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Sample records for anhydrase inhibitors inhibition

  1. Carbonic anhydrase inhibitors: the membrane-associated isoform XV is highly inhibited by inorganic anions.

    Science.gov (United States)

    Innocenti, Alessio; Hilvo, Mika; Parkkila, Seppo; Scozzafava, Andrea; Supuran, Claudiu T

    2009-02-15

    The membrane-associated mouse isozyme of carbonic anhydrase XV (mCA XV), has been investigated for its interaction with anion inhibitors. mCA XV is an isoforms possessing a very particular inhibition profile by anions, dissimilar to that of all other mammalian CAs investigated earlier. Many simple inorganic anions (thiocyanate, cyanide, azide, bicarbonate, hydrogen sulfide, bisulfite and sulfate) showed low micromolar inhibition constants against mCA XV (K(I)s of 8.2-10.1 microM), whereas they acted as much weaker (usually millimolar) inhibitors of other isoforms. Halides, nitrate, nitrite, carbonate, sulfamate, sulfamide and phenylboronic/arsonic acid were weaker inhibitors, with inhibition constants in the range of 27.6-288 microM. Our data may be useful for the design of more potent inhibitors of mCA XV (considering various zinc binding groups present in the anions investigated here, e.g., the sulfonate one) and for understanding some physiologic/pharmacologic consequences of mCA XV inhibition by anions such as bicarbonate or sulfate which show quite high affinity for it.

  2. Nanoemulsions of sulfonamide carbonic anhydrase inhibitors strongly inhibit the growth of Trypanosoma cruzi.

    Science.gov (United States)

    Vermelho, Alane Beatriz; da Silva Cardoso, Verônica; Ricci Junior, Eduardo; Dos Santos, Elisabete Pereira; Supuran, Claudiu T

    2018-12-01

    Sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors targeting the α-class enzyme from the protozoan pathogen Trypanosoma cruzi, responsible of Chagas disease, were recently reported. Although many such derivatives showed low nanomolar activity in vitro, they were inefficient anti-T. cruzi agents in vivo. Here, we show that by formulating such sulfonamides as nanoemulsions in clove (Eugenia caryophyllus) oil, highly efficient anti-protozoan effects are observed against two different strains of T. cruzi. These effects are probably due to an enhanced permeation of the enzyme inhibitor through the nanoemulsion formulation, interfering in this way with the life cycle of the pathogen either by inhibiting pH regulation or carboxylating reactions in which bicarbonate/CO 2 are involved. This type of formulation of sulfonamides with T. cruzi CA inhibitory effects may lead to novel therapeutic approaches against this orphan disease.

  3. Determination of activities of human carbonic anhydrase II inhibitors ...

    African Journals Online (AJOL)

    Purpose: To evaluate the activities of new curcumin analogs as carbonic anhydrase II (CA-II) inhibitor. Methods: Carbonic anhydrase II (CA-II) inhibition was determined by each ligand capability to inhibit the esterase activity of CA-II using 4-NPA as a substrate in 96-well plates. Dimethyl sulfoxide was used to dissolve each ...

  4. Carborane-based carbonic anhydrase inhibitors

    Czech Academy of Sciences Publication Activity Database

    Brynda, Jiří; Mader, Pavel; Šícha, Václav; Fábry, Milan; Poncová, Kristýna; Bakardjiev, Mario; Grüner, Bohumír; Cígler, Petr; Řezáčová, Pavlína

    2013-01-01

    Roč. 52, č. 51 (2013), s. 13760-13763 ISSN 1433-7851 R&D Projects: GA TA ČR(CZ) TE01020028; GA AV ČR IAAX00320901 Institutional support: RVO:68378050 ; RVO:61388963 ; RVO:61388980 Keywords : carbonic anhydrases * carboranes * drug discovery * inhibitors * structure elucidation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 11.336, year: 2013

  5. New coumarin derivatives as carbonic anhydrase inhibitors.

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    Karataş, Mert Olgun; Alici, Bülent; Cakir, Umit; Cetinkaya, Engin; Demir, Dudu; Ergün, Adem; Gençer, Nahit; Arslan, Oktay

    2014-06-01

    In the current study, a series of 4-chloromethyl-7-hydroxy-coumarin derivatives containing imidazolium, benzimidazolium, bisbenzimidazolium and quaternary ammonium salts were synthesized, characterized and the inhibition effects of the derivatives on human carbonic anhydrases (hCA I and hCA II) were investigated as in vitro. Structures of these coumarins were confirmed by FT-IR, (1)H NMR, (13)C NMR and LC-MS analyses. Structure activity relationship study showed that 3d (IC50: 79 μM for hCA I and 88 μM for hCA II) performed higher inhibitory activity than others.

  6. Carbonic anhydrase inhibition increases retinal oxygen tension and dilates retinal vessels

    DEFF Research Database (Denmark)

    Pedersen, Daniella Bach; Koch Jensen, Peter; la Cour, Morten

    2005-01-01

    Carbonic anhydrase inhibitors (CAIs) increase blood flow in the brain and probably also in the optic nerve and retina. Additionally they elevate the oxygen tension in the optic nerve in the pig. We propose that they also raise the oxygen tension in the retina. We studied the oxygen tension...... in the pig retina and optic nerve before and after dorzolamide injection. Also the retinal vessel diameters during carbonic anhydrase inhibition were studied....

  7. Carbonic anhydrase inhibitors: inhibition of the beta-class enzymes from the fungal pathogens Candida albicans and Cryptococcus neoformans with simple anions.

    Science.gov (United States)

    Innocenti, Alessio; Mühlschlegel, Fritz A; Hall, Rebecca A; Steegborn, Clemens; Scozzafava, Andrea; Supuran, Claudiu T

    2008-09-15

    The catalytic activity and inhibition of the beta-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic fungi Candida albicans (Nce103) and Cryptococcus neoformans (Can2) with inorganic anions such as halogenides, pseudohalogenides, bicarbonate, carbonate, nitrate, nitrite, hydrogen sulfide, bisulfite, perchlorate, sulfate were investigated. The two enzymes showed appreciable CO(2) hydrase activity (k(cat) in the range of (3.9-8.0)x10(5)s(-1), and k(cat)/K(m) in the range of (4.3-9.7)x10(7)M(-1)s(-1)). Can2 was weakly inhibited by cyanide and sulfamic acid (K(I)s of 8.22-13.56 mM), while all other anions displayed more potent inhibition. Nce103 was strongly inhibited by cyanide and carbonate (K(I)s of 10-11 microM), and weakly inhibited by sulfate, phenylboronic, and phenyl arsonic acid (K(I)s of 14.15-30.85 mM). These data demonstrate that pathogenic, fungal beta-CAs may be targets for the development of antifungals that have a novel mechanism of action.

  8. Carborane-based inhibitors of carbonic anhydrases

    Czech Academy of Sciences Publication Activity Database

    Brynda, Jiří; Pachl, Petr; Šícha, Václav; Fábry, Milan; Grüner, Bohumír; Cígler, Petr; Řezáčová, Pavlína

    2015-01-01

    Roč. 22, č. 1 (2015), s. 3 ISSN 1211-5894. [Discussions in Structural Molecular Biology. Annual Meeting of the Czech Society for Structural Biology /13./. 19.03.2015-21.03.2015, Nové Hrady] R&D Projects: GA ČR GA15-05677S Institutional support: RVO:61388963 ; RVO:68378050 ; RVO:61388980 Keywords : carboranes * carbonic anhydrase Subject RIV: CE - Biochemistry

  9. Lansoprazole and carbonic anhydrase IX inhibitors sinergize against human melanoma cells.

    Science.gov (United States)

    Federici, Cristina; Lugini, Luana; Marino, Maria Lucia; Carta, Fabrizio; Iessi, Elisabetta; Azzarito, Tommaso; Supuran, Claudiu T; Fais, Stefano

    2016-01-01

    Proton Pump Inhibitors (PPIs) reduce tumor acidity and therefore resistance of tumors to drugs. Carbonic Anhydrase IX (CA IX) inhibitors have proven to be effective against tumors, while tumor acidity might impair their full effectiveness. To analyze the effect of PPI/CA IX inhibitors combined treatment against human melanoma cells. The combination of Lansoprazole (LAN) and CA IX inhibitors (FC9-399A and S4) has been investigated in terms of cell proliferation inhibition and cell death in human melanoma cells. The combination of these inhibitors was more effective than the single treatments in both inhibiting cell proliferation and in inducing cell death in human melanoma cells. These results represent the first successful attempt in combining two different proton exchanger inhibitors. This is the first evidence on the effectiveness of a new approach against tumors based on the combination of PPI and CA IX inhibitors, thus providing an alternative strategy against tumors.

  10. Inhibition of Malassezia globosa carbonic anhydrase with phenols.

    Science.gov (United States)

    Entezari Heravi, Yeganeh; Bua, Silvia; Nocentini, Alessio; Del Prete, Sonia; Saboury, Ali Akbar; Sereshti, Hassan; Capasso, Clemente; Gratteri, Paola; Supuran, Claudiu T

    2017-05-01

    A panel of 22 phenols was investigated as inhibitors of the β-class carbonic anhydrase (CAs, EC 4.2.1.1) from the fungal parasite Malassezia globosa (MgCA), a validated anti-dandruff drug target. The displayed inhibitory activities were compared to the ones previously reported against the off-target widely distributed human (h) isoforms hCA I and II. All tested phenols possessed a better efficacy in inhibiting MgCA than the clinically used sulfonamide acetazolamide, with K I s in the range of 2.5 and 65.0μM. A homology-built model of MgCA was also used for understanding the binding mode of phenols to the fungal enzyme. Indeed, a wide network of hydrogen bonds and hydrophobic interactions between the phenol and active site residues were evidenced. The OH moiety of the inhibitor was observed anchored to the zinc-coordinated water, also making hydrogen bonds with Ser48 and Asp49. The diverse substituents at the phenolic scaffold were observed to interact with different portions of the hydrophobic pocket according to their nature and position. Considering the effective MgCA inhibitory properties of phenols, beside to the rather low inhibition against the off-target hCA I and II, this class of compounds might be of considerable interest in the cosmetics field as potential anti-dandruff drugs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Structural Basis for the Inhibition of Helicobacter pylori α-Carbonic Anhydrase by Sulfonamides.

    Directory of Open Access Journals (Sweden)

    Joyanta K Modak

    Full Text Available Periplasmic α-carbonic anhydrase of Helicobacter pylori (HpαCA, an oncogenic bacterium in the human stomach, is essential for its acclimation to low pH. It catalyses the conversion of carbon dioxide to bicarbonate using Zn(II as the cofactor. In H. pylori, Neisseria spp., Brucella suis and Streptococcus pneumoniae this enzyme is the target for sulfonamide antibacterial agents. We present structural analysis correlated with inhibition data, on the complexes of HpαCA with two pharmacological inhibitors of human carbonic anhydrases, acetazolamide and methazolamide. This analysis reveals that two sulfonamide oxygen atoms of the inhibitors are positioned proximal to the putative location of the oxygens of the CO2 substrate in the Michaelis complex, whilst the zinc-coordinating sulfonamide nitrogen occupies the position of the catalytic water molecule. The structures are consistent with acetazolamide acting as site-directed, nanomolar inhibitors of the enzyme by mimicking its reaction transition state. Additionally, inhibitor binding provides insights into the channel for substrate entry and product exit. This analysis has implications for the structure-based design of inhibitors of bacterial carbonic anhydrases.

  12. Carbonic anhydrase inhibitors; phosphoryl-sulfonamides--a new class of high affinity inhibitors of isozymes I and II.

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    Fenesan, I; Popescu, R; Scozzafava, A; Crucin, V; Mateiciuc, E; Bauer, R; Ilies, M A; Supuran, C T

    2000-01-01

    A series of phosphorylated aromatic/heterocyclic sulfonamides with the general formula ArSO2NHPO3H2 have been prepared by condensing ArSO2NH2 with phosphorus pentachloride, followed by controlled hydrolysis in the presence of formic acid. The new derivatives generally act as stronger inhibitors of two carbonic anhydrase (CA) isozymes, CA I and CA II, as compared to the parent unsubstituted sulfonamides from which they were obtained. The inhibition mechanism by this new class of CA inhibitors, as well as structure activity correlations for the series of investigated derivatives, are also discussed.

  13. Azobenzene-based inhibitors of human carbonic anhydrase II

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    Leander Simon Runtsch

    2015-07-01

    Full Text Available Aryl sulfonamides are a widely used drug class for the inhibition of carbonic anhydrases. In the context of our program of photochromic pharmacophores we were interested in the exploration of azobenzene-containing sulfonamides to block the catalytic activity of human carbonic anhydrase II (hCAII. Herein, we report the synthesis and in vitro evaluation of a small library of nine photochromic sulfonamides towards hCAII. All molecules are azobenzene-4-sulfonamides, which are substituted by different functional groups in the 4´-position and were characterized by X-ray crystallography. We aimed to investigate the influence of electron-donating or electron-withdrawing substituents on the inhibitory constant Ki. With the aid of an hCAII crystal structure bound to one of the synthesized azobenzenes, we found that the electronic structure does not strongly affect inhibition. Taken together, all compounds are strong blockers of hCAII with Ki = 25–65 nM that are potentially photochromic and thus combine studies from chemical synthesis, crystallography and enzyme kinetics.

  14. Exploring new Probenecid-based carbonic anhydrase inhibitors: Synthesis, biological evaluation and docking studies.

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    Mollica, Adriano; Costante, Roberto; Akdemir, Atilla; Carradori, Simone; Stefanucci, Azzurra; Macedonio, Giorgia; Ceruso, Mariangela; Supuran, Claudiu T

    2015-09-01

    Novel Probenecid-based amide derivatives, incorporating different natural amino acids, were synthesized and assayed to test their effect on the human carbonic anhydrase (hCA, EC 4.2.1.1) transmembrane isoforms hCA IX and XII over the ubiquitous isoforms hCA I and II. Most of them presented a complete loss of hCA II inhibition (K(i)s > 10,000 nM) and strong inhibitory activity against hCA IX and XII in the nanomolar range with respect to the parent compound. A residual activity against hCA I was observed for some of them. These biological results have been explained by docking studies within the active sites of the four studied human carbonic anhydrases (with or without the zinc-bound water) and helped us to better comprehend the rationale behind the design of tertiary sulfonamide compounds as potent but atypical inhibitors of specific isoforms of human carbonic anhydrase. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Optic nerve oxygen tension in pigs and the effect of carbonic anhydrase inhibitors

    DEFF Research Database (Denmark)

    Stefánsson, E; Jensen, P K; Eysteinsson, T

    1999-01-01

    To evaluate how the oxygen tension of the optic nerve (ONP(O)2) is affected by the administration of the carbonic anhydrase inhibitors dorzolamide and acetazolamide and by alterations in oxygen and carbon dioxide in the breathing mixture....

  16. Optic nerve oxygen tension in pigs and the effect of carbonic anhydrase inhibitors

    DEFF Research Database (Denmark)

    Stefánsson, E; Jensen, P K; Eysteinsson, T

    1999-01-01

    To evaluate how the oxygen tension of the optic nerve (ONP(O)2) is affected by the administration of the carbonic anhydrase inhibitors dorzolamide and acetazolamide and by alterations in oxygen and carbon dioxide in the breathing mixture.......To evaluate how the oxygen tension of the optic nerve (ONP(O)2) is affected by the administration of the carbonic anhydrase inhibitors dorzolamide and acetazolamide and by alterations in oxygen and carbon dioxide in the breathing mixture....

  17. Antipsychotic agents screened as human carbonic anhydrase I and II inhibitors.

    Science.gov (United States)

    Erzengin, Mahmut; Bilen, Cigdem; Ergun, Adem; Gencer, Nahit

    2014-02-01

    The antipsychotic drugs currently used to treat schizophrenia can be divided into two distinct classes, typical and atypical antipsychotics. Many drug molecules are enzyme inhibitors that bind reversibly or irreversibly to their target through intermolecular interactions. That's why enzyme inhibition studies are an important issue for drug design and biochemical applications. In this study, in vitro inhibition effect of some antipsychotic drugs on the purified carbonic anhydrase (CA) I and II isoenzymes were investigated by using CO2 as a substrate. CA I and II were purified from human erythrocytes by a simple one step procedure using Sepharose 4B-L-tyrosine-sulfonamide affinity column. The results showed that all the drugs inhibited the cytosolic carbonic anhydrases enzyme activity in a concentration-dependent fashion. Among the studied drugs, aripiprazole and pramipexole were found to be the most active one for hCA I (IC50: 3.64 and 5.37 μM) and hCA II (IC50: 4.16 and 4.81 μM) activity, respectively.

  18. Intrinsic thermodynamics of inhibitor binding to human carbonic anhydrase IX.

    Science.gov (United States)

    Linkuvienė, Vaida; Matulienė, Jurgita; Juozapaitienė, Vaida; Michailovienė, Vilma; Jachno, Jelena; Matulis, Daumantas

    2016-04-01

    Human carbonic anhydrase 9th isoform (CA IX) is an important marker of numerous cancers and is increasingly interesting as a potential anticancer drug target. Various synthetic aromatic sulfonamide-bearing compounds are being designed as potent inhibitors of CA IX. However, sulfonamide compound binding to CA IX is linked to several reactions, the deprotonation of the sulfonamide amino group and the protonation of the CA active site Zn(II)-bound hydroxide. These linked reactions significantly affect the affinities and other thermodynamic parameters such as enthalpies and entropies of binding. The observed and intrinsic affinities of compound binding to CA IX were determined by the fluorescent thermal shift assay. The enthalpies and entropies of binding were determined by the isothermal titration calorimetry. The pKa of CA IX was determined to be 6.8 and the enthalpy of CA IX-Zn(II)-bound hydroxide protonation was -24 kJ/mol. These values enabled the analysis of intrinsic thermodynamics of a library of compounds binding to CA IX. The most strongly binding compounds exhibited the intrinsic affinity of 0.01 nM and the observed affinity of 2 nM. The intrinsic thermodynamic parameters of compound binding to CA IX helped to draw the compound structure to thermodynamics relationship. It is important to distinguish the intrinsic from observed parameters of any disease target protein interaction with its inhibitors as drug candidates when drawing detailed compound structure to thermodynamics correlations. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Carbonic anhydrase from Apis mellifera: purification and inhibition by pesticides.

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    Soydan, Ercan; Güler, Ahmet; Bıyık, Selim; Şentürk, Murat; Supuran, Claudiu T; Ekinci, Deniz

    2017-12-01

    Carbonic anhydrase (CA) enzymes have been shown to play an important role in ion transport and in pH regulation in several organisms. Despite this information and the wealth of knowledge regarding the significance of CA enzymes, few studies have been reported about bee CA enzymes and the hazardous effects of chemicals. Using Apis mellifera as a model, this study aimed to determine the risk of pesticides on Apis mellifera Carbonic anhydrase enzyme (Am CA). CA was initially purified from Apis mellifera spermatheca for the first time in the literature. The enzyme was purified with an overall purification of ∼35-fold with a molecular weight of ∼32 kDa. The enzyme was then exposed to pesticides, including tebuconazole, propoxur, carbaryl, carbofuran, simazine and atrazine. The six pesticides dose-dependently inhibited in vitro AmCA activity at low micromolar concentrations. IC 50 values for the pesticides were 0.0030, 0.0321, 0.0031, 0.0087, 0.0273 and 0.0165 μM, respectively. The AmCA inhibition mechanism of these compounds is unknown at this moment.

  20. Cyclic secondary sulfonamides: unusually good inhibitors of cancer-related carbonic anhydrase enzymes.

    Science.gov (United States)

    Moeker, Janina; Peat, Thomas S; Bornaghi, Laurent F; Vullo, Daniela; Supuran, Claudiu T; Poulsen, Sally-Ann

    2014-04-24

    Carbonic anhydrase IX (CA IX) is a target for hypoxic cancer therapies, and the discovery of CA IX selective ligands is imperative for the development of these agents. Primary sulfonamides are broad specificity inhibitors of CA enzymes, while secondary sulfonamides are generally poor CA inhibitors. However, saccharin, a cyclic secondary sulfonamide, has unusually good inhibition of CA IX (Ki = 103 nM). In this study, we demonstrate that the affinity and selectivity of saccharin for CA IX can be further modulated when linked to hydrophobic or hydrophilic substituents. The hydrophilic glycoconjugate derivative (12) showed improved inhibition of CA IX (Ki = 49.5 nM) and extremely poor inhibition of the predominant off-target CAs (Ki > 50000 nM) compared to saccharin. This >1000-fold selectivity for CA IX over off-target CAs is unprecedented for classical primary sulfonamide CA inhibitors. Our study highlights the potential of cyclic secondary sulfonamides to be exploited for the discovery of potent, cancer-selective CA inhibitors.

  1. Comparison of inhibition effects of some benzoic acid derivatives on sheep heart carbonic anhydrase

    Science.gov (United States)

    Kiliç, Deryanur; Yildiz, Melike; Şentürk, Murat; Erdoǧan, Orhan; Küfrevioǧlu, Ömer Irfan

    2016-04-01

    Carbonic anhydrase (CA) is a family of metalloenzymes that requires Zn as a cofactor and catalyze the quick conversion of CO2 to HCO3- and H+. Inhibitors of the carbonic anhydrases (CAs) have medical usage of significant diseases such as glaucoma, epilepsy, gastroduodenal ulcers, acid-base disequilibria and neurological disorders. In the present study, inhibition of CA with some benzoic derivatives (1-6) were investigated. Sheep heart CA (shCA) enzyme was isolated by means of designed affinity chromatography gel (cellulose-benzyl-sulfanylamide) 42.45-fold in a yield of 44 % with 564.65 EU/mg. Purified shCA enzyme was used in vitro studies. In the studies, IC50 values were calculated for 3-aminobenzoic acid (1), 4-aminobenzoic acid (2), 2-hydroxybenzoic acid (3), 2-benzoylbenzoic acid (4), 2,3-dimethoxybenzoic acid (5), and 3,4,5-trimethoxybenzoic acid (6), showing the inhibition effects on the purified enzyme. Such molecules can be used as pioneer for discovery of novel effective CA inhibitors for medicinal chemistry applications.

  2. Capsaicin: A Potent Inhibitor of Carbonic Anhydrase Isoenzymes

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    Betul Arabaci

    2014-07-01

    Full Text Available Carbonic anhydrase (CA, EC 4.2.1.1 is a zinc containing metalloenzyme that catalyzes the rapid and reversible conversion of carbon dioxide (CO2 and water (H2O into a proton (H+ and bicarbonate (HCO3– ion. On the other hand, capsaicin is the main component in hot chili peppers and is used extensively used in spices, food additives and drugs; it is responsible for their spicy flavor and pungent taste. There are sixteen known CA isoforms in humans. Human CA isoenzymes I, and II (hCA I and hCA II are ubiquitous cytosolic isoforms. In this study, the inhibition properties of capsaicin against the slow cytosolic isoform hCA I, and the ubiquitous and dominant rapid cytosolic isozymes hCA II were studied. Both CA isozymes were inhibited by capsaicin in the micromolar range. This naturally bioactive compound has a Ki of 696.15 µM against hCA I, and of 208.37 µM against hCA II.

  3. Diuretics: from classical carbonic anhydrase inhibitors to novel applications of the sulfonamides.

    Science.gov (United States)

    Supuran, Claudiu T

    2008-01-01

    The widely clinically used benzothiadiazines and high ceiling diuretics, such as hydrochlorothiazide, hydroflumethiazide, quinethazone, metolazone, chlorthalidone, indapamide, furosemide and bumetanide, contain SO(2)NH(2) moieties acting as an effective zinc-binding function in carbonic anhydrases (CAs, EC 4.2.1.1) inhibitors. These drugs were launched in a period when only isoform CA II was known and considered physiologically/pharmacologically relevant. Although acting as moderate-weak inhibitors of CA II, all these drugs considerably inhibit other CA isozymes known nowadays to be involved in critical physiologic processes, among the 16 CAs present in vertebrates. Some low nanomolar (or even subnanomolar) inhibitors against such isoforms were recently detected, such as metholazone against CA VII, XII and XIII, chlorthalidone against CA VB, VII, IX, XII and XIII, indapamide against CA VII, IX, XII and XIII, furosemide against CA I, II and XIV, and bumethanide against CA IX and XII. The X-ray crystal structure of the CA II-indapamide adduct was also reported recently, revealing interesting aspects useful for the drug design of CA inhibitors. It has also been proposed that the recently observed beneficial effect of indapamide for the treatment of patients with hypertension and type 2 diabetes might be due to its potent inhibition of CA isoforms present in kidneys and blood vessels, which would thus explain both the blood pressure lowering effects as well as organ-protective activity of the drug. Thus, these old drugs may be useful as leads for new applications.

  4. Carbon- versus sulphur-based zinc binding groups for carbonic anhydrase inhibitors?

    Science.gov (United States)

    Supuran, Claudiu T

    2018-12-01

    A set of compounds incorporating carbon-based zinc-binding groups (ZBGs), of the type PhX (X = COOH, CONH 2 , CONHNH 2 , CONHOH, CONHOMe), and the corresponding derivatives with sulphur(VI)-based ZBGs (X = SO 3 H, SO 2 NH 2 , SO 2 NHNH 2 , SO 2 NHOH, SO 2 NHOMe) were tested as inhibitors of all mammalian isoforms of carbonic anhydrase (CA, EC 4.2.1.1), CA I-XV. Three factors connected with the ZBG influenced the efficacy as CA inhibitor (CAI) of the investigated compounds: (i) the pKa of the ZBG; (ii) its geometry (tetrahedral, i.e. sulphur-based, versus trigonal, i.e. carbon-based ZBGs), and (iii) orientation of the organic scaffold induced by the nature of the ZBG. Benzenesulphonamide was the best inhibitor of all isoforms, but other ZBGs led to interesting inhibition profiles, although with an efficacy generally reduced when compared to the sulphonamide. The nature of the ZBG also influenced the CA inhibition mechanism. Most of these derivatives were zinc binders, but some of them (sulfonates, carboxylates) may interact with the enzyme by anchoring to the zinc-coordinated water molecule or by other inhibition mechanisms (occlusion of the active site entrance, out of the active site binding, etc.). Exploring structurally diverse ZBGs may lead to interesting new developments in the field of CAIs.

  5. Inhibition of beta-carbonic anhydrases from the bacterial pathogen Brucella suis with inorganic anions.

    Science.gov (United States)

    Maresca, Alfonso; Scozzafava, Andrea; Köhler, Stephan; Winum, Jean-Yves; Supuran, Claudiu T

    2012-05-01

    The bacterial pathogen Brucella suis encodes two carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the β-class, bsCA1 and bsCA2, which are crucial for its life cycle. Sulfonamides, strong inhibitors of these enzymes, were shown to block the growth of the pathogen in vitro. Here we report the inhibition of these two CAs by inorganic and complex anions and other molecules interacting with zinc proteins, such as sulfamide, sulfamic acid, and phenylboronic/arsonic acids. The enzyme bsCA1 was inhibited in the low micromolar range by sulfamide, sulfamic acid, phenylboronic/arsonic acid, and in the submillimolar range by diethyldithiocarbamate. Isoform bsCA2 generally showed a stronger inhibition with most of these anions, with several low micromolar and many submillimolar inhibitors detected. Micromolar inhibition against bsCA2 was observed for sulfamide and sulfamic acid, whereas diethyldithiocarbamate, perruthenate, pyrovanadate, tellurate and phenylarsonic acid showed inhibition constants in the range of 0.29-1.52mM. These inhibitors may be used as leads for developing anti-Brucella agents with a diverse mechanism of action compared to clinically used antibiotics. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. New amide derivatives of Probenecid as selective inhibitors of carbonic anhydrase IX and XII: biological evaluation and molecular modelling studies.

    Science.gov (United States)

    Carradori, Simone; Mollica, Adriano; Ceruso, Mariangela; D'Ascenzio, Melissa; De Monte, Celeste; Chimenti, Paola; Sabia, Rocchina; Akdemir, Atilla; Supuran, Claudiu T

    2015-07-01

    Novel amide derivatives of Probenecid were synthesized and discovered to act as potent and selective inhibitors of the human carbonic anhydrase (hCA, EC 4.2.1.1) transmembrane isoforms hCA IX and XII. The proposed chemical transformation of the carboxylic acid into an amide group led to a complete loss of hCA I and II inhibition (Kis >10,000nM) and enhanced the inhibitory activity against hCA IX and XII, with respect to the parent compound (incorporating a COOH function). These promising biological results have been corroborated by molecular modelling studies within the active sites of the four studied human carbonic anhydrases, which enabled us to rationalize both the isoform selectivity and high activity against the tumor-associated isoforms hCA IX/XII. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Ultrastructural changes in the membrane system of isolated chloroplasts of spinach under the influence of carbonic anhydrase inhibitors AA and EA

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    Marina V. Vodka

    2013-04-01

    Full Text Available The effects of carbonic anhydrase inhibitors (АА and EA on the membrane system of isolated chloroplasts of spinach were investigated. Under the influence of AA the considerable alterations in granal structure occurred, the thickness of the granal thylakoids increased by 36% and the interspace between thylakoids by 10% comparable with the control. As a result of EA treatment, the thickness of granal thylakoids enhanced by 31% and the interspace between thylakoids increased by 8% in comparison to the control. It was shown that structure of the granal system of the chloroplast was more sensitive to AA than EA. The data obtained can indicate a decrease in the activity of the thylakoid carbonic anhydrase, inhibition of electron transport and photosynthetic process as a whole in the presence of carbonic anhydrase inhibitors (AA and EA.

  8. Anion inhibition studies of a beta carbonic anhydrase from the malaria mosquito Anopheles gambiae.

    Science.gov (United States)

    Vullo, Daniela; Syrjänen, Leo; Kuuslahti, Marianne; Parkkila, Seppo; Supuran, Claudiu T

    2018-12-01

    An anion inhibition study of the β-class carbonic anhydrase, AgaCA, from the malaria mosquito Anopheles gambiae is reported. A series of simple as well as complex inorganic anions, and small molecules known to interact with CAs were included in the study. Bromide, iodide, bisulphite, perchlorate, perrhenate, perruthenate, and peroxydisulphate were ineffective AgaCA inhibitors, with K I s > 200 mM. Fluoride, chloride, cyanate, thiocyanate, cyanide, bicarbonate, carbonate, nitrite, nitrate, sulphate, stannate, selenate, tellurate, diphosphate, divanadate, tetraborate, selenocyanide, and trithiocarbonate showed K I s in the range of 1.80-9.46 mM, whereas N,N-diethyldithiocarbamate was a submillimolar AgaCA inhibitor (K I of 0.65 mM). The most effective AgaCA inhibitors were sulphamide, sulphamic acid, phenylboronic acid and phenylarsonic acid, with inhibition constants in the range of 21-84 µM. The control of insect vectors responsible of the transmission of many protozoan diseases is rather difficult nowadays, and finding agents which can interfere with these processes, as the enzyme inhibitors investigated here, may arrest the spread of these diseases worldwide.

  9. Variable involvement of the perivascular retinal tissue in carbonic anhydrase inhibitor induced relaxation of porcine retinal arterioles in vitro

    DEFF Research Database (Denmark)

    Kehler, Anne Katrine; Holmgaard, Kim; Hessellund, Anders

    2007-01-01

    PURPOSE: Inhibition of carbonic anhydrase in the eye is an important treatment modality for reducing the intraocular pressure in glaucoma. However, evidence suggests that carbonic anhydrase inhibition also exerts a relaxing effect on the vessels in the optic nerve, and it has been suggested...

  10. Development of sulfonamides incorporating phenylacrylamido functionalities as carbonic anhydrase isoforms I, II, IX and XII inhibitors.

    Science.gov (United States)

    Angapelly, Srinivas; Ramya, P V Sri; Angeli, Andrea; Del Prete, Sonia; Capasso, Clemente; Arifuddin, Mohammed; Supuran, Claudiu T

    2017-10-15

    A series of novel sulfonamides incorporating phenylacrylamido functionalities were synthesized and investigated for the inhibition of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The physiologically and pharmacologically relevant human (h) isoforms hCA I and II (cytosolic isozymes), as well as the transmembrane tumor-associated hCA IX and XII were included in the study. These compounds showed low nanomolar or sub-nanomolar inhibition constants against hCA II (K I s in the range of 0.50-50.5nM), hCA IX (K I s of 1.8-228.5nM), and hCA XII (K I s of 3.5-96.2nM) being less effective as inhibitors of the off target isoform hCA I. A detailed structure-activity relationship study demonstrates that the nature and position of substituents present on the aromatic part of the scaffold strongly influence the inhibition of CA isoforms. As hCA II, IX and XII are involved in pathologies such as glaucoma and hypoxic, and metastatic tumors, compounds of the type reported in this work may be useful preclinical candidates. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Coumarin or benzoxazinone based novel carbonic anhydrase inhibitors: synthesis, molecular docking and anticonvulsant studies.

    Science.gov (United States)

    Karataş, Mert Olgun; Uslu, Harun; Sarı, Suat; Alagöz, Mehmet Abdullah; Karakurt, Arzu; Alıcı, Bülent; Bilen, Cigdem; Yavuz, Emre; Gencer, Nahit; Arslan, Oktay

    2016-10-01

    Among many others, coumarin derivatives are known to show human carbonic anhydrase (hCA) inhibitory activity. Since hCA inhibition is one of the underlying mechanisms that account for the activities of some antiepileptic drugs (AEDs), hCA inhibitors are expected to have anti-seizure properties. There are also several studies reporting compounds with an imidazole and/or benzimidazole moiety which exert these pharmacological properties. In this study, we prepared fifteen novel coumarin-bearing imidazolium and benzimidazolium chloride, nine novel benzoxazinone-bearing imidazolium and benzimidazolium chloride derivatives and evaluated their hCA inhibitory activities and along with fourteen previously synthesized derivatives we scanned their anticonvulsant effects. As all compounds inhibited purified hCA isoforms I and II, some of them also proved protective against Maximal electroshock seizure (MES) and ScMet induced seizures in mice. Molecular docking studies with selected coumarin derivatives have revealed that these compounds bind to the active pocket of the enzyme in a similar fashion to that previously described for coumarin derivatives.

  12. Captopril/enalapril inhibit promiscuous esterase activity of carbonic anhydrase at micromolar concentrations: An in vitro study.

    Science.gov (United States)

    Esmaeili, Sajjad; Ashrafi-Kooshk, Mohammad Reza; Adibi, Hadi; Khodarahmi, Reza

    2017-03-01

    The inhibitory activity of captopril, a thiol-containing competitive inhibitor of the angiotensin-converting enzyme, ACE, against esterase activity of carbonic anhydrase, CA was investigated. This small molecule, as well as enalapril, was selected in order to represents both thiol and carboxylate, as two well-known metal binding functional groups of metalloprotein inhibitors. Since captopril, has also been observed to inhibit other metalloenzymes such as tyrosinase and metallo-beta lactamase through binding to the catalytic metal ions and regarding CA as a zinc-containing metallo-enzyme, in the current study, we set out to determine whether captopril/enalapril inhibit CA esterase activity of the purified human CA II or not? Then, we revealed the inhibitors' potencies (IC 50 , K i and K diss values) and also mode of inhibition. Our results also showed that enalapril is more potent CA inhibitor than captopril. Since enalapril represents no sulfhydryl moiety, thus carboxylate group may have a determinant role in inhibiting of CA esterase activity, the conclusion confirmed by molecular docking studies. Additionally, since CA inhibitory potencies of captopril/enalapril were much lower than those of classic sulfonamide drugs, the findings of the current study may explain why these drugs exhibit no effective CA inhibition at the concentrations reached in vivo and also may shed light on the way of generating new class of inhibitors that will discriminately inhibit various CA isoforms. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Dithiocarbamates effectively inhibit the β-carbonic anhydrase from the dandruff-producing fungus Malassezia globosa.

    Science.gov (United States)

    Vullo, Daniela; Del Prete, Sonia; Nocentini, Alessio; Osman, Sameh M; AlOthman, Zeid; Capasso, Clemente; Bozdag, Murat; Carta, Fabrizio; Gratteri, Paola; Supuran, Claudiu T

    2017-02-01

    A series of dithiocarbamates (DTCs) was investigated for the inhibition of the β-class carbonic anhydrase (CAs, EC 4.2.1.1) from the fungal parasite Malassezia globosa, MgCA, a validated anti-dandruff drug target. These DTCs incorporate various scaffold, among which those of N,N-dimethylaminoethylenediamine, the aminoalcohols with 3-5 carbon atoms in their molecule, 3-amino-quinuclidine, piperidine, morpholine and piperazine derivatives, as well as phenethylamine and its 4-sulfamoylated derivative. Several DTCs resulted more effective in inhibiting MgCA compared to the standard sulfonamide drug acetazolamide (K I of 74μM), with K I s ranging between 383 and 6235nM. A computational approach, involving a homology modeling of the enzyme and docking inhibitors within its active site, helped us rationalize the results. This study may contribute to better understand the inhibition profile of MgCA, and offer new ideas for the design of modulators of activity which belong to less investigated chemical classes, thus potentially useful to combat dandruff and other fungal infections. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Carbonic anhydrase inhibition increases retinal oxygen tension and dilates retinal vessels

    DEFF Research Database (Denmark)

    Pedersen, Daniella Bach; Koch Jensen, Peter; la Cour, Morten

    2005-01-01

    Carbonic anhydrase inhibitors (CAIs) increase blood flow in the brain and probably also in the optic nerve and retina. Additionally they elevate the oxygen tension in the optic nerve in the pig. We propose that they also raise the oxygen tension in the retina. We studied the oxygen tension in the...

  15. Carbonic Anhydrase Inhibitors Induce Developmental Toxicity During Zebrafish Embryogenesis, Especially in the Inner Ear.

    Science.gov (United States)

    Matsumoto, Hiroko; Fujiwara, Shoko; Miyagi, Hisako; Nakamura, Nobuhiro; Shiga, Yasuhiro; Ohta, Toshihiro; Tsuzuki, Mikio

    2017-10-01

    In vertebrates, carbonic anhydrases (CAs) play important roles in ion transport and pH regulation in many organs, including the eyes, kidneys, central nervous system, and inner ear. In aquatic organisms, the enzyme is inhibited by various chemicals present in the environment, such as heavy metals, pesticides, and pharmaceuticals. In this study, the effects of CA inhibitors, i.e., sulfonamides [ethoxyzolamide (EZA), acetazolamide (AZA), and dorzolamide (DZA)], on zebrafish embryogenesis were investigated. In embryos treated with the sulfonamides, abnormal development, such as smaller otoliths, an enlarged heart, an irregular pectoral fin, and aberrant swimming behavior, was observed. Especially, the development of otoliths and locomotor activity was severely affected by all the sulfonamides, and EZA was a consistently stronger inhibitor than AZA or DZA. In the embryos treated with EZA, inner ear hair cells containing several CA isoforms, which provide HCO 3 - to the endolymph for otolith calcification and maintain an appropriate pH there, were affected. Acridine orange/ethidium bromide staining indicated that the hair cell damage in the inner ear and pectral fin is due to apoptosis. Moreover, RNA measurement demonstrated that altered gene expression of cell cycle arrest- and apoptosis-related proteins p53, p21, p27, and Bcl-2 occurred even at 0.08 ppm with which normal development was observed. This finding suggests that a low concentration of EZA may affect embryogenesis via the apoptosis pathway. Thus, our findings demonstrated the importance of potential risk assessment of CA inhibition, especially regarding the formation of otoliths as a one of the most sensitive organs in embryogenesis.

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Inhibition of the α-carbonic anhydrase from Vibrio cholerae with amides and sulfonamides incorporating imidazole moieties.

    Science.gov (United States)

    De Vita, Daniela; Angeli, Andrea; Pandolfi, Fabiana; Bortolami, Martina; Costi, Roberta; Di Santo, Roberto; Suffredini, Elisabetta; Ceruso, Mariangela; Del Prete, Sonia; Capasso, Clemente; Scipione, Luigi; Supuran, Claudiu T

    2017-12-01

    We discovered novel and selective sulfonamides/amides acting as inhibitors of the α-carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic bacterium Vibrio cholerae (VchCA). This Gram-negative bacterium is the causative agent of cholera and colonises the upper small intestine where sodium bicarbonate is present at a high concentration. The secondary sulfonamides and amides investigated here were potent, low nanomolar VchCA inhibitors whereas their inhibition of the human cytosolic isoforms CA I and II was in the micromolar range or higher. The molecules represent an interesting lead for antibacterial agents with a possibly new mechanism of action, although their CA inhibition mechanism is unknown for the moment.

  18. Inhibition of carbonic anhydrase from Trypanosoma cruzi for the management of Chagas disease: an underexplored therapeutic opportunity.

    Science.gov (United States)

    Supuran, Claudiu T

    2016-01-01

    An α-carbonic anhydrases (CAs, EC 4.2.1.1) was recently discovered, cloned and characterized in the genome of the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease, a neglected but widespread tropical disease. Inhibition of this α-CAs (TcCA) with anions, sulfonamides, sulfamates, thiols and hydroxamates has been investigated in detail, with several low nanomolar in vitro inhibitors. Although the sulfonamides were the best in vitro inhibitors, they showed no ex vivo anti-T. cruzi activity, due to poor penetration. However, some thiols and hydroxamates acting as low nanomolar TcCA inhibitors also showed significant antitrypanosomal ex vivo activity, making this enzyme an attractive yet underexplored drug target for the management of Chagas disease.

  19. Strong topical steroid, NSAID, and carbonic anhydrase inhibitor cocktail for treatment of cystoid macular edema

    Directory of Open Access Journals (Sweden)

    Asahi MG

    2015-12-01

    Full Text Available Masumi G Asahi, Gabriela L Bobarnac Dogaru, Spencer M Onishi, Ron P GallemoreRetina Macula Institute, Torrance, CA, USA Purpose: To report the combination cocktail of strong steroid, non-steroidal anti-inflammatory drug (NSAID, and carbonic anhydrase inhibitor drops for treatment of cystoid macular edema. Methods: This is a retrospective case series of patients with cystoid macular edema managed with a topical combination of strong steroid (difluprednate, NSAID, and carbonic anhydrase inhibitor drops. The patients were followed with optical coherence tomography and fluorescein angiography. Results: In our six cases, resolution of the cystic edema with improvement in visual acuity was achieved with the use of a combination cocktail of drops. Leakage on fluorescein angiography and cystic edema on optical coherence tomography both responded to treatment with the topical cocktail of drops. Conclusion: A topical cocktail of strong steroid, NSAID, and carbonic anhydrase inhibitor drops are effective for managing cystoid macular edema. Further studies comparing this combination with more invasive treatments should be undertaken to determine the efficacy of this cocktail over other treatment options. Keywords: birdshot chorioretinopathy, diabetic macular edema, retinal vein occlusion

  20. Investigations into the carbonic anhydrase inhibition of COS-releasing donor core motifs.

    Science.gov (United States)

    Steiger, Andrea K; Zhao, Yu; Choi, Won Jin; Crammond, Alder; Tillotson, McKinna R; Pluth, Michael D

    2018-03-01

    Carbonyl sulfide (COS) releasing scaffolds are gaining popularity as hydrogen sulfide (H 2 S) donors through exploitation of the carbonic anhydrase (CA)-mediated hydrolysis of COS to H 2 S. The majority of compounds in this emerging class of donors undergo triggerable decomposition (often referred to as self-immolation) to release COS, and a handful of different COS-releasing structures have been reported. One benefit of this donation strategy is that numerous caged COS-containing core motifs are possible and are poised for development into self-immolative COS/H 2 S donors. Because the intermediate release of COS en route to H 2 S donation requires CA, it is important that the COS donor motifs do not inhibit CA directly. In this work, we investigate the cytotoxicity and CA inhibition properties of different caged COS donor cores, as well as caged CO 2 and CS 2 motifs and non-self-immolative control compounds. None of the compounds investigated exhibited significant cytotoxicity or enhanced cell proliferation at concentrations up to 100 μM in A549 cells, but we identified four core structures that function as CA inhibitors, thus providing a roadmap for the future development of self-immolative COS/H 2 S donor motifs. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Carbonic anhydrase inhibitor attenuates ischemia-reperfusion induced acute lung injury.

    Directory of Open Access Journals (Sweden)

    Chou-Chin Lan

    Full Text Available Ischemia-reperfusion (IR-induced acute lung injury (ALI is implicated in several clinical conditions including lung transplantation, cardiopulmonary bypass surgery, re-expansion of collapsed lung from pneumothorax or pleural effusion and etc. IR-induced ALI remains a challenge in the current treatment. Carbonic anhydrase has important physiological function and influences on transport of CO2. Some investigators suggest that CO2 influences lung injury. Therefore, carbonic anhydrase should have the role in ALI. This study was undertaken to define the effect of a carbonic anhydrase inhibitor, acetazolamide (AZA, in IR-induced ALI, that was conducted in a rat model of isolated-perfused lung with 30 minutes of ischemia and 90 minutes of reperfusion. The animals were divided into six groups (n = 6 per group: sham, sham + AZA 200 mg/kg body weight (BW, IR, IR + AZA 100 mg/kg BW, IR + AZA 200 mg/kg BW and IR+ AZA 400 mg/kg BW. IR caused significant pulmonary micro-vascular hyper-permeability, pulmonary edema, pulmonary hypertension, neutrophilic sequestration, and an increase in the expression of pro-inflammatory cytokines. Increases in carbonic anhydrase expression and perfusate pCO2 levels were noted, while decreased Na-K-ATPase expression was noted after IR. Administration of 200mg/kg BW and 400mg/kg BW AZA significantly suppressed the expression of pro-inflammatory cytokines (TNF-α, IL-1, IL-6 and IL-17 and attenuated IR-induced lung injury, represented by decreases in pulmonary hyper-permeability, pulmonary edema, pulmonary hypertension and neutrophilic sequestration. AZA attenuated IR-induced lung injury, associated with decreases in carbonic anhydrase expression and pCO2 levels, as well as restoration of Na-K-ATPase expression.

  2. Effects of the carbonic anhydrase inhibitor acetazolamide on splanchnic blood flow in anaesthetized rats.

    Science.gov (United States)

    Carlsson, P O; Lindberg, M; Jansson, L

    1998-12-01

    The aim of the present study was to evaluate whether inhibition of the enzyme carbonic anhydrase with acetazolamide interfered with pancreatic islet and whole splanchnic blood perfusion in rats. Carbonic anhydrase is present both in the endocrine cells and, in particular, the endothelium of the pancreatic islet. Thiobutabarbital-anaesthetized, male Sprague-Dawley rats were used in all experiments, and acetazolamide (50 mg/kg body weight) was given to untreated control rats or rats pretreated with glucose, i.e. to normoglycaemic and hyperglycaemic animals. No acetazolamide-induced effects on blood glucose or serum insulin concentrations, mean arterial blood pressure, whole pancreatic or islet blood flow were seen in any of the animals. There were no effects on duodenal or colonic blood flow recorded in the control rats, whereas an increase in duodenal blood flow (P<0.02) was observed in the hyperglycaemic animals. A tendency to an increase was seen in colonic blood flow in hyperglycaemic animals, although this was not statistically significant (P = 0.069). Inhibition of carbonic anhydrase seems to induce only minor effects on pancreatic blood flow, while duodenal blood flow is slightly enhanced in hyperglycaemic animals.

  3. Selective inhibition of human carbonic anhydrases by novel amide derivatives of probenecid: synthesis, biological evaluation and molecular modelling studies.

    Science.gov (United States)

    D'Ascenzio, Melissa; Carradori, Simone; Secci, Daniela; Vullo, Daniela; Ceruso, Mariangela; Akdemir, Atilla; Supuran, Claudiu T

    2014-08-01

    Novel amide derivatives of probenecid, a well-known uricosuric agent, were synthesized and evaluated as inhibitors of human carbonic anhydrases (hCAs, EC 4.2.1.1). The transmembrane isoforms (hCA IX and XII) were potently and selectively inhibited by some of them. The proposed chemical modification led to a complete loss of hCA II inhibition (K(i)s>10,000 nM) and enhanced the inhibitory activity against the tumour-associated hCA XII (compound 4 showed a K(i) value of 15.3 nM). The enzyme inhibitory data have also been validated by docking studies of the compounds within the active site of hCA XII. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Determination of activities of human carbonic anhydrase II inhibitors ...

    African Journals Online (AJOL)

    intensive search for novel drugs is ongoing, through synthesis ... previously synthesized using microwave irradiation [10], were evaluated for their potential as inhibitors of human CA-II. The basic structure of all 44 curcumin analogs is depicted in Figure. 1. Figure 1: .... algorithm was used for exploring ligand poses inside the ...

  5. Structural elucidation of the hormonal inhibition mechanism of the bile acid cholate on human carbonic anhydrase II

    International Nuclear Information System (INIS)

    Boone, Christopher D.; Tu, Chingkuang; McKenna, Robert

    2014-01-01

    The structure of human carbonic anhydrase II in complex with cholate has been determined to 1.54 Å resolution. Elucidation of the novel inhibition mechanism of cholate will aid in the development of a nonsulfur-containing, isoform-specific therapeutic agent. The carbonic anhydrases (CAs) are a family of mostly zinc metalloenzymes that catalyze the reversible hydration/dehydration of CO 2 into bicarbonate and a proton. Human isoform CA II (HCA II) is abundant in the surface epithelial cells of the gastric mucosa, where it serves an important role in cytoprotection through bicarbonate secretion. Physiological inhibition of HCA II via the bile acids contributes to mucosal injury in ulcerogenic conditions. This study details the weak biophysical interactions associated with the binding of a primary bile acid, cholate, to HCA II. The X-ray crystallographic structure determined to 1.54 Å resolution revealed that cholate does not make any direct hydrogen-bond interactions with HCA II, but instead reconfigures the well ordered water network within the active site to promote indirect binding to the enzyme. Structural knowledge of the binding interactions of this nonsulfur-containing inhibitor with HCA II could provide the template design for high-affinity, isoform-specific therapeutic agents for a variety of diseases/pathological states, including cancer, glaucoma, epilepsy and osteoporosis

  6. Structural elucidation of the hormonal inhibition mechanism of the bile acid cholate on human carbonic anhydrase II

    Energy Technology Data Exchange (ETDEWEB)

    Boone, Christopher D. [University of Florida, PO Box 100267, Gainesville, FL 32610 (United States); Tu, Chingkuang [University of Florida, PO Box 100245, Gainesville, FL 32610 (United States); McKenna, Robert, E-mail: rmckenna@ufl.edu [University of Florida, PO Box 100267, Gainesville, FL 32610 (United States)

    2014-06-01

    The structure of human carbonic anhydrase II in complex with cholate has been determined to 1.54 Å resolution. Elucidation of the novel inhibition mechanism of cholate will aid in the development of a nonsulfur-containing, isoform-specific therapeutic agent. The carbonic anhydrases (CAs) are a family of mostly zinc metalloenzymes that catalyze the reversible hydration/dehydration of CO{sub 2} into bicarbonate and a proton. Human isoform CA II (HCA II) is abundant in the surface epithelial cells of the gastric mucosa, where it serves an important role in cytoprotection through bicarbonate secretion. Physiological inhibition of HCA II via the bile acids contributes to mucosal injury in ulcerogenic conditions. This study details the weak biophysical interactions associated with the binding of a primary bile acid, cholate, to HCA II. The X-ray crystallographic structure determined to 1.54 Å resolution revealed that cholate does not make any direct hydrogen-bond interactions with HCA II, but instead reconfigures the well ordered water network within the active site to promote indirect binding to the enzyme. Structural knowledge of the binding interactions of this nonsulfur-containing inhibitor with HCA II could provide the template design for high-affinity, isoform-specific therapeutic agents for a variety of diseases/pathological states, including cancer, glaucoma, epilepsy and osteoporosis.

  7. A novel library of saccharin and acesulfame derivatives as potent and selective inhibitors of carbonic anhydrase IX and XII isoforms.

    Science.gov (United States)

    Carradori, Simone; Secci, Daniela; De Monte, Celeste; Mollica, Adriano; Ceruso, Mariangela; Akdemir, Atilla; Sobolev, Anatoly P; Codispoti, Rossella; De Cosmi, Federica; Guglielmi, Paolo; Supuran, Claudiu T

    2016-03-01

    Small libraries of N-substituted saccharin and N-/O-substituted acesulfame derivatives were synthesized and tested as atypical and selective inhibitors of four different isoforms of human carbonic anhydrase (hCA I, II, IX and XII, EC 4.2.1.1). Most of them inhibited hCA XII in the low nanomolar range, hCA IX with KIs ranging between 19 and 2482nM, whereas they were poorly active against hCA II (KIs >10μM) and hCA I (KIs ranging between 318nM and 50μM). Since hCA I and II are ubiquitous off-target isoforms, whereas the cancer-related isoforms hCA IX and XII were recently validated as drug targets, these results represent an encouraging achievement in the development of new anticancer candidates. Moreover, the lack of a classical zinc binding group in the structure of these inhibitors opens innovative, yet unexplored scenarios for different mechanisms of inhibition that could explain the high inhibitory selectivity. A computational approach has been carried out to further rationalize the biological data and to characterize the binding mode of some of these inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Indomethacin lowers optic nerve oxygen tension and reduces the effect of carbonic anhydrase inhibition and carbon dioxide breathing

    OpenAIRE

    Pedersen, D B; Eysteinsson, T; Stefánsson, E; Kiilgaard, J F; la Cour, M; Bang, K; Jensen, P K

    2004-01-01

    Background/aims: Prostaglandins are important in blood flow regulation. Carbon dioxide (CO2) breathing and carbonic anhydrase inhibition increase the oxygen tension in the retina and optic nerve. To study the mechanism of this effect and the role of cyclo-oxygenase in the regulation of optic nerve oxygen tension (ONPO2), the authors investigated how indomethacin affects ONPO2 and the ONPO2 increases caused by CO2 breathing and carbonic anhydrase inhibition in the pig.

  9. Indomethacin lowers optic nerve oxygen tension and reduces the effect of carbonic anhydrase inhibition and carbon dioxide breathing

    DEFF Research Database (Denmark)

    Pedersen, D B; Eysteinsson, T; Stefánsson, E

    2004-01-01

    Prostaglandins are important in blood flow regulation. Carbon dioxide (CO(2)) breathing and carbonic anhydrase inhibition increase the oxygen tension in the retina and optic nerve. To study the mechanism of this effect and the role of cyclo-oxygenase in the regulation of optic nerve oxygen tension...... (ONPO(2)), the authors investigated how indomethacin affects ONPO(2) and the ONPO(2) increases caused by CO(2) breathing and carbonic anhydrase inhibition in the pig....

  10. Pharmacotherapy of intraocular pressure - part II. Carbonic anhydrase inhibitors, prostaglandin analogues and prostamides.

    Science.gov (United States)

    Costagliola, Ciro; dell'Omo, Roberto; Romano, Mario R; Rinaldi, Michele; Zeppa, Lucia; Parmeggiani, Francesco

    2009-12-01

    The second part of this two part review (please see Expert Opinion on Pharmacotherapy 10(16)) reports the characteristics of other antiglaucoma medications: systemic (acetazomide) and topical (dorzolamide and brinzolamide) carbonic anhydrase inhibitors, which suppress aqueous humour formation; and prostaglandin analogues (latanoprost and travoprost) and prostamides (bimatoprost), which raise aqueous humour outflow. The pharmacologic properties of each compound and its efficacy in the medical treatment of glaucoma, mainly the primary open-angle form, are discussed briefly, focusing on the clinical evidence supporting their use.

  11. Saccharin: a lead compound for structure-based drug design of carbonic anhydrase IX inhibitors.

    Science.gov (United States)

    Mahon, Brian P; Hendon, Alex M; Driscoll, Jenna M; Rankin, Gregory M; Poulsen, Sally-Ann; Supuran, Claudiu T; McKenna, Robert

    2015-02-15

    Carbonic anhydrase IX (CA IX) is a key modulator of aggressive tumor behavior and a prognostic marker and target for several cancers. Saccharin (SAC) based compounds may provide an avenue to overcome CA isoform specificity, as they display both nanomolar affinity and preferential binding, for CA IX compared to CA II (>50-fold for SAC and >1000-fold when SAC is conjugated to a carbohydrate moiety). The X-ray crystal structures of SAC and a SAC-carbohydrate conjugate bound to a CA IX-mimic are presented and compared to CA II. The structures provide substantial new insight into the mechanism of SAC selective CA isoform inhibition. Published by Elsevier Ltd.

  12. Carbonic anhydrase inhibitors: Design, synthesis and structural characterization of new heteroaryl-N-carbonylbenzenesulfonamides targeting druggable human carbonic anhydrase isoforms

    Czech Academy of Sciences Publication Activity Database

    Buemi, M. R.; De Luca, L.; Ferro, S.; Bruno, E.; Ceruso, M.; Supuran, C. T.; Pospíšilová, K.; Brynda, Jiří; Řezáčová, Pavlína; Gitto, R.

    2015-01-01

    Roč. 102, Sep 18 (2015), s. 223-232 ISSN 0223-5234 Institutional support: RVO:61388963 Keywords : human carbonic anhydrase * isoquinoline * quinoline * X-ray * molecular docking Subject RIV: CE - Biochemistry Impact factor: 3.902, year: 2015

  13. Sulfonamide inhibition studies of the β-carbonic anhydrase from the newly discovered bacterium Enterobacter sp. B13.

    Science.gov (United States)

    Eminoğlu, Ayşenur; Vullo, Daniela; Aşık, Aycan; Çolak, Dilşat Nigar; Çanakçı, Sabriye; Beldüz, Ali Osman; Supuran, Claudiu T

    2016-04-01

    The genome of the newly identified bacterium Enterobacter sp. B13 encodes for a β-class carbonic anhydrases (CAs, EC 4.2.1.1), EspCA. This enzyme was recently cloned, and characterized kinetically by this group (J. Enzyme Inhib. Med. Chem. 2016, 31). Here we report an inhibition study with sulfonamides and sulfamates of this enzyme. The best EspCA inhibitors were some sulfanylated sulfonamides with elongated molecules, metanilamide, 4-aminoalkyl-benzenesulfonamides, acetazolamide, and deacetylated methazolamide (KIs in the range of 58.7-96.5nM). Clinically used agents such as methazolamide, ethoxzolamide, dorzolamide, brinzolamide, benzolamide, zonisamide, sulthiame, sulpiride, topiramate and valdecoxib were slightly less effective inhibitors (KIs in the range of 103-138nM). Saccharin, celecoxib, dichlorophenamide and many simple benzenesulfonamides were even less effective as EspCA inhibitors, with KIs in the range of 384-938nM. Identification of effective inhibitors of this bacterial enzyme may lead to pharmacological tools useful for understanding the physiological role(s) of the β-class CAs in bacterial pathogenicity/virulence. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Cloning, characterization and anion inhibition study of a β-class carbonic anhydrase from the caries producing pathogen Streptococcus mutans.

    Science.gov (United States)

    Dedeoglu, Nurcan; De Luca, Viviana; Isik, Semra; Yildirim, Hatice; Kockar, Feray; Capasso, Clemente; Supuran, Claudiu T

    2015-07-01

    The oral pathogenic bacterium involved in human dental caries formation Streptococcus mutans, encodes for two carbonic anhydrase (CA, EC 4.2.1.1) one belonging to the α- and the other one to the β-class. This last enzyme (SmuCA) has been cloned, characterized and investigated for its inhibition profile with a major class of CA inhibitors, the inorganic anions. Here we show that SmuCA has a good catalytic activity for the CO2 hydration reaction, with kcat 4.2×10(5)s(-1) and kcat/Km of 5.8×10(7)M(-1)×s(-1), being inhibited by cyanate, carbonate, stannate, divannadate and diethyldithiocarbamate in the submillimolar range (KIs of 0.30-0.64mM) and more efficiently by sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid (KIs of 15-46μM). The anion inhibition profile of the S. mutans enzyme is very different from other α- and β-CAs investigated earlier. Identification of effective inhibitors of this new enzyme may lead to pharmacological tools useful for understanding the role of S. mutans CAs in dental caries formation, and eventually the development of pharmacological agents with a new mechanism of antibacterial action. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Carbonic anhydrase inhibitors: Design, synthesis and structural characterization of new heteroaryl-N-carbonylbenzenesulfonamides targeting druggable human carbonic anhydrase isoforms

    Czech Academy of Sciences Publication Activity Database

    Buemi, M. R.; De Luca, L.; Ferro, S.; Bruno, E.; Ceruso, M.; Supuran, C. T.; Pospíšilová, K.; Brynda, Jiří; Řezáčová, Pavlína; Gitto, R.

    2015-01-01

    Roč. 102, SEP 18 (2015), s. 223-232 ISSN 0223-5234 R&D Projects: GA ČR GA15-05677S Grant - others:Fondo di Ateneo per la Ricerca (PRA)(IT) ORME09SPNC Institutional support: RVO:68378050 Keywords : Human carbonic anhydrase * Isoquinoline * Quinoline * X-ray * Molecular docking Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.902, year: 2015

  16. Synthesis and discovery of potent carbonic anhydrase, acetylcholinesterase, butyrylcholinesterase, and α-glycosidase enzymes inhibitors: The novel N,N'-bis-cyanomethylamine and alkoxymethylamine derivatives.

    Science.gov (United States)

    Taslimi, Parham; Caglayan, Cuneyt; Farzaliyev, Vagif; Nabiyev, Oruj; Sujayev, Afsun; Turkan, Fikret; Kaya, Ruya; Gulçin, İlhami

    2018-04-01

    During this investigation, N,N'-bis-azidomethylamines, N,N'-bis-cyanomethylamine, new alkoxymethylamine and chiral derivatives, which are considered to be a new generation of multifunctional compounds, were synthesized, functional properties were investigated, and anticholinergic and antidiabetic properties of those compounds were studied through the laboratory tests, and it was approved that they contain physiologically active compounds rather than analogues. Novel N-bis-cyanomethylamine and alkoxymethylamine derivatives were effective inhibitors of the α-glycosidase, cytosolic carbonic anhydrase I and II isoforms, butyrylcholinesterase (BChE), and acetylcholinesterase (AChE) with K i values in the range of 0.15-13.31 nM for α-glycosidase, 2.77-15.30 nM for human carbonic anhydrase isoenzymes I (hCA I), 3.12-21.90 nM for human carbonic anhydrase isoenzymes II (hCA II), 23.33-73.23 nM for AChE, and 3.84-48.41 nM for BChE, respectively. Indeed, the inhibition of these metabolic enzymes has been considered as a promising factor for pharmacologic intervention in a diversity of disturbances. © 2018 Wiley Periodicals, Inc.

  17. Update and critical appraisal of combined timolol and carbonic anhydrase inhibitors and the effect on ocular blood flow in glaucoma patients.

    Science.gov (United States)

    Moss, Adam M; Harris, Alon; Siesky, Brent; Rusia, Deepam; Williamson, Kathleen M; Shoshani, Yochai

    2010-04-26

    Topical hypotensive therapy with both timolol and carbonic anhydrase inhibitors has been shown to be efficacious at reducing intraocular pressure. Many prospective studies have also suggested that carbonic anhydrase inhibitors augment ocular blood flow and vascular regulation independent of their hypotensive effects. Although consistent in their findings, these studies must be cautiously interpreted due to the limitations of study design and specific blood flow imaging modalities. The purpose of this review is to appraise and critically evaluate the current body of literature investigating the effects of combined treatment with topical carbonic anhydrase inhibitors and timolol in patients with glaucoma with respect to ocular blood flow, visual function, and optic nerve head structure.

  18. Comparison of the Sulfonamide Inhibition Profiles of the β- and γ-Carbonic Anhydrases from the Pathogenic Bacterium Burkholderia pseudomallei

    Directory of Open Access Journals (Sweden)

    Daniela Vullo

    2017-03-01

    Full Text Available We have cloned, purified, and characterized a β-carbonic anhydrase (CA, EC 4.2.1.1, BpsCAβ, from the pathogenic bacterium Burkholderia pseudomallei, responsible for the tropical disease melioidosis. The enzyme showed high catalytic activity for the physiologic CO2 hydration reaction to bicarbonate and protons, with the following kinetic parameters: kcat of 1.6 × 105 s−1 and kcat/KM of 3.4 × 107 M−1 s−1. An inhibition study with a panel of 38 sulfonamides and one sulfamate—including 15 compounds that are used clinically—revealed an interesting structure–activity relationship for the interaction of this enzyme with these inhibitors. Many simple sulfonamides and clinically used agents such as topiramate, sulpiride, celecoxib, valdecoxib, and sulthiame were ineffective BpsCAβ inhibitors (KI > 50 µM. Other drugs, such as ethoxzolamide, dorzolamide, brinzolamide, zonisamide, indisulam, and hydrochlorothiazide were moderately potent micromolar inhibitors. The best inhibition was observed with benzene-1,3-disulfonamides—benzolamide and its analogs acetazolamide and methazolamide—which showed KI in the range of 185–745 nM. The inhibition profile of BpsCAβ is very different from that of the γ-class enzyme from the same pathogen, BpsCAγ. Thus, identifying compounds that would effectively interact with both enzymes is relatively challenging. However, benzolamide was one of the best inhibitors of both of these CAs with KI of 653 and 185 nM, respectively, making it an interesting lead compound for the design of more effective agents, which may be useful tools for understanding the pathogenicity of this bacterium.

  19. A new class of quinazoline-sulfonamides acting as efficient inhibitors against the α-carbonic anhydrase from Trypanosoma cruzi.

    Science.gov (United States)

    Alafeefy, Ahmed M; Ceruso, Mariangela; Al-Jaber, Nabila A; Parkkila, Seppo; Vermelho, Alane Beatriz; Supuran, Claudiu T

    2015-01-01

    The protozoan parasite Trypanosoma cruzi is the agent responsible for trypanosomiasis (Chagas disease) in humans and other animals. It has been recently reported that this pathogen encodes for an α-class carbonic anhydrase (CA, EC 4.2.1.1), denominated TcCA, which was shown to be crucial for its life cycle. Inhibition studies of a class of 4-oxoquinazoline containing a benzensulfonamide moiety and their 4-thioxo bioisosteres against the protozoan enzyme TcCA are described here. Most of 4-oxoquinazoline sulfonamides showed nanomolar TcCA inhibition activity with K(I)s in the same order of magnitude of acetazolamide (AAZ), whereas their thioxo bioisosters showed moderate anti-Trypanosoma CA potency with K(I)s in the micromolar range. The discovery of compounds incorporating a 4-oxoquinazoline ring as a low-nanomolar TcCA inhibitor is quite promising and it may be useful for developing anti-Trypanosoma agents with a novel mechanism of action compared to the clinically used drugs (such as benznidazole, nifurtimox) for which significant resistance and serious adverse effects due to their high-toxicity appeared.

  20. Saccharin Sulfonamides as Inhibitors of Carbonic Anhydrases I, II, VII, XII, and XIII

    Directory of Open Access Journals (Sweden)

    Vaida Morkūnaitė

    2014-01-01

    Full Text Available A series of modified saccharin sulfonamides have been designed as carbonic anhydrase (CA inhibitors and synthesized. Their binding to CA isoforms I, II, VII, XII, and XIII was measured by the fluorescent thermal shift assay (FTSA and isothermal titration calorimetry (ITC. Saccharin bound the CAs weakly, exhibiting the affinities of 1–10 mM for four CAs except CA I where binding could not be detected. Several sulfonamide-bearing saccharines exhibited strong affinities of 1–10 nM towards particular CA isoforms. The functional group binding Gibbs free energy additivity maps are presented which may provide insights into the design of compounds with increased affinity towards selected CAs.

  1. Crystal structure correlations with the intrinsic thermodynamics of human carbonic anhydrase inhibitor binding

    Science.gov (United States)

    Smirnov, Alexey; Zubrienė, Asta; Manakova, Elena; Gražulis, Saulius

    2018-01-01

    The structure-thermodynamics correlation analysis was performed for a series of fluorine- and chlorine-substituted benzenesulfonamide inhibitors binding to several human carbonic anhydrase (CA) isoforms. The total of 24 crystal structures of 16 inhibitors bound to isoforms CA I, CA II, CA XII, and CA XIII provided the structural information of selective recognition between a compound and CA isoform. The binding thermodynamics of all structures was determined by the analysis of binding-linked protonation events, yielding the intrinsic parameters, i.e., the enthalpy, entropy, and Gibbs energy of binding. Inhibitor binding was compared within structurally similar pairs that differ by para- or meta-substituents enabling to obtain the contributing energies of ligand fragments. The pairs were divided into two groups. First, similar binders—the pairs that keep the same orientation of the benzene ring exhibited classical hydrophobic effect, a less exothermic enthalpy and a more favorable entropy upon addition of the hydrophobic fragments. Second, dissimilar binders—the pairs of binders that demonstrated altered positions of the benzene rings exhibited the non-classical hydrophobic effect, a more favorable enthalpy and variable entropy contribution. A deeper understanding of the energies contributing to the protein-ligand recognition should lead toward the eventual goal of rational drug design where chemical structures of ligands could be designed based on the target protein structure. PMID:29503769

  2. Metallocene-based inhibitors of cancer-associated carbonic anhydrase enzymes IX and XII.

    Science.gov (United States)

    Salmon, Adam J; Williams, Michael L; Wu, Quoc K; Morizzi, Julia; Gregg, Daniel; Charman, Susan A; Vullo, Daniela; Supuran, Claudiu T; Poulsen, Sally-Ann

    2012-06-14

    In this study, 20 metallocene-based compounds comprising extensive structural diversity were synthesized and evaluated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. These compounds proved moderate to good CA inhibitors in vitro, with several compounds displaying selectivity for cancer-associated isozymes CA IX and CA XII compared to off-target CA I and CA II. Compound 6 was the most potent ferrocene-based inhibitor with K(i)s of 5.9 and 6.8 nM at CA IX and XII, respectively. A selection of key drug-like parameters comprising Log P, Log D, solubility, and in vitro metabolic stability and permeability were measured for two of the ferrocene-based compounds, regioisomers 1 and 5. Compounds 1 and 5 were found to have characteristics consistent with lipophilic compounds, however, our findings show that the lipophilicity of the ferrocene moiety is not well modeled by replacement with either a naphthyl or a phenyl moiety in software prediction tools.

  3. Potentiation of the effect of thiazide derivatives by carbonic anhydrase inhibitors: molecular mechanisms and potential clinical implications.

    Directory of Open Access Journals (Sweden)

    Kamyar Zahedi

    Full Text Available Carbonic anhydrase inhibitors (CAI are mild diuretics, hence not widely used in fluid overloaded states. They are however the treatment of choice for certain non-kidney conditions. Thiazides, specific inhibitors of Na-Cl cotransport (NCC, are mild agents and the most widely used diuretics in the world for control of mild hypertension.In addition to inhibiting the salt reabsorption in the proximal tubule, CAIs down-regulate pendrin, therefore leaving NCC as the major salt absorbing transporter in the distal nephron, and hence allowing for massive diuresis by the inhibitors of NCC in the setting of increased delivery of salt from the proximal tubule.Daily treatment of rats with acetazolamide (ACTZ, a known CAI, for 10 days caused mild diuresis whereas daily treatment with hydrochlorothiazide (HCTZ for 4 days caused hardly any diuresis. However, treatment of rats that were pretreated with ACTZ for 6 days with a combination of ACTZ plus HCTZ for 4 additional days increased the urine output by greater than 2 fold (p<0.001, n = 5 compared to ACTZ-treated animals. Sodium excretion increased by 80% in the ACTZ plus HCTZ group and animals developed significant volume depletion, metabolic alkalosis and pre-renal failure. Molecular studies demonstrated ∼75% reduction in pendrin expression by ACTZ. The increased urine output in ACTZ/HCTZ treated rats was associated with a significant reduction in urine osmolality and reduced membrane localization of AQP-2 (aquaporin2.These results indicate that ACTZ down-regulates pendrin expression and leaves NCC as the major salt absorbing transporter in the distal nephron in the setting of increased delivery of salt from the proximal tubule. Despite being considered mild agents individually, we propose that the combination of ACTZ and HCTZ is a powerful diuretic regimen.

  4. Synthesis and carbonic anhydrase I, II, VII, and IX inhibition studies with a series of benzo[d]thiazole-5- and 6-sulfonamides.

    Science.gov (United States)

    Abdoli, Morteza; Angeli, Andrea; Bozdag, Murat; Carta, Fabrizio; Kakanejadifard, Ali; Saeidian, Hamid; Supuran, Claudiu T

    2017-12-01

    A series of benzo[d]thiazole-5- and 6-sulfonamides has been synthesized and investigated for the inhibition of several human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoforms, using ethoxzolamide (EZA) as lead molecule. 2-Amino-substituted, 2-acylamino- and halogenated (bromo-and iodo-derivatives at the heterocyclic ring) compounds led to several interesting inhibitors against the cytosolic hCA I, II and VII, as well as the transmembrane, tumor-associated hCA IX isoforms. Several subnanomolar/low nanomolar, isoform-selective sulfonamide inhibitors targeting hCA II, VII and IX were detected. The sharp structure-activity relationship for CA inhibition with this small series of derivatives, with important changes of activity observed even after minor changes in the scaffold or at the 2-amino moiety, make this class of scarcely investigated sulfonamides of particular interest for further investigations.

  5. Design and Synthesis of Novel Nonsteroidal Anti-Inflammatory Drugs and Carbonic Anhydrase Inhibitors Hybrids (NSAIDs-CAIs) for the Treatment of Rheumatoid Arthritis.

    Science.gov (United States)

    Bua, Silvia; Di Cesare Mannelli, Lorenzo; Vullo, Daniela; Ghelardini, Carla; Bartolucci, Gianluca; Scozzafava, Andrea; Supuran, Claudiu T; Carta, Fabrizio

    2017-02-09

    We report the synthesis of a series of hybrid compounds incorporating 6- and 7-substituted coumarins (carbonic anhydrase, CA inhibitors) derivatized with clinically used NSAIDs (indomethacin, sulindac, ketoprofen, ibuprofen, diclofenac, ketorolac, etc., cyclooxygenase inhibitors) as agents for the management of rheumatoid arthritis (RA). Most compounds were effective in inhibiting the RA overexpressed hCA IX and XII, with K I values in the low nanomolar-subnanomolar ranges. The antihyperalgesic activity of such compounds was assessed by means of the paw-pressure and incapacitance tests using an in vivo RA model. Among all tested compounds, the 7-coumarine hybrid with ibuprofen showed potent and persistent antihyperalgesic effect up to 60 min after administration.

  6. Synthesis, structure-activity relationship studies, and X-ray crystallographic analysis of arylsulfonamides as potent carbonic anhydrase inhibitors

    Czech Academy of Sciences Publication Activity Database

    Gitto, R.; Damiano, F.; Mader, Pavel; De Luca, L.; Ferro, S.; Supuran, C. T.; Vullo, D.; Brynda, Jiří; Řezáčová, Pavlína; Chimirri, A.

    2012-01-01

    Roč. 55, č. 8 (2012), s. 3891-3899 ISSN 0022-2623 R&D Projects: GA ČR GA203/09/0820 Grant - others:MiUR(IT) 20085HR5JK_002; EK(XE) 222741 Institutional research plan: CEZ:AV0Z50520514; CEZ:AV0Z40550506 Keywords : carbonic anhydrase * inhibitors * arylsulfonamides Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.614, year: 2012

  7. Purification and inhibition studies with anions and sulfonamides of an α-carbonic anhydrase from the Antarctic seal Leptonychotes weddellii.

    Science.gov (United States)

    Cincinelli, Alessandra; Martellini, Tania; Innocenti, Alessio; Scozzafava, Andrea; Supuran, Claudiu T

    2011-03-15

    A high activity α-carbonic anhydrase (CA, EC 4.2.1.1) has been purified from various tissues of the Antarctic seal Leptonychotes weddellii. The new enzyme, denominated lwCA, has a catalytic activity for the physiologic CO(2) hydration to bicarbonate reaction, similar to that of the high activity human isoform hCA II, with a k(cat) of 1.1×10(6) s(-1), and a k(cat)/K(m) of 1.4×10(8) M(-1) s(-1). The enzyme was highly inhibited by cyanate, thiocyanate, cyanide, bicarbonate, carbonate, as well as sulfamide, sulfamate, phenylboronic/phenylarsonic acids (K(I)s in the range of 46-100 μM). Many clinically used sulfonamides, such as acetazolamide, methazolamide, dorzolamide, brinzolamide and benzolamide were low nanomolar inhibitors, with K(I)s in the range of 5.7-67 nM. Dichlorophenamide, zonisamide, saccharin and hydrochlorothiazide were weaker inhibitors, with K(I)s in the range of 513-5390 nM. The inhibition profile with anions and sulfonamides of the seal enzyme was rather different from those of the human isoforms hCA I and II. The high sensitivity to bicarbonate inhibition of lwCA, unlike that of the human enzymes, may reflect an evolutionary adaptation to the deep water, high CO(2) partial pressure and hypoxic conditions in which Weddell seals spend much of their life. Copyright © 2011 Elsevier Ltd. All rights reserved.

  8. Indomethacin lowers optic nerve oxygen tension and reduces the effect of carbonic anhydrase inhibition and carbon dioxide breathing

    DEFF Research Database (Denmark)

    Pedersen, D B; Eysteinsson, T; Stefánsson, E

    2004-01-01

    Prostaglandins are important in blood flow regulation. Carbon dioxide (CO(2)) breathing and carbonic anhydrase inhibition increase the oxygen tension in the retina and optic nerve. To study the mechanism of this effect and the role of cyclo-oxygenase in the regulation of optic nerve oxygen tension...

  9. Hypokalemia and suspected renal tubular acidosis associated with topical carbonic anhydrase inhibitor therapy in a cat.

    Science.gov (United States)

    Thiessen, Charlotte E; Tofflemire, Kyle L; Makielski, Kelly M; Ben-Shlomo, Gil; Whitley, R David; Allbaugh, Rachel A

    2016-11-01

    To describe the occurrence of hypokalemia, metabolic acidosis, and suspected renal tubular acidosis associated with the administration of topical ophthalmic carbonic anhydrase inhibitor (CAI) in a cat. A 2-year-old, 5.3 kg, male, castrated, domestic short-haired cat developed hyporexia 6 weeks after starting topical ophthalmic dorzolamide 2% therapy for treatment of ocular hypertension. Two weeks later, the cat was evaluated for severe weakness, cervical ventroflexion, and anorexia. Plasma electrolyte and acid-base measurement revealed hypokalemia (K + = 2.9 mmol/L; reference interval 3.8-5.4 mmol/L) and metabolic acidosis (plasma HCO 3 - = 9.8 mmol/L; reference interval 15-23 mmol/L) in the presence of a urine pH of 7.5 (reference interval 6.5-7.5). The pH abnormalities were consistent with a renal tubular acidosis. Clinical and biochemical abnormalities resolved with short-term supportive care, potassium supplementation, and discontinuation of dorzolamide therapy. This is the first report of hypokalemia and metabolic acidosis associated with topical CAI therapy in a cat. © Veterinary Emergency and Critical Care Society 2015.

  10. Carbonic anhydrase inhibition and cytotoxicity studies of Mannich base derivatives of thymol.

    Science.gov (United States)

    Inci Gul, Halise; Yamali, Cem; Tugce Yasa, Asiye; Unluer, Elif; Sakagami, Hiroshi; Tanc, Muhammet; Supuran, Claudiu T

    2016-12-01

    Mannich bases of thymol were synthesized. The aminomethylation reaction was realised in the ortho position of the phenol for compounds 2 (dipropylamine), 3 (benzylamine), and 4 (dibenzylamine) while it was from para position for 1 (dimethylamine), 5 (piperidine), 6 (morpholine) and 7 (N-methylpiperazine). The carbonic anhydrase (CA, EC 4.2.1.1) inhibitory effects of the compounds were asssessed against hCA I and hCA II. All compounds moderately inhibited hCA I and hCA II. The cytotoxicity of the compounds against four human oral squamous cell carcinoma cell lines were compared those against three normal oral cells. Tumor specificity values were about 2 or slightly more for the compounds 2, 3, 4, 5 and 6. Compound 2 showed cytostatic activity against OSCC cell lines at 16 to 32-fold lower concentrations as compared with normal cells. This suggests that compound 2 can be considered as cytotoxicity enhancing drug candidate for further investigations.

