Science.gov

Sample records for active site inhibitors

  1. Activation of Inhibitors by Sortase Triggers Irreversible Modification of the Active Site*S

    PubMed Central

    Maresso, Anthony W.; Wu, Ruiying; Kern, Justin W.; Zhang, Rongguang; Janik, Dorota; Missiakas, Dominique M.; Duban, Mark-Eugene; Joachimiak, Andrzej; Schneewind, Olaf

    2011-01-01

    Sortases anchor surface proteins to the cell wall of Gram-positive pathogens through recognition of specific motif sequences. Loss of sortase leads to large reductions in virulence, which identifies sortase as a target for the development of antibacterials. By screening 135,625 small molecules for inhibition, we report here that aryl (β-amino)ethyl ketones inhibit sortase enzymes from staphylococci and bacilli. Inhibition of sortases occurs through an irreversible, covalent modification of their active site cysteine. Sortases specifically activate this class of molecules via β-elimination, generating a reactive olefin intermediate that covalently modifies the cysteine thiol. Analysis of the three-dimensional structure of Bacillus anthracis sortase B with and without inhibitor provides insights into the mechanism of inhibition and reveals binding pockets that can be exploited for drug discovery. PMID:17545669

  2. Active site - a site of binding of affinity inhibitors in baker's yeast inorganic pyrophosphatase

    SciTech Connect

    Svyato, I.E.; Sklyankina, V.A.; Avaeva, S.M.

    1986-03-20

    The interaction of the enzyme-substrate complex with methyl phosphate, O-phosphoethanolamine, O-phosphopropanolamine, N-acetylphosphoserine, and phosphoglyolic acid, as well as pyrophosphatase, modified by monoesters of phosphoric acid, with pyrophosphate and tripolyphosphate, was investigated. It was shown that the enzyme containing the substrate in the active site does not react with monophosphates, but modified pyrophosphatase entirely retains the ability to bind polyanions to the regulatory site. It is concluded that the inactivation of baker's yeast inorganic pyrophosphatase by monoesters of phosphoric acid, which are affinity inhibitors of it, is the result of modification of the active site of the enzyme.

  3. Identification of inhibitors against the potential ligandable sites in the active cholera toxin.

    PubMed

    Gangopadhyay, Aditi; Datta, Abhijit

    2015-04-01

    The active cholera toxin responsible for the massive loss of water and ions in cholera patients via its ADP ribosylation activity is a heterodimer of the A1 subunit of the bacterial holotoxin and the human cytosolic ARF6 (ADP Ribosylation Factor 6). The active toxin is a potential target for the design of inhibitors against cholera. In this study we identified the potential ligandable sites of the active cholera toxin which can serve as binding sites for drug-like molecules. By employing an energy-based approach to identify ligand binding sites, and comparison with the results of computational solvent mapping, we identified two potential ligandable sites in the active toxin which can be targeted during structure-based drug design against cholera. Based on the probe affinities of the identified ligandable regions, docking-based virtual screening was employed to identify probable inhibitors against these sites. Several indole-based alkaloids and phosphates showed strong interactions to the important residues of the ligandable region at the A1 active site. On the other hand, 26 top scoring hits were identified against the ligandable region at the A1 ARF6 interface which showed strong hydrogen bonding interactions, including guanidines, phosphates, Leucopterin and Aristolochic acid VIa. This study has important implications in the application of hybrid structure-based and ligand-based methods against the identified ligandable sites using the identified inhibitors as reference ligands, for drug design against the active cholera toxin.

  4. Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol*

    PubMed Central

    Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.; Fiamengo, Bryan A.; Foley, Sage E.; Frank, Kristine E.; George, Jonathan S.; Harris, Christopher M.; Hobson, Adrian D.; Ihle, David C.; Marcotte, Douglas; Merta, Philip J.; Michalak, Mark E.; Murdock, Sara E.; Tomlinson, Medha J.; Voss, Jeffrey W.

    2015-01-01

    The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC50 < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases. PMID:25552479

  5. Detection of allosteric kinase inhibitors by displacement of active site probes.

    PubMed

    Lebakken, Connie S; Reichling, Laurie J; Ellefson, Jason M; Riddle, Steven M

    2012-07-01

    Non-adenosine triphosphate (ATP) competitive, allosteric inhibitors provide a promising avenue to develop highly selective small-molecule kinase inhibitors. Although this class of compounds is growing, detection of such inhibitors can be challenging as standard kinase activity assays preferentially detect compounds that bind to active kinases in an ATP competitive manner. We have previously described a time-resolved fluorescence resonance energy transfer (TR-FRET)-based kinase binding assay using the competitive displacement of ATP competitive active site fluorescent probes ("tracers"). Although this format has gained acceptance, published data with this and related formats are almost entirely without examples of non-ATP competitive compounds. Thus, this study addresses whether this format is useful for non-ATP competitive inhibitors. To this end, 15 commercially available non-ATP competitive inhibitors were tested for their ability to displace ATP competitive probes. Despite the diversity of both compound structures and their respective targets, 14 of the 15 compounds displaced the tracers with IC(50) values comparable to literature values. We conclude that such binding assays are well suited for the study of non-ATP competitive inhibitors. In addition, we demonstrate that allosteric inhibitors of BCR-Abl and MEK bind preferentially to the nonphosphorylated (i.e., inactive) form of the kinase, indicating that binding assays may be a preferred format in some cases.

  6. Complement receptor 2-mediated targeting of complement inhibitors to sites of complement activation.

    PubMed

    Song, Hongbin; He, Chun; Knaak, Christian; Guthridge, Joel M; Holers, V Michael; Tomlinson, Stephen

    2003-06-01

    In a strategy to specifically target complement inhibitors to sites of complement activation and disease, recombinant fusion proteins consisting of a complement inhibitor linked to a C3 binding region of complement receptor (CR) 2 were prepared and characterized. Natural ligands for CR2 are C3 breakdown products deposited at sites of complement activation. Fusion proteins were prepared consisting of a human CR2 fragment linked to either the N terminus or C terminus of soluble forms of the membrane complement inhibitors decay accelerating factor (DAF) or CD59. The targeted complement inhibitors bound to C3-opsonized cells, and all were significantly more effective (up to 20-fold) than corresponding untargeted inhibitors at protecting target cells from complement. CR2 fusion proteins also inhibited CR3-dependent adhesion of U937 cells to C3 opsonized erythrocytes, indicating a second potential anti-inflammatory mechanism of CR2 fusion proteins, since CR3 is involved in endothelial adhesion and diapedesis of leukocytes at inflammatory sites. Finally, the in vivo validity of the targeting strategy was confirmed by the demonstration that CR2-DAF, but not soluble DAF, targets to the kidney in mouse models of lupus nephritis that are associated with renal complement deposition.

  7. Complement receptor 2-mediated targeting of complement inhibitors to sites of complement activation.

    PubMed

    Song, Hongbin; He, Chun; Knaak, Christian; Guthridge, Joel M; Holers, V Michael; Tomlinson, Stephen

    2003-06-01

    In a strategy to specifically target complement inhibitors to sites of complement activation and disease, recombinant fusion proteins consisting of a complement inhibitor linked to a C3 binding region of complement receptor (CR) 2 were prepared and characterized. Natural ligands for CR2 are C3 breakdown products deposited at sites of complement activation. Fusion proteins were prepared consisting of a human CR2 fragment linked to either the N terminus or C terminus of soluble forms of the membrane complement inhibitors decay accelerating factor (DAF) or CD59. The targeted complement inhibitors bound to C3-opsonized cells, and all were significantly more effective (up to 20-fold) than corresponding untargeted inhibitors at protecting target cells from complement. CR2 fusion proteins also inhibited CR3-dependent adhesion of U937 cells to C3 opsonized erythrocytes, indicating a second potential anti-inflammatory mechanism of CR2 fusion proteins, since CR3 is involved in endothelial adhesion and diapedesis of leukocytes at inflammatory sites. Finally, the in vivo validity of the targeting strategy was confirmed by the demonstration that CR2-DAF, but not soluble DAF, targets to the kidney in mouse models of lupus nephritis that are associated with renal complement deposition. PMID:12813023

  8. Calorimetric studies of the interactions of metalloenzyme active site mimetics with zinc-binding inhibitors.

    PubMed

    Robinson, Sophia G; Burns, Philip T; Miceli, Amanda M; Grice, Kyle A; Karver, Caitlin E; Jin, Lihua

    2016-07-19

    The binding of drugs to metalloenzymes is an intricate process that involves several interactions, including binding of the drug to the enzyme active site metal, as well as multiple interactions between the drug and the enzyme residues. In order to determine the free energy contribution of Zn(2+) binding by known metalloenzyme inhibitors without the other interactions, valid active site zinc structural mimetics must be formed and binding studies need to be performed in biologically relevant conditions. The potential of each of five ligands to form a structural mimetic with Zn(2+) was investigated in buffer using Isothermal Titration Calorimetry (ITC). All five ligands formed strong 1 : 1 (ligand : Zn(2+)) binary complexes. The complexes were used in further ITC experiments to study their interaction with 8-hydroxyquinoline (8-HQ) and/or acetohydroxamic acid (AHA), two bidentate anionic zinc-chelating enzyme inhibitors. It was found that tetradentate ligands were not suitable for creating zinc structural mimetics for inhibitor binding in solution due to insufficient coordination sites remaining on Zn(2+). A stable binary complex, [Zn(BPA)](2+), which was formed by a tridentate ligand, bis(2-pyridylmethyl)amine (BPA), was found to bind one AHA in buffer or a methanol : buffer mixture (60 : 40 by volume) at pH 7.25 or one 8-HQ in the methanol : buffer mixture at pH 6.80, making it an effective structural mimetic for the active site of zinc metalloenzymes. These results are consistent with the observation that metalloenzyme active site zinc ions have three residues coordinated to them, leaving one or two sites open for inhibitors to bind. Our findings indicate that Zn(BPA)X2 can be used as an active site structural mimetic for zinc metalloenzymes for estimating the free energy contribution of zinc binding to the overall inhibitor active site interactions. Such use will help aid in the rational design of inhibitors to a variety of zinc metalloenzymes

  9. A new family of covalent inhibitors block nucleotide binding to the active site of pyruvate kinase

    PubMed Central

    Morgan, Hugh P.; Walsh, Martin J.; Blackburn, Elizabeth A.; Wear, Martin A.; Boxer, Matthew B.; Shen, Min; Mcnae, Iain W.; Nowicki, Matthew W.; Michels, Paul A. M.; Auld, Douglas S.; Fothergill-Gilmore, Linda A.; Walkinshaw, Malcolm D.

    2012-01-01

    SYNOPSIS Pyruvate kinase (PYK) plays a central role in the metabolism of many organisms and cell types, but the elucidation of the details of its function in a systems biology context has been hampered by the lack of specific high-affinity small molecule inhibitors. High-throughput screening has been used to identify a family of saccharin derivatives which inhibit Leishmania mexicana PYK (LmPYK) activity in a time- (and dose-) dependent manner; a characteristic of irreversible inhibition. The crystal structure of 4-[(1,1-dioxo-1,2-benzothiazol-3-yl)sulfanyl]benzoic acid (DBS) complexed with LmPYK shows that the saccharin moiety reacts with an active-site lysine residue (Lys335), forming a covalent bond and sterically hindering the binding of ADP/ATP. Mutation of the lysine residue to an arginine residue eliminated the effect of the inhibitor molecule, providing confirmation of the proposed inhibitor mechanism. This lysine residue is conserved in the active sites of the four human PYK isoenzymes, which were also found to be irreversibly inhibited by DBS. X-ray structures of PYK isoforms show structural differences at the DBS binding pocket, and this covalent inhibitor of PYK provides a chemical scaffold for the design of new families of potentially isoform-specific irreversible inhibitors. PMID:22906073

  10. Effect of reactive site loop elongation on the inhibitory activity of C1-inhibitor.

    PubMed

    Bos, Ineke G A; Lubbers, Yvonne T P; Eldering, Eric; Abrahams, Jan Pieter; Hack, C Erik

    2004-06-01

    The serine protease inhibitor C1-Inhibitor (C1-Inh) inhibits several complement- and contact-system proteases, which play an important role in inflammation. C1-Inh has a short reactive site loop (RSL) compared to other serpins. RSL length determines the inhibitory activity of serpins. We investigated the effect of RSL elongation on inhibitory activity of C1-Inh by insertion of one or two alanine residues in the RSL. One of five mutants had an increased association rate with kallikrein, but was nevertheless a poor inhibitor because of a simultaneous high stoichiometry of inhibition (>10). The association rate of the other variants was lower than that of wild-type C1-Inh. These data suggest that the relatively weak inhibitory activity of C1-Inh is not the result of its short RSL. The short RSL of C1-Inh has, surprisingly, the optimal length for inhibition.

  11. Structure-guided inhibitor design for human FAAH by interspecies active site conversion

    SciTech Connect

    Mileni, Mauro; Johnson, Douglas S.; Wang, Zhigang; Everdeen, Daniel S.; Liimatta, Marya; Pabst, Brandon; Bhattacharya, Keshab; Nugent, Richard A.; Kamtekar, Satwik; Cravatt, Benjamin F.; Ahn, Kay; Stevens, Raymond C.

    2008-11-24

    The integral membrane enzyme fatty acid amide hydrolase (FAAH) hydrolyzes the endocannabinoid anandamide and related amidated signaling lipids. Genetic or pharmacological inactivation of FAAH produces analgesic, anxiolytic, and antiinflammatory phenotypes but not the undesirable side effects of direct cannabinoid receptor agonists, indicating that FAAH may be a promising therapeutic target. Structure-based inhibitor design has, however, been hampered by difficulties in expressing the human FAAH enzyme. Here, we address this problem by interconverting the active sites of rat and human FAAH using site-directed mutagenesis. The resulting humanized rat (h/r) FAAH protein exhibits the inhibitor sensitivity profiles of human FAAH but maintains the high-expression yield of the rat enzyme. We report a 2.75-{angstrom} crystal structure of h/rFAAH complexed with an inhibitor, N-phenyl-4-(quinolin-3-ylmethyl)piperidine-1-carboxamide (PF-750), that shows strong preference for human FAAH. This structure offers compelling insights to explain the species selectivity of FAAH inhibitors, which should guide future drug design programs.

  12. A comparative structure-function analysis of active-site inhibitors of Vibrio cholerae cholix toxin.

    PubMed

    Lugo, Miguel R; Merrill, A Rod

    2015-09-01

    Cholix toxin from Vibrio cholerae is a novel mono-ADP-ribosyltransferase (mART) toxin that shares structural and functional properties with Pseudomonas aeruginosa exotoxin A and Corynebacterium diphtheriae diphtheria toxin. Herein, we have used the high-resolution X-ray structure of full-length cholix toxin in the apo form, NAD(+) bound, and 10 structures of the cholix catalytic domain (C-domain) complexed with several strong inhibitors of toxin enzyme activity (NAP, PJ34, and the P-series) to study the binding mode of the ligands. A pharmacophore model based on the active pose of NAD(+) was compared with the active conformation of the inhibitors, which revealed a cationic feature in the side chain of the inhibitors that may determine the active pose. Moreover, a conformational search was conducted for the missing coordinates of one of the main active-site loops (R-loop). The resulting structural models were used to evaluate the interaction energies and for 3D-QSAR modeling. Implications for a rational drug design approach for mART toxins were derived.

  13. Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

    SciTech Connect

    Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele; Goedken, Eric R.; Gum, Rebecca J.; Borhani, David W.; Argiriadi, Maria; Groebe, Duncan R.; Jia, Yong; Clampit, Jill E.; Haasch, Deanna L.; Smith, Harriet T.; Wang, Sanyi; Song, Danying; Coen, Michael L.; Cloutier, Timothy E.; Tang, Hua; Cheng, Xueheng; Quinn, Christopher; Liu, Bo; Xin, Zhili; Liu, Gang; Fry, Elizabeth H.; Stoll, Vincent; Ng, Teresa I.; Banach, David; Marcotte, Doug; Burns, David J.; Calderwood, David J.; Hajduk, Philip J.

    2012-03-02

    Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in

  14. Molecular recognition at the active site of subtilisin BPN': crystallographic studies using genetically engineered proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor).

    PubMed

    Takeuchi, Y; Noguchi, S; Satow, Y; Kojima, S; Kumagai, I; Miura, K; Nakamura, K T; Mitsui, Y

    1991-06-01

    Unlike trypsin-like serine proteases having only one conspicuous binding pocket in the active site, subtilisin BPN' has two such pockets, the S1 and S4 pockets, which accommodate the P1 and P4 residues of ligands (after Schechter and Berger notation) respectively. Using computer graphics, the geometrical nature of the two pockets was carefully examined and strategies for site-directed mutagenesis studies were set up against a protein SSI (Streptomyces subtilisin inhibitor), which is a strong proteinaceous inhibitor (or a substrate analogue) of subtilisin BPN'. It was decided to convert the P1 residue, methionine 73, into lysine (M73K) with or without additional conversion of the P4 residue, methionine 70, into glycine (M70G). The crystal structures of the two complexes of subtilisin BPN', one with the single mutant SSI (M73K) and the other with the double mutant SSI (M73K, M70G) were solved showing that (i) small 'electrostatic induced-fit movement' occurs in the S1 pocket upon introducing the terminal plus charge of the lysine side chain, and (ii) large 'mechanical induced-fit movement' occurs in the S4 pocket upon reducing the size of the P4 side chain from methionine to glycine. In both (i) and (ii), the induced-fit movement occurred in a concerted fashion involving both the enzyme and 'substrate' amino acid residues. The term 'substrate-assisted stabilization' was coined to stress the cooperative nature of the induced-fit movements. PMID:1891457

  15. Active site-directed plasmin inhibitors: Extension on the P2 residue.

    PubMed

    Hidaka, Koushi; Gohda, Keigo; Teno, Naoki; Wanaka, Keiko; Tsuda, Yuko

    2016-02-15

    Based on the structure of YO-2 [N-(trans-4-aminomethylcyclohexanecarbonyl)-l-Tyr(O-picolyl)-NH-octyl], active site-directed plasmin (Plm) inhibitors were explored. The picolyl moiety in the Tyr(O-picolyl) residue (namely, the P2 residue) was replaced with smaller or larger groups, such as hydrogen, tert-butyl, benzyl, (2-naphthyl)methyl, and (quinolin-2-yl)methyl. Those efforts produced compound 17 {N-(trans-4-aminomethylcyclohexanecarbonyl)-l-Tyr[O-(quinolin-2-yl)methyl]-NH-octyl} [IC50=0.22 and 77μM for Plm and urokinase (UK), respectively], which showed not only 2.4-fold greater Plm inhibition than YO-2, but also an improvement in selectivity (Plm/UK) by 35-fold. The docking experiments of the Plm-17 complexes disclosed that the amino group of the tranexamyl moiety interacted with the side-chain of Asp753 which formed S1 site.

  16. Structure of HIV-1 Reverse Transcriptase with the Inhibitor -thujaplicinol Bound at the RNase H Active Site

    SciTech Connect

    Himmel, D.; Maegley, K; Pauly, T; Bauman, J; Das, K; Dharia, C; Clark, Jr., A; Ryan, K; Hickey, M; et al.

    2009-01-01

    Novel inhibitors are needed to counteract the rapid emergence of drug-resistant HIV variants. HIV-1 reverse transcriptase (RT) has both DNA polymerase and RNase H (RNH) enzymatic activities, but approved drugs that inhibit RT target the polymerase. Inhibitors that act against new targets, such as RNH, should be effective against all of the current drug-resistant variants. Here, we present 2.80 {angstrom} and 2.04 {angstrom} resolution crystal structures of an RNH inhibitor, {beta}-thujaplicinol, bound at the RNH active site of both HIV-1 RT and an isolated RNH domain. {beta}-thujaplicinol chelates two divalent metal ions at the RNH active site. We provide biochemical evidence that {beta}-thujaplicinol is a slow-binding RNH inhibitor with noncompetitive kinetics and suggest that it forms a tropylium ion that interacts favorably with RT and the RNA:DNA substrate.

  17. Synthesis and evaluation of M. tuberculosis salicylate synthase (MbtI) inhibitors designed to probe plasticity in the active site.

    PubMed

    Manos-Turvey, Alexandra; Cergol, Katie M; Salam, Noeris K; Bulloch, Esther M M; Chi, Gamma; Pang, Angel; Britton, Warwick J; West, Nicholas P; Baker, Edward N; Lott, J Shaun; Payne, Richard J

    2012-12-14

    Mycobacterium tuberculosis salicylate synthase (MbtI) catalyses the first committed step in the biosynthesis of mycobactin T, an iron-chelating siderophore essential for the virulence and survival of M. tuberculosis. Co-crystal structures of MbtI with members of a first generation inhibitor library revealed large inhibitor-induced rearrangements within the active site of the enzyme. This plasticity of the MbtI active site was probed via the preparation of a library of inhibitors based on a 2,3-dihydroxybenzoate scaffold with a range of substituted phenylacrylate side chains appended to the C3 position. Most compounds exhibited moderate inhibitory activity against the enzyme, with inhibition constants in the micromolar range, while several dimethyl ester variants possessed promising anti-tubercular activity in vitro. PMID:23108268

  18. Non-empirical analysis of the nature of the inhibitor active-site interactions in leucine aminopeptidase

    NASA Astrophysics Data System (ADS)

    Grembecka, J.; K ȩdzierski, P.; Sokalski, W. A.

    1999-11-01

    Non-empirical analysis of the physical nature of the intermolecular interactions between several leucine aminopeptidase inhibitors and various constituents of the enzyme active site has been performed using a direct version of the hybrid variation-perturbation decomposition of SCF and MP2 interaction energies. The interaction energy terms obtained at different theory levels have been correlated with experimentally measured activities of the inhibitors, indicating that the more advanced the quantum-chemical method and, the larger the active-site model, the better is the correlation between calculated and measured binding energies. The electrostatic multipole term constitutes the dominant contribution in the total interaction energy, whereas Zn 2+488 and Lys +262 enzyme residues play the crucial role in the binding of these inhibitors by leucine aminopeptidase.

  19. Implications of binding mode and active site flexibility for inhibitor potency against the salicylate synthase from Mycobacterium tuberculosis.

    PubMed

    Chi, Gamma; Manos-Turvey, Alexandra; O'Connor, Patrick D; Johnston, Jodie M; Evans, Genevieve L; Baker, Edward N; Payne, Richard J; Lott, J Shaun; Bulloch, Esther M M

    2012-06-19

    MbtI is the salicylate synthase that catalyzes the first committed step in the synthesis of the iron chelating compound mycobactin in Mycobacterium tuberculosis. We previously developed a series of aromatic inhibitors against MbtI based on the reaction intermediate for this enzyme, isochorismate. The most potent of these inhibitors had hydrophobic substituents, ranging in size from a methyl to a phenyl group, appended to the terminal alkene of the enolpyruvyl group. These compounds exhibited low micromolar inhibition constants against MbtI and were at least an order of magnitude more potent than the parental compound for the series, which carries a native enolpyruvyl group. In this study, we sought to understand how the substituted enolpyruvyl group confers greater potency, by determining cocrystal structures of MbtI with six inhibitors from the series. A switch in binding mode at the MbtI active site is observed for inhibitors carrying a substituted enolpyruvyl group, relative to the parental compound. Computational studies suggest that the change in binding mode, and higher potency, is due to the effect of the substituents on the conformational landscape of the core inhibitor structure. The crystal structures and fluorescence-based thermal shift assays indicate that substituents larger than a methyl group are accommodated in the MbtI active site through significant but localized flexibility in the peptide backbone. These findings have implications for the design of improved inhibitors of MbtI, as well as other chorismate-utilizing enzymes from this family. PMID:22607697

  20. A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior.

    PubMed

    Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H; Muyldermans, Serge; Declerck, Paul J; Huang, Mingdong; Andreasen, Peter A; Ngo, Jacky Chi Ki

    2016-07-15

    A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30-40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors.

  1. Structures of Clostridium Botulinum Neurotoxin Serotype A Light Chain Complexed with Small-Molecule Inhibitors Highlight Active-Site Flexibility

    SciTech Connect

    Silvaggi,N.; Boldt, G.; Hixon, M.; Kennedy, J.; Tzipori, S.; Janda, K.; Allen, K.

    2007-01-01

    The potential for the use of Clostridial neurotoxins as bioweapons makes the development of small-molecule inhibitors of these deadly toxins a top priority. Recently, screening of a random hydroxamate library identified a small-molecule inhibitor of C. botulinum Neurotoxin Serotype A Light Chain (BoNT/A-LC), 4-chlorocinnamic hydroxamate, a derivative of which has been shown to have in vivo efficacy in mice and no toxicity. We describe the X-ray crystal structures of BoNT/A-LC in complexes with two potent small-molecule inhibitors. The structures of the enzyme with 4-chlorocinnamic hydroxamate or 2,4-dichlorocinnamic hydroxamate bound are compared to the structure of the enzyme complexed with L-arginine hydroxamate, an inhibitor with modest affinity. Taken together, this suite of structures provides surprising insights into the BoNT/A-LC active site, including unexpected conformational flexibility at the S1' site that changes the electrostatic environment of the binding pocket. Information gained from these structures will inform the design and optimization of more effective small-molecule inhibitors of BoNT/A-LC.

  2. Novel human D-amino acid oxidase inhibitors stabilize an active-site lid-open conformation

    PubMed Central

    Terry-Lorenzo, Ryan T.; Chun, Lawrence E.; Brown, Scott P.; Heffernan, Michele L. R.; Fang, Q. Kevin; Orsini, Michael A.; Pollegioni, Loredano; Hardy, Larry W.; Spear, Kerry L.; Large, Thomas H.

    2014-01-01

    The NMDAR (N-methyl-D-aspartate receptor) is a central regulator of synaptic plasticity and learning and memory. hDAAO (human D-amino acid oxidase) indirectly reduces NMDAR activity by degrading the NMDAR co-agonist D-serine. Since NMDAR hypofunction is thought to be a foundational defect in schizophrenia, hDAAO inhibitors have potential as treatments for schizophrenia and other nervous system disorders. Here, we sought to identify novel chemicals that inhibit hDAAO activity. We used computational tools to design a focused, purchasable library of compounds. After screening this library for hDAAO inhibition, we identified the structurally novel compound, ‘compound 2’ [3-(7-hydroxy-2-oxo-4-phenyl-2H-chromen-6-yl)propanoic acid], which displayed low nM hDAAO inhibitory potency (Ki=7 nM). Although the library was expected to enrich for compounds that were competitive for both D-serine and FAD, compound 2 actually was FAD uncompetitive, much like canonical hDAAO inhibitors such as benzoic acid. Compound 2 and an analog were independently co-crystalized with hDAAO. These compounds stabilized a novel conformation of hDAAO in which the active-site lid was in an open position. These results confirm previous hypotheses regarding active-site lid flexibility of mammalian D-amino acid oxidases and could assist in the design of the next generation of hDAAO inhibitors. PMID:25001371

  3. Structural Basis for the Inhibition of RNase H Activity of HIV-1 Reverse Transcriptase by RNase H Active Site-Directed Inhibitors

    SciTech Connect

    Su, Hua-Poo; Yan, Youwei; Prasad, G. Sridhar; Smith, Robert F.; Daniels, Christopher L.; Abeywickrema, Pravien D.; Reid, John C.; Loughran, H. Marie; Kornienko, Maria; Sharma, Sujata; Grobler, Jay A.; Xu, Bei; Sardana, Vinod; Allison, Timothy J.; Williams, Peter D.; Darke, Paul L.; Hazuda, Daria J.; Munshi, Sanjeev

    2010-09-02

    HIV/AIDS continues to be a menace to public health. Several drugs currently on the market have successfully improved the ability to manage the viral burden in infected patients. However, new drugs are needed to combat the rapid emergence of mutated forms of the virus that are resistant to existing therapies. Currently, approved drugs target three of the four major enzyme activities encoded by the virus that are critical to the HIV life cycle. Although a number of inhibitors of HIV RNase H activity have been reported, few inhibit by directly engaging the RNase H active site. Here, we describe structures of naphthyridinone-containing inhibitors bound to the RNase H active site. This class of compounds binds to the active site via two metal ions that are coordinated by catalytic site residues, D443, E478, D498, and D549. The directionality of the naphthyridinone pharmacophore is restricted by the ordering of D549 and H539 in the RNase H domain. In addition, one of the naphthyridinone-based compounds was found to bind at a second site close to the polymerase active site and non-nucleoside/nucleotide inhibitor sites in a metal-independent manner. Further characterization, using fluorescence-based thermal denaturation and a crystal structure of the isolated RNase H domain reveals that this compound can also bind the RNase H site and retains the metal-dependent binding mode of this class of molecules. These structures provide a means for structurally guided design of novel RNase H inhibitors.

  4. Structural basis for the inhibition of RNase H activity of HIV-1 reverse transcriptase by RNase H active site-directed inhibitors.

    PubMed

    Su, Hua-Poo; Yan, Youwei; Prasad, G Sridhar; Smith, Robert F; Daniels, Christopher L; Abeywickrema, Pravien D; Reid, John C; Loughran, H Marie; Kornienko, Maria; Sharma, Sujata; Grobler, Jay A; Xu, Bei; Sardana, Vinod; Allison, Timothy J; Williams, Peter D; Darke, Paul L; Hazuda, Daria J; Munshi, Sanjeev

    2010-08-01

    HIV/AIDS continues to be a menace to public health. Several drugs currently on the market have successfully improved the ability to manage the viral burden in infected patients. However, new drugs are needed to combat the rapid emergence of mutated forms of the virus that are resistant to existing therapies. Currently, approved drugs target three of the four major enzyme activities encoded by the virus that are critical to the HIV life cycle. Although a number of inhibitors of HIV RNase H activity have been reported, few inhibit by directly engaging the RNase H active site. Here, we describe structures of naphthyridinone-containing inhibitors bound to the RNase H active site. This class of compounds binds to the active site via two metal ions that are coordinated by catalytic site residues, D443, E478, D498, and D549. The directionality of the naphthyridinone pharmacophore is restricted by the ordering of D549 and H539 in the RNase H domain. In addition, one of the naphthyridinone-based compounds was found to bind at a second site close to the polymerase active site and non-nucleoside/nucleotide inhibitor sites in a metal-independent manner. Further characterization, using fluorescence-based thermal denaturation and a crystal structure of the isolated RNase H domain reveals that this compound can also bind the RNase H site and retains the metal-dependent binding mode of this class of molecules. These structures provide a means for structurally guided design of novel RNase H inhibitors.

  5. INTERACTIONS OF DIFFERENT INHIBITORS WITH ACTIVE-SITE ASPARTYL RESIDUES OF HIV-1 PROTEASE AND POSSIBLE RELEVANCE TO PEPSINS

    PubMed Central

    Sayer, Jane M.; Louis, John M.

    2008-01-01

    The importance of the active site region aspartyl residues 25 and 29 of the mature HIV-1 protease (PR) for the binding of five clinical and three experimental protease inhibitors (symmetric cyclic urea inhibitor DMP323, non-hydrolysable substrate analog (RPB) and the generic aspartic protease inhibitor acetyl-pepstatin (Ac-PEP)) was assessed by differential scanning calorimetry. ΔTm values, defined as the difference in Tm for a given protein in the presence and absence of inhibitor, for PR with DRV, ATV, SQV, RTV, APV, DMP323, RPB and Ac-PEP are 22.4, 20.8, 19.3, 15.6, 14.3, 14.7, 8.7, and 6.5 °C, respectively. Binding of APV and Ac-PEP is most sensitive to the D25N mutation, as shown by ΔTm ratios [ΔTm(PR)/ΔTm(PRD25N)] of 35.8 and 16.3, respectively, whereas binding of DMP323 and RPB (ΔTm ratios of 1-2) is least affected. Binding of the substrate-like inhibitors RPB and Ac-PEP is nearly abolished (ΔTm(PR)/ΔTm(PRD29N) ≥ 44) by the D29N mutation, whereas this mutation only moderately affects binding of the smaller inhibitors (ΔTm ratios of 1.4-2.2). Of the 9 FDA approved clinical HIV-1 protease inhibitors screened, APV, RTV and DRV competitively inhibit porcine pepsin with Ki values of 0.3, 0.6 and 2.14 μM, respectively. DSC results were consistent with this relatively weak binding of APV (ΔTm 2.7 °C) compared with the tight binding of AcPEP (ΔTm ≥17 °C). Comparison of superimposed structures of the PR/APV complex with those of PR/Ac-PEP and pepsin/pepstatin A complexes suggests a role for Asp215, Asp32 and Ser219 in pepsin, equivalent to Asp25, Asp25′ and Asp29 in PR, in the binding and stabilization of the pepsin/APV complex. PMID:18951411

  6. Active site characterization and structure based 3D-QSAR studies on non-redox type 5-lipoxygenase inhibitors.

    PubMed

    Ul-Haq, Zaheer; Khan, Naveed; Zafar, Syed Kashif; Moin, Syed Tarique

    2016-06-10

    Structure-based 3D-QSAR study was performed on a class of 5-benzylidene-2-phenylthiazolinones non-redox type 5-LOX inhibitors. In this study, binding pocket of 5-Lipoxygenase (pdb id 3o8y) was identified by manual docking using 15-LOX (pdb id 2p0m) as a reference structure. Additionally, most of the binding site residues were found conserved in both structures. These non-redox inhibitors were then docked into the binding site of 5-LOX. To generate reliable CoMFA and CoMSIA models, atom fit data base alignment method using docked conformation of the most active compound was employed. The q(2)cv and r(2)ncv values for CoMFA model were found to be 0.549 and 0.702, respectively. The q(2)cv and r(2)ncv values for the selected CoMSIA model comprised four descriptors steric, electrostatic, hydrophobic and hydrogen bond donor fields were found to be 0.535 and 0.951, respectively. Obtained results showed that our generated model was statistically reliable. Furthermore, an external test set validates the reliability of the predicted model by calculating r(2)pred i.e.0.787 and 0.571 for CoMFA and CoMSIA model, respectively. 3D contour maps generated from CoMFA and CoMSIA models were utilized to determine the key structural features of ligands responsible for biological activities. The applied protocol will be helpful to design more potent and selective inhibitors of 5-LOX. PMID:27044904

  7. Conversed mutagenesis of an inactive peptide to ASIC3 inhibitor for active sites determination.

    PubMed

    Osmakov, Dmitry I; Koshelev, Sergey G; Andreev, Yaroslav A; Dyachenko, Igor A; Bondarenko, Dmitry A; Murashev, Arkadii N; Grishin, Eugene V; Kozlov, Sergey A

    2016-06-15

    Peptide Ugr9-1 from the venom of sea anemone Urticina grebelnyi selectively inhibits the ASIC3 channel and significantly reverses inflammatory and acid-induced pain in vivo. A close homolog peptide Ugr 9-2 does not have these features. To find the pharmacophore residues and explore structure-activity relationships of Ugr 9-1, we performed site-directed mutagenesis of Ugr 9-2 and replaced several positions by the corresponding residues from Ugr 9-1. Mutant peptides Ugr 9-2 T9F and Ugr 9-2 Y12H were able to inhibit currents of the ASIC3 channels 2.2 times and 1.3 times weaker than Ugr 9-1, respectively. Detailed analysis of the spatial models of Ugr 9-1, Ugr 9-2 and both mutant peptides revealed the presence of the basic-aromatic clusters on opposite sides of the molecule, each of which is responsible for the activity. Additionally, Ugr9-1 mutant with truncated N- and C-termini retained similar with the Ugr9-1 action in vitro and was equally potent in vivo model of thermal hypersensitivity. All together, these results are important for studying the structure-activity relationships of ligand-receptor interaction and for the future development of peptide drugs from animal toxins. PMID:26686983

  8. Implication of crystal water molecules in inhibitor binding at ALR2 active site.

    PubMed

    Hymavati; Kumar, Vivek; Sobhia, M Elizabeth

    2012-01-01

    Water molecules play a crucial role in mediating the interaction between a ligand and a macromolecule. The solvent environment around such biomolecule controls their structure and plays important role in protein-ligand interactions. An understanding of the nature and role of these water molecules in the active site of a protein could greatly increase the efficiency of rational drug design approaches. We have performed the comparative crystal structure analysis of aldose reductase to understand the role of crystal water in protein-ligand interaction. Molecular dynamics simulation has shown the versatile nature of water molecules in bridge H bonding during interaction. Occupancy and life time of water molecules depend on the type of cocrystallized ligand present in the structure. The information may be useful in rational approach to customize the ligand, and thereby longer occupancy and life time for bridge H-bonding. PMID:22649481

  9. X-ray structure at 1.75 resolution of a norovirus 3C protease linked to an active site-directed peptide inhibitor

    SciTech Connect

    Cooper, Jon; Coates, Leighton; Hussey, Robert

    2010-01-01

    Noroviruses are recognized universally as the most important cause of human epidemic non-bacterial gastroenteritis. Viral replication requires a 3C cysteine protease that cleaves a 200kDa viral polyprotein into its constituent functional proteins. Here we describe the X-ray structure of the Southampton norovirus 3C protease (SV3CP) bound to an active site-directed peptide inhibitor (MAPI) which has been refined at 1.75 resolution, following initial MAD phasing with a selenomethionine derivative. The inhibitor, acetyl-Glu-Phe-Gln-Leu-Gln-X, based on a 3C protease cleavage recognition sequences in the 200kDa polyprotein substrate, reacts covalently through its propenylethylester group (X) with the active site nucleophile, Cys 139. The 3C protease-inhibitor structure permits, for the first time, the identification of substrate recognition and binding groups and provides important new information for the development of antiviral prophylactics.

  10. Rapid binding of a cationic active site inhibitor to wild type and mutant mouse acetylcholinesterase: Brownian dynamics simulation including diffusion in the active site gorge.

    PubMed

    Tara, S; Elcock, A H; Kirchhoff, P D; Briggs, J M; Radic, Z; Taylor, P; McCammon, J A

    1998-12-01

    It is known that anionic surface residues play a role in the long-range electrostatic attraction between acetylcholinesterase and cationic ligands. In our current investigation, we show that anionic residues also play an important role in the behavior of the ligand within the active site gorge of acetylcholinesterase. Negatively charged residues near the gorge opening not only attract positively charged ligands from solution to the enzyme, but can also restrict the motion of the ligand once it is inside of the gorge. We use Brownian dynamics techniques to calculate the rate constant kon, for wild type and mutant acetylcholinesterase with a positively charged ligand. These calculations are performed by allowing the ligand to diffuse within the active site gorge. This is an extension of previously reported work in which a ligand was allowed to diffuse only to the enzyme surface. By setting the reaction criteria for the ligand closer to the active site, better agreement with experimental data is obtained. Although a number of residues influence the movement of the ligand within the gorge, Asp74 is shown to play a particularly important role in this function. Asp74 traps the ligand within the gorge, and in this way helps to ensure a reaction.

  11. Active site binding modes of inhibitors of Staphylococcus aureus mevalonate diphosphate decarboxylase from docking and molecular dynamics simulations.

    PubMed

    Addo, James K; Skaff, D Andrew; Miziorko, Henry M

    2016-01-01

    Bacterial mevalonate diphosphate decarboxylase (MDD) is an attractive therapeutic target for antibacterial drug development. In this work, we discuss a combined docking and molecular dynamics strategy toward inhibitor binding to bacterial MDD. The docking parameters utilized in this study were first validated with observations for the inhibitors 6-fluoromevalonate diphosphate (FMVAPP) and diphosphoglycolylproline (DPGP) using existing structures for the Staphylococcus epidermidis enzyme. The validated docking protocol was then used to predict structures of the inhibitors bound to Staphylococcus aureus MDD using the unliganded crystal structure of Staphylococcus aureus MDD. We also investigated a possible interactions improvement by combining this docking method with molecular dynamics simulations. Thus, the predicted docking structures were analyzed in a molecular dynamics trajectory to generate dynamic models and reinforce the predicted binding modes. FMVAPP is predicted to make more extensive contacts with S. aureus MDD, forming stable hydrogen bonds with Arg144, Arg193, Lys21, Ser107, and Tyr18, as well as making stable hydrophobic interactions with Tyr18, Trp19, and Met196. The differences in predicted binding are supported by experimentally determined Ki values of 0.23 ± 0.02 and 34 ± 8 μM, for FMVAPP and DPGP, respectively. The structural information coupled with the kinetic characterization obtained from this study should be useful in defining the requirements for inhibition as well as in guiding the selection of active compounds for inhibitor optimization.

  12. Beta-D-xylosidase from Selenomonas ruminantium: Role of Glutamate 186 in Catalysis Revealed by Site-Directed Mutagenesis, Alternate Substrates, and Active-site Inhibitor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Beta-D-xylosidase/alpha-L-arabinofuranosidase from Selenomonas ruminantium (SXA) is the most active enzyme known for catalyzing hydrolysis of 1,4-beta-D-xylooligosaccharides to D xylose. Catalysis and inhibitor binding by the GH43 beta-xylosidase are governed by the protonation states of catalytic ...

  13. Probing the Active Site of Candida Glabrata Dihydrofolate Reductase with High Resolution Crystal Structures and the Synthesis of New Inhibitors

    SciTech Connect

    Liu, J.; Bolstad, D; Smith, A; Priestley, N; Wright, D; Anderson, A

    2009-01-01

    Candida glabrata, a fungal strain resistant to many commonly administered antifungal agents, has become an emerging threat to human health. In previous work, we validated that the essential enzyme, dihydrofolate reductase, is a drug target in C. glabrata. Using a crystal structure of dihydrofolate reductase from C. glabrata bound to an initial lead compound, we designed a class of biphenyl antifolates that potently and selectively inhibit both the enzyme and the growth of the fungal culture. In this work, we explore the structure-activity relationships of this class of antifolates with four new high resolution crystal structures of enzyme:inhibitor complexes and the synthesis of four new inhibitors. The designed inhibitors are intended to probe key hydrophobic pockets visible in the crystal structure. The crystal structures and an evaluation of the new compounds reveal that methyl groups at the meta and para positions of the distal phenyl ring achieve the greatest number of interactions with the pathogenic enzyme and the greatest degree of selectivity over the human enzyme. Additionally, antifungal activity can be tuned with substitution patterns at the propargyl and para-phenyl positions.

  14. Design and discovery of flavonoid-based HIV-1 integrase inhibitors targeting both the active site and the interaction with LEDGF/p75.

    PubMed

    Li, Bo-Wen; Zhang, Feng-Hua; Serrao, Erik; Chen, Huan; Sanchez, Tino W; Yang, Liu-Meng; Neamati, Nouri; Zheng, Yong-Tang; Wang, Hui; Long, Ya-Qiu

    2014-06-15

    HIV integrase (IN) is an essential enzyme for the viral replication. Currently, three IN inhibitors have been approved for treating HIV-1 infection. All three drugs selectively inhibit the strand transfer reaction by chelating a divalent metal ion in the enzyme active site. Flavonoids are a well-known class of natural products endowed with versatile biological activities. Their β-ketoenol or catechol structures can serve as a metal chelation motif and be exploited for the design of novel IN inhibitors. Using the metal chelation as a common pharmacophore, we introduced appropriate hydrophobic moieties into the flavonol core to design natural product-based novel IN inhibitors. We developed selective and efficient syntheses to generate a series of mono 3/5/7/3'/4'-substituted flavonoid derivatives. Most of these new compounds showed excellent HIV-1 IN inhibitory activity in enzyme-based assays and protected against HIV-1 infection in cell-based assays. The 7-morpholino substituted 7c showed effective antiviral activity (EC50=0.826 μg/mL) and high therapeutic index (TI>242). More significantly, these hydroxyflavones block the IN-LEDGF/p75 interaction with low- to sub-micromolar IC50 values and represent a novel scaffold to design new generation of drugs simultaneously targeting the catalytic site as well as protein-protein interaction domains.

  15. Carbonic anhydrase inhibitors with dual-tail moieties to match the hydrophobic and hydrophilic halves of the carbonic anhydrase active site.

    PubMed

    Tanpure, Rajendra P; Ren, Bin; Peat, Thomas S; Bornaghi, Laurent F; Vullo, Daniela; Supuran, Claudiu T; Poulsen, Sally-Ann

    2015-02-12

    We present a new approach to carbonic anhydrase II (CA II) inhibitor design that enables close interrogation of the regions of the CA active site where there is the greatest variability in amino acid residues among the different CA isozymes. By appending dual tail groups onto the par excellence CA inhibitor acetazolamide, compounds that may interact with the distinct hydrophobic and hydrophilic halves of the CA II active site were prepared. The dual-tail combinations selected included (i) two hydrophobic moieties, (ii) two hydrophilic moieties, and (iii) one hydrophobic and one hydrophilic moiety. The CA enzyme inhibition profile as well as the protein X-ray crystal structure of compound 3, comprising one hydrophobic and one hydrophilic tail moiety, in complex with CA II is described. This novel dual-tail approach has provided an enhanced opportunity to more fully exploit interactions with the CA active site by enabling these molecules to interact with the distinct halves of the active site. In addition to the dual-tail compounds, a corresponding set of single-tail derivatives was synthesized, enabling a comparative analysis of the single-tail versus dual-tail compound CA inhibition profile.

  16. Targeting the active site of the placental isozyme of alkaline phosphatase by phage-displayed scFv antibodies selected by a specific uncompetitive inhibitor

    PubMed Central

    Saini, Deepti; Kala, Mrinalini; Jain, Vishal; Sinha, Subrata

    2005-01-01

    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. Conclusion The results

  17. Insights into the fine architecture of the active site of chicory fructan 1-exohydrolase: 1-kestose as substrate vs sucrose as inhibitor.

    PubMed

    Verhaest, Maureen; Lammens, Willem; Le Roy, Katrien; De Ranter, Camiel J; Van Laere, André; Rabijns, Anja; Van den Ende, Wim

    2007-01-01

    * Invertases and fructan exohydrolases (FEHs) fulfil important physiological functions in plants. Sucrose is the typical substrate for invertases and bacterial levansucrases but not for plant FEHs, which are usually inhibited by sucrose. * Here we report on complexes between chicory (Cichorium intybus) 1-FEH IIa with the substrate 1-kestose and the inhibitors sucrose, fructose and 2,5 dideoxy-2,5-imino-D-mannitol. Comparisons with other family GH32 and 68 enzyme-substrate complexes revealed that sucrose can bind as a substrate (invertase/levansucrase) or as an inhibitor (1-FEH IIa). * Sucrose acts as inhibitor because the O2 of the glucose moiety forms an H-linkage with the acid-base catalyst E201, inhibiting catalysis. By contrast, the homologous O3 of the internal fructose in the substrate 1-kestose forms an intramolecular H-linkage and does not interfere with the catalytic process. Mutagenesis showed that W82 and S101 are important for binding sucrose as inhibitor. * The physiological implications of the essential differences in the active sites of FEHs and invertases/levansucrases are discussed. Sucrose-inhibited FEHs show a K(i) (inhibition constant) well below physiological sucrose concentrations and could be rapidly activated under carbon deprivation.

  18. Non-coding nucleotides and amino acids near the active site regulate peptide deformylase expression and inhibitor susceptibility in Chlamydia trachomatis

    PubMed Central

    Bao, Xiaofeng; Pachikara, Niseema D.; Oey, Christopher B.; Balakrishnan, Amit; Westblade, Lars F.; Tan, Ming; Chase, Theodore; Nickels, Bryce E.

    2011-01-01

    Chlamydia trachomatis, an obligate intracellular bacterium, is a highly prevalent human pathogen. Hydroxamic-acid-based matrix metalloprotease inhibitors can effectively inhibit the pathogen both in vitro and in vivo, and have exhibited therapeutic potential. Here, we provide genome sequencing data indicating that peptide deformylase (PDF) is the sole target of the inhibitors in this organism. We further report molecular mechanisms that control chlamydial PDF (cPDF) expression and inhibition efficiency. In particular, we identify the σ66-dependent promoter that controls cPDF gene expression and demonstrate that point mutations in this promoter lead to resistance by increasing cPDF transcription. Furthermore, we show that substitution of two amino acids near the active site of the enzyme alters enzyme kinetics and protein stability. PMID:21719536

  19. Probing the location and function of the conserved histidine residue of phosphoglucose isomerase by using an active site directed inhibitor N-bromoacetylethanolamine phosphate.

    PubMed Central

    Meng, M.; Chane, T. L.; Sun, Y. J.; Hsiao, C. D.

    1999-01-01

    Phosphoglucose isomerase (EC 5.3.1.9) catalyzes the interconversion of D-glucopyranose-6-phosphate and D-fructofuranose-6-phosphate by promoting an intrahydrogen transfer between C1 and C2. A conserved histidine exists throughout all phosphoglucose isomerases and was hypothesized to be the base catalyzing the isomerization reaction. In the present study, this conserved histidine, His311, of the enzyme from Bacillus stearothermophilus was subjected to mutational analysis, and the mutational effect on the inactivation kinetics by N-bromoacetylethanolamine phosphate was investigated. The substitution of His311 with alanine, asparagine, or glutamine resulted in the decrease of activity, in k(cat)/K(M), by a factor of 10(3), indicating the importance of this residue. N-bromoacetylethanolamine phosphate inactivated irreversibly the activity of wild-type phosphoglucose isomerase; however, His311 --> Ala became resistant to this inhibitor, indicating that His311 is located in the active site and is responsible for the inactivation of the enzyme by this active site-directed inhibitor. The pKa of His311 was estimated to be 6.31 according to the pH dependence of the inactivation. The proximity of this value with the pKa value of 6.35, determined from the pH dependence of k(cat)/K(M), supports a role of His311 as a general base in the catalysis. PMID:10595547

  20. Amino-steroids as inhibitors and probes of the active site of cytochrome P-450scc. Effects on the enzyme from different sources.

    PubMed

    Kellis, J T; Sheets, J J; Vickery, L E

    1984-02-01

    A series of analogues of cholesterol, each having a primary amine attached to a shortened side chain, were tested for their effects on cytochrome P-450scc from several different sources. Reconstituted enzyme systems using disrupted mitochondria from bovine adrenal and placenta, adult human adrenal and placenta, neonatal human adrenal, and rat adrenal and testis were used to assay for inhibitory effects on the side chain cleavage of cholesterol to pregnenolone. Two of the derivatives tested, 22-amino-23,24-bisnor-5-cholen-3 beta-ol and 23-amino-24-nor-5-cholen-3 beta-ol, were found to be potent inhibitors of this reaction; the derivatives in which the amine was attached closer to or further from the steroid ring, (20 R and S)-20-amino-5-pregnen-3 beta-ol and 24-amino-5-cholen-3 beta-ol, were much weaker inhibitors. In addition, spectral studies with rat adrenal mitochondria and a soluble preparation of human placental cytochrome P-450scc showed that binding of the 22-amine derivative to the enzyme produces difference spectra characteristic of nitrogen bonding to the heme; this indicates that the heme is positioned close to C-22 in the steroid-enzyme complex. These findings on the relative effectiveness of the amino-steroid inhibitors and the type of complex formed are similar to results obtained with purified bovine adrenocortical cytochrome P-450scc. This establishes that the proximity of the substrate binding site and the heme-iron catalytic site is a feature common to the enzyme from several sources and is therefore likely to be a necessary property of the active site structure.

  1. Sulfated Low Molecular Weight Lignins, Allosteric Inhibitors of Coagulation Proteinases via the Heparin Binding Site, Significantly Alter the Active Site of Thrombin and Factor Xa Compared to Heparin

    PubMed Central

    Henry, Brian L.; Desai, Umesh R.

    2014-01-01

    Sulfated low molecular weight lignins (LMWLs) have been found to bind in the heparin binding sites of coagulation proteinases. LMWLs represent a library of diverse non-carbohydrate, aromatic molecules which are structures different from heparin, but still potently inhibit thrombin and factor Xa. To better understand their mechanism of action, we studied the effects of three sulfated LMWLs (CDSO3, FDSO3, and SDSO3) on the active sites of thrombin and factor Xa. LMWLs were found to uniformly inhibit the catalytic activity of thrombin and factor Xa, regardless of the substrate used. Michaelis-Menten kinetic studies indicate that maximal velocity of hydrolysis of each chromogenic substrate decreases significantly in the presence of sulfated LMWLs, while the effect on Michaelis constant is dependent on the nature of the substrate. These studies indicate that LMWLs inhibit thrombin and factor Xa through allosteric disruption of the catalytic apparatus, specifically through the catalytic step. As opposed to heparin, LMWLs significantly alter the binding of the active site fluorescent ligand p-aminobenzamidine. LMWLs also had a greater effect on the molecular orientation of fluorescein-labeled His 57 than heparin. The molecular geometry surrounding the most important catalytic amino acid, Ser 195, was significantly altered by the binding of LMWLs while heparin had no measurable effect on Ser 195. These results further advance the concept of sulfated LMWLs as heparin mimics and will aid the design of anticoagulants based on their novel scaffold. PMID:25242245

  2. Sulfated low molecular weight lignins, allosteric inhibitors of coagulation proteinases via the heparin binding site, significantly alter the active site of thrombin and factor xa compared to heparin.

    PubMed

    Henry, Brian L; Desai, Umesh R

    2014-11-01

    Sulfated low molecular weight lignins (LMWLs) have been found to bind in the heparin binding sites of coagulation proteinases. LMWLs represent a library of diverse non-carbohydrate, aromatic molecules which are structures different from heparin, but still potently inhibit thrombin and factor Xa. To better understand their mechanism of action, we studied the effects of three sulfated LMWLs (CDSO3, FDSO3, and SDSO3) on the active sites of thrombin and factor Xa. LMWLs were found to uniformly inhibit the catalytic activity of thrombin and factor Xa, regardless of the substrate used. Michaelis-Menten kinetic studies indicate that maximal velocity of hydrolysis of each chromogenic substrate decreases significantly in the presence of sulfated LMWLs, while the effect on Michaelis constant is dependent on the nature of the substrate. These studies indicate that LMWLs inhibit thrombin and factor Xa through allosteric disruption of the catalytic apparatus, specifically through the catalytic step. As opposed to heparin, LMWLs significantly alter the binding of the active site fluorescent ligand p-aminobenzamidine. LMWLs also had a greater effect on the molecular orientation of fluorescein-labeled His 57 than heparin. The molecular geometry surrounding the most important catalytic amino acid, Ser 195, was significantly altered by the binding of LMWLs while heparin had no measurable effect on Ser 195. These results further advance the concept of sulfated LMWLs as heparin mimics and will aid the design of anticoagulants based on their novel scaffold. PMID:25242245

  3. Crystal Structures of Human Choline Kinase Isoforms in Complex with Hemicholinium-3 Single Amino Acid near the Active Site Influences Inhibitor Sensitivity

    SciTech Connect

    Hong, Bum Soo; Allali-Hassani, Abdellah; Tempel, Wolfram; Finerty, Jr., Patrick J.; MacKenzie, Farrell; Dimov, Svetoslav; Vedadi, Masoud; Park, Hee-Won

    2010-07-06

    Human choline kinase (ChoK) catalyzes the first reaction in phosphatidylcholine biosynthesis and exists as ChoK{alpha} ({alpha}1 and {alpha}2) and ChoK{beta} isoforms. Recent studies suggest that ChoK is implicated in tumorigenesis and emerging as an attractive target for anticancer chemotherapy. To extend our understanding of the molecular mechanism of ChoK inhibition, we have determined the high resolution x-ray structures of the ChoK{alpha}1 and ChoK{beta} isoforms in complex with hemicholinium-3 (HC-3), a known inhibitor of ChoK. In both structures, HC-3 bound at the conserved hydrophobic groove on the C-terminal lobe. One of the HC-3 oxazinium rings complexed with ChoK{alpha}1 occupied the choline-binding pocket, providing a structural explanation for its inhibitory action. Interestingly, the HC-3 molecule co-crystallized with ChoK{beta} was phosphorylated in the choline binding site. This phosphorylation, albeit occurring at a very slow rate, was confirmed experimentally by mass spectroscopy and radioactive assays. Detailed kinetic studies revealed that HC-3 is a much more potent inhibitor for ChoK{alpha} isoforms ({alpha}1 and {alpha}2) compared with ChoK{beta}. Mutational studies based on the structures of both inhibitor-bound ChoK complexes demonstrated that Leu-401 of ChoK{alpha}2 (equivalent to Leu-419 of ChoK{alpha}1), or the corresponding residue Phe-352 of ChoK{beta}, which is one of the hydrophobic residues neighboring the active site, influences the plasticity of the HC-3-binding groove, thereby playing a key role in HC-3 sensitivity and phosphorylation.

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

    PubMed

    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. PMID:25111069

  5. Recombinant plasmepsin 1 from the human malaria parasite Plasmodium falciparum: Enzymatic characterization, active site inhibitor design, and structural analysis

    PubMed Central

    Liu, Peng; Marzahn, Melissa R.; Robbins, Arthur H.; Gutiérrez-de-Terán, Hugo; Rodríguez, David; McClung, Scott; Stevens, Stanley M.; Yowell, Charles A.; Dame, John B.; McKenna, Robert; Dunn, Ben M.

    2009-01-01

    A mutated form of truncated proplasmepsin 1 (proPfPM1) from the human malaria parasite Plasmodium falciparum, proPfPM1 K110pN, was generated and overexpressed in E. coli. The auto-maturation process was carried out at pH 4.0 and 4.5, and the optimal catalytic pH of the resulting mature PfPM1 was determined to be pH 5.5. This mature PfPM1 showed comparable binding affinity to peptide substrates and inhibitors with the naturally-occurring form isolated from parasites. The S3-S3’ subsite preferences of the recombinant mature PfPM1 were explored using combinatorial chemistry based peptide libraries. Based on the results, a peptidomimetic inhibitor (compound 1) was designed and yielded 5-fold selectivity for binding to PfPM1 versus the homologous human cathepsin D (hcatD). The 2.8 Å structure of the PfPMP2-compound 1 complex is reported. Modeling studies were conducted using a series of peptidomimetic inhibitors (compounds 1–6, Table 3) and three plasmepsins: the crystal structure of PfPM2, and homology derived models of PfPM1 and PfPM4. PMID:19271776

  6. Molecular analysis and structure-activity relationship modeling of the substrate/inhibitor interaction site of plasma membrane monoamine transporter.

    PubMed

    Ho, Horace T B; Pan, Yongmei; Cui, Zhiyi; Duan, Haichuan; Swaan, Peter W; Wang, Joanne

    2011-11-01

    Plasma membrane monoamine transporter (PMAT) is a new polyspecific transporter that interacts with a wide range of structurally diverse organic cations. To map the physicochemical descriptors of cationic compounds that allow interaction with PMAT, we systematically analyzed the interactions between PMAT and three series of structural analogs of known organic cation substrates including phenylalkylamines, n-tetraalkylammonium (n-TAA) compounds, and β-carbolines. Our results showed that phenylalkylamines with a distance between the aromatic ring and the positively charged amine nitrogen atom of ∼6.4 Å confer optimal interactions with PMAT, whereas studies with n-TAA compounds revealed an excellent correlation between IC(50) values and hydrophobicity. The five β-carbolines that we tested, which possess a pyridinium-like structure and are structurally related to the neurotoxin 1-methyl-4-phenylpyridinium, inhibited PMAT with high affinity (IC(50) values of 39.1-65.5 μM). Cytotoxicity analysis further showed that cells expressing PMAT are 14- to 15-fold more sensitive to harmalan and norharmanium, suggesting that these two β-carbolines are also transportable substrates of PMAT. We then used computer-aided modeling to generate qualitative and quantitative three-dimensional pharmacophore models on the basis of 23 previously reported and currently identified PMAT inhibitors and noninhibitors. These models are characterized by a hydrogen bond donor and two to three hydrophobic features with distances between the hydrogen bond donor and hydrophobic features ranging between 5.20 and 7.02 Å. The consistency between the mapping results and observed PMAT affinity of a set of test compounds indicates that the models performed well in inhibitor prediction and could be useful for future virtual screening of new PMAT inhibitors. PMID:21816955

  7. High resolution crystal structures of triosephosphate isomerase complexed with its suicide inhibitors: The conformational flexibility of the catalytic glutamate in its closed, liganded active site

    PubMed Central

    Venkatesan, Rajaram; Alahuhta, Markus; Pihko, Petri M; Wierenga, Rik K

    2011-01-01

    The key residue of the active site of triosephosphate isomerase (TIM) is the catalytic glutamate, which is proposed to be important (i) as a catalytic base, for initiating the reaction, as well as (ii) for the subsequent proton shuttling steps. The structural properties of this glutamate in the liganded complex have been investigated by studying the high resolution crystal structures of typanosomal TIM, complexed with three suicide inhibitors: (S)-glycidol phosphate ((S)-GOP, at 0.99 Å resolution), (R)-glycidol phosphate, ((R)-GOP, at 1.08 Å resolution), and bromohydroxyacetone phosphate (BHAP, at 1.97 Å resolution). The structures show that in the (S)-GOP active site this catalytic glutamate is in the well characterized, competent conformation. However, an unusual side chain conformation is observed in the (R)-GOP and BHAP complexes. In addition, Glu97, salt bridged to the catalytic lysine in the competent active site, adopts an unusual side chain conformation in these two latter complexes. The higher chemical reactivity of (S)-GOP compared with (R)-GOP, as known from solution studies, can be understood: the structures indicate that in the case of (S)-GOP, Glu167 can attack the terminal carbon of the epoxide in a stereoelectronically favored, nearly linear O–C–O arrangement, but this is not possible for the (R)-GOP isomer. These structures confirm the previously proposed conformational flexibility of the catalytic glutamate in its closed, liganded state. The importance of this conformational flexibility for the proton shuttling steps in the TIM catalytic cycle, which is apparently achieved by a sliding motion of the side chain carboxylate group above the enediolate plane, is also discussed. PMID:21633986

  8. Orally active 7-substituted (4-benzylphthalazin-1-yl)-2-methylpiperazin-1-yl]nicotinonitriles as active-site inhibitors of sphingosine 1-phosphate lyase for the treatment of multiple sclerosis.

    PubMed

    Weiler, Sven; Braendlin, Nadine; Beerli, Christian; Bergsdorf, Christian; Schubart, Anna; Srinivas, Honnappa; Oberhauser, Berndt; Billich, Andreas

    2014-06-26

    Sphingosine 1-phosphate (S1P) lyase has recently been implicated as a therapeutic target for the treatment of multiple sclerosis (MS), based on studies in a genetic mouse model. Potent active site directed inhibitors of the enzyme are not known so far. Here we describe the discovery of (4-benzylphthalazin-1-yl)-2-methylpiperazin-1-yl]nicotinonitrile 5 in a high-throughput screen using a biochemical assay, and its further optimization. This class of compounds was found to inhibit catalytic activity of S1PL by binding to the active site of the enzyme, as seen in the cocrystal structure of derivative 31 with the homodimeric human S1P lyase. 31 induces profound reduction of peripheral T cell numbers after oral dosage and confers pronounced protection in a rat model of multiple sclerosis. In conclusion, this novel class of direct S1P lyase inhibitors provides excellent tools to further explore the therapeutic potential of T cell-targeted therapies in multiple sclerosis and other autoimmune and inflammatory diseases.

  9. Site-specific interaction of thrombin and inhibitors observed by fluorescence correlation spectroscopy.

    PubMed Central

    Klingler, J; Friedrich, T

    1997-01-01

    We report on the application of fluorescence correlation spectroscopy (FCS) to observe the interaction between thrombin and thrombin inhibitors. Two site-specific fluorescent labels were used to distinguish between inhibitors directed to the active site, the exosite, or both binding sites of thrombin. For several well-known inhibitors of thrombin, the binding sites observed by FCS correspond to previous studies. The interaction of the recently discovered thrombin inhibitor ornithodorin from the tick Ornithodorus moubata with thrombin was investigated. It was found that this inhibitor, like hirudin and rhodniin, binds to both the active site and exosite of thrombin simultaneously. This study shows the feasibility of FCS as a sensitive and selective method for observing protein-ligand interactions. As an additional technique, simultaneous labeling with both fluorescent labels was successfully demonstrated. Images FIGURE 1 PMID:9336216

  10. Human γ-Glutamyl Transpeptidase 1: STRUCTURES OF THE FREE ENZYME, INHIBITOR-BOUND TETRAHEDRAL TRANSITION STATES, AND GLUTAMATE-BOUND ENZYME REVEAL NOVEL MOVEMENT WITHIN THE ACTIVE SITE DURING CATALYSIS.

    PubMed

    Terzyan, Simon S; Burgett, Anthony W G; Heroux, Annie; Smith, Clyde A; Mooers, Blaine H M; Hanigan, Marie H

    2015-07-10

    γ-Glutamyl transpeptidase 1 (GGT1) is a cell surface, N-terminal nucleophile hydrolase that cleaves glutathione and other γ-glutamyl compounds. GGT1 expression is essential in cysteine homeostasis, and its induction has been implicated in the pathology of asthma, reperfusion injury, and cancer. In this study, we report four new crystal structures of human GGT1 (hGGT1) that show conformational changes within the active site as the enzyme progresses from the free enzyme to inhibitor-bound tetrahedral transition states and finally to the glutamate-bound structure prior to the release of this final product of the reaction. The structure of the apoenzyme shows flexibility within the active site. The serine-borate-bound hGGT1 crystal structure demonstrates that serine-borate occupies the active site of the enzyme, resulting in an enzyme-inhibitor complex that replicates the enzyme's tetrahedral intermediate/transition state. The structure of GGsTop-bound hGGT1 reveals its interactions with the enzyme and why neutral phosphonate diesters are more potent inhibitors than monoanionic phosphonates. These structures are the first structures for any eukaryotic GGT that include a molecule in the active site covalently bound to the catalytic Thr-381. The glutamate-bound structure shows the conformation of the enzyme prior to release of the final product and reveals novel information regarding the displacement of the main chain atoms that form the oxyanion hole and movement of the lid loop region when the active site is occupied. These data provide new insights into the mechanism of hGGT1-catalyzed reactions and will be invaluable in the development of new classes of hGGT1 inhibitors for therapeutic use.

  11. Preclinical activity of the novel B-cell-specific Moloney murine leukemia virus integration site 1 inhibitor PTC-209 in acute myeloid leukemia: Implications for leukemia therapy.

    PubMed

    Nishida, Yuki; Maeda, Aya; Chachad, Dhruv; Ishizawa, Jo; Qiu, Yi Hua; Kornblau, Steven M; Kimura, Shinya; Andreeff, Michael; Kojima, Kensuke

    2015-12-01

    Curing patients with acute myeloid leukemia (AML) remains a therapeutic challenge. The polycomb complex protein B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) is required for the self-renewal and maintenance of leukemia stem cells. We investigated the prognostic significance of BMI-1 in AML and the effects of a novel small molecule selective inhibitor of BMI-1, PTC-209. BMI-1 protein expression was determined in 511 newly diagnosed AML patients together with 207 other proteins using reverse-phase protein array technology. Patients with unfavorable cytogenetics according to Southwest Oncology Group criteria had higher levels of BMI-1 compared to those with favorable (P = 0.0006) or intermediate cytogenetics (P = 0.0061), and patients with higher levels of BMI-1 had worse overall survival (55.3 weeks vs. 42.8 weeks, P = 0.046). Treatment with PTC-209 reduced protein level of BMI-1 and its downstream target mono-ubiquitinated histone H2A and triggered several molecular events consistent with the induction of apoptosis, this is, loss of mitochondrial membrane potential, caspase-3 cleavage, BAX activation, and phosphatidylserine externalization. PTC-209 induced apoptosis in patient-derived CD34(+)CD38(low/-) AML cells and, less prominently, in CD34(-) differentiated AML cells. BMI-1 reduction by PTC-209 directly correlated with apoptosis induction in CD34(+) primary AML cells (r = 0.71, P = 0.022). However, basal BMI-1 expression was not a determinant of AML sensitivity. BMI-1 inhibition, which targets a primitive AML cell population, might offer a novel therapeutic strategy for AML. PMID:26450753

  12. Syntheses of coumarin-tacrine hybrids as dual-site acetylcholinesterase inhibitors and their activity against butylcholinesterase, Aβ aggregation, and β-secretase.

    PubMed

    Sun, Qi; Peng, Da-Yong; Yang, Sheng-Gang; Zhu, Xiao-Lei; Yang, Wen-Chao; Yang, Guang-Fu

    2014-09-01

    Exploring small-molecule acetylcholinesterase (AChE) inhibitors to slow the breakdown of acetylcholine (Ach) represents the mainstream direction for Alzheimer's disease (AD) therapy. As the first acetylcholinesterase inhibitor approved for the clinical treatment of AD, tacrine has been widely used as a pharmacophore to design hybrid compounds in order to combine its potent AChE inhibition with other multi-target profiles. In present study, a series of novel tacrine-coumarin hybrids were designed, synthesized and evaluated as potent dual-site AChE inhibitors. Moreover, compound 1g was identified as the most potent candidate with about 2-fold higher potency (Ki=16.7nM) against human AChE and about 2-fold lower potency (Ki=16.1nM) against BChE than tacrine (Ki=35.7nM for AChE, Ki=8.7nM for BChE), respectively. In addition, some of the tacrine-coumarin hybrids showed simultaneous inhibitory effects against both Aβ aggregation and β-secretase. We therefore conclude that tacrine-coumarin hybrid is an interesting multifunctional lead for the AD drug discovery.

  13. Synthesis of potent and broad genotypically active NS5B HCV non-nucleoside inhibitors binding to the thumb domain allosteric site 2 of the viral polymerase.

    PubMed

    Pierra Rouvière, Claire; Amador, Agnès; Badaroux, Eric; Convard, Thierry; Da Costa, Daniel; Dukhan, David; Griffe, Ludovic; Griffon, Jean-François; LaColla, Massimiliano; Leroy, Frédéric; Liuzzi, Michel; Loi, Anna Giulia; McCarville, Joe; Mascia, Valeria; Milhau, Julien; Onidi, Loredana; Paparin, Jean-Laurent; Rahali, Rachid; Sais, Efisio; Seifer, Maria; Surleraux, Dominique; Standring, David; Dousson, Cyril

    2016-09-15

    The hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp) plays a central role in virus replication. NS5B has no functional equivalent in mammalian cells and, as a consequence, is an attractive target for selective inhibition. This Letter describes the discovery of a new family of HCV NS5B non-nucleoside inhibitors, based on the bioisosterism between amide and phosphonamidate functions. As part of this program, SAR in this new series led to the identification of IDX17119, a potent non-nucleoside inhibitor, active on the genotypes 1b, 2a, 3a and 4a. The structure and binding domain of IDX17119 were confirmed by X-ray co-crystallization study. PMID:27520942

  14. Synthesis of potent and broad genotypically active NS5B HCV non-nucleoside inhibitors binding to the thumb domain allosteric site 2 of the viral polymerase.

    PubMed

    Pierra Rouvière, Claire; Amador, Agnès; Badaroux, Eric; Convard, Thierry; Da Costa, Daniel; Dukhan, David; Griffe, Ludovic; Griffon, Jean-François; LaColla, Massimiliano; Leroy, Frédéric; Liuzzi, Michel; Loi, Anna Giulia; McCarville, Joe; Mascia, Valeria; Milhau, Julien; Onidi, Loredana; Paparin, Jean-Laurent; Rahali, Rachid; Sais, Efisio; Seifer, Maria; Surleraux, Dominique; Standring, David; Dousson, Cyril

    2016-09-15

    The hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp) plays a central role in virus replication. NS5B has no functional equivalent in mammalian cells and, as a consequence, is an attractive target for selective inhibition. This Letter describes the discovery of a new family of HCV NS5B non-nucleoside inhibitors, based on the bioisosterism between amide and phosphonamidate functions. As part of this program, SAR in this new series led to the identification of IDX17119, a potent non-nucleoside inhibitor, active on the genotypes 1b, 2a, 3a and 4a. The structure and binding domain of IDX17119 were confirmed by X-ray co-crystallization study.

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

    PubMed

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

    2015-03-01

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

  16. Active site-directed inhibitors of cytochrome P-450scc. Structural and mechanistic implications of a side chain-substituted series of amino-steroids.

    PubMed

    Sheets, J J; Vickery, L E

    1983-10-10

    A series of analogues of cholesterol, each having a shortened side chain and a primary amine group, were prepared and tested for their effects on bovine adrenocortical cholesterol side chain cleavage cytochrome P-450 (P-450scc). A previous study had shown that one derivative, 22-amino-23,24-bisnor-5-cholen-3 beta-ol, is a potent competitive inhibitor of the enzyme and forms a complex in which the steroid ring binds to the cholesterol site and the side chain amine forms a bond with the heme iron (Sheets, J. J., and Vickery, L. E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 5773-5777). In the studies reported here, the 23-amine derivative, 23-amino-24-nor-5-cholen-3 beta-ol, was found to be an equally potent inhibitor and to be competitive with respect to cholesterol (Ki = 38 nM). Binding of the 23-amine to P-450scc also caused formation of a low spin complex with an absorption maximum at 422 nm, indicative of a nitrogen-donor ligand. Other derivatives in which the side chain amine was linked closer to the steroid, 17 beta-amino-5-androsten-3 beta-ol and (20 R + S)-20-amino-5-pregnen-3 beta-ol, were found to be only very weak inhibitors (I50 greater than 100 microM) and did not produce the 422 nm spectral form when bound. Derivatives in which the amine was attached a greater distance from the steroid ring, 24-amino-5-cholen-3 beta-ol and 25-amino-26,27-bisnor-5-cholesten-3 beta-ol, caused a progressive decrease in inhibitory potency and a failure to produce the 422 nm form on binding. The dependence of the type of interaction of these amino-steroids with P-450scc upon the amine position establishes that the steroid binding site and the heme catalytic site of the enzyme are fixed within a specific distance of one another. The heme appears to be located sufficiently close to the position that the side chain of cholesterol would occupy to allow for direct attack of an iron-bound oxidant to occur during hydroxylation and side chain cleavage.

  17. A novel strategy for the development of selective active-site inhibitors of the protein tyrosine phosphatase-like proteins islet-cell antigen 512 (IA-2) and phogrin (IA-2beta).

    PubMed

    Drake, Paul G; Peters, Günther H; Andersen, Henrik Sune; Hendriks, Wiljan; Møller, Niels Peter H

    2003-07-15

    Islet-cell antigen 512 (IA-2) and phogrin (IA-2beta) are atypical members of the receptor protein tyrosine phosphatase (PTP) family that are characterized by a lack of activity against conventional PTP substrates. The physiological role(s) of these proteins remain poorly defined, although recent studies indicate that IA-2 may be involved in granule trafficking and exocytosis. To further understand their function, we have embarked upon developing low-molecular-mass inhibitors of IA-2 and IA-2beta. Previously, we have shown that a general PTP inhibitor, 2-(oxalylamino)benzoic acid (OBA), can be developed into highly selective and potent inhibitors of PTP1B. However, since wild-type IA-2 and IA-2beta lack conventional PTP activity, a novel strategy was designed whereby catalytically active species were generated by 'back-mutating' key non-consensus catalytic region residues to those of PTP1B. These mutants were then used as tools with which to test the potency and selectivity of OBA and a variety of its derivatives. Catalytically competent IA-2 and IA-2beta species were generated by 'back-mutation' of only three key residues (equivalent to Tyr(46), Asp(181) and Ala(217) using the human PTP1B numbering) to those of PTP1B. Importantly, enzyme kinetic analyses indicated that the overall fold of both mutant and wild-type IA-2 and IA-2beta was similar to that of classic PTPs. In particular, one derivative of OBA, namely 7-(1,1-dioxo-1 H -benzo[ d ]isothiazol-3-yloxymethyl)-2-(oxalylamino)-4,7-dihydro-5 H -thieno[2,3- c ]pyran-3 -carboxylic acid ('Compound 6 ' shown in the main paper), which inhibited IA-2beta((S762Y/Y898P/D933A)) (IA-2beta in which Ser(762) has been mutated to tyrosine, Tyr(898) to proline, and Asp(933) to alanine) with a K (i) value of approximately 8 microM, appeared ideal for future lead optimization. Thus molecular modelling of this classical, competitive inhibitor in the catalytic site of wild-type IA-2beta identified two residues (Ser(762) and Asp

  18. Selectivity for strand-transfer over 3'-processing and susceptibility to clinical resistance of HIV-1 integrase inhibitors are driven by key enzyme-DNA interactions in the active site.

    PubMed

    Métifiot, Mathieu; Johnson, Barry C; Kiselev, Evgeny; Marler, Laura; Zhao, Xue Zhi; Burke, Terrence R; Marchand, Christophe; Hughes, Stephen H; Pommier, Yves

    2016-08-19

    Integrase strand transfer inhibitors (INSTIs) are highly effective against HIV infections. Co-crystal structures of the prototype foamy virus intasome have shown that all three FDA-approved drugs, raltegravir (RAL), elvitegravir and dolutegravir (DTG), act as interfacial inhibitors during the strand transfer (ST) integration step. However, these structures give only a partial sense for the limited inhibition of the 3'-processing reaction by INSTIs and how INSTIs can be modified to overcome drug resistance, notably against the G140S-Q148H double mutation. Based on biochemical experiments with modified oligonucleotides, we demonstrate that both the viral DNA +1 and -1 bases, which flank the 3'-processing site, play a critical role for 3'-processing efficiency and inhibition by RAL and DTG. In addition, the G140S-Q148H (SH) mutant integrase, which has a reduced 3'-processing activity, becomes more active and more resistant to inhibition of 3'-processing by RAL and DTG in the absence of the -1 and +1 bases. Molecular modeling of HIV-1 integrase, together with biochemical data, indicate that the conserved residue Q146 in the flexible loop of HIV-1 integrase is critical for productive viral DNA binding through specific contacts with the virus DNA ends in the 3'-processing and ST reactions. The potency of integrase inhibitors against 3'-processing and their ability to overcome resistance is discussed. PMID:27369381

  19. Vibrational Stark Effects of Carbonyl Probes Applied to Reinterpret IR and Raman Data for Enzyme Inhibitors in Terms of Electric Fields at the Active Site.

    PubMed

    Schneider, Samuel H; Boxer, Steven G

    2016-09-15

    IR and Raman frequency shifts have been reported for numerous probes of enzyme transition states, leading to diverse interpretations. In the case of the model enzyme ketosteroid isomerase (KSI), we have argued that IR spectral shifts for a carbonyl probe at the active site can provide a connection between the active site electric field and the activation free energy (Fried et al. Science 2014, 346, 1510-1514). Here we generalize this approach to a much broader set of carbonyl probes (e.g., oxoesters, thioesters, and amides), first establishing the sensitivity of each probe to an electric field using vibrational Stark spectroscopy, vibrational solvatochromism, and MD simulations, and then applying these results to reinterpret data already in the literature for enzymes such as 4-chlorobenzoyl-CoA dehalogenase and serine proteases. These results demonstrate that the vibrational Stark effect provides a general framework for estimating the electrostatic contribution to the catalytic rate and may provide a metric for the design or modification of enzymes. Opportunities and limitations of the approach are also described. PMID:27541577

  20. Location of inhibitor binding sites in the human inducible prostaglandin E synthase, MPGES1.

    PubMed

    Prage, Edward B; Pawelzik, Sven-Christian; Busenlehner, Laura S; Kim, Kwangho; Morgenstern, Ralf; Jakobsson, Per-Johan; Armstrong, Richard N

    2011-09-01

    The inducible microsomal prostaglandin E(2) synthase 1 (MPGES1) is an integral membrane protein coexpressed with and functionally coupled to cyclooxygenase 2 (COX-2) generating the pro-inflammatory molecule PGE(2). The development of effective inhibitors of MPGES1 holds promise as a highly selective route for controlling inflammation. In this paper, we describe the use of backbone amide H/D exchange mass spectrometry to map the binding sites of different types of inhibitors of MPGES1. The results reveal the locations of specific inhibitor binding sites that include the GSH binding site and a hydrophobic cleft in the protein thought to accommodate the prostaglandin H(2) substrate. In the absence of three-dimensional crystal structures of the enzyme-bound inhibitors, the results provide clear physical evidence that three pharmacologically active inhibitors bind in a hydrophobic cleft composed of sections of transmembrane helices Ia, IIb, IIIb, and IVb at the interface of subunits in the trimer. In principle, the H/D exchange behavior of the protein can be used as a preliminary guide for optimization of inhibitor efficacy. Finally, a comparison of the structures and H/D exchange behavior of MPGES1 and the related enzyme MGST1 in the presence of glutathione and the inhibitor glutathione sulfonate confirms the unusual observation that two proteins from the same superfamily harbor GSH binding sites in different locations.

