants that are blocked in internalization and degradation

    SciTech Connect

    Robach, Jessica G.; Lamb, Robert A.

    2010-10-25

    The PIV-5 hemagglutinin-neuraminidase (HN) protein is a multifunctional protein with sialic acid binding, neuraminidase and fusion promotion activity. HN is internalized by clathrin-mediated endocytosis and degraded. HN lacks internalization signals in its cytoplasmic tail but a single glutamic acid present at residue 37 at the putative transmembrane/ectodomain boundary is critical. We rescued rPIV-5 with mutations E37D or E37K, which have been shown to impair or abolish HN internalization, respectively. These viruses exhibited growth properties similar to wild-type (wt) virus but are impaired for fitness in tissue culture. Biochemical analysis of HN activities showed differences between HN E37D and HN E37K in fusion promotion and incorporation of HN and F into virions. Furthermore, oligomeric analyses indicate that HN E37 mutants perturb the tetrameric organization of HN, probably by destabilizing the dimer-of-dimers interface.

  15. Influenza Neuraminidase Subtype N1: Immunobiological Properties and Functional Assays for Specific Antibody Response

    PubMed Central

    Changsom, Don; Lerdsamran, Hatairat; Wiriyarat, Witthawat; Chakritbudsabong, Warunya; Siridechadilok, Bunpote; Prasertsopon, Jarunee; Noisumdaeng, Pirom; Masamae, Wanibtisam; Puthavathana, Pilaipan

    2016-01-01

    Influenza neuraminidase (NA) proteins expressed in TK− cells infected with recombinant vaccinia virus carrying NA gene of highly pathogenic avian influenza H5N1 virus or 2009 pandemic H1N1 (H1N1pdm) virus were characterized for their biological properties, i.e., cell localization, molecular weight (MW), glycosylation and sialidase activity. Immune sera collected from BALB/c mice immunized with these recombinant viruses were assayed for binding and functional activities of anti-NA antibodies. Recombinant NA proteins were found localized in cytoplasm and cytoplasmic membrane of the infected cells. H1N1pdm NA protein had MW at about 75 kDa while it was 55 kDa for H5N1 NA protein. Hyperglycosylation was more pronounced in H1N1pdm NA compared to H5N1 NA according to N-glycosidase F treatment. Three dimensional structures also predicted that H1N1 NA globular head contained 4 and that of H5N1 contained 2 potential glycosylation sites. H5N1 NA protein had higher sialidase activity than H1N1pdm NA protein as measured by both MUNANA-based assay and fetuin-based enzyme-linked lectin assay (ELLA). Plaque reduction assay demonstrated that anti-NA antibody could reduce number of plaques and plaque size through inhibiting virus release, not virus entry. Assay for neuraminidase-inhibition (NI) antibody by ELLA showed specific and cross reactivity between H5N1 NA and H1N1pdm NA protein derived from reverse genetic viruses or wild type viruses. In contrast, replication-inhibition assay in MDCK cells showed that anti-H1N1 NA antibody moderately inhibited viruses with homologous NA gene only, while anti-H5N1 NA antibody modestly inhibited the replication of viruses containing homologous NA gene and NA gene derived from H1N1pdm virus. Anti-H1N1 NA antibody showed higher titers of inhibiting virus replication than anti-H5N1 NA antibody, which are consistent with the results on reduction in plaque numbers and sizes as well as in inhibiting NA enzymatic activity. No assay showed cross

  16. Influenza A(H7N9) virus acquires resistance-related neuraminidase I222T substitution when infected mallards are exposed to low levels of oseltamivir in water.

    PubMed

    Gillman, Anna; Nykvist, Marie; Muradrasoli, Shaman; Söderström, Hanna; Wille, Michelle; Daggfeldt, Annika; Bröjer, Caroline; Waldenström, Jonas; Olsen, Björn; Järhult, Josef D

    2015-09-01

    Influenza A virus (IAV) has its natural reservoir in wild waterfowl, and new human IAVs often contain gene segments originating from avian IAVs. Treatment options for severe human influenza are principally restricted to neuraminidase inhibitors (NAIs), among which oseltamivir is stockpiled in preparedness for influenza pandemics. There is evolutionary pressure in the environment for resistance development to oseltamivir in avian IAVs, as the active metabolite oseltamivir carboxylate (OC) passes largely undegraded through sewage treatment to river water where waterfowl reside. In an in vivo mallard (Anas platyrhynchos) model, we tested if low-pathogenic avian influenza A(H7N9) virus might become resistant if the host was exposed to low levels of OC. Ducks were experimentally infected, and OC was added to their water, after which infection and transmission were maintained by successive introductions of uninfected birds. Daily fecal samples were tested for IAV excretion, genotype, and phenotype. Following mallard exposure to 2.5 μg/liter OC, the resistance-related neuraminidase (NA) I222T substitution, was detected within 2 days during the first passage and was found in all viruses sequenced from subsequently introduced ducks. The substitution generated 8-fold and 2.4-fold increases in the 50% inhibitory concentration (IC50) for OC (P < 0.001) and zanamivir (P = 0.016), respectively. We conclude that OC exposure of IAV hosts, in the same concentration magnitude as found in the environment, may result in amino acid substitutions, leading to changed antiviral sensitivity in an IAV subtype that can be highly pathogenic to humans. Prudent use of oseltamivir and resistance surveillance of IAVs in wild birds are warranted. PMID:26077257

  17. Influenza A(H7N9) virus acquires resistance-related neuraminidase I222T substitution when infected mallards are exposed to low levels of oseltamivir in water.

    PubMed

    Gillman, Anna; Nykvist, Marie; Muradrasoli, Shaman; Söderström, Hanna; Wille, Michelle; Daggfeldt, Annika; Bröjer, Caroline; Waldenström, Jonas; Olsen, Björn; Järhult, Josef D

    2015-09-01

    Influenza A virus (IAV) has its natural reservoir in wild waterfowl, and new human IAVs often contain gene segments originating from avian IAVs. Treatment options for severe human influenza are principally restricted to neuraminidase inhibitors (NAIs), among which oseltamivir is stockpiled in preparedness for influenza pandemics. There is evolutionary pressure in the environment for resistance development to oseltamivir in avian IAVs, as the active metabolite oseltamivir carboxylate (OC) passes largely undegraded through sewage treatment to river water where waterfowl reside. In an in vivo mallard (Anas platyrhynchos) model, we tested if low-pathogenic avian influenza A(H7N9) virus might become resistant if the host was exposed to low levels of OC. Ducks were experimentally infected, and OC was added to their water, after which infection and transmission were maintained by successive introductions of uninfected birds. Daily fecal samples were tested for IAV excretion, genotype, and phenotype. Following mallard exposure to 2.5 μg/liter OC, the resistance-related neuraminidase (NA) I222T substitution, was detected within 2 days during the first passage and was found in all viruses sequenced from subsequently introduced ducks. The substitution generated 8-fold and 2.4-fold increases in the 50% inhibitory concentration (IC50) for OC (P < 0.001) and zanamivir (P = 0.016), respectively. We conclude that OC exposure of IAV hosts, in the same concentration magnitude as found in the environment, may result in amino acid substitutions, leading to changed antiviral sensitivity in an IAV subtype that can be highly pathogenic to humans. Prudent use of oseltamivir and resistance surveillance of IAVs in wild birds are warranted.

  18. Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay.

    PubMed

    Gao, Jin; Couzens, Laura; Eichelberger, Maryna C

    2016-01-01

    Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 °C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in ≥50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination. PMID:27684188

  19. Minimal Features of Efficient Incorporation of the Hemagglutinin-Neuraminidase Protein into Sendai Virus Particles

    PubMed Central

    Essaidi-Laziosi, Manel; Shevtsova, Anastasia S.

    2014-01-01

    Two transmembrane glycoproteins form spikes on the surface of Sendai virus, a member of the Respirovirus genus of the Paramyxovirinae subfamily of the Paramyxoviridae family: the hemagglutinin-neuraminidase (HN) and the fusion (F) proteins. HN, in contrast to F, is dispensable for viral particle production, as normal amounts of particles can be produced with highly reduced levels of HN. This HN reduction can result from mutation of an SYWST motif in its cytoplasmic tail to AFYKD. HNAFYKD accumulates at the infected cell surface but does not get incorporated into particles. In this work, we derived experimental tools to rescue HNAFYKD incorporation. We found that coexpression of a truncated HN harboring the wild-type cytoplasmic tail, the transmembrane domain, and at most 80 amino acids of the ectodomain was sufficient to complement defective HNAFYKD incorporation into particles. This relied on formation of disulfide-bound heterodimers carried out by the two cysteines present in the HN 80-amino-acid (aa) ectodomain. Finally, the replacement of the measles virus H cytoplasmic and transmembrane domains with the corresponding HN domains promoted measles virus H incorporation in Sendai virus particles. PMID:24155372

  20. Contribution of antibody production against neuraminidase to the protection afforded by influenza vaccines

    PubMed Central

    Marcelin, Glendie; Sandbulte, Matthew R.; Webby, Richard J.

    2012-01-01

    SUMMARY Vaccines are instrumental in controlling the burden of influenza virus infection in humans and animals. Antibodies raised against both major viral surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), can contribute to protective immunity. Vaccine-induced HA antibodies have been characterized extensively, and they generally confer protection by blocking the attachment and fusion of a homologous virus onto host cells. Though not as well characterized, some functions of NA antibodies in influenza vaccine-mediated immunity have been recognized for many years. In this review we summarize the case for NA antibodies in influenza vaccine-mediated immunity. In the absence of well-matched HA antibodies, NA antibodies can provide varying degrees of protection against disease. NA proteins of seasonal influenza vaccines have been shown in some instances to elicit serum antibodies with cross-reactivity to avian- and swine-origin influenza strains, in addition to HA drift variants. NA-mediated immunity has been linked to [i] conserved NA epitopes amongst otherwise antigenically distinct strains, partly attributable to the segmented influenza viral genome; [ii] inhibition of NA enzymatic activity; and [iii] the NA content in vaccine formulations. There is potential to enhance the effectiveness of existing and future influenza vaccines by focusing greater attention on the antigenic characteristics and potency of the NA protein. PMID:22438243

  1. Variable ligand- and receptor-binding hot spots in key strains of influenza neuraminidase

    PubMed Central

    Votapka, Lane; Demir, Özlem; Swift, Robert V; Walker, Ross C; Amaro, Rommie E

    2012-01-01

    Influenza A continues to be a major public health concern due to its ability to cause epidemic and pandemic disease outbreaks in humans. Computational investigations of structural dynamics of the major influenza glycoproteins, especially the neuraminidase (NA) enzyme, are able to provide key insights beyond what is currently accessible with standard experimental techniques. In particular, all-atom molecular dynamics simulations reveal the varying degrees of flexibility for such enzymes. Here we present an analysis of the relative flexibility of the ligand- and receptor-binding area of three key strains of influenza A: highly pathogenic H5N1, the 2009 pandemic H1N1, and a human N2 strain. Through computational solvent mapping, we investigate the various ligand- and receptor-binding “hot spots” that exist on the surface of NA which interacts with both sialic acid receptors on the host cells and antiviral drugs. This analysis suggests that the variable cavities found in the different strains and their corresponding capacities to bind ligand functional groups may play an important role in the ability of NA to form competent reaction encounter complexes with other species of interest, including antiviral drugs, sialic acid receptors on the host cell surface, and the hemagglutinin protein. Such considerations may be especially useful for the prediction of how such complexes form and with what binding capacity. PMID:22872804

  2. Development of neuraminidase detection using gold nanoparticles boron-doped diamond electrodes.

    PubMed

    Wahyuni, Wulan T; Ivandini, Tribidasari A; Saepudin, Endang; Einaga, Yasuaki

    2016-03-15

    Gold nanoparticles-modified boron-doped diamond (AuNPs-BDD) electrodes, which were prepared with a self-assembly deposition of AuNPs at amine-terminated boron-doped diamond, were examined for voltammetric detection of neuraminidase (NA). The detection method was performed based on the difference of electrochemical responses of zanamivir at gold surface before and after the reaction with NA in phosphate buffer solution (PBS, pH 5.5). A linear calibration curve for zanamivir in 0.1 M PBS in the absence of NA was achieved in the concentration range of 1 × 10(-6) to 1 × 10(-5) M (R(2) = 0.99) with an estimated limit of detection (LOD) of 2.29 × 10(-6) M. Furthermore, using its reaction with 1.00 × 10(-5) M zanamivir, a linear calibration curve of NA can be obtained in the concentration range of 0-12 mU (R(2) = 0.99) with an estimated LOD of 0.12 mU. High reproducibility was shown with a relative standard deviation (RSD) of 1.14% (n = 30). These performances could be maintained when the detection was performed in mucin matrix. Comparison performed using gold-modified BDD (Au-BDD) electrodes suggested that the good performance of the detection method is due to the stability of the gold particles position at the BDD surface.

  3. Core-6 fucose and the oligomerization of the 1918 pandemic influenza viral neuraminidase.

    PubMed

    Wu, Zhengliang L; Zhou, Hui; Ethen, Cheryl M; N Reinhold, Vernon

    2016-04-29

    The 1918 H1N1 influenza virus was responsible for one of the most deadly pandemics in human history. Yet to date, the structure component responsible for its virulence is still a mystery. In order to search for such a component, the neuraminidase (NA) antigen of the virus was expressed, which led to the discovery of an active form (tetramer) and an inactive form (dimer and monomer) of the protein due to different glycosylation. In this report, the N-glycans from both forms were released and characterized by mass spectrometry. It was found that the glycans from the active form had 26% core-6 fucosylated, while the glycans from the inactive form had 82% core-6 fucosylated. Even more surprisingly, the stalk region of the active form was almost completely devoid of core-6-linked fucose. These findings were further supported by the results obtained from in vitro incorporation of azido fucose and (3)H-labeled fucose using core-6 fucosyltransferase, FUT8. In addition, the incorporation of fucose did not change the enzymatic activity of the active form, implying that core-6 fucose is not directly involved in the enzymatic activity. It is postulated that core-6 fucose prohibits the oligomerization and subsequent activation of the enzyme.

  4. The serologic behaviour of neuraminidase-treated lymphoid cells: alloantigenicity and complement sensitivity

    PubMed Central

    Ray, P. K.; Gewurz, H.; Simmons, R. L.

    1972-01-01

    Vibrio cholerae neuraminidase (VCN) renders mouse lymphoid cells highly susceptible to the cytolytic effects of alloantibody and complement (C). This increased susceptibility to lysis is not due to unmasking of alloantigens since no increase in alloantibody-binding capacity of VCN-treated cells could be detected. However, VCN-treated cells can be lysed by normal rabbit serum, human serum, and guinea-pig serum even if specific antibody is not added to the incubation mixture. VCN, therefore, while not unmasking strong H-2 histocompatibility antigens, may be capable of unmasking other antigens with which heterologous sera can react. The increased susceptibility to immune cytolysis of VCN-treated cells appears to be related, at least partially, to its extreme susceptibility to C. Complement inactivation by heat totally abrogated the lytic effect, as did C inactivation by ammonium hydroxide, viscarin, and zymosan. In addition, activation of the autologous serum C within the fluid phase by cobra venom factor produced cytolysis only in VCN-treated cells. Thus, VCN renders nucleated cells highly susceptible to lysis by C. PMID:5042921

  5. Activation of brain endothelium by Pneumococcal neuraminidase NanA promotes bacterial internalization

    PubMed Central

    Banerjee, Anirban; van Sorge, Nina M.; Sheen, Tamsin R.; Uchiyama, Satoshi; Mitchell, Tim J.; Doran, Kelly S.

    2010-01-01

    Streptococcus pneumoniae (SPN), the leading cause of meningitis in children and adults worldwide, is associated with an overwhelming host inflammatory response and subsequent brain injury. Here we examine the global response of the blood-brain barrier to SPN infection and the role of neuraminidase A (NanA), a SPN surface anchored protein recently described to promote central nervous system tropism. Microarray analysis of human brain microvascular endothelial cells (hBMEC) during infection with SPN or an isogenic NanA-deficient (ΔnanA) mutant revealed differentially activated genes, including neutrophil chemoattractants IL-8, CXCL-1, CXCL-2. Studies using bacterial mutants, purified recombinant NanA proteins and in vivo neutrophil chemotaxis assays indicated that pneumococcal NanA is necessary and sufficient to activate host chemokine expression and neutrophil recruitment during infection. Chemokine induction was mapped to the NanA N-terminal lectin-binding domain with a limited contribution of the sialidase catalytic activity, and was not dependent on the invasive capability of the organism. Further, pretreatment of hBMEC with recombinant NanA protein significantly increased bacterial invasion suggesting that NanA-mediated activation of hBMEC is a prerequisite for efficient SPN invasion. These findings were corroborated in an acute murine infection model where we observed less inflammatory infiltrate and decreased chemokine expression following infection with the ΔnanA mutant. PMID:20557315

  6. Structural Characterization of the 1918 Influenza H1N1 Neuraminidase

    SciTech Connect

    Xu, X.; Zhu, X.; Dwek, R.A.; Stevens, J.; Wilson, I.A.

    2009-05-28

    Influenza virus neuraminidase (NA) plays a crucial role in facilitating the spread of newly synthesized virus in the host and is an important target for controlling disease progression. The NA crystal structure from the 1918 'Spanish flu' (A/Brevig Mission/1/18 H1N1) and that of its complex with zanamivir (Relenza) at 1.65-{angstrom} and 1.45-{angstrom} resolutions, respectively, corroborated the successful expression of correctly folded NA tetramers in a baculovirus expression system. An additional cavity adjacent to the substrate-binding site is observed in N1, compared to N2 and N9 NAs, including H5N1. This cavity arises from an open conformation of the 150 loop (Gly147 to Asp151) and appears to be conserved among group 1 NAs (N1, N4, N5, and N8). It closes upon zanamivir binding. Three calcium sites were identified, including a novel site that may be conserved in N1 and N4. Thus, these high-resolution structures, combined with our recombinant expression system, provide new opportunities to augment the limited arsenal of therapeutics against influenza.

  7. Core-6 fucose and the oligomerization of the 1918 pandemic influenza viral neuraminidase.

    PubMed

    Wu, Zhengliang L; Zhou, Hui; Ethen, Cheryl M; N Reinhold, Vernon

    2016-04-29

    The 1918 H1N1 influenza virus was responsible for one of the most deadly pandemics in human history. Yet to date, the structure component responsible for its virulence is still a mystery. In order to search for such a component, the neuraminidase (NA) antigen of the virus was expressed, which led to the discovery of an active form (tetramer) and an inactive form (dimer and monomer) of the protein due to different glycosylation. In this report, the N-glycans from both forms were released and characterized by mass spectrometry. It was found that the glycans from the active form had 26% core-6 fucosylated, while the glycans from the inactive form had 82% core-6 fucosylated. Even more surprisingly, the stalk region of the active form was almost completely devoid of core-6-linked fucose. These findings were further supported by the results obtained from in vitro incorporation of azido fucose and (3)H-labeled fucose using core-6 fucosyltransferase, FUT8. In addition, the incorporation of fucose did not change the enzymatic activity of the active form, implying that core-6 fucose is not directly involved in the enzymatic activity. It is postulated that core-6 fucose prohibits the oligomerization and subsequent activation of the enzyme. PMID:27012207

  8. Comparative Efficacy of Monoclonal Antibodies That Bind to Different Epitopes of the 2009 Pandemic H1N1 Influenza Virus Neuraminidase

    PubMed Central

    Jiang, Lianlian; Fantoni, Giovanna; Couzens, Laura; Gao, Jin; Plant, Ewan; Ye, Zhiping

    2015-01-01

    ABSTRACT Antibodies against the neuraminidase (NA) of influenza virus correlate with resistance against disease, but the effectiveness of antibodies against different NA epitopes has not been compared. In the present study, we evaluated the in vitro and in vivo efficacies of four monoclonal antibodies (MAbs): HF5 and CD6, which are specific to two different epitopes in the NA of 2009 pandemic H1N1 (pH1N1) virus, and 4E9 and 1H5, which are specific to a conserved epitope in the NA of both H1N1 and H5N1 viruses. In the in vitro assays, HF5 and CD6 inhibited virus spread and growth more effectively than 4E9 and 1H5, with HF5 being the most effective inhibitor. When administered prophylactically at 5 mg/kg of body weight, HF5 and CD6 protected ∼90 to 100% of DBA/2 mice against lethal wild-type pH1N1 virus challenge; however, at a lower dose (1 mg/kg), HF5 protected ∼90% of mice, whereas CD6 protected only 25% of mice. 4E9 and 1H5 were less effective than HF5 and CD6, as indicated by the partial protection achieved even at doses as high as 15 mg/kg. When administered therapeutically, HF5 protected a greater proportion of mice against lethal pH1N1 challenge than CD6. However, HF5 quickly selected pH1N1 virus escape mutants in both prophylactic and therapeutic treatments, while CD6 did not. Our findings confirm the important role of NA-specific antibodies in immunity to influenza virus and provide insight into the properties of NA antibodies that may serve as good candidates for therapeutics against influenza. IMPORTANCE Neuraminidase (NA) is one of the major surface proteins of influenza virus, serving as an important target for antivirals and therapeutic antibodies. The impact of NA-specific antibodies on NA activity and virus replication is likely to depend on where the antibody binds. Using in vitro assays and the mouse model, we compared the inhibitory/protective efficacy of four mouse monoclonal antibodies (MAbs) that bind to different sites within the 2009

  9. Optimization and comparative characterization of neuraminidase activities from Pseudomonas aeruginosa with Klebsiella pneumoniae, Hep-2 cell, sheep kidney and rat liver lysosome

    PubMed Central

    Ghazaei, C; Ahmadi, M; Hosseini Jazani, N

    2010-01-01

    Background and Objectives The properties of neuraminidase produced by P. aeruginosa strain PAO1 during growth in a defined medium (BHI) was examined and compared with some neuraminidase features of K. pneumoniae in this investigation. Materials and Methods The enzyme was isolated from concentrated culture supernatants of P. aeruginosa which was used in a sensitive fluorometric assay by using 2′-(4-methylumbelliferyl) α-D-N acetylneuraminic acid as substrate. Results Neuraminidase production in P. aeruginosa PAO1 paralleled bacterial growth in defined medium (BHI) and was maximal in the late logarithmic phase of growth but decreased during the stationary phase, probably owing to protease production or thermal instability. Highest production of P. aeruginosa PAO1 neuraminidase was in BHI culture media. The neuraminidase of P. aeruginosa PAO1 possessed an optimum temperature of activity at 56°C and the activity was maximal at pH 5. Heating the enzyme to 56°C for 45 min., in the presence of bovine serum albumin destroyed 33.1% of it's activity and addition of Ca+2, EDTA and NANA also decreased activity markedly. Conclusion The results revealed that the highest specific activity is for p. aeruginosa PAO1. PMID:22347548

  10. Computational study of interdependence between hemagglutinin and neuraminidase of pandemic 2009 H1N1.

    PubMed

    Hu, Wei

    2015-03-01

    Influenza type A viruses are classified into subtypes based on their two surface proteins, hemagglutinin (HA) and neuraminidase (NA). The HA protein facilitates the viral binding and entering a host cell and the NA protein helps the release of viral progeny from the infected cell. The complementary roles of HA and NA entail their collaboration, which has important implications for viral replication and fitness. The HA protein from early strains of pandemic 2009 H1N1 of swine origin preferentially binds to human type receptors with a weak binding to avian type receptors. This virus caused several human deaths in December 2013 in Texas, USA, which motivated us to investigate the changes of genetic features that might contribute to the surged virulence of the virus. Our time series analysis on the strains of this virus collected from 2009 to 2013 implied that the HA binding preference of this virus in USA, Europe, and Asia has been the characteristic of swine H1N1 virus since 2009. However, its characteristic of seasonal human H1N1 and its binding avidity for avian type receptors both were on steady rise and had a clear increase in 2013 with American strains having the sharpest surge. The first change could enhance the viral transmission and replication in humans and the second could increase its ability to cause infection deep in lungs, which might account for the recent human deaths in Texas. In light of HA and NA coadaptation and evolutionary interactions, we also explored the NA activity of this virus to reveal the functional balance between HA and NA during the course of virus evolution. Finally we identified amino acid substitutions in HA and NA of the virus that were critical for the observed evolution. PMID:25751873

  11. Cloning, expression and characterization of potential immunogenic recombinant hemagglutinin-neuraminidase protein of Porcine rubulavirus.

    PubMed

    Cuevas-Romero, Julieta Sandra; Rivera-Benítez, José Francisco; Hernández-Baumgarten, Eliseo; Hernández-Jaúregui, Pablo; Vega, Marco; Blomström, Anne-Lie; Berg, Mikael; Baule, Claudia

    2016-12-01

    Blue eye disease caused by Porcine rubulavirus (PorPV) is an endemic viral infection of swine causing neurological and respiratory disease in piglets, and reproductive failure in sows and boars. The hemagglutinin-neuraminidase (HN) glycoprotein of PorPV is the most abundant component in the viral envelope and the main target of the immune response in infected animals. In this study, we expressed the HN-PorPV-recombinant (rHN-PorPV) protein in an Escherichia coli system and analyzed the immune responses in mice. The HN gene was cloned from the reference strain PorPV-La Piedad Michoacan Virus (GenBank accession number BK005918), into the pDual expression vector. The expressed protein was identified at a molecular weight of 61.7 kDa. Three-dimensional modeling showed that the main conformational and functional domains of the rHN-PorPV protein were preserved. The antigenicity of the expressed protein was confirmed by Western blot with a monoclonal antibody recognizing the HN, and by testing against serum samples from pigs experimentally infected with PorPV. The immunogenicity of the rHN-PorPV protein was tested by inoculation of BALB/c mice with AbISCO-100(®) as adjuvant. Analysis of the humoral immune responses in mice showed an increased level of specific antibodies 14 days after the first immunization, compared to the control group (P < 0.0005). The results show the ability of the rHN-PorPV protein to induce an antibody response in mice. Due to its immunogenic potential, the rHN-PorPV protein will be further evaluated in pig trials for its suitability for prevention and control of blue eye disease. PMID:27496728

  12. Neuraminidase hemadsorption activity, conserved in avian influenza A viruses, does not influence viral replication in ducks.

    PubMed Central

    Kobasa, D; Rodgers, M E; Wells, K; Kawaoka, Y

    1997-01-01

    The N1 and N9 neuraminidase (NA) subtypes of influenza A viruses exhibit significant hemadsorption activity that localizes to a site distinct from that of the enzymatic active site. To determine the conservation of hemadsorption activity among different NAs, we have examined most of the NA subtypes from avian, swine, equine, and human virus isolates. All subtypes of avian virus NAs examined and one equine virus N8 NA possessed high levels of hemadsorption activity. A swine virus N1 NA exhibited only weak hemadsorption activity, while in human virus N1 and N2 NAs, the activity was detected at a much lower level than in avian virus NAs. NAs which possessed hemadsorption activity for chicken erythrocytes (RBCs) were similarly able to adsorb human RBCs. However, none of the hemadsorption-positive NAs could bind equine, swine, or bovine RBCs, suggesting that RBCs from these species lack molecules, recognized by the NA hemadsorption site, present on human and chicken RBCs. Mutagenesis of the putative hemadsorption site of A/duck/Hong Kong/7/75 N2 NA abolished the high level of hemadsorption activity exhibited by the wild-type protein but also resulted in a 50% reduction of the NA enzymatic activity. A transfectant virus, generated by reverse genetics, containing this mutated NA replicated 10-fold less efficiently in chicken embryo fibroblast cultures than did a transfectant virus expressing the wild-type NA. However, both viruses replicated equally well in Peking ducks. Although conservation of NA hemadsorption activity among avian virus NAs suggests the maintenance of a required function of NA, loss of the activity does not preclude the replication of the virus in an avian host. PMID:9261394

  13. Simultaneous quantification of hemagglutinin and neuraminidase of influenza virus using isotope dilution mass spectrometry.

    PubMed

    Williams, Tracie L; Pirkle, James L; Barr, John R

    2012-03-23

    Influenza vaccination is the primary method for preventing influenza and its severe complications. Licensed inactivated vaccines for seasonal or pandemic influenza are formulated to contain a preset amount of hemagglutinin (HA), the critical antigen to elicit protection. There is currently no regulatory method that quantifies neuraminidase (NA), the other major membrane-bound protein thought to have protective capability. This is primarily due to the limitations both in sensitivity and in selectivity of current means to quantify these antigens. Current methods to establish the HA concentration of vaccines rely on indirect measurements that are subject to considerable experimental variability. We present a liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the absolute quantification of viral proteins in a complex mixture. Through use of an isotope dilution approach, HA and NA from viral subtypes H1N1, H3N2, and B were determined both directly and rapidly. Three peptides of each subtype were used in the analysis of HA to ensure complete digestion of the protein and accuracy of the measurement. This method has been applied to purified virus preparations, to monovalent bulk concentrates, to trivalent inactivated influenza vaccines, and even crude allantoic fluid with improved speed, sensitivity, precision, and accuracy. Detection of 1 μg/mL of protein is easily obtained using this method. The sensitivity of the method covers the range expected in vaccine preparations, including adjuvant-based vaccine. This LC/MS/MS approach substantially increases the selectivity, accuracy and precision used to quantify the amount of viral proteins in seasonal and pandemic influenza vaccines and reduce the time and effort to deliver influenza vaccines for public health use during the next influenza pandemic.

  14. Cloning, expression and characterization of potential immunogenic recombinant hemagglutinin-neuraminidase protein of Porcine rubulavirus.

    PubMed

    Cuevas-Romero, Julieta Sandra; Rivera-Benítez, José Francisco; Hernández-Baumgarten, Eliseo; Hernández-Jaúregui, Pablo; Vega, Marco; Blomström, Anne-Lie; Berg, Mikael; Baule, Claudia

    2016-12-01

    Blue eye disease caused by Porcine rubulavirus (PorPV) is an endemic viral infection of swine causing neurological and respiratory disease in piglets, and reproductive failure in sows and boars. The hemagglutinin-neuraminidase (HN) glycoprotein of PorPV is the most abundant component in the viral envelope and the main target of the immune response in infected animals. In this study, we expressed the HN-PorPV-recombinant (rHN-PorPV) protein in an Escherichia coli system and analyzed the immune responses in mice. The HN gene was cloned from the reference strain PorPV-La Piedad Michoacan Virus (GenBank accession number BK005918), into the pDual expression vector. The expressed protein was identified at a molecular weight of 61.7 kDa. Three-dimensional modeling showed that the main conformational and functional domains of the rHN-PorPV protein were preserved. The antigenicity of the expressed protein was confirmed by Western blot with a monoclonal antibody recognizing the HN, and by testing against serum samples from pigs experimentally infected with PorPV. The immunogenicity of the rHN-PorPV protein was tested by inoculation of BALB/c mice with AbISCO-100(®) as adjuvant. Analysis of the humoral immune responses in mice showed an increased level of specific antibodies 14 days after the first immunization, compared to the control group (P < 0.0005). The results show the ability of the rHN-PorPV protein to induce an antibody response in mice. Due to its immunogenic potential, the rHN-PorPV protein will be further evaluated in pig trials for its suitability for prevention and control of blue eye disease.

  15. Preterm human milk contains a large pool of latent TGF-β, which can be activated by exogenous neuraminidase.

    PubMed

    Namachivayam, Kopperuncholan; Blanco, Cynthia L; Frost, Brandy L; Reeves, Aaron A; Jagadeeswaran, Ramasamy; MohanKumar, Krishnan; Safarulla, Azif; Mandal, Partha; Garzon, Steven A; Raj, J Usha; Maheshwari, Akhil

    2013-06-15

    Human milk contains substantial amounts of transforming growth factor (TGF)-β, particularly the isoform TGF-β2. We previously showed in preclinical models that enterally administered TGF-β2 can protect against necrotizing enterocolitis (NEC), an inflammatory bowel necrosis of premature infants. In this study we hypothesized that premature infants remain at higher risk of NEC than full-term infants, even when they receive their own mother's milk, because preterm human milk contains less bioactive TGF-β than full-term milk. Our objective was to compare TGF-β bioactivity in preterm vs. full-term milk and identify factors that activate milk-borne TGF-β. Mothers who delivered between 23 0/7 and 31 6/7 wk or at ≥37 wk of gestation provided milk samples at serial time points. TGF-β bioactivity and NF-κB signaling were measured using specific reporter cells and in murine intestinal tissue explants. TGF-β1, TGF-β2, TGF-β3, and various TGF-β activators were measured by real-time PCR, enzyme immunoassays, or established enzymatic activity assays. Preterm human milk showed minimal TGF-β bioactivity in the native state but contained a large pool of latent TGF-β. TGF-β2 was the predominant isoform of TGF-β in preterm milk. Using a combination of several in vitro and ex vivo models, we show that neuraminidase is a key regulator of TGF-β bioactivity in human milk. Finally, we show that addition of bacterial neuraminidase to preterm human milk increased TGF-β bioactivity. Preterm milk contains large quantities of TGF-β, but most of it is in an inactive state. Addition of neuraminidase can increase TGF-β bioactivity in preterm milk and enhance its anti-inflammatory effects.

  16. Proteasome inhibitors.

    PubMed

    Teicher, Beverly A; Tomaszewski, Joseph E

    2015-07-01

    Proteasome inhibitors have a 20 year history in cancer therapy. The first proteasome inhibitor, bortezomib (Velcade, PS-341), a break-through multiple myeloma treatment, moved rapidly through development from bench in 1994 to first approval in 2003. Bortezomib is a reversible boronic acid inhibitor of the chymotrypsin-like activity of the proteasome. Next generation proteasome inhibitors include carfilzomib and oprozomib which are irreversible epoxyketone proteasome inhibitors; and ixazomib and delanzomib which are reversible boronic acid proteasome inhibitors. Two proteasome inhibitors, bortezomib and carfilzomib are FDA approved drugs and ixazomib and oprozomib are in late stage clinical trials. All of the agents are potent cytotoxics. The disease focus for all the proteasome inhibitors is multiple myeloma. This focus arose from clinical observations made in bortezomib early clinical trials. Later preclinical studies confirmed that multiple myeloma cells were indeed more sensitive to proteasome inhibitors than other tumor cell types. The discovery and development of the proteasome inhibitor class of anticancer agents has progressed through a classic route of serendipity and scientific investigation. These agents are continuing to have a major impact in their treatment of hematologic malignancies and are beginning to be explored as potential treatment agent for non-cancer indications. PMID:25935605

  17. Acid-active neuraminidases in the growth media from cultures of pathogenic Naegleria fowleri and in sonicates of rabbit alveolar macrophages.

    PubMed

    Eisen, D; Franson, R C

    1987-05-19

    Using bovine mucin and isolated human myelin as sources of sialic acid, we demonstrate the presence of neuraminidase activities in the growth media of pathogenic, but not nonpathogenic, Naegleria sp. and in sonicates of rabbit alveolar macrophages. Neuraminidase activity was maximal at pH 4.5 and 5.0, and the specific activity for sialic acid release was up to 13-fold greater with mucin than with human myelin. Activity in the growth media from cultures of pathogenic Naegleria fowleri was ion-independent, while that of macrophage sonicates required divalent cation; optimal activity was noted with 2.5 mM Zn2+, while Mg2+ and Mn2+ supported activity to a lesser extent. Such acid-active neuraminidases may contribute to the reported glycolipid alterations associated with demyelinating diseases.

  18. Chimeric neuraminidase and mutant PB1 gene constellation improves growth and yield of H5N1 vaccine candidate virus.

    PubMed

    Plant, Ewan P; Ye, Zhiping

    2015-04-01

    We previously showed that a mutated PB1 gene improved the growth kinetics of a H3N2 influenza reassortant. Here, we showed that the same mutations improved the growth kinetics of a virus containing the A/Vietnam/1203/2004 (H5N1) haemagglutinin and neuraminidase (NA). Total protein yield and NA activity were increased when a chimeric NA was included. These increases indicated that the synergistic effect was due to the gene constellation containing both the altered PB1 gene and the chimeric NA gene.

  19. IgA1 desialylated by microbial neuraminidase forms immune complex with naturally occurring anti-T antibody in human serum.

    PubMed

    Anuradha; Jayakumari, N; Appukuttan, P S

    2008-01-29

    IgA1 was identified as the most prominent O-glycosylated protein of human serum. Desialylation by bacterial (Clostridium perfringens) neuraminidase rendered dot-blotted IgA1 recognizable by the naturally occurring serum antibody (anti-T) directed against Thomsen-Friedenreich antigen, Galbeta1-->3GalNAc-alpha-. On Western blot of serum O-glycosylated proteins anti-T recognized nearly all the bands including IgA1 as did the T antigen-specific animal lectin galectin-1 but only after their desialylation. Agglutination of desialylated human erythrocytes by anti-T was effectively inhibited by desialylated IgA1, but not by native IgA1 or other immunoglobulins. Desialylation of serum by neuraminidase led to significantly increased formation of immune complexes containing IgM, the major immunoglobulin type in anti-T on one hand and O-glycosylated proteins/IgA1 on the other. In further evidence for anti-T-desialylated IgA1 immune complex formation, purified anti-T added to desialylated, but not native serum led to formation of additional IgA-IgM immune complexes. Also neuraminidase treatment significantly reduced the titre of free (non-immune complexed) anti-T in serum, while selective removal of anti-T by affinity absorption resulted in considerable decrease in the amount of IgA1 that got converted to immune complexes following enzymatic desialylation of serum. Formation of immune complex between anti-T and neuraminidase-treated IgA1 in serum may be significant since many disease pathogens release neuraminidase and since IgA1 is a powerful ligand for tissue galectin-1 more so after desialylation. Diabetes also raises serum IgA and neuraminidase levels.

  20. Platelet Inhibitors.

    PubMed

    Shifrin, Megan M; Widmar, S Brian

    2016-03-01

    Antithrombotic medications have become standard of care for management of acute coronary syndrome. Platelet adhesion, activation, and aggregation are essential components of platelet function; platelet-inhibiting medications interfere with these components and reduce incidence of thrombosis. Active bleeding is a contraindication for administration of platelet inhibitors. There is currently no reversal agent for platelet inhibitors, although platelet transfusion may be used to correct active bleeding after administration of platelet inhibitors. PMID:26897422

  1. An extract from Taxodium distichum targets hemagglutinin- and neuraminidase-related activities of influenza virus in vitro

    PubMed Central

    Hsieh, Chung-Fan; Chen, Yu-Li; Lin, Chwan-Fwu; Ho, Jin-Yuan; Huang, Chun-Hsun; Chiu, Cheng-Hsun; Hsieh, Pei-Wen; Horng, Jim-Tong

    2016-01-01

    Influenza virus remains an emerging virus and causes pandemics with high levels of fatality. After screening different plant extracts with potential anti-influenza activity, a water extract of Taxodium distichum stems (TDSWex) showed excellent activity against influenza viruses. The EC50 of TDSWex was 0.051 ± 0.024 mg/mL against influenza virus A/WSN/33. TDSWex had excellent antiviral efficacy against various strains of human influenza A and B viruses, particularly oseltamivir-resistant clinical isolates and a swine-origin influenza strain. We observed that the synthesis of viral RNA and protein were inhibited in the presence of TDSWex. The results of the time-of-addition assay suggested that TDSWex inhibited viral entry and budding. In the hemagglutination inhibition assay, TDSWex inhibited the hemagglutination of red blood cells, implying that the extract targeted hemagglutin-related functions such as viral entry. In the attachment and penetration assay, TDSWex showed antiviral activity with EC50s of 0.045 ± 0.026 and 0.012 ± 0.003 mg/mL, respectively. In addition, TDSWex blocked neuraminidase activity. We conclude that TDSWex has bimodal activities against both hemagglutinin and neuraminidase during viral replication. PMID:27796330

  2. In the Shadow of Hemagglutinin: A Growing Interest in Influenza Viral Neuraminidase and Its Role as a Vaccine Antigen

    PubMed Central

    Wohlbold, Teddy John; Krammer, Florian

    2014-01-01

    Despite the availability of vaccine prophylaxis and antiviral therapeutics, the influenza virus continues to have a significant, annual impact on the morbidity and mortality of human beings, highlighting the continued need for research in the field. Current vaccine strategies predominantly focus on raising a humoral response against hemagglutinin (HA)—the more abundant, immunodominant glycoprotein on the surface of the influenza virus. In fact, anti-HA antibodies are often neutralizing, and are used routinely to assess vaccine immunogenicity. Neuraminidase (NA), the other major glycoprotein on the surface of the influenza virus, has historically served as the target for antiviral drug therapy and is much less studied in the context of humoral immunity. Yet, the quest to discern the exact importance of NA-based protection is decades old. Also, while antibodies against the NA glycoprotein fail to prevent infection of the influenza virus, anti-NA immunity has been shown to lessen the severity of disease, decrease viral lung titers in animal models, and reduce viral shedding. Growing evidence is intimating the possible gains of including the NA antigen in vaccine design, such as expanded strain coverage and increased overall immunogenicity of the vaccine. After giving a tour of general influenza virology, this review aims to discuss the influenza A virus neuraminidase while focusing on both the historical and present literature on the use of NA as a possible vaccine antigen. PMID:24960271

  3. Structure of the Ulster Strain Newcastle Disease Virus Hemagglutinin-Neuraminidase Reveals Auto-Inhibitory Interactions Associated with Low Virulence

    SciTech Connect

    Yuan, Ping; Paterson, Reay G.; Leser, George P.; Lamb, Robert A.; Jardetzky, Theodore S.

    2012-09-06

    Paramyxovirus hemagglutinin-neuraminidase (HN) plays roles in viral entry and maturation, including binding to sialic acid receptors, activation of the F protein to drive membrane fusion, and enabling virion release during virus budding. HN can thereby directly influence virulence and in a subset of avirulent Newcastle disease virus (NDV) strains, such as NDV Ulster, HN must be proteolytically activated to remove a C-terminal extension not found in other NDV HN proteins. Ulster HN is 616 amino acids long and the 45 amino acid C-terminal extension present in its precursor (HN0) form has to be cleaved to render HN biologically active. Here we show that Ulster HN contains an inter-subunit disulfide bond within the C-terminal extension at residue 596, which regulates HN activities and neuraminidase (NA) domain dimerization. We determined the crystal structure of the dimerized NA domain containing the C-terminal extension, which extends along the outside of the sialidase {beta}-propeller domain and inserts C-terminal residues into the NA domain active site. The C-terminal extension also engages a secondary sialic acid binding site present in NDV HN proteins, which is located at the NA domain dimer interface, that most likely blocks its attachment function. These results clarify how the Ulster HN C-terminal residues lead to an auto-inhibited state of HN, the requirement for proteolytic activation of HN{sub 0} and associated reduced virulence.

  4. Functional balance of the hemagglutinin and neuraminidase activities accompanies the emergence of the 2009 H1N1 influenza pandemic.

    PubMed

    Xu, Rui; Zhu, Xueyong; McBride, Ryan; Nycholat, Corwin M; Yu, Wenli; Paulson, James C; Wilson, Ian A

    2012-09-01

    The 2009 H1N1 influenza pandemic is the first human pandemic in decades and was of swine origin. Although swine are believed to be an intermediate host in the emergence of new human influenza viruses, there is still little known about the host barriers that keep swine influenza viruses from entering the human population. We surveyed swine progenitors and human viruses from the 2009 pandemic and measured the activities of the hemagglutinin (HA) and neuraminidase (NA), which are the two viral surface proteins that interact with host glycan receptors. A functional balance of these two activities (HA binding and NA cleavage) is found in human viruses but not in the swine progenitors. The human 2009 H1N1 pandemic virus exhibited both low HA avidity for glycan receptors as a result of mutations near the receptor binding site and weak NA enzymatic activity. Thus, a functional match between the hemagglutinin and neuraminidase appears to be necessary for efficient transmission between humans and may be an indicator of the pandemic potential of zoonotic viruses.

  5. Corrosion inhibitor

    SciTech Connect

    Wisotsky, M.J.; Metro, S.J.

    1989-10-31

    A corrosion inhibitor for use in synthetic ester lubricating oils is disclosed. It comprises an effective amount of: at least one aromatic amide; and at least one hydroxy substituted aromatic compound. The corrosion inhibitor thus formed is particularly useful in synthetic ester turbo lubricating oils.

  6. Impact of the H275Y and I223V Mutations in the Neuraminidase of the 2009 Pandemic Influenza Virus In Vitro and Evaluating Experimental Reproducibility

    PubMed Central

    Paradis, Eric G.; Pinilla, Lady Tatiana; Holder, Benjamin P.; Abed, Yacine; Boivin, Guy; Beauchemin, Catherine A.A.

    2015-01-01

    The 2009 pandemic H1N1 (H1N1pdm09) influenza virus is naturally susceptible to neuraminidase (NA) inhibitors, but mutations in the NA protein can cause oseltamivir resistance. The H275Y and I223V amino acid substitutions in the NA of the H1N1pdm09 influenza strain have been separately observed in patients exhibiting oseltamivir-resistance. Here, we apply mathematical modelling techniques to compare the fitness of the wild-type H1N1pdm09 strain relative to each of these two mutants. We find that both the H275Y and I223V mutations in the H1N1pdm09 background significantly lengthen the duration of the eclipse phase (by 2.5 h and 3.6 h, respectively), consistent with these NA mutations delaying the release of viral progeny from newly infected cells. Cells infected by H1N1pdm09 virus carrying the I223V mutation display a disadvantageous, shorter infectious lifespan (17 h shorter) than those infected with the wild-type or MUT-H275Y strains. In terms of compensating traits, the H275Y mutation in the H1N1pdm09 background results in increased virus infectiousness, as we reported previously, whereas the I223V exhibits none, leaving it overall less fit than both its wild-type counterpart and the MUT-H275Y strain. Using computer simulated competition experiments, we determine that in the presence of oseltamivir at doses even below standard therapy, both the MUT-H275Y and MUT-I223V dominate their wild-type counterpart in all aspects, and the MUT-H275Y outcompetes the MUT-I223V. The H275Y mutation should therefore be more commonly observed than the I223V mutation in circulating H1N1pdm09 strains, assuming both mutations have a similar impact or no significant impact on between-host transmission. We also show that mathematical modelling offers a relatively inexpensive and reliable means to quantify inter-experimental variability and assess the reproducibility of results. PMID:25992792

  7. Large-scale FMO-MP3 calculations on the surface proteins of influenza virus, hemagglutinin (HA) and neuraminidase (NA)

    NASA Astrophysics Data System (ADS)

    Mochizuki, Yuji; Yamashita, Katsumi; Fukuzawa, Kaori; Takematsu, Kazutomo; Watanabe, Hirofumi; Taguchi, Naoki; Okiyama, Yoshio; Tsuboi, Misako; Nakano, Tatsuya; Tanaka, Shigenori

    2010-06-01

    Two proteins on the influenza virus surface have been well known. One is hemagglutinin (HA) associated with the infection to cells. The fragment molecular orbital (FMO) calculations were performed on a complex consisting of HA trimer and two Fab-fragments at the third-order Møller-Plesset perturbation (MP3) level. The numbers of residues and 6-31G basis functions were 2351 and 201276, and thus a massively parallel-vector computer was utilized to accelerate the processing. This FMO-MP3 job was completed in 5.8 h with 1024 processors. Another protein is neuraminidase (NA) involved in the escape from infected cells. The FMO-MP3 calculation was also applied to analyze the interactions between oseltamivir and surrounding residues in pharmacophore.

  8. Influenza A virus nucleoprotein selectively decreases neuraminidase gene-segment packaging while enhancing viral fitness and transmissibility

    PubMed Central

    Brooke, Christopher B.; Ince, William L.; Wei, Jiajie; Bennink, Jack R.; Yewdell, Jonathan W.

    2014-01-01

    The influenza A virus (IAV) genome is divided into eight distinct RNA segments believed to be copackaged into virions with nearly perfect efficiency. Here, we describe a mutation in IAV nucleoprotein (NP) that enhances replication and transmission in guinea pigs while selectively reducing neuraminidase (NA) gene segment packaging into virions. We show that incomplete IAV particles lacking gene segments contribute to the propagation of the viral population through multiplicity reactivation under conditions of widespread coinfection, which we demonstrate commonly occurs in the upper respiratory tract of guinea pigs. NP also dramatically altered the functional balance of the viral glycoproteins on particles by selectively decreasing NA expression. Our findings reveal novel functions for NP in selective control of IAV gene packaging and balancing glycoprotein expression and suggest a role for incomplete gene packaging during host adaptation and transmission. PMID:25385602

  9. Hemagglutinin-neuraminidase balance confers respiratory-droplet transmissibility of the pandemic H1N1 influenza virus in ferrets.

    PubMed

    Yen, Hui-Ling; Liang, Chi-Hui; Wu, Chung-Yi; Forrest, Heather L; Ferguson, Angela; Choy, Ka-Tim; Jones, Jeremy; Wong, Diana Dik-Yan; Cheung, Peter Pak-Hang; Hsu, Che-Hsiung; Li, Olive T; Yuen, Kit M; Chan, Renee W Y; Poon, Leo L M; Chan, Michael C W; Nicholls, John M; Krauss, Scott; Wong, Chi-Huey; Guan, Yi; Webster, Robert G; Webby, Richard J; Peiris, Malik

    2011-08-23

    A novel reassortant derived from North American triple-reassortant (TRsw) and Eurasian swine (EAsw) influenza viruses acquired sustained human-to-human transmissibility and caused the 2009 influenza pandemic. To identify molecular determinants that allowed efficient transmission of the pandemic H1N1 virus among humans, we evaluated the direct-contact and respiratory-droplet transmissibility in ferrets of representative swine influenza viruses of different lineages obtained through a 13-y surveillance program in southern China. Whereas all viruses studied were transmitted by direct contact with varying efficiency, respiratory-droplet transmissibility (albeit inefficient) was observed only in the TRsw-like A/swine/Hong Kong/915/04 (sw915) (H1N2) virus. The sw915 virus had acquired the M gene derived from EAsw and differed from the gene constellation of the pandemic H1N1 virus by the neuraminidase (NA) gene alone. Glycan array analysis showed that pandemic H1N1 virus A/HK/415742/09 (HK415742) and sw915 possess similar receptor-binding specificity and affinity for α2,6-linked sialosides. Sw915 titers in differentiated normal human bronchial epithelial cells and in ferret nasal washes were lower than those of HK415742. Introducing the NA from pandemic HK415742 into sw915 did not increase viral replication efficiency but increased respiratory-droplet transmissibility, despite a substantial amino acid difference between the two viruses. The NA of the pandemic HK415742 virus possessed significantly higher enzyme activity than that of sw915 or other swine influenza viruses. Our results suggest that a unique gene constellation and hemagglutinin-neuraminidase balance play a critical role in acquisition of efficient and sustained human-to-human transmissibility.

  10. Hemagglutinin–neuraminidase balance confers respiratory-droplet transmissibility of the pandemic H1N1 influenza virus in ferrets

    PubMed Central

    Yen, Hui-Ling; Liang, Chi-Hui; Wu, Chung-Yi; Forrest, Heather L.; Ferguson, Angela; Choy, Ka-Tim; Jones, Jeremy; Wong, Diana Dik-Yan; Cheung, Peter Pak-Hang; Hsu, Che-Hsiung; Li, Olive T.; Yuen, Kit M.; Chan, Renee W. Y.; Poon, Leo L. M.; Chan, Michael C. W.; Nicholls, John M.; Krauss, Scott; Wong, Chi-Huey; Guan, Yi; Webster, Robert G.; Webby, Richard J.; Peiris, Malik

    2011-01-01

    A novel reassortant derived from North American triple-reassortant (TRsw) and Eurasian swine (EAsw) influenza viruses acquired sustained human-to-human transmissibility and caused the 2009 influenza pandemic. To identify molecular determinants that allowed efficient transmission of the pandemic H1N1 virus among humans, we evaluated the direct-contact and respiratory-droplet transmissibility in ferrets of representative swine influenza viruses of different lineages obtained through a 13-y surveillance program in southern China. Whereas all viruses studied were transmitted by direct contact with varying efficiency, respiratory-droplet transmissibility (albeit inefficient) was observed only in the TRsw-like A/swine/Hong Kong/915/04 (sw915) (H1N2) virus. The sw915 virus had acquired the M gene derived from EAsw and differed from the gene constellation of the pandemic H1N1 virus by the neuraminidase (NA) gene alone. Glycan array analysis showed that pandemic H1N1 virus A/HK/415742/09 (HK415742) and sw915 possess similar receptor-binding specificity and affinity for α2,6-linked sialosides. Sw915 titers in differentiated normal human bronchial epithelial cells and in ferret nasal washes were lower than those of HK415742. Introducing the NA from pandemic HK415742 into sw915 did not increase viral replication efficiency but increased respiratory-droplet transmissibility, despite a substantial amino acid difference between the two viruses. The NA of the pandemic HK415742 virus possessed significantly higher enzyme activity than that of sw915 or other swine influenza viruses. Our results suggest that a unique gene constellation and hemagglutinin–neuraminidase balance play a critical role in acquisition of efficient and sustained human-to-human transmissibility. PMID:21825167

  11. Structure determination of the 1918 H1N1 neuraminidase from a crystal with lattice-translocation defects

    SciTech Connect

    Zhu, Xueyong; Xu, Xiaojin; Wilson, Ian A.

    2008-08-01

    The structure of the 1918 H1N1 neuraminidase was determined to 1.65 Å from crystals with a lattice-translocation defect using uncorrected, as well as corrected, diffraction data. Few examples of macromolecular crystals containing lattice-translocation defects have been published in the literature. Lattice translocation and twinning are believed to be two common but different crystal-growth anomalies. While the successful use of twinned data for structure determination has become relatively routine in recent years, structure determination of crystals with lattice-translocation defects has not often been reported. To date, only four protein crystal structures containing such a crystal defect have been determined, using corrected, but not uncorrected, intensity data. In this report, the crystallization, structure determination and refinement of N1 neuraminidase derived from the 1918 H1N1 influenza virus (18NA) at 1.65 Å resolution are described. The crystal was indexed in space group C222{sub 1}, with unit-cell parameters a = 117.7, b = 138.5, c = 117.9 Å, and the structure was solved by molecular replacement. The lattice-translocation vector in the 18NA crystal was (0, 1/2, 1/2) or its equivalent vector (1/2, 0, 1/2) owing to the C lattice symmetry. Owing to this special lattice-translocation vector in space group C222{sub 1}, structure refinement could be achieved in two different ways: using corrected or uncorrected diffraction data. In the refinement with uncorrected data, a composite model was built to represent the molecules in the translated and untranslated layers, respectively. This composite structure model provided a unique example to examine how the molecules were arranged in the two lattice domains resulting from lattice-translocation defects.

  12. Structure of the Newcastle disease virus hemagglutinin-neuraminidase (HN) ectodomain reveals a four-helix bundle stalk

    SciTech Connect

    Yuan, Ping; Swanson, Kurt A.; Leser, George P.; Paterson, Reay G.; Lamb, Robert A.; Jardetzky, Theodore S.

    2014-10-02

    The paramyxovirus hemagglutinin-neuraminidase (HN) protein plays multiple roles in viral entry and egress, including binding to sialic acid receptors, activating the fusion (F) protein to activate membrane fusion and viral entry, and cleaving sialic acid from carbohydrate chains. HN is an oligomeric integral membrane protein consisting of an N-terminal transmembrane domain, a stalk region, and an enzymatically active neuraminidase (NA) domain. Structures of the HN NA domains have been solved previously; however, the structure of the stalk region has remained elusive. The stalk region contains specificity determinants for F interactions and activation, underlying the requirement for homotypic F and HN interactions in viral entry. Mutations of the Newcastle disease virus HN stalk region have been shown to affect both F activation and NA activities, but a structural basis for understanding these dual affects on HN functions has been lacking. Here, we report the structure of the Newcastle disease virus HN ectodomain, revealing dimers of NA domain dimers flanking the N-terminal stalk domain. The stalk forms a parallel tetrameric coiled-coil bundle (4HB) that allows classification of extensive mutational data, providing insight into the functional roles of the stalk region. Mutations that affect both F activation and NA activities map predominantly to the 4HB hydrophobic core, whereas mutations that affect only F-protein activation map primarily to the 4HB surface. Two of four NA domains interact with the 4HB stalk, and residues at this interface in both the stalk and NA domain have been implicated in HN function.

  13. Evidence for sialylated type 1 blood group chains on human erythrocyte membranes revealed by agglutination of neuraminidase-treated erythrocytes with Waldenström's macroglobulin IgMWOO and hybridoma antibody FC 10.2.

    PubMed

    Picard, J K; Loveday, D; Feizi, T

    1985-01-01

    Haemagglutination studies have been performed with untreated and neuraminidase-treated human erythrocytes of the three Lewis antigen types Le(a-b-), Le(a+b-) and Le(a-b+) using two monoclonal antibodies, IgMWOO and FC 10.2, which were previously shown to recognize the type 1 based blood group chains: Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc/GlcNAc (for explanation of abbreviations see table IV legend). Both antibodies behaved as cold agglutinins with neuraminidase-treated but not with untreated erythrocytes of the three Lewis antigen types. Neuraminidase-treated erythrocytes of i antigen type were similarly agglutinated. This haemagglutination was specifically inhibited by the type 1 based milk oligosaccharide lacto-N-tetraose. Thus, there is strong evidence for the occurrence of sialylated type 1 chains on human erythrocyte membranes of I and i antigen types. In addition, evidence for the presence of type 1 chains which are both sialylated and fucosylated was obtained by (1) haemagglutination of Le(a+b-) erythrocytes with the monoclonal antibody 19.9; (2) increased haemagglutination of neuraminidase-treated erythrocytes with anti-H antibodies of Bombay serum; (3) increased haemagglutination of neuraminidase-treated Le(a+b-) cells with anti-Lea antibodies, and (4) the appearance of Lea antigen activity on neuraminidase-treated erythrocytes of Le(a-b+) type.