  11. Keratitis in six dogs after topical treatment with carbonic anhydrase inhibitors for glaucoma.

    Science.gov (United States)

    Beckwith-Cohen, Billie; Bentley, Ellison; Gasper, David J; McLellan, Gillian J; Dubielzig, Richard R

    2015-12-15

    6 dogs (10 eyes) with keratitis following long-term topical treatment with a carbonic anhydrase inhibitor (CAI) were evaluated. In 4 dogs (6 eyes), CAI treatment was discontinued. Three dogs (4 eyes) underwent enucleation because of end-stage corneal disease. One dog was treated differently in each eye and thus was represented in both aforementioned groups. Following initiation of treatment with a CAI (ie, brinzolamide or dorzolamide), the median time to development of severe ocular signs was 266 days (range, 133 to 679 days). Clinically severe ocular signs included ulcerative and nonulcerative perilimbal keratitis or severe diffuse keratitis with marked vascularization. The keratitis was refractory to treatment with anti-inflammatory medications. Histologic and immunohistochemical examination of enucleated globes was performed in 3 affected dogs and in 1 dog with keratitis that recovered. Corneal lesions included 2 distinct inflammatory infiltrates with plasma cells predominating in the anterior stroma and both T cells and neutrophils in the epithelium. Stromal plasma cells and overlying epithelium exhibited strong positive immunoreactivity for IgG. Topical CAI treatment was discontinued in 4 dogs after a median of 209 days (range, 44 to 433 days), and in these dogs, clinical improvement was evident within 2 to 4 days of CAI treatment cessation. Signs of keratitis resolved in 12 to 25 days in these 4 dogs, and median follow-up time after CAI discontinuation was 25.5 months (range, 6 to 42 months), during which time signs of corneal disease did not recur. On the basis of this small series, presumed topical CAI-associated keratitis in dogs appeared to be an uncommon immune-mediated disease that was not responsive to corticosteroid treatment. Affected patients improved rapidly, but only after discontinuation of CAI treatment. In dogs with glaucoma, clinicians should consider the development of punctate keratopathy and severe diffuse keratitis as potential adverse

  12. Structural insight into activity enhancement and inhibition of H64A carbonic anhydrase II by imidazoles.

    Science.gov (United States)

    Aggarwal, Mayank; Kondeti, Bhargav; Tu, Chingkuang; Maupin, C Mark; Silverman, David N; McKenna, Robert

    2014-03-01

    Human carbonic anhydrases (CAs) are zinc metalloenzymes that catalyze the hydration and dehydration of CO2 and HCO3 (-), respectively. The reaction follows a ping-pong mechanism, in which the rate-limiting step is the transfer of a proton from the zinc-bound solvent (OH(-)/H2O) in/out of the active site via His64, which is widely believed to be the proton-shuttling residue. The decreased catalytic activity (∼20-fold lower with respect to the wild type) of a variant of CA II in which His64 is replaced with Ala (H64A CA II) can be enhanced by exogenous proton donors/acceptors, usually derivatives of imidazoles and pyridines, to almost the wild-type level. X-ray crystal structures of H64A CA II in complex with four imidazole derivatives (imidazole, 1--methylimidazole, 2--methylimidazole and 4-methylimidazole) have been determined and reveal multiple binding sites. Two of these imidazole binding sites have been identified that mimic the positions of the 'in' and 'out' rotamers of His64 in wild-type CA II, while another directly inhibits catalysis by displacing the zinc-bound solvent. The data presented here not only corroborate the importance of the imidazole side chain of His64 in proton transfer during CA catalysis, but also provide a complete structural understanding of the mechanism by which imidazoles enhance (and inhibit when used at higher concentrations) the activity of H64A CA II.

  13. Anion inhibition studies of the α-carbonic anhydrase from the protozoan pathogen Trypanosoma cruzi, the causative agent of Chagas disease.

    Science.gov (United States)

    Pan, Peiwen; Vermelho, Alane Beatriz; Scozzafava, Andrea; Parkkila, Seppo; Capasso, Clemente; Supuran, Claudiu T

    2013-08-01

    The protozoan pathogen Trypanosoma cruzi, the causative agent of Chagas disease, encodes an α-class carbonic anhydrase (CA, EC 4.2.1.1), TcCA, which was recently shown to be crucial for its life cycle. Thiols, a class of strong TcCA inhibitors, were also shown to block the growth of the pathogen in vitro. Here we report the inhibition of TcCA by inorganic and complex anions and other molecules interacting with zinc proteins, such as sulfamide, sulfamic acid, phenylboronic/arsonic acids. TcCA was inhibited in the low micromolar range by iodide, cyanate, thiocyanate, hydrogensulfide and trithiocarbonate (KIs in the range of 44-93 μM), but the best inhibitor was diethyldithiocarbamate (KI=5 μM). Sulfamide showed an inhibition constant of 120 μM, but sulfamic acid was much less effective (KI of 10.6 mM). The discovery of diethyldithiocarbamate as a low micromolar TcCA inhibitor may be useful to detect leads for developing anti-Trypanosoma agents with a diverse mechanism of action compared to clinically used drugs (benznidazole, nifurtimox) for which significant resistance emerged. Copyright © 2013. Published by Elsevier Ltd.

  14. Open saccharin-based secondary sulfonamides as potent and selective inhibitors of cancer-related carbonic anhydrase IX and XII isoforms.

    Science.gov (United States)

    D'Ascenzio, Melissa; Guglielmi, Paolo; Carradori, Simone; Secci, Daniela; Florio, Rosalba; Mollica, Adriano; Ceruso, Mariangela; Akdemir, Atilla; Sobolev, Anatoly P; Supuran, Claudiu T

    2017-12-01

    A large number of novel secondary sulfonamides based on the open saccharin scaffold were synthesized and evaluated as selective inhibitors of four different isoforms of human carbonic anhydrase (hCA I, II, IX and XII, EC 4.2.1.1). They were obtained by reductive ring opening of the newly synthesized N-alkylated saccharin derivatives and were shown to be inactive against the two cytosolic off-target hCA I and II (K i s > 10 µM). Interestingly, these compounds inhibited hCA IX in the low nanomolar range with K i s ranging between 20 and 298 nM and were extremely potent inhibitors of hCA XII isoenzyme (K i s ranging between 4.3 and 432 nM). Since hCA IX and XII are the cancer-related isoforms recently validated as drug targets, these results represent an important goal in the development of new anticancer candidates. Finally, a computational approach has been performed to better correlate the biological data to the binding mode of these inhibitors.

  15. In silico modeling of β-carbonic anhydrase inhibitors from the fungus Malassezia globosa as antidandruff agents.

    Science.gov (United States)

    Singh, Shalini; Supuran, Claudiu T

    2016-01-01

    A quantitative structure-activity relationship (QSAR) study of sulfonamide inhibitors targeting the β-carbonic anhydrase (CA, EC 4.2.1.1) from the fungus Malassezia globosa is reported. A large set of PRECLAV descriptors has been used to obtain four parametric models. This study presents QSAR data on a pool of 28 compounds. The quality of prediction is high enough (SE = 0.3446, r(2) = 0.8687, F = 39.6921, Q = 0.7446). A heuristic algorithm selected the best multiple linear regression (MLR) equation which showed the correlation between the observed values and the calculated values of activity. The proposed prediction set included new, not yet synthesized, 23 molecules having various structures. Many compounds in the prediction set seem to possess higher computed activity compared to the presently available M. globosa β-CA inhibitors.

  16. Carbonic anhydrase inhibitors modify intracellular pH transients and contractions of rat middle cerebral arteries during CO2/HCO3- fluctuations.

    Science.gov (United States)

    Rasmussen, Jacob K; Boedtkjer, Ebbe

    2018-03-01

    The CO 2 /HCO 3 - buffer minimizes pH changes in response to acid-base loads, HCO 3 - provides substrate for Na + ,HCO 3 - -cotransporters and Cl - /HCO 3 - -exchangers, and H + and HCO 3 - modify vasomotor responses during acid-base disturbances. We show here that rat middle cerebral arteries express cytosolic, mitochondrial, extracellular, and secreted carbonic anhydrase isoforms that catalyze equilibration of the CO 2 /HCO 3 - buffer. Switching from CO 2 /HCO 3 - -free to CO 2 /HCO 3 - -containing extracellular solution results in initial intracellular acidification due to hydration of CO 2 followed by gradual alkalinization due to cellular HCO 3 - uptake. Carbonic anhydrase inhibition decelerates the initial acidification and attenuates the associated transient vasoconstriction without affecting intracellular pH or artery tone at steady-state. Na + ,HCO 3 - -cotransport and Na + /H + -exchange activity after NH 4 + -prepulse-induced intracellular acidification are unaffected by carbonic anhydrase inhibition. Extracellular surface pH transients induced by transmembrane NH 3 flux are evident under CO 2 /HCO 3 - -free conditions but absent when the buffer capacity and apparent H + mobility increase in the presence of CO 2 /HCO 3 - even after the inhibition of carbonic anhydrases. We conclude that (a) intracellular carbonic anhydrase activity accentuates pH transients and vasoconstriction in response to acute elevations of pCO 2 , (b) CO 2 /HCO 3 - minimizes extracellular surface pH transients without requiring carbonic anhydrase activity, and (c) carbonic anhydrases are not rate limiting for acid-base transport across cell membranes during recovery from intracellular acidification.

  17. Structural insight into activity enhancement and inhibition of H64A carbonic anhydrase II by imidazoles

    Directory of Open Access Journals (Sweden)

    Mayank Aggarwal

    2014-03-01

    Full Text Available Human carbonic anhydrases (CAs are zinc metalloenzymes that catalyze the hydration and dehydration of CO2 and HCO3−, respectively. The reaction follows a ping-pong mechanism, in which the rate-limiting step is the transfer of a proton from the zinc-bound solvent (OH−/H2O in/out of the active site via His64, which is widely believed to be the proton-shuttling residue. The decreased catalytic activity (∼20-fold lower with respect to the wild type of a variant of CA II in which His64 is replaced with Ala (H64A CA II can be enhanced by exogenous proton donors/acceptors, usually derivatives of imidazoles and pyridines, to almost the wild-type level. X-ray crystal structures of H64A CA II in complex with four imidazole derivatives (imidazole, 1-methylimidazole, 2-methylimidazole and 4-methylimidazole have been determined and reveal multiple binding sites. Two of these imidazole binding sites have been identified that mimic the positions of the `in' and `out' rotamers of His64 in wild-type CA II, while another directly inhibits catalysis by displacing the zinc-bound solvent. The data presented here not only corroborate the importance of the imidazole side chain of His64 in proton transfer during CA catalysis, but also provide a complete structural understanding of the mechanism by which imidazoles enhance (and inhibit when used at higher concentrations the activity of H64A CA II.

  18. Inhibition studies on a panel of human carbonic anhydrases with N1-substituted secondary sulfonamides incorporating thiazolinone or imidazolone-indole tails.

    Science.gov (United States)

    Awadallah, Fadi M; Bua, Silvia; Mahmoud, Walaa R; Nada, Hossam H; Nocentini, Alessio; Supuran, Claudiu T

    2018-12-01

    Being the primary sulfonamide among the most efficient zinc binding group (ZBG) to design inhibitors for the metallo-enzymes carbonic anhydrases (CA, EC 4.2.1.1), herein, we propose an investigation on four physiologically important human (h) CAs (hCA I, II, IV, and IX) with N 1 -substituted secondary sulfonamides incorporating thiazolinone or imidazolone-indole tails. The effect of the functionalisation of the sulfonamide group with five different substitution patterns, namely acetyl, pyridine, thiazole, pyrimidine, and carbamimidoyl, was evaluated in relation to the inhibition profile of the corresponding primary sulfonamide analogues. With most of these latter being nanomolar inhibitors of all four considered isoforms, a totally counterproductive effect on the inhibition potency can be ascribed to N 1 -functionalisations of the ZBG primary sulfonamide structure with pyridine, thiazole, and pyrimidine moieties. On the other hand, incorporation of less hindered groups, such as sulfonylacetamides and sulfonylguanidines, maintained a certain degree of activity dependent on the tailing moiety, with K I s spanning in the low micromolar range.

  19. Carbonic anhydrase inhibitors. Comparison of chlorthalidone, indapamide, trichloromethiazide, and furosemide X-ray crystal structures in adducts with isozyme II, when several water molecules make the difference.

    Science.gov (United States)

    Temperini, Claudia; Cecchi, Alessandro; Scozzafava, Andrea; Supuran, Claudiu T

    2009-02-01

    Thiazide and high ceiling diuretics were recently shown to inhibit all mammalian isoforms of carbonic anhydrase (CA, EC 4.2.1.1) with a very different profile as compared to classical inhibitors, such as acetazolamide, methazolamide, and ethoxzolamide. Some of these structurally related compounds have a very different behavior against the widespread isozyme CA II, with chlorthalidone, trichloromethiazide, and furosemide being efficient inhibitors against CA II (K(I)s of 65-138 nM), whereas indapamide is a much weaker one (K(I) of 2520 nM). Furthermore, some of these diuretics are quite efficient (low nanomolar) inhibitors of other isoforms, for example, chlorthalidone against hCA VB, VII, IX, and XIII; indapamide against CA VII, IX, XII, and XIII, trichloromethiazide against CA VII and IX, and furosemide against CA I and XIV. Examining the four X-ray crystal structures of their CA II adducts, we observed several (2-3) active site water molecules interacting with the chlorthalidone, trichloromethiazide, and furosemide scaffolds which may be responsible for this important difference of activity. Indeed, indapamide bound to CA II has no interactions with active site water molecules. Chlorthalidone bound within the CA II active site is in an enolic (lactimic) tautomeric form, with the enolic OH also participating in two strong hydrogen bonds with Asn67 and a water molecule. The newly evidenced binding modes of these diuretics may be exploited for designing better CA II inhibitors as well as compounds with selectivity/affinity for various isoforms with medicinal chemistry applications.

  20. The history and rationale of using carbonic anhydrase inhibitors in the treatment of peptic ulcers. In memoriam Ioan Puşcaş (1932-2015).

    Science.gov (United States)

    Buzás, György M; Supuran, Claudiu T

    2016-08-01

    Carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) started to be used in the treatment of peptic ulcers in the 1970s, and for more than two decades, a group led by Ioan Puşcaş used them for this purpose, assuming that by inhibiting the gastric mucosa CA isoforms, hydrochloric acid secretion is decreased. Although acetazolamide and other sulfonamide CAIs are indeed effective in healing ulcers, the inhibition of CA isoforms in other organs than the stomach led to a number of serious side effects which made this treatment obsolete when the histamine H2 receptor antagonists and the proton pump inhibitors became available. Decades later, in 2002, it has been discovered that Helicobacter pylori, the bacterial pathogen responsible for gastric ulcers and cancers, encodes for two CAs, one belonging to the α-class and the other one to the β-class of these enzymes. These enzymes are crucial for the life cycle of the bacterium and its acclimation within the highly acidic environment of the stomach. Inhibition of the two bacterial CAs with sulfonamides such as acetazolamide, a low-nanomolar H. pylori CAI, is lethal for the pathogen, which explains why these compounds were clinically efficient as anti-ulcer drugs. Thus, the approach promoted by Ioan Puşcaş for treating this disease was a good one although the rationale behind it was wrong. In this review, we present a historical overview of the sulfonamide CAIs as anti-ulcer agents, in memoriam of the scientist who was in the first line of this research trend.

  1. 4-Amino-substituted Benzenesulfonamides as Inhibitors of Human Carbonic Anhydrases

    Directory of Open Access Journals (Sweden)

    Kęstutis Rutkauskas

    2014-10-01

    Full Text Available A series of N-aryl-β-alanine derivatives and diazobenzenesulfonamides containing aliphatic rings were designed, synthesized, and their binding to carbonic anhydrases (CA I, II, VI, VII, XII, and XIII was studied by the fluorescent thermal shift assay and isothermal titration calorimetry. The results showed that 4-substituted diazobenzenesulfonamides were more potent CA binders than N-aryl-β-alanine derivatives. Most of the N-aryl-β-alanine derivatives showed better affinity for CA II while diazobenzenesulfonamides possessed nanomolar affinities towards CA I isozyme. X-ray crystallographic structures showed the modes of binding of both compound groups.

  2. Design and synthesis of novel 1,3-diaryltriazene-substituted sulfonamides as potent and selective carbonic anhydrase II inhibitors.

    Science.gov (United States)

    Lolak, Nabih; Akocak, Suleyman; Bua, Silvia; Koca, Murat; Supuran, Claudiu T

    2018-04-01

    A series of novel 1,3-diaryltriazene-substituted sulfonamides was synthesized by reaction of diazonium salt of 4-amino benzenesulfonamide with substituted aromatic amines. The obtained 1,3-diaryltriazene-substituted sulfonamides were investigated as inhibitors of four selected human carbonic anhydrase (CA, EC 4.2.1.1) isoforms (hCA I, hCA II, hCA VII and hCA IX) are involved in various diseases such as glaucoma, epilepsy, retinitis pigmentosa, cancer, obesity, etc. All these sulfonamides were found to be potent inhibitors of the cytosolic isoform hCA II with low nanomolar to sub-nanomolar K i s in the range of 0.2-21.5 nM, as well as a moderate selectivity against other cytosolic isoforms hCA I and hCA VII, and great selectivity against membrane-bound isoform hCA IX was observed. Since hCA II is an important drug target for antiglaucoma agents and diuretics, these isoform-selective inhibitors may be considered of interest as tools for the development of new candidates for these conditions. Copyright © 2018 Elsevier Inc. All rights reserved.

  3. The changes in the chloroplast membranes of pea leaves under the influence of carbonic anhydrase inhibitors (ions of copper and zinc

    Directory of Open Access Journals (Sweden)

    M.V. Vodka

    2014-04-01

    Full Text Available Тhe effects of carbonic anhydrase inhibitors, such as ions Cu2+ and Zn2+, on the membrane system of chloroplasts in pea leaves were investigated. After treatment of pea leaves with 250 mM Cu2+ or 400 mM Zn2+ we observed changes in the granal structure and compactness of the thylakoids in granae. It was shown that the thickness of granal thylakoids and the interspace between thylakoids increased comparing to control. Changes of the size and structure of thylakoids and granae in treated leaves may be associated with the enhanced accumulation of CO2 in the membrane. It is suggested that the carbonic anhydrase may also play a structural role in chloroplast granae.

  4. Inhibition of the β-carbonic anhydrase from the dandruff-producing fungus Malassezia globosa with monothiocarbamates.

    Science.gov (United States)

    Nocentini, Alessio; Vullo, Daniela; Del Prete, Sonia; Osman, Sameh M; Alasmary, Fatmah A S; AlOthman, Zeid; Capasso, Clemente; Carta, Fabrizio; Gratteri, Paola; Supuran, Claudiu T

    2017-12-01

    A series of monothiocarbamates (MTCs) was investigated for the inhibition of the β-class carbonic anhydrase (CAs, EC 4.2.1.1) from the fungal parasite Malassezia globosa, MgCA. These MTCs incorporate various scaffolds, among which aliphatic amine with 1-4 carbons atom in their molecule, morpholine, piperazine, as well as phenethylamine and benzylamine derivatives. All the reported MTCs displayed a better efficacy in inhibiting MgCA compared to the clinically used sulphonamide drug acetazolamide (K I of 74 μM), with K I s spanning between 1.85 and 18.9 μM. The homology model of the enzyme previously reported by us was used to rationalize the results by docking some of these MTCs within the fungal CA active site. This study might be useful to enrich the knowledge of the MgCA inhibition profile, eliciting novel ideas pertaining the design of modulators with potential efficacy in combatting dandruff or other fungal infections.

  5. Tumor microenvironmental changes induced by the sulfamate carbonic anhydrase IX inhibitor S4 in a laryngeal tumor model.

    Directory of Open Access Journals (Sweden)

    Tineke W H Meijer

    Full Text Available BACKGROUND AND PURPOSE: Carbonic anhydrase IX (CAIX plays a pivotal role in pH homeostasis, which is essential for tumor cell survival. We examined the effect of the CAIX inhibitor 4-(3'(3",5"-dimethylphenyl-ureidophenyl sulfamate (S4 on the tumor microenvironment in a laryngeal tumor model by analyzing proliferation, apoptosis, necrosis, hypoxia, metabolism and CAIX ectodomain shedding. METHODS: SCCNij202 tumor bearing-mice were treated with S4 for 1, 3 or 5 days. CAIX ectodomain shedding was measured in the serum after therapy. Effects on tumor cell proliferation, apoptosis, necrosis, hypoxia (pimonidazole and CAIX were investigated with quantitative immunohistochemistry. Metabolic transporters and enzymes were quantified with qPCR. RESULTS: CAIX ectodomain shedding decreased after treatment with S4 (p<0.01. S4 therapy did neither influence tumor cell proliferation nor the amount of apoptosis and necrosis. Hypoxia (pimonidazole and CAIX expression were also not affected by S4. CHOP and MMP9 mRNA as a reference of intracellular pH did not change upon treatment with S4. Compensatory mechanisms of pH homeostasis at the mRNA level were not observed. CONCLUSION: As the clinical and biological meaning of the decrease in CAIX ectodomain shedding after S4 therapy is not clear, studies are required to elucidate whether the CAIX ectodomain has a paracrine or autocrine signaling function in cancer biology. S4 did not influence the amount of proliferation, apoptosis, necrosis and hypoxia. Therefore, it is unlikely that S4 can be used as single agent to influence tumor cell kill and proliferation, and to target primary tumor growth.

  6. Sulfonamides containing curcumin scaffold: Synthesis, characterization, carbonic anhydrase inhibition and molecular docking studies.

    Science.gov (United States)

    Ahmed, Mahmood; Qadir, Muhammad Abdul; Hameed, Abdul; Arshad, Muhammad Nadeem; Asiri, Abdullah M; Muddassar, Muhammad

    2018-02-01

    Curcumin is a multi-functional pharmacologically safe natural agent with proven cytoprotective effects to healthy human cells. In this study, a new series of sulfonamides with curcumin scaffold were synthesized, characterized and investigated for their carbonic anhydrase isoenzyme I (human) and II (bovine) isoforms. The structures of newly synthesized compounds were described by IR, 1 H NMR and 13 C NMR spectral data. Compound 14 showed the K i value of 0.99 µM with highest inhibitory activity among all other synthesized compounds against hCA-I enzyme. Similarly enzyme kinetic studies of compound 14, 16 and 30 against bCAII enzyme showed Ki values of 0.71, 0.67 and 0.71 µM respectively. Our biological assays results showed that most of active compounds have similar inhibitory activities compared to standard acetazolamide drug. The molecular docking predicted binding modes showed that these compounds bind with hCA-1 enzyme in similar fashion. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Carbonic anhydrase inhibition of Schiff base derivative of imino-methyl-naphthalen-2-ol: Synthesis, structure elucidation, molecular docking, dynamic simulation and density functional theory calculations

    Science.gov (United States)

    Abbas, Saghir; Nasir, Hafiza Huma; Zaib, Sumera; Ali, Saqib; Mahmood, Tariq; Ayub, Khurshid; Tahir, Muhammad Nawaz; Iqbal, Jamshed

    2018-03-01

    In the present study, we have designed and synthesized a Schiff base derivative 3 and characterized by FT-IR, 1H and 13C NMR spectroscopy. Single crystal X-ray diffraction and NMR studies were also performed. The synthetic compound was screened for its inhibitory potential against carbonic anhydrase II. The experimental results were validated by molecular docking and dynamic simulations of compound 3 in the active pocket of enzyme. Important binding interactions with the key residues in the active site of the carbonic anhydrase enzyme were revealed. Moreover, supramolecular assembly of the title compound was analyzed by density functional theory (DFT) calculations. These studies rendered a more clear understanding for the demonstration of novel molecular mechanism involved in CA II inhibition by the synthesized compound.

  8. Synthesis, characterization, antimicrobial activity and carbonic anhydrase enzyme inhibitor effects of salicilaldehyde-N-methyl p-toluenesulfonylhydrazone and its Palladium(II), Cobalt(II) complexes

    Science.gov (United States)

    Alyar, Saliha; Adem, Şevki

    2014-10-01

    We report the synthesis of the ligand, salicilaldehyde-N-methyl p-toluenesulfonylhydrazone (salptsmh) derived from p-toluenesulfonicacid-1-methylhydrazide (ptsmh) and its Pd(II) and Co(II) metal complexes were synthesized for the first time. The structure of the ligand and their complexes were investigated using elemental analysis, magnetic susceptibility, molar conductance and spectral (IR, NMR and LC-MS) measurements. Salptsmh has also been characterized by single crystal X-ray diffraction. 1H and 13C shielding tensors for crystal structure were calculated with GIAO/DFT/B3LYP/6-311++G(d,p) methods in CDCl3. The complexes were found to have general composition [ML2]. The results of elemental analysis showed 1:2 (metal/ligand) stoichiometry for all the complex. Magnetic and spectral data indicate a square planar geometry for Pd(II) complex and a distorted tetrahedral geometry for Co(II) complexes. The ligand and its metal chelates have been screened for their antimicrobial activities using the disk diffusion method against the selected Gram positive bacteria: Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Enterococcus faecalis, Gram negative bacteria: Eschericha coli, Pseudomonas aeruginosa, Klebsiella pneumonia. The inhibition activities of these compounds on carbonic anhydrase II (CA II) and carbonic anhydrase I (CA I) have been investigated by comparing IC50 and Ki values and it has been found that Pd(II) complex have more enzyme inhibition efficiency than salptsmh and Co(II) complex.

  9. Pharmacological inhibition of carbonic anhydrase XII interferes with cell proliferation and induces cell apoptosis in T-cell lymphomas

    NARCIS (Netherlands)

    Lounnas, Nadia; Rosilio, Célia; Nebout, Marielle; Mary, Didier; Griessinger, Emmanuel; Neffati, Zouhour; Chiche, Johanna; Spits, Hergen; Hagenbeek, Thijs J.; Asnafi, Vahid; Poulsen, Sally-Ann; Supuran, Claudiu T.; Peyron, Jean-François; Imbert, Véronique

    2013-01-01

    The membrane-bound carbonic anhydrase isoforms CAIX and CAXII, underpin a pH-regulating system that enables hypoxic tumor cell survival. Here, we observed for the first time an upregulation of CAXII in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LL) cells. First we showed that CAXII is

  10. Inhibition stereochemistry of hydroxamate inhibitors for thermolysin.

    Science.gov (United States)

    Jin, Y; Kim, D H

    1998-12-15

    N-Acyl-N-hydroxy-beta-amino acid derivatives were prepared and tested as inhibitors for thermolysin to find that these inhibitors show the L-stereospecificity in contrast to the corresponding hydroxamates prepared from alpha-amino acid, which exhibit the D-stereochemistry. N-Formyl-N-hydroxy-beta-L-Phe-NHMe is the most potent inhibitor having the Ki value of 1.66 microM.

  11. Selective serotonin reuptake inhibitor sertraline inhibits voltage ...

    Indian Academy of Sciences (India)

    2016-10-04

    701. South Korea. *Corresponding author (Email, parkws@kangwon.ac.kr). We examined the effects of the selective serotonin reuptake inhibitor (SSRI) sertraline on voltage-dependent K+ (Kv) channels in freshly isolated ...

  12. Degradation products of the artificial azo dye, Allura red, inhibit esterase activity of carbonic anhydrase II: A basic in vitro study on the food safety of the colorant in terms of enzyme inhibition.

    Science.gov (United States)

    Esmaeili, Sajjad; Ashrafi-Kooshk, Mohammad Reza; Khaledian, Koestan; Adibi, Hadi; Rouhani, Shohre; Khodarahmi, Reza

    2016-12-15

    Allura red is a widely used food colorant, but there is debate on its potential security risk. In the present study, we found that degradation products of the dye were more potent agents with higher carbonic anhydrase inhibitory action than the parent dye. The mechanism by which the compounds inhibit the enzyme activity has been determined as competitive mode. In addition, the enzyme binding properties of the compounds were investigated employing different spectroscopic techniques and molecular docking. The analyses of fluorescence quenching data revealed the existence of the same binding site for the compounds on the enzyme molecule. The thermodynamic parameters of ligand binding were not similar, which indicates that different interactions are responsible in binding of the parent dye and degradation products to the enzyme. It appears that enzyme inhibition should be considered, more seriously, as a new opened dimension in food safety. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Inhibition of matrix metalloproteinase-2 by PARP inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Nicolescu, Adrian C.; Holt, Andrew; Kandasamy, Arulmozhi D. [Departments of Pharmacology and Pediatrics, Cardiovascular Research Centre, University of Alberta, Edmonton, Alta., Canada T6G 2S2 (Canada); Pacher, Pal [National Institutes of Health, NIAAA, Laboratory of Physiologic Studies, Bethesda, MD (United States); Schulz, Richard, E-mail: richard.schulz@ualberta.ca [Departments of Pharmacology and Pediatrics, Cardiovascular Research Centre, University of Alberta, Edmonton, Alta., Canada T6G 2S2 (Canada)

    2009-10-02

    Matrix metalloproteinase-2 (MMP-2), a ubiquitously expressed zinc-dependent endopeptidase, and poly(ADP-ribosyl) polymerase (PARP), a nuclear enzyme regulating DNA repair, are activated by nitroxidative stress associated with various pathologies. As MMP-2 plays a detrimental role in heart injuries resulting from enhanced nitroxidative stress, where PARP and MMP inhibitors are beneficial, we hypothesized that PARP inhibitors may affect MMP-2 activity. Using substrate degradation assays to determine MMP-2 activity we found that four PARP inhibitors (3-AB, PJ-34, 5-AIQ, and EB-47) inhibited 64 kDa MMP-2 in a concentration-dependent manner. The IC{sub 50} values of PJ-34 and 5-AIQ were in the high micromolar range and comparable to those of known MMP-2 inhibitors doxycycline, minocycline or o-phenanthroline, whereas those for 3-AB and EB-47 were in the millimolar range. Co-incubation of PARP inhibitors with doxycycline showed an additive inhibition of MMP-2 that was significant for 3-AB alone. These data demonstrate that the protective effects of some PARP inhibitors may include inhibition of MMP-2 activity.

  14. Inhibition of matrix metalloproteinase-2 by PARP inhibitors

    International Nuclear Information System (INIS)

    Nicolescu, Adrian C.; Holt, Andrew; Kandasamy, Arulmozhi D.; Pacher, Pal; Schulz, Richard

    2009-01-01

    Matrix metalloproteinase-2 (MMP-2), a ubiquitously expressed zinc-dependent endopeptidase, and poly(ADP-ribosyl) polymerase (PARP), a nuclear enzyme regulating DNA repair, are activated by nitroxidative stress associated with various pathologies. As MMP-2 plays a detrimental role in heart injuries resulting from enhanced nitroxidative stress, where PARP and MMP inhibitors are beneficial, we hypothesized that PARP inhibitors may affect MMP-2 activity. Using substrate degradation assays to determine MMP-2 activity we found that four PARP inhibitors (3-AB, PJ-34, 5-AIQ, and EB-47) inhibited 64 kDa MMP-2 in a concentration-dependent manner. The IC 50 values of PJ-34 and 5-AIQ were in the high micromolar range and comparable to those of known MMP-2 inhibitors doxycycline, minocycline or o-phenanthroline, whereas those for 3-AB and EB-47 were in the millimolar range. Co-incubation of PARP inhibitors with doxycycline showed an additive inhibition of MMP-2 that was significant for 3-AB alone. These data demonstrate that the protective effects of some PARP inhibitors may include inhibition of MMP-2 activity.

  15. Anion inhibition studies of two α-carbonic anhydrases from Lotus japonicus, LjCAA1 and LjCAA2.

    Science.gov (United States)

    Vullo, Daniela; Flemetakis, Emmanouil; Scozzafava, Andrea; Capasso, Clemente; Supuran, Claudiu T

    2014-07-01

    The model organism for the investigation of symbiotic nitrogen fixation in legumes Lotus japonicus encodes two carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α-class, LjCAA1 and LjCAA2. Here we report the kinetic characterization and inhibition of these two CAs with inorganic and complex anions and other molecules interacting with zinc proteins, such as sulfamide, sulfamic acid, and phenylboronic/arsonic acids. LjCAA1 showed a high catalytic activity for the CO2 hydration reaction, with a k(cat) of 7.4∗10(5) s(-1) and a k(cat)/K(m) of 9.6∗10(7) M(-1) s(-1) and was inhibited in the low micromolar range by N,N-diethyldithiocarbamate, sulfamide, sulfamic acid, phenylboronic/arsonic acid (K(I)s of 4-62 μM). LjCAA2 showed a moderate catalytic activity for the physiologic reaction, with a k(cat) of 4.0∗10(5) s(-1) and a k(cat)/K(m) of 4.9∗10(7) M(-1) s(-1). The same anions mentioned above for the inhibition of LjCAA1 showed the best activity against LjCAA2 (K(I)s of 7-29 μM). Nitrate and nitrite, anions involved in nitrogen fixation, showed lower affinity for the two enzymes, with inhibition constants in the range of 3.7-7.0 mM. Halides and sulfate also behaved in a distinct manner towards the two enzymes investigated here. As LjCAA1/2 participate in the pH regulation processes and CO2 metabolism within the nitrogen-fixing nodules of the plant, our studies may shed some light regarding these complex biochemical processes. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Rho kinase inhibitors block melanoma cell migration and inhibit metastasis.

    Science.gov (United States)

    Sadok, Amine; McCarthy, Afshan; Caldwell, John; Collins, Ian; Garrett, Michelle D; Yeo, Maggie; Hooper, Steven; Sahai, Erik; Kuemper, Sandra; Mardakheh, Faraz K; Marshall, Christopher J

    2015-06-01

    There is an urgent need to identify new therapeutic opportunities for metastatic melanoma. Fragment-based screening has led to the discovery of orally available, ATP-competitive AKT kinase inhibitors, AT13148 and CCT129254. These compounds also inhibit the Rho-kinases ROCK 1 and ROCK 2 and we show they potently inhibit ROCK activity in melanoma cells in culture and in vivo. Treatment of melanoma cells with CCT129254 or AT13148 dramatically reduces cell invasion, impairing both "amoeboid-like" and mesenchymal-like modes of invasion in culture. Intravital imaging shows that CCT129254 or AT13148 treatment reduces the motility of melanoma cells in vivo. CCT129254 inhibits melanoma metastasis when administered 2 days after orthotopic intradermal injection of the cells, or when treatment starts after metastases have arisen. Mechanistically, our data suggest that inhibition of ROCK reduces the ability of melanoma cells to efficiently colonize the lungs. These results suggest that these novel inhibitors of ROCK may be beneficial in the treatment of metastasis. ©2015 American Association for Cancer Research.

  17. Lignocellulosic hydrolysate inhibitors selectively inhibit/deactivate cellulase performance.

    Science.gov (United States)

    Mhlongo, Sizwe I; den Haan, Riaan; Viljoen-Bloom, Marinda; van Zyl, Willem H

    2015-12-01

    In this study, we monitored the inhibition and deactivation effects of various compounds associated with lignocellulosic hydrolysates on individual and combinations of cellulases. Tannic acid representing polymeric lignin residues strongly inhibited cellobiohydrolase 1 (CBH1) and β-glucosidase 1 (BGL1), but had a moderate inhibitory effect on endoglucanase 2 (EG2). Individual monomeric lignin residues had little or no inhibitory effect on hydrolytic enzymes. However, coniferyl aldehyde and syringaldehyde substantially decreased the activity of CBH1 and deactivated BGL1. Acetic and formic acids also showed strong inhibition of BGL1 but not CBH1 and EG2, whereas tannic, acetic and formic acid strongly inhibited a combination of CBH1 and EG2 during Avicel hydrolysis. Diminishing enzymatic hydrolysis is largely a function of inhibitor concentration and the enzyme-inhibitor relationship, rather than contact time during the hydrolysis process (i.e. deactivation). This suggests that decreased rates of hydrolysis during the enzymatic depolymerisation of lignocellulosic hydrolysates may be imparted by other factors related to substrate crystallinity and accessibility. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Synthesis and inhibitory properties of some carbamates on carbonic anhydrase and acetylcholine esterase.

    Science.gov (United States)

    Yılmaz, Süleyman; Akbaba, Yusuf; Özgeriş, Bünyamin; Köse, Leyla Polat; Göksu, Süleyman; Gülçin, İlhami; Alwasel, Saleh H; Supuran, Claudiu T

    2016-12-01

    A series of carbamate derivatives were synthesized and their carbonic anhydrase I and II isoenzymes and acetylcholinesterase enzyme (AChE) inhibitory effects were investigated. All carbamates were synthesized from the corresponding carboxylic acids via the Curtius reactions of the acids with diphenyl phosphoryl azide followed by addition of benzyl alcohol. The carbamates were determined to be very good inhibitors against for AChE and hCA I, and II isoenzymes. AChE inhibition was determined in the range 0.209-0.291 nM. On the other hand, tacrine, which is used in the treatment of Alzheimer's disease possessed lower inhibition effect (Ki: 0.398 nM). Also, hCA I and II isoenzymes were effectively inhibited by the carbamates, with inhibition constants (Ki) in the range of 4.49-5.61 nM for hCA I, and 4.94-7.66 nM for hCA II, respectively. Acetazolamide, which was clinically used carbonic anhydrase (CA) inhibitor demonstrated Ki values of 281.33 nM for hCA I and 9.07 nM for hCA II. The results clearly showed that AChE and both CA isoenzymes were effectively inhibited by carbamates at the low nanomolar levels.

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

    Directory of Open Access Journals (Sweden)

    Christina Gavegnano

    2017-12-01

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

  20. DNA methyltransferase inhibitor CDA-II inhibits myogenic differentiation

    International Nuclear Information System (INIS)

    Chen, Zirong; Jin, Guorong; Lin, Shuibin; Lin, Xiumei; Gu, Yumei; Zhu, Yujuan; Hu, Chengbin; Zhang, Qingjiong; Wu, Lizi; Shen, Huangxuan

    2012-01-01

    Highlights: ► CDA-II inhibits myogenic differentiation in a dose-dependent manner. ► CDA-II repressed expression of muscle transcription factors and structural proteins. ► CDA-II inhibited proliferation and migration of C2C12 myoblasts. -- Abstract: CDA-II (cell differentiation agent II), isolated from healthy human urine, is a DNA methyltransferase inhibitor. Previous studies indicated that CDA-II played important roles in the regulation of cell growth and certain differentiation processes. However, it has not been determined whether CDA-II affects skeletal myogenesis. In this study, we investigated effects of CDA-II treatment on skeletal muscle progenitor cell differentiation, migration and proliferation. We found that CDA-II blocked differentiation of murine myoblasts C2C12 in a dose-dependent manner. CDA-II repressed expression of muscle transcription factors, such as Myogenin and Mef2c, and structural proteins, such as myosin heavy chain (Myh3), light chain (Mylpf) and MCK. Moreover, CDA-II inhibited C1C12 cell migration and proliferation. Thus, our data provide the first evidence that CDA-II inhibits growth and differentiation of muscle progenitor cells, suggesting that the use of CDA-II might affect skeletal muscle functions.

  1. Stabilization versus inhibition of TAFIa by competitive inhibitors in vitro

    NARCIS (Netherlands)

    Walker, J.B.; Hughes, B.; James, I.; Haddock, P.; Kluft, C.; Bajzar, L.

    2003-01-01

    Two competitive inhibitors of TAFIa (activated thrombin-activable fibrinolysis inhibitor), 2-guanidinoethyl-mercaptosuccinic acid and potato tuber carboxypeptidase inhibitor, variably affect fibrinolysis of clotted human plasma. Depending on their concentration, the inhibitors shortened, prolonged,

  2. Discovery of a new family of carbonic anhydrases in the malaria pathogen Plasmodium falciparum--the η-carbonic anhydrases.

    Science.gov (United States)

    Del Prete, Sonia; Vullo, Daniela; Fisher, Gillian M; Andrews, Katherine T; Poulsen, Sally-Ann; Capasso, Clemente; Supuran, Claudiu T

    2014-09-15

    The genome of the protozoan parasite Plasmodium falciparum, the causative agent of the most lethal type of human malaria, contains a single gene annotated as encoding a carbonic anhydrase (CAs, EC 4.2.1.1) thought to belong to the α-class, PfCA. Here we demonstrate the kinetic properties of PfCA for the CO2 hydration reaction, as well as an inhibition study of this enzyme with inorganic and complex anions and other molecules known to interact with zinc proteins, including sulfamide, sulfamic acid, and phenylboronic/arsonic acids, detecting several low micromolar inhibitors. A closer examination of the sequence of this and the CAs from other Plasmodium spp., as well as a phylogenetic analysis, revealed that these protozoa encode for a yet undisclosed, new genetic family of CAs termed the η-CA class. The main features of the η-CAs are described in this report. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Carbonic anhydrase and acetylcholinesterase inhibitory effects of carbamates and sulfamoylcarbamates.

    Science.gov (United States)

    Göçer, Hülya; Akincioğlu, Akın; Göksu, Süleyman; Gülçin, İlhami; Supuran, Claudiu T

    2015-04-01

    Carbonic anhydrases (CA), as a family of metalloenzymes, are found in almost every type of tissue and play an important role in catalyzing the equilibration of carbon dioxide and carbonic acid. In this study, a series of carbamate derivative was synthesized, and their inhibition effects on hCA I, hCA II and acetylcholinesterase (AChE) enzymes were investigated. They were determined to be very good inhibitor against for both isoenzymes (hCA I and hCA II) and AChE. The hCA I and hCA II were effectively inhibited by the carbamate derivatives, with inhibition constants (Ki) in the range of 194.4-893.5 nM (for hCA I) and 103.9-835.7 nM (for hCA II). On the other hand, Ki parameters of these compounds for AChE enzyme inhibition were determined in the range of 12.0-61.3 nM. The results clearly showed that both CA isoenzymes and AChE were inhibited by carbamate derivatives at the nM levels.

  4. Bile acids and their oxo derivatives: Potential inhibitors of carbonic anhydrase I and II, androgen receptor antagonists and CYP3A4 substrates.

    Science.gov (United States)

    Trifunović, Jovana; Borčić, Vladan; Mikov, Momir

    2017-05-01

    Some biological properties of bile acids and their oxo derivatives have not been sufficiently investigated, although the interest in bile acids as signaling molecules is rising. The aim of this work was to evaluate physico-chemical parametar b (slope) that represents the lipophilicity of the examined molecules and to investigate interactions of bile acids with carbonic anhydrase I, II, androgen receptor and CYP450s. Thirteen candidates were investigated using normal-phase thin-layer chromatography in two solvent systems. Retention parameters were used in further quantitative structure-activity relationship analysis and docking studies to predict interactions and binding affinities of examined molecules with enzymes and receptors. Prediction of activity on androgen receptor showed that compounds 3α-hydroxy-12-oxo-5β-cholanoic and 3α-hydroxy-7-oxo-5β-cholanoic acid have stronger antiandrogen activity than natural bile acids. The inhibitory potential for carbonic anhydrase I and II was tested and it was concluded that molecules 3α-hydroxy-12-oxo-5β-cholanoic, 3α-hydroxy-7-oxo-5β-cholanoic, 3,7,12-trioxo-5β-cholanoic acid and hyodeoxycholic acid show the best results. Substrate behavior for CYP3A4 was confirmed for all investigated compounds. Oxo derivatives of bile acids show stronger interactions with enzymes and receptors as classical bile acids and lower membranolytic activity compared with them. These significant observations could be valuable in consideration of oxo derivatives as building blocks in medicinal chemistry. Copyright © 2016 John Wiley & Sons, Ltd.

  5. Resynthesis of reactive site peptide bond and temporary inhibition of Streptomyces metalloproteinase inhibitor.

    Science.gov (United States)

    Seeram, S S; Hiraga, K; Oda, K

    1997-10-01

    Streptomyces metalloproteinase inhibitor (SMPI) is a small proteinaceous inhibitor which inhibits metalloproteinases such as thermolysin (Ki =1.14 x 10(-10) M). When incubated with the enzyme, it is gradually hydrolyzed at the Cys64-Val65 peptide bond, which was identified as the reactive site by mutational analysis. To achieve a further understanding of the inhibition mechanism, we attempted to resynthesize the cleaved reactive site by using the enzyme catalytic action. The native inhibitor was resynthesized from the modified inhibitor (Ki =2.18 x 10(-8) M) by incubation with a catalytic amount of thermolysin under the same conditions as used for hydrolysis (pH 7.5, 25 degrees C), suggesting that SMPI follows the standard mechanism of inhibition of serine proteinase inhibitors. Temporary inhibition was observed when the native inhibitor and thermolysin were incubated at a 1:100 (mol/mol) enzyme-inhibitor ratio at 37 degrees C. SMPI showed temporary inhibition towards all the enzymes it inhibited. The inhibitory spectrum of SMPI was analyzed with various metalloproteinases based on the Ki values and limited proteolysis patterns. Pseudomonas elastase and Streptomyces griseus metalloproteinase II formed more stable complexes and showed much lower Ki values (approximately 2 pM) than thermolysin. In the limited proteolysis experiments weak inhibitors were degraded by the enzymes. SMPI did not inhibit almelysin, Streptomyces caespitosus neutral proteinase or matrix metalloproteinases. SMPI specifically inhibits metalloproteinases which are sensitive to phosphoramidon.

  6. Rapid redistribution and inhibition of renal sodium transporters during acute pressure natriuresis

    DEFF Research Database (Denmark)

    Zhang, Y; Mircheff, A K; Hensley, C B

    1996-01-01

    and basolateral Na+ pumps to internal membranes. Arterial pressure was increased 50 mmHg by constricting various arteries. We also tested whether transporter internalization occurred when PT Na+ reabsorption was inhibited with the carbonic anhydrase inhibitor benzolamide. Five minutes after initiating either...

  7. Autophagy induction by histone deacetylase inhibitors inhibits HIV type 1.

    Science.gov (United States)

    Campbell, Grant R; Bruckman, Rachel S; Chu, Yen-Lin; Spector, Stephen A

    2015-02-20

    Histone deacetylase inhibitors (HDACi) are being evaluated in a "shock-and-kill" therapeutic approach to reverse human immunodeficiency virus type-1 (HIV) latency from CD4(+) T cells. Using this approach, HDACi have induced HIV RNA synthesis in latently infected cells from some patients. The hope is that the increase in viral production will lead to killing of the infected cell either by the virus itself or by the patient's immune system, a "sterilizing cure." Although administered within the context of combination antiretroviral therapy, the infection of bystander cells remains a concern. In this study, we investigated the effect of HDACi (belinostat, givinostat, panobinostat, romidepsin, and vorinostat) on the productive infection of macrophages. We demonstrate that the HDACi tested do not alter the initial susceptibility of macrophages to HIV infection. However, we demonstrate that HDACi decrease HIV release from macrophages in a dose-dependent manner (belinostat the canonical autophagy pathway. This mechanism involves unc-51-like autophagy-activating kinase 1 (ULK1) and the inhibition of the mammalian target of rapamycin and requires the formation of autophagosomes and their maturation into autolysosomes in the absence of increased cell death. These data provide further evidence in support of a role for autophagy in the control of HIV infection and suggest that careful consideration of off-target effects will be essential if HDACi are to be a component of a multipronged approach to eliminate latently infected cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Effects of cryoprotectants on the structure and thermostability of the human carbonic anhydrase II–acetazolamide complex

    Energy Technology Data Exchange (ETDEWEB)

    Aggarwal, Mayank; Boone, Christopher D.; Kondeti, Bhargav; Tu, Chingkuang; Silverman, David N.; McKenna, Robert, E-mail: rmckenna@ufl.edu [University of Florida, 1600 SW Archer Road, PO Box 100245, Gainesville, FL 32610 (United States)

    2013-05-01

    Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Protein X-ray crystallography has seen a progressive shift from data collection at cool/room temperature (277–298 K) to data collection at cryotemperature (100 K) because of its ease of crystal preparation and the lessening of the detrimental effects of radiation-induced crystal damage, with 20–25%(v/v) glycerol (GOL) being the preferred choice of cryoprotectant. Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Comparative studies of crystal structure, kinetics, inhibition and thermostability were performed on CA II and its complex with AZM in the presence of either GOL or sucrose. These results suggest that even though the cryoprotectant GOL was previously shown to be directly bound in the active site and to interact with AZM, it affects neither the thermostability of CA II nor the binding of AZM in the crystal structure or in solution. However, addition of GOL does affect the kinetics of CA II, presumably as it displaces the water proton-transfer network in the active site.

  9. Effects of cryoprotectants on the structure and thermostability of the human carbonic anhydrase II–acetazolamide complex

    International Nuclear Information System (INIS)

    Aggarwal, Mayank; Boone, Christopher D.; Kondeti, Bhargav; Tu, Chingkuang; Silverman, David N.; McKenna, Robert

    2013-01-01

    Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Protein X-ray crystallography has seen a progressive shift from data collection at cool/room temperature (277–298 K) to data collection at cryotemperature (100 K) because of its ease of crystal preparation and the lessening of the detrimental effects of radiation-induced crystal damage, with 20–25%(v/v) glycerol (GOL) being the preferred choice of cryoprotectant. Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Comparative studies of crystal structure, kinetics, inhibition and thermostability were performed on CA II and its complex with AZM in the presence of either GOL or sucrose. These results suggest that even though the cryoprotectant GOL was previously shown to be directly bound in the active site and to interact with AZM, it affects neither the thermostability of CA II nor the binding of AZM in the crystal structure or in solution. However, addition of GOL does affect the kinetics of CA II, presumably as it displaces the water proton-transfer network in the active site

  10. Combined Pan-RAF and MEK Inhibition Overcomes Multiple Resistance Mechanisms to Selective RAF Inhibitors.

    Science.gov (United States)

    Whittaker, Steven R; Cowley, Glenn S; Wagner, Steve; Luo, Flora; Root, David E; Garraway, Levi A

    2015-12-01

    RAF and MEK inhibitors are effective in BRAF-mutant melanoma but not in BRAF-mutant colorectal cancer. To gain additional insights into this difference, we performed a genome-scale pooled shRNA enhancer screen in a BRAF-mutant, RAF inhibitor-resistant colorectal cancer cell line exposed to the selective RAF inhibitor PLX4720. We identified multiple genes along the receptor tyrosine kinase (RTK)/mitogen-activated protein kinase (MAPK) signaling axis that, when suppressed, either genetically or pharmacologically, sensitized cells to the selective RAF inhibitor through sustained inhibition of MAPK signaling. Strikingly, CRAF was a key mediator of resistance that could be overcome by the use of pan-RAF inhibitors in combination with a MEK inhibitor. Furthermore, the combination of pan-RAF and MEK inhibitors displayed strong synergy in melanoma and colorectal cancer cell lines with RAS-activating events such as RTK activation, KRAS mutation, or NF1 loss-of-function mutations. Combinations of selective RAF inhibitors, such as PLX4720 or dabrafenib, with MEK inhibitors did not incur such profound synergy, suggesting that inhibition of CRAF by pan-RAF inhibitors plays a key role in determining cellular response. Importantly, in contrast to the modest activity seen with single-agent treatment, dual pan-RAF and MEK inhibition results in the induction of apoptosis, greatly enhancing efficacy. Notably, combined pan-RAF and MEK inhibition can overcome intrinsic and acquired resistance to single-agent RAF/MEK inhibition, supporting dual pan-RAF and MEK inhibition as a novel therapeutic strategy for BRAF- and KRAS-mutant cancers. ©2015 American Association for Cancer Research.

  11. Understanding the Origins of Time-Dependent Inhibition by Polypeptide Deformylase Inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Totoritis, Rachel; Duraiswami, Chaya; Taylor, Amy N.; Kerrigan, John J.; Campobasso, Nino; Smith, Katherine J.; Ward, Paris; King, Bryan W.; Murrayz-Thompson, Monique; Jones, Amber D.; Van Aller, Glenn S.; Aubart, Kelly M.; Zalacain, Magdalena; Thrall, Sara H.; Meek, Thomas D.; Schwartz, Benjamin (GSKPA)

    2012-03-15

    The continual bacterial adaptation to antibiotics creates an ongoing medical need for the development of novel therapeutics. Polypeptide deformylase (PDF) is a highly conserved bacterial enzyme, which is essential for viability. It has previously been shown that PDF inhibitors represent a promising new area for the development of antimicrobial agents, and that many of the best PDF inhibitors demonstrate slow, time-dependent binding. To improve our understanding of the mechanistic origin of this time-dependent inhibition, we examined in detail the kinetics of PDF catalysis and inhibition by several different PDF inhibitors. Varying pH and solvent isotope led to clear changes in time-dependent inhibition parameters, as did inclusion of NaCl, which binds to the active site metal of PDF. Quantitative analysis of these results demonstrated that the observed time dependence arises from slow binding of the inhibitors to the active site metal. However, we also found several metal binding inhibitors that exhibited rapid, non-time-dependent onset of inhibition. By a combination of structural and chemical modification studies, we show that metal binding is only slow when the rest of the inhibitor makes optimal hydrogen bonds within the subsites of PDF. Both of these interactions between the inhibitor and enzyme were found to be necessary to observe time-dependent inhibition, as elimination of either leads to its loss.

  12. Inhibition of neuraminidase by Ganoderma triterpenoids and implications for neuraminidase inhibitor design

    Science.gov (United States)

    Zhu, Qinchang; Bang, Tran Hai; Ohnuki, Koichiro; Sawai, Takashi; Sawai, Ken; Shimizu, Kuniyoshi

    2015-01-01

    Neuraminidase (NA) inhibitors are the dominant antiviral drugs for treating influenza in the clinic. Increasing prevalence of drug resistance makes the discovery of new NA inhibitors a high priority. Thirty-one triterpenoids from the medicinal mushroom Ganoderma lingzhi were analyzed in an in vitro NA inhibition assay, leading to the discovery of ganoderic acid T-Q and TR as two inhibitors of H5N1 and H1N1 NAs. Structure-activity relationship studies revealed that the corresponding triterpenoid structure is a potential scaffold for the design of NA inhibitors. Using these triterpenoids as probes we found, through further in silico docking and interaction analysis, that interactions with the amino-acid residues Arg292 and/or Glu119 of NA are critical for the inhibition of H5N1 and H1N1. These findings should prove valuable for the design and development of NA inhibitors. PMID:26307417

  13. Evaluation of the inhibition of other metalloproteinases by matrix metalloproteinase inhibitors.

    Science.gov (United States)

    Marcotte, P A; Elmore, I N; Guan, Z; Magoc, T J; Albert, D H; Morgand, D W; Curtin, M L; Garland, R B; Guo, Y; Heyman, H R; Holms, J H; Sheppard, G S; Steinman, D H; Wada, C K; Davidsen, S K

    1999-01-01

    Two series of compounds synthesized as specific matrix metalloproteinase (MMP) inhibitors have been evaluated for their inhibition of non-MMPs. In a series of substituted succinyl hydroxamic acids, some were found to be significant (IC50 inhibitors of leucine (microsomal) aminopeptidase, neprilysin (3.4.24.11), and thermolysin. Macrocyclic compounds in which the alpha carbon of the succinyl hydroxamate is linked to the side chain of the P2' amino acid were found to be good inhibitors of aminopeptidase, but not of neprilysin or thermolysin. Compounds of neither series were found to be significant inhibitors of angiotensin converting enzyme or carboxypeptidase A.

  14. What determines the inhibition effectiveness of ATA, BTAH, and BTAOH corrosion inhibitors on copper?

    Science.gov (United States)

    Kokalj, Anton; Peljhan, Sebastijan; Finsgar, Matjaz; Milosev, Ingrid

    2010-11-24

    Three corrosion inhibitors for copper-3-amino-1,2,4-triazole (ATA), benzotriazole (BTAH), and 1-hydroxybenzotriazole (BTAOH)-were investigated by corrosion experiments and atomistic computer simulations. The trend of corrosion inhibition effectiveness of the three inhibitors on copper in near-neutral chloride solution is determined experimentally as BTAH ≳ ATA ≫ BTAOH. A careful analysis of the results of computer simulations based on density functional theory allowed to pinpoint the superior inhibiting action of BTAH and ATA as a result of their ability to form strong N-Cu chemical bonds in deprotonated form. While these bonds are not as strong as the Cl-Cu bonds, the presence of solvent favors the adsorption of inhibitor molecules onto the surface due to stronger solvation of the Cl(-) anions. Moreover, benzotriazole displays the largest affinity among the three inhibitors to form intermolecular aggregates, such as [BTA-Cu](n) polymeric complex. This is another factor contributing to the stability of the protective inhibitor film on the surface, thus making benzotriazole an outstanding corrosion inhibitor for copper. These findings cannot be anticipated on the basis of inhibitors' molecular electronic properties alone, thus emphasizing the importance of a rigorous modeling of the interactions between the components of the corrosion system in corrosion inhibition studies.

  15. Carbonic anhydrase activity of integral-functional complexes of thylakoid membranes of spinach chloroplasts

    Directory of Open Access Journals (Sweden)

    A. V. Semenihin

    2015-06-01

    Full Text Available Isolated thylakoid membranes were disrupted by treatment with nonionic detergents digitonin or dodecyl maltoside. Solubilized polypeptide complexes were separated by native gel charge shift electrophoresis. The position of ATP-synthase complex and its isolated catalytic part (CF1 within gel was determined using the color reaction for ATPase activity. Due to the presence of cytochromes, the red band in unstained gels corresponded to the cytochrome b6f complex. Localization of the cytochrome b6f complex, ATP synthase and coupling CF1 in the native gel was confirmed by their subunit composition determined after SDS-electrophoretic analysis. Carbonic anhydrase (CA activity in polypeptide zones of PS II, cytochrome b6f complex, and ATP-synthase CF1 was identified in native gels using indicator bromothymol blue. CA activity of isolated CF1 in solution was determined by infrared gas analysis as the rate of bicarbonate dehydration. The water-soluble acetazolamide, an inhibitor of CA, unlike lipophilic ethoxyzolamide inhibited CA activity of CF1. Thus, it was shown for the first time that ATP-synthase has a component which is capable of catalyzing the interconversion of forms of carbonic acid associated with proton exchange. The data obtained suggest the presence of multiple forms of carbonic anhydrase in the thylakoid membranes of spinach chloroplasts and confirm their involvement in the proton transfer to the ATP synthase.

  16. The "SWOT" of BRAF inhibition in melanoma: RAF inhibitors, MEK inhibitors or both?

    Science.gov (United States)

    Nissan, Moriah H; Solit, David B

    2011-12-01

    Activating mutations in the BRAF gene are among the most prevalent kinase mutations in human cancer. BRAF mutations are most frequent in patients with melanoma where they occur in approximately 50% of patients with advanced disease. Remarkable clinical activity has recently been reported with highly selective RAF inhibitors in melanoma patients whose tumors harbor V600E BRAF mutations. The response rates of RAF inhibitors in patients with BRAF-mutant melanomas far exceed the activity level of any prior therapy studied in this disease. The results suggest that we have entered an era of personalized therapy for patients with metastatic melanoma in which treatment selection will be guided by BRAF mutational status. This review will discuss the strengths, weaknesses, opportunities and threats ("SWOT") of developing RAF and MEK selective inhibitors as anti-cancer therapies, recent insights into the mechanisms of intrinsic and acquired resistance to these agents, and current efforts to develop mechanism-based combination therapies.

  17. Inhibition of natural gas hydrates by means of kinetic inhibitors; Inhibierung von Erdgashydraten mit kinetischen Inhibitoren

    Energy Technology Data Exchange (ETDEWEB)

    Eberhardt, E.; Froemmel, J.; Hase, A. [Inst. fuer Erdoel- und Erdgasforschung, Clausthal-Zellerfeld (Germany)

    1997-12-31

    The use of kinetic inhibitors saves considerable costs as compared with thermodynamic inhibition. The effectivity of kinetic inhibitors can be examined by means of screening tests using an agitated autoclave. This contribution describes the experimental set-up and measuring methods used for this purpose and discusses the results obtained. (MSK) [Deutsch] Der Einsatz von kinetischen Inhibitoren fuehrt im Vergleich zur thermodynamischen Inhibition zu einer erheblichen Kostenreduzierung. Die Effektivitaet wird anhand von Screening-Versuchen in einem Ruehrautoklaven geprueft.Im Folgenden werden die Versuchsapparatur und die Messmethodik beschrieben. Ebenso werden die Ergebnisse diskutiert.

  18. Inhibition of xanthine oxidase by the aldehyde oxidase inhibitor raloxifene: implications for identifying molybdopterin nitrite reductases.

    Science.gov (United States)

    Weidert, E R; Schoenborn, S O; Cantu-Medellin, N; Choughule, K V; Jones, J P; Kelley, E E

    2014-02-15

    Sources of nitric oxide alternative to nitric oxide synthases are gaining significant traction as crucial mediators of vessel function under hypoxic inflammatory conditions. For example, capacity to catalyze the one electron reduction of nitrite (NO2-) to ·NO has been reported for hemoglobin, myoglobin and molybdopterin-containing enzymes including xanthine oxidoreductase (XOR) and aldehyde oxidase (AO). For XOR and AO, use of selective inhibition strategies is therefore crucial when attempting to assign relative contributions to nitrite-mediated ·NO formation in cells and tissue. To this end, XOR inhibition has been accomplished with application of classic pyrazolopyrimidine-based inhibitors allo/oxypurinol or the newly FDA-approved XOR-specific inhibitor, Uloric® (febuxostat). Likewise, raloxifene, an estrogen receptor antagonist, has been identified as a potent (Ki=1.0 nM) inhibitor of AO. Herein, we characterize the inhibition kinetics of raloxifene for XOR and describe the resultant effects on inhibiting XO-catalyzed ·NO formation. Exposure of purified XO to raloxifene (PBS, pH 7.4) resulted in a dose-dependent (12.5-100 μM) inhibition of xanthine oxidation to uric acid. Dixon plot analysis revealed a competitive inhibition process with a Ki=13 μM. This inhibitory process was more effective under acidic pH; similar to values encountered under hypoxic/inflammatory conditions. In addition, raloxifene also inhibited anoxic XO-catalyzed reduction of NO2- to NO (EC50=64 μM). In contrast to having no effect on XO-catalyzed uric acid production, the AO inhibitor menadione demonstrated potent inhibition of XO-catalyzed NO2- reduction (EC50=60 nM); somewhat similar to the XO-specific inhibitor, febuxostat (EC50=4 nM). Importantly, febuxostat was found to be a very poor inhibitor of human AO (EC50=613 μM) suggesting its usefulness for validating XO-dependent contributions to NO2- reduction in biological systems. Combined, these data indicate care should be taken

  19. N-acetyl lysyltyrosylcysteine amide inhibits myeloperoxidase, a novel tripeptide inhibitor1[S

    OpenAIRE

    Zhang, Hao; Jing, Xigang; Shi, Yang; Xu, Hao; Du, Jianhai; Guan, Tongju; Weihrauch, Dorothee; Jones, Deron W.; Wang, Weiling; Gourlay, David; Oldham, Keith T.; Hillery, Cheryl A.; Pritchard, Kirkwood A.

    2013-01-01

    Myeloperoxidase (MPO) plays important roles in disease by increasing oxidative and nitrosative stress and oxidizing lipoproteins. Here we report N-acetyl lysyltyrosylcysteine amide (KYC) is an effective inhibitor of MPO activity. We show KYC inhibits MPO-mediated hypochlorous acid (HOCl) formation and nitration/oxidation of LDL. Disulfide is the major product of MPO-mediated KYC oxidation. KYC (⩽4,000 μM) does not induce cytotoxicity in bovine aortic endothelial cells (BAECs). KYC inhibits HO...

  20. Inhibition of YAP function overcomes BRAF inhibitor resistance in melanoma cancer stem cells

    Science.gov (United States)

    Fisher, Matthew L.; Grun, Daniel; Adhikary, Gautam; Xu, Wen; Eckert, Richard L.

    2017-01-01

    Treating BRAF inhibitor-resistant melanoma is an important therapeutic goal. Thus, it is important to identify and target mechanisms of resistance to improve therapy. The YAP1 and TAZ proteins of the Hippo signaling pathway are important drivers of cancer cell survival, and are BRAF inhibitor resistant factors in melanoma. We examine the role of YAP1/TAZ in melanoma cancer stem cells (MCS cells). We demonstrate that YAP1, TAZ and TEAD (TEA domain transcription factor) levels are elevated in BRAF inhibitor resistant MCS cells and enhance cell survival, spheroid formation, matrigel invasion and tumor formation. Moreover, increased YAP1, TAZ and TEAD are associated with sustained ERK1/2 activity that is not suppressed by BRAF inhibitor. Xenograft studies show that treating BRAF inhibitor-resistant tumors with verteporfin, an agent that interferes with YAP1 function, reduces YAP1/TAZ level, restores BRAF inhibitor suppression of ERK1/2 signaling and reduces tumor growth. Verteporfin is highly effective as concentrations of verteporfin that do not impact tumor formation restore BRAF inhibitor suppression of tumor formation, suggesting that co-treatment with agents that inhibit YAP1 and BRAF(V600E) may be a viable therapy for cancer stem cell-derived BRAF inhibitor-resistant melanoma. PMID:29299145

  1. Dipeptidyl peptidase IV inhibitors in diabetes: more than inhibition of glucagon-like peptide-1 metabolism?

    NARCIS (Netherlands)

    Michel, Martin C.; Fliers, Eric; van Noorden, Cornelis J. F.

    2008-01-01

    Inhibitors of the protease dipeptidyl peptidase IV (DPP-IV) are promising new drugs for the treatment of type 2 diabetes. They are thought to act by inhibiting the breakdown of glucagon-like peptide-1 and, thereby, selectively enhancing insulin release under conditions when it is physiologically

  2. A specific and potent inhibitor of glucosylceramide synthase for substrate inhibition therapy of Gaucher disease

    NARCIS (Netherlands)

    McEachern, Kerry Anne; Fung, John; Komarnitsky, Svetlana; Siegel, Craig S.; Chuang, Wei-Lien; Hutto, Elizabeth; Shayman, James A.; Grabowski, Gregory A.; Aerts, Johannes M. F. G.; Cheng, Seng H.; Copeland, Diane P.; Marshall, John

    2007-01-01

    An approach to treating Gaucher disease is substrate inhibition therapy which seeks to abate the aberrant lysosomal accumulation of glucosylceramide. We have identified a novel inhibitor of glucosylceramide synthase (Genz-112638) and assessed its activity in a murine model of Gaucher disease

  3. 5-Substituted-benzylsulfanyl-thiophene-2-sulfonamides with effective carbonic anhydrase inhibitory activity: Solution and crystallographic investigations.

    Science.gov (United States)

    Ivanova, Jekaterīna; Balode, Agnese; Žalubovskis, Raivis; Leitans, Janis; Kazaks, Andris; Vullo, Daniela; Tars, Kaspars; Supuran, Claudiu T

    2017-02-01

    A series of 5-substituted-benzylsulfanyl-thiophene-2-sulfonamides was prepared by reacting 5-bromo-thiophene-2-sulfonamide with 5-substituted-benzyl mercaptans. The new compounds were investigated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. The cytosolic human (h) isoforms hCA I was poorly inhibited by the new sulfonamides (K I s in the range of 683-4250nM), whereas hCA II, and the transmembrane, tumor associated isoforms hCA IX and XII were effectively inhibited in the subnanomolar-nanomolar range. A high resolution X-ray crystal structure of the adduct of hCA II with one of the new sulfonamides allowed us to rationalize the excellent inhibitory activity of these heterocyclic sulfonamides. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Lipoyl-Homotaurine Derivative (ADM_12) Reverts Oxaliplatin-Induced Neuropathy and Reduces Cancer Cells Malignancy by Inhibiting Carbonic Anhydrase IX (CAIX).

    Science.gov (United States)

    Fragai, Marco; Comito, Giuseppina; Di Cesare Mannelli, Lorenzo; Gualdani, Roberta; Calderone, Vito; Louka, Alexandra; Richichi, Barbara; Francesconi, Oscar; Angeli, Andrea; Nocentini, Alessio; Gratteri, Paola; Chiarugi, Paola; Ghelardini, Carla; Tadini-Buoninsegni, Francesco; Supuran, Claudiu T; Nativi, Cristina

    2017-11-09

    Oxaliplatin (OXA) is a valuable and largely used cancer drug which induces a serious and intractable neuropathy. The lipoyl-homotaurine derivative (ADM_12) reverts in vivo OXA-induced neuropathy, and it is an effective antagonist of the nociceptive sensor channel TRPA1. Unprecedentedly, this safe analgesic showed a synergy with OXA in vitro and proved to inhibit CA IX, a relevant therapeutic target, clearly interfering with pancreatic cancer cells' aggressiveness.

  5. Protein C inhibitor acts as a procoagulant by inhibiting the thrombomodulin-induced activation of protein C in human plasma

    NARCIS (Netherlands)

    Elisen, M. G.; von dem Borne, P. A.; Bouma, B. N.; Meijers, J. C.

    1998-01-01

    Protein C inhibitor (PCI), which was originally identified as an inhibitor of activated protein C, also efficiently inhibits coagulation factors such as factor Xa and thrombin. Recently it was found, using purified proteins, that the anticoagulant thrombin-thrombomodulin complex was also inhibited

  6. Lovastatin inhibits VEGFR and AKT activation: synergistic cytotoxicity in combination with VEGFR inhibitors.

    Directory of Open Access Journals (Sweden)

    Tong T Zhao

    Full Text Available 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.

  7. General amyloid inhibitors? A critical examination of the inhibition of IAPP amyloid formation by inositol stereoisomers.

    Directory of Open Access Journals (Sweden)

    Hui Wang

    Full Text Available Islet amyloid polypeptide (IAPP or amylin forms amyloid deposits in the islets of Langerhans; a process that is believed to contribute to the progression of type 2 diabetes and to the failure of islet transplants. An emerging theme in amyloid research is the hypothesis that the toxic species produced during amyloid formation by different polypeptides share common features and exert their effects by common mechanisms. If correct, this suggests that inhibitors of amyloid formation by one polypeptide might be effective against other amyloidogenic sequences. IAPP and Aβ, the peptide responsible for amyloid formation in Alzheimer's disease, are particularly interesting in this regard as they are both natively unfolded in their monomeric states and share some common characteristics. Comparatively little effort has been expended on the design of IAPP amyloid inhibitors, thus it is natural to inquire if Aβ inhibitors are effective against IAPP, especially since no IAPP inhibitors have been clinically approved. A range of compounds inhibit Aβ amyloid formation, including various stereoisomers of inositol. Myo-, scyllo-, and epi-inositol have been shown to induce conformational changes in Aβ and prevent Aβ amyloid fibril formation by stabilizing non-fibrillar β-sheet structures. We investigate the ability of inositol stereoisomers to inhibit amyloid formation by IAPP. The compounds do not induce a conformational change in IAPP and are ineffective inhibitors of IAPP amyloid formation, although some do lead to modest apparent changes in IAPP amyloid fibril morphology. Thus not all classes of Aβ inhibitors are effective against IAPP. This work provides a basis of comparison to work on polyphenol based inhibitors of IAPP amyloid formation and helps provide clues as to the features which render them effective. The study also helps provide information for further efforts in rational inhibitor design.

  8. General Amyloid Inhibitors? A Critical Examination of the Inhibition of IAPP Amyloid Formation by Inositol Stereoisomers

    Science.gov (United States)

    Wang, Hui; Raleigh, Daniel P.

    2014-01-01

    Islet amyloid polypeptide (IAPP or amylin) forms amyloid deposits in the islets of Langerhans; a process that is believed to contribute to the progression of type 2 diabetes and to the failure of islet transplants. An emerging theme in amyloid research is the hypothesis that the toxic species produced during amyloid formation by different polypeptides share common features and exert their effects by common mechanisms. If correct, this suggests that inhibitors of amyloid formation by one polypeptide might be effective against other amyloidogenic sequences. IAPP and Aβ, the peptide responsible for amyloid formation in Alzheimer's disease, are particularly interesting in this regard as they are both natively unfolded in their monomeric states and share some common characteristics. Comparatively little effort has been expended on the design of IAPP amyloid inhibitors, thus it is natural to inquire if Aβ inhibitors are effective against IAPP, especially since no IAPP inhibitors have been clinically approved. A range of compounds inhibit Aβ amyloid formation, including various stereoisomers of inositol. Myo-, scyllo-, and epi-inositol have been shown to induce conformational changes in Aβ and prevent Aβ amyloid fibril formation by stabilizing non-fibrillar β-sheet structures. We investigate the ability of inositol stereoisomers to inhibit amyloid formation by IAPP. The compounds do not induce a conformational change in IAPP and are ineffective inhibitors of IAPP amyloid formation, although some do lead to modest apparent changes in IAPP amyloid fibril morphology. Thus not all classes of Aβ inhibitors are effective against IAPP. This work provides a basis of comparison to work on polyphenol based inhibitors of IAPP amyloid formation and helps provide clues as to the features which render them effective. The study also helps provide information for further efforts in rational inhibitor design. PMID:25260075

  9. MEK inhibition potentiates the activity of Hsp90 inhibitor 17-AAG against pancreatic cancer cells.

    Science.gov (United States)

    Zhang, Tao; Li, Yanyan; Zhu, Zhenkun; Gu, Mancang; Newman, Bryan; Sun, Duxin

    2010-10-04

    The Ras/Raf/MEK/ERK signaling has been implicated in uncontrolled cell proliferation and tumor progression in pancreatic cancer. The purpose of this study is to evaluate the antitumor activity of MEK inhibitor U0126 in combination with Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) in pancreatic cancer cells. Western blotting showed that 17-AAG caused a 2- to 3-fold transient activation of MEK/ERK signaling in pancreatic cancer cells. The activation sustained for 6 h before phospho-ERK (p-ERK) destabilization. The selective MEK inhibitor U0126 completely abolished 17-AAG induced ERK1/2 activation and resulted in more than 80% of phospho-ERK degradation after only 15 min treatment. Moreover, U0126 had complementary effect on 17-AAG regulated oncogenic and cell cycle related proteins. Although 17-AAG downregulated cyclin D1, cyclin E, CDK4 and CDK6, it led to cyclin A and CDK2 accumulation, which was reversed by the addition of U0126. Antiproliferation assay showed that combination of U0126 and 17-AAG resulted in synergistic cytotoxic effect. More importantly, 17-AAG alone only exhibited moderate inhibition of cell migration in vitro, while addition of U0126 dramatically enhanced the inhibitory effect by 2- to 5-fold. Taken together, these data demonstrate that MEK inhibitor U0126 potentiates the activity of Hsp90 inhibitor 17-AAG against pancreatic cancer cells. The combination of Hsp90 and MEK inhibition could provide a promising avenue for the treatment of pancreatic cancer.

  10. [Cyclooxygenase inhibitors in some dietary vegetables inhibit platelet aggregation function induced by arachidonic acid].

    Science.gov (United States)

    Wang, Xin-Hua; Shao, Dong-Hua; Liang, Guo-Wei; Zhang, Ru; Xin, Qin; Zhang, Tao; Cao, Qing-Yun

    2011-10-01

    The study was purposed to investigate whether the cyclooxygenase inhibitors from some dietary vegetables can inhibit platelet aggregation function by the arachidonic acid (AA). The vegetable juice was mixed with platelet rich plasma (PRP), and asprin was used as positive control. The maximum ratio of platelet aggregation induced by AA was measured on the aggregometer; heme and cyclooxygenase-1 (COX(1)) or cyclooxygenase-2 (COX(2)) were added to test tubes containing COX reaction buffer, the mixture was vortex-mixed and exposed to aspirin or vegetable juice, followed by addition of AA and then hydrochloric acid (1 mol/L) was added to stop the COX reaction, followed by chemical reduction with stannous chloride solution. The concentration of COX inhibitors was detected by the enzyme immunoassay kit; vegetable juice (aspirin as positive control) was mixed with whole blood, which was followed by the addition of AA, and then the reaction was stopped by adding indomethacin, centrifuged, then the supernatant was collected, and the plasma thromboxane B(2) (TXB(2)) was measured by radioimmunoassay. The results showed that spinach juice, garlic bolt juice, blanched garlic leave juice and Chinese leek juice could inhibit by 80% human platelet aggregation induced by AA. 4 kinds of vegetables were all found a certain amount of cyclooxygenase inhibitors, which COX(1) and COX(2) inhibitor concentrations of spinach were higher than that of aspirin; 4 vegetable juice could significantly reduce the human plasma concentrations of TXB(2) induced by AA (p < 0.05). It is concluded that 4 kinds of raw vegetables containing cyclooxygenase inhibitors inhibit the production of TXA(2) and thus hinder platelet aggregation. Raw spinach, garlic bolt, blanched garlic and chinese leek inhibit significantly AA-induced human platelet aggregation in vitro. 4 kinds of vegetables may have a good potential perspective of anti-platelet aggregation therapy or prevention of thrombosis.

  11. The acetylcholinesterase inhibitor galantamine inhibits d-amphetamine-induced psychotic-like behavior in Cebus monkeys

    DEFF Research Database (Denmark)

    Andersen, Maibritt B; Werge, Thomas; Fink-Jensen, Anders

    2007-01-01

    allosterically potentiates nicotinic receptor stimulation. To this end, we investigated its ability to antagonize d-amphetamine-induced psychotic-like behavior in extrapyramidal side effects (EPS)-primed Cebus monkeys. Galantamine inhibited d-amphetamine-induced unrest, arousal, and stereotypy. Side effects...... such as emesis, sedation, and EPS were minor or not existing. The results indicate that AChE inhibitors have antipsychotic potentials and suggest that clinical trials investigating antipsychotic effects of AChE inhibitors as monotherapy would be of interest....

  12. 4- and 6-(p-Sulphamoylphenyl)androstenediones: Studies of aromatase inhibitor-based oestrone sulphatase inhibition.

    Science.gov (United States)

    Watari, Yoko; Yamaguchi, Satoshi; Takahashi, Madoka; Nagaoka, Masao; Numazawa, Mitsuteru

    2010-12-01

    4-(p-Sulphamoylphenyl)androstenedione (3) and 6alpha-p-sulphamoylphenyl analogues 12-14 were synthesised and tested as aromatase inhibitors as well as oestrone sulphatase inhibitors in human placental microsomes. All of the p-sulphamoylphenyl compounds synthesised were powerful inhibitors of aromatase with apparent K(i) values ranging between 30 and 97nM. In addition, the aromatase inhibitory activities of 6alpha-p-hydroxyphenyl compounds 9-11, which may be produced from their respective sulphamoylphenyl compounds by action of oestrone sulphatase, were also high in a range of 23 and 75nM of the K(i) values. On the other hand, all of the sulphamoylphenyl compounds were poor inhibitors of oestrone sulphatase with more than about 200microM of IC(25) values. Although the present findings of the oestrone sulphatase inhibition are disappointing, such attempts may be valuable to develop a new class of drugs having a dual function, aromatase inhibitor and oestrone sulphatase inhibitor, for the treatment of oestrogen-dependent breast cancer. Copyright 2010 Elsevier Inc. All rights reserved.

  13. N-acetyl lysyltyrosylcysteine amide inhibits myeloperoxidase, a novel tripeptide inhibitor1[S

    Science.gov (United States)

    Zhang, Hao; Jing, Xigang; Shi, Yang; Xu, Hao; Du, Jianhai; Guan, Tongju; Weihrauch, Dorothee; Jones, Deron W.; Wang, Weiling; Gourlay, David; Oldham, Keith T.; Hillery, Cheryl A.; Pritchard, Kirkwood A.

    2013-01-01

    Myeloperoxidase (MPO) plays important roles in disease by increasing oxidative and nitrosative stress and oxidizing lipoproteins. Here we report N-acetyl lysyltyrosylcysteine amide (KYC) is an effective inhibitor of MPO activity. We show KYC inhibits MPO-mediated hypochlorous acid (HOCl) formation and nitration/oxidation of LDL. Disulfide is the major product of MPO-mediated KYC oxidation. KYC (⩽4,000 μM) does not induce cytotoxicity in bovine aortic endothelial cells (BAECs). KYC inhibits HOCl generation by phorbol myristate acetate (PMA)-stimulated neutrophils and human promyelocytic leukemia (HL-60) cells but not superoxide generation by PMA-stimulated HL-60 cells. KYC inhibits MPO-mediated HOCl formation in BAEC culture and protects BAECs from MPO-induced injury. KYC inhibits MPO-mediated lipid peroxidation of LDL whereas tyrosine (Tyr) and tryptophan (Trp) enhance oxidation. KYC is unique as its isomers do not inhibit MPO activity, or are much less effective. Ultraviolet-visible spectral studies indicate KYC binds to the active site of MPO and reacts with compounds I and II. Docking studies show the Tyr of KYC rests just above the heme of MPO. Interestingly, KYC increases MPO-dependent H2O2 consumption. These data indicate KYC is a novel and specific inhibitor of MPO activity that is nontoxic to endothelial cell cultures. Accordingly, KYC may be useful for treating MPO-mediated vascular disease. PMID:23883583

  14. N-acetyl lysyltyrosylcysteine amide inhibits myeloperoxidase, a novel tripeptide inhibitor.

    Science.gov (United States)

    Zhang, Hao; Jing, Xigang; Shi, Yang; Xu, Hao; Du, Jianhai; Guan, Tongju; Weihrauch, Dorothee; Jones, Deron W; Wang, Weiling; Gourlay, David; Oldham, Keith T; Hillery, Cheryl A; Pritchard, Kirkwood A

    2013-11-01

    Myeloperoxidase (MPO) plays important roles in disease by increasing oxidative and nitrosative stress and oxidizing lipoproteins. Here we report N-acetyl lysyltyrosylcysteine amide (KYC) is an effective inhibitor of MPO activity. We show KYC inhibits MPO-mediated hypochlorous acid (HOCl) formation and nitration/oxidation of LDL. Disulfide is the major product of MPO-mediated KYC oxidation. KYC (≤4,000 μM) does not induce cytotoxicity in bovine aortic endothelial cells (BAECs). KYC inhibits HOCl generation by phorbol myristate acetate (PMA)-stimulated neutrophils and human promyelocytic leukemia (HL-60) cells but not superoxide generation by PMA-stimulated HL-60 cells. KYC inhibits MPO-mediated HOCl formation in BAEC culture and protects BAECs from MPO-induced injury. KYC inhibits MPO-mediated lipid peroxidation of LDL whereas tyrosine (Tyr) and tryptophan (Trp) enhance oxidation. KYC is unique as its isomers do not inhibit MPO activity, or are much less effective. Ultraviolet-visible spectral studies indicate KYC binds to the active site of MPO and reacts with compounds I and II. Docking studies show the Tyr of KYC rests just above the heme of MPO. Interestingly, KYC increases MPO-dependent H₂O₂ consumption. These data indicate KYC is a novel and specific inhibitor of MPO activity that is nontoxic to endothelial cell cultures. Accordingly, KYC may be useful for treating MPO-mediated vascular disease.

  15. Peptide Inhibitor of Complement C1 Inhibits the Peroxidase Activity of Hemoglobin and Myoglobin

    Directory of Open Access Journals (Sweden)

    Pamela S. Hair

    2017-01-01

    Full Text Available Hemoglobin is the natural carrier of oxygen in red blood cells (RBCs. While intracellular hemoglobin provides life-sustaining oxygen transport, extracellular free hemoglobin displays toxicity due to inherent peroxidase activity generating reactive oxygen species that subsequently react with the hemoglobin molecule to produce toxic heme degradation products resulting in free radicals, oxidative stress damage, and lipid peroxidation. We have recently demonstrated that Peptide Inhibitor of Complement C1 (PIC1 inhibits peroxidase activity of the heme-based enzyme myeloperoxidase. To elucidate whether PIC1 could inhibit peroxidase activity of hemoglobin, we evaluated the consequence of PIC1 on RBC lysates, methemoglobin, and myoglobin using tetramethylbenzidine (TMB as an oxidation target. PIC1 reversibly and dose-dependently prevented TMB oxidation to tetramethylbenzidine diimine by RBC lysates, methemoglobin, and myoglobin, having comparable activity to the inhibitor 4-aminobenzoic acid hydrazide. PIC1 inhibited TMB oxidation of RBC lysates similar to L-cysteine suggesting that the two cysteine residues contained in PIC1 may mediate peroxidase activity. PIC1 also inhibited heme destruction by NaOCl for RBC lysates, hemoglobin, and myoglobin as assayed by preservation of the Soret absorbance peak in the presence of NaOCl and reduction in free iron release. In conclusion, PIC1 inhibits peroxidase activity of hemoglobin and myoglobin likely via an antioxidant mechanism.

