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Sample records for specific peptide inhibitors

  1. Chemical probing of the human sirtuin 5 active site reveals its substrate acyl specificity and peptide-based inhibitors.

    Science.gov (United States)

    Roessler, Claudia; Nowak, Theresa; Pannek, Martin; Gertz, Melanie; Nguyen, Giang T T; Scharfe, Michael; Born, Ilona; Sippl, Wolfgang; Steegborn, Clemens; Schutkowski, Mike

    2014-09-26

    Sirtuins are NAD(+)-dependent deacetylases acting as sensors in metabolic pathways and stress response. In mammals there are seven isoforms. The mitochondrial sirtuin 5 is a weak deacetylase but a very efficient demalonylase and desuccinylase; however, its substrate acyl specificity has not been systematically analyzed. Herein, we investigated a carbamoyl phosphate synthetase 1 derived peptide substrate and modified the lysine side chain systematically to determine the acyl specificity of Sirt5. From that point we designed six potent peptide-based inhibitors that interact with the NAD(+) binding pocket. To characterize the interaction details causing the different substrate and inhibition properties we report several X-ray crystal structures of Sirt5 complexed with these peptides. Our results reveal the Sirt5 acyl selectivity and its molecular basis and enable the design of inhibitors for Sirt5. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Investigating PKA-RII specificity using analogs of the PKA:AKAP peptide inhibitor STAD-2.

    Science.gov (United States)

    Bendzunas, N George; Dörfler, Sabrina; Autenrieth, Karolin; Bertinetti, Daniela; Machal, Erik M F; Kennedy, Eileen J; Herberg, Friedrich W

    2018-03-15

    Generation of the second messenger molecule cAMP mediates a variety of cellular responses which are essential for critical cellular processes. In response to elevated cAMP levels, cAMP dependent protein kinase (PKA) phosphorylates serine and threonine residues on a wide variety of target substrates. In order to enhance the precision and directionality of these signaling events, PKA is localized to discrete locations within the cell by A-kinase anchoring proteins (AKAPs). The interaction between PKA and AKAPs is mediated via an amphipathic α-helix derived from AKAPs which binds to a stable hydrophobic groove formed in the dimerization/docking (D/D) domain of PKA-R in an isoform-specific fashion. Although numerous AKAP disruptors have previously been identified that can inhibit either RI- or RII-selective AKAPs, no AKAP disruptors have been identified that have isoform specificity for RIα versus RIβ or RIIα versus RIIβ. As a strategy to identify isoform-specific AKAP inhibitors, a library of chemically stapled protein-protein interaction (PPI) disruptors was developed based on the RII-selective AKAP disruptor, STAD-2. An alanine was substituted at each position in the sequence, and from this library it was possible to delineate the importance of longer aliphatic residues in the formation of a region which complements the hydrophobic cleft formed by the D/D domain. Interestingly, lysine residues that were added to both terminal ends of the peptide sequence to facilitate water solubility appear to contribute to isoform specificity for RIIα over RIIβ while having only weak interaction with RI. This work supports current hypotheses on the mechanisms of AKAP binding and highlights the significance of particular residue positions that aid in distinguishing between the RII isoforms and may provide insight into future design of isoform-selective AKAP disruptors. Copyright © 2018 Elsevier Ltd. All rights reserved.

  3. Peptide aptamers: The versatile role of specific protein function inhibitors in plant biotechnology.

    Science.gov (United States)

    Colombo, Monica; Mizzotti, Chiara; Masiero, Simona; Kater, Martin M; Pesaresi, Paolo

    2015-11-01

    In recent years, peptide aptamers have emerged as novel molecular tools that have attracted the attention of researchers in various fields of basic and applied science, ranging from medicine to analytical chemistry. These artificial short peptides are able to specifically bind, track, and inhibit a given target molecule with high affinity, even molecules with poor immunogenicity or high toxicity, and represent a remarkable alternative to antibodies in many different applications. Their use is on the rise, driven mainly by the medical and pharmaceutical sector. Here we discuss the enormous potential of peptide aptamers in both basic and applied aspects of plant biotechnology and food safety. The different peptide aptamer selection methods available both in vivo and in vitro are introduced, and the most important possible applications in plant biotechnology are illustrated. In particular, we discuss the generation of broad-based virus resistance in crops, "reverse genetics" and aptasensors in bioassays for detecting contaminations in food and feed. Furthermore, we suggest an alternative to the transfer of peptide aptamers into plant cells via genetic transformation, based on the use of cell-penetrating peptides that overcome the limits imposed by both crop transformation and Genetically Modified Organism commercialization. © 2015 Institute of Botany, Chinese Academy of Sciences.

  4. Cyclic peptide inhibitors of lysine-specific demethylase 1 with improved potency identified by alanine scanning mutagenesis.

    Science.gov (United States)

    Kumarasinghe, Isuru R; Woster, Patrick M

    2018-03-25

    Lysine-specific demethylase 1 (LSD1) is a chromatin-remodeling enzyme that plays an important role in cancer. Over-expression of LSD1 decreases methylation at histone 3 lysine 4, and aberrantly silences tumor suppressor genes. Inhibitors of LSD1 have been designed as chemical probes and potential antitumor agents. We recently reported the cyclic peptide 9, which potently and reversibly inhibits LSD1 (IC 50 2.1 μM; K i 385 nM). Systematic alanine mutagenesis of 9 revealed residues that are critical for LSD1 inhibition, and these mutated peptides were evaluated as LSD1 inhibitors. Alanine substitution at positions 2, 3, 4, 6 and 11-17 preserved inhibition, while substitution of alanine at positions 8 and 9 resulted in complete loss of activity. Cyclic mutant peptides 11 and 16 produced the greatest LSD1 inhibition, and 11, 16, 27 and 28 increased global H3K4me2 in K562 cells. In addition, 16, 27 and 28 promoted significant increases in H3K4me2 levels at the promoter sites of the genes IGFBP2 and FEZ1. Data from these LSD1 inhibitors will aid in the design of peptidomimetics with improved stability and pharmacokinetics. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  5. A cyclic peptidic serine protease inhibitor

    DEFF Research Database (Denmark)

    Zhao, Baoyu; Xu, Peng; Jiang, Longguang

    2014-01-01

    Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase...... pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending......, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity...

  6. Peptide aldehyde inhibitors of bacterial peptide deformylases.

    Science.gov (United States)

    Durand, D J; Gordon Green, B; O'Connell, J F; Grant, S K

    1999-07-15

    Bacterial peptide deformylases (PDF, EC 3.5.1.27) are metalloenzymes that cleave the N-formyl groups from N-blocked methionine polypeptides. Peptide aldehydes containing a methional or norleucinal inhibited recombinant peptide deformylase from gram-negative Escherichia coli and gram-positive Bacillus subtilis. The most potent inhibitor was calpeptin, N-CBZ-Leu-norleucinal, which was a competitive inhibitor of the zinc-containing metalloenzymes, E. coli and B. subtilis PDF with Ki values of 26.0 and 55.6 microM, respectively. Cobalt-substituted E. coli and B. subtilis deformylases were also inhibited by these aldehydes with Ki values for calpeptin of 9.5 and 12.4 microM, respectively. Distinct spectral changes were observed upon binding of calpeptin to the Co(II)-deformylases, consistent with the noncovalent binding of the inhibitor rather than the formation of a covalent complex. In contrast, the chelator 1,10-phenanthroline caused the time-dependent inhibition of B. subtilis Co(II)-PDF activity with the loss of the active site metal. The fact that calpeptin was nearly equipotent against deformylases from both gram-negative and gram-positive bacterial sources lends further support to the idea that a single deformylase inhibitor might have broad-spectrum antibacterial activity. Copyright 1999 Academic Press.

  7. Effective intracellular inhibition of the cAMP-dependent protein kinase by microinjection of a modified form of the specific inhibitor peptide PKi in living fibroblasts.

    Science.gov (United States)

    Fernandez, A; Mery, J; Vandromme, M; Basset, M; Cavadore, J C; Lamb, N J

    1991-08-01

    In order to obtain a peptide retaining its biological activity following microinjection into living cells, we have modified a synthetic peptide [PKi(m)(6-24)], derived from the specific inhibitor protein of the cAMP-dependent protein kinase (A-kinase) in two ways: (1) substitution of the arginine at position 18 for a D-arginine; (2) blockade of the side chain on the C-terminal aspartic acid by a cyclohexyl ester group. In an in vitro assay, PKi(m) has retained a specific inhibitory activity against A-kinase as assessed against six other kinases, with similar efficiency to that of the unmodified PKi(5-24) peptide. Microinjection of PKi(m) into living fibroblasts reveals its capacity to prevent the changes in cell morphology and cytoskeleton induced by drugs which activate endogenous A-kinase, whereas the original PKi peptide failed to do so. This inhibition of A-kinase in vivo by PKi(m) lasts between 4 and 6 h after injection. In light of its effective half-life, this modified peptide opens a route for the use of biologically active peptides in vivo, an approach which has been hampered until now by the exceedingly short half-life of peptides inside living cells. By providing a direct means of inhibiting A-kinase activity for sufficiently long periods to observe effects on cellular functions in living cells, PKi(m) represents a powerful tool in studying the potential role of cAMP-dependent phosphorylation in vivo.

  8. Therapeutic peptides for cancer therapy. Part I - peptide inhibitors of signal transduction cascades.

    Science.gov (United States)

    Bidwell, Gene L; Raucher, Drazen

    2009-10-01

    Therapeutic peptides have great potential as anticancer agents owing to their ease of rational design and target specificity. However, their utility in vivo is limited by low stability and poor tumor penetration. The authors review the development of peptide inhibitors with potential for cancer therapy. Peptides that inhibit signal transduction cascades are discussed. The authors searched Medline for articles concerning the development of therapeutic peptides and their delivery. Given our current knowledge of protein sequences, structures and interaction interfaces, therapeutic peptides that inhibit interactions of interest are easily designed. These peptides are advantageous because they are highly specific for the interaction of interest, and they are much more easily developed than small molecule inhibitors of the same interactions. The main hurdle to application of peptides for cancer therapy is their poor pharmacokinetic and biodistribution parameters. Therefore, successful development of peptide delivery vectors could potentially make possible the use of this new and very promising class of anticancer agents.

  9. The binding mechanism of a peptidic cyclic serine protease inhibitor

    DEFF Research Database (Denmark)

    Jiang, Longguang; Svane, Anna Sigrid P.; Sørensen, Hans Peter

    2011-01-01

    Serine proteases are classical objects for studies of catalytic and inhibitory mechanisms as well as interesting as therapeutic targets. Since small-molecule serine protease inhibitors generally suffer from specificity problems, peptidic inhibitors, isolated from phage-displayed peptide libraries......, have attracted considerable attention. Here, we have investigated the mechanism of binding of peptidic inhibitors to serine protease targets. Our model is upain-1 (CSWRGLENHRMC), a disulfide-bond-constrained competitive inhibitor of human urokinase-type plasminogen activator with a noncanonical...... inhibitory mechanism and an unusually high specificity. Using a number of modified variants of upain-1, we characterised the upain-1-urokinase-type plasminogen activator complex using X-ray crystal structure analysis, determined a model of the peptide in solution by NMR spectroscopy, and analysed binding...

  10. Potent peptidic fusion inhibitors of influenza virus

    Energy Technology Data Exchange (ETDEWEB)

    Kadam, Rameshwar U.; Juraszek, Jarek; Brandenburg, Boerries; Buyck, Christophe; Schepens, Wim B. G.; Kesteleyn, Bart; Stoops, Bart; Vreeken, Rob J.; Vermond, Jan; Goutier, Wouter; Tang, Chan; Vogels, Ronald; Friesen, Robert H. E.; Goudsmit, Jaap; van Dongen, Maria J. P.; Wilson, Ian A.

    2017-09-28

    Influenza therapeutics with new targets and mechanisms of action are urgently needed to combat potential pandemics, emerging viruses, and constantly mutating strains in circulation. We report here on the design and structural characterization of potent peptidic inhibitors of influenza hemagglutinin. The peptide design was based on complementarity-determining region loops of human broadly neutralizing antibodies against the hemagglutinin (FI6v3 and CR9114). The optimized peptides exhibit nanomolar affinity and neutralization against influenza A group 1 viruses, including the 2009 H1N1 pandemic and avian H5N1 strains. The peptide inhibitors bind to the highly conserved stem epitope and block the low pH–induced conformational rearrangements associated with membrane fusion. These peptidic compounds and their advantageous biological properties should accelerate the development of new small molecule– and peptide-based therapeutics against influenza virus.

  11. Novel peptide-based protease inhibitors

    DEFF Research Database (Denmark)

    Roodbeen, Renée

    of novel peptide-based protease inhibitors, efforts were made towards improved methods for peptide synthesis. The coupling of Fmoc-amino acids onto N-methylated peptidyl resins was investigated. These couplings can be low yielding and the effect of the use of microwave heating combined with the coupling...

  12. Structural Principles in the Development of Cyclic Peptidic Enzyme Inhibitors

    Science.gov (United States)

    Xu, Peng; Andreasen, Peter A.; Huang, Mingdong

    2017-01-01

    This review summarizes our studies in the development of small cyclic peptides for specifically modulating enzyme activity. Serine proteases share highly similar active sites but perform diverse physiological and pathological functions. From a phage-display peptide library, we isolated two mono-cyclic peptides, upain-1 (CSWRGLENHRMC) and mupain-1 (CPAYSRYLDC), which inhibit the activity of human and murine urokinase-type plasminogen activators (huPA and muPA) with Ki values in the micromolar or sub-micromolar range, respectively. The following affinity maturations significantly enhanced the potencies of the two peptides, 10-fold and >250-fold for upain-1 and mupain-1, respectively. The most potent muPA inhibitor has a potency (Ki = 2 nM) and specificity comparable to mono-clonal antibodies. Furthermore, we also found an unusual feature of mupain-1 that its inhibitory potency can be enhanced by increasing the flexibility, which challenges the traditional viewpoint that higher rigidity leading to higher affinity. Moreover, by changing a few key residues, we converted mupain-1 from a uPA inhibitor to inhibitors of other serine proteases, including plasma kallikrein (PK) and coagulation factor XIa (fXIa). PK and fXIa inhibitors showed Ki values in the low nanomolar range and high specificity. Our studies demonstrate the versatility of small cyclic peptides to engineer inhibitory potency against serine proteases and to provide a new strategy for generating peptide inhibitors of serine proteases. PMID:29104489

  13. Proposal for novel curcumin derivatives as potent inhibitors against Alzheimer's disease: Ab initio molecular simulations on the specific interactions between amyloid-beta peptide and curcumin

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    Ota, Shintaro; Fujimori, Mitsuki; Ishimura, Hiromi; Shulga, Sergiy; Kurita, Noriyuki

    2017-10-01

    Accumulation of amyloid-β (Aβ) peptides in a brain is closely related with the pathogenesis of Alzheimer's disease. To suppress the production of Aβ peptides, we propose novel curcumin derivatives and investigate their binding properties with the amyloid precursor protein (APP), using protein-ligand docking as well as ab initio molecular simulations. Our proposed derivative (curcumin XIV) is found to have a large binding energy with APP and interacts strongly with the cleavage site Ala19 by secretase. It is thus expected that curcumin XIV can protect APP from the secretase attack and be a potent inhibitor against the production of Aβ peptides.

  14. In silico panning for a non-competitive peptide inhibitor

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    Ikebukuro Kazunori

    2007-01-01

    Full Text Available Abstract Background Peptide ligands have tremendous therapeutic potential as efficacious drugs. Currently, more than 40 peptides are available in the market for a drug. However, since costly and time-consuming synthesis procedures represent a problem for high-throughput screening, novel procedures to reduce the time and labor involved in screening peptide ligands are required. We propose the novel approach of 'in silico panning' which consists of a two-stage screening, involving affinity selection by docking simulation and evolution of the peptide ligand using genetic algorithms (GAs. In silico panning was successfully applied to the selection of peptide inhibitor for water-soluble quinoprotein glucose dehydrogenase (PQQGDH. Results The evolution of peptide ligands for a target enzyme was achieved by combining a docking simulation with evolution of the peptide ligand using genetic algorithms (GAs, which mimic Darwinian evolution. Designation of the target area as next to the substrate-binding site of the enzyme in the docking simulation enabled the selection of a non-competitive inhibitor. In all, four rounds of selection were carried out on the computer; the distribution of the docking energy decreased gradually for each generation and improvements in the docking energy were observed over the four rounds of selection. One of the top three selected peptides with the lowest docking energy, 'SERG' showed an inhibitory effect with Ki value of 20 μM. PQQGDH activity, in terms of the Vmax value, was 3-fold lower than that of the wild-type enzyme in the presence of this peptide. The mechanism of the SERG blockage of the enzyme was identified as non-competitive inhibition. We confirmed the specific binding of the peptide, and its equilibrium dissociation constant (KD value was calculated as 60 μM by surface plasmon resonance (SPR analysis. Conclusion We demonstrate an effective methodology of in silico panning for the selection of a non

  15. Novel nonpeptidic inhibitors of peptide deformylase.

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    Jayasekera, M M; Kendall, A; Shammas, R; Dermyer, M; Tomala, M; Shapiro, M A; Holler, T P

    2000-09-15

    A novel series of nonpeptidic compounds structurally related to the known anticholesteremic thyropropic acid were found to inhibit Escherichia coli peptide deformylase (PDF), with IC50 values in the low-micromolar range. Kinetic analysis of [4-(4-hydroxyphenoxy)-3,5-diiodophenyl]acetic acid reveals competitive inhibition, with a Ki value of 0.66 +/- 0.007 microM. A structure-activity relationship study demonstrates that the carboxylate is required for activity, while the distal phenolic function can be methylated without significant effect. Either decreasing the number of iodine atoms on the molecule to one or increasing the number of iodine atoms to four results in the loss of an order of magnitude in potency. These compounds are the first nonpeptidic inhibitors disclosed and represent a template from which better inhibitors might be designed.

  16. Bicyclic peptide inhibitor of urokinase-type plasminogen activator

    DEFF Research Database (Denmark)

    Roodbeen, Renée; Jensen, Berit Paaske; Jiang, Longguang

    2013-01-01

    The development of protease inhibitors for pharmacological intervention has taken a new turn with the use of peptide-based inhibitors. Here, we report the rational design of bicyclic peptide inhibitors of the serine protease urokinase-type plasminogen activator (uPA), based on the established...... investigated the solution structures of the bicyclic peptide by NMR spectroscopy to map possible conformations. An X-ray structure of the bicyclic-peptide-uPA complex confirmed an interaction similar to that for the previous upain-1/upain-2-uPA complexes. These physical studies of the peptide...

  17. Peptide-based proteasome inhibitors in anticancer drug design.

    Science.gov (United States)

    Micale, Nicola; Scarbaci, Kety; Troiano, Valeria; Ettari, Roberta; Grasso, Silvana; Zappalà, Maria

    2014-09-01

    The identification of the key role of the eukaryotic 26S proteasome in regulated intracellular proteolysis and its importance as a target in many pathological conditions wherein the proteasomal activity is defective (e.g., malignancies, autoimmune diseases, neurodegenerative diseases, etc.) prompted several research groups to the development of specific inhibitors of this multicatalytic complex with the aim of obtaining valid drug candidates. In regard to the anticancer therapy, the peptide boronate bortezomib (Velcade®) represents the first molecule approved by FDA for the treatment of multiple myeloma in 2003 and mantle cell lymphoma in 2006. Since then, a plethora of molecules targeting the proteasome have been identified as potential anticancer agents and a few of them reached clinical trials or are already in the market (i.e., carfilzomib; Kyprolis®). In most cases, the design of new proteasome inhibitors (PIs) takes into account a proven peptide or pseudopeptide motif as a base structure and places other chemical entities throughout the peptide skeleton in such a way to create an efficacious network of interactions within the catalytic sites. The purpose of this review is to provide an in-depth look at the current state of the research in the field of peptide-based PIs, specifically those ones that might find an application as anticancer agents. © 2014 Wiley Periodicals, Inc.

  18. Sifuvirtide, a potent HIV fusion inhibitor peptide

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  19. Peptide inhibitors of botulinum neurotoxin by mRNA display

    International Nuclear Information System (INIS)

    Yiadom, Kwabena P.A.B.; Muhie, Seid; Yang, David C.H.

    2005-01-01

    Botulinum neurotoxins (BoNTs) are extremely toxic. The metalloproteases associated with the toxins cleave proteins essential for neurotransmitter secretion. Inhibitors of the metalloprotease are currently sought to control the toxicity of BoNTs. Toward that goal, we produced a synthetic cDNA for the expression and purification of the metalloprotease of BoNT/A in Escherichia coli as a biotin-ubiquitin fusion protein, and constructed a combinatorial peptide library to screen for BoNT/A light chain inhibitors using mRNA display. A protease assay was developed using immobilized intact SNAP-25 as the substrate. The new peptide inhibitors showed a 10-fold increase in affinity to BoNT/A light chain than the parent peptide. Interestingly, the sequences of the new peptide inhibitors showed abundant hydrophobic residues but few hydrophilic residues. The results suggest that mRNA display may provide a general approach in developing peptide inhibitors of BoNTs

  20. Synthetic peptide inhibitors of DNA replication in Staphylococcus aureus

    DEFF Research Database (Denmark)

    Løbner-Olesen, Anders; Kjelstrup, Susanne

    F counterselection was developed to directly select for compounds able to disrupt selected interactions. We have subsequently constructed a cyclic peptide library for intracellular synthesis of cyclic peptides using known technology. Several cyclic peptides were able to interfere with oligomerization of Dna......N (), DnaB and DnaX (). Three peptides identified as inhibitors of DnaN have been purified. Two of these peptides inhibited growth as well as DNA replication in S. aureus. The minimal inhibitory concentration (MIC) of the peptides was approximately 50 g/ml. Overexpression of DnaN reduced the inhibitory...

  1. De novo peptide design and experimental validation of histone methyltransferase inhibitors.

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    James Smadbeck

    Full Text Available Histones are small proteins critical to the efficient packaging of DNA in the nucleus. DNA–protein complexes, known as nucleosomes, are formed when the DNA winds itself around the surface of the histones. The methylation of histone residues by enhancer of zeste homolog 2 (EZH2 maintains gene repression over successive cell generations. Overexpression of EZH2 can silence important tumor suppressor genes leading to increased invasiveness of many types of cancers. This makes the inhibition of EZH2 an important target in the development of cancer therapeutics. We employed a three-stage computational de novo peptide design method to design inhibitory peptides of EZH2. The method consists of a sequence selection stage and two validation stages for fold specificity and approximate binding affinity. The sequence selection stage consists of an integer linear optimization model that was solved to produce a rank-ordered list of amino acid sequences with increased stability in the bound peptide-EZH2 structure. These sequences were validated through the calculation of the fold specificity and approximate binding affinity of the designed peptides. Here we report the discovery of novel EZH2 inhibitory peptides using the de novo peptide design method. The computationally discovered peptides were experimentally validated in vitro using dose titrations and mechanism of action enzymatic assays. The peptide with the highest in vitro response, SQ037, was validated in nucleo using quantitative mass spectrometry-based proteomics. This peptide had an IC50 of 13.5 mM, demonstrated greater potency as an inhibitor when compared to the native and K27A mutant control peptides, and demonstrated competitive inhibition versus the peptide substrate. Additionally, this peptide demonstrated high specificity to the EZH2 target in comparison to other histone methyltransferases. The validated peptides are the first computationally designed peptides that directly inhibit EZH2

  2. De novo peptide design and experimental validation of histone methyltransferase inhibitors.

    Directory of Open Access Journals (Sweden)

    James Smadbeck

    Full Text Available Histones are small proteins critical to the efficient packaging of DNA in the nucleus. DNA-protein complexes, known as nucleosomes, are formed when the DNA winds itself around the surface of the histones. The methylation of histone residues by enhancer of zeste homolog 2 (EZH2 maintains gene repression over successive cell generations. Overexpression of EZH2 can silence important tumor suppressor genes leading to increased invasiveness of many types of cancers. This makes the inhibition of EZH2 an important target in the development of cancer therapeutics. We employed a three-stage computational de novo peptide design method to design inhibitory peptides of EZH2. The method consists of a sequence selection stage and two validation stages for fold specificity and approximate binding affinity. The sequence selection stage consists of an integer linear optimization model that was solved to produce a rank-ordered list of amino acid sequences with increased stability in the bound peptide-EZH2 structure. These sequences were validated through the calculation of the fold specificity and approximate binding affinity of the designed peptides. Here we report the discovery of novel EZH2 inhibitory peptides using the de novo peptide design method. The computationally discovered peptides were experimentally validated in vitro using dose titrations and mechanism of action enzymatic assays. The peptide with the highest in vitro response, SQ037, was validated in nucleo using quantitative mass spectrometry-based proteomics. This peptide had an IC50 of 13.5 [Formula: see text]M, demonstrated greater potency as an inhibitor when compared to the native and K27A mutant control peptides, and demonstrated competitive inhibition versus the peptide substrate. Additionally, this peptide demonstrated high specificity to the EZH2 target in comparison to other histone methyltransferases. The validated peptides are the first computationally designed peptides that directly

  3. Biological Applications of Designed Hairpin Peptides: As Antimicrobials and as Inhibitors of Amyloidogenesis

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    Sivanesam, Kalkena

    More than 40 diseases have been associated with the misfolding of peptides (or proteins) that form fibrils with a very specific morphology. These peptides classified as amyloidogenic peptides have been implicated in the development of Alzheimer's Disease, Parkinson's Disease, Type II Diabetes, Hungtinton's Disease etc. To date, these diseases have no cure, only therapies that can ameliorate the symptoms to a degree. Inhibition of the amyloidogenesis of these peptides has been proposed as a possible treatment option. While small molecules have been heavily tested as inhibitors of amyloidogenesis, peptides have emerged as potential inhibitors. In this work, the ability of a set of designed hairpin peptides to inhibit the amyloidogenesis of two different systems, alpha-synuclein (implicated in Parkinson's Disease) and human amylin (implicated in Type II Diabetes) is tested. Using circular dichroism and thioflavin T fluorescence, the ability of these peptides to inhibit amyloidogenesis is tested. The binding loci of these inhibitors to alpha-synuclein are also explored. The use of peptides as antimicrobials on the other hand is not a novel concept. However, most antimicrobial peptides, both natural and designed, rely heavily on covalent stabilizations in order to maintain secondary structure. In this study, non-covalent stabilizations are applied to a couple of natural as well as designed antimicrobials in order to study the effects of secondary structure stabilization on biological activity.

  4. Selection of specific inhibitor peptides in enzyme-linked immunosorbent assay (ELISA) of cardiac troponin I using immuno-dominant epitopes as competitor.

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    Rezaee, Majid Asiabanha; Rasaee, Mohammad Javad; Mohammadnejad, Javad

    2017-01-01

    Human cardiac troponin I (cTni) is the gold marker for early diagnosis of myocardial infarction. In this regard, four immune-dominant epitopes of cTni were predicted and their 3D structures were determined. Thereafter, the competitive performance of the peptides was monitored with the developed polyclonal antibody-based indirect competitive ELISA; a half-maximal inhibitory concentration (IC50) of 0.49 (µg/mL) and detection limit of 0.037 (µg/mL) were achieved for recombinant cTni. The competitive ELISA determined sensitivity levels of 0.306, 0.141, 0.960, and 0.155 (µg/mL), respectively, for each peptide as competitor. We indicated that two of the selected epitopes have significant sensitivity scales and inhibition ability.

  5. Novel Inhibitor Cystine Knot Peptides from Momordica charantia

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    Clark, Richard J.; Tang, Jun; Zeng, Guang-Zhi; Franco, Octavio L.; Cantacessi, Cinzia; Craik, David J.; Daly, Norelle L.; Tan, Ning-Hua

    2013-01-01

    Two new peptides, MCh-1 and MCh-2, along with three known trypsin inhibitors (MCTI-I, MCTI-II and MCTI-III), were isolated from the seeds of the tropical vine Momordica charantia. The sequences of the peptides were determined using mass spectrometry and NMR spectroscopy. Using a strategy involving partial reduction and stepwise alkylation of the peptides, followed by enzymatic digestion and tandem mass spectrometry sequencing, the disulfide connectivity of MCh-1 was elucidated to be CysI-CysIV, CysII-CysV and CysIII-CysVI. The three-dimensional structures of MCh-1 and MCh-2 were determined using NMR spectroscopy and found to contain the inhibitor cystine knot (ICK) motif. The sequences of the novel peptides differ significantly from peptides previously isolated from this plant. Therefore, this study expands the known peptide diversity in M. charantia and the range of sequences that can be accommodated by the ICK motif. Furthermore, we show that a stable two-disulfide intermediate is involved in the oxidative folding of MCh-1. This disulfide intermediate is structurally homologous to the proposed ancestral fold of ICK peptides, and provides a possible pathway for the evolution of this structural motif, which is highly prevalent in nature. PMID:24116036

  6. Novel inhibitor cystine knot peptides from Momordica charantia.

    Directory of Open Access Journals (Sweden)

    Wen-Jun He

    Full Text Available Two new peptides, MCh-1 and MCh-2, along with three known trypsin inhibitors (MCTI-I, MCTI-II and MCTI-III, were isolated from the seeds of the tropical vine Momordica charantia. The sequences of the peptides were determined using mass spectrometry and NMR spectroscopy. Using a strategy involving partial reduction and stepwise alkylation of the peptides, followed by enzymatic digestion and tandem mass spectrometry sequencing, the disulfide connectivity of MCh-1 was elucidated to be CysI-CysIV, CysII-CysV and CysIII-CysVI. The three-dimensional structures of MCh-1 and MCh-2 were determined using NMR spectroscopy and found to contain the inhibitor cystine knot (ICK motif. The sequences of the novel peptides differ significantly from peptides previously isolated from this plant. Therefore, this study expands the known peptide diversity in M. charantia and the range of sequences that can be accommodated by the ICK motif. Furthermore, we show that a stable two-disulfide intermediate is involved in the oxidative folding of MCh-1. This disulfide intermediate is structurally homologous to the proposed ancestral fold of ICK peptides, and provides a possible pathway for the evolution of this structural motif, which is highly prevalent in nature.

  7. Multimerized CHR-derived peptides as HIV-1 fusion inhibitors.

    Science.gov (United States)

    Nomura, Wataru; Hashimoto, Chie; Suzuki, Takaharu; Ohashi, Nami; Fujino, Masayuki; Murakami, Tsutomu; Yamamoto, Naoki; Tamamura, Hirokazu

    2013-08-01

    To date, several HIV-1 fusion inhibitors based on the carboxy-terminal leucine/isoleucine heptad repeat (CHR) region of an HIV-1 envelope protein gp41 have been discovered. We have shown that a synthetic peptide mimetic of a trimer form of the CHR-derived peptide C34 has potent inhibitory activity against the HIV-1 fusion mechanism, compared to a monomer C34 peptide. The present study revealed that a dimeric form of C34 is evidently structurally critical for fusion inhibitors, and that the activity of multimerized CHR-derived peptides in fusion inhibition is affected by the properties of the unit peptides C34, SC34EK, and T20. The fluorescence-based study suggested that the N36-interactive sites of the C34 trimer, including hydrophobic residues, are exposed outside the trimer and that trimerization of C34 caused a remarkable increase in fusion inhibitory activity. The present results could be useful in the design of fusion inhibitors against viral infections which proceed via membrane fusion with host cells. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

  8. Peptide binding specificity of the chaperone calreticulin

    DEFF Research Database (Denmark)

    Sandhu, N.; Duus, K.; Jorgensen, C.S.

    2007-01-01

    Calreticulin is a molecular chaperone with specificity for polypeptides and N-linked monoglucosylated glycans. In order to determine the specificity of polypeptide binding, the interaction of calreticulin with polypeptides was investigated using synthetic peptides of different length and composit......Calreticulin is a molecular chaperone with specificity for polypeptides and N-linked monoglucosylated glycans. In order to determine the specificity of polypeptide binding, the interaction of calreticulin with polypeptides was investigated using synthetic peptides of different length...... than 5 amino acids showed binding and a clear correlation with hydrophobicity was demonstrated for oligomers of different hydrophobic amino acids. Insertion of hydrophilic amino acids in a hydrophobic sequence diminished or abolished binding. In conclusion our results show that calreticulin has...

  9. Computational Studies of Difference in Binding Modes of Peptide and Non-Peptide Inhibitors to MDM2/MDMX Based on Molecular Dynamics Simulations

    Directory of Open Access Journals (Sweden)

    Yuxin Zhang

    2012-02-01

    Full Text Available Inhibition of p53-MDM2/MDMX interaction is considered to be a promising strategy for anticancer drug design to activate wild-type p53 in tumors. We carry out molecular dynamics (MD simulations to study the binding mechanisms of peptide and non-peptide inhibitors to MDM2/MDMX. The rank of binding free energies calculated by molecular mechanics generalized Born surface area (MM-GBSA method agrees with one of the experimental values. The results suggest that van der Waals energy drives two kinds of inhibitors to MDM2/MDMX. We also find that the peptide inhibitors can produce more interaction contacts with MDM2/MDMX than the non-peptide inhibitors. Binding mode predictions based on the inhibitor-residue interactions show that the π–π, CH–π and CH–CH interactions dominated by shape complimentarity, govern the binding of the inhibitors in the hydrophobic cleft of MDM2/MDMX. Our studies confirm the residue Tyr99 in MDMX can generate a steric clash with the inhibitors due to energy and structure. This finding may theoretically provide help to develop potent dual-specific or MDMX inhibitors.

  10. Lipid raft-like liposomes used for targeted delivery of a chimeric entry-inhibitor peptide with anti-HIV-1 activity.

    Science.gov (United States)

    Gómara, María José; Pérez-Pomeda, Ignacio; Gatell, José María; Sánchez-Merino, Victor; Yuste, Eloisa; Haro, Isabel

    2017-02-01

    The work reports the design and synthesis of a chimeric peptide that is composed of the peptide sequences of two entry inhibitors which target different sites of HIV-1 gp41. The chimeric peptide offers the advantage of targeting two gp41 regions simultaneously: the fusion peptide and the loop both of which are membrane active and participate in the membrane fusion process. We therefore use lipid raft-like liposomes as a tool to specifically direct the chimeric inhibitor peptide to the membrane domains where the HIV-1 envelope protein is located. Moreover, the liposomes that mimic the viral membrane composition protect the chimeric peptide against proteolytic digestion thereby increasing the stability of the peptide. The described liposome preparations are suitable nanosystems for managing hydrophobic entry-inhibitor peptides as putative therapeutics. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Accessing Specific Peptide Recognition by Combinatorial Chemistry

    DEFF Research Database (Denmark)

    Li, Ming

    Molecular recognition is at the basis of all processes for life, and plays a central role in many biological processes, such as protein folding, the structural organization of cells and organelles, signal transduction, and the immune response. Hence, my PhD project is entitled “Accessing Specific...... Peptide Recognition by Combinatorial Chemistry”. Molecular recognition is a specific interaction between two or more molecules through noncovalent bonding, such as hydrogen bonding, metal coordination, van der Waals forces, π−π, hydrophobic, or electrostatic interactions. The association involves kinetic....... Combinatorial chemistry was invented in 1980s based on observation of functional aspects of the adaptive immune system. It was employed for drug development and optimization in conjunction with high-throughput synthesis and screening. (chapter 2) Combinatorial chemistry is able to rapidly produce many thousands...

  12. Plasmin substrate binding site cooperativity guides the design of potent peptide aldehyde inhibitors.

    Science.gov (United States)

    Swedberg, Joakim E; Harris, Jonathan M

    2011-10-04

    Perioperative bleeding is a cause of major blood loss and is associated with increased rates of postoperative morbidity and mortality. To combat this, antifibrinolytic inhibitors of the serine protease plasmin are commonly used to reduce bleeding during surgery. The most effective and previously widely used of these is the broad range serine protease inhibitor aprotinin. However, adverse clinical outcomes have led to use of alternative serine lysine analogues to inhibit plasmin. These compounds suffer from low selectivity and binding affinity. Consequently, a concerted effort to discover potent and selective plasmin inhibitors has developed. This study used a noncombinatorial peptide library to define plasmin's extended substrate specificity and guide the design of potent transition state analogue inhibitors. The various substrate binding sites of plasmin were found to exhibit a higher degree of cooperativity than had previously been appreciated. Peptide sequences capitalizing on these features produced high-affinity inhibitors of plasmin. The most potent of these, Lys-Met(sulfone)-Tyr-Arg-H [KM(O(2))YR-H], inhibited plasmin with a K(i) of 3.1 nM while maintaining 25-fold selectivity over plasma kallikrein. Furthermore, 125 nM (0.16 μg/mL) KM(O(2))YR-H attenuated fibrinolysis in vitro with an efficacy similar to that of 15 nM (0.20 μg/mL) aprotinin. To date, this is the most potent peptide inhibitor of plasmin that exhibits selectivity against plasma kallikrein, making this compound an attractive candidate for further therapeutic development.

  13. Production of peptide antisera specific for mouse and rat proinsulin C-peptide 2

    DEFF Research Database (Denmark)

    Blume, N; Madsen, O D; Kofod, Hans

    1990-01-01

    for antibody binding to the immunizing antigen. Antisera to C-peptide 2, stained islet beta-cells on mouse and rat, but not monkey pancreas sections in immunocytochemical analysis. Preabsorption to the synthetic C-peptide 2, but not the synthetic mouse and rat C-peptide 1 abolished staining. In conclusion we......Mice and rats have two functional non-allelic insulin genes. By using a synthetic peptide representing a common sequence in mouse and rat C-peptide 2 as antigen, we have produced rabbit antisera specific for an epitope which is not present in mouse or rat C-peptide 1. Long-term immunization did...... not seem to increase the end point titre as tested in direct ELISA. The specificity of the antiserum was determined by competitive ELISA and histochemistry on pancreas sections. Only the synthetic C-peptide 2, but not the homologous synthetic C-peptide 1 from mouse and rat competed efficiently in ELISA...

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

    Directory of Open Access Journals (Sweden)

    Matthew P Wood

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

  15. Drug forecast - the peptide deformylase inhibitors as antibacterial agents.

    Science.gov (United States)

    Guay, David R P

    2007-08-01

    The relatively rapid development of microbial resistance after the entry of every new antimicrobial into the marketplace necessitates a constant supply of new agents to maintain effective pharmacotherapy. Despite extensive efforts to identify novel lead compounds from molecular targets, only the peptide deformylase inhibitors (PDIs) have shown any real promise, with some advancing to phase I human trials. Bacterial peptide deformylase, which catalyzes the removal of the N-formyl group from N-terminal methionine following translation, is essential for bacterial protein synthesis, growth, and survival. The majority of PDIs are pseudopeptide hydroxamic acids and two of these (IV BB-83698 and oral NVP LBM-415) entered phase I human trials. However, agents to the present have suffered from major potential liabilities. Their in vitro activity has been limited to gram-positive aerobes and some anaerobes and has been quite modest against the majority of such species (MIC(90) values ranging from 1-8 mg/L). They have exerted bacteriostatic, not bacteriocidal, activity, thus reducing their potential usefulness in the management of serious infections in the immunocompromised. The relative ease with which microorganisms have been able to develop resistance and the multiple available mechanisms of resistance (mutations in fmt, defB, folD genes; AcrAB/TolC efflux pump; overexpression of peptide deformylase) are worrisome. These could portend a short timespan of efficacy after marketing. Despite these current liabilities, further pursuit of more potent and broader spectrum PDIs which are less susceptible to bacterial mechanisms of resistance is still warranted.

  16. Peptide Based Radiopharmaceuticals: Specific Construct Approach

    Energy Technology Data Exchange (ETDEWEB)

    Som, P; Rhodes, B A; Sharma, S S

    1997-10-21

    The objective of this project was to develop receptor based peptides for diagnostic imaging and therapy. A series of peptides related to cell adhesion molecules (CAM) and immune regulation were designed for radiolabeling with 99mTc and evaluated in animal models as potential diagnostic imaging agents for various disease conditions such as thrombus (clot), acute kidney failure, and inflection/inflammation imaging. The peptides for this project were designed by the industrial partner, Palatin Technologies, (formerly Rhomed, Inc.) using various peptide design approaches including a newly developed rational computer assisted drug design (CADD) approach termed MIDAS (Metal ion Induced Distinctive Array of Structures). In this approach, the biological function domain and the 99mTc complexing domain are fused together so that structurally these domains are indistinguishable. This approach allows construction of conformationally rigid metallo-peptide molecules (similar to cyclic peptides) that are metabolically stable in-vivo. All the newly designed peptides were screened in various in vitro receptor binding and functional assays to identify a lead compound. The lead compounds were formulated in a one-step 99mTc labeling kit form which were studied by BNL for detailed in-vivo imaging using various animals models of human disease. Two main peptides usingMIDAS approach evolved and were investigated: RGD peptide for acute renal failure and an immunomodulatory peptide derived from tuftsin (RMT-1) for infection/inflammation imaging. Various RGD based metallopeptides were designed, synthesized and assayed for their efficacy in inhibiting ADP-induced human platelet aggregation. Most of these peptides displayed biological activity in the 1-100 µM range. Based on previous work by others, RGD-I and RGD-II were evaluated in animal models of acute renal failure. These earlier studies showed that after acute ischemic injury the renal cortex displays

  17. A novel affinity purification method to isolate peptide specific antibodies

    DEFF Research Database (Denmark)

    Karlsen, Alan E; Lernmark, A; Kofod, Hans

    1990-01-01

    Site-specific, high affinity polyclonal antisera are effectively and successfully produced by immunizing rabbits with synthetic peptides. The use of these antisera in subsequent immune analysis is often limited because of non-specific binding. We describe a new and simple method to effectively...... affinity-purify anti-peptide antibodies. To test our system, rabbits were immunized with model peptides representing sequences of the putative rabbit growth hormone receptor and several HLA-DQ beta-chain molecules. Polystyrene plastic beads were coated with peptides. Immune serum was incubated...... with the beads and after a wash step the bound antibodies were eluted in 1 M acetic acid. The eluted material was composed predominantly of intact immunoglobulin as evidenced by the presence of heavy and light chain bands in SDS-PAGE. The eluted antibodies were peptide specific in ELISA and bound only to intact...

  18. Potent and Selective Peptidyl Boronic Acid Inhibitors of the Serine Protease Prostate-Specific Antigen

    Science.gov (United States)

    LeBeau, Aaron M.; Singh, Pratap; Isaacs, John T.; Denmeade, Samuel R.

    2012-01-01

    SUMMARY Prostate cancer cells produce high (microgram to milligram/milliliter) levels of the serine protease Prostate-Specific Antigen (PSA). PSA is enzymatically active in the extracellular fluid surrounding prostate cancers but is found at 1,000- to 10,000-fold lower concentrations in the circulation, where it is inactivated due to binding to abundant serum protease inhibitors. The exclusive presence of high levels of active PSA within prostate cancer sites makes PSA an attractive candidate for targeted imaging and therapeutics. A synthetic approach based on a peptide substrate identified first peptide aldehyde and then boronic acid inhibitors of PSA. The best of these had the sequence Cbz-Ser-Ser-Lys-Leu-(boro)Leu, with a Ki for PSA of 65 nM. The inhibitor had a 60-fold higher Ki for chymotrypsin. A validated model of PSA’s catalytic site confirmed the critical interactions between the inhibitor and residues within the PSA enzyme. PMID:18635003

  19. Peptide-Based Selective Inhibitors of Matrix Metalloproteinase-Mediated Activities

    Directory of Open Access Journals (Sweden)

    Margaret W. Ndinguri

    2012-11-01

    Full Text Available The matrix metalloproteinases (MMPs exhibit a broad array of activities, some catalytic and some non-catalytic in nature. An overall lack of selectivity has rendered small molecule, active site targeted MMP inhibitors problematic in execution. Inhibitors that favor few or individual members of the MMP family often take advantage of interactions outside the enzyme active site. We presently focus on peptide-based MMP inhibitors and probes that do not incorporate conventional Zn2+ binding groups. In some cases, these inhibitors and probes function by binding only secondary binding sites (exosites, while others bind both exosites and the active site. A myriad of MMP mediated-activities beyond selective catalysis can be inhibited by peptides, particularly cell adhesion, proliferation, motility, and invasion. Selective MMP binding peptides comprise highly customizable, unique imaging agents. Areas of needed improvement for MMP targeting peptides include binding affinity and stability.

  20. Identification of novel inhibitors of Pseudomonas aeruginosa MurC enzyme derived from phage-displayed peptide libraries.

    Science.gov (United States)

    El Zoeiby, Ahmed; Sanschagrin, François; Darveau, André; Brisson, Jean-Robert; Levesque, Roger C

    2003-03-01

    The machinery of peptidoglycan biosynthesis is an ideal site at which to look for novel antimicrobial targets. Phage display was used to develop novel peptide inhibitors for MurC, an essential enzyme involved in the early steps of biosynthesis of peptidoglycan monomer. We cloned and overexpressed the murA, -B and -C genes from Pseudomonas aeruginosa in the pET expression vector, adding a His-tag to their C termini. The three proteins were overproduced in Escherichia coli and purified to homogeneity in milligram quantities. MurA and -B were combinatorially used to synthesize the MurC substrate UDP-N-acetylmuramate, the identity of which was confirmed by mass spectrometry and nuclear magnetic resonance analysis. Two phage-display libraries were screened against MurC in order to identify peptide ligands to the enzyme. Three rounds of biopanning were carried out, successively increasing elution specificity from round 1 to 3. The third round was accomplished with both non-specific elution and competitive elution with each of the three MurC substrates, UDP-N-acetylmuramic acid (UNAM), ATP and L-alanine. The DNA of 10 phage, selected randomly from each group, was extracted and sequenced, and consensus peptide sequences were elucidated. Peptides were synthesized and tested for inhibition of the MurC-catalysed reaction, and two peptides were shown to be inhibitors of MurC activity with IC(50)s of 1.5 and 0.9 mM, respectively. The powerful selection technique of phage display allowed us to identify two peptide inhibitors of the essential bacterial enzyme MurC. The peptide sequences represent the basis for the synthesis of inhibitory peptidomimetic molecules.

  1. [Ala12]MCD peptide: a lead peptide to inhibitors of immunoglobulin E binding to mast cell receptors.

    Science.gov (United States)

    Buku, A; Condie, B A; Price, J A; Mezei, M

    2005-09-01

    An effort was made to discover mast cell degranulating (MCD) peptide analogs that bind with high affinity to mast cell receptors without triggering secretion of histamine or other mediators of the allergic reaction initiated by immunoglobulin E (IgE) after mast cell activation. Such compounds could serve as inhibitors of IgE binding to mast cell receptors. An alanine scan of MCD peptide reported previously showed that the analog [Ala12]MCD was 120-fold less potent in histamine-releasing activity and fivefold more potent in binding affinity to mast cell receptors than the parent MCD peptide. Because this analog showed marginal intrinsic activity and good binding affinity it was subsequently tested in the present study as an IgE inhibitor. In contrast to MCD peptide, [Ala12]MCD showed a 50% inhibition of IgE binding to the Fc epsilon RI alpha mast cell receptor by using rat basophilic leukemia (RBL-2H3) mast cells and fluorescence polarization. Furthermore, in a beta-hexosaminidase secretory assay, the peptide also showed a 50% inhibition of the secretion of this enzyme caused by IgE. An attempt was made to relate structural changes and biologic differences between the [Ala12]MCD analog and the parent MCD peptide. The present results show that [Ala12]MCD may provide a base for designing agents to prevent IgE/Fc epsilon RI alpha interactions and, consequently, allergic conditions.

  2. Snake Venom Metalloproteinases and Their Peptide Inhibitors from Myanmar Russell’s Viper Venom

    Directory of Open Access Journals (Sweden)

    Khin Than Yee

    2016-12-01

    Full Text Available Russell’s viper bites are potentially fatal from severe bleeding, renal failure and capillary leakage. Snake venom metalloproteinases (SVMPs are attributed to these effects. In addition to specific antivenom therapy, endogenous inhibitors from snakes are of interest in studies of new treatment modalities for neutralization of the effect of toxins. Two major snake venom metalloproteinases (SVMPs: RVV-X and Daborhagin were purified from Myanmar Russell’s viper venom using a new purification strategy. Using the Next Generation Sequencing (NGS approach to explore the Myanmar RV venom gland transcriptome, mRNAs of novel tripeptide SVMP inhibitors (SVMPIs were discovered. Two novel endogenous tripeptides, pERW and pEKW were identified and isolated from the crude venom. Both purified SVMPs showed caseinolytic activity. Additionally, RVV-X displayed specific proteolytic activity towards gelatin and Daborhagin showed potent fibrinogenolytic activity. These activities were inhibited by metal chelators. Notably, the synthetic peptide inhibitors, pERW and pEKW, completely inhibit the gelatinolytic and fibrinogenolytic activities of respective SVMPs at 5 mM concentration. These complete inhibitory effects suggest that these tripeptides deserve further study for development of a therapeutic candidate for Russell’s viper envenomation.

  3. Potentiation of peptide receptor radionuclide therapy by the PARP inhibitor olaparib

    NARCIS (Netherlands)

    J. Nonnekens (Julie); M. van Kranenburg (Melissa); C.E.M.T. Beerens (Cecile); M. Suker (Mustafa); M. Doukas (Michael); C.H.J. van Eijck (Casper); M. de Jong (Marcel); D.C. van Gent (Dik)

    2016-01-01

    textabstractMetastases expressing tumor-specific receptors can be targeted and treated by binding of radiolabeled peptides (peptide receptor radionuclide therapy or PRRT). For example, patients with metastasized somatostatin receptor-positive neuroendocrine tumors (NETs) can be treated with

  4. Improving the representation of peptide-like inhibitor and antibiotic molecules in the Protein Data Bank.

    Science.gov (United States)

    Dutta, Shuchismita; Dimitropoulos, Dimitris; Feng, Zukang; Persikova, Irina; Sen, Sanchayita; Shao, Chenghua; Westbrook, John; Young, Jasmine; Zhuravleva, Marina A; Kleywegt, Gerard J; Berman, Helen M

    2014-06-01

    With the accumulation of a large number and variety of molecules in the Protein Data Bank (PDB) comes the need on occasion to review and improve their representation. The Worldwide PDB (wwPDB) partners have periodically updated various aspects of structural data representation to improve the integrity and consistency of the archive. The remediation effort described here was focused on improving the representation of peptide-like inhibitor and antibiotic molecules so that they can be easily identified and analyzed. Peptide-like inhibitors or antibiotics were identified in over 1000 PDB entries, systematically reviewed and represented either as peptides with polymer sequence or as single components. For the majority of the single-component molecules, their peptide-like composition was captured in a new representation, called the subcomponent sequence. A novel concept called "group" was developed for representing complex peptide-like antibiotics and inhibitors that are composed of multiple polymer and nonpolymer components. In addition, a reference dictionary was developed with detailed information about these peptide-like molecules to aid in their annotation, identification and analysis. Based on the experience gained in this remediation, guidelines, procedures, and tools were developed to annotate new depositions containing peptide-like inhibitors and antibiotics accurately and consistently. © 2013 The Authors Biopolymers Published by Wiley Periodicals, Inc.

  5. Elapid Snake Venom Analyses Show the Specificity of the Peptide Composition at the Level of Genera Naja and Notechis

    Directory of Open Access Journals (Sweden)

    Aisha Munawar

    2014-02-01

    Full Text Available Elapid snake venom is a highly valuable, but till now mainly unexplored, source of pharmacologically important peptides. We analyzed the peptide fractions with molecular masses up to 10 kDa of two elapid snake venoms—that of the African cobra, N. m. mossambica (genus Naja, and the Peninsula tiger snake, N. scutatus, from Kangaroo Island (genus Notechis. A combination of chromatographic methods was used to isolate the peptides, which were characterized by combining complimentary mass spectrometric techniques. Comparative analysis of the peptide compositions of two venoms showed specificity at the genus level. Three-finger (3-F cytotoxins, bradykinin-potentiating peptides (BPPs and a bradykinin inhibitor were isolated from the Naja venom. 3-F neurotoxins, Kunitz/basic pancreatic trypsin inhibitor (BPTI-type inhibitors and a natriuretic peptide were identified in the N. venom. The inhibiting activity of the peptides was confirmed in vitro with a selected array of proteases. Cytotoxin 1 (P01467 from the Naja venom might be involved in the disturbance of cellular processes by inhibiting the cell 20S-proteasome. A high degree of similarity between BPPs from elapid and viperid snake venoms was observed, suggesting that these molecules play a key role in snake venoms and also indicating that these peptides were recruited into the snake venom prior to the evolutionary divergence of the snakes.

  6. Turning a Substrate Peptide into a Potent Inhibitor for the Histone Methyltransferase SETD8

    Energy Technology Data Exchange (ETDEWEB)

    Judge, Russell A.; Zhu, Haizhong; Upadhyay, Anup K.; Bodelle, Pierre M.; Hutchins, Charles W.; Torrent, Maricel; Marin, Violeta L.; Yu, Wenyu; Vedadi, Masoud; Li, Fengling; Brown, Peter J.; Pappano, William N.; Sun, Chaohong; Petros, Andrew M.

    2016-12-08

    SETD8 is a histone H4–K20 methyltransferase that plays an essential role in the maintenance of genomic integrity during mitosis and in DNA damage repair, making it an intriguing target for cancer research. While some small molecule inhibitors for SETD8 have been reported, the structural binding modes for these inhibitors have not been revealed. Using the complex structure of the substrate peptide bound to SETD8 as a starting point, different natural and unnatural amino acid substitutions were tested, and a potent (Ki 50 nM, IC50 0.33 μM) and selective norleucine containing peptide inhibitor has been obtained.

  7. Potent and Selective BACE-1 Peptide Inhibitors Lower Brain Aβ Levels Mediated by Brain Shuttle Transport

    Directory of Open Access Journals (Sweden)

    Nadine Ruderisch

    2017-10-01

    Full Text Available Therapeutic approaches to fight Alzheimer's disease include anti-Amyloidβ (Aβ antibodies and secretase inhibitors. However, the blood-brain barrier (BBB limits the brain exposure of biologics and the chemical space for small molecules to be BBB permeable. The Brain Shuttle (BS technology is capable of shuttling large molecules into the brain. This allows for new types of therapeutic modalities engineered for optimal efficacy on the molecular target in the brain independent of brain penetrating properties. To this end, we designed BACE1 peptide inhibitors with varying lipid modifications with single-digit picomolar cellular potency. Secondly, we generated active-exosite peptides with structurally confirmed dual binding mode and improved potency. When fused to the BS via sortase coupling, these BACE1 inhibitors significantly reduced brain Aβ levels in mice after intravenous administration. In plasma, both BS and non-BS BACE1 inhibitor peptides induced a significant time- and dose-dependent decrease of Aβ. Our results demonstrate that the BS is essential for BACE1 peptide inhibitors to be efficacious in the brain and active-exosite design of BACE1 peptide inhibitors together with lipid modification may be of therapeutic relevance.

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

    Science.gov (United States)

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

    1996-09-01

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

  9. Specificity of B-type natriuretic peptide assays

    DEFF Research Database (Denmark)

    Saenger, Amy K.; Rodriguez-Fraga, Olaia; Ler, Ranka

    2017-01-01

    BACKGROUND: B-type natriuretic peptides (BNPs) are used clinically to diagnose and monitor heart failure and are present in the circulation as multiple proBNP-derived fragments. We investigated the specificity of BNP immunoassays with glycosylated and nonglycosylated BNP, N-terminal proBNP (NT......-proBNP), and proBNP peptides to probe the cross-reactivity of each assay. METHODS: Nine B-type natriuretic peptides were studied, including synthetic and recombinant BNP (Shionogi, Scios, Mayo), human and synthetic glycosylated and nonglycosylated NT-proBNP (HyTest, Roche Diagnostics), and human glycosylated......-Rad, Goetze] were evaluated. Specificity was assessed by calculating the recovery between baseline and peptide-spiked human plasma pools at target concentrations of 100 ng/L BNP, 300 ng/L proBNP, or 450 ng/L NT-proBNP. All assays were performed in duplicate. RESULTS: BNP and NT-proBNP assays demonstrated...

  10. Peptide inhibitors of appressorium development in Glomerella cingulata.

    Science.gov (United States)

    Al-Samarrai, Taha H; Sullivan, Patrick A; Templeton, Matthew D; Farley, Peter C

    2002-04-09

    The phytopathogen Glomerella cingulata (anamorph: Colletotrichum gloeosporioides) infects host tissue by means of a specialised infection structure, the appressorium. The Saccharomyces cerevisiae alpha-mating factor pheromone, the Saccharomyces kluyveri alpha-mating factor pheromone and a hendecapeptide produced by G. cingulata inhibit appressorium development. The amino acid sequence of the G. cingulata peptide, GYFSYPHGNLF, is different from that of the mature pheromone peptides of other filamentous fungi. The peptide has sequence similarity with the Saccharomyces alpha-mating factor pheromones, but is unable to elicit growth arrest in S. cerevisiae.

  11. A cGMP kinase mutant with increased sensitivity to the protein kinase inhibitor peptide PKI(5-24).

    Science.gov (United States)

    Ruth, P; Kamm, S; Nau, U; Pfeifer, A; Hofmann, F

    1996-01-01

    Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein PKI are very potent inhibitors of cAMP-dependent protein kinase, but are extremely weak inhibitors of cGMP-dependent protein kinase. In this study, we tried to confer PKI sensitivity to cGMP kinase by site-directed mutagenesis. The molecular requirements for high affinity inhibition by PKI were deduced from the crystal structure of the cAMP kinase/PKI complex. A prominent site of interaction are residues Tyr235 and Phe239 in the catalytic subunit, which from a sandwich-like structure with Phe10 of the PKI(5-24) peptide. To increase the sensitivity for PKI, the cGMP kinase codons at the corresponding sites, Ser555 and Ser559, were changed to Tyr and Phe. The mutant cGMP kinase was stimulated half maximally by cGMP at 3-fold higher concentrations (240 nM) than the wild type (77 nM). Wild type and mutant cGMP kinase did not differ significantly in their Km and Vmax for three different substrate peptides. The PKI(5-24) peptide inhibited phosphotransferase activity of the mutant cGMP kinase with higher potency than that of wild type, with Ki values of 42 +/- .3 microM and 160 +/- .7 microM, respectively. The increased affinity of the mutant cGMP kinase was specific for the PKI(5-24) peptide. Mutation of the essential Phe10 in the PKI(5-24) sequence to an Ala yielded a peptide that inhibited mutant and wild type cGMP kinase with similar potency, with Ki values of 160 +/- 11 and 169 +/- 27 microM, respectively. These results suggest that the mutations Ser555Tyr and Ser559Phe are required, but not sufficient, for high affinity inhibition of cGMP kinase by PKI.

  12. Interaction of amyloid inhibitor proteins with amyloid beta peptides: insight from molecular dynamics simulations.

    Directory of Open Access Journals (Sweden)

    Payel Das

    Full Text Available Knowledge of the detailed mechanism by which proteins such as human αB- crystallin and human lysozyme inhibit amyloid beta (Aβ peptide aggregation is crucial for designing treatment for Alzheimer's disease. Thus, unconstrained, atomistic molecular dynamics simulations in explicit solvent have been performed to characterize the Aβ17-42 assembly in presence of the αB-crystallin core domain and of lysozyme. Simulations reveal that both inhibitor proteins compete with inter-peptide interaction by binding to the peptides during the early stage of aggregation, which is consistent with their inhibitory action reported in experiments. However, the Aβ binding dynamics appear different for each inhibitor. The binding between crystallin and the peptide monomer, dominated by electrostatics, is relatively weak and transient due to the heterogeneous amino acid distribution of the inhibitor surface. The crystallin-bound Aβ oligomers are relatively long-lived, as they form more extensive contact surface with the inhibitor protein. In contrast, a high local density of arginines from lysozyme allows strong binding with Aβ peptide monomers, resulting in stable complexes. Our findings not only illustrate, in atomic detail, how the amyloid inhibitory mechanism of human αB-crystallin, a natural chaperone, is different from that of human lysozyme, but also may aid de novo design of amyloid inhibitors.

  13. Buckwheat trypsin inhibitor with helical hairpin structure belongs to a new family of plant defence peptides.

    Science.gov (United States)

    Oparin, Peter B; Mineev, Konstantin S; Dunaevsky, Yakov E; Arseniev, Alexander S; Belozersky, Mikhail A; Grishin, Eugene V; Egorov, Tsezi A; Vassilevski, Alexander A

    2012-08-15

    A new peptide trypsin inhibitor named BWI-2c was obtained from buckwheat (Fagopyrum esculentum) seeds by sequential affinity, ion exchange and reversed-phase chromatography. The peptide was sequenced and found to contain 41 amino acid residues, with four cysteine residues involved in two intramolecular disulfide bonds. Recombinant BWI-2c identical to the natural peptide was produced in Escherichia coli in a form of a cleavable fusion with thioredoxin. The 3D (three-dimensional) structure of the peptide in solution was determined by NMR spectroscopy, revealing two antiparallel α-helices stapled by disulfide bonds. Together with VhTI, a trypsin inhibitor from veronica (Veronica hederifolia), BWI-2c represents a new family of protease inhibitors with an unusual α-helical hairpin fold. The linker sequence between the helices represents the so-called trypsin inhibitory loop responsible for direct binding to the active site of the enzyme that cleaves BWI-2c at the functionally important residue Arg(19). The inhibition constant was determined for BWI-2c against trypsin (1.7×10(-1)0 M), and the peptide was tested on other enzymes, including those from various insect digestive systems, revealing high selectivity to trypsin-like proteases. Structural similarity shared by BWI-2c, VhTI and several other plant defence peptides leads to the acknowledgement of a new widespread family of plant peptides termed α-hairpinins.

  14. Design and characterization of hirulogs: A novel class of bivalent peptide inhibitors of thrombin

    Energy Technology Data Exchange (ETDEWEB)

    Maraganore, J.M.; Bourdon, P.; Jablonski, J.; Ramachandran, K.L. (Biogen, Inc., Cambridge, MA (USA)); Fenton, J.W. II (New York State Department of Health, Albany (USA))

    1990-07-31

    A novel class of synthetic peptides has been designed that inhibit the thrombin catalytic site and exhibit specificity for the anion-binding exosite (ABE) of {alpha}-thrombin. These peptides, called hirulogs, consist of (i) an active-site specificity sequence with a restricted Arg-Pro scissile bond, (ii) a polymeric linker of glycyl residues from 6 to 18 {angstrom} in length, and (iii) an ABE recognition sequence such as that in the hirudin C-terminus. Hirulog-1 ((D-Phe)-Pro-Arg-Pro-(Gly){sub 4}-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Tyr-Leu) inhibits the thrombin-catalyzed hydrolysis of a tripeptide p-nitroanilide substrate with K{sub i} = 2.3 nM. In contrast, the synthetic C-terminal hirudin peptide S-Hir{sub 53-64}, which binds to the thrombin ABE, blocked the fibrinogen clotting activity of the enzyme with K{sub i} = 144 nM but failed to inhibit the hydrolysis of p-nitroanilide substrates at concentrations as high as 1 mM. Hirulog-1, but not S-Hir{sub 53-64}, was found to inhibit the incorporation of ({sup 14}C)diisopropyl fluorophosphate in thrombin. Hirulog-1 appears specific for thrombin as it lacks inhibitory activities toward human factor Xa, human plasmin, and bovine trypsin at inhibitor:enzyme concentrations 3 orders of magnitude higher than those required to inhibit thrombin. The optimal inhibitory activity of hirulog-1 depends upon all three components of its structure. Comparison of anticoagulant activities of hirulog-1, hirudin, and S-Hir{sub 53-64} showed that the synthetic hirulog-1 is 2-fold more potent than hirudin and 100-fold more active than S-Hir{sub 53-64} in increasing the activated partial thromboplastin time of normal human plasma.

  15. In-silico screening and validation of high-affinity tetra-peptide inhibitor of Leishmania donovani O-acetyl serine sulfhydrylase (OASS).

    Science.gov (United States)

    Kant, Vishnu; Vijayakumar, Saravanan; Sahoo, Ganesh Chandra; Chaudhery, Shailendra S; Das, Pradeep

    2018-02-07

    OASS is a specific enzyme that helps Leishmania parasite to survive the oxidative stress condition in human macrophages. SAT C-terminal peptides in several organisms, including Leishmania, were reported to inhibit or reduce the activity of OASS. Small peptide and small molecules mimicking the SAT C-terminal residues are designed and tested for the inhibition of OASS in different organisms. Hence, in this study, all the possible tetra-peptide combinations were designed and screened based on the docking ability with Leishmania donovani OASS (Ld-OASS). The top ranked peptides were further validated for the stability using 50 ns molecular dynamic simulation. In order to identify the better binding capability of the peptides, the top peptides complexed with Ld-OASS were also subjected to molecular dynamic simulation. The docking and simulation results favored the peptide EWSI to possess greater advantage than previously reported peptide (DWSI) in binding with Ld-OASS active site. Also, screening of non-peptide inhibitor of Asinex Biodesign library based on the shape similarity of EWSI and DWSI was performed. The top similar molecules of each peptides were docked on to Ld-OASS active site and subsequently simulated for 20 ns. The results suggested that the ligand that shares high shape similarity with EWSI possess better binding capability than the ligand that shares high shape similarity with DWSI. This study revealed that the tetra-peptide EWSI had marginal advantage over DWSI in binding with Ld-OASS, thereby providing basis for defining a pharmacophoric scaffold for the design of peptidomimetic inhibitors as well as non-peptide inhibitors of Ld-OASS.

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

    Science.gov (United States)

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

    2017-05-01

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

  17. A phage display selected 7-mer peptide inhibitor of the Tannerella forsythia metalloprotease-like enzyme Karilysin can be truncated to Ser-Trp-Phe-Pro.

    Science.gov (United States)

    Skottrup, Peter Durand; Sørensen, Grete; Ksiazek, Miroslaw; Potempa, Jan; Riise, Erik

    2012-01-01

    Tannerella forsythia is a gram-negative bacteria, which is strongly associated with the development of periodontal disease. Karilysin is a newly identified metalloprotease-like enzyme, that is secreted from T. forsythia. Karilysin modulates the host immune response and is therefore considered a likely drug target. In this study peptides were selected towards the catalytic domain from Karilysin (Kly18) by phage display. The peptides were linear with low micromolar binding affinities. The two best binders (peptide14 and peptide15), shared the consensus sequence XWFPXXXGGG. A peptide15 fusion with Maltose Binding protein (MBP) was produced with peptide15 fused to the N-terminus of MBP. The peptide15-MBP was expressed in E. coli and the purified fusion-protein was used to verify Kly18 specific binding. Chemically synthesised peptide15 (SWFPLRSGGG) could inhibit the enzymatic activity of both Kly18 and intact Karilysin (Kly48). Furthermore, peptide15 could slow down the autoprocessing of intact Kly48 to Kly18. The WFP motif was important for inhibition and a truncation study further demonstrated that the N-terminal serine was also essential for Kly18 inhibition. The SWFP peptide had a Ki value in the low micromolar range, which was similar to the intact peptide15. In conclusion SWFP is the first reported inhibitor of Karilysin and can be used as a valuable tool in structure-function studies of Karilysin.

  18. A phage display selected 7-mer peptide inhibitor of the Tannerella forsythia metalloprotease-like enzyme Karilysin can be truncated to Ser-Trp-Phe-Pro.

    Directory of Open Access Journals (Sweden)

    Peter Durand Skottrup

    Full Text Available Tannerella forsythia is a gram-negative bacteria, which is strongly associated with the development of periodontal disease. Karilysin is a newly identified metalloprotease-like enzyme, that is secreted from T. forsythia. Karilysin modulates the host immune response and is therefore considered a likely drug target. In this study peptides were selected towards the catalytic domain from Karilysin (Kly18 by phage display. The peptides were linear with low micromolar binding affinities. The two best binders (peptide14 and peptide15, shared the consensus sequence XWFPXXXGGG. A peptide15 fusion with Maltose Binding protein (MBP was produced with peptide15 fused to the N-terminus of MBP. The peptide15-MBP was expressed in E. coli and the purified fusion-protein was used to verify Kly18 specific binding. Chemically synthesised peptide15 (SWFPLRSGGG could inhibit the enzymatic activity of both Kly18 and intact Karilysin (Kly48. Furthermore, peptide15 could slow down the autoprocessing of intact Kly48 to Kly18. The WFP motif was important for inhibition and a truncation study further demonstrated that the N-terminal serine was also essential for Kly18 inhibition. The SWFP peptide had a Ki value in the low micromolar range, which was similar to the intact peptide15. In conclusion SWFP is the first reported inhibitor of Karilysin and can be used as a valuable tool in structure-function studies of Karilysin.

  19. Glyceraldehyde-3-phosphate dehydrogenase aggregation inhibitor peptide: A potential therapeutic strategy against oxidative stress-induced cell death.

    Science.gov (United States)

    Itakura, Masanori; Nakajima, Hidemitsu; Semi, Yuko; Higashida, Shusaku; Azuma, Yasu-Taka; Takeuchi, Tadayoshi

    2015-11-13

    The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has multiple functions, including mediating oxidative stress-induced neuronal cell death. This process is associated with disulfide-bonded GAPDH aggregation. Some reports suggest a link between GAPDH and the pathogenesis of several oxidative stress-related diseases. However, the pathological significance of GAPDH aggregation in disease pathogenesis remains unclear due to the lack of an effective GAPDH aggregation inhibitor. In this study, we identified a GAPDH aggregation inhibitor (GAI) peptide and evaluated its biological profile. The decapeptide GAI specifically inhibited GAPDH aggregation in a concentration-dependent manner. Additionally, the GAI peptide did not affect GAPDH glycolytic activity or cell viability. The GAI peptide also exerted a protective effect against oxidative stress-induced cell death in SH-SY5Y cells. This peptide could potentially serve as a tool to investigate GAPDH aggregation-related neurodegenerative and neuropsychiatric disorders and as a possible therapy for diseases associated with oxidative stress-induced cell death. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Structural Insights into the Mechanisms of Action of Short-Peptide HIV-1 Fusion Inhibitors Targeting the Gp41 Pocket

    Directory of Open Access Journals (Sweden)

    Xiujuan Zhang

    2018-02-01

    Full Text Available The deep hydrophobic pocket of HIV-1 gp41 has been considered a drug target, but short-peptides targeting this site usually lack potent antiviral activity. By applying the M-T hook structure, we previously generated highly potent short-peptide fusion inhibitors that specifically targeted the pocket site, such as MT-SC22EK, HP23L, and LP-11. Here, the crystal structures of HP23L and LP-11 bound to the target mimic peptide N36 demonstrated the critical intrahelical and interhelical interactions, especially verifying that the hook-like conformation was finely adopted while the methionine residue was replaced by the oxidation-less prone residue leucine, and that addition of an extra glutamic acid significantly enhanced the binding and inhibitory activities. The structure of HP23L bound to N36 with two mutations (E49K and L57R revealed the critical residues and motifs mediating drug resistance and provided new insights into the mechanism of action of inhibitors. Therefore, the present data help our understanding for the structure-activity relationship (SAR of HIV-1 fusion inhibitors and facilitate the development of novel antiviral drugs.

  1. Synthesis and Biological Evaluation of a Chitobiose-Based Peptide N-Glycanase Inhibitor Library

    NARCIS (Netherlands)

    Witte, Martin D.; Horst, Danielle; Wiertz, Emmanuel J.H.J.; Marel, Gijsbert A. van der; Overkleeft, Herman S.

    2009-01-01

    Peptide N-glycanase (PNGase), the enzyme responsible for the deglycosylation of N-linked glycoproteins, has an active site related to that of cysteine proteases. Chitiobiose was equipped with electrophilic traps often used in cysteine protease inhibitors, and the resulting compounds were evaluated

  2. Design and synthesis of macrocyclic peptidyl hydroxamates as peptide deformylase inhibitors.

    Science.gov (United States)

    Shen, Gang; Zhu, Jinge; Simpson, Anthony M; Pei, Dehua

    2008-05-15

    Macrocyclic peptidyl hydroxamates were designed, synthesized, and evaluated as peptide deformylase (PDF) inhibitors. The most potent compound exhibited tight, slow-binding inhibition of Escherichia coli PDF (K(I)(*)=4.4 nM) and had potent antibacterial activity against Gram-positive bacterium Bacillus subtilis (MIC=2-4 microg/mL).

  3. Identification of a peptide inhibitor for the histone methyltransferase WHSC1.

    Directory of Open Access Journals (Sweden)

    Michael J Morrison

    Full Text Available WHSC1 is a histone methyltransferase that is responsible for mono- and dimethylation of lysine 36 on histone H3 and has been implicated as a driver in a variety of hematological and solid tumors. Currently, there is a complete lack of validated chemical matter for this important drug discovery target. Herein we report on the first fully validated WHSC1 inhibitor, PTD2, a norleucine-containing peptide derived from the histone H4 sequence. This peptide exhibits micromolar affinity towards WHSC1 in biochemical and biophysical assays. Furthermore, a crystal structure was solved with the peptide in complex with SAM and the SET domain of WHSC1L1. This inhibitor is an important first step in creating potent, selective WHSC1 tool compounds for the purposes of understanding the complex biology in relation to human disease.

  4. Structure-Based Design of Peptidic Inhibitors of the Interaction between CC Chemokine Ligand 5 (CCL5) and Human Neutrophil Peptides 1 (HNP1)

    NARCIS (Netherlands)

    Wichapong, Kanin; Alard, Jean-Eric; Ortega-Gomez, Almudena; Weber, Christian; Hackeng, Tilman M.; Soehnlein, Oliver; Nicolaes, Gerry A. F.

    2016-01-01

    Protein-protein interactions (PPIs) are receiving increasing interest, much sparked by the realization that they represent druggable targets. Recently, we successfully developed a peptidic inhibitor, RRYGTSKYQ ("SKY" peptide), that shows high potential in vitro and in vivo to interrupt a PPI between

  5. Screening for Selective Protein Inhibitors by Using the IANUS Peptide Array.

    Science.gov (United States)

    Erdmann, Frank; Prell, Erik; Jahreis, Günther; Fischer, Gunter; Malešević, Miroslav

    2018-04-16

    Finding new road blacks: A peptidic inhibitor of calcineurin (CaN)-mediated nuclear factor of activated T cells (NFAT) dephosphorylation, which is developed through a template-assisted IANUS (Induced orgANisation of strUcture by matrix-assisted togethernesS) peptide array, is cell permeable and able to block the translocation of green fluorescent protein-NFAT fusion protein (GFP-NFAT) into the nucleus after stimulation. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. PinaColada: peptide-inhibitor ant colony ad-hoc design algorithm.

    Science.gov (United States)

    Zaidman, Daniel; Wolfson, Haim J

    2016-08-01

    Design of protein-protein interaction (PPI) inhibitors is a major challenge in Structural Bioinformatics. Peptides, especially short ones (5-15 amino acid long), are natural candidates for inhibition of protein-protein complexes due to several attractive features such as high structural compatibility with the protein binding site (mimicking the surface of one of the proteins), small size and the ability to form strong hotspot binding connections with the protein surface. Efficient rational peptide design is still a major challenge in computer aided drug design, due to the huge space of possible sequences, which is exponential in the length of the peptide, and the high flexibility of peptide conformations. In this article we present PinaColada, a novel computational method for the design of peptide inhibitors for protein-protein interactions. We employ a version of the ant colony optimization heuristic, which is used to explore the exponential space ([Formula: see text]) of length n peptide sequences, in combination with our fast robotics motivated PepCrawler algorithm, which explores the conformational space for each candidate sequence. PinaColada is being run in parallel, on a DELL PowerEdge 2.8 GHZ computer with 20 cores and 256 GB memory, and takes up to 24 h to design a peptide of 5-15 amino acids length. An online server available at: http://bioinfo3d.cs.tau.ac.il/PinaColada/. danielza@post.tau.ac.il; wolfson@tau.ac.il. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  7. C-terminal peptides of tissue factor pathway inhibitor are novel host defense molecules.

    Science.gov (United States)

    Papareddy, Praveen; Kalle, Martina; Kasetty, Gopinath; Mörgelin, Matthias; Rydengård, Victoria; Albiger, Barbara; Lundqvist, Katarina; Malmsten, Martin; Schmidtchen, Artur

    2010-09-03

    Tissue factor pathway inhibitor (TFPI) inhibits tissue factor-induced coagulation, but may, via its C terminus, also modulate cell surface, heparin, and lipopolysaccharide interactions as well as participate in growth inhibition. Here we show that C-terminal TFPI peptide sequences are antimicrobial against the gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungi Candida albicans and Candida parapsilosis. Fluorescence studies of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted membrane-breaking effects similar to those seen for the "classic" human antimicrobial peptide LL-37. The killing of E. coli, but not P. aeruginosa, by the C-terminal peptide GGLIKTKRKRKKQRVKIAYEEIFVKNM (GGL27), was enhanced in human plasma and largely abolished in heat-inactivated plasma, a phenomenon linked to generation of antimicrobial C3a and activation of the classic pathway of complement activation. Furthermore, GGL27 displayed anti-endotoxic effects in vitro and in vivo in a mouse model of LPS shock. Importantly, TFPI was found to be expressed in the basal layers of normal epidermis, and was markedly up-regulated in acute skin wounds as well as wound edges of chronic leg ulcers. Furthermore, C-terminal fragments of TFPI were associated with bacteria present in human chronic leg ulcers. These findings suggest a new role for TFPI in cutaneous defense against infections.