  14. A Viable Recombinant Rhabdovirus Lacking Its Glycoprotein Gene and Expressing Influenza Virus Hemagglutinin and Neuraminidase Is a Potent Influenza Vaccine

    PubMed Central

    Ryder, Alex B.; Buonocore, Linda; Vogel, Leatrice; Nachbagauer, Raffael; Krammer, Florian

    2014-01-01

    ABSTRACT The emergence of novel influenza viruses that cause devastating human disease is an ongoing threat and serves as an impetus for the continued development of novel approaches to influenza vaccines. Influenza vaccine development has traditionally focused on producing humoral and/or cell-mediated immunity, often against the viral surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Here, we describe a new vaccine candidate that utilizes a replication-defective vesicular stomatitis virus (VSV) vector backbone that lacks the native G surface glycoprotein gene (VSVΔG). The expression of the H5 HA of an H5N1 highly pathogenic avian influenza virus (HPAIV), A/Vietnam/1203/04 (VN1203), and the NA of the mouse-adapted H1N1 influenza virus A/Puerto Rico/8/34 (PR8) in the VSVΔG vector restored the ability of the recombinant virus to replicate in cell culture, without the requirement for the addition of trypsin. We show here that this recombinant virus vaccine candidate was nonpathogenic in mice when given by either the intramuscular or intranasal route of immunization and that the in vivo replication of VSVΔG-H5N1 is profoundly attenuated. This recombinant virus also provided protection against lethal H5N1 infection after a single dose. This novel approach to vaccination against HPAIVs may be widely applicable to other emerging strains of influenza virus. IMPORTANCE Preparation for a potentially catastrophic influenza pandemic requires novel influenza vaccines that are safe, can be produced and administered quickly, and are effective, both soon after administration and for a long duration. We have created a new influenza vaccine that utilizes an attenuated vesicular stomatitis virus (VSV) vector, to deliver and express influenza virus proteins against which vaccinated animals develop potent antibody responses. The influenza virus hemagglutinin and neuraminidase proteins, expressed on the surface of VSV particles, allowed this vaccine to grow in cell

  15. Interaction between the Hemagglutinin-Neuraminidase and Fusion Glycoproteins of Human Parainfluenza Virus Type III Regulates Viral Growth In Vivo

    PubMed Central

    Xu, Rui; Palmer, Samantha G.; Porotto, Matteo; Palermo, Laura M.; Niewiesk, Stefan; Wilson, Ian A.; Moscona, Anne

    2013-01-01

    ABSTRACT Paramyxoviruses, enveloped RNA viruses that include human parainfluenza virus type 3 (HPIV3), cause the majority of childhood viral pneumonia. HPIV3 infection starts when the viral receptor-binding protein engages sialic acid receptors in the lung and the viral envelope fuses with the target cell membrane. Fusion/entry requires interaction between two viral surface glycoproteins: tetrameric hemagglutinin-neuraminidase (HN) and fusion protein (F). In this report, we define structural correlates of the HN features that permit infection in vivo. We have shown that viruses with an HN-F that promotes growth in cultured immortalized cells are impaired in differentiated human airway epithelial cell cultures (HAE) and in vivo and evolve in HAE into viable viruses with less fusogenic HN-F. In this report, we identify specific structural features of the HN dimer interface that modulate HN-F interaction and fusion triggering and directly impact infection. Crystal structures of HN, which promotes viral growth in vivo, show a diminished interface in the HN dimer compared to the reference strain’s HN, consistent with biochemical and biological data indicating decreased dimerization and decreased interaction with F protein. The crystallographic data suggest a structural explanation for the HN’s altered ability to activate F and reveal properties that are critical for infection in vivo. IMPORTANCE Human parainfluenza viruses cause the majority of childhood cases of croup, bronchiolitis, and pneumonia worldwide. Enveloped viruses must fuse their membranes with the target cell membranes in order to initiate infection. Parainfluenza fusion proceeds via a multistep reaction orchestrated by the two glycoproteins that make up its fusion machine. In vivo, viruses adapt for survival by evolving to acquire a set of fusion machinery features that provide key clues about requirements for infection in human beings. Infection of the lung by parainfluenzavirus is determined by

  16. Arterial Stiffening in Western Diet-Fed Mice Is Associated with Increased Vascular Elastin, Transforming Growth Factor-β, and Plasma Neuraminidase

    PubMed Central

    Foote, Christopher A.; Castorena-Gonzalez, Jorge A.; Ramirez-Perez, Francisco I.; Jia, Guanghong; Hill, Michael A.; Reyes-Aldasoro, Constantino C.; Sowers, James R.; Martinez-Lemus, Luis A.

    2016-01-01

    Consumption of excess fat and carbohydrate (Western diet, WD) is associated with alterations in the structural characteristics of blood vessels. This vascular remodeling contributes to the development of cardiovascular disease, particularly as it affects conduit and resistance arteries. Vascular remodeling is often associated with changes in the elastin-rich internal elastic lamina (IEL) and the activation of transforming growth factor (TGF)-β. In addition, obesity and type II diabetes have been associated with increased serum neuraminidase, an enzyme known to increase TGF-β cellular output. Therefore, we hypothesized that WD-feeding would induce structural modifications to the IEL of mesenteric resistance arteries in mice, and that these changes would be associated with increased levels of circulating neuraminidase and the up-regulation of elastin and TGF-β in the arterial wall. To test this hypothesis, a WD, high in fat and sugar, was used to induce obesity in mice, and the effect of this diet on the structure of mesenteric resistance arteries was investigated. 4-week old, Post-weaning mice were fed either a normal diet (ND) or WD for 16 weeks. Mechanically, arteries from WD-fed mice were stiffer and less distensible, with marginally increased wall stress for a given strain, and a significantly increased Young's modulus of elasticity. Structurally, the wall cross-sectional area and the number of fenestrae found in the internal elastic lamina (IEL) of mesenteric arteries from mice fed a WD were significantly smaller than those of arteries from the ND-fed mice. There was also a significant increase in the volume of elastin, but not collagen in arteries from the WD cohort. Plasma levels of neuraminidase and the amount of TGF-β in mesenteric arteries were elevated in mice fed a WD, while ex vivo, cultured vascular smooth muscle cells exposed to neuraminidase secreted greater amounts of tropoelastin and TGF-β than those exposed to vehicle. These data suggest that

  17. Hemagglutinin-Neuraminidase Balance Influences the Virulence Phenotype of a Recombinant H5N3 Influenza A Virus Possessing a Polybasic HA0 Cleavage Site

    PubMed Central

    Diederich, Sandra; Berhane, Yohannes; Embury-Hyatt, Carissa; Hisanaga, Tamiko; Handel, Katherine; Cottam-Birt, Colleen; Ranadheera, Charlene; Kobasa, Darwyn

    2015-01-01

    ABSTRACT Although a polybasic HA0 cleavage site is considered the dominant virulence determinant for highly pathogenic avian influenza (HPAI) H5 and H7 viruses, naturally occurring virus isolates possessing a polybasic HA0 cleavage site have been identified that are low pathogenic in chickens. In this study, we generated a reassortant H5N3 virus that possessed the hemagglutinin (HA) gene from H5N1 HPAI A/swan/Germany/R65/2006 and the remaining gene segments from low pathogenic A/chicken/British Columbia/CN0006/2004 (H7N3). Despite possessing the HA0 cleavage site GERRRKKR/GLF, this rH5N3 virus exhibited a low pathogenic phenotype in chickens. Although rH5N3-inoculated birds replicated and shed virus and seroconverted, transmission to naive contacts did not occur. To determine whether this virus could evolve into a HPAI form, it underwent six serial passages in chickens. A progressive increase in virulence was observed with the virus from passage number six being highly transmissible. Whole-genome sequencing demonstrated the fixation of 12 nonsynonymous mutations involving all eight gene segments during passaging. One of these involved the catalytic site of the neuraminidase (NA; R293K) and is associated with decreased neuraminidase activity and resistance to oseltamivir. Although introducing the R293K mutation into the original low-pathogenicity rH5N3 increased its virulence, transmission to naive contact birds was inefficient, suggesting that one or more of the remaining changes that had accumulated in the passage number six virus also play an important role in transmissibility. Our findings show that the functional linkage and balance between HA and NA proteins contributes to expression of the HPAI phenotype. IMPORTANCE To date, the contribution that hemagglutinin-neuraminidase balance can have on the expression of a highly pathogenic avian influenza virus phenotype has not been thoroughly examined. Reassortment, which can result in new hemagglutinin-neuraminidase

  18. Molecular modeling studies demonstrate key mutations that could affect the ligand recognition by influenza AH1N1 neuraminidase.

    PubMed

    Ramírez-Salinas, Gema L; García-Machorro, J; Quiliano, Miguel; Zimic, Mirko; Briz, Verónica; Rojas-Hernández, Saul; Correa-Basurto, J

    2015-11-01

    The goal of this study was to identify neuraminidase (NA) residue mutants from human influenza AH1N1 using sequences from 1918 to 2012. Multiple alignment studies of complete NA sequences (5732) were performed. Subsequently, the crystallographic structure of the 1918 influenza (PDB ID: 3BEQ-A) was used as a wild-type structure and three-dimensional (3-D) template for homology modeling of the mutated selected NA sequences. The 3-D mutated NAs were refined using molecular dynamics (MD) simulations (50 ns). The refined 3-D models were used to perform docking studies using oseltamivir. Multiple sequence alignment studies showed seven representative mutations (A232V, K262R, V263I, T264V, S367L, S369N, and S369K). MD simulations applied to 3-D NAs showed that each NA had different active-site shapes according to structural surface visualization and docking results. Moreover, Cartesian principal component analyses (cPCA) show structural differences among these NA structures caused by mutations. These theoretical results suggest that the selected mutations that are located outside of the active site of NA could affect oseltamivir recognition and could be associated with resistance to oseltamivir.

  19. What adaptive changes in hemagglutinin and neuraminidase are necessary for emergence of pandemic influenza virus from its avian precursor?

    PubMed

    Gambaryan, A S; Matrosovich, M N

    2015-07-01

    Wild ducks serve as the primary host for numerous and various influenza type A viruses. Occasionally, viruses from this reservoir can be transferred to other host species and cause outbreaks of influenza in fowl, swine, and horses, as well as result in novel human pandemics. Cellular tropism and range of susceptible host species are determined by interaction between virus and receptor molecules on cells. Here we discuss modern data regarding molecular features underlying interactions of influenza viruses with cellular receptors as well as a role for receptor specificity in interspecies transmission. By analyzing the earliest available pandemic influenza viruses (1918, 1957, 1968, 2009), we found that hemagglutinin reconfigured to recognize 2-6 sialic acid-containing receptors in the human upper airway tract together with altered enzymatic activity of neuraminidase necessary for maintaining functional balance with hemagglutinin are responsible for effective spread of influenza viruses in human populations. Resistance to low pH also contributes to this. Thus, a combination of such parameters makes it possible that influenza viruses give rise to novel pandemics.

  20. Analyzing swine sera for functional antibody titers against influenza A neuraminidase proteins using an enzyme-linked lectin assay (ELLA).

    PubMed

    Sandbulte, Matthew R; Eichelberger, Maryna C

    2014-01-01

    Neuraminidase (NA) is an envelope glycoprotein of influenza viruses, including swine-lineage influenza A viruses. NA possesses sialidase activity, which is functionally important at multiple points in viral replication, counter-balancing the sialic acid receptor binding activity of the hemagglutinin (HA), the other major envelope glycoprotein. The NA proteins of influenza A viruses have been classified into nine serological subtypes, and they undergo antigenic drift variation similar to that of HA. Antibodies to NA are analyzed much less often than antibodies to HA. The conventional assay for NA inhibition (NI) antibody titration, established decades ago, is widely considered unwieldy and inefficient for routine use. In recent years, a few new formats have been developed which still measure inhibition of NA enzymatic function, but more efficiently and with less chemical waste produced. Described here is the enzyme-linked lectin assay (ELLA), which is performed in 96-well plates and analyzed on a spectrophotometric plate reader. An important factor in adoption of the ELLA technique for animal studies, such as swine, is the choice of NA antigen, which may be purified protein or whole virus containing an antigenically irrelevant HA protein. This NI assay, in conjunction with the hemagglutination inhibiting (HI) antibody assay, offers a practical way to characterize viral isolates more fully and to quantify antibodies induced by infection or vaccination.

  1. Molecular modeling studies demonstrate key mutations that could affect the ligand recognition by influenza AH1N1 neuraminidase.

    PubMed

    Ramírez-Salinas, Gema L; García-Machorro, J; Quiliano, Miguel; Zimic, Mirko; Briz, Verónica; Rojas-Hernández, Saul; Correa-Basurto, J

    2015-11-01

    The goal of this study was to identify neuraminidase (NA) residue mutants from human influenza AH1N1 using sequences from 1918 to 2012. Multiple alignment studies of complete NA sequences (5732) were performed. Subsequently, the crystallographic structure of the 1918 influenza (PDB ID: 3BEQ-A) was used as a wild-type structure and three-dimensional (3-D) template for homology modeling of the mutated selected NA sequences. The 3-D mutated NAs were refined using molecular dynamics (MD) simulations (50 ns). The refined 3-D models were used to perform docking studies using oseltamivir. Multiple sequence alignment studies showed seven representative mutations (A232V, K262R, V263I, T264V, S367L, S369N, and S369K). MD simulations applied to 3-D NAs showed that each NA had different active-site shapes according to structural surface visualization and docking results. Moreover, Cartesian principal component analyses (cPCA) show structural differences among these NA structures caused by mutations. These theoretical results suggest that the selected mutations that are located outside of the active site of NA could affect oseltamivir recognition and could be associated with resistance to oseltamivir. PMID:26499499

  2. Discordant antigenic drift of neuraminidase and hemagglutinin in H1N1 and H3N2 influenza viruses

    PubMed Central

    Sandbulte, Matthew R.; Westgeest, Kim B.; Gao, Jin; Xu, Xiyan; Klimov, Alexander I.; Russell, Colin A.; Burke, David F.; Smith, Derek J.; Fouchier, Ron A. M.; Eichelberger, Maryna C.

    2011-01-01

    Seasonal epidemics caused by influenza virus are driven by antigenic changes (drift) in viral surface glycoproteins that allow evasion from preexisting humoral immunity. Antigenic drift is a feature of not only the hemagglutinin (HA), but also of neuraminidase (NA). We have evaluated the antigenic evolution of each protein in H1N1 and H3N2 viruses used in vaccine formulations during the last 15 y by analysis of HA and NA inhibition titers and antigenic cartography. As previously shown for HA, genetic changes in NA did not always lead to an antigenic change. The noncontinuous pattern of NA drift did not correspond closely with HA drift in either subtype. Although NA drift was demonstrated using ferret sera, we show that these changes also impact recognition by NA-inhibiting antibodies in human sera. Remarkably, a single point mutation in the NA of A/Brisbane/59/2007 was primarily responsible for the lack of inhibition by polyclonal antibodies specific for earlier strains. These data underscore the importance of NA inhibition testing to define antigenic drift when there are sequence changes in NA. PMID:22143798

  3. Hemagglutinin-neuraminidase enhances F protein-mediated membrane fusion of reconstituted Sendai virus envelopes with cells.

    PubMed Central

    Bagai, S; Puri, A; Blumenthal, R; Sarkar, D P

    1993-01-01

    Reconstituted Sendai virus envelopes containing both the fusion (F) protein and the hemagglutinin-neuraminidase (HN) (F,HN-virosomes) or only the F protein (F-virosomes) were prepared by solubilization of the intact virus with Triton X-100 followed by its removal by using SM2 Bio-Beads. Viral envelopes containing HN whose disulfide bonds were irreversibly reduced (HNred) were also prepared by treating the envelopes with dithiothreitol followed by dialysis (F,HNred-virosomes). Both F-virosomes and F,HNred-virosomes induced hemolysis of erythrocytes in the presence of wheat germ agglutinin, but the rates and extents were markedly lower than those for hemolysis induced by F,HN-virosomes. Using an assay based on the relief of self-quenching of a lipid probe incorporated in the Sendai virus envelopes, we demonstrate the fusion of both F,HN-virosomes and F-virosomes with cultured HepG2 cells containing the asialoglycoprotein receptor, which binds to a terminal galactose moiety of F. By desialylating the HepG2 cells, the entry mediated by HN-terminal sialic acid receptor interactions was bypassed. We show that both F-virosomes and F,HN-virosomes fuse with desialylated HepG2 cells, although the rate was two- to threefold higher if HN was included in the viral envelope. We also observed enhancement of fusion rates when both F and HN envelope proteins were attached to their specific receptors. Images PMID:8388501

  4. Long-Lasting Cross-Protection Against Influenza A by Neuraminidase and M2e-based immunization strategies

    PubMed Central

    Schotsaert, Michael; Ysenbaert, Tine; Smet, Anouk; Schepens, Bert; Vanderschaeghe, Dieter; Stegalkina, Svetlana; Vogel, Thorsten U.; Callewaert, Nico; Fiers, Walter; Saelens, Xavier

    2016-01-01

    There is mounting evidence that in the absence of neutralizing antibodies cross-reactive T cells provide protection against pandemic influenza viruses. Here, we compared protection and CD8+ T cell responses following challenge with H1N1 2009 pandemic and H3N2 viruses of mice that had been immunized with hemagglutinin (HA), neuraminidase (NA) and the extracellular domain of matrix protein 2 (M2e) fused to a virus-like particle (VLP). Mice were challenged a first time with a sublethal dose of H1N1 2009 pandemic virus and, four weeks later, challenged again with an H3N2 virus. Mice that had been vaccinated with HA, NA, NA + M2e-VLP and HA + NA + M2e-VLP were protected against homologous H1N1 virus challenge. Challenged NA and NA + M2e-VLP vaccinated mice mounted CD8+ T cell responses that correlated with protection against secondary H3N2 challenge. HA-vaccinated mice were fully protected against challenge with homologous H1N1 2009 virus, failed to mount cross-reactive CD8+ T cells and succumbed to the second challenge with heterologous H3N2 virus. In summary, NA- and M2e-based immunity can protect against challenge with (homologous) virus without compromising the induction of robust cross-reactive CD8+ T cell responses upon exposure to virus. PMID:27072615

  5. What adaptive changes in hemagglutinin and neuraminidase are necessary for emergence of pandemic influenza virus from its avian precursor?

    PubMed

    Gambaryan, A S; Matrosovich, M N

    2015-07-01

    Wild ducks serve as the primary host for numerous and various influenza type A viruses. Occasionally, viruses from this reservoir can be transferred to other host species and cause outbreaks of influenza in fowl, swine, and horses, as well as result in novel human pandemics. Cellular tropism and range of susceptible host species are determined by interaction between virus and receptor molecules on cells. Here we discuss modern data regarding molecular features underlying interactions of influenza viruses with cellular receptors as well as a role for receptor specificity in interspecies transmission. By analyzing the earliest available pandemic influenza viruses (1918, 1957, 1968, 2009), we found that hemagglutinin reconfigured to recognize 2-6 sialic acid-containing receptors in the human upper airway tract together with altered enzymatic activity of neuraminidase necessary for maintaining functional balance with hemagglutinin are responsible for effective spread of influenza viruses in human populations. Resistance to low pH also contributes to this. Thus, a combination of such parameters makes it possible that influenza viruses give rise to novel pandemics. PMID:26542001

  6. Punctuated Evolution of Influenza Virus Neuraminidase (A/H1N1) under Opposing Migration and Vaccination Pressures

    PubMed Central

    Phillips, J. C.

    2014-01-01

    Influenza virus contains two highly variable envelope glycoproteins, hemagglutinin (HA) and neuraminidase (NA). The structure and properties of HA, which is responsible for binding the virus to the cell that is being infected, change significantly when the virus is transmitted from avian or swine species to humans. Here we focus first on the simpler problem of the much smaller human individual evolutionary amino acid mutational changes in NA, which cleaves sialic acid groups and is required for influenza virus replication. Our thermodynamic panorama shows that very small amino acid changes can be monitored very accurately across many historic (1945–2011) Uniprot and NCBI strains using hydropathicity scales to quantify the roughness of water film packages. Quantitative sequential analysis is most effective with the fractal differential hydropathicity scale based on protein self-organized criticality (SOC). Our analysis shows that large-scale vaccination programs have been responsible for a very large convergent reduction in common influenza severity in the last century. Hydropathic analysis is capable of interpreting and even predicting trends of functional changes in mutation prolific viruses directly from amino acid sequences alone. An engineered strain of NA1 is described which could well be significantly less virulent than current circulating strains. PMID:25143953

  7. A monoclonal antibody targeting a highly conserved epitope in influenza B neuraminidase provides protection against drug resistant strains.

    PubMed

    Doyle, Tracey M; Li, Changgui; Bucher, Doris J; Hashem, Anwar M; Van Domselaar, Gary; Wang, Junzhi; Farnsworth, Aaron; She, Yi-Min; Cyr, Terry; He, Runtao; Brown, Earl G; Hurt, Aeron C; Li, Xuguang

    2013-11-01

    All influenza viral neuraminidases (NA) of both type A and B viruses have only one universally conserved sequence located between amino acids 222-230. A monoclonal antibody against this region has been previously reported to provide broad inhibition against all nine subtypes of influenza A NA; yet its inhibitory effect against influenza B viral NA remained unknown. Here, we report that the monoclonal antibody provides a broad inhibition against various strains of influenza B viruses of both Victoria and Yamagata genetic lineage. Moreover, the growth and NA enzymatic activity of two drug resistant influenza B strains (E117D and D197E) are also inhibited by the antibody even though these two mutations are conformationally proximal to the universal epitope. Collectively, these data suggest that this unique, highly-conserved linear sequence in viral NA is exposed sufficiently to allow access by inhibitory antibody during the course of infection; it could represent a potential target for antiviral agents and vaccine-induced immune responses against diverse strains of type B influenza virus. PMID:24140051

  8. Revertant analysis of a temperature-sensitive mutant of Newcastle disease virus with defective glycoproteins: implication of the fusion glycoprotein in cell killing and isolation of a neuraminidase-deficient hemagglutinating virus.

    PubMed Central

    Smith, G W; Hightower, L E

    1982-01-01

    Biological and molecular properties of a temperature-sensitive mutant (C1) of Newcastle disease virus and its revertants were analyzed. C1 exhibited three temperature-sensitive alterations (plaque formation, virion assembly, and cytopathogenicity) and several defects which were also present at the permissive temperature. C1 virions contained low amounts of hemagglutinin-neuraminidase glycopeptides and consequently were deficient in hemagglutinating and neuraminidase activities. These virions also contained defective fusion glycoproteins which rendered them poorly hemolytic and slow to penetrate cultured chicken embryo cells. The biological activities of the membrane glycoproteins were recovered sequentially in a series of plaque-forming revertants. The coreversion of hemolysis, membrane-penetrating activities, and cytopathogenicity in the first-step revertant (S1) suggested that fusion glycoproteins were major contributors to cellular destruction. This revertant also provided evidence of a role for fusion glycoproteins in virion assembly. From S1 we isolated a large-plaque-forming revertant (L1) that assembled wild-type amounts of biologically active hemagglutinin-neuraminidase glycoproteins into virions. Although it was normal for hemagglutination, L1 had less than 3% of the neuraminidase activity of the wild type, demonstrating that these two activities can be uncoupled genetically. The neuraminidase deficiency of L1 did not impair its virulence in ovo or its reproduction in cultured cells. PMID:6896347

  9. Active influenza virus neuraminidase is expressed in monkey cells from cDNA cloned in simian virus 40 vectors.

    PubMed Central

    Davis, A R; Bos, T J; Nayak, D P

    1983-01-01

    We have replaced the late genes of simian virus 40 (SV40) with a cloned cDNA copy of the neuraminidase (NA; EC 3.2.1.18) gene of the WSN (H1N1) strain of human influenza virus. When the SV40-NA recombinant virus was complemented in a lytic infection of monkey cells with a helper virus containing an early region deletion mutant, influenza NA was expressed and readily detected by immunofluorescence as well as by immunoprecipitation of in vivo labeled proteins with monoclonal antibodies against NA. In addition, the expressed NA exhibited enzymatic activity by cleaving the sialic acid residue from alpha-2,3-sialyllactitol. The expressed protein was glycosylated and transported to the cell surface, and it possessed the same molecular weight as the NA of WSN virus grown in monkey cells. Because the structure of NA is quite different from that of other integral membrane proteins and includes an anchoring region at the NH2 terminus consisting of hydrophobic amino acids, we also constructed deletion mutants of NA in this region. Replacement of DNA coding for the first 10 NH2-terminal amino acids with SV40 and linker sequences had no apparent effect on NA expression, glycosylation, transport to the cell surface, or enzymatic activity. However, further deletion of NA DNA encoding the first 26 amino acids abolished NA expression. These data suggest that the hydrophobic NH2-terminal region is multifunctional and is important in biosynthesis and translocation of NA across the membrane as well as in anchoring the protein. Images PMID:6306656

  10. A Genetically Engineered Waterfowl Influenza Virus with a Deletion in the Stalk of the Neuraminidase Has Increased Virulence for Chickens▿

    PubMed Central

    Munier, S.; Larcher, T.; Cormier-Aline, F.; Soubieux, D.; Su, B.; Guigand, L.; Labrosse, B.; Cherel, Y.; Quéré, P.; Marc, D.; Naffakh, N.

    2010-01-01

    A deletion of about 20 amino acids in the stalk of the neuraminidase (NA) is frequently detected upon transmission of influenza A viruses from waterfowl to domestic poultry. Using reverse genetics, a recombinant virus derived from a wild duck influenza virus isolate, A/Mallard/Marquenterre/Z237/83 (MZ), and an NA stalk deletion variant (MZ-delNA) were produced. Compared to the wild type, the MZ-delNA virus showed a moderate growth advantage on avian cultured cells. In 4-week-old chickens inoculated intratracheally with the MZ-delNA virus, viral replication in the lungs, liver, and kidneys was enhanced and interstitial pneumonia lesions were more severe than with the wild-type virus. The MZ-delNA-inoculated chickens showed significantly increased levels of mRNAs encoding interleukin-6 (IL-6), transforming growth factor-β4 (TGF-β4), and CCL5 in the lungs and a higher frequency of apoptotic cells in the liver than did their MZ-inoculated counterparts. Molecular mechanisms possibly underlying the growth advantage of the MZ-delNA virus were explored. The measured enzymatic activities toward a small substrate were similar for the wild-type and deleted NA, but the MZ-delNA virus eluted from chicken erythrocytes at reduced rates. Pseudoviral particles expressing the MZ hemagglutinin in combination with the MZ-NA or MZ-delNA protein were produced from avian cultured cells with similar efficiencies, suggesting that the deletion in the NA stalk does not enhance the release of progeny virions and probably affects an earlier step of the viral cycle. Overall, our data indicate that a shortened NA stalk is a strong determinant of adaptation and virulence of waterfowl influenza viruses in chickens. PMID:19889765

  11. Outside-binding site mutations modify the active site's shapes in neuraminidase from influenza A H1N1.

    PubMed

    Tolentino-Lopez, Luis; Segura-Cabrera, Aldo; Reyes-Loyola, Paola; Zimic, Mirko; Quiliano, Miguel; Briz, Veronica; Muñoz-Fernández, Angeles; Rodríguez-Pérez, Mario; Ilizaliturri-Flores, Ian; Correa-Basurto, Jose

    2013-01-01

    The recent occurrence of 2009 influenza A (H1N1) pandemic as well as others has raised concern of a far more dangerous outcome should this virus becomes resistant to current drug therapies. The number of clinical cases that are resistant to oseltamivir (Tamiflu®) is larger than the limited number of neuraminidase (NA) mutations (H275Y, N295S, and I223R) that have been identified at the active site and that are associated to oseltamivir resistance. In this study, we have performed a comparative analysis between a set of NAs that have the most representative mutations located outside the active site. The recently crystallized NA-oseltamivir complex (PDB ID: 3NSS) was used as a wild-type structure. After selecting the target NA sequences, their three-dimensional (3D) structure was built using 3NSS as a template by homology modeling. The 3D NA models were refined by molecular dynamics (MD) simulations. The refined models were used to perform a docking study, using oseltamivir as a ligand. Furthermore, the docking results were refined by free-energy analysis using the MM-PBSA method. The analysis of the MD simulation results showed that the NA models reached convergence during the first 10 ns. Visual inspection and structural measures showed that the mutated NA active sites show structural variations. The docking and MM-PBSA results from the complexes showed different binding modes and free energy values. These results suggest that distant mutations located outside the active site of NA affect its structure and could be considered to be a new source of resistance to oseltamivir, which agrees with reports in the clinical literature.

  12. Neuraminidase inhibiting antibody responses in pigs differ between influenza A virus N2 lineages and by vaccine type.

    PubMed

    Sandbulte, Matthew R; Gauger, Phillip C; Kitikoon, Pravina; Chen, Hongjun; Perez, Daniel R; Roth, James A; Vincent, Amy L

    2016-07-19

    The neuraminidase (NA) protein of influenza A viruses (IAV) has important functional roles in the viral replication cycle. Antibodies specific to NA can reduce viral replication and limit disease severity, but are not routinely measured. We analyzed NA inhibiting (NI) antibody titers in serum and respiratory specimens of pigs vaccinated with intramuscular whole-inactivated virus (WIV), intranasal live-attenuated influenza virus (LAIV), and intranasal wild type (WT) IAV. NI titers were also analyzed in sera from an investigation of piglet vaccination in the presence of passive maternally-derived antibodies. Test antigens contained genetically divergent swine-lineage NA genes homologous or heterologous to the vaccines with mismatched hemagglutinin genes (HA). Naïve piglets responded to WIV and LAIV vaccines and WT infection with strong homologous serum NI titers. Cross-reactivity to heterologous NAs depended on the degree of genetic divergence between the NA genes. Bronchoalveolar lavage specimens of LAIV and WT-immunized groups also had significant NI titers against the homologous antigen whereas the WIV group did not. Piglets of vaccinated sows received high levels of passive NI antibody, but their NI responses to homologous LAIV vaccination were impeded. These data demonstrate the utility of the enzyme-linked lectin assay for efficient NI antibody titration of serum as well as respiratory tract secretions. Swine IAV vaccines that induce robust NI responses are likely to provide broader protection against the diverse and rapidly evolving IAV strains that circulate in pig populations. Mucosal antibodies to NA may be one of the protective immune mechanisms induced by LAIV vaccines.

  13. Structure Determination of the 1918 H1N1 Neuraminidase From a Crystal With Lattice-Translocation Defects

    SciTech Connect

    Zhu, X.; Xu, X.; Wilson, I.A.

    2009-05-28

    Few examples of macromolecular crystals containing lattice-translocation defects have been published in the literature. Lattice translocation and twinning are believed to be two common but different crystal-growth anomalies. While the successful use of twinned data for structure determination has become relatively routine in recent years, structure determination of crystals with lattice-translocation defects has not often been reported. To date, only four protein crystal structures containing such a crystal defect have been determined, using corrected, but not uncorrected, intensity data. In this report, the crystallization, structure determination and refinement of N1 neuraminidase derived from the 1918 H1N1 influenza virus (18NA) at 1.65 {angstrom} resolution are described. The crystal was indexed in space group C222{sub 1}, with unit-cell parameters a = 117.7, b = 138.5, c = 117.9 {angstrom}, and the structure was solved by molecular replacement. The lattice-translocation vector in the 18NA crystal was (0, 1/2, 1/2) or its equivalent vector (1/2, 0, 1/2) owing to the C lattice symmetry. Owing to this special lattice-translocation vector in space group C222{sub 1}, structure refinement could be achieved in two different ways: using corrected or uncorrected diffraction data. In the refinement with uncorrected data, a composite model was built to represent the molecules in the translated and untranslated layers, respectively. This composite structure model provided a unique example to examine how the molecules were arranged in the two lattice domains resulting from lattice-translocation defects.

  14. Lectin histochemistry of an ovine lysosomal storage disease with deficiencies of beta-galactosidase and alpha-neuraminidase.

    PubMed Central

    Murnane, R. D.; Ahern-Rindell, A. J.; Prieur, D. J.

    1989-01-01

    Lectin histochemistry is a useful technique to identify and to localize in cells and tissues the terminal carbohydrate moieties of glycoproteins and glycolipids. The specific diagnosis of some glycoprotein storage diseases was accomplished using lectin staining patterns, and such methods of diagnosis have been attempted for some glycolipid storage diseases. This technique was applied to formalin-fixed paraffin-embedded and frozen neural, hepatic, and renal tissues of sheep with an inherited lysosomal storage disease with deficiencies of beta-galactosidase and alpha-neuraminidase. The cytoplasm of central nervous system neurons of affected sheep in paraffin-embedded sections stained with peanut agglutinin (PNA), Ricinus communis agglutinin-I (RCA-I), Dolichos biflorus agglutinin (DBA), and soybean agglutinin (SBA). The cytoplasm of neurons in frozen sections of these tissues stained with PNA, RCA-I, wheat germ agglutinin (WGA), and Ulex europaeus agglutinin-I (UEA-I). The cytoplasm of frozen and paraffin-embedded sections of liver and kidney of affected sheep stained with PNA, whereas paraffin-embedded sections also stained with RCA-I. These results suggest the stored material in this disease has terminal saccharide moieties consisting of beta-galactose, N-acetylneuraminic acid, and N-acetylgalactosamine. Paraffin processing altered lectin staining patterns. Although the staining pattern in this glycolipid storage disease was complex, lectin histochemistry may prove to be a useful technique for the characterization of storage products and for the diagnosis of lysosomal storage diseases. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:2508478

  15. Influenza virus neuraminidase contributes to the dextran sulfate-dependent suppressive replication of some influenza A virus strains.

    PubMed

    Yamada, Hiroshi; Moriishi, Eiko; Haredy, Ahmad M; Takenaka, Nobuyuki; Mori, Yasuko; Yamanishi, Koichi; Okamoto, Shigefumi

    2012-12-01

    Dextran sulfate (DS), a negatively charged, sulfated polysaccharide, suppresses the replication of an influenza A virus strain, and this suppression is associated with inhibition of the hemagglutinin (HA)-dependent fusion activity. However, it remains unknown whether the replication of all or just some influenza A virus strains is suppressed by DS, or whether HA is the only target for the replication suppression. In the present study, we found that DS inhibited the replication of some, but not all influenza A virus strains. The suppression in the DS-sensitive strains was dose-dependent and neutralized by diethylaminoethyl-dextran (DD), which has a positive charge. The suppression by DS was observed not only at the initial stage of viral infection, which includes viral attachment and entry, but also at the late stage, which includes virus assembly and release from infected cells. Electron microscopy revealed that the DS induced viral aggregation at the cell surface. The neuraminidase (NA) activity of the strains whose viral replication was inhibited at the late stage was also more suppressed by DS than that of the strains whose replication was not inhibited, and this inhibition of NA activity was also neutralized by adding positively charged DD. Furthermore, we found that replacing the NA gene of a strain in which viral replication was inhibited by DS at the late stage with the NA gene from a strain in which viral replication was not inhibited, eliminated the DS-dependent suppression. These results suggest that the influenza virus NA contributes to the DS-suppressible virus release from infected cells at the late stage, and the suppression may involve the inhibition of NA activity by DS's negative charge.

  16. Vaccination with Adjuvanted Recombinant Neuraminidase Induces Broad Heterologous, but Not Heterosubtypic, Cross-Protection against Influenza Virus Infection in Mice

    PubMed Central

    Wohlbold, Teddy John; Nachbagauer, Raffael; Xu, Haoming; Tan, Gene S.; Hirsh, Ariana; Brokstad, Karl A.; Cox, Rebecca J.; Palese, Peter

    2015-01-01

    ABSTRACT In an attempt to assess the cross-protective potential of the influenza virus neuraminidase (NA) as a vaccine antigen, different subtypes of recombinant NA were expressed in a baculovirus system and used to vaccinate mice prior to lethal challenge with homologous, heterologous, or heterosubtypic viruses. Mice immunized with NA of subtype N2 were completely protected from morbidity and mortality in a homologous challenge and displayed significantly reduced viral lung titers. Heterologous challenge with a drifted strain resulted in morbidity but no mortality. Similar results were obtained for challenge experiments with N1 NA. Mice immunized with influenza B virus NA (from B/Yamagata/16/88) displayed no morbidity when sublethally infected with the homologous strain and, importantly, were completely protected from morbidity and mortality when lethally challenged with the prototype Victoria lineage strain or a more recent Victoria lineage isolate. Upon analyzing the NA content in 4 different inactivated-virus vaccine formulations from the 2013-2014 season via Western blot assay and enzyme-linked immunosorbent assay quantification, we found that the amount of NA does indeed vary across vaccine brands. We also measured hemagglutinin (HA) and NA endpoint titers in pre- and postvaccination human serum samples from individuals who received a trivalent inactivated seasonal influenza vaccine from the 2004-2005 season; the induction of NA titers was statistically less pronounced than the induction of HA titers. The demonstrated homologous and heterologous protective capacity of recombinant NA suggests that supplementing vaccine formulations with a standard amount of NA may offer increased protection against influenza virus infection. PMID:25759506

  17. Deliberate reduction of hemagglutinin and neuraminidase expression of influenza virus leads to an ultraprotective live vaccine in mice.

    PubMed

    Yang, Chen; Skiena, Steven; Futcher, Bruce; Mueller, Steffen; Wimmer, Eckard

    2013-06-01

    A long-held dogma posits that strong presentation to the immune system of the dominant influenza virus glycoprotein antigens neuraminidase (NA) and hemagglutinin (HA) is paramount for inducing protective immunity against influenza virus infection. We have deliberately violated this dogma by constructing a recombinant influenza virus strain of A/PR8/34 (H1N1) in which expression of NA and HA genes was suppressed. We down-regulated NA and HA expression by recoding the respective genes with suboptimal codon pair bias, thereby introducing hundreds of nucleotide changes while preserving their codon use and protein sequence. The variants PR8-NA(Min), PR8-HA(Min), and PR8-(NA+HA)(Min) (Min, minimal expression) were used to assess the contribution of reduced glycoprotein expression to growth in tissue culture and pathogenesis in BALB/c mice. All three variants proliferated in Madin-Darby canine kidney cells to nearly the degree as WT PR8. In mice, however, they expressed explicit attenuation phenotypes, as revealed by their LD50 values: PR8, 32 plaque-forming units (PFU); HA(Min), 1.7 × 10(3) PFU; NA(Min), 2.4 × 10(5) PFU; (NA+HA)(Min), ≥3.16 × 10(6) PFU. Remarkably, (NA+HA)(Min) was attenuated >100,000-fold, with NA(Min) the major contributor to attenuation. In vaccinated mice (NA+HA)(Min) was highly effective in providing long-lasting protective immunity against lethal WT challenge at a median protective dose (PD50) of 2.4 PFU. Moreover, at a PD50 of only 147 or 237, (NA+HA)(Min) conferred protection against heterologous lethal challenges with two mouse-adapted H3N2 viruses. We conclude that the suppression of HA and NA is a unique strategy in live vaccine development. PMID:23690603

  18. Transforming Growth Factor-β: Activation by Neuraminidase and Role in Highly Pathogenic H5N1 Influenza Pathogenesis

    PubMed Central

    Moser, Lindsey A.; O'Brien, Kevin B.; Cline, Troy D.; Jones, Jeremy C.; Tumpey, Terrence M.; Katz, Jacqueline M.; Kelley, Laura A.; Gauldie, Jack; Schultz-Cherry, Stacey

    2010-01-01

    Transforming growth factor-beta (TGF-β), a multifunctional cytokine regulating several immunologic processes, is expressed by virtually all cells as a biologically inactive molecule termed latent TGF-β (LTGF-β). We have previously shown that TGF-β activity increases during influenza virus infection in mice and suggested that the neuraminidase (NA) protein mediates this activation. In the current study, we determined the mechanism of activation of LTGF-β by NA from the influenza virus A/Gray Teal/Australia/2/1979 by mobility shift and enzyme inhibition assays. We also investigated whether exogenous TGF-β administered via a replication-deficient adenovirus vector provides protection from H5N1 influenza pathogenesis and whether depletion of TGF-β during virus infection increases morbidity in mice. We found that both the influenza and bacterial NA activate LTGF-β by removing sialic acid motifs from LTGF-β, each NA being specific for the sialic acid linkages cleaved. Further, NA likely activates LTGF-β primarily via its enzymatic activity, but proteases might also play a role in this process. Several influenza A virus subtypes (H1N1, H1N2, H3N2, H5N9, H6N1, and H7N3) except the highly pathogenic H5N1 strains activated LTGF-β in vitro and in vivo. Addition of exogenous TGF-β to H5N1 influenza virus–infected mice delayed mortality and reduced viral titers whereas neutralization of TGF-β during H5N1 and pandemic 2009 H1N1 infection increased morbidity. Together, these data show that microbe-associated NAs can directly activate LTGF-β and that TGF-β plays a pivotal role protecting the host from influenza pathogenesis. PMID:20949074

  19. Deliberate reduction of hemagglutinin and neuraminidase expression of influenza virus leads to an ultraprotective live vaccine in mice

    PubMed Central

    Yang, Chen; Skiena, Steven; Futcher, Bruce; Mueller, Steffen; Wimmer, Eckard

    2013-01-01

    A long-held dogma posits that strong presentation to the immune system of the dominant influenza virus glycoprotein antigens neuraminidase (NA) and hemagglutinin (HA) is paramount for inducing protective immunity against influenza virus infection. We have deliberately violated this dogma by constructing a recombinant influenza virus strain of A/PR8/34 (H1N1) in which expression of NA and HA genes was suppressed. We down-regulated NA and HA expression by recoding the respective genes with suboptimal codon pair bias, thereby introducing hundreds of nucleotide changes while preserving their codon use and protein sequence. The variants PR8-NAMin, PR8-HAMin, and PR8-(NA+HA)Min (Min, minimal expression) were used to assess the contribution of reduced glycoprotein expression to growth in tissue culture and pathogenesis in BALB/c mice. All three variants proliferated in Madin–Darby canine kidney cells to nearly the degree as WT PR8. In mice, however, they expressed explicit attenuation phenotypes, as revealed by their LD50 values: PR8, 32 plaque-forming units (PFU); HAMin, 1.7 × 103 PFU; NAMin, 2.4 × 105 PFU; (NA+HA)Min, ≥3.16 × 106 PFU. Remarkably, (NA+HA)Min was attenuated >100,000-fold, with NAMin the major contributor to attenuation. In vaccinated mice (NA+HA)Min was highly effective in providing long-lasting protective immunity against lethal WT challenge at a median protective dose (PD50) of 2.4 PFU. Moreover, at a PD50 of only 147 or 237, (NA+HA)Min conferred protection against heterologous lethal challenges with two mouse-adapted H3N2 viruses. We conclude that the suppression of HA and NA is a unique strategy in live vaccine development. PMID:23690603

  20. Evolutionary History and Phylodynamics of Influenza A and B Neuraminidase (NA) Genes Inferred from Large-Scale Sequence Analyses

    PubMed Central

    Xu, Jianpeng; Davis, C. Todd; Christman, Mary C.; Rivailler, Pierre; Zhong, Haizhen; Donis, Ruben O.; Lu, Guoqing

    2012-01-01

    Background Influenza neuraminidase (NA) is an important surface glycoprotein and plays a vital role in viral replication and drug development. The NA is found in influenza A and B viruses, with nine subtypes classified in influenza A. The complete knowledge of influenza NA evolutionary history and phylodynamics, although critical for the prevention and control of influenza epidemics and pandemics, remains lacking. Methodology/Principal findings Evolutionary and phylogenetic analyses of influenza NA sequences using Maximum Likelihood and Bayesian MCMC methods demonstrated that the divergence of influenza viruses into types A and B occurred earlier than the divergence of influenza A NA subtypes. Twenty-three lineages were identified within influenza A, two lineages were classified within influenza B, and most lineages were specific to host, subtype or geographical location. Interestingly, evolutionary rates vary not only among lineages but also among branches within lineages. The estimated tMRCAs of influenza lineages suggest that the viruses of different lineages emerge several months or even years before their initial detection. The dN/dS ratios ranged from 0.062 to 0.313 for influenza A lineages, and 0.257 to 0.259 for influenza B lineages. Structural analyses revealed that all positively selected sites are at the surface of the NA protein, with a number of sites found to be important for host antibody and drug binding. Conclusions/Significance The divergence into influenza type A and B from a putative ancestral NA was followed by the divergence of type A into nine NA subtypes, of which 23 lineages subsequently diverged. This study provides a better understanding of influenza NA lineages and their evolutionary dynamics, which may facilitate early detection of newly emerging influenza viruses and thus improve influenza surveillance. PMID:22808012

  1. Reassortment between Avian H5N1 and Human Influenza Viruses Is Mainly Restricted to the Matrix and Neuraminidase Gene Segments

    PubMed Central

    Schrauwen, Eefje J. A.; Bestebroer, Theo M.; Rimmelzwaan, Guus F.; Osterhaus, Albert D. M. E.; Fouchier, Ron A. M.; Herfst, Sander

    2013-01-01

    Highly pathogenic avian influenza H5N1 viruses have devastated the poultry industry in many countries of the eastern hemisphere. Occasionally H5N1 viruses cross the species barrier and infect humans, sometimes with a severe clinical outcome. When this happens, there is a chance of reassortment between H5N1 and human influenza viruses. To assess the potential of H5N1 viruses to reassort with contemporary human influenza viruses (H1N1, H3N2 and pandemic H1N1), we used an in vitro selection method to generate reassortant viruses, that contained the H5 hemagglutinin gene, and that have a replication advantage in vitro. We found that the neuraminidase and matrix gene segments of human influenza viruses were preferentially selected by H5 viruses. However, these H5 reassortant viruses did not show a marked increase in replication in MDCK cells and human bronchial epithelial cells. In ferrets, inoculation with a mixture of H5N1-pandemic H1N1 reassortant viruses resulted in outgrowth of reassortant H5 viruses that had incorporated the neuraminidase and matrix gene segment of pandemic 2009 H1N1. This virus was not transmitted via aerosols or respiratory droplets to naïve recipient ferrets. Altogether, these data emphasize the potential of avian H5N1 viruses to reassort with contemporary human influenza viruses. The neuraminidase and matrix gene segments of human influenza viruses showed the highest genetic compatibility with HPAI H5N1 virus. PMID:23527283

  2. Reassortment between Avian H5N1 and human influenza viruses is mainly restricted to the matrix and neuraminidase gene segments.

    PubMed

    Schrauwen, Eefje J A; Bestebroer, Theo M; Rimmelzwaan, Guus F; Osterhaus, Albert D M E; Fouchier, Ron A M; Herfst, Sander

    2013-01-01

    Highly pathogenic avian influenza H5N1 viruses have devastated the poultry industry in many countries of the eastern hemisphere. Occasionally H5N1 viruses cross the species barrier and infect humans, sometimes with a severe clinical outcome. When this happens, there is a chance of reassortment between H5N1 and human influenza viruses. To assess the potential of H5N1 viruses to reassort with contemporary human influenza viruses (H1N1, H3N2 and pandemic H1N1), we used an in vitro selection method to generate reassortant viruses, that contained the H5 hemagglutinin gene, and that have a replication advantage in vitro. We found that the neuraminidase and matrix gene segments of human influenza viruses were preferentially selected by H5 viruses. However, these H5 reassortant viruses did not show a marked increase in replication in MDCK cells and human bronchial epithelial cells. In ferrets, inoculation with a mixture of H5N1-pandemic H1N1 reassortant viruses resulted in outgrowth of reassortant H5 viruses that had incorporated the neuraminidase and matrix gene segment of pandemic 2009 H1N1. This virus was not transmitted via aerosols or respiratory droplets to naïve recipient ferrets. Altogether, these data emphasize the potential of avian H5N1 viruses to reassort with contemporary human influenza viruses. The neuraminidase and matrix gene segments of human influenza viruses showed the highest genetic compatibility with HPAI H5N1 virus.

  3. Isolation of novel triple-reassortant swine H3N2 influenza viruses possessing the hemagglutinin and neuraminidase genes of a seasonal influenza virus in Vietnam in 2010.

    PubMed

    Ngo, Long Thanh; Hiromoto, Yasuaki; Pham, Vu Phong; Le, Ha Thi Hong; Nguyen, Ha Truc; Le, Vu Tri; Takemae, Nobuhiro; Saito, Takehiko

    2012-01-01

    Surveillance of swine influenza viruses (SIVs) in 31 pig farms in northern and southern parts of Vietnam was conducted. Six H3N2 influenza A viruses were isolated from a pig farm in southern Vietnam. They were novel genetic reassortants between a triple-reassortant SIV and a human seasonal H3N2 virus. Their hemagglutinin and neuraminidase genes were derived from a human virus circulating around 2004-2006 and the remaining genes from a triple-reassortant SIV that originated in North America. This is the first report describing the isolation of a novel triple-reassortant SIV in Vietnam.

  4. Design of new inhibitors for H5N1 avian influenza using a molecular dynamics simulation

    NASA Astrophysics Data System (ADS)

    Park, Jin Woo; Jo, Won Ho

    2008-03-01

    Recently, there has been a growing interest in the treatment of H5N1 avian influenza. One of the most widely used antiviral agents is oseltamivir. However, it has been reported that oseltamivir is not as effective against the neuraminidase subtype N1 as it is against subtypes N2 and N9. In our research we addressed this problem by designing new inhibitors and these altered inhibitor's binding affinities were calculated. In this study, we introduced chemical groups to the existing oseltamivir, so to fit into the newly discovered cavity in the subtype N1. When the binding strengths of the oseltamivir and the newly designed inhibitors for N1 were calculated to examine the drug efficiency through a molecular dynamics simulation, then compared with each other, it was found that one of the designed molecules exhibited a strong binding affinity, with more than twice the binding strength than that of oseltamivir. Since the aforementioned designed inhibitor appears to have the possibility for oral activity according to the criteria of human oral bioavailability, we propose that the inhibitor is a promising antiviral drug for H5N1 avian influenza.

  5. Using Common Spatial Distributions of Atoms to Relate Functionally Divergent Influenza Virus N10 and N11 Protein Structures to Functionally Characterized Neuraminidase Structures, Toxin Cell Entry Domains, and Non-Influenza Virus Cell Entry Domains

    PubMed Central

    Weininger, Arthur; Weininger, Susan

    2015-01-01

    The ability to identify the functional correlates of structural and sequence variation in proteins is a critical capability. We related structures of influenza A N10 and N11 proteins that have no established function to structures of proteins with known function by identifying spatially conserved atoms. We identified atoms with common distributed spatial occupancy in PDB structures of N10 protein, N11 protein, an influenza A neuraminidase, an influenza B neuraminidase, and a bacterial neuraminidase. By superposing these spatially conserved atoms, we aligned the structures and associated molecules. We report spatially and sequence invariant residues in the aligned structures. Spatially invariant residues in the N6 and influenza B neuraminidase active sites were found in previously unidentified spatially equivalent sites in the N10 and N11 proteins. We found the corresponding secondary and tertiary structures of the aligned proteins to be largely identical despite significant sequence divergence. We found structural precedent in known non-neuraminidase structures for residues exhibiting structural and sequence divergence in the aligned structures. In N10 protein, we identified staphylococcal enterotoxin I-like domains. In N11 protein, we identified hepatitis E E2S-like domains, SARS spike protein-like domains, and toxin components shared by alpha-bungarotoxin, staphylococcal enterotoxin I, anthrax lethal factor, clostridium botulinum neurotoxin, and clostridium tetanus toxin. The presence of active site components common to the N6, influenza B, and S. pneumoniae neuraminidases in the N10 and N11 proteins, combined with the absence of apparent neuraminidase function, suggests that the role of neuraminidases in H17N10 and H18N11 emerging influenza A viruses may have changed. The presentation of E2S-like, SARS spike protein-like, or toxin-like domains by the N10 and N11 proteins in these emerging viruses may indicate that H17N10 and H18N11 sialidase-facilitated cell

  6. Using common spatial distributions of atoms to relate functionally divergent influenza virus N10 and N11 protein structures to functionally characterized neuraminidase structures, toxin cell entry domains, and non-influenza virus cell entry domains.

    PubMed

    Weininger, Arthur; Weininger, Susan

    2015-01-01

    The ability to identify the functional correlates of structural and sequence variation in proteins is a critical capability. We related structures of influenza A N10 and N11 proteins that have no established function to structures of proteins with known function by identifying spatially conserved atoms. We identified atoms with common distributed spatial occupancy in PDB structures of N10 protein, N11 protein, an influenza A neuraminidase, an influenza B neuraminidase, and a bacterial neuraminidase. By superposing these spatially conserved atoms, we aligned the structures and associated molecules. We report spatially and sequence invariant residues in the aligned structures. Spatially invariant residues in the N6 and influenza B neuraminidase active sites were found in previously unidentified spatially equivalent sites in the N10 and N11 proteins. We found the corresponding secondary and tertiary structures of the aligned proteins to be largely identical despite significant sequence divergence. We found structural precedent in known non-neuraminidase structures for residues exhibiting structural and sequence divergence in the aligned structures. In N10 protein, we identified staphylococcal enterotoxin I-like domains. In N11 protein, we identified hepatitis E E2S-like domains, SARS spike protein-like domains, and toxin components shared by alpha-bungarotoxin, staphylococcal enterotoxin I, anthrax lethal factor, clostridium botulinum neurotoxin, and clostridium tetanus toxin. The presence of active site components common to the N6, influenza B, and S. pneumoniae neuraminidases in the N10 and N11 proteins, combined with the absence of apparent neuraminidase function, suggests that the role of neuraminidases in H17N10 and H18N11 emerging influenza A viruses may have changed. The presentation of E2S-like, SARS spike protein-like, or toxin-like domains by the N10 and N11 proteins in these emerging viruses may indicate that H17N10 and H18N11 sialidase-facilitated cell

  7. HDAC Inhibitors.

    PubMed

    Olzscha, Heidi; Bekheet, Mina E; Sheikh, Semira; La Thangue, Nicholas B

    2016-01-01

    Lysine acetylation in proteins is one of the most abundant posttranslational modifications in eukaryotic cells. The dynamic homeostasis of lysine acetylation and deacetylation is dictated by the action of histone acetyltransferases (HAT) and histone deacetylases (HDAC). Important substrates for HATs and HDACs are histones, where lysine acetylation generally leads to an open and transcriptionally active chromatin conformation. Histone deacetylation forces the compaction of the chromatin with subsequent inhibition of transcription and reduced gene expression. Unbalanced HAT and HDAC activity, and therefore aberrant histone acetylation, has been shown to be involved in tumorigenesis and progression of malignancy in different types of cancer. Therefore, the development of HDAC inhibitors (HDIs) as therapeutic agents against cancer is of great interest. However, treatment with HDIs can also affect the acetylation status of many other non-histone proteins which play a role in different pathways including angiogenesis, cell cycle progression, autophagy and apoptosis. These effects have led HDIs to become anticancer agents, which can initiate apoptosis in tumor cells. Hematological malignancies in particular are responsive to HDIs, and four HDIs have already been approved as anticancer agents. There is a strong interest in finding adequate biomarkers to predict the response to HDI treatment. This chapter provides information on how to assess HDAC activity in vitro and determine the potency of HDIs on different HDACs. It also gives information on how to analyze cellular markers following HDI treatment and to analyze tissue biopsies from HDI-treated patients. Finally, a protocol is provided on how to detect HDI sensitivity determinants in human cells, based on a pRetroSuper shRNA screen upon HDI treatment. PMID:27246222

  8. A Contributing Role for Anti-Neuraminidase Antibodies on Immunity to Pandemic H1N1 2009 Influenza A Virus

    PubMed Central

    Marcelin, Glendie; DuBois, Rebecca; Rubrum, Adam; Russell, Charles J.; McElhaney, Janet E.; Webby, Richard J.