  16. Inhibition of TGF-β Signaling in Tumor Cells by Small Molecule Src Family Kinase Inhibitors.

    Science.gov (United States)

    Bartscht, Tobias; Rosien, Benjamin; Rades, Dirk; Kaufmann, Roland; Biersack, Harald; Lehnerta, Hendrik; Ungefroren, Hendrik

    2017-01-01

    In a series of studies carried out over the last couple of years in various cell types, it was observed that the experimentally used Src family kinase inhibitors PP1 and PP2 and the clinically used Src/Abl inhibitors AZM475271 and dasatinib are potent inhibitors of TGF-β mediated cellular responses such as Smad and p38 mitogen-activated protein kinase phosphorylation, Smad-dependent transcriptional activation, growth inhibition, epithelial-mesenchymal transition (EMT), and cell motility. While for PP1/PP2 it was demonstrated that these agents directly inhibit the kinase activity of the TGF-β type I receptor activin receptor-like kinase 5, the mechanism of the anti-TGF-β effect of AZM475271 and dasatinib is less clear. In contrast, the anti-TGF-β effect of yet another Src/Abl inhibitor, bosutinib, is more variable with respect to the type of the TGF-β response and the cell type affected, and lacks a clear dose-dependency. In the light of their strong anti-activin receptor-like kinase 5 kinase effect, PP1 and PP2 should not be used when studying the role of c-Src as downstream mediators in TGF-β/activin receptor-like kinase 5 signaling. On the other hand, based upon in vitro findings, it is conceivable that part of the therapeutic effects of AZM475271 and dasatinib seen in preclinical and clinical studies with solid tumors was caused by inhibition of prometastatic TGF-β rather than Src signaling. If AZM475271 and dasatinib can indeed act as dual Src / TGF-β inhibitors in vivo, this may be beneficial for prevention of metastatic disease in more advanced tumor stages. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  17. Renin-angiotensin-aldosterone system inhibition: overview of the therapeutic use of angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, mineralocorticoid receptor antagonists, and direct renin inhibitors.

    Science.gov (United States)

    Mercier, Kelly; Smith, Holly; Biederman, Jason

    2014-12-01

    Angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) therapy in hypertensive diabetic patients with macroalbuminuria, microalbuminuria, or normoalbuminuria has been repeatedly shown to improve cardiovascular mortality and reduce the decline in glomerular filtration rate. Renin-angiotensin-aldosterone system (RAAS) blockade in normotensive diabetic patients with normoalbuminuria or microalbuminuria cannot be advocated at present. Dual RAAS inhibition with ACE inhibitors plus ARBs or ACE inhibitors plus direct renin inhibitors has failed to improve cardiovascular or renal outcomes but has predisposed patients to serious adverse events. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Inhibition of receptor-bound urokinase by plasminogen-activator inhibitors

    DEFF Research Database (Denmark)

    Ellis, Vincent John; Wun, T C; Behrendt, N

    1990-01-01

    activator inhibitors (PAI-1 and PAI-2). uPA bound to its receptor on human U937 monocyte-like cells was inhibited by PAI-1 (in its active form in the presence of vitronectin fragments) with an association rate constant of 4.5 x 10(6) M-1 s-1, which was 40% lower than that obtained for uPA in solution (7.9 x...

  19. Mechanisms of inhibition of HIV replication by nonnucleoside reverse transcriptase inhibitors

    OpenAIRE

    Sluis-Cremer, Nicolas; Tachedjian, Gilda

    2008-01-01

    The nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are a therapeutic class of compounds that are routinely used, in combination with other antiretroviral drugs, to treat HIV-1 infection. NNRTIs primarily block HIV-1 replication by preventing RT from completing reverse transcription of the viral single-stranded RNA genome into DNA. However, some NNRTIs, such as efavirenz, have been shown to inhibit the late stages of HIV-1 replication by interfering with HIV-1 Gag-Pol polyprotein...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-04-18

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

  1. A Pharmacological Inhibitor of the Protease Taspase1 Effectively Inhibits Breast and Brain Tumor Growth

    Science.gov (United States)

    Chen, David Y.; Lee, Yishan; Van Tine, Brian A.; Searleman, Adam C.; Westergard, Todd D.; Liu, Han; Tu, Ho-Chou; Takeda, Shugaku; Dong, Yiyu; Piwnica-Worms, David R.; Oh, Kyoung J.; Korsmeyer, Stanley J.; Hermone, Ann; Gussio, Richard; Shoemaker, Robert H.; Cheng, Emily H.-Y.; Hsieh, James J.-D.

    2011-01-01

    The threonine endopeptidase Taspase1 has a critical role in cancer cell proliferation and apoptosis. In this study, we developed and evaluated small molecule inhibitors of Taspase1 as a new candidate class of therapeutic modalities. Genetic deletion of Taspase1 in the mouse produced no overt deficiencies, suggesting the possibility of a wide therapeutic index for use of Taspase1 inhibitors in cancers. We defined the peptidyl motifs recognized by Taspase1 and conducted a cell-based dual-fluorescent proteolytic screen of the NCI diversity library to identify Taspase1 inhibitors (TASPINs). Based on secondary and tertiary screens the 4-[(4-arsonophenyl)methyl]phenyl]arsonic acid NSC48300 was determined to be the most specific active compound. Structure-activity relationship studies indicated a crucial role for the arsenic acid moiety in mediating Taspase1 inhibition. Additional FRET-based kinetic analysis characterized NSC48300 as a reversible, non-competitive inhibitor of Taspase1 (KI = 4.22 μM). In the MMTV-neu mouse model of breast cancer and the U251 xenograft model of brain cancer NSC48300 produced effective tumor growth inhibition. Our results offer an initial preclinical proof of concept to develop TASPINs for cancer therapy. PMID:22166309

  2. A pharmacologic inhibitor of the protease Taspase1 effectively inhibits breast and brain tumor growth.

    Science.gov (United States)

    Chen, David Y; Lee, Yishan; Van Tine, Brian A; Searleman, Adam C; Westergard, Todd D; Liu, Han; Tu, Ho-Chou; Takeda, Shugaku; Dong, Yiyu; Piwnica-Worms, David R; Oh, Kyoung J; Korsmeyer, Stanley J; Hermone, Ann; Gussio, Richard; Shoemaker, Robert H; Cheng, Emily H-Y; Hsieh, James J-D

    2012-02-01

    The threonine endopeptidase Taspase1 has a critical role in cancer cell proliferation and apoptosis. In this study, we developed and evaluated small molecule inhibitors of Taspase1 as a new candidate class of therapeutic modalities. Genetic deletion of Taspase1 in the mouse produced no overt deficiencies, suggesting the possibility of a wide therapeutic index for use of Taspase1 inhibitors in cancers. We defined the peptidyl motifs recognized by Taspase1 and conducted a cell-based dual-fluorescent proteolytic screen of the National Cancer Institute diversity library to identify Taspase1 inhibitors (TASPIN). On the basis of secondary and tertiary screens the 4-[(4-arsonophenyl)methyl]phenyl] arsonic acid NSC48300 was determined to be the most specific active compound. Structure-activity relationship studies indicated a crucial role for the arsenic acid moiety in mediating Taspase1 inhibition. Additional fluorescence resonance energy transfer-based kinetic analysis characterized NSC48300 as a reversible, noncompetitive inhibitor of Taspase1 (K(i) = 4.22 μmol/L). In the MMTV-neu mouse model of breast cancer and the U251 xenograft model of brain cancer, NSC48300 produced effective tumor growth inhibition. Our results offer an initial preclinical proof-of-concept to develop TASPINs for cancer therapy. ©2011 AACR.

  3. Inhibition of estrone sulfatase by aromatase inhibitor-based estrogen 3-sulfamates.

    Science.gov (United States)

    Numazawa, Mitsuteru; Tominaga, Takako; Watari, Yoko; Tada, Yasue

    2006-05-01

    Our rationale is based on the finding that estrone 3-sulfamate (EMATE, 2d), a typical estrone sulfatase (ES) inhibitor, can be hydrolyzed and the pharmacological effect of the free estrogen contributes to the bioactivity of the sulfamate. A number of 3-sulfamoylated derivatives of the good aromatase inhibitors, 2- and 4-halogeno (F, Cl, and Br) estrones and their estradiol analogs as well as 6beta-methyl and phenyl estrones, were synthesized and evaluated as inhibitors of ES in human placental microsomes in comparison with the lead compound EMATE. Among them, 2-chloro- and 2-bromoestrone 3-sulfamates (2b and 2c), along with their estradiol analogs 3b and 3c, were powerful competitive inhibitors with K(i)'s ranging between 4.0 and 11.3 nM (K(i) for EMATE, 73 nM). These four sulfamates as well as the 2-fluoro analogs 2a and 3a inactivated ES in a time-dependent manner more efficiently than EMATE, and 2-halogeno estrone sulfamates 2 also caused a concentration-dependent loss of ES activity. The results may be useful for developing a new class of drugs having a dual function, ES inhibition and aromatase inhibition, for the treatment of breast cancer.

  4. STRUCTURAL ASPECTS OF STRONG INHIBITION AND ROLE OF SCAFFOLD FOR SERINE PROTEASE INHIBITORS

    Directory of Open Access Journals (Sweden)

    Jhimli Dasgupta

    2011-12-01

    Full Text Available Canonical serine protease inhibitors inhibit their cognate enzymes by binding tightly at the enzyme active site in a substrate-like manner, being cleaved extremely slowly compared to a true substrate. They interact with cognate enzymes through P3-P2 region of the inhibitory loop while the scaffold hardly makes any contact. Neighbouring scaffolding residues like arginine or asparagine shape-up the inhibitory loop and religate the cleaved scissile bond. The specificity of the inhibitor can be altered by mutating the hyper solvent accessible P1 residue without changing loop-scaffold interactions. To understand the loop-scaffold compatibility, we prepared three chimeric proteins ECIL-WCIS , ETIL-WCIS , and STIL-WCIS , where the inhibitory loops of ECI, ETI, and STI were placed on the scaffold of their homologue WCI. Results showed that although ECIL-WCIS and STIL-WCIS behave like inhibitors, ETIL-WCIS behaves like a substrate. Crystal structure of ETIL-WCIS and its comparison with ETI indicated that three novel scaffolding residues Trp88, Arg74, and Tyr113 in ETI act as barrier to confine the inhibitory loop to canonical conformation. Absence of this barrier in the scaffold of WCI makes the inhibitory loop flexible in ETIL-WCIS leading to a loss of canonical conformation, explaining its substrate-like behaviour. Furthermore, complex structures of the inhibitors with their cognate enzymes indicate that rigidification of the inhibitory loop at the enzyme active site is necessary for efficient inhibition.

  5. Beyond topoisomerase inhibition: antitumor 1,4-naphthoquinones as potential inhibitors of human monoamine oxidase.

    Science.gov (United States)

    Coelho-Cerqueira, Eduardo; Netz, Paulo A; do Canto, Vanessa P; Pinto, Angelo C; Follmer, Cristian

    2014-04-01

    Monoamine oxidase (MAO) action has been involved in the regulation of neurotransmitters levels, cell signaling, cellular growth, and differentiation as well as in the balance of the intracellular polyamine levels. Although so far obscure, MAO inhibitors are believed to have some effect on tumors progression. 1,4-naphthoquinone (1,4-NQ) has been pointed out as a potential pharmacophore for inhibition of both MAO and DNA topoisomerase activities, this latter associated with antitumor activity. Herein, we demonstrated that certain antitumor 1,4-NQs, including spermidine-1,4-NQ, lapachol, and nor-lapachol display inhibitory activity on human MAO-A and MAO-B. Kinetic studies indicated that these compounds are reversible and competitive MAO inhibitors, being the enzyme selectivity greatly affected by substitutions on 1,4-NQ ring. Molecular docking studies suggested that the most potent MAO inhibitors are capable to bind to the MAO active site in close proximity of flavin moiety. Furthermore, ability to inhibit both MAO-A and MAO-B can be potentialized by the formation of hydrogen bonds between these compounds and FAD and/or the residues in the active site. Although spermidine-1,4-NQs exhibit antitumor action primarily by inhibiting topoisomerase via DNA intercalation, our findings suggest that their effect on MAO activity should be taken into account when their application in cancer therapy is considered. © 2013 John Wiley & Sons A/S.

  6. Hedgehog pathway inhibition in chondrosarcoma using the smoothened inhibitor IPI-926 directly inhibits sarcoma cell growth.

    Science.gov (United States)

    Campbell, Veronica T; Nadesan, Puviindran; Ali, S Amanda; Wang, Chang Ye Yale; Whetstone, Heather; Poon, Raymond; Wei, Qingxia; Keilty, John; Proctor, Jennifer; Wang, Lauren W; Apte, Suneel S; McGovern, Karen; Alman, Benjamin A; Wunder, Jay S

    2014-05-01

    Hedgehog (Hh) pathway inhibition in cancer has been evaluated in both the ligand-independent and ligand-dependent settings, where Hh signaling occurs either directly within the cancer cells or within the nonmalignant cells of the tumor microenvironment. Chondrosarcoma is a malignant tumor of cartilage in which there is ligand-dependent activation of Hh signaling. IPI-926 is a potent, orally delivered small molecule that inhibits Hh pathway signaling by binding to Smoothened (SMO). Here, the impact of Hh pathway inhibition on primary chondrosarcoma xenografts was assessed. Mice bearing primary human chondrosarcoma xenografts were treated with IPI-926. The expression levels of known Hh pathway genes, in both the tumor and stroma, and endpoint tumor volumes were measured. Gene expression profiling of tumors from IPI-926-treated mice was conducted to identify potential novel Hh target genes. Hh target genes were studied to determine their contribution to the chondrosarcoma neoplastic phenotype. IPI-926 administration results in downmodulation of the Hh pathway in primary chondrosarcoma xenografts, as demonstrated by evaluation of the Hh target genes GLI1 and PTCH1, as well as inhibition of tumor growth. Chondrosarcomas exhibited autocrine and paracrine Hh signaling, and both were affected by IPI-926. Decreased tumor growth is accompanied by histopathologic changes, including calcification and loss of tumor cells. Gene profiling studies identified genes differentially expressed in chondrosarcomas following IPI-926 treatment, one of which, ADAMTSL1, regulates chondrosarcoma cell proliferation. These studies provide further insight into the role of the Hh pathway in chondrosarcoma and provide a scientific rationale for targeting the Hh pathway in chondrosarcoma.

  7. Multifaceted mechanisms of HIV inhibition and resistance to CCR5 inhibitors PSC-RANTES and Maraviroc.

    Science.gov (United States)

    Lobritz, Michael A; Ratcliff, Annette N; Marozsan, Andre J; Dudley, Dawn M; Tilton, John C; Arts, Eric J

    2013-06-01

    Small-molecule CCR5 antagonists, such as maraviroc (MVC), likely block HIV-1 through an allosteric, noncompetitive inhibition mechanism, whereas inhibition by agonists such as PSC-RANTES is less defined and may involve receptor removal by cell surface downregulation, competitive inhibition by occluding the HIV-1 envelope binding, and/or allosteric effects by altering CCR5 conformation. We explored the inhibitory mechanisms of maraviroc and PSC-RANTES by employing pairs of virus clones with differential sensitivities to these inhibitors. Intrinsic PSC-RANTES-resistant virus (YA versus RT) or those selected in PSC-RANTES treated macaques (M584 versus P3-4) only displayed resistance in multiple-cycle assays or with a CCR5 mutant that cannot be downregulated. In single-cycle assays, these HIV-1 clones displayed equal sensitivity to PSC-RANTES inhibition, suggesting effective receptor downregulation. Prolonged PSC-RANTES exposure resulted in desensitization of the receptor to internalization such that increasing virus concentration (substrate) could saturate the receptors and overcome PSC-RANTES inhibition. In contrast, resistance to MVC was observed with the MVC-resistant HIV-1 (R3 versus S2) in both multiple- and single-cycle assays and with altered virus concentrations, which is indicative of allosteric inhibition. MVC could also mediate inhibition and possibly resistance through competitive mechanisms.

  8. Pulse treatment with the proteasome inhibitor bortezomib inhibits osteoclast resorptive activity in clinically relevant conditions

    DEFF Research Database (Denmark)

    Boissy, P; Levin Andersen, Thomas; Lund, T

    2008-01-01

    Myeloma bone disease is due to bone degradation by osteoclasts, and absence of repair by bone forming osteoblasts. Recent observations suggest that the anti-myeloma drug bortezomib, a proteasome inhibitor, stimulates bone formation and may inhibit bone resorption. Here, we tested bortezomib...... on cultured osteoclasts in conditions mimicking the pulse treatment used in the clinic, thereby avoiding continuous proteasome inhibition and unselective toxicity. A 3h pulse with 25nM bortezomib followed by a 3-day culture in its absence markedly inhibited osteoclast activity as evaluated through bone...... cells drastically reduced their survival. We measured next the levels of two bone resorption markers in patients during the 3 days following five and seven therapeutic bortezomib administrations, respectively. These levels decreased significantly already 1-2 days after injection, and then increased...

  9. Proton pump inhibitors inhibit metformin uptake by organic cation transporters (OCTs.

    Directory of Open Access Journals (Sweden)

    Anne T Nies

    Full Text Available Metformin, an oral insulin-sensitizing drug, is actively transported into cells by organic cation transporters (OCT 1, 2, and 3 (encoded by SLC22A1, SLC22A2, or SLC22A3, which are tissue specifically expressed at significant levels in various organs such as liver, muscle, and kidney. Because metformin does not undergo hepatic metabolism, drug-drug interaction by inhibition of OCT transporters may be important. So far, comprehensive data on the interaction of proton pump inhibitors (PPIs with OCTs are missing although PPIs are frequently used in metformin-treated patients. Using in silico modeling and computational analyses, we derived pharmacophore models indicating that PPIs (i.e. omeprazole, pantoprazole, lansoprazole, rabeprazole, and tenatoprazole are potent OCT inhibitors. We then established stably transfected cell lines expressing the human uptake transporters OCT1, OCT2, or OCT3 and tested whether these PPIs inhibit OCT-mediated metformin uptake in vitro. All tested PPIs significantly inhibited metformin uptake by OCT1, OCT2, and OCT3 in a concentration-dependent manner. Half-maximal inhibitory concentration values (IC(50 were in the low micromolar range (3-36 µM and thereby in the range of IC(50 values of other potent OCT drug inhibitors. Finally, we tested whether the PPIs are also transported by OCTs, but did not identify PPIs as OCT substrates. In conclusion, PPIs are potent inhibitors of the OCT-mediated metformin transport in vitro. Further studies are needed to elucidate the clinical relevance of this drug-drug interaction with potential consequences on metformin disposition and/or efficacy.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-07-25

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

  11. BRAF/MEK inhibitors promote CD47 expression that is reversible by ERK inhibition in melanoma.

    Science.gov (United States)

    Liu, Fen; Jiang, Chen Chen; Yan, Xu Guang; Tseng, Hsin-Yi; Wang, Chun Yan; Zhang, Yuan Yuan; Yari, Hamed; La, Ting; Farrelly, Margaret; Guo, Su Tang; Thorne, Rick F; Jin, Lei; Wang, Qi; Zhang, Xu Dong

    2017-09-19

    The expression of CD47 on the cancer cell surface transmits "don't eat me" signalling that not only inhibits phagocytosis of cancer cells by phagocytes but also impairs anti-cancer T cell responses. Here we report that oncogenic activation of ERK plays an important role in transcriptional activation of CD47 through nuclear respiratory factor 1 (NRF-1) in melanoma cells. Treatment with BRAF/MEK inhibitors upregulated CD47 in cultured melanoma cells and fresh melanoma isolates. Similarly, melanoma cells selected for resistance to the BRAF inhibitor vemurafenib expressed higher levels of CD47. The increase in CD47 expression was mediated by ERK signalling, as it was associated with rebound activation of ERK and co-knockdown of ERK1/2 by siRNA diminished upregulation of CD47 in melanoma cells after exposure to BRAF/MEK inhibitors. Furthermore, ERK1/2 knockdown also reduced the constitutive expression of CD47 in melanoma cells. We identified a DNA fragment that was enriched with the consensus binding sites for NRF-1 and was transcriptionally responsive to BRAF/MEK inhibitor treatment. Knockdown of NRF-1 inhibited the increase in CD47, indicating that NRF-1 has a critical role in transcriptional activation of CD47 by ERK signalling. Functional studies showed that melanoma cells resistant to vemurafenib were more susceptible to macrophage phagocytosis when CD47 was blocked. So these results suggest that NRF-1-mediated regulation of CD47 expression is a novel mechanism by which ERK signalling promotes the pathogenesis of melanoma, and that the combination of CD47 blockade and BRAF/MEK inhibitors may be a useful approach for improving their therapeutic efficacy.

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

    International Nuclear Information System (INIS)

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

    2014-01-01

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

  13. Drug design studies of the novel antitumor targets carbonic anhydrase IX and XII.

    Science.gov (United States)

    Guler, O Ozensoy; De Simone, G; Supuran, C T

    2010-01-01

    The carbonic anhydrase (CA, EC 4.2.1.1) isozymes IX and XII are predominantly found in tumor cells and show a restricted expression in normal tissues. By efficiently hydrating carbon dioxide to protons and bicarbonate, these CAs contribute significantly to the extracellular acidification of solid tumors. CA IX and XII are overexpressed in many such tumors in response to the hypoxia inducible factor (HIF) pathway, and research on the involvement of these isozymes in cancer has progressed in recent years. The report of the X-ray crystal structure of CA IX, which is a dimeric protein with a quaternary structure not evidenced earlier for this family of enzymes, allows for structure-based drug design campaigns of inhibitors against this novel antitumor target. Indeed, it has been known for some time that aromatic/ heterocyclic sulfonamides and sulfamates have good affinity for this isoform, but generally they do not show specificity for the inhibition of the tumor-associated isoform versus the remaining CA isozymes (CA I-VII, and XII-XV) found in mammals. Recently, we reported several classes of compounds with good selectivity for the tumor-associated CAs, being shown that CA IX/XII inhibition reverses the effect of tumor acidification, leading to inhibition of the cancer cells growth. CA IX/XII are now proposed as novel therapeutic antitumor targets. Furthermore, as some types of CA inhibitors (CAIs), such as the fluorescent sulfonamides accumulate only in hypoxic tumor cells overexpressing these enzymes, CAIs may be also used as diagnostic tools for imaging of hypoxic cancer cells. Work from several laboratories recently reported the proof-of-concept studies for the use of CA IX/XII inhibitors as well as antibodies both in the therapy and imaging of hypoxic tumors.

  14. Apyrase inhibitors enhance the ability of diverse fungicides to inhibit the growth of different plant-pathogenic fungi.

    Science.gov (United States)

    Kumar Tripathy, Manas; Weeraratne, Gayani; Clark, Greg; Roux, Stanley J

    2017-09-01

    A previous study has demonstrated that the treatment of Arabidopsis plants with chemical inhibitors of apyrase enzymes increases their sensitivity to herbicides. In this study, we found that the addition of the same or related apyrase inhibitors could potentiate the ability of different fungicides to inhibit the growth of five different pathogenic fungi in plate growth assays. The growth of all five fungi was partially inhibited by three commonly used fungicides: copper octanoate, myclobutanil and propiconazole. However, when these fungicides were individually tested in combination with any one of four different apyrase inhibitors (AI.1, AI.10, AI.13 or AI.15), their potency to inhibit the growth of five fungal pathogens was increased significantly relative to their application alone. The apyrase inhibitors were most effective in potentiating the ability of copper octanoate to inhibit fungal growth, and least effective in combination with propiconazole. Among the five pathogens assayed, that most sensitive to the fungicide-potentiating effects of the inhibitors was Sclerotinia sclerotiorum. Overall, among the 60 treatment combinations tested (five pathogens, four apyrase inhibitors, three fungicides), the addition of apyrase inhibitors increased significantly the sensitivity of fungi to the fungicide treatments in 53 of the combinations. Consistent with their predicted mode of action, inhibitors AI.1, AI.10 and AI.13 each increased the level of propiconazole retained in one of the fungi, suggesting that they could partially block the ability of efflux transporters to remove propiconazole from these fungi. © 2016 BSPP AND JOHN WILEY & SONS LTD.

  15. Enhanced Inhibition of ERK Signaling by a Novel Allosteric MEK Inhibitor, CH5126766, That Suppresses Feedback Reactivation of RAF Activity

    Science.gov (United States)

    Joseph, Eric W.; Ohara, Kazuhiro; Miura, Takaaki; Sakamoto, Hiroshi; Matsuda, Yutaka; Tomii, Yasushi; Tachibana-Kondo, Yukako; Iikura, Hitoshi; Aoki, Toshihiro; Shimma, Nobuo; Arisawa, Mikio; Sowa, Yoshihiro; Poulikakos, Poulikos I.; Rosen, Neal; Aoki, Yuko; Sakai, Toshiyuki

    2014-01-01

    Tumors with mutant RAS are often dependent on extracellular signal–regulated kinase (ERK) signaling for growth; however, MEK inhibitors have only marginal antitumor activity in these tumors. MEK inhibitors relieve ERK-dependent feedback inhibition of RAF and cause induction of MEK phosphorylation. We have now identified a MEK inhibitor, CH5126766 (RO5126766), that has the unique property of inhibiting RAF kinase as well. CH5126766 binding causes MEK to adopt a conformation in which it cannot be phosphorylated by and released from RAF. This results in formation of a stable MEK/RAF complex and inhibition of RAF kinase. Consistent with this mechanism, this drug does not induce MEK phosphorylation. CH5126766 inhibits ERK signaling output more effectively than a standard MEK inhibitor that induces MEK phosphorylation and has potent antitumor activity as well. These results suggest that relief of RAF feedback limits pathway inhibition by standard MEK inhibitors. CH5126766 represents a new type of MEK inhibitor that causes MEK to become a dominant-negative inhibitor of RAF and that, in doing so, may have enhanced therapeutic activity in ERK-dependent tumors with mutant RAS. PMID:23667175

  16. Inhibition of dihydroceramide desaturase activity by the sphingosine kinase inhibitor SKI II.

    Science.gov (United States)

    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. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

  17. Characteristics of the Inhibition of Potato (Solanum tuberosum) Invertase by an Endogenous Proteinaceous Inhibitor in Potatoes

    Science.gov (United States)

    Bracho, Geracimo E.; Whitaker, John R.

    1990-01-01

    Effect of several parameters on inhibition of potato (Solanum tuberosum) invertase by its endogenous proteinaceous inhibitor was determined using homogeneous preparations of both proteins. The inhibitor and invertase formed an inactive complex with an observed association rate constant at pH 4.70 and 37°C of 8.82 × 102 per molar per second and a dissociation rate constant of 3.3 × 10−3 per minute. The inhibitor appeared to bind to invertase in more than one step. Initial interaction (measured by loss of invertase activity) was rapid, relatively weak, readily reversible (Ki of 2 × 10−6 molar) and noncompetitive with substrate at pH 4.70. Initial interaction was probably followed by isomerization to a tighter (Ki of 6.23 × 10−8 molar) complex, which dissociated slowly with a half-time of 3.5 hour. Interaction between enzyme and inhibitor appeared to be of ionic character and essentially pH independent between pH 3.5 and 7.4. PMID:16667286

  18. [Inhibition of aggregation of dissociated embryonic Amphibian cells by inhibitors of protein and RNA synthesis].

    Science.gov (United States)

    Grunz, Horst

    1969-06-01

    1. Aggregation of embryonic Amphibian cells dissociated by Trypsin or EDTA is supressed reversibly by actidion (cycloheximid 0.5-2 Μg/ml), an inhibitor of protein synthesis. Protein synthesis is inhibited more than 90% at these concentrations. The inhibition by actidion was still reversible after 48 hours, when the cells were transferred to normal medium. Actidion added to cells, cultured in Flickinger solution for 3 h, was no longer able to stop further aggregation. 2. Puromycin reversibly suppresses aggregation at a concentration of 20 (Μg/ml, when protein synthesis is inhibited to 66 %. 3. After EDTA-dissociation the formation of initial small clusters of 5-10 cells (primary aggregation) occurred even in the presence of inhibitors of protein synthesis. The primary aggregation is however suppressed if actidion or puromycin are already added during the dissociation procedure. 4. In the presence of actinomycin-D (1-2 Μg/ml) the cells continue to aggregate like the controls. After 30 h further aggregation is stopped and the aggregates loose single cells. 5. Differences in the aggregation of cells after EDTA and Trypsin treatment are discussed.

  19. EFFECTS OF PESTICIDES AND PLANT HORMONES ON CARBONIC ANHYDRASE ACTIVITY IN SPINACH (Spinacia oleraceae L. cv. Gladiatör)

    OpenAIRE

    ODABAŞOĞLU, Fehmi; KÜFREVİOĞLU Ö., İrfan

    2001-01-01

    In this study, the 6-7 weeks old spinach (Spinacia oleraceae L. cv. Gladiatör) seedlings were grown up for 15-days after pesticide, hormone and pesticide+hormone had sprayed, and carbonic anhydrase activities were established in the extracted leaves. The established carbonic anhydrase (CA) activities, were compared with treated plants and control plants. We determined that CA activity was inhibited by all pesticides. All hormones which were applied in three different concentrations except 10&...

  20. The histone deacetylase inhibitor, Vorinostat, represses hypoxia inducible factor 1 alpha expression through translational inhibition.

    Directory of Open Access Journals (Sweden)

    Darren M Hutt

    Full Text Available Hypoxia inducible factor 1α (HIF-1α is a master regulator of tumor angiogenesis being one of the major targets for cancer therapy. Previous studies have shown that Histone Deacetylase Inhibitors (HDACi block tumor angiogenesis through the inhibition of HIF-1α expression. As such, Vorinostat (Suberoylanilide Hydroxamic Acid/SAHA and Romidepsin, two HDACis, were recently approved by the Food and Drug Administration (FDA for the treatment of cutaneous T cell lymphoma. Although HDACis have been shown to affect HIF-1α expression by modulating its interactions with the Hsp70/Hsp90 chaperone axis or its acetylation status, the molecular mechanisms by which HDACis inhibit HIF-1α expression need to be further characterized. Here, we report that the FDA-approved HDACi Vorinostat/SAHA inhibits HIF-1α expression in liver cancer-derived cell lines, by a new mechanism independent of p53, prolyl-hydroxylases, autophagy and proteasome degradation. We found that SAHA or silencing of HDAC9 mechanism of action is due to inhibition of HIF-1α translation, which in turn, is mediated by the eukaryotic translation initiation factor--eIF3G. We also highlighted that HIF-1α translation is dramatically inhibited when SAHA is combined with eIF3H silencing. Taken together, we show that HDAC activity regulates HIF-1α translation, with HDACis such as SAHA representing a potential novel approach for the treatment of hepatocellular carcinoma.

  1. Diuretics with carbonic anhydrase inhibitory action: a patent and literature review (2005 - 2013).

    Science.gov (United States)

    Carta, Fabrizio; Supuran, Claudiu T

    2013-01-01

    The benzothiadiazines and high ceiling diuretics (hydrochlorothiazide, hydroflumethiazide, quinethazone, metolazone, chlorthalidone, indapamide, furosemide and bumetanide) contain primary sulfamoyl moieties acting as zinc-binding groups in the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). These drugs are widely used clinically and were recently shown to weakly inhibit isoforms CA I and II, but to possess stronger activity against isoforms involved in other important pathologies, for example, obesity, cancer, epilepsy and hypertension. The class of clinically used diuretics, with CA inhibitory properties, is the main topic of the review. A patent literature review covering the period from 2005 to 2013 is presented. This section presents an overview of the patent literature in the sulfonamide diuretic field. Most of the patents deal with the combination of diuretic sulfonamide CA inhibitors with other agents useful in the management of cardiovascular diseases and obesity. Such combinations exert a better therapeutic activity compared to similar diuretics that do not inhibit CAs, raising the question of the polypharmacological and drug repositioning effects of these old drugs. These effects seem to be due to the potent inhibition of such drugs against CA isoforms present in kidneys and blood vessels, which explain both the blood pressure lowering effects as well as organ-protective activity of the drugs. An explanation of these data is provided by the fact that inhibition of the renal CAs leads to a large increase of the nitrite excretion in urine, suggesting that renal CAs are involved in nitrite reabsorption in humans. Important lessons for the drug design of sulfonamide CA inhibitors (CAIs) can be drawn from these data.

  2. In vivo effects of radioactive properties of Tl-201 on human carbonic anhydrase activity

    Science.gov (United States)

    Sahin, Ali; Senturk, Murat

    2017-04-01

    Carbonic anhydrase (CA) is a family of metalloenzymes that requires Zn as a cofactor and catalyze the quick conversion of CO2 to HCO3- and H+. Inhibitors of the carbonic anhydrases (CAs) have medical usage of significant diseases such as glaucoma, epilepsy, gastroduodenal ulcers, acid-base disequilibria and neurological disorders. The most useful radioisotope, Tl-201, decays by electron capture, emitting Hg X-rays ( 70-80 keV), and photons of 135 and 167 keV in 10% total abundance. Therefore, it has good imaging characteristics without excessive patient radiation dose. It is the most popular isotope used for thallium 201 nuclear cardiac stress tests. In the present study, In vivo inhibitory effect of Tl-201 (Thallium-201) on human erythrocyte carbonic anhydrase (CA) activity were investigated.

  3. Carbonic anhydrase activity from the gilthead sea bream (Sparus aurata) liver: the toxicological effects of heavy metals.

    Science.gov (United States)

    Kaya, Elif Duygu; Söyüt, Hakan; Beydemir, Şükrü

    2013-09-01

    Many studies have shown that metal ions may lead to oxidative stress in biological systems. Accordingly, DNA damage, protein modification, enzyme inhibition and activation, lipid peroxidation and many other effects may occur in living organisms. Many different formations of metal ions may enter human cells along with water, air, and various foods, and humans are negatively affected by these conditions, either directly or indirectly. These effects may cause irreversible damage to human metabolism. In this study, the toxicological effects of heavy metals on carbonic anhydrase enzyme activity from the gilthead sea bream liver were investigated. The carbonic anhydrase enzyme was purified via affinity chromatography and had a specific activity of 6775.5EUmg(-1). The kinetics and characteristic properties, such as optimum pH, stable pH, optimum temperature, activation energy (Ea), activation enthalpy (ΔH), Q10, Km, and Vmax, were determined for the purified enzyme SDS-polyacrylamide gel electrophoresis showed a single band and molecular weight of the subunit was approximately 25kDa. Cd(II), Cu(II), Ni(II) and Ag(I) inhibited the enzyme activity in vitro. The type of inhibition and Ki values for these metals were calculated from Lineweaver-Burk plots as 17.74mM, 36.20mM, 12.85mM and 0.025mM for Cd(II), Cu(II), Ni(II) and Ag(I), respectively. All the metals were noncompetitive inhibitors. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

  4. The M358R variant of α{sub 1}-proteinase inhibitor inhibits coagulation factor VIIa

    Energy Technology Data Exchange (ETDEWEB)

    Sheffield, William P., E-mail: sheffiel@mcmaster.ca [Canadian Blood Services, Centre for Innovation, Hamilton, Ontario (Canada); Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario (Canada); Bhakta, Varsha [Canadian Blood Services, Centre for Innovation, Hamilton, Ontario (Canada)

    2016-02-12

    The naturally occurring M358R mutation of the plasma serpin α{sub 1}-proteinase inhibitor (API) changes both its cleavable reactive centre bond to Arg–Ser and the efficacy with which it inhibits different proteases, reducing the rate of inhibition of neutrophil elastase, and enhancing that of thrombin, factor XIa, and kallikrein, by several orders of magnitude. Although another plasma serpin with an Arg–Ser reactive centre, antithrombin (AT), has been shown to inhibit factor VIIa (FVIIa), no published data are available with respect to FVIIa inhibition by API M358R. Recombinant bacterially-expressed API M358R and plasma-derived AT were therefore compared using gel-based and kinetic assays of FVIIa integrity and activity. Under pseudo-first order conditions of excess serpin over protease, both AT and API M358R formed denaturation-resistant inhibitory complexes with FVIIa in reactions accelerated by TF; AT, but not API M358R, also required heparin for maximal activity. The second order rate constant for heparin-independent API M358R-mediated FVIIa inhibition was determined to be 7.8 ± 0.8 × 10{sup 2} M{sup −1}sec{sup −1}. We conclude that API M358R inhibits FVIIa by forming inhibitory complexes of the serpin type more rapidly than AT in the absence of heparin. The likely 20-fold excess of API M358R over AT in patient plasma during inflammation raises the possibility that it could contribute to the hemorrhagic tendencies manifested by rare individuals expressing this mutant serpin. - Highlights: • The inhibitory specificity of the serpin alpha-1-proteinase inhibitor (API) is sharply altered in the M358R variant. • API M358R forms denaturation-resistant complexes with coagulation factor VIIa at a rate accelerated by tissue factor but unaffected by heparin. • Complex formation was shown by gel-based assays and quantified kinetically by inhibition of FVIIa-dependent amidolysis.

  5. Intracellular serine protease inhibitor SERPINB4 inhibits granzyme M-induced cell death.

    Directory of Open Access Journals (Sweden)

    Pieter J A de Koning

    Full Text Available Granzyme-mediated cell death is the major pathway for cytotoxic lymphocytes to kill virus-infected and tumor cells. In humans, five different granzymes (i.e. GrA, GrB, GrH, GrK, and GrM are known that all induce cell death. Expression of intracellular serine protease inhibitors (serpins is one of the mechanisms by which tumor cells evade cytotoxic lymphocyte-mediated killing. Intracellular expression of SERPINB9 by tumor cells renders them resistant to GrB-induced apoptosis. In contrast to GrB, however, no physiological intracellular inhibitors are known for the other four human granzymes. In the present study, we show that SERPINB4 formed a typical serpin-protease SDS-stable complex with both recombinant and native human GrM. Mutation of the P2-P1-P1' triplet in the SERPINB4 reactive center loop completely abolished complex formation with GrM and N-terminal sequencing revealed that GrM cleaves SERPINB4 after P1-Leu. SERPINB4 inhibited GrM activity with a stoichiometry of inhibition of 1.6 and an apparent second order rate constant of 1.3×10(4 M(-1 s(-1. SERPINB4 abolished cleavage of the macromolecular GrM substrates α-tubulin and nucleophosmin. Overexpression of SERPINB4 in tumor cells inhibited recombinant GrM-induced as well as NK cell-mediated cell death and this inhibition depended on the reactive center loop of the serpin. As SERPINB4 is highly expressed by squamous cell carcinomas, our results may represent a novel mechanism by which these tumor cells evade cytotoxic lymphocyte-induced GrM-mediated cell death.

  6. Inhibition of receptor-bound urokinase by plasminogen-activator inhibitors

    DEFF Research Database (Denmark)

    Ellis, Vincent John; Wun, T C; Behrendt, N

    1990-01-01

    Urokinase-type plasminogen activator (uPA) binds to a specific receptor on various cell types, the bound molecule retaining its enzymatic activity against plasminogen. We have now investigated whether receptor-bound uPA also retains the ability to react with and be inhibited by plasminogen...... activator inhibitors (PAI-1 and PAI-2). uPA bound to its receptor on human U937 monocyte-like cells was inhibited by PAI-1 (in its active form in the presence of vitronectin fragments) with an association rate constant of 4.5 x 10(6) M-1 s-1, which was 40% lower than that obtained for uPA in solution (7.9 x...... 10(6) M-1 s-1). The inhibition of uPA by PAI-2 was decreased to a similar extent by receptor binding, falling from 5.3 x 10(5) to 3.3 x 10(5) M-1 s-1. Stimulation of U937 cells with phorbol 12-myristate 13-acetate was accompanied by a further reduction in receptor-bound uPA inhibition by PAI-1...

  7. Factor VII activating protease (FSAP) promotes the proteolysis and inhibition of tissue factor pathway inhibitor (TFPI)

    Science.gov (United States)

    Kanse, Sandip M.; Declerck, Paul J.; Ruf, Wolfram; Broze, George; Etscheid, Michael

    2013-01-01

    Objectives Factor VII activating protease (FSAP) activates FVII as well as pro-urokinase and inhibits platelet-derived growth factor-BB, thus regulating haemostasis- and remodeling-associated processes in the vasculature. A genetic variant of FSAP (Marburg I polymorphism) results in low enzymatic activity and is associated with an enhanced risk for carotid stenosis and stroke. We postulate that there are additional substrates for FSAP that will help to explain its role in vascular biology and have searched for such a substrate. Results and Methods Using screening procedures to determine the influence of FSAP on various haemostasis-related processes on endothelial cells we discovered that FSAP inhibited tissue factor pathway inhibitor (TFPI), a major anti-coagulant secreted by these cells. Proteolytic degradation of TFPI by FSAP could also be demonstrated by Western blotting and the exact cleavage sites were determined by N-terminal sequencing. The Marburg I variant of FSAP had a diminished ability to inhibit TFPI. A monoclonal antibody to FSAP, that specifically inhibited FSAP binding to TFPI, reversed the inhibitory effect of FSAP on TFPI. Conclusions The identification of TFPI as a sensitive substrate for FSAP increases our understanding of its role in regulating haemostasis and proliferative remodeling events in the vasculature. PMID:22116096

  8. An update on anticancer drug development and delivery targeting carbonic anhydrase IX

    Directory of Open Access Journals (Sweden)

    Justina Kazokaitė

    2017-11-01

    Full Text Available The expression of carbonic anhydrase (CA IX is up-regulated in many types of solid tumors in humans under hypoxic and acidic microenvironment. Inhibition of CA IX enzymatic activity with selective inhibitors, antibodies or labeled probes has been shown to reverse the acidic environment of solid tumors and reduce the tumor growth establishing the significant role of CA IX in tumorigenesis. Thus, the development of potent antitumor drugs targeting CA IX with minimal toxic effects is important for the target-specific tumor therapy. Recently, several promising antitumor agents against CA IX have been developed to treat certain types of cancers in combination with radiation and chemotherapy. Here we review the inhibition of CA IX by small molecule compounds and monoclonal antibodies. The methods of enzymatic assays, biophysical methods, animal models including zebrafish and Xenopus oocytes, and techniques of diagnostic imaging to detect hypoxic tumors using CA IX-targeted conjugates are discussed with the aim to overview the recent progress related to novel therapeutic agents that target CA IX in hypoxic tumors.

  9. Inhibitors

    Science.gov (United States)

    ... Icon View public health webinars on blood disorders Inhibitors Language: English (US) Español (Spanish) Recommend on Facebook ... because treatment of bleeds becomes less effective. About Inhibitors People with hemophilia, and many with VWD type ...

  10. Isthmin is a novel secreted angiogenesis inhibitor that inhibits tumour growth in mice

    Science.gov (United States)

    Xiang, Wei; Ke, Zhiyuan; Zhang, Yong; Ho-Yuet Cheng, Grace; Irwan, Ishak Darryl; Sulochana, K N; Potturi, Padma; Wang, Zhengyuan; Yang, He; Wang, Jingyu; Zhuo, Lang; Kini, R Manjunatha; Ge, Ruowen

    2011-01-01

    Abstract Anti-angiogenesis represents a promising therapeutic strategy for the treatment of various malignancies. Isthmin (ISM) is a gene highly expressed in the isthmus of the midbrain–hindbrain organizer in Xenopus with no known functions. It encodes a secreted 60 kD protein containing a thrombospondin type 1 repeat domain in the central region and an adhesion-associated domain in MUC4 and other proteins (AMOP) domain at the C-terminal. In this work, we demonstrate that ISM is a novel angiogenesis inhibitor. Recombinant mouse ISM inhibited endothelial cell (EC) capillary network formation on Matrigel through its C-terminal AMOP domain. It also suppressed vascular endothelial growth factor (VEGF)-basic fibroblast growth factor (bFGF) induced in vivo angiogenesis in mouse. It mitigated VEGF-stimulated EC proliferation without affecting EC migration. Furthermore, ISM induced EC apoptosis in the presence of VEGF through a caspase-dependent pathway. ISM binds to αvβ5 integrin on EC surface and supports EC adhesion. Overexpression of ISM significantly suppressed mouse B16 melanoma tumour growth through inhibition of tumour angiogenesis without affecting tumour cell proliferation. Knockdown of isthmin in zebrafish embryos using morpholino antisense oligonucleotides led to disorganized intersegmen-tal vessels in the trunk. Our results demonstrate that ISM is a novel endogenous angiogenesis inhibitor with functions likely in physiological as well as pathological angiogenesis. PMID:19874420

  11. Small molecule inhibitor regorafenib inhibits RET signaling in neuroblastoma cells and effectively suppresses tumor growthin vivo.

    Science.gov (United States)

    Chen, Zhenghu; Zhao, Yanling; Yu, Yang; Pang, Jonathan C; Woodfield, Sarah E; Tao, Ling; Guan, Shan; Zhang, Huiyuan; Bieerkehazhi, Shayahati; Shi, Yan; Patel, Roma; Vasudevan, Sanjeev A; Yi, Joanna S; Muscal, Jodi A; Xu, Guo-Tong; Yang, Jianhua

    2017-11-28

    Neuroblastoma (NB), the most common extracranial pediatric solid tumor, continues to cause significant cancer-related morbidity and mortality in children. Dysregulation of oncogenic receptor tyrosine kinases (RTKs) has been shown to contribute to tumorigenesis in various human cancers and targeting these RTKs has had therapeutic benefit. RET is an RTK which is commonly expressed in NB, and high expression of RET correlates with poor outcomes in patients with NB. Herein we report that RET is required for NB cell proliferation and that the small molecule inhibitor regorafenib (BAY 73-4506) blocks glial cell derived neurotrophic factor (GDNF)-induced RET signaling in NB cells and inhibits NB growth both in vitro and in vivo . We found that regorafenib significantly inhibited cell proliferation and colony formation ability of NB cells. Moreover, regorafenib suppressed tumor growth in both an orthotopic xenograft NB mouse model and a TH-MYCN transgenic NB mouse model. Finally, regorafenib markedly improved the overall survival of TH-MYCN transgenic tumor-bearing mice. In summary, our study suggests that RET is a potential therapeutic target in NB, and that using a novel RET inhibitor, like regorafenib, should be investigated as a therapeutic treatment option for children with NB.

  12. Inhibition of hydrate formation by kinetic inhibitors. Literature study; Inhibierung von Erdgashydraten durch kinetische Inhibitoren. Literaturstudie

    Energy Technology Data Exchange (ETDEWEB)

    Eberhardt, E.; Meyn, V.; Rahimian, I. [Institut fuer Erdoel- und Erdgasforschung, Clausthal-Zellerfeld (Germany)

    2000-04-01

    The aim of this study was to represent the state-of-the art of the inhibition of gas hydrates. Corresponding to recent publications the kinetic inhibition was considered in particular. Special inhibitors were validated using a set of criteria derived from different experimental test methods. Best results were obtained by the application of terpolymer VC-713 especially in relation to nucleation and crystal growth, followed by PVCap (polyvinylcaprolactame) and THI (threshold hydrate inhibitor), the chemical structure of which is derived from the antifreeze glycopeptids of antarcitc winter flounder. (orig.) [German] Die vorliegende Literaturstudie gibt den derzeitigen Stand der Kenntnis zur Inhibierung von Gashydraten wieder. Entsprechend der neueren Literatur wird insbesondere auf die kinetische Inhibierung eingegangen. Zur Beurteilung der verschiedenen Inhibitoren werden Bewertungskriterien zur Validierung der mit unterschiedlichen Untersuchungsmethoden erzielten experimentellen Ergebnisse angegeben. Anhand dieser Vorgehensweise zeigte sich, dass mit dem Terpolymer VC-713 die besten Ergebnisse, insbesondere im Hinblick auf Keimbildung und Wachstum, erzielt werden konnten. Sehr gute Ergebnisse wurden auch mit dem Polyvinylcaprolactam (PVCap) und den aus den Antigefrierpeptiden der antarktischen Winterflunder abgeleiteten Threshold Hydrate Inhibitoren (THI) erhalten. (orig.)

  13. GS143, an IκB ubiquitination inhibitor, inhibits allergic airway inflammation in mice

    International Nuclear Information System (INIS)

    Hirose, Koichi; Wakashin, Hidefumi; Oki, Mie; Kagami, Shin-ichiro; Suto, Akira; Ikeda, Kei; Watanabe, Norihiko; Iwamoto, Itsuo; Furuichi, Yasuhiro; Nakajima, Hiroshi

    2008-01-01

    Asthma is characterized by airway inflammation with intense eosinophil infiltration and mucus hyper-production, in which antigen-specific Th2 cells play critical roles. Nuclear factor-κB (NF-κB) pathway has been demonstrated to be essential for the production of Th2 cytokines and chemokines in the airways in murine asthma models. In the present study, we examined the effect of GS143, a novel small-molecule inhibitor of IκB ubiquitination, on antigen-induced airway inflammation and Th2 cytokine production in mice. Intranasal administration of GS143 prior to antigen challenge suppressed antigen-induced NF-κB activation in the lung of sensitized mice. Intranasal administration of GS143 also inhibited antigen-induced eosinophil and lymphocyte recruitment into the airways as well as the expression of Th2 cytokines and eotaxin in the airways. Moreover, GS143 inhibited antigen-induced differentiation of Th2 cells but not of Th1 cells in vitro. Taken together, these results suggest that IκB ubiquitination inhibitor may have therapeutic potential against asthma

  14. Therapeutic TNF Inhibitors can Differentially Stabilize Trimeric TNF by Inhibiting Monomer Exchange

    Science.gov (United States)

    van Schie, Karin A.; Ooijevaar-de Heer, Pleuni; Dijk, Lisanne; Kruithof, Simone; Wolbink, Gertjan; Rispens, Theo

    2016-01-01

    Tumor necrosis factor (TNF) is a homotrimeric cytokine that is a key mediator of inflammation. It is unstable at physiological concentrations and slowly converts into an inactive form. Here, we investigated the mechanism of this process by using a Förster resonance energy transfer (FRET) assay that allowed monitoring of monomeric subunit exchange in time. We observed continuous exchange of monomeric subunits even at concentrations of TNF high enough to maintain its bioactivity. The kinetics of this process closely corresponds with the appearance of monomeric subunits and disappearance of trimeric TNF in time at ng/ml concentrations as monitored by high-performance size-exclusion chromatography (HP-SEC). Furthermore, of the five therapeutic TNF inhibitors that are currently used in the clinic, three (adalimumab, infliximab, etanercept) were found to completely inhibit the monomer exchange reaction and stabilize TNF trimers, whereas golimumab and certolizumab could not prevent monomer exchange, but did slow down the exchange process. These differences were not correlated with the affinities of the TNF inhibitors, measured with both surface plasmon resonance (SPR) and in fluid phase using fluorescence-assisted HP-SEC. The stabilizing effect of these TNF inhibitors might result in prolonged residual TNF bioactivity under conditions of incomplete blocking, as observed in vitro for adalimumab. PMID:27605058

  15. Vanadium(V) complexes in enzyme systems: aqueous chemistry, inhibition and molecular modeling in inhibitor design.

    Science.gov (United States)

    Bhattacharyya, S; Tracey, A S

    2001-05-01

    Vanadate in aqueous solution is known to influence a number of enzyme-catalyzed reactions. Such effects are well known to carry over to living systems where numerous responses to the influence of vanadium have been well-documented; perhaps the most studied being the insulin-mimetic effect. Studies of the aqueous chemistry of vanadate provide an insight into the mechanisms by which vanadate affects enzyme systems and suggests methods for the elucidation of specific types of responses. Studies of the corresponding enzymes provide complementary information that suggests model vanadate systems be studied and provides clues as to functional groups that might be utilized in the development of selective enzyme inhibition. The insulin-mimetic effect is thought by many workers to originate in the effectiveness of vanadium as an inhibitor of protein tyrosine phosphatase (PTPase) activity. One, or more PTPases regulate the phosphotyrosine levels of the insulin receptor kinase domain. Appropriate ligands allow modification of the reactivity and function of vanadate. For instance, although the complex, ((CH(3))(2)NO)(2)V(O)OH, is not quite as good an inhibitor of PTPase activity as is vanadate, it is much more effective in cell cultures for increasing glucose transport and glycogen synthesis. Studies of the chemistry of this complex provide an explanation of the efficacy of this compound as a PTPase inhibitor that is supported by computer modeling studies. Computer calculations using X-ray data of known PTPases as a basis for homology modeling then suggests functionality that needs to be addressed in developing selective PTPase inhibitors.

  16. Plant-derived protease inhibitors LC-pi (Lavatera cashmeriana) inhibit human lung cancer cell proliferation in vitro.

    Science.gov (United States)

    Rakashanda, Syed; Qazi, Asif Khurshid; Majeed, Rabiya; Andrabi, Syed Mubashir; Hamid, Abid; Sharma, P R; Amin, Shajrul

    2015-01-01

    The objective of this study was to check the anticancer activity of purified protease inhibitors of Lavatera cashmeriana viz LC-pi I, II, III, and IV (Lavatera cashmeriana protease inhibitors) on A549 (lung) cell. It was found that LC-pi I and II significantly inhibited the proliferation of A549 cells with IC₅₀ value of 54 μg/ml and 38 μg/ml, respectively, whereas inhibition by LC-pi III and IV was negligible. LC-pi I and II were further found to inhibit formation of colonies in a dose-dependent manner. Also, both inhibitors were found to induce apoptosis causing chromatin condensation and DNA fragmentation, without loss of mitochondrial membrane potential. Cell cycle revealed a significant increase of subG₀/G₁ phase cells that are apoptotic cells. We also demonstrated a dose-dependent decrease in migration of A549 cells on cell migration assay by both inhibitors. Taken together, we demonstrate that LC-pi I and II inhibited proliferation through arresting cells before apoptosis, inducing apoptosis and inhibiting cell migration in human lung cancer cells, but the study warrants further investigation. Our results support the notion that plant protease inhibitors may have the potential to advance as chemopreventive agents.

  17. Sorafenib enhances proteasome inhibitor-mediated cytotoxicity via inhibition of unfolded protein response and keratin phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Honma, Yuichi; Harada, Masaru, E-mail: msrharada@med.uoeh-u.ac.jp

    2013-08-15

    Hepatocellular carcinoma (HCC) is highly resistant to conventional systemic therapies and prognosis for advanced HCC patients remains poor. Recent studies of the molecular mechanisms responsible for tumor initiation and progression have identified several potential molecular targets in HCC. Sorafenib is a multi-kinase inhibitor shown to have survival benefits in advanced HCC. It acts by inhibiting the serine/threonine kinases and the receptor type tyrosine kinases. In preclinical experiments sorafenib had anti-proliferative activity in hepatoma cells and it reduced tumor angiogenesis and increased apoptosis. Here, we demonstrate for the first time that the cytotoxic mechanisms of sorafenib include its inhibitory effects on protein ubiquitination, unfolded protein response (UPR) and keratin phosphorylation in response to endoplasmic reticulum (ER) stress. Moreover, we show that combined treatment with sorafenib and proteasome inhibitors (PIs) synergistically induced a marked increase in cell death in hepatoma- and hepatocyte-derived cells. These observations may open the way to potentially interesting treatment combinations that may augment the effect of sorafenib, possibly including drugs that promote ER stress. Because sorafenib blocked the cellular defense mechanisms against hepatotoxic injury not only in hepatoma cells but also in hepatocyte-derived cells, we must be careful to avoid severe liver injury. -- Graphical abstract: Display Omitted -- Highlights: •We examined the cytotoxic mechanisms of sorafenib in hepatoma cells. •Sorafenib induces cell death via apoptotic and necrotic fashion. •Sorafenib inhibits protein ubiquitination and unfolded protein response. •Autophagy induced by sorafenib may affect its cytotoxicity. •Sorafenib inhibits keratin phosphorylation and cytoplasmic inclusion formation.

  18. Inhibition of chlorine-induced lung injury by the type 4 phosphodiesterase inhibitor rolipram

    International Nuclear Information System (INIS)

    Chang, Weiyuan; Chen, Jing; Schlueter, Connie F.; Rando, Roy J.; Pathak, Yashwant V.; Hoyle, Gary W.

    2012-01-01

    Chlorine is a highly toxic respiratory irritant that when inhaled causes epithelial cell injury, alveolar-capillary barrier disruption, airway hyperreactivity, inflammation, and pulmonary edema. Chlorine is considered a chemical threat agent, and its release through accidental or intentional means has the potential to result in mass casualties from acute lung injury. The type 4 phosphodiesterase inhibitor rolipram was investigated as a rescue treatment for chlorine-induced lung injury. Rolipram inhibits degradation of the intracellular signaling molecule cyclic AMP. Potential beneficial effects of increased cyclic AMP levels include inhibition of pulmonary edema, inflammation, and airway hyperreactivity. Mice were exposed to chlorine (whole body exposure, 228–270 ppm for 1 h) and were treated with rolipram by intraperitoneal, intranasal, or intramuscular (either aqueous or nanoemulsion formulation) delivery starting 1 h after exposure. Rolipram administered intraperitoneally or intranasally inhibited chlorine-induced pulmonary edema. Minor or no effects were observed on lavage fluid IgM (indicative of plasma protein leakage), KC (Cxcl1, neutrophil chemoattractant), and neutrophils. All routes of administration inhibited chlorine-induced airway hyperreactivity assessed 1 day after exposure. The results of the study suggest that rolipram may be an effective rescue treatment for chlorine-induced lung injury and that both systemic and targeted administration to the respiratory tract were effective routes of delivery. -- Highlights: ► Chlorine causes lung injury when inhaled and is considered a chemical threat agent. ► Rolipram inhibited chlorine-induced pulmonary edema and airway hyperreactivity. ► Post-exposure rolipram treatments by both systemic and local delivery were effective. ► Rolipram shows promise as a rescue treatment for chlorine-induced lung injury.

  19. Inhibition of chlorine-induced lung injury by the type 4 phosphodiesterase inhibitor rolipram

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Weiyuan; Chen, Jing; Schlueter, Connie F. [Department of Environmental and Occupational Health Sciences, School of Public Health and Information Sciences, University of Louisville, Louisville, KY (United States); Rando, Roy J. [Department of Environmental Health Sciences, School of Public Health and Tropical Medicine, Tulane University Health Sciences Center, New Orleans, LA (United States); Pathak, Yashwant V. [College of Pharmacy, University of South Florida, Tampa, FL (United States); Hoyle, Gary W., E-mail: Gary.Hoyle@louisville.edu [Department of Environmental and Occupational Health Sciences, School of Public Health and Information Sciences, University of Louisville, Louisville, KY (United States)

    2012-09-01

    Chlorine is a highly toxic respiratory irritant that when inhaled causes epithelial cell injury, alveolar-capillary barrier disruption, airway hyperreactivity, inflammation, and pulmonary edema. Chlorine is considered a chemical threat agent, and its release through accidental or intentional means has the potential to result in mass casualties from acute lung injury. The type 4 phosphodiesterase inhibitor rolipram was investigated as a rescue treatment for chlorine-induced lung injury. Rolipram inhibits degradation of the intracellular signaling molecule cyclic AMP. Potential beneficial effects of increased cyclic AMP levels include inhibition of pulmonary edema, inflammation, and airway hyperreactivity. Mice were exposed to chlorine (whole body exposure, 228–270 ppm for 1 h) and were treated with rolipram by intraperitoneal, intranasal, or intramuscular (either aqueous or nanoemulsion formulation) delivery starting 1 h after exposure. Rolipram administered intraperitoneally or intranasally inhibited chlorine-induced pulmonary edema. Minor or no effects were observed on lavage fluid IgM (indicative of plasma protein leakage), KC (Cxcl1, neutrophil chemoattractant), and neutrophils. All routes of administration inhibited chlorine-induced airway hyperreactivity assessed 1 day after exposure. The results of the study suggest that rolipram may be an effective rescue treatment for chlorine-induced lung injury and that both systemic and targeted administration to the respiratory tract were effective routes of delivery. -- Highlights: ► Chlorine causes lung injury when inhaled and is considered a chemical threat agent. ► Rolipram inhibited chlorine-induced pulmonary edema and airway hyperreactivity. ► Post-exposure rolipram treatments by both systemic and local delivery were effective. ► Rolipram shows promise as a rescue treatment for chlorine-induced lung injury.

  20. Synthesis, characterization, computational studies, antimicrobial activities and carbonic anhydrase inhibitor effects of 2-hydroxy acetophenone-N-methyl p-toluenesulfonylhydrazone and its Co(II), Pd(II), Pt(II) complexes

    Science.gov (United States)

    Özbek, Neslihan; Alyar, Saliha; Memmi, Burcu Koçak; Gündüzalp, Ayla Balaban; Bahçeci, Zafer; Alyar, Hamit

    2017-01-01

    2-Hydroxyacetophenone-N-methyl p-toluenesulfonylhydrazone (afptsmh) derived from p-toluenesulfonicacid-1-methylhydrazide (ptsmh) and its Co(II), Pd(II), Pt(II) complexes were synthesized for the first time. Synthesized compounds were characterized by spectroscopic methods (FT-IR, 1Hsbnd 13C NMR, LC-MS, UV-vis), magnetic susceptibility and conductivity measurements. 1H and 13C shielding tensors for crystal structure of ligand were calculated with GIAO/DFT/B3LYP/6-311++G(d,p) methods in CDCl3. The vibrational band assignments were performed at B3LYP/6-311++G(d,p) theory level combined with scaled quantum mechanics force field (SQMFF) methodology. The antibacterial activities of synthesized compounds were studied against some Gram positive and Gram negative bacteria by using microdilution and disc diffusion methods. In vitro enzyme inhibitory effects of the compounds were measured by UV-vis spectrophotometer. The enzyme activities against human carbonic anhydrase II (hCA II) were evaluated as IC50 (the half maximal inhibitory concentration) values. It was found that afptsmh and its metal complexes have inhibitory effects on hCA II isoenzyme. General esterase activities were determined using alpha and beta naphtyl acetate substrates (α- and β-NAs) of Drosophila melanogaster (D. melanogaster). Activity results show that afptsmh does not strongly affect the bacteria strains and also shows poor inhibitory activity against hCAII isoenzyme whereas all complexes posses higher biological activities.

  1. Genetics Home Reference: carbonic anhydrase VA deficiency

    Science.gov (United States)

    ... bicarbonate. Bicarbonate is necessary to maintain the proper acid-base balance in the body, which is necessary for most biological reactions to proceed properly. The carbonic anhydrase VA enzyme ...

  2. Kinetic analysis of the inhibition of matrix metalloproteinases: lessons from the study of tissue inhibitors of metalloproteinases.

    Science.gov (United States)

    Willenbrock, Frances; Thomas, Daniel A; Amour, Augustin

    2010-01-01

    Tissue inhibitors of metalloproteinases (TIMPs) are a group of highly potent inhibitors of matrix metalloproteinases (MMPs) and disintegrin metalloproteinases (ADAMs). The high affinity and "tight-binding" nature of the inhibition of MMPs or ADAMs by TIMPs presents challenges for the determination of both equilibrium and dissociation rate constants of these inhibitory events. Methodologies that enable some of these challenges to be overcome are described in this chapter and represent valuable lessons for the in vitro assessment of MMP or ADAM inhibitors within a drug discovery context.

  3. Oral administration of FAK inhibitor TAE226 inhibits the progression of peritoneal dissemination of colorectal cancer

    Energy Technology Data Exchange (ETDEWEB)

    Hao, Hui-fang [Department of Gastroenterological Surgery, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikatacho, Kita-ku, Okayama 700-8558 (Japan); Takaoka, Munenori [Department of General Surgery, Kawasaki Medical School, 2-1-80 Nakasange, Kita-ku, Okayama 700-8505 (Japan); Bao, Xiao-hong [Department of Gastroenterological Surgery, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikatacho, Kita-ku, Okayama 700-8558 (Japan); Wang, Zhi-gang [College of Life Science, Inner Mongolia University, The Key Laboratory of Mammal Reproductive Biology and Biotechnology, Ministry of Education, Hohhot 010021 (China); Tomono, Yasuko [Division of Molecular and Cell Biology, Shigei Medical Research Institute, 2117 Yamada, Okayama 700-0202 (Japan); Sakurama, Kazufumi; Ohara, Toshiaki [Department of Gastroenterological Surgery, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikatacho, Kita-ku, Okayama 700-8558 (Japan); Fukazawa, Takuya; Yamatsuji, Tomoki [Department of General Surgery, Kawasaki Medical School, 2-1-80 Nakasange, Kita-ku, Okayama 700-8505 (Japan); Fujiwara, Toshiyoshi [Department of Gastroenterological Surgery, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikatacho, Kita-ku, Okayama 700-8558 (Japan); Naomoto, Yoshio, E-mail: ynaomoto@med.kawasaki-m.ac.jp [Department of General Surgery, Kawasaki Medical School, 2-1-80 Nakasange, Kita-ku, Okayama 700-8505 (Japan)

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer A novel FAK inhibitor TAE226 suppressed FAK activity in HCT116 colon cancer cells. Black-Right-Pointing-Pointer TAE226 suppressed proliferation and migration, with a modest effect on adhesion. Black-Right-Pointing-Pointer Silencing of FAK by siRNA made no obvious difference on cancer cell attachment. Black-Right-Pointing-Pointer TAE226 treatment suppressed the progression of peritoneal dissemination. Black-Right-Pointing-Pointer Oral administration of TAE226 prolonged the survival of tumor-bearing mice. -- Abstract: Peritoneal dissemination is one of the most terrible types of colorectal cancer progression. Focal adhesion kinase (FAK) plays a crucial role in the biological processes of cancer, such as cell attachment, migration, proliferation and survival, all of which are essential for the progression of peritoneal dissemination. Since we and other groups have reported that the inhibition of FAK activity exhibited a potent anticancer effect in several cancer models, we hypothesized that TAE226, a novel ATP-competitive tyrosine kinase inhibitor designed to target FAK, can prevent the occurrence and progression of peritoneal dissemination. In vitro, TAE226 greatly inhibited the proliferation and migration of HCT116 colon cancer cells, while their adhesion on the matrix surface was minimally inhibited when FAK activity and expression was suppressed by TAE226 and siRNA. In vivo, when HCT116 cells were intraperitoneally inoculated in mice, the cells could attach to the peritoneum and begin to grow within 24 h regardless of the pretreatment of cells with TAE226 or FAK-siRNA, suggesting that FAK is not essential, at least for the initial integrin-matrix contact. Interestingly, the treatment of mice before and after inoculation significantly suppressed cell attachment to the peritoneum. Furthermore, oral administration of TAE226 greatly reduced the size of disseminated tumors and prolonged survival in tumor-bearing mice. Taken

  4. Second Generation Grp94-Selective Inhibitors Provide Opportunities for the Inhibition of Metastatic Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Crowley, Vincent M. [Department of Medicinal Chemistry, The University of Kansas, 1251 Wescoe Hall Dr. Malott 4070 Lawrence KS 66045 USA; Huard, Dustin J. E. [School of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta GA 30332 USA; Lieberman, Raquel L. [School of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta GA 30332 USA; Blagg, Brian S. J. [Warren Family Research Center for Drug Discovery and Development, and Department of Chemistry & Biochemistry, University of Notre Dame, 305 McCourtney Hall Notre Dame IN 46556 USA

    2017-09-27

    Glucose regulated protein 94 (Grp94) is the endoplasmic reticulum (ER) resident isoform of the 90 kDa heat shock protein (Hsp90) family and its inhibition represents a promising therapeutic target for the treatment of many diseases. Modification of the first generation cis-amide bioisostere imidazole to alter the angle between the resorcinol ring and the benzyl side chain via cis-amide replacements produced compounds with improved Grp94 affinity and selectivity. Structure–activity relationship studies led to the discovery of compound 30, which exhibits 540 nm affinity and 73-fold selectivity towards Grp94. Grp94 is responsible for the maturation and trafficking of proteins associated with cell signaling and motility, including select integrins. The Grp94-selective inhibitor 30 was shown to exhibit potent anti-migratory effects against multiple aggressive and metastatic cancers.

  5. The p38 MAPK inhibitor, SB203580, inhibits cell invasion by Neospora caninum.

    Science.gov (United States)

    Jin, Xiaoxia; Gong, Pengtao; Li, Guojiang; Zhang, Xichen; Li, Jianhua

    2017-02-01

    The Apicomplexan parasite Neospora caninum is an obligate intracellular parasitic protozoan. It can cause severe diseases in a number of animals throughout the world. Infection with N. caninum leads to abortions in pregnant animals and neuromuscular disorders of newborns which cause great economic losses to animal husbandry. However, the mechanism of cell invasion by N. caninum is still unclear. This paper aims to investigate the impact of SB203580, a p38 MAPK inhibitor, on host cell invasion by N. caninum. The results suggested the presence of putative p38 MAPK homologues in N. caninum, and incubation of N. caninum with SB203580 markedly reduced the tachyzoite motility and microneme exocytosis (NcMIC2, 3, and 6). Furthermore, treatment or pretreatment of MDBK cells with SB203580 effectively reduced cell invasion by N. caninum. Therefore, SB203580 affected both, parasites and host cells, resulting in inhibition of cell invasion by N. caninum.

  6. Synergistic Inhibition of Carbon Steel Corrosion by Inhibitor-Blends in Chloride-Containing Simulated Cooling Water

    Energy Technology Data Exchange (ETDEWEB)

    Shaban, Abdul; Felhosi, Ilona [Hungarian Academy of Sciences, Budapest (Hungary); Vastag, Gyongyi [University of Novi Sad, Novi Sad (Serbia)

    2017-06-15

    The objective of this work was to develop efficient synergistic inhibitor combinations comprising sodium nitrite (NaNO{sub 2}) and an inhibitor-blend code named (SN-50), keeping in view of their application in industrial cooling water systems. The electrochemical characteristics of the carbon steel working electrode in simulated cooling water (SCW), without and with the addition of different combinations of the inhibitors, were investigated using electrochemical impedance spectroscopy (EIS), open circuit potential (OCP). The electrode surface changes were followed by visual characterization methods. It was demonstrated in this study that all the combinations of the inhibitors exhibited synergistic benefit and higher inhibition efficiencies than did either of the individual inhibitors. The addition of SN-50 inhibitor to the SCW shifted the OCP to more anodic values and increased the polarization resistance (R{sub p}) values of carbon steel at all applied concentrations. The higher the applied sodium nitrite concentration (in the protection concentration range), the higher the obtained R{sub p} values and the inhibition efficiency improved by increasing the inhibitor concentration.

  7. Soybean-derived Bowman-Birk inhibitor inhibits neurotoxicity of LPS-activated macrophages

    Directory of Open Access Journals (Sweden)

    Persidsky Yuri

    2011-02-01

    Full Text Available Abstract Background Lipopolysaccharide (LPS, the major component of the outer membrane of gram-negative bacteria, can activate immune cells including macrophages. Activation of macrophages in the central nervous system (CNS contributes to neuronal injury. Bowman-Birk inhibitor (BBI, a soybean-derived protease inhibitor, has anti-inflammatory properties. In this study, we examined whether BBI has the ability to inhibit LPS-mediated macrophage activation, reducing the release of pro-inflammatory cytokines and subsequent neurotoxicity in primary cortical neural cultures. Methods Mixed cortical neural cultures from rat were used as target cells for testing neurotoxicity induced by LPS-treated macrophage supernatant. Neuronal survival was measured using a cell-based ELISA method for expression of the neuronal marker MAP-2. Intracellular reactive oxygen species (ROS production in macrophages was measured via 2', 7'-dichlorofluorescin diacetate (DCFH2DA oxidation. Cytokine expression was determined by quantitative real-time PCR. Results LPS treatment of macrophages induced expression of proinflammatory cytokines (IL-1β, IL-6 and TNF-α and of ROS. In contrast, BBI pretreatment (1-100 μg/ml of macrophages significantly inhibited LPS-mediated induction of these cytokines and ROS. Further, supernatant from BBI-pretreated and LPS-activated macrophage cultures was found to be less cytotoxic to neurons than that from non-BBI-pretreated and LPS-activated macrophage cultures. BBI, when directly added to the neuronal cultures (1-100 μg/ml, had no protective effect on neurons with or without LPS-activated macrophage supernatant treatment. In addition, BBI (100 μg/ml had no effect on N-methyl-D-aspartic acid (NMDA-mediated neurotoxicity. Conclusions These findings demonstrate that BBI, through its anti-inflammatory properties, protects neurons from neurotoxicity mediated by activated macrophages.

  8. Allosteric Inhibition of SHP2: Identification of a Potent, Selective, and Orally Efficacious Phosphatase Inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Fortanet, Jorge Garcia; Chen, Christine Hiu-Tung; Chen, Ying-Nan P.; Chen, Zhouliang; Deng, Zhan; Firestone, Brant; Fekkes, Peter; Fodor, Michelle; Fortin, Pascal D.; Fridrich, Cary; Grunenfelder, Denise; Ho, Samuel; Kang, Zhao B.; Karki, Rajesh; Kato, Mitsunori; Keen, Nick; LaBonte, Laura R.; Larrow, Jay; Lenoir, Francois; Liu, Gang; Liu, Shumei; Lombardo, Franco; Majumdar, Dyuti; Meyer, Matthew J.; Palermo, Mark; Perez, Lawrence; Pu, Minying; Ramsey, Timothy; Sellers, William R.; Shultz, Michael D.; Stams, Travis; Towler, Christopher; Wang, Ping; Williams, Sarah L.; Zhang, Ji-Hu; LaMarche, Matthew J. (Novartis)

    2016-09-08

    SHP2 is a nonreceptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also purportedly plays an important role in the programmed cell death pathway (PD-1/PD-L1). Because it is an oncoprotein associated with multiple cancer-related diseases, as well as a potential immunomodulator, controlling SHP2 activity is of significant therapeutic interest. Recently in our laboratories, a small molecule inhibitor of SHP2 was identified as an allosteric modulator that stabilizes the autoinhibited conformation of SHP2. A high throughput screen was performed to identify progressable chemical matter, and X-ray crystallography revealed the location of binding in a previously undisclosed allosteric binding pocket. Structure-based drug design was employed to optimize for SHP2 inhibition, and several new protein–ligand interactions were characterized. These studies culminated in the discovery of 6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine (SHP099, 1), a potent, selective, orally bioavailable, and efficacious SHP2 inhibitor.

  9. Farnesyltransferase inhibitor tipifarnib (R115777) preferentially inhibits in vitro autonomous erythropoiesis of polycythemia vera patient cells.

    Science.gov (United States)

    Larghero, Jérôme; Gervais, Nathalie; Cassinat, Bruno; Rain, Jean-Didier; Schlageter, Marie-Hélène; Padua, Rose Ann; Chomienne, Christine; Rousselot, Philippe

    2005-05-01

    Polycythemia vera (PV) is an acquired myeloproliferative disorder with primary expansion of the red cell mass leading to an increased risk of thrombosis and less frequently to myelofibrosis and secondary acute leukemia. Standard therapies include cytoreduction with either phlebotomy or chemotherapeutic agents and antithrombotic drugs. Because long-term exposure to cytotoxic chemotherapy may increase the risk of acute transformation, new therapeutic options are needed. Tipifarnib is a nonpeptidomimetic inhibitor of farnesyl transferase that was developed as a potential inhibitor of RAS signaling. In the present study we report that tipifarnib used at pharmacologically achievable concentrations strongly inhibits the erythroid burst-forming unit (BFU-E) autonomous growth that characterizes patients with PV. Moreover, at low tipifarnib concentrations (0.15 muM), the inhibitory effect was preferentially observed in PV BFU-E progenitors and not in normal BFU-E progenitors and was not rescued by erythropoietin (EPO). Thus tipifarnib may specifically target PV stem cells and may be of clinical interest in the treatment of patients with PV.

  10. Inhibition of thermolysin with nitrone-bearing substrate analogs: a new type of thermolysin inhibitors.

    Science.gov (United States)

    Lee, K J; Kim, D H

    1998-02-17

    Nitrones are utilized as the active site zinc coordinating functionality in the design of inhibitors for thermolysin. This new type of thermolysin inhibitors are as potent as the existing inhibitors bearing a carboxylate or hydroxamate zinc ligating moiety.

  11. Switch in Site of Inhibition: A Strategy for Structure-Based Discovery of Human Topoisomerase IIα Catalytic Inhibitors.

    Science.gov (United States)

    Baviskar, Ashish T; Amrutkar, Suyog M; Trivedi, Neha; Chaudhary, Vikas; Nayak, Anmada; Guchhait, Sankar K; Banerjee, Uttam C; Bharatam, Prasad V; Kundu, Chanakya N

    2015-04-09

    A study of structure-based modulation of known ligands of hTopoIIα, an important enzyme involved in DNA processes, coupled with synthesis and in vitro assays led to the establishment of a strategy of rational switch in mode of inhibition of the enzyme's catalytic cycle. 6-Arylated derivatives of known imidazopyridine ligands were found to be selective inhibitors of hTopoIIα, while not showing TopoI inhibition and DNA binding. Interestingly, while the parent imidazopyridines acted as ATP-competitive inhibitors, arylated derivatives inhibited DNA cleavage similar to merbarone, indicating a switch in mode of inhibition from ATP-hydrolysis to the DNA-cleavage stage of catalytic cycle of the enzyme. The 6-aryl-imidazopyridines were relatively more cytotoxic than etoposide in cancer cells and less toxic to normal cells. Such unprecedented strategy will encourage research on "choice-based change" in target-specific mode of action for rapid drug discovery.

  12. Studies on bicarbonate transporters and carbonic anhydrase in porcine non-pigmented ciliary epithelium

    Science.gov (United States)

    Shahidullah, Mohammad; C-H, To; Pelis, Ryan M.; Delamere, Nicholas A

    2009-01-01

    Purpose Bicarbonate transport plays a role in aqueous humor (AH) secretion. Here, we examined bicarbonate transport mechanisms and carbonic anhydrase (CA) in porcine non-pigmented ciliary epithelium (NPE). Methods Cytoplasmic pH (pHi) was measured in cultured porcine NPE loaded with BCECF. Anion exchanger (AE), sodium bicarbonate cotransporter (NBC) and CA were examined by RT-PCR and immunolocalization. AH secretion was measured in the intact porcine eye using a fluorescein dilution technique. Results Anion exchanger AE2, CAII and CAIV were abundant in the NPE layer. In cultured NPE superfused with a CO2/HCO3− free HEPES buffer, exposure to a CO2/HCO3−-containing buffer caused a rapid acidification followed by a gradual pHi increase. Subsequent removal of CO2/HCO3− with HEPES buffer caused rapid alkalinization followed by gradual pHi decrease. The rate of gradual alkalinization after addition of HCO3−/CO2 was inhibited by sodium-free conditions, DIDS, CA inhibitors acetazolamide and methazolamide but not by Na-H exchange inhibitor dimethylamiloride or low chloride buffer. The phase of gradual acidification after removal of HCO3−/CO2 was inhibited by DIDS, acetazolamide, methazolamide and by low chloride buffer. DIDS reduced baseline pHi. In the intact eye, DIDS and acetazolamide reduced AH secretion by 25% and 44% respectively. Conclusion The results suggest the NPE uses a Na+-HCO3− cotransporter to import bicarbonate and a Cl−/HCO3− exchanger to export bicarbonate. CA influences the rate of bicarbonate transport. AE2, CAII and CAIV are enriched in the NPE layer of the ciliary body and their coordinated function may contribute to AH secretion by effecting bicarbonate transport into the eye. PMID:19011010

  13. Concurrent Autophagy Inhibition Overcomes the Resistance of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Human Bladder Cancer Cells

    Directory of Open Access Journals (Sweden)

    Minyong Kang

    2017-02-01

    Full Text Available Despite the potential therapeutic efficacy of epithelial growth factor receptor (EGFR inhibitors in the treatment of advanced stage bladder cancer, there currently is no clear evidence to support this hypothesis. In this study, we investigate whether the concurrent treatment of autophagy-blocking agents with EGFR inhibitors exerts synergistic anti-cancer effects in T24 and J82 human bladder cancer cells. Lapatinib and gefitinib were used as EGFR inhibitors, and bafilomycin A1 (BFA1, chloroquine (CQ and 3-methyladenine (3-MA were used as the pharmacologic inhibitors of autophagy activities. To assess the proliferative and self-renewal capabilities, the Cell Counting Kit-8 (CCK-8 assay and a clonogenic assay were performed, respectively. To examine apoptotic cell death, flow cytometry using annexin-V/propidium iodide (PI was used. To measure the autophagy activities, the expression levels of LC3I and II was determined by Western blot analysis. To validate the synergistic effects of autophagy inhibition with EGFR inhibitors, we specifically blocked key autophagy regulatory gene ATG12 by transfection of small interference RNA and examined the phenotypic changes. Of note, lapatinib and gefitinib triggered autophagy activities in T24 and J82 human bladder cancer cells, as indicated by upregulation of LC3II. More importantly, inhibiting autophagy activities with pharmacologic inhibitors (BFA1, CQ or 3-MA remarkably reduced the cell viabilities and clonal proliferation of T24 and J82 cells, compared to those treated with either of the agents alone. We also obtained similar results of the enhanced anti-cancer effects of EGFR inhibitors by suppressing the expression of ATG12. Notably, the apoptotic assay showed that synergistic anti-cancer effects were induced via the increase of apoptotic cell death. In summary, concomitant inhibition of autophagy activities potentiated the anti-cancer effects of EGFR inhibitors in human bladder cancer cells, indicating

  14. Regulation of expression and biochemical characterization of a beta-class carbonic anhydrase from the plant growth-promoting rhizobacterium, Azospirillum brasilense Sp7.