  8. Screening and Identification of Peptides Specifically Targeted to Gastric Cancer Cells from a Phage Display Peptide Library

    Science.gov (United States)

    Sahin, Deniz; Taflan, Sevket Onur; Yartas, Gizem; Ashktorab, Hassan; Smoot, Duane T

    2018-04-25

    Background: Gastric cancer is the second most common cancer among the malign cancer types. Inefficiency of traditional techniques both in diagnosis and therapy of the disease makes the development of alternative and novel techniques indispensable. As an alternative to traditional methods, tumor specific targeting small peptides can be used to increase the efficiency of the treatment and reduce the side effects related to traditional techniques. The aim of this study is screening and identification of individual peptides specifically targeted to human gastric cancer cells using a phage-displayed peptide library and designing specific peptide sequences by using experimentally-eluted peptide sequences. Methods: Here, MKN-45 human gastric cancer cells and HFE-145 human normal gastric epithelial cells were used as the target and control cells, respectively. 5 rounds of biopannning with a phage display 12-peptide library were applied following subtraction biopanning with HFE-145 control cells. The selected phage clones were established by enzyme-linked immunosorbent assay and immunofluorescence detection. We first obtain random phage clones after five biopanning rounds, determine the binding levels of each individual clone. Then, we analyze the frequencies of each amino acid in best binding clones to determine positively overexpressed amino acids for designing novel peptide sequences. Results: DE532 (VETSQYFRGTLS) phage clone was screened positive, showing specific binding on MKN-45 gastric cancer cells. DE-Obs (HNDLFPSWYHNY) peptide, which was designed by using amino acid frequencies of experimentally selected peptides in the 5th round of biopanning, showed specific binding in MKN-45 cells. Conclusion: Selection and characterization of individual clones may give us specifically binding peptides, but more importantly, data extracted from eluted phage clones may be used to design theoretical peptides with better binding properties than even experimentally selected ones

  9. Hydroxamic acid derivatives as potent peptide deformylase inhibitors and antibacterial agents.

    Science.gov (United States)

    Apfel, C; Banner, D W; Bur, D; Dietz, M; Hirata, T; Hubschwerlen, C; Locher, H; Page, M G; Pirson, W; Rossé, G; Specklin, J L

    2000-06-15

    Low-molecular-weight beta-sulfonyl- and beta-sulfinylhydroxamic acid derivatives have been synthesized and found to be potent inhibitors of Escherichia coli peptide deformylase (PDF). Most of the compounds synthesized and tested displayed antibacterial activities that cover several pathogens found in respiratory tract infections, including Chlamydia pneumoniae, Mycoplasma pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. The potential of these compounds as antibacterial agents is discussed with respect to selectivity, intracellular concentrations in bacteria, and potential for resistance development.

  10. Antistaphylococcal activity of LBM415, a new peptide deformylase inhibitor, compared with those of other agents.

    Science.gov (United States)

    Credito, Kim; Lin, Gengrong; Ednie, Lois M; Appelbaum, Peter C

    2004-10-01

    The MICs of LBM415, a new peptide diformylase inhibitor, were

  11. Study of plasma binding of receptor-specific peptides

    OpenAIRE

    Gregor, David

    2008-01-01

    The binding ability of two receptor specific peptides namely 90Y-DOTA-TATE and 111In-DOTA-TATE was studied in therm of interspecies comparison by the method of equilibrium dialysis. This plasma protein binding was different for the chosen animal species (human, rat, rabbit, bovine eventually pork) whereas binding of 90Y-DOTA- TATE was higher than binding of 111In-DOTA-TATE. KEYWORDS: Protein binding, radiofarmaceuticals, equilibrium dialysis, 90Y-DOTA-TATE, 111In- DOTA-TATE

  12. Radiolabelled peptides and nanoparticles for specific molecular targeting in oncology

    International Nuclear Information System (INIS)

    Helbok, A.

    2011-01-01

    The aim of this thesis is the development of radiolabelled peptides and nanoparticles (NP) for specific molecular targeting in oncology. Three different types of NP were investigated in this study: lipid - based NP (liposomes and micelles), human serum albumin - based NP (albumin NP) and protamine - oligonucleotide - based NP (proticles). In a first step, radiolabelling protocols were set up for the different NP - formulations. The variety of radioisotopes used, covers the whole spectrum of applications in nuclear medicine: SPECT (111In, 99mTc), (2) PET (68Ga) and therapeutic applications (177Lu, 90Y) opening a manifold administration potential for these NP aiming towards multiple targeting and hybrid imaging strategies (combined SPECT / PET and MRI). Radiolabelling quality was analyzed by instant thin layer chromatography (ITLC). High radiochemical yields (RCY >90 %) and high specific activity (SA) were achieved. NP - formulations were derivatized with the chelating agent Diethylenetriaminepentaacetic acid (DTPA) allowing complexation of trivalent radiometals, and potentially nonradioactive metals, such as Gd3+, for MRI imaging leading to the development of multifunctionalized NP for a unified labelling approach. Furthermore, NP were derivatized with the pharmacokinetic modifier polyethylene glycol (PEG) to maintain NP with long circulating ability. Stability assessments included incubation in different media (serum, 4 mM DTPA - solution and PBS pH 7.4, at 37 o C for a period of 24 h). For the in vivo biodistribution of the NP, static and / or dynamic SPECT / PET imaging studies were performed at different time points with Lewis rats and correlated to results from quantification of tissue - uptake. Results indicate differences in stability and general pharmacokinetic behaviour depended on the NP - formulation. However, a positive influence expressed in a prolonged retention time in circulation was investigated for all different NP - formulations due to PEG

  13. Grb7 SH2 domain structure and interactions with a cyclic peptide inhibitor of cancer cell migration and proliferation

    Directory of Open Access Journals (Sweden)

    Pero Stephanie C

    2007-09-01

    Full Text Available Abstract Background Human growth factor receptor bound protein 7 (Grb7 is an adapter protein that mediates the coupling of tyrosine kinases with their downstream signaling pathways. Grb7 is frequently overexpressed in invasive and metastatic human cancers and is implicated in cancer progression via its interaction with the ErbB2 receptor and focal adhesion kinase (FAK that play critical roles in cell proliferation and migration. It is thus a prime target for the development of novel anti-cancer therapies. Recently, an inhibitory peptide (G7-18NATE has been developed which binds specifically to the Grb7 SH2 domain and is able to attenuate cancer cell proliferation and migration in various cancer cell lines. Results As a first step towards understanding how Grb7 may be inhibited by G7-18NATE, we solved the crystal structure of the Grb7 SH2 domain to 2.1 Å resolution. We describe the details of the peptide binding site underlying target specificity, as well as the dimer interface of Grb 7 SH2. Dimer formation of Grb7 was determined to be in the μM range using analytical ultracentrifugation for both full-length Grb7 and the SH2 domain alone, suggesting the SH2 domain forms the basis of a physiological dimer. ITC measurements of the interaction of the G7-18NATE peptide with the Grb7 SH2 domain revealed that it binds with a binding affinity of Kd = ~35.7 μM and NMR spectroscopy titration experiments revealed that peptide binding causes perturbations to both the ligand binding surface of the Grb7 SH2 domain as well as to the dimer interface, suggesting that dimerisation of Grb7 is impacted on by peptide binding. Conclusion Together the data allow us to propose a model of the Grb7 SH2 domain/G7-18NATE interaction and to rationalize the basis for the observed binding specificity and affinity. We propose that the current study will assist with the development of second generation Grb7 SH2 domain inhibitors, potentially leading to novel inhibitors of

  14. Evaluation of MAP-specific peptides following vaccination of goats

    DEFF Research Database (Denmark)

    Lybeck, Kari; Sjurseth, Siri K.; Melvang, Heidi Mikkelsen

    species or 2) selected based on “experience”. Peptides predicted to bind bovine MHC II by in silico analysis were included in further studies, resulting in two panels 1) genome-based and 2) selected. Initially, two groups of 15 healthy goats were vaccinated with one of the two panels (50 µg/peptide in CAF......01 adjuvant/CAF04 for boosting). Four MAP-infected goats were also vaccinated. In a second vaccination trail, groups of 8 healthy goat kids were vaccinated with genome-based peptides, selected peptides or selected peptides linked together in a recombinant protein (20 µg/peptide or 50 µg protein...... peptides. IFN-γ responses in healthy goats after the first vaccination were low, but testing of T cell lines from MAP-infected goats identified peptides inducing strong proliferative responses. Peptides for a second vaccination were selected by combining results from this study with a parallel cattle study...

  15. Cytological profile of antibacterial FtsZ inhibitors and synthetic peptide MciZ

    Directory of Open Access Journals (Sweden)

    Lidia Araujo-Bazan

    2016-10-01

    Full Text Available Cell division protein FtsZ is the organizer of the cytokinetic ring in almost all bacteria and a target for the discovery of new antibacterial agents that are needed to counter widespread antibiotic resistance. Bacterial cytological profiling, using quantitative microscopy, is a powerful approach for identifying the mechanism of action of antibacterial molecules affecting different cellular pathways. We have determined the cytological profile on Bacillus subtilis cells of a selection of small molecule inhibitors targeting FtsZ on different binding sites. FtsZ inhibitors lead to long undivided cells, impair the normal assembly of FtsZ into the midcell Z-rings, induce aberrant ring distributions, punctate FtsZ foci, membrane spots and also modify nucleoid length. Quantitative analysis of cell and nucleoid length combined, or the Z-ring distribution, allows categorizing FtsZ inhibitors and to distinguish them from antibiotics with other mechanisms of action, which should be useful for identifying new antibacterial FtsZ inhibitors. Biochemical assays of FtsZ polymerization and GTPase activity combined explain the cellular effects of the FtsZ polymer stabilizing agent PC190723 and its fragments. MciZ is a 40-aminoacid endogenous inhibitor of cell division normally expressed during sporulation in B. subtilis. Using FtsZ cytological profiling we have determined that exogenous synthetic MciZ is an effective inhibitor of B. subtilis cell division, Z-ring formation and localization. This finding supports our cell-based approach to screen for FtsZ inhibitors and opens new possibilities for peptide inhibitors of bacterial cell division.

  16. Rationally Designed Peptides and Peptidomimetics as Inhibitors of Amyloid-β (Aβ) Aggregation: Potential Therapeutics of Alzheimer's Disease.

    Science.gov (United States)

    Goyal, Deepti; Shuaib, Suniba; Mann, Sukhmani; Goyal, Bhupesh

    2017-02-13

    Alzheimer's disease (AD) is a progressive neurodegenerative disease with no clinically accepted treatment to cure or halt its progression. The worldwide effort to develop peptide-based inhibitors of amyloid-β (Aβ) aggregation can be considered an unplanned combinatorial experiment. An understanding of what has been done and achieved may advance our understanding of AD pathology and the discovery of effective therapeutic agents. We review here the history of such peptide-based inhibitors, including those based on the Aβ sequence and those not derived from that sequence, containing both natural and unnatural amino acid building blocks. Peptide-based aggregation inhibitors hold significant promise for future AD therapy owing to their high selectivity, effectiveness, low toxicity, good tolerance, low accumulation in tissues, high chemical and biological diversity, possibility of rational design, and highly developed methods for analyzing their mode of action, proteolytic stability (modified peptides), and blood-brain barrier (BBB) permeability.

  17. Computational design of a PDZ domain peptide inhibitor that rescues CFTR activity.

    Directory of Open Access Journals (Sweden)

    Kyle E Roberts

    Full Text Available The cystic fibrosis transmembrane conductance regulator (CFTR is an epithelial chloride channel mutated in patients with cystic fibrosis (CF. The most prevalent CFTR mutation, ΔF508, blocks folding in the endoplasmic reticulum. Recent work has shown that some ΔF508-CFTR channel activity can be recovered by pharmaceutical modulators ("potentiators" and "correctors", but ΔF508-CFTR can still be rapidly degraded via a lysosomal pathway involving the CFTR-associated ligand (CAL, which binds CFTR via a PDZ interaction domain. We present a study that goes from theory, to new structure-based computational design algorithms, to computational predictions, to biochemical testing and ultimately to epithelial-cell validation of novel, effective CAL PDZ inhibitors (called "stabilizers" that rescue ΔF508-CFTR activity. To design the "stabilizers", we extended our structural ensemble-based computational protein redesign algorithm K* to encompass protein-protein and protein-peptide interactions. The computational predictions achieved high accuracy: all of the top-predicted peptide inhibitors bound well to CAL. Furthermore, when compared to state-of-the-art CAL inhibitors, our design methodology achieved higher affinity and increased binding efficiency. The designed inhibitor with the highest affinity for CAL (kCAL01 binds six-fold more tightly than the previous best hexamer (iCAL35, and 170-fold more tightly than the CFTR C-terminus. We show that kCAL01 has physiological activity and can rescue chloride efflux in CF patient-derived airway epithelial cells. Since stabilizers address a different cellular CF defect from potentiators and correctors, our inhibitors provide an additional therapeutic pathway that can be used in conjunction with current methods.

  18. Acetylation site specificities of lysine deacetylase inhibitors in human cells

    DEFF Research Database (Denmark)

    Schölz, Christian; Weinert, Brian Tate; Wagner, Sebastian A

    2015-01-01

    Lysine deacetylases inhibitors (KDACIs) are used in basic research, and many are being investigated in clinical trials for treatment of cancer and other diseases. However, their specificities in cells are incompletely characterized. Here we used quantitative mass spectrometry (MS) to obtain acety......1-α, providing a possible mechanistic explanation of its adverse, pro-inflammatory effects. Our results offer a systems view of KDACI specificities, providing a framework for studying function of acetylation and deacetylases....

  19. Pre-equilibrium competitive library screening for tuning inhibitor association rate and specificity toward serine proteases.

    Science.gov (United States)

    Cohen, Itay; Naftaly, Si; Ben-Zeev, Efrat; Hockla, Alexandra; Radisky, Evette S; Papo, Niv

    2018-04-16

    High structural and sequence similarity within protein families can pose significant challenges to the development of selective inhibitors, especially toward proteolytic enzymes. Such enzymes usually belong to large families of closely similar proteases and may also hydrolyze, with different rates, protein- or peptide-based inhibitors. To address this challenge, we employed a combinatorial yeast surface display library approach complemented with a novel pre-equilibrium, competitive screening strategy for facile assessment of the effects of multiple mutations on inhibitor association rates and binding specificity. As a proof of principle for this combined approach, we utilized this strategy to alter inhibitor/protease association rates and to tailor the selectivity of the amyloid β-protein precursor Kunitz protease inhibitor domain (APPI) for inhibition of the oncogenic protease mesotrypsin, in the presence of three competing serine proteases, anionic trypsin, cationic trypsin and kallikrein-6. We generated a variant, designated APPI P13W/M17G/I18F/F34V , with up to 30-fold greater specificity relative to the parental APPI M17G/I18F/F34V protein, and 6500- to 230 000-fold improved specificity relative to the wild-type APPI protein in the presence of the other proteases tested. A series of molecular docking simulations suggested a mechanism of interaction that supported the biochemical results. These simulations predicted that the selectivity and specificity are affected by the interaction of the mutated APPI residues with nonconserved enzyme residues located in or near the binding site. Our strategy will facilitate a better understanding of the binding landscape of multispecific proteins and will pave the way for design of new drugs and diagnostic tools targeting proteases and other proteins. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  20. Gold nanoparticles-based electrochemical method for the detection of protein kinase with a peptide-like inhibitor as the bioreceptor

    Directory of Open Access Journals (Sweden)

    Sun K

    2017-03-01

    Full Text Available Kai Sun, Yong Chang, Binbin Zhou, Xiaojin Wang, Lin Liu Henan Province of Key Laboratory of New Optoelectronic Functional Materials, College of Chemistry and Chemical Engineering, Anyang Normal University, Anyang, Henan, People’s Republic of China Abstract: This article presents a general method for the detection of protein kinase with a peptide-like kinase inhibitor as the bioreceptor, and it was done by converting gold nanoparticles (AuNPs-based colorimetric assay into sensitive electrochemical analysis. In the colorimetric assay, the kinase-specific aptameric peptide triggered the aggregation of AuNPs in solution. However, the specific binding of peptide to the target protein (kinase inhibited its ability to trigger the assembly of AuNPs. In the electrochemical analysis, peptides immobilized on a gold electrode and presented as solution triggered together the in situ formation of AuNPs-based network architecture on the electrode surface. Nevertheless, the formation of peptide–kinase complex on the electrode surface made the peptide-triggered AuNPs assembly difficult. Electrochemical impedance spectroscopy was used to measure the change in surface property in the binding events. When a ferrocene-labeled peptide (Fc-peptide was used in this design, the network of AuNPs/Fc-peptide produced a good voltammetric signal. The competitive assay allowed for the detection of protein kinase A with a detection limit of 20 mU/mL. This work should be valuable for designing novel optical or electronic biosensors and likely lead to many detection applications. Keywords: electrochemical biosensor, colorimetric assay, gold nanoparticle, aptameric peptide, protein kinase A, signal amplification 

  1. Structure of a retro-binding peptide inhibitor complexed with human alpha-thrombin.

    Science.gov (United States)

    Tabernero, L; Chang, C Y; Ohringer, S L; Lau, W F; Iwanowicz, E J; Han, W C; Wang, T C; Seiler, S M; Roberts, D G; Sack, J S

    1995-02-10

    The crystallographic structure of the ternary complex between human alpha-thrombin, hirugen and the peptidyl inhibitor Phe-alloThr-Phe-O-CH3, which is acylated at its N terminus with 4-guanidino butanoic acid (BMS-183507), has been determined at 2.6 A resolution. The structure reveals a unique "retro-binding" mode for this tripeptide active site inhibitor. The inhibitor binds with its alkyl-guanidine moiety in the primary specificity pocket and its two phenyl rings occupying the hydrophobic proximal and distal pockets of the thrombin active site. In this arrangement the backbone of the tripeptide forms a parallel beta-strand to the thrombin main-chain at the binding site. This is opposite to the orientation of the natural substrate, fibrinogen, and all the small active site-directed thrombin inhibitors whose bound structures have been previously reported. BMS-183507 is the first synthetic inhibitor proved to bind in a retro-binding fashion to thrombin, in a fashion similar to that of the N-terminal residues of the natural inhibitor hirudin. Furthermore, this new potent thrombin inhibitor (Ki = 17.2 nM) is selective for thrombin over other serine proteases tested and may be a template to be considered in designing hirudin-based thrombin inhibitors with interactions at the specificity pocket.

  2. Epoxyethylglycyl peptides as inhibitors of oligosaccharyltransferase: double-labelling of the active site.

    Science.gov (United States)

    Bause, E; Wesemann, M; Bartoschek, A; Breuer, W

    1997-02-15

    Pig liver oligosaccharyltransferase (OST) is inactivated irreversibly by a hexapeptide in which threonine has been substituted by epoxyethylglycine in the Asn-Xaa-Thr glycosylation triplet. Incubation of the enzyme in the presence of Dol-PP-linked [14C]oligosaccharides and the N-3,5-dinitrobenzoylated epoxy derivative leads to the double-labelling of two subunits (48 and 66 kDa) of the oligomeric OST complex, both of which are involved in the catalytic activity. Labelling of both subunits was blocked competitively by the acceptor peptide N-benzoyl-Asu-Gly-Thr-NHCH3 and by the OST inhibitor N-benzoyl-alpha,gamma-diaminobutyric acid-Gly-Thr-NHCH3, but not by an analogue derived from the epoxy-inhibitor by replacing asparagine with glutamine. Our data clearly show that double-labelling is an active-site-directed modification, involving inhibitor glycosylation at asparagine and covalent attachment of the glycosylated inhibitor, via the epoxy group, to the enzyme. Double-labelling of OST can occur as the result of either a consecutive or a syn-catalytic reaction sequence. The latter mechanism, during the course of which OST catalyses its own 'suicide' inactivation, is more likely, as suggested by indirect experimental evidence. The syn-catalytic mechanism corresponds with our current view of the functional role of the acceptor site Thr/Ser acting as a hydrogen-bond acceptor, not a donor, during transglycosylation.

  3. Preparation and crystallization of the Grb7 SH2 domain in complex with the G7-18NATE nonphosphorylated cyclic inhibitor peptide

    International Nuclear Information System (INIS)

    Yap, Min Y.; Wilce, Matthew C. J.; Clayton, Daniel J.; Perlmutter, Patrick; Aguilar, Marie-Isabel; Wilce, Jacqueline A.

    2010-01-01

    The preparation and successful crystallization of the Grb7 SH2 domain in complex with the specific cyclic peptide inhibitor G7-18NATE are reported. This structure is anticipated to reveal the basis of the binding affinity and specificity and to assist with the development of second-generation inhibitors of Grb7, which is involved in cancer progression. Grb7 is an adapter protein that is involved in signalling pathways that mediate eukaryotic cell proliferation and migration. Its overexpression in several cancer types has implicated it in cancer progression and led to the development of the G7-18NATE cyclic peptide inhibitor. Here, the preparation of crystals of G7-18NATE in complex with its Grb7 SH2 domain target is reported. Crystals of the complex were grown by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant at room temperature. X-ray diffraction data were collected from crystals to 2.4 Å resolution using synchrotron X-ray radiation at 100 K. The diffraction was consistent with space group P2 1 , with unit-cell parameters a = 52.7, b = 79.1, c = 54.7 Å, α = γ = 90.0, β = 104.4°. The structure of the G7-18NATE peptide in complex with its target will facilitate the rational development of Grb7-targeted cancer therapeutics

  4. Protein kinase inhibitor peptide (PKI): a family of endogenous neuropeptides that modulate neuronal cAMP-dependent protein kinase function.

    Science.gov (United States)

    Dalton, George D; Dewey, William L

    2006-02-01

    Signal transduction cascades involving cAMP-dependent protein kinase are highly conserved among a wide variety of organisms. Given the universal nature of this enzyme it is not surprising that cAMP-dependent protein kinase plays a critical role in numerous cellular processes. This is particularly evident in the nervous system where cAMP-dependent protein kinase is involved in neurotransmitter release, gene transcription, and synaptic plasticity. Protein kinase inhibitor peptide (PKI) is an endogenous thermostable peptide that modulates cAMP-dependent protein kinase function. PKI contains two distinct functional domains within its amino acid sequence that allow it to: (1) potently and specifically inhibit the activity of the free catalytic subunit of cAMP-dependent protein kinase and (2) export the free catalytic subunit of cAMP-dependent protein kinase from the nucleus. Three distinct PKI isoforms (PKIalpha, PKIbeta, PKIgamma) have been identified and each isoform is expressed in the brain. PKI modulates neuronal synaptic activity, while PKI also is involved in morphogenesis and symmetrical left-right axis formation. In addition, PKI also plays a role in regulating gene expression induced by cAMP-dependent protein kinase. Future studies should identify novel physiological functions for endogenous PKI both in the nervous system and throughout the body. Most interesting will be the determination whether functional differences exist between individual PKI isoforms which is an intriguing possibility since these isoforms exhibit: (1) cell-type specific tissue expression patterns, (2) different potencies for the inhibition of cAMP-dependent protein kinase activity, and (3) expression patterns that are hormonally, developmentally and cell-cycle regulated. Finally, synthetic peptide analogs of endogenous PKI will continue to be invaluable tools that are used to elucidate the role of cAMP-dependent protein kinase in a variety of cellular processes throughout the nervous

  5. Dual function of a bee (Apis cerana) inhibitor cysteine knot peptide that acts as an antifungal peptide and insecticidal venom toxin.

    Science.gov (United States)

    Park, Hee Geun; Kyung, Seung Su; Lee, Kwang Sik; Kim, Bo Yeon; Choi, Yong Soo; Yoon, Hyung Joo; Kwon, Hyung Wook; Je, Yeon Ho; Jin, Byung Rae

    2014-12-01

    Inhibitor cysteine knot (ICK) peptides exhibit ion channel blocking, insecticidal, and antimicrobial activities, but currently, no functional roles for bee-derived ICK peptides have been identified. In this study, a bee (Apis cerana) ICK peptide (AcICK) that acts as an antifungal peptide and as an insecticidal venom toxin was identified. AcICK contains an ICK fold that is expressed in the epidermis, fat body, or venom gland and is present as a 6.6-kDa peptide in bee venom. Recombinant AcICK peptide (expressed in baculovirus-infected insect cells) bound directly to Beauveria bassiana and Fusarium graminearum, but not to Escherichia coli or Bacillus thuringiensis. Consistent with these findings, AcICK showed antifungal activity, indicating that AcICK acts as an antifungal peptide. Furthermore, AcICK expression is induced in the fat body and epidermis after injection with B. bassiana. These results provide insight into the role of AcICK during the innate immune response following fungal infection. Additionally, we show that AcICK has insecticidal activity. Our results demonstrate a functional role for AcICK in bees: AcICK acts as an antifungal peptide in innate immune reactions in the body and as an insecticidal toxin in venom. The finding that the AcICK peptide functions with different mechanisms of action in the body and in venom highlights the two-pronged strategy that is possible with the bee ICK peptide. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Affinity Purification and Comparative Biosensor Analysis of Citrulline-Peptide-Specific Antibodies in Rheumatoid Arthritis

    Directory of Open Access Journals (Sweden)

    Eszter Szarka

    2018-01-01

    Full Text Available Background: In rheumatoid arthritis (RA, anti-citrullinated protein/peptide antibodies (ACPAs are responsible for disease onset and progression, however, our knowledge is limited on ligand binding affinities of autoantibodies with different citrulline-peptide specificity. Methods: Citrulline-peptide-specific ACPA IgGs were affinity purified and tested by ELISA. Binding affinities of ACPA IgGs and serum antibodies were compared by surface plasmon resonance (SPR analysis. Bifunctional nanoparticles harboring a multi-epitope citrulline-peptide and a complement-activating peptide were used to induce selective depletion of ACPA-producing B cells. Results: KD values of affinity-purified ACPA IgGs varied between 10−6 and 10−8 M and inversely correlated with disease activity. Based on their cross-reaction with citrulline-peptides, we designed a novel multi-epitope peptide, containing Cit-Gly and Ala-Cit motifs in two–two copies, separated with a short, neutral spacer. This peptide detected antibodies in RA sera with 66% sensitivity and 98% specificity in ELISA and was recognized by 90% of RA sera, while none of the healthy samples in SPR. When coupled to nanoparticles, the multi-epitope peptide specifically targeted and depleted ACPA-producing B cells ex vivo. Conclusions: The unique multi-epitope peptide designed based on ACPA cross-reactivity might be suitable to develop better diagnostics and novel therapies for RA.

  7. Alterations in brain Protein Kinase A activity and reversal of morphine tolerance by two fragments of native Protein Kinase A inhibitor peptide (PKI).

    Science.gov (United States)

    Dalton, George D; Smith, Forrest L; Smith, Paul A; Dewey, William L

    2005-04-01

    Two peptide fragments of native Protein Kinase A inhibitor (PKI), PKI-(6-22)-amide and PKI-(Myr-14-22)-amide, significantly reversed low-level morphine antinociceptive tolerance in mice. The inhibition of Protein Kinase A (PKA) activity by both peptide fragments was then measured in specific brain regions (thalamus, periaqueductal gray (PAG), and medulla) and in lumbar spinal cord (LSC), which in previous studies have been shown to play a role in morphine-induced analgesia. In drug naive animals, cytosolic PKA activity was greater than particulate PKA activity in each region, while cytosolic and particulate PKA activities were greater in thalamus and PAG compared to medulla and LSC. The addition of both peptides to homogenates from each region completely abolished cytosolic and particulate PKA activities in vitro. Following injection into the lateral ventricle of the brain of drug naive mice and morphine-tolerant mice, both peptides inhibited PKA activity in the cytosolic, but not the particulate fraction of LSC. In addition, cytosolic and particulate PKA activities were inhibited by both peptides in thalamus. These results demonstrate that the inhibition of PKA reverses morphine tolerance. Moreover, the inhibition of PKA activity in specific brain regions and LSC from morphine-tolerant mice by PKI analogs administered i.c.v. is evidence that PKA plays a role in morphine tolerance.

  8. Incretin-based treatment of type 2 diabetes: glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors

    DEFF Research Database (Denmark)

    Deacon, Carolyn F

    2007-01-01

    Incretins are gut peptides that potentiate nutrient-stimulated insulin secretion following meal ingestion. Activities of the dominant incretins, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide, include glucose-dependent stimulation of insulin secretion and, in preclin...... and liraglutide) and DPP-4 inhibitors that act to increase concentrations of endogenous intact incretins (e.g. sitagliptin and vildagliptin). Clinical trials of these incretin-based therapies have shown them to be effective in improving glycaemic control in patients with T2DM....

  9. Structure-Based Drug Design of Small Molecule Peptide Deformylase Inhibitors to Treat Cancer

    Directory of Open Access Journals (Sweden)

    Jian Gao

    2016-03-01

    Full Text Available Human peptide deformylase (HsPDF is an important target for anticancer drug discovery. In view of the limited HsPDF, inhibitors were reported, and high-throughput virtual screening (HTVS studies based on HsPDF for developing new PDF inhibitors remain to be reported. We reported here on diverse small molecule inhibitors with excellent anticancer activities designed based on HTVS and molecular docking studies using the crystal structure of HsPDF. The compound M7594_0037 exhibited potent anticancer activities against HeLa, A549 and MCF-7 cell lines with IC50s of 35.26, 29.63 and 24.63 μM, respectively. Molecular docking studies suggested that M7594_0037 and its three derivatives could interact with HsPDF by several conserved hydrogen bonds. Moreover, the pharmacokinetic and toxicity properties of M7594_0037 and its derivatives were predicted using the OSIRIS property explorer. Thus, M7594_0037 and its derivatives might represent a promising scaffold for the further development of novel anticancer drugs.

  10. Ligand and Structure-Based Approaches for the Identification of Peptide Deformylase Inhibitors as Antibacterial Drugs.

    Science.gov (United States)

    Gao, Jian; Liang, Li; Zhu, Yasheng; Qiu, Shengzhi; Wang, Tao; Zhang, Ling

    2016-07-15

    Peptide deformylase (PDF) is a metalloprotease catalyzing the removal of a formyl group from newly synthesized proteins, which makes it an important antibacterial drug target. Given the importance of PDF inhibitors like actinonin in antibacterial drug discovery, several reported potent PDF inhibitors were used to develop pharmacophore models using the Galahad module of Sybyl 7.1 software. Generated pharmacophore models were composed of two donor atom centers, four acceptor atom centers and two hydrophobic groups. Model-1 was screened against the Zinc database and several compounds were retrieved as hits. Compounds with Qfit values of more than 60 were employed to perform a molecular docking study with the receptor Escherichia coli PDF, then compounds with docking score values of more than 6 were used to predict the in silico pharmacokinetic and toxicity risk via OSIRIS property explorer. Two known PDF inhibitors were also used to perform a molecular docking study with E. coli PDF as reference molecules. The results of the molecular docking study were validated by reproducing the crystal structure of actinonin. Molecular docking and in silico pharmacokinetic and toxicity prediction studies suggested that ZINC08740166 has a relatively high docking score of 7.44 and a drug score of 0.78.

  11. Designing peptide inhibitor of insulin receptor to induce diabetes mellitus type 2 in animal model Mus musculus.

    Science.gov (United States)

    Permatasari, Galuh W; Utomo, Didik H; Widodo

    2016-10-01

    A designing peptide as agent for inducing diabetes mellitus type 2 (T2DM) in an animal model is challenging. The computational approach provides a sophisticated tool to design a functional peptide that may block the insulin receptor activity. The peptide that able to inhibit the binding between insulin and insulin receptor is a warrant for inducing T2DM. Therefore, we designed a potential peptide inhibitor of insulin receptor as an agent to generate T2DM animal model by bioinformatics approach. The peptide has been developed based on the structure of insulin receptor binding site of insulin and then modified it to obtain the best properties of half life, hydrophobicity, antigenicity, and stability binding into insulin receptor. The results showed that the modified peptide has characteristics 100h half-life, high-affinity -95.1±20, and high stability 28.17 in complex with the insulin receptor. Moreover, the modified peptide has molecular weight 4420.8g/Mol and has no antigenic regions. Based on the molecular dynamic simulation, the complex of modified peptide-insulin receptor is more stable than the commercial insulin receptor blocker. This study suggested that the modified peptide has the promising performance to block the insulin receptor activity that potentially induce diabetes mellitus type 2 in mice. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Non-peptidic cruzain inhibitors with trypanocidal activity discovered by virtual screening and in vitro assay.

    Directory of Open Access Journals (Sweden)

    Helton J Wiggers

    (compound Nequimed176 is highlighted as a novel non-peptidic, non-covalent cruzain inhibitor as a trypanocidal agent candidate for optimization.

  13. Non-peptidic cruzain inhibitors with trypanocidal activity discovered by virtual screening and in vitro assay.

    Science.gov (United States)

    Wiggers, Helton J; Rocha, Josmar R; Fernandes, William B; Sesti-Costa, Renata; Carneiro, Zumira A; Cheleski, Juliana; da Silva, Albérico B F; Juliano, Luiz; Cezari, Maria H S; Silva, João S; McKerrow, James H; Montanari, Carlos A

    2013-01-01

    Nequimed176) is highlighted as a novel non-peptidic, non-covalent cruzain inhibitor as a trypanocidal agent candidate for optimization.

  14. Specific Affinity Enrichment of Electrochemically Cleaved Peptides Based on Cu(II)-Mediated Spirolactone Tagging

    NARCIS (Netherlands)

    Zhang, Tao; de Vries, Marcel P.; Permentier, Hjalmar P.; Bischoff, Rainer

    2017-01-01

    Specific digestion of proteins is an essential step for mass spectrometry-based proteomics, and the chemical labeling of the resulting peptides is often used for peptide enrichment or the introduction of desirable tags. Electrochemical oxidation yielding specific cleavage C-terminal to tyrosine

  15. Peptide Inhibitor of Complement C1 (PIC1 Rapidly Inhibits Complement Activation after Intravascular Injection in Rats.

    Directory of Open Access Journals (Sweden)

    Julia A Sharp

    Full Text Available The complement system has been increasingly recognized to play a pivotal role in a variety of inflammatory and autoimmune diseases. Consequently, therapeutic modulators of the classical, lectin and alternative pathways of the complement system are currently in pre-clinical and clinical development. Our laboratory has identified a peptide that specifically inhibits the classical and lectin pathways of complement and is referred to as Peptide Inhibitor of Complement C1 (PIC1. In this study, we determined that the lead PIC1 variant demonstrates a salt-dependent binding to C1q, the initiator molecule of the classical pathway. Additionally, this peptide bound to the lectin pathway initiator molecule MBL as well as the ficolins H, M and L, suggesting a common mechanism of PIC1 inhibitory activity occurs via binding to the collagen-like tails of these collectin molecules. We further analyzed the effect of arginine and glutamic acid residue substitution on the complement inhibitory activity of our lead derivative in a hemolytic assay and found that the original sequence demonstrated superior inhibitory activity. To improve upon the solubility of the lead derivative, a pegylated, water soluble variant was developed, structurally characterized and demonstrated to inhibit complement activation in mouse plasma, as well as rat, non-human primate and human serum in vitro. After intravenous injection in rats, the pegylated derivative inhibited complement activation in the blood by 90% after 30 seconds, demonstrating extremely rapid function. Additionally, no adverse toxicological effects were observed in limited testing. Together these results show that PIC1 rapidly inhibits classical complement activation in vitro and in vivo and is functional for a variety of animal species, suggesting its utility in animal models of classical complement-mediated diseases.

  16. Anti-Hemagglutinin Antibody Derived Lead Peptides for Inhibitors of Influenza Virus Binding.

    Directory of Open Access Journals (Sweden)

    Henry Memczak

    Full Text Available Antibodies against spike proteins of influenza are used as a tool for characterization of viruses and therapeutic approaches. However, development, production and quality control of antibodies is expensive and time consuming. To circumvent these difficulties, three peptides were derived from complementarity determining regions of an antibody heavy chain against influenza A spike glycoprotein. Their binding properties were studied experimentally, and by molecular dynamics simulations. Two peptide candidates showed binding to influenza A/Aichi/2/68 H3N2. One of them, termed PeB, with the highest affinity prevented binding to and infection of target cells in the micromolar region without any cytotoxic effect. PeB matches best the conserved receptor binding site of hemagglutinin. PeB bound also to other medical relevant influenza strains, such as human-pathogenic A/California/7/2009 H1N1, and avian-pathogenic A/Mute Swan/Rostock/R901/2006 H7N1. Strategies to improve the affinity and to adapt specificity are discussed and exemplified by a double amino acid substituted peptide, obtained by substitutional analysis. The peptides and their derivatives are of great potential for drug development as well as biosensing.

  17. Cleavage specificity analysis of six type II transmembrane serine proteases (TTSPs using PICS with proteome-derived peptide libraries.

    Directory of Open Access Journals (Sweden)

    Olivier Barré

    Full Text Available Type II transmembrane serine proteases (TTSPs are a family of cell membrane tethered serine proteases with unclear roles as their cleavage site specificities and substrate degradomes have not been fully elucidated. Indeed just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors.To profile the active site specificities of the TTSPs, we applied Proteomic Identification of protease Cleavage Sites (PICS. Human proteome-derived database searchable peptide libraries were assayed with six human TTSPs (matriptase, matriptase-2, matriptase-3, HAT, DESC and hepsin to simultaneously determine sequence preferences on the N-terminal non-prime (P and C-terminal prime (P' sides of the scissile bond. Prime-side cleavage products were isolated following biotinylation and identified by tandem mass spectrometry. The corresponding non-prime side sequences were derived from human proteome databases using bioinformatics. Sequencing of 2,405 individual cleaved peptides allowed for the development of the family consensus protease cleavage site specificity revealing a strong specificity for arginine in the P1 position and surprisingly a lysine in P1' position. TTSP cleavage between R↓K was confirmed using synthetic peptides. By parsing through known substrates and known structures of TTSP catalytic domains, and by modeling the remainder, structural explanations for this strong specificity were derived.Degradomics analysis of 2,405 cleavage sites revealed a similar and characteristic TTSP family specificity at the P1 and P1' positions for arginine and lysine in unfolded peptides. The prime side is important for cleavage specificity, thus making these proteases unusual within the tryptic-enzyme class that generally has overriding non-prime side specificity.

  18. Two-dimensional NMR studies of squash family inhibitors. Sequence-specific proton assignments and secondary structure of reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor III

    Energy Technology Data Exchange (ETDEWEB)

    Krisnamoorthi, R.; Yuxi Gong; Chanlan Sun Lin (Kansas State Univ., Manhattan (United States)); VanderVelde, D. (Univ. of Kansas, Lawrence (United States))

    1992-01-28

    The solution structure of reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor III (CMTI-III*) was investigated by two-dimensional proton nuclear magnetic resonance (2D NMR) spectroscopy. CMTI-III*, prepared by reacting CMTI-III with trypsin which cleaved the Arg5-Ile6 peptide bond, had the two fragments held together by a disulfide linkage. Sequence-specific {sup 1}H NMR resonance assignments were made for all the 29 amino acid residues of the protein. The secondary structure of CMTI-III*, as deduced from NOESY cross peaks and identification of slowly exchanging hydrogens, contains two turns, a 3{sub 10}-helix, and a triple-stranded {beta}-sheet. Sequential proton assignments were also made for the virgin inhibitor, CMTI-III, at pH 4.71, 30C. Comparison of backbone hydrogen chemical shifts of CMTI-III and CMTI-III* revealed significant changes for residues located far away from the reactive-site region as well as for those located near it, indicating tertiary structural changes that are transmitted through most of the 29 residues of the inhibitor protein. These chemical shift changes were relatively small compared to changes that occurred upon hydrolysis of the reactive-site peptide bond between Arg 5 and Ile6 in CMTI-III.

  19. Comparative in vitro activities of investigational peptide deformylase inhibitor NVP LBM-415 and other agents against human mycoplasmas and ureaplasmas.

    Science.gov (United States)

    Waites, Ken B; Reddy, Nipun B; Crabb, Donna M; Duffy, Lynn B

    2005-06-01

    Peptide deformylase inhibitor LBM-415 and seven other drugs were tested against Mycoplasma pneumoniae (100 isolates), Mycoplasma hominis (20 isolates), Mycoplasma fermentans (10 isolates), and Ureaplasma species (50 isolates). LBM-415 was active against M. pneumoniae (MICs,

  20. Glucagon-like peptide 1 and inhibitors of dipeptidyl peptidase IV in the treatment of type 2 diabetes mellitus

    DEFF Research Database (Denmark)

    Holst, Jens Juul; Deacon, Carolyn F

    2004-01-01

    Proof-of-concept for the efficacy of a glucagon-like peptide 1 (GLP-1)-based therapy of patients with type 2 diabetes was provided in 2002 by means of prolonged continuous subcutaneous infusion of native GLP-1. Since then, several long-acting analogues of GLP-1, as well as inhibitors of dipeptidyl...

  1. Structural Study of a New HIV-1 Entry Inhibitor and Interaction with the HIV-1 Fusion Peptide in Dodecylphosphocholine Micelles.

    Science.gov (United States)

    Pérez, Yolanda; Gómara, Maria José; Yuste, Eloísa; Gómez-Gutierrez, Patricia; Pérez, Juan Jesús; Haro, Isabel

    2017-08-25

    Previous studies support the hypothesis that the envelope GB virus C (GBV-C) E1 protein interferes the HIV-1 entry and that a peptide, derived from the region 139-156 of this protein, has been defined as a novel HIV-1 entry inhibitor. In this work, we firstly focus on the characterization of the structural features of this peptide, which are determinant for its anti-HIV-1 activity and secondly, on the study of its interaction with the proposed viral target (i.e., the HIV-1 fusion peptide). We report the structure of the peptide determined by NMR spectroscopy in dodecylphosphocholine (DPC) micelles solved by using restrained molecular dynamics calculations. The acquisition of different NMR experiments in DPC micelles (i.e., peptide-peptide titration, diffusion NMR spectroscopy, and addition of paramagnetic relaxation agents) allows a proposal of an inhibition mechanism. We conclude that a 18-mer peptide from the non-pathogenic E1 GBV-C protein, with a helix-turn-helix structure inhibits HIV-1 by binding to the HIV-1 fusion peptide at the membrane level, thereby interfering with those domains in the HIV-1, which are critical for stabilizing the six-helix bundle formation in a membranous environment. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Rapid Analysis of Protein Farnesyltransferase Substrate Specificity Using Peptide Libraries and Isoprenoid Diphosphate Analogues

    OpenAIRE

    Wang, Yen-Chih; Dozier, Jonathan K.; Beese, Lorena S.; Distefano, Mark D.

    2014-01-01

    Protein farnesytransferase (PFTase) catalyzes the farnesylation of proteins with a carboxy-terminal tetrapeptide sequence denoted as a Ca1a2X box. To explore the specificity of this enzyme, an important therapeutic target, solid-phase peptide synthesis in concert with a peptide inversion strategy was used to prepare two libraries, each containing 380 peptides. The libraries were screened using an alkyne-containing isoprenoid analogue followed by click chemistry with biotin azide and subsequen...

  3. Describing the Peptide Binding Specificity of HLA-C

    DEFF Research Database (Denmark)

    Rasmussen, Michael; Harndahl, Mikkel Nors; Nielsen, Morten

    for 5 HLA-C molecules and for all, but one, molecule we find a high frequency of binders, >70%, among these peptides. To extend the examined peptide space, we use bioinformatic prediction tools to search for additional binders. Finally, we update our prediction tool, NetMHCpan, with the HLA-C affinity......Human leukocyte antigen (HLA) presents peptides to T-cells for immune scrutiny. Whereas HLA-A and -B have been described in great detail, HLA-C has received much less attention. Here, to increase the coverage of HLA-C and the accuracy of the corresponding tools, we have generated HLA-C molecules...... data and show that the predictive performance for HLA-C molecules now is increased to a level comparable withthat of HLA-A and -B. These novel HLA-C molecules and predictors are successfully used to generate HLA-C tetramers and validate HLA-C-restricted T cell responses....

  4. Reversal of the Hofmeister Series: Specific Ion Effects on Peptides

    Czech Academy of Sciences Publication Activity Database

    Paterová, Jana; Rembert, K. B.; Heyda, J.; Kurra, Y.; Okur, H. I.; Liu, W. R.; Hilty, Ch.; Cremer, P. S.; Jungwirth, Pavel

    2013-01-01

    Roč. 117, č. 27 (2013), s. 8150-8158 ISSN 1520-6106 R&D Projects: GA ČR GBP208/12/G016 Institutional support: RVO:61388963 Keywords : ions * peptide * NMR * molecular dynamics Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.377, year: 2013

  5. A Simple PB/LIE Free Energy Function Accurately Predicts the Peptide Binding Specificity of the Tiam1 PDZ Domain.

    Science.gov (United States)

    Panel, Nicolas; Sun, Young Joo; Fuentes, Ernesto J; Simonson, Thomas

    2017-01-01

    PDZ domains generally bind short amino acid sequences at the C-terminus of target proteins, and short peptides can be used as inhibitors or model ligands. Here, we used experimental binding assays and molecular dynamics simulations to characterize 51 complexes involving the Tiam1 PDZ domain and to test the performance of a semi-empirical free energy function. The free energy function combined a Poisson-Boltzmann (PB) continuum electrostatic term, a van der Waals interaction energy, and a surface area term. Each term was empirically weighted, giving a Linear Interaction Energy or "PB/LIE" free energy. The model yielded a mean unsigned deviation of 0.43 kcal/mol and a Pearson correlation of 0.64 between experimental and computed free energies, which was superior to a Null model that assumes all complexes have the same affinity. Analyses of the models support several experimental observations that indicate the orientation of the α 2 helix is a critical determinant for peptide specificity. The models were also used to predict binding free energies for nine new variants, corresponding to point mutants of the Syndecan1 and Caspr4 peptides. The predictions did not reveal improved binding; however, they suggest that an unnatural amino acid could be used to increase protease resistance and peptide lifetimes in vivo . The overall performance of the model should allow its use in the design of new PDZ ligands in the future.

  6. A Simple PB/LIE Free Energy Function Accurately Predicts the Peptide Binding Specificity of the Tiam1 PDZ Domain

    Directory of Open Access Journals (Sweden)

    Nicolas Panel

    2017-09-01

    Full Text Available PDZ domains generally bind short amino acid sequences at the C-terminus of target proteins, and short peptides can be used as inhibitors or model ligands. Here, we used experimental binding assays and molecular dynamics simulations to characterize 51 complexes involving the Tiam1 PDZ domain and to test the performance of a semi-empirical free energy function. The free energy function combined a Poisson-Boltzmann (PB continuum electrostatic term, a van der Waals interaction energy, and a surface area term. Each term was empirically weighted, giving a Linear Interaction Energy or “PB/LIE” free energy. The model yielded a mean unsigned deviation of 0.43 kcal/mol and a Pearson correlation of 0.64 between experimental and computed free energies, which was superior to a Null model that assumes all complexes have the same affinity. Analyses of the models support several experimental observations that indicate the orientation of the α2 helix is a critical determinant for peptide specificity. The models were also used to predict binding free energies for nine new variants, corresponding to point mutants of the Syndecan1 and Caspr4 peptides. The predictions did not reveal improved binding; however, they suggest that an unnatural amino acid could be used to increase protease resistance and peptide lifetimes in vivo. The overall performance of the model should allow its use in the design of new PDZ ligands in the future.

  7. Peptide specific expansion of CD8(+) T cells by recombinant plate bound MHC/peptide complexes

    DEFF Research Database (Denmark)

    Schmidt, Esben G W; Buus, Soren; Thorn, Mette

    2009-01-01

    to in vitro T cell stimulation was investigated. By use of an antigenic peptide derived from the cytomegalovirus (CMVp) we tested the stimulatory efficacy of recombinant plate bound MHC molecules (PB-MHC), being immobilized in culture plates. A single stimulation of non-adherent peripheral blood mononuclear...

  8. Identification of Four-Jointed Box 1 (FJX1-Specific Peptides for Immunotherapy of Nasopharyngeal Carcinoma.

    Directory of Open Access Journals (Sweden)

    San Jiun Chai

    Full Text Available Nasopharyngeal carcinoma (NPC is highly prevalent in South East Asia and China. The poor outcome is due to late presentation, recurrence, distant metastasis and limited therapeutic options. For improved treatment outcome, immunotherapeutic approaches focusing on dendritic and autologous cytotoxic T-cell based therapies have been developed, but cost and infrastructure remain barriers for implementing these in low-resource settings. As our prior observations had found that four-jointed box 1 (FJX1, a tumor antigen, is overexpressed in NPCs, we investigated if short 9-20 amino acid sequence specific peptides matching to FJX1 requiring only intramuscular immunization to train host immune systems would be a better treatment option for this disease. Thus, we designed 8 FJX1-specific peptides and implemented an assay system to first, assess the binding of these peptides to HLA-A2 molecules on T2 cells. After, ELISPOT assays were used to determine the peptides immunogenicity and ability to induce potential cytotoxicity activity towards cancer cells. Also, T-cell proliferation assay was used to evaluate the potential of MHC class II peptides to stimulate the expansion of isolated T-cells. Our results demonstrate that these peptides are immunogenic and peptide stimulated T-cells were able to induce peptide-specific cytolytic activity specifically against FJX1-expressing cancer cells. In addition, we demonstrated that the MHC class II peptides were capable of inducing T-cell proliferation. Our results suggest that these peptides are capable of inducing specific cytotoxic cytokines secretion against FJX1-expressing cancer cells and serve as a potential vaccine-based therapy for NPC patients.

  9. Identification of Four-Jointed Box 1 (FJX1)-Specific Peptides for Immunotherapy of Nasopharyngeal Carcinoma

    Science.gov (United States)

    Chai, San Jiun; Yap, Yoke Yeow; Foo, Yoke Ching; Yap, Lee Fah; Ponniah, Sathibalan; Teo, Soo Hwang; Cheong, Sok Ching; Patel, Vyomesh; Lim, Kue Peng

    2015-01-01

    Nasopharyngeal carcinoma (NPC) is highly prevalent in South East Asia and China. The poor outcome is due to late presentation, recurrence, distant metastasis and limited therapeutic options. For improved treatment outcome, immunotherapeutic approaches focusing on dendritic and autologous cytotoxic T-cell based therapies have been developed, but cost and infrastructure remain barriers for implementing these in low-resource settings. As our prior observations had found that four-jointed box 1 (FJX1), a tumor antigen, is overexpressed in NPCs, we investigated if short 9–20 amino acid sequence specific peptides matching to FJX1 requiring only intramuscular immunization to train host immune systems would be a better treatment option for this disease. Thus, we designed 8 FJX1-specific peptides and implemented an assay system to first, assess the binding of these peptides to HLA-A2 molecules on T2 cells. After, ELISPOT assays were used to determine the peptides immunogenicity and ability to induce potential cytotoxicity activity towards cancer cells. Also, T-cell proliferation assay was used to evaluate the potential of MHC class II peptides to stimulate the expansion of isolated T-cells. Our results demonstrate that these peptides are immunogenic and peptide stimulated T-cells were able to induce peptide-specific cytolytic activity specifically against FJX1-expressing cancer cells. In addition, we demonstrated that the MHC class II peptides were capable of inducing T-cell proliferation. Our results suggest that these peptides are capable of inducing specific cytotoxic cytokines secretion against FJX1-expressing cancer cells and serve as a potential vaccine-based therapy for NPC patients. PMID:26536470

  10. Sequence-Specific Model for Peptide Retention Time Prediction in Strong Cation Exchange Chromatography.

    Science.gov (United States)

    Gussakovsky, Daniel; Neustaeter, Haley; Spicer, Victor; Krokhin, Oleg V

    2017-11-07

    The development of a peptide retention prediction model for strong cation exchange (SCX) separation on a Polysulfoethyl A column is reported. Off-line 2D LC-MS/MS analysis (SCX-RPLC) of S. cerevisiae whole cell lysate was used to generate a retention dataset of ∼30 000 peptides, sufficient for identifying the major sequence-specific features of peptide retention mechanisms in SCX. In contrast to RPLC/hydrophilic interaction liquid chromatography (HILIC) separation modes, where retention is driven by hydrophobic/hydrophilic contributions of all individual residues, SCX interactions depend mainly on peptide charge (number of basic residues at acidic pH) and size. An additive model (incorporating the contributions of all 20 residues into the peptide retention) combined with a peptide length correction produces a 0.976 R 2 value prediction accuracy, significantly higher than the additive models for either HILIC or RPLC. Position-dependent effects on peptide retention for different residues were driven by the spatial orientation of tryptic peptides upon interaction with the negatively charged surface functional groups. The positively charged N-termini serve as a primary point of interaction. For example, basic residues (Arg, His, Lys) increase peptide retention when located closer to the N-terminus. We also found that hydrophobic interactions, which could lead to a mixed-mode separation mechanism, are largely suppressed at 20-30% of acetonitrile in the eluent. The accuracy of the final Sequence-Specific Retention Calculator (SSRCalc) SCX model (∼0.99 R 2 value) exceeds all previously reported predictors for peptide LC separations. This also provides a solid platform for method development in 2D LC-MS protocols in proteomics and peptide retention prediction filtering of false positive identifications.

  11. Peptide ligands specific to the oxidized form of escherichia coli thioredoxin.

    Energy Technology Data Exchange (ETDEWEB)

    Scholle, M. D.; Banach, B. S.; Hamdan, S. M.; Richardson, C. C.; Kay, B. K.; Biosciences Division; Amunix, Inc.; Univ. of Illinois at Chicago; Harvard Medical School

    2008-11-01

    Thioredoxin (Trx) is a highly conserved redox protein involved in several essential cellular processes. In this study, our goal was to isolate peptide ligands to Escherichia coli Trx that mimic protein-protein interactions, specifically the T7 polymerase-Trx interaction. To do this, we subjected Trx to affinity selection against a panel of linear and cysteine-constrained peptides using M13 phage display. A novel cyclized conserved peptide sequence, with a motif of C(D/N/S/T/G)D(S/T)-hydrophobic-C-X-hydrophobic-P, was isolated to Trx. These peptides bound specifically to the E. coli Trx when compared to the human and spirulina homologs. An alanine substitution of the active site cysteines (CGPC) resulted in a significant loss of peptide binding affinity to the Cys-32 mutant. The peptides were also characterized in the context of Trx's role as a processivity factor of the T7 DNA polymerase (gp5). As the interaction between gp5 and Trx normally takes place under reducing conditions, which might interfere with the conformation of the disulfide-bridged peptides, we made use of a 22 residue deletion mutant of gp5 in the thioredoxin binding domain (gp5{Delta}22) that bypassed the requirements of reducing conditions to interact with Trx. A competition study revealed that the peptide selectively inhibits the interaction of gp5{Delta}22 with Trx, under oxidizing conditions, with an IC50 of {approx} 10 {micro}M.

  12. Rapid discovery of peptide capture candidates with demonstrated specificity for structurally similar toxins

    Science.gov (United States)

    Sarkes, Deborah A.; Hurley, Margaret M.; Coppock, Matthew B.; Farrell, Mikella E.; Pellegrino, Paul M.; Stratis-Cullum, Dimitra N.

    2016-05-01

    Peptides have emerged as viable alternatives to antibodies for molecular-based sensing due to their similarity in recognition ability despite their relative structural simplicity. Various methods for peptide capture reagent discovery exist, including phage display, yeast display, and bacterial display. One of the primary advantages of peptide discovery by bacterial display technology is the speed to candidate peptide capture agent, due to both rapid growth of bacteria and direct utilization of the sorted cells displaying each individual peptide for the subsequent round of biopanning. We have previously isolated peptide affinity reagents towards protective antigen of Bacillus anthracis using a commercially available automated magnetic sorting platform with improved enrichment as compared to manual magnetic sorting. In this work, we focus on adapting our automated biopanning method to a more challenging sort, to demonstrate the specificity possible with peptide capture agents. This was achieved using non-toxic, recombinant variants of ricin and abrin, RiVax and abrax, respectively, which are structurally similar Type II ribosomal inactivating proteins with significant sequence homology. After only two rounds of biopanning, enrichment of peptide capture candidates binding abrax but not RiVax was achieved as demonstrated by Fluorescence Activated Cell Sorting (FACS) studies. Further sorting optimization included negative sorting against RiVax, proper selection of autoMACS programs for specific sorting rounds, and using freshly made buffer and freshly thawed protein target for each round of biopanning for continued enrichment over all four rounds. Most of the resulting candidates from biopanning for abrax binding peptides were able to bind abrax but not RiVax, demonstrating that short peptide sequences can be highly specific even at this early discovery stage.

  13. Complementary roles in cancer prevention: protease inhibitor makes the cancer preventive peptide lunasin bioavailable.

    Directory of Open Access Journals (Sweden)

    Chia-Chien Hsieh

    Full Text Available BACKGROUND: The lower incidence of breast cancer among Asian women compared with Western countries has been partly attributed to soy in the Asian diet, leading to efforts to identify the bioactive components that are responsible. Soy Bowman Birk Inhibitor Concentrate (BBIC is a known cancer preventive agent now in human clinical trials. METHODOLOGY/PRINCIPAL FINDINGS: The objectives of this work are to establish the presence and delineate the in vitro activity of lunasin and BBI found in BBIC, and study their bioavailability after oral administration to mice and rats. We report that lunasin and BBI are the two main bioactive ingredients of BBIC based on inhibition of foci formation, lunasin being more efficacious than BBI on an equimolar basis. BBI and soy Kunitz Trypsin Inhibitor protect lunasin from in vitro digestion with pancreatin. Oral administration of (3H-labeled lunasin with lunasin-enriched soy results in 30% of the peptide reaching target tissues in an intact and bioactive form. In a xenograft model of nude mice transplanted with human breast cancer MDA-MB-231 cells, intraperitoneal injections of lunasin, at 20 mg/kg and 4 mg/kg body weight, decrease tumor incidence by 49% and 33%, respectively, compared with the vehicle-treated group. In contrast, injection with BBI at 20 mg/kg body weight shows no effect on tumor incidence. Tumor generation is significantly reduced with the two doses of lunasin, while BBI is ineffective. Lunasin inhibits cell proliferation and induces cell death in the breast tumor sections. CONCLUSIONS/SIGNIFICANCE: We conclude that lunasin is actually the bioactive cancer preventive agent in BBIC, and BBI simply protects lunasin from digestion when soybean and other seed foods are eaten by humans.

  14. Porcine major histocompatibility complex (MHC) class I molecules and analysis of their peptide-binding specificities

    DEFF Research Database (Denmark)

    Pedersen, Lasse Eggers; Harndahl, Mikkel; Rasmussen, Michael

    2011-01-01

    a HLA-I molecule (HLA-A*11:01), thereby generating recombinant human/swine chimeric MHC-I molecules as well as the intact SLA-1*0401 molecule. Biochemical peptide-binding assays and positional scanning combinatorial peptide libraries were used to analyze the peptide-binding motifs of these molecules....... A pan-specific predictor of peptide–MHC-I binding, NetMHCpan, which was originally developed to cover the binding specificities of all known HLA-I molecules, was successfully used to predict the specificities of the SLA-1*0401 molecule as well as the porcine/human chimeric MHC-I molecules. These data......In all vertebrate animals, CD8+ cytotoxic T lymphocytes (CTLs) are controlled by major histocompatibility complex class I (MHC-I) molecules. These are highly polymorphic peptide receptors selecting and presenting endogenously derived epitopes to circulating CTLs. The polymorphism of the MHC...

  15. Augmentation of Cationic Antimicrobial Peptide Production with Histone Deacetylase Inhibitors as a Novel Epigenetic Therapy for Bacterial Infections

    Directory of Open Access Journals (Sweden)

    Roshan D. Yedery

    2015-01-01

    Full Text Available The emergence of antibiotic resistance seriously threatens our ability to treat many common and medically important bacterial infections. Novel therapeutics are needed that can be used alone or in conjunction with antibiotics. Cationic antimicrobial peptides (CAMPs are important effectors of the host innate defense that exhibit broad-spectrum activity against a wide range of microorganisms. CAMPs are carried within phagocytic granules and are constitutively or inducibly expressed by multiple cell types, including epithelial cells. The role of histone modification enzymes, specifically the histone deacetylases (HDAC, in down-regulating the transcription of CAMP-encoding genes is increasingly appreciated as is the capacity of HDAC inhibitors (HDACi to block the action of HDACs to increase CAMP expression. The use of synthetic and natural HDACi molecules to increase CAMPs on mucosal surfaces, therefore, has potential therapeutic applications. Here, we review host and pathogen regulation of CAMP expression through the induction of HDACs and assess the therapeutic potential of natural and synthetic HDACi based on evidence from tissue culture systems, animal models, and clinical trials.

  16. A MAM7 peptide-based inhibitor of Staphylococcus aureus adhesion does not interfere with in vitro host cell function.

    Directory of Open Access Journals (Sweden)

    Catherine Alice Hawley

    Full Text Available Adhesion inhibitors that block the attachment of pathogens to host tissues may be used synergistically with or as an alternative to antibiotics. The wide-spread bacterial adhesin Multivalent Adhesion Molecule (MAM 7 has recently emerged as a candidate molecule for a broad-spectrum adhesion inhibitor which may be used to prevent bacterial colonization of wounds. Here we have tested if the antibacterial properties of a MAM-based inhibitor could be used to competitively inhibit adhesion of methicillin-resistant Staphylococcus aureus (MRSA to host cells. Additionally, we analyzed its effect on host cellular functions linked to the host receptor fibronectin, such as migration, adhesion and matrix formation in vitro, to evaluate potential side effects prior to advancing our studies to in vivo infection models. As controls, we used inhibitors based on well-characterized bacterial adhesin-derived peptides from F1 and FnBPA, which are known to affect host cellular functions. Inhibitors based on F1 or FnBPA blocked MRSA attachment but at the same time abrogated important cellular functions. A MAM7-based inhibitor did not interfere with host cell function while showing good efficacy against MRSA adhesion in a tissue culture model. These observations provide a possible candidate for a bacterial adhesion inhibitor that does not cause adverse effects on host cells while preventing bacterial infection.

  17. Glucagon-like peptide-1 7-36 amide and peptide YY from the L-cell of the ileal mucosa are potent inhibitors of vagally induced gastric acid secretion in man

    DEFF Research Database (Denmark)

    Wettergren, A; Petersen, H; Orskov, C

    1994-01-01

    BACKGROUND: Glucagon-like peptide (GLP-1) 7-36 amide and peptide YY (PYY) from the L-cell of the ileal mucosa are potent inhibitors of gastric acid secretion in man. It is not clear, however, by which mechanism(s) they inhibit acid secretion. In dogs the inhibitory effect of PYY on acid secretion...

  18. Fusion peptides from oncogenic chimeric proteins as putative specific biomarkers of cancer.

    Science.gov (United States)

    Conlon, Kevin P; Basrur, Venkatesha; Rolland, Delphine; Wolfe, Thomas; Nesvizhskii, Alexey I; MacCoss, Michael J; Lim, Megan S; Elenitoba-Johnson, Kojo S J

    2013-10-01

    Chromosomal translocations encoding chimeric fusion proteins constitute one of the most common mechanisms underlying oncogenic transformation in human cancer. Fusion peptides resulting from such oncogenic chimeric fusions, though unique to specific cancer subtypes, are unexplored as cancer biomarkers. Here we show, using an approach termed fusion peptide multiple reaction monitoring mass spectrometry, the direct identification of different cancer-specific fusion peptides arising from protein chimeras that are generated from the juxtaposition of heterologous genes fused by recurrent chromosomal translocations. Using fusion peptide multiple reaction monitoring mass spectrometry in a clinically relevant scenario, we demonstrate the specific, sensitive, and unambiguous detection of a specific diagnostic fusion peptide in clinical samples of anaplastic large cell lymphoma, but not in a diverse array of benign lymph nodes or other forms of primary malignant lymphomas and cancer-derived cell lines. Our studies highlight the utility of fusion peptides as cancer biomarkers and carry broad implications for the use of protein biomarkers in cancer detection and monitoring.

  19. Novel Peptide with Specific Calcium-Binding Capacity from Schizochytrium sp. Protein Hydrolysates and Calcium Bioavailability in Caco-2 Cells

    Directory of Open Access Journals (Sweden)

    Xixi Cai

    2016-12-01

    Full Text Available Peptide-calcium can probably be a suitable supplement to improve calcium absorption in the human body. In this study, a specific peptide Phe-Tyr (FY with calcium-binding capacity was purified from Schizochytrium sp. protein hydrolysates through gel filtration chromatography and reversed phase HPLC. The calcium-binding capacity of FY reached 128.77 ± 2.57 μg/mg. Results of ultraviolet spectroscopy, fluorescence spectroscopy, and infrared spectroscopy showed that carboxyl groups, amino groups, and amido groups were the major chelating sites. FY-Ca exhibited excellent thermal stability and solubility, which were beneficial to be absorbed and transported in the basic intestinal tract of the human body. Moreover, the calcium bioavailability in Caco-2 cells showed that FY-Ca could enhance calcium uptake efficiency by more than three times when compared with CaCl2, and protect calcium ions against dietary inhibitors, such as tannic acid, oxalate, phytate, and Zn2+. Our findings further the progress of algae-based peptide-calcium, suggesting that FY-Ca has the potential to be developed as functionally nutraceutical additives.

  20. LC-MS/MS Identification of Species-Specific Muscle Peptides in Processed Animal Proteins.

    Science.gov (United States)

    Marchis, Daniela; Altomare, Alessandra; Gili, Marilena; Ostorero, Federica; Khadjavi, Amina; Corona, Cristiano; Ru, Giuseppe; Cappelletti, Benedetta; Gianelli, Silvia; Amadeo, Francesca; Rumio, Cristiano; Carini, Marina; Aldini, Giancarlo; Casalone, Cristina

    2017-12-06

    An innovative analytical strategy has been applied to identify signature peptides able to distinguish among processed animal proteins (PAPs) derived from bovine, pig, fish, and milk products. Proteomics was first used to elucidate the proteome of each source. Starting from the identified proteins and using a funnel based approach, a set of abundant and well characterized peptides with suitable physical-chemical properties (signature peptides) and specific for each source was selected. An on-target LC-ESI-MS/MS method (MRM mode) was set up using standard peptides and was then applied to selectively identify the PAP source and also to distinguish proteins from bovine carcass and milk proteins. We believe that the method described meets the request of the European Commission which has developed a strategy for gradually lifting the "total ban" toward "species to species ban", therefore requiring official methods for species-specific discrimination in feed.

  1. Recombinant heat shock protein 70 functional peptide and alpha-fetoprotein epitope peptide vaccine elicits specific anti-tumor immunity.

    Science.gov (United States)

    Wang, Xiao-Ping; Wang, Qiao-Xia; Lin, Huan-Ping; Xu, Bing; Zhao, Qian; Chen, Kun

    2016-11-01

    Alpha-fetoprotein (AFP) is a marker of hepatocellular carcinoma (HCC) and serves as a target for immunotherapy. However, current treatments targeting AFP are not reproducible and do not provide complete protection against cancer. This issue may be solved by developing novel therapeutic vaccines with enhanced immunogenicity that could effectively target AFP-expressing tumors. In this study, we report construction of a therapeutic peptide vaccine by linking heat shock protein 70 (HSP70) functional peptide to the AFP epitope to obtain HSP70-P/AFP-P. This novel peptide was administered into BALB/c mice to observe the effects. Quantification of AFP-specific CD8 + T cells that secrete IFN-γ in these mice via ELISPOT revealed the synergistic effects of HSP70-P/AFP-P with increased numbers of AFP-specific CD8 + T cells. Similarly, ELISA analysis showed increased granzyme B and perforin released by natural killer cells. Moreover, in vitro cytotoxic T-lymphocyte assays and in vivo tumor preventive experiments clearly showed the higher antitumor effects of HSP70-P/AFP-P against AFP-expressing tumors. These results show that treatment of BALB/c mice with HSP70-P/AFP-P induced stronger T-cells responses and improved protective immunity. Our data suggest that HSP70-P/AFP-P may be used as a therapeutic approach in the treatment of AFP-expressing cancers.

  2. Vaccination with map specific peptides reduces map burden in tissues of infected goats

    DEFF Research Database (Denmark)

    Melvang, Heidi Mikkelsen; Hassan, Sufia Butt; Thakur, Aneesh

    As an alternative to protein-based vaccines, we investigated the effect of post-exposure vaccination with Map specific peptides in a goat model aiming at developing a Map vaccine that will neither interfere with diagnosis of paratuberculosis nor bovine tuberculosis. Peptides were initially select...... in the unvaccinated control group seroconverted in ID Screen® ELISA at last sampling prior to euthanasia. These results indicate that a subunit vaccine against Map can induce a protective immune response against paratuberculosis in goats....

  3. Transthyretin protects against A-beta peptide toxicity by proteolytic cleavage of the peptide: a mechanism sensitive to the Kunitz protease inhibitor.

    Directory of Open Access Journals (Sweden)

    Rita Costa

    Full Text Available Alzheimer's disease (AD is a neurodegenerative disorder characterized by the deposition of amyloid beta-peptide (A-Beta in the brain. Transthyretin (TTR is a tetrameric protein of about 55 kDa mainly produced in the liver and choroid plexus of the brain. The known physiological functions of TTR are the transport of thyroid hormone T(4 and retinol, through binding to the retinol binding protein. TTR has also been established as a cryptic protease able to cleave ApoA-I in vitro. It has been described that TTR is involved in preventing A-Beta fibrilization, both by inhibiting and disrupting A-Beta fibrils, with consequent abrogation of toxicity. We further characterized the nature of the TTR/A-Beta interaction and found that TTR, both recombinant or isolated from human sera, was able to proteolytically process A-Beta, cleaving the peptide after aminoacid residues 1, 2, 3, 10, 13, 14,16, 19 and 27, as determined by mass spectrometry, and reversed phase chromatography followed by N-terminal sequencing. A-Beta peptides (1-14 and (15-42 showed lower amyloidogenic potential than the full length counterpart, as assessed by thioflavin binding assay and ultrastructural analysis by transmission electron microscopy. A-Beta cleavage by TTR was inhibited in the presence of an alphaAPP peptide containing the Kunitz Protease Inhibitor (KPI domain but not in the presence of the secreted alphaAPP derived from the APP isoform 695 without the KPI domain. TTR was also able to degrade aggregated forms of A-Beta peptide. Our results confirmed TTR as a protective molecule in AD, and prompted A-Beta proteolysis by TTR as a protective mechanism in this disease. TTR may prove to be a useful therapeutic agent for preventing or retarding the cerebral amyloid plaque formation implicated in AD pathology.

  4. Insight into the Selectivity of the G7-18NATE Inhibitor Peptide for the Grb7-SH2 Domain Target.

    Science.gov (United States)

    Watson, Gabrielle M; Lucas, William A H; Gunzburg, Menachem J; Wilce, Jacqueline A

    2017-01-01

    Growth factor receptor bound protein 7 (Grb7) is an adaptor protein with established roles in the progression of both breast and pancreatic cancers. Through its C-terminal SH2 domain, Grb7 binds to phosphorylated tyrosine kinases to promote proliferative and migratory signaling. Here, we investigated the molecular basis for the specificity of a Grb7 SH2-domain targeted peptide inhibitor. We identified that arginine 462 in the BC loop is unique to Grb7 compared to Grb2, another SH2 domain bearing protein that shares the same consensus binding motif as Grb7. Using surface plasmon resonance we demonstrated that Grb7-SH2 binding to G7-18NATE is reduced 3.3-fold when the arginine is mutated to the corresponding Grb2 amino acid. The reverse mutation in Grb2-SH2 (serine to arginine), however, was insufficient to restore binding of G7-18NATE to Grb2-SH2. Further, using a microarray, we confirmed that G7-18NATE is specific for Grb7 over a panel of 79 SH2 domains, and identified that leucine at the βD6 position may also be a requirement for Grb7-SH2 binding. This study provides insight into the specificity defining features of Grb7 for the inhibitor molecule G7-18NATE, that will assist in the development of improved Grb7 targeted inhibitors.

  5. Insight into the Selectivity of the G7-18NATE Inhibitor Peptide for the Grb7-SH2 Domain Target

    Directory of Open Access Journals (Sweden)

    Gabrielle M. Watson

    2017-09-01

    Full Text Available Growth factor receptor bound protein 7 (Grb7 is an adaptor protein with established roles in the progression of both breast and pancreatic cancers. Through its C-terminal SH2 domain, Grb7 binds to phosphorylated tyrosine kinases to promote proliferative and migratory signaling. Here, we investigated the molecular basis for the specificity of a Grb7 SH2-domain targeted peptide inhibitor. We identified that arginine 462 in the BC loop is unique to Grb7 compared to Grb2, another SH2 domain bearing protein that shares the same consensus binding motif as Grb7. Using surface plasmon resonance we demonstrated that Grb7-SH2 binding to G7-18NATE is reduced 3.3-fold when the arginine is mutated to the corresponding Grb2 amino acid. The reverse mutation in Grb2-SH2 (serine to arginine, however, was insufficient to restore binding of G7-18NATE to Grb2-SH2. Further, using a microarray, we confirmed that G7-18NATE is specific for Grb7 over a panel of 79 SH2 domains, and identified that leucine at the βD6 position may also be a requirement for Grb7-SH2 binding. This study provides insight into the specificity defining features of Grb7 for the inhibitor molecule G7-18NATE, that will assist in the development of improved Grb7 targeted inhibitors.

  6. Insight into the Selectivity of the G7-18NATE Inhibitor Peptide for the Grb7-SH2 Domain Target

    Science.gov (United States)

    Watson, Gabrielle M.; Lucas, William A. H.; Gunzburg, Menachem J.; Wilce, Jacqueline A.

    2017-01-01

    Growth factor receptor bound protein 7 (Grb7) is an adaptor protein with established roles in the progression of both breast and pancreatic cancers. Through its C-terminal SH2 domain, Grb7 binds to phosphorylated tyrosine kinases to promote proliferative and migratory signaling. Here, we investigated the molecular basis for the specificity of a Grb7 SH2-domain targeted peptide inhibitor. We identified that arginine 462 in the BC loop is unique to Grb7 compared to Grb2, another SH2 domain bearing protein that shares the same consensus binding motif as Grb7. Using surface plasmon resonance we demonstrated that Grb7-SH2 binding to G7-18NATE is reduced 3.3-fold when the arginine is mutated to the corresponding Grb2 amino acid. The reverse mutation in Grb2-SH2 (serine to arginine), however, was insufficient to restore binding of G7-18NATE to Grb2-SH2. Further, using a microarray, we confirmed that G7-18NATE is specific for Grb7 over a panel of 79 SH2 domains, and identified that leucine at the βD6 position may also be a requirement for Grb7-SH2 binding. This study provides insight into the specificity defining features of Grb7 for the inhibitor molecule G7-18NATE, that will assist in the development of improved Grb7 targeted inhibitors. PMID:29018805

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

    Science.gov (United States)

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

    2002-01-01

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

  8. Characterization of the fine specificity of peptide antibodies to HLA-DQ beta-chain molecules

    DEFF Research Database (Denmark)

    Petersen, J S; Atar, D; Karlsen, Alan E

    1990-01-01

    In an attempt to produce epitope specific antisera which could distinguish two closely associated HLA-DQ beta-chain alleles, we immunized 20 rabbits with synthetic peptides representing sequences from the first domain of the HLA-DQw8 and -DQw7 beta-chain molecules, differing only by one amino acid...... in position 57. Several of the antisera in immunoblotting specifically recognized either the HLA-DQw7 or the HLA-DQw8 beta-chain allele as previously reported. The fine specificity of the antisera was tested in ELISA using synthetic peptides of varying length as solid phase antigen. Two out of the 20 antisera...

  9. Inhibition of neurotensin-stimulated mast cell secretion and carboxypeptidase A activity by the peptide inhibitor of carboxypeptidase A and neurotensin-receptor antagonist SR 48692.

    Science.gov (United States)

    Miller, L A; Cochrane, D E; Feldberg, R S; Carraway, R E

    1998-06-01

    Neurotensin (NT), a peptide found in brain and several peripheral tissues, is a potent stimulus for mast cell secretion and its actions are blocked by the specific NT receptor antagonist, SR 48692. Subsequent to stimulation, NT is rapidly degraded by mast cell carboxypeptidase A (CPA). In the experiments described here, we tested for the involvement of CPA activity in the activation of mast cell secretion by the peptide, NT. Mast cells were isolated from the peritoneal and pleural cavities of rats, purified over metrizamide gradients and incubated at 37 degrees C in Locke solution or Locke containing the appropriate inhibitors. For some experiments, media derived from mast cells stimulated by compound 48/80 were used as a source of mast cell CPA activity. Treatment of mast cells with the highly specific peptide inhibitor of CPA derived from potato (PCI) inhibited histamine release in response to NT and NT8-13 (the biologically active region of NT). This inhibition required some 20 min to develop and was only partially reversed by a 20-min wash period. PCI (10 microM) did not inhibit histamine release in response to NT1-12, bradykinin, compound 48/80, the calcium ionophore, A23187, or anti-IgE serum. PCI also inhibited mast cell CPA activity. SR 48692, a highly selective antagonist of the brain NT receptor and of NT-stimulated mast cell secretion, also inhibited mast cell CPA activity as well as bovine pancreatic CPA activity in a concentration-dependent manner. It is suggested that the mast cell binding site for NT and the active site for CPA may share similar characteristics. The results are discussed in terms of NT mechanism of action on the mast cell.