    2011-01-01

    Background Exposure to contemporary seasonal influenza A viruses affords partial immunity to pandemic H1N1 2009 influenza A virus (pH1N1) infection. The impact of antibodies to the neuraminidase (NA) of seasonal influenza A viruses to cross-immunity against pH1N1 infection is unknown. Methods and Results Antibodies to the NA of different seasonal H1N1 influenza strains were tested for cross-reactivity against A/California/04/09 (pH1N1). A panel of reverse genetic (rg) recombinant viruses was generated containing 7 genes of the H1N1 influenza strain A/Puerto Rico/08/34 (PR8) and the NA gene of either the pandemic H1N1 2009 strain (pH1N1) or one of the following contemporary seasonal H1N1 strains: A/Solomon/03/06 (rg Solomon) or A/Brisbane/59/07 (rg Brisbane). Convalescent sera collected from mice infected with recombinant viruses were measured for cross-reactive antibodies to pH1N1 via Hemagglutinin Inhibition (HI) or Enzyme-Linked Immunosorbent Assay (ELISA). The ectodomain of a recombinant NA protein from the pH1N1 strain (pNA-ecto) was expressed, purified and used in ELISA to measure cross-reactive antibodies. Analysis of sera from elderly humans immunized with trivalent split-inactivated influenza (TIV) seasonal vaccines prior to 2009 revealed considerable cross-reactivity to pNA-ecto. High titers of cross-reactive antibodies were detected in mice inoculated with either rg Solomon or rg Brisbane. Convalescent sera from mice inoculated with recombinant viruses were used to immunize naïve recipient Balb/c mice by passive transfer prior to challenge with pH1N1. Mice receiving rg California sera were better protected than animals receiving rg Solomon or rg Brisbane sera. Conclusions The NA of contemporary seasonal H1N1 influenza strains induces a cross-reactive antibody response to pH1N1 that correlates with reduced lethality from pH1N1 challenge, albeit less efficiently than anti-pH1N1 NA antibodies. These findings demonstrate that seasonal NA antibodies

  9. Hemagglutinin and neuraminidase containing virus-like particles produced in HEK-293 suspension culture: An effective influenza vaccine candidate.

    PubMed

    Venereo-Sanchez, Alina; Gilbert, Renald; Simoneau, Melanie; Caron, Antoine; Chahal, Parminder; Chen, Wangxue; Ansorge, Sven; Li, Xuguang; Henry, Olivier; Kamen, Amine

    2016-06-17

    Virus-like particles (VLPs) constitute a promising alternative as influenza vaccine. They are non-replicative particles that mimic the morphology of native viruses which make them more immunogenic than classical subunit vaccines. In this study, we propose HEK-293 cells in suspension culture in serum-free medium as an efficient platform to produce large quantities of VLPs. For this purpose, a stable cell line expressing the main influenza viral antigens hemagglutinin (HA) and neuraminidase (NA) (subtype H1N1) under the regulation of a cumate inducible promoter was developed (293HA-NA cells). The production of VLPs was evaluated by transient transfection of plasmids encoding human immunodeficiency virus (HIV) Gag or M1 influenza matrix protein. To facilitate the monitoring of VLPs production, Gag was fused to the green fluorescence protein (GFP). The transient transfection of the gag containing plasmid in 293HA-NA cells increased the release of HA and NA seven times more than its counterpart transfected with the M1 encoding plasmid. Consequently, the production of HA-NA containing VLPs using Gag as scaffold was evaluated in a 3-L controlled stirred tank bioreactor. The VLPs secreted in the culture medium were recovered by ultracentrifugation on a sucrose cushion and ultrafiltered by tangential flow filtration. Transmission electron micrographs of final sample revealed the presence of particles with the average typical size (150-200nm) and morphology of HIV-1 immature particles. The concentration of the influenza glycoproteins on the Gag-VLPs was estimated by single radial immunodiffusion and hemagglutination assay for HA and by Dot-Blot for HA and NA. More significantly, intranasal immunization of mice with influenza Gag-VLPs induced strong antigen-specific mucosal and systemic antibody responses and provided full protection against a lethal intranasal challenge with the homologous virus strain. These data suggest that, with further optimization and characterization

  10. Detection of neuraminidase stalk motifs associated with enhanced N1 subtype influenza A virulence via Raman spectroscopy.

    PubMed

    Choi, JooYoung; Martin, Sharon J H; Tripp, Ralph A; Tompkins, S Mark; Dluhy, Richard A

    2015-11-21

    Oligonucleotides corresponding to neuraminidase (NA) stalk motifs that have been associated with enhanced influenza virulence have been identified using surface-enhanced Raman spectroscopy (SERS). 5'-Thiolated ssDNA oligonucleotides were immobilized onto a hexadecyltrimethylammonium bromide (CTAB) coated Au nanoparticles (AuNP). Three synthetic RNA sequences corresponding to specific amino acid deletions in the influenza NA stalk region were attached to the CTAB-modified AuNPs. Two of these sequences were specific to sequences with amino acid deletions associated with increased virulence, and one was a low virulence sequence with no amino acid deletions. Hybridization of synthetic matched and mismatched DNA-RNA complexes were detected based on the intrinsic SERS spectra. In addition, this platform was used to analyze RNA sequences isolated from laboratory grown influenza viruses having the NA stalk motif associated with enhanced virulence, including A/WSN/33/H1N1, A/Anhui/1/2005/H5N, and A/Vietnam/1203/2004/H5N1 strains. Multivariate feature selection methods were employed to determine the specific wavenumbers in the Raman spectra that contributed the most information for class discrimination. A one-way analysis of variance (ANOVA) test identified 884 and 1196 wavenumbers as being highly significant in the high and low virulence spectra, respectively (p < 0.01). A post-hoc Tukey Honestly Significance Difference (HSD) test identified the wavenumbers that played a major role in differentiating the DNA-RNA hybrid classes. An estimate of the spectral variability, based on the Wilcoxon rank sum test, found the major source of variation to be predominately between the different classes, and not within the classes, thus confirming that the spectra reflected real class differences and not sampling artifacts. The multivariate classification methods partial least squares discriminant analysis (PLS-DA) and support vector machine discriminant analysis (SVM-DA) were able to

  11. [Drug susceptibility of wild-type and mutant H7N9 neuraminidase to zanamivir and oseltamivir].

    PubMed

    Wei, Yan-Nan; Zhang, Chao; Chen, Qing; Guo, Ying

    2014-07-01

    This study aimed to investigate the drug susceptibility of wild-type and mutant avian influenza A (H7N9) virus neuraminidase (NA) to oseltamivir and zanamivir. Codon optimized DNA of H7N9 (A/ Hangzhou/1/2013) NA was synthesized and constructed into the pcDNA3.1/His vector (NA(H7N9-WT)). Mutant NA(H7N9-H274Y) and NA(H7N9-R292K) plasmids were constructed by directed mutagenesis PCR using NA(H7N9-WT) plasmid as the template followed by sequencing. NA plasmids were transfected into 293T cells and cell lysates containing NAs were collected 48 h post-transfection. Wild-type and mutant NAs were analyzed by Western blotting and their activities were tested by the 4-MUNANA-based assay. All three NAs were expressed and enzymatic activities were confirmed. The effects of oseltamivir and zanamivir on all three NAs were then tested. It showed that the half maximal inhibitory concentrations (IC50s) of oseltamivir carboxylate on NA(H7N9-WT), NA(H7N9-H274Y) and NA(H7N9-R292K) were 1.6 nM, 15.1 nM, and > 1 000 nM with fold changes of 9 and > 625, respectively. The IC50 values of zanamivir on NA(H7N9-WT), NA(H7N9-H274Y), and NA(H7N9-R292K) were 1.1 nM, 1.4 nM, and 38.0 nM with fold changes of 1.3 and 34, respectively. These results indicated that oseltamivir and zanamivir could significantly inhibit NA(H7N9-WT). NA(H7N9-R292K) showed high-level resistance to both drugs (34-fold and 625-fold) and NA(H7N9-H274Y) was sensitive to both (1.3-fold and 9-fold). These results indicated that both oseltamivir and zanamivir could be used for patients infected with the H7N9 virus. However, when patients carried the H7N9 virus with a NA R292K mutation, other medications would be preferred over oseltamivir or zanamivir. PMID:25272593

  12. Roles of the Fusion and Hemagglutinin-Neuraminidase Proteins in Replication, Tropism, and Pathogenicity of Avian Paramyxoviruses▿

    PubMed Central

    Kim, Shin-Hee; Subbiah, Madhuri; Samuel, Arthur S.; Collins, Peter L.; Samal, Siba K.

    2011-01-01

    Virulent and moderately virulent strains of Newcastle disease virus (NDV), representing avian paramyxovirus serotype 1 (APMV-1), cause respiratory and neurological disease in chickens and other species of birds. In contrast, APMV-2 is avirulent in chickens. We investigated the role of the fusion (F) and hemagglutinin-neuraminidase (HN) envelope glycoproteins in these contrasting phenotypes by designing chimeric viruses in which the F and HN glycoproteins or their ectodomains were exchanged individually or together between the moderately virulent, neurotropic NDV strain Beaudette C (BC) and the avirulent APMV-2 strain Yucaipa. When we attempted to exchange the complete F and HN glycoproteins individually and together between the two viruses, the only construct that could be recovered was recombinant APMV-2 strain Yucaipa (rAPMV-2), containing the NDV F glycoprotein in place of its own. This substitution of NDV F into APMV-2 was sufficient to confer the neurotropic, neuroinvasive, and neurovirulent phenotypes, in spite of all being at reduced levels compared to what was seen for NDV-BC. When the ectodomains of F and HN were exchanged individually and together, two constructs could be recovered: NDV, containing both the F and HN ectodomains of APMV-2; and APMV-2, containing both ectodomains of NDV. This supported the idea that homologous cytoplasmic tails and matched F and HN ectodomains are important for virus replication. Analysis of these viruses for replication in vitro, syncytium formation, mean embryo death time, intracerebral pathogenicity index, and replication and tropism in 1-day-old chicks and 2-week-old chickens showed that the two contrasting phenotypes of NDV and APMV-2 could largely be transferred between the two backbones by transfer of homotypic F and HN ectodomains. Further analysis provided evidence that the homologous stalk domain of NDV HN is essential for virus replication, while the globular head domain of NDV HN could be replaced with that of

  13. Detection of neuraminidase stalk motifs associated with enhanced N1 subtype influenza A virulence via Raman spectroscopy.

    PubMed

    Choi, JooYoung; Martin, Sharon J H; Tripp, Ralph A; Tompkins, S Mark; Dluhy, Richard A

    2015-11-21

    Oligonucleotides corresponding to neuraminidase (NA) stalk motifs that have been associated with enhanced influenza virulence have been identified using surface-enhanced Raman spectroscopy (SERS). 5'-Thiolated ssDNA oligonucleotides were immobilized onto a hexadecyltrimethylammonium bromide (CTAB) coated Au nanoparticles (AuNP). Three synthetic RNA sequences corresponding to specific amino acid deletions in the influenza NA stalk region were attached to the CTAB-modified AuNPs. Two of these sequences were specific to sequences with amino acid deletions associated with increased virulence, and one was a low virulence sequence with no amino acid deletions. Hybridization of synthetic matched and mismatched DNA-RNA complexes were detected based on the intrinsic SERS spectra. In addition, this platform was used to analyze RNA sequences isolated from laboratory grown influenza viruses having the NA stalk motif associated with enhanced virulence, including A/WSN/33/H1N1, A/Anhui/1/2005/H5N, and A/Vietnam/1203/2004/H5N1 strains. Multivariate feature selection methods were employed to determine the specific wavenumbers in the Raman spectra that contributed the most information for class discrimination. A one-way analysis of variance (ANOVA) test identified 884 and 1196 wavenumbers as being highly significant in the high and low virulence spectra, respectively (p < 0.01). A post-hoc Tukey Honestly Significance Difference (HSD) test identified the wavenumbers that played a major role in differentiating the DNA-RNA hybrid classes. An estimate of the spectral variability, based on the Wilcoxon rank sum test, found the major source of variation to be predominately between the different classes, and not within the classes, thus confirming that the spectra reflected real class differences and not sampling artifacts. The multivariate classification methods partial least squares discriminant analysis (PLS-DA) and support vector machine discriminant analysis (SVM-DA) were able to

  14. Effects of Water Models on Binding Affinity: Evidence from All-Atom Simulation of Binding of Tamiflu to A/H5N1 Neuraminidase

    PubMed Central

    Nguyen, Trang Truc; Viet, Man Hoang

    2014-01-01

    The influence of water models SPC, SPC/E, TIP3P, and TIP4P on ligand binding affinity is examined by calculating the binding free energy ΔGbind of oseltamivir carboxylate (Tamiflu) to the wild type of glycoprotein neuraminidase from the pandemic A/H5N1 virus. ΔGbind is estimated by the Molecular Mechanic-Poisson Boltzmann Surface Area method and all-atom simulations with different combinations of these aqueous models and four force fields AMBER99SB, CHARMM27, GROMOS96 43a1, and OPLS-AA/L. It is shown that there is no correlation between the binding free energy and the water density in the binding pocket in CHARMM. However, for three remaining force fields ΔGbind decays with increase of water density. SPC/E provides the lowest binding free energy for any force field, while the water effect is the most pronounced in CHARMM. In agreement with the popular GROMACS recommendation, the binding score obtained by combinations of AMBER-TIP3P, OPLS-TIP4P, and GROMOS-SPC is the most relevant to the experiments. For wild-type neuraminidase we have found that SPC is more suitable for CHARMM than TIP3P recommended by GROMACS for studying ligand binding. However, our study for three of its mutants reveals that TIP3P is presumably the best choice for CHARMM. PMID:24672329

  15. Optimization of an enzyme-linked lectin assay suitable for rapid antigenic characterization of the neuraminidase of human influenza A(H3N2) viruses

    PubMed Central

    Westgeest, Kim; Bestebroer, Theo M.; Spronken, Monique I.J.; Gao, Jin; Couzens, Laura; Osterhaus, Albert D.M.E.; Eichelberger, Maryna; Fouchier, Ron A.M.; de Graaf, Miranda

    2015-01-01

    Antibodies to neuraminidase (NA), the second most abundant surface protein of the influenza virus, contribute to protection against influenza virus infection. Although traditional and miniaturized thiobarbituric acid (TBA) neuraminidase inhibition (NI) assays have been successfully used to characterize the antigenic properties of NA, these methods are cumbersome and not easily amendable to rapid screening. An additional difficulty of the NI assay is the interference by hemagglutinin (HA)-specific antibodies. To prevent interference of HA-specific antibodies, most NI assays are performed with recombinant viruses containing a mismatched HA. However, generation of these viruses is time consuming and unsuitable for large-scale surveillance. The feasibility of using the recently developed enzyme-linked lectin assay (ELLA) to evaluate the antigenic relatedness of NA of wild type A(H3N2) viruses was assessed. Rather than using recombinant viruses, wild type A(H3N2) viruses were used as antigen with ferret sera elicited against recombinant viruses with a mismatched HA. In this study, details of the critical steps that are needed to modify and optimize the NI ELLA in a format that is reproducible, highly sensitive, and useful for influenza virus surveillance to monitor antigenic drift of NA are provided. PMID:25712563

  16. Optimization of an enzyme-linked lectin assay suitable for rapid antigenic characterization of the neuraminidase of human influenza A(H3N2) viruses.

    PubMed

    Westgeest, Kim B; Bestebroer, Theo M; Spronken, Monique I J; Gao, Jin; Couzens, Laura; Osterhaus, Albert D M E; Eichelberger, Maryna; Fouchier, Ron A M; de Graaf, Miranda

    2015-06-01

    Antibodies to neuraminidase (NA), the second most abundant surface protein of the influenza virus, contribute to protection against influenza virus infection. Although traditional and miniaturized thiobarbituric acid (TBA) neuraminidase inhibition (NI) assays have been successfully used to characterize the antigenic properties of NA, these methods are cumbersome and not easily amendable to rapid screening. An additional difficulty of the NI assay is the interference by hemagglutinin (HA)-specific antibodies. To prevent interference of HA-specific antibodies, most NI assays are performed with recombinant viruses containing a mismatched HA. However, generation of these viruses is time consuming and unsuitable for large-scale surveillance. The feasibility of using the recently developed enzyme-linked lectin assay (ELLA) to evaluate the antigenic relatedness of NA of wild type A(H3N2) viruses was assessed. Rather than using recombinant viruses, wild type A(H3N2) viruses were used as antigen with ferret sera elicited against recombinant viruses with a mismatched HA. In this study, details of the critical steps that are needed to modify and optimize the NI ELLA in a format that is reproducible, highly sensitive, and useful for influenza virus surveillance to monitor antigenic drift of NA are provided.

  17. Synthetic Mucus Nanobarriers for Identification of Glycan-Dependent Primary Influenza A Infection Inhibitors

    PubMed Central

    2016-01-01

    Current drugs against the influenza A virus (IAV) act by inhibiting viral neuraminidase (NA) enzymes responsible for the release of budding virions from sialoglycans on infected cells. Here, we describe an approach focused on a search for inhibitors that reinforce the protective functions of mucosal barriers that trap viruses en route to the target cells. We have generated mimetics of sialo-glycoproteins that insert into the viral envelope to provide a well-defined mucus-like environment encapsulating the virus. By introducing this barrier, which the virus must breach using its NA enzymes to infect a host cell, into a screening platform, we have been able to identify compounds that provide significant protection against IAV infection. This approach may facilitate the discovery of potent new IAV prophylactics among compounds with NA activities too weak to emerge from traditional drug screens. PMID:27800553

  18. Proton pump inhibitors

    MedlinePlus

    Proton pump inhibitors (PPIs) are medicines that work by reducing the amount of stomach acid made by ... Proton pump inhibitors are used to: Relieve symptoms of acid reflux, or gastroesophageal reflux disease (GERD). This ...

  19. Inactivated Seasonal Influenza Vaccines Increase Serum Antibodies to the Neuraminidase of Pandemic Influenza A(H1N1) 2009 Virus in an Age-Dependent Manner

    PubMed Central

    Marcelin, Glendie; Bland, Hilliary M.; Negovetich, Nicholas J.; Sandbulte, Matthew R.; Ellebedy, Ali H.; Webb, Ashley D.; Griffin, Yolanda S.; DeBeauchamp, Jennifer L.; McElhaney, Janet E.; Webby, Richard J.

    2010-01-01

    Levels of preexisting antibodies to the hemagglutinin of pandemic influenza A(H1N1) 2009 (hereafter pandemic H1N1) virus positively correlate with age. The impact of contemporary seasonal influenza vaccines on establishing immunity to other pandemic H1N1 proteins is unknown. We measured serum antibodies to the neuraminidase (NA) of pandemic H1N1 in adults prior to and after vaccination with seasonal trivalent inactivated influenza vaccines. Serum antibodies to pandemic H1N1 NA were observed in all age groups; however, vaccination elevated levels of pandemic H1N1 NA antibodies predominately in elderly individuals (age,⩾60 years). Therefore, contemporary seasonal vaccines likely contribute to reduction of pandemic H1N1-associated disease in older individuals. PMID:20979454

  20. The interaction of selective plant lectins with neuraminidase-treated and untreated IgA1 from the sera of IgA nephropathy patients.

    PubMed

    Hashim, O H; Shuib, A S; Chua, C T

    2001-02-01

    A study on the binding interaction of lectins from Artocarpus heterophyllus (jacalin), Glycine max and Sambucus nigra with standardised quantity of IgA from the IgA nephropathy patients and normal controls was performed. The Glycine max lectin demonstrated higher affinity towards the serum IgA of IgAN patients as compared to normal controls. However, the affinity binding was lower in cases ofjacalin and the Sambucus nigra lectin. When serum samples were treated with neuraminidase, the differential jacalin affinity binding between IgA1 of patients and normal controls was abrogated. Our data are in support of the view that the O-linked oligosaccharide moieties of the patients IgA1 were generally lacking in galactose and sialic acid residues.

  1. Filament-producing mutants of influenza A/Puerto Rico/8/1934 (H1N1) virus have higher neuraminidase activities than the spherical wild-type.

    PubMed

    Seladi-Schulman, Jill; Campbell, Patricia J; Suppiah, Suganthi; Steel, John; Lowen, Anice C

    2014-01-01

    Influenza virus exhibits two morphologies - spherical and filamentous. Strains that have been grown extensively in laboratory substrates are comprised predominantly of spherical virions while clinical or low passage isolates produce a mixture of spheres and filamentous virions of varying lengths. The filamentous morphology can be lost upon continued passage in embryonated chicken eggs, a common laboratory substrate for influenza viruses. The fact that the filamentous morphology is maintained in nature but lost in favor of a spherical morphology in ovo suggests that filaments confer a selective advantage within the infected host that is not necessary for growth in laboratory substrates. Indeed, we have recently shown that filament-producing variant viruses are selected upon passage of the spherical laboratory strain A/Puerto Rico/8/1934 (H1N1) [PR8] in guinea pigs. Toward determining the nature of the selective advantage conferred by filaments, we sought to identify functional differences between spherical and filamentous particles. We compared the wild-type PR8 virus to two previously characterized recombinant PR8 viruses in which single point mutations within M1 confer a filamentous morphology. Our results indicate that these filamentous PR8 mutants have higher neuraminidase activities than the spherical PR8 virus. Conversely, no differences were observed in HAU:PFU or HAU:RNA ratios, binding avidity, sensitivity to immune serum in hemagglutination inhibition assays, or virion stability at elevated temperatures. Based on these results, we propose that the pleomorphic nature of influenza virus particles is important for the optimization of neuraminidase functions in vivo.

  2. Filament-Producing Mutants of Influenza A/Puerto Rico/8/1934 (H1N1) Virus Have Higher Neuraminidase Activities than the Spherical Wild-Type

    PubMed Central

    Seladi-Schulman, Jill; Campbell, Patricia J.; Suppiah, Suganthi; Steel, John; Lowen, Anice C.

    2014-01-01

    Influenza virus exhibits two morphologies – spherical and filamentous. Strains that have been grown extensively in laboratory substrates are comprised predominantly of spherical virions while clinical or low passage isolates produce a mixture of spheres and filamentous virions of varying lengths. The filamentous morphology can be lost upon continued passage in embryonated chicken eggs, a common laboratory substrate for influenza viruses. The fact that the filamentous morphology is maintained in nature but lost in favor of a spherical morphology in ovo suggests that filaments confer a selective advantage within the infected host that is not necessary for growth in laboratory substrates. Indeed, we have recently shown that filament-producing variant viruses are selected upon passage of the spherical laboratory strain A/Puerto Rico/8/1934 (H1N1) [PR8] in guinea pigs. Toward determining the nature of the selective advantage conferred by filaments, we sought to identify functional differences between spherical and filamentous particles. We compared the wild-type PR8 virus to two previously characterized recombinant PR8 viruses in which single point mutations within M1 confer a filamentous morphology. Our results indicate that these filamentous PR8 mutants have higher neuraminidase activities than the spherical PR8 virus. Conversely, no differences were observed in HAU:PFU or HAU:RNA ratios, binding avidity, sensitivity to immune serum in hemagglutination inhibition assays, or virion stability at elevated temperatures. Based on these results, we propose that the pleomorphic nature of influenza virus particles is important for the optimization of neuraminidase functions in vivo. PMID:25383873

  3. In situ molecular identification of the Influenza A (H1N1) 2009 Neuraminidase in patients with severe and fatal infections during a pandemic in Mexico City

    PubMed Central

    2013-01-01

    Background In April 2009, public health surveillance detected an increased number of influenza-like illnesses in Mexico City’s hospitals. The etiological agent was subsequently determined to be a spread of a worldwide novel influenza A (H1N1) triple reassortant. The purpose of the present study was to demonstrate that molecular detection of pandemic influenza A (H1N1) 2009 strains is possible in archival material such as paraffin-embedded lung samples. Methods In order to detect A (H1N1) virus sequences in archived biological samples, eight paraffin-embedded lung samples from patients who died of pneumonia and respiratory failure were tested for influenza A (H1N1) Neuraminidase (NA) RNA using in situ RT-PCR. Results We detected NA transcripts in 100% of the previously diagnosed A (H1N1)-positive samples as a cytoplasmic signal. No expression was detected by in situ RT-PCR in two Influenza-like Illness A (H1N1)-negative patients using standard protocols nor in a non-related cervical cell line. In situ relative transcription levels correlated with those obtained when in vitro RT-PCR assays were performed. Partial sequences of the NA gene from A (H1N1)-positive patients were obtained by the in situ RT-PCR-sequencing method. Sequence analysis showed 98% similarity with influenza viruses reported previously in other places. Conclusions We have successfully amplified specific influenza A (H1N1) NA sequences using stored clinical material; results suggest that this strategy could be useful when clinical RNA samples are quantity limited, or when poor quality is obtained. Here, we provide a very sensitive method that specifically detects the neuraminidase viral RNA in lung samples from patients who died from pneumonia caused by Influenza A (H1N1) outbreak in Mexico City. PMID:23327529

  4. Comparability of neuraminidase inhibition antibody titers measured by enzyme-linked lectin assay (ELLA) for the analysis of influenza vaccine immunogenicity.

    PubMed

    Eichelberger, Maryna C; Couzens, Laura; Gao, Yonghong; Levine, Min; Katz, Jacqueline; Wagner, Ralf; Thompson, Catherine I; Höschler, Katja; Laurie, Karen; Bai, Tian; Engelhardt, Othmar G; Wood, John

    2016-01-20

    Neuraminidase-inhibition (NI) antibody titers can be used to evaluate the immunogenicity of inactivated influenza vaccines and have provided evidence of serologic cross-reactivity between seasonal and pandemic H1N1 viruses. The traditional thiobarbituric acid assay is impractical for large serologic analyses, and therefore many laboratories use an enzyme-linked lectin assay (ELLA) to determine serum NI antibody titers. The comparability of ELLA NI antibody titers when measured in different laboratories was unknown. Here we report a study conducted through the Consortium for the Standardisation of Influenza SeroEpidemiology (CONSISE) to evaluate the variability of the ELLA. NI antibody titers of a set of 12 samples were measured against both N1 and N2 neuraminidase antigens in 3 independent assays by each of 23 laboratories. For a sample repeated in the same assay, ≥96% of N1 and N2 assays had less than a 4-fold difference in titer. Comparison of the titers measured in assays conducted on 3 different days in the same laboratory showed that a four-fold difference in titer was uncommon. Titers of the same sera measured in different laboratories spanned 3 to 6 two-fold dilutions (i.e., 8-64 fold difference in titer), with an average percent geometric coefficient of variation (%GCV) of 112 and 82% against N1 and N2 antigens, respectively. The difference in titer as indicated by fold range and %GCV was improved by normalizing the NI titers to a standard that was included in each assay. This study identified background signal and the amount of antigen in the assay as critical factors that influence titer, providing important information toward development of a consensus ELLA protocol.

  5. A new in vitro hemagglutinin inhibitor screening system based on a single-vesicle fusion assay

    PubMed Central

    Lee, Hanki; Jin, Wook; Jeong, Byeong-Chul; Suh, Joo-Won

    2016-01-01

    Hemagglutinin (HA) from the influenza virus plays a pivotal role in the infection of host mammalian cells and is, therefore, a druggable target, similar to neuraminidase. However, research involving the influenza virus must be conducted in facilities certified at or above Biosafety Level 2 because of the potential threat of the contagiousness of this virus. To develop a new HA inhibitor screening system without intact influenza virus, we conceived a single-vesicle fusion assay using full-length recombinant HA. In this study, we first showed that full-length recombinant HA can mediate membrane fusion in ensemble and single-vesicle fusion assays. The fluorescence resonance energy transfer (FRET) frequency pattern of single-vesicle complexes completely differed when the inhibitors targeted the HA1 or HA2 domain of HA. This result indicates that analysing the FRET patterns in this assay can provide information regarding the domains of HA inhibited by compounds and compounds’ inhibitory activities. Therefore, our results suggest that the assay developed here is a promising tool for the discovery of anti-influenza virus drug candidates as a new in vitro inhibitor screening system against HA from the influenza virus. PMID:27469068

  6. Novel corrosion inhibitor technology

    SciTech Connect

    Van de Ven, P.; Fritz, P.; Pellet, R.

    1999-11-01

    A novel, patented corrosion inhibitor technology has been identified for use in heat transfer applications such as automotive and heavy-duty coolant. The new technology is based on a low-toxic, virtually depletion-free carboxylic acid corrosion inhibitor package that performs equally well in mono ethylene glycol and in less toxic propylene glycol coolants. An aqueous inhibitor concentrate is available to provide corrosion protection where freezing protection is not an issue. In the present paper, this inhibitor package is evaluated in the different base fluids: mono ethylene glycol, mono propylene glycol and water. Results are obtained in both standardized and specific corrosion tests as well as in selected field trials. These results indicate that the inhibitor package remains effective and retains the benefits previously identified in automotive engine coolant applications: excellent corrosion protection under localized conditions, general corrosion conditions as well as at high temperature.

  7. Production of an enzymatically active and immunogenic form of ectodomain of Porcine rubulavirus hemagglutinin-neuraminidase in the yeast Pichia pastoris.

    PubMed

    Cerriteño-Sánchez, José Luis; Santos-López, Gerardo; Rosas-Murrieta, Nora Hilda; Reyes-Leyva, Julio; Cuevas-Romero, Sandra; Herrera-Camacho, Irma

    2016-04-10

    Blue-eye disease (BED) of swine is a viral disease endemic in Mexico. The etiological agent is a paramyxovirus classified as Porcine rubulavirus (PoRV-LPMV), which exhibits in its envelope the hemagglutinin-neuraminidase (HN) glycoprotein, the most immunogenic and a major target for vaccine development. We report in this study the obtaining of ectodomain of PoRV HN (eHN) through the Pichia pastoris expression system. The expression vector (pPICZαB-HN) was integrated by displacement into the yeast chromosome and resulted in a Mut(+) phenotype. Expressed eHN in the P. pastoris X33 strain was recovered from cell-free medium, featuring up to 67 nmol/min/mg after 6 days of expression. eHN was recognized by the serum of infected pigs with strains currently circulating in the Mexican Bajio region. eHN induces antibodies in mice after 28 days of immunization with specific recognition in ELISA test. These antibodies were able to inhibit >80% replication by viral neutralization assays in cell culture. These studies show the obtaining of a protein with similar characteristics to the native HN and which may be a candidate to propose a vaccine or to use the antigen in a serologic diagnostic test.

  8. Amino acid substitutions in the neuraminidase protein of an H9N2 avian influenza virus affect its airborne transmission in chickens.

    PubMed

    Lv, Jing; Wei, Liangmeng; Yang, Yan; Wang, Bingxiao; Liang, Wei; Gao, Yuwei; Xia, Xianzhu; Gao, Lili; Cai, Yumei; Hou, Peiqiang; Yang, Huili; Wang, Airong; Huang, Rong; Gao, Jing; Chai, Tongjie

    2015-01-01

    Cases of H9N2 avian influenza virus (AIV) in poultry are increasing throughout many Eurasian countries, and co-infections with other pathogens have resulted in high morbidity and mortality in poultry. Few studies have investigated the genetic factors of virus airborne transmission which determine the scope of this epidemic. In this study, we used specific-pathogen-free chickens housed in isolators to investigate the airborne transmissibility of five recombinant H9N2 AIV rescued by reverse genetic technology. The results show that airborne transmission of A/Chicken/Shandong/01/2008 (SD01) virus was related to the neuraminidase (NA) gene, and four amino acid mutations (D368E, S370L, E313K and G381D) within the head region of the SD01 NA, reduced virus replication in the respiratory tract of chickens, reduced virus NA activity, and resulted in a loss of airborne transmission ability in chickens. Similarly, reverse mutations of these four amino acids in the NA protein of r01/NASS virus, conferred an airborne transmission ability to the recombinant virus. We conclude that these four NA residues may be significant genetic markers for evaluating potential disease outbreak of H9N2 AIV, and propose that immediate attention should be paid to the airborne transmission of this virus. PMID:25928577

  9. Combined quantum mechanics/molecular mechanics (QM/MM) simulations for protein-ligand complexes: free energies of binding of water molecules in influenza neuraminidase.

    PubMed

    Woods, Christopher J; Shaw, Katherine E; Mulholland, Adrian J

    2015-01-22

    The applicability of combined quantum mechanics/molecular mechanics (QM/MM) methods for the calculation of absolute binding free energies of conserved water molecules in protein/ligand complexes is demonstrated. Here, we apply QM/MM Monte Carlo simulations to investigate binding of water molecules to influenza neuraminidase. We investigate five different complexes, including those with the drugs oseltamivir and peramivir. We investigate water molecules in two different environments, one more hydrophobic and one hydrophilic. We calculate the free-energy change for perturbation of a QM to MM representation of the bound water molecule. The calculations are performed at the BLYP/aVDZ (QM) and TIP4P (MM) levels of theory, which we have previously demonstrated to be consistent with one another for QM/MM modeling. The results show that the QM to MM perturbation is significant in both environments (greater than 1 kcal mol(-1)) and larger in the more hydrophilic site. Comparison with the same perturbation in bulk water shows that this makes a contribution to binding. The results quantify how electronic polarization differences in different environments affect binding affinity and also demonstrate that extensive, converged QM/MM free-energy simulations, with good levels of QM theory, are now practical for protein/ligand complexes.

  10. Preparation and diagnostic utility of a hemagglutination inhibition test antigen derived from the baculovirus-expressed hemagglutinin-neuraminidase protein gene of Newcastle disease virus.

    PubMed

    Choi, Kang-Seuk; Kye, Soo-Jeong; Jeon, Woo-Jin; Park, Mi-Ja; Kim, Saeromi; Seul, Hee-Jung; Kwon, Jun-Hun

    2013-01-01

    A recombinant hemagglutinin-neuraminidase (rHN) protein from Newcastle disease virus (NDV) with hemagglutination (HA) activity was expressed in Spodoptera frugiperda cells using a baculovirus expression system. The rHN protein extracted from infected cells was used as an antigen in a hemagglutination inhibition (HI) test for the detection and titration of NDV-specific antibodies present in chicken sera. The rHN antigen produced high HA titers of 2(13) per 25 μL, which were similar to those of the NDV antigen produced using chicken eggs, and it remained stable without significant loss of the HA activity for at least 12 weeks at 4°C. The rHN-based HI assay specifically detected NDV antibodies, but not the sera of other avian pathogens, with a specificity and sensitivity of 100% and 98.0%, respectively, in known positive and negative chicken sera (n = 430). Compared with an NDV-based HI assay, the rHN-based HI assay had a relative sensitivity and specificity of 96.1% and 95.5%, respectively, when applied to field chicken sera. The HI titers of the rHN-based HI assay were highly correlated with those in an NDV-based HI assay (r = 0.927). Overall, these results indicate that rHN protein provides a useful alternative to NDV antigen in HI assays.

  11. Restricted Infectivity of a Human-Lineage H3N2 Influenza A Virus in Pigs Is Hemagglutinin and Neuraminidase Gene Dependent

    PubMed Central

    Landolt, Gabriele A.; Karasin, Alexander I.; Schutten, Melissa M.; Olsen, Christopher W.

    2006-01-01

    Influenza A viruses cause pandemics at sporadic intervals. Pandemic viruses can potentially be introduced into the human population through in toto transfer of an avian influenza virus or through reassortment between avian and human strains. Pigs are believed to play a central role in the creation of pandemic viruses through reassortment because of their susceptibility to infection with both avian and human influenza viruses. However, we recently found that a human-lineage H3N2 influenza virus was highly restricted in its ability to infect pigs after intranasal inoculation. We hypothesized that this restricted infectivity phenotype was controlled by the hemagglutinin (HA) and neuraminidase (NA). To test this, we infected pigs with reverse genetics-created HA plus NA reassortant viruses. Specifically, introduction of the HA and NA genes of a contemporary H3N2 swine virus into the genetic background of the wholly human virus resulted in a significant increase in virus shedding and pathogenicity. These data indicate that the HA/NA can play important roles in controlling human influenza virus infectivity in pigs. The results further support the premise that a barrier exists to human influenza virus infection in pigs, which may limit the role of pigs in pandemic virus creation through reassortment of human and avian influenza viruses. PMID:16455873

  12. Expression and Characterization of Recombinant, Tetrameric and Enzymatically Active Influenza Neuraminidase for the Setup of an Enzyme-Linked Lectin-Based Assay

    PubMed Central

    Prevato, Marua; Ferlenghi, Ilaria; Bonci, Alessandra; Uematsu, Yasushi; Anselmi, Giulia; Giusti, Fabiola; Bertholet, Sylvie; Legay, Francois; Telford, John Laird; Settembre, Ethan C.; Maione, Domenico; Cozzi, Roberta

    2015-01-01

    Developing a universal influenza vaccine that induces broad spectrum and longer-term immunity has become an important potentially achievable target in influenza vaccine research and development. Hemagglutinin (HA) and neuraminidase (NA) are the two major influenza virus antigens. Although antibody responses against influenza virus are mainly directed toward HA, NA is reported to be more genetically stable; hence NA-based vaccines have the potential to be effective for longer time periods. NA-specific immunity has been shown to limit the spread of influenza virus, thus reducing disease symptoms and providing cross-protection against heterosubtypic viruses in mouse challenge experiments. The production of large quantities of highly pure and stable NA could be beneficial for the development of new antivirals, subunit-based vaccines, and novel diagnostic tools. In this study, recombinant NA (rNA) was produced in mammalian cells at high levels from both swine A/California/07/2009 (H1N1) and avian A/turkey/Turkey/01/2005 (H5N1) influenza viruses. Biochemical, structural, and immunological characterizations revealed that the soluble rNAs produced are tetrameric, enzymatically active and immunogenic, and finally they represent good alternatives to conventionally used sources of NA in the Enzyme-Linked Lectin Assay (ELLA). PMID:26280677

  13. Thio-ketosides of sialic acid containing aryl azides: potential photo-affinity probes for analysis of neuraminidases and sialic acid binding proteins

    SciTech Connect

    Warner, T.G.; Lee, L.A.

    1986-05-01

    To date, only a single report describing the synthesis of thio-ketosides of sialic acid has appeared. In this procedure, the pseudo thiourea of acetoneuraminic acid methyl ester (NTU) was used to prepare the sodium thiolate salt. However, in their hands, the preparation of NTU was not straight-forward, and in subsequent reactions thio glycosides were not obtained. Therefore, they have developed an alternate route for introduction of the sulfhydryl group and have prepared novel thio-ketosides with aryl azides. The thio linkage is advantageous since it is not easily cleaved by neuraminidases and it allows incorporation of /sup 35/S as a convenient radioactive label. 2-deoxy-2-S-acetyl-4,7,8,9,- tetra-0-acetyl-N-acetyl neuraminic acid methyl ester was prepared (70% yield) from 2-chloro aceto- neuraminic acid methyl ester and potassium thioacetate in acetone at room temperature (RT) for 90 min. Selective hydrolysis of the thio acetate group was accomplished with equimolar sodium methoxide in DMF. After 10 min at RT, 4-fluoro-3-nitrophenyl azide was added and reaction continued for 60 min. Silicic acid purification, base hydrolysis, and gel filtration chromatography, gave 2'-deoxy-2'-(2-nitro-4-azido-thiophenyl)-..cap alpha..-D-N-acetyl neuraminic acid (35% yield). Other thio-arylazido ketosides were prepared similarly.

  14. Permissive changes in the neuraminidase play a dominant role in improving the viral fitness of oseltamivir-resistant seasonal influenza A(H1N1) strains.

    PubMed

    Abed, Yacine; Pizzorno, Andrés; Bouhy, Xavier; Boivin, Guy

    2015-02-01

    Permissive neuraminidase (NA) substitutions such as R222Q, V234M and D344N have facilitated the emergence and worldwide spread of oseltamivir-resistant influenza A/Brisbane/59/2007 (H1N1)-H275Y viruses. However, the potential contribution of genetic changes in other viral segments on viral fitness remains poorly investigated. A series of recombinant A(H1N1)pdm09 and A/WSN/33 7:1 reassortants containing the wild-type (WT) A/Brisbane/59/2007 NA gene or its single (H275Y) and double (H275Y/Q222R, H275Y/M234V and H275Y/N344D) variants were generated and their replicative properties were assessed in vitro. The Q222R reversion substitution significantly reduced viral titers when evaluated in both A(H1N1)pdm09 and A/WSN/33 backgrounds. The permissive role of the R222Q was further confirmed using A/WSN/33 7:1 reassortants containing the NA gene of the oseltamivir-susceptible or oseltamivir-resistant influenza A/Mississippi/03/2001 strains. Therefore, NA permissive substitutions play a dominant role for improving viral replication of oseltamivir-resistant A (H1N1)-H275Y viruses in vitro.

  15. Preclinical activity of VX-787, a first-in-class, orally bioavailable inhibitor of the influenza virus polymerase PB2 subunit.

    PubMed

    Byrn, Randal A; Jones, Steven M; Bennett, Hamilton B; Bral, Chris; Clark, Michael P; Jacobs, Marc D; Kwong, Ann D; Ledeboer, Mark W; Leeman, Joshua R; McNeil, Colleen F; Murcko, Mark A; Nezami, Azin; Perola, Emanuele; Rijnbrand, Rene; Saxena, Kumkum; Tsai, Alice W; Zhou, Yi; Charifson, Paul S

    2015-03-01

    VX-787 is a novel inhibitor of influenza virus replication that blocks the PB2 cap-snatching activity of the influenza viral polymerase complex. Viral genetics and X-ray crystallography studies provide support for the idea that VX-787 occupies the 7-methyl GTP (m(7)GTP) cap-binding site of PB2. VX-787 binds the cap-binding domain of the PB2 subunit with a KD (dissociation constant) of 24 nM as determined by isothermal titration calorimetry (ITC). The cell-based EC50 (the concentration of compound that ensures 50% cell viability of an uninfected control) for VX-787 is 1.6 nM in a cytopathic effect (CPE) assay, with a similar EC50 in a viral RNA replication assay. VX-787 is active against a diverse panel of influenza A virus strains, including H1N1pdm09 and H5N1 strains, as well as strains with reduced susceptibility to neuraminidase inhibitors (NAIs). VX-787 was highly efficacious in both prophylaxis and treatment models of mouse influenza and was superior to the neuraminidase inhibitor, oseltamivir, including in delayed-start-to-treat experiments, with 100% survival at up to 96 h postinfection and partial survival in groups where the initiation of therapy was delayed up to 120 h postinfection. At different doses, VX-787 showed a 1-log to >5-log reduction in viral load (relative to vehicle controls) in mouse lungs. Overall, these favorable findings validate the PB2 subunit of the viral polymerase as a drug target for influenza therapy and support the continued development of VX-787 as a novel antiviral agent for the treatment of influenza infection.

  16. A Balance between Inhibitor Binding and Substrate Processing Confers Influenza Drug Resistance.

    PubMed

    Jiang, Li; Liu, Ping; Bank, Claudia; Renzette, Nicholas; Prachanronarong, Kristina; Yilmaz, Lutfu S; Caffrey, Daniel R; Zeldovich, Konstantin B; Schiffer, Celia A; Kowalik, Timothy F; Jensen, Jeffrey D; Finberg, Robert W; Wang, Jennifer P; Bolon, Daniel N A

    2016-02-13

    The therapeutic benefits of the neuraminidase (NA) inhibitor oseltamivir are dampened by the emergence of drug resistance mutations in influenza A virus (IAV). To investigate the mechanistic features that underlie resistance, we developed an approach to quantify the effects of all possible single-nucleotide substitutions introduced into important regions of NA. We determined the experimental fitness effects of 450 nucleotide mutations encoding positions both surrounding the active site and at more distant sites in an N1 strain of IAV in the presence and absence of oseltamivir. NA mutations previously known to confer oseltamivir resistance in N1 strains, including H275Y and N295S, were adaptive in the presence of drug, indicating that our experimental system captured salient features of real-world selection pressures acting on NA. We identified mutations, including several at position 223, that reduce the apparent affinity for oseltamivir in vitro. Position 223 of NA is located adjacent to a hydrophobic portion of oseltamivir that is chemically distinct from the substrate, making it a hotspot for substitutions that preferentially impact drug binding relative to substrate processing. Furthermore, two NA mutations, K221N and Y276F, each reduce susceptibility to oseltamivir by increasing NA activity without altering drug binding. These results indicate that competitive expansion of IAV in the face of drug pressure is mediated by a balance between inhibitor binding and substrate processing.

  17. Tolerability, safety, and pharmacokinetics of the novel cathepsin A inhibitor SAR164653 in healthy subjects.

    PubMed

    Tillner, Joachim; Lehmann, Anne; Paehler, Tobias; Lukacs, Zoltan; Ruf, Sven; Sadowski, Thorsten; Pinquier, Jean-Louis; Ruetten, Hartmut

    2016-01-01

    Cathepsin A (CathA) is a lysosomal protein where it forms a stable complex with neuraminidase and ß-galactosidase. CathA also has enzymatic activity and is involved in the degradation of many peptides. CathA was recently discovered as a target for heart failure, fostering the development of CathA inhibitors with SAR164653 as a frontrunner. The first-in-man study investigated single oral doses from 20 to 800 mg of SAR164653 followed by repeat dose studies at doses up to 800 mg in healthy young and elderly subjects. SAR164653 was safe and well tolerated at doses up to 800 mg in healthy subjects, and a maximum tolerated dose could not be determined from the study. Activity of ß-galactosidase measured in leukocytes did not show any abnormalities. The tmax was 1.0 to 2.5 hours, and the t1/2 was ∼5-11 after single dosing; exposure increased less than dose proportional. Following multiple dosing, accumulation was not observed, Cmax and AUC0-24 increased in a dose-proportional manner, and t1/2 was around 14-20 hours. The novel CathA inhibitor SAR164653 was found to have a favorable safety profile in these early phase 1 studies, but further studies are required to confirm if SAR164653 is equally safe in patients undergoing long-term treatment. PMID:27119579

  18. Pathway modulators and inhibitors.

    PubMed

    Smith, John A

    2009-07-01

    Inhibitors of specific cellular pathways are useful for investigating the roles of proteins of unknown function, and for selectively inhibiting a protein in complex pathways to uncover its relationships to other proteins in this and other interacting pathways. This appendix provides links to Web sites that describe cellular processes and pathways along with the various classes of inhibitors, numerous references, downloadable diagrams, and technical tips.

  19. Update on TNF Inhibitors.

    PubMed

    Kerdel, Francisco A

    2016-06-01

    The introduction of tumor necrosis factor (TNF)-α inhibitors dramatically improved the management of psoriasis. Some newer or investigational biologics with different mechanisms of action have demonstrated noninferiority or superiority to etanercept, the first self-injectable anti-TNF-α agent to become available in the United States. Nonetheless, TNF-α inhibitors are likely to remain a mainstay of therapy for many years.

  20. Synthetic inhibitors of elastase.

    PubMed

    Edwards, P D; Bernstein, P R

    1994-03-01

    For more than two decades investigators around the world, in both academic and industrial institutions, have been developing inhibitors of human neutrophil elastase. A number of very elegant and insightful strategies have been reported. In the case of reversible peptidic inhibitors, this has resulted in the identification of some extremely potent compounds with dissociation constants in the 10(-11) M range. This is quite an accomplishment considering that these low molecular-weight inhibitors are only tri- and tetrapeptides. In the case of the heterocyclic-based inhibitors, the challenge of balancing the heterocycle's inherent reactivity and aqueous stability with the stability of the enzyme-inhibitor adduct has been meet by either using a latent, reactive functionality which is only activated within the enzyme, or by incorporating features which selectively obstruct deacylation but have little effect on the enzyme acylation step. The underlying goal of this research has been the identification of agents to treat diseases associated with HNE. Several animal models have been developed for evaluating the in vivo activity of elastase inhibitors, and compounds have been shown to be effective in all of these models by the intravenous, intratrachael or oral routes of administration. However, only a very small percentage of compounds have possessed all the necessary properties, including lack of toxicity, for progression into the clinic. The peptidyl TFMK ICI 200,880 (25-12) has many of the desired characteristics of a drug to treat the diseases associated with HNE: chemical stability, in vitro and in vivo activity, a long duration of action, and adequate metabolic stability. Currently ICI 200,880 is the only low molecular-weight HNE inhibitor known to be undergoing clinical trials, and may be the compound which finally demonstrates the clinical utility of a synthetic HNE inhibitor. PMID:8189835

  1. Independent-Trajectories Thermodynamic-Integration Free-Energy Changes for Biomolecular Systems: Determinants of H5N1 Avian Influenza Virus Neuraminidase Inhibition by Peramivir

    PubMed Central

    2009-01-01

    Free-energy changes are essential physicochemical quantities for understanding most biochemical processes. Yet, the application of accurate thermodynamic-integration (TI) computation to biological and macromolecular systems is limited by finite-sampling artifacts. In this paper, we employ independent-trajectories thermodynamic-integration (IT-TI) computation to estimate improved free-energy changes and their uncertainties for (bio)molecular systems. IT-TI aids sampling statistics of the thermodynamic macrostates for flexible associating partners by ensemble averaging of multiple, independent simulation trajectories. We study peramivir (PVR) inhibition of the H5N1 avian influenza virus neuraminidase flexible receptor (N1). Binding site loops 150 and 119 are highly mobile, as revealed by N1-PVR 20-ns molecular dynamics. Due to such heterogeneous sampling, standard TI binding free-energy estimates span a rather large free-energy range, from a 19% underestimation to a 29% overestimation of the experimental reference value (−62.2 ± 1.8 kJ mol−1). Remarkably, our IT-TI binding free-energy estimate (−61.1 ± 5.4 kJ mol−1) agrees with a 2% relative difference. In addition, IT-TI runs provide a statistics-based free-energy uncertainty for the process of interest. Using ∼800 ns of overall sampling, we investigate N1-PVR binding determinants by IT-TI alchemical modifications of PVR moieties. These results emphasize the dominant electrostatic contribution, particularly through the N1 E277−PVR guanidinium interaction. Future drug development may be also guided by properly tuning ligand flexibility and hydrophobicity. IT-TI will allow estimation of relative free energies for systems of increasing size, with improved reliability by employing large-scale distributed computing. PMID:19461872

  2. Chimeric Bovine Respiratory Syncytial Virus with Attachment and Fusion Glycoproteins Replaced by Bovine Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase and Fusion Proteins

    PubMed Central

    Stope, Matthias B.; Karger, Axel; Schmidt, Ulrike; Buchholz, Ursula J.

    2001-01-01

    Chimeric bovine respiratory syncytial viruses (BRSV) expressing glycoproteins of bovine parainfluenza virus type 3 (BPIV-3) instead of BRSV glycoproteins were generated from cDNA. In the BRSV antigenome cDNA, the open reading frames of the major BRSV glycoproteins, attachment protein G and fusion protein F, were replaced individually or together by those of the BPIV-3 hemagglutinin-neuraminidase (HN) and/or fusion (F) glycoproteins. Recombinant virus could not be recovered from cDNA when the BRSV F open reading frame was replaced by the BPIV-3 F open reading frame. However, cDNA recovery of the chimeric virus rBRSV-HNF, with both glycoproteins replaced simultaneously, and of the chimeric virus rBRSV-HN, with the BRSV G protein replaced by BPIV-3 HN, was successful. The replication rates of both chimeras were similar to that of standard rBRSV. Moreover, rBRSV-HNF was neutralized by antibodies specific for BPIV-3, but not by antibodies specific to BRSV, demonstrating that the BRSV glycoproteins can be functionally replaced by BPIV-3 glycoproteins. In contrast, rBRSV-HN was neutralized by BRSV-specific antisera, but not by BPIV-3 specific sera, showing that infection of rBRSV-HN is mediated by BRSV F. Hemadsorption of cells infected with rBRSV-HNF and rBRSV-HN proved that BPIV-3 HN protein expressed by rBRSV is functional. Colocalization of the BPIV-3 glycoproteins with BRSV M protein was demonstrated by confocal laser scan microscopy. Moreover, protein analysis revealed that the BPIV-3 glycoproteins were present in chimeric virions. Taken together, these data indicate that the heterologous glycoproteins were not only expressed but were incorporated into the envelope of recombinant BRSV. Thus, the envelope glycoproteins derived from a member of the Respirovirus genus can together functionally replace their homologs in a Pneumovirus background. PMID:11533200

  3. The neuraminidase and matrix genes of the 2009 pandemic influenza H1N1 virus cooperate functionally to facilitate efficient replication and transmissibility in pigs

    PubMed Central

    Liu, Qinfang; Bawa, Bhupinder; Qiao, Chuanling; Qi, Wenbao; Shen, Huigang; Chen, Ying; Ma, Jingqun; Li, Xi; Webby, Richard J.; García-Sastre, Adolfo

    2012-01-01

    The 2009 pandemic H1N1 virus (pH1N1) contains neuraminidase (NA) and matrix (M) genes from Eurasian avian-like swine influenza viruses (SIVs), with the remaining six genes from North American triple-reassortant SIVs. To characterize the role of the pH1N1 NA and M genes in pathogenesis and transmission, their impact was evaluated in the background of an H1N1 triple-reassortant (tr1930) SIV in which the HA (H3) and NA (N2) of influenza A/swine/Texas/4199-2/98 virus were replaced with those from the classical H1N1 A/swine/Iowa/15/30 (1930) virus. The laboratory-adapted 1930 virus did not shed nor transmit in pigs, but tr1930 was able to shed in infected pigs. The NA, M or both genes of the tr1930 virus were then substituted by those of pH1N1. The resulting virus with both NA and M from pH1N1 grew to significantly higher titre in cell cultures than the viruses with single NA or M from pH1N1. In a pig model, only the virus containing both NA and M from pH1N1 was transmitted to and infected sentinels, whereas the viruses with single NA or M from pH1N1 did not. These results demonstrate that the right combination of NA and M genes is critical for the replication and transmissibility of influenza viruses in pigs. PMID:22337640

  4. Theoretical analysis of the neuraminidase epitope of the Mexican A H1N1 influenza strain, and experimental studies on its interaction with rabbit and human hosts.

    PubMed

    Loyola, Paola Kinara Reyes; Campos-Rodríguez, R; Bello, Martiniano; Rojas-Hernández, S; Zimic, Mirko; Quiliano, Miguel; Briz, Verónica; Muñoz-Fernández, M Angeles; Tolentino-López, Luis; Correa-Basurto, Jose

    2013-05-01

    The neuraminidase (NA) epitope from the Mexican AH1N1 influenza virus was identified by using sequences registered at the GenBank during the peak of a pandemic (from April 2009 to October 2010). First, NA protein sequences were submitted for multiple alignment analysis, and their three-dimensional models (3-D) were then built by using homology modeling. The most common sequence (denominated wild-type) and its mutants were submitted to linear and nonlinear epitope predictors, which included the major histocompatibility complex type II (MHC II) and B-cell peptides. The epitope prediction was in accordance with evolutionary behavior and some protein structural properties. The latter included a low NA mutation rate, NA 3-D surface exposure, and the presence of high hindrance side chain residues. After selecting the epitope, docking studies and molecular dynamics (MD) simulations were used to explore interactions between the epitope and MHC II. Afterward, several experimental assays were performed to validate the theoretical study by using antibodies from humans (infected by pandemic H1N1) and rabbits (epitope vaccination). The results show 119 complete sequences that were grouped into 28 protein sequences according to their identity (one wild-type and 27 representative mutants (1-5 mutations)). The predictors yielded several epitopes, with the best fit being the one located in the C-terminal region. Theoretical methods demonstrated that the selected epitope reached the P4, P6, P7, and P9 pockets of MHC II, whereas the experimental evidence indicates that the epitope is recognized by human antibodies and also by rabbit antibodies immunized with the peptide.

  5. The amino-terminal region of the neuraminidase protein from avian H5N1 influenza virus is important for its biosynthetic transport to the host cell surface.

    PubMed

    Qian, Guomin; Wang, Song; Chi, Xiaojuan; Li, Hua; Wei, Haitao; Zhu, Xiaomei; Chen, Yuhai; Chen, Ji-Long

    2014-12-01

    Influenza virus neuraminidase (NA) is a major viral envelope glycoprotein, which plays a critical role in viral infection. Although NA functional domains have been determined previously, the precise role of the amino acids located at the N-terminus of avian H5N1 NA for protein expression and intracellular transport to the host plasma membrane is not fully understood. In the present study, a series of N-terminal truncation or deletion mutants of H5N1 NA were generated and their expression and intracellular trafficking were investigated. Protein expression from mutants NAΔ20, NAΔ35, NAΔ40, NAΔ7-20 and NAΔ7-35 was undetectable by immunoblotting and by performing NA activity assays. Mutants NAΔ6, NAΔ11 and NAΔ15-20 showed a marked decreased in protein expression, whereas mutants NAΔ7-15 and NAΔ15 displayed a slight increase in protein expression, compared with that of the native NA protein. These data suggest that amino acid residues 16-20 are vital for NA protein expression, while amino acids 7-15 might suppress NA protein expression. In deletion mutants NAΔ7-15 and NAΔ15 there was an accumulation of NA protein at the juxta-nuclear region, with reduced expression of NA at the cell surface. Although active Cdc42 could promote transport of wild-type NA to the host cell surface, this member of the Rho family of GTPases failed to regulate transport of mutants NAΔ7-15 and NAΔ15. The results of the study reveal that amino acid residues 7-15 of H5N1 NA are critical for its biosynthetic transport to the host cell surface.

  6. Protection against avian influenza H9N2 virus challenge by immunization with hemagglutinin- or neuraminidase-expressing DNA in BALB/c mice

    SciTech Connect

    Qiu Meizhen; Fang Fang; Chen Yan; Wang Hualin; Chen Quanjiao; Chang Haiyan; Wang Fuyan; Wang Hanzhong; Zhang Ran; Chen Ze . E-mail: chenze2005@263.net

    2006-05-19

    Avian influenza viruses of H9N2 subtype are widely spread in avian species. The viruses have recently been transmitted to mammalian species, including humans, accelerating the efforts to devise protective strategies against them. In this study, an avian influenza H9N2 virus strain (A/Chicken/Jiangsu/7/2002), isolated in Jiangsu Province, China, was used to infect BALB/c mice for adaptation. After five lung-to-lung passages, the virus was stably proliferated in a large quantity in the murine lung and caused the deaths of mice. In addition, we explored the protection induced by H9N2 virus hemagglutinin (HA)- and neuraminidase (NA)-expressing DNAs in BALB/c mice. Female BALB/c mice aged 6-8 weeks were immunized once or twice at a 3-week interval with HA-DNA and NA-DNA by electroporation, respectively, each at a dose of 3, 10 or 30 {mu}g. The mice were challenged with a lethal dose (40x LD{sub 5}) of influenza H9N2 virus four weeks after immunization once or one week after immunization twice. The protections of DNA vaccines were evaluated by the serum antibody titers, residual lung virus titers, and survival rates of the mice. The result showed that immunization once with not less than 10 {mu}g or twice with 3 {mu}g HA-DNA or NA-DNA provided effective protection against homologous avian influenza H9N2 virus.