    Science.gov (United States)

    Kaur, Simarjot; Mishra, Mukti Nath; Tripathi, Anil K

    2009-10-01

    Carbonic anhydrase (CA; [EC 4.2.1.1]) is a ubiquitous enzyme catalysing the reversible hydration of CO(2) to bicarbonate, a reaction that supports various biochemical and physiological functions. Genome analysis of Azospirillum brasilense, a nonphotosynthetic, nitrogen-fixing, rhizobacterium, revealed an ORF with homology to beta-class carbonic anhydrases (CAs). Biochemical characteristics of the beta-class CA of A. brasilense, analysed after cloning the gene (designated as bca), overexpressing in Escherichia coli and purifying the protein by affinity purification, revealed that the native recombinant enzyme is a homotetramer, inhibited by the known CA inhibitors. CA activity in A. brasilense cell extracts, reverse transcriptase (RT)-PCR and Western blot analyses showed that bca was constitutively expressed under aerobic conditions. Lower beta-galactosidase activity in A. brasilense cells harbouring bca promoter: lacZ fusion during the stationary phase or during growth on 3% CO(2) enriched air or at acidic pH indicated that the transcription of bca was downregulated by the stationary phase, elevated CO(2) levels and acidic pH conditions. These observations were also supported by RT-PCR analysis. Thus, beta-CA in A. brasilense seems to be required for scavenging CO(2) from the ambient air and the requirement of CO(2) hydration seems to be higher for the cultures growing exponentially at neutral to alkaline pH.

  15. The selective serotonin reuptake inhibitor, escitalopram, enhances inhibition of prepotent responding and spatial reversal learning

    Science.gov (United States)

    Brown, Holden D.; Amodeo, Dionisio A.; Sweeney, John A.; Ragozzino, Michael E.

    2011-01-01

    Previous findings indicate treatment with a selective serotonin reuptake inhibitor (SSRI) facilitates behavioral flexibility when conditions require inhibition of a learned response pattern. The present experiment investigated whether acute treatment with the SSRI, escitalopram, affects behavioral flexibility when conditions require inhibition of a naturally-biased response pattern (elevated conflict test) and/or reversal of a learned response pattern (spatial reversal learning). An additional experiment was carried out to determine whether escitalopram, at doses that affected behavioral flexibility, also reduced anxiety as tested in the elevated plus-maze. In each experiment, Long-Evans rats received an intraperitoneal injection of either saline or escitalopram (0.03, 0.3 or 1.0 mg/kg) 30 minutes prior to behavioral testing. Escitalopram, at all doses tested, enhanced acquisition in the elevated conflict test, but did not affect performance in the elevated plus-maze. Escitalopram (0.3 and 1.0 mg/kg) did not alter acquisition of the spatial discrimination, but facilitated reversal learning. In the elevated conflict and spatial reversal learning test, escitalopram enhanced the ability to maintain the relevant strategy after being initially selected. The present findings suggest that enhancing serotonin transmission with a SSRI facilitates inhibitory processes when conditions require a shift away from either a naturally-biased response pattern or a learned choice pattern. PMID:22219222

  16. [Effect of BRD4 Inhibitor JQ1 on Proliferation Inhibition and Apotosis Induction in Jurkat Cells].

    Science.gov (United States)

    Sun, Xiao-Xia; Ma, Liang-Ming; Wang, Tao

    2016-08-01

    To investigate the effect and possible mechanism of bromo-domain inhibitors (JQ1) on proliferation inhibition and inducing apoptosis of acute T lymphocyte leukemia cell line (Jurkat) . Jurkat cell line was treated by JQ1 at different concentrations. MTT was used to detect the cell proliferation inhibition rate. The flow cytometry with AnnexinV-FITC/PI fluorescence staining was used to detect the changes of apoptosis rate, and real-time fluorescent quantitative PCR was used to detect c-Myc/Notch1 gene expression levels. With the increasing of drug concentration and prolonging of time, the inhibitory rate of Jurkat cell growth was enhanced in time-dose dependent manner; Jurkat cells was treated by 0.8, 1.6, and 4 µ mol/LJQ1 for 48 h and 72 h, the cell apoptosis rate was enhanced with the increase of drug concentration and prolonging of time, and the difference was statistically different in comparison with the control group(PJurkat cell line, and potentially induce apoptosis through Notch1 and c-Myc gene. Hence JQ1 may be one of new methods used to treat T-ALL.

  17. Inhibition of pseudolysin and thermolysin by hydroxamate-based MMP inhibitors.

    Science.gov (United States)

    Adekoya, Olayiwola A; Sjøli, Stian; Wuxiuer, Yimingjiang; Bilto, Irina; Marques, Sérgio M; Santos, M Amélia; Nuti, Elisa; Cercignani, Giovanni; Rossello, Armando; Winberg, Jan-Olof; Sylte, Ingebrigt

    2015-01-07

    In the present study, we have investigated the inhibition of thermolysin and pseudolysin by a series of compounds previously identified as matrix metalloproteinase (MMP) inhibitors using experimental binding studies and theoretical calculations. The experimental studies showed that some of the compounds were able to inhibit thermolysin and pseudolysin in the low μM range. The studies revealed that, in general, the compounds bound in the order MMPs > pseudolysin > thermolysin, and the strongest pseudolysin and thermolysin binders were compounds 8-12. Furthermore, compounds 8 and 9 were unique in that they bound much stronger to the two bacterial enzymes than to the MMPs. The docking calculations suggested that the phenyl group of the strongest binders (compounds 8 and 9) occupy the S2(')-subpocket, while a second ring system occupy the S1-subpocket in both thermolysin and pseudolysin. When the compounds possess two ring systems, the largest and most electron rich ring system seems to occupy the S1-subpocket. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  18. Fasting potentiates the anticancer activity of tyrosine kinase inhibitors by strengthening MAPK signaling inhibition

    Science.gov (United States)

    Caffa, Irene; D'Agostino, Vito; Damonte, Patrizia; Soncini, Debora; Cea, Michele; Monacelli, Fiammetta; Odetti, Patrizio; Ballestrero, Alberto; Provenzani, Alessandro; Longo, Valter D.; Nencioni, Alessio

    2015-01-01

    Tyrosine kinase inhibitors (TKIs) are now the mainstay of treatment in many types of cancer. However, their benefit is frequently short-lived, mandating the search for safe potentiation strategies. Cycles of fasting enhance the activity of chemo-radiotherapy in preclinical cancer models and dietary approaches based on fasting are currently explored in clinical trials. Whether combining fasting with TKIs is going to be potentially beneficial remains unknown. Here we report that starvation conditions increase the ability of commonly administered TKIs, including erlotinib, gefitinib, lapatinib, crizotinib and regorafenib, to block cancer cell growth, to inhibit the mitogen-activated protein kinase (MAPK) signaling pathway and to strengthen E2F-dependent transcription inhibition. In cancer xenografts models, both TKIs and cycles of fasting slowed tumor growth, but, when combined, these interventions were significantly more effective than either type of treatment alone. In conclusion, cycles of fasting or of specifically designed fasting-mimicking diets should be evaluated in clinical studies as a means to potentiate the activity of TKIs in clinical use. PMID:25909220

  19. Carbonic anhydrases and their functional differences in human and mouse sperm physiology.

    Science.gov (United States)

    José, O; Torres-Rodríguez, P; Forero-Quintero, L S; Chávez, J C; De la Vega-Beltrán, J L; Carta, F; Supuran, C T; Deitmer, J W; Treviño, C L

    2015-12-25

    Fertilization is a key reproductive event in which sperm and egg fuse to generate a new individual. Proper regulation of certain parameters (such as intracellular pH) is crucial for this process. Carbonic anhydrases (CAs) are among the molecular entities that control intracellular pH dynamics in most cells. Unfortunately, little is known about the function of CAs in mammalian sperm physiology. For this reason, we re-explored the expression of CAI, II, IV and XIII in human and mouse sperm. We also measured the level of CA activity, determined by mass spectrometry, and found that it is similar in non-capacitated and capacitated mouse sperm. Importantly, we found that CAII activity accounts for half of the total CA activity in capacitated mouse sperm. Using the general CA inhibitor ethoxyzolamide, we studied how CAs participate in fundamental sperm physiological processes such as motility and acrosome reaction in both species. We found that capacitated human sperm depend strongly on CA activity to support normal motility, while capacitated mouse sperm do not. Finally, we found that CA inhibition increases the acrosome reaction in capacitated human sperm, but not in capacitated mouse sperm. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Garcinol, a Histone Acetyltransferase Inhibitor, Radiosensitizes Cancer Cells by Inhibiting Non-Homologous End Joining

    Energy Technology Data Exchange (ETDEWEB)

    Oike, Takahiro [Division of Multistep Carcinogenesis, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi, Gunma (Japan); Ogiwara, Hideaki [Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Torikai, Kohta [Gunma University Heavy Ion Medical Center, Maebashi, Gunma (Japan); Nakano, Takashi [Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi, Gunma (Japan); Yokota, Jun [Division of Multistep Carcinogenesis, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Kohno, Takashi, E-mail: tkkohno@ncc.go.jp [Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan)

    2012-11-01

    Purpose: Non-homologous end joining (NHEJ), a major pathway used to repair DNA double-strand breaks (DSBs) generated by ionizing radiation (IR), requires chromatin remodeling at DSB sites through the acetylation of histones by histone acetyltransferases (HATs). However, the effect of compounds with HAT inhibitory activities on the DNA damage response (DDR), including the NHEJ and cell cycle checkpoint, as well as on the radiosensitivity of cancer cells, remains largely unclear. Here, we investigated whether garcinol, a HAT inhibitor found in the rinds of Garcinia indica fruit (called mangosteens), has effects on DDR, and whether it can be used for radiosensitization. Methods and Materials: The following assays were used to examine the effect of garcinol on the inhibition of DSB repair, including the following: a conventional neutral comet assay; a cell-based assay recently developed by us, in which NHEJ repair of DSBs on chromosomal DNA was evaluated; the micrococcal nuclease sensitivity assay; and immunoblotting for autophosphorylation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs). We assessed the effect of garcinol on the cell cycle checkpoint after IR treatment by analyzing the phosphorylation levels of checkpoint kinases CHK1 and CHK2 and histone H3, and by cell cycle profile analysis using flow cytometry. The radiosensitizing effect of garcinol was assessed by a clonogenic survival assay, whereas its effects on apoptosis and senescence were examined by annexin V and senescence-associated {beta}-galactosidase (SA-{beta}-Gal) staining, respectively. Results: We found that garcinol inhibits DSB repair, including NHEJ, without affecting cell cycle checkpoint. Garcinol radiosensitized A549 lung and HeLa cervical carcinoma cells with dose enhancement ratios (at 10% surviving fraction) of 1.6 and 1.5, respectively. Cellular senescence induced by IR was enhanced by garcinol. Conclusion: These results suggest that garcinol is a radiosensitizer that

  1. Computational study of physical and chemical flame inhibition. [Ar, N/sub 2/, and HBr as inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Brown, N.J.; Schefer, R.W.

    1978-04-01

    This paper reports a set of modelling studies that were undertaken to acquire a more detailed knowledge of combustion inhibition mechanisms. H/sub 2//O/sub 2//Ar mixtures reacting in the idealized perfectly stirred reactor were investigated. Three H/sub 2//O/sub 2/ kinetic mechanisms were considered, differing from one another by the number of HO/sub 2/ reactions included. Two physical inhibitors, Ar and N/sub 2/, and one chemical inhibitor, HBr, were investigated. Additional parameters considered were pressure, equivalence ratio, inhibitor concentration and rate coefficient variation. HBr was the most effective inhibitor and acted chemically in that it caused substantial reduction in radical concentrations in the mixtures considered. Ar and N/sub 2/ acted as physical diluents with N/sub 2/ the more effective of the two due to its larger heat capacity.

  2. Kinetics of Corrosion Inhibition of Aluminum in Acidic Media by Water-Soluble Natural Polymeric Pectates as Anionic Polyelectrolyte Inhibitors

    Directory of Open Access Journals (Sweden)

    Refat M. Hassan

    2013-06-01

    Full Text Available Corrosion inhibition of aluminum (Al in hydrochloric acid by anionic polyeletrolyte pectates (PEC as a water-soluble natural polymer polysaccharide has been studied using both gasometric and weight loss techniques. The results drawn from these two techniques are comparable and exhibit negligible differences. The inhibition efficiency was found to increase with increasing inhibitor concentration and decrease with increasing temperature. The inhibition action of PEC on Al metal surface was found to obey the Freundlich isotherm. Factors such as the concentration and geometrical structure of the inhibitor, concentration of the corrosive medium, and temperature affecting the corrosion rates were examined. The kinetic parameters were evaluated and a suitable corrosion mechanism consistent with the kinetic results is discussed in the paper.

  3. Kinetics of Corrosion Inhibition of Aluminum in Acidic Media by Water-Soluble Natural Polymeric Pectates as Anionic Polyelectrolyte Inhibitors.

    Science.gov (United States)

    Hassan, Refat M; Zaafarany, Ishaq A

    2013-06-17

    Corrosion inhibition of aluminum (Al) in hydrochloric acid by anionic polyeletrolyte pectates (PEC) as a water-soluble natural polymer polysaccharide has been studied using both gasometric and weight loss techniques. The results drawn from these two techniques are comparable and exhibit negligible differences. The inhibition efficiency was found to increase with increasing inhibitor concentration and decrease with increasing temperature. The inhibition action of PEC on Al metal surface was found to obey the Freundlich isotherm. Factors such as the concentration and geometrical structure of the inhibitor, concentration of the corrosive medium, and temperature affecting the corrosion rates were examined. The kinetic parameters were evaluated and a suitable corrosion mechanism consistent with the kinetic results is discussed in the paper.

  4. Targeting carbonic anhydrase to treat diabetic retinopathy: Emerging evidences and encouraging results

    Energy Technology Data Exchange (ETDEWEB)

    Weiwei, Zhang [Department of Endocrinology and Metabolism, HuaShan Hospital, Institute of Endocrinology and Diabetology, Shanghai Medical College, Fudan University, No. 12 Wulumuqi Road, Shanghai 200040 (China); Hu, Renming, E-mail: taylorzww@gmail.com [Department of Endocrinology and Metabolism, HuaShan Hospital, Institute of Endocrinology and Diabetology, Shanghai Medical College, Fudan University, No. 12 Wulumuqi Road, Shanghai 200040 (China)

    2009-12-18

    Diabetic retinopathy (DR) is the leading cause of vision loss among working-age populations in developed countries. Current treatment options are limited to tight glycemic, blood pressure control and destructive laser surgery. Carbonic anhydrases (CAs) are a group of enzymes involving in the rapid conversion of carbon dioxide to bicarbonate and protons. Emerging evidences reveal CA inhibitors hold the promise for the treatment of DR. This article summarizes encouraging results from clinical and animal studies, and reviews the possible mechanisms.

  5. Hepatocellular Toxicity Associated with Tyrosine Kinase Inhibitors: Mitochondrial Damage and Inhibition of Glycolysis

    Directory of Open Access Journals (Sweden)

    Franziska Paech

    2017-06-01

    Full Text Available Tyrosine kinase inhibitors (TKIs are anticancer drugs with a lesser toxicity than classical chemotherapeutic agents but still with a narrow therapeutic window. While hepatotoxicity is known for most TKIs, underlying mechanisms remain mostly unclear. We therefore aimed at investigating mechanisms of hepatotoxicity for imatinib, sunitinib, lapatinib and erlotinib in vitro. We treated HepG2 cells, HepaRG cells and mouse liver mitochondria with TKIs (concentrations 1–100 μM for different periods of time and assessed toxicity. In HepG2 cells maintained with glucose (favoring glycolysis, all TKIs showed a time- and concentration-dependent cytotoxicity and, except erlotinib, a drop in intracellular ATP. In the presence of galactose (favoring mitochondrial metabolism, imatinib, sunitinib and erlotinib showed a similar toxicity profile as for glucose whereas lapatinib was less toxic. For imatinib, lapatinib and sunitinib, cytotoxicity increased in HepaRG cells induced with rifampicin, suggesting formation of toxic metabolites. In contrast, erlotinib was more toxic in HepaRG cells under basal than CYP-induced conditions. Imatinib, sunitinib and lapatinib reduced the mitochondrial membrane potential in HepG2 cells and in mouse liver mitochondria. In HepG2 cells, these compounds increased reactive oxygen species production, impaired glycolysis, and induced apoptosis. In addition, imatinib and sunitinib impaired oxygen consumption and activities of complex I and III (only imatinib, and reduced the cellular GSH pool. In conclusion, imatinib and sunitinib are mitochondrial toxicants after acute and long-term exposure and inhibit glycolysis. Lapatinib affected mitochondria only weakly and inhibited glycolysis, whereas the cytotoxicity of erlotinib could not be explained by a mitochondrial mechanism.

  6. Flower inhibition in Kalanchoe blossfeldiana. Bioassay of an endogenous long-day inhibitor and inhibition by (±) abscisic acid and xanthoxin.

    Science.gov (United States)

    Schwabe, W W

    1972-03-01

    The inhibition of flowering in Kalanchoe by crude sap expressed from leaves held in non-inductive long-day conditions is described, using a bioassay technique of leaf injection, which confirms the existence of a transferable inhibitor.This technique has also revealed that ± abscisic acid and Xanthoxin are inhibitory to flowering at 50 and 100 ppm respectively. The previously known inhibitory effects of gibberellic acid on flowering have also been confirmed.

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

    Science.gov (United States)

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

    2013-01-01

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

  8. NAD+-dependent HDAC inhibitor stimulates Monascus pigment production but inhibit citrinin.

    Science.gov (United States)

    Hu, Yan; Zhou, Youxiang; Mao, Zejing; Li, Huihui; Chen, Fusheng; Shao, Yanchun

    2017-08-23

    Monascus species are edible fungi due to the production of food colorant and other beneficial compounds. Hence, it has been an attractive thesis to improve their productivities. Increasing numbers of investigations revealed that regulating the activities of histone deacetylases can significantly perturb secondary metabolites (SM) production at a global level. In this study, dihydrocoumarin (DHC, an inhibitor of the Sirtuin family of NAD + -dependent deacetylases) was used to treat Monascus ruber for evaluating its effects on organism growth and SM production. The results revealed that the variation trends of colonial sizes, biomass and mycotoxin were in a dose-dependent manner. Generally, they decreased with the increased DHC concentrations in the designed range. But the variation trend of pigment was different. Comparison of SM profile, three new peaks occurred to the mycelia extractions from DHC-treated strain corresponding to molecular weights 402, 416 and 444, respectively. These three compounds were identified as Monasfluol B, Monascus azaphilone C and acetyl-monasfluol B (a new Monascus chemical pigment structure). In short, DHC can stimulate M. ruber strain to produce more pigment-like polyketides but inhibition of mycotoxin (citrinin).

  9. Cloning, expression and biochemical characterization of a β-carbonic anhydrase from the soil bacterium Enterobacter sp. B13.

    Science.gov (United States)

    Eminoğlu, Ayşenur; Vullo, Daniela; Aşık, Aycan; Çolak, Dilşat Nigar; Supuran, Claudiu T; Çanakçı, Sabriye; Osman Beldüz, Ali

    2016-12-01

    A recombinant carbonic anhydrase (CA, EC 4.2.1.1) from the soil-dwelling bacterium Enterobacter sp. B13 was cloned and purified by Co(2+) affinity chromatography. Bioinformatic analysis showed that the new enzyme (denominated here B13-CA) belongs to the β-class CAs and to possess 95% homology with the ortholog enzyme from Escherichia coli encoded by the can gene, whereas its sequence homology with the other such enzyme from E. coli (encoded by the cynT gene) was of 33%. B13-CA was characterized kinetically as a catalyst for carbon dioxide hydration to bicarbonate and protons. The enzyme shows a significant catalytic activity, with the following kinetic parameters at 20 °C and pH of 8.3: kcat of 4.8 × 10(5) s(-1) and kcat/Km of 5.6 × 10(7) M(-1) × s(-1). This activity was potently inhibited by acetazolamide which showed a KI of 78.9 nM. Although only this compound was investigated for the moment as B13-CA inhibitor, further studies may reveal new classes of inhibitors/activators of this enzyme which may show biomedical or environmental applications, considering the posssible role of this enzyme in CaCO3 biomineralization processes.

  10. Enfuvirtide (T20)-Based Lipopeptide Is a Potent HIV-1 Cell Fusion Inhibitor: Implications for Viral Entry and Inhibition.

    Science.gov (United States)

    Ding, Xiaohui; Zhang, Xiujuan; Chong, Huihui; Zhu, Yuanmei; Wei, Huamian; Wu, Xiyuan; He, Jinsheng; Wang, Xinquan; He, Yuxian

    2017-09-15

    The peptide drug enfuvirtide (T20) is the only viral fusion inhibitor used in combination therapy for HIV-1 infection, but it has relatively low antiviral activity and easily induces drug resistance. Emerging studies demonstrate that lipopeptide-based fusion inhibitors, such as LP-11 and LP-19, which mainly target the gp41 pocket site, have greatly improved antiviral potency and in vivo stability. In this study, we focused on developing a T20-based lipopeptide inhibitor that lacks pocket-binding sequence and targets a different site. First, the C-terminal tryptophan-rich motif (TRM) of T20 was verified to be essential for its target binding and inhibition; then, a novel lipopeptide, termed LP-40, was created by replacing the TRM with a fatty acid group. LP-40 showed markedly enhanced binding affinity for the target site and dramatically increased inhibitory activity on HIV-1 membrane fusion, entry, and infection. Unlike LP-11 and LP-19, which required a flexible linker between the peptide sequence and the lipid moiety, addition of a linker to LP-40 sharply reduced its potency, implying different binding modes with the extended N-terminal helices of gp41. Also, interestingly, LP-40 showed more potent activity than LP-11 in inhibiting HIV-1 Env-mediated cell-cell fusion while it was less active than LP-11 in inhibiting pseudovirus entry, and the two inhibitors displayed synergistic antiviral effects. The crystal structure of LP-40 in complex with a target peptide revealed their key binding residues and motifs. Combined, our studies have not only provided a potent HIV-1 fusion inhibitor, but also revealed new insights into the mechanisms of viral inhibition. IMPORTANCE T20 is the only membrane fusion inhibitor available for treatment of viral infection; however, T20 requires high doses and has a low genetic barrier for resistance, and its inhibitory mechanism and structural basis remain unclear. Here, we report the design of LP-40, a T20-based lipopeptide inhibitor

  11. Combination of verteporfin-PDT and PI3K inhibitors enhances cell growth inhibition and apoptosis in endothelial cells

    Science.gov (United States)

    Kraus, Daniel; Chen, Bin

    2016-03-01

    Vascular targeted photodynamic therapy is a promising cancer treatment modality by ablating tumor vasculature. The effectiveness of this treatment is often compromised by regrowth of endothelial cells, which causes tumor recurrence. In this preliminary report, we showed that activated PI3K signaling was involved in endothelial cell regrowth after PDT with verteporfin and combination between verteporfin-PDT and PI3K pathway inhibitor BEZ235 induced more cell apoptosis and greater inhibition in cell proliferation. These results suggest that rational combination of verteporfin-PDT and PI3K inhibitors result in enhanced treatment outcomes.

  12. Suppression of Homologous Recombination by insulin-like growth factor-1 inhibition sensitizes cancer cells to PARP inhibitors

    International Nuclear Information System (INIS)

    Amin, Oreekha; Beauchamp, Marie-Claude; Nader, Paul Abou; Laskov, Ido; Iqbal, Sanaa; Philip, Charles-André; Yasmeen, Amber; Gotlieb, Walter H.

    2015-01-01

    Impairment of homologous recombination (HR) is found in close to 50 % of ovarian and breast cancer. Tumors with BRCA1 mutations show increased expression of the Insulin-like growth factor type 1 receptor (IGF-1R). We previously have shown that inhibition of IGF-1R results in growth inhibition and apoptosis of ovarian tumor cells. In the current study, we aimed to investigate the correlation between HR and sensitivity to IGF-1R inhibition. Further, we hypothesized that IGF-1R inhibition might sensitize HR proficient cancers to Poly ADP ribose polymerase (PARP) inhibitors. Using ovarian and breast cancer cellular models with known BRCA1 status, we evaluated their HR functionality by RAD51 foci formation assay. The 50 % lethal concentration (LC50) of Insulin-like growth factor type 1 receptor kinase inhibitor (IGF-1Rki) in these cells was assessed, and western immunoblotting was performed to determine the expression of proteins involved in the IGF-1R pathway. Moreover, IGF-1R inhibitors were added on HR proficient cell lines to assess mRNA and protein expression of RAD51 by qPCR and western blot. Also, we explored the interaction between RAD51 and Insulin receptor substance 1 (IRS-1) by immunoprecipitation. Next, combination effect of IGF-1R and PARP inhibitors was evaluated by clonogenic assay. Cells with mutated/methylated BRCA1 showed an impaired HR function, and had an overactivation of the IGF-1R pathway. These cells were more sensitive to IGF-1R inhibition compared to HR proficient cells. In addition, the IGF-IR inhibitor reduced RAD51 expression at mRNA and protein levels in HR proficient cells, and sensitized these cells to PARP inhibitor. Targeting IGF-1R might lead to improved personalized therapeutic approaches in cancer patients with HR deficiency. Targeting both PARP and IGF-1R might increase the clinical efficacy in HR deficient patients and increase the population of patients who may benefit from PARP inhibitors

  13. Mdm2 inhibition confers protection of p53-proficient cells from the cytotoxic effects of Wee1 inhibitors.

    Science.gov (United States)

    Li, Yizhu; Saini, Priyanka; Sriraman, Anusha; Dobbelstein, Matthias

    2015-10-20

    Pharmacological inhibition of the cell cycle regulatory kinase Wee1 represents a promising strategy to eliminate cancer cells. Wee1 inhibitors cooperate with chemotherapeutics, e. g. nucleoside analogues, pushing malignant cells from S phase towards premature mitosis and death. However, considerable toxicities are observed in preclinical and clinical trials. A high proportion of tumor cells can be distinguished from all other cells of a patient's body by inactivating mutations in the tumor suppressor p53. Here we set out to develop an approach for the selective protection of p53-proficient cells against the cytotoxic effects of Wee1 inhibitors. We pretreated such cells with Nutlin-3a, a prototype inhibitor of the p53-antagonist Mdm2. The resulting transient cell cycle arrest effectively increased the survival of cells that were subsequently treated with combinations of the Wee1 inhibitor MK-1775 and/or the nucleoside analogue gemcitabine. In this constellation, Nutlin-3a reduced caspase activation and diminished the phosphorylation of Histone 2AX, an indicator of the DNA damage response. Both effects were strictly dependent on the presence of p53. Moreover, Nutlin pre-treatment reduced the fraction of cells that were undergoing premature mitosis in response to Wee1 inhibition. We conclude that the pre-activation of p53 through Mdm2 antagonists serves as a viable option to selectively protect p53-proficient cells against the cytotoxic effects of Wee1 inhibitors, especially when combined with a nucleoside analogue. Thus, Mdm2 antagonists might prove useful to avoid unwanted side effects of Wee1 inhibitors. On the other hand, when a tumor contains wild type p53, care should be taken not to induce its activity before applying Wee1 inhibitors.

  14. Ascaris lumbricoides β carbonic anhydrase: a potential target enzyme for treatment of ascariasis.

    Science.gov (United States)

    Zolfaghari Emameh, Reza; Kuuslahti, Marianne; Vullo, Daniela; Barker, Harlan R; Supuran, Claudiu T; Parkkila, Seppo

    2015-09-18

    A parasitic roundworm, Ascaris lumbricoides, is the causative agent of ascariasis, with approximately 760 million cases around the world. Helminthic infections occur with a high prevalence mostly in tropical and developing xcountries. Therefore, design of affordable broad-spectrum anti-helminthic agents against a variety of pathogens, including not only A. lumbricoides but also hookworms and whipworms, is desirable. Beta carbonic anhydrases (β-CAs) are considered promising targets of novel anthelminthics because these enzymes are present in various parasites, while completely absent in vertebrates. In this study, we identified an A. lumbricoides β-CA (AIBCA) protein from protein sequence data using bioinformatics tools. We used computational biology resources and methods (including InterPro, CATH/Gene3D, KEGG, and METACYC) to analyze AlBCA and define potential roles of this enzyme in biological pathways. The AlBCA gene was cloned into pFastBac1, and recombinant AIBCA was produced in sf-9 insect cells. Kinetics of AlBCA were analyzed by a stopped-flow method. Multiple sequence alignment revealed that AIBCA contains the two sequence motifs, CXDXR and HXXC, typical for β-CAs. Recombinant AIBCA showed significant CA catalytic activity with kcat of 6.0 × 10(5) s(-1) and kcat/KM of 4.3 × 10(7) M(-1) s(-1). The classical CA inhibitor, acetazolamide, showed an inhibition constant of 84.1 nM. Computational modeling suggests that the molecular architecture of AIBCA is highly similar to several other known β-CA structures. Functional predictions suggest that AIBCA might play a role in bicarbonate-mediated metabolic pathways, such as gluconeogenesis and removal of metabolically produced cyanate. These results open new avenues to further investigate the precise functions of β-CAs in parasites and suggest that novel β-CA specific inhibitors should be developed and tested against helminthic diseases.

  15. Thermostable Carbonic Anhydrases in Biotechnological Applications

    Directory of Open Access Journals (Sweden)

    Anna Di Fiore

    2015-07-01

    Full Text Available Carbonic anhydrases are ubiquitous metallo-enzymes which catalyze the reversible hydration of carbon dioxide in bicarbonate ions and protons. Recent years have seen an increasing interest in the utilization of these enzymes in CO2 capture and storage processes. However, since this use is greatly limited by the harsh conditions required in these processes, the employment of thermostable enzymes, both those isolated by thermophilic organisms and those obtained by protein engineering techniques, represents an interesting possibility. In this review we will provide an extensive description of the thermostable carbonic anhydrases so far reported and the main processes in which these enzymes have found an application.

  16. Protease inhibitors from processed legumes effectively inhibit superoxide generation in response to TPA.

    Science.gov (United States)

    Yavelow, J; Gidlund, M; Troll, W

    1982-01-01

    Crude extracts containing protease inhibitors from edible legumes (canned chick-peas, canned kidney beans and bean curd) were capable of blocking the superoxide response to human polymorphonuclear leukocytes produced by the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Protease inhibitors purified from crude extracts more effectively blocked the superoxide response produced by TPA. Bowman-Birk soybean inhibitor was more effective in blocking this effect of the tumor promoter than Kunitz soybean inhibitor. The significance of protease inhibitors in edible legumes and the possible role of free oxygen radicals in tumor promotion are discussed.

  17. Identification of 3,4-Dihydroisoquinoline-2(1H)-sulfonamides as Potent Carbonic AnhydraseInhibitors: Synthesis, Biological Evaluation, and Enzyme-Ligand X-ray Studies

    Czech Academy of Sciences Publication Activity Database

    Gitto, R.; Agnello, S.; Ferro, S.; De Luca, L.; Vullo, D.; Brynda, Jiří; Mader, Pavel; Supuran, C. T.; Chimirri, A.

    2010-01-01

    Roč. 53, č. 6 (2010), s. 2401-2408 ISSN 0022-2623 R&D Projects: GA ČR GA203/09/0820 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520514 Keywords : inhibitor design * crystal structure * X-ray analysis * dehalogenase Subject RIV: CC - Organic Chemistry Impact factor: 5.207, year: 2010

  18. Organic compounds as corrosion inhibitors for mild steel in acidic media: correlation between inhibition efficiency and chemical structure

    Energy Technology Data Exchange (ETDEWEB)

    Elias, Elizandra C.S.; Chrisman, Erika C.A.N. [Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ (Brazil). Escola de Quimica

    2009-12-19

    The use of inhibitors for mild steels corrosion control which are in contact with aggressive environment is an accepted practice in acid treatment of oil-wells. Organic compounds have been studied to evaluate their corrosion inhibition potential. Film-forming corrosion inhibitors, commonly used to protect oil-field equipment, can be absorbed on the steel surface to give structurally ordered layers. Therefore, the electrons should act as an important role for this adsorption. Studies reveal that organic compounds show significant inhibition efficiency. For this purpose, their molecules should contain N, O and S heteroatoms in various functional groups, long hydrocarbon linear or branched radical and anion and cation active components. However, most of these compounds are not only expensive but also toxic to living beings. According to the 'Green Chemistry' rules, corrosion inhibitors based on organic compounds should be cheap, with low toxicity and have high inhibition efficiency. In this study, the effects of some organic compounds with different groups such as amide, ether, phenyldiamine, anime and aminophenol on the corrosion behavior of mild steel in acidic media have been investigated. The experimental data were obtained by gravimetric measurements. The results show that these compounds reveal a promising corrosion inhibition where phenyldiamine is the most efficient. The effect of molecular structure on the corrosion inhibition efficiency was investigated by semi-empirical quantum chemical calculations. The electronic properties such as highest occupied molecular orbital (HOMO), lowest unoccupied molecular orbital (LUMO) energy levels, and LUMO-HOMO energy gap orbital density were calculated. The relations between the inhibition efficiency and some quantum parameters are discussed and correlations are proposed. The highest values for the HOMO densities were found in the vicinity nitrogen atom, indicating that it is the most probable adsorption center

  19. Inhibitors of the mitochondrial cytochrome b-c1 complex inhibit the cyanide-insensitive respiration of Trypanosoma brucei.

    Science.gov (United States)

    Turrens, J F; Bickar, D; Lehninger, A L

    1986-06-01

    The cyanide-insensitive respiration of bloodstream trypomastigote forms of Trypanosoma brucei (75 +/- 8 nmol O2 min-1(mg protein)-1) is completely inhibited by the mitochondrial ubiquinone-like inhibitors 2-hydroxy-3-undecyl-1,4-naphthoquinone (UHNQ) and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT). The Ki values for UHDBT (30 nM) and UHNQ (2 microM) are much lower than the reported Ki for salicylhydroxamic acid (SHAM) (5 microM), a widely used inhibitor of the cyanide-insensitive oxidase. UHNQ also stimulated the glycerol-3-phosphate-dependent reduction of phenazine methosulfate, demonstrating that the site of UHNQ inhibition is on the terminal oxidase of the cyanide-insensitive respiration of T. brucei. These results suggest that a ubiquinone-like compound may act as an electron carrier between the two enzymatic components of the cyanide-insensitive glycerol-3-phosphate oxidase.

  20. mTOR inhibitors block Kaposi sarcoma growth by inhibiting essential autocrine growth factors and tumor angiogenesis.

    Science.gov (United States)

    Roy, Debasmita; Sin, Sang-Hoon; Lucas, Amy; Venkataramanan, Raman; Wang, Ling; Eason, Anthony; Chavakula, Veenadhari; Hilton, Isaac B; Tamburro, Kristen M; Damania, Blossom; Dittmer, Dirk P

    2013-04-01

    Kaposi sarcoma originates from endothelial cells and it is one of the most overt angiogenic tumors. In Sub-Saharan Africa, where HIV and the Kaposi sarcoma-associated herpesvirus (KSHV) are endemic, Kaposi sarcoma is the most common cancer overall, but model systems for disease study are insufficient. Here, we report the development of a novel mouse model of Kaposi sarcoma, where KSHV is retained stably and tumors are elicited rapidly. Tumor growth was sensitive to specific allosteric inhibitors (rapamycin, CCI-779, and RAD001) of the pivotal cell growth regulator mTOR. Inhibition of tumor growth was durable up to 130 days and reversible. mTOR blockade reduced VEGF secretion and formation of tumor vasculature. Together, the results show that mTOR inhibitors exert a direct anti-Kaposi sarcoma effect by inhibiting angiogenesis and paracrine effectors, suggesting their application as a new treatment modality for Kaposi sarcoma and other cancers of endothelial origin. ©2012 AACR.

  1. Genetic and Pharmacological Inhibition of PDK1 in Cancer Cells: Characterization of a Selective Allosteric Kinase Inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Nagashima, Kumiko; Shumway, Stuart D.; Sathyanarayanan, Sriram; Chen, Albert H.; Dolinski, Brian; Xu, Youyuan; Keilhack, Heike; Nguyen, Thi; Wiznerowicz, Maciej; Li, Lixia; Lutterbach, Bart A.; Chi, An; Paweletz, Cloud; Allison, Timothy; Yan, Youwei; Munshi, Sanjeev K.; Klippel, Anke; Kraus, Manfred; Bobkova, Ekaterina V.; Deshmukh, Sujal; Xu, Zangwei; Mueller, Uwe; Szewczak, Alexander A.; Pan, Bo-Sheng; Richon, Victoria; Pollock, Roy; Blume-Jensen, Peter; Northrup, Alan; Andersen, Jannik N. (Merck)

    2013-11-20

    Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1-5, which are classical ATP-competitive kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems.

  2. Human monoamine oxidase is inhibited by tobacco smoke: β-carboline alkaloids act as potent and reversible inhibitors

    International Nuclear Information System (INIS)

    Herraiz, Tomas; Chaparro, Carolina

    2005-01-01

    Monoamine oxidase (MAO) is a mitochondrial outer-membrane flavoenzyme involved in brain and peripheral oxidative catabolism of neurotransmitters and xenobiotic amines, including neurotoxic amines, and a well-known target for antidepressant and neuroprotective drugs. Recently, positron emission tomography imaging has shown that smokers have a much lower activity of peripheral and brain MAO-A (30%) and -B (40%) isozymes compared to non-smokers. This MAO inhibition results from a pharmacological effect of smoke, but little is known about its mechanism. Working with mainstream smoke collected from commercial cigarettes we confirmed that cigarette smoke is a potent inhibitor of human MAO-A and -B isozymes. MAO inhibition was partly reversible, competitive for MAO-A, and a mixed-type inhibition for MAO-B. Two β-carboline alkaloids, norharman (β-carboline) and harman (1-methyl-β-carboline), were identified by GC-MS, quantified, and isolated from the mainstream smoke by solid phase extraction and HPLC. Kinetics analysis revealed that β-carbolines from cigarette smoke were competitive, reversible, and potent inhibitors of MAO enzymes. Norharman was an inhibitor of MAO-A (K i = 1.2 ± 0.18 μM) and MAO-B (K i = 1.12 ± 0.19 μM), and harman of MAO-A (K i = 55.54 ± 5.3 nM). β-Carboline alkaloids are psychopharmacologically active compounds that may occur endogenously in human tissues, including the brain. These results suggest that β-carboline alkaloids from cigarette smoke acting as potent reversible inhibitors of MAO enzymes may contribute to the MAO-reduced activity produced by tobacco smoke in smokers. The presence of MAO inhibitors in smoke like β-carbolines and others may help us to understand some of the purported neuropharmacological effects associated with smoking

  3. Inhibition of SRC-3 enhances sensitivity of human cancer cells to histone deacetylase inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Zou, Zhengzhi, E-mail: zouzhengzhi@m.scnu.edu.cn [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510000 (China); Luo, Xiaoyong [Department of Oncology, The Affiliated Luoyang Central Hospital of Zhengzhou University, Luoyang 471000 (China); Nie, Peipei [KingMed Diagnostics and KingMed School of Laboratory Medicine, Guangzhou Medical University, Guangzhou 510000 (China); Wu, Baoyan; Zhang, Tao; Wei, Yanchun [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510000 (China); Wang, Wenyi [Xiamen Cancer Center, Department of Medical Oncology, The First Affiliated Hospital of Xiamen University, Xiamen 361000 (China); Geng, Guojun; Jiang, Jie [Xiamen Cancer Center, Department of Thoracic Surgery, The First Affiliated Hospital of Xiamen University, Xiamen 361000 (China); Mi, Yanjun, E-mail: myjgj_77@163.com [Xiamen Cancer Center, Department of Medical Oncology, The First Affiliated Hospital of Xiamen University, Xiamen 361000 (China)

    2016-09-09

    SRC-3 is widely expressed in multiple tumor types and involved in cancer cell proliferation and apoptosis. Histone deacetylase (HDAC) inhibitors are promising antitumor drugs. However, the poor efficacy of HDAC inhibitors in solid tumors has restricted its further clinical application. Here, we reported the novel finding that depletion of SRC-3 enhanced sensitivity of breast and lung cancer cells to HDAC inhibitors (SAHA and romidepsin). In contrast, overexpression of SRC-3 decreased SAHA-induced cancer cell apoptosis. Furthermore, we found that SRC-3 inhibitor bufalin increased cancer cell apoptosis induced by HDAC inhibitors. The combination of bufalin and SAHA was particular efficient in attenuating AKT activation and reducing Bcl-2 levels. Taken together, these accumulating data might guide development of new breast and lung cancer therapies. - Highlights: • Depletion of SRC-3 enhanced sensitivity of breast and lung cancer cells to HDAC inhibitors. • Overexpression of SRC-3 enhanced cancer cell resistance to HDAC inhibitors. • SRC-3 inhibitor bufalin increased cancer cell apoptosis induced by HDAC inhibitors. • Bufalin synergized with HDAC inhibitor attenuated AKT activation and reduced Bcl-2 levels in human cancer cell.

  4. Human carbonic anhydrase inhibitory profile of mono- and bis-sulfonamides synthesized via a direct sulfochlorination of 3- and 4-(hetero)arylisoxazol-5-amine scaffolds.

    Science.gov (United States)

    Krasavin, Mikhail; Korsakov, Mikhail; Zvonaryova, Zhanna; Semyonychev, Evgenii; Tuccinardi, Tiziano; Kalinin, Stanislav; Tanç, Muhammet; Supuran, Claudiu T

    2017-03-15

    Three distinct series of isoxazole-based primary mono- and bis-sulfonamides have been synthesized via direct sulfochlorination, each of them delivering nanomolar inhibitors of human carbonic anhydrase. Certain pronounced SAR trends have been established and rationalized by in silico docking. These findings expand the structure-activity knowledge base for heterocycle-containing sulfonamide carbonic anhydrase inhibitors and further validate the power of direct electrophilic sulfochlorination as a means of introducing the pharmacophoric primary sulfonamide group into structurally diverse aromatic precursors. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. A Revisit to the Corrosion Inhibition of Aluminum in Aqueous Alkaline Solutions by Water-Soluble Alginates and Pectates as Anionic Polyelectrolyte Inhibitors

    Directory of Open Access Journals (Sweden)

    Refat Hassan

    2013-01-01

    Full Text Available The corrosion behavior of aluminum (Al in alkaline media in presence of some natural polymer inhibitors has been reinvestigated. The inhibition action of the tested inhibitors was found to obey both Langmuir and Freundlich isotherms models. The inhibition efficiency was found to increase with increasing the inhibitors concentration and decrease with increasing the temperature, suggesting physical adsorption mechanism. Factors such as the concentration and geometrical structure of the inhibitor, concentration of the corrosive medium, and temperature affecting the corrosion rates were examined. The kinetic parameters were evaluated, and a suitable corrosion mechanism consistent with the kinetic results obtained is suggested and discussed.

  6. Hsp90 inhibitor geldanamycin attenuates the cytotoxicity of sunitinib in cardiomyocytes via inhibition of the autophagy pathway.

    Science.gov (United States)

    Kimura, Takayuki; Uesugi, Mai; Takase, Kazuma; Miyamoto, Norimasa; Sawada, Kohei

    2017-08-15

    Sunitinib malate (sunitinib) is an orally available, multitargeted tyrosine kinase inhibitor with antitumor and antiangiogenic activities. Although sunitinib is effective for the treatment of patients with gastrointestinal stromal tumor, advanced renal cell carcinoma, or pancreatic neuroendocrine tumor, adverse cardiac events associated with sunitinib administration have been reported. Here, we examined the effect of geldanamycin, an inhibitor of heat shock protein (Hsp) 90, on sunitinib-induced cytotoxicity in cardiomyocytes. First, we found that treatment with geldanamycin or other Hsp90 inhibitors (tanespimycin, ganetespib, or BIIB021) significantly attenuated sunitinib-induced cytotoxicity in rat H9c2 cardiomyocytes, suggesting a drug-class effect of Hsp90 inhibitors. We then examined the mechanisms underlying sunitinib-induced cytotoxicity and found that sunitinib induced autophagy in H9c2 cells and that pretreatment with geldanamycin inhibited the induction of autophagy by promoting degradation of the autophagy-related proteins Atg7, Beclin-1, and ULK1. Pharmacological assessment with autophagy inhibitors confirmed that geldanamycin attenuated the cytotoxicity of sunitinib by interfering with autophagy. In addition, we found that the molecular chaperone Hsp70, which is induced by geldanamycin, was not involved in the attenuation of sunitinib-induced cytotoxicity. Finally, to provide more clinically relevant data, we confirmed that geldanamycin attenuated sunitinib-induced cytotoxicity in human induced pluripotent stem cell-derived cardiomyocytes. Together, these data suggest that geldanamycin attenuates sunitinib-induced cytotoxicity in cardiomyocytes by inhibiting the autophagy pathway. Thus, the further investigation of combination or sequential treatment with an Hsp90 inhibitor and sunitinib is warranted as a potential strategy of attenuating the cardiotoxicity associated with sunitinib administration in the clinical setting. Copyright © 2017 Elsevier Inc

  7. SphK1 inhibitor II (SKI-II) inhibits acute myelogenous leukemia cell growth in vitro and in vivo.

    Science.gov (United States)

    Yang, Li; Weng, Wei; Sun, Zhi-Xin; Fu, Xian-Jie; Ma, Jun; Zhuang, Wen-Fang

    2015-05-15

    Previous studies have identified sphingosine kinase 1 (SphK1) as a potential drug target for treatment of acute myeloid leukemia (AML). In the current study, we investigated the potential anti-leukemic activity of a novel and specific SphK1 inhibitor, SKI-II. We demonstrated that SKI-II inhibited growth and survival of human AML cell lines (HL-60 and U937 cells). SKI-II was more efficient than two known SphK1 inhibitors SK1-I and FTY720 in inhibiting AML cells. Meanwhile, it induced dramatic apoptosis in above AML cells, and the cytotoxicity by SKI-II was almost reversed by the general caspase inhibitor z-VAD-fmk. SKI-II treatment inhibited SphK1 activation, and concomitantly increased level of sphingosine-1-phosphate (S1P) precursor ceramide in AML cells. Conversely, exogenously-added S1P protected against SKI-II-induced cytotoxicity, while cell permeable short-chain ceramide (C6) aggravated SKI-II's lethality against AML cells. Notably, SKI-II induced potent apoptotic death in primary human AML cells, but was generally safe to the human peripheral blood mononuclear cells (PBMCs) isolated from healthy donors. In vivo, SKI-II administration suppressed growth of U937 leukemic xenograft tumors in severe combined immunodeficient (SCID) mice. These results suggest that SKI-II might be further investigated as a promising anti-AML agent. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Optimization of nutritional constituents for carbonic anhydrase ...

    African Journals Online (AJOL)

    Jane

    2011-08-08

    Aug 8, 2011 ... design. INTRODUCTION. Carbonic anhydrase (CA), which catalyzes the hydration of carbon dioxide and dehydration of dicarbonate, exists in most living organism ... The optimal design of the culture medium is one of the most important ... The reactive mixture consisted of 1 mL enzyme solution and 1 mL ...

  9. Coral Carbonic Anhydrases: Regulation by Ocean Acidification

    Directory of Open Access Journals (Sweden)

    Didier Zoccola

    2016-06-01

    Full Text Available Global change is a major threat to the oceans, as it implies temperature increase and acidification. Ocean acidification (OA involving decreasing pH and changes in seawater carbonate chemistry challenges the capacity of corals to form their skeletons. Despite the large number of studies that have investigated how rates of calcification respond to ocean acidification scenarios, comparatively few studies tackle how ocean acidification impacts the physiological mechanisms that drive calcification itself. The aim of our paper was to determine how the carbonic anhydrases, which play a major role in calcification, are potentially regulated by ocean acidification. For this we measured the effect of pH on enzyme activity of two carbonic anhydrase isoforms that have been previously characterized in the scleractinian coral Stylophora pistillata. In addition we looked at gene expression of these enzymes in vivo. For both isoforms, our results show (1 a change in gene expression under OA (2 an effect of OA and temperature on carbonic anhydrase activity. We suggest that temperature increase could counterbalance the effect of OA on enzyme activity. Finally we point out that caution must, thus, be taken when interpreting transcriptomic data on carbonic anhydrases in ocean acidification and temperature stress experiments, as the effect of these stressors on the physiological function of CA will depend both on gene expression and enzyme activity.

  10. Coral Carbonic Anhydrases: Regulation by Ocean Acidification.

    Science.gov (United States)

    Zoccola, Didier; Innocenti, Alessio; Bertucci, Anthony; Tambutté, Eric; Supuran, Claudiu T; Tambutté, Sylvie

    2016-06-03

    Global change is a major threat to the oceans, as it implies temperature increase and acidification. Ocean acidification (OA) involving decreasing pH and changes in seawater carbonate chemistry challenges the capacity of corals to form their skeletons. Despite the large number of studies that have investigated how rates of calcification respond to ocean acidification scenarios, comparatively few studies tackle how ocean acidification impacts the physiological mechanisms that drive calcification itself. The aim of our paper was to determine how the carbonic anhydrases, which play a major role in calcification, are potentially regulated by ocean acidification. For this we measured the effect of pH on enzyme activity of two carbonic anhydrase isoforms that have been previously characterized in the scleractinian coral Stylophora pistillata. In addition we looked at gene expression of these enzymes in vivo. For both isoforms, our results show (1) a change in gene expression under OA (2) an effect of OA and temperature on carbonic anhydrase activity. We suggest that temperature increase could counterbalance the effect of OA on enzyme activity. Finally we point out that caution must, thus, be taken when interpreting transcriptomic data on carbonic anhydrases in ocean acidification and temperature stress experiments, as the effect of these stressors on the physiological function of CA will depend both on gene expression and enzyme activity.

  11. Optimization of nutritional constituents for carbonic anhydrase ...

    African Journals Online (AJOL)

    Jane

    2011-08-08

    Aug 8, 2011 ... The objective of this study was to optimize the submerged cultured conditions of carbonic anhydrase. (CA) production from the strain of Bacillus mucilaginosus K02 by mono-factor tests with blank control and orthogonal design methodology. When the bacteria was cultured under the condition of mono-.

  12. Optimization of nutritional constituents for carbonic anhydrase ...

    African Journals Online (AJOL)

    The objective of this study was to optimize the submerged cultured conditions of carbonic anhydrase (CA) production from the strain of Bacillus mucilaginosus K02 by mono-factor tests with blank control and orthogonal design methodology. When the bacteria was cultured under the condition of monofactor tests with blank ...

  13. 4-Bromophenylhydrazinyl benzenesulfonylphenylureas as indoleamine 2,3-dioxygenase inhibitors with in vivo target inhibition and anti-tumor efficacy.

    Science.gov (United States)

    Lin, Shu-Yu; Yeh, Teng-Kuang; Song, Jen-Shin; Hung, Ming-Shiu; Cheng, Ming-Fu; Liao, Fang-Yu; Li, An-Shiou; Cheng, Shu-Ying; Lin, Li-Mei; Chiu, Chun-Hsien; Wu, Mine-Hsine; Lin, Yi-Jyun; Hsiao, Wenchi; Sun, Manwu; Wang, Yi-Hsin; Huang, Chin-Hsiang; Tang, Ya-Chu; Chang, Hsin-Huei; Huang, Zih-Ting; Chao, Yu-Sheng; Shih, Chuan; Pan, Shiow-Lin; Wu, Su-Ying; Kuo, Ching-Chuan; Ueng, Shau-Hua

    2018-02-12

    Indoleamine 2,3-dioxygenase is a heme-containing enzyme implicated in the down regulation of the anti-tumor immune response, and considered a promising anti-cancer drug target. Several pharmaceutical companies, including Pfizer, Merck, and Bristol-Myers Squibb, are known to be in pursuit of IDO inhibitors, and Incyte recently reported good results in the phase II clinical trial of the IDO inhibitor Epacadostat. In previous work, we developed a series of IDO inhibitors based on a sulfonylhydrazide core structure, and explored how they could serve as potent IDO inhibitors with good drug profiles. Herein, we disclose the development of the 4-bromophenylhydrazinyl benzenesulfonylphenylurea 5k, a potent IDO inhibitor which demonstrated 25% tumor growth inhibition in a murine CT26 syngeneic model on day 18 with 100 mg/kg oral administration twice daily, and a 30% reduction in tumor weight. Pharmacodynamic testing of 5k found it to cause a 25% and 21% reduction in kyn/trp ratio at the plasma and tumor, respectively. In the CT26 tumor model, 5k was found to slightly increase the percentage of CD3 + T cells and lymphocyte responsiveness, indicating that 5k may have potential in modulating anti-tumor immunity. These data suggest 5k to be worthy of further investigation in the development of anti-tumor drugs. Copyright © 2018 Elsevier Inc. All rights reserved.

  14. Benzothiazole Derivative as a Novel Mycobacterium tuberculosis Shikimate Kinase Inhibitor: Identification and Elucidation of Its Allosteric Mode of Inhibition.

    Science.gov (United States)

    Mehra, Rukmankesh; Rajput, Vikrant Singh; Gupta, Monika; Chib, Reena; Kumar, Amit; Wazir, Priya; Khan, Inshad Ali; Nargotra, Amit

    2016-05-23

    Mycobacterium tuberculosis shikimate kinase (Mtb-SK) is a key enzyme involved in the biosynthesis of aromatic amino acids through the shikimate pathway. Since it is proven to be essential for the survival of the microbe and is absent from mammals, it is a promising target for anti-TB drug discovery. In this study, a combined approach of in silico similarity search and pharmacophore building using already reported inhibitors was used to screen a procured library of 20,000 compounds of the commercially available ChemBridge database. From the in silico screening, 15 hits were identified, and these hits were evaluated in vitro for Mtb-SK enzyme inhibition. Two compounds presented significant enzyme inhibition with IC50 values of 10.69 ± 0.9 and 46.22 ± 1.2 μM. The best hit was then evaluated for the in vitro mode of inhibition where it came out to be an uncompetitive and noncompetitive inhibitor with respect to shikimate (SKM) and ATP, respectively, suggesting its binding at an allosteric site. Potential binding sites of Mtb-SK were identified which confirmed the presence of an allosteric binding pocket apart from the ATP and SKM binding sites. The docking simulations were performed at this pocket in order to find the mode of binding of the best hit in the presence of substrates and the products of the enzymatic reaction. Molecular dynamics (MD) simulations elucidated the probability of inhibitor binding at the allosteric site in the presence of ADP and shikimate-3-phosphate (S-3-P), that is, after the formation of products of the reaction. The inhibitor binding may prevent the release of the product from Mtb-SK, thereby inhibiting its activity. The binding stability and the key residue interactions of the inhibitor to this product complex were also revealed by the MD simulations. Residues ARG43, ILE45, and PHE57 were identified as crucial that were involved in interactions with the best hit. This is the first report of an allosteric binding site of Mtb-SK, which

  15. Env-glycoprotein heterogeneity as a source of apparent synergy and enhanced cooperativity in inhibition of HIV-1 infection by neutralizing antibodies and entry inhibitors

    Science.gov (United States)

    Ketas, Thomas J.; Holuigue, Sophie; Matthews, Katie; Moore, John P.

    2011-01-01

    We measured the inhibition of infectivity of HIV-1 isolates and derivative clones by combinations of neutralizing antibodies (NAbs) and other entry inhibitors in a single-cycle-replication assay. Synergy was analyzed both by the current linear and a new nonlinear method. The new method reduced spurious indications of synergy and antagonism. Synergy between NAbs was overall weaker than between other entry inhibitors, and no stronger where one ligand is known to enhance the binding of another. However, synergy was stronger for a genetically heterogeneous HIV-1 R5 isolate than for its derivative clones. Enhanced cooperativity in inhibition by combinations, compared with individual inhibitors, correlated with increased synergy at higher levels of inhibition, while being less variable. Again, cooperativity enhancement was stronger for isolates than clones. We hypothesize that genetic, post-translational or conformational heterogeneity of the Env protein and of other targets for inhibitors can yield apparent synergy and increased cooperativity between inhibitors. PMID:22018634

  16. Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain

    Science.gov (United States)

    Mistry, Pragnesh; Laird, Michelle H. W.; Schwarz, Ryan S.; Greene, Shannon; Dyson, Tristan; Snyder, Greg A.; Xiao, Tsan Sam; Chauhan, Jay; Fletcher, Steven; Toshchakov, Vladimir Y.; MacKerell, Alexander D.; Vogel, Stefanie N.

    2015-01-01

    Toll-like receptor (TLR) signaling is initiated by dimerization of intracellular Toll/IL-1 receptor resistance (TIR) domains. For all TLRs except TLR3, recruitment of the adapter, myeloid differentiation primary response gene 88 (MyD88), to TLR TIR domains results in downstream signaling culminating in proinflammatory cytokine production. Therefore, blocking TLR TIR dimerization may ameliorate TLR2-mediated hyperinflammatory states. The BB loop within the TLR TIR domain is critical for mediating certain protein–protein interactions. Examination of the human TLR2 TIR domain crystal structure revealed a pocket adjacent to the highly conserved P681 and G682 BB loop residues. Using computer-aided drug design (CADD), we sought to identify a small molecule inhibitor(s) that would fit within this pocket and potentially disrupt TLR2 signaling. In silico screening identified 149 compounds and 20 US Food and Drug Administration-approved drugs based on their predicted ability to bind in the BB loop pocket. These compounds were screened in HEK293T-TLR2 transfectants for the ability to inhibit TLR2-mediated IL-8 mRNA. C16H15NO4 (C29) was identified as a potential TLR2 inhibitor. C29, and its derivative, ortho-vanillin (o-vanillin), inhibited TLR2/1 and TLR2/6 signaling induced by synthetic and bacterial TLR2 agonists in human HEK-TLR2 and THP-1 cells, but only TLR2/1 signaling in murine macrophages. C29 failed to inhibit signaling induced by other TLR agonists and TNF-α. Mutagenesis of BB loop pocket residues revealed an indispensable role for TLR2/1, but not TLR2/6, signaling, suggesting divergent roles. Mice treated with o-vanillin exhibited reduced TLR2-induced inflammation. Our data provide proof of principle that targeting the BB loop pocket is an effective approach for identification of TLR2 signaling inhibitors. PMID:25870276

  17. Caractérisation d’inhibiteurs d’anhydrase carbonique IX, études de complexes supramoléculaires et interactions moléculaires par résonance plasmonique de surface

    OpenAIRE

    Florent, Tiphaine

    2014-01-01

    Carbonic anhydrase (CA) IX expression is increased upon hypoxia and has been proposed as a therapeutic target since it has been associated with poor prognosis, tumor progression and pH regulation. A new class of human carbonic anhydrase IX (hCA IX) inhibitors, diarylpyrazole sulfonamide derivatives, has been synthesized in our team. These compounds have a very limited water solubility which limits their pharmaceutical development. The complexation with cyclodextrins (CDs) offers the possibili...

  18. Salicylate, a catalytic inhibitor of topoisomerase II, inhibits DNA cleavage and is selective for the α isoform.

    Science.gov (United States)

    Bau, Jason T; Kang, Zhili; Austin, Caroline A; Kurz, Ebba U

    2014-02-01

    Topoisomerase II (topo II) is a ubiquitous enzyme that is essential for cell survival through its role in regulating DNA topology and chromatid separation. Topo II can be poisoned by common chemotherapeutics (such as doxorubicin and etoposide), leading to the accumulation of cytotoxic enzyme-linked DNA double-stranded breaks. In contrast, nonbreak-inducing topo II catalytic inhibitors have also been described and have more limited use in clinical chemotherapy. These agents, however, may alter the efficacy of regimens incorporating topo II poisons. We previously identified salicylate, the primary metabolite of aspirin, as a novel catalytic inhibitor of topo II. We have now determined the mechanism by which salicylate inhibits topo II. As catalytic inhibitors can act at a number of steps in the topo II catalytic cycle, we used multiple independent, biochemical approaches to interrogate the catalytic cycle. Furthermore, as mammalian cells express two isoforms of topo II (α and β), we examined whether salicylate was isoform selective. Our results demonstrate that salicylate is unable to intercalate DNA, and does not prevent enzyme-DNA interaction, nor does it promote stabilization of topo IIα in closed clamps on DNA. Although salicylate decreased topo IIα ATPase activity in a dose-dependent noncompetitive manner, this was secondary to salicylate-mediated inhibition of DNA cleavage. Surprisingly, comparison of salicylate's effects using purified human topo IIα and topo IIβ revealed that salicylate selectively inhibits the α isoform. These findings provide a definitive mechanism for salicylate-mediated inhibition of topo IIα and provide support for further studies determining the basis for its isoform selectivity.

  19. On determinants of glomerular filtration rate after inhibition of proximal tubular reabsorption

    DEFF Research Database (Denmark)

    Leyssac, P P; Karlsen, F M; Holstein-Rathlou, N H

    1994-01-01

    The carbonic anhydrase inhibitor acetazolamide (ACZ) inhibits the absolute rate of proximal reabsorption (APR), causes a reduction in glomerular filtration rate (GFR), and activates the tubuloglomerular feedback mechanism (TGF) resulting in afferent vasoconstriction. The quantitative importance...... of the afferent vasoconstriction for the reduced GFR was tested by addition of a vasodilator during continuous infusion of ACZ. Dopamine caused an increase in renal blood flow (RBF) to pre-ACZ levels. Glomerular capillary pressure (Pgc) and proximal tubular pressure (Pprox) increased in parallel (by 3.1 and 3...

  20. Mammalian Target of Rapamycin Inhibitors Induce Tumor Cell Apoptosis In Vivo Primarily by Inhibiting VEGF Expression and Angiogenesis

    Directory of Open Access Journals (Sweden)

    Patrick Frost

    2013-01-01

    Full Text Available We found that rapalog mTOR inhibitors induce G1 arrest in the PTEN-null HS Sultan B-cell lymphoma line in vitro, but that administration of rapalogs in a HS Sultan xenograft model resulted in significant apoptosis, and that this correlated with induction of hypoxia and inhibition of neoangiogenesis and VEGF expression. Mechanistically, rapalogs prevent cap-dependent translation, but studies have shown that cap-independent, internal ribosome entry site (IRES-mediated translation of genes, such as c-myc and cyclin D, can provide a fail-safe mechanism that regulates tumor survival. Therefore, we tested if IRES-dependent expression of VEGF could likewise regulate sensitivity of tumor cells in vivo. To achieve this, we developed isogenic HS Sultan cell lines that ectopically express the VEGF ORF fused to the p27 IRES, an IRES sequence that is insensitive to AKT-mediated inhibition of IRES activity and effective in PTEN-null tumors. Mice challenged with p27-VEGF transfected tumor cells were more resistant to the antiangiogenic and apoptotic effects of the rapalog, temsirolimus, and active site mTOR inhibitor, pp242. Our results confirm the critical role of VEGF expression in tumors during treatment with mTOR inhibitors and underscore the importance of IRES activity as a resistance mechanism to such targeted therapy.

  1. Inhibitory effects of cadmium on carbonic anhydrase activity and ionic regulation of the estuarine crab Chasmagnathus granulata (Decapoda, Grapsidae)

    International Nuclear Information System (INIS)

    Vitale, A.M.; Monserrat, J.M.; Castilho, P.; Rodriguez, E.M.

    1999-01-01

    This work was aimed at evaluating the gill carbonic anhydrase (CA) activity of the estuarine crab Chasmagnathus granulata exposed in vivo to cadmium, at different salinities. The in vivo effect of the specific inhibitor acetazolamide (AZ) was also assayed. Besides, the inhibition of CA activity by different heavy metals (cadmium, copper, zinc) and AZ were evaluated under in vitro conditions. For the in vivo assays, adult males were acclimated to salinities of 2.5 or 30per thousand. The corresponding 96-h LC 50 of cadmium was 2.69 mg l -1 50 mg l -1 at 30per thousand. Cadmium only caused a significant lower CA activity than control at 2.5per thousand. EC 50 for CA inhibition was estimated to be 1.59 mg l -1 at 2.5per thousand. Statistical differences in Na + hemolymphatic levels (P -1 of cadmium, but no statistical differences were observed for Cl - levels at any assayed salinity. As CA inhibition registered at 2.5per thousand was followed by only changes in Na + concentration, it is likely that cadmium exposure could differentially affect ions permeability, among others factors. The concentrations that inhibited in vitro 50% of enzymatic activity (IC 50 ) were 2.15x10 -5 , 1.62x10 -5 , 3.75x10 -6 and 4.4x10 -10 M for cadmium, copper, zinc and AZ, respectively. The comparison with IC 50 values of other aquatic species, indicates a higher CA sensitivity for C. granulata to pollutants. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

  2. Molecular modeling study for inhibition mechanism of human chymase and its application in inhibitor design.

    Directory of Open Access Journals (Sweden)

    Mahreen Arooj

    Full Text Available Human chymase catalyzes the hydrolysis of peptide bonds. Three chymase inhibitors with very similar chemical structures but highly different inhibitory profiles towards the hydrolase function of chymase were selected with the aim of elucidating the origin of disparities in their biological activities. As a substrate (angiotensin-I bound crystal structure is not available, molecular docking was performed to dock the substrate into the active site. Molecular dynamics simulations of chymase complexes with inhibitors and substrate were performed to calculate the binding orientation of inhibitors and substrate as well as to characterize conformational changes in the active site. The results elucidate details of the 3D chymase structure as well as the importance of K40 in hydrolase function. Binding mode analysis showed that substitution of a heavier Cl atom at the phenyl ring of most active inhibitor produced a great deal of variation in its orientation causing the phosphinate group to interact strongly with residue K40. Dynamics simulations revealed the conformational variation in region of V36-F41 upon substrate and inhibitor binding induced a shift in the location of K40 thus changing its interactions with them. Chymase complexes with the most active compound and substrate were used for development of a hybrid pharmacophore model which was applied in databases screening. Finally, hits which bound well at the active site, exhibited key interactions and favorable electronic properties were identified as possible inhibitors for chymase. This study not only elucidates inhibitory mechanism of chymase inhibitors but also provides key structural insights which will aid in the rational design of novel potent inhibitors of the enzyme. In general, the strategy applied in the current study could be a promising computational approach and may be generally applicable to drug design for other enzymes.

  3. Data on the inhibition of RNase inhibitor activity by a monoclonal antibody as assessed by microfluidics-based RNA electrophoresis.

    Science.gov (United States)

    Wang, Xiao; Teferedegne, Belete; Shatzkes, Kenneth; Tu, Wei; Murata, Haruhiko

    2016-12-01

    Using purified reaction components, a commercial monoclonal antibody (Ab) specific to RNase inhibitor (RI) was found to interfere with the activity of RI. Total RNA was mixed with a monoclonal Ab specific to either RI (clone 3F11) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNase A, RI, or a combination of the above. Following incubation for 1 h at 22 °C or 37 °C, RNA integrity of the mixtures was assessed using microfluidics-based Bio-Rad Experion RNA electrophoresis. The addition of Ab 3F11 prevented RI from effectively inhibiting RNase A and therefore resulted in extensive RNA degradation. The data presented are associated with the research article entitled "Endogenous RNase Inhibitor Contributes to Stability of RNA in Crude Cell Lysates: Applicability to Reverse Transcription Quantitative PCR (RT-qPCR)" (Wang et al., 2016) [1].

  4. Data on the inhibition of RNase inhibitor activity by a monoclonal antibody as assessed by microfluidics-based RNA electrophoresis

    Directory of Open Access Journals (Sweden)

    Xiao Wang

    2016-12-01

    Full Text Available Using purified reaction components, a commercial monoclonal antibody (Ab specific to RNase inhibitor (RI was found to interfere with the activity of RI. Total RNA was mixed with a monoclonal Ab specific to either RI (clone 3F11 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, RNase A, RI, or a combination of the above. Following incubation for 1 h at 22 °C or 37 °C, RNA integrity of the mixtures was assessed using microfluidics-based Bio-Rad Experion RNA electrophoresis. The addition of Ab 3F11 prevented RI from effectively inhibiting RNase A and therefore resulted in extensive RNA degradation. The data presented are associated with the research article entitled “Endogenous RNase Inhibitor Contributes to Stability of RNA in Crude Cell Lysates: Applicability to Reverse Transcription Quantitative PCR (RT-qPCR” (Wang et al., 2016 [1].

  5. Regulatory T-Cell Augmentation or Interleukin-17 Inhibition Prevents Calcineurin Inhibitor-Induced Hypertension in Mice.

    Science.gov (United States)

    Chiasson, Valorie L; Pakanati, Abhinandan R; Hernandez, Marcos; Young, Kristina J; Bounds, Kelsey R; Mitchell, Brett M

    2017-07-01

    The immunosuppressive calcineurin inhibitors cyclosporine A and tacrolimus alter T-cell subsets and can cause hypertension, vascular dysfunction, and renal toxicity. We and others have reported that cyclosporine A and tacrolimus decrease anti-inflammatory regulatory T cells and increase proinflammatory interleukin-17-producing T cells; therefore, we hypothesized that inhibition of these effects using noncellular therapies would prevent the hypertension, endothelial dysfunction, and renal glomerular injury induced by calcineurin inhibitor therapy. Daily treatment of mice with cyclosporine A or tacrolimus for 1 week significantly decreased CD4 + /FoxP3 + regulatory T cells in the spleen and lymph nodes, as well as induced hypertension, vascular injury and dysfunction, and glomerular mesangial expansion in mice. Daily cotreatment with all-trans retinoic acid reported to increase regulatory T cells and decrease interleukin-17-producing T cells, prevented all of the detrimental effects of cyclosporine A and tacrolimus. All-trans retinoic acid also increased regulatory T cells and prevented the hypertension, endothelial dysfunction, and glomerular injury in genetically modified mice that phenocopy calcineurin inhibitor-treated mice (FKBP12-Tie2 knockout). Treatment with an interleukin-17-neutralizing antibody also increased regulatory T-cell levels and prevented the hypertension, endothelial dysfunction, and glomerular injury in cyclosporine A-treated and tacrolimus-treated mice and FKBP12-Tie2 knockout mice, whereas an isotype control had no effect. Augmenting regulatory T cells and inhibiting interleukin-17 signaling using noncellular therapies prevents the cardiovascular and renal toxicity of calcineurin inhibitors in mice. © 2017 American Heart Association, Inc.

  6. Discovery of a highly selective chemical inhibitor of matrix metalloproteinase-9 (MMP-9) that allosterically inhibits zymogen activation.

    Science.gov (United States)

    Scannevin, Robert H; Alexander, Richard; Haarlander, Tara Mezzasalma; Burke, Sharon L; Singer, Monica; Huo, Cuifen; Zhang, Yue-Mei; Maguire, Diane; Spurlino, John; Deckman, Ingrid; Carroll, Karen I; Lewandowski, Frank; Devine, Eric; Dzordzorme, Keli; Tounge, Brett; Milligan, Cindy; Bayoumy, Shariff; Williams, Robyn; Schalk-Hihi, Celine; Leonard, Kristi; Jackson, Paul; Todd, Matthew; Kuo, Lawrence C; Rhodes, Kenneth J

    2017-10-27

    Aberrant activation of matrix metalloproteinases (MMPs) is a common feature of pathological cascades observed in diverse disorders, such as cancer, fibrosis, immune dysregulation, and neurodegenerative diseases. MMP-9, in particular, is highly dynamically regulated in several pathological processes. Development of MMP inhibitors has therefore been an attractive strategy for therapeutic intervention. However, a long history of failed clinical trials has demonstrated that broad-spectrum MMP inhibitors have limited clinical utility, which has spurred the development of inhibitors selective for individual MMPs. Attaining selectivity has been technically challenging because of sequence and structural conservation across the various MMPs. Here, through a biochemical and structural screening paradigm, we have identified JNJ0966, a highly selective compound that inhibited activation of MMP-9 zymogen and subsequent generation of catalytically active enzyme. JNJ0966 had no effect on MMP-1, MMP-2, MMP-3, MMP-9, or MMP-14 catalytic activity and did not inhibit activation of the highly related MMP-2 zymogen. The molecular basis for this activity was characterized as an interaction of JNJ0966 with a structural pocket in proximity to the MMP-9 zymogen cleavage site near Arg-106, which is distinct from the catalytic domain. JNJ0966 was efficacious in reducing disease severity in a mouse experimental autoimmune encephalomyelitis model, demonstrating the viability of this therapeutic approach. This discovery reveals an unprecedented pharmacological approach to MMP inhibition, providing an opportunity to improve selectivity of future clinical drug candidates. Targeting zymogen activation in this manner may also allow for pharmaceutical exploration of other enzymes previously viewed as intractable drug targets. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Reversible Inhibitors of Monoamine Oxidase-A (RIMAs): Robust, Reversible Inhibition of Human Brain MAO-A by CX157

    Science.gov (United States)

    Fowler, Joanna S; Logan, Jean; Azzaro, Albert J; Fielding, Robert M; Zhu, Wei; Poshusta, Amy K; Burch, Daniel; Brand, Barry; Free, James; Asgharnejad, Mahnaz; Wang, Gene-Jack; Telang, Frank; Hubbard, Barbara; Jayne, Millard; King, Payton; Carter, Pauline; Carter, Scott; Xu, Youwen; Shea, Colleen; Muench, Lisa; Alexoff, David; Shumay, Elena; Schueller, Michael; Warner, Donald; Apelskog-Torres, Karen

    2010-01-01

    Reversible inhibitors of monoamine oxidase-A (RIMA) inhibit the breakdown of three major neurotransmitters, serotonin, norepinephrine and dopamine, offering a multi-neurotransmitter strategy for the treatment of depression. CX157 (3-fluoro-7-(2,2,2-trifluoroethoxy)phenoxathiin-10,10-dioxide) is a RIMA, which is currently in development for the treatment of major depressive disorder. We examined the degree and reversibility of the inhibition of brain monoamine oxidase-A (MAO-A) and plasma CX157 levels at different times after oral dosing to establish a dosing paradigm for future clinical efficacy studies, and to determine whether plasma CX157 levels reflect the degree of brain MAO-A inhibition. Brain MAO-A levels were measured with positron emission tomography (PET) imaging and [11C]clorgyline in 15 normal men after oral dosing of CX157 (20–80 mg). PET imaging was conducted after single and repeated doses of CX157 over a 24-h time course. We found that 60 and 80 mg doses of CX157 produced a robust dose-related inhibition (47–72%) of [11C]clorgyline binding to brain MAO-A at 2 h after administration and that brain MAO-A recovered completely by 24 h post drug. Plasma CX157 concentration was highly correlated with the inhibition of brain MAO-A (EC50: 19.3 ng/ml). Thus, CX157 is the first agent in the RIMA class with documented reversible inhibition of human brain MAO-A, supporting its classification as a RIMA, and the first RIMA with observed plasma levels that can serve as a biomarker for the degree of brain MAO-A inhibition. These data were used to establish the dosing regimen for a current clinical efficacy trial with CX157. PMID:19890267

  8. Inhibition of nitric oxide and inflammatory cytokines in LPS-stimulated murine macrophages by resveratrol, a potent proteasome inhibitor

    Directory of Open Access Journals (Sweden)

    Qureshi Asaf A

    2012-07-01

    Full Text Available Abstract Background Altered immune function during ageing results in increased production of nitric oxide (NO and other inflammatory mediators. Recently, we have reported that NO production was inhibited by naturally-occurring proteasome inhibitors (quercetin, δ-tocotrienol, and riboflavin in lipopolysaccharide (LPS-stimulated RAW264.7 cells, and thioglycolate-elicited peritoneal macrophages from C57BL/6 mice. In a continuous effort to find more potent, non-toxic, commercially available, naturally-occurring proteasome inhibitors that suppress inflammation, the present study was carried out to describe the inhibition of NF-κB activation and NO, TNF-α, IL-6, IL-1β, and iNOS expression by trans-resveratrol, trans-pterostilbene, morin hydrate, and nicotinic acid in LPS-induced RAW 264.7 cells and thioglycolate-elicited peritoneal macrophages from C57BL/6 and BALB/c mice. Results The present results indicate that resveratrol, pterostilbene, and morin hydrate caused significant inhibition (>70% to 90%; P 40%; P 60%; P 40%; P P  Conclusions The present results clearly demonstrate that resveratrol and pterostilbene are particularly potent proteasome inhibitors that suppress expression of genes, and production of inflammatory products in LPS-stimulated RAW 264.7 cells, and macrophages from C57BL/6 and BALB/c mice. Resveratrol and pterostilbene which are present in grapes, blueberries, and red wine, have been implicated as contributing factors to the lower incidence of cardiovascular disease in the French population, despite their relatively high dietary fat intake. Consequently, it appears likely that the beneficial nutritional effects of resveratrol and pterostilbene are due at least in part, to their ability to inhibit NF-κB activation by the proteasome, thereby suppressing activation of pro-inflammatory cytokines and iNOS genes, resulting in decreased secretion of TNF-α, IL-1β, IL-6, and NO levels, in response to inflammatory stimuli

  9. Theoretical study of inhibition efficiencies of some amino acids on corrosion of carbon steel in acidic media: green corrosion inhibitors.

    Science.gov (United States)

    Dehdab, Maryam; Shahraki, Mehdi; Habibi-Khorassani, Sayyed Mostafa

    2016-01-01

    Inhibition efficiencies of three amino acids [tryptophan (B), tyrosine (c), and serine (A)] have been studied as green corrosion inhibitors on corrosion of carbon steel using density functional theory (DFT) method in gas and aqueous phases. Quantum chemical parameters such as EH OMO (highest occupied molecular orbital energy), E LUMO (lowest unoccupied molecular orbital energy), hardness (η), polarizability ([Formula: see text]), total negative charges on atoms (TNC), molecular volume (MV) and total energy (TE) have been calculated at the B3LYP level of theory with 6-311++G** basis set. Consistent with experimental data, theoretical results showed that the order of inhibition efficiency is tryptophan (B) > tyrosine (C) > serine (A). In order to determine the possible sites of nucleophilic and electrophilic attacks, local reactivity has been evaluated through Fukui indices.

  10. Binding and Inhibition of Spermidine Synthase from Plasmodium falciparum and Implications for In Vitro Inhibitor Testing.

    Directory of Open Access Journals (Sweden)

    Janina Sprenger

    Full Text Available The aminopropyltransferase spermidine synthase (SpdS is a promising drug target in cancer and in protozoan diseases including malaria. Plasmodium falciparum SpdS (PfSpdS transfers the aminopropyl group of decarboxylated S-adenosylmethionine (dcAdoMet to putrescine or to spermidine to form spermidine or spermine, respectively. In an effort to understand why efficient inhibitors of PfSpdS have been elusive, the present study uses enzyme activity assays and isothermal titration calorimetry with verified or predicted inhibitors of PfSpdS to analyze the relationship between binding affinity as assessed by KD and inhibitory activity as assessed by IC50. The results show that some predicted inhibitors bind to the enzyme with high affinity but are poor inhibitors. Binding studies with PfSpdS substrates and products strongly support an ordered sequential mechanism in which the aminopropyl donor (dcAdoMet site must be occupied before the aminopropyl acceptor (putrescine site can be occupied. Analysis of the results also shows that the ordered sequential mechanism adequately accounts for the complex relationship between IC50 and KD and may explain the limited success of previous efforts at structure-based inhibitor design for PfSpdS. Based on PfSpdS active-site occupancy, we suggest a classification of ligands that can help to predict the KD-IC50 relations in future design of new inhibitors. The present findings may be relevant for other drug targets that follow an ordered sequential mechanism.

  11. Development of ATX and DUSP inhibitors : inhibiting phosphate ester hydrolysis in biology

    NARCIS (Netherlands)

    Albers, Harald Matheas Henricus Gerardus

    2012-01-01

    The first part of this thesis describes the development of inhibitors of autotaxin (ATX or ENPP2), a phosphodiesterase that is responsible for the production of the lipid lysophosphatidic acid (LPA) in the circulation. ATX is implicated in several diseases including inflammation, fibrotic disease

  12. The digestion of phagocytosed collagen is inhibited by the proteinase inhibitors leupeptin and E-64

    NARCIS (Netherlands)

    Everts, V.; Beertsen, W.; Tigchelaar-Gutter, W.

    1985-01-01

    Using morphometric methods the effects of the thiol-proteinase inhibitors leupeptin and E-64 on the digestion of intracytoplasmic collagen fibrils were studied in cultured mouse bone explants. Both drugs caused a dose-dependent increase of lysosomal structures containing cross-banded collagen

  13. Electrochemically reduced graphene and iridium oxide nanoparticles for inhibition-based angiotensin-converting enzyme inhibitor detection.

    Science.gov (United States)

    Kurbanoglu, Sevinc; Rivas, Lourdes; Ozkan, Sibel A; Merkoçi, Arben

    2017-02-15

    In this work, a novel biosensor based on electrochemically reduced graphene oxide and iridium oxide nanoparticles for the detection of angiotensin-converting enzyme inhibitor drug, captopril, is presented. For the preparation of the biosensor, tyrosinase is immobilized onto screen printed electrode by using 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-Hydroxysuccinimide coupling reagents, in electrochemically reduced graphene oxide and iridium oxide nanoparticles matrix. Biosensor response is characterized towards catechol, in terms of graphene oxide concentration, number of cycles to reduce graphene oxide, volume of iridium oxide nanoparticles and tyrosinase solution. The designed biosensor is used to inhibit tyrosinase activity by Captopril, which is generally used to treat congestive heart failure. It is an angiotensin-converting enzyme inhibitor that operates via chelating copper at the active site of tyrosinase and thioquinone formation. The captopril detections using both inhibition ways are very sensitive with low limits of detection: 0.019µM and 0.008µM for chelating copper at the active site of tyrosinase and thioquinone formation, respectively. The proposed methods have been successfully applied in captopril determination in spiked human serum and pharmaceutical dosage forms with acceptable recovery values. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Peptide inhibitors of botulinum neurotoxin serotype A: design, inhibition, cocrystal structures, structure-activity relationship and pharmacophore modeling

    Energy Technology Data Exchange (ETDEWEB)

    Kumar G.; Swaminathan S.; Kumaran, D.; Ahmed, S. A.