  10. Substrate and inhibitor specificity of kynurenine monooxygenase from Cytophaga hutchinsonii.

    Science.gov (United States)

    Phillips, Robert S; Anderson, Andrew D; Gentry, Harvey G; Güner, Osman F; Bowen, J Phillip

    2017-04-15

    Kynurenine monooxygenase (KMO) is a potential drug target for treatment of neurodegenerative disorders such as Huntington's and Alzheimer's diseases. We have evaluated substituted kynurenines as substrates or inhibitors of KMO from Cytophaga hutchinsonii. Kynurenines substituted with a halogen at the 5-position are excellent substrates, with values of k cat and k cat /K m comparable to or higher than kynurenine. However, kynurenines substituted in the 3-position are competitive inhibitors, with K I values lower than the K m for kynurenine. Bromination also enhances inhibition, and 3,5-dibromokynurenine is a potent competitive inhibitor with a K I value of 1.5μM. A pharmacophore model of KMO was developed, and predicted that 3,4-dichlorohippuric acid would be an inhibitor. The K I for this compound was found to be 34μM, thus validating the pharmacophore model. We are using these results and our model to design more potent inhibitors of KMO. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Uncovering the Peptide-Binding Specificities of HLA-C: A General Strategy To Determine the Specificity of Any MHC Class I Molecule

    DEFF Research Database (Denmark)

    Rasmussen, Michael; Harndahl, Mikkel; Stryhn, Anette

    2014-01-01

    MHC class I molecules (HLA-I in humans) present peptides derived from endogenous proteins to CTLs. Whereas the peptide-binding specificities of HLA-A and -B molecules have been studied extensively, little is known about HLA-C specificities. Combining a positional scanning combinatorial peptide...... library approach with a peptide-HLA-I dissociation assay, in this study we present a general strategy to determine the peptide-binding specificity of any MHC class I molecule. We applied this novel strategy to 17 of the most common HLA-C molecules, and for 16 of these we successfully generated matrices...... representing their peptide-binding motifs. The motifs prominently shared a conserved C-terminal primary anchor with hydrophobic amino acid residues, as well as one or more diverse primary and auxiliary anchors at P1, P2, P3, and/or P7. Matrices were used to generate a large panel of HLA-C-specific peptide...

  12. Stapled Voltage-Gated Calcium Channel (CaV) α-Interaction Domain (AID) Peptides Act As Selective Protein-Protein Interaction Inhibitors of CaV Function.

    Science.gov (United States)

    Findeisen, Felix; Campiglio, Marta; Jo, Hyunil; Abderemane-Ali, Fayal; Rumpf, Christine H; Pope, Lianne; Rossen, Nathan D; Flucher, Bernhard E; DeGrado, William F; Minor, Daniel L

    2017-06-21

    For many voltage-gated ion channels (VGICs), creation of a properly functioning ion channel requires the formation of specific protein-protein interactions between the transmembrane pore-forming subunits and cystoplasmic accessory subunits. Despite the importance of such protein-protein interactions in VGIC function and assembly, their potential as sites for VGIC modulator development has been largely overlooked. Here, we develop meta-xylyl (m-xylyl) stapled peptides that target a prototypic VGIC high affinity protein-protein interaction, the interaction between the voltage-gated calcium channel (Ca V ) pore-forming subunit α-interaction domain (AID) and cytoplasmic β-subunit (Ca V β). We show using circular dichroism spectroscopy, X-ray crystallography, and isothermal titration calorimetry that the m-xylyl staples enhance AID helix formation are structurally compatible with native-like AID:Ca V β interactions and reduce the entropic penalty associated with AID binding to Ca V β. Importantly, electrophysiological studies reveal that stapled AID peptides act as effective inhibitors of the Ca V α 1 :Ca V β interaction that modulate Ca V function in an Ca V β isoform-selective manner. Together, our studies provide a proof-of-concept demonstration of the use of protein-protein interaction inhibitors to control VGIC function and point to strategies for improved AID-based Ca V modulator design.

  13. Therapeutic peptides for cancer therapy. Part II - cell cycle inhibitory peptides and apoptosis-inducing peptides.

    Science.gov (United States)

    Raucher, Drazen; Moktan, Shama; Massodi, Iqbal; Bidwell, Gene L

    2009-10-01

    Therapeutic peptides have great potential as anticancer agents owing to their ease of rational design and target specificity. However, their utility in vivo is limited by low stability and poor tumor penetration. The authors review the development of peptide inhibitors with potential for cancer therapy. Peptides that arrest the cell cycle by mimicking CDK inhibitors or induce apoptosis directly are discussed. The authors searched Medline for articles concerning the development of therapeutic peptides and their delivery. Inhibition of cancer cell proliferation directly using peptides that arrest the cell cycle or induce apoptosis is a promising strategy. Peptides can be designed that interact very specifically with cyclins and/or cyclin-dependent kinases and with members of apoptotic cascades. Use of these peptides is not limited by their design, as a rational approach to peptide design is much less challenging than the design of small molecule inhibitors of specific protein-protein interactions. However, the limitations of peptide therapy lie in the poor pharmacokinetic properties of these large, often charged molecules. Therefore, overcoming the drug delivery hurdles could open the door for effective peptide therapy, thus making an entirely new class of molecules useful as anticancer drugs.

  14. Characterizing SH2 Domain Specificity and Network Interactions Using SPOT Peptide Arrays.

    Science.gov (United States)

    Liu, Bernard A

    2017-01-01

    Src Homology 2 (SH2) domains are protein interaction modules that recognize and bind tyrosine phosphorylated ligands. Their ability to distinguish binding to over thousands of potential phosphotyrosine (pTyr) ligands within the cell is critical for the fidelity of receptor tyrosine kinase (RTK) signaling. Within humans there are over a hundred SH2 domains with more than several thousand potential ligands across many cell types and cell states. Therefore, defining the specificity of individual SH2 domains is critical for predicting and identifying their physiological ligands. Here, in this chapter, I describe the broad use of SPOT peptide arrays for examining SH2 domain specificity. An orientated peptide array library (OPAL) approach can uncover both favorable and non-favorable residues, thus providing an in-depth analysis to SH2 specificity. Moreover, I discuss the application of SPOT arrays for paneling SH2 ligand binding with physiological peptides.

  15. Bacillus subtilis RapA phosphatase domain interaction with its substrate, phosphorylated Spo0F, and its inhibitor, the PhrA peptide.

    Science.gov (United States)

    Diaz, Alejandra R; Core, Leighton J; Jiang, Min; Morelli, Michela; Chiang, Christina H; Szurmant, Hendrik; Perego, Marta

    2012-03-01

    Rap proteins in Bacillus subtilis regulate the phosphorylation level or the DNA-binding activity of response regulators such as Spo0F, involved in sporulation initiation, or ComA, regulating competence development. Rap proteins can be inhibited by specific peptides generated by the export-import processing pathway of the Phr proteins. Rap proteins have a modular organization comprising an amino-terminal alpha-helical domain connected to a domain formed by six tetratricopeptide repeats (TPR). In this study, the molecular basis for the specificity of the RapA phosphatase for its substrate, phosphorylated Spo0F (Spo0F∼P), and its inhibitor pentapeptide, PhrA, was analyzed in part by generating chimeric proteins with RapC, which targets the DNA-binding domain of ComA, rather than Spo0F∼P, and is inhibited by the PhrC pentapeptide. In vivo analysis of sporulation efficiency or competence-induced gene expression, as well as in vitro biochemical assays, allowed the identification of the amino-terminal 60 amino acids as sufficient to determine Rap specificity for its substrate and the central TPR3 to TPR5 (TPR3-5) repeats as providing binding specificity toward the Phr peptide inhibitor. The results allowed the prediction and testing of key residues in RapA that are essential for PhrA binding and specificity, thus demonstrating how the widespread structural fold of the TPR is highly versatile, using a common interaction mechanism for a variety of functions in eukaryotic and prokaryotic organisms.

  16. A small molecule inhibitor of signal peptide peptidase inhibits Plasmodium development in the liver and decreases malaria severity.

    Directory of Open Access Journals (Sweden)

    Iana Parvanova

    Full Text Available The liver stage of Plasmodium's life cycle is the first, obligatory step in malaria infection. Decreasing the hepatic burden of Plasmodium infection decreases the severity of disease and constitutes a promising strategy for malaria prophylaxis. The efficacy of the gamma-secretase and signal peptide peptidase inhibitor LY411,575 in targeting Plasmodium liver stages was evaluated both in human hepatoma cell lines and in mouse primary hepatocytes. LY411,575 was found to prevent Plasmodium's normal development in the liver, with an IC(50 of approximately 80 nM, without affecting hepatocyte invasion by the parasite. In vivo results with a rodent model of malaria showed that LY411,575 decreases the parasite load in the liver and increases by 55% the resistance of mice to cerebral malaria, one of the most severe malaria-associated syndromes. Our data show that LY411,575 does not exert its effect via the Notch signaling pathway suggesting that it may interfere with Plasmodium development through an inhibition of the parasite's signal peptide peptidase. We therefore propose that selective signal peptide peptidase inhibitors could be potentially used for preventive treatment of malaria in humans.

  17. Two-dimensional NMR studies of squash family inhibitors. Sequence-specific proton assignments and secondary structure of reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor III.

    Science.gov (United States)

    Krishnamoorthi, R; Gong, Y X; Lin, C L; VanderVelde, D

    1992-01-28

    The solution structure of reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor III (CMTI-III*) was investigated by two-dimensional proton nuclear magnetic resonance (2D NMR) spectroscopy. CMTI-III*, prepared by reacting CMTI-III with trypsin which cleaved the Arg5-Ile6 peptide bond, had the two fragments held together by a disulfide linkage. Sequence-specific 1H NMR resonance assignments were made for all the 29 amino acid residues of the protein. The secondary structure of CMTI-III*, as deduced from NOESY cross peaks and identification of slowly exchanging hydrogens, contains two turns (residues 8-12 and 24-27), a 3(10)-helix (residues 13-16), and a triple-stranded beta-sheet (residues 8-10, 29-27, and 21-25). This secondary structure is similar to that of CMTI-I [Holak, T. A., Gondol, D., Otlewski, J., & Wilusz, T. (1989) J. Mol. Biol. 210, 635-648], which has a Glu instead of a Lys at position 9. Sequential proton assignments were also made for the virgin inhibitor, CMTI-III, at pH 4.71, 30 degrees C. Comparison of backbone hydrogen chemical shifts of CMTI-III and CMTI-III* revealed significant changes for residues located far away from the reactive-site region as well as for those located near it, indicating tertiary structural changes that are transmitted through most of the 29 residues of the inhibitor protein. Many of these residues are functionally important in that they make contact with atoms of the enzyme in the trypsin-inhibitor complex, as revealed by X-ray crystallography [Bode, W., Greyling, H. J., Huber, R., Otlewski, J., & Wilusz, T. (1989) FEBS Lett. 242, 285-292].(ABSTRACT TRUNCATED AT 250 WORDS)

  18. CD25 targeted therapy of chemotherapy resistant leukemic stem cells using DR5 specific TRAIL peptide

    Directory of Open Access Journals (Sweden)

    Jayaprakasam Madhumathi

    2017-03-01

    Full Text Available Chemotherapy resistant leukemic stem cells (LSCs are being targeted as a modern therapeutic approach to prevent disease relapse. LSCs isolated from methotrexate resistant side population (SP of leukemic cell lines HL60 and MOLT4 exhibited high levels of CD25 and TRAIL R2/DR5 which are potential targets. Recombinant immunotoxin conjugating IL2α with TRAIL peptide mimetic was constructed for DR5 receptor specific targeting of LSCs and were tested in total cell population and LSCs. IL2-TRAIL peptide induced apoptosis in drug resistant SP cells from cell lines and showed potent cytotoxicity in PBMCs derived from leukemic patients with an efficacy of 81.25% in AML and 100% in CML, ALL and CLL. IL2-TRAIL peptide showed cytotoxicity in relapsed patient samples and was more effective than TRAIL or IL2-TRAIL proteins. Additionally, DR5 specific IL2-TRAIL peptide was effective in targeting and killing LSCs purified from cell lines [IC50: 952 nM in HL60, 714 nM in MOLT4] and relapsed patient blood samples with higher efficacy (85% than IL2-TRAIL protein (46%. Hence, CD25 and DR5 specific targeting by IL2-TRAIL peptide may be an effective strategy for targeting drug resistant leukemic cells and LSCs.

  19. Mammalian protein secretion without signal peptide removal. Biosynthesis of plasminogen activator inhibitor-2 in U-937 cells

    International Nuclear Information System (INIS)

    Ye, R.D.; Wun, T.C.; Sadler, J.E.

    1988-01-01

    Plasminogen activator inhibitor-2 (PAI-2) is a serine protease inhibitor that regulates plasmin generation by inhibiting urokinase and tissue plasminogen activator. The primary structure of PAI-2 suggests that it may be secreted without cleavage of a single peptide. To confirm this hypothesis we have studied the glycosylation and secretion of PAI-2 in human monocytic U-937 cells by metabolic labeling, immunoprecipitation, glycosidase digestion, and protein sequencing. PAI-2 is variably glycosylated on asparagine residues to yield intracellular intermediates with zero, one, two, or three high mannose-type oligosaccharide units. Secretion of the N-glycosylated species began by 1 h of chase and the secreted molecules contained both complex-type N-linked and O-linked oligosaccharides. Enzymatically deglycosylated PAI-2 had an electrophoretic mobility identical to that of the nonglycosylated precursor and also to that of PAI-2 synthesized in vitro in a rabbit reticulocyte lysate from synthetic mRNA derived from full length PAI-2 cDNA. The amino-terminal protein sequence of secreted PAI-2 began with the initiator methionine residue. These results indicate that PAI-2 is glycosylated and secreted efficiently without the cleavage of a signal peptide. PAI-2 shares this property with its nearest homologue in the serine protease inhibitor family, chicken ovalbumin, and appears to be the first well characterized example of this phenomenon among natural mammalian proteins

  20. Sequence-specific inhibition of duck hepatitis B virus reverse transcription by peptide nucleic acids (PNA)

    DEFF Research Database (Denmark)

    Robaczewska, Magdalena; Narayan, Ramamurthy; Seigneres, Beatrice

    2005-01-01

    BACKGROUND/AIMS: Peptide nucleic acids (PNAs) appear as promising new antisense agents, that have not yet been examined as hepatitis B virus (HBV) inhibitors. Our aim was to study the ability of PNAs targeting the duck HBV (DHBV) encapsidation signal epsilon to inhibit reverse transcription (RT...... in primary duck hepatocytes (PDH). RESULTS: Both PNAs reproducibly inhibited DHBV RT in a dose-dependent manner with IC(50) of 10nM, whereas up to 600-fold higher concentration of S-ODNs was required for similar inhibition. The PNA targeting the bulge and upper stem of epsilon appeared as more efficient RT...

  1. Discovery of non-peptidic small molecule inhibitors of cyclophilin D as neuroprotective agents in Aβ-induced mitochondrial dysfunction

    Science.gov (United States)

    Park, Insun; Londhe, Ashwini M.; Lim, Ji Woong; Park, Beoung-Geon; Jung, Seo Yun; Lee, Jae Yeol; Lim, Sang Min; No, Kyoung Tai; Lee, Jiyoun; Pae, Ae Nim

    2017-10-01

    Cyclophilin D (CypD) is a mitochondria-specific cyclophilin that is known to play a pivotal role in the formation of the mitochondrial permeability transition pore (mPTP).The formation and opening of the mPTP disrupt mitochondrial homeostasis, cause mitochondrial dysfunction and eventually lead to cell death. Several recent studies have found that CypD promotes the formation of the mPTP upon binding to β amyloid (Aβ) peptides inside brain mitochondria, suggesting that neuronal CypD has a potential to be a promising therapeutic target for Alzheimer's disease (AD). In this study, we generated an energy-based pharmacophore model by using the crystal structure of CypD—cyclosporine A (CsA) complex and performed virtual screening of ChemDiv database, which yielded forty-five potential hit compounds with novel scaffolds. We further tested those compounds using mitochondrial functional assays in neuronal cells and identified fifteen compounds with excellent protective effects against Aβ-induced mitochondrial dysfunction. To validate whether these effects derived from binding to CypD, we performed surface plasmon resonance (SPR)—based direct binding assays with selected compounds and discovered compound 29 was found to have the equilibrium dissociation constants (KD) value of 88.2 nM. This binding affinity value and biological activity correspond well with our predicted binding mode. We believe that this study offers new insights into the rational design of small molecule CypD inhibitors, and provides a promising lead for future therapeutic development.

  2. A nanoengineered peptidic delivery system with specificity for human brain capillary endothelial cells

    DEFF Research Database (Denmark)

    Wu, Linping; Moghimi, Seyed Moein

    2016-01-01

    , without manipulating the integrity of the BBB. This may be achieved by simultaneous and appropriate nanoparticle surface decoration with polymers that protect nanoparticles against rapid interception by body's defenses and ligands specific for cerebral capillary endothelial cells. To date, the binding...... avidity of the majority of the so-called ‘brain-specific’ nanoparticles to the brain capillary endothelial cells has been poor, even during in vitro conditions. We have addressed this issue and designed a versatile peptidic nanoplatform with high binding avidity to the human cerebral capillary endothelial...... cells. This was achieved by selecting an appropriate phage-derived peptide with high specificity for human brain capillary endothelial cells, which following careful structural modifications spontaneously formed a nanoparticle-fiber network. The peptidic network was characterized fully and its uptake...

  3. Deep convolutional neural networks for pan-specific peptide-MHC class I binding prediction.

    Science.gov (United States)

    Han, Youngmahn; Kim, Dongsup

    2017-12-28

    Computational scanning of peptide candidates that bind to a specific major histocompatibility complex (MHC) can speed up the peptide-based vaccine development process and therefore various methods are being actively developed. Recently, machine-learning-based methods have generated successful results by training large amounts of experimental data. However, many machine learning-based methods are generally less sensitive in recognizing locally-clustered interactions, which can synergistically stabilize peptide binding. Deep convolutional neural network (DCNN) is a deep learning method inspired by visual recognition process of animal brain and it is known to be able to capture meaningful local patterns from 2D images. Once the peptide-MHC interactions can be encoded into image-like array(ILA) data, DCNN can be employed to build a predictive model for peptide-MHC binding prediction. In this study, we demonstrated that DCNN is able to not only reliably predict peptide-MHC binding, but also sensitively detect locally-clustered interactions. Nonapeptide-HLA-A and -B binding data were encoded into ILA data. A DCNN, as a pan-specific prediction model, was trained on the ILA data. The DCNN showed higher performance than other prediction tools for the latest benchmark datasets, which consist of 43 datasets for 15 HLA-A alleles and 25 datasets for 10 HLA-B alleles. In particular, the DCNN outperformed other tools for alleles belonging to the HLA-A3 supertype. The F1 scores of the DCNN were 0.86, 0.94, and 0.67 for HLA-A*31:01, HLA-A*03:01, and HLA-A*68:01 alleles, respectively, which were significantly higher than those of other tools. We found that the DCNN was able to recognize locally-clustered interactions that could synergistically stabilize peptide binding. We developed ConvMHC, a web server to provide user-friendly web interfaces for peptide-MHC class I binding predictions using the DCNN. ConvMHC web server can be accessible via http://jumong.kaist.ac.kr:8080/convmhc

  4. Interactions of a potent cyclic peptide inhibitor with the light chain of botulinum neurotoxin A: Insights from X-ray crystallography.

    Science.gov (United States)

    Kumaran, Desigan; Adler, Michael; Levit, Matthew; Krebs, Michael; Sweeney, Richard; Swaminathan, Subramanyam

    2015-11-15

    The seven antigenically distinct serotypes (A-G) of botulinum neurotoxin (BoNT) are responsible for the deadly disease botulism. BoNT serotype A (BoNT/A) exerts its lethal action by cleaving the SNARE protein SNAP-25, leading to inhibition of neurotransmitter release, flaccid paralysis and autonomic dysfunction. BoNTs are dichain proteins consisting of a ∼ 100 kDa heavy chain and a ∼ 50 kDa light chain; the former is responsible for neurospecific binding, internalization and translocation, and the latter for cleavage of neuronal SNARE proteins. Because of their extreme toxicity and history of weaponization, the BoNTs are regarded as potential biowarfare/bioterrorism agents. No post-symptomatic therapeutic interventions are available for BoNT intoxication other than intensive care; therefore it is imperative to develop specific antidotes against this neurotoxin. To this end, a cyclic peptide inhibitor (CPI-1) was evaluated in a FRET assay for its ability to inhibit BoNT/A light chain (Balc). CPI was found to be highly potent, exhibiting a Ki of 12.3 nM with full-length Balc448 and 39.2 nM using a truncated crystallizable form of the light chain (Balc424). Cocrystallization studies revealed that in the Balc424-CPI-1 complex, the inhibitor adopts a helical conformation, occupies a high percentage of the active site cavity and interacts in an amphipathic manner with critical active site residues. The data suggest that CPI-1 prevents SNAP-25 from accessing the Balc active site by blocking both the substrate binding path at the surface and the Zn(2+) binding region involved in catalysis. This differs from linear peptide inhibitors described to date which block only the latter. Published by Elsevier Ltd.

  5. Oral peptide specific egg antibody to intestinal sodium-dependent phosphate co-transporter-2b is effective at altering phosphate transport in vitro and in vivo.

    Science.gov (United States)

    Bobeck, Elizabeth A; Hellestad, Erica M; Sand, Jordan M; Piccione, Michelle L; Bishop, Jeff W; Helvig, Christian; Petkovich, Martin; Cook, Mark E

    2015-06-01

    Hyperimmunized hens are an effective means of generating large quantities of antigen specific egg antibodies that have use as oral supplements. In this study, we attempted to create a peptide specific antibody that produced outcomes similar to those of the human pharmaceutical, sevelamer HCl, used in the treatment of hyperphosphatemia (a sequela of chronic renal disease). Egg antibodies were generated against 8 different human intestinal sodium-dependent phosphate cotransporter 2b (NaPi2b) peptides, and hNaPi2b peptide egg antibodies were screened for their ability to inhibit phosphate transport in human intestinal Caco-2 cell line. Antibody produced against human peptide sequence TSPSLCWT (anti-h16) was specific for its peptide sequence, and significantly reduced phosphate transport in human Caco-2 cells to 25.3±11.5% of control nonspecific antibody, when compared to nicotinamide, a known inhibitor of phosphate transport (P≤0.05). Antibody was then produced against the mouse-specific peptide h16 counterpart (mouse sequence TSPSYCWT, anti-m16) for further analysis in a murine model. When anti-m16 was fed to mice (1% of diet as dried egg yolk powder), egg yolk immunoglobulin (IgY) was detected using immunohistochemical staining in mouse ileum, and egg anti-m16 IgY colocalized with a commercial goat anti-NaPi2b antibody. The effectiveness of anti-m16 egg antibody in reducing serum phosphate, when compared to sevelamer HCl, was determined in a mouse feeding study. Serum phosphate was reduced 18% (Pegg yolk powder) and 30% (Pegg immunoglobulin. The methods described and the findings reported show that oral egg antibodies are useful and easy to prepare reagents for the study and possible treatment of select diseases. © 2015 Poultry Science Association Inc.

  6. Cross-reactive microbial peptides can modulate HIV-specific CD8+ T cell responses.

    Directory of Open Access Journals (Sweden)

    Christopher W Pohlmeyer

    Full Text Available Heterologous immunity is an important aspect of the adaptive immune response. We hypothesized that this process could modulate the HIV-1-specific CD8+ T cell response, which has been shown to play an important role in HIV-1 immunity and control. We found that stimulation of peripheral blood mononuclear cells (PBMCs from HIV-1-positive subjects with microbial peptides that were cross-reactive with immunodominant HIV-1 epitopes resulted in dramatic expansion of HIV-1-specific CD8+ T cells. Interestingly, the TCR repertoire of HIV-1-specific CD8+ T cells generated by ex vivo stimulation of PBMCs using HIV-1 peptide was different from that of cells stimulated with cross-reactive microbial peptides in some HIV-1-positive subjects. Despite these differences, CD8+ T cells stimulated with either HIV-1 or cross-reactive peptides effectively suppressed HIV-1 replication in autologous CD4+ T cells. These data suggest that exposure to cross-reactive microbial antigens can modulate HIV-1-specific immunity.

  7. A library of fluorescent peptides for exploring the substrate specificities of prolyl isomerases

    NARCIS (Netherlands)

    Zoldak, G.; Aumuller, T.; Lucke, C.; Hritz, J.; Oostenbrink, C.; Fischer, G.; Schmid, F.X.

    2009-01-01

    To fully explore the substrate specificities of prolyl isomerases, we synthesized a library of 20 tetrapeptides that are labeled with a 2-aminobenzoyl (Abz) group at the amino terminus and a p-nitroanilide (pNA) group at the carboxy terminus. In this peptide library of the general formula

  8. Collision-Induced Dissociation of Deprotonated Peptides. Relative Abundance of Side-Chain Neutral Losses, Residue-Specific Product Ions, and Comparison with Protonated Peptides.

    Science.gov (United States)

    Liang, Yuxue; Neta, Pedatsur; Yang, Xiaoyu; Stein, Stephen E

    2018-03-01

    High-accuracy MS/MS spectra of deprotonated ions of 390 dipeptides and 137 peptides with three to six residues are studied. Many amino acid residues undergo neutral losses from their side chains. The most abundant is the loss of acetaldehyde from threonine. The abundance of losses from the side chains of other amino acids is estimated relative to that of threonine. While some amino acids lose the whole side chain, others lose only part of it, and some exhibit two or more different losses. Side-chain neutral losses are less abundant in the spectra of protonated peptides, being significant mainly for methionine and arginine. In addition to the neutral losses, many amino acid residues in deprotonated peptides produce specific negative ions after peptide bond cleavage. An expanded list of fragment ions from protonated peptides is also presented and compared with those of deprotonated peptides. Fragment ions are mostly different for these two cases. These lists of fragments are used to annotate peptide mass spectral libraries and to aid in the confirmation of specific amino acids in peptides. Graphical Abstract ᅟ.

  9. A Specific Peptide with Calcium-Binding Capacity from Defatted Schizochytrium sp. Protein Hydrolysates and the Molecular Properties.

    Science.gov (United States)

    Cai, Xixi; Yang, Qian; Lin, Jiaping; Fu, Nanyan; Wang, Shaoyun

    2017-03-29

    Marine microorganisms have been proposed as a new kind of protein source. Efforts are needed in order to transform the protein-rich biological wastes left after lipid extraction into value-added bio-products. Thus, the utilization of protein recovered from defatted Schizochytrium sp. by-products presents an opportunity. A specific peptide Tyr-Leu (YL) with calcium-binding capacity was purified from defatted Schizochytrium sp. protein hydrolysates through gel filtration chromatography and RP-HPLC. The calcium-binding activity of YL reached 126.34 ± 3.40 μg/mg. The calcium-binding mechanism was investigated through ultraviolet, fluorescence and infrared spectroscopy. The results showed that calcium ions could form dative bonds with carboxyl oxygen atoms and amino nitrogen atoms as well as the nitrogen and oxygen atoms of amide bonds. YL-Ca exhibited excellent thermal stability and solubility, which was beneficial for its absorption and transport in the basic intestinal tract of the human body. Moreover, the cellular uptake of calcium in Caco-2 cells showed that YL-Ca could enhance calcium uptake efficiency and protect calcium ions against precipitation caused by dietary inhibitors such as tannic acid, oxalate, phytate and metal ions. The findings indicate that the by-product of Schizochytrium sp. is a promising source for making peptide-calcium bio-products as algae-based functional supplements for human beings.

  10. A Specific Peptide with Calcium-Binding Capacity from Defatted Schizochytrium sp. Protein Hydrolysates and the Molecular Properties

    Directory of Open Access Journals (Sweden)

    Xixi Cai

    2017-03-01

    Full Text Available Marine microorganisms have been proposed as a new kind of protein source. Efforts are needed in order to transform the protein-rich biological wastes left after lipid extraction into value-added bio-products. Thus, the utilization of protein recovered from defatted Schizochytrium sp. by-products presents an opportunity. A specific peptide Tyr-Leu (YL with calcium-binding capacity was purified from defatted Schizochytrium sp. protein hydrolysates through gel filtration chromatography and RP-HPLC. The calcium-binding activity of YL reached 126.34 ± 3.40 μg/mg. The calcium-binding mechanism was investigated through ultraviolet, fluorescence and infrared spectroscopy. The results showed that calcium ions could form dative bonds with carboxyl oxygen atoms and amino nitrogen atoms as well as the nitrogen and oxygen atoms of amide bonds. YL-Ca exhibited excellent thermal stability and solubility, which was beneficial for its absorption and transport in the basic intestinal tract of the human body. Moreover, the cellular uptake of calcium in Caco-2 cells showed that YL-Ca could enhance calcium uptake efficiency and protect calcium ions against precipitation caused by dietary inhibitors such as tannic acid, oxalate, phytate and metal ions. The findings indicate that the by-product of Schizochytrium sp. is a promising source for making peptide-calcium bio-products as algae-based functional supplements for human beings.

  11. Potential clinical usefulness of new glandular and circulating parathyroid peptides illuminated by sequence specific radioimmunoassay

    International Nuclear Information System (INIS)

    Lindall, A.W.; Cecchettin, M.

    1981-01-01

    It is now well known that human PTH peptides constitute a heterogeneous population of fragments of the eighty-four amino acid molecules. Over the years, considerable confusion has resulted from measurements made in both clinical and experimental settings with radioimmunoassays of either undefined or partially-defined specificity for a particular region of the intact molecule. The approach of the authors has been to make use of modern technology to synthesize peptide fragments that mimic portions of native molecules, and to use these fragments in RIA. A more detailed description of a portion of this work will appear in the Journal of Clinical Endocrinology and Metabolism. (Auth.)

  12. Lactoferrin-derived Peptides Active towards Influenza: Identification of Three Potent Tetrapeptide Inhibitors.

    Science.gov (United States)

    Scala, Maria Carmina; Sala, Marina; Pietrantoni, Agostina; Spensiero, Antonia; Di Micco, Simone; Agamennone, Mariangela; Bertamino, Alessia; Novellino, Ettore; Bifulco, Giuseppe; Gomez-Monterrey, Isabel M; Superti, Fabiana; Campiglia, Pietro

    2017-09-06

    Bovine lactoferrin is a biglobular multifunctional iron binding glycoprotein that plays an important role in innate immunity against infections. We have previously demonstrated that selected peptides from bovine lactoferrin C-lobe are able to prevent both Influenza virus hemagglutination and cell infection. To deeper investigate the ability of lactoferrin derived peptides to inhibit Influenza virus infection, in this study we identified new bovine lactoferrin C-lobe derived sequences and corresponding synthetic peptides were synthesized and assayed to check their ability to prevent viral hemagglutination and infection. We identified three tetrapeptides endowed with broad anti-Influenza activity and able to inhibit viral infection in a concentration range femto- to picomolar. Our data indicate that these peptides may constitute a non-toxic tool for potential applications as anti-Influenza therapeutics.

  13. Impact of epitope specificity and precursor maturation in pro-B-type natriuretic peptide measurement

    DEFF Research Database (Denmark)

    Goetze, J.P.; Dahlstrom, U.; Alehagen, U.

    2008-01-01

    with different epitope specificities in a cohort of elderly patients presenting with symptoms associated with heart failure (n = 415). RESULTS: Comparison of N-terminal proBNP with proBNP 1-76 measurement in plasma revealed a high correlation on regression analysis (r(2) = 0.91, P ..., the proBNP 1-76 assay measured lower concentrations in the high range than the N-terminal proBNP assay. Correlations between assay measurements in a clinical setting were comparable for all the assays (r(2) approximately 0.57-0.83), and ROC analyses revealed area-under-the-curve values ranging between 0......BACKGROUND: Cardiac-derived natriuretic peptides are sensitive plasma markers of cardiac dysfunction. Recent reports have disclosed a more complex molecular heterogeneity of B-type natriuretic peptide precursor (proBNP)-derived peptides than previously suggested. In this study, we examined...

  14. Perturbation of estrogen receptor α localization with synthetic nona-arginine LXXLL-peptide coactivator binding inhibitors

    NARCIS (Netherlands)

    Carraz, M.; Zwart, W.; Phan, T.; Michalides, R.; Brunsveld, L.

    2009-01-01

    The interaction of estrogen receptor a (ERa) with the consensus LXXLL motifs of transcriptional coactivators provides an entry for functional ERa inhibition. Here, synthetic cell-permeable LXXLL peptide probes are brought forward that allow evaluation of the interaction of specific recognition

  15. Epitope and functional specificity of monoclonal antibodies to mouse gamma interferon: the synthetic peptide approach

    International Nuclear Information System (INIS)

    Russell, J.K.; Hayes, M.P.; Carter, J.M.; Torres, B.A.; Dunn, B.M.; Johnson, H.M.

    1986-01-01

    Four anti-recombinant mouse gamma interferon (α-IFNγ) monoclonal antibodies were generated using hamster spleen cells. Binding of 125 I-IFNγ by these protein A-bound antibodies was specifically blocked by cold IFNγ. Binding by three of these antibodies was also blocked by a synthetic peptide corresponding to the N-terminal 1-39 amino acids of IFNγ, while a corresponding C-terminal (95-133) peptide had no effect on binding. One of the N-terminal specific monoclonal antibodies inhibited both the antiviral and macrophage priming (for tumor cell killing) activities of IFNγ, while the other two had no effect on either biological function. Blocking experiments with cold IFNγ and N-terminal peptide suggest that the epitope specificities of the monoclonal antibodies could be determined by the conformational or topographic structure of IFNγ. Polyclonal antibodies to either the N-terminal or C-terminal peptides also inhibited both the antiviral and macrophage priming activities of IFNγ. All of the antibodies that inhibited IFNγ function also blocked binding of IFNγ to membrane receptor on cells, while antibodies that did not inhibit function also did not block binding. The data suggest that both the N-terminal and C-terminal domains of IFNγ play an important role in its antiviral and macrophage priming functions, possibly in a cooperative manner

  16. Nitrite and nitroso compounds can serve as specific catalase inhibitors.

    Science.gov (United States)

    Titov, Vladimir Yu; Osipov, Anatoly N

    2017-03-01

    We present evidence that nitrite and nitrosothiols, nitrosoamines and non-heme dinitrosyl iron complexes can reversibly inhibit catalase with equal effectiveness. Catalase activity was evaluated by the permanganatometric and calorimetric assays. This inhibition is not the result of chemical transformations of these compounds to a single inhibitor, as well as it is not the result of NO release from these substances (as NO traps have no effect on the extent of inhibition). It was found that chloride and bromide in concentration above 80 mM and thiocyanate in concentration above 20 μM enhance catalase inhibition by nitrite and the nitroso compounds more than 100 times. The inhibition degree in this case is comparable with that induced by azide. We propose that the direct catalase inhibitor is a positively charged NO-group. This group acquires a positive charge in the active center of enzyme by interaction of nitrite or nitroso compounds with some enzyme groups. Halides and thiocyanate protect the NO + group from hydration and thus increase its inhibition effect. It is probable that a comparatively low chloride concentration in many cells is the main factor to protect catalase from inhibition by nitrite and nitroso compounds.

  17. Macrocyclic peptide inhibitors for the protein-protein interaction of Zaire Ebola virus protein 24 and karyopherin alpha 5.

    Science.gov (United States)

    Song, Xiao; Lu, Lu-Yi; Passioura, Toby; Suga, Hiroaki

    2017-06-21

    Ebola virus infection leads to severe hemorrhagic fever in human and non-human primates with an average case fatality rate of 50%. To date, numerous potential therapies are in development, but FDA-approved drugs or vaccines are yet unavailable. Ebola viral protein 24 (VP24) is a multifunctional protein that plays critical roles in the pathogenesis of Ebola virus infection, e.g. innate immune suppression by blocking the interaction between KPNA and PY-STAT1. Here we report macrocyclic peptide inhibitors of the VP24-KPNA5 protein-protein interaction (PPI) by means of the RaPID (Random non-standard Peptides Integrated Discovery) system. These macrocyclic peptides showed remarkably high affinity to recombinant Zaire Ebola virus VP24 (eVP24), with a dissociation constant in the single digit nanomolar range, and could also successfully disrupt the eVP24-KPNA interaction. This work provides for the first time a chemical probe capable of modulating this PPI interaction and is the starting point for the development of unique anti-viral drugs against the Ebola virus.

  18. An integrated in silico approach to design specific inhibitors targeting human poly(a-specific ribonuclease.

    Directory of Open Access Journals (Sweden)

    Dimitrios Vlachakis

    Full Text Available Poly(A-specific ribonuclease (PARN is an exoribonuclease/deadenylase that degrades 3'-end poly(A tails in almost all eukaryotic organisms. Much of the biochemical and structural information on PARN comes from the human enzyme. However, the existence of PARN all along the eukaryotic evolutionary ladder requires further and thorough investigation. Although the complete structure of the full-length human PARN, as well as several aspects of the catalytic mechanism still remain elusive, many previous studies indicate that PARN can be used as potent and promising anti-cancer target. In the present study, we attempt to complement the existing structural information on PARN with in-depth bioinformatics analyses, in order to get a hologram of the molecular evolution of PARNs active site. In an effort to draw an outline, which allows specific drug design targeting PARN, an unequivocally specific platform was designed for the development of selective modulators focusing on the unique structural and catalytic features of the enzyme. Extensive phylogenetic analysis based on all the publicly available genomes indicated a broad distribution for PARN across eukaryotic species and revealed structurally important amino acids which could be assigned as potentially strong contributors to the regulation of the catalytic mechanism of PARN. Based on the above, we propose a comprehensive in silico model for the PARN's catalytic mechanism and moreover, we developed a 3D pharmacophore model, which was subsequently used for the introduction of DNP-poly(A amphipathic substrate analog as a potential inhibitor of PARN. Indeed, biochemical analysis revealed that DNP-poly(A inhibits PARN competitively. Our approach provides an efficient integrated platform for the rational design of pharmacophore models as well as novel modulators of PARN with therapeutic potential.

  19. Novel phospholipase A2 inhibitors from python serum are potent peptide antibiotics.

    Science.gov (United States)

    Samy, Ramar Perumal; Thwin, Maung Maung; Stiles, Brad G; Satyanarayana-Jois, Seetharama; Chinnathambi, Arunachalam; Zayed, M E; Alharbi, Sulaiman Ali; Siveen, Kodappully Sivaraman; Sikka, Sakshi; Kumar, Alan Prem; Sethi, Gautam; Lim, Lina Hsiu Kim

    2015-04-01

    Antimicrobial peptides (AMPs) play a vital role in defense against resistant bacteria. In this study, eight different AMPs synthesized from Python reticulatus serum protein were tested for bactericidal activity against various Gram-positive and Gram-negative bacteria (Staphylococcus aureus, Burkholderia pseudomallei (KHW and TES strains), and Proteus vulgaris) using a disc-diffusion method (20 μg/disc). Among the tested peptides, phospholipase A2 inhibitory peptide (PIP)-18[59-76], β-Asp65-PIP[59-67], D-Ala66-PNT.II, and D60,65E-PIP[59-67] displayed the most potent bactericidal activity against all tested pathogens in a dose-dependent manner (100-6.8 μg/ml), with a remarkable activity noted against S. aureus at 6.8 μg/ml dose within 6 h of incubation. Determination of minimum inhibitory concentrations (MICs) by a micro-broth dilution method at 100-3.125 μg/ml revealed that PIP-18[59-76], β-Asp65-PIP[59-67] and D-Ala66-PNT.II peptides exerted a potent inhibitory effect against S. aureus and B. pseudomallei (KHW) (MICs 3.125 μg/ml), while a much less inhibitory potency (MICs 12.5 μg/ml) was noted for β-Asp65-PIP[59-67] and D-Ala66-PNT.II peptides against B. pseudomallei (TES). Higher doses of peptides had no effect on the other two strains (i.e., Klebsiella pneumoniae and Streptococcus pneumoniae). Overall, PIP-18[59-76] possessed higher antimicrobial activity than that of chloramphenicol (CHL), ceftazidime (CF) and streptomycin (ST) (30 μg/disc). When the two most active peptides, PIP-18[59-76] and β-Asp65-PIP[59-67], were applied topically at a 150 mg/kg dose for testing wound healing activity in a mouse model of S. aureus infection, the former accelerates faster wound healing than the latter peptide at 14 days post-treatment. The western blot data suggest that the topical application of peptides (PIP-18[59-67] and β-Asp65-PIP[59-67]) modulates NF-kB mediated wound repair in mice with relatively little haemolytic (100-1.56 μg/ml) and cytotoxic (1000

  20. Effects of phosphodiesterase inhibitors on atrial dynamics induced by C-type natriuretic peptide in isolated beating rabbit atria

    International Nuclear Information System (INIS)

    Ding Dazhi; Cui Xun; Jin Xiunan; Lan Ying; Liu Liping; Hong Lan

    2010-01-01

    Objective: To investigate the effects of phosphodiesterase inhibitors (PDEI) on atrial dynamics induced by C-type natriuretic peptide (CNP) and the contents of cyclic nucleotide (cAMP, cGMP) in isolated beating rabbit atria. Methods: After the rabbits had been anesthetized, the hearts were removed rapidly. The left auricles were isolated and fixed on the atrial perfusion system. The atrial stroke volume and the pulse pressure were observed by CNP with or without PDEIs pretreatment. The contents of cAMP and cGMP were measured by radioimmunoassay. Results: (1)Compared with control cycle group, CNP (30.0 nmol · L -1 ) obviously decreased the atrial stroke volume and pulse pressure (P 0.05). (2)Compared with control cycle group, IBMX(1000.0 nmol · L -1 ), a non-selective inhibitor of PDE, significantly increased the atrial stroke volume, pulse pressure, cAMP and cGMP contents (P -1 ) plus CNP (30.0 nmol · L -1 )group and IBMX group (P>0.05). (3)Compared with control cycle group, EHNA(30.0 nmol · L -1 ), an inhibitor of PDE2, obviously decreased the atrial stroke volume and pulse pressure (P 0.05). EHNA(30.0 nmol · L -1 ) plus CNP (30.0 nmol · L -1 ) showed similar roles with EHNA only. (4)Compared with control cycle group, milrinone (1.0 nmol · L -1 ), an inhibitor of PDE3, significantly increased the content of cAMP (P 0.05). CNP (30.0 nmol · L -1 ) obviously decreased the atrial stroke volume and pulse pressure (P 0.05). Conclusion: CNP can inhibit atrial dynamics by increasing the content of cGMP, the different inhibitors of PDEs play different roles in the CNP-induced inhibition of atrial dynamics in isolated beating rabbit atria. (authors)

  1. Peptide-targeted, stimuli-responsive polymersomes for delivering a cancer stemness inhibitor to cancer stem cell microtumors.

    Science.gov (United States)

    Karandish, Fataneh; Froberg, James; Borowicz, Pawel; Wilkinson, John C; Choi, Yongki; Mallik, Sanku

    2018-03-01

    Often cancer relapses after an initial response to chemotherapy because of the tumor's heterogeneity and the presence of progenitor stem cells, which can renew. To overcome drug resistance, metastasis, and relapse in cancer, a promising approach is the inhibition of cancer stemness. In this study, the expression of the neuropilin-1 receptor in both pancreatic and prostate cancer stem cells was identified and targeted with a stimuli-responsive, polymeric nanocarrier to deliver a stemness inhibitor (napabucasin) to cancer stem cells. Reduction-sensitive amphiphilic block copolymers PEG 1900 -S-S-PLA 6000 and the N 3 -PEG 1900 -PLA 6000 were synthesized. The tumor penetrating iRGD peptide-hexynoic acid conjugate was linked to the N 3 -PEG 1900 -PLA 6000 polymer via a Cu 2+ catalyzed "Click" reaction. Subsequently, this peptide-polymer conjugate was incorporated into polymersomes for tumor targeting and tissue penetration. We prepared polymersomes containing 85% PEG 1900 -S-S-PLA 6000 , 10% iRGD-polymer conjugate, and 5% DPPE-lissamine rhodamine dye. The iRGD targeted polymersomes encapsulating the cancer stemness inhibitor napabucasin were internalized in both prostate and pancreatic cancer stem cells. The napabucasin encapsulated polymersomes significantly (p < .05) reduced the viability of both prostate and pancreatic cancer stem cells and decreased the stemness protein expression notch-1 and nanog compared to the control and vesicles without any drug. The napabucasin encapsulated polymersome formulations have the potential to lead to a new direction in prostate and pancreatic cancer therapy by penetrating deeply into the tumors, releasing the encapsulated stemness inhibitor, and killing cancer stem cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Peptide degradation and the role of DPP-4 inhibitors in the treatment of type 2 diabetes

    DEFF Research Database (Denmark)

    Deacon, Carolyn F

    2018-01-01

    -1 is an intestinal hormone with potent insulinotropic and glucagonostatic effects and can normalise blood glucose levels in patients with type 2 diabetes, but the native peptide is not therapeutically useful because of its inherent metabolic instability. Using the GLP-1/DPP-4 system and type 2...... diabetes as an example, this review summarises how knowledge of a peptide's biological effects coupled with an understanding of the pathways involved in its metabolic clearance can be exploited in a rational, step-by-step manner to develop a therapeutic agent, which is effective and well tolerated, and any...

  3. Residual DNA-bound proteins are a source of in vitro transcription inhibitor peptides

    International Nuclear Information System (INIS)

    Venanzi, F.M.

    1989-01-01

    Enzymatic breakdown of residual proteins occurs at mild alkaline pH (pH optimum 8.5) as monitored by using radioiodinated, purified genomic DNA from calf thymus. These DNA fibers also possess a differential ability to hydrolyze added exogenous small and linker histones. The results described argue strongly that a putative protease activity, co-purified with DNA, is the source of short chain peptides which inhibit transcription in vitro. Therefore, we propose that RNA repressor peptides must be of higher molecular weight than previously reported

  4. In vitro comparison of renal handling and uptake of two somatostatin receptor-specific peptides labeled with indium-111

    International Nuclear Information System (INIS)

    Trejtnar, F.; Novy, Z.; Petrik, M.; Laznickova, A.; Melicharova, L.; Vankova, M.; Laznicek, M.

    2008-01-01

    Radiolabeled receptor-specific somatostatin analogs labeled with gamma- or beta-emitting radionuclides are useful for scintigraphic imaging and/or therapy of selected neuroendocrine tumors. However, significant renal uptake may result in radiotoxicological injury of the kidney and can limit clinical application of the agents. The aim of the study was to analyze renal handling, rate, and mechanism of renal accumulation of two somatostatin receptor-targeted peptides, [DOTA 0 , Tyr 3 , Thr 8 ]-octreotide (DOTA-TATE) and [DOTA 0 , 1-Nal 3 ]-octreotide (DOTA-NOC), labeled with indium-111 using in vitro methods. The perfused rat kidney and freshly isolated rat renal cells were used as experimental models. The perfusion was performed in a recirculation regimen at constant pressure with solution containing bovine albumin, erythrocytes, and a mixture of essential substrates. The renal cells were isolated from rat kidneys using two-phase collagenase perfusion. Accumulation studies were used to evaluate the renal uptake of the peptides and to compare their accumulation with that of passively or actively transported model drugs. The influence of selected inhibitors of receptor-mediated endocytosis and the inhibition of energy-dependent transport processes on the uptake were also investigated using isolated renal cells. The renal clearance of 111 In-DOTA-NOC in the perfused rat kidney was significantly lower than that of 111 In-DOTA-TATE. Reverse situation was found in the case of renal retention. Pretreatment of the perfused kidney with maleate markedly decreased the renal retention. 111 In-DOTA-NOC was accumulated in the isolated renal cells at a higher rate than 111 In-DOTA-TATE (ratio 3:1). The uptake of the radiopeptides in renal cells was higher than the uptake of not only the passively transported sucrose but also actively transported and accumulated methylglucose. The rank order of potency to inhibit the uptake by active endocytosis was approximately aprotinin

  5. Inhibitors

    Science.gov (United States)

    ... JM, and the Hemophilia Inhibitor Research Study Investigators. Validation of Nijmegen-Bethesda assay modifications to allow inhibitor ... webinars on blood disorders Language: English (US) Español (Spanish) File Formats Help: How do I view different ...

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

    International Nuclear Information System (INIS)

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

    2012-01-01

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

  7. Preliminary study on the inhibition of nuclear internalization of Tat peptides by conjugation with a receptor-specific peptide and fluorescent dyes

    Science.gov (United States)

    Shen, Duanwen; Liang, Kexiang; Ye, Yunpeng; Tetteh, Elizabeth; Achilefu, Samuel

    2006-02-01

    Numerous studies have shown that basic Tat peptide (48-57) internalized non-specifically in cells and localized in the nucleus. However, localization of imaging agents in cellular nucleus is not desirable because of the potential mutagenesis. When conjugated to the peptides that undergo receptor-mediated endocytosis, Tat peptide could target specific cells or pathologic tissue. We tested this hypothesis by incorporating a somatostatin receptor-avid peptide (octreotate, Oct) and two different fluorescent dyes, Cypate 2 (Cy2) and fluorescein 5'-carboxlic acid (5-FAM), into the Tat-peptide sequence. In addition to the Cy2 or 5-FAM-labeled Oct conjugated to Tat peptide (Tat) to produce Tat-Oct-Cypate2 or Tat-Oct-5-FAM, we also labeled the Tat the Tat peptide with these dyes (Tat-Cy2 and Tat-5-FAM) to serve as positive control. A somatostatin receptor-positive pancreatic tumor cell line, AR42J, was used to assess cell internalization. The results show that Tat-5-FAM and Tat-Cypate2 localized in both nucleus and cytoplasm of the cells. In contrast to Tat-Oct-Cypate2, which localized in both the cytoplasm and nucleus, Tat-Oct-5-FAM internalized in the cytoplasm but not in the nucleus of AR42J cells. The internalizations were inhibited by adding non-labeled corresponding peptides, suggesting that the endocytoses of each group of labeled and the corresponding unlabeled compounds occurred through a common pathway. Thus, fluorescent probes and endocytosis complex between octreotate and somatostatin receptors in cytoplasm could control nuclear internalization of Tat peptides.

  8. Penetration of polymeric nanoparticles loaded with an HIV-1 inhibitor peptide derived from GB virus C in a vaginal mucosa model.

    Science.gov (United States)

    Ariza-Sáenz, Martha; Espina, Marta; Bolaños, Nuria; Calpena, Ana Cristina; Gomara, María José; Haro, Isabel; García, María Luisa

    2017-11-01

    Despite the great effort to decrease the HIV infectivity rate, current antiretroviral therapy has several weaknesses; poor bioavailability, development of drug resistance and poor ability to access tissues. However, molecules such as peptides have emerged asa new expectative to HIV eradication. The vaginal mucosa is the main spreading point of HIV. There are natural barriers such as the vaginal fluid which protects the vaginal epithelium from any foreign agents reaching it. This work has developed and characterized Nanoparticles (NPs) coated with glycol chitosan (GC), loaded with an HIV-1 inhibitor peptide (E2). In vitro release and ex vivo studies were carried out using the vaginal mucosa of swine and the peptide was determined by HPLC MS/MS validated method. Moreover, the peptide was labeled with 5(6)-carboxyfluoresceine and entrapped into the NPs to carried out in vivo studies and to evaluate the NPs penetration and toxicity in the vaginal mucosa of the swine. The mean size of the NPs, ξ and the loading percentage were fundamental features for to reach the vaginal tissue and to release the peptide within intercellular space. The obtained results suggesting that the fusion inhibitor peptides loaded into the NPs coated with GC might be a new way to fight the HIV-1, due to the formulation might reach the human epithelial mucosa and release peptide without any side effects. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Synthesis and characterisation of substrate-based peptides as inhibitors of histone demethylase KDM4C

    DEFF Research Database (Denmark)

    Nielsen, Simon D; Leurs, Ulrike; Bergner, Magnus

    2016-01-01

    different series of peptides were designed and synthesized. One series contained N-acylated H3K9 and two series introduced triazoles in this position via click chemistry to enable facile variation of headgroups. As the click reaction is compatible with free amino acids this was performed on an azido...

  10. Solid-Phase Synthesis of Modified Peptides as Putative Inhibitors of Histone Modifying Enzymes

    DEFF Research Database (Denmark)

    Cohrt, Anders Emil O'Hanlon

    to be compatible with all 20 naturally occurring amino acids, and were furthermore feasible on several commonly used polymeric supports. By using dilute SnCl4 for N -Boc deprotection, and NaOH for the release of material from the solid support, N -modified peptides were cleanly obtained in excellent yields...

  11. Trends in the Design and Development of Specific Aptamers Against Peptides and Proteins.

    Science.gov (United States)

    Tabarzad, Maryam; Jafari, Marzieh

    2016-04-01

    Aptamers are single stranded oligonucleotides, comparable to monoclonal antibodies (mAbs) in selectivity and affinity and have significant strategic properties in design, development and applications more than mAbs. Ease of design and development, simple chemical modification and the attachment of functional groups, easily handling and more adaptability with analytical methods, small size and adaptation with nanostructures are the valuable characteristics of aptamers in comparison to large protein based ligands. Among a broad range of targets that their specific aptamers developed, proteins and peptides have significant position according to the number of related studies performed so far. Since proteins control many of important physiological and pathological incidents in the living organisms, particularly human beings and because of the benefits of aptamers in clinical and analytical applications, aptamer related technologies in the field of proteins and peptides are under progress, exclusively. Currently, there is only one FDA approved therapeutic aptamer in the pharmaceutical market, which is specific to vascular endothelial growth factor and is prescribed for age related macular degenerative disease. Additionally, there are several aptamers in the different phases of clinical trials. Almost all of these aptamers are specific to clinically important peptide or protein targets. In addition, the application of protein specific aptamers in the design and development of targeted drug delivery systems and diagnostic biosensors is another interesting field of aptamer technology. In this review, significant efforts related to development and applications of aptamer technologies in proteins and peptides sciences were considered to emphasis on the importance of aptamers in medicinal and clinical applications.

  12. Studies on peptide amidase-catalysed C-terminal peptide amidation in organic media with respect to its substrate specificity

    Czech Academy of Sciences Publication Activity Database

    Čeřovský, Václav; Kula, M. R.

    2001-01-01

    Roč. 33, - (2001), s. 183-187 ISSN 0885-4513 R&D Projects: GA ČR GA203/99/1458 Keywords : enzymic amidation * peptide amides * peptide synthesis Subject RIV: CC - Organic Chemistry Impact factor: 1.408, year: 2001

  13. Study of pharmacokinetics and biodistribution of radiolabelled receptor specific peptides in laboratory animals

    International Nuclear Information System (INIS)

    Laznickova, A.; Laznicek, M.; Trejtnar, F.; Maecke, H.R.; Mather, S.J.

    2001-01-01

    Somatostatin analogues labelled with different radionuclides could be employed for visualization or treatment of somatostatin receptor-positive tumours. An octapeptide 111 In [DTPA] octreotide is a synthetic radiolabelled somatostatin analogue which is currently in clinical use for detecting small neuroendocrine tumours and metastases not detectable by conventional means. However, several other somatostatin analogues have been under development and testing. The aim of this study was to radiolabel selected somatostatin receptor-binding octapeptides by different radionuclides and to report the results of their biodistribution in rats. The study was focused on the direct labelling of vapreotide (RC-160) with 99m Tc, on the conjugates of octreotide with DFO (desferrioxamine) for labelling with 67 Ga, and on the conjugates of octreotide and TOC with DOTA (tetraazacyclo-dodecane tetraacetic acid) for labelling with 88 Y. In the present study, 88 Y isotope instead of 90 Y was used as a label as 88 Y exhibits a longer half life of decay and emits gamma radiation which can be much more easily detected in biological samples than beta emission. The labelling of octreotide analogues with metal radionuclides using derived bifunctional chelates was simple, straightforward and consistently resulted in high radiochemical purity of the product. On the other hand, the application of the direct labelling method for labelling of RC-160 with 99m Tc was difficult because all procedures had to be made under nitrogen atmosphere and an attainment of high yield proved to be highly dependent on the accurate observation of reaction conditions. The labelling efficiency makes an immediate use of the radiolabelled RC-160 for biological studies impossible and it is necessary to involve the purification step into the labelling procedure. All radiolabelled receptor specific peptides under study exhibited rapid radioactivity clearance from the blood and most organs and tissues. On the other hand

  14. Extraction and identification of α-amylase inhibitor peptides from Nephelium lappacheum and Nephelium mutabile seed protein using gastro-digestive enzymes.

    Science.gov (United States)

    Evaristus, Natashya Anak; Wan Abdullah, Wan Nadiah; Gan, Chee-Yuen

    2018-04-01

    The potential of N. lappacheum and N. mutabile seed as a source of α-amylase inhibitor peptides was explored based on the local traditional practice of using the seed. Different gastro-digestive enzymes (i.e. pepsin or chymotrypsin) or a sequential digestion were used to extract the peptides. The effects of digestion time and enzyme to substrate (E:S) ratio on the α-amylase inhibitory activity were investigated. Results showed that chymotrypsin was effective in producing the inhibitor peptides from rambutan seed protein at E:S ratio 1:20 for 1 h, whereas pepsin was more effective for pulasan seed protein under the same condition. A total of 20 and 31 novel inhibitor peptides were identified, respectively. These peptides could bind with the subsites of α-amylase (i.e. Trp58, Trp59, Tyr62, Asp96, Arg195, Asp197, Glu233, His299, Asp300, and His305) and formed a sliding barrier that preventing the formation of enzyme/substrate intermediate leading to lower α-amylase activity. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. Bowman-Birk Protease Inhibitor from Vigna unguiculata Seeds Enhances the Action of Bradykinin-Related Peptides

    Directory of Open Access Journals (Sweden)

    Alice da Cunha M. Álvares

    2014-10-01

    Full Text Available The hydrolysis of bradykinin (Bk by different classes of proteases in plasma and tissues leads to a decrease in its half-life. Here, Bk actions on smooth muscle and in vivo cardiovascular assays in association with a protease inhibitor, Black eyed-pea trypsin and chymotrypsin inhibitor (BTCI and also under the effect of trypsin and chymotrypsin were evaluated. Two synthetic Bk-related peptides, Bk1 and Bk2, were used to investigate the importance of additional C-terminal amino acid residues on serine protease activity. BTCI forms complexes with Bk and analogues at pH 5.0, 7.4 and 9.0, presenting binding constants ranging from 103 to 104 M−1. Formation of BTCI-Bk complexes is probably driven by hydrophobic forces, coupled with slight conformational changes in BTCI. In vitro assays using guinea pig (Cavia porcellus ileum showed that Bk retains the ability to induce smooth muscle contraction in the presence of BTCI. Moreover, no alteration in the inhibitory activity of BTCI in complex with Bk and analogous was observed. When the BTCI and BTCI-Bk complexes were tested in vivo, a decrease of vascular resistance and consequent hypotension and potentiating renal and aortic vasodilatation induced by Bk and Bk2 infusions was observed. These results indicate that BTCI-Bk complexes may be a reliable strategy to act as a carrier and protective approach for Bk-related peptides against plasma serine proteases cleavage, leading to an increase in their half-life. These findings also indicate that BTCI could remain stable in some tissues to inhibit chymotrypsin or trypsin-like enzymes that cleave and inactivate bradykinin in situ.

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

    OpenAIRE

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

    1986-01-01

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

  17. Glucagon-like peptide receptor agonists and dipeptidyl peptidase-4 inhibitors in the treatment of diabetes: a review of clinical trials

    DEFF Research Database (Denmark)

    Madsbad, Sten; Krarup, Thure; Deacon, Carolyn F

    2008-01-01

    -acting glucagon-like peptide-1 receptor agonists liraglutide and exenatide long-acting release reduce haemoglobin A1c by about 1.0-2.0% and have fewer gastrointestinal side-effects. The orally available dipeptidyl peptidase-4 inhibitors, that is sitagliptin and vildagliptin reduce haemoglobin A1c by 0...

  18. Discovery of Peptide-based Inhibitors against Dendrotoxin B from Black Mamba through Phage Display Screening

    DEFF Research Database (Denmark)

    O. Cob, Saioa; Munk, Andreas; Laustsen, Andreas Hougaard

    The black mamba (Dendroaspis polylepis) is Africa’s most feared snake due to its potent, rapidacting venom and its speed of attack. The most abundant toxins in D. polylepis venom are the Kunitz-type proteinase inhibitors, dendrotoxins, which are unique for mamba. Dendrotoxinsare poorly neutralized...

  19. Macrocyclic Peptoid–Peptide Hybrids as Inhibitors of Class I Histone Deacetylases

    DEFF Research Database (Denmark)

    Olsen, Christian Adam; Montero, Ana; Leman, Luke J.

    2012-01-01

    We report the design, synthesis, and biological evaluation of the first macrocyclic peptoid-containing histone deacetylase (HDAC) inhibitors. The compounds selectively inhibit human class I HDAC isoforms in vitro, with no inhibition of the tubulin deacetylase activity associated with class IIb HDAC...

  20. Effects of matrix metalloproteinase inhibitor doxycycline and CD147 antagonist peptide-9 on gallbladder carcinoma cell lines.

    Science.gov (United States)

    Wang, Shihang; Liu, Chao; Liu, Xinjiang; He, Yanxin; Shen, Dongfang; Luo, Qiankun; Dong, Yuxi; Dong, Haifeng; Pang, Zhigang

    2017-10-01

    Gallbladder carcinoma is the most common and aggressive malignancy of the biliary tree and highly expresses CD147, which is closely related to disease prognosis in a variety of human cancers. Doxycycline exhibited anti-tumor properties in many cancer cells. CD147 antagonist peptide-9 is a polypeptide and can specifically bind to CD147. The effect of these two drugs on gallbladder cancer cells has not been studied. The aim of this study is to investigate the effect of doxycycline and antagonist peptide-9 on gallbladder carcinoma cells and the possible mechanism of inhibition on cancer cell of doxycycline. To investigate the effects of doxycycline and antagonist peptide-9 on gallbladder carcinoma cells (GBC-SD and SGC-996), cell proliferation, CD147 expression, and early-stage apoptosis rate were measured after treated with doxycycline. Matrix metalloproteinase-2 and matrix metalloproteinase-9 activities were measured after treated with different concentrations of doxycycline, antagonist peptide-9, and their combination. The results demonstrated that doxycycline inhibited cell proliferation, reduced CD147 expression level, and induced an early-stage apoptosis response in GBC-SD and SGC-996 cells. The matrix metalloproteinase-2 and matrix metalloproteinase-9 activities were inhibited by antagonist peptide-9 and doxycycline, and the inhibitory effects were enhanced by combined drugs in gallbladder carcinoma cell lines. Taken together, doxycycline showed inhibitory effects on gallbladder carcinoma cell lines and reduced the expression of CD147, and this may be the mechanism by which doxycycline inhibits cancer cells. This study provides new information and tries to implement the design of adjuvant therapy method for gallbladder carcinoma.

  1. Human lactoferricin derived di-peptides deploying loop structures induce apoptosis specifically in cancer cells through targeting membranous phosphatidylserine.

    Science.gov (United States)

    Riedl, Sabrina; Leber, Regina; Rinner, Beate; Schaider, Helmut; Lohner, Karl; Zweytick, Dagmar

    2015-11-01

    Host defense-derived peptides have emerged as a novel strategy for the development of alternative anticancer therapies. In this study we report on characteristic features of human lactoferricin (hLFcin) derivatives which facilitate specific killing of cancer cells of melanoma, glioblastoma and rhabdomyosarcoma compared with non-specific derivatives and the synthetic peptide RW-AH. Changes in amino acid sequence of hLFcin providing 9-11 amino acids stretched derivatives LF11-316, -318 and -322 only yielded low antitumor activity. However, the addition of the repeat (di-peptide) and the retro-repeat (di-retro-peptide) sequences highly improved cancer cell toxicity up to 100% at 20 μM peptide concentration. Compared to the complete parent sequence hLFcin the derivatives showed toxicity on the melanoma cell line A375 increased by 10-fold and on the glioblastoma cell line U-87mg by 2-3-fold. Reduced killing velocity, apoptotic blebbing, activation of caspase 3/7 and formation of apoptotic DNA fragments proved that the active and cancer selective peptides, e.g. R-DIM-P-LF11-322, trigger apoptosis, whereas highly active, though non-selective peptides, such as DIM-LF11-318 and RW-AH seem to kill rapidly via necrosis inducing membrane lyses. Structural studies revealed specific toxicity on cancer cells by peptide derivatives with loop structures, whereas non-specific peptides comprised α-helical structures without loop. Model studies with the cancer membrane mimic phosphatidylserine (PS) gave strong evidence that PS only exposed by cancer cells is an important target for specific hLFcin derivatives. Other negatively charged membrane exposed molecules as sialic acid, heparan and chondroitin sulfate were shown to have minor impact on peptide activity. Copyright © 2015. Published by Elsevier B.V.

  2. Shape-specific nanostructured protein mimics from de novo designed chimeric peptides.

    Science.gov (United States)

    Jiang, Linhai; Yang, Su; Lund, Reidar; Dong, He

    2018-01-30

    Natural proteins self-assemble into highly-ordered nanoscaled architectures to perform specific functions. The intricate functions of proteins have provided great impetus for researchers to develop strategies for designing and engineering synthetic nanostructures as protein mimics. Compared to the success in engineering fibrous protein mimetics, the design of discrete globular protein-like nanostructures has been challenging mainly due to the lack of precise control over geometric packing and intermolecular interactions among synthetic building blocks. In this contribution, we report an effective strategy to construct shape-specific nanostructures based on the self-assembly of chimeric peptides consisting of a coiled coil dimer and a collagen triple helix folding motif. Under salt-free conditions, we showed spontaneous self-assembly of the chimeric peptides into monodisperse, trigonal bipyramidal-like nanoparticles with precise control over the stoichiometry of two folding motifs and the geometrical arrangements relative to one another. Three coiled coil dimers are interdigitated on the equatorial plane while the two collagen triple helices are located in the axial position, perpendicular to the coiled coil plane. A detailed molecular model was proposed and further validated by small angle X-ray scattering experiments and molecular dynamics (MD) simulation. The results from this study indicated that the molecular folding of each motif within the chimeric peptides and their geometric packing played important roles in the formation of discrete protein-like nanoparticles. The peptide design and self-assembly mechanism may open up new routes for the construction of highly organized, discrete self-assembling protein-like nanostructures with greater levels of control over assembly accuracy.

  3. Shared fine specificity between T-cell receptors and an antibody recognizing a peptide/major histocompatibility class I complex

    DEFF Research Database (Denmark)

    Stryhn, A; Andersen, P S; Pedersen, L O

    1996-01-01

    Cytotoxic T cells recognize mosaic structures consisting of target peptides embedded within self-major histocompatibility complex (MHC) class I molecules. This structure has been described in great detail for several peptide-MHC complexes. In contrast, how T-cell receptors recognize peptide...... each other showing that peptide residues 1, 3, 4, 6, and 7 were exposed on the MHC surface and recognized by the T cells. Thus, the majority, and perhaps all, of the side chains of the non-primary anchor residues may be available for T-cell recognition, and contribute to the stringent specificity of T...... cells. A striking similarity between the specificity of the T cells and that of the pSAN antibody was found and most of the peptide residues, which could be recognized by the T cells, could also be recognized by the antibody....

  4. Dual-functioning peptides discovered by phage display increase the magnitude and specificity of BMSC attachment to mineralized biomaterials.

    Science.gov (United States)

    Ramaraju, Harsha; Miller, Sharon J; Kohn, David H

    2017-07-01

    Design of biomaterials for cell-based therapies requires presentation of specific physical and chemical cues to cells, analogous to cues provided by native extracellular matrices (ECM). We previously identified a peptide sequence with high affinity towards apatite (VTKHLNQISQSY, VTK) using phage display. The aims of this study were to identify a human MSC-specific peptide sequence through phage display, combine it with the apatite-specific sequence, and verify the specificity of the combined dual-functioning peptide to both apatite and human bone marrow stromal cells. In this study, a combinatorial phage display identified the cell binding sequence (DPIYALSWSGMA, DPI) which was combined with the mineral binding sequence to generate the dual peptide DPI-VTK. DPI-VTK demonstrated significantly greater binding affinity (1/K D ) to apatite surfaces compared to VTK, phosphorylated VTK (VTK phos ), DPI-VTK phos , RGD-VTK, and peptide-free apatite surfaces (p biomaterial surfaces and subsequently increase cell proliferation and differentiation. These new peptides expand biomaterial design methodology for cell-based regeneration of bone defects. This strategy of combining cell and material binding phage display derived peptides is broadly applicable to a variety of systems requiring targeted adhesion of specific cell populations, and may be generalized to the engineering of any adhesion surface. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Cyclic peptide inhibitors of the β-sliding clamp in Staphylococcus aureus

    DEFF Research Database (Denmark)

    Kjelstrup, Susanne; Hansen, Paula Melo Paulon; Thomsen, Line Elnif

    2013-01-01

    Interaction between pairs of Staphylococcus aureus replication proteins was detected in an Escherichia coli based two-hybrid analysis. A reverse two-hybrid system was constructed for selection of compounds that hindered interaction between interacting protein pairs. A number of cyclic peptides, f....... The minimum inhibitory concentration was ∼50 μg/ml for S. aureus cells. These compounds may serve as lead candidates for future development into novel classes of antibiotics as well as provide information on the function of the S. aureus replication process....

  6. Rostafuroxin: an ouabain-inhibitor counteracting specific forms of hypertension.

    Science.gov (United States)

    Ferrari, Patrizia

    2010-12-01

    An innovative approach to the therapy of essential hypertension (EH) and the related complications has been pursued by our group with the aim of defining specific genetic-molecular mechanisms underlying the disease in sub-sets of patients. This approach is anticipated to have a major effect on the clinical practice, diagnostics and development of new drugs able to selectively target such mechanisms. The final achievement is the definition of biomarkers for identifying patients who more likely should benefit for a given therapy both in terms of efficacy and reduction of the adverse reactions. Among many, two mechanisms have been defined and addressed:Both alterations lead to hypertension, organ hypertrophy, negative vascular remodeling and increased cardiovascular risk by affecting the renal Na(+) handling, through the up-regulation of the Na(+)-K(+) pump and the activation of the Src-dependent signal transduction pathway. A novel antihypertensive agent, rostafuroxin (PST2238), has been selected and developed for its ability to correct the renal Na(+)-K(+) pump abnormalities sustained by the mutant adducin and EO-dependent mechanisms. It is endowed with high potency and efficacy in reducing blood pressure (BP) and preventing organ hypertrophy in animal models representative of both adducin and EO mechanisms. At molecular level, in the kidney, rostafuroxin normalizes the enhanced activity of the Na(+)-K(+) pump induced by mutant adducin and antagonizes the EO triggering of the Src-EGFr-dependent signaling pathway leading to renal Na(+)-K(+) pump and ERK phosphorylation and activation. In the vasculature, it normalizes the increased myogenic tone caused by ouabain. A very high safety ratio and the absence of interaction with other mechanisms involved in BP regulation, together with evidence of high tolerability and efficacy in hypertensive patients indicate rostafuroxin as the first example of a new class of antihypertensive agents designed to antagonize adducin and

  7. Characterization of reaction conditions providing rapid and specific cysteine alkylation for peptide-based mass spectrometry.

    Science.gov (United States)

    Paulech, Jana; Solis, Nestor; Cordwell, Stuart J

    2013-01-01

    Alkylation converts Cys thiols to thioethers and prevents unwanted side reactions, thus facilitating mass spectrometric identification of Cys-containing peptides. Alkylation occurs preferentially at Cys due to its high nucleophilicity, however reactions at other such sites are possible. N-ethylmaleimide (NEM) shows rapid reaction kinetics with Cys and careful definition of reaction conditions results in little reactivity at other sites. Analysis of a protein standard alkylated under differing reaction conditions (pH, NEM concentrations and reaction times) was performed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and selected reaction monitoring (SRM) of NEM-modified and unmodified peptide pairs. Mis-alkylation sites at primary and secondary amines were identified and limited to one equivalent of NEM. No evidence for hydroxyl or thioether alkylation was observed. Improved specificity was achieved by restricting the pH below neutral, NEM concentration below 10mM and/or reaction time to below 5min. Maximal removal of Cys activity was observed in tissue homogenates at 40mM NEM within 1min, dependent upon efficient protein denaturation. SRM assays identified peptide-specific levels of mis-alkylation, indicating that NEM-modified to unmodified ratios did not exceed 10%, with the exception of Cys alkylation that proceeded to 100%, and some Lys residues that resulted in tryptic missed cleavages. High reactivity was observed for His residues considering their relatively low abundance. These data indicate that rapid and specific Cys alkylation is possible with NEM under relatively mild conditions, with more abrasive conditions leading to increased non-specific alkylation without appreciable benefit for MS-based proteomics. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. A dual-specificity isoform of the protein kinase inhibitor PKI produced by alternate gene splicing.

    Science.gov (United States)

    Kumar, Priyadarsini; Walsh, Donal A

    2002-03-15

    We have previously shown that the protein kinase inhibitor beta (PKIbeta) form of the cAMP-dependent protein kinase inhibitor exists in multiple isoforms, some of which are specific inhibitors of the cAMP-dependent protein kinase, whereas others also inhibit the cGMP-dependent enzyme [Kumar, Van Patten and Walsh (1997), J. Biol. Chem. 272, 20011-20020]. We have now demonstrated that the switch from a cAMP-dependent protein kinase (PKA)-specific inhibitor to one with dual specificity arises as a consequence of alternate gene splicing. We have confirmed using bacterially produced pure protein that a single inhibitor species has dual specificity for both PKA and cGMP-dependent protein kinase (PKG), inhibiting each with very high and closely similar inhibitory potencies. The gene splicing converted a protein with 70 amino acids into one of 109 amino acids, and did not change the inhibitory potency to PKA, but changed it from a protein that had no detectable PKG inhibitory activity to one that now inhibited PKG in the nanomolar range.

  9. A specific pharmacophore model of sodium-dependent glucose co-transporter 2 (SGLT2) inhibitors.

    Science.gov (United States)

    Tang, Chunlei; Zhu, Xiaoyun; Huang, Dandan; Zan, Xin; Yang, Baowei; Li, Ying; Du, Xiaoyong; Qian, Hai; Huang, Wenlong

    2012-06-01

    Sodium-dependent glucose co-transporter 2 (SGLT2) plays a pivotal role in maintaining glucose equilibrium in the human body, emerging as one of the most promising targets for the treatment of diabetes mellitus type 2. Pharmacophore models of SGLT2 inhibitors have been generated with a training set of 25 SGLT2 inhibitors using Discovery Studio V2.1. The best hypothesis (Hypo1(SGLT2)) contains one hydrogen bond donor, five excluded volumes, one ring aromatic and three hydrophobic features, and has a correlation coefficient of 0.955, cost difference of 68.76, RMSD of 0.85. This model was validated by test set, Fischer randomization test and decoy set methods. The specificity of Hypo1(SGLT2) was evaluated. The pharmacophore features of Hypo1(SGLT2) were different from the best pharmacophore model (Hypo1(SGLT1)) of SGLT1 inhibitors we developed. Moreover, Hypo1(SGLT2) could effectively distinguish selective inhibitors of SGLT2 from those of SGLT1. These results indicate that a highly predictive and specific pharmacophore model of SGLT2 inhibitors has been successfully obtained. Then Hypo1(SGLT2) was used as a 3D query to screen databases including NCI and Maybridge for identifying new inhibitors of SGLT2. The hit compounds were subsequently subjected to filtering by Lipinski's rule of five. And several compounds selected from the top ranked hits have been suggested for further experimental assay studies.

  10. Novel Protein-Protein Inhibitor Based Approach to Control Plant Ethylene Responses: Synthetic Peptides for Ripening Control

    Directory of Open Access Journals (Sweden)

    Mareike Kessenbrock

    2017-09-01

    and phenotypic studies reveal that peptide biologicals could contribute to the development of a novel family of ripening inhibitors and innovative ripening control in climacteric fruit.