  7. Protection of pigs against pandemic swine origin H1N1 influenza A virus infection by hemagglutinin- or neuraminidase-expressing attenuated pseudorabies virus recombinants.

    PubMed

    Klingbeil, Katharina; Lange, Elke; Blohm, Ulrike; Teifke, Jens P; Mettenleiter, Thomas C; Fuchs, Walter

    2015-03-01

    Influenza is an important respiratory disease of pigs, and may lead to novel human pathogens like the 2009 pandemic H1N1 swine-origin influenza virus (SoIV). Therefore, improved influenza vaccines for pigs are required. Recently, we demonstrated that single intranasal immunization with a hemagglutinin (HA)-expressing pseudorabies virus recombinant of vaccine strain Bartha (PrV-Ba) protected pigs from H1N1 SoIV challenge (Klingbeil et al., 2014). Now we investigated enhancement of efficacy by prime-boost vaccination and/or intramuscular administration. Furthermore, a novel PrV-Ba recombinant expressing codon-optimized N1 neuraminidase (NA) was included. In vitro replication of this virus was only slightly affected compared to parental virus. Unlike HA, the abundantly expressed NA was efficiently incorporated into PrV particles. Immunization of pigs with the two PrV recombinants, either singly or in combination, induced B cell proliferation and the expected SoIV-specific antibodies, whose titers increased substantially after boost vaccination. After immunization of animals with either PrV recombinant H1N1 SoIV challenge virus replication was significantly reduced compared to PrV-Ba vaccinated or naïve controls. Protective efficacy of HA-expressing PrV was higher than of NA-expressing PrV, and not significantly enhanced by combination. Despite higher serum antibody titers obtained after intramuscular immunization, transmission of challenge virus to naïve contact animals was only prevented after intranasal prime-boost vaccination with HA-expressing PrV-Ba.

  8. Small-molecule caspase inhibitors

    NASA Astrophysics Data System (ADS)

    Zhenodarova, S. M.

    2010-02-01

    The review considers low-molecular weight inhibitors of caspases, cysteine proteases being key contributors to apoptosis (programmed cell death). The inhibitors with aspartic acid residues or various heterocyclic systems (both synthetic and natural) are covered. Their possible mechanisms of action are discussed. Data on inhibitor structure-activity relationship studies are systematically surveyed. The interactions of the non-peptide fragments of an inhibitor with the enzymes are examined. Examples of the use of some inhibitors for apoptosis suppression are provided.

  9. Natural inhibitors of thrombin.

    PubMed

    Huntington, James A

    2014-04-01

    The serine protease thrombin is the effector enzyme of blood coagulation. It has many activities critical for the formation of stable clots, including cleavage of fibrinogen to fibrin, activation of platelets and conversion of procofactors to active cofactors. Thrombin carries-out its multiple functions by utilising three special features: a deep active site cleft and two anion binding exosites (exosite I and II). Similarly, thrombin inhibitors have evolved to exploit the unique features of thrombin to achieve rapid and specific inactivation of thrombin. Exogenous thrombin inhibitors come from several different protein families and are generally found in the saliva of haematophagous animals (blood suckers) as part of an anticoagulant cocktail that allows them to feed. Crystal structures of several of these inhibitors reveal how peptides and proteins can be targeted to thrombin in different and interesting ways. Thrombin activity must also be regulated by endogenous inhibitors so that thrombi do not occlude blood flow and cause thrombosis. A single protein family, the serpins, provides all four of the endogenous thrombin inhibitors found in man. The crystal structures of these serpins bound to thrombin have been solved, revealing a similar exosite-dependence on complex formation. In addition to forming the recognition complex, serpins destroy the structure of thrombin, allowing them to be released from cofactors and substrates for clearance. This review examines how the special features of thrombin have been exploited by evolution to achieve inhibition of the ultimate coagulation protease.

  10. SGLT2 inhibitors.

    PubMed

    Dardi, I; Kouvatsos, T; Jabbour, S A

    2016-02-01

    Diabetes mellitus is a serious health issue and an economic burden, rising in epidemic proportions over the last few decades worldwide. Although several treatment options are available, only half of the global diabetic population achieves the recommended or individualized glycemic targets. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a new class of antidiabetic agents with a novel insulin-independent action. SGLT2 is a transporter found in the proximal renal tubules, responsible for the reabsorption of most of the glucose filtered by the kidney. Inhibition of SGLT2 lowers the blood glucose level by promoting the urinary excretion of excess glucose. Due to their insulin-independent action, SGLT2 inhibitors can be used with any degree of beta-cell dysfunction or insulin resistance, related to a very low risk of hypoglycemia. In addition to improving glycemic control, SGLT2 inhibitors have been associated with a reduction in weight and blood pressure when used as monotherapy or in combination with other antidiabetic agents in patients with type 2 diabetes mellitus (T2DM). Treatment with SGLT2 inhibitors is usually well tolerated; however, they have been associated with an increased incidence of urinary tract and genital infections, although these infections are usually mild and easy to treat. SGLT2 inhibitors are a promising new option in the armamentarium of drugs for patients with T2DM. PMID:26362302

  11. Cholinesterase inhibitors from botanicals

    PubMed Central

    Ahmed, Faiyaz; Ghalib, Raza Murad; Sasikala, P.; Ahmed, K. K. Mueen

    2013-01-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disease, wherein a progressive loss of cholinergic synapses occurs in hippocampus and neocortex. Decreased concentration of the neurotransmitter, acetylcholine (ACh), appears to be critical element in the development of dementia, and the most appropriate therapeutic approach to treat AD and other form of dementia is to restore acetylcholine levels by inhibiting both major form of cholinesterase: Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Consequently, researches have focused their attention towards finding cholinesterase inhibitors from natural products. A large number of such inhibitors have been isolated from medicinal plants. This review presents a comprehensive account of the advances in field of cholinesterase inhibitor phytoconstituents. The structures of some important phytoconstituents (collected through www.Chemspider.com) are also presented and the scope for future research is discussed. PMID:24347920

  12. Cholinesterase inhibitors from botanicals.

    PubMed

    Ahmed, Faiyaz; Ghalib, Raza Murad; Sasikala, P; Ahmed, K K Mueen

    2013-07-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disease, wherein a progressive loss of cholinergic synapses occurs in hippocampus and neocortex. Decreased concentration of the neurotransmitter, acetylcholine (ACh), appears to be critical element in the development of dementia, and the most appropriate therapeutic approach to treat AD and other form of dementia is to restore acetylcholine levels by inhibiting both major form of cholinesterase: Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Consequently, researches have focused their attention towards finding cholinesterase inhibitors from natural products. A large number of such inhibitors have been isolated from medicinal plants. This review presents a comprehensive account of the advances in field of cholinesterase inhibitor phytoconstituents. The structures of some important phytoconstituents (collected through www.Chemspider.com) are also presented and the scope for future research is discussed. PMID:24347920

  13. Phosphodiesterase-5 inhibitors.

    PubMed

    Cockrill, Barbara A; Waxman, Aaron B

    2013-01-01

    Nitric oxide (NO) signaling plays a key role in modulating vascular tone and remodeling in the pulmonary circulation. The guanylate cyclase/cyclic guanylate monophosphate-signaling pathway primarily mediates nitric oxide signaling. This pathway is critical in normal regulation of the pulmonary vasculature, and is an important target for therapy in patients with pulmonary hypertension. In the pulmonary vasculature, degradation of cGMP is primarily regulated by PDE-5, and inhibition of this enzyme has important effects on pulmonary vasculature smooth muscle tone. Large randomized placebo-controlled trials of PDE-5 inhibitors demonstrated improved exercise capacity, hemodynamics and quality of life in adult patients with PAH. This chapter will discuss the mechanisms of NO signaling in the vasculature, characteristics of the PDE5-inhibitors approved for treatment of PH, and review available data on the use of phosphodiesterase inhibitors in PH. PMID:24092343

  14. Human plasma alpha-cysteine proteinase inhibitor. Purification by affinity chromatography, characterization and isolation of an active fragment.

    PubMed Central

    Gounaris, A D; Brown, M A; Barrett, A J

    1984-01-01

    Human plasma alpha-cysteine proteinase inhibitor (alpha CPI) was purified by a two-stage method: affinity chromatography on S-carboxymethyl-papain-Sepharose, and high-resolution anion-exchange chromatography. The protein was obtained as a form of Mr about 64 000 and material of higher Mr (about 100 000). In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with reduction, both forms showed a major component of Mr 64 000. An antiserum was raised against alpha CPI, and 'rocket' immunoassays showed the mean concentration in sera from 19 individuals to be 35.9 mg/dl. Both low-Mr and high-Mr forms of alpha CPI were confirmed to be sialoglycoproteins by the decrease in electrophoretic mobility after treatment with neuraminidase. alpha CPI was shown immunologically to be distinct from antithrombin III and alpha 1-antichymotrypsin, two serine proteinase inhibitors from plasma with somewhat similar Mr values. alpha CPI was also distinct from cystatins A and B, the two intracellular low-Mr cysteine proteinase inhibitors from human liver. Complexes of alpha CPI with papain were detectable in immunoelectrophoresis, but dissociated to free enzyme and intact inhibitor in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The stoichiometry of binding of papain was close to 1:1 for both low-Mr and high-Mr forms. alpha CPI was found to be a tight-binding inhibitor of papain and human cathepsins H and L (Ki 34 pM, 1.1 nM and 62 pM respectively). By contrast, inhibition of cathepsin B was much weaker, Ki being about 35 microM. Dipeptidyl peptidase I also was weakly inhibited. Digestion of alpha CPI with bromelain gave rise to an inhibitory fragment of Mr about 22 000, which was isolated. Images Fig. 2. Fig. 3. Fig. 4. PMID:6548132

  15. Pectin methylesterase inhibitor.

    PubMed

    Giovane, A; Servillo, L; Balestrieri, C; Raiola, A; D'Avino, R; Tamburrini, M; Ciardiello, M A; Camardella, L

    2004-02-12

    Pectin methylesterase (PME) is the first enzyme acting on pectin, a major component of plant cell wall. PME action produces pectin with different structural and functional properties, having an important role in plant physiology. Regulation of plant PME activity is obtained by the differential expression of several isoforms in different tissues and developmental stages and by subtle modifications of cell wall local pH. Inhibitory activities from various plant sources have also been reported. A proteinaceous inhibitor of PME (PMEI) has been purified from kiwi fruit. The kiwi PMEI is active against plant PMEs, forming a 1:1 non-covalent complex. The polypeptide chain comprises 152 amino acid residues and contains five Cys residues, four of which are connected by disulfide bridges, first to second and third to fourth. The sequence shows significant similarity with the N-terminal pro-peptides of plant PME, and with plant invertase inhibitors. In particular, the four Cys residues involved in disulfide bridges are conserved. On the basis of amino acid sequence similarity and Cys residues conservation, a large protein family including PMEI, invertase inhibitors and related proteins of unknown function has been identified. The presence of at least two sequences in the Arabidopsis genome having high similarity with kiwi PMEI suggests the ubiquitous presence of this inhibitor. PMEI has an interest in food industry as inhibitor of endogenous PME, responsible for phase separation and cloud loss in fruit juice manufacturing. Affinity chromatography on resin-bound PMEI can also be used to concentrate and detect residual PME activity in fruit and vegetable products.

  16. Estimating the fitness advantage conferred by permissive neuraminidase mutations in recent oseltamivir-resistant A(H1N1)pdm09 influenza viruses.

    PubMed

    Butler, Jeff; Hooper, Kathryn A; Petrie, Stephen; Lee, Raphael; Maurer-Stroh, Sebastian; Reh, Lucia; Guarnaccia, Teagan; Baas, Chantal; Xue, Lumin; Vitesnik, Sophie; Leang, Sook-Kwan; McVernon, Jodie; Kelso, Anne; Barr, Ian G; McCaw, James M; Bloom, Jesse D; Hurt, Aeron C

    2014-04-01

    Oseltamivir is relied upon worldwide as the drug of choice for the treatment of human influenza infection. Surveillance for oseltamivir resistance is routinely performed to ensure the ongoing efficacy of oseltamivir against circulating viruses. Since the emergence of the pandemic 2009 A(H1N1) influenza virus (A(H1N1)pdm09), the proportion of A(H1N1)pdm09 viruses that are oseltamivir resistant (OR) has generally been low. However, a cluster of OR A(H1N1)pdm09 viruses, encoding the neuraminidase (NA) H275Y oseltamivir resistance mutation, was detected in Australia in 2011 amongst community patients that had not been treated with oseltamivir. Here we combine a competitive mixtures ferret model of influenza infection with a mathematical model to assess the fitness, both within and between hosts, of recent OR A(H1N1)pdm09 viruses. In conjunction with data from in vitro analyses of NA expression and activity we demonstrate that contemporary A(H1N1)pdm09 viruses are now more capable of acquiring H275Y without compromising their fitness, than earlier A(H1N1)pdm09 viruses circulating in 2009. Furthermore, using reverse engineered viruses we demonstrate that a pair of permissive secondary NA mutations, V241I and N369K, confers robust fitness on recent H275Y A(H1N1)pdm09 viruses, which correlated with enhanced surface expression and enzymatic activity of the A(H1N1)pdm09 NA protein. These permissive mutations first emerged in 2010 and are now present in almost all circulating A(H1N1)pdm09 viruses. Our findings suggest that recent A(H1N1)pdm09 viruses are now more permissive to the acquisition of H275Y than earlier A(H1N1)pdm09 viruses, increasing the risk that OR A(H1N1)pdm09 will emerge and spread worldwide. PMID:24699865

  17. Acyclic peptide inhibitors of amylases.

    PubMed

    Pohl, Nicola

    2005-12-01

    In this issue of Chemistry and Biology, a library screening approach reveals a linear octapeptide inhibitor of alpha-amylases reached by de novo design . The selected molecule shares characteristics with naturally occurring protein inhibitors -- a result that suggests general rules for the design of peptide-based amylase inhibitors may be achievable.

  18. Potential of Peptides as Inhibitors and Mimotopes: Selection of Carbohydrate-Mimetic Peptides from Phage Display Libraries

    PubMed Central

    Matsubara, Teruhiko

    2012-01-01

    Glycoconjugates play various roles in biological processes. In particular, oligosaccharides on the surface of animal cells are involved in virus infection and cell-cell communication. Inhibitors of carbohydrate-protein interactions are potential antiviral drugs. Several anti-influenza drugs such as oseltamivir and zanamivir are derivatives of sialic acid, which inhibits neuraminidase. However, it is very difficult to prepare a diverse range of sugar derivatives by chemical synthesis or by the isolation of natural products. In addition, the pathogenic capsular polysaccharides of bacteria are carbohydrate antigens, for which a safe and efficacious method of vaccination is required. Phage-display technology has been improved to enable the identification of peptides that bind to carbohydrate-binding proteins, such as lectins and antibodies, from a large repertoire of peptide sequences. These peptides are known as “carbohydrate-mimetic peptides (CMPs)” because they mimic carbohydrate structures. Compared to carbohydrate derivatives, it is easy to prepare mono- and multivalent peptides and then to modify them to create various derivatives. Such mimetic peptides are available as peptide inhibitors of carbohydrate-protein interactions and peptide mimotopes that are conjugated with adjuvant for vaccination. PMID:23094142

  19. [JAK2 inhibitors].

    PubMed

    Hernández Boluda, Juan Carlos; Gómez, Montse; Pérez, Ariadna

    2016-07-15

    Pharmacological inhibition of the kinase activity of JAK proteins can interfere with the signaling of immunomodulatory cytokines and block the constitutive activation of the JAK-STAT pathway that characterizes certain malignancies, including chronic myeloproliferative neoplasms. JAK inhibitors may, therefore, be useful to treat malignancies as well as inflammatory or immune disorders. Currently, the most significant advances have been made in the treatment of myelofibrosis, where these drugs may lead to a remarkable improvement in the control of hyperproliferative manifestations. However, available data suggest that this treatment is not curative of myelofibrosis. In general, JAK2 inhibition induces cytopaenias, with this being considered a class side-effect. By contrast, the extrahaematologic toxicity profile varies significantly among the different JAK inhibitors. At present, there are several clinical trials evaluating the combination of ruxolitinib with other drugs, in order to improve its therapeutic activity as well as reducing haematologic toxicity. PMID:27033437

  20. Coagulation inhibitors in inflammation.

    PubMed

    Esmon, C T

    2005-04-01

    Coagulation is triggered by inflammatory mediators in a number of ways. However, to prevent unwanted clot formation, several natural anticoagulant mechanisms exist, such as the antithrombin-heparin mechanism, the tissue factor pathway inhibitor mechanism and the protein C anticoagulant pathway. This review examines the ways in which these pathways are down-regulated by inflammation, thus limiting clot formation and decreasing the natural anti-inflammatory mechanisms that these pathways possess. PMID:15787615

  1. Neutrophil Elastase Inhibitors

    PubMed Central

    Groutas, William C.; Dou, Dengfeng; Alliston, Kevin R.

    2011-01-01

    Introduction Chronic obstructive pulmonary disease (COPD) constitutes a worldwide health problem. There is currently an urgent and unmet need for the development of small molecule therapeutics capable of blocking and/or reversing the progression of the disorder. Recent studies have greatly illuminated our understanding of the multiple pathogenic processes associated with COPD. Of paramount importance is the key role played by proteases, oxidative stress, apoptosis, and inflammation. Insights gained from these studies have made possible the exploration of new therapeutic approaches. Areas covered An overview of major developments in COPD research with emphasis on low molecular weight neutrophil elastase inhibitors is described in this review. Expert opinion Great strides have been made toward our understanding of the biochemical and cellular events associated with COPD. However, our knowledge regarding the inter-relationships among the multiple pathogenic mechanisms and their mediators involved is till limited. The problem is further compounded by the unavailability of suitable validated biomarkers for assessing the efficacy of potential therapeutic interventions. The complexity of COPD suggests that effective therapeutic interventions may require the administration of more than one agent such as, for instance, an HNE or MMP-12 inhibitor with an anti-inflammatory agent such as a phosphodiesterase-4 inhibitor, or a dual function agent capable of disrupting the cycle of proteolysis, apoptosis, inflammation and oxidative stress PMID:21235378

  2. Protein farnesyltransferase inhibitors.

    PubMed

    Ayral-Kaloustian, Semiramis; Salaski, Edward J

    2002-05-01

    Specific mutations in the ras gene impair the guanosine triphophatase (GTPase) activity of Ras proteins, which play a fundamental role in the signaling cascade, leading to uninterrupted growth signals and to the transformation of normal cells into malignant phenotypes. It has been shown that normal cells transfected with mutant ras gene become cancerous and that unfarnesylated, cytosolic mutant Ras protein does not anchor onto cell membranes and cannot induce this transformation. Posttranslational modification and plasma membrane association of mutant Ras is necessary for this transforming activity. Since its identification, the enzyme protein farnesyltransferase (FTase) that catalyzes the first and essential step of the three Ras-processing steps has emerged as the most promising target for therapeutic intervention. FTase has been implicated as a potential target in inhibiting the prenylation of a variety of proteins, thus in controlling varied disease states (e.g. cancer, neurofibromatosis, restenosis, viral hepatitis, bone resorption, parasitic infections, corneal inflammations, and diabetes) associated with prenyl modifications of Ras and other proteins. Furthermore, it has been suggested that FTase inhibitors indirectly help in inhibiting tumors via suppression of angiogenesis and induction of apoptosis. Major milestones have been achieved with small-molecule FTase inhibitors that show efficacy without toxicity in vitro, as well as in mouse models bearing ras-dependent tumors. With the determination of the crystal structure of mammalian FTase, existent leads have been fine-tuned and new potent molecules of diverse structural classes have been designed. A few of these molecules are currently in the clinic, with at least three drug candidates in Phase II studies and one in Phase III. This article will review the progress that has been reported with FTase inhibitors in drug discovery and in the clinic. PMID:12733981

  3. Novel Ranking System for Identifying Efficacious Anti-Influenza Virus PB2 Inhibitors

    PubMed Central

    McNeil, Colleen F.; Leeman, Joshua R.; Bennett, Hamilton B.; Nti-Addae, Kwame; Huang, Cassey; Germann, Ursula A.; Byrn, Randal A.; Berlioz-Seux, Francoise; Rijnbrand, Rene; Clark, Michael P.; Charifson, Paul S.; Jones, Steven M.

    2015-01-01

    Through antigenic drift and shifts, influenza virus infections continue to be an annual cause of morbidity in healthy populations and of death among elderly and at-risk patients. The emergence of highly pathogenic avian influenza viruses such as H5N1 and H7N9 and the rapid spread of the swine-origin H1N1 influenza virus in 2009 demonstrate the continued need for effective therapeutic agents for influenza. While several neuraminidase inhibitors have been developed for the treatment of influenza virus infections, these have shown a limited window for treatment initiation, and resistant variants have been noted in the population. In addition, an older class of antiviral drugs for influenza, the adamantanes, are no longer recommended for treatment due to widespread resistance. There remains a need for new influenza therapeutic agents with improved efficacy as well as an expanded window for the initiation of treatment. Azaindole compounds targeting the influenza A virus PB2 protein and demonstrating excellent in vitro and in vivo properties have been identified. To evaluate the in vivo efficacy of these PB2 inhibitors, we utilized a mouse influenza A virus infection model. In addition to traditional endpoints, i.e., death, morbidity, and body weight loss, we measured lung function using whole-body plethysmography, and we used these data to develop a composite efficacy score that takes compound exposure into account. This model allowed the rapid identification and ranking of molecules relative to each other and to oseltamivir. The ability to identify compounds with enhanced preclinical properties provides an opportunity to develop more-effective treatments for influenza in patients. PMID:26169418

  4. Synthesis of Lysine Methyltransferase Inhibitors

    NASA Astrophysics Data System (ADS)

    Ye, Tao; Hui, Chunngai

    2015-07-01

    Lysine methyltransferase which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and has emerged as a promising target for the development of various human diseases, including cancer, inflammation, and psychiatric disorders. However, inhibiting Lysine methyltransferases selectively has presented many challenges to medicinal chemists. During the past decade, lysine methyltransferase inhibitors covering many different structural classes have been designed and developed. In this review, we describe the development of selective, small-molecule inhibitors of lysine methyltransferases with an emphasis on their discovery and chemical synthesis. We highlight the current state of lysine methyltransferase inhibitors and discuss future directions and opportunities for lysine methyltransferase inhibitor discovery.

  5. High performance oilfield scale inhibitors

    SciTech Connect

    Duccini, Y.; Dufour, A.; Hann, W.M.; Sanders, T.W.; Weinstein, B.

    1997-08-01

    Sea water often reacts with the formation water in offshore fields to produce barium, calcium and strontium sulfate deposits that hinder oil production. Newer fields often have more difficult to control scale problems than older ones, and current technology scale inhibitors are not able to control the deposits as well as needed. In addition, ever more stringent regulations designed to minimize the impact of inhibitors on the environment are being enacted. Three new inhibitors are presented that overcome many of the problems of older technology scale inhibitors.

  6. Evaluation of the antigenic relatedness and cross-protective immunity of the neuraminidase between human influenza A (H1N1) virus and highly pathogenic avian influenza A (H5N1) virus.

    PubMed

    Lu, Xiuhua; Liu, Feng; Zeng, Hui; Sheu, Tiffany; Achenbach, Jenna E; Veguilla, Vic; Gubareva, Larisa V; Garten, Rebecca; Smith, Catherine; Yang, Hua; Stevens, James; Xu, Xiyan; Katz, Jacqueline M; Tumpey, Terrence M

    2014-04-01

    To determine the genetic and antigenic relatedness as well as the cross-protective immunity of human H1N1 and avian H5N1 influenza virus neuraminidase (NA), we immunized rabbits with either a baculovirus-expressed recombinant NA from A/Beijing/262/95 (BJ/262) H1N1 or A/Hong Kong/483/97 (HK/483) H5N1 virus. Cross-reactive antibody responses were evaluated by multiple serological assays and cross-protection against H5N1 virus challenge was evaluated in mice. In a neuraminidase inhibition (NI) test, the antisera exhibited substantial inhibition of NA activity of the homologous virus, but failed to inhibit the NA activity of heterologous virus. However, these antisera exhibited low levels of cross-reactivity measured by plaque size reduction, replication inhibition, single radial hemolysis, and ELISA assays. Passive immunization with HK/483 NA-specific antisera significantly reduced virus replication and disease, and afforded almost complete protection against lethal homologous virus challenge in mice. However, passive immunization with BJ/262 (H1N1) NA-specific antisera was ineffective at providing cross-protection against lethal H5N1 virus challenge and only slightly reduced weight loss. Substantial amino acid variation among the NA antigenic sites was observed between BJ/262 and HK/483 virus, which was consistent with the lack of cross-reactive NI activity by the antibody and limited cross-protective immunity in mice. These results show a strong correlation between the lack of cross-protective immunity and low structural similarities of NA from a human seasonal H1N1 virus and an avian H5N1 influenza virus.

  7. Osteocompatibility of Biofilm Inhibitors

    PubMed Central

    Rawson, Monica; Haggard, Warren; Jennings, Jessica A

    2014-01-01

    The demand for infection prevention therapies has led to the discovery of several biofilm inhibitors. These inhibiting signals are released by bacteria, fungi, or marine organisms to signal biofilm dispersal or disruption in Gram-positive, Gram-negative, and fungal microorganisms. The purpose of this study was to test the biocompatibility of five different naturally-produced biofilm chemical dispersal and inhibition signals with osteoblast-like cells: D-amino acids (D-AA), lysostaphin (LS), farnesol, cis-2-decenoic acid (C2DA), and desformyl flustrabromine (dFBr). In this preliminary study, compatibility of these anti-biofilm agents with differentiating osteoblasts was examined over a 21 days period at levels above and below concentrations active against bacterial biofilm. Anti-biofilm compounds listed above were serially diluted in osteogenic media and added to cultures of MC3T3 cells. Cell viability and cytotoxicity, after exposure to each anti-biofilm agent, were measured using a DNA assay. Differentiation characteristics of osteoblasts were determined qualitatively by observing staining of mineral deposits and quantitatively with an alkaline phosphatase assay. D-AA, LS, and C2DA were all biocompatible within the reported biofilm inhibitory concentration ranges and supported osteoblast differentiation. Farnesol and dFBr induced cytotoxic responses within the reported biofilm inhibitory concentration range and low doses of dFBr were found to inhibit osteoblast differentiation. At high concentrations, such as those that may be present after local delivery, many of these biofilm inhibitors can have effects on cellular viability and osteoblast function. Concentrations at which negative effects on osteoblasts occur should serve as upper limits for delivery to orthopaedic trauma sites and guide development of these potential therapeutics for orthopaedics. PMID:25505496

  8. Osteocompatibility of biofilm inhibitors.

    PubMed

    Rawson, Monica; Haggard, Warren; Jennings, Jessica A

    2014-01-01

    The demand for infection prevention therapies has led to the discovery of several biofilm inhibitors. These inhibiting signals are released by bacteria, fungi, or marine organisms to signal biofilm dispersal or disruption in Gram-positive, Gram-negative, and fungal microorganisms. The purpose of this study was to test the biocompatibility of five different naturally-produced biofilm chemical dispersal and inhibition signals with osteoblast-like cells: D-amino acids (D-AA), lysostaphin (LS), farnesol, cis-2-decenoic acid (C2DA), and desformyl flustrabromine (dFBr). In this preliminary study, compatibility of these anti-biofilm agents with differentiating osteoblasts was examined over a 21 days period at levels above and below concentrations active against bacterial biofilm. Anti-biofilm compounds listed above were serially diluted in osteogenic media and added to cultures of MC3T3 cells. Cell viability and cytotoxicity, after exposure to each anti-biofilm agent, were measured using a DNA assay. Differentiation characteristics of osteoblasts were determined qualitatively by observing staining of mineral deposits and quantitatively with an alkaline phosphatase assay. D-AA, LS, and C2DA were all biocompatible within the reported biofilm inhibitory concentration ranges and supported osteoblast differentiation. Farnesol and dFBr induced cytotoxic responses within the reported biofilm inhibitory concentration range and low doses of dFBr were found to inhibit osteoblast differentiation. At high concentrations, such as those that may be present after local delivery, many of these biofilm inhibitors can have effects on cellular viability and osteoblast function. Concentrations at which negative effects on osteoblasts occur should serve as upper limits for delivery to orthopaedic trauma sites and guide development of these potential therapeutics for orthopaedics. PMID:25505496

  9. Biological abatement of cellulase inhibitors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bio-abatement uses a fungus to metabolize and remove fermentation inhibitors. To determine whether bio-abatement could alleviate enzyme inhibitor effects observed in biomass liquors after pretreatment, corn stover at 10% (w/v) solids was pretreated with either dilute acid or liquid hot water. The ...

  10. Anthranilamide inhibitors of factor Xa.

    PubMed

    Mendel, David; Marquart, Angela L; Joseph, Sajan; Waid, Philip; Yee, Ying K; Tebbe, Anne Louise; Ratz, Andrew M; Herron, David K; Goodson, Theodore; Masters, John J; Franciskovich, Jeffry B; Tinsley, Jennifer M; Wiley, Michael R; Weir, Leonard C; Kyle, Jeffrey A; Klimkowski, Valentine J; Smith, Gerald F; Towner, Richard D; Froelich, Larry L; Buben, John; Craft, Trelia J

    2007-09-01

    SAR about the B-ring of a series of N(2)-aroyl anthranilamide factor Xa (fXa) inhibitors is described. B-ring o-aminoalkylether and B-ring p-amine probes of the S1' and S4 sites, respectively, afforded picomolar fXa inhibitors that performed well in in vitro anticoagulation assays.

  11. Proteinaceous alpha-amylase inhibitors.

    PubMed

    Svensson, Birte; Fukuda, Kenji; Nielsen, Peter K; Bønsager, Birgit C

    2004-02-12

    Proteins that inhibit alpha-amylases have been isolated from plants and microorganisms. These inhibitors can have natural roles in the control of endogenous alpha-amylase activity or in defence against pathogens and pests; certain inhibitors are reported to be antinutritional factors. The alpha-amylase inhibitors belong to seven different protein structural families, most of which also contain evolutionary related proteins without inhibitory activity. Two families include bifunctional inhibitors acting both on alpha-amylases and proteases. High-resolution structures are available of target alpha-amylases in complex with inhibitors from five families. These structures indicate major diversity but also some similarity in the structural basis of alpha-amylase inhibition. Mutational analysis of the mechanism of inhibition was performed in a few cases and various protein engineering and biotechnological approaches have been outlined for exploitation of the inhibitory function. PMID:14871655

  12. Authentic HIV-1 integrase inhibitors

    PubMed Central

    Liao, Chenzhong; Marchand, Christophe; Burke, Terrence R; Pommier, Yves; Nicklaus, Marc C

    2010-01-01

    HIV-1 integrase (IN) is indispensable for HIV-1 replication and has become a validated target for developing anti-AIDS agents. In two decades of development of IN inhibition-based anti-HIV therapeutics, a significant number of compounds were identified as IN inhibitors, but only some of them showed antiviral activity. This article reviews a number of patented HIV-1 IN inhibitors, especially those that possess high selectivity for the strand transfer reaction. These compounds generally have a polar coplanar moiety, which is assumed to chelate two magnesium ions in the binding site. Resistance to those compounds, when given to patients, can develop as a result of IN mutations. We refer to those compounds as authentic IN inhibitors. Continued drug development has so far delivered one authentic IN inhibitor to the market (raltegravir in 2007). Current and future attention will be focused on the development of novel authentic IN inhibitors with the goal of overcoming viral resistance. PMID:21426159

  13. Oxidized mucus proteinase inhibitor: a fairly potent neutrophil elastase inhibitor.

    PubMed Central

    Boudier, C; Bieth, J G

    1994-01-01

    N-chlorosuccinimide oxidizes one of the methionine residues of mucus proteinase inhibitor with a second-order rate constant of 1.5 M-1.s-1. Cyanogen bromide cleavage and NH2-terminal sequencing show that the modified residue is methionine-73, the P'1 component of the inhibitor's active centre. Oxidation of the inhibitor decreases its neutrophil elastase inhibitory capacity but does not fully abolish it. The kinetic parameters describing the elastase-oxidized inhibitor interaction are: association rate constant kass. = 2.6 x 10(5) M-1.s-1, dissociation rate constant kdiss. = 2.9 x 10(-3) s-1 and equilibrium dissociation constant Ki = 1.1 x 10(-8) M. Comparison with the native inhibitor indicates that oxidation decreases kass. by a factor of 18.8 and increases kdiss. by a factor of 6.4, and therefore leads to a 120-fold increase in Ki. Yet, the oxidized inhibitor may still act as a potent elastase inhibitor in the upper respiratory tract where its concentration is 500-fold higher than Ki, i.e. where the elastase inhibition is pseudo-irreversible. Experiments in vitro with fibrous human lung elastin, the most important natural substrate of elastase, support this view: 1.35 microM elastase is fully inhibited by 5-6 microM oxidized inhibitor whether the enzyme-inhibitor complex is formed in the presence or absence of elastin and whether elastase is pre-adsorbed on elastin or not. PMID:7945266

  14. A new charge-tagged proline-based organocatalyst for mechanistic studies using electrospray mass spectrometry

    PubMed Central

    Willms, J Alexander; Beel, Rita; Schmidt, Martin L; Mundt, Christian

    2014-01-01

    Summary A new 4-hydroxy-L-proline derivative with a charged 1-ethylpyridinium-4-phenoxy substituent has been synthesized with the aim of facilitating mechanistic studies of proline-catalyzed reactions by ESI mass spectrometry. The charged residue ensures a strongly enhanced ESI response compared to neutral unmodified proline. The connection by a rigid linker fixes the position of the charge tag far away from the catalytic center in order to avoid unwanted interactions. The use of a charged catalyst leads to significantly enhanced ESI signal abundances for every catalyst-derived species which are the ones of highest interest present in a reacting solution. The new charged proline catalyst has been tested in the direct asymmetric inverse aldol reaction between aldehydes and diethyl ketomalonate. Two intermediates in accordance with the List–Houk mechanism for enamine catalysis have been detected and characterized by gas-phase fragmentation. In addition, their temporal evolution has been followed using a microreactor continuous-flow technique. PMID:25246962

  15. Identification of pyrrolo[3,2-c]pyridin-4-amine compounds as a new class of entry inhibitors against influenza viruses in vitro.

    PubMed

    Chang, So Young; Cruz, Deu John M; Ko, Yoonae; Min, Ji-Young

    2016-09-30

    Various influenza virus entry inhibitors are being developed as therapeutic antiviral agents in ongoing preparation for emerging influenza viruses, particularly those that may possess drug resistance to the current FDA-approved neuraminidase inhibitors. In this study, small molecules having the pyrrolopyridinamine (PPA), aminothiadiazole (ATD), dihydrofuropyridine carboxamide (HPC), or imidazopyridinamine (IPA) moiety were selected from a target-focused chemical library for their inhibitory activity against influenza A virus by high-throughput screening using the PR8GFP assay. Activity was evaluated by measuring changes the proportion of GFP-expressing cells as a reflection of influenza virus infection. Among them, PPA showed broad-spectrum activity against multiple influenza A viruses and influenza B virus. PPA was found to block the early stages of influenza virus infection using a time-of-addition assay. Using additional phenotypic assays that dissect the virus entry process, it appears that the antiviral activity of PPA against influenza virus can be attributed to interference of the post-fusion process: namely, virus uncoating and nuclear import of viral nucleoprotein complexes. Based on these results, PPA is an attractive chemical moiety that can be used to develop new antiviral drug candidates against influenza viruses. PMID:27586275

  16. Flavivirus Entry Inhibitors.

    PubMed

    Wang, Qing-Yin; Shi, Pei-Yong

    2015-09-11

    Many flaviviruses are significant human pathogens that are transmitted by mosquitoes and ticks. Although effective vaccines are available for yellow fever virus, Japanese encephalitic virus, and tick-borne encephalitis virus, these and other flaviviruses still cause thousands of human deaths and millions of illnesses each year. No clinically approved antiviral therapy is available for flavivirus treatment. To meet this unmet medical need, industry and academia have taken multiple approaches to develop antiflavivirus therapy, among which targeting viral entry has been actively pursued in the past decade. Here we review the current knowledge of flavivirus entry and its use for small molecule drug discovery. Inhibitors of two major steps of flaviviral entry have been reported: (i) molecules that block virus-receptor interaction; (ii) compounds that prevent conformational change of viral envelope protein during virus-host membrane fusion. We also discuss the advantages and disadvantages of targeting viral entry for treatment of flavivirus infection as compared to targeting viral replication proteins. PMID:27617926

  17. Small molecules inhibitors of plasminogen activator inhibitor-1 - an overview.

    PubMed

    Rouch, Anne; Vanucci-Bacqué, Corinne; Bedos-Belval, Florence; Baltas, Michel

    2015-03-01

    PAI-1, a glycoprotein from the serpin family and the main inhibitor of tPA and uPA, plays an essential role in the regulation of intra and extravascular fibrinolysis by inhibiting the formation of plasmin from plasminogen. PAI-1 is also involved in pathological processes such as thromboembolic diseases, atherosclerosis, fibrosis and cancer. The inhibition of PAI-1 activity by small organic molecules has been observed in vitro and with some in vivo models. Based on these findings, PAI-1 appears as a potential therapeutic target for several pathological conditions. Over the past decades, many efforts have therefore been devoted to developing PAI-1 inhibitors. This article provides an overview of the publishing activity on small organic molecules used as PAI-1 inhibitors. The chemical synthesis of the most potent inhibitors as well as their biological and biochemical evaluations is also presented.

  18. Synthetic conversion of ACAT inhibitor to acetylcholinesterase inhibitor.

    PubMed

    Obata, R; Sunazuka, T; Otoguro, K; Tomoda, H; Harigaya, Y; Omura, S

    2000-06-19

    Natural product acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor pyripyropene A was synthetically converted to acetylcholinesterase (AChE) inhibitor via heterolitic cleavage of the 2-pyrone ring, followed by gamma-acylation/cyclization with several aroyl chlorides. The 4-pyridyl analogue selectively showed AChE inhibitory activity (IC50 7.9 microM) and no ACAT inhibitory activity IC50 = >1000 microM. PMID:10890154

  19. Glycosylasparaginase inhibition studies: competitive inhibitors, transition state mimics, noncompetitive inhibitors.

    PubMed

    Risley, J M; Huang, D H; Kaylor, J J; Malik, J J; Xia, Y Q

    2001-01-01

    Glycosylasparaginase catalyzes the hydrolysis of the N-glycosylic bond between asparagine and N-acetylglucosamine in the catabolism of N-linked glycoproteins. Previously only three competitive inhibitors, one noncompetitive inhibitor, and one irreversible inhibitor of glycosylasparaginase activity had been reported. Using human glycosylasparaginase from human amniotic fluid, L-aspartic acid and four of its analogues, where the alpha-amino group was substituted with a chloro, bromo, methyl or hydrogen, were competitive inhibitors having Ki values between 0.6-7.7 mM. These results provide supporting evidence for a proposed intramolecular autoproteolytic activation reaction. A proposed phosphono transition state mimic and a sulfo transition state mimic were competitive inhibitors with Ki values 0.9 mM and 1.4 mM, respectively. These results support a mechanism for the enzyme-catalyzed reaction involving formation of a tetrahedral high-energy intermediate. Three analogues of the natural substrate were noncompetitive inhibitors with Ki values between 0.56-0.75 mM, indicating the presence of a second binding site that may recognize (substituted)acetamido groups.

  20. Selective Inhibitors of Protein Methyltransferases

    PubMed Central

    2015-01-01

    Mounting evidence suggests that protein methyltransferases (PMTs), which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and human diseases. In particular, PMTs have been recognized as major players in regulating gene expression and chromatin state. PMTs are divided into two categories: protein lysine methyltransferases (PKMTs) and protein arginine methyltransferases (PRMTs). There has been a steadily growing interest in these enzymes as potential therapeutic targets and therefore discovery of PMT inhibitors has also been pursued increasingly over the past decade. Here, we present a perspective on selective, small-molecule inhibitors of PMTs with an emphasis on their discovery, characterization, and applicability as chemical tools for deciphering the target PMTs’ physiological functions and involvement in human diseases. We highlight the current state of PMT inhibitors and discuss future directions and opportunities for PMT inhibitor discovery. PMID:25406853

  1. Synthesis of lysine methyltransferase inhibitors

    PubMed Central

    Hui, Chunngai; Ye, Tao

    2015-01-01

    Lysine methyltransferase which catalyze methylation of histone and non-histone proteins, play a crucial role in diverse biological processes and has emerged as a promising target for the development of various human diseases, including cancer, inflammation, and psychiatric disorders. However, inhibiting lysine methyltransferases selectively has presented many challenges to medicinal chemists. During the past decade, lysine methyltransferase inhibitors covering many different structural classes have been designed and developed. In this review, we describe the development of selective, small-molecule inhibitors of lysine methyltransferases with an emphasis on their discovery and chemical synthesis. We highlight the current state of lysine methyltransferase inhibitors and discuss future directions and opportunities for lysine methyltransferase inhibitor discovery. PMID:26258118

  2. [Pharmacology of bone resorption inhibitor].

    PubMed

    Menuki, Kunitaka; Sakai, Akinori

    2015-10-01

    Currently, bone resorption inhibitor is mainly used for osteoporosis. A number of these agents have been developed. These pharmacological action are various. Bisphosphonate inhibit functions of the osteoclasts by inducing apoptosis. On the one hand, RANK-ligand inhibitor and selective estrogen receptor modulator inhibit formation of osteoclasts. It is important to understand these pharmacological action for the selection of the appropriate medicine. PMID:26529923

  3. The NEU1-selective sialidase inhibitor, C9-butyl-amide-DANA, blocks sialidase activity and NEU1-mediated bioactivities in human lung in vitro and murine lung in vivo.

    PubMed

    Hyun, Sang W; Liu, Anguo; Liu, Zhenguo; Cross, Alan S; Verceles, Avelino C; Magesh, Sadagopan; Kommagalla, Yadagiri; Kona, Chandrababunaidu; Ando, Hiromune; Luzina, Irina G; Atamas, Sergei P; Piepenbrink, Kurt H; Sundberg, Eric J; Guang, Wei; Ishida, Hideharu; Lillehoj, Erik P; Goldblum, Simeon E

    2016-08-01

    Neuraminidase-1 (NEU1) is the predominant sialidase expressed in human airway epithelia and lung microvascular endothelia where it mediates multiple biological processes. We tested whether the NEU1-selective sialidase inhibitor, C9-butyl-amide-2-deoxy-2,3-dehydro-N-acetylneuraminic acid (C9-BA-DANA), inhibits one or more established NEU1-mediated bioactivities in human lung cells. We established the IC50 values of C9-BA-DANA for total sialidase activity in human airway epithelia, lung microvascular endothelia and lung fibroblasts to be 3.74 µM, 13.0 µM and 4.82 µM, respectively. In human airway epithelia, C9-BA-DANA dose-dependently inhibited flagellin-induced, NEU1-mediated mucin-1 ectodomain desialylation, adhesiveness for Pseudomonas aeruginosa and shedding. In lung microvascular endothelia, C9-BA-DANA reversed NEU1-driven restraint of cell migration into a wound and disruption of capillary-like tube formation. NEU1 and its chaperone/transport protein, protective protein/cathepsin A (PPCA), were differentially expressed in these same cells. Normalized NEU1 protein expression correlated with total sialidase activity whereas PPCA expression did not. In contrast to eukaryotic sialidases, C9-BA-DANA exerted far less inhibitory activity for three selected bacterial neuraminidases (IC50 > 800 µM). Structural modeling of the four human sialidases and three bacterial neuraminidases revealed a loop between the seventh and eighth strands of the β-propeller fold, that in NEU1, was substantially shorter than that seen in the six other enzymes. Predicted steric hindrance between this loop and C9-BA-DANA could explain its selectivity for NEU1. Finally, pretreatment of mice with C9-BA-DANA completely protected against flagellin-induced increases in lung sialidase activity. Our combined data indicate that C9-BA-DANA inhibits endogenous and ectopically expressed sialidase activity and established NEU1-mediated bioactivities in human airway epithelia, lung microvascular

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

    SciTech Connect

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

    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.

  5. Peptidomimetic inhibitors of HIV protease.

    PubMed

    Randolph, John T; DeGoey, David A

    2004-01-01

    There are currently (July, 2002) six protease inhibitors approved for the treatment of HIV infection, each of which can be classified as peptidomimetic in structure. These agents, when used in combination with other antiretroviral agents, produce a sustained decrease in viral load, often to levels below the limits of quantifiable detection, and a significant reconstitution of the immune system. Therapeutic regimens containing one or more HIV protease inhibitors thus provide a highly effective method for disease management. The important role of protease inhibitors in HIV therapy, combined with numerous challenges remaining in HIV treatment, have resulted in a continued effort both to optimize regimens using the existing agents and to identify new protease inhibitors that may provide unique properties. This review will provide an overview of the discovery and clinical trials of the currently approved HIV protease inhibitors, followed by an examination of important aspects of therapy, such as pharmacokinetic enhancement, resistance and side effects. A description of new peptidomimetic compounds currently being investigated in the clinic and in preclinical discovery will follow. PMID:15193140

  6. Microbial inhibitors of cysteine proteases.

    PubMed

    Kędzior, Mateusz; Seredyński, Rafał; Gutowicz, Jan

    2016-08-01

    Cysteine proteases are one of the major classes of proteolytic enzymes involved in a number of physiological and pathological processes in plants, animals and microorganisms. When their synthesis, activity and localization in mammalian cells are altered, they may contribute to the development of many diseases, including rheumatoid arthritis, osteoporosis and cancer. Therefore, cysteine proteases have become promising drug targets for the medical treatment of these disorders. Inhibitors of cysteine proteases are also produced by almost every group of living organisms, being responsible for the control of intracellular proteolytic activity. Microorganisms synthesize cysteine protease inhibitors not only to regulate the activity of endogenous, often virulent enzymes, but also to hinder the host's proteolytic defense system and evade its immune responses against infections. Present work describes known to date microbial inhibitors of cysteine proteases in terms of their structure, enzyme binding mechanism, specificity and pathophysiological roles. The overview of both proteinaceous and small-molecule inhibitors produced by all groups of microorganisms (bacteria, archaea, fungi, protists) and viruses is provided. Subsequently, possible applications of microbial inhibitors in science, medicine and biotechnology are also highlighted. PMID:27048482

  7. Evolutionary families of peptidase inhibitors.

    PubMed Central

    Rawlings, Neil D; Tolle, Dominic P; Barrett, Alan J

    2004-01-01

    The proteins that inhibit peptidases are of great importance in medicine and biotechnology, but there has never been a comprehensive system of classification for them. Some of the terminology currently in use is potentially confusing. In the hope of facilitating the exchange, storage and retrieval of information about this important group of proteins, we now describe a system wherein the inhibitor units of the peptidase inhibitors are assigned to 48 families on the basis of similarities detectable at the level of amino acid sequence. Then, on the basis of three-dimensional structures, 31 of the families are assigned to 26 clans. A simple system of nomenclature is introduced for reference to each clan, family and inhibitor. We briefly discuss the specificities and mechanisms of the interactions of the inhibitors in the various families with their target enzymes. The system of families and clans of inhibitors described has been implemented in the MEROPS peptidase database (http://merops.sanger.ac.uk/), and this will provide a mechanism for updating it as new information becomes available. PMID:14705960

  8. Microbial inhibitors of cysteine proteases.

    PubMed

    Kędzior, Mateusz; Seredyński, Rafał; Gutowicz, Jan

    2016-08-01

    Cysteine proteases are one of the major classes of proteolytic enzymes involved in a number of physiological and pathological processes in plants, animals and microorganisms. When their synthesis, activity and localization in mammalian cells are altered, they may contribute to the development of many diseases, including rheumatoid arthritis, osteoporosis and cancer. Therefore, cysteine proteases have become promising drug targets for the medical treatment of these disorders. Inhibitors of cysteine proteases are also produced by almost every group of living organisms, being responsible for the control of intracellular proteolytic activity. Microorganisms synthesize cysteine protease inhibitors not only to regulate the activity of endogenous, often virulent enzymes, but also to hinder the host's proteolytic defense system and evade its immune responses against infections. Present work describes known to date microbial inhibitors of cysteine proteases in terms of their structure, enzyme binding mechanism, specificity and pathophysiological roles. The overview of both proteinaceous and small-molecule inhibitors produced by all groups of microorganisms (bacteria, archaea, fungi, protists) and viruses is provided. Subsequently, possible applications of microbial inhibitors in science, medicine and biotechnology are also highlighted.

  9. Targeting a cluster of arginine residues of neuraminidase to avoid oseltamivir resistance in influenza A (H1N1): a theoretical study.

    PubMed

    Gema, L Ramírez-Salinas; Tolentino-Lopez, L E; Martínez-Ramos, F; Padilla-Martínez, I; García-Machorro, J; Correa-Basurto, J

    2015-01-01

    Following the influenza A (H1N1) pandemic in Mexico and around the world in 2009, the numbers of oseltamivir-resistant clinical cases have increased through a mechanism that remains unclear. In this work, we focus on studying the mutated NA structures ADA71175 (GenBank) and 3CKZ (PDB ID). Recently crystallized NA (PDB ID: 3NSS) was used as a wild-type structure and template to construct the three-dimensional (3D) structure of ADA71175. Then, the NA mutants and 3NSS natives as well as their refined monomer structures as determined through MD simulations (snapshots at 50 ns) were used as models to perform a docking study using a set of aryl-oseltamivir derivatives. These aryl-oseltamivir derivatives have better recognition properties than oseltamivir because of cation-π interactions with a cluster of Arg residues (118, 292, and 371) at the binding site. This cluster of Arg residues represents a potential binding site for aryl-oseltamivir derivatives that are potentially new NA inhibitors.

  10. Positron emitter labeled enzyme inhibitors

    SciTech Connect

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.; Langstrom, B.

    1990-04-03

    This invention involves a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography.

  11. Electrochemical studies of corrosion inhibitors

    NASA Technical Reports Server (NTRS)

    Danford, M. D.

    1990-01-01

    The effect of single salts, as well as multicomponent mixtures, on corrosion inhibition was studied for type 1010 steel; for 5052, 1100, and 2219-T87 aluminum alloys; and for copper. Molybdate-containing inhibitors exhibit an immediate, positive effect for steel corrosion, but an incubation period may be required for aluminum before the effect of a given inhibitor can be determined. The absence of oxygen was found to provide a positive effect (smaller corrosion rate) for steel and copper, but a negative effect for aluminum. This is attributed to the two possible mechanisms by which aluminum can oxidize. Corrosion inhibition is generally similar for oxygen-rich and oxygen-free environments. The results show that the electrochemical method is an effective means of screening inhibitors for the corrosion of single metals, with caution to be exercised in the case of aluminum.

  12. Positron emitter labeled enzyme inhibitors

    DOEpatents

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.

    1987-05-22

    This invention involved a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide in activators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography. 2 figs.

  13. Positron emitter labeled enzyme inhibitors

    DOEpatents

    Fowler, Joanna S.; MacGregor, Robert R.; Wolf, Alfred P.; Langstrom, Bengt

    1990-01-01

    This invention involves a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography.

  14. Corrosion inhibitors from expired drugs.

    PubMed

    Vaszilcsin, Nicolae; Ordodi, Valentin; Borza, Alexandra

    2012-07-15

    This paper presents a method of expired or unused drugs valorization as corrosion inhibitors for metals in various media. Cyclic voltammograms were drawn on platinum in order to assess the stability of pharmaceutically active substances from drugs at the metal-corrosive environment interface. Tafel slope method was used to determine corrosion rates of steel in the absence and presence of inhibitors. Expired Carbamazepine and Paracetamol tablets were used to obtain corrosion inhibitors. For the former, the corrosion inhibition of carbon steel in 0.1 mol L(-1) sulfuric acid solution was about 90%, whereas for the latter, the corrosion inhibition efficiency of the same material in the 0.25 mol L(-1) acetic acid-0.25 mol L(-1) sodium acetate buffer solution was about 85%.

  15. An environmentally friendly scale inhibitor

    SciTech Connect

    Dobbs, J.B.; Brown, J.M.

    1999-11-01

    This paper describes a method of inhibiting the formation of scales such as barium and strontium sulfate in low pH aqueous systems, and calcium carbonate in systems containing high concentrations of dissolved iron. The solution, chemically, involves treating the aqueous system with an inhibitor designed to replace organic-phosphonates. Typical low pH aqueous systems where the inhibitor is particularly useful are oilfield produced-water, resin bed water softeners that form scale during low pH, acid regeneration operations. Downhole applications are recommended where high concentrations of dissolved iron are present in the produced water. This new approach to inhibition replaces typical organic phosphonates and polymers with a non-toxic, biodegradable scale inhibitor that performs in harsh environments.

  16. Diverse inhibitors of aflatoxin biosynthesis.

    PubMed

    Holmes, Robert A; Boston, Rebecca S; Payne, Gary A

    2008-03-01

    Pre-harvest and post-harvest contamination of maize, peanuts, cotton, and tree nuts by members of the genus Aspergillus and subsequent contamination with the mycotoxin aflatoxin pose a widespread food safety problem for which effective and inexpensive control strategies are lacking. Since the discovery of aflatoxin as a potently carcinogenic food contaminant, extensive research has been focused on identifying compounds that inhibit its biosynthesis. Numerous diverse compounds and extracts containing activity inhibitory to aflatoxin biosynthesis have been reported. Only recently, however, have tools been available to investigate the molecular mechanisms by which these inhibitors affect aflatoxin biosynthesis. Many inhibitors are plant-derived and a few may be amenable to pathway engineering for tissue-specific expression in susceptible host plants as a defense against aflatoxin contamination. Other compounds show promise as protectants during crop storage. Finally, inhibitors with different modes of action could be used in comparative transcriptional and metabolomic profiling experiments to identify regulatory networks controlling aflatoxin biosynthesis.

  17. Nonnucleoside inhibitors of adenosine kinase.

    PubMed

    Gomtsyan, Arthur; Lee, Chih-Hung

    2004-01-01

    Adenosine (ADO) is an endogenous inhibitory neuromodulator that increases nociceptive thresholds in response to tissue trauma and inflammation. Adenosine kinase (AK) is a key intracellular enzyme regulating intra- and extracellular concentrations of ADO. AK inhibition selectively amplifies extracellular ADO levels at cell and tissue sites where accelerated release of ADO occurs. AK inhibitors have been shown to provide effective antinociceptive, antiinflammatory and anticonvulsant activity in animal models, thus suggesting their potential therapeutic utility for pain, inflammation, epilepsy and possibly other central and peripheral nervous system diseases associated with cellular trauma and inflammation. This beneficial outcome may potentially lack nonspecific effects associated with the systemic administration of ADO receptor agonists. Until recently all of the reported AK inhibitors contained adenosine-like structural motif. The present review will discuss design, synthesis and analgesic and antiinflammatory properties of the novel nonnucleoside AK inhibitors that do not have close structural resemblance with the natural substrate ADO. Two classes of the nonnucleoside AK inhibitors are built on pyridopyrimidine and alkynylpyrimidine cores.

  18. C1-inhibitor and transplantation.

    PubMed

    Kirschfink, Michael

    2002-09-01

    Excessive activation of the protein cascade systems has been associated with post-transplantation inflammatory disorders. There is increasing evidence that complement not only significantly contributes to ischemia/reperfusion injury upon cold storage of the organ but also, although to a different degree, to allograft rejection. Complement activation is most fulminant in hyperacute rejection but seems also to contribute to acute transplant rejection. Therapeutic substitution of appropriate regulators, therefore, appears to be a reasonable approach to reduce undesirable inflammatory reactions in the grafted organ. C1-inhibitor, a multifunctional regulator of the various kinin-generating cascade systems (for review see: E. Hack, chapter in this issue), is frequently reduced in patients suffering from severe inflammatory disorders. Studies applying pathophysiologically relevant animal models of allo- and xenotransplantation as well as promising first clinical results from successful allotransplantation now provide evidence that C1-inhibitor may also serve as an effective means to protect the grafted organ against inflammatory tissue injury. In xenotransplantation, complement inhibition by specific regulators such as C1-inhibitor may help to overcome hyperacute graft rejection. After a brief introduction on the significance of complement to allo- and xenotransplantation the following review will focus on the impact of C1-inhibitor treatment on transplantation-associated inflammatory disorders, where complement contributes to the pathogenesis.