    2012-05-01

    Clostridium botulinum neurotoxins are classified as Category A bioterrorism agents by the Centers for Disease Control and Prevention (CDC). The seven serotypes (A-G) of the botulinum neurotoxin, the causative agent of the disease botulism, block neurotransmitter release by specifically cleaving one of the three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and induce flaccid paralysis. Using a structure-based drug-design approach, a number of peptide inhibitors were designed and their inhibitory activity against botulinum serotype A (BoNT/A) protease was determined. The most potent peptide, RRGF, inhibited BoNT/A protease with an IC{sub 50} of 0.9 {micro}M and a K{sub i} of 358 nM. High-resolution crystal structures of various peptide inhibitors in complex with the BoNT/A protease domain were also determined. Based on the inhibitory activities and the atomic interactions deduced from the cocrystal structures, the structure-activity relationship was analyzed and a pharmacophore model was developed. Unlike the currently available models, this pharmacophore model is based on a number of enzyme-inhibitor peptide cocrystal structures and improved the existing models significantly, incorporating new features.

  15. Flavonoids as noncompetitive inhibitors of Dengue virus NS2B-NS3 protease: inhibition kinetics and docking studies.

    Science.gov (United States)

    de Sousa, Lorena Ramos Freitas; Wu, Hongmei; Nebo, Liliane; Fernandes, João Batista; da Silva, Maria Fátima das Graças Fernandes; Kiefer, Werner; Kanitz, Manuel; Bodem, Jochen; Diederich, Wibke E; Schirmeister, Tanja; Vieira, Paulo Cezar

    2015-02-01

    NS2B-NS3 is a serine protease of the Dengue virus considered a key target in the search for new antiviral drugs. In this study flavonoids were found to be inhibitors of NS2B-NS3 proteases of the Dengue virus serotypes 2 and 3 with IC50 values ranging from 15 to 44 μM. Agathisflavone (1) and myricetin (4) turned out to be noncompetitive inhibitors of dengue virus serotype 2 NS2B-NS3 protease with Ki values of 11 and 4.7 μM, respectively. Docking studies propose a binding mode of the flavonoids in a specific allosteric binding site of the enzyme. Analysis of biomolecular interactions of quercetin (5) with NT647-NHS-labeled Dengue virus serotype 3 NS2B-NS3 protease by microscale thermophoresis experiments, yielded a dissociation constant KD of 20 μM. Our results help to understand the mechanism of inhibition of the Dengue virus serine protease by flavonoids, which is essential for the development of improved inhibitors. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Screening of E. coli β-clamp Inhibitors Revealed that Few Inhibit Helicobacter pylori More Effectively: Structural and Functional Characterization.

    Science.gov (United States)

    Pandey, Preeti; Verma, Vijay; Dhar, Suman Kumar; Gourinath, Samudrala

    2018-01-11

    The characteristic of interaction with various enzymes and processivity-promoting nature during DNA replication makes β-clamp an important drug target. Helicobacter pylori ( H. pylori ) have several unique features in DNA replication machinery that makes it different from other microorganisms. To find out whether difference in DNA replication proteins behavior accounts for any difference in drug response when compared to E. coli , in the present study, we have tested E. coli β-clamp inhibitor molecules against H. pylori β-clamp. Various approaches were used to test the binding of inhibitors to H. pylori β-clamp including docking, surface competition assay, complex structure determination, as well as antimicrobial assay. Out of five shortlisted inhibitor molecules on the basis of docking score, three molecules, 5-chloroisatin, carprofen, and 3,4-difluorobenzamide were co-crystallized with H. pylori β-clamp and the structures show that they bind at the protein-protein interaction site as expected. In vivo studies showed only two molecules, 5-chloroisatin, and 3,4-difluorobenzamide inhibited the growth of the pylori with MIC values in micro molar range, which is better than the inhibitory effect of the same drugs on E. coli . Therefore, the evaluation of such drugs against H. pylori may explore the possibility to use to generate species-specific pharmacophore for development of new drugs against H. pylori .

  17. Application of calculated NMR parameters, aromaticity indices and wavefunction properties for evaluation of corrosion inhibition efficiency of pyrazine inhibitors

    Science.gov (United States)

    Behzadi, Hadi; Manzetti, Sergio; Dargahi, Maryam; Roonasi, Payman; Khalilnia, Zahra

    2018-01-01

    In light of the importance of developing novel corrosion inhibitors, a series of quantum chemical calculations were carried out to evaluate 15N chemical shielding CS tensors as well as aromaticity indexes including NICS, HOMA, FLU, and PDI of three pyrazine derivatives, 2-methylpyrazine (MP), 2-aminopyrazine (AP) and 2-amino-5-bromopyrazine (ABP). The NICS parameters have been shown in previous studies to be paramount to the prediction of anti-corrosion properties, and have been combined here with HOMA, FLU and PDI and detailed wavefunction analysis to determine the effects from bromination and methylation on pyrazine. The results show that the electron density around the nitrogens, represented by CS tensors, can be good indicators of anti-corrosion efficiency. Additionally, the NICS, FLU and PDI, as aromaticity indicators of molecule, are well correlated with experimental corrosion inhibition efficiencies of the studied inhibitors. Bader sampling and detailed wavefunction analysis shows that the major effects from bromination on the pyrazine derivatives affect the Laplacian of the electron density of the ring, delocalizing the aromatic electrons of the carbon atoms into lone pairs and increasing polarization of the Laplacian values. This feature is well agreement with empirical studies, which show that ABP is the most efficient anti-corrosion compound followed by AP and MP, a property which can be attributed and predicted by derivation of the Laplacian of the electron density of the ring nuclei. This study shows the importance of devising DFT methods for development of new corrosion inhibitors, and the strength of electronic and nuclear analysis, and depicts most importantly how corrosion inhibitors composed of aromatic moieties may be modified to increase anti-corrosive properties.

  18. The effect of corn trypsin inhibitor and inhibiting antibodies for FXIa and FXIIa on coagulation of plasma and whole blood.

    Science.gov (United States)

    Hansson, K M; Nielsen, S; Elg, M; Deinum, J

    2014-10-01

    Corn trypsin inhibitor (CTI), an inhibitor of FXIIa, is used to prevent plasma coagulation by contact activation, to specifically investigate tissue factor (TF)-initiated coagulation. In the present work the specificity of CTI for factor (F) XIIa is questioned. In the commercial available plasma coagulation assays CTI was found to double activated partial thromboplastin time (APTT) at a plasma concentration of 7.3 ± 1.5 μm CTI (assay concentration 2.4 μm). No effect was found on the prothrombin time (PT) when high TF concentrations were used. Also, with specific antibodies for FXIIa and for FXIa only APTT was found to be extended but not PT. With specific enzyme assays using chromogenic substrates CTI was shown to be a strong inhibitor of FXIIa and a competitive inhibitor of FXIa with Ki  = 8.1 ± 0.3 μm, without effect on the coagulation factors FVIIa, FIXa, FXa and thrombin. In thrombin generation and coagulation (free oscillation rheometry, FOR) assays, initiated with low TF concentrations, no effect of CTI (plasma concentrations of 4.4 and 13.6 μm CTI, 25 resp. 100 mg L(-1) in blood) was found with ≥ 1 pm TF. At ≤ 0.1 pm TF in the FOR whole blood assay the coagulation time (CT) concentration dependently increased while the plasma CT became longer than the observation time. To avoid inhibition of FXIa and the thrombin feedback loop we recommend that for coagulation assays the concentration of CTI in blood should be below 20 mg L(-1) (1.6 μm) and in plasma below 3 μm. © 2014 International Society on Thrombosis and Haemostasis.

  19. Inhibition of glucagon secretion by GLP-1 agonists and DPP4 inhibitors

    DEFF Research Database (Denmark)

    Hansen, Morten; Juul Hare, Kristine; Holst, Jens Juul

    2011-01-01

    with emphasis on their glucagon-lowering effects. Finally, we review available glucagon data from current clinical studies on incretin-based treatment modalities (dipeptidyl peptidase 4 [DPP4] inhibitors and GLP-1 receptor agonists). Most of these studies suggest that both DPP4 inhibitors and GLP-1 receptor......*both known to contribute significantly to the hyperglycemic state of patients with T2DM. This article outlines the role of hyperglucagonemia in type 2 diabetic pathophysiology, summarizes the physiologic effects of glucagon-like peptide-1 (GLP-1), and gives an introduction to incretin-based treatments...... agonists lower fasting and postprandial plasma glucagon, and recent data suggest that these effects contribute importantly to the glucose-lowering effect of these treatments....

  20. Inhibition of hydrogen sulfide generation from disposed gypsum drywall using chemical inhibitors.

    Science.gov (United States)

    Xu, Qiyong; Townsend, Timothy; Bitton, Gabriel

    2011-07-15

    Disposal of gypsum drywall in landfills has been demonstrated to elevate hydrogen sulfide (H(2)S) concentrations in landfill gas, a problem with respect to odor, worker safety, and deleterious effect on gas-to-energy systems. Since H(2)S production in landfills results from biological activity, the concept of inhibiting H(2)S production through the application of chemical agents to drywall during disposal was studied. Three possible inhibition agents - sodium molybdate (Na(2)MoO(4)), ferric chloride (FeCl(3)), and hydrated lime (Ca(OH)(2)) - were evaluated using flask and column experiments. All three agents inhibited H(2)S generation, with Na(2)MoO(4) reducing H(2)S generation by interrupting the biological sulfate reduction process and Ca(OH)(2) providing an unfavorable pH for biological growth. Although FeCl(3) was intended to provide an electron acceptor for a competing group of bacteria, the mechanism found responsible for inhibiting H(2)S production in the column experiment was a reduction in pH. Application of both Na(2)MoO(4) and FeCl(3) inhibited H(2)S generation over a long period (over 180 days), but the impact of Ca(OH)(2) decreased with time as the alkalinity it contributed was neutralized by the generated H(2)S. Practical application and potential environmental implications need additional exploration. Copyright © 2011 Elsevier B.V. All rights reserved.

  1. Triterpenoids from Ganoderma lucidum inhibit the activation of EBV antigens as telomerase inhibitors.

    Science.gov (United States)

    Zheng, Dong-Shu; Chen, Liang-Shu

    2017-10-01

    Nasopharyngeal carcinoma (NPC) is a malignant disease that threatens the health of humans. To find effective agents for the inhibition of Epstein-Barr virus (EBV) infection, which is associated with NPC, a phytochemical investigation of Ganoderma lucidum was carried out in the present study. Five triterpenoids were identified, including ganoderic acid A (compound 1), ganoderic acid B (compound 2), ganoderol B (compound 3), ganodermanontriol (compound 4), and ganodermanondiol (compound 5), on the basis of spectroscopic analysis. An inhibition of EBV antigens activation assay was implemented to elucidate the triterpenoids from G. lucidum and potentially prevent NPC. All the triterpenoids showed significant inhibitory effects on both EBV EA and CA activation at 16 nmol. At 3.2 nmol, all the compounds moderately inhibited the activation of the two antigens. The activity of telomerase was inhibited by these triterpenoids at 10 µM. Molecular docking demonstrated that compound 1 was able to inhibit telomerase as a ligand. In addition, the physicochemical properties of these compounds were calculated to elucidate their drug-like properties. These results provided evidence for the application of these triterpenoids and whole G. lucidum in the treatment of NPC.

  2. Characterization of a small molecule inhibitor of melanogenesis that inhibits tyrosinase activity and scavenges nitric oxide (NO).

    Science.gov (United States)

    Chung, Ki Wung; Jeong, Hyoung Oh; Jang, Eun Ji; Choi, Yeon Ja; Kim, Dae Hyun; Kim, So Ra; Lee, Kyung Jin; Lee, Hye Jin; Chun, Pusoon; Byun, Youngjoo; Moon, Hyung Ryong; Chung, Hae Young

    2013-10-01

    Excessive melanin production and accumulation are characteristics of a large number of skin diseases, including melasma, and post-inflammatory hyperpigmentation. During our on-going search for new agents with an inhibitory effect on tyrosinase, we synthesized a new type of tyrosinase inhibitor, 4-(thiazolidin-2-yl)benzene-1,2-diol (MHY-794), which directly inhibits mushroom tyrosinase. The inhibitory effect of MHY-794 on tyrosinase activity and nitric oxide (NO) scavenging activity was evaluated in cell free system. Additional experiments were performed using B16F10 melanoma cells to demonstrate the effects of MHY-794 in vitro. HRM2 hairless mice were used to evaluate anti-melanogenic effects of MHY-794 in vivo. MHY-794 effectively inhibited mushroom tyrosinase activity in cell free system. In silico docking simulation also supported the inhibitory effects of MHY-794 on mushroom tyrosinase. MHY-794 also proved to be effective at scavenging nitric oxide (NO), which serves as an important modulator in the melanogenesis signaling pathway. In addition, MHY-794 effectively inhibited SNP (NO donor)-induced melanogenesis by directly inhibiting tyrosinase and diminishing NO-mediated melanogenesis signaling in B16 melanoma cells. The anti-melanogenic effects of MHY-794 were further confirmed in HRM2 hairless mice. Ultraviolet light (UV) significantly up-regulated NO-mediated melanogenesis signaling in HRM2 hairless mice, but MHY-794 effectively inhibited both melanogenesis and diminished UV-induced NO-signaling. Our results indicate that MHY-794 is highly effective at inhibiting NO-mediated melanogenesis in vitro and in vivo by direct NO scavenging and directly inhibiting tyrosinase activity, and suggest that MHY-794 be considered a new developmental candidate for the treatment of hyper-pigmentation disorders. MHY-794, which showed great efficacy on NO-mediated melanogenesis by direct NO scavenging as well as direct inhibition of tyrosinase catalytic activity, might be

  3. Corrosion inhibition of Q235 steel in 1 M HCl using quaternized tetraaniline as a corrosion inhibitor

    Science.gov (United States)

    Li, Xiangyu; Ye, Yuwei; Liu, Tong; Zheng, Wenru; Yang, Feng; Zhao, Haichao; Wang, Liping

    2017-12-01

    Novel quaternary ammonium cation containing tetraaniline (QATA) was successfully synthesized by condensation of amine-capped tetraaniline with 6-bromohexanoic acid, followed by quaternarization with triethylamine. The corrosion inhibition performance of QATA with their adsorption mechanisms for Q235 steel was studied in 1 M HCl solution by a series of methods such as weight loss measurements, potentiodynamic polarization, electrochemical impedance spectroscopy, scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and atomic force microscopy (AFM). The inhibition efficiency (IE%) increased with increasing concentrations of QATA, reaching a value up to 97.47% at a concentration of 150 mg l‑1. Potentiodynamic polarization curves showed that the QATA affected both cathodic and anodic protection and was a mixed type inhibitor in 1 M HCl corrosive medium. Adsorption isotherm studies confirmed that the absorption of QATA on the Q235 steel surface in 1 M HCl solution obeyed Langmuir adsorption isotherm, and the adsorption process of corrosion inhibition on Q235 steel surface involved both the physical and chemical adsorption. The EDS analysis determined the adsorption of QATA molecules on the steel surface, the surface morphologies before and after immersion in 1 M HCl medium were also investigated by SEM and AFM.

  4. Cerebrovascular effects of angiotensin converting enzyme inhibition involve large artery dilatation in rats

    DEFF Research Database (Denmark)

    Postiglione, A; Bobkiewicz, T; Vinholdt-Pedersen, E

    1991-01-01

    The aim of the study was to selectively examine the effects of converting enzyme inhibition on the large brain arteries by using concomitant inhibition of carbonic anhydrase to cause severe dilatation of mainly parenchymal resistance vessels....

  5. The Src inhibitor AZD0530 reversibly inhibits the formation and activity of human osteoclasts

    NARCIS (Netherlands)

    de Vries, T.J.; Mullender, M.G.; van Duin, M.A.; Semeins, C.M.; James, N.; Green, T.P.; Everts, V.; Klein Nulend, J.

    2009-01-01

    Tumor cells in the bone microenvironment are able to initiate a vicious cycle of bone degradation by mobilizing osteoclasts, multinucleated cells specialized in bone degradation. c-Src is highly expressed both in tumors and in osteoclasts. Therefore, drugs like AZD0530, designed to inhibit Src

  6. A broad range quorum sensing inhibitor working through sRNA inhibition

    DEFF Research Database (Denmark)

    Jakobsen, Tim H.; Warming, Anders N.; Vejborg, Rebecca M.

    2017-01-01

    that the repressing effect of ajoene on quorum sensing occurs by inhibition of small regulatory RNAs (sRNA) in P. aeruginosa as well as in Staphylococcus aureus, another important human pathogen that employs quorum sensing to control virulence gene expression. Using various reporter constructs, we found that ajoene...

  7. Checkpoint Inhibition: Programmed Cell Death 1 and Programmed Cell Death 1 Ligand Inhibitors in Hodgkin Lymphoma.

    Science.gov (United States)

    Villasboas, Jose Caetano; Ansell, Stephen

    2016-01-01

    Hodgkin lymphoma (HL) is a lymphoid malignancy characterized by a reactive immune infiltrate surrounding relatively few malignant cells. In this scenario, active immune evasion seems to play a central role in allowing tumor progression. Immune checkpoint inhibitor pathways are normal mechanisms of T-cell regulation that suppress immune effector function following an antigenic challenge. Hodgkin lymphoma cells are able to escape immune surveillance by co-opting these mechanisms. The programmed cell death 1 (PD-1) pathway in particular is exploited in HL as the malignant Hodgkin and Reed-Sternberg cells express on their surface cognate ligands (PD-L1/L2) for the PD-1 receptor and thereby dampen the T-cell-mediated antitumoral response. Monoclonal antibodies that interact with and disrupt the PD-1:PD-L1/L2 axis have now been developed and tested in early-phase clinical trials in patients with advanced HL with encouraging results. The remarkable clinical activity of PD-1 inhibitors in HL highlights the importance of immune checkpoint pathways as therapeutic targets in HL. In this review, we discuss the rationale for targeting PD-1 and PD-L1 in the treatment of HL. We will evaluate the published clinical data on the different agents and highlight the safety profile of this class of agents. We discuss the available evidence on the use of biomarkers as predictors of response to checkpoint blockade and summarize the areas under active investigation in the use of PD-1/PD-L1 inhibitors for the treatment of HL.

  8. Paralysis Episodes in Carbonic Anhydrase II Deficiency.

    Science.gov (United States)

    Al-Ibrahim, Alia; Al-Harbi, Mosa; Al-Musallam, Sulaiman

    2003-01-01

    Carbonic anhydrase II (CAII) deficiency is an autosomal recessive disorder manifest by osteopetrosis, renal tubular acidosis, and cerebral calcification. Other features include growth failure and mental retardation. Complications of the osteopetrosis include frequent bone fractures, cranial nerve compression, and dental mal-occlusion. A hyper-chloremic metabolic acidosis, sometimes with hypokalemia, occurs due to renal tubular acidosis that may be proximal, distal, or more commonly, the combined type. Such patients may present with global hypotonia, muscle weakness or paralysis. We report a case of CA II deficiency with recurrent attacks of acute paralysis which was misdiagnosed initially as Guillian-Barre syndrome.

  9. Reduction of mouse atherosclerosis by urokinase inhibition or with a limited-spectrum matrix metalloproteinase inhibitor

    DEFF Research Database (Denmark)

    Hu, Jie Hong; Touch, Phanith; Zhang, Jingwan

    2015-01-01

    ) or a control antibody. mU1-treated mice were also compared with PBS-treated non-uPA-overexpressing Apoe-null mice. Other SR-uPA mice were treated with one of three doses of a limited-spectrum synthetic MMP inhibitor (XL784) or vehicle. mU1 reduced aortic root intimal lesion area (20%; P = 0.05) and aortic root...... circumference (12%; P = 0.01). All XL784 doses reduced aortic root intimal lesion area (22-29%) and oil-red-O-positive lesion area (36-42%; P

  10. Inhibitor Ranking Through QM based Chelation Calculations for Virtual Screening of HIV-1 RNase H inhibition

    DEFF Research Database (Denmark)

    Poongavanam, Vasanthanathan; Svendsen, Casper Steinmann; Kongsted, Jacob

    2014-01-01

    of the methods based on the use of a training set of molecules, QM based chelation calculations were used as filter in virtual screening of compounds in the ZINC database. By this, we find, compared to regular docking, QM based chelation calculations to significantly reduce the large number of false positives......Quantum mechanical (QM) calculations have been used to predict the binding affinity of a set of ligands towards HIV-1 RT associated RNase H (RNH). The QM based chelation calculations show improved binding affinity prediction for the inhibitors compared to using an empirical scoring function...

  11. E2F1-mediated transcriptional inhibition of the plasminogen activator inhibitor type 1 gene

    DEFF Research Database (Denmark)

    Koziczak, M; Müller, H; Helin, K

    2001-01-01

    Gene expression of the plasminogen activation system is cell-cycle dependent. Previously, we showed that ectopic expression of E2F1 repressed the plasminogen activator inhibitor type 1 (PAI-1) promoter in a manner dependent on the presence of DNA-binding and transactivation domains of E2F1....... These results all indicate that endogenous E2F can directly repress the PAI-1 gene. DNase I hypersensitive-site analysis of the PAI-1 promoter suggested the involvement of conformation changes in chromatin structure of the PAI-1 promoter. 5' deletion analysis of the PAI-1 promoter showed that multiple sites......-regulation of PAI-1 gene expression correlates with an increase in endogenous E2F activity. When cells were treated with a cdk2/4-specific inhibitor, which maintains E2F in an inactive state, the decline of serum-induced PAI-1 mRNA levels was suppressed. In mutant U2OS cells expressing a temperature...

  12. A photoactivable multi-inhibitor nanoliposome for tumour control and simultaneous inhibition of treatment escape pathways

    Science.gov (United States)

    Spring, Bryan Q.; Bryan Sears, R.; Zheng, Lei Zak; Mai, Zhiming; Watanabe, Reika; Sherwood, Margaret E.; Schoenfeld, David A.; Pogue, Brian W.; Pereira, Stephen P.; Villa, Elizabeth; Hasan, Tayyaba

    2016-04-01

    Nanoscale drug delivery vehicles can facilitate multimodal therapies of cancer by promoting tumour-selective drug release. However, few are effective because cancer cells develop ways to resist and evade treatment. Here, we introduce a photoactivable multi-inhibitor nanoliposome (PMIL) that imparts light-induced cytotoxicity in synchrony with a photoinitiated and sustained release of inhibitors that suppress tumour regrowth and treatment escape signalling pathways. The PMIL consists of a nanoliposome doped with a photoactivable chromophore (benzoporphyrin derivative, BPD) in the lipid bilayer, and a nanoparticle containing cabozantinib (XL184)—a multikinase inhibitor—encapsulated inside. Near-infrared tumour irradiation, following intravenous PMIL administration, triggers photodynamic damage of tumour cells and microvessels, and simultaneously initiates release of XL184 inside the tumour. A single PMIL treatment achieves prolonged tumour reduction in two mouse models and suppresses metastatic escape in an orthotopic pancreatic tumour model. The PMIL offers new prospects for cancer therapy by enabling spatiotemporal control of drug release while reducing systemic drug exposure and associated toxicities.

  13. The myosin II ATPase inhibitor blebbistatin prevents thrombin-induced inhibition of intercellular calcium wave propagation in corneal endothelial cells.

    Science.gov (United States)

    Ponsaerts, Raf; D'hondt, Catheleyne; Bultynck, Geert; Srinivas, Sangly P; Vereecke, Johan; Himpens, Bernard

    2008-11-01

    Thrombin inhibits intercellular Ca(2+) wave propagation in bovine corneal endothelial cells (BCECs) through a mechanism dependent on myosin light chain (MLC) phosphorylation. In this study, blebbistatin, a selective myosin II ATPase inhibitor, was used to investigate whether the effect of thrombin is mediated by enhanced actomyosin contractility. BCECs were exposed to thrombin (2 U/mL) for 5 minutes. MLC phosphorylation was assayed by immunocytochemistry. Ca(2+) waves were visualized by confocal microscopy with Fluo-4AM. Fluorescence recovery after photobleaching (FRAP) was used to investigate intercellular communication (IC) via gap junctions. ATP release was measured by luciferin-luciferase assay. Lucifer yellow (LY) uptake was used to investigate hemichannel activity, and Fura-2 was used to assay thrombin- and ATP-mediated Ca(2+) responses. Pretreatment with blebbistatin (5 microM for 20 minutes) or its nitro derivative prevented the thrombin-induced inhibition of the Ca(2+) wave. Neither photo-inactivated blebbistatin nor the inactive enantiomers prevented the thrombin effect. Blebbistatin also prevented thrombin-induced inhibition of LY uptake, ATP release and FRAP, indicating that it prevented the thrombin effect on paracrine and gap junctional IC. In the absence of thrombin, blebbistatin had no significant effect on paracrine or gap junctional IC. The drug had no influence on MLC phosphorylation or on [Ca(2+)](i) transients in response to thrombin or ATP. Blebbistatin prevents the inhibitory effects of thrombin on intercellular Ca(2+) wave propagation. The findings demonstrate that myosin II-mediated actomyosin contractility plays a central role in thrombin-induced inhibition of gap junctional IC and of hemichannel-mediated paracrine IC.

  14. Radicicol, a heat shock protein 90 inhibitor, inhibits differentiation and adipogenesis in 3T3-L1 preadipocytes

    Energy Technology Data Exchange (ETDEWEB)

    He, Yonghan [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223 (China); Li, Ying [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Zhang, Shuocheng [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Perry, Ben [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Department of Biomedical Sciences, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, Canada C1A 4P3 (Canada); Zhao, Tiantian [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Department of Psychology, University of Toronto, 1265 Military Trail, Toronto, ON, Canada M1C 1A4 (Canada); Wang, Yanwen, E-mail: yanwen.wang@nrc.ca [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Department of Biomedical Sciences, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, Canada C1A 4P3 (Canada); Sun, Changhao, E-mail: sun2002changhao@yahoo.com [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China)

    2013-06-28

    Highlights: •Radicicol suppressed intracellular fat accumulation in 3T3-L1 adipocytes. •Radicicol inhibited the expression of FAS and FABP4. •Radicicol blocked cell cycle at the G1-S phase during cell differentiation. •Radicicol inhibited the PDK1/Akt pathway in adipocyte differentiation. -- Abstract: Heat shock protein 90 (Hsp90) is involved in various cellular processes, such as cell proliferation, differentiation and apoptosis. As adipocyte differentiation plays a critical role in obesity development, the present study investigated the effect of an Hsp90 inhibitor radicicol on the differentiation of 3T3-L1 preadipocytes and potential mechanisms. The cells were treated with different concentrations of radicicol during the first 8 days of cell differentiation. Adipogenesis, the expression of adipogenic transcriptional factors, differentiation makers and cell cycle were determined. It was found that radicicol dose-dependently decreased intracellular fat accumulation through down-regulating the expression of peroxisome proliferator-activated receptor γ (PPAR{sub γ}) and CCAAT element binding protein α (C/EBP{sub α}), fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Flow cytometry analysis revealed that radicicol blocked cell cycle at G1-S phase. Radicicol redcued the phosphorylation of Akt while showing no effect on β-catenin expression. Radicicol decreased the phosphorylation of phosphoinositide-dependent kinase 1 (PDK1). The results suggest that radicicol inhibited 3T3-L1 preadipocyte differentiation through affecting the PDK1/Akt pathway and subsequent inhibition of mitotic clonal expansion and the expression/activity of adipogenic transcriptional factors and their downstream adipogenic proteins.

  15. Radicicol, a heat shock protein 90 inhibitor, inhibits differentiation and adipogenesis in 3T3-L1 preadipocytes

    International Nuclear Information System (INIS)

    He, Yonghan; Li, Ying; Zhang, Shuocheng; Perry, Ben; Zhao, Tiantian; Wang, Yanwen; Sun, Changhao

    2013-01-01

    Highlights: •Radicicol suppressed intracellular fat accumulation in 3T3-L1 adipocytes. •Radicicol inhibited the expression of FAS and FABP4. •Radicicol blocked cell cycle at the G1-S phase during cell differentiation. •Radicicol inhibited the PDK1/Akt pathway in adipocyte differentiation. -- Abstract: Heat shock protein 90 (Hsp90) is involved in various cellular processes, such as cell proliferation, differentiation and apoptosis. As adipocyte differentiation plays a critical role in obesity development, the present study investigated the effect of an Hsp90 inhibitor radicicol on the differentiation of 3T3-L1 preadipocytes and potential mechanisms. The cells were treated with different concentrations of radicicol during the first 8 days of cell differentiation. Adipogenesis, the expression of adipogenic transcriptional factors, differentiation makers and cell cycle were determined. It was found that radicicol dose-dependently decreased intracellular fat accumulation through down-regulating the expression of peroxisome proliferator-activated receptor γ (PPAR γ ) and CCAAT element binding protein α (C/EBP α ), fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Flow cytometry analysis revealed that radicicol blocked cell cycle at G1-S phase. Radicicol redcued the phosphorylation of Akt while showing no effect on β-catenin expression. Radicicol decreased the phosphorylation of phosphoinositide-dependent kinase 1 (PDK1). The results suggest that radicicol inhibited 3T3-L1 preadipocyte differentiation through affecting the PDK1/Akt pathway and subsequent inhibition of mitotic clonal expansion and the expression/activity of adipogenic transcriptional factors and their downstream adipogenic proteins

  16. A Jak1/2 inhibitor, baricitinib, inhibits osteoclastogenesis by suppressing RANKL expression in osteoblasts in vitro.

    Directory of Open Access Journals (Sweden)

    Kohei Murakami

    Full Text Available The Janus kinases (Jaks are hubs in the signaling process of more than 50 cytokine or hormone receptors. However, the function of Jak in bone metabolism remains to be elucidated. Here, we showed that the inhibition of Jak1 and/or Jak2 in osteoblast-lineage cells led to impaired osteoclastogenesis due to the reduced expression of receptor activator of nuclear factor-κB ligand (RANKL. Murine calvaria-derived osteoblasts induced differentiation of bone marrow cells into osteoclasts in the presence of 1,25-dihydroxyvitamin D3 (1,25D3 and prostaglandin E2 (PGE2 in vitro. However, treatment with the Jak1/2 inhibitor, baricitinib, markedly inhibited osteoclastogenesis in the co-culture. On the other hand, baricitinib did not inhibit RANKL-induced osteoclast differentiation of bone marrow macrophages. These results indicated that baricitinib acted on osteoblasts, but not on bone marrow macrophages. Baricitinib suppressed 1,25D3 and PGE2-induced up-regulation of RANKL in osteoblasts, but not macrophage colony-stimulating factor expression. Moreover, the addition of recombinant RANKL to co-cultures completely rescued baricitinib-induced impairment of osteoclastogenesis. shRNA-mediated knockdown of Jak1 or Jak2 also suppressed RANKL expression in osteoblasts and inhibited osteoclastogenesis. Finally, cytokine array revealed that 1,25D3 and PGE2 stimulated secretion of interleukin-6 (IL-6, IL-11, and leukemia inhibitory factor in the co-culture. Hence, Jak1 and Jak2 represent novel therapeutic targets for osteoporosis as well as inflammatory bone diseases including rheumatoid arthritis.

  17. A Jak1/2 inhibitor, baricitinib, inhibits osteoclastogenesis by suppressing RANKL expression in osteoblasts in vitro.

    Science.gov (United States)

    Murakami, Kohei; Kobayashi, Yasuhiro; Uehara, Shunsuke; Suzuki, Takako; Koide, Masanori; Yamashita, Teruhito; Nakamura, Midori; Takahashi, Naoyuki; Kato, Hiroyuki; Udagawa, Nobuyuki; Nakamura, Yukio

    2017-01-01

    The Janus kinases (Jaks) are hubs in the signaling process of more than 50 cytokine or hormone receptors. However, the function of Jak in bone metabolism remains to be elucidated. Here, we showed that the inhibition of Jak1 and/or Jak2 in osteoblast-lineage cells led to impaired osteoclastogenesis due to the reduced expression of receptor activator of nuclear factor-κB ligand (RANKL). Murine calvaria-derived osteoblasts induced differentiation of bone marrow cells into osteoclasts in the presence of 1,25-dihydroxyvitamin D3 (1,25D3) and prostaglandin E2 (PGE2) in vitro. However, treatment with the Jak1/2 inhibitor, baricitinib, markedly inhibited osteoclastogenesis in the co-culture. On the other hand, baricitinib did not inhibit RANKL-induced osteoclast differentiation of bone marrow macrophages. These results indicated that baricitinib acted on osteoblasts, but not on bone marrow macrophages. Baricitinib suppressed 1,25D3 and PGE2-induced up-regulation of RANKL in osteoblasts, but not macrophage colony-stimulating factor expression. Moreover, the addition of recombinant RANKL to co-cultures completely rescued baricitinib-induced impairment of osteoclastogenesis. shRNA-mediated knockdown of Jak1 or Jak2 also suppressed RANKL expression in osteoblasts and inhibited osteoclastogenesis. Finally, cytokine array revealed that 1,25D3 and PGE2 stimulated secretion of interleukin-6 (IL-6), IL-11, and leukemia inhibitory factor in the co-culture. Hence, Jak1 and Jak2 represent novel therapeutic targets for osteoporosis as well as inflammatory bone diseases including rheumatoid arthritis.

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

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    Julia A Sharp

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

  19. HIV protease inhibitors disrupt lipid metabolism by activating endoplasmic reticulum stress and inhibiting autophagy activity in adipocytes.

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    Beth S Zha

    Full Text Available HIV protease inhibitors (PI are core components of Highly Active Antiretroviral Therapy (HAART, the most effective treatment for HIV infection currently available. However, HIV PIs have now been linked to lipodystrophy and dyslipidemia, which are major risk factors for cardiovascular disease and metabolic syndrome. Our previous studies have shown that HIV PIs activate endoplasmic reticulum (ER stress and disrupt lipid metabolism in hepatocytes and macrophages. Yet, little is known on how HIV PIs disrupt lipid metabolism in adipocytes, a major cell type involved in the pathogenesis of metabolic syndrome.Cultured and primary mouse adipocytes and human adipocytes were used to examine the effect of frequently used HIV PIs in the clinic, lopinavir/ritonavir, on adipocyte differentiation and further identify the underlying molecular mechanism of HIV PI-induced dysregulation of lipid metabolism in adipocytes. The results indicated that lopinavir alone or in combination with ritonavir, significantly activated the ER stress response, inhibited cell differentiation, and induced cell apoptosis in adipocytes. In addition, HIV PI-induced ER stress was closely linked to inhibition of autophagy activity. We also identified through the use of primary adipocytes of CHOP(-/- mice that CHOP, the major transcriptional factor of the ER stress signaling pathway, is involved in lopinavir/ritonavir-induced inhibition of cell differentiation in adipocytes. In addition, lopinavir/ritonavir-induced ER stress appears to be associated with inhibition of autophagy activity in adipocytes.Activation of ER stress and impairment of autophagy activity are involved in HIV PI-induced dysregulation of lipid metabolism in adipocytes. The key components of ER stress and autophagy signaling pathways are potential therapeutic targets for HIV PI-induced metabolic side effects in HIV patients.

  20. Histone deacetylase inhibitor upregulates peroxisomal fatty acid oxidation and inhibits apoptotic cell death in abcd1-deficient glial cells.

    Science.gov (United States)

    Singh, Jaspreet; Khan, Mushfiquddin; Pujol, Aurora; Baarine, Mauhamad; Singh, Inderjit

    2013-01-01

    In X-ALD, mutation/deletion of ALD gene (ABCD1) and the resultant very long chain fatty acid (VLCFA) derangement has dramatically opposing effects in astrocytes and oligodendrocytes. While loss of Abcd1 in astrocytes produces a robust inflammatory response, the oligodendrocytes undergo cell death leading to demyelination in X-linked adrenoleukodystrophy (X-ALD). The mechanisms of these distinct pathways in the two cell types are not well understood. Here, we investigated the effects of Abcd1-knockdown and the subsequent alteration in VLCFA metabolism in human U87 astrocytes and rat B12 oligodendrocytes. Loss of Abcd1 inhibited peroxisomal β-oxidation activity and increased expression of VLCFA synthesizing enzymes, elongase of very long chain fatty acids (ELOVLs) (1 and 3) in both cell types. However, higher induction of ELOVL's in Abcd1-deficient B12 oligodendrocytes than astrocytes suggests that ELOVL pathway may play a prominent role in oligodendrocytes in X-ALD. While astrocytes are able to maintain the cellular homeostasis of anti-apoptotic proteins, Abcd1-deletion in B12 oligodendrocytes downregulated the anti-apototic (Bcl-2 and Bcl-xL) and cell survival (phospho-Erk1/2) proteins, and upregulated the pro-apoptotic proteins (Bad, Bim, Bax and Bid) leading to cell loss. These observations provide insights into different cellular signaling mechanisms in response to Abcd1-deletion in two different cell types of CNS. The apoptotic responses were accompanied by activation of caspase-3 and caspase-9 suggesting the involvement of mitochondrial-caspase-9-dependent mechanism in Abcd1-deficient oligodendrocytes. Treatment with histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) corrected the VLCFA derangement both in vitro and in vivo, and inhibited the oligodendrocytes loss. These observations provide a proof-of principle that HDAC inhibitor SAHA may have a therapeutic potential for X-ALD.

  1. Histone deacetylase inhibitor upregulates peroxisomal fatty acid oxidation and inhibits apoptotic cell death in abcd1-deficient glial cells.

    Directory of Open Access Journals (Sweden)

    Jaspreet Singh

    Full Text Available In X-ALD, mutation/deletion of ALD gene (ABCD1 and the resultant very long chain fatty acid (VLCFA derangement has dramatically opposing effects in astrocytes and oligodendrocytes. While loss of Abcd1 in astrocytes produces a robust inflammatory response, the oligodendrocytes undergo cell death leading to demyelination in X-linked adrenoleukodystrophy (X-ALD. The mechanisms of these distinct pathways in the two cell types are not well understood. Here, we investigated the effects of Abcd1-knockdown and the subsequent alteration in VLCFA metabolism in human U87 astrocytes and rat B12 oligodendrocytes. Loss of Abcd1 inhibited peroxisomal β-oxidation activity and increased expression of VLCFA synthesizing enzymes, elongase of very long chain fatty acids (ELOVLs (1 and 3 in both cell types. However, higher induction of ELOVL's in Abcd1-deficient B12 oligodendrocytes than astrocytes suggests that ELOVL pathway may play a prominent role in oligodendrocytes in X-ALD. While astrocytes are able to maintain the cellular homeostasis of anti-apoptotic proteins, Abcd1-deletion in B12 oligodendrocytes downregulated the anti-apototic (Bcl-2 and Bcl-xL and cell survival (phospho-Erk1/2 proteins, and upregulated the pro-apoptotic proteins (Bad, Bim, Bax and Bid leading to cell loss. These observations provide insights into different cellular signaling mechanisms in response to Abcd1-deletion in two different cell types of CNS. The apoptotic responses were accompanied by activation of caspase-3 and caspase-9 suggesting the involvement of mitochondrial-caspase-9-dependent mechanism in Abcd1-deficient oligodendrocytes. Treatment with histone deacetylase (HDAC inhibitor suberoylanilide hydroxamic acid (SAHA corrected the VLCFA derangement both in vitro and in vivo, and inhibited the oligodendrocytes loss. These observations provide a proof-of principle that HDAC inhibitor SAHA may have a therapeutic potential for X-ALD.

  2. SNX-25a, a novel Hsp90 inhibitor, inhibited human cancer growth more potently than 17-AAG.

    Science.gov (United States)

    Wang, Shaoxiang; Wang, Xiao; Du, Zhan; Liu, Yuting; Huang, Dane; Zheng, Kai; Liu, Kaisheng; Zhang, Yi; Zhong, Xueyun; Wang, Yifei

    2014-07-18

    17-Allylamino-17-demethoxygeldanamycin (17-AAG), a typical Hsp90 inhibitor derived from geldanamycin (GA), has entered Phase III clinical trials for cancer therapy. However, it has several significant limitations such as poor solubility, limited bioavailability and unacceptable hepatotoxicity. In this study, the anticancer activity and mechanism of SNX-25a, a novel Hsp90 inhibitor, was investigated comparing with that of 17-AAG. We showed that SNX-25a triggered growth inhibition more sensitively than 17-AAG against many human cancer cells, including K562, SW-620, A375, Hep-2, MCF-7, HepG2, HeLa, and A549 cell lines, especially at low concentrations (AAG, SNX-25a was more potent in arresting the cell cycle at G2 phase, and displayed more potent effects on human cancer cell apoptosis and Hsp90 client proteins. It also exhibited a stronger binding affinity to Hsp90 than 17-AAG using molecular docking. Considering the superiority effects on Hsp90 affinity, cell growth, cell cycle, apoptosis, and Hsp90 client proteins, SNX-25a is supposed as a potential anticancer agent that needs to be explored in detail. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Wound and methyl jasmonate induced pigeon pea defensive proteinase inhibitor has potency to inhibit insect digestive proteinases.

    Science.gov (United States)

    Lomate, Purushottam R; Hivrale, Vandana K

    2012-08-01

    Wounding of plants by chewing insects or other damage induces the synthesis of defensive proteinase inhibitors (PI) in both wounded and distal unwounded leaves. In the present paper we report the characterization of inducible defensive PI from pigeon pea (Cajanus cajan) and its in vitro interaction with Helicoverpa armigera gut proteinases (HGP). We found that PI activity was induced in local as well as systemic leaves of pigeon pea by the wounding and methyl jasmonate (MeJA) application. Consistent induction of PI was observed in two wild cultivars of pigeon pea at various growth stages. The estimated molecular weight of inducible PI was ~16.5 kDa. Electrophoretic analysis and enzyme assays revealed that the induced PI significantly inhibited total gut proteinase as well as trypsin-like activity from the midgut of H. armigera. The induced PI was found to be inhibitor of trypsin as well as chymotrypsin. Study could be important to know the further roles of defensive PIs. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  4. Chemically modified carbonic anhydrases useful in carbon capture systems

    Science.gov (United States)

    Novick, Scott; Alvizo, Oscar

    2013-01-15

    The present disclosure relates to chemically modified carbonic anhydrase polypeptides and soluble compositions, homogenous liquid formulations comprising them. The chemically modified carbonic anhydrase polypeptides have improved properties relative to the same carbonic anhydrase polypeptide that is not chemically modified including the improved properties of increased activity and/or stability in the presence of amine compounds, ammonia, or carbonate ion. The present disclosure also provides methods of preparing the chemically modified polypeptides and methods of using the chemically modified polypeptides for accelerating the absorption of carbon dioxide from a gas stream into a solution as well as for the release of the absorbed carbon dioxide for further treatment and/or sequestering.

  5. Chemically modified carbonic anhydrases useful in carbon capture systems

    Energy Technology Data Exchange (ETDEWEB)

    Novick, Scott J; Alvizo, Oscar

    2013-10-29

    The present disclosure relates to chemically modified carbonic anhydrase polypeptides and soluble compositions, homogenous liquid formulations comprising them. The chemically modified carbonic anhydrase polypeptides have improved properties relative to the same carbonic anhydrase polypeptide that is not chemically modified including the improved properties of increased activity and/or stability in the presence of amine compounds, ammonia, or carbonate ion. The present disclosure also provides methods of preparing the chemically modified polypeptides and methods of using the chemically modified polypeptides for accelerating the absorption of carbon dioxide from a gas stream into a solution as well as for the release of the absorbed carbon dioxide for further treatment and/or sequestering.

  6. Molecular modeling study on the allosteric inhibition mechanism of HIV-1 integrase by LEDGF/p75 binding site inhibitors.

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    Weiwei Xue

    Full Text Available HIV-1 integrase (IN is essential for the integration of viral DNA into the host genome and an attractive therapeutic target for developing antiretroviral inhibitors. LEDGINs are a class of allosteric inhibitors targeting LEDGF/p75 binding site of HIV-1 IN. Yet, the detailed binding mode and allosteric inhibition mechanism of LEDGINs to HIV-1 IN is only partially understood, which hinders the structure-based design of more potent anti-HIV agents. A molecular modeling study combining molecular docking, molecular dynamics simulation, and binding free energy calculation were performed to investigate the interaction details of HIV-1 IN catalytic core domain (CCD with two recently discovered LEDGINs BI-1001 and CX14442, as well as the LEDGF/p75 protein. Simulation results demonstrated the hydrophobic domain of BI-1001 and CX14442 engages one subunit of HIV-1 IN CCD dimer through hydrophobic interactions, and the hydrophilic group forms hydrogen bonds with HIV-1 IN CCD residues from other subunit. CX14442 has a larger tert-butyl group than the methyl of BI-1001, and forms better interactions with the highly hydrophobic binding pocket of HIV-1 IN CCD dimer interface, which can explain the stronger affinity of CX14442 than BI-1001. Analysis of the binding mode of LEDGF/p75 with HIV-1 IN CCD reveals that the LEDGF/p75 integrase binding domain residues Ile365, Asp366, Phe406 and Val408 have significant contributions to the binding of the LEDGF/p75 to HIV1-IN. Remarkably, we found that binding of BI-1001 and CX14442 to HIV-1 IN CCD induced the structural rearrangements of the 140 s loop and oration displacements of the side chains of the three conserved catalytic residues Asp64, Asp116, and Glu152 located at the active site. These results we obtained will be valuable not only for understanding the allosteric inhibition mechanism of LEDGINs but also for the rational design of allosteric inhibitors of HIV-1 IN targeting LEDGF/p75 binding site.

  7. Synthesis of 3-alkyl enol mimics inhibitors of type II dehydroquinase: factors influencing their inhibition potency.

    Science.gov (United States)

    Blanco, Beatriz; Sedes, Antía; Peón, Antonio; Lamb, Heather; Hawkins, Alastair R; Castedo, Luis; González-Bello, Concepción

    2012-05-14

    Several 3-alkylaryl mimics of the enol intermediate in the reaction catalyzed by type II dehydroquinase were synthesized to investigate the effect on the inhibition potency of replacing the oxygen atom in the side chain by a carbon atom. The length and the rigidity of the spacer was also studied. The inhibitory properties of the reported compounds against type II dehydroquinase from Mycobacterium tuberculosis and Helicobacter pylori are also reported. The binding modes of these analogs in the active site of both enzymes were studied by molecular docking using GOLD 5.0 and dynamic simulations studies.

  8. Inhibition of plasmin, urokinase, tissue plasminogen activator, and C1S by a myxoma virus serine proteinase inhibitor.

    Science.gov (United States)

    Lomas, D A; Evans, D L; Upton, C; McFadden, G; Carrell, R W

    1993-01-05

    The myxoma and malignant rabbit fibroma poxviruses are lethal tumorigenic viruses of rabbits whose virulence is modulated by the production of a virus-encoded secreted serine proteinase inhibitor, SERP-1. This viral protein was detected in medium harvested from myxoma and malignant rabbit fibroma virus-infected cells, and its inhibitory profile has been characterized by gel and kinetic analysis. SERP-1 forms complexes with and inhibits the human fibrinolytic enzymes plasmin, urokinase, and two-chain tissue-type plasminogen activator (association rate constants 3.4 x 10(4), 4.3 x 10(4), and 3.6 x 10(4) M-1 s-1 respectively). It is also able to inhibit C1S, the first enzyme in the complement cascade with an association rate constant which was unaffected by the addition of heparin (1.3 x 10(3) M-1 s-1). SERP-1 acts as a substrate for and is cleaved by thrombin, porcine trypsin, human neutrophil elastase, porcine pancreatic elastase, thermolysin, subtilisin, bovine alpha-chymotrypsin, and factor Xa. Incubation with kallikrein and cathepsin G had no effect. The structure of SERP-1 has been modeled on other members of the serpin family which revealed the characteristic serpin architecture apart from the absence of the D-helix. Structural analysis and kinetic assays demonstrate that the absence of this region does not prevent inhibitory activity and furthermore allow the identification of cysteine residues involved in internal and intermolecular disulfide bonding.

  9. Inhibition of homologous recombination repair in irradiated tumor cells pretreated with Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin

    International Nuclear Information System (INIS)

    Noguchi, Miho; Yu, Dong; Hirayama, Ryoichi; Ninomiya, Yasuharu; Sekine, Emiko; Kubota, Nobuo; Ando, Koichi; Okayasu, Ryuichi

    2006-01-01

    In order to investigate the mechanism of radio-sensitization by an Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), we studied repair of DNA double strand breaks (DSBs) in irradiated human cells pre-treated with 17-AAG. DSBs are thought to be the critical target for radiation-induced cell death. Two human tumor cell lines DU145 and SQ-5 which showed clear radio-sensitization by 17-AAG revealed a significant inhibition of DSB repair, while normal human cells which did not show radio-sensitization by the drug indicated no change in the DSB repair kinetics with 17-AAG. We further demonstrated that BRCA2 was a novel client protein for Hsp90, and 17-AAG caused the degradation of BRCA2 and in turn altered the behavior of Rad51, a critical protein for homologous recombination (HR) pathway of DSB repair. Our data demonstrate for the first time that 17-AAG inhibits the HR repair process and could provide a new therapeutic strategy to selectively result in higher tumor cell killing

  10. Hsp90 inhibitor 17-AAG inhibits progression of LuCaP35 xenograft prostate tumors to castration resistance.

    Science.gov (United States)

    O'Malley, Katherine J; Langmann, Gabrielle; Ai, Junkui; Ramos-Garcia, Raquel; Vessella, Robert L; Wang, Zhou

    2012-07-01

    Advanced prostate cancer is currently treated with androgen deprivation therapy (ADT). ADT initially results in tumor regression; however, all patients eventually relapse with castration-resistant prostate cancer. New approaches to delay the progression of prostate cancer to castration resistance are in desperate need. This study addresses whether targeting Heat shock protein 90 (HSP90) regulation of androgen receptor (AR) can inhibit prostate cancer progression to castration resistance. The HSP90 inhibitor 17-AAG was injected intraperitoneally into nude mice bearing LuCaP35 xenograft tumors to determine the effect of HSP90 inhibition on prostate cancer progression to castration resistance and host survival. Administration of 17-AAG maintained androgen-sensitivity, delayed the progression of LuCaP35 xenograft tumors to castration resistance, and prolonged the survival of host. In addition, 17-AAG prevented nuclear localization of endogenous AR in LuCaP35 xenograft tumors in castrated nude mice. Targeting Hsp90 or the mechanism by which HSP90 regulates androgen-independent AR nuclear localization and activation may lead to new approaches to prevent and/or treat castration-resistant prostate cancer. Copyright © 2011 Wiley Periodicals, Inc.

  11. Pectinesterase Inhibitor from Jelly Fig (Ficus awkeotsang Makino Achene Inhibits Surface Antigen Expression by Human Hepatitis B Virus

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    Yu-Chuen Huang

    2013-01-01

    Full Text Available Pectinesterase inhibitor (PEI isolated from jelly fig (Ficus awkeotsang Makino is an edible component of a popular drink consumed in Asia. Hepatitis B virus (HBV infection is prevalent in Asia, and current treatments for HBV infection need improvement. This study aimed to evaluate the effect of PEI on the surface antigen expression by HBV (HBsAg. Human hepatoma cell lines Hep3B and Huh7 served as in vitro models for assessing the cytotoxicity and HBsAg expression. A culture of primary hepatocytes cultured from mice served as the normal counterpart. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT colorimetric assay. HBsAg expression was evaluated by measuring HBsAg secretion into the culture medium using an enzyme-linked immunosorbent assay. The results showed that PEI did not affect the viability of the human hepatoma cell lines or primary mouse hepatocytes. PEI inhibited the expression of HBsAg in hepatoma cell lines harboring endogenous (Hep3B and integrated (Huh7 HBV genomes in a concentration- and time-dependent manner, thus implicating a universal activity against HBV gene expression. In conclusion, it suggests that PEI from jelly fig inhibits the expression of human HBsAg in host cells without toxic effects on normal primary hepatocytes.

  12. Pectinesterase Inhibitor from Jelly Fig (Ficus awkeotsang Makino) Achene Inhibits Surface Antigen Expression by Human Hepatitis B Virus.

    Science.gov (United States)

    Huang, Yu-Chuen; Jiang, Chii-Ming; Chen, Yu-Jen; Chen, Yu-Yawn

    2013-01-01

    Pectinesterase inhibitor (PEI) isolated from jelly fig (Ficus awkeotsang Makino) is an edible component of a popular drink consumed in Asia. Hepatitis B virus (HBV) infection is prevalent in Asia, and current treatments for HBV infection need improvement. This study aimed to evaluate the effect of PEI on the surface antigen expression by HBV (HBsAg). Human hepatoma cell lines Hep3B and Huh7 served as in vitro models for assessing the cytotoxicity and HBsAg expression. A culture of primary hepatocytes cultured from mice served as the normal counterpart. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. HBsAg expression was evaluated by measuring HBsAg secretion into the culture medium using an enzyme-linked immunosorbent assay. The results showed that PEI did not affect the viability of the human hepatoma cell lines or primary mouse hepatocytes. PEI inhibited the expression of HBsAg in hepatoma cell lines harboring endogenous (Hep3B) and integrated (Huh7) HBV genomes in a concentration- and time-dependent manner, thus implicating a universal activity against HBV gene expression. In conclusion, it suggests that PEI from jelly fig inhibits the expression of human HBsAg in host cells without toxic effects on normal primary hepatocytes.

  13. Inhibition of hydroxycinnamoyl-CoA thioesterases in ginger (Zingiber officinale Rosc.) and turmeric (Curcuma longa L.) by lipase inhibitors.

    Science.gov (United States)

    Flores-Sanchez, Isvett Josefina; Gang, David Roger

    2013-11-01

    Ginger (Zingiber officinale Rosc.) and turmeric (Curcuma longa L.), members of the Zingiberaceae, are widely used in traditional Asian cuisines and herbal medicine. Gingerols and diarylheptanoids, important compounds from these plants, appear to be produced by enzymes of the type III polyketide synthase class. Previous efforts to detect activity of such enzymes in tissues from these plants were only marginally successful in turmeric and completely unsuccessful in ginger because of very rapid hydrolysis of the hydroxycinnamoyl-CoA substrates (p-coumaroyl-CoA, feruloyl-CoA and caffeoyl-CoA) in these assays, presumably due to the presence of thioesterases in these tissues. In order to determine whether such thioesterase activities were specific and could be reduced so that the polyketide synthase activities could be better characterized, three inhibitors of the thioesterase domain of fatty acid synthase were tested in assays with leaf and rhizome crude protein extracts from these plants: orlistat, a reduced form of lipstatin, and peptide 1 and peptide 2 from hydrolysates of soybean β-conglycinin. Results of these analyses indicated that specific thioesterases do exist in these plants and that they could indeed be inhibited, with highest inhibition occurring with a mixture of these three compounds, leading for example to a reduction of caffeoyl-CoA hydrolysis in leaves and rhizomes of ginger by 40-fold and 27-fold, respectively. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  14. Inhibition of multiple pathogenic pathways by histone deacetylase inhibitor SAHA in a corneal alkali-burn injury model

    Science.gov (United States)

    Li, Xinyu; Zhou, Qinbo; Hanus, Jakub; Anderson, Chastain; Zhang, Hongmei; Dellinger, Michael; Brekken, Rolf; Wang, Shusheng

    2013-01-01

    Neovascularization (NV) in the cornea is a major cause of vision impairment and corneal blindness. Hemangiogenesis and lymphangiogenesis induced by inflammation underlie the pathogenesis of corneal NV. The current mainstay treatment, corticosteroid, treats the inflammation associated with corneal NV, but is not satisfactory due to such side effects as cataract and the increase in intraocular pressure. It is imperative to develop a novel therapy that specifically targets the hemangiogenesis, lymphangiogenesis and inflammation pathways underlying corneal NV. Histone deacetylase inhibitors (HDACi) have been in clinical trials for cancer and other diseases. In particular, HDACi suberoylanilide hydroxamic acid (SAHA, vorinostat, Zolinza) has been approved by the FDA for the treatment of cutaneous T-cell lymphoma. The functional mechanism of SAHA in cancer and especially in corneal NV remains unclear. Here, we show that topical application of SAHA inhibits neovascularization in an alkali-burn corneal injury model. Mechanistically, SAHA inhibits corneal NV by repressing hemangiogenesis, inflammation pathways and previously overlooked lymphangiogenesis. Topical SAHA is well tolerated on the ocular surface. In addition, the potency of SAHA in corneal NV appears to be comparable to the current steroid therapy. SAHA may possess promising therapeutic potential in alkali-burn corneal injury and other inflammatory neovascularization disorders. PMID:23186311

  15. Characterization of Carbonic Anhydrase 9 in the Alimentary Canal of Aedes aegypti and Its Relationship to Homologous Mosquito Carbonic Anhydrases

    Directory of Open Access Journals (Sweden)

    Daniel P. Dixon

    2017-02-01

    Full Text Available In the mosquito midgut, luminal pH regulation and cellular ion transport processes are important for the digestion of food and maintenance of cellular homeostasis. pH regulation in the mosquito gut is affected by the vectorial movement of the principal ions including bicarbonate/carbonate and protons. As in all metazoans, mosquitoes employ the product of aerobic metabolism carbon dioxide in its bicarbonate/carbonate form as one of the major buffers of cellular and extracellular pH. The conversion of metabolic carbon dioxide to bicarbonate/carbonate is accomplished by a family of enzymes encoded by the carbonic anhydrase gene family. This study characterizes Aedes aegypti carbonic anhydrases using bioinformatic, molecular, and immunohistochemical methods. Our analyses show that there are fourteen Aedes aegypti carbonic anhydrase genes, two of which are expressed as splice variants. The carbonic anhydrases were classified as either integral membrane, peripheral membrane, mitochondrial, secreted, or soluble cytoplasmic proteins. Using polymerase chain reaction and Western blotting, one of the carbonic anhydrases, Aedes aegypti carbonic anhydrase 9, was analyzed and found in each life stage, male/female pupae, male/female adults, and in the female posterior midgut. Next, carbonic anhydrase 9 was analyzed in larvae and adults using confocal microscopy and was detected in the midgut regions. According to our analyses, carbonic anhydrase 9 is a soluble cytoplasmic enzyme found in the alimentary canal of larvae and adults and is expressed throughout the life cycle of the mosquito. Based on previous physiological analyses of adults and larvae, it appears AeCA9 is one of the major carbonic anhydrases involved in producing bicarbonate/carbonate which is involved in pH regulation and ion transport processes in the alimentary canal. Detailed understanding of the molecular bases of ion homeostasis in mosquitoes will provide targets for novel mosquito control

  16. Characterization of Carbonic Anhydrase 9 in the Alimentary Canal of Aedes aegypti and Its Relationship to Homologous Mosquito Carbonic Anhydrases.

    Science.gov (United States)

    Dixon, Daniel P; Van Ekeris, Leslie; Linser, Paul J

    2017-02-21

    In the mosquito midgut, luminal pH regulation and cellular ion transport processes are important for the digestion of food and maintenance of cellular homeostasis. pH regulation in the mosquito gut is affected by the vectorial movement of the principal ions including bicarbonate/carbonate and protons. As in all metazoans, mosquitoes employ the product of aerobic metabolism carbon dioxide in its bicarbonate/carbonate form as one of the major buffers of cellular and extracellular pH. The conversion of metabolic carbon dioxide to bicarbonate/carbonate is accomplished by a family of enzymes encoded by the carbonic anhydrase gene family. This study characterizes Aedes aegypti carbonic anhydrases using bioinformatic, molecular, and immunohistochemical methods. Our analyses show that there are fourteen Aedes aegypti carbonic anhydrase genes, two of which are expressed as splice variants. The carbonic anhydrases were classified as either integral membrane, peripheral membrane, mitochondrial, secreted, or soluble cytoplasmic proteins. Using polymerase chain reaction and Western blotting, one of the carbonic anhydrases, Aedes aegypti carbonic anhydrase 9, was analyzed and found in each life stage, male/female pupae, male/female adults, and in the female posterior midgut. Next, carbonic anhydrase 9 was analyzed in larvae and adults using confocal microscopy and was detected in the midgut regions. According to our analyses, carbonic anhydrase 9 is a soluble cytoplasmic enzyme found in the alimentary canal of larvae and adults and is expressed throughout the life cycle of the mosquito. Based on previous physiological analyses of adults and larvae, it appears AeCA9 is one of the major carbonic anhydrases involved in producing bicarbonate/carbonate which is involved in pH regulation and ion transport processes in the alimentary canal. Detailed understanding of the molecular bases of ion homeostasis in mosquitoes will provide targets for novel mosquito control strategies into the

  17. Mechanistic Studies and Modeling Reveal the Origin of Differential Inhibition of Gag Polymorphic Viruses by HIV-1 Maturation Inhibitors.

    Science.gov (United States)

    Lin, Zeyu; Cantone, Joseph; Lu, Hao; Nowicka-Sans, Beata; Protack, Tricia; Yuan, Tian; Yang, Hong; Liu, Zheng; Drexler, Dieter; Regueiro-Ren, Alicia; Meanwell, Nicholas A; Cockett, Mark; Krystal, Mark; Lataillade, Max; Dicker, Ira B

    2016-11-01

    HIV-1 maturation inhibitors (MIs) disrupt the final step in the HIV-1 protease-mediated cleavage of the Gag polyprotein between capsid p24 capsid (CA) and spacer peptide 1 (SP1), leading to the production of infectious virus. BMS-955176 is a second generation MI with improved antiviral activity toward polymorphic Gag variants compared to a first generation MI bevirimat (BVM). The underlying mechanistic reasons for the differences in polymorphic coverage were studied using antiviral assays, an LC/MS assay that quantitatively characterizes CA/SP1 cleavage kinetics of virus like particles (VLPs) and a radiolabel binding assay to determine VLP/MI affinities and dissociation kinetics. Antiviral assay data indicates that BVM does not achieve 100% inhibition of certain polymorphs, even at saturating concentrations. This results in the breakthrough of infectious virus (partial antagonism) regardless of BVM concentration. Reduced maximal percent inhibition (MPI) values for BVM correlated with elevated EC50 values, while rates of HIV-1 protease cleavage at CA/SP1 correlated inversely with the ability of BVM to inhibit HIV-1 Gag polymorphic viruses: genotypes with more rapid CA/SP1 cleavage kinetics were less sensitive to BVM. In vitro inhibition of wild type VLP CA/SP1 cleavage by BVM was not maintained at longer cleavage times. BMS-955176 exhibited greatly improved MPI against polymorphic Gag viruses, binds to Gag polymorphs with higher affinity/longer dissociation half-lives and exhibits greater time-independent inhibition of CA/SP1 cleavage compared to BVM. Virological (MPI) and biochemical (CA/SP1 cleavage rates, MI-specific Gag affinities) data were used to create an integrated semi-quantitative model that quantifies CA/SP1 cleavage rates as a function of both MI and Gag polymorph. The model outputs are in accord with in vitro antiviral observations and correlate with observed in vivo MI efficacies. Overall, these findings may be useful to further understand antiviral

  18. Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin inhibits the proliferation of ARPE-19 cells

    Directory of Open Access Journals (Sweden)

    Wang Lin

    2010-04-01

    Full Text Available Abstract Background The antiproliferative effect of the Hsp90 inhibitor 17-AAG (17-allylamino-17-demethoxygeldanamycin on human retinal pigment epithelial cells is investigated. Methods MTT and flow cytometry were used to study the antiproliferative effects of the 17-AAG treatment of ARPE-19 cells. 2D gel electrophoresis (2-DE and mass spectrometry were applied to detect the altered expression of proteins, which was verified by real-time PCR. Gene Ontology analysis and Ingenuity Pathway Analysis (IPA were utilized to analyze the signaling pathways, cellular location, function, and network connections of the identified proteins. And SOD assay was employed to confirm the analysis. Results 17-AAG suppressed the proliferation of ARPE-19 cells by inducing cell cycle arrest and apoptosis. Proteomic analysis revealed that the expression of 94 proteins was altered by a factor of more than 1.5 following exposure to 17-AAG. Of these 94, 87 proteins were identified. Real-time PCR results indicated that Hsp90 and Hsp70, which were not identified by proteomic analysis, were both upregulated upon 17-AAG treatment. IPA revealed that most of the proteins have functions that are related to oxidative stress, as verified by SOD assay, while canonical pathway analysis revealed glycolysis/gluconeogenesis. Conclusions 17-AAG suppressed the proliferation of ARPE-19 cells by inducing cell cycle arrest and apoptosis, and possibly by oxidative stress.

  19. 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis inhibitors increase erythritol production in Torula corallina, and DHN-melanin inhibits erythrose reductase.

    Science.gov (United States)

    Lee, Jung-Kul; Jung, Hyung-Moo; Kim, Sang-Yong

    2003-06-01

    The yeast Torula corallina is a strong erythritol producer that is used in the industrial production of erythritol. However, melanin accumulation during culture represents a serious problem for the purification of erythritol from the fermentation broth. Melanin biosynthesis inhibitors such as 3,4-dihydroxyphenylalanine and 1,8-dihydroxynaphthalene (DHN)-melanin inhibitors were added to the T. corallina cultures. Only the DHN-melanin inhibitors showed an effect on melanin production, which suggests that the melanin formed during the culturing of T. corallina is derived from DHN. This finding was confirmed by the detection of a shunt product of the pentaketide pathway, flaviolin, and elemental analysis. Among the DHN-melanin inhibitors, tricyclazole was the most effective. Supplementation with tricyclazole enhanced the production of erythritol while significantly inhibiting the production of DHN-melanin and DHN-melanin biosynthetic enzymes, such as trihydroxynaphthalene reductase. The erythrose reductase from T. corallina was purified to homogeneity by ion-exchange and affinity chromatography. Purified erythrose reductase was significantly inhibited in vitro in a noncompetitive manner by elevated levels of DHN-melanin. In contrast, the level of erythrose reductase activity was unaffected by increasing concentrations of tricyclazole. These results suggest that supplemental tricyclazole reduces the production of DHN-melanin, which may lead to a reduction in the inhibition of erythrose reductase and a higher yield of erythritol. This is the first report to demonstrate that melanin biosynthesis inhibitors increase the production of a sugar alcohol in T. corallina.

  20. Gene expression for carbonic anhydrase isoenzymes in human nasal mucosa.

    Science.gov (United States)

    Tarun, Alice S; Bryant, Bruce; Zhai, Wenwu; Solomon, Colin; Shusterman, Dennis

    2003-09-01

    Carbonic anhydrase (CA) is physiologically important in the reversible hydration reaction of CO(2); it is expressed in a number of isoforms (CA I-XIV) with varying degrees of enzymatic activity. In nasal chemesthesis, CA inhibition decreases the electrophysiologic response to CO(2), a common irritant test compound. CA enzymatic activity has been demonstrated in the human nasal mucosa using enzyme histochemical methods, but no systematic study of nasal mucosal CA isoenzyme gene expression has been published. We examined CA gene expression in superficial nasal mucosal scrapings from 15 subjects (6 females; 6 allergic rhinitics; age range, 21-56 years). Both non-quantitative and quantitative reverse transcription polymerase chain reaction (RT-PCR) were performed using primers for each gene coding for the 11 catalytically active CA isoenzymes and the housekeeping gene GADPH. Amplification products of GADPH and 10 of the 11 CA genes were detected in the specimens (CA VA was not detected). Relative expression of the CA genes was quantified using real-time PCR. Averaged across subjects, the relative abundance of the CA isoenzyme transcripts is as follows: CA XII > CA II > CA VB > CA IV > CA IX > CA III > CA XIV > CA I > CA VI > CA VII. Limited qualitative validation of gene expression was obtained by immunohistochemistry for CA I, CA II and CA IV. We also observed inter-individual variability in the expression of CA isoenzymes in human nasal mucosa, potentially contributing to differences in nasal chemosensitivity to CO(2) between individuals

  1. SphK1 inhibitor II (SKI-II) inhibits acute myelogenous leukemia cell growth in vitro and in vivo

    International Nuclear Information System (INIS)

    Yang, Li; Weng, Wei; Sun, Zhi-Xin; Fu, Xian-Jie; Ma, Jun; Zhuang, Wen-Fang

    2015-01-01

    Previous studies have identified sphingosine kinase 1 (SphK1) as a potential drug target for treatment of acute myeloid leukemia (AML). In the current study, we investigated the potential anti-leukemic activity of a novel and specific SphK1 inhibitor, SKI-II. We demonstrated that SKI-II inhibited growth and survival of human AML cell lines (HL-60 and U937 cells). SKI-II was more efficient than two known SphK1 inhibitors SK1-I and FTY720 in inhibiting AML cells. Meanwhile, it induced dramatic apoptosis in above AML cells, and the cytotoxicity by SKI-II was almost reversed by the general caspase inhibitor z-VAD-fmk. SKI-II treatment inhibited SphK1 activation, and concomitantly increased level of sphingosine-1-phosphate (S1P) precursor ceramide in AML cells. Conversely, exogenously-added S1P protected against SKI-II-induced cytotoxicity, while cell permeable short-chain ceramide (C6) aggravated SKI-II's lethality against AML cells. Notably, SKI-II induced potent apoptotic death in primary human AML cells, but was generally safe to the human peripheral blood mononuclear cells (PBMCs) isolated from healthy donors. In vivo, SKI-II administration suppressed growth of U937 leukemic xenograft tumors in severe combined immunodeficient (SCID) mice. These results suggest that SKI-II might be further investigated as a promising anti-AML agent. - Highlights: • SKI-II inhibits proliferation and survival of primary and transformed AML cells. • SKI-II induces apoptotic death of AML cells, but is safe to normal PBMCs. • SKI-II is more efficient than two known SphK1 inhibitors in inhibiting AML cells. • SKI-II inhibits SphK1 activity, while increasing ceramide production in AML cells. • SKI-II dose-dependently inhibits U937 xenograft growth in SCID mice

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-09-07

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

  3. Carbonic Anhydrases and Their Biotechnological Applications

    Directory of Open Access Journals (Sweden)

    Robert McKenna

    2013-08-01

    Full Text Available The carbonic anhydrases (CAs are mostly zinc-containing metalloenzymes which catalyze the reversible hydration/dehydration of carbon dioxide/bicarbonate. The CAs have been extensively studied because of their broad physiological importance in all kingdoms of life and clinical relevance as drug targets. In particular, human CA isoform II (HCA II has a catalytic efficiency of 108 M−1 s−1, approaching the diffusion limit. The high catalytic rate, relatively simple procedure of expression and purification, relative stability and extensive biophysical studies of HCA II has made it an exciting candidate to be incorporated into various biomedical applications such as artificial lungs, biosensors and CO2 sequestration systems, among others. This review highlights the current state of these applications, lists their advantages and limitations, and discusses their future development.

  4. Insights into the mechanism of inhibition of novel bacterial topoisomerase inhibitors from characterization of resistant mutants of Staphylococcus aureus.

    Science.gov (United States)

    Lahiri, Sushmita D; Kutschke, Amy; McCormack, Kathy; Alm, Richard A

    2015-09-01

    The type II topoisomerases DNA gyrase and topoisomerase IV are clinically validated bacterial targets that catalyze the modulation of DNA topology that is vital to DNA replication, repair, and decatenation. Increasing resistance to fluoroquinolones, which trap the topoisomerase-DNA complex, has led to significant efforts in the discovery of novel inhibitors of these targets. AZ6142 is a member of the class of novel bacterial topoisomerase inhibitors (NBTIs) that utilizes a distinct mechanism to trap the protein-DNA complex. AZ6142 has very potent activity against Gram-positive organisms, including Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes. In this study, we determined the frequencies of resistance to AZ6142 and other representative NBTI compounds in S. aureus and S. pneumoniae. The frequencies of selection of resistant mutants at 4× the MIC were 1.7 × 10(-8) for S. aureus and topoisomerase subunits were identified. Many of these substitutions were located outside the NBTI binding pocket and impact the susceptibility of AZ6142, resulting in a 4- to 32-fold elevation in the MIC over the wild-type parent strain. Data on cross-resistance with other NBTIs and fluoroquinolones enabled the differentiation of scaffold-specific changes from compound-specific variations. Our results suggest that AZ6142 inhibits both type II topoisomerases in S. aureus but that DNA gyrase is the primary target. Further, the genotype of the resistant mutants suggests that domain conformations and DNA interactions may uniquely impact NBTIs compared to fluoroquinolones. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  5. Pim kinase inhibition sensitizes FLT3-ITD acute myeloid leukemia cells to topoisomerase 2 inhibitors through increased DNA damage and oxidative stress

    Science.gov (United States)

    Doshi, Kshama A.; Trotta, Rossana; Natarajan, Karthika; Rassool, Feyruz V.; Tron, Adriana E.; Huszar, Dennis; Perrotti, Danilo; Baer, Maria R.

    2016-01-01

    Internal tandem duplication of fms-like tyrosine kinase-3 (FLT3-ITD) is frequent (30 percent) in acute myeloid leukemia (AML), and is associated with short disease-free survival following chemotherapy. The serine threonine kinase Pim-1 is a pro-survival oncogene transcriptionally upregulated by FLT3-ITD that also promotes its signaling in a positive feedback loop. Thus inhibiting Pim-1 represents an attractive approach in targeting FLT3-ITD cells. Indeed, co-treatment with the pan-Pim kinase inhibitor AZD1208 or expression of a kinase-dead Pim-1 mutant sensitized FLT3-ITD cell lines to apoptosis triggered by chemotherapy drugs including the topoisomerase 2 inhibitors daunorubicin, etoposide and mitoxantrone, but not the nucleoside analog cytarabine. AZD1208 sensitized primary AML cells with FLT3-ITD to topoisomerase 2 inhibitors, but did not sensitize AML cells with wild-type FLT3 or remission bone marrow cells, supporting a favorable therapeutic index. Mechanistically, the enhanced apoptosis observed with AZD1208 and topoisomerase 2 inhibitor combination treatment was associated with increased DNA double-strand breaks and increased levels of reactive oxygen species (ROS), and co-treatment with the ROS scavenger N-acetyl cysteine rescued FLT3-ITD cells from AZD1208 sensitization to topoisomerase 2 inhibitors. Our data support testing of Pim kinase inhibitors with topoisomerase 2 inhibitors, but not with cytarabine, to improve treatment outcomes in AML with FLT3-ITD. PMID:27374090

  6. L-carnitine is an endogenous HDAC inhibitor selectively inhibiting cancer cell growth in vivo and in vitro.

    Directory of Open Access Journals (Sweden)

    Hongbiao Huang

    Full Text Available L-carnitine (LC is generally believed to transport long-chain acyl groups from fatty acids into the mitochondrial matrix for ATP generation via the citric acid cycle. Based on Warburg's theory that most cancer cells mainly depend on glycolysis for ATP generation, we hypothesize that, LC treatment would lead to disturbance of cellular metabolism and cytotoxicity in cancer cells. In this study, Human hepatoma HepG2, SMMC-7721 cell lines, primary cultured thymocytes and mice bearing HepG2 tumor were used. ATP content was detected by HPLC assay. Cell cycle, cell death and cell viability were assayed by flow cytometry and MTS respectively. Gene, mRNA expression and protein level were detected by gene microarray, Real-time PCR and Western blot respectively. HDAC activities and histone acetylation were detected both in test tube and in cultured cells. A molecular docking study was carried out with CDOCKER protocol of Discovery Studio 2.0 to predict the molecular interaction between L-carnitine and HDAC. Here we found that (1 LC treatment selectively inhibited cancer cell growth in vivo and in vitro; (2 LC treatment selectively induces the expression of p21(cip1 gene, mRNA and protein in cancer cells but not p27(kip1; (4 LC increases histone acetylation and induces accumulation of acetylated histones both in normal thymocytes and cancer cells; (5 LC directly inhibits HDAC I/II activities via binding to the active sites of HDAC and induces histone acetylation and lysine-acetylation accumulation in vitro; (6 LC treatment induces accumulation of acetylated histones in chromatin associated with the p21(cip1 gene but not p27(kip1 detected by ChIP assay. These data support that LC, besides transporting acyl group, works as an endogenous HDAC inhibitor in the cell, which would be of physiological and pathological importance.

  7. Inhibition of P-glycoprotein by HIV protease inhibitors increases intracellular accumulation of berberine in murine and human macrophages.

    Directory of Open Access Journals (Sweden)

    Weibin Zha

    Full Text Available HIV protease inhibitor (PI-induced inflammatory response in macrophages is a major risk factor for cardiovascular diseases. We have previously reported that berberine (BBR, a traditional herbal medicine, prevents HIV PI-induced inflammatory response through inhibiting endoplasmic reticulum (ER stress in macrophages. We also found that HIV PIs significantly increased the intracellular concentrations of BBR in macrophages. However, the underlying mechanisms of HIV PI-induced BBR accumulation are unknown. This study examined the role of P-glycoprotein (P-gp in HIV PI-mediated accumulation of BBR in macrophages.Cultured mouse RAW264.7 macrophages, human THP-1-derived macrophages, Wild type MDCK (MDCK/WT and human P-gp transfected (MDCK/P-gp cells were used in this study. The intracellular concentration of BBR was determined by HPLC. The activity of P-gp was assessed by measuring digoxin and rhodamine 123 (Rh123 efflux. The interaction between P-gp and BBR or HIV PIs was predicated by Glide docking using Schrodinger program. The results indicate that P-gp contributed to the efflux of BBR in macrophages. HIV PIs significantly increased BBR concentrations in macrophages; however, BBR did not alter cellular HIV PI concentrations. Although HIV PIs did not affect P-gp expression, P-gp transport activities were significantly inhibited in HIV PI-treated macrophages. Furthermore, the molecular docking study suggests that both HIV PIs and BBR fit the binding pocket of P-gp, and HIV PIs may compete with BBR to bind P-gp.HIV PIs increase the concentration of BBR by modulating the transport activity of P-gp in macrophages. Understanding the cellular mechanisms of potential drug-drug interactions is critical prior to applying successful combinational therapy in the clinic.

  8. L-carnitine is an endogenous HDAC inhibitor selectively inhibiting cancer cell growth in vivo and in vitro.

    Science.gov (United States)

    Huang, Hongbiao; Liu, Ningning; Guo, Haiping; Liao, Siyan; Li, Xiaofen; Yang, Changshan; Liu, Shouting; Song, Wenbin; Liu, Chunjiao; Guan, Lixia; Li, Bing; Xu, Li; Zhang, Change; Wang, Xuejun; Dou, Q Ping; Liu, Jinbao

    2012-01-01

    L-carnitine (LC) is generally believed to transport long-chain acyl groups from fatty acids into the mitochondrial matrix for ATP generation via the citric acid cycle. Based on Warburg's theory that most cancer cells mainly depend on glycolysis for ATP generation, we hypothesize that, LC treatment would lead to disturbance of cellular metabolism and cytotoxicity in cancer cells. In this study, Human hepatoma HepG2, SMMC-7721 cell lines, primary cultured thymocytes and mice bearing HepG2 tumor were used. ATP content was detected by HPLC assay. Cell cycle, cell death and cell viability were assayed by flow cytometry and MTS respectively. Gene, mRNA expression and protein level were detected by gene microarray, Real-time PCR and Western blot respectively. HDAC activities and histone acetylation were detected both in test tube and in cultured cells. A molecular docking study was carried out with CDOCKER protocol of Discovery Studio 2.0 to predict the molecular interaction between L-carnitine and HDAC. Here we found that (1) LC treatment selectively inhibited cancer cell growth in vivo and in vitro; (2) LC treatment selectively induces the expression of p21(cip1) gene, mRNA and protein in cancer cells but not p27(kip1); (4) LC increases histone acetylation and induces accumulation of acetylated histones both in normal thymocytes and cancer cells; (5) LC directly inhibits HDAC I/II activities via binding to the active sites of HDAC and induces histone acetylation and lysine-acetylation accumulation in vitro; (6) LC treatment induces accumulation of acetylated histones in chromatin associated with the p21(cip1) gene but not p27(kip1) detected by ChIP assay. These data support that LC, besides transporting acyl group, works as an endogenous HDAC inhibitor in the cell, which would be of physiological and pathological importance.

  9. Beta carbonic anhydrases: novel targets for pesticides and anti-parasitic agents in agriculture and livestock husbandry.

    Science.gov (United States)

    Zolfaghari Emameh, Reza; Barker, Harlan; Hytönen, Vesa P; Tolvanen, Martti E E; Parkkila, Seppo

    2014-08-29

    The genomes of many insect and parasite species contain beta carbonic anhydrase (β-CA) protein coding sequences. The lack of β-CA proteins in mammals makes them interesting target proteins for inhibition in treatment of some infectious diseases and pests. Many insects and parasites represent important pests for agriculture and cause enormous economic damage worldwide. Meanwhile, pollution of the environment by old pesticides, emergence of strains resistant to them, and their off-target effects are major challenges for agriculture and society. In this study, we analyzed a multiple sequence alignment of 31 β-CAs from insects, some parasites, and selected plant species relevant to agriculture and livestock husbandry. Using bioinformatics tools a phylogenetic tree was generated and the subcellular localizations and antigenic sites of each protein were predicted. Structural models for β-CAs of Ancylostoma caninum, Ascaris suum, Trichinella spiralis, and Entamoeba histolytica, were built using Pisum sativum and Mycobacterium tuberculosis β-CAs as templates. Six β-CAs of insects and parasites and six β-CAs of plants are predicted to be mitochondrial and chloroplastic, respectively, and thus may be involved in important metabolic functions. All 31 sequences showed the presence of the highly conserved β-CA active site sequence motifs, CXDXR and HXXC (C: cysteine, D: aspartic acid, R: arginine, H: histidine, X: any residue). We discovered that these two motifs are more antigenic than others. Homology models suggested that these motifs are mostly buried and thus not well accessible for recognition by antibodies. The predicted mitochondrial localization of several β-CAs and hidden antigenic epitopes within the protein molecule, suggest that they may not be considered major targets for vaccines. Instead, they are promising candidate enzymes for small-molecule inhibitors which can easily penetrate the cell membrane. Based on current knowledge, we conclude that

  10. Enzyme inhibition studies by integrated Michaelis-Menten equation considering simultaneous presence of two inhibitors when one of them is a reaction product.