  1. Metalloprotein-inhibitor binding: human carbonic anhydrase II as a model for probing metal-ligand interactions in a metalloprotein active site.

    PubMed

    Martin, David P; Hann, Zachary S; Cohen, Seth M

    2013-11-01

    An ever-increasing number of metalloproteins are being discovered that play essential roles in physiological processes. Inhibitors of these proteins have significant potential for the treatment of human disease, but clinical success of these compounds has been limited. Herein, zinc(II)-dependent metalloprotein inhibitors in clinical use are reviewed, and the potential for using novel metal-binding groups (MBGs) in the design of these inhibitors is discussed. By using human carbonic anhydrase II as a model system, the nuances of MBG-metal interactions in the context of a protein environment can be probed. Understanding how metal coordination influences inhibitor binding may help in the design of new therapeutics targeting metalloproteins.

  2. The High Affinity Binding Site on Plasminogen Activator Inhibitor-1 (PAI-1) for the Low Density Lipoprotein Receptor-related Protein (LRP1) Is Composed of Four Basic Residues.

    PubMed

    Gettins, Peter G W; Dolmer, Klavs

    2016-01-01

    Plasminogen activator inhibitor 1 (PAI-1) is a serpin inhibitor of the plasminogen activators urokinase-type plasminogen activator (uPA) and tissue plasminogen activator, which binds tightly to the clearance and signaling receptor low density lipoprotein receptor-related protein 1 (LRP1) in both proteinase-complexed and uncomplexed forms. Binding sites for PAI-1 within LRP1 have been localized to CR clusters II and IV. Within cluster II, there is a strong preference for the triple CR domain fragment CR456. Previous mutagenesis studies to identify the binding site on PAI-1 for LRP1 have given conflicting results or implied small binding contributions incompatible with the high affinity PAI-1/LRP1 interaction. Using a highly sensitive solution fluorescence assay, we have examined binding of CR456 to arginine and lysine variants of PAI-1 and definitively identified the binding site as composed of four basic residues, Lys-69, Arg-76, Lys-80, and Lys-88. These are highly conserved among mammalian PAI-1s. Individual mutations result in a 13-800-fold increase in Kd values. We present evidence that binding involves engagement of CR4 by Lys-88, CR5 by Arg-76 and Lys-80, and CR6 by Lys-69, with the strongest interactions to CR5 and CR6. Collectively, the individual binding contributions account quantitatively for the overall PAI-1/LRP1 affinity. We propose that the greater efficiency of PAI-1·uPA complex binding and clearance by LRP1, compared with PAI-1 alone, is due solely to simultaneous binding of the uPA moiety in the complex to its receptor, thereby making binding of the PAI-1 moiety to LRP1 a two-dimensional surface-localized association.

  3. The High Affinity Binding Site on Plasminogen Activator Inhibitor-1 (PAI-1) for the Low Density Lipoprotein Receptor-related Protein (LRP1) Is Composed of Four Basic Residues.

    PubMed

    Gettins, Peter G W; Dolmer, Klavs

    2016-01-01

    Plasminogen activator inhibitor 1 (PAI-1) is a serpin inhibitor of the plasminogen activators urokinase-type plasminogen activator (uPA) and tissue plasminogen activator, which binds tightly to the clearance and signaling receptor low density lipoprotein receptor-related protein 1 (LRP1) in both proteinase-complexed and uncomplexed forms. Binding sites for PAI-1 within LRP1 have been localized to CR clusters II and IV. Within cluster II, there is a strong preference for the triple CR domain fragment CR456. Previous mutagenesis studies to identify the binding site on PAI-1 for LRP1 have given conflicting results or implied small binding contributions incompatible with the high affinity PAI-1/LRP1 interaction. Using a highly sensitive solution fluorescence assay, we have examined binding of CR456 to arginine and lysine variants of PAI-1 and definitively identified the binding site as composed of four basic residues, Lys-69, Arg-76, Lys-80, and Lys-88. These are highly conserved among mammalian PAI-1s. Individual mutations result in a 13-800-fold increase in Kd values. We present evidence that binding involves engagement of CR4 by Lys-88, CR5 by Arg-76 and Lys-80, and CR6 by Lys-69, with the strongest interactions to CR5 and CR6. Collectively, the individual binding contributions account quantitatively for the overall PAI-1/LRP1 affinity. We propose that the greater efficiency of PAI-1·uPA complex binding and clearance by LRP1, compared with PAI-1 alone, is due solely to simultaneous binding of the uPA moiety in the complex to its receptor, thereby making binding of the PAI-1 moiety to LRP1 a two-dimensional surface-localized association. PMID:26555266

  4. Structure of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase in complex with the feedback inhibitor CoA reveals only one active-site conformation

    SciTech Connect

    Wubben, T.; Mesecar, A.D.

    2014-10-02

    Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in the coenzyme A (CoA) biosynthetic pathway, reversibly transferring an adenylyl group from ATP to 4'-phosphopantetheine to form dephosphocoenzyme A (dPCoA). To complement recent biochemical and structural studies on Mycobacterium tuberculosis PPAT (MtPPAT) and to provide further insight into the feedback regulation of MtPPAT by CoA, the X-ray crystal structure of the MtPPAT enzyme in complex with CoA was determined to 2.11 {angstrom} resolution. Unlike previous X-ray crystal structures of PPAT-CoA complexes from other bacteria, which showed two distinct CoA conformations bound to the active site, only one conformation of CoA is observed in the MtPPAT-CoA complex.

  5. Identification of Serpin Determinants of Specificity and Selectivity for Furin Inhibition through Studies of α1PDX (α1-Protease Inhibitor Portland)-Serpin B8 and Furin Active-site Loop Chimeras*

    PubMed Central

    Izaguirre, Gonzalo; Qi, Lixin; Lima, Mary; Olson, Steven T.

    2013-01-01

    α1-Protease inhibitor Portland (α1PDX) is an engineered serpin family inhibitor of the proprotein convertase (PC), furin, that exhibits high specificity but limited selectivity for inhibiting furin over other PC family proteases. Here, we characterize serpin B8, a natural inhibitor of furin, together with α1PDX-serpin B8 and furin-PC chimeras to identify determinants of serpin specificity and selectivity for furin inhibition. Replacing reactive center loop (RCL) sequences of α1PDX with those of serpin B8 demonstrated that both the P4–P1 RXXR recognition sequence as well as the P1′–P5′ sequence are critical determinants of serpin specificity for furin. Alignments of PC catalytic domains revealed four variable active-site loops whose role in furin reactivity with serpin B8 was tested by engineering furin-PC loop chimeras. The furin(298–300) loop but not the other loops differentially affected furin reactivity with serpin B8 and α1PDX in a manner that depended on the serpin RCL-primed sequence. Modeling of the serpin B8-furin Michaelis complex identified serpin exosites in strand 3C close to the 298–300 loop whose substitution in α1PDX differentially affected furin reactivity depending on the furin loop and serpin RCL-primed sequences. These studies demonstrate that RCL-primed residues, strand 3C exosites, and the furin(298–300) loop are critical determinants of serpin reactivity with furin, which may be exploited in the design of specific and selective α1PDX inhibitors of PCs. PMID:23744066

  6. A Conserved Hydrogen-Bonding Network of P2 bis-Tetrahydrofuran-Containing HIV-1 Protease Inhibitors (PIs) with a Protease Active-Site Amino Acid Backbone Aids in Their Activity against PI-Resistant HIV

    PubMed Central

    Yedidi, Ravikiran S.; Garimella, Harisha; Aoki, Manabu; Aoki-Ogata, Hiromi; Desai, Darshan V.; Chang, Simon B.; Davis, David A.; Fyvie, W. Sean; Kaufman, Joshua D.; Smith, David W.; Das, Debananda; Wingfield, Paul T.; Maeda, Kenji; Ghosh, Arun K.

    2014-01-01

    In the present study, GRL008, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI), and darunavir (DRV), both of which contain a P2-bis-tetrahydrofuranyl urethane (bis-THF) moiety, were found to exert potent antiviral activity (50% effective concentrations [EC50s], 0.029 and 0.002 μM, respectively) against a multidrug-resistant clinical isolate of HIV-1 (HIVA02) compared to ritonavir (RTV; EC50, >1.0 μM) and tipranavir (TPV; EC50, 0.364 μM). Additionally, GRL008 showed potent antiviral activity against an HIV-1 variant selected in the presence of DRV over 20 passages (HIVDRVRP20), with a 2.6-fold increase in its EC50 (0.097 μM) compared to its corresponding EC50 (0.038 μM) against wild-type HIV-1NL4-3 (HIVWT). Based on X-ray crystallographic analysis, both GRL008 and DRV showed strong hydrogen bonds (H-bonds) with the backbone-amide nitrogen/carbonyl oxygen atoms of conserved active-site amino acids G27, D29, D30, and D30′ of HIVA02 protease (PRA02) and wild-type PR in their corresponding crystal structures, while TPV lacked H-bonds with G27 and D30′ due to an absence of polar groups. The P2′ thiazolyl moiety of RTV showed two conformations in the crystal structure of the PRA02-RTV complex, one of which showed loss of contacts in the S2′ binding pocket of PRA02, supporting RTV's compromised antiviral activity (EC50, >1 μM). Thus, the conserved H-bonding network of P2-bis-THF-containing GRL008 with the backbone of G27, D29, D30, and D30′ most likely contributes to its persistently greater antiviral activity against HIVWT, HIVA02, and HIVDRVRP20. PMID:24752271

  7. Unexpected Activity of a Novel Kunitz-type Inhibitor

    PubMed Central

    Smith, David; Tikhonova, Irina G.; Jewhurst, Heather L.; Drysdale, Orla C.; Dvořák, Jan; Robinson, Mark W.; Cwiklinski, Krystyna; Dalton, John P.

    2016-01-01

    Kunitz-type (KT) protease inhibitors are low molecular weight proteins classically defined as serine protease inhibitors. We identified a novel secreted KT inhibitor associated with the gut and parenchymal tissues of the infective juvenile stage of Fasciola hepatica, a helminth parasite of medical and veterinary importance. Unexpectedly, recombinant KT inhibitor (rFhKT1) exhibited no inhibitory activity toward serine proteases but was a potent inhibitor of the major secreted cathepsin L cysteine proteases of F. hepatica, FhCL1 and FhCL2, and of human cathepsins L and K (Ki = 0.4-27 nm). FhKT1 prevented the auto-catalytic activation of FhCL1 and FhCL2 and formed stable complexes with the mature enzymes. Pulldown experiments from adult parasite culture medium showed that rFhKT1 interacts specifically with native secreted FhCL1, FhCL2, and FhCL5. Substitution of the unusual P1 Leu15 within the exposed reactive loop of FhKT1 for the more commonly found Arg (FhKT1Leu15/Arg15) had modest adverse effects on the cysteine protease inhibition but conferred potent activity against the serine protease trypsin (Ki = 1.5 nm). Computational docking and sequence analysis provided hypotheses for the exclusive binding of FhKT1 to cysteine proteases, the importance of the Leu15 in anchoring the inhibitor into the S2 active site pocket, and the inhibitor's selectivity toward FhCL1, FhCL2, and human cathepsins L and K. FhKT1 represents a novel evolutionary adaptation of KT protease inhibitors by F. hepatica, with its prime purpose likely in the regulation of the major parasite-secreted proteases and/or cathepsin L-like proteases of its host. PMID:27422822

  8. Kinetics of activated thrombin-activatable fibrinolysis inhibitor (TAFIa)-catalyzed cleavage of C-terminal lysine residues of fibrin degradation products and removal of plasminogen-binding sites.

    PubMed

    Foley, Jonathan H; Cook, Paul F; Nesheim, Michael E

    2011-06-01

    Partial digestion of fibrin by plasmin exposes C-terminal lysine residues, which comprise new binding sites for both plasminogen and tissue-type plasminogen activator (tPA). This binding increases the catalytic efficiency of plasminogen activation by 3000-fold compared with tPA alone. The activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates fibrinolysis by removing these residues, which causes a 97% reduction in tPA catalytic efficiency. The aim of this study was to determine the kinetics of TAFIa-catalyzed lysine cleavage from fibrin degradation products and the kinetics of loss of plasminogen-binding sites. We show that the k(cat) and K(m) of Glu(1)-plasminogen (Glu-Pg)-binding site removal are 2.34 s(-1) and 142.6 nm, respectively, implying a catalytic efficiency of 16.21 μm(-1) s(-1). The corresponding values of Lys(77)/Lys(78)-plasminogen (Lys-Pg)-binding site removal are 0.89 s(-1) and 96 nm implying a catalytic efficiency of 9.23 μm(-1) s(-1). These catalytic efficiencies of plasminogen-binding site removal by TAFIa are the highest of any TAFIa-catalyzed reaction with a biological substrate reported to date and suggest that plasmin-modified fibrin is a primary physiological substrate for TAFIa. We also show that the catalytic efficiency of cleavage of all C-terminal lysine residues, whether they are involved in plasminogen binding or not, is 1.10 μm(-1) s(-1). Interestingly, this value increases to 3.85 μm(-1) s(-1) in the presence of Glu-Pg. These changes are due to a decrease in K(m). This suggests that an interaction between TAFIa and plasminogen comprises a component of the reaction mechanism, the plausibility of which was established by showing that TAFIa binds both Glu-Pg and Lys-Pg. PMID:21467042

  9. Protease Inhibitors from Plants with Antimicrobial Activity

    PubMed Central

    Kim, Jin-Young; Park, Seong-Cheol; Hwang, Indeok; Cheong, Hyeonsook; Nah, Jae-Woon; Hahm, Kyung-Soo; Park, Yoonkyung

    2009-01-01

    Antimicrobial proteins (peptides) are known to play important roles in the innate host defense mechanisms of most living organisms, including plants, insects, amphibians and mammals. They are also known to possess potent antibiotic activity against bacteria, fungi, and even certain viruses. Recently, the rapid emergence of microbial pathogens that are resistant to currently available antibiotics has triggered considerable interest in the isolation and investigation of the mode of action of antimicrobial proteins (peptides). Plants produce a variety of proteins (peptides) that are involved in the defense against pathogens and invading organisms, including ribosome-inactivating proteins, lectins, protease inhibitors and antifungal peptides (proteins). Specially, the protease inhibitors can inhibit aspartic, serine and cysteine proteinases. Increased levels of trypsin and chymotrypsin inhibitors correlated with the plants resistance to the pathogen. Usually, the purification of antimicrobial proteins (peptides) with protease inhibitor activity was accomplished by salt-extraction, ultrafiltration and C18 reverse phase chromatography, successfully. We discuss the relation between antimicrobial and anti-protease activity in this review. Protease inhibitors from plants potently inhibited the growth of a variety of pathogenic bacterial and fungal strains and are therefore excellent candidates for use as the lead compounds for the development of novel antimicrobial agents. PMID:19582234

  10. Discovery of Novel Nonactive Site Inhibitors of the Prothrombinase Enzyme Complex.

    PubMed

    Kapoor, Karan; McGill, Nicole; Peterson, Cynthia B; Meyers, Harold V; Blackburn, Michael N; Baudry, Jerome

    2016-03-28

    The risk of serious bleeding is a major liability of anticoagulant drugs that are active-site competitive inhibitors targeting the Factor Xa (FXa) prothrombin (PT) binding site. The present work identifies several new classes of small molecule anticoagulants that can act as nonactive site inhibitors of the prothrombinase (PTase) complex composed of FXa and Factor Va (FVa). These new classes of anticoagulants were identified, using a novel agnostic computational approach to identify previously unrecognized binding pockets at the FXa-FVa interface. From about three million docking calculations of 281,128 compounds in a conformational ensemble of FXa heavy chains identified by molecular dynamics (MD) simulations, 97 compounds and their structural analogues were selected for experimental validation, through a series of inhibition assays. The compound selection was based on their predicted binding affinities to FXa and their ability to successfully bind to multiple protein conformations while showing selectivity for particular binding sites at the FXa/FVa interface. From these, thirty-one (31) compounds were experimentally identified as nonactive site inhibitors. Concentration-based assays further identified 10 compounds represented by four small-molecule families of inhibitors that achieve dose-independent partial inhibition of PTase activity in a nonactive site-dependent and self-limiting mechanism. Several compounds were identified for their ability to bind to protein conformations only seen during MD, highlighting the importance of accounting for protein flexibility in structure-based drug discovery approaches.

  11. Discovery of Novel Nonactive Site Inhibitors of the Prothrombinase Enzyme Complex.

    PubMed

    Kapoor, Karan; McGill, Nicole; Peterson, Cynthia B; Meyers, Harold V; Blackburn, Michael N; Baudry, Jerome

    2016-03-28

    The risk of serious bleeding is a major liability of anticoagulant drugs that are active-site competitive inhibitors targeting the Factor Xa (FXa) prothrombin (PT) binding site. The present work identifies several new classes of small molecule anticoagulants that can act as nonactive site inhibitors of the prothrombinase (PTase) complex composed of FXa and Factor Va (FVa). These new classes of anticoagulants were identified, using a novel agnostic computational approach to identify previously unrecognized binding pockets at the FXa-FVa interface. From about three million docking calculations of 281,128 compounds in a conformational ensemble of FXa heavy chains identified by molecular dynamics (MD) simulations, 97 compounds and their structural analogues were selected for experimental validation, through a series of inhibition assays. The compound selection was based on their predicted binding affinities to FXa and their ability to successfully bind to multiple protein conformations while showing selectivity for particular binding sites at the FXa/FVa interface. From these, thirty-one (31) compounds were experimentally identified as nonactive site inhibitors. Concentration-based assays further identified 10 compounds represented by four small-molecule families of inhibitors that achieve dose-independent partial inhibition of PTase activity in a nonactive site-dependent and self-limiting mechanism. Several compounds were identified for their ability to bind to protein conformations only seen during MD, highlighting the importance of accounting for protein flexibility in structure-based drug discovery approaches. PMID:26848511

  12. Discovering Anti-platelet Drug Combinations with an Integrated Model of Activator-Inhibitor Relationships, Activator-Activator Synergies and Inhibitor-Inhibitor Synergies

    PubMed Central

    Lombardi, Federica; Golla, Kalyan; Fitzpatrick, Darren J.; Casey, Fergal P.; Moran, Niamh; Shields, Denis C.

    2015-01-01

    Identifying effective therapeutic drug combinations that modulate complex signaling pathways in platelets is central to the advancement of effective anti-thrombotic therapies. However, there is no systems model of the platelet that predicts responses to different inhibitor combinations. We developed an approach which goes beyond current inhibitor-inhibitor combination screening to efficiently consider other signaling aspects that may give insights into the behaviour of the platelet as a system. We investigated combinations of platelet inhibitors and activators. We evaluated three distinct strands of information, namely: activator-inhibitor combination screens (testing a panel of inhibitors against a panel of activators); inhibitor-inhibitor synergy screens; and activator-activator synergy screens. We demonstrated how these analyses may be efficiently performed, both experimentally and computationally, to identify particular combinations of most interest. Robust tests of activator-activator synergy and of inhibitor-inhibitor synergy required combinations to show significant excesses over the double doses of each component. Modeling identified multiple effects of an inhibitor of the P2Y12 ADP receptor, and complementarity between inhibitor-inhibitor synergy effects and activator-inhibitor combination effects. This approach accelerates the mapping of combination effects of compounds to develop combinations that may be therapeutically beneficial. We integrated the three information sources into a unified model that predicted the benefits of a triple drug combination targeting ADP, thromboxane and thrombin signaling. PMID:25875950

  13. Structures of Aquifex aeolicus KDO8P synthase in complex with R5P and PEP, and with a bisubstrate inhibitor: role of active site water in catalysis.

    PubMed

    Wang, J; Duewel, H S; Woodard, R W; Gatti, D L

    2001-12-25

    We have determined the crystal structures of the metalloenzyme 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase from Aquifex aeolicus in complex with phosphoenolpyruvate (PEP) and ribose 5-phosphate (R5P), and with a bisubstrate inhibitor that mimics the postulated linear reaction intermediate. R5P, which is not a substrate for KDO8P synthase, binds in a manner similar to that of arabinose 5-phosphate (A5P), which is the natural substrate. The lack of reactivity of R5P appears to be primarily a consequence of the loss of a water molecule coordinated to Cd(2+) and located on the si side of PEP. This water molecule is no longer present because it cannot form a hydrogen bond with C2-OH(R5P), which is oriented in a different direction from C2-OH(A5P). The bisubstrate inhibitor binds with its phosphate and phosphonate moieties occupying the positions of the phosphate groups of A5P and PEP, respectively. One of the inhibitor hydroxyls replaces water as a ligand of Cd(2+). The current work supports a mechanism for the synthesis of KDO8P, in which a hydroxide ion on the si side of PEP attacks C2(PEP), forming a tetrahedral-like intermediate with a buildup of negative charge at C3(PEP). The ensuing condensation of C3(PEP) with C1(A5P) would be favored by a proton transfer from the phosphate moiety of PEP to the aldehyde carbonyl of A5P to generate the hydroxyl. Overall, the process can be described as a syn addition of water and A5P to the si side of PEP.

  14. Functional Stability of Plasminogen Activator Inhibitor-1

    PubMed Central

    Kuru, Pinar; Toksoy Oner, Ebru; Agirbasli, Mehmet

    2014-01-01

    Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of plasminogen activators, such as tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), and a major regulator of the fibrinolytic system. PAI-1 plays a pivotal role in acute thrombotic events such as deep vein thrombosis (DVT) and myocardial infarction (MI). The biological effects of PAI-1 extend far beyond thrombosis including its critical role in fibrotic disorders, atherosclerosis, renal and pulmonary fibrosis, type-2 diabetes, and cancer. The conversion of PAI-1 from the active to the latent conformation appears to be unique among serpins in that it occurs spontaneously at a relatively rapid rate. Latency transition is believed to represent a regulatory mechanism, reducing the risk of thrombosis from a prolonged antifibrinolytic action of PAI-1. Thus, relying solely on plasma concentrations of PAI-1 without assessing its function may be misleading in interpreting the role of PAI-1 in many complex diseases. Environmental conditions, interaction with other proteins, mutations, and glycosylation are the main factors that have a significant impact on the stability of the PAI-1 structure. This review provides an overview on the current knowledge on PAI-1 especially importance of PAI-1 level and stability and highlights the potential use of PAI-1 inhibitors for treating cardiovascular disease. PMID:25386620

  15. A complement C3 inhibitor specifically targeted to sites of complement activation effectively ameliorates collagen-induced arthritis in DBA/1J mice.

    PubMed

    Song, Hongbin; Qiao, Fei; Atkinson, Carl; Holers, V Michael; Tomlinson, Stephen

    2007-12-01

    Collagen-induced arthritis (CIA) represents an animal model of autoimmune polyarthritis with similarities to human rheumatoid arthritis, and therapy with various systemic complement-inhibitory proteins has been investigated in this model with varying results. We investigated the use of complement receptor 2 (CR2)-Crry, a complement inhibitor with the ability to target C3 breakdown products deposited in a rheumatic joint. Following induction of CIA in DBA/1J mice, animals were treated with either PBS or CR2-Crry (every other day, every 4 days, or with a single injection). The severity of clinical disease was significantly reduced in all CR2-Crry-treated groups compared with controls. Joints from mice receiving multiple doses of CR2-Crry showed significantly decreased inflammatory cell infiltrate, cartilage damage, pannus formation, and bone damage. CR2-Crry treatment also significantly decreased production of anti-collagen IgG and the inflammatory cytokines TNF-alpha and IL-1beta. IL-10 and IL-1Ra levels were increased in CR2-Crry-treated mice. CR2-Crry localized preferentially in the joints of mice with CIA. Analysis of IgG and C3 deposition in the joints of treated animals indicated that both complement regulation and the modulation of anti-collagen Ab production contributed to the protective effects of CR2-Crry. Of interest, a previous study reported that Crry-Ig, an untargeted counterpart of CR2-Crry, had minimal effect on disease, even when administered at a sufficiently high dose to maintain chronic complement inhibition. PMID:18025232

  16. Exploiting an Allosteric Binding Site of PRMT3 Yields Potent and Selective Inhibitors

    PubMed Central

    Liu, Feng; Li, Fengling; Ma, Anqi; Dobrovetsky, Elena; Dong, Aiping; Gao, Cen; Korboukh, Ilia; Liu, Jing; Smil, David; Brown, Peter J.; Frye, Stephen V.; Arrowsmith, Cheryl H.; Schapira, Matthieu; Vedadi, Masoud; Jin, Jian

    2015-01-01

    Protein arginine methyltransferases (PRMTs) play an important role in diverse biological processes. Among the nine known human PRMTs, PRMT3 has been implicated in ribosomal biosynthesis via asymmetric dimethylation of the 40S ribosomal protein S2 and in cancer via interaction with the DAL-1 tumor suppressor protein. However, few selective inhibitors of PRMTs have been discovered. We recently disclosed the first selective PRMT3 inhibitor, which occupies a novel allosteric binding site and is noncompetitive with both the peptide substrate and cofactor. Here we report comprehensive structure–activity relationship studies of this series, which resulted in the discovery of multiple PRMT3 inhibitors with submicromolar potencies. An X-ray crystal structure of compound 14u in complex with PRMT3 confirmed that this inhibitor occupied the same allosteric binding site as our initial lead compound. These studies provide the first experimental evidence that potent and selective inhibitors can be created by exploiting the allosteric binding site of PRMT3. PMID:23445220

  17. Arginine mimetic structures in biologically active antagonists and inhibitors.

    PubMed

    Masic, Lucija Peterlin

    2006-01-01

    Peptidomimetics have found wide application as bioavailable, biostable, and potent mimetics of naturally occurring biologically active peptides. L-Arginine is a guanidino group-containing basic amino acid, which is positively charged at neutral pH and is involved in many important physiological and pathophysiological processes. Many enzymes display a preference for the arginine residue that is found in many natural substrates and in synthetic inhibitors of many trypsin-like serine proteases, e.g. thrombin, factor Xa, factor VIIa, trypsin, and in integrin receptor antagonists, used to treat many blood-coagulation disorders. Nitric oxide (NO), which is produced by oxidation of L-arginine in an NADPH- and O(2)-dependent process catalyzed by isoforms of nitric oxide synthase (NOS), exhibits diverse roles in both normal and pathological physiologies and has been postulated to be a contributor to the etiology of various diseases. Development of NOS inhibitors as well as analogs and mimetics of the natural substrate L-arginine, is desirable for potential therapeutic use and for a better understanding of their conformation when bound in the arginine binding site. The guanidino residue of arginine in many substrates, inhibitors, and antagonists forms strong ionic interactions with the carboxylate of an aspartic acid moiety, which provides specificity for the basic amino acid residue in the active side. However, a highly basic guanidino moiety incorporated in enzyme inhibitors or receptor antagonists is often associated with low selectivity and poor bioavailability after peroral application. Thus, significant effort is focused on the design and preparation of arginine mimetics that can confer selective inhibition for specific trypsin-like serine proteases and NOS inhibitors as well as integrin receptor antagonists and possess reduced basicity for enhanced oral bioavailability. This review will describe the survey of arginine mimetics designed to mimic the function of the

  18. Aldose and aldehyde reductases : structure-function studies on the coenzyme and inhibitor-binding sites.

    SciTech Connect

    El-Kabbani, O.; Old, S. E.; Ginell, S. L.; Carper, D. A.; Biosciences Division; Monash Univ.; NIH

    1999-09-03

    PURPOSE: To identify the structural features responsible for the differences in coenzyme and inhibitor specificities of aldose and aldehyde reductases. METHODS: The crystal structure of porcine aldehyde reductase in complex with NADPH and the aldose reductase inhibitor sorbinil was determined. The contribution of each amino acid lining the coenzyme-binding site to the binding of NADPH was calculated using the Discover package. In human aldose reductase, the role of the non-conserved Pro 216 (Ser in aldehyde reductase) in the binding of coenzyme was examined by site-directed mutagenesis. RESULTS: Sorbinil binds to the active site of aldehyde reductase and is hydrogen-bonded to Trp 22, Tyr 50, His 113, and the non-conserved Arg 312. Unlike tolrestat, the binding of sorbinil does not induce a change in the side chain conformation of Arg 312. Mutation of Pro 216 to Ser in aldose reductase makes the binding of coenzyme more similar to that of aldehyde reductase. CONCLUSIONS: The participation of non-conserved active site residues in the binding of inhibitors and the differences in the structural changes required for the binding to occur are responsible for the differences in the potency of inhibition of aldose and aldehyde reductases. We report that the non-conserved Pro 216 in aldose reductase contributes to the tight binding of NADPH.

  19. Catalytic site remodelling of the DOT1L methyltransferase by selective inhibitors

    SciTech Connect

    Yu, Wenyu; Chory, Emma J.; Wernimont, Amy K.; Tempel, Wolfram; Scopton, Alex; Federation, Alexander; Marineau, Jason J.; Qi, Jun; Barsyte-Lovejoy, Dalia; Yi, Joanna; Marcellus, Richard; Iacob, Roxana E.; Engen, John R.; Griffin, Carly; Aman, Ahmed; Wienholds, Erno; Li, Fengling; Pineda, Javier; Estiu, Guillermina; Shatseva, Tatiana; Hajian, Taraneh; Al-awar, Rima; Dick, John E.; Vedadi, Masoud; Brown, Peter J.; Arrowsmith, Cheryl H.; Bradner, James E.; Schapira, Matthieu

    2012-12-18

    Selective inhibition of protein methyltransferases is a promising new approach to drug discovery. An attractive strategy towards this goal is the development of compounds that selectively inhibit binding of the cofactor, S-adenosylmethionine, within specific protein methyltransferases. Here we report the three-dimensional structure of the protein methyltransferase DOT1L bound toEPZ004777, the first S-adenosylmethionine-competitive inhibitor of a protein methyltransferase with in vivo efficacy. This structure and those of four new analogues reveal remodelling of the catalytic site. EPZ004777 and a brominated analogue, SGC0946, inhibit DOT1L in vitro and selectively kill mixed lineage leukaemia cells, in which DOT1L is aberrantly localized via interaction with an oncogenic MLL fusion protein. These data provide important new insight into mechanisms of cell-active S-adenosylmethionine-competitive protein methyltransferase inhibitors, and establish a foundation for the further development of drug-like inhibitors of DOT1L for cancer therapy.

  20. Low dielectric response in enzyme active site

    PubMed Central

    Mertz, Edward L.; Krishtalik, Lev I.

    2000-01-01

    The kinetics of charge transfer depend crucially on the dielectric reorganization of the medium. In enzymatic reactions that involve charge transfer, atomic dielectric response of the active site and of its surroundings determines the efficiency of the protein as a catalyst. We report direct spectroscopic measurements of the reorganization energy associated with the dielectric response in the active site of α-chymotrypsin. A chromophoric inhibitor of the enzyme is used as a spectroscopic probe. We find that water strongly affects the dielectric reorganization in the active site of the enzyme in solution. The reorganization energy of the protein matrix in the vicinity of the active site is similar to that of low-polarity solvents. Surprisingly, water exhibits an anomalously high dielectric response that cannot be described in terms of the dielectric continuum theory. As a result, sequestering the active site from the aqueous environment inside low-dielectric enzyme body dramatically reduces the dielectric reorganization. This reduction is particularly important for controlling the rate of enzymatic reactions. PMID:10681440

  1. Selective small molecule inhibitor of the Mycobacterium tuberculosis fumarate hydratase reveals an allosteric regulatory site

    PubMed Central

    Kasbekar, Monica; Fischer, Gerhard; Mott, Bryan T.; Yasgar, Adam; Hyvönen, Marko; Boshoff, Helena I. M.; Abell, Chris; Barry, Clifton E.; Thomas, Craig J.

    2016-01-01

    Enzymes in essential metabolic pathways are attractive targets for the treatment of bacterial diseases, but in many cases, the presence of homologous human enzymes makes them impractical candidates for drug development. Fumarate hydratase, an essential enzyme in the tricarboxylic acid (TCA) cycle, has been identified as one such potential therapeutic target in tuberculosis. We report the discovery of the first small molecule inhibitor, to our knowledge, of the Mycobacterium tuberculosis fumarate hydratase. A crystal structure at 2.0-Å resolution of the compound in complex with the protein establishes the existence of a previously unidentified allosteric regulatory site. This allosteric site allows for selective inhibition with respect to the homologous human enzyme. We observe a unique binding mode in which two inhibitor molecules interact within the allosteric site, driving significant conformational changes that preclude simultaneous substrate and inhibitor binding. Our results demonstrate the selective inhibition of a highly conserved metabolic enzyme that contains identical active site residues in both the host and the pathogen. PMID:27325754

  2. Plasminogen activator inhibitor-1 and diabetic nephropathy.

    PubMed

    Lee, Hi Bahl; Ha, Hunjoo

    2005-10-01

    Diabetic nephropathy is characterized by excessive accumulation of extracellular matrix (ECM) in the kidney. Decreased ECM degradation as well as increased ECM synthesis plays an important role in ECM remodeling that favours tissue fibrosis. Plasminogen activator (PA)/plasmin/PA inhibitor (PAI) system is involved in ECM degradation and PAI-1 plays a critical role in ECM remodeling in the kidney. Normal human kidneys do not express PAI-1 but PAI-1 is overexpressed in pathologic conditions associated with renal fibrosis including diabetic nephropathy. Reactive oxygen species mediate PAI-1 up-regulation in renal cells cultured under high glucose, hypoxia, and TGF-beta1. Recent studies utilizing PAI-1 deficient mice suggest that PAI-1 induce ECM deposition in diabetic kidney through increased ECM synthesis by TGF-beta1 up-regulation as well as through decreased ECM degradation by suppression of plasmin and MMP-2 activity.

  3. Discovery of a novel allosteric inhibitor-binding site in ERK5: comparison with the canonical kinase hinge ATP-binding site

    PubMed Central

    Chen, Hongming; Tucker, Julie; Wang, Xiaotao; Gavine, Paul R.; Phillips, Chris; Augustin, Martin A.; Schreiner, Patrick; Steinbacher, Stefan; Preston, Marian; Ogg, Derek

    2016-01-01

    MAP kinases act as an integration point for multiple biochemical signals and are involved in a wide variety of cellular processes such as proliferation, differentiation, regulation of transcription and development. As a member of the MAP kinase family, ERK5 (MAPK7) is involved in the downstream signalling pathways of various cell-surface receptors, including receptor tyrosine kinases and G protein-coupled receptors. In the current study, five structures of the ERK5 kinase domain co-crystallized with ERK5 inhibitors are reported. Interestingly, three of the compounds bind at a novel allosteric binding site in ERK5, while the other two bind at the typical ATP-binding site. Binding of inhibitors at the allosteric site is accompanied by displacement of the P-loop into the ATP-binding site and is shown to be ATP-competitive in an enzymatic assay of ERK5 kinase activity. Kinase selectivity data show that the most potent allosteric inhibitor exhibits superior kinase selectivity compared with the two inhibitors that bind at the canonical ATP-binding site. An analysis of these structures and comparison with both a previously published ERK5–inhibitor complex structure (PDB entry 4b99) and the structures of three other kinases (CDK2, ITK and MEK) in complex with allosteric inhibitors are presented. PMID:27139631

  4. Discovery of a novel allosteric inhibitor-binding site in ERK5: comparison with the canonical kinase hinge ATP-binding site.

    PubMed

    Chen, Hongming; Tucker, Julie; Wang, Xiaotao; Gavine, Paul R; Phillips, Chris; Augustin, Martin A; Schreiner, Patrick; Steinbacher, Stefan; Preston, Marian; Ogg, Derek

    2016-05-01

    MAP kinases act as an integration point for multiple biochemical signals and are involved in a wide variety of cellular processes such as proliferation, differentiation, regulation of transcription and development. As a member of the MAP kinase family, ERK5 (MAPK7) is involved in the downstream signalling pathways of various cell-surface receptors, including receptor tyrosine kinases and G protein-coupled receptors. In the current study, five structures of the ERK5 kinase domain co-crystallized with ERK5 inhibitors are reported. Interestingly, three of the compounds bind at a novel allosteric binding site in ERK5, while the other two bind at the typical ATP-binding site. Binding of inhibitors at the allosteric site is accompanied by displacement of the P-loop into the ATP-binding site and is shown to be ATP-competitive in an enzymatic assay of ERK5 kinase activity. Kinase selectivity data show that the most potent allosteric inhibitor exhibits superior kinase selectivity compared with the two inhibitors that bind at the canonical ATP-binding site. An analysis of these structures and comparison with both a previously published ERK5-inhibitor complex structure (PDB entry 4b99) and the structures of three other kinases (CDK2, ITK and MEK) in complex with allosteric inhibitors are presented.

  5. Plasminogen activator inhibitor-1 in aging.

    PubMed

    Yamamoto, Koji; Takeshita, Kyosuke; Saito, Hidehiko

    2014-09-01

    Plasminogen activator inhibitor-1 (PAI-1), a principal inhibitor of fibrinolysis, is induced in thrombotic, fibrotic, and cardiovascular diseases, which in turn primarily afflict the older population. This induction of PAI-1 may play an important role in the pathology of these diseases as PAI-1 can regulate the dissolution of fibrin and also inhibit the degradation of the extracellular matrix by reducing plasmin generation. PAI-1 expression is elevated in aged individuals and is significantly upregulated in a variety of pathologies associated with the process of aging, including myocardial and cerebral infarction, vascular (athero) sclerosis, cardiac and lung fibrosis, metabolic syndromes (e.g., hypertension, hyperlipidemia, and insulin resistance), cancer, and inflammatory/stress responses. Thus, PAI-1 may play a critical role in the development of aging-associated pathological changes. In addition, PAI-1 is recognized as a marker of senescence and a key member of a group of proteins collectively known as the senescence-messaging secretome. In this review, we highlight the role of PAI-1 in the pathophysiology of aging and aging-associated disorders.

  6. Engineering Factor Xa Inhibitor with Multiple Platelet-Binding Sites Facilitates its Platelet Targeting

    PubMed Central

    Zhu, Yuanjun; Li, Ruyi; Lin, Yuan; Shui, Mengyang; Liu, Xiaoyan; Chen, Huan; Wang, Yinye

    2016-01-01

    Targeted delivery of antithrombotic drugs centralizes the effects in the thrombosis site and reduces the hemorrhage side effects in uninjured vessels. We have recently reported that the platelet-targeting factor Xa (FXa) inhibitors, constructed by engineering one Arg-Gly-Asp (RGD) motif into Ancylostoma caninum anticoagulant peptide 5 (AcAP5), can reduce the risk of systemic bleeding than non-targeted AcAP5 in mouse arterial injury model. Increasing the number of platelet-binding sites of FXa inhibitors may facilitate their adhesion to activated platelets, and further lower the bleeding risks. For this purpose, we introduced three RGD motifs into AcAP5 to generate a variant NR4 containing three platelet-binding sites. NR4 reserved its inherent anti-FXa activity. Protein-protein docking showed that all three RGD motifs were capable of binding to platelet receptor αIIbβ3. Molecular dynamics simulation demonstrated that NR4 has more opportunities to interact with αIIbβ3 than single-RGD-containing NR3. Flow cytometry analysis and rat arterial thrombosis model further confirmed that NR4 possesses enhanced platelet targeting activity. Moreover, NR4-treated mice showed a trend toward less tail bleeding time than NR3-treated mice in carotid artery endothelium injury model. Therefore, our data suggest that engineering multiple binding sites in one recombinant protein is a useful tool to improve its platelet-targeting efficiency. PMID:27432161

  7. Identification of a new JNK inhibitor targeting the JNK-JIP interaction site

    PubMed Central

    Stebbins, John L.; De, Surya K.; Machleidt, Thomas; Becattini, Barbara; Vazquez, Jesus; Kuntzen, Christian; Chen, Li-Hsing; Cellitti, Jason F.; Riel-Mehan, Megan; Emdadi, Aras; Solinas, Giovanni; Karin, Michael; Pellecchia, Maurizio

    2008-01-01

    JNK is a stress-activated protein kinase that modulates pathways implicated in a variety of disease states. JNK-interacting protein-1 (JIP1) is a scaffolding protein that enhances JNK signaling by creating a proximity effect between JNK and upstream kinases. A minimal peptide region derived from JIP1 is able to inhibit JNK activity both in vitro and in cell. We report here a series of small molecules JIP1 mimics that function as substrate competitive inhibitors of JNK. One such compound, BI-78D3, dose-dependently inhibits the phosphorylation of JNK substrates both in vitro and in cell. In animal studies, BI-78D3 not only blocks JNK dependent Con A-induced liver damage but also restores insulin sensitivity in mouse models of type 2 diabetes. Our findings open the way for the development of protein kinase inhibitors targeting substrate specific docking sites, rather than the highly conserved ATP binding sites. In view of its favorable inhibition profile, selectivity, and ability to function in the cellular milieu and in vivo, BI-78D3 represents not only a JNK inhibitor, but also a promising stepping stone toward the development of an innovative class of therapeutics. PMID:18922779

  8. 3-Nitropropionic Acid is a Suicide Inhibitor of MitochondrialRespiration that, Upon Oxidation by Complex II, Forms a Covalent AdductWith a Catalytic Base Arginine in the Active Site of the Enzyme

    SciTech Connect

    Huang, Li-shar; Sun, Gang; Cobessi, David; Wang, Andy C.; Shen,John T.; Tung, Eric Y.; Anderson, Vernon E.; Berry, Edward A.

    2005-12-01

    We report three new structures of mitochondrial respiratory Complex II (succinate ubiquinone oxidoreductase, E.C. 1.3.5.1) at up to 2.1 {angstrom} resolution, with various inhibitors. The structures define the conformation of the bound inhibitors and suggest the residues involved in substrate binding and catalysis at the dicarboxylate site. In particular they support the role of Arg297 as a general base catalyst accepting a proton in the dehydrogenation of succinate. The dicarboxylate ligand in oxaloacetate-containing crystals appears to be the same as that reported for Shewanella flavocytochrome c treated with fumarate. The plant and fungal toxin 3-nitropropionic acid, an irreversible inactivator of succinate dehydrogenase, forms a covalent adduct with the side chain of Arg297. The modification eliminates a trypsin cleavage site in the flavoprotein, and tandem mass spectroscopic analysis of the new fragment shows the mass of Arg 297 to be increased by 83 Da and to have potential of losing 44 Da, consistent with decarboxylation, during fragmentation.

  9. New potent inhibitors of tyrosinase: novel clues to binding of 1,3,4-thiadiazole-2(3H)-thiones, 1,3,4-oxadiazole-2(3H)-thiones, 4-amino-1,2,4-triazole-5(4H)-thiones, and substituted hydrazides to the dicopper active site.

    PubMed

    Ghani, Usman; Ullah, Nisar

    2010-06-01

    A series of 1,3,4-thiadiazole-2(3H)-thiones, 1,3,4-oxadiazole-2(3H)-thiones, 4-amino-1,2,4-triazole-5(4H)-thiones, and substituted hydrazides were tailored and synthesized as new potent inhibitors of tyrosinase. The rationale for inhibitor design was based on the active site structural evidence from the crystal structures of bacterial tyrosinase and potato catechol oxidase enzymes. Kinetic and active site binding studies suggested mono-dentate binding of thiadiazole, oxadiazole, and triazole rings to the active site dicopper center of tyrosinase including hydrophobicity contributing to the potent inhibition. Kinetic plots showed mixed-type of inhibition by all 25 compounds. Substitutions at C3 of the triazole ring and C5 of the thiadiazole/oxadiazole rings were found to be playing a major role in the high binding affinity to tyrosinase. The current work may help develop new potent tyrosinase inhibitors against hyperpigmentation including potential insecticides. PMID:20452224

  10. Discovery of dual binding site acetylcholinesterase inhibitors identified by pharmacophore modeling and sequential virtual screening techniques.

    PubMed

    Gupta, Shikhar; Fallarero, Adyary; Järvinen, Päivi; Karlsson, Daniela; Johnson, Mark S; Vuorela, Pia M; Mohan, C Gopi

    2011-02-15

    Dual binding site acetylcholinesterase (AChE) inhibitors are promising for the treatment of Alzheimer's disease (AD). They alleviate the cognitive deficits and AD-modifying agents, by inhibiting the β-amyloid (Aβ) peptide aggregation, through binding to both the catalytic and peripheral anionic sites, the so called dual binding site of the AChE enzyme. In this Letter, chemical features based 3D-pharmacophore models were developed based on the eight potent and structurally diverse AChE inhibitors (I-VIII) obtained from high-throughput in vitro screening technique. The best 3D-pharmacophore model, Hypo1, consists of two hydrogen-bond acceptor lipid, one hydrophobe, and two hydrophobic aliphatic features obtained by Catalyst/HIPHOP algorithm adopted in Discovery studio program. Hypo1 was used as a 3D query in sequential virtual screening study to filter three small compound databases. Further, a total of nine compounds were selected and followed on in vitro analysis. Finally, we identified two leads--Specs1 (IC(50)=3.279 μM) and Spec2 (IC(50)=5.986 μM) dual binding site compounds from Specs database, having good AChE enzyme inhibitory activity. PMID:21273074

  11. Discovery of dual binding site acetylcholinesterase inhibitors identified by pharmacophore modeling and sequential virtual screening techniques.

    PubMed

    Gupta, Shikhar; Fallarero, Adyary; Järvinen, Päivi; Karlsson, Daniela; Johnson, Mark S; Vuorela, Pia M; Mohan, C Gopi

    2011-02-15

    Dual binding site acetylcholinesterase (AChE) inhibitors are promising for the treatment of Alzheimer's disease (AD). They alleviate the cognitive deficits and AD-modifying agents, by inhibiting the β-amyloid (Aβ) peptide aggregation, through binding to both the catalytic and peripheral anionic sites, the so called dual binding site of the AChE enzyme. In this Letter, chemical features based 3D-pharmacophore models were developed based on the eight potent and structurally diverse AChE inhibitors (I-VIII) obtained from high-throughput in vitro screening technique. The best 3D-pharmacophore model, Hypo1, consists of two hydrogen-bond acceptor lipid, one hydrophobe, and two hydrophobic aliphatic features obtained by Catalyst/HIPHOP algorithm adopted in Discovery studio program. Hypo1 was used as a 3D query in sequential virtual screening study to filter three small compound databases. Further, a total of nine compounds were selected and followed on in vitro analysis. Finally, we identified two leads--Specs1 (IC(50)=3.279 μM) and Spec2 (IC(50)=5.986 μM) dual binding site compounds from Specs database, having good AChE enzyme inhibitory activity.

  12. Design, Synthesis, and Biological Evaluation of Stable Colchicine Binding Site Tubulin Inhibitors as Potential Anticancer Agents

    PubMed Central

    2015-01-01

    To block the metabolically labile sites of novel tubulin inhibitors targeting the colchicine binding site based on SMART, ABI, and PAT templates, we have designed, synthesized, and biologically tested three focused sets of new derivatives with modifications at the carbonyl linker, the para-position in the C ring of SMART template, and modification of A ring of the PAT template. Structure–activity relationships of these compounds led to the identification of new benzimidazole and imidazo[4,5-c]pyridine-fused ring templates, represented by compounds 4 and 7, respectively, which showed enhanced antitumor activity and substantially improved the metabolic stability in liver microsomes compared to SMART. MOM group replaced TMP C ring and generated a potent analogue 15, which showed comparable potency to the parent SMART compound. Further modification of PAT template yielded another potent analogue 33 with 5-indolyl substituent at A ring. PMID:25122533

  13. The granzyme B inhibitor, protease inhibitor 9, is mainly expressed by dendritic cells and at immune-privileged sites.

    PubMed

    Bladergroen, B A; Strik, M C; Bovenschen, N; van Berkum, O; Scheffer, G L; Meijer, C J; Hack, C E; Kummer, J A

    2001-03-01

    Granzyme B is released from CTLs and NK cells and an important mediator of CTL/NK-induced apoptosis in target cells. The human intracellular serpin proteinase inhibitor (PI)9 is the only human protein able to inhibit the activity of granzyme B. As a first step to elucidate the physiological role of PI9, PI9 protein expression in various human tissues was studied. A mAb directed against human PI9 was developed, which specifically stained PI9-transfected COS-7 cells, and was used for immunohistochemistry. Both in primary lymphoid organs and in inflammatory infiltrates, PI9 was present in different subsets of dendritic cells. Also T-lymphocytes in primary and organ-associated lymphoid tissues were PI9 positive. Endothelial cells of small vessels in most organs tested as well as the endothelial layer of large veins and arteries showed strong PI9 staining. Surprisingly, high PI9 protein expression was also found at immune-privileged sites like the placenta, the testis, the ovary, and the eye. These data fit with the hypothesis that PI9 is expressed at sites where degranulation of CTL or NK cells is potentially deleterious.

  14. Salt site performance assessment activities

    SciTech Connect

    Kircher, J.F.; Gupta, S.K.

    1983-01-01

    During this year the first selection of the tools (codes) for performance assessments of potential salt sites have been tentatively selected and documented; the emphasis has shifted from code development to applications. During this period prior to detailed characterization of a salt site, the focus is on bounding calculations, sensitivity and with the data available. The development and application of improved methods for sensitivity and uncertainty analysis is a focus for the coming years activities and the subject of a following paper in these proceedings. Although the assessments to date are preliminary and based on admittedly scant data, the results indicate that suitable salt sites can be identified and repository subsystems designed which will meet the established criteria for protecting the health and safety of the public. 36 references, 5 figures, 2 tables.

  15. Structure-activity relationships by mass spectrometry: identification of novel MMP-3 inhibitors.

    PubMed

    Ockey, Denise A; Dotson, Jenna L; Struble, Martin E; Stults, John T; Bourell, James H; Clark, Kevin R; Gadek, Thomas R

    2004-01-01

    A novel class of nonpeptide inhibitors of stromelysin (MMP-3) has been discovered with the use of mass spectrometry. The method relies on the development of structure-activity relationships by mass spectrometry (SAR by MS) and utilizes information derived from the binding of known inhibitors to identify novel inhibitors of a target protein with a minimum of synthetic effort. Noncovalent complexes of known inhibitors with a target protein are analyzed; these inhibitors are deconstructed into sets of fragments which compete for common or overlapping binding sites on the target protein. The binding of each fragment set can be studied independently. With the use of competition studies, novel members of each fragment set are identified from compound libraries that bind to the same site on the target protein. A novel inhibitor of the target protein was then constructed by chemically linking a combination of members of each fragment set in a manner guided by the proximity and orientation of the fragments derived from the known inhibitors. In the case of stromelysin, a novel inhibitor composed of favorably linked fragments was observed to form a 1:1 complex with stromelysin. Compounds that were not linked appropriately formed higher order complexes with stoichiometries of 2:1 or greater. These linked molecules were subsequently assessed for their ability to block stromelysin function in a chromogenic substrate assay.

  16. Two sites of action for PLD2 inhibitors: The enzyme catalytic center and an allosteric, phosphoinositide biding pocket.

    PubMed

    Ganesan, Ramya; Mahankali, Madhu; Alter, Gerald; Gomez-Cambronero, Julian

    2015-03-01

    Phospholipase D (PLD) has been implicated in many physiological functions, such as chemotaxis and phagocytosis, as well as pathological functions, such as cancer cell invasion and metastasis. New inhibitors have been described that hamper the role of PLD in those pathologies but their site of action is not known. We have characterized the biochemical and biological behavior of the PLD1/2 dual inhibitor 5-Fluoro-2-indolyl des-chlorohalopemide (FIPI), and the specific PLD2 inhibitor, N-[2-[1-(3-Fluorophenyl)-4-oxo-1,3,-8-triazaspiro[4.5]dec-8-yl]ethyl]-2-naphthalenecarboxamide (NFOT), and found that both FIPI and NFOT are mixed-kinetics inhibitors. Mutagenesis studies indicate that FIPI binds at S757 of PLD2, which is within the HKD2 catalytic site of the enzyme, whereas NFOT binds to PLD2 at two different sites, one being at S757/S648 and another to an allosteric site that is a natural site occupied by PIP2 (R210/R212). This latter site, along with F244/L245/L246, forms a hydrophobic pocket in the PH domain. The mechanism of action of FIPI is a direct effect on the catalytic site (and as such inhibits both PLD1 and PLD2 isoforms), whereas PLD2 affects both the catalytic site (orthosteric) and blocks PIP2 binding to PLD2 (allosteric), which negates the natural enhancing role of PIP2. Moreover, NFOT prevents cell invasion of cancer cells, which does not occur in cells overexpressing PLD2-F244A/L245A/L246A, or PLD2-R210A/R212A, or PLD2-S757/S648 mutants. This study provides new specific knowledge of enzyme regulation and mechanisms of activation and inhibition of PLD2 that are necessary to understand its role in cell signaling and to develop new inhibitors for cancer cell invasion and metastasis.

  17. Development and structural analysis of adenosine site binding tankyrase inhibitors.

    PubMed

    Haikarainen, Teemu; Waaler, Jo; Ignatev, Alexander; Nkizinkiko, Yves; Venkannagari, Harikanth; Obaji, Ezeogo; Krauss, Stefan; Lehtiö, Lari

    2016-01-15

    Tankyrases 1 and 2, the specialized members of the ARTD protein family, are druggable biotargets whose inhibition may have therapeutic potential against cancer, metabolic disease, fibrotic disease, fibrotic wound healing and HSV viral infections. We have previously identified a novel tankyrase inhibitor scaffold, JW55, and showed that it reduces mouse colon adenoma formation in vivo. Here we expanded the scaffold and profiled the selectivity of the compounds against a panel of human ARTDs. The scaffold also enables a fine modulation of selectivity towards either tankyrase 1 or tankyrase 2. In order to get insight about the binding mode of the inhibitors, we solved crystal structures of the compounds in complex with tankyrase 2. The compounds bind to the adenosine pocket of the catalytic domain and cause changes in the protein structure that are modulated by the chemical modifications of the compounds. The structural analysis allows further rational development of this compound class as a potent and selective tankyrase inhibitor. PMID:26706174

  18. Development and structural analysis of adenosine site binding tankyrase inhibitors.

    PubMed

    Haikarainen, Teemu; Waaler, Jo; Ignatev, Alexander; Nkizinkiko, Yves; Venkannagari, Harikanth; Obaji, Ezeogo; Krauss, Stefan; Lehtiö, Lari

    2016-01-15

    Tankyrases 1 and 2, the specialized members of the ARTD protein family, are druggable biotargets whose inhibition may have therapeutic potential against cancer, metabolic disease, fibrotic disease, fibrotic wound healing and HSV viral infections. We have previously identified a novel tankyrase inhibitor scaffold, JW55, and showed that it reduces mouse colon adenoma formation in vivo. Here we expanded the scaffold and profiled the selectivity of the compounds against a panel of human ARTDs. The scaffold also enables a fine modulation of selectivity towards either tankyrase 1 or tankyrase 2. In order to get insight about the binding mode of the inhibitors, we solved crystal structures of the compounds in complex with tankyrase 2. The compounds bind to the adenosine pocket of the catalytic domain and cause changes in the protein structure that are modulated by the chemical modifications of the compounds. The structural analysis allows further rational development of this compound class as a potent and selective tankyrase inhibitor.

  19. Transforming growth factor beta 1-responsive element: closely associated binding sites for USF and CCAAT-binding transcription factor-nuclear factor I in the type 1 plasminogen activator inhibitor gene.

    PubMed Central

    Riccio, A; Pedone, P V; Lund, L R; Olesen, T; Olsen, H S; Andreasen, P A

    1992-01-01

    Transforming growth factor beta (TGF-beta) is the name of a group of closely related polypeptides characterized by a multiplicity of effects, including regulation of extracellular proteolysis and turnover of the extracellular matrix. Its cellular mechanism of action is largely unknown. TGF-beta 1 is a strong and fast inducer of type 1 plasminogen activator inhibitor gene transcription. We have identified a TGF-beta 1-responsive element in the 5'-flanking region of the human type 1 plasminogen activator inhibitor gene and shown that it is functional both in its natural context and when fused to a heterologous nonresponsive promoter. Footprinting and gel retardation experiments showed that two different nuclear factors, present in extracts from both TGF-beta 1-treated and nontreated cells, bind to adjacent sequences contained in the responsive unit. A palindromic sequence binds a trans-acting factor(s) of the CCAAT-binding transcription factor-nuclear factor I family. A partially overlapping dyad symmetry interacts with a second protein that much evidence indicates to be USF. USF is a transactivator belonging to the basic helix-loop-helix family of transcription factors. Mutations which abolish the binding of either CCAAT-binding transcription factor-nuclear factor I or USF result in reduction of transcriptional activation upon exposure to TGF-beta 1, thus showing that both elements of the unit are necessary for the TGF-beta 1 response. We discuss the possible relationship of these findings to the complexity of the TGF-beta action. Images PMID:1549130

  20. Discovery of an Aurora kinase inhibitor through site-specific dynamic combinatorial chemistry.

    PubMed

    Cancilla, Mark T; He, Molly M; Viswanathan, Nina; Simmons, Robert L; Taylor, Meggin; Fung, Amy D; Cao, Kathy; Erlanson, Daniel A

    2008-07-15

    We demonstrate a fragment-based lead discovery method that combines site-directed ligand discovery with dynamic combinatorial chemistry. Our technique targets dynamic combinatorial screening to a specified region of a protein by using reversible disulfide chemistry. We have used this technology to rapidly identify inhibitors of the drug target Aurora A that span the purine-binding site and the adaptive pocket of the kinase. The binding mode of a noncovalent inhibitor has been further characterized through crystallography.

  1. Discovery of An Aurora Kinase Inhibitor Through Site-Specific Dynamic Combinatorial Chemistry

    SciTech Connect

    Cancilla, M.T.; He, M.M.; Viswanathan, N.; Simmons, R.L.; Taylor, M.; Fung, A.D.; Cao, K.; Erlanson, D.A.

    2009-05-12

    We demonstrate a fragment-based lead discovery method that combines site-directed ligand discovery with dynamic combinatorial chemistry. Our technique targets dynamic combinatorial screening to a specified region of a protein by using reversible disulfide chemistry. We have used this technology to rapidly identify inhibitors of the drug target Aurora A that span the purine-binding site and the adaptive pocket of the kinase. The binding mode of a noncovalent inhibitor has been further characterized through crystallography.

  2. Turing pattern formation in fractional activator-inhibitor systems

    NASA Astrophysics Data System (ADS)

    Henry, B. I.; Langlands, T. A. M.; Wearne, S. L.

    2005-08-01

    Activator-inhibitor systems of reaction-diffusion equations have been used to describe pattern formation in numerous applications in biology, chemistry, and physics. The rate of diffusion in these applications is manifest in the single parameter of the diffusion constant, and stationary Turing patterns occur above a critical value of d representing the ratio of the diffusion constants of the inhibitor to the activator. Here we consider activator-inhibitor systems in which the diffusion is anomalous subdiffusion; the diffusion rates are manifest in both a diffusion constant and a diffusion exponent. A consideration of this problem in terms of continuous-time random walks with sources and sinks leads to a reaction-diffusion system with fractional order temporal derivatives operating on the spatial Laplacian. We have carried out an algebraic stability analysis of the homogeneous steady-state solution in fractional activator-inhibitor systems, with Gierer-Meinhardt reaction kinetics and with Brusselator reaction kinetics. For each class of reaction kinetics we identify a Turing instability bifurcation curve in the two-dimensional diffusion parameter space. The critical value of d , for Turing instabilities, decreases monotonically with the anomalous diffusion exponent between unity (standard diffusion) and zero (extreme subdiffusion). We have also carried out numerical simulations of the governing fractional activator-inhibitor equations and we show that the Turing instability precipitates the formation of complex spatiotemporal patterns. If the diffusion of the activator and inhibitor have the same anomalous scaling properties, then the surface profiles of these patterns for values of d slightly above the critical value varies from smooth stationary patterns to increasingly rough and nonstationary patterns as the anomalous diffusion exponent varies from unity towards zero. If the diffusion of the activator is anomalous subdiffusion but the diffusion of the inhibitor

  3. Turing pattern formation in fractional activator-inhibitor systems.

    PubMed

    Henry, B I; Langlands, T A M; Wearne, S L

    2005-08-01

    Activator-inhibitor systems of reaction-diffusion equations have been used to describe pattern formation in numerous applications in biology, chemistry, and physics. The rate of diffusion in these applications is manifest in the single parameter of the diffusion constant, and stationary Turing patterns occur above a critical value of d representing the ratio of the diffusion constants of the inhibitor to the activator. Here we consider activator-inhibitor systems in which the diffusion is anomalous subdiffusion; the diffusion rates are manifest in both a diffusion constant and a diffusion exponent. A consideration of this problem in terms of continuous-time random walks with sources and sinks leads to a reaction-diffusion system with fractional order temporal derivatives operating on the spatial Laplacian. We have carried out an algebraic stability analysis of the homogeneous steady-state solution in fractional activator-inhibitor systems, with Gierer-Meinhardt reaction kinetics and with Brusselator reaction kinetics. For each class of reaction kinetics we identify a Turing instability bifurcation curve in the two-dimensional diffusion parameter space. The critical value of d , for Turing instabilities, decreases monotonically with the anomalous diffusion exponent between unity (standard diffusion) and zero (extreme subdiffusion). We have also carried out numerical simulations of the governing fractional activator-inhibitor equations and we show that the Turing instability precipitates the formation of complex spatiotemporal patterns. If the diffusion of the activator and inhibitor have the same anomalous scaling properties, then the surface profiles of these patterns for values of d slightly above the critical value varies from smooth stationary patterns to increasingly rough and nonstationary patterns as the anomalous diffusion exponent varies from unity towards zero. If the diffusion of the activator is anomalous subdiffusion but the diffusion of the inhibitor

  4. Structure of a small-molecule inhibitor complexed with GlmU from Haemophilus influenzae reveals an allosteric binding site

    SciTech Connect

    Mochalkin, Igor; Lightle, Sandra; Narasimhan, Lakshmi; Bornemeier, Dirk; Melnick, Michael; VanderRoest, Steven; McDowell, Laura

    2008-04-02

    N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) is an essential enzyme in aminosugars metabolism and an attractive target for antibiotic drug discovery. GlmU catalyzes the formation of uridine-diphospho-N-acetylglucosamine (UDP-GlcNAc), an important precursor in the peptidoglycan and lipopolisaccharide biosynthesis in both Gram-negative and Gram-positive bacteria. Here we disclose a 1.9 {angstrom} resolution crystal structure of a synthetic small-molecule inhibitor of GlmU from Haemophilus influenzae (hiGlmU). The compound was identified through a high-throughput screening (HTS) configured to detect inhibitors that target the uridyltransferase active site of hiGlmU. The original HTS hit exhibited a modest micromolar potency (IC{sub 50} - 18 {mu}M in a racemic mixture) against hiGlmU and no activity against Staphylococcus aureus GlmU (saGlmU). The determined crystal structure indicated that the inhibitor occupies an allosteric site adjacent to the GlcNAc-1-P substrate-binding region. Analysis of the mechanistic model of the uridyltransferase reaction suggests that the binding of this allosteric inhibitor prevents structural rearrangements that are required for the enzymatic reaction, thus providing a basis for structure-guided design of a new class of mechanism-based inhibitors of GlmU.

  5. Inhibition and active-site modelling of prolidase.

    PubMed

    King, G F; Crossley, M J; Kuchel, P W

    1989-03-15

    Consideration of the active-site model of prolidase led us to examine azetidine, pyrrolidine and piperidine substrate analogs as potential in vivo inhibitors of the enzyme. One of these, N-benzyloxycarbonyl-L-proline, was shown to be a potent competitive inhibitor of porcine kidney prolidase (Ki = 90 microM); its rapid protein-mediated permeation of human and sheep erythrocytes suggests that it may be effective in vivo. The higher homolog, N-benzyloxycarbonyl-L-pipecolic acid, was also a potent inhibitor of the enzyme while the antihypertensive drugs, captopril and enalaprilat, were shown to have mild and no inhibitory effects, respectively. Analysis of inhibitor action and consideration of X-ray crystallographic data of relevant Mn2+ complexes allowed the active-site model of prolidase to be further refined; a new model is presented in which the substrate acts as a bidentate ligand towards the active-site manganous ion. Various aspects of the new model help to explain why Mn2+ has been 'chosen' by the enzyme in preference to other biologically available metal ions. PMID:2924773

  6. Hydroxamate based inhibitors of adenylyl cyclase. Part 1: the effect of acyclic linkers on P-site binding.

    PubMed

    Levy, Daniel; Marlowe, Charles; Kane-Maguire, Kim; Bao, Ming; Cherbavaz, Diana; Tomlinson, James; Sedlock, David; Scarborough, Robert

    2002-11-01

    The adenylyl cyclases (ACs) are a family of enzymes that are key elements of signal transduction by virtue of their ability to convert ATP to cAMP. The catalytic mechanism of this transformation proceeds through initial binding of ATP to the purine binding site (P-site) followed by metal mediated cyclization with loss of pyrophosphate. Crystallographic analysis of ACs with known inhibitors reveals the presence of two metals in the active site. Presently, nine isoforms of adenylyl cyclase are known and unique isoform combinations are expressed in a tissue specific manner. The development of isoform specific inhibitors of adenylyl cyclase may prove to be a useful strategy toward the design of novel therapeutic agents. In order to develop novel AC inhibitors, we have chosen a design approach utilizing molecules with the adenine ring system joined to a metal-coordinating hydroxamic acid via flexible acyclic linkers. The designed inhibitors were assayed against type V AC with the size and heteroatom content of the linkers varied to probe the interaction of the nucleotide and metal binding sites within the enzyme. PMID:12372507

  7. Cutaneous wound healing through paradoxical MAPK activation by BRAF inhibitors

    PubMed Central

    Escuin-Ordinas, Helena; Li, Shuoran; Xie, Michael W.; Sun, Lu; Hugo, Willy; Huang, Rong Rong; Jiao, Jing; de-Faria, Felipe Meira; Realegeno, Susan; Krystofinski, Paige; Azhdam, Ariel; Komenan, Sara Marie D.; Atefi, Mohammad; Comin-Anduix, Begoña; Pellegrini, Matteo; Cochran, Alistair J.; Modlin, Robert L.; Herschman, Harvey R.; Lo, Roger S.; McBride, William H.; Segura, Tatiana; Ribas, Antoni

    2016-01-01

    BRAF inhibitors are highly effective therapies for the treatment of BRAFV600-mutated melanoma, with the main toxicity being a variety of hyperproliferative skin conditions due to paradoxical activation of the mitogen-activated protein kinase (MAPK) pathway in BRAF wild-type cells. Most of these hyperproliferative skin changes improve when a MEK inhibitor is co-administered, as it blocks paradoxical MAPK activation. Here we show how the BRAF inhibitor vemurafenib accelerates skin wound healing by inducing the proliferation and migration of human keratinocytes through extracellular signal-regulated kinase (ERK) phosphorylation and cell cycle progression. Topical treatment with vemurafenib in two wound-healing mice models accelerates cutaneous wound healing through paradoxical MAPK activation; addition of a mitogen-activated protein kinase kinase (MEK) inhibitor reverses the benefit of vemurafenib-accelerated wound healing. The same dosing regimen of topical BRAF inhibitor does not increase the incidence of cutaneous squamous cell carcinomas in mice. Therefore, topical BRAF inhibitors may have clinical applications in accelerating the healing of skin wounds. PMID:27476449

  8. Cutaneous wound healing through paradoxical MAPK activation by BRAF inhibitors.

    PubMed

    Escuin-Ordinas, Helena; Li, Shuoran; Xie, Michael W; Sun, Lu; Hugo, Willy; Huang, Rong Rong; Jiao, Jing; de-Faria, Felipe Meira; Realegeno, Susan; Krystofinski, Paige; Azhdam, Ariel; Komenan, Sara Marie D; Atefi, Mohammad; Comin-Anduix, Begoña; Pellegrini, Matteo; Cochran, Alistair J; Modlin, Robert L; Herschman, Harvey R; Lo, Roger S; McBride, William H; Segura, Tatiana; Ribas, Antoni

    2016-08-01

    BRAF inhibitors are highly effective therapies for the treatment of BRAF(V600)-mutated melanoma, with the main toxicity being a variety of hyperproliferative skin conditions due to paradoxical activation of the mitogen-activated protein kinase (MAPK) pathway in BRAF wild-type cells. Most of these hyperproliferative skin changes improve when a MEK inhibitor is co-administered, as it blocks paradoxical MAPK activation. Here we show how the BRAF inhibitor vemurafenib accelerates skin wound healing by inducing the proliferation and migration of human keratinocytes through extracellular signal-regulated kinase (ERK) phosphorylation and cell cycle progression. Topical treatment with vemurafenib in two wound-healing mice models accelerates cutaneous wound healing through paradoxical MAPK activation; addition of a mitogen-activated protein kinase kinase (MEK) inhibitor reverses the benefit of vemurafenib-accelerated wound healing. The same dosing regimen of topical BRAF inhibitor does not increase the incidence of cutaneous squamous cell carcinomas in mice. Therefore, topical BRAF inhibitors may have clinical applications in accelerating the healing of skin wounds.

  9. In silico, in vitro and cellular analysis with a kinome-wide inhibitor panel correlates cellular LRRK2 dephosphorylation to inhibitor activity on LRRK2.

    PubMed

    Vancraenenbroeck, Renée; De Raeymaecker, Joren; Lobbestael, Evy; Gao, Fangye; De Maeyer, Marc; Voet, Arnout; Baekelandt, Veerle; Taymans, Jean-Marc

    2014-01-01

    Leucine-rich repeat kinase 2 (LRRK2) is a complex, multidomain protein which is considered a valuable target for potential disease-modifying therapeutic strategies for Parkinson's disease (PD). In mammalian cells and brain, LRRK2 is phosphorylated and treatment of cells with inhibitors of LRRK2 kinase activity can induce LRRK2 dephosphorylation at a cluster of serines including Ser910/935/955/973. It has been suggested that phosphorylation levels at these sites reflect LRRK2 kinase activity, however kinase-dead variants of LRRK2 or kinase activating variants do not display altered Ser935 phosphorylation levels compared to wild type. Furthermore, Ser910/935/955/973 are not autophosphorylation sites, therefore, it is unclear if inhibitor induced dephosphorylation depends on the activity of compounds on LRRK2 or on yet to be identified upstream kinases. Here we used a panel of 160 ATP competitive and cell permeable kinase inhibitors directed against all branches of the kinome and tested their activity on LRRK2 in vitro using a peptide-substrate-based kinase assay. In neuronal SH-SY5Y cells overexpressing LRRK2 we used compound-induced dephosphorylation of Ser935 as readout. In silico docking of selected compounds was performed using a modeled LRRK2 kinase structure. Receiver operating characteristic plots demonstrated that the obtained docking scores to the LRRK2 ATP binding site correlated with in vitro and cellular compound activity. We also found that in vitro potency showed a high degree of correlation to cellular compound induced LRRK2 dephosphorylation activity across multiple compound classes. Therefore, acute LRRK2 dephosphorylation at Ser935 in inhibitor treated cells involves a strong component of inhibitor activity on LRRK2 itself, without excluding a role for upstream kinases. Understanding the regulation of LRRK2 phosphorylation by kinase inhibitors aids our understanding of LRRK2 signaling and may lead to development of new classes of LRRK2 kinase

  10. The natural product dihydrotanshinone I provides a prototype for uncharged inhibitors that bind specifically to the acetylcholinesterase peripheral site with nanomolar affinity.

    PubMed

    Beri, Veena; Wildman, Scott A; Shiomi, Kazuro; Al-Rashid, Ziyad F; Cheung, Jonah; Rosenberry, Terrone L

    2013-10-22

    Cholinergic synaptic transmission often requires extremely rapid hydrolysis of acetylcholine by acetylcholinesterase (AChE). AChE is inactivated by organophosphates (OPs) in chemical warfare nerve agents. The resulting accumulation of acetylcholine disrupts cholinergic synaptic transmission and can lead to death. A potential long-term strategy for preventing AChE inactivation by OPs is based on evidence that OPs must pass through a peripheral site or P-site near the mouth of the AChE active site gorge before reacting with a catalytic serine in an acylation site or A-site at the base of the gorge. An ultimate goal of this strategy is to design compounds that bind tightly at or near the P-site and exclude OPs from the active site while interfering minimally with the passage of acetylcholine. However, to target the AChE P-site with ligands and potential drugs that selectively restrict access, much more information must be gathered about the structure-activity relationships of ligands that bind specifically to the P-site. We apply here an inhibitor competition assay that can correctly determine whether an AChE inhibitor binds to the P-site, the A-site, or both sites. We have used this assay to examine three uncharged, natural product inhibitors of AChE, including aflatoxin B1, dihydrotanshinone I, and territrem B. The first two of these inhibitors are predicted by the competition assay to bind selectively to the P-site, while territrem B is predicted to span both the P- and A-sites. These predictions have recently been confirmed by X-ray crystallography. Dihydrotanshinone I, with an observed binding constant (KI) of 750 nM, provides a good lead compound for the development of high-affinity, uncharged inhibitors with specificity for the P-site. PMID:24040835

  11. Increasing the binding affinity of VEGFR-2 inhibitors by extending their hydrophobic interaction with the active site: Design, synthesis and biological evaluation of 1-substituted-4-(4-methoxybenzyl)phthalazine derivatives.

    PubMed

    Eldehna, Wagdy M; Abou-Seri, Sahar M; El Kerdawy, Ahmed M; Ayyad, Rezk R; Hamdy, Abdallah M; Ghabbour, Hazem A; Ali, Mamdouh M; Abou El Ella, Dalal A

    2016-05-01

    A series of anilinophthalazine derivatives 4a-j was initially synthesized and tested for its VEGFR-2 inhibitory activity where it showed promising activity (IC50 = 0.636-5.76 μM). Molecular docking studies guidance was used to improve the binding affinity for series 4a-j towards VEGFR-2 active site. This improvement was achieved by increasing the hydrophobic interaction with the hydrophobic back pocket of the VEGFR-2 active site lined with the hydrophobic side chains of Ile888, Leu889, Ile892, Val898, Val899, Leu1019 and Ile1044. Increasing the hydrophobic interaction was accomplished by extending the anilinophthalazine scaffold with a substituted phenyl moiety through an uriedo linker which should give this extension the flexibility required to accommodate itself deeply into the hydrophobic back pocket. As planned, the designed uriedo-anilinophthalazines 7a-i showed superior binding affinity than their anilinophthalazine parents (IC50 = 0.083-0.473 μM). In particular, compounds 7g-i showed IC50 of 0.086, 0.083 and 0.086 μM, respectively, which are better than that of the reference drug sorafenib (IC50 = 0.09 μM).

  12. Antihormonal activities of 5 alpha-reductase and aromatase inhibitors.

    PubMed

    Zoppi, S; Cocconi, M; Lechuga, M J; Messi, E; Zanisi, M; Motta, M

    1988-10-01

    The problem of developing androgen antagonists has been tackled so far only by synthesizing steroids able to displace testosterone and other androgens from their specific receptor sites. The observation that testosterone has to be converted intracellularly either to 5 alpha-reduced metabolites (DHT, 3 alpha-diol, etc.) or to estrogens, in order to become fully active on androgen-dependent structures (both central and peripheral), has opened the possibility of creating molecules which prevent these conversions, and which could then block the actions of testosterone. The availability of these new compounds has allowed a better understanding of the selective physiological role of each of the metabolites of testosterone, and to provide the basis for the development of new hormone antagonists to be used in those clinical conditions for which an inhibition of the actions of testosterone is foreseen. The usefulness of these enzyme inhibitors is underlined by some examples described in this paper. The results obtained may permit the formulation of the following conclusions: (1) The conversion of testosterone to its 5 alpha-reduced metabolites occurring in the neuroendocrine structures may represent an essential step for the appearance of the inhibitory feedback effect testosterone exerts on LH secretion; (2) Testosterone exhibits its negative feedback effect on FSH secretion as such and not following the local aromatization to estrogens; (3) Testosterone exerts its effect on the intrahypothalamic stores of LHRH acting as such and not following its local conversion either to 5 alpha-reduced metabolites or to estrogenic molecules; (4) Some of the new enzyme inhibitors (e.g. 4-OH-A) may represent an interesting tool for the treatment and/or the prevention of BPH and possibly of other androgen-dependent diseases (prostate carcinoma, acne etc.), as shown by their ability to prevent the in vitro conversion of testosterone to its 5 alpha-reduced metabolites both in the normal

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

    PubMed Central

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

    2013-01-01

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

  14. Type 1 plaminogen activator inhibitor gene: Functional analysis and glucocorticoid regulation of its promoter

    SciTech Connect

    Van Zonneveld, A.J.; Curriden, S.A.; Loskutoff, D.J. )

    1988-08-01

    Plasminogen activator inhibitor type 1 is an important component of the fibrinolytic system and its biosynthesis is subject to complex regulation. To study this regulation at the level of transcription, the authors have identified and sequenced the promoter of the human plasminogen activator inhibitor type 1 gene. Nuclease protection experiments were performed by using endothelial cell mRNA and the transcription initiation (cap) site was established. Sequence analysis of the 5{prime} flanking region of the gene revealed a perfect TATA box at position {minus}28 to position {minus}23, the conserved distance from the cap site. Comparative functional studies with the firefly luciferase gene as a reporter gene showed that fragments derived from this 5{prime} flanking region exhibited high promoter activity when transfected into bovine aortic endothelial cells and mouse Ltk{sup {minus}} fibroblasts but were inactive when introduced into HeLa cells. These studies indicate that the fragments contain the plasminogen activator inhibitor type 1 promoter and that it is expressed in a tissue-specific manner. Although the fragments were also silent in rat FTO2B hepatoma cells, their promoter activity could be induced up to 40-fold with the synthetic glucocorticoid dexamethasone. Promoter deletion mapping experiments and studies involving the fusion of promoter fragments to a heterologous gene indicated that dexamethasone induction is mediated by a glucocorticoid responsive element with enhancer-like properties located within the region between nucleotides {minus}305 and +75 of the plasminogen activator inhibitor type 1 gene.