  11. Histone peptide AKRHRK enhances H2O2-induced DNA damage and alters its site specificity

    International Nuclear Information System (INIS)

    Midorikawa, Kaoru; Murata, Mariko; Kawanishi, Shosuke

    2005-01-01

    Histone proteins are involved in compaction of DNA and the protection of cells from oxygen toxicity. However, several studies have demonstrated that the metal-binding histone reacts with H 2 O 2 , leading to oxidative damage to a nucleobase. We investigated whether histone can accelerate oxidative DNA damage, using a minimal model for the N-terminal tail of histone H4, CH 3 CO-AKRHRK-CONH 2 , which has a metal-binding site. This histone peptide enhanced DNA damage induced by H 2 O 2 and Cu(II), especially at cytosine residues, and induced additional DNA cleavage at the 5'-guanine of GGG sequences. The peptide also enhanced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine and ESR spin-trapping signal from H 2 O 2 and Cu(II). Cyclic redox reactions involving histone-bound Cu(II) and H 2 O 2 , may give rise to multiple production of radicals leading to multiple hits in DNA. It is noteworthy that the histone H4 peptide with specific sequence AKRHRK can cause DNA damage rather than protection under metal-overloaded condition

  12. Effective delivery of a nematode-repellent peptide using a root-cap-specific promoter.

    Science.gov (United States)

    Lilley, Catherine J; Wang, Dong; Atkinson, Howard J; Urwin, Peter E

    2011-02-01

    The potential of the MDK4-20 promoter of Arabidopsis thaliana to direct effective transgenic expression of a secreted nematode-repellent peptide was investigated. Its expression pattern was studied in both transgenic Arabidopsis and Solanum tuberosum (potato) plants. It directed root-specific β-glucuronidase expression in both species that was chiefly localized to cells of the root cap. Use of the fluorescent timer protein dsRED-E5 established that the MDK4-20 promoter remains active for longer than the commonly used constitutive promoter CaMV35S in separated potato root border cells. Transgenic Arabidopsis lines that expressed the nematode-repellent peptide under the control of either AtMDK4-20 or CaMV35S reduced the establishment of the beet cyst nematode Heterodera schachtii. The best line using the AtMDK4-20 promoter displayed a level of resistance >80%, comparable to that of lines using the CaMV35S promoter. In transgenic potato plants, 94.9 ± 0.8% resistance to the potato cyst nematode Globodera pallida was achieved using the AtMDK4-20 promoter, compared with 34.4 ± 8.4% resistance displayed by a line expressing the repellent peptide from the CaMV35S promoter. These results establish the potential of the AtMDK4-20 promoter to limit expression of a repellent peptide whilst maintaining or even improving the efficacy of the cyst-nematode defence. © 2010 The Authors. Plant Biotechnology Journal © 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

  13. A complicated complex: Ion channels, voltage sensing, cell membranes and peptide inhibitors.

    Science.gov (United States)

    Zhang, Alan H; Sharma, Gagan; Undheim, Eivind A B; Jia, Xinying; Mobli, Mehdi

    2018-04-21

    Voltage-gated ion channels (VGICs) are specialised ion channels that have a voltage dependent mode of action, where ion conduction, or gating, is controlled by a voltage-sensing mechanism. VGICs are critical for electrical signalling and are therefore important pharmacological targets. Among these, voltage-gated sodium channels (Na V s) have attracted particular attention as potential analgesic targets. Na V s, however, comprise several structurally similar subtypes with unique localisations and distinct functions, ranging from amplification of action potentials in nociception (e.g. Na V 1.7) to controlling electrical signalling in cardiac function (Na V 1.5). Understanding the structural basis of Na V function is therefore of great significance, both to our knowledge of electrical signalling and in development of subtype and state selective drugs. An important tool in this pursuit has been the use of peptides from animal venoms as selective Na V modulators. In this review, we look at peptides, particularly from spider venoms, that inhibit Na V s by binding to the voltage sensing domain (VSD) of this channel, known as gating modifier toxins (GMT). In the first part of the review, we look at the structural determinants of voltage sensing in VGICs, the gating cycle and the conformational changes that accompany VSD movement. Next, the modulation of the analgesic target Na V 1.7 by GMTs is reviewed to develop bioinformatic tools that, based on sequence information alone, can identify toxins that are likely to inhibit this channel. The same approach is also used to define VSD sequences, other than that from Na V 1.7, which are likely to be sensitive to this class of toxins. The final section of the review focuses on the important role of the cellular membrane in channel modulation and also how the lipid composition affects measurements of peptide-channel interactions both in binding kinetics measurements in solution and in cell-based functional assays. Copyright © 2018

  14. Type II collagen in cartilage evokes peptide-specific tolerance and skews the immune response.

    Science.gov (United States)

    Malmström, V; Kjellén, P; Holmdahl, R

    1998-06-01

    T cell recognition of type II collagen (CII) is a crucial event in the induction of collagen-induced arthritis in the mouse. Several CII peptides have been shown to be of importance, dependent on which MHC haplotype the mouse carries. By sequencing the rat CII and comparing the sequence with mouse, human, bovine and chicken CII, we have found that the immunodominant peptides all differ at critical positions compared with the autologous mouse sequence. Transgenic expression of the immunodominant Aq-restricted heterologous CII 256-270 epitope inserted into type I collagen (TSC mice) or type II collagen (MMC-1 mice) led to epitope-specific tolerance. Immunization of TSC mice with chick CII led to arthritis and immune responses, dependent on the subdominant, Aq-restricted and chick-specific CII 190-200 epitope. Immunization of F1 mice, expressing both H-2q and H-2r as well as transgenic expression of the Aq-restricted CII 256-270 epitope in cartilage, with bovine CII, led to arthritis, dependent on the Ar-restricted, bovine-specific epitope CII 607-621. These data show that the immunodominance of CII recognition is directed towards heterologous determinants, and that T cells directed towards the corresponding autologous epitopes are tolerated without evidence of active suppression.

  15. Handling a tricycle: Orthogonal versus random oxidation of the tricyclic inhibitor cystine knotted peptide gurmarin

    DEFF Research Database (Denmark)

    Eliasen, Rasmus; Andresen, Thomas L.; Conde-Frieboes, Kilian W.

    2012-01-01

    random oxidation strategy. We compared two oxidation procedures to form the three disulfide bridges. In the first, based on random oxidation, reduced gurmarin was synthesized using trityl for cysteine protection, and oxidized for 48h in a Tris–HCl buffer containing cystamine and reduced glutathione...... to facilitate disulfide scrambling. The second was based on step-wise deprotection followed by oxidation in which the cysteine pairs are orthogonally protected with tert-Butylthio, trityl and acetamidomethyl. To verify that the native gurmarin oxidation product was obtained, thermolysin cleavage was used...... UPLC. It was found that the random oxidation procedure leads to native gurmarin in high yield. Thus, the synthetic route was simple and significantly more efficient than previously reported syntheses of gurmarin and other cysteine rich peptides. Importantly, native gurmarin was obtained by random...

  16. Emulsified phosphatidylserine, simple and effective peptide carrier for induction of potent epitope-specific T cell responses.

    Directory of Open Access Journals (Sweden)

    Toru Ichihashi

    Full Text Available BACKGROUND: To induce potent epitope-specific T cell immunity by a peptide-based vaccine, epitope peptides must be delivered efficiently to antigen-presenting cells (APCs in vivo. Therefore, selecting an appropriate peptide carrier is crucial for the development of an effective peptide vaccine. In this study, we explored new peptide carriers which show enhancement in cytotoxic T lymphocyte (CTL induction capability. METHODOLOGY/PRINCIPAL FINDINGS: Data from an epitope-specific in vivo CTL assay revealed that phosphatidylserine (PS has a potent adjuvant effect among candidate materials tested. Further analyses showed that PS-conjugated antigens were preferentially and efficiently captured by professional APCs, in particular, by CD11c(+CD11b(+MHCII(+ conventional dendritic cells (cDCs compared to multilamellar liposome-conjugates or unconjugated antigens. In addition, PS demonstrated the stimulatory capacity of peptide-specific helper T cells in vivo. CONCLUSIONS/SIGNIFICANCE: This work indicates that PS is the easily preparable efficient carrier with a simple structure that delivers antigen to professional APCs effectively and induce both helper and cytotoxic T cell responses in vivo. Therefore, PS is a promising novel adjuvant for T cell-inducing peptide vaccines.

  17. Emulsified phosphatidylserine, simple and effective peptide carrier for induction of potent epitope-specific T cell responses.

    Science.gov (United States)

    Ichihashi, Toru; Satoh, Toshifumi; Sugimoto, Chihiro; Kajino, Kiichi

    2013-01-01

    To induce potent epitope-specific T cell immunity by a peptide-based vaccine, epitope peptides must be delivered efficiently to antigen-presenting cells (APCs) in vivo. Therefore, selecting an appropriate peptide carrier is crucial for the development of an effective peptide vaccine. In this study, we explored new peptide carriers which show enhancement in cytotoxic T lymphocyte (CTL) induction capability. Data from an epitope-specific in vivo CTL assay revealed that phosphatidylserine (PS) has a potent adjuvant effect among candidate materials tested. Further analyses showed that PS-conjugated antigens were preferentially and efficiently captured by professional APCs, in particular, by CD11c(+)CD11b(+)MHCII(+) conventional dendritic cells (cDCs) compared to multilamellar liposome-conjugates or unconjugated antigens. In addition, PS demonstrated the stimulatory capacity of peptide-specific helper T cells in vivo. This work indicates that PS is the easily preparable efficient carrier with a simple structure that delivers antigen to professional APCs effectively and induce both helper and cytotoxic T cell responses in vivo. Therefore, PS is a promising novel adjuvant for T cell-inducing peptide vaccines.

  18. Chagas disease-specific antigens: characterization of epitopes in CRA/FRA by synthetic peptide mapping and evaluation by ELISA-peptide assay.

    Science.gov (United States)

    Bottino, Carolina G; Gomes, Luciano P; Pereira, José B; Coura, José R; Provance, David William; De-Simone, Salvatore G

    2013-12-03

    The identification of epitopes in proteins recognized by medically relevant antibodies is useful for the development of peptide-based diagnostics and vaccines. In this study, epitopes in the cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) proteins from Trypanosoma cruzi were identified using synthetic peptide techniques and pooled sera from Chagasic patients. The epitopes were further assayed with an ELISA assay based on synthetic peptides. Twenty-two overlapping synthetic peptides representing the coding sequence of the T. cruzi CRA and FRA proteins were assessed by a Spot-synthesis array analysis using sera donated by patients with Chagas disease. Shorter peptides were selected that represented the determined epitopes and synthesized by solid phase synthesis to evaluate the patterns of cross-reactivities and discrimination through an ELISA-diagnostic assay. The peptide Spot-synthesis array successfully identified two IgG antigenic determinants in the CRA protein and four in FRA. Bioinformatics suggested that the CRA antigens were unique to T. cruzi while the FRA antigen showed similarity with sequences present within various proteins from Leishmania sp. Subsequently, shorter peptides representing the CRA-1, CRA-2 and FRA-1 epitopes were synthesized by solid phase synthesis and assayed by an ELISA-diagnostic assay. The CRA antigens gave a high discrimination between Chagasic, Leishmaniasis and T. cruzi-uninfected serum. A sensitivity and specificity of 100% was calculated for CRA. While the FRA antigen showed a slightly lower sensitivity (91.6%), its specificity was only 60%. The epitopes recognized by human anti-T. cruzi antibodies have been precisely located in two biomarkers of T. cruzi, CRA and FRA. The results from screening a panel of patient sera through an ELISA assay based on peptides representing these epitopes strongly suggest that the sequences from CRA would be useful for the development of diagnostic reagents that could

  19. Molecular Docking Simulation of Neuraminidase Influenza A Subtype H1N1 with Potential Inhibitor of Disulfide Cyclic Peptide (DNY, NNY, LRL)

    Science.gov (United States)

    Putra, R. P.; Imaniastuti, R.; Nasution, M. A. F.; Kerami, Djati; Tambunan, U. S. F.

    2018-04-01

    Oseltamivir resistance as an inhibitor of neuraminidase influenza A virus subtype H1N1 has been reported lately. Therefore, to solve this problem, several kinds of research has been conducted to design and discover disulfide cyclic peptide ligands through molecular docking method, to find the potential inhibitors for neuraminidase H1N1 which then can disturb the virus replication. This research was studied and evaluated the interaction of ligands toward enzyme using molecular docking simulation, which was performed on three disulfide cyclic peptide inhibitors (DNY, LRL, and NNT), along with oseltamivir and zanamivir as the standard ligands using MOE 2008.10 software. The docking simulation shows that all disulfide cyclic peptide ligands have lower Gibbs free binding energies (ΔGbinding) than the standard ligands, with DNY ligand has the lowest ΔGbinding at -7.8544 kcal/mol. Furthermore, these ligands were also had better molecular interactions with neuraminidase than the standards, owing by the hydrogen bonds that were formed during the docking simulation. In the end, we concluded that DNY, LRL and NNT ligands have the potential to be developed as the inhibitor of neuraminidase H1N1.

  20. Safety, efficacy, and molecular mechanism of claudin-1-specific peptides to enhance blood-nerve-barrier permeability.

    Science.gov (United States)

    Sauer, Reine-Solange; Krug, Susanne M; Hackel, Dagmar; Staat, Christian; Konasin, Natalia; Yang, Shaobing; Niedermirtl, Benedikt; Bosten, Judith; Günther, Ramona; Dabrowski, Sebastian; Doppler, Kathrin; Sommer, Claudia; Blasig, Ingolf E; Brack, Alexander; Rittner, Heike L

    2014-07-10

    The blood-nerve barrier consists of the perineurium and endoneurial vessels. The perineurial barrier is composed of a basal membrane and a layer of perineurial cells sealed by tight junction proteins preventing e.g. application of analgesics for selective regional pain control. One of the barrier-sealing proteins in the blood-nerve barrier is claudin-1. Therefore, the claudin-1-peptidomimetics (C1C2), derived from the first extracellular loop (ECL1) on claudin-1 was developed. In this study, we further evaluated the expression of tight junction proteins in the perineurium in Wistar rats and characterized the specificity, in vivo applicability, mechanism of action as well as the biocompatibility of C1C2. In the perineurium, claudin-19, tricellulin and ZO-1, but no claudin-2, 3, 8 and -11 were expressed. C1C2 specifically bound to the ECL1 of claudin-1 and fluorescent 5,6-carboxytetramethylrhodamine-C1C2 was rapidly internalized. Opening the perineurium with C1C2 reduced the mRNA and protein expression of claudin-1 and increased small and macromolecule permeability into the peripheral nerve. Application of C1C2 facilitated regional analgesia using μ-opioid receptor agonists like DAMGO or morphine without motor impairment in naïve rats as well as rats with hind paw inflammation. In contrast the control peptide C2C2 derived from ECL1 on claudin-2 did neither open the barrier nor facilitated opioid-mediated regional analgesia. C1C2 delivery was well tolerated and caused no morphological and functional nerve damage. C1C2 effects could be reversed by interference with the wnt-signal-transduction pathway, specifically the homeobox transcription factor cdx2, using a glycogen-synthase-kinase-3 inhibitor. In summary, we describe the composition of and a pathway to open the perineurial barrier employing a peptide to deliver hydrophilic substances to the peripheral nerve. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Identification of STAT1 and STAT3 specific inhibitors using comparative virtual screening and docking validation.

    Directory of Open Access Journals (Sweden)

    Malgorzata Szelag

    Full Text Available Signal transducers and activators of transcription (STATs facilitate action of cytokines, growth factors and pathogens. STAT activation is mediated by a highly conserved SH2 domain, which interacts with phosphotyrosine motifs for specific STAT-receptor contacts and STAT dimerization. The active dimers induce gene transcription in the nucleus by binding to a specific DNA-response element in the promoter of target genes. Abnormal activation of STAT signaling pathways is implicated in many human diseases, like cancer, inflammation and auto-immunity. Searches for STAT-targeting compounds, exploring the phosphotyrosine (pTyr-SH2 interaction site, yielded many small molecules for STAT3 but sparsely for other STATs. However, many of these inhibitors seem not STAT3-specific, thereby questioning the present modeling and selection strategies of SH2 domain-based STAT inhibitors. We generated new 3D structure models for all human (hSTATs and developed a comparative in silico docking strategy to obtain further insight into STAT-SH2 cross-binding specificity of a selection of previously identified STAT3 inhibitors. Indeed, by primarily targeting the highly conserved pTyr-SH2 binding pocket the majority of these compounds exhibited similar binding affinity and tendency scores for all STATs. By comparative screening of a natural product library we provided initial proof for the possibility to identify STAT1 as well as STAT3-specific inhibitors, introducing the 'STAT-comparative binding affinity value' and 'ligand binding pose variation' as selection criteria. In silico screening of a multi-million clean leads (CL compound library for binding of all STATs, likewise identified potential specific inhibitors for STAT1 and STAT3 after docking validation. Based on comparative virtual screening and docking validation, we developed a novel STAT inhibitor screening tool that allows identification of specific STAT1 and STAT3 inhibitory compounds. This could increase our

  2. PEGylated DX-1000: Pharmacokinetics and Antineoplastic Activity of a Specific Plasmin Inhibitor

    Directory of Open Access Journals (Sweden)

    Laetitia Devy

    2007-11-01

    Full Text Available Novel inhibitors of the urokinase-mediated plasminogen (plg activation system are potentially of great clinical benefit as anticancer treatments. Using phage display, we identified DX-1000 a tissue factor pathway inhibitor-derived Kunitz domain protein which is a specific high-affinity inhibitor of plasmin (pin (Ki = 99 pM. When tested in vitro, DX-1000 blocks plasminmediated pro-matrix metal loproteinase-9 (proMMP-9 activation on cells and dose-dependently inhibits tube formation, while not significantly affecting hemostasis and coagulation. However, this low-molecular weight protein inhibitor (~ 7 kDa exhibits rapid plasma clearance in mice and rabbits, limiting its potential clinical use in chronic diseases. After site-specific PEGylation, DX-1000 retains its activity and exhibits a decreased plasma clearance. This PEGylated derivative is effective in vitro, as well as potent in inhibiting tumor growth of green fluorescent protein (GFP-labeled MDA-MB-231 cells. 4PEG-DX-1000 treatment causes a significant reduction of urokinase-type plasminogen activator (uPA and plasminogen expressions, a reduction of tumor proliferation, and vascularization. 4PEG-DX-1000 treatment significantly decreases the level of active mitogenactivated protein kinase (MAPK in the primary tumors and reduces metastasis incidence. Together, our results demonstrate the potential value of plasmin inhibitors as therapeutic agents for blocking breast cancer growth and metastasis.

  3. Selective serotonin reuptake inhibitor antidepressant use in first trimester pregnancy and risk of specific congenital anomalies

    DEFF Research Database (Denmark)

    Wemakor, Anthony; Casson, Karen; Garne, Ester

    2015-01-01

    Evidence of an association between early pregnancy exposure to selective serotonin reuptake inhibitors (SSRI) and congenital heart defects (CHD) has contributed to recommendations to weigh benefits and risks carefully. The objective of this study was to determine the specificity of association be...

  4. Scorpion Toxins Specific for Potassium (K+ Channels: A Historical Overview of Peptide Bioengineering

    Directory of Open Access Journals (Sweden)

    Zachary L. Bergeron

    2012-11-01

    Full Text Available Scorpion toxins have been central to the investigation and understanding of the physiological role of potassium (K+ channels and their expansive function in membrane biophysics. As highly specific probes, toxins have revealed a great deal about channel structure and the correlation between mutations, altered regulation and a number of human pathologies. Radio- and fluorescently-labeled toxin isoforms have contributed to localization studies of channel subtypes in expressing cells, and have been further used in competitive displacement assays for the identification of additional novel ligands for use in research and medicine. Chimeric toxins have been designed from multiple peptide scaffolds to probe channel isoform specificity, while advanced epitope chimerization has aided in the development of novel molecular therapeutics. Peptide backbone cyclization has been utilized to enhance therapeutic efficiency by augmenting serum stability and toxin half-life in vivo as a number of K+-channel isoforms have been identified with essential roles in disease states ranging from HIV, T-cell mediated autoimmune disease and hypertension to various cardiac arrhythmias and Malaria. Bioengineered scorpion toxins have been monumental to the evolution of channel science, and are now serving as templates for the development of invaluable experimental molecular therapeutics.

  5. Glucagon-like peptide-1 is specifically involved in sweet taste transmission.

    Science.gov (United States)

    Takai, Shingo; Yasumatsu, Keiko; Inoue, Mayuko; Iwata, Shusuke; Yoshida, Ryusuke; Shigemura, Noriatsu; Yanagawa, Yuchio; Drucker, Daniel J; Margolskee, Robert F; Ninomiya, Yuzo

    2015-06-01

    Five fundamental taste qualities (sweet, bitter, salty, sour, umami) are sensed by dedicated taste cells (TCs) that relay quality information to gustatory nerve fibers. In peripheral taste signaling pathways, ATP has been identified as a functional neurotransmitter, but it remains to be determined how specificity of different taste qualities is maintained across synapses. Recent studies demonstrated that some gut peptides are released from taste buds by prolonged application of particular taste stimuli, suggesting their potential involvement in taste information coding. In this study, we focused on the function of glucagon-like peptide-1 (GLP-1) in initial responses to taste stimulation. GLP-1 receptor (GLP-1R) null mice had reduced neural and behavioral responses specifically to sweet compounds compared to wild-type (WT) mice. Some sweet responsive TCs expressed GLP-1 and its receptors were expressed in gustatory neurons. GLP-1 was released immediately from taste bud cells in response to sweet compounds but not to other taste stimuli. Intravenous administration of GLP-1 elicited transient responses in a subset of sweet-sensitive gustatory nerve fibers but did not affect other types of fibers, and this response was suppressed by pre-administration of the GLP-1R antagonist Exendin-4(3-39). Thus GLP-1 may be involved in normal sweet taste signal transmission in mice. © FASEB.

  6. A Tetrameric Peptide Derived from Bovine Lactoferricin Exhibits Specific Cytotoxic Effects against Oral Squamous-Cell Carcinoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Víctor A. Solarte

    2015-01-01

    Full Text Available Several short linear peptides derived from cyclic bovine lactoferricin were synthesized and tested for their cytotoxic effect against the oral cavity squamous-cell carcinoma (OSCC cell lines CAL27 and SCC15. As a control, an immortalized and nontumorigenic cell line, Het-1A, was used. Linear peptides based on the RRWQWR core sequence showed a moderate cytotoxic effect and specificity towards tumorigenic cells. A tetrameric peptide, LfcinB(20–254, containing the RRWQWR motif, exhibited greater cytotoxic activity (>90% in both OSCC cell lines compared to the linear lactoferricin peptide or the lactoferrin protein. Additionally, this tetrameric peptide showed the highest specificity towards tumorigenic cells among the tested peptides. Interestingly, this effect was very fast, with cell shrinkage, severe damage to cell membrane permeability, and lysis within one hour of treatment. Our results are consistent with a necrotic effect rather than an apoptotic one and suggest that this tetrameric peptide could be considered as a new candidate for the therapeutic treatment of OSCC.

  7. A Tetrameric Peptide Derived from Bovine Lactoferricin Exhibits Specific Cytotoxic Effects against Oral Squamous-Cell Carcinoma Cell Lines.

    Science.gov (United States)

    Solarte, Víctor A; Rosas, Jaiver E; Rivera, Zuly J; Arango-Rodríguez, Martha L; García, Javier E; Vernot, Jean-Paul

    2015-01-01

    Several short linear peptides derived from cyclic bovine lactoferricin were synthesized and tested for their cytotoxic effect against the oral cavity squamous-cell carcinoma (OSCC) cell lines CAL27 and SCC15. As a control, an immortalized and nontumorigenic cell line, Het-1A, was used. Linear peptides based on the RRWQWR core sequence showed a moderate cytotoxic effect and specificity towards tumorigenic cells. A tetrameric peptide, LfcinB(20-25)4, containing the RRWQWR motif, exhibited greater cytotoxic activity (>90%) in both OSCC cell lines compared to the linear lactoferricin peptide or the lactoferrin protein. Additionally, this tetrameric peptide showed the highest specificity towards tumorigenic cells among the tested peptides. Interestingly, this effect was very fast, with cell shrinkage, severe damage to cell membrane permeability, and lysis within one hour of treatment. Our results are consistent with a necrotic effect rather than an apoptotic one and suggest that this tetrameric peptide could be considered as a new candidate for the therapeutic treatment of OSCC.

  8. Anti-trypanosomal activity of non-peptidic nitrile-based cysteine protease inhibitors.

    Science.gov (United States)

    Burtoloso, Antonio C B; de Albuquerque, Sérgio; Furber, Mark; Gomes, Juliana C; Gonçalez, Cristiana; Kenny, Peter W; Leitão, Andrei; Montanari, Carlos A; Quilles, José Carlos; Ribeiro, Jean F R; Rocha, Josmar R

    2017-02-01

    The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the treatment of Chagas disease. Anti-trypanosomal activity against the CL Brener strain of T. cruzi was observed in the 0.1 μM to 1 μM range for three nitrile-based cysteine protease inhibitors based on two scaffolds known to be associated with cathepsin K inhibition. The two compounds showing the greatest potency against the trypanosome were characterized by EC50 values (0.12 μM and 0.25 μM) that were an order of magnitude lower than the corresponding Ki values measured against cruzain, a recombinant form of cruzipain, in an enzyme inhibition assay. This implies that the anti-trypanosomal activity of these two compounds may not be explained only by the inhibition of the cruzain enzyme, thereby triggering a putative polypharmacological profile towards cysteine proteases.

  9. Design, Synthesis, and Biological Evaluation of Vanillin Hydroxamic Acid Derivatives as Novel Peptide Deformylase Inhibitors.

    Science.gov (United States)

    Gao, Jian; Qiu, Shengzhi; Liang, Li; Hao, Zhixiang; Zhou, Qianqian; Wang, Fanfan; Mou, Jie; Lin, Qisi

    2018-01-01

    Infectious disease is increasingly hampering human health, which challenge the discovery of new antibacterial target. Peptide deformylase (PDF), a metalloenzyme responsible for catalyzing the removal of the N-formyl group from nascent proteins, was considered as an important target in antibacterial drug discovery. Reported here are the design, synthesis and biological evaluation of vanillin hydroxamic acid derivatives. Analysis of the structure-activity relationships lead to the discovery of compound 8, which exhibits promising antibacterial activity against Escherichia coli, Staphylococcus aureus, Aspergillus oryzae, and Aspergillus foetidus with the MIC value of 0.32 µg/ml, 0.32 µg/ml, 0.16 µg/ml and 0.16 µg/ml, respectively. Furthermore, molecular docking study was applied to elucidate binding interaction between compound 8 and PDF, which indicate that compound 8 not only shares the same binding pocket with actinonin, but also has a similar binding pattern. In silico pharmacokinetic and toxicity prediction studies also suggested that compound 8 has a relatively high drug score of 0.80, and has no risk of toxicity. Compound 8 might represent a promising scaffold for the further development of novel antibacterial drugs. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  10. 2D-QSAR in hydroxamic acid derivatives as peptide deformylase inhibitors and antibacterial agents.

    Science.gov (United States)

    Gupta, Manish K; Mishra, Pradeep; Prathipati, Philip; Saxena, Anil K

    2002-12-01

    Peptide deformylase catalyzes the removal of N-formyl group from the N-formylmethionine of ribosome synthesized polypeptide in eubacteria. Quantitative structure-activity relationship (QSAR) studies have been carried out in a series of beta-sulfonyl and beta-sulfinyl hydroxamic acid derivatives for their PDF enzyme inhibitory and antibacterial activities against Escherichia coli DC2 and Moraxella catarrhalis RA21 which demonstrate that the PDF inhibitory activity in cell free and whole cell system increases with increase in molar refractivity and hydrophobicity. The comparison of the QSARs between the cell free and whole cell system indicate that the active binding sites in PDF isolated from E. coli and in M. catarrhalis RA21 are similar and the whole cell antibacterial activity is mainly due to the inhibition of PDF. Apart from this the QSARs on some matrixmetelloproteins (COL-1, COL-3, MAT and HME) and natural endopeptidase (NEP) indicate the possibilities of introducing selectivity in these hydroxamic acid derivatives for their PDF inhibitory activity.

  11. Kinetic analysis of the mechanism and specificity of protein-disulfide isomerase using fluorescence-quenched peptides

    DEFF Research Database (Denmark)

    Westphal, V; Spetzler, J C; Meldal, M

    1998-01-01

    Protein-disulfide isomerase (PDI) is an abundant folding catalyst in the endoplasmic reticulum of eukaryotic cells. PDI introduces disulfide bonds into newly synthesized proteins and catalyzes disulfide bond isomerizations. We have synthesized a library of disulfide-linked fluorescence......-quenched peptides, individually linked to resin beads, for two purposes: 1) to probe PDI specificity, and 2) to identify simple, sensitive peptide substrates of PDI. Using this library, beads that became rapidly fluorescent by reduction by human PDI were selected. Amino acid sequencing of the bead-linked peptides...

  12. The development and application of a quantitative peptide microarray platform to SH2 domain specificity space

    Science.gov (United States)

    Engelmann, Brett Warren

    The Src homology 2 (SH2) domains evolved alongside protein tyrosine kinases (PTKs) and phosphatases (PTPs) in metazoans to recognize the phosphotyrosine (pY) post-translational modification. The human genome encodes 121 SH2 domains within 111 SH2 domain containing proteins that represent the primary mechanism for cellular signal transduction immediately downstream of PTKs. Despite pY recognition contributing to roughly half of the binding energy, SH2 domains possess substantial binding specificity, or affinity discrimination between phosphopeptide ligands. This specificity is largely imparted by amino acids (AAs) adjacent to the pY, typically from positions +1 to +4 C-terminal to the pY. Much experimental effort has been undertaken to construct preferred binding motifs for many SH2 domains. However, due to limitations in previous experimental methodologies these motifs do not account for the interplay between AAs. It was therefore not known how AAs within the context of individual peptides function to impart SH2 domain specificity. In this work we identified the critical role context plays in defining SH2 domain specificity for physiological ligands. We also constructed a high quality interactome using 50 SH2 domains and 192 physiological ligands. We next developed a quantitative high-throughput (Q-HTP) peptide microarray platform to assess the affinities four SH2 domains have for 124 physiological ligands. We demonstrated the superior characteristics of our platform relative to preceding approaches and validated our results using established biophysical techniques, literature corroboration, and predictive algorithms. The quantitative information provided by the arrays was leveraged to investigate SH2 domain binding distributions and identify points of binding overlap. Our microarray derived affinity estimates were integrated to produce quantitative interaction motifs capable of predicting interactions. Furthermore, our microarrays proved capable of resolving

  13. Engineering the substrate and inhibitor specificities of human coagulation Factor VIIa

    DEFF Research Database (Denmark)

    Larsen, Katrine S; Østergaard, Henrik; Bjelke, Jais R

    2007-01-01

    The remarkably high specificity of the coagulation proteases towards macromolecular substrates is provided by numerous interactions involving the catalytic groove and remote exosites. For FVIIa [activated FVII (Factor VII)], the principal initiator of coagulation via the extrinsic pathway, several...... for FVIIa by marked changes in primary substrate specificity and decreased rates of antithrombin III inhibition. Interestingly, these changes do not necessarily coincide with an altered ability to activate Factor X, demonstrating that inhibitor and macromolecular substrate selectivity may be engineered...

  14. 99mTc-UBI Biokinetics: A Specific Peptide for Infection Detection

    Science.gov (United States)

    Rodríguez-Cortés, J.; Meléndez-Alafort, L.; Herrera-Rodríguez, R.; Ferro-Flores, G.; Mitsoura, E.; Martínez-Duncker, C.

    2003-09-01

    Recently, antimicrobial peptides have been proposed as new agents to distinguish between bacterial infections and sterile inflammatory processes. Based on these considerations, Ubiquicidin peptide 29-41 (UBI) has been labeled with 99mTc using a new direct method showing a radiochemical purity > 97 %, high stability in human serum, and low protein binding. In addition 99mTc-UBI showed a specific in vitro and in vivo binding to bacteria. However its biokinetic parameters have not been evaluated since it is one of the new generation radiopharmaceuticals based on peptide structures. Therefore the aim of this project was to establish the biokinetic model for 99mTc-UBI. An activity from 74 to 148 MBq was injected to patients with bone infection and 5 whole body scans were taken at 1, 30, 120, 240 min and 24 h after radiopharmaceutical administration, with a dual head scanner. Urine was collected for 24 h. An antropomorphic phantom was previously used to calculate the effect of attenuation and scattered radiation on the gamma camera acquisition images. ROIs of the selected organs in patients (kidney, liver, heart, bone, soft tissue and lesion) were drawn, and attenuation and scatter corrected. The % urine elimination at 24 h and time integrated ROIs (cpm/pixel/ROIs) were used to obtain the residence time (τ) in each tissue and to establish the biokinetic model. Pharmacokinetic data show that blood clearance is biexponential with a mean residence time in the central compartment of 0.52 h. The images showed non-accumulation in metabolic organs. More than 75 % of the injected activity was eliminated by renal clearance 4 h after 99mTc-UBI administration. The mean radiation dose calculated according to the MIRD formalism was 0.130 mGy/MBq for kidney and the effective dose was 4.29 × 10-3 mSv/MBq.

  15. Propiconazole is a specific and accessible brassinosteroid (BR) biosynthesis inhibitor for Arabidopsis and maize.

    Science.gov (United States)

    Hartwig, Thomas; Corvalan, Claudia; Best, Norman B; Budka, Joshua S; Zhu, Jia-Ying; Choe, Sunghwa; Schulz, Burkhard

    2012-01-01

    Brassinosteroids (BRs) are steroidal hormones that play pivotal roles during plant development. In addition to the characterization of BR deficient mutants, specific BR biosynthesis inhibitors played an essential role in the elucidation of BR function in plants. However, high costs and limited availability of common BR biosynthetic inhibitors constrain their key advantage as a species-independent tool to investigate BR function. We studied propiconazole (Pcz) as an alternative to the BR inhibitor brassinazole (Brz). Arabidopsis seedlings treated with Pcz phenocopied BR biosynthetic mutants. The steady state mRNA levels of BR, but not gibberellic acid (GA), regulated genes increased proportional to the concentrations of Pcz. Moreover, root inhibition and Pcz-induced expression of BR biosynthetic genes were rescued by 24epi-brassinolide, but not by GA(3) co-applications. Maize seedlings treated with Pcz showed impaired mesocotyl, coleoptile, and true leaf elongation. Interestingly, the genetic background strongly impacted the tissue specific sensitivity towards Pcz. Based on these findings we conclude that Pcz is a potent and specific inhibitor of BR biosynthesis and an alternative to Brz. The reduced cost and increased availability of Pcz, compared to Brz, opens new possibilities to study BR function in larger crop species.

  16. Novel tetra-peptide insertion in Gag-p6 ALIX-binding motif in HIV-1 subtype C associated with protease inhibitor failure in Indian patients.

    Science.gov (United States)

    Neogi, Ujjwal; Rao, Shwetha D; Bontell, Irene; Verheyen, Jens; Rao, Vasudev R; Gore, Sagar C; Soni, Neelesh; Shet, Anita; Schülter, Eugen; Ekstrand, Maria L; Wondwossen, Amogne; Kaiser, Rolf; Madhusudhan, Mallur S; Prasad, Vinayaka R; Sonnerborg, Anders

    2014-09-24

    A novel tetra-peptide insertion was identified in Gag-p6 ALIX-binding region, which appeared in protease inhibitor failure Indian HIV-1C sequences (odds ratio=17.1, P < 0.001) but was naturally present in half of untreated Ethiopian HIV-1C sequences. The insertion is predicted to restore ALIX-mediated virus release pathway, which is lacking in HIV-1C. The clinical importance of the insertion needs to be evaluated in HIV-1C dominating regions wherein the use of protease inhibitor drugs are being scaled up.

  17. Peptide-Based Membrane Fusion Inhibitors Targeting HCoV-229E Spike Protein HR1 and HR2 Domains

    Directory of Open Access Journals (Sweden)

    Shuai Xia

    2018-02-01

    Full Text Available Human coronavirus 229E (HCoV-229E infection in infants, elderly people, and immunocompromised patients can cause severe disease, thus calling for the development of effective and safe therapeutics to treat it. Here we reported the design, synthesis and characterization of two peptide-based membrane fusion inhibitors targeting HCoV-229E spike protein heptad repeat 1 (HR1 and heptad repeat 2 (HR2 domains, 229E-HR1P and 229E-HR2P, respectively. We found that 229E-HR1P and 229E-HR2P could interact to form a stable six-helix bundle and inhibit HCoV-229E spike protein-mediated cell-cell fusion with IC50 of 5.7 and 0.3 µM, respectively. 229E-HR2P effectively inhibited pseudotyped and live HCoV-229E infection with IC50 of 0.5 and 1.7 µM, respectively. In a mouse model, 229E-HR2P administered intranasally could widely distribute in the upper and lower respiratory tracts and maintain its fusion-inhibitory activity. Therefore, 229E-HR2P is a promising candidate for further development as an antiviral agent for the treatment and prevention of HCoV-229E infection.

  18. Crystallization and Preliminary Diffraction Analysis of the CAL PDZ Domain in Complex with a Selective Peptide Inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    J Amacher; P Cushing; J Weiner; D Madden

    2011-12-31

    Cystic fibrosis (CF) is associated with loss-of-function mutations in the CF transmembrane conductance regulator (CFTR), which regulates epithelial fluid and ion homeostasis. The CFTR cytoplasmic C-terminus interacts with a number of PDZ (PSD-95/Dlg/ZO-1) proteins that modulate its intracellular trafficking and chloride-channel activity. Among these, the CFTR-associated ligand (CAL) has a negative effect on apical-membrane expression levels of the most common disease-associated mutant {Delta}F508-CFTR, making CAL a candidate target for the treatment of CF. A selective peptide inhibitor of the CAL PDZ domain (iCAL36) has recently been developed and shown to stabilize apical expression of {Delta}F508-CFTR, enhancing net chloride-channel activity, both alone and in combination with the folding corrector corr-4a. As a basis for structural studies of the CAL-iCAL36 interaction, a purification protocol has been developed that increases the oligomeric homogeneity of the protein. Here, the cocrystallization of the complex in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 35.9, b = 47.7, c = 97.3 {angstrom}, is reported. The crystals diffracted to 1.4 {angstrom} resolution. Based on the calculated Matthews coefficient (1.96 {angstrom}{sup 3} Da{sup -1}), it appears that the asymmetric unit contains two complexes.

  19. Patient-specific dosimetry in peptide receptor radionuclide therapy: a clinical review

    International Nuclear Information System (INIS)

    Chalkia, M.T.; Stefanoyiannis, A.P.; Chatziioannou, S.N.; Efstathopoulos, E.P.; Round, W.H.; Nikiforidis, G.C.

    2015-01-01

    Neuroendocrine tumours (NETs) belong to a relatively rare class of neoplasms. Nonetheless, their prevalence has increased significantly during the last decades. Peptide receptor radionuclide therapy (PRRT) is a relatively new treatment approach for inoperable or metastasised NETs. The therapeutic effect is based on the binding of radiolabelled somatostatin analogue peptides with NETs’ somatostatin receptors, resulting in internal irradiation of tumours. Pre-therapeutic patient-specific dosimetry is essential to ensure that a treatment course has high levels of safety and efficacy. This paper reviews the methods applied for PRRT dosimetry, as well as the dosimetric results presented in the literature. Focus is given on data concerning the therapeutic somatostatin analogue radiopeptides 111 In-[DTPA o , D -Phe 1 ]-octreotide ( 111 In-DTPA-octreotide), 90 Y-[DOTA o ,Tyr 3 ]-octreotide ( 90 Y-DOTATOC) and 177 Lu-[DOTA o ,Tyr 3 ,Thr 8 ]-octreotide ( 177 Lu-DOTATATE). Following the Medical Internal Radiation Dose (MIRD) Committee formalism, dosimetric analysis demonstrates large interpatient variability in tumour and organ uptake, with kidneys and bone marrow being the critical organs. The results are dependent on the image acquisition and processing protocol, as well as the dosimetric imaging radiopharmaceutical.

  20. Sequence specificity for uridylylation of the viral peptide linked to the genome (VPg) of enteroviruses.

    Science.gov (United States)

    Schein, Catherine H; Ye, Mengyi; Paul, Aniko V; Oberste, M Steven; Chapman, Nora; van der Heden van Noort, Gerbrand J; Filippov, Dmitri V; Choi, Kyung H

    2015-10-01

    Enteroviruses (EV) uridylylate a peptide, VPg, as the first step in their replication. VPgpUpU, found free in infected cells, serves as the primer for RNA elongation. The abilities of four polymerases (3D(pol)), from EV-species A-C, to uridylylate VPgs that varied by up to 60% of their residues were compared. Each 3D(pol) was able to uridylylate all five VPgs using polyA RNA as template, while showing specificity for its own genome encoded peptide. All 3D(pol) uridylylated a consensus VPg representing the physical chemical properties of 31 different VPgs. Thus the residues required for uridylylation and the enzymatic mechanism must be similar in diverse EV. As VPg-binding sites differ in co-crystal structures, the reaction is probably done by a second 3D(pol) molecule. The conservation of polymerase residues whose mutation reduces uridylylation but not RNA elongation is compared. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Conformation-specific spectroscopy of capped glutamine-containing peptides: role of a single glutamine residue on peptide backbone preferences.

    Science.gov (United States)

    Walsh, Patrick S; Dean, Jacob C; McBurney, Carl; Kang, Hyuk; Gellman, Samuel H; Zwier, Timothy S

    2016-04-28

    The conformational preferences of a series of short, aromatic-capped, glutamine-containing peptides have been studied under jet-cooled conditions in the gas phase. This work seeks a bottom-up understanding of the role played by glutamine residues in directing peptide structures that lead to neurodegenerative diseases. Resonant ion-dip infrared (RIDIR) spectroscopy is used to record single-conformation infrared spectra in the NH stretch, amide I and amide II regions. Comparison of the experimental spectra with the predictions of calculations carried out at the DFT M05-2X/6-31+G(d) level of theory lead to firm assignments for the H-bonding architectures of a total of eight conformers of four molecules, including three in Z-Gln-OH, one in Z-Gln-NHMe, three in Ac-Gln-NHBn, and one in Ac-Ala-Gln-NHBn. The Gln side chain engages actively in forming H-bonds with nearest-neighbor amide groups, forming C8 H-bonds to the C-terminal side, C9 H-bonds to the N-terminal side, and an amide-stacked geometry, all with an extended (C5) peptide backbone about the Gln residue. The Gln side chain also stabilizes an inverse γ-turn in the peptide backbone by forming a pair of H-bonds that bridge the γ-turn and stabilize it. Finally, the entire conformer population of Ac-Ala-Gln-NHBn is funneled into a single structure that incorporates the peptide backbone in a type I β-turn, stabilized by the Gln side chain forming a C7 H-bond to the central amide group in the β-turn not otherwise involved in a hydrogen bond. This β-turn backbone structure is nearly identical to that observed in a series of X-(AQ)-Y β-turns in the protein data bank, demonstrating that the gas-phase structure is robust to perturbations imposed by the crystalline protein environment.

  2. Changes in glucose-induced plasma active glucagon-like peptide-1 levels by co-administration of sodium–glucose cotransporter inhibitors with dipeptidyl peptidase-4 inhibitors in rodents

    Directory of Open Access Journals (Sweden)

    Takahiro Oguma

    2016-12-01

    Full Text Available We investigated whether structurally different sodium–glucose cotransporter (SGLT 2 inhibitors, when co-administered with dipeptidyl peptidase-4 (DPP4 inhibitors, could enhance glucagon-like peptide-1 (GLP-1 secretion during oral glucose tolerance tests (OGTTs in rodents. Three different SGLT inhibitors—1-(β-d-Glucopyranosyl-4-chloro-3-[5-(6-fluoro-2-pyridyl-2-thienylmethyl]benzene (GTB, TA-1887, and canagliflozin—were examined to assess the effect of chemical structure. Oral treatment with GTB plus a DPP4 inhibitor enhanced glucose-induced plasma active GLP-1 (aGLP-1 elevation and suppressed glucose excursions in both normal and diabetic rodents. In DPP4-deficient rats, GTB enhanced glucose-induced aGLP-1 elevation without affecting the basal level, whereas metformin, previously reported to enhance GLP-1 secretion, increased both the basal level and glucose-induced elevation. Oral treatment with canagliflozin and TA-1887 also enhanced glucose-induced aGLP-1 elevation when co-administered with either teneligliptin or sitagliptin. These data suggest that structurally different SGLT2 inhibitors enhance plasma aGLP-1 elevation and suppress glucose excursions during OGTT when co-administered with DPP4 inhibitors, regardless of the difference in chemical structure. Combination treatment with DPP4 inhibitors and SGLT2 inhibitors having moderate SGLT1 inhibitory activity may be a promising therapeutic option for improving glycemic control in patients with type 2 diabetes mellitus.

  3. Single-subunit oligosaccharyltransferases of Trypanosoma brucei display different and predictable peptide acceptor specificities.

    Science.gov (United States)

    Jinnelov, Anders; Ali, Liaqat; Tinti, Michele; Güther, Maria Lucia S; Ferguson, Michael A J

    2017-12-08

    Trypanosoma brucei causes African trypanosomiasis and contains three full-length oligosaccharyltransferase (OST) genes; two of which, Tb STT3A and Tb STT3B, are expressed in the bloodstream form of the parasite. These OSTs have different peptide acceptor and lipid-linked oligosaccharide donor specificities, and trypanosomes do not follow many of the canonical rules developed for other eukaryotic N -glycosylation pathways, raising questions as to the basic architecture and detailed function of trypanosome OSTs. Here, we show by blue-native gel electrophoresis and stable isotope labeling in cell culture proteomics that the Tb STT3A and Tb STT3B proteins associate with each other in large complexes that contain no other detectable protein subunits. We probed the peptide acceptor specificities of the OSTs in vivo using a transgenic glycoprotein reporter system and performed glycoproteomics on endogenous parasite glycoproteins using sequential endoglycosidase H and peptide: N -glycosidase-F digestions. This allowed us to assess the relative occupancies of numerous N -glycosylation sites by endoglycosidase H-resistant N -glycans originating from Man 5 GlcNAc 2 -PP-dolichol transferred by Tb STT3A, and endoglycosidase H-sensitive N -glycans originating from Man 9 GlcNAc 2 -PP-dolichol transferred by Tb STT3B. Using machine learning, we assessed the features that best define Tb STT3A and Tb STT3B substrates in vivo and built an algorithm to predict the types of N -glycan most likely to predominate at all the putative N -glycosylation sites in the parasite proteome. Finally, molecular modeling was used to suggest why Tb STT3A has a distinct preference for sequons containing and/or flanked by acidic amino acid residues. Together, these studies provide insights into how a highly divergent eukaryote has re-wired protein N -glycosylation to provide protein sequence-specific N -glycan modifications. Data are available via ProteomeXchange with identifiers PXD007236, PXD007267

  4. Vaccination with lipid core peptides fails to induce epitope-specific T cell responses but confers non-specific protective immunity in a malaria model.

    Directory of Open Access Journals (Sweden)

    Simon H Apte

    Full Text Available Vaccines against many pathogens for which conventional approaches have failed remain an unmet public health priority. Synthetic peptide-based vaccines offer an attractive alternative to whole protein and whole organism vaccines, particularly for complex pathogens that cause chronic infection. Previously, we have reported a promising lipid core peptide (LCP vaccine delivery system that incorporates the antigen, carrier, and adjuvant in a single molecular entity. LCP vaccines have been used to deliver several peptide subunit-based vaccine candidates and induced high titre functional antibodies and protected against Group A streptococcus in mice. Herein, we have evaluated whether LCP constructs incorporating defined CD4(+ and/or CD8(+ T cell epitopes could induce epitope-specific T cell responses and protect against pathogen challenge in a rodent malaria model. We show that LCP vaccines failed to induce an expansion of antigen-specific CD8(+ T cells following primary immunization or by boosting. We further demonstrated that the LCP vaccines induced a non-specific type 2 polarized cytokine response, rather than an epitope-specific canonical CD8(+ T cell type 1 response. Cytotoxic responses of unknown specificity were also induced. These non-specific responses were able to protect against parasite challenge. These data demonstrate that vaccination with lipid core peptides fails to induce canonical epitope-specific T cell responses, at least in our rodent model, but can nonetheless confer non-specific protective immunity against Plasmodium parasite challenge.

  5. T-cell libraries allow simple parallel generation of multiple peptide-specific human T-cell clones.

    Science.gov (United States)

    Theaker, Sarah M; Rius, Cristina; Greenshields-Watson, Alexander; Lloyd, Angharad; Trimby, Andrew; Fuller, Anna; Miles, John J; Cole, David K; Peakman, Mark; Sewell, Andrew K; Dolton, Garry

    2016-03-01

    Isolation of peptide-specific T-cell clones is highly desirable for determining the role of T-cells in human disease, as well as for the development of therapies and diagnostics. However, generation of monoclonal T-cells with the required specificity is challenging and time-consuming. Here we describe a library-based strategy for the simple parallel detection and isolation of multiple peptide-specific human T-cell clones from CD8(+) or CD4(+) polyclonal T-cell populations. T-cells were first amplified by CD3/CD28 microbeads in a 96U-well library format, prior to screening for desired peptide recognition. T-cells from peptide-reactive wells were then subjected to cytokine-mediated enrichment followed by single-cell cloning, with the entire process from sample to validated clone taking as little as 6 weeks. Overall, T-cell libraries represent an efficient and relatively rapid tool for the generation of peptide-specific T-cell clones, with applications shown here in infectious disease (Epstein-Barr virus, influenza A, and Ebola virus), autoimmunity (type 1 diabetes) and cancer. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  6. Sensing site-specific structural characteristics and chirality using vibrational circular dichroism of isotope labeled peptides.

    Science.gov (United States)

    Keiderling, Timothy A

    2017-12-01

    Isotope labeling has a long history in chemistry as a tool for probing structure, offering enhanced sensitivity, or enabling site selection with a wide range of spectroscopic tools. Chirality sensitive methods such as electronic circular dichroism are global structural tools and have intrinsically low resolution. Consequently, they are generally insensitive to modifications to enhance site selectivity. The use of isotope labeling to modify vibrational spectra with unique resolvable frequency shifts can provide useful site-specific sensitivity, and these methods have been recently more widely expanded in biopolymer studies. While the spectral shifts resulting from changes in isotopic mass can provide resolution of modes from specific parts of the molecule and can allow detection of local change in structure with perturbation, these shifts alone do not directly indicate structure or chirality. With vibrational circular dichroism (VCD), the shifted bands and their resultant sign patterns can be used to indicate local conformations in labeled biopolymers, particularly if multiple labels are used and if their coupling is theoretically modeled. This mini-review discusses selected examples of the use of labeling specific amides in peptides to develop local structural insight with VCD spectra. © 2017 Wiley Periodicals, Inc.

  7. Baicalin and scutellarin are proteasome inhibitors that specifically target chymotrypsin-like catalytic activity.

    Science.gov (United States)

    Wu, Yi-Xin; Sato, Eiji; Kimura, Wataru; Miura, Naoyuki

    2013-09-01

    Baicalin and scutellarin are the major active principal flavonoids extracted from the Chinese herbal medicines Scutellaria baicalensis and Erigeron breviscapus (Vant.) Hand-Mazz. It has recently been reported that baicalin and scutellarin have antitumor activity. However, the mechanisms of action are unknown. We previously reported that some flavonoids have a specific role in the inhibition of the activity of proteasome subunits and induced apoptosis in tumor cells. To further investigate these pharmacological effects, we examined the inhibitory activity of baicalin and scutellarin on the extracted proteasomes from mice and cancer cells. Using fluorogenic substrates for proteasome catalytic subunits, we found that baicalin and scutellarin specifically inhibited chymotrypsin-like activity but did not inhibit trypsin-like and peptidyl-glutamyl peptide hydrolyzing activities. These data suggested that baicalin and scutellarin specifically inhibit chymotrypsin-like catalytic activity in the proteasome. Copyright © 2012 John Wiley & Sons, Ltd.

  8. Structural Basis for Specific Inhibition of tRNA Synthetase by an ATP Competitive Inhibitor

    OpenAIRE

    Fang, Pengfei; Han, Hongyan; Wang, Jing; Chen, Kaige; Chen, Xin; Guo, Min

    2015-01-01

    Pharmaceutical inhibitors of aminoacyl-tRNA synthetases demand high species and family specificity. The antimalarial ATP-mimetic cladosporin selectively inhibits P. falciparum LysRS (PfLysRS). How the binding to a universal ATP site achieves the specificity is unknown. Here we report 3 crystal structures of cladosporin with human LysRS, PfLysRS, and a Pf-like human LysRS mutant. In all 3 structures, cladosporin occupies the class defining ATP-binding pocket, replacing the adenosine portion of...

  9. Matrix- and plasma-derived peptides promote tissue-specific injury responses and wound healing in diabetic swine.

    Science.gov (United States)

    Sheets, Anthony R; Massey, Conner J; Cronk, Stephen M; Iafrati, Mark D; Herman, Ira M

    2016-07-02

    , peptide treatment increases mRNA expression of several pro-angiogenic, epithelializing, and matrix-remodeling factors, importantly including balanced inductions in matrix metalloproteinase-2, -9, and tissue inhibitor of metalloproteinases-1, lending further insight into their mechanisms. Comb1 and UN3 stimulate wound resolution in diabetic Yorkshire swine through upregulation of multiple reparative growth factors and cytokines, especially matrix metalloproteinases and inhibitors that may aid in reversing the proteolytic imbalance characteristic of chronically inflamed non-healing wounds. Together, these peptides should have great therapeutic potential for all patients in need of healing, regardless of injury etiology.

  10. Specificity of RSG-1.2 peptide binding to RRE-IIB RNA element of HIV-1 over Rev peptide is mainly enthalpic in origin.

    Science.gov (United States)

    Kumar, Santosh; Bose, Debojit; Suryawanshi, Hemant; Sabharwal, Harshana; Mapa, Koyeli; Maiti, Souvik

    2011-01-01

    Rev is an essential HIV-1 regulatory protein which binds to the Rev responsive element (RRE) present within the env gene of HIV-1 RNA genome. This binding facilitates the transport of the RNA to the cytoplasm, which in turn triggers the switch between viral latency and active viral replication. Essential components of this complex have been localized to a minimal arginine rich Rev peptide and stem IIB region of RRE. A synthetic peptide known as RSG-1.2 binds with high binding affinity and specificity to the RRE-IIB than the Rev peptide, however the thermodynamic basis of this specificity has not yet been addressed. The present study aims to probe the thermodynamic origin of this specificity of RSG-1.2 over Rev Peptide for RRE-IIB. The temperature dependent melting studies show that RSG-1.2 binding stabilizes the RRE structure significantly (ΔT(m) = 4.3°C), in contrast to Rev binding. Interestingly the thermodynamic signatures of the binding have also been found to be different for both the peptides. At pH 7.5, RSG-1.2 binds RRE-IIB with a K(a) = 16.2±0.6×10(7) M(-1) where enthalpic change ΔH = -13.9±0.1 kcal/mol is the main driving force with limited unfavorable contribution from entropic change TΔS = -2.8±0.1 kcal/mol. A large part of ΔH may be due to specific stacking between U72 and Arg15. In contrast binding of Rev (K(a) = 3.1±0.4×10(7) M(-1)) is driven mainly by entropy (ΔH = 0 kcal/mol and TΔS = 10.2±0.2 kcal/mol) which arises from major conformational changes in the RNA upon binding.

  11. Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours

    Energy Technology Data Exchange (ETDEWEB)

    Lasko, Loren M.; Jakob, Clarissa G.; Edalji, Rohinton P.; Qiu, Wei; Montgomery, Debra; Digiammarino, Enrico L.; Hansen, T. Matt; Risi, Roberto M.; Frey, Robin; Manaves, Vlasios; Shaw, Bailin; Algire, Mikkel; Hessler, Paul; Lam, Lloyd T.; Uziel, Tamar; Faivre, Emily; Ferguson, Debra; Buchanan, Fritz G.; Martin, Ruth L.; Torrent, Maricel; Chiang, Gary G.; Karukurichi, Kannan; Langston, J. William; Weinert, Brian T.; Choudhary, Chunaram; de Vries, Peter; Van Drie, John H.; McElligott, David; Kesicki, Ed; Marmorstein, Ronen; Sun, Chaohong; Cole, Philip A.; Rosenberg, Saul H.; Michaelides, Michael R.; Lai, Albert; Bromberg, Kenneth D. (AbbVie); (UCopenhagen); (Petra Pharma); (UPENN); (JHU); (Van Drie); (Faraday)

    2017-09-27

    The dynamic and reversible acetylation of proteins, catalysed by histone acetyltransferases (HATs) and histone deacetylases (HDACs), is a major epigenetic regulatory mechanism of gene transcription1 and is associated with multiple diseases. Histone deacetylase inhibitors are currently approved to treat certain cancers, but progress on the development of drug-like histone actyltransferase inhibitors has lagged behind2. The histone acetyltransferase paralogues p300 and CREB-binding protein (CBP) are key transcriptional co-activators that are essential for a multitude of cellular processes, and have also been implicated in human pathological conditions (including cancer3). Current inhibitors of the p300 and CBP histone acetyltransferase domains, including natural products4, bi-substrate analogues5 and the widely used small molecule C6466,7, lack potency or selectivity. Here, we describe A-485, a potent, selective and drug-like catalytic inhibitor of p300 and CBP. We present a high resolution (1.95 Å) co-crystal structure of a small molecule bound to the catalytic active site of p300 and demonstrate that A-485 competes with acetyl coenzyme A (acetyl-CoA). A-485 selectively inhibited proliferation in lineage-specific tumour types, including several haematological malignancies and androgen receptor-positive prostate cancer. A-485 inhibited the androgen receptor transcriptional program in both androgen-sensitive and castration-resistant prostate cancer and inhibited tumour growth in a castration-resistant xenograft model. These results demonstrate the feasibility of using small molecule inhibitors to selectively target the catalytic activity of histone acetyltransferases, which may provide effective treatments for transcriptional activator-driven malignancies and diseases.

  12. Specific CLK inhibitors from a novel chemotype for regulation of alternative splicing

    DEFF Research Database (Denmark)

    Fedorov, Oleg; Huber, Kilian; Eisenreich, Andreas

    2011-01-01

    There is a growing recognition of the importance of protein kinases in the control of alternative splicing. To define the underlying regulatory mechanisms, highly selective inhibitors are needed. Here, we report the discovery and characterization of the dichloroindolyl enaminonitrile KH-CB19......, a potent and highly specific inhibitor of the CDC2-like kinase isoforms 1 and 4 (CLK1/CLK4). Cocrystal structures of KH-CB19 with CLK1 and CLK3 revealed a non-ATP mimetic binding mode, conformational changes in helix aC and the phosphate binding loop and halogen bonding to the kinase hinge region. KH-CB19...... effectively suppressed phosphorylation of SR (serine/arginine) proteins in cells, consistent with its expected mechanism of action. Chemical inhibition of CLK1/CLK4 generated a unique pattern of splicing factor dephosphorylation and had at low nM concentration a profound effect on splicing of the two tissue...

  13. Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours

    DEFF Research Database (Denmark)

    Lasko, Loren M; Jakob, Clarissa G; Edalji, Rohinton P

    2017-01-01

    -specific tumour types, including several haematological malignancies and androgen receptor-positive prostate cancer. A-485 inhibited the androgen receptor transcriptional program in both androgen-sensitive and castration-resistant prostate cancer and inhibited tumour growth in a castration-resistant xenograft...... to treat certain cancers, but progress on the development of drug-like histone actyltransferase inhibitors has lagged behind. The histone acetyltransferase paralogues p300 and CREB-binding protein (CBP) are key transcriptional co-activators that are essential for a multitude of cellular processes, and have...... also been implicated in human pathological conditions (including cancer). Current inhibitors of the p300 and CBP histone acetyltransferase domains, including natural products, bi-substrate analogues and the widely used small molecule C646, lack potency or selectivity. Here, we describe A-485, a potent...

  14. Inhibitors of serotonin reuptake and specific imipramine binding in human blood plasma

    International Nuclear Information System (INIS)

    Brusov, O.S.; Fomenko, A.M.; Katasonov, A.B.; Lidemann, R.R.

    1985-01-01

    This paper describes a method of extraction of endogenous inhibitors of specific IMI binding and of 5-HT reuptake, from human blood plasma and the heterogeneity of these compounds is demonstrated. Specific binding was determined as the difference between binding of 3 H-IMI in the absence and in the presence of 50 microM IMI. Under these conditions, specific binding amounted to 70-80% of total binding of 3 H-IMI. It is shown that extract obtained from human blood contains a material which inhibits dose-dependently both 5-HT reuptake and specific binding of 3 H-IMI. Gel-chromatography of extracts of human blood plasma on Biogel P-2 is also shown

  15. PLGA nanoparticles modified with a BBB-penetrating peptide co-delivering Aβ generation inhibitor and curcumin attenuate memory deficits and neuropathology in Alzheimer's disease mice.

    Science.gov (United States)

    Huang, Na; Lu, Shuai; Liu, Xiao-Ge; Zhu, Jie; Wang, Yu-Jiong; Liu, Rui-Tian

    2017-10-06

    Alzheimer's disease (AD) is the most common form of dementia, characterized by the formation of extracellular senile plaques and neuronal loss caused by amyloid β (Aβ) aggregates in the brains of AD patients. Conventional strategies failed to treat AD in clinical trials, partly due to the poor solubility, low bioavailability and ineffectiveness of the tested drugs to cross the blood-brain barrier (BBB). Moreover, AD is a complex, multifactorial neurodegenerative disease; one-target strategies may be insufficient to prevent the processes of AD. Here, we designed novel kind of poly(lactide-co-glycolic acid) (PLGA) nanoparticles by loading with Aβ generation inhibitor S1 (PQVGHL peptide) and curcumin to target the detrimental factors in AD development and by conjugating with brain targeting peptide CRT (cyclic CRTIGPSVC peptide), an iron-mimic peptide that targets transferrin receptor (TfR), to improve BBB penetration. The average particle size of drug-loaded PLGA nanoparticles and CRT-conjugated PLGA nanoparticles were 128.6 nm and 139.8 nm, respectively. The results of Y-maze and new object recognition test demonstrated that our PLGA nanoparticles significantly improved the spatial memory and recognition in transgenic AD mice. Moreover, PLGA nanoparticles remarkably decreased the level of Aβ, reactive oxygen species (ROS), TNF-α and IL-6, and enhanced the activities of super oxide dismutase (SOD) and synapse numbers in the AD mouse brains. Compared with other PLGA nanoparticles, CRT peptide modified-PLGA nanoparticles co-delivering S1 and curcumin exhibited most beneficial effect on the treatment of AD mice, suggesting that conjugated CRT peptide, and encapsulated S1 and curcumin exerted their corresponding functions for the treatment.

  16. Optimizing fluorescently tethered Hsp90 inhibitor dose for maximal specific uptake by breast tumors

    Science.gov (United States)

    Crouch, Brian T.; Duer, Joy; Wang, Roujia; Gallagher, Jennifer; Hall, Allison; Soo, Mary Scott; Hughes, Philip; Haystead, Timothy A. J.; Ramanujam, Nirmala

    2018-03-01

    Despite improvements in surgical resection, 20-40% of patients undergoing breast conserving surgery require at least one additional re-excision. Leveraging the unique surface expression of heat shock protein 90 (Hsp90), a chaperone protein involved in several key hallmarks of cancer, in breast cancer provides an exciting opportunity to identify residual disease during surgery. We developed a completely non-destructive strategy using HS-27, a fluorescently-tethered Hsp90 inhibitor, to assay surface Hsp90 expression on intact tissue specimens using a fluorescence microendoscope with a field of view of 750 μm and subcellular resolution of 4 μm. HS-27 consists of an FDA approved Hsp90 inhibitor tethered to fluorescein isothiocyanate (EX 488nm, EM 525nm). Here, we optimized ex vivo HS-27 administration in pre-clinical breast cancer models and validated our approach on 21 patients undergoing standard of care ultrasound guided core needle biopsy. HS-27 administration time was fixed at 1- minute to minimize imaging impact on clinical workflow. HS-27 and HS-217 (non-specific control) doses were modulated from 1 μM up to 100 μM to identify the dose maximizing the ratio of specific uptake (HS-27 fluorescence) to non-specific uptake (HS-217 fluorescence). The specificity ratio was maximized at 100 μM and was significantly greater than all other doses (pcancer makes this technology attractive for assessing tumor margins, as one agent can be used for all subtypes.

  17. The Hydrophobic Region of the DmsA Twin-Arginine Leader Peptide Determines Specificity with Chaperone DmsD

    OpenAIRE

    Winstone, Tara M. L.; Tran, Vy A.; Turner, Raymond J.

    2013-01-01

    The system specific chaperone DmsD plays a role in the maturation of the catalytic subunit of dimethyl sulfoxide (DMSO) reductase, DmsA. Pre-DmsA contains a 45-amino acid twin-arginine leader peptide that is important for targeting and translocation of folded and cofactor-loaded DmsA by the twin-arginine translocase. DmsD has previously been shown to interact with the complete twin-arginine leader peptide of DmsA. In this study, isothermal titration calorimetry was used to investigate the the...

  18. Design of potent inhibitors of human RAD51 recombinase based on BRC motifs of BRCA2 protein: modeling and experimental validation of a chimera peptide.

    KAUST Repository

    Nomme, Julian; Renodon-Corniè re, Axelle; Asanomi, Yuya; Sakaguchi, Kazuyasu; Stasiak, Alicja Z; Stasiak, Andrzej; Norden, Bengt; Tran, Vinh; Takahashi, Masayuki

    2010-01-01

    We have previously shown that a 28-amino acid peptide derived from the BRC4 motif of BRCA2 tumor suppressor inhibits selectively human RAD51 recombinase (HsRad51). With the aim of designing better inhibitors for cancer treatment, we combined an in silico docking approach with in vitro biochemical testing to construct a highly efficient chimera peptide from eight existing human BRC motifs. We built a molecular model of all BRC motifs complexed with HsRad51 based on the crystal structure of the BRC4 motif-HsRad51 complex, computed the interaction energy of each residue in each BRC motif, and selected the best amino acid residue at each binding position. This analysis enabled us to propose four amino acid substitutions in the BRC4 motif. Three of these increased the inhibitory effect in vitro, and this effect was found to be additive. We thus obtained a peptide that is about 10 times more efficient in inhibiting HsRad51-ssDNA complex formation than the original peptide.

  19. Design of potent inhibitors of human RAD51 recombinase based on BRC motifs of BRCA2 protein: modeling and experimental validation of a chimera peptide.

    KAUST Repository

    Nomme, Julian

    2010-08-01

    We have previously shown that a 28-amino acid peptide derived from the BRC4 motif of BRCA2 tumor suppressor inhibits selectively human RAD51 recombinase (HsRad51). With the aim of designing better inhibitors for cancer treatment, we combined an in silico docking approach with in vitro biochemical testing to construct a highly efficient chimera peptide from eight existing human BRC motifs. We built a molecular model of all BRC motifs complexed with HsRad51 based on the crystal structure of the BRC4 motif-HsRad51 complex, computed the interaction energy of each residue in each BRC motif, and selected the best amino acid residue at each binding position. This analysis enabled us to propose four amino acid substitutions in the BRC4 motif. Three of these increased the inhibitory effect in vitro, and this effect was found to be additive. We thus obtained a peptide that is about 10 times more efficient in inhibiting HsRad51-ssDNA complex formation than the original peptide.

  20. A+-Helix of Protein C Inhibitor (PCI) Is a Cell-penetrating Peptide That Mediates Cell Membrane Permeation of PCI*

    Science.gov (United States)

    Yang, Hanjiang; Wahlmüller, Felix Christof; Sarg, Bettina; Furtmüller, Margareta; Geiger, Margarethe

    2015-01-01

    Protein C inhibitor (PCI) is a serpin with broad protease reactivity. It binds glycosaminoglycans and certain phospholipids that can modulate its inhibitory activity. PCI can penetrate through cellular membranes via binding to phosphatidylethanolamine. The exact mechanism of PCI internalization and the intracellular role of the serpin are not well understood. Here we showed that testisin, a glycosylphosphatidylinositol-anchored serine protease, cleaved human PCI and mouse PCI (mPCI) at their reactive sites as well as at sites close to their N terminus. This cleavage was observed not only with testisin in solution but also with cell membrane-anchored testisin on U937 cells. The cleavage close to the N terminus released peptides rich in basic amino acids. Synthetic peptides corresponding to the released peptides of human PCI (His1–Arg11) and mPCI (Arg1–Ala18) functioned as cell-penetrating peptides. Because intact mPCI but not testisin-cleaved mPCI was internalized by Jurkat T cells, a truncated mPCI mimicking testisin-cleaved mPCI was created. The truncated mPCI lacking 18 amino acids at the N terminus was not taken up by Jurkat T cells. Therefore our model suggests that testisin or other proteases could regulate the internalization of PCI by removing its N terminus. This may represent one of the mechanisms regulating the intracellular functions of PCI. PMID:25488662

  1. Anti-tumor effects of dehydroaltenusin, a specific inhibitor of mammalian DNA polymerase α

    International Nuclear Information System (INIS)

    Maeda, Naoki; Kokai, Yasuo; Ohtani, Seiji; Sahara, Hiroeki; Kuriyama, Isoko; Kamisuki, Shinji; Takahashi, Shunya; Sakaguchi, Kengo; Sugawara, Fumio; Yoshida, Hiromi; Sato, Noriyuki; Mizushina, Yoshiyuki

    2007-01-01

    In the screening of selective inhibitors of eukaryotic DNA polymerases (pols), dehydroaltenusin was found to be an inhibitor of pol α from a fungus (Alternaria tennuis). We succeeded in chemically synthesizing dehydroaltenusin, and the compound inhibited only mammalian pol α with IC 50 value of 0.5 μM, and did not influence the activities of other replicative pols such as pols δ and ε, but also showed no effect on pol α activity from another vertebrate, fish, or from a plant species. Dehydroaltenusin also had no influence on the other pols and DNA metabolic enzymes tested. The compound also inhibited the proliferation of human cancer cells with LD 50 values of 38.0-44.4 μM. In an in vivo anti-tumor assay on nude mice bearing solid tumors of HeLa cells, dehydroaltenusin was shown to be a promising suppressor of solid tumors. Histopathological examination revealed that increased tumor necrosis and decreased mitotic index were apparently detected by the compound in vivo. Therefore, dehydroaltenusin could be of interest as not only a mammalian pol α-specific inhibitor, but also as a candidate drug for anti-cancer treatment

  2. The role of secretory leukocyte proteinase inhibitor and elafin (elastase-specific inhibitor/skin-derived antileukoprotease as alarm antiproteinases in inflammatory lung disease

    Directory of Open Access Journals (Sweden)

    Sallenave Jean-Michel

    2000-08-01

    Full Text Available Abstract Secretory leukocyte proteinase inhibitor and elafin are two low-molecular-mass elastase inhibitors that are mainly synthesized locally at mucosal sites. It is thought that their physicochemical properties allow them to efficiently inhibit target enzymes, such as neutrophil elastase, released into the interstitium. Historically, in the lung, these inhibitors were first purified from secretions of patients with chronic obstructive pulmonary disease and cystic fibrosis. This suggested that they might be important in controlling excessive neutrophil elastase release in these pathologies. They are upregulated by 'alarm signals' such as bacterial lipopolysaccharides, and cytokines such as interleukin-1 and tumor necrosis factor and have been shown to be active against Gram-positive and Gram-negative bacteria, so that they have joined the growing list of antimicrobial 'defensin-like' peptides produced by the lung. Their site of synthesis and presumed functions make them very attractive candidates as potential therapeutic agents under conditions in which the excessive release of elastase by neutrophils might be detrimental. Because of its natural tropism for the lung, the use of adenovirus-mediated gene transfer is extremely promising in such applications.

  3. A mucin-like peptide from Fasciola hepatica induces parasite-specific Th1-type cell immunity.

    Science.gov (United States)

    Noya, Verónica; Brossard, Natalie; Berasaín, Patricia; Rodríguez, Ernesto; Chiale, Carolina; Mazal, Daniel; Carmona, Carlos; Freire, Teresa

    2016-03-01

    Fasciolosis, caused by the liver fluke Fasciola hepatica, is a major parasitic disease of livestock that causes significant economic losses worldwide. Although drugs are effective against liver flukes, they do not prevent reinfection, and continuous treatment is costly. Moreover, resistant fluke strains are emerging. In this context, vaccination is a good alternative since it provides a cost-effective long-term prevention strategy to control fasciolosis. In this paper, we evaluate the Fhmuc peptide as a potential vaccine against fasciolosis. This peptide derives from a mucin-like protein highly expressed in the infective stage of Fasciola hepatica. Mucin-like molecules expressed by parasites can contribute to several infection processes by protecting the parasite from host proteases and recognition by the immune system. We show that the Fhmuc peptide induces Th1-like immune responses specific for F. hepatica excretion-secretion products (FhESP) with a high production of IFNγ. We also investigated whether this peptide could protect animals from infection, and present preliminary data indicating that animals treated with Fhmuc exhibited reduced liver damage compared to non-immunised animals and that this protection was associated with a recruitment of B and T lymphocytes in the peritoneum, as well as eosinophils and mature dendritic cells. These results suggest that the mucin-like peptide Fhmuc could constitute a potential vaccine candidate against fasciolosis and pave the way towards the development of vaccines against parasites.

  4. Enzyme-Linked Immunosorbent Assay Using a Virus Type-Specific Peptide Based on a Subdomain of Envelope Protein Erns for Serologic Diagnosis of Pestivirus Infections in Swine

    Science.gov (United States)

    Langedijk, J. P. M.; Middel, W. G. J.; Meloen, R. H.; Kramps, J. A.; de Smit, J. A.

    2001-01-01

    Peptides deduced from the C-terminal end (residues 191 to 227) of pestivirus envelope protein Erns were used to develop enzyme-linked immunosorbent assays (ELISAs) to measure specifically antibodies against different types of pestiviruses. The choice of the peptide was based on the modular structure of the Erns protein, and the peptide was selected for its probable independent folding and good exposure, which would make it a good candidate for an antigenic peptide to be used in a diagnostic test. A solid-phase peptide ELISA which was cross-reactive for several types of pestivirus antibodies and which can be used for the general detection of pestivirus antibodies was developed. To identify type-specific pestivirus antibodies, a liquid-phase peptide ELISA, with a labeled, specific classical swine fever virus (CSFV) peptide and an unlabeled bovine viral diarrhea virus peptide to block cross-reactivity, was developed. Specificity and sensitivity of the liquid-phase peptide ELISA for CSFV were 98 and 100%, respectively. Because the peptide is a fragment of the Erns protein, it can be used to differentiate between infected and vaccinated animals when a vaccine based on the E2 protein, which is another pestivirus envelope protein, is used. PMID:11230402

  5. Endogenous collagen peptide activation of CD1d-restricted NKT cells ameliorates tissue-specific inflammation in mice

    DEFF Research Database (Denmark)

    Liu, Yawei; Teige, Anna; Mondoc, Emma

    2011-01-01

    NKT cells in the mouse recognize antigen in the context of the MHC class I-like molecule CD1d and play an important role in peripheral tolerance and protection against autoimmune and other diseases. NKT cells are usually activated by CD1d-presented lipid antigens. However, peptide recognition...... in the context of CD1 has also been documented, although no self-peptide ligands have been reported to date. Here, we have identified an endogenous peptide that is presented by CD1d to activate mouse NKT cells. This peptide, the immunodominant epitope from mouse collagen type II (mCII707-721), was not associated...... with either MHC class I or II. Activation of CD1d-restricted mCII707-721-specific NKT cells was induced via TCR signaling and classical costimulation. In addition, mCII707-721-specific NKT cells induced T cell death through Fas/FasL, in an IL-17A-independent fashion. Moreover, mCII707-721-specific NKT cells...

  6. TNYL peptide functional chitosan-g-stearate conjugate micelles for tumor specific targeting

    Directory of Open Access Journals (Sweden)

    Chen FY

    2014-09-01

    Full Text Available Feng-Ying Chen,1 Jing-Jing Yan,1 Han-Xi Yi,2 Fu-Qiang Hu,2 Yong-Zhong Du,2 Hong Yuan,2 Jian You,2 Meng-Dan Zhao1 1Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, People’s Republic of China; 2College of Pharmaceutical Science, Zhejiang University, Hangzhou, People’s Republic of China Abstract: Nowadays, a real challenge in cancer therapy is to design drug delivery systems that can achieve high concentrations of drugs at the target site for improved therapeutic effect with reduced side effects. In this research, we designed and synthesized a homing peptide-(TNYLFSPNGPIA, TNYL modified chitosan-g-stearate (CS polymer micelle (named T-CS for targeting delivery. The peptide displayed specific binding affinity to EphB4 which is a member of the Eph family of receptor tyrosine protein kinases. The amphiphilic polymer T-CS can gather into micelles by themselves in an aqueous environment with a low critical micelle concentration value (91.2 µg/L and nano-scaled size (82.1±2.8 nm. The drug encapsulation efficiency reached 86.43% after loading the hydrophobic drug doxorubicin (DOX. The cytotoxicity of T-CS/DOX against SKOV3 cells was enhanced by approximately 2.3-fold when compared with CS/DOX. The quantitative and qualitative analysis for cellular uptake indicated that TNYL modification can markedly increase cellular internalization in the EphB4-overexpressing SKOV3 cell line, especially with a short incubation time. It is interesting that relatively higher uptake of the T-CS/DOX micelles by SKOV3 cells (positive-EphB4 than A549 cells (negative-EphB4 was observed when the two cells were co-incubated. Furthermore, in vivo distribution experiment using a bilateral-tumor model showed that there was more fluorescence accumulation in the SKOV3 tumor than in the A549 tumor over the whole experiment. These results suggest that TNYL-modified CS micelles may be promising drug carriers as targeting therapy for the EphB4-overexpressing

  7. Isolation and characterization of a specific endogenous Na+, K+-ATPase inhibitor from bovine adrenal

    International Nuclear Information System (INIS)

    Tamura, M.; Lam, T.T.; Inagami, T.