    Science.gov (United States)

    Bezerra, Rui M F; Pinto, Paula A; Fraga, Irene; Dias, Albino A

    2016-03-01

    To determine initial velocities of enzyme catalyzed reactions without theoretical errors it is necessary to consider the use of the integrated Michaelis-Menten equation. When the reaction product is an inhibitor, this approach is particularly important. Nevertheless, kinetic studies usually involved the evaluation of other inhibitors beyond the reaction product. The occurrence of these situations emphasizes the importance of extending the integrated Michaelis-Menten equation, assuming the simultaneous presence of more than one inhibitor because reaction product is always present. This methodology is illustrated with the reaction catalyzed by alkaline phosphatase inhibited by phosphate (reaction product, inhibitor 1) and urea (inhibitor 2). The approach is explained in a step by step manner using an Excel spreadsheet (available as a template in Appendix). Curve fitting by nonlinear regression was performed with the Solver add-in (Microsoft Office Excel). Discrimination of the kinetic models was carried out based on Akaike information criterion. This work presents a methodology that can be used to develop an automated process, to discriminate in real time the inhibition type and kinetic constants as data (product vs. time) are achieved by the spectrophotometer. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  11. Suppression of human T cell proliferation by the caspase inhibitors, z-VAD-FMK and z-IETD-FMK is independent of their caspase inhibition properties

    Energy Technology Data Exchange (ETDEWEB)

    Lawrence, C.P. [Medical Research Council Toxicology Unit, Hodgkin Building, Lancaster Road, University of Leicester, Leicester LE1 9HN (United Kingdom); Chow, S.C., E-mail: chow.sek.chuen@monash.edu [School of Science, Monash University Sunway Campus, Jalan Lagoon Selatan, Bandar Sunway, 46150 Selangor Darul Ehsan (Malaysia)

    2012-11-15

    The caspase inhibitors, benzyloxycarbony (Cbz)-l-Val-Ala-Asp (OMe)-fluoromethylketone (z-VAD-FMK) and benzyloxycarbonyl (Cbz)-Ile-Glu (OMe)-Thr-Asp (OMe)-FMK (z-IETD-FMK) at non-toxic doses were found to be immunosuppressive and inhibit human T cell proliferation induced by mitogens and IL-2 in vitro. Both caspase inhibitors were shown to block NF-κB in activated primary T cells, but have little inhibitory effect on the secretion of IL-2 and IFN-γ during T cell activation. However, the expression of IL-2 receptor α-chain (CD25) in activated T cells was inhibited by both z-VAD-FMK and z-IETD-FMK, whereas the expression of the early activated T cell marker, CD69 was unaffected. During primary T cell activation via the antigen receptor, both caspase-8 and caspase-3 were activated and processed to their respective subunits, but neither caspase inhibitors had any effect on the processing of these two caspases. In sharp contrast both caspase inhibitors readily blocked apoptosis and the activation of caspases during FasL-induced apoptosis in activated primary T cells and Jurkat T cells. Collectively, the results demonstrate that both z-VAD-FMK and z-IETD-FMK are immunosuppressive in vitro and inhibit T cell proliferation without blocking the processing of caspase-8 and caspase-3. -- Highlights: ► Caspase-8 and caspase-3 were activated during T cell activation and proliferation. ► T cell proliferation was blocked by caspase inhibitors. ► Caspase activation during T cell proliferation was not block by caspase inhibitors.

  12. Histone deacetylase inhibition modulates deoxyribonucleotide pools and enhances the antitumor effects of the ribonucleotide reductase inhibitor 3'-C-methyladenosine in leukaemia cells.

    Science.gov (United States)

    Meli, Maria; Tolomeo, Manlio; Grifantini, Mario; Mai, Antonello; Cappellacci, Loredana; Petrelli, Riccardo; Rotili, Dante; Ferro, Arianna; Saiko, Philipp; Szekeres, Thomas; Dusonchet, Luisa

    2011-05-01

    Histone deacetylase (HDAC) inhibitors are a new class of epigenetic agents that were reported to enhance the cytotoxic effects of classical anticancer drugs through multiple mechanisms. However, which of the possible drug combinations would be the most effective and clinically useful are to be determined. We treated the HL60 and NB4 promyelocytic leukaemia cells with a combination of the ribonucleotide reductase (RR) inhibitor 3'-C-methyladenosine (3'-Me-Ado) and several hydroxamic acid-derived HDAC inhibitors, including two recently synthesized molecules, MC1864 and MC1879, and the reference compound trichostatin A (TSA). The results showed significant growth inhibitory and apoptotic synergistic effects with the combinations. Hence, we evaluated the effects of the combinations on cell cycle distribution and on the level of several proteins involved in the apoptotic process (p21, caspase-3, Bcl-2, Bax, AIF). Since HDAC inhibitors increased the G1-S transition block induced by 3'-Me-Ado, an effect on RR activity was hypothesized. Indeed, the HPLC evaluation of intracellular deoxyribonucleotide (dNTP) pools showed that both TSA and MC1864 induced a decrease in dNTPs, even if with a somewhat different pattern, suggesting that RR inhibition contributes to the observed synergism. Furthermore, while TSA was shown to activate the intrinsic apoptotic pathway, MC1864 induced a dose-dependent increase in ROS and AIF levels. Moreover, the treatment with the radical scavenger N-acetylcysteine determined a significant inhibition of MC1864- but not TSA-mediated synergistic effects. Hence, our findings are consistent with a possible role of HDAC inhibitor mediated-ROS induction in RR inhibition and in the potentiation of RR inhibitor-mediated apoptosis.

  13. Insight to structural subsite recognition in plant thiol protease-inhibitor complexes : Understanding the basis of differential inhibition and the role of water

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    Mukhopadhayay Bishnu P

    2001-09-01

    Full Text Available Abstract Background This work represents an extensive MD simulation / water-dynamics studies on a series of complexes of inhibitors (leupeptin, E-64, E-64-C, ZPACK and plant cysteine proteases (actinidin, caricain, chymopapain, calotropin DI of papain family to understand the various interactions, water binding mode, factors influencing it and the structural basis of differential inhibition. Results The tertiary structure of the enzyme-inhibitor complexes were built by visual interactive modeling and energy minimization followed by dynamic simulation of 120 ps in water environment. DASA study with and without the inhibitor revealed the potential subsite residues involved in inhibition. Though the interaction involving main chain atoms are similar, critical inspection of the complexes reveal significant differences in the side chain interactions in S2-P2 and S3-P3 pairs due to sequence differences in the equivalent positions of respective subsites leading to differential inhibition. Conclusion The key finding of the study is a conserved site of a water molecule near oxyanion hole of the enzyme active site, which is found in all the modeled complexes and in most crystal structures of papain family either native or complexed. Conserved water molecules at the ligand binding sites of these homologous proteins suggest the structural importance of the water, which changes the conventional definition of chemical geometry of inhibitor binding domain, its shape and complimentarity. The water mediated recognition of inhibitor to enzyme subsites (Pn...H2O....Sn of leupeptin acetyl oxygen to caricain, chymopapain and calotropinDI is an additional information and offer valuable insight to potent inhibitor design.

  14. Inhibition of 5-Lipoxygenase inhibitor zileuton in high-fat diet-induced nonalcoholic fatty liver disease progression model

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    Kuifen Ma

    2017-11-01

    Full Text Available Objective(s: Arachidonic Acid/5-lipoxygenase (AA/5-LOX pathway connects lipid metabolism and proinflammatory cytokine, which are both related to the development and progression of nonalcoholic fatty liver disease (NAFLD. Therefore, the present study was designed to investigate the role of AA/5-LOX pathway in progression of NAFLD, and the effect of zileuton, an inhibitor of 5-LOX, in this model. Materials and Methods: Animal model for progression of NAFLD was established via feeding high saturated fat diet (HFD. Liver function, HE staining, NAFLD activity score (NAS were used to evaluate NAFLD progression. We detected the lipid metabolism substrates: free fatty acids (FFA and AA, products: cysteinyl-leukotrienes (CysLTs, and changes in gene and protein level of key enzyme in AA/5-LOX pathway including PLA2 and 5-LOX. Furthermore, we determined whether NAFLD progression pathway was delayed or reversed when zileuton (1-[1-(1-benzothiophen-2-ylethyl]-1-hydroxyurea was administrated. Results: Rat model for progression of NAFLD was well established as analyzed by liver transaminase activities, hematoxylin-eosin (HE staining and NAS. The concentrations of substrates and products in AA/5-LOX pathway were increased with the progression of NAFLD. mRNA and protein expression of PLA2 and 5-LOX were all enhanced. Moreover, administration of zileuton inhibited AA/5-LOX pathway and reversed the increased transamine activities and NAS. Conclusion: AA/5-LOX pathway promotes the progression of NAFLD, which can be reversed by zileuton.

  15. 17-AAG, an Hsp90 inhibitor, causes kinetochore defects: a novel mechanism by which 17-AAG inhibits cell proliferation.

    Science.gov (United States)

    Niikura, Y; Ohta, S; Vandenbeldt, K J; Abdulle, R; McEwen, B F; Kitagawa, K

    2006-07-13

    The Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG), which is currently in clinical trials, is thought to exert antitumor activity by simultaneously targeting several oncogenic signaling pathways. Here we report a novel mechanism by which 17-AAG inhibits cell proliferation, and we provide the first evidence that HSP90 is required for the assembly of kinetochore protein complexes in humans. 17-AAG caused delocalization of several kinetochore proteins including CENP-I and CENP-H but excluding CENP-B and CENP-C. Consistently, 17-AAG induced a mitotic arrest that depends on the spindle checkpoint and induced misalignment of chromosomes and aneuploidy. We found that HSP90 associates with SGT1 (suppressor of G2 allele of skp1; SUGT1) in human cells and that depletion of SGT1 sensitizes HeLa cells to 17-AAG. Overexpression of SGT1 restored the localization of specific kinetochore proteins and chromosome alignment in cells treated with 17-AAG. Biochemical and genetic results suggest that HSP90, through its interaction with SGT1 (SUGT1), is required for kinetochore assembly. Furthermore, time-course experiments revealed that transient treatment with 17-AAG between late S and G2/M phases causes substantial delocalization of CENP-H and CENP-I, a finding that strongly suggests that HSP90 participates in kinetochore assembly in a cell cycle-dependent manner.

  16. 1,25-dihydroxyvitamin D(3) and PI3K/AKT inhibitors synergistically inhibit growth and induce senescence in prostate cancer cells.

    Science.gov (United States)

    Axanova, Linara S; Chen, Yong Q; McCoy, Thomas; Sui, Guangchao; Cramer, Scott D

    2010-11-01

    1-Alpha, 25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) inhibits proliferation of multiple cancer cell types including prostate cells and upregulates p21 and/or p27, while loss of Pten and PI3K/AKT activation stimulates survival and downregulates p21 and p27. We hypothesized that inhibition of the PI3K/AKT pathway synergizes with the antiproliferative signaling of 1,25(OH)(2)D(3). Viability, cell cycle and senescence of cells were evaluated upon combinational treatment with 1,25(OH)(2)D(3) and pharmacological PI3K/AKT inhibitors. Pharmacological inhibitors of PI3K or Akt and 1,25(OH)(2)D(3) synergistically inhibited growth of DU145, LNCaP, primary human prostate cancer cell strains and Pten null mouse prostatic epithelial cells (MPEC). The inhibitors used included API-2 (Triciribine) and GSK690693 which are currently in clinical trials for treatment of cancer. A novel mechanism for antiproliferative effects of 1,25(OH)(2)D(3) in prostate cells, induction of senescence, was discovered. Combination of 1,25(OH)(2)D(3) and AKT inhibitor cooperated to induce G(1) arrest, senescence, and p21 levels in prostate cancer cells. As AKT is commonly activated by PTEN loss, we evaluated the role of Pten in responsiveness to 1,25(OH)(2)D(3) using shRNA knockdown and by in vitro knockout of Pten. MPEC that lost Pten expression remained sensitive to the antiproliferative action of 1,25(OH)(2)D(3), and showed higher degree of synergism between AKT inhibitor and 1,25(OH)(2)D(3) compared to Pten-expressing counterparts. These findings provide the rationale for the development of therapies utilizing 1,25(OH)(2)D(3) or its analogs combined with inhibition of PI3K/AKT for the treatment of prostate cancer.

  17. 1,25-Dihydroxyvitamin D3 and PI3K/AKT Inhibitors Synergistically Inhibit Growth and Induce Senescence in Prostate Cancer Cells

    Science.gov (United States)

    Axanova, Linara S.; Chen, Yong Q.; McCoy, Thomas; Sui, Guangchao; Cramer, Scott D.

    2011-01-01

    BACKGROUND 1-Alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3) inhibits proliferation of multiple cancer cell types including prostate cells and upregulates p21 and/or p27, while loss of Pten and PI3K/AKT activation stimulates survival and down regulates p21 and p27. We hypothesized that inhibition of the PI3K/AKT pathway synergizes with the antiproliferative signaling of 1,25(OH)2D3. METHODS Viability, cell cycle and senescence of cells were evaluated upon combinational treatment with 1,25(OH)2D3 and pharmacological PI3K/AKT inhibitors. RESULTS Pharmacological inhibitors of PI3K or Akt and 1,25(OH)2D3 synergistically inhibited growth of DU145, LNCaP, primary human prostate cancer cell strains and Pten null mouse prostatic epithelial cells (MPEC). The inhibitors used included API-2 (Triciribine) and GSK690693 which are currently in clinical trials for treatment of cancer. A novel mechanism for antiproliferative effects of 1,25(OH)2D3 in prostate cells, induction of senescence, was discovered. Combination of 1,25(OH)2D3 and AKT inhibitor cooperated to induce G1 arrest, senescence, and p21 levels in prostate cancer cells. As AKT is commonly activated by PTEN loss, we evaluated the role of Pten in responsiveness to 1,25(OH)2D3 using shRNA knockdown and by in vitro knockout of Pten. MPEC that lost Pten expression remained sensitive to the antiproliferative action of 1,25(OH)2D3, and showed higher degree of synergism between AKT inhibitor and 1,25(OH)2D3 compared to Pten-expressing counterparts. CONCLUSIONS These findings provide the rationale for the development of therapies utilizing 1,25(OH)2D3 or its analogs combined with inhibition of PI3K/AKT for the treatment of prostate cancer. PMID:20583132

  18. Prevention of wear particle-induced osteolysis by a novel V-ATPase inhibitor saliphenylhalamide through inhibition of osteoclast bone resorption.

    Directory of Open Access Journals (Sweden)

    An Qin

    Full Text Available Wear particle-induced peri-implant loosening (Aseptic prosthetic loosening is one of the most common causes of total joint arthroplasty. It is well established that extensive bone destruction (osteolysis by osteoclasts is responsible for wear particle-induced peri-implant loosening. Thus, inhibition of osteoclastic bone resorption should prevent wear particle induced osteolysis and may serve as a potential therapeutic avenue for prosthetic loosening. Here, we demonstrate for the first time that saliphenylhalamide, a new V-ATPase inhibitor attenuates wear particle-induced osteolysis in a mouse calvarial model. In vitro biochemical and morphological assays revealed that the inhibition of osteolysis is partially attributed to a disruption in osteoclast acidification and polarization, both a prerequisite for osteoclast bone resorption. Interestingly, the V-ATPase inhibitor also impaired osteoclast differentiation via the inhibition of RANKL-induced NF-κB and ERK signaling pathways. In conclusion, we showed that saliphenylhalamide affected multiple physiological processes including osteoclast differentiation, acidification and polarization, leading to inhibition of osteoclast bone resorption in vitro and wear particle-induced osteolysis in vivo. The results of the study provide proof that the new generation V-ATPase inhibitors, such as saliphenylhalamide, are potential anti-resorptive agents for treatment of peri-implant osteolysis.

  19. Synthesis and biological evaluation of benzenesulphonamide-bearing 1,4,5-trisubstituted-1,2,3-triazoles possessing human carbonic anhydrase I, II, IV, and IX inhibitory activity.

    Science.gov (United States)

    Kumar, Rajiv; Sharma, Vikas; Bua, Silvia; Supuran, Claudiu T; Sharma, Pawan K

    2017-12-01

    A library of benzenesulphonamides incorporating 1,2,3-triazole rings functionalised with ester, carboxylic acid, carboxamide, carboxyhydrazide, and hydroxymethyl moieties were synthesised. The carbonic anhydrase (CAs, EC 4.2.1.1) inhibitory activity of the new compounds was assessed against four human (h) isoforms, hCA I, hCA II, hCA IV, and hCA IX. Among them, hCA II and IV are anti-glaucoma drug targets, being involved in aqueous humour secretion within the eye. hCA I was inhibited with Ki's ranging between 8.3 nM and 0.8737 µM. hCA II, the physiologically dominant cytosolic isoform, was excellently inhibited by these compounds, with Ki's in the range of 1.6-9.4 nM, whereas hCA IV was effectively inhibited by most of them, with Ki's in the range of 1.4-55.3 nM. Thirteen of the twenty sulphonamides were found to be excellent inhibitors of tumour associated hCA IX with Ki's ≤ 9.5 nM. Many of the new compounds reported here showed low nM inhibitory action against hCA II, IV, and IX, isoforms involved in glaucoma and some tumours, making them interesting candidates for further medicinal chemistry/pharmacologic studies.

  20. A new peptide ligand for targeting human carbonic anhydrase IX, identified through the phage display technology.

    Directory of Open Access Journals (Sweden)

    Vasileios Askoxylakis

    2010-12-01

    Full Text Available Carbonic anhydrase IX (CAIX is a transmembrane enzyme found to be overexpressed in various tumors and associated with tumor hypoxia. Ligands binding this target may be used to visualize hypoxia, tumor manifestation or treat tumors by endoradiotherapy.Phage display was performed with a 12 amino acid phage display library by panning against a recombinant extracellular domain of human carbonic anhydrase IX. The identified peptide CaIX-P1 was chemically synthesized and tested in vitro on various cell lines and in vivo in Balb/c nu/nu mice carrying subcutaneously transplanted tumors. Binding, kinetic and competition studies were performed on the CAIX positive human renal cell carcinoma cell line SKRC 52, the CAIX negative human renal cell carcinoma cell line CaKi 2, the human colorectal carcinoma cell line HCT 116 and on human umbilical vein endothelial cells (HUVEC. Organ distribution studies were carried out in mice, carrying SKRC 52 tumors. RNA expression of CAIX in HCT 116 and HUVEC cells was investigated by quantitative real time PCR.In vitro binding experiments of (125I-labeled-CaIX-P1 revealed an increased uptake of the radioligand in the CAIX positive renal cell carcinoma cell line SKRC 52. Binding of the radioligand in the colorectal carcinoma cell line HCT 116 increased with increasing cell density and correlated with the mRNA expression of CAIX. Radioligand uptake was inhibited up to 90% by the unlabeled CaIX-P1 peptide, but not by the negative control peptide octreotide at the same concentration. No binding was demonstrated in CAIX negative CaKi 2 and HUVEC cells. Organ distribution studies revealed a higher accumulation in SKRC 52 tumors than in heart, spleen, liver, muscle, intestinum and brain, but a lower uptake compared to blood and kidney.These data indicate that CaIX-P1 is a promising candidate for the development of new ligands targeting human carbonic anhydrase IX.

  1. Release Profile and Inhibition Test of The Nanoparticles A. Paniculata Extract as Inhibitor of α-Glucosidase in The Process of Carbohydrates Breakdown Into Glucose Diabetes Mellitus

    Science.gov (United States)

    Imansari, Farisa; Sahlan, Muhammad; Arbianti, Rita

    2017-07-01

    Andrographis paniculata (A.paniculata) contain the main active substances Andrographolide which helps lower glucose levels in diabetics by inhibiting the enzyme α-glucosidase. The ability of the extract A.paniculata in lowering glucose levels will increase with the technique encapsulation with a coating of composition Chitosan-STPP as a drug delivery to the target organ. This study aimed to get an overview of A.paniculata release profile of nanoparticles in a synthetic fluid media with various concentrations of coating and inhibition testing nasty shard extract in inhibiting the enzyme α-glucosidase. This research resulted in nanoparticles by coating efficiency and loading capacity of chitosan greatest variation of 2% and 1% STPP 60% and 46.29%. chitosan greatest variation of 2% and 1% STPP 60% and 46.29%. The ability of A.paniculata extracts as α-glucosidase enzyme inhibitors has been demonstrated in this study, the percent inhibition of 33.17%.

  2. A c-Src Inhibitor Peptide Based on Connexin43 Exerts Neuroprotective Effects through the Inhibition of Glial Hemichannel Activity.

    Science.gov (United States)

    Gangoso, Ester; Talaverón, Rocío; Jaraíz-Rodríguez, Myriam; Domínguez-Prieto, Marta; Ezan, Pascal; Koulakoff, Annette; Medina, José M; Giaume, Christian; Tabernero, Arantxa

    2017-01-01

    The non-receptor tyrosine kinase c-Src is an important mediator in several signaling pathways related to neuroinflammation. Our previous study showed that cortical injection of kainic acid (KA) promoted a transient increase in c-Src activity in reactive astrocytes surrounding the neuronal lesion. As a cell-penetrating peptide based on connexin43 (Cx43), specifically TAT-Cx43 266-283 , inhibits Src activity, we investigated the effect of TAT-Cx43 266-283 on neuronal death promoted by cortical KA injections in adult mice. As expected, KA promoted neuronal death, estimated by the reduction in NeuN-positive cells and reactive gliosis, characterized by the increase in glial fibrillary acidic protein (GFAP) expression. Interestingly, TAT-Cx43 266-283 injected with KA diminished neuronal death and reactive gliosis compared to KA or KA+TAT injections. In order to gain insight into the neuroprotective mechanism, we used in vitro models. In primary cultured neurons, TAT-Cx43 266-283 did not prevent neuronal death promoted by KA, but when neurons were grown on top of astrocytes, TAT-Cx43 266-283 prevented neuronal death promoted by KA. These observations demonstrate the participation of astrocytes in the neuroprotective effect of TAT-Cx43 266-283 . Furthermore, the neuroprotective effect was also present in non-contact co-cultures, suggesting the contribution of soluble factors released by astrocytes. As glial hemichannel activity is associated with the release of several factors, such as ATP and glutamate, that cause neuronal death, we explored the participation of these channels on the neuroprotective effect of TAT-Cx43 266-283. Our results confirmed that inhibitors of ATP and NMDA receptors prevented neuronal death in co-cultures treated with KA, suggesting the participation of astrocyte hemichannels in neurotoxicity. Furthermore, TAT-Cx43 266-283 reduced hemichannel activity promoted by KA in neuron-astrocyte co-cultures as assessed by ethidium bromide (EtBr) uptake assay

  3. A c-Src Inhibitor Peptide Based on Connexin43 Exerts Neuroprotective Effects through the Inhibition of Glial Hemichannel Activity

    Directory of Open Access Journals (Sweden)

    Ester Gangoso

    2017-12-01

    Full Text Available The non-receptor tyrosine kinase c-Src is an important mediator in several signaling pathways related to neuroinflammation. Our previous study showed that cortical injection of kainic acid (KA promoted a transient increase in c-Src activity in reactive astrocytes surrounding the neuronal lesion. As a cell-penetrating peptide based on connexin43 (Cx43, specifically TAT-Cx43266–283, inhibits Src activity, we investigated the effect of TAT-Cx43266–283 on neuronal death promoted by cortical KA injections in adult mice. As expected, KA promoted neuronal death, estimated by the reduction in NeuN-positive cells and reactive gliosis, characterized by the increase in glial fibrillary acidic protein (GFAP expression. Interestingly, TAT-Cx43266–283 injected with KA diminished neuronal death and reactive gliosis compared to KA or KA+TAT injections. In order to gain insight into the neuroprotective mechanism, we used in vitro models. In primary cultured neurons, TAT-Cx43266–283 did not prevent neuronal death promoted by KA, but when neurons were grown on top of astrocytes, TAT-Cx43266–283 prevented neuronal death promoted by KA. These observations demonstrate the participation of astrocytes in the neuroprotective effect of TAT-Cx43266–283. Furthermore, the neuroprotective effect was also present in non-contact co-cultures, suggesting the contribution of soluble factors released by astrocytes. As glial hemichannel activity is associated with the release of several factors, such as ATP and glutamate, that cause neuronal death, we explored the participation of these channels on the neuroprotective effect of TAT-Cx43266–283. Our results confirmed that inhibitors of ATP and NMDA receptors prevented neuronal death in co-cultures treated with KA, suggesting the participation of astrocyte hemichannels in neurotoxicity. Furthermore, TAT-Cx43266–283 reduced hemichannel activity promoted by KA in neuron-astrocyte co-cultures as assessed by ethidium

  4. Dual Inhibition of PI3K-AKT-mTOR- and RAF-MEK-ERK-signaling is synergistic in cholangiocarcinoma and reverses acquired resistance to MEK-inhibitors.

    Science.gov (United States)

    Ewald, Florian; Nörz, Dominik; Grottke, Astrid; Hofmann, Bianca T; Nashan, Björn; Jücker, Manfred

    2014-12-01

    Until today, there is no systemic treatment available for advanced cholangiocarcinoma (CCA). Recent studies have shown a frequent upregulation of the PI3K-AKT-mTOR and RAF-MEK-ERK pathways in this type of cancer. However, considering their high extend of redundancy and cross-talk, targeting only one pathway is likely to result in therapy failure and emergence of resistances. To provide a rationale for treatment of CCA with inhibitors of these respective pathways, we analyzed the effects of AKT inhibitor MK-2206, MEK inhibitor AZD6244 (ARRY-142886) and mTOR kinase inhibitor AZD8055 on three CCA cell lines in vitro, concerning proliferation, cell signaling and apoptosis. Furthermore, AZD6244 resistant cell lines have been generated to investigate, how their response may be affected by prolonged treatment with only a single inhibitor. Our data demonstrates that co-targeting of both, the PI3K/AKT/mTOR and RAF-MEK-ERK pathway, as well as vertical targeting of AKT and mTOR results in strong synergistic effects on proliferation and cell survival with combination indices below 0.3. Mechanistically, the combinatorial treatment with MK-2206 in addition to AZD8055 is necessary because AKT kinase activity was quickly restored after mTOR kinase inhibition. Interestingly, acquired MEK inhibitor resistance to AZD6244 was reversed by combined treatment with AZD6244 and either MK-2206 or AZD8055. Our data suggest that a combination of inhibitors targeting those respective pathways may be a viable approach for future application in patients with cholangiocarcinoma. AKT, mTOR and MEK are promising targets for a combinatorial treatment of cholangiocarcinoma cells even after acquisition of MEK inhibitor resistance.

  5. In vivo and in vitro inhibition of Spodoptera littoralis gut-serine protease by protease inhibitors isolated from maize and sorghum seeds.

    Science.gov (United States)

    El-latif, Ashraf Oukasha Abd

    2014-11-01

    Seeds of cereals (Gramineae) are a rich source of serine proteinase inhibitors of most of the several inhibitor families. In the present study, trypsin and chymotrypsin inhibitory activities was detected in the seed flour extracts of three varieties of maize (Zea maize) and six varieties of sorghum (Sorghum bicolor). The maize variety, Hi Teck 2031 and the sorghum variety, Giza 10 were found to have higher trypsin and chymotrypsin inhibitory potentials compared to other tested varieties for which they have been selected for further purification studies using ammonium sulfate fractionation and DEAE-Sephadex A-25 column. Maize and sorghum purified proteins showed a single band on SDS-PAGE corresponding to molecular mass of 20.0 and 15.2 kDa for maize and sorghum PIs respectively. The purified inhibitors were stable at temperature below 60 °C and were active at wide range of pH from 2 to 12 pH. The kinetic analysis revealed non-competitive type of inhibition for both inhibitors against both enzymes. The inhibitor constant (Ki) values suggested high affinity between inhibitors and enzymes. Purified inhibitors were found to have deep and negative effects on the mean larval weight, larval mortality, pupation and mean pupal weight of S.littoralis where maize PI was more effective than sorghum PI. It may be concluded that maize and sorghum protease inhibitor gene(s) could be potential targets for future studies in developing insect resistant transgenic plants. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Lidocaine inhibits NIH-3T3 cell multiplication by increasing the expression of cyclin-dependent kinase inhibitor 1A (p21).

    Science.gov (United States)

    Desai, Sukumar P; Kojima, Koji; Vacanti, Charles A; Kodama, Shohta

    2008-11-01

    We explored molecular mechanisms by which lidocaine inhibits growth in the murine embryonic fibroblast cell line NIH-3T3. Local anesthetics can adversely affect cell growth in vitro. Their effects on wound healing are controversial. We examined the effects and novel mechanisms by which lidocaine affects in vitro multiplication of the murine fibroblast cell line NIH-3T3. NIH-3T3 cells were grown in culture with lidocaine [0, 0.05, 0.5, 1, 2, and 5 mM]. Cell multiplication was assessed by determining cell counts on subsequent days, while mechanisms by which inhibition occurred were evaluated by bromodeoxyuridine uptake, gene expression using polymerase chain reaction array, and Western blot analysis to verify increased levels of affected proteins. Lidocaine caused dose-dependent inhibition of multiplication of NIH-3T3 cells. Effects ranged from no inhibition [0.05 and 0.5 mM] and mild inhibition [1 mM], to severe inhibition [2 and 5 mM] [P = 0.006]. Lidocaine 2 mM inhibited bromodeoxyuridine uptake at day 3.5 [P = 0.02 versus control, and P = 0.0495 vs 1 mM lidocaine]. On day 1.5, lidocaine upregulated expression of cyclin-D1 and cyclin-dependent kinase inhibitor 1A [p21]. On day 2.5, lidocaine increased the levels of p21 protein. Low concentrations of lidocaine, as would be seen in plasma after spinal, epidural, or plexus anesthesia, do not significantly affect multiplication of fibroblasts. Higher doses of lidocaine arrest cell multiplication at the S-phase of the growth cycle by upregulation of p21, an extremely potent inhibitor of cell multiplication. Higher concentrations, as would be seen after tissue infiltration, severely inhibit fibroblast multiplication and thus may impair wound healing.

  7. Inhibition of peripheral aromatization in baboons by an enzyme-activated aromatase inhibitor (MDL 18,962)

    International Nuclear Information System (INIS)

    Longcope, C.; Femino, A.; Johnston, J.O.

    1988-01-01

    The peripheral aromatization ([rho]BM) of androstenedione (A) and testosterone (T) was measured before and after administration of the aromatase inhibitor 10-(2 propynyl)estr-4-ene-3,17-dione (MDL-18,962) to five mature female baboons, Papio annubis. The measurements were made by infusing [3H]androstenedione/[14C]estrone or [3H]testosterone/[14C]estradiol for 3.5 h and collecting blood samples during the infusions and all urine for 96 h from the start of the infusion. Blood samples were analyzed for radioactivity as infused and product steroids, and the data were used to calculate MCRs. An aliquot of the pooled urine was analyzed for the glucuronides of estrone and estradiol and used to calculate the [rho]BM. MDL-18,962 was administered as a pulse in polyethylene glycol-400 (1-5 ml) either iv or via gastric tube 30 min before administration of the radiolabeled steroids. Control studies were done with and without polyethylene glycol-400 administration. When MDL-18,962 was given iv at 4 mg/kg, the aromatization of A was decreased 91.8 +/- 0.9% from the control value of 1.23 +/- 0.13% to 0.11 +/- 0.01%. At the same dose, aromatization of T was decreased 82.0 +/- 7.1%, from a control value of 0.20 +/- 0.03% to 0.037 +/- 0.018%. When MDL-18,962 was given iv at doses of 0.4, 0.1, 0.04, and 0.01 mg/kg, the values for aromatization of A were 0.16 +/- 0.03%, 0.18 +/- 0.06%, 0.37 +/- 11%, and 0.65 +/- 0.09%, respectively. The administration of MDL-18,962 via gastric tube at 4 mg/kg as a pulse decreased the aromatization of A from 1.35 +/- 0.06% to 0.43 +/- 0.12%, an inhibition of 67.2 +/- 10.7%. When administered via gastric tube daily for 5 days at 4 mg/kg, the aromatization of A fell from 1.35 +/- 0.06% to 0.063 +/- 0.003%, an inhibition of 84.4 +/- 0.5%

  8. Mode of inhibition of HIV-1 reverse transcriptase by polyacetylenetriol, a novel inhibitor of RNA- and DNA-directed DNA polymerases.

    Science.gov (United States)

    Loya, Shoshana; Rudi, Amira; Kashman, Yoel; Hizi, Amnon

    2002-03-15

    Polyacetylenetriol (PAT), a natural marine product from the Mediterranean sea sponge Petrosia sp., was found to be a novel general potent inhibitor of DNA polymerases. It inhibits equally well the RNA- and DNA-dependent DNA polymerase activities of retroviral reverse transcriptases (RTs) (i.e. of HIV, murine leukaemia virus and mouse mammary tumour virus) as well as cellular DNA polymerases (i.e. DNA polymerases alpha and beta and Escherichia coli polymerase I). A study of the mode and mechanism of the polymerase inhibition by PAT has been conducted with HIV-1 RT. PAT was shown to be a reversible non-competitive inhibitor. PAT binds RT independently and at a site different from that of the primer-template and dNTP substrates with high affinity (K(i)=0.51 microM and K(i)=0.53 microM with dTTP and with dGTP as the variable substrates respectively). Blocking the polar hydroxy groups of PAT has only a marginal effect on the inhibitory capacity, thus hydrophobic interactions are likely to play a major role in inhibiting RT. Preincubation of RT with the primer-template substrate prior to the interaction with PAT reduces substantially the inhibition capacity, probably by preventing these contacts. PAT does not interfere with the first step of polymerization, the binding of RT to DNA, nor does the inhibitor interfere with the binding of dNTP to RT/DNA complex, as evident from the steady-state kinetic study, whereby K(m) remains unchanged. We assume, therefore, that PAT interferes with subsequent catalytic steps of DNA polymerization. The inhibitor may alter the optimal stereochemistry of the polymerase active site relative to the primer terminus, bound dNTP and the metal ions that are crucial for efficient catalysis or, alternatively, may interfere with the thumb sub-domain movement and, thus, with the translocation of the primer-template following nucleotide incorporation.

  9. An innovative strategy for dual inhibitor design and its application in dual inhibition of human thymidylate synthase and dihydrofolate reductase enzymes.

    Directory of Open Access Journals (Sweden)

    Mahreen Arooj

    Full Text Available Due to the diligence of inherent redundancy and robustness in many biological networks and pathways, multitarget inhibitors present a new prospect in the pharmaceutical industry for treatment of complex diseases. Nevertheless, to design multitarget inhibitors is concurrently a great challenge for medicinal chemists. We have developed a novel computational approach by integrating the affinity predictions from structure-based virtual screening with dual ligand-based pharmacophore to discover potential dual inhibitors of human Thymidylate synthase (hTS and human dihydrofolate reductase (hDHFR. These are the key enzymes in folate metabolic pathway that is necessary for the biosynthesis of RNA, DNA, and protein. Their inhibition has found clinical utility as antitumor, antimicrobial, and antiprotozoal agents. A druglike database was utilized to perform dual-target docking studies. Hits identified through docking experiments were mapped over a dual pharmacophore which was developed from experimentally known dual inhibitors of hTS and hDHFR. Pharmacophore mapping procedure helped us in eliminating the compounds which do not possess basic chemical features necessary for dual inhibition. Finally, three structurally diverse hit compounds that showed key interactions at both active sites, mapped well upon the dual pharmacophore, and exhibited lowest binding energies were regarded as possible dual inhibitors of hTS and hDHFR. Furthermore, optimization studies were performed for final dual hit compound and eight optimized dual hits demonstrating excellent binding features at target systems were also regarded as possible dual inhibitors of hTS and hDHFR. In general, the strategy used in the current study could be a promising computational approach and may be generally applicable to other dual target drug designs.

  10. Anorectic activities of serotonin uptake inhibitors: correlation with their potencies at inhibiting serotonin uptake in vivo and /sup 3/H-mazindol binding in vitro

    Energy Technology Data Exchange (ETDEWEB)

    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.

  11. Anorectic activities of serotonin uptake inhibitors: correlation with their potencies at inhibiting serotonin uptake in vivo and 3H-mazindol binding in vitro

    International Nuclear Information System (INIS)

    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 50 values of anorectic action and the ED 50 values of serotonin uptake inhibition in vivo (but not in vitro) for several specific serotonin uptake inhibitors. Most of the drugs tested displaced [ 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 [ 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 [ 3 H]-mazindol is discussed in connection with the serotonergic regulation of carbohydrate intake

  12. AT-406, an orally active antagonist of multiple inhibitor of apoptosis proteins, inhibits progression of human ovarian cancer.

    Science.gov (United States)

    Brunckhorst, Melissa K; Lerner, Dimitry; Wang, Shaomeng; Yu, Qin

    2012-07-01

    Ovarian carcinoma is the most deadly gynecological malignancy. Current chemotherapeutic drugs are only transiently effective and patients with advance disease often develop resistance despite significant initial responses. Mounting evidence suggests that anti-apoptotic proteins, including those of the inhibitor of apoptosis protein (IAP) family, play important roles in the chemoresistance. There has been a recent emergence of compounds that block the IAP functions. Here, we evaluated AT-406, a novel and orally active antagonist of multiple IAP proteins, in ovarian cancer cells as a single agent and in the combination with carboplatin for therapeutic efficacy and mechanism of action. We demonstrate that AT-406 has significant single agent activity in 60% of human ovarian cancer cell lines examined in vitro and inhibits ovarian cancer progression in vivo and that 3 out of 5 carboplatin-resistant cell lines are sensitive to AT-406, highlighting the therapeutic potential of AT-406 for patients with inherent or acquired platinum resistance. Additionally, our in vivo studies show that AT-406 enhances the carboplatin-induced ovarian cancer cell death and increases survival of the experimental mice, suggesting that AT-406 sensitizes the response of these cells to carboplatin. Mechanistically, we demonstrate that AT-406 induced apoptosis is correlated with its ability to down-regulate XIAP whereas AT-406 induces cIAP1 degradation in both AT-406 sensitive and resistance cell lines. Together, these results demonstrate, for the first time, the anti-ovarian cancer efficacy of AT-406 as a single agent and in the combination with carboplatin, suggesting that AT-406 has potential as a novel therapy for ovarian cancer patients, especially for patients exhibiting resistance to the platinum-based therapies.

  13. Extramitochondrial domain rich in carbonic anhydrase activity improves myocardial energetics.

    Science.gov (United States)

    Schroeder, Marie A; Ali, Mohammad A; Hulikova, Alzbeta; Supuran, Claudiu T; Clarke, Kieran; Vaughan-Jones, Richard D; Tyler, Damian J; Swietach, Pawel

    2013-03-05

    CO2 is produced abundantly by cardiac mitochondria. Thus an efficient means for its venting is required to support metabolism. Carbonic anhydrase (CA) enzymes, expressed at various sites in ventricular myocytes, may affect mitochondrial CO2 clearance by catalyzing CO2 hydration (to H(+) and HCO3(-)), thereby changing the gradient for CO2 venting. Using fluorescent dyes to measure changes in pH arising from the intracellular hydration of extracellularly supplied CO2, overall CA activity in the cytoplasm of isolated ventricular myocytes was found to be modest (2.7-fold above spontaneous kinetics). Experiments on ventricular mitochondria demonstrated negligible intramitochondrial CA activity. CA activity was also investigated in intact hearts by (13)C magnetic resonance spectroscopy from the rate of H(13)CO3(-) production from (13)CO2 released specifically from mitochondria by pyruvate dehydrogenase-mediated metabolism of hyperpolarized [1-(13)C]pyruvate. CA activity measured upon [1-(13)C]pyruvate infusion was fourfold higher than the cytoplasm-averaged value. A fluorescent CA ligand colocalized with a mitochondrial marker, indicating that mitochondria are near a CA-rich domain. Based on immunoreactivity, this domain comprises the nominally cytoplasmic CA isoform CAII and sarcoplasmic reticulum-associated CAXIV. Inhibition of extramitochondrial CA activity acidified the matrix (as determined by fluorescence measurements in permeabilized myocytes and isolated mitochondria), impaired cardiac energetics (indexed by the phosphocreatine-to-ATP ratio measured by (31)P magnetic resonance spectroscopy of perfused hearts), and reduced contractility (as measured from the pressure developed in perfused hearts). These data provide evidence for a functional domain of high CA activity around mitochondria to support CO2 venting, particularly during elevated and fluctuating respiratory activity. Aberrant distribution of CA activity therefore may reduce the heart's energetic

  14. Molecular and biochemical characterization of carbonic anhydrases of Paracoccidioides.

    Science.gov (United States)

    Tomazett, Mariana Vieira; Zanoelo, Fabiana Fonseca; Bailão, Elisa Flávia Cardoso; Bailão, Alexandre Melo; Borges, Clayton Luiz; Soares, Célia Maria de Almeida

    2016-01-01

    Carbonic anhydrases (CA) belong to the family of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate. In the present work, we characterized the cDNAs of four Paracoccidioides CAs (CA1, CA2, CA3, and CA4). In the presence of CO2, there was not a significant increase in fungal ca1, ca2 and ca4 gene expression. The ca1 transcript was induced during the mycelium-to-yeast transition, while ca2 and ca4 gene expression was much higher in yeast cells, when compared to mycelium and mycelium-to-yeast transition. The ca1 transcript was induced in yeast cells recovered directly from liver and spleen of infected mice, while transcripts for ca2 and ca4 were down-regulated. Recombinant CA1 (rCA1) and CA4 (rCA4), with 33 kDa and 32 kDa respectively, were obtained from bacteria. The enzymes rCA1 (β-class) and rCA4 (α-class) were characterized regarding pH, temperature, ions and amino acids addition influence. Both enzymes were stable at pHs 7.5-8.5 and temperatures of 30-35 °C. The enzymes were dramatically inhibited by Hg+2 and activated by Zn+2, while only rCA4 was stimulated by Fe2+. Among the amino acids tested (all in L configuration), arginine, lysine, tryptophan and histidine enhanced residual activity of rCA1 and rCA4.

  15. Molecular and biochemical characterization of carbonic anhydrases of Paracoccidioides

    Directory of Open Access Journals (Sweden)

    Mariana Vieira Tomazett

    Full Text Available Abstract Carbonic anhydrases (CA belong to the family of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate. In the present work, we characterized the cDNAs of four Paracoccidioides CAs (CA1, CA2, CA3, and CA4. In the presence of CO2, there was not a significant increase in fungal ca1, ca2 and ca4 gene expression. The ca1 transcript was induced during the mycelium-to-yeast transition, while ca2 and ca4 gene expression was much higher in yeast cells, when compared to mycelium and mycelium-to-yeast transition. The ca1 transcript was induced in yeast cells recovered directly from liver and spleen of infected mice, while transcripts for ca2 and ca4 were down-regulated. Recombinant CA1 (rCA1 and CA4 (rCA4, with 33 kDa and 32 kDa respectively, were obtained from bacteria. The enzymes rCA1 (β-class and rCA4 (α-class were characterized regarding pH, temperature, ions and amino acids addition influence. Both enzymes were stable at pHs 7.5-8.5 and temperatures of 30-35 °C. The enzymes were dramatically inhibited by Hg+2 and activated by Zn+2, while only rCA4 was stimulated by Fe2+. Among the amino acids tested (all in L configuration, arginine, lysine, tryptophan and histidine enhanced residual activity of rCA1 and rCA4.

  16. The Protease Inhibitor CI2c Gene Induced by Bird Cherry-Oat Aphid in Barley Inhibits Green Peach Aphid Fecundity in Transgenic Arabidopsis.

    Science.gov (United States)

    Losvik, Aleksandra; Beste, Lisa; Mehrabi, Sara; Jonsson, Lisbeth

    2017-06-20

    Aphids are phloem feeders that cause large damage globally as pest insects. They induce a variety of responses in the host plant, but not much is known about which responses are promoting or inhibiting aphid performance. Here, we investigated whether one of the responses induced in barley by the cereal aphid, bird cherry-oat aphid ( Rhopalosiphum padi L.) affects aphid performance in the model plant Arabidopsis thaliana L. A barley cDNA encoding the protease inhibitor CI2c was expressed in A. thaliana and aphid performance was studied using the generalist green peach aphid ( Myzus persicae Sulzer). There were no consistent effects on aphid settling or preference or on parameters of life span and long-term fecundity. However, short-term tests with apterous adult aphids showed lower fecundity on three of the transgenic lines, as compared to on control plants. This effect was transient, observed on days 5 to 7, but not later. The results suggest that the protease inhibitor is taken up from the tissue during probing and weakly inhibits fecundity by an unknown mechanism. The study shows that a protease inhibitor induced in barley by an essentially monocot specialist aphid can inhibit a generalist aphid in transgenic Arabidopsis.

  17. The Protease Inhibitor CI2c Gene Induced by Bird Cherry-Oat Aphid in Barley Inhibits Green Peach Aphid Fecundity in Transgenic Arabidopsis

    Directory of Open Access Journals (Sweden)

    Aleksandra Losvik

    2017-06-01

    Full Text Available Aphids are phloem feeders that cause large damage globally as pest insects. They induce a variety of responses in the host plant, but not much is known about which responses are promoting or inhibiting aphid performance. Here, we investigated whether one of the responses induced in barley by the cereal aphid, bird cherry-oat aphid (Rhopalosiphum padi L. affects aphid performance in the model plant Arabidopsis thaliana L. A barley cDNA encoding the protease inhibitor CI2c was expressed in A. thaliana and aphid performance was studied using the generalist green peach aphid (Myzus persicae Sulzer. There were no consistent effects on aphid settling or preference or on parameters of life span and long-term fecundity. However, short-term tests with apterous adult aphids showed lower fecundity on three of the transgenic lines, as compared to on control plants. This effect was transient, observed on days 5 to 7, but not later. The results suggest that the protease inhibitor is taken up from the tissue during probing and weakly inhibits fecundity by an unknown mechanism. The study shows that a protease inhibitor induced in barley by an essentially monocot specialist aphid can inhibit a generalist aphid in transgenic Arabidopsis.

  18. Insulin-stimulated lipid accumulation is inhibited by ROS-scavenging chemicals, but not by the Drp1 inhibitor Mdivi-1.

    Science.gov (United States)

    Kim, Jung-Hak; Park, Sun-Ji; Kim, Bokyung; Choe, Young-Geun; Lee, Dong-Seok

    2017-01-01

    Adipocyte differentiation is regulated by intracellular reactive oxygen species (ROS) generation and mitochondrial fission and fusion processes. However, the correlation between intracellular ROS generation and mitochondrial remodeling during adipocyte differentiation is still unknown. Here, we investigated the effect on adipocyte differentiation of 3T3-L1 cells of intracellular ROS inhibition using N-acetyl cysteine (Nac) and Mito-TEMPO and of mitochondrial fission inhibition using Mdivi-1. Differentiated 3T3-L1 adipocytes displayed an increase in mitochondrial fission, ROS generation, and the expression of adipogenic and mitochondrial dynamics-related proteins. ROS scavenger (Nac or Mito-TEMPO) treatment inhibited ROS production, lipid accumulation, the expression of adipogenic and mitochondrial dynamics-related proteins, and mitochondrial fission during adipogenesis of 3T3-L1 cells. On the other hand, treatment with the mitochondrial fission inhibitor Mdivi-1 inhibited mitochondrial fission but did not inhibit ROS production, lipid accumulation, or the expression of adipogenic and mitochondrial dynamics-related proteins, with the exception of phosphorylated Drp1 (Ser616), in differentiated 3T3-L1 adipocytes. The inhibition of mitochondrial fission did not affect adipocyte differentiation, while intracellular ROS production decreased in parallel with inhibition of adipocyte differentiation. Therefore, our results indicated that ROS are an essential regulator of adipocyte differentiation in 3T3-L1 cells.

  19. Inhibition of Plasminogen Activator Inhibitor-1 Attenuates Transforming Growth Factor-β-Dependent Epithelial Mesenchymal Transition and Differentiation of Fibroblasts to Myofibroblasts.

    Directory of Open Access Journals (Sweden)

    Keitaro Omori

    Full Text Available Transforming growth factor-β (TGF-β is central during the pathogenesis of pulmonary fibrosis, in which the plasminogen activator inhibitor-1 (PAI-1 also has an established role. TGF-β is also known to be the strongest inducer of PAI-1. To investigate the link between PAI-1 and TGF-β in fibrotic processes, we evaluated the effect of SK-216, a PAI-1-specific inhibitor, in TGF-β-dependent epithelial-mesenchymal transition (EMT and fibroblast to myofibroblast differentiation. In human alveolar epithelial A549 cells, treatment with TGF-β induced EMT, whereas co-treatment with SK-216 attenuated the occurrence of EMT. The inhibition of TGF-β-induced EMT by SK-216 was also confirmed in the experiment using murine epithelial LA-4 cells. Blocking EMT by SK-216 inhibited TGF-β-induced endogenous production of PAI-1 and TGF-β in A549 cells as well. These effects of SK-216 were not likely mediated by suppressing either Smad or ERK pathways. Using human lung fibroblast MRC-5 cells, we demonstrated that SK-216 inhibited TGF-β-dependent differentiation of fibroblasts to myofibroblasts. We also observed this inhibition by SK-216 in human primary lung fibroblasts. Following these in vitro results, we tested oral administration of SK-216 into mice injected intratracheally with bleomycin. We found that SK-216 reduced the degree of bleomycin-induced pulmonary fibrosis in mice. Although the precise mechanisms underlying the link between TGF-β and PAI-1 regarding fibrotic process were not determined, PAI-1 seems to act as a potent downstream effector on the pro-fibrotic property of TGF-β. In addition, inhibition of PAI-1 activity by a PAI-1 inhibitor exerts an antifibrotic effect even in vivo. These data suggest that targeting PAI-1 as a downstream effector of TGF-β could be a promising therapeutic strategy for pulmonary fibrosis.

  20. The Crystal Structure of BRAF in Complex with an Organoruthenium Inhibitor Reveals a Mechanism for Inhibition of an Active Form of BRAF Kinase

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Peng; Streu, Craig; Qin, Jie; Bregman, Howard; Pagano, Nicholas; Meggers, Eric; Marmorstein, Ronen (Wistar); (UPENN)

    2012-06-19

    Substitution mutations in the BRAF serine/threonine kinase are found in a variety of human cancers. Such mutations occur in 70% of human malignant melanomas, and a single hyperactivating V600E mutation is found in the activation segment of the kinase domain and accounts for more than 90% of these mutations. Given this correlation, the molecular mechanism for BRAF regulation as well as oncogenic activation has attracted considerable interest, and activated forms of BRAF, such as BRAF{sup V600E}, have become attractive targets for small molecule inhibition. Here we report on the identification and subsequent optimization of a potent BRAF inhibitor, CS292, based on an organometallic kinase inhibitor scaffold. A cocrystal structure of CS292 in complex with the BRAF kinase domain reveals that CS292 binds to the ATP binding pocket of the kinase and is an ATP competitive inhibitor. The structure of the kinase-inhibitor complex also demonstrates that CS292 binds to BRAF in an active conformation and suggests a mechanism for regulation of BRAF by phosphorylation and BRAF{sup V600E} oncogene-induced activation. The structure of CS292 bound to the active form of the BRAF kinase also provides a novel scaffold for the design of BRAF{sup V600E} oncogene selective BRAF inhibitors for therapeutic application.

  1. Inhibiting properties and adsorption of an amine based fatty acid corrosion inhibitor on carbon steel in aqueous carbon dioxide solutions

    Energy Technology Data Exchange (ETDEWEB)

    Buchweishaija, Joseph

    1997-12-31

    Carbon dioxide corrosion is a major corrosion problem in oil and gas production systems and many organic inhibitors have been tested and used to protect the substrate from corrosion. This thesis studies the mechanism of interaction of the inhibitor molecule with the metallic substrate and how this affects the dissolution rate of the metal. The performance of a commercial amine based fatty acid corrosion inhibitor has been investigated using rotating cylinder electrodes and carbon steel electrodes in CO{sub 2} saturated formation water in the temperature range between 35 to 80{sup o}C. The corrosion process was monitored by electrochemical impedance measurements, and at the end of each experiment full polarization curves were recorded. When the inhibitor was applied on noncorroded electrodes, high inhibitor performance, over 99.7%, was observed independent of temperature. On precorroded electrodes inhibitor performance was found to depend on temperature and time of precorrosion. Above 60{sup o}C, the inhibitor performance decreased with increasing time of precorrosion, presumably because of the formation of a corrosion film of either iron carbonate or a combination of iron carbonate and iron carbide which prevent the inhibitor from reaching the surface. The inhibitor protection efficiency was assumed to be associated with the degree of inhibitor coverage at the material surface, and adsorption isotherms have been calculated in the concentration range between 0.1 ppm and 100 ppm. A Langmuir isotherm was found to give the best fit. The inhibitor performance on a 2 days precorroded rotating electrode was investigated at different solution pH ranging between 4.5 and 6.5 at 35{sup o}C. 130 refs., 80 figs., 22 tabs.

  2. L-Chicoric acid inhibits human immunodeficiency virus type 1 integration in vivo and is a noncompetitive but reversible inhibitor of HIV-1 integrase in vitro

    International Nuclear Information System (INIS)

    Reinke, Ryan A.; Lee, Deborah J.; McDougall, Brenda R.; King, Peter J.; Victoria, Joseph; Mao Yingqun; Lei Xiangyang; Reinecke, Manfred G.; Robinson, W. Edward

    2004-01-01

    The human immunodeficiency virus (HIV) integrase (IN) must covalently join the viral cDNA into a host chromosome for productive HIV infection. L-Chicoric acid (L-CA) enters cells poorly but is a potent inhibitor of IN in vitro. Using quantitative real-time polymerase chain reaction (PCR), L-CA inhibits integration at concentrations from 500 nM to 10 μM but also inhibits entry at concentrations above 1 μM. Using recombinant HIV IN, steady-state kinetic analyses with L-CA were consistent with a noncompetitive or irreversible mechanism of inhibition. IN, in the presence or absence of L-CA, was successively washed. Inhibition of IN diminished, demonstrating that L-CA was reversibly bound to the protein. These data demonstrate that L-CA is a noncompetitive but reversible inhibitor of IN in vitro and of HIV integration in vivo. Thus, L-CA likely interacts with amino acids other than those which bind substrate

  3. Multi-tyrosine kinase inhibitors in preclinical studies for pediatric CNS AT/RT: Evidence for synergy with Topoisomerase-I inhibition

    Directory of Open Access Journals (Sweden)

    Jayanthan Aarthi

    2011-12-01

    Full Text Available Abstract Background Currently, Atypical Teratoid Rhabdoid Tumor (AT/RT constitutes one of the most difficult to treat malignancies in pediatrics. Hence, new knowledge of potential targets for therapeutics and the development of novel treatment approaches are urgently needed. We have evaluated the presence of cytokine pathways and the effects of two clinically available multi-tyrosine kinase inhibitors for cytotoxicity, target modulation and drug combinability against AT/RT cell lines. Results AT/RT cell lines expressed measurable quantities of VEGF, FGF, PDGF and SDF-1, although the absolute amounts varied between the cell lines. The targeted receptor tyrosine kinase inhibitor sorafenib inhibited the key signaling molecule Erk, which was activated following the addition of own conditioned media, suggesting the existence of autocrine/paracrine growth stimulatory pathways. The multi-tyrosine kinase inhibitors sorafenib and sunitinib also showed significant growth inhibition of AT/RT cells and their activity was enhanced by combination with the topoisomerase inhibitor, irinotecan. The loss of cytoplasmic NF-kappa-B in response to irinotecan was diminished by sorafenib, providing evidence for a possible benefit for this drug combination. Conclusions In addition to previously described involvement of insulin like growth factor (IGF family of cytokines, a multitude of other growth factors may contribute to the growth and survival of AT/RT cells. However, consistent with the heterogeneous nature of this tumor, quantitative and qualitative differences may exist among different tumor samples. Multi-tyrosine kinase inhibitors appear to have effective antitumor activity against all cell lines studied. In addition, the target modulation studies and drug combinability data provide the groundwork for additional studies and support the evaluation of these agents in future treatment protocols.

  4. PI-273, a Substrate-Competitive, Specific Small-Molecule Inhibitor of PI4KIIα, Inhibits the Growth of Breast Cancer Cells.

    Science.gov (United States)

    Li, Jiangmei; Gao, Zhen; Zhao, Dan; Zhang, Lunfeng; Qiao, Xinhua; Zhao, Yingying; Ding, Hong; Zhang, Panpan; Lu, Junyan; Liu, Jia; Jiang, Hualiang; Luo, Cheng; Chen, Chang

    2017-11-15

    While phosphatidylinositol 4-kinase (PI4KIIα) has been identified as a potential target for antitumor therapy, the clinical applications of PI4KIIα are limited by a lack of specific inhibitors. Here we report the first small-molecule inhibitor (SMI) of human PI4KIIα. Docking-based and ligand-based virtual screening strategies were first employed to identify promising hits, followed by two rounds of kinase activity inhibition validation. 2-(3-(4-Chlorobenzoyl)thioureido)-4-ethyl-5-methylthiophene-3-carboxamide (PI-273) exhibited the greatest inhibitory effect on PI4KIIα kinase activity (IC 50 = 0.47 μmol/L) and suppressed cell proliferation. Surface plasmon resonance and thermal shift assays indicated that PI-273 interacted directly with PI4KIIα. Kinetic analysis identified PI-273 as a reversible competitive inhibitor with respect to the substrate phosphatidylinositol (PI), which contrasted with most other PI kinase inhibitors that bind the ATP binding site. PI-273 reduced PI4P content, cell viability, and AKT signaling in wild-type MCF-7 cells, but not in PI4KIIα knockout MCF-7 cells, indicating that PI-273 is highly selective for PI4KIIα. Mutant analysis revealed a role of palmitoylation insertion in the selectivity of PI-273 for PI4KIIα. In addition, PI-273 treatment retarded cell proliferation by blocking cells in G 2 -M, inducing cell apoptosis and suppressing colony-forming ability. Importantly, PI-273 significantly inhibited MCF-7 cell-induced breast tumor growth without toxicity. PI-273 is the first substrate-competitive, subtype-specific inhibitor of PI4KIIα, the use of which will facilitate evaluations of PI4KIIα as a cancer therapeutic target. Cancer Res; 77(22); 6253-66. ©2017 AACR . ©2017 American Association for Cancer Research.

  5. Allosteric inhibition enhances the efficacy of ABL kinase inhibitors to target unmutated BCR-ABL and BCR-ABL-T315I

    Directory of Open Access Journals (Sweden)

    Mian Afsar

    2012-09-01

    Full Text Available Abstract Background Chronic myelogenous leukemia (CML and Philadelphia chromosome-positive (Ph+ acute lymphatic leukemia (Ph + ALL are caused by the t(9;22, which fuses BCR to ABL resulting in deregulated ABL-tyrosine kinase activity. The constitutively activated BCR/ABL-kinase “escapes” the auto-inhibition mechanisms of c-ABL, such as allosteric inhibition. The ABL-kinase inhibitors (AKIs Imatinib, Nilotinib or Dasatinib, which target the ATP-binding site, are effective in Ph + leukemia. Another molecular therapy approach targeting BCR/ABL restores allosteric inhibition. Given the fact that all AKIs fail to inhibit BCR/ABL harboring the ‘gatekeeper’ mutation T315I, we investigated the effects of AKIs in combination with the allosteric inhibitor GNF2 in Ph + leukemia. Methods The efficacy of this approach on the leukemogenic potential of BCR/ABL was studied in Ba/F3 cells, primary murine bone marrow cells, and untransformed Rat-1 fibroblasts expressing BCR/ABL or BCR/ABL-T315I as well as in patient-derived long-term cultures (PDLTC from Ph + ALL-patients. Results Here, we show that GNF-2 increased the effects of AKIs on unmutated BCR/ABL. Interestingly, the combination of Dasatinib and GNF-2 overcame resistance of BCR/ABL-T315I in all models used in a synergistic manner. Conclusions Our observations establish a new approach for the molecular targeting of BCR/ABL and its resistant mutants using a combination of AKIs and allosteric inhibitors.

  6. Similarly potent inhibition of adenylyl cyclase by P-site inhibitors in hearts from wild type and AC5 knockout mice.

    Directory of Open Access Journals (Sweden)

    Joerg H Braeunig

    Full Text Available Adenylyl cyclase type 5 (AC5 was described as major cardiac AC isoform. The knockout of AC5 (AC5KO exerted cardioprotective effects in heart failure. Our study explored the impact of AC5KO on mouse heart AC activities and evaluated putative AC5-selective inhibitors. In cardiac membranes from AC5KO mice, basal AC activity was decreased, while AC stimulation was intact. The putative AC5-selective P-site inhibitors SQ22,536 [9-(tetra-hydro-2-furanyl-9H-purin-6-amine], vidarabine (9-β-D-arabinosyladenine and NKY80 [2-amino-7-(2-furanyl-7,8-dihydro-5(6H-quinazolinone] inhibited recombinant AC5 more potently than AC2 and AC1, but selectivity was only modest (∼4-40-fold. These compounds inhibited cardiac AC from WT and AC5KO mice with similar potencies. In conclusion, AC regulation in AC5KO hearts was unimpaired, questioning the supposed dominant role of AC5 in the heart. Moreover, the AC inhibitors SQ22,536, NKY80 and vidarabine lack adequate selectivity for AC5 and, therefore, do not present suitable tools to study AC5-specific functions.

  7. BZML, a novel colchicine binding site inhibitor, overcomes multidrug resistance in A549/Taxol cells by inhibiting P-gp function and inducing mitotic catastrophe.

    Science.gov (United States)

    Bai, Zhaoshi; Gao, Meiqi; Zhang, Huijuan; Guan, Qi; Xu, Jingwen; Li, Yao; Qi, Huan; Li, Zhengqiang; Zuo, Daiying; Zhang, Weige; Wu, Yingliang

    2017-08-28

    Multidrug resistance (MDR) interferes with the efficiency of chemotherapy. Therefore, developing novel anti-cancer agents that can overcome MDR is necessary. Here, we screened a series of colchicine binding site inhibitors (CBSIs) and found that 5-(3, 4, 5-trimethoxybenzoyl)-4-methyl-2-(p-tolyl) imidazol (BZML) displayed potent cytotoxic activity against both A549 and A549/Taxol cells. We further explored the underlying mechanisms and found that BZML caused mitosis phase arrest by inhibiting tubulin polymerization in A549 and A549/Taxol cells. Importantly, BZML was a poor substrate for P-glycoprotein (P-gp) and inhibited P-gp function by decreasing P-gp expression at the protein and mRNA levels. Cell morphology changes and the expression of cycle- or apoptosis-related proteins indicated that BZML mainly drove A549/Taxol cells to die by mitotic catastrophe (MC), a p53-independent apoptotic-like cell death, whereas induced A549 cells to die by apoptosis. Taken together, our data suggest that BZML is a novel colchicine binding site inhibitor and overcomes MDR in A549/Taxol cells by inhibiting P-gp function and inducing MC. Our study also offers a new strategy to solve the problem of apoptosis-resistance. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. The growth hormone dependent serine protease inhibitor, Spi 2.1 inhibits the des (1-3) insulin-like growth factor-I generating protease.

    Science.gov (United States)

    Maake, C; Yamamoto, H; Murphy, L J

    1997-12-01

    The conversion of insulin-like growth factor-I (IGF-I) to the biologically more active des (1-3) IGF-I variant is catalyzed by a ubiquitous protease. This proteolytic activity is inhibited by human alpha1-antitrypsin and soy-bean trypsin inhibitor and is up-regulated in serum and tissue extracts of hypophysectomized rats. These observations lead us to investigate whether the growth hormone regulated, serine protease inhibitor, Spi 2.1 was able to inhibit the des (1-3) IGF-I generating protease. Dihydrofolate reductase deficient Chinese hamster ovary (CHO(dhfr-ve)) cells were transfected with a rat Spi 2.1 expression vector containing the dhfr and neomycin resistance gene. Stable transfectants were selected using G418 and amplified using methotrexate. Conditioned medium from Spi 2.1 transfected CHO cells potently inhibited proteolytic activity directed against a synthetic hexa-peptide with a sequence identical to the N-terminal of IGF-I. In contrast conditioned medium from wild-type CHO cells had little effect. Based upon these observations we suggest that our previous finding of enhanced des (1-3) IGF-I generating protease activity in growth hormone deficient rats may be, at least partly explained by reduced levels of Spi 2.1. Furthermore, we propose that the regulation of the generation of des (1-3) IGF-I may be an additional potential site of growth hormone regulation of IGF-I action.

  9. Inhibition of ErbB2 by receptor tyrosine kinase inhibitors causes myofibrillar structural damage without cell death in adult rat cardiomyocytes

    International Nuclear Information System (INIS)

    Pentassuglia, Laura; Graf, Michael; Lane, Heidi; Kuramochi, Yukio; Cote, Gregory; Timolati, Francesco; Sawyer, Douglas B.; Zuppinger, Christian; Suter, Thomas M.

    2009-01-01

    Inhibition of ErbB2 (HER2) with monoclonal antibodies, an effective therapy in some forms of breast cancer, is associated with cardiotoxicity, the pathophysiology of which is poorly understood. Recent data suggest, that dual inhibition of ErbB1 (EGFR) and ErbB2 signaling is more efficient in cancer therapy, however, cardiac safety of this therapeutic approach is unknown. We therefore tested an ErbB1-(CGP059326) and an ErbB1/ErbB2-(PKI166) tyrosine kinase inhibitor in an in-vitro system of adult rat ventricular cardiomyocytes and assessed their effects on 1. cell viability, 2. myofibrillar structure, 3. contractile function, and 4. MAPK- and Akt-signaling alone or in combination with Doxorubicin. Neither CGP nor PKI induced cardiomyocyte necrosis or apoptosis. PKI but not CGP caused myofibrillar structural damage that was additive to that induced by Doxorubicin at clinically relevant doses. These changes were associated with an inhibition of excitation-contraction coupling. PKI but not CGP decreased p-Erk1/2, suggesting a role for this MAP-kinase signaling pathway in the maintenance of myofibrils. These data indicate that the ErbB2 signaling pathway is critical for the maintenance of myofibrillar structure and function. Clinical studies using ErbB2-targeted inhibitors for the treatment of cancer should be designed to include careful monitoring for cardiac dysfunction.

  10. NHE1 inhibition by amiloride- and benzoylguanidine-type compounds. Inhibitor binding loci deduced from chimeras of NHE1 homologues with endogenous differences in inhibitor sensitivity

    DEFF Research Database (Denmark)

    Pedersen, Stine F; King, Scott A; Nygaard, Eva B

    2007-01-01

    NHE1). Although highly homologous to the amiloride- and HOE-sensitive human NHE1 (hNHE1), AtNHE1 is insensitive to HOE-type and PaNHE1 to both amiloride- and HOE-type compounds. Here we generated chimeras to "knock in" amiloride and HOE sensitivity to PaNHE1, and we thereby identified several NHE1...... regions involved in inhibitor interaction. The markedly different inhibitor sensitivities of hNHE1, AtNHE1, and PaNHE1 could not be accounted for by differences in transmembrane (TM) region 9. Replacing TM10 through the C-terminal tail of PaNHE1 with the corresponding region of AtNHE1 partially restored...... sensitivity to amiloride and the related compound 5'-(N-ethyl-N-isopropyl)amiloride (EIPA) but not to HOE694. This effect was not due to the tail region, but it was dependent on TM10-11, because replacing only this region with that of AtNHE1 also partially restored amiloride and EIPA but not HOE sensitivity...

  11. Two-pronged attack: dual inhibition of Plasmodium falciparum M1 and M17 metalloaminopeptidases by a novel series of hydroxamic acid-based inhibitors.

    Science.gov (United States)

    Mistry, Shailesh N; Drinkwater, Nyssa; Ruggeri, Chiara; Sivaraman, Komagal Kannan; Loganathan, Sasdekumar; Fletcher, Sabine; Drag, Marcin; Paiardini, Alessandro; Avery, Vicky M; Scammells, Peter J; McGowan, Sheena

    2014-11-13

    Plasmodium parasites, the causative agents of malaria, have developed resistance to most of our current antimalarial therapies, including artemisinin combination therapies which are widely described as our last line of defense. Antimalarial agents with a novel mode of action are urgently required. Two Plasmodium falciparum aminopeptidases, PfA-M1 and PfA-M17, play crucial roles in the erythrocytic stage of infection and have been validated as potential antimalarial targets. Using compound-bound crystal structures of both enzymes, we have used a structure-guided approach to develop a novel series of inhibitors capable of potent inhibition of both PfA-M1 and PfA-M17 activity and parasite growth in culture. Herein we describe the design, synthesis, and evaluation of a series of hydroxamic acid-based inhibitors and demonstrate the compounds to be exciting new leads for the development of novel antimalarial therapeutics.

  12. A small molecule inhibitor of dengue virus type 2 protease inhibits the replication of all four dengue virus serotypes in cell culture.

    Science.gov (United States)

    Raut, Rajendra; Beesetti, Hemalatha; Tyagi, Poornima; Khanna, Ira; Jain, Swatantra K; Jeankumar, Variam U; Yogeeswari, Perumal; Sriram, Dharmarajan; Swaminathan, Sathyamangalam

    2015-02-08

    Dengue has emerged as the most significant of arboviral diseases in the 21st century. It is endemic to >100 tropical and sub-tropical countries around the world placing an estimated 3.6 billion people at risk. It is caused by four genetically similar but antigenically distinct, serotypes of dengue viruses. There is neither a vaccine to prevent nor a drug to treat dengue infections, at the present time. The major objective of this work was to explore the possibility of identifying a small molecule inhibitor of the dengue virus protease and assessing its ability to suppress viral replication in cultured cells. We cloned, expressed and purified recombinant dengue virus type 2 protease. Using an optimized and validated fluorogenic peptide substrate cleavage assay to monitor the activity of this cloned dengue protease we randomly screened ~1000 small molecules from an 'in-house' library to identify potential dengue protease inhibitors. A benzimidazole derivative, named MB21, was found to be the most potent in inhibiting the cloned protease (IC₅₀ = 5.95 μM). In silico docking analysis indicated that MB21 binds to the protease in the vicinity of the active site. Analysis of kinetic parameters of the enzyme reaction suggested that MB21 presumably functions as a mixed type inhibitor. Significantly, this molecule identified as an inhibitor of dengue type 2 protease was also effective in inhibiting each one of the four serotypes of dengue viruses in infected cells in culture, based on analysis of viral antigen synthesis and infectious virus production. Interestingly, MB21 did not manifest any discernible cytotoxicity. This work strengthens the notion that a single drug molecule can be effective against all four dengue virus serotypes. The molecule MB21 could be a potential candidate for 'hit-to-lead' optimization, and may pave the way towards developing a pan-dengue virus antiviral drug.

  13. Inhibition of Heat-Stable Toxin-Induced Intestinal Salt and Water Secretion by a Novel Class of Guanylyl Cyclase C Inhibitors.

    Science.gov (United States)

    Bijvelds, Marcel J C; Loos, Michaela; Bronsveld, Inez; Hellemans, Ann; Bongartz, Jean-Pierre; Ver Donck, Luc; Cox, Eric; de Jonge, Hugo R; Schuurkes, Jan A J; De Maeyer, Joris H

    2015-12-01

    Many enterotoxigenic Escherichia coli strains produce the heat-stable toxin, STa, which, by activation of the intestinal receptor-enzyme guanylyl cyclase (GC) C, triggers an acute, watery diarrhea. We set out to identify GCC inhibitors that may be of benefit for the treatment of infectious diarrheal disease. Compounds that inhibit STa-induced cyclic guanosine 3',5'-monophosphate (cGMP) production were selected by performing cyclase assays on cells and membranes containing GCC, or the related GCA. The effect of leads on STa/GCC-dependent activation of the cystic fibrosis transmembrane conductance regulator anion channel was investigated in T84 cells, and in porcine and human intestinal tissue. Their effect on STa-provoked fluid transport was assessed in ligated intestinal loops in piglets. Four N-2-(propylamino)-6-phenylpyrimidin-4-one-substituted piperidines were shown to inhibit GCC-mediated cellular cGMP production. The half maximal inhibitory concentrations were ≤ 5 × 10(-7) mol/L, whereas they were >10 times higher for GCA. In T84 monolayers, these leads blocked STa/GCC-dependent, but not forskolin/adenylyl cyclase-dependent, cystic fibrosis transmembrane conductance regulator activity. GCC inhibition reduced STa-provoked anion secretion in pig jejunal tissue, and fluid retention and cGMP levels in STa-exposed loops. These GCC inhibitors blocked STa-provoked anion secretion in rectal biopsy specimens. We have identified a novel class of GCC inhibitors that may form the basis for development of future therapeutics for (infectious) diarrheal disease. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  14. Soluble epoxide hydrolase inhibitor AUDA decreases bleomycin-induced pulmonary toxicity in mice by inhibiting the p38/Smad3 pathways.

    Science.gov (United States)

    Dong, Xin-Wei; Jia, Yong-Liang; Ge, Ling-Tian; Jiang, Bo; Jiang, Jun-Xia; Shen, Jian; Jin, Ya-Chao; Guan, Yan; Sun, Yun; Xie, Qiang-Min

    2017-08-15

    Bleomycin (BLM) has potent tumor cell-killing properties that have given it an important place in cancer chemotherapy, but pulmonary toxicity is its major adverse effect. Soluble epoxide hydrolase (sEH) inhibitors have been reported to have protective effects in fibrosis models, but the effects of AUDA, an sEH inhibitor of BLM-induced pulmonary toxicity and fibrosis, remain to be researched. In this study, we assessed the effects of AUDA on the BLM-induced pulmonary fibrosis in a mouse model, and transforming growth factor (TGF)-β 1 -induced epithelial proliferation and epithelial-mesenchymal transition (EMT) in vitro by monitoring changes in pulmonary function, inflammatory response, fibrotic remodeling, and signaling pathways. AUDA was administered by intragastric administration (i.g) daily for three weeks, starting at seven days after intratracheal instillation of BLM. All examinations were performed 24h after the last i.g. In vivo, AUDA significantly improved BLM-induced decline in lung function and body weight, and inhibited inflammatory cell accumulation and the mRNA and protein expression of interleukin (IL)-1β, TGF-β 1 , and matrix metalloproteinase 9 (MMP-9) in lung tissue. Moreover, AUDA attenuated BLM-induced deposition of collagen fibers, destruction of alveolar structures, and pulmonary parenchyma. Additionally, AUDA regulated the expression of α-smooth muscle actin (α-SMA) and E-cadherin by inhibiting the Smad3/p38 signaling pathway. In vitro, AUDA significantly inhibited TGF-β 1 -induced epithelial cells and fibroblast proliferation, reduced sEH expression and α-SMA expression, and increased epoxyeicosatrienoic acid (EET) levels and E-cadherin expression in epithelial cells. These effects were blocked by AUDA by downregulating the Smad3 and p38 signaling pathways. Taken together, these data indicate that treatment with sEH inhibitors may improve BLM-induced pulmonary toxicity. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic proteases: Could the aspartic protease inhibition have evolved from a canonical serine protease-binding loop?

    Science.gov (United States)

    Guerra, Yasel; Valiente, Pedro A; Pons, Tirso; Berry, Colin; Rudiño-Piñera, Enrique

    2016-08-01

    Bi-functional inhibitors from the Kunitz-type soybean trypsin inhibitor (STI) family are glycosylated proteins able to inhibit serine and aspartic proteases. Here we report six crystal structures of the wild-type and a non-glycosylated mutant of the bifunctional inhibitor E3Ad obtained at different pH values and space groups. The crystal structures show that E3Ad adopts the typical β-trefoil fold of the STI family exhibiting some conformational changes due to pH variations and crystal packing. Despite the high sequence identity with a recently reported potato cathepsin D inhibitor (PDI), three-dimensional structures obtained in this work show a significant conformational change in the protease-binding loop proposed for aspartic protease inhibition. The E3Ad binding loop for serine protease inhibition is also proposed, based on structural similarity with a novel non-canonical conformation described for the double-headed inhibitor API-A from the Kunitz-type STI family. In addition, structural and sequence analyses suggest that bifunctional inhibitors of serine and aspartic proteases from the Kunitz-type STI family are more similar to double-headed inhibitor API-A than other inhibitors with a canonical protease-binding loop. Copyright © 2016. Published by Elsevier Inc.

  16. The broad-spectrum metalloproteinase inhibitor BB-94 inhibits growth, HER3 and Erk activation in fulvestrant-resistant breast cancer cell lines

    DEFF Research Database (Denmark)

    Kirkegaard, Tove; Yde, Christina Westmose; Kveiborg, Marie

    2014-01-01

    and consequently increased cell growth. In this study, we investigated the importance of HER receptors, in particular HER3, and HER ligand shedding for growth and signaling in human MCF-7 breast cancer cells and MCF-7-derived sublines resistant to the antiestrogen fulvestrant. The HER3/HER4 ligand heregulin 1β...... induced phosphorylation of HER3, Akt and Erk, and partly rescued fulvestrant-inhibited growth of MCF-7 cells. HER3 ligands were found to be produced and shed from the fulvestrant-resistant cells as conditioned medium from fulvestrant-resistant MCF-7 cells induced phosphorylation of HER3 and Akt in MCF-7......-resistant cells, was able to inhibit growth and activation of HER3 and Erk in resistant cells. Compared to MCF-7, fulvestrant-resistant cells have increased HER3 phosphorylation, but knockdown of HER3 had no inhibitory effect on resistant cell growth. The EGFR inhibitor gefitinib exhibited only a minor growth...

  17. Verrucarin A, a protein synthesis inhibitor, induces growth inhibition and apoptosis in breast cancer cell lines MDA-MB-231 and T47D.

    Science.gov (United States)

    Palanivel, Kandasamy; Kanimozhi, Veerasamy; Kadalmani, Balamuthu; Akbarsha, Mohammad Abdulkader

    2013-09-01

    Verrucarin A (VA), a protein synthesis inhibitor, derived from the pathogen fungus Myrothecium verrucaria, inhibits growth of leukemia cell lines and activates caspases and apoptosis and inflammatory signaling in macrophages. We have investigated VA-induced growth inhibition in breast cancer cells MDA-MB-231 and T47D and, particularly, the mechanism of VA-induced apoptosis. VA treatment brought about apoptotic cell death in a dose- and time-dependent manner which was associated with chromatin condensation, cell shrinkage, nuclear fragmentation and intracellular ROS production. Mitochondrial membrane depolarization, activation of caspase-3, down-regulation of Bcl-2 expression and up-regulation of Bax and p53 expression were observed. VA thus affects the viability of both the breast cancer cells by triggering ROS-mediated intrinsic mechanism of apoptosis.

  18. INHIBITION OF THE GROWTH OF TOLERANT YEAST Saccharomyces cerevisiae STRAIN I136 BY A MIXTURE OF SYNTHETIC INHIBITORS

    Directory of Open Access Journals (Sweden)

    Eny Ida Riyanti

    2017-09-01

    Full Text Available Biomass from lignocellulosic wastes is a potential source for biobased products.  However, one of the constraints in utilization of biomass hydrolysate is the presence of inhibitors. Therefore, the use of inhibitor-tolerant microorganisms in the fermentation is required. The study aimed to investigate the effect of a mixture of inhibitors on the growth of Saccharomyces cerevisiae strain I136 grown in medium containing synthetic inhibitors (acetic acid, formic acid, furfural, 5-hydroxymethyl furfural/5-HMF, and levulinic acid in four different concentrations with a mixture of carbon sources, glucose  (50 g.l-1 and xylose (50 g.l-1 at 30oC. The parameters related to growth and fermentation products were observed. Results showed that the strain was able to grow in media containing natural inhibitors (BSL medium with µmax of 0.020/h. Higher level of synthetic inhibitors prolonged the lag phase, decreased the cell biomass and ethanol production, and specific growth rate. The strain could detoxify furfural and 5-HMF and produced the highest ethanol (Y(p/s of 0.32 g.g-1 when grown in BSL. Glucose was utilized as its level decreased in a result of increase in cell biomass, in contrast to xylose which was not consumed. The highest cell biomass was produced in YNB with Y (x/s value of 0.25 g.g-1. The strain produced acetic acid as a dominant side product and could convert furfural into a less toxic compound, hydroxyl furfural. This robust tolerant strain provides basic information on resistance mechanism and would be useful for bio-based cell factory using lignocellulosic materials. 

  19. Role of Protein Phosphatase 1 and Inhibitor of Protein Phosphatase-1 in Nitric Oxide-Dependent Inhibition of the DNA Damage Response in Pancreatic β-Cells.

    Science.gov (United States)

    Oleson, Bryndon J; Naatz, Aaron; Proudfoot, Sarah C; Yeo, Chay Teng; Corbett, John A

    2018-02-14

    Nitric oxide is produced at micromolar levels by pancreatic β-cells during exposure to proinflammatory cytokines. While classically viewed as damaging, nitric oxide also activates pathways that promote β-cell survival. We have shown that nitric oxide, in a cell type selective manner, inhibits the DNA damage response (DDR) and, in doing so, protects β-cells from DNA damage-induced apoptosis. This study explores potential mechanisms by which nitric oxide inhibits DDR signaling. We show that inhibition of DDR signaling (measured by γH2AX formation and the phosphorylation of KAP1) is selective for nitric oxide, as other forms of reactive oxygen/nitrogen species do not impair DDR signaling. The kinetics and broad range of DDR substrates that are inhibited suggest that protein phosphatase activation may be one mechanism by which nitric oxide attenuates DDR signaling in β-cells. While protein phosphatase 1 (PP1) is a primary regulator of DDR signaling and an inhibitor of protein phosphatase-1 (IPP-1) is selectively expressed only in β-cells, disruption of either IPP-1 or PP1 does not modify the inhibitory actions of nitric oxide on DDR signaling in β-cells. These findings support a PP1-independent mechanism by which nitric oxide selectively impairs DDR signaling and protects β-cells from DNA damage-induced apoptosis. © 2018 by the American Diabetes Association.