  15. A FluoPol-ABPP PAD2 High-Throughput Screen Identifies the First Calcium Site Inhibitor Targeting the PADs

    PubMed Central

    2015-01-01

    The protein arginine deiminases (PADs) catalyze the post-translational hydrolysis of peptidyl-arginine to form peptidyl-citrulline in a process termed deimination or citrullination. PADs likely play a role in the progression of a range of disease states because dysregulated PAD activity is observed in a host of inflammatory diseases and cancer. For example, recent studies have shown that PAD2 activates ERα target gene expression in breast cancer cells by citrullinating histone H3 at ER target promoters. To date, all known PAD inhibitors bind directly to the enzyme active site. PADs, however, also require calcium ions to drive a conformational change between the inactive apo-state and the fully active calcium bound holoenzyme, suggesting that it would be possible to identify inhibitors that bind the apoenzyme and prevent this conformational change. As such, we set out to develop a screen that can identify PAD2 inhibitors that bind to either the apo or calcium bound form of PAD2. Herein, we provide definitive proof of concept for this approach and report the first PAD inhibitor, ruthenium red (Ki of 17 μM), to preferentially bind the apoenzyme. PMID:24467619

  16. Designed Inhibitors of Insulin-Degrading Enzyme Regulate the Catabolism and Activity of Insulin

    PubMed Central

    Leissring, Malcolm A.; Malito, Enrico; Hedouin, Sabrine; Reinstatler, Lael; Sahara, Tomoko; Abdul-Hay, Samer O.; Choudhry, Shakeel; Maharvi, Ghulam M.; Fauq, Abdul H.; Huzarska, Malwina; May, Philip S.; Choi, Sungwoon; Logan, Todd P.; Turk, Benjamin E.; Cantley, Lewis C.; Manolopoulou, Marika; Tang, Wei-Jen; Stein, Ross L.; Cuny, Gregory D.; Selkoe, Dennis J.

    2010-01-01

    Background Insulin is a vital peptide hormone that is a central regulator of glucose homeostasis, and impairments in insulin signaling cause diabetes mellitus. In principle, it should be possible to enhance the activity of insulin by inhibiting its catabolism, which is mediated primarily by insulin-degrading enzyme (IDE), a structurally and evolutionarily distinctive zinc-metalloprotease. Despite interest in pharmacological inhibition of IDE as an attractive anti-diabetic approach dating to the 1950s, potent and selective inhibitors of IDE have not yet emerged. Methodology/Principal Findings We used a rational design approach based on analysis of combinatorial peptide mixtures and focused compound libraries to develop novel peptide hydroxamic acid inhibitors of IDE. The resulting compounds are ∼106 times more potent than existing inhibitors, non-toxic, and surprisingly selective for IDE vis-à-vis conventional zinc-metalloproteases. Crystallographic analysis of an IDE-inhibitor complex reveals a novel mode of inhibition based on stabilization of IDE's “closed,” inactive conformation. We show further that pharmacological inhibition of IDE potentiates insulin signaling by a mechanism involving reduced catabolism of internalized insulin. Conclusions/Significance The inhibitors we describe are the first to potently and selectively inhibit IDE or indeed any member of this atypical zinc-metalloprotease superfamily. The distinctive structure of IDE's active site, and the mode of action of our inhibitors, suggests that it may be possible to develop inhibitors that cross-react minimally with conventional zinc-metalloproteases. Significantly, our results reveal that insulin signaling is normally regulated by IDE activity not only extracellularly but also within cells, supporting the longstanding view that IDE inhibitors could hold therapeutic value for the treatment of diabetes. PMID:20498699

  17. Designed Inhibitors of Insulin-Degrading Enzyme Regulate the Catabolism and Activity of Insulin

    SciTech Connect

    Leissring, Malcolm A.; Malito, Enrico; Hedouin, Sabrine; Reinstatler, Lael; Sahara, Tomoko; Abdul-Hay, Samer O.; Choudhry, Shakeel; Maharvi, Ghulam M.; Fauq, Abdul H.; Huzarska, Malwina; May, Philip S.; Choi, Sungwoon; Logan, Todd P.; Turk, Benjamin E.; Cantley, Lewis C.; Manolopoulou, Marika; Tang, Wei-Jen; Stein, Ross L.; Cuny, Gregory D.; Selkoe, Dennis J.

    2010-09-20

    Insulin is a vital peptide hormone that is a central regulator of glucose homeostasis, and impairments in insulin signaling cause diabetes mellitus. In principle, it should be possible to enhance the activity of insulin by inhibiting its catabolism, which is mediated primarily by insulin-degrading enzyme (IDE), a structurally and evolutionarily distinctive zinc-metalloprotease. Despite interest in pharmacological inhibition of IDE as an attractive anti-diabetic approach dating to the 1950s, potent and selective inhibitors of IDE have not yet emerged. We used a rational design approach based on analysis of combinatorial peptide mixtures and focused compound libraries to develop novel peptide hydroxamic acid inhibitors of IDE. The resulting compounds are {approx} 10{sup 6} times more potent than existing inhibitors, non-toxic, and surprisingly selective for IDE vis-a-vis conventional zinc-metalloproteases. Crystallographic analysis of an IDE-inhibitor complex reveals a novel mode of inhibition based on stabilization of IDE's 'closed,' inactive conformation. We show further that pharmacological inhibition of IDE potentiates insulin signaling by a mechanism involving reduced catabolism of internalized insulin. Conclusions/Significance: The inhibitors we describe are the first to potently and selectively inhibit IDE or indeed any member of this atypical zinc-metalloprotease superfamily. The distinctive structure of IDE's active site, and the mode of action of our inhibitors, suggests that it may be possible to develop inhibitors that cross-react minimally with conventional zinc-metalloproteases. Significantly, our results reveal that insulin signaling is normally regulated by IDE activity not only extracellularly but also within cells, supporting the longstanding view that IDE inhibitors could hold therapeutic value for the treatment of diabetes.

  18. Therapeutic potential of an orally effective small molecule inhibitor of plasminogen activator inhibitor for asthma.

    PubMed

    Liu, Rui-Ming; Eldridge, Stephanie; Watanabe, Nobuo; Deshane, Jessy; Kuo, Hui-Chien; Jiang, Chunsun; Wang, Yong; Liu, Gang; Schwiebert, Lisa; Miyata, Toshio; Thannickal, Victor J

    2016-02-15

    Asthma is one of the most common respiratory diseases. Although progress has been made in our understanding of airway pathology and many drugs are available to relieve asthma symptoms, there is no cure for chronic asthma. Plasminogen activator inhibitor 1 (PAI-1), a primary inhibitor of tissue-type and urokinase-type plasminogen activators, has pleiotropic functions besides suppression of fibrinolysis. In this study, we show that administration of TM5275, an orally effective small-molecule PAI-1 inhibitor, 25 days after ovalbumin (OVA) sensitization-challenge, significantly ameliorated airway hyperresponsiveness in an OVA-induced chronic asthma model. Furthermore, we show that TM5275 administration significantly attenuated OVA-induced infiltration of inflammatory cells (neutrophils, eosinophils, and monocytes), the increase in the levels of OVA-specific IgE and Th2 cytokines (IL-4 and IL-5), the production of mucin in the airways, and airway subepithelial fibrosis. Together, the results suggest that the PAI-1 inhibitor TM5275 may have therapeutic potential for asthma through suppressing eosinophilic allergic response and ameliorating airway remodeling. PMID:26702150

  19. Therapeutic potential of an orally effective small molecule inhibitor of plasminogen activator inhibitor for asthma.

    PubMed

    Liu, Rui-Ming; Eldridge, Stephanie; Watanabe, Nobuo; Deshane, Jessy; Kuo, Hui-Chien; Jiang, Chunsun; Wang, Yong; Liu, Gang; Schwiebert, Lisa; Miyata, Toshio; Thannickal, Victor J

    2016-02-15

    Asthma is one of the most common respiratory diseases. Although progress has been made in our understanding of airway pathology and many drugs are available to relieve asthma symptoms, there is no cure for chronic asthma. Plasminogen activator inhibitor 1 (PAI-1), a primary inhibitor of tissue-type and urokinase-type plasminogen activators, has pleiotropic functions besides suppression of fibrinolysis. In this study, we show that administration of TM5275, an orally effective small-molecule PAI-1 inhibitor, 25 days after ovalbumin (OVA) sensitization-challenge, significantly ameliorated airway hyperresponsiveness in an OVA-induced chronic asthma model. Furthermore, we show that TM5275 administration significantly attenuated OVA-induced infiltration of inflammatory cells (neutrophils, eosinophils, and monocytes), the increase in the levels of OVA-specific IgE and Th2 cytokines (IL-4 and IL-5), the production of mucin in the airways, and airway subepithelial fibrosis. Together, the results suggest that the PAI-1 inhibitor TM5275 may have therapeutic potential for asthma through suppressing eosinophilic allergic response and ameliorating airway remodeling.

  20. Determinants of the activity of beta-lactamase inhibitor combinations.

    PubMed

    Livermore, D M

    1993-01-01

    Inhibitor combinations provide one strategy to overcome beta-lactamase-mediated resistance. Their success depends, obviously, on the inhibitor being able to bind and inactivate the beta-lactamase molecules. Clavulanate, sulbactam and tazobactam are irreversible inactivators of many beta-lactamases, forming covalent complexes which resist hydrolysis. 'Suicide' kinetics are seen with some, but not all, enzymes. All three compounds inactivate staphylococcal penicillinase, the chromosomal beta-lactamases of Proteus vulgaris and Bacteroides spp., and the Class IV beta-lactamases present in some klebsiellae. Tazobactam, but not the other compounds, has moderate activity against some Class I (AmpC) chromosomal beta-lactamases, notably that of Morganella morganii, but not that of Enterobacter cloacae. Both clavulanate and tazobactam are strong inhibitors of the widely distributed TEM and SHV plasmid-mediated beta-lactamases; sulbactam is a weaker inhibitor. Other factors, aside from the affinity of the inhibitor for the enzyme, co-determine the success or failure of inhibition. Potentiation is most readily achieved if little enzyme is produced, and if the organism is very permeable to the inhibitor. Thus, resistance to inhibitor combinations is rare in strains of Haemophilus influenzae and Neisseria gonorrhoeae that produce TEM-beta-lactamase, but is commoner in enterobacteria that produce this enzyme, since these are less permeable and sometimes manufacture very large amounts of enzyme. The partner beta-lactam agent is also important. Irrespective of the inhibitor used, piperacillin is easier to protect against TEM beta-lactamases and the M. morganii Class I enzyme than are ampicillin, amoxycillin or ticarcillin. This may relate to the lower affinity of piperacillin for these enzymes, or to its greater affinity for the bacterial penicillin-binding proteins. Finally, pH can affect the degree of inhibition achieved with sulphones for some beta-lactamases, notably TEM-1

  1. A New Class of Rhomboid Protease Inhibitors Discovered by Activity-Based Fluorescence Polarization

    PubMed Central

    Wolf, Eliane V.; Zeißler, Annett; Vosyka, Oliver; Zeiler, Evelyn; Sieber, Stephan; Verhelst, Steven H. L.

    2013-01-01

    Rhomboids are intramembrane serine proteases that play diverse biological roles, including some that are of potential therapeutical relevance. Up to date, rhomboid inhibitor assays are based on protein substrate cleavage. Although rhomboids have an overlapping substrate specificity, substrates cannot be used universally. To overcome the need for substrates, we developed a screening assay using fluorescence polarization activity-based protein profiling (FluoPol ABPP) that is compatible with membrane proteases. With FluoPol ABPP, we identified new inhibitors for the E. coli rhomboid GlpG. Among these was a structural class that has not yet been reported as rhomboid inhibitors: β-lactones. They form covalent and irreversible complexes with the active site serine of GlpG. The presence of alkyne handles on the β-lactones also allowed activity-based labeling. Overall, these molecules represent a new scaffold for future inhibitor and activity-based probe development, whereas the assay will allow inhibitor screening of ill-characterized membrane proteases. PMID:23991088

  2. Systematic assessment of coordinated activity cliffs formed by kinase inhibitors and detailed characterization of activity cliff clusters and associated SAR information.

    PubMed

    Dimova, Dilyana; Stumpfe, Dagmar; Bajorath, Jürgen

    2015-01-27

    From currently available kinase inhibitors and their activity data, clusters of coordinated activity cliffs were systematically derived and subjected to cluster index and index map analysis. Type I-like inhibitors with well-defined IC50 measurements were found to provide a large knowledge base of activity cliff clusters for 266 targets from nine kinase groups. On the basis of index map analysis, these clusters were systematically organized according to structural similarity of inhibitors and activity cliff diversity and prioritized for structure-activity relationship (SAR) analysis. From prioritized clusters, interpretable SAR information can be extracted. It is also shown that activity cliff clusters formed by ATP site-directed inhibitors often represent local SAR environments of rather different complexity and interpretability. In addition, activity cliff clusters including promiscuous kinase inhibitors have been determined. Only a small subset of inhibitors was found to change activity cliff roles in different clusters. The activity cliff clusters described herein and their index map organization substantially enrich SAR information associated with kinase inhibitors in compound subsets of limited size. The cluster and index map information is made available upon request to provide opportunities for further SAR exploration. On the basis of our analysis and the data provided, activity cliff clusters and corresponding inhibitor series for kinase targets of interest can be readily selected.

  3. Small Molecule Inhibitors Targeting Activator Protein 1 (AP-1)

    PubMed Central

    2015-01-01

    Activator protein 1 (AP-1) is a pivotal transcription factor that regulates a wide range of cellular processes including proliferation, apoptosis, differentiation, survival, cell migration, and transformation. Accumulating evidence supports that AP-1 plays an important role in several severe disorders including cancer, fibrosis, and organ injury, as well as inflammatory disorders such as asthma, psoriasis, and rheumatoid arthritis. AP-1 has emerged as an actively pursued drug discovery target over the past decade. Excitingly, a selective AP-1 inhibitor T-5224 (51) has been investigated in phase II human clinical trials. Nevertheless, no effective AP-1 inhibitors have yet been approved for clinical use. Despite significant advances achieved in understanding AP-1 biology and function, as well as the identification of small molecules modulating AP-1 associated signaling pathways, medicinal chemistry efforts remain an urgent need to yield selective and efficacious AP-1 inhibitors as a viable therapeutic strategy for human diseases. PMID:24831826

  4. Discovery of an Allosteric Inhibitor Binding Site in 3-Oxo-acyl-ACP Reductase from Pseudomonas aeruginosa

    PubMed Central

    2013-01-01

    3-Oxo-acyl-acyl carrier protein (ACP) reductase (FabG) plays a key role in the bacterial fatty acid synthesis II system in pathogenic microorganisms, which has been recognized as a potential drug target. FabG catalyzes reduction of a 3-oxo-acyl-ACP intermediate during the elongation cycle of fatty acid biosynthesis. Here, we report gene deletion experiments that support the essentiality of this gene in P. aeruginosa and the identification of a number of small molecule FabG inhibitors with IC50 values in the nanomolar to low micromolar range and good physicochemical properties. Structural characterization of 16 FabG-inhibitor complexes by X-ray crystallography revealed that the compounds bind at a novel allosteric site located at the FabG subunit–subunit interface. Inhibitor binding relies primarily on hydrophobic interactions, but specific hydrogen bonds are also observed. Importantly, the binding cavity is formed upon complex formation and therefore would not be recognized by virtual screening approaches. The structure analysis further reveals that the inhibitors act by inducing conformational changes that propagate to the active site, resulting in a displacement of the catalytic triad and the inability to bind NADPH. PMID:24015914

  5. Novel Mps1 Kinase Inhibitors with Potent Antitumor Activity.

    PubMed

    Wengner, Antje M; Siemeister, Gerhard; Koppitz, Marcus; Schulze, Volker; Kosemund, Dirk; Klar, Ulrich; Stoeckigt, Detlef; Neuhaus, Roland; Lienau, Philip; Bader, Benjamin; Prechtl, Stefan; Raschke, Marian; Frisk, Anna-Lena; von Ahsen, Oliver; Michels, Martin; Kreft, Bertolt; von Nussbaum, Franz; Brands, Michael; Mumberg, Dominik; Ziegelbauer, Karl

    2016-04-01

    Monopolar spindle 1 (Mps1) has been shown to function as the key kinase that activates the spindle assembly checkpoint (SAC) to secure proper distribution of chromosomes to daughter cells. Here, we report the structure and functional characterization of two novel selective Mps1 inhibitors, BAY 1161909 and BAY 1217389, derived from structurally distinct chemical classes. BAY 1161909 and BAY 1217389 inhibited Mps1 kinase activity with IC50 values below 10 nmol/L while showing an excellent selectivity profile. In cellular mechanistic assays, both Mps1 inhibitors abrogated nocodazole-induced SAC activity and induced premature exit from mitosis ("mitotic breakthrough"), resulting in multinuclearity and tumor cell death. Both compounds efficiently inhibited tumor cell proliferation in vitro (IC50 nmol/L range). In vivo, BAY 1161909 and BAY 1217389 achieved moderate efficacy in monotherapy in tumor xenograft studies. However, in line with its unique mode of action, when combined with paclitaxel, low doses of Mps1 inhibitor reduced paclitaxel-induced mitotic arrest by the weakening of SAC activity. As a result, combination therapy strongly improved efficacy over paclitaxel or Mps1 inhibitor monotreatment at the respective MTDs in a broad range of xenograft models, including those showing acquired or intrinsic paclitaxel resistance. Both Mps1 inhibitors showed good tolerability without adding toxicity to paclitaxel monotherapy. These preclinical findings validate the innovative concept of SAC abrogation for cancer therapy and justify clinical proof-of-concept studies evaluating the Mps1 inhibitors BAY 1161909 and BAY 1217389 in combination with antimitotic cancer drugs to enhance their efficacy and potentially overcome resistance. Mol Cancer Ther; 15(4); 583-92. ©2016 AACR.

  6. Diversity-Oriented Synthesis Probe Targets Plasmodium falciparum Cytochrome b Ubiquinone Reduction Site and Synergizes With Oxidation Site Inhibitors

    PubMed Central

    Lukens, Amanda K.; Heidebrecht, Richard W.; Mulrooney, Carol; Beaudoin, Jennifer A.; Comer, Eamon; Duvall, Jeremy R.; Fitzgerald, Mark E.; Masi, Daniela; Galinsky, Kevin; Scherer, Christina A.; Palmer, Michelle; Munoz, Benito; Foley, Michael; Schreiber, Stuart L.; Wiegand, Roger C.; Wirth, Dyann F.

    2015-01-01

    Background. The emergence and spread of drug resistance to current antimalarial therapies remains a pressing concern, escalating the need for compounds that demonstrate novel modes of action. Diversity-Oriented Synthesis (DOS) libraries bridge the gap between conventional small molecule and natural product libraries, allowing the interrogation of more diverse chemical space in efforts to identify probes of novel parasite pathways. Methods. We screened and optimized a probe from a DOS library using whole-cell phenotypic assays. Resistance selection and whole-genome sequencing approaches were employed to identify the cellular target of the compounds. Results. We identified a novel macrocyclic inhibitor of Plasmodium falciparum with nanomolar potency and identified the reduction site of cytochrome b as its cellular target. Combination experiments with reduction and oxidation site inhibitors showed synergistic inhibition of the parasite. Conclusions. The cytochrome b oxidation center is a validated antimalarial target. We show that the reduction site of cytochrome b is also a druggable target. Our results demonstrating a synergistic relationship between oxidation and reduction site inhibitors suggests a future strategy for new combination therapies in the treatment of malaria. PMID:25336726

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

    SciTech Connect

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

    2007-01-01

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

  8. Investigating the Conformational Structure and Potential Site Interactions of SOD Inhibitors on Ec-SOD in Marine Mud Crab Scylla serrata: A Molecular Modeling Approach.

    PubMed

    Paital, Biswaranjan; Sablok, Gaurav; Kumar, Sunil; Singh, Sanjeev Kumar; Chainy, G B N

    2016-09-01

    Superoxide dismutases (SODs) act as a first line of the enzymatic antioxidant defense system to control cellular superoxide anion toxicity. Previously, several inhibitors have been widely identified and catalogued for inhibition of SOD activity; however, still the information about the mechanism of interaction and points toward the inhibitor interactions in structures of SODs in general and in extracellular (Ec)-SOD in particular is still in naive. In the present research, we present an insight to elucidate the molecular basis of interactions of SOD inhibitors with Ec-SOD in mud crab Scylla serrata using molecular modeling and docking approaches. Different inhibitors of SOD such as hydrogen peroxide [Formula: see text], potassium cyanide, sodium dodecyl sulfate (SDS), [Formula: see text]-mercaptoethanol and dithiocarbamate were screened to understand the potential sites that may act as sites for cleavage or blocking in the protein. SOD-SDS and [Formula: see text] complex interactions indicate residues Pro72 and Asp102 of the predicted crab Ec-SOD as common targets. The GOLD result indicates that Pro72, Asp102 and Thr103 are commonly acting as the site of interaction in Ec-SOD of S. serrata with SOD inhibitors. For the first time, the results of this study provide an insight into the structural properties of Ec-SOD of S. serrata and define the possible involvements between the amino acids present in its active sites, i.e., in the regions from 70 to 84 and from 101 to 103 and different inhibitors.

  9. Novel Plasminogen Activator Inhibitor-1 Inhibitors Prevent Diabetic Kidney Injury in a Mouse Model

    PubMed Central

    Park, Jong Hee; Lee, Jung Hwa; Lee, Hi Bahl; Miyata, Toshio; Ha, Hunjoo

    2016-01-01

    Diabetic nephropathy is the leading cause of end-stage renal disease worldwide, but no effective therapeutic strategy is available. Because plasminogen activator inhibitor-1 (PAI-1) is increasingly recognized as a key factor in extracellular matrix (ECM) accumulation in diabetic nephropathy, this study examined the renoprotective effects of TM5275 and TM5441, two novel orally active PAI-1 inhibitors that do not trigger bleeding episodes, in streptozotocin (STZ)-induced diabetic mice. TM5275 (50 mg/kg) and TM5441 (10 mg/kg) were administered orally for 16 weeks to STZ-induced diabetic and age-matched control mice. Relative to the control mice, the diabetic mice showed significantly increased (p < 0.05) plasma glucose and creatinine levels, urinary albumin excretion, kidney-to-bodyweight ratios, glomerular volume, and fractional mesangial area. Markers of fibrosis and inflammation along with PAI-1 were also upregulated in the kidney of diabetic mice, and treatment with TM5275 and TM5441 effectively inhibited albuminuria, mesangial expansion, ECM accumulation, and macrophage infiltration in diabetic kidneys. Furthermore, in mouse proximal tubular epithelial (mProx24) cells, both TM5275 and TM5441 effectively inhibited PAI-1-induced mRNA expression of fibrosis and inflammation markers and also reversed PAI-1-induced inhibition of plasmin activity, which confirmed the efficacy of the TM compounds as PAI-1 inhibitors. These data suggest that TM compounds could be used to prevent diabetic kidney injury. PMID:27258009

  10. Unraveling the Pivotal Role of Bradykinin in ACE Inhibitor Activity.

    PubMed

    Taddei, Stefano; Bortolotto, L

    2016-10-01

    Historically, the first described effect of an angiotensin converting enzyme (ACE) inhibitor was an increased activity of bradykinin, one of the substrates of ACE. However, in the subsequent years, molecular models describing the mechanism of action of ACE inhibitors in decreasing blood pressure and cardiovascular risk have focused mostly on the renin-angiotensin system. Nonetheless, over the last 20 years, the importance of bradykinin in regulating vasodilation, natriuresis, oxidative stress, fibrinolysis, inflammation, and apoptosis has become clearer. The affinity of ACE appears to be higher for bradykinin than for angiotensin I, thereby suggesting that ACE inhibitors may be more effective inhibitors of bradykinin degradation than of angiotensin II production. Data describing the effect of ACE inhibition on bradykinin signaling support the hypothesis that the most cardioprotective benefits attributed to ACE inhibition may be due to increased bradykinin signaling rather than to decreased angiotensin II signaling, especially when high dosages of ACE inhibitors are considered. In particular, modulation of bradykinin in the endothelium appears to be a major target of ACE inhibition. These new mechanistic concepts may lead to further development of strategies enhancing the bradykinin signaling. PMID:27260014

  11. Design, Synthesis and Inhibitory Activity of Photoswitchable RET Kinase Inhibitors

    NASA Astrophysics Data System (ADS)

    Ferreira, Rubén; Nilsson, Jesper R.; Solano, Carlos; Andréasson, Joakim; Grøtli, Morten

    2015-05-01

    REarranged during Transfection (RET) is a transmembrane receptor tyrosine kinase required for normal development and maintenance of neurons of the central and peripheral nervous systems. Deregulation of RET and hyperactivity of the RET kinase is intimately connected to several types of human cancers, most notably thyroid cancers, making it an attractive therapeutic target for small-molecule kinase inhibitors. Novel approaches, allowing external control of the activity of RET, would be key additions to the signal transduction toolbox. In this work, photoswitchable RET kinase inhibitors based on azo-functionalized pyrazolopyrimidines were developed, enabling photonic control of RET activity. The most promising compound displays excellent switching properties and stability with good inhibitory effect towards RET in cell-free as well as live-cell assays and a significant difference in inhibitory activity between its two photoisomeric forms. As the first reported photoswitchable small-molecule kinase inhibitor, we consider the herein presented effector to be a significant step forward in the development of tools for kinase signal transduction studies with spatiotemporal control over inhibitor concentration in situ.

  12. Design, Synthesis and Inhibitory Activity of Photoswitchable RET Kinase Inhibitors

    PubMed Central

    Ferreira, Rubén; Nilsson, Jesper R.; Solano, Carlos; Andréasson, Joakim; Grøtli, Morten

    2015-01-01

    REarranged during Transfection (RET) is a transmembrane receptor tyrosine kinase required for normal development and maintenance of neurons of the central and peripheral nervous systems. Deregulation of RET and hyperactivity of the RET kinase is intimately connected to several types of human cancers, most notably thyroid cancers, making it an attractive therapeutic target for small-molecule kinase inhibitors. Novel approaches, allowing external control of the activity of RET, would be key additions to the signal transduction toolbox. In this work, photoswitchable RET kinase inhibitors based on azo-functionalized pyrazolopyrimidines were developed, enabling photonic control of RET activity. The most promising compound displays excellent switching properties and stability with good inhibitory effect towards RET in cell-free as well as live-cell assays and a significant difference in inhibitory activity between its two photoisomeric forms. As the first reported photoswitchable small-molecule kinase inhibitor, we consider the herein presented effector to be a significant step forward in the development of tools for kinase signal transduction studies with spatiotemporal control over inhibitor concentration in situ. PMID:25944708

  13. Discovery of Pyrrolopyridine−Pyridone Based Inhibitors of Met Kinase: Synthesis, X-ray Crystallographic Analysis, and Biological Activities

    SciTech Connect

    Kim, Kyoung Soon; Zhang, Liping; Schmidt, Robert; Cai, Zhen-Wei; Wei, Donna; Williams, David K.; Lombardo, Louis J.; Trainor, George L.; Xie, Dianlin; Zhang, Yaquan; An, Yongmi; Sack, John S.; Tokarski, John S.; Darienzo, Celia; Kamath, Amrita; Marathe, Punit; Zhang, Yueping; Lippy, Jonathan; Jeyaseelan, Sr., Robert; Wautlet, Barri; Henley, Benjamin; Gullo-Brown, Johnni; Manne, Veeraswamy; Hunt, John T.; Fargnoli, Joseph; Borzilleri, Robert M.

    2008-10-02

    Conformationally constrained 2-pyridone analogue 2 is a potent Met kinase inhibitor with an IC50 value of 1.8 nM. Further SAR of the 2-pyridone based inhibitors of Met kinase led to potent 4-pyridone and pyridine N-oxide inhibitors such as 3 and 4. The X-ray crystallographic data of the inhibitor 2 bound to the ATP binding site of Met kinase protein provided insight into the binding modes of these inhibitors, and the SAR of this series of analogues was rationalized. Many of these analogues showed potent antiproliferative activities against the Met dependent GTL-16 gastric carcinoma cell line. Compound 2 also inhibited Flt-3 and VEGFR-2 kinases with IC{sub 50} values of 4 and 27 nM, respectively. It possesses a favorable pharmacokinetic profile in mice and demonstrates significant in vivo antitumor activity in the GTL-16 human gastric carcinoma xenograft model.

  14. The EMT-activator ZEB1 induces bone metastasis associated genes including BMP-inhibitors

    PubMed Central

    Mock, Kerstin; Preca, Bogdan-Tiberius; Brummer, Tilman; Brabletz, Simone; Stemmler, Marc P.; Brabletz, Thomas

    2015-01-01

    Tumor cell invasion, dissemination and metastasis is triggered by an aberrant activation of epithelial-to-mesenchymal transition (EMT), often mediated by the transcription factor ZEB1. Disseminating tumor cells must acquire specific features that allow them to colonize at different organ sites. Here we identify a set of genes that is highly expressed in breast cancer bone metastasis and activated by ZEB1. This gene set includes various secreted factors, e.g. the BMP-inhibitor FST, that are described to reorganize the bone microenvironment. By inactivating BMP-signaling, BMP-inhibitors are well-known to induce osteolysis in development and disease. We here demonstrate that the expression of ZEB1 and BMP-inhibitors is correlated with bone metastasis, but not with brain or lung metastasis of breast cancer patients. In addition, we show that this correlated expression pattern is causally linked, as ZEB1 induces the expression of the BMP-inhibitors NOG, FST and CHRDL1 both by directly increasing their gene transcription, as well as by indirectly suppressing their reduction via miR-200 family members. Consequently, ZEB1 stimulates BMP-inhibitor mediated osteoclast differentiation. These findings suggest that ZEB1 is not only driving EMT, but also contributes to the formation of osteolytic bone metastases in breast cancer. PMID:25973542

  15. Parathyroid hormone is not an inhibitor of lipoprotein lipase activity.

    PubMed

    Arnadottir, M; Nilsson-Ehle, P

    1994-01-01

    The reduced lipoprotein lipase (LPL) activities in uraemia are reflected by increased serum triglyceride concentrations and reduced HDL cholesterol concentrations. Both hyperparathyroidism and circulating inhibitor(s) of LPL have been associated with the disturbances of lipid metabolism in uraemia. The aim of the present study was to investigate if parathyroid hormone (PTH) had an inhibitory effect on LPL activity. Plasma post-heparin LPL activities, plasma LPL inhibitory activities, serum PTHintact and serum PTHC-terminal concentrations were analysed in 20 patients on haemodialysis and 20 healthy controls. The effects of purified, human PTHintact and a carboxyterminal fragment of PTH (PTH39-84) on LPL activities in post-heparin plasma from healthy individuals and on the enzyme activity of purified, bovine milk LPL, activated with apolipoprotein CII, were studied. Patients had significantly higher plasma LPL inhibitory activities than controls, but there was no correlation between plasma LPL inhibitory activities and serum PTH concentrations. Neither PTHintact nor PTH39-84 had a significant effect on LPL activities in vitro. Thus there was no evidence of a direct inhibition of LPL activity by PTH under the present in-vivo or in-vitro conditions.

  16. Parathyroid hormone is not an inhibitor of lipoprotein lipase activity.

    PubMed

    Arnadottir, M; Nilsson-Ehle, P

    1994-01-01

    The reduced lipoprotein lipase (LPL) activities in uraemia are reflected by increased serum triglyceride concentrations and reduced HDL cholesterol concentrations. Both hyperparathyroidism and circulating inhibitor(s) of LPL have been associated with the disturbances of lipid metabolism in uraemia. The aim of the present study was to investigate if parathyroid hormone (PTH) had an inhibitory effect on LPL activity. Plasma post-heparin LPL activities, plasma LPL inhibitory activities, serum PTHintact and serum PTHC-terminal concentrations were analysed in 20 patients on haemodialysis and 20 healthy controls. The effects of purified, human PTHintact and a carboxyterminal fragment of PTH (PTH39-84) on LPL activities in post-heparin plasma from healthy individuals and on the enzyme activity of purified, bovine milk LPL, activated with apolipoprotein CII, were studied. Patients had significantly higher plasma LPL inhibitory activities than controls, but there was no correlation between plasma LPL inhibitory activities and serum PTH concentrations. Neither PTHintact nor PTH39-84 had a significant effect on LPL activities in vitro. Thus there was no evidence of a direct inhibition of LPL activity by PTH under the present in-vivo or in-vitro conditions. PMID:7870347

  17. Activator-inhibitor systems on heterogeneous ecological networks

    NASA Astrophysics Data System (ADS)

    Nicolaides, C.; Cueto-Felgueroso, L.; Juanes, R.

    2012-12-01

    The consideration of activator-inhibitor systems as complex networks has broadened our knowledge of non-equilibrium reaction-diffusion processes in heterogeneous systems. For example, the Turing mechanism represents a classical model for the formation of self-organized spatial structures in non-equilibrium activator-inhibitor systems. The study of Turing patterns in networks with heterogeneous connectivity has revealed that, contrary to other models and systems, the segregation process takes place mainly in vertices of low degree. In this paper, we study the formation of vegetation patterns in semiarid ecosystems from the perspective of a heterogeneous interacting ecological network. The structure of ecological networks yields fundamental insight into the ecosystem self-organization. Using simple rules for the short-range activation and global inhibition, we reconstruct the observed power-law distribution of vegetation patch size that has been observed in semiarid ecosystems like the Kalahari transect.

  18. Antimalarial activity of HIV-1 protease inhibitor in chromone series.

    PubMed

    Lerdsirisuk, Pradith; Maicheen, Chirattikan; Ungwitayatorn, Jiraporn

    2014-12-01

    Increasing parasite resistance to nearly all available antimalarial drugs becomes a serious problem to human health and necessitates the need to continue the search for new effective drugs. Recent studies have shown that clinically utilized HIV-1 protease (HIV-1 PR) inhibitors can inhibit the in vitro and in vivo growth of Plasmodium falciparum. In this study, a series of chromone derivatives possessing HIV-1 PR inhibitory activity has been tested for antimalarial activity against P. falciparum (K1 multi-drug resistant strain). Chromone 15, the potent HIV-1 PR inhibitor (IC50=0.65μM), was found to be the most potent antimalarial compound with IC50=0.95μM while primaquine and tafenoquine showed IC50=2.41 and 1.95μM, respectively. Molecular docking study of chromone compounds against plasmepsin II, an aspartic protease enzyme important in hemoglobin degradation, revealed that chromone 15 exhibited the higher binding affinity (binding energy=-13.24kcal/mol) than the known PM II inhibitors. Thus, HIV-1 PR inhibitor in chromone series has the potential to be a new class of antimalarial agent. PMID:25462990

  19. Novel Triazole-Quinoline Derivatives as Selective Dual Binding Site Acetylcholinesterase Inhibitors.

    PubMed

    Mantoani, Susimaire P; Chierrito, Talita P C; Vilela, Adriana F L; Cardoso, Carmen L; Martínez, Ana; Carvalho, Ivone

    2016-02-05

    Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder worldwide. Currently, the only strategy for palliative treatment of AD is to inhibit acetylcholinesterase (AChE) in order to increase the concentration of acetylcholine in the synaptic cleft. Evidence indicates that AChE also interacts with the β-amyloid (Aβ) protein, acting as a chaperone and increasing the number and neurotoxicity of Aβ fibrils. It is known that AChE has two binding sites: the peripheral site, responsible for the interactions with Aβ, and the catalytic site, related with acetylcholine hydrolysis. In this work, we reported the synthesis and biological evaluation of a library of new tacrine-donepezil hybrids, as a potential dual binding site AChE inhibitor, containing a triazole-quinoline system. The synthesis of hybrids was performed in four steps using the click chemistry strategy. These compounds were evaluated as hAChE and hBChE inhibitors, and some derivatives showed IC50 values in the micro-molar range and were remarkably selective towards hAChE. Kinetic assays and molecular modeling studies confirm that these compounds block both catalytic and peripheral AChE sites. These results are quite interesting since the triazole-quinoline system is a new structural scaffold for AChE inhibitors. Furthermore, the synthetic approach is very efficient for the preparation of target compounds, allowing a further fruitful new chemical library optimization.

  20. Conformation-selective ATP-competitive inhibitors control regulatory interactions and noncatalytic functions of mitogen-activated protein kinases.

    PubMed

    Hari, Sanjay B; Merritt, Ethan A; Maly, Dustin J

    2014-05-22

    Most potent protein kinase inhibitors act by competing with ATP to block the phosphotransferase activity of their targets. However, emerging evidence demonstrates that ATP-competitive inhibitors can affect kinase interactions and functions in ways beyond blocking catalytic activity. Here, we show that stabilizing alternative ATP-binding site conformations of the mitogen-activated protein kinases (MAPKs) p38α and Erk2 with ATP-competitive inhibitors differentially, and in some cases divergently, modulates the abilities of these kinases to interact with upstream activators and deactivating phosphatases. Conformation-selective ligands are also able to modulate Erk2's ability to allosterically activate the MAPK phosphatase DUSP6, highlighting how ATP-competitive ligands can control noncatalytic kinase functions. Overall, these studies underscore the relationship between the ATP-binding and regulatory sites of MAPKs and provide insight into how ATP-competitive ligands can be designed to confer graded control over protein kinase function.

  1. Differential regulation of the Cdk5-dependent phosphorylation sites of inhibitor-1 and DARPP-32 by depolarization.

    PubMed

    Nguyen, Chan; Hosokawa, Tomohisa; Kuroiwa, Mahomi; Ip, Nancy Y; Nishi, Akinori; Hisanaga, Shin-Ichi; Bibb, James A

    2007-11-01

    While cyclin-dependent kinase 5 (Cdk5) is of growing importance to neuronal signaling, its regulation remains relatively unexplored. Examination of the mechanism by which NMDA modulates the phosphorylation of protein phosphatase inhibitor-1 at Ser6 and Ser67 and dopamine- and cAMP-regulated phosphoprotein M(r) 32 000 at Thr75 revealed that generalized depolarization, rather than specific activation of NMDA receptors, was sufficient to induce decreases in these Cdk5 sites. Although no evidence for the involvement of the Cdk5 cofactors p35 or p39, or for L- and T-type voltage-gated Ca(2+) channels, was found, evaluation of the role of phosphatases and extracellular cations revealed differential regulation of the three sites. NMDA-induced decreases in the phosphorylation of Thr75 of dopamine- and cAMP-regulated phosphoprotein M(r) 32 000 required protein phosphatase 1/2A activity and extracellular Ca(2+). In contrast, the effects on Ser6 and Ser67 of inhibitor-1 were not cation specific; either Na(+) or Ca(2+) sufficed. Furthermore, while the decrease in phosphorylation of Ser6 was partially dependent on protein phosphatase 2B, that of Ser67 was independent of the major protein serine/threonine phosphatases, likely indicating the presence of a pathway by which NMDA inhibits Cdk5 activity. Thus, in the striatum the regulation of phosphorylation of Cdk5-dependent sites by NMDA occurs through multiple distinct pathways.

  2. Activating PTEN by COX-2 inhibitors antagonizes radiation-induced AKT activation contributing to radiosensitization

    SciTech Connect

    Meng, Zhen; Gan, Ye-Hua

    2015-05-01

    Radiotherapy is still one of the most effective nonsurgical treatments for many tumors. However, radioresistance remains a major impediment to radiotherapy. Although COX-2 inhibitors can induce radiosensitization, the underlying mechanism is not fully understood. In this study, we showed that COX-2 selective inhibitor celecoxib enhanced the radiation-induced inhibition of cell proliferation and apoptosis in HeLa and SACC-83 cells. Treatment with celecoxib alone dephosphorylated phosphatase and tensin homolog deleted on chromosome ten (PTEN), promoted PTEN membrane translocation or activation, and correspondingly dephosphorylated or inactivated protein kinase B (AKT). By contrast, treatment with radiation alone increased PTEN phosphorylation, inhibited PTEN membrane translocation and correspondingly activated AKT in the two cell lines. However, treatment with celecoxib or another COX-2 selective inhibitor (valdecoxib) completely blocked radiation-induced increase of PTEN phosphorylation, rescued radiation-induced decrease in PTEN membrane translocation, and correspondingly inactivated AKT. Moreover, celecoxib could also upregulate PTEN protein expression by downregulating Sp1 expression, thereby leading to the activation of PTEN transcription. Our results suggested that COX-2 inhibitors could enhance radiosensitization at least partially by activating PTEN to antagonize radiation-induced AKT activation. - Highlights: • COX-2 inhibitor, celecoxib, could enhance radiosensitization. • Radiation induced PTEN inactivation (phosphorylation) and AKT activation. • COX-2 inhibitor induced PTEN expression and activation, and inactivated AKT. • COX-2 inhibitor enhanced radiosensitization through activating PTEN.

  3. A rapid and direct method for the determination of active site accessibility in proteins based on ESI-MS and active site titrations.

    PubMed

    O'Farrell, Norah; Kreiner, Michaela; Moore, Barry D; Parker, Marie-Claire

    2006-11-01

    We have developed an electrospray ionisation mass spectrometry (ESI-MS) technique that can be applied to rapidly determine the number of intact active sites in proteins. The methodology relies on inhibiting the protein with an active-site irreversible inhibitor and then using ESI-MS to determine the extent of inhibition. We have applied this methodology to a test system: a serine protease, subtilisin Carlsberg, and monitored the extent of inhibition by phenylmethylsulfonyl fluoride (PMSF), an irreversible serine hydrolase inhibitor as a function of the changes in immobilisation and hydration conditions. Two types of enzyme preparation were investigated, lyophilised enzymes and protein-coated microcrystals (PCMC).

  4. Characterization of novel MPS1 inhibitors with preclinical anticancer activity

    PubMed Central

    Jemaà, M; Galluzzi, L; Kepp, O; Senovilla, L; Brands, M; Boemer, U; Koppitz, M; Lienau, P; Prechtl, S; Schulze, V; Siemeister, G; Wengner, A M; Mumberg, D; Ziegelbauer, K; Abrieu, A; Castedo, M; Vitale, I; Kroemer, G

    2013-01-01

    Monopolar spindle 1 (MPS1), a mitotic kinase that is overexpressed in several human cancers, contributes to the alignment of chromosomes to the metaphase plate as well as to the execution of the spindle assembly checkpoint (SAC). Here, we report the identification and functional characterization of three novel inhibitors of MPS1 of two independent structural classes, N-(4-{2-[(2-cyanophenyl)amino][1,2,4]triazolo[1,5-a]pyridin-6-yl}phenyl)-2-phenylacetamide (Mps-BAY1) (a triazolopyridine), N-cyclopropyl-4-{8-[(2-methylpropyl)amino]-6-(quinolin-5-yl)imidazo[1,2-a]pyrazin-3-yl}benzamide (Mps-BAY2a) and N-cyclopropyl-4-{8-(isobutylamino)imidazo[1,2-a]pyrazin-3-yl}benzamide (Mps-BAY2b) (two imidazopyrazines). By selectively inactivating MPS1, these small inhibitors can arrest the proliferation of cancer cells, causing their polyploidization and/or their demise. Cancer cells treated with Mps-BAY1 or Mps-BAY2a manifested multiple signs of mitotic perturbation including inefficient chromosomal congression during metaphase, unscheduled SAC inactivation and severe anaphase defects. Videomicroscopic cell fate profiling of histone 2B-green fluorescent protein-expressing cells revealed the capacity of MPS1 inhibitors to subvert the correct timing of mitosis as they induce a premature anaphase entry in the context of misaligned metaphase plates. Hence, in the presence of MPS1 inhibitors, cells either divided in a bipolar (but often asymmetric) manner or entered one or more rounds of abortive mitoses, generating gross aneuploidy and polyploidy, respectively. In both cases, cells ultimately succumbed to the mitotic catastrophe-induced activation of the mitochondrial pathway of apoptosis. Of note, low doses of MPS1 inhibitors and paclitaxel (a microtubular poison) synergized at increasing the frequency of chromosome misalignments and missegregations in the context of SAC inactivation. This resulted in massive polyploidization followed by the activation of mitotic catastrophe. A

  5. Characterization of novel MPS1 inhibitors with preclinical anticancer activity.

    PubMed

    Jemaà, M; Galluzzi, L; Kepp, O; Senovilla, L; Brands, M; Boemer, U; Koppitz, M; Lienau, P; Prechtl, S; Schulze, V; Siemeister, G; Wengner, A M; Mumberg, D; Ziegelbauer, K; Abrieu, A; Castedo, M; Vitale, I; Kroemer, G

    2013-11-01

    Monopolar spindle 1 (MPS1), a mitotic kinase that is overexpressed in several human cancers, contributes to the alignment of chromosomes to the metaphase plate as well as to the execution of the spindle assembly checkpoint (SAC). Here, we report the identification and functional characterization of three novel inhibitors of MPS1 of two independent structural classes, N-(4-{2-[(2-cyanophenyl)amino][1,2,4]triazolo[1,5-a]pyridin-6-yl}phenyl)-2-phenylacetamide (Mps-BAY1) (a triazolopyridine), N-cyclopropyl-4-{8-[(2-methylpropyl)amino]-6-(quinolin-5-yl)imidazo[1,2-a]pyrazin-3-yl}benzamide (Mps-BAY2a) and N-cyclopropyl-4-{8-(isobutylamino)imidazo[1,2-a]pyrazin-3-yl}benzamide (Mps-BAY2b) (two imidazopyrazines). By selectively inactivating MPS1, these small inhibitors can arrest the proliferation of cancer cells, causing their polyploidization and/or their demise. Cancer cells treated with Mps-BAY1 or Mps-BAY2a manifested multiple signs of mitotic perturbation including inefficient chromosomal congression during metaphase, unscheduled SAC inactivation and severe anaphase defects. Videomicroscopic cell fate profiling of histone 2B-green fluorescent protein-expressing cells revealed the capacity of MPS1 inhibitors to subvert the correct timing of mitosis as they induce a premature anaphase entry in the context of misaligned metaphase plates. Hence, in the presence of MPS1 inhibitors, cells either divided in a bipolar (but often asymmetric) manner or entered one or more rounds of abortive mitoses, generating gross aneuploidy and polyploidy, respectively. In both cases, cells ultimately succumbed to the mitotic catastrophe-induced activation of the mitochondrial pathway of apoptosis. Of note, low doses of MPS1 inhibitors and paclitaxel (a microtubular poison) synergized at increasing the frequency of chromosome misalignments and missegregations in the context of SAC inactivation. This resulted in massive polyploidization followed by the activation of mitotic catastrophe. A

  6. Inhibitors

    MedlinePlus

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

  7. Molecular Docking Guided Comparative GFA, G/PLS, SVM and ANN Models of Structurally Diverse Dual Binding Site Acetylcholinesterase Inhibitors.

    PubMed

    Gupta, Shikhar; Fallarero, Adyary; Vainio, Mikko J; Saravanan, P; Santeri Puranen, J; Järvinen, Päivi; Johnson, Mark S; Vuorela, Pia M; Mohan, C Gopi

    2011-08-01

    Recently discovered 42 AChE inhibitors binding at the catalytic and peripheral anionic site were identified on the basis of molecular docking approach, and its comparative quantitative structure-activity relationship (QSAR) models were developed. These structurally diverse inhibitors were obtained by our previously reported high-throughput in vitro screening technique using 384-well plate's assay based on colorimetric method of Ellman. QSAR models were developed using (i) genetic function algorithm, (ii) genetic partial least squares, (iii) support vector machine and (iv) artificial neural network techniques. The QSAR model robustness and significance was critically assessed using different cross-validation techniques on test data set. The generated QSAR models using thermodynamic, electrotopological and electronic descriptors showed that nonlinear methods are more robust than linear methods, and provide insight into the structural features of compounds that are important for AChE inhibition.

  8. Discovering Echinococcus granulosus thioredoxin glutathione reductase inhibitors through site-specific dynamic combinatorial chemistry.

    PubMed

    Saiz, Cecilia; Castillo, Valerie; Fontán, Pablo; Bonilla, Mariana; Salinas, Gustavo; Rodríguez-Haralambides, Alejandra; Mahler, S Graciela

    2014-02-01

    In this study, we report a strategy using dynamic combinatorial chemistry for targeting the thioredoxin (Trx)-reductase catalytic site on Trx glutathione reductase (TGR), a pyridine nucleotide thiol-disulfide oxido-reductase. We chose Echinococcus granulosus TGR since it is a bottleneck enzyme of platyhelminth parasites and a validated pharmacological target. A dynamic combinatorial library (DCL) was constructed based on thiol-disulfide reversible exchange. We demonstrate the use of 5-thio-2-nitrobenzoic acid (TNB) as a non-covalent anchor fragment in a DCL templated by E. granulosus TGR. The heterodimer of TNB and bisthiazolidine (2af) was identified, upon library analysis by HPLC (IC50 = 24 μM). Furthermore, 14 analogs were synthetically prepared and evaluated against TGR. This allowed the study of a structure-activity relationship and the identification of a disulfide TNB-tricyclic bisthiazolidine (2aj) as the best enzyme inhibitor in these series, with an IC50 = 24 μM. Thus, our results validate the use of DCL for targeting thiol-disulfide oxido-reductases.

  9. Nanoscale liposomal formulation of a SYK P-site inhibitor against B-precursor leukemia

    PubMed Central

    Qazi, Sanjive; Cely, Ingrid; Sahin, Kazim; Shahidzadeh, Anoush; Ozercan, Ibrahim; Yin, Qian; Gaynon, Paul; Termuhlen, Amanda; Cheng, Jianjun

    2013-01-01

    We report preclinical proof of principle for effective treatment of B-precursor acute lymphoblastic leukemia (ALL) by targeting the spleen tyrosine kinase (SYK)–dependent antiapoptotic blast cell survival machinery with a unique nanoscale pharmaceutical composition. This nanoscale liposomal formulation (NLF) contains the pentapeptide mimic 1,4-Bis (9-O dihydroquinidinyl) phthalazine/hydroquinidine 1,4-phathalazinediyl diether (C61) as the first and only selective inhibitor of the substrate binding P-site of SYK. The C61 NLF exhibited a very favorable pharmacokinetic and safety profile in mice, induced apoptosis in primary B-precursor ALL blast cells taken directly from patients as well as in vivo clonogenic ALL xenograft cells, destroyed the in vivo clonogenic fraction of ALL blast cells, and, at nontoxic dose levels, exhibited potent in vivo antileukemic activity against patient-derived ALL cells in xenograft models of aggressive B-precursor ALL. Our findings establish SYK as an attractive molecular target for therapy of B-precursor ALL. Further development of the C61 NLF may provide the foundation for therapeutic innovation against therapy-refractory B-precursor ALL. PMID:23568490

  10. Mapping the Binding Site of the Inhibitor Tariquidar That Stabilizes the First Transmembrane Domain of P-glycoprotein*

    PubMed Central

    Loo, Tip W.; Clarke, David M.

    2015-01-01

    ABC (ATP-binding cassette) transporters are clinically important because drug pumps like P-glycoprotein (P-gp, ABCB1) confer multidrug resistance and mutant ABC proteins are responsible for many protein-folding diseases such as cystic fibrosis. Identification of the tariquidar-binding site has been the subject of intensive molecular modeling studies because it is the most potent inhibitor and corrector of P-gp. Tariquidar is a unique P-gp inhibitor because it locks the pump in a conformation that blocks drug efflux but activates ATPase activity. In silico docking studies have identified several potential tariquidar-binding sites. Here, we show through cross-linking studies that tariquidar most likely binds to sites within the transmembrane (TM) segments located in one wing or at the interface between the two wings (12 TM segments form 2 divergent wings). We then introduced arginine residues at all positions in the 12 TM segments (223 mutants) of P-gp. The rationale was that a charged residue in the drug-binding pocket would disrupt hydrophobic interaction with tariquidar and inhibit its ability to rescue processing mutants or stimulate ATPase activity. Arginines introduced at 30 positions significantly inhibited tariquidar rescue of a processing mutant and activation of ATPase activity. The results suggest that tariquidar binds to a site within the drug-binding pocket at the interface between the TM segments of both structural wings. Tariquidar differed from other drug substrates, however, as it stabilized the first TM domain. Stabilization of the first TM domain appears to be a key mechanism for high efficiency rescue of ABC processing mutants that cause disease. PMID:26507655

  11. Metals in the active site of native protein phosphatase-1.

    PubMed

    Heroes, Ewald; Rip, Jens; Beullens, Monique; Van Meervelt, Luc; De Gendt, Stefan; Bollen, Mathieu

    2015-08-01

    Protein phosphatase-1 (PP1) is a major protein Ser/Thr phosphatase in eukaryotic cells. Its activity depends on two metal ions in the catalytic site, which were identified as manganese in the bacterially expressed phosphatase. However, the identity of the metal ions in native PP1 is unknown. In this study, total reflection X-ray fluorescence (TXRF) was used to detect iron and zinc in PP1 that was purified from rabbit skeletal muscle. Metal exchange experiments confirmed that the distinct substrate specificity of recombinant and native PP1 is determined by the nature of their associated metals. We also found that the iron level associated with native PP1 is decreased by incubation with inhibitor-2, consistent with a function of inhibitor-2 as a PP1 chaperone. PMID:25890482

  12. Inhibition of dog and human gastric lipases by enantiomeric phosphonate inhibitors: a structure-activity study.

    PubMed

    Miled, Nabil; Roussel, Alain; Bussetta, Cécile; Berti-Dupuis, Liliane; Rivière, Mireille; Buono, Gérard; Verger, Robert; Cambillau, Christian; Canaan, Stéphane

    2003-10-14

    The crystal structures of gastric lipases in the apo form [Roussel, A., et al. (1999) J. Biol. Chem. 274, 16995-17002] or in complex with the (R(P))-undecyl butyl phosphonate [C(11)Y(4)(+)] [Roussel, A., et al. (2002) J. Biol. Chem. 277, 2266-2274] have improved our understanding of the structure-activity relationships of acid lipases. In this report, we have performed a kinetic study with dog and human gastric lipases (DGL and HGL, respectively) using several phosphonate inhibitors by varying the absolute configuration of the phosphorus atom and the chain length of the alkyl/alkoxy substituents. Using the two previously determined structures and that of a new crystal structure obtained with the other (S(P))-phosphonate enantiomer [C(11)Y(4)(-)], we constructed models of phosphonate inhibitors fitting into the active site crevices of DGL and HGL. All inhibitors with a chain length of fewer than 12 carbon atoms were found to be completely buried in the catalytic crevice, whereas longer alkyl/alkoxy chains were found to point out of the cavity. The main stereospecific determinant explaining the stronger inhibition of the S(P) enantiomers is the presence of a hydrogen bond involving the catalytic histidine as found in the DGL-C(11)Y(4)(-) complex. On the basis of these results, we have built a model of the first tetrahedral intermediate corresponding to the tristearoyl-lipase complex. The triglyceride molecule completely fills the active site crevice of DGL, in contrast with what is observed with other lipases such as pancreatic lipases which have a shallower and narrower active site. For substrate hydrolysis, the supply of water molecules to the active site might be achieved through a lateral channel identified in the protein core.

  13. The pan-PI3K inhibitor GDC-0941 activates canonical WNT signaling to confer resistance in TNBC cells: resistance reversal with WNT inhibitor.

    PubMed

    Tzeng, Huey-En; Yang, Lixin; Chen, Kemin; Wang, Yafan; Liu, Yun-Ru; Pan, Shiow-Lin; Gaur, Shikha; Hu, Shuya; Yen, Yun

    2015-05-10

    The pan-PI3K inhibitors are one treatment option for triple-negative breast cancer (TNBC). However, this treatment is ineffective for unknown reasons. Here, we report that aberrant expression of wingless-type MMTV integration site family (WNT) and activated WNT signals, which crosstalk with the PI3K-AKT-mTOR signaling pathway through GSK3β, plays the most critical role in resistance to pan-PI3K inhibitors in TNBC cells. GDC-0941 is a pan-PI3K inhibitor that activates the WNT/beta-catenin pathway in TNBC cells through stimulation of WNT secretion. GDC-0941-triggered WNT/beta-catenin pathway activation was observed in MDA-MB-231 and HCC1937 cells, which are TNBC cell lines showing aberrant WNT/beta-catenin activation, and not in SKBR3 and MCF7 cells. This observation is further investigated in vivo. GDC-0941 exhibited minimal tumor inhibition in MDA-MB-231 cells, but it significantly suppressed tumor growth in HER-positive SK-BR3 cells. In vivo mechanism study revealed the activation of WNT/beta-catenin pathway by GDC-0941. A synergistic effect was observed when combined treatment with GDC-0941 and the WNT inhibitor LGK974 at low concentrations in MDA-MB-231 cells. These findings indicated that WNT pathway activation conferred resistance in TNBC cells treated with GDC-0941. This resistance may be further circumvented through combined treatment with pan-PI3K and WNT inhibitors. Future clinical trials of these two inhibitors are warranted.

  14. The pan-PI3K inhibitor GDC-0941 activates canonical WNT signaling to confer resistance in TNBC cells: resistance reversal with WNT inhibitor

    PubMed Central

    Tzeng, Huey-En; Yang, Lixin; Chen, Kemin; Wang, Yafan; Liu, Yun-Ru; Pan, Shiow-Lin; Gaur, Shikha; Hu, Shuya; Yen, Yun

    2015-01-01

    The pan-PI3K inhibitors are one treatment option for triple-negative breast cancer (TNBC). However, this treatment is ineffective for unknown reasons. Here, we report that aberrant expression of wingless-type MMTV integration site family (WNT) and activated WNT signals, which crosstalk with the PI3K-AKT-mTOR signaling pathway through GSK3β, plays the most critical role in resistance to pan-PI3K inhibitors in TNBC cells. GDC-0941 is a pan-PI3K inhibitor that activates the WNT/beta-catenin pathway in TNBC cells through stimulation of WNT secretion. GDC-0941-triggered WNT/beta-catenin pathway activation was observed in MDA-MB-231 and HCC1937 cells, which are TNBC cell lines showing aberrant WNT/beta-catenin activation, and not in SKBR3 and MCF7 cells. This observation is further investigated in vivo. GDC-0941 exhibited minimal tumor inhibition in MDA-MB-231 cells, but it significantly suppressed tumor growth in HER-positive SK-BR3 cells. In vivo mechanism study revealed the activation of WNT/beta-catenin pathway by GDC-0941. A synergistic effect was observed when combined treatment with GDC-0941 and the WNT inhibitor LGK974 at low concentrations in MDA-MB-231 cells. These findings indicated that WNT pathway activation conferred resistance in TNBC cells treated with GDC-0941. This resistance may be further circumvented through combined treatment with pan-PI3K and WNT inhibitors. Future clinical trials of these two inhibitors are warranted. PMID:25857298

  15. Druggability analysis and classification of protein tyrosine phosphatase active sites

    PubMed Central

    Ghattas, Mohammad A; Raslan, Noor; Sadeq, Asil; Al Sorkhy, Mohammad; Atatreh, Noor

    2016-01-01

    Protein tyrosine phosphatases (PTP) play important roles in the pathogenesis of many diseases. The fact that no PTP inhibitors have reached the market so far has raised many questions about their druggability. In this study, the active sites of 17 PTPs were characterized and assessed for its ability to bind drug-like molecules. Consequently, PTPs were classified according to their druggability scores into four main categories. Only four members showed intermediate to very druggable pocket; interestingly, the rest of them exhibited poor druggability. Particularly focusing on PTP1B, we also demonstrated the influence of several factors on the druggability of PTP active site. For instance, the open conformation showed better druggability than the closed conformation, while the tight-bound water molecules appeared to have minimal effect on the PTP1B druggability. Finally, the allosteric site of PTP1B was found to exhibit superior druggability compared to the catalytic pocket. This analysis can prove useful in the discovery of new PTP inhibitors by assisting researchers in predicting hit rates from high throughput or virtual screening and saving unnecessary cost, time, and efforts via prioritizing PTP targets according to their predicted druggability. PMID:27757011

  16. Novel cinnoline-based inhibitors of LRRK2 kinase activity.

    PubMed

    Garofalo, Albert W; Adler, Marc; Aubele, Danielle L; Bowers, Simeon; Franzini, Maurizio; Goldbach, Erich; Lorentzen, Colin; Neitz, R Jeffrey; Probst, Gary D; Quinn, Kevin P; Santiago, Pam; Sham, Hing L; Tam, Danny; Truong, Anh P; Ye, Xiaocong M; Ren, Zhao

    2013-01-01

    Leucine rich repeat kinase 2 (LRRK2) has been implicated in the pathogenesis of Parkinson's disease (PD). Inhibition of LRRK2 kinase activity is a therapeutic approach that may lead to new treatments for PD. Herein we report the discovery of a series of cinnoline-3-carboxamides that are potent against both wild-type and mutant LRRK2 kinase activity in biochemical assays. These compounds are also shown to be potent inhibitors in a cellular assay and to have good to excellent CNS penetration. PMID:23219325

  17. Pyrrolopyridine inhibitors of mitogen-activated protein kinase-activated protein kinase 2 (MK-2).

    PubMed

    Anderson, David R; Meyers, Marvin J; Vernier, William F; Mahoney, Matthew W; Kurumbail, Ravi G; Caspers, Nicole; Poda, Gennadiy I; Schindler, John F; Reitz, David B; Mourey, Robert J

    2007-05-31

    A new class of potent kinase inhibitors selective for mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2 or MK-2) for the treatment of rheumatoid arthritis has been prepared and evaluated. These inhibitors have IC50 values as low as 10 nM against the target and have good selectivity profiles against a number of kinases including CDK2, ERK, JNK, and p38. These MK-2 inhibitors have been shown to suppress TNFalpha production in U397 cells and to be efficacious in an acute inflammation model. The structure-activity relationships of this series, the selectivity for MK-2 and their activity in both in vitro and in vivo models are discussed. The observed selectivity is discussed with the aid of an MK-2/inhibitor crystal structure.

  18. Novel histone deacetylase inhibitor NCH-51 activates latent HIV-1 gene expression.

    PubMed

    Victoriano, Ann Florence B; Imai, Kenichi; Togami, Hiroaki; Ueno, Takaharu; Asamitsu, Kaori; Suzuki, Takayoshi; Miyata, Naoki; Ochiai, Kuniyasu; Okamoto, Takashi

    2011-04-01

    Pharmacological manipulations to purge human immunodeficiency virus (HIV) from latent reservoirs have been considered as an adjuvant therapeutic approach to highly-active antiretroviral therapy for the eradication of HIV. Our novel histone deacetylase inhibitor NCH-51 induced expression of latent HIV-1 with minimal cytotoxicity. Using chromatin immunoprecipitation assays, we observed a reduction of HDAC1 occupancy, histone hyperacetylation and the recruitment of positive transcription factors at the HIV-1 promoter in latently infected-cells under the treatment with NCH-51. Mutation studies of the long terminal repeat (LTR) revealed NCH-51 mediated gene expression through the Sp1 sites. When Sp1 expression was knocked-down by small interfering RNA, the NCH-51-mediated activation of a stably integrated HIV-1 LTR was attenuated. Moreover, the Sp1 inhibitor mithramycin A abolished the effects of NCH-51.

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

    PubMed

    Swedberg, Joakim E; Harris, Jonathan M

    2011-10-01

    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. PMID:21877690

  20. Catalysis: Elusive active site in focus

    NASA Astrophysics Data System (ADS)

    Labinger, Jay A.

    2016-08-01

    The identification of the active site of an iron-containing catalyst raises hopes of designing practically useful catalysts for the room-temperature conversion of methane to methanol, a potential fuel for vehicles. See Letter p.317

  1. Uncoupling of RNA binding and PKR kinase activation by viral inhibitor RNAs.

    PubMed

    McKenna, Sean A; Kim, Insil; Liu, Corey W; Puglisi, Joseph D

    2006-05-19

    Protein kinase RNA-activated (PKR) is a serine/threonine kinase that contains an N-terminal RNA-binding domain and a C-terminal kinase domain. Upon binding double-stranded RNA (dsRNA), PKR can become activated and phosphorylate cellular targets, such as eukaryotic translation initiation factor 2alpha (eIF-2alpha). Phosphorylation of eIF-2alpha results in attenuation of protein translation by the ribosome in either a general or an mRNA-specific manner. Therefore, the interaction between PKR and dsRNAs represents a crucial host cell defense mechanism against viral infection. Viruses can circumvent PKR function by transcription of virus-encoded dsRNA inhibitors that bind to and inactivate PKR. We present here a biophysical characterization of the interactions between human PKR and two viral inhibitor RNAs, EBER(I) (from Epstein-Barr virus) and VA(I) (from human adenovirus). Autophosphorylation assays confirmed that both EBER(I) and VA(I) are inhibitors of PKR activation, and profiled the kinetics of the inhibition. Binding affinities of dsRNAs to PKR double-stranded RNA-binding domains (dsRBDs) were determined by isothermal titration calorimetry and gel electrophoresis. A single stem-loop domain from each inhibitory RNA mediates the interaction with both dsRBDs of PKR. The binding sites on inhibitor RNAs and the dsRBDs of PKR have been mapped by NMR chemical shift perturbation experiments, which indicate that inhibitors of PKR employ similar surfaces of interaction as activators. Finally, we show that dsRNA binding and inactivation are non-equivalent; regions other than the dsRBD stem-loops of inhibitory RNA are required for inhibition.

  2. Fragment-based discovery of mexiletine derivatives as orally bioavailable inhibitors of urokinase-type plasminogen activator.

    PubMed

    Frederickson, Martyn; Callaghan, Owen; Chessari, Gianni; Congreve, Miles; Cowan, Suzanna R; Matthews, Julia E; McMenamin, Rachel; Smith, Donna-Michelle; Vinković, Mladen; Wallis, Nicola G

    2008-01-24

    Fragment-based lead discovery has been applied to urokinase-type plasminogen activator (uPA). The (R)-enantiomer of the orally active drug mexiletine 5 (a fragment hit from X-ray crystallographic screening) was the chemical starting point. Structure-aided design led to elaborated inhibitors that retained the key interactions of (R)-5 while gaining extra potency by simultaneously occupying neighboring regions of the active site. Subsequent optimization led to 15, a potent, selective, and orally bioavailable inhibitor of uPA. PMID:18163548

  3. Structural Interactions between Inhibitor and Substrate Docking Sites Give Insight into Mechanisms of Human PS1 Complexes

    PubMed Central

    Li, Yi; Lu, Stephen Hsueh-Jeng; Tsai, Ching-Ju; Bohm, Christopher; Qamar, Seema; Dodd, Roger B.; Meadows, William; Jeon, Amy; McLeod, Adam; Chen, Fusheng; Arimon, Muriel; Berezovska, Oksana; Hyman, Bradley T.; Tomita, Taisuke; Iwatsubo, Takeshi; Johnson, Christopher M.; Farrer, Lindsay A.; Schmitt-Ulms, Gerold; Fraser, Paul E.; St George-Hyslop, Peter H.

    2014-01-01

    Summary Presenilin-mediated endoproteolysis of transmembrane proteins plays a key role in physiological signaling and in the pathogenesis of Alzheimer disease and some cancers. Numerous inhibitors have been found via library screens, but their structural mechanisms remain unknown. We used several biophysical techniques to investigate the structure of human presenilin complexes and the effects of peptidomimetic γ-secretase inhibitors. The complexes are bilobed. The head contains nicastrin ectodomain. The membrane-embedded base has a central channel and a lateral cleft, which may represent the initial substrate docking site. Inhibitor binding induces widespread structural changes, including rotation of the head and closure of the lateral cleft. These changes block substrate access to the catalytic pocket and inhibit the enzyme. Intriguingly, peptide substrate docking has reciprocal effects on the inhibitor binding site. Similar reciprocal shifts may underlie the mechanisms of other inhibitors and of the “lateral gate” through which substrates access to the catalytic site. PMID:24210759

  4. Computational discovery of picomolar Q(o) site inhibitors of cytochrome bc1 complex.

    PubMed

    Hao, Ge-Fei; Wang, Fu; Li, Hui; Zhu, Xiao-Lei; Yang, Wen-Chao; Huang, Li-Shar; Wu, Jia-Wei; Berry, Edward A; Yang, Guang-Fu

    2012-07-11

    A critical challenge to the fragment-based drug discovery (FBDD) is its low-throughput nature due to the necessity of biophysical method-based fragment screening. Herein, a method of pharmacophore-linked fragment virtual screening (PFVS) was successfully developed. Its application yielded the first picomolar-range Q(o) site inhibitors of the cytochrome bc(1) complex, an important membrane protein for drug and fungicide discovery. Compared with the original hit compound 4 (K(i) = 881.80 nM, porcine bc(1)), the most potent compound 4f displayed 20 507-fold improved binding affinity (K(i) = 43.00 pM). Compound 4f was proved to be a noncompetitive inhibitor with respect to the substrate cytochrome c, but a competitive inhibitor with respect to the substrate ubiquinol. Additionally, we determined the crystal structure of compound 4e (K(i) = 83.00 pM) bound to the chicken bc(1) at 2.70 Å resolution, providing a molecular basis for understanding its ultrapotency. To our knowledge, this study is the first application of the FBDD method in the discovery of picomolar inhibitors of a membrane protein. This work demonstrates that the novel PFVS approach is a high-throughput drug discovery method, independent of biophysical screening techniques. PMID:22690928

  5. Methylene bisphosphonates as the inhibitors of HIV RT phosphorolytic activity.

    PubMed

    Yanvarev, D V; Korovina, A N; Usanov, N N; Khomich, O A; Vepsäläinen, J; Puljula, E; Kukhanova, M K; Kochetkov, S N

    2016-08-01

    The structure-function analysis of 36 methylenebisphosphonates (BPs) as inhibitors of the phosphorolytic activity of native and drug-resistant forms of HIV-1 reverse transcriptase (RT) was performed. It was shown that with the increase of the inhibitory potential of BPs towards the phosphorolytic activity raises their ability to inhibit the RT-catalyzed DNA elongation. Herein, we report the impact of the thymidine analog mutations (TAM) on the activity of bisphosphonates, as well as some structural features of the BPs, allowing them to maintain the inhibitory activity on the enzyme resistant to nucleoside analog therapy. We estimated the Mg(2+)-coordinating group structure, the linker and the aromatic pharmacophore influence on the inhibitory potential of the BPs. Based on the 31 BPs SAR, several BPs with improved inhibitory properties were designed and synthesized. PMID:27230835

  6. Methylene bisphosphonates as the inhibitors of HIV RT phosphorolytic activity.

    PubMed

    Yanvarev, D V; Korovina, A N; Usanov, N N; Khomich, O A; Vepsäläinen, J; Puljula, E; Kukhanova, M K; Kochetkov, S N

    2016-08-01

    The structure-function analysis of 36 methylenebisphosphonates (BPs) as inhibitors of the phosphorolytic activity of native and drug-resistant forms of HIV-1 reverse transcriptase (RT) was performed. It was shown that with the increase of the inhibitory potential of BPs towards the phosphorolytic activity raises their ability to inhibit the RT-catalyzed DNA elongation. Herein, we report the impact of the thymidine analog mutations (TAM) on the activity of bisphosphonates, as well as some structural features of the BPs, allowing them to maintain the inhibitory activity on the enzyme resistant to nucleoside analog therapy. We estimated the Mg(2+)-coordinating group structure, the linker and the aromatic pharmacophore influence on the inhibitory potential of the BPs. Based on the 31 BPs SAR, several BPs with improved inhibitory properties were designed and synthesized.

  7. Pseudomonas syringae pv. syringae uses proteasome inhibitor syringolin A to colonize from wound infection sites.

    PubMed

    Misas-Villamil, Johana C; Kolodziejek, Izabella; Crabill, Emerson; Kaschani, Farnusch; Niessen, Sherry; Shindo, Takayuki; Kaiser, Markus; Alfano, James R; van der Hoorn, Renier A L

    2013-03-01

    Infection of plants by bacterial leaf pathogens at wound sites is common in nature. Plants defend wound sites to prevent pathogen invasion, but several pathogens can overcome spatial restriction and enter leaf tissues. The molecular mechanisms used by pathogens to suppress containment at wound infection sites are poorly understood. Here, we studied Pseudomonas syringae strains causing brown spot on bean and blossom blight on pear. These strains exist as epiphytes that can cause disease upon wounding caused by hail, sand storms and frost. We demonstrate that these strains overcome spatial restriction at wound sites by producing syringolin A (SylA), a small molecule proteasome inhibitor. Consequently, SylA-producing strains are able to escape from primary infection sites and colonize adjacent tissues along the vasculature. We found that SylA diffuses from the primary infection site and suppresses acquired resistance in adjacent tissues by blocking signaling by the stress hormone salicylic acid (SA). Thus, SylA diffusion creates a zone of SA-insensitive tissue that is prepared for subsequent colonization. In addition, SylA promotes bacterial motility and suppresses immune responses at the primary infection site. These local immune responses do not affect bacterial growth and were weak compared to effector-triggered immunity. Thus, SylA facilitates colonization from wounding sites by increasing bacterial motility and suppressing SA signaling in adjacent tissues. PMID:23555272

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

    SciTech Connect

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

    1987-05-01

    BCKDH is phosphorylated by a copurifying kinase at two serine residues on the El..cap alpha.. 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, ..cap alpha..-chloroisocaproate (CIC) and branched chain ..cap alpha..-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/sup 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.

  9. Classification of ADAMTS binding sites: The first step toward selective ADAMTS7 inhibitors.

    PubMed

    Müller, Michaela; Kessler, Thorsten; Schunkert, Heribert; Erdmann, Jeanette; Tennstedt, Stephanie

    2016-03-11

    Genome-wide association studies identified ADAMTS7 as a risk locus for coronary artery disease. In carotid arteries of rats, neointima formation after balloon-mediated injury goes along with enhanced Adamts7 expression. Vice versa, Adamts7-deficient mice display reduced neointima formation following vascular injury. Although a causal link between ADAMTS7 and coronary artery disease remains to be proven, inhibition of ADAMTS7 represents a potential new target for intervention in this disease. ADAMTS7, a member of the 'a disintegrin and metalloproteinase with thrombospondin motifs' (ADAMTS) family of proteins, contains a catalytic zinc ion in the binding site of its metalloproteinase domain. The structure of ADAMTS7 and its inhibitors are unknown. In this study, we used in silico methods, including homology modeling and pharmacophore modeling, to analyze the ADAMTS7 metalloproteinase domain, particularly its binding site. The results revealed structural and sequence differences relative to the binding sites of the other ADAMTS proteins; these non-conserved regions represent potential binding regions for selective ADAMTS7 inhibitors. The main contribution of this study is the proposal of a pharmacophore for ADAMTS7. The characterization of the ADAMTS7 binding site and definition of a pharmacophore are the first step toward developing a new therapeutic target for coronary artery disease. PMID:26872430

  10. Crystal structure of plasminogen activator inhibitor-1 in an active conformation with normal thermodynamic stability.

    PubMed

    Jensen, Jan K; Thompson, Lawrence C; Bucci, Joel C; Nissen, Poul; Gettins, Peter G W; Peterson, Cynthia B; Andreasen, Peter A; Morth, J Preben

    2011-08-26

    The serpin plasminogen activator inhibitor-1 (PAI-1) is a crucial regulator in fibrinolysis and tissue remodeling. PAI-1 has been associated with several pathological conditions and is a validated prognostic marker in human cancers. However, structural information about the native inhibitory form of PAI-1 has been elusive because of its inherent conformational instability and rapid conversion to a latent, inactive structure. Here we report the crystal structure of PAI-1 W175F at 2.3 Å resolution as the first model of the metastable native molecule. Structural comparison with a quadruple mutant (14-1B) previously used as representative of the active state uncovered key differences. The most striking differences occur near the region that houses three of the four mutations in the 14-1B PAI-1 structure. Prominent changes are localized within a loop connecting β-strand 3A with the F helix, in which a previously observed 3(10)-helix is absent in the new structure. Notably these structural changes are found near the binding site for the cofactor vitronectin. Because vitronectin is the only known physiological regulator of PAI-1 that slows down the latency conversion, the structure of this region is important. Furthermore, the previously identified chloride-binding site close to the F-helix is absent from the present structure and likely to be artifactual, because of its dependence on the 14-1B mutations. Instead we found a different chlorine-binding site that is likely to be present in wild type PAI-1 and that more satisfactorily accounts for the chlorine stabilizing effect on PAI-1.

  11. Crystal Structure of Plasminogen Activator Inhibitor-1 in an Active Conformation with Normal Thermodynamic Stability*

    PubMed Central

    Jensen, Jan K.; Thompson, Lawrence C.; Bucci, Joel C.; Nissen, Poul; Gettins, Peter G. W.; Peterson, Cynthia B.; Andreasen, Peter A.; Morth, J. Preben

    2011-01-01

    The serpin plasminogen activator inhibitor-1 (PAI-1) is a crucial regulator in fibrinolysis and tissue remodeling. PAI-1 has been associated with several pathological conditions and is a validated prognostic marker in human cancers. However, structural information about the native inhibitory form of PAI-1 has been elusive because of its inherent conformational instability and rapid conversion to a latent, inactive structure. Here we report the crystal structure of PAI-1 W175F at 2.3 Å resolution as the first model of the metastable native molecule. Structural comparison with a quadruple mutant (14-1B) previously used as representative of the active state uncovered key differences. The most striking differences occur near the region that houses three of the four mutations in the 14-1B PAI-1 structure. Prominent changes are localized within a loop connecting β-strand 3A with the F helix, in which a previously observed 310-helix is absent in the new structure. Notably these structural changes are found near the binding site for the cofactor vitronectin. Because vitronectin is the only known physiological regulator of PAI-1 that slows down the latency conversion, the structure of this region is important. Furthermore, the previously identified chloride-binding site close to the F-helix is absent from the present structure and likely to be artifactual, because of its dependence on the 14-1B mutations. Instead we found a different chlorine-binding site that is likely to be present in wild type PAI-1 and that more satisfactorily accounts for the chlorine stabilizing effect on PAI-1. PMID:21697084

  12. Formation of Tankyrase Inhibitor-Induced Degradasomes Requires Proteasome Activity

    PubMed Central

    Pedersen, Nina Marie; Thorvaldsen, Tor Espen; Schultz, Sebastian Wolfgang; Wenzel, Eva Maria; Stenmark, Harald

    2016-01-01

    In canonical Wnt signaling, the protein levels of the key signaling mediator β-catenin are under tight regulation by the multimeric destruction complex that mediates proteasomal degradation of β-catenin. In colorectal cancer, destruction complex activity is often compromised due to mutations in the multifunctional scaffolding protein Adenomatous Polyposis Coli (APC), leading to a stabilization of β-catenin. Recently, tankyrase inhibitors (TNKSi), a novel class of small molecule inhibitors, were shown to re-establish a functional destruction complex in APC-mutant cancer cell lines by stabilizing AXIN1/2, whose protein levels are usually kept low via poly(ADP-ribosyl)ation by the tankyrase enzymes (TNKS1/2). Surprisingly, we found that for the formation of the morphological correlates of destruction complexes, called degradasomes, functional proteasomes are required. In addition we found that AXIN2 is strongly upregulated after 6 h of TNKS inhibition. The proteasome inhibitor MG132 counteracted TNKSi-induced degradasome formation and AXIN2 stabilization, and this was accompanied by reduced transcription of AXIN2. Mechanistically we could implicate the transcription factor FoxM1 in this process, which was recently shown to be a transcriptional activator of AXIN2. We observed a substantial reduction in TNKSi-induced stabilization of AXIN2 after siRNA-mediated depletion of FoxM1 and found that proteasome inhibition reduced the active (phosphorylated) fraction of FoxM1. This can explain the decreased protein levels of AXIN2 after MG132 treatment. Our findings have implications for the design of in vitro studies on the destruction complex and for clinical applications of TNKSi. PMID:27482906

  13. Anti-diabetic activity of insulin-degrading enzyme inhibitors mediated by multiple hormones.

    PubMed

    Maianti, Juan Pablo; McFedries, Amanda; Foda, Zachariah H; Kleiner, Ralph E; Du, Xiu Quan; Leissring, Malcolm A; Tang, Wei-Jen; Charron, Maureen J; Seeliger, Markus A; Saghatelian, Alan; Liu, David R

    2014-07-01

    Despite decades of speculation that inhibiting endogenous insulin degradation might treat type-2 diabetes, and the identification of IDE (insulin-degrading enzyme) as a diabetes susceptibility gene, the relationship between the activity of the zinc metalloprotein IDE and glucose homeostasis remains unclear. Although Ide(-/-) mice have elevated insulin levels, they exhibit impaired, rather than improved, glucose tolerance that may arise from compensatory insulin signalling dysfunction. IDE inhibitors that are active in vivo are therefore needed to elucidate IDE's physiological roles and to determine its potential to serve as a target for the treatment of diabetes. Here we report the discovery of a physiologically active IDE inhibitor identified from a DNA-templated macrocycle library. An X-ray structure of the macrocycle bound to IDE reveals that it engages a binding pocket away from the catalytic site, which explains its remarkable selectivity. Treatment of lean and obese mice with this inhibitor shows that IDE regulates the abundance and signalling of glucagon and amylin, in addition to that of insulin. Under physiological conditions that augment insulin and amylin levels, such as oral glucose administration, acute IDE inhibition leads to substantially improved glucose tolerance and slower gastric emptying. These findings demonstrate the feasibility of modulating IDE activity as a new therapeutic strategy to treat type-2 diabetes and expand our understanding of the roles of IDE in glucose and hormone regulation.