    1988-01-01

    In order to identify a specific endogenous Na + ,K + -ATPase inhibitor which could possibly be related to salt-dependent hypertension, the authors looked for substances in the methanol extract of bovine whole adrenal which show all of the following properties: (i) inhibitory activity for Na + ,K + -ATPase; (ii) competitive displacing activity against [ 3 H]ouabain binding to the enzyme; (iii) inhibitory activity for 86 Rb uptake into intact human erythrocytes; and (iv) cross-reactivity with sheep anti-digoxin-specific antibody. After stepwise fractionation of the methanol extract of bovine adrenal glands by chromatography on a C 18 open column, a 0-15% acetonitrile fraction was fractionated by high-performance liquid chromatography on a Zorbax octadecylsilane column. One of the most active fractions in 0-15% acetonitrile was found to exhibit all of the four types of the activities. It was soluble in water and was distinct from various substances which have been known to inhibit Na + ,K + -ATPase. These results strongly suggest that this water-soluble nonpeptidic Na + ,K + -ATPase inhibitor may be a specific endogenous regulator for the ATPase

  8. Endogenous collagen peptide activation of CD1d-restricted NKT cells ameliorates tissue-specific inflammation in mice.

    Science.gov (United States)

    Liu, Yawei; Teige, Anna; Mondoc, Emma; Ibrahim, Saleh; Holmdahl, Rikard; Issazadeh-Navikas, Shohreh

    2011-01-01

    NKT cells in the mouse recognize antigen in the context of the MHC class I-like molecule CD1d and play an important role in peripheral tolerance and protection against autoimmune and other diseases. NKT cells are usually activated by CD1d-presented lipid antigens. However, peptide recognition in the context of CD1 has also been documented, although no self-peptide ligands have been reported to date. Here, we have identified an endogenous peptide that is presented by CD1d to activate mouse NKT cells. This peptide, the immunodominant epitope from mouse collagen type II (mCII707-721), was not associated with either MHC class I or II. Activation of CD1d-restricted mCII707-721-specific NKT cells was induced via TCR signaling and classical costimulation. In addition, mCII707-721-specific NKT cells induced T cell death through Fas/FasL, in an IL-17A-independent fashion. Moreover, mCII707-721-specific NKT cells suppressed a range of in vivo inflammatory conditions, including delayed-type hypersensitivity, antigen-induced airway inflammation, collagen-induced arthritis, and EAE, which were all ameliorated by mCII707-721 vaccination. The findings presented here offer new insight into the intrinsic roles of NKT cells in health and disease. Given the results, endogenous collagen peptide activators of NKT cells may offer promise as novel therapeutics in tissue-specific autoimmune and inflammatory diseases.

  9. Novel tetra-peptide insertion in Gag-p6 ALIX-binding motif in HIV-1 subtype C associated with protease inhibitor failure

    Science.gov (United States)

    Neogi, Ujjwal; RAO, Shwetha D; BONTELL, Irene; VERHEYEN, Jens; RAO, Vasudev R; GORE, Sagar C; SONI, Neelesh; SHET, Anita; SCHÜLTER, Eugen; EKSTRAND, Maria L.; WONDWOSSEN, Amogne; KAISER, Rolf; MADHUSUDHAN, Mallur S.; PRASAD, Vinayaka R; SONNERBORG, Anders

    2014-01-01

    A novel tetra-peptide insertion was identified in Gag-p6 ALIX-binding region which is appears in protease inhibitor (PI) failure Indian HIV-1C sequences (Odds Ratio 17.1, p<0.001) but naturally present in half of untreated Ethiopian sequences. The insertion will probably restore the ALIX mediated virus release pathway, which is lacking in HIV-1C. The clinical importance of such insertion need to be evaluated in HIV-1C dominating regions were PI-drugs are being scaled up as second line treatment options. PMID:25102091

  10. Investigation of SP94 Peptide as a Specific Probe for Hepatocellular Carcinoma Imaging and Therapy

    Science.gov (United States)

    Li, Yanli; Hu, Yan; Xiao, Jie; Liu, Guobing; Li, Xiao; Zhao, Yanzhao; Tan, Hui; Shi, Hongcheng; Cheng, Dengfeng

    2016-01-01

    SP94 (SFSIIHTPILPL), a novel peptide, has shown specific binding to hepatocellular carcinoma (HCC) cells. We aimed to investigate the capability of SP94 as a targeting probe for HCC imaging and therapy following labeling with technetium-99m (99mTc) and rhenium-188 (188Re). HYNIC-SP94 was prepared by solid phase synthesis and then labeled with 99mTc. Cell competitive binding, internalization assay, in vitro and in vivo stability, biodistribution and micro-single photon emission computed tomography /computed tomography (SPECT/CT) imaging studies were performed to investigate the capability of 99mTc tricine-EDDA/HYNIC-SP94 as a specific HCC imaging probe. Initial promising targeting results inspired evaluation of its therapeutic effect when labeled by 188Re. HYNIC-SP94 was then labeled again with 188Re to perform cell apoptosis, microSPECT/CT imaging evaluation and immunohistochemistry. Huh-7 cells exhibited typical apoptotic changes after 188Re irradiation. According to 99mTc tricine-EDDA/HYNIC-SP94 microSPECT/CT imaging, tumor uptake was significantly decreased compared with that of pre-treatment with 188Re-HYNIC-SP94. The immunohistochemistry also displayed obvious necrosis and apoptosis as well as inhibition of proliferation in the 188Re-HYNIC-SP94 treatment group. The results supported that 99mTc tricine-EDDA/HYNIC-SP94 is able to target HCC cells and 188Re-HYNIC- SP94 holds potential as a therapeutic agent for HCC, making 99mTc/188Re-HYNIC-SP94 a promising targeting probe for HCC imaging and therapy. PMID:27649935

  11. Pharmacological characterization of potent and selective NaV1.7 inhibitors engineered from Chilobrachys jingzhao tarantula venom peptide JzTx-V.

    Directory of Open Access Journals (Sweden)

    Bryan D Moyer

    Full Text Available Identification of voltage-gated sodium channel NaV1.7 inhibitors for chronic pain therapeutic development is an area of vigorous pursuit. In an effort to identify more potent leads compared to our previously reported GpTx-1 peptide series, electrophysiology screening of fractionated tarantula venom discovered the NaV1.7 inhibitory peptide JzTx-V from the Chinese earth tiger tarantula Chilobrachys jingzhao. The parent peptide displayed nominal selectivity over the skeletal muscle NaV1.4 channel. Attribute-based positional scan analoging identified a key Ile28Glu mutation that improved NaV1.4 selectivity over 100-fold, and further optimization yielded the potent and selective peptide leads AM-8145 and AM-0422. NMR analyses revealed that the Ile28Glu substitution changed peptide conformation, pointing to a structural rationale for the selectivity gains. AM-8145 and AM-0422 as well as GpTx-1 and HwTx-IV competed for ProTx-II binding in HEK293 cells expressing human NaV1.7, suggesting that these NaV1.7 inhibitory peptides interact with a similar binding site. AM-8145 potently blocked native tetrodotoxin-sensitive (TTX-S channels in mouse dorsal root ganglia (DRG neurons, exhibited 30- to 120-fold selectivity over other human TTX-S channels and exhibited over 1,000-fold selectivity over other human tetrodotoxin-resistant (TTX-R channels. Leveraging NaV1.7-NaV1.5 chimeras containing various voltage-sensor and pore regions, AM-8145 mapped to the second voltage-sensor domain of NaV1.7. AM-0422, but not the inactive peptide analog AM-8374, dose-dependently blocked capsaicin-induced DRG neuron action potential firing using a multi-electrode array readout and mechanically-induced C-fiber spiking in a saphenous skin-nerve preparation. Collectively, AM-8145 and AM-0422 represent potent, new engineered NaV1.7 inhibitory peptides derived from the JzTx-V scaffold with improved NaV selectivity and biological activity in blocking action potential firing in both

  12. Immunogenicity of Mycobacterium avium subsp. paratuberculosis specific peptides for inclusion in a subunit vaccine against paratuberculosis

    DEFF Research Database (Denmark)

    Mikkelsen, Heidi; Tollefsen, S.; Olsen, I.

    Paratuberculosis in ruminants is caused by an infection with Mycobacterium avium subspecies paratuberculosis (MAP) and is a chronic disease characterized by granulomatous enteritis. Available vaccines against paratuberculosis consist of variations of whole bacteria with adjuvant showing various...... efficacies. The main problem with available vaccines is their interference with surveillance and diagnosis of bovine tuberculosis and paratuberculosis. Our ultimate aim is to develop a subunit vaccine consisting of selected MAP peptides, which allow differentiation of infected from vaccinated animals. Here......, 118 peptides were identified by in silico analysis and synthesized chemically. Peptides were tested for reactivity and immunogenicity with T-cell lines generated from PBMCs isolated from MAP infected goats and with blood samples from MAP infected calves. Immunogenicity of peptides was evaluated using...

  13. Novel chimeric peptide with enhanced cell specificity and anti-inflammatory activity.

    Science.gov (United States)

    Kim, Young-Min; Kim, Nam-Hong; Lee, Jong-Wan; Jang, Jin-Sun; Park, Yung-Hoon; Park, Seong-Cheol; Jang, Mi-Kyeong

    2015-07-31

    An antimicrobial peptide (AMP), Hn-Mc, was designed by combining the N-terminus of HPA3NT3 and the C-terminus of melittin. This chimeric AMP exhibited potent antibacterial activity with low minimal inhibitory concentrations (MICs), ranging from 1 to 2 μM against four drug-susceptible bacteria and ten drug-resistant bacteria. Moreover, the hemolysis and cytotoxicity was reduced significantly compared to those of the parent peptides, highlighting its high cell selectivity. The morphological changes in the giant unilamellar vesicles and bacterial cell surfaces caused by the Hn-Mc peptide suggested that it killed the microbial cells by damaging the membrane envelope. An in vivo study also demonstrated the antibacterial activity of the Hn-Mc peptide in a mouse model infected with drug-resistant bacteria. In addition, the chimeric peptide inhibited the expression of lipopolysaccharide (LPS)-induced cytokines in RAW 264.7 cells by preventing the interaction between LPS and Toll-like receptors. These results suggest that this chimeric peptide is an antimicrobial and anti-inflammatory candidate as a pharmaceutic agent. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Opportunities to Target Specific Contractile Abnormalities with Smooth Muscle Protein Kinase Inhibitors

    Directory of Open Access Journals (Sweden)

    Annegret Ulke-Lemée

    2010-05-01

    Full Text Available Smooth muscle is a major component of most hollow organ systems (e.g., airways, vasculature, bladder and gut/gastrointestine; therefore, the coordinated regulation of contraction is a key property of smooth muscle. When smooth muscle functions normally, it contributes to general health and wellness, but its dysfunction is associated with morbidity and mortality. Rho-associated protein kinase (ROCK is central to calcium-independent, actomyosin-mediated contractile force generation in the vasculature, thereby playing a role in smooth muscle contraction, cell motility and adhesion. Recent evidence supports an important role for ROCK in the increased vasoconstriction and remodeling observed in various models of hypertension. This review will provide a commentary on the development of specific ROCK inhibitors and their clinical application. Fasudil will be discussed as an example of bench-to-bedside development of a clinical therapeutic that is used to treat conditions of vascular hypercontractility. Due to the wide spectrum of biological processes regulated by ROCK, many additional clinical indications might also benefit from ROCK inhibition. Apart from the importance of ROCK in smooth muscle contraction, a variety of other protein kinases are known to play similar roles in regulating contractile force. The zipper-interacting protein kinase (ZIPK and integrin-linked kinase (ILK are two well-described regulators of contraction. The relative contribution of each kinase to contraction depends on the muscle bed as well as hormonal and neuronal stimulation. Unfortunately, specific inhibitors for ZIPK and ILK are still in the development phase, but the success of fasudil suggests that inhibitors for these other kinases may also have valuable clinical applications. Notably, the directed inhibition of ZIPK with a pseudosubstrate molecule shows unexpected effects on the contractility of gastrointestinal smooth muscle.

  15. A preliminary electron microscopic investigation into the interaction between Aβ1-42 peptide and a novel nanoliposome- coupled retro-inverso peptide inhibitor, developed as a potential treatment for Alzheimer's disease

    Science.gov (United States)

    Sherer, M.; Fullwood, N. J.; Taylor, M.; Allsop, D.

    2015-10-01

    Alzheimer's disease (AD) is a progressive neurodegenerative condition that results in severe cognitive and functional decline in sufferers and for which there are currently no effective treatments to halt or reverse disease progression. AD is the most common form of dementia and age is the major risk factor for this disease. With worldwide population structures changing as increasing number of individuals survive into old age, there is urgent need for novel disease modifying treatments for this condition, which has profound effects upon sufferers in addition to those around them. Some of us have previously developed a peptide inhibitor of Aβ1-42 aggregation (RI-OR2-TAT) that has been shown to reduce Aβ1-42 pathology in vivo in mouse models of AD. ∼1690 copies of RI-OR2-TAT have been covalently attached to nanoliposome carrier particles forming Peptide Inhibitor NanoParticles (PINPs), and this study investigated the effect of PINPs upon Aβ1-42 aggregation at the molecular level. Our results show that PINPs are able to reduce Aβ1-42 aggregation and do so by binding early (oligomers) and late (fibrillar) stage aggregates. These results highlight the ability of PINPs to disrupt the formation of multiple Aβ1-42 aggregates capable of causing neurotoxicity and thus provide a strong case for PINPs to be carried forward into early stage clinical trials as a novel therapeutic option for the treatment of AD.

  16. A preliminary electron microscopic investigation into the interaction between Aβ1-42 peptide and a novel nanoliposome- coupled retro-inverso peptide inhibitor, developed as a potential treatment for Alzheimer's disease

    International Nuclear Information System (INIS)

    Sherer, M; Fullwood, N J; Taylor, M; Allsop, D

    2015-01-01

    Alzheimer's disease (AD) is a progressive neurodegenerative condition that results in severe cognitive and functional decline in sufferers and for which there are currently no effective treatments to halt or reverse disease progression. AD is the most common form of dementia and age is the major risk factor for this disease. With worldwide population structures changing as increasing number of individuals survive into old age, there is urgent need for novel disease modifying treatments for this condition, which has profound effects upon sufferers in addition to those around them. Some of us have previously developed a peptide inhibitor of Aβ 1-42 aggregation (RI-OR2-TAT) that has been shown to reduce Aβ 1-42 pathology in vivo in mouse models of AD. ∼1690 copies of RI-OR2-TAT have been covalently attached to nanoliposome carrier particles forming Peptide Inhibitor NanoParticles (PINPs), and this study investigated the effect of PINPs upon Aβ 1-42 aggregation at the molecular level. Our results show that PINPs are able to reduce Aβ 1-42 aggregation and do so by binding early (oligomers) and late (fibrillar) stage aggregates. These results highlight the ability of PINPs to disrupt the formation of multiple Aβ 1-42 aggregates capable of causing neurotoxicity and thus provide a strong case for PINPs to be carried forward into early stage clinical trials as a novel therapeutic option for the treatment of AD. (paper)

  17. Grafting of a peptide probe for Prostate-Specific Antigen detection using diazonium electroreduction and click chemistry.

    Science.gov (United States)

    Strzemińska, I; Sainte Rose Fanchine, S; Anquetin, G; Reisberg, S; Noël, V; Pham, M C; Piro, B

    2016-07-15

    The main objective of this work was to validate a label-free electrochemical method of protein detection using peptides as capture probes. As a proof-of-concept, we used a 7 amino acids sequence (HSSKLQL) specific for Prostate Specific Antigen. We investigated various electrografting conditions of two anilines (2-[(4-aminophenyl)sulfanyl]-8-hydroxy-1,4-naphthoquinone and 4-azidoaniline) further converted in situ into their corresponding diazonium salts on glassy carbon electrodes. It was demonstrated that the best method to obtain a mixed layer is the simultaneous electroreduction of the two diazonium salts. 4-azidoaniline was used to covalently immobilize the ethynyl-functionalized peptide probe by click coupling, and the hydroxynaphthoquinone derivative plays the role of electrochemical transducer of the peptide-protein recognition. The proteolytic activity of PSA towards a small peptide substrate carrying streptavidin at its distal end was also investigated to design an original sensing architecture leading to a reagentless, label free, and "signal-on" PSA sensor. Without optimization, the limit of quantification can be estimated in the nM to pM range. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. A novel method for screening the glutathione transferase inhibitors

    Directory of Open Access Journals (Sweden)

    Węgrzyn Grzegorz

    2009-03-01

    Full Text Available Abstract Background Glutathione transferases (GSTs belong to the family of Phase II detoxification enzymes. GSTs catalyze the conjugation of glutathione to different endogenous and exogenous electrophilic compounds. Over-expression of GSTs was demonstrated in a number of different human cancer cells. It has been found that the resistance to many anticancer chemotherapeutics is directly correlated with the over-expression of GSTs. Therefore, it appears to be important to find new GST inhibitors to prevent the resistance of cells to anticancer drugs. In order to search for glutathione transferase (GST inhibitors, a novel method was designed. Results Our results showed that two fragments of GST, named F1 peptide (GYWKIKGLV and F2 peptide (KWRNKKFELGLEFPNL, can significantly inhibit the GST activity. When these two fragments were compared with several known potent GST inhibitors, the order of inhibition efficiency (measured in reactions with 2,4-dinitrochlorobenzene (CDNB and glutathione as substrates was determined as follows: tannic acid > cibacron blue > F2 peptide > hematin > F1 peptide > ethacrynic acid. Moreover, the F1 peptide appeared to be a noncompetitive inhibitor of the GST-catalyzed reaction, while the F2 peptide was determined as a competitive inhibitor of this reaction. Conclusion It appears that the F2 peptide can be used as a new potent specific GST inhibitor. It is proposed that the novel method, described in this report, might be useful for screening the inhibitors of not only GST but also other enzymes.

  19. Large-scale detection of antigen-specific T cells using peptide-MHC-I multimers labeled with DNA barcodes

    DEFF Research Database (Denmark)

    Bentzen, Amalie Kai; Marquard, Andrea Marion; Lyngaa, Rikke Birgitte

    2016-01-01

    -major histocompatibility complex (MHC) multimers labeled with individual DNA barcodes to screen >1,000 peptide specificities in a single sample, and detect low-frequency CD8 T cells specific for virus- or cancer-restricted antigens. When analyzing T-cell recognition of shared melanoma antigens before and after adoptive...... cell therapy in melanoma patients, we observe a greater number of melanoma-specific T-cell populations compared with cytometry-based approaches. Furthermore, we detect neoepitope-specific T cells in tumor-infiltrating lymphocytes and peripheral blood from patients with non-small cell lung cancer...

  20. Inhibition of xyloglucanase from an alkalothermophilic Thermomonospora sp. by a peptidic aspartic protease inhibitor from Penicillium sp. VM24.

    Science.gov (United States)

    Menon, Vishnu; Rao, Mala

    2012-11-01

    A bifunctional inhibitor from Penicillium sp VM24 causing inactivation of xyloglucanase from Thermomonospora sp and an aspartic protease from Aspergillus saitoi was identified. Steady state kinetics studies of xyloglucanase and the inhibitor revealed an irreversible, non-competitive, two-step inhibition mechanism with IC(50) and K(i) values of 780 and 500nM respectively. The interaction of o-phthalaldehyde (OPTA)-labeled xyloglucanase with the inhibitor revealed that the inhibitor binds to the active site of the enzyme. Far- and near-UV spectrophotometric analysis suggests that the conformational changes induced in xyloglucanase by the inhibitor may be due to irreversible denaturation of enzyme. The bifunctional inhibitor may have potential as a biocontrol agent for the protection of plants against phytopathogenic fungi. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. Selection of SARS-Coronavirus-specific B cell epitopes by phage peptide library screening and evaluation of the immunological effect of epitope-based peptides on mice

    International Nuclear Information System (INIS)

    Yu Hua; Jiang Lifang; Fang Danyun; Yan Huijun; Zhou Jingjiao; Zhou Junmei; Liang Yu; Gao Yang; Zhao, Wei; Long Beiguo

    2007-01-01

    Antibodies to SARS-Coronavirus (SARS-CoV)-specific B cell epitopes might recognize the pathogen and interrupt its adherence to and penetration of host cells. Hence, these epitopes could be useful for diagnosis and as vaccine constituents. Using the phage-displayed peptide library screening method and purified Fab fragments of immunoglobulin G (IgG Fab) from normal human sera and convalescent sera from SARS-CoV-infected patients as targets, 11 B cell epitopes of SARS-CoV spike glycoprotein (S protein) and membrane protein (M protein) were screened. After a bioinformatics tool was used to analyze these epitopes, four epitope-based S protein dodecapeptides corresponding to the predominant epitopes were chosen for synthesis. Their antigenic specificities and immunogenicities were studied in vitro and in vivo. Flow cytometry and ELISPOT analysis of lymphocytes as well as a serologic analysis of antibody showed that these peptides could trigger a rapid, highly effective, and relatively safe immune response in BALB/c mice. These findings might aid development of SARS diagnostics and vaccines. Moreover, the role of S and M proteins as important surface antigens is confirmed

  2. Generation and characterization of peptide-specific, MHC-restricted cytotoxic T lymphocyte (CTL) and helper T cell lines from unprimed T cells under microculture conditions.

    Science.gov (United States)

    Sambhara, S R; Upadhya, A G; Miller, R G

    1990-06-12

    We describe a microculture system for the generation of CTL and T helper cells against peptides. Tryptic digest and cyanogen bromide fragments of chicken ovalbumin and synthetic peptides of ovalbumin (323-339) and influenza virus (NP 365-380) were used to generate CTL and T helper lines from unprimed T cells. These lines were both peptide-specific and MHC-restricted. The relative ease of generating peptide-specific, MHC-restricted CTL and helper T cell lines with as few as 10(6) unprimed lymphocytes can be an efficient method of detecting potential immunogenic determinants of an antigen.

  3. Wheat Subtilisin/Chymotrypsin Inhibitor (WSCI) as a scaffold for novel serine protease inhibitors with a given specificity.

    Science.gov (United States)

    Tedeschi, Francesca; Di Maro, Antimo; Facchiano, Angelo; Costantini, Susan; Chambery, Angela; Bruni, Natalia; Capuzzi, Valeria; Ficca, Anna Grazia; Poerio, Elia

    2012-10-30

    WSCI (Wheat Subtilisin/Chymotrypsin Inhibitor) is a small protein belonging to the Potato inhibitor I family exhibiting a high content of essential amino acid. In addition to bacterial subtilisins and mammalian chymotrypsins, WSCI inhibits chymotrypsin-like activities isolated from digestive traits of a number of insect larvae. In vivo, as suggested for many plant proteinase inhibitors, WSCI seems to play a role of natural defence against attacks of pests and pathogens. The functional region of WSCI, containing the inhibitor reactive site (Met48-Glu49), corresponds to an extended flexible loop (Val42-Asp53) whose architecture is somehow stabilized by a number of secondary interactions established with a small β-sheet located underneath. The aim of this study was to employ a WSCI molecule as a stable scaffold to obtain recombinant inhibitors with new acquired anti-proteinase activity or, alternatively, inactive WSCI variants. A gene sequence coding for the native WSCI, along with genes coding for muteins with different specficities, could be exploited to obtain transformed non-food use plants with improved insect resistance. On the other hand, the genetic transformation of cereal plants over-expressing inactive WSCI muteins could represent a possible strategy to improve the nutritional quality of cereal-based foods, without risk of interference with human or animal digestive enzymes. Here, we described the characterization of four muteins containing single/multiple amino acid substitutions at the WSCI reactive site and/or at its proximity. Modalities of interaction of these muteins with proteinases (subtilisin, trypsin and chymotrypsin) were investigated by time course hydrolysis and molecular simulations studies.

  4. Crystal structure of the N domain of human somatic angiotensin I-converting enzyme provides a structural basis for domain-specific inhibitor design.

    Science.gov (United States)

    Corradi, Hazel R; Schwager, Sylva L U; Nchinda, Aloysius T; Sturrock, Edward D; Acharya, K Ravi

    2006-03-31

    Human somatic angiotensin I-converting enzyme (sACE) is a key regulator of blood pressure and an important drug target for combating cardiovascular and renal disease. sACE comprises two homologous metallopeptidase domains, N and C, joined by an inter-domain linker. Both domains are capable of cleaving the two hemoregulatory peptides angiotensin I and bradykinin, but differ in their affinities for a range of other substrates and inhibitors. Previously we determined the structure of testis ACE (C domain); here we present the crystal structure of the N domain of sACE (both in the presence and absence of the antihypertensive drug lisinopril) in order to aid the understanding of how these two domains differ in specificity and function. In addition, the structure of most of the inter-domain linker allows us to propose relative domain positions for sACE that may contribute to the domain cooperativity. The structure now provides a platform for the design of "domain-specific" second-generation ACE inhibitors.

  5. Specificity Characterization of SLA Class I Molecules Binding to Swine-Origin Viral Cytotoxic T Lymphocyte Epitope Peptides in Vitro

    Directory of Open Access Journals (Sweden)

    Caixia Gao

    2017-12-01

    Full Text Available Swine leukocyte antigen (SLA class I molecules play a crucial role in generating specific cellular immune responses against viruses and other intracellular pathogens. They mainly bind and present antigens of intracellular origin to circulating MHC I-restricted cytotoxic T lymphocytes (CTLs. Binding of an appropriate epitope to an SLA class I molecule is the single most selective event in antigen presentation and the first step in the killing of infected cells by CD8+ CTLs. Moreover, the antigen epitopes are strictly restricted to specific SLA molecules. In this study, we constructed SLA class I complexes in vitro comprising viral epitope peptides, the extracellular region of the SLA-1 molecules, and β2-microglobulin (β2m using splicing overlap extension polymerase chain reaction (SOE-PCR. The protein complexes were induced and expressed in an Escherichia coli prokaryotic expression system and subsequently purified and refolded. Specific binding of seven SLA-1 proteins to one classical swine fever virus (CSFV and four porcine reproductive and respiratory syndrome virus (PRRSV epitope peptides was detected by enzyme-linked immunosorbent assay (ELISA-based method. The SLA-1∗13:01, SLA-1∗11:10, and SLA-1∗11:01:02 proteins were able to bind specifically to different CTL epitopes of CSFV and PRRSV and the MHC restrictions of the five epitopes were identified. The fixed combination of Asn151Val152 residues was identified as the potentially key amino acid residues influencing the binding of viral several CTL epitope peptides to SLA-1∗13:01 and SLA-1∗04:01:01 proteins. The more flexible pocket E in the SLA-1∗13:01 protein might have fewer steric limitations and therefore be able to accommodate more residues of viral CTL epitope peptides, and may thus play a critical biochemical role in determining the peptide-binding motif of SLA-1∗13:01. Characterization of the binding specificity of peptides to SLA class I molecules provides an

  6. Monitoring multiple myeloma by idiotype-specific peptide binders of tumor-derived exosomes.

    Science.gov (United States)

    Iaccino, Enrico; Mimmi, Selena; Dattilo, Vincenzo; Marino, Fabiola; Candeloro, Patrizio; Di Loria, Antonio; Marimpietri, Danilo; Pisano, Antonio; Albano, Francesco; Vecchio, Eleonora; Ceglia, Simona; Golino, Gaetanina; Lupia, Antonio; Fiume, Giuseppe; Quinto, Ileana; Scala, Giuseppe

    2017-10-13

    Tumor-derived exosomes (TDEs) play a pivotal role in tumor establishment and progression, and are emerging biomarkers for tumor diagnosis in personalized medicine. To date, there is a lack of efficient technology platforms for exosome isolation and characterization. Multiple myeloma (MM) is an incurable B-cell malignancy due to the rapid development of drug-resistance. MM-released exosomes express the immunoglobulin B-cell receptor (Ig-BCR) of the tumor B-cells, which can be targeted by Idiotype-binding peptides (Id-peptides). In this study, we analyzed the production of MM-released exosomes in the murine 5T33MM multiple myeloma model as biomarkers of tumor growth. To this end, we selected Id-peptides by screening a phage display library using as bait the Ig-BCR expressed by 5T33MM cells. By FACS, the FITC-conjugated Id-peptides detected the MM-released exosomes in the serum of 5T33MM-engrafted mice, levels of which are correlated with tumor progression at an earlier time point compared to serum paraprotein. These results indicate that Id-peptide-based recognition of MM-released exosomes may represent a very sensitive diagnostic approach for clinical evaluation of disease progression.

  7. CD4-specific designed ankyrin repeat proteins are novel potent HIV entry inhibitors with unique characteristics.

    Directory of Open Access Journals (Sweden)

    Andreas Schweizer

    2008-07-01

    Full Text Available Here, we describe the generation of a novel type of HIV entry inhibitor using the recently developed Designed Ankyrin Repeat Protein (DARPin technology. DARPin proteins specific for human CD4 were selected from a DARPin DNA library using ribosome display. Selected pool members interacted specifically with CD4 and competed with gp120 for binding to CD4. DARPin proteins derived in the initial selection series inhibited HIV in a dose-dependent manner, but showed a relatively high variability in their capacity to block replication of patient isolates on primary CD4 T cells. In consequence, a second series of CD4-specific DARPins with improved affinity for CD4 was generated. These 2nd series DARPins potently inhibit infection of genetically divergent (subtype B and C HIV isolates in the low nanomolar range, independent of coreceptor usage. Importantly, the actions of the CD4 binding DARPins were highly specific: no effect on cell viability or activation, CD4 memory cell function, or interference with CD4-independent virus entry was observed. These novel CD4 targeting molecules described here combine the unique characteristics of DARPins-high physical stability, specificity and low production costs-with the capacity to potently block HIV entry, rendering them promising candidates for microbicide development.

  8. Structural Basis for Specific Inhibition of tRNA Synthetase by an ATP Competitive Inhibitor.

    Science.gov (United States)

    Fang, Pengfei; Han, Hongyan; Wang, Jing; Chen, Kaige; Chen, Xin; Guo, Min

    2015-06-18

    Pharmaceutical inhibitors of aminoacyl-tRNA synthetases demand high species and family specificity. The antimalarial ATP-mimetic cladosporin selectively inhibits Plasmodium falciparum LysRS (PfLysRS). How the binding to a universal ATP site achieves the specificity is unknown. Here we report three crystal structures of cladosporin with human LysRS, PfLysRS, and a Pf-like human LysRS mutant. In all three structures, cladosporin occupies the class defining ATP-binding pocket, replacing the adenosine portion of ATP. Three residues holding the methyltetrahydropyran moiety of cladosporin are critical for the specificity of cladosporin against LysRS over other class II tRNA synthetase families. The species-exclusive inhibition of PfLysRS is linked to a structural divergence beyond the active site that mounts a lysine-specific stabilizing response to binding cladosporin. These analyses reveal that inherent divergence of tRNA synthetase structural assembly may allow for highly specific inhibition even through the otherwise universal substrate binding pocket and highlight the potential for structure-driven drug development. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Potential Therapeutic Applications of Mucuna pruriens Peptide Fractions Purified by High-Performance Liquid Chromatography as Angiotensin-Converting Enzyme Inhibitors, Antioxidants, Antithrombotic and Hypocholesterolemic Agents.

    Science.gov (United States)

    Herrera-Chalé, Francisco; Ruiz-Ruiz, Jorge Carlos; Betancur-Ancona, David; Segura-Campos, Maira Rubi

    2016-02-01

    A Mucuna pruriens protein concentrate was hydrolyzed with a digestive (pepsin-pancreatin) enzymatic system. The soluble portion of the hydrolysate was fractionated by ultrafiltration and the ultrafiltered peptide fraction (PF) with lower molecular weight was purified by reversed-phase high-performance liquid chromatography. The PF obtained were evaluated by testing the biological activity in vitro. Fractions showed that the ability to inhibit the angiotensin-converting enzyme had IC50 values that ranged from 2.7 to 6.2 μg/mL. Trolox equivalent antioxidant capacity values ranged from 132.20 to 507.43 mM/mg. The inhibition of human platelet aggregation ranged from 1.59% to 11.11%, and the inhibition of cholesterol micellar solubility ranged from 0.24% to 0.47%. Hydrophobicity, size, and amino acid sequence could be factors in determining the biological activity of peptides contained in fractions. This is the first report that M. pruriens peptides act as antihypertensives, antioxidants, and inhibitors for human platelet aggregation and cholesterol micellar solubility in vitro.

  10. The Sam-Sam interaction between Ship2 and the EphA2 receptor: design and analysis of peptide inhibitors.

    Science.gov (United States)

    Mercurio, Flavia Anna; Di Natale, Concetta; Pirone, Luciano; Iannitti, Roberta; Marasco, Daniela; Pedone, Emilia Maria; Palumbo, Rosanna; Leone, Marilisa

    2017-12-12

    The lipid phosphatase Ship2 represents a drug discovery target for the treatment of different diseases, including cancer. Its C-terminal sterile alpha motif domain (Ship2-Sam) associates with the Sam domain from the EphA2 receptor (EphA2-Sam). This interaction is expected to mainly induce pro-oncogenic effects in cells therefore, inhibition of the Ship2-Sam/EphA2-Sam complex may represent an innovative route to discover anti-cancer therapeutics. In the present work, we designed and analyzed several peptide sequences encompassing the interaction interface of EphA2-Sam for Ship2-Sam. Peptide conformational analyses and interaction assays with Ship2-Sam conducted through diverse techniques (CD, NMR, SPR and MST), identified a positively charged penta-amino acid native motif in EphA2-Sam, that once repeated three times in tandem, binds Ship2-Sam. NMR experiments show that the peptide targets the negatively charged binding site of Ship2-Sam for EphA2-Sam. Preliminary in vitro cell-based assays indicate that -at 50 µM concentration- it induces necrosis of PC-3 prostate cancer cells with more cytotoxic effect on cancer cells than on normal dermal fibroblasts. This work represents a pioneering study that opens further opportunities for the development of inhibitors of the Ship2-Sam/EphA2-Sam complex for therapeutic applications.

  11. Pharmacological and safety evaluation of CIGB-300, a casein kinase 2 inhibitor peptide, administered intralesionally to patients with cervical cancer stage IB2/II

    Directory of Open Access Journals (Sweden)

    Soriano-García JL

    2013-08-01

    Full Text Available CIGB-300 is a pro-apoptotic casein kinase 2 inhibitor peptide with potential anticancer action. An open-label and dose scaling Phase I trial was carried out to investigate the peptide tumor uptake, pharmacokinetics, toxicity, and levels of a CIGB-300 response biomarker in patients with cervical cancer stage IB2/II. Fourteen patients were included; six of them received 35 mg, 6 received 70 mg and the two remaining patients received 245 mg of CIGB-300 prior chemoradiotherapy. CIGB-300 was applied by intratumor injections during 5-consecutive days. For pharmacokinetic and biodistribution studies, the peptide was radiolabeled with 99mTc in the first administration and whole body gammagraphy and plasma testing were done during 48 h. Data showed that the maximum tolerated dose was 70 mg for CIGB-300 in this clinical setting. Furthermore, an allergic-like syndrome was identified as the dose limiting toxicity, which was well-correlated with plasmatic histamine levels. Importantly, the mean tumor uptake was 14.9 mg and 10.4 mg for CIGB-300 doses of 35 and 70 mg, respectively. Also, the kidneys were the main target organ for drug elimination. Finally, treatment with CIGB-300 significantly reduced the B23/nucleophosmin levels in tumor specimens. CIGB-300 meets potentialities to be tested in future trials in a neoadjuvant setting prior to chemoradiotherapy in cervical cancer.

  12. Rotavirus NSP4114-135 peptide has no direct, specific effect on chloride transport in rabbit brush-border membrane

    Directory of Open Access Journals (Sweden)

    Vasseur Monique

    2006-11-01

    Full Text Available Abstract The direct effect of the rotavirus NSP4114-135 and Norovirus NV464-483 peptides on 36Cl uptake was studied by using villus cell brush border membrane (BBM isolated from young rabbits. Both peptides inhibited the Cl-/H+ symport activity about equally and partially. The interaction involved one peptide-binding site per carrier unit. Whereas in vitro NSP4114-135 caused nonspecific inhibition of the Cl-/H+ symporter, the situation in vivo is different. Because rotavirus infection in young rabbits accelerated both Cl- influx and Cl- efflux rates across villi BBM without stimulating Cl- transport in crypt BBM, we conclude that the NSP4114-135 peptide, which causes diarrhea in young rodents, did not have any direct, specific effect on either intestinal absorption or secretion of chloride. The lack of direct effect of NSP4 on chloride transport strengthens the hypothesis that NSP4 would trigger signal transduction pathways to enhance net chloride secretion at the onset of rotavirus diarrhea.

  13. Proteolytic activity of prostate-specific antigen (PSA towards protein substrates and effect of peptides stimulating PSA activity.

    Directory of Open Access Journals (Sweden)

    Johanna M Mattsson

    Full Text Available Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3 exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA.

  14. Proteolytic Activity of Prostate-Specific Antigen (PSA) towards Protein Substrates and Effect of Peptides Stimulating PSA Activity

    Science.gov (United States)

    Mattsson, Johanna M.; Ravela, Suvi; Hekim, Can; Jonsson, Magnus; Malm, Johan; Närvänen, Ale; Stenman, Ulf-Håkan; Koistinen, Hannu

    2014-01-01

    Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3) exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA. PMID:25237904

  15. How much of Virus-Specific CD8 T Cell Reactivity is Detected with a Peptide Pool when Compared to Individual Peptides?

    Directory of Open Access Journals (Sweden)

    Ramu A. Subbramanian

    2012-10-01

    Full Text Available Immune monitoring of T cell responses increasingly relies on the use of peptide pools. Peptides, when restricted by the same HLA allele, and presented from within the same peptide pool, can compete for HLA binding sites. What impact such competition has on functional T cell stimulation, however, is not clear. Using a model peptide pool that is comprised of 32 well-defined viral epitopes from Cytomegalovirus, Epstein-Barr virus, and Influenza viruses (CEF peptide pool, we assessed peptide competition in PBMC from 42 human subjects. The magnitude of the peptide pool-elicited CD8 T cell responses was a mean 79% and a median 77% of the sum of the CD8 T cell responses elicited by the individual peptides. Therefore, while the effect of peptide competition was evident, it was of a relatively minor magnitude. By studying the dose-response curves for individual CEF peptides, we show that several of these peptides are present in the CEF-pool at concentrations that are orders of magnitude in excess of what is needed for the activation threshold of the CD8 T cells. The presence of such T cells with very high functional avidity for the viral antigens can explain why the effect of peptide competition is relatively minor within the CEF-pool.

  16. Efficacy of peptide nucleic acid and selected conjugates against specific cellular pathologies of amyotrophic lateral sclerosis.

    Science.gov (United States)

    Browne, Elisse C; Parakh, Sonam; Duncan, Luke F; Langford, Steven J; Atkin, Julie D; Abbott, Belinda M

    2016-04-01

    Cellular studies have been undertaken on a nonamer peptide nucleic acid (PNA) sequence, which binds to mRNA encoding superoxide dismutase 1, and a series of peptide nucleic acids conjugated to synthetic lipophilic vitamin analogs including a recently prepared menadione (vitamin K) analog. Reduction of both mutant superoxide dismutase 1 inclusion formation and endoplasmic reticulum stress, two of the key cellular pathological hallmarks in amyotrophic lateral sclerosis, by two of the prepared PNA oligomers is reported for the first time. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

  17. Stereochemical determinants of C-terminal specificity in PDZ peptide-binding domains: a novel contribution of the carboxylate-binding loop.

    Science.gov (United States)

    Amacher, Jeanine F; Cushing, Patrick R; Bahl, Christopher D; Beck, Tobias; Madden, Dean R

    2013-02-15

    PDZ (PSD-95/Dlg/ZO-1) binding domains often serve as cellular traffic engineers, controlling the localization and activity of a wide variety of binding partners. As a result, they play important roles in both physiological and pathological processes. However, PDZ binding specificities overlap, allowing multiple PDZ proteins to mediate distinct effects on shared binding partners. For example, several PDZ domains bind the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), an epithelial ion channel mutated in CF. Among these binding partners, the CFTR-associated ligand (CAL) facilitates post-maturational degradation of the channel and is thus a potential therapeutic target. Using iterative optimization, we previously developed a selective CAL inhibitor peptide (iCAL36). Here, we investigate the stereochemical basis of iCAL36 specificity. The crystal structure of iCAL36 in complex with the CAL PDZ domain reveals stereochemical interactions distributed along the peptide-binding cleft, despite the apparent degeneracy of the CAL binding motif. A critical selectivity determinant that distinguishes CAL from other CFTR-binding PDZ domains is the accommodation of an isoleucine residue at the C-terminal position (P(0)), a characteristic shared with the Tax-interacting protein-1. Comparison of the structures of these two PDZ domains in complex with ligands containing P(0) Leu or Ile residues reveals two distinct modes of accommodation for β-branched C-terminal side chains. Access to each mode is controlled by distinct residues in the carboxylate-binding loop. These studies provide new insights into the primary sequence determinants of binding motifs, which in turn control the scope and evolution of PDZ interactomes.

  18. Cell penetrating peptides to dissect host-pathogen protein-protein interactions in Theileria -transformed leukocytes

    KAUST Repository

    Haidar, Malak; de Laté , Perle Latré ; Kennedy, Eileen J.; Langsley, Gordon

    2017-01-01

    One powerful application of cell penetrating peptides is the delivery into cells of molecules that function as specific competitors or inhibitors of protein-protein interactions. Ablating defined protein-protein interactions is a refined way

  19. A novel synthetic peptide microarray assay detects Chlamydia species-specific antibodies in animal and human sera.

    Science.gov (United States)

    Sachse, Konrad; Rahman, Kh Shamsur; Schnee, Christiane; Müller, Elke; Peisker, Madlen; Schumacher, Thomas; Schubert, Evelyn; Ruettger, Anke; Kaltenboeck, Bernhard; Ehricht, Ralf

    2018-03-16

    Serological analysis of Chlamydia (C.) spp. infections is still mainly based on micro-immunofluorescence and ELISA. To overcome the limitations of conventional serology, we have designed a novel microarray carrying 52 synthetic peptides representing B-cell epitopes from immunodominant proteins of all 11 chlamydial species. The new assay has been validated using monospecific mouse hyperimmune sera. Subsequently, serum samples from cattle, sheep and humans with a known history of chlamydial infection were examined. For instance, the specific humoral response of sheep to treatment with a C. abortus vaccine has been visualized against a background of C. pecorum carriership. In samples from humans, dual infection with C. trachomatis and C. pneumoniae could be demonstrated. The experiments revealed that the peptide microarray assay was capable of simultaneously identifying specific antibodies to each Chlamydia spp. The actual assay represents an open platform test that can be complemented through future advances in Chlamydia proteome research. The concept of the highly parallel multi-antigen microarray proven in this study has the potential to enhance our understanding of antibody responses by defining not only a single quantitative response, but also the pattern of this response. The added value of using peptide antigens will consist in unprecedented serodiagnostic specificity.

  20. Covalent cross-linking of insulin-like growth factor-1 to a specific inhibitor from human serum

    International Nuclear Information System (INIS)

    Ooi, G.T.; Herington, A.C.

    1986-01-01

    Previous studies have shown that a specific inhibitor of insulin-like growth factor (IGF) action in vitro can be isolated from normal human serum and subsequently partially purified on an IGF-affinity column. The ability of the inhibitor to bind the IGFs has now been confirmed directly using covalent cross-linking techniques. When 125 I-IGF-1 was cross-linked to inhibitor using disuccinimidyl suberate, five specifically labelled bands were seen on SDS-PAGE and autoradiography. Two bands (MW 21.5 K and 25.5 K) were intensely labelled, while the remaining three (MW 37 K, 34 K and 18 K) appeared as minor bands only. Inhibitor bioactivity, following further analysis by hydrophobic interaction chromatography or Con A-Sepharose affinity chromatography, was always associated with the presence of the 21.5 K and/or 25.5 K bands

  1. Purification of charybdotoxine, a specific inhibitor of the high-conductance Ca2+-activated K+ channel

    International Nuclear Information System (INIS)

    Smith, C.; Phillips, M.; Miller, C.

    1986-01-01

    Charybdotoxim is a high-affinity specific inhibitor of the high-conductance Ca 2+ -activated K + channel found in the plasma membranes of many vertebrate cell types. Using Ca 2+ -activated K + channels reconstituted into planar lipid bilayer membranes as an assay, the authors have purified the toxin from the venom of the scorpion Leiurus quinquestriatus by a two-step procedure involving chromatofocusing on SP-Sephadex, followed by reversed-phase high-performance liquid chromatography. Charybdotoxin is shown to be a highly basic protein with a mass of 10 kDa. Under the standard assay conditions, the purified toxin inhibits the Ca 2+ -activated K + channel with an apparent dissociation constant of 3.5 nM. The protein is unusually stable, with inhibitory potency being insensitive to boiling or exposure to organic solvents. The toxin's activity is sensitive to chymotrypsin treatment and to acylation of lysine groups. The protein may be radioiodinated without loss of activity

  2. Diphenyleneiodonium, an inhibitor of NOXes and DUOXes, is also an iodide-specific transporter

    Directory of Open Access Journals (Sweden)

    C. Massart

    2014-01-01

    Full Text Available NADPH oxidases (NOXes and dual oxidases (DUOXes generate O2.− and H2O2. Diphenyleneiodonium (DPI inhibits the activity of these enzymes and is often used as a specific inhibitor. It is shown here that DPI, at concentrations similar to those which inhibit the generation of O2 derivatives, activated the efflux of radioiodide but not of its analog 99mTcO4− nor of the K+ cation mimic 86Rb+ in thyroid cells, in the PCCl3 rat thyroid cell line and in COS cell lines expressing the iodide transporter NIS. Effects obtained with DPI, especially in thyroid cells, should therefore be interpreted with caution.

  3. Task-specific ionic liquid as a new green inhibitor of mild steel corrosion

    Energy Technology Data Exchange (ETDEWEB)

    Kowsari, E., E-mail: kowsarie@aut.ac.ir [Department of Chemistry, Amirkabir University of Technology, No. 424, Hafez Avenue, 1591634311 Tehran (Iran, Islamic Republic of); Payami, M. [Department of Chemistry, Amirkabir University of Technology, No. 424, Hafez Avenue, 1591634311 Tehran (Iran, Islamic Republic of); Amini, R.; Ramezanzadeh, B. [Department of Surface Coating and Corrosion, Institute for Color Science and Technology (ICST), PO 16765-654 Tehran (Iran, Islamic Republic of); Javanbakht, M. [Department of Chemistry, Amirkabir University of Technology, No. 424, Hafez Avenue, 1591634311 Tehran (Iran, Islamic Republic of)

    2014-01-15

    The corrosion inhibition effects of an imidazolium-based task specific ionic liquid (TSIL) were investigated on a low carbon steel in 1 M HCl solution. Samples were exposed to 1 M HCl solution without and with different concentrations of TSIL. Weight loss measurements, potentiodynamic polarization test, electrochemical impedance spectroscopy (EIS), scanning electron microscope (SEM), atomic force microscope (AFM) and contact angle measurements were utilized to investigate the inhibition effects of TSIL. The results obtained from the polarization studies revealed that both the anodic and cathodic branches slopes affected in the presence of TSIL. It was shown that TSIL behaved as a mixed type inhibitor with a dominant effect on the anodic reaction rate depression. It was shown that the increase in polarization resistance and the decrease in corrosion current density were more pronounced using 100 mg/L of TSIL after 2 h immersion time. It was also shown that the adsorption of TSIL followed a Langmuir adsorption isotherm.

  4. Computational discovery of specificity-conferring sites in non-ribosomal peptide synthetases

    DEFF Research Database (Denmark)

    Knudsen, Michael; Søndergaard, Dan Ariel; Tofting-Olesen, Claus

    2016-01-01

    Motivation: By using a class of large modular enzymes known as Non-Ribosomal Peptide Synthetases (NRPS), bacteria and fungi are capable of synthesizing a large variety of secondary metabolites, many of which are bioactive and have potential, pharmaceutical applications as e.g.~antibiotics. There ...

  5. Identification of brain-specific angiogenesis inhibitor 2 as an interaction partner of glutaminase interacting protein

    International Nuclear Information System (INIS)

    Zencir, Sevil; Ovee, Mohiuddin; Dobson, Melanie J.; Banerjee, Monimoy; Topcu, Zeki; Mohanty, Smita

    2011-01-01

    Highlights: → Brain-specific angiogenesis inhibitor 2 (BAI2) is a new partner protein for GIP. → BAI2 interaction with GIP was revealed by yeast two-hybrid assay. → Binding of BAI2 to GIP was characterized by NMR, CD and fluorescence. → BAI2 and GIP binding was mediated through the C-terminus of BAI2. -- Abstract: The vast majority of physiological processes in living cells are mediated by protein-protein interactions often specified by particular protein sequence motifs. PDZ domains, composed of 80-100 amino acid residues, are an important class of interaction motif. Among the PDZ-containing proteins, glutaminase interacting protein (GIP), also known as Tax Interacting Protein TIP-1, is unique in being composed almost exclusively of a single PDZ domain. GIP has important roles in cellular signaling, protein scaffolding and modulation of tumor growth and interacts with a number of physiological partner proteins, including Glutaminase L, β-Catenin, FAS, HTLV-1 Tax, HPV16 E6, Rhotekin and Kir 2.3. To identify the network of proteins that interact with GIP, a human fetal brain cDNA library was screened using a yeast two-hybrid assay with GIP as bait. We identified brain-specific angiogenesis inhibitor 2 (BAI2), a member of the adhesion-G protein-coupled receptors (GPCRs), as a new partner of GIP. BAI2 is expressed primarily in neurons, further expanding GIP cellular functions. The interaction between GIP and the carboxy-terminus of BAI2 was characterized using fluorescence, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy assays. These biophysical analyses support the interaction identified in the yeast two-hybrid assay. This is the first study reporting BAI2 as an interaction partner of GIP.

  6. Construction of a multifunctional coating consisting of phospholipids and endothelial progenitor cell-specific peptides on titanium substrates

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Huiqing; Li, Xiaojing [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Zhao, Yuancong, E-mail: zhaoyc7320@163.com [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Li, Jingan; Chen, Jiang [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Yang, Ping, E-mail: yangping8@263.net [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Maitz, Manfred F. [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Max Bergmann Center of Biomaterials Dresden, Leibniz of Polymer Research Dresden, 01069 Dresden (Germany); Huang, Nan [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China)

    2015-08-30

    Graphical abstract: The phospholipid groups of PMMDP can inhibit platele adhesion, and the EPCs-specific peptide of the PMMDP showed special recognition and capture for EPCs. The catechol groups of PMMDP play a critical role as molecular anchor for balancing the binding between the coating and the substrate. - Highlights: • The uniform coating of PMMDP can be constructed on titanium surface successfully through the catechol groups. • The phospholipid groups of PMMDP can inhibit platele adhesion, fibrinogen denaturation and improve the hydrophilicity of substrate. • The EPCs-specific peptide of the PMMDP showed special recognition and capture for EPCs. - Abstract: A phospholipid/peptide polymer (PMMDP) with phosphorylcholine groups, endothelial progenitor cell (EPC)-specific peptides and catechol groups was anchored onto a titanium (Ti) surface to fabricate a biomimetic multifunctional surface. The PMMDP coating was characterized by X-ray photoelectron spectroscopy (XPS), water contact angle measurements and atomic force microscopy (AFM), respectively. The amount of PMMDP coating on the Ti surface was quantified by using the quartz crystal microbalance with dissipation (QCM-D). Interactions between blood components and the coated and bare Ti substrates were evaluated by platelet adhesion and activation assays and fibrinogen denaturation test using platelet rich plasma (PRP). The results revealed that the PMMDP-modified surface inhibited fibrinogen denaturation and reduced platelet adhesion and activation. EPC cell culture on the PMMDP-modified surface showed increased adhesion and proliferation of EPCs when compared to the cells cultured on untreated Ti surface. The inhibition of fibrinogen denaturation and platelet adhesion and support of EPCs attachment and proliferation indicated that this coating might be beneficial for future applications in blood-contacting implants, such as vascular stents.

  7. d-Amino acid mutation of PMI as potent dual peptide inhibitors of p53-MDM2/MDMX interactions.

    Science.gov (United States)

    Li, Xiang; Liu, Chao; Chen, Si; Hu, Honggang; Su, Jiacan; Zou, Yan

    2017-10-15

    According to the previously reported potent dual l-peptide PMI of p53-MDM2/MDMX interactions, a series of d-amino acid mutational PMI analogues, PMI-1-4, with enhanced proteolytic resistence and in vitro tumor cell inhibitory activities were reported, of which Liposome-PMI-1 showed a stronger inhibitory activity against the U87 cell lines than Nutlin-3. This d-amino acid mutation strategy may give a hand for enhancing the potential of peptide drugs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Rational Design of a Highly Potent and Selective Peptide Inhibitor of PACE4 by Salt Bridge Interaction with D160 at Position P3.

    Science.gov (United States)

    Dianati, Vahid; Shamloo, Azar; Kwiatkowska, Anna; Desjardins, Roxane; Soldera, Armand; Day, Robert; Dory, Yves L

    2017-08-08

    PACE4, a member of the proprotein convertases (PCs) family of serine proteases, is a validated target for prostate cancer. Our group has developed a potent and selective PACE4 inhibitor: Ac-LLLLRVKR-NH 2 . In seeking for modifications to increase the selectivity of this ligand toward PACE4, we replaced one of its P3 Val methyl groups with a basic group capable of forming a salt bridge with D160 of PACE4. The resulting inhibitor is eight times more potent than the P3 Val parent inhibitor and two times more selective over furin, because the equivalent salt bridge with furin E257 is not optimal. Moreover, the β-branched nature of the new P3 residue favors the extended β-sheet conformation usually associated with substrates of proteases. This work provides new insight for better understanding of β-sheet backbone-backbone interactions between serine proteases and their peptidic ligands. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Self-Assembling Peptide Nanofiber Scaffold Enhanced with RhoA Inhibitor CT04 Improves Axonal Regrowth in the Transected Spinal Cord

    Directory of Open Access Journals (Sweden)

    Weiwei Zhang

    2012-01-01

    Full Text Available The present study was designed to explore the therapeutic potential of self-assembling peptide nanofiber scaffold (SAPNS delivered RhoA inhibitor to ameliorate the hostile microenvironment of injured spinal cord for axonal regeneration. After a transection was applied to the thoracic spinal cord of mice, the combination of SAPNS and CT04 (a cell permeable RhoA inhibitor, single SAPNS with vehicle, or saline was transplanted into the lesion cavity. Results showed that SAPNS+CT04 implants achieved the best therapeutic outcomes among treatment groups. The novel combination not only reconstructed the injured nerve gap but also elicited significant axonal regeneration and motor functional recovery. Additionally, the combination also effectively reduced the apoptosis and infiltration of activated macrophages in the injured spinal cord. Collectively, the present study demonstrated that SAPNS-based delivery of RhoA inhibitor CT04 presented a highly potential therapeutic strategy for spinal cord injury with reknitting lesion gap, attenuating secondary injury, and improving axonal regrowth.

  10. Self-Assembling Peptide Nanofiber Scaffold Enhanced with RhoA Inhibitor CT04 Improves Axonal Regrowth in the Transected Spinal Cord

    International Nuclear Information System (INIS)

    Weiwei, Z.; Xiaoduo, Z.; Zhongying, L.

    2012-01-01

    The present study was designed to explore the therapeutic potential of self-assembling peptide nano fiber scaffold (SAPNS) delivered RhoA inhibitor to ameliorate the hostile microenvironment of injured spinal cord for axonal regeneration. After a transection was applied to the thoracic spinal cord of mice, the combination of SAPNS and CT04 (a cell permeable RhoA inhibitor), single SAPNS with vehicle, or saline was transplanted into the lesion cavity. Results showed that SAPNS+CT04 implants achieved the best therapeutic outcomes among treatment groups. The novel combination not only reconstructed the injured nerve gap but also elicited significant axonal regeneration and motor functional recovery. Additionally, the combination also effectively reduced the apoptosis and infiltration of activated macrophages in the injured spinal cord. Collectively, the present study demonstrated that SAPNS-based delivery of RhoA inhibitor CT04 presented a highly potential therapeutic strategy for spinal cord injury with reknitting lesion gap, attenuating secondary injury, and improving axonal regrowth.

  11. Isozyme-specific fluorescent inhibitor of glutathione s-transferase omega 1.

    Science.gov (United States)

    Son, Junghyun; Lee, Jae-Jung; Lee, Jun-Seok; Schüller, Andreas; Chang, Young-Tae

    2010-05-21

    Recently, the glutathione S-transferase omega 1 (GSTO1) is suspected to be involved in certain cancers and neurodegenerative diseases. However, profound investigation on the pathological roles of GSTO1 has been hampered by the lack of specific methods to determine or modulate its activity in biological systems containing other isoforms with similar catalytic function. Here, we report a fluorescent compound that is able to inhibit and monitor the activity of GSTO1. We screened 43 fluorescent chemicals and found a compound (6) that binds specifically to the active site of GSTO1. We observed that compound 6 inhibits GSTO1 by covalent modification but spares other isoforms in HEK293 cells and demonstrated that compound 6 could report the activity of GSTO1 in NIH/3T3 or HEK293 cells by measuring the fluorescence intensity of the labeled amount of GSTO1 in SDS-PAGE. Compound 6 is a useful tool to study GSTO1, applicable as a specific inhibitor and an activity reporter.

  12. Effects of kinase inhibitors and potassium phosphate (KPi) on site-specific phosphorylation of branched chain α-ketoacid dehydrogenase (BCKDH)

    International Nuclear Information System (INIS)

    Kuntz, M.J.; Shimomura, Y.; Ozawa, T.; Harris, R.A.

    1987-01-01

    BCKDH is phosphorylated by a copurifying kinase at two serine residues on the Elα subunit. Phosphorylation of both sites occurs at about the same rate initially, but inactivation is believed associated only with site 1 phosphorylation. The effects of KPi and known inhibitors of BCKDH kinase, α-chloroisocaproate (CIC) and branched chain α-ketoacids (BCKA), on the phosphorylation of purified rat liver BCKDH were studied. Site-specific phosphorylation was quantitated by thin-layer electrophoresis of tryptic peptides followed by densitometric scanning of autoradiograms. Addition of 5 mM KPi was found necessary to stabilize the BCKDH activity at 37 0 C. Increasing the KPi to 50 mM dramatically increased the CIC and BCKA inhibition of site 1 and site 2 phosphorylation. The finding of enhanced sensitivity of inhibitors with 50 mM KPi may facilitate identification of physiologically important kinase effectors. Regardless of the KPi concentration, CIC and the BCKA showed much more effective inhibition of site 2 than site 1 phosphorylation. Although site 1 is the primary inactivating site, predominant inhibition of site 2 phosphorylation may provide a means of modulating kinase/phosphatase control of BCKDH activity under steady state conditions

  13. Optimal Classes of Chemotherapeutic Agents Sensitized by Specific Small-Molecule Inhibitors of Akt In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Yan Shi

    2005-11-01

    Full Text Available Akt is a serine/threonine kinase that transduces survival signals from survival/growth factors. Deregulation and signal imbalance in cancer cells make them prone to apoptosis. Upregulation or activation of Akt to aid the survival of cancer cells is a common theme in human malignancies. We have developed small-molecule Akt inhibitors that are potent and specific. These Akt inhibitors can inhibit Akt activity and block phosphorylation by Akt on multiple downstream targets in cells. Synergy in apoptosis induction was observed when Akt inhibitors were combined with doxorubicin or camptothecin. Akt inhibitor-induced enhancement of topoisomerase inhibitor cytotoxicity was also evident in long-term cell survival assay. Synergy with paclitaxel in apoptosis induction was evident in cells pretreated with paclitaxel, and enhancement of tumor delay by paclitaxel was demonstrated through cotreatment with Akt inhibitor Compound A (A-443654. Combination with other classes of chemotherapeutic agents did not yield any enhancement of cytotoxicity. These findings provide important guidance in selecting appropriate classes of chemotherapeutic agents for combination with Akt inhibitors in cancer treatment.

  14. Characterization of Foodborne Strains of Staphylococcus aureus by Shotgun Proteomics: Functional Networks, Virulence Factors and Species-Specific Peptide Biomarkers

    Science.gov (United States)

    Carrera, Mónica; Böhme, Karola; Gallardo, José M.; Barros-Velázquez, Jorge; Cañas, Benito; Calo-Mata, Pilar

    2017-01-01

    In the present work, we applied a shotgun proteomics approach for the fast and easy characterization of 20 different foodborne strains of Staphylococcus aureus (S. aureus), one of the most recognized foodborne pathogenic bacteria. A total of 644 non-redundant proteins were identified and analyzed via an easy and rapid protein sample preparation procedure. The results allowed the differentiation of several proteome datasets from the different strains (common, accessory, and unique datasets), which were used to determine relevant functional pathways and differentiate the strains into different Euclidean hierarchical clusters. Moreover, a predicted protein-protein interaction network of the foodborne S. aureus strains was created. The whole confidence network contains 77 nodes and 769 interactions. Most of the identified proteins were surface-associated proteins that were related to pathways and networks of energy, lipid metabolism and virulence. Twenty-seven virulence factors were identified, and most of them corresponded to autolysins, N-acetylmuramoyl-L-alanine amidases, phenol-soluble modulins, extracellular fibrinogen-binding proteins and virulence factor EsxA. Potential species-specific peptide biomarkers were screened. Twenty-one species-specific peptide biomarkers, belonging to eight different proteins (nickel-ABC transporter, N-acetylmuramoyl-L-alanine amidase, autolysin, clumping factor A, gram-positive signal peptide YSIRK, cysteine protease/staphopain, transcriptional regulator MarR, and transcriptional regulator Sar-A), were proposed to identify S. aureus. These results constitute the first major dataset of peptides and proteins of foodborne S. aureus strains. This repository may be useful for further studies, for the development of new therapeutic treatments for S. aureus food intoxications and for microbial source-tracking in foodstuffs. PMID:29312172

  15. A screen for kinase inhibitors identifies antimicrobial imidazopyridine aminofurazans as specific inhibitors of the Listeria monocytogenes PASTA kinase PrkA.

    Science.gov (United States)

    Schaenzer, Adam J; Wlodarchak, Nathan; Drewry, David H; Zuercher, William J; Rose, Warren E; Striker, Rob; Sauer, John-Demian

    2017-10-13

    Bacterial signaling systems such as protein kinases and quorum sensing have become increasingly attractive targets for the development of novel antimicrobial agents in a time of rising antibiotic resistance. The family of bacterial P enicillin-binding-protein A nd S erine/ T hreonine kinase- A ssociated (PASTA) kinases is of particular interest due to the role of these kinases in regulating resistance to β-lactam antibiotics. As such, small-molecule kinase inhibitors that target PASTA kinases may prove beneficial as treatments adjunctive to β-lactam therapy. Despite this interest, only limited progress has been made in identifying functional inhibitors of the PASTA kinases that have both activity against the intact microbe and high kinase specificity. Here, we report the results of a small-molecule screen that identified GSK690693, an imidazopyridine aminofurazan-type kinase inhibitor that increases the sensitivity of the intracellular pathogen Listeria monocytogenes to various β-lactams by inhibiting the PASTA kinase PrkA. GSK690693 potently inhibited PrkA kinase activity biochemically and exhibited significant selectivity for PrkA relative to the Staphylococcus aureus PASTA kinase Stk1. Furthermore, other imidazopyridine aminofurazans could effectively inhibit PrkA and potentiate β-lactam antibiotic activity to varying degrees. The presence of the 2-methyl-3-butyn-2-ol (alkynol) moiety was important for both biochemical and antimicrobial activity. Finally, mutagenesis studies demonstrated residues in the back pocket of the active site are important for GSK690693 selectivity. These data suggest that targeted screens can successfully identify PASTA kinase inhibitors with both biochemical and antimicrobial specificity. Moreover, the imidazopyridine aminofurazans represent a family of PASTA kinase inhibitors that have the potential to be optimized for selective PASTA kinase inhibition.

  16. Effects of specific inhibitors on anammox and denitrification in marine sediments.

    Science.gov (United States)

    Jensen, Marlene Mark; Thamdrup, Bo; Dalsgaard, Tage

    2007-05-01

    The effects of three metabolic inhibitors (acetylene, methanol, and allylthiourea [ATU]) on the pathways of N2 production were investigated by using short anoxic incubations of marine sediment with a 15N isotope technique. Acetylene inhibited ammonium oxidation through the anammox pathway as the oxidation rate decreased exponentially with increasing acetylene concentration; the rate decay constant was 0.10+/-0.02 microM-1, and there was 95% inhibition at approximately 30 microM. Nitrous oxide reduction, the final step of denitrification, was not sensitive to acetylene concentrations below 10 microM. However, nitrous oxide reduction was inhibited by higher concentrations, and the sensitivity was approximately one-half the sensitivity of anammox (decay constant, 0.049+/-0.004 microM-1; 95% inhibition at approximately 70 microM). Methanol specifically inhibited anammox with a decay constant of 0.79+/-0.12 mM-1, and thus 3 to 4 mM methanol was required for nearly complete inhibition. This level of methanol stimulated denitrification by approximately 50%. ATU did not have marked effects on the rates of anammox and denitrification. The profile of inhibitor effects on anammox agreed with the results of studies of the process in wastewater bioreactors, which confirmed the similarity between the anammox bacteria in bioreactors and natural environments. Acetylene and methanol can be used to separate anammox and denitrification, but the effects of these compounds on nitrification limits their use in studies of these processes in systems where nitrification is an important source of nitrate. The observed differential effects of acetylene and methanol on anammox and denitrification support our current understanding of the two main pathways of N2 production in marine sediments and the use of 15N isotope methods for their quantification.

  17. Breast Tumor Specific Peptides: Development of Breast Carcinoma Diagnostic and Therapeutic Agents

    Science.gov (United States)

    2000-11-01

    Columbia, Missouri 65211; La Jolla Institute for Allergy and Inmunology [M. E. H.], San Diego, California 92121; and Sidney Kimmel Cancer Center and...spontaneous homotypic aggregation of breast The costs of publication of this article were defrayed in part by the payment of page cancer cells, then a T...antigen-binding peptide may likewise inhibit charges. This article must therefore be hereby marked advertisement in accordance with this aggregation

  18. Specific expression of GFPuv-β1,3-N-acetylglucosaminyltransferase 2 fusion protein in fat body of Bombyx mori silkworm larvae using signal peptide

    International Nuclear Information System (INIS)

    Kato, Tatsuya; Park, Enoch Y.

    2007-01-01

    Bombyxin (bx) and prophenoloxidase-activating enzyme (ppae) signal peptides from Bombyx mori, their modified signal peptides, and synthetic signal peptides were investigated for the secretion of GFP uv -β1,3-N-acetylglucosaminyltransferase 2 (GGT2) fusion protein in B. mori Bm5 cells and silkworm larvae using cysteine protease deficient B. mori multiple nucleopolyhedrovirus (BmMNPV-CP - ) and its bacmid. The secretion efficiencies of all signal peptides were 15-30% in Bm5 cells and 24-30% in silkworm larvae, while that of the +16 signal peptide was 0% in Bm5 cells and 1% in silkworm larvae. The fusion protein that contained the +16 signal peptide was expressed specifically in the endoplasmic reticulum (ER) and in the fractions of cell precipitations. Ninety-four percent of total intracellular β1,3-N-acetylglucosaminyltransferase (β3GnT) activity was detected in cell precipitations following the 600, 8000, and 114,000g centrifugations. In the case of the +38 signal peptide, 60% of total intracellular activity was detected in the supernatant from the 114,000g spin, and only 1% was found in the precipitate. Our results suggest that the +16 signal peptide might be situated in the transmembrane region and not cleaved by signal peptidase in silkworm or B. mori cells. Therefore, the fusion protein connected to the +16 signal peptide stayed in the fat body of silkworm larvae with biological function, and was not secreted extracellularly

  19. Effect of specific enzyme inhibitors on replication, total genome DNA repair and on gene-specific DNA repair after UV irradiation in CHO cells

    Energy Technology Data Exchange (ETDEWEB)

    Jones, J.C.; Stevsner, Tinna; Bohr, Vilhelm A. (National Cancer Institute, NIH, Bethesda, MD (USA). Division of Cancer Treatment, Laboratory of Molecular Pharmacology); Mattern, M.R. (Smith Kline Beecham Pharmaceuticals, King of Prussia, PA (USA). Department of Biomolecular Discovery)

    1991-09-01

    The effects were studied of some specific enzyme inhibitors on DNA repair and replication after UV damage in Chinese hamster ovary cells. The DNA repair was studied at the level of the average, overall genome and also in the active dihydrofolate reductase gene. Replication was measured in the overall genome. The inhibitors were tested of DNA poly-merase {alpha} and {delta} (aphidicolin), of poly(ADPr) polymerase (3-aminobenzamide), of ribonucleotide reductase (hydroxyurea), of topo-isomerase I (camptothecin), and of topoisomerase II (merbarone, VP-16). In addition, the effects were tested of the potential topoisomerase I activator, {beta}-lapachone. All of these compounds inhibited genome replication and all topoisomerase inhibitors affected the overall genome repair; {beta}-lapachone stimulated it. None of these compounds had any effect on the gene-specific repair. (author). 36 refs.; 3 figs.; 2 tabs.

  20. Evaluation of the site specific protein glycation and antioxidant capacity of rare sugar-protein/peptide conjugates.

    Science.gov (United States)

    Sun, Yuanxia; Hayakawa, Shigeru; Ogawa, Masahiro; Izumori, Ken

    2005-12-28

    Protein-sugar conjugates generated in nonenzymatic glycation of alpha-lactalbumin (LA) with rare sugars [D-allose (All) and D-psicose (Psi)] and alimentary sugars as controls [D-glucose (Glc) and D-fructose (Fru)] were qualitatively determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Mass spectra revealed that the extent of glycation at lysine residues on LA with D-aldose molecules was very much higher than that of glycation with d-ketose molecules. To identify the specific site of glycation, the peptide mapping was established from protease V8 digestion, using a combination of computational cutting of proteins and MALDI-TOF-MS. As compared to peptide mapping, three and seven glycation sites were located in the primary structure of LA-ketose and LA-aldose conjugates, respectively. On the other hand, the antioxidant activities of protein-sugar conjugates and their peptic hydrolysates were investigated by 1,1-diphenyl-2-picrylhydrazyl radical scavenging method. The antioxidant activities of proteins/peptides glycated with rare sugars were significantly higher than those modified with the control sugars. The results indicated that the glycation degree and position were not markedly different between rare sugar and corresponding control sugar, but the antioxidant properties of protein and its hydrolysate were significantly enhanced by modifying with rare sugar.

  1. Casein Hydrolysates by Lactobacillus brevis and Lactococcus lactis Proteases: Peptide Profile Discriminates Strain-Dependent Enzyme Specificity.

    Science.gov (United States)

    Bounouala, Fatima Zohra; Roudj, Salima; Karam, Nour-Eddine; Recio, Isidra; Miralles, Beatriz

    2017-10-25

    Casein from ovine and bovine milk were hydrolyzed with two extracellular protease preparations from Lactobacillus brevis and Lactococcus lactis. The hydrolysates were analyzed by HPLC-MS/MS for peptide identification. A strain-dependent peptide profile could be observed, regardless of the casein origin, and the specificity of these two proteases could be computationally ascribed. The cleavage pattern yielding phenylalanine, leucine, or tyrosine at C-terminal appeared both at L. lactis and Lb. brevis hydrolysates. However, the cleavage C-terminal to lysine was favored with Lb. brevis protease. The hydrolysates showed ACE-inhibitory activity with IC 50 in the 16-70 μg/mL range. Ovine casein hydrolysates yielded greater ACE-inhibitory activity. Previously described antihypertensive and opioid peptides were found in these ovine and bovine casein hydrolysates and prediction of the antihypertensive activity of the sequences based on quantitative structure and activity relationship (QSAR) was performed. This approach might represent a useful classification tool regarding health-related properties prior to further purification.

  2. Characterization of an insulin-like growth factor-I/somatomedin-C radioimmunoassay specific for the C-peptide region

    International Nuclear Information System (INIS)

    Hintz, R.L.; Liu, F.; Seegan, G.

    1982-01-01

    Insulin-like growth factor-I (IGF-I) and somatomedin-C (SM-C) have been shown to be functionally identical by a number of criteria. We have synthesized the 12 amino acid C-peptide region of IGF-I (Gly-Tyr-Gly-Ser-Ser-Ser-Arg-Arg-Ala-Pro-Glu-Thr) and developed a RIA based on antibodies against this synthetic peptide. IGF-I and SM-C were indistinguishable in this RIA. No other peptides competed for this antiserum. The SM-C/IGF-I values of acid-chromatographed serum were strongly age dependent. The mean of children 1-5 yr old was 0.67 +/- 0.033 U/ml (mean +/- sD; n = 23), whereas the mean of children 12-17 yr old was 2.01 +/- 0.66 U/ml (n = 39) and the mean of 38 adults 26-85 yr old was 1.05 +/- 0.34. The SM-C/IGF-I values measured by this RIA were also growth hormone dependent. Thus, this region-specific RIA provides a clinically useful assessment of serum SM-C/IGF-I levels

  3. Influence of prosthetic radioiodination on the chemical and biological behavior of chemotactic peptides labeled at high specific activity

    International Nuclear Information System (INIS)

    Pozzi, Oscar R.; Sajaroff, Elisa O.; Edreira, Martin M.

    2006-01-01

    The influence of radioiodination made through prosthetic group N-succinimidyl-3-[ 131 I]iodo-benzoate ([ 131 I]SIB) on the behavior of small peptides was investigated using as model the chemotactic hexapeptide Nα-for-Nle-Leu-Phe-Nle-Tyr-Lys. No carrier added labeled peptide was isolated by reverse-phase HPLC (RP-HPLC) with coupling efficiencies up to 59-75%. Biodistribution in normal and infected C57 mice showed mainly a hepatobiliary clearance, a very low thyroid uptake and the highest uptake at the infection site was within 1h of injection. Superoxide production and competitive binding assays studies in human polymorphonuclear leukocytes showed a preserved biological activity and high-affinity specific binding. However, the results indicated that the changes observed in the receptor-binding properties with an IC 50 almost twice than the unlabeled peptide and the increasing in the hepatobiliary excretion could be the consequence of the increased lipophicity observed due to the presence of the prosthetic group together with a strong influence of the radioisotope per se

  4. Influence of prosthetic radioiodination on the chemical and biological behavior of chemotactic peptides labeled at high specific activity

    Energy Technology Data Exchange (ETDEWEB)

    Pozzi, Oscar R. [National Atomic Energy Commission, Ezeiza Atomic Centre, Buenos Aires (Argentina)]. E-mail: oscar.pozzi@duke.edu; Sajaroff, Elisa O. [National Atomic Energy Commission, Ezeiza Atomic Centre, Buenos Aires (Argentina); Edreira, Martin M. [National Atomic Energy Commission, Ezeiza Atomic Centre, Buenos Aires (Argentina)

    2006-06-15

    The influence of radioiodination made through prosthetic group N-succinimidyl-3-[{sup 131}I]iodo-benzoate ([{sup 131}I]SIB) on the behavior of small peptides was investigated using as model the chemotactic hexapeptide N{alpha}-for-Nle-Leu-Phe-Nle-Tyr-Lys. No carrier added labeled peptide was isolated by reverse-phase HPLC (RP-HPLC) with coupling efficiencies up to 59-75%. Biodistribution in normal and infected C57 mice showed mainly a hepatobiliary clearance, a very low thyroid uptake and the highest uptake at the infection site was within 1h of injection. Superoxide production and competitive binding assays studies in human polymorphonuclear leukocytes showed a preserved biological activity and high-affinity specific binding. However, the results indicated that the changes observed in the receptor-binding properties with an IC{sub 50} almost twice than the unlabeled peptide and the increasing in the hepatobiliary excretion could be the consequence of the increased lipophicity observed due to the presence of the prosthetic group together with a strong influence of the radioisotope per se.

  5. Commercial broth microdilution panel validation and reproducibility trials for NVP PDF-713 (LBM 415), a novel inhibitor of bacterial peptide deformylase.

    Science.gov (United States)

    Fritsche, T R; Moet, G J; Jones, R N

    2004-09-01

    NVP PDF-713 (LBM 415) is a peptide deformylase inhibitor being progressed into clinical trials. Dry-form broth microdilution panels of NVP PDF-713 were compared to reference MIC panels of 552 recent clinical isolates. Most (99.2%) dry-form MIC results were within +/- 1 log(2) dilution of the reference panel MICs. Of the bacteria tested, Streptococcus pneumoniae and Haemophilus influenzae showed a bias towards higher and lower MICs, respectively. Same-day and between-day reproducibility tests showed that 98.9% and 96.7% of MIC values, respectively, were within +/- 1 log(2) dilution step, thereby demonstrating a high degree of reliability of the dry-form MIC product for clinical studies.

  6. Dipeptidyl peptidase-4 (DPP-4) inhibitors are favourable to glucagon-like peptide-1 (GLP-1) agonists

    DEFF Research Database (Denmark)

    Madsbad, Sten

    2012-01-01

    Incretin-based therapies, which include the GLP-1 receptor agonists and DPP-4 inhibitors, use the antidiabetic properties of potentiating the GLP-1 receptor signalling via the regulation of insulin and glucagon secretion, inhibition of gastric emptying and suppression of appetite. Most physicians...... will start antidiabetic treatment with metformin, but adding a GLP-1 receptor agonist as the second drug seems to be optimal since more patients will reach an HbA1c below 7% than with a DPP-4 inhibitor or another oral antidiabetic agents and with minimal risk of hypoglycaemia. The GLP-1 receptor agonists...