  20. Inhibition of human platelet aggregation by dihydropyrano- and dihydrofuranocoumarins, a new class of cAMP-phosphodiesterase inhibitors

    DEFF Research Database (Denmark)

    Thastrup, Ole; Knudsen, J B; Lemmich, J

    1985-01-01

    Certain esters of dihydropyranocoumarin and dihydrofuranocoumarin alcohols have previously been shown to inhibit the cAMP-phosphodiesterase from bovine heart. We now report that these naturally occurring coumarins inhibit the high affinity (Km = 1.1 microM) cAMP-phosphodiesterase from human...... platelets with activities that closely correlate with those obtained using phosphodiesterase from bovine heart tissue. Additionally the coumarins inhibit the aggregation of human platelets induced with ADP, adrenaline and collagen with activities comparable to those of dipyridamole. A lack of significant...

  1. Selective inhibition of ADAM12 catalytic activity through engineering of tissue inhibitor of metalloproteinase 2 (TIMP-2)

    DEFF Research Database (Denmark)

    Kveiborg, Marie; Jacobsen, Jonas; Lee, Meng-Huee

    2010-01-01

    activity of TIMPs against the transmembrane ADAM12-L (full-length ADAM12), verifying the distinctive inhibitory abilities of N-TIMP-2 and engineered N-TIMP-2 mutants in a cellular environment. Taken together, our findings support the idea that a distinctive ADAM12 inhibitor with future therapeutic...

  2. Inhibition of Post-Transcriptional RNA Processing by CDK Inhibitors and Its Implication in Anti-Viral Therapy

    Czech Academy of Sciences Publication Activity Database

    Holčáková, J.; Müller, P.; Tomasec, P.; Hrstka, R.; Nekulová, M.; Kryštof, Vladimír; Strnad, Miroslav; Wilkinson, G. W. G.; Vojtěšek, B.

    2014-01-01

    Roč. 9, č. 2 (2014) E-ISSN 1932-6203 R&D Projects: GA ČR GBP206/12/G151 Institutional support: RVO:61389030 Keywords : IMMUNODEFICIENCY-VIRUS TYPE-1 * DEPENDENT KINASE INHIBITORS * LARGE T-ANTIGEN Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.234, year: 2014

  3. A new scaffold for inhibition of cysteine proteases: Structural and functional characterization of Kunitz inhibitors from potato

    Czech Academy of Sciences Publication Activity Database

    Mishra, Manasi; Brynda, Jiří; Mareš, Michael

    2015-01-01

    Roč. 24, Suppl 1 (2015), s. 13 ISSN 0961-8368. [Annual Symposium of the Protein Society /29./. 22.07.2015-25.07.2015, Barcelona] Institutional support: RVO:61388963 Keywords : cysteine proteases * protease inhibitors Subject RIV: CE - Biochemistry

  4. Inhibition of canonical WNT signaling pathway by β-catenin/CBP inhibitor ICG-001 ameliorates liver fibrosis in vivo through suppression of stromal CXCL12.

    Science.gov (United States)

    Akcora, Büsra Öztürk; Storm, Gert; Bansal, Ruchi

    2018-03-01

    Quiescent hepatic stellate cells (HSCs), in response to liver injury, undergo characteristic morphological transformation into proliferative, contractile and ECM-producing myofibroblasts. In this study, we investigated the implication of canonical Wnt signaling pathway in HSCs and liver fibrogenesis. Canonical Wnt signaling pathway activation and inhibition using β-catenin/CBP inhibitor ICG001 was examined in-vitro in TGFβ-activated 3T3, LX2, primary human HSCs, and in-vivo in CCl 4 -induced acute liver injury mouse model. Fibroblasts-conditioned medium studies were performed to assess the Wnt-regulated paracrine factors involved in crosstalk between HSCs-macrophages and HSCs-endothelial cells. Canonical Wnt signaling pathway components were significantly up-regulated in-vitro and in-vivo. In-vitro, ICG-001 significantly inhibited fibrotic parameters, 3D-collagen contractility and wound healing. Conditioned medium induced fibroblasts-mediated macrophage and endothelial cells activation was significantly inhibited by ICG-001. In-vivo, ICG-001 significantly attenuated collagen accumulation and HSC activation. Interestingly, ICG-001 drastically inhibited macrophage infiltration, intrahepatic inflammation and angiogenesis. We further analyzed the paracrine factors involved in Wnt-mediated effects and found CXCL12 was significantly suppressed both in-vitro and in-vivo following Wnt inhibition. Wnt-regulated CXCL12 secretion from activated HSCs potentiated macrophage infiltration and activation, and angiogenesis. Pharmacological inhibition of canonical Wnt signaling pathway via suppression of stromal CXCL12 suggests a potential therapeutic approach targeting activated HSCs in liver fibrosis. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  5. Discovery of highly potent and selective small molecule ADAMTS-5 inhibitors that inhibit human cartilage degradation via encoded library technology (ELT).

    Science.gov (United States)

    Deng, Hongfeng; O'Keefe, Heather; Davie, Christopher P; Lind, Kenneth E; Acharya, Raksha A; Franklin, G Joseph; Larkin, Jonathan; Matico, Rosalie; Neeb, Michael; Thompson, Monique M; Lohr, Thomas; Gross, Jeffrey W; Centrella, Paolo A; O'Donovan, Gary K; Bedard, Katie L Sargent; van Vloten, Kurt; Mataruse, Sibongile; Skinner, Steven R; Belyanskaya, Svetlana L; Carpenter, Tiffany Y; Shearer, Todd W; Clark, Matthew A; Cuozzo, John W; Arico-Muendel, Christopher C; Morgan, Barry A

    2012-08-23

    The metalloprotease ADAMTS-5 is considered a potential target for the treatment of osteoarthritis. To identify selective inhibitors of ADAMTS-5, we employed encoded library technology (ELT), which enables affinity selection of small molecule binders from complex mixtures by DNA tagging. Selection of ADAMTS-5 against a four-billion member ELT library led to a novel inhibitor scaffold not containing a classical zinc-binding functionality. One exemplar, (R)-N-((1-(4-(but-3-en-1-ylamino)-6-(((2-(thiophen-2-yl)thiazol-4-yl)methyl)amino)-1,3,5-triazin-2-yl)pyrrolidin-2-yl)methyl)-4-propylbenzenesulfonamide (8), inhibited ADAMTS-5 with IC(50) = 30 nM, showing >50-fold selectivity against ADAMTS-4 and >1000-fold selectivity against ADAMTS-1, ADAMTS-13, MMP-13, and TACE. Extensive SAR studies showed that potency and physicochemical properties of the scaffold could be further improved. Furthermore, in a human osteoarthritis cartilage explant study, compounds 8 and 15f inhibited aggrecanase-mediated (374)ARGS neoepitope release from aggrecan and glycosaminoglycan in response to IL-1β/OSM stimulation. This study provides the first small molecule evidence for the critical role of ADAMTS-5 in human cartilage degradation.

  6. A Dual Non-ATP Analogue Inhibitor of Aurora Kinases A and B, Derived from Resorcinol with a Mixed Mode of Inhibition.

    Science.gov (United States)

    Karthigeyan, Dhanasekaran; Surabhi, Sudhevan; Mizar, Pushpak; Soumik, Siddhanta; Banerjee, Amrita; Sinha, Sarmistha Halder; Dasgupta, Dipak; Narayana, Chandrabhas; Kundu, Tapas K

    2016-06-01

    Aurora kinases are the most commonly targeted mitotic kinases in the intervention of cancer progression. Here, we report a resorcinol derivative, 5-methyl-4-(2-thiazolylazo) resorcinol (PTK66), a dual inhibitor of Aurora A and Aurora B kinases. PTK66 is a surface binding non-ATP analogue inhibitor that shows a mixed pattern of inhibition against both of Aurora A and B kinases. The in vitro IC50 is approximately 47 and 40 μm for Aurora A and Aurora B kinases, respectively. In cellular systems, PTK66 exhibits a substantially low cytotoxicity at micromolar concentrations but it can induce aneuploidy under similar dosages as a consequence of Aurora kinase inhibition. This result was corroborated by a drop in the histone H3 (S10) phosphorylation level detected via Western blot analysis using three different cell types. Altogether, our findings indicate that the ligand containing resorcinol backbone is one of the novel scaffolds targeting the Aurora family of kinases, which could be a target for antineoplastic drug development. © 2016 John Wiley & Sons A/S.

  7. Discovery of novel PDE9 inhibitors capable of inhibiting Aβ aggregation as potential candidates for the treatment of Alzheimer’s disease

    Science.gov (United States)

    Su, Tao; Zhang, Tianhua; Xie, Shishun; Yan, Jun; Wu, Yinuo; Li, Xingshu; Huang, Ling; Luo, Hai-Bin

    2016-02-01

    Recently, phosphodiesterase-9 (PDE9) inhibitors and biometal-chelators have received much attention as potential therapeutics for the treatment of Alzheimer’s disease (AD). Here, we designed, synthesized, and evaluated a novel series of PDE9 inhibitors with the ability to chelate metal ions. The bioassay results showed that most of these molecules strongly inhibited PDE9 activity. Compound 16 showed an IC50 of 34 nM against PDE9 and more than 55-fold selectivity against other PDEs. In addition, this compound displayed remarkable metal-chelating capacity and a considerable ability to halt copper redox cycling. Notably, in comparison to the reference compound clioquinol, it inhibited metal-induced Aβ1-42 aggregation more effectively and promoted greater disassembly of the highly structured Aβ fibrils generated through Cu2+-induced Aβ aggregation. These activities of 16, together with its favorable blood-brain barrier permeability, suggest that 16 may be a promising compound for treatment of AD.

  8. Partial contribution of Rho-kinase inhibition to the bioactivity of Ganoderma lingzhi and its isolated compounds: insights on discovery of natural Rho-kinase inhibitors.

    Science.gov (United States)

    Amen, Yhiya; Zhu, Qinchang; Tran, Hai-Bang; Afifi, Mohamed S; Halim, Ahmed F; Ashour, Ahmed; Shimizu, Kuniyoshi

    2017-04-01

    Recent studies identified Rho-kinase enzymes (ROCK-I and ROCK-II) as important targets that are involved in a variety of diseases. Synthetic Rho-kinase inhibitors have emerged as potential therapeutic agents to treat disorders such as hypertension, stroke, cancer, diabetes, glaucoma, etc. Our study is the first to screen the total ethanol extract of the medicinal mushroom Ganoderma lingzhi with thirty-five compounds for Rho-kinase inhibitory activity. Moreover, a molecular binding experiment was designed to investigate the binding affinity of the compounds at the active sites of Rho-kinase enzymes. The structure-activity relationship analysis was investigated. Our results suggest that the traditional uses of G. lingzhi might be in part due to the ROCK-I and ROCK-II inhibitory potential of this mushroom. Structure-activity relationship studies revealed some interesting features of the lanostane triterpenes that potentiate their Rho-kinase inhibition. These findings would be helpful for further studies on the design of Rho-kinase inhibitors from natural sources and open the door for contributions from other researchers for optimizing the development of natural Rho-kinase inhibitors.

  9. Structural basis for the inhibition of poly(ADP-ribose) polymerases 1 and 2 by BMN 673, a potent inhibitor derived from dihydropyridophthalazinone

    Energy Technology Data Exchange (ETDEWEB)

    Aoyagi-Scharber, Mika, E-mail: maoyagi@bmrn.com [BioMarin Pharmaceutical Inc., 105 Digital Drive, Novato, CA 94949 (United States); Gardberg, Anna S. [Emerald BioStructures, 7869 NE Day Road West, Bainbridge Island, WA 98110 (United States); Yip, Bryan K.; Wang, Bing; Shen, Yuqiao; Fitzpatrick, Paul A. [BioMarin Pharmaceutical Inc., 105 Digital Drive, Novato, CA 94949 (United States)

    2014-08-29

    BMN 673, a novel PARP1/2 inhibitor in clinical development with substantial tumor cytotoxicity, forms extensive hydrogen-bonding and π-stacking in the nicotinamide pocket, with its unique disubstituted scaffold extending towards the less conserved edges of the pocket. These interactions might provide structural insight into the ability of BMN 673 to both inhibit catalysis and affect DNA-binding activity. Poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2), which are involved in DNA damage response, are targets of anticancer therapeutics. BMN 673 is a novel PARP1/2 inhibitor with substantially increased PARP-mediated tumor cytotoxicity and is now in later-stage clinical development for BRCA-deficient breast cancers. In co-crystal structures, BMN 673 is anchored to the nicotinamide-binding pocket via an extensive network of hydrogen-bonding and π-stacking interactions, including those mediated by active-site water molecules. The novel di-branched scaffold of BMN 673 extends the binding interactions towards the outer edges of the pocket, which exhibit the least sequence homology among PARP enzymes. The crystallographic structural analyses reported here therefore not only provide critical insights into the molecular basis for the exceptionally high potency of the clinical development candidate BMN 673, but also new opportunities for increasing inhibitor selectivity.

  10. Structural basis for the inhibition of poly(ADP-ribose) polymerases 1 and 2 by BMN 673, a potent inhibitor derived from dihydropyridophthalazinone

    International Nuclear Information System (INIS)

    Aoyagi-Scharber, Mika; Gardberg, Anna S.; Yip, Bryan K.; Wang, Bing; Shen, Yuqiao; Fitzpatrick, Paul A.

    2014-01-01

    BMN 673, a novel PARP1/2 inhibitor in clinical development with substantial tumor cytotoxicity, forms extensive hydrogen-bonding and π-stacking in the nicotinamide pocket, with its unique disubstituted scaffold extending towards the less conserved edges of the pocket. These interactions might provide structural insight into the ability of BMN 673 to both inhibit catalysis and affect DNA-binding activity. Poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2), which are involved in DNA damage response, are targets of anticancer therapeutics. BMN 673 is a novel PARP1/2 inhibitor with substantially increased PARP-mediated tumor cytotoxicity and is now in later-stage clinical development for BRCA-deficient breast cancers. In co-crystal structures, BMN 673 is anchored to the nicotinamide-binding pocket via an extensive network of hydrogen-bonding and π-stacking interactions, including those mediated by active-site water molecules. The novel di-branched scaffold of BMN 673 extends the binding interactions towards the outer edges of the pocket, which exhibit the least sequence homology among PARP enzymes. The crystallographic structural analyses reported here therefore not only provide critical insights into the molecular basis for the exceptionally high potency of the clinical development candidate BMN 673, but also new opportunities for increasing inhibitor selectivity

  11. Glycogen synthase kinase-3 inhibitors suppress the AR-V7-mediated transcription and selectively inhibit cell growth in AR-V7-positive prostate cancer cells.

    Science.gov (United States)

    Nakata, Daisuke; Koyama, Ryokichi; Nakayama, Kazuhide; Kitazawa, Satoshi; Watanabe, Tatsuya; Hara, Takahito

    2017-06-01

    Recent evidence suggests that androgen receptor (AR) splice variants, including AR-V7, play a pivotal role in resistance to androgen blockade in prostate cancer treatment. The development of new therapeutic agents that can suppress the transcriptional activities of AR splice variants has been anticipated as the next generation treatment of castration-resistant prostate cancer. High-throughput screening of AR-V7 signaling inhibitors was performed using an AR-V7 reporter system. The effects of a glycogen synthase kinase-3 (GSK3) inhibitor, LY-2090314, on endogenous AR-V7 signaling were evaluated in an AR-V7-positive cell line, JDCaP-hr, by quantitative reverse transcription polymerase chain reaction. The relationship between AR-V7 signaling and β-catenin signaling was assessed using RNA interference. The effect of LY-2090314 on cell growth in various prostate cancer cell lines was also evaluated. We identified GSK3 inhibitors as transcriptional suppressors of AR-V7 using a high-throughput screen with an AR-V7 reporter system. LY-2090314 suppressed the reporter activity and endogenous AR-V7 activity in JDCaP-hr cells. Because silencing of β-catenin partly rescued the suppression, it was evident that the suppression was mediated, at least partially, via the activation of β-catenin signaling. AR-V7 signaling and β-catenin signaling reciprocally regulate each other in JDCaP-hr cells, and therefore, GSK3 inhibition can repress AR-V7 transcriptional activity by accumulating intracellular β-catenin. Notably, LY-2090314 selectively inhibited the growth of AR-V7-positive prostate cancer cells in vitro. Our findings demonstrate the potential of GSK3 inhibitors in treating advanced prostate cancer driven by AR splice variants. In vivo evaluation of AR splice variant-positive prostate cancer models will help illustrate the overall significance of GSK3 inhibitors in treating prostate cancer. © 2017 Wiley Periodicals, Inc.

  12. A novel small molecular STAT3 inhibitor, LY5, inhibits cell viability, cell migration, and angiogenesis in medulloblastoma cells.

    Science.gov (United States)

    Xiao, Hui; Bid, Hemant Kumar; Jou, David; Wu, Xiaojuan; Yu, Wenying; Li, Chenglong; Houghton, Peter J; Lin, Jiayuh

    2015-02-06

    Signal transducers and activators of transcription 3 (STAT3) signaling is persistently activated and could contribute to tumorigenesis of medulloblastoma. Numerous studies have demonstrated that inhibition of the persistent STAT3 signaling pathway results in decreased proliferation and increased apoptosis in human cancer cells, indicating that STAT3 is a viable molecular target for cancer therapy. In this study, we investigated a novel non-peptide, cell-permeable small molecule, named LY5, to target STAT3 in medulloblastoma cells. LY5 inhibited persistent STAT3 phosphorylation and induced apoptosis in human medulloblastoma cell lines expressing constitutive STAT3 phosphorylation. The inhibition of STAT3 signaling by LY5 was confirmed by down-regulating the expression of the downstream targets of STAT3, including cyclin D1, bcl-XL, survivin, and micro-RNA-21. LY5 also inhibited the induction of STAT3 phosphorylation by interleukin-6 (IL-6), insulin-like growth factor (IGF)-1, IGF-2, and leukemia inhibitory factor in medulloblastoma cells, but did not inhibit STAT1 and STAT5 phosphorylation stimulated by interferon-γ (IFN-γ) and EGF, respectively. In addition, LY5 blocked the STAT3 nuclear localization induced by IL-6, but did not block STAT1 and STAT5 nuclear translocation mediated by IFN-γ and EGF, respectively. A combination of LY5 with cisplatin or x-ray radiation also showed more potent effects than single treatment alone in the inhibition of cell viability in human medulloblastoma cells. Furthermore, LY5 demonstrated a potent inhibitory activity on cell migration and angiogenesis. Taken together, these findings indicate LY5 inhibits persistent and inducible STAT3 phosphorylation and suggest that LY5 is a promising therapeutic drug candidate for medulloblastoma by inhibiting persistent STAT3 signaling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. The angiotensin-converting enzyme inhibitor captopril inhibits poly(ADP-ribose)polymerase activation and exerts beneficial effects in an ovine model of burn and smoke injury

    Science.gov (United States)

    Asmussen, Sven; Bartha, Eva; Olah, Gabor; Sbrana, Elena; Rehberg, Sebastian W.; Yamamoto, Yusuke; Enkhbaatar, Perenlei; Hawkins, Hal K.; Ito, Hiroshi; Cox, Robert A.; Traber, Lillian D.; Traber, Daniel L.; Szabo, Csaba

    2011-01-01

    We investigated the effect of the angiotensin converting enzyme (ACE) inhibitor captopril in a clinically relevant ovine model of smoke and burn injury, with special reference to oxidative stress, activation of poly(ADP-ribose) polymerase in the lung and in circulating leukocytes. Female, adult sheep (28–40 kg) were divided into 3 groups. After tracheostomy and under deep anesthesia both vehicle-control (n=5) and captopril (20 mg/kg/d, iv., starting 0.5 hour before the injury) treated (n=5) groups were subjected to 2×20%, third degree burn injury and were insufflated with 48 breaths of cotton smoke. A sham group not receiving burn/smoke was also studied (n=5). Animals were mechanically ventilated and fluid resuscitated for 24 h in the awake state. Burn and smoke injury resulted in an upregulation of ACE in the lung, evidenced by immunohistochemical determination and Western blotting. Burn and smoke injury resulted in pulmonary dysfunction, as well as systemic hemodynamic alterations. Captopril treatment of burn and smoke animals improved PaO2/FiO2 ratio and pulmonary shunt fraction and reduced the degree of lung edema. There was a marked increase in PAR levels in circulating leukocytes after burn/smoke injury, which was significantly decreased by captopril. The pulmonary level of ACE and the elevated pulmonary levels of TGF-β in response to burn and smoke injury were significantly decreased by captopril treatment. Our results suggest that the ACE inhibitor captopril exerts beneficial effects on the pulmonary function in burn/smoke injury. The effects of the ACE inhibitor may be related to the prevention of ROS-induced PARP over-activation. ACE inhibition may also exert additional beneficial effects by inhibiting the expression of the pro-fibrotic mediator TGF-β. PMID:21701415

  14. The novel Akt inhibitor API-1 induces c-FLIP degradation and synergizes with TRAIL to augment apoptosis independent of Akt inhibition.

    Science.gov (United States)

    Li, Bo; Ren, Hui; Yue, Ping; Chen, Mingwei; Khuri, Fadlo R; Sun, Shi-Yong

    2012-04-01

    API-1 (pyrido[2,3-d]pyrimidines) is a novel small-molecule inhibitor of Akt, which acts by binding to Akt and preventing its membrane translocation and has promising preclinical antitumor activity. In this study, we reveal a novel function of API-1 in regulation of cellular FLICE-inhibitory protein (c-FLIP) levels and TRAIL-induced apoptosis, independent of Akt inhibition. API-1 effectively induced apoptosis in tested cancer cell lines including activation of caspase-8 and caspase-9. It reduced the levels of c-FLIP without increasing the expression of death receptor 4 (DR4) or DR5. Accordingly, it synergized with TRAIL to induce apoptosis. Enforced expression of ectopic c-FLIP did not attenuate API-1-induced apoptosis but inhibited its ability to enhance TRAIL-induced apoptosis. These data indicate that downregulation of c-FLIP mediates enhancement of TRAIL-induced apoptosis by API-1 but is not sufficient for API-1-induced apoptosis. API-1-induced reduction of c-FLIP could be blocked by the proteasome inhibitor MG132. Moreover, API-1 increased c-FLIP ubiquitination and decreased c-FLIP stability. These data together suggest that API-1 downregulates c-FLIP by facilitating its ubiquitination and proteasome-mediated degradation. Because other Akt inhibitors including API-2 and MK2206 had minimal effects on reducing c-FLIP and enhancement of TRAIL-induced apoptosis, it is likely that API-1 reduces c-FLIP and enhances TRAIL-induced apoptosis independent of its Akt-inhibitory activity. 2012 AACR

  15. Donepezil, an acetylcholinesterase inhibitor, attenuates LPS-induced inflammatory response in murine macrophage cell line RAW 264.7 through inhibition of nuclear factor kappa B translocation.

    Science.gov (United States)

    Arikawa, Mikihiko; Kakinuma, Yoshihiko; Noguchi, Tatsuya; Todaka, Hiroshi; Sato, Takayuki

    2016-10-15

    We have previously demonstrated that the pharmacotherapy with donepezil, an acetylcholinesterase inhibitor, suppresses cardiac remodeling in a mouse model of ischemic heart failure after myocardial infarction (MI). However, the precise mechanisms of the cardioprotective effect of donepezil have not been completely delineated. Because post-ischemic inflammation is a pathological key event in the cardiac remodeling process following MI, we investigated the hypothesis that donepezil acts as an inhibitor of inflammatory mediators. RAW 264.7 murine macrophage cells were pretreated with donepezil (100µM) prior to a pro-inflammatory stimulation by administration of lipopolysaccharide (LPS, 10ng/ml). Donepezil significantly reduced intra- and extracellular levels of various kinds of inflammatory mediators such as TNF-α, IL-1β, IL-2, IL-6 and IL-18 after the LPS stimulation, and attenuated LPS-induced nuclear translocation of nuclear factor-kappa B (NF-κB). These results indicate that donepezil possesses an anti-inflammatory property. However, the inhibitory effect of donepezil on the macrophage inflammatory responses was never reproduced by ACh, nor was disrupted by ACh receptor blockers. Moreover, other kinds of acetylcholinesterase inhibitors failed to inhibit the inflammatory responses in LPS-stimulated macrophage cells. These results suggest that a cholinergic anti-inflammatory pathway would not be involved in the anti-inflammatory effect of donepezil and that the specific characteristics of donepezil in suppressing the LPS-induced cytokine release and the NF-κB activation would be independent of its acetylcholinesterase inhibition. The present study showed that donepezil exerts an anti-inflammatory effect independently of acetylcholinesterase inhibitory action, thereby donepezil may contribute to cardioprotection during cardiac remodeling process in an ischemic heart failure after MI. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Inhibition of p70S6K1 Activation by Pdcd4 Overcomes the Resistance to an IGF-1R/IR Inhibitor in Colon Carcinoma Cells.

    Science.gov (United States)

    Zhang, Yan; Wang, Qing; Chen, Li; Yang, Hsin-Sheng

    2015-03-01

    Agents targeting insulin-like growth factor 1 receptor (IGF-1R) are being actively examined in clinical trials. Although there has been some initial success of single-agent targeting IGF-1R, attempts in later studies failed because of resistance. This study aimed to understand the effects of programmed cell death 4 (Pdcd4) on the chemosensitivity of the IGF-1R inhibitor OSI-906 in colorectal cancer cells and the mechanism underlying this impact. Using OSI-906-resistant and -sensitive colorectal cancer cells, we found that the Pdcd4 level directly correlates with cell chemosensitivity to OSI-906. In addition, tumors derived from Pdcd4 knockdown cells resist the growth inhibitory effect of OSI-906 in a colorectal cancer xenograft mouse model. Moreover, Pdcd4 enhances the antiproliferative effect of OSI-906 in resistant cells through suppression of p70S6K1 activation. Knockdown of p70S6K1, but not p70S6K2, significantly increases the chemosensitivity of OSI-906 in cultured colorectal cancer cells. Furthermore, the combination of OSI-906 and PF-4708671, a p70S6K1 inhibitor, efficiently suppresses the growth of OSI-906-resistant colon tumor cells in vitro and in vivo. Taken together, activation of p70S6K1 that is inhibited by Pdcd4 is essential for resistance to the IGF-1R inhibitor in colon tumor cells, and the combinational treatment of OSI-906 and PF-4708671 results in enhanced antiproliferation effects in colorectal cancer cells in vitro and in vivo, providing a novel venue to overcome the resistance to the IGF-1R inhibitor in treating colorectal cancer. ©2015 American Association for Cancer Research.

  17. Coumarin or benzoxazinone bearing benzimidazolium and bis(benzimidazolium salts; involvement in transfer hydrogenation of acetophenone derivatives and hCA inhibition

    Directory of Open Access Journals (Sweden)

    Mert Olgun Karataş

    2015-10-01

    Full Text Available Four new salts of benzimidazolium and bis(benzimidazolium which include coumarin or benzoxazinone moieties were synthesized and the structures of the newly synthesized compounds were elucidated on the basis of spectral analyses such as 1H-NMR, 13C-NMR, HSQC, IR, LC-MS and elemental analysis. Benzimidazolium salts were used intensively as N-heterocyclic carbene (NHC precursors in the various catalytic reactions such as transfer hydrogenation (TH, C-H bond activation, Heck, Suzuki reaction etc. With the prospect of potential NHC precursor properties of the synthesized compounds, they were employed in the (TH reaction of p-substitute acetophenones (acetophenone, p-methyl acetophenone, p-chloro acetophenone and good yields were observed. Coumarin compounds are known as inhibitor of carbonic anhydrase and inhibition effects of the synthesized compounds on human carbonic anhydrases (hCA were investigated as in vitro. The in vitro results demonstrated that all compounds inhibited hCA I and hCA II activity. Among the synthesized compounds 1,4-bis(1-((6,8-dimethyl-2H-chromen-2-one-4-ylmethylbenzimidazolium-3-ylbutane dichloride was found to be the most active IC50= 5.55 mM and 6.06 mM for hCA I and hCA II, respectively.

  18. Identification of a small-molecule inhibitor of the PICK1 PDZ domain that inhibits hippocampal LTP and LTD

    DEFF Research Database (Denmark)

    Thorsen, Thor S; Madsen, Kenneth L; Rebola, Nelson

    2010-01-01

    Proteins containing PSD-95/Discs-large/ZO-1 homology (PDZ) domains play key roles in the assembly and regulation of cellular signaling pathways and represent putative targets for new pharmacotherapeutics. Here we describe the first small-molecule inhibitor (FSC231) of the PDZ domain in protein in...... the proposed role of the PICK1/AMPA receptor interaction in neuropathic pain, excitotoxicity, and cocaine addiction, FSC231 might serve as a lead in the future development of new therapeutics against these conditions....

  19. Synthetic matrix metalloproteinase inhibitors inhibit growth of established breast cancer osteolytic lesions and prolong survival in mice

    DEFF Research Database (Denmark)

    Winding, Bent; NicAmhlaoibh, Róisín; Misander, Henriette

    2002-01-01

    Breast cancer frequently leads to incurable bone metastasis. Essential requirements for the development of bone metastasis are cell-cell and cell-matrix interactions, release of bioactive growth factors and cytokines, and removal of large amounts of bone matrix. Matrix metalloproteinases (MMPs) p......) play an important role in all of these processes, but the possibility of using synthetic MMP inhibitors to decrease bone metastasis has received little attention....

  20. Combined inhibition of IGFR enhances the effects of gefitinib in H1650: a lung cancer cell line with EGFR mutation and primary resistance to EGFR-TK inhibitors.

    Science.gov (United States)

    Choi, Yun Jung; Rho, Jin Kyung; Jeon, Byung-suk; Choi, Su Jin; Park, Su Cheol; Lee, Seung Sook; Kim, Hye-Ryoun; Kim, Cheol Hyeon; Lee, Jae Cheol

    2010-07-01

    H1650 non-small cell lung cancer (NSCLC) cells display primary resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) although they have a deletion mutation on exon 19 of the EGFR gene. We investigated the effect of inhibition of both insulin-like growth factor receptor (IGFR) and EGFR signaling considering that IGFR signaling pathway has been implicated in the development and progression with therapeutic resistance of various cancers including lung cancer. Three human NSCLC cell lines with an EGFR mutation of PC-9, HCC827 and H1650 were used for experiment. Cell viability and proliferative activity were assessed by MTT and three-dimensional culture assay. Combination index was obtained by CalcuSyn software. The change of EGFR- and IGFR-related signals was evaluated by western blots. H1650 cells were 1,000 times more resistant to gefitinib and erlotinib than HCC827 and PC-9 cells possessing the same EGFR mutation. Phosphatase and tensin homolog loss and sustained phosphorylation of Akt in spite of treatment with gefitinib were evident only in H1650 cells. Interestingly, IGFR phosphorylation was decreased by gefitinib in HCC827 and PC-9 cells while being maintained in H1650 cells. Combined treatment with the IGFR inhibitors alpha-IR3 and AG1024 enhanced gefitinib-induced growth inhibition and apoptosis, and down-regulated phosphorylation of Akt, EGFR and IGFR. Combined inhibition of IGFR signaling enhances the growth inhibitory and apoptosis-inducing effects of gefitinib, suggesting that this approach could be useful to overcome the primary resistance to EGFR-TKIs in lung cancer.

  1. Estrogens, selective estrogen receptor modulators, and a selective estrogen receptor down-regulator inhibit endothelial production of tissue factor pathway inhibitor 1

    Directory of Open Access Journals (Sweden)

    Ree Anne

    2006-10-01

    Full Text Available Abstract Background Hormone therapy, oral contraceptives, and tamoxifen increase the risk of thrombotic disease. These compounds also reduce plasma content of tissue factor pathway inhibitor-1 (TFPI, which is the physiological inhibitor of the tissue factor pathway of coagulation. The current aim was to study if estrogens and estrogen receptor (ER modulators may inhibit TFPI production in cultured endothelial cells and, if so, identify possible mechanisms involved. Methods Human endothelial cell cultures were treated with 17β-estradiol (E2, 17α-ethinylestradiol (EE2, tamoxifen, raloxifene, or fulvestrant. Protein levels of TFPI in cell media and cell lysates were measured by an enzyme-linked immunosorbent assay, and TFPI mRNA levels were assessed by quantitative PCR. Expression of ERα was analysed by immunostaining. Results All compounds (each in a concentration of 10 nM reduced TFPI in cell medium, by 34% (E2, 21% (EE2, 16% (tamoxifen, and 28% (raloxifene, respectively, with identical inhibitory effects on cellular TFPI levels. Expression of TFPI mRNA was principally unchanged. Treatment with fulvestrant, which was also associated with down-regulation of secreted TFPI (9% with 10 nM and 26% with 1000 nM, abolished the TFPI-inhibiting effect of raloxifene, but not of the other compounds. Notably, the combination of 1000 nM fulvestrant and 10 nM raloxifene increased TFPI secretion, and, conversely, 10 nM of either tamoxifen or raloxifene seemed to partly (tamoxifen or fully (raloxifene counteract the inhibitory effect of 1000 nM fulvestrant. The cells did not express the regular nuclear 66 kDa ERα, but instead a 45 kDa ERα, which was not regulated by estrogens or ER modulators. Conclusion E2, EE2, tamoxifen, raloxifene, and fulvestrant inhibited endothelial production of TFPI by a mechanism apparently independent of TFPI transcription.

  2. MCPIP1 contributes to the toxicity of proteasome inhibitor MG-132 in HeLa cells by the inhibition of NF-κB.

    Science.gov (United States)

    Skalniak, Lukasz; Dziendziel, Monika; Jura, Jolanta

    2014-10-01

    Recently, we have shown that the treatment of cells with proteasome inhibitor MG-132 results in the induction of expression of monocyte chemotactic protein-1 induced protein 1 (MCPIP1). MCPIP1 is a ribonuclease, responsible for the degradation of transcripts encoding certain pro-inflammatory cytokines. The protein is also known as an inhibitor of NF-κB transcription factor. Thanks to its molecular properties, MCPIP1 is considered as a regulator of inflammation, differentiation, and survival. Using siRNA technology, we show here that MCPIP1 expression contributes to the toxic properties of MG-132 in HeLa cells. The inhibition of proteasome by MG-132 and epoxomicin markedly increased MCPIP1 expression. While MG-132 induces HeLa cell death, down-regulation of MCPIP1 expression by siRNA partially protects HeLa cells from MG-132 toxicity and restores Nuclear factor-κB (NF-κB) activity, inhibited by MG-132 treatment. Inversely, overexpression of MCPIP1 decreased constitutive activity of NF-κB and limited the survival of HeLa cells, as we have shown in the previous study. Interestingly, although MG-132 decreased the expression of IκBα and increased p65 phosphorylation, the inhibition of constitutive NF-κB activity was observed in MG-132-treated cells. Since the elevated constitutive activity of NF-κB is one of the mechanisms providing increased survival of cancer cells, including HeLa cells, we propose that death-promoting properties of MCPIP1 in MG-132-treated HeLa cells may, at least partially, derive from the negative effect on the constitutive NF-κB activity.

  3. [Forkhead domain inhibitor-6 (FDI-6) increases apoptosis and inhibits invasion and migration of laryngeal carcinoma cells by down-regulating nuclear FoxM1].

    Science.gov (United States)

    Liu, Yanan; Zhu, Lin; Wen, Taoyu; Wan, Jie; Lei, Yue; Chen, Hongyan

    2017-05-01

    Objective To study the effects of new small molecular inhibitor, forkhead domain inhibitor-6 (FDI-6), on proliferation, apoptosis, invasion and migration in human laryngeal carcinoma Hep-2 cell line and the related mechanism. Methods MTT assay was used to test the proliferation rate of Hep-2 cells before and 12, 24 hours after treated with (5, 10, 20, 40, 80) μmol/L of FDI-6. Flow cytometry (FCM) and Transwell TM chamber assay were respectively carried out to detect the apoptosis rate, cell invasion and migration in Hep-2 cells after treated by 10, 20 μmol/L FDI-6 for 24 hours. Real-time quantitative PCR (qRT-PCR) and Western blotting were performed to determine the mRNA and protein levels of FoxM1, Bcl-2 and BAX, respectively. Results Cell proliferation rate was inhibited by FDI-6 in a dose- and time-dependent manner. Twenty-four hours after 10, 20 μmol/L FDI-6 treatment, the apoptosis rate in Hep-2 cells was elevated and the ability of cell invasion and migration was inhibited in a dose-dependent manner. The qRT-PCR showed that there was no significant change in FoxM1 mRNA expression with or without FDI-6 treatment. Western blotting showed that the total protein level of FoxM1 was not obviously changed, but Bcl-2 was down-regulated, BAX was up-regulated. However, in the nuclear FoxM1 protein level decreased along with the ascent of FDI-6 concentration. Conclusion FDI-6 could induce cell apoptosis and inhibit cell proliferation, invasion and migration in Hep-2 cells. This may be related to the down-regulation of FoxM1 in the nucleus.

  4. Transport inhibition of digoxin using several common P-gp expressing cell lines is not necessarily reporting only on inhibitor binding to P-gp.

    Directory of Open Access Journals (Sweden)

    Annie Albin Lumen

    Full Text Available We have reported that the P-gp substrate digoxin required basolateral and apical uptake transport in excess of that allowed by digoxin passive permeability (as measured in the presence of GF120918 to achieve the observed efflux kinetics across MDCK-MDR1-NKI (The Netherlands Cancer Institute confluent cell monolayers. That is, GF120918 inhibitable uptake transport was kinetically required. Therefore, IC50 measurements using digoxin as a probe substrate in this cell line could be due to inhibition of P-gp, of digoxin uptake transport, or both. This kinetic analysis is now extended to include three additional cell lines: MDCK-MDR1-NIH (National Institute of Health, Caco-2 and CPT-B2 (Caco-2 cells with BCRP knockdown. These cells similarly exhibit GF120918 inhibitable uptake transport of digoxin. We demonstrate that inhibition of digoxin transport across these cell lines by GF120918, cyclosporine, ketoconazole and verapamil is greater than can be explained by inhibition of P-gp alone. We examined three hypotheses for this non-P-gp inhibition. The inhibitors can: (1 bind to a basolateral digoxin uptake transporter, thereby inhibiting digoxin's cellular uptake; (2 partition into the basolateral membrane and directly reduce membrane permeability; (3 aggregate with digoxin in the donor chamber, thereby reducing the free concentration of digoxin, with concomitant reduction in digoxin uptake. Data and simulations show that hypothesis 1 was found to be uniformly acceptable. Hypothesis 2 was found to be uniformly unlikely. Hypothesis 3 was unlikely for GF120918 and cyclosporine, but further studies are needed to completely adjudicate whether hetero-dimerization contributes to the non-P-gp inhibition for ketoconazole and verapamil. We also find that P-gp substrates with relatively low passive permeability such as digoxin, loperamide and vinblastine kinetically require basolateral uptake transport over that allowed by +GF120918 passive permeability, while

  5. Btk Inhibitor RN983 Delivered by Dry Powder Nose-only Aerosol Inhalation Inhibits Bronchoconstriction and Pulmonary Inflammation in the Ovalbumin Allergic Mouse Model of Asthma.

    Science.gov (United States)

    Phillips, Jonathan E; Renteria, Lorena; Burns, Lisa; Harris, Paul; Peng, Ruoqi; Bauer, Carla M T; Laine, Dramane; Stevenson, Christopher S

    2016-06-01

    In allergen-induced asthma, activated mast cells start the lung inflammatory process with degranulation, cytokine synthesis, and mediator release. Bruton's tyrosine kinase (Btk) activity is required for the mast cell activation during IgE-mediated secretion. This study characterized a novel inhaled Btk inhibitor RN983 in vitro and in ovalbumin allergic mouse models of the early (EAR) and late (LAR) asthmatic response. RN983 potently, selectively, and reversibly inhibited the Btk enzyme. RN983 displayed functional activities in human cell-based assays in multiple cell types, inhibiting IgG production in B-cells with an IC50 of 2.5 ± 0.7 nM and PGD2 production from mast cells with an IC50 of 8.3 ± 1.1 nM. RN983 displayed similar functional activities in the allergic mouse model of asthma when delivered as a dry powder aerosol by nose-only inhalation. RN983 was less potent at inhibiting bronchoconstriction (IC50(RN983) = 59 μg/kg) than the β-agonist salbutamol (IC50(salbutamol) = 15 μg/kg) in the mouse model of the EAR. RN983 was more potent at inhibiting the antigen induced increase in pulmonary inflammation (IC50(RN983) = <3 μg/kg) than the inhaled corticosteroid budesonide (IC50(budesonide) = 27 μg/kg) in the mouse model of the LAR. Inhalation of aerosolized RN983 may be effective as a stand-alone asthma therapy or used in combination with inhaled steroids and β-agonists in severe asthmatics due to its potent inhibition of mast cell activation.

  6. The papain inhibitor (SPI) of Streptomyces mobaraensis inhibits bacterial cysteine proteases and is an antagonist of bacterial growth.

    Science.gov (United States)

    Zindel, Stephan; Kaman, Wendy E; Fröls, Sabrina; Pfeifer, Felicitas; Peters, Anna; Hays, John P; Fuchsbauer, Hans-Lothar

    2013-07-01

    A novel papain inhibitory protein (SPI) from Streptomyces mobaraensis was studied to measure its inhibitory effect on bacterial cysteine protease activity (Staphylococcus aureus SspB) and culture supernatants (Porphyromonas gingivalis, Bacillus anthracis). Further, growth of Bacillus anthracis, Staphylococcus aureus, Pseudomonas aeruginosa, and Vibrio cholerae was completely inhibited by 10 μM SPI. At this concentration of SPI, no cytotoxicity was observed. We conclude that SPI inhibits bacterial virulence factors and has the potential to become a novel therapeutic treatment against a range of unrelated pathogenic bacteria.

  7. Wnt/β-Catenin Small-Molecule Inhibitor CWP232228 Preferentially Inhibits the Growth of Breast Cancer Stem-like Cells.

    Science.gov (United States)

    Jang, Gyu-Beom; Hong, In-Sun; Kim, Ran-Ju; Lee, Su-Youn; Park, Se-Jin; Lee, Eun-Sook; Park, Jung Hyuck; Yun, Chi-Ho; Chung, Jae-Uk; Lee, Kyoung-June; Lee, Hwa-Yong; Nam, Jeong-Seok

    2015-04-15

    Breast cancer stem cells (BCSC) are resistant to conventional chemotherapy and radiotherapy, which may destroy tumor masses but not all BCSC that can mediate relapses. In the present study, we showed that the level of Wnt/β-catenin signaling in BCSC is relatively higher than in bulk tumor cells, contributing to a relatively higher level of therapeutic resistance. We designed a highly potent small-molecule inhibitor, CWP232228, which antagonizes binding of β-catenin to T-cell factor (TCF) in the nucleus. Notably, although CWP232228 inhibited the growth of both BCSC and bulk tumor cells by inhibiting β-catenin-mediated transcription, BCSC exhibited greater growth inhibition than bulk tumor cells. We also documented evidence of greater insulin-like growth factor-I (IGF-I) expression by BCSC than by bulk tumor cells and that CWP232228 attenuated IGF-I-mediated BCSC functions. These results suggested that the inhibitory effect of CWP232228 on BCSC growth might be achieved through the disruption of IGF-I activity. Taken together, our findings indicate that CWP232228 offers a candidate therapeutic agent for breast cancer that preferentially targets BCSC as well as bulk tumor cells. ©2015 American Association for Cancer Research.

  8. Development of Classification Models for Identifying “True” P-glycoprotein (P-gp Inhibitors Through Inhibition, ATPase Activation and Monolayer Efflux Assays

    Directory of Open Access Journals (Sweden)

    Anna Maria Bianucci

    2012-06-01

    Full Text Available P-glycoprotein (P-gp is an efflux pump involved in the protection of tissues of several organs by influencing xenobiotic disposition. P-gp plays a key role in multidrug resistance and in the progression of many neurodegenerative diseases. The development of new and more effective therapeutics targeting P-gp thus represents an intriguing challenge in drug discovery. P-gp inhibition may be considered as a valid approach to improve drug bioavailability as well as to overcome drug resistance to many kinds of tumours characterized by the over-expression of this protein. This study aims to develop classification models from a unique dataset of 59 compounds for which there were homogeneous experimental data on P-gp inhibition, ATPase activation and monolayer efflux. For each experiment, the dataset was split into a training and a test set comprising 39 and 20 molecules, respectively. Rational splitting was accomplished using a sphere-exclusion type algorithm. After a two-step (internal/external validation, the best-performing classification models were used in a consensus predicting task for the identification of compounds named as “true” P-gp inhibitors, i.e., molecules able to inhibit P-gp without being effluxed by P-gp itself and simultaneously unable to activate the ATPase function.

  9. Synthesis, SAR study, and biological evaluation of novel quinoline derivatives as phosphodiesterase 10A inhibitors with reduced CYP3A4 inhibition.

    Science.gov (United States)

    Hamaguchi, Wataru; Masuda, Naoyuki; Miyamoto, Satoshi; Shiina, Yasuhiro; Kikuchi, Shigetoshi; Mihara, Takuma; Moriguchi, Hiroyuki; Fushiki, Hiroshi; Murakami, Yoshihiro; Amano, Yasushi; Honbou, Kazuya; Hattori, Kouji

    2015-01-15

    A novel class of phosphodiesterase 10A inhibitors with potent PDE10A inhibitory activity and reduced CYP3A4 inhibition was designed and synthesized starting from 2-[4-({[1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl]oxy}methyl)phenyl]quinoline (1). Replacement of pyridine ring of 1 with N-methyl pyridone ring drastically improved CYP3A4 inhibition, and further optimization of these quinoline analogues identified 1-methyl-5-(1-methyl-3-{[4-(quinolin-2-yl)phenoxy]methyl}-1H-pyrazol-4-yl)pyridin-2(1H)-one (42b), which showed potent PDE10A inhibitory activity and a good CYP3A4 inhibition profile. A PET study with (11)C-labeled 42b indicated that 42b exhibited good brain penetration and specifically accumulated in the rodent striatum. Further, oral administration of 42b dose-dependently attenuated phencyclidine-induced hyperlocomotion in mice with an ED50 value of 2.0mg/kg and improved visual-recognition memory impairment at 0.1 and 0.3mg/kg in mice novel object recognition test. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Inhibition of adipose triglyceride lipase (ATGL) by the putative tumor suppressor G0S2 or a small molecule inhibitor attenuates the growth of cancer cells.

    Science.gov (United States)

    Zagani, Rachid; El-Assaad, Wissal; Gamache, Isabelle; Teodoro, Jose G

    2015-09-29

    The G0/G1 switch gene 2 (G0S2) is methylated and silenced in a wide range of human cancers. The protein encoded by G0S2 is an endogenous inhibitor of lipid catabolism that directly binds adipose triglyceride lipase (ATGL). ATGL is the rate-limiting step in triglyceride metabolism. Although the G0S2 gene is silenced in cancer, the impact of ATGL in the growth and survival of cancer cells has never been addressed. Here we show that ectopic expression of G0S2 in non-small cell lung carcinomas (NSCL) inhibits triglyceride catabolism and results in lower cell growth. Similarly, knockdown of ATGL increased triglyceride levels, attenuated cell growth and promoted apoptosis. Conversely, knockdown of endogenous G0S2 enhanced the growth and invasiveness of cancer cells. G0S2 is strongly induced in acute promyelocytic leukemia (APL) cells in response to all trans retinoic acid (ATRA) and we show that inhibition of ATGL in these cells by G0S2 is required for efficacy of ATRA treatment. Our data uncover a novel tumor suppressor mechanism by which G0S2 directly inhibits activity of a key intracellular lipase. Our results suggest that elevated ATGL activity may be a general property of many cancer types and potentially represents a novel target for chemotherapy.

  11. A group-specific inhibitor of lysosomal cysteine proteinases selectively inhibits both proteolytic degradation and presentation of the antigen dinitrophenyl-poly-L-lysine by guinea pig accessory cells to T cells

    DEFF Research Database (Denmark)

    Buus, S; Werdelin, O

    1986-01-01

    A limited intralysosomal proteolytic degradation is probably a key event in the accessory cell processing of large protein antigens before their presentation to T cells. With the aid of highly specific inhibitors of proteinases, we have examined the role of proteolysis in the presentation...... of antigens by guinea pig accessory cells. The proteinase inhibitor benzyloxycarbonyl-phenylalanylalanine-diazomethyl-ketone, which selectively inhibits cysteine proteinases, was used to block this set of enzymes in cultured cells. We demonstrate that the selective inhibition of the cysteine proteinases...... inhibitor. Another inhibitor, pepstatin A, which selectively blocks aspartic proteinases, did not block the presentation of dinitrophenyl-poly-L-lysine. The results identify cysteine proteinases, probably lysosomal, as one of the groups of enzymes involved in antigen processing....

  12. Inhibitors of angiotensin-converting enzyme modulate mitosis and gene expression in pancreatic cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Reddy, M.K.; Baskaran, K.; Molteni, A. [Northwestern Univ. Medical School, Chicago, IL (United States)

    1995-12-01

    The angiotensin-converting enzyme (ACE) inhibitor captopril inhibits mitosis in several cell types that contain ACE and renin activity. In the present study, we evaluated the effect of the ACE inhibitors captopril and CGS 13945 (10{sup {minus}8} to 10{sup {minus}2}M) on proliferation and gene expression in hamster pancreatic duct carcinoma cells in culture. These cells lack renin and ACE activity. Both ACE inhibitors produced a dose-dependent reduction in tumor cell proliferation within 24 hr. Captopril at a concentration of 0.36 mM and CGS 13945 at 150 {mu}M decreased cellular growth rate to approximately half that of the control. Neither drug influenced the viability or the cell cycle distribution of the tumor cells. Slot blot analysis of mRNA for four genes, proliferation associated cell nuclear antigen (PCNA), K-ras, protein kinase C-{Beta} (PKC-{Beta}) and carbonic anhydrase II (CA II) was performed. Both ACE inhibitors increased K-ras expression by a factor of 2, and had no effect on CA II mRNA levels. Captopril also lowered PCNA by 40% and CGS 13945 lowered PKC-{Beta} gene expression to 30% of the control level. The data demonstrate that ACE inhibitors exhibit antimitotic activity and differential gene modulation in hamster pancreatic duct carcinoma cells. The absence of renin and ACE activity in these cells suggests that the antimitotic action of captopril and CGS 13945 is independent of renin-angiotensin regulation. The growth inhibition may occur through downregulation of growth-related gene expression. 27 refs., 5 figs.

  13. DPP-4 inhibitors

    DEFF Research Database (Denmark)

    Deacon, Carolyn F.

    2016-01-01

    Dipeptidyl peptidase (DPP)-4 inhibitors inhibit the activity of the enzyme responsible for the initial rapid degradation of the incretin hormones, thereby enhancing their antihyperglycemic effects.......Dipeptidyl peptidase (DPP)-4 inhibitors inhibit the activity of the enzyme responsible for the initial rapid degradation of the incretin hormones, thereby enhancing their antihyperglycemic effects....

  14. Glycosphingolipid synthesis inhibitor AMP-DNM lowers plasma cholesterol levels by promoting fecal cholesterol excretion without inhibiting cholesterol absorption

    NARCIS (Netherlands)

    Vrins, Carlos L. J.; Bietrix, Florence; Lombardo, Elisa; van Roomen, Cindy P. A. A.; Ottenhoff, Roelof; Overkleeft, Herman S.; Aerts, Johannes M.

    2012-01-01

    Inhibition of glycosphingolipid synthesis with iminosugar N-(5'-adamantane-1'-yl-methoxy)-pentyl-1-deoxynojirimycin (AMP-DNM) increases fecal neutral sterol output in mice. To investigate which pathways were involved in this increase, C57BI/6J mice were treated with AMP-DNM and/or ezetimibe. Fecal

  15. Effects of 17β-estradiol on radiation transformation in vitro; inhibition of effects by protease inhibitors

    International Nuclear Information System (INIS)

    Kennedy, A.R.; Weichselbaum, R.R.

    1981-01-01

    The effects of 17β-estradiol, given either alone or with X-radiation, on the induction of malignant transformation were investigated in vitro. Treatment with 10 -6 M 17β-estradiol for 6 weeks, or 10 -5 M 17β-estradiol for only 5 days, induced malignant transformation in C3H 10T1/2 cells. Estradiol also acted as a cocarcinogen for X-ray induced transformation; the results indicated an additive effect when the cells were exposed to both agents together. The protease inhibitors antipain and leupeptin suppressed estradiol induced transformation as well as the additive effect observed for estradiol-radiation transformation. (author)

  16. Ovostatin: a novel proteinase inhibitor from chicken egg white. II. Mechanism of inhibition studied with collagenase and thermolysin.

    Science.gov (United States)

    Nagase, H; Harris, E D

    1983-06-25

    The inhibition mechanism of ovostatin was studied using rabbit synovial collagenase and thermolysin. When enzymes were complexed with ovostatin, only the proteolytic activity towards high molecular weight substrates was inhibited. Activity towards low molecular weight substrates was partially modified: the catalytic activity of collagenase bound to ovostatin was inhibited by only 40% towards 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg and that of thermolysin bound to ovostatin was activated about 2.6-fold towards benzyloxycarbonyl-Gly-Leu-NH2 and benzyloxycarbonyl-Gly-Phe-NH2. Collagenase-ovostatin complexes failed to react with anti-(collagenase) antibody. Saturation of ovostatin with thermolysin prevented the subsequent binding of collagenase. Ovostatin-proteinase complexes ran faster than free ovostatin on 5% polyacrylamide gel electrophoresis. Complexing ovostatin with either collagenase or thermolysin resulted in the cleavage of the quarter-subunit of ovostatin (Mr = 165,000) into two fragments with Mr = 88,000 and 78,000. On the other hand, when the inhibitory capacity of ovostatin was tested with trypsin, chymotrypsin, and papain, only partial inhibition of their proteolytic activities was observed towards azocasein. Stronger inhibition was noted when Azocoll was a substrate, however. Analyses of ovostatin-enzyme complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the quarter-subunit of ovostatin was cleaved into several fragments by those enzymes. These results led us to propose that ovostatin inhibits metalloproteinases in preference to proteinases of other classes in a manner similar to alpha 2-macroglobulin; hydrolysis of a peptide bond by a proteinase in the susceptible region of the ovostatin polypeptide chain triggers a conformational change in the ovostatin molecule and the enzyme becomes bound to ovostatin in such a way that the proteinase is sterically hindered from access to large protein substrates and yet

  17. Brown Algae Polyphenol, a Prolyl Isomerase Pin1 Inhibitor, Prevents Obesity by Inhibiting the Differentiation of Stem Cells into Adipocytes.

    Science.gov (United States)

    Suzuki, Atsuko; Saeki, Toshiyuki; Ikuji, Hiroko; Uchida, Chiyoko; Uchida, Takafumi

    2016-01-01

    While screening for an inhibitor of the peptidyl prolyl cis/trans isomerase, Pin1, we came across a brown algae polyphenol that blocks the differentiation of fibroblasts into adipocytes. However, its effectiveness on the accumulation of fat in the body has never been studied. Oral administration of brown algae polyphenol to mice fed with a high fat diet, suppressed the increase in fat volume to a level observed in mice fed with a normal diet. We speculate that Pin1 might be required for the differentiation of stem cell to adipocytes. We established wild type (WT) and Pin1-/- (Pin1-KO) adipose-derived mesenchymal stem cell (ASC) lines and found that WT ASCs differentiate to adipocytes but Pin1-KO ASCs do not. Oral administration of brown algae polyphenol, a Pin1 inhibitor, reduced fat buildup in mice. We showed that Pin1 is required for the differentiation of stem cells into adipocytes. We propose that oral intake of brown algae polyphenol is useful for the treatment of obesity.

  18. Brown Algae Polyphenol, a Prolyl Isomerase Pin1 Inhibitor, Prevents Obesity by Inhibiting the Differentiation of Stem Cells into Adipocytes.

    Directory of Open Access Journals (Sweden)

    Atsuko Suzuki

    Full Text Available While screening for an inhibitor of the peptidyl prolyl cis/trans isomerase, Pin1, we came across a brown algae polyphenol that blocks the differentiation of fibroblasts into adipocytes. However, its effectiveness on the accumulation of fat in the body has never been studied.Oral administration of brown algae polyphenol to mice fed with a high fat diet, suppressed the increase in fat volume to a level observed in mice fed with a normal diet. We speculate that Pin1 might be required for the differentiation of stem cell to adipocytes. We established wild type (WT and Pin1-/- (Pin1-KO adipose-derived mesenchymal stem cell (ASC lines and found that WT ASCs differentiate to adipocytes but Pin1-KO ASCs do not.Oral administration of brown algae polyphenol, a Pin1 inhibitor, reduced fat buildup in mice. We showed that Pin1 is required for the differentiation of stem cells into adipocytes. We propose that oral intake of brown algae polyphenol is useful for the treatment of obesity.

  19. The farnesyltransferase inhibitor, LB42708, inhibits growth and induces apoptosis irreversibly in H-ras and K-ras-transformed rat intestinal epithelial cells

    International Nuclear Information System (INIS)

    Kim, Han-Soo; Kim, Ju Won; Gang, Jingu; Wen, Jing; Koh, Sang Seok; Koh, Jong Sung; Chung, Hyun-Ho; Song, Si Young

    2006-01-01

    LB42708 (LB7) and LB42908 (LB9) are pyrrole-based orally active farnesyltransferase inhibitors (FTIs) that have similar structures. The in vitro potencies of these compounds against FTase and GGTase I are remarkably similar, and yet they display different activity in apoptosis induction and morphological reversion of ras-transformed rat intestinal epithelial (RIE) cells. Both FTIs induced cell death despite K-ras prenylation, implying the participation of Ras-independent mechanism(s). Growth inhibition by these two FTIs was accompanied by G1 and G2/M cell cycle arrests in H-ras and K-ras-transformed RIE cells, respectively. We identified three key markers, p21 CIP1/WAF1 , RhoB and EGFR, that can explain the differences in the molecular mechanism of action between two FTIs. Only LB7 induced the upregulation of p21 CIP1/WAF1 and RhoB above the basal level that led to the cell cycle arrest and to distinct morphological alterations of ras-transformed RIE cells. Both FTIs successfully inhibited the ERK and activated JNK in RIE/K-ras cells. While the addition of conditioned medium from RIE/K-ras reversed the growth inhibition of ras-transformed RIE cells by LB9, it failed to overcome the growth inhibitory effect of LB7 in both H-ras- and K-ras-transformed RIE cells. We found that LB7, but not LB9, decreased the expression of EGFRs that confers the cellular unresponsiveness to EGFR ligands. These results suggest that LB7 causes the induction of p21 CIP1/WAF1 and RhoB and downregulation of EGFR that may serve as critical steps in the mechanism by which FTIs trigger irreversible inhibitions on the cell growth and apoptosis in ras-transformed cells

  20. Novel Insights Into The Mode of Inhibition of Class A SHV-1 Beta-Lactamases Revealed by Boronic Acid Transition State Inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    W Ke; J Sampson; C Ori; F Prati; S Drawz; C Bethel; R Bonomo; F van den Akker

    2011-12-31

    Boronic acid transition state inhibitors (BATSIs) are potent class A and C {beta}-lactamase inactivators and are of particular interest due to their reversible nature mimicking the transition state. Here, we present structural and kinetic data describing the inhibition of the SHV-1 {beta}-lactamase, a clinically important enzyme found in Klebsiella pneumoniae, by BATSI compounds possessing the R1 side chains of ceftazidime and cefoperazone and designed variants of the latter, compounds 1 and 2. The ceftazidime and cefoperazone BATSI compounds inhibit the SHV-1 {beta}-lactamase with micromolar affinity that is considerably weaker than their inhibition of other {beta}-lactamases. The solved crystal structures of these two BATSIs in complex with SHV-1 reveal a possible reason for SHV-1's relative resistance to inhibition, as the BATSIs adopt a deacylation transition state conformation compared to the usual acylation transition state conformation when complexed to other {beta}-lactamases. Active-site comparison suggests that these conformational differences might be attributed to a subtle shift of residue A237 in SHV-1. The ceftazidime BATSI structure revealed that the carboxyl-dimethyl moiety is positioned in SHV-1's carboxyl binding pocket. In contrast, the cefoperazone BATSI has its R1 group pointing away from the active site such that its phenol moiety moves residue Y105 from the active site via end-on stacking interactions. To work toward improving the affinity of the cefoperazone BATSI, we synthesized two variants in which either one or two extra carbons were added to the phenol linker. Both variants yielded improved affinity against SHV-1, possibly as a consequence of releasing the strain of its interaction with the unusual Y105 conformation.

  1. Design and Synthesis of New Dual Binding Site Cholinesterase Inhibitors: in vitro Inhibition Studies with in silico Docking.

    Science.gov (United States)

    Yar, Muhammad; Bajda, Marek; Mehmood, Rana Atif; Sidra, Lala Rukh; Ullah, Nisar; Shahzadi, Lubna; Ashraf, Muhammad; Ismail, Tayaba; Shahzad, Sohail Anjum; Khan, Zulfiqar Ali; Naqvi, Syed Ali Raza; Mahmood, Nasir

    2014-03-01

    Cholinesterases (ChEs) play a vital role in the regulation of cholinergic transmission. The inhibition of ChEs is considered to be involved in increasing acetylcholine level in the brain and thus has been implicated in the treatment of Alzheimer's disease. We have designed and synthesized a series of novel indole derivatives and screened them for inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Most of the tested compounds exhibited inhibitory activity against AChE and BChE. Among them 4f and 6e showed the highest AChE inhibitory activity with IC 50 91.21±0.06 and 68.52±0.04 μM, respectively. However compound 5a exhibited the highest inhibitory activity against BChE (IC 50 55.21±0.12 μM).

  2. PHA665752, a small-molecule inhibitor of c-Met, inhibits hepatocyte growth factor-stimulated migration and proliferation of c-Met-positive neuroblastoma cells

    International Nuclear Information System (INIS)

    Crosswell, Hal E; Dasgupta, Anindya; Alvarado, Carlos S; Watt, Tanya; Christensen, James G; De, Pradip; Durden, Donald L; Findley, Harry W

    2009-01-01

    c-Met is a tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF), and both c-Met and its ligand are expressed in a variety of tissues. C-Met/HGF/SF signaling is essential for normal embryogenesis, organogenesis, and tissue regeneration. Abnormal c-Met/HGF/SF signaling has been demonstrated in different tumors and linked to aggressive and metastatic tumor phenotypes. In vitro and in vivo studies have demonstrated inhibition of c-Met/HGF/SF signaling by the small-molecule inhibitor PHA665752. This study investigated c-Met and HGF expression in two neuroblastoma (NBL) cell lines and tumor tissue from patients with NBL, as well as the effects of PHA665752 on growth and motility of NBL cell lines. The effect of the tumor suppressor protein PTEN on migration and proliferation of tumor cells treated with PHA665752 was also evaluated. Expression of c-Met and HGF in NBL cell lines SH-EP and SH-SY5Y and primary tumor tissue was assessed by immunohistochemistry and quantitative RT-PCR. The effect of PHA665752 on c-Met/HGF signaling involved in NBL cell proliferation and migration was evaluated in c-Met-positive cells and c-Met-transfected cells. The transwell chemotaxis assay and the MTT assay were used to measure migration and proliferation/cell-survival of tumor cells, respectively. The PPAR-γ agonist rosiglitazone was used to assess the effect of PTEN on PHA665752-induced inhibition of NBL cell proliferation/cell-survival and migration High c-Met expression was detected in SH-EP cells and primary tumors from patients with advanced-stage disease. C-Met/HGF signaling induced both migration and proliferation of SH-EP cells. Migration and proliferation/cell-survival were inhibited by PHA665752 in a dose-dependent manner. We also found that induced overexpression of PTEN following treatment with rosiglitazone significantly enhanced the inhibitory effect of PHA665752 on NBL-cell migration and proliferation. c-Met is highly expressed in most tumors from

  3. Mitochondrial toxicity of selective COX-2 inhibitors via inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria

    DEFF Research Database (Denmark)

    Syed, Muzeeb; Skonberg, Christian; Hansen, Steen Honoré

    2016-01-01

    : 238.4 ± 79.2 μM)>etoricoxib (IC50: 405.1 ± 116.3 μM). Mechanism based inhibition of ATP synthesis (Kinact 0.078 min(-1) and KI 21.46 μM and Kinact/KI ratio 0.0036 min(-1)μM(-1)) was shown by lumiracoxib and data suggest that the opening of the MPT pore may not be the mechanism of toxicity. A positive...

  4. Carbonic anhydrase expression in kidney and renal cancer: implications for diagnosis and treatment

    NARCIS (Netherlands)

    Oosterwijk, E.

    2014-01-01

    Four different carbonic anhydrases are expressed in the human nephron, the functional unit of the kidney. These are specifically expressed in different nephron segments, emphasizing the critical role carbonic anhydrases play in maintaining the homeostasis of this crucial organ.Whereas the

  5. 35Cl and 81Br nuclear magnetic resonance studies of carbonic anhydrase

    International Nuclear Information System (INIS)

    Ward, R.L.

    1979-02-01

    35 Cl NMR studies substantiated the binding of Cl - to the Zn(II) of carbonic anhydrase. Zinc-free carbonic anhydrase was prepared and it exhibited essentially no effect on the Cl - line width. The net Cl - line width increased with temperature. 81 Br NMR was quite similar to 35 Cl in that its relaxation is dominated by quadrupolar interactions

  6. A trypsin inhibitor from Tecoma stans leaves inhibits growth and promotes ATP depletion and lipid peroxidation in Candida albicans and Candida krusei

    Directory of Open Access Journals (Sweden)

    Leydianne Leite de Siqueira Patriota

    2016-04-01

    Full Text Available Tecoma stans (yellow elder has shown medicinal properties and antimicrobial activity. Previous reports on antifungal activity of T. stans preparations and presence of trypsin inhibitor activity from T. stans leaves stimulated the investigation reported here. In this work, we proceeded to the purification and characterization of a trypsin inhibitor (TesTI, which was investigated for anti-Candida activity. Finally, in order to determine the potential of TesTI as a new natural chemotherapeutic product, its cytotoxicity to human peripheral blood mononuclear cells (PBMCs was evaluated. TesTI was isolated from saline extract by ammonium sulphate fractionation followed by ion exchange and gel filtration chromatographies. Antifungal activity was evaluated by determining the minimal inhibitory (MIC and fungicide (MFC concentrations using fungal cultures containing only yeast form or both yeast and hyphal forms. Candida cells treated with TesTI were evaluated for intracellular ATP levels and lipid peroxidation. Cytotoxicity of TesTI to PBMCs was evaluated by MTT assay. TesTI (39.8 kDa, pI 3.41, Ki 43 nM inhibited similarly the growth of both C. albicans and C. krusei culture types at MIC of 100 µg/mL. The MFCs were 200 µg/mL for C. albicans and C. krusei. Time-response curves revealed that TesTI (at MIC was more effective at inhibiting the replication of C. albicans cells. At MIC, TesTI promoted reduction of ATP levels and lipid peroxidation in the Candida cells, being not cytotoxic to PBMCs. In conclusion, TesTI is an antifungal agent against C. albicans and C. krusei, without toxicity to human cells.

  7. The dual mTORC1 and mTORC2 inhibitor AZD8055 inhibits head and neck squamous cell carcinoma cell growth in vivo and in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Li, Qiang; Song, Xin-mao; Ji, Yang-yang; Jiang, Hui; Xu, Lin-gen, E-mail: drlingenxu@126.com

    2013-11-01

    Highlights: •AZD8055 induces significant cytotoxic effects in cultured HNSCC cells. •AZD8055 blocks mTORC1 and mTORC2 activation in cultured HNSCC cells. •JNK activation is required for AZD8055-induced HNSCC cell death. •AZD8055 inhibits Hep-2 cell growth in vivo, and was more efficient than rapamycin. -- Abstract: The serine/threonine kinase mammalian target of rapamycin (mTOR) promotes cell survival and proliferation, and is constitutively activated in head and neck squamous cell carcinoma (HNSCC). Thus mTOR is an important target for drug development in this disease. Here we tested the anti-tumor ability of AZD8055, the novel mTOR inhibitor, in HNSCC cells. AZD8055 induced dramatic cell death of HNSCC lines (Hep-2 and SCC-9) through autophagy. AZD8055 blocked both mTOR complex (mTORC) 1 and mTORC2 activation without affecting Erk in cultured HNSCC cells. Meanwhile, AZD8055 induced significant c-Jun N-terminal kinase (JNK) activation, which was also required for cancer cell death. JNK inhibition by its inhibitors (SP 600125 and JNK-IN-8), or by RNA interference (RNAi) alleviated AZD8055-induced cell death. Finally, AZD8055 markedly increased the survival of Hep-2 transplanted mice through a significant reduction of tumor growth, without apparent toxicity, and its anti-tumor ability was more potent than rapamycin. Meanwhile, AZD8055 administration activated JNK while blocking mTORC1/2 in Hep-2 tumor engrafts. Our current results strongly suggest that AZD8055 may be further investigated for HNSCC treatment in clinical trials.

  8. The guanylhydrazone CNI-1493: an inhibitor with dual activity against malaria-inhibition of host cell pro-inflammatory cytokine release and parasitic deoxyhypusine synthase.

    Science.gov (United States)

    Specht, Sabine; Sarite, Salem Ramadan; Hauber, Ilona; Hauber, Joachim; Görbig, Ulf F; Meier, Chris; Bevec, Dorian; Hoerauf, Achim; Kaiser, Annette

    2008-05-01

    Malaria is still a major cause of death in the tropics. There is an urgent need for new anti-malarial drugs because drug-resistant plasmodia frequently occur. Over recent years, we elucidated the biosynthesis of hypusine, a novel amino acid contained in eukaryotic initiation factor 5A (eIF-5A) in Plasmodium. Hypusine biosynthesis involves catalysis of deoxyhypusine synthase (DHS) in the first step of post-translational modification. In a screen for new inhibitors of purified plasmodium DHS, CNI-1493, a novel selective pro-inflammatory cytokine inhibitor used in clinical phase II for the treatment of Crohn's disease, inhibited the enzyme of the parasite 3-fold at a concentration of 2 microM. In vitro experiments with 200 microM CNI-1493 in Plasmodium-infected erythrocytes, which lack nuclei and DHS protein, showed a parasite clearance within 2 days. This can presumably be attributed to an anti-proliferating effect because of the inhibition of DHS by the parasite. The determined IC50 of CNI-1493 was 135.79 microM after 72 h. In vivo application of this substance in Plasmodium berghei ANKA-infected C57BL/6 mice significantly reduced parasitemia after dosage of 1 mg/kg or 4 mg/kg/body weight and prevented death of mice with cerebral malaria. This effect was paralleled by a decrease in serum TNF levels of the mice. We suggest that the new mechanism of CNI-1493 is caused by a decrease in modified eIF-5A biosynthesis with a downstream effect on the TNF synthesis of the host. From the current data, we consider CNI-1493 to be a promising drug for anti-malarial therapy because of its combined action, i.e., the decrease in eIF-5A biosynthesis of the parasite and host cell TNF biosynthesis.

  9. Alpha-amylase inhibitor-1 gene from Phaseolus vulgaris expressed in Coffea arabica plants inhibits alpha-amylases from the coffee berry borer pest.

    Science.gov (United States)

    Barbosa, Aulus E A D; Albuquerque, Erika V S; Silva, Maria C M; Souza, Djair S L; Oliveira-Neto, Osmundo B; Valencia, Arnubio; Rocha, Thales L; Grossi-de-Sa, Maria F

    2010-06-17

    Coffee is an important crop and is crucial to the economy of many developing countries, generating around US$70 billion per year. There are 115 species in the Coffea genus, but only two, C. arabica and C. canephora, are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer (Hypotheneumus hampei), is responsible for worldwide annual losses of around US$500 million. The coffee berry borer exclusively damages the coffee berries, and it is mainly controlled by organochlorine insecticides that are both toxic and carcinogenic. Unfortunately, natural resistance in the genus Coffea to H. hampei has not been documented. To overcome these problems, biotechnological strategies can be used to introduce an alpha-amylase inhibitor gene (alpha-AI1), which confers resistance against the coffee berry borer insect-pest, into C. arabica plants. We transformed C. arabica with the alpha-amylase inhibitor-1 gene (alpha-AI1) from the common bean, Phaseolus vulgaris, under control of the seed-specific phytohemagglutinin promoter (PHA-L). The presence of the alpha-AI1 gene in six regenerated transgenic T1 coffee plants was identified by PCR and Southern blotting. Immunoblotting and ELISA experiments using antibodies against alpha-AI1 inhibitor showed a maximum alpha-AI1 concentration of 0.29% in crude seed extracts. Inhibitory in vitro assays of the alpha-AI1 protein against H. hampei alpha-amylases in transgenic seed extracts showed up to 88% inhibition of enzyme activity. This is the first report showing the production of transgenic coffee plants with the biotechnological potential to control the coffee berry borer, the most important insect-pest of crop coffee.

  10. α-Amylase inhibitor-1 gene from Phaseolus vulgaris expressed in Coffea arabica plants inhibits α-amylases from the coffee berry borer pest

    Directory of Open Access Journals (Sweden)

    Oliveira-Neto Osmundo B

    2010-06-01

    Full Text Available Abstract Background Coffee is an important crop and is crucial to the economy of many developing countries, generating around US$70 billion per year. There are 115 species in the Coffea genus, but only two, C. arabica and C. canephora, are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer (Hypotheneumus hampei, is responsible for worldwide annual losses of around US$500 million. The coffee berry borer exclusively damages the coffee berries, and it is mainly controlled by organochlorine insecticides that are both toxic and carcinogenic. Unfortunately, natural resistance in the genus Coffea to H. hampei has not been documented. To overcome these problems, biotechnological strategies can be used to introduce an α-amylase inhibitor gene (α-AI1, which confers resistance against the coffee berry borer insect-pest, into C. arabica plants. Results We transformed C. arabica with the α-amylase inhibitor-1 gene (α-AI1 from the common bean, Phaseolus vulgaris, under control of the seed-specific phytohemagglutinin promoter (PHA-L. The presence of the α-AI1 gene in six regenerated transgenic T1 coffee plants was identified by PCR and Southern blotting. Immunoblotting and ELISA experiments using antibodies against α-AI1 inhibitor showed a maximum α-AI1 concentration of 0.29% in crude seed extracts. Inhibitory in vitro assays of the α-AI1 protein against H. hampei α-amylases in transgenic seed extracts showed up to 88% inhibition of enzyme activity. Conclusions This is the first report showing the production of transgenic coffee plants with the biotechnological potential to control the coffee berry borer, the most important insect-pest of crop coffee.

  11. Nitrifier-induced denitrification is an important source of soil nitrous oxide and can be inhibited by a nitrification inhibitor 3,4-dimethylpyrazole phosphate.

    Science.gov (United States)

    Shi, Xiuzhen; Hu, Hang-Wei; Zhu-Barker, Xia; Hayden, Helen; Wang, Juntao; Suter, Helen; Chen, Deli; He, Ji-Zheng

    2017-12-01

    Soil ecosystem represents the largest contributor to global nitrous oxide (N 2 O) production, which is regulated by a wide variety of microbial communities in multiple biological pathways. A mechanistic understanding of these N 2 O production biological pathways in complex soil environment is essential for improving model performance and developing innovative mitigation strategies. Here, combined approaches of the 15 N- 18 O labelling technique, transcriptome analysis, and Illumina MiSeq sequencing were used to identify the relative contributions of four N 2 O pathways including nitrification, nitrifier-induced denitrification (nitrifier denitrification and nitrification-coupled denitrification) and heterotrophic denitrification in six soils (alkaline vs. acid soils). In alkaline soils, nitrification and nitrifier-induced denitrification were the dominant pathways of N 2 O production, and application of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) significantly reduced the N 2 O production from these pathways; this is probably due to the observed reduction in the expression of the amoA gene in ammonia-oxidizing bacteria (AOB) in the DMPP-amended treatments. In acid soils, however, heterotrophic denitrification was the main source for N 2 O production, and was not impacted by the application of DMPP. Our results provide robust evidence that the nitrification inhibitor DMPP can inhibit the N 2 O production from nitrifier-induced denitrification, a potential significant source of N 2 O production in agricultural soils. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  12. Endocrine disruptors and other inhibitors of 11β-hydroxysteroid dehydrogenase 1 and 2: Tissue-specific consequences of enzyme inhibition.

    Science.gov (United States)

    Vitku, Jana; Starka, Luboslav; Bicikova, Marie; Hill, Martin; Heracek, Jiri; Sosvorova, Lucie; Hampl, Richard

    2016-01-01

    Numerous chemicals in the environment have the ability to interact with the endocrine system. These compounds are called endocrine disruptors (EDs). Exposure to EDs represents one of the hypotheses for decreasing fertility, the increased risk of numerous cancers and obesity, metabolic syndrome and type 2 diabetes. There are various mechanisms of ED action, one of which is their interference in the action of 11β-hydroxysteroid dehydrogenase (11βHSD) that maintains a balance between active and inactive glucocorticoids on the intracellular level. This enzyme has two isoforms and is expressed in various tissues. Inhibition of 11βHSD in various tissues can have different consequences. In the case of EDs, the results of exposure are mainly adverse; on the other hand pharmaceutically developed inhibitors of 11βHSD type 1 are evaluated as an option for treating metabolic syndrome, as well as related diseases and depressive disorders. This review focuses on the effects of 11βHSD inhibitors in the testis, colon, adipose tissue, kidney, brain and placenta. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. New structure–activity relationships of chalcone inhibitors of breast cancer resistance protein: polyspecificity toward inhibition and critical substitutions against cytotoxicity

    Science.gov (United States)

    Rangel, Luciana Pereira; Winter, Evelyn; Gauthier, Charlotte; Terreux, Raphaël; Chiaradia-Delatorre, Louise D; Mascarello, Alessandra; Nunes, Ricardo J; Yunes, Rosendo A; Creczynski-Pasa, Tania B; Macalou, Sira; Lorendeau, Doriane; Baubichon-Cortay, Hélène; Ferreira-Pereira, Antonio; Di Pietro, Attilio

    2013-01-01

    Adenosine triphosphate-binding cassette subfamily G member 2 (ABCG2) plays a major role in cancer cell multidrug resistance, which contributes to low eifficacy of chemotherapy. Chalcones were recently found to be potent and specific inhibitors, but unfortunately display a significant cytotoxicity. A cellular screening against ABCG2-mediated mitoxantrone efflux was performed here by flow cytometry on 54 chalcone derivatives from three different series with a wide panel of substituents. The identified leads, with submicromolar IC50 (half maximal inhibitory concentration) values, showed that the previously identified 2′-OH-4′,6′-dimethoxyphenyl, as A-ring, could be efficiently replaced by a 2′-naphthyl group, or a 3′,4′-methylenedioxyphenyl with lower affinity. Such a structural variability indicates 3polyspecificity of the multidrug transporter for inhibitors. At least two methoxyl groups were necessary on B-ring for optimal inhibition, but substitution at positions 3, 4, and 5 induced cytotoxicity. The presence of a large O-benzyl substituent at position 4 and a 2′-naphthyl as A-ring markedly decreased the cytotoxicity, giving a high therapeutic ratio, which constitutes a critical requirement for future in-vivo assays in animal models. PMID:24109177

  14. The gap junction inhibitor 2-aminoethoxy-diphenyl-borate protects against acetaminophen hepatotoxicity by inhibiting cytochrome P450 enzymes and c-jun N-terminal kinase activation

    Energy Technology Data Exchange (ETDEWEB)

    Du, Kuo; Williams, C. David; McGill, Mitchell R.; Xie, Yuchao [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Farhood, Anwar [Department of Pathology, St. David' s North Austin Medical Center, Austin, TX 78756 (United States); Vinken, Mathieu [Department of Toxicology, Center for Pharmaceutical Sciences, Vrije Universiteit Brussels, 1090 Brussels (Belgium); Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States)

    2013-12-15

    Acetaminophen (APAP) hepatotoxicity is the leading cause of acute liver failure in the US. Although many aspects of the mechanism are known, recent publications suggest that gap junctions composed of connexin32 function as critical intercellular communication channels which transfer cytotoxic mediators into neighboring hepatocytes and aggravate liver injury. However, these studies did not consider off-target effects of reagents used in these experiments, especially the gap junction inhibitor 2-aminoethoxy-diphenyl-borate (2-APB). In order to assess the mechanisms of protection of 2-APB in vivo, male C56Bl/6 mice were treated with 400 mg/kg APAP to cause extensive liver injury. This injury was prevented when animals were co-treated with 20 mg/kg 2-APB and was attenuated when 2-APB was administered 1.5 h after APAP. However, the protection was completely lost when 2-APB was given 4–6 h after APAP. Measurement of protein adducts and c-jun-N-terminal kinase (JNK) activation indicated that 2-APB reduced both protein binding and JNK activation, which correlated with hepatoprotection. Although some of the protection was due to the solvent dimethyl sulfoxide (DMSO), in vitro experiments clearly demonstrated that 2-APB directly inhibits cytochrome P450 activities. In addition, JNK activation induced by phorone and tert-butylhydroperoxide in vivo was inhibited by 2-APB. The effects against APAP toxicity in vivo were reproduced in primary cultured hepatocytes without use of DMSO and in the absence of functional gap junctions. We conclude that the protective effect of 2-APB was caused by inhibition of metabolic activation of APAP and inhibition of the JNK signaling pathway and not by blocking connexin32-based gap junctions. - Highlights: • 2-APB protected against APAP-induced liver injury in mice in vivo and in vitro • 2-APB protected by inhibiting APAP metabolic activation and JNK signaling pathway • DMSO inhibited APAP metabolic activation as the solvent of 2-APB

  15. Effects of 17 beta-estradiol on radiation transformation in vitro; inhibition of effects by protease inhibitors

    International Nuclear Information System (INIS)

    Kennedy, A.R.; Weichselbaum, R.R.