  14. Conformation-selective inhibitors reveal differences in the activation and phosphate-binding loops of the tyrosine kinases Abl and Src.

    PubMed

    Hari, Sanjay B; Perera, B Gayani K; Ranjitkar, Pratistha; Seeliger, Markus A; Maly, Dustin J

    2013-12-20

    Over the past decade, an increasingly diverse array of potent and selective inhibitors that target the ATP-binding sites of protein kinases have been developed. Many of these inhibitors, like the clinically approved drug imatinib (Gleevec), stabilize a specific catalytically inactive ATP-binding site conformation of their kinases targets. Imatinib is notable in that it is highly selective for its kinase target, Abl, over other closely related tyrosine kinases, such as Src. In addition, imatinib is highly sensitive to the phosphorylation state of Abl's activation loop, which is believed to be a general characteristic of all inhibitors that stabilize a similar inactive ATP-binding site conformation. In this report, we perform a systematic analysis of a diverse series of ATP-competitive inhibitors that stabilize a similar inactive ATP-binding site conformation as imatinib with the tyrosine kinases Src and Abl. In contrast to imatinib, many of these inhibitors have very similar potencies against Src and Abl. Furthermore, only a subset of this class of inhibitors is sensitive to the phosphorylation state of the activation loop of these kinases. In attempting to explain this observation, we have uncovered an unexpected correlation between Abl's activation loop and another flexible active site feature, called the phosphate-binding loop (p-loop). These studies shed light on how imatinib is able to obtain its high target selectivity and reveal how the conformational preference of flexible active site regions can vary between closely related kinases.

  15. Computer-based design of novel HIV-1 entry inhibitors: neomycin conjugated to arginine peptides at two specific sites.

    PubMed

    Berchanski, Alexander; Lapidot, Aviva

    2009-03-01

    Aminoglycoside-arginine conjugates (AAC and APAC) are multi-target inhibitors of human immunodeficiency virus type-1 (HIV-1). Here, we predict new conjugates of neomycin with two arginine peptide chains binding at specific sites on neomycin [poly-arginine-neomycin-poly-arginine (PA-Neo-PA)]. The rationale for the design of such compounds is to separate two short arginine peptides with neomycin, which may extend the binding region of the CXC chemokine receptor type 4 (CXCR4). We used homology models of CXCR4 and unliganded envelope glycoprotein 120 (HIV-1(IIIB) gp120) and docked PA-Neo-PAs and APACs to these using a multistep docking procedure. The results indicate that PA-Neo-PAs spread over two negatively charged patches of CXCR4. PA-Neo-PA-CXCR4 complexes are energetically more favorable than AACs/APAC-CXCR4 complexes. Notably, our CXCR4 model and docking procedure can be applied to predict new compounds that are either inhibitors of gp120-CXCR4 binding without affecting stromal cell-derived factor 1 alpha (SDF-1 alpha) chemotaxis activity, or inhibitors of SDF-1 alpha-CXCR4 binding resulting in an anti-metastasis effect. We also predict that PA-Neo-PAs and APACs can interfere with CD4-gp120 binding in unliganded conformation.

  16. A novel dipyridodiazepinone inhibitor of HIV-1 reverse transcriptase acts through a nonsubstrate binding site

    SciTech Connect

    Wu, J.C.; Warren, T.C.; Adams, J.; Proudfoot, J.; Skiles, J.; Raghavan, P.; Perry, C.; Potocki, I.; Farina, P.R.; Grob, P.M. )

    1991-02-26

    A novel dipyridodiazepinone, 6,11-dihydro-11-cyclopropyl-4-methyldipyrido(2,3-b:2{prime},3{prime}-e)-(1,4)diazepin-6-one (BI-RG-587), is a selective noncompetitive inhibitor of HIV-1 reverse transcriptase (RT-1). An azido photoaffinity analogue of BI-RG-587 was synthesized and found to irreversibly inhibit the enzyme upon UV irradiation. BI-RG-587 and close structural analogues competitively protected RT-1 from inactivation by the photoaffinity label. A thiobenzimidazolone (TIBO) derivative, a nonnucleoside inhibitor of RT-1, also protected the enzyme from photoinactivation, which suggests a common binding site for these compounds. Substrates dGTP, template-primer, and tRNA afforded no protection from enzyme inactivation. A tritiated photoaffinity probe was found to stoichiometrically and selectively label p66 such that 1 mol of probe inactivates 1 mol of RT-1.

  17. P-glycoprotein efflux and other factors limit brain amyloid beta reduction by beta-site amyloid precursor protein-cleaving enzyme 1 inhibitors in mice.

    PubMed

    Meredith, Jere E; Thompson, Lorin A; Toyn, Jeremy H; Marcin, Lawrence; Barten, Donna M; Marcinkeviciene, Jovita; Kopcho, Lisa; Kim, Young; Lin, Alan; Guss, Valerie; Burton, Catherine; Iben, Lawrence; Polson, Craig; Cantone, Joe; Ford, Michael; Drexler, Dieter; Fiedler, Tracey; Lentz, Kimberley A; Grace, James E; Kolb, Janet; Corsa, Jason; Pierdomenico, Maria; Jones, Kelli; Olson, Richard E; Macor, John E; Albright, Charles F

    2008-08-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disease. Amyloid beta (Abeta) peptides are hypothesized to cause the initiation and progression of AD based on pathologic data from AD patients, genetic analysis of mutations that cause early onset forms of AD, and preclinical studies. Based on this hypothesis, beta-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) inhibitors are an attractive therapeutic approach for AD because cleavage of the APP by BACE1 is required to form Abeta. In this study, three potent BACE1 inhibitors are characterized. All three inhibitors decrease Abeta formation in cultured cells with IC(50) values less than 10 nM. Analysis of APP C-terminal fragments by immunoblotting and Abeta peptides by mass spectrometry showed that these inhibitors decreased Abeta by inhibiting BACE1. An assay for Abeta1-40 in mice was developed and used to show that these BACE1 inhibitors decreased plasma Abeta1-40, but not brain Abeta1-40, in wild-type mice. Because these BACE1 inhibitors were substrates for P-glycoprotein (P-gp), a member of the ATP-binding cassette superfamily of efflux transporters, these inhibitors were administered to P-gp knockout (KO) mice. These studies showed that all three BACE1 inhibitors decreased brain Abeta1-40 in P-gp KO mice, demonstrating that P-gp is a major limitation for development of BACE1 inhibitors to test the amyloid hypothesis. A comparison of plasma Abeta1-40 and brain Abeta1-40 dose responses for these three compounds revealed differences in relative ED(50) values, indicating that factors other than P-gp can also contribute to poor brain activity by BACE1 inhibitors.

  18. Lumican: a new inhibitor of matrix metalloproteinase-14 activity.

    PubMed

    Pietraszek, Katarzyna; Chatron-Colliet, Aurore; Brézillon, Stéphane; Perreau, Corinne; Jakubiak-Augustyn, Anna; Krotkiewski, Hubert; Maquart, François-Xavier; Wegrowski, Yanusz

    2014-11-28

    We previously showed that lumican regulates MMP-14 expression. The aim of this study was to compare the effect of lumican and decorin on MMP-14 activity. In contrast to decorin, the glycosylated form of lumican was able to significantly decrease MMP-14 activity in B16F1 melanoma cells. Our results suggest that a direct interaction occurs between lumican and MMP-14. Lumican behaves as a competitive inhibitor which leads to a complete blocking of the activity of MMP-14. It binds to the catalytic domain of MMP-14 with moderate affinity (KD∼275 nM). Lumican may protect collagen against MMP-14 proteolysis, thus influencing cell-matrix interaction in tumor progression. PMID:25304424

  19. Structure-activity relationships of organofluorine inhibitors of β-amyloid self-assembly.

    PubMed

    Török, Béla; Sood, Abha; Bag, Seema; Kulkarni, Aditya; Borkin, Dmitry; Lawler, Elizabeth; Dasgupta, Sujaya; Landge, Shainaz; Abid, Mohammed; Zhou, Weihong; Foster, Michelle; LeVine, Harry; Török, Marianna

    2012-05-01

    A broad group of structurally diverse small organofluorine compounds were synthesized and evaluated as inhibitors of β-amyloid (Aβ) self-assembly. The main goal was to generate a diverse library of compounds with the same functional group and to observe general structural features that characterize inhibitors of Aβ oligomer and fibril formation, ultimately identifying structures for further focused inhibitor design. The common structural motifs in these compounds are CF(3) -C-OH and CF(3) -C-NH groups that were proposed to be binding units in our previous studies. A broad range of potential small-molecule inhibitors were synthesized by combining various carbocyclic and heteroaromatic rings with an array of substituents, generating a total of 106 molecules. The compounds were tested by standard methods such as thioflavin-T fluorescence spectroscopy for monitoring fibril formation, biotinyl Aβ(1-42) single-site streptavidin-based assays for observing oligomer formation, and atomic force microscopy for morphological studies. These assays revealed a number of structures that show significant inhibition against either Aβ fibril or oligomer formation. A detailed analysis of the structure-activity relationship of anti-fibril and -oligomer properties is provided. These data present further experimental evidence for the distinct nature of fibril versus oligomer formation and indicate that the interaction of the Aβ peptide with chiral small molecules is not stereospecific in nature.

  20. NMR reveals the allosteric opening and closing of Abelson tyrosine kinase by ATP-site and myristoyl pocket inhibitors

    PubMed Central

    Skora, Lukasz; Mestan, Jürgen; Fabbro, Doriano; Jahnke, Wolfgang; Grzesiek, Stephan

    2013-01-01

    Successful treatment of chronic myelogenous leukemia is based on inhibitors binding to the ATP site of the deregulated breakpoint cluster region (Bcr)–Abelson tyrosine kinase (Abl) fusion protein. Recently, a new type of allosteric inhibitors targeting the Abl myristoyl pocket was shown in preclinical studies to overcome ATP-site inhibitor resistance arising in some patients. Using NMR and small-angle X-ray scattering, we have analyzed the solution conformations of apo Abelson tyrosine kinase (c-Abl) and c-Abl complexes with ATP-site and allosteric inhibitors. Binding of the ATP-site inhibitor imatinib leads to an unexpected open conformation of the multidomain SH3-SH2-kinase c-Abl core, whose relevance is confirmed by cellular assays on Bcr-Abl. The combination of imatinib with the allosteric inhibitor GNF-5 restores the closed, inactivated state. Our data provide detailed insights on the poorly understood combined effect of the two inhibitor types, which is able to overcome drug resistance. PMID:24191057

  1. RASA3 is a critical inhibitor of RAP1-dependent platelet activation

    PubMed Central

    Stefanini, Lucia; Paul, David S.; Robledo, Raymond F.; Chan, E. Ricky; Getz, Todd M.; Campbell, Robert A.; Kechele, Daniel O.; Casari, Caterina; Piatt, Raymond; Caron, Kathleen M.; Mackman, Nigel; Weyrich, Andrew S.; Parrott, Matthew C.; Boulaftali, Yacine; Adams, Mark D.; Peters, Luanne L.; Bergmeier, Wolfgang

    2015-01-01

    The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the GEF CalDAG-GEFI and an unknown regulator that operates downstream of the adenosine diphosphate (ADP) receptor, P2Y12, a target of antithrombotic therapy. Here, we provide evidence that the GAP, RASA3, inhibits platelet activation and provides a link between P2Y12 and activation of the RAP1 signaling pathway. In mice, reduced expression of RASA3 led to premature platelet activation and markedly reduced the life span of circulating platelets. The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of the gene encoding CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Moreover, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Together, our results indicate that RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling and suggest that P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation at sites of vascular injury. These findings provide insight into the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders. PMID:25705885

  2. Chronic monoamine oxidase-B inhibitor treatment blocks monoamine oxidase-A enzyme activity.

    PubMed

    Bartl, Jasmin; Müller, Thomas; Grünblatt, Edna; Gerlach, Manfred; Riederer, Peter

    2014-04-01

    Patients with Parkinson's disease receive selective irreversible monoamine oxidase (MAO)-B inhibitors, but their effects on MAO-A activity are not known during long-term application. We determined MAO-A inhibition in plasma samples from patients with MAO-B inhibitor intake or without MAO-B inhibitor treatment and from healthy controls. We detected a 70 % reduction of MAO-A activity in patients with MAO-B inhibitor therapy in comparison to the other groups. Our results suggest that treatment with MAO-B inhibitor may also influence MAO-A activity in vivo, when administered daily.

  3. Shp2 protein tyrosine phosphatase inhibitor activity of estramustine phosphate and its triterpenoid analogs

    PubMed Central

    Scott, Latanya M.; Chen, Liwei; Daniel, Kenyon G.; Brooks, Wesley H.; Guida, Wayne C.; Lawrence, Harshani R.; Sebti, Said M.; Lawrence, Nicholas J.; Wu, Jie

    2010-01-01

    Shp2 protein tyrosine phosphate (PTP) is a novel target for anticancer drug discovery. We identified estramustine phosphate as a Shp2 PTP inhibitor from the National Cancer Institute Approved Oncology Drug set. A focused structure-activity relationship study indicated that the 17- phosphate group is required for the Shp2 PTP inhibitor activity of estramustine phosphate. A search for estramustine phosphate analogs led to identification of two triperpenoids, enoxolone and celastrol, having Shp2 PTP inhibitor activity. With the previously reported PTP1B inhibitor trodusquemine, our study reveals steroids and triterpenoids with negatively charged phosphate, carboxylate, or sulfonate groups as novel pharmacophores of selective PTP inhibitors. PMID:21193311

  4. Identification of Novel Plasmodium falciparum Hexokinase Inhibitors with Antiparasitic Activity.

    PubMed

    Davis, Mindy I; Patrick, Stephen L; Blanding, Walker M; Dwivedi, Varun; Suryadi, Jimmy; Golden, Jennifer E; Coussens, Nathan P; Lee, Olivia W; Shen, Min; Boxer, Matthew B; Hall, Matthew D; Sharlow, Elizabeth R; Drew, Mark E; Morris, James C

    2016-10-01

    Plasmodium falciparum, the deadliest species of malaria parasites, is dependent on glycolysis for the generation of ATP during the pathogenic red blood cell stage. Hexokinase (HK) catalyzes the first step in glycolysis, transferring the γ-phosphoryl group of ATP to glucose to yield glucose-6-phosphate. Here, we describe the validation of a high-throughput assay for screening small-molecule collections to identify inhibitors of the P. falciparum HK (PfHK). The assay, which employed an ADP-Glo reporter system in a 1,536-well-plate format, was robust with a signal-to-background ratio of 3.4 ± 1.2, a coefficient of variation of 6.8% ± 2.9%, and a Z'-factor of 0.75 ± 0.08. Using this assay, we screened 57,654 molecules from multiple small-molecule collections. Confirmed hits were resolved into four clusters on the basis of structural relatedness. Multiple singleton hits were also identified. The most potent inhibitors had 50% inhibitory concentrations as low as ∼1 μM, and several were found to have low-micromolar 50% effective concentrations against asexual intraerythrocytic-stage P. falciparum parasites. These molecules additionally demonstrated limited toxicity against a panel of mammalian cells. The identification of PfHK inhibitors with antiparasitic activity using this validated screening assay is encouraging, as it justifies additional HTS campaigns with more structurally amenable libraries for the identification of potential leads for future therapeutic development. PMID:27458230

  5. Chitinase inhibitors: extraction of the active framework from natural argifin and use of in situ click chemistry.

    PubMed

    Hirose, Tomoyasu; Sunazuka, Toshiaki; Sugawara, Akihiro; Endo, Ayako; Iguchi, Kanami; Yamamoto, Tsuyoshi; Ui, Hideaki; Shiomi, Kazuro; Watanabe, Takeshi; Sharpless, K Barry; Omura, Satoshi

    2009-05-01

    In situ click chemistry is a target-guided synthesis technique for discovering potent protein ligands by assembling azides and alkynes into triazoles inside the affinity site of a target protein. We report the rapid discovery of a new and potent inhibitor of bacterial chitinases by the use of in situ click chemistry. We observed a target-templated formation of a potent triazole inhibitor of the chitinase-catalyzed chitin hydrolysis, through in situ click chemistry between a biologically active azide-containing scaffold and structurally unrelated alkyne fragments. Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides and antiasthmatics. Argifin, which has been isolated and characterized as a cyclopentapeptide natural product by our research group, shows strong inhibitory activity against chitinases. As a result of our efforts at developing a chitinase inhibitor from an azide-bearing argifin fragment and the application of the chitinase template and a library of alkynes, we rapidly obtained a very potent and new 1,5-disubstituted triazole inhibitor against Serratia marcescens chitinase (SmChi) B. The new inhibitor expressed 300-fold increase in the inhibitory activity against SmChiB compared with that of argifin. To the best of our knowledge, our finding of an enzyme-made 1,5-disubstituted triazole, using in situ click chemistry is the second example reported in the literature.

  6. Binding site residues control inhibitor selectivity in the human norepinephrine transporter but not in the human dopamine transporter

    PubMed Central

    Andersen, Jacob; Ringsted, Kristoffer B.; Bang-Andersen, Benny; Strømgaard, Kristian; Kristensen, Anders S.

    2015-01-01

    The transporters for norepinephrine and dopamine (NET and DAT, respectively) constitute the molecular targets for recreational drugs and therapeutics used in the treatment of psychiatric disorders. Despite a strikingly similar amino acid sequence and predicted topology between these transporters, some inhibitors display a high degree of selectivity between NET and DAT. Here, a systematic mutational analysis of non-conserved residues within the extracellular entry pathway and the high affinity binding site in NET and DAT was performed to examine their role for selective inhibitor recognition. Changing the six diverging residues in the central binding site of NET to the complementary residues in DAT transferred a DAT-like pharmacology to NET, showing that non-conserved binding site residues in NET are critical determinants for inhibitor selectivity. In contrast, changing the equivalent residues in the central site of DAT to the corresponding residues in NET had modest effects on the same inhibitors, suggesting that non-conserved binding site residues in DAT play a minor role for selective inhibitor recognition. Our data points towards distinct structural determinants governing inhibitor selectivity in NET and DAT, and provide important new insight into the molecular basis for NET/DAT selectivity of therapeutic and recreational drugs. PMID:26503701

  7. Characterization of inhibitors acting at the synthetase site of Escherichia coli asparagine synthetase B.

    PubMed

    Boehlein, S K; Nakatsu, T; Hiratake, J; Thirumoorthy, R; Stewart, J D; Richards, N G; Schuster, S M

    2001-09-18

    Asparagine synthetase catalyzes the ATP-dependent formation of L-asparagine from L-aspartate and L-glutamine, via a beta-aspartyl-AMP intermediate. Since interfering with this enzyme activity might be useful for treating leukemia and solid tumors, we have sought small-molecule inhibitors of Escherichia coli asparagine synthetase B (AS-B) as a model system for the human enzyme. Prior work showed that L-cysteine sulfinic acid competitively inhibits this enzyme by interfering with L-aspartate binding. Here, we demonstrate that cysteine sulfinic acid is also a partial substrate for E. coli asparagine synthetase, acting as a nucleophile to form the sulfur analogue of beta-aspartyl-AMP, which is subsequently hydrolyzed back to cysteine sulfinic acid and AMP in a futile cycle. While cysteine sulfinic acid did not itself constitute a clinically useful inhibitor of asparagine synthetase B, these results suggested that replacing this linkage by a more stable analogue might lead to a more potent inhibitor. A sulfoximine reported recently by Koizumi et al. as a competitive inhibitor of the ammonia-dependent E. coli asparagine synthetase A (AS-A) [Koizumi, M., Hiratake, J., Nakatsu, T., Kato, H., and Oda, J. (1999) J. Am. Chem. Soc. 121, 5799-5800] can be regarded as such a species. We found that this sulfoximine also inhibited AS-B, effectively irreversibly. Unlike either the cysteine sulfinic acid interaction with AS-B or the sulfoximine interaction with AS-A, only AS-B productively engaged in asparagine synthesis could be inactivated by the sulfoximine; free enzyme was unaffected even after extended incubation with the sulfoximine. Taken together, these results support the notion that sulfur-containing analogues of aspartate can serve as platforms for developing useful inhibitors of AS-B. PMID:11551215

  8. Design, synthesis and molecular docking of α,β-unsaturated cyclohexanone analogous of curcumin as potent EGFR inhibitors with antiproliferative activity.

    PubMed

    Xu, Yun-Yun; Cao, Yi; Ma, Hailkuo; Li, Huan-Qiu; Ao, Gui-Zhen

    2013-01-15

    A type of novel α,β-unsaturated cyclohexanone analogous, which designed based on the curcumin core structure, have been discovered as potential EGFR inhibitors. These compounds exhibit potent antiproliferative activity in two human tumor cell lines (Hep G2 and B16-F10). Among them, compounds I(3) and I(12) displayed the most potent EGFR inhibitory activity (IC(50) = 0.43 μM and 1.54 μM, respectively). Molecular docking of I(12) into EGFR TK active site was also performed. This inhibitor nicely fitting the active site might well explain its excellent inhibitory activity.

  9. Extravascular fibrin, plasminogen activator, plasminogen activator inhibitors, and airway hyperresponsiveness

    PubMed Central

    Wagers, Scott S.; Norton, Ryan J.; Rinaldi, Lisa M.; Bates, Jason H.T.; Sobel, Burton E.; Irvin, Charles G.

    2004-01-01

    Mechanisms underlying airway hyperresponsiveness are not yet fully elucidated. One of the manifestations of airway inflammation is leakage of diverse plasma proteins into the airway lumen. They include fibrinogen and thrombin. Thrombin cleaves fibrinogen to form fibrin, a major component of thrombi. Fibrin inactivates surfactant. Surfactant on the airway surface maintains airway patency by lowering surface tension. In this study, immunohistochemically detected fibrin was seen along the luminal surface of distal airways in a patient who died of status asthmaticus and in mice with induced allergic airway inflammation. In addition, we observed altered airway fibrinolytic system protein balance consistent with promotion of fibrin deposition in mice with allergic airway inflammation. The airways of mice were exposed to aerosolized fibrinogen, thrombin, or to fibrinogen followed by thrombin. Only fibrinogen followed by thrombin resulted in airway hyperresponsiveness compared with controls. An aerosolized fibrinolytic agent, tissue-type plasminogen activator, significantly diminished airway hyperresponsiveness in mice with allergic airway inflammation. These results are consistent with the hypothesis that leakage of fibrinogen and thrombin and their accumulation on the airway surface can contribute to the pathogenesis of airway hyperresponsiveness. PMID:15232617

  10. Protein inhibitor of activated STAT3 inhibits adipogenic gene expression

    SciTech Connect

    Deng Jianbei; Hua Kunjie; Caveney, Erica J.; Takahashi, Nobuyuki; Harp, Joyce B. . E-mail: jharp@unc.edu

    2006-01-20

    Protein inhibitor of activated STAT3 (PIAS3), a cytokine-induced repressor of signal transducer and activator of transcription 3 (STAT3) and a modulator of a broad array of nuclear proteins, is expressed in white adipose tissue, but its role in adipogenesis is not known. Here, we determined that PIAS3 was constitutively expressed in 3T3-L1 cells at all stages of adipogenesis. However, it translocated from the nucleus to the cytoplasm 4 days after induction of differentiation by isobutylmethylxanthine, dexamethasone, and insulin (MDI). In ob/ob mice, PIAS3 expression was increased in white adipose tissue depots compared to lean mice and was found in the cytoplasm of adipocytes. Overexpression of PIAS3 in differentiating preadipocytes, which localized primarily to the nucleus, inhibited mRNA level gene expression of adipogenic transcription factors C/EBP{alpha} and PPAR{gamma}, as well as their downstream target genes aP2 and adiponectin. PIAS3 also inhibited C/EBP{alpha} promoter activation mediated specifically by insulin, but not dexamethasone or isobutylmethylxanthine. Taken together, these data suggest that PIAS3 may play an inhibitory role in adipogenesis by modulating insulin-activated transcriptional activation events. Increased PIAS3 expression in adipose tissue may play a role in the metabolic disturbances of obesity.

  11. Inhibition of proteolytic activity of poliovirus and rhinovirus 2A proteinases by elastase-specific inhibitors.

    PubMed Central

    Molla, A; Hellen, C U; Wimmer, E

    1993-01-01

    A polyprotein cleavage assay has been developed to assay the proteolytic activities in vitro of the 2A proteinases encoded by poliovirus and human rhinovirus 14, which are representative members of the Enterovirus and Rhinovirus genera of picornaviruses, respectively. The elastase-specific substrate-based inhibitors elastatinal and methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (MPCMK) inhibited both 2A proteinases in vitro. The electrophoretic mobilities of both 2A proteinases were reduced upon incubation with elastatinal, whereas the mobility of a Cys-109-->Ala poliovirus 2Apro mutant was unchanged, an observation suggesting that this inhibitor may have formed a covalent bond with the active-site Cys-109 nucleophile. Iodoacetamide, calpain inhibitor 1, and antipain inhibited poliovirus 2Apro. MPCMK caused a reduction in the yields of the enteroviruses poliovirus type 1 and coxsackievirus A21 and of human rhinovirus 2 in infected HeLa cells but did not affect the growth of encephalomyocarditis virus, a picornavirus of the Cardiovirus genus. MPCMK abrogated the shutoff of host cell protein synthesis that is induced by enterovirus and rhinovirus infection and reduced the synthesis of virus-encoded polypeptides in infected cells. These results indicate that the determinants of substrate recognition by 2A proteinases resemble those of pancreatic and leukocyte elastases. These results may be relevant to the development of broad-range chemotherapeutic agents against entero- and rhinoviruses. Images PMID:8392608

  12. Grassypeptolides As Natural Inhibitors of Dipeptidyl Peptidase 8 and T-Cell Activation

    PubMed Central

    Kwan, Jason C.; Liu, Yanxia; Ratnayake, Ranjala; Hatano, Ryo; Kuribara, Akiko; Morimoto, Chiko; Ohnuma, Kei; Paul, Valerie J.; Ye, Tao

    2014-01-01

    Natural products made by marine cyanobacteria are often highly modified peptides and depsipeptides that have the potential to act as inhibitors for proteases. In the interest of finding novel protease inhibition activity and selectivity grassypeptolide A (1) was screened against a panel of proteases and found to selectively inhibit DPP8 over DPP4. Grassypeptolides were also found to inhibit IL-2 production and proliferation in activated T-cells, consistent with a putative role of DPP8 in the immune system. These effects were also observed in Jurkat cells, and DPP activity in Jurkat cell cytosol was shown to be inhibited by grassypeptolides. In silico docking suggests two possible binding modes of grassypeptolides – both at the active site of DPP8 and at one of the entrances to the internal cavity. Collectively these results suggest that grassypeptolides may be useful tool compounds in the study of DPP8 function. PMID:24591193

  13. Unexpected Activity of a Novel Kunitz-type Inhibitor: INHIBITION OF CYSTEINE PROTEASES BUT NOT SERINE PROTEASES.

    PubMed

    Smith, David; Tikhonova, Irina G; Jewhurst, Heather L; Drysdale, Orla C; Dvořák, Jan; Robinson, Mark W; Cwiklinski, Krystyna; Dalton, John P

    2016-09-01

    Kunitz-type (KT) protease inhibitors are low molecular weight proteins classically defined as serine protease inhibitors. We identified a novel secreted KT inhibitor associated with the gut and parenchymal tissues of the infective juvenile stage of Fasciola hepatica, a helminth parasite of medical and veterinary importance. Unexpectedly, recombinant KT inhibitor (rFhKT1) exhibited no inhibitory activity toward serine proteases but was a potent inhibitor of the major secreted cathepsin L cysteine proteases of F. hepatica, FhCL1 and FhCL2, and of human cathepsins L and K (Ki = 0.4-27 nm). FhKT1 prevented the auto-catalytic activation of FhCL1 and FhCL2 and formed stable complexes with the mature enzymes. Pulldown experiments from adult parasite culture medium showed that rFhKT1 interacts specifically with native secreted FhCL1, FhCL2, and FhCL5. Substitution of the unusual P1 Leu(15) within the exposed reactive loop of FhKT1 for the more commonly found Arg (FhKT1Leu(15)/Arg(15)) had modest adverse effects on the cysteine protease inhibition but conferred potent activity against the serine protease trypsin (Ki = 1.5 nm). Computational docking and sequence analysis provided hypotheses for the exclusive binding of FhKT1 to cysteine proteases, the importance of the Leu(15) in anchoring the inhibitor into the S2 active site pocket, and the inhibitor's selectivity toward FhCL1, FhCL2, and human cathepsins L and K. FhKT1 represents a novel evolutionary adaptation of KT protease inhibitors by F. hepatica, with its prime purpose likely in the regulation of the major parasite-secreted proteases and/or cathepsin L-like proteases of its host.

  14. Creating Novel Activated Factor XI Inhibitors through Fragment Based Lead Generation and Structure Aided Drug Design

    PubMed Central

    Fjellström, Ola; Akkaya, Sibel; Beisel, Hans-Georg; Eriksson, Per-Olof; Erixon, Karl; Gustafsson, David; Jurva, Ulrik; Kang, Daiwu; Karis, David; Knecht, Wolfgang; Nerme, Viveca; Nilsson, Ingemar; Olsson, Thomas; Redzic, Alma; Roth, Robert; Sandmark, Jenny; Tigerström, Anna; Öster, Linda

    2015-01-01

    Activated factor XI (FXIa) inhibitors are anticipated to combine anticoagulant and profibrinolytic effects with a low bleeding risk. This motivated a structure aided fragment based lead generation campaign to create novel FXIa inhibitor leads. A virtual screen, based on docking experiments, was performed to generate a FXIa targeted fragment library for an NMR screen that resulted in the identification of fragments binding in the FXIa S1 binding pocket. The neutral 6-chloro-3,4-dihydro-1H-quinolin-2-one and the weakly basic quinolin-2-amine structures are novel FXIa P1 fragments. The expansion of these fragments towards the FXIa prime side binding sites was aided by solving the X-ray structures of reported FXIa inhibitors that we found to bind in the S1-S1’-S2’ FXIa binding pockets. Combining the X-ray structure information from the identified S1 binding 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment and the S1-S1’-S2’ binding reference compounds enabled structure guided linking and expansion work to achieve one of the most potent and selective FXIa inhibitors reported to date, compound 13, with a FXIa IC50 of 1.0 nM. The hydrophilicity and large polar surface area of the potent S1-S1’-S2’ binding FXIa inhibitors compromised permeability. Initial work to expand the 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment towards the prime side to yield molecules with less hydrophilicity shows promise to afford potent, selective and orally bioavailable compounds. PMID:25629509

  15. Quantitative High-Throughput Screening Identifies 8-Hydroxyquinolines as Cell-Active Histone Demethylase Inhibitors

    PubMed Central

    Kawamura, Akane; Rose, Nathan R.; Ng, Stanley S.; Quinn, Amy M.; Rai, Ganesha; Mott, Bryan T.; Beswick, Paul; Klose, Robert J.; Oppermann, Udo; Jadhav, Ajit; Heightman, Tom D.; Maloney, David J.; Schofield, Christopher J.; Simeonov, Anton

    2010-01-01

    Background Small molecule modulators of epigenetic processes are currently sought as basic probes for biochemical mechanisms, and as starting points for development of therapeutic agents. Nε-Methylation of lysine residues on histone tails is one of a number of post-translational modifications that together enable transcriptional regulation. Histone lysine demethylases antagonize the action of histone methyltransferases in a site- and methylation state-specific manner. Nε-Methyllysine demethylases that use 2-oxoglutarate as co-factor are associated with diverse human diseases, including cancer, inflammation and X-linked mental retardation; they are proposed as targets for the therapeutic modulation of transcription. There are few reports on the identification of templates that are amenable to development as potent inhibitors in vivo and large diverse collections have yet to be exploited for the discovery of demethylase inhibitors. Principal Findings High-throughput screening of a ∼236,000-member collection of diverse molecules arrayed as dilution series was used to identify inhibitors of the JMJD2 (KDM4) family of 2-oxoglutarate-dependent histone demethylases. Initial screening hits were prioritized by a combination of cheminformatics, counterscreening using a coupled assay enzyme, and orthogonal confirmatory detection of inhibition by mass spectrometric assays. Follow-up studies were carried out on one of the series identified, 8-hydroxyquinolines, which were shown by crystallographic analyses to inhibit by binding to the active site Fe(II) and to modulate demethylation at the H3K9 locus in a cell-based assay. Conclusions These studies demonstrate that diverse compound screening can yield novel inhibitors of 2OG dependent histone demethylases and provide starting points for the development of potent and selective agents to interrogate epigenetic regulation. PMID:21124847

  16. Discovery of Cyclic Acylguanidines as Highly Potent and Selective β-Site Amyloid Cleaving Enzyme (BACE) Inhibitors: Part I-Inhibitor Design and Validation

    SciTech Connect

    Zhu, Zhaoning; Sun, Zhong-Yue; Ye, Yuanzan; Voigt, Johannes; Strickland, Corey; Smith, Elizabeth M; Cumming, Jared; Wang, Lingyan; Wong, Jesse; Wang, Yu-Sen; Wyss, Daniel F; Chen, Xia; Kuvelkar, Reshma; Kennedy, Matthew E; Favreau, Leonard; Parker, Eric; McKittrick, Brian A; Stamford, Andrew; Czarniecki, Michael; Greenlee, William; Hunter, John C

    2010-10-18

    A number of novel amidine containing heterocycles were designed to reproduce the unique interaction pattern, revealed by X-ray crystallography, between the BACE-1 catalytic diad and a weak NMR screening hit (3), with special attention paid to maintaining the appropriate basicity and limiting the number of H-bonding donors of these scaffolds. The iminohydantoin cores (10 and 23) were examined first and found to interact with the catalytic diad in one of two binding modes (A and B), each with the iminohydantoin core flipped 180º in relation to the other. The amidine structural motif within each core forms a bidentate interaction with a different aspartic acid of the catalytic diad. Both modes reproduced a highly conserved interaction pattern between the inhibitors and the catalytic aspartates, as revealed by 3. Potent iminohydantoin BACE-1 inhibitors have been obtained, validating the molecular design as aspartyl protease catalytic site inhibitors. Brain penetrant small molecule BACE inhibitors with high ligand efficiencies have been discovered, enabling multiple strategies for further development of these inhibitors into highly potent, selective and in vivo efficacious BACE inhibitors.

  17. Discovery of Cyclic Acylguanidines as Highly Potent and Selective β-Site Amyloid Cleaving Enzyme (BACE) Inhibitors: Part I–Inhibitor Design and Validation

    SciTech Connect

    Zhu, Zhaoning; Sun, Zhong-Yue; Ye, Yuanzan; Voigt, Johannes; Strickland, Corey; Smith, Elizabeth M.; Cumming, Jared; Wang, Lingyan; Wong, Jesse; Wang, Yu-Sen; Wyss, Daniel F.; Chen, Xia; Kuvelkar, Reshma; Kennedy, Matthew E.; Favreau, Leonard; Parker, Eric; McKittrick, Brian A.; Stamford, Andrew; Czarniecki, Michael; Greenlee, William; Hunter, John C.

    2013-11-20

    A number of novel amidine containing heterocycles were designed to reproduce the unique interaction pattern, revealed by X-ray crystallography, between the BACE-1 catalytic diad and a weak NMR screening hit, with special attention paid to maintaining the appropriate basicity and limiting the number of H-bonding donors of these scaffolds. The iminohydantoin cores were examined first and found to interact with the catalytic diad in one of two binding modes (A and B), each with the iminohydantoin core flipped 180° in relation to the other. The amidine structural motif within each core forms a bidentate interaction with a different aspartic acid of the catalytic diad. Both modes reproduced a highly conserved interaction pattern between the inhibitors and the catalytic aspartates. Potent iminohydantoin BACE-1 inhibitors have been obtained, validating the molecular design as aspartyl protease catalytic site inhibitors. Brain penetrant small molecule BACE inhibitors with high ligand efficiencies have been discovered, enabling multiple strategies for further development of these inhibitors into highly potent, selective and in vivo efficacious BACE inhibitors.

  18. Effects of protein phosphatase and kinase inhibitors on the cardiac L- type Ca current suggest two sites are phosphorylated by protein kinase A and another protein kinase

    PubMed Central

    1995-01-01

    We previously showed (Frace, A.M. and H.C. Hartzell. 1993. Journal of Physiology. 472:305-326) that internal perfusion of frog atrial myocytes with the nonselective protein phosphatase inhibitors microcystin or okadaic acid produced an increase in the L-type Ca current (ICa) and a decrease in the delayed rectifier K current (IK). We hypothesized that microcystin revealed the activity of a protein kinase (PKX) that was basally active in the cardiac myocyte that could phosphorylate the Ca and K channels or regulators of the channels. The present studies were aimed at determining the nature of PKX and its phosphorylation target. The effect of internal perfusion with microcystin on ICa or IK was not attenuated by inhibitors of protein kinase A (PKA). However, the effect of microcystin on ICa was largely blocked by the nonselective protein kinase inhibitors staurosporine (10- 30 nM), K252a (250 nM), and H-7 (10 microM). Staurosporine and H-7 also decreased the stimulation of ICa by isoproterenol, but K252a was more selective and blocked the ability of microcystin to stimulate ICa without significantly reducing isoproterenol-stimulated current. Internal perfusion with selective inhibitors of protein kinase C (PKC), including the autoinhibitory pseudosubstrate PKC peptide (PKC(19-31)) and a myristoylated derivative of this peptide had no effect. External application of several PKC inhibitors had negative side effects that prevented their use as selective PKC inhibitors. Nevertheless, we conclude that PKX is not PKC. PKA and PKX phosphorylate sites with different sensitivities to the phosphatase inhibitors calyculin A and microcystin. In contrast to the results with ICa, the effect of microcystin on IK was not blocked by any of the kinase inhibitors tested, suggesting that the effect of microcystin on IK may not be mediated by a protein kinase but may be due to a direct effect of microcystin on the IK channel. PMID:8786340

  19. Potentiation of C1 inhibitor by glycosaminoglycans: dextran sulfate species are effective inhibitors of in vitro complement activation in plasma.

    PubMed

    Wuillemin, W A; te Velthuis, H; Lubbers, Y T; de Ruig, C P; Eldering, E; Hack, C E

    1997-08-15

    Activation of the complement system may contribute to the pathogenesis of many diseases. Hence, an effective inhibitor of complement might be useful to reduce tissue damage. Some glycosaminoglycans (GAG), such as heparin, are known to inhibit the interaction of C1q with activators and the assembly of the classical and the alternative pathway C3 convertases. Furthermore, they may potentiate C1 inhibitor-mediated inactivation of C1s. To search for potential complement inhibitors, we systematically investigated the complement inhibitory properties of various synthetic and naturally occurring GAG (dextran sulfates 500,000 and 5,000, heparin, N-acetylheparin, heparan sulfate, dermatan sulfate, and chondroitin sulfates A and C). First, we assessed the effect of GAG on the second-order rate constant of the inactivation of C1s by C1 inhibitor. This rate constant increased 6- to 130-fold in the presence of the GAG, dextran sulfate being the most effective. Second, all tested GAG were found to reduce deposition of C4 and C3 on immobilized aggregated human IgG (AHG) and to reduce fluid phase formation of C4b/c and C3b/c in recalcified plasma upon incubation with AHG. Dextran sulfate again was found to be most effective. We conclude that GAG modulate complement activation in vitro and that the low molecular weight dextran sulfate (m.w. 5000) may be a candidate for pharmacologic manipulation of complement activation via potentiation of C1 inhibitor.

  20. Carbohydrate scaffolds as glycosyltransferase inhibitors with in vivo antibacterial activity.

    PubMed

    Zuegg, Johannes; Muldoon, Craig; Adamson, George; McKeveney, Declan; Le Thanh, Giang; Premraj, Rajaratnam; Becker, Bernd; Cheng, Mu; Elliott, Alysha G; Huang, Johnny X; Butler, Mark S; Bajaj, Megha; Seifert, Joachim; Singh, Latika; Galley, Nicola F; Roper, David I; Lloyd, Adrian J; Dowson, Christopher G; Cheng, Ting-Jen; Cheng, Wei-Chieh; Demon, Dieter; Meyer, Evelyne; Meutermans, Wim; Cooper, Matthew A

    2015-01-01

    The rapid rise of multi-drug-resistant bacteria is a global healthcare crisis, and new antibiotics are urgently required, especially those with modes of action that have low-resistance potential. One promising lead is the liposaccharide antibiotic moenomycin that inhibits bacterial glycosyltransferases, which are essential for peptidoglycan polymerization, while displaying a low rate of resistance. Unfortunately, the lipophilicity of moenomycin leads to unfavourable pharmacokinetic properties that render it unsuitable for systemic administration. In this study, we show that using moenomycin and other glycosyltransferase inhibitors as templates, we were able to synthesize compound libraries based on novel pyranose scaffold chemistry, with moenomycin-like activity, but with improved drug-like properties. The novel compounds exhibit in vitro inhibition comparable to moenomycin, with low toxicity and good efficacy in several in vivo models of infection. This approach based on non-planar carbohydrate scaffolds provides a new opportunity to develop new antibiotics with low propensity for resistance induction. PMID:26194781

  1. Carbohydrate scaffolds as glycosyltransferase inhibitors with in vivo antibacterial activity

    PubMed Central

    Zuegg, Johannes; Muldoon, Craig; Adamson, George; McKeveney, Declan; Le Thanh, Giang; Premraj, Rajaratnam; Becker, Bernd; Cheng, Mu; Elliott, Alysha G.; Huang, Johnny X.; Butler, Mark S.; Bajaj, Megha; Seifert, Joachim; Singh, Latika; Galley, Nicola F.; Roper, David I.; Lloyd, Adrian J.; Dowson, Christopher G.; Cheng, Ting-Jen; Cheng, Wei-Chieh; Demon, Dieter; Meyer, Evelyne; Meutermans, Wim; Cooper, Matthew A.

    2015-01-01

    The rapid rise of multi-drug-resistant bacteria is a global healthcare crisis, and new antibiotics are urgently required, especially those with modes of action that have low-resistance potential. One promising lead is the liposaccharide antibiotic moenomycin that inhibits bacterial glycosyltransferases, which are essential for peptidoglycan polymerization, while displaying a low rate of resistance. Unfortunately, the lipophilicity of moenomycin leads to unfavourable pharmacokinetic properties that render it unsuitable for systemic administration. In this study, we show that using moenomycin and other glycosyltransferase inhibitors as templates, we were able to synthesize compound libraries based on novel pyranose scaffold chemistry, with moenomycin-like activity, but with improved drug-like properties. The novel compounds exhibit in vitro inhibition comparable to moenomycin, with low toxicity and good efficacy in several in vivo models of infection. This approach based on non-planar carbohydrate scaffolds provides a new opportunity to develop new antibiotics with low propensity for resistance induction. PMID:26194781

  2. Isolation and characterization of a protease inhibitor from Acacia karroo with a common combining loop and overlapping binding sites for chymotrypsin and trypsin.

    PubMed

    Patthy, András; Molnár, Tamás; Porrogi, Pálma; Naudé, Ryno; Gráf, László

    2015-01-01

    By using affinity and reversed-phase HPLC (RP-HPLC) chromatographies two chymotrypsin-trypsin inhibitors were isolated from seeds of Acacia karroo, a legume of the subfamily Mimosoideae. The primary structure of one of these inhibitors, named AkCI/1, was determined. The inhibitor consists of two polypeptide chains, 139 and 44 residues respectively, which are linked by a single disulfide bridge. The amino acid sequence of AkCI/1 is homologous to and showed more than 60% sequence similarity with other protease inhibitors isolated earlier from the group of Mimosoideae. AkCI/1 inhibits both chymotrypsin (EC 3.4.21.1) and trypsin (EC 3.4.21.4) in a 1:1M ratio with Ki values of 2.8 × 10(-12)M and 1.87 × 10(-12)M, respectively. The P1-P1' residues for trypsin were identified as Arg68-Ile69 by selective hydrolysis of the inhibitor at this site, with bovine trypsin and human trypsin IV. The cleavage did not affect the inhibition of trypsin, but fully abolished the chymotrypsin inhibitory activity of AkCI/1. This finding together with our studies on competition of the two enzymes for the same combining loop suggests that the same loop has to contain the binding sites for both proteases. The most likely P1 residue of AkCI/1 for chymotrypsin is Tyr67. PMID:25447841

  3. Isolation and characterization of a protease inhibitor from Acacia karroo with a common combining loop and overlapping binding sites for chymotrypsin and trypsin.

    PubMed

    Patthy, András; Molnár, Tamás; Porrogi, Pálma; Naudé, Ryno; Gráf, László

    2015-01-01

    By using affinity and reversed-phase HPLC (RP-HPLC) chromatographies two chymotrypsin-trypsin inhibitors were isolated from seeds of Acacia karroo, a legume of the subfamily Mimosoideae. The primary structure of one of these inhibitors, named AkCI/1, was determined. The inhibitor consists of two polypeptide chains, 139 and 44 residues respectively, which are linked by a single disulfide bridge. The amino acid sequence of AkCI/1 is homologous to and showed more than 60% sequence similarity with other protease inhibitors isolated earlier from the group of Mimosoideae. AkCI/1 inhibits both chymotrypsin (EC 3.4.21.1) and trypsin (EC 3.4.21.4) in a 1:1M ratio with Ki values of 2.8 × 10(-12)M and 1.87 × 10(-12)M, respectively. The P1-P1' residues for trypsin were identified as Arg68-Ile69 by selective hydrolysis of the inhibitor at this site, with bovine trypsin and human trypsin IV. The cleavage did not affect the inhibition of trypsin, but fully abolished the chymotrypsin inhibitory activity of AkCI/1. This finding together with our studies on competition of the two enzymes for the same combining loop suggests that the same loop has to contain the binding sites for both proteases. The most likely P1 residue of AkCI/1 for chymotrypsin is Tyr67.

  4. Plasminogen activator inhibitor-1 antisense oligodeoxynucleotides abrogate mesangial fibronectin accumulation.

    PubMed

    Park, Jehyun; Seo, Ji Yeon; Ha, Hunjoo

    2010-12-01

    Excessive extracellular matrix (ECM) accumulation is the main feature of chronic renal disease including diabetic nephropathy. Plasminogen activator inhibitor (PAI)-1 is known to play an important role in renal ECM accumulation in part through suppression of plasmin generation and matrix metalloproteinase (MMP) activation. The present study examined the effect of PAI-1 antisense oligodeoxynucleotide (ODN) on fibronectin upregulation and plasmin/MMP suppression in primary mesangial cells cultured under high glucose (HG) or transforming growth factor (TGF)-β1, major mediators of diabetic renal ECM accumulation. Growth arrested and synchronized rat primary mesangial cells were transfected with 1 µM phosphorothioate-modified antisense or control mis-match ODN for 24 hours with cationic liposome and then stimulated with 30 mM D-glucose or 2 ng/ml TGF-β1. PAI-1 or fibronectin protein was measured by Western blot analysis. Plasmin activity was determined using a synthetic fluorometric plasmin substrate and MMP-2 activity analyzed using zymography. HG and TGF-β1 significantly increased PAI-1 and fibronectin protein expression as well as decreased plasmin and MMP-2 activity. Transient transfection of mesangial cells with PAI-1 antisense ODN, but not mis-match ODN, effectively reversed basal as well as HG- and TGF-β1-induced suppression of plasmin and MMP-2 activity. Both basal and upregulated fibronectin secretion were also inhibited by PAI-1 antisense ODN. These data confirm that PAI-1 plays an important role in ECM accumulation in diabetic mesangium through suppression of protease activity and suggest that PAI-1 antisense ODN would be an effective therapeutic strategy for prevention of renal fibrosis including diabetic nephropathy.

  5. Lethal Factor Active-Site Mutations Affect Catalytic Activity In Vitro

    PubMed Central

    Hammond, S. E.; Hanna, P. C.

    1998-01-01

    The lethal factor (LF) protein of Bacillus anthracis lethal toxin contains the thermolysin-like active-site and zinc-binding consensus motif HEXXH (K. R. Klimpel, N. Arora, and S. H. Leppla, Mol. Microbiol. 13:1093–1100, 1994). LF is hypothesized to act as a Zn2+ metalloprotease in the cytoplasm of macrophages, but no proteolytic activities have been previously shown on any target substrate. Here, synthetic peptides are hydrolyzed by LF in vitro. Mass spectroscopy and peptide sequencing of isolated cleavage products separated by reverse-phase high-pressure liquid chromatography indicate that LF seems to prefer proline-containing substrates. Substitution mutations within the consensus active-site residues completely abolish all in vitro catalytic functions, as does addition of 1,10-phenanthroline, EDTA, and certain amino acid hydroxamates, including the novel zinc metalloprotease inhibitor ZINCOV. In contrast, the protease inhibitors bestatin and lysine CMK, previously shown to block LF activity on macrophages, did not block LF activity in vitro. These data provide the first direct evidence that LF may act as an endopeptidase. PMID:9573135

  6. Activating PTEN by COX-2 inhibitors antagonizes radiation-induced AKT activation contributing to radiosensitization.

    PubMed

    Meng, Zhen; Gan, Ye-Hua

    2015-05-01

    Radiotherapy is still one of the most effective nonsurgical treatments for many tumors. However, radioresistance remains a major impediment to radiotherapy. Although COX-2 inhibitors can induce radiosensitization, the underlying mechanism is not fully understood. In this study, we showed that COX-2 selective inhibitor celecoxib enhanced the radiation-induced inhibition of cell proliferation and apoptosis in HeLa and SACC-83 cells. Treatment with celecoxib alone dephosphorylated phosphatase and tensin homolog deleted on chromosome ten (PTEN), promoted PTEN membrane translocation or activation, and correspondingly dephosphorylated or inactivated protein kinase B (AKT). By contrast, treatment with radiation alone increased PTEN phosphorylation, inhibited PTEN membrane translocation and correspondingly activated AKT in the two cell lines. However, treatment with celecoxib or another COX-2 selective inhibitor (valdecoxib) completely blocked radiation-induced increase of PTEN phosphorylation, rescued radiation-induced decrease in PTEN membrane translocation, and correspondingly inactivated AKT. Moreover, celecoxib could also upregulate PTEN protein expression by downregulating Sp1 expression, thereby leading to the activation of PTEN transcription. Our results suggested that COX-2 inhibitors could enhance radiosensitization at least partially by activating PTEN to antagonize radiation-induced AKT activation.

  7. Benzimidazole-Based Quinazolines: In Vitro Evaluation, Quantitative Structure-Activity Relationship, and Molecular Modeling as Aurora Kinase Inhibitors.

    PubMed

    Sharma, Alka; Luxami, Vijay; Saxena, Sanjai; Paul, Kamaldeep

    2016-03-01

    A series of benzimidazole-based quinazoline derivatives with different substitutions of primary and secondary amines at the C2 position (1-12) were evaluated for their Aurora kinase inhibitory activities. All compounds except for 3 and 6 showed good activity against Aurora kinase inhibitors, with IC50 values in the range of 0.035-0.532 μM. The ligand efficiency (LE) of the compounds with Aurora A kinase was also determined. The structure-activity relationship and the quantitative structure-activity relationship revealed that the Aurora inhibitory activities of these derivatives primarily depend on the different substitutions of the amine present at the C2 position of the quinazoline core. Molecular docking studies in the active binding site also provided theoretical support for the experimental biological data acquired. The current study identifies a novel class of Aurora kinase inhibitors, which can further be used for the treatment of cancer.

  8. Turing patterns in network-organized activator-inhibitor systems

    NASA Astrophysics Data System (ADS)

    Nakao, Hiroya; Mikhailov, Alexander S.

    2010-07-01

    Turing instability in activator-inhibitor systems provides a paradigm of non-equilibrium self-organization; it has been extensively investigated for biological and chemical processes. Turing instability should also be possible in networks, and general mathematical methods for its treatment have been formulated previously. However, only examples of regular lattices and small networks were explicitly considered. Here we study Turing patterns in large random networks, which reveal striking differences from the classical behaviour. The initial linear instability leads to spontaneous differentiation of the network nodes into activator-rich and activator-poor groups. The emerging Turing patterns become furthermore strongly reshaped at the subsequent nonlinear stage. Multiple coexisting stationary states and hysteresis effects are observed. This peculiar behaviour can be understood in the framework of a mean-field theory. Our results offer a new perspective on self-organization phenomena in systems organized as complex networks. Potential applications include ecological metapopulations, synthetic ecosystems, cellular networks of early biological morphogenesis, and networks of coupled chemical nanoreactors.

  9. Discovery of novel STAT3 small molecule inhibitors via in silico site-directed fragment-based drug design.

    PubMed

    Yu, Wenying; Xiao, Hui; Lin, Jiayuh; Li, Chenglong

    2013-06-13

    Constitutive activation of signal transducer and activator of transcription 3 (STAT3) has been validated as an attractive therapeutic target for cancer therapy. To stop both STAT3 activation and dimerization, a viable strategy is to design inhibitors blocking its SH2 domain phosphotyrosine binding site that is responsible for both actions. A new fragment-based drug design (FBDD) strategy, in silico site-directed FBDD, was applied in this study. A designed novel compound, 5,8-dioxo-6-(pyridin-3-ylamino)-5,8-dihydronaphthalene-1-sulfonamide (LY5), was confirmed to bind to STAT3 SH2 by fluorescence polarization assay. In addition, four out of the five chosen compounds have IC50 values lower than 5 μM for the U2OS cancer cells. 8 (LY5) has an IC50 range in 0.5-1.4 μM in various cancer cell lines. 8 also suppresses tumor growth in an in vivo mouse model. This study has demonstrated the utility of this approach and could be used to other drug targets in general. PMID:23651330

  10. Novel quinolinonyl diketo acid derivatives as HIV-1 integrase inhibitors: design, synthesis, and biological activities.

    PubMed

    Di Santo, Roberto; Costi, Roberta; Roux, Alessandra; Miele, Gaetano; Crucitti, Giuliana Cuzzucoli; Iacovo, Alberto; Rosi, Federica; Lavecchia, Antonio; Marinelli, Luciana; Di Giovanni, Carmen; Novellino, Ettore; Palmisano, Lucia; Andreotti, Mauro; Amici, Roberta; Galluzzo, Clementina Maria; Nencioni, Lucia; Palamara, Anna Teresa; Pommier, Yves; Marchand, Christophe

    2008-08-14

    Novel quinolinonyl diketo acids were designed to obtain integrase (IN) inhibitors selectively active against the strand transfer (ST) step of the HIV integration process. Those new compounds are characterized by a single aryl diketo acid (DKA) chain in comparison to 4, a bifunctional diketo acid reported by our group as an anti-IN agent highly potent against both the 3'-processing and ST steps. Compound 6d was the most potent derivative in IN enzyme assays, while 6i showed the highest potency against HIV-1 in acutely infected cells. The selective inhibition of ST suggested the newly designed monofunctional DKAs bind the IN-DNA acceptor site without affecting the DNA donor site.

  11. Nonpeptidic HIV protease inhibitors possessing excellent antiviral activities and therapeutic indices. PD 178390: a lead HIV protease inhibitor.

    PubMed

    Prasad, J V; Boyer, F E; Domagala, J M; Ellsworth, E L; Gajda, C; Hamilton, H W; Hagen, S E; Markoski, L J; Steinbaugh, B A; Tait, B D; Humblet, C; Lunney, E A; Pavlovsky, A; Rubin, J R; Ferguson, D; Graham, N; Holler, T; Hupe, D; Nouhan, C; Tummino, P J; Urumov, A; Zeikus, E; Zeikus, G; Gracheck, S J; Erickson, J W

    1999-12-01

    With the insight generated by the availability of X-ray crystal structures of various 5,6-dihydropyran-2-ones bound to HIV PR, inhibitors possessing various alkyl groups at the 6-position of 5,6-dihydropyran-2-one ring were synthesized. The inhibitors possessing a 6-alkyl group exhibited superior antiviral activities when compared to 6-phenyl analogues. Antiviral efficacies were further improved upon introduction of a polar group (hydroxyl or amino) on the 4-position of the phenethyl moiety as well as the polar group (hydroxymethyl) on the 3-(tert-butyl-5-methyl-phenylthio) moiety. The polar substitution is also advantageous for decreasing toxicity, providing inhibitors with higher therapeutic indices. The best inhibitor among this series, (S)-6-[2-(4-aminophenyl)-ethyl]-(3-(2-tert-butyl-5-methyl-phenylsulfa nyl)-4-hydroxy-6-isopropyl-5,6-dihydro-pyran-2-one (34S), exhibited an EC50 of 200 nM with a therapeutic index of > 1000. More importantly, these non-peptidic inhibitors, 16S and 34S, appear to offer little cross-resistance to the currently marketed peptidomimetic PR inhibitors. The selected inhibitors tested in vitro against mutant HIV PR showed a very small increase in binding affinities relative to wild-type HIV PR. Cmax and absolute bioavailability of 34S were higher and half-life and time above EC95 were longer compared to 16S. Thus 34S, also known as PD 178390, which displays good antiviral efficacy, promising pharmacokinetic characteristics and favorable activity against mutant enzymes and CYP3A4, has been chosen for further preclinical evaluation.

  12. MAPK pathway activation leads to Bim loss and histone deacetylase inhibitor resistance: rationale to combine romidepsin with an MEK inhibitor.

    PubMed

    Chakraborty, Arup R; Robey, Robert W; Luchenko, Victoria L; Zhan, Zhirong; Piekarz, Richard L; Gillet, Jean-Pierre; Kossenkov, Andrew V; Wilkerson, Julia; Showe, Louise C; Gottesman, Michael M; Collie, Nathan L; Bates, Susan E

    2013-05-16

    To identify molecular determinants of histone deacetylase inhibitor (HDI) resistance, we selected HuT78 cutaneous T-cell lymphoma (CTCL) cells with romidepsin in the presence of P-glycoprotein inhibitors to prevent transporter upregulation. Resistant sublines were 250- to 385-fold resistant to romidepsin and were resistant to apoptosis induced by apicidin, entinostat, panobinostat, belinostat, and vorinostat. A custom TaqMan array identified increased insulin receptor (INSR) gene expression; immunoblot analysis confirmed increased protein expression and a four- to eightfold increase in mitogen-activated protein kinase (MAPK) kinase (MEK) phosphorylation in resistant cells compared with parental cells. Resistant cells were exquisitely sensitive to MEK inhibitors, and apoptosis correlated with restoration of proapoptotic Bim. Romidepsin combined with MEK inhibitors yielded greater apoptosis in cells expressing mutant KRAS compared with romidepsin treatment alone. Gene expression analysis of samples obtained from patients with CTCL enrolled on the NCI1312 phase 2 study of romidepsin in T-cell lymphoma suggested perturbation of the MAPK pathway by romidepsin. Immunohistochemical analysis of Bim expression demonstrated decreased expression in some skin biopsies at disease progression. These findings implicate increased activation of MEK and decreased Bim expression as a resistance mechanism to HDIs, supporting combination of romidepsin with MEK inhibitors in clinical trials.

  13. Structure-activity relationship of cyanine tau aggregation inhibitors

    PubMed Central

    Chang, Edward; Congdon, Erin E.; Honson, Nicolette S.; Duff, Karen E.; Kuret, Jeff

    2009-01-01

    A structure-activity relationship for symmetrical cyanine inhibitors of human tau aggregation was elaborated using a filter trap assay. Antagonist activity depended on cyanine heterocycle, polymethine bridge length, and the nature of meso- and N-substituents. One potent member of the series, 3,3’-diethyl-9-methylthiacarbocyanine iodide (compound 11), retained submicromolar potency and had calculated physical properties consistent with blood-brain barrier and cell membrane penetration. Exposure of organotypic slices prepared from JNPL3 transgenic mice (which express human tau harboring the aggregation prone P301L tauopathy mutation) to compound 11 for one week revealed a biphasic dose response relationship. Low nanomolar concentrations decreased insoluble tau aggregates to half those observed in slices treated with vehicle alone. In contrast, high concentrations (≥300 nM) augmented tau aggregation and produced abnormalities in tissue tubulin levels. These data suggest that certain symmetrical carbocyanine dyes can modulate tau aggregation in the slice biological model at concentrations well below those associated with toxicity. PMID:19432420

  14. Direct inhibitors of InhA active against Mycobacterium tuberculosis

    PubMed Central

    Manjunatha, Ujjini H.; Rao, Srinivasa P. S.; Kondreddi, Ravinder Reddy; Noble, Christian G.; Camacho, Luis R.; Tan, Bee H.; Ng, Seow H.; Ng, Pearly Shuyi; Ma, N. L.; Lakshminarayana, Suresh B.; Herve, Maxime; Barnes, S. Whitney; Yu, Weixuan; Kuhen, Kelli; Blasco, Francesca; Beer, David; Walker, John R.; Tonge, Peter J.; Glynne, Richard; Smith, Paul W.; Diagana, Thierry T.

    2015-01-01

    New chemotherapeutic agents are urgently required to combat the global spread of multi-drug resistant tuberculosis (MDR-TB). The mycobacterial enoyl reductase, InhA, is one of the few clinically-validated targets in tuberculosis drug discovery. Here, we report the identification of a new class of direct InhA inhibitors, the 4-hydroxy-2-pyridones, using phenotypic high-throughput whole-cell screening. This class of orally-active compounds showed potent bactericidal activity against common isoniazid-resistant TB clinical isolates. Biophysical studies revealed that 4-hydroxy-2-pyridones bound specifically to InhA in an NADH-dependent manner and blocked the enoyl-substrate binding pocket. The lead compound NITD-916 directly blocked InhA in a dose-dependent manner and showed in vivo efficacy in acute and established mouse models of infection by Mycobacterium tuberculosis. Collectively, our structural and biochemical data open up new avenues for rational structure-guided optimization of the 4-hydroxy-2-pyridone class of compounds for the treatment of MDR-TB. PMID:25568071

  15. Relationships between plasma insulin triglyceride, body mass index, and plasminogen activator inhibitor 1.

    PubMed

    Juhan-Vague, I; Vague, P; Alessi, M C; Badier, C; Valadier, J; Aillaud, M F; Atlan, C

    1987-07-01

    Low fibrinolytic activity, as measured by euglobulin (EFA), has been observed in obese subjects, and hypofibrinolysis may play a role in the pathogenesis of atherosclerosis and its complications. Blood fibrinolytic activity is regulated through a complex system of activators and inhibitors, especially plasminogen activator inhibitors (PA Inhibitors). In a group of 35 non-diabetic subjects with a wide range of body mass index (BMI), EFA was negatively correlated, and PA Inhibitor activity positively correlated, with BMI and plasma insulin levels. In a population of 49 non-diabetic obese women (differing from a control group of normal weight by lower EFA and higher level, of PA Inhibitor activity, plasma insulin and triglyceride), the PA Inhibitor activity was positively correlated with BMI, insulin and triglyceride. The increase in PA Inhibitor activity was associated with a high value of PA Inhibitor 1 antigen measured by an immuno-radiometric assay, indicating that the increased activity was due to a high level of circulating PA Inhibitor 1. Plasma insulin was lowered in obese non-diabetic subjects, without modification of the body weight, by a 24 hour fast or by treatment with Metformin. After 24 hours' fast, ten obese subjects had lower levels of insulin and PA Inhibitor activity and an increase in EFA. Treatment for 15 days by 1.75 g Metformin (or placebo), on a weight maintaining diet, induced, in the Metformin group, a decrease in plasma insulin, triglyceride and PA Inhibitor activity and an increase in EFA, while no change was observed in the placebo group.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. Active Sites Environmental Monitoring Program: Action levels

    SciTech Connect

    Ashwood, J.S.; Ashwood, T.L.

    1991-10-01

    The Active Sites Environmental Monitoring Program (ASEMP) was established at Oak Ridge National Laboratory to provide for early leak detection and to monitor performance of the active low-level waste disposal facilities in Solid Waste Storage Area (SWSA) 6 and the transuranic waste storage areas in SWSA 5 North. Early leak detection is accomplished by sampling runoff, groundwater, and perched water in burial trenches. Sample results are compared to action levels that represent background contamination by naturally occurring and fallout-derived radionuclides. 15 refs., 3 figs., 12 tabs.

  17. Virtual Screening and Optimization Yield Low-Nanomolar Inhibitors of the Tautomerase Activity of Plasmodium falciparum Macrophage Migration Inhibitory Factor

    PubMed Central

    Dahlgren, Markus K.; Garcia, Alvaro Baeza; Hare, Alissa A.; Tirado-Rives, Julian; Leng, Lin; Bucala, Richard; Jorgensen, William L.

    2012-01-01

    The Plasmodium falciparum ortholog of the human cytokine, macrophage migratory inhibitory factor (PfMIF), is produced by the parasite during malaria infection and modulates the host’s immune response. As for other MIF orthologs, PfMIF has tautomerase activity, whose inhibition may influence the cytokine activity. To identify small-molecule inhibitors of the tautomerase activity of PfMIF, virtual screening has been performed by docking 2.1 million compounds into the enzymatic site. Assaying of 17 compounds identified four as active. Substructure search for the most potent of these compounds, a 4-phenoxypyridine analogue, identified four additional compounds that were purchased and also shown to be active. Thirty-one additional analogues were then designed, synthesized, and assayed. Three were found to be potent PfMIF tautomerase inhibitors with Ki values of ca. 40 nM; they are also highly selective with Ki values of >100 μM for human MIF. PMID:23067344

  18. Accounting for target flexibility and water molecules by docking to ensembles of target structures: the HCV NS5B palm site I inhibitors case study.

    PubMed

    Barreca, Maria Letizia; Iraci, Nunzio; Manfroni, Giuseppe; Gaetani, Rosy; Guercini, Chiara; Sabatini, Stefano; Tabarrini, Oriana; Cecchetti, Violetta

    2014-02-24

    The introduction of new anti-HCV drugs in therapy is an imperative need and is necessary with a view to develop an interferon-free therapy. Thus, the discovery and development of novel small molecule inhibitors of the viral NS5B polymerase represent an exciting area of research for many pharmaceutical companies and academic groups. This study represents a contribution to this field and relies on the identification of the best NS5B model(s) to be used in structure-based computational approaches aimed at identifying novel non-nucleoside inhibitors of one of the protein allosteric sites, namely, palm site I. First, the NS5B inhibitors at palm site I were classified as water-mediated or nonwater-mediated ligands depending on their ability to interact with or displace a specific water molecule. Then, we took advantage of the available X-ray structures of the NS5B/ligand complexes to build different models of protein/water combinations, which were used to investigate the influence on docking studies of solvent sites as well as of the influence of the protein conformations. As the overall trend, we observed improved performance in the docking results of the water-mediated inhibitors by inclusion of explicit water molecules, with an opposite behavior generally happening for the nonwater-mediated inhibitors. The best performing target structures for the two ligand sets were then used for virtual screening simulations of a library containing the known NS5B inhibitors along with related decoys to assess the best performing targets ensembles on the basis of their ability to discriminate active and inactive compounds as well as to generate the correct binding modes. The parallel use of different protein structures/water sets outperformed the use of a single target structure, with the two-protein 3H98/2W-2FVC/7W and 3HKY/NoW-3SKE/NoW models resulting in the best performing ensembles for water-mediated inhibitors and nonwater-mediated inhibitors, respectively. The information

  19. Preclinical Profile of BI 224436, a Novel HIV-1 Non-Catalytic-Site Integrase Inhibitor

    PubMed Central

    Amad, Ma'an; Bailey, Murray D.; Bethell, Richard; Bös, Michael; Bonneau, Pierre; Cordingley, Michael; Coulombe, René; Duan, Jianmin; Edwards, Paul; Faucher, Anne-Marie; Garneau, Michel; Jakalian, Araz; Kawai, Stephen; Lamorte, Louie; LaPlante, Steven; Luo, Laibin; Mason, Steve; Poupart, Marc-André; Rioux, Nathalie; Schroeder, Patricia; Simoneau, Bruno; Tremblay, Sonia; Tsantrizos, Youla; Witvrouw, Myriam; Yoakim, Christiane

    2014-01-01

    BI 224436 is an HIV-1 integrase inhibitor with effective antiviral activity that acts through a mechanism that is distinct from that of integrase strand transfer inhibitors (INSTIs). This 3-quinolineacetic acid derivative series was identified using an enzymatic integrase long terminal repeat (LTR) DNA 3′-processing assay. A combination of medicinal chemistry, parallel synthesis, and structure-guided drug design led to the identification of BI 224436 as a candidate for preclinical profiling. It has antiviral 50% effective concentrations (EC50s) of <15 nM against different HIV-1 laboratory strains and cellular cytotoxicity of >90 μM. BI 224436 also has a low, ∼2.1-fold decrease in antiviral potency in the presence of 50% human serum and, by virtue of a steep dose-response curve slope, exhibits serum-shifted EC95 values ranging between 22 and 75 nM. Passage of virus in the presence of inhibitor selected for either A128T, A128N, or L102F primary resistance substitutions, all mapping to a conserved allosteric pocket on the catalytic core of integrase. BI 224436 also retains full antiviral activity against recombinant viruses encoding INSTI resistance substitutions N155S, Q148H, and E92Q. In drug combination studies performed in cellular antiviral assays, BI 224436 displays an additive effect in combination with most approved antiretrovirals, including INSTIs. BI 224436 has drug-like in vitro absorption, distribution, metabolism, and excretion (ADME) properties, including Caco-2 cell permeability, solubility, and low cytochrome P450 inhibition. It exhibited excellent pharmacokinetic profiles in rat (clearance as a percentage of hepatic flow [CL], 0.7%; bioavailability [F], 54%), monkey (CL, 23%; F, 82%), and dog (CL, 8%; F, 81%). Based on the excellent biological and pharmacokinetic profile, BI 224436 was advanced into phase 1 clinical trials. PMID:24663024

  20. Design and synthesis of conformationally restricted inhibitors of active thrombin activatable fibrinolysis inhibitor (TAFIa).

    PubMed

    Brink, Mikael; Dahlén, Anders; Olsson, Thomas; Polla, Magnus; Svensson, Tor

    2014-04-01

    A series of 4,5,6,7-tetrahydro-1H-benzimidazole-5-carboxylic acid and 5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-7-carboxylic acid derivatives designed as inhibitors of TAFIa has been prepared via a common hydrogenation-alkylation sequence starting from the appropriate benzimidazole and imidazopyridine system. We present a successful design strategy using a conformational restriction approach resulting in potent and selective inhibitors of TAFIa. The X-ray structure of compound 5 in complex with a H333Y/H335Q double mutant TAFI indicate that the conformational restriction is responsible for the observed potency increase. PMID:24588961

  1. Control of DNA replication in a transformed lymphoid cell line: coexistence of activator and inhibitor activities.

    PubMed

    Coffman, F D; Fresa, K L; Oglesby, I; Cohen, S

    1991-12-01

    Proliferating lymphocytes contain an intracellular factor, ADR (activator of DNA replication), which can initiate DNA synthesis in isolated quiescent nuclei. Resting lymphocytes lack ADR activity and contain an intracellular inhibitory factor that suppresses DNA synthesis in normal but not transformed nuclei. In this study we describe a MOLT-4 subline that produces both the activator and inhibitory activities which can be separated by ammonium sulfate fractionation. The inhibitor is heat stable and inhibits ADR-mediated DNA replication in a dose-dependent manner. It does not inhibit DNA polymerase alpha activity. The inhibitor must be present at the initiation of DNA replication to be effective, as it loses most of its effectiveness if it is added after replication has begun. The presence of inhibitory activity in proliferating MOLT-4 cells, taken with the previous observation that inhibitor derived from normal resting cells does not affect DNA synthesis by MOLT-4 nuclei, suggests that failure of a down-regulating signal may play an important role in proliferative disorder. PMID:1934078

  2. The tyrosine kinase inhibitor nilotinib selectively inhibits CYP2C8 activities in human liver microsomes.

    PubMed

    Kim, Min-Jung; Lee, Jae-Won; Oh, Kyung-Suk; Choi, Chang-Soo; Kim, Kwang Hee; Han, Won Seok; Yoon, Chang-No; Chung, Eun Sook; Kim, Dong-Hyun; Shin, Jae-Gook

    2013-01-01

    The tyrosine kinase inhibitor nilotinib was examined for its inhibition of cytochrome P450s (CYPs) in human liver microsomes and in human CYPs expressed in a baculovirus-insect cell system. Nilotinib demonstrated preferential inhibition of CYP2C8-mediated paclitaxel 6α-hydroxylation, rosiglitazone hydroxylation and amodiaquine N-deethylation in human liver microsomes, with IC₅₀ values of 0.4, 7.5 and 0.7 µM, respectively. The IC₅₀ value of nilotinib for paclitaxel 6α-hydroxylation was 20-fold lower than that of the other five tyrosine-kinase inhibitors tested. Nilotinib appears to display competitive inhibition against paclitaxel 6α-hydroxylation and amodiaquine N-deethylation, with estimated mean Ki values of 0.90 and 0.15 µM in human liver microsomes and 0.10 and 0.61 µM in recombinant human CYP2C8, respectively. These results are consistent with those of molecular docking simulations, where paclitaxel could not access the CYP2C8 catalytic site in the presence of nilotinib, but the binding of midazolam, a substrate of CYP3A4, to the catalytic site of CYP3A4 was not affected by nilotinib. The demonstrated inhibitory activity of nilotinib against CYP2C8 at concentrations less than those observed in patients who received nilotinib therapy is of potential clinical relevance and further in vivo exploration is warranted.

  3. Characterization of active sites in zeolite catalysts

    SciTech Connect

    Eckert, J.; Bug, A.; Nicol, J.M.

    1997-11-01

    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). Atomic-level details of the interaction of adsorbed molecules with active sites in catalysts are urgently needed to facilitate development of more effective and/or environmentally benign catalysts. To this end the authors have carried out neutron scattering studies combined with theoretical calculations of the dynamics of small molecules inside the cavities of zeolite catalysts. The authors have developed the use of H{sub 2} as a probe of adsorption sites by observing the hindered rotations of the adsorbed H{sub 2} molecule, and they were able to show that an area near the four-rings is the most likely adsorption site for H{sub 2} in zeolite A while adsorption of H{sub 2} near cations located on six-ring sites decreases in strength as Ni {approximately} Co > Ca > Zn {approximately} Na. Vibrational and rotational motions of ethylene and cyclopropane adsorption complexes were used as a measure for zeolite-adsorbate interactions. Preliminary studies of the binding of water, ammonia, and methylamines were carried out in a number of related guest-host materials.

  4. Angiotensin-Converting Enzyme Inhibitors and Active Tuberculosis

    PubMed Central

    Wu, Jiunn-Yih; Lee, Meng-Tse Gabriel; Lee, Si-Huei; Lee, Shih-Hao; Tsai, Yi-Wen; Hsu, Shou-Chien; Chang, Shy-Shin; Lee, Chien-Chang

    2016-01-01

    Abstract Numerous epidemiological data suggest that the use of angiotensin-converting enzyme inhibitors (ACEis) can improve the clinical outcomes of pneumonia. Tuberculosis (TB) is an airborne bacteria like pneumonia, and we aimed to find out whether the use of ACEis can decrease the risk of active TB. We conducted a nested case–control analysis by using a 1 million longitudinally followed cohort, from Taiwan national health insurance research database. The rate ratios (RRs) for TB were estimated by conditional logistic regression, and adjusted using a TB-specific disease risk score (DRS) with 71 TB-related covariates. From January, 1997 to December, 2011, a total of 75,536 users of ACEis, and 7720 cases of new active TB were identified. Current use (DRS adjusted RR, 0.87 [95% CI, 0.78–0.97]), but not recent and past use of ACEis, was associated with a decrease in risk of active TB. Interestingly, it was found that chronic use (>90 days) of ACEis was associated with a further decrease in the risk of TB (aRR, 0.74, [95% CI, 0.66–0.83]). There was also a duration response effect, correlating decrease in TB risk with longer duration of ACEis use. The decrease in TB risk was also consistent across all patient subgroups (age, sex, heart failure, cerebrovascular diseases, myocardial infraction, renal diseases, and diabetes) and patients receiving other cardiovascular medicine. In this large population-based study, we found that subjects with recent and chronic use of ACEis were associated with decrease in TB risk. PMID:27175655

  5. Oximes: inhibitors of human recombinant acetylcholinesterase. A structure-activity relationship (SAR) study.

    PubMed

    Sepsova, Vendula; Karasova, Jana Zdarova; Korabecny, Jan; Dolezal, Rafael; Zemek, Filip; Bennion, Brian J; Kuca, Kamil

    2013-08-16

    Acetylcholinesterase (AChE) reactivators were developed for the treatment of organophosphate intoxication. Standard care involves the use of anticonvulsants (e.g., diazepam), parasympatolytics (e.g., atropine) and oximes that restore AChE activity. However, oximes also bind to the active site of AChE, simultaneously acting as reversible inhibitors. The goal of the present study is to determine how oxime structure influences the inhibition of human recombinant AChE (hrAChE). Therefore, 24 structurally different oximes were tested and the results compared to the previous eel AChE (EeAChE) experiments. Structural factors that were tested included the number of pyridinium rings, the length and structural features of the linker, and the number and position of the oxime group on the pyridinium ring.

  6. The regulatory region of the human plasminogen activator inhibitor type-1 (PAI-1) gene.

    PubMed Central

    Riccio, A; Lund, L R; Sartorio, R; Lania, A; Andreasen, P A; Danø, K; Blasi, F

    1988-01-01

    The human gene for plasminogen activator inhibitor type-1 (PAI-1) has been isolated and its promoter region characterized. PAI-1 regulation by glucocorticoids, transforming growth factor-beta (TGF-beta) and the phorbol ester PMA is shown to be exerted at the promoter level. A fragment spanning 805 nucleotides of the 5' flanking and 72 of the 5' untranslated region contain information enough to promote transcription and to respond to glucocorticoids when fused to a reporter gene and transfected into human fibrosarcoma cells. A moderately repetitive DNA sequence, containing a TATA box, a GRE consensus, a Z-DNA forming sequence and two imperfect direct repeats at the extremities, is present a few nucleotides 5' of the human PAI-1 gene transcription start site, raising the possibility that this gene could have been activated by DNA insertion during evolution. Images PMID:3130610

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

    SciTech Connect

    Krisnamoorthi, R.; Yuxi Gong; Chanlan Sun Lin ); 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 {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.