  19. Bivalent Inhibitors of Protein Kinases

    PubMed Central

    Gower, Carrie M.; Chang, Matthew E. K.; Maly, Dustin J.

    2015-01-01

    Protein kinases are key players in a large number of cellular signaling pathways. Dysregulated kinase activity has been implicated in a number of diseases, and members of this enzyme family are of therapeutic interest. However, due to the fact that most inhibitors interact with the highly conserved ATP-binding sites of kinases, it is a significant challenge to develop pharmacological agents that target only one of the greater than 500 kinases present in humans. A potential solution to this problem is the development of bisubstrate and bivalent kinase inhibitors, in which an active site-directed moiety is tethered to another ligand that targets a location outside of the ATP-binding cleft. Because kinase signaling specificity is modulated by regions outside of the ATP-binding site, strategies that exploit these interactions have the potential to provide reagents with high target selectivity. This review highlights examples of kinase interaction sites that can potentially be exploited by bisubstrate and bivalent inhibitors. Furthermore, an overview of efforts to target these interactions with bisubstrate and bivalent inhibitors is provided. Finally, several examples of the successful application of these reagents in a cellular setting are described. PMID:24564382

  20. PDE-5 inhibitors: clinical points.

    PubMed

    Doumas, Michael; Lazaridis, Antonios; Katsiki, Niki; Athyros, Vasilios

    2015-01-01

    Erectile dysfunction is usually of vascular origin and is frequently encountered in men with cardiovascular disease. The introduction of phosphodiesterase-5 inhibitors has revolutionized the management of patients with erectile dysfunction. Currently available phosphodiesterase-5 inhibitors have distinct pharmacokinetic and pharmacodynamic properties, thus permitting for tailoring sexual therapy according to patient characteristics and needs. Phosphodiesterase-5 inhibitors possess vasorelaxing properties and exert systemic hemodynamic effects, which need to be taken into account when other cardiovascular drugs are co-administered. Special caution is needed with alpha-blockers, while the co-administration with nitrates is contra-indicated due to the risk of life-threatening hypotension. This review presents the advent of sexual therapy, describes the mechanism of action and the specific characteristics of commercially available phosphodiesterase-5 inhibitors, summarizes the efficacy and safety of these drugs with special emphasis on the cardiovascular system, and discusses the clinical criteria used for the selection of each drug for the individual patient. PMID:25392015

  1. Acetylcholinesterase Inhibitors: Pharmacology and Toxicology

    PubMed Central

    Čolović, Mirjana B; Krstić, Danijela Z; Lazarević-Pašti, Tamara D; Bondžić, Aleksandra M; Vasić, Vesna M

    2013-01-01

    Acetylcholinesterase is involved in the termination of impulse transmission by rapid hydrolysis of the neurotransmitter acetylcholine in numerous cholinergic pathways in the central and peripheral nervous systems. The enzyme inactivation, induced by various inhibitors, leads to acetylcholine accumulation, hyperstimulation of nicotinic and muscarinic receptors, and disrupted neurotransmission. Hence, acetylcholinesterase inhibitors, interacting with the enzyme as their primary target, are applied as relevant drugs and toxins. This review presents an overview of toxicology and pharmacology of reversible and irreversible acetylcholinesterase inactivating compounds. In the case of reversible inhibitors being commonly applied in neurodegenerative disorders treatment, special attention is paid to currently approved drugs (donepezil, rivastigmine and galantamine) in the pharmacotherapy of Alzheimer’s disease, and toxic carbamates used as pesticides. Subsequently, mechanism of irreversible acetylcholinesterase inhibition induced by organophosphorus compounds (insecticides and nerve agents), and their specific and nonspecific toxic effects are described, as well as irreversible inhibitors having pharmacological implementation. In addition, the pharmacological treatment of intoxication caused by organophosphates is presented, with emphasis on oxime reactivators of the inhibited enzyme activity administering as causal drugs after the poisoning. Besides, organophosphorus and carbamate insecticides can be detoxified in mammals through enzymatic hydrolysis before they reach targets in the nervous system. Carboxylesterases most effectively decompose carbamates, whereas the most successful route of organophosphates detoxification is their degradation by corresponding phosphotriesterases. PMID:24179466

  2. Biocatalysts with enhanced inhibitor tolerance

    DOEpatents

    Yang, Shihui; Linger, Jeffrey; Franden, Mary Ann; Pienkos, Philip T.; Zhang, Min

    2015-12-08

    Disclosed herein are biocatalysts for the production of biofuels, including microorganisms that contain genetic modifications conferring tolerance to growth and fermentation inhibitors found in many cellulosic feedstocks. Methods of converting cellulose-containing materials to fuels and chemicals, as well as methods of fermenting sugars to fuels and chemicals, using these biocatalysts are also disclosed.

  3. Proton pump inhibitor-induced hypomagnesemic hypoparathyroidism.

    PubMed

    Swaminathan, Krishnan

    2015-01-01

    Proton pump inhibitors are the one of the most widely used drugs in the world. Hypomagnesemic hypoparathyroidism has been reported with different proton pump inhibitors with prolonged oral use. We report the first reported case of possible such effect with intravenous preparation of proton pump inhibitor. This case report raises awareness among physicians worldwide of this often unknown association, as life-threatening cardiac and neuromuscular complications can arise with unrecognized hypocalcemia and hypomagnesemia with proton pump inhibitors.

  4. KID, a Kinase Inhibitor Database project.

    PubMed

    Collin, O; Meijer, L

    1999-01-01

    The Kinase Inhibitor Database is a small specialized database dedicated to the gathering of information on protein kinase inhibitors. The database is accessible through the World Wide Web system and gives access to structural and bibliographic information on protein kinase inhibitors. The data in the database will be collected and submitted by researchers working in the kinase inhibitor field. The submitted data will be checked by the curator of the database before entry.

  5. Sialyltransferase and Neuraminidase Levels/Ratios and Sialic Acid Levels in Peripheral Blood B Cells Correlate with Measures of Disease Activity in Patients with Systemic Lupus Erythematosus and Rheumatoid Arthritis: A Pilot Study

    PubMed Central

    Liou, Lieh-bang; Huang, Che-ching

    2016-01-01

    Objective We attempted to determine whether the level of enzymes sialyltransferase (ST) and neuraminidase (Neu) and sialic acid (SIA) in patients with systemic lupus erythematosus (SLE) correlates with the SLE Disease Activity Index (SLEDAI) and in patients with rheumatoid arthritis (RA) correlates with the Disease Activity Score28 (DAS28). Methods We examined cell-surface levels of ST6Gal-1, Neu1, ST3Gal-1, Neu3, α-2,6-SIA, and α-2,3-SIA by using fluorescent anti-enzyme antibodies, fluorescent-conjugated Sambucus nigra lectin, and fluorescent-conjugated Maackia amurensis lectin on blood cells in SLE and RA patients and assessed correlations of these levels with SLEDAI and with DAS28. Areas under the curve (AUC) were calculated for different variables against SLEDAI. Results The B-cell ST3Gal-1/Neu3 ratio positively correlated with SLEDAI scores (ρ = 0.409 and P = 0.002, statistically significant after Bonferroni’ correction for multiple analyses.). It was supported by the inverse correlation of B-cell Neu3 levels with SLEDAI scores (ρ = −0.264, P = 0.048). The B-cell ST3Gal-1/Neu3 ratio against SLEDAI yielded an AUC of 0.689, which was comparable to that of anti-dsDNA levels at 0.635. In contrast, both ST3Gal-1 and Neu3 levels of RA B cells (r = 0.376, P = 0.013; r = 0.425, P = 0.005, respectively) correlated positively with high disease-activity DAS28 scores. Conclusion B-cell ST3Gal-1/Neu3 ratios in SLE and B-cell ST3Gal-1 and Neu3 levels in RA with high disease-activity DAS28 scores correlated with disease activity measures and may be useful in monitoring disease activities. PMID:26981635

  6. Enhanced replication of avian-origin H3N2 canine influenza virus in eggs, cell cultures and mice by a two-amino acid insertion in neuraminidase stalk.

    PubMed

    Lin, Yan; Xie, Xing; Zhao, Yanbing; Kalhoro, Dildar Hussain; Lu, Chengping; Liu, Yongjie

    2016-01-01

    Canine influenza virus (CIV) is a newly identified, highly contagious respiratory pathogen in dogs. Recent studies indicate that avian-origin H3N2 CIV are circulating in Chinese dogs. To investigate the effects of a two-amino acid (2-aa) insertion naturally occurring at the distal end of the neuraminidase (NA) stalk found in Chinese isolates since 2010 on virus replication and virulence, we rescued the CIV strain, A/canine/Jiangsu/06/2011(H3N2) and its NA mutant without the 2-aa insertion using reverse genetics. The NA stalk length affected virus growth in cell culture. Compared to the short stalk strain (without 2-aa insertion), the long stalk strain (with 2-aa insertion) exhibited higher peak titers and greater yields in Madin-Darby canine kidney (MDCK) cells, chicken embryo fibroblasts and canine bronchiolar epithelial cells, as well as much larger plaques in MDCK cell monolayers. Furthermore, mice inoculated with the long stalk strain showed more severe pathologic damage in lung and higher proportion of detectable viral RNA in tissues. The long stalk strain induced local IFN-γ production with faster kinetics and higher levels in mice. However, in chickens, the two viral strains showed no significant difference with nearly the same proportion of detectable viral RNA loads in tissues. These observations suggest that the 2-aa insertion in the NA stalk acquired by avian-origin H3N2 CIV helps to enhance viral replication and is likely a result of adaptive evolution in canine hosts. PMID:27160077

  7. SYBR green-based real-time reverse transcription-PCR for typing and subtyping of all hemagglutinin and neuraminidase genes of avian influenza viruses and comparison to standard serological subtyping tests

    USGS Publications Warehouse

    Tsukamoto, K.; Javier, P.C.; Shishido, M.; Noguchi, D.; Pearce, J.; Kang, H.-M.; Jeong, O.M.; Lee, Y.-J.; Nakanishi, K.; Ashizawa, T.

    2012-01-01

    Continuing outbreaks of H5N1 highly pathogenic (HP) avian influenza virus (AIV) infections of wild birds and poultry worldwide emphasize the need for global surveillance of wild birds. To support the future surveillance activities, we developed a SYBR green-based, real-time reverse transcriptase PCR (rRT-PCR) for detecting nucleoprotein (NP) genes and subtyping 16 hemagglutinin (HA) and 9 neuraminidase (NA) genes simultaneously. Primers were improved by focusing on Eurasian or North American lineage genes; the number of mixed-base positions per primer was set to five or fewer, and the concentration of each primer set was optimized empirically. Also, 30 cycles of amplification of 1:10 dilutions of cDNAs from cultured viruses effectively reduced minor cross- or nonspecific reactions. Under these conditions, 346 HA and 345 NA genes of 349 AIVs were detected, with average sensitivities of NP, HA, and NA genes of 10 1.5, 10 2.3, and 10 3.1 50% egg infective doses, respectively. Utility of rRT-PCR for subtyping AIVs was compared with that of current standard serological tests by using 104 recent migratory duck virus isolates. As a result, all HA genes and 99% of the NA genes were genetically subtyped, while only 45% of HA genes and 74% of NA genes were serologically subtyped. Additionally, direct subtyping of AIVs in fecal samples was possible by 40 cycles of amplification: approximately 70% of HA and NA genes of NP gene-positive samples were successfully subtyped. This validation study indicates that rRT-PCR with optimized primers and reaction conditions is a powerful tool for subtyping varied AIVs in clinical and cultured samples. Copyright ?? 2012, American Society for Microbiology. All Rights Reserved.

  8. Salicylanilide inhibitors of Toxoplasma gondii.

    PubMed

    Fomovska, Alina; Wood, Richard D; Mui, Ernest; Dubey, Jitenter P; Ferreira, Leandra R; Hickman, Mark R; Lee, Patricia J; Leed, Susan E; Auschwitz, Jennifer M; Welsh, William J; Sommerville, Caroline; Woods, Stuart; Roberts, Craig; McLeod, Rima

    2012-10-11

    Toxoplasma gondii (T. gondii) is an apicomplexan parasite that can cause eye disease, brain disease, and death, especially in congenitally infected and immune-compromised people. Novel medicines effective against both active and latent forms of the parasite are greatly needed. The current study focused on the discovery of such medicines by exploring a family of potential inhibitors whose antiapicomplexan activity has not been previously reported. Initial screening efforts revealed that niclosamide, a drug approved for anthelmintic use, possessed promising activity in vitro against T. gondii. This observation inspired the evaluation of the activity of a series of salicylanilides and derivatives. Several inhibitors with activities in the nanomolar range with no appreciable in vitro toxicity to human cells were identified. An initial structure-activity relationship was explored. Four compounds were selected for evaluation in an in vivo model of infection, and two derivatives with potentially enhanced pharmacological parameters demonstrated the best activity profiles.

  9. Salicylanilide Inhibitors of Toxoplasma gondii

    PubMed Central

    Fomovska, Alina; Wood, Richard D.; Mui, Ernest; Dubey, Jitenter P.; Ferriera, Leandra R.; Hickman, Mark R.; Lee, Patricia J.; Leed, Susan E.; Auschwitz, Jennifer M.; Welsh, William J.; Sommerville, Caroline; Woods, Stuart; Roberts, Craig; McLeod, Rima

    2012-01-01

    Toxoplasma gondii(T. gondii) is an apicomplexan parasite that can cause eye disease, brain disease, and death, especially in congenitally infected and immune-compromised people. Novel medicines effective against both active and latent forms of the parasite are greatly needed. The current study focused on the discovery of such medicines by exploring a family of potential inhibitors whose anti-apicomplexan activity has not been previously reported. Initial screening efforts revealed that niclosamide, a drug approved for anthelmintic use, possessed promising activity in vitro against T. gondii. This observation inspired the evaluation of the activity of a series of salicylanilides and derivatives. Several inhibitors with activities in the nanomolar range with no appreciable in vitro toxicity to human cells were identified. An initial structure-activity relationship was explored. Four compounds were selected for evaluation in an in vivo model of infection, and two derivatives with potentially enhanced pharmacological parameters demonstrated the best activity profiles. PMID:22970937

  10. Macrocyclic compounds as corrosion inhibitors

    SciTech Connect

    Quraishi, M.A.; Rawat, J.; Ajmal, M.

    1998-12-01

    The influence of three macrocyclic compounds on corrosion of mild steel (MS) in hydrochloric acid (HCl) was investigated using weight loss, potentiodynamic polarization, alternating current (AC) impedance, and hydrogen permeation techniques. All the investigated compounds showed significant efficiencies and reduced permeation of hydrogen through MS in HCl. Inhibition efficiency (IE) varied with the nature and concentrations of the inhibitors, temperature, and concentrations of the acid solutions. The addition of iodide ions (I{sup {minus}}) increased IE of all the tested compounds as a result of the synergistic effect. Potentiodynamic polarization results revealed that macrocyclic compounds acted as mixed inhibitors in 1 M HCl to 5 M HCl. Adsorption on the metal surface obeyed Temkin`s adsorption isotherm. Auger electron spectroscopy (AES) of the polished MS surface, exposed with tetraphenyldithia-octaazacyclotetradeca-hexaene (PTAT) proved adsorption of this compound on the surface through nitrogen and sulfur atoms.

  11. [Proteasome inhibitors in cancer therapy].

    PubMed

    Romaniuk, Wioletta; Ołdziej, Agnieszka Ewa; Zińczuk, Justyna; Kłoczko, Janusz

    2015-01-01

    Proteasomes are multisubunit enzyme complexes. They contain three enzymatic active sites which are termed chymotrypsin-like, trypsin-like, and caspase-like. The elementary function of the proteasomes is degradation of damaged proteins. Proteasome inhibition leads to accumulation of damaged protein, which leads to caspase activation and cell death. This relationship is used in cancer therapy. Bortezomib is the first proteasome inhibitor approved by the US Food and Drug Administration for the treatment of relapsed/refractory multiple myeloma. Carfilzomib belongs to the second generation of drugs, which was approved by the US FDA in 2012. Currently in the study phase there are four new inhibitors: ixazomib (MLN9780/MLN2238), delanzomib (CEP-18770), oprozomib (ONX0912/PR-047) and marizomib (NPI-0052). PMID:27259216

  12. Aromatase Inhibitors and Other Compounds for Lowering Breast Cancer Risk

    MedlinePlus

    ... References Aromatase inhibitors and other compounds for lowering breast cancer risk Aromatase inhibitors (drugs that lower estrogen levels) ... day. Can aromatase inhibitors lower the risk of breast cancer? Aromatase inhibitors are used mainly to treat hormone ...

  13. Conformation-specific inhibitors of Raf kinases.

    PubMed

    Wang, Xiaolun; Schleicher, Kristin

    2013-01-01

    Since the discovery linking B-Raf mutations to human tumors in 2002, significant advances in the development of Raf inhibitors have been made, leading to the recent approval of two Raf inhibitor drugs. This chapter includes a brief introduction to B-Raf as a validated target and focuses on the three different binding modes observed with Raf small-molecule inhibitors. These various binding modes lock the Raf kinase in different conformations that impact the toxicity profiles of the inhibitors. Possible solutions to mitigate the side effects caused by inhibitor-induced dimerization are also discussed.

  14. Thioredoxin Reductase and its Inhibitors

    PubMed Central

    Saccoccia, Fulvio; Angelucci, Francesco; Boumis, Giovanna; Carotti, Daniela; Desiato, Gianni; Miele, Adriana E; Bellelli, Andrea

    2014-01-01

    Thioredoxin plays a crucial role in a wide number of physiological processes, which span from reduction of nucleotides to deoxyriboucleotides to the detoxification from xenobiotics, oxidants and radicals. The redox function of Thioredoxin is critically dependent on the enzyme Thioredoxin NADPH Reductase (TrxR). In view of its indirect involvement in the above mentioned physio/pathological processes, inhibition of TrxR is an important clinical goal. As a general rule, the affinities and mechanisms of binding of TrxR inhibitors to the target enzyme are known with scarce precision and conflicting results abound in the literature. A relevant analysis of published results as well as the experimental procedures is therefore needed, also in view of the critical interest of TrxR inhibitors. We review the inhibitors of TrxR and related flavoreductases and the classical treatment of reversible, competitive, non competitive and uncompetitive inhibition with respect to TrxR, and in some cases we are able to reconcile contradictory results generated by oversimplified data analysis. PMID:24875642

  15. Carbonic anhydrase inhibitors drug design.

    PubMed

    McKenna, Robert; Supuran, Claudiu T

    2014-01-01

    Inhibition of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) has pharmacologic applications in the field of antiglaucoma, anticonvulsant, antiobesity, and anticancer agents but is also emerging for designing anti-infectives (antifungal and antibacterial agents) with a novel mechanism of action. As a consequence, the drug design of CA inhibitors (CAIs) is a very dynamic field. Sulfonamides and their isosteres (sulfamates/sulfamides) constitute the main class of CAIs which bind to the metal ion in the enzyme active site. Recently the dithiocarbamates, possessing a similar mechanism of action, were reported as a new class of inhibitors. Other families of CAIs possess a distinct mechanism of action: phenols, polyamines, some carboxylates, and sulfocoumarins anchor to the zinc-coordinated water molecule. Coumarins and five/six-membered lactones are prodrug inhibitors, binding in hydrolyzed form at the entrance of the active site cavity. Novel drug design strategies have been reported principally based on the tail approach for obtaining all these types of CAIs, which exploit more external binding regions within the enzyme active site (in addition to coordination to the metal ion), leading thus to isoform-selective compounds. Sugar-based tails as well as click chemistry were the most fruitful developments of the tail approach. Promising compounds that inhibit CAs from bacterial and fungal pathogens, of the dithiocarbamate, phenol and carboxylate types have also been reported. PMID:24146385

  16. ANTIDEPRESSANT ACTIONS OF HDAC INHIBITORS

    PubMed Central

    Covington, Herbert E.; Maze, Ian; LaPlant, Quincey C.; Vialou, Vincent F.; Yoshinori, Ohnishi N.; Berton, Olivier; Fass, Dan M.; Renthal, William; Rush, Augustus J.; Wu, Emma Y.; Ghose, Subroto; Krishnan, Vaishnav; Russo, Scott J.; Tamminga, Carol; Haggarty, Stephen J.; Nestler, Eric J.

    2009-01-01

    Persistent symptoms of depression suggest the involvement of stable molecular adaptations in brain, which may be reflected at the level of chromatin remodeling. We find that chronic social defeat stress in mice causes a transient decrease, followed by a persistent increase, in levels of acetylated histone H3 in the nucleus accumbens, an important limbic brain region. This persistent increase in H3 acetylation is associated with decreased levels of histone deacetylase 2 (HDAC2) in the nucleus accumbens. Similar effects were observed in the nucleus accumbens of depressed humans studied postmortem. These changes in H3 acetylation and HDAC2 expression mediate long-lasting positive neuronal adaptations, since infusion of HDAC inhibitors into the nucleus accumbens, which increases histone acetylation, exerts robust antidepressant-like effects in the social defeat paradigm and other behavioral assays. HDAC inhibitor (MS-275) infusion also reverses the effects of chronic defeat stress on global patterns of gene expression in the nucleus accumbens, as determined by microarray analysis, with striking similarities to the effects of the standard antidepressant, fluoxetine. Stress-regulated genes whose expression is normalized selectively by MS-275 may provide promising targets for the future development of novel antidepressant treatments. Together, these findings provide new insight into the underlying molecular mechanisms of depression and antidepressant action, and support the antidepressant potential of HDAC inhibitors and perhaps other agents that act at the level of chromatin structure. PMID:19759294

  17. Investigating the selectivity of metalloenzyme inhibitors.

    PubMed

    Day, Joshua A; Cohen, Seth M

    2013-10-24

    The inhibitory activity of a broad group of known metalloenzyme inhibitors against a panel of metalloenzymes was evaluated. Clinically approved inhibitors were selected as well as several other reported metalloprotein inhibitors in order to represent a broad range of metal binding groups (MBGs), including hydroxamic acid, carboxylate, hydroxypyridinonate, thiol, and N-hydroxyurea functional groups. A panel of metalloenzymes, including carbonic anhydrase (hCAII), several matrix metalloproteinases (MMPs), angiotensin converting enzyme (ACE), histone deacetylase (HDAC-2), and tyrosinase (TY), was selected based on their clinical importance for a range of pathologies. In addition, each inhibitor was evaluated for its ability to remove Fe(3+) from holo-transferrin to gauge the ability of the inhibitors to access Fe(3+) from a primary transport protein. The results show that the metalloenzyme inhibitors are quite selective for their intended targets, suggesting that despite their ability to bind metal ions, metalloprotein inhibitors are not prone to widespread off-target enzyme inhibition activity.

  18. The burden of inhibitors in haemophilia patients.

    PubMed

    Walsh, Christopher E; Jiménez-Yuste, Víctor; Auerswald, Guenter; Grancha, Salvador

    2016-08-31

    The burden of disease in haemophilia patients has wide ranging implications for the family and to society. There is evidence that having a current inhibitor increases the risk of morbidity and mortality. Morbidity is increased by the inability to treat adequately and its consequent disabilities, which then equates to a poor quality of life compared with non-inhibitor patients. The societal cost of care, or `burden of inhibitors', increases with the ongoing presence of an inhibitor. Therefore, it is clear that successful eradication of inhibitors by immune tolerance induction (ITI) is the single most important milestone one can achieve in an inhibitor patient. The type of factor VIII (FVIII) product used in ITI regimens varies worldwide. Despite ongoing debate, there is in vitro and retrospective clinical evidence to support the use of plasma-derived VWF-containing FVIII concentrates in ITI regimens in order to achieve early and high inhibitor eradication success rates. PMID:27528280

  19. Investigating the Selectivity of Metalloenzyme Inhibitors

    PubMed Central

    Day, Joshua A.; Cohen, Seth M.

    2013-01-01

    The inhibitory activity of a broad group of known metalloenzyme inhibitors against a panel of metalloenzymes was evaluated. Clinically approved inhibitors were selected as well as several other reported metalloprotein inhibitors, in order to represent a broad range of metal binding groups (MBGs), including hydroxamic acid, carboxylate, hydroxypyridinonate, thiol, and N-hydroxyurea functional groups. A panel of metalloenzymes, including carbonic anhydrase (hCAII), several matrix metalloproteinases (MMPs), angiotensin converting enzyme (ACE), histone deacetylase (HDAC-2), and tyrosinase (TY) was selected based on their clinical importance for a range of pathologies. In addition, each inhibitor was evaluated for its ability to remove Fe3+ from holo-transferrin to gauge the ability of the inhibitors to access Fe3+ from a primary transport protein. The results show that the metalloenzyme inhibitors are quite selective for their intended targets, suggesting that despite their ability to bind metal ions, metalloprotein inhibitors are not prone to widespread off-target enzyme inhibition activity. PMID:24074025

  20. Non-ATP competitive protein kinase inhibitors.

    PubMed

    Garuti, L; Roberti, M; Bottegoni, G

    2010-01-01

    Protein kinases represent an attractive target in oncology drug discovery. Most of kinase inhibitors are ATP-competitive and are called type I inhibitors. The ATP-binding pocket is highly conserved among members of the kinase family and it is difficult to find selective agents. Moreover, the ATP-competitive inhibitors must compete with high intracellular ATP levels leading to a discrepancy between IC50s measured by biochemical versus cellular assays. The non-ATP competitive inhibitors, called type II and type III inhibitors, offer the possibility to overcome these problems. These inhibitors act by inducing a conformational shift in the target enzyme such that the kinase is no longer able to function. In the DFG-out form, the phenylalanine side chain moves to a new position. This movement creates a hydrophobic pocket available for occupation by the inhibitor. Some common features are present in these inhibitors. They contain a heterocyclic system that forms one or two hydrogen bonds with the kinase hinge residue. They also contain a hydrophobic moiety that occupies the pocket formed by the shift of phenylalanine from the DFG motif. Moreover, all the inhibitors bear a hydrogen bond donor-acceptor pair, usually urea or amide, that links the hinge-binding portion to the hydrophobic moiety and interacts with the allosteric site. Examples of non ATP-competitive inhibitors are available for various kinases. In this review small molecules capable of inducing the DFG-out conformation are reported, especially focusing on structural feature, SAR and biological properties.

  1. Monoamine Oxidase Inhibitors: Clinical Review

    PubMed Central

    Remick, Ronald A.; Froese, Colleen

    1990-01-01

    Monoamine oxidase inhibitors (MAOIs) are effective antidepressant agents. They are increasingly and effectively used in a number of other psychiatric and non-psychiatric medical syndromes. Their potential for serious toxicity (i.e., hypertensive reaction) is far less than original reports suggest, and newer reversible substrate-specific MAOIs may offer even less toxicity. The author reviews the pharmacology, mechanism of action, clinical indications, and dosing strategies of MAOIs. The common MAOI side-effects (hypotension, weight gain, sexual dysfunction, insomnia, daytime sedation, myoclonus, and hypertensive episodes) are described and management techniques suggested. Recent clinical developments involving MAOIs are outlined. PMID:21233984

  2. Techniques for Screening Translation Inhibitors

    PubMed Central

    Osterman, Ilya A.; Bogdanov, Alexey A.; Dontsova, Olga A.; Sergiev, Petr V.

    2016-01-01

    The machinery of translation is one of the most common targets of antibiotics. The development and screening of new antibiotics usually proceeds by testing antimicrobial activity followed by laborious studies of the mechanism of action. High-throughput methods for new antibiotic screening based on antimicrobial activity have become routine; however, identification of molecular targets is usually a challenge. Therefore, it is highly beneficial to combine primary screening with the identification of the mechanism of action. In this review, we describe a collection of methods for screening translation inhibitors, with a special emphasis on methods which can be performed in a high-throughput manner. PMID:27348012

  3. Antibody responses and protection against influenza virus infection in different congenic strains of mice immunized intranasally with adjuvant-combined A/Beijing/262/95 (H1N1) virus hemagglutinin or neuraminidase.

    PubMed

    Yoshikawa, Tomoki; Suzuki, Yujiro; Nomoto, Akio; Sata, Tetutaro; Kurata, Takeshi; Tamura, Shin ichi

    2002-11-22

    Antibody (Ab) responses and protection against influenza virus infection in mice immunized intranasally with hemagglutinin (HA) or neuraminidase (NA) purified from the A/Beijing/262/95 (A/Beijing) (H1N1) virus were compared among B10 congenic mouse strains. Mice were immunized intranasally with 0.1, 0.3 or 1microg of HA or NA together with the cholera toxin adjuvant, and then boosted intranasally with 0.3 microg of the adjuvant-combined HA or NA 4 weeks later. Two weeks after the second immunization, the mice were challenged by an infection of the upper respiratory tract with the homologous virus. After 3 days, nasal wash and serum specimens were collected for virus and Ab titration. The HA immunization induced HA-specific IgG Ab responses against A/Beijing HA, which depended on the H-2 haplotype of the strain: The B10.A (H-2(a)), B10.D2 (H-2(d)), B10.BR (H-2(k)) and B10 (H-2(b)) strains were the highest, high, intermediate and low responders, respectively. The nasal IgA responses were induced in the B10.A, B10.D2 and B10.BR strains, but not in the B10 strain. In parallel with Ab responses, the B10.A, B10.BR and B10.D2 strains were conferred significant protection at any dose of primary immunization, but the B10 strain was provided protection only at 1microg of HA. On the other hand, the NA immunization induced NA-specific Ab responses, which depended on the the H-2 haplotype of the strain: the B10.A, B10.D2, B10 and B10.BR strains were the highest, high, intermediate and low responders, respectively. In parallel with Ab responses, all the strains were conferred significant protection at any dose of primary immunization. These results indicate that the MHC-restricted responsiveness of mice to HA is different from that to NA, suggesting that the use of high-HA dose or NA as a component of the nasal influenza A (H1N1 subtype) virus vaccine improves the protective efficacy against influenza among low responder populations.

  4. Improvement of influenza A/Fujian/411/02 (H3N2) virus growth in embryonated chicken eggs by balancing the hemagglutinin and neuraminidase activities, using reverse genetics.

    PubMed

    Lu, Bin; Zhou, Helen; Ye, Dan; Kemble, George; Jin, Hong

    2005-06-01

    The H3N2 influenza A/Fujian/411/02-like virus strains that circulated during the 2003-2004 influenza season caused influenza epidemics. Most of the A/Fujian/411/02 virus lineages did not replicate well in embryonated chicken eggs and had to be isolated originally by cell culture. The molecular basis for the poor replication of A/Fujian/411/02 virus was examined in this study by the reverse genetics technology. Two antigenically related strains that replicated well in embryonated chicken eggs, A/Sendai-H/F4962/02 and A/Wyoming/03/03, were compared with the prototype A/Fujian/411/02 virus. A/Sendai differed from A/Fujian by three amino acids in the neuraminidase (NA), whereas A/Wyoming differed from A/Fujian by five amino acids in the hemagglutinin (HA). The HA and NA segments of these three viruses were reassorted with cold-adapted A/Ann Arbor/6/60, the master donor virus for the live attenuated type A influenza vaccines (FluMist). The HA and NA residues differed between these three H3N2 viruses evaluated for their impact on virus replication in MDCK cells and in embryonated chicken eggs. It was determined that replication of A/Fujian/411/02 in eggs could be improved by either changing minimum of two HA residues (G186V and V226I) to increase the HA receptor-binding ability or by changing a minimum of two NA residues (E119Q and Q136K) to lower the NA enzymatic activity. Alternatively, recombinant A/Fujian/411/02 virus could be adapted to grow in eggs by two amino acid substitutions in the HA molecule (H183L and V226A), which also resulted in the increased HA receptor-binding activity. Thus, the balance between the HA and NA activities is critical for influenza virus replication in a different host system. The HA or NA changes that increased A/Fujian/411/02 virus replication in embryonated chicken eggs were found to have no significant impact on antigenicity of these recombinant viruses. This study demonstrated that the reverse genetics technology could be used to

  5. The E119D neuraminidase mutation identified in a multidrug-resistant influenza A(H1N1)pdm09 isolate severely alters viral fitness in vitro and in animal models.

    PubMed

    Abed, Yacine; Bouhy, Xavier; L'Huillier, Arnaud G; Rhéaume, Chantal; Pizzorno, Andrés; Retamal, Miguel; Fage, Clément; Dubé, Karen; Joly, Marie-Hélène; Beaulieu, Edith; Mallett, Corey; Kaiser, Laurent; Boivin, Guy

    2016-08-01

    We recently isolated an influenza A(H1N1)pdm09 E119D/H275Y neuraminidase (NA) variant from an immunocompromised patient who received oseltamivir and zanamivir therapies. This variant demonstrated cross resistance to zanamivir, oseltamivir, peramivir and laninamivir. In this study, the viral fitness of the recombinant wild-type (WT), E119D and E119D/H275Y A(H1N1)pdm09 viruses was evaluated in vitro and in experimentally-infected C57BL/6 mice and guinea pigs. In replication kinetics experiments, viral titers obtained with the E119D and E119D/H275Y recombinants were up to 2- and 4-log lower compared to the WT virus in MDCK and ST6GalI-MDCK cells, respectively. Enzymatic studies revealed that the E119D mutation significantly decreased the surface NA activity. In experimentally-infected mice, a 50% mortality rate was recorded in the group infected with the WT recombinant virus whereas no mortality was observed in the E119D and E119D/H275Y groups. Mean lung viral titers on day 5 post-inoculation for the WT (1.2 ± 0.57 × 10(8) PFU/ml) were significantly higher than those of the E119D (9.75 ± 0.41 × 10(5) PFU/ml, P < 0.01) and the E119D/H275Y (1.47 ± 0.61 × 10(6) PFU/ml, P < 0.01) groups. In guinea pigs, comparable seroconversion rates and viral titers in nasal washes (NW) were obtained for the WT and mutant index and contact groups. However, the D119E reversion was observed in most NW samples of the E119D and E119D/H275Y animals. In conclusion, the E119D NA mutation that could emerge in A(H1N1)pdm09 viruses during zanamivir therapy has a significant impact on viral fitness and such mutant is unlikely to be highly transmissible. PMID:27185624

  6. Oligopeptide cyclophilin inhibitors: a reassessment.

    PubMed

    Schumann, Michael; Jahreis, Günther; Kahlert, Viktoria; Lücke, Christian; Fischer, Gunter

    2011-11-01

    Potent cyclophilin A (CypA) inhibitors such as non-immunosuppressive cyclosporin A (CsA) derivatives have been already used in clinical trials in patients with viral infections. CypA is a peptidyl prolyl cis/trans isomerase (PPIase) that catalyzes slow prolyl bond cis/trans interconversions of the backbone of substrate peptides and proteins. In this study we investigate whether the notoriously low affinity inhibitory interaction of linear proline-containing peptides with the active site of CypA can be increased through a combination of a high cis/trans ratio and a negatively charged C-terminus as has been recently reported for Trp-Gly-Pro. Surprisingly, isothermal titration calorimetry did not reveal formation of an inhibitory CypA/Trp-Gly-Pro complex previously described within a complex stability range similar to CsA, a nanomolar CypA inhibitor. Moreover, despite of cis content of 41% at pH 7.5 Trp-Gly-Pro cannot inhibit CypA-catalyzed standard substrate isomerization up to high micromolar concentrations. However, in the context of the CsA framework a net charge of -7 clustered at the amino acid side chain of position 1 resulted in slightly improved CypA inhibition.

  7. Carborane-based carbonic anhydrase inhibitors.

    PubMed

    Brynda, Jiří; Mader, Pavel; Šícha, Václav; Fábry, Milan; Poncová, Kristýna; Bakardiev, Mario; Grüner, Bohumír; Cígler, Petr; Řezáčová, Pavlína

    2013-12-16

    CA inhibitors: Human carbonic anhydrases (CAs) are diagnostic and therapeutic targets. Various carborane cages are shown to act as active-site-directed inhibitors, and substitution with a sulfamide group and other substituents leads to compounds with high selectivity towards the cancer-specific isozyme IX. Crystal structures of the carboranes in the active site provide information that can be applied to the structure-based design of specific inhibitors. PMID:24307504

  8. KH-30 Parafin Inhibitor Treatment

    SciTech Connect

    Rochelle, J.

    2001-09-30

    United Energy Corporation (UNRG) and the U.S. Department of Energy personnel tested KH-30 at the Rocky Mountain Oilfield Testing Center (RMOTC) outside Casper, Wyoming on two separate occasions. KH-30 is a non-toxic, non-hazardous product, which combines the functions of a solvent dispersant, crystal modifier and inhibitor into a single solution. The first test was held in March of 2001, wherein five wells were treated with a mixture of KH-30 and brine water, heated to 180 degrees F. No increase in production was attained in these tests. In June, 2001, three shallow, low pressure RMOTC wells with 30 years of production were treated with a mixture of 40% KH-30 and 60% diesel. Increases were seen in three wells. The wells then returned to their original rates.

  9. Natural Products as Aromatase Inhibitors

    PubMed Central

    Balunas, Marcy J.; Su, Bin; Brueggemeier, Robert W.; Kinghorn, A. Douglas

    2010-01-01

    With the clinical success of several synthetic aromatase inhibitors (AIs) in the treatment of postmenopausal estrogen receptor-positive breast cancer, researchers have also been investigating also the potential of natural products as AIs. Natural products from terrestrial and marine organisms provide a chemically diverse array of compounds not always available through current synthetic chemistry techniques. Natural products that have been used traditionally for nutritional or medicinal purposes (e.g., botanical dietary supplements) may also afford AIs with reduced side effects. A thorough review of the literature regarding natural product extracts and secondary metabolites of plant, microbial, and marine origin that have been shown to exhibit aromatase inhibitory activity is presented herein. PMID:18690828

  10. Tyrosine Kinase Inhibitors and Pregnancy

    PubMed Central

    Abruzzese, Elisabetta; Trawinska, Malgorzata Monika; Perrotti, Alessio Pio; De Fabritiis, Paolo

    2014-01-01

    The management of patients with chronic myeloid leukemia (CML) during pregnancy has become recently a matter of continuous debate. The introduction of the Tyrosine Kinase Inhibitors (TKIs) in clinical practice has dramatically changed the prognosis of CML patients; in fact, patients diagnosed in chronic phase can reasonably expect many years of excellent disease control and good quality of life, as well as a normal life expectancy, including the necessity to address issues relating to fertility and pregnancy. Physicians are frequently being asked for advice regarding the need for, and/or the appropriateness of, stopping treatment in order to conceive. In this report, we will review the data published in terms of fertility, conception, pregnancy, pregnancy outcome and illness control for TKI treated CML patients, as well as how to manage a planned and/or unplanned pregnancy. PMID:24804001

  11. Glycine Transporters and Their Inhibitors

    NASA Astrophysics Data System (ADS)

    Gilfillan, Robert; Kerr, Jennifer; Walker, Glenn; Wishart, Grant

    Glycine plays a ubiquitous role in many biological processes. In the central nervous system it serves as an important neurotransmitter acting as an agonist at strychnine-sensitive glycine receptors and as an essential co-agonist with glutamate at the NMDA receptor complex. Control of glycine concentrations in the vicinity of these receptors is mediated by the specific glycine transporters, GlyT1 and GlyT2. Inhibition of these transporters has been postulated to be of potential benefit in several therapeutic indications including schizophrenia and pain. In this review we discuss our current knowledge of glycine transporters and focus on recent advances in the medicinal chemistry of GlyT1 and GlyT2 inhibitors.

  12. Enzyme-Inhibitor Association Thermodynamics

    PubMed Central

    Resat, Haluk; Marrone, Tami J.; McCammon, J. Andrew

    1997-01-01

    Studying the thermodynamics of biochemical association reactions at the microscopic level requires efficient sampling of the configurations of the reactants and solvent as a function of the reaction pathways. In most cases, the associating ligand and receptor have complementary interlocking shapes. Upon association, loosely connected or disconnected solvent cavities at and around the binding site are formed. Disconnected solvent regions lead to severe statistical sampling problems when simulations are performed with explicit solvent. It was recently proposed that, when such limitations are encountered, they might be overcome by the use of the grand canonical ensemble. Here we investigate one such case and report the association free energy profile (potential of mean force) between trypsin and benzamidine along a chosen reaction coordinate as calculated using the grand canonical Monte Carlo method. The free energy profile is also calculated for a continuum solvent model using the Poisson equation, and the results are compared to the explicit water simulations. The comparison shows that the continuum solvent approach is surprisingly successful in reproducing the explicit solvent simulation results. The Monte Carlo results are analyzed in detail with respect to solvation structure. In the binding site channel there are waters bridging the carbonyl oxygen groups of Asp189 with the NH2 groups of benzamidine, which are displaced upon inhibitor binding. A similar solvent-bridging configuration has been seen in the crystal structure of trypsin complexed with bovine pancreatic trypsin inhibitor. The predicted locations of other internal waters are in very good agreement with the positions found in the crystal structures, which supports the accuracy of the simulations. ImagesFIGURE 5 PMID:9017183

  13. Pyridopyrimidine analogues as novel adenosine kinase inhibitors.

    PubMed

    Zheng, G Z; Lee, C; Pratt, J K; Perner, R J; Jiang, M Q; Gomtsyan, A; Matulenko, M A; Mao, Y; Koenig, J R; Kim, K H; Muchmore, S; Yu, H; Kohlhaas, K; Alexander, K M; McGaraughty, S; Chu, K L; Wismer, C T; Mikusa, J; Jarvis, M F; Marsh, K; Kowaluk, E A; Bhagwat, S S; Stewart, A O

    2001-08-20

    A novel series of pyridopyrimidine analogues 9 was identified as potent adenosine kinase inhibitors based on the SAR and computational studies. Substitution of the C7 position of the pyridopyrimidino core with C2' substituted pyridino moiety increased the in vivo potency and enhanced oral bioavailability of these adenosine kinase inhibitors.

  14. Rust inhibitor and oil composition containing same

    SciTech Connect

    Bialy, J.J.; Cullen, W.P.; Dorn, P.; Nebzydoski, J.W.; Sung, R.L.

    1981-04-21

    A rust inhibitor comprising the reaction product of a hydrocarbylsuccinic anhydride in which the hydrocarbyl radical has from about 6 to 30 carbon atoms and an aminotriazole is provided. The rust inhibitor is effective in motor fuel and lubricating oil compositions.

  15. Intellectual property issues of immune checkpoint inhibitors

    PubMed Central

    Storz, Ulrich

    2016-01-01

    Immune checkpoint inhibitors are drugs that interfere with tumor escape responses. Some members of this class are already approved, and expected to be blockbusters in the future. Many companies have developed patent activities in this field. This article focuses on the patent landscape, and discusses key players and cases related to immune checkpoint inhibitors. PMID:26466763

  16. Intellectual property issues of immune checkpoint inhibitors.

    PubMed

    Storz, Ulrich

    2016-01-01

    Immune checkpoint inhibitors are drugs that interfere with tumor escape responses. Some members of this class are already approved, and expected to be blockbusters in the future. Many companies have developed patent activities in this field. This article focuses on the patent landscape, and discusses key players and cases related to immune checkpoint inhibitors.

  17. Computer simulation of inhibitor application -- A review

    SciTech Connect

    Banerjee, G.; Vasanth, K.L.

    1997-12-01

    The rapid development of powerful software as well as hardware in computer technology has changed the traditional approach to all areas of science and technology. In the field of corrosion inhibitors, computers are used to model, simulate, analyze and monitor inhibitor applications in both laboratory and industrial environments. This paper will present an up-to-date critical review of such simulation studies.

  18. Intellectual property issues of immune checkpoint inhibitors.

    PubMed

    Storz, Ulrich

    2016-01-01

    Immune checkpoint inhibitors are drugs that interfere with tumor escape responses. Some members of this class are already approved, and expected to be blockbusters in the future. Many companies have developed patent activities in this field. This article focuses on the patent landscape, and discusses key players and cases related to immune checkpoint inhibitors. PMID:26466763

  19. Aminofurazans as potent inhibitors of AKT kinase

    SciTech Connect

    Rouse, Meagan B.; Seefeld, Mark A.; Leber, Jack D.; McNulty, Kenneth C.; Sun, Lihui; Miller, William H.; Zhang, ShuYun; Minthorn, Elisabeth A.; Concha, Nestor O.; Choudhry, Anthony E.; Schaber, Michael D.; Heerding, Dirk A.

    2009-06-24

    AKT inhibitors containing an imidazopyridine aminofurazan scaffold have been optimized. We have previously disclosed identification of the AKT inhibitor GSK690693, which has been evaluated in clinical trials in cancer patients. Herein we describe recent efforts focusing on investigating a distinct region of this scaffold that have afforded compounds (30 and 32) with comparable activity profiles to that of GSK690693.

  20. Trypsin inhibitors of buffalo seminal plasma.

    PubMed

    Ahmed, N; Ramesh, V

    1992-03-01

    Two trypsin inhibitors from acid-treated buffalo seminal plasma were purified by gel filtration and affinity chromatography. These acid-stable trypsin inhibitors having charge heterogeneity were homogeneous with respect to size as revealed by gel filtration and SDS-PAGE. Gel filtration data suggest molecular weight value of 9,900 Da for inhibitor I and 10,900 Da for inhibitor II. Molecular weight estimated by SDS-PAGE was found to be 10,600 Da and 11,200 Da for inhibitors I and II, respectively. The hydrodynamic properties such as Stokes radii (1.58 nm and 1.62 nm); intrinsic viscosity (2.5725 ml/g and 2.5025 ml/g) and diffusion coefficient (13.499 x 10(-11) m2/sec. and 13.166X10(-11) m2/sec) respectively for inhibitor I and II were determined by analytical gel filtration. These inhibitors were fairly thermostable and could not be stained by PAS reagent. Both the inhibitors were found to inhibit buffalo acrosin but not bovine chymotrypsin.

  1. MAO inhibitors: risks, benefits, and lore.

    PubMed

    Wimbiscus, Molly; Kostenko, Olga; Malone, Donald

    2010-12-01

    Monoamine oxidase (MAO) inhibitors were the first antidepressants introduced, but their use has dwindled because of their reported side effects, their food and drug interactions, and the introduction of other classes of agents. However, interest in MAO inhibitors is reviving. Here, we discuss their use, risks, and benefits in clinical medicine.

  2. Exploring the scaffold universe of kinase inhibitors.

    PubMed

    Hu, Ye; Bajorath, Jürgen

    2015-01-01

    The scaffold concept was applied to systematically determine, analyze, and compare core structures of kinase inhibitors. From publicly available inhibitors of the human kinome, scaffolds and cyclic skeletons were systematically extracted and organized taking activity data, structural relationships, and retrosynthetic criteria into account. Scaffold coverage varied greatly across the kinome, and many scaffolds representing compounds with different activity profiles were identified. The majority of kinase inhibitor scaffolds were involved in well-defined yet distinct structural relationships, which had different consequences on compound activity. Scaffolds exclusively representing highly potent compounds were identified as well as structurally analogous scaffolds with very different degrees of promiscuity. Scaffold relationships presented herein suggest a variety of hypotheses for inhibitor design. Our detailed organization of the kinase inhibitor scaffold universe with respect to different activity and structural criteria, all scaffolds, and the original compound data assembled for our analysis are made freely available.

  3. A Spider-Derived Kunitz-Type Serine Protease Inhibitor That Acts as a Plasmin Inhibitor and an Elastase Inhibitor

    PubMed Central

    Wan, Hu; Lee, Kwang Sik; Kim, Bo Yeon; Zou, Feng Ming; Yoon, Hyung Joo; Je, Yeon Ho; Li, Jianhong; Jin, Byung Rae

    2013-01-01

    Kunitz-type serine protease inhibitors are involved in various physiological processes, such as ion channel blocking, blood coagulation, fibrinolysis, and inflammation. While spider-derived Kunitz-type proteins show activity in trypsin or chymotrypsin inhibition and K+ channel blocking, no additional role for these proteins has been elucidated. In this study, we identified the first spider (Araneus ventricosus) Kunitz-type serine protease inhibitor (AvKTI) that acts as a plasmin inhibitor and an elastase inhibitor. AvKTI possesses a Kunitz domain consisting of a 57-amino-acid mature peptide that displays features consistent with Kunitz-type inhibitors, including six conserved cysteine residues and a P1 lysine residue. Recombinant AvKTI, expressed in baculovirus-infected insect cells, showed a dual inhibitory activity against trypsin (Ki 7.34 nM) and chymotrypsin (Ki 37.75 nM), defining a role for AvKTI as a spider-derived Kunitz-type serine protease inhibitor. Additionally, AvKTI showed no detectable inhibitory effects on factor Xa, thrombin, or tissue plasminogen activator; however, AvKTI inhibited plasmin (Ki 4.89 nM) and neutrophil elastase (Ki 169.07 nM), indicating that it acts as an antifibrinolytic factor and an antielastolytic factor. These findings constitute molecular evidence that AvKTI acts as a plasmin inhibitor and an elastase inhibitor and also provide a novel view of the functions of a spider-derived Kunitz-type serine protease inhibitor. PMID:23308198

  4. Designing Inhibitors of Anthrax Toxin

    PubMed Central

    Nestorovich, Ekaterina M.; Bezrukov, Sergey M.

    2014-01-01

    Introduction Present-day rational drug design approaches are based on exploiting unique features of the target biomolecules, small- or macromolecule drug candidates, and physical forces that govern their interactions. The 2013 Nobel Prize in chemistry awarded “for the development of multiscale models for complex chemical systems” once again demonstrated the importance of the tailored drug discovery that reduces the role of the trial and error approach to a minimum. The “rational drug design” term is rather comprehensive as it includes all contemporary methods of drug discovery where serendipity and screening are substituted by the information-guided search for new and existing compounds. Successful implementation of these innovative drug discovery approaches is inevitably preceded by learning the physics, chemistry, and physiology of functioning of biological structures under normal and pathological conditions. Areas covered This article provides an overview of the recent rational drug design approaches to discover inhibitors of anthrax toxin. Some of the examples include small-molecule and peptide-based post-exposure therapeutic agents as well as several polyvalent compounds. The review also directs the reader to the vast literature on the recognized advances and future possibilities in the field. Expert opinion Existing options to combat anthrax toxin lethality are limited. With the only anthrax toxin inhibiting therapy (PA-targeting with a monoclonal antibody, raxibacumab) approved to treat inhalational anthrax, in our view, the situation is still insecure. The FDA’s animal rule for drug approval, which clears compounds without validated efficacy studies on humans, creates a high level of uncertainty, especially when a well-characterized animal model does not exist. Besides, unlike PA, which is known to be unstable, LF remains active in cells and in animal tissues for days. Therefore, the effectiveness of the post-exposure treatment of the individuals

  5. High-affinity Cyclic Peptide Matriptase Inhibitors*

    PubMed Central

    Quimbar, Pedro; Malik, Uru; Sommerhoff, Christian P.; Kaas, Quentin; Chan, Lai Y.; Huang, Yen-Hua; Grundhuber, Maresa; Dunse, Kerry; Craik, David J.; Anderson, Marilyn A.; Daly, Norelle L.

    2013-01-01

    The type II transmembrane serine protease matriptase is a key activator of multiple signaling pathways associated with cell proliferation and modification of the extracellular matrix. Deregulated matriptase activity correlates with a number of diseases, including cancer and hence highly selective matriptase inhibitors may have therapeutic potential. The plant-derived cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), is a promising drug scaffold with potent matriptase inhibitory activity. In the current study we have analyzed the structure-activity relationships of SFTI-1 and Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II), a structurally divergent trypsin inhibitor from Momordica cochinchinensis that also contains a cyclic backbone. We show that MCoTI-II is a significantly more potent matriptase inhibitor than SFTI-1 and that all alanine mutants of both peptides, generated using positional scanning mutagenesis, have decreased trypsin affinity, whereas several mutations either maintain or result in enhanced matriptase inhibitory activity. These intriguing results were used to design one of the most potent matriptase inhibitors known to date with a 290 pm equilibrium dissociation constant, and provide the first indication on how to modulate affinity for matriptase over trypsin in cyclic peptides. This information might be useful for the design of more selective and therapeutically relevant inhibitors of matriptase. PMID:23548907

  6. Leflunomide, a Reversible Monoamine Oxidase Inhibitor.

    PubMed

    Petzer, Jacobus P; Petzer, Anél

    2016-01-01

    A screening study aimed at identifying inhibitors of the enzyme, monoamine oxidase (MAO), among clinically used drugs have indicated that the antirheumatic drug, leflunomide, is an inhibitor of both MAO isoforms. Leflunomide inhibits human MAO-A and MAO-B and exhibits IC50 values of 19.1 μM and 13.7 μM, respectively. The corresponding Ki values are 17.7 μM (MAO-A) and 10.1 μM (MAO-B). Dialyses of mixtures of the MAO enzymes and leflunomide show that inhibition of the MAOs by leflunomide is reversible. The principal metabolite of leflunomide, teriflunomide (A77 1726), in contrast is not an MAO inhibitor. This study concludes that, although leflunomide is only moderately potent as an MAO inhibitor, isoxazole derivatives may represent a general class of MAO inhibitors and this heterocycle may find application in MAO inhibitor design. In this respect, MAO inhibitors are used in the clinic for the treatment of depressive illness and Parkinson's disease, and are under investigation as therapy for certain types of cancer, Alzheimer's disease and age-related impairment of cardiac function. PMID:26299850

  7. SGLT2 Inhibitors May Predispose to Ketoacidosis

    PubMed Central

    Blau, Jenny E.; Rother, Kristina I.

    2015-01-01

    Context: Sodium glucose cotransporter 2 (SGLT2) inhibitors are antidiabetic drugs that increase urinary excretion of glucose, thereby improving glycemic control and promoting weight loss. Since approval of the first-in-class drug in 2013, data have emerged suggesting that these drugs increase the risk of diabetic ketoacidosis. In May 2015, the Food and Drug Administration issued a warning that SGLT2 inhibitors may lead to ketoacidosis. Evidence Acquisition: Using PubMed and Google, we conducted Boolean searches including terms related to ketone bodies or ketoacidosis with terms for SGLT2 inhibitors or phlorizin. Priority was assigned to publications that shed light on molecular mechanisms whereby SGLT2 inhibitors could affect ketone body metabolism. Evidence Synthesis: SGLT2 inhibitors trigger multiple mechanisms that could predispose to diabetic ketoacidosis. When SGLT2 inhibitors are combined with insulin, it is often necessary to decrease the insulin dose to avoid hypoglycemia. The lower dose of insulin may be insufficient to suppress lipolysis and ketogenesis. Furthermore, SGLT2 is expressed in pancreatic α-cells, and SGLT2 inhibitors promote glucagon secretion. Finally, phlorizin, a nonselective inhibitor of SGLT family transporters decreases urinary excretion of ketone bodies. A decrease in the renal clearance of ketone bodies could also increase the plasma ketone body levels. Conclusions: Based on the physiology of SGLT2 and the pharmacology of SGLT2 inhibitors, there are several biologically plausible mechanisms whereby this class of drugs has the potential to increase the risk of developing diabetic ketoacidosis. Future research should be directed toward identifying which patients are at greatest risk for this side effect and also to optimizing pharmacotherapy to minimize the risk to patients. PMID:26086329

  8. Thrombin-activatable fibrinolysis inhibitor.

    PubMed

    Marx, Pauline F

    2004-09-01

    The coagulation system is a potent mechanism that prevents blood loss after vascular injury. It consists of a number of linked enzymatic reactions resulting in thrombin generation. Thrombin converts soluble fibrinogen into a fibrin clot. The clot is subsequently removed by the fibrinolytic system upon wound healing. Thrombin-activatable fibrinolysis inhibitor (TAFI), which is identical to the previously identified proteins procarboxypeptidase B, R, and U, forms a link between blood coagulation and fibrinolysis. TAFI circulates as an inactive proenzyme in the bloodstream, and becomes activated during blood clotting. The active form, TAFIa, inhibits fibrinolysis by cleaving off C-terminal lysine residues from partially degraded fibrin that stimulates the tissue-type plasminogen activator-mediated conversion of plasminogen to plasmin. Consequently, removal of these lysines leads to less plasmin formation and subsequently to protection of the fibrin clot from break down. Moreover, TAFI may also play a role in other processes such as, inflammation and tissue repair. In this review, recent developments in TAFI research are discussed. PMID:15379716

  9. HIV Protease Inhibitors and Obesity

    PubMed Central

    Anuurad, Erdembileg; Bremer, Andrew; Berglund, Lars

    2011-01-01

    Purpose of review To review the current scientific literature and recent clinical trials on HIV protease inhibitors (PIs) and their potential role in the pathogenesis of lipodystrophy and metabolic disorders. Recent findings HIV PI treatment may affect the normal stimulatory effect of insulin on glucose and fat storage. Further, chronic inflammation from HIV infection and PI treatment trigger cellular homeostatic stress responses with adverse effects on intermediary metabolism. The physiologic outcome is such that total adipocyte storage capacity is decreased, and the remaining adipocytes resist further fat storage. This process leads to a pathologic cycle of lipodystrophy and lipotoxicity, a pro-atherogenic lipid profile, and a clinical phenotype of increased central body fat distribution similar to the metabolic syndrome. Summary PIs are a key component of antiretroviral therapy and have dramatically improved the life expectancy of HIV-infected individuals. However, they are also associated with abnormalities in glucose/lipid metabolism and body fat distribution. Further studies are needed to better define the pathogenesis of PI-associated metabolic and body fat changes and their potential treatment. PMID:20717021

  10. MMP Inhibitors: Past, present and future.

    PubMed

    Cathcart, Jillian M; Cao, Jian

    2015-01-01

      Development of inhibitors of matrix metalloproteinases (MMPs) has been fraught with challenges. Early compounds largely failed due to poor selectivity and bioavailability. Dose-limiting side effects, off-target interactions, and improperly designed clinical trials significantly impeded clinical success. As information becomes available and technology evolves, tools to combat these obstacles have been developed. Improved methods for high throughput screening and drug design have led to identification of compounds exhibiting high potency, binding affinity, and favorable pharmacokinetic profiles. Current research into MMP inhibitors employs innovative approaches for drug delivery methods and allosteric inhibitors. Such innovation is key for development of clinically successful compounds.