  7. Measles Virus Nucleocapsid (MVNP) Gene Expression and RANK Receptor Signaling in Osteoclast Precursors, Osteoclast Inhibitors Peptide Therapy for Pagets Disease

    Science.gov (United States)

    2008-10-01

    PS, Zurbriggen A, Cosby SL, Dickson GR, Fraser WD, Ooi CG, Selby PL, Crisp AJ, Wallace RG, Kahn S, Ralston SH 2000 A negative search for a...Zurbriggen A, Cosby SL, Dickson GR, Fraser WD, Ooi CG, Selby PL, Crisp AJ, Wallace RG, Kahn S, Ralston SH. 2000. A negative search for a paramyxoviral...van Hul W, Whyte MP, Nakatsuka K, Hovy L, Anderson DM . 2000. Mutations in TNFRSF11A, affecting the signal peptide of RANK, cause familial expansile

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

    Science.gov (United States)

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

    2015-01-01

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

  9. The transferrin receptor of Actinobacillus pleuropneumoniae: Quantitation of expression and structural characterization using a peptide-specific monoclonal antibody

    DEFF Research Database (Denmark)

    Bøg, Yang S.; Andresen, Lars Ole; Bastholm, L.

    2001-01-01

    transferrin. This complex was studied using a monoclonal antibody (Mab 1.48) raised against a synthetic peptide corresponding to a hydrophilic domain of Tbp2 common to several A. pp serotypes. The antibody reacted specifically with a 60-70 kDa Tbp2-antigen found in all serotypes of A. pp obtained from iron...... expressing Tbp2 and in wild type A. pp grown under iron restricted conditions. The subcellular location of Tbp2 in A. pp was studied by immunoelectron microscopy using the Mab 1.48. Interestingly, all antibody binding was found inside the A. pp cells, while Tbp2 expressed in recombinant E. coli was found...

  10. A specific and potent inhibitor of glucosylceramide synthase for substrate inhibition therapy of Gaucher disease.

    Science.gov (United States)

    McEachern, Kerry Anne; Fung, John; Komarnitsky, Svetlana; Siegel, Craig S; Chuang, Wei-Lien; Hutto, Elizabeth; Shayman, James A; Grabowski, Gregory A; Aerts, Johannes M F G; Cheng, Seng H; Copeland, Diane P; Marshall, John

    2007-07-01

    An approach to treating Gaucher disease is substrate inhibition therapy which seeks to abate the aberrant lysosomal accumulation of glucosylceramide. We have identified a novel inhibitor of glucosylceramide synthase (Genz-112638) and assessed its activity in a murine model of Gaucher disease (D409V/null). Biochemical characterization of Genz-112638 showed good potency (IC(50) approximately 24nM) and specificity against the target enzyme. Mice that received drug prior to significant accumulation of substrate (10 weeks of age) showed reduced levels of glucosylceramide and number of Gaucher cells in the spleen, lung and liver when compared to age-matched control animals. Treatment of older mice that already displayed significant amounts of tissue glucosylceramide (7 months old) resulted in arrest of further accumulation of the substrate and appearance of additional Gaucher cells in affected organs. These data indicate that substrate inhibition therapy with Genz-112638 represents a viable alternate approach to enzyme therapy to treat the visceral pathology in Gaucher disease.

  11. From viral genome to specific peptide epitopes: methods for identifying porcine T cell epitopes based on in silico predictions, in vitro identification and ex vivo verification

    DEFF Research Database (Denmark)

    Pedersen, Lasse Eggers; Rasmussen, Michael; Harndah, Mikkel

    2013-01-01

    to predict likely candidates for peptide-SLA binding. These results were combined with binding predictions generated by the algorithm, NetMHCpan (http://www.cbs.dtu.dk/services/NetMHCpan/) in order to select peptide candidates for in vitro analysis. The correlation between high affinity and high stability.......000 peptides. T cell epitopes were identified using peptide-SLA complexes assembled into fluorescent tetramers to stain swine influenza specific CTLs derived from immunized animals and MHC-defined pigs vaccinated against foot-and-mouth disease virus. These results demonstrate the broad applicability of methods...... originally developed for analysis of human leukocyte antigen (HLA) presentation of peptides. The methods presented provide a timely and cost-effective approach to CTL epitope discovery that can be applied to diseases of swine and of other mammalian species of interest....

  12. Design and synthesis of 4'-((5-benzylidene-2,4-dioxothiazolidin-3-yl)methyl)biphenyl-2-carbonitrile analogs as bacterial peptide deformylase inhibitors.

    Science.gov (United States)

    Khan, Firoz A Kalam; Patil, Rajendra H; Shinde, Devanand B; Sangshetti, Jaiprakash N

    2016-12-01

    Herein, we report the synthesis and screening of 4'-((5-benzylidene-2,4-dioxothiazolidin-3-yl)methyl)biphenyl-2-carbonitrile analogs 11(a-j) as bacterial peptide deformylase (PDF) enzyme inhibitors. The compounds 11b (IC 50 value = 139.28 μm), 11g (IC 50 value = 136.18 μm), and 11h (IC 50 value = 131.65 μm) had shown good PDF inhibition activity. The compounds 11b (MIC range = 103.36-167.26 μg/mL), 11g (MIC range = 93.75-145.67 μg/mL), and 11h (MIC range = 63.61-126.63 μg/mL) had also shown potent antibacterial activity when compared with standard ampicillin (MIC range = 100.00-250.00 μg/mL). Thus, the active derivatives were not only PDF inhibitors but also efficient antibacterial agents. To gain more insight on the binding mode of the compounds with PDF enzyme, the synthesized compounds 11(a-j) were docked against PDF enzyme of Escherichia coli and compounds exhibited good binding properties. The results suggest that this class of compounds has potential for development and use in future as antibacterial drugs. © 2016 John Wiley & Sons A/S.

  13. Evaluation of the specificity of antigen assays for plasminogen activator inhibitor 1 : Comparison of two new commercial kits

    NARCIS (Netherlands)

    Huisman, L.G.M.; Meijer, P.; Griensven, J. van; Kluft, C.

    1992-01-01

    t-PA depleted citrated plasma was used to prepare standards of different molecular forms of plasminogen activator inhibitor 1 (PAI-1). These standards were used to evaluate the specificity of two new PAI-1 antigen assays: the TintElize PAI-1 antigen assay (cat. no. 210221) and the Innotest PAI-1.

  14. A novel peptide derived from human apolipoprotein E is an inhibitor of tumor growth and ocular angiogenesis.

    Directory of Open Access Journals (Sweden)

    Partha S Bhattacharjee

    2011-01-01

    Full Text Available Angiogenesis is a hallmark of tumor development and metastasis and now a validated target for cancer treatment. We previously reported that a novel dimer peptide (apoEdp derived from the receptor binding region of human apolipoprotein E (apoE inhibits virus-induced angiogenesis. However, its role in tumor anti-angiogenesis is unknown. This study demonstrates that apoEdp has anti-angiogenic property in vivo through reduction of tumor growth in a mouse model and ocular angiogenesis in a rabbit eye model. Our in vitro studies show that apoEdp inhibits human umbilical vein endothelial cell proliferation, migration, invasion and capillary tube formation. We document that apoEdp inhibits vascular endothelial growth factor-induced Flk-1 activation as well as downstream signaling pathways that involve c-Src, Akt, eNOS, FAK, and ERK1/2. These in vitro data suggest potential sites of the apoE dipeptide inhibition that could occur in vivo.This is the first evidence that a synthetic dimer peptide mimicking human apoE has anti-angiogenesis functions and could be an anti-tumor drug candidate.

  15. Molecular identification of a Drosophila G protein-coupled receptor specific for crustacean cardioactive peptide

    DEFF Research Database (Denmark)

    Cazzamali, Giuseppe; Hauser, Frank; Kobberup, Sune

    2003-01-01

    The Drosophila Genome Project website (www.flybase.org) contains the sequence of an annotated gene (CG6111) expected to code for a G protein-coupled receptor. We have cloned this receptor and found that its gene was not correctly predicted, because an annotated neighbouring gene (CG14547) was also...... part of the receptor gene. DNA corresponding to the corrected gene CG6111 was expressed in Chinese hamster ovary cells, where it was found to code for a receptor that could be activated by low concentrations of crustacean cardioactive peptide, which is a neuropeptide also known to occur in Drosophila...... and other insects (EC(50), 5.4 x 10(-10)M). Other known Drosophila neuropeptides, such as adipokinetic hormone, did not activate the receptor. The receptor is expressed in all developmental stages from Drosophila, but only very weakly in larvae. In adult flies, the receptor is mainly expressed in the head...

  16. Massively parallel amplicon sequencing reveals isotype-specific variability of antimicrobial peptide transcripts in Mytilus galloprovincialis.

    Directory of Open Access Journals (Sweden)

    Umberto Rosani

    Full Text Available BACKGROUND: Effective innate responses against potential pathogens are essential in the living world and possibly contributed to the evolutionary success of invertebrates. Taken together, antimicrobial peptide (AMP precursors of defensin, mytilin, myticin and mytimycin can represent about 40% of the hemocyte transcriptome in mussels injected with viral-like and bacterial preparations, and unique profiles of myticin C variants are expressed in single mussels. Based on amplicon pyrosequencing, we have ascertained and compared the natural and Vibrio-induced diversity of AMP transcripts in mussel hemocytes from three European regions. METHODOLOGY/PRINCIPAL FINDINGS: Hemolymph was collected from mussels farmed in the coastal regions of Palavas (France, Vigo (Spain and Venice (Italy. To represent the AMP families known in M. galloprovincialis, nine transcript sequences have been selected, amplified from hemocyte RNA and subjected to pyrosequencing. Hemolymph from farmed (offshore and wild (lagoon Venice mussels, both injected with 10(7 Vibrio cells, were similarly processed. Amplicon pyrosequencing emphasized the AMP transcript diversity, with Single Nucleotide Changes (SNC minimal for mytilin B/C and maximal for arthropod-like defensin and myticin C. Ratio of non-synonymous vs. synonymous changes also greatly differed between AMP isotypes. Overall, each amplicon revealed similar levels of nucleotidic variation across geographical regions, with two main sequence patterns confirmed for mytimycin and no substantial changes after immunostimulation. CONCLUSIONS/SIGNIFICANCE: Barcoding and bidirectional pyrosequencing allowed us to map and compare the transcript diversity of known mussel AMPs. Though most of the genuine cds variation was common to the analyzed samples we could estimate from 9 to 106 peptide variants in hemolymph pools representing 100 mussels, depending on the AMP isoform and sampling site. In this study, no prevailing SNC patterns related

  17. A PKA survival pathway inhibited by DPT-PKI, a new specific cell permeable PKA inhibitor, is induced by T. annulata in parasitized B-lymphocytes.

    Science.gov (United States)

    Guergnon, Julien; Dessauge, Frederic; Traincard, François; Cayla, Xavier; Rebollo, Angelita; Bost, Pierre Etienne; Langsley, Gordon; Garcia, Alphonse

    2006-08-01

    T. annulata, an intracellular pathogenic parasite of the Aplicomplexa protozoan family infects bovine B-lymphocytes and macrophages. Parasitized cells that become transformed survive and proliferate independently of exogenous growth factors. In the present study, we used the isogenic non parasitized BL3 and parasitized TBL3 B cell lines, as a model to evaluate the contribution of two-major PI3-K- and PKA-dependent anti-apoptotic pathways in the survival of T. annulata parasitized B lymphocytes. We found that T. annulata increases PKA activity, induces over-expression of the catalytic subunit and down-regulates the pro-survival phosphorylation state of Akt/PKB. Consistent with a role of PKA activation in survival, two pharmacological inhibitors H89 and KT5720 ablate PKA-dependent survival of parasitized cells. To specifically inhibit PKA pro-survival pathways we linked the DPTsh1 peptide shuttle sequence to PKI(5-24) and we generated DPT-PKI, a cell permeable PKI. DPT-PKI specifically inhibited PKA activity in bovine cell extracts and, as expected, also inhibited the PKA-dependent survival of T. annulata parasitized TBL3 cells. Thus, parasite-dependent constitutive activation of PKA in TBL3 cells generates an anti-apoptotic pathway that can protect T. annulata-infected B cells from apoptosis. These results also indicate that DPT-PKI could be a powerful tool to inhibit PKA pathways in other cell types.

  18. Oligopeptidase B from Serratia proteamaculans. III. Inhibition analysis. Specific interactions with metalloproteinase inhibitors.

    Science.gov (United States)

    Mikhailova, A G; Khairullin, R F; Kolomijtseva, G Ya; Rumsh, L D

    2012-03-01

    Inhibition of the novel oligopeptidase B from Serratia proteamaculans (PSP) by basic pancreatic trypsin inhibitor, Zn2+ ions, and o- and m-phenanthroline was investigated. A pronounced effect of calcium ions on the interaction of PSP with inhibitors was demonstrated. Inversion voltamperometry and atomic absorption spectrometry revealed no zinc ions in the PSP molecule. Hydrophobic nature of the enzyme inhibition by o- and m-phenanthroline was established.

  19. Intracellular lysyl oxidase: Effect of a specific inhibitor on nuclear mass in proliferating cells

    Energy Technology Data Exchange (ETDEWEB)

    Saad, Fawzy A. [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States); Torres, Marie [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Wang, Hao [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States); Graham, Lila, E-mail: lilagraham@cs.com [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States)

    2010-06-11

    LOX, the principal enzyme involved in crosslinking of collagen, was the first of several lysyl oxidase isotypes to be characterized. Its active form was believed to be exclusively extracellular. Active LOX was later reported to be present in cell nuclei; its function there is unknown. LOX expression opposes the effect of mutationally activated Ras, which is present in about 30% of human cancers. The mechanism of LOX in countering the action of Ras is also unknown. In the present work, assessment of nuclear protein for possible effects of lysyl oxidase activity led to the discovery that proliferating cells dramatically increase their nuclear protein content when exposed to BAPN ({beta}-aminopropionitrile), a highly specific lysyl oxidase inhibitor that reportedly blocks LOX inhibition of Ras-induced oocyte maturation. In three cell types (PC12 cells, A7r5 smooth muscle cells, and NIH 3T3 fibroblasts), BAPN caused a 1.8-, 1.7-, and 2.1-fold increase in total nuclear protein per cell, respectively, affecting all major components in both nuclear matrix and chromatin fractions. Since nuclear size is correlated with proliferative status, enzyme activity restricting nuclear growth may be involved in the lysyl oxidase tumor suppressive effect. Evidence is also presented for the presence of apparent lysyl oxidase isotype(s) containing a highly conserved LOX active site sequence in the nuclei of PC12 cells, which do not manufacture extracellular lysyl oxidase substrates. Results reported here support the hypothesis that nuclear lysyl oxidase regulates nuclear growth, and thereby modulates cell proliferation.

  20. Characterization of HIV-Specific CD4+T Cell Responses against Peptides Selected with Broad Population and Pathogen Coverage

    DEFF Research Database (Denmark)

    Buggert, Marcus; Norstrom, Melissa M.; Czarnecki, Chris

    2012-01-01

    for the identification of HIV-specific CD4+ T cells targeting broadly reactive epitopes in populations with diverse ethnic background stems from the vast genomic variation of HIV and the diversity of the host cellular immune system. Here, we describe a novel epitope selection strategy, PopCover, that aims to resolve...... this challenge, and identify a set of potential HLA class II-restricted HIV epitopes that in concert will provide optimal viral and host coverage. Using this selection strategy, we identified 64 putative epitopes (peptides) located in the Gag, Nef, Env, Pol and Tat protein regions of HIV. In total, 73...... II-restricted epitopes. All together, selection strategies, such as PopCover, might with success be used for the evaluation of antigen-specific CD4+ T cell responses and design of future vaccines....

  1. Changing the inhibitory specificity and function of Cucurbita maxima trypsin inhibitor-V by site-directed mutagenesis.

    Science.gov (United States)

    Wen, L; Lee, I; Chen, G; Huang, J K; Gong, Y; Krishnamoorthi, R

    1995-02-27

    Cucurbita maxima trypsin inhibitor-V (CMTI-V) is also a specific inhibitor of human blood coagulation factor beta-factor XIIa. A recombinant version of CMTI-V has allowed probing of roles of individual amino acid residues including the reactive site residue, lysine (P1), by site-directed mutagenesis. The K44R showed at least a 5-fold increase in inhibitory activity toward human beta-factor XIIa, while there was no change toward bovine trypsin. This result demonstrates that beta-factor-XIIa prefers an arginine residue over lysine residue, while trypsin is non-specific to lysine or arginine in its binding pocket. On the other hand, the specificity of CMTI-V could be changed from trypsin to chymotrypsin inhibition by mutation of the P1 residue to either leucine or methionine (K44L or K44M).

  2. Generation in vivo of peptide-specific cytotoxic T cells and presence of regulatory T cells during vaccination with hTERT (class I and II peptide-pulsed DCs

    Directory of Open Access Journals (Sweden)

    Satthaporn Sukchai

    2009-03-01

    Full Text Available Abstract Background Optimal techniques for DC generation for immunotherapy in cancer are yet to be established. Study aims were to evaluate: (i DC activation/maturation milieu (TNF-α +/- IFN-α and its effects on CD8+ hTERT-specific T cell responses to class I epitopes (p540 or p865, (ii CD8+ hTERT-specific T cell responses elicited by vaccination with class I alone or both class I and II epitope (p766 and p672-pulsed DCs, prepared without IFN-α, (iii association between circulating T regulatory cells (Tregs and clinical responses. Methods Autologous DCs were generated from 10 patients (HLA-0201 with advanced cancer by culturing CD14+ blood monocytes in the presence of GM-CSF and IL-4 supplemented with TNF-α [DCT] or TNF-α and IFN-α [DCTI]. The capacity of the DCs to induce functional CD8+ T cell responses to hTERT HLA-0201 restricted nonapeptides was assessed by MHC tetramer binding and peptide-specific cytotoxicity. Each DC preparation (DCT or DCTI was pulsed with only one type of hTERT peptide (p540 or p865 and both preparations were injected into separate lymph node draining regions every 2–3 weeks. This vaccination design enabled comparison of efficacy between DCT and DCTI in generating hTERT peptide specific CD8+ T cells and comparison of class I hTERT peptide (p540 or p865-loaded DCT with or without class II cognate help (p766 and p672 in 6 patients. T regulatory cells were evaluated in 8 patients. Results (i DCTIs and DCTs, pulsed with hTERT peptides, were comparable (p = 0.45, t-test in inducing peptide-specific CD8+ T cell responses. (ii Class II cognate help, significantly enhanced (p (iii Clinical responders had significantly lower (p Conclusion Addition of IFN-α to ex vivo monocyte-derived DCs, did not significantly enhance peptide-specific T cell responses in vivo, compared with TNF-α alone. Class II cognate help significantly augments peptide-specific T cell responses. Clinically favourable responses were seen in patients

  3. Development of peptide-based lineage-specific serology for chronic Chagas disease: geographical and clinical distribution of epitope recognition.

    Directory of Open Access Journals (Sweden)

    Tapan Bhattacharyya

    2014-05-01

    Full Text Available BACKGROUND: Chagas disease, caused by infection with the protozoan Trypanosoma cruzi, remains a serious public health issue in Latin America. Genetically diverse, the species is sub-divided into six lineages, known as TcI-TcVI, which have disparate geographical and ecological distributions. TcII, TcV, and TcVI are associated with severe human disease in the Southern Cone countries, whereas TcI is associated with cardiomyopathy north of the Amazon. T. cruzi persists as a chronic infection, with cardiac and/or gastrointestinal symptoms developing years or decades after initial infection. Identifying an individual's history of T. cruzi lineage infection directly by genotyping of the parasite is complicated by the low parasitaemia and sequestration in the host tissues. METHODOLOGY/PRINCIPAL FINDINGS: We have applied here serology against lineage-specific epitopes of the T. cruzi surface antigen TSSA, as an indirect approach to allow identification of infecting lineage. Chagasic sera from chronic patients from a range of endemic countries were tested by ELISA against synthetic peptides representing lineage-specific TSSA epitopes bound to avidin-coated ELISA plates via a biotin labelled polyethylene glycol-glycine spacer to increase rotation and ensure each amino acid side chain could freely interact with their antibodies. 79/113 (70% of samples from Brazil, Bolivia, and Argentina recognised the TSSA epitope common to lineages TcII/TcV/TcVI. Comparison with clinical information showed that a higher proportion of Brazilian TSSApep-II/V/VI responders had ECG abnormalities than non-responders (38% vs 17%; p<0.0001. Among northern chagasic sera 4/20 (20% from Ecuador reacted with this peptide; 1/12 Venezuelan and 1/34 Colombian samples reacted with TSSApep-IV. In addition, a proposed TcI-specific epitope, described elsewhere, was demonstrated here to be highly conserved across lineages and therefore not applicable to lineage-specific serology. CONCLUSIONS

  4. Development of peptide-based lineage-specific serology for chronic Chagas disease: geographical and clinical distribution of epitope recognition.

    Science.gov (United States)

    Bhattacharyya, Tapan; Falconar, Andrew K; Luquetti, Alejandro O; Costales, Jaime A; Grijalva, Mario J; Lewis, Michael D; Messenger, Louisa A; Tran, Trang T; Ramirez, Juan-David; Guhl, Felipe; Carrasco, Hernan J; Diosque, Patricio; Garcia, Lineth; Litvinov, Sergey V; Miles, Michael A

    2014-05-01

    Chagas disease, caused by infection with the protozoan Trypanosoma cruzi, remains a serious public health issue in Latin America. Genetically diverse, the species is sub-divided into six lineages, known as TcI-TcVI, which have disparate geographical and ecological distributions. TcII, TcV, and TcVI are associated with severe human disease in the Southern Cone countries, whereas TcI is associated with cardiomyopathy north of the Amazon. T. cruzi persists as a chronic infection, with cardiac and/or gastrointestinal symptoms developing years or decades after initial infection. Identifying an individual's history of T. cruzi lineage infection directly by genotyping of the parasite is complicated by the low parasitaemia and sequestration in the host tissues. We have applied here serology against lineage-specific epitopes of the T. cruzi surface antigen TSSA, as an indirect approach to allow identification of infecting lineage. Chagasic sera from chronic patients from a range of endemic countries were tested by ELISA against synthetic peptides representing lineage-specific TSSA epitopes bound to avidin-coated ELISA plates via a biotin labelled polyethylene glycol-glycine spacer to increase rotation and ensure each amino acid side chain could freely interact with their antibodies. 79/113 (70%) of samples from Brazil, Bolivia, and Argentina recognised the TSSA epitope common to lineages TcII/TcV/TcVI. Comparison with clinical information showed that a higher proportion of Brazilian TSSApep-II/V/VI responders had ECG abnormalities than non-responders (38% vs 17%; p<0.0001). Among northern chagasic sera 4/20 (20%) from Ecuador reacted with this peptide; 1/12 Venezuelan and 1/34 Colombian samples reacted with TSSApep-IV. In addition, a proposed TcI-specific epitope, described elsewhere, was demonstrated here to be highly conserved across lineages and therefore not applicable to lineage-specific serology. These results demonstrate the considerable potential for synthetic

  5. MUSI: an integrated system for identifying multiple specificity from very large peptide or nucleic acid data sets.

    Science.gov (United States)

    Kim, Taehyung; Tyndel, Marc S; Huang, Haiming; Sidhu, Sachdev S; Bader, Gary D; Gfeller, David; Kim, Philip M

    2012-03-01

    Peptide recognition domains and transcription factors play crucial roles in cellular signaling. They bind linear stretches of amino acids or nucleotides, respectively, with high specificity. Experimental techniques that assess the binding specificity of these domains, such as microarrays or phage display, can retrieve thousands of distinct ligands, providing detailed insight into binding specificity. In particular, the advent of next-generation sequencing has recently increased the throughput of such methods by several orders of magnitude. These advances have helped reveal the presence of distinct binding specificity classes that co-exist within a set of ligands interacting with the same target. Here, we introduce a software system called MUSI that can rapidly analyze very large data sets of binding sequences to determine the relevant binding specificity patterns. Our pipeline provides two major advances. First, it can detect previously unrecognized multiple specificity patterns in any data set. Second, it offers integrated processing of very large data sets from next-generation sequencing machines. The results are visualized as multiple sequence logos describing the different binding preferences of the protein under investigation. We demonstrate the performance of MUSI by analyzing recent phage display data for human SH3 domains as well as microarray data for mouse transcription factors.

  6. Sequence-specific unusual (1-->2)-type helical turns in alpha/beta-hybrid peptides.

    Science.gov (United States)

    Prabhakaran, Panchami; Kale, Sangram S; Puranik, Vedavati G; Rajamohanan, P R; Chetina, Olga; Howard, Judith A K; Hofmann, Hans-Jörg; Sanjayan, Gangadhar J

    2008-12-31

    This article describes novel conformationally ordered alpha/beta-hybrid peptides consisting of repeating l-proline-anthranilic acid building blocks. These oligomers adopt a compact, right-handed helical architecture determined by the intrinsic conformational preferences of the individual amino acid residues. The striking feature of these oligomers is their ability to display an unusual periodic pseudo beta-turn network of nine-membered hydrogen-bonded rings formed in the forward direction of the sequence by 1-->2 amino acid interactions both in solid-state and in solution. Conformational investigations of several of these oligomers by single-crystal X-ray diffraction, solution-state NMR, and ab initio MO theory suggest that the characteristic steric and dihedral angle restraints exerted by proline are essential for stabilizing the unusual pseudo beta-turn network found in these oligomers. Replacing proline by the conformationally flexible analogue alanine (Ala) or by the conformationally more constrained alpha-amino isobutyric acid (Aib) had an adverse effect on the stabilization of this structural architecture. These findings increase the potential to design novel secondary structure elements profiting from the steric and dihedral angle constraints of the amino acid constituents and help to augment the conformational space available for synthetic oligomer design with diverse backbone structures.

  7. Crystal structure of a polyhistidine-tagged recombinant catalytic subunit of cAMP-dependent protein kinase complexed with the peptide inhibitor PKI(5-24) and adenosine.

    Science.gov (United States)

    Narayana, N; Cox, S; Shaltiel, S; Taylor, S S; Xuong, N

    1997-04-15

    The crystal structure of the hexahistidine-tagged mouse recombinant catalytic subunit (H6-rC) of cAMP-dependent protein kinase (cAPK), complexed with a 20-residue peptide inhibitor from the heat-stable protein kinase inhibitor PKI(5-24) and adenosine, was determined at 2.2 A resolution. Novel crystallization conditions were required to grow the ternary complex crystals. The structure was refined to a final crystallographic R-factor of 18.2% with good stereochemical parameters. The "active" enzyme adopts a "closed" conformation as found in rC:PKI(5-24) [Knighton et al. (1991a,b) Science 253, 407-414, 414-420] and packs in a similar manner with the peptide providing a major contact surface. This structure clearly defines the subsites of the unique nucleotide binding site found in the protein kinase family. The adenosine occupies a mostly hydrophobic pocket at the base of the cleft between the two lobes and is completely buried. The missing triphosphate moiety of ATP is filled with a water molecule (Wtr 415) which replaces the gamma-phosphate of ATP. The glycine-rich loop between beta1 and beta2 helps to anchor the phosphates while the ribose ring is buried beneath beta-strand 2. Another ordered water molecule (Wtr 375) is pentacoordinated with polar atoms from adenosine, Leu 49 in beta-strand 1, Glu 127 in the linker strand between the two lobes, Tyr 330, and a third water molecule, Wtr 359. The conserved nucleotide fold can be defined as a lid comprised of beta-strand 1, the glycine-rich loop, and beta-strand 2. The adenine ring is buried beneath beta-strand 1 and the linker strand (120-127) that joins the small and large lobes. The C-terminal tail containing Tyr 330, a segment that lies outside the conserved core, covers this fold and anchors it in a closed conformation. The main-chain atoms of the flexible glycine-rich loop (residues 50-55) in the ATP binding domain have a mean B-factor of 41.4 A2. This loop is quite mobile, in striking contrast to the other

  8. Identification of tyrosinase specific inhibitors from Xanthium strumarium fruit extract using ultrafiltration-high performance liquid chromatography.

    Science.gov (United States)

    Wang, Zhiqiang; Hwang, Seung Hwan; Huang, Bo; Lim, Soon Sung

    2015-10-01

    In this study, a strategy based on ultrafiltration-high performance liquid chromatography coupled with diode array detection (UF-HPLC-DAD) was proposed for screening tyrosinase specific inhibitors in Xanthii fructus. The false negatives were distinguished by optimizing the UF-HPLC-DAD parameters to reduce the background noise; the false positives were distinguished by introducing a blocked tyrosinase in the control group for comparison. To obtain the best blocker, the competitive experiments were performed using various known ligands. Using this strategy, three competitive inhibitors (protocatechuic acid; 3,5-di-O-caffeoylquinic acid; and 1,5-di-O-caffeoylquinic acid) and one mixed-type inhibitor (chlorogenic acid) were identified. These results were verified using tyrosinase inhibition assay, kinetic analysis, and structural simulation of the complex. Our experimental results suggest that the proposed strategy could be useful for high-throughput identification of tyrosinase specific inhibitors in natural products. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Pan-specific prediction of peptide-MHC Class I complex stability, a correlate of T cell immunogenicity

    DEFF Research Database (Denmark)

    Rasmussen, Michael; Fenoy, Emilio; Harndahl, Mikkel

    2016-01-01

    Binding of peptides to MHC class I (MHC-I) molecules is the most selective event in the processing and presentation of Ags to CTL, and insights into the mechanisms that govern peptide-MHC-I binding should facilitate our understanding of CTL biology. Peptide-MHC-I interactions have traditionally b...

  10. The Structural Basis of Cryptosporidium-Specific IMP Dehydrogenase Inhibitor Selectivity

    Energy Technology Data Exchange (ETDEWEB)

    MacPherson, Iain S.; Kirubakaran, Sivapriya; Gorla, Suresh Kumar; Riera, Thomas V.; D’Aquino, J. Alejandro; Zhang, Minjia; Cuny, Gregory D.; Hedstrom, Lizbeth (BWH); (Brandeis)

    2010-03-29

    Cryptosporidium parvum is a potential biowarfare agent, an important AIDS pathogen, and a major cause of diarrhea and malnutrition. No vaccines or effective drug treatment exist to combat Cryptosporidium infection. This parasite relies on inosine 5{prime}-monophosphate dehydrogenase (IMPDH) to obtain guanine nucleotides, and inhibition of this enzyme blocks parasite proliferation. Here, we report the first crystal structures of CpIMPDH. These structures reveal the structural basis of inhibitor selectivity and suggest a strategy for further optimization. Using this information, we have synthesized low-nanomolar inhibitors that display 10{sup 3} selectivity for the parasite enzyme over human IMPDH2.

  11. Albumin-derived peptides efficiently reduce renal uptake of radiolabelled peptides

    International Nuclear Information System (INIS)

    Vegt, Erik; Eek, Annemarie; Oyen, Wim J.G.; Gotthardt, Martin; Boerman, Otto C.; Jong, Marion de

    2010-01-01

    In peptide-receptor radionuclide therapy (PRRT), the maximum activity dose that can safely be administered is limited by high renal uptake and retention of radiolabelled peptides. The kidney radiation dose can be reduced by coinfusion of agents that competitively inhibit the reabsorption of radiolabelled peptides, such as positively charged amino acids, Gelofusine, or trypsinised albumin. The aim of this study was to identify more specific and potent inhibitors of the kidney reabsorption of radiolabelled peptides, based on albumin. Albumin was fragmented using cyanogen bromide and six albumin-derived peptides with different numbers of electric charges were selected and synthesised. The effect of albumin fragments (FRALB-C) and selected albumin-derived peptides on the internalisation of 111 In-albumin, 111 In-minigastrin, 111 In-exendin and 111 In-octreotide by megalin-expressing cells was assessed. In rats, the effect of Gelofusine and albumin-derived peptides on the renal uptake and biodistribution of 111 In-minigastrin, 111 In-exendin and 111 In-octreotide was determined. FRALB-C significantly reduced the uptake of all radiolabelled peptides in vitro. The albumin-derived peptides showed different potencies in reducing the uptake of 111 In-albumin, 111 In-exendin and 111 In-minigastrin in vitro. The most efficient albumin-derived peptide (peptide 6), was selected for in vivo testing. In rats, 5 mg of peptide 6 very efficiently inhibited the renal uptake of 111 In-minigastrin, by 88%. Uptake of 111 In-exendin and 111 In-octreotide was reduced by 26 and 33%, respectively. The albumin-derived peptide 6 efficiently inhibited the renal reabsorption of 111 In-minigastrin, 111 In-exendin and 111 In-octreotide and is a promising candidate for kidney protection in PRRT. (orig.)

  12. Synthetic Curcumin Analogs as Inhibitors of β -Amyloid Peptide Aggregation: Potential Therapeutic and Diagnostic Agents for Alzheimer's Disease.

    Science.gov (United States)

    Bukhari, Syed Nasir Abbas; Jantan, Ibrahim

    2015-01-01

    There is a crucial need to develop new effective drugs for Alzheimer's disease (AD) as the currently available AD treatments provide only momentary and incomplete symptomatic relief. Amongst natural products, curcumin, a major constituent of turmeric, has been intensively investigated for its neuroprotective effect against β-amyloid (Aβ)-induced toxicity in cultured neuronal cells. The ability of curcumin to attach to Aβ peptide and prevent its accumulation is attributed to its three structural characteristics such as the presence of two aromatic end groups and their co-planarity, the length and rigidity of the linker region and the substitution conformation of these aromatics. However, curcumin failed to reach adequate brain levels after oral absorption in AD clinical trials due to its low water solubility and poor oral bioavailability. A number of new curcumin analogs that mimic the active site of the compound along with analogs that mimic the curcumin anti-amyloid effect combined with anticholinesterase effect have been developed to enhance the bioavailability, pharmacokinetics, water solubility, stability at physiological conditions and delivery of curcumin. In this article, we have summarized all reported synthetic analogs of curcumin showing effects on β-amyloid and discussed their potential as therapeutic and diagnostic agents for AD.

  13. A multimodal RAGE-specific inhibitor reduces amyloid β–mediated brain disorder in a mouse model of Alzheimer disease

    Science.gov (United States)

    Deane, Rashid; Singh, Itender; Sagare, Abhay P.; Bell, Robert D.; Ross, Nathan T.; LaRue, Barbra; Love, Rachal; Perry, Sheldon; Paquette, Nicole; Deane, Richard J.; Thiyagarajan, Meenakshisundaram; Zarcone, Troy; Fritz, Gunter; Friedman, Alan E.; Miller, Benjamin L.; Zlokovic, Berislav V.

    2012-01-01

    In Alzheimer disease (AD), amyloid β peptide (Aβ) accumulates in plaques in the brain. Receptor for advanced glycation end products (RAGE) mediates Aβ-induced perturbations in cerebral vessels, neurons, and microglia in AD. Here, we identified a high-affinity RAGE-specific inhibitor (FPS-ZM1) that blocked Aβ binding to the V domain of RAGE and inhibited Aβ40- and Aβ42-induced cellular stress in RAGE-expressing cells in vitro and in the mouse brain in vivo. FPS-ZM1 was nontoxic to mice and readily crossed the blood-brain barrier (BBB). In aged APPsw/0 mice overexpressing human Aβ-precursor protein, a transgenic mouse model of AD with established Aβ pathology, FPS-ZM1 inhibited RAGE-mediated influx of circulating Aβ40 and Aβ42 into the brain. In brain, FPS-ZM1 bound exclusively to RAGE, which inhibited β-secretase activity and Aβ production and suppressed microglia activation and the neuroinflammatory response. Blockade of RAGE actions at the BBB and in the brain reduced Aβ40 and Aβ42 levels in brain markedly and normalized cognitive performance and cerebral blood flow responses in aged APPsw/0 mice. Our data suggest that FPS-ZM1 is a potent multimodal RAGE blocker that effectively controls progression of Aβ-mediated brain disorder and that it may have the potential to be a disease-modifying agent for AD. PMID:22406537

  14. Mechanism of action of hypoglycemic effects of an intestine-specific inhibitor of microsomal triglyceride transfer protein (MTP) in obese rats.

    Science.gov (United States)

    Sakata, Shohei; Katsumi, Sohei; Mera, Yasuko; Kuroki, Yukiharu; Nashida, Reiko; Kakutani, Makoto; Ohta, Takeshi

    2015-01-01

    Diminished insulin sensitivity in the peripheral tissues and failure of pancreatic beta cells to secrete insulin are known major determinants of type 2 diabetes mellitus. JTT-130, an intestine-specific microsomal transfer protein inhibitor, has been shown to suppress high fat-induced obesity and ameliorate impaired glucose tolerance while enhancing glucagon-like peptide-1 (GLP-1) secretion. We investigated the effects of JTT-130 on glucose metabolism and elucidated the mechanism of action, direct effects on insulin sensitivity and glucose-stimulated insulin secretion in a high fat diet-induced obesity rat model. Male Sprague Dawley rats fed a high-fat diet were treated with a single administration of JTT-130. Glucose tolerance, hyperglycemic clamp and hyperinsulinemic-euglycemic testing were performed to assess effects on insulin sensitivity and glucose-stimulated insulin secretion, respectively. Plasma GLP-1 and tissue triglyceride content were also determined under the same conditions. A single administration of JTT-130 suppressed plasma glucose elevations after oral glucose loading and increased the disposition index while elevating GLP-1. JTT-130 also enhanced glucose-stimulated insulin secretion in hyperglycemic clamp tests, whereas increased insulin sensitivity was observed in hyperinsulinemic-euglycemic clamp tests. Single-dose administration of JTT-130 decreased lipid content in the liver and skeletal muscle. JTT-130 demonstrated acute and direct hypoglycemic effects by enhancing insulin secretion and/or insulin sensitivity. Copyright © 2014 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

  15. Ion-specific weak adsorption of salts and water/octanol transfer free energy of a model amphiphilic hexa-peptide

    International Nuclear Information System (INIS)

    Dejugnat, Ch.; Dufreche, J.F.; Zemb, Th.; Dejugnat, Ch.

    2011-01-01

    An amphiphilic hexa-peptide has been used as a model to quantify how specific ion effects induced by addition of four salts tune the hydrophilic/hydrophobic balance and induce temperature-dependant coacervate formation from aqueous solution. The hexa-peptide chosen is present as a dimer with low transfer energy from water to octanol. Taking sodium chloride as the reference state in the Hofmeister scale, we identify water activity effects and therefore measure the free energy of transfer from water to octanol and separately the free energy associated to the adsorption of chaotropic ions or the desorption of kosmotropic ions for the same amphiphilic peptide. These effects have the same order of magnitude: therefore, both energies of solvation as well as transfer into octanol strongly depend on the nature of the electrolytes used to formulate any buffer. Model peptides could be used on separation processes based on criteria linked to 'Hofmeister' but different from volume and valency. (authors)

  16. Critical Data-Based Re-Evaluation of Minocycline as a Putative Specific Microglia Inhibitor

    NARCIS (Netherlands)

    Moller, Thomas; Bard, Frederique; Bhattacharya, Anindya; Biber, Knut; Campbell, Brian; Dale, Elena; Eder, Claudia; Gan, Li; Garden, Gwenn A.; Hughes, Zoe A.; Pearse, Damien D.; Staal, Roland G. W.; Sayed, Faten A.; Wes, Paul D.; Boddeke, Hendrikus W. G. M.

    2016-01-01

    Minocycline, a second generation broad-spectrum antibiotic, has been frequently postulated to be a "microglia inhibitor." A considerable number of publications have used minocycline as a tool and concluded, after achieving a pharmacological effect, that the effect must be due to "inhibition" of

  17. Molecular dynamics of conformation-specific dopamine transporter-inhibitor complexes.

    Science.gov (United States)

    Jean, Bernandie; Surratt, Christopher K; Madura, Jeffry D

    2017-09-01

    The recreational psychostimulant cocaine inhibits dopamine reuptake from the synapse, resulting in excessive stimulation of postsynaptic dopamine receptors in brain areas associated with reward and addiction. Cocaine binds to and stabilizes the outward- (extracellular-) facing conformation of the dopamine transporter (DAT) protein, while the low abuse potential DAT inhibitor benztropine prefers the inward- (cytoplasmic-) facing conformation. A correlation has been previously postulated between psychostimulant abuse potential and preference for the outward-facing DAT conformation. The 3β-aryltropane cocaine analogs LX10 and LX11, however, differ only in stereochemistry and share a preference for the outward-facing DAT, yet are reported to vary widely in abuse potential in an animal model. In search of the molecular basis for DAT conformation preference, complexes of cocaine, benztropine, LX10 or LX11 bound to each DAT conformation were subjected to 100ns of all-atom molecular dynamics simulation. Results were consistent with previous findings from cysteine accessibility assays used to assess an inhibitor's DAT conformation preference. The respective 2β- and 2α-substituted phenyltropanes of LX10 and LX11 interacted with hydrophobic regions of the DAT S1 binding site that were inaccessible to cocaine. Solvent accessibility measurements also revealed subtle differences in inhibitor positioning within a given DAT conformation. This work serves to advance our understanding of the conformational selectivity of DAT inhibitors and suggests that MD may be useful in antipsychostimulant therapeutic design. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Residue-specific membrane location of peptides and proteins using specifically and extensively deuterated lipids and {sup 13}C-{sup 2}H rotational-echo double-resonance solid-state NMR

    Energy Technology Data Exchange (ETDEWEB)

    Xie Li; Ghosh, Ujjayini; Schmick, Scott D.; Weliky, David P., E-mail: weliky@chemistry.msu.edu [Michigan State University, Department of Chemistry (United States)

    2013-01-15

    Residue-specific location of peptides in the hydrophobic core of membranes was examined using {sup 13}C-{sup 2}H REDOR and samples in which the lipids were selectively deuterated. The transmembrane topology of the KALP peptide was validated with this approach with substantial dephasing observed for deuteration in the bilayer center and reduced or no dephasing for deuteration closer to the headgroups. Insertion of {beta} sheet HIV and helical and {beta} sheet influenza virus fusion peptides into the hydrophobic core of the membrane was validated in samples with extensively deuterated lipids.

  19. Fasxiator, a novel factor XIa inhibitor from snake venom, and its site-specific mutagenesis to improve potency and selectivity.

    Science.gov (United States)

    Chen, W; Carvalho, L P D; Chan, M Y; Kini, R M; Kang, T S

    2015-02-01

    Bleeding remains a major limitation of standard anticoagulant drugs that target the extrinsic and common coagulation pathways. Recently, intrinsic coagulation factors are increasingly being investigated as alternative targets for developing anticoagulant drugs with lower bleeding risk. Goals were to (i) identify novel anticoagulants selectively targeting intrinsic coagulation pathway and (ii) characterize and further improve the properties of the identified anticoagulants. We have isolated and sequenced a specific factor XIa (FXIa) inhibitor, henceforth named Fasxiator, from the venom of the banded krait snake, Bungarus fasciatus. It is a Kunitz-type protease inhibitor that prolonged activated partial thromboplastin time without significant effects on prothrombin time. Fasxiator was recombinantly expressed (rFasxiator), purified, and characterized to be a slow-type inhibitor of FXIa that exerts its anticoagulant activities (doubled activated partial thromboplastin time at ~ 3 μmol L(-1) ) by selectively inhibiting human FXIa in in vitro assays. A series of mutants were subsequently generated to improve the potency and selectivity of recombinant rFasxiator. rFasxiatorN17R,L19E showed the best balance between potency (IC50 ~ 1 nmol L(-1) ) and selectivity (> 100 times). rFasxiatorN17R,L19E is a competitive slow-type inhibitor of FXIa (Ki  = 0.86 nmol L(-1) ), possesses anticoagulant activity that is ~ 10 times stronger in human plasma than in murine plasma, and prolonged the occlusion time of mice carotid artery in FeCl3 -induced thrombosis models. We have isolated an exogenous FXIa specific inhibitor, engineered it to improve its potency by ~ 1000 times and demonstrated its in vitro and in vivo efficacy. These proof-of-principle data supported the further development of Fasxiator as a novel anticoagulant candidate. © 2014 International Society on Thrombosis and Haemostasis.

  20. Selective assay for CyPA and CyPB in human blood using highly specific anti-peptide antibodies.

    Science.gov (United States)

    Allain, F; Boutillon, C; Mariller, C; Spik, G

    1995-01-13

    Cyclophilins A and B (CyPA and CyPB) are known to be the main binding proteins for cyclosporin A (CsA), a potent immunosuppressive drug. Due to the high homology between the two proteins, antibodies to CyPB were found to cross-react with CyPA. In order to avoid this phenomenon, we raised specific antibodies against peptides copying the most divergent parts of the two sequences. These antibodies allowed us to develop an ELISA capture assay selective for either isotype. Thus, we showed that leukocyte CyPB concentration was almost ten times lower than that of CyPA, and that in contrast to the results described in the literature, only CyPB was released in plasma. Moreover, CyPB levels in leukocytes and plasma were found to correlate for the same donor, but no relationship was found with CyPA level.

  1. Heterologous Coproduction of Enterocin A and Pediocin PA-1 by Lactococcus lactis: Detection by Specific Peptide-Directed Antibodies

    Science.gov (United States)

    Martínez, José M.; Kok, Jan; Sanders, Jan W.; Hernández, Pablo E.

    2000-01-01

    Antibodies against enterocin A were obtained by immunization of rabbits with synthetic peptides PH4 and PH5 designed, respectively, on the N- and C-terminal amino acid sequences of enterocin A and conjugated to the carrier protein KLH. Anti-PH4-KLH antibodies not only recognized enterocin A but also pediocin PA-1, enterocin P, and sakacin A, three bacteriocins which share the N-terminal class IIa consensus motif (YGNGVXC) that is contained in the sequence of the peptide PH4. In contrast, anti-PH5-KLH antibodies only reacted with enterocin A because the amino acid sequences of the C-terminal parts of class IIa bacteriocins are highly variable. Enterocin A and/or pediocin PA-1 structural and immunity genes were introduced in Lactococcus lactis IL1403 to achieve (co)production of the bacteriocins. The level of production of the two bacteriocins was significantly lower than that obtained by the wild-type producers, a fact that suggests a low efficiency of transport and/or maturation of these bacteriocins by the chromosomally encoded bacteriocin translocation machinery of IL1403. Despite the low production levels, both bacteriocins could be specifically detected and quantified with the anti-PH5-KLH (anti-enterocin A) antibodies isolated in this study and the anti-PH2-KLH (anti-pediocin PA-1) antibodies previously generated (J. M. Martínez, M. I. Martínez, A. M. Suárez, C. Herranz, P. Casaus, L. M. Cintas, J. M. Rodríguez, and P. E. Hernández, Appl. Environ. Microbiol. 64:4536–4545, 1998). In this work, the availability of antibodies for the specific detection and quantification of enterocin A and pediocin PA-1 was crucial to demonstrate coproduction of both bacteriocins by L. lactis IL1403(pJM04), because indicator strains that are selectively inhibited by each bacteriocin are not available. PMID:10919819

  2. In silico design and biological evaluation of a dual specificity kinase inhibitor targeting cell cycle progression and angiogenesis.

    Directory of Open Access Journals (Sweden)

    Antony M Latham

    Full Text Available Protein kinases play a central role in tumor progression, regulating fundamental processes such as angiogenesis, proliferation and metastasis. Such enzymes are an increasingly important class of drug target with small molecule kinase inhibitors being a major focus in drug development. However, balancing drug specificity and efficacy is problematic with off-target effects and toxicity issues.We have utilized a rational in silico-based approach to demonstrate the design and study of a novel compound that acts as a dual inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2 and cyclin-dependent kinase 1 (CDK1. This compound acts by simultaneously inhibiting pro-angiogenic signal transduction and cell cycle progression in primary endothelial cells. JK-31 displays potent in vitro activity against recombinant VEGFR2 and CDK1/cyclin B proteins comparable to previously characterized inhibitors. Dual inhibition of the vascular endothelial growth factor A (VEGF-A-mediated signaling response and CDK1-mediated mitotic entry elicits anti-angiogenic activity both in an endothelial-fibroblast co-culture model and a murine ex vivo model of angiogenesis.We deduce that JK-31 reduces the growth of both human endothelial cells and human breast cancer cells in vitro. This novel synthetic molecule has broad implications for development of similar multi-kinase inhibitors with anti-angiogenic and anti-cancer properties. In silico design is an attractive and innovative method to aid such drug discovery.

  3. In silico design and biological evaluation of a dual specificity kinase inhibitor targeting cell cycle progression and angiogenesis.

    Science.gov (United States)

    Latham, Antony M; Kankanala, Jayakanth; Fearnley, Gareth W; Gage, Matthew C; Kearney, Mark T; Homer-Vanniasinkam, Shervanthi; Wheatcroft, Stephen B; Fishwick, Colin W G; Ponnambalam, Sreenivasan

    2014-01-01

    Protein kinases play a central role in tumor progression, regulating fundamental processes such as angiogenesis, proliferation and metastasis. Such enzymes are an increasingly important class of drug target with small molecule kinase inhibitors being a major focus in drug development. However, balancing drug specificity and efficacy is problematic with off-target effects and toxicity issues. We have utilized a rational in silico-based approach to demonstrate the design and study of a novel compound that acts as a dual inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2) and cyclin-dependent kinase 1 (CDK1). This compound acts by simultaneously inhibiting pro-angiogenic signal transduction and cell cycle progression in primary endothelial cells. JK-31 displays potent in vitro activity against recombinant VEGFR2 and CDK1/cyclin B proteins comparable to previously characterized inhibitors. Dual inhibition of the vascular endothelial growth factor A (VEGF-A)-mediated signaling response and CDK1-mediated mitotic entry elicits anti-angiogenic activity both in an endothelial-fibroblast co-culture model and a murine ex vivo model of angiogenesis. We deduce that JK-31 reduces the growth of both human endothelial cells and human breast cancer cells in vitro. This novel synthetic molecule has broad implications for development of similar multi-kinase inhibitors with anti-angiogenic and anti-cancer properties. In silico design is an attractive and innovative method to aid such drug discovery.

  4. Synergistic Protective Effects of Mitochondrial Division Inhibitor 1 and Mitochondria-Targeted Small Peptide SS31 in Alzheimer's Disease.

    Science.gov (United States)

    Reddy, P Hemachandra; Manczak, Maria; Yin, XiangLing; Reddy, Arubala P

    2018-01-01

    The purpose of our study was to determine the synergistic protective effects of mitochondria-targeted antioxidant SS31 and mitochondria division inhibitor 1 (Mdivi1) in Alzheimer's disease (AD). Using biochemical methods, we assessed mitochondrial function by measuring the levels of hydrogen peroxide, lipid peroxidation, cytochrome c oxidase activity, mitochondrial ATP, and GTPase Drp1 enzymatic activity in mutant AβPP cells. Using biochemical methods, we also measured cell survival and apoptotic cell death. Amyloid-β (Aβ) levels were measured using sandwich ELISA, and using real-time quantitative RT-PCR, we assessed mtDNA (mtDNA) copy number in relation to nuclear DNA (nDNA) in all groups of cells. We found significantly reduced levels of Aβ40 and Aβ42 in mutant AβPP cells treated with SS31, Mdivi1, and SS31+Mdivi1, and the reduction of Aβ42 levels were much higher in SS31+Mdivi1 treated cells than individual treatments of SS31 and Mdivi1. The levels of mtDNA copy number and cell survival were significantly increased in SS31, Mdivi1, and SS31+Mdivi1 treated mutant AβPP cells; however, the increased levels of mtDNA copy number and cell survival were much higher in SS31+Mdivi1 treated cells than individual treatments of SS31 and Mdivi1. Mitochondrial dysfunction is significantly reduced in SS31, Mdivi1, and SS31+Mdivi1 treated mutant AβPP cells; however, the reduction is much higher in cells treated with both SS31+Mdvi1. Similarly, GTPase Drp1 activity is reduced in all treatments, but reduced much higher in SS31+Mdivi1 treated cells. These observations strongly suggest that combined treatment of SS31+Mdivi1 is effective than individual treatments of SS31 and Mdivi1. Therefore, we propose that combined treatment of SS31+Mdivi1 is a better therapeutic strategy for AD. Ours is the first study to investigate combined treatment of mitochondria-targeted antioxidant SS31 and mitochondrial division inhibitor 1 in AD neurons.

  5. CART (cocaine- and amphetamine-regulated transcript) peptide specific binding sites in PC12 cells have characteristics of CART peptide receptors

    Czech Academy of Sciences Publication Activity Database

    Nagelová, Veronika; Pirnik, Z.; Železná, Blanka; Maletínská, Lenka

    2014-01-01

    Roč. 1547, Feb 14 (2014), s. 16-24 ISSN 0006-8993 R&D Projects: GA ČR GAP303/10/1368 Institutional support: RVO:61388963 Keywords : CART peptide * PC12 cell * differentiation * binding * signaling * c-Jun Subject RIV: CE - Biochemistry Impact factor: 2.843, year: 2014

  6. The simultaneous ex vivo detection of low-frequency antigen-specific CD4+ and CD8+ T-cell responses using overlapping peptide pools.

    Science.gov (United States)

    Singh, Satwinder Kaur; Meyering, Maaike; Ramwadhdoebe, Tamara H; Stynenbosch, Linda F M; Redeker, Anke; Kuppen, Peter J K; Melief, Cornelis J M; Welters, Marij J P; van der Burg, Sjoerd H

    2012-11-01

    The ability to measure antigen-specific T cells at the single-cell level by intracellular cytokine staining (ICS) is a promising immunomonitoring tool and is extensively applied in the evaluation of immunotherapy of cancer. The protocols used to detect antigen-specific CD8+ T-cell responses generally work for the detection of antigen-specific T cells in samples that have undergone at least one round of in vitro pre-stimulation. Application of a common protocol but now using long peptides as antigens was not suitable to simultaneously detect antigen-specific CD8+ and CD4+ T cells directly ex vivo in cryopreserved samples. CD8 T-cell reactivity to monocytes pulsed with long peptides as antigens ranged between 5 and 25 % of that observed against monocytes pulsed with a direct HLA class I fitting minimal CTL peptide epitope. Therefore, we adapted our ICS protocol and show that the use of tenfold higher concentration of long peptides to load APC, the use of IFN-α and poly(I:C) to promote antigen processing and improve T-cell stimulation, does allow for the ex vivo detection of low-frequency antigen-specific CD8+ and CD4+ T cells in an HLA-independent setting. While most of the improvements were related to increasing the ability to measure CD8+ T-cell reactivity following stimulation with long peptides to at least 50 % of the response detected when using a minimal peptide epitope, the final analysis of blood samples from vaccinated patients successfully showed that the adapted ICS protocol also increases the ability to ex vivo detect low-frequency p53-specific CD4+ T-cell responses in cryopreserved PBMC samples.

  7. [New Radiopharmaceuticals Based on Prostate-Specific Inhibitors of Membrane Antigen for Diagnostics and Therapy of Metastatic Prostate Cancer].

    Science.gov (United States)

    Vlasova, O P; German, K E; Krilov, V V; Petriev, V M; Epstein, N B

    2015-01-01

    About 10.7% cases of prostate cancer were registered in Russia in 2011 (40,000 patients). More than half of cancer cases were revealed in advanced (III-IV) stages when metastases inevitably developed quickly. Clinical problem of early diagnostics and treatment of metastatic prostate cancer is still not solved. Anatomical imaging techniques have low sensitivity and specificity for the detection of this disease. Metabolic visualization methods which use prostate specific antigen (PSA) as a marker are also ineffective. This article describes prostate-specific membrane antigens (PSMA) that are proposed as a marker for diagnostics and therapy of prostate cancer. The most promising PSMA-based radiopharmaceutical agent for diagnostics has been developed and clinically tested in the European countries. These pharmaceuticals are based on small peptide molecules modified with urea, and have the highest affinity to PSMA. Favorable phannacokinetics, rapid accumulation in the tumor and rapid excretion from the body are beneficial features of these pharmaceuticals.

  8. Specificity and sensitivity of commercially available assays for glucagon-like peptide-1 (GLP-1)

    DEFF Research Database (Denmark)

    Bak, Monika Judyta; Albrechtsen, Nicolai Jacob Wewer; Pedersen, Jens

    2014-01-01

    assessed with 3 commercial kits. RESULTS: The USCN LIFE assay detected none of the GLP-1 isoforms. The active GLP-1 ELISAs from Millipore and DRG seemed identical and were specific for intact GLP-1 in buffer and plasma. The Meso Scale Discovery Total GLP-1 kit detected all six GLP-1 isoforms, although...

  9. [Specific inhibitors of cyclooxygenase-2 (COX-2): current knowledge and perspectives].

    Science.gov (United States)

    Rioda, W T; Nervetti, A

    2001-01-01

    The Authors summarize the current knowledge on a new class of nonsteroidal anti-inflammatory drugs (NSAIDs), the coxib (celecoxib and rofecoxib), in the treatment of rheumatic diseases. Celecoxib and rofecoxib are selective cyclooxygenase-2 (COX-2) inhibitors which possess the same anti-inflammatory and analgesic activities, but a better gastric tolerability compared to the non-selective COX-1 and COX-2 inhibitors. The Authors also report other possible therapeutic effects of these NSADIs as evidenced by the more recent data of the literature. Celecoxib seems to reduce the incidence of new polyps in patients with familial adenomatous polyposis. It has been suggested the use of celecoxib as a protective drug against the development of colorectal cancer. Other (neoplastic) or pre-neoplastic conditions, such as bladder dysplasia, Barret esophagus, attinic keratosis and Alzheimer's disease seem to have benefit from this class of drugs.

  10. Investigation of the substrate specificity of the proton coupled peptide transporter PepTSo from Shewanella oneidensis

    DEFF Research Database (Denmark)

    Prabhala, Bala Krishna; Aduri, Nanda Gowtham; Hald, Helle

    2015-01-01

    a strikingly high sequence identity, can be used to rationalize its mechanism and substrate preference. However, very little is known about the substrate specificity of PepTSo. To elaborate on this, the natural peptide specificity of PepTSo was investigated. Di and tri-peptides were found to be substrates...... for PepTSo in contrast to mono- and tetrapeptides as was indicated by previous competition studies. Interestingly, a negatively charged side chain was better accommodated on the dipeptide N- than the C-terminus position. Inversely, a positive charged side chain appeared to be tolerated better...

  11. Amyloid-β peptide-specific DARPins as a novel class of potential therapeutics for Alzheimer disease.

    Science.gov (United States)

    Hanenberg, Michael; McAfoose, Jordan; Kulic, Luka; Welt, Tobias; Wirth, Fabian; Parizek, Petra; Strobel, Lisa; Cattepoel, Susann; Späni, Claudia; Derungs, Rebecca; Maier, Marcel; Plückthun, Andreas; Nitsch, Roger M

    2014-09-26

    Passive immunization with anti-amyloid-β peptide (Aβ) antibodies is effective in animal models of Alzheimer disease. With the advent of efficient in vitro selection technologies, the novel class of designed ankyrin repeat proteins (DARPins) presents an attractive alternative to the immunoglobulin scaffold. DARPins are small and highly stable proteins with a compact modular architecture ideal for high affinity protein-protein interactions. In this report, we describe the selection, binding profile, and epitope analysis of Aβ-specific DARPins. We further showed their ability to delay Aβ aggregation and prevent Aβ-mediated neurotoxicity in vitro. To demonstrate their therapeutic potential in vivo, mono- and trivalent Aβ-specific DARPins (D23 and 3×D23) were infused intracerebroventricularly into the brains of 11-month-old Tg2576 mice over 4 weeks. Both D23 and 3×D23 treatments were shown to result in improved cognitive performance and reduced soluble Aβ levels. These findings demonstrate the therapeutic potential of Aβ-specific DARPins for the treatment of Alzheimer disease. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Breakdown of the FLT3-ITD/STAT5 axis and synergistic apoptosis induction by the histone deacetylase inhibitor panobinostat and FLT3-specific inhibitors.

    Science.gov (United States)

    Pietschmann, Kristin; Bolck, Hella Anna; Buchwald, Marc; Spielberg, Steffi; Polzer, Harald; Spiekermann, Karsten; Bug, Gesine; Heinzel, Thorsten; Böhmer, Frank-Dietmar; Krämer, Oliver H

    2012-11-01

    Activating mutations of the class III receptor tyrosine kinase FLT3 are the most frequent molecular aberration in acute myeloid leukemia (AML). Mutant FLT3 accelerates proliferation, suppresses apoptosis, and correlates with poor prognosis. Therefore, it is a promising therapeutic target. Here, we show that RNA interference against FLT3 with an internal tandem duplication (FLT3-ITD) potentiates the efficacy of the histone deacetylase inhibitor (HDACi) panobinostat (LBH589) against AML cells expressing FLT3-ITD. Similar to RNA interference, tyrosine kinase inhibitors (TKI; AC220/cpd.102/PKC412) in combination with LBH589 exhibit superior activity against AML cells. Median dose-effect analyses of drug-induced apoptosis rates of AML cells (MV4-11 and MOLM-13) revealed combination index (CI) values indicating strong synergism. AC220, the most potent and FLT3-specific TKI, shows highest synergism with LBH589 in the low nanomolar range. A 4-hour exposure to LBH589 + AC220 already generates more than 50% apoptosis after 24 hours. Different cell lines lacking FLT3-ITD as well as normal peripheral blood mononuclear cells are not significantly affected by LBH589 + TKI, showing the specificity of this treatment regimen. Immunoblot analyses show that LBH589 + TKI induce apoptosis via degradation of FLT3-ITD and its prosurvival target STAT5. Previously, we showed the LBH589-induced proteasomal degradation of FLT3-ITD. Here, we show that activated caspase-3 also contributes to the degradation of FLT3-ITD and that STAT5 is a direct target of this protease. Our data strongly emphasize HDACi/TKI drug combinations as promising modality for the treatment of FLT3-ITD-positive AMLs. ©2012 AACR.

  13. LC-QTOF-MS identification of porcine-specific peptide in heat treated pork identifies candidate markers for meat species determination.

    Science.gov (United States)

    Sarah, S A; Faradalila, W N; Salwani, M S; Amin, I; Karsani, S A; Sazili, A Q

    2016-05-15

    The purpose of this study was to identify porcine-specific peptide markers from thermally processed meat that could differentiate pork from beef, chevon and chicken meat. In the initial stage, markers from tryptic digested protein of chilled, boiled and autoclaved pork were identified using LC-QTOF-MS. An MRM method was then established for verification. A thorough investigation of LC-QTOF-MS data showed that only seven porcine-specific peptides were consistently detected. Among these peptides, two were derived from lactate dehydrogenase, one from creatine kinase, and four from serum albumin protein. However, MRM could only detect four peptides (EVTEFAK, LVVITAGAR, FVIER and TVLGNFAAFVQK) that were consistently present in pork samples. In conclusion, meat species determination through a tandem mass spectrometry platform shows high potential in providing scientifically valid and reliable results even at peptide level. Besides, the specificity and selectivity offered by the proteomics approach also provide a robust platform for Halal authentication. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. A cancer specific cell-penetrating peptide, BR2, for the efficient delivery of an scFv into cancer cells.

    Directory of Open Access Journals (Sweden)

    Ki Jung Lim

    Full Text Available Cell-penetrating peptides (CPPs have proven very effective as intracellular delivery vehicles for various therapeutics. However, there are some concerns about non-specific penetration and cytotoxicity of CPPs for effective cancer treatments. Herein, based on the cell-penetrating motif of an anticancer peptide, buforin IIb, we designed several CPP derivatives with cancer cell specificity. Among the derivatives, a 17-amino acid peptide (BR2 was found to have cancer-specificity without toxicity to normal cells. After specifically targeting cancer cells through interaction with gangliosides, BR2 entered cells via lipid-mediated macropinocytosis. Moreover, BR2 showed higher membrane translocation efficiency than the well-known CPP Tat (49-57. The capability of BR2 as a cancer-specific drug carrier was demonstrated by fusion of BR2 to a single-chain variable fragment (scFv directed toward a mutated K-ras (G12V. BR2-fused scFv induced a higher degree of apoptosis than Tat-fused scFv in K-ras mutated HCT116 cells. These results suggest that the novel cell-penetrating peptide BR2 has great potential as a useful drug delivery carrier with cancer cell specificity.

  15. A cancer specific cell-penetrating peptide, BR2, for the efficient delivery of an scFv into cancer cells.

    Science.gov (United States)

    Lim, Ki Jung; Sung, Bong Hyun; Shin, Ju Ri; Lee, Young Woong; Kim, Da Jung; Yang, Kyung Seok; Kim, Sun Chang

    2013-01-01

    Cell-penetrating peptides (CPPs) have proven very effective as intracellular delivery vehicles for various therapeutics. However, there are some concerns about non-specific penetration and cytotoxicity of CPPs for effective cancer treatments. Herein, based on the cell-penetrating motif of an anticancer peptide, buforin IIb, we designed several CPP derivatives with cancer cell specificity. Among the derivatives, a 17-amino acid peptide (BR2) was found to have cancer-specificity without toxicity to normal cells. After specifically targeting cancer cells through interaction with gangliosides, BR2 entered cells via lipid-mediated macropinocytosis. Moreover, BR2 showed higher membrane translocation efficiency than the well-known CPP Tat (49-57). The capability of BR2 as a cancer-specific drug carrier was demonstrated by fusion of BR2 to a single-chain variable fragment (scFv) directed toward a mutated K-ras (G12V). BR2-fused scFv induced a higher degree of apoptosis than Tat-fused scFv in K-ras mutated HCT116 cells. These results suggest that the novel cell-penetrating peptide BR2 has great potential as a useful drug delivery carrier with cancer cell specificity.

  16. A Novel Screen for Suppressors of Breast Tumor Cell Growth Using an Oriented Random Peptide Library Method to Identify Inhibitors of the ErbB2 Tyrosine Kinase

    National Research Council Canada - National Science Library

    Carraway, Kermit

    1998-01-01

    .... To identify potential antagonists, the extracellular ligand binding domain of the ErbB2 is immobilized on a column support, and used to affinity purify cyclic peptides from oriented random peptide libraries...

  17. A Novel Screen for Suppressors of Breast Tumor Cell Growth Using an Oriented Random Peptide Library Method to Identify Inhibitors of the ErbB2 Tyrosine Kinase

    National Research Council Canada - National Science Library

    Carraway, Kermit

    1999-01-01

    .... To identify potential antagonists, the extracellular ligand binding domain of the ErbB2 is immobilized on a column support, and used to affinity purify cyclic peptides from oriented random peptide libraries...

  18. Peptides for Specific Intracellular Delivery and Targeting of Nanoparticles: Implications for Developing Nanoparticle-Mediated Drug Delivery

    Science.gov (United States)

    2010-01-01

    less in size and span an array of compositions including met- als, semiconductor quantum dots (QDs), oxides, polymers , vesicles (e.g., micelles...93,98] Polymers Poly(lactic-co-glycolic acid), poly(amido amine) and dendrimers Biocompatibility, biodegradability and controlled release High drug...Secondary interactions Covalent atachment to surface ligand Thiolated peptide Biotinylated peptide Aminated peptide Sulfo-NHS Tetravalent streptavidin

  19. Detection of specific IgA antibodies against a novel deamidated 8-Mer gliadin peptide in blood plasma samples from celiac patients.

    Directory of Open Access Journals (Sweden)

    Sara Vallejo-Diez

    Full Text Available We studied whether celiac disease (CD patients produce antibodies against a novel gliadin peptide specifically generated in the duodenum of CD patients by a previously described pattern of CD-specific duodenal proteases. Fingerprinting and ion-trap mass spectrometry of CD-specific duodenal gliadin-degrading protease pattern revealed a new 8-mer gliadin-derived peptide. An ELISA against synthetic deamidated 8-mer peptides (DGP 8-mer was used to study the presence of IgA anti-DGP 8-mer antibodies in plasma samples from 81 children (31 active CD patients (aCD, 17 CD patients on a gluten-free diet (GFD, 10 healthy controls (C and 23 patients with other gastrointestinal pathology (GP and 101 adults (16 aCD, 12 GFD, 27 C and 46 GP-patients. Deamidation of the 8-mer peptide significantly increased the reactivity of the IgA antibodies from CD patients against the peptide. Significant IgA anti-DGP 8-mer antibodies levels were detected in 93.5% of aCD-, 11.8% of GFD- and 4.3% of GP-patients in children. In adults, antibodies were detected in 81.3% of aCD-patients and 8.3% of GFD-patients while were absent in 100% of C- and GP-patients. Duodenal CD-specific gliadin degrading proteases release an 8-mer gliadin peptide that once deamidated is an antigen for specific IgA antibodies in CD patients which may provide a new accurate diagnostic tool in CD.

  20. Induction of human immunodeficiency virus (HIV-1 envelope specific cell-mediated immunity by a non-homologous synthetic peptide.

    Directory of Open Access Journals (Sweden)

    Ammar Achour

    2007-11-01

    Full Text Available Cell mediated immunity, including efficient CTL response, is required to prevent HIV-1 from cell-to-cell transmission. In previous investigations, we have shown that B1 peptide derived by Fourier transformation of HIV-1 primary structures and sharing no sequence homology with the parent proteins was able to generate antiserum which recognizes envelope and Tat proteins. Here we have investigated cellular immune response towards a novel non-homologous peptide, referred to as cA1 peptide.The 20 amino acid sequence of cA1 peptide was predicted using the notion of peptide hydropathic properties; the peptide is encoded by the complementary anti-sense DNA strand to the sense strand of previously described non-homologous A1 peptide. In this report we demonstrate that the cA1 peptide can be a target for major histocompatibility complex (MHC class I-restricted cytotoxic T lymphocytes in HIV-1-infected or envelope-immunized individuals. The cA1 peptide is recognized in association with different MHC class I allotypes and could prime in vitro CTLs, derived from gp160-immunized individuals capable to recognize virus variants.For the first time a theoretically designed immunogen involved in broad-based cell-immune memory activation is described. Our findings may thus contribute to the advance in vaccine research by describing a novel strategy to develop a synthetic AIDS vaccine.

  1. Aborted germinal center reactions and B cell memory by follicular T cells specific for a B cell receptor V region peptide.

    Science.gov (United States)

    Heiser, Ryan A; Snyder, Christopher M; St Clair, James; Wysocki, Lawrence J

    2011-07-01

    A fundamental problem in immunoregulation is how CD4(+) T cells react to immunogenic peptides derived from the V region of the BCR that are created by somatic mechanisms, presented in MHC II, and amplified to abundance by B cell clonal expansion during immunity. BCR neo Ags open a potentially dangerous avenue of T cell help in violation of the principle of linked Ag recognition. To analyze this issue, we developed a murine adoptive transfer model using paired donor B cells and CD4 T cells specific for a BCR-derived peptide. BCR peptide-specific T cells aborted ongoing germinal center reactions and impeded the secondary immune response. Instead, they induced the B cells to differentiate into short-lived extrafollicular plasmablasts that secreted modest quantities of Ig. These results uncover an immunoregulatory process that restricts the memory pathway to B cells that communicate with CD4 T cells via exogenous foreign Ag.

  2. Magnetic microparticle-based SELEX process for the identification of highly specific aptamers of heart marker--brain natriuretic peptide

    International Nuclear Information System (INIS)

    Wang, Ying; Cao, Jinxuan; Wu, Jingjing; Xue, Feng; Teng, Jun; Chen, Wei; Chen, Yinji; Lu, Chunxia

    2015-01-01

    The brain natriuretic peptide (BNP) is known to be an effective indicator of heart failure. It has been widely adopted as a parameter for the evaluation of heart function of cardiovascular and cerebrovascular diseases (CVDs). Current immune-recognition based methods for the detection of BNP are limited, to a certain extent, by the poor stability of the antibody and by high costs. The availability of an aptamer specific for BNP would greatly assist in the rapid and early diagnosis of CVDs. In order to screen for such an aptamer by the SELEX method, we have used magnetic microparticles (m-MPs) as the separation substrate for immobilization of target BNP. The use of m-MPs for rapid separation of combined aptamers enables bound oligonucleotides to be separated directly, quickly, and with high efficiency. After 14 rounds of selection, a panel of six aptamers against BNP was identified. Their dissociation constants range from 12.5 to 139 nM. The classical technique for conjugation of a target to m-MPs is known to be applicable to various fields, and we conclude that this m-MP-based SELEX process provides a general strategy for screening of specific aptamers against various analytes. (author)

  3. pH-dependence of the specific binding of Cu(II) and Zn(II) ions to the amyloid-β peptide

    International Nuclear Information System (INIS)

    Ghalebani, Leila; Wahlström, Anna; Danielsson, Jens; Wärmländer, Sebastian K.T.S.; Gräslund, Astrid

    2012-01-01

    Highlights: ► Cu(II) and Zn(II) display pH-dependent binding to the Aβ(1–40) peptide. ► At pH 7.4 both metal ions display residue-specific binding to the Aβ peptide. ► At pH 5.5 the binding specificity is lost for Zn(II). ► Differential Cu(II) and Zn(II) binding may help explain metal-induced AD toxicity. -- Abstract: Metal ions like Cu(II) and Zn(II) are accumulated in Alzheimer’s disease amyloid plaques. The amyloid-β (Aβ) peptide involved in the disease interacts with these metal ions at neutral pH via ligands provided by the N-terminal histidines and the N-terminus. The present study uses high-resolution NMR spectroscopy to monitor the residue-specific interactions of Cu(II) and Zn(II) with 15 N- and 13 C, 15 N-labeled Aβ(1–40) peptides at varying pH levels. At pH 7.4 both ions bind to the specific ligands, competing with one another. At pH 5.5 Cu(II) retains its specific histidine ligands, while Zn(II) seems to lack residue-specific interactions. The low pH mimics acidosis which is linked to inflammatory processes in vivo. The results suggest that the cell toxic effects of redox active Cu(II) binding to Aβ may be reversed by the protective activity of non-redox active Zn(II) binding to the same major binding site under non-acidic conditions. Under acidic conditions, the protective effect of Zn(II) may be decreased or changed, since Zn(II) is less able to compete with Cu(II) for the specific binding site on the Aβ peptide under these conditions.

  4. Specificity of the second binding protein of the peptide ABC-transporter (Dpp) of Lactococcus lactis IL1403

    NARCIS (Netherlands)

    Sanz, Y; Toldra, F; Renault, P; Poolman, B

    2003-01-01

    The genome sequence of Lactococcus lactis IL1403 revealed the presence of a putative peptide-binding protein-dependent ABC-transporter (Dpp). The genes for two peptide-binding proteins (dppA and dppP) precede the membrane components, which include two transmembrane protein genes (dppB and dppC) and

  5. Identification of novel peptide ligands for the cancer-specific receptor mutation EFGRvIII using a mixture-based synthetic combinatorial library

    DEFF Research Database (Denmark)

    Denholt, Charlotte Lund; Hansen, Paul Robert; Pedersen, Nina

    2009-01-01

    We report here, the design and synthesis of a positional scanning synthetic combinatorial library for the identification of novel peptide ligands targeted against the cancer-specific epidermal growth factor tyrosine kinase receptor mutation variant III (EGFRvIII). This receptor is expressed in se...

  6. Early detection of response in small cell bronchogenic carcinoma by changes in serum concentrations of creatine kinase, neuron specific enolase, calcitonin, ACTH, serotonin and gastrin releasing peptide

    DEFF Research Database (Denmark)

    Bork, E; Hansen, M; Urdal, P

    1988-01-01

    Creatine kinase (CK-BB), neuron specific enolase (NSE), ACTH, calcitonin, serotonin and gastrin releasing peptide (GRP) were measured in serum or plasma before and immediately after initiation of treatment in patients with small cell lung cancer (SCC). Pretherapeutic elevated concentrations of CK...

  7. Peptide microarray analysis of substrate specificity of the transmembrane Ser/Thr kinase KPI-2 reveals reactivity with cystic fibrosis transmembrane conductance regulator and phosphorylase.

    Science.gov (United States)

    Wang, Hong; Brautigan, David L

    2006-11-01

    Human lemur (Lmr) kinases are predicted to be Tyr kinases based on sequences and are related to neurotrophin receptor Trk kinases. This study used homogeneous recombinant KPI-2 (Lmr2, LMTK2, Cprk, brain-enriched protein kinase) kinase domain and a library of 1,154 peptides on a microarray to analyze substrate specificity. We found that KPI-2 is strictly a Ser/Thr kinase that reacts with Ser either preceded by or followed by Pro residues but unlike other Pro-directed kinases does not strictly require an adjacent Pro residue. The most reactive peptide in the library corresponds to Ser-737 of cystic fibrosis transmembrane conductance regulator, and the recombinant R domain of cystic fibrosis transmembrane conductance regulator was a preferred substrate. Furthermore the KPI-2 kinase phosphorylated peptides corresponding to the single site in phosphorylase and purified phosphorylase b, making this only the second known phosphorylase b kinase. Phosphorylase was used as a specific substrate to show that KPI-2 is inhibited in living cells by addition of nerve growth factor or serum. The results demonstrate the utility of the peptide library to probe specificity and discover kinase substrates and offer a specific assay that reveals hormonal regulation of the activity of this unusual transmembrane kinase.

  8. Discovery of cofactor-specific, bactericidal Mycobacterium tuberculosis InhA inhibitors using DNA-encoded library technology.