  8. Role of plasminogen activator inhibitor-1 in senescence and aging.

    PubMed

    Eren, Mesut; Boe, Amanda E; Klyachko, Ekaterina A; Vaughan, Douglas E

    2014-09-01

    The average age of the US population continues to increase. Age is the most important determinant of disease and disability in humans, but the fundamental mechanisms of aging remain largely unknown. Many age-related diseases are associated with an impaired fibrinolytic system. Elevated plasminogen activator inhibitor-1 (PAI-1) levels are reported in age-associated clinical conditions including cardiovascular diseases, type 2 diabetes, obesity and inflammation. PAI-1 levels are also elevated in animal models of aging. While the association of PAI-1 with physiological aging is well documented, it is only recently that its critical role in the regulation of aging and senescence has become evident. PAI-1 is synthesized and secreted in senescent cells and contributes directly to the development of senescence by acting downstream of p53 and upstream of insulin-like growth factor binding protein-3. Pharmacologic inhibition or genetic deficiency of PAI-1 was shown to be protective against senescence and the aging-like phenotypes in kl/kl and N(ω)-nitro-l-arginine methyl ester-treated wild-type mice. Further investigation into PAI-1's role in senescence and aging will likely contribute to the prevention and treatment of aging-related pathologies.

  9. Effect of wine inhibitors on free pineapple stem bromelain activity in a model wine system.

    PubMed

    Esti, Marco; Benucci, Ilaria; Liburdi, Katia; Garzillo, Anna Maria Vittoria

    2011-04-13

    The influence of potential inhibitors, naturally present in wine, on the activity of stem bromelain was investigated in order to evaluate the applicability of this enzyme for protein stabilization in white wine. Bromelain proteolytic activity was tested against a synthetic substrate (Bz-Phe-Val-Arg-pNA) in a model wine system after adding ethanol, sulfur dioxide (SO(2)), skin, seed, and gallic and ellagic tannins at the average range of their concentration in wine. All the inhibitors of stem bromelain activity tested turned out to be reversible. Ethanol was a competitive inhibitor with a rather limited effect. Gallic and ellagic tannins have no inhibitory effect on stem bromelain activity, while both seed and skin tannins were uncompetitive inhibitors. The strongest inhibition effect was revealed for sulfur dioxide, which was a mixed-type inhibitor for the enzyme activity. This study provides useful information relative to a future biotechnological application of stem bromelain in winemaking.

  10. Synergistic Activity of Combined NS5A Inhibitors

    PubMed Central

    Nower, Peter T.; Gao, Min; Fridell, Robert; Wang, Chunfu; Hewawasam, Piyasena; Lopez, Omar; Tu, Yong; Meanwell, Nicholas A.; Belema, Makonen; Roberts, Susan B.; Cockett, Mark; Sun, Jin-Hua

    2015-01-01

    Daclatasvir (DCV) is a first-in-class hepatitis C virus (HCV) nonstructural 5A replication complex inhibitor (NS5A RCI) that is clinically effective in interferon-free combinations with direct-acting antivirals (DAAs) targeting alternate HCV proteins. Recently, we reported NS5A RCI combinations that enhance HCV inhibitory potential in vitro, defining a new class of HCV inhibitors termed NS5A synergists (J. Sun, D. R. O’Boyle II, R. A. Fridell, D. R. Langley, C. Wang, S. Roberts, P. Nower, B. M. Johnson F. Moulin, M. J. Nophsker, Y. Wang, M. Liu, K. Rigat, Y. Tu, P. Hewawasam, J. Kadow, N. A. Meanwell, M. Cockett, J. A. Lemm, M. Kramer, M. Belema, and M. Gao, Nature 527:245–248, 2015, doi:10.1038/nature15711). To extend the characterization of NS5A synergists, we tested new combinations of DCV and NS5A synergists against genotype (gt) 1 to 6 replicons and gt 1a, 2a, and 3a viruses. The kinetics of inhibition in HCV-infected cells treated with DCV, an NS5A synergist (NS5A-Syn), or a combination of DCV and NS5A-Syn were distinctive. Similar to activity observed clinically, DCV caused a multilog drop in HCV, followed by rebound due to the emergence of resistance. DCV–NS5A-Syn combinations were highly efficient at clearing cells of viruses, in line with the trend seen in replicon studies. The retreatment of resistant viruses that emerged using DCV monotherapy with DCV–NS5A-Syn resulted in a multilog drop and rebound in HCV similar to the initial decline and rebound observed with DCV alone on wild-type (WT) virus. A triple combination of DCV, NS5A-Syn, and a DAA targeting the NS3 or NS5B protein cleared the cells of viruses that are highly resistant to DCV. Our data support the observation that the cooperative interaction of DCV and NS5A-Syn potentiates both the genotype coverage and resistance barrier of DCV, offering an additional DAA option for combination therapy and tools for explorations of NS5A function. PMID:26711745

  11. Inhibitors of urokinase type plasminogen activator and cytostatic activity from crude plants extracts.

    PubMed

    Zha, Xueqiang; Diaz, Ricardo; Franco, Jose Javier Rosado; Sanchez, Veronica Forbes; Fasoli, Ezio; Barletta, Gabriel; Carvajal, Augusto; Bansal, Vibha

    2013-01-01

    In view of the clear evidence that urokinase type plasminogen activator (uPA) plays an important role in the processes of tumor cell metastasis, aortic aneurysm, and multiple sclerosis, it has become a target of choice for pharmacological intervention. The goal of this study was thus to determine the presence of inhibitors of uPA in plants known traditionally for their anti-tumor properties. Crude methanol extracts were prepared from the leaves of plants (14) collected from the subtropical dry forest (Guanica, Puerto Rico), and tested for the presence of inhibitors of uPA using the fibrin plate assay. The extracts that tested positive (6) were then partitioned with petroleum ether, chloroform, ethyl acetate and n-butanol, in a sequential manner. The resulting fractions were then tested again using the fibrin plate assay. Extracts from leaves of Croton lucidus (C. lucidus) showed the presence of a strong uPA inhibitory activity. Serial dilutions of these C. lucidus partitions were performed to determine the uPA inhibition IC₅₀ values. The chloroform extract showed the lowest IC₅₀ value (3.52 µg/mL) and hence contained the most potent uPA inhibitor. Further investigations revealed that the crude methanol extract and its chloroform and n-butanol partitions did not significantly inhibit closely related proteases such as the tissue type plasminogen activator (tPA) and plasmin, indicating their selectivity for uPA, and hence superior potential for medicinal use with fewer side effects. In a further evaluation of their therapeutic potential for prevention of cancer metastasis, the C. lucidus extracts displayed cytostatic activity against human pancreatic carcinoma (PaCa-2) cells, as determined through an MTS assay. The cytostatic activities recorded for each of the partitions correlated with their relative uPA inhibitory activities. There are no existing reports of uPA inhibitors being present in any of the plants reported in this study.

  12. Inhibitors of Urokinase Type Plasminogen Activator and Cytostatic Activity from Crude Plants Extracts

    PubMed Central

    Zha, Xueqiang; Diaz, Ricardo; Franco, Jose Javier Rosado; Sanchez, Veronica Forbes; Fasoli, Ezio; Barletta, Gabriel; Carvajal, Augusto; Bansal, Vibha

    2014-01-01

    In view of the clear evidence that urokinase type plasminogen activator (uPA) plays an important role in the processes of tumor cell metastasis, aortic aneurysm, and multiple sclerosis, it has become a target of choice for pharmacological intervention. The goal of this study was thus to determine the presence of inhibitors of uPA in plants known traditionally for their anti-tumor properties. Crude methanol extracts were prepared from the leaves of plants (14) collected from the subtropical dry forest (Guanica, Puerto Rico), and tested for the presence of inhibitors of uPA using the fibrin plate assay. The extracts that tested positive (6) were then partitioned with petroleum ether, chloroform, ethyl acetate and n-butanol, in a sequential manner. The resulting fractions were then tested again using the fibrin plate assay. Extracts from leaves of Croton lucidus (C. lucidus) showed the presence of a strong uPA inhibitory activity. Serial dilutions of these C. lucidus partitions were performed to determine the uPA inhibition IC50 values. The chloroform extract showed the lowest IC50 value (3.52 μg/mL) and hence contained the most potent uPA inhibitor. Further investigations revealed that the crude methanol extract and its chloroform and n-butanol partitions did not significantly inhibit closely related proteases such as the tissue type plasminogen activator (tPA) and plasmin, indicating their selectivity for uPA, and hence superior potential for medicinal use with fewer side effects. In a further evaluation of their therapeutic potential for prevention of cancer metastasis, the C. lucidus extracts displayed cytostatic activity against human pancreatic carcinoma (PaCa-2) cells, as determined through an MTS assay. The cytostatic activities recorded for each of the partitions correlated with their relative uPA inhibitory activities. There are no existing reports of uPA inhibitors being present in any of the plants reported in this study. PMID:23896619

  13. Characterization of Lassa Virus Cell Entry Inhibitors: Determination of the Active Enantiomer by Asymmetric Synthesis

    PubMed Central

    Whitby, Landon R.; Lee, Andrew M.; Kunz, Stefan; Oldstone, Michael B. A.; Boger, Dale L.

    2009-01-01

    The comparative characterization of a series of 4-acyl-1,6-dialkylpiperazin-2-ones as potent cell entry inhibitors of the hemorrhagic fever arenavirus Lassa (LASV) is disclosed. The resolution and examination of the individual enantiomers of the prototypical LASV cell entry inhibitor 3 (16G8) is reported and the more potent (–)-enantiomer was found to be 15-fold more active than the corresponding (+)-enantiomer. The absolute configuration of (–)-3 was established by asymmetric synthesis of the active inhibitor (–)-(S)-3 (lassamycin-1). A limited deletion scan of lassamycin-1 defined key structural features required of the prototypical inhibitors. PMID:19428249

  14. Non-target-site resistance to ALS inhibitors in waterhemp (Amaranthus tuberculatus)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A waterhemp population (MCR) previously characterized as resistant to 4-hyroxyphenylpyruvate dioxygenase (HPPD) and photosystem II (PSII) inhibitors was found to have two different resistance responses to acetolactate synthase (ALS) inhibitors. Plants from the MCR population exhibiting high resistan...

  15. Distinct Effects of Two HIV-1 Capsid Assembly Inhibitor Families That Bind the Same Site within the N-Terminal Domain of the Viral CA Protein

    PubMed Central

    Titolo, Steve; von Schwedler, Uta; Goudreau, Nathalie; Mercier, Jean-François; Wardrop, Elizabeth; Faucher, Anne-Marie; Coulombe, René; Banik, Soma S. R.; Fader, Lee; Gagnon, Alexandre; Kawai, Stephen H.; Rancourt, Jean; Tremblay, Martin; Yoakim, Christiane; Simoneau, Bruno; Archambault, Jacques; Sundquist, Wesley I.

    2012-01-01

    The emergence of resistance to existing classes of antiretroviral drugs necessitates finding new HIV-1 targets for drug discovery. The viral capsid (CA) protein represents one such potential new target. CA is sufficient to form mature HIV-1 capsids in vitro, and extensive structure-function and mutational analyses of CA have shown that the proper assembly, morphology, and stability of the mature capsid core are essential for the infectivity of HIV-1 virions. Here we describe the development of an in vitro capsid assembly assay based on the association of CA-NC subunits on immobilized oligonucleotides. This assay was used to screen a compound library, yielding several different families of compounds that inhibited capsid assembly. Optimization of two chemical series, termed the benzodiazepines (BD) and the benzimidazoles (BM), resulted in compounds with potent antiviral activity against wild-type and drug-resistant HIV-1. Nuclear magnetic resonance (NMR) spectroscopic and X-ray crystallographic analyses showed that both series of inhibitors bound to the N-terminal domain of CA. These inhibitors induce the formation of a pocket that overlaps with the binding site for the previously reported CAP inhibitors but is expanded significantly by these new, more potent CA inhibitors. Virus release and electron microscopic (EM) studies showed that the BD compounds prevented virion release, whereas the BM compounds inhibited the formation of the mature capsid. Passage of virus in the presence of the inhibitors selected for resistance mutations that mapped to highly conserved residues surrounding the inhibitor binding pocket, but also to the C-terminal domain of CA. The resistance mutations selected by the two series differed, consistent with differences in their interactions within the pocket, and most also impaired virus replicative capacity. Resistance mutations had two modes of action, either directly impacting inhibitor binding affinity or apparently increasing the overall

  16. Small molecule inhibitor screen identifies synergistic activity of the bromodomain inhibitor CPI203 and bortezomib in drug resistant myeloma

    PubMed Central

    Siegel, Matthew B.; Liu, Selina Qiuying; Davare, Monika A.; Spurgeon, Stephen E.; Loriaux, Marc M.; Druker, Brian J.

    2015-01-01

    Purpose Despite significant therapeutic progress in multiple myeloma, drug resistance is uniformly inevitable and new treatments are needed. Our aim was to identify novel, efficacious small-molecule combinations for use in drug resistant multiple myeloma. Experimental Design A panel of 116 small molecule inhibitors was used to screen resistant myeloma cell lines for potential therapeutic targets. Agents found to have enhanced activity in the bortezomib or melphalan resistant myeloma cell lines were investigated further in combination. Synergistic combinations of interest were evaluated in primary patient cells. Results The overall single-agent drug sensitivity profiles were dramatically different between melphalan and bortezomib resistant cells, however, the bromodomain inhibitor, CPI203, was observed to have enhanced activity in both the bortezomib and melphalan resistant lines compared to their wild-type counterparts. The combination of bortezomib and CPI203 was found to be synergistic in both the bortezomib and melphalan resistant cell lines as well as in a primary multiple myeloma sample from a patient refractory to recent proteasome inhibitor treatment. The CPI203-bortezomib combination led to enhanced apoptosis and anti-proliferative effects. Finally, in contrast to prior reports of synergy between bortezomib and other epigenetic modifying agents, which implicated MYC downregulation or NOXA induction, our analyses suggest that CPI203-bortezomib synergy is independent of these events. Conclusion Our preclinical data supports a role for the clinical investigation of the bromodomain inhibitor CPI203 combined with bortezomib or alkylating agents in resistant multiple myeloma. PMID:26254279

  17. Thumb Site 2 Inhibitors of Hepatitis C Viral RNA-dependent RNA Polymerase Allosterically Block the Transition from Initiation to Elongation.

    PubMed

    Li, Jiawen; Johnson, Kenneth A

    2016-05-01

    Replication of the hepatitis C viral genome is catalyzed by the NS5B (nonstructural protein 5B) RNA-dependent RNA polymerase, which is a major target of antiviral drugs currently in the clinic. Prior studies established that initiation of RNA replication could be facilitated by starting with a dinucleotide (pGG). Here we establish conditions for efficient initiation from GTP to form the dinucleotide and subsequent intermediates leading to highly processive elongation, and we examined the effects of four classes of nonnucleoside inhibitors on each step of the reaction. We show that palm site inhibitors block initiation starting from GTP but not when starting from pGG. In addition we show that nonnucleoside inhibitors binding to thumb site-2 (NNI2) lead to the accumulation of abortive intermediates three-five nucleotides in length. Our kinetic analysis shows that NNI2 do not significantly block initiation or elongation of RNA synthesis; rather, they block the transition from initiation to elongation, which is thought to proceed with significant structural rearrangement of the enzyme-RNA complex including displacement of the β-loop from the active site. Direct measurement in single turnover kinetic studies show that pyrophosphate release is faster than the chemistry step, which appears to be rate-limiting during processive synthesis. These results reveal important new details to define the steps involved in initiation and elongation during viral RNA replication, establish the allosteric mechanisms by which NNI2 inhibitors act, and point the way to the design of more effective allosteric inhibitors that exploit this new information. PMID:26851276

  18. Diarylsulfonamides and their bioisosteres as dual inhibitors of alkaline phosphatase and carbonic anhydrase: Structure activity relationship and molecular modelling studies.

    PubMed

    Al-Rashida, Mariya; Ejaz, Syeda Abida; Ali, Sharafat; Shaukat, Aisha; Hamayoun, Mehwish; Ahmed, Maqsood; Iqbal, Jamshed

    2015-05-15

    The effect of bioisosteric replacement of carboxamide linking group with sulfonamide linking group, on alkaline phosphatase (AP) and carbonic anhydrase (CA) inhibition activity of aromatic benzenesulfonamides was investigated. A series of carboxamide linked aromatic benzenesulfonamides 1a-1c, 2a-2d and their sulfonamide linked bioisosteres 3a-3d, 4a-4d was synthesized and evaluated for inhibitory activity against bovine tissue non-specific alkaline phosphatase (TNAP), intestinal alkaline phosphatase (IAP) and bCA II. A significant increase in CA inhibition activity was observed upon bioisosteric replacement of carboxamide linking group with a sulfonamide group. Some of these compounds were identified as highly potent and selective AP inhibitors. Compounds 1b, 2b, 3d, 4d 5b and 5c were found to be selective bTNAP inhibitors, whereas compounds 1a, 1c, 2a, 2c, 2d, 3a, 3c, 4a, 4b, 4c, 5a were found to be selective bIAP inhibitors. For most active AP inhibitor 3b, detailed kinetic studies indicated a competitive mode of inhibition against tissue non-specific alkaline phosphatase (TNAP) and non-competitive mode of inhibition against intestinal alkaline phosphatase (IAP). Molecular docking studies were carried out to rationalize important binding site interactions. PMID:25865133

  19. Diarylsulfonamides and their bioisosteres as dual inhibitors of alkaline phosphatase and carbonic anhydrase: Structure activity relationship and molecular modelling studies.

    PubMed

    Al-Rashida, Mariya; Ejaz, Syeda Abida; Ali, Sharafat; Shaukat, Aisha; Hamayoun, Mehwish; Ahmed, Maqsood; Iqbal, Jamshed

    2015-05-15

    The effect of bioisosteric replacement of carboxamide linking group with sulfonamide linking group, on alkaline phosphatase (AP) and carbonic anhydrase (CA) inhibition activity of aromatic benzenesulfonamides was investigated. A series of carboxamide linked aromatic benzenesulfonamides 1a-1c, 2a-2d and their sulfonamide linked bioisosteres 3a-3d, 4a-4d was synthesized and evaluated for inhibitory activity against bovine tissue non-specific alkaline phosphatase (TNAP), intestinal alkaline phosphatase (IAP) and bCA II. A significant increase in CA inhibition activity was observed upon bioisosteric replacement of carboxamide linking group with a sulfonamide group. Some of these compounds were identified as highly potent and selective AP inhibitors. Compounds 1b, 2b, 3d, 4d 5b and 5c were found to be selective bTNAP inhibitors, whereas compounds 1a, 1c, 2a, 2c, 2d, 3a, 3c, 4a, 4b, 4c, 5a were found to be selective bIAP inhibitors. For most active AP inhibitor 3b, detailed kinetic studies indicated a competitive mode of inhibition against tissue non-specific alkaline phosphatase (TNAP) and non-competitive mode of inhibition against intestinal alkaline phosphatase (IAP). Molecular docking studies were carried out to rationalize important binding site interactions.

  20. Structural and Kinetic Analyses of Macrophage Migration Inhibitory Factor Active Site Interactions

    SciTech Connect

    Crichlow, G.; Lubetsky, J; Leng, L; Bucala, R; Lolis, E

    2009-01-01

    Macrophage migration inhibitory factor (MIF) is a secreted protein expressed in numerous cell types that counters the antiinflammatory effects of glucocorticoids and has been implicated in sepsis, cancer, and certain autoimmune diseases. Interestingly, the structure of MIF contains a catalytic site resembling the tautomerase/isomerase sites of microbial enzymes. While bona fide physiological substrates remain unknown, model substrates have been identified. Selected compounds that bind in the tautomerase active site also inhibit biological functions of MIF. It had previously been shown that the acetaminophen metabolite, N-acetyl-p-benzoquinone imine (NAPQI), covalently binds to the active site of MIF. In this study, kinetic data indicate that NAPQI inhibits MIF both covalently and noncovalently. The structure of MIF cocrystallized with NAPQI reveals that the NAPQI has undergone a chemical alteration forming an acetaminophen dimer (bi-APAP) and binds noncovalently to MIF at the mouth of the active site. We also find that the commonly used protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), forms a covalent complex with MIF and inhibits the tautomerase activity. Crystallographic analysis reveals the formation of a stable, novel covalent bond for PMSF between the catalytic nitrogen of the N-terminal proline and the sulfur of PMSF with complete, well-defined electron density in all three active sites of the MIF homotrimer. Conclusions are drawn from the structures of these two MIF-inhibitor complexes regarding the design of novel compounds that may provide more potent reversible and irreversible inhibition of MIF.

  1. Effects of inhibitors of tumoricidal activity upon schistosomulum killing by activated macrophages.

    PubMed

    James, S L; Glaven, J A

    1987-12-01

    Larvae of the helminth parasite Schistosoma mansoni are efficiently killed in vitro by lymphokine-activated macrophages, leading to the hypothesis that these cells may participate in the effector mechanism of protective immunity against schistosomiasis. Larvacidal activity has also been demonstrated in the IC-21 macrophage cell line in the absence of a demonstrable respiratory burst, indicating that macrophages possess nonoxidative mechanisms of schistosomulum killing. In this study, we demonstrated that IC-21 larval killing was most effective when contact was allowed between cells and target. Nonoxidative larvacidal activity was prevented by protein synthesis inhibitors, by the inhibition of microtubule polymerization, and by tosyllysylchloromethylketone but not by other inhibitors or substrates of tryptic or chymotryptic protease activity. The addition of excess iron to the culture also prevented IC-21-mediated larval killing, suggesting that the production of an iron-binding molecule may be involved. In contrast, the addition of excess thymidine or arginine did not reverse macrophage larvacidal activity, nor did lysosomotropic agents that depress the activity of acid hydrolases. Under appropriate conditions of activation and surface membrane stimulation, IC-21 cells could be induced to release soluble cytotoxic factors retaining larvacidal activity. These observations provide insight into the mechanism of macrophage-mediated schistosome killing, in comparison to the cytotoxic mechanisms described in the better-studied tumoricidal models, and supply a basis for further biochemical investigation of macrophage function against a multicellular target. PMID:3119500

  2. Effects of inhibitors of tumoricidal activity upon schistosomulum killing by activated macrophages.

    PubMed Central

    James, S L; Glaven, J A

    1987-01-01

    Larvae of the helminth parasite Schistosoma mansoni are efficiently killed in vitro by lymphokine-activated macrophages, leading to the hypothesis that these cells may participate in the effector mechanism of protective immunity against schistosomiasis. Larvacidal activity has also been demonstrated in the IC-21 macrophage cell line in the absence of a demonstrable respiratory burst, indicating that macrophages possess nonoxidative mechanisms of schistosomulum killing. In this study, we demonstrated that IC-21 larval killing was most effective when contact was allowed between cells and target. Nonoxidative larvacidal activity was prevented by protein synthesis inhibitors, by the inhibition of microtubule polymerization, and by tosyllysylchloromethylketone but not by other inhibitors or substrates of tryptic or chymotryptic protease activity. The addition of excess iron to the culture also prevented IC-21-mediated larval killing, suggesting that the production of an iron-binding molecule may be involved. In contrast, the addition of excess thymidine or arginine did not reverse macrophage larvacidal activity, nor did lysosomotropic agents that depress the activity of acid hydrolases. Under appropriate conditions of activation and surface membrane stimulation, IC-21 cells could be induced to release soluble cytotoxic factors retaining larvacidal activity. These observations provide insight into the mechanism of macrophage-mediated schistosome killing, in comparison to the cytotoxic mechanisms described in the better-studied tumoricidal models, and supply a basis for further biochemical investigation of macrophage function against a multicellular target. PMID:3119500

  3. Active site of ribulosebisphosphate carboxylase/oxygenase

    SciTech Connect

    Hartman, F.C.; Stringer, C.D.; Milanez, S.; Lee, E.H.

    1985-01-01

    Previous affinity labeling studies and comparative sequence analyses have identified two different lysines at the active site of ribulosebisphosphate carboxylase/oxygenase and have suggested their essentiality to function. The essential lysines occupy positions 166 and 329 in the Rhodospirillum rubrum enzyme and positions 175 and 334 in the spinach enzyme. Based on the pH-dependencies of inactivations of the two enzymes by trinitrobenzene sulfonate, Lys-166 (R. rubrum enzyme) exhibits a pK/sub a/ of 7.9 and Lys-334 (spinach enzyme) exhibits a pK/sub a/ of 9.0. These low pK/sub a/ values as well as the enhanced nucleophilicities of the lysyl residues argue that both are important to catalysis rather than to substrate binding. Lys-166 may correspond to the essential base that initiates catalysis and that displays a pK/sub a/ of 7.5 in the pH-curve for V/sub max//K/sub m/. Cross-linking experiments with 4,4'-diisothiocyano-2,2'-disulfonate stilbene demonstrate that the two active-site lysines are within 12 A. 50 refs., 7 figs., 1 tab.

  4. The ACE inhibitor ( sup 3 H)SQ29,852 identifies a high affinity recognition site located in the human temporal cortex

    SciTech Connect

    Barnes, N.M.; Costall, B.; Egli, P.; Horovitz, Z.P.; Ironside, J.W.; Naylor, R.J.; Williams, T.J. )

    1990-07-01

    The angiotensin converting enzyme (ACE) inhibitor ({sup 3}H)SQ29,852 identified a single high affinity recognition site (defined by 10.0 microM captopril) in the human temporal cortex (pKD 8.62 +/- 0.03; Bmax 248 +/- 24 fmol mg-1 protein, mean +/- S.E.M., n = 4). ACE inhibitors and thiorphan competed to a similar level for the ({sup 3}H)SQ29,852 binding site in the human temporal cortex with a rank order of affinity (pKi values mean +/- S.E.M., n = 3), lisinopril (9.49 +/- 0.02), captopril (9.16 +/- 0.08), SQ29,852 (8.58 +/- 0.04), epicaptopril (7.09 +/- 0.08), fosinopril (7.08 +/- 0.05) and thiorphan (6.40 +/- 0.04). Since this rank order of affinity is similar to the affinity of these compounds to inhibit brain ACE activity it is concluded that ({sup 3}H)SQ29,852 selectively labels the inhibitor recognition site of ACE in the human temporal cortex.

  5. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1992-02-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of transuranic (TRU) waste and active low-level waste (LLW) facilities at Oak Ridge National Laboratory (ORNL) in accordance with US Department of Energy (DOE) Order 5820.2A. Active LLW facilities in Solid Waste Storage Area (SWSA) 6 include Tumulus I and Tumulus II, the Interim Waste Management Facility (IWMF), LLW silos, high-range wells, asbestos silos, and fissile wells. The tumulus pads and IWMF are aboveground, high-strength concrete pads on which concrete vaults containing metal boxes of LLW are placed; the void space between the boxes and vaults is filled with grout. Eventually, these pads and vaults will be covered by an engineered multilayered cap. All other LLW facilities in SWSA 6 are below ground. In addition, this plan includes monitoring of the Hillcut Disposal Test Facility (HDTF) in SWSA 6, even though this facility was completed prior to the data of the DOE order. In SWSA 5 North, the TRU facilities include below-grade engineered caves, high-range wells, and unlined trenches. All samples from SWSA 6 are screened for alpha and beta activity, counted for gamma-emitting isotopes, and analyzed for tritium. In addition to these analytes, samples from SWSA 5 North are analyzed for specific transuranic elements.

  6. Design and prediction of new acetylcholinesterase inhibitor via quantitative structure activity relationship of huprines derivatives.

    PubMed

    Zhang, Shuqun; Hou, Bo; Yang, Huaiyu; Zuo, Zhili

    2016-05-01

    Acetylcholinesterase (AChE) is an important enzyme in the pathogenesis of Alzheimer's disease (AD). Comparative quantitative structure-activity relationship (QSAR) analyses on some huprines inhibitors against AChE were carried out using comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis (CoMSIA), and hologram QSAR (HQSAR) methods. Three highly predictive QSAR models were constructed successfully based on the training set. The CoMFA, CoMSIA, and HQSAR models have values of r (2) = 0.988, q (2) = 0.757, ONC = 6; r (2) = 0.966, q (2) = 0.645, ONC = 5; and r (2) = 0.957, q (2) = 0.736, ONC = 6. The predictabilities were validated using an external test sets, and the predictive r (2) values obtained by the three models were 0.984, 0.973, and 0.783, respectively. The analysis was performed by combining the CoMFA and CoMSIA field distributions with the active sites of the AChE to further understand the vital interactions between huprines and the protease. On the basis of the QSAR study, 14 new potent molecules have been designed and six of them are predicted to be more active than the best active compound 24 described in the literature. The final QSAR models could be helpful in design and development of novel active AChE inhibitors.

  7. A thermostable trypsin inhibitor with antiproliferative activity from small pinto beans.

    PubMed

    Chan, Yau Sang; Zhang, Yanbo; Sze, Stephen Cho Wing; Ng, Tzi Bun

    2014-08-01

    Small pinto bean is a cultivar of Phaseolus vulgaris. It produces a 16-kDa trypsin inhibitor that could be purified using anion exchange and size chromatography. Q-Sepharose, Mono Q and Superdex 75 columns were employed for the isolation process. Small pinto bean trypsin inhibitor demonstrated moderate pH stability (pH 2-10) and marked heat stability, with its trypsin inhibitory activity largely retained after exposure to 100 °C for half an hour. The activity was abolished in the presence of dithiothreitol, in a dose-dependent manner, implying that disulfide bonds in small pinto bean trypsin inhibitor are crucial for the activity. The trypsin inhibitor showed a blocked N-terminus. The trypsin inhibitor only slightly inhibited the viability of breast cancer MCF7 and hepatoma HepG2 cells at 125 μM. PMID:23859150

  8. Plasminogen Activator Inhibitor-1 in Cancer: Rationale and Insight for Future Therapeutic Testing.

    PubMed

    Placencio, Veronica R; DeClerck, Yves A

    2015-08-01

    Despite its function as an inhibitor of urokinase and tissue-type plasminogen activator (PA), PA inhibitor-1 (PAI-1) has a paradoxical protumorigenic role in cancer, promoting angiogenesis and tumor cell survival. In this review, we summarize preclinical evidence in support of the protumorigenic function of PAI-1 that has led to the testing of small-molecule PAI-1 inhibitors, initially developed as antithrombotic agents, in animal models of cancer. The review discusses the challenges and the opportunities that lay ahead to the development of efficacious and nontoxic PAI-1 inhibitors as anticancer agents.

  9. Antiviral activity of a Rac GEF inhibitor characterized with a sensitive HIV/SIV fusion assay

    SciTech Connect

    Pontow, Suzanne; Harmon, Brooke; Campbell, Nancy; Ratner, Lee

    2007-11-10

    A virus-dependent fusion assay was utilized to examine the activity of a panel of HIV-1, -2, and SIV isolates of distinct coreceptor phenotypes. This assay allowed identification of entry inhibitors, and characterization of an antagonist of a Rac guanine nucleotide exchange factor, as an inhibitor of HIV-mediated fusion.

  10. The activity and location of cathepsin D inhibitor in seeds of common vetch (Vicia sativa L.).

    PubMed

    Roszkowska-Jakimiec, W; Leśniewska, J

    2004-01-01

    The activity of cathepsin D inhibitor is markedly higher in common vetch seed coat than in embryo cotyledons. The occurrence of considerable amounts of the inhibitor in the seed coat of vetch was confirmed by the fluorescent microscopic technique, with the use of fluorescein-marked cathepsin D.

  11. A mutant trypsin-like enzyme from Streptomyces fradiae, created by site-directed mutagenesis, improves affinity chromatography for protein trypsin inhibitors.

    PubMed

    Katoh, T; Kikuchi, N; Nagata, K; Yoshida, N

    1996-08-01

    The Ser-170 residue of a trypsin-like enzyme from Streptomyces fradiae (SFT), which is considered to be the active-site serine, was replaced with alanine by site-directed mutagenesis to improve the affinity chromatography step for a Kazal-type trypsin inhibitor pancreatic secretory trypsin inhibitor (PSTI). The resulting mutant SFT, designated as [S170A]SFT, was expressed in Streptomyces lividans and purified to homogeneity. [S170A]SFT was catalytically inactive, but still had the ability to bind tightly to PSTI and to soybean trypsin inhibitor with dissociation constants of 3.1 x 10(-7) M and 1.9 x 10(-8) M respectively. We further demonstrated that recombinant human PSTI secreted into Saccharomyces cerevisiae culture broth could be purified to homogeneity with a one-step [S170A]SFT-affinity column. The purified PSTI contained no molecules intramolecularly cleaved by active trypsin, which are found when trypsin-affinity chromatography is used for the purification. This eliminated the need for further separation of intact PSTI from intramolecularly cleaved PSTI by high-performance liquid chromatography, thus simplifying and improving its purification process.

  12. In Vitro Activity and Resistance Profile of Dasabuvir, a Nonnucleoside Hepatitis C Virus Polymerase Inhibitor

    PubMed Central

    Koev, Gennadiy; Irvin, Michelle; Beyer, Jill; Liu, Yaya; Krishnan, Preethi; Reisch, Thomas; Mondal, Rubina; Wagner, Rolf; Molla, Akhteruzzaman; Maring, Clarence; Collins, Christine

    2014-01-01

    Dasabuvir (ABT-333) is a nonnucleoside inhibitor of the RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) NS5B gene. Dasabuvir inhibited recombinant NS5B polymerases derived from HCV genotype 1a and 1b clinical isolates, with 50% inhibitory concentration (IC50) values between 2.2 and 10.7 nM, and was at least 7,000-fold selective for the inhibition of HCV genotype 1 polymerases over human/mammalian polymerases. In the HCV subgenomic replicon system, dasabuvir inhibited genotype 1a (strain H77) and 1b (strain Con1) replicons with 50% effective concentration (EC50) values of 7.7 and 1.8 nM, respectively, with a 13-fold decrease in inhibitory activity in the presence of 40% human plasma. This level of activity was retained against a panel of chimeric subgenomic replicons that contained HCV NS5B genes from 22 genotype 1 clinical isolates from treatment-naive patients, with EC50s ranging between 0.15 and 8.57 nM. Maintenance of replicon-containing cells in medium containing dasabuvir at concentrations 10-fold or 100-fold greater than the EC50 resulted in selection of resistant replicon clones. Sequencing of the NS5B coding regions from these clones revealed the presence of variants, including C316Y, M414T, Y448C, Y448H, and S556G, that are consistent with binding to the palm I site of HCV polymerase. Consequently, dasabuvir retained full activity against replicons known to confer resistance to other polymerase inhibitors, including the S282T variant in the nucleoside binding site and the M423T, P495A, P495S, and V499A single variants in the thumb domain. The use of dasabuvir in combination with inhibitors targeting HCV NS3/NS4A protease (ABT-450 with ritonavir) and NS5A (ombitasvir) is in development for the treatment of HCV genotype 1 infections. PMID:25534735

  13. Identification and Structure-Activity Relationships of Diarylhydrazides as Novel Potent and Selective Human Enterovirus Inhibitors.

    PubMed

    Han, Xin; Sun, Ningyuan; Wu, Haoming; Guo, Deyin; Tien, Po; Dong, Chune; Wu, Shuwen; Zhou, Hai-Bing

    2016-03-10

    Enterovirus 71 (EV71) plays an important role in hand-foot-and-mouth disease. In this study, a series of diarylhydrazide analogues was synthesized, and the systematic exploration of SAR led to potent enterovirus inhibitors, of which compound 15 exhibits significant improvements in inhibition potency with an EC50 value of 0.02 μM against EV71. It is very interesting that this class of diarylhydrazides exhibits activities against a series of human enteroviruses at the picomolar level, including EV71 and Coxsackieviruses B1 (CVB1), CVB2, CVB3, CVB4, CVB5, and CVB6 (EC50 as low as 0.5 nM). Compared with the reference antienterovirus drug 1 (enviroxime) and known inhibitor 5 (WIN 51711), the four highly selective compounds 15, 27, 41 and 47 inhibited EV71 replication with EC50 values of 0.17-0.02 μM and SI values in a range of 978.4-12338. A preliminary mechanistic study indicated that VP1 might be the target site for this type of compound.

  14. A nonnucleoside reverse transcriptase inhibitor active on human immunodeficiency virus type 1 isolates resistant to related inhibitors.

    PubMed Central

    Goldman, M E; O'Brien, J A; Ruffing, T L; Schleif, W A; Sardana, V V; Byrnes, V W; Condra, J H; Hoffman, J M; Emini, E A

    1993-01-01

    Pyridinone derivatives are potent and specific inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and HIV-1 replication in cell culture. However, the potential clinical usefulness of these compounds as monotherapeutic agents may be limited by the selection of inhibitor-resistant viral variants. Resistance in cell culture is due primarily to mutational alterations at RT amino acid residues 103 and 181. A recombinant HIV-1 RT containing both of these mutations was used to screen a panel of pyridinone analogs for inhibitory activity. L-696,229 and L-697,661, pyridinones currently undergoing clinical evaluation, were more than 4,000-fold weaker against the mutant enzyme than against the wild-type enzyme. In contrast, one derivative of L-696,229, L-702,019 (3-[2-(4,7-dichlorobenzoxazol-2-yl)ethyl]-5-ethyl-6-methylpyrid in-2(1H)-thione), showed only three-fold different potencies against the two enzymes. L-702,019 was also a potent inhibitor of the replication of mutant HIV-1 containing the individual mutations at amino acid 103 or 181 as well as of clinical isolates resistant to L-697,661 and L-696,229. Isolation and analysis of resistant viral variants in cell culture showed that significant resistance to L-702,019 could be engendered only by multiple amino acid substitutions in RT. Accordingly, these studies demonstrated the potential of identifying second-generation specific HIV-1 RT inhibitors that can overcome the viral resistance selected by the first generation of inhibitors. PMID:7685996

  15. Flavonoids as CDK1 Inhibitors: Insights in Their Binding Orientations and Structure-Activity Relationship.

    PubMed

    Navarro-Retamal, Carlos; Caballero, Julio

    2016-01-01

    In the last years, the interactions of flavonoids with protein kinases (PKs) have been described by using crystallographic experiments. Interestingly, different orientations have been found for one flavonoid inside different PKs and different chemical substitutions lead to different orientations of the flavonoid scaffold inside one PK. Accordingly, orientation predictions of novel analogues could help to the design of flavonoids with high PK inhibitory activities. With this in mind, we studied the binding modes of 37 flavonoids (flavones and chalcones) inside the cyclin-dependent PK CDK1 using docking experiments. We found that the compounds under study adopted two different orientations into the active site of CDK1 (orientations I and II in the manuscript). In addition, quantitative structure-activity relationship (QSAR) models using CoMFA and CoMSIA methodologies were constructed to explain the trend of the CDK1 inhibitory activities for the studied flavonoids. Template-based and docking-based alignments were used. Models developed starting from docking-based alignment were applied for describing the whole dataset and compounds with orientation I. Adequate R2 and Q2 values were obtained by each method; interestingly, only hydrophobic and hydrogen bond donor fields describe the differential potency of the flavonoids as CDK1 inhibitors for both defined alignments and subsets. Our current application of docking and QSAR together reveals important elements to be drawn for the design of novel flavonoids with increased PK inhibitory activities. PMID:27517610

  16. Docking and quantitative structure-activity relationship of oxadiazole derivates as inhibitors of GSK3β.

    PubMed

    Quesada-Romero, Luisa; Caballero, Julio

    2014-02-01

    The binding modes of 42 oxadiazole derivates inside glycogen synthase kinase 3 beta (GSK3β were determined using docking experiments; thus, the preferred active conformations of these inhibitors are proposed. We found that these compounds adopt a scorpion-shaped conformation and they accept a hydrogen bond (HB) from the residue Val135 of the GSK3β ATP-binding site hinge region. In addition, quantitative structure-activity relationship (QSAR) models were constructed to explain the trend of the GSK3β inhibitory activities for the studied compounds. In a first approach, three-dimensional (3D) vectors were calculated using docking conformations and, by using multiple-linear regression, we assessed that GETAWAY vectors were able to describe the reported biological activities. In other QSAR approach, SMILES-based optimal descriptors were calculated. The best model included three-SMILES elements SSSβ leading to the identification of key molecular features that contribute to a high GSK3β inhibitory activity.

  17. Docking and quantitative structure-activity relationship of oxadiazole derivates as inhibitors of GSK3β.

    PubMed

    Quesada-Romero, Luisa; Caballero, Julio

    2014-02-01

    The binding modes of 42 oxadiazole derivates inside glycogen synthase kinase 3 beta (GSK3β were determined using docking experiments; thus, the preferred active conformations of these inhibitors are proposed. We found that these compounds adopt a scorpion-shaped conformation and they accept a hydrogen bond (HB) from the residue Val135 of the GSK3β ATP-binding site hinge region. In addition, quantitative structure-activity relationship (QSAR) models were constructed to explain the trend of the GSK3β inhibitory activities for the studied compounds. In a first approach, three-dimensional (3D) vectors were calculated using docking conformations and, by using multiple-linear regression, we assessed that GETAWAY vectors were able to describe the reported biological activities. In other QSAR approach, SMILES-based optimal descriptors were calculated. The best model included three-SMILES elements SSSβ leading to the identification of key molecular features that contribute to a high GSK3β inhibitory activity. PMID:24081608

  18. Flavonoids as CDK1 Inhibitors: Insights in Their Binding Orientations and Structure-Activity Relationship

    PubMed Central

    Navarro-Retamal, Carlos

    2016-01-01

    In the last years, the interactions of flavonoids with protein kinases (PKs) have been described by using crystallographic experiments. Interestingly, different orientations have been found for one flavonoid inside different PKs and different chemical substitutions lead to different orientations of the flavonoid scaffold inside one PK. Accordingly, orientation predictions of novel analogues could help to the design of flavonoids with high PK inhibitory activities. With this in mind, we studied the binding modes of 37 flavonoids (flavones and chalcones) inside the cyclin-dependent PK CDK1 using docking experiments. We found that the compounds under study adopted two different orientations into the active site of CDK1 (orientations I and II in the manuscript). In addition, quantitative structure–activity relationship (QSAR) models using CoMFA and CoMSIA methodologies were constructed to explain the trend of the CDK1 inhibitory activities for the studied flavonoids. Template-based and docking-based alignments were used. Models developed starting from docking-based alignment were applied for describing the whole dataset and compounds with orientation I. Adequate R2 and Q2 values were obtained by each method; interestingly, only hydrophobic and hydrogen bond donor fields describe the differential potency of the flavonoids as CDK1 inhibitors for both defined alignments and subsets. Our current application of docking and QSAR together reveals important elements to be drawn for the design of novel flavonoids with increased PK inhibitory activities. PMID:27517610

  19. Discovery of HDAC Inhibitors with Potent Activity Against Multiple Malaria Parasite Life Cycle Stages

    PubMed Central

    Hansen, Finn K.; Sumanadasa, Subathdrage D. M.; Stenzel, Katharina; Duffy, Sandra; Meister, Stephan; Marek, Linda; Schmetter, Rebekka; Kuna, Krystina; Hamacher, Alexandra; Mordmüller, Benjamin; Kassack, Matthias U.; Winzeler, Elizabeth A.; Avery, Vicky M.; Andrews, Katherine T.; Kurz, Thomas

    2015-01-01

    In this work we investigated the antiplasmodial activity of a series of HDAC inhibitors containing an alkoxyamide connecting-unit linker region. HDAC inhibitor 1a (LMK235), previously shown to be a novel and specific inhibitor of human HDAC4 and 5, was used as a starting point to rapidly construct a mini-library of HDAC inhibitors using a straightforward solid-phase supported synthesis. Several of these novel HDAC inhibitors were found to have potent in vitro activity against asexual stage P. falciparum malaria parasites. Representative compounds were shown to hyperacetylate P. falciparum histones and to inhibit deacetylase activity of recombinant PfHDAC1 and P. falciparum nuclear extracts. All compounds were also screened in vitro for activity against P. berghei exo-erythrocytic stages and selected compounds were further tested against late stage (IV and V) P. falciparum gametocytes. Of note, some compounds showed nanomolar activity against all three life cycle stages tested (asexual, exo-erythrocytic and gametocyte stages) and several compounds displayed significantly increased parasite selectivity compared to the reference HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). These data suggest that it may be possible to develop HDAC inhibitors that target multiple malaria parasite life cycle stages. PMID:24904967

  20. Thermodynamic linkage between the S1 site, the Na+ site, and the Ca2+ site in the protease domain of human coagulation factor xa. Studies on catalytic efficiency and inhibitor binding.

    PubMed

    Underwood, M C; Zhong, D; Mathur, A; Heyduk, T; Bajaj, S P

    2000-11-24

    The serine protease domain of factor Xa (FXa) contains a sodium as well as a calcium-binding site. Here, we investigated the functional significance of these two cation-binding sites and their thermodynamic links to the S1 site. Kinetic data reveal that Na(+) binds to the substrate bound FXa with K(d) approximately 39 mm in the absence and approximately 9.5 mm in the presence of Ca(2+). Sodium-bound FXa (sodium-Xa) has approximately 18-fold increased catalytic efficiency ( approximately 4.5-fold decrease in K(m) and approximately 4-fold increase in k(cat)) in hydrolyzing S-2222 (benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide), and Ca(2+) further increases this k(cat) approximately 1.4-fold. Ca(2+) binds to the protease domain of substrate bound FXa with K(d) approximately 705 microm in the absence and approximately 175 microm in the presence of Na(+). Ca(2+) binding to the protease domain of FXa (Xa-calcium) has no effect on the K(m) but increases the k(cat) approximately 4-fold in hydrolyzing S-2222, and Na(+) further increases this k(cat) approximately 1.4-fold. In agreement with the K(m) data, sodium-Xa has approximately 5-fold increased affinity in its interaction with p-aminobenzamidine (S1 site probe) and approximately 4-fold increased rate in binding to the two-domain tissue factor pathway inhibitor; Ca(2+) (+/-Na(+)) has no effect on these interactions. Antithrombin binds to Xa-calcium with a approximately 4-fold faster rate, to sodium-Xa with a approximately 24-fold faster rate and to sodium-Xa-calcium with a approximately 28-fold faster rate. Thus, Ca(2+) and Na(+) together increase the catalytic efficiency of FXa approximately 28-fold. Na(+) enhances Ca(2+) binding, and Ca(2+) enhances Na(+) binding. Further, Na(+) enhances S1 site occupancy, and S1 site occupancy enhances Na(+) binding. Therefore, Na(+) site is thermodynamically linked to the S1 site as well as to the protease domain Ca(2+) site, whereas Ca(2+) site is only linked to the Na(+) site. The

  1. Correlated structural kinetics and retarded solvent dynamics at the metalloprotease active site

    SciTech Connect

    Grossman, Moran; Born, Benjamin; Heyden, Matthias; Tworowski, Dmitry; Fields, Gregg B.; Sagi, Irit; Havenith, Martina

    2011-09-18

    Solvent dynamics can play a major role in enzyme activity, but obtaining an accurate, quantitative picture of solvent activity during catalysis is quite challenging. Here, we combine terahertz spectroscopy and X-ray absorption analyses to measure changes in the coupled water-protein motions during peptide hydrolysis by a zinc-dependent human metalloprotease. These changes were tightly correlated with rearrangements at the active site during the formation of productive enzyme-substrate intermediates and were different from those in an enzyme–inhibitor complex. Molecular dynamics simulations showed a steep gradient of fast-to-slow coupled protein-water motions around the protein, active site and substrate. Our results show that water retardation occurs before formation of the functional Michaelis complex. We propose that the observed gradient of coupled protein-water motions may assist enzyme-substrate interactions through water-polarizing mechanisms that are remotely mediated by the catalytic metal ion and the enzyme active site.

  2. Structural basis for the active site inhibition mechanism of human kidney-type glutaminase (KGA).

    PubMed

    Thangavelu, K; Chong, Qing Yun; Low, Boon Chuan; Sivaraman, J

    2014-01-01

    Glutaminase is a metabolic enzyme responsible for glutaminolysis, a process harnessed by cancer cells to feed their accelerated growth and proliferation. Among the glutaminase isoforms, human kidney-type glutaminase (KGA) is often upregulated in cancer and is thus touted as an attractive drug target. Here we report the active site inhibition mechanism of KGA through the crystal structure of the catalytic domain of KGA (cKGA) in complex with 6-diazo-5-oxo-L-norleucine (DON), a substrate analogue of glutamine. DON covalently binds with the active site Ser286 and interacts with residues such as Tyr249, Asn335, Glu381, Asn388, Tyr414, Tyr466 and Val484. The nucleophilic attack of Ser286 sidechain on DON releases the diazo group (N2) from the inhibitor and results in the formation of an enzyme-inhibitor complex. Mutational studies confirmed the key role of these residues in the activity of KGA. This study will be important in the development of KGA active site inhibitors for therapeutic interventions.

  3. Structural Basis for the Active Site Inhibition Mechanism of Human Kidney-Type Glutaminase (KGA)

    PubMed Central

    Thangavelu, K.; Chong, Qing Yun; Low, Boon Chuan; Sivaraman, J.

    2014-01-01

    Glutaminase is a metabolic enzyme responsible for glutaminolysis, a process harnessed by cancer cells to feed their accelerated growth and proliferation. Among the glutaminase isoforms, human kidney-type glutaminase (KGA) is often upregulated in cancer and is thus touted as an attractive drug target. Here we report the active site inhibition mechanism of KGA through the crystal structure of the catalytic domain of KGA (cKGA) in complex with 6-diazo-5-oxo-L-norleucine (DON), a substrate analogue of glutamine. DON covalently binds with the active site Ser286 and interacts with residues such as Tyr249, Asn335, Glu381, Asn388, Tyr414, Tyr466 and Val484. The nucleophilic attack of Ser286 sidechain on DON releases the diazo group (N2) from the inhibitor and results in the formation of an enzyme-inhibitor complex. Mutational studies confirmed the key role of these residues in the activity of KGA. This study will be important in the development of KGA active site inhibitors for therapeutic interventions. PMID:24451979

  4. Regulation of protein C inhibitor (PCI) activity by specific oxidized and negatively charged phospholipids.

    PubMed

    Malleier, Julia M; Oskolkova, Olga; Bochkov, Valery; Jerabek, Ingrid; Sokolikova, Barbora; Perkmann, Thomas; Breuss, Johannes; Binder, Bernd R; Geiger, Margarethe

    2007-06-01

    Protein C inhibitor (PCI) is a serpin with affinity for heparin and phosphatidylethanolamine (PE). We analyzed the interaction of PCI with different phospholipids and their oxidized forms. PCI bound to oxidized PE (OxPE), and oxidized and unoxidized phosphatidylserine (PS) immobilized on microtiter plates and in aqueous suspension. Binding to OxPE and PS was competed by heparin, but not by the aminophospholipid-binding protein annexin V or the PCI-binding lipid retinoic acid. PS and OxPE stimulated the inhibition of activated protein C (aPC) by PCI in a Ca(++)-dependent manner, indicating that binding of both, aPC (Ca(++) dependent) and PCI (Ca(++) independent), to phospholipids is necessary. A peptide corresponding to the heparin-binding site of PCI abolished the stimulatory effect of PS on aPC inhibition. No stimulatory effect of phospholipids on aPC inhibition was seen with a PCI mutant lacking the heparin-binding site. A heparin-like effect of phospholipids (OxPE) was not seen with antithrombin III, another heparin-binding serpin, suggesting that it is specific for PCI. PCI and annexin V were found to be endogenously colocalized in atherosclerotic plaques, supporting the hypothesis that exposure of oxidized PE and/or PS may be important for the local regulation of PCI activity in vivo.

  5. Identification of quinazolinyloxy biaryl urea as a new class of SUMO activating enzyme 1 inhibitors.

    PubMed

    Kumar, Ashutosh; Ito, Akihiro; Hirohama, Mikako; Yoshida, Minoru; Zhang, Kam Y J

    2013-09-15

    SUMO activating enzyme 1 (SUMO E1) is the first enzyme in sumoylation pathway and an important cancer drug target. However, only a few inhibitors were reported up to now that includes three natural products, semi-synthetic protein inhibitors and one AMP mimic. Here, we report the identification of quinazolinyloxy biaryl urea as a new class of SUMO E1 inhibitors. The most active compound of this class inhibited the in vitro sumoylation with an IC50 of 13.4 μM. This compound inhibits sumoylation by blocking the formation of SUMOE1-SUMO thioester intermediate. The biological activity of the most active compound is comparable to previously reported inhibitors with properties suitable for medicinal chemistry optimization for potency and druggability.

  6. Pattern formation in a (2 + 1)-species activator-inhibitor-immobilizer system

    NASA Astrophysics Data System (ADS)

    Pearson, John E.

    1992-09-01

    The necessary and sufficient conditions for a Turing instability in a (2 + 1)-component reaction-diffusion system are derived. The 2-component subsystem consists of an arbitrary activator-inhibitor system in which the activator and the inhibitor diffuse at identical rates. The activator reacts with an immobile substrate to form an immobile complex which does not diffuse. It is found that the critical wavenumber and the location of the instability in parameter space are independent of the initial substrate concentration. As a special case, the (2 + 1)-variable activator-inhibitor-immobilizer system can be reduced to an activator-inhibitor system in which the activator diffuses more slowly than the inhibitor. This occurs when the time scales in the system satisfy certain constraints. The general results are applied to a (2 + 1)-variable model of the chlorite-iodide-starch reaction recently proposed by Epstein and Lengyel to explain the experimental observation of Turing patterns in a related system. I find an intrinsic wavelength which is within a factor of two of the experimentally observed wavelength, but the necessary conditions for the (2 + 1)-variable system to reduce to a two-variable activator-inhibitor system with rescaled diffusion coefficients are not satisfied.

  7. Combined rational design and a high throughput screening platform for identifying chemical inhibitors of a Ras-activating enzyme.

    PubMed

    Evelyn, Chris R; Biesiada, Jacek; Duan, Xin; Tang, Hong; Shang, Xun; Papoian, Ruben; Seibel, William L; Nelson, Sandra; Meller, Jaroslaw; Zheng, Yi

    2015-05-15

    The Ras family small GTPases regulate multiple cellular processes, including cell growth, survival, movement, and gene expression, and are intimately involved in cancer pathogenesis. Activation of these small GTPases is catalyzed by a special class of enzymes, termed guanine nucleotide exchange factors (GEFs). Herein, we developed a small molecule screening platform for identifying lead hits targeting a Ras GEF enzyme, SOS1. We employed an ensemble structure-based virtual screening approach in combination with a multiple tier high throughput experimental screen utilizing two complementary fluorescent guanine nucleotide exchange assays to identify small molecule inhibitors of GEF catalytic activity toward Ras. From a library of 350,000 compounds, we selected a set of 418 candidate compounds predicted to disrupt the GEF-Ras interaction, of which dual wavelength GDP dissociation and GTP-loading experimental screening identified two chemically distinct small molecule inhibitors. Subsequent biochemical validations indicate that they are capable of dose-dependently inhibiting GEF catalytic activity, binding to SOS1 with micromolar affinity, and disrupting GEF-Ras interaction. Mutagenesis studies in conjunction with structure-activity relationship studies mapped both compounds to different sites in the catalytic pocket, and both inhibited Ras signaling in cells. The unique screening platform established here for targeting Ras GEF enzymes could be broadly useful for identifying lead inhibitors for a variety of small GTPase-activating GEF reactions.

  8. Combined Rational Design and a High Throughput Screening Platform for Identifying Chemical Inhibitors of a Ras-activating Enzyme*

    PubMed Central

    Evelyn, Chris R.; Biesiada, Jacek; Duan, Xin; Tang, Hong; Shang, Xun; Papoian, Ruben; Seibel, William L.; Nelson, Sandra; Meller, Jaroslaw; Zheng, Yi

    2015-01-01

    The Ras family small GTPases regulate multiple cellular processes, including cell growth, survival, movement, and gene expression, and are intimately involved in cancer pathogenesis. Activation of these small GTPases is catalyzed by a special class of enzymes, termed guanine nucleotide exchange factors (GEFs). Herein, we developed a small molecule screening platform for identifying lead hits targeting a Ras GEF enzyme, SOS1. We employed an ensemble structure-based virtual screening approach in combination with a multiple tier high throughput experimental screen utilizing two complementary fluorescent guanine nucleotide exchange assays to identify small molecule inhibitors of GEF catalytic activity toward Ras. From a library of 350,000 compounds, we selected a set of 418 candidate compounds predicted to disrupt the GEF-Ras interaction, of which dual wavelength GDP dissociation and GTP-loading experimental screening identified two chemically distinct small molecule inhibitors. Subsequent biochemical validations indicate that they are capable of dose-dependently inhibiting GEF catalytic activity, binding to SOS1 with micromolar affinity, and disrupting GEF-Ras interaction. Mutagenesis studies in conjunction with structure-activity relationship studies mapped both compounds to different sites in the catalytic pocket, and both inhibited Ras signaling in cells. The unique screening platform established here for targeting Ras GEF enzymes could be broadly useful for identifying lead inhibitors for a variety of small GTPase-activating GEF reactions. PMID:25825487

  9. The Appearance of New Active Forms of Trypsin Inhibitor in Germinating Mung Bean (Vigna radiata) Seeds.

    PubMed

    Lorensen, E; Prevosto, R; Wilson, K A

    1981-07-01

    Ungerminated seeds of mung bean contain a single major species (F) of trypsin inhibitor with five minor species (A-E) separable on diethylaminoethyl-cellulose. During germination the level of trypsin inhibitory activity decreases from 1.8 units/grams dry weight in ungerminated cotyledons to 1.2 units/grams in cotyledons from seeds germinated 5 days. This decrease is accompanied by major changes in the distribution of inhibitory activity among the inhibitor species. By 48 hours of germination, inhibitor F has largely disappeared with an accompanying rapid increase in inhibitor C. Similarly, though less rapidly, inhibitor E decreases while inhibitor A increases. A similar sequence of changes is found in vitro when purified inhibitor F is incubated with extracts from seeds germinated 96 hours. The combined in vivo and in vitro data suggest a conversion sequence of: F --> E --> C --> A. The in vitro conversion is inhibited by phenylmethyl sulfonyl fluoride but not by iodoacetamide, indicating that at least the initial phases of inhibitor conversion are not catalyzed by the mung bean vicilin peptidohydrolase.

  10. Metavanadate at the active site of the phosphatase VHZ.

    PubMed

    Kuznetsov, Vyacheslav I; Alexandrova, Anastassia N; Hengge, Alvan C

    2012-09-01

    Vanadate is a potent modulator of a number of biological processes and has been shown by crystal structures and NMR spectroscopy to interact with numerous enzymes. Although these effects often occur under conditions where oligomeric forms dominate, the crystal structures and NMR data suggest that the inhibitory form is usually monomeric orthovanadate, a particularly good inhibitor of phosphatases because of its ability to form stable trigonal-bipyramidal complexes. We performed a computational analysis of a 1.14 Å structure of the phosphatase VHZ in complex with an unusual metavanadate species and compared it with two classical trigonal-bipyramidal vanadate-phosphatase complexes. The results support extensive delocalized bonding to the apical ligands in the classical structures. In contrast, in the VHZ metavanadate complex, the central, planar VO(3)(-) moiety has only one apical ligand, the nucleophilic Cys95, and a gap in electron density between V and S. A computational analysis showed that the V-S interaction is primarily ionic. A mechanism is proposed to explain the formation of metavanadate in the active site from a dimeric vanadate species that previous crystallographic evidence has shown to be able to bind to the active sites of phosphatases related to VHZ. Together, the results show that the interaction of vanadate with biological systems is not solely reliant upon the prior formation of a particular inhibitory form in solution. The catalytic properties of an enzyme may act upon the oligomeric forms primarily present in solution to generate species such as the metavanadate ion observed in the VHZ structure. PMID:22876963

  11. Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase

    SciTech Connect

    Scott, A.I.; Roessner, C.A.; Stolowich, N.J.; Karuso, P.; Williams, H.J.; Grant, S.K.; Gonzalez, M.D.; Hoshino, T. )

    1988-10-18

    The active site of porphobilinogen (PBG){sup 1} deaminase from Escherichia coli has been found to contain an unusual dipyrromethane derived from four molecules of 5-aminolevulinic acid (ALA) covalently linked to Cys-242, one of the two cysteine residues conserved in E. coli and human deaminase. By use of a hemA{sup {minus}} strain of E. coli the enzyme was enriched from (5-{sup 13}C)ALA and examined by {sup 1}H-detected multiple quantum coherence spectroscopy, which revealed all of the salient features of a dipyrromethane composed of two PBG units linked heat to tail and terminating in a CH{sub 2}-S bond to a cysteine residue. Site-specific mutagenesis of Cys-99 and Cys-242, respectively, has shown that substitution of Ser for Cys-99 does not affect the enzymatic activity, whereas substitution of Ser for Cys-242 removes essentially all of the catalytic activity as measured by the conversion of the substrate PBG to uro'gen I. The NMR spectrum of the covalent complex of deaminase with the suicide inhibitor 2-bromo-(2,11-{sup 13}C{sub 2})PBG reveals that the aminomethyl terminus of the inhibitor reacts with the enzyme's cofactor at the {alpha}-free pyrrole. NMR spectroscopy of the ES{sub 2} complex confirmed a PBG-derived head-to-tail dipyrromethane attached to the {alpha}-free pyrrole position of the enzyme. A mechanistic rationale for deaminase is presented.

  12. Activity of the HIV-1 attachment inhibitor BMS-626529, the active component of the prodrug BMS-663068, against CD4-independent viruses and HIV-1 envelopes resistant to other entry inhibitors.

    PubMed

    Li, Zhufang; Zhou, Nannan; Sun, Yongnian; Ray, Neelanjana; Lataillade, Max; Hanna, George J; Krystal, Mark

    2013-09-01

    BMS-626529 is a novel small-molecule HIV-1 attachment inhibitor active against both CCR5- and CXCR4-tropic viruses. BMS-626529 functions by preventing gp120 from binding to CD4. A prodrug of this compound, BMS-663068, is currently in clinical development. As a theoretical resistance pathway to BMS-663068 could be the development of a CD4-independent phenotype, we examined the activity of BMS-626529 against CD4-independent viruses and investigated whether resistance to BMS-626529 could be associated with a CD4-independent phenotype. Finally, we evaluated whether cross-resistance exists between BMS-626529 and other HIV-1 entry inhibitors. Two laboratory-derived envelopes with a CD4-independent phenotype (one CXCR4 tropic and one CCR5 tropic), five envelopes from clinical isolates with preexisting BMS-626529 resistance, and several site-specific mutant BMS-626529-resistant envelopes were examined for their dependence on CD4 for infectivity or susceptibility to BMS-626529. Viruses resistant to other entry inhibitors (enfuvirtide, maraviroc, and ibalizumab) were also examined for susceptibility to BMS-626529. Both CD4-independent laboratory isolates retained sensitivity to BMS-626529 in CD4(-) cells, while HIV-1 envelopes from viruses resistant to BMS-626529 exhibited no evidence of a CD4-independent phenotype. BMS-626529 also exhibited inhibitory activity against ibalizumab- and enfuvirtide-resistant envelopes. While there appeared to be some association between maraviroc resistance and reduced susceptibility to BMS-626529, an absolute correlation cannot be presumed, since some CCR5-tropic maraviroc-resistant envelopes remained sensitive to BMS-626529. Clinical use of the prodrug BMS-663068 is unlikely to promote resistance via generation of CD4-independent virus. No cross-resistance between BMS-626529 and other HIV entry inhibitors was observed, which could allow for sequential or concurrent use with different classes of entry inhibitors.

  13. Protumorigenic Activity of Plasminogen Activator Inhibitor-1 Through an Antiapoptotic Function

    PubMed Central

    2012-01-01

    Background Plasminogen activator inhibitor-1 (PAI-1) is a protease inhibitor but is paradoxically associated with poor outcomes in cancer patients. However, the mechanisms of its effects on tumor cells have not been explored. Methods Endogenous PAI-1 in human tumor cell lines (HT-1080, A549, HCT-116, and MDA-MB-231) was suppressed by small interfering RNAs (siRNAs) and PAI-039, a small molecule inhibitor of PAI-1, and the effects on apoptosis were examined. Tumorigenicity of PAI-1 knockdown (KD) tumor cells was examined in immunodeficient PAI-1 wild-type and knockout (KO) mice (9–15 per group), and event-free survival was analyzed by the Kaplan–Meier method. The effect of PAI-1 suppression on HT-1080 xenotransplanted tumors was evaluated for cell proliferation, apoptosis, and angiogenesis. All statistical tests were two-sided. Results Genetic and pharmacological inhibition of PAI-1 in the four tumor cell lines increased spontaneous apoptosis (mean fold increase relative to control: HT-1080, siRNA#1, mean = 4.0, 95% CI = 2.6 to 5.3, P < .001; siRNA#2, mean = 2.6, 95% CI = 2.4 to 2.9, P < .001, Student t test), which was blocked in the presence of recombinant PAI-1, a caspase-8 inhibitor, or Fas/FasL neutralizing antibodies and was partially attenuated by a plasmin inhibitor-aprotinin. PAI-1 KO mice implanted with PAI-1 KD HT-1080 cells had decreased tumorigenesis and prolonged survival compared with control mice (P = .002, log-rank test), and their tumors exhibited decreased cell proliferation and angiogenesis and increased apoptosis. Furthermore, five of 15 PAI-1 KO mice implanted with PAI-1 KD HT-1080 cells never developed tumors. Conclusions These data suggest that PAI-1 exerts a protective effect against tumor cell apoptosis by a mechanism that, in part, involves plasmin activation and inhibition of Fas/Fas-L-mediated apoptosis and may be a promising therapeutic target. PMID:22984202

  14. Caspase Inhibitors of the P35 Family Are More Active When Purified from Yeast than Bacteria

    PubMed Central

    Brand, Ingo L.; Civciristov, Srgjan; Taylor, Nicole L.; Talbo, Gert H.; Pantaki-Eimany, Delara; Levina, Vita; Clem, Rollie J.; Perugini, Matthew A.; Kvansakul, Marc; Hawkins, Christine J.

    2012-01-01

    Many insect viruses express caspase inhibitors of the P35 superfamily, which prevent defensive host apoptosis to enable viral propagation. The prototypical P35 family member, AcP35 from Autographa californica M nucleopolyhedrovirus, has been extensively studied. Bacterially purified AcP35 has been previously shown to inhibit caspases from insect, mammalian and nematode species. This inhibition occurs via a pseudosubstrate mechanism involving caspase-mediated cleavage of a “reactive site loop” within the P35 protein, which ultimately leaves cleaved P35 covalently bound to the caspase's active site. We observed that AcP35 purifed from Saccharomyces cerevisae inhibited caspase activity more efficiently than AcP35 purified from Escherichia coli. This differential potency was more dramatic for another P35 family member, MaviP35, which inhibited human caspase 3 almost 300-fold more potently when purified from yeast than bacteria. Biophysical assays revealed that MaviP35 proteins produced in bacteria and yeast had similar primary and secondary structures. However, bacterially produced MaviP35 possessed greater thermal stability and propensity to form higher order oligomers than its counterpart purified from yeast. Caspase 3 could process yeast-purified MaviP35, but failed to detectably cleave bacterially purified MaviP35. These data suggest that bacterially produced P35 proteins adopt subtly different conformations from their yeast-expressed counterparts, which hinder caspase access to the reactive site loop to reduce the potency of caspase inhibition, and promote aggregation. These data highlight the differential caspase inhibition by recombinant P35 proteins purified from different sources, and caution that analyses of bacterially produced P35 family members (and perhaps other types of proteins) may underestimate their activity. PMID:22720082

  15. Structure-based repurposing of FDA-approved drugs as inhibitors of NEDD8-activating enzyme.

    PubMed

    Zhong, Hai-Jing; Liu, Li-Juan; Chan, Daniel Shiu-Hin; Wang, Hui-Min; Chan, Philip Wai Hong; Ma, Dik-Lung; Leung, Chung-Hang

    2014-07-01

    We report the discovery of an inhibitor of NEDD8-activating enzyme (NAE) by an integrated virtual screening approach. Piperacillin 1 inhibited NAE activity in cell-free and cell-based systems with high selectivity. Furthermore, piperacillin 1 was able to inhibit the degradation of the NAE downstream protein substrate p27(kip1). Our molecular modeling and kinetic studies suggested that this compound may act as a non-covalent ATP-competitive inhibitor of NAE.

  16. Isolation, Co-Crystallization and Structure-Based Characterization of Anabaenopeptins as Highly Potent Inhibitors of Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa)

    PubMed Central

    Schreuder, Herman; Liesum, Alexander; Lönze, Petra; Stump, Heike; Hoffmann, Holger; Schiell, Matthias; Kurz, Michael; Toti, Luigi; Bauer, Armin; Kallus, Christopher; Klemke-Jahn, Christine; Czech, Jörg; Kramer, Dan; Enke, Heike; Niedermeyer, Timo H. J.; Morrison, Vincent; Kumar, Vasant; Brönstrup, Mark

    2016-01-01

    Mature thrombin activatable fibrinolysis inhibitor (TAFIa) is a carboxypeptidase that stabilizes fibrin clots by removing C-terminal arginines and lysines from partially degraded fibrin. Inhibition of TAFIa stimulates the degradation of fibrin clots and may help to prevent thrombosis. Applying a lead finding approach based on literature-mining, we discovered that anabaenopeptins, cyclic peptides produced by cyanobacteria, were potent inhibitors of TAFIa with IC50 values as low as 1.5 nM. We describe the isolation and structure elucidation of 20 anabaenopeptins, including 13 novel congeners, as well as their pronounced structure-activity relationships (SAR) with respect to inhibition of TAFIa. Crystal structures of the anabaenopeptins B, C and F bound to the surrogate protease carboxypeptidase B revealed the binding modes of these large (~850 Da) compounds in detail and explained the observed SAR, i.e. the strong dependence of the potency on a basic (Arg, Lys) exocyclic residue that addressed the S1’ binding pocket, and a broad tolerance towards substitutions in the pentacyclic ring that acted as a plug of the active site. PMID:27604544

  17. Isolation, Co-Crystallization and Structure-Based Characterization of Anabaenopeptins as Highly Potent Inhibitors of Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa).

    PubMed

    Schreuder, Herman; Liesum, Alexander; Lönze, Petra; Stump, Heike; Hoffmann, Holger; Schiell, Matthias; Kurz, Michael; Toti, Luigi; Bauer, Armin; Kallus, Christopher; Klemke-Jahn, Christine; Czech, Jörg; Kramer, Dan; Enke, Heike; Niedermeyer, Timo H J; Morrison, Vincent; Kumar, Vasant; Brönstrup, Mark

    2016-01-01

    Mature thrombin activatable fibrinolysis inhibitor (TAFIa) is a carboxypeptidase that stabilizes fibrin clots by removing C-terminal arginines and lysines from partially degraded fibrin. Inhibition of TAFIa stimulates the degradation of fibrin clots and may help to prevent thrombosis. Applying a lead finding approach based on literature-mining, we discovered that anabaenopeptins, cyclic peptides produced by cyanobacteria, were potent inhibitors of TAFIa with IC50 values as low as 1.5 nM. We describe the isolation and structure elucidation of 20 anabaenopeptins, including 13 novel congeners, as well as their pronounced structure-activity relationships (SAR) with respect to inhibition of TAFIa. Crystal structures of the anabaenopeptins B, C and F bound to the surrogate protease carboxypeptidase B revealed the binding modes of these large (~850 Da) compounds in detail and explained the observed SAR, i.e. the strong dependence of the potency on a basic (Arg, Lys) exocyclic residue that addressed the S1' binding pocket, and a broad tolerance towards substitutions in the pentacyclic ring that acted as a plug of the active site. PMID:27604544

  18. Immunomodulatory activity of a chymotrypsin inhibitor from Momordica cochinchinensis seeds.

    PubMed

    Tsoi, Alex Yuen-Kam; Ng, Tzi-Bun; Fong, Wing-Ping

    2006-09-01

    Serine protease inhibitors are widely distributed in the plant kingdom. Many of them have been purified and characterized from different species. While the physicochemical properties of these protease inhibitors have been extensively investigated, their biological effects, e.g. immunomodulatory effect, remain relatively unexplored. Recently, we isolated a chymotrypsin-specific inhibitor (MCoCI) from the seeds of Momordica cochinchinensis (Lour) Spreng (Family Cucurbitaceae), the traditional Chinese medicine known as Mubiezhi, which has been used as an antiinflammatory agent. In the present study, the effects of MCoCI on different types of cells of the immune system, including splenocytes, splenic lymphocytes, neutrophils, bone marrow cells and macrophages, were investigated. MCoCI was shown to possess immuno-enhancing and antiinflammatory effects. MCoCI could stimulate the proliferation of different cells of the immune system, e.g. splenocytes, splenic lymphocytes and bone marrow cells, in a manner comparable to that of Concanavalin A. Moreover, MCoCI could also suppress the formation of hydrogen peroxide in neutrophils and macrophages. These immunomodulatory effects may explain some of the therapeutic actions of Mubiezhi. PMID:16733830

  19. Immunomodulatory activity of a chymotrypsin inhibitor from Momordica cochinchinensis seeds.

    PubMed

    Tsoi, Alex Yuen-Kam; Ng, Tzi-Bun; Fong, Wing-Ping

    2006-09-01

    Serine protease inhibitors are widely distributed in the plant kingdom. Many of them have been purified and characterized from different species. While the physicochemical properties of these protease inhibitors have been extensively investigated, their biological effects, e.g. immunomodulatory effect, remain relatively unexplored. Recently, we isolated a chymotrypsin-specific inhibitor (MCoCI) from the seeds of Momordica cochinchinensis (Lour) Spreng (Family Cucurbitaceae), the traditional Chinese medicine known as Mubiezhi, which has been used as an antiinflammatory agent. In the present study, the effects of MCoCI on different types of cells of the immune system, including splenocytes, splenic lymphocytes, neutrophils, bone marrow cells and macrophages, were investigated. MCoCI was shown to possess immuno-enhancing and antiinflammatory effects. MCoCI could stimulate the proliferation of different cells of the immune system, e.g. splenocytes, splenic lymphocytes and bone marrow cells, in a manner comparable to that of Concanavalin A. Moreover, MCoCI could also suppress the formation of hydrogen peroxide in neutrophils and macrophages. These immunomodulatory effects may explain some of the therapeutic actions of Mubiezhi.

  20. Suppressive Effects of the Site 1 Protease (S1P) Inhibitor, PF-429242, on Dengue Virus Propagation.

    PubMed

    Uchida, Leo; Urata, Shuzo; Ulanday, Gianne Eduard L; Takamatsu, Yuki; Yasuda, Jiro; Morita, Kouichi; Hayasaka, Daisuke

    2016-02-01

    Dengue virus (DENV) infection causes one of the most widespread mosquito-borne diseases in the world. Despite the great need, effective vaccines and practical antiviral therapies are still under development. Intracellular lipid levels are regulated by sterol regulatory elements-binding proteins (SREBPs), which are activated by serine protease, site 1 protease (S1P). Small compound PF-429242 is known as a S1P inhibitor and the antivirus effects have been reported in some viruses. In this study, we examined the anti-DENV effects of PF-429242 using all four serotypes of DENV by several primate-derived cell lines. Moreover, emergence of drug-resistant DENV mutants was assessed by sequential passages with the drug. DENV dependency on intracellular lipids during their infection was also evaluated by adding extracellular lipids. The addition of PF-429242 showed suppression of viral propagation in all DENV serotypes. We showed that drug-resistant DENV mutants are unlikely to emerge after five times sequential passages through treatment with PF-429242. Although the levels of intracellular cholesterol and lipid droplets were reduced by PF-429242, viral propagations were not recovered by addition of exogenous cholesterol or fatty acids, indicating that the reduction of LD and cholesterol caused by PF-429242 treatment is not related to its mechanism of action against DENV propagation. Our results suggest that PF-429242 is a promising candidate for an anti-DENV agent. PMID:26875984

  1. Suppressive Effects of the Site 1 Protease (S1P) Inhibitor, PF-429242, on Dengue Virus Propagation

    PubMed Central

    Uchida, Leo; Urata, Shuzo; Ulanday, Gianne Eduard L.; Takamatsu, Yuki; Yasuda, Jiro; Morita, Kouichi; Hayasaka, Daisuke

    2016-01-01

    Dengue virus (DENV) infection causes one of the most widespread mosquito-borne diseases in the world. Despite the great need, effective vaccines and practical antiviral therapies are still under development. Intracellular lipid levels are regulated by sterol regulatory elements-binding proteins (SREBPs), which are activated by serine protease, site 1 protease (S1P). Small compound PF-429242 is known as a S1P inhibitor and the antivirus effects have been reported in some viruses. In this study, we examined the anti-DENV effects of PF-429242 using all four serotypes of DENV by several primate-derived cell lines. Moreover, emergence of drug-resistant DENV mutants was assessed by sequential passages with the drug. DENV dependency on intracellular lipids during their infection was also evaluated by adding extracellular lipids. The addition of PF-429242 showed suppression of viral propagation in all DENV serotypes. We showed that drug-resistant DENV mutants are unlikely to emerge after five times sequential passages through treatment with PF-429242. Although the levels of intracellular cholesterol and lipid droplets were reduced by PF-429242, viral propagations were not recovered by addition of exogenous cholesterol or fatty acids, indicating that the reduction of LD and cholesterol caused by PF-429242 treatment is not related to its mechanism of action against DENV propagation. Our results suggest that PF-429242 is a promising candidate for an anti-DENV agent. PMID:26875984

  2. Suppressive Effects of the Site 1 Protease (S1P) Inhibitor, PF-429242, on Dengue Virus Propagation.

    PubMed

    Uchida, Leo; Urata, Shuzo; Ulanday, Gianne Eduard L; Takamatsu, Yuki; Yasuda, Jiro; Morita, Kouichi; Hayasaka, Daisuke

    2016-02-10

    Dengue virus (DENV) infection causes one of the most widespread mosquito-borne diseases in the world. Despite the great need, effective vaccines and practical antiviral therapies are still under development. Intracellular lipid levels are regulated by sterol regulatory elements-binding proteins (SREBPs), which are activated by serine protease, site 1 protease (S1P). Small compound PF-429242 is known as a S1P inhibitor and the antivirus effects have been reported in some viruses. In this study, we examined the anti-DENV effects of PF-429242 using all four serotypes of DENV by several primate-derived cell lines. Moreover, emergence of drug-resistant DENV mutants was assessed by sequential passages with the drug. DENV dependency on intracellular lipids during their infection was also evaluated by adding extracellular lipids. The addition of PF-429242 showed suppression of viral propagation in all DENV serotypes. We showed that drug-resistant DENV mutants are unlikely to emerge after five times sequential passages through treatment with PF-429242. Although the levels of intracellular cholesterol and lipid droplets were reduced by PF-429242, viral propagations were not recovered by addition of exogenous cholesterol or fatty acids, indicating that the reduction of LD and cholesterol caused by PF-429242 treatment is not related to its mechanism of action against DENV propagation. Our results suggest that PF-429242 is a promising candidate for an anti-DENV agent.

  3. Demethylase Inhibitor Fungicide Resistance in Pyrenophora teres f. sp. teres Associated with Target Site Modification and Inducible Overexpression of Cyp51.