  11. An updated review of tyrosinase inhibitors.

    PubMed

    Chang, Te-Sheng

    2009-06-01

    Tyrosinase is a multifunctional, glycosylated, and copper-containing oxidase, which catalyzes the first two steps in mammalian melanogenesis and is responsible for enzymatic browning reactions in damaged fruits during post-harvest handling and processing. Neither hyperpigmentation in human skin nor enzymatic browning in fruits are desirable. These phenomena have encouraged researchers to seek new potent tyrosinase inhibitors for use in foods and cosmetics. This article surveys tyrosinase inhibitors newly discovered from natural and synthetic sources. The inhibitory strength is compared with that of a standard inhibitor, kojic acid, and their inhibitory mechanisms are discussed. PMID:19582213

  12. An Updated Review of Tyrosinase Inhibitors

    PubMed Central

    Chang, Te-Sheng

    2009-01-01

    Tyrosinase is a multifunctional, glycosylated, and copper-containing oxidase, which catalyzes the first two steps in mammalian melanogenesis and is responsible for enzymatic browning reactions in damaged fruits during post-harvest handling and processing. Neither hyperpigmentation in human skin nor enzymatic browning in fruits are desirable. These phenomena have encouraged researchers to seek new potent tyrosinase inhibitors for use in foods and cosmetics. This article surveys tyrosinase inhibitors newly discovered from natural and synthetic sources. The inhibitory strength is compared with that of a standard inhibitor, kojic acid, and their inhibitory mechanisms are discussed. PMID:19582213

  13. Musical Hallucinations Treated with Acetylcholinesterase Inhibitors

    PubMed Central

    Blom, Jan Dirk; Coebergh, Jan Adriaan F.; Lauw, René; Sommer, Iris E. C.

    2015-01-01

    Musical hallucinations are relatively rare auditory percepts which, due to their intrusive nature and the accompanying fear of impending mental decline, tend to cause significant distress and impairment. Although their etiology and pathophysiology appear to be heterogeneous and no evidence-based treatment methods are available, case reports indicate that acetylcholinesterase inhibitors may yield positive results in patients with comorbid hearing loss. We present two female patients (aged 76 and 78 years) both of whom suffered from hearing impairment and practically incessant musical hallucinations. Both patients were successfully treated with the acetylcholinesterase inhibitor rivastigmine. Based on these two case descriptions and an overview of studies describing the use of acetylcholinesterase inhibitors in similar patients, we discuss possible mechanisms and propose further research on the use of acetylcholinesterase inhibitors for musical hallucinations experienced in concordance with hearing loss. PMID:25904872

  14. A tyrosinase inhibitor from Aspergillus niger.

    PubMed

    Vasantha, K Y; Murugesh, C S; Sattur, A P

    2014-10-01

    Tyrosinase, in the presence of oxygen, is the main culprit in post harvest browning of food products, resulting in the drop in its commercial value. In an effort to seek natural tyrosinase inhibitors for food applications, a screening programme was undertaken. Of the 26 fungal cultures isolated from soil samples of Agumbe forest, India, one isolate S16, identified as Aspergillus niger, gave an inhibition of 84 % against the enzyme. The inhibitor was isolated by following an enzyme inhibition assay guided purification protocol. The structure of the inhibitor was elucidated and found to be kojic acid. The IC50 of the Competitive inhibitor was found to be 8.8 μg with a Ki of 0.085 mM.

  15. Inhibitors of acetylcholinesterase and butyrylcholinesterase meet immunity.

    PubMed

    Pohanka, Miroslav

    2014-06-02

    Acetylcholinesterase (AChE) inhibitors are widely used for the symptomatic treatment of Alzheimer's disease and other dementias. More recent use is for myasthenia gravis. Many of these inhibitors interact with the second known cholinesterase, butyrylcholinesterase (BChE). Further, evidence shows that acetylcholine plays a role in suppression of cytokine release through a "cholinergic anti-inflammatory pathway" which raises questions about the role of these inhibitors in the immune system. This review covers research and discussion of the role of the inhibitors in modulating the immune response using as examples the commonly available drugs, donepezil, galantamine, huperzine, neostigmine and pyridostigmine. Major attention is given to the cholinergic anti-inflammatory pathway, a well-described link between the central nervous system and terminal effector cells in the immune system.

  16. Ocular Toxicity of Tyrosine Kinase Inhibitors

    PubMed Central

    Davis, Mary Elizabeth

    2016-01-01

    Purpose/Objectives To review common tyrosine kinase inhibitors, as well as their ocular side effects and management. Data Sources A comprehensive literature search was conducted using cINahl®, Pubmed, and cochrane databases for articles published since 2004 with the following search terms: ocular toxicities, tyrosine kinase inhibitors, ophthalmology, adverse events, eye, and vision. Data Synthesis Tyrosine kinase inhibitors can cause significant eye toxicity. Conclusions Given the prevalence of new tyrosine kinase inhibitor therapies and the complexity of possible pathogenesis of ocular pathology, oncology nurses can appreciate the occurrence of ocular toxicities and the role of nursing in the management of these problems. Implications for Nursing Knowledge of the risk factors and etiology of ocular toxicity of targeted cancer therapies can guide nursing assessment, enhance patient education, and improve care management. Including a review of eye symptoms and vision issues in nursing assessment can enhance early detection and treatment of ocular toxicity. PMID:26906134

  17. Small-Molecule Inhibitors of Urea Transporters

    PubMed Central

    Verkman, Alan S.; Esteva-Font, Cristina; Cil, Onur; Anderson, Marc O.; Li, Fei; Li, Min; Lei, Tianluo; Ren, Huiwen; Yang, Baoxue

    2015-01-01

    Urea transporter (UT) proteins, which include isoforms of UT-A in kidney tubule epithelia and UT-B in vasa recta endothelia and erythrocytes, facilitate urinary concentrating function. Inhibitors of urea transporter function have potential clinical applications as sodium-sparing diuretics, or ‘urearetics,’ in edema from different etiologies, such as congestive heart failure and cirrhosis, as well as in syndrome of inappropriate antidiuretic hormone (SIADH). High-throughput screening of drug-like small molecules has identified UT-A and UT-B inhibitors with nanomolar potency. Inhibitors have been identified with different UT-A versus UT-B selectivity profiles and putative binding sites on UT proteins. Studies in rodent models support the utility of UT inhibitors in reducing urinary concentration, though testing in clinically relevant animal models of edema has not yet been done. PMID:25298345

  18. Small-molecule inhibitors of myosin proteins

    PubMed Central

    Bond, Lisa M; Tumbarello, David A; Kendrick-Jones, John; Buss, Folma

    2014-01-01

    Advances in screening and computational methods have enhanced recent efforts to discover/design small-molecule protein inhibitors. One attractive target for inhibition is the myosin family of motor proteins. Myosins function in a wide variety of cellular processes, from intracellular trafficking to cell motility, and are implicated in several human diseases (e.g., cancer, hypertrophic cardiomyopathy, deafness and many neurological disorders). Potent and selective myosin inhibitors are, therefore, not only a tool for understanding myosin function, but are also a resource for developing treatments for diseases involving myosin dysfunction or overactivity. This review will provide a brief overview of the characteristics and scientific/therapeutic applications of the presently identified small-molecule myosin inhibitors before discussing the future of myosin inhibitor and activator design. PMID:23256812

  19. Polyaspartate scale inhibitors -- Biodegradable alternatives to polyacrylates

    SciTech Connect

    Ross, R.J.; Low, K.C.; Shannon, J.E.

    1997-04-01

    Polyaspartates are highly biodegradable alternatives to polyacrylate-based scale inhibitors. This article presents laboratory testing data on polyaspartate inhibitors of calcium and barium mineral scales. The optimum molecular weight (Mw) for polyaspartate inhibitors of calcium carbonate, calcium sulfate, and barium sulfate mineral scales was determined to be between 1,000 Mw and 4,000 Mw. For inhibition of calcium carbonate and barium sulfate, polyaspartates in the range of 3,000 Mw to 4,000 Mw were most effective. For calcium sulfate inhibition, the optimum Mw lies in the 1,000 Mw to 2,000 Mw range. Biodegradability data (OECD 301B Ready Biodegradability) on polyaspartates of a variety of Mw is also presented, which demonstrates the high biodegradability of this class of mineral scale inhibitors.

  20. Drug design from the cryptic inhibitor envelope

    PubMed Central

    Lee, Chul-Jin; Liang, Xiaofei; Wu, Qinglin; Najeeb, Javaria; Zhao, Jinshi; Gopalaswamy, Ramesh; Titecat, Marie; Sebbane, Florent; Lemaitre, Nadine; Toone, Eric J.; Zhou, Pei

    2016-01-01

    Conformational dynamics plays an important role in enzyme catalysis, allosteric regulation of protein functions and assembly of macromolecular complexes. Despite these well-established roles, such information has yet to be exploited for drug design. Here we show by nuclear magnetic resonance spectroscopy that inhibitors of LpxC—an essential enzyme of the lipid A biosynthetic pathway in Gram-negative bacteria and a validated novel antibiotic target—access alternative, minor population states in solution in addition to the ligand conformation observed in crystal structures. These conformations collectively delineate an inhibitor envelope that is invisible to crystallography, but is dynamically accessible by small molecules in solution. Drug design exploiting such a hidden inhibitor envelope has led to the development of potent antibiotics with inhibition constants in the single-digit picomolar range. The principle of the cryptic inhibitor envelope approach may be broadly applicable to other lead optimization campaigns to yield improved therapeutics. PMID:26912110

  1. Inhibitors of Acetylcholinesterase and Butyrylcholinesterase Meet Immunity

    PubMed Central

    Pohanka, Miroslav

    2014-01-01

    Acetylcholinesterase (AChE) inhibitors are widely used for the symptomatic treatment of Alzheimer’s disease and other dementias. More recent use is for myasthenia gravis. Many of these inhibitors interact with the second known cholinesterase, butyrylcholinesterase (BChE). Further, evidence shows that acetylcholine plays a role in suppression of cytokine release through a “cholinergic anti-inflammatory pathway” which raises questions about the role of these inhibitors in the immune system. This review covers research and discussion of the role of the inhibitors in modulating the immune response using as examples the commonly available drugs, donepezil, galantamine, huperzine, neostigmine and pyridostigmine. Major attention is given to the cholinergic anti-inflammatory pathway, a well-described link between the central nervous system and terminal effector cells in the immune system. PMID:24893223

  2. Selective Phosphodiesterase 4B Inhibitors: A Review

    PubMed Central

    Azam, Mohammed Afzal; Tripuraneni, Naga Srinivas

    2014-01-01

    Abstract Phosphodiesterase 4B (PDE4B) is a member of the phosphodiesterase family of proteins that plays a critical role in regulating intracellular levels of cyclic adenosine monophosphate (cAMP) by controlling its rate of degradation. It has been demonstrated that this isoform is involved in the orchestra of events which includes inflammation, schizophrenia, cancers, chronic obstructive pulmonary disease, contractility of the myocardium, and psoriatic arthritis. Phosphodiesterase 4B has constituted an interesting target for drug development. In recent years, a number of PDE4B inhibitors have been developed for their use as therapeutic agents. In this review, an up-to-date status of the inhibitors investigated for the inhibition of PDE4B has been given so that this rich source of structural information of presently known PDE4B inhibitors could be helpful in generating a selective and potent inhibitor of PDE4B. PMID:25853062

  3. Inhibitors of acetylcholinesterase and butyrylcholinesterase meet immunity.

    PubMed

    Pohanka, Miroslav

    2014-01-01

    Acetylcholinesterase (AChE) inhibitors are widely used for the symptomatic treatment of Alzheimer's disease and other dementias. More recent use is for myasthenia gravis. Many of these inhibitors interact with the second known cholinesterase, butyrylcholinesterase (BChE). Further, evidence shows that acetylcholine plays a role in suppression of cytokine release through a "cholinergic anti-inflammatory pathway" which raises questions about the role of these inhibitors in the immune system. This review covers research and discussion of the role of the inhibitors in modulating the immune response using as examples the commonly available drugs, donepezil, galantamine, huperzine, neostigmine and pyridostigmine. Major attention is given to the cholinergic anti-inflammatory pathway, a well-described link between the central nervous system and terminal effector cells in the immune system. PMID:24893223

  4. Update on TNF Inhibitors in Dermatology.

    PubMed

    Sobell, Jeffrey M

    2016-06-01

    Emerging data describe new potential indications for tumor necrosis factor (TNF) inhibitors in dermatology, including pediatric psoriasis and hidradenitis suppurativa. New biosimilar TNF agents are in late stages of development and may be available in the United States in the near future. Biosimilar agents are similar but not identical to available TNF inhibitors, and approval requires extensive analytic, toxicity, pharmacokinetic, pharmacodynamic, and clinical testing. Semin Cutan Med Surg 35(supp6):S104-S106. PMID:27537073

  5. Heterocyclics as corrosion inhibitors for acid media

    SciTech Connect

    Ajmal, M.; Khan, M.A.W.; Ahmad, S.; Quraishi, M.A.

    1996-12-01

    The available literature on the use of heterocyclic compounds as corrosion inhibitors in acid media has been reviewed. It has been noted that the workers in this field have either used sulfur or nitrogen containing heterocyclic compounds for studying inhibition action. The authors have synthesized compounds containing sulfur and nitrogen both in the same ring and studied their inhibition action in acid media. These compounds were found to be better inhibitors than those containing either atoms alone.

  6. 2,4-Diaminopyrimidine MK2 inhibitors. Part I: Observation of an unexpected inhibitor binding mode

    SciTech Connect

    Argiriadi, Maria A.; Ericsson, Anna M.; Harris, Christopher M.; Banach, David L.; Borhani, David W.; Calderwood, David J.; Demers, Megan D.; DiMauro, Jennifer; Dixon, Richard W.; Hardman, Jennifer; Kwak, Silvia; Li, Biqin; Mankovich, John A.; Marcotte, Douglas; Mullen, Kelly D.; Ni, Baofu; Pietras, M.; Sadhukhan, Ramkrishna; Sousa, Silvino; Tomlinson, Medha J.; Wang, L.; Xiang, T.; Talanian, R.V.

    2010-09-17

    MK2 is a Ser/Thr kinase of significant interest as an anti-inflammatory drug discovery target. Here we describe the development of in vitro tools for the identification and characterization of MK2 inhibitors, including validation of inhibitor interactions with the crystallography construct and determination of the unique binding mode of 2,4-diaminopyrimidine inhibitors in the MK2 active site.

  7. Aromatase inhibitors in the treatment of endometriosis

    PubMed Central

    Męczekalski, Błażej

    2016-01-01

    Endometriosis is a chronic inflammatory condition in which foci of endometrial tissue grow outside of the uterine cavity. Endometriosis was estimated to affect 176 million women of childbearing potential all over the world in 2010. The presence of extrauterine endometrial tissue is associated with pain and infertility. Typical symptoms of endometriosis include dysmenorrhoea, dyspareunia, heavy menstrual periods (menorrhagia), pelvic pain that is not related to menstrual cycles, dysuria, and chronic fatigue. Medical treatments for endometriosis include combined oral contraceptive pills, danazol, gestrinone, medroxyprogesterone acetate, and gonadotropin-releasing hormone agonists (aGnRHs). A new class of medications called aromatase inhibitors has been identified in recent years as potential therapeutic agents for endometriosis. This article provides general information about aromatase inhibitors, their use in gynaecology, and their adverse effects. In particular, the paper discusses the use of aromatase inhibitors in the treatment of endometriosis in postmenopausal women. Unlike oral contraceptives, gestagens, aGnRHs, and danazol, which suppress ovarian oestrogen synthesis, aromatase inhibitors inhibit mainly extra-ovarian synthesis of oestrogens. Therefore, the use of aromatase inhibitors seems to be particularly relevant in older patients, as most of the body's oestrogen is produced outside the ovaries after menopause. The paper discusses also the use of aromatase inhibitors in the treatment of pain associated with endometriosis and infertility caused by endometriosis. PMID:27095958

  8. CHK1 Inhibitors in Combination Chemotherapy

    PubMed Central

    Dent, Paul; Tang, Yong; Yacoub, Adly; Dai, Yun; Fisher, Paul B.; Grant, Steven

    2011-01-01

    Cellular sensing of DNA damage, along with concomitant cell cycle arrest, is mediated by a great many proteins and enzymes. One focus of pharmaceutical development has been the inhibition of DNA damage signaling, and checkpoint kinases (Chks) in particular, as a means to sensitize proliferating tumor cells to chemotherapies that damage DNA. 7-Hydroxystaurosporine, or UCN-01, is a clinically relevant and well-studied kinase activity inhibitor that exerts chemosensitizing effects by inhibition of Chk1, and a multitude of Chk1 inhibitors have entered development. Clinical development of UCN-01 has overcome many initial obstacles, but the drug has nevertheless failed to show a high level of clinical activity when combined with chemotherapeutic agents. One very likely reason for the lack of clinical efficacy of Chk1 inhibitors may be that the inhibition of Chk1 causes the compensatory activation of ATM and ERK1/2 pathways. Indeed, inhibition of many enzyme activities, not necessarily components of cell cycle regulation, may block Chk1 inhibitor–induced ERK1/2 activation and enhance the toxicity of Chk1 inhibitors. This review examines the rationally hypothesized actions of Chk1 inhibitors as cell cycle modulatory drugs as well as the impact of Chk1 inhibition upon other cell survival signaling pathways. An understanding of Chk1 inhibition in multiple signaling contexts will be essential to the therapeutic development of Chk1 inhibitors. PMID:21540473

  9. Novel inhibitors of advanced glycation endproducts.

    PubMed

    Rahbar, Samuel; Figarola, James L

    2003-11-01

    A number of natural or synthetic compounds as AGE inhibitors have been proposed, discovered or currently being advanced by others and us. We have identified two new classes of aromatic compounds; aryl- (and heterocyclic) ureido and aryl (and heterocyclic) carboxamido phenoxyisobutyric acids, and benzoic acid derivatives and related compounds, as potential inhibitors of glycation and AGE formation. Some of these novel compounds also showed "AGE-breaking" activities in vitro. Current evidence is that chelation of transition metals and/or trapping or indirect inhibition of formation of reactive carbonyl compounds are involved in the mechanisms of action of these novel AGE inhibitors and breakers. Here, we review the inhibitors of glycation and AGE-breakers published to date and present the results of our in vitro and in vivo investigations on a number of these novel AGE inhibitors. These AGE-inhibitors and AGE-breakers may find therapeutic use in the treatment of diseases that AGE formation and accumulation may be responsible for their pathogenesis such as diabetes, Alzheimer's, rheumatoid arthritis, and atherosclerosis. PMID:14568010

  10. Aromatase inhibitors in the treatment of endometriosis.

    PubMed

    Słopień, Radosław; Męczekalski, Błażej

    2016-03-01

    Endometriosis is a chronic inflammatory condition in which foci of endometrial tissue grow outside of the uterine cavity. Endometriosis was estimated to affect 176 million women of childbearing potential all over the world in 2010. The presence of extrauterine endometrial tissue is associated with pain and infertility. Typical symptoms of endometriosis include dysmenorrhoea, dyspareunia, heavy menstrual periods (menorrhagia), pelvic pain that is not related to menstrual cycles, dysuria, and chronic fatigue. Medical treatments for endometriosis include combined oral contraceptive pills, danazol, gestrinone, medroxyprogesterone acetate, and gonadotropin-releasing hormone agonists (aGnRHs). A new class of medications called aromatase inhibitors has been identified in recent years as potential therapeutic agents for endometriosis. This article provides general information about aromatase inhibitors, their use in gynaecology, and their adverse effects. In particular, the paper discusses the use of aromatase inhibitors in the treatment of endometriosis in postmenopausal women. Unlike oral contraceptives, gestagens, aGnRHs, and danazol, which suppress ovarian oestrogen synthesis, aromatase inhibitors inhibit mainly extra-ovarian synthesis of oestrogens. Therefore, the use of aromatase inhibitors seems to be particularly relevant in older patients, as most of the body's oestrogen is produced outside the ovaries after menopause. The paper discusses also the use of aromatase inhibitors in the treatment of pain associated with endometriosis and infertility caused by endometriosis. PMID:27095958

  11. Discovery of Novel Haloalkane Dehalogenase Inhibitors

    PubMed Central

    Buryska, Tomas; Daniel, Lukas; Kunka, Antonin; Brezovsky, Jan; Damborsky, Jiri

    2016-01-01

    Haloalkane dehalogenases (HLDs) have recently been discovered in a number of bacteria, including symbionts and pathogens of both plants and humans. However, the biological roles of HLDs in these organisms are unclear. The development of efficient HLD inhibitors serving as molecular probes to explore their function would represent an important step toward a better understanding of these interesting enzymes. Here we report the identification of inhibitors for this enzyme family using two different approaches. The first builds on the structures of the enzymes' known substrates and led to the discovery of less potent nonspecific HLD inhibitors. The second approach involved the virtual screening of 150,000 potential inhibitors against the crystal structure of an HLD from the human pathogen Mycobacterium tuberculosis H37Rv. The best inhibitor exhibited high specificity for the target structure, with an inhibition constant of 3 μM and a molecular architecture that clearly differs from those of all known HLD substrates. The new inhibitors will be used to study the natural functions of HLDs in bacteria, to probe their mechanisms, and to achieve their stabilization. PMID:26773086

  12. Discovering Anti-platelet Drug Combinations with an Integrated Model of Activator-Inhibitor Relationships, Activator-Activator Synergies and Inhibitor-Inhibitor Synergies

    PubMed Central

    Lombardi, Federica; Golla, Kalyan; Fitzpatrick, Darren J.; Casey, Fergal P.; Moran, Niamh; Shields, Denis C.

    2015-01-01

    Identifying effective therapeutic drug combinations that modulate complex signaling pathways in platelets is central to the advancement of effective anti-thrombotic therapies. However, there is no systems model of the platelet that predicts responses to different inhibitor combinations. We developed an approach which goes beyond current inhibitor-inhibitor combination screening to efficiently consider other signaling aspects that may give insights into the behaviour of the platelet as a system. We investigated combinations of platelet inhibitors and activators. We evaluated three distinct strands of information, namely: activator-inhibitor combination screens (testing a panel of inhibitors against a panel of activators); inhibitor-inhibitor synergy screens; and activator-activator synergy screens. We demonstrated how these analyses may be efficiently performed, both experimentally and computationally, to identify particular combinations of most interest. Robust tests of activator-activator synergy and of inhibitor-inhibitor synergy required combinations to show significant excesses over the double doses of each component. Modeling identified multiple effects of an inhibitor of the P2Y12 ADP receptor, and complementarity between inhibitor-inhibitor synergy effects and activator-inhibitor combination effects. This approach accelerates the mapping of combination effects of compounds to develop combinations that may be therapeutically beneficial. We integrated the three information sources into a unified model that predicted the benefits of a triple drug combination targeting ADP, thromboxane and thrombin signaling. PMID:25875950

  13. Corrosion inhibitors for solar heating and cooling systems

    NASA Technical Reports Server (NTRS)

    Humphries, T. S.

    1978-01-01

    Inhibitors which appeared promising in previous tests and additional inhibitors including several proprietary products were evaluated. Evaluation of the inhibitors was based on corrosion protection afforded an aluminum-mild steel-copper-stainless steel assembly in a hot corrosive water. Of the inhibitors tested two were found to be effective and show promise for protecting multimetallic solar heating systems.

  14. Potent pyrrolidine- and piperidine-based BACE-1 inhibitors

    SciTech Connect

    Iserloh, U.; Wu, Y.; Cumming, J.N.; Pan, J.; Wang, L.Y.; Stamford, A.W.; Kennedy, M.E.; Kuvelkar, R.; Chen, X.; Parker, E.M.; Strickland, C.; Voigt, J.

    2008-08-18

    Based on lead compound 1 identified from the patent literature, we developed novel patentable BACE-1 inhibitors by introducing a cyclic amine scaffold. Extensive SAR studies on both pyrrolidines and piperidines ultimately led to inhibitor 2f, one of the most potent inhibitors synthesized to date. The discovery and development of novel BACE-1 inhibitors incorporating a cyclic amine scaffold is described.

  15. Selective serotonin reuptake inhibitor exposure.

    PubMed

    Fitzgerald, Kevin T; Bronstein, Alvin C

    2013-02-01

    Many antidepressants inhibit serotonin or norepinephrine reuptake or both to achieve their clinical effect. The selective serotonin reuptake inhibitor class of antidepressants (SSRIs) includes citalopram, escitalopram (active enantiomer of citalopram), fluoxetine, fluvoxamine, paroxetine, and sertraline. The SSRIs are as effective as tricyclic antidepressants in treatment of major depression with less significant side effects. As a result, they have become the largest class of medications prescribed to humans for depression. They are also used to treat obsessive-compulsive disorder, panic disorders, alcoholism, obesity, migraines, and chronic pain. An SSRI (fluoxetine) has been approved for veterinary use in treatment of canine separation anxiety. SSRIs act specifically on synaptic serotonin concentrations by blocking its reuptake in the presynapse and increasing levels in the presynaptic membrane. Clinical signs of SSRI overdose result from excessive amounts of serotonin in the central nervous system. These signs include nausea, vomiting, mydriasis, hypersalivation, and hyperthermia. Clinical signs are dose dependent and higher dosages may result in the serotonin syndrome that manifests itself as ataxia, tremors, muscle rigidity, hyperthermia, diarrhea, and seizures. Current studies reveal no increase in appearance of any specific clinical signs of serotonin toxicity with regard to any SSRI medication. In people, citalopram has been reported to have an increased risk of electrocardiographic abnormalities. Diagnosis of SSRI poisoning is based on history, clinical signs, and response to therapy. No single clinical test is currently available to confirm SSRI toxicosis. The goals of treatment in this intoxication are to support the animal, prevent further absorption of the drug, support the central nervous system, control hyperthermia, and halt any seizure activity. The relative safety of the SSRIs in overdose despite the occurrence of serotonin syndrome makes them

  16. Selective serotonin reuptake inhibitor exposure.

    PubMed

    Fitzgerald, Kevin T; Bronstein, Alvin C

    2013-02-01

    Many antidepressants inhibit serotonin or norepinephrine reuptake or both to achieve their clinical effect. The selective serotonin reuptake inhibitor class of antidepressants (SSRIs) includes citalopram, escitalopram (active enantiomer of citalopram), fluoxetine, fluvoxamine, paroxetine, and sertraline. The SSRIs are as effective as tricyclic antidepressants in treatment of major depression with less significant side effects. As a result, they have become the largest class of medications prescribed to humans for depression. They are also used to treat obsessive-compulsive disorder, panic disorders, alcoholism, obesity, migraines, and chronic pain. An SSRI (fluoxetine) has been approved for veterinary use in treatment of canine separation anxiety. SSRIs act specifically on synaptic serotonin concentrations by blocking its reuptake in the presynapse and increasing levels in the presynaptic membrane. Clinical signs of SSRI overdose result from excessive amounts of serotonin in the central nervous system. These signs include nausea, vomiting, mydriasis, hypersalivation, and hyperthermia. Clinical signs are dose dependent and higher dosages may result in the serotonin syndrome that manifests itself as ataxia, tremors, muscle rigidity, hyperthermia, diarrhea, and seizures. Current studies reveal no increase in appearance of any specific clinical signs of serotonin toxicity with regard to any SSRI medication. In people, citalopram has been reported to have an increased risk of electrocardiographic abnormalities. Diagnosis of SSRI poisoning is based on history, clinical signs, and response to therapy. No single clinical test is currently available to confirm SSRI toxicosis. The goals of treatment in this intoxication are to support the animal, prevent further absorption of the drug, support the central nervous system, control hyperthermia, and halt any seizure activity. The relative safety of the SSRIs in overdose despite the occurrence of serotonin syndrome makes them

  17. Combined effects of EGFR tyrosine kinase inhibitors and vATPase inhibitors in NSCLC cells

    SciTech Connect

    Jin, Hyeon-Ok; Hong, Sung-Eun; Kim, Chang Soon; Park, Jin-Ah; Kim, Jin-Hee; Kim, Ji-Young; Kim, Bora; Chang, Yoon Hwan; Hong, Seok-Il; Hong, Young Jun; Park, In-Chul; Lee, Jin Kyung

    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.

  18. Three Decades of β-Lactamase Inhibitors

    PubMed Central

    Drawz, Sarah M.; Bonomo, Robert A.

    2010-01-01

    Summary: Since the introduction of penicillin, β-lactam antibiotics have been the antimicrobial agents of choice. Unfortunately, the efficacy of these life-saving antibiotics is significantly threatened by bacterial β-lactamases. β-Lactamases are now responsible for resistance to penicillins, extended-spectrum cephalosporins, monobactams, and carbapenems. In order to overcome β-lactamase-mediated resistance, β-lactamase inhibitors (clavulanate, sulbactam, and tazobactam) were introduced into clinical practice. These inhibitors greatly enhance the efficacy of their partner β-lactams (amoxicillin, ampicillin, piperacillin, and ticarcillin) in the treatment of serious Enterobacteriaceae and penicillin-resistant staphylococcal infections. However, selective pressure from excess antibiotic use accelerated the emergence of resistance to β-lactam-β-lactamase inhibitor combinations. Furthermore, the prevalence of clinically relevant β-lactamases from other classes that are resistant to inhibition is rapidly increasing. There is an urgent need for effective inhibitors that can restore the activity of β-lactams. Here, we review the catalytic mechanisms of each β-lactamase class. We then discuss approaches for circumventing β-lactamase-mediated resistance, including properties and characteristics of mechanism-based inactivators. We next highlight the mechanisms of action and salient clinical and microbiological features of β-lactamase inhibitors. We also emphasize their therapeutic applications. We close by focusing on novel compounds and the chemical features of these agents that may contribute to a “second generation” of inhibitors. The goal for the next 3 decades will be to design inhibitors that will be effective for more than a single class of β-lactamases. PMID:20065329

  19. Cardiovascular effects of selective cyclooxygenase-2 inhibitors.

    PubMed

    Krum, Henry; Liew, Danny; Aw, Juan; Haas, Steven

    2004-03-01

    Selective cyclooxygenase-2 inhibitors represent a significant advance in the management of inflammatory disorders. They have similar efficacy to nonselective 'conventional' nonsteroidal anti-inflammatory drugs, but a superior gastrointestinal safety profile. However, a significant caveat is the perceived potential of cyclooxygenase-2 inhibitors to cause adverse cardiovascular effects, an issue first raised by the Vioxx Gastrointestinal Outcomes Research (VIGOR) study of rofecoxib (Vioxx, Merck & Co. Inc.). Mechanisms by which cyclooxygenase-2 inhibitors may increase cardiovascular risk are selective inhibition of prostaglandin I2 over thromboxane A2 within the eicosanoid pathway, which promotes thrombosis, and inhibition of prostaglandins E2 and I2 within the kidney, which leads to sodium and water retention and blood pressure elevation. In spite of this, the cardiovascular findings from VIGOR are not firmly supported by observations from large cohort studies and other clinical trials of selective cyclooxygenase-2 inhibitors, including the Celecoxib Long-term Arthritis Safety Study. The two main theories that explain the VIGOR findings are that the comparator used (naproxen; Naprosyn, Roche) is cardioprotective and that very high doses of rofecoxib were used, but at present neither is backed by firm evidence. Indeed, there is now early evidence that selective cyclooxygenase-2 inhibition with celecoxib may even protect against the progression of cardiovascular disease, on the basis that cyclooxygenase-2 mediates key processes in atherothrombosis. Currently, it is not clear what the net cardiovascular effects of cyclooxygenase-2 inhibitors are. The data are inconsistent and at best, speculative. It may be also that celecoxib and rofecoxib differ in their cardiovascular effects. Clarification of these issues is of vital importance given the vast number of patients presently taking both types of cyclooxygenase-2 inhibitors. Therefore, what is clear in this situation is

  20. Versatile templates for the development of novel kinase inhibitors: Discovery of novel CDK inhibitors

    SciTech Connect

    Dwyer, Michael P.; Paruch, Kamil; Alvarez, Carmen; Doll, Ronald J.; Keertikar, Kerry; Duca, Jose; Fischmann, Thierry O.; Hruza, Alan; Madison, Vincent; Lees, Emma; Parry, David; Seghezzi, Wolfgang; Sgambellone, Nicole; Shanahan, Frances; Wiswell, Derek; Guzi, Timothy J.

    2008-06-30

    A series of four bicyclic cores were prepared and evaluated as cyclin-dependent kinase-2 (CDK2) inhibitors. From the in-vitro and cell-based analysis, the pyrazolo[1,5-a]pyrimidine core (represented by 9) emerged as the superior core for further elaboration in the identification of novel CDK2 inhibitors.

  1. Polyphenol oxidase inhibitor(s) from German cockroach (Blattella germanica) extract

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An extract from German cockroach appears effective in inhibiting browning on apples and potatoes. Successful identification of inhibitor(s) of PPO from German cockroach would be useful to the fruit and vegetable segments of the food industry, due to the losses they incur from enzymatic browning. Ide...

  2. Epidemiological aspects of inhibitor development redefine the clinical importance of inhibitors.

    PubMed

    van den Berg, H M

    2014-05-01

    Inhibitor development is a serious complication of treatment with coagulation products. Presently, 25-30% of all newly diagnosed patients with severe haemophilia A are diagnosed with inhibitors. An increasing number of genetic and non-genetic risk factors have been reported to be involved, although the impact of them in understanding the aetiology is still limited. Much attention has been focused on factor VIII products, but more recent studies show very little, if any, difference between class plasma and recombinant factor VIII products. More intensive treatment and higher dosing are probably more important factors. More than 10% of the inhibitors diagnosed in the last decade are of low titre. A first goal should be to understand their importance. It is argued that the impact of different risk factors should be studied in high-titre inhibitors to prevent dilution by non-significant low-titre inhibitors.

  3. Inhibitors Selective for Mycobacterial Versus Human Proteasomes

    SciTech Connect

    Lin, G.; Li, D; Sorio de Carvalho, L; Deng, H; Tao, H; Vogt, G; Wu, K; Schneider, J; Chidawanyika, T; et. al.

    2009-01-01

    Many anti-infectives inhibit the synthesis of bacterial proteins, but none selectively inhibits their degradation. Most anti-infectives kill replicating pathogens, but few preferentially kill pathogens that have been forced into a non-replicating state by conditions in the host. To explore these alternative approaches we sought selective inhibitors of the proteasome of Mycobacterium tuberculosis. Given that the proteasome structure is extensively conserved, it is not surprising that inhibitors of all chemical classes tested have blocked both eukaryotic and prokaryotic proteasomes, and no inhibitor has proved substantially more potent on proteasomes of pathogens than of their hosts. Here we show that certain oxathiazol-2-one compounds kill non-replicating M.?tuberculosis and act as selective suicide-substrate inhibitors of the M.?tuberculosis proteasome by cyclocarbonylating its active site threonine. Major conformational changes protect the inhibitor-enzyme intermediate from hydrolysis, allowing formation of an oxazolidin-2-one and preventing regeneration of active protease. Residues outside the active site whose hydrogen bonds stabilize the critical loop before and after it moves are extensively non-conserved. This may account for the ability of oxathiazol-2-one compounds to inhibit the mycobacterial proteasome potently and irreversibly while largely sparing the human homologue.

  4. Inhibitors in LPE growth of garnets

    NASA Astrophysics Data System (ADS)

    De Roode, W. H.; Robertson, J. M.

    1983-09-01

    The growth rate of LPE growth garnets can be reduced considerably by the addition of small amounts of group II oxides. This effect can be helpful for the controlled growth of very thin garnet films for sub-micron bubbles and optical devices. The largest effect was found with the addition of Mg 2+ and Ca 2+, resulting in a maximum decrease of the growth rate of approximately 70%. A semi-empirical formula was used to describe the growth rate as a function of the dipping temperature. The change in the growth rate on the addition of the inhibitor ion at constant temperature was found to be proportional to ( aMO)/( aMO+2 Ln 2O 3), where M is a group II element, Ln 2O 2 is the sum of the yttrium and RE oxides in the melt, and a is the inhibitor factor. The value of the inhibitor factor depends on both the inhibitor ion as well as the composition of the garnet. The lowering of the growth rate on the addition of an inhibitor ion is explained by the introduction of an extra growth resistance due to the charge compensation mechanism of the divalent ions. The influence of the different charge compensation possibilities in the garnet system is examined and the relative importance of these possibilities for charge compensation is discussed.

  5. Inhibitors of the Metalloproteinase Anthrax Lethal Factor.

    PubMed

    Goldberg, Allison B; Turk, Benjamin E

    2016-01-01

    Bacillus anthracis, a rod shaped, spore forming, gram positive bacteria, is the etiological agent of anthrax. B. anthracis virulence is partly attributable to two secreted bipartite protein toxins, which act inside host cells to disrupt signaling pathways important for host defense against infection. These toxins may also directly contribute to mortality in late stage infection. The zinc-dependent metalloproteinase anthrax lethal factor (LF) is a critical component of one of these protein toxins and a prime target for inhibitor development to produce anthrax therapeutics. Here, we describe recent efforts to identify specific and potent LF inhibitors. Derivatization of peptide substrate analogs bearing zinc-binding groups has produced potent and specific LF inhibitors, and X-ray crystallography of LFinhibitor complexes has provided insight into features required for high affinity binding. Novel inhibitor scaffolds have been identified through several approaches, including fragment-based drug discovery, virtual screening, and highthroughput screening of diverse compound libraries. Lastly, efforts to discover LF inhibitors have led to the development of new screening strategies, such as the use of full-length proteins as substrates, that may prove useful for other proteases as well. Overall, these efforts have led to a collection of chemically and mechanistically diverse molecules capable of inhibiting LF activity in vitro and in cells, as well as in animal models of anthrax infection. PMID:27072692

  6. Inhibitors of human immunodeficiency virus integrase.

    PubMed Central

    Fesen, M R; Kohn, K W; Leteurtre, F; Pommier, Y

    1993-01-01

    In an effort to further extend the number of targets for development of antiretroviral agents, we have used an in vitro integrase assay to investigate a variety of chemicals, including topoisomerase inhibitors, antimalarial agents, DNA binders, naphthoquinones, the flavone quercetin, and caffeic acid phenethyl ester as potential human immunodeficiency virus type 1 integrase inhibitors. Our results show that although several topoisomerase inhibitors--including doxorubicin, mitoxantrone, ellipticines, and quercetin--are potent integrase inhibitors, other topoisomerase inhibitors--such as amsacrine, etoposide, teniposide, and camptothecin--are inactive. Other intercalators, such as chloroquine and the bifunctional intercalator ditercalinium, are also active. However, DNA binding does not correlate closely with integrase inhibition. The intercalator 9-aminoacridine and the polyamine DNA minor-groove binders spermine, spermidine, and distamycin have no effect, whereas the non-DNA binders primaquine, 5,8-dihydroxy-1,4-naphthoquinone, and caffeic acid phenethyl ester inhibit the integrase. Caffeic acid phenethyl ester was the only compound that inhibited the integration step to a substantially greater degree than the initial cleavage step of the enzyme. A model of 5,8-dihydroxy-1,4-naphthoquinone interaction with the zinc finger region of the retroviral integrase protein is proposed. Images Fig. 2 PMID:8460151

  7. Resistance to AHAS inhibitor herbicides: current understanding.

    PubMed

    Yu, Qin; Powles, Stephen B

    2014-09-01

    Acetohydroxyacid synthase (AHAS) inhibitor herbicides currently comprise the largest site-of-action group (with 54 active ingredients across five chemical groups) and have been widely used in world agriculture since they were first introduced in 1982. Resistance evolution in weeds to AHAS inhibitors has been rapid and identified in populations of many weed species. Often, evolved resistance is associated with point mutations in the target AHAS gene; however non-target-site enhanced herbicide metabolism occurs as well. Many AHAS gene resistance mutations can occur and be rapidly enriched owing to a high initial resistance gene frequency, simple and dominant genetic inheritance and lack of major fitness cost of the resistance alleles. Major advances in the elucidation of the crystal structure of the AHAS (Arabidopsis thaliana) catalytic subunit in complex with various AHAS inhibitor herbicides have greatly improved current understanding of the detailed molecular interactions between AHAS, cofactors and herbicides. Compared with target-site resistance, non-target-site resistance to AHAS inhibitor herbicides is less studied and hence less understood. In a few well-studied cases, non-target-site resistance is due to enhanced rates of herbicide metabolism (metabolic resistance), mimicking that occurring in tolerant crop species and often involving cytochrome P450 monooxygenases. However, the specific herbicide-metabolising, resistance-endowing genes are yet to be identified in resistant weed species. The current state of mechanistic understanding of AHAS inhibitor herbicide resistance is reviewed, and outstanding research issues are outlined.

  8. Protease Inhibitors from Plants with Antimicrobial Activity

    PubMed Central

    Kim, Jin-Young; Park, Seong-Cheol; Hwang, Indeok; Cheong, Hyeonsook; Nah, Jae-Woon; Hahm, Kyung-Soo; Park, Yoonkyung

    2009-01-01

    Antimicrobial proteins (peptides) are known to play important roles in the innate host defense mechanisms of most living organisms, including plants, insects, amphibians and mammals. They are also known to possess potent antibiotic activity against bacteria, fungi, and even certain viruses. Recently, the rapid emergence of microbial pathogens that are resistant to currently available antibiotics has triggered considerable interest in the isolation and investigation of the mode of action of antimicrobial proteins (peptides). Plants produce a variety of proteins (peptides) that are involved in the defense against pathogens and invading organisms, including ribosome-inactivating proteins, lectins, protease inhibitors and antifungal peptides (proteins). Specially, the protease inhibitors can inhibit aspartic, serine and cysteine proteinases. Increased levels of trypsin and chymotrypsin inhibitors correlated with the plants resistance to the pathogen. Usually, the purification of antimicrobial proteins (peptides) with protease inhibitor activity was accomplished by salt-extraction, ultrafiltration and C18 reverse phase chromatography, successfully. We discuss the relation between antimicrobial and anti-protease activity in this review. Protease inhibitors from plants potently inhibited the growth of a variety of pathogenic bacterial and fungal strains and are therefore excellent candidates for use as the lead compounds for the development of novel antimicrobial agents. PMID:19582234

  9. Monoamine Reuptake Inhibitors in Parkinson's Disease

    PubMed Central

    Huot, Philippe; Fox, Susan H.; Brotchie, Jonathan M.

    2015-01-01

    The motor manifestations of Parkinson's disease (PD) are secondary to a dopamine deficiency in the striatum. However, the degenerative process in PD is not limited to the dopaminergic system and also affects serotonergic and noradrenergic neurons. Because they can increase monoamine levels throughout the brain, monoamine reuptake inhibitors (MAUIs) represent potential therapeutic agents in PD. However, they are seldom used in clinical practice other than as antidepressants and wake-promoting agents. This review article summarises all of the available literature on use of 50 MAUIs in PD. The compounds are divided according to their relative potency for each of the monoamine transporters. Despite wide discrepancy in the methodology of the studies reviewed, the following conclusions can be drawn: (1) selective serotonin transporter (SERT), selective noradrenaline transporter (NET), and dual SERT/NET inhibitors are effective against PD depression; (2) selective dopamine transporter (DAT) and dual DAT/NET inhibitors exert an anti-Parkinsonian effect when administered as monotherapy but do not enhance the anti-Parkinsonian actions of L-3,4-dihydroxyphenylalanine (L-DOPA); (3) dual DAT/SERT inhibitors might enhance the anti-Parkinsonian actions of L-DOPA without worsening dyskinesia; (4) triple DAT/NET/SERT inhibitors might exert an anti-Parkinsonian action as monotherapy and might enhance the anti-Parkinsonian effects of L-DOPA, though at the expense of worsening dyskinesia. PMID:25810948

  10. SHH inhibitors for the treatment of medulloblastoma.

    PubMed

    Samkari, Ayman; White, Jason; Packer, Roger

    2015-01-01

    Medulloblastoma is the most common malignant brain tumor of childhood. It is currently stratified into four molecular variants through the advances in transcriptional profiling. They include: wingless, sonic hedgehog (SHH), Group III, and Group IV. The SHH group is characterized by constitutive activation of the SHH signaling pathway, and genetically characterized by mutations in patched homolog 1 (PTCH1) or other downstream pathway mutations. SHH inhibitors have become of great clinical interest in treating SHH-driven medulloblastoma. Many inhibitors are currently in different stages of development, some already approved for other SHH-driven cancers, such as basal cell carcinoma. In vitro and in vivo medulloblastoma studies have shown efficacy and these findings have been translated into Phase I and II clinical trials. In this review, we present an overview of SHH medulloblastoma, as well as a discussion of currently available SHH inhibitors, and the challenges associated with their use. PMID:26027634

  11. LDL Cholesterol, Statins And PCSK 9 Inhibitors

    PubMed Central

    Gupta, Sanjiv

    2015-01-01

    Reduction of low density lipoprotein cholesterol (LDLc) is of vital importance for the prevention of atherosclerotic cardiovascular disease (ASCVD). Statin is the most effective therapy today to lower LDLc by inhibiting HMG-CoA-reductase. However despite intensive statin therapy, there remains a residual risk of recurrent myocardial infarction in about 20–30% cases. Moreover a few patients develop statin intolerance. For severe hypercholesterolemia, statins alone or in combination of ezetimibe, niacin and fenofibrate have been advocated. For homozygous familial hypercholesterolemia (HOFH), a microsomal triglyceride transfer protein MTP inhibitor (Lopitamide) and antisense oligonucleotide (ASO) (Mipomersen) have recently been approved by FDA, USA through ‘Risk evaluation and Mitigation Strategy (REMS)’. Possible future therapies include PCSK-9 inhibitors which have excellent lipid lowering properties. Three monoclonal antibodies (PCSK 9 Inhibitors) alirocumab, evolocumab and Bococizumab are under advanced clinical stage IV trials and awaiting approval by FDA and European Medicines Agency. PMID:26432726

  12. LDL cholesterol, statins and PCSK 9 inhibitors.

    PubMed

    Gupta, Sanjiv

    2015-01-01

    Reduction of low density lipoprotein cholesterol (LDLc) is of vital importance for the prevention of atherosclerotic cardiovascular disease (ASCVD). Statin is the most effective therapy today to lower LDLc by inhibiting HMG-CoA-reductase. However despite intensive statin therapy, there remains a residual risk of recurrent myocardial infarction in about 20-30% cases. Moreover a few patients develop statin intolerance. For severe hypercholesterolemia, statins alone or in combination of ezetimibe, niacin and fenofibrate have been advocated. For homozygous familial hypercholesterolemia (HOFH), a microsomal triglyceride transfer protein MTP inhibitor (Lopitamide) and antisense oligonucleotide (ASO) (Mipomersen) have recently been approved by FDA, USA through 'Risk evaluation and Mitigation Strategy (REMS)'. Possible future therapies include PCSK-9 inhibitors which have excellent lipid lowering properties. Three monoclonal antibodies (PCSK 9 Inhibitors) alirocumab, evolocumab and Bococizumab are under advanced clinical stage IV trials and awaiting approval by FDA and European Medicines Agency. PMID:26432726

  13. Calcification inhibitors in human ligamentum flavum.

    PubMed

    Maruta, K; Ichimura, K; Matsui, H; Yamagami, T; Sano, A; Tsuji, H

    1993-01-01

    To examine the presence of substances which inhibit calcification in human ligamentum flavum, the inhibitory effect of an Na2HPO4 extract of the flavum was determined in terms of the in vitro calcium uptake of the ligamentum flavum matrix. Additionally, grafts of extracted and non-extracted dry ligamentum flavum matrices were transplanted into the dorsal muscles of rats, and calcification in the grafts was examined radiologically and histochemically. In order to determine if component cells of human ligamentum flavum produce calcification inhibitors, ligamentum flavum cells were cultured, and the crystal inhibitor activity of the culture medium was measured by a seed test which used hydroxyapatite as the nucleus of precipitation. The calcification reaction system demonstrated that the ligamentum flavum extract contains an inhibitory factor for calcium uptake by the ligamentum flavum matrix. The seed test revealed that human ligamentum flavum cells produce calcification inhibitor activity.

  14. Orally Bioavailable Potent Soluble Epoxide Hydrolase Inhibitors

    PubMed Central

    Hwang, Sung Hee; Tsai, Hsing-Ju; Liu, Jun-Yan; Morisseau, Christophe; Hammock, Bruce D.

    2008-01-01

    A series of N,N′-disubstituted ureas having a conformationally restricted cis- or trans-1,4-cyclohexane α to the urea were prepared and tested as soluble epoxide hydrolase (sEH) inhibitors. This series of compounds showed low nanomolar to picomolar activities against recombinant human sEH. Both isomers showed similar potencies, but the trans isomers were more metabolically stable in human hepatic microsomes. Furthermore, these new potent inhibitors show a greater metabolic stability in vivo than previously described sEH inhibitors. We demonstrated that trans-4-[4-(3-adamantan-1-ylureido)cyclohexyloxy]benzoic acid 13g (t-AUCB, IC50 = 1.3 ± 0.05 nM) had excellent oral bioavailability (98%, n = 2) and blood area under the curve in dogs and was effective in vivo to treat hypotension in lipopolysaccharide challenged murine models. PMID:17616115

  15. Serotonin norepinephrine reuptake inhibitors: a pharmacological comparison.

    PubMed

    Sansone, Randy A; Sansone, Lori A

    2014-03-01

    The serotonin norepinephrine reuptake inhibitors are a family of antidepressants that inhibit the reuptake of both serotonin and norepinephrine. While these drugs are traditionally considered a group of inter-related antidepressants based upon reuptake inhibition, they generally display different chemical structures as well as different pharmacological properties. In this article, we discuss these and other differences among the serotonin norepinephrine reuptake inhibitors, including the year of approval by the United States Food and Drug Administration, generic availability, approved clinical indications, half-lives, metabolism and excretion, presence or not of active metabolites, dosing schedules, proportionate effects on serotonin and norepinephrine, and the timing of serotonin and norepinephrine reuptake (i.e., sequential or simultaneous). Again, while serotonin norepinephrine reuptake inhibitors are grouped as a family of antidepressants, they exhibit a surprising number of differences- differences that may ultimately relate to clinical nuances in patient care. PMID:24800132

  16. Cyclooxygenase-2 inhibitors: promise or peril?

    PubMed Central

    Mengle-Gaw, Laurel J; Schwartz, Benjamin D

    2002-01-01

    The discovery of two isoforms of the cyclooxygenase enzyme, COX-1 and COX-2, and the development of COX-2-specific inhibitors as anti-inflammatories and analgesics have offered great promise that the therapeutic benefits of NSAIDs could be optimized through inhibition of COX-2, while minimizing their adverse side effect profile associated with inhibition of COX-1. While COX-2 specific inhibitors have proven to be efficacious in a variety of inflammatory conditions, exposure of large numbers of patients to these drugs in postmarketing studies have uncovered potential safety concerns that raise questions about the benefit/risk ratio of COX-2-specific NSAIDs compared to conventional NSAIDs. This article reviews the efficacy and safety profiles of COX-2-specific inhibitors, comparing them with conventional NSDAIDs. PMID:12467519

  17. Transgenic inhibitors of RNA interference in Drosophila.

    PubMed

    Chou, Yu-ting; Tam, Bergin; Linay, Fabien; Lai, Eric C

    2007-01-01

    RNA silencing functions as an adaptive antiviral defense in both plants and animals. In turn, viruses commonly encode suppressors of RNA silencing, which enable them to mount productive infection. These inhibitor proteins may be exploited as reagents with which to probe mechanisms and functions of RNA silencing pathways. In this report, we describe transgenic Drosophila strains that allow inducible expression of the viral RNA silencing inhibitors Flock House virus-B2, Nodamura virus-B2, vaccinia virus-E3L, influenza A virus-NS1 and tombusvirus P19. Some of these, especially the B2 proteins, are effective transgenic inhibitors of double strand RNA-induced gene silencing in flies. On the other hand, none of them is effective against the Drosophila microRNA pathway. Their functional selectivity makes these viral silencing proteins useful reagents with which to study biological functions of the Drosophila RNA interference pathway.

  18. Current and Novel Inhibitors of HIV Protease

    PubMed Central

    Pokorná, Jana; Machala, Ladislav; Řezáčová, Pavlína; Konvalinka, Jan

    2009-01-01

    The design, development and clinical success of HIV protease inhibitors represent one of the most remarkable achievements of molecular medicine. This review describes all nine currently available FDA-approved protease inhibitors, discusses their pharmacokinetic properties, off-target activities, side-effects, and resistance profiles. The compounds in the various stages of clinical development are also introduced, as well as alternative approaches, aiming at other functional domains of HIV PR. The potential of these novel compounds to open new way to the rational drug design of human viruses is critically assessed. PMID:21994591

  19. Identification of potent, selective KDM5 inhibitors.

    PubMed

    Gehling, Victor S; Bellon, Steven F; Harmange, Jean-Christophe; LeBlanc, Yves; Poy, Florence; Odate, Shobu; Buker, Shane; Lan, Fei; Arora, Shilpi; Williamson, Kaylyn E; Sandy, Peter; Cummings, Richard T; Bailey, Christopher M; Bergeron, Louise; Mao, Weifeng; Gustafson, Amy; Liu, Yichin; VanderPorten, Erica; Audia, James E; Trojer, Patrick; Albrecht, Brian K

    2016-09-01

    This communication describes the identification and optimization of a series of pan-KDM5 inhibitors derived from compound 1, a hit initially identified against KDM4C. Compound 1 was optimized to afford compound 20, a 10nM inhibitor of KDM5A. Compound 20 is highly selective for the KDM5 enzymes versus other histone lysine demethylases and demonstrates activity in a cellular assay measuring the increase in global histone 3 lysine 4 tri-methylation (H3K4me3). In addition compound 20 has good ADME properties, excellent mouse PK, and is a suitable starting point for further optimization. PMID:27476424

  20. Presence of aromatase inhibitors in cycads.

    PubMed

    Kowalska, M T; Itzhak, Y; Puett, D

    1995-07-28

    Cycads, the most primitive of the living gymnosperms, have been used and continue to be used for food and medicinal purposes by many cultures, although toxins must be removed before ingestion. In our quest to identify tropical plants that contain inhibitors of the cytochrome P-450 aromatase and thus may be efficacious in treating estrogen-dependent tumors, we have screened extracts from 5 species of cycad folia encompassing 3 genera: Cycas cairnsiana F. Muell., Cycas revoluta Thunb., Cycas rumphii Miq., Dioon spinulosum Dyer and Encephalartos ferox Bertol. All extracts were found to contain inhibitors of the human enzyme.

  1. Inhibition of elastase by a synthetic cotton-bound serine protease inhibitor: in vitro kinetics and inhibitor release.

    PubMed

    Edwards, J V; Bopp, A F; Batiste, S; Ullah, A J; Cohen, I K; Diegelmann, R F; Montante, S J

    1999-01-01

    A cotton-bound serine protease inhibitor of elastase (fiber-inhibitor) has been formulated for in vitro evaluation in chronic wound fluid. As a model to understand the properties of the inhibitor in wound dressings, the kinetic profile and in vitro release of the fiber-inhibitor formulation have been examined. The elastase inhibitor N-Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone was modified onto cotton cellulose fibers and assayed as a colloidal system. Amino acid analysis and reversed phase high performance liquid chromatography were compared as semiquantitative methods to assess elastase inhibitor release from the cotton fibers. The kinetics of inhibition was assessed on treated fibers of synthetic dressings such that a colloidal suspension of the fiber-inhibitor and elastase was employed as an assay. A dose-response relationship was observed in the kinetics of substrate hydrolysis catalyzed by three elastases: porcine pancreatic elastase, which was employed to model this approach; human leukocyte elastase; and elastase in human chronic wound fluid. Both freely dissolved and fiber-bound inhibitors were studied. The initial rates of substrate hydrolysis were inversely linear with freely dissolved inhibitor dose. The apparent first order rate constants, kobs, for the elastase-inhibitor complex were calculated from the kinetic profiles. The kobs for inhibitor bound enzyme varied as a function of inhibitor vs. enzyme concentration and based on the order of mixing of substrate, inhibitor and enzyme in the assay. Enzyme inhibition by the fiber-inhibitor was measured as inhibitor concentration at 50% inhibition (I50). I50 values measured from the colloidal assay with fiber-released inhibitor were within the same range to those for freely dissolved inhibitor. Inhibition of elastase activity in chronic wound fluid was observed with 1-5 mg of fiber-inhibitor formulation. This approach constitutes an in vitro assessment of synthetic serine protease inhibitors on

  2. Cyclic amidine sugars as transition-state analogue inhibitors of glycosidases: potent competitive inhibitors of mannosidases.