    1981-01-01

    We have investigated the effects of 17 beta-estradiol, given both alone and with X-irradiation, on the induction of malignant transformation in vitro. Treatment with 10(-6)M 17 beta-estradiol for 6 weeks, or 10(-5)M 17 beta-estradiol for only 5 days, induced malignant transformation in C3H 10T1/2 cells. Estradiol also acted as a cocarcinogen for X-ray induced transformation; the results indicate an additive effect when the cells were exposed to both agents together. The protease inhibitors antipain and leupeptin suppressed estradiol induced transformation as well as the additive effect observed for estradiol-radiation transformation

  16. Effects of 17 beta-estradiol on radiation transformation in vitro; inhibition of effects by protease inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Kennedy, A.R.; Weichselbaum, R.R.

    1981-01-01

    We have investigated the effects of 17 beta-estradiol, given both alone and with X-irradiation, on the induction of malignant transformation in vitro. Treatment with 10(-6)M 17 beta-estradiol for 6 weeks, or 10(-5)M 17 beta-estradiol for only 5 days, induced malignant transformation in C3H 10T1/2 cells. Estradiol also acted as a cocarcinogen for X-ray induced transformation; the results indicate an additive effect when the cells were exposed to both agents together. The protease inhibitors antipain and leupeptin suppressed estradiol induced transformation as well as the additive effect observed for estradiol-radiation transformation.

  17. The H+/K+ ATPase Inhibitor SCH-28080 Inhibits Insulin Secretion and Induces Cell Death in INS-1E Rat Insulinoma Cells

    Directory of Open Access Journals (Sweden)

    Martin Jakab

    2017-10-01

    Full Text Available Background/Aims: Glucose-stimulated insulin secretion (GSIS of pancreatic β-cells involves glucose uptake and metabolism, closure of KATP channels and depolarization of the cell membrane potential (Vmem, activation of voltage-activated Ca2+ currents (ICav and influx of Ca2+, which eventually triggers hormone exocytosis. Beside this classical pathway, KATP-independent mechanisms such as changes in intracellular pH (pHi or cell volume, which also affect β-cell viability, can elicit or modify insulin release. In β-cells the regulation of pHi is mainly accomplished by Na+/H+ exchangers (NHEs. To investigate if other proton extrusion mechanisms than NHEs are involved in pH regulation, we tested for the presence of the non-gastric H+/K+ ATPase in rat insulinoma cells and assessed effects of the H+/K+ ATPase inhibitor SCH-28080 on insulin secretion, cell viability and apoptosis. Methods: In INS-1E cell cultures, H+/K+ ATPase gene and protein expression was analyzed by reverse transcription PCR and Western blotting. Intracellular pH (pHi recovery after acute acidic load was measured by NH4Cl prepulsing using BCECF. Insulin secretion was determined by ELISA from the cell culture supernatant. Vmem, K+ and Ca2+ currents were recorded using patch clamp. Overall cell responses were determined using resazurin (viability and cytotoxicity assays. The mean cell volume (MCV, cell granularity (side-scatter; SSC, phosphatidylserine (PS exposure, cell membrane integrity, caspase activity and the mitochondrial membrane potential (ΔΨm were measured by flow cytometry. Results: We found that the α-subunit of the non-gastric H+/K+ ATPase (HKα2 is expressed on mRNA and protein level. However, compared to rat colon tissue, in INS-1E cells mRNA abundance was very low. In NH4Cl prepulsing experiments no K+-dependent pHi recovery was observed under Na+-free extracellular conditions. Nonetheless within 1 h, 20 µM SCH-28080 inhibited GSIS by ∼50%, while basal release

  18. A Jak1/2 inhibitor, baricitinib, inhibits osteoclastogenesis by suppressing RANKL expression in osteoblasts in vitro

    OpenAIRE

    Murakami, Kohei; Kobayashi, Yasuhiro; Uehara, Shunsuke; Suzuki, Takako; Koide, Masanori; Yamashita, Teruhito; Nakamura, Midori; Takahashi, Naoyuki; Kato, Hiroyuki; Udagawa, Nobuyuki; Nakamura, Yukio

    2017-01-01

    The Janus kinases (Jaks) are hubs in the signaling process of more than 50 cytokine or hormone receptors. However, the function of Jak in bone metabolism remains to be elucidated. Here, we showed that the inhibition of Jak1 and/or Jak2 in osteoblast-lineage cells led to impaired osteoclastogenesis due to the reduced expression of receptor activator of nuclear factor-κB ligand (RANKL). Murine calvaria-derived osteoblasts induced differentiation of bone marrow cells into osteoclasts in the pres...

  19. Enzyme inhibition by iminosugars

    DEFF Research Database (Denmark)

    López, Óscar; Qing, Feng-Ling; Pedersen, Christian Marcus

    2013-01-01

    Imino- and azasugar glycosidase inhibitors display pH dependant inhibition reflecting that both the inhibitor and the enzyme active site have groups that change protonation state with pH. With the enzyme having two acidic groups and the inhibitor one basic group, enzyme-inhibitor complexes...

  20. Rituximab effectively reverses Tyrosine kinase inhibitors (TKIs) resistance through inhibiting the accumulation of rictor on mitochondria-associated ER-membrane (MAM).

    Science.gov (United States)

    Xu, Zhi-Hong; Liu, Cai-Hong; Hang, Jun-Biao; Gao, Bei-Li; Hu, Jia-An

    2017-12-06

    Tyrosine kinase inhibitors (TKIs), a novel group of target-specific anti lung cancer drugs, have recently been found to resistant to some NSCLC cells which have the T790M EGFR mutation. However, recent investigations on the therapies of resistance to EGFR-TKIs are very limited. Therefore, it is important to develop more effective therapies to reverse EGFR-TKIs resistance. In our present study, erlotinib was used as the TKIs drug and the effects of the erlotinib on cell growth were evaluated. Cell viability and concentration dependent studies were performed using HCI-H1975 and HCI-H1299 cells alone with erlotinib, respectively. Further combined with rituximab, the results showed that erlotinib and rituximab were significantly inhibited the cell growth. Furthermore, the combination of erlotinib and rituximab greatly decreased the expression of p-mTOR and p-EGFR. Additional results from western blotting and immunofluorescence assays demonstrated that the accumulation of rictor was also decreased on MAM. Thus, all these results suggested that EGFR-TKIs combined with CD20 mono-antibody significantly decrease the cell growth of H1975 cells and H1299, with T790M EGFR mutation, and inhibit the localization of the key mTOR pathway proteins to MAM. So, it may be a promising strategy for overcoming EGFR TKI resistance in NSCLC patients.

  1. 1-L-MT, an IDO inhibitor, prevented colitis-associated cancer by inducing CDC20 inhibition-mediated mitotic death of colon cancer cells.

    Science.gov (United States)

    Liu, Xiuting; Zhou, Wei; Zhang, Xin; Ding, Yang; Du, Qianming; Hu, Rong

    2018-04-01

    Indoleamine 2,3-dioxygenase 1 (IDO1), known as IDO, catabolizes tryptophan through kynurenine pathway, whose activity is correlated with impaired clinical outcome of colorectal cancer. Here we showed that 1-L-MT, a canonical IDO inhibitor, suppressed proliferation of human colorectal cancer cells through inducing mitotic death. Our results showed that inhibition of IDO decreased the transcription of CDC20, which resulted in G2/M cycle arrest of HCT-116 and HT-29. Furthermore, 1-L-MT induced mitochondria injuries and caused apoptotic cancer cells. Importantly, 1-L-MT protected mice from azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colon carcinogenesis, with reduced mortality, tumor number and size. What is more, IDO1-/- mice exhibited fewer tumor burdens and reduced proliferation in the neoplastic epithelium, while, 1-L-MT did not exhibit any further protective effects on IDO-/- mice, confirming the critical role of IDO and the protective effect of 1-L-MT-mediated IDO inhibition in CRC. Furthermore, 1-L-MT also alleviated CRC in Rag1-/- mice, demonstrating the modulatory effects of IDO independent of its role in modulating adaptive immunity. Taken together, our findings validated that the anti-proliferation effect of 1-L-MT in vitro and the prevention of CRC in vivo were through IDO-induced cell cycle disaster of colon cancer cells. Our results identified 1-L-MT as a promising candidate for the chemoprevention of CRC. © 2018 UICC.

  2. XRP44X, an Inhibitor of Ras/Erk Activation of the Transcription Factor Elk3, Inhibits Tumour Growth and Metastasis in Mice.

    Directory of Open Access Journals (Sweden)

    Kostyantyn Semenchenko

    Full Text Available Transcription factors have an important role in cancer but are difficult targets for the development of tumour therapies. These factors include the Ets family, and in this study Elk3 that is activated by Ras oncogene /Erk signalling, and is involved in angiogenesis, malignant progression and epithelial-mesenchymal type processes. We previously described the identification and in-vitro characterisation of an inhibitor of Ras / Erk activation of Elk3 that also affects microtubules, XRP44X. We now report an initial characterisation of the effects of XRP44X in-vivo on tumour growth and metastasis in three preclinical models mouse models, subcutaneous xenografts, intra-cardiac injection-bone metastasis and the TRAMP transgenic mouse model of prostate cancer progression. XRP44X inhibits tumour growth and metastasis, with limited toxicity. Tumours from XRP44X-treated animals have decreased expression of genes containing Elk3-like binding motifs in their promoters, Elk3 protein and phosphorylated Elk3, suggesting that perhaps XRP44X acts in part by inhibiting the activity of Elk3. Further studies are now warranted to develop XRP44X for tumour therapy.

  3. Discovery of Potent Cysteine-Containing Dipeptide Inhibitors against Tyrosinase: A Comprehensive Investigation of 20 × 20 Dipeptides in Inhibiting Dopachrome Formation.

    Science.gov (United States)

    Tseng, Tien-Sheng; Tsai, Keng-Chang; Chen, Wang-Chuan; Wang, Yeng-Tseng; Lee, Yu-Ching; Lu, Chung-Kuang; Don, Ming-Jaw; Chang, Chang-Yu; Lee, Ching-Hsiao; Lin, Hui-Hsiung; Hsu, Hung-Ju; Hsiao, Nai-Wan

    2015-07-15

    Tyrosinase is an essential copper-containing enzyme required for melanin synthesis. The overproduction and abnormal accumulation of melanin cause hyperpigmentation and neurodegenerative diseases. Thus, tyrosinase is promising for use in medicine and cosmetics. Our previous study identified a natural product, A5, resembling the structure of the dipeptide WY and apparently inhibiting tyrosinase. Here, we comprehensively estimated the inhibitory capability of 20 × 20 dipeptides against mushroom tyrosinase. We found that cysteine-containing dipeptides, directly blocking the active site of tyrosinase, are highly potent in inhibition; in particular, N-terminal cysteine-containing dipeptides markedly outperform the C-terminal-containing ones. The cysteine-containing dipeptides, CE, CS, CY, and CW, show comparative bioactivities, and tyrosine-containing dipeptides are substrate-like inhibitors. The dipeptide PD attenuates 16.5% melanin content without any significant cytotoxicity. This study reveals the functional role of cysteine residue positional preference and the selectivity of specific amino acids in cysteine-containing dipeptides against tyrosinase, aiding in developing skin-whitening products.

  4. ASP5878, a Novel Inhibitor of FGFR1, 2, 3, and 4, Inhibits the Growth of FGF19-Expressing Hepatocellular Carcinoma.

    Science.gov (United States)

    Futami, Takashi; Okada, Hidetsugu; Kihara, Rumi; Kawase, Tatsuya; Nakayama, Ayako; Suzuki, Tomoyuki; Kameda, Minoru; Shindoh, Nobuaki; Terasaka, Tadashi; Hirano, Masaaki; Kuromitsu, Sadao

    2017-01-01

    Hepatocellular carcinoma is an aggressive cancer with poor prognosis. Fibroblast growth factor 19, a member of the fibroblast growth factor family, is a ligand for fibroblast growth factor receptor 4. Moreover, it plays a crucial role in the progression of hepatocellular carcinoma. ASP5878 is a novel inhibitor of fibroblast growth factor receptors 1, 2, 3, and 4 that is under development. It inhibits fibroblast growth factor receptor 4 kinase activity with an IC 50 of 3.5 nmol/L. ASP5878 potently suppressed the growth of the fibroblast growth factor 19-expressing hepatocellular carcinoma cell lines Hep3B2.1-7, HuH-7, and JHH-7. In the Hep3B2.1-7 cell line, ASP5878 inhibited the phosphorylation of fibroblast growth factor receptor 4 and its downstream signaling molecules as well as induced apoptosis. Oral administration of ASP5878 at 3 mg/kg induced sustained tumor regression in a subcutaneous xenograft mouse model using Hep3B2.1-7. In HuH-7, an orthotopic xenograft mouse model, ASP5878 induced complete tumor regression and dramatically extended the survival of the mice. These results suggest that ASP5878 is a potentially effective therapeutic agent for hepatocellular carcinoma patients with tumors expressing fibroblast growth factor 19. Mol Cancer Ther; 16(1); 68-75. ©2016 AACR. ©2016 American Association for Cancer Research.

  5. The lysine deacetylase inhibitor givinostat inhibits ß-cell IL-1ß induced IL-1ß transcription and processing

    DEFF Research Database (Denmark)

    Dahllöf, Mattias Salling; Christensen, Dan P; Lundh, Morten

    2012-01-01

    breaks an autoinflammatory circuit by differentially preventing ß-cell expression of the ß-cell toxic inflammatory molecules IL-1ß and CXCL10 induced by single cytokines. Results: CXCL10 did not induce transcription of IL-1ß mRNA. IL-1ß induced ß-cell IL-1ß mRNA and both IL-1ß and IFN¿ individually...... induced Cxcl10 mRNA transcription. Givinostat inhibited IL-1ß-induced IL-1ß mRNA expression in INS-1 and rat islets and IL-1ß processing in INS-1 cells. Givinostat also reduced IFN¿ induced Cxcl10 transcription in INS-1 cells but not in rat islets, while IL-1ß induced Cxcl10 transcription was unaffected....../Interpretation: Inhibition of ß-cell IL-1ß expression and processing and Cxcl10 transcription contributes to the ß-cell protective actions of KDACi. In vitro ß-cell destructive effects of CXCL10 are not mediated via IL-1ß transcription. The differential proinflammatory actions of KDACs may be attractive novel drug targets...

  6. [HSP90 Inhibitor 17-AAG Inhibits Multiple Myeloma Cell Proliferation by Down-regulating Wnt/β-Catenin Signaling Pathway].

    Science.gov (United States)

    Chen, Kan-Kan; He, Zheng-Mei; Ding, Bang-He; Chen, Yue; Zhang, Li-Juan; Yu, Liang; Gao, Jian

    2016-02-01

    To investigate the inhibitory effect of HSP90 inhibitory 17-AAG on proliferation of multiple myeloma cells and its main mechanism. The multiple myeloma cells U266 were treated with 17-AAG of different concentrations (200, 400, 600 and 800 nmol/L) for 24, 48, and 72 hours respectively, then the proliferation rate, expression levels of β-catenin and C-MYC protein, as well as cell cycle of U266 cells were treated with 17-AAG and were detected by MTT method, Western blot and flow cytometry, respectively. The 17-AAG showed inhibitory effect on the proliferation of U266 cells in dose- and time-depetent manners (r = -0.518, P AAG displayed no inhibitory effect on proliferation of U266 cells (P > 0.05). The result of culturing U266 cells for 72 hours by 17-AAG of different concentrations showed that the more high of 17-AAG concentration, the more low level of β-catenin and C-MYC proteins (P AAG concentration, the more high of cell ratio in G1 phase (P AAG, the more long time of culture, the more high of cell ratio in G1 phase (P AAG can inhibit the proliferation of multiple myeloma cells, the down-regulation of Wnt/β-catenin signaling pathway and inhibition of HSP90 expression may be the main mechnisms of 17-AAG effect.

  7. Inhibition of Human Steroid 5-Reductase (AKR1D1) by Finasteride and Structure of the Enzyme-Inhibitor Complex

    Energy Technology Data Exchange (ETDEWEB)

    Drury, J.; Di Costanzo, L; Penning, T; Christianson, D

    2009-01-01

    The {Delta}{sup 4}-3-ketosteroid functionality is present in nearly all steroid hormones apart from estrogens. The first step in functionalization of the A-ring is mediated in humans by steroid 5{alpha}- or 5{beta}-reductase. Finasteride is a mechanism-based inactivator of 5{alpha}-reductase type 2 with subnanomolar affinity and is widely used as a therapeutic for the treatment of benign prostatic hyperplasia. It is also used for androgen deprivation in hormone-dependent prostate carcinoma, and it has been examined as a chemopreventive agent in prostate cancer. The effect of finasteride on steroid 5{beta}-reductase (AKR1D1) has not been previously reported. We show that finasteride competitively inhibits AKR1D1 with low micromolar affinity but does not act as a mechanism-based inactivator. The structure of the AKR1D1 {center_dot} NADP{sup +} {center_dot} finasteride complex determined at 1.7 {angstrom} resolution shows that it is not possible for NADPH to reduce the {Delta}{sup 1-2}-ene of finasteride because the cofactor and steroid are not proximal to each other. The C3-ketone of finasteride accepts hydrogen bonds from the catalytic residues Tyr-58 and Glu-120 in the active site of AKR1D1, providing an explanation for the competitive inhibition observed. This is the first reported structure of finasteride bound to an enzyme involved in steroid hormone metabolism.

  8. Carbonic anhydrase generates CO2 and H+ that drive spider silk formation via opposite effects on the terminal domains.

    Directory of Open Access Journals (Sweden)

    Marlene Andersson

    2014-08-01

    Full Text Available Spider silk fibers are produced from soluble proteins (spidroins under ambient conditions in a complex but poorly understood process. Spidroins are highly repetitive in sequence but capped by nonrepetitive N- and C-terminal domains (NT and CT that are suggested to regulate fiber conversion in similar manners. By using ion selective microelectrodes we found that the pH gradient in the silk gland is much broader than previously known. Surprisingly, the terminal domains respond in opposite ways when pH is decreased from 7 to 5: Urea denaturation and temperature stability assays show that NT dimers get significantly stabilized and then lock the spidroins into multimers, whereas CT on the other hand is destabilized and unfolds into ThT-positive β-sheet amyloid fibrils, which can trigger fiber formation. There is a high carbon dioxide pressure (pCO2 in distal parts of the gland, and a CO2 analogue interacts with buried regions in CT as determined by nuclear magnetic resonance (NMR spectroscopy. Activity staining of histological sections and inhibition experiments reveal that the pH gradient is created by carbonic anhydrase. Carbonic anhydrase activity emerges in the same region of the gland as the opposite effects on NT and CT stability occur. These synchronous events suggest a novel CO2 and proton-dependent lock and trigger mechanism of spider silk formation.

  9. Functional interaction between bicarbonate transporters and carbonic anhydrase modulates lactate uptake into mouse cardiomyocytes.

    Science.gov (United States)

    Peetz, Jan; Barros, L Felipe; San Martín, Alejandro; Becker, Holger M

    2015-07-01

    Blood-derived lactate is a precious energy substrate for the heart muscle. Lactate is transported into cardiomyocytes via monocarboxylate transporters (MCTs) together with H(+), which couples lactate uptake to cellular pH regulation. In this study, we have investigated how the interplay between different acid/base transporters and carbonic anhydrases (CA), which catalyze the reversible hydration of CO2, modulates the uptake of lactate into isolated mouse cardiomyocytes. Lactate transport was estimated both as lactate-induced acidification and as changes in intracellular lactate levels measured with a newly developed Förster resonance energy transfer (FRET) nanosensor. Recordings of intracellular pH showed an increase in the rate of lactate-induced acidification when CA was inhibited by 6-ethoxy-2-benzothiazolesulfonamide (EZA), while direct measurements of lactate flux demonstrated a decrease in MCT transport activity, when CA was inhibited. The data indicate that catalytic activity of extracellular CA increases lactate uptake and counteracts intracellular lactate-induced acidification. We propose a hypothetical model, in which HCO3 (-), formed from cell-derived CO2 at the outer surface of the cardiomyocyte plasma membrane by membrane-anchored, extracellular CA, is transported into the cell via Na(+)/HCO3 (-) cotransport to counteract intracellular acidification, while the remaining H(+) stabilizes extracellular pH at the surface of the plasma membrane during MCT activity to enhance lactate influx into cardiomyocytes.

  10. Adenosine 3',5'-cyclic monophosphate (cAMP)-dependent phosphoregulation of mitochondrial complex I is inhibited by nucleoside reverse transcriptase inhibitors

    International Nuclear Information System (INIS)

    Lund, Kaleb C.; Wallace, Kendall B.

    2008-01-01

    Nucleoside analog reverse transcriptase inhibitors (NRTIs) are known to directly inhibit mitochondrial complex I activity as well as various mitochondrial kinases. Recent observations that complex I activity and superoxide production are modulated through cAMP-dependent phosphorylation suggests a mechanism through which NRTIs may affect mitochondrial respiration via kinase-dependent protein phosphorylation. In the current study, we examine the potential for NRTIs to inhibit the cAMP-dependent phosphorylation of complex I and the associated NADH:CoQ oxidoreductase activities and rates of superoxide production using HepG2 cells. Phosphoprotein staining of immunocaptured complex I revealed that 3'-azido-3'-deoxythymidine (AZT; 10 and 50 μM), AZT monophosphate (150 μM), and 2',3'-dideoxycytidine (ddC; 1 μM) prevented the phosphorylation of the NDUFB11 subunit of complex I. This was associated with a decrease in complex I activity with AZT and AZT monophosphate only. In the presence of succinate, superoxide production was increased with 2',3'-dideoxyinosine (ddI; 10 μM) and ddC (1 μM). In the presence of succinate + cAMP, AZT showed an inverse dose-dependent effect on superoxide production. None of the NRTIs examined inhibit PKA activity suggesting that the observed effects are due to a direct interaction with complex I. These data demonstrate a direct effect of NRTIs on cAMP-dependent regulation of mitochondrial bioenergetics independent of DNA polymerase-γ activity; in the case of AZT, these observations may provide a mechanism for the observed long-term toxicity with this drug

  11. The inhibitor of connexin Cx36 channels, mefloquine, inhibits voltage-dependent Ca2+channels and insulin secretion.

    Science.gov (United States)

    Seemann, Nele; Welling, Andrea; Rustenbeck, Ingo

    2017-12-05

    The antimalarial agent, mefloquine, inhibits the function of connexin Cx36 gap junctions and hemichannels and has thus become a tool to investigate their physiological relevance in pancreatic islets. In view of earlier reports on a K ATP channel-block by mefloquine, the specificity of mefloquine as a pharmacological tool was investigated. Mouse pancreatic islets and single beta cells were used to measure membrane potential, whole cell currents, Ca 2+ channel activity, cytosolic Ca 2+ concentration ([Ca 2+ ] i ) and insulin secretion. Mefloquine was tested in the concentration range of 5-50 μM 25 μM mefloquine was as effective as 500 μM tolbutamide to depolarize the plasma membrane of beta cells, but did not induce action potentials. Rather, it abolished tolbutamide-induced action potentials and the associated increase of [Ca 2+ ] i . In the range of 5-50 μM mefloquine inhibited voltage-dependent Ca 2+ currents in primary beta cells as effectively as 1 μM nisoldipine, a specific blocker of L-type Ca 2+ channels. The Ca 2+ channel opening effect of Bay K8644 was completely antagonized by mefloquine. Likewise, the increase of [Ca 2+ ] i and of insulin secretion stimulated by 40 mM KCl, but not that by 30 mM glucose was antagonized by 50 μM mefloquine. Neither at 5 μM nor at 50 μM did mefloquin stimulate insulin secretion at basal glucose. In conclusion, mefloquine blocks K ATP channels and L-type Ca 2+ channels in pancreatic beta cells in the range from 5 to 50 μM. Thus it inhibits depolarization-induced insulin secretion, but in the presence of a stimulatory glucose concentration additional effects of mefloquine, possibly on intracellular Ca 2+ mobilization, and the metabolic amplification by glucose permit a sustained rate of secretion. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. The lysine deacetylase inhibitor Givinostat inhibits β-cell IL-1β induced IL-1β transcription and processing.

    Science.gov (United States)

    Dahllöf, Mattias S; Christensen, Dan P; Lundh, Morten; Dinarello, Charles A; Mascagni, Paolo; Grunnet, Lars G; Mandrup-Poulsen, Thomas

    2012-01-01

    Pro-inflammatory cytokines and chemokines, in particular IL-1β, IFNγ, and CXCL10, contribute to β-cell failure and loss in DM via IL-1R, IFNγR, and TLR4 signaling. IL-1 signaling deficiency reduces diabetes incidence, islet IL-1β secretion, and hyperglycemia in animal models of diabetes. Further, IL-1R antagonism improves normoglycemia and β-cell function in type 2 diabetic patients. Inhibition of lysine deacetylases (KDACi) counteracts β-cell toxicity induced by the combination of IL-1 and IFNγ and reduces diabetes incidence in non-obese diabetic (NOD) mice. We hypothesized that KDACi breaks an autoinflammatory circuit by differentially preventing β-cell expression of the β-cell toxic inflammatory molecules IL-1β and CXCL10 induced by single cytokines. CXCL10 did not induce transcription of IL-1β mRNA. IL-1β induced β-cell IL-1β mRNA and both IL-1β and IFNγ individually induced Cxcl10 mRNA transcription. Givinostat inhibited IL-1β-induced IL-1β mRNA expression in INS-1 and rat islets and IL-1β processing in INS-1 cells. Givinostat also reduced IFNγ induced Cxcl10 transcription in INS-1 cells but not in rat islets, while IL-1β induced Cxcl10 transcription was unaffected in both. INS-1 cells and rat islets of Langerhans were exposed to IL-1β, IFNγ or CXCL10 in the presence or absence of KDACi (givinostat). Cytokine and chemokine mRNA expressions were quantified by real-time qPCR, and IL-1β processing by western blotting of cell lysates. Inhibition of β-cell IL-1β expression and processing and Cxcl10 transcription contributes to the β-cell protective actions of KDACi. In vitro β-cell destructive effects of CXCL10 are not mediated via IL-1β transcription. The differential proinflammatory actions of KDACs may be attractive novel drug targets in DM.

  13. Aurora kinase inhibitors attached to iron oxide nanoparticles enhances inhibition of the growth of liver cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xiquan [Southeast University, State Key Laboratory of Bioelectronics and Jiangsu Key Laboratory for Biomaterials and Devices, School of Biological Science & Medical Engineering (China); Xie, Li [Southeast University, Zhongda Hospital, School of Medicine (China); Zheng, Ming; Yao, Juan [Jiangsu Chai Tai Tianqing Pharmaceutical Co. Ltd. (China); Song, Lina [Southeast University, State Key Laboratory of Bioelectronics and Jiangsu Key Laboratory for Biomaterials and Devices, School of Biological Science & Medical Engineering (China); Chang, Weiwei [Jiangsu Chai Tai Tianqing Pharmaceutical Co. Ltd. (China); Zhang, Yu; Ji, Min, E-mail: minji888@hotmail.com; Gu, Ning, E-mail: guning@seu.edu.cn [Southeast University, State Key Laboratory of Bioelectronics and Jiangsu Key Laboratory for Biomaterials and Devices, School of Biological Science & Medical Engineering (China); Zhan, Xi, E-mail: zhan01@gmail.com [University of Maryland School of Medicine, The Center of Vascular and Inflammatory Diseases, The Department of Pathology (United States)

    2015-06-15

    We have developed a novel Aurora kinase inhibitor (AKI) AM-005, an analogue of pan-AKI AT-9283. To improve the intracellular efficacy of AM-005 and AT-9283, we utilized magnetite nanoparticles (NPs) to deliver AM-005 and AT-9283 into human SMMC-7721 and HepG2 liver cancer cells. The drug-loaded NPs were prepared through quasi-emulsion solvent diffusion of magnetite NPs with AM-005 or AT-9283. The encapsulated drugs were readily released from NPs, preferentially at low pHs. Upon exposure, cancer cells effectively internalized drug-loaded NPs into lysosome-like vesicles, which triggered a series of cellular changes, including the formation of enlarged cytoplasm, the significant increase of membrane permeability, and the generation of reactive oxygen species (ROS). The increased ROS synthesis sustained over 72 h, whereas that in the cells treated with free-form drugs declined rapidly after 48 h. However, chemical sequestration of the iron core of NPs had a minor influence on the generation of intracellular ROS. On the other hand, uncoupling of AM-005 uptake with NP internalization into cells failed to induce ROS synthesis. Overall, our approach achieved two-fold increase in suppressing the viability of tumor cells in vitro and the growth of tumors in vivo. We conclude that magnetite NPs can be used as pH responsive nanocarriers that are able to improve the efficacy of AKIs.

  14. Effect of temperature on the corrosion inhibition of iron in liquid lead using oxygen inhibitor: studied by MD simulation

    Science.gov (United States)

    Arkundato, Artoto; Monado, Fiber; Su'ud, Zaki

    2017-05-01

    For corrosion mitigation of steels used in a fast nuclear reactor power plant, oxygen gas is one of promising candidates of inhibitors. Many experiments have been conducted to reveal the mechanism of corrosion and mechanism of how to overcome the corrosion. In the previous work, we had shown computationally that the oxygen atom can be used to reduce the corrosion and we had predicted the oxygen contents. In the current work, not only to explore deeeper the ability of oxygen gas to reduce the corrosion, but also to include the variation of used temperature. We still used iron material to represent a real steels. Using MD (molecular dynamics) simulation based on the Lennard-Jones interaction potential, we sought to understand the concentration of oxygen gas as variation of temperature used in the reactor for the best corrosion mitigation. From this work, we conclude that the temperature does not give effect in related with how concentration of injected oxygen. The temparature merely affects to rise the diffusion coefficient of iron in liquid lead, yet it does not influence how much oxygen needed for corrosion mitigation. In this work, all simulations on different series of temperatures (1023°K, 1073°K, 1123°K, 1173°K) reveals that oxygen content of 0.1151wt% will cause the lowest corrosion level of iron in liquid lead.

  15. Nuclear import inhibitor N-(4-hydroxyphenyl) retinamide targets Zika virus (ZIKV) nonstructural protein 5 to inhibit ZIKV infection.

    Science.gov (United States)

    Wang, Chunxiao; Yang, Sundy N Y; Smith, Kate; Forwood, Jade K; Jans, David A

    2017-12-02

    In the absence of approved therapeutics, Zika virus (ZIKV)'s recent prolific outbreaks in the Americas, together with impacts on unborn fetuses of infected mothers, make it a pressing human health concern worldwide. Although a key player in viral replication in the infected host cell cytoplasm, ZIKV non-structural protein 5 (NS5) appears to contribute integrally to pathogenesis by localising in the host cell nucleus, in similar fashion to NS5 from Dengue virus (DENV). We show here for the first time that ZIKV NS5 is recognized with high nanomolar affinity by the host cell importin α/β1 heterodimer, and that this interaction can be blocked by the novel DENV NS5 targeting inhibitor N-(4-hydroxyphenyl) retinamide (4-HPR). Importantly, we show that 4-HPR has potent anti-ZIKV activity at low μM concentrations. With an established safety profile for human use, 4-HPR represents an exciting possibility as an anti-ZIKV agent. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. The Plasmodium PI(4)K inhibitor KDU691 selectively inhibits dihydroartemisinin-pretreated Plasmodium falciparum ring-stage parasites.

    Science.gov (United States)

    Dembele, L; Ang, X; Chavchich, M; Bonamy, G M C; Selva, J J; Lim, M Yi-Xiu; Bodenreider, C; Yeung, B K S; Nosten, F; Russell, B M; Edstein, M D; Straimer, J; Fidock, D A; Diagana, T T; Bifani, P

    2017-05-24

    Malaria control and elimination are threatened by the emergence and spread of resistance to artemisinin-based combination therapies (ACTs). Experimental evidence suggests that when an artemisinin (ART)-sensitive (K13 wild-type) Plasmodium falciparum strain is exposed to ART derivatives such as dihydroartemisinin (DHA), a small population of the early ring-stage parasites can survive drug treatment by entering cell cycle arrest or dormancy. After drug removal, these parasites can resume growth. Dormancy has been hypothesized to be an adaptive physiological mechanism that has been linked to recrudescence of parasites after monotherapy with ART and, possibly contributes to ART resistance. Here, we evaluate the in vitro drug sensitivity profile of normally-developing P. falciparum ring stages and DHA-pretreated dormant rings (DP-rings) using a panel of antimalarial drugs, including the Plasmodium phosphatidylinositol-4-OH kinase (PI4K)-specific inhibitor KDU691. We report that while KDU691 shows no activity against rings, it is highly inhibitory against DP-rings; a drug effect opposite to that of ART. Moreover, we provide evidence that KDU691 also kills DP-rings of P. falciparum ART-resistant strains expressing mutant K13.

  17. Radioimmunoassay of human muscle carbonic anhydrase III in dystrophic states

    International Nuclear Information System (INIS)

    Heath, R.; Jeffery, S.; Carter, N.

    1982-01-01

    A radioimmunoassay for the human isozyme carbonic anhydrase III (CAIII) has been developed. The assay can detect levels as low as 4μg/l of sample. Plasma CAIII levels in patients suffering from Duchenne muscular dystrophy were found to be up to 39 times greater than in a control group. Urine CAIII levels in patients suffering from Duchenne muscular dystrophy were not significantly different from the levels found in urine from normal adults. Measurement of plasma CAIII levels may be useful in prenatal diagnosis of Duchenne muscular dystrophy, and in investigation of adult skeletal muscle disease. (Auth.)

  18. Generation of nitric oxide from nitrite by carbonic anhydrase

    DEFF Research Database (Denmark)

    Aamand, Rasmus; Dalsgaard, Thomas; Jensen, Frank B

    2009-01-01

    In catalyzing the reversible hydration of CO2 to bicarbonate and protons, the ubiquitous enzyme carbonic anhydrase (CA) plays a crucial role in CO2 transport, in acid-base balance, and in linking local acidosis to O2 unloading from hemoglobin. Considering the structural similarity between...... bicarbonate and nitrite, we hypothesized that CA uses nitrite as a substrate to produce the potent vasodilator nitric oxide (NO) to increase local blood flow to metabolically active tissues. Here we show that CA readily reacts with nitrite to generate NO, particularly at low pH, and that the NO produced...

  19. Microbial Carbonic Anhydrases in Biomimetic Carbon Sequestration for Mitigating Global Warming: Prospects and Perspectives.

    Science.gov (United States)

    Bose, Himadri; Satyanarayana, Tulasi

    2017-01-01

    All the leading cities in the world are slowly becoming inhospitable for human life with global warming playing havoc with the living conditions. Biomineralization of carbon dioxide using carbonic anhydrase (CA) is one of the most economical methods for mitigating global warming. The burning of fossil fuels results in the emission of large quantities of flue gas. The temperature of flue gas is quite high. Alkaline conditions are necessary for CaCO 3 precipitation in the mineralization process. In order to use CAs for biomimetic carbon sequestration, thermo-alkali-stable CAs are, therefore, essential. CAs must be stable in the presence of various flue gas contaminants too. The extreme environments on earth harbor a variety of polyextremophilic microbes that are rich sources of thermo-alkali-stable CAs. CAs are the fastest among the known enzymes, which are of six basic types with no apparent sequence homology, thus represent an elegant example of convergent evolution. The current review focuses on the utility of thermo-alkali-stable CAs in biomineralization based strategies. A variety of roles that CAs play in various living organisms, the use of CA inhibitors as drug targets and strategies for overproduction of CAs to meet the demand are also briefly discussed.

  20. Microbial Carbonic Anhydrases in Biomimetic Carbon Sequestration for Mitigating Global Warming: Prospects and Perspectives

    Directory of Open Access Journals (Sweden)

    Himadri Bose

    2017-08-01

    Full Text Available All the leading cities in the world are slowly becoming inhospitable for human life with global warming playing havoc with the living conditions. Biomineralization of carbon dioxide using carbonic anhydrase (CA is one of the most economical methods for mitigating global warming. The burning of fossil fuels results in the emission of large quantities of flue gas. The temperature of flue gas is quite high. Alkaline conditions are necessary for CaCO3 precipitation in the mineralization process. In order to use CAs for biomimetic carbon sequestration, thermo-alkali-stable CAs are, therefore, essential. CAs must be stable in the presence of various flue gas contaminants too. The extreme environments on earth harbor a variety of polyextremophilic microbes that are rich sources of thermo-alkali-stable CAs. CAs are the fastest among the known enzymes, which are of six basic types with no apparent sequence homology, thus represent an elegant example of convergent evolution. The current review focuses on the utility of thermo-alkali-stable CAs in biomineralization based strategies. A variety of roles that CAs play in various living organisms, the use of CA inhibitors as drug targets and strategies for overproduction of CAs to meet the demand are also briefly discussed.

  1. Purification and characterization of carbonic anhydrase from sheep kidney and effects of sulfonamides on enzyme activity.

    Science.gov (United States)

    Demirdağ, Ramazan; Çomaklı, Veysel; Şentürk, Murat; Ekinci, Deniz; İrfan Küfrevioğlu, Ö; Supuran, Claudiu T

    2013-03-15

    Carbonic anhydrase (CA, EC: 4.2.1.1) was purified from sheep kidney by affinity chromatography on a Sepharose 4B-tyrosine-sulfanilamide column. By means of two consecutive procedures, the enzyme (sCA) was purified 227.61-fold with a yield of 60.75%, and a specific activity of 838.89U/mg proteins. The optimum temperature, ionic strength and pH were determined to be 35°C, 20mM and 8.5, respectively. The molecular weight determined by SDS-PAGE was found to be 29kDa. The kinetic parameters, KM and Vmax values were determined for the 4-nitrophenyl acetate (p-NpA) hydrolysis reaction. Some sulfonamides were tested as inhibitors against the purified CAs enzyme. The Ki constants for benzenesulfonamide (1), sulfanilamide (2), mafenide (3), 4-(2-aminoethyl) benzenesulfonamide (4), 4-methyl-benzenesulfonamide (5), 2-bromo-benzenesulfonamide (6), naphthalene-2-sulfonamide (7), 4-amino-6-chlorobenzene-1,3-disulfonamide (8) and saccharin (9) were in the range 1.348-69.31μM. Copyright © 2012 Elsevier Ltd. All rights reserved.

  2. The identification of secreted carbonic anhydrase VI as a constitutive glycoprotein of human and rat milk.

    Science.gov (United States)

    Karhumaa, P; Leinonen, J; Parkkila, S; Kaunisto, K; Tapanainen, J; Rajaniemi, H

    2001-09-25

    In addition to essential nutrients, human milk contains several classes of bioactive factors such as enzymes, hormones, and growth factors, many of which are implicated in infantile growth and development. Secretory carbonic anhydrase isoenzyme VI (CA VI) has been identified earlier as an essential component of mammalian saliva, and we demonstrate here by using biochemical and immunohistochemical techniques that it is also an elementary component of milk. The 42-kDa glycopolypeptide purified from human milk in CA inhibitor affinity chromatography shared 100% homology with salivary CA VI in the protein sequence analysis (40% coverage), and its digestion with PNGase F resulted in a polypeptide backbone similar in size to salivary CA VI. Quantification of CA VI in milk by using a time-resolved immunofluorometric assay revealed an approximately eight-times-higher concentration in human colostrum than in mature milk, the latter corresponding to the levels previously detected in human saliva. The high concentration in the colostrum, in particular its functional and structural stability in an acidic milieu, and its growth-supporting role in the taste buds suggest that milk CA VI is an essential factor in normal growth and development of the infant alimentary tract.

  3. Synthesis of isoxazole-containing sulfonamides with potent carbonic anhydrase II and VII inhibitory properties.

    Science.gov (United States)

    Altug, Cevher; Güneş, Hanife; Nocentini, Alessio; Monti, Simona Maria; Buonanno, Martina; Supuran, Claudiu T

    2017-02-15

    Two series of benzenesulfonamide containing isoxazole compounds were prepared by using conventional and microwave (MW) methods. 5-Amino-3-aryl-N-(4-sulfamoylphenyl)isoxazole-4-carboxamide derivatives were synthesized by the reaction of hydroxymoyl chlorides with 2-cyano-N-(4-sulfamoylphenyl)acetamide in the presence of triethylamine. The synthesized 5-amino isoxazoles were reacted with various benzoyl chlorides in order to obtain 5-amidoisoxazoles. The novel compounds were screened in vitro as inhibitors of four human (h) isoforms of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1): hCA I, hCA II, hCA IV and hCA VII. The derivatives of the first series were shown to possess excellent inhibitory activity against the cytosolic isoform hCA II, an antiglaucoma drug target, with K I s in the range of 0.5-49.3nM and hCA VII, a recently validated anti-neuropathic pain target with K I s in the range of 4.3-51.9nM. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Inhibition of dengue virus replication by novel inhibitors of RNA-dependent RNA polymerase and protease activities.

    Science.gov (United States)

    Pelliccia, Sveva; Wu, Yu-Hsuan; Coluccia, Antonio; La Regina, Giuseppe; Tseng, Chin-Kai; Famiglini, Valeria; Masci, Domiziana; Hiscott, John; Lee, Jin-Ching; Silvestri, Romano

    2017-12-01

    Dengue virus (DENV) is the leading mosquito-transmitted viral infection in the world. With more than 390 million new infections annually, and up to 1 million clinical cases with severe disease manifestations, there continues to be a need to develop new antiviral agents against dengue infection. In addition, there is no approved anti-DENV agents for treating DENV-infected patients. In the present study, we identified new compounds with anti-DENV replication activity by targeting viral replication enzymes - NS5, RNA-dependent RNA polymerase (RdRp) and NS3 protease, using cell-based reporter assay. Subsequently, we performed an enzyme-based assay to clarify the action of these compounds against DENV RdRp or NS3 protease activity. Moreover, these compounds exhibited anti-DENV activity in vivo in the ICR-suckling DENV-infected mouse model. Combination drug treatment exhibited a synergistic inhibition of DENV replication. These results describe novel prototypical small anti-DENV molecules for further development through compound modification and provide potential antivirals for treating DENV infection and DENV-related diseases.

  5. Broad-spectrum inhibition of common respiratory RNA viruses by a pyrimidine synthesis inhibitor with involvement of the host antiviral response.

    Science.gov (United States)

    Cheung, Nam Nam; Lai, Kin Kui; Dai, Jun; Kok, Kin Hang; Chen, Honglin; Chan, Kwok-Hung; Yuen, Kwok-Yung; Kao, Richard Yi Tsun

    2017-05-01

    Our previous screening of 50 240 structurally diverse compounds led to the identification of 39 influenza A virus infection inhibitors (Kao R.Y., Yang D., Lau L.S., Tsui W.H., Hu L. et al. Nat Biotechnol 2010;28:600-605). Further screening of these compounds against common respiratory viruses led to the discovery of compound FA-613. This inhibitor exhibited low micromolar antiviral activity against various influenza A and B virus strains, including the highly pathogenic influenza A strains H5N1 and H7N9, enterovirus A71, respiratory syncytial virus, human rhinovirus A, SARS- and MERS-coronavirus. No significant cellular toxicity was observed at the effective concentrations. Animal studies showed an improved survival rate in BALB/c mice that received intranasal FA-613 treatments against a lethal dose infection of A/HK/415742Md/2009 (H1N1). Further cell-based assays indicated that FA-613 interfer with the de novo pyrimidine biosynthesis pathway by targeting the dihydroorotate dehydrogenase. Surprisingly, FA-613 lost its antiviral potency in the interferon-deficient Vero cell line, while maintaining its inhibitory activity in an interferon-competent cell line which showed elevated expression of host antiviral genes when infected in the presence of FA-613. Further investigation of the specific connection between pyrimidine synthesis inhibition and the induction of host innate immunity might aid clinical development of this type of drug in antiviral therapies. Therefore, in acute cases of respiratory tract infections, when rapid diagnostics of the causative agent are not readily available, an antiviral drug with properties like FA-613 could prove to be very valuable.

  6. Role of 5'TG3'-interacting factors (TGIFs) in Vorinostat (HDAC inhibitor)-mediated Corneal Fibrosis Inhibition.

    Science.gov (United States)

    Sharma, Ajay; Sinha, Nishant R; Siddiqui, Saad; Mohan, Rajiv R

    2015-01-01

    We have previously reported that vorinostat, an FDA-approved, clinically used histone deacetylase (HDAC) inhibitor, attenuates corneal fibrosis in vivo in rabbits by blocking transforming growth factor β (TGFβ). The 5'TG3'-interacting factors (TGIFs) are transcriptional repressors of TGFβ1 signaling via the Smad pathway. The present study was designed to explore the expression of TGIFs in human corneal fibroblasts and to investigate their role in mediating the antifibrotic effect of vorinostat. Human corneal fibroblast cultures were generated from donor corneas. RNA isolation, cDNA preparation, and PCR were performed to detect the presence of TGIF1 and TGIF2 transcripts. The cultures were exposed to vorinostat (2.5 µM) to test its effect on TGIF mRNA and protein levels using qPCR and immunoblotting. Myofibroblast formation was induced with TGFβ1 (5 ng/ml) treatment under serum-free conditions. The changes in fibrosis parameters were quantified by measuring fibrosis marker α-smooth muscle actin (αSMA) mRNA and protein levels with qPCR, immunostaining, and immunoblotting. Smad2/3/4 and TGIF knockdowns were performed using pre-validated RNAi/siRNAs and a commercially available transfection reagent. Human corneal fibroblasts showed the expression of TGIF1 and TGIF2. Vorinostat (2.5 µM) caused a 2.8-3.3-fold increase in TGIF1 and TGIF2 mRNA levels and a 1.4-1.8-fold increase in TGIF1 and TGIF2 protein levels. Vorinostat treatment also caused a significant increase in acetylhistone H3 and acetylhistone H4. Vorinostat-induced increases in TGIF1 and TGIF2 were accompanied by a concurrent decrease in corneal fibrosis, as indicated by a decrease in αSMA mRNA by 83±7.7% and protein levels by 97±5%. The RNAi-mediated knockdown of Smad2, Smad3, and Smad4 markedly attenuated TGFβ1-evoked transdifferentiation of fibroblasts to myofibroblasts. The siRNA-mediated knockdown of TGIF1 and TGIF2 neutralized vorinostat-evoked decreases in αSMA mRNA by 31%-45% and protein

  7. HET0016, a selective inhibitor of 20-HETE synthesis, decreases pro-angiogenic factors and inhibits growth of triple negative breast cancer in mice.

    Directory of Open Access Journals (Sweden)

    Thaiz Ferraz Borin

    Full Text Available A selective inhibitor of 20-HETE synthesis, HET0016, has been reported to inhibit angiogenesis. 20-HETE has been known as a second mitogenic messenger of angiogenesis inducing growth factors. HET0016 effects were analyzed on MDA-MB-231 derived breast cancer in mouse and in vitro cell line. MDA-MB-231 tumor cells were implanted in animals' right flank and randomly assigned to early (1 and 2, starting treatments on day 0, or delayed groups (3 and 4 on day 8 after implantation of tumor. Animals received HET0016 (10 mg/kg treatment via intraperitoneal injection for 5 days/week for either 3 or 4 weeks. Control group received vehicle treatment. Tumor sizes were measured on days 7, 14, 21, and 28 and the animals were euthanized on day 22 and 29. Proteins were extracted from the whole tumor and from cells treated with 10 µM HET0016 for 4 and 24 hrs. Protein array kits of 20 different cytokines/factors were used. ELISA was performed to observe the HIF-1α and MMP-2 protein expression. Other markers were confirmed by IHC. HET0016 significantly inhibited tumor growth in all treatment groups at all-time points compared to control (p<0.05. Tumor growth was completely inhibited on three of ten animals on early treatment group. Treatment groups showed significantly lower expression of pro-angiogenic factors compared to control at 21 days; however, there was no significant difference in HIF-1α expression after treatments. Similar results were found in vitro at 24 hrs of HET0016 treatment. After 28 days, significant increase of angiogenin, angiopoietin-1/2, EGF-R and IGF-1 pro-angiogenic factors were found (p<0.05 compared to control, as well as an higher intensity of all factors were found when compared to that of 21 day's data, suggesting a treatment resistance. HET0016 inhibited tumor growth by reducing expression of different set of pro-angiogenic factors; however, a resistance to treatment seemed to happen after 21 days.

  8. A group-specific inhibitor of lysosomal cysteine proteinases selectively inhibits both proteolytic degradation and presentation of the antigen dinitrophenyl-poly-L-lysine by guinea pig accessory cells to T cells

    DEFF Research Database (Denmark)

    Buus, S; Werdelin, O

    1986-01-01

    A limited intralysosomal proteolytic degradation is probably a key event in the accessory cell processing of large protein antigens before their presentation to T cells. With the aid of highly specific inhibitors of proteinases, we have examined the role of proteolysis in the presentation...... of antigens by guinea pig accessory cells. The proteinase inhibitor benzyloxycarbonyl-phenylalanylalanine-diazomethyl-ketone, which selectively inhibits cysteine proteinases, was used to block this set of enzymes in cultured cells. We demonstrate that the selective inhibition of the cysteine proteinases...... of antigen-presenting cells causes a profound inhibition of both the proteolytic degradation and the presentation of the synthetic antigen dinitrophenyl-poly-L-lysine. In contrast, the presentation of another synthetic antigen, the copolymer of L-glutamic acid and L-alanine, was enhanced by the same...

  9. Carbonic anhydrase 5 regulates acid-base homeostasis in zebrafish.

    Directory of Open Access Journals (Sweden)

    Ruben Postel

    Full Text Available The regulation of the acid-base balance in cells is essential for proper cellular homeostasis. Disturbed acid-base balance directly affects cellular physiology, which often results in various pathological conditions. In every living organism, the protein family of carbonic anhydrases regulate a broad variety of homeostatic processes. Here we describe the identification, mapping and cloning of a zebrafish carbonic anhydrase 5 (ca5 mutation, collapse of fins (cof, which causes initially a collapse of the medial fins followed by necrosis and rapid degeneration of the embryo. These phenotypical characteristics can be mimicked in wild-type embryos by acetazolamide treatment, suggesting that CA5 activity in zebrafish is essential for a proper development. In addition we show that CA5 regulates acid-base balance during embryonic development, since lowering the pH can compensate for the loss of CA5 activity. Identification of selective modulators of CA5 activity could have a major impact on the development of new therapeutics involved in the treatment of a variety of disorders.

  10. Role of Zinc in Catalytic Activity of Carbonic Anhydrase IX

    Science.gov (United States)

    Tu, Chingkuang; Foster, Lauren; Alvarado, Andrea; McKenna, Robert; Silverman, David N.; Frost, Susan C.

    2012-01-01

    The carbonic anhydrases (CAs) in the α class are zinc-dependent metalloenzymes. Previous studies have reported that recombinant forms of carbonic anhydrase IX (CAIX), a membrane-bound form of CA expressed in solid tumors, appear to be activated by low levels of zinc independent of its well-studied role at the catalytic site. In this study, we sought to determine if CAIX is stimulated by zinc in its native environment. MDA-MB-231 breast cancer cells express CAIX in response to hypoxia. We compared CAIX activity associated with membrane ghosts isolated from hypoxic cells with that in intact hypoxic cells. We measured CA activity directly using 18O exchange from 13CO2 into water determined by membrane inlet mass spectrometry. In membrane ghosts, there was little effect of zinc at low concentrations on CAIX activity, although at high concentration zinc was inhibitory. In intact cells, zinc had no significant effect on CAIX activity. This suggests that there is an appreciable decrease in sensitivity to zinc when CAIX is in its natural membrane milieu compared to the purified forms. PMID:22465027

  11. SphK1 inhibitor SKI II inhibits the proliferation of human hepatoma HepG2 cells via the Wnt5A/β-catenin signaling pathway.

    Science.gov (United States)

    Liu, Hong; Zhang, Cai-Xia; Ma, Yan; He, Hong-Wei; Wang, Jia-Ping; Shao, Rong-Guang

    2016-04-15

    Sphingosine 1-phosphate (S1P) promotes cell growth, proliferation and survival. Sphingosine kinase 1 (SphK1), which converts sphingosine to S1P, is a key promoter in cancer. We previously found that the SphK1 inhibitor II (SKI II), suppresses the cell growth and induces apoptosis in human hepatoma HepG2 cells. However, the precise regulatory mechanism and signaling pathway on SKI II inhibiting tumor growth remains unknown. The expressions of β-catenin and related molecules of Wnt/β-catenin signal were detected by western blot in HepG2 cells. And the mRNA expression of β-catenin was detected by RT-PCR. The Wnt5A gene was silenced by siRNA. The colony formation was determined by staining with crystal violet. And the cell growth was examined by SRB assay and BrdU assay. We found that SKI II decreased the expression of β-catenin and the downstream molecules of β-catenin signal pathway and promotes the β-catenin degradation. In addition, SKI II induced the expression of Wnt5A, and then triggered β-catenin degradation. Furthermore, silencing Wnt5A decreased the anti-tumor effects of SKI II through recovering the expressions of β-catenin and downstream molecules of β-catenin signal pathway. SKI II-induced downregulation of HepG2 cell proliferation was associated with Wnt signaling pathway through Wnt5A-mediated β-catenin degradation. Our study revealed that a novel signal pathway was involved in SKI II-inhibited cell proliferation in human hepatoma cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Deoxyhypusine hydroxylase from Plasmodium vivax, the neglected human malaria parasite: molecular cloning, expression and specific inhibition by the 5-LOX inhibitor zileuton.

    Directory of Open Access Journals (Sweden)

    Veronika Anyigoh Atemnkeng

    Full Text Available Primaquine, an 8-aminoquinoline, is the only drug which cures the dormant hypnozoites of persistent liver stages from P. vivax. Increasing resistance needs the discovery of alternative pathways as drug targets to develop novel drug entities. Deoxyhypusine hydroxylase (DOHH completes hypusine biosynthesis in eukaryotic initiation factor (eIF-5A which is the only cellular protein known to contain the unusual amino acid hypusine. Modified EIF-5A is important for proliferation of the malaria parasite. Here, we present the first successful cloning and expression of DOHH from P. vivax causing tertiary malaria. The nucleic acid sequence of 1041 bp encodes an open reading frame of 346 amino acids. Histidine tagged expression of P. vivax DOHH detected a protein of 39.01 kDa in E. coli. The DOHH protein from P. vivax shares significant amino acid identity to the simian orthologues from P. knowlesi and P. yoelii strain H. In contrast to P. falciparum only four E-Z-type HEAT-like repeats are present in P. vivax DOHH with different homology to phycocyanin lyase subunits from cyanobacteria and in proteins participating in energy metabolism of Archaea and Halobacteria. However, phycocyanin lyase activity is absent in P. vivax DOHH. The dohh gene is present as a single copy gene and transcribed throughout the whole erythrocytic cycle. Specific inhibition of recombinant P. vivax DOHH is possible by complexing the ferrous iron with zileuton, an inhibitor of mammalian 5-lipoxygenase (5-LOX. Ferrous iron in the active site of 5-LOX is coordinated by three conserved histidines and the carboxylate of isoleucine(673. Zileuton inhibited the P. vivax DOHH protein with an IC50 of 12,5 nmol determined by a relative quantification by GC/MS. By contrast, the human orthologue is only less affected with an IC50 of 90 nmol suggesting a selective iron-complexing strategy for the parasitic enzyme.

  13. Insight into the molecular basis of pathogen abundance: group A Streptococcus inhibitor of complement inhibits bacterial adherence and internalization into human cells.

    Science.gov (United States)

    Hoe, Nancy P; Ireland, Robin M; DeLeo, Frank R; Gowen, Brian B; Dorward, David W; Voyich, Jovanka M; Liu, Mengyao; Burns, Eugene H; Culnan, Derek M; Bretscher, Anthony; Musser, James M

    2002-05-28

    Streptococcal inhibitor of complement (Sic) is a secreted protein made predominantly by serotype M1 Group A Streptococcus (GAS), which contributes to persistence in the mammalian upper respiratory tract and epidemics of human disease. Unexpectedly, an isogenic sic-negative mutant adhered to human epithelial cells significantly better than the wild-type parental strain. Purified Sic inhibited the adherence of a sic negative serotype M1 mutant and of non-Sic-producing GAS strains to human epithelial cells. Sic was rapidly internalized by human epithelial cells, inducing cell flattening and loss of microvilli. Ezrin and moesin, human proteins that functionally link the cytoskeleton to the plasma membrane, were identified as Sic-binding proteins by affinity chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis. Sic colocalized with ezrin inside epithelial cells and bound to the F-actin-binding site region located in the carboxyl terminus of ezrin and moesin. Synthetic peptides corresponding to two regions of Sic had GAS adherence-inhibitory activity equivalent to mature Sic and inhibited binding of Sic to ezrin. In addition, the sic mutant was phagocytosed and killed by human polymorphonuclear leukocytes significantly better than the wild-type strain, and Sic colocalized with ezrin in discrete regions of polymorphonuclear leukocytes. The data suggest that binding of Sic to ezrin alters cellular processes critical for efficient GAS contact, internalization, and killing. Sic enhances bacterial survival by enabling the pathogen to avoid the intracellular environment. This process contributes to the abundance of M1 GAS in human infections and their ability to cause epidemics.

  14. Fluorocitrate, an inhibitor of glial metabolism, inhibits the up-regulation of NOS expression, activity and NO production in the spinal cord induced by formalin test in rats.

    Science.gov (United States)

    Sun, Xiao-Cai; Chen, Wei-Na; Li, Shu-Qin; Cai, Jin-Song; Li, Wen-Bin; Xian, Xiao-Hui; Hu, Yu-Yan; Zhang, Min; Li, Qing-Jun

    2009-02-01

    Previous experiments have suggested that nitric oxide plays an important role in nociceptive transmission in the spinal cord. In order to explore the involvement of glia in the NO-mediated nociceptive transmission, the present study was undertaken to investigate the effect of fluorocitrate (FC), an inhibitor of glial metabolism, on NOS expression and activity and NO production in the spinal cord during the process of peripheral inflammatory pain and hyperalgesia induced by formalin test in rats. Sixty adult male Sprague-Dawley rats were randomly assigned into sham, formalin, formalin + normal saline (NS), and formalin + FC groups. The NOS expression, NOS activity and NO production was detected by NADPH-d histochemistry staining, NOS and NO assay kit, respectively. It was found that formalin test significantly up-regulated NOS expression and activity and NO production in the laminae I-II of the dorsal horn and the grey matter around the central canal in the lumbar spinal cord at 1 h after the formalin test. Selective inhibition of glia metabolism with intrathecal administration of FC (1 nmol) significantly inhibited the up-regulation in NOS expression and activity and NO production normally induced by the formalin test, which was represented with decreases in the number and density of the NADPH-d positive cells in the dorsal horn and grey matter around the central canal, and decrease in density of NADPH-d positive neuropil in the dorsal horn in formalin + FC group compared with formalin group. The results suggested that glia may be involved in the NO-mediated nociceptive transmission in the spinal cord.

  15. Suppression of CHRN endocytosis by carbonic anhydrase CAR3 in the pathogenesis of myasthenia gravis.

    Science.gov (United States)

    Du, Ailian; Huang, Shiqian; Zhao, Xiaonan; Feng, Kuan; Zhang, Shuangyan; Huang, Jiefang; Miao, Xiang; Baggi, Fulvio; Ostrom, Rennolds S; Zhang, Yanyun; Chen, Xiangjun; Xu, Congfeng

    2017-01-01

    Myasthenia gravis is an autoimmune disorder of the neuromuscular junction manifested as fatigable muscle weakness, which is typically caused by pathogenic autoantibodies against postsynaptic CHRN/AChR (cholinergic receptor nicotinic) in the endplate of skeletal muscle. Our previous studies have identified CA3 (carbonic anhydrase 3) as a specific protein insufficient in skeletal muscle from myasthenia gravis patients. In this study, we investigated the underlying mechanism of how CA3 insufficiency might contribute to myasthenia gravis. Using an experimental autoimmune myasthenia gravis animal model and the skeletal muscle cell C2C12, we find that inhibition of CAR3 (the mouse homolog of CA3) promotes CHRN internalization via a lipid raft-mediated pathway, leading to accelerated degradation of postsynaptic CHRN. Activation of CAR3 reduces CHRN degradation by suppressing receptor endocytosis. CAR3 exerts this effect by suppressing chaperone-assisted selective autophagy via interaction with BAG3 (BCL2-associated athanogene 3) and by dampening endoplasmic reticulum stress. Collectively, our study illustrates that skeletal muscle cell CAR3 is critical for CHRN homeostasis in the neuromuscular junction, and its deficiency leads to accelerated degradation of CHRN and development of myasthenia gravis, potentially revealing a novel therapeutic approach for this disorder.

  16. The toxicological impacts of some heavy metals on carbonic anhydrase from gilthead sea bream (Sparus aurata) gills.

    Science.gov (United States)

    Kaya, Elif Duygu; Söyüt, Hakan; Beydemir, Şükrü

    2015-03-01

    It is known that heavy metals have toxic effects on fish. Insufficient measures are a serious problem in our country and around the world. This problem can threaten human health in areas where it is common for people to obtain nutrition from local bodies of water. In this study, the toxicological impacts of some heavy metals were investigated on carbonic anhydrase activity in gilthead gills. Carbonic anhydrase (CA) was purified from gilthead sea bream (Sparus aurata) gills with a specific activity of 2872.92 EU mg(-1) and a yield of 32.84% using affinity chromatography. The overall purification was approximately ∼ 84-fold. SDS-polyacrylamide gel electrophoresis showed a single band, and the MW was approximately 30.5 kDa (Soyut et al., 2008, 2012; Soyut and Beydemir, 2008, 2012; Kaya et al., 2013). The kinetic and characteristic properties of CA such as the optimum pH, stable pH, optimum temperature, activation energy (Ea), activation enthalpy (ΔH), Q10, Km and Vmax were determined. Cadmium (Cd(2+)), copper (Cu(2+)), nickel (Ni(2+)) and silver (Ag(+)) inhibited CA activity in in vitro conditions. Ki values were calculated for these metals. Ki values were 31.20mM for cadmium (Cd(2+)), 161.96 mM for copper (Cu(2+)), 10.79 mM for nickel (Ni(2+)) and 0.0082 mM for silver (Ag(+)) based on Lineweaver-Burk plots. Except for cadmium, heavy metals had the same inhibition mechanism. Cadmium was competitive, and the others were noncompetitive. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. New bioactive silver(I) complexes: Synthesis, characterization, anticancer, antibacterial and anticarbonic anhydrase II activities

    Science.gov (United States)

    Ozdemir, Ummuhan O.; Ozbek, Neslihan; Genc, Zuhal Karagoz; İlbiz, Firdevs; Gündüzalp, Ayla Balaban

    2017-06-01

    Silver(I) complexes of alkyl sulfonic acide hydrazides were newly synthesized as homologous series. Methanesulfonic acide hydrazide (L1), ethanesulfonic acide hydrazide (L2), propanesulfonic acide hydrazide (L3) and butanesulfonic acide hydrazide (L4) were used for complexation with Ag(I) ions. The silver complexes obtained in the mol ratio of 1:2 have the structural formula as Ag(L1)2NO3 (I), Ag(L2)2NO3 (II), Ag(L3)2NO3(III), (Ag(L4)2NO3 (IV). The Ag(I) complexes exhibit distorted linear two-fold coordination in [AgL2]+ cations with uncoordinated nitrates. Ligands are chelated with silver(I) ions through unsubstituted primary nitrogen in hydrazide group. Ag(I) complexes were characterized by using elemental analysis, spectroscopic methods (FT-IR, LC-MS), magnetic susceptibility and conductivity measurements. Silver(I) complexes were optimized using PBEPBE/LanL2DZ/DEF2SV basic set performed by DFT method with the Gaussian 09 program package. The geometrical parameters, frontier molecular orbitals (HOMOs and LUMOs) and molecular electrostatic potential (MEP) mapped surfaces of the optimized geometries were also determined by this quantum set. The anticancer activities of silver(I) complexes on MCF-7 human breast cancer cell line were investigated by comparing IC50 values. The antibacterial activities of complexes were studied against Gram positive bacteria; S. aureus ATCC 6538, B. subtilis ATCC 6633, B. cereus NRRL-B-3711, E. faecalis ATCC 29212 and Gram negative bacteria; E. coli ATCC 11230, P. aeruginosa ATCC 15442, K. pneumonia ATCC 70063 by using disc diffusion method. The inhibition activities of Ag(I) complexes on carbonic anhydrase II enzyme (hCA II) were also investigated by comparing IC50 and Ki values. The biological activity screening shows that Ag(I) complex of butanesulfonicacidehydrazide (IV) has the highest activity against tested breast cancer cell lines MCF-7, Gram positive/Gram negative bacteria and carbonic anhydrase II (hCA II) isoenzyme.

  18. The effect of L-carnitine on carbonic anhydrase level in rats exposed ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-07-06

    characterized pH ... In the present study, the effect of L-carnitine on carbonic anhydrase levels in rats exposed to hypothermic stress was investigated. ..... exercise done hypothermic and hyperthermic ambient. (Jansens et al., 1998) ...

  19. Fluorescence lifetime components reveal kinetic intermediate states upon equilibrium denaturation of carbonic anhydrase II.

    Science.gov (United States)

    Nemtseva, Elena V; Lashchuk, Olesya O; Gerasimova, Marina A; Melnik, Tatiana N; Nagibina, Galina S; Melnik, Bogdan S

    2017-12-21

    In most cases, intermediate states of multistage folding proteins are not 'visible' under equilibrium conditions but are revealed in kinetic experiments. Time-resolved fluorescence spectroscopy was used in equilibrium denaturation studies. The technique allows for detecting changes in the conformation and environment of tryptophan residues in different structural elements of carbonic anhydrase II which in its turn has made it possible to study the intermediate states of carbonic anhydrase II under equilibrium conditions. The results of equilibrium and kinetic experiments using wild-type bovine carbonic anhydrase II and its mutant form with the substitution of leucine for alanine at position 139 (L139A) were compared. The obtained lifetime components of intrinsic tryptophan fluorescence allowed for revealing that, the same as in kinetic experiments, under equilibrium conditions the unfolding of carbonic anhydrase II ensues through formation of intermediate states.

  20. Precipitation of hydrated Mg carbonate with the aid of carbonic anhydrase for CO2 sequestration

    Science.gov (United States)

    Power, I. M.; Harrison, A. L.; Dipple, G. M.

    2011-12-01

    Strategies for sequestering CO2 directly from the atmosphere are likely required to achieve the desired reduction in CO2 concentration and avoid the most damaging effects of climate change [1]. Numerous studies have demonstrated the accelerated precipitation of calcium carbonate minerals with the aid of carbonic anhydrase (CA) as a means of sequestering CO2 in solid carbonate form; however, no study has examined precipitation of magnesium carbonate minerals using CA. Precipitation of magnesite (MgCO3) is kinetically inhibited [2]; therefore, Mg2+ must be precipitated as hydrated carbonate minerals. In laboratory experiments, the uptake of atmospheric CO2 into brine solutions (0.1 M Mg) was rate-limiting for the precipitation of dypingite [Mg5(CO3)4(OH)2-5H2O] with initial precipitation requiring 15 days [3]. It was also found that dypingite precipitation outpaced the uptake of CO2 gas into solution. CO2 uptake is limited by the hydration of CO2 to form carbonate ions [4]. Carbonic anhydrase (CA) enzymes are among the fastest known in nature and are able to catalyze the hydration of CO2, i.e., converting CO2(aq) to CO32- and HCO3- [5]. CA plays an important role in the carbon concentrating mechanism of photoautotrophic, chemoautotrophic, and heterotrophic prokaryotes and is involved in pH homeostasis, facilitated diffusion of CO2, ion transport, and the interconversion of CO2 and HCO3- [6]. Introducing CA into buffered Mg-rich solutions should allow for more rapid precipitation of hydrated magnesium carbonate minerals. Batch experiments were conducted using 125 mL flasks containing 100 mL of Millipore deionized water with 0.2 M of MgCl2-6H2O. To buffer pH, 1.0 g of pulverized brucite [Mg(OH)2] or 1.0 g of NaOH was added to the systems, which were amended with Bovine carbonic anhydrase (BCA) (Sigma-Aldrich). Solutions were stirred continuously and kept at room temperature (~22°C) with laboratory air introduced by bubbling. Temperature and pH were measured routinely

  1. Evaluation of a carbonic anhydrase mimic for industrial carbon capture.

    Science.gov (United States)

    Floyd, William C; Baker, Sarah E; Valdez, Carlos A; Stolaroff, Joshuah K; Bearinger, Jane P; Satcher, Joe H; Aines, Roger D

    2013-09-03

    Zinc(II) cyclen, a small molecule mimic of the enzyme carbonic anhydrase, was evaluated under rigorous conditions resembling those in an industrial carbon capture process: high pH (>12), nearly saturated salt concentrations (45% K2CO3) and elevated temperatures (100-130 °C). We found that the catalytic activity of zinc cyclen increased with increasing temperature and pH and was retained after exposure to a 45% w/w K2CO3 solution at 130 °C for 6 days. However, high bicarbonate concentrations markedly reduced the activity of the catalyst. Our results establish a benchmark level of stability and provide qualitative insights for the design of improved small-molecule carbon capture catalysts.

  2. Pilot absorption experiments with carbonic anhydrase enhanced MDEA

    DEFF Research Database (Denmark)

    Gladis, Arne; F. Lomholdt, Niels; Fosbøl, Philip Loldrup

    2017-01-01

    was determined by both a density method and the BaCl2 method. After the solvent was loaded to equilibrium it was heated up and reintroduced into the column, where CO2 was stripped off using air as stripping gas. The addition of CA increased the mass transfer significantly in all experiments. Lower absorption......Mass transfer experiments were carried out on DTU’s pilot absorber unit, a 10 m high column packed with 250 Y Mellapak structured packing. The influence of temperature, solvent loading, column height and liquid flow rates on absorption performance were determined for a 30 wt% N......-methyl-diethanolamine (MDEA) solvent, with and without the enzyme carbonic anhydrase (CA). The absorption experiments were performed at atmospheric pressure and agas phase carbon dioxide mole fraction of 0.13. During experiments liquid samples were withdrawn at each meter of column height and the solvent loading...

  3. Carbonic Anhydrase: An Efficient Enzyme with Possible Global Implications

    Directory of Open Access Journals (Sweden)

    Christopher D. Boone

    2013-01-01

    Full Text Available As the global atmospheric emissions of carbon dioxide (CO2 and other greenhouse gases continue to grow to record-setting levels, so do the demands for an efficient and inexpensive carbon sequestration system. Concurrently, the first-world dependence on crude oil and natural gas provokes concerns for long-term availability and emphasizes the need for alternative fuel sources. At the forefront of both of these research areas are a family of enzymes known as the carbonic anhydrases (CAs, which reversibly catalyze the hydration of CO2 into bicarbonate. CAs are among the fastest enzymes known, which have a maximum catalytic efficiency approaching the diffusion limit of 108 M−1s−1. As such, CAs are being utilized in various industrial and research settings to help lower CO2 atmospheric emissions and promote biofuel production. This review will highlight some of the recent accomplishments in these areas along with a discussion on their current limitations.

  4. X-linked inhibitor of apoptosis inhibition sensitizes acute myeloid leukemia cell response to TRAIL and chemotherapy through potentiated induction of proapoptotic machinery.

    Science.gov (United States)

    Zhou, Jianbiao; Lu, Xiao; Tan, Tuan Zea; Chng, Wee-Joo

    2018-01-01

    Acute myeloid leukemia (AML) is an aggressive disease with an increasing incidence and relatively low 5-year survival rate. Unfortunately, the underlying mechanism of leukemogenesis is poorly known, and there has been little progress in the treatment for AML. Studies have shown that X-linked inhibitor of apoptosis (XIAP), one of the inhibitors of apoptosis proteins (IAPs), is highly expressed and contributes to chemoresistance in AML. Hence, a novel drug, RO6867520 (RO-BIR2), developed by Roche targeting the BIR2 domain in XIAP to reactivate blocked apoptosis, is a promising therapy for AML. The monotherapy of RO-BIR2 had minimal effect on most of the AML cell lines tested except U-937. In contrast to AML cell lines, in general, RO-BIR2 alone has been shown to inhibit the proliferation of primary AML patient samples effectively and induced apoptosis in a dose-dependent manner. A combination of RO-BIR2 with TNF-related apoptosis-inducing ligand (TRAIL) led to highly synergistic effect on AML cell lines and AML patient samples. This combination therapy is capable of inducing apoptosis, thereby leading to an increase in specific apoptotic cell population, along with the activation of caspase 3/7. A number of apoptotic-related proteins such as XIAP, cleavage of caspase 3, cleavage of caspase 7, and cleaved PARP were changed upon combination therapy. Combination of RO-BIR2 with Ara-C had similar effect as the TRAIL combination. Ara-C combination also led to synergistic effect on AML cell lines and AML patient samples with low combination indexes (CIs). We conclude that the combination of RO-BIR2 with either TRAIL or Ara-C represents a potent therapeutic strategy for AML and is warranted for further clinical trials to validate the synergistic benefits in patients with AML, especially for the elderly who are abstaining from intensive chemotherapy. © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

  5. New structure–activity relationships of chalcone inhibitors of breast cancer resistance protein: polyspecificity toward inhibition and critical substitutions against cytotoxicity

    Directory of Open Access Journals (Sweden)

    Rangel LP

    2013-09-01

    Full Text Available Luciana Pereira Rangel,1,2,* Evelyn Winter,1,3,* Charlotte Gauthier,1 Raphaël Terreux,4 Louise D Chiaradia-Delatorre,5 Alessandra Mascarello,5 Ricardo J Nunes,5 Rosendo A Yunes,5 Tania B Creczynski-Pasa,3 Sira Macalou,1 Doriane Lorendeau,1 Hélène Baubichon-Cortay,1 Antonio Ferreira-Pereira,2 Attilio Di Pietro11Equipe Labellisée Ligue 2013, BMSSI UMR 5086 CNRS/Université Lyon 1, Institut de Biologie et Chimie des Protéines, Lyon, France; 2Department of General Microbiology, Institute of Microbiology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; 3Department of Pharmaceutical Sciences, PPGFAR, Federal University of Santa Catarina, Florianopolis, Santa Catarina, Brazil; 4Equipe BISI, BMSSI UMR 5086 CNRS/Université Lyon 1, Institut de Biologie et Chimie des Protéines, Lyon, France; 5Department of Chemistry, Federal University of Santa Catarina, Florianopolis, Santa Catarina, Brazil*These authors contributed equally to this workAbstract: Adenosine triphosphate-binding cassette subfamily G member 2 (ABCG2 plays a major role in cancer cell multidrug resistance, which contributes to low efficacy of chemotherapy. Chalcones were recently found to be potent and specific inhibitors, but unfortunately display a significant cytotoxicity. A cellular screening against ABCG2-mediated mitoxantrone efflux was performed here by flow cytometry on 54 chalcone derivatives from three different series with a wide panel of substituents. The identified leads, with submicromolar IC50 (half maximal inhibitory concentration values, showed that the previously identified 2'-OH-4',6'-dimethoxyphenyl, as A-ring, could be efficiently replaced by a 2'-naphthyl group, or a 3',4'-methylenedioxyphenyl with lower affinity. Such a structural variability indicates polyspecificity of the multidrug transporter for inhibitors. At least two methoxyl groups were necessary on B-ring for optimal inhibition, but substitution at positions 3, 4, and 5 induced cytotoxicity

  6. Inhibition of gamma-ray dose-rate effects by D/sup 2/O and inhibitors of poly(ADP-ribose) synthetase in cultured mammalian L5178Y cells

    Energy Technology Data Exchange (ETDEWEB)

    Ueno, A.M.; Tanaka, O.; Matsudaira, H.

    1984-06-01

    Effects of deuterium oxide (D/sub 2/O) and 3-aminobenzamide, an inhibitor of poly(ADP-ribose) synthetase, on cell proliferation and survival were studied in cultured mammalian L5178Y cells under growing conditions and after acute and low-dose-rate irradiation at about 0.1 to 0.4 Gy/hr of ..gamma.. rays. Growth of irradiated and unirradiated cells was inhibited by 45% D/sub 2/O but not by 3-aminobenzamide at 10mM, except for treatments longer than 30 hr. The presence of these agents either alone or in combination during irradiation at low dose rates suppressed almost totally the decrease in cell killing due to the decrease in dose rate. Among other inhibitors tested, theobromine and theophylline were found to be effective in eliminating the dose-rate effects of ..gamma.. rays. Possible mechanisms underlying the inhibition are discussed.

  7. Diclofenac, a selective COX-2 inhibitor, inhibits DMH-induced colon tumorigenesis through suppression of MCP-1, MIP-1α and VEGF.

    Science.gov (United States)

    Kaur, Jasmeet; Sanyal, S N

    2011-09-01

    Angiogenesis is a physiological process involving growth of new blood vessels from pre-existing ones; however, it also plays a critical role in tumor progression. It favors the transition from hyperplasia to neoplasia, that is, from a state of cellular multiplication to uncontrolled proliferation. Therefore targeting angiogenesis will be profitable as a mechanism to inhibit tumor's lifeline. Further, it is important to understand the cross-communication between vascular endothelial growth factor (VEGF)-master switch in angiogenesis and other molecules in the neoplastic and pro-inflammatory milieu. We studied the role of two important chemokines [monocyte chemoattractant protein (MCP)-1 and macrophage inflammatory protein (MIP)-lα] alongwith VEGF and matrix metalloproteinases (MMPs) in non-steroidal anti-inflammatory drugs (NSAIDs)-induced chemopreventive effect in experimental colon cancer in rat. 1,2-Dimethylhydrazine (DMH, 30 mg/kg body weight, subcutaneously (s.c.) once-a-week) for 18 wk was used as pro-carcinogen and diclofenac (8 mg/kg body weight, orally daily) as the preferential cyclooxygenase-2 (COX-2) inhibitor. Expression of COX-2 and VEGF was found to be significantly elevated in the DMH-treated group as compared to the control, which was lowered notably by Diclofenac co-administration with DMH. Gelatin zymography showed prominent MMP-9 activity in the DMH-treated rats, while the activity was nearly absent in all the other groups. Expression of MCP-1 was found to be markedly increased whereas MIP-1α expression was found to be decreased in colonic mucosa from DMH-treated rats, which was reversed in the DMH + Diclofenac group. Our results indicate potential role of chemokines alongwith VEGF in angiogenesis in DMH-induced cancer and its chemoprevention with diclofenac. Copyright ©2011 Wiley-Liss, Inc.

  8. Methionine-restricted diet inhibits growth of MCF10AT1-derived mammary tumors by increasing cell cycle inhibitors in athymic nude mice

    International Nuclear Information System (INIS)

    Hens, J. R.; Sinha, I.; Perodin, F.; Cooper, T.; Sinha, R.; Plummer, J.; Perrone, C. E.; Orentreich, D.

    2016-01-01

    Dietary methionine restriction (MR) improves healthspan in part by reducing adiposity and by increasing insulin sensitivity in rodent models. The purpose of this study was to determine whether MR inhibits tumor progression in breast cancer xenograft model and breast cancer cell lines. Athymic nude mice were injected with MCF10AT1 cells in Matrigel® and fed a diet containing either 0.86 % methionine (control fed, CF), or 0.12 % methionine (MR) for 12 weeks. Plasma amino acid concentrations were measured by UPLC, and proliferation and apoptosis were examined using RT-PCR, immunohistochemistry, and Cell Titer 96® Aqueous One Solution Cell Proliferation assay. Mice on the MR diet had reduced body weight and decreased adiposity. They also had smaller tumors when compared to the mice bearing tumors on the CF diet. Plasma concentrations of the sulfur amino acids (methionine, cysteine, and taurine) were reduced, whereas ornithine, serine, and glutamate acid were increased in mice on the MR diet. MR mice exhibited decreased proliferation and increased apoptosis in cells that comprise the mammary glands and tumors of mice. Elevated expression of P21 occurred in both MCF10AT1-derived tumor tissue and endogenously in mammary gland tissue of MR mice. Breast cancer cell lines MCF10A and MDA-MB-231 grown in methionine-restricted cysteine-depleted media for 24 h also up-regulated P21 and P27 gene expression, and MDA-MB-231 cells had decreased proliferation. MR hinders cancer progression by increasing cell cycle inhibitors that halt cell cycle progression. The application of MR in a clinical setting may provide a delay in the progression of cancer, which would provide more time for conventional cancer therapies to be effective. The online version of this article (doi:10.1186/s12885-016-2367-1) contains supplementary material, which is available to authorized users

  9. Deubiquitinase inhibitor b-AP15 activates endoplasmic reticulum (ER) stress and inhibits Wnt/Notch1 signaling pathway leading to the reduction of cell survival in hepatocellular carcinoma cells.

    Science.gov (United States)

    Ding, Youming; Chen, Xiaoyan; Wang, Bin; Yu, Bin; Ge, Jianhui

    2018-04-15

    b-AP15, a potent and selective inhibitor of the ubiquitin-specific peptidase 14 (USP14), displays in vitro and in vivo antitumor abilities on some types of cancer cells. However, the mechanism underlying its action is not well elucidated. The purposes of the present study are to observe the potential impacts of b-AP15 on cell survival of hepatocellular carcinoma cells and to investigate whether and how this compound inhibits some survival-promoting signaling pathways. We found that b-AP15 significantly decreased cell viability and increased cell apop