    PubMed

    Mair, Wesley J; Deng, Weiwei; Mullins, Jonathan G L; West, Samuel; Wang, Penghao; Besharat, Naghmeh; Ellwood, Simon R; Oliver, Richard P; Lopez-Ruiz, Francisco J

    2016-01-01

    Pyrenophora teres f. sp. teres is the cause of net form of net blotch (NFNB), an economically important foliar disease in barley (Hordeum vulgare). Net and spot forms of net blotch are widely controlled using site-specific systemic fungicides. Although resistance to succinate dehydrogenase inhibitors and quinone outside inhibitors has been addressed before in net blotches, mechanisms controlling demethylation inhibitor resistance have not yet been reported at the molecular level. Here we report the isolation of strains of NFNB in Australia since 2013 resistant to a range of demethylase inhibitor fungicides. Cyp51A:KO103-A1, an allele with the mutation F489L, corresponding to the archetype F495I in Aspergillus fumigatus, was only present in resistant strains and was correlated with resistance factors to various demethylase inhibitors ranging from 1.1 for epoxiconazole to 31.7 for prochloraz. Structural in silico modeling of the sensitive and resistant CYP51A proteins docked with different demethylase inhibitor fungicides showed how the interaction of F489L within the heme cavity produced a localized constriction of the region adjacent to the docking site that is predicted to result in lower binding affinities. Resistant strains also displayed enhanced induced expression of the two Cyp51A paralogs and of Cyp51B genes. While Cyp51B was found to be constitutively expressed in the absence of fungicide, Cyp51A was only detected at extremely low levels. Under fungicide induction, expression of Cyp51B, Cyp51A2, and Cyp51A1 was shown to be 1.6-, 3,- and 5.3-fold higher, respectively in the resistant isolate compared to the wild type. These increased levels of expression were not supported by changes in the promoters of any of the three genes. The implications of these findings on demethylase inhibitor activity will require current net blotch management strategies to be reconsidered in order to avoid the development of further resistance and preserve the lifespan of

  4. Demethylase Inhibitor Fungicide Resistance in Pyrenophora teres f. sp. teres Associated with Target Site Modification and Inducible Overexpression of Cyp51

    PubMed Central

    Mair, Wesley J.; Deng, Weiwei; Mullins, Jonathan G. L.; West, Samuel; Wang, Penghao; Besharat, Naghmeh; Ellwood, Simon R.; Oliver, Richard P.; Lopez-Ruiz, Francisco J.

    2016-01-01

    Pyrenophora teres f. sp. teres is the cause of net form of net blotch (NFNB), an economically important foliar disease in barley (Hordeum vulgare). Net and spot forms of net blotch are widely controlled using site-specific systemic fungicides. Although resistance to succinate dehydrogenase inhibitors and quinone outside inhibitors has been addressed before in net blotches, mechanisms controlling demethylation inhibitor resistance have not yet been reported at the molecular level. Here we report the isolation of strains of NFNB in Australia since 2013 resistant to a range of demethylase inhibitor fungicides. Cyp51A:KO103-A1, an allele with the mutation F489L, corresponding to the archetype F495I in Aspergillus fumigatus, was only present in resistant strains and was correlated with resistance factors to various demethylase inhibitors ranging from 1.1 for epoxiconazole to 31.7 for prochloraz. Structural in silico modeling of the sensitive and resistant CYP51A proteins docked with different demethylase inhibitor fungicides showed how the interaction of F489L within the heme cavity produced a localized constriction of the region adjacent to the docking site that is predicted to result in lower binding affinities. Resistant strains also displayed enhanced induced expression of the two Cyp51A paralogs and of Cyp51B genes. While Cyp51B was found to be constitutively expressed in the absence of fungicide, Cyp51A was only detected at extremely low levels. Under fungicide induction, expression of Cyp51B, Cyp51A2, and Cyp51A1 was shown to be 1.6-, 3,- and 5.3-fold higher, respectively in the resistant isolate compared to the wild type. These increased levels of expression were not supported by changes in the promoters of any of the three genes. The implications of these findings on demethylase inhibitor activity will require current net blotch management strategies to be reconsidered in order to avoid the development of further resistance and preserve the lifespan of

  5. Demethylase Inhibitor Fungicide Resistance in Pyrenophora teres f. sp. teres Associated with Target Site Modification and Inducible Overexpression of Cyp51

    PubMed Central

    Mair, Wesley J.; Deng, Weiwei; Mullins, Jonathan G. L.; West, Samuel; Wang, Penghao; Besharat, Naghmeh; Ellwood, Simon R.; Oliver, Richard P.; Lopez-Ruiz, Francisco J.

    2016-01-01

    Pyrenophora teres f. sp. teres is the cause of net form of net blotch (NFNB), an economically important foliar disease in barley (Hordeum vulgare). Net and spot forms of net blotch are widely controlled using site-specific systemic fungicides. Although resistance to succinate dehydrogenase inhibitors and quinone outside inhibitors has been addressed before in net blotches, mechanisms controlling demethylation inhibitor resistance have not yet been reported at the molecular level. Here we report the isolation of strains of NFNB in Australia since 2013 resistant to a range of demethylase inhibitor fungicides. Cyp51A:KO103-A1, an allele with the mutation F489L, corresponding to the archetype F495I in Aspergillus fumigatus, was only present in resistant strains and was correlated with resistance factors to various demethylase inhibitors ranging from 1.1 for epoxiconazole to 31.7 for prochloraz. Structural in silico modeling of the sensitive and resistant CYP51A proteins docked with different demethylase inhibitor fungicides showed how the interaction of F489L within the heme cavity produced a localized constriction of the region adjacent to the docking site that is predicted to result in lower binding affinities. Resistant strains also displayed enhanced induced expression of the two Cyp51A paralogs and of Cyp51B genes. While Cyp51B was found to be constitutively expressed in the absence of fungicide, Cyp51A was only detected at extremely low levels. Under fungicide induction, expression of Cyp51B, Cyp51A2, and Cyp51A1 was shown to be 1.6-, 3,- and 5.3-fold higher, respectively in the resistant isolate compared to the wild type. These increased levels of expression were not supported by changes in the promoters of any of the three genes. The implications of these findings on demethylase inhibitor activity will require current net blotch management strategies to be reconsidered in order to avoid the development of further resistance and preserve the lifespan of

  6. Demethylase Inhibitor Fungicide Resistance in Pyrenophora teres f. sp. teres Associated with Target Site Modification and Inducible Overexpression of Cyp51.

    PubMed

    Mair, Wesley J; Deng, Weiwei; Mullins, Jonathan G L; West, Samuel; Wang, Penghao; Besharat, Naghmeh; Ellwood, Simon R; Oliver, Richard P; Lopez-Ruiz, Francisco J

    2016-01-01

    Pyrenophora teres f. sp. teres is the cause of net form of net blotch (NFNB), an economically important foliar disease in barley (Hordeum vulgare). Net and spot forms of net blotch are widely controlled using site-specific systemic fungicides. Although resistance to succinate dehydrogenase inhibitors and quinone outside inhibitors has been addressed before in net blotches, mechanisms controlling demethylation inhibitor resistance have not yet been reported at the molecular level. Here we report the isolation of strains of NFNB in Australia since 2013 resistant to a range of demethylase inhibitor fungicides. Cyp51A:KO103-A1, an allele with the mutation F489L, corresponding to the archetype F495I in Aspergillus fumigatus, was only present in resistant strains and was correlated with resistance factors to various demethylase inhibitors ranging from 1.1 for epoxiconazole to 31.7 for prochloraz. Structural in silico modeling of the sensitive and resistant CYP51A proteins docked with different demethylase inhibitor fungicides showed how the interaction of F489L within the heme cavity produced a localized constriction of the region adjacent to the docking site that is predicted to result in lower binding affinities. Resistant strains also displayed enhanced induced expression of the two Cyp51A paralogs and of Cyp51B genes. While Cyp51B was found to be constitutively expressed in the absence of fungicide, Cyp51A was only detected at extremely low levels. Under fungicide induction, expression of Cyp51B, Cyp51A2, and Cyp51A1 was shown to be 1.6-, 3,- and 5.3-fold higher, respectively in the resistant isolate compared to the wild type. These increased levels of expression were not supported by changes in the promoters of any of the three genes. The implications of these findings on demethylase inhibitor activity will require current net blotch management strategies to be reconsidered in order to avoid the development of further resistance and preserve the lifespan of

  7. p53 Small Molecule Inhibitor Enhances Temozolomide Cytotoxic Activity against Intracranial Glioblastoma Xenografts

    PubMed Central

    Dinca, Eduard B.; Lu, Kan V.; Sarkaria, Jann N.; Pieper, Russell O.; Prados, Michael D.; Haas-Kogan, Daphne A.; VandenBerg, Scott R.; Berger, Mitchel S.; James, C. David

    2010-01-01

    In this study we investigated corresponding precursor and active forms of a p53 small molecule inhibitor for effect on temozolomide (TMZ) anti-tumor activity against glioblastoma (GBM), using both in vitro and in vivo experimental approaches. Results from in vitro cell viability analysis showed that the cytotoxic activity of TMZ was substantially increased when GBMs with wild-type p53 were co-treated with the active form of p53 inhibitor, and this heightened cytotoxic response was accompanied by increased PARP cleavage as well as elevated cellular phospho-H2AX. Analysis of the same series of GBMs, as intracranial xenografts in athymic mice, and administering corresponding p53 inhibitor precursor, that is converted to the active compound in vivo, yielded results consistent with the in vitro analyses: i.e., TMZ + p53 inhibitor precursor co-treatment, of three distinct wild-type p53 GBM xenografts, resulted in significant enhancement of TMZ anti-tumor effect relative to treatment with TMZ alone, as indicated by serial bioluminescence monitoring as well as survival analysis (p < 0.001 for co-treatment survival benefit in each case). Mice receiving intracranial injection with p53 null GBM showed similar survival benefit from TMZ treatment regardless of the presence or absence of p53 inhibitor precursor. In total, our results indicate that the p53 active and precursor inhibitor pair enhance TMZ cytotoxicity in vitro and in vivo, respectively, and do so in a p53-dependent manner. PMID:19074867

  8. p53 Small-molecule inhibitor enhances temozolomide cytotoxic activity against intracranial glioblastoma xenografts.

    PubMed

    Dinca, Eduard B; Lu, Kan V; Sarkaria, Jann N; Pieper, Russell O; Prados, Michael D; Haas-Kogan, Daphne A; Vandenberg, Scott R; Berger, Mitchel S; James, C David

    2008-12-15

    In this study, we investigated the precursor and active forms of a p53 small-molecule inhibitor for their effects on temozolomide (TMZ) antitumor activity against glioblastoma (GBM), using both in vitro and in vivo experimental approaches. Results from in vitro cell viability analysis showed that the cytotoxic activity of TMZ was substantially increased when p53 wild-type (p53(wt)) GBMs were cotreated with the active form of p53 inhibitor, and this heightened cytotoxic response was accompanied by increased poly(ADP-ribose) polymerase cleavage as well as elevated cellular phospho-H2AX. Analysis of the same series of GBMs, as intracranial xenografts in athymic mice, and administering corresponding p53 inhibitor precursor, which is converted to the active compound in vivo, yielded results consistent with the in vitro analyses: TMZ + p53 inhibitor precursor cotreatment of three distinct p53(wt) GBM xenografts resulted in significant enhancement of TMZ antitumor effect relative to treatment with TMZ alone, as indicated by serial bioluminescence monitoring as well as survival analysis (P < 0.001 for cotreatment survival benefit in each case). Mice receiving intracranial injection with p53(null) GBM showed similar survival benefit from TMZ treatment regardless of the presence or absence of p53 inhibitor precursor. In total, our results indicate that the p53 active and precursor inhibitor pair enhances TMZ cytotoxicity in vitro and in vivo, respectively, and do so in a p53-dependent manner.

  9. The HMG-CoA reductase inhibitor rosuvastatin inhibits plasminogen activator inhibitor-1 expression and secretion in human adipocytes.

    PubMed

    Laumen, Helmut; Skurk, Thomas; Hauner, Hans

    2008-02-01

    Human preadipocytes and adipocytes are known to produce the proatherogenic factor PAI-1 and proinflammatory cytokines, and obesity was found to be state of increased adipose production of these factors. In the present study, we investigated the effect of rosuvastatin on the regulation of PAI-1 gene expression in human adipocytes. Human preadipocytes, adipocytes in primary culture and the SGBS cell line were used as cell models. Cells were transfected using various constructs and promoter activity was measured as luciferase activity. PAI-1 expression was measured by quantitative RT-PCR and ELISA. Rosuvastatin inhibited PAI-1 mRNA expression and secretion of the protein in a concentration-dependent manner. This effect was reversed by isoprenoids. Addition of MEK-inhibitors and NFkappaB inhibitors also reduced PAI-1 expression and PAI-1 promoter luciferase activity. Further experiments revealed that rosuvastatin down-regulated the MEKK-1 mediated activation of the PAI-1 promoter. In conclusion our data suggest that rosuvastatin inhibits PAI-1 expression and release from human adipocytes via a MEKK-1-dependent but not a NFkappaB-dependent mechanism.

  10. Protease inhibitor in scorpion (Mesobuthus eupeus) venom prolongs the biological activities of the crude venom.

    PubMed

    Ma, Hakim; Xiao-Peng, Tang; Yang, Shi-Long; Lu, Qiu-Min; Lai, Ren

    2016-08-01

    It is hypothesized that protease inhibitors play an essential role in survival of venomous animals through protecting peptide/protein toxins from degradation by proteases in their prey or predators. However, the biological function of protease inhibitors in scorpion venoms remains unknown. In the present study, a trypsin inhibitor was purified and characterized from the venom of scorpion Mesobuthus eupeus, which enhanced the biological activities of crude venom components in mice when injected in combination with crude venom. This protease inhibitor, named MeKTT-1, belonged to Kunitz-type toxins subfamily. Native MeKTT-1 selectively inhibited trypsin with a Kivalue of 130 nmol·L(-1). Furthermore, MeKTT-1 was shown to be a thermo-stable peptide. In animal behavioral tests, MeKTT-1 prolonged the pain behavior induced by scorpion crude venom, suggesting that protease inhibitors in scorpion venom inhibited proteases and protect the functionally important peptide/protein toxins from degradation, consequently keeping them active longer. In conclusion, this was the first experimental evidence about the natural existence of serine protease inhibitor in the venom of scorpion Mesobuthus eupeus, which preserved the activity of venom components, suggests that scorpions may use protease inhibitors for survival. PMID:27608950

  11. Development of an activity assay for discovery of inhibitors of lipopolysaccharide transport.

    PubMed

    Gronenberg, Luisa S; Kahne, Daniel

    2010-03-01

    The outer membrane of gram-negative bacteria contains an outer leaflet composed of lipopolysaccharide (LPS) that is transported to this location by a pathway that is essential for viability. It has been suggested that inhibitors of this pathway could be useful antibiotics. Herein we reconstitute the activity of the ATPase component (LptB) of the ABC transporter that initiates LPS transport and assembly. We developed a high-throughput assay and screened a library of kinase inhibitors against LptB. We identified two classes of ATP-competitive inhibitors. These are the first inhibitors of the ATPase component of any bacterial ABC transporter. The small-molecule inhibitors will be very useful tools for further biochemical studies of the proteins involved in LPS transport and assembly.

  12. Raman chemical mapping reveals site of action of HIV protease inhibitors in HPV16 E6 expressing cervical carcinoma cells.

    PubMed

    Kim, Dong-Hyun; Jarvis, Roger M; Allwood, J William; Batman, Gavin; Moore, Rowan E; Marsden-Edwards, Emma; Hampson, Lynne; Hampson, Ian N; Goodacre, Royston

    2010-12-01

    It has been shown that the HIV protease inhibitors indinavir and lopinavir may have activity against the human papilloma virus (HPV) type 16 inhibiting HPV E6-mediated proteasomal degradation of p53 in cultured cervical carcinoma cells. However, their mode and site of action is unknown. HPV-negative C33A cervical carcinoma cells and the same cells stably transfected with E6 (C33AE6) were exposed to indinavir and lopinavir at concentrations of 1 mM and 30 μM, respectively. The intracellular distribution of metabolites and metabolic changes induced by these treatments were investigated by Raman microspectroscopic imaging combined with the analysis of cell fractionation products by liquid chromatography-mass spectrometry (LC-MS). A uniform cellular distribution of proteins was found in drug-treated cells irrespective of cell type. Indinavir was observed to co-localise with nucleic acid in the nucleus, but only in E6 expressing cells. Principal components analysis (PCA) score maps generated on the full Raman hypercube and the corresponding PCA loadings plots revealed that the majority of metabolic variations influenced by the drug exposure within the cells were associated with changes in nucleic acids. Analysis of cell fractionation products by LC-MS confirmed that the level of indinavir in nuclear extracts was approximately eight-fold greater than in the cytoplasm. These data demonstrate that indinavir undergoes enhanced nuclear accumulation in E6-expressing cells, which suggests that this is the most likely site of action for this compound against HPV.

  13. Control of active sites in flocculation: Concept of equivalent active sites''

    SciTech Connect

    Behl, S.; Moudgil, B.M. . Dept. of Materials Science and Engineering)

    1993-12-01

    Flocculation and dispersion of solids are strong functions of the amount and conformation of the adsorbed polymer. Regions of dispersion and flocculation of solids with particular polymer molecules may be deduced from saturation adsorption data. The concept of equivalent active sites'' is proposed to explain flocculation and dispersion behavior irrespective of the amount or conformation of the adsorbed polymer. The concept has been further extended to study the selective flocculation process.

  14. Purification and characterization of a Kunitz inhibitor from Poincianella pyramidalis with insecticide activity against the Mediterranean flour moth.

    PubMed

    Guimarães, Lays Cordeiro; de Oliveira, Caio Fernando Ramalho; Marangoni, Sergio; de Oliveira, Daniella Gorete Lourenço; Macedo, Maria Lígia Rodrigues

    2015-02-01

    This paper describes the characterization of a trypsin inhibitor from Poincianella pyramidalis seeds (PpyTI). The partial sequencing of PpyTI revealed homology to Kunitz inhibitors, clustered as a member of Family I03 in MEROPS database. PpyTI has a single polypeptide chain of 19,042 Da and presents stability at high temperatures (up to 70 °C) and a wide range of pH. In vitro assays showed that disulfide bridges have an important stabilization role of reactive site in PpyTI, a characteristic shared among several Kunitz inhibitors. Bioassays carried out with the Mediterranean flour moth (Anagasta kuehniella) revealed a significant decrease in both larval weight and survival of PpyTI-fed larvae, besides a larval stage extension. Through biochemical analysis, we demonstrated that the PpyTI insecticide effects were triggered by digestion process commitment, through the inhibition of trypsin and chymotrypsin activities, the major digestive enzymes in this species. The insecticide effects and biochemical characterization of PpyTI encourage further studies using this inhibitor for insect pest control.

  15. PTP1B inhibitor promotes endothelial cell motility by activating the DOCK180/Rac1 pathway

    PubMed Central

    Wang, Yuan; Yan, Feng; Ye, Qing; Wu, Xiao; Jiang, Fan

    2016-01-01

    Promoting endothelial cell (EC) migration is important not only for therapeutic angiogenesis, but also for accelerating re-endothelialization after vessel injury. Several recent studies have shown that inhibition of protein tyrosine phosphatase 1B (PTP1B) may promote EC migration and angiogenesis by enhancing the vascular endothelial growth factor receptor-2 (VEGFR2) signalling. In the present study, we demonstrated that PTP1B inhibitor could promote EC adhesion, spreading and migration, which were abolished by the inhibitor of Rac1 but not RhoA GTPase. PTP1B inhibitor significantly increased phosphorylation of p130Cas, and the interactions among p130Cas, Crk and DOCK180; whereas the phosphorylation levels of focal adhesion kinase, Src, paxillin, or Vav2 were unchanged. Gene silencing of DOCK180, but not Vav2, abrogated the effects of PTP1B inhibitor on EC motility. The effects of PTP1B inhibitor on EC motility and p130Cas/DOCK180 activation persisted in the presence of the VEGFR2 antagonist. In conclusion, we suggest that stimulation of the DOCK180 pathway represents an alternative mechanism of PTP1B inhibitor-stimulated EC motility, which does not require concomitant VEGFR2 activation as a prerequisite. Therefore, PTP1B inhibitor may be a useful therapeutic strategy for promoting EC migration in cardiovascular patients in which the VEGF/VEGFR functions are compromised. PMID:27052191

  16. Carborane-based carbonic anhydrase inhibitors.

    PubMed

    Brynda, Jiří; Mader, Pavel; Šícha, Václav; Fábry, Milan; Poncová, Kristýna; Bakardiev, Mario; Grüner, Bohumír; Cígler, Petr; Řezáčová, Pavlína

    2013-12-16

    CA inhibitors: Human carbonic anhydrases (CAs) are diagnostic and therapeutic targets. Various carborane cages are shown to act as active-site-directed inhibitors, and substitution with a sulfamide group and other substituents leads to compounds with high selectivity towards the cancer-specific isozyme IX. Crystal structures of the carboranes in the active site provide information that can be applied to the structure-based design of specific inhibitors. PMID:24307504

  17. The Effect of Clade-Specific Sequence Polymorphisms on HIV-1 Protease Activity and Inhibitor Resistance Pathways

    SciTech Connect

    Bandaranayake, Rajintha M.; Kolli, Madhavi; King, Nancy M.; Nalivaika, Ellen A.; Heroux, Annie; Kakizawa, Junko; Sugiura, Wataru; Schiffer, Celia A.

    2010-09-08

    The majority of HIV-1 infections around the world result from non-B clade HIV-1 strains. The CRF01{_}AE (AE) strain is seen principally in Southeast Asia. AE protease differs by {approx}10% in amino acid sequence from clade B protease and carries several naturally occurring polymorphisms that are associated with drug resistance in clade B. AE protease has been observed to develop resistance through a nonactive-site N88S mutation in response to nelfinavir (NFV) therapy, whereas clade B protease develops both the active-site mutation D30N and the nonactive-site mutation N88D. Structural and biochemical studies were carried out with wild-type and NFV-resistant clade B and AE protease variants. The relationship between clade-specific sequence variations and pathways to inhibitor resistance was also assessed. AE protease has a lower catalytic turnover rate than clade B protease, and it also has weaker affinity for both NFV and darunavir (DRV). This weaker affinity may lead to the nonactive-site N88S variant in AE, which exhibits significantly decreased affinity for both NFV and DRV. The D30N/N88D mutations in clade B resulted in a significant loss of affinity for NFV and, to a lesser extent, for DRV. A comparison of crystal structures of AE protease shows significant structural rearrangement in the flap hinge region compared with those of clade B protease and suggests insights into the alternative pathways to NFV resistance. In combination, our studies show that sequence polymorphisms within clades can alter protease activity and inhibitor binding and are capable of altering the pathway to inhibitor resistance.

  18. Exploration of the nicotinamide-binding site of the tankyrases, identifying 3-arylisoquinolin-1-ones as potent and selective inhibitors in vitro.

    PubMed

    Paine, Helen A; Nathubhai, Amit; Woon, Esther C Y; Sunderland, Peter T; Wood, Pauline J; Mahon, Mary F; Lloyd, Matthew D; Thompson, Andrew S; Haikarainen, Teemu; Narwal, Mohit; Lehtiö, Lari; Threadgill, Michael D

    2015-09-01

    Tankyrases-1 and -2 (TNKS-1 and TNKS-2) have three cellular roles which make them important targets in cancer. Using NAD(+) as a substrate, they poly(ADP-ribosyl)ate TRF1 (regulating lengths of telomeres), NuMA (facilitating mitosis) and axin (in wnt/β-catenin signalling). Using molecular modelling and the structure of the weak inhibitor 5-aminoiso quinolin-1-one, 3-aryl-5-substituted-isoquinolin-1-ones were designed as inhibitors to explore the structure-activity relationships (SARs) for binding and to define the shape of a hydrophobic cavity in the active site. 5-Amino-3-arylisoquinolinones were synthesised by Suzuki-Miyaura coupling of arylboronic acids to 3-bromo-1-methoxy-5-nitro-isoquinoline, reduction and O-demethylation. 3-Aryl-5-methylisoquinolin-1-ones, 3-aryl-5-fluoroisoquinolin-1-ones and 3-aryl-5-methoxyisoquinolin-1-ones were accessed by deprotonation of 3-substituted-N,N,2-trimethylbenzamides and quench with an appropriate benzonitrile. SAR around the isoquinolinone core showed that aryl was required at the 3-position, optimally with a para-substituent. Small meta-substituents were tolerated but groups in the ortho-positions reduced or abolished activity. This was not due to lack of coplanarity of the rings, as shown by the potency of 4,5-dimethyl-3-phenylisoquinolin-1-one. Methyl and methoxy were optimal at the 5-position. SAR was rationalised by modelling and by crystal structures of examples with TNKS-2. The 3-aryl unit was located in a large hydrophobic cavity and the para-substituents projected into a tunnel leading to the exterior. Potency against TNKS-1 paralleled potency against TNKS-2. Most inhibitors were highly selective for TNKSs over PARP-1 and PARP-2. A range of highly potent and selective inhibitors is now available for cellular studies. PMID:26189030

  19. Targeting the RAS pathway by mitogen-activated protein kinase inhibitors.

    PubMed

    Kiessling, Michael K; Rogler, Gerhard

    2015-01-01

    Targeting of oncogenic driver mutations with small-molecule inhibitors resulted in powerful treatment options for cancer patients in recent years. The RAS (rat sarcoma) pathway is among the most frequently mutated pathways in human cancer. Whereas targeting mutant Kirsten RAS (KRAS) remains difficult, mutant B rapidly accelerated fibrosarcoma (BRAF) kinase is an established drug target in cancer. Now data show that neuroblastoma RAS (NRAS) and even Harvey RAS (HRAS) mutations could be predictive markers for treatment with mitogen-activated protein kinase (MEK) inhibitors. This review discusses recent preclinical and clinical studies of MEK inhibitors in BRAF and RAS mutant cancer. PMID:26691679

  20. Inhibitors of enzymes catalyzing modifications to histone lysine residues: structure, function and activity.

    PubMed

    Lillico, Ryan; Stesco, Nicholas; Khorshid Amhad, Tina; Cortes, Claudia; Namaka, Mike P; Lakowski, Ted M

    2016-05-01

    Gene expression is partly controlled by epigenetic mechanisms including histone-modifying enzymes. Some diseases are caused by changes in gene expression that can be mitigated by inhibiting histone-modifying enzymes. This review covers the enzyme inhibitors targeting histone lysine modifications. We summarize the enzymatic mechanisms of histone lysine acetylation, deacetylation, methylation and demethylation and discuss the biochemical roles of these modifications in gene expression and in disease. We discuss inhibitors of lysine acetylation, deacetylation, methylation and demethylation defining their structure-activity relationships and their potential mechanisms. We show that there are potentially indiscriminant off-target effects on gene expression even with the use of selective epigenetic enzyme inhibitors.

  1. Proteasome inhibitors reduce luciferase and beta-galactosidase activity in tissue culture cells.

    PubMed

    Deroo, Bonnie J; Archer, Trevor K

    2002-06-01

    Reporter enzymes are commonly used in cell biology to study transcriptional activity of genes. Recently, reporter enzymes in combination with compounds that inhibit proteasome function have been used to study the effect of blocking transcription factor degradation on gene activation. While investigating the effect of proteasome inhibition on steroid receptor activation of the mouse mammary tumor virus (MMTV) promoter, we found that treatment with proteasome inhibitors enhanced glucocorticoid activation of the promoter attached to a chloramphenicol acetyltransferase (CAT) reporter, but inhibited activation of MMTV attached to a firefly luciferase or beta-galactosidase reporter. MMTV RNA levels under these conditions correlated with the promoter activity observed using the CAT reporter, suggesting that proteasome inhibitor treatment interfered with luciferase or beta-galactosidase reporter assays. Washout experiments demonstrated that the majority of luciferase activity was lost if the proteasome inhibitor was added at the same time luciferase was produced, not once the functional protein was made, suggesting that proteasome inhibition interferes with production of luciferase protein. Indeed, we found that proteasome inhibitor treatment dramatically reduced the levels of luciferase and beta-galactosidase protein produced, as determined by Western blot. Thus, treatment with proteasome inhibitors interferes with luciferase and beta-galactosidase reporter assays, possibly by inhibiting production of a functional reporter protein.

  2. A straightforward ninhydrin-based method for collagenase activity and inhibitor screening of collagenase using spectrophotometry.

    PubMed

    Zhang, Yanfang; Fu, Yun; Zhou, Sufeng; Kang, Lixia; Li, Changzheng

    2013-06-01

    Currently protease assay kits, requiring substrate that is either radiolabeled or fluorescence labeled and specialized instruments, are all expensive. A simple, reliable assay of protease activity and its inhibitor screening for general laboratory is rare. Here we demonstrated a straightforward ninhydrin-based method for assay of collagenase activity and its inhibitor screening using spectrophotometry. In the method, without multistep sample treatments and substrate labeling, the hydrolytic products were directly traced by ninhydrin. The method is expected to be suitable for not only the assay of collagenase activity but also the others matrix metalloproteinases activities, and can be used for kinetic study.

  3. Feedback Activation of Leukemia Inhibitory Factor Receptor Limits Response to Histone Deacetylase Inhibitors in Breast Cancer.

    PubMed

    Zeng, Hanlin; Qu, Jia; Jin, Nan; Xu, Jun; Lin, Chenchu; Chen, Yi; Yang, Xinying; He, Xiang; Tang, Shuai; Lan, Xiaojing; Yang, Xiaotong; Chen, Ziqi; Huang, Min; Ding, Jian; Geng, Meiyu

    2016-09-12

    Histone deacetylase (HDAC) inhibitors have demonstrated clinical benefits in subtypes of hematological malignancies. However, the efficacy of HDAC inhibitors in solid tumors remains uncertain. This study takes breast cancer as a model to understand mechanisms accounting for limited response of HDAC inhibitors in solid tumors and to seek combination solutions. We discover that feedback activation of leukemia inhibitory factor receptor (LIFR) signaling in breast cancer limits the response to HDAC inhibition. Mechanistically, HDAC inhibition increases histone acetylation at the LIFR gene promoter, which recruits bromodomain protein BRD4, upregulates LIFR expression, and activates JAK1-STAT3 signaling. Importantly, JAK1 or BRD4 inhibition sensitizes breast cancer to HDAC inhibitors, implicating combination inhibition of HDAC with JAK1 or BRD4 as potential therapies for breast cancer. PMID:27622335

  4. Modulation of activation-loop phosphorylation by JAK inhibitors is binding mode dependent

    PubMed Central

    Bonenfant, Débora; Rubert, Joëlle; Vangrevelinghe, Eric; Scheufler, Clemens; Marque, Fanny; Régnier, Catherine H.; De Pover, Alain; Ryckelynck, Hugues; Bhagwat, Neha; Koppikar, Priya; Goel, Aviva; Wyder, Lorenza; Tavares, Gisele; Baffert, Fabienne; Pissot-Soldermann, Carole; Manley, Paul W.; Gaul, Christoph; Voshol, Hans; Levine, Ross L.; Sellers, William R.; Hofmann, Francesco; Radimerski, Thomas

    2016-01-01

    JAK inhibitors are being developed for the treatment of rheumatoid arthritis, psoriasis, myeloproliferative neoplasms and leukemias. Most of these drugs target the ATP-binding pocket and stabilize the active conformation of the JAK kinases. This type-I binding mode leads to an increase in JAK activation-loop phosphorylation, despite blockade of kinase function. Here we report that stabilizing the inactive state via type-II inhibition acts in the opposite manner, leading to a loss of activation-loop phosphorylation. We used X-ray crystallography to corroborate the binding mode and report for the first time the crystal structure of the JAK2 kinase domain in an inactive conformation. Importantly, JAK inhibitor-induced activation-loop phosphorylation requires receptor interaction, as well as intact kinase and pseudokinase domains. Hence, depending on the respective conformation stabilized by a JAK inhibitor, hyperphosphorylation of the activation-loop may or may not be elicited. PMID:22684457

  5. Characterization of inhibitor(s) of lymphocyte activation in serum from rats with adjuvant arthritis.

    PubMed

    Binderup, L; Bramm, E; Arrigoni-Martelli, E

    1978-01-01

    Serum from adjuvant arthritic rats inhibits the concanavalin A- (Con A) and lipopolysaccharide-induced stimulation of lymph node cells, leaving the basal and phytohemagglutinin-stimulated 3H-thymidine incorporation unaffected. Con A-stimulated 3H-thymidine uptake is also inhibited in rat spleen and peripheral blood lymphocytes and in dog peripheral blood lymphocytes. The intensity of the inhibitory activity in serum is positively correlated with the intensity of the secondary lesions of adjuvant arthritis. Inhibitory activity was not found in serum from rats bearing nystatin-induced inflammation. Serum fractionation studies indicated that the inhibitory activity cannot be attributed to low molecular weight alpha2-glycoproteins or to gamma-globulins and alpha2-macroglobulins, but it is present in a fraction migrating with beta-globulins. The inhibitory activity in arthritic rat serum is reduced by treatment with non-steroidal anti-inflammatory drugs, but is unaltered by D-penicillamine. It is suggested that this inhibitory activity is part of the systemic response to an immunologically mediated inflammation. PMID:151867

  6. Study of the Differential Activity of Thrombin Inhibitors Using Docking, QSAR, Molecular Dynamics, and MM-GBSA

    PubMed Central

    Mena-Ulecia, Karel; Tiznado, William; Caballero, Julio

    2015-01-01

    Non-peptidic thrombin inhibitors (TIs; 177 compounds) with diverse groups at motifs P1 (such as oxyguanidine, amidinohydrazone, amidine, amidinopiperidine), P2 (such as cyanofluorophenylacetamide, 2-(2-chloro-6-fluorophenyl)acetamide), and P3 (such as phenylethyl, arylsulfonate groups) were studied using molecular modeling to analyze their interactions with S1, S2, and S3 subsites of the thrombin binding site. Firstly, a protocol combining docking and three dimensional quantitative structure–activity relationship was performed. We described the orientations and preferred active conformations of the studied inhibitors, and derived a predictive CoMSIA model including steric, donor hydrogen bond, and acceptor hydrogen bond fields. Secondly, the dynamic behaviors of some selected TIs (compounds 26, 133, 147, 149, 162, and 177 in this manuscript) that contain different molecular features and different activities were analyzed by creating the solvated models and using molecular dynamics (MD) simulations. We used the conformational structures derived from MD to accomplish binding free energetic calculations using MM-GBSA. With this analysis, we theorized about the effect of van der Waals contacts, electrostatic interactions and solvation in the potency of TIs. In general, the contents reported in this article help to understand the physical and chemical characteristics of thrombin-inhibitor complexes. PMID:26599107

  7. Study of the Differential Activity of Thrombin Inhibitors Using Docking, QSAR, Molecular Dynamics, and MM-GBSA.

    PubMed

    Mena-Ulecia, Karel; Tiznado, William; Caballero, Julio

    2015-01-01

    Non-peptidic thrombin inhibitors (TIs; 177 compounds) with diverse groups at motifs P1 (such as oxyguanidine, amidinohydrazone, amidine, amidinopiperidine), P2 (such as cyanofluorophenylacetamide, 2-(2-chloro-6-fluorophenyl)acetamide), and P3 (such as phenylethyl, arylsulfonate groups) were studied using molecular modeling to analyze their interactions with S1, S2, and S3 subsites of the thrombin binding site. Firstly, a protocol combining docking and three dimensional quantitative structure-activity relationship was performed. We described the orientations and preferred active conformations of the studied inhibitors, and derived a predictive CoMSIA model including steric, donor hydrogen bond, and acceptor hydrogen bond fields. Secondly, the dynamic behaviors of some selected TIs (compounds 26, 133, 147, 149, 162, and 177 in this manuscript) that contain different molecular features and different activities were analyzed by creating the solvated models and using molecular dynamics (MD) simulations. We used the conformational structures derived from MD to accomplish binding free energetic calculations using MM-GBSA. With this analysis, we theorized about the effect of van der Waals contacts, electrostatic interactions and solvation in the potency of TIs. In general, the contents reported in this article help to understand the physical and chemical characteristics of thrombin-inhibitor complexes. PMID:26599107

  8. Antiatherogenic activity of fungal beauveriolides, inhibitors of lipid droplet accumulation in macrophages

    PubMed Central

    Namatame, Ichiji; Tomoda, Hiroshi; Ishibashi, Shun; Ōmura, Satoshi

    2004-01-01

    Beauveriolides I and III, isolated from the culture broth of fungal Beauveria sp. FO-6979, showed potent inhibitory activity of lipid droplet accumulation in primary mouse peritoneal macrophages. The cellular molecular target of this inhibitory activity was studied in macrophages. Beauveriolides I and III strongly inhibited the cholesteryl ester (CE) synthesis with IC50 values of 0.78 and 0.41 μM, respectively, without showing significant effects on the triacylglycerol and phospholipid synthesis. Furthermore, lysosomal cholesterol metabolism to CE in macrophages was inhibited by the compounds, indicating that the inhibition site lies within steps between cholesterol departure from the lysosome and CE synthesis in the endoplasmic reticulum. Therefore, acyl-CoA:cholesterol acyltransferase (ACAT) activity in the membrane fractions prepared from mouse macrophages was studied, resulting in a dose-dependent inhibition by beauveriolides I and III with IC50 values of 6.0 and 5.5 μM, respectively. Thus, we showed that the beauveriolides inhibit macrophage ACAT activity specifically, resulting in blockage of the CE synthesis, leading to a reduction of lipid droplets in macrophages. ACAT activity in the membrane fractions prepared from mouse liver and Caco-2 cells was also inhibited, indicating that the beauveriolides block both ACAT-1 and -2. Moreover, beauveriolides I and III exert antiatherogenic activity in both low-density lipoprotein receptor- and apolipoprotein E-knockout mice without any side effects such as diarrhea or cytotoxicity to adrenal tissues as observed for many synthetic ACAT inhibitors. Beauveriolides I and III are the first microbial cyclodepsipeptides having an in vivo antiatherosclerotic effect and show promise as potential lead compounds for antiatherosclerotic agents. PMID:14718664

  9. 'Unconventional' coordination chemistry by metal chelating fragments in a metalloprotein active site.

    PubMed

    Martin, David P; Blachly, Patrick G; Marts, Amy R; Woodruff, Tessa M; de Oliveira, César A F; McCammon, J Andrew; Tierney, David L; Cohen, Seth M

    2014-04-01

    The binding of three closely related chelators: 5-hydroxy-2-methyl-4H-pyran-4-thione (allothiomaltol, ATM), 3-hydroxy-2-methyl-4H-pyran-4-thione (thiomaltol, TM), and 3-hydroxy-4H-pyran-4-thione (thiopyromeconic acid, TPMA) to the active site of human carbonic anhydrase II (hCAII) has been investigated. Two of these ligands display a monodentate mode of coordination to the active site Zn(2+) ion in hCAII that is not recapitulated in model complexes of the enzyme active site. This unprecedented binding mode in the hCAII-thiomaltol complex has been characterized by both X-ray crystallography and X-ray spectroscopy. In addition, the steric restrictions of the active site force the ligands into a 'flattened' mode of coordination compared with inorganic model complexes. This change in geometry has been shown by density functional computations to significantly decrease the strength of the metal-ligand binding. Collectively, these data demonstrate that the mode of binding by small metal-binding groups can be significantly influenced by the protein active site. Diminishing the strength of the metal-ligand bond results in unconventional modes of metal coordination not found in typical coordination compounds or even carefully engineered active site models, and understanding these effects is critical to the rational design of inhibitors that target clinically relevant metalloproteins.

  10. Discovery, synthesis and structure-activity analysis of symmetrical 2,7-disubstituted fluorenones as urea transporter inhibitors

    PubMed Central

    Lee, Sujin; Esteva-Font, Cristina; Phuan, Puay-Wah; Anderson, Marc O.; Verkman, A.S

    2015-01-01

    Kidney urea transporters are targets for development of small-molecule inhibitors with action as salt-sparing diuretics. A cell-based, functional high-throughput screen identified 2,7-bisacetamido fluorenone 3 as a novel inhibitor of urea transporters UT-A1 and UT-B. Here, we synthesized twenty-two 2,7-disubstituted fluorenone analogs by acylation. Structure-activity relationship analysis revealed: (a) the carbonyl moiety at C9 is required for UT inhibition; (b) steric limitation on C2, 7-substituents; and (c) the importance of a crescent-shape structure. The most potent fluorenones inhibited UT-A1 and UT-B urea transport with IC50 ~ 1 μM. Analysis of in vitro metabolic stability in hepatic microsomes indicated metabolism of 2,7-disubstituted fluorenones by reductase and subsequent elimination. Computational docking to a homology model of UT-A1 suggested UT inhibitor binding to the UT cytoplasmic domain at a site that does not overlap with the putative urea binding site. PMID:26191399

  11. Transforming growth factor-beta requires its target plasminogen activator inhibitor-1 for cytostatic activity.

    PubMed

    Kortlever, Roderik M; Nijwening, Jeroen H; Bernards, René

    2008-09-01

    The cytokine transforming growth factor beta (TGFbeta) has strong antiproliferative activity in most normal cells but contributes to tumor progression in the later stages of oncogenesis. It is not fully understood which TGFbeta target genes are causally involved in mediating its cytostatic activity. We report here that suppression of the TGFbeta target gene encoding plasminogen activator inhibitor-1 (PAI-1) by RNA interference leads to escape from the cytostatic activity of TGFbeta both in human keratinocytes (HaCaTs) and primary mouse embryo fibroblasts. Consistent with this, PAI-1 knock-out mouse embryo fibroblasts are also resistant to TGFbeta growth arrest. Conversely, we show that ectopic expression of PAI-1 in proliferating HaCaT cells induces a growth arrest. PAI-1 knockdown does not interfere with canonical TGFbeta signaling as judged by SMAD phosphorylation and induction of bona fide TGFbeta target genes. Instead, knockdown of PAI-1 results in sustained activation of protein kinase B. Significantly, we find that constitutive protein kinase B activity leads to evasion of the growth-inhibitory action of TGFbeta. Our data are consistent with a model in which induction of PAI-1 by TGFbeta is critical for the induction of proliferation arrest.

  12. Novel peptidomimetic inhibitors of signal transducer and activator of transcription 3 dimerization and biological activity.

    PubMed

    Turkson, James; Kim, Joon S; Zhang, Shumin; Yuan, Jing; Huang, Mei; Glenn, Matthew; Haura, Eric; Sebti, Said; Hamilton, Andrew D; Jove, Richard

    2004-03-01

    The critical role of signal transducer and activator of transcription 3 (Stat3) in the growth and survival of human tumor cells identifies it as a promising target for cancer drug discovery. We previously identified a Stat3 SH2 domain-binding phosphopeptide, PY*LKTK, and its tripeptide derivatives, PY*L and AY*L (where Y* represents phosphotyrosine), which inhibit Stat3 biochemical activity and biological function. Here, we report novel peptidomimetic compounds based on PY*L (or AY*L) with substitution of the Y-1 residue by benzyl, pyridyl, or pyrazinyl derivatives that are selective and greater than 5-fold more potent in disrupting Stat3 activity in vitro than lead tripeptides. The biological activities of these derivatives mirror that originally observed for peptides. In this context, the representative peptidomimetic ISS 610 with 4-cyanobenzoate substitution inhibits constitutive Stat3 activity in Src-transformed mouse fibroblasts and human breast and lung carcinoma cells. This effect is not evident with the non-phosphorylated counterpart, ISS 610NP, consistent with interaction of peptidomimetics with the SH2 domain of Stat3. Moreover, ISS 610 induces cell growth inhibition and apoptosis of Src-transformed fibroblasts that contain persistently active Stat3. We present the first report of a peptidomimetic approach to design of small-molecule inhibitors of Stat3 that are also among the first examples of disruptors of transcription factor dimerization with the potential for novel cancer therapy.

  13. Low-volume multiplexed proteolytic activity assay and inhibitor analysis through a pico-injector array.

    PubMed

    Ng, Ee Xien; Miller, Miles A; Jing, Tengyang; Lauffenburger, Doug A; Chen, Chia-Hung

    2015-02-21

    Secreted active proteases, from families of enzymes such as matrix metalloproteinases (MMPs) and ADAMs (a disintegrin and metalloproteinases), participate in diverse pathological processes. To simultaneously measure multiple specific protease activities, a series of parallel enzyme reactions combined with a series of inhibitor analyses for proteolytic activity matrix analysis (PrAMA) are essential but limited due to the sample quantity requirements and the complexity of performing multiple reactions. To address these issues, we developed a pico-injector array to generate 72 different reactions in picoliter-volume droplets by controlling the sequence of combinational injections, which allowed simultaneous recording of a wide range of multiple enzyme reactions and measurement of inhibitor effects using small sample volumes (~10 μL). Multiple MMP activities were simultaneously determined by 9 different substrates and 2 inhibitors using injections from a pico-injector array. Due to the advantages of inhibitor analysis, the MMP/ADAM activities of MDA-MB-231, a breast cancer cell line, were characterized with high MMP-2, MMP-3 and ADAM-10 activity. This platform could be customized for a wide range of applications that also require multiple reactions with inhibitor analysis to enhance the sensitivity by encapsulating different chemical sensors.

  14. P38 MAPK inhibitors suppress biomarkers of hypertension end-organ damage, osteopontin and plasminogen activator inhibitor-1.

    PubMed

    Nerurkar, S S; Olzinski, A R; Frazier, K S; Mirabile, R C; O'Brien, S P; Jing, J; Rajagopalan, D; Yue, T L; Willette, R N

    2007-01-01

    The assessment of target organ damage is important in defining the optimal treatment of hypertension and blood pressure-related cardiovascular disease. The aims of the present study were (1) to investigate candidate biomarkers of target organ damage, osteopontin (OPN) and plasminogen activator inhibitor-1 (PAI-1), in models of malignant hypertension with well characterized end-organ pathology; and (2) to evaluate the effects of chronic treatment with a p38 MAPK inhibitor. Gene expression, plasma concentrations, and renal immunohistochemical localization of OPN and PAI-1 were measured in stroke-prone spontaneously hypertensive rats on a salt-fat diet (SFD SHR-SP) and in spontaneously hypertensive rats receiving N(omega)-nitro-L-arginine methyl ester (L-NAME SHR). Plasma concentrations of OPN and PAI-1 increased significantly in SFD SHR-SP and L-NAME SHR as compared with controls, (2.5-4.5-fold for OPN and 2.0-9.0-fold for PAI-1). The plasma levels of OPN and PAI-1 were significantly correlated with the urinary excretion of albumin (p < 0.0001). Elevations in urinary albumin, plasma OPN and PAI-1 were abolished by chronic treatment (4-8 weeks) with a specific p38 MAPK inhibitor, SB-239063AN. OPN immunoreactivity was localized predominantly in the apical portion of tubule epithelium, while PAI-1 immunoreactivity was robust in glomeruli, tubules and renal artery endothelium. Treatment with the p38 MAPK inhibitor significantly reduced OPN and PAI-1 protein expression in target organs. Kidney gene expression was increased for OPN (4.9- and 7.9-fold) and PAI-1 (2.8- and 11.5-fold) in SFD SHR-SP and L-NAME SHR, respectively. In-silico pathway analysis revealed that activation of p38 MAPK was linked to OPN and PAI-1 via SPI, c-fos and c-jun; suggesting that these pathways may play an important role in p38 MAPK-dependent hypertensive renal dysfunction. The results suggest that enhanced OPN and PAI-1 expression reflects end-organ damage in hypertension and that suppression

  15. Selective COX-2 Inhibitors: A Review of Their Structure-Activity Relationships

    PubMed Central

    Zarghi, Afshin; Arfaei, Sara

    2011-01-01

    Non-steroidal anti-inflammatory drugs (NSAIDs) are the competitive inhibitors of cyclooxygenase (COX), the enzyme which mediates the bioconversion of arachidonic acid to inflammatory prostaglandins (PGs). Their use is associated with the side effects such as gastrointestinal and renal toxicity. The therapeutic anti-inflammatory action of NSAIDs is produced by the inhibition of COX-2, while the undesired side effects arise from inhibition of COX-1 activity. Thus, it was though that more selective COX-2 inhibitors would have reduced side effects. Based upon a number of selective COX-2 inhibitors (rofecoxib, celecoxib, valdecoxibetc.) were developed as safer NSAIDs with improved gastric safety profile. However, the recent market removal of some COXIBs such as rofecoxib due to its adverse cardiovascular side effects clearly encourages the researchers to explore and evaluate alternative templates with COX-2 inhibitory activity. Recognition of new avenues for selective COX-2 inhibitors in cancer chemotherapy and neurological diseases such as Parkinson and Alzheimer’s diseases still continues to attract investigations on the development of COX-2 inhibitors. This review highlights the various structural classes of selective COX-2 inhibitors with special emphasis on their structure-activity relationships. PMID:24250402

  16. 4-Anilino-6-phenyl-quinoline inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK2).

    PubMed

    Olsson, Henric; Sjö, Peter; Ersoy, Oguz; Kristoffersson, Anna; Larsson, Joakim; Nordén, Bo

    2010-08-15

    A class of inhibitors of mitogen activated protein kinase-activated kinase 2 (MK2) was discovered via high-throughput screening. This compound class demonstrates activity against the enzyme with sub-microM IC(50) values, and suppresses LPS-induced TNFalpha levels in THP-1 cells. MK2 inhibition kinetic measurements indicated mixed binding approaching non-ATP competitive inhibition.

  17. Dissecting the active site of a photoreceptor protein

    NASA Astrophysics Data System (ADS)

    Hoff, Wouter; Hara, Miwa; Ren, Jie; Moghadam, Farzaneh; Xie, Aihua; Kumauchi, Masato

    While enzymes are quite large molecules, functionally important chemical events are often limited to a small region of the protein: the active site. The physical and chemical properties of residues at such active sites are often strongly altered compared to the same groups dissolved in water. Understanding such effects is important for unraveling the mechanisms underlying protein function and for protein engineering, but has proven challenging. Here we report on our ongoing efforts on using photoactive yellow protein (PYP), a bacterial photoreceptor, as a model system for such effects. We will report on the following questions: How many residues affect active site properties? Are these residues in direct physical contact with the active site? Can functionally important residues be recognized in the crystal structure of a protein? What structural resolution is needed to understand active sites? What spectroscopic techniques are most informative? Which weak interactions dominate active site properties?

  18. Microglial activation decreases retention of the protease inhibitor saquinavir: implications for HIV treatment

    PubMed Central

    2013-01-01

    Background Active HIV infection within the central nervous system (CNS) is confined primarily to microglia. The glial cell compartment acts as a viral reservoir behind the blood-brain barrier. It provides an additional roadblock to effective pharmacological treatment via expression of multiple drug efflux transporters, including P-glycoprotein. HIV/AIDS patients frequently suffer bacterial and viral co-infections, leading to deregulation of glial cell function and release of pro-inflammatory mediators including cytokines, chemokines, and nitric oxide. Methods To better define the role of inflammation in decreased HIV drug accumulation into CNS targets, accumulation of the antiretroviral saquinavir was examined in purified cultures of rodent microglia exposed to the prototypical inflammatory mediator lipopolysaccharide (LPS). Results [3H]-Saquinavir accumulation by microglia was rapid, and was increased up to two-fold in the presence of the specific P-glycoprotein inhibitor, PSC833. After six or 24 hours of exposure to 10 ng/ml LPS, saquinavir accumulation was decreased by up to 45%. LPS did not directly inhibit saquinavir transport, and did not affect P-glycoprotein protein expression. LPS exposure did not alter RNA and/or protein expression of other transporters including multidrug resistance-associated protein 1 and several solute carrier uptake transporters. Conclusions The decrease in saquinavir accumulation in microglia following treatment with LPS is likely multi-factorial, since drug accumulation was attenuated by inhibitors of NF-κβ and the MEK1/2 pathway in the microglia cell line HAPI, and in primary microglia cultures from toll-like receptor 4 deficient mice. These data provide new pharmacological insights into why microglia act as a difficult-to-treat viral sanctuary site. PMID:23642074

  19. Effect of mimetic CDK9 inhibitors on HIV-1 activated transcription

    PubMed Central

    Van Duyne, Rachel; Guendel, Irene; Jaworski, Elizabeth; Sampey, Gavin; Klase, Zachary; Chen, Hao; Zeng, Chen; Kovalskyy, Dmytro; el Kouni, Mahmoud H.; Lepene, Benjamin; Patanarut, Alexis; Nekhai, Sergei; Price, David H.; Kashanchi, Fatah

    2013-01-01

    Potent antiretroviral therapy (ART) has transformed HIV-1 infection into a chronic manageable disease; however drug resistance remains a common problem that limits the effectiveness and clinical benefits of this type of treatment. The discovery of viral reservoirs in the body, in which HIV-1 may persist, has helped to explain why therapeutic eradication of HIV-1 has proved so difficult. In the current study we utilized a combination of structure based analysis of Cyclin/CDK complexes with our previously published Tat peptide derivatives. We modeled the Tat peptide inhibitors with CDKs and found a particular pocket which showed the most stable binding site (Cavity 1) using in silico analysis. Furthermore, we were able to find peptide mimetics that bound to similar regions using in silico searches of a chemical library, followed by cell based biological assays. Using these methods we obtained the first generation mimetic drugs and tested these compounds on HIV-1 LTR activated transcription. Using biological assays followed by similar in silico analysis to find a 2nd generation drugs resembling the original mimetic, we found the new targets of Cavity 1 and Cavity 2 regions on CDK9. We examined the 2nd generation mimetic against various viral isolates, and observed a generalized suppression of most HIV-1 isolates. Finally, the drug inhibited viral replication in humanized mouse models of Rag2-/-γc-/- with no toxicity to the animals at tested concentrations. Our results suggest that it may be possible to model peptide inhibitors into available crystal structures and further find drug mimetics using in silico analysis. PMID:23247501

  20. Structure–Activity Relationships of a Novel Capsid Targeted Inhibitor of HIV-1 Replication

    PubMed Central

    2015-01-01

    Despite the considerable successes of highly active antiretroviral therapy (HAART) for the treatment of HIV/AIDS, cumulative drug toxicities and the development of multidrug-resistant virus necessitate the search for new classes of antiretroviral agents with novel modes of action. The HIV-1 capsid (CA) protein has been structurally and functionally characterized as a druggable target. We have recently designed a novel small molecule inhibitor I-XW-053 using the hybrid structure based method to block the interface between CA N-terminal domains (NTD–NTD interface) with micromolar affinity. In an effort to optimize and improve the efficacy of I-XW-053, we have developed the structure activity relationship of I-XW-053 compound series using ligand efficiency methods. Fifty-six analogues of I-XW-053 were designed that could be subclassified into four different core domains based on their ligand efficiency values computed as the ratio of binding efficiency (BEI) and surface efficiency (SEI) indices. Compound 34 belonging to subcore-3 showed an 11-fold improvement over I-XW-053 in blocking HIV-1 replication in primary human peripheral blood mononuclear cells (PBMCs). Surface plasmon resonance experiments confirmed the binding of compound 34 to purified HIV-1 CA protein. Molecular docking studies on compound 34 and I-XW-053 to HIV-1 CA protein suggested that they both bind to NTD–NTD interface region but with different binding modes, which was further validated using site-directed mutagenesis studies. PMID:25302989

  1. Statins Increase Plasminogen Activator Inhibitor Type 1 Gene Transcription through a Pregnane X Receptor Regulated Element

    PubMed Central

    Stanley, Frederick M.; Linder, Kathryn M.; Cardozo, Timothy J.

    2015-01-01

    Plasminogen activator inhibitor type 1 (PAI-1) is a multifunctional protein that has important roles in inflammation and wound healing. Its aberrant regulation may contribute to many disease processes such as heart disease. The PAI-1 promoter is responsive to multiple inputs including cytokines, growth factors, steroids and oxidative stress. The statin drugs, atorvastatin, mevastatin and rosuvastatin, increased basal and stimulated expression of the PAI-1 promoter 3-fold. A statin-responsive, nuclear hormone response element was previously identified in the PAI-1 promoter, but it was incompletely characterized. We characterized this direct repeat (DR) of AGGTCA with a 3-nucleotide spacer at -269/-255 using deletion and directed mutagenesis. Deletion or mutation of this element increased basal transcription from the promoter suggesting that it repressed PAI-1 transcription in the unliganded state. The half-site spacing and the ligand specificity suggested that this might be a pregnane X receptor (PXR) responsive element. Computational molecular docking showed that atorvastatin, mevastatin and rosuvastatin were structurally compatible with the PXR ligand-binding pocket in its agonist conformation. Experiments with Gal4 DNA binding domain fusion proteins showed that Gal4-PXR was activated by statins while other DR + 3 binding nuclear receptor fusions were not. Overexpression of PXR further enhanced PAI-1 transcription in response to statins. Finally, ChIP experiments using Halo-tagged PXR and RXR demonstrated that both components of the PXR-RXR heterodimer bound to this region of the PAI-1 promoter. PMID:26379245

  2. Aminoalcohols as Probes of the Two-subsite Active Site of Beta-D-xylosidase from Selenomonas ruminantium

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Catalysis and inhibitor binding by the GH43 beta-xylosidase are governed by the protonation state of catalytic base (D14, pKa 5.0) and catalytic acid (E186, pKa 7.2) which reside in subsite -1 of the two-subsite active site. Cationic aminoalcohols are shown to bind exclusively to subsite -1 of the ...

  3. The Three Mycobacterium tuberculosis Antigen 85 Isoforms Have Unique Substrates and Activities Determined by Non-active Site Regions*

    PubMed Central

    Backus, Keriann M.; Dolan, Michael A.; Barry, Conor S.; Joe, Maju; McPhie, Peter; Boshoff, Helena I. M.; Lowary, Todd L.; Davis, Benjamin G.; Barry, Clifton E.

    2014-01-01

    The three isoforms of antigen 85 (A, B, and C) are the most abundant secreted mycobacterial proteins and catalyze transesterification reactions that synthesize mycolated arabinogalactan, trehalose monomycolate (TMM), and trehalose dimycolate (TDM), important constituents of the outermost layer of the cellular envelope of Mycobacterium tuberculosis. These three enzymes are nearly identical at the active site and have therefore been postulated to exist to evade host immunity. Distal to the active site is a second putative carbohydrate-binding site of lower homology. Mutagenesis of the three isoforms at this second site affected both substrate selectivity and overall catalytic activity in vitro. Using synthetic and natural substrates, we show that these three enzymes exhibit unique selectivity; antigen 85A more efficiently mycolates TMM to form TDM, whereas C (and to a lesser extent B) has a higher rate of activity using free trehalose to form TMM. This difference in substrate selectivity extends to the hexasaccharide fragment of cell wall arabinan. Mutation of secondary site residues from the most active isoform (C) into those present in A or B partially interconverts this substrate selectivity. These experiments in combination with molecular dynamics simulations reveal that differences in the N-terminal helix α9, the adjacent Pro216–Phe228 loop, and helix α5 are the likely cause of changes in activity and substrate selectivity. These differences explain the existence of three isoforms and will allow for future work in developing inhibitors. PMID:25028517

  4. Structure-activity relationships of substituted oxyoxalamides as inhibitors of the human soluble epoxide hydrolase.

    PubMed

    Kim, In-Hae; Lee, In-Hee; Nishiwaki, Hisashi; Hammock, Bruce D; Nishi, Kosuke

    2014-02-01

    We explored both structure-activity relationships among substituted oxyoxalamides used as the primary pharmacophore of inhibitors of the human sEH and as a secondary pharmacophore to improve water solubility of inhibitors. When the oxyoxalamide function was modified with a variety of alkyls or substituted alkyls, compound 6 with a 2-adamantyl group and a benzyl group was found to be a potent sEH inhibitor, suggesting that the substituted oxyoxalamide function is a promising primary pharmacophore for the human sEH, and compound 6 can be a novel lead structure for the development of further improved oxyoxalamide or other related derivatives. In addition, introduction of substituted oxyoxalamide to inhibitors with an amide or urea primary pharmacophore produced significant improvements in inhibition potency and water solubility. In particular, the N,N,O-trimethyloxyoxalamide group in amide or urea inhibitors (26 and 31) was most effective among those tested for both inhibition and solubility. The results indicate that substituted oxyoxalamide function incorporated into amide or urea inhibitors is a useful secondary pharmacophore, and the resulting structures will be an important basis for the development of bioavailable sEH inhibitors.

  5. Structure–activity studies with high-affinity inhibitors of pyroglutamyl-peptidase II

    PubMed Central

    2005-01-01

    Inhibitors of PPII (pyroglutamyl-peptidase II) (EC 3.4.19.6) have potential applications as investigative and therapeutic agents. The rational design of inhibitors is hindered, however, by the lack of an experimental structure for PPII. Previous studies have demonstrated that replacement of histidine in TRH (thyrotropin-releasing hormone) with asparagine produces a competitive PPII inhibitor (Ki 17.5 μM). To gain further insight into which functional groups are significant for inhibitory activity, we investigated the effects on inhibition of structural modifications to Glp-Asn-ProNH2 (pyroglutamyl-asparaginyl-prolineamide). Synthesis and kinetic analysis of a diverse series of carboxamide and C-terminally extended Glp-Asn-ProNH2 analogues were undertaken. Extensive quantitative structure–activity relationships were generated, which indicated that key functionalities in the basic molecular structure of the inhibitors combine in a unique way to cause PPII inhibition. Data from kinetic and molecular modelling studies suggest that hydrogen bonding between the asparagine side chain and PPII may provide a basis for the inhibitory properties of the asparagine-containing peptides. Prolineamide appeared to be important for interaction with the S2′ subsite, but some modifications were tolerated. Extension of Glp-Asn-ProNH2 with hydrophobic amino acids at the C-terminus led to a novel set of PPII inhibitors active in vitro at nanomolar concentrations. Such inhibitors were shown to enhance recovery of TRH released from rat brain slices. Glp-Asn-Pro-Tyr-Trp-Trp-7-amido-4-methylcoumarin displayed a Ki of 1 nM, making it the most potent competitive PPII inhibitor described to date. PPII inhibitors with this level of potency should find application in exploring the biological functions of TRH and PPII, and potentially provide a basis for development of novel therapeutics. PMID:15799721

  6. Regulatory elements involved in constitutive and phorbol ester-inducible expression of the plasminogen activator inhibitor type 2 gene promoter.

    PubMed Central

    Cousin, E; Medcalf, R L; Bergonzelli, G E; Kruithof, E K

    1991-01-01

    Gene transcription rates and mRNA levels of plasminogen activator inhibitor type 2 (PAI-2) are markedly induced by the tumor promoting agent phorbol 12-myristate 13-acetate (PMA) in human HT1080 fibrosarcoma cells. To identify promoter elements required for basal-, and phorbol ester-inducible expression, deletion mutants of the PAI-1 promoter fused to the chloramphenicol acetyl transferase (CAT) reporter gene, were transiently expressed in HT1080 cells. Constitutive CAT activity was expressed from constructs containing more than 215 bp of promoter sequence, whereas deletion to position -91 bp abolished CAT gene expression. Treatment of transfected cells with PMA resulted in a three- to ten-fold increase in CAT expression from all constructs except from the construct shortened to position -91. DNAse1 protection analysis of the promoter region between -215 and the transcription initiation site revealed numerous protected regions, including two AP1-like binding sites (AP1a and AP1b) and one CRE-like element. Site-directed mutagenesis of the AP1a site or of the CRE-like site resulted in the loss of basal CAT activity and abolished the PMA effect, whereas mutagenesis of AP1b only partially inhibited basal and PMA-mediated expression. Our results suggest that the PAI-2 promoter contains at least two elements required for basal gene transcription and PMA-mediated induction. Images PMID:1650454

  7. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

    Gulick, Virginia C.; Briggs, Geoffrey; Saunders, R. Stephen; Gilmore, Martha; Soderblom, Larry

    1999-01-01

    The Mars Surveyor Program --now a cooperative program led by NASA and CNES along with other international partners -- is underway. It has the primary science objective of furthering our understanding of the biological potential and possible biological history of Mars and has the complementary objective of improving our understanding of martian climate evolution and planetary history The missions will develop technology and acquire data necessary for eventual human Exploration. Launches of orbiters, landers and rovers will take place in 2001 and in 2003; in 2005 a complete system will be launched capable of returning samples to Earth by 2008. A key aspect of the program is the selection of landing sites. This abstract 1) reports on the status of the landing site selection process that begins with the 2001 lander mission and 2) outlines be opportunities for the Mars community to provide input into the landing site selection process.

  8. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

    Gulick, V. C.; Briggs, Geoffrey; Saunders, R. Stephen; Gilmore, Martha; Soderblom, Larry

    1999-01-01

    The Mars Surveyor Program -- now a cooperative program led by NASA and CNES along with other international partners -- is underway. It has the primary science objective of furthering our understanding of the biological potential and possible biological history of Mars and has the complementary objective of improving our understanding of martian climate evolution and planetary history. The missions will develop technology and acquire data necessary for eventual human exploration. Launches of orbiters, landers and rovers will take place in 2001 and in 2003; in 2005 a complete system will be launched capable of returning samples to Earth by 2008. A key aspect of the program is the selection of landing sites. This abstract 1) reports on the status of the landing site selection process that begins with the 2001 lander mission and 2) outlines the opportunities for the Mars community to provide input into the landing site selection process.

  9. Discovery of orally active pyrrolopyridine- and aminopyridine-based Met kinase inhibitors

    SciTech Connect

    Cai, Zhen-Wei; Wei, Donna; Schroeder, Gretchen M.; Cornelius, Lyndon A.M.; Kim, Kyoung; Chen, Xiao-Tao; Schmidt, Robert J.; Williams, David K.; Tokarski, John S.; An, Yongmi; Sack, John S.; Manne, Veeraswamy; Kamath, Amrita; Zhang, Yueping; Marathe, Punit; Hunt, John T.; Lombardo, Louis J.; Fargnoli, Joseph; Borzilleri, Robert M.

    2008-09-10

    A series of acylurea analogs derived from pyrrolopyridine and aminopyridine scaffolds were identified as potent inhibitors of Met kinase activity. The SAR at various positions of the two kinase scaffolds was investigated. These studies led to the discovery of compounds 3b and 20b, which demonstrated favorable pharmacokinetic properties in mice and significant antitumor activity in a human gastric carcinoma xenograft model.

  10. Acylprolinamides: a new class of peptide deformylase inhibitors with in vivo antibacterial activity.

    PubMed

    Axten, Jeffrey M; Medina, Jesús R; Blackledge, Charles W; Duquenne, Céline; Grant, Seth W; Bobko, Mark A; Peng, Tony; Miller, William H; Pinckney, Theresa; Gallagher, Timothy F; Kulkarni, Swarupa; Lewandowski, Thomas; Van Aller, Glenn S; Zonis, Rimma; Ward, Paris; Campobasso, Nino

    2012-06-15

    A new class of PDF inhibitor with potent, broad spectrum antibacterial activity is described. Optimization of blood stability and potency provided compounds with improved pharmacokinetics that were suitable for in vivo experiments. Compound 5c, which has robust antibacterial activity, demonstrated efficacy in two respiratory tract infection models.

  11. The bifunctional active site of s-adenosylmethionine synthetase. Roles of the active site aspartates.

    PubMed

    Taylor, J C; Markham, G D

    1999-11-12

    S-Adenosylmethionine (AdoMet) synthetase catalyzes the biosynthesis of AdoMet in a unique enzymatic reaction. Initially the sulfur of methionine displaces the intact tripolyphosphate chain (PPP(i)) from ATP, and subsequently PPP(i) is hydrolyzed to PP(i) and P(i) before product release. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site contains four aspartate residues. Aspartate residues Asp-16* and Asp-271 individually provide the sole protein ligand to one of the two required Mg(2+) ions (* denotes a residue from a second subunit); aspartates Asp-118 and Asp-238* are proposed to interact with methionine. Each aspartate has been changed to an uncharged asparagine, and the metal binding residues were also changed to alanine, to assess the roles of charge and ligation ability on catalytic efficiency. The resultant enzyme variants all structurally resemble the wild type enzyme as indicated by circular dichroism spectra and are tetramers. However, all have k(cat) reductions of approximately 10(3)-fold in AdoMet synthesis, whereas the MgATP and methionine K(m) values change by less than 3- and 8-fold, respectively. In the partial reaction of PPP(i) hydrolysis, mutants of the Mg(2+) binding residues have >700-fold reduced catalytic efficiency (k(cat)/K(m)), whereas the D118N and D238*N mutants are impaired less than 35-fold. The catalytic efficiency for PPP(i) hydrolysis by Mg(2+) site mutants is improved by AdoMet, like the wild type enzyme. In contrast AdoMet reduces the catalytic efficiency for PPP(i) hydrolysis by the D118N and D238*N mutants, indicating that the events involved in AdoMet activation are hindered in these methionyl binding site mutants. Ca(2+) uniquely activates the D271A mutant enzyme to 15% of the level of Mg(2+), in contrast to the approximately 1% Ca(2+) activation of the wild type enzyme. This indicates that the Asp-271 side chain size is a discriminator between the activating ability of Ca(2+) and the

  12. The murine Spi-2 proteinase inhibitor locus: a multigene family with a hypervariable reactive site domain.

    PubMed Central

    Inglis, J D; Hill, R E

    1991-01-01

    We have isolated 10 closely linked members of a proteinase inhibitor multigene family from the inbred mouse strain 129. These sequences, termed the Serine Proteinase Inhibitor 2 (Spi-2) genes, appear to have been derived from a common ancestor represented in man by the single copy alpha 1-antichymotrypsin gene. The genes are clustered on two cloned genomic DNA segments spanning 220 kb, and have at least partially retained the intragenic structure of the ancestral Spi-2 gene. Sequence analysis from the final coding exon indicates that most of the mouse genes may be competent to encode functional proteins, some with a predictable inhibitory spectrum, and several representing novel inhibitor types. An oligonucleotide probe designed to one reactive centre sequence enabled the isolation of the cognate expressed transcript from a liver cDNA library. However, whether expressed or not, the reactive centre regions of all the sequences have diverged at a rapid rate relative to structurally defined flanking sequences. The divergence is also appreciably greater than that occurring in an adjacent non-coding sequence. This phenomenon has established novel potential inhibitory specificities, while maintaining a functional inhibitor structure. PMID:1991447

  13. Introduction of {alpha}-hydroxymethyamino acid residues in substrate specificity P1 position of trypsin inhibitor SFTI-1 from sunflower seeds retains its activity

    SciTech Connect

    Zablotna, Ewa; Kret, Agnieszka; Jaskiewicz, Anna; Olma, Aleksandra; Leplawy, Miroslaw T.; Rolka, Krzysztof . E-mail: krzys@chem.univ.gda.pl

    2006-02-17

    In many complexes formed by serine proteinases and their inhibitors, the hydroxyl group provided by water molecule or by the inhibitor Ser residue is located close to the inhibitor P{sub 1}-P{sub 1}{sup '} reactive site. In order to investigate the role of this group, we synthesized analogues of trypsin inhibitor SFTI-1 isolated from the seeds of sunflower modified in P{sub 1} by {alpha}-hydroxymethylserine (HmSer) and both enantiomers of {alpha}-hydroxymethylvaline (HmVal). All the synthesized analogues inhibited bovine {beta}-trypsin and human leukocyte elastase. SFTI-1 analogues with HmVal and HmSer appear to be potent inhibitors of bovine {beta}-trypsin, whereas [Val{sup 5}]SFTI-1 is practically inactive. Also trypsin inhibitory activity of [Ser{sup 5}]SFTI-1 is significantly lower. Since the electrostatic interaction between protonated {epsilon}-NH{sub 2} group of the inhibitor P{sub 1} position and {beta}-carboxylate of trypsin Asp{sup 189} is the main driving force for interaction of both molecules, the results obtained are very interesting. We believe that these SFTI-1 analogues belong to a novel class of serine proteinase inhibitors.

  14. Hydroxamate based inhibitors of adenylyl cyclase. Part 2: the effect of cyclic linkers on P-site binding.

    PubMed

    Levy, Daniel; Bao, Ming; Tomlinson, James; Scarborough, Robert

    2002-11-01

    The adenylyl cyclases (ACs) are a family of enzymes that are key elements of signal transduction by virtue of their ability to convert ATP to cAMP. The catalytic mechanism of this transformation proceeds through initial binding of ATP to the purine binding site (P-site) followed by metal mediated cyclization with loss of pyrophosphate. Previous work in our group identified novel inhibitors which possess an adenine ring joined to a metal-coordinating hydroxamic acid through flexible linkers. Considering the spatial positioning of the metals with respect to the adenine binding site coupled with potentially favorable entropic factors, conformational restriction of the tether through a stereochemistry based SAR employing a rigid cyclic scaffold was explored. PMID:12372508

  15. Novel Protein Disulfide Isomerase Inhibitor with Anticancer Activity in Multiple Myeloma.

    PubMed

    Vatolin, Sergei; Phillips, James G; Jha, Babal K; Govindgari, Shravya; Hu, Jennifer; Grabowski, Dale; Parker, Yvonne; Lindner, Daniel J; Zhong, Fei; Distelhorst, Clark W; Smith, Mitchell R; Cotta, Claudiu; Xu, Yan; Chilakala, Sujatha; Kuang, Rebecca R; Tall, Samantha; Reu, Frederic J

    2016-06-01

    Multiple myeloma cells secrete more disulfide bond-rich proteins than any other mammalian cell. Thus, inhibition of protein disulfide isomerases (PDI) required for protein folding in the endoplasmic reticulum (ER) should increase ER stress beyond repair in this incurable cancer. Here, we report the mechanistically unbiased discovery of a novel PDI-inhibiting compound with antimyeloma activity. We screened a 30,355 small-molecule library using a multilayered multiple myeloma cell-based cytotoxicity assay that modeled disease niche, normal liver, kidney, and bone marrow. CCF642, a bone marrow-sparing compound, exhibited a submicromolar IC50 in 10 of 10 multiple myeloma cell lines. An active biotinylated analog of CCF642 defined binding to the PDI isoenzymes A1, A3, and A4 in MM cells. In vitro, CCF642 inhibited PDI reductase activity about 100-fold more potently than the structurally distinct established inhibitors PACMA 31 and LOC14. Computational modeling suggested a novel covalent binding mode in active-site CGHCK motifs. Remarkably, without any further chemistry optimization, CCF642 displayed potent efficacy in an aggressive syngeneic mouse model of multiple myeloma and prolonged the lifespan of C57BL/KaLwRij mice engrafted with 5TGM1-luc myeloma, an effect comparable to the first-line multiple myeloma therapeutic bortezomib. Consistent with PDI inhibition, CCF642 caused acute ER stress in multiple myeloma cells accompanied by apoptosis-inducing calcium release. Overall, our results provide an illustration of the utility of simple in vivo simulations as part of a drug discovery effort, along with a sound preclinical rationale to develop a new small-molecule therapeutic to treat multiple myeloma. Cancer Res; 76(11); 3340-50. ©2016 AACR. PMID:27197150

  16. Highly improved antiparasitic activity after introduction of an N-benzylimidazole moiety on protein farnesyltransferase inhibitors.

    PubMed

    Bosc, Damien; Mouray, Elisabeth; Cojean, Sandrine; Franco, Caio Haddad; Loiseau, Philippe M; Freitas-Junior, Lucio H; Moraes, Carolina Borsoi; Grellier, Philippe; Dubois, Joëlle

    2016-02-15

    In our search for new protein farnesyltransferase inhibitors with improved antiparasitic activities, we modified our previously developed 3-arylthiophene series of inhibitors by replacing the thioisopropyl group by different substituted imidazolylmethanamino moieties. Twenty four new derivatives were synthesized and evaluated against human and parasite farnesyltransferases, and their anti-parasitic activity was determined against Plasmodium falciparum, Trypanosoma brucei, Trypanosoma cruzi, and Leishmania donovani. Introduction of a N-p-substituted-benzylimidazole led to significantly increase the inhibition of parasite proliferation in the submicromolar range. The structure of the best inhibitors was parasite dependent. Three compounds possess IC50 values at the same range as the reference miltefosine against L. donovani proliferation and other new derivatives display high level of anti-trypanosomal activity against T. cruzi, higher or in the same order of magnitude as the reference compounds benznidazole and nifurtimox. PMID:26774924

  17. PHOTOREGULATION OF BIOLOGICAL ACTIVITY BY PHOTOCROMIC REAGENTS, II. INHIBITORS OF ACETYLCHOLINESTERASE*†

    PubMed Central

    Bieth, Joseph; Vratsanos, Spyros M.; Wassermann, Norbert; Erlanger, Bernard F.

    1969-01-01

    The enzymic activity of acetylcholinesterase can be photoregulated through the mediation of photochromic inhibitors of the enzyme. N-p-phenylazophenyl-N-phenylcarbamyl fluoride, an irreversible inhibitor of acetylcholinesterase, exists as two geometric isomers which are interconvertible through the action of light. The cis isomer, which predominates after exposure to light of 320 nm, is more active than the trans isomer, which results from exposure to light of 420 nm. It was possible, therefore, to use light energy to regulate the inactivation of the enzyme. Similarly, levels of acetylcholinesterase activity could be photo-regulated in a completely reversible manner by means of the photochromic reversible inhibitor p-phenylazophenyltrimethylammonium chloride. These experiments can serve as models for similar phenomena observed in nature, particularly in photoperiodic rhythms of higher animals. Images PMID:5264140

  18. A sucrose-binding site provides a lead towards an isoform-specific inhibitor of the cancer-associated enzyme carbonic anhydrase IX.

    PubMed

    Pinard, Melissa A; Aggarwal, Mayank; Mahon, Brian P; Tu, Chingkuang; McKenna, Robert

    2015-10-01

    Human carbonic anhydrase (CA; EC 4.2.1.1) isoform IX (CA IX) is an extracellular zinc metalloenzyme that catalyzes the reversible hydration of CO2 to HCO3(-), thereby playing a role in pH regulation. The majority of normal functioning cells exhibit low-level expression of CA IX. However, in cancer cells CA IX is upregulated as a consequence of a metabolic transition known as the Warburg effect. The upregulation of CA IX for cancer progression has drawn interest in it being a potential therapeutic target. CA IX is a transmembrane protein, and its purification, yield and crystallization have proven challenging to structure-based drug design, whereas the closely related cytosolic soluble isoform CA II can be expressed and crystallized with ease. Therefore, we have utilized structural alignments and site-directed mutagenesis to engineer a CA II that mimics the active site of CA IX. In this paper, the X-ray crystal structure of this CA IX mimic in complex with sucrose is presented and has been refined to a resolution of 1.5 Å, an Rcryst of 18.0% and an Rfree of 21.2%. The binding of sucrose at the entrance to the active site of the CA IX mimic, and not CA II, in a non-inhibitory mechanism provides a novel carbohydrate moiety binding site that could be further exploited to design isoform-specific inhibitors of CA IX.

  19. A sucrose-binding site provides a lead towards an isoform-specific inhibitor of the cancer-associated enzyme carbonic anhydrase IX.

    PubMed

    Pinard, Melissa A; Aggarwal, Mayank; Mahon, Brian P; Tu, Chingkuang; McKenna, Robert

    2015-10-01

    Human carbonic anhydrase (CA; EC 4.2.1.1) isoform IX (CA IX) is an extracellular zinc metalloenzyme that catalyzes the reversible hydration of CO2 to HCO3(-), thereby playing a role in pH regulation. The majority of normal functioning cells exhibit low-level expression of CA IX. However, in cancer cells CA IX is upregulated as a consequence of a metabolic transition known as the Warburg effect. The upregulation of CA IX for cancer progression has drawn interest in it being a potential therapeutic target. CA IX is a transmembrane protein, and its purification, yield and crystallization have proven challenging to structure-based drug design, whereas the closely related cytosolic soluble isoform CA II can be expressed and crystallized with ease. Therefore, we have utilized structural alignments and site-directed mutagenesis to engineer a CA II that mimics the active site of CA IX. In this paper, the X-ray crystal structure of this CA IX mimic in complex with sucrose is presented and has been refined to a resolution of 1.5 Å, an Rcryst of 18.0% and an Rfree of 21.2%. The binding of sucrose at the entrance to the active site of the CA IX mimic, and not CA II, in a non-inhibitory mechanism provides a novel carbohydrate moiety binding site that could be further exploited to design isoform-specific inhibitors of CA IX. PMID:26457530

  20. Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor

    PubMed Central

    Spatzal, Thomas; Perez, Kathryn A; Howard, James B; Rees, Douglas C

    2015-01-01

    Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32–1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis. DOI: http://dx.doi.org/10.7554/eLife.11620.001 PMID:26673079

  1. Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor.