    PubMed

    Heck, Marie-Pierre; Vincent, Stéphane P; Murray, Brion W; Bellamy, François; Wong, Chi-Huey; Mioskowski, Charles

    2004-02-25

    A series of monocyclic glycoamidines bearing different exocyclic amine, alcohol, or alkyl functionalities and bicyclic amidines derived from D-glucose and D-mannose were synthesized and tested as inhibitors of various glycosidases. All the prepared compounds demonstrated good to excellent inhibition toward glycosidases. In particular, the biscationic D-mannoamidine 9b bearing an exocyclic ethylamine moiety proved to be a selective competitive inhibitor of alpha- and beta-mannosidases (K(i) = 6 nM) making it the most potent inhibitor of these glycosidases reported to date. A favorable B(2,5) boat conformation might explain the selectivity of mannosidase inhibition compared to other glycosidases.

  3. Effect of Chirality of Small Molecule Organofluorine Inhibitors of Amyloid Self-Assembly on Inhibitor Potency

    PubMed Central

    Sood, Abha; Abid, Mohammed; Hailemichael, Samson; Foster, Michelle; Török, Béla

    2009-01-01

    The effect of enantiomeric trifluromethyl-indolyl-acetic acid ethyl esters on the fibrillogenesis of Alzheimer's amyloid β (Aβ) peptide is described. These compounds have been previously identified as effective inhibitors of the Aβ self-assembly in their racemic form. Thioflavin-T Fluorescence Spectroscopy and Atomic Force Microscopy were applied to assess the potency of the chiral target compounds. Both enantiomers showed significant inhibition in the in vitro assays. The potency of the enantiomeric inhibitors appeared to be very similar to each other suggesting the lack of the stereospecific binding interactions between these small molecule inhibitors and the Aβ peptide. PMID:19880318

  4. Polyphenolic Compounds as Pancreatic Lipase Inhibitors.

    PubMed

    Buchholz, Tina; Melzig, Matthias F

    2015-07-01

    Obesity and its associated diseases such as diabetes mellitus and coronary heart diseases are a major challenge for our society. An important target for the treatment of obesity includes the development of inhibitors of nutrient digestion and absorption. Inhibition of pancreatic lipase and the associated reduction of lipid absorption is an attractive approach for the discovery of potent agents. Currently, the only clinically approved pharmacologic agent as pancreatic lipase inhibitor is Orlistat. However, its usage is compromised by unpleasant gastrointestinal adverse reactions (oily stools, oily spotting, flatulence). The use of botanical materials as a potential source of new drugs is of increasing importance and application. Natural products that are interesting for obesity treatment are generally considered to have less toxic and side effects than totally synthetic drugs. One of the most important sources of potential pancreatic lipase inhibitors represents the class of polyphenols. This article summarizes most studied subclasses of polyphenols including flavonoids, hydroxycinnamic acids, hydroxybenzoic acids and lignans with pancreatic lipase inhibitory effects. A structural comparison of potent inhibitors shows an increased inhibitory effect depending on number and position of phenolic hydroxyl groups, degree of polymerization and elimination of glycosylation during digestion. PMID:26132857

  5. Small Molecule Inhibitor of AICAR Transformylase Homodimerization

    PubMed Central

    Spurr, Ian B.; Birts, Charles N.; Cuda, Francesco; Benkovic, Stephen J; Blaydes, Jeremy P.; Tavassoli, Ali

    2012-01-01

    Aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) is a bifunctional homodimeric enzyme that catalyses the last two steps of de novo purine biosynthesis. Homodimerization of ATIC, a protein-protein interaction with an interface of over 5000 Å2, is required for its aminoimidazole carboxamide ribonucleotide (AICAR) transformylase activity, with the active sites forming at the interface of the interacting proteins. Here, we report the development of a small-molecule inhibitor of AICAR transformylase that functions by preventing the homodimerization of ATIC. The compound is derived from a previously reported cyclic hexa-peptide inhibitor of AICAR transformylase (with a Ki of 17 μM), identified by high-throughput screening. The active motif of the cyclic peptide is identified as an arginine-tyrosine dipeptide, a capped analogue of which inhibits AICAR transformylase with a Ki of 84 μM. A library of non-natural analogues of this dipeptide was designed, synthesized, and assayed. The most potent compound inhibits AICAR transformylase with a Ki of 685 nM, a 25-fold improvement in activity from the parent cyclic peptide. The potential for this AICAR transformylase inhibitor in cancer therapy is assessed by studying its effect on the proliferation of a model breast cancer cell line. Using a non-radioactive proliferation assay and live cell imaging, a dose-dependent reduction in cell numbers and cell division rates was observed in cells treated with our ATIC dimerization inhibitor. PMID:22764122

  6. Alcalase rapeseed inhibitors: purification and partial characterization.

    PubMed

    Vioque, J; Sánchez-Vioque, R; Clemente, A; Pedroche, J; Mar Yust, M; Millán, F

    2001-01-01

    Extensive rapeseed protein hydrolysate obtained sequentially with Alcalase and Flavourzyme showed inhibitory activity towards Alcalase. Inhibitory activity decreased as the hydrolytic process progressed probably by heat denaturation and/or partial protease degradation. Alcalase rapeseed inhibitors were purified by gel filtration and subsequent ion exchange chromatography. They are composed of peptides of 8.4 and 6.1 kDa linked by interchain disulphide bonds, as observed by reducing SDS-PAGE, with a native molecular weight of 18 kDa. Aminoacid composition of the inhibitors was characterized by the high proportion of methionine (4.2%) and cysteine (4.6%). Alcalase inhibitors were partially resistant to heat treatment; after heating at 70 degrees C for 45 minutes more than 50% of the original inhibitory activity remained in the purified protein but after heating at 90 degrees C for 5 minutes, inhibitory activity decreased very fast to a basal level. The possible relation of these protease inhibitors with the 2S albumin storage proteins is discussed.

  7. Curcumin derivatives as HIV-1 protease inhibitors

    SciTech Connect

    Sui, Z.; Li, J.; Craik, C.S.; Ortiz de Montellano, P.R.

    1993-12-31

    Curcumin, a non-toxic natural compound from Curcuma longa, has been found to be an HIV-1 protease inhibitor. Some of its derivatives were synthesized and their inhibitory activity against the HIV-1 protease was tested. Curcumin analogues containing boron enhanced the inhibitory activity. At least of the the synthesized compounds irreversibly inhibits the HIV-1 protease.

  8. Therapeutic potential of peptide deformylase inhibitors.

    PubMed

    Chen, D; Yuan, Z

    2005-09-01

    Peptide deformylase (PDF) is an attractive target for antibacterial drug discovery. Progress in the biological characterisation of the enzyme, coupled with newly obtained mechanistic and structural insight, enabled the pharmaceutical industry to discover potent PDF inhibitors that can be considered as clinical development candidates for this new class of antibacterial agents. The in vitro and in vivo data for several lead PDF inhibitors suggest that the current PDF inhibitors are most suitable for the treatment of respiratory tract infections and they are not cross-resistant to the current clinically used antibiotics. Two PDF inhibitors, BB-83698 and VIC-104959, have progressed to Phase I clinical trials by intravenous and oral administration, respectively. Both of these compounds show promising in vitro and in vivo efficacy and an excellent safety profile. The pharmacokinetics in humans for both of the compounds suggest the possibility of a twice-daily dosing regimen for clinical use. Thus far, all of the data suggest a promising future for this new class of antibacterial agents.

  9. Cellulose biosynthesis inhibitors - a multifunctional toolbox.

    PubMed

    Tateno, Mizuki; Brabham, Chad; DeBolt, Seth

    2016-01-01

    In the current review, we examine the growing number of existing Cellulose Biosynthesis Inhibitors (CBIs) and based on those that have been studied with live cell imaging we group their mechanism of action. Attention is paid to the use of CBIs as tools to ask fundamental questions about cellulose biosynthesis.

  10. Novel proteinase inhibitor promotes resistance to insects

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A novel Beta vulgaris serine proteinase inhibitor gene (BvSTI) and its protein are identified in response to insect feeding on B. vulgaris seedlings. BvSTI is cloned into an expression vector with constitutive promoter and transformed into Nicotiana benthamiana plants to assess BvSTI’s ability to ...

  11. Resistant mechanisms to BRAF inhibitors in melanoma

    PubMed Central

    Layos, Laura; Bugés, Cristina; de los Llanos Gil, María; Vila, Laia; Martínez-Balibrea, Eva; Martínez-Cardús, Anna

    2016-01-01

    Patients with advanced melanoma have traditionally had very poor prognosis. However, since 2011 better understanding of the biology and epidemiology of this disease has revolutionized its treatment, with newer therapies becoming available. These newer therapies can be classified into immunotherapy and targeted therapy. The immunotherapy arsenal includes inhibitors of CTLA4, PD-1 and PDL-1, while targeted therapy focuses on BRAF and MEK. BRAF inhibitors (vemurafenib, dabrafenib) have shown benefit in terms of overall survival (OS) compared to chemotherapy, and their combination with MEK inhibitors has recently been shown to improve progression-free survival (PFS), compared with monotherapy with BRAF inhibitors. However, almost 20% of patients initially do not respond, due to intrinsic resistance to therapy and, of those who do, most eventually develop mechanisms of acquired resistance, including reactivation of the MAP kinase pathway, persistent activation of receptor tyrosine kinase (RTKS) receptor, activation of phosphatidyinositol-3OH kinase, overexpression of epidermal growth factor receptor (EGFR), and interactions with the tumor microenvironment. Herein we comment in detail on mechanisms of resistance to targeted therapy and discuss the strategies to overcome them. PMID:27429963

  12. FAAH inhibitors in the limelight, but regrettably

    PubMed Central

    Mallet, Christophe; Dubray, Claude; Dualé, Christian

    2016-01-01

    Abstract. This short review focuses on the recent drug development of FAAH inhibitors, as recent serious adverse events have been reported in a phase I study with a compound of this class. The authors overview the potential interest in targeting FAAH inhibition, the current programs, and the available information on the recent dramatic events. PMID:27191771

  13. Cellulose biosynthesis inhibitors - a multifunctional toolbox.

    PubMed

    Tateno, Mizuki; Brabham, Chad; DeBolt, Seth

    2016-01-01

    In the current review, we examine the growing number of existing Cellulose Biosynthesis Inhibitors (CBIs) and based on those that have been studied with live cell imaging we group their mechanism of action. Attention is paid to the use of CBIs as tools to ask fundamental questions about cellulose biosynthesis. PMID:26590309

  14. Novel Cytoprotective Inhibitors for Apoptotic Endonuclease G

    PubMed Central

    Jang, Dae Song; Penthala, Narsimha R.; Apostolov, Eugene O.; Wang, Xiaoying; Crooks, Peter A.

    2015-01-01

    Apoptotic endonuclease G (EndoG) is responsible for DNA fragmentation both during and after cell death. Previous studies demonstrated that genetic inactivation of EndoG is cytoprotective against various pro-apoptotic stimuli; however, specific inhibitors for EndoG are not available. In this study, we have developed a high-throughput screening assay for EndoG and have used it to screen a chemical library. The screening resulted in the identification of two potent EndoG inhibitors, PNR-3-80 and PNR-3-82, which are thiobarbiturate analogs. As determined by their IC50s, the inhibitors are more potent than ZnCl2 or EDTA. They inhibit EndoG at one or two orders of magnitude greater than another apoptotic endonuclease, DNase I, and do not inhibit the other five tested cell death-related enzymes: DNase II, RNase A, proteinase, lactate dehydrogenase, and superoxide dismutase 1. Exposure of natural EndoG-expressing 22Rv1 or EndoG-overexpressing PC3 cells rendered them significantly resistant to Cisplatin and Docetaxel, respectively. These novel EndoG inhibitors have the potential to be utilized for amelioration of cell injuries in which participation of EndoG is essential. PMID:25401220

  15. Tetra- versus Pentavalent Inhibitors of Cholera Toxin**

    PubMed Central

    Fu, Ou; Pukin, Aliaksei V; van Ufford, H C Quarles; Branson, Thomas R; Thies-Weesie, Dominique M E; Turnbull, W Bruce; Visser, Gerben M; Pieters, Roland J

    2015-01-01

    The five B-subunits (CTB5) of the Vibrio cholerae (cholera) toxin can bind to the intestinal cell surface so the entire AB5 toxin can enter the cell. Simultaneous binding can occur on more than one of the monosialotetrahexosylganglioside (GM1) units present on the cell surface. Such simultaneous binding arising from the toxins multivalency is believed to enhance its affinity. Thus, blocking the initial attachment of the toxin to the cell surface using inhibitors with GM1 subunits has the potential to stop the disease. Previously we showed that tetravalent GM1 molecules were sub-nanomolar inhibitors of CTB5. In this study, we synthesized a pentavalent version and compared the binding and potency of penta- and tetravalent cholera toxin inhibitors, based on the same scaffold, for the first time. The pentavalent geometry did not yield major benefits over the tetravalent species, but it was still a strong inhibitor, and no major steric clashes occurred when binding the toxin. Thus, systems which can adopt more geometries, such as those described here, can be equally potent, and this may possibly be due to their ability to form higher-order structures or simply due to more statistical options for binding. PMID:26478842

  16. Haemophilia pseudotumours in patients with inhibitors.

    PubMed

    Caviglia, H; Candela, M; Landro, M E; Douglas Price, A L; Neme, D; Galatro, G A

    2015-09-01

    Development of inhibitors against factor VIII (FVIII) or FIX is the most serious complication of replacement therapy in patients with haemophilia. Haemophilic pseudotumours in a patient with inhibitors can lead to devastating consequences. The aim of this study is to show our experience in the treatment of 10 pseudotumours in 7 patients with inhibitors who were treated by the same multidisciplinary team in the period between January 2000 and March 2013. Seven severe haemophilia A patients were treated at the Haemophilia Foundation in Buenos Aires, Argentina, for 10 pseudotumours. Eight were bone pseudotumours and two soft tissue. All patients underwent imaging studies at baseline to assess the size and content of the lesion. The patients received Buenos Aires protocol as conservative treatment of their pseudotumours for 6 weeks, after which they were evaluated. Only one patient responded to conservative treatment. Surgery was performed on the others six patients, since their pseudotumours did not shrink to less than half their original size. Any bleeding in the musculoskeletal system must be treated promptly in order to prevent pseudotumours. When pseudotumours do appear in inhibitor patients, they can be surgically removed when patients received proper haemostatic coverage, improving the quality of life of these patients.

  17. Synthesis and Assays of Inhibitors of Methyltransferases.

    PubMed

    Cai, X-C; Kapilashrami, K; Luo, M

    2016-01-01

    Epigenetic regulation requires site-specific modification of the genome and is involved in multiple physiological processes and disease etiology. Methyltransferases, which catalyze the transfer of a methyl group from S-adenosyl-l-methionine (SAM) to various substrates, are critical components of the epigenetic machinery. This group of enzymes can methylate diverse substrates including DNA, RNA, proteins, and small-molecule metabolites. Their dysregulation has also been implicated in multiple disease states such as cancer, neurological, and cardiovascular disorders. Developing potent and selective small-molecule inhibitors of methyltransferases is valuable not only for therapeutic intervention but also for investigating the roles of these enzymes in disease progression. In this chapter, we will discuss the strategies of designing and synthesizing methyltransferases inhibitors based on the SAM scaffold. Following the section of inhibitor design, we will briefly review representative assays that are available to evaluate the potency of these inhibitors along with a detailed description of the most commonly used radiometric assay. PMID:27423865

  18. [Cyclooxygenase 2 inhibitors and colorectal cancer].

    PubMed

    Bernardeau-Mozer, Marianne; Chaussade, Stanislas

    2004-05-01

    Cyclooxygenase-2 (Cox2) is an inductible isoenzyme of cyclooxygenase undetectable in normal colonic mucosa and overexpressed in 80% colonic tumor. Several works in vitro and in vivo showed that Cox2 plays a key role in the multistep process of colorectal tumorigenesis such apoptosis inhibition of cellular proliferation and angiogenesis induction. So that Cox2 represent a potential molecular target in colorectal management and specific Cox2 inhibitors may be useful as chemopreventive as well as therapeutic agent in humans. In animals study Cox2 inhibitors was shown to be effective and in humans Cox2 inhibitors are approved by the Food and Drug Administration as an adjunct to endoscopic surveillance and surgery in patients with Familial Adenomatous Polyposis (FAP). The purpose of this article is to review the relationship between Cox2/Cox2 inhibitors and differents signaling pathways of colorectal carcinogenesis and to precise their possible molecular mechanisms of action. This work although review clinicals data of their efficacy as chemopreventive agent as well as therapeutic in the differents group at risk for colorectal cancer. PMID:15239336

  19. Novel in vitro inhibitory functions of potato tuber proteinaceous inhibitors.

    PubMed

    Fischer, Matthias; Kuckenberg, Markus; Kastilan, Robin; Muth, Jost; Gebhardt, Christiane

    2015-02-01

    Plant protease inhibitors are a structurally highly diverse and ubiquitous class of small proteins, which play various roles in plant development and defense against pests and pathogens. Particular isoforms inhibit in vitro proteases and other enzymes that are not their natural substrates, for example proteases that have roles in human diseases. Mature potato tubers are a rich source of several protease inhibitor families. Different cultivars have different inhibitor profiles. With the objective to explore the functional diversity of the natural diversity of potato protease inhibitors, we randomly selected and sequenced 9,600 cDNA clones originated from mature tubers of ten potato cultivars. Among these, 120 unique inhibitor cDNA clones were identified by homology searches. Eighty-eight inhibitors represented novel sequence variants of known plant protease inhibitor families. Most frequent were Kunitz-type inhibitors (KTI), potato protease inhibitors I and II (PIN), pectin methylesterase inhibitors, metallocarboxypeptidase inhibitors and defensins. Twenty-three inhibitors were functionally characterized after heterologous expression in the yeast Pichia pastoris. The purified recombinant proteins were tested for inhibitory activity on trypsin, eleven pharmacological relevant proteases and the non-proteolytic enzyme 5-lipoxygenase. Members of the KTI and PIN families inhibited pig pancreas elastase, β-Secretase, Cathepsin K, HIV-1 protease and potato 5-lipoxygenase. Our results demonstrate in vitro inhibitory diversity of small potato tuber proteins commonly known as protease inhibitors, which might have biotechnological or medical applications.

  20. Novel in vitro inhibitory functions of potato tuber proteinaceous inhibitors.

    PubMed

    Fischer, Matthias; Kuckenberg, Markus; Kastilan, Robin; Muth, Jost; Gebhardt, Christiane

    2015-02-01

    Plant protease inhibitors are a structurally highly diverse and ubiquitous class of small proteins, which play various roles in plant development and defense against pests and pathogens. Particular isoforms inhibit in vitro proteases and other enzymes that are not their natural substrates, for example proteases that have roles in human diseases. Mature potato tubers are a rich source of several protease inhibitor families. Different cultivars have different inhibitor profiles. With the objective to explore the functional diversity of the natural diversity of potato protease inhibitors, we randomly selected and sequenced 9,600 cDNA clones originated from mature tubers of ten potato cultivars. Among these, 120 unique inhibitor cDNA clones were identified by homology searches. Eighty-eight inhibitors represented novel sequence variants of known plant protease inhibitor families. Most frequent were Kunitz-type inhibitors (KTI), potato protease inhibitors I and II (PIN), pectin methylesterase inhibitors, metallocarboxypeptidase inhibitors and defensins. Twenty-three inhibitors were functionally characterized after heterologous expression in the yeast Pichia pastoris. The purified recombinant proteins were tested for inhibitory activity on trypsin, eleven pharmacological relevant proteases and the non-proteolytic enzyme 5-lipoxygenase. Members of the KTI and PIN families inhibited pig pancreas elastase, β-Secretase, Cathepsin K, HIV-1 protease and potato 5-lipoxygenase. Our results demonstrate in vitro inhibitory diversity of small potato tuber proteins commonly known as protease inhibitors, which might have biotechnological or medical applications. PMID:25260821

  1. DNA Methyltransferases Inhibitors from Natural Sources.

    PubMed

    Zwergel, Clemens; Valente, Sergio; Mai, Antonello

    2016-01-01

    DNA methyltransferases (DNMTs) catalyze the methylation at cytosine-C5 mainly in a CpG dinucleotide context. Although DNA methylation is essential for fundamental processes like embryonic development or differentiation, aberrant expression and/or activities of DNMTs are involved in several pathologies, from neurodegeneration to cancer. DNMTs inhibition can arrest tumor growth, cells invasiveness and induce differentiation, whereas their increased expression is shown in numerous cancer types. Moreover, hypermethylated promoters of tumor suppressor genes lead to their silencing. Hence, the use of specific inhibitors of DNMT might reactivate those genes and stop or even reverse the aberrant cell processes. To date, the only approved DNMTs inhibitors for therapy belong to the nucleoside-based family of drugs, but they display relevant side effects as well as high chemical instability. Thus, there is a keen interest actually exists to develop novel, potent and safe inhibitors possessing a nonnucleoside structure. Increasing literature evidence is highlighting that natural sources could help the researchers to achieve this goal. Indeed, several polyphenols, flavonoids, antraquinones, and others are described able to inhibit DNMTs activity and/or expression, thus decreasing the methylation/silencing of different genes involved in tumorigenesis. These events can lead to re-expression of such genes and to cell death in diverse cancer cell lines. Epigallocatechin-3-gallate (1) and laccaic acid A (11) resulted the most effective DNMT1 inhibitors with submicromolar IC50 values, acting as competitive inhibitors. Compound 1 and 11 both displayed gene demethylation and re-activation in several cancers. However, all of the natural compounds described in this review showed important results, from gene reactivation to cell growth inhibition. Moreover, some of them displayed interesting activity even in rodent cancer models and very recently entered clinical trials. PMID:26303417

  2. Cost of care of haemophilia with inhibitors.

    PubMed

    Di Minno, M N D; Di Minno, G; Di Capua, M; Cerbone, A M; Coppola, A

    2010-01-01

    In Western countries, the treatment of patients with inhibitors is presently the most challenging and serious issue in haemophilia management, direct costs of clotting factor concentrates accounting for >98% of the highest economic burden absorbed for the healthcare of patients in this setting. Being designed to address questions of resource allocation and effectiveness, decision models are the golden standard to reliably assess the overall economic implications of haemophilia with inhibitors in terms of mortality, bleeding-related morbidity, and severity of arthropathy. However, presently, most data analyses stem from retrospective short-term evaluations, that only allow for the analysis of direct health costs. In the setting of chronic diseases, the cost-utility analysis, that takes into account the beneficial effects of a given treatment/healthcare intervention in terms of health-related quality of life, is likely to be the most appropriate approach. To calculate net benefits, the quality adjusted life year, that significantly reflects such health gain, has to be compared with specific economic impacts. Differences in data sources, in medical practice and/or in healthcare systems and costs, imply that most current pharmacoeconomic analyses are confined to a narrow healthcare payer perspective. Long-term/lifetime prospective or observational studies, devoted to a careful definition of when to start a treatment; of regimens (dose and type of product) to employ, and of inhibitor population (children/adults, low-responding/high responding inhibitors) to study, are thus urgently needed to allow for newer insights, based on reliable data sources into resource allocation, effectiveness and cost-utility analysis in the treatment of haemophiliacs with inhibitors.

  3. Peptidyl cyclopropenones: Reversible inhibitors, irreversible inhibitors, or substrates of cysteine proteases?

    PubMed Central

    Cohen, Meital; Bretler, Uriel; Albeck, Amnon

    2013-01-01

    Peptidyl cyclopropenones were previously introduced as selective cysteine protease reversible inhibitors. In the present study we synthesized one such peptidyl cyclopropenone and investigated its interaction with papain, a prototype cysteine protease. A set of kinetics, biochemical, HPLC, MS, and 13C-NMR experiments revealed that the peptidyl cyclopropenone was an irreversible inhibitor of the enzyme, alkylating the catalytic cysteine. In parallel, this cyclopropenone also behaved as an alternative substrate of the enzyme, providing a product that was tentatively suggested to be either a spiroepoxy cyclopropanone or a gamma-lactone. Thus, a single family of compounds exhibits an unusual variety of activities, being reversible inhibitors, irreversible inhibitors and alternative substrates towards enzymes of the same family. PMID:23553793

  4. Therapeutic substitution post-patent expiry: the cases of ACE inhibitors and proton pump inhibitors.

    PubMed

    Vandoros, Sotiris

    2014-05-01

    This paper examines whether there is a switch in total (originator and generic) consumption after generic entry from molecules that face generic competition towards other molecules of the same class, which are still in-patent. Data from six European countries for the time period 1991 to 2006 are used to study the cases of angiotensin-converting enzyme inhibitors and proton pump inhibitors. Empirical evidence shows that patent expiry of captopril and enalapril led to a switch in total (off-patent originator and generic) consumption towards other in-patent angiotensin-converting enzyme inhibitors, whereas patent expiry of omeprazole led to a switch in consumption towards other proton pump inhibitors. This phenomenon makes generic policies ineffective and results in an increase in pharmaceutical expenditure due to the absence of generic alternatives in the market of in-patent molecules.

  5. Neuroprotective Tri- and Tetracyclic BChE Inhibitors Releasing Reversible Inhibitors upon Carbamate Transfer

    PubMed Central

    2012-01-01

    Tri- and tetracyclic nitrogen-bridgehead compounds were designed and synthesized to yield micromolar cholinesterase (ChE) inhibitors. Structure–activity relationships identified potent compounds with butyrylcholinesterase selectivity. These compounds were selected as starting points for the design and synthesis of carbamate-based (pseudo)irreversible inhibitors. Compounds with superior inhibitory activity and selectivity were obtained and kinetically characterized also with regard to the velocity of enzyme carbamoylation. Structural elements were identified and introduced that additionally showed neuroprotective properties on a hippocampal neuronal cell line (HT-22) after glutamate-induced intracellular reactive oxygen species generation. We have identified potent and selective pseudoirreversible butyrylcholinesterase inhibitors that release reversible inhibitors with neuroprotective properties after carbamate transfer to the active site of cholinesterases. PMID:24900407

  6. Dermatologic adverse events to chemotherapeutic agents, Part 2: BRAF inhibitors, MEK inhibitors, and ipilimumab.

    PubMed

    Choi, Jennifer Nam

    2014-03-01

    The advent of novel targeted chemotherapeutic agents and immunotherapies has dramatically changed the arena of cancer treatment in recent years. BRAF inhibitors, MEK inhibitors, and ipilimumab are among the newer chemotherapy drugs that are being used at an increasing rate. Dermatologic adverse events to these medications are common, and it is important for dermatologists and oncologists alike to learn to recognize and treat such side effects in order to maintain both patients' quality of life and their anticancer treatment. This review describes the cutaneous side effects seen with BRAF inhibitors (eg, maculopapular eruption, photosensitivity, squamoproliferative growths, melanocytic proliferations), MEK inhibitors (eg, papulopustular eruption), and ipilimumab (eg, maculopapular eruption, vitiligo), with a mention of vismodegib and anti-PD-1 agents.

  7. Development of covalent inhibitors that can overcome resistance to first-generation FGFR kinase inhibitors

    PubMed Central

    Tan, Li; Wang, Jun; Tanizaki, Junko; Huang, Zhifeng; Aref, Amir R.; Rusan, Maria; Zhu, Su-Jie; Zhang, Yiyun; Ercan, Dalia; Liao, Rachel G.; Capelletti, Marzia; Zhou, Wenjun; Hur, Wooyoung; Kim, NamDoo; Sim, Taebo; Gaudet, Suzanne; Barbie, David A.; Yeh, Jing-Ruey Joanna; Yun, Cai-Hong; Hammerman, Peter S.; Mohammadi, Moosa; Jänne, Pasi A.; Gray, Nathanael S.

    2014-01-01

    The human FGF receptors (FGFRs) play critical roles in various human cancers, and several FGFR inhibitors are currently under clinical investigation. Resistance usually results from selection for mutant kinases that are impervious to the action of the drug or from up-regulation of compensatory signaling pathways. Preclinical studies have demonstrated that resistance to FGFR inhibitors can be acquired through mutations in the FGFR gatekeeper residue, as clinically observed for FGFR4 in embryonal rhabdomyosarcoma and neuroendocrine breast carcinomas. Here we report on the use of a structure-based drug design to develop two selective, next-generation covalent FGFR inhibitors, the FGFR irreversible inhibitors 2 (FIIN-2) and 3 (FIIN-3). To our knowledge, FIIN-2 and FIIN-3 are the first inhibitors that can potently inhibit the proliferation of cells dependent upon the gatekeeper mutants of FGFR1 or FGFR2, which confer resistance to first-generation clinical FGFR inhibitors such as NVP-BGJ398 and AZD4547. Because of the conformational flexibility of the reactive acrylamide substituent, FIIN-3 has the unprecedented ability to inhibit both the EGF receptor (EGFR) and FGFR covalently by targeting two distinct cysteine residues. We report the cocrystal structure of FGFR4 with FIIN-2, which unexpectedly exhibits a “DFG-out” covalent binding mode. The structural basis for dual FGFR and EGFR targeting by FIIN3 also is illustrated by crystal structures of FIIN-3 bound with FGFR4 V550L and EGFR L858R. These results have important implications for the design of covalent FGFR inhibitors that can overcome clinical resistance and provide the first example, to our knowledge, of a kinase inhibitor that covalently targets cysteines located in different positions within the ATP-binding pocket. PMID:25349422

  8. Inhibitor analysis for a solar heating and cooling system

    NASA Technical Reports Server (NTRS)

    Tabony, J. H.

    1977-01-01

    A study of potential corrosion inhibitors for the NASA solar heating and cooling system which uses aluminum solar panels is provided. Research consisted of testing using a dynamic corrosion system, along with an economic analysis of proposed corrosion inhibitors. Very good progress was made in finding a suitable inhibitor for the system.

  9. Controlled-release scale inhibitor for use in fracturing treatments

    SciTech Connect

    Powell, R.J.; Gdanski, R.D.; McCabe, M.A.; Buster, D.C.

    1995-11-01

    This paper describes results of laboratory and field testing of a solid, controlled-release scale inhibitor for use in fracturing treatments. Laboratory testing with a continuous flow apparatus has yielded inhibitor release rates under dynamic conditions. The inhibitor was tested to determine the minimum inhibitor concentration required to inhibit the formation of CaCO{sub 3}, CaSO{sub 4}, and BaSO{sub 4} scales in a brine. A model to predict the long-term release rate of the inhibitor was developed from data collected on the continuous flow apparatus. Data from treated wells will be compared with predictions of the model. Inhibitor release-rate testing in a continuous-flow apparatus shows that a solid, calcium-magnesium polyphosphate inhibitor has a sustained release profile. Release-rate testing shows that the inhibitor can be used up to 175 F. The inhibitor is compatible with both borate and zirconium crosslinked fracturing fluids and foamed fluids. The effective lifetime of the scale treatment can be predicted based on a model developed from laboratory data. The input variables required for the prediction include: temperature, water production, amount of inhibitor, minimum effective concentration of inhibitor for the specific brine. The model can be used to aid in the design of the scale inhibitor treatment.

  10. Deletion Mutations Keep Kinase Inhibitors in the Loop

    PubMed Central

    Freed, Daniel M.; Park, Jin H.; Radhakrishnan, Ravi; Lemmon, Mark A.

    2016-01-01

    Effective clinical application of conformationally selective kinase inhibitors requires tailoring drug choice to the tumor's activating mutation(s). In this issue of Cancer Cell, Foster et al. (2016) describe how activating deletions in BRAF, EGFR, and HER2 cause primary resistance to common inhibitors, suggesting strategies for improved inhibitor selection. PMID:27070691

  11. Serine protease inhibitors of parasitic helminths.

    PubMed

    Molehin, Adebayo J; Gobert, Geoffrey N; McManus, Donald P

    2012-05-01

    Serine protease inhibitors (serpins) are a superfamily of structurally conserved proteins that inhibit serine proteases and play key physiological roles in numerous biological systems such as blood coagulation, complement activation and inflammation. A number of serpins have now been identified in parasitic helminths with putative involvement in immune regulation and in parasite survival through interference with the host immune response. This review describes the serpins and smapins (small serine protease inhibitors) that have been identified in Ascaris spp., Brugia malayi, Ancylostoma caninum Onchocerca volvulus, Haemonchus contortus, Trichinella spiralis, Trichostrongylus vitrinus, Anisakis simplex, Trichuris suis, Schistosoma spp., Clonorchis sinensis, Paragonimus westermani and Echinococcus spp. and discusses their possible biological functions, including roles in host-parasite interplay and their evolutionary relationships. PMID:22310379

  12. Penicillin inhibitors of purple acid phosphatase.

    PubMed

    Faridoon; Hussein, Waleed M; Ul Islam, Nazar; Guddat, Luke W; Schenk, Gerhard; McGeary, Ross P

    2012-04-01

    Purple acid phosphatases (PAPs) are binuclear metallohydrolases that have a multitude of biological functions and are found in fungi, bacteria, plants and animals. In mammals, PAP activity is linked with bone resorption and over-expression can lead to bone disorders such as osteoporosis. PAP is therefore an attractive target for the development of drugs to treat this disease. A series of penicillin conjugates, in which 6-aminopenicillanic acid was acylated with aromatic acid chlorides, has been prepared and assayed against pig PAP. The binding mode of most of these conjugates is purely competitive, and some members of this class have potencies comparable to the best PAP inhibitors yet reported. The structurally related penicillin G was shown to be neither an inhibitor nor a substrate for pig PAP. Molecular modelling has been used to examine the binding modes of these compounds in the active site of the enzyme and to rationalise their activities.

  13. mTOR inhibitors in cancer therapy

    PubMed Central

    Xie, Jianling; Wang, Xuemin; Proud, Christopher G.

    2016-01-01

    The mammalian target of rapamycin, mTOR, plays key roles in cell growth and proliferation, acting at the catalytic subunit of two protein kinase complexes: mTOR complexes 1 and 2 (mTORC1/2). mTORC1 signaling is switched on by several oncogenic signaling pathways and is accordingly hyperactive in the majority of cancers. Inhibiting mTORC1 signaling has therefore attracted great attention as an anti-cancer therapy. However, progress in using inhibitors of mTOR signaling as therapeutic agents in oncology has been limited by a number of factors, including the fact that the classic mTOR inhibitor, rapamycin, inhibits only some of the effects of mTOR; the existence of several feedback loops; and the crucial importance of mTOR in normal physiology.

  14. Replacing sulfa drugs with novel DHPS inhibitors.

    PubMed

    Hammoudeh, Dalia I; Zhao, Ying; White, Stephen W; Lee, Richard E

    2013-07-01

    More research effort needs to be invested in antimicrobial drug development to address the increasing threat of multidrug-resistant organisms. The enzyme DHPS has been a validated drug target for over 70 years as the target for the highly successful sulfa drugs. The use of sulfa drugs has been compromised by the widespread presence of resistant organisms and the adverse side effects associated with their use. Despite the large amount of structural information available for DHPS, few recent publications address the possibility of using this knowledge for novel drug design. This article reviews the relevant papers and patents that report promising new small-molecule inhibitors of DHPS, and discuss these data in light of new insights into the DHPS catalytic mechanism and recently determined crystal structures of DHPS bound to potent small-molecule inhibitors. This new functional understanding confirms that DHPS deserves further consideration as an antimicrobial drug target. PMID:23859210

  15. New tyrosinase inhibitors, (+)-catechin-aldehyde polycondensates.

    PubMed

    Kim, Young-Jin; Chung, Joo Eun; Kurisawa, Motoichi; Uyama, Hiroshi; Kobayashi, Shiro

    2004-01-01

    In this study, new tyrosinase inhibitors, (+)-catechin-aldehyde polycondensates, have been developed. Tyrosinase is a copper-containing enzyme that catalyzes the hydroxylation of a monophenol (monophenolase activity) and the oxidation of an o-diphenol (diphenolase activity). In the measurement of tyrosinase inhibition activity, (+)-catechin acted as substrate and cofactor of tyrosinase. On the other hand, the polycondensates inhibited the tyrosine hydroxylation and L-DOPA oxidation by chelation to the active site of tyrosinase. The UV-visible spectrum of a mixture of tyrosinase and the polycondensate exhibited a characteristic shoulder peak ascribed to the chelation of the polycondensate to the active site of tyrosinase. Furthermore, circular dichroism measurement showed a small red shift of the band due to the interaction between tyrosinase and the polycondensate. These data support that the polycondensate acts as an inhibitor of tyrosinase. PMID:15003008

  16. mTOR inhibitors in cancer therapy

    PubMed Central

    Xie, Jianling; Wang, Xuemin; Proud, Christopher G.

    2016-01-01

    The mammalian target of rapamycin, mTOR, plays key roles in cell growth and proliferation, acting at the catalytic subunit of two protein kinase complexes: mTOR complexes 1 and 2 (mTORC1/2). mTORC1 signaling is switched on by several oncogenic signaling pathways and is accordingly hyperactive in the majority of cancers. Inhibiting mTORC1 signaling has therefore attracted great attention as an anti-cancer therapy. However, progress in using inhibitors of mTOR signaling as therapeutic agents in oncology has been limited by a number of factors, including the fact that the classic mTOR inhibitor, rapamycin, inhibits only some of the effects of mTOR; the existence of several feedback loops; and the crucial importance of mTOR in normal physiology. PMID:27635236

  17. Automated colorimetric screen for apyrase inhibitors.

    PubMed

    Windsor, J B; Thomas, C; Hurley, L; Roux, S J; Lloyd, A M

    2002-11-01

    Apyrases are enzymes that efficiently hydrolyze ATP and ADP and may operate both inside and outside the cell. Although apyrases are important to a variety of cellular mechanisms and uses in industry, there are no available apyrase-specific inhibitors. Colorimetric assays based on the Fiske-Subbarow method for measuring inorganic phosphate are able to detect the release of inorganic phosphate from ATP and other nucleotides. We found that this type of assay could be automated and used to screen for apyrase-inhibiting compounds by assaying for a reduction in released phosphate in the presence of potential inhibitors. The automation of this assay allowed for the successful screening of a commercially available compound library. Several low molecular weight compounds were identified that, when used at micromolar concentrations, effectively inhibited apyrase activity.

  18. Replacing sulfa drugs with novel DHPS inhibitors.

    PubMed

    Hammoudeh, Dalia I; Zhao, Ying; White, Stephen W; Lee, Richard E

    2013-07-01

    More research effort needs to be invested in antimicrobial drug development to address the increasing threat of multidrug-resistant organisms. The enzyme DHPS has been a validated drug target for over 70 years as the target for the highly successful sulfa drugs. The use of sulfa drugs has been compromised by the widespread presence of resistant organisms and the adverse side effects associated with their use. Despite the large amount of structural information available for DHPS, few recent publications address the possibility of using this knowledge for novel drug design. This article reviews the relevant papers and patents that report promising new small-molecule inhibitors of DHPS, and discuss these data in light of new insights into the DHPS catalytic mechanism and recently determined crystal structures of DHPS bound to potent small-molecule inhibitors. This new functional understanding confirms that DHPS deserves further consideration as an antimicrobial drug target.

  19. SGLT2 Inhibitors: Benefit/Risk Balance.

    PubMed

    Scheen, André J

    2016-10-01

    Inhibitors of sodium-glucose cotransporters type 2 (SGLT2) reduce hyperglycemia by increasing urinary glucose excretion. They have been evaluated in patients with type 2 diabetes treated with diet/exercise, metformin, dual oral therapy or insulin. Three agents are available in Europe and the USA (canagliflozin, dapagliflozin, empagliflozin) and others are commercialized in Japan or in clinical development. SGLT2 inhibitors reduce glycated hemoglobin, with a minimal risk of hypoglycemia. They exert favorable effects beyond glucose control with consistent body weight, blood pressure, and serum uric acid reductions. Empagliflozin showed remarkable reductions in cardiovascular/all-cause mortality and in hospitalization for heart failure in patients with previous cardiovascular disease. Positive renal outcomes were also shown with empagliflozin. Mostly reported adverse events are genital mycotic infections, while urinary tract infections and events linked to volume depletion are rather rare. Concern about a risk of ketoacidosis and bone fractures has been recently raised, which deserves caution and further evaluation.

  20. Proteasome Inhibitors Block Development of Plasmodium spp.

    PubMed Central

    Gantt, Soren M.; Myung, Joon Mo; Briones, Marcelo R. S.; Li, Wei Dong; Corey, E. J.; Omura, Satoshi; Nussenzweig, Victor; Sinnis, Photini

    1998-01-01

    Proteasomes degrade most of the proteins inside eukaryotic cells, including transcription factors and regulators of cell cycle progression. Here we show that nanomolar concentrations of lactacystin, a specific irreversible inhibitor of the 20S proteasome, inhibit development of the exoerythrocytic and erythrocytic stages of the malaria parasite. Although lactacystin-treated Plasmodium berghei sporozoites are still invasive, their development into exoerythrocytic forms (EEF) is inhibited in vitro and in vivo. Erythrocytic schizogony of P. falciparum in vitro is also profoundly inhibited when drug treatment of the synchronized parasites is prior, but not subsequent, to the initiation of DNA synthesis, suggesting that the inhibitory effect of lactacystin is cell cycle specific. Lactacystin reduces P. berghei parasitemia in rats, but the therapeutic index is very low. Along with other studies showing that lactacystin inhibits stage-specific transformation in Trypanosoma and Entamoeba spp., these findings highlight the potential of proteasome inhibitors as drugs for the treatment of diseases caused by protozoan parasites. PMID:9756786

  1. Prospective therapeutic applications of p53 inhibitors

    SciTech Connect

    Gudkov, Andrei V. . E-mail: gudkov@ccf.org; Komarova, Elena A.

    2005-06-10

    p53, in addition to being a key cancer preventive factor, is also a determinant of cancer treatment side effects causing excessive apoptotic death in several normal tissues during cancer therapy. p53 inhibitory strategy has been suggested to protect normal tissues from chemo- and radiotherapy, and to treat other pathologies associated with stress-mediated activation of p53. This strategy was validated by isolation and testing of small molecule p53 inhibitor pifithrin-{alpha} that demonstrated broad tissue protecting capacity. However, in some normal tissues and tumors p53 plays protective role by inducing growth arrest and preventing cells from premature entrance into mitosis and death from mitotic catastrophe. Inhibition of this function of p53 can sensitize tumor cells to chemo- and radiotherapy, thus opening new potential application of p53 inhibitors and justifying the need in pharmacological agents targeting specifically either pro-apoptotic or growth arrest functions of p53.

  2. Protein synthesis inhibitor from potato tuber

    SciTech Connect

    Romaen, R. )

    1989-04-01

    A protein fraction capable of inhibit in vitro protein synthesis was found in potato tubers in fresh and wounded tissue. Inhibitor activity from fresh tissue decays with wounding. Inhibition activity was detected absorbed to ribsomal fraction and cytosol of potato tuber tissue by a partially reconstituted in vitro system from potato tuber and wheat germ. Adsorbed ribosomal fraction was more suitable of purification. This fraction was washed from ribosomes with 0.3M KCl, concentrated with ammonium sulfate precipitation and purified through sephadex G100 and sephadex G-75 columns chromatography. After 61 fold purification adsorbed protein fraction can inhibit germination of maize, wheat and sesame seeds, as well as {sup 3}H-leucine incorporation into protein by imbibed maize embryos. Inhibition activity was lost by temperature, alkali and protease-K hydrolysis. Preliminar analysis could not show presence of reductor sugars. Physiological role of this inhibitor in relation to rest and active tissue remains to be studied.

  3. mTOR inhibitors in cancer therapy.

    PubMed

    Xie, Jianling; Wang, Xuemin; Proud, Christopher G

    2016-01-01

    The mammalian target of rapamycin, mTOR, plays key roles in cell growth and proliferation, acting at the catalytic subunit of two protein kinase complexes: mTOR complexes 1 and 2 (mTORC1/2). mTORC1 signaling is switched on by several oncogenic signaling pathways and is accordingly hyperactive in the majority of cancers. Inhibiting mTORC1 signaling has therefore attracted great attention as an anti-cancer therapy. However, progress in using inhibitors of mTOR signaling as therapeutic agents in oncology has been limited by a number of factors, including the fact that the classic mTOR inhibitor, rapamycin, inhibits only some of the effects of mTOR; the existence of several feedback loops; and the crucial importance of mTOR in normal physiology. PMID:27635236

  4. Replacing sulfa drugs with novel DHPS inhibitors

    PubMed Central

    Hammoudeh, Dalia I; Zhao, Ying; White, Stephen W; Lee, Richard E

    2013-01-01

    More research effort needs to be invested in antimicrobial drug development to address the increasing threat of multidrug-resistant organisms. The enzyme DHPS has been a validated drug target for over 70 years as the target for the highly successful sulfa drugs. The use of sulfa drugs has been compromised by the widespread presence of resistant organisms and the adverse side effects associated with their use. Despite the large amount of structural information available for DHPS, few recent publications address the possibility of using this knowledge for novel drug design. This article reviews the relevant papers and patents that report promising new small-molecule inhibitors of DHPS, and discuss these data in light of new insights into the DHPS catalytic mechanism and recently determined crystal structures of DHPS bound to potent small-molecule inhibitors. This new functional understanding confirms that DHPS deserves further consideration as an antimicrobial drug target. PMID:23859210

  5. Inhibitor prevents corrosion, scale in Chinese waterflood

    SciTech Connect

    Yong, W.; Jianhua, W. )

    1994-03-14

    An imidazoline derivative-based series inhibitor has prevented both corrosion and scale formation in produced-water treatment and water-injection equipment in China National Petroleum Co.'s (CNPC) Shengli oil field. Development of the inhibitor started in 1986, and after successful field trials the chemical is now being extensively applied. To increase oil recovery, water injection is widely used in China's onshore oil fields. Oil production in the Shengli oil field, for example, requires injection of about 4 bbl of water/1 bbl of oil produced. The large volumes of produced formation water contain many substances that can cause serious corrosion and scale. Also, the makeup water from other sources, subsurface or surface, complicates water handling. The paper discusses the following: corrosion and scale, oxygen, carbon dioxide, H[sub 2]S and sulfur reducing bacteria, temperature, inhibition, field tests, applications, and economics.

  6. A Bacterial Cell Shape-Determining Inhibitor.

    PubMed

    Liu, Yanjie; Frirdich, Emilisa; Taylor, Jennifer A; Chan, Anson C K; Blair, Kris M; Vermeulen, Jenny; Ha, Reuben; Murphy, Michael E P; Salama, Nina R; Gaynor, Erin C; Tanner, Martin E

    2016-04-15

    Helicobacter pylori and Campylobacter jejuni are human pathogens and causative agents of gastric ulcers/cancer and gastroenteritis, respectively. Recent studies have uncovered a series of proteases that are responsible for maintaining the helical shape of these organisms. The H. pylori metalloprotease Csd4 and its C. jejuni homologue Pgp1 cleave the amide bond between meso-diaminopimelate and iso-d-glutamic acid in truncated peptidoglycan side chains. Deletion of either csd4 or pgp1 results in bacteria with a straight rod phenotype, a reduced ability to move in viscous media, and reduced pathogenicity. In this work, a phosphinic acid-based pseudodipeptide inhibitor was designed to act as a tetrahedral intermediate analog against the Csd4 enzyme. The phosphinic acid was shown to inhibit the cleavage of the alternate substrate, Ac-l-Ala-iso-d-Glu-meso-Dap, with a Ki value of 1.5 μM. Structural analysis of the Csd4-inhibitor complex shows that the phosphinic acid displaces the zinc-bound water and chelates the metal in a bidentate fashion. The phosphinate oxygens also interact with the key acid/base residue, Glu222, and the oxyanion-stabilizing residue, Arg86. The results are consistent with the "promoted-water pathway" mechanism for carboxypeptidase A catalysis. Studies on cultured bacteria showed that the inhibitor causes significant cell straightening when incubated with H. pylori at millimolar concentrations. A diminished, yet observable, effect on the morphology of C. jejuni was also apparent. Cell straightening was more pronounced with an acapsular C. jejuni mutant strain compared to the wild type, suggesting that the capsule impaired inhibitor accessibility. These studies demonstrate that a highly polar compound is capable of crossing the outer membrane and altering cell shape, presumably by inhibiting cell shape determinant proteases. Peptidoglycan proteases acting as cell shape determinants represent novel targets for the development of antimicrobials

  7. Inhibitors of the AAA+ Chaperone p97

    PubMed Central

    Chapman, Eli; Maksim, Nick; de la Cruz, Fabian; La Clair, James J.

    2015-01-01

    It is remarkable that a pathway as ubiquitous as protein quality control can be targeted to treat cancer. Bortezomib, an inhibitor of the proteasome, was first approved by the US Food and Drug Administration (FDA) more than 10 years ago to treat refractory myeloma and later extended to lymphoma. Its use has increased the survival rate of myeloma patients by as much as three years. This success was followed with the recent accelerated approval of the natural product derived proteasome inhibitor carfilzomib (Kyprolis®), which is used to treat patients with bortezomib-resistant multiple myeloma. The success of these two drugs has validated protein quality control as a viable target to fight select cancers, but begs the question why are proteasome inhibitors limited to lymphoma and myeloma? More recently, these limitations have encouraged the search for additional targets within the protein quality control system that might offer heightened cancer cell specificity, enhanced clinical utility, a lower rate of resistance, reduced toxicity, and mitigated side effects. One promising target is p97, an ATPase associated with various cellular activities (AAA+) chaperone. p97 figures prominently in protein quality control as well as serving a variety of other cellular functions associated with cancer. More than a decade ago, it was determined that up-regulation of p97 in many forms of cancer correlates with a poor clinical outcome. Since these initial discoveries, a mechanistic explanation for this observation has been partially illuminated, but details are lacking. Understandably, given this clinical correlation, myriad roles within the cell, and its importance in protein quality control, p97 has emerged as a potential therapeutic target. This review provides an overview of efforts towards the discovery of small molecule inhibitors of p97, offering a synopsis of efforts that parallel the excellent reviews that currently exist on p97 structure, function, and physiology. PMID

  8. Corrosion protection with eco-friendly inhibitors

    NASA Astrophysics Data System (ADS)

    Shahid, Muhammad

    2011-12-01

    Corrosion occurs as a result of the interaction of a metal with its environment. The extent of corrosion depends on the type of metal, the existing conditions in the environment and the type of aggressive ions present in the medium. For example, CO3‑2 and NO‑3 produce an insoluble deposit on the surface of iron, resulting in the isolation of metal and consequent decrease of corrosion. On the other hand, halide ions are adsorbed selectively on the metal surface and prevent formation of the oxide phase on the metal surface, resulting in continuous corrosion. Iron, aluminum and their alloys are widely used, both domestically and industrially. Linear alkylbenzene and linear alkylbenzene sulfonate are commonly used as detergents. They have also been found together in waste water. It is claimed that these chemicals act as inhibitors for stainless steel and aluminum. Release of toxic gases as a result of corrosion in pipelines may lead in certain cases to air pollution and possible health hazards. Therefore, there are two ways to look at the relationship between corrosion and pollution: (i) corrosion of metals and alloys due to environmental pollution and (ii) environmental pollution as a result of corrosion protection. This paper encompasses the two scenarios and possible remedies for various cases, using 'green' inhibitors obtained either from plant extracts or from pharmaceutical compounds. In the present study, the effect of piperacillin sodium as a corrosion inhibitor for mild steel was investigated using a weight-loss method as well as a three-electrode dc electrochemical technique. It was found that the corrosion rate decreased as the concentration of the inhibitor increased up to 9×10‑4 M 93% efficiency was exhibited at this concentration.

  9. Monoamine oxidase inhibitors from Gentiana lutea.

    PubMed

    Haraguchi, Hiroyuki; Tanaka, Yasumasa; Kabbash, Amal; Fujioka, Toshihiro; Ishizu, Takashi; Yagi, Akira

    2004-08-01

    Three monoamine oxidase (MAO) inhibitors were isolated from Gentiana lutea. Their structures were elucidated to be 3-3''linked-(2'-hydroxy-4-O-isoprenylchalcone)-(2'''-hydroxy-4''-O-isoprenyldihydrochalcone) (1), 2-methoxy-3-(1,1'-dimethylallyl)-6a,10a-dihydrobenzo(1,2-c)chroman-6-one and 5-hydroxyflavanone. These compounds, and the hydrolysis product of 1, displayed competitive inhibitory properties against MAO-B which was more effective than MAO-A.

  10. Trial Watch: Proteasomal inhibitors for anticancer therapy

    PubMed Central

    Obrist, Florine; Manic, Gwenola; Kroemer, Guido; Vitale, Ilio; Galluzzi, Lorenzo

    2015-01-01

    The so-called “ubiquitin-proteasome system” (UPS) is a multicomponent molecular apparatus that catalyzes the covalent attachment of several copies of the small protein ubiquitin to other proteins that are generally (but not always) destined to proteasomal degradation. This enzymatic cascade is crucial for the maintenance of intracellular protein homeostasis (both in physiological conditions and in the course of adaptive stress responses), and regulates a wide array of signaling pathways. In line with this notion, defects in the UPS have been associated with aging as well as with several pathological conditions including cardiac, neurodegenerative, and neoplastic disorders. As transformed cells often experience a constant state of stress (as a result of the hyperactivation of oncogenic signaling pathways and/or adverse microenvironmental conditions), their survival and proliferation are highly dependent on the integrity of the UPS. This rationale has driven an intense wave of preclinical and clinical investigation culminating in 2003 with the approval of the proteasomal inhibitor bortezomib by the US Food and Drug Administration for use in multiple myeloma patients. Another proteasomal inhibitor, carfilzomib, is now licensed by international regulatory agencies for use in multiple myeloma patients, and the approved indications for bortezomib have been extended to mantle cell lymphoma. This said, the clinical activity of bortezomib and carfilzomib is often limited by off-target effects, innate/acquired resistance, and the absence of validated predictive biomarkers. Moreover, the antineoplastic activity of proteasome inhibitors against solid tumors is poor. In this Trial Watch we discuss the contribution of the UPS to oncogenesis and tumor progression and summarize the design and/or results of recent clinical studies evaluating the therapeutic profile of proteasome inhibitors in cancer patients. PMID:27308423

  11. Quinoxaline derivatives: novel and selective butyrylcholinesterase inhibitors.

    PubMed

    Zeb, Aurang; Hameed, Abdul; Khan, Latifullah; Khan, Imran; Dalvandi, Kourosh; Choudhary, M Iqbal; Basha, Fatima Z

    2014-01-01

    Alzheimer's disease (AD) is a progressive brain disorder which occurs due to lower levels of acetylcholine (ACh) neurotransmitters, and results in a gradual decline in memory and other cognitive processes. Acetycholinesterase (AChE) and butyrylcholinesterase (BChE) are considered to be primary regulators of the ACh levels in the brain. Evidence shows that AChE activity decreases in AD, while activity of BChE does not change or even elevate in advanced AD, which suggests a key involvement of BChE in ACh hydrolysis during AD symptoms. Therefore, inhibiting the activity of BChE may be an effective way to control AD associated disorders. In this regard, a series of quinoxaline derivatives 1-17 was synthesized and biologically evaluated against cholinesterases (AChE and BChE) and as well as against α- chymotrypsin and urease. The compounds 1-17 were found to be selective inhibitors for BChE, as no activity was found against other enzymes. Among the series, compounds 6 (IC50 = 7.7 ± 1.0 µM) and 7 (IC50 = 9.7 ± 0.9 µM) were found to be the most active inhibitors against BChE. Their IC50 values are comparable to the standard, galantamine (IC50 = 6.6 ± 0.38 µM). Their considerable BChE inhibitory activity makes them selective candidates for the development of BChE inhibitors. Structure-activity relationship (SAR) of this new class of selective BChE inhibitors has been discussed.

  12. Serendipity in discovery of proteasome inhibitors.

    PubMed

    Dunn, Derek; Iqbal, Mohamed; Husten, Jean; Ator, Mark A; Chatterjee, Sankar

    2012-05-15

    Among its various catalytic activities, the 'chymotrypsin-like' activity of the proteasome, a large multicatalytic proteinase complex has emerged as the focus of drug discovery efforts in cancer therapy. Herein, a series of first generation (2S, 3R)-2-amino-3-hydroxybutyric acid derived proteasome inhibitors that were discovered serendipitously en route to original goal of generating a series of sterically constrained oxazoline derivatives has been reported. PMID:22503349

  13. Rust Inhibitor And Fungicide For Cooling Systems

    NASA Technical Reports Server (NTRS)

    Adams, James F.; Greer, D. Clay

    1988-01-01

    Mixture of benzotriazole, benzoic acid, and fungicide prevents growth of rust and fungus. Water-based cooling mixture made from readily available materials prevents formation of metallic oxides and growth of fungi in metallic pipes. Coolant remains clear and does not develop thick sludge tending to collect in low points in cooling systems with many commercial rust inhibitors. Coolant compatible with iron, copper, aluminum, and stainless steel. Cannot be used with cadmium or cadmium-plated pipes.