    Science.gov (United States)

    Soutter, Holly H; Centrella, Paolo; Clark, Matthew A; Cuozzo, John W; Dumelin, Christoph E; Guie, Marie-Aude; Habeshian, Sevan; Keefe, Anthony D; Kennedy, Kaitlyn M; Sigel, Eric A; Troast, Dawn M; Zhang, Ying; Ferguson, Andrew D; Davies, Gareth; Stead, Eleanor R; Breed, Jason; Madhavapeddi, Prashanti; Read, Jon A

    2016-12-06

    Millions of individuals are infected with and die from tuberculosis (TB) each year, and multidrug-resistant (MDR) strains of TB are increasingly prevalent. As such, there is an urgent need to identify novel drugs to treat TB infections. Current frontline therapies include the drug isoniazid, which inhibits the essential NADH-dependent enoyl-acyl-carrier protein (ACP) reductase, InhA. To inhibit InhA, isoniazid must be activated by the catalase-peroxidase KatG. Isoniazid resistance is linked primarily to mutations in the katG gene. Discovery of InhA inhibitors that do not require KatG activation is crucial to combat MDR TB. Multiple discovery efforts have been made against InhA in recent years. Until recently, despite achieving high potency against the enzyme, these efforts have been thwarted by lack of cellular activity. We describe here the use of DNA-encoded X-Chem (DEX) screening, combined with selection of appropriate physical properties, to identify multiple classes of InhA inhibitors with cell-based activity. The utilization of DEX screening allowed the interrogation of very large compound libraries (10 11 unique small molecules) against multiple forms of the InhA enzyme in a multiplexed format. Comparison of the enriched library members across various screening conditions allowed the identification of cofactor-specific inhibitors of InhA that do not require activation by KatG, many of which had bactericidal activity in cell-based assays.

  9. In vivo dual-delivery of glucagon like peptide-1 (GLP-1) and dipeptidyl peptidase-4 (DPP4) inhibitor through composites prepared by microfluidics for diabetes therapy

    Science.gov (United States)

    Araújo, F.; Shrestha, N.; Gomes, M. J.; Herranz-Blanco, B.; Liu, D.; Hirvonen, J. J.; Granja, P. L.; Santos, H. A.; Sarmento, B.

    2016-05-01

    Oral delivery of proteins is still a challenge in the pharmaceutical field. Nanoparticles are among the most promising carrier systems for the oral delivery of proteins by increasing their oral bioavailability. However, most of the existent data regarding nanosystems for oral protein delivery is from in vitro studies, lacking in vivo experiments to evaluate the efficacy of these systems. Herein, a multifunctional composite system, tailored by droplet microfluidics, was used for dual delivery of glucagon like peptide-1 (GLP-1) and dipeptidyl peptidase-4 inhibitor (iDPP4) in vivo. Oral delivery of GLP-1 with nano- or micro-systems has been studied before, but the simultaneous nanodelivery of GLP-1 with iDPP4 is a novel strategy presented here. The type 2 diabetes mellitus (T2DM) rat model, induced through the combined administration of streptozotocin and nicotinamide, a non-obese model of T2DM, was used. The combination of both drugs resulted in an increase in the hypoglycemic effects in a sustained, but prolonged manner, where the iDPP4 improved the therapeutic efficacy of GLP-1. Four hours after the oral administration of the system, blood glucose levels were decreased by 44%, and were constant for another 4 h, representing half of the glucose area under the curve when compared to the control. An enhancement of the plasmatic insulin levels was also observed 6 h after the oral administration of the dual-drug composite system and, although no statistically significant differences existed, the amount of pancreatic insulin was also higher. These are promising results for the oral delivery of GLP-1 to be pursued further in a chronic diabetic model study.

  10. Systemic co-delivery of doxorubicin and siRNA using nanoparticles conjugated with EGFR-specific targeting peptide to enhance chemotherapy in ovarian tumor bearing mice

    Energy Technology Data Exchange (ETDEWEB)

    Liu, C. W.; Lin, W. J., E-mail: wjlin@ntu.edu.tw [National Taiwan University, Graduate Institute of Pharmaceutical Sciences, School of Pharmacy (China)

    2013-10-15

    This aim of this study was to develop peptide-conjugated nanoparticles (NPs) for systemic co-delivery of siRNA and doxorubicin to enhance chemotherapy in epidermal growth factor receptor (EGFR) high-expressed ovarian tumor bearing mice. The active targeting NPs were prepared using heptapeptide-conjugated poly(d,l-lactic-co-glycolic acid)-poly(ethylene glycol). The particle sizes of peptide-free and peptide-conjugated NPs were 159.3 {+-} 32.5 and 184.0 {+-} 52.9 nm, respectively, with zeta potential -21.3 {+-} 3.8 and -15.3 {+-} 2.8 mV. The peptide-conjugated NPs uptake were more efficient in EGFR high-expressed SKOV3 cells than in EGFR low-expressed HepG2 cells due to heptapeptide specificity. The NPs were used to deliver small molecule anticancer drug (e.g., doxorubicin) and large molecule genetic agent (e.g., siRNA). The IC{sub 50} of doxorubicin-loaded peptide-conjugated NPs (0.09 {+-} 0.06 {mu}M) was significantly lower than peptide-free NPs (5.72 {+-} 2.64 {mu}M). The similar result was observed in siRNA-loaded NPs. The peptide-conjugated NPs not only served as a nanocarrier to efficiently deliver doxorubicin and siRNA to EGFR high-expressed ovarian cancer cells but also increased the intracellular accumulation of the therapeutic agents to induce assured anti-tumor growth effect in vivo.

  11. Isolation and characterization of a specific endogenous Na/sup +/, K/sup +/-ATPase inhibitor from bovine adrenal

    Energy Technology Data Exchange (ETDEWEB)

    Tamura, M.; Lam, T.T.; Inagami, T.

    1988-06-14

    In order to identify a specific endogenous Na/sup +/,K/sup +/-ATPase inhibitor which could possibly be related to salt-dependent hypertension, the authors looked for substances in the methanol extract of bovine whole adrenal which show all of the following properties: (i) inhibitory activity for Na/sup +/,K/sup +/-ATPase; (ii) competitive displacing activity against (/sup 3/H)ouabain binding to the enzyme; (iii) inhibitory activity for /sup 86/Rb uptake into intact human erythrocytes; and (iv) cross-reactivity with sheep anti-digoxin-specific antibody. After stepwise fractionation of the methanol extract of bovine adrenal glands by chromatography on a C/sub 18/ open column, a 0-15% acetonitrile fraction was fractionated by high-performance liquid chromatography on a Zorbax octadecylsilane column. One of the most active fractions in 0-15% acetonitrile was found to exhibit all of the four types of the activities. It was soluble in water and was distinct from various substances which have been known to inhibit Na/sup +/,K/sup +/-ATPase. These results strongly suggest that this water-soluble nonpeptidic Na/sup +/,K/sup +/-ATPase inhibitor may be a specific endogenous regulator for the ATPase.

  12. In silico simulations of STAT1 and STAT3 inhibitors predict SH2 domain cross-binding specificity.

    Science.gov (United States)

    Szelag, Malgorzata; Sikorski, Krzysztof; Czerwoniec, Anna; Szatkowska, Katarzyna; Wesoly, Joanna; Bluyssen, Hans A R

    2013-11-15

    Signal transducers and activators of transcription (STATs) comprise a family of transcription factors that are structurally related and which participate in signaling pathways activated by cytokines, growth factors and pathogens. Activation of STAT proteins is mediated by the highly conserved Src homology 2 (SH2) domain, which interacts with phosphotyrosine motifs for specific contacts between STATs and receptors and for STAT dimerization. By generating new models for human (h)STAT1, hSTAT2 and hSTAT3 we applied comparative in silico docking to determine SH2-binding specificity of the STAT3 inhibitor stattic, and of fludarabine (STAT1 inhibitor). Thus, we provide evidence that by primarily targeting the highly conserved phosphotyrosine (pY+0) SH2 binding pocket stattic is not a specific hSTAT3 inhibitor, but is equally effective towards hSTAT1 and hSTAT2. This was confirmed in Human Micro-vascular Endothelial Cells (HMECs) in vitro, in which stattic inhibited interferon-α-induced phosphorylation of all three STATs. Likewise, fludarabine inhibits both hSTAT1 and hSTAT3 phosphorylation, but not hSTAT2, by competing with the highly conserved pY+0 and pY-X binding sites, which are less well-preserved in hSTAT2. Moreover we observed that in HMECs in vitro fludarabine inhibits cytokine and lipopolysaccharide-induced phosphorylation of hSTAT1 and hSTAT3 but does not affect hSTAT2. Finally, multiple sequence alignment of STAT-SH2 domain sequences confirmed high conservation between hSTAT1 and hSTAT3, but not hSTAT2, with respect to stattic and fludarabine binding sites. Together our data offer a molecular basis that explains STAT cross-binding specificity of stattic and fludarabine, thereby questioning the present selection strategies of SH2 domain-based competitive small inhibitors. © 2013 Elsevier B.V. All rights reserved.

  13. Melanoma inhibitor of apoptosis protein (ML-IAP) specific cytotoxic T lymphocytes cross-react with an epitope from the auto-antigen SS56

    DEFF Research Database (Denmark)

    Baek Sørensen, Rikke; Faurschou, Mikkel; Troelsen, Lone

    2009-01-01

    A large proportion of melanoma patients host a spontaneous T-cell response specifically against ML-IAP-derived peptides. In this study, we describe that some ML-IAP-specific cytotoxic T cells isolated from melanoma patients cross react with an epitope from the auto-antigen SS56. SS56 is a recentl...

  14. Design of Tail-Clamp Peptide Nucleic Acid Tethered with Azobenzene Linker for Sequence-Specific Detection of Homopurine DNA

    Directory of Open Access Journals (Sweden)

    Shinjiro Sawada

    2017-10-01

    Full Text Available DNA carries genetic information in its sequence of bases. Synthetic oligonucleotides that can sequence-specifically recognize a target gene sequence are a useful tool for regulating gene expression or detecting target genes. Among the many synthetic oligonucleotides, tail-clamp peptide nucleic acid (TC-PNA offers advantages since it has two homopyrimidine PNA strands connected via a flexible ethylene glycol-type linker that can recognize complementary homopurine sequences via Watson-Crick and Hoogsteen base pairings and form thermally-stable PNA/PNA/DNA triplex structures. Here, we synthesized a series of TC-PNAs that can possess different lengths of azobenzene-containing linkers and studied their binding behaviours to homopurine single-stranded DNA. Introduction of azobenzene at the N-terminus amine of PNA increased the thermal stability of PNA-DNA duplexes. Further extension of the homopyrimidine PNA strand at the N-terminus of PNA-AZO further increased the binding stability of the PNA/DNA/PNA triplex to the target homopurine sequence; however, it induced TC-PNA/DNA/TC-PNA complex formation. Among these TC-PNAs, 9W5H-C4-AZO consisting of nine Watson-Crick bases and five Hoogsteen bases tethered with a beta-alanine conjugated azobenzene linker gave a stable 1:1 TC-PNA/ssDNA complex and exhibited good mismatch recognition. Our design for TC-PNA-AZO can be utilized for detecting homopurine sequences in various genes.

  15. Loss of the nodule-specific cysteine rich peptide, NCR169, abolishes symbiotic nitrogen fixation in the Medicago truncatula dnf7 mutant.

    Science.gov (United States)

    Horváth, Beatrix; Domonkos, Ágota; Kereszt, Attila; Szűcs, Attila; Ábrahám, Edit; Ayaydin, Ferhan; Bóka, Károly; Chen, Yuhui; Chen, Rujin; Murray, Jeremy D; Udvardi, Michael K; Kondorosi, Éva; Kaló, Péter

    2015-12-08

    Host compatible rhizobia induce the formation of legume root nodules, symbiotic organs within which intracellular bacteria are present in plant-derived membrane compartments termed symbiosomes. In Medicago truncatula nodules, the Sinorhizobium microsymbionts undergo an irreversible differentiation process leading to the development of elongated polyploid noncultivable nitrogen fixing bacteroids that convert atmospheric dinitrogen into ammonia. This terminal differentiation is directed by the host plant and involves hundreds of nodule specific cysteine-rich peptides (NCRs). Except for certain in vitro activities of cationic peptides, the functional roles of individual NCR peptides in planta are not known. In this study, we demonstrate that the inability of M. truncatula dnf7 mutants to fix nitrogen is due to inactivation of a single NCR peptide, NCR169. In the absence of NCR169, bacterial differentiation was impaired and was associated with early senescence of the symbiotic cells. Introduction of the NCR169 gene into the dnf7-2/NCR169 deletion mutant restored symbiotic nitrogen fixation. Replacement of any of the cysteine residues in the NCR169 peptide with serine rendered it incapable of complementation, demonstrating an absolute requirement for all cysteines in planta. NCR169 was induced in the cell layers in which bacteroid elongation was most pronounced, and high expression persisted throughout the nitrogen-fixing nodule zone. Our results provide evidence for an essential role of NCR169 in the differentiation and persistence of nitrogen fixing bacteroids in M. truncatula.

  16. Human immunodeficiency virus contains an epitope immunoreactive with thymosin α1 and the 30-amino acid synthetic p17 group-specific antigen peptide HGP-30

    International Nuclear Information System (INIS)

    Naylor, P.H.; Naylor, C.W.; Badamchian, M.; Wada, S.; Goldstein, A.L.; Wang, S.S.; Sun, D.K.; Thornton, A.H.; Sarin, P.S.

    1987-01-01

    The authors have reported that an antiserum prepared against thymosin α 1 [which shares a region of homology with the p17 protein of the acquired immunodeficiency syndrome (AIDS)-associated human immunodeficiency virus] effectively neutralized the AIDs virus and prevented its replication in H9 cells. Using HPLC and immunoblot analysis, they have identified from a clone B, type III human T-lymphotropic virus (HTLV-IIIB) extracts a protein with a molecular weight of 17,000 that is immunoreactive with thymosin α 1 . In contrast, no immunoreactivity was found in retroviral extracts from a number of nonhuman species including feline, bovine, simian, gibbon, and murine retroviruses. Heterologous antiserum prepared against a 30-amino acid synthetic peptide analogue (HGP-30) does not cross-react with thymosin α 1 but does react specifically with the p17 protein of the AIDS virus in a manner identical to that seen with an HTLV-IIIB p17-specific monoclonal antibody. The demonstration that this synthetic analogue is immunogenic and that antibodies to HGP-30 cross-react not only with synthetic peptide but also with the HTLV-IIIB p17 viral protein provides an additional, and potentially more specific, candidate for development of a synthetic peptide vaccine for AIDS. In addition, the p17 synthetic peptide (HGP-3) may prove to be useful in a diagnostic assay for the detection of AIDS virus infection in seronegative individuals

  17. HLArestrictor-a tool for patient-specific predictions of HLA restriction elements and optimal epitopes within peptides

    DEFF Research Database (Denmark)

    Larsen, Malene Erup; Kloverpris, H.; Stryhn, A.

    2011-01-01

    ://www.cbs.dtu.dk/services/HLArestrictor ), which is based on the highly versatile and accurate NetMHCpan predictor, which here has been optimized for the identification of both the MHC restriction element and the corresponding minimal epitope of a T cell response in a given individual. As input, it requires high-resolution (i.e., 4-digit) HLA...... HLA restrictions and minimal epitopes for about 90% of the positive peptide/patient pairs while rejecting more than 95% of the negative peptide-HLA pairs. Furthermore, for 18 peptide/HLA tetramer validated responses, HLArestrictor in all cases predicted both the HLA restriction element and minimal...

  18. Discovery of Specific Inhibitors for Intestinal E. coli  β-Glucuronidase through In Silico Virtual Screening

    Directory of Open Access Journals (Sweden)

    Ta-Chun Cheng

    2015-01-01

    Full Text Available Glucuronidation is a major metabolism process of detoxification for carcinogens, 4-(methylnitrosamino-1-(3-pyridy-1-butanone (NNK and 1,2-dimethylhydrazine (DMH, of reactive oxygen species (ROS. However, intestinal E. coli   β-glucuronidase (eβG has been considered pivotal to colorectal carcinogenesis. Specific inhibition of eβG may prevent reactivating the glucuronide-carcinogen and protect the intestine from ROS-mediated carcinogenesis. In order to develop specific eβG inhibitors, we found that 59 candidate compounds obtained from the initial virtual screening had high inhibition specificity against eβG but not human βG. In particular, we found that compounds 7145 and 4041 with naphthalenylidene-benzenesulfonamide (NYBS are highly effective and selective to inhibit eβG activity. Compound 4041  (IC50=2.8 μM shows a higher inhibiting ability than compound 7145  (IC50=31.6 μM against eβG. Furthermore, the molecular docking analysis indicates that compound 4041 has two hydrophobic contacts to residues L361 and I363 in the bacterial loop, but 7145 has one contact to L361. Only compound 4041 can bind to key residue (E413 at active site of eβG via hydrogen-bonding interactions. These novel NYBS-based eβG specific inhibitors may provide as novel candidate compounds, which specifically inhibit eβG to reduce eβG-based carcinogenesis and intestinal injury.

  19. 'Multi-epitope-targeted' immune-specific therapy for a multiple sclerosis-like disease via engineered multi-epitope protein is superior to peptides.

    Directory of Open Access Journals (Sweden)

    Nathali Kaushansky

    Full Text Available Antigen-induced peripheral tolerance is potentially one of the most efficient and specific therapeutic approaches for autoimmune diseases. Although highly effective in animal models, antigen-based strategies have not yet been translated into practicable human therapy, and several clinical trials using a single antigen or peptidic-epitope in multiple sclerosis (MS yielded disappointing results. In these clinical trials, however, the apparent complexity and dynamics of the pathogenic autoimmunity associated with MS, which result from the multiplicity of potential target antigens and "epitope spread", have not been sufficiently considered. Thus, targeting pathogenic T-cells reactive against a single antigen/epitope is unlikely to be sufficient; to be effective, immunospecific therapy to MS should logically neutralize concomitantly T-cells reactive against as many major target antigens/epitopes as possible. We investigated such "multi-epitope-targeting" approach in murine experimental autoimmune encephalomyelitis (EAE associated with a single ("classical" or multiple ("complex" anti-myelin autoreactivities, using cocktail of different encephalitogenic peptides vis-a-vis artificial multi-epitope-protein (designated Y-MSPc encompassing rationally selected MS-relevant epitopes of five major myelin antigens, as "multi-epitope-targeting" agents. Y-MSPc was superior to peptide(s in concomitantly downregulating pathogenic T-cells reactive against multiple myelin antigens/epitopes, via inducing more effective, longer lasting peripheral regulatory mechanisms (cytokine shift, anergy, and Foxp3+ CTLA4+ regulatory T-cells. Y-MSPc was also consistently more effective than the disease-inducing single peptide or peptide cocktail, not only in suppressing the development of "classical" or "complex EAE" or ameliorating ongoing disease, but most importantly, in reversing chronic EAE. Overall, our data emphasize that a "multi-epitope-targeting" strategy is required for

  20. Dissection of the BCR-ABL signaling network using highly specific monobody inhibitors to the SHP2 SH2 domains.

    Science.gov (United States)

    Sha, Fern; Gencer, Emel Basak; Georgeon, Sandrine; Koide, Akiko; Yasui, Norihisa; Koide, Shohei; Hantschel, Oliver

    2013-09-10

    The dysregulated tyrosine kinase BCR-ABL causes chronic myelogenous leukemia in humans and forms a large multiprotein complex that includes the Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2). The expression of SHP2 is necessary for BCR-ABL-dependent oncogenic transformation, but the precise signaling mechanisms of SHP2 are not well understood. We have developed binding proteins, termed monobodies, for the N- and C-terminal SH2 domains of SHP2. Intracellular expression followed by interactome analysis showed that the monobodies are essentially monospecific to SHP2. Two crystal structures revealed that the monobodies occupy the phosphopeptide-binding sites of the SH2 domains and thus can serve as competitors of SH2-phosphotyrosine interactions. Surprisingly, the segments of both monobodies that bind to the peptide-binding grooves run in the opposite direction to that of canonical phosphotyrosine peptides, which may contribute to their exquisite specificity. When expressed in cells, monobodies targeting the N-SH2 domain disrupted the interaction of SHP2 with its upstream activator, the Grb2-associated binder 2 adaptor protein, suggesting decoupling of SHP2 from the BCR-ABL protein complex. Inhibition of either N-SH2 or C-SH2 was sufficient to inhibit two tyrosine phosphorylation events that are critical for SHP2 catalytic activity and to block ERK activation. In contrast, targeting the N-SH2 or C-SH2 revealed distinct roles of the two SH2 domains in downstream signaling, such as the phosphorylation of paxillin and signal transducer and activator of transcription 5. Our results delineate a hierarchy of function for the SH2 domains of SHP2 and validate monobodies as potent and specific antagonists of protein-protein interactions in cancer cells.

  1. Dendritic cells pulsed with a tumor-specific peptide induce long-lasting immunity and are effective against murine intracerebral melanoma.

    Science.gov (United States)

    Heimberger, Amy B; Archer, Gary E; Crotty, Laura E; McLendon, Roger E; Friedman, Allan H; Friedman, Henry S; Bigner, Darell D; Sampson, John H

    2002-01-01

    Dendritic cells (DCs) are specialized cells of the immune system that are capable of generating potent immune responses that are active even within the "immunologically privileged" central nervous system. However, immune responses generated by DCs have also been demonstrated to produce clinically significant autoimmunity. Targeting the epidermal growth factor receptor variant III (EGFRvIII), which is a mutation specific to tumor tissue, could eliminate this risk. The purpose of this study was to demonstrate that DC-based immunizations directed solely against this tumor-specific antigen, which is commonly found on tumors that originate within or metastasize to the brain, could be efficacious. C3H mice were vaccinated with DCs mixed with a keyhole limpet hemocyanin conjugate of the tumor-specific peptide, PEP-3, which spans the EGFRvIII mutation, or the random-sequence peptide, PEP-1, and were intracerebrally challenged with a syngeneic melanoma expressing a murine homologue of EGFRvIII. Systemic immunization with DCs mixed with PEP-3-keyhole limpet hemocyanin generated antigen-specific immunity. Among mice challenged with intracerebral tumors, this resulted in an approximately 600% increase in the median survival time (>300 d, P < 0.0016), relative to control values. Sixty-three percent of mice treated with DCs mixed with the tumor-specific peptide survived in the long term and 100% survived rechallenge with tumor, indicating that antitumor immunological memory was also induced. In a murine melanoma model, immunization with DCs mixed with tumor-specific peptide results in an antigen-specific immunological response that recognizes the EGFRvIII mutation, has potent antitumor efficacy against intracerebral tumors that express EGFRvIII, and results in long-lasting antitumor immunity.

  2. Evaluation of peptide designing strategy against subunit reassociation in mucin 1: A steered molecular dynamics approach.

    Directory of Open Access Journals (Sweden)

    J Lesitha Jeeva Kumari

    Full Text Available Subunit reassociation in mucin 1, a breast cancer tumor marker, is reported as one of the critical factors for its cytoplasmic activation. Inhibition of its heterodimeric association would therefore result in loss of its function and alter disease progression. The present study aimed at evaluating peptide inhibitor designing strategies that may serve as antagonist against this receptor-ligand alliance. Several peptides and their derivatives were designed based on native residues, subunit interface, hydrogen bonding and secondary structure. Docking studies with the peptides were carried on the receptor subunit and their binding affinities were evaluated using steered molecular dynamics simulation and umbrella sampling. Our results showed that among all the different classes of peptides evaluated, the receptor based peptide showed the highest binding affinity. This result was concurrent with the experimental observation that the receptor-ligand alliance in mucin 1 is highly specific. Our results also show that peptide ligand against this subunit association is only stabilized through native residue inter-protein interaction irrespective of the peptide structure, peptide length and number of hydrogen bonds. Consistency in binding affinity, pull force and free energy barrier was observed with only the receptor derived peptides which resulted in favorable interprotein interactions at the interface. Several observations were made and discussed which will eventually lead to designing efficient peptide inhibitors against mucin 1 heterodimeric subunit reassociation.

  3. Tracking of peptide-specific CD4+ T-cell responses after an acute resolving viral infection: a study of parvovirus B19

    DEFF Research Database (Denmark)

    Kasprowicz, Victoria; Isa, Adiba; Tolfvenstam, Thomas

    2006-01-01

    The evolution of peptide-specific CD4(+) T-cell responses to acute viral infections of humans is poorly understood. We analyzed the response to parvovirus B19 (B19), a ubiquitous and clinically significant pathogen with a compact and conserved genome. The magnitude and breadth of the CD4(+) T......-cell response to the two B19 capsid proteins were investigated using a set of overlapping peptides and gamma interferon-specific enzyme-linked immunospot assays of peripheral blood mononuclear cells (PBMCs) from a cohort of acutely infected individuals who presented with acute arthropathy. These were compared...... to those for a cohort of B19-specific immunoglobulin M-negative (IgM(-)), IgG(+) remotely infected individuals. Both cohorts of individuals were found to make broad CD4(+) responses. However, while the responses following acute infection were detectable ex vivo, responses in remotely infected individuals...

  4. Peptides of presenilin-1 bind the amyloid precursor protein ectodomain and offer a novel and specific therapeutic approach to reduce ß-amyloid in Alzheimer's disease.

    Science.gov (United States)

    Dewji, Nazneen N; Singer, S Jonathan; Masliah, Eliezer; Rockenstein, Edward; Kim, Mihyun; Harber, Martha; Horwood, Taylor

    2015-01-01

    β-Amyloid (Aβ) accumulation in the brain is widely accepted to be critical to the development of Alzheimer's disease (AD). Current efforts at reducing toxic Aβ40 or 42 have largely focused on modulating γ-secretase activity to produce shorter, less toxic Aβ, while attempting to spare other secretase functions. In this paper we provide data that offer the potential for a new approach for the treatment of AD. The method is based on our previous findings that the production of Aβ from the interaction between the β-amyloid precursor protein (APP) and Presenilin (PS), as part of the γ-secretase complex, in cell culture is largely inhibited if the entire water-soluble NH2-terminal domain of PS is first added to the culture. Here we demonstrate that two small, non-overlapping water-soluble peptides from the PS-1 NH2-terminal domain can substantially and specifically inhibit the production of total Aβ as well as Aβ40 and 42 in vitro and in vivo in the brains of APP transgenic mice. These results suggest that the inhibitory activity of the entire amino terminal domain of PS-1 on Aβ production is largely focused in a few smaller sequences within that domain. Using biolayer interferometry and confocal microscopy we provide evidence that peptides effective in reducing Aβ give a strong, specific and biologically relevant binding with the purified ectodomain of APP 695. Finally, we demonstrate that the reduction of Aβ by the peptides does not affect the catalytic activities of β- or γ-secretase, or the level of APP. P4 and P8 are the first reported protein site-specific small peptides to reduce Aβ production in model systems of AD. These peptides and their derivatives offer new potential drug candidates for the treatment of AD.

  5. Peptides of presenilin-1 bind the amyloid precursor protein ectodomain and offer a novel and specific therapeutic approach to reduce ß-amyloid in Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Nazneen N Dewji

    Full Text Available β-Amyloid (Aβ accumulation in the brain is widely accepted to be critical to the development of Alzheimer's disease (AD. Current efforts at reducing toxic Aβ40 or 42 have largely focused on modulating γ-secretase activity to produce shorter, less toxic Aβ, while attempting to spare other secretase functions. In this paper we provide data that offer the potential for a new approach for the treatment of AD. The method is based on our previous findings that the production of Aβ from the interaction between the β-amyloid precursor protein (APP and Presenilin (PS, as part of the γ-secretase complex, in cell culture is largely inhibited if the entire water-soluble NH2-terminal domain of PS is first added to the culture. Here we demonstrate that two small, non-overlapping water-soluble peptides from the PS-1 NH2-terminal domain can substantially and specifically inhibit the production of total Aβ as well as Aβ40 and 42 in vitro and in vivo in the brains of APP transgenic mice. These results suggest that the inhibitory activity of the entire amino terminal domain of PS-1 on Aβ production is largely focused in a few smaller sequences within that domain. Using biolayer interferometry and confocal microscopy we provide evidence that peptides effective in reducing Aβ give a strong, specific and biologically relevant binding with the purified ectodomain of APP 695. Finally, we demonstrate that the reduction of Aβ by the peptides does not affect the catalytic activities of β- or γ-secretase, or the level of APP. P4 and P8 are the first reported protein site-specific small peptides to reduce Aβ production in model systems of AD. These peptides and their derivatives offer new potential drug candidates for the treatment of AD.

  6. Use of a specific MMP inhibitor (Galardin) for preservation of hybrid layer

    Science.gov (United States)

    Breschi, Lorenzo; Martin, Patrizia; Mazzoni, Annalisa; Nato, Fernando; Carrilho, Marcela; Tjäderhane, Leo; Visintini, Erika; Cadenaro, Milena; Tay, Franklin R; De Stefano Dorigo, Elettra; Pashley, David H

    2013-01-01

    Objective Dentinal MMPs have been claimed to contribute to the auto-degradation of collagen fibrils within incompletely resin-infiltrated hybrid layers and their inhibition may, therefore, slow the degradation of hybrid layer. This study aimed to determine the contribution of a synthetic MMPs inhibitor (Galardin) to the proteolytic activity of dentinal MMPs and to the morphological and mechanical features of hybrid layers after aging. Methods Dentin powder obtained from human molars was treated with Galardin or chlorhexidine digluconate and zymographically analyzed. Microtensile bond strength was also evaluated in extracted human teeth. Exposed dentin was etched with 35% phosphoric acid and specimens were assigned to (1) pre-treatment with Galardin as additional primer for 30s; (2) no pre-treatment. A two-step etch-and-rinse adhesive (Adper Scotchbond 1XT, 3M ESPE) was then applied in accordance with manufacturer's instructions and resin composite build-ups were created. Specimens were immediately tested for their microtensile bond strength or stored in artificial saliva for 12 months prior to being tested. Data were evaluated by two-way ANOVA and Tukey's tests (〈=0.05). Additional specimens were prepared for interfacial nanoleakage analysis under light microscopy and TEM, quantified by two independent observers and statistically analyzed (|2 test, 〈=0.05). Results The inhibitory effect of Galardin on dentinal MMPs was confirmed by zymographic analysis, as complete inhibition of both MMP-2 and -9 was observed. The use of Galardin had no effect on immediate bond strength, while it significantly decreased bond degradation after 1 year (padhesive after artificial aging. PMID:20299089

  7. HAEdb: a novel interactive, locus-specific mutation database for the C1 inhibitor gene.

    Science.gov (United States)

    Kalmár, Lajos; Hegedüs, Tamás; Farkas, Henriette; Nagy, Melinda; Tordai, Attila

    2005-01-01

    Hereditary angioneurotic edema (HAE) is an autosomal dominant disorder characterized by episodic local subcutaneous and submucosal edema and is caused by the deficiency of the activated C1 esterase inhibitor protein (C1-INH or C1INH; approved gene symbol SERPING1). Published C1-INH mutations are represented in large universal databases (e.g., OMIM, HGMD), but these databases update their data rather infrequently, they are not interactive, and they do not allow searches according to different criteria. The HAEdb, a C1-INH gene mutation database (http://hae.biomembrane.hu) was created to contribute to the following expectations: 1) help the comprehensive collection of information on genetic alterations of the C1-INH gene; 2) create a database in which data can be searched and compared according to several flexible criteria; and 3) provide additional help in new mutation identification. The website uses MySQL, an open-source, multithreaded, relational database management system. The user-friendly graphical interface was written in the PHP web programming language. The website consists of two main parts, the freely browsable search function, and the password-protected data deposition function. Mutations of the C1-INH gene are divided in two parts: gross mutations involving DNA fragments >1 kb, and micro mutations encompassing all non-gross mutations. Several attributes (e.g., affected exon, molecular consequence, family history) are collected for each mutation in a standardized form. This database may facilitate future comprehensive analyses of C1-INH mutations and also provide regular help for molecular diagnostic testing of HAE patients in different centers.

  8. Beta-blockers influence the short-term and long-term prognostic information of natriuretic peptides and catecholamines in chronic heart failure independent from specific agents.

    Science.gov (United States)

    Frankenstein, Lutz; Nelles, Manfred; Slavutsky, Maxim; Schellberg, Dieter; Doesch, Andreas; Katus, Hugo; Remppis, Andrew; Zugck, Christian

    2007-10-01

    In chronic heart failure (CHF), the physiologic effects of natriuretic peptides and catecholamines are interdependent. Furthermore, reports state an agent-dependent effect of individual beta-blockers on biomarkers. Data on the short-term and long-term predictive power comparing these biomarkers as well as accounting for the influence of beta-blocker treatment both on the marker or the resultant prognostic information are scarce. We included 513 consecutive patients with systolic CHF, measured atrial natriuretic peptide (ANP), N-terminal prohormone brain natriuretic peptide (NTproBNP), noradrenaline, and adrenaline, and monitored them for 90 +/- 25 months. Death or the combination of death and cardiac transplantation at 1 year, 5 years, and overall follow-up were considered end points. Compared with patients not taking beta-blockers, patients taking beta-blockers had significantly lower levels of catecholamines but not natriuretic peptides. Only for adrenaline was the amount of this effect related to the specific beta-blocker chosen. Receiver operating characteristic curves demonstrated superior prognostic accuracy for NTproBNP both at the 1- and 5-year follow-up compared with ANP, noradrenaline, and adrenaline. In multivariate analysis including established risk markers (New York Heart Association functional class, left ventricular ejection fraction, peak oxygen uptake, and 6-minute walk test), of all neurohumoral parameters, only NTproBNP remained an independent predictor for both end points. Long-term beta-blocker therapy is associated with decreased levels of plasma catecholamines but not natriuretic peptides. This effect is independent from the actual beta-blocker chosen for natriuretic peptides and noradrenaline. In multivariate analysis, both for short-term and long-term prediction of mortality or the combined end point of death and cardiac transplantation, only NTproBNP remained independent from established clinical risk markers.

  9. Artificial 64-Residue HIV-1 Enhancer-Binding Peptide Is a Potent Inhibitor of Viral Replication in HIV-1-Infected Cells.

    Science.gov (United States)

    Oufir, Mouhssin; Bisset, Leslie R; Hoffmann, Stefan R K; Xue, Gongda; Klauser, Stephan; Bergamaschi, Bianca; Gervaix, Alain; Böni, Jürg; Schüpbach, Jörg; Gutte, Bernd

    2011-01-01

    An artificial HIV-1 enhancer-binding peptide was extended by nine consecutive arginine residues at the C-terminus and by the nuclear localization signal of SV40 large T antigen at the N-terminus. The resulting synthetic 64-residue peptide was found to bind to the two enhancers of the HIV-1 long terminal repeat, cross the plasma membrane and the nuclear envelope of human cells, and suppress the HIV-1 enhancer-controlled expression of a green fluorescent protein reporter gene. Moreover, HIV-1 replication is inhibited by this peptide in HIV-1-infected CEM-GFP cells as revealed by HIV-1 p24 ELISA and real-time RT-PCR of HIV-1 RNA. Rapid uptake of this intracellular stable and inhibitory peptide into the cells implies that this peptide may have the potential to attenuate HIV-1 replication in vivo.

  10. Artificial 64-Residue HIV-1 Enhancer-Binding Peptide Is a Potent Inhibitor of Viral Replication in HIV-1-Infected Cells

    Directory of Open Access Journals (Sweden)

    Mouhssin Oufir

    2011-01-01

    Full Text Available An artificial HIV-1 enhancer-binding peptide was extended by nine consecutive arginine residues at the C-terminus and by the nuclear localization signal of SV40 large T antigen at the N-terminus. The resulting synthetic 64-residue peptide was found to bind to the two enhancers of the HIV-1 long terminal repeat, cross the plasma membrane and the nuclear envelope of human cells, and suppress the HIV-1 enhancer-controlled expression of a green fluorescent protein reporter gene. Moreover, HIV-1 replication is inhibited by this peptide in HIV-1-infected CEM-GFP cells as revealed by HIV-1 p24 ELISA and real-time RT-PCR of HIV-1 RNA. Rapid uptake of this intracellular stable and inhibitory peptide into the cells implies that this peptide may have the potential to attenuate HIV-1 replication in vivo.

  11. Strategies to overcome HBV-specific T cell exhaustion: checkpoint inhibitors and metabolic re-programming.

    Science.gov (United States)

    Fisicaro, Paola; Boni, Carolina; Barili, Valeria; Laccabue, Diletta; Ferrari, Carlo

    2018-01-29

    HBV-specific T cells play a key role in antiviral protection and failure to control HBV is associated with severely dysfunctional T cell responses. Therefore, functional T cell reconstitution represents a potential way to treat chronically infected patients. The growing understanding of the dysregulated transcriptional/epigenetic and metabolic programs underlying T cell exhaustion allows to envisage functional T cell reconstitution strategies based on the combined/sequential use of compounds able to induce decline of antigen load, checkpoint modulation, metabolic and epigenetic reprogramming with possible boosting of functionally restored responses by specific vaccines. Copyright © 2018 Elsevier B.V. All rights reserved.

  12. Caracemide, a site-specific irreversible inhibitor of protein R1 of Escherichia coli ribonucleotide reductase

    DEFF Research Database (Denmark)

    Larsen, I. K.; Cornett, Claus; Karlsson, M.

    1992-01-01

    The anticancer drug caracemide, N-acetyl-N,O-di(methylcarbamoyl)hydroxylamine, and one of its degradation products, N-acetyl-O-methylcarbamoyl-hydroxylamine, were found to inhibit the enzyme ribonucleotide reductase of Escherichia coli by specific interaction with its larger component protein R1....

  13. A specific scenario for the origin of life and the genetic code based on peptide/oligonucleotide interdependence.

    Science.gov (United States)

    Griffith, Robert W

    2009-12-01

    Among various scenarios that attempt to explain how life arose, the RNA world is currently the most widely accepted scientific hypothesis among biologists. However, the RNA world is logistically implausible and doesn't explain how translation arose and DNA became incorporated into living systems. Here I propose an alternative hypothesis for life's origin based on cooperation between simple nucleic acids, peptides and lipids. Organic matter that accumulated on the prebiotic Earth segregated into phases in the ocean based on density and solubility. Synthesis of complex organic monomers and polymerization reactions occurred within a surface hydrophilic layer and at its aqueous and atmospheric interfaces. Replication of nucleic acids and translation of peptides began at the emulsified interface between hydrophobic and aqueous layers. At the core of the protobiont was a family of short nucleic acids bearing arginine's codon and anticodon that added this amino acid to pre-formed peptides. In turn, the survival and replication of nucleic acid was aided by the peptides. The arginine-enriched peptides served to sequester and transfer phosphate bond energy and acted as cohesive agents, aggregating nucleic acids and keeping them at the interface.

  14. Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

    International Nuclear Information System (INIS)

    Fernández-Sainz, I.J.; Largo, E.; Gladue, D.P.; Fletcher, P.; O’Donnell, V.; Holinka, L.G.; Carey, L.B.; Lu, X.; Nieva, J.L.; Borca, M.V.

    2014-01-01

    E2, along with E rns and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions: cell attachment, host range susceptibility and virulence in natural hosts. Here we evaluate the role of a specific E2 region, 818 CPIGWTGVIEC 828 , containing a putative fusion peptide (FP) sequence. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how individual amino acid substitutions within this region of E2 may affect replication of BICv. A synthetic peptide representing the complete E2 FP amino acid sequence adopted a β-type extended conformation in membrane mimetics, penetrated into model membranes, and perturbed lipid bilayer integrity in vitro. Similar peptides harboring amino acid substitutions adopted comparable conformations but exhibited different membrane activities. Therefore, a preliminary characterization of the putative FP 818 CPIGWTGVIEC 828 indicates a membrane fusion activity and a critical role in virus replication. - Highlights: • A putative fusion peptide (FP) region in CSFV E2 protein was shown to be critical for virus growth. • Synthetic FPs were shown to efficiently penetrate into lipid membranes using an in vitro model. • Individual residues in the FP affecting virus replication were identified by reverse genetics. • The same FP residues are also responsible for mediating membrane fusion

  15. Efficient preparation of {sup 99m}Tc(III) '4+1' mixed-ligand complexes for peptide labeling with high specific activity

    Energy Technology Data Exchange (ETDEWEB)

    Kunstler, Jens-Uwe [Biotectid GmbH, Deutscher Platz 5c, 04103 Leipzig (Germany); Seidel, Gesine [Institute of Radiopharmacy, Forschungszentrum Dresden-Rossendorf, P.O. Box 510 119, 01314 Dresden (Germany); Pietzsch, Hans-Jurgen, E-mail: h.j.pietzsch@fzd.d [Institute of Radiopharmacy, Forschungszentrum Dresden-Rossendorf, P.O. Box 510 119, 01314 Dresden (Germany)

    2010-09-15

    An improved labeling procedure for peptides attached to organometallic {sup 99m}Tc(III) '4+1' mixed-ligand complexes in which the radiometal is coordinated by a tripodal tetradentate chelator 2,2',2''-nitrilotriethanethiol (NS{sub 3}) and a monodentate isocyanide ligand is presented. The labeling procedure was evaluated by the synthesis of [{sup 99m}Tc(NS{sub 3})(L2-RGD)]. The containing radiopharmaceutically interesting RGD-peptide cyclo[Arg-Gly-Asp-D-Tyr-Lys] was modified with 4-isocyanobutanoic acid (L2) as linker conjugated to N{sup 6}-Lys to get the monodentate ligand L2-RGD. The structural identity of the {sup 99m}Tc-conjugate was confirmed by comparison to a Re reference compound. The Tc- and Re-conjugates had matching retention times under identical HPLC conditions. The {sup 99m}Tc-labeling was performed in a novel one-step procedure using the eluate of a {sup 99}Mo/{sup 99m}Tc generator, NS{sub 3}, the isocyanide modified peptide, SnCl{sub 2}, Na{sub 2}EDTA, mannitol and ascorbic acid in the reaction mixture. Using optimized reagents it is possible to label 50 nmol peptide with {sup 99m}Tc within 60 min at room temperature with a radiochemical yield higher than 95% and a specific activity of {approx}20 GBq/{mu}mol.

  16. Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

    Energy Technology Data Exchange (ETDEWEB)

    Fernández-Sainz, I.J. [Plum Island Animal Disease Center, ARS, USDA (United States); Largo, E. [Biophysics Unit (CSIC-UPV/EHU), Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), P.O. Box 644, 48080 Bilbao (Spain); Gladue, D.P.; Fletcher, P. [Plum Island Animal Disease Center, ARS, USDA (United States); O’Donnell, V. [Plum Island Animal Disease Center, ARS, USDA (United States); Plum Island Animal Disease Center, DHS, Greenport, NY 11944 (United States); Holinka, L.G. [Plum Island Animal Disease Center, ARS, USDA (United States); Carey, L.B. [Department of Experimental and Health Sciences, Universitat Pompeu Fabra (UPF), E-08003 Barcelona (Spain); Lu, X. [Plum Island Animal Disease Center, DHS, Greenport, NY 11944 (United States); Nieva, J.L. [Biophysics Unit (CSIC-UPV/EHU), Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), P.O. Box 644, 48080 Bilbao (Spain); Borca, M.V., E-mail: manuel.borca@ars.usda.gov [Plum Island Animal Disease Center, ARS, USDA (United States)

    2014-05-15

    E2, along with E{sup rns} and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions: cell attachment, host range susceptibility and virulence in natural hosts. Here we evaluate the role of a specific E2 region, {sup 818}CPIGWTGVIEC{sup 828}, containing a putative fusion peptide (FP) sequence. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how individual amino acid substitutions within this region of E2 may affect replication of BICv. A synthetic peptide representing the complete E2 FP amino acid sequence adopted a β-type extended conformation in membrane mimetics, penetrated into model membranes, and perturbed lipid bilayer integrity in vitro. Similar peptides harboring amino acid substitutions adopted comparable conformations but exhibited different membrane activities. Therefore, a preliminary characterization of the putative FP {sup 818}CPIGWTGVIEC{sup 828} indicates a membrane fusion activity and a critical role in virus replication. - Highlights: • A putative fusion peptide (FP) region in CSFV E2 protein was shown to be critical for virus growth. • Synthetic FPs were shown to efficiently penetrate into lipid membranes using an in vitro model. • Individual residues in the FP affecting virus replication were identified by reverse genetics. • The same FP residues are also responsible for mediating membrane fusion.

  17. The v-Src and c-Src tyrosine kinases immunoprecipitated from Rous sarcoma virus-transformed cells display different peptide substrate specificities

    Czech Academy of Sciences Publication Activity Database

    Vojtěchová, Martina; Tuháčková, Zdena; Hlaváček, Jan; Velek, Jiří; Sovová, Vlasta

    2004-01-01

    Roč. 421, č. 2 (2004), s. 277-282 ISSN 0003-9861 R&D Projects: GA ČR GV312/96/K205; GA ČR GA301/00/0269 Institutional research plan: CEZ:AV0Z4055905; CEZ:AV0Z1003909 Keywords : Src kinase, in vitro phosphorylation, peptide substrate specificity Subject RIV: CE - Biochemistry Impact factor: 2.657, year: 2004

  18. A new generation of topical chronic wound treatments containing specific MMP inhibitors

    Directory of Open Access Journals (Sweden)

    Shrivastava R

    2014-09-01

    Full Text Available Ravi Shrivastava, Nathalie Cucuat, Monika Rousse, Thomas Weigand, Pedro Neto, Claire Janicot, Christiane Shrivastava VITROBIO Research Institute, Issoire, France Purpose: Incidence of chronic wounds is constantly rising worldwide, but all currently available treatments are intended either to provide symptomatic relief or to assist cicatrization to some extent, but not to directly stimulate cellular growth. Physiologically, chronic wound healing simply requires cell growth to fill the injured cavity. To grow, our cells need to attach onto a cushion, called extracellular matrix (ECM, secreted by the mother cells and composed of multiple proteins. Recent scientific works prove that the concentration of certain matrix metalloproteinases (MMPs is extremely high in all chronic wounds and, because of their proteolytic nature, some MMPs completely degrade the ECM, hindering cell attachment and cell growth. The aim of this study was to identify, neutralize, and eliminate these MMPs from the wound surface so as to design an effective treatment for chronic wounds. Methods: Acute and chronic models of human epithelial and fibroblast cells were prepared on a defined ECM cushion in vitro and MMPs were added in the culture medium to identify the MMPs causing ECM disintegration for each cell type. ECM-degrading MMPs were then incubated with selected procyanidin-rich plant extracts (PCDs and cell growth was reanalyzed. Results: It was observed that: 1 multiple MMPs are involved in cellular matrix destruction; 2 ECM-destroying MMPs are specific with respect to cell type; and 3 specific PCDs may bind and neutralize selected MMPs. Conclusion: Topical application of specific plant PCDs to selectively neutralize ECM-destroying MMPs in acute and chronic wounds represents a novel approach for the treatment of superficial and deep skin wounds. Keywords: extra cellular matrix (ECM, matrix metalloproteinases, procyanidins (PCDs, tannins, ulcers

  19. Substrate Specificity and Inhibitor Sensitivity of Plant UDP-Sugar Producing Pyrophosphorylases

    Directory of Open Access Journals (Sweden)

    Daniel Decker

    2017-09-01

    Full Text Available UDP-sugars are essential precursors for glycosylation reactions producing cell wall polysaccharides, sucrose, glycoproteins, glycolipids, etc. Primary mechanisms of UDP sugar formation involve the action of at least three distinct pyrophosphorylases using UTP and sugar-1-P as substrates. Here, substrate specificities of barley and Arabidopsis (two isozymes UDP-glucose pyrophosphorylases (UGPase, Arabidopsis UDP-sugar pyrophosphorylase (USPase and Arabidopsis UDP-N-acetyl glucosamine pyrophosphorylase2 (UAGPase2 were investigated using a range of sugar-1-phosphates and nucleoside-triphosphates as substrates. Whereas all the enzymes preferentially used UTP as nucleotide donor, they differed in their specificity for sugar-1-P. UGPases had high activity with D-Glc-1-P, but could also react with Fru-1-P and Fru-2-P (Km values over 10 mM. Contrary to an earlier report, their activity with Gal-1-P was extremely low. USPase reacted with a range of sugar-1-phosphates, including D-Glc-1-P, D-Gal-1-P, D-GalA-1-P (Km of 1.3 mM, β-L-Ara-1-P and α-D-Fuc-1-P (Km of 3.4 mM, but not β-L-Fuc-1-P. In contrast, UAGPase2 reacted only with D-GlcNAc-1-P, D-GalNAc-1-P (Km of 1 mM and, to some extent, D-Glc-1-P (Km of 3.2 mM. Generally, different conformations/substituents at C2, C4, and C5 of the pyranose ring of a sugar were crucial determinants of substrate specificity of a given pyrophosphorylase. Homology models of UDP-sugar binding to UGPase, USPase and UAGPase2 revealed more common amino acids for UDP binding than for sugar binding, reflecting differences in substrate specificity of these proteins. UAGPase2 was inhibited by a salicylate derivative that was earlier shown to affect UGPase and USPase activities, consistent with a common structural architecture of the three pyrophosphorylases. The results are discussed with respect to the role of the pyrophosphorylases in sugar activation for glycosylated end-products.

  20. Seed-Specific Stable Expression of the ?-AI1 Inhibitor in Coffee Grains and the In Vivo Implications for the Development of the Coffee Berry Borer

    OpenAIRE

    Albuquerque, ?rika V. S.; Bezerra, Caroline A.; Romero, Juan V.; Valencia, Jorge W. A.; Valencia-Jim?nez, Arnubio; Pimenta, Lucas M.; Barbosa, Aulus E. A. D.; Silva, Maria C. M.; Meneguim, Ana M.; S?, Maria Eug?nia L.; Engler, Gilbert; de Almeida-Engler, Janice; Fernandez, Diana; Grossi-de-S?, Maria F.

    2015-01-01

    Genetic transformation of coffee (Coffea spp.), the second most traded commodity worldwide, is an alternative approach to introducing features that cannot be introgressed by traditional crossings. The transgenic stability, heritability and quantitative and spatial expression patterns of the seed-specific promoter phytohemagglutinin (PHA-L) from Phaseolus vulgaris were characterized in genetically modified C. arabica expressing the ?-amylase inhibitor-1 (?-AI1) gene. The ?-AI1 inhibitor shows ...

  1. Alignment independent 3D-QSAR, quantum calculations and molecular docking of Mer specific tyrosine kinase inhibitors as anticancer drugs.

    Science.gov (United States)

    Shiri, Fereshteh; Pirhadi, Somayeh; Ghasemi, Jahan B

    2016-03-01

    Mer receptor tyrosine kinase is a promising novel cancer therapeutic target in many human cancers, because abnormal activation of Mer has been implicated in survival signaling and chemoresistance. 3D-QSAR analyses based on alignment independent descriptors were performed on a series of 81 Mer specific tyrosine kinase inhibitors. The fractional factorial design (FFD) and the enhanced replacement method (ERM) were applied and tested as variable selection algorithms for the selection of optimal subsets of molecular descriptors from a much greater pool of such regression variables. The data set was split into 65 molecules as the training set and 16 compounds as the test set. All descriptors were generated by using the GRid INdependent descriptors (GRIND) approach. After variable selection, GRIND were correlated with activity values (pIC50) by PLS regression. Of the two applied variable selection methods, ERM had a noticeable improvement on the statistical parameters of PLS model, and yielded a q (2) value of 0.77, an [Formula: see text] of 0.94, and a low RMSEP value of 0.25. The GRIND information contents influencing the affinity on Mer specific tyrosine kinase were also confirmed by docking studies. In a quantum calculation study, the energy difference between HOMO and LUMO (gap) implied the high interaction of the most active molecule in the active site of the protein. In addition, the molecular electrostatic potential energy at DFT level confirmed results obtained from the molecular docking. The identified key features obtained from the molecular modeling, enabled us to design novel kinase inhibitors.

  2. Enzyme immunoassay for measurement of murine plasminogen activator inhibitor-1, employing a specific antibody produced by the DNA vaccine method.

    Science.gov (United States)

    Yamada, Takayuki; Takagi, Akira; Takeshita, Kyosuke; Yamamoto, Koji; Ito, Masafumi; Matsushita, Tadashi; Murate, Takashi; Saito, Hidehiko; Kojima, Tetsuhito

    2003-01-01

    We developed a sensitive immunoassay to determine the concentration of mouse plasminogen activator inhibitor-1. The assay was a non-competitive sandwich enzyme-linked immunosorbent assay (ELISA) based on the production of a specific polyclonal antibody against mouse plasminogen activator inhibitor type-1 (PAI-1) used both as a trapping and detecting antibody. This antibody was raised in a rabbit by direct introduction of the expression vector plasmid DNA encoding mouse PAI-1, instead of conventional immunization with the purified protein. The standard curve was constructed with a recombinant glutathione S-transferase (GST)-mouse PAI-1 fusion protein (GST-mPAI-1) and dose-response of the assay was linear for GST-mPAI-1 between 6.25 and 100 pM. In order to assess the consistency of the assay, we measured PAI-1 antigen in normal mouse pooled plasma several times. We found that the intra-assay and inter-assay coefficients of variation (CV) were 4.8% and 9.2%, respectively, indicating that the ELISA would be sufficiently repeatable and reproducible. In this assay, lipopolysaccharide (LPS)-injected mice showed substantially higher levels (22-fold) of plasma PAI-1 antigen than did control mice (12.5+/-2.4 vs. 0.58+/-0.16 nM), similar to results reported elsewhere. Taken together, the DNA vaccine method is extremely useful for preparing specific antibodies against mouse PAI-1, which can be utilized to establish the ELISA and analyze the profile of PAI-1 distributions in mice under various conditions. This approach might also be useful for immunological investigation of other coagulation factors and related proteins.

  3. Alignment independent 3D-QSAR, quantum calculations and molecular docking of Mer specific tyrosine kinase inhibitors as anticancer drugs

    Directory of Open Access Journals (Sweden)

    Fereshteh Shiri

    2016-03-01

    Full Text Available Mer receptor tyrosine kinase is a promising novel cancer therapeutic target in many human cancers, because abnormal activation of Mer has been implicated in survival signaling and chemoresistance. 3D-QSAR analyses based on alignment independent descriptors were performed on a series of 81 Mer specific tyrosine kinase inhibitors. The fractional factorial design (FFD and the enhanced replacement method (ERM were applied and tested as variable selection algorithms for the selection of optimal subsets of molecular descriptors from a much greater pool of such regression variables. The data set was split into 65 molecules as the training set and 16 compounds as the test set. All descriptors were generated by using the GRid INdependent descriptors (GRIND approach. After variable selection, GRIND were correlated with activity values (pIC50 by PLS regression. Of the two applied variable selection methods, ERM had a noticeable improvement on the statistical parameters of PLS model, and yielded a q2 value of 0.77, an rpred2 of 0.94, and a low RMSEP value of 0.25. The GRIND information contents influencing the affinity on Mer specific tyrosine kinase were also confirmed by docking studies. In a quantum calculation study, the energy difference between HOMO and LUMO (gap implied the high interaction of the most active molecule in the active site of the protein. In addition, the molecular electrostatic potential energy at DFT level confirmed results obtained from the molecular docking. The identified key features obtained from the molecular modeling, enabled us to design novel kinase inhibitors.

  4. The in vivo disposition and in vitro transmembrane transport of two model radiometabolites of DOTA-conjugated receptor-specific peptides labelled with (177) Lu.

    Science.gov (United States)

    Volková, Marie; Mandíková, Jana; Bárta, Pavel; Navrátilová, Lucie; Lázníčková, Alice; Trejtnar, František

    2015-01-01

    In vivo metabolism of the radiolabelled receptor-specific peptides has been described; however, information regarding the pharmacokinetic behaviour of the degradation products within the body is very scarce. The present study was designed to obtain new knowledge on the disposition and elimination of low-molecular radiometabolites of receptor-specific peptides in the organism and to reveal the potential involvement of selected membrane transport mechanisms in the cellular uptake of radiometabolites, especially in the kidney. The study compared pharmacokinetics of two radiometabolites: a final metabolite of somatostatin analogues, (177)Lu-DOTA-DPhe, and a tripeptide metabolite of (177)Lu-DOTA-minigastrin 11, (177)Lu-DOTA-DGlu-Ala-Tyr. Their pharmacokinetics was compared with that of respective parent (177)Lu-radiopeptide. Both radiometabolites exhibited relative rapid clearing from most body tissues in rats in vivo along with predominant renal excretion. The long-term renal retention of the smaller radiometabolite (177)Lu-DOTA-DPhe was lower than that of (177)Lu-DOTA-DGlu-Ala-Tyr. An uptake of (177)Lu-DOTA-DPhe by human renal influx transporter organic cation transporter 2 was found in vitro in a cellular model. The study brings the first experimental data on the in vivo pharmacokinetics of radiometabolites of receptor-specific somatostatin and gastrin analogues. The found results may indicate a negative correlation between the degree of decomposition of the parent peptide chain and the renal retention of the metabolite. Copyright © 2015 John Wiley & Sons, Ltd.

  5. Clinical-grade generation of peptide-stimulated CMV/EBV-specific T cells from G-CSF mobilized stem cell grafts.

    Science.gov (United States)

    Gary, Regina; Aigner, Michael; Moi, Stephanie; Schaffer, Stefanie; Gottmann, Anja; Maas, Stefanie; Zimmermann, Robert; Zingsem, Jürgen; Strobel, Julian; Mackensen, Andreas; Mautner, Josef; Moosmann, Andreas; Gerbitz, Armin

    2018-05-09

    A major complication after allogeneic hematopoietic stem cell transplantation (aSCT) is the reactivation of herpesviruses such as cytomegalovirus (CMV) and Epstein-Barr virus (EBV). Both viruses cause significant mortality and compromise quality of life after aSCT. Preventive transfer of virus-specific T cells can suppress reactivation by re-establishing functional antiviral immune responses in immunocompromised hosts. We have developed a good manufacturing practice protocol to generate CMV/EBV-peptide-stimulated T cells from leukapheresis products of G-CSF mobilized and non-mobilized donors. Our procedure selectively expands virus-specific CD8+ und CD4+ T cells over 9 days using a generic pool of 34 CMV and EBV peptides that represent well-defined dominant T-cell epitopes with various HLA restrictions. For HLA class I, this set of peptides covers at least 80% of the European population. CMV/EBV-specific T cells were successfully expanded from leukapheresis material of both G-CSF mobilized and non-mobilized donors. The protocol allows administration shortly after stem cell transplantation (d30+), storage over liquid nitrogen for iterated applications, and protection of the stem cell donor by avoiding a second leukapheresis. Our protocol allows for rapid and cost-efficient production of T cells for early transfusion after aSCT as a preventive approach. It is currently evaluated in a phase I/IIa clinical trial.

  6. Heterologous coproduction of enterocin A and pediocin PA-1 by Lactococcus lactis : Detection by specific peptide-directed antibodies

    NARCIS (Netherlands)

    Martinez, JM; Kok, J; Sanders, JW; Hernandez, PE

    Antibodies against enterocin A were obtained by immunization of rabbits with synthetic peptides PH4 and PH5 designed, respectively, on the N- and C-terminal amino acid sequences of enterocin A and conjugated to the carrier protein KLH, Anti-PH4-KLH antibodies not only recognized enterocin A but also

  7. Cell type-specific sorting of neuropeptides : a mechanism to modulate peptide composition of large dense core vesicles

    NARCIS (Netherlands)

    Klumperman, J.; Spijker, S.; Minnen, J. van; Sharp-Baker, H.; Smit, A.B.; Geraerts, W.P.M.

    1996-01-01

    The CNS of Lymnaea stagnalis contains two populations of egg-laying hormone (ELH)-producing neurons that differ in size and topology. In type I neurons, all peptides located C-terminally from the cleavage site Arg-Ser-Arg-Arg180-183 are sorted into secretory large dense-core vesicles (LDCV), whereas

  8. A virosomal formulated Her-2/neu multi-peptide vaccine induces Her-2/neu-specific immune responses in patients with metastatic breast cancer: a phase I study.

    Science.gov (United States)

    Wiedermann, Ursula; Wiltschke, C; Jasinska, J; Kundi, M; Zurbriggen, R; Garner-Spitzer, E; Bartsch, R; Steger, G; Pehamberger, H; Scheiner, O; Zielinski, C C

    2010-02-01

    We have previously shown in mice that vaccination with three Her-2-peptides representing B-cell epitopes of the extracellular domain of Her-2/neu induces Her-2/neu-specific IgG antibodies with strong anti-tumor activity in vitro and in vivo. We have now finalized a phase I clinical trial with an anti-Her-2/neu vaccine-construct of immunopotentiating reconstituted influenza virosomes with the three peptides in patients with metastatic breast cancer (MBC). Ten MBC patients with low protein overexpression of Her-2/neu of MBC (+ or ++ upon immunohistochemistry, FISH negative) and positive hormone receptor status were enrolled in a single center phase I study. The virosomal formulated vaccine, consisting of 10 microg/peptide, was intramuscularly applied three times on days 1, 28, and 56. The primary endpoint of the study, which lasted 12 weeks, was safety, the secondary endpoint immunogenicity. Local erythema at the injection site was the only vaccine-related side effect occurring in four patients. In 8 of 10 patients an increase in peptide-specific antibody titer measured by ELISA was found. Importantly, the induced antibodies were also directed against the native Her-2/neu protein. Cellular immune responses, as measured by in vitro production of IL-2, IFN-c, and TNF-a of PBMCs showed a marked increase after vaccination in the majority of vaccinees. Notably, the number of CD4+CD25+Foxp3+T regulatory cells, which were significantly increased compared to healthy controls prior to vaccination, was markedly reduced following vaccination. In all, the immunological responses after vaccination indicated that the patients in stage IV of disease were immunocompetent and susceptible to vaccination. The Her-2/neu multipeptide vaccine was safe, well tolerated and effective in overcoming immunological tolerance to Her-2/neu. The induction of anti-Her-2-specific antibodies could result in clinical benefit comparable to passive anti-Her-2 antibody therapy.

  9. A novel CXCR4-targeted near-infrared (NIR) fluorescent probe (Peptide R-NIR750) specifically detects CXCR4 expressing tumors.

    Science.gov (United States)

    Santagata, Sara; Portella, Luigi; Napolitano, Maria; Greco, Adelaide; D'Alterio, Crescenzo; Barone, Maria Vittoria; Luciano, Antonio; Gramanzini, Matteo; Auletta, Luigi; Arra, Claudio; Zannetti, Antonella; Scala, Stefania

    2017-05-31

    C-X-C chemokine receptor 4 (CXCR4) is over-expressed in multiple human cancers and correlates with tumor aggressiveness, poor prognosis and increased risk for distant metastases. Imaging agents for CXCR4 are thus highly desirable. We developed a novel CXCR4-targeted near-infrared (NIR) fluorescent probe (Peptide R-NIR750) conjugating the new developed CXCR4 peptidic antagonist Peptide R with the NIR fluorescent dye VivoTag-S750. Specific CXCR4 binding was obtained in cells overexpressing human CXCR4 (B16-hCXCR4 and human melanoma cells PES43), but not in CXCR4 low expressing cells (FB-1). Ex vivo evaluation demonstrated that PepR-NIR750 specifically detects B16-hCXCR4-derived subcutaneous tumors and lung metastases. Fluorescence Molecular Tomography (FMT) in vivo imaging was performed on mice carrying subcutaneous CHO and CHO-CXCR4 tumors. PepR-NIR750 accumulates only in CXCR4-positive expressing subcutaneous tumors. Additionally, an intense NIR fluorescence signal was detected in PES43-derived lung metastases of nude mice injected with PepR-NIR750 versus mice injected with VivoTag-S750. With a therapeutic intent, mice bearing PES43-derived lung metastases were treated with Peptide R. A the dramatic reduction in PES43-derived lung metastases was detected through a decrease of the PepR-NIR750 signal. PepR-NIR750 is a specific probe for non-invasive detection of human high CXCR4-expressing tumors and metastatic lesion and thus a valuable tool for cancer molecular imaging.

  10. Enzyme-linked immunosorbent assay using a virus type-specific peptide based on a subdomain of envelope protein e(rns) for serologic diagnosis of pestivirus infections in swine

    NARCIS (Netherlands)

    Langedijk, J.P.; Middel, W.G.; Meloen, R.H.; Kramps, J.A.; Smit, de J.A.

    2001-01-01

    Peptides deduced from the C-terminal end (residues 191 to 227) of pestivirus envelope protein Erns were used to develop enzyme-linked immunosorbent assays (ELISAs) to measure specifically antibodies against different types of pestiviruses. The choice of the peptide was based on the modular structure

  11. Human leukocyte and porcine pancreatic elastase: X-ray crystal structures, mechanism, substrate specificity, and mechanism-based inhibitors

    International Nuclear Information System (INIS)

    Bode, W.; Meyer, E. Jr.; Powers, J.C.

    1989-01-01

    The serine protease family of enzymes is one of the most widely studied group of enzymes, as evidenced by the fact that more crystal structures are available for individuals of this superfamily than for any other homologous group of enzymes. These enzymes contain a conserved triad of catalytic residues including Ser-195, His-57, and Asp-102. The active-site serine is very nucleophilic, and serine proteases are inhibited by specific serine protease reagents such as diisopropyl phosphorofluoridate (DFP), phenylmethanesulfonyl fluoride, and 3,4-dichloroisocoumarin. Elastases are a group of proteases that possess the ability to cleave the important connective tissue protein elastin. Elastin has the unique property of elastic recoil, is widely distributed in vertebrate tissue, and is particularly abundant in the lungs, arteries, skin, and ligaments. Human neutrophil elastase and pancreatic elastase are two major serine proteases that cleave elastin. Neutrophil elastase is found in the dense granules of polymorphonuclear leukycytes and is essential for phagocytosis and defense against infection by invading microorganisms. Pancreatic elastase is stored as an inactive zymogen in the pancreas and is secreted into the intestines where it becomes activated by trypsin and then participates in digestion. Both elastases cleave substrates at peptide bonds where the P 1 residue is an amino acid residue with a small alkyl side chain

  12. Establishment of the cross-clade antigen detection system for H5 subtype influenza viruses using peptide monoclonal antibodies specific for influenza virus H5 hemagglutinin.

    Science.gov (United States)

    Takahashi, Hitoshi; Nagata, Shiho; Odagiri, Takato; Kageyama, Tsutomu

    2018-04-15

    The H5 subtype of highly pathogenic avian influenza (H5 HPAI) viruses is a threat to both animal and human public health and has the potential to cause a serious future pandemic in humans. Thus, specific and rapid detection of H5 HPAI viruses is required for infection control in humans. To develop a simple and rapid diagnostic system to detect H5 HPAI viruses with high specificity and sensitivity, we attempted to prepare monoclonal antibodies (mAbs) that specifically recognize linear epitopes in hemagglutinin (HA) of H5 subtype viruses. Nine mAb clones were obtained from mice immunized with a synthetic partial peptide of H5 HA molecules conserved among various H5 HPAI viruses. The antigen-capture enzyme-linked immunosorbent assay using the most suitable combination of these mAbs, which bound specifically to lysed H5 HA under an optimized detergent condition, was specific for H5 viruses and could broadly detect H5 viruses in multiple different clades. Taken together, these peptide mAbs, which recognize linear epitopes in a highly conserved region of H5 HA, may be useful for specific and highly sensitive detection of H5 HPAI viruses and can help in the rapid diagnosis of human, avian, and animal H5 virus infections. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. Tofogliflozin, a potent and highly specific sodium/glucose cotransporter 2 inhibitor, improves glycemic control in diabetic rats and mice.

    Science.gov (United States)

    Suzuki, Masayuki; Honda, Kiyofumi; Fukazawa, Masanori; Ozawa, Kazuharu; Hagita, Hitoshi; Kawai, Takahiro; Takeda, Minako; Yata, Tatsuo; Kawai, Mio; Fukuzawa, Taku; Kobayashi, Takamitsu; Sato, Tsutomu; Kawabe, Yoshiki; Ikeda, Sachiya

    2012-06-01

    Sodium/glucose cotransporter 2 (SGLT2) is the predominant mediator of renal glucose reabsorption and is an emerging molecular target for the treatment of diabetes. We identified a novel potent and selective SGLT2 inhibitor, tofogliflozin (CSG452), and examined its efficacy and pharmacological properties as an antidiabetic drug. Tofogliflozin competitively inhibited SGLT2 in cells overexpressing SGLT2, and K(i) values for human, rat, and mouse SGLT2 inhibition were 2.9, 14.9, and 6.4 nM, respectively. The selectivity of tofogliflozin toward human SGLT2 versus human SGLT1, SGLT6, and sodium/myo-inositol transporter 1 was the highest among the tested SGLT2 inhibitors under clinical development. Furthermore, no interaction with tofogliflozin was observed in any of a battery of tests examining glucose-related physiological processes, such as glucose uptake, glucose oxidation, glycogen synthesis, hepatic glucose production, glucose-stimulated insulin secretion, and glucosidase reactions. A single oral gavage of tofogliflozin increased renal glucose clearance and lowered the blood glucose level in Zucker diabetic fatty rats. Tofogliflozin also improved postprandial glucose excursion in a meal tolerance test with GK rats. In db/db mice, 4-week tofogliflozin treatment reduced glycated hemoglobin and improved glucose tolerance in the oral glucose tolerance test 4 days after the final administration. No blood glucose reduction was observed in normoglycemic SD rats treated with tofogliflozin. These findings demonstrate that tofogliflozin inhibits SGLT2 in a specific manner, lowers blood glucose levels by increasing renal glucose clearance, and improves pathological conditions of type 2 diabetes with a low hypoglycemic potential.

  14. MRM screening/biomarker discovery with linear ion trap MS: a library of human cancer-specific peptides

    International Nuclear Information System (INIS)

    Yang, Xu; Lazar, Iulia M

    2009-01-01

    The discovery of novel protein biomarkers is essential in the clinical setting to enable early disease diagnosis and increase survivability rates. To facilitate differential expression analysis and biomarker discovery, a variety of tandem mass spectrometry (MS/MS)-based protein profiling techniques have been developed. For achieving sensitive detection and accurate quantitation, targeted MS screening approaches, such as multiple reaction monitoring (MRM), have been implemented. MCF-7 breast cancer protein cellular extracts were analyzed by 2D-strong cation exchange (SCX)/reversed phase liquid chromatography (RPLC) separations interfaced to linear ion trap MS detection. MS data were interpreted with the Sequest-based Bioworks software (Thermo Electron). In-house developed Perl-scripts were used to calculate the spectral counts and the representative fragment ions for each peptide. In this work, we report on the generation of a library of 9,677 peptides (p < 0.001), representing ~1,572 proteins from human breast cancer cells, that can be used for MRM/MS-based biomarker screening studies. For each protein, the library provides the number and sequence of detectable peptides, the charge state, the spectral count, the molecular weight, the parameters that characterize the quality of the tandem mass spectrum (p-value, DeltaM, Xcorr, DeltaCn, Sp, no. of matching a, b, y ions in the spectrum), the retention time, and the top 10 most intense product ions that correspond to a given peptide. Only proteins identified by at least two spectral counts are listed. The experimental distribution of protein frequencies, as a function of molecular weight, closely matched the theoretical distribution of proteins in the human proteome, as provided in the SwissProt database. The amino acid sequence coverage of the identified proteins ranged from 0.04% to 98.3%. The highest-abundance proteins in the cellular extract had a molecular weight (MW)<50,000. Preliminary experiments have

  15. Can free energy calculations be fast and accurate at the same time? Binding of low-affinity, non-peptide inhibitors to the SH2 domain of the src protein

    Science.gov (United States)

    Chipot, Christophe; Rozanska, Xavier; Dixit, Surjit B.

    2005-11-01

    The usefulness of free-energy calculations in non-academic environments, in general, and in the pharmaceutical industry, in particular, is a long-time debated issue, often considered from the angle of cost/performance criteria. In the context of the rational drug design of low-affinity, non-peptide inhibitors to the SH2 domain of the pp60src tyrosine kinase, the continuing difficulties encountered in an attempt to obtain accurate free-energy estimates are addressed. free-energy calculations can provide a convincing answer, assuming that two key-requirements are fulfilled: (i) thorough sampling of the configurational space is necessary to minimize the statistical error, hence raising the question: to which extent can we sacrifice the computational effort, yet without jeopardizing the precision of the free-energy calculation? (ii) the sensitivity of binding free-energies to the parameters utilized imposes an appropriate parametrization of the potential energy function, especially for non-peptide molecules that are usually poorly described by multipurpose macromolecular force fields. Employing the free-energy perturbation method, accurate ranking, within ±0.7 kcal/mol, is obtained in the case of four non-peptide mimes of a sequence recognized by the pp60src SH2 domain.

  16. Regulation of the number of cell division rounds by tissue-specific transcription factors and Cdk inhibitor during ascidian embryogenesis.

    Directory of Open Access Journals (Sweden)

    Mami Kuwajima

    Full Text Available Mechanisms that regulate the number of cell division rounds during embryogenesis have remained largely elusive. To investigate this issue, we used the ascidian, which develops into a tadpole larva with a small number of cells. The embryonic cells divide 11.45 times on average from fertilization to hatching. The number of cell division rounds varies depending on embryonic lineages. Notochord and muscle consist of large postmitotic cells and stop dividing early in developing embryos. Here we show that conversion of mesenchyme to muscle cell fates by inhibition of inductive FGF signaling or mis-expression of a muscle-specific key transcription factor for muscle differentiation, Tbx6, changed the number of cell divisions in accordance with the altered fate. Tbx6 likely activates a putative mechanism to halt cell division at a specific stage. However, precocious expression of Tbx6 has no effect on progression of the developmental clock itself. Zygotic expression of a cyclin-dependent kinase inhibitor, CKI-b, is initiated in muscle and then in notochord precursors. CKI-b is possibly downstream of tissue-specific key transcription factors of notochord and muscle. In the two distinct muscle lineages, postmitotic muscle cells are generated after 9 and 8 rounds of cell division depending on lineage, but the final cell divisions occur at a similar developmental stage. CKI-b gene expression starts simultaneously in both muscle lineages at the 110-cell stage, suggesting that CKI-b protein accumulation halts cell division at a similar stage. The difference in the number of cell divisions would be due to the cumulative difference in cell cycle length. These results suggest that muscle cells do not count the number of cell division rounds, and that accumulation of CKI-b protein triggered by tissue-specific key transcription factors after cell fate determination might act as a kind of timer that measures elapsed time before cell division termination.

  17. Peptides corresponding to the predicted heptad repeat 2 domain of the feline coronavirus spike protein are potent inhibitors of viral infection.

    Directory of Open Access Journals (Sweden)

    I-Jung Liu

    Full Text Available BACKGROUND: Feline infectious peritonitis (FIP is a lethal immune-mediated disease caused by feline coronavirus (FCoV. Currently, no therapy with proven efficacy is available. In searching for agents that may prove clinically effective against FCoV infection, five analogous overlapping peptides were designed and synthesized based on the putative heptad repeat 2 (HR2 sequence of the spike protein of FCoV, and the antiviral efficacy was evaluated. METHODS: Plaque reduction assay and MTT (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide cytotoxicity assay were performed in this study. Peptides were selected using a plaque reduction assay to inhibit Feline coronavirus infection. RESULTS: The results demonstrated that peptide (FP5 at concentrations below 20 μM inhibited viral replication by up to 97%. The peptide (FP5 exhibiting the most effective antiviral effect was further combined with a known anti-viral agent, human interferon-α (IFN-α, and a significant synergistic antiviral effect was observed. CONCLUSION: Our data suggest that the synthetic peptide FP5 could serve as a valuable addition to the current FIP prevention methods.

  18. High resolution crystal structure of rat long chain hydroxy acid oxidase in complex with the inhibitor 4-carboxy-5-[(4-chlorophenyl)sulfanyl]-1, 2, 3-thiadiazole. Implications for inhibitor specificity and drug design

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Zhi-wei; Vignaud, Caroline; Jaafar, Adil; Lévy, Bernard; Guéritte, Françoise; Guénard, Daniel; Lederer, Florence; Mathews, F. Scott (CNRS-UMR); (WU-MED)

    2012-05-24

    Long chain hydroxy acid oxidase (LCHAO) is responsible for the formation of methylguanidine, a toxic compound with elevated serum levels in patients with chronic renal failure. Its isozyme glycolate oxidase (GOX), has a role in the formation of oxalate, which can lead to pathological deposits of calcium oxalate, in particular in the disease primary hyperoxaluria. Inhibitors of these two enzymes may have therapeutic value. These enzymes are the only human members of the family of FMN-dependent L-2-hydroxy acid-oxidizing enzymes, with yeast flavocytochrome b{sub 2} (Fcb2) among its well studied members. We screened a chemical library for inhibitors, using in parallel rat LCHAO, human GOX and the Fcb2 flavodehydrogenase domain (FDH). Among the hits was an inhibitor, CCPST, with an IC{sub 50} in the micromolar range for all three enzymes. We report here the crystal structure of a complex between this compound and LCHAO at 1.3 {angstrom} resolution. In comparison with a lower resolution structure of this enzyme, binding of the inhibitor induces a conformational change in part of the TIM barrel loop 4, as well as protonation of the active site histidine. The CCPST interactions are compared with those it forms with human GOX and those formed by two other inhibitors with human GOX and spinach GOX. These compounds differ from CCPST in having the sulfur replaced with a nitrogen in the five-membered ring as well as different hydrophobic substituents. The possible reason for the {approx}100-fold difference in affinity between these two series of inhibitors is discussed. The present results indicate that specificity is an issue in the quest for therapeutic inhibitors of either LCHAO or GOX, but they may give leads for this quest.