    PubMed

    Spatzal, Thomas; Perez, Kathryn A; Howard, James B; Rees, Douglas C

    2015-12-16

    Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32-1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis.

  2. The active site of ribulose-bisphosphate carboxylase/oxygenase

    SciTech Connect

    Hartman, F.C.

    1991-01-01

    The active site of ribulose-bisphosphate carboxylase/oxygenase requires interacting domains of adjacent, identical subunits. Most active-site residues are located within the loop regions of an eight-stranded {beta}/{alpha}-barrel which constitutes the larger C-terminal domain; additional key residues are located within a segment of the smaller N-terminal domain which partially covers the mouth of the barrel. Site-directed mutagenesis of the gene encoding the enzyme from Rhodospirillum rubrum has been used to delineate functions of active-site residues. 6 refs., 2 figs.

  3. Discovery of small molecule inhibitors of xyloglucan endotransglucosylase (XET) activity by high-throughput screening

    PubMed Central

    Chormova, Dimitra; Franková, Lenka; Defries, Andrew; Cutler, Sean R.; Fry, Stephen C.

    2015-01-01

    Small molecules (xenobiotics) that inhibit cell-wall-localised enzymes are valuable for elucidating the enzymes’ biological roles. We applied a high-throughput fluorescent dot-blot screen to search for inhibitors of Petroselinum xyloglucan endotransglucosylase (XET) activity in vitro. Of 4216 xenobiotics tested, with cellulose-bound xyloglucan as donor-substrate, 18 inhibited XET activity and 18 promoted it (especially anthraquinones and flavonoids). No compounds promoted XET in quantitative assays with (cellulose-free) soluble xyloglucan as substrate, suggesting that promotion was dependent on enzyme–cellulose interactions. With cellulose-free xyloglucan as substrate, we found 22 XET-inhibitors – especially compounds that generate singlet oxygen (1O2) e.g., riboflavin (IC50 29 μM), retinoic acid, eosin (IC50 27 μM) and erythrosin (IC50 36 μM). The riboflavin effect was light-dependent, supporting 1O2 involvement. Other inhibitors included tannins, sulphydryl reagents and triphenylmethanes. Some inhibitors (vulpinic acid and brilliant blue G) were relatively specific to XET, affecting only two or three, respectively, of nine other wall-enzyme activities tested; others [e.g. (−)-epigallocatechin gallate and riboflavin] were non-specific. In vivo, out of eight XET-inhibitors bioassayed, erythrosin (1 μM) inhibited cell expansion in Rosa and Zea cell-suspension cultures, and 40 μM mycophenolic acid and (−)-epigallocatechin gallate inhibited Zea culture growth. Our work showcases a general high-throughput strategy for discovering wall-enzyme inhibitors, some being plant growth inhibitors potentially valuable as physiological tools or herbicide leads. PMID:26093490

  4. Discovery of small molecule inhibitors of xyloglucan endotransglucosylase (XET) activity by high-throughput screening.

    PubMed

    Chormova, Dimitra; Franková, Lenka; Defries, Andrew; Cutler, Sean R; Fry, Stephen C

    2015-09-01

    Small molecules (xenobiotics) that inhibit cell-wall-localised enzymes are valuable for elucidating the enzymes' biological roles. We applied a high-throughput fluorescent dot-blot screen to search for inhibitors of Petroselinum xyloglucan endotransglucosylase (XET) activity in vitro. Of 4216 xenobiotics tested, with cellulose-bound xyloglucan as donor-substrate, 18 inhibited XET activity and 18 promoted it (especially anthraquinones and flavonoids). No compounds promoted XET in quantitative assays with (cellulose-free) soluble xyloglucan as substrate, suggesting that promotion was dependent on enzyme-cellulose interactions. With cellulose-free xyloglucan as substrate, we found 22 XET-inhibitors - especially compounds that generate singlet oxygen ((1)O2) e.g., riboflavin (IC50 29 μM), retinoic acid, eosin (IC50 27 μM) and erythrosin (IC50 36 μM). The riboflavin effect was light-dependent, supporting (1)O2 involvement. Other inhibitors included tannins, sulphydryl reagents and triphenylmethanes. Some inhibitors (vulpinic acid and brilliant blue G) were relatively specific to XET, affecting only two or three, respectively, of nine other wall-enzyme activities tested; others [e.g. (-)-epigallocatechin gallate and riboflavin] were non-specific. In vivo, out of eight XET-inhibitors bioassayed, erythrosin (1 μM) inhibited cell expansion in Rosa and Zea cell-suspension cultures, and 40 μM mycophenolic acid and (-)-epigallocatechin gallate inhibited Zea culture growth. Our work showcases a general high-throughput strategy for discovering wall-enzyme inhibitors, some being plant growth inhibitors potentially valuable as physiological tools or herbicide leads. PMID:26093490

  5. The potent Cdc7-Dbf4 (DDK) kinase inhibitor XL413 has limited activity in many cancer cell lines and discovery of potential new DDK inhibitor scaffolds.

    PubMed

    Sasi, Nanda Kumar; Tiwari, Kanchan; Soon, Fen-Fen; Bonte, Dorine; Wang, Tong; Melcher, Karsten; Xu, H Eric; Weinreich, Michael

    2014-01-01

    Cdc7-Dbf4 kinase or DDK (Dbf4-dependent kinase) is required to initiate DNA replication by phosphorylating and activating the replicative Mcm2-7 DNA helicase. DDK is overexpressed in many tumor cells and is an emerging chemotherapeutic target since DDK inhibition causes apoptosis of diverse cancer cell types but not of normal cells. PHA-767491 and XL413 are among a number of potent DDK inhibitors with low nanomolar IC50 values against the purified kinase. Although XL413 is highly selective for DDK, its activity has not been extensively characterized on cell lines. We measured anti-proliferative and apoptotic effects of XL413 on a panel of tumor cell lines compared to PHA-767491, whose activity is well characterized. Both compounds were effective biochemical DDK inhibitors but surprisingly, their activities in cell lines were highly divergent. Unlike PHA-767491, XL413 had significant anti-proliferative activity against only one of the ten cell lines tested. Since XL413 did not effectively inhibit DDK in multiple cell lines, this compound likely has limited bioavailability. To identify potential leads for additional DDK inhibitors, we also tested the cross-reactivity of ∼400 known kinase inhibitors against DDK using a DDK thermal stability shift assay (TSA). We identified 11 compounds that significantly stabilized DDK. Several inhibited DDK with comparable potency to PHA-767491, including Chk1 and PKR kinase inhibitors, but had divergent chemical scaffolds from known DDK inhibitors. Taken together, these data show that several well-known kinase inhibitors cross-react with DDK and also highlight the opportunity to design additional specific, biologically active DDK inhibitors for use as chemotherapeutic agents.

  6. The Potent Cdc7-Dbf4 (DDK) Kinase Inhibitor XL413 Has Limited Activity in Many Cancer Cell Lines and Discovery of Potential New DDK Inhibitor Scaffolds

    PubMed Central

    Sasi, Nanda Kumar; Tiwari, Kanchan; Soon, Fen-Fen; Bonte, Dorine; Wang, Tong; Melcher, Karsten; Xu, H. Eric; Weinreich, Michael

    2014-01-01

    Cdc7-Dbf4 kinase or DDK (Dbf4-dependent kinase) is required to initiate DNA replication by phosphorylating and activating the replicative Mcm2-7 DNA helicase. DDK is overexpressed in many tumor cells and is an emerging chemotherapeutic target since DDK inhibition causes apoptosis of diverse cancer cell types but not of normal cells. PHA-767491 and XL413 are among a number of potent DDK inhibitors with low nanomolar IC50 values against the purified kinase. Although XL413 is highly selective for DDK, its activity has not been extensively characterized on cell lines. We measured anti-proliferative and apoptotic effects of XL413 on a panel of tumor cell lines compared to PHA-767491, whose activity is well characterized. Both compounds were effective biochemical DDK inhibitors but surprisingly, their activities in cell lines were highly divergent. Unlike PHA-767491, XL413 had significant anti-proliferative activity against only one of the ten cell lines tested. Since XL413 did not effectively inhibit DDK in multiple cell lines, this compound likely has limited bioavailability. To identify potential leads for additional DDK inhibitors, we also tested the cross-reactivity of ∼400 known kinase inhibitors against DDK using a DDK thermal stability shift assay (TSA). We identified 11 compounds that significantly stabilized DDK. Several inhibited DDK with comparable potency to PHA-767491, including Chk1 and PKR kinase inhibitors, but had divergent chemical scaffolds from known DDK inhibitors. Taken together, these data show that several well-known kinase inhibitors cross-react with DDK and also highlight the opportunity to design additional specific, biologically active DDK inhibitors for use as chemotherapeutic agents. PMID:25412417

  7. Synthesis of non-competitive inhibitors of sphingomyelinases with significant activity.

    PubMed

    Yokomatsu, Tsutomu; Murano, Tetsuo; Akiyama, Takeshi; Koizumi, Junichi; Shibuya, Shiroshi; Tsuji, Yoshiaki; Soeda, Shinji; Shimeno, Hiroshi

    2003-01-20

    A series of short-chain analogues of N-palmitoylsphingosine-1-phosphate, modified by replacement of the phosphate and the long alkenyl side chain with hydrolytically stable difluoromethylene phosphonate and phenyl, respectively, were prepared to study the structure-activity relationship for inhibition of sphingomyelinase. The study revealed that inhibition is highly dependent upon the stereochemistry of the asymmetric centers of the acylamino moiety, and resulted in identification of a non-competitive inhibitor with the same level of inhibitory activity of schyphostatin, the most potent of the few known small molecular inhibitors of sphingomyelinase.

  8. Synthesis and structure-activity relationships of potent 4-fluoro-2-cyanopyrrolidine dipeptidyl peptidase IV inhibitors.

    PubMed

    Fukushima, Hiroshi; Hiratate, Akira; Takahashi, Masato; Mikami, Ayako; Saito-Hori, Masako; Munetomo, Eiji; Kitano, Kiyokazu; Chonan, Sumi; Saito, Hidetaka; Suzuki, Akio; Takaoka, Yuji; Yamamoto, Koji

    2008-04-01

    Dipeptidyl peptidase IV (DPP-IV) inhibitors are promising antidiabetic drugs, and several drugs are in the developmental stage. We previously reported that the introduction of fluorine to the 4-position of 2-cyanopyrrolidine enhanced the DPP-IV inhibitory effect. In the present report, we examined the structure-activity relationship (SAR) of 2-cyano-4-fluoropyrrolidine with N-substituted glycine at the 1-position. We report the identification of a potent and stable DPP-IV inhibitor (TS-021) with a long-term persistent plasma drug concentration and a potent antihyperglycemic activity.

  9. New direct inhibitors of InhA with antimycobacterial activity based on a tetrahydropyran scaffold.

    PubMed

    Pajk, Stane; Živec, Matej; Šink, Roman; Sosič, Izidor; Neu, Margarete; Chung, Chun-wa; Martínez-Hoyos, María; Pérez-Herrán, Esther; Álvarez-Gómez, Daniel; Álvarez-Ruíz, Emilio; Mendoza-Losana, Alfonso; Castro-Pichel, Julia; Barros, David; Ballell-Pages, Lluís; Young, Robert J; Convery, Maire A; Encinas, Lourdes; Gobec, Stanislav

    2016-04-13

    Tetrahydropyran derivative 1 was discovered in a high-throughput screening campaign to find new inhibitors of mycobacterial InhA. Following initial in-vitro profiling, a structure-activity relationship study was initiated and a focused library of analogs was synthesized and evaluated. This yielded compound 42 with improved antimycobacterial activity and low cytotoxicity. Additionally, the crystal structure of InhA in complex with inhibitor 1 was resolved, to reveal the binding mode and provide hints for further optimization. PMID:26900657

  10. A Trypsin Inhibitor from Clitoria fairchildiana Cotyledons is Active Against Digestive Enzymes of Aedes aegypti Larvae.

    PubMed

    de Oliveira, Lucilene O; Fernandes, Kátia V S; Pádua, Dayanni de Souza; Carvalho, André de O; Lemos, Francisco J A; Gomes, Valdirene M; Oliveira, Antônia E A; Ferreira, André T da Silva; Perales, Jonas

    2015-01-01

    Aedes aegypti, the principal mosquito vector of yellow fever, dengue fever and chikungunya fever virus-transmitted diseases, is an insect closely associated with humans and their housing habitats. As there is no commercially available vaccine, prevention is the most suggested form of avoiding disease spreading and a number of studies are being developed in order to give support to vector control operations. The present study reports on the identification of a trypsin inhibitor isolated from cotyledons of the Clitoria fairchildiana amazonic tree seeds, which was able to reduce by 87.93 % the activity of digestive enzymes of fourth instar A. aegypti larva. A partial amino acid sequence showed strong similarity with sequences from several trypsin inhibitors already reported in the literature. The 13,000 Da isolated inhibitor was seen to be active solely against trypsin-like enzymes, neither acting on papain, α-amylase nor on other serine proteases, such as elastase, chymotrypsin or subtilisin. At least six from seven active digestive proteases from A. aegypti larvae, visualized by zymography, were severely affected soon after exposed to the inhibitor. The strong and specific action of the isolated inhibitor against trypsin digestive enzymes of this insect vector led us to believe that this protein may be a good candidate for a prospective alternative biocontrol method. PMID:26156641

  11. Polycomb repressive complex 2 structure with inhibitor reveals a mechanism of activation and drug resistance

    PubMed Central

    Brooun, Alexei; Gajiwala, Ketan S.; Deng, Ya-Li; Liu, Wei; Bolaños, Ben; Bingham, Patrick; He, You-Ai; Diehl, Wade; Grable, Nicole; Kung, Pei-Pei; Sutton, Scott; Maegley, Karen A.; Yu, Xiu; Stewart, Al E.

    2016-01-01

    Polycomb repressive complex 2 (PRC2) mediates gene silencing through chromatin reorganization by methylation of histone H3 lysine 27 (H3K27). Overexpression of the complex and point mutations in the individual subunits of PRC2 have been shown to contribute to tumorigenesis. Several inhibitors of the PRC2 activity have shown efficacy in EZH2-mutated lymphomas and are currently in clinical development, although the molecular basis of inhibitor recognition remains unknown. Here we report the crystal structures of the inhibitor-bound wild-type and Y641N PRC2. The structures illuminate an important role played by a stretch of 17 residues in the N-terminal region of EZH2, we call the activation loop, in the stimulation of the enzyme activity, inhibitor recognition and the potential development of the mutation-mediated drug resistance. The work presented here provides new avenues for the design and development of next-generation PRC2 inhibitors through establishment of a structure-based drug design platform. PMID:27122193

  12. p38 Mitogen-activated protein kinase inhibitors: a review on pharmacophore mapping and QSAR studies.

    PubMed

    Gangwal, Rahul P; Bhadauriya, Anuseema; Damre, Mangesh V; Dhoke, Gaurao V; Sangamwar, Abhay T

    2013-01-01

    p38 mitogen-activated protein (MAP) kinases are the serine/threonine protein kinases, which play a vital role in cellular responses to external stress signals. p38 MAP kinase inhibitors have shown anti-inflammatory effects in the preclinical disease models, primarily through inhibition of the expression of inflammatory mediators. A number of structurally diverse p38 MAP kinase inhibitors have been developed as potential anti-inflammatory agents. Most of the inhibitors have failed in the clinical trials either due to poor pharmacokinetic profile or selectivity issue, which makes p38 MAP kinase a promising target for molecular modelling studies. Several quantitative structure activity relationships (QSAR) and pharmacophore models have been developed to identify the structural requirements essential for p38 MAP kinase inhibitory activity. In this review, we provide an overview of the presently known p38 MAP kinase inhibitors and how QSAR analyses among series of compounds have led to the development of molecular models and pharmacophores, allowing the design of novel inhibitors.

  13. Savannah River Site prioritization of transition activities

    SciTech Connect

    Finley, R.H.

    1993-11-01

    Effective management of SRS conversion from primarily a production facility to other missions (or Decontamination and Decommissioning (D&D)) requires a systematic and consistent method of prioritizing the transition activities. This report discusses the design of a prioritizing method developed to achieve systematic and consistent methods of prioritizing these activities.

  14. DOE site performance assessment activities. Radioactive Waste Technical Support Program

    SciTech Connect

    Not Available

    1990-07-01

    Information on performance assessment capabilities and activities was collected from eight DOE sites. All eight sites either currently dispose of low-level radioactive waste (LLW) or plan to dispose of LLW in the near future. A survey questionnaire was developed and sent to key individuals involved in DOE Order 5820.2A performance assessment activities at each site. The sites surveyed included: Hanford Site (Hanford), Idaho National Engineering Laboratory (INEL), Los Alamos National Laboratory (LANL), Nevada Test Site (NTS), Oak Ridge National Laboratory (ORNL), Paducah Gaseous Diffusion Plant (Paducah), Portsmouth Gaseous Diffusion Plant (Portsmouth), and Savannah River Site (SRS). The questionnaire addressed all aspects of the performance assessment process; from waste source term to dose conversion factors. This report presents the information developed from the site questionnaire and provides a comparison of site-specific performance assessment approaches, data needs, and ongoing and planned activities. All sites are engaged in completing the radioactive waste disposal facility performance assessment required by DOE Order 5820.2A. Each site has achieved various degrees of progress and have identified a set of critical needs. Within several areas, however, the sites identified common needs and questions.

  15. Safety Oversight of Decommissioning Activities at DOE Nuclear Sites

    SciTech Connect

    Zull, Lawrence M.; Yeniscavich, William

    2008-01-15

    The Defense Nuclear Facilities Safety Board (Board) is an independent federal agency established by Congress in 1988 to provide nuclear safety oversight of activities at U.S. Department of Energy (DOE) defense nuclear facilities. The activities under the Board's jurisdiction include the design, construction, startup, operation, and decommissioning of defense nuclear facilities at DOE sites. This paper reviews the Board's safety oversight of decommissioning activities at DOE sites, identifies the safety problems observed, and discusses Board initiatives to improve the safety of decommissioning activities at DOE sites. The decommissioning of former defense nuclear facilities has reduced the risk of radioactive material contamination and exposure to the public and site workers. In general, efforts to perform decommissioning work at DOE defense nuclear sites have been successful, and contractors performing decommissioning work have a good safety record. Decommissioning activities have recently been completed at sites identified for closure, including the Rocky Flats Environmental Technology Site, the Fernald Closure Project, and the Miamisburg Closure Project (the Mound site). The Rocky Flats and Fernald sites, which produced plutonium parts and uranium materials for defense needs (respectively), have been turned into wildlife refuges. The Mound site, which performed R and D activities on nuclear materials, has been converted into an industrial and technology park called the Mound Advanced Technology Center. The DOE Office of Legacy Management is responsible for the long term stewardship of these former EM sites. The Board has reviewed many decommissioning activities, and noted that there are valuable lessons learned that can benefit both DOE and the contractor. As part of its ongoing safety oversight responsibilities, the Board and its staff will continue to review the safety of DOE and contractor decommissioning activities at DOE defense nuclear sites.

  16. Combinatorial active-site variants confer sustained clavulanate resistance in BlaC β-lactamase from Mycobacterium tuberculosis

    PubMed Central

    Egesborg, Philippe; Carlettini, Hélène; Volpato, Jordan P; Doucet, Nicolas

    2015-01-01

    Bacterial resistance to β-lactam antibiotics is a global issue threatening the success of infectious disease treatments worldwide. Mycobacterium tuberculosis has been particularly resilient to β-lactam treatment, primarily due to the chromosomally encoded BlaC β-lactamase, a broad-spectrum hydrolase that renders ineffective the vast majority of relevant β-lactam compounds currently in use. Recent laboratory and clinical studies have nevertheless shown that specific β-lactam–BlaC inhibitor combinations can be used to inhibit the growth of extensively drug-resistant strains of M. tuberculosis, effectively offering new tools for combined treatment regimens against resistant strains. In the present work, we performed combinatorial active-site replacements in BlaC to demonstrate that specific inhibitor-resistant (IRT) substitutions at positions 69, 130, 220, and/or 234 can act synergistically to yield active-site variants with several thousand fold greater in vitro resistance to clavulanate, the most common clinical β-lactamase inhibitor. While most single and double variants remain sensitive to clavulanate, double mutants R220S-K234R and S130G-K234R are substantially less affected by time-dependent clavulanate inactivation, showing residual β-lactam hydrolytic activities of 46% and 83% after 24 h incubation with a clinically relevant inhibitor concentration (5 μg/ml, 25 µM). These results demonstrate that active-site alterations in BlaC yield resistant variants that remain active and stable over prolonged bacterial generation times compatible with mycobacterial proliferation. These results also emphasize the formidable adaptive potential of inhibitor-resistant substitutions in β-lactamases, potentially casting a shadow on specific β-lactam–BlaC inhibitor combination treatments against M. tuberculosis. PMID:25492589

  17. Electron transport chain inhibitors induce microglia activation through enhancing mitochondrial reactive oxygen species production.

    PubMed

    Ye, Junli; Jiang, Zhongxin; Chen, Xuehong; Liu, Mengyang; Li, Jing; Liu, Na

    2016-01-15

    Reactive oxygen species (ROS) are believed to be mediators of excessive microglial activation, yet the resources and mechanism are not fully understood. Here we stimulated murine microglial BV-2 cells and primary microglial cells with different inhibitors of electron transport chain (ETC), rotenone, thenoyltrifluoroacetone (TTFA), antimycin A, and NaN3 to induce mitochondrial ROS production and we observed the role of mitochondrial ROS in microglial activation. Our results showed that ETC inhibitors resulted in significant changes in cell viability, microglial morphology, cell cycle arrest and mitochondrial ROS production in a dose-dependent manner in both primary cultural microglia and BV-2 cell lines. Moreover, ETC inhibitors, especially rotenone and antimycin A stimulated secretion of interleukin 1β (IL-1β), interleukin 6 (IL-6), interleukin 12 (IL-12) and tumor necrosis factor α (TNF-α) by microglia with marked activation of mitogen-activated proteinkinases (MAPKs) and nuclear factor κB (NF-κB), which could be blocked by specific inhibitors of MAPK and NF-κB and mitochondrial antioxidants, Mito-TEMPO. Taken together, our results demonstrated that inhibition of mitochondrial respiratory chain in microglia led to production of mitochondrial ROS and therefore may activate MAPK/NF-кB dependent inflammatory cytokines release in microglia, which indicated that mitochondrial-derived ROS were contributed to microglial activation.

  18. Identification of Selective and Potent Inhibitors of Fibroblast Activation Protein and Prolyl Oligopeptidase

    PubMed Central

    Poplawski, Sarah E.; Lai, Jack H.; Li, Youhua; Jin, Zhiping; Liu, Yuxin; Wu, Wengen; Wu, Yong; Zhou, Yuhong; Sudmeier, James L.; Sanford, David G.; Bachovchin, William W.

    2014-01-01

    Fibroblast activation protein (FAP) is a serine protease selectively expressed on reactive stromal fibroblasts of epithelial carcinomas. It is widely believed to play a role in tumor invasion and metastasis and therefore to represent a potential new drug target for cancer. Investigation into its biological function, however, has been hampered by the current unavailability of selective inhibitors. The challenge has been in identifying inhibitors that are selective for FAP over both the dipeptidyl peptidases (DPPs), with which it shares exopeptidase specificity, and prolyl oligopeptidase (PREP), with which it shares endopeptidase specificity. Here, we report the first potent FAP inhibitor with selectivity over both the DPPs and PREP, N-(pyridine-4-carbonyl)-d-Ala-boroPro (ARI-3099, 6). We also report a similarly potent and selective PREP inhibitor, N-(pyridine-3-carbonyl)-Val-boroPro (ARI-3531, 22). Both are boronic acid based inhibitors, demonstrating that high selectivity can be achieved using this electrophile. The inhibitors are stable, easy to synthesize, and should prove to be useful in helping to elucidate the biological functions of these two unique and interesting enzymes, as well as their potential as drug targets. PMID:23594271

  19. Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site.

    PubMed Central

    Weaver, T.; Lees, M.; Banaszak, L.

    1997-01-01

    Two mutant forms of fumarase C from E. coli have been made using PCR and recombinant DNA. The recombinant form of the protein included a histidine arm on the C-terminal facilitating purification. Based on earlier studies, two different carboxylic acid binding sites, labeled A- and B-, were observed in crystal structures of the wild type and inhibited forms of the enzyme. A histidine at each of the sites was mutated to an asparagine. H188N at the A-site resulted in a large decrease in specific activity, while the H129N mutation at the B-site had essentially no effect. From the results, we conclude that the A-site is indeed the active site, and a dual role for H188 as a potential catalytic base is proposed. Crystal structures of the two mutant proteins produced some unexpected results. Both mutations reduced the affinity for the carboxylic acids at their respective sites. The H129N mutant should be particularly useful in future kinetic studies because it sterically blocks the B-site with the carboxyamide of asparagine assuming the position of the ligand's carboxylate. In the H188N mutation at the active site, the new asparagine side chain still interacts with an active site water that appears to have moved slightly as a result of the mutation. PMID:9098893

  20. Hepatitis C virus NS3 protease is activated by low concentrations of protease inhibitors.

    PubMed

    Dahl, Göran; Arenas, Omar Gutiérrez; Danielson, U Helena

    2009-12-01

    The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) is a bifunctional enzyme with a protease and a helicase functionality located in each of the two domains of the single peptide chain. There is little experimental evidence for a functional role of this unexpected arrangement since artificial single domain forms of both enzymes are catalytically competent. We have observed that low concentrations of certain protease inhibitors activate the protease of full-length NS3 from HCV genotype 1a with up to 100%, depending on the preincubation time and the inhibitor used. The activation was reduced, but not eliminated, by increased ionic strength, lowered glycerol concentration, or lowered pH. In all cases, it was at the expense of a significant loss of activity. Activation was not seen with the artificial protease domain of genotype 1b NS3 fused with a fragment of the NS4A cofactor. This truncated and covalently modified enzyme form was much less active and exhibited fundamentally different catalytic properties to the full-length NS3 protease without the fused cofactor. The most plausible explanation for the activation was found to involve a slow transition between two enzyme conformations, which differed in their catalytic ability and affinity for inhibitors. Equations derived based on this assumption resulted in better fits to the experimental data than the equation for simple competitive inhibition. The mechanism may involve an inhibitor-induced stabilization of the helicase domain in a conformation that enhances the protease activity, or an improved alignment of the catalytic triad in the protease. The proposed mnemonic mechanism and derived equations are viable for both these explanations and can serve as a basic framework for future studies of enzymes activated by inhibitors or other ligands.

  1. Computational characterization of ketone-ketal transformations at the active site of matrix metalloproteinases.

    PubMed

    Khrenova, Maria G; Nemukhin, Alexander V; Savitsky, Alexander P

    2014-04-24

    We modeled the first steps of hydrolysis reactions of a natural oligopeptide substrate of matrix metalloproteinase MMP-2 as well as of a substrate analogue. In the latter, the scissile amide group is substituted by a ketomethylene group which can be transformed to the ketal group upon binding of this compound to the enzyme active site. According to our quantum mechanical-molecular mechanical (QM/MM) calculations, the reaction of the ketone-ketal transformation proceeds with a low energy barrier (3.4 kcal/mol) and a high equilibrium constant (10(4)). The reaction product with the ketal group formed directly at the active site of the enzyme works as an inhibitor that chelates the zinc ion. On the other hand, the oligopeptide mimetic retains molecular groups responsible for binding of this compound to the enzyme active site. This example illustrates a strategy to design MMP inhibitors in situ by using data on binding specificity of substrates to a particular type of MMP and details of the reaction mechanism. PMID:24684684

  2. Benzothiophene inhibitors of MK2. Part 1: structure-activity relationships, assessments of selectivity and cellular potency.

    PubMed

    Anderson, David R; Meyers, Marvin J; Kurumbail, Ravi G; Caspers, Nicole; Poda, Gennadiy I; Long, Scott A; Pierce, Betsy S; Mahoney, Matthew W; Mourey, Robert J

    2009-08-15

    Identification of potent benzothiophene inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK2), structure-activity relationship (SAR) studies, selectivity assessments against CDK2, cellular potency and mechanism of action are presented. Crystallographic data provide a rationale for the observed MK2 potency as well as selectivity over CDK2 for this class of inhibitors.

  3. Ionizable Side Chains at Catalytic Active Sites of Enzymes

    PubMed Central

    Jimenez-Morales, David; Liang, Jie

    2012-01-01

    Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1072 Å3. The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes. PMID:22484856

  4. Molecular Design, Synthesis and Trypanocidal Activity of Dipeptidyl Nitriles as Cruzain Inhibitors

    PubMed Central

    Avelar, Leandro A. A.; Camilo, Cristian D.; de Albuquerque, Sérgio; Fernandes, William B.; Gonçalez, Cristiana; Kenny, Peter W.; Leitão, Andrei; McKerrow, James H.; Montanari, Carlos A.; Orozco, Erika V. Meñaca; Ribeiro, Jean F. R.; Rocha, Josmar R.; Rosini, Fabiana; Saidel, Marta E.

    2015-01-01

    A series of compounds based on the dipeptidyl nitrile scaffold were synthesized and assayed for their inhibitory activity against the T. cruzi cysteine protease cruzain. Structure activity relationships (SARs) were established using three, eleven and twelve variations respectively at the P1, P2 and P3 positions. A Ki value of 16 nM was observed for the most potent of these inhibitors which reflects a degree of non-additivity in the SAR. An X-ray crystal structure was determined for the ligand-protein complex for the structural prototype for the series. Twenty three inhibitors were also evaluated for their anti-trypanosomal effects and an EC50 value of 28 μM was observed for the most potent of these. Although there remains scope for further optimization, the knowledge gained from this study is also transferable to the design of cruzain inhibitors based on warheads other than nitrile as well as alternative scaffolds. PMID:26173110

  5. Design, synthesis, and biological evaluation of novel, highly active soft ROCK inhibitors.

    PubMed

    Boland, Sandro; Bourin, Arnaud; Alen, Jo; Geraets, Jacques; Schroeders, Pieter; Castermans, Karolien; Kindt, Nele; Boumans, Nicki; Panitti, Laura; Fransen, Silke; Vanormelingen, Jessica; Stassen, Jean Marie; Leysen, Dirk; Defert, Olivier

    2015-05-28

    ROCK1 and ROCK2 play important roles in numerous cellular functions, including smooth muscle cell contraction, cell proliferation, adhesion, and migration. Consequently, ROCK inhibitors are of interest for treating multiple indications including cardiovascular diseases, inflammatory and autoimmune diseases, lung diseases, and eye diseases. However, systemic inhibition of ROCK is expected to result in significant side effects. Strategies allowing reduced systemic exposure are therefore of interest. In a continuing effort toward identification of ROCK inhibitors, we here report the design, synthesis, and evaluation of novel soft ROCK inhibitors displaying an ester function allowing their rapid inactivation in the systemic circulation. Those compounds display subnanomolar activity against ROCK and strong differences of functional activity between parent compounds and expected metabolites. The binding mode of a representative compound was determined experimentally in a single-crystal X-ray diffraction study. Enzymes responsible for inactivation of these compounds once they enter systemic circulation are also discussed.

  6. Bioreductively Activated Reactive Oxygen Species (ROS) Generators as MRSA Inhibitors.

    PubMed

    Khodade, Vinayak S; Sharath Chandra, Mallojjala; Banerjee, Ankita; Lahiri, Surobhi; Pulipeta, Mallikarjuna; Rangarajan, Radha; Chakrapani, Harinath

    2014-07-10

    The number of cases of drug resistant Staphylococcus aureus infections is on the rise globally and new strategies to identify drug candidates with novel mechanisms of action are in urgent need. Here, we report the synthesis and evaluation of a series of benzo[b]phenanthridine-5,7,12(6H)-triones, which were designed based on redox-active natural products. We find that the in vitro inhibitory activity of 6-(prop-2-ynyl)benzo[b]phenanthridine-5,7,12(6H)-trione (1f) against methicillin-resistant Staphylococcus aureus (MRSA), including a panel of patient-derived strains, is comparable or better than vancomycin. We show that the lead compound generates reactive oxygen species (ROS) in the cell, contributing to its antibacterial activity. PMID:25050164

  7. Gene expression and activity of digestive proteases in Daphnia: effects of cyanobacterial protease inhibitors

    PubMed Central

    2010-01-01

    Background The frequency of cyanobacterial blooms has increased worldwide, and these blooms have been claimed to be a major factor leading to the decline of the most important freshwater herbivores, i.e. representatives of the genus Daphnia. This suppression of Daphnia is partly attributed to the presence of biologically active secondary metabolites in cyanobacteria. Among these metabolites, protease inhibitors are found in almost every natural cyanobacterial bloom and have been shown to specifically inhibit Daphnia's digestive proteases in vitro, but to date no physiological responses of these serine proteases to cyanobacterial protease inhibitors in Daphnia have been reported in situ at the protein and genetic levels. Results Nine digestive proteases were detected in D. magna using activity-stained SDS-PAGE. Subsequent analyses by LC-MS/MS and database search led to the identification of respective protease genes. D. magna responded to dietary protease inhibitors by up-regulation of the expression of these respective proteases at the RNA-level and by the induction of new and less sensitive protease isoforms at the protein level. The up-regulation in response to dietary trypsin- and chymotrypsin-inhibitors ranged from 1.4-fold to 25.6-fold. These physiological responses of Daphnia, i.e. up-regulation of protease expression and the induction of isoforms, took place even after feeding on 20% cyanobacterial food for only 24 h. These physiological responses proved to be independent from microcystin effects. Conclusion Here for the first time it was shown in situ that a D. magna clone responds physiologically to dietary cyanobacterial protease inhibitors by phenotypic plasticity of the targets of these specific inhibitors, i.e. Daphnia gut proteases. These regulatory responses are adaptive for D. magna, as they increase the capacity for protein digestion in the presence of dietary protease inhibitors. The type and extent of these responses in protease expression might

  8. Host immunity contributes to the anti-melanoma activity of BRAF inhibitors.

    PubMed

    Knight, Deborah A; Ngiow, Shin Foong; Li, Ming; Parmenter, Tiffany; Mok, Stephen; Cass, Ashley; Haynes, Nicole M; Kinross, Kathryn; Yagita, Hideo; Koya, Richard C; Graeber, Thomas G; Ribas, Antoni; McArthur, Grant A; Smyth, Mark J

    2013-03-01

    The BRAF mutant, BRAF(V600E), is expressed in nearly half of melanomas, and oral BRAF inhibitors induce substantial tumor regression in patients with BRAF(V600E) metastatic melanoma. The inhibitors are believed to work primarily by inhibiting BRAF(V600E)-induced oncogenic MAPK signaling; however, some patients treated with BRAF inhibitors exhibit increased tumor immune infiltration, suggesting that a combination of BRAF inhibitors and immunotherapy may be beneficial. We used two relatively resistant variants of Braf(V600E)-driven mouse melanoma (SM1 and SM1WT1) and melanoma-prone mice to determine the role of host immunity in type I BRAF inhibitor PLX4720 antitumor activity. We found that PLX4720 treatment downregulated tumor Ccl2 gene expression and decreased tumor CCL2 expression in both Braf(V600E) mouse melanoma transplants and in de novo melanomas in a manner that was coincident with reduced tumor growth. While PLX4720 did not directly increase tumor immunogenicity, analysis of SM1 tumor-infiltrating leukocytes in PLX4720-treated mice demonstrated a robust increase in CD8(+) T/FoxP3(+)CD4(+) T cell ratio and NK cells. Combination therapy with PLX4720 and anti-CCL2 or agonistic anti-CD137 antibodies demonstrated significant antitumor activity in mouse transplant and de novo tumorigenesis models. These data elucidate a role for host CCR2 in the mechanism of action of type I BRAF inhibitors and support the therapeutic potential of combining BRAF inhibitors with immunotherapy.

  9. Effective virtual screening strategy toward covalent ligands: identification of novel NEDD8-activating enzyme inhibitors.

    PubMed

    Zhang, Shengping; Tan, Jiani; Lai, Zhonghui; Li, Ying; Pang, Junxia; Xiao, Jianhu; Huang, Zhangjian; Zhang, Yihua; Ji, Hui; Lai, Yisheng

    2014-06-23

    The NEDD8-activating enzyme (NAE) is an emerging target for cancer therapy, which regulates the degradation and turnover of a variety of cancer-related proteins by activating the cullin-RING E3 ubiquitin ligases. Among a limited number of known NAE inhibitors, the covalent inhibitors have demonstrated the most potent efficacy through their covalently linked adducts with NEDD8. Inspired by this unique mechanism, in this study, a novel combined strategy of virtual screening (VS) was adopted with the aim to identify diverse covalent inhibitors of NAE. To be specific, a docking-enabled pharmacophore model was first built from the possible active conformations of chosen covalent inhibitors. Meanwhile, a dynamic structure-based phamacophore was also established based on the snapshots derived from molecular dynamic simulation. Subsequent screening of a focused ZINC database using these pharmacophore models combined with covalent docking discovered three novel active compounds. Among them, compound LZ3 exhibited the most potent NAE inhibitory activity with an IC50 value of 1.06 ± 0.18 μM. Furthermore, a cell-based washout experiment proved the proposed covalent binding mechanism for compound LZ3, which confirmed the successful application of our combined VS strategy, indicating it may provide a viable solution to systematically discover novel covalent ligands.

  10. Measuring the serotonin uptake site using (/sup 3/H)paroxetine--a new serotonin uptake inhibitor

    SciTech Connect

    Gleiter, C.H.; Nutt, D.J.

    1988-01-01

    Serotonin is an important neurotransmitter that may be involved in ethanol preference and dependence. It is possible to label the serotonin uptake site in brain using the tricyclic antidepressant imipramine, but this also binds to other sites. We have used the new high-affinity uptake blocker paroxetine to define binding to this site and report it to have advantages over imipramine as a ligand.

  11. Epirubicin, Identified Using a Novel Luciferase Reporter Assay for Foxp3 Inhibitors, Inhibits Regulatory T Cell Activity

    PubMed Central

    Kashima, Hajime; Momose, Fumiyasu; Umehara, Hiroshi; Miyoshi, Nao; Ogo, Naohisa; Muraoka, Daisuke; Shiku, Hiroshi; Harada, Naozumi; Asai, Akira

    2016-01-01

    Forkhead box protein p3 (Foxp3) is crucial to the development and suppressor function of regulatory T cells (Tregs) that have a significant role in tumor-associated immune suppression. Development of small molecule inhibitors of Foxp3 function is therefore considered a promising strategy to enhance anti-tumor immunity. In this study, we developed a novel cell-based assay system in which the NF-κB luciferase reporter signal is suppressed by the co-expressed Foxp3 protein. Using this system, we screened our chemical library consisting of approximately 2,100 compounds and discovered that a cancer chemotherapeutic drug epirubicin restored the Foxp3-inhibited NF-κB activity in a concentration-dependent manner without influencing cell viability. Using immunoprecipitation assay in a Treg-like cell line Karpas-299, we found that epirubicin inhibited the interaction between Foxp3 and p65. In addition, epirubicin inhibited the suppressor function of murine Tregs and thereby improved effector T cell stimulation in vitro. Administration of low dose epirubicin into tumor-bearing mice modulated the function of immune cells at the tumor site and promoted their IFN-γ production without direct cytotoxicity. In summary, we identified the novel action of epirubicin as a Foxp3 inhibitor using a newly established luciferase-based cellular screen. Our work also demonstrated our screen system is useful in accelerating discovery of Foxp3 inhibitors. PMID:27284967

  12. Antiplasmodial Activity and Mechanism of Action of RSM-932A, a Promising Synergistic Inhibitor of Plasmodium falciparum Choline Kinase

    PubMed Central

    Zimmerman, Tahl; Moneriz, Carlos; Diez, Amalia; Bautista, José Manuel; Gómez del Pulgar, Teresa; Cebrián, Arancha

    2013-01-01

    We have investigated the mechanism of action of inhibition of the choline kinase of P. falciparum (p.f.-ChoK) by two inhibitors of the human ChoKα, MN58b and RSM-932A, which have previously been shown to be potent antitumoral agents. The efficacy of these inhibitors against p.f.-ChoK is investigated using enzymatic and in vitro assays. While MN58b may enter the choline/phosphocholine binding site, RSM-932A appears to have an altogether novel mechanism of inhibition and is synergistic with respect to both choline and ATP. A model of inhibition for RSM-932A in which this inhibitor traps p.f.-ChoK in a phosphorylated intermediate state blocking phosphate transfer to choline is presented. Importantly, MN58b and RSM-932A have in vitro inhibitory activity in the low nanomolar range and are equally effective against chloroquine-sensitive and chloroquine-resistant strains. RSM-932A and MN58b significantly reduced parasitemia and induced the accumulation of trophozoites and schizonts, blocking intraerythrocytic development and interfering with parasite egress or invasion, suggesting a delay of the parasite maturation stage. The present data provide two new potent structures for the development of antimalarial compounds and validate p.f.-ChoK as an accessible drug target against the parasite. PMID:24041883

  13. Plasminogen activator inhibitor type 1 interacts with alpha3 subunit of proteasome and modulates its activity.

    PubMed

    Boncela, Joanna; Przygodzka, Patrycja; Papiewska-Pajak, Izabela; Wyroba, Elzbieta; Osinska, Magdalena; Cierniewski, Czeslaw S

    2011-02-25

    Plasminogen activator inhibitor type-1 (PAI-1), a multifunctional protein, is an important physiological regulator of fibrinolysis, extracellular matrix homeostasis, and cell motility. Recent observations show that PAI-1 may also be implicated in maintaining integrity of cells, especially with respect to cellular proliferation or apoptosis. In the present study we provide evidence that PAI-1 interacts with proteasome and affects its activity. First, by using the yeast two-hybrid system, we found that the α3 subunit of proteasome directly interacts with PAI-1. Then, to ensure that the PAI-1-proteasome complex is formed in vivo, both proteins were coimmunoprecipitated from endothelial cells and identified with specific antibodies. The specificity of this interaction was evidenced after transfection of HeLa cells with pCMV-PAI-1 and coimmunoprecipitation of both proteins with anti-PAI-1 antibodies. Subsequently, cellular distribution of the PAI-1-proteasome complexes was established by immunogold staining and electron microscopy analyses. Both proteins appeared in a diffuse cytosolic pattern but also could be found in a dense perinuclear and nuclear location. Furthermore, PAI-1 induced formation of aggresomes freely located in endothelial cytoplasm. Increased PAI-1 expression abrogated degradation of degron analyzed after cotransfection of HeLa cells with pCMV-PAI-1 and pd2EGFP-N1 and prevented degradation of p53 as well as IκBα, as evidenced both by confocal microscopy and Western immunoblotting.

  14. Plasminogen Activator Inhibitor Type 1 Interacts with α3 Subunit of Proteasome and Modulates Its Activity*

    PubMed Central

    Boncela, Joanna; Przygodzka, Patrycja; Papiewska-Pajak, Izabela; Wyroba, Elzbieta; Osinska, Magdalena; Cierniewski, Czeslaw S.

    2011-01-01

    Plasminogen activator inhibitor type-1 (PAI-1), a multifunctional protein, is an important physiological regulator of fibrinolysis, extracellular matrix homeostasis, and cell motility. Recent observations show that PAI-1 may also be implicated in maintaining integrity of cells, especially with respect to cellular proliferation or apoptosis. In the present study we provide evidence that PAI-1 interacts with proteasome and affects its activity. First, by using the yeast two-hybrid system, we found that the α3 subunit of proteasome directly interacts with PAI-1. Then, to ensure that the PAI-1-proteasome complex is formed in vivo, both proteins were coimmunoprecipitated from endothelial cells and identified with specific antibodies. The specificity of this interaction was evidenced after transfection of HeLa cells with pCMV-PAI-1 and coimmunoprecipitation of both proteins with anti-PAI-1 antibodies. Subsequently, cellular distribution of the PAI-1-proteasome complexes was established by immunogold staining and electron microscopy analyses. Both proteins appeared in a diffuse cytosolic pattern but also could be found in a dense perinuclear and nuclear location. Furthermore, PAI-1 induced formation of aggresomes freely located in endothelial cytoplasm. Increased PAI-1 expression abrogated degradation of degron analyzed after cotransfection of HeLa cells with pCMV-PAI-1 and pd2EGFP-N1 and prevented degradation of p53 as well as IκBα, as evidenced both by confocal microscopy and Western immunoblotting. PMID:21135093

  15. Structure–Activity Relationship for the First-in-Class Clinical Steroid Sulfatase Inhibitor Irosustat (STX64, BN83495)

    PubMed Central

    Woo, L W Lawrence; Ganeshapillai, Dharshini; Thomas, Mark P; Sutcliffe, Oliver B; Malini, Bindu; Mahon, Mary F; Purohit, Atul; Potter, Barry V L

    2011-01-01

    Structure–activity relationship studies were conducted on Irosustat (STX64, BN83495), the first steroid sulfatase (STS) inhibitor to enter diverse clinical trials for patients with advanced hormone-dependent cancer. The size of its aliphatic ring was expanded; its sulfamate group was N,N-dimethylated, relocated to another position and flanked by an adjacent methoxy group; and series of quinolin-2(1H)-one and quinoline derivatives of Irosustat were explored. The STS inhibitory activities of the synthesised compounds were assessed in a preparation of JEG-3 cells. Stepwise enlargement of the aliphatic ring from 7 to 11 members increases potency, although a further increase in ring size is detrimental. The best STS inhibitors in vitro had IC50 values between 0.015 and 0.025 nm. Other modifications made to Irosustat were found to either abolish or significantly weaken its activity. An azomethine adduct of Irosustat with N,N-dimethylformamide (DMF) was isolated, and crystal structures of Irosustat and this adduct were determined. Docking studies were conducted to explore the potential interactions between compounds and the active site of STS, and suggest a sulfamoyl group transfer to formylglycine 75 during the inactivation mechanism. PMID:21990014

  16. HSP90 inhibitors decrease AID levels and activity in mice and in human cells.

    PubMed

    Montamat-Sicotte, Damien; Litzler, Ludivine C; Abreu, Cecilia; Safavi, Shiva; Zahn, Astrid; Orthwein, Alexandre; Müschen, Markus; Oppezzo, Pablo; Muñoz, Denise P; Di Noia, Javier M

    2015-08-01

    Activation induced deaminase (AID) initiates somatic hypermutation and class switch recombination of the Ig genes in antigen-activated B cells, underpinning antibody affinity maturation and isotype switching. AID can also be pathogenic by contributing to autoimmune diseases and oncogenic mutations. Moreover, AID can exert noncanonical functions when aberrantly expressed in epithelial cells. The lack of specific inhibitors prevents therapeutic applications to modulate AID functions. Here, we have exploited our previous finding that the HSP90 molecular chaperoning pathway stabilizes AID in B cells, to test whether HSP90 inhibitors could target AID in vivo. We demonstrate that chronic administration of HSP90 inhibitors decreases AID protein levels and isotype switching in immunized mice. HSP90 inhibitors also reduce disease severity in a mouse model of acute B-cell lymphoblastic leukemia in which AID accelerates disease progression. We further show that human AID protein levels are sensitive to HSP90 inhibition in normal and leukemic B cells, and that HSP90 inhibition prevents AID-dependent epithelial to mesenchymal transition in a human breast cancer cell line in vitro. Thus, we provide proof-of-concept that HSP90 inhibitors indirectly target AID in vivo and that endogenous human AID is widely sensitive to them, which could have therapeutic applications.

  17. Herbacetin Is a Novel Allosteric Inhibitor of Ornithine Decarboxylase with Antitumor Activity.

    PubMed

    Kim, Dong Joon; Roh, Eunmiri; Lee, Mee-Hyun; Oi, Naomi; Lim, Do Young; Kim, Myoung Ok; Cho, Yong-Yeon; Pugliese, Angelo; Shim, Jung-Hyun; Chen, Hanyong; Cho, Eun Jin; Kim, Jong-Eun; Kang, Sun Chul; Paul, Souren; Kang, Hee Eun; Jung, Ji Won; Lee, Sung-Young; Kim, Sung-Hyun; Reddy, Kanamata; Yeom, Young Il; Bode, Ann M; Dong, Zigang

    2016-03-01

    Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the first step of polyamine biosynthesis that is associated with cell growth and tumor formation. Existing catalytic inhibitors of ODC have lacked efficacy in clinical testing or displayed unacceptable toxicity. In this study, we report the identification of an effective and nontoxic allosteric inhibitor of ODC. Using computer docking simulation and an in vitro ODC enzyme assay, we identified herbacetin, a natural compound found in flax and other plants, as a novel ODC inhibitor. Mechanistic investigations defined aspartate 44 in ODC as critical for binding. Herbacetin exhibited potent anticancer activity in colon cancer cell lines expressing high levels of ODC. Intraperitoneal or oral administration of herbacetin effectively suppressed HCT116 xenograft tumor growth and also reduced the number and size of polyps in a mouse model of APC-driven colon cancer (ApcMin/+). Unlike the well-established ODC inhibitor DFMO, herbacetin treatment was not associated with hearing loss. Taken together, our findings defined the natural product herbacetin as an allosteric inhibitor of ODC with chemopreventive and antitumor activity in preclinical models of colon cancer, prompting its further investigation in clinical trials. PMID:26676750

  18. Inhibitors of SRC kinases impair antitumor activity of anti-CD20 monoclonal antibodies

    PubMed Central

    Winiarska, Magdalena; Bojarczuk, Kamil; Pyrzynska, Beata; Bil, Jacek; Siernicka, Marta; Dwojak, Michal; Bobrowicz, Malgorzata; Miazek, Nina; Zapala, Piotr; Zagozdzon, Agnieszka; Krol, Magdalena; Syta, Aleksandra; Podszywalow-Bartnicka, Paulina; Pilch, Zofia; Dabrowska-Iwanicka, Anna; Juszczynski, Przemyslaw; Efremov, Dimitar G; Slabicki, Mikolaj; Zenz, Thorsten; Roy, Aude Le; Olive, Daniel; Rygiel, Tomasz P; Leusen, Jeanette HW; Golab, Jakub

    2014-01-01

    Clinical trials with SRC family kinases (SFKs) inhibitors used alone or in a combination with anti-CD20 monoclonal antibodies (mAbs) are currently underway in the treatment of B-cell tumors. However, molecular interactions between these therapeutics have not been studied so far. A transcriptional profiling of tumor cells incubated with SFKs inhibitors revealed strong downregulation of MS4A1 gene encoding CD20 antigen. In a panel of primary and established B-cell tumors we observed that SFKs inhibitors strongly affect CD20 expression at the transcriptional level, leading to inhibition of anti-CD20 mAbs binding and increased resistance of tumor cells to complement-dependent cytotoxicity. Activation of the AKT signaling pathway significantly protected cells from dasatinib-triggered CD20 downregulation. Additionally, SFKs inhibitors suppressed antibody-dependent cell-mediated cytotoxicity by direct inhibition of natural killer cells. Abrogation of antitumor activity of rituximab was also observed in vivo in a mouse model. Noteworthy, the effects of SFKs inhibitors on NK cell function are largely reversible. The results of our studies indicate that development of optimal combinations of novel treatment modalities with anti-CD20 mAbs should be preceded by detailed preclinical evaluation of their effects on target cells. PMID:25517315

  19. HSP90 inhibitors decrease AID levels and activity in mice and in human cells

    PubMed Central

    Montamat-Sicotte, Damien; Liztler, Ludivine C; Abreu, Cecilia; Safavi, Shiva; Zahn, Astrid; Orthwein, Alexandre; Muschen, Markus; Oppezzo, Pablo; Muñoz, Denise P; Di Noia, Javier M

    2015-01-01

    Activation induced deaminase (AID) initiates somatic hypermutation and class switch recombination of the Ig genes in antigen-activated B cells, underpinning antibody affinity maturation and isotype switching. AID can also be pathogenic by contributing to autoimmune diseases and oncogenic mutations. Moreover, AID can exert non-canonical functions when aberrantly expressed in epithelial cells. The lack of specific inhibitors prevents therapeutic applications to modulate AID functions. Here, we have exploited our previous finding that the HSP90 molecular chaperoning pathway stabilizes AID in B cells, to test whether HSP90 inhibitors could target AID in vivo. We demonstrate that chronic administration of HSP90 inhibitors decreases AID protein levels and isotype switching in immunized mice. HSP90 inhibitors also reduce disease severity in a mouse model of acute B-cell lymphoblastic leukemia in which AID accelerates disease progression. We further show that human AID protein levels are sensitive to HSP90 inhibition in normal and leukemic B cells, and that HSP90 inhibition prevents AID-dependent epithelial to mesenchymal transition in a human breast cancer cell line in vitro. Thus, we provide proof-of-concept that HSP90 inhibitors indirectly target AID in vivo and that endogenous human AID is widely sensitive to them, which could have therapeutic applications. PMID:25912253

  20. Effect of dipeptidyl peptidase-4 inhibitor, vildagliptin on plasminogen activator inhibitor-1 in patients with diabetes mellitus.

    PubMed

    Tani, Shigemasa; Takahashi, Atsuhiko; Nagao, Ken; Hirayama, Atsushi

    2015-02-15

    Dipeptidyl peptidase-4 (DPP-4) inhibitors may affect the serum levels of plasminogen activator inhibitor-1 (PAI-1) associated with triglyceride (TG) metabolism, which is a prognostic factor for cardiovascular disease, in diabetic patients. We conducted an 8-week, prospective, randomized study in which we assigned type 2 diabetic patients who were inadequately controlled with antidiabetic therapy to the vildagliptin group (50 mg bid, n = 49) or the control group (n = 49). The primary efficacy parameter was the change in the serum level of PAI-1, and the secondary end point was the change in the serum levels of TG-rich lipoproteins. In the vildagliptin group, significant decrease of the serum PAI-1 level by 16.3% (p <0.0001) and significant decreases of the serum TG, remnant-like particle cholesterol, and apolipoprotein B levels by 12.1% (p = 0.002), 13.9% (p = 0.003), and 9.5% (p <0.0001), respectively, were observed. No such changes were observed in the control group. Multivariate regression analyses identified the absolute change from the baseline (Δ) of the PAI-1, but not that of the fasting blood glucose or hemoglobin A1c, as independent predictors of the ΔTG, Δ remnant-like particle cholesterol, and Δ apolipoprotein B. In conclusion, treatment of type 2 diabetes with vildagliptin might prevent the progression of atherosclerotic cardiovascular disease in diabetic patients by decreasing the serum PAI-1 levels and improving TG metabolism.

  1. Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis).

    PubMed

    Samac, D; Storey, R

    1981-12-01

    Changes in proteolytic activity (aminopeptidase, carboxypeptidase, endopeptidase) were followed during germination (imbibition through seedling development) in extracts from cotyledons of jojoba seeds (Simmondsia chinensis). After imbibition, the cotyledons contained high levels of sulfhydryl aminopeptidase activity (APA) but low levels of serine carboxypeptidase activity (CPA). CPA increased with germination through the apparent loss of a CPA inhibitor substance in the seed. Curves showing changes in endopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 during germination were distinctly different. EPA at pH 4, 5, 6, and 7 showed characteristics of sulfhydryl enzymes while activity at pH 8 was probably due to a serine type enzyme. EPA at pH 6 was inhibited early in germination by one or more substances in the seed. Activities at pH 5 and later at pH 6 were the highest of all EPA throughout germination and increases in these activities were associated with a rapid loss of protein from the cotyledons of the developing seedling.Jojoba cotyledonary extracts were found to inhibit the enzymic activity of trypsin, chymotrypsin, and pepsin but not the protease from Aspergillus saotoi. The heat-labile trypsin inhibitor substance(s) was found in commercially processed jojoba seed meal and the albumin fraction of seed proteins. Trypsin inhibitor activity decreased with germination.

  2. Differential regulation of plasminogen activator and inhibitor gene transcription by the tumor suppressor p53.

    PubMed Central

    Kunz, C; Pebler, S; Otte, J; von der Ahe, D

    1995-01-01

    The ability of p53 to activate or repress transcription suggests that its biological function as tumor suppressor is in part accomplished by regulating a number of genes including such required for inhibition of cell growth. We here give evidence that p53 also may regulate genes responsible for the proteolytic degradation of the extracellular matrix, which is considered a crucial feature for local invasion and metastasis of neoplastic cells. An important and highly regulated cascade of such proteolytic events involves the plasminogen activator system. We show that wild-type p53 represses transcription from the enhancer and promoter of the human urokinase-type (u-PA) and the tissue-type plasminogen activator (t-PA) gene through a non-DNA binding mechanism. Oncogenic mutants lost the repressing activity. In contrast, wild-type but not mutant p53 specifically binds to and activates the promoter of the plasminogen activator inhibitor type-1 (PAI-1) gene. Interestingly, one of the p53 mutants (273his) inhibited PAI-1 promoter activity. Our results suggest that altered function of oncogenic forms of p53 may lead to altered expression of the plasminogen activators and their inhibitor(s) and thus to altered activation of the plasminogen/plasmin system during tumor progression. Images PMID:7479001

  3. The Inhibitor DBMIB Provides Insight into the Functional Architecture of the Qo Site in the Cytochrome b(6)f Complex

    SciTech Connect

    Roberts, Arthur G.; Bowman, Michael K.; Kramer, David M.

    2004-06-22

    Previously, we showed that two equivalents of the quinone analog, 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB), could occupy the Qo site of the cytochrome (cyt) b6f complex simultaneously. In this work, study of electron paramagnetic resonance (EPR) spectra from oriented cyt b6f complex shows that the Rieske iron-sulfur protein (ISP) is in distinct orientations, depending on the stoichiometry of the inhibitor at the Qo site. With a single DBMIB at the Qo site, the ISP is oriented with the 2Fe2S cluster toward cyt f, which is similar to the orientation of the ISP in the x-ray crystal structure of the cyt b6f complex from thermophilic cyanobacteria, Mastigocladus laminosus, in the presence of DBMIB, as well as that of the chicken mitochondrial cyt bc1 complex in the presence of the class II inhibitor myxothiazol, which binds in the so-called ''proximal niche,'' near the cyt bL heme. These data suggest that the high affinity DBMIB site is at the proximal niche Qo pocket. With 2 equivalents or more of DBMIB bound, the Rieske ISP is in a position that resembles the ISPB position of chicken mitochondrial cyt bc1 complex in the presence of stigmatellin and Chlamydamonas reinhardtii cyt b6f complex in the presence of tridecyl-stigmatellin (TDS), which suggests that the low affinity DBMIB site is at the distal niche. The close interaction of DBMIB bound at the distal niche with the ISP induced the well-known effects on the 2Fe2S EPR spectrum and redox potential. To further test the effects of DBMIB on the ISP, the extents of cyt f oxidation after flash excitation in the presence of photosystem II inhibitor DCMU were measured as a function of DBMIB concentration in thylakoids. Addition of DBMIB concentrations where single binding was expected, did not markedly affect the extent of cyt f oxidation, whereas higher concentrations, where double occupancy was expected, increased the extent of cyt f oxidation to levels similar to cyt f oxidation in the presence of

  4. Small Molecule Inhibitors of the LEDGF Site of Human Immunodeficiency Virus Integrase Identified by Fragment Screening and Structure Based Design

    PubMed Central

    Peat, Thomas S.; Rhodes, David I.; Vandegraaff, Nick; Le, Giang; Smith, Jessica A.; Clark, Lisa J.; Jones, Eric D.; Coates, Jonathan A. V.; Thienthong, Neeranat; Newman, Janet; Dolezal, Olan; Mulder, Roger; Ryan, John H.; Savage, G. Paul; Francis, Craig L.; Deadman, John J.

    2012-01-01

    A fragment-based screen against human immunodeficiency virus type 1 (HIV) integrase led to a number of compounds that bound to the lens epithelium derived growth factor (LEDGF) binding site of the integrase catalytic core domain. We determined the crystallographic structures of complexes of the HIV integrase catalytic core domain for 10 of these compounds and quantitated the binding by surface plasmon resonance. We demonstrate that the compounds inhibit the interaction of LEDGF with HIV integrase in a proximity AlphaScreen assay, an assay for the LEDGF enhancement of HIV integrase strand transfer and in a cell based assay. The compounds identified represent a potential framework for the development of a new series of HIV integrase inhibitors that do not bind to the catalytic site of the enzyme. PMID:22808106

  5. MK-886, an inhibitor of the 5-lipoxygenase-activating protein, inhibits cyclooxygenase-1 activity and suppresses platelet aggregation.

    PubMed

    Koeberle, Andreas; Siemoneit, Ulf; Northoff, Hinnak; Hofmann, Bettina; Schneider, Gisbert; Werz, Oliver

    2009-04-17

    MK-886, an inhibitor of the 5-lipoxygenase-activating protein (FLAP), potently suppresses leukotriene biosynthesis in intact cells and is frequently used to define a role of the 5-lipoxygenase (EC 1.13.11.34) pathway in cellular or animal models of inflammation, allergy, cancer, and cardiovascular disease. Here we show that MK-886 also interferes with the activities of cyclooxygenases (COX, EC 1.14.99.1). MK-886 inhibited isolated COX-1 (IC(50)=8 microM) and blocked the formation of the COX-1-derived products 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid (12-HHT) and thromboxane B(2) in washed human platelets in response to collagen as well as from exogenous arachidonic acid (IC(50)=13-15 microM). Isolated COX-2 was less affected (IC(50)=58 microM), and in A549 cells, MK-886 (33 microM) failed to suppress COX-2-dependent 6-keto-prostaglandin (PG)F(1alpha) formation. The distinct susceptibility of MK-886 towards COX-1 and -2 is apparent in automated molecular docking studies that indicate a preferred binding of MK-886 to COX-1 into the active site. MK-886 (10 microM) inhibited COX-1-mediated platelet aggregation induced by collagen or arachidonic acid whereas thrombin- or U-46619-induced (COX-independent) aggregation was not affected. Since leukotrienes and prostaglandins share (patho)physiological properties in the development and regulation of carcinogenesis, inflammation, and vascular functions, caution should be used when interpreting data where MK-886 is used as tool to determine the involvement of FLAP and/or the 5-lipoxygenase pathway in respective experimental models.

  6. Structure of the binding site for nonnucleoside inhibitors of the reverse transcriptase of human immunodeficiency virus type 1.

    PubMed Central

    Smerdon, S J; Jäger, J; Wang, J; Kohlstaedt, L A; Chirino, A J; Friedman, J M; Rice, P A; Steitz, T A

    1994-01-01

    The dipyridodiazepinone Nevirapine is a potent and highly specific inhibitor of the reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1). It is a member of an important class of nonnucleoside drugs that appear to share part or all of the same binding site on the enzyme but are susceptible to a variety of spontaneous drug-resistance mutations. The co-crystal-structure of HIV-1 RT and Nevirapine has been solved previously at 3.5-A resolution and now is partially refined against data extending to 2.9-A spacing. The drug is bound in a hydrophobic pocket and in contact with some 38 protein atoms from the p66 palm and thumb subdomains. Most, but not all, nonnucleoside drug-resistance mutations map to residues in close contact with Nevirapine. The major effects of these mutations are to introduce steric clashes with the drug molecule or to remove favorable protein-drug contacts. Additionally, four residues (Phe-227, Trp-229, Leu-234, and Tyr-319) in contact with Nevirapine have not been selected as sites of drug-resistance mutations, implying that there may be limitations on the number and types of resistance mutations that yield viable virus. Strategies of inhibitor design that target interactions with these conserved residues may yield drugs that are less vulnerable to escape mutations. Images PMID:7513427

  7. Administration of C1 inhibitor reduces neutrophil activation in patients with sepsis.

    PubMed

    Zeerleder, Sacha; Caliezi, Christoph; van Mierlo, Gerard; Eerenberg-Belmer, Anke; Sulzer, Irmela; Hack, C Erik; Wuillemin, Walter A

    2003-07-01

    Forty patients with severe sepsis or septic shock recently received C1 inhibitor. In the present study we studied the effect of C1 inhibitor therapy on circulating elastase-alpha(1)-antitrypsin complex (EA) and lactoferrin (LF) levels in these patients to gain further insight about agonists involved in the activation of neutrophils in human sepsis. Elevated levels of EA and LF were found in 65 and 85% of the septic patients, respectively. Patients with elevated EA levels had higher organ dysfunction scores, higher levels of cytokines, and higher levels of complement activation products than patients with normal EA levels. C1 inhibitor therapy reduced EA as well as complement activation and IL-8 release in the patients with elevated EA on admission. We conclude that neutrophil activation in human sepsis correlates with the severity of organ dysfunction and involves complement and interleukin-8 as agonists. The effect of C1 inhibitor therapy on neutrophils may provide an explanation for the beneficial, although mild, effects of this treatment on organ dysfunction in sepsis.

  8. Metabolic factors, adipose tissue, and plasminogen activator inhibitor-1 levels in Type 2 diabetes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plasminogen activator inhibitor-1 (PAI-1) production by adipose tissue is increased in obesity, and its circulating levels are high in type 2 diabetes. PAI-1 increases cardiovascular risk by favoring clot stability, interfering with vascular remodeling, or both. We investigated in obese diabetic per...

  9. The biological activity of a-mangostin, a larvicidal botanic mosquito sterol carrier protein-2 inhibitor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alpha-mangostin derived from mangosteen was identified as a mosquito sterol carrier protein-2 inhibitor via high throughput insecticide screening. Alpha-mangostin was tested for its larvicidal activity against 3rd instar larvae of six mosquito species and the LC50 values range from 0.84 to 2.90 ppm....

  10. In Vitro Antimalarial Activity of Different Inhibitors of the Plasmodial Isoprenoid Synthesis Pathway.

    PubMed

    da Silva, Marcia F; Saito, Alexandre Y; Peres, Valnice J; Oliveira, Antonio C; Katzin, Alejandro M

    2015-08-01

    Previous studies have shown that fosmidomycin, risedronate, and nerolidol exert antimalarial activity in vitro. We included squalestatin, an inhibitor of the isoprenoid metabolism in Erwinia uredovora, and found that combinations of compounds which act on different targets of the plasmodial isoprenoid pathway possess important supra-additivity effects. PMID:26055383

  11. In Vitro Antimalarial Activity of Different Inhibitors of the Plasmodial Isoprenoid Synthesis Pathway

    PubMed Central

    da Silva, Marcia F.; Saito, Alexandre Y.; Peres, Valnice J.; Oliveira, Antonio C.

    2015-01-01

    Previous studies have shown that fosmidomycin, risedronate, and nerolidol exert antimalarial activity in vitro. We included squalestatin, an inhibitor of the isoprenoid metabolism in Erwinia uredovora, and found that combinations of compounds which act on different targets of the plasmodial isoprenoid pathway possess important supra-additivity effects. PMID:26055383

  12. Hydrogen/Deuterium Exchange Kinetics Demonstrate Long Range Allosteric Effects of Thumb Site 2 Inhibitors of Hepatitis C Viral RNA-dependent RNA Polymerase.

    PubMed

    Deredge, Daniel; Li, Jiawen; Johnson, Kenneth A; Wintrode, Patrick L

    2016-05-01

    New nonnucleoside analogs are being developed as part of a multi-drug regimen to treat hepatitis C viral infections. Particularly promising are inhibitors that bind to the surface of the thumb domain of the viral RNA-dependent RNA polymerase (NS5B). Numerous crystal structures have been solved showing small molecule non-nucleoside inhibitors bound to the hepatitis C viral polymerase, but these structures alone do not define the mechanism of inhibition. Our prior kinetic analysis showed that nonnucleoside inhibitors binding to thumb site-2 (NNI2) do not block initiation or elongation of RNA synthesis; rather, they block the transition from the initiation to elongation, which is thought to proceed with significant structural rearrangement of the enzyme-RNA complex. Here we have mapped the effect of three NNI2 inhibitors on the conformational dynamics of the enzyme using hydrogen/deuterium exchange kinetics. All three inhibitors rigidify an extensive allosteric network extending >40 Å from the binding site, thus providing a structural rationale for the observed disruption of the transition from distributive initiation to processive elongation. The two more potent inhibitors also suppress slow cooperative unfolding in the fingers extension-thumb interface and primer grip, which may contribute their stronger inhibition. These results establish that NNI2 inhibitors act through long range allosteric effects, reveal important conformational changes underlying normal polymerase function, and point the way to the design of more effective allosteric inhibitors that exploit this new information. PMID:27006396

  13. Inhibitor focusing: direct selection of drug targets from proteomes using activity-based probes.

    PubMed

    Nomanbhoy, Tyzoon K; Rosenblum, Jonathan; Aban, Arwin; Burbaum, Jonathan J

    2003-02-01

    In the latter stages of drug discovery and development, assays that establish drug selectivity and toxicity are important when side effects, which are often due to lack of specificity, determine drug candidate viability. There has been no comprehensive or systematic methodology to measure these factors outside of whole-animal assays, and such phenomenological assays generally fail to establish the additional targets of a given small molecule, or the molecular origin of toxicity. Consequently, small-molecule development programs destined for failure often reach advanced stages of testing, and the money and time invested in such programs could be saved if information on selectivity were available early in the process. Here, we present a methodology that utilizes chemical ABPs in combination with small-molecule inhibitors to selectively label small-molecule binding sites in whole proteomic samples. In principle, the ABP and small molecule will compete for similar binding sites, such that the small molecule will protect against modification by the ABP. Thus, after removal of the small molecule, the binding site for the ABP will be revealed, and a second probe can then be used to label the small-molecule binding sites selectively. To demonstrate this experimentally, we mapped the binding sites of the DPP4 inhibitor, IT, in a number of different tissue types. PMID:15090140

  14. Enhanced release of plasminogen activator inhibitor(s) but not of plasminogen activators by cultured rat glial cells treated with interleukin-1.

    PubMed

    Rogister, B; Leprince, P; Delree, P; Van Damme, J; Billiau, A; Moonen, G

    1990-01-01

    Astroglial cells are known to proliferate during development of the nervous system, as well as during post-traumatic gliosis. We have previously shown that the proliferation of cultured astrocytes can be stimulated by the urokinase-type (uPA) of plasminogen activator (PA) and that astrocytes are able to release such uPA upon stimulation with basic fibroblast growth factor, which is known to act as a mitogen for these cells. Here we report studies on the effects of human interleukin-1 (IL-1) on the release of PA activity by cultured newborn rat astroglial cells. Whereas there is controversy in the literature as to whether IL-1 stimulates multiplication of astroglial cells, we failed to observe such an effect in our system. We did observe, however, a dose-dependent decrease in PA activity in the supernatant of the IL-1 treated cultures. Further analysis revealed that this apparent decrease in PA release was in fact due to an increased release of plasminogen activator inhibitor (PAI). A similar IL-1 induced increase in PAI release was also found to occur in cultures of transformed astrocytes (human glioma LN18) and in cultured Schwann cells, but not in cultures of neurons or neuronal tumour cells. Since protease inhibitors are known to possess neuritogenic properties, our results suggest that IL-1, by its capacity to induce PAI, may promote neuritogenesis.

  15. Active site conformational changes of prostasin provide a new mechanism of protease regulation by divalent cations

    SciTech Connect

    Spraggon, Glen; Hornsby, Michael; Shipway, Aaron; Tully, David C.; Bursulaya, Badry; Danahay, Henry; Harris, Jennifer L.; Lesley, Scott A.

    2010-01-12

    Prostasin or human channel-activating protease 1 has been reported to play a critical role in the regulation of extracellular sodium ion transport via its activation of the epithelial cell sodium channel. Here, the structure of the extracellular portion of the membrane associated serine protease has been solved to high resolution in complex with a nonselective d-FFR chloromethyl ketone inhibitor, in an apo form, in a form where the apo crystal has been soaked with the covalent inhibitor camostat and in complex with the protein inhibitor aprotinin. It was also crystallized in the presence of the divalent cation Ca{sup +2}. Comparison of the structures with each other and with other members of the trypsin-like serine protease family reveals unique structural features of prostasin and a large degree of conformational variation within specificity determining loops. Of particular interest is the S1 subsite loop which opens and closes in response to basic residues or divalent ions, directly binding Ca{sup +2} cations. This induced fit active site provides a new possible mode of regulation of trypsin-like proteases adapted in particular to extracellular regions with variable ionic concentrations such as the outer membrane layer of the epithelial cell.

  16. Altered binding of thioflavin t to the peripheral anionic site of acetylcholinesterase after phosphorylation of the active site by chlorpyrifos oxon or dichlorvos

    SciTech Connect

    Sultatos, L.G. Kaushik, R.

    2008-08-01

    The peripheral anionic site of acetylcholinesterase, when occupied by a ligand, is known to modulate reaction rates at the active site of this important enzyme. The current report utilized the peripheral anionic site specific fluorogenic probe thioflavin t to determine if the organophosphates chlorpyrifos oxon and dichlorvos bind to the peripheral anionic site of human recombinant acetylcholinesterase, since certain organophosphates display concentration-dependent kinetics when inhibiting this enzyme. Incubation of 3 nM acetylcholinesterase active sites with 50 nM or 2000 nM inhibitor altered both the B{sub max} and K{sub d} for thioflavin t binding to the peripheral anionic site. However, these changes resulted from phosphorylation of Ser203 since increasing either inhibitor from 50 nM to 2000 nM did not alter further thioflavin t binding kinetics. Moreover, the organophosphate-induced decrease in B{sub max} did not represent an actual reduction in binding sites, but instead likely resulted from conformational interactions between the acylation and peripheral anionic sites that led to a decrease in the rigidity of bound thioflavin t. A drop in fluorescence quantum yield, leading to an apparent decrease in B{sub max}, would accompany the decreased rigidity of bound thioflavin t molecules. The organophosphate-induced alterations in K{sub d} represented changes in binding affinity of thioflavin t, with diethylphosphorylation of Ser203 increasing K{sub d}, and dimethylphosphorylation of Ser203 decreasing K{sub d}. These results indicate that chlorpyrifos oxon and dichlorvos do not bind directly to the peripheral anionic site of acetylcholinesterase, but can affect binding to that site through phosphorylation of Ser203.

  17. Reduction of Urease Activity by Interaction with the Flap Covering the Active Site

    PubMed Central

    Macomber, Lee; Minkara, Mona S.; Hausinger, Robert P.; Merz, Kenneth M.

    2015-01-01

    With the increasing appreciation for the human microbiome coupled with the global rise of antibiotic resistant organisms, it is imperative that new methods be developed to specifically target pathogens. To that end, a novel computational approach was devised to identify compounds that reduce the activity of urease, a medically important enzyme of Helicobacter pylori, Proteus mirabilis, and many other microorganisms. Urease contains a flexible loop that covers its active site; Glide was used to identify small molecules predicted to lock this loop in an open conformation. These compounds were screened against the model urease from Klebsiella aerogenes and the natural products epigallocatechin and quercetin were shown to inhibit at low and high micromolar concentrations, respectively. These molecules exhibit a strong time-dependent inactivation of urease that was not due to their oxygen sensitivity. Rather, these compounds appear to inactivate urease by reacting with a specific Cys residue located on the flexible loop. Substitution of this cysteine by alanine in the C319A variant increased the urease resistance to both epigallocatechin and quercetin, as predicted by the computational studies. Protein dynamics are integral to the function of many enzymes; thus, identification of compounds that lock an enzyme into a single conformation presents a useful approach to define potential inhibitors. PMID:25594724

  18. Antiepileptic Activity of Preferential Inhibitors of Persistent Sodium Current

    PubMed Central

    Anderson, Lyndsey L.; Thompson, Christopher H.; Hawkins, Nicole A.; Nath, Ravi D.; Petersohn, Adam A.; Rajamani, Sridharan; Bush, William S.; Frankel, Wayne N.; Vanoye, Carlos G.; Kearney, Jennifer A.; George, Alfred L.

    2014-01-01

    Objective Evidence from basic neurophysiology and molecular genetics has implicated persistent sodium current conducted by voltage-gated sodium (NaV) channels as a contributor to the pathogenesis of epilepsy. Many antiepileptic drugs target NaV channels and modulate neuronal excitability mainly by a use-dependent block of transient sodium current, although suppression of persistent current may also contribute to the efficacy of these drugs. We hypothesized that a drug or compound capable of preferential inhibition of persistent sodium current would have antiepileptic activity. Methods We examined the antiepileptic activity of two selective persistent sodium current blockers ranolazine, an FDA-approved drug for treatment of angina pectoris, and GS967, a novel compound with more potent effects on persistent current, in the epileptic Scn2aQ54 mouse model. We also examined the effect of GS967 in the maximal electroshock model and evaluated effects of the compound on neuronal excitability, propensity for hilar neuron loss, development of mossy fiber sprouting and survival of Scn2aQ54 mice. Results We found that ranolazine was capable of reducing seizure frequency by ~50% in Scn2aQ54 mice. The more potent persistent current blocker GS967 reduced seizure frequency by greater than 90% in Scn2aQ54 mice and protected against induced seizures in the maximal electroshock model. GS967 greatly attenuated abnormal spontaneous action potential firing in pyramidal neurons acutely isolated from Scn2aQ54 mice. In addition to seizure suppression in vivo, GS967 treatment greatly improved the survival of Scn2aQ54 mice, prevented hilar neuron loss, and suppressed the development of hippocampal mossy fiber sprouting. Significance Our findings indicate that the selective persistent sodium current blocker GS967 has potent antiepileptic activity and this compound could inform development of new agents. PMID:24862204

  19. Targeting Large Kinase Active Site with Rigid, Bulky Octahedral Ruthenium Complexes

    SciTech Connect

    Maksimoska, Jasna; Feng, Li; Harms, Klaus; Yi, Chunling; Kissil, Joseph; Marmorstein, Ronen; Meggers, Eric

    2009-09-02

    A strategy for targeting protein kinases with large ATP-binding sites by using bulky and rigid octahedral ruthenium complexes as structural scaffolds is presented. A highly potent and selective GSK3 and Pim1 half-sandwich complex NP309 was successfully converted into a PAK1 inhibitor by making use of the large octahedral compounds {Lambda}-FL172 and {Lambda}-FL411 in which the cyclopentadienyl moiety of NP309 is replaced by a chloride and sterically demanding diimine ligands. A 1.65 {angstrom}cocrystal structure of PAK1 with {Lambda}-FL172 reveals how the large coordination sphere of the ruthenium complex matches the size of the active site and serves as a yardstick to discriminate between otherwise closely related binding sites.

  20. Structure of recombinant Leishmania donovani pteridine reductase reveals a disordered active site

    PubMed Central

    Barrack, Keri L.; Tulloch, Lindsay B.; Burke, Lynsey-Ann; Fyfe, Paul K.; Hunter, William N.