  14. Immune Checkpoint Inhibitors in Older Adults.

    PubMed

    Elias, Rawad; Morales, Joshua; Rehman, Yasser; Khurshid, Humera

    2016-08-01

    Cancer is primarily a disease of older adults. The treatment of advanced stage tumors usually involves the use of systemic agents that may be associated with significant risk of toxicity, especially in older patients. Immune checkpoint inhibitors are newcomers to the oncology world with improved efficacy and better safety profiles when compared to traditional cytotoxic drugs. This makes them an attractive treatment option. While there are no elderly specific trials, this review attempts to look at the current available data from a geriatric oncology perspective. We reviewed data from phase III studies that led to newly approved indications of checkpoint inhibitors in non-small cell lung cancer, melanoma, and renal cell cancer. Data were reviewed with respect to response, survival, and toxicity according to three groups: <65 years, 65-75 years, and >75 years. Current literature does not allow one to draw definitive conclusions regarding the role of immune checkpoint inhibitors in older adults. However, they may offer a potentially less toxic but equally efficacious treatment option for the senior adult oncology patient. PMID:27287329

  15. PTEN inhibitors: an evaluation of current compounds.

    PubMed

    Spinelli, Laura; Lindsay, Yvonne E; Leslie, Nicholas R

    2015-01-01

    Small molecule inhibitors of many classes of enzymes, including phosphatases, have widespread use as experimental tools and as therapeutics. Efforts to develop inhibitors against the lipid phosphatase and tumour suppressor, PTEN, was for some time limited by concerns that their use as therapy could result in increased risk of cancer. However, the accumulation of evidence that short term PTEN inhibition may be valuable in conditions such as nerve injury has raised interest. Here we investigate the inhibition of PTEN by four available PTEN inhibitors, bpV(phen), bpV(pic), VO-OHpic and SF1670 and compared this inhibition with that of only 3 other related enzymes, the tyrosine phosphatase SHP1 and the phosphoinositide phosphatases INPP4A and INPP4B. Even with this very small number of comparators, for all compounds, inhibition of multiple enzymes was observed and with all three vanadate compounds, this was similar or more potent than the inhibition of PTEN. In particular, the bisperoxovanadate compounds were found to inhibit PTEN poorly in the presence of reducing agents including the cellular redox buffer glutathione.

  16. Histone deacetylase inhibitors as cancer therapeutics.

    PubMed

    Clawson, Gary A

    2016-08-01

    Cancer cells contain significant alterations in their epigenomic landscape, which several enzyme families reversibly contribute to. One class of epigenetic modifying enzymes is that of histone deacetylases (HDAC), which are receiving considerable scrutiny clinically as a therapeutic target in many cancers. The underlying rationale is that inhibiting HDACs will reverse dysregulated target gene expression by modulating functional histone (or other) acetylation marks. This perspective will discuss a recent paper by Markozashvili and co-workers which appeared in Gene, which indicates that the mechanisms by which HDAC inhibitors (HDACis) alter the epigenetic landscape include widespread alternative effects beyond simply controlling regional epigenetic marks. HDACs are involved in many processes/diseases, and it is not surprising that HDACis have considerable off-target effects, and thus a major effort is being directed toward identification of inhibitors which are selective for HDAC isoforms often uniquely implicated in various cancers. This Perspective will also discuss some representative work with inhibitors targeting individual HDAC classes or isoforms. At present, it is not really clear that isoform-specific HDACis will avoid non-selective effects on other unrecognized activities of HDACs. PMID:27568481

  17. Optogenetic Inhibitor of the Transcription Factor CREB.

    PubMed

    Ali, Ahmed M; Reis, Jakeb M; Xia, Yan; Rashid, Asim J; Mercaldo, Valentina; Walters, Brandon J; Brechun, Katherine E; Borisenko, Vitali; Josselyn, Sheena A; Karanicolas, John; Woolley, G Andrew

    2015-11-19

    Current approaches for optogenetic control of transcription do not mimic the activity of endogenous transcription factors, which act at numerous sites in the genome in a complex interplay with other factors. Optogenetic control of dominant negative versions of endogenous transcription factors provides a mechanism for mimicking the natural regulation of gene expression. Here we describe opto-DN-CREB, a blue-light-controlled inhibitor of the transcription factor CREB created by fusing the dominant negative inhibitor A-CREB to photoactive yellow protein (PYP). A light-driven conformational change in PYP prevents coiled-coil formation between A-CREB and CREB, thereby activating CREB. Optogenetic control of CREB function was characterized in vitro, in HEK293T cells, and in neurons where blue light enabled control of expression of the CREB targets NR4A2 and c-Fos. Dominant negative inhibitors exist for numerous transcription factors; linking these to optogenetic domains offers a general approach for spatiotemporal control of native transcriptional events. PMID:26590638

  18. Functional Stability of Plasminogen Activator Inhibitor-1

    PubMed Central

    Kuru, Pinar; Toksoy Oner, Ebru; Agirbasli, Mehmet

    2014-01-01

    Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of plasminogen activators, such as tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), and a major regulator of the fibrinolytic system. PAI-1 plays a pivotal role in acute thrombotic events such as deep vein thrombosis (DVT) and myocardial infarction (MI). The biological effects of PAI-1 extend far beyond thrombosis including its critical role in fibrotic disorders, atherosclerosis, renal and pulmonary fibrosis, type-2 diabetes, and cancer. The conversion of PAI-1 from the active to the latent conformation appears to be unique among serpins in that it occurs spontaneously at a relatively rapid rate. Latency transition is believed to represent a regulatory mechanism, reducing the risk of thrombosis from a prolonged antifibrinolytic action of PAI-1. Thus, relying solely on plasma concentrations of PAI-1 without assessing its function may be misleading in interpreting the role of PAI-1 in many complex diseases. Environmental conditions, interaction with other proteins, mutations, and glycosylation are the main factors that have a significant impact on the stability of the PAI-1 structure. This review provides an overview on the current knowledge on PAI-1 especially importance of PAI-1 level and stability and highlights the potential use of PAI-1 inhibitors for treating cardiovascular disease. PMID:25386620

  19. The hunt for HIV-1 integrase inhibitors.

    PubMed

    Lataillade, Max; Kozal, Michael J

    2006-07-01

    Currently, there are three distinct mechanistic classes of antiretrovirals: inhibitors of the HIV- 1 reverse transcriptase and protease enzymes and inhibitors of HIV entry, including receptor and coreceptor binding and cell fusion. A new drug class that inhibits the HIV-1 integrase enzyme (IN) is in development and may soon be available in the clinic. IN is an attractive drug target because it is essential for a stable and productive HIV-1 infection and there is no mammalian homologue of IN. Inhibitors of integrase enzyme (INI) block the integration of viral double-stranded DNA into the host cell's chromosomal DNA. HIV-1 integration has many potential steps that can be inhibited and several new compounds that target specific integration steps have been identified by drug developers. Recently, two INIs, GS-9137 and MK-0518, demonstrated promising early clinical trial results and have been advanced into later stage trials. In this review, we describe how IN facilitates HIV-1 integration, the needed enzyme cofactors, and the resultant byproducts created during integration. Furthermore, we review the different INIs under development, their mechanism of actions, site of IN inhibition, potency, resistance patterns, and discuss the early clinical trial results.

  20. Histone deacetylase inhibitors as cancer therapeutics

    PubMed Central

    2016-01-01

    Cancer cells contain significant alterations in their epigenomic landscape, which several enzyme families reversibly contribute to. One class of epigenetic modifying enzymes is that of histone deacetylases (HDAC), which are receiving considerable scrutiny clinically as a therapeutic target in many cancers. The underlying rationale is that inhibiting HDACs will reverse dysregulated target gene expression by modulating functional histone (or other) acetylation marks. This perspective will discuss a recent paper by Markozashvili and co-workers which appeared in Gene, which indicates that the mechanisms by which HDAC inhibitors (HDACis) alter the epigenetic landscape include widespread alternative effects beyond simply controlling regional epigenetic marks. HDACs are involved in many processes/diseases, and it is not surprising that HDACis have considerable off-target effects, and thus a major effort is being directed toward identification of inhibitors which are selective for HDAC isoforms often uniquely implicated in various cancers. This Perspective will also discuss some representative work with inhibitors targeting individual HDAC classes or isoforms. At present, it is not really clear that isoform-specific HDACis will avoid non-selective effects on other unrecognized activities of HDACs. PMID:27568481

  1. Selective Water-Soluble Gelatinase Inhibitor Prodrugs

    PubMed Central

    Gooyit, Major; Lee, Mijoon; Schroeder, Valerie A.; Ikejiri, Masahiro; Suckow, Mark A.; Mobashery, Shahriar; Chang, Mayland

    2011-01-01

    SB-3CT (1), a selective and potent thiirane-based gelatinase inhibitor, is effective in animal models of cancer metastasis and stroke; however, it is limited by poor aqueous solubility and extensive metabolism. We addressed these issues by blocking the primary site of metabolism and capitalizing on a prodrug strategy to achieve >5000-fold increased solubility. The amide prodrugs were quantitatively hydrolyzed in human blood to a potent gelatinase inhibitor, ND-322 (3). The arginyl amide prodrug (ND-478, 5d) was metabolically stable in mouse, rat, and human liver microsomes. Both 5d and 3 were non-mutagenic in the Ames II mutagenicity assay. The prodrug 5d showed moderate clearance of 0.0582 L/min/kg, remained mostly in the extracellular fluid compartment (Vd = 0.0978 L/kg), and had a terminal half-life of >4 h. The prodrug 5d had superior pharmacokinetic properties than 3, making the thiirane class of selective gelatinase inhibitors suitable for intravenous administration in treatment of acute gelatinase-dependent diseases. PMID:21866961

  2. Salicylanilide diethyl phosphates as cholinesterases inhibitors.

    PubMed

    Krátký, Martin; Štěpánková, Šárka; Vorčáková, Katarína; Vinšová, Jarmila

    2015-02-01

    Based on the presence of dialkyl phosphate moiety, we evaluated twenty-seven salicylanilide diethyl phosphates (diethyl [2-(phenylcarbamoyl)phenyl] phosphates) for the inhibition of acetylcholinesterase (AChE) from electric eel (Electrophorus electricus L.) and butyrylcholinesterase (BChE) from equine serum. Ellman's spectrophotometric method was used. The inhibitory activity (expressed as IC50 values) was compared with that of the established drugs galantamine and rivastigmine. Salicylanilide diethyl phosphates showed significant activity against both cholinesterases with IC50 values from 0.903 to 86.3 μM. IC50s for BChE were comparatively lower than those obtained for AChE. All of the investigated compounds showed higher inhibition of AChE than rivastigmine, and six of them inhibited BChE more effectively than both rivastigmine and galantamine. In general, derivatives of 4-chlorosalicylic acid showed enhanced activity when compared to derivatives of 5-halogenated salicylic acids, especially against BChE. The most effective inhibitor of AChE was O-{5-chloro-2-[(3-bromophenyl)carbamoyl]phenyl} O,O-diethyl phosphate with IC50 of 35.4 μM, which is also one of the most potent inhibitors of BChE. O-{5-Chloro-2-[(3,4-dichlorophenyl)carbamoyl]phenyl} O,O-diethyl phosphate exhibited in vitro the strongest inhibition of BChE (0.90 μM). Salicylanilide diethyl phosphates act as pseudo-irreversible cholinesterases inhibitors. PMID:25462625

  3. Immune Checkpoint Inhibitors in Older Adults.

    PubMed

    Elias, Rawad; Morales, Joshua; Rehman, Yasser; Khurshid, Humera

    2016-08-01

    Cancer is primarily a disease of older adults. The treatment of advanced stage tumors usually involves the use of systemic agents that may be associated with significant risk of toxicity, especially in older patients. Immune checkpoint inhibitors are newcomers to the oncology world with improved efficacy and better safety profiles when compared to traditional cytotoxic drugs. This makes them an attractive treatment option. While there are no elderly specific trials, this review attempts to look at the current available data from a geriatric oncology perspective. We reviewed data from phase III studies that led to newly approved indications of checkpoint inhibitors in non-small cell lung cancer, melanoma, and renal cell cancer. Data were reviewed with respect to response, survival, and toxicity according to three groups: <65 years, 65-75 years, and >75 years. Current literature does not allow one to draw definitive conclusions regarding the role of immune checkpoint inhibitors in older adults. However, they may offer a potentially less toxic but equally efficacious treatment option for the senior adult oncology patient.

  4. Inhibitory spectrum of alpha 2-plasmin inhibitor.

    PubMed

    Saito, H; Goldsmith, G H; Moroi, M; Aoki, N

    1979-04-01

    alpha 2-Plasmin inhibitor (alpha 2PI) has been recently characterized as a fast-reacting inhibitor of plasmin in human plasma and appears to play an important role in the regulation of fibrinolysis in vivo. We have studied the effect of purified alpha 2PI upon various proteases participating in human blood coagulation and kinin generation. At physiological concentration (50 microgram/ml), alpha 2PI inhibited the clot-promoting and prekallikrein-activating activity of Hageman factor fragments, the amidolytic, kininogenase, and clot-promoting activities of plasma kallikrein, and the clot-promoting properties of activated plasma thromboplastin antecedent (PTA, Factor XIa) and thrombin. alpha 2PI had minimal inhibitory effect on surface-bound activated PTA and activated Stuart factor (Factor Xa). alpha 2PI did not inhibit the activity of activated Christmas factor (Factor IXa) or urinary kallikrein. Heparin (1.5-2.0 units/ml) did not enhance the inhibitory function of alpha 2PI. These results suggest that, like other plasma protease inhibitors, alpha 2PI possesses a broad in vitro spectrum of inhibitory properties.

  5. Recent advances in designing substrate-competitive protein kinase inhibitors.

    PubMed

    Han, Ki-Cheol; Kim, So Yeon; Yang, Eun Gyeong

    2012-01-01

    Protein kinases play central roles in cellular signaling pathways and their abnormal phosphorylation activity is inseparably linked with various human diseases. Therefore, modulation of kinase activity using potent inhibitors is an attractive strategy for the treatment of human disease. While most protein kinase inhibitors in clinical development are mainly targeted to the highly conserved ATP-binding sites and thus likely promiscuously inhibit multiple kinases including kinases unrelated to diseases, protein substrate-competitive inhibitors are more selective and expected to be promising therapeutic agents. Most substrate-competitive inhibitors mimic peptides derived from substrate proteins, or from inhibitory domains within kinases or inhibitor proteins. In addition, bisubstrate inhibitors are generated by conjugating substrate-competitive peptide inhibitors to ATP-competitive inhibitors to improve affinity and selectivity. Although structural information on protein kinases provides invaluable guidance in designing substrate-competitive inhibitors, other strategies including bioinformatics, computational modeling, and high-throughput screening are often employed for developing specific substrate-competitive kinase inhibitors. This review focuses on recent advances in the design and discovery of substrate-competitive inhibitors of protein kinases.

  6. Cyclin-Dependent Kinase Inhibitors as Anticancer Therapeutics.

    PubMed

    Law, Mary E; Corsino, Patrick E; Narayan, Satya; Law, Brian K

    2015-11-01

    Cyclin-dependent kinases (CDKs) have been considered promising drug targets for a number of years, but most CDK inhibitors have failed rigorous clinical testing. Recent studies demonstrating clear anticancer efficacy and reduced toxicity of CDK4/6 inhibitors such as palbociclib and multi-CDK inhibitors such as dinaciclib have rejuvenated the field. Favorable results with palbociclib and its recent U.S. Food and Drug Administration approval demonstrate that CDK inhibitors with narrow selectivity profiles can have clinical utility for therapy based on individual tumor genetics. A brief overview of results obtained with ATP-competitive inhibitors such as palbociclib and dinaciclib is presented, followed by a compilation of new avenues that have been pursued toward the development of novel, non-ATP-competitive CDK inhibitors. These creative ways to develop CDK inhibitors are presented along with crystal structures of these agents complexed with CDK2 to highlight differences in their binding sites and mechanisms of action. The recent successes of CDK inhibitors in the clinic, combined with the potential for structure-based routes to the development of non-ATP-competitive CDK inhibitors, and evidence that CDK inhibitors may have use in suppressing chromosomal instability and in synthetic lethal drug combinations inspire optimism that CDK inhibitors will become important weapons in the fight against cancer.

  7. Solution structures of stromelysin complexed to thiadiazole inhibitors.

    PubMed Central

    Stockman, B. J.; Waldon, D. J.; Gates, J. A.; Scahill, T. A.; Kloosterman, D. A.; Mizsak, S. A.; Jacobsen, E. J.; Belonga, K. L.; Mitchell, M. A.; Mao, B.; Petke, J. D.; Goodman, L.; Powers, E. A.; Ledbetter, S. R.; Kaytes, P. S.; Vogeli, G.; Marshall, V. P.; Petzold, G. L.; Poorman, R. A.

    1998-01-01

    Unregulated or overexpressed matrix metalloproteinases (MMPs), including stromelysin, collagenase, and gelatinase. have been implicated in several pathological conditions including arthritis and cancer. Small-molecule MMP inhibitors may have therapeutic value in the treatment of these diseases. In this regard, the solution structures of two stromelysin/ inhibitor complexes have been investigated using 1H, 13C, and 15N NMR spectroscopy. Both-inhibitors are members of a novel class of matrix metalloproteinase inhibitor that contain a thiadiazole group and that interact with stromelysin in a manner distinct from other classes of inhibitors. The inhibitors coordinate the catalytic zinc atom through their exocyclic sulfur atom, with the remainder of the ligand extending into the S1-S3 side of the active site. The binding of inhibitor containing a protonated or fluorinated aromatic ring was investigated using 1H and 19F NMR spectroscopy. The fluorinated ring was found to have a reduced ring-flip rate compared to the protonated version. A strong, coplanar interaction between the fluorinated ring of the inhibitor and the aromatic ring of Tyr155 is proposed to account for the reduced ring-flip rate and for the increase in binding affinity observed for the fluorinated inhibitor compared to the protonated inhibitor. Binding interactions observed for the thiadiazole class of ligands have implications for the design of matrix metalloproteinase inhibitors. PMID:9827994

  8. Molecular mechanism of respiratory syncytial virus fusion inhibitors.

    PubMed

    Battles, Michael B; Langedijk, Johannes P; Furmanova-Hollenstein, Polina; Chaiwatpongsakorn, Supranee; Costello, Heather M; Kwanten, Leen; Vranckx, Luc; Vink, Paul; Jaensch, Steffen; Jonckers, Tim H M; Koul, Anil; Arnoult, Eric; Peeples, Mark E; Roymans, Dirk; McLellan, Jason S

    2016-02-01

    Respiratory syncytial virus (RSV) is a leading cause of pneumonia and bronchiolitis in young children and the elderly. Therapeutic small molecules have been developed that bind the RSV F glycoprotein and inhibit membrane fusion, yet their binding sites and molecular mechanisms of action remain largely unknown. Here we show that these inhibitors bind to a three-fold-symmetric pocket within the central cavity of the metastable prefusion conformation of RSV F. Inhibitor binding stabilizes this conformation by tethering two regions that must undergo a structural rearrangement to facilitate membrane fusion. Inhibitor-escape mutations occur in residues that directly contact the inhibitors or are involved in the conformational rearrangements required to accommodate inhibitor binding. Resistant viruses do not propagate as well as wild-type RSV in vitro, indicating a fitness cost for inhibitor escape. Collectively, these findings provide new insight into class I viral fusion proteins and should facilitate development of optimal RSV fusion inhibitors.

  9. Advances in algal drug research with emphasis on enzyme inhibitors.

    PubMed

    Rengasamy, Kannan R R; Kulkarni, Manoj G; Stirk, Wendy A; Van Staden, Johannes

    2014-12-01

    Enzyme inhibitors are now included in all kinds of drugs essential to treat most of the human diseases including communicable, metabolic, cardiovascular, neurological diseases and cancer. Numerous marine algae have been reported to be a potential source of novel enzyme inhibitors with various pharmaceutical values. Thus, the purpose of this review is to brief the enzyme inhibitors from marine algae of therapeutic potential to treat common diseases. As per our knowledge this is the first review for the potential enzyme inhibitors from marine origin. This review contains 86 algal enzyme inhibitors reported during 1989-2013 and commercial enzyme inhibitors available in the market. Compounds in the review are grouped according to the disease conditions in which they are involved; diabetes, obesity, dementia, inflammation, melanogenesis, AIDS, hypertension and other viral diseases. The structure-activity relationship of most of the compounds are also discussed. In addition, the drug likeness properties of algal inhibitors were evaluated using Lipinski's 'Rule of Five'. PMID:25195189

  10. Evaluation of Encapsulated Inhibitor for Autonomous Corrosion Protection

    NASA Technical Reports Server (NTRS)

    Johnsey, M. N.; Li, W.; Buhrow, J. W.; Calle, L. M.; Pearman, B. P.; Zhang, X.

    2015-01-01

    This work concerns the development of smart coating technologies based on microencapsulation for the autonomous control of corrosion. Microencapsulation allows the incorporation of corrosion inhibitors into coating which provides protection through corrosion-controlled release of these inhibitors.One critical aspect of a corrosion protective smart coating is the selection of corrosion inhibitor for encapsulation and comparison of the inhibitor function before and after encapsulation. For this purpose, a systematic approach is being used to evaluate free and encapsulated corrosion inhibitors by salt immersion. Visual, optical microscope, and Scanning Electron Microscope (with low-angle backscatter electron detector) are used to evaluate these inhibitors. It has been found that the combination of different characterization tools provide an effective method for evaluation of early stage localized corrosion and the effectiveness of corrosion inhibitors.

  11. Phosphorylation of protein phosphatase inhibitor-1 by protein kinase C.

    PubMed

    Sahin, Bogachan; Shu, Hongjun; Fernandez, Joseph; El-Armouche, Ali; Molkentin, Jeffery D; Nairn, Angus C; Bibb, James A

    2006-08-25

    Inhibitor-1 becomes a potent inhibitor of protein phosphatase 1 when phosphorylated by cAMP-dependent protein kinase at Thr(35). Moreover, Ser(67) of inhibitor-1 serves as a substrate for cyclin-dependent kinase 5 in the brain. Here, we report that dephosphoinhibitor-1 but not phospho-Ser(67) inhibitor-1 was efficiently phosphorylated by protein kinase C at Ser(65) in vitro. In contrast, Ser(67) phosphorylation by cyclin-dependent kinase 5 was unaffected by phospho-Ser(65). Protein kinase C activation in striatal tissue resulted in the concomitant phosphorylation of inhibitor-1 at Ser(65) and Ser(67), but not Ser(65) alone. Selective pharmacological inhibition of protein phosphatase activity suggested that phospho-Ser(65) inhibitor-1 is dephosphorylated by protein phosphatase 1 in the striatum. In vitro studies confirmed these findings and suggested that phospho-Ser(67) protects phospho-Ser(65) inhibitor-1 from dephosphorylation by protein phosphatase 1 in vivo. Activation of group I metabotropic glutamate receptors resulted in the up-regulation of diphospho-Ser(65)/Ser(67) inhibitor-1 in this tissue. In contrast, the activation of N-methyl-d-aspartate-type ionotropic glutamate receptors opposed increases in striatal diphospho-Ser(65)/Ser(67) inhibitor-1 levels. Phosphomimetic mutation of Ser(65) and/or Ser(67) did not convert inhibitor-1 into a protein phosphatase 1 inhibitor. On the other hand, in vitro and in vivo studies suggested that diphospho-Ser(65)/Ser(67) inhibitor-1 is a poor substrate for cAMP-dependent protein kinase. These observations extend earlier studies regarding the function of phospho-Ser(67) and underscore the possibility that phosphorylation in this region of inhibitor-1 by multiple protein kinases may serve as an integrative signaling mechanism that governs the responsiveness of inhibitor-1 to cAMP-dependent protein kinase activation.

  12. Studies on bacterial cell wall inhibitors. VI. Screening method for the specific inhibitors of peptidoglycan synthesis.

    PubMed

    Omura, S; Tanaka, H; Oiwa, R; Nagai, T; Koyama, Y; Takahashi, Y

    1979-10-01

    A screening method was established for selecting new specific inhibitors of bacterial cell wall peptidoglycan synthesis. In the primary test, culture broths of soil isolates were selected based on relative microbial activity. A culture, to be retained, must be active against Bacillus subtilis and lack activities against Acholeplasma laidawii. In the secondary test, inhibitors of bacterial cell wall synthesis were identified by their ability to prevent the incorporation of meso-[3H]diaminopimelic acid but not to prevent the incorporation of L-[4C]leucine into the acid-insoluble macromolecular fraction of growing cells of Bacillus sp. ATCC 21206 (Dpm-). As the tertiary test, inhibitors with molecular weights under 1,000 were selected by passage through a Diaflo UM-2 membrane. By this screening procedure, six known antibiotics and one new one were picked out from ten thousand soil isolates. PMID:528376

  13. The "SWOT" of BRAF inhibition in melanoma: RAF inhibitors, MEK inhibitors or both?

    PubMed

    Nissan, Moriah H; Solit, David B

    2011-12-01

    Activating mutations in the BRAF gene are among the most prevalent kinase mutations in human cancer. BRAF mutations are most frequent in patients with melanoma where they occur in approximately 50% of patients with advanced disease. Remarkable clinical activity has recently been reported with highly selective RAF inhibitors in melanoma patients whose tumors harbor V600E BRAF mutations. The response rates of RAF inhibitors in patients with BRAF-mutant melanomas far exceed the activity level of any prior therapy studied in this disease. The results suggest that we have entered an era of personalized therapy for patients with metastatic melanoma in which treatment selection will be guided by BRAF mutational status. This review will discuss the strengths, weaknesses, opportunities and threats ("SWOT") of developing RAF and MEK selective inhibitors as anti-cancer therapies, recent insights into the mechanisms of intrinsic and acquired resistance to these agents, and current efforts to develop mechanism-based combination therapies. PMID:21997758

  14. Designing Inhibitors Against Fructose 1,6-bisphosphatase: Exploring Natural Products for Novel Inhibitor Scaffolds

    PubMed Central

    Heng, Sabrina; Harris, Katharine M.; Kantrowitz, Evan R.

    2010-01-01

    Natural products often contain unusual scaffold structures that may be elaborated by combinatorial methods to develop new drug-like molecules. Visual inspection of more than 128 natural products with some type of anti-diabetic activity suggested that a subset might provide novel scaffolds for designing potent inhibitors against fructose 1,6-bisphosphatase (FBPase), an enzyme critical in the control of gluconeogenesis. Using in silico docking methodology, these were evaluated to determine those that exhibited affinity for the AMP binding site. Achyrofuran from the South American plant Achyrocline satureoides, was selected for further investigation. Using the achyrofuran scaffold, inhibitors against FBPase were developed. Compounds 15 and 16 inhibited human liver and pig kidney FBPases at IC50 values comparable to that of AMP, the natural allosteric inhibitor. PMID:20116906

  15. The "SWOT" of BRAF inhibition in melanoma: RAF inhibitors, MEK inhibitors or both?

    PubMed

    Nissan, Moriah H; Solit, David B

    2011-12-01

    Activating mutations in the BRAF gene are among the most prevalent kinase mutations in human cancer. BRAF mutations are most frequent in patients with melanoma where they occur in approximately 50% of patients with advanced disease. Remarkable clinical activity has recently been reported with highly selective RAF inhibitors in melanoma patients whose tumors harbor V600E BRAF mutations. The response rates of RAF inhibitors in patients with BRAF-mutant melanomas far exceed the activity level of any prior therapy studied in this disease. The results suggest that we have entered an era of personalized therapy for patients with metastatic melanoma in which treatment selection will be guided by BRAF mutational status. This review will discuss the strengths, weaknesses, opportunities and threats ("SWOT") of developing RAF and MEK selective inhibitors as anti-cancer therapies, recent insights into the mechanisms of intrinsic and acquired resistance to these agents, and current efforts to develop mechanism-based combination therapies.

  16. Structural analysis of Golgi alpha-mannosidase II inhibitors identified from a focused glycosidase inhibitor screen.

    PubMed

    Kuntz, Douglas A; Tarling, Chris A; Withers, Stephen G; Rose, David R

    2008-09-23

    The N-glycosylation pathway is a target for pharmaceutical intervention in a number of pathological conditions including cancer. Golgi alpha-mannosidase II (GMII) is the final glycoside hydrolase in the pathway and has been the target for a number of synthetic efforts aimed at providing more selective and effective inhibitors. Drosophila GMII (dGMII) has been extensively studied due to the ease of obtaining high resolution structural data, allowing the observation of substrate distortion upon binding and after formation of a trapped covalent reaction intermediate. However, attempts to find new inhibitor leads by high-throughput screening of large commercial libraries or through in silico docking were unsuccessful. In this paper we provide a kinetic and structural analysis of five inhibitors derived from a small glycosidase-focused library. Surprisingly, four of these were known inhibitors of beta-glucosidases. X-ray crystallographic analysis of the dGMII:inhibitor complexes highlights the ability of the zinc-containing GMII active site to deform compounds, even ones designed as conformationally restricted transition-state mimics of beta-glucosidases, into binding entities that have inhibitory activity. Although these deformed conformations do not appear to be on the expected conformational itinerary of the enzyme, and are thus not transition-state mimics of GMII, they allow positioning of the three vicinal hydroxyls of the bound gluco-inhibitors into similar locations to those found with mannose-containing substrates, underlining the importance of these hydrogen bonds for binding. Further, these studies show the utility of targeting the acid-base catalyst using appropriately positioned positively charged nitrogen atoms, as well as the challenges associated with aglycon substitutions.

  17. F8 haplotype and inhibitor risk: results from the Hemophilia Inhibitor Genetics Study (HIGS) Combined Cohort

    PubMed Central

    Schwarz, John; Astermark, Jan; Menius, Erika D.; Carrington, Mary; Donfield, Sharyne M.; Gomperts, Edward D.; Nelson, George W.; Oldenburg, Johannes; Pavlova, Anna; Shapiro, Amy D.; Winkler, Cheryl A.; Berntorp, Erik

    2012-01-01

    Background Ancestral background, specifically African descent, confers higher risk for development of inhibitory antibodies to factor VIII (FVIII) in hemophilia A. It has been suggested that differences in the distribution of factor VIII gene (F8) haplotypes, and mismatch between endogenous F8 haplotypes and those comprising products used for treatment could contribute to risk. Design and Methods Data from the HIGS Combined Cohort were used to determine the association between F8 haplotype 3 (H3) vs. haplotypes 1 and 2 (H1+H2) and inhibitor risk among individuals of genetically-determined African descent. Other variables known to affect inhibitor risk including type of F8 mutation and HLA were included in the analysis. A second research question regarding risk related to mismatch in endogenous F8 haplotype and recombinant FVIII products used for treatment was addressed. Results H3 was associated with higher inhibitor risk among those genetically-identified (N=49) as of African ancestry, but the association did not remain significant after adjustment for F8 mutation type and the HLA variables. Among subjects of all racial ancestries enrolled in HIGS who reported early use of recombinant products (N=223), mismatch in endogenous haplotype and the FVIII proteins constituting the products used did not confer greater risk for inhibitor development. Conclusion H3 was not an independent predictor of inhibitor risk. Further, our findings did not support a higher risk of inhibitors in the presence of a haplotype mismatch between the FVIII molecule infused and that of the individual. PMID:22958194

  18. Design and synthesis of conformationally restricted inhibitors of active thrombin activatable fibrinolysis inhibitor (TAFIa).

    PubMed

    Brink, Mikael; Dahlén, Anders; Olsson, Thomas; Polla, Magnus; Svensson, Tor

    2014-04-01

    A series of 4,5,6,7-tetrahydro-1H-benzimidazole-5-carboxylic acid and 5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-7-carboxylic acid derivatives designed as inhibitors of TAFIa has been prepared via a common hydrogenation-alkylation sequence starting from the appropriate benzimidazole and imidazopyridine system. We present a successful design strategy using a conformational restriction approach resulting in potent and selective inhibitors of TAFIa. The X-ray structure of compound 5 in complex with a H333Y/H335Q double mutant TAFI indicate that the conformational restriction is responsible for the observed potency increase. PMID:24588961

  19. Protocol for rational design of covalently interacting inhibitors.

    PubMed

    Schmidt, Thomas C; Welker, Armin; Rieger, Max; Sahu, Prabhat K; Sotriffer, Christoph A; Schirmeister, Tanja; Engels, Bernd

    2014-10-20

    The inhibition potencies of covalent inhibitors mainly result from the formation of a covalent bond to the enzyme during the inhibition mechanism. This class of inhibitors has essentially been ignored in previous target-directed drug discovery projects because of concerns about possible side effects. However, their advantages, such as higher binding energies and longer drug-target residence times moved them into the focus of recent investigations. While the rational design of non-covalent inhibitors became standard the corresponding design of covalent inhibitors is still in its early stages. Potent covalent inhibitors can be retrieved from large compound libraries by covalent docking approaches but protocols are missing that can reliably predict the influence of variations in the substitution pattern on the affinity and/or reactivity of a given covalent inhibitor. Hence, the wanted property profile can only be obtained from trial-and-error proceedings. This paper presents an appropriate protocol which is able to predict improved covalent inhibitors. It uses hybrid approaches, which mix quantum mechanical (QM) and molecular mechanical (MM) methods to predict variations in the reactivity of the inhibitor. They are also used to compute the required information about the non-covalent enzyme-inhibitor complex. Docking tools are employed to improve the inhibitor with respect to the non-covalent interactions formed in the binding site. PMID:25251382

  20. Homologous inhibitors from potato tubers of serine endopeptidases and metallocarboxypeptidases.

    PubMed Central

    Hass, C M; Venkatakrishnan, R; Ryan, C A

    1976-01-01

    A potent polypeptide inhibitor of chymotrypsin has been purified from Russett Burbank potatoes. The inhibitor has no effect on bovine carboxypeptidases A or B but exhibits homology with a carboxypeptidase inhibitor that is also present in potato tubers. The chymotrypsin inhibitor has a molecular weight of approximately 5400 as estimated by gel filtration, amino acid analysis, and titration with chymotrypsin. The polypeptide chain consists of 49 amino acid residues, of which six are half-cystine, forming three disulfide bonds. Its size is similar to that of the carboxypeptidase inhibitor, which contains 39 amino acid residues and also has three disulfide bridges. In immunological double diffusion assays, the chymotrypsin inhibitor and the carboxypeptidase inhibitor do not crossreact; however, automatic Edman degradation of reduced and alkylated derivatives of the chymotrypsin inhibitor, yielding a partial sequence of 18 amino acid residues at the NH2-terminus, reveals a similarity in sequence to that of the carboxypeptidase inhibitor. Thus, inhibitors directed toward two distinct classes of proteases, the serine endopeptidases and the metallocarboxypeptidases, appear to have evolved from a common ancestor. Images PMID:1064864

  1. Inhibitor development in non-severe haemophilia across Europe.

    PubMed

    Fischer, Kathelijn; Iorio, Alfonso; Lassila, Riitta; Peyvandi, Flora; Calizzani, Gabriele; Gatt, Alex; Lambert, Thierry; Windyga, Jerzy; Gilman, Estelle A; Hollingsworth, R; Makris, Michael

    2015-10-01

    Evidence about inhibitor formation in non-severe haemophilia and the potential role for clotting factor concentrate type is scant. It was the aim of this study to report inhibitor development in non-severe haemophilia patients enrolled in the European Haemophilia Safety Surveillance (EUHASS) study. Inhibitors are reported quarterly and total treated patients annually. Incidence rates and 95% confidence intervals (95% CI) were calculated according to diagnosis and concentrate used. Between 1-10-2008 and 31-12-2012, 68 centres reported on 7,969 patients with non-severe haemophilia A and 1,863 patients with non-severe haemophilia B. For haemophilia A, 37 inhibitors occurred in 8,622 treatment years, resulting in an inhibitor rate of 0.43/100 treatment years (95% CI 0.30-0.59). Inhibitors occurred at a median age of 35 years, after a median of 38 exposure days (EDs; P25-P75: 20-80); with 72% occurring within the first 50 EDs. In haemophilia B, one inhibitor was detected in 2,149 treatment years, resulting in an inhibitor rate of 0.05/100 years (95% CI 0.001-0.26). This inhibitor developed at the age of six years, after six EDs. The rate of inhibitors appeared similar across recombinant and plasma derived factor VIII (FVIII) concentrates. Rates for individual concentrates could not be calculated at this stage due to low number of events. In conclusion, inhibitors in non-severe haemophilia occur three times more frequently than in previously treated patients with severe haemophilia at a rate of 0.43/100 patient years (haemophilia A) and 0.05/100 years (haemophilia B). Although the majority of inhibitors developed in the first 50 EDs, inhibitor development continued with increasing exposure to FVIII.

  2. Structural determinants of Tau aggregation inhibitor potency.

    PubMed

    Schafer, Kelsey N; Cisek, Katryna; Huseby, Carol J; Chang, Edward; Kuret, Jeff

    2013-11-01

    Small-molecule Tau aggregation inhibitors are under investigation as potential therapeutic agents against Alzheimer disease. Many such inhibitors have been identified in vitro, but their potency-driving features, and their molecular targets in the Tau aggregation pathway, have resisted identification. Previously we proposed ligand polarizability, a measure of electron delocalization, as a candidate descriptor of inhibitor potency. Here we tested this hypothesis by correlating the ground state polarizabilities of cyanine, phenothiazine, and arylmethine derivatives calculated using ab initio quantum methods with inhibitory potency values determined in the presence of octadecyl sulfate inducer under reducing conditions. A series of rhodanine analogs was analyzed as well using potency values disclosed in the literature. Results showed that polarizability and inhibitory potency directly correlated within all four series. To identify putative binding targets, representative members of the four chemotypes were added to aggregation reactions, where they were found to stabilize soluble, but SDS-resistant Tau species at the expense of filamentous aggregates. Using SDS resistance as a secondary assay, and a library of Tau deletion and missense mutants as targets, interaction with cyanine was localized to the microtubule binding repeat region. Moreover, the SDS-resistant phenotype was completely dependent on the presence of octadecyl sulfate inducer, but not intact PHF6/PH6* hexapeptide motifs, indicating that cyanine interacted with a species in the aggregation pathway prior to nucleus formation. Together the data suggest that flat, highly polarizable ligands inhibit Tau aggregation by interacting with folded species in the aggregation pathway and driving their assembly into soluble but highly stable Tau oligomers.

  3. Structural Determinants of Tau Aggregation Inhibitor Potency*

    PubMed Central

    Schafer, Kelsey N.; Cisek, Katryna; Huseby, Carol J.; Chang, Edward; Kuret, Jeff

    2013-01-01

    Small-molecule Tau aggregation inhibitors are under investigation as potential therapeutic agents against Alzheimer disease. Many such inhibitors have been identified in vitro, but their potency-driving features, and their molecular targets in the Tau aggregation pathway, have resisted identification. Previously we proposed ligand polarizability, a measure of electron delocalization, as a candidate descriptor of inhibitor potency. Here we tested this hypothesis by correlating the ground state polarizabilities of cyanine, phenothiazine, and arylmethine derivatives calculated using ab initio quantum methods with inhibitory potency values determined in the presence of octadecyl sulfate inducer under reducing conditions. A series of rhodanine analogs was analyzed as well using potency values disclosed in the literature. Results showed that polarizability and inhibitory potency directly correlated within all four series. To identify putative binding targets, representative members of the four chemotypes were added to aggregation reactions, where they were found to stabilize soluble, but SDS-resistant Tau species at the expense of filamentous aggregates. Using SDS resistance as a secondary assay, and a library of Tau deletion and missense mutants as targets, interaction with cyanine was localized to the microtubule binding repeat region. Moreover, the SDS-resistant phenotype was completely dependent on the presence of octadecyl sulfate inducer, but not intact PHF6/PH6* hexapeptide motifs, indicating that cyanine interacted with a species in the aggregation pathway prior to nucleus formation. Together the data suggest that flat, highly polarizable ligands inhibit Tau aggregation by interacting with folded species in the aggregation pathway and driving their assembly into soluble but highly stable Tau oligomers. PMID:24072703

  4. Sifuvirtide, a potent HIV fusion inhibitor peptide

    SciTech Connect

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

    2009-05-08

    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{sub 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{sub 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{sub 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.

  5. Tools for Characterizing Bacterial Protein Synthesis Inhibitors

    PubMed Central

    Orelle, Cédric; Carlson, Skylar; Kaushal, Bindiya; Almutairi, Mashal M.; Liu, Haipeng; Ochabowicz, Anna; Quan, Selwyn; Pham, Van Cuong; Squires, Catherine L.; Murphy, Brian T.

    2013-01-01

    Many antibiotics inhibit the growth of sensitive bacteria by interfering with ribosome function. However, discovery of new protein synthesis inhibitors is curbed by the lack of facile techniques capable of readily identifying antibiotic target sites and modes of action. Furthermore, the frequent rediscovery of known antibiotic scaffolds, especially in natural product extracts, is time-consuming and expensive and diverts resources that could be used toward the isolation of novel lead molecules. In order to avoid these pitfalls and improve the process of dereplication of chemically complex extracts, we designed a two-pronged approach for the characterization of inhibitors of protein synthesis (ChIPS) that is suitable for the rapid identification of the site and mode of action on the bacterial ribosome. First, we engineered antibiotic-hypersensitive Escherichia coli strains that contain only one rRNA operon. These strains are used for the rapid isolation of resistance mutants in which rRNA mutations identify the site of the antibiotic action. Second, we show that patterns of drug-induced ribosome stalling on mRNA, monitored by primer extension, can be used to elucidate the mode of antibiotic action. These analyses can be performed within a few days and provide a rapid and efficient approach for identifying the site and mode of action of translation inhibitors targeting the bacterial ribosome. Both techniques were validated using a bacterial strain whose culture extract, composed of unknown metabolites, exhibited protein synthesis inhibitory activity; we were able to rapidly detect the presence of the antibiotic chloramphenicol. PMID:24041905

  6. Kynurenine Aminotransferase Isozyme Inhibitors: A Review

    PubMed Central

    Nematollahi, Alireza; Sun, Guanchen; Jayawickrama, Gayan S.; Church, W. Bret

    2016-01-01

    Kynurenine aminotransferase isozymes (KATs 1–4) are members of the pyridoxal-5’-phosphate (PLP)-dependent enzyme family, which catalyse the permanent conversion of l-kynurenine (l-KYN) to kynurenic acid (KYNA), a known neuroactive agent. As KATs are found in the mammalian brain and have key roles in the kynurenine pathway, involved in different categories of central nervous system (CNS) diseases, the KATs are prominent targets in the quest to treat neurodegenerative and cognitive impairment disorders. Recent studies suggest that inhibiting these enzymes would produce effects beneficial to patients with these conditions, as abnormally high levels of KYNA are observed. KAT-1 and KAT-3 share the highest sequence similarity of the isozymes in this family, and their active site pockets are also similar. Importantly, KAT-2 has the major role of kynurenic acid production (70%) in the human brain, and it is considered therefore that suitable inhibition of this isozyme would be most effective in managing major aspects of CNS diseases. Human KAT-2 inhibitors have been developed, but the most potent of them, chosen for further investigations, did not proceed in clinical studies due to the cross toxicity caused by their irreversible interaction with PLP, the required cofactor of the KAT isozymes, and any other PLP-dependent enzymes. As a consequence of the possibility of extensive undesirable adverse effects, it is also important to pursue KAT inhibitors that reversibly inhibit KATs and to include a strategy that seeks compounds likely to achieve substantial interaction with regions of the active site other than the PLP. The main purpose of this treatise is to review the recent developments with the inhibitors of KAT isozymes. This treatise also includes analyses of their crystallographic structures in complex with this enzyme family, which provides further insight for researchers in this and related studies. PMID:27314340

  7. Sifuvirtide, a potent HIV fusion inhibitor peptide.

    PubMed

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

    2009-05-01

    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.

  8. Nanomolar Inhibitors of Trypanosoma brucei RNA Triphosphatase

    PubMed Central

    Smith, Paul; Ho, C. Kiong; Takagi, Yuko; Djaballah, Hakim

    2016-01-01

    ABSTRACT Eukaryal taxa differ with respect to the structure and mechanism of the RNA triphosphatase (RTPase) component of the mRNA capping apparatus. Protozoa, fungi, and certain DNA viruses have a metal-dependent RTPase that belongs to the triphosphate tunnel metalloenzyme (TTM) superfamily. Because the structures, active sites, and chemical mechanisms of the TTM-type RTPases differ from those of mammalian RTPases, the TTM RTPases are potential targets for antiprotozoal, antifungal, and antiviral drug discovery. Here, we employed RNA interference (RNAi) knockdown methods to show that Trypanosoma brucei RTPase Cet1 (TbCet1) is necessary for proliferation of procyclic cells in culture. We then conducted a high-throughput biochemical screen for small-molecule inhibitors of the phosphohydrolase activity of TbCet1. We identified several classes of chemicals—including chlorogenic acids, phenolic glycopyranosides, flavonoids, and other phenolics—that inhibit TbCet1 with nanomolar to low-micromolar 50% inhibitory concentrations (IC50s). We confirmed the activity of these compounds, and tested various analogs thereof, by direct manual assays of TbCet1 phosphohydrolase activity. The most potent nanomolar inhibitors included tetracaffeoylquinic acid, 5-galloylgalloylquinic acid, pentagalloylglucose, rosmarinic acid, and miquelianin. TbCet1 inhibitors were less active (or inactive) against the orthologous TTM-type RTPases of mimivirus, baculovirus, and budding yeast (Saccharomyces cerevisiae). Our results affirm that a TTM RTPase is subject to potent inhibition by small molecules, with the caveat that parallel screens against TTM RTPases from multiple different pathogens may be required to fully probe the chemical space of TTM inhibition. PMID:26908574

  9. Hypomagnesemia associated with a proton pump inhibitor.

    PubMed

    Matsuyama, Jun; Tsuji, Kunihiro; Doyama, Hisashi; Kim, Fae; Takeda, Yasuhito; Kito, Yosuke; Ito, Renma; Nakanishi, Hiroyoshi; Hayashi, Tomoyuki; Waseda, Yohei; Tsuji, Shigetsugu; Takemura, Kenichi; Yamada, Shinya; Okada, Toshihide; Kanaya, Honin

    2012-01-01

    Severe hypomagnesemia is a serious clinical condition. Proton pump inhibitor (PPI) induced hypomagnesemia has been recognized since 2006. In March 2011 the U.S. Food and Drug Administration advised that long-term use of PPI can induce hypomagnesemia. We report the first Japanese case of hypomagnesemia associated with chronic use of PPIs in a 64-year-old man hospitalized for nausea, bilateral ankle arthritis, and tremor of the extremities who had convulsions 3 days after admission. Blood analysis showed severe hypomagnesemia. He had been taking rabeprazole (10 mg/day) for 5 years. After stopping rabeprazole and correcting the electrolytes imbalances, his symptoms improved without recurrence.

  10. Inhibitors of glycogen synthase 3 kinase

    DOEpatents

    Schultz, Peter; Ring, David B.; Harrison, Stephen D.; Bray, Andrew M.

    2006-05-30

    Compounds of formula 1: ##STR00001## wherein R.sub.1 is alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl, substituted with 0 3 substituents selected from lower alkyl, halo, hydroxy, lower alkoxy, amino, lower alkyl-amino, and nitro; R.sub.2 is hydroxy, amino, or lower alkoxy; R.sub.3 is H, lower alkyl, lower acyl, lower alkoxy-acyl, or amino-acyl; R.sub.4 is H or lower alkyl; and pharmaceutically acceptable salts and esters thereof; are effective inhibitors of GSK3.

  11. Inhibitors of glycogen synthase 3 kinase

    DOEpatents

    Schultz, Peter; Ring, David B.; Harrison, Stephen D.; Bray, Andrew M.

    2000-01-01

    Compounds of formula 1: ##STR1## wherein R.sub.1 is alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl, substituted with 0-3 substituents selected from lower alkyl, halo, hydroxy, lower alkoxy, amino, lower alkyl-amino, and nitro; R.sub.2 is hydroxy, amino, or lower alkoxy; R.sub.3 is H, lower alkyl, lower acyl, lower alkoxy-acyl, or amnino-acyl; R.sub.4 is H or lower alkyl; and pharmaceutically acceptable salts and esters thereof; are effective inhibitors of GSK3.

  12. A new urease inhibitor from Viola betonicifolia.

    PubMed

    Muhammad, Naveed; Saeed, Muhammad; Khan, Ajmal; Adhikari, Achyut; Wadood, Abdul; Khan, Khalid Mohammed; De Feo, Vincenzo

    2014-10-17

    Urease has attracted much attention, as it is directly involved in the formation of infection stones and contributes to the pathogenesis of urolithiasis, pyelonephritis, ammonia and hepatic encephalopathy, hepatic coma and urinary catheter encrustation. Moreover, urease is the major cause of pathologies induced by H. pylori, such as gastritis and peptic ulcer. In the present work, the new natural compound, 3-methoxydalbergione, was isolated from Viola betonicifolia. A mechanistic study of this compound as a natural urease inhibitor was performed by using enzyme kinetics and docking studies. 3-Methoxydalbergione could be considered as a lead molecule for drugs useful in the urease associated diseases.

  13. Inhibitors of platelet lipoxygenase from Ponkan fruit.

    PubMed

    Nogata, Y; Sekiya, K; Ohta, H; Kusumoto, K; Ishizu, T

    2001-04-01

    An activity-guided separation for inhibitors of rat platelet 12-lipoxygenase led to the isolation of two compounds, 4-O-feruloyl-5-O-caffeoylquinic acid (IC50; 5.5 microM) and methyl 4-O-feruloyl-5-O-caffeoylquinate (IC50; 1.9 microM) from the peel of Ponkan fruit (Citrus reticulata). The complete structure of each phenolic ester was determined by NMR spectroscopy [1H and 13C NMR spectra, 1H-1H correlation spectroscopy (COSY), 1H-detected heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond connectivity (HMBC) spectroscopies] and other spectral methods. PMID:11314960

  14. PDE-10A inhibitors as insulin secretagogues.

    PubMed

    Cantin, Louis-David; Magnuson, Steven; Gunn, David; Barucci, Nicole; Breuhaus, Marina; Bullock, William H; Burke, Jennifer; Claus, Thomas H; Daly, Michelle; Decarr, Lynn; Gore-Willse, Ann; Hoover-Litty, Helana; Kumarasinghe, Ellalahewage S; Li, Yaxin; Liang, Sidney X; Livingston, James N; Lowinger, Timothy; Macdougall, Margit; Ogutu, Herbert O; Olague, Alan; Ott-Morgan, Ronda; Schoenleber, Robert W; Tersteegen, Adrian; Wickens, Philip; Zhang, Zhonghua; Zhu, Jian; Zhu, Lei; Sweet, Laurel J

    2007-05-15

    Modulation of cAMP levels has been linked to insulin secretion in preclinical animal models and in humans. The high expression of PDE-10A in pancreatic islets suggested that inhibition of this enzyme may provide the necessary modulation to elicit increased insulin secretion. Using an HTS approach, we have identified quinoline-based PDE-10A inhibitors as insulin secretagogues in vitro. Optimized compounds were evaluated in vivo where improvements in glucose tolerance and increases in insulin secretion were measured. PMID:17400452

  15. Theoretical study of classical acetylcholinesterase inhibitors

    NASA Astrophysics Data System (ADS)

    Nascimento, Érica C. M.; Martins, João B. L.; dos Santos, Maria L.; Gargano, R.

    2008-06-01

    Semi-empirical, RHF and DFT calculations were carried out to study well known acetylcholinesterase inhibitors, i.e., tacrine, donepezil, galantamine, physostigmine, and tacrine dimer (bis-tacrine). Electronic and structural parameters were used in order to correlate the acetylcholinesterase inhibition activity with their molecular structure. The optimized geometries of these drugs were analyzed by multivariate PCA statistical method. Frontier orbital energies (HOMO and LUMO), the (HOMO-LUMO) gap and the distance between more acidic hydrogen species were used to determine principal components. The PCA results indicated that these drugs were ordered into three groups according to the first principal component: galantamine/physostigmine, donepezil/tacrine dimer and tacrine.

  16. Radical scavengers as ribonucleotide reductase inhibitors.

    PubMed

    Basu, Arijit; Sinha, Barij Nayan

    2012-01-01

    This paper compiled all the previous reports on radical scavengers, an interesting class of ribonucleotide reductase inhibitors. We have highlighted three key research areas: chemical classification of radical scavengers, structural and functional aspects of the radical site, and progress in drug designing for radical scavengers. Under the chemical classification section, we have recorded the discovery of hydroxyurea followed by discussions on hydroxamic acids, amidoximes, hydroxyguanidines, and phenolic compounds. In the next section, we have compiled the structural information for the radical site obtained from different crystallographic and theoretical studies. Finally, we have included the reported ligand based and structure based drug-designing studies.

  17. Inside HDACs with more selective HDAC inhibitors.

    PubMed

    Roche, Joëlle; Bertrand, Philippe

    2016-10-01

    Inhibitors of histone deacetylases (HDACs) are nowadays part of the therapeutic arsenal mainly against cancers, with four compounds approved by the Food and Drug Administration. During the last five years, several groups have made continuous efforts to improve this class of compounds, designing more selective compounds or compounds with multiple capacities. After a survey of the HDAC biology and structures, this review summarizes the results of the chemists working in this field, and highlights when possible the behavior of the molecules inside their targets.

  18. Protease Inhibitors Targeting Coronavirus and Filovirus Entry

    PubMed Central

    Zhou, Yanchen; Vedantham, Punitha; Lu, Kai; Agudelo, Juliet; Carrion, Ricardo; Nunneley, Jerritt W.; Barnard, Dale; Pöhlmann, Stefan; McKerrow, James H.; Renslo, Adam R.; Simmons, Graham

    2016-01-01

    In order to gain entry into cells, diverse viruses, including Ebola virus, SARS-coronavirus and the emerging MERS-coronavirus, depend on activation of their envelope glycoproteins by host cell proteases. The respective enzymes are thus excellent targets for antiviral intervention. In cell culture, activation of Ebola virus, as well as SARS- and MERS-coronavirus can be accomplished by the endosomal cysteine proteases, cathepsin L (CTSL) and cathepsin B (CTSB). In addition, SARS- and MERS-coronavirus can use serine proteases localized at the cell surface, for their activation. However, it is currently unclear which protease(s) facilitate viral spread in the infected host. We report here that the cysteine protease inhibitor K11777, ((2S)-N-[(1E,3S)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl]-2-{[(E)-4-methylpiperazine-1-carbonyl]amino}-3-phenylpropanamide) and closely-related vinylsulfones act as broad-spectrum antivirals by targeting cathepsin-mediated cell entry. K11777 is already in advanced stages of development for a number of parasitic diseases, such as Chagas disease, and has proven to be safe and effective in a range of animal models. K11777 inhibition of SARS-CoV and Ebola virus entry was observed in the sub-nanomolar range. In order to assess, whether cysteine or serine proteases promote viral spread in the host, we compared the antiviral activity of an optimized K11777-derivative with that of camostat, an inhibitor of TMPRSS2 and related serine proteases. Employing a pathogenic animal model of SARS-CoV infection, we demonstrated that viral spread and pathogenesis of SARS-CoV is driven by serine rather than cysteine proteases and can be effectively prevented by camostat. Camostat has been clinically used to treat chronic pancreatitis, and thus represents an exciting potential therapeutic for respiratory coronavirus infections. Our results indicate that camostat, or similar serine protease inhibitors, might be an effective option for treatment of SARS and

  19. Modified 5-fluorouracil: Uridine phosphorylase inhibitor

    NASA Astrophysics Data System (ADS)

    Lashkov, A. A.; Shchekotikhin, A. A.; Shtil, A. A.; Sotnichenko, S. E.; Mikhailov, A. M.

    2016-09-01

    5-Fluorouracil (5-FU) is a medication widely used in chemotherapy to treat various types of cancer. Being a substrate for the reverse reaction catalyzed by uridine phosphorylase (UPase), 5-FU serves as a promising prototype molecule (molecular scaffold) for the design of a selective UPase inhibitor that enhances the antitumor activity of 5-FU and exhibits intrinsic cytostatic effects on cancer cells. The chemical formula of the new compound, which binds to the uracil-binding site and, in the presence of a phosphate anion, to the phosphate-binding site of UPase, is proposed and investigated by molecular simulation methods.

  20. Inactivation of C-1 inhibitor by proteases: demonstration by a monoclonal antibody of a neodeterminant on inactivated, non-complexed C-1 inhibitor.

    PubMed

    Nuijens, J H; Huijbregts, C C; van Mierlo, G M; Hack, C E

    1987-07-01

    Monoclonal antibodies were raised against kallikrein-C-1 inhibitor and factor XIIa-C-1 inhibitor complexes. One of the monoclonal antibodies (KII) appeared to react predominantly with C-1 inhibitor complexes in an ELISA. However, the apparent binding of KII to C-1 inhibitor complexes was probably due to the presence of proteolytically inactivated C-1 inhibitor in the complex mixture used for the coating:KII did not bind either kallikrein-C-1 inhibitor or factor XIIa-C-1 inhibitor complexes generated in plasma by dextran sulphate. SDS-PAGE analysis of C-1 inhibitor incubated with proteases revealed that KII-reactive C-1 inhibitor has a lower molecular weight than native C-1 inhibitor. We propose that the determinant that reacts with KII is exposed after cleavage of C-1 inhibitor in its reactive site. The monoclonal antibody KII will enable us to study the inactivation of C-1 inhibitor in human inflammatory disease.