  19. EPOR-Based Purification and Analysis of Erythropoietin Mimetic Peptides from Human Urine by Cys-Specific Cleavage and LC/MS/MS

    Science.gov (United States)

    Vogel, Matthias; Thomas, Andreas; Schänzer, Wilhelm; Thevis, Mario

    2015-09-01

    The development of a new class of erythropoietin mimetic agents (EMA) for treating anemic conditions has been initiated with the discovery of oligopeptides capable of dimerizing the erythropoietin (EPO) receptor and thus stimulating erythropoiesis. The most promising amino acid sequences have been mounted on various different polymeric structures or carrier molecules to obtain highly active EPO-like drugs exhibiting beneficial and desirable pharmacokinetic profiles. Concomitant with creating new therapeutic options, erythropoietin mimetic peptide (EMP)-based drug candidates represent means to artificially enhance endurance performance and necessitate coverage by sports drug testing methods. Therefore, the aim of the present study was to develop a strategy for the comprehensive detection of EMPs in doping controls, which can be used complementary to existing protocols. Three model EMPs were used to provide proof-of-concept data. Following EPO receptor-facilitated purification of target analytes from human urine, the common presence of the cysteine-flanked core structure of EMPs was exploited to generate diagnostic peptides with the aid of a nonenzymatic cleavage procedure. Sensitive detection was accomplished by targeted-SIM/data-dependent MS2 analysis. Method characterization was conducted for the EMP-based drug peginesatide concerning specificity, linearity, precision, recovery, stability, ion suppression/enhancement, and limit of detection (LOD, 0.25 ng/mL). Additionally, first data for the identification of the erythropoietin mimetic peptides EMP1 and BB68 were generated, demonstrating the multi-analyte testing capability of the presented approach.

  20. Immunohistochemical localization of gastrin-releasing peptide, neuronal nitric oxide synthase and neurone-specific enolase in the uterus of the North American opossum, Didelphis virginiana.

    Science.gov (United States)

    Kumano, A; Sasaki, M; Budipitojo, T; Kitamura, N; Krause, W J; Yamada, J

    2005-08-01

    The present study has demonstrated the immunohistochemical localization of gastrin-releasing peptide (GRP), neuronal nitric oxide synthase (nNOS) and neurone-specific enolase (NSE) in the uterus of the North American opossum. Although the presence of GRP, nNOS and NSE has been reported recently in the uterus of eutherian species this is the first description of these peptides in a metatherian species. Metatherian mammals are of interest because in these species it is the prolonged lactation phase of development that is the period of primary reproductive investment rather than intrauterine development as is true of eutherian mammals. The opossum, like other marsupial species, has a very abbreviated gestation period which in Didelphis lasts only 12.5 days. GRP was localized in the cytoplasm of cells forming the surface lining epithelium and the glandular epithelium of the opossum endometrium late in pregnancy, at 11.5 days of gestation. Likewise, immunoreactivities of nNOS and NSE were found primarily within the epithelial cells of the endometrium at 11.5 days of gestation. As these peptides and enzymes appear primarily at the time of establishment of the yolk sac placenta (between day 10 and day 12.5 gestation), the present results strongly suggest that these factors may play a fundamental role in the placentation of the opossum.

  1. Characterization of the peptide binding specificity of the HLA class I alleles B*38:01 and B*39:06.

    Science.gov (United States)

    Sidney, John; Schloss, Jennifer; Moore, Carrie; Lindvall, Mikaela; Wriston, Amanda; Hunt, Donald F; Shabanowitz, Jeffrey; DiLorenzo, Teresa P; Sette, Alessandro

    2016-03-01

    B*38:01 and B*39:06 are present with phenotypic frequencies 39:06 is the B allele most associated with type 1 diabetes susceptibility and 38:01 is most protective. A previous study derived putative main anchor motifs for both alleles based on peptide elution data. The present study has utilized panels of single amino acid substitution peptide libraries to derive detailed quantitative motifs accounting for both primary and secondary influences on peptide binding. From these analyses, both alleles were confirmed to utilize the canonical position 2/C-terminus main anchor spacing. B*38:01 preferentially bound peptides with the positively charged or polar residues H, R, and Q in position 2 and the large hydrophobic residues I, F, L, W, and M at the C-terminus. B*39:06 had a similar preference for R in position 2, but also well-tolerated M, Q, and K. A more dramatic contrast between the two alleles was noted at the C-terminus, where the specificity of B*39:06 was clearly for small residues, with A as most preferred, followed by G, V, S, T, and I. Detailed position-by-position and residue-by-residue coefficient values were generated from the panels to provide detailed quantitative B*38:01 and B*39:06 motifs. It is hoped that these detailed motifs will facilitate the identification of T cell epitopes recognized in the context of two class I alleles associated with dramatically different dispositions towards type 1 diabetes, offering potential avenues for the investigation of the role of CD8 T cells in this disease.

  2. Crystallization and preliminary X-ray diffraction analysis of the Fab fragment of WO2, an antibody specific for the Aβ peptides associated with Alzheimer’s disease

    Energy Technology Data Exchange (ETDEWEB)

    Wun, Kwok S. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Miles, Luke A. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Department of Chemical and Biomolecular Engineering, The University of Melbourne, Victoria 3010 (Australia); Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010 (Australia); Crespi, Gabriela A. N. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Wycherley, Kaye [WEHI Biotechnology Centre, La Trobe R& D Park, Bundoora, Victoria 3086 (Australia); Ascher, David B. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Barnham, Kevin J.; Cappai, Roberto [Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010 (Australia); Department of Pathology, The University of Melbourne, Victoria 3010 (Australia); The Mental Health Research Institute of Victoria, Parkville, Victoria 3052 (Australia); Beyreuther, Konrad [ZMBH, University of Heidelberg, Heidelberg (Germany); Masters, Colin L. [Department of Pathology, The University of Melbourne, Victoria 3010 (Australia); The Mental Health Research Institute of Victoria, Parkville, Victoria 3052 (Australia); Parker, Michael W. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010 (Australia); McKinstry, William J., E-mail: wjmckinstry@hotmail.com [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Department of Medicine (St Vincent’s Hospital), The University of Melbourne, 41 Victoria Parade, Fitzroy 3065 (Australia)

    2008-05-01

    Crystallization and X-ray diffraction data collection of the Fab fragment of the monoclonal antibody WO2 in the absence or presence of amyloid β peptides associated with Alzheimer’s disease are reported. The murine monoclonal antibody WO2 specifically binds the N-terminal region of the amyloid β peptide (Aβ) associated with Alzheimer’s disease. This region of Aβ has been shown to be the immunodominant B-cell epitope of the peptide and hence is considered to be a basis for the development of immunotherapeutic strategies against this prevalent cause of dementia. Structural studies have been undertaken in order to characterize the molecular basis for antibody recognition of this important epitope. Here, details of the crystallization and X-ray analysis of the Fab fragment of the unliganded WO2 antibody in two crystal forms and of the complexes that it forms with the truncated Aβ peptides Aβ{sub 1–16} and Aβ{sub 1–28} are presented. These crystals were all obtained using the hanging-drop vapour-diffusion method at 295 K. Crystals of WO2 Fab were grown in polyethylene glycol solutions containing ZnSO{sub 4}; they belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1} and diffracted to 1.6 Å resolution. The complexes of WO2 Fab with either Aβ{sub 1–@}@{sub 16} or Aβ{sub 1–28} were cocrystallized from polyethylene glycol solutions. These two complex crystals grew in the same space group, P2{sub 1}2{sub 1}2{sub 1}, and diffracted to 1.6 Å resolution. A second crystal form of WO2 Fab was grown in the presence of the sparingly soluble Aβ{sub 1–42} in PEG 550 MME. This second form belonged to space group P2{sub 1} and diffracted to 1.9 Å resolution.

  3. Effect of catalase-specific inhibitor 3-amino-1,2,4-triazole on yeast peroxisomal catalase in vivo.

    Science.gov (United States)

    Ueda, Mitsuyoshi; Kinoshita, Hiroshi; Yoshida, Tomoko; Kamasawa, Naomi; Osumi, Masako; Tanaka, Atsuo

    2003-02-14

    3-Amino-1,2,4-triazole (3-AT) is known as an inhibitor of catalase to whose active center it specifically and covalently binds. Subcellular fractionation and immunoelectronmicroscopic observation of the yeast Candida tropicalis revealed that, in 3-AT-treated cells in which the 3-AT was added to the n-alkane medium from the beginning of cultivation, catalase transported into peroxisomes was inactivated and was present as insoluble aggregated forms in the organelle. The aggregation of catalase in peroxisomes occurred only in these 3-AT-treated cells and not in cells in which 3-AT was added at the late exponential growth phase. Furthermore, 3-AT did not affect the transportation of catalase into peroxisomes. The appearance of aggregation only in cells to which 3-AT was added from the beginning of cultivation suggests that, in the process of catalase transportation into yeast peroxisomes, some conformational change may take place and that correct folding may be inhibited by the binding of 3-AT to the active center of catalase. Accordingly, 3-AT will be an interesting compound for investigation of the transport machinery of the peroxisomal tetrameric catalase.

  4. Placenta-specific novel splice variants of Rho GDP dissociation inhibitor β are highly expressed in cancerous cells

    Directory of Open Access Journals (Sweden)

    Hatakeyama Keiichi

    2012-12-01

    Full Text Available Abstract Background Alternative splicing of pre-mRNA transcripts not only plays a role in normal molecular processes but is also associated with cancer development. While normal transcripts are ubiquitously expressed in normal tissues, splice variants created through abnormal alternative splicing events are often expressed in cancer cells. Although the Rho GDP dissociation inhibitor β (ARHGDIB gene has been found to be ubiquitously expressed in normal tissues and involved in cancer development, the presence of splice variants of ARHGDIB has not yet been investigated. Results Validation analysis for the presence of and exon structures of splice variants of ARHGDIB, performed using reverse-transcriptase polymerase chain reaction and DNA sequencing, successfully identified novel splice variants of ARHGDIB, that is, 6a, 6b, and 6c, in colon, pancreas, stomach, and breast cancer cell lines. Quantitative real-time polymerase chain reaction analysis showed that these variants were also highly expressed in normal placental tissue but not in other types of normal tissue. Conclusions Expression of ARHGDIB variants 6a, 6b, and 6c appears to be restricted to cancer cells and normal placental tissue, suggesting that these variants possess cancer-specific functions and, as such, are potential cancer-related biomarkers.

  5. Proton Pump Inhibitors and the Risk for Fracture at Specific Sites: Data Mining of the FDA Adverse Event Reporting System.

    Science.gov (United States)

    Wang, Liwei; Li, Mei; Cao, Yuying; Han, Zhengqi; Wang, Xueju; Atkinson, Elizabeth J; Liu, Hongfang; Amin, Shreyasee

    2017-07-17

    Proton pump inhibitors (PPIs) are widely used to treat gastric acid-related disorders. Concerns have been raised about potential fracture risk, especially at the hip, spine and wrist. However, fracture risk at other bone sites has not been as well studied. We investigated the association between PPIs and specific fracture sites using an aggregated knowledge-enhanced database, the Food and Drug Administration Adverse Event Reporting System Data Mining Set (AERS-DM). Proportional reporting ratio (PRR) was used to detect statistically significant associations (signals) between PPIs and fractures. We analyzed both high level terms (HLT) and preferred terms (PT) for fracture sites, defined by MedDRA (Medical Dictionary for Regulatory Activities). Of PPI users reporting fractures, the mean age was 65.3 years and the female to male ratio was 3.4:1. Results revealed signals at multiple HLT and PT fracture sites, consistent for both sexes. These included fracture sites with predominant trabecular bone, not previously reported as being associated with PPIs, such as 'rib fractures', where signals were detected for overall PPIs as well as for each of 5 generic ingredients (insufficient data for dexlansoprazole). Based on data mining from AERS-DM, PPI use appears to be associated with an increased risk for fractures at multiple sites.

  6. Studying the Specific Activity of the Amide Form of HLDF-6 Peptide using the Transgenic Model of Alzheimer's Disease.

    Science.gov (United States)

    Bogachouk, A P; Storozheva, Z I; Telegin, G B; Chernov, A S; Proshin, A T; Sherstnev, V V; Zolotarev, Yu A; Lipkin, V M

    2017-01-01

    The neuroprotective and nootropic activities of the amide form (AF) of the HLDF-6 peptide (TGENHR-NH 2 ) were studied in transgenic mice of the B6C3-Tg(APPswe,PSEN1de9)85Dbo (Tg+) line (the animal model of familial Alzheimer's disease (AD)). The study was performed in 4 mouse groups: group 1 (study group): Tg+ mice intranasally injected with the peptide at a dose of 250 μg/kg; group 2 (active control): Tg+ mice intranasally injected with normal saline; group 3 (control 1): Tg- mice; and group 4 (control 2): C57Bl/6 mice. The cognitive functions were evaluated using three tests: the novel object recognition test, the conditioned passive avoidance task, and the Morris water maze. The results testify to the fact that the pharmaceutical substance (PhS) based on the AF of HLDF-6 peptide at a dose of 250 μg/kg administered intranasally efficiently restores the disturbed cognitive functions in transgenic mice. These results are fully consistent with the data obtained in animal models of Alzheimer's disease induced by the injection of the beta-amyloid (βA) fragment 25-35 into the giant-cell nucleus basalis of Meynert or by co-injection of the βA fragment 25-35 and ibotenic acid into the hippocampus, and the model of ischemia stroke (chronic bilateral occlusion of carotids, 2VO). According to the overall results, PhS based on AF HLDF-6 was chosen as an object for further investigation; the dose of 250 μg/kg was used as an effective therapeutic dose. Intranasal administration was the route for delivery.

  7. The dipeptidyl peptidase 4 inhibitor vildagliptin does not accentuate glibenclamide-induced hypoglycemia but reduces glucose-induced glucagon-like peptide 1 and gastric inhibitory polypeptide secretion

    DEFF Research Database (Denmark)

    El-Ouaghlidi, Andrea; Rehring, Erika; Holst, Jens Juul

    2007-01-01

    BACKGROUND/AIMS: Inhibition of dipeptidyl peptidase 4 by vildagliptin enhances the concentrations of the active form of the incretin hormones glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP). The present study asked whether vildagliptin accentuates glibenclamide-induced hy...

  8. Adding an Artificial Tail—Anchor to a Peptide-Based HIV-1 Fusion Inhibitor for Improvement of Its Potency and Resistance Profile

    Directory of Open Access Journals (Sweden)

    Shan Su

    2017-11-01

    Full Text Available Peptides derived from the C-terminal heptad repeat (CHR of human immunodeficiency virus type 1 (HIV-1 envelope protein transmembrane subunit gp41, such as T20 (enfuvirtide, can bind to the N-terminal heptad repeat (NHR of gp41 and block six-helix bundle (6-HB formation, thus inhibiting HIV-1 fusion with the target cell. However, clinical application of T20 is limited because of its low potency and genetic barrier to resistance. HP23, the shortest CHR peptide, exhibits better anti-HIV-1 activity than T20, but the HIV-1 strains with E49K mutations in gp41 will become resistant to it. Here, we modified HP23 by extending its C-terminal sequence using six amino acid residues (E6 and adding IDL (Ile-Asp-Leu to the C-terminus of E6, which is expected to bind to the shallow pocket in the gp41 NHR N-terminal region. The newly designed peptide, designated HP23-E6-IDL, was about 2- to 16-fold more potent than HP23 against a broad spectrum of HIV-1 strains and more than 12-fold more effective against HIV-1 mutants resistant to HP23. These findings suggest that addition of an anchor–tail to the C-terminus of a CHR peptide will allow binding with the pocket in the gp41 NHR that may increase the peptide’s antiviral efficacy and its genetic barrier to resistance.

  9. Inhibition of plasminogen activator inhibitor-1 binding to endocytosis receptors of the low density lipoprotein receptor family by a peptide isolated from a phage displayed library

    DEFF Research Database (Denmark)

    Jensen, Jan K.; Malmendal, Anders; Schiøtt, Birgit

    2006-01-01

    (DVPCFGWCQDA) was determined by NMR. A binding site in the so-called flexible joint region of PAI-1 was suggested by molecular modelling and validated through binding studies with various competitors and site-directed mutagenesis of PAI-1. The peptide with an N-terminal biotin inhibited the binding of the u...

  10. Purification and characterization of peptides from Capsicum annuum fruits which are α-amylase inhibitors and exhibit high antimicrobial activity against fungi of agronomic importance.

    Science.gov (United States)

    Dos Santos, Layrana de Azevedo; Taveira, Gabriel Bonan; Ribeiro, Suzanna de Fátima Ferreira; Pereira, Lídia da Silva; Carvalho, André de Oliveira; Rodrigues, Rosana; Oliveira, Antônia Elenir Amâncio; Machado, Olga Lima Tavares; Araújo, Jucélia da Silva; Vasconcelos, Ilka Maria; Gomes, Valdirene Moreira

    2017-04-01

    Proteins extracted from Capsicum annuum L. fruits were initially subjected to reversed-phase chromatography on HPLC, resulting in eight peptide-rich fractions. All the fractions obtained were tested for their ability to inhibit porcine trypsin and amylase from both human saliva and from larval insect in vitro. All fractions were also tested for their ability to inhibit growth of the phytopathogenic fungi. Several fractions inhibited the activity of human salivary amylase and larval insect amylase, especially fraction Fa5. No fraction tested was found to inhibit trypsin activity, being Fa2 fraction an exception. Interestingly fraction Fa5 also displayed high antimicrobial activity against the species of the Fusarium genus. Fraction Fa5 was found to have two major protein bands of 17 and 6.5 kDa, and these were sequenced by mass spectrometry. Two peptides were obtained from the 6.5-kDa band, which showed similarity to antimicrobial peptides. Fraction Fa5 was also tested for its ability to permeabilize membranes and induce ROS. Fraction Fa5 was able to permeabilize the membranes of all the fungi tested. Fungi belonging to the genus Fusarium also showed an increase in the endogenous production of ROS when treated with this fraction. Antimicrobial peptides were also identified in the fruits from other Capsicum species. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Peptides derived from specific interaction sites of the fibroblast growth factor 2 - FGF receptor complexes induce receptor activation and signaling

    DEFF Research Database (Denmark)

    Manfè, Valentina; Kochoyan, Artur; Bock, Elisabeth

    2010-01-01

    J. Neurochem. (2010) 10.1111/j.1471-4159.2010.06718.x Abstract Basic fibroblast growth factor (FGF2, bFGF) is the most extensively studied member of the FGF family and is involved in neurogenesis, differentiation, neuroprotection, and synaptic plasticity in the CNS. FGF2 executes its pleiotropic...... biologic actions by binding, dimerizing, and activating FGF receptors (FGFRs). The present study reports the physiologic impact of various FGF2-FGFR1 contact sites employing three different synthetic peptides, termed canofins, designed based on structural analysis of the interactions between FGF2 and FGFR1...

  12. Ca2+-dependent proteolytic activity in crab claw muscle: effects of inhibitors and specificity for myofibrillar proteins

    International Nuclear Information System (INIS)

    Mykles, D.L.; Skinner, D.M.

    1983-01-01

    The claw closer muscle of the Bermuda land crab, Gecarcinus lateralis, undergoes a sequential atrophy and restoration during each molting cycle. The role of Ca 2+ -dependent proteinases in the turn-over of myofibrillar protein in normal anecdysial (intermolt) claw muscle is described. Crab Ca 2+ -dependent proteinase degrades the myofibrillar proteins actin, myosin heavy and light chains, paramyosin, tropomyosin, and troponin-T and -I. Ca 2+ -dependent proteinase activity in whole homogenates and 90,000 x g supernatant fractions from muscle homogenates has been characterized with respect to Ca 2+ requirement, substrate specificity, and effects of proteinase inhibitors. The enzyme is inhibited by antipain, leupeptin, E-64, and iodoacetamide; it is insensitive to pepstatin A. The specificity of crab Ca 2+ -dependent proteinase was examined with native myosin with normal ATPase activity as well as with radioiodinated myosin and radioiodinated hemolymph proteins. Hydrolysis of 125 I-myosin occurs in two phases, both Ca 2+ -dependent: (1) heavy chain (M/sub r/ = 200,000) is cleaved into four large fragments (M/sub r/ = 160,000, 110,000, 73,000, 60,000) and numerous smaller fragments; light chain (M/sub r/ = 18,000) is cleaved to a 15,000-Da fragment; (2) the fragments produced in the first phase are hydrolyzed to acid-soluble material. Although radioiodinated native hemolymph proteins are not susceptible to the Ca 2+ -dependent proteinase, those denatured by carboxymethylation are degraded. These data suggest that crab Ca 2+ -dependent proteinase is involved in turnover of myofibrillar protein in normal muscle and muscle undergoing proecdysial atrophy

  13. CD8+ T Cells Specific to Apoptosis-Associated Antigens Predict the Response to Tumor Necrosis Factor Inhibitor Therapy in Rheumatoid Arthritis.

    Directory of Open Access Journals (Sweden)

    Alessandra Citro

    Full Text Available CD8+ T cells specific to caspase-cleaved antigens derived from apoptotic T cells (apoptotic epitopes represent a principal player in chronic immune activation, which is known to amplify immunopathology in various inflammatory diseases. The purpose of the present study was to investigate the relationship involving these autoreactive T cells, the rheumatoid arthritis immunopathology, and the response to tumor necrosis factor-α inhibitor therapy. The frequency of autoreactive CD8+ T cells specific to various apoptotic epitopes, as detected by both enzyme-linked immunospot assay and dextramers of major histocompatibility complex class I molecules complexed with relevant apoptotic epitopes, was longitudinally analyzed in the peripheral blood of rheumatoid arthritis patients who were submitted to etanercept treatment (or other tumor necrosis factor inhibitors as a control. The percentage of apoptotic epitope-specific CD8+ T cells was significantly higher in rheumatoid arthritis patients than in healthy donors, and correlated with the disease activity. More important, it was significantly more elevated in responders to tumor necrosis factor-α inhibitor therapy than in non-responders before the start of therapy; it significantly dropped only in the former following therapy. These data indicate that apoptotic epitope-specific CD8+ T cells may be involved in rheumatoid arthritis immunopathology through the production of inflammatory cytokines and that they may potentially represent a predictive biomarker of response to tumor necrosis factor-α inhibitor therapy to validate in a larger cohort of patients.

  14. Insights into the substrate specificity of plant peptide deformylase, an essential enzyme with potential for the development of novel biotechnology applications in agriculture.

    Science.gov (United States)

    Dirk, Lynnette M A; Schmidt, Jack J; Cai, Yiying; Barnes, Jonathan C; Hanger, Katherine M; Nayak, Nihar R; Williams, Mark A; Grossman, Robert B; Houtz, Robert L; Rodgers, David W

    2008-08-01

    The crystal structure of AtPDF1B [Arabidopsis thaliana PDF (peptide deformylase) 1B; EC 3.5.1.88], a plant specific deformylase, has been determined at a resolution of 2.4 A (1 A=0.1 nm). The overall fold of AtPDF1B is similar to other peptide deformylases that have been reported. Evidence from the crystal structure and gel filtration chromatography indicates that AtPDF1B exists as a symmetric dimer. PDF1B is essential in plants and has a preferred substrate specificity towards the PS II (photosystem II) D1 polypeptide. Comparative analysis of AtPDF1B, AtPDF1A, and the type 1B deformylase from Escherichia coli, identifies a number of differences in substrate binding subsites that might account for variations in sequence preference. A model of the N-terminal five amino acids from the D1 polypeptide bound in the active site of AtPDF1B suggests an influence of Tyr(178) as a structural determinant for polypeptide substrate specificity through hydrogen bonding with Thr(2) in the D1 sequence. Kinetic analyses using a polypeptide mimic of the D1 N-terminus was performed on AtPDF1B mutated at Tyr(178) to alanine, phenylalanine or arginine (equivalent residue in AtPDF1A). The results suggest that, whereas Tyr(178) can influence catalytic activity, other residues contribute to the overall preference for the D1 polypeptide.

  15. Specific degradation of the mucus adhesion-promoting protein (MapA) of Lactobacillus reuteri to an antimicrobial peptide.

    Science.gov (United States)

    Bøhle, Liv Anette; Brede, Dag Anders; Diep, Dzung B; Holo, Helge; Nes, Ingolf F

    2010-11-01

    The intestinal flora of mammals contains lactic acid bacteria (LAB) that may provide positive health effects for the host. Such bacteria are referred to as probiotic bacteria. From a pig, we have isolated a Lactobacillus reuteri strain that produces an antimicrobial peptide (AMP). The peptide was purified and characterized, and it was unequivocally shown that the AMP was a well-defined degradation product obtained from the mucus adhesion-promoting protein (MapA); it was therefore termed AP48-MapA. This finding demonstrates how large proteins might inherit unexpected pleiotropic functions by conferring antimicrobial capacities on the producer. The MapA/AP48-MapA system is the first example where a large protein of an intestinal LAB is shown to give rise to such an AMP. It is also of particular interest that the protein that provides this AMP is associated with the binding of the bacterium producing it to the surface/lining of the gut. This finding gives us new perspective on how some probiotic bacteria may successfully compete in this environment and thereby contribute to a healthy microbiota.

  16. Specific Degradation of the Mucus Adhesion-Promoting Protein (MapA) of Lactobacillus reuteri to an Antimicrobial Peptide

    Science.gov (United States)

    Bøhle, Liv Anette; Brede, Dag Anders; Diep, Dzung B.; Holo, Helge; Nes, Ingolf F.

    2010-01-01

    The intestinal flora of mammals contains lactic acid bacteria (LAB) that may provide positive health effects for the host. Such bacteria are referred to as probiotic bacteria. From a pig, we have isolated a Lactobacillus reuteri strain that produces an antimicrobial peptide (AMP). The peptide was purified and characterized, and it was unequivocally shown that the AMP was a well-defined degradation product obtained from the mucus adhesion-promoting protein (MapA); it was therefore termed AP48-MapA. This finding demonstrates how large proteins might inherit unexpected pleiotropic functions by conferring antimicrobial capacities on the producer. The MapA/AP48-MapA system is the first example where a large protein of an intestinal LAB is shown to give rise to such an AMP. It is also of particular interest that the protein that provides this AMP is associated with the binding of the bacterium producing it to the surface/lining of the gut. This finding gives us new perspective on how some probiotic bacteria may successfully compete in this environment and thereby contribute to a healthy microbiota. PMID:20833791

  17. Pharmacokinetics of gene recombined angiogenesis inhibitor Kringle 5 in vivo using 131I specific markers and SPECT/CT

    Directory of Open Access Journals (Sweden)

    Ge Yan

    2016-10-01

    Full Text Available The previous pharmacokinetic methods can be only limited to drug analysis in vitro, which provide less information on the distribution and metabolismof drugs, and limit the interpretation and assessment of pharmacokinetics, the determination of metabolic principles, and evaluation of treatment effect. The objective of the study was to investigate the pharmacokinetic characteristics of gene recombination angiogenesis inhibitor Kringle 5 in vivo. The SPECT/CT and specific 131I-Kringle 5 marked by Iodogen method were both applied to explore the pharmacokinetic characteristics of 131I-Kringle 5 in vivo, and to investigate the dynamic distributions of 131I-Kringle 5 in target organs. Labeling recombinant angiogenesis inhibitor Kringle 5 using 131I with longer half-life and imaging in vivo using SPECT instead of PET, could overcome the limitations of previous methods. When the doses of 131I-Kringle 5 were 10.0, 7.5 and 5.0 g/kg, respectively, the two-compartment open models can be determined within all the metabolic process in vivo. There were no significant differences in t1/2α, t1/2β, apparent volume of distribution and CL between those three levels. The ratio of AUC(0~∞ among three different groups of 10.0, 7.5 and 5.0 g/kg was 2.56:1.44:1.0, which was close to the ratio (2:1.5:1.0. It could be clear that in the range of 5.0–10.0 g/kg, Kringle 5 was characterized by the first-order pharmacokinetics. Approximately 30 min after 131I-Kringle 5 was injected, 131I-Kringle 5 could be observed to concentrate in the heart, kidneys, liver and other organs by means of planar imaging and tomography. After 1 h of being injected, more radionuclide retained in the bladder, but not in intestinal. It could be concluded that 131I-Kringle 5 is mainly excreted through the kidneys. About 2 h after the injection of 131I-Kringle 5, the radionuclide in the heart, kidneys, liver and other organs was gradually reduced, while more radionuclide was concentrated

  18. Molecular Determinants Underlying Binding Specificities of the ABL Kinase Inhibitors: Combining Alanine Scanning of Binding Hot Spots with Network Analysis of Residue Interactions and Coevolution.

    Directory of Open Access Journals (Sweden)

    Amanda Tse

    Full Text Available Quantifying binding specificity and drug resistance of protein kinase inhibitors is of fundamental importance and remains highly challenging due to complex interplay of structural and thermodynamic factors. In this work, molecular simulations and computational alanine scanning are combined with the network-based approaches to characterize molecular determinants underlying binding specificities of the ABL kinase inhibitors. The proposed theoretical framework unveiled a relationship between ligand binding and inhibitor-mediated changes in the residue interaction networks. By using topological parameters, we have described the organization of the residue interaction networks and networks of coevolving residues in the ABL kinase structures. This analysis has shown that functionally critical regulatory residues can simultaneously embody strong coevolutionary signal and high network centrality with a propensity to be energetic hot spots for drug binding. We have found that selective (Nilotinib and promiscuous (Bosutinib, Dasatinib kinase inhibitors can use their energetic hot spots to differentially modulate stability of the residue interaction networks, thus inhibiting or promoting conformational equilibrium between inactive and active states. According to our results, Nilotinib binding may induce a significant network-bridging effect and enhance centrality of the hot spot residues that stabilize structural environment favored by the specific kinase form. In contrast, Bosutinib and Dasatinib can incur modest changes in the residue interaction network in which ligand binding is primarily coupled only with the identity of the gate-keeper residue. These factors may promote structural adaptability of the active kinase states in binding with these promiscuous inhibitors. Our results have related ligand-induced changes in the residue interaction networks with drug resistance effects, showing that network robustness may be compromised by targeted mutations

  19. Molecular Determinants Underlying Binding Specificities of the ABL Kinase Inhibitors: Combining Alanine Scanning of Binding Hot Spots with Network Analysis of Residue Interactions and Coevolution

    Science.gov (United States)

    Tse, Amanda; Verkhivker, Gennady M.

    2015-01-01

    Quantifying binding specificity and drug resistance of protein kinase inhibitors is of fundamental importance and remains highly challenging due to complex interplay of structural and thermodynamic factors. In this work, molecular simulations and computational alanine scanning are combined with the network-based approaches to characterize molecular determinants underlying binding specificities of the ABL kinase inhibitors. The proposed theoretical framework unveiled a relationship between ligand binding and inhibitor-mediated changes in the residue interaction networks. By using topological parameters, we have described the organization of the residue interaction networks and networks of coevolving residues in the ABL kinase structures. This analysis has shown that functionally critical regulatory residues can simultaneously embody strong coevolutionary signal and high network centrality with a propensity to be energetic hot spots for drug binding. We have found that selective (Nilotinib) and promiscuous (Bosutinib, Dasatinib) kinase inhibitors can use their energetic hot spots to differentially modulate stability of the residue interaction networks, thus inhibiting or promoting conformational equilibrium between inactive and active states. According to our results, Nilotinib binding may induce a significant network-bridging effect and enhance centrality of the hot spot residues that stabilize structural environment favored by the specific kinase form. In contrast, Bosutinib and Dasatinib can incur modest changes in the residue interaction network in which ligand binding is primarily coupled only with the identity of the gate-keeper residue. These factors may promote structural adaptability of the active kinase states in binding with these promiscuous inhibitors. Our results have related ligand-induced changes in the residue interaction networks with drug resistance effects, showing that network robustness may be compromised by targeted mutations of key mediating

  20. Squash inhibitor family of serine proteinases

    International Nuclear Information System (INIS)

    Otlewski, J.; Krowarsch, D.

    1996-01-01

    Squash inhibitors of serine proteinases form an uniform family of small proteins. They are built of 27-33 amino-acid residues and cross-linked with three disulfide bridges. The reactive site peptide bond (P1-P1') is between residue 5 (Lys, Arg or Leu) and 6 (always Ile). High resolution X-ray structures are available for two squash inhibitors complexed with trypsin. NMR solution structures have also been determined for free inhibitors. The major structural motif is a distorted, triple-stranded antiparallel beta-sheet. A similar folding motif has been recently found in a number of proteins, including: conotoxins from fish-hunting snails, carboxypeptidase inhibitor from potato, kalata B1 polypeptide, and in some growth factors (e.g. nerve growth factor, transforming growth factor β2, platelet-derived growth factor). Squash inhibitors are highly stable and rigid proteins. They inhibit a number of serine proteinases: trypsin, plasmin, kallikrein, blood clotting factors: X a and XII a , cathepsin G. The inhibition spectrum can be much broadened if specific amino-acid substitutions are introduced, especially at residues which contact proteinase. Squash inhibitors inhibit proteinases via the standard mechanism. According to the mechanism, inhibitors are substrates which exhibit at neutral pH a high k cat /K m index for hydrolysis and resynthesis of the reactive site, and a low value of the hydrolysis constant. (author)

  1. The gastric acid secretagogue gastrin-releasing peptide and the inhibitor oxyntomodulin do not exert their effect directly on the parietal cell in the rat

    DEFF Research Database (Denmark)

    Poulsen, Steen Seier; Holst, J J

    1988-01-01

    in vitro by measuring [14C]-aminopyrine accumulation, a reliable index of H+ generation, in isolated rat parietal cells. However, neither gastrin-releasing peptide nor oxyntomodulin influenced basal acid secretion or histamine-stimulated gastric acid secretion. Electron-microscopic studies of unstimulated...... and histamine-stimulated parietal cells confirmed that the cells retained the normal morphology of intracellular organelles and that the cells responded to physiological stimulation by marked expansion of the intracellular canaliculi....

  2. IMD-4690, a novel specific inhibitor for plasminogen activator inhibitor-1, reduces allergic airway remodeling in a mouse model of chronic asthma via regulating angiogenesis and remodeling-related mediators.

    Directory of Open Access Journals (Sweden)

    Toshifumi Tezuka

    Full Text Available Plasminogen activator inhibitor (PAI-1 is the principal inhibitor of plasminogen activators, and is responsible for the degradation of fibrin and extracellular matrix. IMD-4690 is a newly synthesized inhibitor for PAI-1, whereas the effect on allergic airway inflammation and remodeling is still unclear. We examined the in vivo effects by using a chronic allergen exposure model of bronchial asthma in mice. The model was generated by an immune challenge for 8 weeks with house dust mite antigen, Dermatophagoides pteronyssinus (Dp. IMD-4690 was intraperitoneally administered during the challenge. Lung histopathology, hyperresponsiveness and the concentrations of mediators in lung homogenates were analyzed. The amount of active PAI-1 in the lungs was increased in mice treated with Dp. Administration with IMD-4690 reduced an active/total PAI-1 ratio. IMD-4690 also reduced the number of bronchial eosinophils in accordance with the decreased expressions of Th2 cytokines in the lung homogenates. Airway remodeling was inhibited by reducing subepithelial collagen deposition, smooth muscle hypertrophy, and angiogenesis. The effects of IMD-4690 were partly mediated by the regulation of TGF-β, HGF and matrix metalloproteinase. These results suggest that PAI-1 plays crucial roles in airway inflammation and remodeling, and IMD-4690, a specific PAI-1 inhibitor, may have therapeutic potential for patients with refractory asthma due to airway remodeling.

  3. IMD-4690, a novel specific inhibitor for plasminogen activator inhibitor-1, reduces allergic airway remodeling in a mouse model of chronic asthma via regulating angiogenesis and remodeling-related mediators.

    Science.gov (United States)

    Tezuka, Toshifumi; Ogawa, Hirohisa; Azuma, Masahiko; Goto, Hisatsugu; Uehara, Hisanori; Aono, Yoshinori; Hanibuchi, Masaki; Yamaguchi, Yoichi; Fujikawa, Tomoyuki; Itai, Akiko; Nishioka, Yasuhiko

    2015-01-01

    Plasminogen activator inhibitor (PAI)-1 is the principal inhibitor of plasminogen activators, and is responsible for the degradation of fibrin and extracellular matrix. IMD-4690 is a newly synthesized inhibitor for PAI-1, whereas the effect on allergic airway inflammation and remodeling is still unclear. We examined the in vivo effects by using a chronic allergen exposure model of bronchial asthma in mice. The model was generated by an immune challenge for 8 weeks with house dust mite antigen, Dermatophagoides pteronyssinus (Dp). IMD-4690 was intraperitoneally administered during the challenge. Lung histopathology, hyperresponsiveness and the concentrations of mediators in lung homogenates were analyzed. The amount of active PAI-1 in the lungs was increased in mice treated with Dp. Administration with IMD-4690 reduced an active/total PAI-1 ratio. IMD-4690 also reduced the number of bronchial eosinophils in accordance with the decreased expressions of Th2 cytokines in the lung homogenates. Airway remodeling was inhibited by reducing subepithelial collagen deposition, smooth muscle hypertrophy, and angiogenesis. The effects of IMD-4690 were partly mediated by the regulation of TGF-β, HGF and matrix metalloproteinase. These results suggest that PAI-1 plays crucial roles in airway inflammation and remodeling, and IMD-4690, a specific PAI-1 inhibitor, may have therapeutic potential for patients with refractory asthma due to airway remodeling.

  4. Tissue- and agonist-specific regulation of human and murine plasminogen activator inhibitor-1 promoters in transgenic mice.

    Science.gov (United States)

    Eren, M; Painter, C A; Gleaves, L A; Schoenhard, J A; Atkinson, J B; Brown, N J; Vaughan, D E

    2003-11-01

    Numerous studies have described regulatory factors and sequences that control transcriptional responses in vitro. However, there is a paucity of information on the qualitative and quantitative regulation of heterologous promoters using transgenic strategies. In order to investigate the physiological regulation of human plasminogen activator inhibitor type-1 (hPAI-1) expression in vivo compared to murine PAI-1 (mPAI-1) and to test the physiological relevance of regulatory mechanisms described in vitro, we generated transgenic mice expressing enhanced green fluorescent protein (EGFP) driven by the proximal -2.9 kb of the hPAI-1 promoter. Transgenic animals were treated with Ang II, TGF-beta1 and lipopolysaccharide (LPS) to compare the relative activation of the human and murine PAI-1 promoters. Ang II increased EGFP expression most effectively in brain, kidney and spleen, while mPAI-1 expression was quantitatively enhanced most prominently in heart and spleen. TGF-beta1 failed to induce activation of the hPAI-1 promoter but potently stimulated mPAI-1 in kidney and spleen. LPS administration triggered robust expression of mPAI-1 in liver, kidney, pancreas, spleen and lung, while EGFP was induced only modestly in heart and kidney. These results indicate that the transcriptional response of the endogenous mPAI-1 promoter varies widely in terms of location and magnitude of response to specific stimuli. Moreover, the physiological regulation of PAI-1 expression likely involves a complex interaction of transcription factors and DNA sequences that are not adequately replicated by in vitro functional studies focused on the proximal -2.9 kb promoter.

  5. RAGE-Specific Inhibitor FPS-ZM1 Attenuates AGEs-Induced Neuroinflammation and Oxidative Stress in Rat Primary Microglia.

    Science.gov (United States)

    Shen, Chao; Ma, Yingjuan; Zeng, Ziling; Yin, Qingqing; Hong, Yan; Hou, Xunyao; Liu, Xueping

    2017-10-01

    Advanced glycation end products (AGEs) enhance microglial activation and intensify the inflammatory response and oxidative stress in the brain. This process may occur due to direct cytotoxicity or interacting with AGEs receptors (RAGE), which are expressed on the surface of microglia. FPS-ZM1 is a high-affinity but nontoxic RAGE-specific inhibitor that has been recently shown to attenuate the Aβ-induced inflammatory response by blocking the ligation of Aβ to RAGE. In this study, we further investigated the effect of FPS-ZM1 on the AGEs/RAGE interaction and downstream elevation of neuroinflammation and oxidative stress in primary microglia cells. The results suggested that FPS-ZM1 significantly suppressed AGEs-induced RAGE overexpression, RAGE-dependent microglial activation, nuclear translocation of nuclear factor kappaB p65 (NF-κB p65), and the expression of downstream inflammatory mediators such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), cyclooxygenase 2 (COX-2)/prostaglandin E2 (PGE2) and inducible nitric oxide synthase (iNOS)/nitric oxide (NO). Furthermore, FPS-ZM1 attenuated AGEs-stimulated NADPH oxidase (NOX) activation and reactive oxygen species (ROS) expression. Finally, FPS-ZM1 elevated the levels of transcription factors nuclear-factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1 (HO-1), as well as decreased antioxidant capacity and increased production of oxidative species. Our results suggest that FPS-ZM1 may be neuroprotective through attenuating microglial activation, oxidative stress and inflammation by blocking RAGE.

  6. Prostate-specific membrane antigen targeted imaging and therapy of prostate cancer using a PSMA inhibitor as a homing ligand.

    Science.gov (United States)

    Kularatne, Sumith A; Wang, Kevin; Santhapuram, Hari-Krishna R; Low, Philip S

    2009-01-01

    Prostate cancer (PCa) is a major cause of mortality and morbidity in Western society today. Current methods for detecting PCa are limited, leaving most early malignancies undiagnosed and sites of metastasis in advanced disease undetected. Major deficiencies also exist in the treatment of PCa, especially metastatic disease. In an effort to improve both detection and therapy of PCa, we have developed a PSMA-targeted ligand that delivers attached imaging and therapeutic agents selectively to PCa cells without targeting normal cells. The PSMA-targeted radioimaging agent (DUPA-(99m)Tc) was found to bind PSMA-positive human PCa cells (LNCaP cell line) with nanomolar affinity (K(D) = 14 nM). Imaging and biodistribution studies revealed that DUPA-(99m)Tc localizes primarily to LNCaP cell tumor xenografts in nu/nu mice (% injected dose/gram = 11.3 at 4 h postinjection; tumor-to-muscle ratio = 75:1). Two PSMA-targeted optical imaging agents (DUPA-FITC and DUPA-rhodamine B) were also shown to efficiently label PCa cells and to internalize and traffic to intracellular endosomes. A PSMA-targeted chemotherapeutic agent (DUPA-TubH) was demonstrated to kill PSMA-positive LNCaP cells in culture (IC(50) = 3 nM) and to eliminate established tumor xenografts in nu/nu mice with no detectable weight loss. Blockade of tumor targeting upon administration of excess PSMA inhibitor (PMPA) and the absence of targeting to PSMA-negative tumors confirmed the specificity of each of the above targeted reagents for PSMA. Tandem use of the imaging and therapeutic agents targeted to the same receptor could allow detection, staging, monitoring, and treatment of PCa with improved accuracy and efficacy.

  7. Peptides in melanoma therapy.

    Science.gov (United States)

    Mocellin, Simone

    2012-01-01

    Peptides derived from tumor associated antigens can be utilized to elicit a therapeutically effective immune response against melanoma in experimental models. However, patient vaccination with peptides - although it is often followed by the induction of melanoma- specific T lymphocytes - is rarely associated with tumor response of clinical relevance. In this review I summarize the principles of peptide design as well as the results so far obtained in the clinical setting while treating cutaneous melanoma by means of this active immunotherapy strategy. I also discuss some immunological and methodological issues that might be helpful for the successful development of peptide-based vaccines.

  8. Homology-guided mutational analysis reveals the functional requirements for antinociceptive specificity of collapsin response mediator protein 2-derived peptides.

    Science.gov (United States)

    Moutal, Aubin; Li, Wennan; Wang, Yue; Ju, Weina; Luo, Shizhen; Cai, Song; François-Moutal, Liberty; Perez-Miller, Samantha; Hu, Jackie; Dustrude, Erik T; Vanderah, Todd W; Gokhale, Vijay; Khanna, May; Khanna, Rajesh

    2017-02-05

    N-type voltage-gated calcium (Ca v 2.2) channels are critical determinants of increased neuronal excitability and neurotransmission accompanying persistent neuropathic pain. Although Ca v 2.2 channel antagonists are recommended as first-line treatment for neuropathic pain, calcium-current blocking gabapentinoids inadequately alleviate chronic pain symptoms and often exhibit numerous side effects. Collapsin response mediator protein 2 (CRMP2) targets Ca v 2.2 channels to the sensory neuron membrane and allosterically modulates their function. A 15-amino-acid peptide (CBD3), derived from CRMP2, disrupts the functional protein-protein interaction between CRMP2 and Ca v 2.2 channels to inhibit calcium influx, transmitter release and acute, inflammatory and neuropathic pain. Here, we have mapped the minimal domain of CBD3 necessary for its antinociceptive potential. Truncated as well as homology-guided mutant versions of CBD3 were generated and assessed using depolarization-evoked calcium influx in rat dorsal root ganglion neurons, binding between CRMP2 and Ca v 2.2 channels, whole-cell voltage clamp electrophysiology and behavioural effects in two models of experimental pain: post-surgical pain and HIV-induced sensory neuropathy induced by the viral glycoprotein 120. The first six amino acids within CBD3 accounted for all in vitro activity and antinociception. Spinal administration of a prototypical peptide (TAT-CBD3-L5M) reversed pain behaviours. Homology-guided mutational analyses of these six amino acids identified at least two residues, Ala1 and Arg4, as being critical for antinociception in two pain models. These results identify an antinociceptive scaffold core in CBD3 that can be used for development of low MW mimetics of CBD3. © 2017 The British Pharmacological Society.

  9. Site-specific Orientation of an α-helical Peptide Ovispirin-1 from Isotope Labeled SFG Spectroscopy

    Science.gov (United States)

    Ding, Bei; Laaser, Jennifer E.; Liu, Yuwei; Wang, Pengrui; Zanni, Martin T.; Chen, Zhan

    2013-01-01

    Sum-frequency generation (SFG) vibrational spectroscopy is often used to probe the backbone structures and orientations of polypeptides at surfaces. Using the ovispirin-1 polypeptide at the solid/liquid interface of polystyrene, we demonstrate for the first time that SFG can probe the polarization response of a single isotope labeled residue. To interpret the spectral intensities, we simulated the spectra using an excitonic Hamiltonian approach. We show that the polarization dependence of either the label or the unlabeled amide I band alone does not provide sufficient structural constraints to obtain both the tilt and the twist of the ovispirin helix at a solid/liquid interface, but that both can be determined from the polarization dependence of the complete spectrum. For ovispirin, the detailed analysis of the polarized SFG experimental data shows that the helix axis is tilted at roughly 138 degrees from the surface normal, and the transition dipole of the isotope labeled C=O group is tilted at 23 degrees from the surface normal, with the hydrophobic region facing the polystyrene surface. We further demonstrated that the Hamiltonian approach is able to address the coupling effect and the structural disorder. For comparison, we also collected the FTIR spectrum of ovispirin under similar conditions, which reveals the enhanced sensitivity of SFG for structural studies of single monolayer peptide surfaces. Our study provides insight into how structural and environmental effects appear in SFG spectra of the amide I band and establishes that SFG of isotope labeled peptides will be a powerful technique for elucidating secondary structures with residue-by-residue resolution. PMID:24228619

  10. Site-specific orientation of an α-helical peptide ovispirin-1 from isotope-labeled SFG spectroscopy.

    Science.gov (United States)

    Ding, Bei; Laaser, Jennifer E; Liu, Yuwei; Wang, Pengrui; Zanni, Martin T; Chen, Zhan

    2013-11-27

    Sum-frequency generation (SFG) vibrational spectroscopy is often used to probe the backbone structures and orientations of polypeptides at surfaces. Using the ovispirin-1 polypeptide at the solid/liquid interface of polystyrene, we demonstrate for the first time that SFG can probe the polarization response of a single-isotope-labeled residue. To interpret the spectral intensities, we simulated the spectra using an excitonic Hamiltonian approach. We show that the polarization dependence of either the label or the unlabeled amide I band alone does not provide sufficient structural constraints to obtain both the tilt and the twist of the ovispirin helix at a solid/liquid interface, but that both can be determined from the polarization dependence of the complete spectrum. For ovispirin, the detailed analysis of the polarized SFG experimental data shows that the helix axis is tilted at roughly 138° from the surface normal, and the transition dipole of the isotope-labeled C═O group is tilted at 23° from the surface normal, with the hydrophobic region facing the polystyrene surface. We further demonstrate that the Hamiltonian approach is able to address the coupling effect and the structural disorder. For comparison, we also collected the FTIR spectrum of ovispirin under similar conditions, which reveals the enhanced sensitivity of SFG for structural studies of single monolayer peptide surfaces. Our study provides insight into how structural and environmental effects appear in SFG spectra of the amide I band and establishes that SFG of isotope-labeled peptides will be a powerful technique for elucidating secondary structures with residue-by-residue resolution.

  11. Specificity Determinants” Improve Therapeutic Indices of Two Antimicrobial Peptides Piscidin 1 and Dermaseptin S4 Against the Gram-negative Pathogens Acinetobacter baumannii and Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Ziqing Jiang

    2014-03-01

    Full Text Available A new class of antimicrobial agents with lower rates of resistance and different targets is urgently needed because of the rapidly increasing resistance to classical antibiotics. Amphipathic cationic α-helical antimicrobial peptides (AMPs represent such a class of compounds. In our previous studies, using a 26-residue de novo designed antimicrobial peptide, we proposed the concept of “specificity determinant(s”: positively charged residue(s in the center of the non-polar face of AMPs that could decrease hemolytic activity/toxicity but increase or maintain the same level of antimicrobial activity to increase dramatically the therapeutic index. In the current study, we used d-enantiomers of two AMPs, Piscidin 1 isolated from fish and dermaseptin S4 isolated from frog. We substituted different positions in the center of the hydrophobic face with one or two lysine residue(s (one or two “specificity determinant(s”. This simple modification not only maintained or improved antimicrobial activity against Gram-negative pathogens Acinetobacter baumannii (11 strains and Pseudomonas aeruginosa (6 strains, but also dramatically decreased hemolytic activity of human red blood cells, as predicted. Therapeutic indices improved by 55-fold and 730-fold for piscidin 1 (I9K and dermaseptin S4 (L7K, A14K, respectively, against A. baumannii. Similarly, the therapeutic indices improved 32-fold and 980-fold for piscidin 1 (I9K and dermaseptin S4 (L7K, A14K, respectively, against P. aeruginosa.

  12. Analysis of binding properties and specificity through identification of the interface forming residues (IFR for serine proteases in silico docked to different inhibitors

    Directory of Open Access Journals (Sweden)

    da Silveira Carlos H

    2010-10-01

    Full Text Available Abstract Background Enzymes belonging to the same super family of proteins in general operate on variety of substrates and are inhibited by wide selection of inhibitors. In this work our main objective was to expand the scope of studies that consider only the catalytic and binding pocket amino acids while analyzing enzyme specificity and instead, include a wider category which we have named the Interface Forming Residues (IFR. We were motivated to identify those amino acids with decreased accessibility to solvent after docking of different types of inhibitors to sub classes of serine proteases and then create a table (matrix of all amino acid positions at the interface as well as their respective occupancies. Our goal is to establish a platform for analysis of the relationship between IFR characteristics and binding properties/specificity for bi-molecular complexes. Results We propose a novel method for describing binding properties and delineating serine proteases specificity by compiling an exhaustive table of interface forming residues (IFR for serine proteases and their inhibitors. Currently, the Protein Data Bank (PDB does not contain all the data that our analysis would require. Therefore, an in silico approach was designed for building corresponding complexes The IFRs are obtained by "rigid body docking" among 70 structurally aligned, sequence wise non-redundant, serine protease structures with 3 inhibitors: bovine pancreatic trypsin inhibitor (BPTI, ecotine and ovomucoid third domain inhibitor. The table (matrix of all amino acid positions at the interface and their respective occupancy is created. We also developed a new computational protocol for predicting IFRs for those complexes which were not deciphered experimentally so far, achieving accuracy of at least 0.97. Conclusions The serine proteases interfaces prefer polar (including glycine residues (with some exceptions. Charged residues were found to be uniquely prevalent at the

  13. Analysis of binding properties and specificity through identification of the interface forming residues (IFR) for serine proteases in silico docked to different inhibitors.

    Science.gov (United States)

    Ribeiro, Cristina; Togawa, Roberto C; Neshich, Izabella A P; Mazoni, Ivan; Mancini, Adauto L; Minardi, Raquel C de Melo; da Silveira, Carlos H; Jardine, José G; Santoro, Marcelo M; Neshich, Goran

    2010-10-20

    Enzymes belonging to the same super family of proteins in general operate on variety of substrates and are inhibited by wide selection of inhibitors. In this work our main objective was to expand the scope of studies that consider only the catalytic and binding pocket amino acids while analyzing enzyme specificity and instead, include a wider category which we have named the Interface Forming Residues (IFR). We were motivated to identify those amino acids with decreased accessibility to solvent after docking of different types of inhibitors to sub classes of serine proteases and then create a table (matrix) of all amino acid positions at the interface as well as their respective occupancies. Our goal is to establish a platform for analysis of the relationship between IFR characteristics and binding properties/specificity for bi-molecular complexes. We propose a novel method for describing binding properties and delineating serine proteases specificity by compiling an exhaustive table of interface forming residues (IFR) for serine proteases and their inhibitors. Currently, the Protein Data Bank (PDB) does not contain all the data that our analysis would require. Therefore, an in silico approach was designed for building corresponding complexes. The IFRs are obtained by "rigid body docking" among 70 structurally aligned, sequence wise non-redundant, serine protease structures with 3 inhibitors: bovine pancreatic trypsin inhibitor (BPTI), ecotine and ovomucoid third domain inhibitor. The table (matrix) of all amino acid positions at the interface and their respective occupancy is created. We also developed a new computational protocol for predicting IFRs for those complexes which were not deciphered experimentally so far, achieving accuracy of at least 0.97. The serine proteases interfaces prefer polar (including glycine) residues (with some exceptions). Charged residues were found to be uniquely prevalent at the interfaces between the "miscellaneous-virus" subfamily

  14. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2004-01-01

    A novel class of compounds known as peptide nucleic acids, bind complementary DNA and RNA strands, and generally do so more strongly than the corresponding DNA or RNA strands while exhibiting increased sequence specificity and solubility. The peptide nucleic acids comprise ligands selected from...

  15. The intestinotrophic peptide, GLP-2, counteracts the gastrointestinal atrophy in mice induced by the epidermal growth factor receptor inhibitor, erlotinib, and cisplatin

    DEFF Research Database (Denmark)

    Rasmussen, Andreas Rosén; Viby, Niels-Erik; Hare, Kristine Juul

    2010-01-01

    Erlotinib, an epidermal-growth-factor receptor inhibitor, belongs to a new generation of targeted cancer therapeutics. Gastrointestinal side-effects are common and have been markedly aggravated when erlotinib is combined with cytostatics. We examined the effects of erlotinib alone and combined wi...

  16. Effect of the renal natriuretic peptide, ularitide, alone or combined ...

    African Journals Online (AJOL)

    Effect of the renal natriuretic peptide, ularitide, alone or combined with ... inhibitor, Omapatrilat, on experimental volume overloadinduced congestive heart failure in ... N-terminal pro–brain natriuretic peptide (NT-proBNP) and high-sensitivity ...

  17. Targeting the active site of the placental isozyme of alkaline phosphatase by phage-displayed scFv antibodies selected by a specific uncompetitive inhibitor

    Directory of Open Access Journals (Sweden)

    Kala Mrinalini

    2005-12-01

    Full Text Available Abstract Background The isozymes of alkaline phosphatase, the tissue non-specific, intestinal and placental, have similar properties and a high degree of identity. The placental isozyme (PLAP is an oncofetal antigen expressed in several malignancies including choriocarcinoma, seminoma and ovarian carcinoma. We had earlier attempted to isolate PLAP-specific scFv from a synthetic human immunoglobulin library but were unable to do so, presumably because of the similarity between the isozymes. In this work, we have employed a PLAP-specific uncompetitive inhibitor, L-Phe-Gly-Gly, to select isozyme specific scFvs. An uncompetitive inhibitor binds to the enzyme in the presence of substrate and stabilizes the enzyme-substrate complex. Several uncompetitive inhibitors have varying degrees of isozyme specificity for human alkaline phosphatase isozymes. A specific uncompetitive inhibitor would be able to unmask conformational differences between the otherwise very similar molecules. Also, such inhibitors would be directed to regions at/close to the active site of the enzyme. In this work, the library was first incubated with PLAP and the bound clones then eluted by incubation with L-Phe-Gly-Gly along with the substrate, para-nitro phenyl phosphate (pNPP. The scFvs were then studied with regard to the biochemical modulation of their binding, isozyme specificity and effect on enzyme activity. Results Of 13 clones studied initially, the binding of 9 was inhibited by L-Phe-Gly-Gly (with pNPP and 2 clones were inhibited by pNPP alone. Two clones had absolute and 2 clones had partial specificity to PLAP. Two clones were cross-reactive with only one other isozyme. Three scFv clones, having an accessible His6-tag, were purified and studied for their modulation of enzyme activity. All the three scFvs inhibited PLAP activity with the kinetics of competitive inhibition. Cell ELISA could demonstrate binding of the specific scFvs to the cell surface expressed PLAP

  18. 99mTc-labeling and evaluation of a HYNIC modified small-molecular inhibitor of prostate-specific membrane antigen

    International Nuclear Information System (INIS)

    Xu, Xiaoping; Zhang, Jianping; Hu, Silong; He, Simin; Bao, Xiao; Ma, Guang; Luo, Jianmin; Cheng, Jingyi; Zhang, Yingjian

    2017-01-01

    Introduction: Prostate-specific membrane antigen (PSMA) is a well-established target in the development of radiopharmaceuticals for the diagnosis and therapy of prostate cancer (PCa). In this study, we evaluated a novel 99m Tc-labeled small molecular inhibitor of PSMA. Methods: This new small-molecular inhibitor of PSMA, 6-hydrazinonicotinate-Aminocaproic acid-Lysine-Urea-Glutamate (HYNIC-ALUG) was radiolabeled by 99m Tc and was evaluated both in vitro and in vivo using PCa models (PC-3 and LNCaP). Radiation dosimetry was assessed in mice. Results: 99m Tc-HYNIC-ALUG showed excellent stability in different media. A cell assay preliminarily displayed its specificity for PSMA. The inhibitor showed good pharmacokinetics making it suitable for in vivo imaging. PC-3-derived tumors showed no obvious radioactive uptake; however, the LNCaP-derived tumors showed very high radioactive uptake which was significantly decreased by the selective PSMA inhibitor 2-PMPA. Biodistribution in LNCaP xenografts showed an optimum tumor-to-blood ratio of 24.23 ± 3.54 at 2 h. Tumor uptake was also decreased in the inhibition experiment with 2-PMPA (19.45 ± 2.14%ID/g versus 1.42 ± 0.15%ID/g at 2 h). The effective dose of the 99m Tc-HYNIC-ALUG was 8.4E-04 mSv/MBq. Conclusions: A new 99m Tc-labeled PSMA inhibitor with specific accumulation in PSMA-positive tumors and low background in other organs was synthesized. The radiopharmaceutical also showed very low radiation dosimetry. This agent may significantly improve the diagnosis, staging, and subsequent monitoring of therapeutic effects in PCa patients.

  19. Study of the peptide length and amino acid specific substitution in the antigenic activity of the chimeric synthetic peptides, containing the p19 core and gp46 envelope proteins of the HTLV-I virus.

    Science.gov (United States)

    Marin, Milenen Hernández; Rodríguez-Tanty, Chryslaine; Higginson-Clarke, David; Bocalandro, Yadaris Márquez; Peña, Lilliam Pozo

    2005-10-28

    Four chimeric synthetic peptides (Q5, Q6, Q7(multiply sign in circle), and Q8(multiply sign in circle)), incorporating immunodominant epitopes of the core p19 (105-124 a.a.) and envelope gp46 proteins (175-205 a.a.), of HTLV-I were obtained. Also, two gp46 monomeric peptides M4 and M5(multiply sign in circle) (Ser at position 192) were synthesized. The analysis of the influence of the peptide lengths and the proline to serine substitution on the chimeric and monomeric peptides' antigenicity, with regard to the chimeric peptides Q1, Q2, Q3(multiply sign in circle), and Q4(multiply sign in circle), reported previously, for HTLV-I was carried out. The peptides' antigenicity was evaluated in an ultramicroenzyme-linked immunosorbent assay (UMELISA) using sera of HTLV-I/II. The peptides' antigenicity was affected appreciably by the change of the peptide length and amino acid substitutions into the immunodominant sequence of gp46 peptide.

  20. Subtype-specific suppression of Shiga toxin 2 released from Escherichia coli upon exposure to protein synthesis inhibitors

    DEFF Research Database (Denmark)

    Pedersen, Malene Gantzhorn; Hansen, Claus; Riise, Erik

    2008-01-01

    Shiga toxins (Stx) are important virulence factors in the pathogenesis of severe disease including hemolytic-uremic syndrome, caused by Stx-producing Escherichia coli (STEC). STEC strains increase the release of Stx in vitro following the addition of fluoroquinolones, whereas protein synthesis...... inhibitors previously have been reported to suppress the release of Stx. The amount of Stx released from wild-type STEC strains incubated with protein synthesis inhibitors was examined by a Vero cell cytotoxicity assay. The amounts released were compared to the Stx type (Stx1 or Stx2) and additionally...... to the individual subtypes and toxin variants of Stx2. In general, Stx2 release was suppressed significantly upon exposure to protein synthesis inhibitors at MICs, which was not observed in the case of Stx1. Also, the average amount of different Stx2 toxin variants released was suppressed to various levels ranging...

  1. Oral administration of type-II collagen peptide 250-270 suppresses specific cellular and humoral immune response in collagen-induced arthritis.

    Science.gov (United States)

    Zhu, Ping; Li, Xiao-Yan; Wang, Hong-Kun; Jia, Jun-Feng; Zheng, Zhao-Hui; Ding, Jin; Fan, Chun-Mei

    2007-01-01

    Oral antigen is an attractive approach for the treatment of autoimmune and inflammatory diseases. Establishment of immune markers and methods in evaluating the effects of antigen-specific cellular and humoral immune responses will help the application of oral tolerance in the treatment of human diseases. The present article observed the effects of chicken collagen II (CII), the recombinant polymerized human collagen II 250-270 (rhCII 250-270) peptide and synthesized human CII 250-270 (syCII 250-270) peptide on the induction of antigen-specific autoimmune response in rheumatoid arthritis (RA) peripheral blood mononuclear cells (PBMC) and on the specific cellular and humoral immune response in collagen-induced arthritis (CIA) and mice fed with CII (250-270) prior to immunization with CII. In the study, proliferation, activation and intracellular cytokine production of antigen-specific T lymphocytes were simultaneously analyzed by bromodeoxyuridine (BrdU) incorporation and flow cytometry at the single-cell level. The antigen-specific antibody and antibody-forming cells were detected by ELISA and ELISPOT, respectively. CII (250-270) was found to have stimulated the response of specific lymphocytes in PBMC from RA patients, including the increase expression of surface activation antigen marker CD69 and CD25, and DNA synthesis. Mice, fed with CII (250-270) before CII immunization, had significantly lower arthritic scores than the mice immunized with CII alone, and the body weight of the former increased during the study period. Furthermore, the specific T cell activity, proliferation and secretion of interferon (IFN)-gamma in spleen cells were actively suppressed in CII (250-270)-fed mice, and the serum anti-CII, anti-CII (250-270) antibody activities and the frequency of specific antibody-forming spleen cells were significantly lower in CII (250-270)-fed mice than in mice immunized with CII alone. These observations suggest that oral administration of CII (250-270) can

  2. Substrate and Inhibitor-Specific Conformational Changes in the Human Serotonin Transporter Revealed by Voltage-Clamp Fluorometry

    DEFF Research Database (Denmark)

    Söderhielm, Pella C; Andersen, Jacob; Munro, Lachlan

    2015-01-01

    of TM6, Ala419 in the interface between TM8 and extracellular loop (EL) 4, and Leu481 in EL5. The reporter positions were used for time-resolved measurement of conformational changes during 5-HT transport and binding of cocaine and the selective serotonin reuptake inhibitors fluoxetine and escitalopram...... changes overall, which included movements within or around TM1b, EL4, and EL5. Taken together, our data lead us to suggest that competitive inhibitors stabilize hSERT in a state that is different from the apo outward-open conformation as well as inward-facing conformations....

  3. Association of Distinct Fine Specificities of Anti-Citrullinated Peptide Antibodies With Elevated Immune Responses to Prevotella intermedia in a Subgroup of Patients With Rheumatoid Arthritis and Periodontitis.

    Science.gov (United States)

    Schwenzer, Anja; Quirke, Anne-Marie; Marzeda, Anna M; Wong, Alicia; Montgomery, Anna B; Sayles, Harlan R; Eick, Sigrun; Gawron, Katarzyna; Chomyszyn-Gajewska, Maria; Łazarz-Bartyzel, Katarzyna; Davis, Simon; Potempa, Jan; Kessler, Benedikt M; Fischer, Roman; Venables, Patrick J; Payne, Jeffrey B; Mikuls, Ted R; Midwood, Kim S

    2017-12-01

    In addition to the long-established link with smoking, periodontitis (PD) is a risk factor for rheumatoid arthritis (RA). This study was undertaken to elucidate the mechanism by which PD could induce antibodies to citrullinated peptides (ACPAs), by examining the antibody response to a novel citrullinated peptide of cytokeratin 13 (CK-13) identified in gingival crevicular fluid (GCF), and comparing the response to 4 other citrullinated peptides in patients with RA who were well-characterized for PD and smoking. The citrullinomes of GCF and periodontal tissue from patients with PD were mapped by mass spectrometry. ACPAs of CK13 (cCK13), tenascin-C (cTNC5), vimentin (cVIM), α-enolase (CEP-1), and fibrinogen β (cFIBβ) were examined by enzyme-linked immunosorbent assay in patients with RA (n = 287) and patients with osteoarthritis (n = 330), and cross-reactivity was assessed by inhibition assays. A novel citrullinated peptide cCK13-1 ( 444 TSNASGR-Cit-TSDV-Cit-RP 458 ) identified in GCF exhibited elevated antibody responses in RA patients (24%). Anti-cCK13-1 antibody levels correlated with anti-cTNC5 antibody levels, and absorption experiments confirmed this was not due to cross-reactivity. Only anti-cCK13-1 and anti-cTNC5 were associated with antibodies to the periodontal pathogen Prevotella intermedia (P = 0.05 and P = 0.001, respectively), but not with antibodies to Porphyromonas gingivalis arginine gingipains. Levels of antibodies to CEP-1, cFIBβ, and cVIM correlated with each other, and with smoking and shared epitope risk factors in RA. This study identifies 2 groups of ACPA fine specificities associated with different RA risk factors. One is predominantly linked to smoking and shared epitope, and the other links anti-cTNC5 and cCK13-1 to infection with the periodontal pathogen P intermedia. © 2017 The Authors. Arthritis & Rheumatology published by Wiley Periodicals, Inc. on behalf of American College of Rheumatology.

  4. Combination of high pressure liquid chromatography and radioimmunoassay is a powerful tool for the specific and quantitative determination of endorphins and related peptides

    NARCIS (Netherlands)

    Loeber, J.G.; Verhoef, J.; Burbach, J.P.H.; Witter, A.

    1979-01-01

    A method for the separation and subsequent quantification of endorphins and related peptides was developed. Separation of the peptides was achieved by high pressure liquid chromatography on a reversed phase column. By virtue of the high resolving capacity of this system peptides differing in only

  5. Isozyme-specific enzyme inhibitors. 14. 5'(R)-C-[(L-homocystein-S-yl)methyl]adenosine 5'-(beta,gamma-imidotriphosphate), a potent inhibitor of rat methionine adenosyltransferases.

    Science.gov (United States)

    Kappler, F; Vrudhula, V M; Hampton, A

    1987-09-01

    The title compound is a covalent adduct of L-methionine (Met) and beta,gamma-imido-ATP. In its synthesis the N-Boc derivative of 5'(R)-C-(aminomethyl)-N6-benzoyl-5'-O-tosyl-2',3'-O- isopropylidenadenosine was converted by the successive actions of CF3CO2H and HNO2 into the corresponding 5'(R)-C-hydroxymethyl derivative. Treatment of this with disodium L-homocysteinate led to attack of sulfur at C6', apparently via a 5',6'-epoxide, and to total stereoselective inversion at C5' to furnish, after debenzoylation, 5'(R)-C-(L-homocystein-S-ylmethyl)-2',3'-O-isopropylidene ade nosine. The 5' configuration was established by conversion of this into the known 5'(S)-C-methyl-2',3'-O-isopropylidene adenosine with Raney nickel. The alpha-amino acid residue was protected as an N-Boc methyl ester, after which the 5'-hydroxyl was phosphorylated with benzyl phosphate and dicyclohexylcarbodiimide. The phosphoanhydride bond with inorganic imidodiphosphate was then created by established methods. Finally, blocking groups were removed under conditions that gave the desired adduct with no racemization of its L-methionine residue. It was a potent inhibitor [KM(ATP)/Ki = 1080; KM(Met)/Ki = 7.7] of the M-2 (normal tissue) form of rat methionine adenosyltransferase and of the M-T (hepatoma tissue) form [KM(ATP)/Ki = 670; KM(Met)/Ki = 22]. Inhibitions were competitive with respect to ATP or to L-methionine, indicating a dual substrate site mode of binding to the enzyme forms.

  6. Inhibitors of dual-specificity tyrosine phosphorylation-regulated kinases (DYRK) exert a strong anti-herpesviral activity.

    Science.gov (United States)

    Hutterer, Corina; Milbradt, Jens; Hamilton, Stuart; Zaja, Mirko; Leban, Johann; Henry, Christophe; Vitt, Daniel; Steingruber, Mirjam; Sonntag, Eric; Zeitträger, Isabel; Bahsi, Hanife; Stamminger, Thomas; Rawlinson, William; Strobl, Stefan; Marschall, Manfred

    2017-07-01

    Infection with human cytomegalovirus (HCMV) is a serious medical problem, particularly in immunocompromised individuals and neonates. The success of (val)ganciclovir therapy is hampered by low drug compatibility and induction of viral resistance. A novel strategy of antiviral treatment is based on the exploitation of cell-directed signaling, e. g. pathways with a known relevance for carcinogenesis and tumor drug development. Here we describe a principle for putative antiviral drugs based on targeting dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs). DYRKs constitute an evolutionarily conserved family of protein kinases with key roles in the control of cell proliferation and differentiation. Members of the DYRK family are capable of phosphorylating a number of substrate proteins, including regulators of the cell cycle, e.g. DYRK1B can induce cell cycle arrest, a critical step for the regulation of HCMV replication. Here we provide first evidence for a critical role of DYRKs during viral replication and the high antiviral potential of DYRK inhibitors (SC84227, SC97202 and SC97208, Harmine and AZ-191). Using established replication assays for laboratory and clinically relevant strains of HCMV, concentration-dependent profiles of inhibition were obtained. Mean inhibitory concentrations (EC50) of 0.98 ± 0.08 μM/SC84227, 0.60 ± 0.02 μM/SC97202, 6.26 ± 1.64 μM/SC97208, 0.71 ± 0.019 μM/Harmine and 0.63 ± 0.23 μM/AZ-191 were determined with HCMV strain AD169-GFP for the infection of primary human fibroblasts. A first analysis of the mode of antiviral action suggested a block of viral replication at the early-late stage of HCMV gene expression. Moreover, rhesus macaque cytomegalovirus (RhCMV), varicella-zoster virus (VZV) and herpes simplex virus (HSV-1) showed a similarly high sensitivity to these compounds. Thus, we conclude that DYRK signaling represents a promising target pathway for the development of novel anti

  7. Antimicrobial Peptides in Reptiles

    Science.gov (United States)

    van Hoek, Monique L.

    2014-01-01

    Reptiles are among the oldest known amniotes and are highly diverse in their morphology and ecological niches. These animals have an evolutionarily ancient innate-immune system that is of great interest to scientists trying to identify new and useful antimicrobial peptides. Significant work in the last decade in the fields of biochemistry, proteomics and genomics has begun to reveal the complexity of reptilian antimicrobial peptides. Here, the current knowledge about antimicrobial peptides in reptiles is reviewed, with specific examples in each of the four orders: Testudines (turtles and tortosises), Sphenodontia (tuataras), Squamata (snakes and lizards), and Crocodilia (crocodilans). Examples are presented of the major classes of antimicrobial peptides expressed by reptiles including defensins, cathelicidins, liver-expressed peptides (hepcidin and LEAP-2), lysozyme, crotamine, and others. Some of these peptides have been identified and tested for their antibacterial or antiviral activity; others are only predicted as possible genes from genomic sequencing. Bioinformatic analysis of the reptile genomes is presented, revealing many predicted candidate antimicrobial peptides genes across this diverse class. The study of how these ancient creatures use antimicrobial peptides within their innate immune systems may reveal new understandings of our mammalian innate immune system and may also provide new and powerful antimicrobial peptides as scaffolds for potential therapeutic development. PMID:24918867

  8. Vicilin-like peptides from Capsicum baccatum L. seeds are α-amylase inhibitors and exhibit antifungal activity against important yeasts in medical mycology.

    Science.gov (United States)

    Vieira Bard, Gabriela C; Nascimento, Viviane V; Oliveira, Antônia Elenir A; Rodrigues, Rosana; Da Cunha, Maura; Dias, Germana B; Vasconcelos, Ilka M; Carvalho, Andre O; Gomes, Valdirene M

    2014-07-01

    The objective of this study was to isolate antimicrobial peptides from Capsicum baccatum seeds and evaluate their antimicrobial activity and inhibitory effects against α-amylase. Initially, proteins from the flour of C. baccatum seeds were extracted in sodium phosphate buffer, pH 5.4, and precipitated with ammonium sulfate at 90% saturation. The D1 and D2 fractions were subjected to antifungal tests against the yeasts Saccharomyces cerevisiae, Candida albicans, Candida tropicalis, and Kluyveromyces marxiannus, and tested against α-amylases from Callosobruchus maculates and human saliva. The D2 fraction presented higher antimicrobial activity and was subjected to further purification and seven new different fractions (H1-H7) were obtained. Peptides in the H4 fraction were sequenced and the N-terminal sequences revealed homology with previously reported storage vicilins from seeds. The H4 fraction exhibited strong antifungal activity and also promoted morphological changes in yeast, including pseudohyphae formation. All fractions, including H4, inhibited mammalian α-amylase activity but only the H4 fraction was able to inhibit C. maculatus α-amylase activity. These results suggest that the fractions isolated from the seeds of C. baccatum can act directly in plant defenses against pathogens and insects. © 2014 Wiley Periodicals, Inc.

  9. A Specific Inhibitor of TGF-β Receptor Kinase, SB-431542, as a Potent Antitumor Agent for Human Cancers

    Directory of Open Access Journals (Sweden)

    Sunil K. Halder

    2005-05-01

    Full Text Available Small molecule inhibitors of signaling pathways have proven to be extremely useful for the development of therapeutic strategies for human cancers. Blocking the tumor-promoting effects of transforming growth factor-β (TGF-β in advanced stage carcinogenesis provides a potentially interesting drug target for therapeutic intervention. Although very few TGF-β receptor kinase inhibitors (TRKI are now emerging in preclinical studies, nothing is known about how these inhibitors might regulate the tumor-suppressive or tumor-promoting effects of TGF-β, or when these inhibitors might be useful for treatment during cancer progression. We have investigated the potential of TRKI in new therapeutic approaches in preclinical models. Here, we demonstrate that the TRKI, SB-431542, inhibits TGF-β-induced transcription, gene expression, apoptosis, and growth suppression. We have observed that SB-431542 attenuates the tumor-promoting effects of TGF-β, including TGF-β-induced EMT, cell motility, migration and invasion, and vascular endothelial growth factor secretion in human cancer cell lines. Interestingly, SB-431542 induces anchorage independent growth of cells that are growth-inhibited by TGF-β, whereas it reduces colony formation by cells that are growth-promoted by TGF-β. However, SB-431542 has no effect on a cell line that failed to respond to TGF-β. This represents a novel potential application of these inhibitors as therapeutic agents for human cancers with the goal of blocking tumor invasion, angiogenesis, and metastasis, when tumors are refractory to TGF-β-induced tumor-suppressor functions but responsive to tumor-promoting effects of TGF-β.

  10. Seed-specific stable expression of the alpha-AI1 inhibitor in coffee grains and the in vivo implications for the development of the coffee berry borer

    OpenAIRE

    Albuquerque, E. V. S.; Bezerra, C. A.; Romero, J. V.; Valencia, J. W. A.; Valencia-Jimenez, A.; Pimenta, L. M.; Barbosa, Aead; Silva, M. C. M.; Meneguim, A. M.; Sa, M. E. L.; Engler, G.; de Almeida-Engler, J.; Fernandez, Diana; Grossi-de-Sa, M. F.

    2015-01-01

    Genetic transformation of coffee (Coffea spp.), the second most traded commodity worldwide, is an alternative approach to introducing features that cannot be introgressed by traditional crossings. The transgenic stability, heritability and quantitative and spatial expression patterns of the seed-specific promoter phytohemagglutinin (PHA-L) from Phaseolus vulgaris were characterized in genetically modified C. arabica expressing the alpha-amylase inhibitor-1 (alpha-AI1) gene. The alpha-AI1 inhi...

  11. Human amyloid β peptide and tau co-expression impairs behavior and causes specific gene expression changes in Caenorhabditis elegans.