    2011-01-01

    Pteridine reductase (PTR1) is a potential target for drug development against parasitic Trypanosoma and Leishmania species, protozoa that are responsible for a range of serious diseases found in tropical and subtropical parts of the world. As part of a structure-based approach to inhibitor development, specifically targeting Leishmania species, well ordered crystals of L. donovani PTR1 were sought to support the characterization of complexes formed with inhibitors. An efficient system for recombinant protein production was prepared and the enzyme was purified and crystallized in an orthorhombic form with ammonium sulfate as the precipitant. Diffraction data were measured to 2.5 Å resolution and the structure was solved by molecular replacement. However, a sulfate occupies a phosphate-binding site used by NADPH and occludes cofactor binding. The nicotinamide moiety is a critical component of the active site and without it this part of the structure is disordered. The crystal form obtained under these conditions is therefore unsuitable for the characterization of inhibitor complexes. PMID:21206018

  1. Identification of Novel Human Dipeptidyl Peptidase-IV Inhibitors of Natural Origin (Part I): Virtual Screening and Activity Assays

    PubMed Central

    Guasch, Laura; Ojeda, Maria José; González-Abuín, Noemí; Sala, Esther; Cereto-Massagué, Adrià; Mulero, Miquel; Valls, Cristina; Pinent, Montserrat; Ardévol, Anna; Garcia-Vallvé, Santiago; Pujadas, Gerard

    2012-01-01

    Background There has been great interest in determining whether natural products show biological activity toward protein targets of pharmacological relevance. One target of particular interest is DPP-IV whose most important substrates are incretins that, among other beneficial effects, stimulates insulin biosynthesis and secretion. Incretins have very short half-lives because of their rapid degradation by DPP-IV and, therefore, inhibiting this enzyme improves glucose homeostasis. As a result, DPP-IV inhibitors are of considerable interest to the pharmaceutical industry. The main goals of this study were (a) to develop a virtual screening process to identify potential DPP-IV inhibitors of natural origin; (b) to evaluate the reliability of our virtual-screening protocol by experimentally testing the in vitro activity of selected natural-product hits; and (c) to use the most active hit for predicting derivatives with higher binding affinities for the DPP-IV binding site. Methodology/Principal Findings We predicted that 446 out of the 89,165 molecules present in the natural products subset of the ZINC database would inhibit DPP-IV with good ADMET properties. Notably, when these 446 molecules were merged with 2,342 known DPP-IV inhibitors and the resulting set was classified into 50 clusters according to chemical similarity, there were 12 clusters that contained only natural products for which no DPP-IV inhibitory activity has been previously reported. Nine molecules from 7 of these 12 clusters were then selected for in vitro activity testing and 7 out of the 9 molecules were shown to inhibit DPP-IV (where the remaining two molecules could not be solubilized, preventing the evaluation of their DPP-IV inhibitory activity). Then, the hit with the highest activity was used as a lead compound in the prediction of more potent derivatives. Conclusions/Significance We have demonstrated that our virtual-screening protocol was successful in identifying novel lead compounds for

  2. Platelet Inhibitors.

    PubMed

    Shifrin, Megan M; Widmar, S Brian

    2016-03-01

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

  3. CR2-mediated targeting of complement inhibitors: bench-to-bedside using a novel strategy for site-specific complement modulation.

    PubMed

    Holers, V Michael; Rohrer, Bärbel; Tomlinson, Stephen

    2013-01-01

    Recent approval of the first human complement pathway-directed therapeutics, along with high-profile genetic association studies, has catalyzed renewed biopharmaceutical interest in developing drugs that modulate the complement system. Substantial challenges remain, however, that must be overcome before widespread application of complement inhibitors in inflammatory and autoimmune diseases becomes possible. Among these challenges are the following: (1) defining the complement pathways and effector mechanisms that cause tissue injury in humans and determining whether the relative importance of each varies by disease, (2) blocking or modulating, using traditional small molecule or biologic approaches, the function of complement proteins whose circulating levels are very high and whose turnover rates are relatively rapid, especially in the setting of acute and chronic autoimmune diseases, and (3) avoiding infectious complications or impairment of other important physiological functions of complement when using systemically active complement-blocking agents. This chapter will review data that address these challenges to therapeutic development, with a focus on the development of a novel strategy of blocking specific complement pathways by targeting inhibitors using a recombinant portion of the human complement receptor type 2 (CR2/CD21) which specifically targets to sites of local complement C3 activation where C3 fragments are covalently fixed. Recently, the first of these CR2-targeted proteins has entered human phase I studies in the human disease paroxysmal nocturnal hemoglobinuria. The results of murine translational studies using CR2-targeted inhibitors strongly suggest that a guiding principle going forward in complement therapeutic development may well be to focus on developing strategies to modulate the pathway as precisely as possible by physically localizing therapeutic inhibitory effects.

  4. CR2-mediated targeting of complement inhibitors: bench-to-bedside using a novel strategy for site-specific complement modulation.

    PubMed

    Holers, V Michael; Rohrer, Bärbel; Tomlinson, Stephen

    2013-01-01

    Recent approval of the first human complement pathway-directed therapeutics, along with high-profile genetic association studies, has catalyzed renewed biopharmaceutical interest in developing drugs that modulate the complement system. Substantial challenges remain, however, that must be overcome before widespread application of complement inhibitors in inflammatory and autoimmune diseases becomes possible. Among these challenges are the following: (1) defining the complement pathways and effector mechanisms that cause tissue injury in humans and determining whether the relative importance of each varies by disease, (2) blocking or modulating, using traditional small molecule or biologic approaches, the function of complement proteins whose circulating levels are very high and whose turnover rates are relatively rapid, especially in the setting of acute and chronic autoimmune diseases, and (3) avoiding infectious complications or impairment of other important physiological functions of complement when using systemically active complement-blocking agents. This chapter will review data that address these challenges to therapeutic development, with a focus on the development of a novel strategy of blocking specific complement pathways by targeting inhibitors using a recombinant portion of the human complement receptor type 2 (CR2/CD21) which specifically targets to sites of local complement C3 activation where C3 fragments are covalently fixed. Recently, the first of these CR2-targeted proteins has entered human phase I studies in the human disease paroxysmal nocturnal hemoglobinuria. The results of murine translational studies using CR2-targeted inhibitors strongly suggest that a guiding principle going forward in complement therapeutic development may well be to focus on developing strategies to modulate the pathway as precisely as possible by physically localizing therapeutic inhibitory effects. PMID:23402024

  5. Structure-activity studies of 5-substituted pyridopyrimidines as adenosine kinase inhibitors.

    PubMed

    Cowart, M; Lee, C H; Gfesser, G A; Bayburt, E K; Bhagwat, S S; Stewart, A O; Yu, H; Kohlhaas, K L; McGaraughty, S; Wismer, C T; Mikusa, J; Zhu, C; Alexander, K M; Jarvis, M F; Kowaluk, E A

    2001-01-01

    The synthesis and SAR of a novel series of non-nucleoside pyridopyrimidine inhibitors of the enzyme adenosine kinase (AK) are described. It was found that pyridopyrimidines with a broad range of medium and large non-polar substituents at the 5-position potently inhibited AK activity. A narrower range of analogues was capable of potently inhibiting adenosine phosphorylation in intact cells indicating an enhanced ability of these analogues to penetrate cell membranes. Potent AK inhibitors were found to effectively reduce nociception in animal models of thermal hyperalgesia and persistent pain.

  6. Synthesis, antiviral activity, and bioavailability studies of gamma-lactam derived HIV protease inhibitors.

    PubMed

    Hungate, R W; Chen, J L; Starbuck, K E; Vacca, J P; McDaniel, S L; Levin, R B; Dorsey, B D; Guare, J P; Holloway, M K; Whitter, W

    1994-09-01

    Incorporation of a gamma-lactam in hydroxyethylene isosteres results in modest inhibitors of HIV-1 protease. Additional structural activity studies have produced significantly more potent inhibitors with the introduction of the trisubstituted cyclopentane (see compound 20) as the optimum substituent for the C-terminus. This new amino acid amide surrogate can be readily prepared in large scale from (R)-pulegone. Optimized compounds (36) and (60) are potent antiviral agents and are well absorbed (15-20%) in a dog model after oral administration. PMID:7712123

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

    PubMed

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

    2015-01-01

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

  8. Characterization of a Kunitz-type serine protease inhibitor from Solanum tuberosum having lectin activity.

    PubMed

    Shah, Kunal R; Patel, Dhaval K; Pappachan, Anju; Prabha, C Ratna; Singh, Desh Deepak

    2016-02-01

    Plant lectins and protease inhibitors constitute a class of proteins which plays a crucial role in plant defense. In our continuing investigations on lectins from plants, we have isolated, purified and characterized a protein of about 20 kDa, named PotHg, showing hemagglutination activity from tubers of Indian potato, Solanum tuberosum. De novo sequencing and MS/MS analysis confirmed that the purified protein was a Kunitz-type serine protease inhibitor having two chains (15 kDa and 5 kDa). SDS and native PAGE analysis showed that the protein was glycosylated and was a heterodimer of about 15 and 5 kDa subunits. PotHg agglutinated rabbit erythrocytes with specific activity of 640 H.U./mg which was inhibited by complex sugars like fetuin. PotHg retained hemagglutination activity over a pH range 4-9 and up to 80°C. Mannose and galactose interacted with the PotHg with a dissociation constant (Kd) of 1.5×10(-3) M and 2.8×10(-3) M, respectively as determined through fluorescence studies. Fluorescence studies suggested the involvement of a tryptophan in sugar binding which was further confirmed through modification of tryptophan residues using N-bromosuccinimide. Circular dichroism (CD) studies showed that PotHg contains mostly β sheets (∼45%) and loops which is in line with previously characterized protease inhibitors and modeling studies. There are previous reports of Kunitz-type protease inhibitors showing lectin like activity from Peltophorum dubium and Labramia bojeri. This is the first report of a Kunitz-type protease inhibitor showing lectin like activity from a major crop plant and this makes PotHg an interesting candidate for further investigation. PMID:26645142

  9. Expression and activity of ovarian tissue inhibitors of metalloproteinases during pseudopregnancy in the rat.

    PubMed

    Nothnick, W B; Edwards, D R; Leco, K J; Curry, T E

    1995-09-01

    The present study examined the role of tissue inhibitors of metalloproteinases (TIMPs) in tissue remodeling that occurs during luteal development and regression throughout pseudopregnancy in the rat. Pseudopregnancy was induced in immature female rats by eCG/hCG priming. Animals (n = 4 per time point) were killed on Days 1, 2, 4, 8, 12, 14, and 16 of pseudopregnancy (post hCG administration), and ovaries were removed and analyzed for metalloproteinase inhibitor activity or TIMP-1, TIMP-2, and TIMP-3 mRNA expression. Inhibitory activity was highest in Day-1 samples (41.35 +/- 6.50 inhibitory units), and inhibitor activity significantly decreased (p < 0.05) thereafter to minimal values at Day 12 (8.14 +/- 2.71 inhibitory units). Methylamine hydrochloride treatment, which inactivates macroglobulin-type inhibitors, revealed that the majority of the inhibitor activity in the Day-1 samples (82.6%) and the Day-16 samples (77.3%) could be attributed to TIMPs. To further distinguish the contribution of each TIMP to this activity, Northern analysis for TIMP-1, -2, and -3 was performed. Analysis of TIMP mRNA expression revealed that TIMP-1 transcript expression was highest (p = 0.00009) at Day 1, decreased approximately 3- to 20-fold from Days 2 to 12, respectively, and again increased at Days 14-16. However, TIMP-2 expression did not change (p > 0.05) over any of the time points studied. In contrast to TIMP-1 and TIMP-2 expression, TIMP-3 mRNA expression was lowest during Days 1 and 2 of pseudopregnancy, increased approximately 4-fold at Day 4, peaked at Day 8, and remained elevated throughout the remainder of pseudopregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Characterization of a Kunitz-type serine protease inhibitor from Solanum tuberosum having lectin activity.

    PubMed

    Shah, Kunal R; Patel, Dhaval K; Pappachan, Anju; Prabha, C Ratna; Singh, Desh Deepak

    2016-02-01

    Plant lectins and protease inhibitors constitute a class of proteins which plays a crucial role in plant defense. In our continuing investigations on lectins from plants, we have isolated, purified and characterized a protein of about 20 kDa, named PotHg, showing hemagglutination activity from tubers of Indian potato, Solanum tuberosum. De novo sequencing and MS/MS analysis confirmed that the purified protein was a Kunitz-type serine protease inhibitor having two chains (15 kDa and 5 kDa). SDS and native PAGE analysis showed that the protein was glycosylated and was a heterodimer of about 15 and 5 kDa subunits. PotHg agglutinated rabbit erythrocytes with specific activity of 640 H.U./mg which was inhibited by complex sugars like fetuin. PotHg retained hemagglutination activity over a pH range 4-9 and up to 80°C. Mannose and galactose interacted with the PotHg with a dissociation constant (Kd) of 1.5×10(-3) M and 2.8×10(-3) M, respectively as determined through fluorescence studies. Fluorescence studies suggested the involvement of a tryptophan in sugar binding which was further confirmed through modification of tryptophan residues using N-bromosuccinimide. Circular dichroism (CD) studies showed that PotHg contains mostly β sheets (∼45%) and loops which is in line with previously characterized protease inhibitors and modeling studies. There are previous reports of Kunitz-type protease inhibitors showing lectin like activity from Peltophorum dubium and Labramia bojeri. This is the first report of a Kunitz-type protease inhibitor showing lectin like activity from a major crop plant and this makes PotHg an interesting candidate for further investigation.

  11. CINPA1 Is an Inhibitor of Constitutive Androstane Receptor That Does Not Activate Pregnane X Receptor

    PubMed Central

    Cherian, Milu T; Lin, Wenwei; Wu, Jing

    2015-01-01

    Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are xenobiotic sensors that enhance the detoxification and elimination of xenobiotics and endobiotics by modulating the expression of genes encoding drug-metabolizing enzymes and transporters. Elevated levels of drug-metabolizing enzymes and efflux transporters, resulting from CAR activation in various cancers, promote the elimination of chemotherapeutic agents, leading to reduced therapeutic effectiveness and acquired drug resistance. CAR inhibitors, in combination with existing chemotherapeutics, could therefore be used to attenuate multidrug resistance in cancers. Interestingly, all previously reported CAR inverse-agonists are also activators of PXR, rendering them mechanistically counterproductive in tissues where both these xenobiotic receptors are present and active. We used a directed high-throughput screening approach, followed by subsequent mechanistic studies, to identify novel, potent, and specific small-molecule CAR inhibitors that do not activate PXR. We describe here one such inhibitor, CINPA1 (CAR inhibitor not PXR activator 1), capable of reducing CAR-mediated transcription with an IC50 of ∼70 nM. CINPA1 1) is a specific xenobiotic receptor inhibitor and has no cytotoxic effects up to 30 µM; 2) inhibits CAR-mediated gene expression in primary human hepatocytes, where CAR is endogenously expressed; 3) does not alter the protein levels or subcellular localization of CAR; 4) increases corepressor and reduces coactivator interaction with the CAR ligand-binding domain in mammalian two-hybrid assays; and 5) disrupts CAR binding to the promoter regions of target genes in chromatin immunoprecipitation assays. CINPA1 could be used as a novel molecular tool for understanding CAR function. PMID:25762023

  12. High-throughput screening identifies novel inhibitors of the acetyltransferase activity of Escherichia coli GlmU.

    PubMed

    Pereira, Mark P; Blanchard, Jan E; Murphy, Cecilia; Roderick, Steven L; Brown, Eric D

    2009-06-01

    The bifunctional GlmU protein catalyzes the formation of UDP-N-acetylglucosamine in a two-step reaction using the substrates glucosamine-1-phosphate, acetyl coenzyme A, and UTP. This metabolite is a common precursor to the synthesis of bacterial cell surface carbohydrate polymers, such as peptidoglycan, lipopolysaccharide, and wall teichoic acid that are involved in the maintenance of cell shape, permeability, and virulence. The C-terminal acetyltransferase domain of GlmU exhibits structural and mechanistic features unique to bacterial UDP-N-acetylglucosamine synthases, making it an excellent target for antibacterial design. In the work described here, we have developed an absorbance-based assay to screen diverse chemical libraries in high throughput for inhibitors to the acetyltransferase reaction of Escherichia coli GlmU. The primary screen of 50,000 drug-like small molecules identified 63 hits, 37 of which were specific to acetyltransferase activity of GlmU. Secondary screening and mode-of-inhibition studies identified potent inhibitors where compound binding within the acetyltransferase active site was requisite on the presence of glucosamine-1-phosphate and were competitive with the substrate acetyl coenzyme A. These molecules may represent novel chemical scaffolds for future antimicrobial drug discovery. In addition, this work outlines the utility of catalytic variants in targeting specific activities of bifunctional enzymes in high-throughput screens.

  13. Novel diphenylamine 2,4'-dicarboxamide based azoles as potential epidermal growth factor receptor inhibitors: synthesis and biological activity.

    PubMed

    Abou-Seri, Sahar Mahmoud; Farag, Nahla Ahmed; Hassan, Ghaneya Sayed

    2011-01-01

    Several hybrid molecules of diphenylamine-2,4'-dicarboxamide with various azolidinones and related heterocyclic rings have been synthesized and explored as epidermal growth factor receptor (EGFR) kinase inhibitors. Most of them displayed promising in vitro tyrosine kinase inhibition as well as potent cellular antiproliferative activity in the EGFR over-expressing breast cancer cell line (MCF-7). Compounds 12b and 13b that exhibited the highest inhibition in the kinase assay (89, 81% inhibition at 10 μM, respectively), showed potent antiproliferative effect against MCF-7 tumor cell line (IC(50) 1.04, 0.91 μM respectively). Molecular docking studies revealed that these compounds can bind to ATP binding site of the EGFR kinase domain and were involved in H-bonding with Met 793, in analogy to the known EGFR tyrosine kinase inhibitors. Moreover, compounds 15a-c possessed profound antitumor activity (IC(50) 0.59-0.73 μM) and significant EGFR-TK inhibition, making them of particular interest. In summary, the newly synthesized compounds provide promising new lead for the future design and development of anticancer agents of potential EGFR-TK inhibitory activity.

  14. Study on the activity of non-purine xanthine oxidase inhibitor by 3D-QSAR modeling and molecular docking

    NASA Astrophysics Data System (ADS)

    Li, Peizhen; Tian, Yueli; Zhai, Honglin; Deng, Fangfang; Xie, Meihong; Zhang, Xiaoyun

    2013-11-01

    Non-purine derivatives have been shown to be promising novel drug candidates as xanthine oxidase inhibitors. Based on three-dimensional quantitative structure-activity relationship (3D-QSAR) methods including comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA), two 3D-QSAR models for a series of non-purine xanthine oxidase (XO) inhibitors were established, and their reliability was supported by statistical parameters. Combined 3D-QSAR modeling and the results of molecular docking between non-purine xanthine oxidase inhibitors and XO, the main factors that influenced activity of inhibitors were investigated, and the obtained results could explain known experimental facts. Furthermore, several new potential inhibitors with higher activity predicted were designed, which based on our analyses, and were supported by the simulation of molecular docking. This study provided some useful information for the development of non-purine xanthine oxidase inhibitors with novel structures.

  15. [Response of N transformation related soil enzyme activities to inhibitor applications].

    PubMed

    Chen, Lijun; Wu, Zhijie; Jiang, Yong; Zhou, Likai

    2002-09-01

    With an aerobic incubation test, this paper studied the response of soil urease, nitrate reductase, nitrite reductase, and hydroxylamine reductase to urease inhibitor hydroquinone (HQ) applied in combination with nitrification inhibitor encapsulated calcium carbide (HQ + ECC) or dicyandiamide (HQ + DCD). The results showed that HQ + DCD could inhibit urease activity and increase activities of nitrate reductase, nitrite reductase, and hydroxylamine reductase significantly in comparison with CK, HQ and HQ + ECC. Under the condition of our test, there existed a significant relationship between soil urease, nitrate reductase, nitrite reductase, and hydroxylamine reductase activities and soil NH4+ and NO3- contents, NH3 volatilization and N2O emission rate, and regression analysis indicated that there were significantly positive relationships between soil urease, nitrite reductase and hydroxylamine reductase activities.

  16. Basis Tetrapeptides as Potent Intracellular Inhibitors of type A Botulinum Neurotoxin Protease Activity

    SciTech Connect

    Hale, M.; Swaminathan, S.; Oyler, G.; Ahmed, S. A.

    2011-01-21

    Botulinum neurotoxins (BoNT) are the most potent of all toxins that cause flaccid muscle paralysis leading to death. They are also potential biothreat agents. A systematic investigation of various short peptide inhibitors of the BoNT protease domain with a 17-residue peptide substrate led to arginine-arginine-glycine-cysteine having a basic tetrapeptide structure as the most potent inhibitor. When assayed in the presence of dithiothreitol (DTT), the inhibitory effect was drastically reduced. Replacing the terminal cysteine with one hydrophobic residue eliminated the DTT effect but with two hydrophobic residues made the pentapeptide a poor inhibitor. Replacing the first arginine with cysteine or adding an additional cysteine at the N terminus did not improve inhibition. When assessed using mouse brain lysates, the tetrapeptides also inhibited BoNT/A cleavage of the endogenous SNAP-25. The peptides penetrated the neuronal cell lines, N2A and BE(2)-M17, without adversely affecting metabolic functions as measured by ATP production and P-38 phosphorylation. Biological activity of the peptides persisted within cultured chick motor neurons and rat and mouse cerebellar neurons for more than 40 h and inhibited BoNT/A protease action inside the neurons in a dose- and time-dependent fashion. Our results define a tetrapeptide as the smallest peptide inhibitor in the backdrop of a large substrate protein of 200+ amino acids having multiple interaction regions with its cognate enzyme. The inhibitors should also be valuable candidates for drug development.

  17. Mixed lineage kinases activate MEK independently of RAF to mediate resistance to RAF inhibitors

    PubMed Central

    Marusiak, Anna A.; Edwards, Zoe C.; Hugo, Willy; Trotter, Eleanor W.; Girotti, Maria R.; Stephenson, Natalie L.; Kong, Xiangju; Gartside, Michael G.; Fawdar, Shameem; Hudson, Andrew; Breitwieser, Wolfgang; Hayward, Nicholas K.; Marais, Richard; Lo, Roger S.; Brognard, John

    2014-01-01

    RAF inhibitor therapy yields significant reductions in tumour burden in the majority of V600E-positive melanoma patients; however, resistance occurs within 2–18 months. Here we demonstrate that the mixed lineage kinases (MLK1–4) are MEK kinases that reactivate the MEK/ERK pathway in the presence of RAF inhibitors. Expression of MLK1–4 mediates resistance to RAF inhibitors and promotes survival in V600E-positive melanoma cell lines. Furthermore, we observe upregulation of the MLKs in 9 of 21 melanoma patients with acquired drug resistance. Consistent with this observation, MLKs promote resistance to RAF inhibitors in mouse models and contribute to acquired resistance in a cell line model. Lastly, we observe that a majority of MLK1 mutations identified in patients are gain-of-function mutations. In summary, our data demonstrate a role for MLKs as direct activators of the MEK/ERK pathway with implications for melanomagenesis and resistance to RAF inhibitors. PMID:24849047

  18. 1,25-Dihydroxyvitamin D3 Suppresses Inflammation-Induced Expression of Plasminogen Activator Inhibitor-1 by Blocking Nuclear Factor-κB Activation

    PubMed Central

    Chen, Yunzi; Kong, Juan; Sun, Tao; Li, George; Szeto, Frances L.; Liu, Weicheng; Deb, Dilip K.; Wang, Youli; Zhao, Qun; Thadhani, Ravi; Li, Yan Chun

    2011-01-01

    Plasminogen activator inhibitor (PAI)-1 is a major fibrinolytic inhibitor. High PAI-1 is associated with increased renal and cardiovascular disease risk. Previous studies demonstrated PAI-1 down-regulation by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), but the molecular mechanism remains unknown. Here we show that exposure of mouse embryonic fibroblasts to TNFα or LPS led to a marked induction of PAI-1, which was blunted by 1,25(OH)2D3, NF-κB inhibitor or p65 siRNA, suggesting the involvement of NF-κB in 1,25(OH)2D3-induced repression. In mouse Pai-1 promoter a putative cis-κB element was identified at −299. EMSA and ChIP assays showed that TNF-α increased p65/p50 binding to this κB site, which was disrupted by 1,25(OH)2D3. Luciferase reporter assays showed that PAI-1 promoter activity was induced by TNFα or LPS, and the induction was blocked by 1,25(OH)2D3. Mutation of the κB site blunted TNFα, LPS or 1,25(OH)2D3 effects. 1,25(OH)2D3 blocked IκBα degradation and arrested p50/p65 nuclear translocation. In mice LPS stimulated PAI-1 expression in the heart and macrophages, and the stimulation was blunted by pre-treatment with a vitamin D analog. Together these data demonstrate that 1,25(OH)2D3 down-regulates PAI-1 by blocking NF-κB activation. Inhibition of PAI-1 production may contribute to the reno- and cardio-protective effects of vitamin D. PMID:21176770

  19. Crystal Structures and Structure–Activity Relationships of Imidazothiazole Derivatives as IDO1 Inhibitors

    PubMed Central

    2014-01-01

    Indoleamine 2,3-dioxygenase 1 (IDO1) is considered as a promising target for the treatment of several diseases, including neurological disorders and cancer. We report here the crystal structures of two IDO1/IDO1 inhibitor complexes, one of which shows that Amg-1 is directly bound to the heme iron of IDO1 with a clear induced fit. We also describe the identification and preliminary optimization of imidazothiazole derivatives as novel IDO1 inhibitors. Using our crystal structure information and structure–activity relationships (SAR) at the pocket-B of IDO1, we found a series of urea derivatives as potent IDO1 inhibitors and revealed that generation of an induced fit and the resulting interaction with Phe226 and Arg231 are essential for potent IDO1 inhibitory activity. The results of this study are very valuable for understanding the mechanism of IDO1 activation, which is very important for structure-based drug design (SBDD) to discover potent IDO1 inhibitors. PMID:25313323

  20. John Montgomery's legacy: carbocyclic adenosine analogues as SAH hydrolase inhibitors with broad-spectrum antiviral activity.

    PubMed

    De Clercq, Erik

    2005-01-01

    Ever since the S-adenosylhomocysteine (AdoHcy, SAH) hydrolase was recognized as a pharmacological target for antiviral agents (J. A. Montgomery et al., J. Med. Chem. 25:626-629, 1982), an increasing number of adenosine, acyclic adenosine, and carbocyclic adenosine analogues have been described as potent SAH hydrolase inhibitors endowed with broad-spectrum antiviral activity. The antiviral activity spectrum of the SAH hydrolase inhibitors include pox-, rhabdo-, filo-, arena-, paramyxo-, reo-, and retroviruses. Among the most potent SAH hydrolase inhibitors and antiviral agents rank carbocyclic 3-deazaadenosine (C-c3 Ado), neplanocin A, 3-deazaneplanocin A, the 5'-nor derivatives of carbocyclic adenosine (C-Ado, aristeromycin), and the 2-halo (i.e., 2-fluoro) and 6'-R-alkyl (i.e., 6'-R-methyl) derivatives of neplanocin A. These compounds are particularly active against poxviruses (i.e., vaccinia virus), and rhabdoviruses (i.e., vesicular stomatitis virus). The in vivo efficacy of C-c3 Ado and 3-deazaneplanocin A has been established in mouse models for vaccinia virus, vesicular stomatitis virus, and Ebola virus. SAH hydrolase inhibitors such as C-c3Ado and 3-deazaneplanocin A should in thefirst place be considered for therapeutic (or prophylactic) use against poxvirus infections, including smallpox, and hemorrhagic fever virus infections such as Ebola. PMID:16438025

  1. Binding mode prediction of biologically active compounds from plant Salvia Miltiorrhiza as integrase inhibitor

    PubMed Central

    Nunthaboot, Nadtanet; Lugsanangarm, Kiattisak; Kokpol, Sirirat; Abd-Elazem, Ibrahim S

    2013-01-01

    Integrase (IN), an essential enzyme for HIV-1 replication, has been targeted in antiretroviral drug therapy. The emergence of HIV-1 variants clinically resistant to antiretroviral agents has lead to the development of alternative IN inhibitors. In the present work, binding modes of a high potent IN inhibitor, M522 and M532, within the catalytic binding site of wild type (WT) IN were determined using molecular docking calculation. Both M522 and M532 displayed similar modes of binding within the IN putative binding pocket and exhibited favorable interactions with the catalytic Mg2+ ions, the nearby amino acids and viral DNA through metal-ligand chelation, hydrogen bonding and π-π stacking interactions. Furthermore, the modes of action of these two compounds against the mutated Y212R, N224H and S217H PFV IN were also predicted. Although the replacement of amino acid could somehow disturb inhibitor binding mode, almost key interactions which detected in the WT complexes were fairly conserved. Detailed information could highlight the application of M522 and M532 as candidate IN inhibitors for drug development against drug resistant strains. PMID:23750093

  2. Computation of Rate Constants for Diffusion of Small Ligands to and from Buried Protein Active Sites.

    PubMed

    Wang, P-H; De Sancho, D; Best, R B; Blumberger, J

    2016-01-01

    The diffusion of ligands to actives sites of proteins is essential to enzyme catalysis and many cellular signaling processes. In this contribution we review our recently developed methodology for calculation of rate constants for diffusion and binding of small molecules to buried protein active sites. The diffusive dynamics of the ligand obtained from molecular dynamics simulation is coarse grained and described by a Markov state model. Diffusion and binding rate constants are then obtained either from the reactive flux formalism or by fitting the time-dependent population of the Markov state model to a phenomenological rate law. The method is illustrated by applications to diffusion of substrate and inhibitors in [NiFe] hydrogenase, CO-dehydrogenase, and myoglobin. We also discuss a recently developed sensitivity analysis that allows one to identify hot spots in proteins, where mutations are expected to have the strongest effects on ligand diffusion rates.

  3. Computation of Rate Constants for Diffusion of Small Ligands to and from Buried Protein Active Sites.

    PubMed

    Wang, P-H; De Sancho, D; Best, R B; Blumberger, J

    2016-01-01

    The diffusion of ligands to actives sites of proteins is essential to enzyme catalysis and many cellular signaling processes. In this contribution we review our recently developed methodology for calculation of rate constants for diffusion and binding of small molecules to buried protein active sites. The diffusive dynamics of the ligand obtained from molecular dynamics simulation is coarse grained and described by a Markov state model. Diffusion and binding rate constants are then obtained either from the reactive flux formalism or by fitting the time-dependent population of the Markov state model to a phenomenological rate law. The method is illustrated by applications to diffusion of substrate and inhibitors in [NiFe] hydrogenase, CO-dehydrogenase, and myoglobin. We also discuss a recently developed sensitivity analysis that allows one to identify hot spots in proteins, where mutations are expected to have the strongest effects on ligand diffusion rates. PMID:27497172

  4. Lovastatin Inhibits VEGFR and AKT Activation: Synergistic Cytotoxicity in Combination with VEGFR Inhibitors

    PubMed Central

    Addison, Christina L.; Dimitroulakos, Jim

    2010-01-01

    Background In a recent study, we demonstrated the ability of lovastatin, a potent inhibitor of mevalonate synthesis, to inhibit the function of the epidermal growth factor receptor (EGFR). Lovastatin attenuated ligand-induced receptor activation and downstream signaling through the PI3K/AKT pathway. Combining lovastatin with gefitinib, a potent EGFR inhibitor, induced synergistic cytotoxicity in a variety of tumor derived cell lines. The vascular endothelial growth factor receptor (VEGFR) and EGFR share similar activation, internalization and downstream signaling characteristics. Methodology/Principal Findings The VEGFRs, particularly VEGFR-2 (KDR, Flt-1), play important roles in regulating tumor angiogenesis by promoting endothelial cell proliferation, survival and migration. Certain tumors, such as malignant mesothelioma (MM), also express both the VEGF ligand and VEGFRs that act in an autocrine loop to directly stimulate tumor cell growth and survival. In this study, we have shown that lovastatin inhibits ligand-induced VEGFR-2 activation through inhibition of receptor internalization and also inhibits VEGF activation of AKT in human umbilical vein endothelial cells (HUVEC) and H28 MM cells employing immunofluorescence and Western blotting. Combinations of lovastatin and a VEGFR-2 inhibitor showed more robust AKT inhibition than either agent alone in the H28 MM cell line. Furthermore, combining 5 µM lovastatin treatment, a therapeutically relevant dose, with two different VEGFR-2 inhibitors in HUVEC and the H28 and H2052 mesothelioma derived cell lines demonstrated synergistic cytotoxicity as demonstrated by MTT cell viability and flow cytometric analyses. Conclusions/Significance These results highlight a novel mechanism by which lovastatin can regulate VEGFR-2 function and a potential therapeutic approach for MM through combining statins with VEGFR-2 inhibitors. PMID:20838437

  5. Evaluating and monitoring the efficacy of recombinant activated factor VIIa in patients with haemophilia and inhibitors.

    PubMed

    Qi, Xue; Zhao, Yongqiang; Li, Kuixing; Fan, Liankai; Hua, Baolai

    2014-10-01

    Although the use of bypassing agents has dramatically improved the management of haemophilia in patients with inhibitors, questions remain regarding optimal dosing regimens and methodology for monitoring their clinical effectiveness. In this study, we evaluated the efficacy and safety of two different doses of recombinant activated factor VIIa (rFVIIa) in patients with haemophilia and inhibitors and assessed the feasibility of using thromboelastography (TEG) and thrombin generation assays (TGA) for monitoring the response to rFVIIa. Six patients aged 9-49 years with congenital or acquired haemophilia with inhibitors who experienced a total of nine bleeding episodes were included. Seven episodes were treated with conventional rFVIIa dosing (72.7-109.1 μg/kg), and two episodes were treated with a single high-dose regimen (254.6-264.0 μg/kg). Clinical and haemostatic responses were evaluated. Haemostasis was assessed by prothrombin time (PT), activated partial thromboplastin time (aPTT), factor VII coagulant activity (FVII:C), TEG, and TGA. Six out of seven (85.7%) bleeding episodes responded to conventional rFVIIa dosing, and half (50%) responded to the high-dose regimen. No relationships between PT, aPTT, and FVII:C levels and clinical outcome were observed. However, changes in TEG and TGA parameters tended to correspond to clinical response, although large inter-individual variation in rFVIIa efficacy was noted. A good response was seen with rFVIIa in treating acute bleeding episodes in patients with haemophilia and inhibitors. Because changes in TEG and TGA may correlate with clinical outcomes of rFVIIa, TEG and TGA may be useful for monitoring rFVIIa activity in inhibitor-positive haemophilia.

  6. Inhibitors of Ketohexokinase: Discovery of Pyrimidinopyrimidines with Specific Substitution that Complements the ATP-Binding Site

    PubMed Central

    2011-01-01

    Attenuation of fructose metabolism by the inhibition of ketohexokinase (KHK; fructokinase) should reduce body weight, free fatty acids, and triglycerides, thereby offering a novel approach to treat diabetes and obesity in response to modern diets. We have identified potent, selective inhibitors of human hepatic KHK within a series of pyrimidinopyrimidines (1). For example, 8, 38, and 47 exhibited KHK IC50 values of 12, 7, and 8 nM, respectively, and also showed potent cellular KHK inhibition (IC50 < 500 nM), which relates to their intrinsic potency vs KHK and their ability to penetrate cells. X-ray cocrystal structures of KHK complexes of 3, 8, and 47 revealed the important interactions within the enzyme's adenosine 5'-triphosphate (ATP)-binding pocket. PMID:24900346

  7. Anorectic activities of serotonin uptake inhibitors: correlation with their potencies at inhibiting serotonin uptake in vivo and /sup 3/H-mazindol binding in vitro

    SciTech Connect

    Angel, I.; Taranger, M.A.; Claustre, Y.; Scatton, B.; Langer, S.Z.

    1988-01-01

    The mechanism of anorectic action of several serotonin uptake inhibitors was investigated by comparing their anorectic potencies with several biochemical and pharmacological properties and in reference to the novel compound SL 81.0385. The anorectic effect of the potent serotonin uptake inhibitor SL 81.0385 was potentiated by pretreatment with 5-hydroxytryptophan and blocked by the serotonin receptor antagonist metergoline. A good correlation was obtained between the ED/sub 50/ values of anorectic action and the ED/sub 50/ values of serotonin uptake inhibition in vivo (but not in vitro) for several specific serotonin uptake inhibitors. Most of the drugs tested displaced (/sup 3/H)-mazindol from its binding to the anorectic recognition site in the hypothalamus, except the pro-drug zimelidine which was inactive. Excluding zimelidine, a good correlation was obtained between the affinities of these drugs for (/sup 3/H)-mazindol binding and their anorectic action indicating that their anorectic activity may be associated with an effect mediated through this site. Taken together these results suggest that the anorectic action of serotonin uptake inhibitors is directly associated to their ability to inhibit serotonin uptake and thus increasing the synaptic levels of serotonin. The interactions of these drugs with the anorectic recognition site labelled with (/sup 3/H)-mazindol is discussed in connection with the serotonergic regulation of carbohydrate intake.

  8. Quantification of active mitochondrial permeability transition pores using GNX-4975 inhibitor titrations provides insights into molecular identity

    PubMed Central

    Richardson, Andrew P.; Halestrap, Andrew P.

    2016-01-01

    Inhibition of the mitochondrial permeability transition pore (MPTP) by the novel inhibitor GNX-4975 was characterized. Titration of MPTP activity in de-energized rat liver mitochondria allowed determination of the number of GNX-4975-binding sites and their dissociation constant (Ki). Binding sites increased in number when MPTP opening was activated by increasing [Ca2+], phenylarsine oxide (PAO) or KSCN, and decreased when MPTP opening was inhibited with bongkrekic acid (BKA) or ADP. Values ranged between 9 and 50 pmol/mg of mitochondrial protein, but the Ki remained unchanged at ∼1.8 nM when the inhibitor was added before Ca2+. However, when GNX-4975 was added after Ca2+ it was much less potent with a Ki of ∼140 nM. These data imply that a protein conformational change is required to form the MPTP complex and generate the GNX-4975-binding site. Occupation of the latter with GNX-4975 prevents the Ca2+ binding that triggers pore opening. We also demonstrated that GNX-4975 stabilizes an interaction between the adenine nucleotide translocase (ANT), held in its ‘c’ conformation with carboxyatractyloside (CAT), and the phosphate carrier (PiC) bound to immobilized PAO. No components of the F1Fo-ATP synthase bound significantly to immobilized PAO. Our data are consistent with our previous proposal that the MPTP may form at an interface between the PiC and ANT (or other similar mitochondrial carrier proteins) when they adopt novel conformations induced by factors that sensitize the MPTP to [Ca2+]. We propose that GNX-4975 binds to this interface preventing a calcium-triggered event that opens the interface into a pore. PMID:26920024

  9. Quantification of active mitochondrial permeability transition pores using GNX-4975 inhibitor titrations provides insights into molecular identity.

    PubMed

    Richardson, Andrew P; Halestrap, Andrew P

    2016-05-01

    Inhibition of the mitochondrial permeability transition pore (MPTP) by the novel inhibitor GNX-4975 was characterized. Titration of MPTP activity in de-energized rat liver mitochondria allowed determination of the number of GNX-4975-binding sites and their dissociation constant (Ki). Binding sites increased in number when MPTP opening was activated by increasing [Ca(2+)], phenylarsine oxide (PAO) or KSCN, and decreased when MPTP opening was inhibited with bongkrekic acid (BKA) or ADP. Values ranged between 9 and 50 pmol/mg of mitochondrial protein, but the Ki remained unchanged at ∼1.8 nM when the inhibitor was added before Ca(2+) However, when GNX-4975 was added after Ca(2+) it was much less potent with a Ki of ∼140 nM. These data imply that a protein conformational change is required to form the MPTP complex and generate the GNX-4975-binding site. Occupation of the latter with GNX-4975 prevents the Ca(2+) binding that triggers pore opening. We also demonstrated that GNX-4975 stabilizes an interaction between the adenine nucleotide translocase (ANT), held in its 'c' conformation with carboxyatractyloside (CAT), and the phosphate carrier (PiC) bound to immobilized PAO. No components of the F1Fo-ATP synthase bound significantly to immobilized PAO. Our data are consistent with our previous proposal that the MPTP may form at an interface between the PiC and ANT (or other similar mitochondrial carrier proteins) when they adopt novel conformations induced by factors that sensitize the MPTP to [Ca(2+)]. We propose that GNX-4975 binds to this interface preventing a calcium-triggered event that opens the interface into a pore.

  10. Selective Sirt2 inhibition by ligand-induced rearrangement of the active site.

    PubMed

    Rumpf, Tobias; Schiedel, Matthias; Karaman, Berin; Roessler, Claudia; North, Brian J; Lehotzky, Attila; Oláh, Judit; Ladwein, Kathrin I; Schmidtkunz, Karin; Gajer, Markus; Pannek, Martin; Steegborn, Clemens; Sinclair, David A; Gerhardt, Stefan; Ovádi, Judit; Schutkowski, Mike; Sippl, Wolfgang; Einsle, Oliver; Jung, Manfred

    2015-01-01

    Sirtuins are a highly conserved class of NAD(+)-dependent lysine deacylases. The human isotype Sirt2 has been implicated in the pathogenesis of cancer, inflammation and neurodegeneration, which makes the modulation of Sirt2 activity a promising strategy for pharmaceutical intervention. A rational basis for the development of optimized Sirt2 inhibitors is lacking so far. Here we present high-resolution structures of human Sirt2 in complex with highly selective drug-like inhibitors that show a unique inhibitory mechanism. Potency and the unprecedented Sirt2 selectivity are based on a ligand-induced structural rearrangement of the active site unveiling a yet-unexploited binding pocket. Application of the most potent Sirtuin-rearranging ligand, termed SirReal2, leads to tubulin hyperacetylation in HeLa cells and induces destabilization of the checkpoint protein BubR1, consistent with Sirt2 inhibition in vivo. Our structural insights into this unique mechanism of selective sirtuin inhibition provide the basis for further inhibitor development and selective tools for sirtuin biology. PMID:25672491

  11. Selective Sirt2 inhibition by ligand-induced rearrangement of the active site

    PubMed Central

    Rumpf, Tobias; Schiedel, Matthias; Karaman, Berin; Roessler, Claudia; North, Brian J.; Lehotzky, Attila; Oláh, Judit; Ladwein, Kathrin I.; Schmidtkunz, Karin; Gajer, Markus; Pannek, Martin; Steegborn, Clemens; Sinclair, David A.; Gerhardt, Stefan; Ovádi, Judit; Schutkowski, Mike; Sippl, Wolfgang; Einsle, Oliver; Jung, Manfred

    2015-01-01

    Sirtuins are a highly conserved class of NAD+-dependent lysine deacylases. The human isotype Sirt2 has been implicated in the pathogenesis of cancer, inflammation and neurodegeneration, which makes the modulation of Sirt2 activity a promising strategy for pharmaceutical intervention. A rational basis for the development of optimized Sirt2 inhibitors is lacking so far. Here we present high-resolution structures of human Sirt2 in complex with highly selective drug-like inhibitors that show a unique inhibitory mechanism. Potency and the unprecedented Sirt2 selectivity are based on a ligand-induced structural rearrangement of the active site unveiling a yet-unexploited binding pocket. Application of the most potent Sirtuin-rearranging ligand, termed SirReal2, leads to tubulin hyperacetylation in HeLa cells and induces destabilization of the checkpoint protein BubR1, consistent with Sirt2 inhibition in vivo. Our structural insights into this unique mechanism of selective sirtuin inhibition provide the basis for further inhibitor development and selective tools for sirtuin biology. PMID:25672491

  12. A novel approach to predict active sites of enzyme molecules.

    PubMed

    Chou, Kuo-Chen; Cai, Yu-dong

    2004-04-01

    Enzymes are critical in many cellular signaling cascades. With many enzyme structures being solved, there is an increasing need to develop an automated method for identifying their active sites. However, given the atomic coordinates of an enzyme molecule, how can we predict its active site? This is a vitally important problem because the core of an enzyme molecule is its active site from the viewpoints of both pure scientific research and industrial application. In this article, a topological entity was introduced to characterize the enzymatic active site. Based on such a concept, the covariant discriminant algorithm was formulated for identifying the active site. As a paradigm, the serine hydrolase family was demonstrated. The overall success rate by jackknife test for a data set of 88 enzyme molecules was 99.92%, and that for a data set of 50 independent enzyme molecules was 99.91%. Meanwhile, it was shown through an example that the prediction algorithm can also be used to find any typographic error of a PDB file in annotating the constituent amino acids of catalytic triad and to suggest a possible correction. The very high success rates are due to the introduction of a covariance matrix in the prediction algorithm that makes allowance for taking into account the coupling effects among the key constituent atoms of active site. It is anticipated that the novel approach is quite promising and may become a useful high throughput tool in enzymology, proteomics, and structural bioinformatics. PMID:14997541

  13. CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil

    NASA Astrophysics Data System (ADS)

    Almqvist, Helena; Axelsson, Hanna; Jafari, Rozbeh; Dan, Chen; Mateus, André; Haraldsson, Martin; Larsson, Andreas; Molina, Daniel Martinez; Artursson, Per; Lundbäck, Thomas; Nordlund, Pär

    2016-03-01

    Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery.

  14. CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil.

    PubMed

    Almqvist, Helena; Axelsson, Hanna; Jafari, Rozbeh; Dan, Chen; Mateus, André; Haraldsson, Martin; Larsson, Andreas; Martinez Molina, Daniel; Artursson, Per; Lundbäck, Thomas; Nordlund, Pär

    2016-01-01

    Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery. PMID:27010513

  15. Enhancement of active corrosion protection via combination of inhibitor-loaded nanocontainers.

    PubMed

    Tedim, J; Poznyak, S K; Kuznetsova, A; Raps, D; Hack, T; Zheludkevich, M L; Ferreira, M G S

    2010-05-01

    The present work reports the synthesis of layered double hydroxides (LDHs) nanocontainers loaded with different corrosion inhibitors (vanadate, phosphate, and 2-mercaptobenzothiazolate) and the characterization of the resulting pigments by X-ray diffraction (XRD) and transmission electron microscopy (TEM). The anticorrosion activity of these nanocontainers with respect to aluminum alloy AA2024 was investigated by electrochemical impedance spectroscopy (EIS). The bare metallic substrates were immersed in dispersions of nanocontainers in sodium chloride solution and tested to understand the inhibition mechanisms and efficiency. The nanocontainers were also incorporated into commercial coatings used for aeronautical applications to study the active corrosion protection properties in systems of industrial relevance. The results show that an enhancement of the active protection effect can be reached when nanocontainers loaded with different inhibitors are combined in the same protective coating system.

  16. Synthesis of Novel Tricyclic Chromenone-Based Inhibitors of IRE-1 RNase Activity

    PubMed Central

    2015-01-01

    Inositol-requiring enzyme 1 (IRE-1) is a kinase/RNase ER stress sensor that is activated in response to excessive accumulation of unfolded proteins, hypoxic conditions, calcium imbalance, and other stress stimuli. Activation of IRE-1 RNase function exerts a cytoprotective effect and has been implicated in the progression of cancer via increased expression of the transcription factor XBP-1s. Here, we describe the synthesis and biological evaluation of novel chromenone-based covalent inhibitors of IRE-1. Preparation of a family of 8-formyltetrahydrochromeno[3,4-c]pyridines was achieved via a Duff formylation that is attended by an unusual cyclization reaction. Biological evaluation in vitro and in whole cells led to the identification of 30 as a potent inhibitor of IRE-1 RNase activity and XBP-1s expression in wild type B cells and human mantle cell lymphoma cell lines. PMID:24749861

  17. Synthesis, biological activities and pharmacokinetic properties of new fluorinated derivatives of selective PDE4D inhibitors.

    PubMed

    Brullo, Chiara; Massa, Matteo; Villa, Carla; Ricciarelli, Roberta; Rivera, Daniela; Pronzato, Maria Adelaide; Fedele, Ernesto; Barocelli, Elisabetta; Bertoni, Simona; Flammini, Lisa; Bruno, Olga

    2015-07-01

    A new series of selective PDE4D inhibitors has been designed and synthesized by replacing 3-methoxy group with 3-difluoromethoxy isoster moiety in our previously reported cathecolic structures. All compounds showed a good PDE4D3 inhibitory activity, most of them being inactive toward other PDE4 isoforms (PDE4A4, PDE4B2 and PDE4C2). Compound 3b, chosen among the synthesized compounds as the most promising in terms of inhibitory activity, selectivity and safety, showed an improved pharmacokinetic profile compared to its non fluorinated analogue. Spontaneous locomotor activity, assessed in an open field apparatus, showed that, differently from rolipram and diazepam, selective PDE4D inhibitors, such as compounds 3b, 5b and 7b, did not affect locomotion, whereas compound 1b showed a tendency to reduce the distance traveled and to prolong the immobility period, possibly due to a poor selectivity.

  18. Structure-Activity Relationships of Novel Tryptamine-Based Inhibitors of Bacterial Transglycosylase.

    PubMed

    Sosič, Izidor; Anderluh, Marko; Sova, Matej; Gobec, Martina; Mlinarič Raščan, Irena; Derouaux, Adeline; Amoroso, Ana; Terrak, Mohammed; Breukink, Eefjan; Gobec, Stanislav

    2015-12-24

    Penicillin-binding proteins represent well-established, validated, and still very promising targets for the design and development of new antibacterial agents. The transglycosylase domain of penicillin-binding proteins is especially important, as it catalyzes polymerization of glycan chains, using the peptidoglycan precursor lipid II as a substrate. On the basis of the previous discovery of a noncovalent small-molecule inhibitor of transglycosylase activity, we systematically explored the structure-activity relationships of these tryptamine-based inhibitors. The main aim was to reduce the nonspecific cytotoxic properties of the initial hit compound and concurrently to retain the mode of its inhibition. A focused library of tryptamine-based compounds was synthesized, characterized, and evaluated biochemically. The results presented here show the successful reduction of the nonspecific cytotoxicity, and the retention of the inhibition of transglycosylase enzymatic activity, as well as the ability of these compounds to bind to lipid II and to have antibacterial actions. PMID:26588190

  19. Activity Based Profiling of Deubiquitylating Enzymes and Inhibitors in Animal Tissues.

    PubMed

    McLellan, Lauren; Forder, Cassie; Cranston, Aaron; Harrigan, Jeanine; Jacq, Xavier

    2016-01-01

    The attachment of ubiquitin or ubiquitin-like modifiers to proteins is an important signal for the regulation of a variety of biological processes including the targeting of substrates for degradation, receptor internalization, regulation of gene expression, and DNA repair. Posttranslational modification of proteins by ubiquitin controls many cellular processes, and aberrant ubiquitylation can contribute to cancer, immunopathologies, and neurodegeneration. Thus, deubiquitylating enzymes (DUBs) that remove ubiquitin from proteins have become attractive therapeutic targets. Monitoring the activity of DUBs in cells or in tissues is critical for understanding the biological function of DUBs in particular pathways and is essential for determining the physiological specificity and potency of small-molecule DUB inhibitors. Here, we describe a method for the homogenization of animal tissues and incubation of tissue lysates with ubiquitin-based activity probes to monitor DUB activity in mouse tissues and target engagement following treatment of animals with small-molecule DUB inhibitors. PMID:27613053

  20. CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil

    PubMed Central

    Almqvist, Helena; Axelsson, Hanna; Jafari, Rozbeh; Dan, Chen; Mateus, André; Haraldsson, Martin; Larsson, Andreas; Molina, Daniel Martinez; Artursson, Per; Lundbäck, Thomas; Nordlund, Pär

    2016-01-01

    Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery. PMID:27010513

  1. Physiological and pathological roles of tissue plasminogen activator and its inhibitor neuroserpin in the nervous system

    PubMed Central

    Lee, Tet Woo; Tsang, Vicky W. K.; Birch, Nigel P.

    2015-01-01

    Although its roles in the vascular space are most well-known, tissue plasminogen activator (tPA) is widely expressed in the developing and adult nervous system, where its activity is believed to be regulated by neuroserpin, a predominantly brain-specific member of the serpin family of protease inhibitors. In the normal physiological state, tPA has been shown to play roles in the development and plasticity of the nervous system. Ischemic damage, however, may lead to excess tPA activity in the brain and this is believed to contribute to neurodegeneration. In this article, we briefly review the physiological and pathological roles of tPA in the nervous system, which includes neuronal migration, axonal growth, synaptic plasticity, neuroprotection and neurodegeneration, as well as a contribution to neurological disease. We summarize tPA's multiple mechanisms of action and also highlight the contributions of the inhibitor neuroserpin to these processes. PMID:26528129

  2. Spiroimidazolidinone NPC1L1 inhibitors. Part 2: structure-activity studies and in vivo efficacy.

    PubMed

    Howell, Kobporn L; DeVita, Robert J; Garcia-Calvo, Margarita; Meurer, Roger D; Lisnock, JeanMarie; Bull, Herbert G; McMasters, Daniel R; McCann, Margaret E; Mills, Sander G

    2010-12-01

    Ezetimibe (Zetia®), a cholesterol-absorption inhibitor (CAI) approved by the FDA for the treatment of hypercholesterolemia, is believed to target the intestine protein Niemann-Pick C1-Like 1 (NPC1L1) or its pathway. A spiroimidazolidinone NPC1L1 inhibitor identified by virtual screening showed moderate binding activity but was not efficacious in an in vivo rodent model of cholesterol absorption. Synthesis of analogs established the structure-activity relationships for binding activity, and resulted in compounds with in vivo efficacy, including 24, which inhibited plasma cholesterol absorption by 67% in the mouse, thereby providing proof-of-concept that non-β-lactams can be effective CAIs.

  3. Antimicrobial Activity of ILTI, a Kunitz-Type Trypsin Inhibitor from Inga laurina (SW.) Willd.

    PubMed

    Macedo, Maria Lígia R; Ribeiro, Suzanna F F; Taveira, Gabriel B; Gomes, Valdirene M; de Barros, Karina M C A; Maria-Neto, Simone

    2016-05-01

    Over the last few years, a growing number of proteinase inhibitors have been isolated from plants and particularly from seeds and have shown antimicrobial activity. A 20,000 Da serine peptidase inhibitor, named ILTI, was isolated from Inga laurina seeds and showed potent inhibitory enzymatic activity against trypsin. The aim of this study was to determine the effects of ILTI on the growth of pathogenic and non-pathogenic microorganisms. We observed that ILTI strongly inhibited in particular the growth of Candida tropicalis and Candida buinensis, inducing cellular agglomeration. However, it was ineffective against human pathogenic bacteria. We also investigated the potential of ILTI to permeabilize the plasma membrane of yeast cells. C. tropicalis and C. buinensis were incubated for 24 h with the ILTI at different concentrations, which showed that this inhibitor induced changes in the membranes of yeast cells, leading to their permeabilization. Interestingly, ILTI induced the production of reactive oxygen species (ROS) in C. tropicalis and C. buinensis cells. Finally, ILTI was coupled with fluorescein isothiocyanate, and subsequent treatment of C. tropicalis and C. buinensis with DAPI revealed the presence of the labeled protein in the intracellular spaces. In conclusion, our results indicated the ability of peptidase inhibitors to induce microbial inhibition; therefore, they might offer templates for the design of new antifungal agents. PMID:26769111

  4. Effect of ace inhibitors and TMOF on growth, development, and trypsin activity of larval Spodoptera littoralis.

    PubMed

    Lemeire, Els; Borovsky, Dov; Van Camp, John; Smagghe, Guy

    2008-12-01

    Angiotensin converting enzyme (ACE) is a zinc metallopeptidase capable of cleaving dipeptide or dipeptideamide moieties at the C-terminal end of peptides. ACE is present in the hemolymph and reproductive tissues of insects. The presence of ACE in the hemolymph and its broad substrate specificity suggests an important role in processing of bioactive peptides. This study reports the effects of ACE inhibitors on larval growth in the cotton leafworm Spodoptera littoralis. Feeding ACE inhibitors ad lib decreased the growth rate, inhibited ACE activity in the larval hemolymph, and down-regulated trypsin activity in the larval gut. These results indicate that S. littoralis ACE may influence trypsin biosynthesis in the larval gut by interacting with a trypsin-modulating oostatic factor (TMOF). Injecting third instar larvae with a combination of Aea-TMOF and the ACE inhibitor captopril, down-regulated trypsin biosynthesis in the larval gut indicating that an Aea-TMOF gut receptor analogue could be present. Injecting captopril and enalapril into newly molted fifth instar larvae stopped larval feeding and decreased weight gain. Together, these results indicate that ACE inhibitors are efficacious in stunting larval growth and ACE plays an important role in larval growth and development. PMID:18949805

  5. Inhibition of dihydroceramide desaturase activity by the sphingosine kinase inhibitor SKI II.

    PubMed

    Cingolani, Francesca; Casasampere, Mireia; Sanllehí, Pol; Casas, Josefina; Bujons, Jordi; Fabrias, Gemma

    2014-08-01

    Sphingosine kinase inhibitor (SKI) II has been reported as a dual inhibitor of sphingosine kinases (SKs) 1 and 2 and has been extensively used to prove the involvement of SKs and sphingosine-1-phosphate (S1P) in cellular processes. Dihydroceramide desaturase (Des1), the last enzyme in the de novo synthesis of ceramide (Cer), regulates the balance between dihydroceramides (dhCers) and Cers. Both SKs and Des1 have interest as therapeutic targets. Here we show that SKI II is a noncompetitive inhibitor (Ki = 0.3 μM) of Des1 activity with effect also in intact cells without modifying Des1 protein levels. Molecular modeling studies support that the SKI II-induced decrease in Des1 activity could result from inhibition of NADH-cytochrome b5 reductase. SKI II, but not the SK1-specific inhibitor PF-543, provoked a remarkable accumulation of dhCers and their metabolites, while both SKI II and PF-543 reduced S1P to almost undetectable levels. SKI II, but not PF543, reduced cell proliferation with accumulation of cells in the G0/G1 phase. SKI II, but not PF543, induced autophagy. These overall findings should be taken into account when using SKI II as a pharmacological tool, as some of the effects attributed to decreased S1P may actually be caused by augmented dhCers and/or their metabolites.

  6. Activation Adverse Events Induced by the Selective Serotonin Reuptake Inhibitor Fluvoxamine in Children and Adolescents

    PubMed Central

    dosReis, Susan; Walkup, John T.; Riddle, Mark A.

    2009-01-01

    Abstract Objective The aim of this study was to examine the prevalence of activation cluster adverse events (AC-AEs) in youths treated with the selective serotonin reuptake inhibitor (SSRI) fluvoxamine for anxiety and the relationship of AC-AEs to SSRI blood levels. Methods Data from the Research Units on Pediatric Psychopharmacology (RUPP) Anxiety Study were examined for 45 youths (22 active fluvoxamine, 23 placebo) treated for Diagnostic and Statistical Manual for Mental Disorders, 4th edition (DSM-IV) anxiety disorders at the Johns Hopkins University site with an 8-week forced-flexible titration schedule. As part of the double-blind placebo-controlled trial, AC-AEs were recorded by clinicians at weekly patient visits. AC-AEs were defined as hyperactivity, activation, and disinhibition. Demographic characteristics, daily doses, and week-8 blood levels were examined in relation to the presence of AC-AEs. The prevalence of AC-AE and time to first event were established for those who experienced this side effect. Results AC-AEs were found in 10 of 22 participants (45%) receiving fluvoxamine and only 1 of 23 in the placebo group (4%). The onset of AC-AEs occurred from week 1 to week 8, with the majority occurring at or before week 4. The mean fluvoxamine blood level at week 8 in subjects with AC-AEs was higher than in subjects without AC-AEs (n = 16, t = −2.61, p = 0.04). Neither the age of the participants nor family history of bipolar or anxiety disorder differed between those who did and did not develop an AC-AE. Conclusions AC-AEs were common side effects of fluvoxamine, often appeared during the first 8 weeks of treatment, and were associated with higher fluvoxamine blood levels. Close monitoring for AC-AEs, not only when initiating SSRI treatment but also throughout dose titration, is recommended for early identification of activation. PMID:19364290

  7. High resolution genetic mapping uncovers chitin synthase-1 as the target-site of the structurally diverse mite growth inhibitors clofentezine, hexythiazox and etoxazole in Tetranychus urticae

    PubMed Central

    Demaeght, Peter; Osborne, Edward J.; Odman-Naresh, Jothini; Grbić, Miodrag; Nauen, Ralf; Merzendorfer, Hans

    2014-01-01

    The acaricides clofentezine, hexythiazox and etoxazole are commonly referred to as ‘mite growth inhibitors’, and clofentezine and hexythiazox have been used successfully for the integrated control of plant mite pests for decades. Although they are still important today, their mode of action has remained elusive. Recently, a mutation in chitin synthase 1 (CHS1) was linked to etoxazole resistance. In this study, we identified and investigated a T. urticae strain (HexR) harboring recessive, monogenic resistance to each of hexythiazox, clofentezine, and etoxazole. To elucidate if there is a common genetic basis for the observed cross-resistance, we adapted a previously developed bulk segregant analysis method to map with high resolution a single, shared resistance locus for all three compounds. This finding indicates that the underlying molecular basis for resistance to all three compounds is identical. This locus is centered on the CHS1 gene, and as supported by additional genetic and biochemical studies, a non-synonymous variant (I1017F) in CHS1 associates with resistance to each of the tested acaricides in HexR. Our findings thus demonstrate a shared molecular mode of action for the chemically diverse mite growth inhibitors clofentezine, hexythiazox and etoxazole as inhibitors of an essential, non-catalytic activity of CHS1. Given the previously documented cross-resistance between clofentezine, hexythiazox and the benzyolphenylurea compounds flufenoxuron and cycloxuron, CHS1 should be also considered as a potential target-site of insecticidal BPUs. PMID:24859419

  8. Discovery of a Series of Acridinones as Mechanism-Based Tubulin Assembly Inhibitors with Anticancer Activity

    PubMed Central

    Magalhaes, Luma G.; Marques, Fernando B.; da Fonseca, Marina B.; Rogério, Kamilla R.; Graebin, Cedric S.; Andricopulo, Adriano D.

    2016-01-01

    Microtubules play critical roles in vital cell processes, including cell growth, division, and migration. Microtubule-targeting small molecules are chemotherapeutic agents that are widely used in the treatment of cancer. Many of these compounds are structurally complex natural products (e.g., paclitaxel, vinblastine, and vincristine) with multiple stereogenic centers. Because of the scarcity of their natural sources and the difficulty of their partial or total synthesis, as well as problems related to their bioavailability, toxicity, and resistance, there is an urgent need for novel microtubule binding agents that are effective for treating cancer but do not have these disadvantages. In the present work, our lead discovery effort toward less structurally complex synthetic compounds led to the discovery of a series of acridinones inspired by the structure of podophyllotoxin, a natural product with important microtubule assembly inhibitory activity, as novel mechanism-based tubulin assembly inhibitors with potent anticancer properties and low toxicity. The compounds were evaluated in vitro by wound healing assays employing the metastatic and triple negative breast cancer cell line MDA-MB-231. Four compounds with IC50 values between 0.294 and 1.7 μM were identified. These compounds showed selective cytotoxicity against MDA-MB-231 and DU-145 cancer cell lines and promoted cell cycle arrest in G2/M phase and apoptosis. Consistent with molecular modeling results, the acridinones inhibited tubulin assembly in in vitro polymerization assays with IC50 values between 0.9 and 13 μM. Their binding to the colchicine-binding site of tubulin was confirmed through competitive assays. PMID:27508497

  9. Discovery of a Series of Acridinones as Mechanism-Based Tubulin Assembly Inhibitors with Anticancer Activity.

    PubMed

    Magalhaes, Luma G; Marques, Fernando B; da Fonseca, Marina B; Rogério, Kamilla R; Graebin, Cedric S; Andricopulo, Adriano D

    2016-01-01

    Microtubules play critical roles in vital cell processes, including cell growth, division, and migration. Microtubule-targeting small molecules are chemotherapeutic agents that are widely used in the treatment of cancer. Many of these compounds are structurally complex natural products (e.g., paclitaxel, vinblastine, and vincristine) with multiple stereogenic centers. Because of the scarcity of their natural sources and the difficulty of their partial or total synthesis, as well as problems related to their bioavailability, toxicity, and resistance, there is an urgent need for novel microtubule binding agents that are effective for treating cancer but do not have these disadvantages. In the present work, our lead discovery effort toward less structurally complex synthetic compounds led to the discovery of a series of acridinones inspired by the structure of podophyllotoxin, a natural product with important microtubule assembly inhibitory activity, as novel mechanism-based tubulin assembly inhibitors with potent anticancer properties and low toxicity. The compounds were evaluated in vitro by wound healing assays employing the metastatic and triple negative breast cancer cell line MDA-MB-231. Four compounds with IC50 values between 0.294 and 1.7 μM were identified. These compounds showed selective cytotoxicity against MDA-MB-231 and DU-145 cancer cell lines and promoted cell cycle arrest in G2/M phase and apoptosis. Consistent with molecular modeling results, the acridinones inhibited tubulin assembly in in vitro polymerization assays with IC50 values between 0.9 and 13 μM. Their binding to the colchicine-binding site of tubulin was confirmed through competitive assays. PMID:27508497

  10. Modulation of lysyl oxidase-like 2 enzymatic activity by an allosteric antibody inhibitor.

    PubMed

    Rodriguez, Hector M; Vaysberg, Maria; Mikels, Amanda; McCauley, Scott; Velayo, Arleene C; Garcia, Carlos; Smith, Victoria

    2010-07-01

    In this report, we assessed the steady-state enzymatic activity of lysyl oxidase-like 2 (LOXL2) against the substrates 1,5-diaminopentane (DAP), spermine, and fibrillar type I collagen. We find that both DAP and spermine are capable of activating LOXL2 to the same extent and have similar Michaelis constants (K(m) approximately 1 mm) and catalytic rates (k(cat) approximately 0.02 s(-1)). We also show that LOXL2 is capable of being inhibited by a known suicide inhibitor of lysyl oxidase (LOX), beta-aminopropionitrile, which we find is a potent inhibitor of LOXL2 activity. The modality of inhibition of beta-aminopropionitrile was also examined and found to be competitive with respect to the substrates DAP and spermine. In addition, we identified an antibody inhibitor (AB0023) of LOXL2 enzymatic function and have found that the inhibition occurs in a non-competitive manner with respect to both spermine and DAP. The binding epitope of AB0023 was mapped to the scavenger receptor cysteine-rich domain four of human LOXL2. AB0023 binds to a region remote from the catalytic domain making AB0023 an allosteric inhibitor of LOXL2. This affords AB0023 several advantages, because it is specific for LOXL2 and inhibits the enzymatic function of LOXL2 in a non-competitive manner thereby allowing inhibition of LOXL2 regardless of substrate concentration. These results suggest that antibody allosteric modulators of enzymatic function represent a novel drug development strategy and, in the context of LOXL2, suggest that inhibitors such as these might be useful therapeutics in oncology, fibrosis, and inflammation.

  11. Computational Design of a PDZ Domain Peptide Inhibitor that Rescues CFTR Activity

    PubMed Central

    Roberts, Kyle E.; Cushing, Patrick R.; Boisguerin, Prisca; Madden, Dean R.; Donald, Bruce R.

    2012-01-01

    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 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. PMID:22532795

  12. Growth exponents in surface models with non-active sites

    NASA Astrophysics Data System (ADS)

    Santos, M.; Figueiredo, W.; Aarão Reis, F. D. A.

    2006-11-01

    In this work, we studied the role played by the inactive sites present on the substrate of a growing surface. In our model, one particle sticks at the surface if the site where it falls is an active site. However, we allow the deposited particle to diffuse along the surface in accordance with some mechanism previously defined. Using Monte Carlo simulations, and some analytical results, we have investigated the model in (1+1) and (2+1) dimensions considering different relaxation mechanisms. We show that the consideration of non-active sites is a crucial point in the model. In fact, we have seen that the saturation regime is not observed for any value of the density of inactive sites. Besides, the growth exponent β turns to be one, at long times, whatever the mechanism of diffusion we consider in one and two dimensions.

  13. Development of a QPatch Automated Electrophysiology Assay for Identifying KCa3.1 Inhibitors and Activators

    PubMed Central

    Jenkins, David Paul; Yu, Weifeng; Brown, Brandon M.; Løjkner, Lars Damgaard

    2013-01-01

    Abstract The intermediate-conductance Ca2+-activated K+ channel KCa3.1 (also known as KCNN4, IK1, or the Gárdos channel) plays an important role in the activation of T and B cells, mast cells, macrophages, and microglia by regulating membrane potential, cellular volume, and calcium signaling. KCa3.1 is further involved in the proliferation of dedifferentiated vascular smooth muscle cells and fibroblast and endothelium-derived hyperpolarization responses in the vascular endothelium. Accordingly, KCa3.1 inhibitors are therapeutically interesting as immunosuppressants and for the treatment of a wide range of fibroproliferative disorders, whereas KCa3.1 activators constitute a potential new class of endothelial function preserving antihypertensives. Here, we report the development of QPatch assays for both KCa3.1 inhibitors and activators. During assay optimization, the Ca2+ sensitivity of KCa3.1 was studied using varying intracellular Ca2+ concentrations. A free Ca2+ concentration of 1 μM was chosen to optimally test inhibitors. To identify activators, which generally act as positive gating modulators, a lower Ca2+ concentration (∼200 nM) was used. The QPatch results were benchmarked against manual patch-clamp electrophysiology by determining the potency of several commonly used KCa3.1 inhibitors (TRAM-34, NS6180, ChTX) and activators (EBIO, riluzole, SKA-31). Collectively, our results demonstrate that the QPatch provides a comparable but much faster approach to study compound interactions with KCa3.1 channels in a robust and reliable assay. PMID:24351043

  14. A small ribozyme with dual-site kinase activity

    PubMed Central

    Biondi, Elisa; Maxwell, Adam W.R.; Burke, Donald H.

    2012-01-01

    Phosphoryl transfer onto backbone hydroxyls is a recognized catalytic activity of nucleic acids. We find that kinase ribozyme K28 possesses an unusually complex active site that promotes (thio)phosphorylation of two residues widely separated in primary sequence. After allowing the ribozyme to radiolabel itself by phosphoryl transfer from [γ-32P]GTP, DNAzyme-mediated cleavage yielded two radiolabeled cleavage fragments, indicating phosphorylation sites within each of the two cleavage fragments. These sites were mapped by alkaline digestion and primer extension pausing. Enzymatic digestion and mutational analysis identified nucleotides important for activity and established the active structure as being a constrained pseudoknot with unusual connectivity that may juxtapose the two reactive sites. Nuclease sensitivities for nucleotides near the pseudoknot core were altered in the presence of GTPγS, indicating donor-induced folding. The 5′ target site was more strongly favored in full-length ribozyme K28 (128 nt) than in truncated RNAs (58 nt). Electrophoretic mobilities of self-thiophosphorylated products on organomercurial gels are distinct from the 5′ mono-thiophosphorylated product produced by reaction with polynucleotide kinase, potentially indicating simultaneous labeling of both sites within individual RNA strands. Our evidence supports a single, compact structure with local dynamics, rather than global rearrangement, as being responsible for dual-site phosphorylation. PMID:22618879

  15. Aromatic inhibitors derived from ammonia-pretreated lignocellulose hinder bacterial ethanologenesis by activating regulatory circuits controlling inhibitor efflux and detoxification

    PubMed Central

    Keating, David H.; Zhang, Yaoping; Ong, Irene M.; McIlwain, Sean; Morales, Eduardo H.; Grass, Jeffrey A.; Tremaine, Mary; Bothfeld, William; Higbee, Alan; Ulbrich, Arne; Balloon, Allison J.; Westphall, Michael S.; Aldrich, Josh; Lipton, Mary S.; Kim, Joonhoon; Moskvin, Oleg V.; Bukhman, Yury V.; Coon, Joshua J.; Kiley, Patricia J.; Bates, Donna M.; Landick, Robert

    2014-01-01

    Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH). To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5-hydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts. PMID:25177315

  16. Comparison of in silico tools for binding site prediction applied for structure-based design of autolysin inhibitors.

    PubMed

    Tibaut, T; Borišek, J; Novič, M; Turk, D

    2016-07-01

    Autolysin E (AtlE) is a bacteriolytic enzyme which plays an important role in division and growth of bacterial cells and therefore represents a promising potential drug target. Its 3D structure has been recently elucidated. We used in silico prediction tools to study substrate or ligand (inhibitor) binding regions of AtlE. We applied several freely available tools and a commercial tool for binding site identification and compared results of the prediction. Calculation time, number of predictions and output data provided by specific software vary according to the different approaches utilized by specific method categories. Despite different approaches, binding sites in similar locations on the protein were predicted. Specific amino acid residues that form these binding sites were predicted as binding residues. The predicted residues, especially those with predicted highest conservation score, could theoretically have catalytic and binding properties. According to our results, we assume that E138, which has the highest conservation score, is the catalytic residue and F161, G162 and Y224, which are also highly conserved, are responsible for substrate binding. Ligands developed with binding specificity towards these residues could inhibit the catalysis and binding of the substrate of AtlE. The molecules with inhibitory potency could therefore represent potential new antibacterial agents. PMID:27686112

  17. Structure based design towards the identification of novel binding sites and inhibitors for the chikungunya virus envelope proteins.

    PubMed

    Rashad, Adel A; Keller, Paul A

    2013-07-01

    Chikungunya virus is an emerging arbovirus that is widespread in tropical regions and is spreading quickly to temperate climates with recent epidemics in Africa, Asia, Europe and the Americas. It is having an increasingly major impact on humans with potentially life-threatening and debilitating arthritis. Thus far, neither vaccines nor medications are available to treat or control the virus and therefore, the development of medicinal chemistry is a vital and immediate issue that needs to be addressed. The viral envelope proteins play a major role during infection through mediation of binding and fusion with the infected cell surfaces. The possible binding target sites of the chikungunya virus envelope proteins have not previously been investigated; we describe here for the first time the identification of novel sites for potential binding on the chikungunya glycoprotein complexes and the identification of possible antagonists for these sites through virtual screening using two successive docking scores; FRED docking for fast precise screening, with the top hits then subjected to a ranking scoring using the AUTODOCK algorithm. Both the immature and the mature forms of the chikungunya envelope proteins were included in the study to increase the probability of finding positive and reliable hits. Some small molecules have been identified as good in silico chikungunya virus envelope proteins inhibitors and these could be good templates for drug design targeting this virus.

  18. Inhibitors of protein synthesis. 4. Studies on the structure-activity relationship of gougerotin and some of its analogs.

    PubMed

    Coutsogeorgopoulos, C; Bloch, A; Watanabe, K A; Fox, J J

    1975-08-01

    Seventeen structural analogs of gougerotin have been compared with the parent compound as inhibitors of the growh of E. coli B and as inhibitors of N-acetylphenylalanylpuromycin formation. The analogs comprise compounds with (1) an intact sarcosyl-D-seryl moiety and modifications at various positions of the carbohydrate moiety, (2) compounds which lack the peptidyl residue, and (3) compounds in which the sarcosyl-D-seryl moiety has been replaced by other aminoacyl or peptidyl moieties and in which various sites in the carbohydrate moiety have been modified in addition. These comparisons revealed that (1) the 3'-hydroxyl group makes a strong contribution to inhibitory activity, (2) the introduction of a double bond between the 2' and 3' positions of the carbohydrate moiety causes a marked decrease in activity; however, if in addition the sarcosyl-D-seryl moiety is replaced by the epsilon-N-methyl-beta-L-arginyl side chain, full activity is restored, (3) there is a marked degree of stereospecificity around the alpha carbon of the seryl moiety (replacement of the sarcosyl-D-seryl residue with the sarcosyl-L-seryl residue results in greatly reduced activity), and (4) replacement of the carboxamide group at position 5' with a hydroxymethyl function resulsts in reduced activity which is further decreased by alterations in the sarcosyl-D-seryl moiety. These alterations include (a) replacement of the D-seryl residue of the sarcosyl-D-seryl moiety by the D-alanyl or the D-phenylalanyl residues and (b) replacement of the whole sarcosyl-D-seryl side chain by the D-seryl, D-alanyl, D-phenylalanyl, p-methoxy-D-phenylalanyl, or p-methoxy-L-phenylalanyl moieties; the replacement of the sarcosyl-D-seryl side chain with the various aminoacyl residues did not lead to acceptor (puromycin-like) activity with N-Ac-Phe-tRNA as the donor.

  19. Structure-activity relationships and molecular modelling of new 5-arylidene-4-thiazolidinone derivatives as aldose reductase inhibitors and potential anti-inflammatory agents.

    PubMed

    Maccari, Rosanna; Vitale, Rosa Maria; Ottanà, Rosaria; Rocchiccioli, Marco; Marrazzo, Agostino; Cardile, Venera; Graziano, Adriana Carol Eleonora; Amodeo, Pietro; Mura, Umberto; Del Corso, Antonella

    2014-06-23

    A series of 5-(carbamoylmethoxy)benzylidene-2-oxo/thioxo-4-thiazolidinone derivatives (6-9) were synthesized as inhibitors of aldose reductase (AR), enzyme which plays a crucial role in the development of diabetes complications as well as in the inflammatory processes associated both to diabetes mellitus and to other pathologies. In vitro inhibitory activity indicated that compounds 6-9a-d were generally good AR inhibitors. Acetic acid derivatives 8a-d and 9a-d were shown to be the best enzyme inhibitors among the tested compounds endowed with significant inhibitory ability levels reaching submicromolar IC50 values. Moreover, some representative AR inhibitors (7a, 7c, 9a, 9c, 9d) were assayed in cultures of human keratinocytes in order to evaluate their capability to reduce NF-kB activation and iNOS expression. Compound 9c proved to be the best derivative endowed with both interesting AR inhibitory effectiveness and ability to reduce NF-kB activation and iNOS expression. Molecular docking and molecular dynamics simulations were undertaken to investigate the binding modes of selected compounds into the active site of AR in order to rationalize the inhibitory effectiveness of these derivatives.

  20. In Vitro Mode of Action and Anti-thrombotic Activity of Boophilin, a Multifunctional Kunitz Protease Inhibitor from the Midgut of a Tick Vector of Babesiosis, Rhipicephalus microplus

    PubMed Central

    Assumpção, Teresa C.; Ma, Dongying; Mizurini, Daniella M.; Kini, R. Manjunatha; Ribeiro, José M. C.; Kotsyfakis, Michail; Monteiro, Robson Q.; Francischetti, Ivo M. B.

    2016-01-01

    Background Hematophagous mosquitos and ticks avoid host hemostatic system through expression of enzyme inhibitors targeting proteolytic reactions of the coagulation and complement cascades. While most inhibitors characterized to date were found in the salivary glands, relatively few others have been identified in the midgut. Among those, Boophilin is a 2-Kunitz multifunctional inhibitor targeting thrombin, elastase, and kallikrein. However, the kinetics of Boophilin interaction with these enzymes, how it modulates platelet function, and whether it inhibits thrombosis in vivo have not been determined. Methodology/Principal Findings Boophilin was expressed in HEK293 cells and purified to homogeneity. Using amidolytic assays and surface plasmon resonance experiments, we have demonstrated that Boophilin behaves as a classical, non-competitive inhibitor of thrombin with respect to small chromogenic substrates by a mechanism dependent on both exosite-1 and catalytic site. Inhibition is accompanied by blockade of platelet aggregation, fibrin formation, and clot-bound thrombin in vitro. Notably, we also identified Boophilin as a non-competitive inhibitor of FXIa, preventing FIX activation. In addition, Boophilin inhibits kallikrein activity and the reciprocal activation, indicating that it targets the contact pathway. Furthermore, Boophilin abrogates cathepsin G- and plasmin-induced platelet aggregation and partially affects elastase-mediated cleavage of Tissue Factor Pathway Inhibitor (TFPI). Finally, B